120 Extended Abstracts
1.0.1
Injury prevention initiative - FIFA 11+
S. Della Villa1, M. Bizzini2, A. Junge2, J. Dvorak2
1
Bologna/Italy, 2Zürich/Switzerland
Introduction: Football is the most popular sport in the world, played
by approximately 200´000 professional and 240 million amateur
players. Comparing the exposure-related incidence of injury in
different team sports, ice hockey, handball, basketball, football and
rugby are clearly types of sport with a relatively high risk of injury. The
incidence of football injury has been investigated in several studies
and varies substantially depending on the definition of injury, the
characteristics of the investigated players and the research design.
The majority of studies focus on adult male professional players.
Epidemiological information on injuries in female and youth football
players is limited. From the data presented, it is estimated that on
average every elite male football player incurs approximately one
performance-limiting injury a year.
Content: Several authors have described risk factors for football
injuries, and discussed possibilities for prevention such as: warm-up
(with more emphasis on stretching), regular cool-down, adequate
rehabilitation with sufficient recovery time, proprioceptive training,
protective equipment, good playing field conditions, and adherence
to the existing rules. However, only few authors have reported
results of preventive interventions in football players. Some studies
have focused on the prevention of injuries in general, and others
have evaluated the prevention of specific types of injury, namely
ankle sprains, severe injuries of the knee and hamstring strains.
Summarising the results of these studies, there is some evidence
that multi-modal intervention programmes result in a reduction of
injuries in general. Proprioceptive or neuromuscular training seems
to prevent severe knee injuries and recurrent ankle sprains. The
use of semi-rigid orthosis should be recommended for players with
previous ankle sprains. The eccentric strength training of hamstrings
has recently been shown to be effective in reducing hamstring strains.
However, football injuries can be prevented only partly by improved
physical condition of players. Knowing that a substantial amount of
football injuries are caused by foul play, the observance of the laws of
the game and especially the regard to Fair Play is an essential aspect
in the prevention of injury. Recently, the effectiveness of specific
exercise-based prevention programs in football has been examined.
Research in amateur football has shown that specific programs
were successfully implemented as “standard warm up” prior to the
“routine” training. Recently, the effectiveness of specific exercise-
based prevention program has been examined: the PEP program in
the field of non-contact ACL injury, and moreover the 11 + program,
were able to significantly reduce the injury incidence. “The 11+” injury
prevention program was developed by F-MARC in cooperation with
the Oslo Sports Trauma & Research Center and the Santa Monica
Orthopaedics & Sports Medicine Center. The preventive exercises
focus on core stabilisation, eccentric strength, neuromuscular
control, agility and plyometrics. Good body control, and proper
technique while performing the exercises are the key to enhance
sensorimotor awareness and performance. The “11+” program
represents an advanced version of the “11”, and is the result of a
teamwork within the above mentioned research centers. In a recent
published RCT study, Soligard et al showed that this program was
effective in reducing the incidence of injuries by 1/3 in young female
football players. The risk of severe injuries, overuse injuries, and
injuries overall was significantly reduced. Another interesting finding
in the study was that the results showed a trend toward a lower risk
of injury among the most complaint players, which underlies the
importance of compliance within the implementation of prevention
programs. The teams in the intervention group performed regularly
the “11+” as a routine warm-up, prior to the technical training. The
program consists of three parts: a running part in the beginning and
at the end to warm up, and six set of exercises focusing on core and
legs strength, balance, plyometrics and agility (each exercises set
with 3 levels of increasing difficulty). The “11+” should be performed
at least two to three times a week, and takes about 20 minutes to
be completed.
References:
Junge A, Dvorak J. Soccer Injuries. A Review on Incidence and
Prevention. Sports Med 2004;34(13):929-938 Gilchrist G et al. A
randomized controlled trial to prevent non contact anterior cruciate
ligament injury in female collegiate soccer players. Am J Sports
Med 2008 Soligard T et al. Comprehensive warm-up programme
to prevent injuries in young female footballers: cluster randomized
controlled trial. BMJ 2008
2.1.1
A novel Polycarbonate-urethane meniscal implant: from bench to
clinical use
E. Linder-Ganz1, J.J. Elsner1, G. Zur1, A. Shterling1, R. Arbel2, V.
Condello3, C. Zorzi3, F. Guilak4, E. Hershman5
1
Netanya/Israel, 2Hod Hasharon/Israel, 3Negrar, Verona/Italy,
4
Durham/United States of America, 5New York/United States of
America
Introduction: The menisci play an important role in knee joint
biomechanics. Clinical studies have shown that the loss of the
meniscus leads to degenerative arthritis due to changes in cartilage
load distribution [14]. In these cases, there is clearly a need to protect
the articular cartilage by either repairing or replacing the meniscus.
Meniscus replacement still represents an unsolved problem in
orthopedics. Meniscal allografts have been shown to heal to the
capsule and relieve pain [23]. However, besides problems related
to availability, size matching, cost and risk of disease transmission,
allograft menisci undergo remodeling after implantation, causing
shrinkage and reduced mechanical strength [15,21]. These may lead
to tearing of the allograft and contribute to uneven distribution of
load, instability and recurrence of degenerative damage. Several
meniscal substitutes based on synthetic and natural polymers have
been described [3,5,10,20]. Most of these prostheses are based
on biodegradable materials, which form temporary scaffolds that
degrade in the body and are replaced gradually by newly formed
tissue. Potential shortcomings of this approach include the lack
of durability associated with most biodegradable materials under
in vivo knee loading conditions [8,10], as well as the variability
in the body response to the implant, limited age of the target
population and the quality of the tissue formed. Traditional
unicompartmental knee arthroplasty (UKA) is regaining popularity
but requires significant bone resection and subsequent activity
modification. Total knee replacement (TKR) is a reliable procedure
but is not usually recommended for relatively young patients, less
than fifty-five years of age, who will probably require subsequent
revision surgery. The concept of a self-centering, non-fixed meniscal
interpositional spacer that does not require osseous resection is
an appealing alternative, designed to bridge the gap between the
abovementioned approaches. Polycarbonate-Urethane (PCU) is a
tough polymer with a low elastic modulus (10-100MPa, [16]). It is
an attractive material for a meniscal implant application since it is
durable and offers good mechanical and tribological properties
that are comparable to those of natural cartilage [6,9,16,17].
Specifically, the natural meniscus is an anisotropic material and
therefore it is suggested that circumferential reinforcement of PCU
with high modulus UHMWPE fibers (Dyneema® Purity, DSM) be
used to further improve its performance in this application. This
is conceptually analogous to the structural characteristics of the
natural meniscus where a highly orientated collagen fiber network
supports the large hoop stresses to produce better distribution of
contact pressures within the knee joint [1]. We hypothesized that a
PCU-based meniscal implant, reinforced with high tensile modulus
fibers, can provide better conformity in the knee joint as compared to
previous approaches using hard materials as interpositional devices.
Furthermore, we believe that such a compliant device can improve
the load distribution by permitting local material deformation and
thus delay degenerative changes and provide significant pain relief
for the younger patient with osteoarthritis (OA). Therefore, the
objectives of this study were (1) to develop a composite PCU-based
meniscal implant that is able to restore the pressure distribution
over the articular surfaces following meniscectomy, and (2) to apply
a series of laboratory, computational and animal studies in order
to evaluate the safety and effectiveness of the proposed implant
towards clinical evaluation.
Content: The following sections will briefly review the development
processes of a novel medial meniscal implant (Fig. 1a), composed of
PCU reinforced with high modulus UHMWPE fibers (Dyneema®
Purity, DSM). Implant Design As mentioned before, smart material
design was considered a prime feature in the making of a compliant
yet durable implant which is able to function as a load distributor on
the medial cartilaginous surface. The implant was designed as a
composite construct, reinforced circumferentially with UHMWPE
fibers, embedded during the process of molding (Fig. 1a) to reproduce
the structural characteristics of the natural meniscus which consists
of a solid matrix embedded with a highly orientated collagen fiber
network [1]. This combination of materials was chosen to enable the
implant to withstand high impact forces while maintaining its form.
The pliable matrix material should provide a damping effect and
distribute pressure by permitting local material deformation whereas
the reinforcement is designed to restrain matrix flow and bear a high
portion of the stresses, i.e., compressive loads exerted on the

implant transformed into tensile loads acting on the fibers. A
representative geometric design for the meniscus shape was formed
based on the analysis of more than 130 human knee MRI-scans. The
lateral side of the implant body was restructured to form a full discoid
shape by creating an artificial “bridge” along the gap between the
original medial insertion points of the meniscus (the region of the
inter-condylar notch). The preservation of the cruciate ligaments and
prevention of undesired impingement were taken into account in the
design, as well as knee alignment and stability. In-vitro biomechanical
evaluation The biomechanical evaluation of the meniscal implant
included more than 1500 in-vitro compression tests in 29 cadaveric
knees, to assess the implants’ ability to distribute load on the tibial
plateau. The compression test protocol described in detail elsewhere
[12],. In brief, the implants were inserted into the medial compartment
of cadaveric knees and were loaded under medial compression
similar to the physiological load occurring during human gait cycle
(1200N). Pressure distribution under the meniscal implant was
measured utilizing flexible pressure sensors (Tekscan Inc., Boston,
MA) and then compared to those attained for the natural meniscus
prior to meniscectomy. Specifically, maximal pressure values and
locations as well as pressure coverage area with respect to the
natural meniscus were analyzed. A Peak-to-Average pressure
relation (PAR) dimensionless ratio was defined, as an additional
measure, which together with total contact area was used to compare
the results to other related studies. Contact pressure distributions
measured on the tibial plateau were in very good agreement to those
measured under the intact natural meniscus of the specific knee
(Fig. 1b). Peak and average pressures developed under the implant
were compared to those measured under the natural meniscus and
were found to be statistically indistinguishable (p≥0.05). Calculation
of PAR (3.1±0.3) and contact area (658±135mm2) for the implant
were also statistically indistinguishable compared to PAR (2.7±0.5)
and contact area (642±96mm2) measured for the natural meniscus.
Fifteen-million cycles fatigue test Cyclic mechanical compression/
compression loading was applied to the implant according to fatigue
tests requirements (ISO14243). The fatigue station was equipped
with a flexible tube that maintained a continuous circulation of the
heated test fluid (saline, 37°C). Six specimens of the smallest implant
size were tested and considered the worst case scenario, since (i) it
possesses the thinnest thickness to be subjected to the
aforementioned compressive load, and (ii) the distance between the
reinforcement fibers and the outer surface is the smallest. Thus, for
the smallest implant, load would be distributed on a smaller area
and may lead to the development of greater internal stresses. MRI-
based UHMWPE replicas of the medial tibial plateau and femoral
condyle were used as the compression surfaces. Fifteen-million
loading cycles were applied on each specimen and the implant’s
structure and functionality were examined before and after the test.
The tests demonstrated that both of the implant’s components, PCU
and UHMWPE fibers were not affected in the long term in respect to
form, fiber-matrix bonding and structure-function relationship.
Specifically, no significant dimensional changes were observed
during the course of the test and pressure distributions post
15-milion loading cycles remained similar to those measured prior to
the test. Finite element analysis A Finite element (FE) model of the
medial knee with the PCU implant inside was developed and internal
strains/stresses developed in the PCU bulk and UHMWPE fibers
were calculated [11]. The model geometry was based on MR-scans of
a cadaveric specimen and analyzed under 1200N compression:
comparable to the biomechanical evaluation and other FE models.
Peak stresses were compared to the allowed values supplied by the
manufacturer for each material. The model was validated by
comparing computational results to analogous tibial plateau contact
pressures, measured in cadaveric knees in vitro [12]. Peak von-Mises,
compressive and tensile stresses in the PCU were all lower than the
maximal allowed stress (15MPa). Similarly, the peak tensile stress
calculated in the fibers was significantly lower than the material’s
yield stress (3.1GPa). Animal study All procedures were approved by
the Institutional Animal Care and Use Committee of the Technion
University Israel (#10-6-11-06) and are described in detail elsewhere
[13]. Six ewes (1-2 years, 60-80 Kg) were allocated for the research.
The sheep underwent a full meniscectomy of the medial meniscus of
their left knee, and were implanted with a PCU meniscus substitute.
Smaller joint tolerance in sheep required the release and
reattachments of the MCL from the epicondyle to ease the insertion
of the implant. Subsequent to the rehabilitation period, the sheep
were relocated to a large pen and were allowed to ambulate freely.
Functionality of the joint was assessed by measuring mobility and
range-of-motion (ROM). Animals were euthanized at 3 (n=3) and 6
(n=3) months. Cartilage and the surrounding soft tissues of both
knees were assessed macroscopically and microscopically, using a
semi-quantitative histological analysis, based on a modified Mankin
scale [4]. The contra-lateral knee served as control. From gross
inspection, the PCU implant remained well-secured throughout the
Extended Abstracts 121
experimental period and showed no visible signs of wear. Gross and
microscopic examinations of the explanted PCU implant’s surfaces
did not reveal any changes in their structural or material properties.
Histological analysis showed relatively mild degenerative changes in
the articular cartilage that were dominated by loss of proteoglycan
content and cartilage structure. However, the total OA score did not
significantly differ between the control and operated knees and
there were no differences in the severity of degenerative changes
between 3 and 6 months post surgery. Surgical procedure
development The device was implanted under operating room
conditions in >30 cadaveric knees, firstly in order to develop tools
and a surgical procedure for the insertion of the device and secondly,
as a part of a training program for surgeons. In general, the surgical
procedure included standard arthroscopic meniscectomy, ~4 cm
incision opening, a trial-based sizing confirmation, implantation,
and closing. After insertion, the implant was found to independently
self-center into its designated location due to its discoid shape. The
insertion was initially performed by pushing the implant manually
into the joint space, though at a later stage, when insertion tools
were produced, the surgeon could decide whether to use the manual
approach or a custom insertion tool. First clinical results Following
all of the necessary laboratory tests and regulatory approvals (CE
Marking 0473), the meniscal implant was implanted in 18 patients,
of May 2010. The arthroscopic implantation procedure was short and
uncomplicated. Arthroscopic observation assured that the device
was located in its intended position between the medial femur and
tibia. In addition, correct sizing was verified in terms of physical
examination, e.g., the implant should rotate smoothly with the knee
as the knee flexes and extends without undesired contact with the
PCL or the ACL. The outcomes used in the study were the KOOS, VAS-
Pain, Lysholm, and IKDC. MRI images were taken after 1.5, 12 and 24
months (Fig. 2a). The averaged KOOS results showed an improvement
of ~65% in pain, ~12% in symptoms, ~35% in daily activity, ~30% in
sports, and ~65% in quality of life, 1.5-months post implantation
(Fig. 2b). When looking at the 12-months results, the averaged KOOS
results showed an improvement of ~75%, ~35%, ~45%, ~250%,
and ~120% in pain, symptoms, daily activity, sport and quality of life
sections, respectively. DISCUSSION In the current study, we
presented the development of a novel PCU meniscal implant for the
medial compartment of the knee, along with an overview of essential
tests. The main benefits claimed for this meniscal implant are pain
relief and preservation of meniscal functionality. In a comprehensive
review of synthetic meniscal implant solutions by van Tienen et al.,
(2009) it was concluded that the material requirements for artificial
total meniscus replacement are not fully addressed to date, implying
that cartilage damage in such case has not yet been prevented [22].
In the current work a new approach in the field of total meniscal
replacement is presented. We suggest the use of a stable (non-
degradable) PCU implant with exceptional mechanical and
tribological properties to restore the missing functions of the
meniscus following meniscectomy, e.g., shock absorption, pressure
distribution and consequent chondroprotection. It is well agreed
that the meniscus’s main biomechanical role is to distribute joint
forces over a wider area to lower the pressure developed in the
articular cartilage [18]. Good resemblance of the natural meniscus
pressure patterns, as resulted from the current study, to other
related works was observed. Specifically, PAR calculations (2.7±0.5)
and contact area measurements for the natural meniscus
(642±96mm2) were in accordance with ‘typical’ normative PAR
values and contact area measurements [7]. Likewise, the implant’s
PAR and contact area were in agreement with those measured for
the natural meniscus. Interestingly, the meniscal implant did not
display a focal stress concentration that was typically observed for
natural menisci where direct femur to tibia contact occurred. This
finding is due to the implant’s closed discoid shape, which is able to
provide a larger, continuous bearing surface compared to the semi-
lunar natural meniscus. The implication of these being that (a) the
implant is able to reduce the overall cartilage load associated with
meniscectomy by effectively distributing joint loads, and (b) the
implant completely prevents contact between opposing cartilage
surfaces. The in-vivo study of an analogous implant configuration in
sheep showed that minimal changes associated with joint remodeling
had occurred within the first 3 months. Such changes can be linked
to the surgical procedure itself (e.g. joint opening and MCL release
and reattachment from the epicondyle), and this is supported by the
longer term (6 months) findings, which were consistent with the 3
months findings showing that on the whole, cartilage was preserved
well [13]. On the other hand, it has recently been shown that total
medial meniscectomy in a sheep model leads to extensive destruction
of articular cartilage on the medial tibial and femoral condyles in as
little as 3.5 months [8]. In light of these findings, the results of the
current study can be considered exceptionally favorable, and support
the hypothesis that a PCU meniscal implant may counter the
occurrence of major degenerative cartilage changes following
122 Extended Abstracts

meniscectomy. Implantation of a non-fixed meniscal implant
provides additional advantages compared to fixed implants reported
to date. Without additional joint preparation or manipulation other
than meniscectomy (e.g., drilling of bone tunnels or suturing to
existing elements). The implantation surgical procedure is relatively
short (20-25 min.), simple and straightforward, and does not require
special expertise. It has been shown, for instance, that the
performance of a fixed meniscus is very much influenced by the
location and method of fixation [2,18,19], and calls for specialized
training. Furthermore, preservation of existing bone stock is expected
to reduce pain and rehabilitation time considerably and to increase
the success rate of future revisions/interventions. In conclusion, this
paper summarizes a series of studies carried out to design, create,
evaluate, and implant a novel free-floating meniscal replacement.
Preliminary clinical studies show a significant beneficial effect of the
KOOS scores in patients receiving this implant. Our findings show
that the device described here can significantly relieve pain post-
injury and may delay the need for more aggressive procedures that
require bone resection such as UKA or TKR among the goal-target
population.
in symptomatic patients less than fifty years old. J Bone Joint Surg
Am 86-A: 1392-1404, 2004.
16. Scholes SC, Burgess IC, Marsden HR, Unsworth A, Jones E, Smith
N. Compliant layer acetabular cups: friction testing of a range of
materials and designs for a new generation of prosthesis that mimics
the natural joint. Proc Inst Mech Eng [H] 220:583-596, 2006.
17. Scholes SC, Unsworth A, Jones E. Polyurethane unicondylar knee
prostheses: simulator wear tests and lubrication studies. Phys Med
Biol 52:197-212, 2007.
18. Sekaran SV, Hull ML, Howell SM. Nonanatomic location of
the posterior horn of a medial meniscal autograft implanted in a
cadaveric knee adversely affects the pressure distribution on the
tibial plateau. Am J Sports Med 30:74-82, 2002.
19. Tienen TG, Verdonschot N, Heijkants RG, Buma P, Scholten JG, van
Kampen A, et al. Prosthetic replacement of the medial meniscus in
cadaveric knees: does the prosthesis mimic the functional behavior
of the native meniscus? Am J Sports Med 32:1182-1188, 2004.

References: 20. Tienen TG, Heijkants RG, de Groot JH, Pennings AJ, Schouten
AJ, Veth RP, et al. Replacement of the knee meniscus by a porous
1. Adams M, Hukins D. The extracellular matrix of the meniscus. New polymer implant: a study in dogs. Am J Sports Med 34:64-71, 2006.
York, NY, Raven Press 1992.
21. van Arkel ER, de Boer HH. Survival analysis of human meniscal
2. Alhalki MM, Howell SM, Hull ML. How three methods for fixing transplantations. J Bone Joint Surg Br 84227-231, 2002.
a medial meniscal autograft affect tibial contact mechanics. Am J
Sports Med 27: 320-328, 1999. 22. van Tienen TG, Hannink G, Buma P. Meniscus replacement using
synthetic materials. Clin Sports Med 28:143-156, 2009.
3. Buma P, van Tienen T, Veth R. The collagen meniscus implant.
Expert Rev Med Devices 4: 507-516, 2007. 23. Verdonk PC, Demurie A, Almqvist KF, Veys EM, Verbruggen G,
Verdonk R. Transplantation of viable meniscal allograft. Survivorship
4. Carlson CS, Guilak F, Vail TP, Gardin JF, Kraus VB. Synovial fluid analysis and clinical outcome of one hundred cases. J Bone Joint
biomarker levels predict articular cartilage damage following Surg Am 87:715-724, 2005.
complete medial meniscectomy in the canine knee. J Orthop Res
20:92-100, 2002.
5. Chiari C, Koller U, Dorotka R, Eder C, Plasenzotti R, Lang S, et al.
A tissue engineering approach to meniscus regeneration in a sheep
model. Osteoarthritis Cartilage 14: 1056-1065, 2006.
6. Elleuch R, Elleuch K, Salah B, Zahouani H. Tribological behavior
of thermoplastic polyurethane elastomers. Materials & Design 28:
824-830, 2007.
7. Fukubayashi T, Kurosawa H. The contact area and pressure
distribution pattern of the knee. A study of normal and osteoarthrotic
knee joints. Acta Orthop Scand 51: 871-879, 1980.
8. Kelly BT, Robertson W, Potter HG, Deng XH, Turner AS, Lyman S,
et al. Hydrogel meniscal replacement in the sheep knee: preliminary
evaluation of chondroprotective effects. Am J Sports Med 35: 43-52,
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9. Khan I, Smith N, Jones E, Finch DS, Cameron RE. Analysis and
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injury. II: animal experiments. Knee 10: 53, 2003.
11. Linder-Ganz E, Elsner JJ, Danino A, Zur G, Guilak F, and Shterling
A. Design of a Polycarbonate-Urethane meniscal implant: Finite
element approach. The 2009 ASME-SBC, June 17-21, Lake Tahoe,
California, USA, 2009.
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novel quantitative approach for evaluating contact mechanics of
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2.2.2
Adult Stem Cells and Nanomaterials in Skeletal Tissue
Engineering
R. Tuan
Pittsburgh/United States of America
Introduction: Nanoscale materials are the fundamental building
blocks and functional subunits of cells, including subcellular
organelles and extracellular matrix components. Currently, there
is growing recognition of the importance of understanding and
incorporating nanobiology into biomedical applications. This issue
is of particular importance in the emerging field of regenerative
medicine, the goal of which is to develop methods to repair,
replace, and regenerate diseased, injured, or non-functional tissues.
Towards this goal, stem or progenitor cells have been considered a
highly desirable candidate cell type, because of their expandability
and potential to be induced toward specific cell differentiation
lineages. A key requirement in tissue engineering and regenerative
medicine is that ultimately the “regenerate tissue” needs to be a
three-dimensional structure. In weight-bearing musculoskeletal
tissues, this requirement is particularly critical. Musculoskeletal
disorders affect one out of seven Americans. This severe disease
burden underscores the need to develop novel and effective
treatment protocols. This lecture will present the promises as well
as the challenges in the field of skeletal tissue engineering and
regeneration, specifically the application of adult stem cells and
nanomaterial scaffolds. The biology of human adult mesenchymal
stem cells, particularly the mechanisms regulating their proliferation
versus differentiation into specific lineages, is intricately regulated
by cell-cell interactions, signaling by extracellular bioactive factors,
and transcriptional and epigenetic activities. More importantly,
the extracellular matrix milieu provides critical cues, both
architectural and structure-dependent, to guide cell-based tissue
morphogenesis. We have developed biomimetic and biodegradable
nanofibrous biomaterials to serve as scaffolds for cell-based tissue
engineering. Information on the fabrication and biological basis
of the scale-dependent bioactivities of the nanofibrous scaffold
will be presented. Cell-nanofibrous constructs are currently being
developed for the engineering of cartilaginous tissues, including
articular cartilage and intervertebral disc. In conclusion, cell-based
tissue engineering and regenerative medicine is an exciting, inter-
disciplinary, and potentially high-impact research field that presents
a natural platform for collaboration among life scientists, engineers,
and clinicians.
Content: Nanoscale materials are the fundamental building blocks
and functional subunits of cells, including subcellular organelles
and extracellular matrix components. Currently, there is growing
recognition of the importance of understanding and incorporating
nanobiology into biomedical applications. This issue is of particular
importance in the emerging field of regenerative medicine, the goal
of which is to develop methods to repair, replace, and regenerate
diseased, injured, or non-functional tissues. Towards this goal, stem
or progenitor cells have been considered a highly desirable candidate
cell type, because of their expandability and potential to be induced
toward specific cell differentiation lineages. A key requirement in
tissue engineering and regenerative medicine is that ultimately
the “regenerate tissue” needs to be a three-dimensional structure.
In weight-bearing musculoskeletal tissues, this requirement is
particularly critical. Musculoskeletal disorders affect one out of
seven Americans. This severe disease burden underscores the need
to develop novel and effective treatment protocols. This lecture will
present the promises as well as the challenges in the field of skeletal
tissue engineering and regeneration, specifically the application
of adult stem cells and nanomaterial scaffolds. The biology of
human adult mesenchymal stem cells, particularly the mechanisms
regulating their proliferation versus differentiation into specific
lineages, is intricately regulated by cell-cell interactions, signaling
by extracellular bioactive factors, and transcriptional and epigenetic
activities. More importantly, the extracellular matrix milieu provides
critical cues, both architectural and structure-dependent, to guide
cell-based tissue morphogenesis. We have developed biomimetic
and biodegradable nanofibrous biomaterials to serve as scaffolds
for cell-based tissue engineering. Information on the fabrication
and biological basis of the scale-dependent bioactivities of the
nanofibrous scaffold will be presented. Cell-nanofibrous constructs
are currently being developed for the engineering of cartilaginous
tissues, including articular cartilage and intervertebral disc. In
conclusion, cell-based tissue engineering and regenerative medicine
is an exciting, inter-disciplinary, and potentially high-impact research
field that presents a natural platform for collaboration among life
scientists, engineers, and clinicians.
Extended Abstracts 123
2.2.3
Growth factors as regulators of chondrogenesis in vitro and in
vivo
G.J.V.M. Van Osch
Rotterdam/Netherlands
Introduction: Cartilage defects do not heal well, despite the fact that
chondrocytes in cartilage wound areas increase the expression of
various growth factors (Bos et al 2001) and during surgical repair
procedures, such as in micro-fracture, endogenous growth factors
are being released. In order to improve the repair of cartilage defects
using surgical techniques, application of growth factors, cells or
bioactive materials more knowledge about the role of growth factors
in cartilage repair is required. We have addressed the following
questions:
· what is the effect of growth factors on chondrogenic differentiation
of progenitor cells?
· what is the effect of growth factors on matrix synthesis and matrix
assembly by chondrocytes? We focused our research on two well
known growth factors in cartilage repair: Transforming Growth
Factor-beta (TGF-beta) and Fibroblast Growth Factor (FGF).
Content: 0Growth factors and chondrogenic differentiation
Mesenchymal progenitor cells derived from bone marrow stroma are
well known to be able to differentiate to the chondrogenic lineage
when cultured in the presence of TGF-beta. Human Bone Marrow
Stroma-derived Cells (hBMSC) cultured in pellets go through phases
similar to embryonic limb development: cellular condensation, early
chondrogenic differentiation, finally inevitably leading to terminal
differentiation (Hellingman et al 2010). Implantation of these cells
in-vivo results in formation of bone tissue (Farrell et al 2009 and
unpublished data). During chondrogenic differentiation of hBMSC in
vitro FGF Receptors (FGFR) 1, 2, 3 are expressed in a stage specific
manner that is comparable to embryonic limb development. During
the condensation phase (N-cadherin expression)FGFR1 drops and
FGFR2 showed in peak in expression. Differentiating chondrocytes
(collagen II positive, collagen X negative) did not express any FGFRs.
During hypertrophy (collagen X positive) all FGFRs were expressed.
Different FGFs are known to have different binding affinities for the
different FGFRs. To examine potential application of our findings,
hBMSC were treated with FGF2 (high affinity for FGFR1) or FGF9
(High affinity for FGFR2 and FGFR3) in a stage specific manner
(Hellingman et al 2010). When added during the entire culture
period, both FGF2 and FGF9 treated pellets contained significantly
less GAG at day 35 than control pellets. Pellets treated with FGF2
from day 3-14 had a lower GAG content at day 35 than control pellets
whereas a trend towards a higher GAG content was seen when FGF
9 was added. Addition of FGF during hypertrophic differentiation is
detrimental for cartilage-matrix production. Although all receptors
are expressed during hypertrophy, FGF2 and FGF9 have differential
effect when added from day 21-35. While FGF2 inhibits further matrix
deposition, FGF9 increases matrix resorption. This suggests that the
FGFRs have specific effects, even during hypertrophy when they are
all expressed. We have also investigated the TGF-beta pathway in
more detail (Hellingman et al unpublished data). TGF-beta signals
through the canonical SMAD pathway (SMAD2/3) but recently it
has been shown that also SMAD1/5/8 pathway can be targeted.
We investigated the role of these different TGF-beta signaling
pathways in chondrogenically differentiating BMSC. Terminally
differentiated BMSC produced in vitro by addition of TGF-beta
stained positive for both Smad2/3P and Smad1/5/8P, similar to
cartilage in murine embryonic limbs. On the other hand, permanent
hyaline cartilage that lacks expression for MMP13 and collagen X
only expressed Smad2/3P. To investigate the role of Smad signaling
pathways, Smad2/3 phosphorylation was blocked by addition of
SB-505124 or Smad1/5/8 phosphorylation was blocked by addition
of dorsomorphin in chondrogenically differentiation hBMSC. When
either of them was added through-out culture, no collagen II
expression was observed, indicating that both pathways are involved
in early chondrogenesis. Distinct functions for these pathways were
demonstrated when Smad signaling was blocked after the onset of
chondrogenesis. Blocking Smad2/3P from day 14-35 resulted in a halt
in collagen II production. On the other hand, blocking Smad1/5/8P
during this time resulted in decreased expression of MMP13,
collagen X and alkaline phosphatase without inhibiting further
collagen II production. Moreover, blocking Smad1/5/8P prevented
mineralization. Growth factors and cartilage matrix assembly TGF-
beta and FGF2 have differential effect on matrix production by
differentiated chondrocytes in alginate (Jenniskens et al 2006;
Bastiaansen-Jenniskens et al 2010). FGF2 inhibits collagen and COMP
deposition whereas TGF-beta inhibits proteoglycan deposition and
124 Extended Abstracts
collagen cross-link formation but stimulates COMP deposition. The
distribution of extra-cellular matrix components over territorial and
inter-territorial matrix is also differentially affected by these growth
factors. In addition, modulation of glycosaminoglycan synthesis
influences the assembly of collagen and vice versa. As a resultant
of all these actions, growth factors can influence the mechanical
properties of the produced matrix. We have demonstrated that the
effect of a growth factor depends on the stage in which it is added in
the medium. Since the effects of growth factors and modulation of
intracellular signalling are mostly studied throughout culture, more
attention to differentiation stage-specific effects and application
may be warranted to improve cartilage repair. It is unpredictable
what happens if different growth factors are present simultaneously
(e.g. during wound healing in vivo). The growth factors may very well
be counteractive. Knowledge about interactions between growth
factors in the various stages and their mechanisms of action is largely
absent. More specific, temporal control of growth factor signaling can
help to optimize cartilage repair.
References:
Bastiaansen-Jenniskens YM, de Bart ACW, Koevoet W, Jansen
KMB, Verhaar JAN, van Osch GJVM, DeGroot J. Stimulation of COMP
Production in Cartilage Matrix Generation Decreases Collagen
Fibril Diameter. Cartilage 2010 in press Bos PK, van Osch GJ, Frenz
DA, Verhaar JA, Verwoerd-Verhoef HL. Growth factor expression in
cartilage wound healing: temporal and spatial immunolocalization
in a rabbit auricular cartilage wound model. Osteoarthritis Cartilage.
2001 May;9(4):382-9. Farrell E, van der Jagt OP, Koevoet W, Kops
N, van Manen CJ, Hellingman CA, Jahr H, O‘Brien FJ, Verhaar JA,
Weinans H, van Osch GJ. Chondrogenic Priming of Human Bone
Marrow Stromal Cells: A Better Route to Bone Repair? Tissue Eng
part C Methods 2009 Jun;15(2):285-95 Hellingman CA, Koevoet
W, Kops N, Farrell E, Jahr H, Liu W, Baatenburg de Jong RJ, Frenz
D, van Osch G. Fibroblast Growth Factor Receptors in in-vitro and
in-vivo chondrogenesis: Relating Tissue Engineering using adult
mesenchymal stem cells to embryonic development. Tissue Eng
Part A. 2009 Sep 3. Hellingman. CA , Blaney Davidson EN, Koevoet
W, Vitters EL, van den Berg EB, van Osch GJVM, van der Kraan PM.
Smad signaling determines chondrogenic differentiation of bone-
marrow derived mesenchymal stem cells. Submitted for publication.
Jenniskens YM, Koevoet W, de Bart AC, Weinans H, Jahr H, Verhaar
JA, DeGroot J, van Osch GJ. Biochemical and functional modulation
of the cartilage collagen network by IGF1, TGFbeta2 and FGF2.
Osteoarthritis Cartilage. 2006 Nov;14(11):1136-46.
Acknowledgments:
This research was financially supported by the Dutch Program for
Tissue Engineering and the Dutch Arthritis Association.
2.3.1
Enhancing tensile and compressive properties of self-assembled
articular cartilage
K. Athanasiou1, J. Hu1, D. Responte2
1
Davis/United States of America, 2Houston/United States of
America
Introduction: Due to articular cartilage’s low repair capacity,
alternate strategies to effect a robust reparative process need to
be developed, providing a strong impetus for tissue engineering.
Tissue engineering strategies can be categorized as scaffold-based
or scaffoldless. Scaffold-based tissue engineering uses a synthetic
and/or natural polymer to create a temporary matrix for cells to
populate. Various synthetic polymers1,2 and natural materials
such as collagen,3,4 hyaluronic acid,5-7 and fibrin8,9 have been
investigated for cartilage regeneration. Additionally, there are
various scaffoldless approaches including pellet culture,10 aggregate
culture,11 and self-assembly.12 The key advantage of these methods
is increased cell-cell interaction, which has been shown to promote
chondrocyte differentiation.13-15 Despite these various approaches,
studies have n1ot yet produced neotissue with the same properties
as native cartilage. To improve the functionality of constructs,
various biochemical and biomechanical stimuli have been studied.
In particular, members of the TGF-β superfamily have been shown
to increase the deposition of GAGs16-18 and collagen.19,20 Insulin
growth factor-1 (IGF-1) has been shown to increase GAG production in
both explants21 and tissue engineered constructs.22-24 Mechanical
stimuli can also be employed to advance tissue engineering efforts.
Direct compression has been used to modulate matrix composition
and concomitantly influence construct properties.25,26 In addition,
hydrostatic pressure application has increased gene transcription,27
collagen production,28,29 and construct tensile properties.30
However, even with the administration of external stimuli,
engineered cartilage has not yet attained the mechanical properties
of native tissue. Our laboratory seeks to develop new scaffoldless
strategies for articular cartilage engineering. In particular, a self-
assembly process has been developed to grow functional neotissue
that could potentially replace degenerated cartilage. Self-assembly
acts as a simple, yet effective, platform technology that can be
enhanced by applying various biochemical and biomechanical
stimuli. Additionally, this versatile methodology can be employed to
engineer a wide spectrum of cartilage types and geometries.
Content: Self-assembly: a new tissue engineering strategy
The problems associated with scaffolds including biocompatibility
issues and exogenous degradation products spurred the investigation
of a novel scaffoldless approach for functional tissue engineering. It
was found that when chondrocytes were cultured at high density in
non-adherent molds, the cells aggregated to form constructs that
not only appeared to be hyaline cartilage-like but also had functional
properties of the same order as native values. This self-aggregation
or self-assembly of chondrocytes provided numerous advantages
over scaffolds including increased retention of phenotype, increased
cell-cell contact, and lack of degradation products. As a result, a
comprehensive tissue engineering strategy was developed, based
on self-assembly and a panoply of assays, to achieve the formation
of cartilage constructs effectively and functionally. The objective
continues to be the development of a clinically feasible method
for engineering cartilage with the biochemical properties and
mechanical integrity of native tissue. Self-assembly was developed
based on the differential adhesion hypothesis to produce robust
cartilage constructs (Fig. 1A). For example, on a dry weight basis
these tissue-engineered constructs contained two thirds more GAG
than native tissue. Collagen reached one third the level of native
tissue, and the compressive stiffness reached more than one third
of native tissue values.7 This progress toward achieving native
biomechanical properties and matrix composition was exciting as it
provided early validation for this scaffoldless approach.
These promising results spurred an investigation of the mechanism
underlying self-assembly (Fig. 1B).19 Increased N-cadherin
expression during neotissue formation suggested that differential
adhesion mediated self-assembly. Also, several biochemical
properties recapitulated cartilage development including an
increased proportion of collagen II, decreased proportion of collagen
type VI, decreased chondroitin 6- to 4- sulfate ratio, and localization
of collagen VI to the pericellular matrix. In addition, the compressive
properties reached a plateau and tensile characteristics peaked
at 4 weeks. These studies showed that the self-assembly method
mimicked tissue development and maturation, suggesting that a
set of exogenous stimuli could then be applied to augment tissue
functional properties. Various growth factors, applied individually

and in combination, were investigated to improve the functionality
of self-assembled constructs. For example, a combination treatment
of BMP-2 and IGF-I resulted in over 1-fold increases in aggregate
modulus, accompanied by increases in GAG production. However,
TGF-β1 was found to be the most potent growth factor, inducing
1-fold increases in both aggregate modulus and tensile modulus,
and increasing GAG and collagen content.13 These findings are
exciting as coupling application of select growth factors with the
self-assembly process resulted in tissue engineered constructs with
substantially improved functional properties.
Hydrostatic pressure stimulation was also advantageous for
self-assembly. After applying hydrostatic pressure of different
magnitudes, durations, and frequencies, a particularly effective
regimen was identified. At the counterintuitive frequency of 0 Hz
(i.e., static), 10 MPa, applied for 1 hour on days 10-14 of a 4 week
culture, significantly increased the aggregate modulus by 1.4-fold.
It was exciting to note that this regimen also affected functional
properties that seem to be difficult to improve upon, namely tensile
modulus and strength along with corresponding collagen content,
which increased over 2-fold.20 For the first time, this study examined
the immediate and long-term effects of hydrostatic pressure on
biomechanical properties, and demonstrated that hydrostatic
pressure has an optimal application time in construct development.
The next logical step was to combine the optimal regimens of the
growth factor and hydrostatic pressure stimuli. The combination
further improved the properties of the tissue engineered constructs.
Thus, the combination of 10 MPa static hydrostatic pressure, applied
for 1 hour a day for 5 days, and 30 ng/ml TGF-β1 had an additive
effect on the mechanical properties, increasing the aggregate
modulus by 164% and the Young’s modulus by 231%, approaching
300 kPa and 2 MPa, respectively (Fig. 2A). Additionally, the combined
treatment had a synergistic effect on collagen content, increasing
it by 173%.18 Thus, the combination of hydrostatic pressure
stimulation with growth factor application resulted in the formation
of tissue engineered constructs with biomechanical and biochemical
properties spanning native articular cartilage values.
Applying direct compression increases the aggregate modulus by
70%
Since hydrostatic pressure was proven to be such a potent stimulator,
it became apparent that other biomechanical stimuli ought to be
examined. Thus, a direct compression instrument was developed
to compress samples at specific frequencies and magnitudes. By
applying dynamic compression to medial meniscal explants, we
showed that aggrecan was up-regulated by 108%.21 The beneficial
effects of dynamic compression have also been observed in self-
assembled constructs, where applying 17%, 0.1 Hz compression,
for example, was found to increase the aggregate modulus by 70%.
These results have shown the potential of direct compression to
further improve tissue engineered constructs. The enzyme C-ABC
was applied to deplete GAG content and subsequently improve
biomechanical properties. C-ABC increased tensile properties of
self-assembled articular cartilage (Fig. 2B) without compromising
compressive properties, as GAG levels return post-treatment.22
Multiple C-ABC treatments further increased tensile properties,
reaching values of 3.4 and 1.4 MPa for the tensile modulus and
ultimate tensile strength, respectively.23 C-ABC represents an
exciting method for engineering functional articular cartilage by
departing from conventional anabolic approaches.
Intracellular Na+ and Ca2+ modulation increases the tensile
properties
The effects of hydrostatic pressure are known to be mediated by
various ion channels.24 Motivated by the mechanism of action
of hydrostatic pressure, inhibitors of Na+ ion transporters and
stimulators of intracellular Ca2+ were investigated as possible
actors in the development of self-assembled constructs.25 We
applied ouabain (Na+/K+-ATPase inhibitor), bumetanide (Na+/
K+/2Cl- tritransporter inhibitor), histamine (cAMP activator), and
ionomycin (a Ca2+ ionophore) to self-assembled constructs for 1
hour daily on days 10-14 of culture and examined the constructs at
2 weeks or 4 weeks. The results of these experiments showed that
20 µM ouabain, 0.3 µM ionomycin, or their combination increased
the tensile modulus by 40-95%. Furthermore, the 20 µM ouabain
treatment increased the ultimate tensile strength by 56-86% at 4
weeks. This study was the first to show that altering intracellular ion
concentrations can increase the mechanical properties of engineered
articular cartilage. In addition, these results have important
relationships to hydrostatic pressure mechanotransduction.
Extended Abstracts 125
Conclusions: Self-assembly shows great promise for cartilage
tissue engineering and various stimuli can be used to improve
the properties of constructs. Growth factor application induces
1-fold increases in both compressive and tensile properties.
Mechanical stimuli also increase the mechanical properties of
constructs; hydrostatic pressure increases tensile properties over
two-fold. Furthermore, combining hydrostatic pressure and TGF-β1
synergistically increases functional properties. Additionally, the
application of chondroitinase-ABC increases the tensile modulus
by 80% without compromising compressive properties. By altering
the concentrations of intracellular ions, the tensile properties can
be increased. These studies illustrate how both mechanical and
biochemical stimuli can be employed to improve the properties of
self-assembled constructs. The straightforward approach of self-
assembly and its versatility render it a highly translatable platform
technology. Although results thus far show great promise, this
process will need to be improved prior to clinical application. Current
studies focus on continuing to optimize the process and improve the
functional properties of constructs.
References:
1. Vacanti, CA, Kim, W, Schloo, B, Upton, J, Vacanti, JP. 1994. Joint
resurfacing with cartilage grown in situ from cell-polymer structures.
Am J Sports Med 22: 485-488.
2. Grande, DA, Halberstadt, C, Naughton, G, Schwartz, R, Manji, R.
1997. Evaluation of matrix scaffolds for tissue engineering of articular
cartilage grafts. J Biomed Mater Res 34: 211-220.
3. Nehrer, S, Domayer, S, Dorotka, R, Schatz, K, Bindreiter, U, Kotz, R.
2006. Three-year clinical outcome after chondrocyte transplantation
using a hyaluronan matrix for cartilage repair. Eur J Radiol 57: 3-8.
4. Kaplonyi, G, Zimmerman, I, Frenyo, AD, Farkas, T, Nemes, G. 1988.
The use of fibrin adhesive in the repair of chondral and osteochondral
injuries. Injury 19: 267-272.
5. Furukawa, KS, Suenaga, H, Toita, K, Numata, A, Tanaka, J, Ushida,
T, Sakai, Y, Tateishi, T. 2003. Rapid and large-scale formation of
chondrocyte aggregates by rotational culture. Cell Transplant 12:
475-479.
6. Stewart, MC, Saunders, KM, Burton-Wurster, N, Macleod, JN.
2000. Phenotypic stability of articular chondrocytes in vitro: the
effects of culture models, bone morphogenetic protein 2, and serum
supplementation. J Bone Miner Res 15: 166-174.
7. Hu, JC, Athanasiou, KA. 2006. A self-assembling process in articular
cartilage tissue engineering. Tissue Eng 12: 969-979.
8. Deng, Y, Zhao, K, Zhang, XF, Hu, P, Chen, GQ. 2002. Study on the
three-dimensional proliferation of rabbit articular cartilage-derived
chondrocytes on polyhydroxyalkanoate scaffolds. Biomaterials 23:
4049-4056.
9. Fortier, LA, Nixon, AJ, Mohammed, HO, Lust, G. 1997. Altered
biological activity of equine chondrocytes cultured in a three-
dimensional fibrin matrix and supplemented with transforming
growth factor beta-1. Am J Vet Res 58: 66-70.
10. Smith, P, Shuler, FD, Georgescu, HI, Ghivizzani, SC, Johnstone, B,
Niyibizi, C, Robbins, PD, Evans, CH. 2000. Genetic enhancement of
matrix synthesis by articular chondrocytes: comparison of different
growth factor genes in the presence and absence of interleukin-1.
Arthritis Rheum 43: 1156-1164.
11. Sah, RL, Chen, AC, Grodzinsky, AJ, Trippel, SB. 1994. Differential
effects of bFGF and IGF-I on matrix metabolism in calf and adult
bovine cartilage explants. Arch Biochem Biophys 308: 137-147.
12. Blunk, T, Sieminski, AL, Gooch, KJ, Courter, DL, Hollander,
AP, Nahir, AM, Langer, R, Vunjak-Novakovic, G, Freed, LE. 2002.
Differential effects of growth factors on tissue-engineered cartilage.
Tissue Eng 8: 73-84.
13. Elder, BD, Athanasiou, KA. 2008. Systematic assessment of growth
factor treatment on biochemical and biomechanical properties of
engineered articular cartilage constructs. Osteoarthritis Cartilage
18: 18.
14. Mauck, RL, Soltz, MA, Wang, CC, Wong, DD, Chao, PH, Valhmu,
WB, Hung, CT, Ateshian, GA. 2000. Functional tissue engineering of
126 Extended Abstracts
articular cartilage through dynamic loading of chondrocyte-seeded
agarose gels. J Biomech Eng 122: 252-260.
15. Pei, M, Solchaga, LA, Seidel, J, Zeng, L, Vunjak-Novakovic, G,
Caplan, AI, Freed, LE. 2002. Bioreactors mediate the effectiveness of
tissue engineering scaffolds. Faseb J
16: 1691-1694. 16. Smith, RL, Lin, J, Trindade, MC, Shida, J, Kajiyama,
G, Vu, T, Hoffman, AR, van der Meulen, MC, Goodman, SB, Schurman,
DJ, Carter, DR. 2000. Time-dependent effects of intermittent
hydrostatic pressure on articular chondrocyte type II collagen and
aggrecan mRNA expression. J Rehabil Res Dev 37: 153-161.
17. Hu, JC, Athanasiou, KA. 2006. The effects of intermittent
hydrostatic pressure on self-assembled articular cartilage constructs.
Tissue Eng 12: 1337-1344.
18. Elder, BD, Athanasiou, KA. 2008. Synergistic and additive effects
of hydrostatic pressure and growth factors on tissue formation. PLoS
ONE 3: e2341.
19. Ofek, G, Revell, CM, Hu, JC, Allison, DD, Grande-Allen, KJ,
Athanasiou, KA. 2008. Matrix development in self-assembly of
articular cartilage. PLoS ONE 3: e2795.
20. Elder, BD, Athanasiou, KA. 2008. Effects of Temporal Hydrostatic
Pressure on Tissue-Engineered Bovine Articular Cartilage Constructs.
Tissue Eng Part A 15: 1151-1158.
21. Aufderheide, AC, Athanasiou, KA. 2006. A direct compression
stimulator for articular cartilage and meniscal explants. Ann Biomed
Eng 34: 1463-1474.
22. Natoli, R, Revell, CM, Athanasiou, K. 2009. Chondroitinase ABC
Treatment Results in Increased Tensile Properties of Self-Assembled
Tissue Engineered Articular Cartilage. Tissue Eng Part A: doi:10.1089/
ten.TEA.2008.0478.
23. Natoli, RM, Responte, DJ, Lu, BY, Athanasiou, KA. 2009. Effects
of multiple chondroitinase ABC applications on tissue engineered
articular cartilage. J Orthop Res.
24. Elder, BD, Athanasiou, KA. 2009. Hydrostatic pressure in
articular cartilage tissue engineering: from chondrocytes to tissue
regeneration. Tissue Eng Part B Rev 15: 43-53.
25. Natoli, RM, Skaalure, S, Bijlani, S, Chen, KX, Hu, J, Athanasiou,
KA. Intracellular Na(+) and Ca(2+) modulation increases the tensile
properties of developing engineered articular cartilage. Arthritis
Rheum 62: 1097-1107.
Acknowledgments:
The authors acknowledge funding from NIH NIAMS R01 AR053286.
3.2.2
Nutraceuticals in the treatment of OA
J. Steinmeyer
Giessen/Germany
Introduction: Osteoarthritis (OA) is the most prevalent disease of
the locomotory apparatus, affects people of all ethnic groups in all
geographic locations, occurs more commonly in women, and is the
most common cause of long-term disability in most populations of
people over 65. The growing population of older age groups and
an increase in risk factors for OA, primarily obesity and an inactive
life style, causes further increase in the estimated number of total
prevalent cases in the USA, Europe and Japan. The fact that OA
is no longer seen as a “battle wound” of becoming old or as an
unavoidable fate has created major demands on its therapy: drugs
used for its treatment should if possible have a causal mode of
action, and in addition, inhibit pain and inflammation, but should
still have only few adverse effects in long-term use, in spite of age-
related comorbidity and eventual polypharmacy.
Content: Alleviation of pain and inhibition of inflammation are the
primary goal of pharmacotherapy whereby the objective is to return
an active or transiently painful, decompensated OA to a latent (silent,
pain-free) condition. This therapeutic goal can almost always be
accomplished by using analgesics, nonsteroidal anti-inflammatory
drugs (NSAIDs) or intra-articularly applied glucocorticoids. NSAIDs,
despite serious adverse effects associated with their long-term
use, remain among the most widely prescribed drugs for OA.
In this context, there is a need for safe and effective alternative
treatments while the absence of any cure reinforces the importance
of prevention.
Such preventions and alternative treatment could come from
nutritional factors and has created an enormous public interest in the
relationship between nutrition and disease. The term “nutraceutical”
was coined from “nutrition” and “pharmaceutical” in 1989 by
DeFelice and was originally defined as “a food (or part of the food)
that provides medical or health benefits, including the prevention
and/or treatment of a disease” (1). In 1999, natural bioactive chemical
compounds derived from whole food and available in a non-food
matrix was used in a policy paper of Zeisel to describe nutraceuticals
(2). Under this newer definition, nutraceuticals are thus functional
ingredients sold as powders, tablets, dragées and other medicinal
forms not generally associated with food. The FDA, however, uses
instead the term dietary supplements (3). Numerous speculative lay
publications advertise the use of a whole range of nutraceuticals,
and health food stores offer a plethora of nutraceutical supplements
represented as therapies for OA. Surveys suggest that up to 5-8%
of adults in the US have used one or another of these products at
some time with glucosamine and chondroitin together ranking third
among all top-selling nutritional products in the US (4).
Nutrition is naturally better positioned to provide long-term rather
than short-term health benefits. This is because, in most cases,
a nutritional compound has only limited effects on its biological
target and clinical relevant effects are only reached over time. For
this reason, chronic diseases such as OA should, in theory, benefit
more from nutrition than acute diseases. However, besides the
general advice about healthy eating, dietary programs have played a
secondary role in the management of this degenerative joint disease.
Articular cartilage is critically dependent upon the regular provision
of nutrients (glucose and amino acids), vitamins (particularly
vitamin C), and essential trace elements (zinc, magnesium, copper).
Depending on the nutraceutical as sold over-the-counter they can
include a sole substance or a mixture of different agents from a wide
variety of different chemical groups including sugars (glucosamine,
chondroitinsulfate), vitamins (vitamin C, D, E, K), fatty acids (omega-
3-polyunsaturated fatty acids, fish oil), proteins, peptides or amino
acids (gelatin, collagen hydrolysate, lactalbon, glycine, histidin,
lysin) and/or plant extracts.
In most cases, the marketing of nutraceutical products has been
extensive and often based on minimal experimental data. It is
clear from the literature that the subject of nutraceuticals is highly
controversial and fraught with confusing and conflicting reports of
efficacy, safety, and mechanism of action (for review see e.g. 4-7).
Thus, it is difficult to form a balanced opinion about nutraceuticals.
However, some nutritional factors seem to improve the symptoms
of declared OA (glucosamine, chondroitinsulfate, collagen
hydrolysate), whereas the role of nutraceuticals in slowing down
progression of the disease remains to be seen. For instance, only very
few randomized controlled trials, which used structure-modifying
variables as primary endpoints, were unable to demonstrate a

benefit for glucosamine, chondroitinsulfate and vitamin E, but the
area deserves further investigation. Evidence for real efficacy is often
lacking (e.g. gelatine, lactalbon, glycin, histidin, lysin, lycopene) or
are based on epidemiological studies (e.g. vitamin C, D, K, calcium,
zinc, boron, selenium); therefore, large, controlled, double-blinded,
randomized clinical studies are required to determine the effect
of nutraceuticals on symptom and structure modification in OA.
Overall, it appears that there is some evidence to support claims for
efficacy for some nutraceuticals, but given the lack of extensive well-
designed clinical studies, we must be cautious in advocating their
widespread use. Perhaps, at present, their best place in the clinical
armory is as adjuncts to conventional therapy.
References:
(1) Kalra EK. Nutraceutical—definition and introduction. AAPS Pharm
Sci 5, E25, 2003
(2) Zeisel SH. Regulation of “nutraceuticals”. Science 285, 1853-
1855, 1999.
(3) Halstead CH. Dietary supplements and functional foods: 2 sides
of a coin? Am. J. Clin. Nutr. 77, 1001S-1007S, 2003.
(4) McAlindon TE. Nutraceuticals: do they work and when could we
use them? Best Practice Res. Clin. Rheum. 20, 99-115, 2006.
(5) Steinmeyer J, Konttinen YT. Oral treatment options for
degenerative joint disease—presence and future. Adv. Drug Del.
Rev. 58, 168-211, 2006.
(6) Rayman MP, Pattison DJ.. Dietary manipulation in musculoskeletal
conditions. Best Practice Res. Clin Rheum. 22, 535-561, 2008.
(7) Ameye LG, Chee WSS. Osteoarthritis and nutrition. From
nutraceuticals to functional foods: a systematic review of the
scientific evidence. Arthritis Res. Ther. 8, R127, 2006.
Extended Abstracts 127
3.2.3
Can drugs affect cartilage regeneration? -Immunosuppressant
FK506 promote chondrogenesis of synovial mesenchymal stem
cells-
N. Nakamura1, K. Tateishi1, W. Ando1, K. Kita2, K. Nakata1, H.
Yoshikawa1
1
Osaka/Japan, 2Suita, Osaka/Japan
Introduction: It is well known that the healing potential of articular
cartilage is limited, in part, due to its avascularity. To compensate
for such poor healing capacity, several cell-based approaches have
been investigated to repair chondral lesions.
Mesenchymal stem cells (MSCs) were first identified in bone
marrow and shown to have the ability to differentiate into a variety
of connective tissue phenotypes including bone, cartilage, and
adipose-tissue. Specifically, MSCs derived from synovium have
been shown to have the ability to proliferate over many passages
without losing their multipotency, and such expansion appears to be
independent of donor age or cryopreservation. In addition, a recent
study has shown that MSCs derived from synovium are superior to
those from bone marrow or adipose tissue in terms of chondrogenic
differentiation and it is a relatively easy and safe procedure to
obtain cells from synovium with minimal donor-site morbidity. Taken
together, synovium-derived MSCs could be a promising source for
future cell-based cartilage repair.
FK506 (tacrolimus) is a widely used immunosuppressive agent
with FDA approval, usually used to prevent graft rejection. The
immunosuppressive effect of FK506 is believed to be related to its
ability to inhibit calcineurin, an enzyme involved in activation of
the nuclear factor of activated T cells (NFAT). Recent studies also
suggest the involvement of NFAT in chondrogenesis. Furthermore, it
has been shown that FK506 can induce chondrogenic differentiation
of clonal mouse embryonic carcinoma cells (ATDC5), although
the molecular target for its effect on chondrogenesis remains
unclear. Such observations raise the possibility that FK506 might
likewise promote chondrogenic differentiation of MSCs. To test this
hypothesis, the effect of FK506 on chondrogenic differentiation of
human synovial mesenchymal stem cells (hSMSCs) was investigated.
Furthermore, the molecular mechanism (s) underlying the promotion
of chondrogenic differentiation by FK506 were explored.
Content: FK506 promotes chondrogenic differentiation of SMSCs.
It has been reported that the degree of chondrogenic differentiation is
related to pellet size and weight, based on increases in proteoglycan
synthesis. SMSC pellets cultured in chondrogenic culture medium
with FK506 were significantly larger than those cultured without
FK506. Increases in pellet size were observed in a dose-dependent
manner, but were most prominent at 1000ng/ml FK506 (mean of
27.5%). Exposure to FK506 also enhanced alcian blue staining of the
cell pellets. Increases in staining were prominent in the center area
of the FK506-treated cell pellets, and the staining intensity exhibited
a dose dependency. Likewise, exposure to FK506 significantly
increased GAG levels in the pellets; and again these exhibited a dose-
dependency with increases of approximately 8-fold when FK506 was
added at 1000ng/ml.
FK506 and BMP2/TGFbeta1 additively promotes chondrogenesis in
human SMSCs.
To investigate whether the effects of FK506 on chondrogenesis were
additive or synergistic with those observed for chondrogenic growth
factors, the pellet culture system in the presence of growth factors
(BMP2 or TGFbeta1) and several doses of FK506 was assessed. In
combination with BMP2 or TGFbeta1, FK506 additively increased both
the size of the cell pellet and GAG levels in a dose dependent manner.
RT-PCR analysis showed that FK506 treated cell pellets exhibited
increased mRNA levels for collagen II and aggrecan irrespective of
the presence of growth factors, and the positive effects of FK506
on chondrogenesis were significantly promoted in combination
with BMP2 or TGFbeta1. Type X collagen gene expression was not
detected after 3 weeks of culture in any of the control or experimental
conditions. However, after 6 weeks of culture, expression of the type
X collagen gene in the BMP2/TGF beta1 supplemented groups was
detected.
Involvement of Smad signaling pathway in FK506 induced SMSCs
differentiation.
Since FK506 appeared to exhibit an additive effect with not only
BMP2, but also TGF beta1, both of which are members of the TGF beta
128 Extended Abstracts
super-family, the potential involvement of Smad signaling pathway
in FK506-mediated chondrogenesis was further investigated. The
phosphorylation of Smad1/5/8 and Smad3 was readily detected
following chondrogenic medium treatment of SMSCs. In FK506-
treated cells, phospho-Smad1/5/8 and phospho-Smad3 levels
were significantly higher than those in non-treated cells by 20-
40% (p<0.01) after 30 minutes, 60 minutes, and 120 minutes of
exposure to the drug. In addition, levels of phospho-Smad1/5/8 and
phospho-Smad3 were significantly elevated in both the nuclear and
cytoplasmic fractions after FK506 treatment. These data suggest
that FK506 treatment has significant effects on the phosphorylation
state of Smad proteins.
Discussion: The present studies demonstrate that a widely used
immunosuppressant, FK506, promotes chondrogenic differentiation
of human SMSCs and moreover, that FK506 additively enhances
chondrogenic differentiation of hSMSCs in the presence of
chondrogenic growth factors, such as BMP2 and TGF beta1. These
results indicated that FK506 treatment especially promoted early
chondrogenic differentiation of hSMSCs. These results indicate that
the Smad signaling pathways are involved in the FK506 effects on
SMSC differentiation towards a chondrogenic phenotype.
Although FK506 is already used patients clinically for therapeutic
purposes, particularly to prevent graft rejection; With further
optimization, FK506 could potentially become a unique therapeutic
reagent to promote cartilage repair, alone or in combination with
other modalities, either in vivo or in vitro.
References:
1. Nishigaki F, Sakuma S, Ogawa T, Miyata S, Ohkubo T, Goto T. FK506
induces chondrogenic differentiation of clonal mouse embryonic
carcinoma cells, ATDC5.: Eur J Pharmacol, 2002;437, 123-8.
2. Kugimiya F, Yano F, Ohba S, Igawa K, Nakamura K, Kawaguchi H,
Chung U. I. Mechanism of osteogenic induction by FK506 via BMP/
Smad pathways.: Biochem Biophys Res Commun, 2005;338, 872-9.
3. Tateishi K, Higuchi C, Ando W, Nakata K, Hashimoto J, Hart
DA, Yoshikawa H, Nakamura N. The immunosuppressant FK506
promotes development of the chondrogenic phenotype in human
synovial stromal cells via modulation of the Smad signaling pathway.
Osteoarthritis Cartilage. 2007 15:709-18.
Acknowledgments:
This work is supported by the Grant-in-Aid for Scientific Research,
Japan Society for the Promotion of Science, and the Nakatomi
Foundation Research Grant
3.3.1
Issues regarding clinical cartilage repair studies: Design, bias
and interpretation
G. Knutsen
Tromsoe/Norway
Introduction: Our group from Norway has lead a randomized trial
aimed at comparing ACI and microfracture[1,2]. This journey has
given me experience and reflections that I would like to share and
discuss. Our study, widely acknowledged as a well organized and
performed trial has also some weak points and certain bias, and for
the designing of a new study today improvements would have been
appropriate.
Content: Over the last two decades we have seen increasing amounts
of efforts to develop approaches to promote biological repair of
articular cartilage defects. The NEJM paper by Brittberg, Peterson et
al[3] introduced the orthopedic community to ACI and since then cell
based therapies have been a “hot” topic and tissue engineering
therapies using cells, growth factors and scaffolds come in different
and newer generations. Even though, Brittbergs study was not a
RCT, it motivated basic and clinical scientists all over the world, and
was also an important motivation factor when we planned our RCT.
However, we still do not have enough evidence to say that newer
generations and therapies are better than standard treatments like
marrow stimulation procedures[4]. New is not synonymous with
improved. It has been estimated that up to 90% of new treatments
introduced in medicine in general are useless (Dr. Karim F Hirji,
personal communication). Randomization Most of the clinical
cartilage repair studies do not answer important therapeutic
questions and do not bring us forward. We are still in the need of
higher quality RCTs and registries. It is extremely important to design
new studies so that we can get valid answers and a solid base for
further improvements. On the other hand every type of study has a
place, even case reports. One of the major problems we are facing in
this field is that we have hundreds of studies not supporting
necessary evidence for superiority. Additionally variations and newer
generations of techniques are introduced before the older ones have
been properly validated. The safety and complication rate have been
well documented in clinical studies. Randomization may be
accomplished using envelopes ore more sophisticated computer
models. It is also important to have an appropriate comparator when
new studies are designed. Bias, blinding and control It is also a fact
that bias is a big problem in cartilage repair studies. Even if stated
that no conflict of interest exists, problematic bias may be present.
Orthopedic surgeons and researchers need to collaborate with the
industry, but industry funded trials have a tendency to be biased. In
general patient based questionnaires are preferred. However, a
general observation is that overloading the participating patients
with too many surveys and tests, the drop out rate may increase. The
drop out rate of a study should be low. My experience is that by
using phone, mail and email you can get information from most
patients. Failures should have their last clinical score before revision-
surgery as their final score (principle of intention to treat). Blinding,
if possible should also as far as possible be used. There is a possibility
to blind the assessor using stockings covering an operated knee. It is
also possible to blind radiologists and pathologists even in case
series. In general bias is reduced by having a control group, and
blinding both assessors and patients. Independent assessors are of
great value. Blinding of the surgeons is seldom used, but may be
possible having e.g. products with and without cells. A major
weakness of RCTs in this field (including our original study) is the
lack of a non operative control group. The non operative control
group could e.g. be offered a detailed rehabilitation and training
program. Including a placebo surgery group is difficult, but very
interesting[5]. It has been argued that placebo effect could reach up
to 30-40% in surgery. It is also accepted that you need fewer patients
(smaller sample size) in a placebo designed study. Power analyses
are necessary to plan for optimal numbers of patients for each study.
Multi center studies are often needed to be able to get enough
power. However, multi center and multi surgeon trials create new
issues to be considered. It is extremely important to standardize the
protocols and provide adequate training for all surgeons. Some
centers may not have the same interests in the trial compared to
others and this may have high impact on the final outcomes.
Motivation of the participating surgeons is essential for success.
Having a non biased sponsor improves the quality enormously.
Participating centers should have written regulations regarding all
details including publication and use of the data. When designing a
new study you should define one primary outcome. Secondary
outcomes are also defined and described in detail before start of the
study. Surrogate endpoints as MRI and histology are additionally
clearly planned in advance. During the study period it is not a good

practice to change the endpoints (it may even be unethical and could
violate the study design). Analyzing subgroups could be problematic
and may confuse the main results of the study. In our study we
probably had too many endpoints and significant findings using
analyses at a given time point could have been interpreted as a false
positive result (Type I error). We concluded at the last follow up that
there was no difference between the two treatments, and if in fact
there was a difference this would have been a false negative result
(Type II error). It is possible to note that non superior trials are less
frequently published (publication bias). Journals also tend to favor
studies showing superior results of new treatments compared to
standard treatments. This fact indirectly represents a big hamper for
progression. We sent our Norwegian randomized trial first to NEJM;
however, this journal rejected the paper almost immediately. The
following years have told us that our trial has been evaluated as one
of the best so far and also rated as the trial with the least bias[6].
Clinical trials could give superior, non-inferior or equivalent profiles.
It is not enough to have one excellent and well performed clinical
RCT showing superiority to have the rights for changing the treatment
strategies. However, surgery is complex and several different
therapies may have equal outcomes for the patients. Before robot
surgery has taken over we have to realize that the surgeon himself
plays a very important role in the outcomes of an operation. Even
though, we need to answer critical questions regarding introduction
of new treatments as well as critical appraisal of standard treatments.
Animal studies are necessary as well as pilot clinical studies followed
by RCTs and registries. I believe it is critical to have easy access to
databases and clinical questionnaires. We need to collaborate and
share our knowledge to achieve clear improvements. I give value to
the Socrates database and others, but would support that these
instruments should be open and of free use for every clinician. I
believe a common platform-database including large numbers of
patients anonymously (both patients and surgeons) could have been
useful. A properly validated patient-based score could have been
the primary outcome, and secondary hard outcomes could have
been failure (new resurfacing cartilage operation) and joint
prosthesis. It is further for all clinical studies in this field essential to
have a detailed rehabilitation protocol, and evaluate the patient
compliance in this context. Interpretation We have so far not been
able to regenerate native-like hyaline cartilage in adults, and our
efforts results in repair cartilage of various qualities. Recently
subchondral bone and the interface between bone and cartilage
have come more into focus[7]. This might be an interesting arena for
research regarding cartilage regeneration approaches. The safety
and complication rate of biological clinically tested procedures
seems to be acceptable. Most procedures seems to have acceptable
short term results in younger active patients, however, osteoarthric
defects in older patients not wanting joint prosthesis are problematic
and long term results (more than 10 years) in quality RCTs are lacking
for all defects. In evaluating clinical studies it is important to
distinguish between acute, subacute and chronic lesions and also
note size, depth and localization of the treated defects. Acute smaller
chondral and subchondral injuries may become asymptomatic
regardless of surgery or not, and particularly in studies including
mainly acute lesions this has to be considered. In conclusion, I am
optimistic, in light of the huge efforts worldwide from scientists and
clinicians. Even though, we still have some steps to go.
References:
1. Knutsen G, Engebretsen L, Ludvigsen TC, Drogset JO, Grontvedt
T, Solheim E et al.: Autologous chondrocyte implantation compared
with microfracture in the knee. A randomized trial. J Bone Joint Surg
Am 2004, 86-A: 455-464.
2. Knutsen G, Drogset JO, Engebretsen L, Grontvedt T, Isaksen
V, Ludvigsen TC et al.: A randomized trial comparing autologous
chondrocyte implantation with microfracture. Findings at five years.
J Bone Joint Surg Am 2007, 89: 2105-2112.
3. Brittberg M, Lindahl A, Nilsson A, Ohlsson C, Isaksson O, Peterson
L: Treatment of deep cartilage defects in the knee with autologous
chondrocyte transplantation. N Engl J Med 1994, 331: 889-895.
4. Jakobsen RB, Engebretsen L, Slauterbeck JR: An analysis of the
quality of cartilage repair studies. J Bone Joint Surg Am 2005, 87:
2232-2239.
5. Moseley JB, O’Malley K, Petersen NJ, Menke TJ, Brody BA,
Kuykendall DH et al.: A controlled trial of arthroscopic surgery for
osteoarthritis of the knee. N Engl J Med 2002, 347: 81-88.
6. Magnussen RA, Dunn WR, Carey JL, Spindler KP: Treatment of
Extended Abstracts 129
focal articular cartilage defects in the knee: a systematic review.
Clin Orthop Relat Res 2008, 466: 952-962.
7. Gomoll AH, Madry H, Knutsen G, van Dijk N, Seil R, Brittberg M
et al.: The subchondral bone in articular cartilage repair: current
problems in the surgical management. Knee Surg Sports Traumatol
Arthrosc 2010, 18: 434-447.
8.1.2
The posterolateral corner of the knee
T. Spalding1, J. Bird2
1
Leamington Spa/United Kingdom, 2Coventry/United Kingdom
Introduction: THE POSTERO-LATERAL CORNER OF THE KNEE Tim
Spalding FRCS Orth. and Jonathan Bird FRCS Orth. ICRS 2010
University Hospital Coventry, UK Introduction Isolated postero
lateral corner (PLC) injuries are uncommon and probably represent
less than 2% of knee injuries. There is however a higher incidence of
PLC injuries in combination with ACL or PCL injuries. This is reported
to be as high as 30-40% depending on the series. The postero
lateral corner should always be examined when diagnosing an ACL
or PCL injury as unrecognised PLC injury can be a cause for failure in
outcome after ACL reconstruction.
Content: Evaluation of Postero-lateral Corner Accurate history is
important for the mechanism of injury which can include: impact
force to the antero medial aspect of the knee when it is nearly
at full extension, non contact or contact hyperextension injury,
varus load on a flexed knee, and knee dislocation. The patient will
usually describe pain in the postero lateral area of the knee and
it is important to ask for associated altered sensation in the lower
leg which may have been transient at the time of injury. There may
be giving way in a rotational activity or an instability feeling when
going downstairs. Symptoms may not necessarily start immediately
following injury but can progress over time as the PLC gradually
‘stretches out’. Examination Key examination features include
those listed below. In evaluation of acute injury it may be necessary
to rest the knee over a pillow in order to reduce guarding and make
it more comfortable to examine the PLC.
Alignment – check for varus alignment and lateral thrust of the knee
on walking.
Pain: there may be pain on hyperextension of the knee and a
reluctance to straighten the leg as this pulls on the injured PLC.
Swelling and bruising: there may be isolated swelling around the
postero lateral corner.
Footdrop: indicates nerve stretching.
Neurovascular examination looking for abnormal nerve function
including numbness and the dorsum of the foot and first web
space.
Detecting abnormal movement is the key to diagnosing PLC injury
and the tests include:
Varus stress test at 0° and 30° with leg in neutral rotation.
Prone external rotation dial test at 30° and 90°
Isolated PLC injury will give increased rotation at 30° only and not
at 90°
Increased rotation at 30° and 90° indicates combined PCL and PLC
injury
External rotation recurvatum test
Reverse pivot shift test
These clinical tests are hard to do as positive findings are infrequent,
making it difficult for the surgeon to build up expertise. The most
sensitive test is the prone dial test and in this test the patient needs
to lie fully prone and relaxed. Often an assistant is needed to hold
the two knees together while the examiner supports the feet and
ankle at 30° and 90°. A positive test is indicated when it is increased
from the other side – usually by 15° or more. There is a fair amount of
subjective interpretation required in this test. Management of Acute
Injuries Once acute injury to the postero lateral corner is recognised
then surgical management is best undertaken early before the
130 Extended Abstracts

tissue becomes too scared and difficult to reattach. The literature is Dowd GS: Combined reconstruction of chronic posterior cruciate
clear that acute reconstruction is better than chronic reconstruction ligament and posterolateral corner deficiency: A two- to nine-year
for isolated injury. (Noyes 1996) In multi ligament reconstruction for follow-up study. J Bone Joint Surg Br 2006;88:1169-1172. Larson RV:
the acutely multi-ligament injured knee Stannard et al (2005) has Isometry of the lateral collateral and popliteofibular ligaments and
identified a high failure rate for primary repair compared to anatomic techniques for reconstruction using a free semitendinosus tendon
reconstruction. Chronic Injuries Injuries beyond six weeks fall under graft. Oper Tech Sports Med 2001;9:84-90. LaPrade RF, Johansen
the category of chronic injuries where significant scarring means S, Wentorf FA, Engebretsen L, Esterberg JL,TsoA:An analysis of
that it is difficult to undertake any primary repair and the tissue is an anatomical posterolateral knee reconstruction: An in vitro
usually not suitable for repair. As previously described evaluation biomechanical study and development of a surgical technique.
of gait and alignment along with the state of the other ligaments in Am J Sports Med 2004;32:1405-1414. Noyes FR, Barber-Westin SD:
the knee is critical to formulate a complete plan for management. Treatment of complex injuries involving the posterior cruciate and
Various surgical techniques have been described over the years and posterolateral ligaments of the knee. Am J Knee Surg 1996;9:200-
these have been broadly categorised into non anatomic and anatomic 214.
reconstruction. (Cooper et al 2006) Non anatomic reconstructions are
generally based on the fibula head, reconstructing ligaments to the Noyes FR, Barber-Westin SD, Albright JC: An analysis of the causes
lateral epicondyle. The most popular repair is the Larson repair and of failure in 57 consecutive posterolateral operative procedures.
there have been many modifications to this over the years (Larson Am J Sports Med 2006;34:1419-1430. Stannard JP, Brown SL, Farris
2001). These techniques are technically easier than the anatomic RC, McGwin G Jr, Volgas DA: The posterolateral corner of the knee:
techniques and continue to be reported with good results (Khanduja Repair versus reconstruction. Am J Sports Med 2005;33:881-888.
2006). Anatomic techniques attempt to reconstruct the primary Yoon KH, Bae DK, Ha JH, Park SW: Anatomic reconstructive surgery
anatomy of the postero-lateral corner including the elements of the for posterolateral instability of the knee. Arthroscopy 2006;22:159-
three functional components – the lateral collateral ligament, the 165.
popliteus and the popliteo-fibula ligament utilising their anatomic
insertion points. In addition plication of the postero lateral capsule
is used to add to the repair. Two key techniques stand out: the
technique published by La Prade et al (La Prade 2004) and by Arciero
et al (Arciero 2005). Published results are scarce and generally tend
to involve surgery for the postero-lateral corner in combination
with posterior cruciate ligament injuries. The optimal technique is
therefore unclear. Long term knee stability still remains an issue
after reconstructive surgery. Yoon et al (2006) published improved
results comparing anatomic against non anatomic reconstruction
techniques. The decision however is difficult as anatomic techniques
are more surgically demanding and may lead to over constraint of
the knee.
La Prade technique: in this technique allograft tendons are used,
either Achilles tendon with bone block at one end or hamstring
tendons. An oblique tunnel is fashioned through the head of fibula
from antero-lateral to postero-medial at the styloid process. An
additional tunnel is made in the tibia from Gerdy’s tubercle to a point
in the popliteal fossa just medial to the superior T-F joint emerging
under the muscle belly of popliteus. The graft is passed from the
lateral epicondyle, under IT band to the antero-lateral fibula head,
through the fibula tunnel and through the tibial tunnel from posterior
to anterior. A second graft is passed from the insertion of the
popliteus on femur in a second tunnel, under the LCL reconstruction
and through the tibia from posterior to anterior. Both grafts are then
fixed with a staple and interference screw to the tibia.
Arciero technique: in this technique no tibial tunnel is drilled but
the same fibula tunnel is drilled. A single graft is passed from lateral
epicondyle, under IT band to antero lateral aspect of fibula, through
fibula tunnel and obliquely anterior to the popliteus tunnel which is
a separate tunnel located 12-15mm distal to the epicondyle (in the
line of the femur).
Grafts are tensioned with the knee at 60° but care needs to be
taken to ensure the knee is not over constrained. Post operative
rehabilitation is slow and the knee is protected in a hinged knee
brace. This is locked in extension when mobilising for the first six
weeks. During that time period the brace is unlocked during the
day for exercises. After six weeks the brace is removed and a sport
protective brace may be used up to six months.
Complications The main structure at risk is the peroneal nerve with
injury reported in 12-17% of acute cases. Fluid extravasation is a
further complication in addition to residual laxity, persistent knee
pain and arthrofibrosis (Noyes 2006). Importance Injury to the PLC
is uncommon and difficult to detect, yet it is vitally important when
reconstructing the ACL or the PCL and persisting joint laxity from a
missed PLC injury will compromise the result of the more central
ligament reconstruction. With practice, the surgical procedures can
become straightforward enough to lead to increasing the indications
for reconstruction and increased patient function.

References:
References Arciero RA. Technical Note Anatomic Posterolateral
Corner Knee Reconstruction. Arthroscopy 2005;21: 1147-1148 Cooper
JM, McAndrews PT, LaPrade RF: Posterolateral corner injuries of the
knee: Anatomy, diagnosis, and treatment. Sports Med Arthrosc
2006;14:213-220. Khanduja V, Somayaji HS, Harnett P, Utukuri M,

8.2.2
Using Biomarkers to Monitor Joint Tissue Pathology and Repair
V. Byers - Kraus
Durham/United States of America
Introduction: Osteoarthritis represents a heterogeneous group of
conditions with a common endstage pattern of joint degeneration
and maladaptive repair. The earlier the treatment likely, the greater
the chance of substantively altering the course of the disease for the
better. Biomarkers have the potential to provide an early warning
of the initiation of matrix breakdown that could prompt earlier
treatment to prevent the cartilage and bone destruction that leads to
disability. Although there are currently no qualified biomarkers that
can be considered as surrogate clinical endpoints, there are many
biomarkers that are both commercially available and shown to be
associated with one or more aspects of osteoarthritis and that fit
one or more categories of the BIPEDS classification scheme. BIPEDS
corresponds to Burden of disease, Investigational, Prognostic,
Efficacy of Intervention, Diagnostic and Safety biomarkers. These six
categories encompass the array of types of biomarkers that could be
used for enhancing clinical trials and repair strategies.
Content: When using biomarkers, the principle tissue source(s) of
a given biomarker should be identified as accurately as possible,
so that the origin(s) of the epitope(s) is clearly understood. This
in turn will facilitate the interpretation of clinical data when repair
strategies are utilized in a trial setting. In this perspective the
most proximal source of information is provided by synovial fluid
analyses. A characterization of the synovial fluid biomarker patterns
ensuing in early joint damage provides a signature of pathology
that can be monitored to determine the efficacy of a repair strategy.
An example will be provided from a recent pilot clinical trial. The
OARSI FDA Biomarkers Working Group recommendations and
current developments to enhance qualification of biomarkers for
osteoarthritis will be described. It is hoped that these endeavors
will provide valuable tools to assist the pursuit of cartilage repair
strategies.
References:
Bauer DC, Hunter DJ, Abramson SB, et al. Classification of
osteoarthritis biomarkers: a proposed approach. Osteoarthritis
Cartilage. Aug 2006;14(8):723-727. [BIPEDS reference] Kraus, VB,
B Burnett, J Coindreau, S Cottrell, D Eyre, M Gendreau, J Gardiner,
P Garnero, J Hardin, Y Henrotin, D Heinegard, A Ko, S Lohmander,
G Matthews, J Menetski, R Moskowitz, S Persiani, AR Poole, JC
Rousseau, M Todman. 2010. Application of biomarkers in the
development of drugs intended for the treatment of osteoarthritis:
OARSI FDA Osteoarthritis Biomarkers Working Group. Osteoarthritis
Cartilage (in press).
Extended Abstracts 131
8.2.3
Can biomarkers be used as outcome measures in cartilage repair?
S.L. Lohmander
Lund/Sweden
Introduction: The challenge of cartilage repair, be it the repair
of a cartilage defect in an otherwise normal joint, or the repair/
regeneration of cartilage in a joint with early-stage osteoarthritic
changes, carries with it the need for outcome measures. Without
appropriate outcome measures, results cannot be documented.
Here, as in other conditions of the joint, we should consider patient-
reported outcomes on symptoms, function and quality of life as the
gold standard, the clinical endpoint. Other outcomes of interest may
include functional tests to determine biomechanical tissue quality,
imaging techniques to monitor structure and quality of joint tissues,
and biomarkers (aka molecular markers) to reflect the turnover,
structure and status of joint tissues. In common with osteoarthritis,
studies on cartilage repair require long observation times and
trials, and there is therefore a great need for measures that predict
the long-term clinical outcome: surrogate markers of outcome.
Considerable efforts are being invested in the development of
biomarkers for osteoarthritis, and much of this work is relevant also
when considering biomarkers for cartilage repair.
Content: For osteoarthritis biomarkers, a terminology named BIPEDS
was proposed (Bauer et al. 26), which classifies these biomarkers
into five categories corresponding to their proposed utility: Burden
of disease, Investigational, Prognostic, Efficacy of Intervention,
Diagnostic and Safety. Biomarkers that are likely to have the
earliest beneficial impact on clinical trials fall into two categories.
The first are those markers that would allow us to select for trials
subjects that are most likely to respond and/or progress (prognostic
markers) within a reasonable time for a clinical study, which for a
cartilage repair or osteoarthritis study could be one to two years.
The second category of biomarkers includes those that provide early
feedback for preclinical decision-making and for trial organizers that
an intervention has the desired effect on the primary target (efficacy
markers). Both types of biomarkers are highly desirable in chronic
conditions where conventional clinical outcomes may take years
to present. These two categories could reduce the burden and risk
of clinical trials by delivering essential early information, speeding
up the product development cycle and making both osteoarthritis
and cartilage repair more manageable targets. Many biomarkers are
currently available that are being tested in relation to their utility
for studies on osteoarthritis, existing in various states of validation.
No biomarkers have been validated for cartilage repair. The term
validation as used here refers to the evidence for a marker in
support of its use as a surrogate endpoint, and includes verification
of analytical performance characteristics and correlation of the
biomarker with a specific biological process. Naturally, validation of
a biomarker against a gold standard endpoint depends critically also
on the performance and specificity of that gold standard endpoint.
So what is the current evidence to support the use of biomarkers as
surrogates of clinical outcomes in osteoarthritis, and might they be
helpful as biomarkers of cartilage repair? A second useful classification
system divides biomarkers into four categories according to their
current level of qualification (Wagner et al. 27). Exploration level
biomarkers are research and development tools with in vitro and/
or preclinical evidence but without consistent information linking
the biomarker to clinical outcomes in humans. Demonstration
level biomarkers are associated with clinical outcomes in humans
but have not been reproducibly demonstrated in clinical studies.
Characterization level biomarkers are reproducibly linked to clinical
outcomes in more than one prospective clinical study in humans.
Surrogacy level biomarkers can substitute for a clinical endpoint,
corresponding to ‘‘surrogate end point’’ as mentioned above, and
requires agreement with regulatory authorities as an FDA registrable
endpoint. The table below lists a selection of commercially available
osteoarthritis-related biomarkers and an approximation with
regard to level of qualification (van Spil et al. 2). However, that
qualification is as always dependent on a specific context, limiting
generalizability in the early development phase. There are currently
no qualified biomarkers that can be considered as surrogate clinical
endpoints in OA. Advances in the field will lead to rapid expansion
of this list and more specificity with regard to qualification. Each of
these biomarkers can be measured from easily available serum or
urine samples. Challenges for the development and validation of
biomarkers with utility for cartilage repair are similar to those for
osteoarthritis, and will require the combination of reproducible and
specific biomarker assays with large prospective, controlled clinical
trials. To demonstrate utility of e.g. efficacy biomarkers, these trials
will need to show structural joint protection, cartilage repair and/
or clinically relevant efficacy. An additional challenge with regard
132 Extended Abstracts

to cartilage repair studies and biomarkers will be that the cartilage 11.1.1
repair site often represents only a small localized process, as
compared to a condition such as osteoarthritis which affects the Physical stimulation of cartilage repair by pulsed
whole or most of the joint. electromagnetic fields
M. Cadossi
Bologna/Italy
Biomarker Proposed process BIPEDS Classification Introduction: Articular cartilage is a complex tissue characterised by
urinary CTX-II type II collagen degradation BPED (characterization) a cellular component, chondrocytes, that are embedded within an
organized dense extracellular matrix of collagen and proteoglycan,
serum COMP cartilage degeneration BPD (exploration) with a high water content: 75% of the weight of articular cartilage
serum HA osteophyte burden, synovitis BPED (demonstration) (Ulrich-Vinther M, 2003). Because of its high hydrostratic pressure,
serum and urine C,2C types I and II collagen degradation D (exploration)
the articular cartilage can withstand a large number of repetitive
strains during the lifetime; in fact, articular cartilage plays the
serum and urine C2C type II collagen degradation ED (demonstration) fundamental role of facilitating movements between the articular
serum and urine Coll2- & heads, reducing friction, it facilitates the sliding of opposite articular
type II collagen degradation D,B,P (exploration) surface, it absorbs and redistributes the mechanical loads on the
Coll2-NO2
joint (Buckwalter JA, 2002). Under physiologic condition, articular
serum CPII type II collagen synthesis D (exploration)
metabolism is slow, but under pathological condition turnover can
PIIANP collagen synthesis BPD (exploration) increase and the matrix undergoes faster mechanical failure and
urine/serum NTX- bone resorption P,E (demonstration) deterioration, resulting in cartilage degeneration and development
of arthritis, with a consecutively loss of the integrity of the collagen
urine/serum CTX- bone resorption B,D,P (exploration) matrix. Among the causes that may damage the cartilaginous tissue,
serum CS846 aggrecan synthesis /turnover P (exploration) we should distinguish between a large number of stimuli, including
serum MMP-3 protease in joint tissue degradation E (characterization)
metabolic, genetic, vascular and mechanical disorders. Mechanical
injuries can occur from a single but severe load or repetitive but
prolonged joint loading, that can cause microdamage, chondral or
osteochondral fracture, ultimately exposing the subchondral bone
References: tissue (Buckwalter JA, 2002; Radin EL, 1986). Moreover, modest
damage of the articular cartilage, resulting from trauma or less
Bauer, D. C. et al. Classification of osteoarthritis biomarkers: a invasive surgical procedure, produces an inflammatory reaction of
proposed approach. Osteoarthritis Cartilage 2006;14:723-7. the joint cartilage, that can cause irreversible degeneration through
the increase of catabolic cytokines synthesis and the decrease
van Spil, W., DeGroot, J., Lems, W., Oostveen, J. & Lafeber, F. Serum of anabolic activity of chondrocytes. Pro-inflammatory cytokines
and urinary biochemical markers for knee and hip osteoarthritis: increase the synthesis of matrix degrading enzymes and limit the
a systematic review applying the consensus BIPED criteria. production of proteoglycans (Schuerwegh AJ, 2003; Pellettier JP,
Osteoarthritis Cartilage in press 2010. 1999; Goldring SR, 2004). Being a hypocellular avascular tissue, the
articular cartilage has minimal reparative potential: “cartilage when
Wagner, J. A., Williams, S. A. & Webster, C. J. Biomarkers and destroyed it is never recovered” (Hunter W, 1743). In the course of
surrogate end points for fit-for-purpose development and regulatory human life, the quality and mechanical properties of the articular
evaluation of new drugs. Clin Pharmacol Ther 2007;81:104-7. cartilage can only decrease. In consideration of such premises, the
availability of a therapeutic technique to prevent degeneration and
to control local inflammation, both in the subchondral bone and
in the articular structures, is of fundamental importance. Therapy
has to directly affect the chondrocytes in the cartilage thickness, to
prevent the catabolic effects of inflammatory cytokines and to favour
anabolic activities and proteoglycans synthesis. The simultaneous
treatment of cartilage, the subchondral bone tissue and the articular
structures, is feasible only through physical means, such as pulsed
electromagnetic fields, whose spatial distribution is able to expose
uniformly the whole articular cartilage in its total thickness and
extension. Massari et al.(Massari L, 2007), summarised the results
of the translational research of the CRES (Cartilage Repair and
Electromagnetic Stimulation) study group on the use of pulsed
electromagnetic fields with specific parameters (I-ONE therapy,
IGEA, Carpi, Italy) to control local joint inflammation and ultimately
to have a chondroprotection effect on articular cartilage.
Content: Biophysical stimulation with I-ONE therapy In vitro and ex
vivo effects: inflammation control and chondroprotection Preclinical
studies for the cartilage repair have shown how biophysical
stimulation with I-ONE therapy has to be considered as a therapeutic
system, comparable to a pharmacological treatment. A recent ex
vivo study, on articular cartilage explants, showed that the
proteoglycan synthesis and the anabolic activity increased
significantly when specific parameters of the pulsed electromagnetic
fields were set: peak intensity of magnetic field 1.5 mT, minimum
stimulation time 4 hours, I-ONE therapy (De Mattei M, 2007). These
effective parameters were subsequently used in experiments on
animal model. With these exposure parameters, the presence of
growth factors in the microenvironment and environmental condition
are able to increase the proliferation response of chondrocytes
(Pezzetti F, 1999; De Mattei M, 2001). On cartilaginous bovine
explants, I-ONE therapy acts in concert with the major anabolic
growth factor (IGF-1) for cartilage to stimulate the synthesis of
essential cartilage matrix components and to increase significantly
proteoglycan synthesis (De Mattei M, 2003; De Mattei M, 2004).
Articular cartilage explants cultivated in the presence of inflammatory
cytokines (IL-1b) show an increase in catabolic activities, accompanied
by the degradation of the cartilaginous matrix. However, if explants
are exposed to biophysical stimulation with I-ONE therapy, the
catabolic effect of the inflammatory cytokine on the matrix is totally
inhibited and the integrity of the cartilaginous matrix is preserved
(De Mattei M, 2003). The anti-inflammatory activity was also tested

on human neutrophils cell membrane, on bovine chondrocytes and
fibroblast-like synoviocytes exposed to I-ONE therapy, where an
upregulation of the A2A adenosine receptors determined: inhibition
of pro-inflammatory cytokine production, an increase of adenalyl
cyclase activity and a reduction of superoxide anion production, as a
result of upregulation of the A2A receptors located on the surface
(Varani K, 2002; Varani K, 2008). Kinetics studies have shown that
the anti-inflammatory effect is dependent on the width, frequency
and shape of the wave of the biophysical stimulus created at the
surface of the cell membrane (Varani K, 2002). Moreover, it has been
demonstrated that drugs with A2A adenosine receptor agonist
activity have shown to protect articular cartilage in animal models of
induced osteoarthritis (Cohen SB, 2004). Together, these findings
show that I-ONE therapy has an A2A adenosine receptor agonist
activity, identifying the A2A adenosine receptor as the
pharmacological molecular target of I-ONE treatment in inflammatory
joint diseases. In a recent study, De Mattei et al. demonstrated how
I-ONE therapy can strongly inhibit the release of PGE2 in bovine
synovial fibroblasts exerting an anti-apoptotic effect on cells (De
Mattei M, 2009). Moreover the effects of I-ONE therapy exposure on
PGE2 production and COX-2 expression closely involved A1 and A2A
adenosine receptors. These results are in line with the known anti-
inflammatory activities of adenosine. The involvement of adenosine
receptors and I-ONE therapy exposure in the inflammatory responses
of synovial fibroblasts open perspectives to develop novel anti-
inflammatory approaches in joint degenerative diseases. In vivo
effects: chondroprotection and bone repair The in vitro results are
confirmed by in vivo experiments. Subchondral bone, bone marrow,
synovial cells, chondrocytes and synovial fluid contribute to the
development of osteoarthritis and to the healing of defects of
articular cartilage; for this reason the use of animal models is
essential to understand the repair process. An animal model which
develops spontaneous and asymptomatic age-related osteoarthritis
of the knee, like Dunkin Hartley guinea pigs, was studied to show the
strong chondroprotective effect of I-ONE therapy. The capability of
biophysical stimulation to modify osteoarthritis progression was
firstly demonstrated by Ciombor (Ciombor DM, 2005) and
subsequently by Fini (Fini M, 2005; Fini M, 2008) in osteoarthritis
lesions of different severity. The values of the cartilage and
subchondral bone thickness, the fibrillation index and the
quantification of the articular cartilage damage, according to
Mankin’s score, highlight that I-ONE therapy exposure preserves the
morphology of articular cartilage, inhibit subchondral bone sclerosis
and retard the development of osteoarthritis lesions in the entire
knee of aged osteoarthritic guinea pig. The degenerative processes
observed in the articular cartilage with aging are inhibited by I-ONE
stimulation. The health of the cartilage and the success of surgery is
largely dependent on the characteristics of the subchondral bone
tissue. Large osteochondral defect can be healed with many
arthroscopic repair strategies, such as osteochondral grafts. In a
sheep model, in which osteochondral grafts were performed, it has
been showed that I-ONE therapy is able to enhance the new
subchondral bone formation, in the interface between the graft and
the host bone, favouring early graft stabilization. Moreover, a smaller
incidence of cyst-like resorption areas, that can compromise the
stability of graft and the success of the technique, was recorded in
the stimulated animal. To support the in vitro results histological
analysis of the synovial fluid were also performed in this animal
model. The amount of inflammatory catabolic cytokines (IL-1b and
TNF-a) in the synovial fluid of I-ONE treated animals was significantly
lower than in control animals. On the contrary, TGF-b1 was
significantly higher in stimulated animals than it was in controls.
These results demonstrate not only the capability of I-ONE therapy
to control the inflammatory reaction, but also its capability to favours
cartilage anabolic activity (Benazzo F. J Orthop Res. 2008). Clinical
trials The above results provide the rational to design clinical studies
to demonstrate the possibility to transfer the treatment to humans.
Two randomized, prospective, double-blind clinical trials (Level I)
were conducted, one in patients treated by arthroscopy with
condroabrasion and/or microfractures in the knee (Zorzi C. 2007)
and the other in patients after anterior cruciate ligament
reconstruction (Benazzo L. Knee Surg Sports Traumatol Arthrosc.
2008). Condroabrasion and/or microfractures. The first randomized,
prospective, double-blind study was performed on patients suffering
from cartilaginous degeneration in the knee that underwent a
microfractures and chondroabrasion arthroscopic treatment. The
patients were randomized in two homogenous groups: half of the
patients were stimulated with active device (I-ONE group) and half
with placebo one. All patients were instructed to use I-ONE device
for 90 days, 4h per day. The patients were evaluated by the Knee
injury and Osteoarthritis Outcome Score (KOOS) test before
arthroscopy, and after 45 and 90 days. The use of NSAIDs to control
pain was also recorded. Patients were interviewed for the long-term
outcome 3 years after arthroscopic surgery. Thirty-one patients were
Extended Abstracts 133
evaluated. KOOS value was the same for both groups at time zero
(p=n.s.); at 45 and 90 days higher KOOS values were observed in the
I-ONE group and the difference became significant at day 90
(p<0.05) and demonstrated that I-ONE therapy leads to complete
patient’s recovery in a significantly shorter time. The percentage of
patient who used of NSAIDs was 26% in the active group and 75% in
the control group (p<0.05). At 3 years follow-up, the number of
patients who completely recovered was higher in the I-ONE group
compared to the control group (p<0.05). Anterior cruciate ligament
reconstruction. Patients with rupture of anterior cruciate ligament at
knee underwent arthroscopically assisted reconstruction with use of
double-looped semitendinosus and gracilis tendon grafts using
biodegradable interference fit fixation. They were randomised in
placebo or active group according to age, sex, smoking status and
pathology. All patients were instructed to use the device for 60 days,
4 hours per day: half of the patients were stimulated with active
device (I-ONE group) and half with placebo one. The assessment was
performed using the International Knee Documentation Committee
form (IKDC) before, 30, 60 and 180 days after arthroscopy. The
control of pain was also recorded by VAS (Visual Analog Scale).
Patients were interviewed for long term follow-up at 1 and 2 years
after arthroscopy. Sixty patients completed the study. Pre-
operatively, no difference was observed between the 2 groups. At
day 30 the percentage of patients requiring NSAID was significantly
lower in the I-ONE group than in the placebo (p<0.05). The results
showed a significant difference (P<0.0001) also between the I-ONE
and placebo groups for VAS. At day 60, joint swelling was observed
only in the placebo group (p<0.05) and limitation of knee range of
motion was less frequent in the I-ONE group than in the placebo
(p<0.05). After surgery, the SF-36 score increased faster in the
I-ONE group than in the placebo (p<0.05). At the 2-year follow-up
86% of the patients in the I-ONE group and 75% in the placebo group
reported complete functional recovery: no knee pain and return to
sport activity. Conclusions. Joint injury involves synovial tissue,
cartilage and subchondral bone leading to joint inflammation,
swelling and pain. Inflammation can cause irreversible degeneration
of the cartilage through increased catabolic functions and decreased
anabolic activity of chondrocytes. Damage of this remarkable tissue
decreases mobility and frequently causes pain with movement and,
in the most severe instance, chronic pain. Our experimental and
clinical results show how the specific biophysical stimulus, I-ONE, is
able to inhibit inflammatory effects of catabolic cytokines and to
favour anabolic activity of cytokines, in the whole extension and
thickness of the articular cartilage. Moreover, I-ONE therapy acts like
a drug, with a specific posology; in fact its efficacy is dependent on
the field amplitude, frequency and duration. The preclinical findings
are the scientific basis for the clinical use of I-ONE therapy
immediately following surgery. I-ONE therapy is an effective
chondroprotective treatment for patients, without any negative side
effects, that limits inflammation, reduces recovery time and,
ultimately, preserves a healthy articular cartilage of the knee.
References:
Benazzo F, Cadossi M, Cavani F, Fini M, Giavaresi G, Setti S, Cadossi R,
Giardino R. Cartilage repair with osteochondral autografts in sheep:
effect of biophysical stimulation with pulsed electromagnetic fields.J
Orthop Res. 2008 May;26(5):631-42. · Benazzo F, Zanon G, Pederzini
L, Modonesi F, Cardile C, Falez F, Ciolli L, La Cava F, Giannini S, Buda
R, Setti S, Caruso G, Massari L. Effects of biophysical stimulation
in patients undergoing arthroscopic reconstruction of anterior
cruciate ligament: prospective, randomized and double blind study.
Knee Surg Sports Traumatol Arthrosc. 2008 Jun;16(6):595-601. ·
Buckwalter JA. Articular cartilage injuries. Clin Orthop Relat Res.
2002 Sep;(402):21-37. Review. · Modification of osteoarthritis by
pulsed electromagnetic field--a morphological study. Osteoarthritis
Cartilage 2003;11(6):455-62. · Cohen SB, Gill SS, Baer GS, Leo BM,
Scheld WM, Diduch DR. Reducing joint destruction due to septic
arthrosis usingan adenosine2A receptor agonist. J Orthop Res
2004;22:427–35. · De Mattei M, Caruso A, Pezzetti F, Pellati A,
Stabellini G, Sollazzo V, Traina GC. Effects of pulsed electromagnetic
fields on human articular chondrocyte proliferation. Connect Tissue
Res. 2001;42(4):269-79. · De Mattei M, Pasello M, Pellati A, Stabellini
G, Massari L, Gemmati D, Caruso A. Effects of electromagnetic fields
on proteoglycan metabolism of bovine articular cartilage explants.
Connect Tissue Res. 2003;44(3-4):154-9. · De Mattei M, Pellati A,
Pasello M, Ongaro A, Setti S, Massari L, Gemmati D, Caruso A. Effects
of physical stimulation with electromagnetic field and insulin growth
factor-I treatment on proteoglycan synthesis of bovine articular
cartilage. Osteoarthritis Cartilage. 2004;12(10):793-800. · De
Mattei M, Fini M, Setti S, Ongaro A, Gemmati D, Stabellini G, Pellati
A, Caruso A. Proteoglycan synthesis in bovine articular cartilage
134 Extended Abstracts
explants exposed to different low-frequency low-energy pulsed
electromagnetic fields. Osteoarthritis Cartilage. 2007;15(2):163-
8. · De Mattei M, Varani K, Masieri FF, Pellati A, Ongaro A, Fini M,
Cadossi R, Vincenzi F, Borea PA, Caruso A. Adenosine analogs and
electromagnetic fields inhibit prostaglandin E(2) release in bovine
synovial fibroblasts. Osteoarthritis Cartilage. 2009 Feb;17(2):252-
62. · Fini M, Giavaresi G, Torricelli P, Cavani F, Setti S, Cane V,
Giardino R. Pulsed electromagnetic fields reduce knee osteoarthritic
lesion progression in the aged Dunkin Hartley guinea pig. J Orthop
Res. 2005;23(4):899-908. · Fini M, Torricelli P, Giavaresi G, Aldini
NN, Cavani F, Setti S, Nicolini A, Carpi A, Giardino R. Effect of pulsed
electromagnetic field stimulation on knee cartilage, subchondral
and epyphiseal trabecular bone of aged Dunkin Hartley guinea
pigs. Biomed Pharmacother. 2008 Dec;62(10):709-15. · Goldring SR,
Goldring MB. The role of cytokines in cartilage matrix degeneration
in osteoarthritis. Clin Orthop Rel Res 2004;427S:S27-S36. ·
Hunter W. Of the structure and diseases of the articular cartilages.
Philosophical Transaction London. 1743;42:514-21. · Massari L,
Benazzo F, De Mattei M, Setti S, Fini M; CRES Study Group. Effects
of electrical physical stimuli on articular cartilage. J Bone Joint Surg
Am. 2007;89 Suppl 3:152-61. Review. · Pellettier JP. The influence
of tissue cross-talking on OA progression: role of nonsteroidal
anti-inflammatory drugs. Osteoarthritis Cartilage1999;7:374-6.
· Pezzetti F, De Mattei M, Caruso A, Cadossi R, Zucchini P, Carinci
F, Traina GC, Sollazzo V. Effects of pulsed electromagnetic fields
on human chondrocytes: an in vitro study. Calcif Tissue Int.
1999;65(5):396-401. · Radin EL, Rose RM: Role of subchondral bone
in the initiation and progression of cartilage damage. Clin Orthop.
1986, Dec;(213):34-40. · Schuerwegh AJ, Dombrecht EJ, Stevens WJ,
Van Offel JF, Bridts CH, De Clerck LS. Influence of pro-inflammatory
(IL-1 alpha, IL-6, TNF-alpha, IFN-gamma) and anti-inflammatory
(IL-4) cytokines on chondrocyte function. Osteoarthritis Cartilage.
2003 Sep;11(9):681-7. · Ulrich-Vinther M, Maloney MD, Schwarz EM,
Rosier R, O’Keefe RJ. Articular cartilage biology. J Am Acad Orthop
Surg. 2003;11(6):421-30. Review. · Varani K, Gessi S, Merighi S,
Iannotta V, Cattabriga E, Spisani S, Cadossi R, Borea PA. Effect of
low frequency electromagnetic fields on A2A adenosine receptors in
human neutrophils. Br J Pharmacol. 2002;136(1):57-66. · Varani K, De
Mattei M, Vincenzi F, Gessi S, Merighi S, Pellati A, Ongaro A, Caruso
A, Cadossi R, Borea PA. Characterization of adenosine receptors in
bovine chondrocytes and fibroblast-like synoviocytes exposed to low
frequency low energy pulsed electromagnetic fields. Osteoarthritis
Cartilage. 2008;16(3):292-304. · Zorzi C, Dall’oca C, Cadossi R, Setti
S. Effects of pulsed electromagnetic fields on patients’ recovery after
arthroscopic surgery: prospective, randomized and double-blind
study. Knee Surg Sports Traumatol Arthrosc. 2007;15(7):830-4.
11.2.1
New insights into cartilage biology and pathology using mouse
cartilage proteomics
J.F. Bateman1, R. Wilson2, S. Zivkovic2
1
Victoria/Australia, 2Melbourne/Australia
Introduction: In recent years there has been a rapid expansion in the
use of proteomics to discover biomarkers of joint disease, unravel
new molecular mechanisms underlying cartilage degeneration and
identify cartilage components and interactions that have evaded
detection by conventional biochemical approaches. Our focus is
to develop and apply techniques for proteomic analysis of mouse
cartilage. Despite the limited amounts of material available,
the benefits of mouse tissues outweigh the disadvantages.
Specifically, the use of mouse models will facilitate comparison
between wild-type tissue and cartilage lacking key elements of the
degenerative machinery (e.g. ADAMTS5), cartilage lacking novel
or important cartilage components or harbouring disease -causing
mutations (eg., chondrodysplasia). 2-D electrophoresis (2-DE)
remains a popular method for differential proteomics; however the
composition of cartilage presents a unique challenge (1-5). Using
centrifugal ultrafiltration to deplete aggrecan and hyaluronan from
the media of femoral head (P21) explant cultures, we identified novel
components of the cartilage “secretome”, including proteolytic
fragments of perlecan/endorepellin, thrombospondin-1, connective
tissue growth factor and chondromodulin. In addition to well-known
markers such as MMP-3 and cartilage gp-39, new markers for
cytokine-induced cartilage degradation, such as lipocalin-2, were
identified and quantified using fluorescent labelling and difference
in-gel electrophoresis (DIGE). More recently we have focused
on proteomic analysis of cartilage tissue extracts using a novel
fractionation approach based on differential protein solubility (6).
Sequential extraction of mouse femoral head cartilage with 1 M NaCl
followed by 4 M GdnHCl generates very distinct but, importantly,
consistent 2-DE profiles. Identification of differentially abundant
protein spots using HPLC-MS/MS (Agilent XCTplus ion trap) revealed
effective partitioning of readily soluble cytosolic components
(e.g. triosephsophate isomerase) from more tightly integrated,
predominantly, matrix components (e.g. decorin, matrilin-1,
lactadherin). Resolution by 2-DE effectively targets differentially
abundant proteins and readily provides a visual map of a tissue
proteome, including modified protein isoforms and proteolytic
fragments. However, recent advances in high-resolution tandem
mass spectrometry have enabled large-scale protein identification
from complex peptide mixtures generated by in-solution tryptic
digestion. Using nanoLC MS/MS (LTQ-Orbitrap) and spectral counting
we identified 600 proteins at high confidence in extracts of 21 day old
mouse cartilage, of which more than 250 proteins were significantly
enriched in either the 1M NaCl or 4M GuHCl extract. Surprisingly, the
solubility-based partitioning of proteins applied equally to cellular
components (e.g. ribonuclear and proteasomal subunits) as ECM
proteins and proteoglycans. Our fractionation approach therefore
facilitates deeper mining of the cartilage proteome whilst retaining
important biochemical information related to the proteins identified.
We are currently using this approach to identify novel components
of cartilage development, using high-density scaffold-free mouse
chondrocyte cultures. While the resulting multi-layered “neo-
cartilage” lacks the stratified organization of other systems (e.g.
bovine), ultrastructural analysis by transmission EM revealed clearly
defined pericellular and territorial zones, dense proteoglycan/
collagen networks and intricate chondrocyte-matrix contacts.
Comparison of 3-week neo-cartilage with 3-day epiphyseal cartilage
by SDS-PAGE revealed major differences in protein composition and
extractability. In juvenile cartilage the greater proportion of proteins
were readily soluble, whereas in the neo-cartilage a higher proportion
of the extract was poorly soluble. Therefore, label-free quantitative
MS/MS, combined with rigorous statistical and bioinformatic
methods, was used to generate “extraction profiles” (NaCl-extracted
versus GdnHCl-extracted) of the juvenile cartilage and neo-cartilage
(7). We identified a panel of proteins involved in maturation of the
neo-cartilage ECM, i.e. components with the greatest differential
in extractability between the two sample types, including many
pericellular and extracellular matrix components (e.g. collagen VI,
nidogen-2, perlecan, matrilin-3 and COMP). Unexpectedly, one of
the guanidine extract specific proteins in the mouse neo-cartilage
was a serine protease inhibitor, protease nexin-1. We confirmed that
PN-1 was a novel component of the developing articular cartilage in
vivo by immunohistochemistry. The potential further application of
cartilage “extraction profiling” in the context of cartilage repair will
be discussed.

References:
1. Belluoccio, D., Wilson, R, Thornton, D.J., Wallis, T.P., Gorman, J.J.
and Bateman, J.F. (2006) Proteomic analysis of mouse growth plate
cartilage. Proteomics 6, 6549-6553.
2. Wilson, R. and Bateman, J.F. (2008) Cartilage Proteomics:
Challenges, solutions and recent advances. Proteomics: Clinical
Applications 2 (2), 251-263
3. Wilson, R., Belluoccio, D and Bateman, J.F. (2008) Proteomic
analysis of cartilage proteins Methods 45 (1), 22-31
4. Wilson, R., Belluoccio, D., Little, C.B., Fosang, A.J. and Bateman,
J.F. (2008) Proteomic characterization of cartilage degradation in
vitro.Arthritis and Rheumatism 58(10): 3120-3131.
5. Wilson, R., Whitelock, J.M. and Bateman, J.F. (2009) Proteomics
makes progress in cartilage and arthritis research Matrix Biology
28(3):121-8.
6. Wilson, R. and Bateman, J.F. (2008) A robust method for proteomic
characterization of mouse cartilage using solubility-based
fractionation and two-dimensional electrophoresis. Matrix Biology
27(8):709-12
7. Wilson, R., Diseberg, A., Gordon, L., Zivkovic, S., Tatarczuch, L.,
Mackie, E., Gorman, J.J. and Bateman, J.F. (2010) Comprehensive
profiling of cartilage extracellular matrix formation and maturation
using sequential extraction and label-free quantitative proteomics.
Molecular and Cellular Proteomics. Feb 26. [Epub ahead of print]
Acknowledgments:
This work was funded by the National Health and Medical Research
Council of Australia.
Extended Abstracts 135
11.2.3
Specific regions of genome contribute to articular cartilage
regeneration and osteoarthritis resistance in mice
M.F. Rai, S. Hashimoto, J. Cheverud, L.J. Sandell
St. Louis/United States of America
Introduction: Articular cartilage injuries heal poorly which is thought
to lead to cartilage degeneration and predispose to the development
of osteoarthritis (OA) [1]. Many attempts have been made to restore
or repair cartilage injuries through surgical interventions including
tissue engineering but have shown unpredictable and variable
outcomes. Recently, a mouse model of cartilage repair has been
characterized which determined a direct correlation between
articular cartilage healing and protection from OA comparing the
healing DBA/1 mouse with the non-healing C57Bl/6 strain [2]. Also
recently, it has been shown that MRL/MpJ strain of mice shows an
ability to regenerate knee articular cartilage over time [3]. The MRL/
MpJ strain has been produced by two final backcrosses of LG/J (Large,
a congenic healer strain for ear wound healing) and shares 75% of its
genome with this strain [4]. We were interested in exploring cartilage
repair ability in healer and non-healer recombinant inbred (RI) lines
generated from LG/J and SM/J (small) mice and thus to discover
the genetic basis for cartilage repair. The RI lines were developed
through brother-sister mating of LG/J x SM/J mice at F7 for over 20
generations [5]. Each strain of the RI line contains unique portions of
the healing strain genome. We predict that the animals that are able
to heal a wound in their ear are more able to repair damaged articular
cartilage and may be more resistant to OA. As OA is considered to
be a complex genetic disease, our present project creates a unique
resource for study of genes that contribute to cartilage healing and
thus to protection from OA in 4 strains of RI line and the parental
strains. This will narrow the genetic site to 10-20 genes responsible
for cartilage healing.
Content: An acute full-thickness cartilage injury was introduced in
the trochlear groove of both knees of 8-weeks old mice from both
sexes according to the method of Fitzgerald et al. [3]. Mice were
sacrificed at 4, 12 and 16 weeks (strain 6 and 33) and at 12 weeks
(strain 35 and N48). SM/J, DBA/1 and MRL/MpJ were used as controls
and sacrificed at 12 weeks. At indicated time points, knee joints were
harvested and promptly fixed in 10% neutral buffered formalin for
12-14 hours. Joints were decalcified using 10% formic acid in 5%
formaldehyde for 48 hours followed by incubation in 0.01% EDTA for
4-6 hours. Samples were paraffin embedded, sagittally sectioned at
5 µm intervals and subsequently stained with 0.03% toluidine blue.
Slides were graded for five parameters including cell morphology,
staining intensity, surface regularity, thickness of hyaline cartilage
and its integration with native cartilage tissue on a scale from 1 to 14.
Selected sections from each strain were subjected to immunostaining
using collagen type II and collagen type I antibodies detected by
immunofluorescence. OA was induced by transection of the medial
meniscal ligament.
Results indicated that strain 6 showed a partial filling of defects up to
12 weeks post-surgery, nonetheless, it demonstrated a significantly
superior healing potential at 16 weeks when the defects were
completely filled with new cartilage tissue. The most striking changes
in the cartilage regeneration in this strain were observed between
12 and 16 weeks after surgery. Further, our preliminary results
revealed that males in this strain show better healing than females
at 16 weeks. In contrast, strain 33 showed poor or no healing at each
time point and the defects remained unfilled even at 16 weeks (Fig.
1). Strain 35, N48 and SM/J did not show healing potential as well.
As expected, DBA/1 and MRL/MpJ control mice showed a complete
healing ability at 12 weeks with DBA/1 healing slightly faster than
the MRL/MpJ. The outcome measures from histological grading
based on five parameters mentioned above are summarized in Fig.
2. Immunostaining of collagen type II revealed that articular cartilage
defects in strains 6, DBA/1 and MRL/MpJ were altogether filled with
a hyaline-like cartilage. It was, however, interesting to note that the
cartilage defect site in the non-healer mice showed no collagen type
I staining. The composition of the repair cartilage is currently under
investigation.
OA was induced by transection of the medial meniscal liagament in
Strains 33 and 6. Strain 6 was protected from OA while strain 33 had
severe OA.
Discussion. Our murine model of cartilage regeneration indicates that
the outcome of full thickness cartilage lesions is strain, sex and age
dependent in RI line. Amongst all RI strains, strain 6 males exhibited
superior healing potential at 16 weeks. Based on our preliminary
finding we have classified the mouse strains in to two categories,
136 Extended Abstracts
super healers (high scorers) and poor healers (low scorers). Among
the first category include strain 6, MRL/MpJ and DBA/1 while the
second category includes strains 33, 35, N48 and SM/J.
We have also demonstrated that strain 6 is protected from OA when
compared to strain 33, which is highly susceptible. Thus we have
established in these recombinant inbred lines, that cartilage healing
is associated with protection from OA. These new lines, 1 healing and
4 non-healing, establish a new set of models that will now be used to
narrow the regions of the genome that contribute to cartilage repair
and thus to OA.
References:
1. Buckwalter JA, Mankin HJ. Articular cartilage. 2: Degeneration and
osteoarthrosis, repair, regeneration, and transplantation. J Bone
Joint Surg 1997;79A:612–32.
2. Eltawil NM, De Bari C, Achan P, Pitzalis C, Dell’accio F. A novel
in vivo murine model of cartilage regeneration. Age and strain-
dependent outcome after joint surface injury. Osteoarthritis
Cartilage. 2009;17:695-704.
3. Fitzgerald J, Rich C, Burkhardt D, Allen J, Herzka AS, Little CB.
Evidence for articular cartilage regeneration in MRL/MpJ mice.
Osteoarthritis Cartilage. 2008;16:1319-26.
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Factor in Strain BXSB. Genetic Control of Autoimmune Disease, eds
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5. Hrbek T, de Brito RA, Wang B, Pletscher LS, Cheverud JM. Genetic
characterization of a new set of recombinant inbred lines (LGXSM)
formed from the inter-cross of SM/J and LG/J inbred mouse strains.
Mamm Genome 2006;17:417–29.
11.3.1
Cartilage repair by joint distraction in experimental models of
joint degeneration
T. Ishii
Tsukuba/Japan
Introduction: Cartilage regeneration in degenerative joint needs
cells to make cartilage, a space for cell differentiation and making
matrix, physiological conditionas growth factors and control of
mechanical condition. We made an experimental model for large full
thickness cartilaginous defect and control of mechanical condition
using hinged joint external fixator.
Content: Experimental Model: Japanese white male rabbits those
were more than 16 weeks old, were used for the experimental model.
Under anesthesia, each rabbit was fixed external fixator, which was
an originally constructed Ilizarov-type half-ring with a hinge using
fine wires. After fixing, an operative approach to the joint was
performed and cruciate and collateral ligaments were divided and
the menisci resected. A 3 mm deep of full- thickness osteochondral
defect was made by excising the entire surface of the tibial plateau
using an oscillating saw. The space formed at the femorotibial joint
by the resected osteochondral defect was maintained by the fixator.
After the operation, voluntary movement was unrestricted.
Experimental groups:
(1) An effect of Joint Distraction and Motion. Control group (Group C)
was killed at 1, 3, 6 and 12 weeks after the external fixation. Weight
bearing group (Group W) was not distracted. Immobilization group
(Group I) was locked the hinge joint. Those two groups were killed
at 12 weeks.
(2) An effect of autologous culture expanded bone-marrow-derived
mesenchymal cell transplantation (ACBMT). In transplantation
group (Group T), ACBMT with atelocollagen gel was injected into
the knee using an 18-gauge needle three weeks after operation. In
vehicle group (Group V), only atelocollagen gel was injected at the
same term. Those two groups were killed at 12 weeks. (3) An effect
of concentrated autologous bone marrow aspirate transplantation
(CABMAT). CABMAT group (Group CAB), CABMAT with autologous
fibrin gel, which was prepared by 10 ml aspirated bone marrow blood
of the femur, was injectd 7 days after the first operation. In another
group, concentrated autologous peripheral blood cell with fibrin gel
(Group PBC), which was prepared by 10ml of peripheral blood, was
also injected in the same manner. Group GEL was underwent only
fibrin gel transplantation from peripheral blood as same. Group CON
was no transplantation. The animals were killed 8 or 12 weeks after
the operation. (4) An effect of long term distraction. In Group A, the
distraction and movement with the fixator were maintained for six
months, and the animals were then killed.
In Group B, the distraction and movement were maintained for six
months. After which the devices were removed and without
restriction for a further six months, rabbits were killed one year after
operation.
(5) An effect of controlled mechanical stress. To clear the effect on
mechanical stress to the regenerated cartilage in the experimental
model, we develop the control system for hinged fixator with
continuous passive motion (CPM) and gradual applied weight-
bearing function. In GWB groups (6W-GWB and 9W-GWB), GWB was
initiated 6 weeks in the 6W-GWB and 9 weeks in the 9W-GWB after
operation. GWB (0.5 kg in the first week; 1 kg, second week; and 2
kg, third week) was applied with CPM for 2 hours every day. In CPM
groups (6W-CPM and 9W-CPM), CPM with fixator was applied in the
same manner but without GWB. The CONTROL groups (6W-CON and
9W-CON) received only joint distraction without GWB or CPM.
Evaluation: The proximal tibia was decalcified, and sagittal sections
at the midportion of the medial and lateral tibial plateaus were
obtained for histological analysis. These sections were stained
with hematoxylin and eosin, safranin-O/fast green (SO), and a
monoclonal antibody for type-II collagen (Col-II). The central one-
third of the regenerated tissue was assessed and scored blindly
using a grading
scale of the International Cartilage Repair Society (ICRS) visual
histological assessment scale [3]. The area of regenerated soft tissue
and that stained by SO and Col-II were measured, and the ratio of
the area stained by SO and Col-II to the area of the regenerated soft
tissue was calculated. Results:
(1) In regenerated process of Group C, immediately after forming
the defect, the trabecular bone was exposed to the joint space.
Seven days later, the articular surface was smooth and remodelled.
Spindle-shaped mesenchymal cells were observed on the surface of
the defect. At three weeks, there was synthesis of the cartilaginous

extracellular matrix, as indicated by the staining of the matrix by SO
and Col-I and II. The matrix was synthesised between the superficial
fibrous stroma and the deeper area of a newly-formed woven bone.
At six weeks, the
regenerated cartilage tissue had reached the surface of some areas
and the cartilage matrix stained strongly with SO. The flat bone
surface of the tibial plateau was remodelled into a concavity adapted
to the shape of each femoral condyle. At 12 weeks, the cells had
a columnar distribution. At six and 12 weeks, Col-II was observed
in the matrix and Col-1 was less intense compared with Col-II. SO
stained area was largest in Group C among three groups, which was
significant
against Group I.
(2) Group T was significantly superior to Group V in ICRS scale II,
scale III and SO-stained positive area. Comparing Group T with Group
C of experimental group (1), there is no significant difference.
(3) Results were more eminent in 12 weeks than 8 weeks in general
tendency. SO-stained positive area with respect to the regenerated
soft tissue area was significantly highest in Group CAB to other
groups.
(4) Group A was significantly inferior in SO and Col-II stained positive
areas to Group C in 12 weeks. Comparing Group A with B, Group A was
also significantly inferior in SO and Col-II stained positive areas.
(5) The area of regenerated soft tissue in the Group 9w-GWB was
statistically larger than that in the 9w-CON group. The ratio of the
SO-stained positive area in the Group 9W-GWB was the largest
among all the groups and significantly larger than those in the
Group 9w-CPM and 6w-GWB. The ratio of the Col-II-stained area in
the 9w-GWB was similar to those of the SO-stained area. The ratios
of the SO and Col-II-stained area in the 6w-CON were significantly
larger than those in the 6w-CPM.
Discussion: Four essential factors for cartilage regeneration, cells to
make cartilage, a space for cell differentiation and making matrix,
physiological condition as growth factors and control of mechanical
condition, are contained in our experimental model. Cells to make
cartilage are introduced from bone marrow by spongialization. Cell
transplantation technique as ACBMT or CABMAT slightly promoted
quality of regenerated cartilage in the experimental model. In human,
cartilage regeneration is difficult than rabbit model and some cell
transplantation technique may be required. Three millimeters
thickness of osteochondral resection of total tibial plateau makes
a space for cell differentiation and making matrix. In large cartilage
defects on weight-bearing joint, the space is lost by weight bearing
without an external fixator. Without joint distraction, Group I were
poor regeneration
especially at the weight bearing area. The space, in other words, is a
culture box supplied growth factors from joint fluid in physiological
articular condition. Control of mechanical condition is the most
difficult understanding factor. Our experimental model is very
interesting in an ability of control of mechanical stress.
Straight cut tibial plateau adapts to remodels in circular orbital
shape centering at the hinged point. Too much time for distraction
reduced the quality of cartilage as in Group A. On the other hand,
excessive mechanical stress induces inflammation in the joint and
progress the joint degeneration. In our gradual weight-bearing
model, 9w-GWB was significant better quality than 6w-GWB. The
result only means 9 weeks is more adequate regenerative condition
for starting to begin weight
bearing than 6 weeks in the experimental model. On the stages of
cartilage regeneration, adequate mechanical stress will be different
in each stage, whose principle never knew.
Conclusion: Control of mechanical stress is essential for the cartilage
regeneration of large cartilage defects. To better understanding of
controlling the mechanical stress lead to clinical application of
cartilage regeneration.
Extended Abstracts 137
11.3.3
Challenge of Repairing Large Cartilage Defects with Minimally
Invasive Technique
M. Ochi
Hiroshima/Japan
Introduction: Several surgical approaches to repair cartilage
defects have been reported such as reattachment of a detached
osteochondral fragment to the lesion, microfracture, mosaicplasty
and ACI. We treated eight cartilage defects with meniscal
transplantation from 1990 to 1995. Then we started to perform
transplantation of tissue-engineered cartilage made ex vivo for
the treatment of osteochondral defects of the joints (110 cases) as
a second generation of chondrocyte transplantation from 1996(1).
Sixty knees who had received transplantation of tissue-engineered
cartilage for cartilage defects were followed up for at least 5 years.
Although the clinical results were satisfactory(2), we need the
surgical approaches to treat large cartilage defects with minimally
invasive technique.
Content: One of the less invasive surgical procedures to treat
large cartilage defects is microfarcture or drilling. However, these
techniques under arthroscopy are not sufficient to repair cartilage
defects with hyaline cartilage. I think that there are two weak points
such as insufficient number of mesenchymal stem cells and early
overloading on the treated area. 1) One recent strategy is by the
implantation of mesenchymal stem cells (MSCs)(3) . MSCs are the
cell population of undifferentiated cells isolated from adult tissue
that have the capacity to differentiate into mesodermal lineages,
such as bone, cartilage, fat, muscle or other tissues. MSCs from bone
marrow can be cultured and differentiated into the desired lineage
in vitro with the application of specific growth factors or bioactive
molecules.Intra-articular injection of too many MSCs, however, can
generate free bodies of scar tissue(4) . We therefore developed a
novel stem cell delivery system for cartilage repair using magnetically
labeled MSCs and an external magnetic device to accumulate a
relatively small number of MSCs to a desired area. Ferumoxides
are dextran-coated super paramagnetic iron oxide nanoparticles
approved by the US Food and Drug Administration as a magnetic
resonance contrast agent for hepatic imaging of humans. By use of
this ferumoxides, it is easy to make magnetically labeled MSCs(5) .
Recently we demonstrated the ability to deliver magnetically labeled
MSCs to a cartilage defect that is a desired place under arthroscopy
in rabbit and swine knee joints using external magnetic device (0.6T)
(5) . This result indicates that this minimally invasive system under
arthroscopy can be applicable for a focal osteochondral defect in
the knee joint. The next step is to examine if this external magnetic
system is effective for osteoarthritis. We investigated if we could
successfully regenerate a cartilage layer on degenerated human
cartilage in vitro using this external magnetic system(6) . MSCs
from human bone marrow were cultured and magnetically labeled.
Degenerated human cartilage was obtained during total knee
arthroplasty. The osteochondral fragments were attached to the
sidewall of tissue culture flasks, and magnetically labeled MSCs
were injected into the flasks. Using an external magnetic device,
a magnetic force was applied for 6 hours to the direction of the
cartilage, and then the degenerated osteochondral fragment was
cultured in chondrogenic differentiation medium for 3 weeks. In the
control group, a magnetic force was not applied. The specimens were
evaluated histologically. A cell layer was formed on the degenerated
cartilage as revealed by hematoxylin and eosin staining. The cell
layer was also stained in Toluidine blue and Safranin O, and with
anti-collagen type II immunostaining, indicating that the cell layer
contained an abundant extracellular matrix. In the control group, a
cell layer was not observed on the cartilage. In conclusion, we could
demonstrate that our system could deliver MSCs onto degenerated
human cartilage, and then form an abundant extracellular matrix on
the degenerated cartilage in vitro. 2) Another technique is to reduce
the load to the repaired area in the knee joint after bone marrow
stimulation to protect immature tissue regenerated at the repaired
area against destruction caused by overloading. We made a new
distraction arthroplasty device (meira, Japan) which allows the
range of motion with knee joint distraction(7)(8) . After drilling or
microfracture under arthroscopy, the new external device was fixed
with four 6-mm pins drilled into the distal femur and the proximal
tibia. After the appropriate distractive tension was applied, the ROM
and the postdistraction and predistraction tibiofemoral joint spaces
at 30° of flexion were measured. Although this device is usually
applied for 3 months, full weight bearing is allowed one month after
surgery. Until now, this device has been demonstrated to function
well to repair cartilage defects. I would like to show my technique
using an external magnetic field to deliver precisely injected cells
with magnetic beads to an articular defect and articulated distraction
device for reducing the load for a large osteochondral defects or
osteoarthritis.
138 Extended Abstracts
References:
(1) Ochi M, Uchio Y, Kawasaki K, Wakitani S, Iwasa J: Transplantation
of cartilage-like tissue made by tissue-engineering for the treatment
of cartilage defects of the knee. Journal of Bone Joint Surgery[Br].
84(4):571-578, 2002
(2) H. Tohyama, K. Yasuda, A. Minami, T. Majima, N. Iwasaki, T.
Muneta , I. Sekiya, K. Yagishita, S. Takahashi, K. Kurokouchi, Y.
Uchio, J. Wasa, M. Deie, N. Adachi, K. Sugawara, and M. Ochi:
Atelocollagen-associated autologous chondrocyte implantation for
the repair of chondral defects of the knee: a prospective multicenter
clinical trial in Japan. Journal of Orthopaedic Science. 14(5):579-588.
Epub Oct 3, 2009
(3) Nishimori M, Deie M, Kanaya A, Exham H, Adachi N, Ochi M:
Repair of chronic osteochondral defects in the rat: The bone
marrow stimulating Procedure with cultured allogenic bone marrow
mesenchymal stromal cells. The Journal of Bone and Joint Surgery
[Br]. 88-B(9):1236-1244, 2006
(4) Muhammad Agung, Mitsuo Ochi, Shinobu Yanada, Nobuo Adachi,
Yasunori Izuta, Takuma Yamasaki, Katsuhiro Toda: Mobilization
of bone marrow-derived mesenchymal stem cells into the injured
tissues after intraaricular injection and their contribution to tissue
regeneration. Knee Surgery Sports Traumatology Arthroscopy.
14(12):1307-1314, 2006
(5) Kobayashi T, Ochi M, Yanada S, Ishikawa M, Adachi N, Deie M,
Arihiro K: A novel cell delivery system using magnetically labeled
mesenchymal stem cells and an external magnetic device for clinical
cartilage repair. Arthroscopy. 24(1):69-76, 2008
(6) Kobayashi T, Ochi M, Yanada S, Ishikawa M, Adachi N, Deie M,
Arihiro K: Augmentation of degenerated human cartilage in vitro
using magnetically labeled mesenchymal stem cells and an external
magnetic device. Arthroscopy. 2009 Dec;25(12):1435-1441. Epub
Nov 6, 2009
(7) Ryoji Kajiwara, Osamu Ishida, Kenzo Kawasaki, Nobuo Adachi,
Yuji Yasunaga, Mitsuo Ochi: Effective repair of a fresh osteochondral
defect in the rabbit knee joint by articulated joint distraction following
subchondral drilling. Journal of Orthopaedic Research. 23:909-915,
2005
(8) Masataka Deie, M.D., Mitsuo Ochi, M.D., Nobuo Adachi, M.D.,
Ryoji Kajiwara, M.D. and Atsushi Kanaya, M.D.: A new articulated
distraction arthroplasty device for treatment of the osteoarthritic
knee joint: a preliminary report. Arthroscopy. 23(8):833-838, 2007
15.0.3
Clinical results with the use of biomaterials for cartilage repair
S. Nehrer, F. Halbwirth
Krems/Austria
Introduction: Cell based therapies in cartilage repair Autologous
Chondrocyte Transplantation with the periosteal flap has changed
the paradigm of the treatment of cartilage defects from repair to
regeneration. This has been demonstrated in randomized trials
proving the concept of regenerating tissue in a cell based therapy
approach. However, the limitations of the periosteal flap concerning
size and thickness and surgical demands with suturing and the
variability of the biological reaction including hypertrophy, as well as
the uncontrolled celltransplatation in a cell suspension has supported
the introduction of biomaterials. The first attempt was to replace the
periosteal flap by membranes which were sutured or glued to the
defect combined with the cellsuspension injected or seeded onto
the membrane during the surgery before implantation. Concerns
about the phenotype of the cultured cells led to the use biomaterial
assisted technologies in sponges or gels to maintain the chondrocytic
function of the cells and allow a more controlled dispersion of the
chondrocytes throughout the defect. The matrix characteristics
concering biochemical composition, biophysical appearance in gel,
foams or sponges, degradation dynamics and products, toxicity,
immunological reactions and general biocompatibility are important
parameters of biomaterial development. The cell-biomaterial
interaction in a biological environment is lastly the decisive process
of a successful cartilage regeneration.
Content:Biomaterials in clinical use Various materials have been
tested in in-vitro laboratory studies and in-vivo animal experiment
to evaluate the best material for human use. Most of the materials
are able to support the chondrocytic phentotype, allow proliferation
and cellmigration and facilitate production of cartilage specific
substances. Animal models show in most cases that cell loaded
constructs show superior results than unloaded matrices. Based
on this results clinical studies were employed to prove feasibility
and safety of these technologies. Most of the reported results
are based on case studies only few randomized controlled trials
were performed. There is still discussion about the approbiate
control group in the study design, since microfracture is performed
arthroscopically and basically a different approach, only very few
studies compare matrix techniques with other cell transplantation
methods, however the benchmark is still autologous chondrocyte
transplantation with the periosteum patch. Collagen membrane
The collagen membrane was the first biomaterial used in clinical
studies. Based on positive results in animal studies, confirming the
biological concept of matrix assisted cell technology a bilayer Type
1/3 collagen membrane was sutured instead of the periosteal flap to
the defect site and the cell suspension is injected underneath. This
method still requires suturing of the membrane and low control of
the seeding process of the cellsuspension. The clinical results of five
years follow-up are compareable to the periosteum technique. In
biopsy studies hyaline-like tissue have been found. The preseeding
of the membrane was the next development, allowing a more
controlled distribution of the cultured cells and also allows glueing
of the graft to the defect, in this technique the chondrocytes are
seeded in a fibrin glue suspension, however the impact of fibringlue
on chondrocytes is still controversial and experimental and studies
on the stability of such fixation show minor mechanical properties.
Hyaluronan Besides the collagen membrane the use of a hyaluronan
based scaffold has been used in the indication of a chronic cartilage
defect. The material consists of a non woven mesh of hyaluronan
fibers modified by esterification by benzylesters to improve the
biocompatibility of the material. Since the hyaluronan is a natural
constituent of cartilage and the material desolves into hyaluronan,
it is a promising candidate for chondrocyte transplantation. The
invitro studies revealed the Hyaff 11 mesh as an appropriate scaffold
for chondrocytes regarding the chondrocytic phenotype, however
the animal studies on the material are sparse and no conclusive
controlled data available. However, the clinical performance of the
material in three- and five-years follow-ups document successful
performance in over 80 % across the study population. Stageing the
data according to age and severity of the cartilage defect, patients
under the age of 35 and single circumscribed defects do a lot better
with success rates over 90 %. The precultured hyaluronan matrix
shows good attachment of the chondrocytes and also allows an
arthroscopic implantation. The preformed shaping of the defect and
the rough surface of the graft allows fixation without glueing and
pressfit technique and reduces joint morbitiy through the arthroscopic
approach. PLA/PGA membranes Simmilar arthroscopic techniques
are performed with precultrred matrices consisting of PLA/PGA
meshes, wich are fixed with technically demanding intraosseous
suture-knot technique or lately with more easy pinfixation. The
cellseeded construct shows compareable results to ACT in midterm
followup in cohort studies and the stable, mechanically resistant
graft allows also to treat circumscribed osteoarthritic lesions with
satisfying outcomes. Recent techniques using minced cartilage
pieces glued to the membrane have been developed, assuming that

outgrowth of chondrocytes from the minced tissue provide adequate
chondrogenic stimulation to facilitate cartilage regeneration. The
minced tissue is harvested with a newly developed shaver system
which allows automized seeding on the artificial membrane.
The 2 years clinical follow up shows better results compared to
microfracture. Collagen Gel The 3D-culture system in gels allows the
chondrocytes to maintain the chondrocytic phenotype, especially
when P1 cells are used. The distribution of the cells resembles more
the sparsely dispersed cells in natural cartilage and a high bioactivity
of the cell is achieved by vigourous cartilage specific protein
production. The soft collagen gel needs a well debrided defect with
stable walls to allow a pressfit stabilisation support by fibrin glue.
The graft shows a complete fill of the defect and a perfect bonding
to the adjacent cartilage. Clinical results of case controlled studies
and multicenter data show comparable results to ACT and better
performance with regards to effusion and surgery time. Fibrin Fibrin
as a biomaterial was used in liquid form in a glue-cellsuspension
or lately also in a spongelike matrix. The fibrin technique allows a
homologous approach to the cell transplantation and the support of
FGF growthfactor stimulates cellproliferation and differentiation to
shorten the necessary culture period. First clinical case series show
promising results of the material, the spongelike appearance serves
as a more stable construct than liquid glues. Other combinations
of gels and membranes are available to combine 3D environment
with a biomechanical stable fixation technique. Clinical performance
The clinical results of matrix techniques are comparable to the
midtermresults of ACT, hence longterm results are not yet available.
Esspecially circumscribed lesions on the condyle in the younger
patients are doing good in all the studies. However, most studies are
case series and only few are controlled trials including controls. The
advantage of the use of biomaterials is the shorter surgery time, the
easier handling and fixation, the secure and biological appropriate
environment for the chondrocytes during the transplantation process,
the smaller incision including arthroscopic techniques and the more
predictable biological regeneration process avoiding overgrowth
like hypertrophy of the periosteum graft. The safety reports reveal
only minor adverse events most of the time and from the ethic
standpoint no bridges are burned for further treatment options
including revisions, mosaicplasty or redos of celltransplantation. The
use of biomaterials without cultured cells is of course the logistical
and legistical easier process and serves economical interest of
companies and healthcare stake holders. All of the matrices were
developed for the cellbased therapies and are now serving as
scaffolds for the bloodclot techniques like microfracture. Some
early midtermresults show increased healing response but –like
microfracture alone- inadequate tissue formation and thinning of the
surface layer by intracartilagenous bone formation. However clinical
and experimental studies are on the way and hopefully will help to
develop the optimal biomaterial based cartilage repair procedure.
Up to now the evidence in experimental studies is low to suggest
successful use of cell-free constructs and most animal experiments
show advantages of the cell augmented techniques, however the
addition of growthhormones, chondrocyte nutrients or application
nanotechologic applications in biomimetic materials may allow such
techniques in the future. However, cellbased therapies without
biomaterials like chondrospheres or stemcells may be a relevant
alternative to biomaterials and make the difference at last.
References:
Literature
1) Bartlett W, Skinner JA, Gooding CR, Canington RW, Flanagan AM,
Briggs TW, Bentley G (2005) Autologous chondrocyte implantation
versus matrix-induced autologous chondrocyte implantation for
osteochondral defects of the knee: a prospective, randomised study.
J Bone Joint Surg Br 87:640-645
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autologous chondrocyte transplantation/implantation (MACT/
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3) Bentley G, Biant LC, Carrington RWT, Akmal M, Goldberg A,
Williams AM, Skinner JA, Pringle J (2003) A prospective, randomized
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4) Brittberg M, Lindahl A, Nilsson A, Ohlsson C, Isaksson O, Peterson
L (1994) Treatment of deep cartilage defects in the knee with
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Nehrer S (2005) Repair of articular cartilage defects treated by
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26:3617-3629
6) Erggelet C, Sittinger M, Lahm A (2003) The arthroscopic
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Extended Abstracts 139
thickness cartilage defects of the knee joint. Arthroscopy 19:108-110
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Ludvigsen TC, Roberts S, Solheim E, Strand T, Johansen O (2007)
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Arthroscopic autologous chondrocyte transplantation: technical
note. Knee Surg Spons Traumatol Arthrosc 10:154-159
15) Marlovits S, Singer P, Zeller P, Mandl I, Haller J, Trattnig S (2006)
Magnetic resonance observation of cartilage repair tissue (MOCART)
for the evaluation of autologous chondrocyte transplantation:
determination of interobserver variability and correlation to clinical
outcome after 2 years. Eur J Radiol 51:16-23
16) Nehrer S, Dorotka R, Domayer S, Stelzeneder D, Kotz R. Treatment
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140 Extended Abstracts
16.1.1
Biologic Unicompartmental Knee Replacement
J. Farr
Indianapolis/United States of America
Introduction: There have been incremental advancements in biologic
unicompartmental knee replacement since the 2007 extended ICRS
abstract on the topic.1 The term applies to resurfacing either one
tibiofemoral compartment or the patellofemoral compartment. The
techniques are extensions of standard cartilage restoration options
applied to both sides of the compartment. That is, bipolar lesion
treatments—often with large area of exposed bone that would
classically be treated with metal and plastic unicompartmental
arthroplasty. At present, biologic unicompartmental arthroplasty
continues to offer limited success, but with growing hope for the
future.
Content: For purposes of expanding discussion for algorithm
development, the knee is typically divided into three uni-
compartments: medial and lateral tibiofemoral and patellofemoral.
The topics will group both tibiofemoral compartments together and the
patellofemoral compartment separately. Biologic unicompartmental
knee replacement requires optimization of the compartment’s
biomechanical environment in addition to resurfacing the bone with
articular cartilage. The patellofemoral (PF) pain patient is obviously
very complex. As articular cartilage is aneural the pain originates in
the bone and/or the soft tissues surrounding the compartment. An
excellent point of reference for PF is a 2006 review by Gomoll et. al.
titled “Treatment of Chondral Defects in the Patellofemoral Joint.”2
Two useful tools in evaluating PF compartment chondrosis are
Fulkerson’s3 classification and Dejour’s4 dysplasia classification.
Fulkerson’s classification may be summarized as: 1) the patella is
located excessively laterally with reference to the trochlea, 2) the
patella is excessively tilted 3) tilt and lateral patellar position 4) and
those with a congruent PF joint. Dejour’s classification A through D
detailed progressive extremes of trochlear dysplasia. In addition,
each of these subtypes may have patellar alta or infera. There are a
different set of consideration at the tibiofemoral (TF) compartment.
The three main considerations at the TF compartment are the
status of the meniscus, ligaments and coronal plane alignment
(varus/valgus). For goal both the TF and PF compartments is to
optimize the biomechanical environment for the cartilage implant.
Results: Minas5 published his PF autologous cultured chondrocyte
implantation (ACI) outcomes, which demonstrated better results than
expected by anteromedialization alone as per the Pidoriano review
of Fulkerson AMZ .6 Of these patients, 29 (64%) had a concomitant
AMZ. His series of 45 patients included 24 bipolar cases with 71%
good or excellent outcomes overall, yet without bipolar subset data
analysis.5 Henderson reported a series with two subgroups: those
with and without AMZ. Overall the groups have 54.5% and 86% good
and excellent results for the ACI alone or with AMZ, respectively, but
without subset analysis of bipolar lesions.7 In Peterson’s series,
there were overall 84% good and excellent results while in the PF
group had 65% good and excellent results without bipolar subset
reporting.8 Farr9 in 2007 reported on 4 bipolar PF ACI patients out
of 39 total PF ACI patients. 81% had good or excellent results. Again,
in light of the small number of patients the bipolar treatments were
not compared to monopolar patients. Cole’s10 subgroup of 5 bipolar
PF ACI patients out of 62 total PF ACI patient were not subanalyzed,
but the group as a whole had significant improvements over multiple
knee scales. In all of these studies the decision to perform AMZ was
empiric based on surgeon preference rather than using the tibial
tuberosity trochlear groove distance. The role of tuberosity surgery
in PF cartilage treatments remains under investigation noting that
finite element analysis by Ateshian and Cohen demonstrated that
AMZ candidates could have quite variable response to elevation and
medialization.11 Their measured decrease in PF forces with AMZ was
on the order of 20% rather than the historically quoted 50% reduction.
Those patients with PF dysplasia and/recurrent patellofemoral lateral
recurrent instability often have medial patellar facet and/or lateral
femoral condyle chondral damage. The essential lesion for patellar
lateral instability is the medial patellofemoral ligament (MPFL).
The MPFL is repaired or reconstructed to prevent further instability
episodes.12 The soft tissue envelope may be further balanced with
a titrated lateral release or lateral retinacular lengthening. There is
consensus that PF stability should be established using an anatomy
based technique without over-constraint. There are few reports of
PF bipolar osteochondral allografts. Bugbee reports a bipolar PF
osteochondral allograft (OCA) shell success of approximately 60%
in 12 bipolar patients compared with an approximately 80% success
rate for PF monopolar patients.13 Bugbee did not alter the position
of the tibial tuberosity. Teitge reported in 2006 on 14 patients, 12 of
which were bipolar.14 His success rate for the bipolar OCA resurfacing
was 58% while none of the monopolar transplants succeeded. In
the TF compartments both OCA and ACI have lower success rates
than unipolar treatments, but use the same basic techniques as
monopolar treatments. The environment for the implant is optimized
in regards to alignment, stability and meniscal function. Farr reported
a similar modest positive result with combined meniscal allograft
transplantation and ACI, but did not separately report the bipolar
outcomes.9 Minas and Peterson both have large ACI series that have
subpopulations with bipolar TF salvage ACI restorations with good
and excellent outcomes of on the order of 80-90% for monopolar
lesions and 60-70% for bipolar lesions.17 Richardson reported on 8
bipolar ACI/MAT patients with 85% good and excellent results and 3
failures.18 In a non peer-reviewed article Gersoff reported acceptable
results after ACI and MAT.16 Cole reported 16 patients treated with
MAT combined with ACI and 15 patients treated with MAT combined
with OCA but did not provide numbers of bipolar treatments.15 The
ACI/MAT patient had 80% satisfaction and the OCA patients had
71% osteochondral allograft satisfaction. The bipolar treatment
outcomes with OCA alone vary from the limited success reported
by Gross who thus abandoned the technique,19 to Bugbee who
has continued to use allograft in selected bipolar salvage patients
with success, but with low patient numbers precluding statistically
analysis20 Conclusion: Although the numbers are small for both PF
and TF bipolar ACI and PF OCA, it appears that biologic arthroplasty
does have a role in the treatment of patients who would otherwise
be suboptimal candidates for metal and plastic endoprosthetics
due to their young age. This group is comprised primarily of salvage
situations in the young. In the future if the success rate of bipolar
biologic treatments improves, the upper age range may be gradually
extended.
References:
1. Farr J. Biologic Unicomparmental Knee Replacement. Interanational
Cartilage Restoration Society; Warsaw, Poland. 2007.
2. Gomoll AH, Minas T, Farr J, Cole BJ. Treatment of chondral defects
in the patellofemoral joint. J Knee Surg. 2006;19:285-295.
3. Fulkerson JP, Shea KP. Disorders of patellofemoral alignment. The
Journal of bone and joint surgery. American volume. 1990;72:1424-
1429.
4. Dejour H, Walch G, Neyret P, Adeleine P. [Dysplasia of the femoral
trochlea]. Rev Chir Orthop Reparatrice Appar Mot. 1990;76:45-54.
5. Minas T, Bryant T. The role of autologous chondrocyte implantation
in the patellofemoral joint. Clin Orthop Relat Res. 2005;30-39.
6. Pidoriano AJ, Weinstein RN, Buuck DA, Fulkerson JP. Correlation
of patellar articular lesions with results from anteromedial tibial
tubercle transfer. Am J Sports Med. 1997;25:533-537.
7. Henderson IJP, Lavigne P. Periosteal autologous chondrocyte
implantation for patellar chondral defect in patients with normal and
abnormal patellar tracking. Knee. 2006;13:274-279.
8. Peterson L, Brittberg M, Kiviranta I, Akerlund EL, Lindahl A.
Autologous chondrocyte transplantation. Biomechanics and long-
term durability. Am J Sports Med. 2002;30:2-12.
9. Farr J, Rawal A, Marberry KM. Concomitant meniscal allograft
transplantation and autologous chondrocyte implantation: minimum
2-year follow-up. Am J Sports Med. 2007;35:1459-1466.
10. Pascual-Garrido C, Slabaugh MA, L’heureux DR, Friel NA, Cole
BJ. Recommendations and treatment outcomes for patellofemoral
articular cartilage defects with autologous chondrocyte implantation:
prospective evaluation at average 4-year follow-up. Am J Sports
Med. 2009;
11. Cohen ZA, Henry JH, McCarthy DM, Mow VC, Ateshian GA. Computer
simulations of patellofemoral joint surgery. Patient-specific models
for tuberosity transfer. Am J Sports Med. 2003;31:87-98.
12. Davis DK, Fithian DC. Techniques of medial retinacular repair and
reconstruction. Clin Orthop Relat Res. 2002;:38-52.
13. Jamali AA, Emmerson BC, Chung C, Convery FR, Bugbee WD.
Fresh osteochondral allografts: results in the patellofemoral joint.
Clin Orthop Relat Res. 2005;:176-185.
14. Torga Spak R, Teitge RA. Fresh osteochondral allografts for
patellofemoral arthritis: long-term followup. Clin Orthop Relat Res.
2006;444:193-200.
15. Rue JPH, Yanke AB, Busam ML, McNickle AG, Cole BJ. Prospective
evaluation of concurrent meniscus transplantation and articular
cartilage repair: minimum 2-year follow-up. Am J Sports Med.
2008;36:1770-1778.

16. Gersoff W. Combined meniscal allograft transplantation and
autologous chondrocyte implantation. Operative Techniques in
Sports Medicine. 2002;10:165-167.
17. Peterson L, Minas T, Brittberg M, Nilsson A, Sjögren-Jansson E,
Lindahl A. Two- to 9-year outcome after autologous chondrocyte
transplantation of the knee. Clin Orthop Relat Res. 2000;212-234.
18. Bhosale AM, Kuiper JH, Johnson WEB, Harrison PE, Richardson
JB. Midterm to long-term longitudinal outcome of autologous
chondrocyte implantation in the knee joint: a multilevel analysis. Am
J Sports Med. 2009;37 Suppl 1:131S-138S.
19. Mahomed MN, Beaver RJ, Gross AE. The long-term success of
fresh, small fragment osteochondral allografts used for intraarticular
post-traumatic defects in the knee joint. Orthopedics. 1992;15:1191-
1199.
20. Emmerson BC, Görtz S, Jamali AA, Chung C, Amiel D, Bugbee WD.
Fresh osteochondral allografting in the treatment of osteochondritis
dissecans of the femoral condyle. Am J Sports Med. 2007;35:907-
914.
Extended Abstracts 141
16.1.2
Treatment of early osteoarthritis with cartilage regeneration
techniques
A.W. Gobbi
Milano/Italy
Introduction: Articular cartilage is a thin layer of specialized
connective tissue lining the articulations of diarthrodial joints,
characterized by unique properties, which enable an almost
frictionless joint movement and protect the underlying bone from
excessive load and trauma, by dissipating the forces produced
during movement. However, articular cartilage has a limited intrinsic
healing potential due the presence of few specialized cells with a
low mitotic activity; furthermore, cartilage is avascular and there
is a lack of source of undifferentiated cells that can promote tissue
repair. Once injury occurs, cartilage gradually degenerates leading
to early Osteoarthritis (OA), which is characterized by full-thickness,
focal loss of the cartilage compaction, sclerosis of the adjacent
subchondral bone, formation of bone cysts, osteophytes and joint
space narrowing thus surgical intervention is necessary in order to
repair chondral defects and protect the joint from OA. The prevalence
of chondral defects is frequent with sporting injuries, especially
in over 40 years of age patients, leaving often-persistent pain. OA
increases steadily with age, affecting 80% of the population over 75
years of age and 12.1% of the population between 25 and 74 years of
age, being the leading cause of physical disability in people over the
age of 65; furthermore community-based studies have shown that
10% of the population over the age of 55 have troublesome knee pain
and, of those, 25% are severely disabled. The social impact of bone
and cartilage pathologies entails high costs in terms of therapeutic
treatments and loss of income: in the US OA therapies cost $5.31
billion dollars in 2007, (Intercontinental Marketing Service data)
and muscoloskeletal conditions including OA results in nearly $86.2
billion per year in direct medical expenses i.e. total joint replacement
procedures and loss of income and production. Accordingly, 2000
to 2010 have been called the “decade of bone and joint” to launch
global awareness and promote further research in prevention,
diagnosis and treatment of joint injuries. For these reasons, the
trend is now going towards preventive interventions and therapeutic
solutions that can lead to an enhancement of tissue regeneration
and the reduction of degenerative mechanisms.
Content: Imaging: The standard radiographic evaluation should
include a standing AP long-leg radiograph, including also hips
and ankles, standing AP/lateral views of both knees, skyline
patellofemoral and standing 45º bend knee views. Among the
diagnostic imaging modalities used, Magnetic Resonance Imaging
(MRI) has been developed for the earlier and more quantitative
detection of articular cartilage changes, having a sensitivity which is
> 95%. Aside from delineating the extent of the articular cartilage
lesions, subchondral bone and associated ligament or meniscal
injuries can also be assessed. The use of fast-spin-echo (with or
without fat suppression) and/or fat-suppressed (or water-selective
excitation) spoiled gradient-echo image for better resolution has
been recommended. Signal properties of articular cartilage are
dependent on: MR pulse sequence utilized, cellular composition of
collagen, proteoglycans and water, orientation of collagen in different
laminae of cartilage, and effective cartilage pulse sequencing.
MRI techniques such as the delayed Gadolinium-Enhanced MRI of
Cartilage (dGEMRIC) and T2 relaxation time mapping have been
available recently, in the evaluation of articular cartilage, providing
the ability of the glycosaminoglycan content visualization, the
measurement of collagen content and the mapping of anatomical
zones of cartilage.
Special Considerations in Decision Making: The arthritic process
may be a result of an inherent biologic failure or secondary to
trauma, remaining knee instability and bone bruising predisposing
to osteoarthritis. Moreover, existing tibio-femoral or patello-femoral
malalignment accelerates the progressive joint degeneration, as
there is asymmetrical distribution of loads. Bone defects following
a subchondral fracture, osteonecrosis or osteochondritis dissecans,
should be considered preoperatively and the depth of the defect
should be estimated by radiography or tomography. Patients with
ongoing inflammatory arthritis or multiple joint involvement might
consider to be addressed with a biologic versus a prosthetic option.
Conversely, patients suffering from tricompartmental arthritis or
osteonecrosis should undergo a total knee replacement (TKR)
rather than a biologic repair of cartilage lesions. Although prosthetic
arthroplasty currently obtains better long-term results and the age at
which it is performed decreases, it is desirable for the young patients
who would like to remain highly active, to avoid it due to associated
activity restrictions after TKR. Several options exist in the treatment of
142 Extended Abstracts
cartilage lesions in young patients with early osteoarthritis: any form
of planned treatment should be based on patient characteristics and
expectations, clinical symptoms and parameters such as lesion size,
depth and associated issues like leg axis alignment, ligament and
meniscal integrity and presence of bone deficiencies. Furthermore,
other factors related to the patient (e.g. age, genetic predisposition,
level of activity and associated pathologies) should not be ignored.
Operative treatment: The coexisting pathologies that need to be
addressed for successful reconstruction are tibio-femoral axial
alignment, patello-femoral alignment, ligamentous insufficiency,
meniscal absence and bone deformities. Knee osteotomies are
used to correct varus or valgus malalignment associated with
unicompartmental osteoarthritis and can be combined with cartilage
repair procedures. Opening wedge high tibial osteotomies are used
extensively for the treatment of varus osteoarthritis as distal femur
osteotomies are indicated mainly to correct valgus deformities.
Correction of the axial deformity should be performed prior to
cartilage defects repair, in a concomitant or staged procedure. When
patello-femoral malalignment is present with a trochlear and/or
patellar chondral lesion, a thorough evaluation must be carried out
primarily in order to restore normal patellar tracking. Ligamentous
insufficiency is a negative factor in chondral graft healing thus,
ACL reconstruction, in a staged or concomitant procedure, must
be performed to ensure graft protection and safe return in daily
life activities or sports. Meniscal absence is also an important
factor to consider and meniscal transplantation might be necessary
furthermore, cartilage degeneration is associated with higher
articular contact stress identified to occur following even partial
meniscectomy. In the literature there is little evidence to support
if concomitant meniscus transplantation following osteotomy will
delay the recurrence of arthritic symptoms however, restoration of
the meniscus it is supposed to be valuable.
Cartilage Repair Techniques: Traditional palliative techniques or
newer reparative treatment options have been utilized to improve
cartilage lesions healing and they have demonstrated variable
results. Lavage and chondroplasty can provide symptomatic pain
relief with no actual hyaline tissue formation. However, these
techniques remove superficial cartilage layers, which include
collagen fibers that are responsible for the tensile strength, creating
a less functional cartilage tissue. Bone marrow stimulation
techniques, such as subchondral plate drilling or microfracture have
been reported to stimulate production of hyaline-like tissue with
variable properties and durability compared to normal cartilage,
decreasing in some cases pain and disability however recent studies
demonstrated that these techniques produce fibrocartilaginous
tissue, which degenerates with time. Osteochondral autologous
transplantation and mosaicplasty can restore normal cartilage
tissue, but they can be applied only to small defects and there are
some concerns regarding donor site morbidity. Autologous
chondrocyte implantation (ACI), which was first introduced by
Peterson, has been proven to be capable of restoring normal hyaline-
like cartilage tissue, which is mechanically and functionally stable
even in athletes at long-term follow up. Defect size ranging from 2-12
cm2 has been shown to be favourable to regeneration.
Osteochondritis Dissecans is not a contraindication for cartilage
transplantation as long as the bone loss does not exceed 8 mm.
However, this two-step procedure and showed local morbidity for
periosteal harvest and uncertain distribution of chondrocytes
solution. Additionally,the possible complication of periosteal patch
hypertrophy prompted the scientific community to develop new
techniques including second generation ACI. The use of a three-
dimensional scaffold for autologous chondrocyte culture was
developed with the aim to improve both the biological performance
of chondrogenic autologous cells as well as renders the surgical
technique easier and surgeons have been enabled to perform this
procedure arthroscopically however this is still a two-step procedure
with arthroscopic evaluation and biopsy followed by implantation,
either using an arthroscopic technique or mini-arthrotomy. Since
2001, we have participated in an ongoing observational multicenter
investigation to evaluate the long-term clinical outcomes of the
treatment with Hyaluronic Acid (HA) scaffold (HYAFF 11® Fidia
Advanced Biopolymers, Abano Terme, Italy). 141 patients with follow
up assessments ranging from 2 to 5 years (average follow up time:
38 months) were evaluated. At follow up 91.5% of patients improved
according to the International Knee Documentation Committee
(IKDC) subjective evaluation 76% and 88% of the patients had no
pain and mobility problems respectively assessed by the Euro Qol-
EQ5D measure. Furthermore, 95.7% of the patients had their treated
knee normal or nearly normal as assessed by the surgeon cartilage
repair was graded arthroscopically as normal or nearly normal in
96.4% of the scored knees the majority of the second-look biopsies
of the grafted site, histologically, were assessed as hyaline-like. A
very limited complication rate was recorded in this study. We also
evaluated 32 patellofemoral full-thickness chondral treated with
Hyalograft C the IKDC and EuroQoL EQ-5D scores demonstrated a
statistically significant improvement (P <. 0001). Objective
preoperative data improved from 6/32 (18.8%) with (IKDC) A or B to
29/32 (90.7%) at 24 months after transplantation. Mean subjective
scores improved from 43.2 points preoperatively to 73.6 points 24
months after implantation. MRI studies at 24 months revealed 71%
to have an almost normal cartilage with positive correlation with
clinical outcomes. Second-look arthroscopies in 6 cases revealed
the repaired surface to be nearly normal with biopsy samples
characterized as hyaline-like in appearance. Characterized
Chondrocyte Implantation (CCI) is a second-generation ACI procedure
that uses ChondroCelect® (Ti-Genix NV, Haasrode, Belgium), which
was developed to limit the chondrocytes&rsquo &ldquode-
differentiation&rdquo and is an expanded population of chondrocytes
that expresses a marker profile predictive of the capacity to form
stable hyaline like cartilage in vivo in a consistent and reproducible
manner. In a prospective, randomized controlled trial, that compared
CCI versus microfracture as treatment for single symptomatic
cartilage defects of the femoral condyle, CCI produced a superior
type of tissue regenerate. It also showed clinical outcome similar in
both treatments. These results suggest CCI may lead to an improved
long-term clinical outcome. Although ACI has the potential to repair
the damaged cartilage and remains one of the most promising
technologies, the expansion of the cell population to obtain a
sufficient cell number appears critical as there are some limitations:
(a) if the damaged surface is particularly wide, the availability of
healthy cartilage to harvest appears limited (b) in vitro expanded
human articular chondrocytes reduce their chondrogenic potential
after 5-6 cell doubling. Therefore, researches are currently underway
to develop alternatives to this technique. Recent directions in
cartilage repair are moving towards the possibility to perform one-
step surgery several groups are analysing the possibility to use
mesenchymal stem cells (MSC) with chondrogenic potential and
growth factors (GF) avoiding the first surgery for cartilage biopsy and
subsequent chondrocyte cell cultivation with a significant reduction
of the cost of the total procedure. Some surgical techniques have
been tested in animals for MSCs implantation including the simple
injection of bone marrow aspirate concentrate cells (BMAC) into the
lesion, which improved full-thickness cartilage repair compared to
microfracture in an equine model of extensive cartilage loss. MSC
have a self-renewal capacity and multi-lineage differentiation
potential and they can be characterized by their cultivation behaviour
and their differentiation potential into adipogenic, osteogenic and
chondrogenic cells, as well as form bone, cartilage and fat therefore,
once MSC are cultured in the appropriate microenvironment, they
can differentiate to chondrocytes and form cartilage. Wakitani et al
have used autologous culture expanded BMSC transplantation for
repair of cartilage defects in osteoarthritic knees and they concluded
that MSC was capable of regenerating a repair tissue for large
chondral defects. Giannini et al. presented their one-step surgery
procedure using MSC and scaffold. We use BMAC and GF combined
with a biologic scaffold (ChondroGide®-Geistlich Wolhusen, CH) for
full thickness cartilage defects repair as the porous structure of the
scaffold facilitates adhesion, proliferation and differentiation of
MSC. We prospectively followed up for 2 yrs 14 patients (15 knees)
operated at our Institution for grade IV cartilage lesions of the knee
all these patients have been transplanted using BMAC covered with
a collagen membrane. Bone marrow was harvested from ipsilateral
iliac crest and subjected to concentration and activation with
Batroxobin solution (Plateltex®act-Plateltex SRO Bratislava, SK).
The patients followed the same specific rehabilitation program for a
minimum of 6 months. All the patients showed improvements in
evaluation scores. Mean pre-op values were: VAS 5, IKDC subjective
41.73, KOOS Scores P=66.6/ S=68.3/ ADL=70/ SP=41.8/ QOL=37.2,
Lysholm 65 and Tegner 2.07. At final follow up mean scores were:
VAS 0.8, IKDC subjective 75.5, KOOS P=89.8/ S=83.6/ ADL=89.6/
SP=58.9/ QOL=68, Lysholm 87.9 and Tegner 4.1. No adverse
reactions or post-op complication were noted. MRI showed good
coverage of the lesions. Good histological findings were reported for
all the specimens analyzed who presented many hyaline-like
features. These data and other Italian authors (Giannini et al.) using
MSC implantation with a one-step procedure seem to be promising,
showing good clinical outcomes at early follow up. The plasma
fraction just above the red and the white cells is called plasma rich in
growth factors (PRGF): it is a plasma fraction with a high number of
platelets - about three times the number of platelets in blood.
Studies have been carried out using PRGF in articular cartilage lesion
treatment can increase total collagen II synthesis and decrease
degradation induce chondrogenesis of MSCs and promote
chondrocyte proliferation, differentiation and adhesion. Kon et al.
have studied a group of 30 patients with symptomatic degenerative
disease of the knee joints treated with three PRP intra-articular

injections weekly the follow up at 6 months showed positive effects
on the function and symptoms. We also prospectively followed up a
group of 50 patients with mean age 46.7, treated with 2 intra-
articular injections (1 monthly) with autologous PRGF (RegenLab-
PRP®). Follow up at 3 &ndash 6-12 months post-treatment showed
improvement in all the scores. Mean pre-treatment values were:
KOOS Scores: P=70.93/ S=69.13/ ADL=76.2/ SP=40.11/ QOL=
38.87, VAS 4, Tegner 3.39, IKDC 50.86 and MARX 3.24.At final follow
up mean scores were: KOOS Scores: P=84.19/ S=82.89 / ADL=91.86/
SP=56.89 /QOL=61.08, VAS 2, Tegner 5.10, IKDC 70.37 and MARX 8.
Recently authors have proven synergistic effects of PRP combined
with MSC. Nishimoto et al suggested that simultaneous concentration
of PRP and BMC could play important role in future regenerative
medicine. Preliminary data are encouraging but further studies on
clinical efficacy will clarify if simultaneous use of PRP and MSC could
represents a real solution for regenerative medicine in cartilage
repair.
Rehabilitation: The conservative postoperative rehabilitation
program follows the principles of cartilage transplantation
and healing of a concomitant osteotomy procedure should be
established by radiographic criteria before making progress to
full-weight bearing. Associated procedures as well as ligamentous
reconstruction or meniscal transplantation should also be
considered. Our rehabilitation protocol is based on functional rather
than temporal criteria the progression of the recovery was related
to the achievement of specific criteria that allow the patient to
proceed to the next rehabilitative phase. The protocol after cartilage
transplantation includes 4 stages:
Phase 1: Protection of the implant (0-6 weeks).
Phase 2: Transition and recovery of gait/ADL (6-12 weeks).
Phase 3: Maturation and functional recovery/running (12-24 weeks).
Phase 4: Turnover and sports recovery (24-52 weeks).
From a functional point of view in our protocol we identified different
phases characterized by micro-objectives, which ended with the
achievement of specific functional criteria as set out at the end
of each stage. The first weeks after surgery are important for the
healing process of the repaired chondral defect and continuous
passive motion (CPM) is prescribed in order to avoid arthrofibrosis.
We suggest a gradual increase of the knee flexion up to 90º and
progressive recovery of range of motion. Partial weight bearing
with crutches is allowed after the first few weeks and our protocol
emphasizes the recovery of strength and proprioceptive abilities. An
important part of the rehabilitation is the recovery of athletic general
conditions and recovery of walking, running and sport activity are
allowed according to specific clinical parameters and functional
objectives.
Conclusions: Articular cartilage has limited intrinsic healing
potential: therefore once injury occurs, may lead to progressive
damage, joint degeneration and early OA. Critical in prevention
of early OA is to restore any existing tibiofemoral axial deformity,
normal patellofemoral tracking and knee stability as well as to
address any bone or meniscal deficiency primarily, in a staged or
concomitant procedure. A number of viable options have been
available over the years to address problems concerning cartilage
lesions each technique has its advantages and disadvantages.
Furthermore, not all lesions can be addressed with a single technique
retropatellar, tibial plateau and posterior condylar lesions remain
a challenge. Development of better instruments and introduction
of new techniques in the near future should be able to solve this
dilemma. Biotechnology on the other hand is progressing at a rapid
pace, allowing the introduction of numerous products for clinical
application. An important issue to be addressed for the future
perspective for cartilage repair is the quality of the bonding and the
integration of the newly formed tissue to the native tissue. Among
the most studied candidates, polypeptide growth factors have
shown promise to enhance the structural quality of the repair tissue,
showing less morbidities and complications inherent to cartilage
surgical techniques however, medium-term prospective randomized
studies are suggested to confirm these premature results. Numerous
studies are currently under way to clarify some of the questions that
still remain unanswered regarding the long-term durability of these
procedures and the possible modifications that can still be made to
achieve better results. However, carefully conducted randomised
prospective studies for each of these innovations should be carried
out to validate their efficacy for cartilage regeneration and prevention
of early OA.
Extended Abstracts 143
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23. Kon E, Buda R, Filardo G et al: Platelet-rich plasma: intra-articular
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9. Epub 2009 Oct 17.
24. Kon E, Filardo G, Delcogliano M, et al: Arthroscopic second-
generation autologous chondrocyte implantation at 48 months
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22.1:44-45.
25. Kon E, Gobbi A, Filardo G et al: Arthroscopic Second-Generation
A.C.I. Compared with Microfractures for chondral lesions of the knee.
AM J Sports Med 2009; 37:33.
26. Mackay AM, Beck SC, Murphy JM et al: Chondrogenic
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Acknowledgments:
The Author acknowledges Georgios Karnatzikos, MD from 2nd
Orthopaedic Dept. of GH “G.Papageorgiou” at Thessaloniki, Greece,
fellow at OASI Bioresearch Foundation, for his contribution.

16.2.1
Growth factor-delivering scaffolds for cartilage tissue
engineering
C. Brochhausen1, R. Zehbe2, B. Watzer3, C.J. Kirkpatrick1
1
Mainz/Germany, 2Berlin/Germany, 3Marburg/Germany
Introduction: Tissue Engineering represents a highly complex
interdisciplinary branch of science which brings the life sciences and
engineering together to design innovative solutions for the treatment
of critical tissue defects after traumatic or degenerative damage. To
achieve this goal an interesting evolution has become apparent.
Although originally understood as a principally biomaterial-based
strategy using natural and synthetic materials as implants to be
integrated into the neighbouring tissue, during the last two decades
a change of paradigm has taken place which involves a movement
away from the structural replacement of damaged tissue towards
strategies to regenerate functional tissue (Kirkpatrick et al., 2006).
In this context, modern tissue engineering approaches integrate the
combined use of cells, scaffolds, and growth factors or signalling
molecules respectively. Growth factors are polypeptides which are
involved in the regulation of important cellular functions such as
proliferation, differentiation, migration and cell adhesion as well
as gene expression. Thus, the rationale for the use of signalling
molecules and growth factors is the fact that such factors are able
to stimulate the controlled proliferation and proper differentiation of
the seeded cells in the scaffolds. Furthermore, growth factors should
also enhance the migration, proliferation, and differentiation of cells
from the edges of the treated defect. In general, such factors can
serve to optimize tissue engineered cell-scaffold constructs in vitro
by providing a suitable biomimetic microenvironment. Furthermore,
growth factors could improve the performance of scaffold-cell
constructs and their integration in vivo.
Content: To introduce growth factor-releasing scaffolds into the
clinic some important requirements should be taken into account
(Biondi et al, 2007). Since the half-life of growth factors is short,
the physiological integration and release profile of such molecules
is of crucial value for effective application. In this context it is of
special interest to know at what time point the activity should reach
its maximum – during the proliferation phase at the beginning of
culture, during the differentiation stage after expansion of cells or
during the integration of the scaffold-cell construct into the patient’s
tissue. The expected time point for maximal efficacy is dependent on
the growth factor itself and its mode of action on cells. To guarantee
maximum release at this time point the release profile must be
carefully controlled. The release profile is highly dependent on the
mode of integration or binding of the growth factor to the scaffold
material. Another important feature is the fact that the activity of the
growth factor is dose-dependent, which means that for an optimal
effect the physiological dose must be liberated at the correct time
point. Furthermore, an accumulation of the growth factor within the
scaffold or the surrounding tissue has to be avoided since there may
be a risk of systemic effects. Taking these points together, it becomes
obvious that for a clinical application of scaffold-cell-growth factor
constructs the mode of action and the role of the candidate molecule
should be well understood. To save time and reduce costs it would of
great interest to use molecules which are well known and which are
already available in pharmaceutical quality. Finally, during scaffold
synthesis and integration a good incorporation efficacy is desirable
to prevent loss of growth factor molecules. For the targeting of such
potential signalling molecules and growth factors it is important
to realize that regeneration in part recapitulates developmental
processes. Therefore, it can be postulated that for the improvement
of regeneration in the context of tissue engineering it is essential
to understand the regulation of developmental processes (Reddi,
2003). With this purpose in mind our group demonstrated that the
growth plate of the long bones represents a suitable developmental
model to target interesting growth factors and signalling
molecules for tissue engineering application of cartilage and bone
(Brochhausen et al., 2009a). In previous work we and others could
demonstrate the role of prostagladin E2 (PGE2) in the metabolism
of chondrocytes (O’Keefe et al., 1998, Brochhausen et al. 2006).
Until now, prostaglandin E2 has not played any significant role as
a potential signalling molecule in tissue engineering applications.
Although it has been reported as being beneficial in numerous tissue
environments ranging from endothelial tissue to cartilage with a
pronounced dose-dependent cell-regulatory mechanism, PGE2 has
not yet achieved a major impact in tissue engineering. Therefore,
one goal of our research was to develop different PGE2-based tissue
engineering solutions. The first approach utilizes an emulsion-
based route to synthesize polymeric (PLGA) microspheres with
incorporated PGE2 (Brochausen et al., 2009b, Watzer et al., 2009).
In this first approach we were able to demonstrate release kinetics
Extended Abstracts 145
of biologically active PGE2 with a burst release of PGE2 over the first
two days and further release over 8 days. These microspheres were
used in a second approach to establish a three-dimensional scaffold
system by distributing PGE2-PLGA-microspheres in a gelatinous
suspension followed by freeze-drying and moderate chemical
crosslinking in a formaldehyde-saturated atmosphere as briefly
described elsewhere (Brochhausen et al. 2009c). The distribution
of growth factor-loaded microspheres allows a gradient of growth
factors due to the spatial distribution of the microspheres. Gradients
of growth factors are of special importance for biphasic scaffolds,
such as scaffolds for osteochondral defects (Wang et al., 2009). In a
third tissue engineering system we developed a PLGA scaffold with
direct integration of the biosignal. In this approach PLGA and PGE2
were prepared in an organic solvent, followed by supercritical fluid
foaming. The integration rate of this scaffold was quite high, which
predicts high release rates. Moreover, the incorporation efficacy
of the fluid-foamed system was higher than different preparations
of microspheres. This third approach demonstrated that that the
direct incorporation of PGE2 into a polymer foam is possible without
extensive loss of the growth factor. Currently, we are testing these
tissue engineering systems extensively in vitro with first results
already being promising with respect to the usefulness of PGE2 in
cell stimulation assays. The development of growth factor-releasing
scaffolds is highly interactive, as it combines aspects from the
biomaterial and life sciences in interaction with important fields of
clinical pharmacology. For clinical use of such systems extensive
in vitro studies and the knowledge of the in situ situation in the
human body are crucial. Therefore, human in situ models such as
the growth plate are of special interest for the targeting of potential
new candidates. In our projects we have used a well understood
molecule –PGE2- which is available in pharmaceutical quality and
offers significant advantage for a possible application in a clinically
relevant scaffold-cell-release system. Future developments should
focus on questions such as to what extent scaffolds are necessary
for the regeneration of functionalized tissue in tissue engineering
and regenerative medicine. In this context, it seems obvious that
different tissues and defect sizes require different tissue engineering
solutions (Chung & Park, 2007). Of particular importance are
investigations which clarify how and under what conditions the
amount of foreign material in scaffolds could be diminished by the
use of controlled release systems which can trigger the endogenous
regenerative potential of the patient’s tissues.
References:
Kirkpatrick, CJ, Fuchs, S, Peters, K, Brochhausen, C, Hermanns, MI,
Unger, RE Visions for regenerative medicine: interface between
scientific fact and science fiction. Artificial Organs. 2006; 30: 822-
827. Biondi M, Ungaro F, Quagila F, Netti PA. Controlled Drug delivery
in Tissue Engineering. Advanced Drug Delivery Reviews. 2007;
60: 229-242. Reddi, AH. Morphogenesis and tissue engineering of
bone and cartilage: inductive signals, stem cells, and biomimetic
biomaterials. Tissue Engineering. 2003; 6, 351-359 Brochhausen,
C, Lehmann, M, Halstenberg, S, Meurer, A, Klaus, G, Kirkpatrick,
CJ. Signalling molecules and growth factors for tissue engineering
of cartilage – What can we learn from the growth plate? Journal of
Tissue Engineering and Regenerative Medicine. 2009a; 3: 416-429.
O‘Keefe RJ, Crabb ID, Puzas JE, Rosier RN: Influence of prostaglandins
on DNA and matrix synthesis in growth plate chondrocytes. Journal
of Bone and Mineral Research. 1992; 7: 397-404. Lowe GN, Fu YH,
McDougall S, Polendo R, Williams A, Benya PD, Hahn TJ: Effects of
prostaglandins on deoxyribonucleic acid and aggrecan synthesis in
the RCJ 3.1C5.18 chondrocyte cell line: role of second messengers.
Endocrinology. 1996; 137: 2208-2216. Schwartz Z, Gilley RM, Sylvia VL,
Dean DD, Boyan BD: The effect of prostaglandin E2 on costochondral
chondrocyte differentiation is mediated by cyclic adenosine
3‘,5‘-monophosphate and protein kinase C. Endocrinology. 1998;
139: 1825-1834. Brochhausen, C, Neuland, P, Kirkpatrick, CJ, Nüsing,
RM, Klaus, G. Cyclooxygenases and prostaglandin E2 receptors in
growth plate chondrocytes in vitro and in situ – PGE2 dependent
proliferation of growth plate chondrocytes. Arthritis Research
and Therapy. 2006; 8: R78. Brochhausen C, Zehbe R, Watzer B,
Halstenberg S, Gabler F, Schubert H, Kirkpatrick CJ, Immobilization
and controlled release of prostaglandin E2 from poly-L-lactide-co-
glycolide microspheres. Journal of Biomedical Materials Research
Part A. 2009b; 91: 454 – 462. Watzer B, Zehbe R, Halstenberg S,
Kirkpatrick CJ, Brochhausen C. Stability of prostaglandin E2 (PGE2)
embedded in poly-D,L-lactide-co-glycolide microspheres - a pre-
conditioning approach for tissue engineering applications. Journal
of Material Science: Materials in Medicine. 2009; 20: 1357-1365
Brochhausen C, Lehmann M, Zehbe R, Watzer B, Grad S, Meurer
A, Kirkpatrick CJ. Targeting und innovative Release Systeme für
146 Extended Abstracts
Wachstumsfaktoren und Signalmoleküle zur Anwendung im Tissue
Engineering, Der Orthopäde. 2009c; 38: 1053-1061. Wang, X, Wenk,
E, Zhang X, Meinel L, Vunjak-Novakovic G, Kaplan DL. Growth factor
gradients via Microsphere Delivery in biopolymer scaffolds for
osteochondral tissue engineering. Journal of Controlled Release.
2009; 134: 81-90. Chung HJ, Park TG. Surface engineered and drug
releasing pre-fabricated scaffolds for tissue engineering. Advanced
Drug Delivery Reviews. 2007; 60: 249-262
Acknowledgments:
This research was supported by the EU Network of Excellence,
EXPERTISSUES, NovoNordisk and the German Research Foundation
(DFG; Se 263/17-1; SCHu 679/27-1, SCHU 679/27-2).
16.2.3
Towards cartilage grafts with zonal organization
J. Malda, W. Schuurman, P.R. van Weeren, W.J.A. Dhert
Utrecht/Netherlands
Introduction: It is well established that articular cartilage has
limited regenerative capacity and successful treatment of cartilage
defects is still a challenge worldwide. The solution to this might be
provided by regenerative medicine, a newly emerging area that aims
to restore tissue function by combining principles of engineering
and life sciences to develop biological substitutes. Functional
reconstruction of articular cartilage defects has been demonstrated
to be feasible using the patients’ own cells[1, 2]. Although such cell
based techniques show superior histology[2] with more hyaline
cartilage formation compared to other standard techniques, such
as micro-fracturing, none of the current treatment modalities has
demonstrated formation of a histologically optimal tissue repair
that mimics the characteristic complex zonal architecture of hyaline
articular cartilage and hence can be supposed to produce the desired
optimal clinical long-term results[3].
Content: Articular cartilage is a hydrogel-like, matrix-rich tissue that
contains only 5 - 10 % of chondrocytes, which maintain the structural
and functional integrity of the matrix. The tissue is organised into
characteristic depth zones, each with distinct physicochemical and
biological properties and functions, which work together to impart
low-friction, wear-resistant behaviour to diarthrodial joints. This
“zonal” structure is typically divided into three zones; superficial
(surface to 10-20% of thickness), middle (20%-70%), and deep
(70%-100%). Cells in each of the different zones are organized
distinctly and express zone-specific markers. In the superficial zone,
the collagen network is aligned parallel to the surface, providing high
tensile strength, whereas the glycosaminoglycan (GAG) content is
low, resulting in compliant compressive properties. In this zone, the
chondrocytes secrete proteoglycan 4 (PRG4 or lubricin), a molecule
important for boundary lubrication and low-friction properties[4, 5].
In the middle zone, the collagen network is randomly oriented and
the mechanical properties are intermediate to the adjacent zones. In
the deep zone (deepest 30-50% of thickness), the collagen network is
oriented perpendicularly to the calcified cartilage and bone, providing
strong integration between dissimilar tissues, and the GAG content
is high, resulting in stiff compressive properties (order of magnitude
higher than the superficial zone [6, 7]). This layered design of articular
cartilage is essential to provide the tissue with the (biomechanical)
characteristics that are required for proper and life-long sustainable
joint function. Providing constructs with zonal organization may
improve long-term outcomes of attempts at cartilage repair via tissue
engineering techniques, since chondrocyte-matrix interactions are
essential for the maintenance of zonal chondrocyte phenotype[8,
9]. Moreover, it has been demonstrated that isolated and expanded
chondrocytes from the different depth zones of the cartilage regain
their zonal differences when they are redifferentiated in alginate
beads, as evidenced by zone-specific reappearance of COMP and
clusterin, as well as significantly higher GAG production by cells from
the deep compared to the superficial zone[10]. To further mimic this
zonal organization, an increasing number of investigations is currently
directed towards the development of zonal tissue-engineered
cartilage implants[11]. To achieve this, we are employing organ
printing technology or bioprinting, which combines the deposition
of specific cell populations with the simultaneous deposition of
biomaterials [12]. This allows the development of zonal cartilaginous
grafts and by using hydrogels a more physiological environment can
be created. Biomechanical strength can be imparted and modified
by mixing cell suspensions into in situ cross-linkable hydrogels
(e.g., gelatin, agarose, alginate or PEG) in a cartridge, after which
the suspension is printed following a programmed 3D pattern[13-15].
Adding biologically active components, such as proteins, peptides,
DNA, hormones and natural or synthetic polymers[16] to these water-
based “bio-inks” will further enhance and direct the behaviour of the
cells. We characterized the use of bioprinting technologies to design
and build heterogeneous cell-laden 3D structures and evaluated
these in vitro and in vivo.
References:
1.Vasiliadis, H., et al., Assessment of clinical outcomes 10-20 years
after autologous chondrocyte implantation. Osteoarthritis Cartilage,
2010. 2.Saris, D.B., et al., Characterized chondrocyte implantation
results in better structural repair when treating symptomatic
cartilage defects of the knee in a randomized controlled trial versus
microfracture. Am J Sports Med, 2008. 36(2): p. 235-46. 3.Haleem,
A.M. and C.R. Chu, Advances in Tissue Engineering Techniques for
Articular Cartilage Repair. Operative Techniques in Otrhopeadics,
2010. 20: p. 76-89. 4.Schumacher, B.L., et al., A novel proteoglycan
synthesized and secreted by chondrocytes of the superficial zone
of articular cartilage. Arch Biochem Biophys, 1994. 311: p. 144-52.
5.Marcelino, J., et al., CACP, encoding a secreted proteoglycan,
is mutated in camptodactyly-arthropathy-coxa vara-pericarditis
syndrome. Nat Genet, 1999. 23(3): p. 319-22. 6.Schinagl, R.M., et al.,
Depth-dependent confined compression modulus of full-thickness
bovine articular cartilage. J Orthop Res, 1997. 15(4): p. 499-506.
7.Chen, A.C., et al., Depth- and strain-dependent mechanical and
electromechanical properties of full-thickness bovine articular
cartilage in confined compression. J Biomech, 2001. 34(1): p. 1-12.
8.Ng, K.W., G.A. Ateshian, and C.T. Hung, Zonal chondrocytes seeded
in a layered agarose hydrogel create engineered cartilage with
depth-dependent cellular and mechanical inhomogeneity. Tissue
Eng Part A, 2009. 15(9): p. 2315-24. 9.Khanarian, N.T., et al., Zonal
Chondrocyte Interactions Regulate Chondrocyte Calcification via
PTHrP, in 56th Annual Meting of the Orthopaedic Research Society.
2010: New Orleans, LO. p. P3. 10.Schuurman, W., et al., Zonal
chondrocyte subpopulations acquire zone-specific characteristics
during in vitro redifferentiation. American Journal of Sports Medicine,
2009. 37: p. S97-S104. 11.Klein, T.J., et al., Tissue Engineering of
Articular Cartilage with Biomimetic Zones. Tissue Engineering, 2009
in press. 12.Mironov, V., N. Reis, and B. Derby, Review: bioprinting:
a beginning. Tissue Eng, 2006. 12(4): p. 631-4. 13.Cohen, D.L., et
al., Direct freeform fabrication of seeded hydrogels in arbitrary
geometries. Tissue Eng, 2006. 12(5): p. 1325-35. 14.Fedorovich,
N.E., et al., Hydrogels as extracellular matrices for skeletal tissue
engineering: state-of-the-art and novel application in organ printing.
Tissue Eng, 2007. 13(8): p. 1905-25. 15.Fedorovich, N.E., et al.,
Three-dimensional fiber deposition of cell-laden, viable, patterned
constructs for bone tissue printing. Tissue Eng Part A, 2008. 14(1):
p. 127-33. 16.Campbell, P.G. and L.E. Weiss, Tissue engineering with
the aid of inkjet printers. Expert Opin Biol Ther, 2007. 7(8): p. 1123-7.
Acknowledgments:
Funding from the AO foundation, the Dutch Technology Foundation
STW, Applied Science Division of NWO and the Technology Program
of the Ministry of Economic Affairs is gratefully acknowledged.

19.0.2
Does cartilage have to be perfect for a good clinical outcome?
E. Kon, G. Filardo, A. Di Martino, S. Patella, L. D’Orazio, B. Di Matteo,
M. Marcacci
Bologna/Italy
Introduction: The ultrastructure of articular cartilage is unique:
chondrocytes are sparsely distributed within the surrounding
matrix, maintaining minimal cell-to-cell contact. The interaction
between the cells, the collagen framework, aggrecan and fluid
constitute a complex biomechanical feature of hyaline cartilage,
making it difficult to repair. Moreover, its isolation from systemic
regulation, and the lack of vessels and nerve supply contribute to
the absence of a spontaneous healing process. The restoration of
damaged cartilage is a difficult challenge for orthopaedic surgeons
because of its limited spontaneous repair capacity and the difficulty
in restoring the damaged areas with hyaline cartilage. Several
techniques have been used in the operative treatment of traumatic
and degenerative cartilage lesions: marrow stimulation techniques,
osteochondral, periosteal or perichondral graft transplantation and
the new procedures based on autologous chondrocyte implantation
or the use of scaffolds to induce an in situ tissue regeneration.
Content: Marrow stimulation procedures, like drilling, abrasion and
microfracturing have been proposed as an easy, rapid and
inexpensive way to restore the articular damaged surface. The
rational of these techniques is to stimulate the formation of
fibrocartilage by facilitating access to the vascular system of the
underlying bone, thus creating a clot populated with growth factors,
platelets and bone marrow-derived progenitor cells capable of
chondrogenesis. Steadman [1] reported highly satisfactory results at
11-year follow-up with the microfracture technique, but patients had
to adjust their activity level to that of their knee function. However,
the repair tissue response can be variable and unpredictable [2].
Moreover, most animal [3], MRI and histological studies [3,4] have
shown the initial fibrocartilage formation with deterioration of the
new –formed tissue over time. Nehrer [4] frequently observed soft,
spongiform, fibrous tissue combined with central degeneration in
the defect. The clinical failure was observed at an average follow-up
of 21 months. Some authors have reported a significant decrease in
clinical outcome at longer follow-up relative to short-term high
satisfaction [5]. Osteochondral autograft transplantation (with an
underlying bone) aims to replace the damaged area with hyaline
cartilage and to capitalize on bone-to-bone healing, since the mature
cartlagineous tissue has limited healing potential and heals
completely with difficulty to surrounding cartilage [6]. However, this
treatment has limited indications [7], due to the fact that a high
number of grafts can promote the formation of a large amount of
fibrous tissue filling the space between the plugs and also increase
the incidence of technical problems, compromising the mechanical
properties of the reconstructed zone. It is also difficult to obtain the
physiological curvature of an articular surface, this could produce an
abnormal stress distribution and subsequent suffering of the
implant. Furthermore, excessive reaming can jeopardize the stability
of the plugs in the subchondral bone, weakened by the fibrous tissue
among them, and the fibrocartilage formed among plugs may reduce
the biomechanical properties of the surface, thus contributing to the
worst results observed in bigger lesions [7]. The limited indications
and the worst results obtained with these reparative procedures
have been mainly attributed to the formation of fibrocartilage, which
presents low biomechanical properties and might not guaranty high
and long-lasting clinical results. The more recent regenerative
technologies aim to offer better results through the formation of
hyaline-like cartilage. Autologous Chondrocyte Implantation (ACI)
proposed by Brittberg in 1994 [8] involves the re-implantation of
autologous cells isolated from cartilage harvested from the patient
and expanded in vitro. Peterson [9] has shown that the early results
obtained with the ACI technique are long-lasting. At 5 to 11-year
follow-up, 51 of 61 patients had satisfactory results. The
biomechanical evaluation of the grafted area by means of an
indentation probe demonstrated greater than 90% normal cartilage
with respect to stiffness measurements. ACI presents some
advantages over other cartilage repair and reconstructive techniques,
including the use of autologous engineered material, reduced donor
site morbidity and no treatment limitations related to defect size.
The most important theoretical advantage of this method is that it
can restore the integrity of the damaged area with cartilage that has
hyaline-like properties [9], whereas all the marrow-stimulation
techniques provide fibrocartilagineous repair tissue that is likely to
be less mechanically stable [10]. The more recently developed
second-generation ACI technique uses a new tissue-engineering
technology to create a cartilage-like tissue in a three-dimensional
culture system, thus presenting all the advantages of the first
Extended Abstracts 147
generation method without the problems that can be observed with
standard ACI procedures, such as the difficulty in handling a delicate
liquid suspension of chondrocytes at implantation surgery, the need
to make a hermetic periosteum seal using sutures, the requirement
of a second open surgery operation, the very long rehabilitation
period, and possible complications associated with the use of a
periosteal flap and large joint exposure. Essentially, the concept is
based on the use of biodegradable polymers as temporary scaffolds
for the in vitro growth of living cells and their subsequent
transplantation into the defect site. However, despite first- and
second-generation autologous chondrocyte implantations have
emerged as promising therapeutic options, and several studies
[11,12] have confirmed the good clinical results and durability of
these treatments, the cell-based regenerative approach has not
been clearly proven to be better than other procedures. At the time
being, there is no agreement about the effective superiority of one
technique to the others, and their indication and results are still
controversial. In fact, most of the studies report clinical outcome at
short- to medium-term follow-up, and many different techniques
may appear satisfactory at the beginning, whereas the quality of the
repair tissue might influence the long-term results. Knutsen et al.
[13,14] didn’t report any differences in clinical outcome between ACI
and microfracture at 2 and 5 years follow-up. Moreover, no
statistically significant differences were detected in the macroscopic
or histological results at 2 years (ACI biopsies tended to have a more
hyaline-like appearance; p=0.08) and radiographic findings at 5
years follow-up, too. However, despite the lack of correlation
observed between histological and clinical results, he also reported
that none of the patients failed after microfracture presented high-
quality repair cartilage, thus suggesting that a worst-quality repair
tissue could increase the risk of failure or present a lower outcome
over time. In another randomized study, Saris et al. [15,16] compared
microfracture with characterized chondrocytes implantation (CCI).
Similarly, comparable clinical outcome was found between the two
procedures at short term follow-up, despite the superior
histomorphometric and histologic score observed in the CCI group.
However, the higher quality of the repair tissue seen with the
structural analysis at short follow-up significantly influenced the
later follow-up. In fact, at three years CCI offered a further
improvement with better clinical results compared with microfracture,
whose results reached a plateau after 18 months. Marrow stimulation
procedures lead to the formation of a fibrous tissue that can not
guaranty good results over time. Oppositely, ACI procedures
regenerate a cartilaginous tissue that undergoes a remodelling
process, thus leading to superior clinical results detectable only
after at least 2 – 3 years follow-up. Therefore, it appears of primary
importance to complete the clinical evaluation with the analysis of
the regenerated tissue quality, but unfortunately only a few studies,
due to practical and ethical difficulties, present a full histological
analysis, thus contributing to the controversies and the lack of
evidence about the potential of the different techniques and the
correlation between histological and clinical results. Despite the
importance of the objective evaluation offered by the histology
analysis, the invasiveness of this procedure does not allow it to be
performed routinely. An objective, non-invasive measure of the
properties of the treated area would be very desirable and helpful
for increasing the knowledge of the regenerative processes. At the
time being, the gold standard method for the imaging evaluation of
cartilage lesions is magnetic resonance. It represents a non-invasive,
multi-planar technique capable of producing high-resolution, high-
contrast images. MRI enables morphological assessment of the
cartilage surface, as well as its internal structure, thickness, volume
and the subchondral bone, other than the biochemical status of
articular cartilage. Furthermore, in contrast to the small biopsy
specimens used for histochemical assessment, which are from a
discrete location and can offer only limited information, MRI can
provide information on the whole area. Moreover, MRI images have
been shown to correlate with cartilage histology, with the biochemical
composition of cartilage in vivo and even in engineered cartilage
generated in a bioreactor [17,]. The only qualitative MRI evaluation is
not sufficient for quantifying the tissue repair characteristics, and a
grading system is required to analyse the maturation processes and
compare the results obtained with different techniques. For this
reason, classification systems for describing articular cartilage repair
tissue have been developed [17,18], one of the most important being
that of Marlovits et al. [18] who in 2004 proposed a definition of
pertinent parameters for the evaluation of chondral repair. This
evaluation system (MOCART) has been shown to be reliable,
reproducible, and with an excellent inter-observer variability [19].
Despite some preliminary results at a 12-month follow-up showing a
poor correlation between MRI findings and clinical outcome,
Marlovits and co-workers found a statistically significant correlation
between the “filling of the defect,” the “structure of repaired tissue”
and most of the subjective and objective outcome scores at two
148 Extended Abstracts
years after ACI. We previously used the MOCART score to evaluate
the results obtained with second-generation, autologous
chondrocyte implantation for the treatment of knee cartilage lesion
after over 5 yrs of follow-up [20]. The MRI findings were interesting
and somehow controversial. The overall MOCART score had a
significant correlation with subjective IKDC scores, but among the
variables, only the structure of the repaired tissue was significantly
correlated to the clinical scores. Most likely, the signal of the repair
tissue represents a most specific MRI parameter. In our previous
experience analysing the long-term outcome of mosaicplasty
technique, strong correlation between the signal of the repair tissue
and the clinical outcome was detected even if a different surgical
procedure was employed [21]. Signal of the repair tissue was
confirmed to be a useful indicator of graft maturation, especially in
comparison with the signal of adjacent normal articular cartilage. All
other parameters did not show a significant correlation with the
clinical outcome. This fact can suggest from one side that we need
more precise tools for clinical and MRI evaluation, and on the other
hand that the MRI findings can be better correlated with the clinical
outcome at longer follow-up, as shown for the histological evaluation.
Moreover, in a subsequent analysis of 78 knee MRIs [22], performed
at least 24 months after second generation autologous chondrocyte
implantation, the larger number of MRIs analyzed allowed to increase
the power to detect any potential correlation between subchondral
changes and clinical outcome, and indeed, the worst results were
found in patients affected by bone marrow oedema, thus suggesting
that the absence of correlations founded between clinical and MRI
findings in some studies can be related to the low number of patients
evaluated. Further longer follow-ups and larger high-level MRI and
histological studies will solve these discrepancies. However, despite
some controversial findings, a better quality tissue seems to be
determinant for the long-term results, as shown by histology and
imaging. In fact, in recent years there is an increasing awareness of
the importance of the quality of the tissue obtained for achieving
better and long-lasting results and several authors have even
highlighted the need to treat the all osteochondral unit. The
subchondral bone plays a role in the etiopathogenetic processes,
and has to be carefully considered in the treatment of articular
surface damage. Articular cartilage and its supporting bone
functional conditions are tightly coupled since injuries of either
adversely affects the all joint mechanical environment [23]. Certain
defects, such as those resulting from osteochondritis dissecans,
osteonecrosis and important trauma may in fact be osteochondral in
nature with involvement of subchondral bone [24,25]. Moreover, in
large cartilage lesions the subchondral bone is involved in the
degenerative process as well, and even focal chondral defects, if left
untreated, may increase in size over time and present concomitant
changes of the underlying subchondral bone plate, either overgrowth
or bone loss [26]. However, most of the bioengineered tissues used
in clinical practice present the problem of promoting only the
cartilage layer, but not the bone regeneration. The development of
biphasic scaffolds aims to regenerate the all articular surface in the
most anatomical way possible, reproducing the different biological
and functional requirements for guiding the growth of the two tissues
[27]. At the time being, despite all the pre-clinical studies reported
[27,28], only two scaffold used for osteochondral regeneration are
currently available for clinical application. Although preliminary
results appear promising, there are no controlled studies, and only
case reports have shown favorable results after implantation of
these osteochondral substitutes [29, 30]. Long term randomized
clinical, histological and MRI studies are need to confirm the potential
of this approach, verifying the regenerative capacity and if the high-
quality tissue obtained will also correspond to good long term
clinical results. Concluding, despite some controversial results at
short term follow-up, at the time being clinical, histological and MRI
evaluation suggest that the higher-quality repair cartilage obtainable
with the more ambitious regenerative approaches can offer better
results at longer follow-up with respect to the traditional reparative
procedures. Surgical goals should always try to re-establish the joint
surface in the most anatomical way possible, restoring the
physiological properties of the chondral tissue or, where necessary,
of the entire osteochondral unit, in order to achieve a more
predictable repair tissue that closely resembles native articular
surface and remains durable over time.
References:
1. Steadman JR Outcomes of microfracture for traumatic chondral
defects of the knee: average 11-year follow-up. Arthroscopy. 2003
May-Jun;19(5):477-84. 2. Shapiro F. Cell origin and differentiation
in the repair of full-thickness defects of articular cartilage. JBJS
1993; 75A:532-53. 3. Mithoefer K. The microfracture technique
for the treatment of articular cartilage lesions in the knee. A
prospective cohort study. JBJS Am. 2005 Sep;87(9):1911-20. 4.
Nehrer S. Hystologic analysis of tissue after failed cartilage repair
procedures. Clin. Orthop. Rel. R-es 365: 149-62, 1999. 5. Kreuz PC
Is microfracture of chondral defects in the knee associated with
different results in patients aged 40 years or younger? Arthroscopy.
2006 Nov;22(11):1180-6. 6. Hangody L: Autologous osteochondral
mosaicplasty for the treatment of full-thickness defects of weight-
bearing joints. J Bone Joint Surg Am 2003;85 A Suppl 2: 25-32. 7.
Marcacci M, Kon E, Delcogliano M, et al: Arthroscopic autologous
osteochondral grafting for cartilage defects of the knee: prospective
study results at a minimum 7-year follow-up. Am J Sports Med. 2007
Dec;35(12):2014-21. 8. Brittberg M: Treatment of deep cartilage
defects in the knee with autologous chondrocyte transplantation. New
Engl J Med 331:889-895, 1994. 9. Peterson L Autologous chondrocyte
transplantation. Biomechanics and long-term durability. Am J Sports
Med. 2002 Jan-Feb;30(1):2-12. 10. Buckwalter J.A Articular Cartilage:
composition, structure, response to injury and methods of repair.
In: Ewin J. W., ed. Articular cartilage and knee joint function: basic
science and arthroscopy. New York, Raven Press 19-56, 1990. 11. Kon
E, et al: Matrix-assisted autologous chondrocyte transplantation for
the repair of cartilage defects of the knee: systematic clinical data
review and study quality analysis. Am J Sports Med. 2009 Nov;37
Suppl 1:156S-66S. 12. Kon E, et al: Second generation issues in
cartilage repair. Sports Med Arthrosc. 2008 Dec;16(4):221-9. 13.
Knutsen G, et al: Autologous chondrocyte implantation compared
with microfracture in the knee. A randomized trial. JBJS Am. 2004
Mar;86-A(3):455-64. 14. Knutsen G. A randomized trial comparing
autologous chondrocyte implantation with microfracture. Findings at
five years. JBJS Am. 2007 Oct;89(10):2105-12. 15. Saris DB. Treatment
of symptomatic cartilage defects of the knee: characterized
chondrocyte implantation results in better clinical outcome at 36
months in a randomized trial compared to microfracture. Am J Sports
Med. 2009 Nov;37 Suppl 1:10S-19S. 16. Saris DB Characterized
chondrocyte implantation results in better structural repair when
treating symptomatic cartilage defects of the knee in a randomized
controlled trial versus microfracture. Am J Sports Med. 2008
Feb;36(2):235-46. 17. Roberts S, et al: Autologous chondrocyte
implantation for cartilage repair: monitoring its success by magnetic
resonance imaging and histology. Arthritis Res Ther. 2003;5(1):R60-
73. 18. Marlovits S. Definition of pertinent parameters for the
evaluation of articular cartilage repair tissue with high-resolution
magnetic resonance imaging. Eur J Radiol 2004; 52, 310-319. 19.
Marlovits S. et al. Magnetic resonance observation of cartilage repair
tissue (MOCART) for the evaluation of autologous chondrocyte
transplantation: determination of interobserver variability and
correlation to clinical outcome after 2 years. Eur J Radiol 2006; 57,
16-23. 20. Kon E.: Eur j rad accepted for publication 21. Tetta C. et
al. Knee Osteochondral Autologous Transplantation: Long-term
MR findings and clinical correlations. Eur J Radiol. 2009 Jun 11. 22.
Gomoll AH The subchondral bone in articular cartilage repair: current
problems in the surgical management. Knee Surg Sports Traumatol
Arthrosc. 2010 Apr;18(4):434-47. 23. Shirazi R, Computational
biomechanics of articular cartilage of human knee joint: effect of
osteochondral defects. J Biomech. 2009 Nov 13;42(15):2458-65.
24. Kocher MS, et al. Management of osteochondritis dissecans
of the knee: current concepts review. Am J Sports Med, 2006 Jul
34(7):1181-91. 25. Pape D, et al: Disease-specific clinical problems
associated with the subchondral bone. Knee Surg Sports Traumatol
Arthrosc. 2010 Apr;18(4):448-62. 26. Gratz KR, et al. The effects of
focal articular defects on cartilage contact mechanics. J Orthop Res.
2009 May;27(5):584-92. 27. Jiang CC Repair of porcine articular
cartilage defect with a biphasic osteochondral composite. J Orthop
Res 2007 Oct; 25(10):1277-90. 28. Kon E. et al: Novel nanostructured
scaffold for osteochondral regeneration: pilot study in horses.
J Tissue Eng Regen Med. 2010 Jan 4. 29. Kon E. et al. Novel nano-
composite multi-layered biomaterial for the treatment of multifocal
degenerative cartilage lesions. Knee Surg Sports Traumatol Arthrosc.
2009 Nov;17(11):1312-5. 30. Kon E. et al: A novel nano-composite
multi-layered biomaterial for treatment of osteochondral lesions:
Technique note and an early stability pilot clinical trial. Injury. 2009
Dec 23.
 Extended Abstracts 149

19.1.1
The patello-femoral joint: What have we been doing so far?
M. Drobnič
Ljubljana/Slovenia
Introduction: The patello-femoral (P-F) joint has become problematic
ever since the humanoids changed their way of walking to the fully
extended knees. During the early extension patella rides proximal to
the trochlear sulcus which makes it, in combination with the lateral
pulling quadriceps forces, unstable in case any of the stabilizers
(osseous-articular geometry, ligaments, retinacula, capsule, and
muscles) fails [1]. High peak loads during activities are another
contributing factor for early cartilage failure besides the intrinsic
instability and incongruence [2]. This makes P-F joint the leader in
the incidence of cartilage lesions encountered in more than 60% of
arthroscopies [3]. In spite of stated problems, the P-F joint has long
been considered as a forgotten compartment of the knee. Most of its
symptoms were uncritically declared as “chondromalacia patellae”.
We have fortunately witnessed a tremendous improvement in the
biomechanical understanding, diagnostics, and therapies of P-F
compartment over the last 2 decades with variable results. We still
lack firm evidence that currently available operative procedures
prevent further cartilage deterioration [1, 4].
References:
1. Aglietti P, Giron F, Cuomo P. Disorders of the patellofemoral joint.
In: WN Scott, ed. Disorders of the patellofemoral joint. Philadelphia,
Pa: Churchill Livingstone Elsevier, 2006; 807-936.
2. Grelsamer RP, Dejour D, Gould J. The pathophysiology of
patellofemoral arthritis. Orthop Clin North Am 2008;39:269-274.
3. Curl WW, Krome J, Gordon ES, Rushing J, Smith BP, Poehling GG.
Cartilage injuries: a review of 31,516 knee arthroscopies. Arthroscopy
1997;13:456-460.
4. Teitge RA. Patellofemoral syndrome a paradigm for current surgical
strategies. Orthop Clin North Am 2008;39:287-311, v.
5. Fithian D, Neyret P, Servien E. Patellar Instability: The Lyon
Experience. Techniques in Knee Surg 2007;6:112-123.
6. Maquet P. Advancement of the tibial tuberosity. Clin Orthop Relat
Res 1976:225-230.
7. Fulkerson JP. Anteromedialization of the tibial tuberosity for
patellofemoral malalignment. Clin Orthop Relat Res 1983:176-181.

Content: The unspecific anterior knee pain is predominantly treated
conservatively. In general, the surgical means are mostly reserved for
objective P-F instability (subluxation or luxation) and symptomatic
cartilage injuries. The cartilage may be injured acutely during a direct
anterior knee trauma (kissing lesions depending on the knee position)
or dislocation (typical lateral trochlea and medial patella lesions),
but also as chronic overload (central trochlea lesions) or maltracking
(distal lateral patellar facet). Several realignment techniques are
available for the treatment of objective P-F instability. In general,
they can be divided into proximal procedures, distal procedures and
trochleoplasties, or a combination of the above [1, 4, 5]. Although
these procedures provide an instant increased stability of the
P-F joint, they often result in an early osteoarthritis [1]. This early
cartilage deterioration may be due to the previous injuries but also
due to altered P-F biomechanics after surgery. We have reviewed the
long-term outcomes of the P-F realignment procedures performed at
our institution between the years 1963 to 1994 by various orthopedic
surgeons using different techniques. Regardless of the realignment
procedure a moderate/severe P-F osteoarthritis was confirmed in the
majority of patients. A relative newcomer in the operative assortment
of P-F instability is the reconstruction of the medial P-F ligament. The
reconstructed ligament needs to be positioned isometrically and it
is expected not to change P-F biomechanics significantly, but we are
still awaiting the long-term outcomes [5].
8. Brittberg M, Lindahl A, Nilsson A, Ohlsson C, Isaksson O, Peterson
L. Treatment of deep cartilage defects in the knee with autologous
chondrocyte transplantation. N Engl J Med 1994;331:889-895.
9. Gobbi A, Kon E, Berruto M, et al. Patellofemoral full-thickness
chondral defects treated with second-generation autologous
chondrocyte implantation: results at 5 years‘ follow-up. Am J Sports
Med 2009;37:1083-1092.
10. Gomoll AH, Minas T, Farr J, Cole BJ. Treatment of chondral defects
in the patellofemoral joint. J Knee Surg 2006;19:285-295.
11. Henderson IJ, Lavigne P. Periosteal autologous chondrocyte
implantation for patellar chondral defect in patients with normal and
abnormal patellar tracking. Knee 2006;13:274-279.
12. Minas T, Bryant T. The role of autologous chondrocyte implantation
in the patellofemoral joint. Clin Orthop Relat Res 2005:30-39.
13. Pascual-Garrido C, Slabaugh MA, L‘Heureux DR, Friel NA, Cole
BJ. Recommendations and treatment outcomes for patellofemoral
articular cartilage defects with autologous chondrocyte implantation:
prospective evaluation at average 4-year follow-up. Am J Sports Med
2009;37 Suppl 1:33S-41S.

Prior to the era of biological resurfacing the symptomatic cartilage 14. Peterson L, Minas T, Brittberg M, Nilsson A, Sjogren-Jansson E,
degeneration was treated by cartilage debridement and P-F Lindahl A. Two- to 9-year outcome after autologous chondrocyte
unloading. A widely used procedure at the time was anteriorization transplantation of the knee. Clin Orthop 2000:212-234.
of the tibial tubercle as proposed by Maquet [6]. This procedure was 15. Farr J. Autologous chondrocyte implantation and
due to a high rate of complications and due to the unpredictable anteromedialization in the treatment of patellofemoral chondrosis.
outcome upgraded to antero-medialization (AMZ) popularized by Orthop Clin North Am 2008;39:329-335.
Fulkerson [7]. AMZ offers simultaneous lateral-central unloading
and medial realignment. The early results were reported to be
clinically 86% to 97% successful; better results were demonstrated
for younger patient with subluxations and without significant
osteoarthritis [1]. The temptations for the cartilage biological
resurfacing have been increasing since 90s; however, the initial
results were inferior to the tibio-femoral compartments [8]. Of the
cartilage repair techniques the only systematic studies on P-F joint
are available for the autologous chondrocyte implantation (ACI).
There have been no systematic published reports for microfractures
alone for the P-F lesions, although they are frequently used. The
results of ACI on P-F joint have heterogeneous results with good
to excellent ranging from 62% to 90% [9-14]. The patients were
not systematically included with regard to size, location, previous
treatments, and additional procedures. The two published studies
that compared patients with ACI alone to ACI with P-F realignment
demonstrated superior outcome with the addition of AMZ [11, 13].
In spite of increasing medical evidence on P-F instability and cartilage
repair, many issues remain unsolved: timing of combined procedures;
treatment modifications with regard to lesion size, location, duration
of symptoms, and patient’s age; degree of AMZ necessary; how to
proceed with medial P-F lesions [15]. Until we get more answers the
two questions need to be cleared and therapy accordingly modified
for a successful cartilage repair: Why did the cartilage lesion occur?
Is the negative impact on cartilage still active?
150 Extended Abstracts
19.1.2
Osteochondritis Dissecans in the Knee: Therapeutic Options
S. Görtz
La Jolla/United States of America
Introduction: Osteochondritis dissecans (OCD) is an acquired
condition primarily affecting the subchondral bone of the femoral
epiphysis which often manifests as progressive disease of the
overlying articular cartilage, ultimately culminating in sequestration
of osteochondral fragments as classified in the ICRS grading scale
for OCD: OCD I - Stable, continuous: Area of softened subchondral
bone covered by intact cartilage. OCD II - Partial discontinuity, stable
on probing. OCD III - Complete discontinuity, ”dead in situ”, not
dislocated. OCD IV - Dislocated fragment, loose within the bed or
empty defect, with lesions of over 10mm in depth constituting the
IVB sub-group.
Content: While the exact incidence and etiology of OCD remains
somewhat controversial, a vast majority of lesions occur in the “classic”
position on the lateral margin of the medial femoral condyle, with a
considerable incidence of bilateral disease. Cumulative microtrauma and
altered biomechanics are often considered causative for OCD, as
evidenced by the association with repetitive impact loading and
conditions such as discoid lateral meniscus. More recently, long axis
varus and valgus malalignment has been implicated as being contributory
to OCD of the medial and lateral femoral condyle, respectively. Generally,
two distinct clinical entities of OCD are recognized, depending on the
maturity of the distal femoral physis: Juvenile OCD characterizes lesions
in skeletally immature patients while adult OCD describes lesions
occurring at or after physeal closure, although many of these are thought
to be progressive sequelae of unresolved juvenile cases. Traditionally,
surgeons have relied on radiographic imaging to localize and characterize
OCD lesions as well as to evaluate skeletal maturity as a prognostic
factor. Standard imaging for OCD should include AP, lateral, Rosenberg
(flexed knee PA) and Merchant (patellofemoral) views. MRI has become
a routine diagnostic tool in the evaluation of OCD lesions, which has
gained further prognostic value with the advent of advanced cartilage
imaging protocols. Besides skeletal maturity and increased lesions size,
a subchondral high T2 signal zone in lesions that present with loss of
surface continuity on T1-weighted images have all been shown to be
possible predictors of failure in non-operative management. Gadolinium
enhancement and bone scintigraphy can be helpful in distinguishing if
subchondral signal enhancement is owing to synovial fluid infiltration or
formation of vascular granulation tissue. The former indicates disease
progression due to loss of surface continuity and increasing instability (a
negative prognostic sign) whereas the latter implies osseous
reconstitution (a positive prognostic sign), especially in patients with
open physes. For stable juvenile OCD lesions with intact cartilage surface
(ICRS grade 1 or 2) and presenting without mechanical symptoms or
effusion, conservative treatment has proven to be a reasonable initial
step in management due to the favorable natural history. Non-operative
treatment usually includes an initial four to six week period of no or
protected weightbearing and range of motion exercises, followed by
progressive low-impact rehabilitation if supported by favorable clinical
presentation and serial imaging. Gradual return to supervised sport
specific activities can be considered after three months if the patient
remains asymptomatic. A repeat MRI can be helpful at that time to
confirm healing and reossification before initiating impact loading. In
juvenile OCD, operative management is indicated for stable lesions that
have failed conservative management as well as more unstable lesions
in patients who are nearing physeal closure. Stable grade 1 or grade 2
lesions with continuous articular cartilage surface are generally amenable
to arthroscopic management via retrograde transarticular drilling.
Antegrade drilling through the epiphysis is technically more challenging,
but has the advantage of avoiding injury to the articular cartilage. Both
techniques aim to stimulate revascularization of the sequestered bony
fragment and have shown good results at short to medium follow up,
with patient age and skeletal maturity being the most important
predictors of outcome. The progressive nature of adult OCD generally
justifies a more aggressive surgical approach, and the presence of
unstable or detached fragments in either adult or juvenile disease
necessitates operative intervention. Partially delaminated grade 1 or
grade 2 lesions can usually be addressed arthroscopically by using
bioabsorbable transchondral fixation devices. While interpositional
fibrous tissue should be removed, extensive subchondral debridement
should be avoided. Possible areas of subchondral bone loss can be
packed with autologous bone graft before definite fixation. Salvageable
sequestered or loose (grade 3 or grade 4) osteochondral fragments with
sufficient (3 mm or more) adherent subchondral bone can be reattached
either through an arthroscopic or mini-open approach if anatomically
congruous. This usually involves removal of fibrous tissue, curettage and
bone grafting of the femoral lesion, with drilling to invite neovascularization
of the OCD bed. Different fixation methods have been described,
including cannulated screws, bioabsorbable devices, or autologous
osteochondral plugs. While reported results have generally been
favorable, these methods all have inherent disadvantages. Metal screws
require eventual removal, bioabsorbable devices have been reported to
cause host bone reaction, and both can damage juxtaposed articular
surfaces or become loose bodies in case fixation is lost. While rare, donor
site morbidity and hemarthrosis are possible complications of autologous
osteochondral graft harvest. Grade 3 or grade 4 lesions with non-
salvagable osteochondral fragments (due to fragmentation or lack of
adherent bone) pose more of a surgical challenge. Simple loose body
removal and debridement has been shown to lead to poor long-term
clinical outcomes and arthritic disease progression and is not
recommended. Due to the subchondral pathology including bone loss
commonly encountered in OCD, marrow stimulating techniques are at a
distinct disadvantage in treating this condition. While least invasive,
microfracture generally leads to only fibrocartilagenous replacement
tissue and has increasingly shown to negatively influence revision
options due to induction of subchondral sclerosis and formation of
intralesional osteophytes. Autologous chondrocyte implantation (ACI)
has led to good clinical results at medium to long-term follow up in
multiple studies. While the use of the ACI sandwich technique (with
chondrocytes implanted between layers of periosteum over autologous
bone graft) to address ICRS grade IVb lesions has been described in the
literature, isolated outcomes of this variant have not been individually
reported. Newer generation matrix-assisted ACI techniques employing
different scaffolds promise to simplify the concurrent application of bone
grafts for use in OCD. Likewise, the advent of multilayered acellular
scaffolds holds promise as biomimetic conduits for true osteochondral
replacement, with encouraging early results. Osteochondral grafting
techniques are attractive for the treatment of OCD because of their ability
to address both the osseous as well as the chondral component of the
disease with a compound graft. Where available, fresh osteochondral
allografts are generally considered the gold standard treatment for high
grade OCD. Allografts reliably restore the appropriate joint anatomy by
providing mature, orthotopic hyaline cartilage with an intact calcified
cartilage tidemark on an osseous scaffold that can restore even advanced
bone loss associated with ICRS grade IVb OCD lesion. Multiple authors
have reported good clinical results with durable outcomes at long-term
follow up for both autologous and allogeneic osteochondral grafts.
Recently, renewed interest has been placed on optimizing the biological
milieu of the joint in cartilage repair, including correction of instability
and mechanical alignment. Since axial varus/valgus malalignment in
particular has been implicated as contributory in the development of
OCD, it stands to reason that failure to correct these factors will negatively
affect outcomes, regardless of technique. Thus, adjuvant osteotomy is
recommended to optimize the biomechanical environment of the knee
with OCD, although this has not been conclusively borne out in the
orthopaedic literature. In summary, osteochondritis dissecans is seen
with increased frequency in adolescent and young adult patients, owing
to increased athletic participation and advances in diagnostic modalities.
Early diagnosis and treatment are important to optimize outcomes. An
initial attempt at conservative treatment, focusing on activity restriction
and modification, is warranted for stable OCD lesions in skeletally
immature patients. Adult OCD lesions have limited healing potential and
usually benefit from early operative treatment. Stable juvenile OCD
lesions that fail conservative treatment, as well as unstable juvenile or
adult OCD lesions, also require surgical management. Operative
treatment options depend on lesion size, site, stage as well as patient
age and include drilling, internal fixation, bone grafting, and different
cartilage repair modalities with possible adjuvant corrective osteotomy.
References:
Management of Osteochondritis Dissecans of the Knee : Current
Concepts Review.
Mininder S. Kocher, Rachael Tucker, Theodore J. Ganley and John M.
Flynn
Am J Sports Med 2006 34: 1181-1192
Treatment of Osteochondritis Dissecans of the Knee with Autologous
Chondrocyte Transplantation: Results at Two to Ten Years.
Lars Peterson, Tom Minas, Mats Brittberg and Anders Lindahl
J Bone Joint Surg Am. 2003;85:17-24.
Fresh Osteochondral Allografting in the Treatment of Osteochondritis
Dissecans of the Femoral Condyle.
Bryan C. Emmerson, Simon Görtz, Amir A. Jamali, Christine Chung, David
Amiel and William D. Bugbee
Am J Sports Med 2007 35: 907-915
 Extended Abstracts 151

19.3.3 then found that 3D expanded HAC could redifferentiate and generate
cartilaginous grafts. Online monitoring of oxygen during perfusion
A 3D-culture model for the streamlined engineering of large- bioreactor culture was used to estimate the number of cells within a
scale human cartilage grafts 3D construct. Given that few non-destructive assays currently exist
I. Martin, B. Tonnarelli, R. Santoro, A. Barbero, D. Wendt which can quantitatively characterize an engineered construct, this
Basel/Switzerland simple and non-invasive assay could represent a significant element
in regulatory compliant manufacturing of tissue grafts meeting
Introduction: While Carticel® and Hyalograft-C® have been well specific quality criteria. This novel streamlined cartilage tissue
established in the clinic for the treatment of traumatic focal cartilage engineering strategy can serve as the basis of a manufacturing
defects, no tissue engineered product is currently available to treat process requiring the minimal number of bioprocesses and unit
large defects or those associated with advanced diseases such operations, thereby facilitating simplified and compact bioreactor
as osteoarthritis. Beyond the biological challenges that must be designs with limited automation requirements, and with the likely
addressed to treat chronic joint disorders, it remains a significant result of lower operating costs and increased compliance to safety
engineering challenge to generate cartilage grafts with dimensions guidelines [2].
that would be sufficient for the repair of large, advanced, and deep
defects, and moreover, to develop a manufacturing process which
is safe, standardized, and ultimately cost-effective. We previously References:
described a perfusion bioreactor system for seeding and culturing
constructs of clinically relevant thickness and demonstrated that [1] Wendt D, Stroebel S, Jakob M, John GT, Martin I. Uniform tissues
highly viable and homogeneous constructs could be generated [1]. In engineered by seeding and culturing cells in 3D scaffolds under
this work, we scaled-up our perfusion bioreactor to engineer human perfusion at defined oxygen tensions. Biorheology 43:481-488
cartilage grafts in dimensions sufficient for uni-compartmental (2006) [2] Martin I, Smith T, Wendt D. A roadmap for the bioreactor-
resurfacing of a human knee joint (i.e., 50mm diameter x 4mm based translation of tissue engineering strategies into clinical
thick). We next aimed to streamline the conventional cartilage products. Trends Biotech 27(9): 495-502 (2009) [3] Moretti M,
tissue engineering production processes, by seeding, expanding, Wendt D, Dickinson SC, Sims TJ, Hollander AP, Kelly DJ, Prendergast
& differentiating isolated primary human articular chondrocytes PJ, Heberer M, Martin I. Effects of in vitro preculture on in vivo
(HAC) entirely within a porous 3D scaffold using a perfusion development of human engineered cartilage in an ectopic model.
bioreactor system. The novel strategy would allow to eliminate the Tissue Eng 11:1421-1428 (2005)
conventional and labor-intensive steps of 2D cell expansion in flasks
prior to loading into the 3D scaffold, facilitating the development of
a simple, automated, and closed bioreactor-based manufacturing Acknowledgments:
process [2].
We acknowledge the EU for financial support (“STEPS”; FP6- #NMP3-
Content: A perfusion bioreactor system was first developed to CT-2005-500465)
generate a uniform velocity profile over the surface of 50 mm diameter
scaffolds. Consistent with theoretical flow simulations, performed
in collaboration with Prof. D. Lacroix (University of Catalonia),
experimental assessments confirmed that cells could be uniformly
seeded throughout the large scaffold volume. Following two weeks
of perfusion culture in the bioreactor, viable, homogeneous and
cartilaginous tissue constructs were generated, which were of similar
quality to smaller “research-scale” control constructs, in spite of the
70-fold larger size (5.9cm3 vs. 0.085cm3). We next established a novel
streamlined and bioreactor-based process, based on the definition
of two different culture phases, namely related to chondrocyte
proliferation and subsequent re-differentiation/extracellular matrix
deposition. In five independent experiments, HAC were isolated from
clinically representative sized biopsies (as obtained in the clinic for
autologous chondrocyte implantations) and perfusion seeded onto
Hyaff-11â non-woven meshes. Seeded meshes were subsequently
perfused with “proliferating” culture medium (containing 1 ng/ml
Transforming Growth Factor-beta1, TGFb, and 5 ng/ml Fibroblast
Growth Factor-2) for approximately two weeks in order to colonize
the scaffold volume and reach a defined cell density. Flow-through
micro-oxygen sensors were integrated into the bioreactor system
to monitor the oxygen levels in the media perfused through the
constructs during bioreactor culture. After initially establishing a
relation between the oxygen measurements and the number of
cells within the 3D constructs, we applied this non-destructive
method to quantitatively and non-invasively monitor the cell growth
throughout the proliferation phase. After the initial proliferation
phase, when the chondrocytes had been sufficiently expanded
within the mesh, chondrocyte re-differentiation was then induced by
perfusing “chondrogenic medium” (containing 10 ng/ml TGFb and
10 mg/ml insulin) under hypoxic conditions (5%O2) for an additional
two weeks. Primary HAC could be seeded and extensively expanded
(about 5 and 9 doublings in respectively 2 and 4 weeks) in the 3D
scaffolds, densely populating the volume of the meshes. Immuno-
staining for the Ki67 marker confirmed the presence of proliferating
cells at day 14. Following subsequent chondrogenic conditions,
constructs engineered by the streamlined bioreactor-based approach
showed evidence of redifferentiation as significant increase in GAG
content (up to about 4.5 ug/mg wet weight), immunohistochemical
staining for collagen type II, and increased expression of collagen
type II mRNA. The work describes a new paradigm for the generation
of large-scale cartilage grafts, where the phases of cell expansion,
cell seeding, and tissue culture are performed directly through 3D
scaffolds and within a single closed perfusion bioreactor system.
The limited number of chondrocytes obtained from a clinically
representative sized biopsy could be extensively expanded directly
in 3D scaffold under perfusion, reaching cell numbers similar to
seeding densities used in conventional cartilage tissue engineering
approaches based on cells previously expanded by 2D culture steps
[3]. When constructs underwent following chondrogenic phase, we
152 Extended Abstracts
20.2.3
Preoperative radiographic evaluation of axis deformities around
the knee
S. Spruijt
Woerden/Netherlands
Introduction: The knee joint is the largest joint in the body and it
has the longest lever arms. These arms produce substantial loading
moments. Deformity in the frontal plane will increase the distance
from the centre of the knee to the mechanical axis and thus create
a moment arm at the knee joint. Axis deformities closer to the knee
produce greater mechanical axis deviations than deformities near
the ankle or the hip1. In a dynamic experimental loading study it was
demonstrated that the position of the loading axis in the frontal plane
has a strong effect on the tibiofemoral cartilage pressure distribution
of the knee. The medial compartment is predominantly loaded in a
varus knee; a neutral mechanical axis slightly loads the lateral more
than the medial compartment. In valgus alignment, the main load
runs through the lateral compartment2. It has been shown that
frontal plane malalignment around the knee is associated not only
with progression of knee osteoarthritis but also with the development
of knee osteoarthritis3. Therefore axis deformities around the knee
are regarded as pre-arthritic deformities. The goal of correctional
osteotomies around the knee is the transfer of mechanical load from
the diseased areas of the joint to areas with healthy, intact cartilage.
Recent biomechanical work has shown that unloading of the medial
compartment can be achieved in high tibial osteotomy by using a
slight valgus overcorrection3, 4. Accurate preoperative evaluation
of axial deformity is mandatory for the success of osteotomies
around the knee. This requires detailed knowledge of normal limb
alignment. Since the most frequent and relevant deformities around
the knee occur in the frontal plane, this paper will primarily, but not
exclusively, focus on the preoperative evaluation of axis deformities
in the frontal plane.
Content: Radiographic work-up A thorough history and physical
examination are absolutely mandatory in the preoperative workup
for osteotomies around the knee. However, to understand the true
nature of the deformity subsequent radiographic examination is
indispensable. Preoperative evaluation for osteotomies around the
knee includes anteroposterior weight-bearing views in full extension,
weight-bearing PA tunnel views in 45° of flexion (Rosenberg view)5,
lateral views and skyline views of the patella. These allow an
assessment of the extent and localisation of knee osteoarthritis. The
anteroposterior whole leg standing radiograph is considered the
gold standard for measuring axial alignment and joint orientation
and serves as the basis for the planning of osteotomies in the frontal
plane. Standardized radiographs with full extension and correct
anterior orientation of the patella are necessary for such
measurements6, 7. For assessment of the sagittal plane of the knee
joint strict lateral knee views are usually sufficient. The lateral whole
leg standing radiograph in full extension is only indicated in the case
of extra-articular deformity. Varus and valgus stress views are not
absolutely necessary, but may be helpful to assess instability of the
collateral knee ligaments or to define the degree of involvement of
the less affected tibiofemoral compartment8. Magnetic resonance
imaging (MRI) may be useful for assessment of meniscal and
ligamentous lesions and cartilage damage. It is however not
mandatory. Especially not if the osteotomy around the knee is
preceded by an arthroscopy. Computer tomography (CT) is only
indicated if torsional malalignment is suspected or in cases with
posttraumatic defects. Scintigraphy is usually not indicated in the
preoperative evaluation for osteotomies around the knee. Causes of
axial deformities The joint orientation angles and axes can be
pathologically altered in any plane (frontal, sagittal, transverse or
oblique) and cause malalignment of the entire leg. The causes may
be congenital or constitutional (skeletal dysplasia, achondroplasia).
They may be develop during childhood as a result of malnutrition
(rickets), metabolic bone disease (hypophosphatemic rickets),
Blount’s disease, osteopathies (renal osteopathy), growth disorders
with premature partial closure of the growth plate, obesity,
iatrogenic, etc. The may also be acquired after trauma (e.g.,
malunited fracture). They may be related do systemic myopathic and
neurogenic disorders. They may be the result of physical trauma.
Furthermore secondary deviation of the axes can be caused by
destruction of the joint surfaces as a result of bone necrosis,
infection, bone tumour, inflammatory arthritis. The most frequent
cause of secondary varus and valgus malalignment is secondary
cartilage damage after meniscectomy9. Mechanical and anatomic
bone axes In long bones anatomical and mechanical axes are
distinguished. In clinical practice radiographs are standard made in
the anteroposterior and lateral direction. Axis lines drawn on these
radiographs are referred to as frontal and sagittal plane axes,
respectively. However, around the knee the deformities in the frontal
plane are the most frequent and most relevant. The anatomical axis
of a bone is the mid-diaphyseal line. The mechanical axis of a bone is
the line that connects the joint centres of the proximal and distal
bone-ends. The mechanical femoral axis in the frontal plane runs
from the centre of the femoral head to the centre of the knee joint
and forms a distally converging angle of 6 ± 1° with the anatomical
axis of the femur9 In the tibia the frontal plane anatomical and
mechanical axes are almost parallel. Physiologically, the angle
between the mechanical axis of the femur and tibia is 1.2 ± 2.2°
varus10. The anatomic tibiofemoral angle is physiologically 5-7°
valgus11.The mechanical axis of the entire leg in the frontal plane
(Mikulicz line or weight bearing line) is the line connecting the centre
of the femoral head and the centre of the ankle joint. This line
normally passes medial to the centre of the knee joint.
Radiographically, this is approximated by the position of the medial
tibial spine. The distance between the mechanical axis line and the
centre of the knee in the frontal plane is the mechanical axis deviation
(MAD). This deviation can be expressed in millimetres or as a
percentage of the tibial plateau width (medial 0% and lateral 100%).
The normal MAD is 8 ± 7mm medial to the centre of the knee joint12.
Joint orientation angles The joint orientation line represent the
position of a joint in a particular plane. These joint lines in the frontal
and sagittal planes have a characteristic orientation to the mechanical
and anatomical axes. Paley has standardised the nomenclature of
these joint orientation angles. He has also reported on the normal
values of these angles relative to the mechanical and anatomic axes
in both the frontal and sagittal planes11, 12. The mechanical lateral
proximal femoral angle (mLPFA) represents the hip joint orientation
in the frontal plane and is defined as the angle between a line from
the centre of the femoral head to the tip of the greater trochanter
and the mechanical axis of the femur. Its standard value is 90° ± 5°.
The distal femoral knee joint orientation is defined as the lateral
distal angle between the mechanical femoral axis and the tangent to
the distal femur (mLDFA) and has a standard value of 87°± 3°. The
proximal tibial knee joint orientation is the medial proximal tibial
angle (MPTA) and is the angle between the tangent to the tibial
plateau and the anatomical or mechanical axes of the tibia and is
87°± 3°. The mechanical lateral distal tibial angle (mLDTA) represents
the ankle joint orientation and is defined as the angle between the
tangent to the dome of the talus and the anatomical or mechanical
axes. Its standard value is 89°± 3°. The angle between joint
orientation lines on opposite sides of the same joint is called the
joint line convergence angle (JLCA). In the knee the JLCA is normally
between 0-2° medial convergence. Increased JLCA-values due to
ligament instability must be taken into account in correctional
osteotomies to avoid overcorrection. The midjoint line (MJL) is a
measure of joint line obliquity 13, 14. The MJL is centred between the
knee base line of the femur and the baseline of the tibia plateau and
forms femorolateral and mediotibial angles of 87°± 3° with the
mechanical axis (Mikulicz line). The MJL thus has a slight varus
inclination. This inclination is valuable because during gait the leg is
held in slight adduction during the stand phase and consequently
the knee joint line becomes parallel to the ground during weight
bearing12. Correctional osteotomy may achieve correction of the
mechanical axis, but may result in a pathological alteration of MJL if
the correction was not performed at the site of the bone deformity.
Pathological MJL leads to increased shear stresses al the joint line,
which is not very well tolerated in the knee. The above described
principles for joint orientation angles in the frontal plane are
applicable to the sagittal plane as well. For the knee the posterior
distal femoral angle (aPDFA = 83°±4°) and the posterior proximal
tibial angle, also known as slope of the tibial plateau. (aPPTA = 81°±
4°) are important12. The sagittal joint orientation angles should not
be increased or decreased during osteotomy in the frontal plane
(valgus or varus correction) in patients with stable ligaments and
normal range of motion. Conversely, increasing tibial plateau slope
(flexion osteotomy) can be used to eliminate hyperextension, to
unload the posterior tibial plateau and to reduce posterior instability.
Alternatively, decreasing the tibial slope (extension osteotomy) can
be used to eliminate extension deficit, to unload the anterior plateau
and to reduce anterior instability15. Systematic evaluation of axis
deformities The most frequent leg deformities occur in the frontal
plane. Malalignment is present if the MAD exceeds its normal range
and it indicates loss of collinearity of the hip, knee and ankle in the
frontal plane. The normal range of MAD is 8 ± 7mm medial12. The
source of MAD is identified with the “malalignment test” (MAT)
which forms the basis in the evaluation of deformities around the
knee11, 16. Hereto, the mLDFA, MPTA and JLCA are measured. If the
mLDFA or MPTA exceeds its normal range respectively the femur or
the tibia is contributing to the MAD. Abnormal values of the JLCA
indicate ligamentous laxity or cartilage loss as causes of
malalignment. Other sources of malalignment include knee joint
subluxation and femoral or tibial condyle deficiency11, 16 The MAT

just identifies which bone or joint contributes to the malalignment. It
does not identify the level of the deformity. Neither does MAT identify
any sagittal plane component of deformity. Malorientation of the
knee very clearly leads to MAD and therefore The MAT is a
“malorientation test” (MOT) of the knee. However, the MAT can be
(almost) normal in the presence of malorientation of the hip and
ankle. To recognize malalignment of hip and ankle it is therefore
necessary to perform separate MOTs for these joint as well11, 16. The
next step is to find the location of deformity within a particular bone.
For this, the anatomical axes, using mid-diaphysial lines, for both
femur and tibia are used. In a deformed bone the proximal and distal
anatomic axes intersect. This point is known as the centre of rotation
of angulation (CORA)17. The mechanical axis can also be used to
determine the CORA. Hereto, the joint centre and a reference angle
are used to draw the mechanical axis of the proximal or distal femur
or tibia. A reference angle is drawn to a reference line. The two
possible reference lines that can be used are the joint orientation
line and the mid-diaphyseal line. If the CORA does not correspond to
the obvious apex of angulation, there is either a translational
deformity or there may be several (multiapical) deformities17.
Furthermore rotational deformity may coexist. Deformity in the limb
may occur in any plane, not just the “anatomical” sagittal or frontal
planes. Uniapical angular deformities for which angulations are
visible on both anteroposterior a lateral radiographs are often
incorrectly referred to as biplanar deformities. These biplanar
angular deformities are actually uniplanar deformities in an oblique
plane (between the frontal and sagittal planes)18. The site of the
deformity may be at the diaphysis, metaphysis or at the level of the
joint. Any angular deformity should be described in terms of its
magnitude, direction, plane and apex location. It is very important to
realise that if angular deformities are corrected by an osteotomy that
passes through CORA realignment occurs without translation. When
the osteotomy is performed at another level than CORA the axis will
not only realign by angulation, but translation will occur as well19.
Genu varum and genu valgum A MAD that lies more medial than the
normal range indicates a varus deformity and a MAD lateral to the
normal range a valgus deformity. If the MPTA is decreased the varus
malalignment is due to tibial deviation, whereas an increased mLDFA
indicates a femoral cause. Conversely, if the mLDFA is smaller than
the standard value, the cause of the valgus deformity is a femoral
one. An increased MPTA indicates a tibial cause of valgus
malalignment9. In a tertiary referral practice it has been demonstrated
that only 31% of patients with varus joint degeneration deformity of
the leg have osseous deformity at the tibia only. In 59% the varus
deformity is located at the femur and in 10% both femur and tibia are
affected. The situation for valgus deformity of the leg is similar. In
these cases the deformity is located in the femur in only 22%. In 45%
the valgus deformity is located at the tibia and in 33% it affects both
femur and tibia. The frequently quoted tenet for correction of varus
malalignment at the tibia and valgus malalignment at the femur is,
consequently, incorrect for 50% of varus and valgus joint
degeneration deformities13. This may lead to joint line obliquity.
Varus and valgus deformities can also be caused by cartilage and
bone loss and ligament instability. The JLCA will identify these
pathologies. The tibial bone varus angle (TBVA) is a prognostic
concept that has been introduced by Levigne in 1991 to differentiate
between a constitutional extra-articular varus deformity of the
proximal tibia and an acquired intra-articular tibial varus secondary
to erosion of the medial plateau24. The TBVA is the angle between
the epiphyseal axis and the mechanical axis of the tibia. The
epiphyseal axis is a line connecting the centre of the tibial spines
with the midpoint of a line connecting the two endpoints of the
physis. In constitutional tibial varus, high tibial osteotomy (HTO)
cures varus malalignment. In acquired tibial varus, HTO is palliative
and creates a tibial valgus position with a poorer clinical outcome
over time24. Patellofemoral joint and rotational deformity of the leg
In the preoperative evaluation of axis deformities around the knee,
the patellofemoral joint should be regarded as well. The height of
the patella is changed in HTO. Using the open wedge technique,
opening of the osteotomy causes distalisation and lateralisation of
the tibial tubercle and the patella that can result in patella infera and
excessive stresses associated with altered patellofemoral joint
mechanics20, 21. In cases of a preexisting patella alta or in large
corrections, an anterior osteotomy of the tibial tubercle extending
distally should be combined with medial open-wedge HTO as this
will leave patellar height unchanged20. Patellar malalignment can
be present in combination with axis deformities around the knee and
often indicates the presence of torsional abnormalities. Evaluation
of patellofemoral and torsional malalignment is beyond the scope of
this paper and the reader is referred to other sources22, 23.
Conclusion Axial deformities around the knee can occur in all 3
anatomical planes of reference. There are frontal plane deformities
(i.e. genu varum and genu valgum), sagittal plane deformities (i.e.
genu procurvatum and genu recurvatum), and transverse plane
Extended Abstracts 153
deformities (translation and torsion). Deformities can even occur in
the so called oblique plane (i.e. in between the frontal and sagittal
planes). Each axis deformity around the knee should be described in
terms of its magnitude, direction, plane and apex location. Deformity
correction should be performed at its source (femur, tibia or both)
and if possible at the CORA. The success of realigning osteotomies
around the knee therefore highly depends on an accurate
preoperative evaluation of axial deformities.
References:
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JV (1991). The effect of simulated fracture-angulations of the tibia on
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· Gibson PH, Goodfellow JW (1986). Stress radiography in
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24.2.2
(EPIC) MicroCT
I. Patsch
Vienna/Austria
Introduction: Microcomputed-tomography (micro-CT) is a high-
resolution imaging technique which is predominantly applied in
osteopororsis research. Depending on geometry and properties of a
dedicated scanner, the resolution can reach up to several micron (ex-
vivo). Similar in-vivo devices with slightly decreased resolution are
available for longitudinal small animal studies. In humans, HR-pQCT
(high-resolution peripheral quantitative computed tomography)
provides comparable image data of the distal radius and the distal
tibia at a resolution of 82 micron. The scanners allow for quantitative
assessment of bone microstructure and their use has significantly
advanced osteoporosis research (e.g. by assessing treatment
effects in animal studies, human iliac crest biopsies or non-invasive
imaging studies of the distal radius or tibia). Microstructural
osteologic parameters include trabecular and cortical thickness,
trabecular number, trabecular separation and connectivity density.
Using the structure model index, it is possible to classify trabecular
bone as either plate-or rod-like. Owing to the outstanding quality
of the images, finite element modelling as well as advanced texture
analyses can be applied. Moreover, cortical porosity has become
quantifiable. Linking 3D-bone-morphometry and biomechanics,
micro-CT has had a major impact on osteoporosis research of the
last decades.
Content: As cartilage can hardly be visualized in normal micro-CT, a
specific technique termed EPIC (equilibrium partitioning of an ionic
contrast) micro-CT has been developed for osteoarthritis research.
Similar to the dGEMRIC (delayed gadolinium enhanced MRI of
cartilage) technique, EPIC-micro-CT is based on the permeation of
cartilage by a ioxaglate-based, negatively charged contrast agent.
In EPIC micro-CT, the contrast material is an iodinated radioopaque
agent, which distributes inversely to GAG content and density.
Regions of low GAG content (i.e. damaged cartilage) will therefore be
targeted by the charged contrast agent. Local increases in contrast
agent deposition cause an increase in x-ray attenuation which is
depicted as difference of a grey-scale image. Data processing includes
sagittal reformation and subsequent semiautomated segmentation
from subchondral bone and bone marrow. Parameters derived from
EPIC micro-CT include cartilage thickness, surface area, volume
and attentuation (i.e. local GAG density). Based on the isotropic,
high resolution of micro-CT, 3D refomations can be easily obtained.
These 3D reformations depict cartilage surface morphology as well
as the spatial distribution of cartilage damage. Moreover, cartilage
thickness maps can be generated .
EPIC micro-CT is applicable to cartilage explants, biopsy specimens,
as well as whole cartilage in situ specimens . It has been used for
longitudinal monitoring in a rat model osteoarthritis study . A recent
study demonstrated the potential of EPIC-micro CT for monitoring
cartilage repair and regeneration of tissue-engineered cartilage in
sternum defects .
Whereas most EPIC research has been performed on the basis of
a negatively charged (anionic) contrast agent, some researchers
suggest the use of positively charged (cationic) agents. Cationic
agents seem to display a higher sensitivity to GAG density than
anionic agents. In a study performed on bovine cartilage plugs, the
concentration of a cationic agent needed for appropriate contrast
was much lower than the concentration of a comparable anionic
agent .
Besides EPIC imaging, it is possible to visualize cartilage thickness
and surface properties by simple micro-CT based arthrography.
After injection into the joint space, the radioopaque contrast agent
provides indirect display of the articular surface. However, micro-CT
arthrography does not enable the assessment of GAG content and
is therefore less sensitive to early osteoarthritic changes than EPIC-
micro CT. However, the use of silicon-based contrast agents seems
to aid in the differentiation of cartilage from synovial fluid and soft
tissue components such as ligaments and menisci. The contrast
between cartilage and adjacent joint fluid obtained from the injection
of silicon-based contrast agents has been reported to be better than
in ioxaglate-based studies .
Certain drawbacks of micro-CT of cartilage, irrespective of the
type of contrast agent, are cartilage layer-dependant contrast up-
take or diffusion into adjacent tissues. For successful application
of this technique, specimens have to be incubated with adequate
concentrations of diluted contrast agent. Optimal incubation times

with the contrast agent seem to be species- and site-specific. Some
researchers even suggest that the incubation times used in the
majority of studies are by far insufficient. Most studies are focusing
on the knee joint, but other locations might display different contrast
media uptake kinetics and imaging characteristics. So far, no ex-vivo
or in vivo studies on drug effects have been published. In addition
the user needs to consider that the resolution of micro-CT depends
on specimen size.
Cartilage and subchondral bone form a functional, metabolic and
biomechanical unit. A certain benefit of EPIC micro-CT and other
micro-CT based cartilage imaging techniques is the option of parallel
imaging of cartilage and subchondral bone .
Cartilagemicro-CT has been carried out in various ex-vivo and also
in some in vivo animal models, including mice, rats, rabbits and
bovine tissue. Studies on human specimens are rare, in vivo data yet
entirely missing. A Finnish research group has undertaken several
approaches to perform EPIC-imaging on pQCT devices. One of these
projects used human cadaveric knee specimens . However, HR-pQCT
or similar high resolution devices which allow for scanning of larger
samples than just biopsies or small animal joints have not yet been
used in EPIC imaging.
To summarize, micro-CT offers a promising new approach to
experimental cartilage and subchondral bone imaging. Longitudinal
animal studies - especially those evaluating treatment options - are
still to be published, and human data are rare. Various contrast
agents are in use, but EPIC micro-CT, using negatively charged
ioxaglate, is most frequently found in the literature.
The possible benefits of (EPIC)-micro-CT cartilage imaging are
1) the very high resolution,
2) the potential of parallel assessment of cartilage and subchodral
bone, as well as
3) its biochemical aspect (i.e. GAG density measurement and
mapping)
References:
Palmer AW. Analysis of cartilage matrix fixed charge density and three-
dimensional morphology via contrast-enhanced microcomputed
tomography. PNAS 2006;103:19255-260.
Xie L. Quantitative assessment of articular cartilage morphology via
EPIC-uCT. Osteoarthritis Cartilage 2009; 17(3):313-20. Epub 2008
Sep 11.
Xie L. Nondestructive assessment of sGAG content and distribution
in normal and degraded rat articular cartilage via EPIC-microCT.
Osteoarthritis Cartilage. 2010; 18: 65-72.
Piscaer TM. Micro-CT Arthrography - A novel in vivo technique for
longitudinal assessment of cartilage alterations in small animal
models. 2010; 56th Annual Meeting of the Orthopaedic Research
Society. Poster No. 1363.
Moyer HR. A new animal model for assessing cartilage repair and
regeneration at a nonarticular site. Tissue Eng Part 2010; 16: 2321-
30.
Stewart RC. The effect of cationic contrast agent concentration on
GAG quantification using computed tomography. 2010; 56th Annual
Meeting of the Orthopaedic Research Society. Poster No. 855.
Gu XI. Contrast Agent Enhanced High Resolution 3D Micro-CT Imaging
of Hard and Soft Tissue in Rat Knee Joint. 2010; 56th Annual Meeting
of the Orthopaedic Research Society. Poster No. 857.
Silvast TS. Diffusion and near-equilibrium distribution of MRI and CT
contrast agents in articular cartilage. Phys Med Biol. 2009; 54:6823-
36.
Aula AS. Simultaneous computed tomography of articular cartilage
and subchonrdal bone. Osteoarthritis Cartilage. 2009; 17: 1583-8.
Silvast TS. pQCT study on diffusion and equilibrium distribution
off iodinated anionic contrast agent in human articular cartilage
- associations to matrix composition and integrity. Osteoarthritis
Cartilage 2009; 17
Extended Abstracts 155
24.2.3
MRI (dGEMRIC and other techniques)
S. Trattnig
Vienna/Austria
Imaging Technologies for MS Tissue Regeneration
MRI (dGEMRIC and other techniques) S.Trattnig MR imaging of the
morphology of cartilage and cartilage repair tissue has significantly
improved in recent years due to the development of clinical high-
field MR systems operating at 3 Tesla. The improved performance
has also been achieved as a result of the higher gradient strengths
and the application of dedicated coils with modern configuration
such as phased array coils. Additionally high resolution isotropic
imaging provides identical resolution with reformatting in all planes.
The combination of these technological advances now allows high-
resolution imaging of cartilage within reasonable scan times. In
addition to the evaluation of gross cartilage morphology by MRI,
there is growing interest in the visualization of ultra structural
components of cartilage by MR in two fields: - Osteoarthritis is
manifested by significant changes in biochemical composition of
articular cartilage. Loss of glycosaminoglycans (GAG) and increased
water content represent the earliest stage of cartilage degeneration,
while the collagenous component of the extracellular matrix remains
mainly intact during this early phase of cartilage degeneration (1). -
During the last years a lot of new surgical techniques for cartilage
repair have been developed based on tissue engineering techniques
such a autologous chondrocyte implantation (ACI) and matrix-
associated autologous chondrocyte transplantation (MACT) (2). In
addition to morphological MR imaging of cartilage repair tissue, an
advanced method to non-destructively and quantitatively monitor
parameters reflecting the biochemical status of cartilage repair
tissue is a necessity for studies which seek to elucidate the natural
maturation of MACT grafts and the efficacy of the technique. For
example GAG are known to be responsible for stiffness properties of
cartilage, which gains even more importance with cartilage implants
and the content and organization of the collagen network reflects
further mechanical properties of cartilage. Therefore, several MR
techniques were developed, which allow detection of biochemical
changes that precede the morphological degeneration in cartilage.
To date, the most promising technique for visualizing the loss of GAG
seems to be the delayed gadolinium-enhanced MRI of cartilage
(dGEMRIC) (3). It is based on the fact that GAG molecules contain
negatively charged side chains which lead to an inverse
proportionality in the distribution of the negatively charged contrast
agent molecules with respect to the concentration of GAG.
Consequently, T1 which is determined by the Gd-DTPA2- concentration
becomes a specific measure of tissue GAG concentration. The
dGEMRIC technique has provided valuable results in studies on hip
dysplasia, in comparative studies with arthroscopically determined
cartilage softening in early osteoarthritis, and demonstrated the
positive effects of moderate exercise on glycosaminoglycan content
in knee cartilage (4). However the main problem was that the
standard dGEMRIC technique is either limited to single slices in 2D
acquisition or is time consuming in 3D sequences such as 3D
inversion recovery prepared fast spoiled gradient recalled acquisition
in the steady state. This therefore limits the attractiveness of
dGEMRIC for clinical use. To overcome these problems a fast T1
determination by using dual flip angle values in gradient echo based
sequence was developed by one vendor. This allowed coverage of
the whole knee joint with two different flip angles within the same
sequence in about four minutes. The 3D dual flip angle dGEMRIC
technique is comparable to standard T1 inversion recovery technique
for T1 mapping and its application in patients after MACT surgery is
feasible and can be performed in clinical acceptable scan times. We
found that the difference between repair tissue and normal hyaline
cartilage was statistically significant (p< 0.007) in patients between
3 and 13 months and patients between 19-42 months after surgery,
the difference between these groups was not statistically significant
(p=0.205). These findings show that the GAG content is significantly
lower in repair tissue than in normal hyaline cartilage and does not
change even after 3 to 4 years. While GAG content reflects stiffness
properties of hyaline cartilage, the organisation of the collagen
matrix in cartilage is important too, as failure within the collageneous
fibre network is considered to entail further cartilage breakdown.. In
a recent study of our group it was possible to evaluate T2 relaxation
times in vivo in normal hyaline cartilage sites and in cartilage repair
tissue after two different cartilage surgical procedures. Obtaining
high resolution MR imaging it was also possible to assess zonal
variations within the ROIs and use the measurement of the
organization of articular cartilage as an additional tool to differentiate
between cartilage repair tissues. We found no differences between
deep, middle and superficial aspects within cartilage repair tissue
156 Extended Abstracts
after microfracture therapy indicating disorganized and more fibrous
tissue. After MACT, zonal variation could be measured, however
compared to healthy cartilage sites the increase from deep to
superficial zones was less pronounced. These findings may indicate
that after MACT, cartilage repair tissue is, in terms of organization,
hyaline like. Quantitative T2 mapping may help to better differentiate
between normal maturation and development of abnormality in ACI
and MACT. This technique may further help in the non-invasive, non
destructive follow-up of patients operated on new generations of
matrix-associated ACT using new scaffold or carriers. Better
knowledge on the macromolecular organization in the implants may
help in the planning of rehabilitative procedures after cartilage repair
surgery. One encouraging alternative to these above mentioned
sequence modalities for the evaluation of cartilage microstructure is
the use of diffusion weighted sequences (9). Diffusion Weighted
Imaging (DWI) is based on molecular motion that is influenced by
intra- and extra-cellular barriers. Consequently, it is possible, by
measuring of the molecular movement, to reflect biochemical
structure and architecture of the tissue. Conventional DWI based on
spin-echo (SE) sequences is relatively insensitive to susceptibility
effects, but diffusion weighted SE sequences require a long
acquisition time, which, for practical reasons, in a clinical examination
is inapplicable. Echo planar imaging (EPI)-based diffusion sequences,
the gold standard of DWI in neuro applications, suffer from image
distortions due to susceptibility changes as well as from limitations
in contrast due to rather long echo times needed. Both renders them
impracticable for low T2 tissues like cartilage and muscles.
Alternatively diffusion imaging can be based on steady state free
precession sequences (SSFP) which realize a diffusion weighting in
relatively short echo times. This is achieved by the application of a
mono-polar diffusion sensitizing gradient, which, under steady-state
conditions, leads to a diffusion weighting of consecutive echoes
(spin echoes and stimulated echoes). For the assessment of diffusion
weighted images, a three-dimensional steady state diffusion
technique, called PSIF (which is a time reversed FISP (Fast Imaging
by Steady State Precession) sequence), has been used (10). In order
to allow a semi-quantitative assessment of the diffusional behaviour
in the cartilage, the diffusion sequence protocol consisted of 2
separate but immediately consecutive measurements using none
(0), and 75 mT*ms*m-1 monopolar diffusion gradient moments for
Diffusion Weighted Imaging (DWI) and otherwise identical imaging
parameters. For evaluation, the quotient image (non-diffusion
weighted / diffusion-weighted image) was calculated on a pixel-by-
pixel basis In a series of 15 patients after MACT we could demonstrate
the feasibility of diffusion-weighted PSIF imaging with high resolution
in vivo. The results show that in the follow up at different time points
after MACT the diffusion behaviour of the transplants is changing. In
the earlier period after MACT the diffusion was higher restricted
which declined in the later follow up, but even after a period of up to
42 months there was still a difference in diffusion values between
repair tissue and normal hyaline cartilage. Diffusion weighted
imaging using the semi-quantitative approach can practicably
complement the information obtained from approaches which rely
on relaxation properties such as dGEMRIC or T2-Mapping. In
comparison to dGEMRIC imaging of cartilage and cartilage repair
procedures, no contrast medium is needed ,coverage and resolution
is improved and scan times reduced. It may be another tool of
biochemical evaluation of cartilage transplants in the near future
and could be added in a clinical setting to dGEMRIC and T2 Mapping
for evaluation of cartilage repair outcomes.
24.3.2
Small animal models in cartilage research: stimulating repair
versus inhibiting degeneration
C.B. Little
St. Leonards, NSW/Australia
Introduction: Small animals (mice, rats, guinea pigs, rabbits) have
been used extensively for studies of cartilage degeneration in arthritis,
and more recently for repair of osteochondral defects. The pros and
cons of the different animal species (small and large) for study of
repair of isolated cartilage defects have recently been well reviewed
(1, 2). In small animals, factors including the physical size of the joint,
articular cartilage cellularity, thickness and ratio of non-calcified to
calcified zones, thickness of subchondral bone, lack of growth plate
closure (mice and rats), and spontaneous repair of cartilage defects
in certain strains (mice, rats, rabbits), are all factors that must be
considered when evaluating use of small animal models and the
translation of findings to humans. Nevertheless, the availability
of immune-compromised mice and rats for xenotransplantation
experiments, and the array of genetically modified mice provide
unique tools to explore specific research questions. Rather than re-
covering the information that has already been presented in these
excellent available reviews, the current presentation will focus on the
relationship between cartilage degradation versus repair and how
the use of genetically modified mice and models of osteoarthritis
(OA) might inform research on cartilage regeneration strategies.
Content: Degradation and erosion of articular cartilage is a central
pathological event of all arthritic conditions. The maintenance of any
tissue is a balance between anabolism and catabolism, both of which
are inevitably occurring simultaneously as part of normal
homeostasis. In musculoskeletal tissues such as cartilage and bone
where the extracellular matrix makes up such a large proportion of
the tissue volume and is key to the critical mechanical properties,
this balance is particularly important. In diseases such as OA, despite
evidence for increased biosynthesis of cartilage matrix components
at least early in the disease, the catabolic processes predominate
and matrix breakdown and cartilage loss occurs. Targeting cartilage
degradative mechanisms has not surprisingly therefore, been a
major focus of OA research, and significant advances have been
made in recent years in identifying important events in OA cartilage
breakdown. Many of these advances have come from using small
animal models and particularly genetically-modified (GM) mice. A
review of the burgeoning literature on cartilage repair highlights that
while the majority of the studies in animal models (both small and
large) have involved evaluation of repair strategies in isolated
chondral or osteochondral defects in otherwise normal joints, a
major focus of this research area is actually targeted towards repair
and/or regeneration of cartilage in OA. It is widely accepted that
cartilage has a limited ability to repair particularly with advancing
age, and this may be even more compromised in diseases such as
OA. A significant research effort has therefore been directed towards
improving the reparative potential of chondrocytes with the view
that this can redress the anabolic/catabolic imbalance in OA cartilage
and facilitate repair. To this end the effect of numerous growth
factors (e.g. IGF-1, TGF-beta, PDGF, CTGF, FGF-2, OP-1) on cartilage
metabolism have been extensively studied. The potential of these
agents to (re)induce a chondrogenic phenotype in chondrocytes or
mesenchymal stem cells for potential repopulation of isolated
cartilage defects or OA is the subject of extensive ongoing study.
Similarly, the chondrogenic potential of different natural and
synthetic scaffolds, ± seeding with different cells with or without
mechanical loading has been investigated. Surprisingly however,
very little attention has been paid in the “cartilage repair literature”
to directly controlling the catabolic pathways that are activated in
diseased joints in order to potentially facilitate cartilage repair. Given
the importance of balance between anabolism and catabolism in
maintenance of cartilage, a valid approach to identifying potential
agents to enhance cartilage repair, may be to identify those
molecules that have been shown to significantly inhibit or enhance
cartilage degeneration in spontaneous or induced models of OA in
genetically modified mice. Particularly in the case of cytokines,
growth factors and enzymes, such proteins would be obvious
therapeutic moieties for cartilage repair, either by augmenting or
inhibiting their function. In a previous study examining the
relationship between cartilage damage and pathology in other joint
tissues in OA, a search in Medline was done using the terms “mouse/
murine/mice” and “osteoarthritis” (3). This data has now been
updated and reexamined with a view towards cartilage repair, and
additional search terms “cartilage repair” and “cartilage
regeneration” were included. A total of 798 entries retrieved dating
from 1952 to 2010. Papers describing GM mice, defined as strains in
which specific genes were either knocked out (KO) or mutated, as

well as those where specific proteins were transgenically (TG) or
virally over-expressed, were further examined to determine if
spontaneous or induced joint disease and/or cartilage degradation
and cartilage repair/regeneration were reported. Studies where only
inflammatory arthritis (e.g. collagen-induced or antigen-induced)
was reported, were excluded except where they provided a useful
comparison for GM mice in which non-inflammatory OA-like cartilage
degradation/disease and repair had also been evaluated in the same
or other reports. No studies were identified where cartilage repair/
regeneration alone was studied (i.e. in the absence of analysis of
cartilage degeneration and OA). A total of 88 different GM mice were
found where an effect on articular cartilage degradation and OA was
specifically described. In 37 of the 88 GM mice, OA was induced
(usually by some form of surgical destabilization of the joint), while
spontaneously occurring joint disease was evaluated in the
remainder. The 88 GM mice do not represent mutations in separate
proteins, as in some cases different mutations in the one gene/
protein, multiple gene KOs, or both increased and decreased
expression of a gene have been reported, and are therefore
considered separately. Over 90% of the GM lines identified were
reported since the year 2001, consistent with the recent explosion in
the use of GM mice as a research tool. The GMs were classified as
those involving cartilage extra-cellular matrix (ECM) proteins (n =
24), matrix degrading enzymes, enzyme inhibitors or mutations in
enzyme cleavage sites in substrates (n = 17), growth factors,
cytokines, effector molecules or their receptors and converting
enzymes (n = 23), cellular proteins and transcription factors (n = 19),
and spontaneous mutations in unknown genes (n = 4). The GM mice
were further separated into those where the manipulation
“increased” (n = 58), “decreased” (n = 10) or had “no effect” (n =
20) on OA cartilage damage. In only 3 of these GM strains of mice
was any analysis of actual “cartilage repair” as opposed to cartilage
degradation examined: tenascin C KO, MRL/MpJ and Jaffa mice (see
below). Recent advances have been made in defining degradative
mechanisms in cartilage, in particular the enzymes responsible for
breakdown of the two principle structural matrix proteins, aggrecan
and type II collagen. Catabolism of aggrecan is an important early
event in pathological cartilage breakdown that likely precedes and
may be prerequisite for collagenolysis. Cleavage within the
interglobular domain (IGD) by the aggrecanases at ..EGE-ARG...
appears to be primarily responsible for aggrecan loss from articular
cartilage in vitro and in vivo particularly in diseased states. Mice with
inactivation of ADAMTS-5 were found to be resistant to IL-1 stimulated
aggrecanolysis in vitro and cartilage degradation following induction
of both inflammatory arthritis and OA in vivo (4, 5). Similarly, mice in
which the IGD sequence was mutated to render it resistant to
ADAMTS-cleavage (but not when made resistant to MMP-cleavage)
also have significant inhibition of cartilage breakdown both in vitro
and in induced models of arthritis (6). Late stage cartilage
degeneration is characterized by extensive proteolysis of the type II
collagen network. Much of the collagenolysis in OA cartilage is due
to the action of MMPs, particularly MMP-13, and mice deficient for
MMP-13 shown chondroprotection in surgically-induced OA (7).
Significant upregulation of MMPs and ADAMTS expression occurs in
focal diseased cartilage in OA. Importantly in the context of cartilage
repair strategies however, we have found that expression of many of
these enzymes is significantly increased in cartilage throughout the
joint, adjacent to and well removed from the focal erosive lesions.
Subsequent activation/release of the latent MMPs and ADAMTS
sequestered in cartilage may significantly impact on cartilage
surrounding a focal repair as well as the repair tissue itself. In a
diseased joint with increased inflammatory cytokines and/or
abnormal biomechanical loading, cells implanted to effect repair of a
defect will have induction of catabolic pathways, and imunostaining
of tissue generated following autologous chondrocyte implantation
(ACI) in humans shows increased MMP-cleavage of collagen and
proteolysis of aggrecan by ADAMTS. There is little/no published
information on the effect of direct “anti-catabolic strategies” (e.g.
proteinase inhibitors, anti-cytokine therapies) on actual cartilage
repair in vivo. As noted above, in only 3 GM mice where OA
degeneration was examined has cartilage regeneration/repair been
described. Following induction of an acute inflammatory arthropathy
in which almost complete and equivalent loss of aggrecan occurs by
day 7 in both wild type mice and those with aggrecan IGD resistant
to ADAMTS or MMP cleavage, subsequent cartilage degeneration or
repair was monitored. We found significant restoration of cartilage
proteoglycan at day 28 only in the Jaffa mouse strain with aggrecan
resistant to ADAMTS cleavage (6). Furthermore, this correlated with
significantly less cartilage fibrillation and structural damage in these
mice compared with the two other strains. MRL/MpJ (“healer”) mice
have a significant inhibition of cartilage proteoglycan loss and
erosion following intra-articular fracture (8). This protection against
post-traumatic OA in MRL/MpJ mice was associated with significantly
lower circulating levels compared with C57BL6 mice of IL-1alpha (but
Extended Abstracts 157
not IL-1beta, TNFalpha or IL-6) both pre- and at 4 and 8 week post-
injury. In a separate study, we found that this same strain of MRL/
MpJ mice had significantly enhanced cartilage regeneration of an
isolated osteochondral defect in the femoral condyle compared with
C57BL6 animals (9). In contrast, there was no difference between
strains in repair, or lack of repair of cartilage injuries that did not
penetrate into the subchondral bone. These two studies support the
hypothesis that factors that inhibit cartilage degeneration in models
of OA may also augment bona fide cartilage repair or regeneration.
In the case of the tenascin C KO mice the corollary was also found to
be true i.e. that a factor (in this case ablation of tenascin C) that
increases cartilage damage in spontaneous (age-related) or
surgically induced OA, also significantly inhibited repair of surgically-
induced isolated osteochondral defects (10). There has often been a
poor overlap between research and publications focused on cartilage
degradation mechanisms in OA and those on cartilage repair. The
above examples highlight that better cross-fertilization between
these two research arms is needed, and further evaluation is
warranted of molecules identified as significantly affecting cartilage
degeneration in models of OA (and even inflammatory arthritis) in
terms of their ability to influence chondral and osteochobdral repair.
In some cases e.g. FGF2 and TGFbeta signaling, there is independent
evidence of a coordinated link between increased cartilage
degeneration in GM mice where these molecules/signaling pathways
have been ablated and beneficial effects on repair of osteochondral
defects in another animal species through administration of the
growth factor. Despite the caveats about use of small animals
described in the opening paragraph, methods to introduce and
evaluate healing of well-controlled osteochondral defects in mice
have recently been reported (11). The current literature survey has
identified at least another 35 GM strains of mice in which a factor
that has a significant effect on OA cartilage degeneration and could
readily be targeted as a therapeutic (i.e. enzyme, cytokine, growth
factor or transcription factor), where cartilage repair/regeneration
should be directly examined. Such studies may provide a significant
and rapid advancement in identifying targets to improve cartilage
repair and tissue engineering.
References:
1. Ahern BJ, Parvizi J, Boston R, Schaer TP. Preclinical animal
models in single site cartilage defect testing: a systematic review.
Osteoarthritis Cartilage 2009;17(6):705-13. 2. Chu CR, Szczodry
M, Bruno S. Animal models for cartilage regeneration and repair.
Tissue Eng Part B Rev 2010;16(1):105-15. 3. Little CB, Fosang AJ.
Is cartilage matrix breakdown an appropriate therapeutic target
in osteoarthritis--insights from studies of aggrecan and collagen
proteolysis? Curr Drug Targets 2010;11(5):561-75. 4. Glasson SS,
Askew R, Sheppard B, Carito B, Blanchet T, Ma HL, et al. Deletion of
active ADAMTS5 prevents cartilage degradation in a murine model of
osteoarthritis. Nature 2005;434(7033):644-8. 5. Stanton H, Rogerson
FM, East CJ, Golub SB, Lawlor KE, Meeker CT, et al. ADAMTS5 is the
major aggrecanase in mouse cartilage in vivo and in vitro. Nature
2005;434(7033):648-52. 6. Little CB, Meeker CT, Golub SB, Lawlor
KE, Farmer PJ, Smith SM, et al. Blocking aggrecanase cleavage in
the aggrecan interglobular domain abrogates cartilage erosion and
promotes cartilage repair. J Clin Invest 2007;117(6):1627-36. 7. Little
CB, Barai A, Burkhardt D, Smith SM, Fosang AJ, Werb Z, et al. Matrix
metalloproteinase 13-deficient mice are resistant to osteoarthritic
cartilage erosion but not chondrocyte hypertrophy or osteophyte
development. Arthritis Rheum 2009;60(12):3723-33. 8. Ward BD,
Furman BD, Huebner JL, Kraus VB, Guilak F, Olson SA. Absence of
posttraumatic arthritis following intraarticular fracture in the MRL/
MpJ mouse. Arthritis Rheum 2008;58(3):744-53. 9. Fitzgerald J, Rich
C, Burkhardt D, Allen J, Herzka AS, Little CB. Evidence for articular
cartilage regeneration in MRL/MpJ mice. Osteoarthritis Cartilage
2008. 10. Okamura N, Hasegawa M, Nakoshi Y, Iino T, Sudo A,
Imanaka-Yoshida K, et al. Deficiency of tenascin-C delays articular
cartilage repair in mice. Osteoarthritis Cartilage 2009. 11. Eltawil NM,
De Bari C, Achan P, Pitzalis C, Dell’accio F. A novel in vivo murine
model of cartilage regeneration. Age and strain-dependent outcome
after joint surface injury. Osteoarthritis Cartilage 2009;17(6):695-704.
158 Extended Abstracts

24.3.3 skeletal maturity, there was no significant difference between the

Skeletal maturity does not affect the repair quality of damaged
articular cartilage using a scaffold-free tissue-engineered
construct (TEC) derived from synovial mesenchymal stem cells
N. Nakamura1, K. Shimomura1, W. Ando1, K. Kita2, H. Fujie3, K.
Nakata4, K. Shino4, H. Yoshikawa4
1
Osaka/Japan, 2Suita, Osaka/Japan, 3Tokyo/Japan, 4Suita/Japan
Introduction: It is widely accepted that chondral injury does not
usually heal spontaneously due to its avascular surroundings and
unique matrix organization. Therefore, a variety of approaches have
been assessed to improve cartilage healing. Among them, stem
cell therapies have been tested to facilitate regenerative cartilage
repair. Mesenchymal stem cells (MSCs) have the capability to
differentiate into a variety of connective tissue cells including bone,
cartilage, tendon, muscle, and adipose tissue. These cells may be
isolated from various tissues such as bone marrow, skeletal muscle,
synovial membrane, adipose tissue, and umbilical cord blood. MSCs
isolated from synovial membrane may be well suited for cell-based
therapies for cartilage based on the relative ease of their harvest
and their strong capability of chondrogenic differentiation. Recent
implantation studies have reported successful cartilage repair using
synovial membrane-derived MSCs.　One of the crucial factors that
may affect the results of cell-based therapies is the age of the donors
and recipients. Specifically, the results of implantation surgery could
be potentially affected by both the characteristics of the cells and the
local environment in the lesions following implantation. Therefore, it
is important to elucidate the influence of skeletal maturity on these
two factors. Regarding the cell proliferation and differentiation
capacities of MSCs, it is controversial as to whether they are age-
dependent or not. In terms of the host tissue reaction, natural
healing responses of osteochondral defects has been compared
between immature and mature animals using rabbit models, and in
this species the studies demonstrated better healing responses in
immature animals. On the other hand, there have been no studies
which compared the results of cell-based repair of chondral defects
between immature and mature animal models.
As a potential MSC-based therapeutic method, we have developed
a scaffold-free three dimensional tissue-engineered construct (TEC)
composed of allogenic mesenchymal stem cells (MSCs) derived
from the synovium and extracellular matrices (ECMs) synthesized
by the cells, and demonstrated the feasibility of the resultant TEC
to facilitate cartilage repair in a large animal study. In the present
study, we compared the results of TEC implantation to the chondral
lesions morphologically and biomechanically in order to elucidate
the influence of the skeletally maturity on subsequent cartilage
repair with the TEC.
modulus of normal cartilage and that of repaired tissue treated with
TEC at either 4μm/s or 100μm/s.
Discussion:
Previous study showed that in vitro expandability and differentiation
capacity of human synovial mesenchymal stem cells do not depend on
donor ages. While, natural healing response of rabbit osteochondral
defect was better in immature model than in mature model.
Therefore, although there has been no study demonstrating the
age dependent healing response of cartilage in large animal model,
skeletal maturity might likewise affect the repair and differentiation
process following the implantation of the TEC. The results of the
present study showed the TEC implantation effectively contributed
to chondrogenic repair of chondral defect with similar viscoelastic
properties to normal cartilage. Notably, such repair response was
equivalently observed in both immature and mature pigs. This
study could be the first demonstration of the comparison of healing
response of cartilage between skeletally mature and immature large
animals. It is notable that the allogenic stem cell-based approach
was proved to be feasible to cartilage repair regardless of the skeletal
maturity. The use of allogenic stem cells could be advantageous
in time- and costsaving without tissue sacrifice of host tissue in
comparison with autologous cell-based approach and therefore the
results of the present study would support the clinical application of
this strategy to promote cartilage repair and regeneration.
References:
1. Ando W, Tateishi K, Katakai D, Hart DA, Higuchi C, Nakata K, et al. In
vitro generation of a scaffold-free tissue-engineered construct (TEC)
derived from human synovial mesenchymal stem cells: biological
and mechanical properties and further chondrogenic potential.
Tissue Eng Part A 2008 Dec;14(12):2041-2049.
2. Ando W, Tateishi K, Hart DA, Katakai D, Tanaka Y, Nakata K, et
al. Cartilage repair using an in vitro generated scaffold-free tissue-
engineered construct derived from porcine synovial mesenchymal
stem cells. Biomaterials 2007 Dec;28(36):5462-5470.
Acknowledgments:
This study was supported by a grant from the New Energy and
Industrial Technology Development Organization, Japan, and Grant-
in-Aid for Scientific Research (B), Japan Society for the promotion of
Science, Japan.

Content: Cell proliferation and chondrogenic differentiation capacity

of immature and mature porcine synovial MSCs
Cell number assessments, as well as WST-1 assays demonstrated
that there were no significant differences in the proliferation capacity
of porcine synovial MSCs derived from immature or mature animals.
In terms of chondrogenic differentiation potential, there were no
significant differences in expression of collagen II detected by RT-
PCR, in Alcian blue staining of the cell pellets, or in GAG synthesis
levels. These results suggested that maturity may not significantly
affect the chondrogenic differentiation capacity of porcine synovial
MSCs.
Histological assessment of the repaired tissue: Regardless of
skeletal maturity, the chondral defect of in the control group
showed osteoarthritic change with loss of cartilage and destruction
of subchondral bone. Conversely, TEC-treated defect was repaired
by chondrogenic tissue with positive Safranin O staining in both
immature and mature models. Higher magnification view showed
that there was good tissue integration to the adjacent cartilage
obtained and the defect treated with the TEC was repaired with
hyaline-like cartilage in both models. In histological scoring, the TEC
group had significantly higher scores than the control group in all the
criteria categories in the immature model. In the mature model, the
TEC group had significantly higher scores than the control group in
all the categories but the category “Matrix” and “Cell distribution”.
Mechanical properties of the repaired tissue: Articular cartilage is
a biphasic viscoelastic material that exhibits strain-rate-dependent
mechanical behaviors. This means that the viscoelasticity of
cartilage which retains interstitial water might be mainly reflected
by faster compression test (@100μm/s) outcomes, while the matrix
viscoelasticity without interstitial water should be mainly reflected
by slower compression test (@4μm/s) outcomes. Regardless of

9.1.2
Predictors of Osteochondral Allograft Failure
W. Bugbee, A. DeYoung, S. Görtz
La Jolla/United States of America
Purpose: To determine risk factors for clinical failure of osteochon-
dral allografting.
Methods and Materials: Between 1983 and 2009, 527 osteochon-
dral allografting procedures of the knee were performed in 467 pa-
tients. Cases were prospectively entered into a clinical database,
including patient demographics, diagnosis, graft characteristics,
clinical outcome, and reoperations. A logistic regression model was
used to determine variables predicting failure of the osteochondral
allograft. Failure was defined as removal (i.e., conversion to knee
arthroplasty) or revision of the allograft. The study included 279
males (59.7%) and 188 females (40.3%) with a mean age of 33.6
years (range, 14–68 years). Primary diagnoses included traumatic
cartilage injury (35.1%), osteochondritis dissecans (29.8%), degen-
erative cartilage lesions (12.0%), osteonecrosis (7.8%), failed al-
lograft (5.7%), and osteochondral fracture (3.2%). Anatomic graft
locations included medial femoral condyle (26.8%), lateral femoral
condyle (20.1%), patella (5.7%), trochlea (4.2%), lateral tibial pla-
teau (4.7%) and medial tibial plateau (0.9%). 28.5% of cases in-
volved multiple grafts to two distinct anatomic locations and 9.1%
of cases involved grafting of three separate surfaces. Average total
graft area was 9.7 cm2 (range, 1.2–57.5 cm2).
Results: Sixty-five knees (15.3%) were reoperated for clinical fail-
ure at a mean of 38.1 months (range, 3–107 months). Forty-four
were converted to arthroplasty and 21 underwent repeat allograft-
ing. Gender, age, total graft area, and number of grafts were signifi-
cant (p<0.05) risk factors for failure. Females were 2.2 times more
likely to fail than males. Compared to patients <30 years, those
who were 30–40 were 5.1 times more likely to fail; those >40 were
6.4 times more likely to fail. Grafts >10 cm2 were more likely to fail
than grafts <5 cm2 (OR=5.1) and knees with two grafts were more
likely to fail than knees with only one graft (OR=58.7).
Conclusions: Understanding the impact of clinical variables on
outcome of osteochondral allografting is useful in clinical decision
making.
9.1.3
The Optimal Temperature for the Long Term ex-Vivo Cartilage
Storage: 11°C is Superior to 4°C, 23°C, and 35°C
A. Alibegovic, R. Blagus, M. Drobni
Ljubljana/Slovenia
Purpose: Several studies confirmed prolonged ex-vivo viability of
human chondrocytes under various environmental conditions. The
longest recorded follow-up of 2 months showed 38% viable cells at
4°C. The objective of presented study was to determine the longest
surviving period of chondrocytes in ex-vivo explants at four ambi-
ent temperatures.
Methods and Materials: Osteochondral cylinders were procured
bilaterally from femoral condyles from 3 male donors (19, 21, and
33 years) within 24 hours after death. They were stored in the cell
culture media (DMEM/F12) parallel at 4°C, 11°C, 23°C, and 35°C.
The middle chondral zone was first stained with Live/Dead Viabil-
ity/Cytotoxicity Kit and analyzed for cell viability by the confocal
laser scanning microscope. Initial analysis was performed 24-36
hours after death, and then repeatedly every 3 days for each tem-
perature until all cells died. Analysis at the individual temperature
level was discontinued after three consecutive viability analyses
failed to detect surviving cells.
Results: The detection of viable chondrocytes was lost after do-
nor’s death as following: 35°C on day 28, 23°C on day 40, 4°C on
day 76, and 11°C on day 100. The statistical analysis with a two
way repeated measures ANOVA performed until day 28 revealed
significant time and ambient temperature dependent chondrocyte
viability. At 28 days were the viabilities following: 20.20% at 35°C,
87.03% at 23°C, 91.38% at 11°C, and 68.90% at 4°C.
Conclusions: The presented study re-confirmed that chondrocyte
viability is dependent of ambient temperature and time of cartilage
ex-vivo storage. Moreover, its results demonstrated the cell sur-
vival up to 100 days, which is much longer as previously known.
A superior survival times at 11°C in comparison to the other three
tested ambient temperatures suggest, that this temperature level
might better preserve osteochondral allografts in tissue banking.
Free Papers 159
9.1.4
Long-Term Survival of Concurrent Meniscus Allograft
Transplantation and Articular Cartilage Repair: A Prospective 2-
to 12-Year Follow-Up Evaluation
K.R. Stone, W. Adelson, J.R. Pelsis, A. Walgenbach, T. Turek
San Francisco/United States of America
Purpose: The purpose of the study was to evaluate the effective-
ness of combined meniscus allograft transplantation and articular
cartilage repair, in patients with meniscus-deficient knees and se-
vere articular cartilage damage, over the course of long-term follow
up (2 - 12 yrs).
Methods and Materials: Clinical examination and subjective pa-
tient outcome assessments were conducted pre-operatively and at
2, 3, 5, 7, and 10-year post-operative intervals (N = 119). Procedure
survival was determined using Kaplan-Meier survival analysis. Cox
proportional hazards model was used to assess the effect of covari-
ates on survival. Secondary analysis of validated subjective patient
outcomes was accomplished using the Wilcoxon rank-sum test.
Results: Average follow-up was 5.80 years (2.1 months - 12.3 years).
25 procedures failed (20.1%) with a mean failure time of 4.65 ± 2.99
years (2.1 months -10.37 years). Age at time of surgery and number
of previous surgeries were significant covariates. Procedure sur-
vival was not affected by sex, severity of cartilage damage, axial
alignment, degree of joint space narrowing, or medial vs lateral
allograft. Cox proportional hazards model showed significant fac-
tors on relative odds of allograft failure were patient age at time
of meniscus allograft (h=1.061;p=0.006) and number of previous
surgeries to the affected knee (h=1.528;p=0.026). Patients expe-
rienced significant improvements in subjective outcome measures
over the course of follow-up (p < 0.05), with exception the 7-year
Tegner Index score.
Conclusions: This study demonstrates that meniscus allograft
transplantation in combination with articular cartilage repair is ef-
fective in patients with severe articular cartilage damage. Outcome
was not affected by the classic contraindications of age, severity of
arthritis, joint space narrowing, and axial alignment. Biologic joint
reconstruction may be an appropriate first step for many people
with severe articular cartilage damage.
9.1.5
Immunosuppressed versus non immunosuppressed bipolar fresh
osteochondral allograft in the treatment of end stage ankle
arthritis: a clinical and histological comparison
F. Vannini, R. Buda, M. Cavallo, A. Ruffilli, R. Ghermandi, B. Grigolo,
S. Neri, S. Giannini
Bologna/Italy
Purpose: Post-traumatic ankle arthritis in young patients rep-
resents a challenge. Fresh bipolar shell osteochondral allograft
(FBOA) is a treatment option, nevertheless radiographic arthritis
occurrence at follow-up is cause of concern and may be correlated
to an immunoreactive response to transplanted cartilage. Aim of
the study is to compare two groups of patients who received FBOA
in association or not to immunosuppression.
Methods and Materials: 2 groups of 16 patients each underwent
FBOA. Only one group (group-B) received an immunosuppressive
therapy. Patients evaluation was carried out clinically by AOFAS,
radiographically by X-Rays, CT scans and MRI up to 2 years and a II
look with biopsy was performed at 1 year follow-up. Samples were
examined histochemically by Safranin-O staining and immunohis-
tochemically by collagen type I, II and caspase-3 evaluations. The
evaluation of the transplanted tissues was performed following the
ICRS II scoring system.
Results: In group-A pre-operative AOFAS score was 26.2 ± 8.2, while
at 24 months was 70.6 ± 17.9 (p<0.005). In group-B pre-operative
AOFAS score was 27.3 ± 6.9 and 72.7 ± 8.5 (p<0.005) at follow-
up. No statistically significant difference in the clinical outcome be-
tween the 2 groups was found. A better morphology of the grafts
was observed in group-B which presented an higher score (mean
~68%) compared to the other (mean 40%)(p<0.05) . In group-B,
cartilage samples showed a regular surface, oval chondrocytes sur-
rounded by metachromatic matrix in the middle and deeper layers,
integration to subchondral bone, moderate positivity for collagen
type II and negativity for caspase-3. Group-A presented instead car-
tilage with low proteoglycan content and hypocellularity.
Conclusions: Although clinical results were comparable in the two
groups, histological results showed that samples from immuno-
suppressed group possess morphological and biochemical charac-
teristics more similar to hyaline articular cartilage compared to the
other group. Further studies are needed to confirm these data.
160 Free Papers
9.1.6
Isolated Osteochondral Allografting of the Femoral Trochlea
W. Bugbee, A. DeYoung, S. Görtz
La Jolla/United States of America
Purpose: Cartilage disease of the patellofemoral joint represents a
difficult clinical challenge. Although osteochondral allografting has
been shown to be effective in the treatment of chondral and osteo-
chondral lesions of the femoral condyle and tibial plateau, there
are few clinical studies on osteochondral allografting of the patel-
lofemoral joint. In this study, we report on the outcome of isolated
osteochondral allografting of the trochlea.
Methods and Materials: Twenty patients (20 knees) underwent
isolated osteochondral allografting (OCA) of the trochlea. Two
were lost to follow-up and the remaining 18 patients (18 knees)
comprise the current study population. The mean age was 31 years
(range, 14–46 years) and 67% were male. 94% had previous sur-
gery (range, 1–13 procedures), including four patients who had sur-
gery for realignment of the extensor mechanism. Major preopera-
tive diagnoses were traumatic chondral injury and osteochondritis
dissecans. Mean allograft area was 6.6 cm2 (range, 2.3–20.0 cm2).
Results: One patient (6%) failed and was converted to a total knee
arthroplasty at 92 months. 11% had further surgery (arthroscopy)
with retention of the allograft. Mean follow-up of the non-failures
was 59 months (range, 26–195 months). Mean IKDC pain scores
improved from 5.7 to 3.4 postoperatively (p= 0.06). Mean IKDC
function scores improved from 3.6 to 7.0 (p< 0.01). 88% reported
less pain and 94% reported better function. 88% reported improve-
ment, were satisfied with their results, and would choose allograft
surgery again under similar circumstances.
Conclusions: Isolated osteochondral allografting of the femoral tro-
chlea resulted in significant functional improvement and less pain.
The majority of patients were satisfied with their treatment. Failure
and reoperation rates were low. We conclude that osteochondral
allografting is a useful procedure for treatment of chondral and os-
teochondral lesions of the femoral trochlea.
9.1.7
Particulated juvenile cartilage allograft transplantation for repair
of high grade chondral lesions
J. Farr1, J. Kercher2, M. Salata2, J. Yao3
1
Indianapolis/United States of America, 2Chicago/United States of
America, 3Austin/United States of America
Purpose: As articular cartilage defects do not spontaneously heal,
multiple techniques are used to restore lesions. Techniques are
typically based on classical treatments such as marrow stimula-
tion, osteochondral grafting, and ACI. This study presents the pre-
liminary results of a new repair technique that utilizes particulated
juvenile cartilage allograft transplantation.
Methods and Materials: 48 patients with ICRS grade 3a,b,c,d or
4a focal chondral defects of the knee were treated with particulated
juvenile cartilage allograft transplantation by a single surgeon. These
lesions were debrided to a stable rim and the calcified cartilage layer
was removed without violating the subchondral bone (in those with
grade 3 lesions). The particulated juvenile cartilage (DeNovoNT from
IsTo [St. Louis, MO USA distributed by Zimmer, Inc-Warsaw, IN USA])
was placed in a monolayer and secured with fibrin glue. For larger le-
sions, the cartilage fragments were protected with a BioGide scaffold
(Geistlich Surgery, Switzerland). A standardized ACI rehabilitation
protocol was utilized. Visual analog scale (VAS) and British Satisfac-
tion (BS) scores were used to evaluate outcomes.
Results: 41/48 patients (85%) were available for follow-up at an
average of 20 months (range 12-35mos). There were 19 right knees
and 22 left knees in the cohort. There were 28 females and 13 males
with a mean age of 36 years (range 17-50). A 1-3 bilayer collagen
patch augmentation was performed in 39/41 knees. 18/41 patients
underwent a concomitant bony realignment procedure. 28 knees
contained unipolar lesions and 13 knees contained multiple lesions.
Average VAS resting scores improved from 2.1 to 0.8 and max pain
scores improved from 6.3 to 4.3. The average BS score improved
from 2.1 to 3.3 post-operatively.
Conclusions: The preliminary results of particulated juvenile carti-
lage allograft transplantation for the treatment of high grade chon-
dral lesions appear to be promising in the short-term and compares
favorably to the currently established methods of chondral restoration.
9.1.8
Chondral defect repair with particulated juvenile cartilage
allograft
J. Farr1, J. Yao2
1
Indianapolis/United States of America, 2Austin/United States of
America
Purpose: This clinical study was conducted to evaluate a cartilage
repair procedure of transplanting particulated juvenile cartilage
tissue allograft.
Methods and Materials: A multi-center, prospective, single arm,
twenty-five-subject clinical study was designed to evaluate clinical
outcomes such as IKDC and KOOS scores as well as extent and qual-
ity of repair with MRI and optional biopsies at various time points
post-implantation (up to five years). Currently, 19 patients with 1 or
2 chondral lesions on the femoral condyles and trochlea have been
enrolled and treated in the prospective study.
Results: The first group of nine patients has been evaluated with up
to 18 months post-op follow-up. No revision has been performed to
date. Significant improvements in clinical outcomes over the pre-
op baseline data have been observed. MRI data indicated good de-
fect filling. No optional biopsy has been obtained to date.
Conclusions: The present report describes, for the first time, clini-
cal data of a cartilage repair procedure of transplanting particu-
lated juvenile cartilage tissue allograft to treat cartilage lesions
of the femoral condyles and/or trochlea. Clinical outcome data as
measured at up to 18 months post-implantation and MRI data in-
dicate early positive outcomes and suggest that this technique is
capable of filling chondral defects and improving symptoms. Con-
tinued clinical evaluation of this technique is needed with longer
follow-up durations.
9.1.9
Skeletal maturity does not affect the healing response of
articular cartilage using scaffold-free tissue-engineered
construct (TEC) derived from allogenic synovial mesenchymal
stem cells. -A large animal study-
K. Shimomura1, W. Ando1, K. Tateishi1, R. Nansai2, H. Fujie2, D.A.
Hart3, T. Kanamoto1, T. Mae1, K. Nakata1, K. Shino1, H. Yoshikawa1,
N. Nakamura4
1
Suita/Japan, 2Tokyo/Japan, 3Calgary/Canada, 4Osaka/Japan
Purpose: One of the crucial factors that may affect the results of
mesenchymal stem cell (MSC)-based therapies is the age of donors
and recipients. However, no basic or clinical research has addressed
this point in detail. Specifically, there have been no controlled stud-
ies which compared the results of MSC-based repair of cartilage be-
tween skeletally immature and mature animal models, particularly
in a large animal model. As a MSC-based therapeutic method, we
previously developed a scaffold-free three dimensional tissue-en-
gineered construct (TEC) composed of synovial membrane-derived
MSCs from immature animals and demonstrated the feasibility of
such TEC to facilitate cartilage repair using similarly aged skeletally
immature recipients in a chondral defect model.
Methods and Materials: Synovial membrane-derived MSCs were
isolated from immature (3-4 months of age) and mature (>12
months of age) domestic pigs. The proliferation and chondrogenic
differentiation capacity of these MSCs were compared. The TEC
from these MSCs were implanted into equivalent chondral defects
in the medial femoral condyle of both immature and mature pigs,
respectively. The results of TEC implantation were morphologically
and biomechanically evaluated at 6 months postoperatively.
Results: Cell proliferation and chondrogenic differentiation capac-
ity showed no significant differences between the MSCs from imma-
ture and mature pigs. Histologically, the TEC implantation effective-
ly contributed to chondrogenic repair of both immature and mature
pigs with good tissue integration to the adjacent cartilage (Fig.1).
The resultant repair tissue exhibited similar viscoelastic properties
to normal cartilage in both immature and mature recipients (Fig.2),
with no evidence of immune reaction to the allogenic TEC implants.
Conclusions: The present study is the first demonstration of the
direct comparison of cartilage repair responses mediated by a cell-
based therapy between skeletally mature and immature large ani-
mals. These findings suggest the use of immature pigs to test the
feasibility of cell-based therapies in chondral lesions has validity.

9.2.2
Chondrocyte gene expression is affected by VSOP-labeling in
long-term in vitro MRI tracking
C.B. Foldager, M. Pedersen, S. Ringgard, C. Bünger, M. Lind
Aarhus/Denmark
Purpose: Autologous chondrocyte transplantation is an essential
part of many treatment procedures for cartilage repair. The aim was
to investigate the effect and dose-response of very-small iron ox-
ide particle (VSOP) labeling of human chondrocytes for long-term
in vitro MRI tracking.
Methods and Materials: Chondrocytes were isolated from cartilage
biopsies from four patients. The cells for the dose-response study
were labeled with 25, 50 or 100 µg/mL VSOP. Quantitative gene
expression and cellular proliferation were compared to unlabeled
controls at day 1, 3, and 7. The cells suited for MRI tracking were
labeled with 50 µg/mL VSOP and embedded in alginate beads,
followed by MRI (using T2-weighted sequences) at day 0, 1, 3, 7, 14,
21, 28, and histology was performed at each time-point.
Results: Histology revealed that VSOP particles were intracellularly
confined at all time-points, whereas no extracellular VSOP™s were
observed. A mean reduction in T2-value of 25.1 ms (±SD 3.5 ms) was
found on T2-maps. The chondrocyte-specific genes aggrecan, colla-
gen type 2, and sox9 were all affected by labeling, the two latter in a
dose-dependent manner. VSOP™s had no effect on proliferation.
Conclusions: VSOP-labeling of chondrocytes affected gene expres-
sion but not proliferation. The labeled chondrocytes could be rec-
ognized by MRI for 4 weeks without significant changes in the T2
relaxation time.
9.2.3
Modelling of tissue formation in chondrocyte filter cultures
C.J. Catt1, W. Schuurman2, B. Sengers1, C.P. Please1, W.J.A. Dhert2,
J. Malda2
1
Southampton/United Kingdom, 2Utrecht/Netherlands
Purpose: The filter culture is a frequently used three-dimensional
(re-)differentiation model to investigate chondrocyte behavior in
cartilage research. In this model, expanded chondrocytes are redif-
ferentiated on collagen coated filters to form a cartilaginous tissue
over time. The understanding of the governing processes involved
in the formation of such a cartilaginous multi-layered structure is
limited. Therefore, this study aims to provide further insight into
these processes by the means of an applied mathematical approach.
Methods and Materials: Filter cultures were generated using ex-
panded adult equine chondrocytes and maintained for up to seven
weeks. Samples were processed for histology and image analysis
was performed. In addition, glycosaminoglycan (GAG) and DNA con-
tent were determined biochemically. A mathematical model was then
constructed based on the analysed data. This consisted of partial
differential equations explaining the distribution of cells and GAG,
as well as the transport of nutrients and overall height of the tissue.
Results: The generated tissue in the filter cultures had a glassy hya-
line appearance and tissue height increased at a constant rate over
a seven week culture period. GAG content of the cultures increased
uniformly, although considerable release of GAG was observed. Af-
ter an initial settling period for cells, growth was restricted to a re-
gion in the upper half of the tissue whilst the density of cells on the
filter membrane and top surface remained constant (Figure 1). The
mathematical model showed that cell behaviour and consequently
growth, was restricted to the concentration of nutrients within a
cell’s local environment.
Conclusions: Redifferentiation of chondrocytes in the filter cul-
ture system resulted in the formation of cartilaginous tissue. The
developed mathematical model could predict the experimental
observations and revealed that the cell’s behaviour is dependent
on its position within the tissue structure, as well as on the local
nutrient concentration.
Free Papers 161
9.2.4
A novel 3D tissue assembly approach for cartilage tissue
engineering applications
S. Stroebel1, J. Malda2, T.B.F. Woodfield1
1
Christchurch/New Zealand, 2Utrecht/Netherlands
Purpose: Cell-scaffold based cartilage tissue engineering strategies
provide the potential to restore long-term function of damaged articu-
lar cartilage. A major hurdle in such strategies is the adequate (uniform
and sufficient) population of porous 3D scaffolds with cells. We de-
scribe a novel approach to engineer cartilage grafts using pre-defined
micro-mass cartilage pellets, integrated into specifically designed 3D
plotted scaffolds.
Methods and Materials: Expanded (P2) human nasal chondrocytes
(HNCs) from 3 donors (age 47-62 years) were micro-mass cell pellet cul-
tivated at 5x105 cells/pellet for 4 days. Subsequently, micro-mass pel-
lets were integrated into 3D plotted poly(ethylene glycol)-terephtha-
late/poly(butylene)-terephthalate (PEGT/PBT) scaffolds with 1mm fibre
spacing. Constructs were cultured dynamically in spinner flasks for a to-
tal of 21 days. As a pellet-free control, expanded HNCs were spinner flask
seeded into PEGT/PBT fibre plotted scaffolds. Constructs were assessed
via histology (Safranin-O staining), biochemistry (glycosaminoglycan
(GAG) and DNA content) and collagen type I and II mRNA expression.
Results: After 4 days, micro-mass cultured pellets could be success-
fully integrated into the fibre plotted scaffolds. DNA content of pellet
integrated constructs was 4.0-fold±1.3 higher compared to single seed-
ed constructs. At day 21, cartilage tissue was uniformly distributed
throughout pellet integrated scaffolds, with minimal cell necrosis ob-
served within the core. GAG/DNA and collagen type II mRNA expression
were significantly higher (2.5-fold±0.5 and 3.1-fold±0.4 respectively) in
pellet versus single cell seeded constructs. Furthermore, compared to
single cell, the pellet seeded constructs contained significantly more
total GAG and DNA (1.6-fold±0.1 and 3.1-fold±1.0 respectively).
Conclusions: We developed a novel 3D tissue assembly approach for
cartilage tissue engineering which significantly improved the seeding
efficiency, as well as tissue uniformity and integrity compared to more
traditional seeding approaches using single cell suspensions. Further-
more, the integration of micro-mass cell pellets into 3D plotted PEGT/
PBT scaffolds significantly improved the amount and quality of tissue
engineered cartilage.
9.2.5
Stem cell surface marker SSEA-4 levels correlate with enhanced
chondrogenic potential of cultured human osteoarthritic chondrocytes
K. Schrobback1, T.B.F. Woodfield2, R. Crawford1, D.W. Hutmacher1, T.
Klein1
1
Brisbane/Australia, 2Christchurch/New Zealand
Purpose: One important challenge for cartilage tissue engineering is to
produce a clinically relevant number of cells with consistent chondrogenic
potential. In vitro expansion of autologous chondrocytes results in a het-
erogeneous population of dedifferentiated cells and variable amounts of
chondroprogenitors. Identification and isolation of chondroprogenitors
could lead to more consistent cartilage formation. Unfortunately, reliable
chondroprogenitor markers have not been identified. Therefore, we eval-
uated SSEA-4, a marker of embryonic and mesenchymal stem cells, as a
potential chondroprogenitor marker in cultured human chondrocytes.
Methods and Materials: Osteoarthritic cartilage was obtained from
four consenting patients (age >60yrs.) undergoing knee replace-
ment. Chondrocytes were isolated from different sites of the joint
(macroscopically normal [ICRS grade 0-1] and degenerated [grade
2-4]) and zones (“normal” superficial and middle/deep) and ex-
panded in monolayers. Immunophenotype was determined by flow
cytometry at passage 3. Propagated cells from two donors were red-
ifferentiated in pellet cultures over four weeks. Glycosaminoglycan
(GAG) and DNA contents were quantified biochemically and expres-
sions of aggrecan, collagen II, I and X were determined by qRT-PCR.
Results: Flow cytometry revealed heterogeneous expression of SSEA-
4 in CD44+/CD45-/CD105+ chondrocyte populations (A). We observed
no differences in surface marker expressions between cells from dif-
ferent sites (B). However, the percentage of SSEA-4-positive cells after
expansion correlated positively with expression levels of chondrogenic
markers aggrecan and collagen type II at day 7, and GAG/DNA levels
at day 28 of pellet culture. Further, SSEA-4 levels were inversely cor-
related with expression of hypertrophic marker collagen type X (C).
Conclusions: We report for the first time that subpopulations of hu-
man osteoarthritic chondrocytes express the stem cell surface antigen
SSEA-4 during in vitro propagation. Our observations suggest that ex-
pression levels of SSEA-4 can be predictive of the cell’s chondrogenic
differentiation capacity. Ongoing research is focussed on the cellular
characterisation of SSEA-4-positive cells to confirm their superior
chondrogenic potential in vitro and in vivo.
162 Free Papers
9.2.6
Polyglycan promotes the intrinsic damage-repair response of
chondrocytes in equine articular cartilage explants.
A. Getgood1, L. Barr1, D. Caborn2, N. Rushton1, F.M. Henson1
1
Cambridge/United Kingdom, 2Louisville/United States of America
Purpose: The aim of this study was to investigate the effect of polyg-
lycan (PG) (hyaluronic acid, N-acetyl glucosamine, chondroitin sul-
phate solution) on the intrinsic damage-repair (IDR) response in ar-
ticular cartilage explants following single impact load (SIL) in-vitro.
Methods and Materials: Equine articular cartilage explants, with-
out subchondral bone, had a SIL of 500g applied from a height of
2.5cm. Explants were then cultured in DMEM in the presence of
10%, 5%, 2%, 1% and 0% PG compared to 50ng/ml FGF-2 as a posi-
tive control for up to 28 days. Unimpacted discs served as negative
controls. Histological and immunohistochemical techniques were
used to quantify and characterise the chondrocyte response to
damage and to the effect of each of the treatments.
Results: Evidence of an IDR was seen in all impacted discs (Fig.1).
This was significantly upregulated by 1% PG compared to FGF-2
and untreated controls, particularly by day 28 (p<0.05); large
amounts of chondrocyte repair cells noted on the cartilage surface
(Fig.2). Within the cartilage both type II collagen and pericellular
type VI collagen was seen to by upregulated following SIL, and
maintained with the addition of 1% PG. The most striking finding
was a significant reduction in dead cells (as detected using TUNEL
staining) with 1% PG (15%) compared to the SIL control group (50%)
(p<0.05). This was not seen in the higher concentrations of PG.
Conclusions: This study again confirms the potential ability for
chondrocytes to migrate to an area of injury and mount an IDR fol-
lowing SIL. The addition of 1% polyglycan has been shown to upreg-
ulate the IDR, to an even greater degree than FGF-2 and to maintain
type II and VI collagen within the cartilage. The addition of polygly-
can to synovial joints following impact injury may have protective
effects by stabilising the extracellular matrix and preventing chon-
drocyte apoptosis thereby preventing early degenerative change.
9.2.7
Redifferentiation in Hyaff scaffolds affects the chondrogenic
potential in vivo
C. Brantsing1, H. Stenhamre1, S. Concaro1, M. Brittberg2, A. Lindahl1
1
Göteborg/Sweden, 2Kungsbacka/Sweden
Purpose: It has been shown that the second generation of autolo-
gous chondrocyte implantation (ACI) has great potential in order
to regenerate cartilage injuries. The purpose of this study was to
evaluate if the degree of redifferentiation of in vitro cultured scaf-
folds could have an impact on neocartilage in vivo in a mice model.
Further, the signaling pattern occurring during the in vitro chondro-
genesis was studied as well as potential chondrogenic markers.
Methods and Materials: Adult human chondrocytes were seeded
into Hyaff scaffolds and cultured for 1, 7, 14 and 21 days in chondro-
genic media and then implanted subcutaneously in Balb C nu/nu
mice model. The constructs were further assessed by histological,
immunohistochemical and molecular methods
Results: The in vitro culture revealed that longer culture time in
redifferentiation culture gave a more mature tissue. The gene ex-
pression during the in vitro showed an uppreglation of Collagen IIA,
Collagen IIB, Aggrecan, Sox 9, SerpinaA3 and COMP. Whereas Col-
lagen I, Versican, Cathepsin B and EGR1 were down regulated. The
markers Tenacin C and Fibronectin increased the first days of cul-
ture and were then down regulated during the further maturation
of the chondrocytes. The in vivo experiments reveal that in vitro
redifferentitaion is needed for further maturation.
Conclusions: The results indicate that a redifferentiation of in vitro
expanded human articular chondrocytes is required to obtain good
quality redifferentiation in vivo. Furthermore the results show that
the chondrocytes cultured in Hyaff scaffolds express a signaling
pattern similar to the one seen during the chondrogenesis during
fetal development. Furthermore, the results might indicate that
Cathepsin B, EGR1 and SerpinA3 are potential markers to monitor
the differentiation status of 3D tissue engineered constructs.
9.2.8
The tumor gene survivin is expressed in primary human
chondrocytes – a novel mechanism of cartilage protection and
regeneration?
P. Lechler1, S. Doukas1, T. Renkawitz1, S. Anders1, J. Grifka1, S.
Balakrishnan2, R.H. Straub3, J. Schaumburger1
1
Bad Abbach/Germany, 2London/United Kingdom, 3Regensburg/
Germany
Purpose: The smallest member of the inhibitor of apoptosis (IAP)
gene family survivin is considered to be of critical importance for
regulation of mitosis and maintenance of cell viability. As such it is
essential for embryonic development and highly relevant in cancer.
Little knowledge exists whether survivin has any physiological or
pathophysiological role in adult differentiated tissues.
Methods and Materials: Survivin expression in primary human
chondrocyte cultures was studied by immunoblotting and real-time
PCR. Subcellular localization was visualized by immunofluorescence.
Knock-down and over-expression experiments were performed to
study survivin function, i.e. cell cycle and apoptosis assays.
Results: Survivin mRNA and protein is expressed in primary human
chondrocytes. Subcellular localization of survivin protein during
interphase showed a predominantly cytoplasmic signal, while in-
volved in the formation of the spindle apparatus during mitosis. RNA-
interference targeting survivin resulted in a G2/M arrest of the cell
cycle and increased the rate of apoptosis in cartilage cultures. Over-
expression of wild-type survivin in primary human chondrocytes in-
creased rates of proliferation and cell viability. During subculturing
of primary chondrocytes, growth stimulatory effects of TGF-beta in
early passages were associated with an up-regulation of survivin.
Conclusions: In summary, we demonstrate for the first time that
survivin is expressed in primary human chondrocytes on mRNA
and protein level. Functional analyzes indicate that survivin is an
important factor for cell division and cell viability in primary chon-
drocytes. In contrast to the reported biologic effect of TGF-beta sig-
naling on malignant cells, in primary human chondrocytes TGF-beta
treatment resulted in a survivin dependent stimulation of prolifera-
tion. Modulation of survivin gene expression should be carefully
evaluated for interventional strategies in cartilage protection and
tissue regeneration.
9.2.9
Beta-1 integrins are crucial for structural organization of the
articular cartilage but dispensable for chondrocyte metabolism.
A. Raducanu1, M. Stolz2, I. Drosse3, E. Hunziker4 , A. Aszodi1
1
Martinsried/Germany, 2Southampton/United Kingdom, 3München/
Germany, 4Bern/Switzerland
Purpose: To explore the role of beta-1 integrins in articular cartilage
(AC) and joint function, we generated mice in which the floxed beta-1
integrin gene was ablated in early limb bud mesenchymal cells using
the Prx1cre transgene.
Methods and Materials: AC of mutant and control mice was exam-
ined by brightfield, confocal and electron microscopy. Chondrocyte
apoptosis and proliferation were evaluated by TUNEL assay and PCNA
immunohistochemistry (IHC), respectively. The expression of matrix
proteins, proteases, degradation products and signaling molecules
were investigated by IHC and/or ELISA assays. Surface topograpy and
AC mechanical properties were assessed by indentation-type atomic
force microscopy (IT-AFM).
Results: The mutant AC was structurally disorganized accompanied
by defects in chondrocyte proliferation, survival and terminal differ-
entiation. Electron micrographs revealed disorganized layers of col-
lagen fibrils in the superficial zone. IT-AFM showed thickened fibrils,
surface roughening and decreased nanostiffness of mutant samples.
No significant differences in cartilage degradation, in the expression of
proteases or in the exposure of matrix degradation neoepitopes were
observed between genotypes. We found no evidence for disturbed
activation of MAP kinases in beta-1-null chondrocytes. Mutant mice
exhibited abnormal gait, reduced flexibility of the knee joints and de-
creased mobility. We frequently observed chondrification of the syn-
ovium and the large vessels. A mouse line with incomplete deletion
of the beta-1 integrin gene exhibited normal motility and joint usage.
The AC of these mice showed mild structural disorganization, soft-
ening and increased destruction compared to the AC of control mice.
Conclusions: Our study indicates that 1) the lack of beta-1 integrins on
AC chondrocytes has no obvious impact on cartilage homeostasis;
2) beta-1 integrins are pivotal for proper development and structural
organization of joint tissues; 3) structural changes lead to softer AC;
4) softening of the AC increases OA severity only upon mechanical
loading; 5) beta-1 integrin signaling is not a good candidate for inter-
vention in osteoarthritis.

9.3.2
Expression patterns of proinflammatory cytokines and matrix
metalloproteinases before and after autologous chondrocyte
transplantation (MACT) in a leporine animal model: The influence
of varying in-vitro settings
M. Sauerschnig1, G.M. Salzmann2, M. Berninger1, T. Sütfels1, M.
Schönfelder1, S. Vogt1, P.B. Schöttle1, A.B. Imhoff1
1
München/Germany, 2Freiburg/Germany
Purpose: We hypothesize that mRNA-expression of TNF-α, IL-1β,
MMP-1 and MMP-3 before and after matrix assisted chondrocyte
transplantation (MACT) could be influenced by variations of the in-
vitro parameter cell-count, cell-passage and time-on-matrix.
Methods and Materials: Chondrocytes of post puberty New Zea-
land White Rabbits were harvested from articular cartilage, cul-
tured under varying combinations of cell-passage (P1,P2,P3) and
cell-count (C: 2x105/matrix=C1, 1x10 6/matrix=C2, 3x10 6/matrix=C3)
and seeded on 3D-matrices (Chondrogide, Geistlich) with varying
time-on-matrix (T: 24 hours=T1, 2 weeks=T2) forming 18 different
groups. Each time two identical cell-seede-matrices (CSM) where
assembled of which one (CSM-i) was analyzed for TNF-α, IL-1β,
MMP-1 and MMP-3 via qPCR directly prior to autologous press-fit-
implantation of the duplicate (CSM-e) into cartilage defects of the
trochlear (n=2/animal; contralateral to harvest-site knee). After 12
weeks CMS-e were explanted and expression of the parameters
were assayed again using qPCR. Data were statistically compared
using a mixed linear model, multiple regression analysis. Signifi-
cance was set P<.05 for all tests.
Results: CSM-i showed higher expression of MMP-1, MMP-3 and IL-
1β compared to CSM-e while expression of TNF-α was found higher
in CSM-e. General linearity between CSM-i and CSM-e values was
found. Expression levels of all parameters showed significant dif-
ferences between all groups in CSM-i while in CSM-e groups this
was found true for MMP-3 only. Comparing CSM-i to CSM-e the
difference for all parameters was significant. MMP-1 and MMP-3
showed stronger expression at T2 combined with P1 while different
C took mild effect. IL-1β expression increased with higher P and T
while for TNF-α the reverse was true.
Conclusions: Results demonstrate expression-levels of cytokines
and proteinases within regenerate-tissue to be influenced by vary-
ing in-vitro-settings and offer routing of inflammatory processes by
optimizing cell-culture and seeding.
9.3.3
The combination of IL-4 and IL-10 protects against blood-induced
cartilage damage
M.E.R. van Meegeren, N.W.D. Jansen, G. Roosendaal, F.P.J.G.
Lafeber
Utrecht/Netherlands
Purpose: Blood-induced cartilage damage can occur after a joint
trauma, during or after major joint surgery, or in haemophilia pa-
tients. We have previously shown that interleukin (IL)-10 limits in
vitro blood-induced cartilage damage. In addition, IL-4 has been
suggested to have cartilage protective properties. Hence our aim
was to study whether IL-4 can support IL-10 in the prevention of
blood-induced damage.
Methods and Materials: Human and canine articular cartilage ex-
plants were cultured for 4 days in the presence or absence of 50%
v/v homologous blood. IL-4, IL-10, or a combination of both was
added in a concentration of 10 or 100 ng/ml during blood exposure.
Cartilage matrix turnover was determined 12 days later.
Results: Human cartilage cultured in the presence of blood showed
a decrease of proteoglycan synthesis rate of 70% and an increase of
proteoglycan release of 60%, resulting in a decrease of proteoglycan
content of 20% (p<0.05). This blood-induced damage of the carti-
lage matrix was limited by IL-4 in a similar way as IL-10 does: proteo-
glycan synthesis rate was approximately 50% enhanced compared
to exposure to blood and the release was 50% reduced (p<0.05,
dose-dependently). The combination of the two cytokines was even
more protective against damage caused by blood. This was espe-
cially evident for the proteoglycan synthesis rate which showed a
twofold increase (p<0.05). Limiting blood-induced cartilage dam-
age by IL-4 and IL-10 was also confirmed in canine cartilage.
Conclusions: Besides IL-10, as shown previously, also IL-4 protects
against blood-induced cartilage damage. The combination of these
two cytokines is clearly the most protective. This justifies further
evaluation of the combination of IL-4 and IL-10 in prevention and
treatment of blood-induced cartilage damage.
Free Papers 163
9.3.4
Platelet-Rich Plasma intra-articular injections versus
viscosupplementation as treatment for early osteoarthritis: a
comparative study
E. Kon1, B. Mandelbaum2, R. Buda1, G. Filardo1, M. Delcogliano3, A.
Timoncini1, A. Di Martino1, S. Patella1, S. Giannini1, M. Marcacci1
1
Bologna/Italy, 2Santa Monica/United States of America, 3Roma/
Italy
Purpose: Aim of this study is to evaluate and compare the efficacy
of Platelet Rich Plasma (PRP) and Viscosupplementation (HA) i.a.
injections for the treatment of severe chondropathy of the knee.
Methods and Materials: The study involved 150 patients affected
by chondropathy and either early stage or severe osteoarthritis.
Fifty symptomatic patients were treated with 3 autologous Platelet-
Rich Plasma (PRP) intra-articular injections and evaluated prospec-
tively. All patients were clinically evaluated at the enrolment, after
the treatment and at 6 months follow up. The results were also com-
pared with two homogeneous groups of patients treated by HA in-
jections in two different centers (High Molecular Weight Hyaluronan
in one group, Low Molecular Weight Hyaluronan in the other). IKDC
and EQ-VAS scores were used to clinically evaluate the patients,
while their satisfaction and functional status were recorded.
Results: Neither complications nor other major adverse events oc-
curred; only some minor adverse events were detected, as mild pain
reaction and effusion after the injections, but they just lasted for a
couple of days maximum. At the follow-up evaluations, all groups
showed a significant improvement in terms of function and quality
of life. The comparison between the outcomes of the three groups
showed a statistically significant difference (p<0.05), reporting a
superiority of the PRP group results.
Conclusions: PRP is a simple, low cost and minimally invasive ap-
proach to osteoarthritis; it leads to a natural concentrate of autolo-
gous growth factors directly from the blood. Our clinical results
are encouraging and suggest PRP may be used to treat the degen-
erative articular pathology of the knee. Autologous PRP injections
demonstrated a longlasting and better efficacy than HA injections
in recovering articular function and reducing symptoms in patients
affected by knee degeneration. Long-term and randomized con-
trolled studies will be needed to confirm the reliability and evaluate
the durability of this promising procedure.
9.3.5
Comparing the chondroprotective capability of curcumin
(Curcuma longa) and curcumin derivatives against IL-1β-
mediated chondrolysis
M.J. Goldstein1, N.V. Shah1, R. Greenwald1, P. Razzano1, L. Golub2,
D. Grande1
1
Manhasset/United States of America, 2Stony Brook/United States
of America
Purpose: The pro-inflammatory cytokine interleukin-1β (IL-1β)
stimulates articular chondrocytes to produce matrix-degrading
enzymes, contributing to the pathogenesis of osteoarthritis (OA)
by promoting lysis of extracellular matrix (ECM) compounds. Tur-
meric-derived curcumin (Curcuma longa) has demonstrated inhi-
bition of inflammatory signaling pathways activated during IL-1β
stimulation. However, poor solubility and rapid metabolism yields
low plasma and tissue levels of curcumin. In this study, we attempt
to identify an improved chondroprotective agent against IL-1β-
mediated chondrolysis by comparing several novel curcumin de-
rivatives. We hypothesize that a curcumin derivative will enhance
chondroprotection compared to curcumin.
Methods and Materials: Full-thickness articular cartilage cores
were isolated from femoral condyles of bovine knee joints. Car-
tilage explants were pulse-labeled with S-35 sulfate for 24 hours
and separated into seven experimental groups: negative control
(growth media alone/D-MEM), positive control (D-MEM+IL-1β, 10
ng/ml), curcumin (10 μM) + IL-1β, and four novel curcumin deriva-
tives (10 μM) + IL-1β, labeled H1.1, H1.2, H2.5, and H2.7. Degree of
S-35 release was evaluated at 24, 48, and 72 hours post-treatment.
Following each time-point, supernatant media was scintillation-
counted. After 72 hours, explants were lyophilized to obtain dry
weights (mg). Counts per minute (CPM)/mg dry weight was calcu-
lated; mean and standard deviation for each group were derived for
statistical analysis.
Results: Media-alone controls (negative controls) displayed a baseline
level of S-35 release throughout the 72-hour period. IL-1β-challenged
explants (positive controls) demonstrated an early release of S-35
significantly higher than that of negative controls at all time-points.
Curcumin derivatives, specifically H2.5 and H2.7, exhibited significant
chondroprotective effects compared to other derivatives at all time-
164 Free Papers
points. Interestingly, native curcumin displayed a similar low release;
however, this is likely attributed to toxicity issues, not chondroprotec-
tive mechanisms based on most recent data.
Conclusions: Results demonstrate improved chondroprotection by
novel derivatives against IL-1β-induced chondrolysis, compared to
no treatment. Curcumin derivatives that improve bioavailability and
solubility may further enhance chondroprotective capability.
9.3.6
Lack of disease modifying activity of celecoxib in osteoarthritis: a
randomized clinical trial
S.C. Mastbergen1, T.N. De Boer1, A.M. Huisman2, A.A. Polak2, J.W.J.
Bijlsma1, F.P.J.G. Lafeber1
1
Utrecht/Netherlands, 2Rotterdam/Netherlands
Purpose: Treating human osteoarthritic (OA) cartilage in vitro and
ex vivo demonstrated a direct chondroprotective (DMOAD) effect
of celecoxib (celebrex), one of the selective COX-2 inhibitors. The
present study aims to validate these findings in a blinded random-
ized in vivo (clinical) study, treating patients with severe knee os-
teoarthritis prior to joint replacement surgery with subsequent ex
vivo detailed evaluation of joint degeneration.
Methods and Materials: Patients (n=172) with knee osteoarthritis
were randomized and treated for at least 4 weeks prior to knee replace-
ment surgery with celecoxib 2dd200mg, celecoxib 2dd200mg stopped
3 days prior to surgery, naproxen 2dd250mg, or received no treatment.
WOMAC questionnaires were used to evaluate pain, stiffness and func-
tion before and after treatment. Cartilage and synovial tissue were col-
lected during replacement surgery and analyzed in detail ex vivo.
Results: Age, gender, weight (BMI) and cartilage damage (X-ray, macro-
scopic or histological) was not different between the treatment groups.
In contrast to all previous findings celecoxib treatment showed no effect
on the proteoglycan release, the primary outcome, of the cartilage com-
pared to the untreated group. Also naproxen showed no effect. Similar
lack of effect was found for proteoglycan content compared to the no-
treatment group. Prostaglandin-E2 and nitric oxide (NO) produced by
the cartilage showed no changes compared to the no-treatment group.
However celecoxib treatment showed an improvement of WOMAC-pain
(p<0.01) compared to the no-treatment group. The ex vivo release of
IL-1β, TNFα, prostaglandin-E2 and NO by the synovial tissue only shows
a statistical significant decrease in NO levels (p<0.04) in the celecoxib
treated group compared to the no-treatment group.
Conclusions: Despite an improvement in WOMAC score no DMOAD
effect of celecoxib could be demonstrated in this blinded randomized
well-powered study.
9.3.7
Treatment of painful bone marrow edema syndrome with
intravenous bisphosphonates
C. Bartl1, G.M. Salzmann2, A.B. Imhoff3, R. Bartl3
1
Ulm/Germany, 2Freiburg/Germany, 3München/Germany
Purpose: The aim of the study was to assess the efficacy of intrave-
nous bisphosphonates (Ibandronate and Zoledronate) in the treatment
of bone marrow edema (BME) syndrome around the knee and ankle in
a prospective, randomised MRI-controlled off-label use study.
Methods and Materials: 30 patients with an ankle-BME and 40 patients
with a knee-BME were randomised to receive either Ibandronate-infu-
sion (3x6 mg – group G1) or zoledronate-infusion (1x 5mg – group G2)
in an outpatient setting following BME diagnosis by MRI. All patients
gave written informed consent and agreed to participate in the study.
Patients were followed-up clinically at baseline, 1, 3 and 6 months after
the first infusion using the Mazur-foot score, the Larson-knee score and
a VAS pain scale. MRI (T1-, T2- and STIR-images) was performed before
and 3 month after the first infusion.
Results: The mean VAS pain-score decreased from 8.1 at baseline to 2.3
at 6 months in G1 (7.9 to 2.1 in G2 respectively – each p<.05). Both the
Mazur (Ankle) and the Larson-score (Knee) increased significantly from
baseline to 6 months (Mazurscore: av. from 57p. to 89p- no sig. diff be-
tween G1 and G2 – each p<.05)( Larsonscore: av. from 52p. to 90p- no
sig. diff between G1 and G2 – each p<.05). MRI revealed a significant
reduction of BME-size or complete normalization in 70% of the cases
in both groups. Acute phase reactions with flue-like symptoms were
higher in group G2 (G1:14% - G2:23%). We did not observe cases of kid-
ney dysfunction or osteonecosis of the jaw during the study period.
Conclusions: Both intravenous bisphosphonates (Ibandronate and
Zoledronate) were very effective for the treatment of BME in the knee
and ankle. After the infusion therapy the majority of patients showed a
fast regain in function and the infusion therapy seems to shorten the
natural course of the disease.
9.3.8
Overexpression of SIRT1 inhibits osteoarthritic gene expression
changes induced by interleukin-1β in human chondrocytes
H. Sasaki, T. Matsushita, R. Kuroda, K. Takayama, K. Ishida, N.
Fujita, S. Kubo, T. Matsumoto, M. Kurosaka
Kobe/Japan
Purpose: Interleukin-1β (IL-1β) has been suggested to be involved
in the pathogenesis of osteoarthritis (OA) by increasing catabolic
enzymes or enhancing apoptosis. Recently it has been reported
that SIRT1, a mammalian homologue of longevity factor sir2, inhib-
its apoptosis and promotes survival of human chondrocytes, sug-
gesting that SIRT1 plays a protective role in human chondrocytes.
Nevertheless, protective roles of SIRT1 in human chondrocytes are
not yet fully understood. In this study, we examined the effect of
overexpression of SIRT1 on IL-1β-treated human chondrocytes to
explore protective roles of SIRT1 in human chondrocytes.
Methods and Materials: Normal Human Articular Cartilage-knee
(NHAC-Kn) was used as normal human chondrocytes. NHAC-kn was
transfected with either control plasmid or SIRT1 expression plas-
mid by a lipofection technique. After the transfection, NHAC-Kn
was treated with 10ng/ml IL-1β for 24 hours. The effects of IL-1β
treatment and the overexpression of SIRT1 were examined by real-
time PCR and western blotting.
Results: The effect of SIRT1 overexpression was confirmed by the
increased SIRT1 protein, decreased acetylated P53 and decreased
acetylated p65 (a member of NF-kB ) in the SIRT1-plasmid-trans-
fected chondrocytes. The stimulation of IL-1β upregulated MMP-13
and ADAMTS-5 while downregulated aggrecan and COL2A1. The
overexpression of SIRT1 inhibited the upregulation of MMP-13 and
ADAMTS-5 caused by the stimulation of IL-1β. On the other hand,
the overexpression of SIRT1 inhibited the downregulation of aggre-
can and COL2A1 caused by the stimulation of IL-1β. In addition, the
overexpression of SIRT1 alone significantly increased the expres-
sion of aggrecan and COL2A1.
Conclusions: SIRT1 overexpression counteracted against the up-
regulations of expressions of catabolic factors induced by IL-1β
while promoted cartilage extracellular matrix gene expressions.
Our observations suggested that SIRT1 can play a protective role
for chondrocytes and SIRT1 overexpression might be a new thera-
peutic approach for OA.
9.3.9
Initial Phase 1 safety of retrovirally transduced human
chondrocytes expressing TGF-β1 (TG-C)
C. Ha
Seoul/Korea, Democratic People‘s Republic of
Purpose: TissueGene-C (TG-C) represents a cell-mediated gene
therapy for localized delivery of allogeneic chondrocytes express-
ing TGF-β1 directly to the damaged knee joint. Untransduced hu-
man chondrocytes (hChonJ cells) have also been incorporated into
the TG-C product in a 3:1 ratio with TGF-β1 expressing chondrocytes
(hChonJb#7) in order to help fill in the defect and as target cells
for the actions of the expressed TGF-β1. A Phase 1 dose escalating
clinical trial was performed to evaluate the safety and biological
activity of TG-C in patients with advanced osteoarthritis of the knee
joint (full thickness cartilage defect) that was refractory to existing
nonoperative therapies.
Methods and Materials: Following a single intraarticular injection
into the joint space of the damaged knee, patients were monitored
for safety, and an evaluation was performed to assess the pharma-
cokinetics and biological activity of TG-C.
Results: There were no treatment related serious adverse events.
Swelling, effusion and minor localized reactions such as warming
sensation or itching were observed in a dose-dependent manner at
the injection site. MRI results and knee evaluation scores seem to
indicate a dose-dependent trend toward efficacy; however patient
numbers were not sufficient to determine statistical significance.
Conclusions: Overall, there were no significant safety issues re-
lated to the administration of TG-C, with only some minor injection
site reactions observed. Additionally, the preliminary data from the
MRI and knee scroring analyses indicate a possibility that TG-C may
contribute to regeneration of articular cartilage and/or improve-
ment of arthritic symptoms. More study is warranted to further
evaluate the safety and determine the potential efficacy of TG-C.

9.4.2
Mechanically Induced in vitro Chondrogenesis under Simulation
of Different Chondrocyte Implantation Techniques
N. Wang1, S. Grad1, M. Stoddart1, M. Alini1, N. Sudkamp2, G.M.
Salzmann2
1
Davos/Switzerland, 2Freiburg/Germany
Purpose: Advanced cell-based cartilage regeneration techniques
are represented by three generations of autologous chondrocyte
implantation (ACI) procedures, which aim to deliver new cells ca-
pable of chondrogenesis into the lesions. This study investigated
the effect of dynamic compression and sliding surface motion on
chondrogenesis in a joint-specific bioreactor to mimic and compare
the performance of first and third generation ACI (matrix-assisted
chondrocyte transplantation, MACT) in vitro.
Methods and Materials: Primary (P0) or third passage (P3) bovine
chondrocytes were seeded in fibrin-polyurethane scaffolds. Con-
structs were cultured either free swelling, under mechanical loading
for 2 or 4 weeks, or free swelling for 2 weeks followed by 2 weeks of
loading (F2L2 group). The latter group simulates the MACT method,
while the directly loaded group mimics conventional ACI. Samples
were collected for DNA and glycosaminoglycan (GAG) quantifica-
tion, expression of chondrogenic marker genes, and histology.
Results: Mechanical loading stimulated GAG synthesis, although a
significant proportion of GAG was released from loaded scaffolds
into the medium. The F2L2 group retained more GAG in scaffolds
than the immediately loaded group. Generally, P0 chondrocytes ap-
peared more responsive to load than P3 cells. Chondrogenic gene
markers, including collagen II, aggrecan, COMP, and lubricin, were
up-regulated after loading in both P0 and P3 chondrocytes; the F2L2
group demonstrated most favorable chondrogenic gene expression
results. Load also increased matrix staining, while most intense
staining was observed in the F2L2 group of P0 chondrocytes.
Conclusions: Different duration and mode of mechanical load can
increase matrix synthesis and chondrogenesis. Our data indicate
that free swelling culture followed by loading (simulating in vivo
MACT) may be most favorable for chondrogenesis. It is likely that a
pre-existing extracellular matrix improves the responsiveness of the
chondrocytes to mechanical stimuli. This in vitro study suggests that
MACT procedures may be preferred, while primary cells show better
responsiveness regarding matrix synthesis than passaged cells.
9.4.3
Chitosan-Glycerol Phosphate/Blood Implants Alter Maturation
and Location of Chondrogenic Foci in Marrow Stimulated
Cartilage Repair
A. Chevrier1, C.D. Hoemann1, J. Sun2, M.D. Buschmann1
1
Montreal/Canada, 2Laval/Canada
Purpose: Bone-marrow stimulation initiates cartilage repair through
the formation of chondrogenic foci. Previous studies have shown that
chitosan-glycerol phosphate/blood implants improve the quantity
and quality of bone marrow derived cartilage repair. The purpose of
this study was to characterize these chondrogenic foci and to investi-
gate the effects of chitosan-GP/blood implants on their formation.
Methods and Materials: Thirty-three New Zealand White rabbits re-
ceived bilateral cartilage defects with four microdrill holes. One knee
per rabbit was treated by applying a chitosan-glycerol phosphate/blood
implant over the defect. Cell morphology and distribution of glycosamin-
oglycan and collagen type II, I and X were characterized. Chondrogenic
markers and proliferation were identified with Patched and Ki-67.
Results: Chondrogenic foci were categorized as nascent (small, ho-
mogenous, GAG and col II positive), mature (larger, col II positive,
stratified cell morphology) or resorbing (extensive vascular invasion
and endochondral bone formation). In mature foci, glycosaminogly-
can and collagen type II were present throughout while the upper
zone expressed collagen type I and the lower zone collagen type X
(Fig 1). Patched and proliferating cells were immunodetected within
foci (Fig 1). Treatment with chitosan-glycerol phosphate/blood im-
plants led to three important and statistically significant modifica-
tions of chondrogenic foci: 1) the initial appearance of foci occurred
later (Day 21 for treated versus Day 14 for control) 2) more nascent
and mature foci were produced than resorbing and 3) foci were closer
to the articular surface (Fig 2).
Conclusions: Chondrogenic foci bear some similarities to growth car-
tilages and can give rise to a repair tissue that has similar zonal strati-
fication as articular cartilage. Treatment with chitosan-GP/blood
implants produce a greater fraction of nascent and mature foci that
are closer to the articular surface and thereby contribute to a more
effective cartilage repair.
Free Papers 165
9.4.4
Presolidified chitosan-based implants for osteochondral repair
in sheep
A. Bell1, M. Lowerison1, J. Sun2, V. Lascau-Coman3, M.B. Hurtig1, C.D.
Hoemann3
1
Guelph/Canada, 2Laval/Canada, 3Montreal/Canada
Purpose: To explore the use of novel pre-solidified chitosan formu-
lations as therapeutic implants for osteochondral repair, using Jam-
shidi bone biopsy as an alternative marrow stimulation approach.
Methods and Materials: Three chitosan formulations (10kDa, 40kDa
and 150kDa MW) were mixed with autologous ovine blood to create
pre-solidified cylindrical implants. Under general anaesthesia using
aseptic technique, six, 2-8 mm deep holes were made with a 2 mm di-
ameter Jamshidi biopsy needle (Cardinal Health) in each medial femo-
ral condyle of five sheep. Each chitosan implant was placed in dupli-
cate holes while controls were left to bleed. Sheep were sacrificed at
1 (n=1), 21 (n=2) or 93 days (n=2). A correlative study was undertaken
using histology images of Safranin O-stained sections and MicroCT
imaging (GE Explore Locus scanner, 27μm resolution) of bone remod-
elling in biopsy holes. Software was created in Python (ver 3.1.1) and
R (www.r-project.org) to determine radial gradients in tissue mineral
density (TMD) and bone volume fraction (BVF) from the central axis of
the hole to beyond the repair tissue-bone interface.
Results: A gradient of increasing BVF and TMD towards the repair
tissue-bone interface was seen after 93 days with a significant dif-
ference compared to day 1 (p<0.05, Fig. 1). There was a significant
difference (p<0.05) in TMD and BVF between the 10kDa and 150kDa
formulations inside the repair tissue-bone interface. The 10kDa chito-
san treatment had the highest increase in BVF and TMD, and control
defects had the lowest increase (Fig. 1). BVF beyond the repair tissue-
bone interface generally decreased after 93 days. Both treated and
control defects developed a mixed angiogenic granulation-cartilage
repair tissue after 93 days (Fig. 2).
Conclusions: These results highlight the potential use of chitosan-
based implants to induce subchondral bone repair of Jamshidi-gen-
erated marrow-stimulation channels. Repair periods longer than
93 days will be required in this model to observe cartilage repair in
treated defects.
9.4.5
An In Vivo Study to Clarify Mechanisms of Spontaneous Cartilage
Regeneration Induced by Implantation of a Novel Double-
network Gel: Determination of the Effect of the Two Component
Gels on the Regeneration
M. Ogawa1, N. Kitamura2, K. Arakaki3, S. Onodera2, T. Kurokawa2,
J.P. Gong2, Y. Tanaka1, Y. Takakura1, K. Yasuda2
1
Kashihara/Japan, 2Sapporo/Japan, 3Naha/Japan
Purpose: We have developed a novel therapy for spontaneous carti-
lage regeneration using the originally developed PAMPS/PDMAAm
double-network (DN) hydrogel. This DN gel was composed of two
single-network (SN) gels; poly(2-acrylamide-2-methylpropanesul-
fonic acid) (PAMPS) and poly(N,N’-dimetyl acrylamide) (PDMAAm).
To clarify the mechanisms for regeneration, we have evaluated the
in vivo effect of these SN gels on the cartilage tissue induction in
comparison with the effect of the DN gel.
Methods and Materials: An osteochondral defect of 4.3-mm diam-
eter was created in the bilateral femoral trochlea of the rabbit knee.
The treatment groups consisted of: 1) PAMPS/PDMAAm DN gel
(Group-DN); 2) PAMPS gel (Group-PAMPS); 3) PDMAAm gel (Group-
PDMAAm); 4) untreated defect (Control). A cylindrical gel plug was
randomly implanted into the defect so that having 2-mm depth re-
mained. At 4 weeks, the regenerated tissue was stained with HE, Sa-
franin-O and immunohistochemical stain (type-2 collagen), and was
quantitatively evaluated with the Wayne’s grading scale. The real
time PCR for type-2 collagen, aggrecan, and Sox9 was performed.
Results: Group-DN was filled with the proteoglycan-rich tissue
stained with Safranin-O and type-2 collagen. Group-PAMPS was
filled with hyaline/fibrocartilage tissue inhomogenously, while
Group-PDMAAm and Control were filled with the fibrous and bone
tissues. Wayne’s scores were significantly higher in Group-DN and
Group-PAMPS than in Group-PDMAAm and control. The histology
score was significantly higher in Group-DN than in Group-PAMPS.
Each mRNAs were highly expressed in Group-DN and Group-PAMPS.
Conclusions: The present study demonstrated that the negatively
charged PAMPS gel may play an important role in induction of the
cartilage regeneration, although the degree of the cartilage regen-
eration was significantly greater in Group-DN than in Group-PAMPS.
We speculate that the unique mechanical properties of the DN gel
improved by the PDMAAm gel enhanced the effect of the PAMPS gel
on spontaneous cartilage regeneration.
166 Free Papers
9.4.6
Testing tissue time dependant mechanical properties by AFM:
experimental procedures and finite element modeling of soft
agarose model gels and articular cartilage
R. Gottardi1, E. Landini1, D. Carnelli2, M. Taffetani2, P. Vena2, R.
Raiteri1
1
Genova/Italy, 2Milano/Italy
Purpose: We recently showed that the atomic force microscope (AFM)
can readily image articular cartilage (AC) morphology and measure its
elastic properties at the nanometer scale, i.e. the scale at which bio-
chemical processes occur and pathological lesions start. This opens
the possibility for detecting pathological features of AC at their early
stages and/or for using AFM as a quality control tool of engineered
tissue (functionality and structure) prior to implantation. Aim of this
study was to develop AFM-based methods for time dependant me-
chanical characterization at different length scales of model materials
(agarose hydrogels) and animal AC through a combined experimen-
tal/numerical approach.
Methods and Materials: An AFM with spherical indenter tip (r=7.5μm,
k=8N/m) was used to test agarose gels (0.8%, 1.5%, 3% w/v) and na-
tive and enzyme (hyaluronidase) treated bovine AC in physiological
conditions. The viscoelastic response was assessed by: (a) frequency
sweep (FS) - a pre-load is applied by the AFM probe followed by an os-
cillation (<10% of the pre-load) over a range of hundreds of hertz; (b)
stress relaxation (SR) - a step-like load is applied by the AFM probe and
the progressive stress relaxation monitored; (c) creep (CR) - a step-
like load is applied and the piezo displacement necessary to maintain
the load constant is monitored. Results were interpreted with the aid
of an axi-symmetric finite element model to simulating both preload
phase and the cyclic load phase during indentation. Equivalent mass,
stiffness and damping were simulated by a lumped parameter model.
Poroelastic properties have been preliminarily disregarded.
Results: Rate-dependant effects related to different gel concentra-
tions were observed for all methods. FS comparison with numerical
modelling suggests a significant influence of mechanical resonance
due to wave propagation and reflexion through the gel. CR testing fol-
lows the same models as millimetre scale creep testing, SR requires
additional modelling to account for AFM force-based controls.
Conclusions: Our preliminary findings open the possibility of using
AFM-based mechanical testing, complemented by appropriate con-
stitutive modelling, in the field of tissue characterization, in particular
testing the functional properties and possible pathologies of both na-
tive and engineered AC.
9.4.7
The application of a chondroitin sulfate-bone marrow adhesive
towards meniscal repair
J.A. Simson, I. Strehin, J. Elisseeff
Baltimore/United States of America
Purpose: Meniscal injuries may lead to joint degeneration and the
development of post-traumatic osteoarthritis. There are few tech-
nologies beyond sutures available for surgeons to repair the menis-
cus, and sutures inherently cause additional damage to the tissue.
Adhesives that work to repair meniscus while fixing the tissue in
place offer an appealing alternative solution. Here, we demonstrate
the use of a NHS-functionalized chondroitin sulfate (CS-NHS) bio-
adhesive for use in meniscal repair.
Methods and Materials: 10% CS-NHS was mixed with 10% Polyeth-
ylene Glycol (PEG) in a 1:1 ratio, and with BM in ratios of 3:7 (70% BM),
1:1 (50% BM), and 7:3 (30% BM). Meniscus cells were encapsulated
in gels to quantify in vitro tissue generation, and bovine meniscus
explants were glued to observe meniscus cell migration into the ad-
hesive. Constructs were analyzed at one and three weeks using live/
dead, H&E staining, and Hoescht dye DNA assays. Glued tissue ex-
plants were sectioned and stained with H&E at two and four weeks.
Results: Live/dead and Hoescht DNA assays showed statistically
significantly higher viability at both time points in CS-BM gels in
comparison to CS-PEG. At one week normalized DNA levels in-
creased with BM concentration, but this effect diminished by week
three. However, extensive clustering of cells was observed at three
weeks in CS-BM gels, indicating cell proliferation. In the explant
study cells were observed proliferating on the surface of 70% BM
at week two, and at week four cells were seen within the gel prolif-
erating and depositing matrix.
Conclusions: These findings indicate that meniscal cell viability in
the CS-BM gel remains high after several weeks in culture, they
proliferate within the gel, and that meniscal cells are capable of
migrating from meniscal tissue first onto the hydrogel surface, and
later into BM gels. These are promising preliminary results for the
use of CS-BM adhesive in regenerating meniscus tissue.
9.4.8
Effects of combined 3D- and hypoxic culturing on cartilage-
specific gene expression in human chondrocytes
C.B. Foldager, A.B. Nielsen, S. Munir, M. Ulrich-Vinther, K. Søballe,
C. Bünger, M. Lind
Aarhus/Denmark
Purpose: In vitro expansion of autologous chondrocytes is an es-
sential part of many clinically used cartilage repair treatments. Na-
tive chondrocytes reside in a 3-dimensional (3D) network and are
exposed to low levels of oxygen. The aim of this study was to inves-
tigate conventional monolayer culturing compared to combined 3D
and hypoxic culturing using quantitative gene expression analysis.
Methods and Materials: Cartilage biopsies were collected from
the intercondylar groove in the distal femur from 12 patients with
healthy cartilage. Cells were divided to either monolayer or scaffold
culture. The scaffold was a clinically available MPEG-PLGA scaffold
(ASEED). After harvesting cells for baseline investigation, the re-
mainders were divided into three groups for incubation in normoxia
(21% oxygen), hypoxia (5% oxygen) or severe hypoxia (1% oxygen).
RNA extractions were performed 1, 2 and 6 days after the baseline
time-point respectively. Quantitative RT-PCR was performed using
assays for collagen type 1 and 2, aggrecan, sox9, ankyrin repeat
domain-37, and glyceraldehyd-3-phosphate dehydrogenase rela-
tive to two hypoxia stable house keeping genes. Statistics were
performed using three-way ANOVA.
Results: Sox9, aggrecan and collagen type 2 expression increased sig-
nificantly with lowered oxygen. The expression of collagen type 2 was
higher after 6 days in 3D compared to monolayer at all levels of oxygen.
Conclusions: These new results suggest a combined positive effect
of 3D and hypoxic culturing on cartilage-specific gene expression.
The positive effects of 3D culture alone were not present until day
6, suggesting a benefit of long-term scaffold culturing for matrix-
assisted chondrocyte implantation.
9.4.9
Biomechanical Optimization of a Novel Polycarbonate-Urethane
Meniscal Implant
J.J. Elsner1, E. Linder-Ganz2, G. Zur2, R. Arbel3, F. Guilak4 , A.
Shterling2
1
Tel Aviv/Israel, 2Netanya/Israel, 3Hod Hasharon/Israel, 4Durham/
United States of America
Purpose: Meniscus replacement represents an unsolved problem in
orthopedics. Graft availability, sizing, and complexity of repairs limit
the use of allografts and may contribute to uneven distribution of
load and degenerative damage. A synthetic substitute, available in
a number of sizes/shapes, offers significant advantages for meniscal
replacement. In this study we present a novel optimization method
for meniscal implant design, and employ it in the development of a
polycarbonate-urethane (PCU) meniscus implant in an ovine model.
Methods and Materials: A gross structure was constructed ac-
cording to MRI scans. Insertion attachments were added later to
the horns (Fig.1). Samples based on this design were produced for
testing. Additional modifications (geometry (surfaces), fibrous re-
inforcement and fixation) were also considered. An experimental
evaluation of the biomechanical performance of different designs
was performed by tibial-plateau (TP) pressure distribution score
(Linder-Ganz et al. 2010), reflecting on the magnitude of peak pres-
sure and contact area coverage with respect to the natural menis-
cus under load (45° flexion, 330N compression).
Results: The all-PCU implant showed limited ability to distribute
pressure. Its pressure score (37%) reflects on a small contact area
(151mm2) subject to high pressures (>1.85MPa, Fig.2b). The pres-
sure distribution improved when reinforcement fibers were added. A
fixation tension of 20N increased the contact area (273mm2) and al-
though a region of focal pressure concentration existed (Fig.2c), the
pressure score increased (77%). Inspection of different pre-tension
values showed that optimal pressure distribution (87%) is attained
when a pre-tension of 30-50N is applied (Fig.2d). In this configura-
tion, peak pressures and coverage area (1.65MPa and 310mm2) are
similar to those of the natural meniscus (1.61MPa and 373 mm2).
Conclusions: The current device can be used as a practical solution
for patients suffering from severe meniscal injury. We conclude that
peripheral reinforcement of the implant, in addition to pretension
of 30-50N, can significantly improve TP pressure distributions.

12.1.2
The small heat-shock protein HSP27, shows a decreased
abundance in OA and mediates IL-1b induced IL-6 secretion in
human articular chondrocytes
S. Lambrecht1, K.F. Almqvist1, P.C. Verdonk2, G. Verbruggen1, D.
Deforce1, D. Elewaut1
1
Gent/Belgium, 2Gent-Zwijnaarde/Belgium
Purpose: Based on two proteome analyses, the differential expres-
sion of two small-heat shock proteins, HSP27 and alphaBcrystallin,
was discovered in osteoarthritis-affected chondrocytes. Recently,
we reported the differential expression and biological function of
alphaBcrystallin. Based on a proteome analysis and the functional
and structural relationship between alphaBcrystallin and HSP27,
we further performed differential expression analysis on HSP27. We
aimed to achieve further insights in the involvement of small heat-
shock proteins, more specific HSP27, in the biology of chondrocytes.
Methods and Materials: Western blot and real-time RT-PCR analy-
sis were performed to determine the expression levels of HSP27
in healthy and OA chondrocytes cultured in alginate beads. RNA-
interference mediated gene knock-down was used to explore the
role of this small heat shock protein in IL-1b activated pathways by
transfecting low concentrations of siRNA in cultured chondrocytes.
Upon knock-down of HSP27, cells were stimulated by IL-1b and IL-6
concentrations were determined by ELISA.
Results: Western blot of healthy and OA chondrocyte lysates
showed a decreased abundance of HSP27 in OA. Moreover, real-
time RT-PCR confirmed the differential expression at the mRNA-lev-
el between chondrocytes isolated from visually intact and visually
damaged zones of OA cartilage. The pro-inflammatory cytokines
IL-1beta and TNF-alpha, both down regulated HP27 expression.
Transfection of low concentrations siRNA in cultured chondrocytes
resulted in an efficient knock down of HSP27 gene expression. This
decreased HSP27 expression results in a reduced secretion of IL-6,
in response to IL-1b (figure 1).
Conclusions: This study adds to the evidence that small heat-shock
proteins may be important mediators in chondrocyte biology during
the development of OA. Our study showed the involvement of HSP27
in IL-1b induced IL-6 secretion in human articular chondrocytes.
12.1.3
Effect of IL-1β on the proteome of chondrocytes derived from
human osteoarthritic cartilage- a pharmacoproteomics approach
for drug screening
H. Zwickl1, E. Niculescu-Morzsa1, V. Haudek2, C. Gerner2, F.
Halbwirth1, M. Berger1, S. Nehrer1
1
Krems/Austria, 2Vienna/Austria
Purpose: Inflammation plays a pivotal role in cartilage tissue de-
struction in osteoarthritis. IL-1β, a key mediator, affects the bal-
ance of biosynthesis and degradation of extracellular matrix (ECM)
constituents by chondrocytes. Indeed, exuberant synthesis of ma-
trix metalloproteinases and reduced expression of collagen II and
aggrecan are well-known cytokine-mediated hallmarks of osteoar-
thritis. In addition, IL-1β induces the production of the pain media-
tor prostaglandin E2 via cyclooxygenase which decisively contrib-
utes to joint dysfunction. Today’s treatment options are restricted
to symptome-modifying drugs. However, analgesics or anti-inflam-
matory drugs partially show just limited efficacy with respect to
pain relief or cause undesirable side effects. Hence, the identifica-
tion of disease-modifying and efficient symptome-modifying drugs
is a main challenge in osteoarthritis research.
Methods and Materials: Pharmacoproteomics is a promising ap-
proach for drug screening. In order to establish a reference system
for monitoring substance effects, we performed proteome profil-
ing of human osteoarthritic chondrocytes. Alterations of the secre-
tion performance and the metabolism of chondrocytes due to IL-1
β treatment were assessed by 2D-PAGE and shotgun proteomics
combined with the mass spectrometrical identification of proteins.
Results: A special focus was laid on the determination of interin-
dividual differences existing per se as well as those arising due to
stimulation by IL-1β. The proteomes were functionally character-
ized and biomarkers/functions reflecting the cytokine effect were
gathered. The resulting database was used to assess the effect of
different derivatives of hyaluronic acid in order to evaluate the ap-
plicability of this approach.
Conclusions: Our approach enabled insights into molecular altera-
tions due to cytokine and substance effects at a comprehensive
systemic level. Furthermore, it is well-suited to identify “real”
substance effects against the background of patient- and disease-
derived heterogeneity of samples.
Free Papers 167
12.1.4
Phenotypic differences between in vitro expanded articular
chondrocytes and mesenchymal stem cells: a proteomic study.
M. Polacek, I. Martinez
Tromsø/Norway
Purpose: One of the remaining conflicts in the field of cartilage re-
construction is linked to the question of which cell type is more suit-
able for attempting cell-based therapies or for the ex-vivo engineer-
ing of cartilage implants. This study was undertaken to compare
the spectrum of proteins released to the spent medium by either
human articular chondrocytes (AC) or bone marrow-derived mes-
enchymal stem cells (MSCs) after cell expansion for several weeks.
Methods and Materials: Cartilage signature gene expression was
measured by real-time PCR on AC and MSC from 6 different patients
after 4 weeks of growth in monolayer cultures. Stable isotope label-
ling of aminoacids in cell culture (SILAC) was applied to investigate
differences between proteins secreted by chondrocytes and MSCs.
Proteins in cell supernatants were resolved by 1D gel electropho-
resis and analysed by LC/MS-MS. The spectrums of identified pro-
teins were contrasted and the results corresponding to relevant
proteins were validated by specific immunoblotting.
Results: Our results show comprehensive list of proteins secreted by
ACs and MSCs after 4 weeks in culture. Qualitative comparison shows
that a larger number matrix components (48% vs. 42%) and growth
factors (15 % vs. 11%) were released into the media by chondrocytes.
However MSCs expressed lower number of matrix catabolic agents
(7% vs. 10%) and higher number of matrix anabolic agents (12%
vs. 5%). In addition some proteins like MMP3, Clusterin, Mimecan,
Proteoglycan 4, Tenascin and Sushi (SVEP1) were expressed only by
chondrocytes, while proteins like serpins, bone morphogenic protein
2 and galectins were expressed exclusively by MSCs.
Conclusions: Our results show that both cell types have developed
a de-differentiated phenotype, and express a similar phenotype
after long term culture periods. However some differentially ex-
pressed proteins could be identified in the culture supernatants.
The biological relevance of the differences revealed in this study
remains an area of investigation.
12.1.5
The effect of cellular passage, quantity and in vitro 3-D matrix
culturing time on gene expression profiles before and after
matrix-assisted chondrocyte transplantation (MACT) in a leporine
animal model
G.M. Salzmann1, M. Sauerschnig2, M. Berninger2, T. Sütfels2, M.
Schönfelder2, P.B. Schöttle2, A.B. Imhoff2
1
Freiburg/Germany, 2München/Germany
Purpose: Matrix-assisted chondrocyte transplantation (MACT) still
lacks any standardization in its execution.
Methods and Materials: Post puberty NZW-rabbit knee articular
chondrocytes were seeded within 3-D matrices (Chondrogide, Gei-
stlich, Switzerland) at different passages (P 1, 3, 5); cellular yields
(C: 2x105/matrix=C1, 1x106/matrix=C2, 3x106/matrix=C3) and in vit-
ro membrane-holding times (T: 24 hours=T1, 2 weeks=T2) to define
18 different groups. Each time, two cell-matrix-constructs (CMC,
n=6/group) were in identical duplicate whereof one was for in vitro
(CMCi) analysis directly prior to re-implantation of the other duplicate
which was press-fit implanted (autologous) (CMCe) into trochlear
full-thickness chondral defects (n=2/animal) of the biopsy-contral-
ateral knee, reproducing a MACT situation. 12 weeks postimplan-
tation the regenerates were analysed for Collagen-1,-2,-10, COMP,
Aggrecan, Sox9 mRNA expression. Data were statistically compared
using a mixed linear model, significance was at P<0.05 for all tests.
Results: Generally, CMCi values were higher than CMCe values for all
differentiation targets, while the opposite was true for dedifferenti-
ation targets. There appeared a general linearity between CMCi and
CMCe values to potentially predict the CMCe outcome based on CMCi
quality. Typically, animals improved, target-specific, a disadvanta-
geous CMCi profile into an advantegeous CMCe expression, while
generally advantageous CMCi values persisted within CMCes. CMCi
values were significantly different between groups for all targets
analysed, while the difference was not significant for Collagen-1,-10,
Aggrecan among CMCes. Usually, interacting P and T resulted in
significant different results, while this was true with much lesser
frequency for C. A combination of low P, of medium C and short T ap-
peared to result in an optimal CMCi and CMCe outcome, while gener-
ally P3, more pronounced P5 and foremost T2 (as well their combina-
tion) strongly impaired the outcome, with much lesser impact of C.
Conclusions: The results demonstrate that both in vitro and in vivo
performance are strongly affected by cellular passage, density,
membrane-holding time.
168 Free Papers
12.1.6
Grem1, FRZb and DKK1 are highly specific markers for articular
cartilage which prevent hypertrophic differentiation
J. Leijten1, J. Emons2, C. Sticht3, E. Dekker4 , G. Rappold4 , J.M. Wit2, C.
Van Blitterswijk1, M. Karperien1
1
Enschede/Netherlands, 2
Leiden/Netherlands, 3
Heidelberg/
Germany, 4Mannheim/Germany
Purpose: Chondrogenically differentiated human mesenchymal
stromal cells (hMSCs) are prone to undergo endochondral ossifica-
tion. Under normal circumstances, this process is limited to growth
plate cartilage (GP) but not articular cartilage (AC). The molecular
mechanisms that prevent AC from terminal differentiation and ossi-
fication are largely unknown. Our key objectives were i) to identify
genetic markers that distinguish AC from GP, and ii) to identify molec-
ular mechanisms that prevent AC from endochondral ossification. We
hypothesized that this information can be utilized to optimize tissue
engineering strategies to obtain AC from differentiating hMSCs.
Methods and Materials: Gene expression profiles of healthy AC
and GP from the same patients were compared in a genome wide
micro-array study.
Results: We identified differential expression of 2915 genes between
GP and AC, from which we generated a marker-set able to distinguish
the two hyaline cartilage subtypes. Furthermore, we showed that
differentiating MSCs acquire a genetic fingerprint resembling GP, but
not AC. Interestingly, the three most differentially expressed genes,
being enriched in the AC, are Wnt- and BMP antagonists: FRZb, DKK1
and Gremlin1. Immunohistochemistry demonstrated the presence of
these antagonists throughout the entire AC. In the GP their expres-
sion was mainly restricted to the resting zone. Chondrogenically dif-
ferentiated hMSCs did not express the 3 antagonists and underwent
hypertrophic differentiation. Remarkably, the addition of recombi-
nant Grem1, FRZb or DKK1 did not affect chondrocyte differentiation
but prevented their hypertrophic differentiation.
Conclusions: Micro-array analysis has identified genetic finger-
prints highly specific for the two types of hyaline cartilage, GP and
AC, which can be used to optimize tissue engineering strategies.
We have identified the Wnt- and BMP-antagonists, FRZb, DKK1 and
Grem1 as specific markers for AC. We show that these antagonists
prevent hypertrophic chondrocyte differentiation. Our data suggest
that Wnt- and BMP-antagonists play a prominent role in establish-
ing a joint micro-environment, which prevents AC from undergoing
endochondral ossification.
12.1.7
Intraarticular platelet rich plasma ( PRP) therapy evaluated in 42
sport horses with Osteoarthrosis (OA)
M. Prades1, I. Abellanet2
1
Cerdanyola/Spain, 2Barcelona/Spain
Purpose: To assess the clinical progression of OA treated by in-
traarticular injection of PRP.
Methods and Materials: 42 sport horses were included in the study
and divided into 2 groups: Group 1:12 control horses chronically
lame unresponsive to conventional therapy . Group 2: 30 horses
with both acute (10 cases) and chronic conditions (20 cases). Com-
plete lameness examination and radiography were performed.
Horses were classified according to chronicity of clinical disease
and OA radiographic findings. All patients were reexamined before
each treatment and at 45 days, 2 months and 4 months following
treatment. Follow-up was from one year up to 3 and a half years.
Preparation of PRP was done by manual double centrifugation
. A paired Student’s T test was used to compare the progression
of lameness before and after treatment and an ANOVA was used
to see the influence of “radiographic changes”, “chronicity” and
“number of PRP doses”.
Results: In group 1, 75% (9/12) returned to previous athletic per-
formance and of these, 33% (3/9) relapsed while in competition.
In group 2 (10 acute lesions and 20 chronic lesions), 70% (21/30)
regained previous athletic level and 9,5% (2/21) relapsed. There
was a negative effect of “chronicity” (P<0.019) and “radiographic
changes” (P<0,021) on athletic prognosis. There was a significant
difference (p<0.005) between 1 or more doses but not between 2
or 3 doses (p<0,532) of PRP on final outcome.
Conclusions: PRP is a safe, effective, simple treatment that allevi-
ates joint pain as evidenced by lameness examination and clini-
cal parameters.
12.1.8
Serum cartilage oligomeric matrix protein (sCOMP) is elevated
in patients with knee osteoarthritis: a systematic review
J.M. Hoch, C.G. Mattacola, J.S. Howard, C. Lattermann
Lexington/United States of America
Purpose: Serum cartilage oligomeric matrix protein (sCOMP) has
been implicated to be a potential biomarker for osteoarthritis
(OA) diagnosis and progression. The purpose of this systematic
review is to assess whether sCOMP is elevated in patients with
radiographically diagnosed knee osteoarthritis (OA) compared to
healthy controls.
Methods and Materials: Data Sources: A systematic search of the
literature was completed from 1966 to March 2010 utilizing 4 dif-
ferent databases: CINAHL, PEDro, Medline, and Sports Discus.
Keywords: knee, osteoarthritis, serum COMP, radiography. Study
Inclusion Criteria: Written in English, comparison of healthy con-
trols and radiographically diagnosed knee OA, Kellgren/Lawrence
(K/L) classification, measured sCOMP in an adult population, and
reported adequate statistics for calculation of effect sizes (means
and associated variability). Data Extraction: Effect sizes (Cohen d)
were calculated. Values were interpreted as weak if they were less
than 0.40, moderate if between 0.41-0.70, and strong if greater
than 0.71. 95% Confidence Intervals (CI) were calculated for each
point estimate.
Results: Five studies met the inclusion criteria resulting in the
calculation of 15 effect sizes and 95% CIs (Figure). All effect sizes
indicated sCOMP was elevated in those with radiographically diag-
nosed OA (range 0.07-5.8). However, the CI at six point estimates
crossed zero indicating a level of caution when interpreting these
results. Level B evidence exists that sCOMP is elevated in patients
with knee OA. This recommendation was reached based on consis-
tent level 3 evidence or higher (as defined by Centre for Evidence
Based Medicine).
Conclusions: Serum COMP is consistently elevated in patients
with knee OA and is sensitive to OA progression as indicated by
greater effect sizes. In conjunction with other properties, such as
its responsiveness to acute knee injury in younger adults, sCOMP
may be useful in identifying early cartilage damage prior to the
development of fully established OA.
12.1.9
Synovial fluid changes related to pathology
R. Esparza1, J. Moya-Angeler2, P. Martinez de Albornoz1, R. Sanchez
Hidalgo1, I. Zapero1, F. Forriol Campos1
1
Majadahonda/Spain, 2Madrid/Spain
Purpose: Synovial fluid (SF) is a joint lubricant that also nourishes
joint tissues and collects catabolic factors that may contribute to
the degradation of cartilage and meniscus.
Methods and Materials: SF was extracted from 123 patients,
males, between 19 and 58 years of age who were operated on dif-
ferent knee pathologies grouped into cartilage, meniscal and liga-
mentous injuries and as well sub classified as acute injuries, if less
than 6 months of evolution, subacute, between 6 and 12 months
evolution and chronic beyond a year of evolution. Aspiration re-
sulted negative in 53 knees. All patients signed informed con-
sent documents. Extracted liquid concentration was performed
with Centricon ® tubes. We used ELISA technique to detect the
presence of IL-2, IL-10 and TNF-a, using an antibody detection kit
(Biolegend). Fluid was incubated with capture antibodies for 16 h
according to instructions. To detect the signal at the plate TECAN
team used a 540 nm wavelength. A multivariant analysis was per-
formed to establish the relation between time, type of injury and
amount of cytokines.
Results: We found no differences in the detection of cytokines (IL-
2, IL-10) with the evolution time, but an increased of TNF-a in the
SF was observed in the group of more than a year of evolution.
Neither difference between meniscus and ligament injuries was
observed, although we note a trend for increased TNF-a in liga-
mentous injuries.
Conclusions: TNF-a is a pro-apoptotic factor related to knee inflam-
matory processed which seems to be elevated in lesions of more
than one-year evolution and also in ligamentous injuries.

12.2.2
Two-year clinical and radiological evaluation following
a prospective, randomized comparison of traditional and
accelerated approaches to post-operative rehabilitation
following matrix-induced autologous chondrocyte implantation
J. Ebert, M. Fallon, W.B. Robertson, D.G. Lloyd, T.R. Ackland, M.H.
Zheng, D.J. Wood
Perth/Australia
Purpose: Policies on post-operative load bearing rehabilitation following
autologous chondrocyte implantation (ACI) are varied and the subject re-
mains controversial. This study aims to determine the safety and efficacy
of an ‘accelerated’ post-operative weight bearing (WB) regime following
matrix-induced autologous chondrocyte implantation (MACI).
Methods and Materials: A randomized controlled study design was used
to assess clinical and radiological outcome in 70 patients (45 males, 25
females) who underwent MACI to the medial or lateral femoral condyle, in
combination with either ‘traditional’ or ‘accelerated’ approaches to post-
operative WB rehabilitation. Apart from the gradient and time to full WB,
components of both rehabilitation interventions were the same. Under the
‘accelerated’ protocol, patients reached full WB at eight weeks post-sur-
gery, compared to 11 weeks for the ‘traditional’ group. Clinical outcomes
were undertaken pre-surgery and at 3, 6, 12 and 24 months post-surgery.
These included the Knee Injury and Osteoarthritis Outcome Score (KOOS),
the visual analogue pain scale (VAS), the six-minute walk test, knee range
of motion and activity level. High resolution magnetic resonance imaging
(MRI) was undertaken at 3, 12 and 24 months post-surgery.
Results: A significant effect (p<0.05) for time existed for all clinical
measures, demonstrating improvement up until 24 months in both
groups. A significant interaction effect (p<0.05) existed for pain sever-
ity and the six-minute walk test, with accelerated group patients report-
ing significantly less severe pain and demonstrating superior six-minute
walk distance over the period. While there was a significant group effect
(p<0.05) for maximal active knee extension range in favor of the accel-
erated regime, no further significant differences existed. Both groups sig-
nificantly improved (p<0.05) in an MRI composite score and pertinent
descriptors of graft repair throughout the post-operative timeline, up
until 24 months post-surgery, while there were no differences (p>0.05)
observed between the two groups.
Conclusions: The ‘accelerated’ WB approach was not detrimental to graft
development at any stage throughout the post-operative assessment
timeline up until 24 months post-surgery, and may accelerate patient
return to normal function, whilst reducing post-operative muscle loss,
intra-articular adhesions and associated gait abnormalities.
12.2.3
Comparison of knee strength pre-operatively, and at 6 and 12
months post-operatively following autologous chondrocyte
implantation(ACI)
C.G. Mattacola, J.S. Howard, J.M. Hoch, C. Lattermann
Lexington/United States of America
Purpose: The goal of rehabilitation programs involve returning patients
to pre-injury or pre-operative status. Achieving pre-operative strength
levels following surgical intervention is necessary to return patients to
their previous levels of function. Our purpose was to compare knee ex-
tension and flexion strength at pre, 6, and 12 months post ACI surgery.
Methods and Materials: 18 (36.6yrs,174cm,89.09kgs) patients under-
going ACI surgery participated. Concentric and eccentric knee exten-
sion (KE) and flexion (KF) were tested on a BTE (Baltimore MD) isokinet-
ic dynamometer at 60 deg/sec. Testing of the involved and uninvolved
limb was performed pre-operatively(n=18), 6(n=12), and 12(n=7) months post-
operatively. Dependent variables were peak force(N) and percentage of
strength of the involved/uninvolved limb. Paired T-tests and Bonferroni
corrections were performed to compare differences between limbs at
pre, 6 and 12 months
Results: Concentric KE strength was significantly different when the
involved and uninvolved limb were compared at pre (331.28 ±148N
vs. 430.53 ±148N) and 6 months, (266.51 ±75.4N vs. 433.4 ±91N,)
(P<.01) but not 12 months, (284.4 ±140N vs. 383.8 ±118N) (P=.09) .
The involved KE strength was less than 60% of the uninvolved leg
12 months post-surgery(Figure). Concentric KF strength was signifi-
cantly different when the involved and uninvolved limb were com-
pared at pre (284.6 ±119N vs. 337.4.53 ±129N) and 6 months(258.8
±80vN vs. 344.54 ±67N,) (P<.01) but not 12 months (313.15 ±95N
vs. 352.5 ±98N) (P=.12). The involved KF strength was less than
80% of the uninvolved leg 12 months post-operatively (Figure).
Conclusions: Although patients may report in the clinic that they subjective-
ly feel stronger 3 and 6 months after ACI, their quadriceps strengths remains
compromised (<80% of uninvolved side) past 1 year postoperatively. The
clinician and patient need to be aware of this and quadriceps strength train-
ing may need to be emphasized and continued past one year.
Free Papers 169
12.2.4
High patient compliance to an identical rehabilitation guideline
after characterized chondrocyte implantation or microfracture is
related to improved patient functional outcome at 2 years
D. Van Assche1, D. Van Caspel2, F. Staes1, D.B. Saris2, J. Bellemans1,
J. Vanlauwe1, F. Luyten1
1
Leuven/Belgium, 2Utrecht/Netherlands
Purpose: Rehabilitation following cartilage repair procedures and
compliance to rehabilitation are often stated as important, but
rarely studied. The major aim of the study was to explore the effect
of patient compliance on the objective functional outcome up to 2
years after surgery. Therefore an identical rehabilitation program
was implemented and followed-up in the RCT comparing character-
ized chondrocyte implantation (CCI) versus microfracture (MF).
Methods and Materials: In a cohort design patient compliance to
an identical rehabilitation protocol was studied. An electronic re-
port form including 18 physiotherapy variables was used to com-
pare physiotherapy management. Patients’ objective functional
outcome was assessed using the pooled symmetry index (SI)
based on one strength and 3 hop tests. The objective outcomes
were evaluated pre-surgery, at 1 and 2 years post-surgery.
Results: During the first 3 months 85% of the physiotherapists reported
weekly. Overall physiotherapists adhered very consistently and showed
comparable management to the protocol in both treatment groups. In
contrast in both treatment groups patients showed great variability in
the amount of time (minutes per day) they were active in low load activi-
ties. Two cohorts were created. Patients with good compliance to the
rehabilitation protocol (Good Comp, n=21) and patients with poor com-
pliance (Poor Comp, n=17) performed activities in low load conditions
for a mean of 45 minutes a day and 7 minutes a day respectively. These
cohorts were not significantly different for any other parameter. At 24
months after surgery the Good Comp cohort performed the objective
functional tests significantly better compared to the Poor Comp cohort.
The mean pooled SI of the Good Comp cohort was 92.4% compared to
78.2% for the Poor Comp cohort (95%CI 1.8 to 26.2).
Conclusions: High patient compliance to the rehabilitation protocol,
which reflects a high amount of low load activities post-surgery, appears
beneficial for the objective functional outcome at 24 months after surgery.
12.2.5
Changes in functional performance during walking, squatting, rising,
and stepping following autologous chondrocyte implantation (ACI)
J.S. Howard, C.G. Mattacola, J.M. Hoch, C. Lattermann
Lexington/United States of America
Purpose: To document the recovery of functional performance over
6 months following ACI.
Methods and Materials: Participantswere18(37.6yrs,172cm,90.03kgs)
patients undergoing ACI to the knee. All patients completed functional
tests simulating walking, squatting, rising from sitting, stepping-up and
stepping-down using the NeuroCom Long Forceplate(Clackamas, OR),
preoperatively and 3, 6, and 12 months postoperatively. Repeated Mea-
sures ANOVAs were used to compare performance(p<.05).
Results: For walking, improvements from preoperative levels were
seen in stride length, width, and speed 3 months post ACI with sig-
nificant increases in stride length(19%) and decreases in stride
width(8%) at 6 months. During squatting(30°, 60°, and 90°of flexion)
asymmetries in weight distribution were minimal preoperatively(3%,
4%, -2%, respectively) but increased significantly 3 months postop-
eratively(8%, 10%, 6%) and persisted 6 months postoperatively(6%,
7%, 5%(Figure1.). The force generated bilaterally to rise from sitting
increased significantly at 6 months(22%>preoperative). Side-to-
side comparison of rise force demonstrated greater force production
by the uninvolved-limb relative to the involved-limb by 5% preopera-
tively, 14% at 3 months, and 11% at 6 months. There were significant
increases in force generated when stepping-up at 3 months(24%) and
at 6 months(33%) relative to preoperative force(Figure2.). Step-down
impact forces increased above preoperative measures at 3(13%) and
6(21%) months, representing a loss of eccentric control as patients
lowered themselves. Preliminary data from 8 subjects at 12 months
suggests continued improvements in speed and stride length and
some reduction in side-to-side differences; however, between limb
discrepancies for weight distribution and force production remain
greater than preoperative observations.
Conclusions: Despite early improvements in some functional tasks,
6 months post-ACI side-to-side differences in weight distribution and
force production during squatting, rising, and stepping-down persist-
ed. Even 12 months postoperatively some elements of function, par-
ticularly those associated with eccentric control and limb symmetry,
remain below preoperative levels, and further emphasis of these ac-
tivities during rehabilitation is necessary.
170 Free Papers
12.2.6
ACI vs Mosaicplasty for Symptomatic Articular Cartilage Lesions
in the Young Adult Knee. Ten-year Results of Randomized
Comparison Study
G. Bentley1, L.C. Biant2, S. Vijayan3, S. MacMull3
1
London/United Kingdom, 2Edinburgh/United Kingdom, 3Stanmore/
United Kingdom
Purpose: Autologous chondrocyte implantation (ACI) and mosa-
icplasty (MP) are two methods of repair of symptomatic articular
cartilage defects in the adult knee. This study represents the only
long-term comparative clinical trial of the two methods.
Methods and Materials: A prospective, randomized comparison of
the two methods involving 100 patients with symptomatic articular
cartilage lesions was undertaken. Patients were followed for ten
years. Pain and function were assessed using the modified Cincin-
nati score, Bentley Stanmore Functional rating system and visual
analogue scores. ‘Failure’ was determined by pain, a poor
outcome score and arthroscopic evidence of graft disintegration.
Patients had a mean age at index operation of 31. There was a long
mean pre-op duration of symptoms of seven years, and the defects
had an average of 1.5 operations (excluding arthroscopy) to the ar-
ticular cartilage lesion prior to the cartilage repair surgery. The aeti-
ology of the articular cartilage defects was mainly trauma, some pa-
tients had osteochondritis dissecans or chondromalacia patellae.
Results: Five patients were lost to follow-up. A total of 23 out of 42
mosaicplasty patients failed, 10 out of 58 ACI patients failed.
Conclusions: At ten years, patients who underwent cartilage re-
pair using ACI fared significantly better than those who under-
went mosaicplasty.
12.2.7
Prospective Evaluation of Meniscal Transplantation Procedure:
Minimum 7-year Follow-Up
E. Daley, S. Bajaj, J. Kercher, E. Strauss, P.B. Lewis, B.J. Cole
Chicago/United States of America
Purpose: Meniscal transplantation is an accepted method of treat-
ment for meniscal deficient patients. Although mid-term results
have been promising there is limited long-term clinical follow-up
data. The purpose of this study is to report post operative out-
comes of meniscal allograft transplantation by a single surgeon at
a minimum of 7 year follow-up.
Methods and Materials: Clinical results from 20 patients who under-
went meniscal allograft transplantation were evaluated at a mean
follow-up of 8.23 years. Average patient age at the time of surgery
was 32.2 years. 10 meniscal transplants were performed in medial
compartment and 10 in the lateral. Post-operatively, patients were
assessed using the International Knee Documentation Committee
(IKDC), Tegner-Lysholm, Knee Injury and Osteoarthritis Outcome
Score (KOOS), and San Francisco – 12 (SF-12) outcome measures.
Results: The mean IKDC score improved from 50.49 to 64.13
(p<0.05). Similarly mean Lysholm improved from 55.83 to 71.84
(p<0.05). SF-12 physical scores improved from 42.24 to 49.74
and SF-12 mental improving from 45.84 to 48.72. In the KOOS scor-
ing system, pain (63.72 to 79.81) (p<0.05), symptoms (63.69 to
67.03), activities of daily living (80.15 to 89.7), and quality of life
(71.15 to 75.0) improved. Subjectively, on a scale of 1-10, with 10
being completely satisfied with surgical outcome, the mean post-
operative satisfaction rate was 8.0. Additionally, 100% of the pa-
tients would elect to have this surgery again if they had the same
problem on the opposite knee.
Conclusions: The average IKDC, Lysholm, SF-12, and KOOS scores
demonstrated improved patient outcomes at a minimum 7-year fol-
low up. Based upon this data, meniscus transplantation is a viable
treatment option for meniscus deficient patients and can reducing
pain, increase range of motion and improve patient function.
12.2.8
Indication of articular cartilage formation inside Trufit® plugs
one year after implantation as analyzed by delayed Gadolinium
Enhanced MRI of Cartilage (dGEMRIC)
J.E.J. Bekkers, L.B. Creemers, W.J.A. Dhert, D.B. Saris
Utrecht/Netherlands
Purpose: Implantation of Trufit® plugs is an option for treatment
of focal osteochondral lesions. This study analyzed the, quality of,
cartilage formation in Trufit® plugs using a dGEMRIC MRI protocol.
Methods and Materials: A total of 5 patients were analyzed by dGEM-
RIC at 12 months (range 9-15 months) after treatment of an osteo-
chondral lesion in their femoral condyle using Trufit® plugs (Smith
& Nephew, UK). Patients were injected with 0.2mmol/kg Magnevist
(Bayer, Germany) 90min prior to scanning and asked to walk for
15min to facilitate contrast uptake into the knee cartilage. A 3D T1
protocol with 5 inversion times (50, 150, 350, 650 and 1650 ms) and 3
mm slice thickness on a sagittal image acquisition at 1.5T was used.
The T1gd was calculated by averaging the T1gd across the manually
segmented region-of-interest (ROI) using in-house developed soft-
ware. Five different ROIs were defined; the Trufit® plug, cartilage
either surrounding or directly opposing the Trufit® plug, non-artic-
ulating tibial cartilage and femur cartilage (Figure 1). Differences in
T1gd were analyzed by a one-way ANOVA and post-hoc-LSD test.
Results: One year after implantation the T1gd in the Trufit® plug
(391±81 ms) is comparable (p=0.812) to the T1gd in the femur car-
tilage (415± ms) and the cartilage surrounding (392±77 ms) the
Trufit® plug (Figure 2). In addition, the T1gd for the directly ar-
ticulating (opposing) cartilage (543±53 ms) at the tibia and non-
articulating tibia cartilage (508±45 ms) is comparable (p=0.535).
Conclusions: This study showed a comparable T1gd for femur car-
tilage and the Trufit® plug, which suggests the presence of carti-
laginous tissue inside the Trufit® plug one year after surgery. In
addition, articulation between the Trufit® plug and tibia cartilage
surface does not introduce damage in the articulating tibial carti-
lage as shown by the similar T1gd signal between the tibial carti-
lage that was articulating against the implant and non articulating
tibial cartilage.
12.2.9
Chondroprotective effects of a polycarbonate-urethane meniscal
implant in a sheep model
G. Zur1, J.J. Elsner2, E. Linder-Ganz1, J. Shani1, O. Brenner1, E.
Hershman3, F. Guilak4 , A. Shterling1
1
Netanya/Israel, 2Tel Aviv/Israel, 3New York/United States of
America, 4Durham/United States of America
Purpose: Meniscus injury leads to alterations in the knee load trans-
fer, associated with degenerative osteoarthritis. While allograft
replacement can improve joint stability and function, it often pro-
vides little benefit in preventing osteoarthritic changes. Thus, the
development of artificial meniscus could provide therapeutic po-
tential for treatment meniscal injury. We hypothesized that a novel
Polycarbonate-Urethane (PCU) meniscus implant could protect the
cartilage under loading.
Methods and Materials: 6 ewes underwent a full medial meniscecto-
my and were implanted with PCU implants (Fig.1). Joint functionality
was assessed by measuring mobility and joint ROM. Animals were
sacrificed at 3 and 6 months. Cartilage was assessed macroscopical-
ly and by H&E and Saf-O staining using a semi-quantitative modified
Mankin score. The contralateral knee served as control. The score
incorporated cartilage structural changes, proteoglycan loss, chon-
drocyte cloning, osteophyte formation and subchondral bone thick-
ening in the tibial plateau (TP), femoral condyles (FC) and patella.
Results: The sheep tolerated the operations well. Full ROM and no
signs of distress were observed. Examinations of the explanted
implants revealed no structural or property changes. Macroscopi-
cally, cartilage in contact with the implant was well preserved.
Remodeling was observed by the presence of minimal to mild ir-
regularity of the medial rim of the FC and the medial TP. Mild his-
tological changes were observed at 3 and 6 months post-op. At 3
months, Mankin scores for the whole joint were increased from 10
in controls to 17 in operated limbs (p=0.05). However, at 6 months,
no progression was observed and modified Mankin scores were
similar to 3 months, and did not significantly differ from control
(p=0.08), (Fig.2).
Conclusions: Histologic changes were generally mild, as previous
animal studies found significant loss of cartilage structure and
properties post meniscectomy. Our findings suggest that a PCU im-
plant may protect, but not completely prevent, cartilage degenera-
tive changes.

12.3-9
Repair tissue assessment after matrix associated autologous
chondrocyte transplantation with CaReS in the knee –
Quantitative MRI evaluation at 3.0 Tesla
D. Stelzeneder1, S. Domayer1, S. Nehrer2, T. Luksch2, R. Dorotka1, S.
Goed1, T.C. Mamisch3, G.H. Welsch1, S. Trattnig1
1
Vienna/Austria, 2Krems/Austria, 3Berne/Switzerland
Purpose: T2-mapping and delayed Gadolinium-Enhanced-MRI of Carti-
lage (dGEMRIC) are helpful for the assessment of cartilage repair tissue.
The purpose of this investigation was to provide biochemical MRI data
for repair tissue after matrix associated autologous chondrocyte trans-
plantation (MACT) with CaReS in the knee.
Methods and Materials: Fourteen knees of 14 patients (age 38.1±10.0
years [mean±standard deviation]) with a mean follow-up of 2.0±0.9 years
(range 0.9-3.6) were investigated. Twelve defects were located on the
medial femoral condyle, 1 at the lateral femoral condyle, and 1 at the tro-
chlea. All MR-examinations were performed on a 3.0-Tesla MR-unit. In
addition to morphological imaging, T2-mapping and a dGEMRIC (T1Gd)
with 60 minutes delay were performed. The region of interest (ROI)-
evaluation was performed manually on 3 adjacent slices covering the
whole transplant-area. Reference regions were evaluated in the healthy
cartilage of the posterior condyle. The deep and superficial layers were
assessed separately, each measuring 50% of full thickness. Repair tissue
area was measured on morphological sequences. The clinical evaluation
was performed on the day of the MRI using the IKDC knee form, IKDC sub-
jective form, Lysholm score, and a modified Cincinnati rating. Descriptive
statistical data is shown as mean±standard deviation or as median and
interquartile range (IQR). Paired t-tests were used for the analysis of zon-
al differences (deep vs. superficial).
Results: The mean repair tissue size was 3.9±1.4cm2. The median of IKDC
rating was B (IQR A;B), IKDC subjektive 68.3 (IQR 43.4;82.2), Cincinnati 7.5
(IQR 4.5;8) and Lysholm 85.5 (IQR 49.5;95.3). Mean T2-values in millisec-
onds for the repair tissue and reference cartilage were as follows (deep/
superficial, p-values for zonal differences are given): 34.8±6.7/39.8±5.7
(p=0.003) and 38.5±5.1/40.7±5.4 (p<0.001). The respective T1Gd-values
were: 630±170/499±114 (p<0.001) und 885±236/697±162 (p<0.001).
Conclusions: Our results suggest a zonal organisation of repair
tissue after MACT with CaReS, with zonal differences in collagen
structure, water and proteoglycan content.
12.3.2
Development of a Flexible Tissue Engineered Ear
M.A. Randolph1, C.A. Sundback1, I. Pomerantseva1, D.A. Bichara1, X.
Zhao1, K.M. Kulig1, L. Zhou1, E. Bassett1, N. Hwang2, R. Langer2, D.G.
Anderson2, M. Cheney1, T. Hadlock1, J. Vacanti1
1
Boston/United States of America, 2Cambridge/United States of
America
Purpose: Current standard approaches for complete auricular recon-
struction include carved autologous rib cartilage and alloplastic im-
plants provide rigid reconstruction of the ear with suboptimal aesthetic
outcomes. Considerable progress in producing tissues from isolated
cells and degradable scaffolds makes engineered cartilage a feasible
option for auricular repair. One of the largest challenges is maintain-
ing the complex 3D shape of the auricle and avoiding distortion and
shrinkage when supporting scaffold is resorbed and the neocartilage
matures. Degradation products of synthetic materials elicit a host in-
flammatory response negatively affecting neocartilage formation in
immunocompetent animals. Fibrillar collagen, although biocompatible,
is promising but does not possess required strength to withstand scar
contraction forces exerted by skin. The objectives of this study were: 1)
to explore neocartilage formation in an immune competent sheep with
FDA-approved materials, and 2) evaluate a flexible internal metallic
structure to maintain 3D shape.
Methods and Materials: Commercially available porous fibrillar colla-
gen were provided by Kensey Nash Corp. and porous poly(L-lactide)/
poly(DL-lactide-co-caprolactone) (PLA/PCL) scaffolds were synthe-
sized by our MIT team. Sheep auricular chondrocytes were seeded onto
the scaffolds. Formulations were screened in a nude mice. Prototypes
of human-shaped ears were manufactured from fibrillar collagen with
an embedded permanent framework to help retain the shape during
neocartilage formation. Seeded constructs were implanted subcutane-
ously on the back of nude mice and in the neck in sheep.
Results: Neocartilage formation was demonstrated in nude mice and,
most importantly, sheep at 6 wks (Fig.1). A surgical model for implanta-
tion of human size and shaped auricles was developed in sheep (Fig.2).
No extrusion of engineered ears was observed. Retention of ear shape
was demonstrated in nude mice and sheep.
Conclusions: Autologous cartilage was successfully engineered in
sheep. Early results show that our enhanced scaffold prevents distor-
tion and allows retention of the original ear shape.
Free Papers 171
12.3.3
MR evaluation of repair tissue in osteochondral defects following
treatment with acellular scaffolds: high resolution MR-
histological correlation in a goat model
C.S. Winalski, M.F. Freire, F.A. Sakamoto, E. Schneider, R. Midura,
A. Vasanji
Cleveland/United States of America
Purpose: Describe morphologic appearances and quantitative MR
measures of repair tissue following treatment of osteochondral
defects with an acellular scaffold, and correlate MR findings with
histology to determine if MR can predict repair tissue type.
Methods and Materials: 12 goats with surgically created 7mm di-
ameter 6mm deep osteochondral defect were treated with TruFitCB
acellular scaffolds (Smith&Nephew). 6 goats per group were sacri-
ficed at 3 or 9 months postoperatively. Anesthetized animals were
imaged in vivo at 1.5x0.2x0.3mm resolution. Following sacrifice the
excised femoral condyles were imaged at 7T with PDW, T2, and T1-
mapping at 1.0x0.1x0.1mm resolution. Condyles were fixed in for-
malin and dGEMRIC was performed with T1 maps repeated before
and after 48 hrs equilibration in 1mM GdDTPA. Specimens were
processed for histology. T1 and T2 maps were created on a pixel by
pixel basis using Matlab and MRImapper software (MIT/BIDMC).
Results: At 3 months, 3 specimens had deep defects and 3 nearly
filled. All had bone openings with a new bone rim covered by hyaline
cartilage repair tissue. Central repair tissue had low glycosamino-
glycan (GAG) on histology. At 9 months, 4 of 6 defects showed near
complete bony closure with overlying repair tissue slightly below
native cartilage. Repair tissue stained for GAG similar to native
cartilage on histology. dGEMRIC values were similar to adjacent
cartilage. Repair tissue T2 had a bilaminar pattern with lower deep
layer T2, similar to, but less uniform than native cartilage. Repairs
with deep defects at 9 months appeared similar to 3 month group.
All specimens had bone cavities larger than initial surgical defects.
Hyaline-like (high-GAG) repair tissue showed significantly longer
post-GdDTPA T1 on dGEMRIC than fibrous-like repair at all time-
points, 525ms vs 383ms, respectively (p<0.01).
Conclusions: MR accurately tracked healing of defects treated with
acellular scaffolds accurately showing bone defect closure, tissue
fill, and cysts. Quantitative techniques may predict tissue type.
12.3.4
Liposomes-enriched fibrin scaffold in cartilage regeneration.
E. Filova1, M. Rampichová1, A. Lytvynets1, A. Míčková1, J. Uhlík1, L.
Vajner1, L. Martinova2, E. Prosecká1, D. Usvald3, J. Motlík3, E. Amler1
1
Prague/Czech Republic, 2Liberec/Czech Republic, 3Liběchov/Czech
Republic
Purpose: Nanofibers are suitable for cell proliferation. Liposomes
have already been used as drug delivery systems.(1) The aim of the
study was to study cartilage regeneration in minipigs using fibrin scaf-
fold with liposomes-enriched nanofibres containing growth factors.
Methods and Materials: Polyvinyl alcohol nanofibers containg lipo-
somes were incubated with basic fibroblast growth factor and in-
sulin, and mixed with Tissucol®. The liposome-enriched composite
scaffold was implanted into eight load-bearing osteochondral de-
fects in miniature pigs. As a control, the defects in the left knees
were left untreated. Animals were sacrified 12 weeks after the sur-
gery, and examined histologically.
Results: In a scaffold group, a regular formation of isogenic lines
of chondrocytes near the defect bases and differentiation towards
hyaline cartilage were observed. Fibrocartilage was found on the
defect surface. The middle and basal zones of the defects were pre-
dominantly alcian blue positive. Type II collagen was positive in the
non-cellular transient zone in the newly formed cartilage and on the
border of young isogenic groups. Weak positivity was in the centre
of the defect. In a control group, fibrocartilage or unorganized fi-
brous tissue with isogenic groups of chondrocytes situated at the
borders, and fibrous tissue on the surface of the defect were ob-
served. The differentiation was predominantly accompanied by vas-
cularization. Alcian blue was positive in the upper part of defects.
Immunohistochemical examination for collagen II was positive in
newly formed cartilage.
Conclusions: The therapy of osteochondral defects with liposomes-
enriched scaffolds supported the hyaline cartilage formation. The
scaffold is suitable for cartilage regeneration. References (1) Panwar
P, et al. Int J Nanomedicine. 9;5:101-8, 2010. Acknowledgements Sup-
ported by Grant Agency of AS CR grant No. IAA500390702, MSMT
CR grants No. 1M0510 (1M6798582302) and NPV II 2B06130, AV0Z –
ASCR, No. AV0Z50390512 and AV0Z50390703, EU project Bioscent ID
number 214539, Grant Agency of Charles university No. 119209.
172 Free Papers
12.3.5
One stage osteochondral repair with cartilage fragments in a
hyaluronic acid/fibrin glue/platelet rich plasma scaffold: in vitro
human and in vivo rabbit and goat animal model
A. Marmotti1, F. Castoldi1, R. Rossi1, C. Realmuto1, M. Bruzzone1,
D.E. Bonasia1, U. Cottino1, B. Peyrone2, M. Mauthe von Degerfeld2,
P. Rossi1
1
Torino/Italy, 2Grugliasco/Italy
Purpose: Our study evaluate a “one-stage” procedure combining
minced autologous cartilage fragments, as a viable cell source,
with a resorbable scaffold composed of hyaluronic acid (Hyaff-11),
fibrin glue and platelet rich plasma.
Methods and Materials: In vitro: minced cartilage fragments
from human knees (<35y and >50y) and rabbit and goat knees
were cultured onto the scaffold for 1, 2 and 3 months and histo-
logically evaluated. Human cultures were also stimulated with TGF-
beta(10ng/ml) and characterized by immunofluorescence. In vivo
unilateral trochlear model: osteochondral defects were created in
32 adult rabbit knees and 15 goat knees and were either treated
with cartilage fragments embedded in the scaffold (Group “one
stage”) or loaded with scaffold alone (Group “scaffold”) or left un-
treated (Group “empty”). Rabbit were sacrificed at 1, 3, 6 months,
goats at 3, 6, 12 months. Repair process was evaluated with histol-
ogy (modified ICRS and O’Driscoll scores) and immunohistochemis-
try. In goat, nanoindentation of defects have also been performed.
Results: In vitro, chondrocytes from cartilage fragments migrate
and proliferated into the scaffold better in animal than in human
explant cultures. In human, outgrowth was time-dependent and
age-dependent; TGF-beta increased outgrowth at 1 and 3 months;
migrating cells were positive for sox9, CD151 and CD49c and nega-
tive for CD105. In vivo, Group “one stage” showed better quality
of repair tissue than control groups and some typical features of
articular cartilage, as presence of collagen type II in extracellular
matrix. Nanoindentation of goat “one stage” samples showed me-
chanical properties similar with normal trochlear goat cartilage.
Conclusions: Minced cartilage fragments in a HA/fibrin/PRP scaf-
fold provide a viable cell source for one-stage cartilage repair in
rabbit and goat model. In-vitro human cultures showed some limi-
tations, as the age-dependence, and some interesting features, as
the positive stimuli of TGF-beta. Further studies are required to op-
timize human model.
12.3.6
Giant cell foreign body reactions to osteobiologic implants: case
reports
S.K. Tabet
Albuquerque/United States of America
Purpose: This paper reports two confirmed cases of giant cell for-
eign body reaction to osteobiologic implants and offers discussion
of an additional three similar cases which occurred in a larger co-
hort of seventy-two cases. Articular surface defects are a major
cause of joint degeneration. They tend to be progressive resulting
in serious debilitation. A number of techniques are aimed at restor-
ing cartilage but none produce the structure of native cartilage.
Some are done at the time the defect is discovered others require
multiple surgeries and a long time to recovery. Failure of any of
these requires extraordinary measures to correct.
Methods and Materials: Having encountered failure of osteo-
biologic implants (Tru-fit, Smith-Nephew), we retrospectively
reviewed charts, x-rays, MRI and path reports of our series of
seventy-two cases done over a period of four years. We found
five cases in which the mechanism of failure was similar. Two
of these cases had pathology examinations showing giant cell
foreign body reaction. We offer case reports of the two cases
that had path confirmation and a summary of the other three.
Results: In two cases pathology confirmed giant cell reactions. We
have found only one other report of this in the literature.
Conclusions: We suspect that all five cases discussed, because of
their similarity, may have involved foreign body reaction. Failure of the
implant is a serious complication resulting in further joint degenera-
tion and requires at least two further surgeries to achieve resolution.
Awareness of this problem may lead to early recognition or prevention.
12.3.7
Use of a biphasic copolymer scaffold for the treatment of isolated
osteochondral defects of the femoral condyle
F.A. Petrigliano, H. Su, I. Solsky, L.F. Foo, S. Rodeo, T.L. Wickiewicz,
R.F. Warren, H.G. Potter, R.J. Williams, III
New York/United States of America
Purpose: The purpose of this study was to report the outcomes
of patients treated with the TruFit biphasic plug for isolated os-
teochondral defects of the knee, and to assess the morphological
characteristics of these scaffolds using cartilage-sensitive MRI.
Methods and Materials: Thirty patients (31 knees; mean age 44.3
years, range: 18 to 66) underwent TruFit scaffold implantation for
isolated osteochondral lesions. Lesions were located on the MFC
(17), trochlea (11), and LFC (3). An average of 3 plugs (range: 1 to
6; size 7-11 mm) were used per lesion. The mean follow-up inter-
val was 32.8 months (range: 24-48). Subjective outcomes were
assessed using the Activity of Daily Living Score (ADL), Marx Ac-
tivity Scale, and IKDC Score. The mean followup for MRI evalua-
tion was 20.8 months (range: 12-40). Implants were evaluated for
cartilage signal, interface morphology, displacement, hypertrophy,
subchondral edema, bony overgrowth, percentage fill, and degree
of incorporation. ANOVA and linear regression analysis were per-
formed with significance set at p<0.05.
Results: The mean ADL score increased from 62.7 to 82.4 (p<0.001).
The mean IKDC score increased from 45.1 to 71.3 (p<0.001). There
was no significant change in Marx score(p=0.13). MRI evaluation
demonstrated that 90% of plugs were isointense or hyperintense.
69% of plugs demonstrated near complete to complete cartilage
fill. Moderate to good bony incorporation was noted in 64% of
plugs, with 49% demonstrating a flush morphology. Subchondral
edema was minimal to absent in 72% of plugs. Plug hypertrophy,
displacement, and bony overgrowth were rare. MRI appearance of
plugs did not correlate with clinical outcomes. There was a trend
towards poorer outcomes with larger plug configurations.
Conclusions: Treatment of isolated femoral cartilage lesions with
the TruFit biphasic implant results in an improvement in clinical
outcome measures at a minimum two-year follow-up. The majority
of the implanted plugs demonstrated a favorable appearance using
cartilage-sensitive MRI.
12.3.8
Nano-composite biomaterial used as salvage procedure for the
treatment of massive osteochondral lesions.
M. Delcogliano, D. Marotta, F. Pezzillo, C.F. De Biase, R. Giacomi, M.
Manili, A. Delcogliano
Roma/Italy
Purpose: Large osteochondral articular defects are difficult to treat
and present a high rate of complications, such as post-traumatic
joint congruity, axial misalignment and possibly arthritis. In the last
20 years many different approaches have been proposed to restore
the cartilage lesions, and satisfactory results have been obtained.
However, the treatment of osteo-cartilaginous lesions is even more
problematic, because tissue damage is extended also to the sub-
chondral bone, involving two different tissues characterized by dif-
ferent intrinsic healing capacity. The objective of this clinical study
was to test safety and performance of a newly developed type-I
collagen-hydroxyapatite (HA) nanostructural bio-mimetic osteo-
chondral (O.C.) scaffold which reproduces cartilage-subchondral
bone morphology.
Methods and Materials: A gradient composite O.C. scaffold, based
on type-I collagen-HA, was obtained by nucleating collagen fibrils
with hydroxyapatite nanoparticles at physiological conditions. 15
cases with large knee osteochondral lesions and osteochondritis
dissecans were treated as salvage procedure with scaffold im-
plantation. The lesions size went from 3 cm2 to 8 cm2. All patients
achieved minimum 1 year follow up and were clinically evaluated
using the International Repair Cartilage Society score.
Results: IKDC objective score improved after 1 year showing a nor-
mal or nearly normal knee in 80% of patients. Similar results were
obtained with the IKDC subjective score MRI evaluation demon-
strated good bone and cartilage formation.
Conclusions: This open one-step surgery was used for the treatment
of large osteochondral defects. The results of this technique at short
follow-up are very encouraging and show satisfactory results even in
massive ostechondral defects where other techniques had filed.

12.4.2
Characterized Chondrocyte Implantation versus microfracture
for the treatment of symptomatic cartilage defects in the knee in
patients with short onset of symptoms, 60 month follow-up
J. Vanlauwe1, D.B. Saris2, J. Victor3, K.F. Almqvist4 , J. Bellemans1, R.
Verdonk4 , F.P. Luyten1
1
Leuven/Belgium, 2Utrecht/Netherlands, 3Assebroek/Belgium,
4
Gent/Belgium
Purpose: A five-year evaluation of clinical efficacy of Characterized
Chondrocyte Implantation (CCI) compared to microfracture (MF) for
the repair of symptomatic cartilage defects of the femoral condyles.
Methods and Materials: In a prospective, randomized, controlled,
multi-center trial, CCI was compared to MF in patients aged 18-
50 years with a single symptomatic ICRS grade III-IV lesion of the
knee. Clinical outcome was measured 60 months post-surgery by
means of the Knee injury and Osteoarthritis Outcome Score (KOOS)
in subgroups with early onset of symptoms.
Results: In patients with time since onset of symptoms <3 years,
the mean improvement from baseline to 60 months in Overall KOOS
was significantly greater with CCI than MF (26.02+/-3.42 vs. 14.26
+/- 3.01 respectively and p 0.012). There were no apparent differ-
ences between treatments in the subgroup of patients with 3 years
or more since onset of symptoms.
Conclusions: Clinical benefit of CCI over MF in patients with short
duration of symptoms was noted at 3 years post-surgery. Time to
treatment has shown to affect the outcome at 5 years post-surgery,
i.e. early treatment with CCI improves long-term clinical outcome.
12.4.3
The full arthroscopic technique of autologous chondrocyte
transplantation using chondrospheres - safety study with 2 years
follow up
S. Roessing1, T. Schreyer2, P. Baum3, H. Thermann4 , H. Paessler4
1
Mannheim/Germany, 2
Darmstadt/Germany, 3
Gundelfingen/
Germany, 4Heidelberg/Germany
Purpose: The most matrix based procedures in ACT require an open
surgical procedure for the cell transplantation. The aim of the study
was to prove the feasibility and safety of the full arthroscopic ap-
proach using spheroids (ACT3D/Codon).
Methods and Materials: Prospective cohort study; Level of evi-
dence 3. multi center study.Mean follow-up was 2 +/- 0.1 years.
42 patients (mean 38,3 years, 18 to 50 yrs) underwent full
arthroscopical autologous chondrocyte implantation with
spheroids (ACT3D Codon) of full size defects in the knee
with a mean defect size of 4,7 cm² (range 2,5 to 9.5cm²).
Knee function was measured by Lysholm- Womac-D score and VAS.
All patients underwent an one year follow up MRI. For MRI imaging
the MOCART score was used.
Results: The study showed no increased risk or severe adverse
events in the arthroscopic application. The two year follow up
showed significant improved results in the Lysholm, Womac D and
tegner activity scoring. No revision surgery was needed during fol-
low up time. We implanted in the mean 38,5 spheroids/cm². This is
corresponding to 7,7 million cells/ cm². The threshold dose of clas-
sical ACT of 1 million cells /cm² (5/spheroids/cm²) was not under
runned. The intraoperative loss of spheroids was estimated by the
surgeons underneath 10% (5 Spheroids). MRI revealed that in the
majority of cases, filling of the defect was accomplished with a non
homogeneous surface and structure of the graft.
Conclusions: The full arthroscopic application of autologous chon-
drocyte implantation using chondrospheres is a safe procedure
that yields to significant functional improvement in the 2 years
follow up time. An arthrotomy causing prolonged rehabilitation
and increased postoperative pain can be avoided. During opera-
tion no conversion to open procedure was necessary. The risk of
intraoperative spheroid loss can be controlled by an experienced
arthroscopic surgeon. Even retropatellar defects can be reached
without everting the patella.
Free Papers 173
12.4.4
Effect of gender in the follow-up after matrix-associated
autologous chondrocyte transplantation (MACT) as assessed by
morphological and biochemical magnetic resonance imaging
G.H. Welsch1, S. Werner1, D. Stelzeneder1, T.C. Mamisch2, F. Hennig3,
S. Domayer1, L. Zak1, S. Marlovits1, A. Kautzky-Willer1, S. Trattnig1
1
Vienna/Austria, 2Berne/Switzerland, 3Erlangen/Germany
Purpose: By means of MRI, the morphological constitution of the re-
pair tissue can be assessed by the magnetic resonance observation
of cartilage repair tissue (MOCART) score, whereas the biochemical
collagen ultra-structure and the water content can be quantified by
T2 mapping. The objective of this study was to determine gender
related differences in the repair tissue after MACT as assessed by
advanced morphological and biochemical MRI.
Methods and Materials: 40 patients (20 female, 20 male) after MACT
of one femoral condyle of the knee were included. Both groups were
matched by age (female:35.1±13.7; male:35.2±8.7 years) and post-
operative follow-up (female:34.0±17.4; male:34.2±16.8 months).
At 3.0 Tesla-MRI, morphological MOCART scoring including nine
variables was based on high-resolution PD-TSE, Dual-FSE and TIRM
sequences; biochemical T2 mapping was based on a multi-echo
spin-echo sequence. A zonal (deep and superficial) region-of-inter-
est analysis was performed to assess the quantitative T2 values in
native control cartilage and cartilage repair tissue.
Results: The MOCART score was comparable in female (69.4±20.0)
and male (69.3±13.0) patients (p=0.982). Seven of the nine vari-
ables did not differ significantly. Whereas the structure of the re-
pair tissue was more homogeneous in female patients (p=0.026),
changes in the subchondral bone and bone marrow edema were
less often visible in male patients (p=0.007). Quantitative T2 values
(ms) showed comparable results for the native control cartilage (fe-
male: deep=47.7±9.6, superficial=53.1±9.2; male: deep=49.8±12.6,
superficial=53.7±10.5; p=0.445(deep), p=0.826(superficial));
whereas the repair tissue showed significant shorter T2 values of
the deep cartilage layer in female compared to male patients ((fe-
male: deep=43.5±9.8, superficial=48.5±9.8; male: deep=48.2±7.7,
superficial=52.6±11.0; p=0.009(deep), p=0.052(superficial)).
Conclusions: Our initial results suggest that there are differences in
the ultra-structure of the repair tissue between female and male pa-
tients after MACT. Based on the morphological and the biochemical
results, these differences might be due to changes in the subchon-
dral bone and the adjacent deep cartilage layer.
12.4.5
Time-line for self-reported changes in function following
autologous chondrocyte implantation (ACI) over one year
C. Lattermann, J.S. Howard, J.M. Hoch, C.G. Mattacola
Lexington/United States of America
Purpose: Functional improvement following ACI is of great interest to
clinicians and patients. The purpose of this study is to describe the pat-
tern of self-reported recovery for patients undergoing ACI to the femo-
ral condyle or patellofemoral joint and provide comparative data for
individuals who failed treatment.
Methods and Materials: Prospectively, 7 patients(38yrs, 176.5cm,
80.2.4kg) undergoing ACI for chondral defects to the femoral condyles
and 10 patients (33yrs, 1746cm, 91.2kg) undergoing ACI for patell-
ofemoral defects were enrolled. All patients completed the IKDC and
Lysholm Scale preoperatively, 3, 6, and 12 months postoperatively. Re-
peated measures ANOVAs were used to evaluate differences between
defect locations and between patients considered clinical successes
(n=13) or clinical failures(n=4, 2 each location) at 12 months.
Results: There were no main effects for location. There were significant
differences(p<.05) between scores for successes and failures (Figures
1&2). At 3 months, IKDC scores for successes improved 8% but decreased
2% among failures. At 6 months, IKDC scores in the success group im-
proved 27% and decreased 4% in the failure group from preoperative
levels. At 12 months success patients improved 35%, while failure pa-
tients decreased 2% from preoperative IKDC scores. For Lysholm scores
successes improved 20% while failures decreased 3% at 3 months. At
6 months successes improved 38% while failures decreased 2% below
preoperative scores. At 12 months, success patients improved 43%
and failure patients improved 1% from preoperative Lysholm scores.
Conclusions: Using the IKDC and Lysholm scale, unique, although not
statistically different, recovery patterns can be observed between fem-
oral and patellofemoral ACIs (Figures1&2). While the overall number of
failures is small, they demonstrate significantly lower IKDC and Lysh-
olm scores as early as 3 months postoperatively. Though there may be
some debate regarding when clinical failure occurs, evidence of failure
was seen as early as 3 months postoperatively, prompting consider-
ation to follow early self-reported outcomes closely.
174 Free Papers
12.4.6
What is the impact of untreated severe cartilage damage on
clinical outcomes and osteoarthritis progression in the knee? A
natural history study.
W. Widuchowski1, G. Kwiatkowski2, R. Faltus2, A. Mroczka2, J.
Widuchowski2
1
Katowice/Poland, 2Piekary Śląskie/Poland
Purpose: Deep cartilage lesions, left with no treatment are considered
to cause permanent knee deterioration and to advance the develop-
ment of osteoarthritis. But, is it always like that? We aimed to evaluate
whether and to what extent isolated deep cartilage lesion localized with-
in tibiofemoral joint and patellofemoral joint has an impact on the clin-
ical outcomes and osteoarthritis progression when it is left untreated.
Methods and Materials: 37 patients with single isolated chondral
lesion of Outerbridge grade 4 located within weight-bearing ar-
eas of femoral and tibial condyles (FT group) and patella (P group)
were included in the study. The lesion size ranged from 2 to 4 cm2.
In 26 cases (70.3%) the lesion was left without any treatment. In
other cases following procedures were performed: debridement
of the lesion, loose body removal, shaving. The mean follow-up
was 15.3 years. Outcomes were collected reported using Lysholm
score, Tegner activity scale and Womac score. Radiographic eval-
uation was performed according to Kellgren-Lawrence scale.
Results: Osteoarthritic changes were found in 39% patients. There was
no difference in OA frequency and severity between injured and unin-
jured knee. In patients of FT group there was a relationship between
the incidence of tibiofemoral OA and patellofemoral OA (p = 0.00075).
Both groups demonstrated good overall clinical results. Worse out-
comes according to all scores were observed in P group (p<0.05).
Conclusions: Severe isolated single chondral damage left with no treat-
ment has a limited influence on clinical outcomes and development of
osteoarthritis. Lesions located within patella may predispose to ad-
vanced knee deterioration. The overall osteoarthritis progression must
be the result of the influence of different local and systemic factors.
12.4.7
Correlation of tissue histomorphometry with ICRS histology
scores in biopsies obtained from a randomized controlled clinical
trial comparing BST-CarGel™ versus microfracture
C.D. Hoemann1, N. Tran-Khanh1, S. Methot2, G. Chen1, C. Marchand1,
V. Lascau-Coman1, E. Rossomacha2, C. Jarry2, A. Restrepo2, W.D.
Stanish3, P. MacDonald4 , N. Mohtadi5, P. Marks6, M. Malo1, R.
McCormack7, J. Desnoyers8, S. Pelet9, F. Lopez-Olivia10, J. Vaquero10,
F. Macule11, M.S. Shive1, M.D. Buschmann1
1
Montreal/Canada, 2Laval/Canada, 3Halifax/Canada, 4Winnipeg/
Canada, 5Calgary/Canada, 6Toronto/Canada, 7Vancouver/Canada,
8
Greenfield Park/Canada, 9Quebec City/Canada, 10Madrid/Spain,
11
Barcelona/Spain
Purpose: BST-CarGel™ (BioSyntech Inc, Laval, Canada) is a novel thera-
py where an in situ-solidifying implant of chitosan and autologous blood
is applied to fresh microfracture defects, to stabilize the blood clot and
stimulate marrow-derived repair. In an 80-patient randomized clinical
trial comparing BST-CarGel™ to microfracture to treat condylar lesions,
an interim analysis was carried out that included 21 osteochondral bi-
opsies obtained from patients with informed consent after 13 months
post-treatment. The aim of this study was to correlate repair tissue
histological quality with histomorphometric staining characteristics.
Methods and Materials: Biopsies were obtained with REB-approved pro-
tocols from the middle of the original condylar lesion at 13 months post-
treatment (N=21), fixed in formalin, decalcified, processed in paraffin and
5 µm sections stained with H&E and Saf-O/Fast Green (SafO), or immu-
nostained for collagen type I (Col1) and collagen type II (Col2). 3 readers
carried out analyses on blinded sections including histological scoring
(ICRS II Overall on a scale of 0=worst to 100=normal hyaline cartilage) and
histomorphometry for each stain (percent stained tissue area). Overall
scores were correlated by calculating Pearson’s correlation coefficients.
Results: A range of repair quality was obtained that could be classified
based on ICRS scores into good/acceptable/poor categories (Fig. 1).
A subgroup within the “poor” category for ICRS scores was found to
have strong SafO and Col2 staining yet very poor structural integrity.
When these 3 disintegrated samples were removed from the analysis,
strong correlations were obtained between histomorphometry and
ICRS evaluations (Fig. 2). These data showed that overall histological
score correlated positively with SafO and Col2 staining and negatively
with Col1 (p<0.05).
Conclusions: Strong correlation between histological scores and quan-
titative histomorphometry in cartilage repair can be obtained when
overall tissue integrity is accounted for.
12.4.8
BST-CarGel™ treatment shows trend of improved collagen
architecture and stratified hyaline structure compared to
microfracture in 13 month biopsies from an interim analysis of a
randomized controlled clinical trial
A. Changoor1, M. Nelea1, N. Tran-Khanh1, S. Methot2, A. Restrepo2,
W.D. Stanish3, P. MacDonald4 , N. Mohtadi5, P. Marks6, M. Malo1, R.
McCormack7, J. Desnoyers8, S. Pelet9, F. Lopez-Olivia10, J. Vaquero10,
F. Macule11, M. Shive2, M.D. Buschmann1
1
Montreal/Canada, 2Laval/Canada, 3Halifax/Canada, 4Winnipeg/
Canada, 5Calgary/Canada, 6Toronto/Canada, 7Vancouver/Canada,
8
Greenfield Park/Canada, 9Quebec City/Canada, 10Madrid/Spain,
11
Barcelona/Spain
Purpose: The biodegradable and natural polymer of glucosamine, chi-
tosan, can be used to physically stabilize an autologous blood implant
above a debrided and microfractured cartilage lesion. This concept is the
basis of the BST-CarGel™ product (BioSyntech Inc., Laval, Canada) that
has been compared to microfracture in a randomized controlled clinical
trial. Here we report biopsy analyses of collagen architecture and zonal
stratification using a novel polarization light microscopy (PLM) method.
Methods and Materials: An interim analysis was carried out on 21
elective condylar biopsies (12 BST-CarGel™, 9 Microfracture) obtained
from the RCT at 13 months post-treatment. They were fixed, decalcified,
embedded and sectioned to 5µm. Blinded consecutive sections were de-
paraffinized and examined unstained by 3 readers using PLM and Scan-
ning Electron Microscopy (SEM) after metallic coating. An ordinal scale
to grade collagen architecture and zonal stratification was applied using
PLM birefringence characteristics: 0=no organization, 1= vertical deep
zone apparent, 2= deep zone well developed, 3= three zones present, 4=
normal zonal proportions. Parametric or non-parametric statistical tests
were used to compare groups depending on distribution normality.
Results: The novel PLM and SEM methods and scores were sensitive to re-
pair tissue collagen structure (Fig. 1) A majority of the BST-CarGel™ treated
biopsies (7 of 12) revealed a well-organized and stratified collagen architec-
ture with a PLM score > 2 (median 2.67) while this desired structure was
only occasionally observed with microfracture (2 of 9) (median 1.33) (Fig. 2).
Due to small sample sizes (12 BST-CarGel™, 9 Microfracture), the effect of
treatment on the PLM score was not statistically significant (p=0.099).
Conclusions: Augmentation of bone-marrow derived cartilage re-
pair using a chitosan scaffold stabilized autologous blood implant
could improve zonal stratification and collagen architecture at 13
months post-treatment compared to microfracture alone. Since
collagen structure is the main determinant of tissue durability, this
treatment may sustain benefits longer than microfracture.
12.4.9
Comparison of the size of the cartilage femoral condyle lesions in
CARTIPATCH® phase III clinical trial versus mosaicplasty
F. Dubrana1, J. Potel2, C. Bussiere3, E. Servien3, H. Robert4 , E.
Stindel1
1
Brest/France, 2Toulouse/France, 3Lyon/France, 4Mayenne/France
Purpose: The tolerance and efficacy of CARTIPATCH®, autologous
chondrocyte implant containing an agarose/alginate gel is fur-
ther demonstrated through a prospective, randomized, controlled
trial versus mosaicplasty, for treatment of femoral condyle lesions.
Methods and Materials: 64 patients, between 18 and 50 years old, with
osteocartilaginous single lesion (2.5 to 7.5cm2) are randomized for
first-line treatment, either into the CARTIPATCH® or the mosaicplasty
arm (Arthrex instruments). CARTIPATCH® superiority is expected on
the basis of IKDC subjective scoring at 24 months. For size evaluation
(MRI and arthroscopy), the lesions were assimilated to rectangles.
Results: 47 patients underwent surgery, 25 in CARTIPATCH® arm and
22 in mosaicplasty arm. The population mean age (15 women, 33 men)
was 28 years old. At inclusion, the mean IKDC subjective scores were 41
(26-55) for CARTIPATCH® arm and 36 (18-53) for mosaicplasty arm. In
CARTIPATCH® arm, the lesion size, evaluated either by the surgeon or by
an independent expert ranged respectively between 2.3-7cm2 (mean:
3.7cm2) and 1.6-4.9cm2 (mean: 2.9cm2, 14 patients). In mosaicplasty
arm, the sizes were respectively between 2.5-7.0cm2 (mean: 4.0cm2)
and 1.0-5.9cm2 (mean: 3.1cm2, 18 patients). During arthroscopy, the
mean size of the lesion was 3.8cm2 for both arms (CARTIPATCH®: 0.5-
8.0cm2, 13 patients; mosaicplasty: 2.0-7.0cm2, 11 patients).
Conclusions: The preliminary characterization of the trial population
shows that a wide range of lesion sizes and associated clinical signs
is represented in this young population of patients. MRI evaluation
of the lesion size seems to be globally lower when evaluated blindly
as compared with the surgeon estimation. The lattest is closer to the
size measured during arthroscopy. This hazard was prevented in the
ongoing phase III clinical trial CARTIPATCH® versus microfracture as
the lesion size is measured during arthroscopy.

17.1.2
ICRS histology scores of biopsies from an interim analysis
of a randomized controlled clinical trial show significant
improvement in tissue quality at 13 months for BST-CarGel™
versus microfracture
S. Méthot1, C.D. Hoemann2, E. Rossomacha1, A. Restrepo1, W.D.
Stanish3, P. MacDonald4 , N. Mohtadi5, P. Marks6, M. Malo2, R.
McCormack7, J. Desnoyers8, S. Pelet9, F. Lopez-Olivia10, J. Vaquero10,
F. Macule11, M.S. Shive2, M.D. Buschmann2
1
Laval/Canada, 2Montreal/Canada, 3Halifax/Canada, 4Winnipeg/
Canada, 5Calgary/Canada, 6Toronto/Canada, 7Vancouver/Canada,
8
Greenfield Park/Canada, 9Quebec City/Canada, 10Madrid/Spain,
11
Barcelona/Spain
Purpose: BST-CarGel™ (BioSyntech Inc., Laval, Canada) is a mix-
ture of a physiological solution of the natural polymer chitosan
(polyglucosamine) and autologous blood that is applied on top of
a debrided and microfractured cartilage lesion to improve cartilage
repair from subchondral bone. The purpose of this study was to
compare the histological quality of cartilage repair by BST-CarGel™
versus microfracture at 13 months in a randomized clinical trial.
Methods and Materials: Voluntary osteochondral biopsies (12 BST-
CarGel™, 9 microfracture) from the central region of the original lesion
on the femoral condyle were obtained after 13 months post treatment
from a total of 41 patients in an interim analysis. They were fixed in For-
malin, decalcified, and 5 μm paraffin sections stained with H&E and
Saf-O/Fast Green. Both the ICRS I and ICRS II histological scores for car-
tilage quality were obtained by 3 readers that were blinded to sample
identity. Scores were unblinded and statistically analysed using para-
metric or non-parametric tests depending on distribution normality.
Results: The ICRS II score contains 14 parameters on a continuous
scale and is an expanded version of the ICRS I system which has 6
parameters evaluated on an ordinal scale. Improvements in qual-
ity that exceeded 10% (range of improvement 10% - 80%) for the
repair tissue of BST-CarGel™ versus microfracture were detected
in 10 of the total 20 parameters (Table 1). Only one parameter was
better in microfracture (Table 1), while 9 were similar (< 10%
difference). Several tissue characteristics were significantly im-
proved (p<0.05) for BST-CarGel™ versus microfracture includ-
ing the Overall Score and Superficial Zone score from ICRS II and
the Surface Score and Cell Viability Score from ICRS I (Figure 1).
Conclusions: A statistically significant improvement in the quality of
cartilage repair was found in patients treated with BST-CarGel™ com-
pared to microfracture, suggesting a greater durability of repair and
sustained clinical benefit.
17.1.3
Mid-term results of Autologous Matrix Induced Chondrogenesis
(AMIC) for treatment of focal cartilage defects in the knee
J. Gille1, J. Wimmer2, E. Schuseil2, J. Gellissen1, S. Wallstabe1, P.
Behrens1
1
Hamburg/Germany, 2Lübeck/Germany
Purpose: Autologous Matrix Induced Chondrogenesis (AMIC) is an
innovative treatment for localized full-thickness cartilage defects
combining the well-known microfracturing with collagen scaffold and
fibrin glue. The purpose of this prospective study was to evaluate the
medium-term results of this enhanced microfracture technique for
the treatment of chondral lesions of the knee.
Methods and Materials: Thirty-two chondral lesions in 27 patients
were treated with AMIC. Within the context of clinical follow-up, these
patients were evaluated for up to 5 years after the intervention. Five dif-
ferent scores (Meyer scoreMeyer score, Tegner score, Lysholm score,
ICRS score, Cincinatti score) were used for outcome analysis. Articu-
lar resurfacing was assessed by magnetic resonance imaging (MRI).
Results: The average age of patients (11 females, 16 males) was 37
years (range 16 to 50 years). The mean defect size of the chondral le-
sions was 4.2 cm2 (range 1.3 to 8.8 cm2). All defects were classified
as grade IV according to the Outerbridge classification. The follow-
up period was between 24 months and 62 months with a mean of 37
months. Twenty out of 23 individuals (87%) questioned were subjec-
tively highly satisfied with the results after surgery. Significant im-
provement (p<0.05) of all scores was observed as early as 12 months
after AMIC and further increased values were notable up to 24 months
postoperatively. MRI analysis showed moderate to complete filling
with a normal to incidentally hyperintense signal in most cases.
Conclusions: AMIC is an effective and safe method of treating symp-
tomatic full-thickness chondral defects of the knee in appropriately
selected cases. However, further studies with long-term follow-up are
needed to determine if the grafted area will maintain structural and
functional integrity over time.
Free Papers 175
17.1.4
TruFit plugs for cartilage repair in the knee. 4 year results and
refining the indications
T. Spalding1, P. Thompson2, M. Dhillon2, J. Bird2
1
Leamington Spa/United Kingdom, 2Coventry/United Kingdom
Purpose: We report the four year results of TruFit plugs for osteo-
chondral and chondral repair of articular cartilage defining the indi-
cations and outlining the gradual regeneration of articular cartilage
surface by ingrowth from the surrounding healthy cartilage and the
from the marrow elements
Methods and Materials: From a consecutive prospective series of
40 patients, 27 have minimum 2 year followup and form the study
group, with clinical scores 6 monthly and MRI scans annually. Mean
defect size was 1.8cm square and the mean patient age was 35.
Three patients had osteochondral defects.
Results: Mean IKDC subjective score improved from 42.5 preop to
69 at 12 months, 76 at 18 months and 81 at 24 months. Tegner ac-
tivity score improved from a mean of 2.8 preop to 5.7 at 2 years
without deterioration. Lysholm score improved from 53 preop to 83
at 24 months. All results are significant (p<0.05). All modalities
of the KOOS score were improved particularly sport and quality of
life domains. MRI showed persistence of bone marrow oedema in
20% up to 12 months and gradual remodelling of subchondral bone
over 24 months. Second Look arthroscopy showed two patterns of
regeneration – ingrowth from surrounding cartilage and from the
subchondral bone marrow elements. No patients were revised in
this series but two patients developed persistent effusions requir-
ing treatment. In both, results improved between 18 and 36 months
post implantation.
Conclusions: TruFit CB plugs remain soft for up to 9 months and are
not indicated where there is no surrounding bone for support. MRI
scans show reformation of the subchondral lamina with no bony
overgrowth. Results are maintained to 4 years without reduction
challenging the results of microfracture which is indicated in similar
size lesions. The plugs have defined indications and are not indi-
cated for larger areas.
17.1.5
New nanostructured biomimetic scaffold for the treatment of
ostechondral defects: pilot clinical study at 3 years follow-up
E. Kon, G. Filardo, A. Di Martino, S. Patella, S. Zaffagnini, L. D’Orazio,
G. Altadonna, M. Marcacci
Bologna/Italy
Purpose: The ideal osteochondral graft should be an off-the-shelf
product. For the clinical pilot study we performed, a newly devel-
oped nanostructured biomimetic scaffold was used to treat chon-
dral and osteochondral lesions of the knee; its safety and manage-
ability, as much as the surgical procedure reproducibility and the
clinical outcome, were evaluated in order to test its intrinsic poten-
tial without any cell culture aid.
Methods and Materials: 30 patients (9F, 21M, mean age 29,3yy)
affected by either chondral or osteochondral lesions of the knee
underwent the scaffold implantation. The sizes of the lesions were
in between 2 and 6 squared cm. All patients and their clinical out-
come were analyzed prospectively at 6, 12, 24 and 36 months using
the Cartilage standard Evaluation Form as proposed by ICRS and an
high resolution MRI.
Results: We observed a statistically significant scores improve-
ment and function recovery comparing the pre-operative to the
follow-up parameters evaluated. Moreover, we noticed a bet-
ter improvement from 12 to 24 months follow-up while the good
results gained at 2yy were confirmed at 3yy follow up evalua-
tion. The MOCART scoring scale was used to analyze the MRIs.
In most of cases we obtained a complete filling of the cartilage de-
fect and in some patients we even appreciated articular surface
congruency. In this series we report 1 failure followed by a re-oper-
ation with different technique.
Conclusions: This new minimally invasive one-step surgical approach
to osteochondral defects seems to be an easy and effective procedure.
176 Free Papers
17.1.6
Influence of the cell differentiation at the timepoint of
transplantation on the clinical outcome of matrix-associated
chondrocyte transplantation (MACT)
C. Albrecht, L. Zak, B. Tichy, S. Hosiner, V. Vecsei, S. Marlovits
Vienna/Austria
Purpose: Chondrocytes lose their specific properties and become
fibroblast-like, when they are propagated in monolayer culture for
MACT. This process is called dedifferentiation. A lot of efforts have
been made to develop different stratagies in order to redifferenti-
ate the cells prior transplantation. The aim of this study has there-
fore been to analyse the effect of chondrocyte differentiation at the
timepoint of transplantation on the clinical outcome of MACT after
6, 12 and 24 months.
Methods and Materials: Gene expression of chondrocyte markers
(Col1, Col2, aggrecan, versican and Gdf-5) were analysed in trans-
plant samples (HYAFF, Bio-Gide, Cares) of 90 patients by quanti-
tative real-time PCR. For the evaluation of cell differentiation two
differentiation indices were calculated as the ratio of Col2/Col1
and aggrecan/versican expression. From the same patients clinical
scores (Brittberg, IKDC, KOOS, Noyes, Tegner, and VAS pain) were
obtained 6, 12 and 24 months after surgery.
Results: The three scaffolds significantly differed in gene expres-
sion. However, no correlation between the analysed factors and
the clinical outcome was found. Also the differentiation indices
showed no significant correlation with clinical outcome. Only a
slight tendency was found that low matrix production resulted in
better IKDC and KOOS scores.
Conclusions: The data of the analysed factors demonstrate that the
differentiation of cells at the time point of transplantation does not
affect the clinical outcome after MACT. In fact other parameters -
such as biological conditions in the joint, weight bearing and after-
treatment - seem to play a much more important role for the suc-
cess of the MACT procedure.
17.1.7
Prospective Evaluation of Autologous Chondrocyte Implantation
Procedure: Minimum 7-year Follow-Up
E. Daley, S. Bajaj, J. Kercher, M. Salata, B.J. Cole
Chicago/United States of America
Purpose: Autologous chondrocyte implantation (ACI) is a common
method for the treatment of full thickness cartilage lesions; how-
ever long-term clinical follow-up data is limited. The purpose of this
study is to report clinical results following ACI from a single sur-
geon at a minimum of 7 years follow-up.
Methods and Materials: Data was collected prospectively on 21 pa-
tients who underwent ACI by a single surgeon. The mean follow-up
time was 8.15 years. Post-operatively, patients were assessed us-
ing the International Knee Documentation Committee (IKDC), Tegn-
er-Lysholm, Knee Injury and Osteoarthritis Outcome Score (KOOS),
and San Francisco – 12 (SF-12) outcome measures.
Results: The mean IKDC score improved from 38.82 to 61.65
(p<0.05). The mean Lysholm score showed an increase from 45.78
to 71.31 (p<0.05). SF-12 physical scores improved from 45.06 to
47.48, with SF-12 mental improving from 40.77 to 44.71 (p<0.05).
In the KOOS scoring system, pain (53.10 to 73.92), symptoms
(49.29 to 71.43), activities of daily living (66.18 to 86.39), and qual-
ity of life (61.16 to 81.25) improved. Subjectively, on a scale of 1-10,
with 10 being completely satisfied with surgical outcome, the mean
post-operative satisfaction rate was 8.18. Additionally, 93% of the
patients would elect to have this surgery again if they had the same
problem on the opposite knee.
Conclusions: The average IKDC, Lysholm, SF-12, and KOOS scores
demonstrated improved patient outcomes at a minimum 7-year fol-
low up. Based upon this data, ACI is a viable treatment option for
patients with symptomatic full thickness cartilage lesions and can
reduce pain, increase range of motion and improve patient function.
17.1.8
Articular cartilage repair using collagen type I hydrogels (CaReS-
technology)– Results of a multicenter study
U. Nöth
Würzburg/Germany
Purpose: Different technologies are available for matrix-based au-
tologous chondrocyte implantation (ACI). Since 2003, over 2000
patients have been treated with a collagen type I hydrogel (CaReS®
technology) in Europe. This study investigated the outcome of ar-
ticular cartilage repair in the knee using collagen type I hydrogels.
Methods and Materials: We performed a prospective multicenter
study in 9 centres including 116 patients to test the outcome of the
CaReS- technology. The International knee documentation score
(IKDC), as well as the patient and doctor satisfaction have been in-
vestigate before, 3, 6, 12, 24, 36, 48, and 60 months after surgery.
Results: The mean follow-up was 30.7 months with an IKDC of 70.5.
Patients with osteochondral defects showed an IKDC of 80.4, pa-
tients with chondral lesions only an IKDC of 68.2. Patients with defect
sizes more than 4 cm2 showed an IKDC of 72.8, while patients with
defects smaller than 4 cm2 showed an IKDC of 69.6. For the defect
site, best results were found at the condyles (IKDC 66.7) compared
to defects of the patella (IKDC 61.7). The patient and doctor satis-
faction showed in 79.4% and 85.3% very good and good results.
Conclusions: Patients with osteochondral lesion showed bet-
ter clinical results compared to chondral lesions. Concerning the
defect size (< 4 cm2 or > 4 cm 2) the results were not statisti-
cally significant different. Defects located at the condyles showed
significant better results when compared to defects located at the
patella. In summary, the results are comparable to those reported
with other scaffold materials for ACI.
17.1.9
Comparison in clinical outcome of first generation ACT with ACT
of third generation with spheroids
T. Schreyer
Darmstadt/Germany
Purpose: The purpose of this retrospective study was to ex-
amine if the better clinical outcome in patients after ACT with
spheroids in arthroscopical technique is due to the new kind of
cells (spheroids) or due to the different operations procedure.
Therefore we compare the clinical outcome of ACTs with perioste-
um flaps to those with spheroids in open procedure and those with
spheroids in arthroscopic procedure.
Methods and Materials: Between 1998 and 2004 we treat-
ed 40 patients with cartilage defects grade 3 -4 acc. to
Outerbridge classification with ACT with perosteum flap
technique (chondrotransplant, codon)= first generation.
Between 2005 and 2009 we treated 15 patients with spheroid tech-
nique (chondrosphere, codon, Teltow, Germany) in open procedure
and 16 patients with arthroscopic procedure = third generation.
We examined after 3 months, 6 months, 12 months and afterwards
every 12 months from 3-7 years with 5 different scores (DGKKT,
HSS, Lysholm, Tegner and Cincinnati - Score).
Results: We show that the improvement in clinical outcome in
the arthroscopic group is much faster than in the open spher-
oid procedure and much faster than in the periosteum group.
The patients of the periosteum group reached almost full score points
after 6 monhts, whereas with open spheroid techique and with perois-
teum technique it lasted up to two years until the get the same level.
Furthermore we can show that the longevity results of spheroid
technique are as good as with the periosteum technique as far as
we can oversee up to now.
Conclusions: We conclude that the big difference in clinical out-
come between the three techniques compared results from the
different operations technique (arthroscopic - open) and not
so much from the different status of the chondrocytes used.
The greater number of cells per scm seems to be subordinate.
We expect the longtime results at least as good as the ones with
the periosteum technique.

17.2.2
Autologous Chondrocyte Implantation for Cartilage Lesions with
Malalignment of the Patellofemoral Joint -Long Term Results
H.S. Vasiliadis1, M. Brittberg2, A. Lindahl3, L. Peterson3
1
Ioaninnina/Greece, 2Kungsbacka/Sweden, 3Gothenburg/Sweden
Purpose: The aim of our current study is to present the long term
follow up of patients with cartilage lesions of the patellofemoral
joint, treated with Autologous Chondrocyte Implantation (ACI) with
the use of periosteum.
Methods and Materials: 92 patients having patella or trochlea le-
sion participated in our study. Lysholm and Tegner-Wallgren ques-
tionnaires were filled 12.6 years in average after the surgery. They
patients were asked whether they feel better, worse or unchanged
compared to previous years and whether they would do the operation
again. Complications or subsequent surgeries were also assessed.
Results: Tegner-Wallgren score was 7.1, improved by 0.95 com-
pared to preoperative values (p=0.01). Lysholm score was 68.1,
improved by 9 points in average (p=0.3). Seventy two % of the pa-
tients were better or unchanged while 93% would do the operation
again. Patients with no kissing lesions appeared to have a better
prognosis. Patients with malalignment or instability that had a re-
alignment procedure had comparable outcomes to the cases which
did not need any additional surgery. Realignment procedures in-
creased the incidence of serious complications but they were as-
sociated with decreased incidence of periosteal hypertrophy. No
association was found between the age at the time of the ACI or the
size per lesion and any of the clinical outcomes.
Conclusions: It seems that correcting the coexisting background
factors with realignment, stabilizing or unloading procedures,
along with the treatment of cartilage lesions, is improving the re-
sults over time. ACI provides a satisfactory result even for the dif-
ficult cases with concomitant patellar instability. Our long term fol-
low up study reveals preservation of good results and of high level
of patients’ activities, even 10 to 20 years after the implantation, in
both isolated trochlea and patella lesions and also in multiple and
in kissing lesions where an intervention could be considered as a
salvage procedure.
17.2.3
Cartilage load before and after opening-wedge high tibial
osteotomy
M. Lind1, J. Mcclelland2, K. Webster2, J. Wittwer2, T. Whitehead3, J.
Feller2
1
Aarhus C/Denmark, 2Bundoora/Australia, 3Richmond/Australia
Purpose: Medial opening wedge high tibial osteotomy (MOW-HTO)
is widely used in the treatment of medial compartment osteoarthri-
tis of the knee. The purpose of the procedure is to alter the coronal
alignment of the knee to a slightly valgus alignment, thereby re-
ducing medial compartment cartilage loading and symptoms. The
aim of this study was to investigate the functional biomechanical
consequences of this alteration in alignment.
Methods and Materials: Eleven male patients with medial com-
partment osteoarthritis underwent 3D gait analysis during level
walking before and 12 months after MOW-HTO. Nine male control
subjects of a similar age were also tested with the same protocol.
Sagittal and coronal angles and moments in both knees were com-
pared. Pre- and postoperative radiographic coronal plane align-
ment was also measured.
Results: Preoperatively the mean maximum varus angle during
stance was 13.5. This reduced to 5.4 postoperatively (p = 0.0001)
and was not different from control subjects (6.8). The mean maxi-
mum adduction moment also reduced from 3.5 to 2.7 (% Bw/ht,
p=0.02), compared to 3.6 in control subjects. Interestingly, the
adductor moments in the non-operated knee increased postopera-
tively. The mean radiological mechanical alignment was changed
from 8.7 varus preoperatively to 0.1 valgus postoperatively.
Conclusions: Gait analysis is a useful tool for functional assessment
following MOW-HTO. The procedure resulted in normalisation of
various aspects of dynamic knee function and specifically reduced
the adduction moment at the knee, which will reduce cartilage me-
dial load and subsequent osteoarthritis development. The finding
of an increased adduction moment in the non-operated knee could
indicate increased cartilage wear in the non-operated knee which
could increase the risk for development of OA in this joint.
Free Papers 177
17.2.4
Patella cartilage – relationship between T2 mapping and
measurements of patello-femoral joint alignment
G. Scheurecker1, M. Vlychou2, S. Apprich1, D. Stelzeneder1, A.
Messner1, G.H. Welsch1, S. Trattnig1
1
Vienna/Austria, 2Larissa/Greece
Purpose: To evaluate the relationship between T2 relaxation times
of patella cartilage and the sulcus angle and the patella tilt angle
Methods and Materials: IRB approval and informed consent was
obtained. Twenty subjects (mean age 27.7y, range 16 - 48y; 10 fe-
males, 10 males) with an ICRS grade 0 cartilage on the whole patella
based on MRI examination. All subjects were examined on a 3T MRI
unit. We employed an axial multi-echo spin-echo sequence for T2
mapping. The T2 maps were analyzed with ROIs for both the deep
and superficial cartilage layer of the medial and lateral patella facet.
We further used an isotropic sagittal PDw sequence for morpholog-
ic analysis of the cartilage according to the ICRS criteria and for vol-
umetric measurements of the sulcus angle of the trochlea and the
patella tilt angle, the latter defined as angle between the posterior
condyle line of the femur and the bony medial-lateral axis of the pa-
tella. Linear regression was used to look for trends in the relationship
between T2 relaxation times and the sulcus or the patella tilt angle.
Results: The regression lines show trends for increased T2 relax-
ation times of patella cartilage with both higher angles of the sulcus
and the patella tilt. These trends were stronger for the lateral versus
the medial facet, the deep versus the superficial layer of the lateral
facet, and the superficial versus the deep layer of the medial facet.
Conclusions: Increased angles of patello-femoral joint alignment
tend to put more strain on the cartilage of the patella as quantified
with T2 mapping.
17.2.5
Psychometric evaluation of the Knee Injury and Osteoarthritis
Outcome Score (KOOS) subscales for patients with articular
cartilage lesions of the knee using pilot study data of the
Cartilage Autograft Implantation System (CAIS)
L. Nelson1, L.D. McLeod1, M.M. Mordin1, J. Farr2, B.J. Cole3, S. Uddin4 ,
L. Engelhart5
1
Research Triangle Park/United States of America, 2Indianapolis/
United States of America, 3Chicago/United States of America,
4
Raynham/United States of America, 5Milford/United States of
America
Purpose: Changes in symptoms, pain, and functioning as mea-
sured by the KOOS subscales are important endpoints in clinical
trials evaluating treatment effectiveness for knee-related injuries
and disease. Psychometric analyses were conducted to provide
evidence of the reliability, validity, responsiveness, and minimal
important difference estimates for the five KOOS subscales (i.e.,
Symptoms, Pain, Activities of Daily Living, Sports/Recreation, and
Quality of Life) for use in clinical studies evaluating patients with
articular cartilage lesions.
Methods and Materials: Two multicenter, randomized pilot studies
evaluated the safety and performance of the Cartilage Autograft
Implantation System (CAIS) for primary surgical treatment of ar-
ticular cartilage lesions of the knee (ranging from ≥ 1 cm2 to ≤ 10
cm2). A total of 54 patients aged 18 to 55 years with articular dam-
age on the femoral condyle were randomized to receive the CAIS
or a microfracture procedure. Data were collected prior to surgery
and at seven post-surgical follow-up visits for up to 12 months. The
internal consistency reliability, construct validity, responsiveness,
discriminating ability, and interpretation statistics were evaluated
for all KOOS subscales.
Results: Participants were predominantly male (59.3%), white
(92.6%) with an average age of 34.1 years. The KOOS subscales
showed excellent internal consistency reliability, ranging from 0.74
to 0.97 at baseline. Construct validity results were supportive of
hypothesized relationships, with statistically significant correla-
tions (r ≥ 0.50) in the expected direction. Responsiveness analy-
ses demonstrated highly satisfactory sensitivity to change, with
standardized response means ranging from 0.8 to 1.2. Discriminant
tests confirmed the utility of the KOOS subscales. Minimal impor-
tant differences were approximately 10 points for each subscale.
Conclusions: Despite the small sample size, the psychometric anal-
yses strongly support the reliability, validity, responsiveness, and
utility of the KOOS subscales in assessing knee-specific improve-
ments in patients after receiving CAIS or microfracture surgery for
treatment of articular cartilage lesions.
178 Free Papers
17.2.6
Improved life quality in patients with hip Osteoarthritis treated
with an implant of cartilage artifical matrix
E. Hermida, L.F. Hermida
Mexico City/Mexico
Purpose: The purpose of this study is to evaluate the clini-
cal outcome of patients with hip Osteoarthritis (OA) after treat-
ment with an intraarticular implant based on cartilage matrix.
Another purpose of this study is to evaluate if a lower cost, less invasive,
ambulatory procedure is a strong option for patients with I-II hip OA.
Methods and Materials: This is a prospective study of 30 patients
with II hip OA that have been treated with NSAID, weight loss and
physical therapy without a good outcome. The treatment consisted
in 5 hip intraarticular injections with a mixture of chondroitin sulfate
and sodium hyaluronate sulfate as follows: day 0,15,30,60 and 90.
This was done in the office. No NSAID was allowed during the treat-
ment, just acetaminophen like analgesic. The follow up was 2 years.
We evaluated the outcome with a patient satisfaction questionare
and the Harris hip score, at the beginning and at the end of follow up.
Results: Of the 30 patients, 2 failed to final follow up. Of the 28 pa-
tients followed, 22 were women and 6 men. Average age was 58.4
years old. The final follow up was 2 years after the first injection
and a physical exam was performed at the office in order to asses
the outcome. Initial Harris Hip Score was 53.25 and at final follow
up was 87.05. Regarding the satisfaction questionare 92% would
definitely go through the treatment all over again.
Conclusions: Although improvement in the range of mo-
tion was not constant in our study (only 40% improved
range of motion), the pain improvement of the hip was
present in all but 1 patient of the study. The fundamental difference
lies in the medium and long term response given by the chondro-
genic induction and Stromelysin enzyme synthesis inhibition with
the implantation of an artificial matrix like device.
17.2.7
Preliminary result of repairing articular cartilage defect by
autogenous chondrocyte implantation: the feasibility study of a
novel biphasic osteochondral composite
H. Chiang1, C. Jiang1, C. Liao2, C. Shen1, C. Shieh1, Y. Huang1
1
Taipei/Taiwan, 2Hsinchu/Taiwan
Purpose: Autologous chondrocyte implantation (ACI) has been re-
cently used to treat cartilage defects. We had previously developed
a biphasic osteochondral composite as the matrix for ACI. We fur-
ther conducted this feasibility study of such device to treat patients
with osteochondral lesion of the knee joints.
Methods and Materials: Nine patients with symptomatic isolated
osteochondritis at the femoral condyle were treated by replacing
the pathological tissue with autologous chondrocyte-laden bipha-
sic cylindrical plug of DL-poly-lactide-co-glycolide, with its lower
body impregnated with b-tricalcium phosphate as the osseous
phase. The osteochondral lesion was drilled to fashion a pit of iden-
tical size and shape as the plug. The chondrocyte-laden plug was
inserted by press-fitting to fill the pit. Outcome of repair was exam-
ined by functional evaluation with KOOS score at 3 and 6 months
postoperatively, and tissue sample was collected with second-look
arthroscopic needle-biopsy at 12 months. The primary outcome
parameter was the postoperative change of KOOS score; and the
secondary outcome parameter was the regeneration of cancellous
bone and hyaline cartilage, in their respective phases, at the repair
site. Mean KOOS scores were compared with paired t-test.
Results: The mean KOOS score at 3 months postoperatively sig-
nificantly improved than the pre-operative baseline. The score kept
improving thereafter, but the change from 3 to 6 months postop-
eratively was insignificant. At 12 months, gross appearance of the
repair site under arthroscopy showed full-filling of the grafted site,
with the surface of regenerate cartilage flush with the surrounding
native joint surface. Microscopic examination of the regenerate tis-
sue showed formation of hyaline cartilage.
Conclusions: The preliminary result of ACI with the novel biphasic
matrix showed successful regeneration of hyaline cartilage and
cancellous bone to repair the osteochondral lesion. The matrix was
feasible for ACI to treat such lesion in the femoral condyle.
17.2.8
Treatment of osteochondritis dissecans of the knee by
arthroscopic one-step repair technique with bone marrow-
derived cells
M. Cavallo, F. Vannini, R. Buda, A. Ruffilli, R. Ghermandi, C. Cavallo,
B. Grigolo, S. Giannini
Bologna/Italy
Purpose: Etiology of osteochondritis dissecans (OCD) of the knee
is still unclear, recognizing a predisposition due to areas of hypo-
vascularization. Although non-surgical management is still a good
option, surgery is often required. Good results were obtained by
fixation of the detached fragment, or by Mosaicplasty and Autolo-
gous Chondrocytes Implantation when a reconstructive procedure
was required. Arthroscopic One step repair technique with bone
marrow derived cells was previously used with satisfactory re-
sults. This option may be particularly suitable for OCD treatment,
since the bone marrow contains different types of cells, including
hemopoietic precursors, useful for neo-angiogenesis. Aim of this
study is to present the application of the “one-step” technique for
the treatment of OCD of the knee and evaluate the results.
Methods and Materials: From April 2006 to May 2007 6 patients
with OCD underwent the One-step procedure. The mean age was 19
yrs (15-24); 4 cases affected the medial condyle and 2 cases the lat-
eral condyle. Bone-marrow was harvested from the posterior iliac
crest, and the cells were concentrated in the operating room and
implanted at the lesion site on a hyaluronan-based scaffold after
detached osteochondral fragment removal. Platelet Rich Fibrin was
added providing growth factors.
Results: The mean preoperative IKDC score was 48±7. The IKDC at
6 months follow-up was 57±6, at 12 months was 83±7 and at 24
months was 90±5. The control MRI at 12 and 24 months follow-up
showed a good regeneration of the subchondral bone and the carti-
laginous tissue. A biopsy of the regenerated tissue performed at 12
months showed a cartilaginous tissue in organization and a newly
formed subchondral bone.
Conclusions: These results demonstrated that the one-step tech-
nique represents a good option for the treatment of OCD in the
knee joint. Further studies are necessary to confirm the angioge-
netic effect of the multipotent cells present in the bone marrow.
17.2.9
Effect of hyperbaric oxygen and compression on chondrocyte
proliferation
B. Sievers1, N. Hoechsmann1, F. Dueren2, C. Melcher1, S. Mayer1, P.E.
Müller1
1
München/Germany, 2Bernau-Felden/Germany
Purpose: Hyperbaric Oxygen (HBO) has been recognized for de-
cades as an appropriate treatment modality for more than a dozen
clinical conditions ranging from decompression-sickness and arte-
rial gas embolism to the treatment of wound problems. Further-
more in in vivo examinations the administration of HBO has also
been shown to have a protective effect on chondrocytes in carti-
lage regeneration. However, the protective in vivo effect of HBO
treatment on chondrocytes has not been examined in vitro. The
purpose of this study was to examine the effect of HBO treatment
on chondrocytes in an in vitro cell culture model concerning growth
and gene expression pattern.
Methods and Materials: Chondrocytes from the 2 passage were
transferred to a HBO chamber and exposed daily to 100% oxygen
for 7 consecutive days. Compressions of 1 and 2atm were used. A
WST-1 assay was performed at 1,3,5, and 7 days after HBO treat-
ment. Gene expression pattern of apoptosis markers as well as car-
tilage specific proteins were detected by real-time-PCR.
Results: In vitro administration of HBO inhibited growth of chon-
drocytes. When the applied compression was increased up to 2atm
the amount of chondrocytes was reduced by half at days 3 and 7.
In association an up regulation of the apoptosis markers PARP and
Casp3 was observed and an increase of cartilage specific proteins
Collagen II and COMP was detected. No significant differences were
monitored between the different times of daily treatment.
Conclusions: In this study the growth of chondrocytes was in-
hibited in vitro by HBO treatment. This inhibitory effect was in-
creased by elevating the applied compression. The molecular re-
sults showed that the administration of HBO may lead to reduced
chondrocytes`growth due to apoptosis. However the increased
compression induced the expression of cartilage specific proteins,
which might cause a redifferentiation of chondrocytes.

17.3.2
Human cultured fibroblasts isolated from anterior cruciate
ligament have different growth rates depending on the age of the
patient and the type of lesion
P. Guillen, E. Rodriguez-Iñigo, I. Guillen-Vicente, R. Caballero-Santos,
M. Guillen-Vicente, E. Santos, F. Garcia-Gomez, T. Fernandez-Jaen,
J.M. Lopez-Alcorocho
Madrid/Spain
Purpose: In the last years, a common approach for replacement of
injured tissue involves cell therapy. An example of a clinical appli-
cation that may benefit from these techniques is the reconstruction
of anterior cruciate ligament (ACL). With this aim, some preliminary
studies have been carried-out in animal models with two main ap-
proaches: a) to determine the efficacy of cells from different sourc-
es and b) the use of new biomaterials combined with cultured cells
to promote the ACL repair by generating a new tissue with the ad-
equate biomechanical properties. The present study compares the
features and behaviour of monolayer cultured fibroblasts extracted
from ACL of patients with different ages and types of lesion.
Methods and Materials: ACL biopsies from 100 patients with ACL
rupture. Forty nine patients had acute ACL rupture (elapsed time
from rupture to surgery shorter than 1 month), twenty nine had
chronic ACL (elapsed time from rupture to surgery longer than 1
month). Normal ACL samples from 22 patients were included as
controls. The median age of these 62 patients was 35 years (range:
18 – 52 years). Fibroblasts were isolated; the number of cells was
estimated and cultured in monolayer.
Results: The fibroblast culture could be established in all cases. First
passage was done at the day 15th and the mean growth rate was dif-
ferent among the three groups: 108.3 ± 47.7 in acute lesions, 79.5 ±
65.5 in healthy ACL and 76.6 ± 41.7 in chronic lesions. Statistical anal-
ysis showed that differences were significant. Negative correlation
was observed between the age of the patient and the growth rate.
Conclusions: The growth rate of ACL fibroblast in culture is higher
in young patients and in patients with acute ACL rupture.
17.3.3
Long-term clinical and radiological follow-up of viable meniscal
allografts.
P.C. Verdonk1, J. van der Maas2, T. Tampere2, K.F. Almqvist2, R.
Verdonk2
1
Gent-Zwijnaarde/Belgium, 2Gent/Belgium
Purpose: There is growing evidence in literature that meniscal allograft
transplantation performed with the right indications results in signifi-
cant pain relief and functional improvement of the involved joint. Long-
term data on clinical and radiological outcome are however scarce.
Methods and Materials: We evaluated 89 transplants (53 lateral
and 36 medial) in 87 patients. Mean time of follow-up was 15,5 ±
2,85 years (range 9,9 - 20,4), mean age at surgery was 35,2 years
(range 22 - 50). Clinically, the patients were evaluated using a
KOOS, SF-36, HSS, VAS, Tegner and Lysholm score. HSS scores
were compared to pre-operative and mid-term follow-up data. Each
patient received radiographs (AP, profile and Rosenberg view). Ra-
diological outcome parameters were joint space width narrowing
and Fairbank changes and were scored according to IKDC. Failures
were defined as patients who were converted to an arthroplasty.
Results: HSS-scores improved significantly from 119 ± 27 pre-oper-
atively to 160 ± 40 at long-term follow-up. Lysholm-score was 69 ±
22, which was defined as a fair result. Mean VAS-score was 3,4 ±
3, mean Tegner was 4 ± 2. There were no significant differences be-
tween following subgroups: left or right knee, medial or lateral al-
lograft, combined procedure with a high tibial osteotomy and male
or female. Nine (25%) of the thirty-six medial and ten (19%) of the
fifty-three lateral grafts failed after a mean of 9,9 years.
Conclusions: Transplantation of a viable meniscal allograft can
significantly relieve pain and improve function of the knee joint.
Survival analysis showed that this beneficial effect remained in ap-
proximately 70% of the patients at fifteen years. This study proves
that meniscal allograft transplantation is a beneficial procedure to
postpone total knee arthroplasty for more than 10 years in young
active patients.
Free Papers 179
17.3.4
The suture of menisceal tears in the avascular zone enforced by
a collagen I/III membrane: Clinical experience over 6 years and
animal experiments
R. Jakob1, M. Jacobi1, D. Nesic2, P. Mainil-Varlet2
1
Fribourg/Switzerland, 2Bern/Switzerland
Purpose: Due to the limited healing potential of meniscal tissue differ-
ent efforts have been done to enhance healing of sutured meniscus.
Methods and Materials: The meniscus wrap technique is based
on the experience with a technique developed by the senior au-
thor (RPJ) and first applied in 2003. Wrapping the meniscus with a
collagen I/III matrix might create some kind of bioreactor, guiding
cell ingrowth and improving suture stability. Thirty patients with
tears in the red-white or white-white zone, complex tears, delayed
traumatic tears with degenerative aspects, or repeat sutures, were
treated with this technique.
Results: After a mean follow-up of 2.5 years (range 1-5) three pa-
tients had a symptomatic failure (10%). In two of them partial me-
niscectomy was performed and in the other patient (a 20-year-old
female with a second suture of a bucket-handle tear) a third suture
combined with an unloading osteotomy was performed, which ul-
timately led to clinical meniscal healing. All other 27 cases (90%)
were asymptomatic. Additionally, the following complications were
noted: arthrofibrosis requiring mobilization under anaesthesia (one
patient), saphenous nerve entrapment necessitating revision (one
patient), and ACL rerupture after reconstruction and a new trauma,
with the meniscus remaining intact (one patient).
Results of an animal experimental study (goats) shall be reported
Conclusions: This repair enhancement technique seems to improve
the chances of healing, even in unfavourable conditions. Although
the evaluation did not include a second-look arthroscopy, 90%
of patients remained asymptomatic after a mean follow-up of 2.5
years. Similar to fascia sheath coverage, this technique has the dis-
advantage of being technically demanding and time-consuming.
17.3.5
TKAMen: A new source for meniscus reconstruction
T. Brune, C. Martin, L. Barnouin
Mions/France
Purpose: Allogeneic transplants and substitutes produced in vari-
ous biomaterials are currently used for partial or complete menis-
cus reconstruction. To combine the advantages of these two so-
lutions (complex matrix structure of allografts and good handling
and safety of substitutes), we explored the opportunity of using
decellularized allogeneic menisci. So as to get a continuous sup-
ply we studied the possibility of considering lateral menisci derived
from total knee arthroplasty (TKAMen) as a potential source.
Methods and Materials: Relying on over 15 years experience in tis-
sue banking, we started in 2006 collecting TKAMen. Around 110
tissues were already collected, fully characterized and classified
in 3 different groups according to criteria describing their integrity
and surfaces aspect. To assess the relevance of our approach, we
compared these macroscopic observations with histological and
mechanical properties for a group of 16 menisci. We determined
their Young modulus by using a compression-testing machine,
while histological sections underwent a zone specific analysis after
standard haematoxylin-eosin staining.
Results: According to our classification, 29% of the tissues are con-
sidered as slightly osteoarthritic, 53% moderately osteoarthritic
and 18% belong to the severely osteoarthritic group. Regarding
the mechanical resistance, we showed a significant decrease of the
mean Young modulus for the severely osteoarthritic tissues (10,62
+/-11,09 N/mm2) compared to both other groups (49,93 +/-8,25 N/
mm2 and 41,54 +/-11,39 N/mm2). Menisci from slightly and mod-
erately osteoarthritic groups displayed a histological pattern simi-
lar to normal tissues or those derived from multi-organ retrievals.
Conversely, severely osteoarthritic TKAMen presented many de-
generative signs, correlating with the macroscopic observations.
Conclusions: Comparing macroscopic observations with mechani-
cal and histological data, we demonstrated the relevance of select-
ing tissues according to well-defined morphological criteria. Our
results showed that 80% of TKAMen display high mechanical prop-
erties and a histological pattern similar to healthy tissues. Further
studies will now investigate the possibility to use them for menis-
cus reconstruction.
180 Free Papers
17.3.6
Lateral Collagen Meniscus Implant (Menaflex): Prospective
European Multicenter Study with 2 Year Minimum Follow-Up
J.C. Monllau1, R. Crespo2, S. Zaffagnini3, M. Marcacci3, R. Berbig4 ,
D.G. Holsten5, P. Bulgheroni6, K. Lagae7
1
Barcelona/Spain, 2Alcazar de San Juan/Spain, 3Bologna/Italy, 4Zürich/
Switzerland, 5Koblenz/Germany, 6Varese/Italy, 7Antwerp/Belgium
Purpose: Our purpose was to determine if Menaflex Collagen Menis-
cus Implants are safe and effective to treat loss of lateral meniscus
tissue and if the implant functions whether or not tissue is missing at
the popliteal hiatus.
Methods and Materials: 60 patients with irreparable lateral meniscus
tears or meniscus loss requiring surgical treatment were enrolled at 7
European centers. Patients were deemed “acute” (no prior surgery) or
“chronic” (1 to 3 prior surgeries on the involved meniscus). Inclusion
criteria included stable knee ligaments, neutral knee alignment and
no untreated Grade IV chondral defects. Menaflex implantation was
performed arthroscopically with all-inside suture fixation +/- inside-out
sutures anteriorly. Patients underwent standardized rehabilitation with
clinical follow-ups (FU) at 6, 12, and 24 months after implantation.
Results: 49 patients (mean age 30.5 years) underwent lateral Mena-
flex implantation. All patients had minimum 24 months FU (mean, 34
months). 11 (22%) patients were acute and 38 (78%) were chronic. 11
(22%) patients had no tissue spanning the popliteal hiatus AT TIME OF
Menaflex implantation. All patients returned to activities of daily living
3 months after surgery. Postoperative scores improved significantly at
1 and 2 years from preoperative. Mean Tegner scores improved from
3.0 to 5.6 at 2 years, mean Lysholm improved from 63 to 93, mean
pain decreased from 36 to 7, and 94% were satisfied. MRIs revealed
no changes to articular surfaces or joint space height postoperatively;
however, the new tissue was not yet mature. Popliteal hiatus tissue
deficiency did not influence device placement or clinical outcomes. 5
patients (10%) underwent reoperation due to pain and swelling.
Conclusions: At 2-year FU, 90% of patients had improved clinical out-
comes compared to preoperative. Menaflex reoperation rates were
similar to reported lateral meniscus repair. Longer FU continues in or-
der to determine extent and duration of the benefits.
17.3.7
Feasibility study of adapting the delayed gadolinium enhanced
magnetic resonance imaging of cartilage (dGEMRIC) technique
for the meniscus
J.E.J. Bekkers1, B. Claassen1, L.B. Creemers1, W.J.A. Dhert1, A.P.
Hollander2, D.B. Saris1
1
Utrecht/Netherlands, 2Bristol/United Kingdom
Purpose: The dGEMRIC technique has successfully been applied to
quantify the quality of articular cartilage. This study analyzed the
feasibility of delayed gadolinium enhanced MRI of meniscal carti-
lage (dGEMRIM).
Methods and Materials: From Feb-Dec 2009, a total of 30 patients
underwent a dGEMRIC for articular cartilage pathology. Magnevist
(Bayer, Germany) was injected 90 min prior to scanning (0.2 mmol/
kg). A 3D T1 MRI protocol, at 1.5T, with 5 inversion times (50, 150, 350,
650 and 1650 ms) and 3 mm slice thickness and sagittal image acquisi-
tion was used. The T1gd (dGEMRIM index) was calculated by averag-
ing the T1gd across the manually segmented region-of-interest (ROI)
using in-house developed software. A total of five different ROIs were
defined for both lateral and medial menisci (Figure1). The menisci
were defined as healthy, degenerated or meniscectomized by con-
ventional MRI and arthroscopy. Statistical analysis was performed by
Pearson correlations and one-way ANOVAs with post-hoc LSD tests.
Results: The meniscus T1gd showed good correlations to the medial
(R=0.699, p=0.001) and lateral (R=0.729, p=0.000) femur cartilage
T1gd. The meniscus as a whole showed no difference (p>0.279)
in the T1gd between those defined healthy or unhealthy. In most
types of menisci there were statistically significant differences in
T1gd between the inner and outer (p=0.064) and upper and lower
(p=0.002) regions. Healthy and degenerate menisci also showed a
statistically significant difference in dGEMRIM index between me-
dial and lateral compartments (p=0.017), however these compart-
ments did not differ after medial meniscectomy (p=0.847).
Conclusions: The meniscus quality (dGEMRIM) shows good cor-
relation with dGEMRIC. The varying T1gd across healthy and de-
generated menisci indicates a heterogeneous glycosaminoglycan
distribution within the tissue. The decrease in T1gd after medial
meniscectomy, compared to healthy lateral menisci, could be due
to leakage of glycosaminoglycans. We conclude that the dGEMRIM
technique offers a new approach to assessing degeneration of the
meniscus after meniscectomy.
17.3.8
New process for the production of an acellular meniscus scaffold
T. Brune, A. Schirlin, C. Martin, L. Barnouin
Mions/France
Purpose: Allogeneic transplants and substitutes produced in vari-
ous biomaterials are currently used for meniscus reconstruction.
To combine the advantages of these two solutions we studied the
possibility of developing an acellular meniscus scaffold. Therefore
we compared the effect of several chemical treatments in combina-
tion with freeze-drying and gamma radiation to find out a process
maintaining the extracellular matrix complexity and leading to a
safe product, sterile and ready to use.
Methods and Materials: Initially, selected chemicals were tested to
remove peripheral tissues as part of a preliminary cleaning. Then
we analyzed the capacity of different chemicals to partly extract gly-
cosaminoglycans (GAG) and eliminate cell residues while preserving
the complex matrix structure of the tissue. This was mainly achieved
by performing biochemical assays (hydroxiproline measurement
and GAG release) and wide-ranging histological studies including
Masson’s trichrome, Feulgen-Rossenbeck and Safranin O staining.
We also addressed the ability of these treatments to inactivate well-
defined viruses. Finally we considered modifications of the menisci
size and weight as a consequence of the process.
Results: Our results suggest that ethanol offers a satisfying solu-
tion to clean out the tissues. Histological data showed that sodium
hydroxide in combination with hydrogen peroxide promotes a com-
plete meniscus decellularization without denaturing collagen struc-
ture, similar to what was obtained with sodium dodecyl sulfate.
Preliminary data also indicated that such a combination turned out
to be effective to inactivate viruses. Moreover the whole process,
especially freeze-drying and sterilization, severely affected menisci
weight and dimensions. However the tissues recovered their initial
physical characteristics following extended rehydration.
Conclusions: Based on a combination of biochemical data, macro-
scopic and histological observations we demonstrated that there
are several alternatives to process successfully menisci with the
intention of getting them acellular. In the ongoing studies we are
now investigating the consequences of such processes in terms of
biocompatibility and alteration of the mechanical properties.
17.3.9
An Ex Vivo Organ Culturing and Loading System for Large Species
Intervertebral Discs; a Feasibily Study With Caprine Lumbar
Discs.
C.P.L. Paul, H.A. Zuiderbaan, B. Zandieh Doulabi, A.J. Veen van der,
T.H. Smit, M. Helder, B.J. Royen van, M.G. Mullender
Amsterdam/Netherlands
Purpose: Mechanical loading is a natural stimulus to chondrocytes
and regarded essential for maintenance and repair of cartilage ex-
tracellular matrix (ECM), yet loading is considered a major extrinsic
component in degenerative disc disease (DDD). To further investi-
gate the interaction between mechanical loading and cell and ma-
trix response we have developed the Loaded Disc Culture System
(LDCS); this ex vivo bioreactor conserves IVD cells in their native
environment and allows close control of tissue loading conditions.
The purpose of the current study is to investigate the feasibility of
culturing goat IVDs with maintenance of their native cellular and ex-
tracellular properties in the LDCS over a 7- and 14-day culture period.
Methods and Materials: IVD’s (L1-6; N=40) were harvested from
goats (N=8) under sterile conditions. IVD’s were cultured in the
LDCS either without loading or with a simulated diurnal physiologi-
cal load. Cell viability was assessed in the nucleus (NP) and annu-
lus (AF) regions. Also, water, glycosaminoglycan (GAG) and total
collagen content of the extracellular matrix (ECM) were measured.
Results: Fresh discs (day 0) displayed a mean cell viability of 79.4%
(NP) and 77.7% (AF). At 7 and 14 days, cell viability of the unloaded
group dropped by half in both NP and AF, while physiological load-
ing maintained cell viability (fig. 1). In the ECM, water-, GAG- and
collagen content of the physiologically loaded IVD’s did not change
significantly after 7 and 14 days, when compared to day 0 (fig. 2).
Conclusions: We were able to maintain the native properties of
goat discs in the LDCS over a 14-day culture period by applying a
simulated diurnal physiological load. The LDCS may serve as a valu-
able platform to study the processes involved in degenerative disc
disease (DDD) along with new treatment strategies for cartilage
repair. Acknowledgements: This study was supported by ZonMw
grant number 11400090. No conflict of interest.

17.4.2
Local anesthetics exposure leads to mitochondrial oxidative
stress and oxidative damage in human chondrocytes.
V. Grishko1, A.W. Pearsall, IV2, G.L. Wilson2
1
36688/United States of America, 2Mobile/United States of
America
Purpose: Purpose: Several different mechanisms have been pro-
posed to explain toxicity of local anesthetics including the block-
ade of potassium channels and mitochondrial injury. The purpose
of this study was to investigate whether oxidative stress is involved
in toxic effects of lidocaine, bupivacaine, and ropivacaine on nor-
mal and OA chondrocytes.
Methods and Materials: Materials and Methods: Primary chondro-
cytes cultures, generated from cartilage from patients undergoing
total knee replacement and normal donors, were exposed for 1 hr
to saline, 2% and 1% of lidocaine, 0.5% and 0.25% of bupivacaine,
0.5% and 0.2% of ropivacaine. Enhanced mitochondrial superoxide
production, oxidative damage to mitochondrial DNA and mitochon-
drial proteins were evaluated immediately after exposure or follow-
ing 72 h of recovery. Mito SOX Red fluorescent dye, highly selective
for mitochondrial superoxide, was used to study the mitochondrial
ROS levels. Protein carbonylation as the result of oxidative stress
was studied by Protein Carbonyl assay. Oxidative damage to mtD-
NA was evaluated using Quantitative Southern blot analysis.
Results: Results: When normal and OA chondrocytes were exposed
to local anesthetics, only small amount of mitochondrial ROS was
accumulated in only OA chondrocytes immediately after exposure.
Following 72 h after treatment, mitochondrial ROS production was
enhanced. OA chondrocytes exhibited significantly higher levels of
mitochondrial superoxide compare to normal donors. Enhanced mi-
tochondrial ROS production in OA chondrocytes dose-dependently
correlated with oxidative damage to mitochondrial DNA and proteins.
Conclusions: Conclusion: The present results demonstrate for the
first time that mitochondrial oxidative stress is involved in toxic ef-
fects of local anesthetics on human chondrocytes. Moreover, OA
chondrocytes exhibit higher susceptibility to this oxidative stress
then chondrocytes obtained from normal donors. These differenc-
es are likely due to already compromised mitochondrial function
and mitochondrial damage in OA chondrocytes.
17.4.3
Matrilin-1 and matrilin-4: new disease causing loci for multiple
epiphyseal dysplasia?
M. Ristiluoma1, A. Aszodi2
1
Oulu/Finland, 2Martinsried/Germany
Purpose: Matrilins (matrilin1-4) are adaptor proteins of connective
tissues and mutations in the matrilin-3 gene (MATN3) are associ-
ated with multiple epiphyseal dysplasia (MED), a skeletal disorder
characterized by mild dwarfism and early-onset osteoarthritis.
Most MATN3 MED mutations are missense and cause the retention
of the missfolded protein in the rER. MED is genetically heteroge-
neous, and in addition to MATN3, mutations in the genes encoding
COMP, COL9A1-A3 and DTDST have been identified, however, the
causative gene in about 50%-80% of the cases is not known. Tak-
ing into account the conserved structure of matrilins, MATN1 and
MATN4 are good candidates for additional MED loci.
Methods and Materials: Human MATN3-like MED mutations were
generated into conserved sites of tagged mouse Matn1, Matn3
and Matn4 cDNAs and transfected into 293-EBNA cells or primary
chondrocytes. Cellular and medium samples were collected and
analyzed by Western blot, or the cells were immunostained using
matrilin- or tag-specific antibodies.
Results: Wild type and polymorph Matn1, Matn3 and Matn3 con-
structs were expressed at high levels in 293-EBNA cells and were
efficiently secreted. MED-type Matn3 mutations were expressed at
lower levels and displayed secretion defects. MATN-3 MED muta-
tions in Matn1 and Matn4 exhibited three secretion patterns: most
mutant proteins were 1) completely missing in the medium or 2)
only moderately secreted; whereas a few 3) were apparently nor-
mally deposited into the supernatant. In chondrocytes, the MATN3
A219D-equivalent Matn1 and Matn4 mutations were present in the
cell fraction but were absent in the medium. Immunofluorescence
analysis revealed that while control constructs were clearly visible
extracellularly, most mutant proteins retained in the cell and ac-
cumulated in the rER.
Conclusions: Most MATN3-MED mutations in Matn1 and Matn4
lead to secretion defect and accumulation of the mutant proteins in
the ER. These results imply the involvement of MATN1 and MATN4
in MED via a similar pathomechanism as described for MATN3.
Free Papers 181
17.4.4
Obesity affects the chondrocyte responsiveness to leptin in
patients with osteoarthritis
P. Francin1, S. Pallu2, C. Guillaume1, P. Pottie1, D. Mainard3, N.
Presle1
1
Vandoeuvre les Nancy/France, 2Orleans/France, 3Nancy/France
Purpose: As hyperleptinemia disrupt the physiological function of
the adipokine in obese individuals, the current study has been un-
dertaken to determine whether elevated leptin levels found in the
joint from patients with osteoarthritis (OA) induces also a defect in
leptin action in chondrocyte.
Methods and Materials: Chondrocytes isolated from OA patients
with various body mass index (BMI) were treated with 100 or 500
ng/ml of leptin. The expression of cartilage-specific components
(aggrecan, type 2 collagen), as well as regulating factors (IGF-1,
TGFβ, MMP-13, TIMP‑2) was investigated by real-time PCR to
evaluate chondrocyte responsiveness to leptin.
Results: The leptin activity was shown to be critically dependent on
both the concentration and the BMI of the patients. A negative as-
sociation between the activation of regulated genes and BMI was
found for the lowest dose of adipokine. Conversely, the cell respon-
siveness increased with the BMI when 500 ng/ml of leptin was used
to stimulate chondrocytes. Additionally, chondrocytes collected
from normal or overweight patients displayed most responsive-
ness with the low concentration of leptin while an elevated dose
of leptin was required to change gene expression in cells obtained
from obese patients. Interestingly, the gene encoding MMP-13 was
identified as a target of leptin for obese patients only. Beside, the
expression of TIMP-2 was up-regulated in leptin-treated cells ob-
tained from OA patients with the lowest BMI.
Conclusions: Our results clearly showed that chondrocytes from
obese OA patients behave differently from chondrocytes isolated
from normal or overweight patients. The cells from obese patients
required elevated levels of leptin to be responsive and the BMI-de-
pendent effect of leptin on the expression of TIMP-2 and MMP-13 may
explain why obesity is associated with an increased risk for OA.
17.4.5
Autologous Matrix-Induced Chondrogenesis (AMIC) on the
patella plus periosteal coverage on the trochlea combined
with mechanical realignment- A New Treatment Option in
Symptomatic Isolated Femoropatellar Osteoarthritis due to
Subluxation of the Patella
T. Kusano, C. Marti, M. Jacobi, R. Jakob
Fribourg/Switzerland
Purpose: Symptomatic isolated femoropatellar osteoarthritis is re-
ported for 8% of women and 2% of men over the age of 55 years.
Surgical options include osteotomy of the tibial tubercle with patel-
lar debridement and realignment (1), patellar hemiarthroplasty and
femoropatellar arthroplasty and patellectomy. We present our re-
sults of a new biological treatment option, in which (1) is combined
with the AMIC procedure (autologous matrix-induced chondro-
genesis) and periosteal resurfacing of the lateral trochlea femoris.
Methods and Materials: Only symptomatic isolated lateral femoro-
patellar osteoarthritis with an unsuccessful conservative treatment
was included in the study. The surgery consisted of an AMIC proce-
dure described by Behrens on the retropatellar cartilage defect and
a periosteal coverage of the trochlear cartilage defect, both naked
surfaces prepared by 1, 1 mm drillings combined with resection of
the lateral pole of the patella with lateral release, medial reefing
and a tibial tubercle medialisation and advancement. Lack of im-
provement and need for reoperation were defined as endpoints.
Results: From 2003 to 2009 a total number of 21 patients underwent
this described new procedure of which two were operated bilater-
ally. 1/4 of patients had a lack of improvement, thus 3/4 were satis-
fied. One patient who underwent bilateral surgery was satisfied at
the last follow-up but died later of an unrelated cause. The other
patient with bilateral surgery had persistent anterior knee pain on
the one knee and a good result on the other. One patient had a
reoperation with the AMIC procedure and is now satisfied. Another
patient underwent partial meniscectomy. His arthroscopy showed
fibrous cartilage coverage. One patient had total knee replacement.
The oldest patient in this series showed radiographic signs of pa-
tellar osteolysis. Patients over the age of 75 years all were failures.
Conclusions: The proposed combination of mechanical and biolog-
ic treatment modalities has proven to be a valid alternative to deb-
ridement and alignment alone or to arthroplasty in patients under
the age of 75 years. It shows that there is a chondroplastic potential
in osteoarthritis and advanced age.
182 Free Papers
17.4.6
Treatment with chondroitin sulfate reduces cartilage volume loss
in knee oa patients assessed by MRI: a randomized, double-
blind, placebo controlled pilot study
J.P. Pelletier1, L.M. Wildi1, J.P. Raynauld1, A.D. Beaulieu2, L. Bessette3,
F. Morin4 , M. Dorais5, J. Martel-Pelletier1
1
Montreal/Canada, 2Quebec/Canada, 3Sainte-Foy/Canada, 4Trois-
Rivières/Canada, 5Notre-Dame de l’Ïlle-Perrot/Canada
Purpose: To determine the effect of chondroitin sulfate (CS) on ex-
tent of cartilage loss, subchondral bone marrow lesion (BML) size
and severity of synovitis in patients with knee OA using magnetic
resonance imaging (MRI).
Methods and Materials: This pilot study was a multicentre, ran-
domized, double-blind, controlled trial of CS (Condrosan®, Bioi-
bérica, Spain) 800 mg daily vs. placebo for 6 months followed by
an open label period of 6 months with all patients receiving CS 800
mg daily. MRI was performed at baseline, 6 and 12 months. The
cartilage volume was quantified using 3D reconstruction of the MR
images. The BML size, severity of synovitis and meniscal extrusion
were assessed on a semi-quantitative scale.
Results: 70 patients were enrolled. Patients receiving CS for 12
months compared to placebo (6 months placebo followed by 6
months CS) experienced a reduction in cartilage volume loss in the
lateral compartment at 6 (-1.1±3.3%, -3.0±4.5%; p=0.05) and 12
months (-1.0±3.8%, -3.9±4.5%; p=0.013). These differences were
more pronounced in OA patients without medial meniscal extrusion
(6 months, -1.1±3.4%, -3.6±4.3%; p=0.02; 12 months, -1.1±3.8%,
-4.3±4.3%; p=0.008). Patients on CS treatment presented a reduc-
tion in BML at 6 months and a marked reduction at 12 months (glob-
al knee, -0.7±2.6, 0.6±2.1; p=0.06; lateral compartment, -0.4±1.1,
0.7±1.4; p=0.002). Patients from CS group, who received concomi-
tant NSAIDs demonstrated a reduction in synovial membrane thick-
ness compared to placebo (1.3±0.3 mm, 1.6±0.3 mm; p=0.03), and
a reduction in synovitis score (3.0±1.9, 4.5±1.6; p=0.09), as well as
lower incidence of joint swelling (p=0.09).
#Conclusions: CS was found to reduce cartilage volume loss in
knee OA starting as early as 6 months treatment. This was asso-
ciated with a reduction in BML size supporting the hypothesis of
their role in cartilage volume loss. CS treatment combined with
NSAIDs also reduced severity of synovitis. These findings support
the DMOAD effect of CS.
17.4.7
Osteoarthritis after intense sports activity
M.I. Iosifidis, D. Neophytou, T. Liakos, I. Melas, K. Apostolidis, T.
Kyriakidis, A. Kyriakidis
Thessaloniki/Greece
Purpose: Intense sport activity, mostly by elite athletes, causes in-
creased loads in the joints. It is hypothesized that the high impact
and torsional loading and also the repeated low magnitude loading
cause cartilage degeneration. We investigated the prevalence of
lower extremities osteoarthritis (OA) in former elite athletes.
Methods and Materials: We studied 218 former athletes (soccer
players, skiers, volleyball players, martial arts athletes, track and
fields athletes, basketball players/ range 40-84 years, mean 50.67
years, SD±10.04) who participated in national championships of
premier league, and in international games. The control group was
181 males (range 40-77 years, mean 50.13 years SD±8.48) who did
not have systematic sport activity. The participants in the study
had not diagnosed or/and operated for significant limb injury. OA
was recorded through questionnaire, clinical examination and ra-
diological evaluation (full weight bearing limbs xrays).
Results: After adjusting the age, height, weight and body mass index
(BMI), the statistical analysis (SPSS, independent samples t-test, z-
test, CI=95%) showed no difference in OA prevalence in former ath-
letes compared with the general population. However, the intensive
sport activity was a significant risk factor for the athletes, as in com-
parison with the control group their body weight increased less, their
occupation was lighter, and also they developed OA in younger age.
Interestingly, the x-rays of the former athletes showed increased
osteoarthritic signs compared with control group (p<0.001).
Conclusions: Even if there was no statistically significant difference
in OA prevalence between elite athletes and general population,
the intensive sports was the major predisposing factor for degen-
erative joint disease in former athletes.
17.4.8
12 year follow-up of ACL Reconstruction: Long Term Outcomes of
Prospectively Studied Osseous and Articular Injuries
B.T. Hanypsiak1, K. Spindler2, R. Parker3, G. Calabrese3, B.
Richmond3, C. Rothrock4 , T. Herrenbruck5
1
Naples/United States of America, 2Nashville/United States of
America, 3Cleveland/United States of America, 4St. Louis/United
States of America, 5Salina/United States of America
Purpose: Bone bruises and articular cartilage injuries sustained
at the time of initial ACL injury would not resolve. Our secondary
hypothesis was that the presence of a bone bruise or articular car-
tilage injury originally identified on magnetic resonance imaging
would not be associated with long-term outcomes after anterior
cruciate ligament reconstruction evaluated by the International
Knee Documentation Committee questionnaire.
Methods and Materials: Cohort study (prognosis); Level of evi-
dence, 1. METHODS: We attempted to contact all patients from an
original cohort (N = 54) for follow-up evaluation, which included
repeat radiographs, magnetic resonance images, physical exami-
nation, and International Knee Documentation Committee ques-
tionnaire more than a decade postoperatively.
Results: Forty-four patients (82% of the original cohort) returned
for on-site follow-up. No patient with a bone bruise identified on
original magnetic resonance imaging had one identified at 12-year
follow-up. The mean ( +/- SD) International Knee Documentation
Committee score at follow-up with no bone bruise originally pres-
ent was 70.6 ( +/- 12.7) versus 70.0 ( +/- 8.1) when a bone bruise
was observed (P > .05). No consistent association was observed
between the presence of an initial articular cartilage lesion with a
lesion on follow-up magnetic resonance images. The mean ( +/- SD)
International Knee Documentation Committee score at follow-up
with no articular cartilage injury was 69.0 ( +/- 11.9) versus 72.8 (
+/- 12.0) with articular cartilage lesion (P > .05).
Conclusions: All bone bruises identified in our study with magnetic
resonance imaging at the time of initial injury had resolved at 12-
year follow-up. The presence of a bone bruise at the time of initial
injury did not significantly alter the patient-oriented outcome by In-
ternational Knee Documentation Committee after anterior cruciate
ligament reconstruction. Additionally, articular cartilage abnormal-
ity on magnetic resonance imaging did not influence the Interna-
tional Knee Documentation Committee score.
17.4.9
Type II collagen fragment HELIX-II is a marker of early cartilage
damage but is insensitive to predict progression of cartilage
destruction in human knee joint synovial fluid
X. Wei1, K. Li2, L. Wei3
1
Taiyuan/China, 2Taiyuan City/China, 3Providence/United States of
America
Purpose: Objectives: To determine whether the level of type II col-
lagen fragment HELIX-II in the knee joint synovial fluid is correlated
to the severity of the articular cartilage damages determined by
arthroscopy or surgical observation directly.
Methods and Materials: Methods: 83 patients who had undergone
knee arthroscopy treatments or total knee replacements were en-
rolled in the study (49% women, age 14-78 year old, mean 49.5 year
old). The content of type II collagen fragment HELIX-II was measured
by enzyme-linked immunosorbent assay (ELISA) in 42 patients with
knee osteoarthritis (OA), 25 patients with meniscus injury, and 14
patients with anterior cruciate ligament (ACL) injury or posterior
cruciate ligament (PCL) injury. Cartilage damage of knee joint was
classified with Outbridge cartilage damage severity scoring system
by arthroscopy or surgical observation directly. Maximum damage
score was defined as the highest score among the six area of the
knee joint cartilage and cumulative score was defined as the sum
of the scores of the six area of the knee joint cartilage. We investi-
gated the relationships between synovial HELIX-II and the cartilage
destruction measured by Outerbridge scoring system.
Results: Results: The level of HELIX-II in the synovial fluid was cor-
related with the cumulative scores (r =0.807, p<0.05) and the
maximum scores (r =0.794, p<0.05) respectively. The level of
HELIX-II in the severe cartilage damage groups (Outbridge score: 2
to 4) was much higher than the slightly cartilage damaged groups
(Outbirdge score: 0 or 1) but there was no significance difference
among the severe groups.
Conclusions: Conclusions: Elevated HELIX-II level is correlated with
cartilage structure changes and is a biomarker for the early diagno-
sis of cartilage damages but is insensitive to predict the severity of
cartilage damage in the knee joint synovial fluid.

25.1.2
Function and structure of cartilage defect repair with frozen and
fresh osteochondral allografts in the goat
A.L. Pallante, S. Görtz, A.C. Chen, S.T. Ball, D. Amiel, R.L. Sah, W.
Bugbee
La Jolla/United States of America
Purpose: Fresh-vs-frozen allografts represent best-vs-worst cases
with respect to cell viability, but difficult-vs-simple with respect to ac-
quisition and distribution. The objective of this study was to compare
load-bearing, geometrical, biochemical, and cellular properties of
such allografts for defect restoration in the adult goat model.
Methods and Materials: Adult Boer goats (n=7, 3yo) were operated in
one knee, with one FROZEN and one FRESH site-matched osteochon-
dral allograft (d=8mm, h=5mm) implanted into alternating medial
femoral condyle (MFC) and lateral trochlea (LT) sites. Contralateral
knees were NON-OPerated controls. At 6months, joint structure was
assessed by micro-Computed Tomography (mCT), quantifying carti-
lage thickness, cartilage contour-fill at the defect site, cartilage sur-
face location, and cartilage-bone interface location. Cartilage stiff-
ness was assessed by indentation testing at the allograft center and
matched Non-Op site. Cellularity was assessed by Live/Dead® stain-
ing overall and in superficial, middle, and deep zones. Matrix fixed
charge was assessed (inversely) with Hexabrix™-enhanced-mCT
(HE-mCT). Effects of treatment (NON-OP, FRESH, FROZEN), site (and
zone) were assessed by ANOVA.
Results: Cartilage cellularity varied with treatment (p<0.001), be-
ing similarly high in NON-OP and FRESH, and ~99% lower in FROZEN
allografts. Cellularity also varied with zone (p<0.001), decreasing
with depth. Cartilage stiffness and HE-mCT absorption varied with
treatment (p<0.001). Cartilage stiffness was ~80% lower in FROZEN
than NON-OP or FRESH allografts. Concomitantly, deep zone HE-mCT
absorption was ~60% higher in FROZEN than NON-OP and FRESH.
Cartilage thickness varied with site (MFC=1.44mm, LT=0.86mm),
but was not affected by treatment (p=0.4). Other allograft geometry
parameters were affected by treatment (p<0.01). In FROZEN MFC
allografts, cartilage fill was less (~50%) in association with surface
depression (~0.7mm), and the cartilage-bone interface tended to be
depressed (~0.9mm, p=0.06).
Conclusions: Maintenance of cartilage load-bearing properties in al-
lografts is associated with maintenance of cartilage cellularity and
fixed charge. Frozen or non-viable allografts may fail due to both car-
tilage softening and graft subsidence.
25.1.3
3D-Printing of cells in gels: a new concept for constructing
osteochondral grafts
W. Schuurman, N.E. Fedorovich, H.M. Wijnberg, H. Prins, P..R. van
Weeren, J. Malda, J. Alblas, W.J.A. Dhert
Utrecht/Netherlands
Purpose: Osteochondral defects are prone to induce osteoarthritic de-
generativechanges.Currentapproachesforosteochondralgraftingsuf-
fer from poor tissue formation and compromised integration. 3D-print-
ing is a novel technique that fabricates organized, cell-laden scaffolds
by layer-by-layer deposition. The aim of this study was to test the appli-
cability of the use of 3D-printing for the production of heterogeneous
cell-laden 3D hydrogel structures as a model for osteochondral grafts.
Methods and Materials: We assessed the effect of the extrusion of cell-
laden hydrogel on cell viability. To construct scaffolds containing two
different cell types, alginate hydrogel was mixed with either human
chondrocytes or human multipotent stromal cells (MSCs) (both fluo-
rescently labeled). Subsequently, porous scaffolds (20x10x1mm) with
in one part MSCs and in the other part chondrocytes were produced.
Extracellular matrix formation was analyzed using (immuno)histology.
Results: The cells survived the deposition process, and were as via-
ble as non-printed controls (mean 89% and 88.9%, respectively). The
MSCs and chondrocytes remained in their printed part of the scaf-
fold for up to three weeks when cultured in vitro. Moreover, heteroge-
neous and cell specific matrix formation occurred in vitro, as shown
macroscopically and by immunocytochemistry. In the chondrocyte-
laden part, cell clusters were observed, as well as cartilage-specific
collagen type II and type VI staining, none of which were noted within
the MSC-laden part. In the MSC-laden part, we showed the presence
of alkaline phosphatase and positive Alizarin red staining, indicative
for respectively osteogenic differentiation and calcium deposition.
Conclusions: Our results demonstrate that 3D-printing can be used
to fabricate viable, heterogeneous cell-laden hydrogel grafts with for-
mation of cartilage-like matrix in the chondrocyte-laden part of the
construct and osteogenous matrix differentiation in the MSC-laden
part, suggesting the potential to produce osteochondral grafts with
this novel technology.
Free Papers 183
25.1.4
Regeneration of osteochondral lesions in the knee with a
combined MACI / Bone Graft - Sandwich Technique
E. Basad, B. Ishaque, H. Stürz
Giessen/Germany
Purpose: Osteochondral defects in the knee are primarily based on
pathologic changes of the subchondral bone, which affect the chon-
dral surface. Repair depends on reconstruction of cartilage and bone.
The Matrix-associated Chondrocyte Implantation (MACI) is a third
generation cartilage regeneration technique where chondrocytes are
seeded onto a collagen I/III membrane. We combined the MACI tech-
nique with an autologous bone graft (BG) in a two-step sandwich pro-
cedure (MACI-BG). Goal of our prospective observational study was
to evaluate the outcomes after two years.
Methods and Materials: In 32 patients with isolated lateral or medial
condylar osteochondral defects (> 3 cm2) first an arthroscopic car-
tilage biopsy was taken and a deep debridement of the sclerotic sub-
chondral bone was performed through a mini arthrotomy, followed by
a press-fit filling of the defect with impacted cancellous iliac bone. In
a second operation, a double-layer MACI was fixed onto the consoli-
dated bone graft with fibrin glue. The clinical outcome was measured
using the Tegner and Lysholm scores after 6, 12 and 24 months
Results: The median Tegner score improved from Level 2 to Level 4
(p= 0.0001). The mean Lysholm score median improved from 57.8
±21.1 to 94.25 ±11.1 (p= 0.0001). There was no significant difference
between the lateral or medial condyle (p=0.349). MRI follow-ups
showed a bi-polar regeneration without dislocation of impacted bone
or hypertrophy of the regenerate tissue.
Conclusions: Cartilage and subchondral bone perform as an interact-
ing unit. Treatment of osteochondral lesions without regeneration of
the subchondral bone is likely to fail. The combination of MACI with
BG is one strategy that addresses this particular problem. Our study
shows promising results pointing out that a rebuilt subchondral bed
plays an important role for the success. The results may also imply that
an early detection and proper treatment of subchondral alterations
is important for the outcome of cartilage regeneration techniques.
25.1.5
Comparison of the results of immature articular cartilage
allograft and mature autograft in osteochondral defect
treatment- an experimental study with rats
A. Koroglu1, K. Memisoglu2, B. Muezzinoglu2, S.U. Muezzinoglu2
1
Sakarya/Turkey, 2Kocaeli/Turkey
Purpose: To address the effect, advantage and superiority of the im-
mature allograft in the treatment of osteochondral defects in rats.
Methods and Materials: A 4x2 mm deep osteochondral lesion was
created in the knee joints - femoral condyles- of 30 mature rats and re-
paired with immature osteochondral allografts and with autografts.
Results were evaluated with Modified Mankin’s histological score at
8 weeks after surgical implantation of the grafts.
Results: All wounds healed without complication. Grossly, almost all de-
fects were filled with healing tissue, but specimens from autograft group
showed rough appearences on the transplantation sites. (p<0.001)
Group 1 Group 2
(Autograft) (Ä°mmature)
Mean ± SD Mean ± SD p value
Articular cartilage structure 8.39±1.66 5.68±2.51 0.001
Superficial cells 2.21±1.55 1.43±0.57 0.001
radial cells 7.36±2.92 4.61±3.2 0.002
Toluidine blue staining 2.96±0.84 1.89±0.99 0.001
Pannus formation 2.29±0.9 1.57±1.07 0.01
Tide mark zone 2.32±1.06 1.75±1.21 0.07
Total 25.54±6.29 17.29±7.9 0.001
Conclusions: Immature cartilage contains high amount of undifferenti-
ated multipotent elements with a marked mitotic activity, paracrine and
autocrine signaling among the cells and high susceptibility to the grow-
ing factors and cytokines. Moreover, the immature cartilage has a double
source of nutrition, it can survive with metabolic support of the synovial
fluid and vascular connection from the base of the graft. Immature carti-
lage allografting group scores differ superiorly from the autograft group
with a very low p value ( p< 0.001). Immature allogenic cartilage with
histopathologino immune response, and due to the superior properties
and well integration with the surrounding tissue the immature cartilage
allograft may be an appropriate graft alternative for chondral defects.
184 Free Papers
25.1.6
Evaluation of a Biphasic Graft for Osteochondral Repair in an
Equine Model
L.A. Fortier1, T.M. McCarrel1, G. Bradica2, E. Castiglione2, R. Saska2,
R.C. Lehman3
1
Ithaca/United States of America, 2Exton/United States of America,
3
Kirkwood/United States of America
Purpose: Osteochondral (OC) and full thickness chondral lesions
heal with biomechanically inferior fibrocartilage. The objective was
to evaluate a biphasic graft (KNC CRD) for OC repair. Our hypoth-
esis was that KNC CRD would be safe and improve repair compared
to microfracture.
Methods and Materials: Twelve horses (2-5 years) had 10mm diam-
eter x 10mm deep OC defects created in the lateral femoral trochlear
ridge arthroscopically. KNC CRD was hydrated in bone marrow and
press-fit into the defect. In the control/contralateral limb, 10mm di-
ameter full thickness chondral defects were created and microfrac-
tured. Radiographs were obtained pre-operatively, post-operative-
ly, and 2-, 4-, and 12-months post-operatively. Repair tissue was
evaluated arthroscopically at 4- and 12-months post-operatively.
Results: No horse had radiographic evidence of lysis, bone cysts,
or osteophytes. KNC CRD had significantly increased sclerosis
(p=0.003) compared to microfracture at all post-operatives time-
points. At 12-months post-operative, the % of defect replaced with
bone for both treatments was significantly improved compared to
all other time points (p<0.001). Percent replaced by bone was sig-
nificantly better for control defects than for KNC CRD at 12-months
(p=0.021). There was no evidence of infection, device fragmen-
tation, synovial inflammation, or blood within any joint at 4- and
12-month arthroscopies. There was no significant difference by
treatment or time for patella, color, surface area repaired, integra-
tion, surface, or total safety score. KNC CRD had significantly better
fill (p<0.0001), total efficacy (p=0.0021), and total arthroscopic
scores (p=0.0069) at 4 months compared to control. Neither treat-
ment changed significantly from the 4- to 12-months.
Conclusions: The study suggests that KNC CRD improves defect fill
earlier, as compared to microfracture, and is able to maintain re-
pair out to 12-months post-operative. Further evaluation to 2 years
post-operative will provide valuable quantitative MRI, biomechani-
cal, and biochemical information regarding the long-term repair tis-
sue formed in KNC CRD or microfracture treated lesions.
25.1.7
Degenerative change and donor-site deterioration of patella-
femoral joint after the harvest of plug in osteochondral
autologous transplantation
S. Mukai, Y. Nakagawa, M. Kobayashi, R. Arai, T. Nakamura
Kyoto/Japan
Purpose: There are very few reports on the radiographic
findings of the donor site of the osteochondral autologous
transplantation(OAT). Our aim is to determine if the donor harvest
directly affect the progression of osteoarthritic(OA) change of pa-
tello-femoral(PF) joint.
Methods and Materials: The materials are patients treated by OAT
against the various knee disorders and followed more than 5 years,
which include 43 persons (17 men, 26 women) 53 knees. The mean
age at the operation was 42.3 ( 14 -76 ) and the follow up was 74
months(60 - 140). We retrospectively evaluated the OA change by
Kellgren-Lawrence classification of PF joint during the post-opera-
tive period. Degree of progression was determined as severe, mild
and no-significant-change (NSC). Then each donor-site was also
evaluated. We assessed age, the size and the location of injured
area, numbers of donor plugs, and primary disorder.
Results: Twenty-one knees (40%) were severe and 16 knees(30%)
were mild, and 16 (30%) were NSC. All cases of steroid-induced
osteonecrosis(5/5) were severe. But excluding these cases, the
patients with NSC were significantly younger. Donors were har-
vested from 49 lateral and 32 medial condyles, and 25 sites were
left as un-operated. The mild or severe OA changes were found on
29 of 49 lateral, 20 of 32 medial donor-sites, but also found on 15
of 25 un-operated sites. Twenty-eight knees were harvested from
both sides, and only 8 of them were bilaterally NSC. It is difficult
to distinguish degenerative change and donor-site deterioration.
We suppose that steroid-induced osteonecrosis strongly concern
to the OA progression. More than half of donor sites deteriorate,
however, more than half of un-operated sites also become worse.
Conclusions: Progression of OA findings in PF joint depends on the
primary disorder. The OA change tend to deteriorate in older peo-
ple. Non-harvested sites as well as donor sites show OA findings
after the donor harvest.
25.1.8
Knee mosaicplasty prospective evaluation: assessment of
functional outcome and joint degeneration progression at 10
years minimum follow-up
G. Filardo, E. Kon, S. Patella, A. Di Martino, L. D’Orazio, B. Di Matteo,
M. Marcacci
Bologna/Italy
Purpose: Articular cartilage lesions, because of their limited heal-
ing potential, are still a challenge for the orthopaedic surgeon. Dif-
ferent approaches have been proposed to treat these lesions, while
both their indications and clinical efficacy are still controversial.
Purpose of this study is the evaluation of the outcome of osteo-
chondral autografts for the treatment of femoral condyle cartilage
lesions at long-term follow-up.
Methods and Materials: We prospectively evaluated 25 patients
(mean age 29.4) affected by full-thickness chondral lesions of the
knee (<2.5 cm2) treated with arthroscopic autologous osteochon-
dral transplantation. 12 out of 25 patients underwent previous sur-
gery. 16 patients underwent associated procedures. All patients were
clinically evaluated at 2 and 7 years and at the final follow-up (10 to 12
years) using the International Knee Documentation Committee form
and the Tegner score. X-rays were also taken at the final follow-up
and used to assess the progression of the articular degeneration.
Results: The International Cartilage Repair Society objective evalu-
ation showed 76% of patients had normal or nearly normal knees at
the latest follow-up. The International Knee Documentation Com-
mittee subjective score significantly improved from preoperative
(37.3) to the final follow-up (76.2). The Tegner evaluation showed
a significant improvement after the surgery at 2- and at the last fol-
low-up (from 2.9 to 6.1 and 5.2, respectively); however, we noticed
reduced sports activity from 2 years to the last follow-up.
Conclusions: The results of this technique at long-term follow-up
are encouraging. This arthroscopic one-step surgery appears to
be a valid solution for treatment of small, grade III to IV cartilage
defects.
25.1.9
Osteochondral allograft: is it an active tissue or just a scaffold?
S. Kirk1, D. Ruta1, A. Meininger1, G. Filardo2, A.A. Hakimiyan1, L.
Rappoport1, B.J. Cole1, A. Kurz3, S. Chubinskaya1
1
Chicago/United States of America, 2Bologna/Italy, 3Centennial/
United States of America
Purpose: Evaluate the effect of pro-inflammatory cytokines on the
metabolism and survival of osteochondral allograft cartilage (OA)
as compared to fresh cartilage (FC).
Methods and Materials: Nine OA specimens (previously refrigerat-
ed) and six fresh hemicondyles (collected within 24h of death) were
obtained from normal donors. 4mm explants from OA and FC were
divided into: cultured control, IL-1b (0.1ng/ml), IL-6 (3ng/ml), IL-1b
(0.1ng/ml)+IL-6, IL-1b (10ng/ml), IL-1b (10ng/ml)+IL-6. IL-6 soluble
receptor (5ng/ml) was added to all IL-6-containing cultures. Cell vi-
ability, apoptosis, histology, proteoglycan (PG) synthesis and con-
tent were tested on days 0, 2, 7, and 14.
Results: At day zero, the viability of OA chondrocytes was 24%
lower and contained 29% more apoptotic cells than fresh chondro-
cytes (p<0.05). Cytokines did not significantly induce cell death
or apoptosis in OA cartilage. In FC, high dose IL-1 alone or in com-
bination with IL-6 decreased chondrocyte viability by day 14 (up to
20%; p=0.047 and p<0.005) and increased apoptosis by more
than 2-fold (p=0.05 and p<0.05) when compared to day 0 or day
14 controls. At day 0, FC showed 2.5 times greater PG synthesis
(p<0.05) and only half as much release of newly synthesized PGs
(p<0.01) when compared to OA chondrocytes. However, FC was
more sensitive to cytokine treatments: by day 14 high dose IL-1
alone or combined with IL-6 inhibited PG synthesis by more than
8-fold (p<0.02) vs 4-fold in OA (p<0.02) and induced higher PG
release, which resulted in elevation of Mankin score for FC.
Conclusions: The viability and metabolism of OA cartilage is sig-
nificantly lower than FC; however, OA cartilage is more resistant
to cytokine treatment. Hypothetically, the reduced metabolism of
OA cartilage might enable its better performance in inflammatory
environment suggesting that OA cartilage acts more like a scaffold
rather than an active tissue.

25.2.2
Hybrid morphological and biochemical T2 evaluation of cartilage
repair tissue based on a recently described double-echo at
steady-state (DESS-T2d) approach
G.H. Welsch1, T.C. Mamisch2, L. Zak1, A. Mauerer3, S. Apprich1, S.
Marlovits1, S. Trattnig1
1
Vienna/Austria, 2Berne/Switzerland, 3Erlangen/Germany
Purpose: Recently, the widely used 3D Double-Echo Steady-State (DESS)
sequence was reported to enable, in addition to the morphological infor-
mation it provides, the generation of biochemical T2 maps in one hybrid
sequence. The aim of this study was to use this new DESS-T2d approach
to assess the morphological Magnetic resonance Observation of Carti-
lage Repair Tissue (MOCART) score, as well as biochemical T2 values in
patients after matrix-associated autologous chondrocyte transplantation
(MACT) of the knee. Furthermore, to correlate this new hybrid approach to
standard morphological sequences as well as to standard T2 mapping.
Methods and Materials: Fifty consecutive MR scans during clinical rou-
tine standard follow-up intervals at 3.0 Tesla in patients (36.1±9.3 years)
after MACT of the knee joint were prospectively included. MOCART scoring
was prepared by i) a set of standard MR sequences and ii) the morphologi-
cal images of the new DESS-T2d sequence. T2 relaxation times were as-
sessed by region-of-interest analysis on basis of i) a standard multi-echo
spin-echo sequence and ii) the biochemical T2d images of the DESS-T2d
sequence. Analysis-of-variance and Pearson correlation was performed.
Results: The MOCART score correlated (0.945; p<0.001) significantly
as assessed with standard morphological sequences (68.8±13.2) and
the morphological images of the DESS T2d sequence (68.7±12.6). T2 and
T2d relaxation times (ms) were comparable in between the control carti-
lage (T2: 52.5±11.4; T2d: 46.6±10.3) and the repair tissue (T2: 54.4±11.4;
T2d: 47.5±13.0) (T2: p=0.157; T2d: p=0.589). As expected, T2d values
were lower than the MSME-T2 values, however both functional relax-
ation times correlated significantly (Pearson:0.429; p<0.001).
Conclusions: Comparable information on the repair tissue could be
achieved at enormous time-savings, with ~20 minutes for the stan-
dard sequences and ~5 minutes for the hybrid DESS-T2d sequence.
Hence the presented approach provides the possibility to combine
morphological and biochemical MRI in one fast 3D sequence, and
thus, may attract for the clinical use of biochemical MRI.
25.2.3
Advanced morphological 3D- magnetic resonance observation of
cartilage repair tissue (3D-MOCART) scoring using different new
isotropic 3D sequences and their multi-planar reconstruction
G.H. Welsch1, L. Zak1, T.C. Mamisch2, A. Mauerer3, S. Marlovits1, S.
Trattnig1
1
Vienna/Austria, 2Berne/Switzerland, 3Erlangen/Germany
Purpose: To use the capabilities of the new isovoxel sequences and
their three-dimensional (3D) multiplanar-reconstruction (MPR) features
in the monitoring after cartilage repair procedures, the 3D magnetic
resonance observation of cartilage repair tissue (MOCART) scoring
system was recently introduced. The purpose of this study was to eval-
uate a new isotropic 3D proton-density, turbo-spin-echo (PD-SPACE)
sequence compared to an isotropic 3D true-fast-imaging with steady-
state-precession (True-FISP) sequence and to a set of 2D standard MR
sequences in their depiction of the 3D-MOCART score.
Methods and Materials: Sixty consecutive MR scans on 37 patients
(32.8±7.9 years) during clinical routine at standard follow-up intervals after
matrix-associated autologous chondrocyte transplantation (MACT) of the
knee at 3 Tesla were prospectively included. The new 3D-MOCART score
with eleven variables was assessed using the standard 2D sequences
(~15minutes) and the multiplanar-reconstruction (MPR) of both isotropic
sequences (3D-PD-SPACE~7minutes; 3D-True-FISP~6minutes). Statisti-
cal correlation as well as subjective quality analysis was performed.
Results: The correlation between the 3D-MOCART scoring performed
by the different sequences was highly significant for the variables 1)de-
fect fill, 2)cartilage interface, 3)bone interface, 4)surface, 7)subchondral
lamina, 8)chondral osteophyte, and 11)effusion (Pearson-coefficients
0.514 to 0.865 (p<0.001)). The variables 5)structure, 6)signal intensity,
9)bone marrow edema, and 10)subchondral bone showed lower correla-
tions with best results in between the standard sequences and the 3D-
PD-SPACE sequence and in between the 3D-True-FISP sequence and the
3D-PD-SPACE sequence (0.307 to 0.633 (p=0.016 to p<0.001). Grading
of subjective quality revealed good results for all sequences (p≥0.05). Ar-
tifacts were most often visible on the 3D-True-FISP sequence (p<0.05).
Conclusions: Different isotropic sequences can be used for the 3D
evaluation of cartilage repair tissue with the benefits of isotropic 3D-
MRI, MPR and a significantly reduced scan time. The 3D-PD-SPACE
sequence reveals best results due to a better performance in the sub-
chondral bone and because of the suppression of susceptibility arti-
facts produced by implantation and previous surgeries.
Free Papers 185
25.2.4
Sodium MRI at 7.0 Tesla in patients after matrix associated
autologous chondrocyte transplantation and microfracturing in
the knee: correlation to morphological scoring and clinical data -
preliminary results
D. Stelzeneder1, S. Zbyn1, L. Negrin1, G.H. Welsch1, V. Juras1, P.
Szomolanyi1, S. Domayer1, R. Dorotka1, T.C. Mamisch2, S. Trattnig1
1
Vienna/Austria, 2Berne/Switzerland
Purpose: Sodium imaging at ultra-high field-strength is able to depict
proteoglycan content in cartilage. The aim was to correlate the rela-
tive sodium content of cartilage repair tissue to morphological MRI
(MOCART score) and clinical evaluation (IKDC subjective form) in pa-
tients after matrix-associated autologous chondrocyte transplanta-
tion (MACT) and microfracturing (MFX).
Methods and Materials: Seventeen patients (mean age 36.7) after
MACT (N=12) and MFX (N=5) were investigated on a 7.0 Tesla whole-
body MR-unit. Mean follow-up time after surgery was 4.7 years, lesion
size 3.0 cm2, and BMI 24.4. In addition to morphological sequences, so-
dium imaging was performed using a dedicated 23Na-knee-coil with an
optimized 3D-gradient-echo sequence. Sodium evaluations were per-
formed on equally sized repair tissue and native cartilage regions. The
normalized sodium-ratio (NSR) was calculated: NSR=healthy cartilage/
repair-tissue. The MOCART-point-scale was evaluated on morphologi-
cal proton-MRI. Clinical assessment was done on the day of MRI using
the IKDC subjective form. Pearson’s correlation was applied.
Results: The mean NSR (±standard deviation) was 1.64±0.37 (range
1.02; 2.42), with no difference between MACT (1.62±0.43) and MFX
(1.67±0.27). The mean MOCART score was 76.4±15.0 and IKDC sub-
jective was 71.7±23.2. There was no correlation between IKDC sub-
jective score and NSR (r=0.18; p=0.48), however there was a trend for
a moderate inverse association of the MOCART-point-scale and the
NSR (r=-0.46; p=0.055).
Conclusions: Sodium imaging at 7.0 Tesla is able to differentiate be-
tween repair tissue and native cartilage. The NSR-values higher than
1.0 reflect a lower sodium content within the repair tissue in compari-
son to healthy cartilage. Our results suggest a trend for an association
of high NSR (i.e. low sodium and proteoglycan content) with low MO-
CART scores. We found no association between the IKDC subjective
score and the NSR. This experimental study correlates sodium imaging
at ultra-high field-strength to clinical parameters and morphological
grading in patients after cartilage repair surgery for the first time.
25.2.5
Correlation between MRI- and clinical assessment after matrix-
associated autologous chondrocyte transplantation on the
femoral condyle of the knee joint
L. Zak1, G.H. Welsch2, S. Trattnig2, S. Marlovits2
1
Baden/Austria, 2Vienna/Austria
Purpose: The aim of this study was to develop a pointing-scale for the
previous published 3D-MOCART-score, to evaluate the correlation
between clinical and MRI-results after matrix-associated autologous
chondrocyte transplantation of the knee (MACT).
Methods and Materials: 41 MRI Follow-up measurements of 41 patients,
with a minimum follow-up of 12 months (Mean: 41.1 month) after MACT
on the medial or lateral femoral condyle of the knee joint were studied
by means of MRI and different clinical Scores. The MRI-measurements
were performed on a 3.0 Tesla unit. A proton-density Turbo Spin-Echo
(PD-TSE) sequence, a T2-weighted Dual Fast Spin Echo (Dual-FSE) se-
quence and a T1-weighted Turbo Inversion Recovery Magnitude (TIRM)
sequence were used for assessing the MOCART-score, and an istotropic
3D-True FISP sequence was used for the 3D-MOCART-score. The sub-
jective clinical assessement was evaluated at the same point than the
MRI measurement, whereas the state of health and state of activity was
ascertained by the Brittberg-Scale, the IKDC-Score, the KOOS-Score, the
Noyes Sports Scale, the Tegner & Lysholm Score and the VAS-Score for
pain. The weighting of the variables in the 3D-MRI-assessement was
calculated by the Regression-coefficients. Furthermore a pointing-scale
for the 3D-MOCART-score was developed and correlated with the origi-
nal MOCART-score as well as with the clinical data.
Results: The comparison between the two scores resulted in a good
correlation with a Spearman Correlation Coefficient of 0.59 (p<0,005).
The MRI-evaluation and clinical scores showed medium, mostly signifi-
cant correlations with coefficients between 0.24 and 0.48 in the 2D-
evaluation and values between 0.26 and 0.5 for the 3D-MOCART.
Conclusions: Within the present approach the newly introduced 3D-
MOCART score was adapted to a point-scaling system based on ac-
cepted clinical parameters. The given results imply that besides the ac-
cepted 2D-MOCART, the 3D-MOCART can be exploited for statements
about the clinical condition of MACT in the knee joint.
186 Free Papers
25.2.6
T2 mapping MRI characterization of articular cartilage in the
ankle joint after bone marrow-derived cells transplantation
repair procedure
M. Battaglia, F. Vannini, R. Buda, M. Cavallo, A. Ruffilli, C. Monti,
S. Giannini
Bologna/Italy
Purpose: Qualitative evaluation of postoperative outcome in carti-
lage repair techniques is an issue. Recently T2 mapping MRI is becom-
ing an increasingly popular instrument to provide information about
the histological and biochemical contents of healthy or reparative tis-
sue. Aim of this study was to investigate by the use of T2 mapping the
quality of the regenerated tissue obtained after bone marrow-derived
cells transplantation (BMDCsT) repair procedure in the ankle.
Methods and Materials: 20 patients affected by osteochondral le-
sions of the talus and previously treated by arthroscopic BMDCsT, were
evaluated by T2 mapping at 24 months post-op. DPFSE with or without
fat suppression, T2FSE with or without fat suppression, 3D SPGR and
T2-Mapping using a dedicated phased array coil and 1.5 T MR scan-
ner were used. 20 healthy patients were used as a control. MRI results
were correlated with clinical score (AOFAS) in all the cases.
Results: The regenerated cartilage showed in all the cases a mean
T2 value of 45 ms (range 34-50) with no significant difference com-
pared to that of normal hyaline cartilage. A reduced mean T2 value
<30 msc expression of fibrocartilage features, was shown in lim-
ited foci at repair sites. A clinical score >90 was correlated with the
presence of hyaline cartilage (p=0,05). Foci of fibrocartilage within
the repaired area negatively affected the clinical results (p=0,045).
Conclusions: T2 mapping MRI evaluation showed that BMDCsT is
able to provide a repair cartilage tissue closely similar to healthy
hyaline cartilage, confirming the validity of the technique. Nev-
ertheless the regenerated cartilage was not qualitatively homo-
geneous in the whole repaired area. Limited foci of fibrocartilage
within the repaired area were shown to be detected by the T2 map-
ping and to significatively correlate with the clinical result. T2 map-
ping showed to be a valuable tool for qualitative cartilage evalua-
tion in the ankle joint.
25.2.7
Delayed Gadolinium Enhanced MRI technique for the long term
evaluation of patients treated with Autologous Chondrocyte
Implantation
H.S. Vasiliadis1, B. Danielson2, M. Ljungberg2, B. McKeon2, A.
Lindahl2, L. Peterson2
1
Ioannina/Greece, 2Gothenburg/Sweden
Purpose: Our purpose is to show whether MRI with dGEMRIC tech-
nique (delayed Gadolinium Enhanced MRI of Cartilage) can give
valuable information regarding the quality and quantity of the re-
paired cartilage lesion after treatment with Autologous Chondro-
cyte Implantation.
Methods and Materials: Thirty six knees in 31 patients were as-
sessed 9 to 18 years after treatment with Autologous Chondrocyte
Implantation. All patients had isolated lesions, 27 on a femoral con-
dyle, 1 on the trochlea and 8 on the patella. The knees were evalu-
ated clinically with KOOS score and with the dGEMRIC (delayed
Gadolinium Enhanced MRI of Cartilage) technique. The T1 value was
measured for two representative regions of interest (ROI), one in the
repair tissue area (ROI1) and one in the surrounding cartilage (RΟΙ2),
giving information of the content of proteoglycans in the tissue.
Results: The T1 value in ROI1 was measured to be on average 467.5
ms (range 440-495) while the mean in ROI2 was 495.3 (range 462.3-
528.2). There was no significant difference between these mean
values of ROI1 and ROI2, suggesting comparable levels of proteogly-
cans in the repair tissue and the surrounding cartilage (p>0.05).
Intralesional osteophytes were found in 64% of the lesions, mainly
in younger patients with osteochondritis dissecans lesions or with
the history of subchondral bone surgeries (abrasion, drilling, mi-
crofractures) (p<0.05). Bone marrow oedema was found in 39%
of the knees. Mean Mean KOOS score was 80 for pain, 85.6 for ADL,
70.7 for symptoms, 48.3 for sport/rec and 57.8 for QOL. There was
no correlation with any of the MRI findings.
Conclusions: MRI with dGEMRIC gives valuable information for the
macroscopic appearance and micro-molecular quality of the repair
tissue after ACI. Nine to 18 years after the ACI, the quality of the
repair tissue is similar to the surrounding normal cartilage. The de-
fect area is restored in most of the cases.
25.2.8
Ultrasonic probe is useful for in-vivo quantitative assessment of
medial femoral condyle articular cartilage
T. Shimizu1, S. Wakitani2, Y. Tanaka2, Y. Yonetani3, Y. Shiozaki2, S.
Horibe2
1
Gifu/Japan, 2Osaka/Japan, 3Sakai/Japan
Purpose: Although objective evaluation of the articular cartilage is
important to assess the outcomes of surgical treatment, a reliable
method has not yet been developed. It has recently been reported
that quantitative ultrasound could assess living human cartilage.
The purpose of this study was to investigate whether quantitative
ultrasound can detect subtle change of the articular cartilage, as
well as age-related changes in the normal cartilage.
Methods and Materials: Thirty patients with an injured knee un-
derwent ultrasonic evaluation of the articular cartilage during ar-
throscopy. During the ultrasonic evaluation, the ultrasonic probe
was inserted into the knee joint using standard anterolateral and
anteromedial portals. The reflex echogram from the cartilage was
transformed into a wavelet map using wavelet transformation. As
quantitative index on the wavelet map, the maximum magnitude
was selected. Whether the cartilage was damaged or not was
judged from the arthroscopic view of the articular surface. The nor-
mal sites (27 sites) and the damaged lesion (outerbridge grade I-II,
10 sites) were measured.
Results: An accurate measurement was possible in the medial fem-
oral condyle, where the ultrasonic probe was easily to be put on
perpendicularly under arthroscopic control. The average maximum
magnitude values of normal and damaged cartilage were 4.06 ±
1.58 and 1.40 ± 0.62, respectively. The maximum magnitude was
significantly higher in the intact cartilage compared to the injured
one (P < 0.01). The maximum magnitude for intact cartilage of me-
dial femoral condyle showed a significant correlation with patient
age (R2 = 0.434, p < 0.01).
Conclusions: Our ultrasound measurement system offers potential
for the detection of subtle change of the cartilage. The maximum
magnitude is useful for quantitative assessment of especially me-
dial femoral condyle articular cartilage. Care should be taken in
interpreting results because the maximum magnitude values de-
crease with age.
25.2.9
Cartilage repair evolution in post-traumatic osteochondral
lesions of the talus: from autologous chondrocyte to bone-
marrow-derived cells transplantation
F. Vannini, R. Buda, M. Cavallo, A. Ruffilli, F. Di Caprio, B. Grigolo,
S. Giannini
Bologna/Italy
Purpose: Cartilage repair in osteochondral lesions is now more
than ever an hot topic for research. Although autologous chondro-
cyte implantation gave well documented satisfactory results, the
need for two operations and high costs has prompted a search for
alternatives. The purposes of this study are to describe evolution
in cartilage repair from open field autologous chondrocyte implan-
tation to regeneration by arthroscopic bone-marrow-derived cells
(BMDCs) “one step” technique; to present the results of a series of
patients consecutively treated and to compare in detail the differ-
ent techniques used in order in order to establish the advantages
obtained with the evolution in cartilage regenerative methods.
Methods and Materials: 81 patients (mean age 30±8 years) were
treated between November 1996 and January 2007. Patient evalu-
ation included clinical AOFAS score, X-Rays and MRI preoperatively
and at different established follow-ups. All the lesions were > 1.5
cm2 and received open autologous chondrocyte implantation (10
cases), arthroscopic autologous chondrocyte implantation (46 cas-
es), and “one step” arthroscopic repair by BMDC transplantation (25
cases). For arthroscopic repair techniques a hyaluronic acid mem-
brane was used to support cells and specifically designed instrumen-
tation was developed. Patients of all the 3 groups underwent a sec-
ond arthroscopy with a bioptic cartilage harvest at 1 year follow-up.
Results: Mean AOFAS score before surgery was 57.1 ± 17.2 and 92.6
±10.5 (p<0.0005) at mean 59.5 ± 26.5 months. A similar pattern
of AOFAS improvement in results was found in the 3 different tech-
niques. Histological evaluations highlighted collagen type II and
proteoglycan expression.
Conclusions: The described techniques were able to provide a repair
tissue which closely approximates the characteristics of the naive
hyaline cartilage. Evolution in surgical technique, new biomaterials
and more recently the use of BMDCs permitted a marked reduction
in procedure morbidity and costs up to a “one step” technique able
to overcome all the drawbacks of previous repair techniques.

25.3.2
Quantitative Evaluation of Spontaneously and Surgically
Repaired Rabbit Articular Cartilage using Ultrasound
Measurements in situ
T. Viren1, S. Saarakkala1, H.J. Pulkkinen1, V. Tiitu1, P. Valonen1, I.
Kiviranta2, M. Lammi1, J. Jurvelin1, J. Töyräs1
1
Kuopio/Finland, 2Helsinki/Finland
Purpose: In this study we investigated the ability of intravascular
ultrasound (IVUS) device to assess structural changes in spontane-
ously and surgically repaired rabbit articular cartilage.
Methods and Materials: Repair tissue of osteochondral lesions (di-
ameter 4 mm) in the rabbit knee joints were investigated within two
groups: 1) surgically repaired (recombinant human type II collagen
gel with chondrocytes (10×10 6/ml), n=8), and 2) spontaneously healed
(n=5) lesion sites. Intact adjacent tissue (n=13) was used as a con-
trol. Histological integrity of the samples was determined with the
O’Driscoll grading, light microscopy (Safranin O staining of proteo-
glycans and immunohistochemical staining of type II collagen) and
polarized light microscopy (Parallelism index). Ultrasound imaging
of the samples was conducted with a high frequency (40 MHz) clini-
cal IVUS (ClearView Ultra, Boston Scientific Corporation, San Jose,
CA, USA) device. Reflection coefficient (R), integrated reflection
coefficient (IRC), apparent integrated backscattering coefficient
(AIB) and ultrasound roughness index (URI) were determined from
radiofrequency ultrasound signal.
Results: URI and AIB was significantly higher in both repair groups
than in intact cartilage (p < 0.05). The reflection parameters (R
and IRC) were significantly lower in surgically repaired cartilage (p
< 0.05) than in intact cartilage. O’Driscoll grading indicated that
surgically and spontaneously repaired tissue were similar with and
structurally inferior to healthy cartilage. Further, repaired tissue
exhibited abnormally organized collagen network, rough surface
and lower type II collagen and proteoglycan contents. The repro-
ducibilities (sCV) of the ultrasound parameters were 1.4 – 9.1%.
Conclusions: Surface roughness and integrity of the intact and re-
paired rabbit cartilage could be quantitatively evaluated with the IVUS
technique. Furthermore, qualitative information about integration of
the repair tissue, integrity of the surface of the repair tissue and the
internal structure could be extracted from the ultrasound images.
25.3.3
The influence of microfracture hole spacing and depth on
cartilage repair in chronic chondral defects: Preliminary results
M.B. Hurtig1, R. Whiteside2, P. Marks2
1
Guelph/Canada, 2Toronto/Canada
Purpose: Approximately 40% of patients with 2-4 cm2chondral defects
do not respond to microfracture. Chronicity and tissue degeneration
must play a role. This experiment was designed to identify operative
parameters that could optimize the repair response after microfrac-
ture in chronic cartilage lesions accompanied by abnormal bone.
Methods and Materials: Adult female sheep (n=24) underwent bi-
lateral femoral condyle contusive impact injuries and partial me-
niscectomy. Three months later the injury sites were debrided to
10 mm in diameter, and three patterns of microfracture holes were
created as per Table 1. The control treatment represents the stan-
dard of care recommended by Steadman et al (2001). Animals were
sacrificed at 3 and 6 months post-microfracture ((n=4/group/time
period). Cartilage repair was assessed by biochemistry (cartilage
total sGAG), ICRS 2 scoring of histological sections and morphom-
etry to determine base attachment, integrative attachment and de-
fect fill. A paired T-test with the significance level set at p<.1 was
used to illustrate differences between treatments.
Results: All condyles developed degenerative cartilage lesions and
sclerotic subchondral bone. Treatment A disrupted of subchondral bone,
and caused cyst formation that delayed elaboration of repair tissue at
both time points. Treatment B improved defect fill at 3 and 6 months
(p<.08) as well as early basal attachment of repair tissue (p<.03).
Conclusions: Dense sclerotic subchondral bone had a negative
effect on repair tissue formation and attachment after microfrac-
ture. Careful resection of the subchondral bone plate followed by
shallow (2 mm deep) microfracture should be considered as an al-
ternative to the standard method. Aggressive microfracture using
closely spaced deep holes is contra-indicated. Drilling may be less
disruptive but without resection of sclerotic bone, attachment may
be a problem.
Free Papers 187
25.3.4
3-D metrics of structure and function of autologous osteochondral
grafts and surrounding host cartilage and bone in the goat knee
at 6 and 12 months
E.F. Chan1, I. Liu2, E. Semler3, H. Aberman4 , T. Simon4 , A.C. Chen2, K.
Truncale3, R.L. Sah2
1
San Diego/United States of America, 2La Jolla/United States of
America, 3Edison/United States of America, 4Los Alamitos/United
States of America
Purpose: Cartilage defect repair is traditionally analyzed by 2-D (im-
muno)histochemistry and biomechanical testing at single locations.
However, bone and cartilage remodeling may vary substantially
within and surrounding the repair site. The objective of this study
was to determine 3-D metrics of repair with autologous osteochon-
dral graft (AOCG) in the goat model.
Methods and Materials: Full-thickness AOCGs (Ø=3.5mm, h=6mm)
were harvested from the lateral trochlea and press-fit into defects of
the medial femoral condyle (MFC) in one knee of adult (3-4yo., n=8)
Spanish goats. Animals were euthanized at 6 and 12 months (n=4
each), and OPerated and NON-OPerated knees were harvested.
Cartilage function was mapped in 63 locations per knee by rapid in-
dentation testing (Ø=0.4mm indenter) and stiffness normalization
based on cartilage thickness. Cartilage structure was determined
by micro-computed tomography (mCT, (45µm)3 voxels) and calcula-
tion of 3-D metrics (volume, thickness, curvature). Data (mean±SD)
were analyzed by 2-way repeated measures ANOVA.
Results: In regions distant from the graft site, OP and contralateral
NON-OP MFC had similar geometry, with bone surface locations dif-
fering by only 0.071±0.023 mm and cartilage thickness differences
of 0.036±0.067 mm. Within and surrounding the OP graft, carti-
lage and bone were different in 3-D geometry from NON-OP. The
OP cartilage surfaces had subsided relative to NON-OP, with carti-
lage volume diminished by 21.3±6.15 mm3 (p<0.05) and cartilage
thickness decreased from 0.92±0.18 to 0.56±0.18 mm in the graft
and from 0.94±0.18 to 0.77± 0.11 mm in the adjacent host cartilage
(p<0.05). The OP bone surfaces were variably elevated relative to
NON-OP and surrounding host bone. Cartilage indentation stiffness
was lower in the OP than NON-OP knees by 68% at the graft site and
28% at the proximal surrounding host site (each, p<0.05).
Conclusions: 3-D analysis may provide increased sensitivity to ef-
ficacy of cartilage defect repair as well as mechanisms underlying
success or failure.
25.3.5
Zonal characterization of Equine Osteochondral Tissue from the
Medial and Lateral Femoral Condyle
K.E.M. Benders1, T. Klein2, J.C. de Grauw1, D. Hutmacher2, P..R. van
Weeren1, M. Kik1, D.B. Saris1, W.J.A. Dhert1, J. Malda1
1
Utrecht/Netherlands, 2Brisbane/Australia
Purpose: The equine species is becoming increasingly popular as a model
for the in vivo evaluation of orthopaedic approaches. As there is also an
increasing body of data suggesting that successful lasting tissue recon-
struction requires an implant that mimics tissue organization, there is a
clear need to investigate characteristics of equine osteochondral tissue.
Therefore, we undertook a comparative study of the characteristics of
equine, human and primate osteochondral tissue of the stifle (knee) joint.
Methods and Materials: Osteochondral cores (4-8 mm) were taken
from the summits of both medial and lateral femoral condyles of horses
(n=16, mean age: 10.5), primates (two Gorillas, a Barbary Macaque and
a Baboon) and humans (n=4, n=8 for cartilage thickness, mean age:
79.0±9.6 yrs). Osteochondral cores were either fixed in 10% formalin
(for histology and image analysis) or frozen at -20°C and sectioned in
200mm slices for zonal biochemical quantification of DNA, glycosamin-
oglycans (GAG) and collagen.
Results: The average Mankin score of the equine samples was 0.9±0.9,
whereas human and primate samples had considerably higher average
scores (3.9±1.1 and 3.5±1.3, respectively). The average equine cartilage
thickness was 2.37±0.58 mm (medial) and 1.30±0.21 mm (lateral). GAG
content was significantly lower in the superficial zone and DNA content
decreased significantly with depth through the articular cartilage. Im-
age analysis of cell distribution yielded a similar distribution with depth.
Consequently, GAG per DNA increased steadily with depth. Bone density
decreased with depth from the bone-cartilage interface. Human and pri-
mate samples demonstrated comparable trends, although more varia-
tion was observed, which was associated with the higher Mankin scores.
Conclusions: Equine and human osteochondral tissue possess similar
geometrical (thickness) and organizational (GAG, DNA distribution,
bone density distribution with depth) features. These similarities fur-
ther highlight the potential value of the equine model in the evaluation
of zonal approaches for cartilage repair.
188 Free Papers
25.3.6
Abnormal tibial torsion may cause an earliest detectable
deformity in STR/OrtCrlj osteoarthritis mouse model
K. Naruse, K. Urabe, K. Uchida, M. Toyama, M. Itoman
Sagamihara/Japan
Purpose: Morbidity caused by osteoarthritis of the knee joint (knee
OA) enormously expands medical expense for the treatment. Physi-
cians and surgeons as well as researchers are anxious to develop treat-
ments for OA patients at early stages of knee OA. Reported model ani-
mals develop detached anterior cruciate ligament or excised menisci,
which mainly reflects post-traumatic late stages of OA. Lack of choice
is apparent among the available models to be used for testing the ef-
ficacy of potential drugs, which target the early stages. A few reported
models, which may be suitable for studying early stages, are those of
spontaneous knee OA in guinea pigs, C57BL/6J and STR/ort mice. Our
histological observation as well as soft x-ray and micro-three dimen-
sional computed tomography (3DCT) profiles supported that early
changes typically observed among OA patients including the internal
tibial torsion are reproduced in STR/ OrtCrlj mice. Abnormality was
detectable even in the 10- to 20-week-old animals.We believe that the
internal tibial torsion reflects mechanisms through which osteoarthritis
develops in STR/OrtCrlj mice.
Methods and Materials: STR/OrtCrlj mice were obtained from Charles
River Laboratories Japan, Inc. (Yokohama, Japan) and analyzed several
weeks of age. The mice were euthanized by intraperitoneal injection of
sodium pentobarbital, and bilateral lower limbs were excised from each
hip joint and cleaned. The excised limbs were immediately reduced with
ice-cold 4% paraformaldehyde in phosphate buffer solution (Wako Pure
Chemical Industries, Ltd., Tokyo, Japan) at 4oC for 48 hours. Samples
were analyzed by X-ray system (SOFTEX-CMB4). The tibiae were ana-
lyzed by a high-resolution micro-CT system (MCT-100CB). For the his-
tochemical studies, dissected bilateral lower limbs were further decal-
cified. Slides were prepared with 4-µm-thick sections, embedded in
paraffin, and stained with classical hematoxylin/eosin or safranine O.
Results: Except for one out of 24 knees in 12 animals of 35-week and 2
out of 4 knees in two 45-week-old mice, all the animals showed subchon-
dral bone sclerotic changes in FT joints. Histological abnormality was a
greater difference in cartilage degeneration between 26 and over 35
weeks. Alterations of cell density and gradient and the decreased stain-
ing with Safranin O were ubiquitously observed in PF joint cartilage of all
the knees of-10 week-old mice. At 20 weeks, the loss of Safranin O stain-
ing was moderate to severe. In 20-week-old STR/OrtCrlj mice with age-
matched C57BL/6J mice as a control, radiographic and micro-CT observa-
tions pointed out internal rotation of tibiae. Morphological measurements
of tibial torsion by micro-CT 3D analysis showed that typical STR/OrtCrlj
tibiae at 35 weeks were twisted by 10 to 27 degrees when the two axes,
the proximal posterior condyle and bimalleolar axes are superimposed.
Conclusions: In conclusion, the earliest histological OA change was ob-
served in the patellofemoral joint prior to similar observations in the femo-
rotibial joint. Internal tibial torsion may be a cause of OA in the patellofem-
oral joints, which leads to the development of medial femorotibial OA.
25.3.7
Efficacy of bi-weekly intra-articular injections of BMP-7 in a
rabbit ACLT/MMT model of osteoarthritis
D. Lickorish, M. Haskell, S. Wood, A. Collett, D. Schrier, E. Goad, C.
Flory
Hopkinton/United States of America
Purpose: To evaluate the effect of bi-weekly, intra-articular injections
of BMP-7 on articular cartilage morphology and bony architecture in
an ACLT/MMT model of osteoarthritis
Methods and Materials: Twenty nine New Zealand White rabbits under-
went unilateral anterior cruciate ligament transection and medial meniscec-
tomy. Intra-articular injections of either control vehicle (n=9) or 5mcg BMP-7
(n=20) were given bi-weekly (every other week) for 8 weeks. Femoral artic-
ular cartilage morphology was assessed by india ink staining and decalci-
fied histology. Osteophyte volume and trabecular bone volume/thickness
in the medial and lateral femoral condyles were quantified using MicroCT.
Results: Compared to vehicle only controls, BMP-7 treated animals
showed consistently superior cartilage lesion scores (Figures 1a, 1b).
Marked fissuring of the articular cartilage and erosion to subchondral
bone was observed in controls but not BMP-7 treated joints. MicroCT
analysis of joints demonstrated no radioopaque material in either the
joint space or surrounding soft tissues in any of the BMP-7 treated ani-
mals. Osteophyte formation, predominant along the medial border of
the medial femoral condyle in control animals, was significantly reduced
or absent in BMP-7 treated animals. Femoral trabecular bone volume
(BV/TV) and trabeculae thickness in both medial and lateral condyles
were both higher in the BMP-7 treated group compared to controls.
Conclusions: Bi-weekly dosing of 5mcg BMP-7 over 8 weeks prevented
progression of cartilage damage in the rabbit ACLT/MMT model and
did not elicit either intra-articular or extra-articular bone formation.
Changes observed in femoral osteophyte formation and the femoral
trabecular bony compartment suggest altered loading conditions of
the BMP-7 treated joints.
25.3.8
Subchondral bone repair following marrow stimulation in a
mature rabbit model
H. Chen1, C.D. Hoemann1, J. Sun2, W. Ouyang1, A. Chevrier1, L.
Dragomir1, V. Lascau-Coman1, N. Tran-Khanh1, M.D. McKee1, M.S.
Shive1, M.B. Hurtig3, M.D. Buschmann1
1
Montreal/Canada, 2Laval/Canada, 3Guelph/Canada
Purpose: Subchondral bone repair in osteochondral defects following
bone marrow stimulation was evaluated at 3 months post-op using sur-
gical techniques of microfracture and drilling to different hole depths.
Methods and Materials: Trochlear cartilage defects 4 mm x 4 mm were
prepared bilaterally in 16 skeletally mature rabbits. Microfracture holes
were made to a depth of 2 mm, and drill holes made to either 2 mm or
6 mm depth under cooled irrigation. Animals were sacrificed 3 months
post-operatively and joints scanned by micro-CT. Bone repair was as-
sessed in a region of interest (ROI) of 3 mm x 3 mm x 2 mm within the
defects and compared to un-operated controls.
Results: Although almost all surgical holes were partially filled with
mineralised tissue, there was incomplete regeneration of normal bone
structure in defects. Abnormalities such as residual holes, cysts and
bony overgrowth were observed (Fig. 1). Subchondral defect repair led
to an average bone volume density (BV/TV) of 53.3%±8.89%, similar
to that in the control (57.1%±7.88%), but the repair bone was more po-
rous and branched as shown by significantly higher bone surface area
density (BS/TV) and connectivity density than native bone (Fig. 2). No
significant differences were found between microfracture and drilling
techniques for BV/TV, BS/TV and connectivity density (Fig. 2, compare
MFX2/I to DRL2/I). However, deeper drilling induced a repair response
in a larger region than shallower drilling (5.4±2.04 mm2 vs 4.4±2.17
mm2), suggesting a greater level of bone remodeling, which also led to
a higher BV/TV in defects (Fig.2A, compare DRL6/II to DRL2/II).
Conclusions: Reconstitution of normal bone structure was incomplete
at 3 months following bone marrow stimulation. Surgical techniques of
microfracture versus drilling had similar effects on subchondral bone
repair. In comparison with shallow drilling, deep drilling appeared to be
advantageous for subchondral bone repair through increased marrow
access and bone remodeling.
25.3.9
Intra-Articular therapy of Hyrdogel scaffold with Chondroitin
Sulphate in porcine cartilage defect model
K. Kannan, R. Xiafei, A. Mohd Hassan, J.H.P. Hui, E.H. Lee
Singapore/Singapore
Purpose: Autologous chondrocyte implantation (ACI), currently, is
the only useful surgical approach for full-thickness cartilage defects
in patients, which utilizes injection of cultured cartilage cells into a
debrided cartilage defect beneath a specially created lid of perioste-
um. Chondroitin sulphate (CS) in hydrogel presents a novel and safe
method of non-cell based treatment modality for cartilage defects. CS
is a direct stimulator of human chondrocytes growth. We studied the
optimal delivery of CS to the defect site using biodegraded injectable
hydrogel medium. A long term release carrier like the hydrogel pro-
vides a scaffold for a more local and direct source of CS to the joint.
Methods and Materials: 18 adolescent male pigs were utilized in the
study that involved the intra-articular injection of CS-hydrogel mix-
tures (CSH) and CS-phosphate buffered solutions (CSPBS) as con-
trols. Full thickness cartilage defects measuring 7mm were created
in the medial distal femoral condyles and the defects were injected.
Histological sectioning of the operated as well as the un-operated
side was performed at 6 weeks and at 12 weeks.
Results: Early results showed significant improvement in cartilage regen-
eration with more native features of hyaline cartilage in the CSH group.
There was no hyaline cartilage detectable at 12 weeks in the CS PBS con-
trol group. Only a few chondrocyte clusters were observed in the control
group while the CSH group exhibited increasing chondroctye morphology.
There were marked improvements in the Wakitani histological scores de-
noting the quality of the repair tissue in both time points of the CSH group.
Conclusions: We conclude that CS delivered in an injectable, in situ
gelable and biodegradeable hydrogel is a safe, convenient and effec-
tive away to treat cartilage defects. The long term and slow release of
CS in the joint could significantly enhance cell proliferation and cel-
lular migration from cartilage and bone to the defect site.

25.4.1
A randomized, multicenter clinical trial comparing BST-CarGel™
to microfracture in repair of focal articular cartilage lesions on
the femoral condyle: interim results from 41 patients
W.D. Stanish1, A. Restrepo2, P. MacDonald3, N. Mohtadi4 , P. Marks5,
M. Malo6, R. McCormack7, J. Desnoyers8, S. Pelet9, F. Lopez-Olivia10,
J. Vaquero10, F. Macule11, M.D. Buschmann6, M.S. Shive6
1
Halifax/Canada, 2Laval/Canada, 3Winnipeg/Canada, 4Calgary/Canada,
5
Toronto/Canada, 6Montreal/Canada, 7Vancouver/Canada, 8Greenfield
Park/Canada, 9Quebec City/Canada, 10Madrid/Spain, 11Barcelona/Spain
Purpose: BST-CarGel™ (BioSyntech Inc., Laval, Canada), a chitosan-
based medical device, is applied in conjunction with bone marrow
stimulation procedures. A randomized clinical trial was conducted to
evaluate the efficacy and safety of BST-CarGel™ treatment compared
to microfracture alone for repair of articular cartilage lesions, and an in-
terim analysis was carried out on the first 41 patients (20 BST-CarGel™,
21 microfracture).
Methods and Materials: The international (Canada, Spain, Korea), mul-
ticenter trial enrolled 80 patients, aged 18 to 55, with BMI≤30 and single
symptomatic grade III/IV focal lesions on femoral condyles. Patients
were randomized (1:1) at the time of surgery to receive either BST-
CarGel+microfracture or microfracture alone, and followed standard-
ized 12 week rehabilitation. The primary endpoint was cartilage repair
structure (quantity and quality), assessed using quantitative MRI at 12
months and analysis of elective biopsies after 13 months. Secondary
endpoints were safety, evaluated through adverse events, and clinical
benefit, determined with WOMAC (VAS) questionnaires.
Results: At 12 months, MRI analyses found increased fill and quality of
repair tissue in BST-CarGel-treated lesions compared to microfracture.
ICRS macroscopic grading of cartilage repair at the time of biopsies was
statistically better (p<0.05) for BST-CarGel™. (Fig1) Analyses of 21
biopsies (12 BST-CarGel™, 9 microfracture) demonstrated statistical
improvement (p<0.05) for BST-CarGel™ compared to microfracture
for Surface Architecture and Cell Viability parameters (ICRS I), the Over-
all Score and Superficial Zone (ICRS II), and repair tissue thickness by
histomorphometry. (Figures 1-2) Collagen organisation via Polarization
Light Microscopy was also improved for BST-CarGel™. WOMAC data
yielded equivalent improvements for both groups for pain, stiffness
and function. Adverse events showed BST-CarGel™ to be comparably
safe to microfracture alone.
Conclusions: BST-CarGel™ treatment improved lesion filling and qual-
ity of repair tissue at 12 months compared to microfracture in this inter-
im analysis. This improvement in tissue quality should result in greater
longer term durability of repair and sustained clinical benefit.
25.4.2
Evaluation of Safety & Efficacy of Mesenchymal Stem Cell
Composite for the Regeneration of Articular Cartilage Defect of
Human Knee Joint
C. Ha
Seoul/Korea, Democratic People‘s Republic of
Purpose: This study is to evaluate the safety and efficacy of human
umbilical cord blood derived mesenchymal stem cell composite for the
regeneration of articular cartilage defects of human knee joint.
Methods and Materials: Arthroscopically proven ICRS grade 4 lesions
were included in this study. MSC concentration of 0.5 × 107cells/ml
were implanted to the lesion as 0.5ml/㎠. Safety parameters included
physical exam (swelling, tenderness, range of motion, pain), vital signs,
lab tests and any advere events which were evaluated by the WHO
common toxicity criteria. Efficacy was evaluated according to the ICRS
Cartilage Repair Assessment , Pain VAS. and histological assessment.
Second look arthroscopy and biopsy was performed in 2 out of 7 cases
in this clinical trial after informed consent was obtained.
Results: There were no significant adverse event of more than grade 3
according to the WHO toxicity criteria. The overall Repair assessment
was improved in 67%. The biopsy result showed highly hyaline like re-
generative tissue by H&E, Saf-O staining and Col II immunostaining.
Conclusions: From the result of this study, the safety of the application
of human umbilical cord derived mesenchymal stem cell composite in
the regenerative treatment of articular cartilage defect of human knee
joint was assured. The efficacy was assessed as reasonable. The prom-
ising result of this study warrants further investigation in this strategy
of articular cartilage repair.
Free Papers 189
25.4.3
Agarose-alginate hydrogel as a scaffold for an allogeneic
Cartipatch®
T. Brune, N. Tan, L. Laganier, L. Barnouin
Mions/France
Purpose: Current strategies for cartilage repair aim at introducing
chondrocytes into scaffolds. An allogeneic source of cells would be
interesting as there would be no donor site morbidity and it would
allow a single-step surgical approach, without any timeout between
cartilage harvest and tissue graft. Therefore we studied the oppor-
tunity to grow arthroplasty-derived chondrocytes in comparison
with chondrocytes from young pathological donors (indicated for
autologous Cartipatch) in a characterized agarose-alginate hydro-
gel for cartilage repair. We also addressed the possibility to main-
tain the constructs throughout extended incubation.
Methods and Materials: Chondrocytes were isolated from macro-
scopically healthy cartilage harvested exclusively in non-weight-
bearing zones, either from young patients (with traumatic lesions or
osteochondritis dissecans) or from total knee arthroplasties. They
were monolayer-expanded, suspended in the agarose-alginate
hydrogel and incubated for 1 to 17 months under orbital agitation
at 37 C. We achieved immunohistochemical staining and RT-PCR
analysis in order to assess the potential of cell redifferentiation. Vi-
ability was also estimated by propidium iodide staining.
Results: Incubation in the hydrogel promoted redifferentiation of
both arthroplasty-derived (n=6) and young donors chondrocytes
(n=23). Indeed histological sections displayed cells with a spheri-
cal morphology surrounded by an extracellular matrix positively
stained for aggrecan and type II collagen. Preliminary RT-PCR
data showed an important increase of type II/I collagen ratio and
a slight increase of aggrecan expression throughout the process,
both higher for arthroplasty-derived chondrocytes. Assessing the
expression of matrix metalloproteinases evidenced the matrix
turnover. Finally following long-term incubation inside the hydrogel
viability remained stable (n=6) and the cells did not proliferate.
Conclusions: This agarose-alginate scaffold allows extracellular
matrix deposition and cell redifferentiation, in a roughly similar
pattern for arthroplasty-derived and young donors chondrocytes,
which had both partly lost their phenotype due to monolayer ex-
pansion. It also maintains cell viability for months, thus offering a
promising “on the shelf” solution for cartilage repair.
25.4.4
Arthroscopic autologous chondrocyte transplantation
A. Di Martino1, E. Kon1, G. Filardo1, S. Patella2, L. D’Orazio1, F.
Balboni, C. Montaperto, M. Marcacci
Bologna/Italy
Purpose: The incidence of articular cartilage pathology has grown
due to the marked increase in sports participation and greater em-
phasis on physical activity in all age groups. Unfortunately, articular
cartilage lesions, with their inherent limited healing potential, are
hard to treat and remain a challenging problem for orthopaedic sur-
geons. In the last years matrix autologous chondrocyte transplan-
tation has become a possible solution in the treatment of chondral
lesions. We used a biodegradable, hyaluronian-based biocompat-
ible scaffold for cell proliferation (Hyalograft C). The easy handling
of Hyalograft C has permitted to develop an arthroscopic procedure
for chondrocyte implant.
Methods and Materials: Arthroscopic technique has been used
from December 2000 in more than 150 cases. All the patients have
been prospectively clinically evaluated using the International Car-
tilage Repair Society score. Actually 83 patients achieved at least
7 years follow-up. Tegner score was applied to evaluate the sport
activity level. MRI evaluation was also performed.
Results: At the 7 years follow-up evaluation we had 8 failed pa-
tients, who were re-operated for the chondral lesion. However, all
the scores analysed still presented good results with a significant
improvement compared to the basal level. The mean IKDC subjec-
tive score obtained was 77.5 at 7 years. Self-assessment of quality
of life, assessed by EQ-VAS, showed a statistically significant im-
provement, too: 85/100.
Conclusions: This matrix autologous chondrocyte transplantation
procedure avoids the use of periosteal flap, simplifies the surgical
procedure and permits to perform the arthroscopic implant reduc-
ing the morbidity of the procedure. The medium-long term clinical
and MRI results at 7 minimum years follow-up are positive, confirm-
ing the positive results previously obtained and the perseverance
of the beneficial outcome offered by this bioengineered approach.
190 Free Papers
25.4.5
Ambulatory treatment of recurrent tenopathies using infiltration
of autholog blood growth factor
M.V. Fernandes Dias, C.H. Bittencourt, P. Bittencourt, C.F.
Bittencourt
Niterói/Brazil
Purpose: .O {font-size:149%;} The use of growth factor on walk-in
patients is considered by us a way of simplifying these procedures
with total safety for patients and also for quality of expected tissue
regeneration. Costs are lowered considerably. Patients become
less anxious as the method is less complex. The procedure is con-
sidered a simple treatment.
Methods and Materials: .O {font-size:149%;} Simplified technique
of infiltration in recurrent tenopathies (Achilles, patellar, elbow,
ischial tuberosity and rotator cuff) using local anesthetic after
evaluation of lesioned area. Seventy six patients were submitted
to this treatment from June 2007 to March 2008. Method consists
in drawing approximately 60ml of total blood from patient. Blood
then undergoes centrifugation for 15 minutes at 3500rpm followed
by removal of gel rich in platelets which are annulled of clotting
functions, adding calcium chlorate. Solution resulting of this pro-
cess is then infiltrated in lesioned area where platelets will release
protein contents during first `hour.
Results: .O {font-size:149%;} Patients informed complete improve-
ment of symptoms in 82% of cases and the other 18% informed
50% to 70% reduction of pain. Significant improvement was seen
when comparing MRIs before treatment and three months after.
Conclusions: .O {font-size:149%;} Chronic tenopathies occur spe-
cially due to repetitive effort lesions associated to deficient irriga-
tion of tendons. Our method proved to be extremely efficient as it
concentrates an extraordinary amount of proteins in the area and
after local gene differentiation, cells multiply and regenerate these
tendons, thus reducing pain and local inflammatory condition, al-
lowing the patient an early return to physical activities.
25.4.6
Image-guided surgical techniques for cartilage repair – an animal
trial
M. Kunz1, M.B. Hurtig2, S. Waldman1, S. Devlin1, J. Rudan1, D.
Bardana1, J. Stewart1
1
Kingston/Canada, 2Guelph/Canada
Purpose: We investigated whether image-guided surgical techniques
can improve the clinical outcome of mosaic arthroplasty. While mo-
saic arthroplasty is a valuable reconstruction option for cartilage
repair, the accuracy to harvest and deliver osteochondral grafts re-
mains problematic. Here we report on the use of two image-guided
systems (opto-electronic and patient-specific instrument guides) to
improve clinical outcome of mosaic arthroplasty in an animal model.
Methods and Materials: Fifteen sheep were randomized into three
groups with impact cartilage defects created in each knee. After
three months, the defect of one knee was repaired using the: (i)
conventional manual surgical approach, (ii) opto-electronic track-
ing, or (iii) patient-specific instrument guides. For both image-guid-
ed groups (ii, iii) harvest and delivery sites were pre-operatively
planned using custom-made software. During opto-electronic guid-
ance (group ii), instrument position and orientation were tracked
and superimposed onto the surgical plan. For the patient-specific
instrument guides (group iii), plastic templates were manufactured
which incorporated mirror images of the articular surface to allow an
exact fit between template and anatomy. Cylindrical holes within the
template guided surgical tools according to the plan. Three months
post-surgery, both knees were harvested and the curvature of the
reconstructed cartilage was compared to the cartilage surface of
a pre-defect arthrogram CT scan. For each repaired defect, macro-
scopic (ICRS) and histological repair (ICRS II) scores were assessed.
All results were statistically compared between the three surgical
approaches using either non-parametric or parametric ANOVA tests.
Results: There were no significant differences found in cartilage
surface reconstruction and macroscopic scores between the im-
age-guided and the conventional surgeries. However, both image-
guided surgical approaches resulted in significantly better histol-
ogy scores compared to the conventional approach (improvement
by 55%, p=0.02).
Conclusions: The results suggest that image-guided systems can
improve the clinical outcome of mosaic arthroplasy for the repair
of cartilage defects.
25.4.7
The biological reconstruction of a chondral defect of the patella-
femoral joint using dry arthroscopy.
B. Sadlik1, A. Solecki1, T. Bielecki2, R. Brzoska1, A. Blasiak1
1
Bielsko-Biala/Poland, 2Sosnowiec/Poland
Purpose: The tissue engineering technique using MSC are promising
for patellar chondral lesions where the blood supply from the sub-
chondral bone is insufficient. We present our method for the biologi-
cal reconstruction of chondral defect of the patello-femoral(PF) joint
with a dry arthroscopy technique (DA). This technique allows us to
perform the AMIC technique in the patella-femoral joint enhanced by
concentrated bone marrow without an open approach. An arthros-
copy conducted with neither a liquid filled joint cavity nor gas hyper-
presure we call dry arthroscopy and this allows us to perform a dry
procedure with a collagen matrix stick into the chondral defect. Such
an MSC implantation needs to be conducted in a dry environment.
Methods and Materials: The lateral border of the patella is sus-
pended by four threads to an Arthromast designed by us to allow
sufficient space for arthroscopic surgery without hyperpressure in
the PF joint. The membrane soaked with MSC is introducted into
the joint using a specially designed sleeve and fixed by Tissucol
glue to the defect crater.
Results: In this preliminary trial five patients ( 2 men and 3 women,
mean age 41Y ) with symptomatic chondral lesion (average 2,3cm2)
in the patello-femoral joint underwent the above described proce-
dure. Clinical results were assessed after 6 and 9 months by clinical
examination IKDC2000 score , VAS scale and MRI. Good and very
good results were obtained and confirmed by MRI in all 5 cases.
Conclusions: We have showed the clinical ability to perform AMIC
procedure with MSC transplantation as an arthroscopic procedure
with good early clinical results. Development of the dry arthrosco-
py technique will allow further application of more dry procedures
which have, up to now, been performed as open techniques.
25.4.8
‘Selective Osteoporosis of Medial Tibial Condyle’, one of the
culprit for onset of Medial Compartment Osteoarthritis Knee.
D. Goyal, A.D. Goyal, V. Thakkar
Ahmedabad/India
Purpose: Majority of Degenerative Osteoarthritis of Knee begins
with Medial Compartment. Medial overload can be due to; Varus de-
formity, Weight bearing axis passing thru Medial Joint in Non-varus
knee, deformities at other bone/joints etc. Literature speaks a lot
about association of Medial Compartment Osteoarthritis (MCOA)
and Varus deformity. But, association between Selective Osteopo-
rosis of Medial Tibial Condyle (MTC) and Varus deformity has never
been reported. Author believes that ‘Selective Osteoporosis of Me-
dial Tibial Condyle’ occurs in some, leading to Proximal Tibia varus
deformity. This in turn causes medial compartment overload, lead-
ing to onset of MCOA. The paper aims to find out if such entity as
“Selective Medial Tibial Condyle Osteoporosis” exists?
Methods and Materials: Presence of Genu Vara overloads the Me-
dial Compartment, thus starting/advancing medial arthritis. MCOA
can also cause Genu vara deformity. So either of them can be pre-
cursor/successor of other. Presence of “Selective Osteoporosis
of MTC” can lead to gradual collapse of MTC architecture causing
‘Proximal Tibial Metaphyseal Varus Deformity’. This deformity, oc-
curring few centimetres below medial joint line causes medial over-
load. The Medial compartment continues to bear the load, till carti-
lage reaches a threshold, and starts degenerating. All Consecutive
patients coming to Senior Author’s Knee Clinic, with Medial Knee
Pain, from Jan2009-Jan2010 were included in study. Patterns of Os-
teoporosis, if present in Proximal Tibia, were studied. Cases with ra-
diological evidence of multiple compartment involvement, Sclerotic
joint margins, multiple osteophytes were excluded from the study.
Results: 27% of cases studied had ‘Selective Osteoporosis of MTC’.
65% of such cases had associated “Proximal Tibial Metaphysis
Varus Deformity”. There is a distinct group with overloaded Medial
Joint, that has Osteoporotic Medial Tibial Condyle as culprit.
Conclusions: “Selective Osteoporosis of Medial Tibial Condyle”
does exist. However, How many progresses to “Proximal Medial Tib-
ial Metaphyseal Vara” and MCOA; remains to be studied further?
 Free Papers 191

25.4.9
Implantation of a novel synthetic, biodegradable Scaffold for
Meniscus Tissue Regeneration
R. Verdonk1, W.C.J. Huysse1, R. Forsyth1, P.C. Verdonk2
1
Gent/Belgium, 2Gent-Zwijnaarde/Belgium
Purpose: To assess the safety and performance of a meniscus scaffold
designed to restore the function of the meniscus after meniscectomy.
Methods and Materials: This was a prospective, non-randomised,
single-arm, multicentre clinical investigation in patients with an ir-
reparable medial or lateral meniscal tear or partial meniscus loss,
with an intact rim and the presence of both horns. The scaffold is
designed to support the body’s own physiological pathways of tis-
sue repair. Clinical efficacy was assessed at up to 24 months post-
surgery using the following scores: Visual Analog Scale (VAS), the
International Knee Documentation Committee (IKDC), the Knee and
Osteoarthritis Outcome Score (KOOS) and the Lysholm score. Follow-
ing implantation, DCE-MRI was performed to assess tissue ingrowth
at 3 months, and anatomic MRI was performed to assess meniscal
defect filling and cartilage scores at 1 week, and 3, 12 and 24 months.
Results: Clinically and statistically significant improvements in all
outcome scores were observed at 6, and 12 months post-implanta-
tion. MRI scans show tissue gain was evident in 97.5% of subjects,
and that there was no evidence of cartilage damage related to the
device, up to 24 months. No safety issues related to the scaffold
were reported. It is anticipated that the complete 24 month data
will be presented here.
Conclusions: Implantation of the meniscal scaffold resulted in
statistically significant and clinically relevant improvements in all
subjective clinical outcome scores at 12 months post-surgery. Tis-
sue ingrowth and stable or improved cartilage scores were demon-
strated by MRI assessments performed up to 24 months after im-
plantation. Actifit Study Group: R Verdonk, P Beaufils, J Bellemans,
P Colombet, R Cugat, P Djian, H Laprell, P Neyret, H Paessler
192 Posters
P1
Histological and Biochemical Characterization of Fresh vs.
Frozen Osteochondral Allografts: A Six Month In Vivo Study.
D. Chase1, A.L. Pallante2, R. Healey2, S. Görtz2, A.C. Chen2, S.T. Ball2,
W. Bugbee2, R.L. Sah2, D. Amiel2
1
8894/United States of America, 2La Jolla/United States of America
Purpose: Osteochondral allografting is a popular articular cartilage repair
procedure. It is empirically believed that the use of fresh allografts conveys
a longevity benefit, as compared to frozen allografts, owing to maintained
tissue homeostasis secondary to preserved chondrocyte viability.
This in vivo study aims to characterize the structural and biochemical
differences between fresh and frozen allografts in a goat model.
Methods and Materials: Frozen (-70°C) osteochondral allografts
(8mm diameter and 5mm deep) and fresh (48hr post-mortem)
allografts were transplanted (site-matched) into the distal femur of
seven adult Boer goats, either in the weightbearing medial femoral
condyle (MFC) or the patella femoral groove (PFG). At six months
the allografts along with contralateral controls were harvested
(28 grafts total). The retrieved allografts and controls underwent
gross and histological analysis with H&E staining. GAG content
was assessed qualitatively with Safranin-O staining. Radioactive
Sulfate (35S) uptake was analyzed as a marker of metabolic activity
and cell viability and was expressed as CPM (Counts Per Million)/
mg. dry weight. Histomorphometry to assess surface roughness
using computer generated idealized curves was also performed.
Results: Grossly, two of the three frozen MFC allografts were markedly
depressed. GAG content was nonexistent in all of the frozen allografts yet
was qualitatively robust in all of the fresh allografts and controls (Figure
2). 35S uptake was significantly decreased in the frozen allograft group
as compared to the fresh allografts (p<0.01) and controls (p<0.05,
(Figure 1). Surface roughness was significantly increased in the frozen
MFC allografts compared to the fresh MFC allografts (p<0.05). However,
roughness was not significantly different between the fresh and frozen
non-weightbearing PFC grafts (p=0.19).
Conclusions: In our study, freezing osteochondral allografts prior
to implantation resulted in gross depression, decreased GAG
content, decreased 35S uptake and increased surface roughness.
Therefore, freezing osteochondral allografts is indeed detrimental
to the biochemical and structural integrity in vivo.
P2
Normalization of Glenohumeral Kinematics in Curved vs. Flat Soft
Tissue Interposition Graft: Implications of Glenoid Reaming
N. Ghodadra1, K. McGill1, M.T. Provencher2, A.A. Romeo1, B.R. Bach,
Jr1, N.N. Verma1
1
Chicago/United States of America, 2San Diego/United States of America
Purpose: Resurfacing of the glenoid with biologic interposition of
soft tissue and isolated reaming of the glenoid to recreate glenoid
concavity have been used to treat young patients with glenohumeral
arthritis. The goal of this study was to determine the change and
location of glenohumeral contact pressures in curved vs. flat
biologic interposition arthroplasty in a glenoid arthritis model. We
hypothesized that biologic interposition with meniscal allograft will
lead to the best normalization of glenoid contact pressure.
Methods and Materials: Twelve cadaveric shoulders were tested in
static positions of humeral abduction (30, 60, 60 with 90 degrees
external rotation) with a 440N compressive load. Glenohumeral
contact area and pressure were determined for 1) intact glenoid,
2) glenoid with cartilage removed, 3) placement of interpositional
lateral meniscus allograft, 4) placement of interpositional achilles
allograft, 5) arthritis model with concentrically reamed glenoid, 6)
reamed glenoid with interpositional lateral meniscal allograft, 7)
reamed glenoid with interpositional Achilles allograft.
Results: Concentrically reamed glenoid with biologic interposition
of lateral meniscal allograft restored mean contact pressure to 80%
(p<0.03) of the intact state compared to 62% (p<0.05) for the
Achilles allograft. Use of the curved allograft (meniscus) in reamed
glenoid resulted in decreased contact pressure in the anteroinferior
quadrant compared to flat allograft (Achilles). Glenoid with cartilage
removed demonstrated statistically higher peak pressures than intact
glenoid and glenoid with interpositional allograft. Concentric reaming
of the glenoid led to decreased peak pressure and edge loading on the
periphery of the glenoid compared to the arthritic glenoid model.
Conclusions: Glenohumeral contact pressure is optimally restored
with biologic interposition of lateral meniscal allograft compared to
Achilles allograft. Our findings suggest that concentric reaming of
the glenoid plays a pivotal role in evenly distributing glenohumeral
contact pressure compared to the unreamed glenoid.
P3
Labral transplantation for hip instability after labrectomy
J.I. Erquicia, J. Miquel, M. Tey Pons, X. Pelfort, J.C. Monllau, E.
Cáceres Palou
Barcelona/Spain
Purpose: Acetabular labrum is important to maintain hip joint
integrity. There is an increasing interest in hip joint related
procedures to preserve the labrum and joint stability. A labral
transplant case is presented.
Methods and Materials: A 27 years old man was visited because
of hip pain. and instability. He was able of daily life activities
but he couldn’t perform heavy works or sports. On clinical exam
impingement test and apprehension test were positive. He was
previously treated because of femoroacetabular impingement
through arthroscopic femoral osteoplasty for three consecutives
times. X ray showed residual cam lesion in the Dunn axial view. MRI
showed absence of anterosuperior labrum. Intraoperative findings
confirmed radiological images. Degenerative labrum lesion around
the defect was eliminated with a 3,5 cm final defect. Acetabular bone
rim was debrided with a round burr. Labral defect was measured
and transplant was fixed with 5 suture anchors. Acetabular labral
transplantation and anterior femoral re-osteoplasty was performed
by an anterior approach.
Results: At two years follow-up patient is pain free and refers no
hip instability. He has returned to sport activity. Postoperative non-
arthritic hip score achieved is 95% and WOMAC score is 90.6%.
Conclusions: At two years follow-up, labral transplantation offers
an excellent result in this case. Effects of labral debridements need
to be considered as a potential risk of hip instability.
P4
Increased articular loading induced by uphill running cause
cartilage lesion: An adjustable cartilage mechanical loading
model in vivo and histological observation
K. Li1, X. Wei2
1
Taiyuan City/China, 2Taiyuan/China
Purpose: To develop a new adjustable in vivo cartilage loading
model and study different effect of uphill and horizontal running
on articular cartilage
Methods and Materials: The training took place on a self-made
adjustable treadmill. 15 rats were divided into three groups: control
group (ambulate freely in cage, n = 5); horizontal running group (1
km/d, 45 days, n = 5); and uphill running group (1 km/d, horizontal
running for 15 day for precondition and then uphill running for
30 days, n = 5).After 45 days, the rats were killed and articular
cartilage of loading area of the knee was observed with Safranin-O
staining and immunohistology.
Results: The cartilage surfaces were intact after the horizontal running
exercise. But the glycosaminoglycan concentration was decreased in
the superficial and middle zones. Pathologic signs consistent with
osteoarthrosis were developed after uphill running exercise. The
OARSI score significantly worsened after uphill running.
Conclusions: The increased mechanical loading during horizontal
running decreased the glycosaminoglycan concentration, and the
more increased mechanical loading induced by running on uphill
slops induced cartilage degeneration. Running on different slops is
an effective way to modulate the in vivo loading on cartilage.
P5
Efficacy and security of a bioengineered meniscus implant.
Experimental study in rabbit model of OA.
A. Izaguirre1, R. Gomez-Garcia1, F. Izaguirre2, G. Gonzalez1, Z. Garcia3,
G. Luna-Barcenas4 , H. Lecona1, H. Garcia-Campillo1, H. Villegas1, L.
Solis-Arrieta1, C. Pineda1, C. Velasquillo1, C. Ibarra1
1
Mexico City/Mexico, 2Tampico/Mexico, 3Queretaro/Mexico,
4
Mexico/Mexico
Purpose: To evaluate the efficacy and security of a bioengineered
meniscus implant and meniscus transplant using meniscectomy as
a control in an OA animal model.
Methods and Materials: Our sample included fifteen New Zealand
rabbits, five in each group. After simple randomization they were
assigned to a control group of lateral meniscectomy (MMx), lateral
Bioengineered Meniscus Implant with a composed chitosan/PVA
scaffold (BEMI), or lateral Meniscus Allograft Transplantation

(MAT). We evaluated the following outcomes: 1) surgical technique
reproducibility; 2) efficacy in terms of Macroscopic Jackson OA
scale, Histologic Modified Mankin OA scale; 3) security in terms
of Electron Microscopy and ultrasonographic assesment of the
implants. We conducted non parametric hypothesis tests with
2-tailed significance set at (p<0.05).
Results: We standardized the fibrochondrocyte cell culture
technique and proved adhesion to the scaffolds. Surgical technique
was feasible for MMx, BEMI, and MAT. We detected no differences
in the OA evaluation between MMx or MAT, and the BEMI group
presented significant inflammatory response.
Conclusions: We standardized surgical techinques of MMx, MAT
and BEMI in a rabbit model of OA. BEMI was neither efficacious nor
secure in preventing OA in this model.
P6
Effect of Hyaluronic Acid on impact-induced chondrocytes
apoptosis
R.B. Barreto, M.U. Rezende, G.B. dos Santos, A.C.F. Bassit, D.
Sadigursky
São Paulo/Brazil
Purpose: To evaluate the suppressive effect of intraarticular
injection of hyaluronic acid immediately after blunt trauma on the
progress of chondrocytes apoptosis in rabbits knees.
Methods and Materials: An experimental model of traumatic-
induced chondrocytes apoptosis was used in a total of twenty
mature New Zealand white rabbits of the study. The rabbits was
anesthetized and in the supine position the Mazieres contusion
model ( Â 1 kg weight released from the top of a cylinder of one-
meter length) was reproduced 3 times in each knee. Immediately
after the trauma was injected in one knee 2ml of Hyaluronic Acid
(HA knees) and in the contralateral knee was injected 2ml of
Saline (saline knees). The medication intervention was repeated 5
times within intervals of 4 days. Postoperatively, the animals was
permitted cage activity without any immobilization. One-week
after the last injection, they were euthanized and articular cartilage
fragment harvested. Apoptosis was detected using the ApopTag®
Peroxidase Kit that uses the TUNEL method. TUNEL-positive cells
was counted under light microscopy.
Results: Two rabbits died before the end of the experiment. There
was no significant difference in gross appearance of cartilage
surface between the HA and saline knees. Blinded histologic
analysis revealed that the HA knees had significant decrease of the
rate of chondrocytes opotosis (apoptosis rate mean + SD, 33.08 +
1.68) compared with the saline knees (54.49 + 3.61) (P<0.001).
Conclusions: This study suggest that the multiple injections of
HA, starting immediately after the trauma, decrease the rates of
impact-induced chondrocytes apoptosis.
P7
The efficacy of a novel double-network hydrogel for spontaneous
articular cartilage regeneration in a sheep model
M. Yokota1, N. Kitamura1, K. Arakaki2, E. Kondo1, S. Onodera1, T.
Kurokawa1, J.P. Gong1, K. Yasuda1
1
Sapporo/Japan, 2Naha/Japan
Purpose: We have developed an innovative method to induce
spontaneous hyaline cartilage regeneration by implanting an
originally developed PAMPS/PDMAAm double-network (DN) gel
at the bottom of an osteochondral defect in a rabbit model. Prior
to clinical application of this method, however, we need to clarify
whether the spontaneous hyaline cartilage regeneration occurs in
vivo in a large animal model.
Methods and Materials: A total of 5 mature female sheep (Suffolk)
were used in this study. An osteochondral defect having a 6.0-mm
diameter was created in the femoral groove of the right patellofemoral
joint. A cylindrical DN gel plug was implanted into the defect so that
a defect having 3-mm depth remained after surgery. In the left knee,
an osteochondral defect was created and remained without any
treatment. At 12 weeks after surgery, the tissue regenerated in the
defect was stained with HE, Safranin-O and immunohistochemical
stain (type-2 collagen). The macroscopic and histological scores
were quantitated using the Wayne’s grading scale.
Results: The defect treated with a DN gel plug was mostly filled
with a white opaque tissue at 12 weeks, while the untreated
(control) defect was filled with white patchy tissues. Histologically,
the defect in the DN gel-implanted knee was filled by a sufficient
Posters 193
volume of the proteoglycan-rich tissue stained with Safranin-O
and type-2 collagen. On the other hand, the untreated defect was
filled with the fibrous and bone tissues (Fig 1). Wayne’s scores
were significantly higher in the DN gel-implanted knees than the
untreated control concerning all gross appearance, histology and
total scores at 12 weeks (Fig 2).
Conclusions: This study demonstrated that implantation of the
PAMPS/PDMAAm DN gel at the bottom of an osteochondral defect
can induce spontaneous hyaline cartilage regeneration in vivo in a
large animal (sheep) model.
P8
Development of a new canine model of osteoarthritis: the groove
model
S.C. Mastbergen, F.P.J.G. Lafeber
Utrecht/Netherlands
Purpose: The frequently used anterior cruciate ligament transection
(ACLT) model of osteoarthritis in the dog makes use of a permanent
trigger (joint instability) for inducing degenerative changes. The
present study evaluates a relatively new canine model of osteoarthritis,
which is induced by a one-time trigger: the groove model.
Methods and Materials: Articular cartilage of the weight-bearing
areas of the femoral condyles in one knee of 30 Beagle-dogs was
damaged by making grooves without damaging the subchondral
bone. Surgery was followed by 3, 10, or 20 weeks intensified loading
of the affected joint. The severity of osteoarthritis was evaluated
at 3 (n=10), 10 (n=10), 20 (n=5) and 40 weeks (n=5) after surgery.
Cartilage integrity, chondrocyte activity, and synovial inflammation
were determined by macroscopy, histochemistry and biochemistry.
Results: Ten weeks post-surgery osteoarthritic features were found.
Proteoglycan synthesis, release of newly formed and resident
proteoglycans, and amount of denatured collagen were enhanced,
whereas proteoglycan content was diminished (all p<0.05). Based
on these parameters there was a slow progression over time from
20 and 40 weeks after induction. Importantly, three weeks post-
surgery these characteristics of osteoarthritis were not yet evident.
In contrast, synovial inflammation was mild at 3 and 10 weeks and
diminished slightly in time.
Conclusions: The present results show that characteristics observed
at 10 weeks or later post-induction of osteoarthritis in the groove
model: · comparable to those found the canine ACLT model. · slowly
progressive over time in the first year. · not (primarily) mediated
by synovial inflammation. · induced by a one-time trigger. · not
just the expression of the surgically applied damage. In this model
the effect of treatment of cartilage damage is not counteracted
by permanent joint instability or hampered by inflammation.
Therefore, the groove model might be more sensitive to effects of
therapy, aimed at cartilage protection and repair.
P9
In vitro effect of Hyaluronic acid, Carnosine and Hyaluronic
Acid+Carnosine on Bovine Articular Cartilage after oxydative
stress (Preliminary Study).
A. Migliore1, E. Bizzi1, U. Massafra1, V. Benetti2, M. Polzella2, D.
Benetti2
1
Rome/Italy, 2Perignano (PI)/Italy
Purpose: To evaluate the effects of Hyaluronic Acid (HA) alone or in
combination with Carnosine on the release of Glucosaminoglycans
(GAG) from a culture of bovine cartilage cells challenged with
hydroxyl radicals.
Methods and Materials: Fragments of articular cartilage were
harvest from 12 months old bovine ankle and cultered in Ham/F12
medium supplemented with antibiotics, fetal calf serum (1%) and
incubated for 24h in a mix of air/CO2 (19:1 v/v). Six different cultures
were then separated. For five cultures the medium was changed with
other mediums containing HA (10 or 50 mg/ml), or Carnosine (350
mg/ml) or HA (10 or 50 mg/ml) and Carnosine (350 mg/ml). Cartilage
was challenged with hydroxyl radicals produced by Femton reaction
with minor modifications and incubated for other 24 hours. At the
end of incubation period, GAG were assayed by DBD method.
Results: Hydroxyl radicals induced a significant release of GAG in the
culture without modified medium, while cultures in medium containing
HA at 10 and 50 and Carnosine 350 mg/ml showed a reduction of
GAG release of 18% and 31% and 15.5% respectively. When HA and
Carnosine were employed together, the efficacy was more evident and
statistically significant at HA 50 mg/ml and Carnosine 350 mg/ml.
194 Posters
Conclusions: This study seems to demonstrate the effects of HA
and Carnosine on cultures of bovine cartilage cells on the release
of GAG caused by oxidative stress induced by hydroxyl radicals.
Such results seem to suggest a biologic role of HA and Carnosine
on bovine cartilage cells in protecting cells from oxidative stress.
P10
Investigation of hyperlipidemic property in STR/Ort mice
K. Uchida, K. Naruse, M. Itoman, K. Urabe
Sagamihara/Japan
Purpose: STR/Ort mice have unique characteristics including
osteoarthritis and hyperlipidemia, and may be a useful model for
investigating the effect of dyslipidemia on the underlying mechanism
of primary osteoarthritis. We investigated the serum biochemical
parameters, and serum adiponectin concentration in STR/Ort mice.
Methods and Materials: Serum biochemical parameters and
serum adiponectin concentration in STR/Ort mice were mesured,
and compared the results with those for C57BL/6J, CBA/JN, and
ICR mice, the genetically obese, diabetic mouse strain BKS.Cg-
m+/+Lepr db/J (db/db) and lean control mice (m/m and db/m)
Results: The serum concentrations of total cholesterol in STR/Ort
mice were higher than those in other strains at 10 weeks. Serum
triglyceride concentrations in STR/Ort mice were also significantly
higher than those in ICR, CBA/JN, m/m, db/m and db/db mice at 10
weeks of age. Serum concentrations of insulin in STR/Ort mice were
higher than those in C57BL/6J, CBA/JN, ICR, m/m, and db/m mice at
10 weeks of age. The serum concentration of adiponectin in STR/Ort
mice was significantly lower than in other strains at 10 weeks.
Conclusions: STR/Ort mice have human hyperlipidemic patient-
like symptoms such as high serum total cholesterol, high serum
triglyceride, hyperinsulinemia, insulin resistance, dysregulation of
NEFA and low serum adiponectin. We hope this information will be
useful for researchers investigating lipid metabolism and primary
osteoarthritis using STR/Ort mice.
P11
In vivo stabilization of chondrogenically differentiated
mesenchymal stem cells by immune-isolation
K. Kleinschmidt1, D. Lichtenberg1, W. Brehm2, E. Steck1, W. Richter1
1
Heidelberg/Germany, 2Leipzig/Germany
Purpose: To determine the cartilaginous potential of human MSC,
immune-deficient mice are used, but their acquirement is expensive.
We investigated if the cartilage developing properties of hMSC can be
analysed in immune-competent mice using encapsulation techniques.
Furthermore, we aimed to analyse whether the reduction of nutrient
supply and thus an approximation to the joint situation may reduce
the mineralisation of MSC based cartilage constructs.
Methods and Materials: MSC pellets were pre-differentiated
chondrogenically for 4 weeks and encapsulated in alginate (AP),
dialysis tubes (DT) or diffusion chambers (DC) in parallel to non-
encapsulated controls. Constructs were transplanted into SCID
and immune-competent BDF1 mice. Explants were analysed by
alcian-blue, alizarin-red, and collagen-II (Col-II) staining as well as
by human specific ALU hybridisation and evaluated using double-
blinded histology scoring.
Results: Control constructs in BDF1 mice surprisingly showed no
signs of destruction and 4/5 spheroids were positive for alcian and
Col-II. In parallel, the degree of mineralisation was reduced in BDF1
compared to SCID (p=0.03). Alginate hampered mineralisation in
BDF1 (0/8) whereas in SCID 7/8 constructs calcified (p=0.001, Figure
A+B). AP resulted in better histological scores for Col-II (p=0.04)
and reduced mineralisation (p=0.03). In addition in SCID alginate
conferred higher alcian levels compared to controls (p=0.003).
DC was inapplicable since constructs lost their cartilage-like
properties. Only in SCID the use of DT resulted in an enhancement
of Col-II (p=0.03) and a reduced calcification (p=0.004).
Conclusions: The BDF1 mouse is an attractive cost-reducing
alternative to evaluate in vivo stability of MSC based cartilage
constructs which obviously conferred a reduced mineralisation
compared to SCID. Possibly, the in vitro generated cartilage
matrix constitutes an environment that inhibited destruction
of transplanted constructs. Whether the cartilage stability is
promoted by an intact immune-system or impaired by a high NK-
cell activity reported for SCID mice has to be evaluated by further
studies in additional mouse models.
P12
Articular cartilage repair by novel CCK2/gastrin receptor
antagonist “AG-041R” using Drug Delivery System
M. Kubo1, S. Imai1, T. Mimura1, K. Nishuzawa1, S. Araki1, H. Kitamura2,
K. Noda2, Y. Hirakura2, Y. Matsusue1
1
Otsu/Japan, 2Tokyo/Japan
Purpose: AG-041R was a novel indolin-2-one derivative and
originally synthesized as a CCK2/gastrin receptor antagonist to
treat gastric ulcers. Oral administration of a high dose of AG-041R
on rats was unexpectedly found to stimulate systemic cartilage
hyperplasia. Beneficial effects on chondrocytes in vitro and also
for cartilage defects in vivo were proven. In this study, we applied
AG-041R for rabbit cartilage defect using Drug Delivery System
(DDS) and examined the effectiveness histologically.
Methods and Materials: Full-thickness articular cartilage defects
(5mm-diameter) at the patellar groove of the rabbit knee were used
as cartilage defect model. PLA microspheres containing several
amounts of AG-041R were prepared by the solvent-evaporation
method. At first, in vivo release test was performed. In actual study,
PLA microsphere containing 40% AG-041R and PLA microsphere
uncontaining AG-041R (control ) embedded collagen gel were
grafted into the left knee (AG group) or right knee (control group),
respectively. The repair tissues were histologically examined by
subsequent staining, Toluidine Blue (TB), the immunostaining
of Bromodeoxyuridine (BrdU), type I and II collagen, and TUNEL
staining, 1, 2, 3, 4, 8, 12 weeks after the surgery.
Results: By in vivo release test, AG-041R was hardly detected in
synovial fluid, however remained in repair tissue for four weeks.
Repair tissue evaluation by TB staining showed significant
improvement of AG group’s repair tissue at 12 weeks compared
with control group’s one. Repair tissues were negative-stained
with type I collagen and positive-stained with type II collagen
immunostaining, i.e. hyaline cartilage. Proliferation cell number by
BrdU staining was significantly large in AG group’s repair tissue at
3 weeks compared with control group’. Apoptotic cell number by
TUNEL staining was not significantly different with each other.
Conclusions: The application of AG-041R using DDS was proved to
be effective for articular cartilage repair.
P13
Experimental Osteoarthritis in a Stable Knee Joint Using a
Critical-Size Defect in an Ovine Model
M. Schinhan1, M. Gruber1, P. Vavken1, R. Dorotka1, L. Samuoh1, C.
Chiari1, S. Nehrer2
1
Vienna/Austria, 2Krems/Austria
Purpose: Animal models simulating osteoarthritis are frequently
associated with irreversible changes in biomechanics. Although
these models successfully induce osteoarthritis, the results of
experimental repair procedures are impaired by biomechanical
problems. The aim of this study was to define the critical size of
a chondral lesion to induce osteoarthritis in a stable joint, thus
permitting correct assessment of cartilage repair procedures.
Methods and Materials: 16 mature Austrian mountain sheep with
a physiological joint were randomly divided into four treatment
groups. In each group, a full-thickness chondral cartilage defect
was created in the weight-bearing zone of the right medial femoral
condyle. The diameter of the defect was 7 or 14 mm. The sheep
were mobilized and permitted full weight-bearing for 6 and 12
weeks. Osteoarthritis was determined by gross assessment, India
ink staining, biomechanical testing, histology (Mankin and OARSI
scores), and COMP was chronologically monitored by ELISA.
Results: In the six-week group, only minor osteoarthritis was
registered for either defect size. After 12 weeks, the seven-millimeter
defect created focal monocompartmental osteoarthritis at the
medial femoral condyle and minor degenerative changes at the
corresponding tibia. The 14-mm defect induced minor osteoarthritis
at the femoral condyle and caused significant degenerative changes
at the tibial articular cartilage and the meniscus.
Conclusions: In an animal model, a 7-mm full-thickness chondral
defect with a weight-bearing regimen of 12 weeks may be considered
suitable to induce local osteoarthritis in an otherwise stable joint.
We conclude that this approach is well suited for cartilage repair
experiments.

P14
Tissue Engineering in Osteoarthritis- Cartilage Repair in an Ovine
Model using a Hyaluronan Matrix
M. Schinhan1, M. Gruber2, R. Dorotka1, N. Roessler1, S. Nehrer2
1
Vienna/Austria, 2Krems/Austria
Purpose: This animal model is aiming to prove the concept of tissue
engineering in a stable osteoarthritic joint in by the use of a hyaluronan
matrix (Hyalograft-C-plus) as a scaffold for chondrocyte transplantation.
Methods and Materials: Osteoarthritis on the medial femoral
condyle was induced using a critical size defect of 7mm and a
weight bearing regime of 12 weeks. At this index procedure cartilage
biopsies were taken for cell culture in the tissue engineering group.
At the second procedure a defect of 20x10mm was created in the
arthritic region of the medial condyle including an abrasion of
the subchondral bone to provide a trough for the implant. Then
the 18 sheep were divided into 3 groups with 6 sheep each: G1:
implantation of a hyaluronacid matrix seeded with chondrocytes,
G2: implantation of an unseeded hyaloronan matrix, G3: only
the bone preparation by abrasion. Sheep were euthanized and
analyzed 16 weeks after the second procedure. Gross examination
and Indian Ink Staining were performed. Histological sections were
stained with HE and Safranin O and assessed with the Mankin and
the O’Driscoll Score.
Results: Comparing the medial femoral condyle gross assessment
showed significant better results in G1 than G2 (p<0,009), which
was worse than G3 but not significant. Histological analysis using the
Mankin score for the regeneration area examined significant better
results of G1 compared to G2 und G3. Scoring the regeneration area with
the O´Driscoll score showed a high significant difference (P<0,0001)
between G1 (15,920±0,519) and G2 (5,042±1,491) and G3 (6,583±0,550).
Conclusions: The four-month results in our ovine model suggest
that matrix associated chondrocyte transplantation is a valid
strategy for osteoarthritis in an early stage.
P15
Modelling of chondral and osteochondral injuries in sheep: a tool
for preclinical studies in regenerative medicine
J. Barrachina1, D. Codina1, D. Peris2, C.S.F.R. Da Fonseca2, M.
Caminal3, X. Moll2, A. Morist2, R. Rabanal2, F. García2, J.J. Cairó2, F.
Gòdia2, J. García3, A. Pla3, J. Vives2
1
Sant Cugat del Vallés/Spain, 2Bellaterra/Spain, 3Barcelona/Spain
Purpose: Animal studies are critically important to developing effective
treatments for cartilage injuries. We herein present our experience, in
an ovine model, on the technical aspects of generation of chondral
and osteochondral lesions and their application for testing the
therapeutical potential of new approaches in regenerative medicine.
Methods and Materials: 2-year old ewes were used in all the
studies. To reduce the number of animals, we first used frozen
legs in order to determine the most convenient tools for each type
of lesion. In living animals, chondral and osteochondral lesions
were generated arthroscopically in the femoral medial and lateral
condyles of the two posterior legs. Animals were monitored by MRI,
echography and X ray and a full necropsy and histological analysis
was performed at the end of the studies.
Results: We describe our arthroscopic technique for the generation of
lesions of different depth for the study of chondral and osteochondral
regeneration. The reproducibility and the clinical evolution of the lesions
were assessed by MRI, ecography and X ray. Also, macroscopic and
histological analysis were performed at the end-point of the studies to
confirm the extent of the lesions and the effect on surrounding tissue.
Conclusions: We demonstrated that we can generate reproducible
lesions in sheep with the same extension and depth for the study of
both partial thickness and full thickness chondral repair, as well as
osteochondral repair.
P16
Establishment of osteoarthritis model using C57BL/6 mice by
controlling their movement
B.J. Kim1, B.H. Choi2, S.R. Park2, D. Minh1, B. Min1
1
Suwon/Korea, 2Incheon/Korea
Purpose: Many osteoarthritis (OA) models have been developed
in mouse to understand the OA progress and evaluating new
therapies. However, the individual variation of the joint lesions
remains as a critical problem in most of the current OA models.
Posters 195
Here, we established an OA model of C57BL/6 mice that is more
reproducible and amenable to therapeutic intervention by
controlling their movement.
Methods and Materials: OA was induced in 9 week-old C57BL/6
mice by destabilizing the medial meniscus (DMM). the mice were
then placed in the big cage for free movement (Group I) or in the
confined cage (4.5x7x20 cm) with no exercise (Group II), with an
exercise of 400 m/day (Group III), and with an exercise of 800 m/
day (Group IV), respectively. The mice were sacrificed 1, 2, and 4
weeks after the surgery. Macroscopic and histological evaluations
with ICRS scoring of cartilage lesions were performed on the medial
femoral condyles and tibial plateaus cartilages.
Results: The Group II mice showed slightly severer but more
reproducible OA lesions than those of Group I mice. The mice in
Groups III and IV showed that the OA lesions were getting severer
depending of the amount of exercise. In all Groups, the degree of
OA lesions increased along with the analysis times of 1, 2 and 4
weeks. Overall, the individual variation of OA lesions was much
less significant in the Groups of the confined cage than in the Group
with the free movement.
Conclusions: The OA lesion of mice could be more reproducible
and regulated by the enforced and periodic exercise of mice in
the confined cage. We speculate that our OA model of mice with
controlled movement could be a useful tool to study the OA process
and efficacy of OA therapy.
P17
Healing process of the fibrocartilaginous insertion of a
supraspinatus tendon tear in primates
H. Ueba, S. Imai, S. Araki, K. Nishizawa, Y. Matsusue
Otsu/Japan
Purpose: It is well known that supraspinatus tendon has a
prominent zone of fibrocatilage at its attachment site. And it
is suggested that it may have some important biomechanical
role to protect the tendon. Microfracture procedure represents
a frequently used technique for repairing of articular cartilage
defects. With this procedure, marrow-derived mesenchymal stem
cells were led to produce a fibrocartilage repair at the defect site.
We compared healing process of the microfracture-group which had
microfracuture procedure after transection of the supraspinatus
tendon to the control group. We chose cynomolgus monkeys as
a model animal because they use their forelimbs for grasping or
holding things rather than walking unlike other animals.
Methods and Materials: Eight cynomolgus monkeys were used in
this study. The shoulders were divided into 4 groups, which were
determined by the size of supraspinatus tear and subsequent
microfractures: 4mm+, 4mm-, 8mm+, and 8mm-. In the 4mm+ and
4mm- groups, the tendon was transected in 4mm length. In the
8mm+ and 8mm- groups, the tendon was transected in 8mm length
to be detached completely from its insertion. Microfracture was
then performed in the 4mm+ and 8mm+ groups. The monkeys were
euthanized on the 6, 12 and 24 weeks after surgery. The specimens
were evaluated with hematoxylin-eosin and Toluidine blue staining
and types I and III collagen immunostaining.
Results: In all groups, torn sites were surrounded by granulation
tissue. Histological analysis revealed progressive maturation and
reorganization of the bone-tendon interface. Compared to the
4mm- group, more collagen fiber continuity and more fibrocartilage
at the insertion site was seen in the 4mm+ group. Same trend was
seen in comparison the 8mm- group to the 8mm+ group.
Conclusions: The result of the present study indicated Marrow-
derived mesenchymal stem cells may play an important role in the
process of rotator cuff tear healing to the bone interface.
P19
Intervertebral disc repair using adipose tissue derived stem and
regenerative cells: Experiments in a canine model
H. Meisel1, T. Ganey2, W. Hutton2, M. Hedrick3, B. Strem3
1
Halle/Germany, 2Atlanta/United States of America, 3San Diego/
United States of America
Purpose: Disc injury can lead to disc degeneration. However, if
a damaged disc could be repaired in the early stages, before the
onslaught of degeneration then the process may be slowed down.
Our goal was to test the hypothesis that repair of a damaged disc
is possible using autologous adipose tissue derived stem and
regenerative cells (ADRCs).
196 Posters
Methods and Materials: Twelve dogs underwent a partial
nucleotomy at three lumbar levels (L3-L4, L4-L5, L5-L6); adjacent
levels served as non-operated controls. The animals (or discs)
were allowed to recover from the surgery for six weeks. At that time
subcutaneous adipose tissue was harvested and adipose tissue
derived stem and regenerative cells (ADRCs) were isolated. The
three experimental discs that had undergone a partial nucleotomy
were randomized to receive: 1) ADRCs in hyaluronic acid carrier
(Cells/HA); 2) HA only; or 3) No Intervention. Assessments of the
three experimental discs plus the two adjacent untouched discs
were made using MRI, radiography, histology and biochemistry.
The animals were euthanized at 6 months, and at 12 months.
Results: Repair in this study was specifically demonstrated
through histology and biochemical analysis. Disc levels receiving
ADRCs more closely resembled the healthy controls as evidenced
in matrix translucency, compartmentalization of the anulus, and in
cell density within the nucleus pulposus. Matrix analysis for Type-
II collagen and aggrecan demonstrated evidence of a statistically
better regenerative stimulation to the disc provided by ADRCs when
compared to either the HA only or the No Intervention treatments.
Conclusions: Autologous adipose tissue derived stem and
regenerative cells, as used in this disc injury model, were effective
in promoting disc regeneration, as evidenced by disc matrix
production and overall disc morphology.
P20
The early administration of PRGF in the ovine ACL-transected
knee osteoarthritis model
M. Sanchez, E. Anitua, J. Guadilla, R. Prado, F. Muruzabal, G. Orive,
N. Fiz, J. Azofra
Vitoria/Spain
Purpose: We assessed the ability of PRGF (plasma rich in growth
factors) to influence OA progression in sheep-model following
trauma to the joint caused by ACL rejection. The treatment was
initiated immediately after the surgery. PRGF system is a 100%
autologous preparation characterized by a 2-3 fold platelet
concentration from whole blood, and is a leukocyte-free plasma.
Methods and Materials: Seven Latxa sheep (4-6 years) and
weighing 61±6.1 kg were used. Body condition score was evaluated
estimating the level of muscling and fat deposition. Neiker-Tecnalia
Animal Care Committee approved all procedures used. Surgeries
were performed bilaterally in the hind limbs. A medial parapatellar
incision was made and the patella was reflected laterally in order to
expose the ACL, which was subsequently transected. Ultrasound-
guided infiltrations were initiated ten days after ACL transection.
Sheep were treated with a series of ten biweekly intra-articular
injections of 4 mL of PRGF in one stifle, and 4 mL of saline in
the contralateral. The platelet concentration was measured.
Sheep were sacrificed 32−37 weeks post-surgery. Knees were
macroscopically studied and the cartilage and subcondral-bone
of the femoral condyles were evaluated by two histological scales.
ANOVA analysis was performed to assess the differences between
the control and PRGF-treated animals. Age, weigh and platelet
number were used as covariables.
Results: Body condition scoring ranged from 2.0 to 3.5 (2.8673±0.5).
PRGF platelet mean content was 319x10 6 (±93x106) platelets/mL and
MPV 4.0±0.3 fL. The macroscopical findings were evident signs
of osteoarthritis, but no significant differences were found in the
scores. In the histological evaluations, the PRGF-treated group
presented an improvement in internal condyle scores (p<0.05)
compared with the controls. Both histological scales showed a high
correlation (Pearson correlation=0.922, p<0.01).
Conclusions: These preliminary data suggests that there may be a
therapeutic benefit associated with intra-articular injection of PRGF
in secondary osteoarthritis, following traumatic injury to the knee.
P21
Potential gene specific markers to proof the purity of human
articular chondrocytes in monolayer culture
C. Concaro1, H. Sterner1, C. Brantsing1, M. Leander1, J. van der Lee1,
M. Brittberg2, T. Dehne3, J. Ringe3, A. Lindahl4
1
Gothenburg/Sweden, 2Kungsbacka/Sweden, 3Berlin/Germany,
4
Göteborg/Sweden
Purpose: Due to the new EC regulations, No 1394/2007 on
Advanced Therapy Medical Products, there is a need to establish
markers to proof the purity of human articular chondrocytes in
monolayer culture before treatment. The most likely contaminantes
in the cultures are synoviocytes due to synovial overgrowth of the
biopsy area. The aim of the study was to (i) study chondrogenic
differentiation of synoviocytes and to (ii) identify genes that could
specifically determine purity regarding synoviocyte contamination.
Methods and Materials: Synoviocytes were isolated from human
tissues (n=5) and the cells were expanded in monolayer culture
(ML) followed by RNA preparation or seeding to a hyaluronan
scaffold (HYAFF 11, Fidia Advanced Polymers) subsequently cultured
for 14 days in a modified differentiation media. The scaffolds
were analyzed regarding handling properties, morphology and
histology. Messenger RNA from ML cultures was subjected to gene
expression analysis using oligonucleotide microarray (Affymetrix).
Expression data was compared to previous microarray data from
human chondrocytes in ML (n=5). Candidate genes selected from
the microarray analysis were confirmed by real-time PCR.
Results: Whencomparingmonolayerchondrocyteswithsynoviocytes
no differences were observed microscopically and synoviocyte
seeded scaffolds showed similar handling characteristics as
chondrocyte seeded scaffolds. However, the histology results
showed slightly higher matrix production in the chondrocyte seeded
scaffolds. The gene expression comparison identified a distinct
set of 4 genes (designated Syn 1- 4) that was barely detected in
chondrocytes but highly expressed in synoviocytes.
Conclusions: Although the articular cartilage and the synovium
tissue can be easily identified in biopsies it is impossible to
exclude potential contaminating synoviocytes in the subsequent
culture process. We here demonstrate that three of the identified
candidate genes have the potential for identifying chondrocyte
purity regarding synoviocyte contamination.
P22
T2 mapping - Does the choice of imaging sequence matter when
using T2 phantoms?
J.K. Riek
Rochester/United States of America
Purpose: The purpose of this study is to examine the use of different
T2 mapping sequences on different manufacturers’ MR scanners and
determine if similar results can be obtained by using T2 phantoms.
Methods and Materials: Two different T2 mapping sequences were
utilized on a Philips Panorama HFO 1T magnet, and on a GE Signa HDxt
1.5T magnet. On the Philips magnet, a 7-echo sagittal T2 mapping
sequence was used (TR=2000ms, TE=16,32,48,64,80,96,112ms,
ETL=7, 3.5mm slices, FatSat), and a dual-echo sagittal FSE
(TR=2000ms, TE=8.9, 85ms, ETL=12, 3.5mm slices, FatSat). On
the GE Magnet, a dual-echo sagittal FSE (TR=3200ms, TE=22.3ms,
78.1ms, ETL=8, 3.5mm slices, FatSat). T2 maps were calculated by
performing a linear regression on the natural log of the intensity of
the images. For the sequences with more than two echoes, where
the signal intensity was not different than the background noise,
the data point was ignored. Vials with varying concentrations of
copper sulfate were placed on the knee. These vials had known T2
relaxation times of 30, 60, 121 and 231 ms. Polynomial interpolation
was used to adjust the calculated T2 values in the image so that the
mean T2 relaxation in the vials corresponded to the known values.
Results: The average T2 value within the femoral cartilage on one sagittal
slice was calculated from both sequences on the Philips machine.
For the dual-echo sequence, the uncalibrated value was 68.1ms.
The calibrated value was 77.8ms. For the seven-echo sequence, the
uncalibrated value was 48.4ms, and the calibrated value was 71.01ms.
The calibrated dual-echo value on the GE machine was 69.5ms.
Conclusions: It has been demonstrated that reasonably similar
T2 values can be calculated across multiple pulse sequences
and multiple vendors utilizing T2 phantoms. Resolution and SNR
become the most important factors, not the pulse sequence.
P23
Expression of microRNAs in peripheral blood mononuclear cells
as a novel biological marker for knee osteoarthritis
A. Okuhara, T. Nakasa, H. Shibuya, T. Niimoto, N. Adachi, M. Deie,
M. Ochi
Hiroshima/Japan
Purpose: MicroRNA (miRNA) is a family of non-coding RNA that
plays an important role in human diseases, including osteoarthritis
(OA). Several studies demonstrated that OA was a systemic disease
with an aggregation of immune cells in synovium. The objective of

this study was to investigate the expression pattern of miRNAs
in peripheral blood mononuclear cells (PBMCs) and explore the
feasibility of using miRNAs as a biomarker to diagnose OA.
Methods and Materials: Thirty six patients with OA, and 10 healthy
controls were included in this study. We obtained PBMCs and
analyzed the expression of miRNA(miR)-146a, 155, 181a, and 223,
which express in immune cells and regulate immune function and
inflammation, in PBMC using quantitative reverse transcription-
polymerase chain reaction. The expression of miRNAs in OA was
compared with that in healthy subjects. We also investigated the
expression pattern of miRNAs expression in OA progression, and
its relationships with parameters that include age, body mass index
(BMI), femoro-tibial angle (FTA), and keratan sulfate.
Results: The average relative expression levels of miR-146a, miR-155
and miR-223 were 5.8-, 7.6-, and 12.6-fold, respectively, higher for OA
patients than for healthy subjects. As for the relative expression levels
of miR-146a, miR-155 and miR-223 according to Kellgren-Lawrence
classification, that of miR-146a, 181a and miR-223 were intensely
expressed in OA PBMCs with a low grade of the Kellgren-Lawrence
classification. There was a significant correlation between the expression
of miR-223 and the serum concentration of the keratan sulfate.
Conclusions: The present study demonstrated that the expression
levels of miR-146a, miR-155, miR-181a, and miR-223 in the PBMC of
OA patients could be a novel biomarker for diagnosis of early stage
OA and its prognosis. This evidence might lead to the elucidation of
OA pathogenesis and a novel therapeutic strategy for OA.
P24
Tailor-made scaffolds for stem cell chondrogenesis? Different
cross-linking of scaffolds from marine collagen affects their
mechanical and physical properties.
J. Renger1, H. Notbohm1, R. Wendlandt1, C. Koch2
1
Lübeck/Germany, 2Kiel/Germany
Purpose: Stiffness of scaffolds for tissue engineering is known to impact
stem cell fate by influencing focal-adhesion structure and cytoskeleton.
For tailor-made scaffolds, one seeks to deterministically change this
parameter to find the optimal growth conditions for chondrogenesis.
From preliminary analyses we can show an initial model for the impact
of UV-irradiation on stiffness and associated physical properties.
Methods and Materials: Beside decreasing its solubility, cross-linking
of collagen by UV-irradiation, heat, or chemicals results in a higher
stiffness. Collagen concentrations and collagen superstructure (i.e.
molecular or fibrillar collagen) have further impact on stiffness of a
matrix. By variing these parameters, one can take this advantage for
tailor-made collagen scaffolds. Pilot study: Scaffolds from molecular
and fibrillar-like marine collagen were produced from several collagen
concentrations. Cross-linking was induced by UV-light at 254 nm in
different doses optionally followed by heat fixation. Levels of cross-
linking were estimated by measuring solubility, enzyme resistance
and heat stability. A mechanical testing machine (Zwick/Roell) was
used to determine the influence of cross-linking degree and scaffold
composition on its mechanical properties, i.e. its elastic modulus.
Results: Increasing levels of cross-linking by UV-light showed
an increase in elastic modulus of matrices, whereas solubility
decreased. Heat fixation (1h) itself had a comparable effect to UV-
irradiation, but scaffolds irradiated with 106 kJ were insoluble even
without heat fixation. Differential scanning calorimetry showed an
increase in collagen denaturation after UV-irradiation, matching
the lower trypsin resistance.
Conclusions: Our studies show that by optimized cross-linking by UV-
irradiation, marine collagen scaffolds can be adjusted to a required
stiffness guiding stem cells to differentiation to chondrocytic
lineage. Cross-linking of the material can be observed by measuring
its solubility, heat- and enzyme resistance. Additionally, fibrillar-
like collagen scaffolds represent nano-designed matrices for tissue
engineering. This approach might be a further step to tailor-made
scaffolds from marine collagen.
P25
Cartilage tissue engineering with recombinant human type II
collagen gel infiltrated in PLDLA-scaffolds
V. Muhonen1, E. Järvinen1, E. Siren1, M. Forsten2, V. Ellä2, M.
Kellomäki2, I. Kiviranta1
1
Helsinki/Finland, 2Tampere/Finland
Purpose: Recombinant human (rh) type II collagen gel has been
shown to be eligible 3D-matrix for chondrocytes. We have used
Posters 197
novel biomaterials based on biodegradable polylactide polymers
in combination with the gel. With these constructs we aim to
provide suitable environment for the maintenance of chondrocyte
phenotype within a matrix that also provides structural stability.
Methods and Materials: Chondrocytes were harvested from
patellofemoral groove cartilage of bovine knees. To fabricate
collagen gels, rh type II collagen (FibroGen Europe, Helsinki,
Finland; 3mg/ml in 10mM HCl) was combined with growth medium
(DMEM/F12 with supplements) containing chondrocytes. After
gelation the gel-cell constructs were moved onto two different type
of non-woven PLDLA-scaffolds (PLDLA 96/4, Tampere University
of Technology, Tampere, Finland), and the hybrids cultured for 9
to 21 days. Different protocols were used to maximize infiltration
of the gel into the PLDLA-scaffold. Cell viability and depth of gel
penetration were analyzed. Chondrocytes were also cultured on the
PLDLA-scaffolds without the rh type II collagen gel, and analyzed
for penetration, viability and collagen type II production.
Results: When cultured in PLDLA-scaffolds without the rh type II
collagen gel, majority of the chondrocytes were alive and maintained
their rounded phenotype while growing parallel to the PLDLA-fibers.
The cells secreted collagen type II, suggesting preservation of
chondrocyte phenotype. Chondrocytes grown inside the gel retained
rounded, and the gel infiltrated into the PLDLA-scaffold. Penetration
depth of the chondrocytes within or without rh type II collagen gel
was 100-500µm. This indicates the tightness of the PLDLA-scaffolds
to allow free infiltration of the gel into the scaffold.
Conclusions: These results suggest that rh type II collagen gel within
biodegrable PLDLA-scaffold can provide mechanically rigid chondrogenic
structures for repair of large articular lesions. Further studies are needed
to increase the penetration of the gel into the PLDLA-scaffold and to
investigate cartilage producing capacity of the hybrid implants.
P26
Chondroinductive potential of subchondral nacre implant in the
sheep knee
M. Rousseau1, O. Delattre2, P. Netter2, P. Gillet1, E. Lopez3
1
Vandoeuvre les Nancy/France, 2Fort de France/Martinique, 3Paris/
France
Purpose: One of the major challenges of orthopedic surgery is the
restoration of bone and cartilage losses by using biomaterials.
Until now, only few molecules and biomaterials have shown their
potential ability to restore osteochondral structure and function.
The present study was thus designed to analyze the intra-articular
behaviour of nacre, a natural biomaterial, when implanted in
the subchondral bone area in the sheep knee. Preliminar results
suggest that this natural biomaterial may provide a potential
alternative source of chondroinductive molecules.
Methods and Materials: We implanted nacre blocks in sheep
trochlea by replacing the halfth of the femoral trochlea (nacre
group). For comparison we used complete cartilage resection
(resection group) down to the subchondral bone. Sheep were
sacrificed after 3, 6 and 9 months for histological and radiological
evaluations of the cartilage/nacre interface.
Results: In the “nacre group”, implants were well tolerated without
any synovial inflammation. In addition, we observed centripetal
regrowth of new hyaline cartilage in 6/9 cases after 3 months as
radiography showed osteointegration. In the “resection group”, no
chondral regrowth was observed, but, in contrast, a thin layer of
fibrous tissue formed. After 6 and 9 months, a new layer of cartilage
covered the nacre implant and cartilaginous islets of hyaline cartilage
were observed in the deep layers. No spontaneous osteochondral
regeneration was found in the “resection group” at these keypoints.
Conclusions: Nacre exerts chondroinductive potential as a
subchondral implant for cartilage regrowth. This new result opens
new perspectives for the use of nacre as a biomaterial for cartilage
repair and regeneration.
P27
Ibuprofen delivery microspheres : efficacy on cyclooxygenase in
activated cartilage and synovial membranes
L. Bédouet1, L. Moine2, F. Pascale3, M. Wassef1, M. Bonneau3, V.
Nguyen2, D. Labarre2, A. Laurent1
1
Paris/France, 2Châtenay-Malabry/France, 3Jouy-en-Josas/France
Purpose: Biodegradable microspheres (40 – 100 µM) containing
ester linked ibuprofen were prepared to provide a sustained release
of the anti-inflammatory drug in the joint cavity for intra-articular
198 Posters
treatment of inflammation and pain during osteoarthritis. The
objective of the study was to determine on cartilage and synovial
membrane explants the efficacy ie the cyclooxygenase (COX)
inhibition of the ibuprofen loaded microspheres.
Methods and Materials: Ibuprofen release from microsphere
Biodegradable microspheres conjugated to ibuprofen (40-100
µM) were incubated in PBS at 37°C. Ibuprofen was measured
using HPLC. Anti-inflammatory assay of microspheres In order
to prove the efficacy of ibuprofen released from microspheres
on COX activity inhibition, cartilage and synovial membrane
explants from sheep shoulder joint were co-cultured (n = 4) and
inflammation was induced with lipopolysaccharides (LPS 10 µg/
mL). To LPS-activated explants were added three doses (25 – 50
and 75 mg) of microspheres conjugated to ibuprofen. As positive
control for COX inhibition, control explants were treated with free
ibuprofen (1 - 10 - 50 and 100 µM). At day 2, PGE2 in the explant
supernatants was measured by ELISA. Statistical analyses were
performed on StatView SAS 2000 (SAS institute, Cary, NC).
Results: HPLC indicated that ~1 % of ibuprofen was released
after one month of incubation in PBS without initial burst release.
Anti-inflammatory testing on joint explants indicated that after
two days of culture, a significant reduction of PGE2 synthesis
was measured at 10 µM (p = 0.0033), 50 µM and 100 µM (p =
0.0008) of free ibuprofen and at every dose of microspheres (p
= 0.0008).
Conclusions: HPLC analysis of the culture medium showed
that 50 mg of microspheres released ibuprofen in medium
at a concentration of 35 µM which was sufficient to inhibit
PGE2 synthesis in explants, authorizing efficacy studies of the
biodegradable microspheres loaded with ibuprofen in an animal
model of osteoarthritis.
P28
Cartilage tissue engineering for auricular reconstruction in based
polyvinyl alcohol polymers
M.I. Garnica1, V. Martinez-Lopez1, Z. Garcia1, C. Ibarra2, F.E. Villalobos
Cordova2, G. Luna-Barcenas1, C. Velasquillo1
1
Mexico/Mexico, 2Mexico City/Mexico
Purpose: The biomaterials used for tissue engineering have
evolved over the last few decades. Natural and synthetic
biopolymers have been investigated for the development of
tissue-engineered cartilage. One obstacle that cartilage tissue
engineering faces is the development of a suitable environment
for growth and cell adhesion, able to promote retention of the
chondrogenic phenotype, production of ECM, and integration of
the scaffold to the native tissue. The objective of this study was
to analyze the viability, tissue organization and cellular adhesion
of auricular chondrocytes seeded onto polymers of PVA-HA-
EDGE (polyvinyl alcohol [PVA], hyaluronic acid-ethylene glycol
diglycidyl ether), Q-PVA-EDGE (chitosan-PVA-EGDE) and Q-PVA-
ECH (Q-PVA-epichlorohydrin), as a step for the clinical application
of tissue engineering in the construction of a pinna.
Methods and Materials: Three polymers, were synthesized as
films: PVA-HA-EDGE, Q-PVA-EDGE and Q-PVA-ECH. Auricular
cartilage was harvested from young New Zealand rabbits. The
perichondrium was carefully removed. Samples were digested to
isolate and to culture chondrocytes at high confluence. The three
aforementioned polymeric materials were seeded and cultured
in standard in vitro conditions for 10 days. The cell viability was
determined by calcein, and morphology characteristics was
studied by hematoxylin staining.Scanning electron microscopy
(SEM) was used in order to analyze cell adhesion to the polymer.
Results: Q-PVA-ECH constructs exhibited growth and cell
adhesion and ECM accumulation, whereas the remained
polymers retained their construct size but induced death cell
and did not provide support to cell adhesion. After 10 days of
culture, analysis by SEM and hematoxylin indicates cell adhesion
to Q-PVA-ECH and calcein test show survival of chondrocytes
seeded onto this film.
Conclusions: According to the results, Q-PVA-ECH is the most
viable to form an auricular structure to be used for reconstruction
of external ear . Nevertheless it is still necessary to perform
the mechanical characterization to determine if it keeps the
properties of the native auricular cartilage.
P29
Surface Modification of Extracellular Matrix Biomembrane for
Cytophilicity
B.R. Song1, Y.J. Kim1, M.S. Kim1, J.H. Yoon1, S.R. Park2, B. Min1
1
Suwon/Korea, 2Incheon/Korea
Purpose: Cell delivery has been getting more popular for tissue
regeneration so that many biomaterials are being developed for cell
delivery vehicle. In our previous study, we developed extracellular
matrix biomembranes with higher biocompatibility, but it needed a
higher cell-compatibility to be used as a vehicle for the cell delivery.
In this study, we examined whether surface charge of extracellular
matrix biomembrane can be changed to be a suitable cell delivery
vehicle by enhancing cytophilicity.
Methods and Materials: The surface charge of the Artifim®
(RegenPrime Co., Ltd., Korea) measured to confirm any changes of
chondrocyte attachment rate/forced with the alternated surface
charge. The surface charge of the Artifim® was changed by the
chemical bond of poly L Lysine and glutaric anhydride. For binding
of electrically positive polymer, poly L Lysine, on Artifim® surface,
the Artifim® was treated with 50mM EDC/NHS for 12hr and reacted
with the poly L Lysine for 24hr. Negative charges were introduced
on Artifim® surface by treating the glutaric anhydride in 0.8M
sodium phosphate buffer (pH8) for 24hr.
Results: Before modification, average surface charge of Artifim®
was -55mV. After modification, poly L lysine 5uM group showed
-30mV and glutaric anhydride 0.45M group showed -66mV. The
glutaric anhydride group significantly enhanced chondrocyte
attachment rate/force and those were getting more along with
higher concentration of glutaric anhydride. However Artifim®
treated with highly concentrated poly L Lysine decreased
chondrocyte attachment rate/force.
Conclusions: The attachment rate/force of chondrocytes can be
affected by the changes of surface charge. Especially the more
negatively charged surface provided suitable condition for the
chondrocyte attachment. From this study, we could modify the
surface of the extracellular matrix biomembrane with improving
cytophilicity.
P30
Substrate Elasticity Modulates TGF beta Stimulated Re-
differentiation of Expanded Human Articular Chondrocytes
D. Vonwil, A. Barbero, A. Trüssel, O. Haupt, I. Martin
Basel/Switzerland
Purpose: Culture of mesenchymal progenitor cells on substrates
with different elasticity has been shown to modulate cell fate/
commitments. We aimed this study at investigating whether
substrate elasticity modulates TGFβ stimulated chondrogenic re-
differentiation of expanded/de-differentiated human articular
chondrocytes (HAC).
Methods and Materials: Expanded HAC from 4 donors (43-77 years)
were seeded onto Type I collagen (CI) functionalized poly acrylamide
(PA) films (100-150 μm thickness) with a Young’s modulus of 0.26±0.08
kPa (soft), 21.32±0.79 kPa (intermediately stiff) and 74.88±5.13 kPa
(stiff) and induced to re-differentiate in a defined serum free medium
containing or not TGFβ-3 for 7 days. CI coated tissue culture treated
plastic was considered as an infinitely stiff substrate and HAC
aggregate cultures served as a standard re-differentitation control.
HAC were assessed for attachment, proliferation, morphology and
mRNA expression (type I and II collagen).
Results: In the presence of TGFβ-3, HAC attached similarly on the
different substrates and accomplished less than one total doubling
within 7 days. On intermediately stiff to infinitely stiff substrates
HAC assumed a fully spread fibroblastic morphology (shape factor
Φ = 0.23-0.27), whereas on the soft substrate, they remained more
spherical (Φ = 0.35±0.02) and had a reduced spreading area (up to
3.2-fold). F-actin organization on the soft substrate was restricted
cortically, while on the stiffer substrates, F-actin assembled into
stress fibres. Type II collagen mRNA expression on the soft substrate
was similar to that in aggregate culture and 18.1-fold higher than on
infinitely stiff substrates.
However, in the absence of TGFβ-3, type II collagen mRNA remained
at levels expressed by expanded/de-differentiated HAC.
Conclusions: Substrate elasticity modulated the re-differentiation
response of expanded/de-differentiated HAC to the chondrogenic
stimulus TGFβ-3, and thus underscores that mechanical compliance
in combination with appropriate soluble signals is an important
parameter in designing biomaterials for cartilage repair.

P31
The Effect of Joint Immobilization On The Spontaneous Hyaline
Cartilage Regeneration Induced By PAMPS/PDMAAm Double-
network Hydrogel
N. Kitamura1, K. Arakaki2, T. Kurokawa1, J.P. Gong1, F. Kanaya2, K.
Yasuda1
1
Sapporo/Japan, 2Naha/Japan
Purpose: We have developed an innovative method to induce
spontaneous hyaline cartilage regeneration in vivo by means of
implanting an originally developed PAMPS/PDMAAm double-network
(DN) gel. However, the mechanisms have not been clarified as of yet. We
have studied the effect of joint immobilization as a kind of mechanically
un-physiological environment on the spontaneous hyaline cartilage
regeneration induced by the PAMPS/PDMAAm DN gel.
Methods and Materials: A total of 20 rabbits were used in this study.
An osteochondral defect having a 4.3-mm diameter was created in
the femoral groove of the patellofemoral joint, and a cylindrical DN
gel plug was implanted into the defect so that a defect having 2-mm
depth remained after surgery. The right knee was immobilized by a
previously validated method, whereas the left knee was left mobile.
Ten rabbits were sacrificed at 4 and 12 weeks after implantation,
respectively. The knee joints were used for histological (HE,
Safranin-O) and immunohistochemical (collagen-2) evaluations,
and real time PCR analysis. The tissue regenerated in the defect
was quantitatively evaluated with the Wayne’s score.
Results: The regenerated tissue was rich in proteoglycan and type-
2 collagen at 4 and 12 weeks, whereas no staining with Safranin-O
or type-2 collagen was found in the defect of the immobilized knees.
Wayne’s scores were significantly higher in the non-immobilized
knees than in the immobilized knees (p<0.05) at 4 weeks. At 12
weeks, the gross appearance and total scores were significantly
higher in the non-immobilized knees than in the immobilized knees
(p<0.05). The mean relative values of type-2 collagen, aggrecan,
and Sox9 mRNAs in the regenerated tissue were higher in the non-
immobilized knees than in the immobilized knees.
Conclusions: The present study suggested that the mechanically
physiological environment is one of the essential factors to induce
the in vivo spontaneous hyaline cartilage regeneration by means of
implanting the PAMPS/PDMAAm DN gel.
P34
Hydrophobic/Hydrophilic Hydrogels for Use as Artificial Cartilage
Materials
B. Thomas, B. Mimnaugh, H. Brinkerhuff
Warsaw/United States of America
Purpose: Cartilage is comprised of both a hydrophobic collagen
segment and a hydrophilic GAG segment. We hypothesized that
hydrogels combining both hydrophilic and hydrophobic structures
may have better mechanical properties than those of produced by a
similar process containing only a hydrophilic segment and that the
hydrophobic segment would not adversely affect the wetability of
these materials.
Methods and Materials: The polymers and chemicals used in this
study included: polyvinylalcohol; poly(ethylene-co-vinyl alcohol);
poly(vinyl pyrrolidone); and dimethyl sulfoxide. A melt processable
miscible blend was created of the polymers utilizing a Leistritz
twinscrew extruder and molded into test samples. Compressive
modulus was measured on an Instron 3345. Contact angle
measurements were performed on a KRÜSS DSA100 Drop Shape
Analysis System using deionized water and bovine serum and a
drop size of 10μl. Bovine cartilage for the study was obtained from
fresh-frozen femoral heads.
Results: Figure 1 shows the compressive modulus of various
hydrogels. Figure 2 shows the contact angle for varying hydrogels
produced from the extruder/injection molding process as compared
to fresh-frozen bovine cartilage.
Conclusions: The data presented demonstrates the usefulness
of producing hydrogels with hydrophilic/hydrophobic segments.
These materials exhibit some of the desired mechanical properties
to be useful as cartilage replacement materials. The introduction of
hydrophobic groups in the materials has provided a positive effect
on mechanical properties. The contact angle has shifted from 32°
for the pure hydrophilic hydrogel to around 60° for the hydrophobic/
hydrophilic hydrogels. Therefore, the materials produced in this
study are more similar in hydrophobic/hydrophilic nature to that of
natural bovine cartilage.
Posters 199
P35
A Novel Double-network Hydrogel Induces Spontaneous Articular
Cartilage Regeneration In A Large Osteochondral Defect
N. Kitamura1, K. Arakaki2, T. Kurokawa1, J.P. Gong1, F. Kanaya2, K.
Yasuda1
1
Sapporo/Japan, 2Naha/Japan
Purpose: We have developed an innovative method to induce
spontaneous hyaline cartilage regeneration in vivo by implanting a
double-network (DN) hydrogel composed of poly-(2-Acrylamido-2-
methylpropanesulfonic acid) and poly-(N,N´-Dimetyl acrylamide).
The purpose of this study is to quantitatively evaluate the
regenerated cartilage with this method in comparison with the
untreated control.
Methods and Materials: A total of 23 mature rabbits were used
in this study. An osteochondral defect having a 4.3-mm diameter
was created in the femoral groove of the right patellofemoral joint.
A cylindrical DN gel plug was implanted into the defect so that a
defect having 2-mm depth remained after surgery. In the left knee,
a defect having 2-mm depth was created and remained without
any treatment. Five rabbits were sacrificed at 1, 2, 3, and 4 weeks
after implantation, respectively. Their knee joints were used for
histological (HE, Safranin-O) and immunohistochemical (collagen-
II) evaluations. The remaining 3 rabbits were sacrificed at 4 weeks
and served for real time PCR analysis.
Results: A proteoglycan-rich tissue appeared in a localized zone close
to the bony wall at 2 weeks, increasing at 3 weeks. The regenerated
tissue was rich in proteoglycan and type-2 collagen at 4 weeks. On the
other hand, the untreated (control) defect was filled with the fibrous
and bone tissues even at 4 weeks. Wayne’s scores were significantly
higher in the gel-treated knees than in the untreated control
(p<0.01). In the real time PCR analysis, type 2 collagen, aggrecan,
and Sox9 mRNAs were highly expressed, while it was seldom seen in
the tissues regenerated in the untreated defect.
Conclusions: The present study demonstrated that the implantation
of the DN gel plug could induce spontaneous hyaline cartilage
regeneration in vivo. We speculate that the in vivo biochemical
and biomechanical environment created by existence of the DN gel
may differentiate some undifferentiated cells including MSCs into
chondrocytes.
P37
Chondrogenic differentiation of human bone marrow
mesenchymal stem cells in chitosan based scaffolds
M. Alves da Silva1, A. Martins2, A.R. Costa-Pinto2, V.M. Correlo2, P.
Sol2, M. Battacharya3, S. Faria4 , R.L. Reis1, N.M. Neves1
1
Caldas das Taipas/Portugal, 2Guimarães/Portugal, 3Minessota/
United States of America, 4Braga/Portugal
Purpose: Herein we studied the effect of the stimulation provided
by a home-made flow perfusion bioreactor in the chondrogenic
differentiation of human bone marrow derived mesenchymal stem
cells (hBM-MSCs). We intended to ascertain if the shear stress
caused by the medium perfusion trough the cell seeded constructs
was capable of augmenting the differentiation process.
Methods and Materials: Human BMSCs were isolated from bone
marrow aspirates from informed consent donors. Cells were
characterized by flow cytometry. After expansion, hBM-MSCs were
seeded statically onto fiber meshes scaffolds, consisting of a blend
of 50/50 chitosan and Poly(Butylene Terephtalate Adipate)– CPBTA.
Constructs were cultured for 4 weeks in a flow perfusion bioreactor,
using complete medium for chondrogenesis supplemented with
TGF-β3. Samples were taken at different time points for DNA
content quantification, SEM analysis, histology, imunolocalisation
of collagens type I and type II and RT-PCR (specific primers for
the marker genes collagen type I, type II, type X, aggrecan, Sox9,
Runx2 and reference gene GAPDH).
Results: We observed enhanced ECM deposition and collagen
type II production in the bioreactor samples, when compared to
the static controls. Moreover, it was observed that hBM-MSCs, in
static cultures, take longer to differentiate. ECM accumulation in
these samples is lower than in the bioreactor sections, and there is
a significant difference in the expression of collagen type I.
Conclusions: We found that shear stress has a beneficial effect
on the chondrogenic differentiation of hMSCs. Thus, shear
stress generated in the bioreactor had a positive influence in the
chondrogenic differentiation of hBM-MSCs cultured in chitosan-
based scaffolds.
200 Posters
P38
Double-Network Hydrogels: candidate Cartilage Repair Materials
with high dynamic Stiffness, Suture Tear Resistance and Tissue
adhesive Bond Strength
M. Arnold1, D. Daniels2, S. Ronken2, N.F. Friederich1, T. Kurokawa3,
J.P. Gong3, D. Wirz2
1
Bruderholz/Switzerland, 2Basel/Switzerland, 3Sapporo/Japan
Purpose: In focal repair of joint cartilage and meniscus, initial
stiffness and strength of repairs is generally much less than
surrounding tissue. This increases early failure potential. Secure
tissue attachment is also a problem. Acrylamide polymer double
network (DN) hydrogels are candidate improved repair materials.
DN gels incorporate a soft, ductile molecular network within a
rigid one. Compared to a single network gel, an optimized DN gel
is markedly stronger and tougher. Previous studies demonstrated
physico-chemical stability and tissue compatibility, plus ability
to foster cartilage formation. Exploratory studies reported here
on PAMPS/PDMAAm and PAMPS/PAAm DN gels determined
mechanical properties related to surgical use.
Methods and Materials: Two different double-network (DN)
hydrogels, known as PAMPS/PDMAAm and PAMPS/PAAm, as
provided from the Japanes Co-authors were tested. Dynamic
stiffness was measured by fast (gait mode) and slow (nutrition-
mode) mm-scale micro-indentation. Suture tear-out and tissue
adhesive forces were measured with an MTS test machine.
Results: The results further support the clinical potential of
the DN-Gel concept. Remarkably these 90+% water, DN gels
exhibited dynamic impact stiffness (E*) values (~1.1 and ~1.5 MPa)
approaching swine meniscus (~2.9 MPa). Dynamic impact energy-
absorbing capability was much lower (median loss angles ~2º)
than swine meniscus (>10º), but it is intriguing that 90+% water
materials can efficiently store energy. Also, fine 4/0 suture tear-
out strength approached cartilage (~2.1 N and ~7.1 N vs. ~13.5 N).
Initial strength of attachment of DN gels to cartilage with acrylic
tissue adhesive was also high (~0.20 and ~0.15 N/mm2).
Conclusions: DN gel strength and toughness properties stem from
optimized entanglement of the two network components. DN gels
have obvious structural parallels with cartilagenous tissues.
P39
Tribological properties of poly(HEMA-co-MMA) materials as
meniscus substitutes.
J.L. Sague Doimeadios1, S. Honold1, Y. Loosli1, J. Vogt2, R. Luginbuehl1
1
Bettlach/Switzerland, 2Flüh/Switzerland
Purpose: Based on their biocompatibility and chemical-physical
properties, hydrogels have found a prominent place in modern
orthopaedic applications in recent years. Most hydrogels under
investigation are composed of a uniform polymeric body. We
demonstrate the potential of tuning hydrogels to comply with
mechanical and tribological requirements for meniscus substitute
materials.
Methods and Materials: Samples were prepared by reacting
2-hydroxyethyl methacrylate (HEMA), methyl-methacrylate (MMA),
distilled water (10%w/w) and 0.01mol% AIBN, as reaction initiator.
Equilibrium water content (EWC) and mechanical characterization
were determined on samples of 10mm height and 12mm diameter. The
unconfined-creep-modulus (Ec) was measured using a Zwicki Z5.0
(Zwick-Roell, Germany). Tribological measurements were done in PBS
with an in-house-built pin-on-plate instrument using a CoCrMo plate
as counterpart.
Results: The EWC decreased linearly as a function of increasing MMA
concentration in the poly(HEMA-co-MMA) (R2=0.99). The Ec values
exhibited a bi-modal relationship: samples with less than 35mol%
MMA had a very low Ec and the influence of MMA was minor whereas
at higher MMA contents, the Ec increased significantly and the MMA
influence was substantially stronger (Fig 1a). The apparent dynamic
friction, µ, increased exponentially as a function of MMA content up to
45mol% MMA. 45mol% MMA content marked a maximum for µ as it
decreased for higher MMA contents significantly.
Conclusions: It was found that an increase in the MMA concentration
yielded higher Ec and µ and lower EWC values as expected for hydrogels
based on PHEMA. Samples with 35mol% MMA content exhibits a
transition point in the Ec values, whereas at MMA content above
50mol% the µ values were similar to PMMA. These HEMA–co–MMA
polymers cover a broad range of Ec and their tribological performance
can be further optimized by mixing different co-polymers. These
approaches allow for tuning the mechanical and tribological properties
to mimic those of human menisci.
P41
Wear Rate Evaluation of Polycarbonate-Urethane Cushion Form
Bearings in Artificial Hip Joints
J.J. Elsner1, Y. Mezape2, G. Zur2, E. Linder-Ganz2, A. Shterling2, N.
Eliaz1
1
Tel Aviv/Israel, 2Netanya/Israel
Purpose: Polycarbonate-urethane (PCU) is an elastic material with a
high resistance to tear. It offers excellent mechanical and tribological
properties, comparable to those of natural cartilage. Thus, it is an
attractive alternative to Polyethylene in load bearing applications
(Fig. 1). The current study aimed to evaluate the wear performance of
a PCU acetabular buffer over 10 million gait cycles. The wear rate was
evaluated using gravimetric, Bio-Ferrography and filtration methods,
simultaneously.
Methods and Materials: Five 46mm buffers were tested, coupled
with 40mm CoCr spherical femoral heads, in anatomical positioning.
Four independently-controlled motions were induced according to
ISO 14242-2 gait pattern for 10 million cycles (Mc). The PCU implants
were examined every 1Mc and weighed to evaluate gravimetric
changes due to wear. The amount of wear particles released into the
lubricant (3:1 diluted bovine serum) was evaluated using a novel Bio-
Ferrography method and conventional filtration.
Results: The wear rate demonstrated by the PCU material in the
M-O-PCU configuration was found to reach a constant rate after ~2
Mc. The initial ‘run-in’ particle generation rate of 1x108 particles/
Mc was reduced gradually to 1-3x106 particles/Mc after 2Mc and
remained steady thereafter. Gravimetric and Bio-Ferrography
methods produced a steady-state volumetric wear rate of 8-10mm3/
mc, whereas filtration yielded a lower wear rate: 5mm3/mc. The wear
particles isolated by filtration and Bio-Ferrography were similar and
mean sizes were found to be 10-14µm
Conclusions: PCU displays promising wear characteristics. The wear
rate was found to be lower than that of UHMWPE and x-UHMWPE,
lying closer to that of MOM bearings (Table 1). The mean PCU wear
particle size was larger than that of UHMWPE, metal and ceramic, and
mostly not in the biologically active range (0.1-10µm). The number of
particles generated was found to be 5-8 orders of magnitude lower
than that reported for the aforementioned materials.
P43
Magnetic scaffold for advanced osteochondral tissue engineering
A. Russo1, S. Panseri1, V. Goranov2, D. Casino1, T. Shelyakova1, A.
Tampieri3, M. Sandri3, C. Dionigi1, V. Dediu1, M. Marcacci1
1
Bologna/Italy, 2Minsk/Belarus, 3Faenza/Italy
Purpose: Tissue engineering has recently emerged as a
multidisciplinary approach for the treatment of bone/osteochondral
defects. Scaffolds with the potential to circumvent the limitations
of autologous and allogenic tissue repair are employed to restore
tissue functions. This project propose magnetic scaffolds that via
magnetic guiding will be able to attract and take up in vivo growth
factors/stem cells bound to magnetic nanoparticles.
Methods and Materials: Magnetic scaffolds are prepared following
two different methods. In the first strategy, apatite/collagen
porous scaffolds are infiltrated with ferrofluids leaving magnetic
nanoparticles entrapped in the construct. The second approach
is based on the direct nucleation of biomimetic apatite on self-
assembling collagen fibrils in presence of magnetite nanoparticles,
thus realizing the magnetization of the scaffold material in situ. The
scaffolds become magnetic maintaining their specific porosity and
shape. In vitro study is performed with human mesenchymal stem
cells (hBMSCs) to test the biocompatibility of magnetic scaffolds.
In vivo biocompatibility of magnetic scaffolds is tested in a rabbit
model. The cylindrical scaffolds are implanted bilaterally in the
tibial diaphyses and femural epiphyses of the animals.
Results: In vitro analysis shows the ability of these new magnetic
scaffolds to sustain cell adhesion and proliferation, since there
are no significant differences in the level of living/dead cells
between control scaffolds and magnetized scaffolds. hBMSCs
adhere and attach firmly to the scaffold surfaces and are shown
to penetrate inside the scaffold. In vivo preliminary results show
good biocompatibility and bone integration with no inflammation
reaction even on organs biopsies.
Conclusions: The proposed scaffolds will work like magnetic
local field amplificators: their relatively strong magnetization
can be aligned in the same direction by moderate external field.
For magnetic targeting, funtionalized nanoparticles with growth
factor/cell introduced in the body will be concentrated in the target
area by means of an externally applied magnetic field.

P44
Comparison of human and animal chondral properties
S.D. Taylor, E. Tsiridis, E. Ingham, Z. Jin, J. Fisher, S. Williams
Leeds/United Kingdom
Purpose: Investigations into tissue preserving orthopaedic
treatments have assessed cartilage tribology; and used samples
from animal joints. However, little is known about the mechanical
differences between human and animal articular cartilage. This
study aimed to characterise the differences in geometry and
mechanical properties of human and animal cartilage samples.
Methods and Materials: Creep indentation was performed on
plugs taken out of the weight bearing portions of porcine (3–6
months old), bovine (18–24 months old), ovine (≈4 years old) and
human femoral heads. The human femoral heads were obtained
from surgery due to femoral neck fracture. Cartilage thickness was
measured by monitoring the resistive force change as a needle
traversed the cartilage and bone at a constant feed rate using a
mechanical testing machine. The percentage deformation over time
was determined by dividing deformation by thickness. A biphasic
finite element model was used to obtain the intrinsic material
properties of each plug.
Results: The femoral head diameters for all animal samples were
considerably different compared to human and all animal cartilage
thickness was remarkably less compared to human (Table 1). When
comparing between animals, the femoral head size was positively
correlated with cartilage thickness, agreeing with previous studies.
Human cartilage showed the least percentage total deformation,
however, ovine cartilage showed the highest modulus and lowest
permeability (Table 1). Ovine showed the most similar mechanical
behaviour to human. Table1: Geometric and mechanical results
(mean±standard deviation). Up to six repeats were tested.
Young‘s
Head Diameter (mm) Thickness (mm) Deformation (%)
Modulus (MPa)
Human 46.84±4.34 1.82±0.15 6.87±0.68 4.95±0.69
Porcine 35.63±1.15 1.22±0.04 28.82±2.57 1.18±0.17
Bovine 64.36±4.96 1.32±0.12 22.47±2.36 1.86±0.42
Ovine 23.25±2.31 0.57±0.07 18.59±1.37 6.73±0.08
Conclusions: Large differences in anatomy and mechanical
properties were established between different species and human
cartilage. Therefore, it is important to consider these differences to
enable more clinically relevant investigations.
P45
Cyclic mechanical stimulation in the generation of cartilage
replacement material
S. Nebelung1, K. Gavenis1, A. Ladenburger1, M. Stoffel1, R. Müller-
Rath2
1
Aachen/Germany, 2Neuss/Germany
Purpose: Adequate cartilage replacement remains a challenge to
orthopedics. Tissue engineering is a promising alternative treatment
for cartilage defects. By specifically stimulating chondrocytes, e.g.
through unconfined dynamic compressive loading, engineered
tissue can be modulated. Numerous bioreactor systems applying
compression have been developed; however, to our knowledge
none is measuring the actual force the probe is exposed to. We
developed a bioreactor system that allows for recording and
analysis of the transmitted forces during the cultivation period
and investigated the influence of cyclic compressive loading on a
chondrocyte-seeded collagen-type-I gel (see Figure 1).
Methods and Materials: Chondrocytes were harvested from
human knees, enzymatically released and seeded in a rat tail
collagen type-I gel at a density of 2x105 chondrocytes/ml gel. The
cell-seeded scaffolds were condensed by factor 20, punched into
disks and cultivated in the afore-mentioned bioreactor system
under continuous cyclic compressive loading (strain: 10 %, force:
30 mN, frequency: 0.3 Hz, duration: 14 days) under standardized
conditions (37° C, 5 % CO2, humidified atmosphere). A load cell
beneath the cultivation chamber recorded the transmitted forces.
Probes were analyzed by means of histology (HE, Safranin O),
immunohistochemistry (col-I, col-II, Ki 67, TUNEL), molecular biology
(RT-PCR of aggrecan, col-I, col-II, MMP-13) and biomechanical
evaluation of tensile and compressive characteristics.
Results: Cyclic compressive loading modulates chondrocytes’
viability, gene expression and biosynthetic activity and thereby the
Posters 201
content of cartilage-specific proteins. Chondrocytes initiated col-II-
secretion, whereas col-I-secretion gradually decreased. Apoptosis
levels after initial preparatory compression remained constant,
while proliferation was slightly elevated in the course of bioreactor
cultivation. The average aggregate modulus did not deteriorate
under cyclic loading, but declined in controls. The transmitted
forces have been recorded and correlated with matrix remodeling.
Conclusions: By compressive cyclic loading engineered cartilage
replacement tissue steadily develops a more cartilage-like
appearance. In the course of tissue remodeling biological outcomes
are well-correlated with mechanical characteristics.
P46
Effects of Serial Sectioning and Repair of Radial Tears in the
Lateral Meniscus
G.E. Ode, S. McArthur, G.S. Van Thiel, J. Kercher, J. Dishkin-Paset, E.
Shewman, V.M. Wang, B.J. Cole
Chicago/United States of America
Purpose: The structural foundation of a functioning meniscus
is the longitudinally oriented peripheral meniscal fibers which
transmit hoop stresses. Radial transection of these fibers could
theoretically render a meniscus non-functional with clinical
sequelae. We hypothesize that sequential radial sectioning of the
lateral meniscus will increase contact pressures, and repair with
either an all-inside or an inside-out technique will restore meniscal
function.
Methods and Materials: Ten paired cadaver knees were dissected
down to the capsule. Each tibia was placed in a Taylor-Spatial-Frame
and the femur was affixed in a custom jig. A vertical osteotomy of
the lateral femoral condyle was performed to access the lateral
compartment. Tekscan sensors were placed sub-meniscal in both
compartments. Knees were loaded at 800N in both extension
and 60 degrees of flexion in an MTS machine. The compartment
was reopened and sequential radial sections approaching the
periphery (50%, 75%, 100% of radial width) were made in the lateral
meniscus, posterior to the popliteal hiatus. Complete sectioning
was followed by a pair matched repair using an all-inside or an
inside-out technique. Each condition was loaded twice to ensure
reproducibility and repaired knees were cycled 50 times to test
structural integrity.
Results: ANOVA testing was completed. We observed overall
no significant difference in contact area or pressure with radial
sectioning up to 75% of the meniscal width but a significant change
from 75% to 100% (p<.05). The two repaired constructs showed
improved pressure profiles when compared to the sectioned state,
but no differences between each other.
Conclusions: Radial meniscal tears that violate the periphery
benefit from repair with either all-inside or inside-out repair at
time zero. Violation of the meniscal periphery results in a pressure
profile identical to total meniscectomy and repair at time zero
approximates the intact condition.
P48
Effect of the sample pre-conditioning on the evaluation of
cartilage mechanical properties
V.A. Acosta, I. Ochoa, J.M. García-Aznar, M. Doblaré
Zaragoza/Spain
Purpose: The mechanical characterization protocol of the articular
cartilage needs to be homogeneous to compare the parameters
in degradated and regenerated cartilage. For the determination
of the cartilage mechanical properties, experimental uniaxial
confined and unconfined compression tests were used to estimate
the Aggregate Modulus, Young Modulus and Poisson’s ratio. To
perform the compression test, a preconditioning protocol has to
be carried out achieving an optimum contact between the test tool
and the sample and eliminating the non-linearities of the cartilage
geometry. This process allows developing all the experiments with
a similar initial strain, obtaining repeatability in the estimated
mechanical properties.
Methods and Materials: The results of applying three different
precondition protocols for load-relaxation tests were presented: A 4
% and 10% predeformation of the total thickness of the sample, and
a preload equivalent to 12.5 kPa. After the precondition protocol, a
mechanical experiment consisting on making a stress–relaxation
ramp with six controlled displacement and relaxation of 15 minutes
until 20% of strain defined from the unloaded thickness.
202 Posters
Results: The 4 and 10% of predeformation have a same tendency
for confined and unconfined tests. For the 10% of predeformation,
the difference is the high degree of deformation and stress.
Although the implementation of preload achieves the contact, the
load-relaxation test starts with different levels of strain what could
alter the results trends. The preconditioning protocols showed
similar tendency where Aggregate Modulus was higher than Young
Modulus. However the variability of the experimental data obtained
by preload shows a statistical difference for Aggregate Modulus
compared with the 10% results.
Conclusions: Performing a predeformation of 4% ensures contact
with the lowest percentage of deformation on the sample, and
makes the compression tests start with a similar initial strain
level. Moreover, despite of the thickness variation, this protocol
allows obtaining a normalized experiments and repeatability in the
estimated mechanical properties.
P49
MRI Analysis of Anteroposterior Stability in the Normal Human
Knee
S. Arno, M. Chaudhary, R. Forman, P. Glassner, R. Regatte, P.
Walker
New York/United States of America
Purpose: We hypothesize that the knee achieves anteroposterior
(AP) stability on the medial side and simultaneously achieves
a high range of flexion by rollback on the lateral side. We also
wish to show that the medial meniscus plays an important role in
maintaining this stability.
Methods and Materials: A Siemens 7 Tesla MRI with T1 weighted
non-fat suppressed settings and isotropic resolution of 0.6mm
was used to scan eight healthy male volunteers with the knee at
15 degrees of flexion. Two scans were obtained: One with an axial
force of 667 N along the tibial long axis and a second with the axial
force and a shear force of 36 N. The forces applied were 25% of
the forces at heel strike determined by D’Lima and Colwell (2005,
2006). Using 3D-Doctor (Able Software Corp, Lexington, MA), solid
models were created of the femur, tibia, and menisci. The solids
were imported into RapidForm (Inus Technology, Seoul, S. Korea)
and a deviation analysis was then performed to determine the
movement of the femur and menisci.
Results: Overall, the lateral side displaced anteriorly (range: 0.5
- 2.5mm) and the medial side even displaced posteriorly (range:
0 - 1.25mm), indicating an axial rotation point between the lateral
and medial condyle, but closer to the medial. In addition, the
medial meniscus was found to exhibit at most 0.5mm of anterior
displacement whereas the maximum anterior displacement of the
lateral meniscus ranged from 0.5mm to 1.00mm, suggesting that
the medial meniscus contributes to medial stability.
Conclusions: These findings can have important implications in the
design of treatments for the restoration of normal knee mechanics.
In addition, the above data stresses the important role of the medial
meniscus in maintaining stability in the knee. Future studies will
look at how a damaged medial meniscus can lead to increased AP
displacements causing long-term damage to cartilage.
P50
Stiffness of cartilage in the normal human knee as determined by
a novel indentation device
L. Kuo, D. John, S. Arno, P. Walker
New York/United States of America
Purpose: The purpose of this study is to determine the resistive
force of normal cartilage on the distal femur and proximal tibia. To
do so, an indentor was modeled and constructed after the Artscan
200 and Actaeon Probe, optimized for lab setting usage to conduct
short duration indentation testing of articular cartilage surface
layer.
Methods and Materials: The indentor was first validated on
samples of agarose and rubber with known properties to show
its capability of providing reproducible readings independent of
the operator. It was then used to test the distal femur (inner and
outer region) and proximal tibia (area covered and uncovered by
the meniscus) of seven fresh-frozen cadaveric knees with visually
normal cartilage. A total of six measurements were made by two
different users and the resistive force was recorded via SensoCom
software. The Friedman test was then used to establish a range of
resistive force per zone tested.
Results: The resistive force varied based on the location in the
knee. For instance, the inner region of the medial femoral condyle
had a lower resistive force than the outer region. The area covered
by the meniscus on the tibial plateau had a higher resistive force
than those uncovered by the meniscus. The ranges per zone are
shown. Similar values were found by Lyrra et al.
Conclusions: The result of this study provides valuable insights
into how mechanical properties vary throughout the knee joint. In
particular, the regions noted to have lower resistive forces are also
regions typically noted to primarily contain osteoarthritic lesions.
With a range of values now established for the normal knee, testing
can be performed on osteoarthritic cartilage to further understand
the mechanical changes caused by this disease.
P51
The Mechanical State of the Human Hip Joint, Implanted with a
Pliable Polycarbonate-Urethane Acetabular Buffer
S. Portnoy1, E. Linder-Ganz1, G. Zur1, Y. Mezape1, F. Guilak2, N.
Shasha3, A. Shterling1
1
Netanya/Israel, 2Durham/United States of America, 3Tel Aviv/
Israel
Purpose: Replacing the hip joint with a prosthetic implant is the
conventional treatment for degenerative hip disorders. Recently, an
acetabular buffer device, made of pliable Polycarbonate-Urethane
(PCU), was developed (TriboFit ®, Active Implants Co., Memphis, TN;
Fig. 1a). The implant’s design aims to mimic the natural behavior
of the cartilage. However, the mechanical state of the pelvis,
implanted with a PCU buffer directly on the acetabular bone, has
never been quantified. Our goal was therefore to compare between
three pelvic states: natural pelvis, pelvis implanted with PCU buffer,
and the buffer on an implanted metal shell.
Methods and Materials: We developed subject-specific 3D CT-
based finite element (FE) models of the natural and implanted
cadaveric human hip joint (Fig. 1b). The models simulated in vitro
experimental trials on the cadaveric sample, where the natural and
implanted hip joint was loaded. Surface pressures between the
acetabulum and the femoral head were measured using pressure
films. Surface strain distributions on the pelvic bone were visualized
using photoelasticity. Principal strains in discrete locations were
measured using strain gauges. The measured and calculated
acetabular contact pressures and surface strains upon the pelvic
bone were compared for model validation and further analyses of
stress/strain distribution in the implanted hip joint (Fig. 2).
Results: The results show similar strain distributions at the natural
acetabulum, compared to the hip joint, implanted with the PCU
buffer. The strains on the bone surface of the hip joint implanted
with the metal shell were distributed across a larger area, compared
to the natural pelvis and buffer-implanted pelvis. The pressure
distribution at the acetabulum-buffer surface was similar to the
pressure distribution in the natural hip joint.
Conclusions: We conclude that the mechanical state of the pelvis,
implanted with a PCU buffer, is similar to the mechanical state of
the natural pelvis.
P52
The effect of clearance to cartilage wear in hip hemiarthroplasty
J. Lizhang, J. Fisher, Z. Jin, S. Williams
Leeds/United Kingdom
Purpose: Hip hemiarthroplasty is used in elderly patients following
femoral neck fracture. However, longevity of this treatment
is limited by the metal or composite prosthesis degrading
the cartilage over time. Inappropriate sizes of implant in hip
hemiarthroplasty may cause early cartilage wear (Beksaç et al,
2008). This study investigated the effect of clearance (between
acetabular cartilage and metal head) on cartilage wear area and
volume. We hypothesised that the outcome of hemiarthroplasty
could be improved if the clearance was optimised.
Methods and Materials: Acetabulums (from 6-month old porcine
hips) were dissected and mounted at 45º in a pendulum friction
simulator. Cobalt Chrome heads were set homocentric to the
acetabulums and applied a dynamic (75~800N) load and a
flexion-extension motion (±15º) in 25% bovine serum for 2 hours.
Components were set up to allow the different radial clearances:
Small (≤0.6mm), Medium (>0.6 & ≤1.2mm), Large (>1.2 &
≤1.8mm), Extra Large (>1.8mm), n=3 per group. A silicon rubber
replicate was made of the acetabular surface after testing to

investigate wear. The wear area was traced onto flexible film and
the area of different cartilage damage grades (followed ICRS wear
grades) measured. Additionally, the replica surface was measured
by a two-dimensional profilometry across the wear area with
approximately 12 traces, and this was multiplied by the wear area
length to provide a volume (Figure 1).
Results: Different cartilage wear grades (1~3) were seen in different
clearances. When clearance increased contact area decreased,
hence the percentage of unworn cartilage area increased. Wear
volume increased significantly with XL due to the increase in contact
stress above a critical threshold and the increase of cartilage wear
depth clearance (Figure 2).
Conclusions: Cartilage wear in hip hemiarthroplasty is affected by
clearance which influences both wear area and wear grade, and
significantly higher wear is produced with extra large clearance.
P53
Loading Effects Intervertebral Disc Cell viability in the Loaded
Disc Culture System.
C.P.L. Paul, H.A. Zuiderbaan, B. Zandieh Doulabi, A.J. Veen van der,
T.H. Smit, M. Helder, B.J. Royen van, M.G. Mullender
Amsterdam/Netherlands
Purpose: We have developed a novel Loaded Disc Culture System
(LDCS) to study the effects of loading on large species IVDs in
relationship with degenerative disc disease (DDD). We are able
to culture and load goat IVDs in the LDCS with maintenance of
their native cellular and extracellular properties over a 14 day
culture (data not published). The purpose of the current study is to
investigate the influence of different loading regimes on goat IVDs
cultured in the LDCS over a 7- and 14-day period.
Methods and Materials: IVD’s (L1-6; N=175), were harvested from
goats (N=35) under sterile conditions. IVD’s were assigned to 1 of
4 diurnal loading groups (fig. 1a). Discs were cultured for 7 or 14
days in the LDCS. Cell viability was measured in the nucleus (NP)
and annulus (AF) regions. IVD stiffness was calculate from loading
force and IVD displacement. Water, glycosaminoglycan (GAG) and
total collagen content were analysed to assess the condition of the
extracellular matrix (ECM).
Results: Static loading showed a drop in cell viability only in the
AF region, when compared to day 0. Low dynamic load maintained
cell viability, whereas high dynamic and prolonged high dynamic
loading showed increasing cell death (fig. 1b). IVD stiffness
increased initially within each cycle, but no significant changes
were observed over the 7 and 14 day culture period (figure 2). In the
ECM, water-, GAG- and collagen content did not change significantly
after 7 and 14 days in any experimental group when compared to
day 0 (data not shown).
Conclusions: Caprine IVD cells show different responses in cell
viability depending on the applied loading condition. Low dynamic
loading maintained cell viability, whereas unloaded, static and
higher dynamic loads induce pathological changes within 14 days
on a cellular level. To determine loading effects on the ECM, longer
culture duration may be needed.
P54
Novel pre-solidified chitosan/blood implant provides local bone-
marrow stimulation-associated biological activity in skeletally
aged rabbits after a three week treatment
C. Lafantaisie Favreau1, J. Guzman-Morales1, J. Sun2, A. Harris1, T.D.
Smith1, A. Carli1, J. Henderson1, W.D. Stanish3, C.D. Hoemann1
1
Montreal/Canada, 2Laval/Canada, 3Halifax/Canada
Purpose: The purpose of this study was to analyze the biological
impact of different formulations of a novel pre-solidified chitosan/
NaCl-blood implant on subchondral bone and cartilage repair after
three weeks in skeletally aged rabbits. We tested the hypothesis
that pre-solidified implants degrade in situ with molecular weight-
specific kinetics, and stimulate subchondral angiogenesis and bone
remodeling, acute reactions previously tied to hyaline cartilage
repair.
Methods and Materials: Hybrid chitosan-autologous blood implants
were made with 150kDa, 40kDa and 10kDa chitosan (80% DDA) with
fluorescently labelled chitosan. Three 1.5mm osteochondral drill
holes were generated bilaterally in femoral trochlea of N=5, 2.5
year old NZW rabbits. One implant of each molecular weight was
press-fit into the 3 drill holes of one knee, while the contralateral
trochlea was used as drill-only controls. After 1 day (N=1) or 21 days
Posters 203
(N=4) post-surgery, the femoral ends were retrieved and scanned
by micro-computed tomography. Histological sections were
Hoechst counter-stained to show nuclei and residual implant in a
representative field with peak chitosan-RITC fluorescence intensity
or stained with Safranin O to detect glycosaminoglycan.
Results: Hoechst staining revealed considerable chitosan-mediated
cellular attraction (Fig. 1). Repair tissue at 21 days in treated defects
had significantly more blood vessels, improved integration and
less areal GAG-positive repair tissue (~0.3% vs ~22.1% control;
p=0.0012) which is, after three weeks, a good indicator of future
repair enhancement. More residual 150kDa implant was detected
compared to 10 kDa implant (p=0.019), however comparison
of hole geometry with both day 1 and day 21 drill-only controls
revealed that all implants elicited a similar level of subchondral
bone remodelling at the edges of the drill holes (Fig. 2).
Conclusions: These results confirm that after three weeks in aged
rabbits, this novel pre-solidified chitosan/NaCl-blood implant can
deliver resident chitosan and stimulate a biological activity in
regenerating subchondral bone that is consistent with previously
successful therapies.
P55
Complete Hole Placement with Microfracture Improves Cartilage
Repair as Compared to Limited Central Hole Placement
M. Suri1, J. Rodrigo2, S.B. Singleton3, D. Wyland4 , J. DesJardins5, M.
Hoyle4 , P. Boatwright6, M. McDonough7
1
Harahan/United States of America, 2Spartanburg/United States of
America, 3Greer/United States of America, 4Greenville/United States
of America, 5Clemson/United States of America, 6Cleveland/United
States of America, 7Washington, DC/United States of America
Purpose: Microfracture is a mainstay in the treatment of articular
cartilage injuries. However, in the cases that do not do well there
may be inadequate surgical technique. We hypothesized that there
would be improved cartilage healing after microfracture in a rabbit
full-thickness trochlear defect model if more holes were distributed
evenly throughout the defect vs. fewer holes placed centrally.
Methods and Materials: Full-thickness cartilage defects measuring
3 x 10mm were made in the articular cartilage of the trochlear grooves
of 30, 52-week old New Zealand white rabbits. Three groups were
assigned and all were sacrificed at 4 months. One group (n=10)
received a 6-hole microfracture, with holes evenly distributed
extending to periphery. The second group (n=10) received a
4-hole microfracture that was central in the lesion, representing
inadequate technique. The third group (n=10) was abrasion only
controls. Photographs of lesions at time of surgery and at sacrifice
were used to calculate percentage fill of the regenerate. H&E
stained slides were graded by the ICRS scoring system and were
used to measure quality of healing. Safranin-O stained slides were
graded to determine the percentage of the regenerate cartilage in
each group that had positive proteoglycan staining.
Results: Percentage fill of the regenerate was significantly (p<0.05)
improved in the 6-hole group compared to the 4-hole group and
the control group. ICRS scores were improved between the 6-hole
group (13.7) and the 4-hole group (9.7) (p<0.05), but not between
the 4-hole group (9.7) and the control group (7.7) (p<0.05). All
of the 6-hole group demonstrated positive proteoglycan staining
as compared to 57% of the 4-hole group and 43% of the abrasion
group (p<0.05).
Conclusions: Placement of microfracture holes extending to the
periphery of the cartilage defect significantly improved percentage
fill and quality of regenerate after microfracture.
P56
Migration of subchondral mesenchymal progenitor cells in
microfracture: Impact of human serum and synovial fluid
M. Endres, G. Kalwitz, K. Andreas, K. Neumann, M. Sittinger, T.
Haeupl, C. Kaps
Berlin/Germany
Purpose: In clinical routine, bone marrow stimulating techniques
like drilling, abrasion, or microfracture (Mfx) are frequently used
for small size focal cartilage defects. In Mfx, the introduction of
multiple perforations into the subchondral bone of the cartilage
defect leads to bleeding, allows mesenchymal stem and progenitor
cells from the subchondral bone to enter the defect and induces
formation of cartilaginous repair tissue. The cellular mechanisms
underlying stem cell migration into the defect and subsequent
204 Posters
tissue formation are not fully understood. In this study, we verify
the assumption that mesenchymal progenitor cells from the
subchondral bone (CSP) can be recruited by chemotactic factors
like blood serum, synovial fluid from normal donors and donors
with rheumatoid arthritis (RA) or osteoarthritis (OA) and selected
chemokines.
Methods and Materials: The presence of chemokines in blood serum
and synovial fluid could be demonstrated using human chemokine
antibody membrane arrays. Human blood serum, synovial fluids
and selected chemokines were tested in combination with CSP
cells in a 96 well chemotaxis assay.
Results: Human blood serum as well as synovial fluid from healthy
donors and OA donors recruited CSP cells. Remarkably, synovial
fluid from RA donors recruited significantly less cells than synovial
fluid from healthy donors or OA donors. Additionally, distinct
chemokine candidates were able to recruit CSP cells.
Conclusions: In conclusion, human serum as well as synovial fluid
recruits CSP during Mfx. For cartilage repair approaches, these
results suggest to use chemotactic factors like serum or potentially
chemokines to improve stem and progenitor cell migration to sites
of defective cartilage.
P57
Analysis of the mid-term effects of chitosan-NaCl/blood pre-
solidified implants in an in vivo osteochondral repair model
J. Guzman-Morales1, C. Lafantaisie Favreau1, J. Sun2, G. Rivard1, C.D.
Hoemann1
1
Montreal/Canada, 2Laval/Canada
Purpose: Strategies are needed to enhance osteochondral
repair with increasing age. Recently, our group developed
novel pre-solidified chitosan/NaCl-blood implants to stimulate
revascularization of subchondral bone in skeletally aged animals.
After 3 weeks, this implant elicits angiogenesis and subchondral
bone remodeling. The purpose of this study was to determine
whether bone repair of treated drill holes is achieved after a longer
repair period.
Methods and Materials: With Institute-approved protocols, 2
osteochondral drill holes (1.5mm diameter, 2mm deep) were
created bilaterally in femoral trochlea of six skeletally mature
NZW rabbits (12 or 32 months old). Drill holes were treated with
pre-solidified implants consisting in a homogeneous mixture of
80% DDA chitosan-NaCl (40 kDa or 10 kDa), autologous blood and
fluorescent chitosan tracer. Contralateral microdrill holes were left
to bleed as surgical controls. During the 10 week repair period, post-
operative knee inflammation was evaluated daily using an in-house
scoring system (0=no inflammation to 4=significant effusion) with
a cumulative score for each day of effusion. At necropsy repair
tissues were assessed macroscopically; femoral ends were fixed
and scanned by micro-computed tomography to quantify 3-D drill
hole bone repair.
Results: Post-operative knee effusion was attenuated in chitosan
implant-treated knees compared to contralateral controls (12
vs 26 average cumulative score, Fig. 1). Treated drill holes were
macroscopically filled with more tissue (p<0.05) compared to
the drill-only controls, and no macroscopic traces of fluorescent
implants remained in treated defects after 10 weeks. New bone
growth filled on average 50% of the untreated drill holes starting
from the base of the hole. Chitosan-treated defects were repaired
with approximately 25% new mineralized bone in the drill holes
especially in the superficial subchondral bone plate (Fig. 2).
Conclusions: Biodegradable pre-solidified chitosan/NaCl-blood
implants suppress post-operative effusion and permit new bone
repair in microdrill holes with a delayed kinetics compared to drill-
only controls.
P58
Subchondral bone plate characteristics in human and animal
bone: Implications for cartilage repair
M. Lowerison1, S. Allendorf1, A. Bell1, L.M. White2, R. Whiteside2, P.
Marks2, M.B. Hurtig1
1
Guelph/Canada, 2Toronto/Canada
Purpose: To characterize the subchondral bone plate (SBP) in normal
and osteoarthritic (OA) femoral condyles. Our hypothesis was that
increased subchondral bone plate thickness in osteoarthritis may
make it necessary to make deeper microfracture holes. Animals were
also considered since they are used as models of cartilage repair.
Methods and Materials: Normal (n=4) and OA (Outerbridge 1-2,
n=4) human medial femoral condyles as well as normal animal
condyles (n=4 each species) were obtained. All underwent microCT
imaging at 45μ resolution and the resulting images were analyzed
using regions of interest (ROIs) created in the SBPs to measure
BMD (bone mineral density), TMD (tissue mineral density), and BVF
(bone volume fraction). A depth-wise analysis of mineral density
was done by expressing the mean voxel densities in each horizontal
slice by depth (Python 3.1.1 and R (www.r-project.org).
Results: Human SBP BMD, TMD was significantly lower (p<.05)
than all animal species except the sheep and dog. Human BVF was
the lowest of all species. Rate of change in BMD by depth showed
that the dog and human have a similar mineralization profile. In
early human osteoarthritis the thickness (>2mm) and density of
the SBP was significantly (p<.01) expanded.
Conclusions: In normal human femoral condyles, the SBP is comprised
of the condensation of approximately two horizontal trabeculae. No
animal species approximates the very thin and porous normal human
SBP; however, the dog and human have a similar mineralization
gradients while other species have properties approximating sclerotic
human bone. In osteoarthritis the sclerotic SBP presents a substantial
barrier to marrow stimulation procedures.
P59
Advance trans-medullary stimulation of mesenchymal stem cells
enhances spontaneous repair of full-thickness articular cartilage
defects.
K. Nishizawa, S. Imai, T. Mimura, M. Kubo, S. Araki, S. Shioji, Y.
Takemura, Y. Matsusue
Otsu/Japan
Purpose: Mesenchymal stem cells (MSCs), especially those close
to cartilage defects, are an important cell source for cartilage
regeneration. We assumed that a larger number of MSCs would
become available, should the bone marrow at the immediate vicinity
of the subchondral bone be stimulated for MSCs in advance of the
microfracture, i.e., creation of the full-thickness articular cartilage
defects in this study.
Methods and Materials: We stimulated the immediate vicinity of the
subchondral bone from outside of the joints 4 days prior to the creation
of cartilage defects (Advance Trans-medullary Stimulation (ATS) group).
In another setting, bFGF was administered through the trans-medullary
passageway in order to augment the stimulation of the MSCs (ATS +
bF group). Finally, we designed a negative control group (non-ATS
group). The rabbits were sacrificed at 1, 2, 3, 4 and 8 weeks after the
creation of cartilage defects. The triple staining of Bromodeoxyuridine
(BrdU), CD44 and CD45, histological evaluation and RT-PCR assay for
assessment of the regenerated cartilage were performed.
Results: A considerable proportion of the proliferating cells were
identified as bone marrow-derived MSCs. The BrdU-positive cell
density of the ATS group increased to a larger extent than that of
the non-ATS group. The recruitment of BrdU-positive cells to the
cartilage defects was even more pronounced in the ATS + bF group
than that in the ATS group. The histological grading score of the
ATS + bF group was superior to the other groups. Type II collagen
mRNA expression also tended to be higher in ATS + bF group.
Conclusions: The advance stimulation of the bone marrow at the
immediate vicinity of the subchondral bone increased the available
MSCs and enhanced the regeneration of the chondral defects at the
early follow-up time point.
P60
Interaction of microfracture awl design with subchondral bone in
early osteoarthritis
S. Allendorf1, M. Lowerison1, A. Bell1, C.D. Hoemann2, P. Bursac3,
M.B. Hurtig1
1
Guelph/Canada, 2Montreal/Canada, 3Alachua/United States of America
Purpose: To explore the relationship between awl design and
performance in osteoarthritic human femoral condyles. Our
hypothesis was that compression and condensation of the
subchondral bone plate may create a barrier to remodeling and
repair in sclerotic bone. Femoral condyles from animals provided a
convenient model of subchondral sclerosis.
Methods and Materials: Human and horse femoral condyles were
scored macroscopically (Outerbridge system 0-5) and microCT
imaged at 27μm voxel resolution. A 2 cm2 full thickness chondral
defect was made, then 3 mm deep microfracture holes were made

with four awls: a 20° Arthrex, a 30° Linvatec, and a 30o Sontec and
half-size 30° Sontec. MicroCT scans were repeated to measure the
depth and shape of each hole. Bone compaction was measured by
a radial analysis of BMD from the central axis of the microfracture
holes at the bone surface and at 0.5 mm intervals mm from the
cartilage surface.
Results: Bone compaction in the microfracture hole wall was
evidenced by increased BMD in the adjacent bone. In human condyles
with low (grade 2 & 3) Outerbridge scores the Linvatec awl created
increased BMD profile from the surface to a depth of 2 mm whereas
other awls were associated with less bone compaction. The Arthrex
awl created larger diameter holes at the surface (1.67 mm) compared
to the other 3 designs (1.12-1.08mm). The dense subchondral bone
plate of the horse resulted in incomplete holes (<2mm deep) holes
that were partially filled with mineralized debris.
Conclusions: All microfracture awls penetrated to the marrow space
of bone affected by early OA but as bone sclerosis increased, bone
compaction and debris resulted in holes with poor connectivity to
marrow spaces. This may lead suboptimal repair tissue formation
and attachment.
P61
Cartilage defects pretreated with microfracture and covered with
a Artelon scaffold give a hyaline like repair tissue.
S. Concaro1, H. Stenhamre2, A. Lindahl3, L. Peterson3
1
Göteburg/Sweden, 2Gäteborg/Sweden, 3Gothenburg/Sweden
Purpose: Microfractures represent the first line of treatment for
cartilage lesions up to 2-3 cm2. This bone marrow stimulating
technique results in a repair tissue that is fibrous. The objective of
this study was to evaluate whether a scaffold implanted into the
defect at the time of microfracture treatment gave a more hyaline
like tissue than microfracture alone in a full-thickness articular
cartilage defect of rabbit.
Methods and Materials: Scaffolds made out of ArtelonÒ, a
poly(urethane urea), was placed in the full-thickness articular
cartilage defects (n=3). Defects treated with microfracture only
served as controls (n=3).
Results: After two weeks of implantation cellular infiltration was
seen in the scaffolds as well as in the controls. Close contact
between the surrounding cartilage and the implanted scaffold was
observed. Six months after implantation, cartilage implants and
controls were assessed by macroscopic evaluation and histological
scoring. ICRS macroscopic evaluation and histological scoring
documented an improvement of repair tissue formation when the
defects treated with microfracture were covered with the Artelon®
scaffold, compared to controls treated with only microfracture.
Conclusions: Implanting a cell-free polymer-based scaffold into
microfractured defects might be a treatment option for cartilage
defects and thus an option to use to improve the regeneration of
articular cartilage.
P62
Beneficial effects of hyperbaric oxygen on human degenerated
intervertebral disc cells via suppression of IL-1 β and p38 MAPK
signal
C. Niu, S. Lin, L. Yuan, C. Yang, W. Chen
1
Kweishan, Taoyuan/Taiwan
Purpose: Nucleus pulposus cells (NPCs) from degenerating discs
produce catabolic and inflammatory factors, including interleukin
(IL)-1, nitric oxide (NO), prostaglandin E2 (PGE-2), and matrix
metalloproteinaes (MMPs). An imbalance between MMPs and tissue
inhibitors of matrix metalloproteinases (TIMPs) has been proposed
to exist in the degenerating disc. This study evaluates the effects of
hyperbaric oxygen (HBO) on the human degenerated NPCs.
Methods and Materials: NPCs were maintained in alginate bead
culture. All hyperoxic cells were exposed to 100% O2 at 2.5 atmospheres
absolute (ATA) in a hyperbaric chamber. p38 MAPK phosphorylation of
the NPCs was detected using the phosphor-kinase array kit. RNA was
isolated for real-time quantitative polymerase chain reaction (Q-PCR)
analysis of aggrecan and type II collagen gene expression. The amounts
of IL-1β, NO, PGE-2, MMP-3, and TIMP-1 in the conditioned media were
quantified by enzyme-linked immunosorbent assay (ELISA).
Results: Our data showed that HBO treatment decreased
expression of IL-1β, increased the gene expression of aggrecan and
type II collagen, suppressed the phosphorylation of p38 MAPK,
decreased NO, PGE-2, and MMP-3, and increased TIMP-1 expression
Posters 205
in NPCs. These results support the hypothesis that IL-1β and the
p38 MAPK signal may be responsible for many of the inflammatory
and catabolic changes seen in the human disc degeneration.
Conclusions: HBO treatment-induced increase of the anabolic factor
(TIMP-1) /catabolic factor (MMP-3) ratio may provide a therapeutic
approach to slow the course of intervertebral disc degeneration.
P63
Digestion-isolated chondrocytes demonstrate superior yield
and chondrogenic potential versus cells migrating from minced
cartilage
M. DiMicco1, S.J. Duguay2, J.P. Wicks2, G. Matthews1
1
Framingham/United States of America, 2Cambridge/United States
of America
Purpose: Autologous chondrocyte implantation (ACI) techniques
currently involve chondrocyte isolation and expansion in culture.
Methods utilizing cell migration from undigested, uncultured
minced cartilage could confer handling and cost advantages to
ACI, perhaps allowing a one-step procedure. Our objective was to
evaluate the suitability of substituting minced cartilage for cultured,
digestion-isolated chondrocytes in cartilage repair procedures.
Methods and Materials: Minced human cartilage (2-3 mm3) was
evaluated ± enzymatic digestion for cell yield at baseline and after
11 days of culture. Separately, minced bovine cartilage was sieved
by size (< 500μm, 500-1000μm, and >1000μm) and cultured.
Cell viability in fragments was observed, and cell migration out of
fragments was assessed at 7 and 12 days. To better understand
the contribution of cell density toward chondrogenic behavior,
aggrecan and collagen II gene expression were determined using
enzymatically-isolated cells seeded at 0.5-4 million cells/cm2 and
cultured on a collagen scaffold.
Results: No cells were detected in supernatants immediately after
mincing. When minced tissue was enzyme-treated for up to 2.5
hours, cell yield was 555±414 cells/mg (mean±SD), approximately
10% of that typical of overnight digestion. Yield from culture of
100-200 mg of minced cartilage (at 10-11 days) was 45,500±40,046
cells, while a 2-hour enzyme treatment of these pieces before
culture increased yield nearly 9-fold. However, these values are
still 2-18% of those expected following overnight enzyme digestion
and culture. Mincing resulted in a zone of cell death at cut tissue
edges, giving smaller pieces more dead cells per unit volume. After
seeding onto a collagen membrane, cells at higher densities had
higher collagen II gene expression, while aggrecan expression did
not vary with density.
Conclusions: Using minced cartilage for cartilage repair is expected
to give fewer implanted cells than enzymatic digestion of tissue,
with potential consequences on the chondrogenic behavior of
these cells.
P64
Human cartilage fragments in a hyaluronic acid/fibrin glue/
platelet rich plasma scaffold for single stage cartilage repair:
how to optimize cell outgrowth. In vitro study
A. Marmotti, C. Realmuto, F. Castoldi, R. Rossi, M. Bruzzone, D.E.
Bonasia, C. Tarella, G. Collo, A. Maiello, P. Rossi
Torino/Italy
Purpose: Cartilage fragments provided a viable cell source for
single stage cartilage repair in previous rabbit and goat models.
Nevertheless, human in vitro explant cultures showed some
limitations as age-dependence, time-dependence and reduced cell
migration and outgrowth from cartilage fragments, compared to
animal model. Purpose of our study is to optimize cell outgrowth
into a hybrid HA/fibrin/PRP scaffold under TGF-beta and G-CSF
exposure and evaluate their effect on chondrocyte behaviour.
Methods and Materials: Articular cartilage from human knees(<
35y and > 50y) was minced into small fragments and loaded
onto the scaffold; constructs were cultured for 1, 2 and 3 months
both in standard culture medium and under exposure to TGF-
beta(10ng/ml) and/or G-CSF(10ng/ml). Explant cultures were
evaluated histologically, with immunohistochemistry and with
immunofluorescence.
Results: Cell migration and outgrowth was time-dependent and
age-dependent; with older donors, increased migration was
observed under exposure to TGF-beta, reaching values similar to
unstimulated younger donors(p<0.05); with younger donors,
outgrowth increased at 1 month in a ratio of 1,6:1 with exposure
206 Posters
to TGF-beta (compared to unstimulated cultures), in a ratio of 2,1:1
with exposure to G-CSF and in a ratio of 1,9:1 with exposure to both
factors(p<0.05); immunofluorescence of migrating cells was
positive for sox9, CD151, CD49c and negative for CD105, consistent
with a predominant chondrogenic phenotype; G-CSF Receptor was
detected on migrating cells with immnunofluorescence; exposure
to G-CSF slightly decreased SOX-9 expression and increased PCNA,
beta-catenin and pan-cadherin expression.
Conclusions: Cell outgrowth into the HA/fibrin/PRP scaffold
increased under exposure to TGF-beta or G-CSF. Higher values have
been obtained with G-CSF, suggesting a possible role of G-CSF in
increasing chondrocyte outgrowth. Further studies are required to
better ascertain the role of G-CSF on chondrocytes migrating from
articular cartilage fragments. These promising results could have
relevance for improving in-vivo single stage cartilage repair with
minced human cartilage fragments.
P65
Reduced chondrocyte viability is associated with the use of
marker pen ink.
A. Getgood1, I. Mcnamara1, S. Kili2, T. Bhullar3, F.M. Henson1
1
Cambridge/United Kingdom, 2Shrewsbury/United Kingdom,
3
Peterborough/United Kingdom
Purpose: The aim of this study was to investigate whether the addition
of two different marker pen inks to a collagen membrane would affect
chondrocyte viability, and therefore be potentially detrimental to
articular cartilage repair in autologous chondrocyte transplantation.
Methods and Materials: Human chondrocytes were applied to
Chondro-Gide® collagen membranes at a volume of 12 million cells/
ml. Two sterile marker pens were used to mark the membranes; one
contained methylene blue, the other contained crystal violet inks.
Three groups of membrane were tested in duplicate for each pen.
Group A consisted of no ink mark, group B had only the uppermost
‘smooth’ layer marked, and group C had the lower ‘porous’ layer
marked. All membranes were then cultured in standard growth media
for 24 hours. Cell viability was assessed at 24 hours on all membranes
using a live/dead cell viability assay. Cell viability was quantified with
florescent microscopy with mean number of cells compared to control
membranes using the students t test (p<0.05).
Results: Table 1 shows the mean number of viable cells in each
group. Control membranes (group A) with no ink showed excellent
cell viability. A statistically significant reduction in cell viability with
both methylene blue (p<0.0001) and crystal violet (p<0.0001) was
found adjacent to the ink mark on the smooth side (group B) and on
the porous side remote from the ink (group C). Marked cell death was
seen with both dyes (p<0.0001) adjacent to the ink on the porous
side. A small number of cells cultured with crystal violet were live – no
live cells were detected in the presence of methylene blue.
Conclusions: Chondrocyte viability is significantly reduced when cells
are cultured in-vitro on collagen membranes marked with methylene
blue and crystal violet pen ink. Surgeons should be aware of the
potential negative impact of marker pens in cell based therapies.
P66
Bone marrow “niche”: evidences which support its use in vivo
B. Grigolo, C. Cavallo, G. Desando, R. Buda, F. Vannini, M. Cavallo,
S. Giannini, A. Facchini
Bologna/Italy
Purpose: Mesenchymal Stem Cells (MSCs) have been indicated as
a new option for regenerative medicine because of their ability to
differentiate into various lineages. However, isolation procedures
are crucial for the functional activity of the transplanted cells and
not easily viable in operatory room. The use of concentrated bone
marrow cells (BMCs) enables to implant a cell population surrounded
by its microenvironment (or “niche”). The cells of the niche regulate
stem cell behaviour through direct physical contact and paracrine
signalling. The aim of the research was to characterize BMCs in
vitro to validate their use to regenerate osteochondral tissues.
Methods and Materials: BMCs were obtained from bone marrow
harvested from iliac crest of 20 patients operated on for focal
osteochondral lesions of the talar dome by “one-step” procedure.
Bone marrow was concentrated in a cell separator (BMAC®;
Harvest Technologies Corp) and BMCs were successively analyzed
by FACS using specific antibodies. BMCs were also grown onto
a hyaluronan based scaffold already used in clinical practice.
Chondrogenic and osteogenic potential were evaluated by Alcian
blue and Alizarin red staining after having stimulated the cells with
specific activating factors. Histological, immunohistochemical and
molecular biological analyses were performed on cells grown in
monolayer and onto the constructs.
Results: Despite significant differences in their cellular composition
BMCs express the same markers as isolated ones but at higher
percentage. They are able to differentiate in osteogenic and
chondrogenic sense after opportune stimuli. The differentiation
process is favoured by the growth of the cells onto the hyluronan-
derivative scaffold.
Conclusions: BMCs represent the ideal candidate to be used in
autologous transplantation to regenerate osteochondral tissues
since are able to differentiate in their main cell populations. This
could be particularly useful for the “one step” procedure allowing
to directly implant the cells in operatory room. The employment of
a hyaluronan scaffold could improve the strategy.
P67
The effect of low-intensity pulsed ultrasound on scaffold-free
chondrocyte plate in rabbit model.
K. Uenaka1, S. Imai2, Y. Matsusue2
1
Otsu City/Japan, 2Otsu/Japan
Purpose: The aim of this study is to evaluate the effect of low-
intensity pulsed ultrasound (LIPUS) for scaffold-free chondrocyte
plate in rabbit full-thickness defect model.
Methods and Materials: Chondrocytes were collected from
articular cartilage of Japanese white rabbits. The cells were then
condensed to the density at 107cells/cm2 (passage 1, P1) on synthetic
membranes with 0.2μm pore. The LIPUS application group was
stimulated for 20 min/day. To investigate effect LIPUS stimulation
on the matrix-synthesis of the constructs, mRNA expression of type
II collagen, aggrecan and type I collagen was studied using real-
time polymerase chain reaction. Synthesis of type II collagen and
proteoglycan was also assessed histochemically in vitro. Moreover
we tried to repair full-thickness defect model using the scaffold-
free plates stimulated LIPUS or not in vivo.
Results: The chondrocytes(P1) prepared at 107cells/cm2 detached
from the membranes to form a plate of chondrocytes around
the 7th day (day 7) of starting P1 culture. The expression of type
II collagen and aggrecan mRNA was significantly higher in the
group by stimulation of LIPUS (LIPUS group) than the group by no
stimulation (sham group). The constructs had resultant matrix of
type II collagen and proteogrycan which confirmed histochemically.
The tissues were stained strongly with safraninO, and there
were partially columnar pattern. Immunohistochemical analysis
revealed that type II collagen was abundantly deposited in the
tissue. Interestingly, LIPUS group was also more strongly stained
than sham group by histology in vitro. However In vivo study,
histology of repair tissue with the chondrocyte plate at 2week was
not significant different between LIPUS group and non-treated
LIPUS (sham) group.
Conclusions: As a result for application of LIPUS, we could stimulate
the matrix synthesis of the scaffold-free chondrocytes plate in
vitro study. In vivo study, we could not show the effect of LIPUS to
stimulate the matrix synthesis of the plate.
P68
Detailed evaluation of Autologous bone marrow-derrived
mesenchmal cells transplantation in Cynomolgus Monkeys.
S. Araki, S. Imai, M. Kubo, T. Mimura, K. Nishizawa, H. Ueba, Y.
Matsusue
Osu/Japan
Purpose: In the field of articular cartilage repair, 5 mm full-
thickness chondral defect is demonstrated to be relatively well
repaired in small animal models (i.e. rabbit models) with tissue
engineering techniques. Although attempts to repair human
chondral defects with tissue engineered methods has been made,
details of histological process is still unknown because therapeutic
and ethical issues make it unable to extract and evaluate human
tissue. In addition, no report of spontaneous repair of primate full-
thickness cartilage defects has ever been made. We have reported
regeneration of full-thickness cartilage defect in cynomolgus
monkey model in our previous studies. In these studies, we have
attempted to determine the critical size that is repairable. Two mm
diameter full-thickness cartilage defect has been demonstrated
to be repaired with hyaline-like cartilage. Three mm diameter

full-thickness cartilage defect was repaired with fibrocartilage.
In contrast, 5 mm diameter full-thickness cartilage defect was
repaired with fibrous tissue. Although up to 3 mm diameter full-
thickness cartilage defect in rabbit models have demonstrated to
be repaired spontaneously, 2 mm diameter is the limit for repair in
primates.
Methods and Materials: With this basic data, in the present study
we assessed the effectiveness of autologous bone marrow-derived
mesenchymal cells transplantation to repair a full-thickness
cartilage defect of 3 mm and 5mm in a cynomolgus monkey model.
Nine skeletally mature Cynomolgus monkeys were used in this
study. The monkeys were anesthetized and approached through
medial parapatellar incision. Full-thickness osteochondral defects
were created in patella groove(3-mm wide, 5-mm deep) and in
medial chondyle(5-mm wide, 5-mm deep). The animals were
devided into three groups: MSC, Gel, and Defect. In the MSC group,
the defects were filled with MSCs that were harvested from bone-
marrow and cultured 4 weeks embedded in collagen gel. In the Gel
group, the defects were filled with Type I collagen. Animals were
sacrificed at 6, 12, and 24 weeks after operation. Samples were
examined histologically.
Results: In the MSC groups 3-mm and 5-mm diameter full-thickness
cartilage defect was repaired with hyaline-like cartilage. Another
2-groups in contrast repaired with fibrocartilage or fibrous tissue.
Conclusions: We decided that Cynomolgus monkeys were useful
in the fields of cartilage repair.We considered that the present
study was useful for regeneration of cartilage defects using bone
marrow-derrived MSCs for clinical cases.
P69
Tendon regeneration using cell seeded scaffolds in a rat model
M.F. Pietschmann, B. Frankewycz, P. Schmitz, D. Docheva, M.
Schieker, V. Jansson, P.E. Müller
Munich/Germany
Purpose: Irreparable tendon ruptures constitute a grave clinical
problem. Our study investigates the effects of scaffold-based tendon
regeneration using different cell types (mesenchymal-stem-cells/
MSC, bone-marrow-stromal-cells/BMSC and tenocytes/TC) in a rat
model. A polyglycol acid (PGA) and a collagen scaffold were used.
Methods and Materials: A 2-3 mm full-thickness-defect in the rats
achilles tendon was created, and filled, with either cell-seeded or
not cell-seeded scaffolds. Cells were harvested from male rats and
then implanted into female rats. After 12 weeks the maximum tensile
strength was determined and histological stainings performed.
Implanted cells were traced by DNA PCR of male Y-chromosomes.
Results: Macroscopically the regenerated tendons were bigger in
diameter, firmer and less elastic than normal tendons. In the MSC
and TC groups the implanted cells could be clearly identified by DNA
PCR. The biomechanical examination revealed a comparable tensile
strength for collagen and PGA without cell-seeding. No positive
influence of using BMSC or MSC on the mechanical stability of the
regenerated tissue could be found. Using tenocytes the highest pull
out strength was found in both scaffolds. Histologically there was a
variable amount of bone formation in all groups, but considerably
less in the TC groups. An inflammatory reaction was seen in PGA
groups but not with collagen.
Conclusions: Our findings indicate that both collagen and
mesenchymal stem cells have stimulating effects on bone
formation. The use of TC improves mechanical stability and
histological appearance of the tendon regenerates. BMSC and MSC
are inferior to TC.
P70
Articular cartilage debrided from focal lesions as a cell source for
cell-based cartilage therapy
J.E.J. Bekkers, L.B. Creemers, M. Rijen, W.J.A. Dhert, D.B. Saris
Utrecht/Netherlands
Purpose: Currently, ACI is based on the expansion of chondrocytes
harvested from a non weight-bearing area in the knee. This study
evaluated the chondrogenic potential of chondrocytes from a focal
lesion as an alternative cell-source for ACI.
Methods and Materials: Articular cartilage was obtained from
focal lesions of patients (n=17) undergoing cartilage surgery and
classified according to the ICRS grading for focal cartilage lesions.
Macroscopically healthy cartilage was harvested from full-weight
bearing (FWB, n=5) and non-weight bearing (NWB, n=5) regions
Posters 207
from knees of deceased patients at the department of pathology.
Cartilage was digested overnight in 0.15% collagenase. Isolated
chondrocytes were, either directly (P0) or after expansion (P2),
pelleted by centrifugation. After 4 weeks of culture, pellets were
digested and analyzed for glycosaminoglycan (GAG) and DNA
content, using a DMMB and Picogreen assay respectively. In
addition, pellets were also paraffin embedded for collagen I, II and
X immunohistochemistry and Safranin-O histology. Differences in
GAG/DNA were analyzed by a one-way ANOVA with post-hoc LSD
test. A p<0.05 was considered statistically significant.
Results: After 4 weeks of culture, P0 pellets from grade III and
FWB chondrocytes contained more (p<0.000) GAG/DNA when
compared to pellets from grade IV and NWB chondrocytes (Figure
1). After expansion, P2 pellets contained less GAG/DNA than
their P0 counterparts and GAG/DNA was not different anymore
between the donor sites. Immunohistochemistry for collagen II
and Safranin-O staining appeared to be increased in the P0 pellets
compared to the P2 pellets. Collagen I and X staining was slightly
positive in the P2 pellets.
Conclusions: The chondrogenic potential of freshly isolated
FWB and chondrocytes from Grade III lesions is higher, and after
expansion similar, compared to NWB and Grade IV chondrocytes.
Thus for ACI based on expanded chondrocytes, cells both from
grade III and grade IV lesions are a suitable alternative for cells
from non-weight bearing areas.
P71
Scaffold-Free, Three-Dimensional Chondrocyte Constructs for
Regeneration of Articular cartilage
J. Lee1, J. Lee1, E. Lee2, J.H. Hwang1, Y. Son2
1
Seoul/Korea, Democratic People’s Republic of, 2Yongin/Korea
Purpose: In this study, we evaluated the potentiality of scaffold-free
pellet-type chondrocytes for regeneration of articular cartilage.
Methods and Materials: Rabbit costal chondrocytes were
expanded at passage 8, and then the expanded cells were seeded
onto commercial collagen scaffold (Ch-COL) or centrifuged to
make scaffold-free pellet constructs (Ch-PEL). After 2-week
chondrogenic three-dimensional culture, the Ch-COL or the
Ch-PEL was transplanted onto cartilage defect of rabbit knee.
Fibrin glue was used as a control. The ability of these constructs
to cartilage defect repair was evaluated by histological and
immunohistological examination of regenerative tissue at 6 and
12 weeks after transplantation.
Results: After chondrogenic culture, the dedifferentiated
chondrocytes formed homogenous hyaline cartilage-like structure
in the scaffold-free pellet culture system and the diameter of
the pellet was approximately 1.0 mm. However, in the Ch-COL
construct, a lacunae-occupied chondrocyte and ECM-rich structure
was only detected at the surface of the construct. In vivo study,
transplantation of Ch-COL as well as Ch-PEL restored hyaline
cartilage in the articular cartilage defect. However, undegradable
scaffold frames were detected in the regenerative tissue of Ch-COL
group even at 12 weeks after transplantation.
Conclusions: In scaffold-free pellet culture system, the cells
generate their own ECM similar to the natural hyaline cartilage
and transplantation of these pellet constructs to articular cartilage
defect is useful to make a good-quality hyaline cartilageous tissue
without immune response.
P72
Repair of partial-thickness chondral defects by gene plugs
A. Ivkovic1, A. Pascher2, D. Hudetz1, D. Maticic1, M. Jelic1, S.C.
Dickinson3, M. Loparic4 , M. Haspl1, R. Windhager5, M. Pecina1
1
Zagreb/Croatia, 2Graz/Austria, 3Bristol/United Kingdom, 4Basel/
Switzerland, 5Austria/Austria
Purpose: The aim of this translational study was to test whether
an abbreviated ex vivo protocol utilizing vector-laden, coagulated
bone marrow aspirates for gene delivery to partial-thickness
cartilage defects may be feasible for clinical application.
Methods and Materials: Autologous bone marrow harvested
from sheep was transduced with adenoviral vectors containing
cDNA for GFP or TGF-β1. The marrow was allowed to clot forming
a gene plug and implanted into partial-thickness defects created
on the medial condyle. After 6 months the quality of repair was
evaluated using biochemical, histological, and biomechanical
parameters.
208 Posters
Results: Assessment of repair showed that the groups treated with
gene plug transplantation contained more cartilage-like tissue
than untreated controls. Improved cartilage repair was observed in
groups treated with unmodified bone marrow plugs and Ad.TGF-β1
transduced plugs, but the repaired tissue from TGF-treated defects
showed significantly higher amounts of collagen II (p<0.001).
Conclusions: The results confirmed that the proposed method is
fairly simple technique for application in clinical settings. It is a
single-step procedure that can be easily implemented in standard
clinical settings, avoids the usual drawbacks associated with
gene therapy because it is administered locally, and excludes
the expensive in vitro production of autologous and engineered
tissues. Gene plugs are sufficient to facilitate articular cartilage
repair of partial thickness defects in vivo. Further studies should
focus on selection of transgene combinations that promote more
natural healing.
P73
Repair of articular cartilage defects with Sox9 gene modified
bone marrow mesenchymal stem cells
Z. Yang, X. Wei
Taiyuan/China
Purpose: to observe the influence of Sox9 gene overexpression
on the chondrogenesis of BMSCs and to repair articular cartilage
defects with Sox9 gene modified BMSCs embedded in alginate.
Methods and Materials: Bone marrow mesenchymal stem
cells of New Zealand Rabbit were transfected with recombinant
eukaryotic expression Sox9 plasmid. The chondrogenesis of Sox9
overexpressing BMSCs were analysised by RT-PCR, western blot,
immunohistology and GAG assay. Cartilage defects of the knee
joints were filled with Sox9 gene modified MSCs, MSCs or alginate
alone. The samples were evaluated histologically with a semi-
quantity scoring system for the repairing of the cartilage .
Results: The Sox9 overexpressing BMSCs expressed chondrogenic
marker molecules and synthesis more GAG than the control groups
. The defects filled with Sox9 gene modified BMSCs showed hyaline
like cartilage tissue with smooth surface and good integrating with
the adjacent cartilage; while most of the defects filled with BMSCs
in alginate showed a fibrocartilage-like regenerate tissue and most
of the defects filled with alginate only showed with fibrous tissues.
The histological scoring system showed that the cartilage repairing
of the experiment groups were better than the two control groups
with statistical significances.
Conclusions: The overexpression of exogenous Sox9 gene can
trigger the chondrogenic differentiation of BMSCs in vitro. Sox9
gene modified BMSCs embedded in alginate may regenerate
hyaline like cartilage in vivo. Gene enhanced tissue engineering
may be a promising way to repair cartilage defects.
P74
Articular cartilage repair with magnetically labeled mesenchymal
stem cells and external magnetic device using porcine model
G. Kamei, N. Adachi, M. Deie, T. Kobayashi, H. Shibuya, S. Ohkawa,
M. Ochi
Hiroshima/Japan
Purpose: The purpose of this study is to evaluate the utility of new
cell delivery system using an external magnetic force to accumulate
a relative small number of magnetically labeled mesenchymal stem
cells (m-MSCs) to the desired area using porcine model.
Methods and Materials: MSCs were cultured from bone marrow of 6
months porcine and labeled with ferucarbotran(superparamagnetic
iron oxide). We made a full-thickness cartilage defect (6mm
diameter) in the center of the patella. 4 weeks after creation of
cartilage defect, for magnetic force group, m-MSCs (5×106 cells)
were accumulated to the cartilage defect using an external magnetic
force (1.5 Tesla) for 10 minutes. For injection group, the patella was
faced upward, filled with MSCs (5×106 cells) and held for 10 minutes.
For the control group, PBS was injected. Porcine were sacrificed at
3 and 6 months, and macroscopic and histological evaluation was
done. We evaluated the maximum magnitude (articular cartilage
stiffness) and echo duration (macroscopic surface roughness) of
the repaired area using ultrasonic pulser system.
Results: Macroscopic and histological findings showed better
cartilage regeneration in magnetic force group at 6 months after
MSC injection. The mean maximum magnitude and echo duration
were 75.1±9.4% and 0.46±0.05㎲at 6 months. The maximum
magnitude was more than 60% and echo duration was almost equal
compared to normal cartilage in magnetic force group (P=0.3734).
Conclusions: This study showed that we could obtain better hyaline-
like cartilage regeneration by accumulating relatively small number
of m-MSCs to the cartilage defect using external magnetic force.
We believe that our cell delivery system using external magnetic
force is a promising option for cartilage regeneration.
P75
Meniscal Repair with Chondrocyte-seeded Flexible Scaffolds
J.J. YOO1, D.A. Bichara2, X. Zhao2, M.A. Randolph2, T.J. Gill2
1
Seoul/Korea, Democratic People’s Republic of, 2Boston/United
States of America
Purpose: A cell-based tissue-engineered construct can be used for
healing of intractable meniscal lesions. Our aims were 1) to assess the
culture conditions (static versus dynamic) and the healing capacity of
the chondrocyte-seeded flexible scaffolds; and 2) to assess in vivo
cell labeling for tracking cells during the reparative process.
Methods and Materials: Swine articular chondrocytes were
labeled with PKH 26 or DiI dye and seeded onto flexible PLGA
scaffold (VicrylTM, Ethicon Inc.) in dynamic oscillating conditions
for 24 hours. Half of cell-seeded scaffolds were cultured in static
conditions and the remaining half were cultured in dynamic
conditions for 7 days. During culture period, live/dead assay and DNA
measurements were performed. Chondrocyte-seeded scaffolds
were placed between devitalized swine meniscal discs and sutured
in place (A, dynamically cultured, B, statically cultured, C, acellular,
and D, no scaffolds). All constructs were implanted subcutaneously
in nude mice for 6 weeks and evaluated histologically.
Results: Labeled chondrocytes covered all scaffolds completely
in both dynamic and static culture. DNA measurement showed no
statistically significant difference in the two culture conditions.
Histologic analysis demonstrated a continuous fibro-cartilagenous
interfacial tissue between meniscal discs in all 12 dynamically
cultured scaffold constructs and 9 of 11 statically cultured scaffold
constructs. There was no evidence of meniscal healing between
discs in both acellular and no scaffold constructs. Both PKH 26-
and DiI-labeled cells were present along the entire interface of the
meniscal discs.
Conclusions: Culture conditions after seeding may not affect
healing outcome. The articular chondrocyte-seeded flexible PLGA
scaffolds induced healing of meniscal discs in nude mice. These
cells are responsible for the healing process in the interface. Based
on these encouraging results, swine experiments are underway to
assess biomechanical bonding properties and immune responses
after meniscal repair with aotologous and allogeneic chondrocyte-
seeded scaffolds.
P76
Optimized Alkylated cyclodextrin polysulphates restore
osteoarthritic chondrocyte extracellular matrix metabolism in
vitro and in vivo
S. Groeneboer1, S. Lambrecht1, A. Dhollander1, P. Jacques1, B.
Vandercruyssen1, K. Devreese1, D. Elewaut1, G. Verbruggen2
1
Gent/Belgium, 2GHENT/Belgium
Purpose: To compare the ability of different cyclodextrin
polysulphate derivatives to affect human articular cartilage cell
metabolism in vitro and to act as Disease Modifying OsteoArthritis
Drugs in vivo.
Methods and Materials: OA chondrocytes were cultured in gelled
alginate and exposed to 5 µg/ml (2-carboxyethyl)-β-cyclodextrin
polysulphate (CE-CDPS), or β-cyclodextrin polysulphate (CDPS)
during 5 days. Effects on IL-1-driven chondrocyte extracellular matrix
(ECM) metabolism was assayed by analysis of the accumulation of
aggrecan in the interterritorial matrix and by the release of IL-6 in the
culture supernatant. The compounds were analyzed for their in vitro
effect on coagulation and their ability to activate platelets in an in vitro
assay to detect possible crossreactivity with HIT (heparin-induced
thrombocytopenia) antibodies. CE-CDPS was selected for further
qPCR analyses, in order to investigate whether this compound directly
influences the gene expression of IL-6 and aggrecan. The effectiveness
of CE-CDPS was further assayed in collagenase-induced OA of the
knee, by injecting 1 mg/kg CE-CDPS or saline subcutaneously once
weekly in a mouse model. Knee joints were scored semiquantitatively
on inflammation, fibrosis, osteophyte growth and loss of articular
cartilage and Safranin-O staining.

Results: The polysulphated cyclodextrin CE-CDPS at 5 µg/ml
concentrations, significantly induced aggrecan production and
repressed IL-6 release by the chondrocytes in culture. Five µg/ml
of CE-CDPS, in contrast to other polysulphated cyclodextrins, did
not significantly activate platelets and thus showed no potential to
induce HIT thromboembolic accidents in vivo. Therefore, CE-CDPS
was selected for further qPCR analyses. These analyses confirmed
the anabolic and anti-catabolic profile of CE-CDPS. CE-CDPS was
therefore tested on collagenase–induced knee OA in mice and
was shown to prevent cartilage proteoglycan depletion but not
osteophyte formation in the OA knee joints of these mice.
Conclusions: CE-CDPS is a new, structurally adjusted sulphated
β-cyclodextrin derivative with preserved chondroprotective
capacity and a promising safety profile.
P77
Pellet culture induces cartilage specific gene expression but does
not alter DNA methylation patterns in bone marrow stromal and
synovial cells
D. Dono, S.J. Duguay, S. Rapko
Cambridge/United States of America
Purpose: To evaluate the capacity of a pellet culture system to
induce chondrocytic gene expression and genomic DNA methylation
patterns in adult human cell cultures derived from bone marrow
stroma, synovium, and hyaline cartilage.
Methods and Materials: Monolayer cultured bone marrow stromal
cells (BMSCs), synovial cells (SCs), and chondrocytes were
seeded into pellet cultures with sampling at days 0 and 20. Gene
expression and DNA methylation levels of cartilage specific genes
were examined using PCR methods.
Results: Gene expression levels of cartilage specific genes were
low or undetectable in all of the day 0 cultures, but significantly
upregulated by day 20 of pellet culture. DNA methylation patterns in
the day 0 cultures were specific to the tissue of origin for each culture
and remained steady throughout the pellet culture period. DNA
methylation did not strongly correlate with expression, as methylated
genes were expressed by MSCs and SCs during pellet culture.
Conclusions: Pellet culture induced the expression of cartilage
specific genes in BMSCs, SCs, and chondrocytes, but did not alter
their original tissue specific DNA methylation patterns. These
observations suggest that MSC and SC DNA methylation patterns
are highly stable. As cartilage possesses specific DNA methylation
patterns presumably established during chondrogenesis, the results
of this work have implications for the selection of cell types and
culture conditions used for cartilage tissue engineering applications.
P78
Valproic acid suppresses interleukin-1b-induced microsomal
prostaglandin E2 Synthase-1 expression in chondrocytes
N. Zayed, N. Chabane, F. El Mansouri, J. Martel-Pelletier, J.P.
Pelletier, H. Fahmi
Montreal/Canada
Purpose: Microsomal prostaglandin E2 Synthase (mPGES)-1
catalyzes the terminal step in the biosynthesis of PGE2. Early
growth response factor-1 (Egr-1) is a key transcription factor in
the regulation of mPGES-1. In the present study we examined
the effects of valproic acid (VA), a histone deacetylase (HDAC)
inhibitor, on interleukin (IL)-1b-induced mPGES-1-expression in
human chondrocytes
Methods and Materials: Chondrocytes were stimulated with IL-1 in
the absence or presence of VA, and the level of mPGES-1 protein and
mRNA expression were evaluated using Western blotting and real-
time reverse-transcription polymerase chain reaction, respectively.
The mPGES-1 promoter activity was analyzed in transient
transfection experiments. Egr-1 recruitment to the mPGES-1
promoter were evaluated using chromatin immunoprecipitation
(ChIP) assays
Results: VA dose-dependently suppressed IL-1b-induced mPGES-1
protein and mRNA expression as well as its promoter activation.
Treatment with VA did not alter IL-1-induced Egr-1 expression,
nor its recruitment to the mPGES-1 promoter, but prevented its
transcriptional activity.
Conclusions: Our study demonstrates that VA inhibits IL-1-induced
mPGES-1 expression in chondrocytes. The suppressive effect of VA
was not due to reduced expression or recruitment of Egr-1 to the
mPGES-1 promoter.
Posters 209
P79
Interleukin-1-induced cyclooxygenase-2 and inducible nitric
oxide synthase expression in human OA chondrocytes is
associated with histone H3K4 methylation
F. El Mansouri, N. Chabane, N. Zayed, J. Martel-Pelletier, J.P.
Pelletier, H. Fahmi
Montreal/Canada
Purpose: Increased expression of inducible NO synthase (iNOS)
and cyclooxygenase (COX)-2 plays a key role in the pathogenesis
of osteoarthritis. Methylation of lysine 4 on histone H3 (H3K4)
was shown to be of fundamental importance in the regulation of
gene expression. In the present study, we investigated the role of
H3K4 methylation in interleukin-1b (IL-1)-induced COX-2 and iNOS
expression in human OA chondrocytes
Methods and Materials: Chondrocytes were stimulated with IL-1 for
various time periods and the expression of iNOS and COX-2 mRNAs
and proteins were evaluated using real-time reverse transcriptase-
polymerase chain reaction (RT-PCR) and Western blotting,
respectively. H3K4 methylation at the iNOS and COX-2 promoters
was evaluated using chromatin immunoprecipitation (ChIP) assays.
The role of histone methylation was further evaluated using the
methyltransferase inhibitor, 5’-deoxy-5’(methylthio) adenosine
(MTA).
Results: IL-1 induced iNOS and COX-2 mRNA and protein in a dose-
and time-dependent manner. The induction of iNOS and COX-2
expression by IL-1 was associated with H3K4 di- and trimethylation
at the iNOS and COX-2 promoters, whereas the levels of H3K4
monomethylation remained unchanged. Treatment with MTA
inhibited IL-1-induced H3K4 methylation as well as IL-1-induced
iNOS and COX-2 expression.
Conclusions: These results indicate that H3K4 methylation
contributes to IL-1-induced iNOS and COX-2 expression and suggest
that this pathway could be a potential target for pharmacological
intervention in the treatment of osteoarthritis.
P80
Glial Fibrillary Acidic Protein Maintains Nuclear Morphology
And Supports the Resistance to Severe Mechanical Stress in the
Chondrocytes of Elastic Cartilage
S. Kanazawa1, S. Nishizawa2, T. Takato2, K. Hoshi2
1
Bunkyo-ku/Japan, 2Tokyo/Japan
Purpose: Glial fibrillary acidic protein (GFAP) is a intermediate
filament that is expressed in astrogrial cells, but is also localized
in chondrocytes of elastic cartilage which is present in auricles or
epiglottis. Among various kinds of cartilage, the elastic type can
maintain tissue function without collapse, even after repeated
bending. This suggests that GFAP may contribute to the resistance
to the severe mechanical stress in the cells and should conserve
the structures and functions of cytoplasmic organelles.
Methods and Materials: To verify the functions of GFAP in
the chondrocytes, we compared the morphology in cultured
chondrocytes of mouse auricles between wild type (Gfap+/+) and
GFAP-deficient mice (Gfap-/-). We also examined the cell responses
to stretch stress for these chondrocytes by cell-stretch devise.
Results: In the auricular chondrocytes (passage 3) of Gfap+/+,
GFAP was densely accumulated in the perinuclear areas, while
the vimentin was generally present in cytoplasmic areas. Nuclei
were shaped columnar, with the size of approximately 12 micron
in diameter and 4 micron in height. In contrast, the Gfap-/-
chondrocytes (passage 3) significantly showed the flattening and
the irregularity of nuclei (approximately 14 micron in diameter and
3 micron in height). When the passage was repeated by passage
8, this tendency was exaggerated in the Gfap-/- chondrocytes,
and, moreover, the multinucleation became evident in that type.
In the cell-stretch studies (30% strain and 24 h), although most of
the Gfap+/+ chondrocytes maintained the cell attachment (approx.
96%) and cell viability (approx. 100%), the Gfap-/- counterparts
were detached at the rate of 60% and decreased the cell viability
to 83%.
Conclusions: Based on these results, GFAP seems to play pivotal
roles in the conservation of cell structures and functions thought
the maintenance of nuclear morphology, and support the resistance
to severe mechanical stress in the tissue which physiologically
suffers from stretch overload.
210 Posters
P81
Trophic effects of mesenchymal stem cells on chondrocytes
increase cartilage formation in co-cultures
L. Wu1, J. Leijten1, N. Georgi2, C.A. van Blitterswijk1, M. Karperien1
1
Enschede/Netherlands, 2Enchede/Netherlands
Purpose: Autologous Chondrocyte Implantation (ACI) requires a large
number of chondrocytes. Replacement of part of the chondrocytes
with Mesenchymal Stem cells can significantly reduce the number
of chondrocytes required for ACI. Previously, it was shown that
cartilage matrix formation is significantly increased in co-cultures
of MSCs and chondrocytes compared to cultures of pure cell
populations. To explain the beneficial effect of MSCs in co-cultures,
it is hypothesized that increased matrix formation is either due to
i) chondrogenic commitment of MSCs induced by chondrocytes; ii)
increase of cartilage matrix formation by chondrocytes stimulated
by the MSCs, or iii) a combination of both.
Methods and Materials: To study cell fate by species specificity, we
used bovine chondrocytes and human MSCs. Pellets were cultured
in a 96-well plate in chondrocyte proliferation medium for 4 weeks.
Glycosaminoglycans (GAG) assay, DNA assay and histological
staining were performed to evaluate cartilage matrix formation.
Real time PCR was carried out to determine the origin of cartilage
matrix. Cell proliferation and apoptosis were also examined.
Results: Co-culture of bovine chondrocytes and human MSCs in
pellets significantly increased glycosaminoglycan production per
initial % of seeded chondrocytes as expected. To avoid donor
variation, we used immortalized MSCs (iMSCs) in the subsequent
experiments. Species specific quantitative PCR indicated
disappearance of human cells after 4 weeks of co-culture. This was
confirmed by species specific qPCR of cartilage matrix components
showing its bovine origin. In co-cultures, chondrocyte, but not MSC,
proliferation was significantly enhanced compared to cultures of
pure chondrocytes. Inversely, TUNEL assay indicated preferential
death of MSCs, most likely by apoptosis, in co-cultures only at
week 1 and week 2. Finally, we show that increased proliferation of
chondrocytes in co-cultures is, at least in part, caused by soluble
factors secreted by MSCs.
Conclusions: Our data indicate that trophic effects of MSCs increase
cartilage matrix formation by chondrocytes in co-cultures.
P82
Prostaglandin D2 enhances interleukin -1beta- induced
cyclooxygenase-2 expression in osteoarthritic chondrocytes
N. Zayed, N. Chabane, F. El Mansouri, J. Martel-Pelletier, J.P.
Pelletier, H. Fahmi
Montreal/Canada
Purpose: To investigate the effects of prostaglandin D2 (PGD2)
on interleukin-1beta (IL-1beta)-induced cyclooxygenase (COX)-2
expression in human chondrocytes and the signalling pathways
involved in these effects.
Methods and Materials: Chondrocytes were stimulated with IL-1 in
the presence or absence of PGD2, and expression of COX-2 protein
was evaluated by western-blotting. Messenger RNA (mRNA)
expression was analyzed by real-time reverse transcription-
polymerase chain reaction. The role of the PGD2 receptors D
prostanoid receptor 1 (DP1) and chemoattractant receptor-like
molecule expressed on Th2 cells (CRTH2) was evaluated using
specific agonists.
Results: PGD2 increased in a dose-dependent manner IL-1-induced
COX-2 protein and mRNA expression. DP1 and CRTH2 were expressed
and functional in chondrocytes. The effect of PGD2 was mimicked
by DK-PGD2 and Indomethacin, selective agonists of CRTH2, but not
by BW245C, a selective agonist of DP1. Furthermore, treatment with
an anti-CRTH2 antibody reversed the effect of PGD2, indicating that
the stimulatory effect of PGD2 is mediated by CRTH2. Activation
of CRTH2 is consistent with the activation of a receptor coupled to
a phosphoinositide-specific phospholipase, suggesting that the
effect of PGD2 is mediated by the CRTH2/PIP2/PKC.
Conclusions: PGD2 enhances IL-1-induced production of COX-2 by
chondrocytes through the CRTH2/PIP2/PKC signalling pathway.
P83
Evaluation of Proliferation and Synthesis of osteoarthritic
Chondrocytes with and without pericellular Matrix
M. Rothdiener, Q. Wang, A. Badke, W.K. Aicher, B. Rolauffs
Tübingen/Germany
Purpose: The pericellular matrix (PCM) surrounding chondrocytes
is responsible for structural and functional connections of cell and
extracellular matrix (ECM) of articular cartilage. PCM is considered to
affect proliferation, phenotype-maintenance and matrix-synthesis. The
aim of this project was the evaluation of chondrocytes with intact PCM
for biological therapy in osteoarthritic (OA) patients. The objectives of the
current studies were a) to investigate the metabolism of OA chondrocytes
under preservation of the intact PCM b) cultivation and proliferation in
the presence or absence of FBS and c) gene expression profiling.
Methods and Materials: Human chondrocytes from knee-replacement
patients (mean: 62 years) were isolated in a collagenase/dispase
digestion at different concentrations and time periods to maintain/
remove PCM. Cells were cultivated as monolayers for 30d (F12:DMEM
1:1, 10% FBS, 0,1% ascorbic acid) and counted every second day.
mRNA was isolated at d0, cDNA was synthesized and quantitative
PCR was performed using the Roche Lightcycler system. OA grades
were assessed with the Kellgren-Lawrence-Score.
Results: Without FBS, chondrocytes +/-PCM showed no proliferation
but remained their typical phenotype. In the presence of FBS, cells
showed spread morphology. With intact PCM, rapid proliferation
was observed (at day 10: 400% of initial) whereas chondrocytes
without PCM remained under 100%. Within this group, fast-growing
and slow-growing populations occurred but the proliferation rate did
not correlate with the OA grades. Gene expression profiles showed a
significantly higher expression of matrix proteins COMP and aggrecan
in chondrocytes with intact PCM. However, no significant differences
in collagen expression occurred. With intact PCM, the chondrogenic
marker FGF-2 was also significantly higher expressed. In contrast, the
cartilage degeneration marker IL-1β was expressed similarly.
Conclusions: Proliferation of OA chondrocytes was vastly superior
with intact PCM. Synthesis of matrix components and chondrogenic
markers were also enhanced with maintained PCM suggesting that
OA cells with PCM may be further evaluated for biological therapy.
P84
Co-culturing of Human Bone Marrow Mononuclear Cells and
Primary Chondrocytes to Improve Cartilage Formation
J.E.J. Bekkers, D.B. Saris, A.I. Tsuchida, W. Verra, W.J.A. Dhert, L.B.
Creemers
Utrecht/Netherlands
Purpose: The combination of freshly isolated chondrocytes with
mesenchymal stem cells has previously been advocated as a possible
alternative for the use of expanded chondrocytes in autologous
chondrocyte implantation (ACI). This study aimed to investigate
whether co-culture of the mononuclear fraction from freshly isolated
human bone marrow mononuclear fraction (MNF) and human
primary chondrocytes (hPC) stimulates chondrogenesis.
Methods and Materials: Knee cartilage was obtained at autopsy, bone
marrow aspirates from patients undergoing hip arthroplasty and human
fibroblasts from the foreskin (hFB). Cartilage was digested overnight
in 0.15% collagenase. After erythrocyte cell lysis, the MNF cells were
combined with hPCs at different ratios (containing 0%, 2%, 5%, 10%,
15%, 20% 50% and 100% hPCs) and pelleted by centrifugation. Pellets
were also created between PCs and hFB in similar ratios as the PC MNF
pellets. In addition, PCs were also pelleted in quantities identical to
those in the co-culture pellets, however, without adding other cells
(PC Alone group). After 4 weeks of culture, the pellets were digested
and analyzed for GAG and DNA content, using a DMMB and PicoGreen
assay respectively. Differences in GAG content per DNA were analyzed
by a one-way ANOVA and post-hoc-LSD test.
Results: The amount of matrix produced per cell (GAG/DNA) after 4
weeks of culture was higher (p<0.05) in the PC/MNF co-cultured
pellets when compared to the PC Alone pellets, in the range 15-
100%hPC (Figure 1). This was not observed in co-cultured pellets
containing PCs and hFB (Figure 2). Surprisingly, low amounts of
chondrocytes (PC Alone 2-5%) show more efficient GAG/DNA
production compared to co-cultured hPCs and MNFs.
Conclusions: Co-culturing of the nonexpanded MNF and hPCs
enhanced matrix producing activity. This stimulation was not achieved
combining hPCs with hFB cells suggesting specific interactions
between PCs and MNF cells. This finding may further pave the way
towards a one-step cell-based regenerative cartilage therapy.

P86
To establish a method for increasing cell seeding rate in PLGA
scaffold
C. Hsieh, J. Tsai-Wu, H. Chiang, W. Lee, C. Jiang
Taipei/Taiwan
Purpose: The application of three-dimensional scaffolds in tissue
engineering has been shown to be important in cell differentiation
induction and extracellular matrix production. Effective cell seeding
and uniform cell distribution throughout the scaffold are the major
issue for the success of three-dimensional culture. Our purpose is to
establish a quick, convenient and efficient method to increase cell-
seeding rate in the 3D sponge scaffold composed of poly DL-lactic-co-
glycolic acid (PLGA).
Methods and Materials: The mesenchymal stem cells (MSCs) from
the green fluorescent protein (GFP) transgenic pigs were used for cell
seeding. Four different methods, including the static surface seeding,
orbital shaker seeding, vacuum seeding and modified vacuum
seeding were examined. The seeding efficiency was compared by cell
survival and distribution of GFP-MSCs after 3-day culture.
Results: The distribution of GFP-MSCs was limited to the cell loading
region by the static surface seeding method, and the seeding rate
was very low. The seeding rate was greatly enhanced by using the
orbital shaker seeding method; however, the cells were not uniformly
distributed. By applying the vacuum seeding method, the cell
distribution was more uniform and the seeding rate was as high as
the orbital shaker seeding. The cell seeding rate and distribution were
further improved by using the modified vacuum seeding method.
Conclusions: Our results showed that the modified vaccum seeding
method was the better method for 3D scaffold culture regardless the
irregular structure of the scaffold. This method could be a useful tool
for the cell-seeding to irregular scaffolds in the future study.
P87
Tumor necrosis factor -α (TNF-α) antagonist inhibits matrix
mettalloproteinase-3 (MMP-3) secretion induced by TNF-α in
human articular chondrocytes in vitro
S. Abe, H. Nochi, T. Ruike, T. Matsuno
Asahikawa/Japan
Purpose: Mouse monoclonal anti-TNF-α antibody, infliximab,
has been used to control inflammation and inhibit bone and joint
destruction in rheumatoid arthritis (RA) We explore the effects of
TNFα-antagonist, infliximab, on human articular chondrocytes
(ACs) in vitro.
Methods and Materials: 1. ACs were isolated from Osteoarthritis
(OA) and RA knee joints under the informed consent. ACs were
cultured in DMEM with 10% human serum and they were used at
the passage 1 or 2. 2. To characterize cultured ACs, flow cytometric
analysis was performed. 3. To measure cell proliferation, [3H]
thymidine was added at the following culture condition. (1) ACs were
cultured with several concentration infliximab (Remicade®, Centcor
Inc.) for 4 days. (2) ACs were treated with 20 ng/ml of TNF-α and
infliximab for 4 days. (3) ACs were treated with 20 ng/ml of TNF-α
and infliximab were added 24 hours later, then they were cultured
for 4 days. 4. The concentration of MMP-3 in conditioned media at
the above assay was determined using ELISA kits.
Results: 1. CD34, CD45, HLA-DR/DP/DQ, CD80 and CD86 were
negative on ACs. HLA-ABC, CD73, CD90, CD105 and CD166 were
positive on ACs. These patterns on surface makers in ACs derived
from OA and RA were same. 2. The proliferation of ACs was not
affected by infliximab. Then, the proliferation of ACs treated by TNF-α
20 ng/ml was neither affected by infliximab statistically. 3. MMP-3
secretion from ACs was not affected by treating infliximab. On the
other hand, MMP-3 secretion was shown an increase in ACs treated
by TNF-α, and infliximab inhibited this effect dose dependently.
Conclusions: In this reports, we demonstrated that infliximab did
not affect ACs proliferation in vitro, but infliximab inhibited MMP-
3 secretion induced by TNF-α in ACs dose dependently in vitro. In
present study demonstrate that infliximab could inhibit MMP-3
secretion and inhibit cartilage degeneration under inflammatory
conditions.
Posters 211
P88
Acid ceramidase, chondrocyte survival and transplantation
S. Sachot, Y. Ge, X. He, E. Eliyahu, E.H. Schuchman, C.M. Simonaro
New York/United States of America
Purpose: The mucopolysaccharidoses (MPS) are inherited joint
and bone disorders due to mutations in specific genes degrading
glycosaminoglycan (GAGs). We have previously shown that GAG
storage in the MPS disorders results in a complex sequence of
downstream events, including the release of numerous inflammatory
cytokines and proteases, and alterations in the metabolism of
several lipid signaling molecules. Acid ceramidase (AC) is a key
enzyme in sphingolipid metabolism, helping to maintain the critical
balance between three important signaling lipids, ceramide,
sphingosine, and sphingosine-1-phosphate (S1P). The purpose of
this research was to evaluate whether AC could be used to improve
the production of bone marrow-derived chondrocytes for the
eventual treatment of MPS patients.
Methods and Materials: Bone marrow-derived mesenchymal
stem cells (BM-MSC) were prepared from normal and MPS VI
(Maroteaux-Lamy disease) rats using standard methods ongoing in
our laboratory, and grown with and without recombinant AC (rAC).
Cell viability, apoptosis rates and stem cell properties, including
the ability of the expanded cells to form chondrocytes, were
evaluated.
Results: Addition of rAC to BM-MSC culture media permitted their
growth and expansion for more than 15 passages. Importantly,
these cells maintained their stem cell properties and could be
differentiated into chondrocytes and other lineages.
Conclusions: rAC can be used to improve the manufacturing of
chondrocytes for transplantation in the MPS disorders and other
diseases with joint injury. Further characterization of these cells is
currently underway in the MPS VI rat model.
P89
Autophagy modulates osteoarthritis-related gene expressions in
human chondrocytes
K. Takayama, T. Matsushita, R. Kuroda, K. Ishida, N. Fujita, S. Kubo,
T. Matsumoto, H. Sasaki, M. Kurosaka
Kobe/Japan
Purpose: Autophagy, an evolutionarily conserved process for the
bulk degradation of cytoplasmic components, serves as a cell
survival mechanism. The purpose of this study is to elucidate the
role of autophagy in human chondrocytes and pathophysiology of
osteoarthritis (OA).
Methods and Materials: Autophagy in articular cartilage tissues
and primary chondrocytes derived from patients with OA was
assessed by immunohistochamistry and immnoblotting using
antibodies for atuophagy markers, LC3II and beclin1. The state
of autophagy in normal chondocytes under catabolic (IL-1β and
NO) and nutritional stresses were examined by real-time PCR. We
also examined the effects of inhibition or induction of autophagy
under the stimulation with IL-1β. Autophagy was inhibited by small
interfering RNA (siRNA) targeting Atg5, an autophagy essential
gene and autophagy was induced by rapamycin under IL-1β
stimulation. The effects of inhibition or induction of autophagy
were examined by real-time PCR for MMP13, ADAMTS5, aggrecan
and COL2A1 mRNA.
Results: Osteoarthritic cartilage strongly expressed LC3II as
compared with non-arthritic cartilage. Catabolic and nutritional
stresses caused increased autophagy. The inhibition of autophagy
by Atg5siRNA under the stimulation of IL-1β caused significant
upregulationofMMP13andADAMTS5expressionswhiletheinduction
of atuophagy by rapamycin reduced these gene expressions.
Additionally, the inhibition of autophagy downregulated aggrecan
and COL2A1 while the induction of autophagy uperegulated the
expressions of aggrecan and COL2A1.
Conclusions: Autophagy was increased in osteoarthritic cartilage.
In addition, the catabolic and nutritional stresses increased
autophagy in normal chondrocytes. Furthermore the inhibition
of autophagy caused OA-like gene expression changes while the
induction of autophagy prevented. These observations suggested
the increased autophagy was an adaptive response to protect
cells from stresses and autophagy may play protective roles in
chondrocytes. Further studies about autophagy in chondrocytes
will provide novel insights into the pathophysiology of OA.
212 Posters
P90
Validation of chondrocytes loading on type I/III collagen
membrane for autologous chondrocyte implantation
Z. Lin1, Q. Zheng1, J. Xu2, M.H. Zheng3
1
Guangzhou/China, 2Perth/Australia, 3City Beach/Australia
Purpose: Matrix Induced Autologous Chondrocyte Implantation
(MACI) relies on the seeding of in vitro expanded cell on type I/
III collagen membrane (Bio-Gide®) as a cell deliver scaffold
prior shipping for clinical implantation. It is always impossible to
standardize the cell loading time on collagen membrane due to the
variation in clinical and cellular manufacture requirement. We have
observed that long term incubation of chondrocyte on collagen
membrane did not favor the maintenance of the cellular phenotype
and function. In this study, we investigated the gene expression
profile and the time-dependent adhesion rate of the chondrocytes
on type I/III collagen membrane, aiming to optimize the pre-surgical
time of cell incubation on Bio-Gide®.
Methods and Materials: Thirty chondrocyte-associated genes
were measured on the chondrocytes loading on Bio-Gide® and
compared to monolayer cultured cells by real-time PCR. Cell
adhesion assay was performed to determine the adhesion rate of
the cells on collagen membrane.
Results: Real-time PCR revealed that human chondrocytes after
seeded on Bio-Gide® for 5 days, the cells display less capability
for the production of cartilage-specific matrix proteins, as
demonstrated by the downregulation of aggrecan, col2a1, link
protein and MMPs. In cell adhesion assay, 80% of the cell attached
to the collagen membrane within 7 mins after loading, and the
adhesion rate raised to 90% after 15 mins. At 40 mins, approximately
99% of the chondrocytes were attached to the membrane.
Conclusions: The study concluded that the pre-surgical time of cell
loading could be optimized to less than 40 mins without loss the
majority of the cell population and proper cellular function.
P91
Effect of the mechanical properties of the substrate on the
expression of cartilage markers in cultured chondrocyte
A. Zabalza-Baranguá1, P. Sanz-Ramos1, P. Ripalda Cemboráin1, C.
Alcaine2, S. Santander2, I. Ochoa2, G. Mora1, I. Izal Azcárate1
1
Pamplona/Spain, 2Zaragoza/Spain
Purpose: Mechanical properties of the substrate may modify the
behaviour and shape of cells in culture. In chondrocytes it have been
studied the effect of mechanical stress, but little is known about the
capacity of cartilage cells to sense the stiffness of the substrate.
Methods and Materials: Sheep chondrocytes were cultured at same
density using standard plastic dishes (1 GPa) and Elastic Supported
Surface dishes (28 kPa). Morphology, adherence, doubling rate and
the expression of cartilage markers (aggrecan and collagens type
I and II) were evaluated alter 3 days in culture. Finaly, the presence
of focal adhesion points was studied by immunofluorescence.
Results: Results did not show any difference according to the
morphology and adherence of cells. The number of doublings suffered
by cells was also similar in both type of plates. RT-PCR analysis of
gene expression showed a significant decrease in the quantity of RNA
for collagen type II and aggrecan, while a significant increase in type
I collagen. Interestingly the addition of blebbistatin (inhibitor of Non
Muscle Myosin II) to the culture prevents for this change, returning
values obtained to those of plastic dishes. The number of focal adhesion
points resulted different according to the stiffness of the substrate.
Conclusions: Chondrocytes have the capacity to sense the stiffness
of the substrate on which they are cultured, and to modify their
phenotype accordingly. Mechanisms for the sensing include the
action of NMMII.
P92
Cell growth of chondrocytes is independent of age and gender.
C. Pronk-Admiraal, B. Ellermeijer, R. Benink
Den Helder/Netherlands
Purpose: Autologous chondrocytes implantation is a very effective
technique for repairing cartilage lesions. The development of new
cartilage is triggered by cells cultivated in vitro. For large surface
defects, more cells are needed, which is especially important for
older patients with more severe defects. We investigated whether
in vitro cell growth depends on age or gender.
Methods and Materials: Cartilage was harvested by arthroscopic
knee surgery and cultivated in 3 stages. After in vitro expansion of the
chondrocytes, which takes place in stages P2 (5 flasks of 75cm²) and P3
(10 flasks of 150cm²), the final number of cultivated cells was harvested
for implantation. Collected data of the mean cell growth factor was
correlated to age and gender, and analyzed with PASW v18.
Results: The gender distribution of 180 patients was 88 female
and 92 male. Age ranged from 18 to 63 (mean 41). Duration of the
culturing stages ranged from 6 to 20 days for P2 (mean 10) and from
13 to 28 days for P3 (mean 19). Mean harvesting resulted in 10,6x10 6
cells at the end of P2 (range 4.2-21, SD 2.9) and 42.5x106 cells at
the end of P3 (range 16.6-87, SD 11.8). The average growth factor
per day was 1.151 in P2 (SD 0.069) and 1.076 in P3 (SD 0.026). No
differences were seen with the independent samples test in respect
to the genders, both in P2 and P3. Comparing the ages over and
under 45 years revealed no statistical significance.
Conclusions: The culturing technique for chondrocytes is suitable
for a wide range of patients. The ability to grow the required cells
in vitro is independent of gender or age up to 60-65 years. No
adaptations of the current cultivating process is required.
P93
Identification and selection of human hyaline chondrocytes with
appropriate levels of differentiation and cell viability for clinical
use
I. Garzón, V. Carriel, A. Morales, M.S. Alaminos, M. Lobo, M.
Alaminos, A. Campos
Granada/Spain
Purpose: Recent reports demonstrated a lack of scientific
evidence supporting the effectiveness of autologous chondrocyte
implantation. This could be due to low cell viability or differentiation
levels of the chondrocyte cultures that are implanted in the patient.
In this work, we have performed a study of seven successive
cell passages of human hyaline chondrocytes to determine the
expression levels of several genes associated to cell death and
chondral differentiation using expression microarrays.
Methods and Materials: Human hyaline chondrocytes were isolated
from the knee joint of healthy donors using collagenase II digestion,
and 7 cell passages were done. Total RNA was extracted from each
cell passage and the RNA expression level was quantified by using
Affymetrix Human-Genome U133 plus 2.0 arrays. Expression of the gene
encoding for caspase 2 and two extracellular matrix-related genes was
analyzed and compared among all the cell passages analyzed here.
Results: Our results showed that CASP2 levels decreased until
the 2nd cell passage and then progressively increased until the
7th passage (Figure 1). In contrast, the extracellular matrix-related
genes matrilin 1 (MATN1, cartilage matrix protein) tended to increase
up to the 3rd passage, and decreased thereafter, with a correlation
with CASP2 of r=-0.7737. Similarly, the extracellular matrix gene
CHST3, encoding for carbohydrate-chondroitin 6-sulfotransferase 3,
showed a sequential increase until the 5th passage and then tended
to progressively decrease until the 7th passage (r=-0.6987).
Conclusions: The equilibrium between cell viability as determined by
CASP2 gene expression and the extracellular matrix differentiation
genes as determined by MATN1 and CHST3 genes expression should
be considered for selection of the most appropriate chondrocyte
cell population for clinical purposes. Since the highest levels of
cell differentiation and the lowest levels of caspase expression are
found in the 3rd passage, we suggest that this passage should be
preferentially selected for therapeutic use. Supported by PAI CTS-
115 (Tissue Engineering Group).
P94
The influence of anaesthetics on in vitro cultured cartilage
cells; a reflection of the effects while performing Autologous
Chondrocyte Implantation (ACI) in the knee?
C. Pronk-Admiraal1, B. Ellermeijer2, R. Benink2
1
den helder/Netherlands, 2Den Helder/Netherlands
Purpose: To repair cartilage lesions the use of autologous
chondrocytes is an upcoming technique. When implantating new
grown cartilage cells back into a knee of the patient, Ropivacaine and
Bupivacaine are sometimes used by wound infiltration. This study
will show which influence these anaesthetics and the povidon-iodine
used as a desinfectant, would have on the growth-rate or further
development of cartilage cells, in case the wound is not properly
closed and the anaesthetic comes into contact with the cells.

Orthopaedists can adapt their methods, when performing ACI into the
knee, to the most optimum with respect to the results of this study.
Methods and Materials: Aside from the regular cultures, culture flasks
have been used to grow cells added with Ropivacaine, Bupivacaine or
Povidon-iodine. All these cells grow under the same circumstances as
the regular culture. The cell growth-rate and cell aspect are evaluated
every three days in all culture flasks.
Results: At a high concentration (1.5 mg/ml Ropivacaine and 0.83
mg/ml Bupivacaine) the cells died immediately and lysated. At lower
concentrations the cells remained alive but they duplicated very
chaotically with respect to the blanco. These cells also formed very
large cores.
When Povidon-iodine was added, the cells could not get attached
to the flask. If the Povidon-iodine was added after the cells had time
to get attached, they had difficulty with duplicating and had a low
growth-rate. (Table 1)
Conclusions: In this study we show strong effects of anaesthetics
and povidon-iodine on the in vitro growth-rate of cartilage cells. With
respect to this results we would advise not to use any form of local
anaesthetics or povidon-iodine, since its use can damage the cells
and lower the growth rate. Further research will evaluate which effect
will remain when no anaesthetics would be added after three days of
culture.
P95
Localization of the Optimal Site for Articular Cartilage Biopsy for
Autologous Chondrocyte Implantation
M. Gibson, J. Elisseeff, M. Trice
Baltimore/United States of America
Purpose: We hypothesized that the quality of cartilage repair tissue in
autologous chondrocyte implantation (ACI) is dictated by the quality
of the articular cartilage biopsy material and have proposed that the
progenitor cells obtained at biopsy for ACI are major determinants of
chondrogenic capacity of redifferentiated chondrocytes. In this study,
we assess the qualitative features, anatomical location, and quantitative
features of articular cartilage biopsy specimens to determine their
relationship to postexpansion chondrogenic capacity.
Methods and Materials: We completed histologic, biochemical, live-
dead, and collagen-type analyses in 10 human articular cartilage
biopsy specimens. Chondrocytes were expended in three-dimensional
peg culture for four to five passages and then studied for cell viability
with WST-I reagent. We assessed chondrogenic capacity by measuring
DNA content through fluorophotometry, glycosaminoglycan (GAG)
content by using a dimethylene blue spectrophotometric assay,
and gene expression by using reverse transcription-polymerase
chain reaction. We analyzed all data with the Microsoft Office Excel
Statistical Package and used one-way single factor analysis of
variance and post hoc Tukey tests. Significance was set at p < 0.05.
Results: A statistically significant correlation (p = 0.001173) was
found between initial GAG concentration in biopsy specimens and
the chondrogenic capacity of redifferentiated chondrocytes. When
the anatomic locations of biopsy site were compared, no difference
was found in cell viability, metabolic activity, histology, biochemical
content, or gene expression before or after in vitro expansion.
Conclusions: Anatomic biopsy location was not a factor in
chondrogenicity of redifferentiated chondrocytes. However,
chondrocytes from locations with higher GAG concentrations
reproducibly produced chondrocytes with higher chondrogenic
capacity as measured by GAG production and collagen type-II markers
in gene expression analysis. A newly proposed measure, GAG to cell
ratio per unit weight of biopsy specimens, proved predictive of the
same ratio in redifferentiated culture. These data have relevance in
choosing optimal biopsy tissue for ACI.
P96
The influence of COX inhibition on primary, human articular
chondrocytes following exposure to IL-1beta
H. Schmal, A.T. Mehlhorn, P. Niemeyer, B. Dirhold, N.P. Suedkamp
Freiburg/Germany
Purpose: COX inhibiting drugs are applied in orthopedics as a
standard analgetic medication for osteoarthritis and posttraumatic
pain. The effects of COX inhibition on differentiation and metabolism
of human articular chondrocytes following cartilage damage are
not yet completely understood.
Methods and Materials: Human femoral heads were obtained during
hip arthroplasties following femoral neck fractures and chondrocytes
Posters 213
enzymatically released. The cells were cultured in alginate beads, exposed
to IL-1β, and treated with the unspecific COX inhibitors ibuprofene,
paracetamol, and diclofenac. Proliferation, bFGF-secretion, rate of apoptosis,
and mRNA-expression of chondrogenic markers were analyzed.
Results: Exposure of chondrocytes to IL-1β for 14 days resulted in
a 4fold increase of metabolic cell activity, and an amplification of
DNA-content by 51% (p<0.05). The addition of ibuprofene induced
a further enhancement by 20% and 24%, respectively (p<0.05). In
contrast, metabolic activity of chondrocytes co-cultured with LPS-
stimulated PBMCs was significantly reduced by 23% (p<0.05).
Despite enhanced proliferation, rate of apoptotic cells increased
following IL-1β exposure by 5%; this effect could significantly
be reduced by COX-inhibition in 2D and 3D chondrocyte cultures
(p<0.05). Correlating with augmented proliferation, bFGF
concentrations measured in the culture supernatants treated with
IL-1β increased 2.85fold at day 3, elevated levels were maintained
up to day 14. Addition of COX-inhibitors caused a further induction
of bFGF between 84% and 245% (p<0.05). Real time PCR showed
the complete suppression of the chondrogenic marker collagen
type II by IL-1β, expression of aggrecan was significantly reduced
by 70% (p<0.05), this was not influenced by COX inhibition.
Conclusions: IL-1β induces cell death of primary human articular
chondrocytes, releasing activating intracellular cytokines as bFGF,
which is associated with an enhanced cell metabolism. Whereas
apoptosis may be reduced by COX inhibition, downregulation of
specific cartilage markers remained unchanged.
P97
Effect of mechanical stress or Interleukin-4 on cartilage-specific
gene expressions of rat chondrocytes in 3-D scaffold
S. Shioji1, S. Imai1, K. Mori1, K. Ando1, K. Uenaka2, K. Nishizawa1, S.
Araki1, Y. Takemura1, Y. Matsusue1
1
Otsu/Japan, 2Otsu City/Japan
Purpose: Interleukin-4 (IL-4) has been suggested to protect the
articular cartilage. Yet, it remains long unclear whether IL-4 acts
on the chondrocytes. In turn, mechanical stress has been known
to influence the chondrocyte function. The aim of this study was
to investigate the role of IL-4 in chondrogenesis and to research
whether IL-4 plays any role in regulating the molecular function
of chondrocyte in response to the mechanical stress. The present
study used the 3-dimentionally (3-D) embedded chondrocytes to
examine the effect of mechanical stress or IL-4 on the expression of
type â…¡ collagen (Col.2) and aggrecan (AGC) mRNA.
Methods and Materials: Chondrocytes were isolated from rat
articular cartilage. On reaching confluence, the cells were 3-D
embedded in type I collagen scaffold. The cell-seeded scaffold was
cultured either under mechanical stress (MS group) or with IL-4 (IL-
4 group). The mechanical stress was a cyclic compression at 5%
compression, 0.33Hz for 1hours, and the IL-4 concentrations were
10ng/ml. The 3-D embedded chondrocytes with neither mechanical
stress nor IL-4 served as non-stressed (NS) cells. These groups
aside, before applying the mechanical stress, IL-4 soluble receptor
(sIL-4R) was added to medium at different concentration. Real-time
PCR was performed for Col.2, AGC, and GAPDH at 1, 7, 13, and 25
hours after the application of the mechanical stress or IL-4.
Results: Expression of AGC and Col.2 was significantly upregulated
in MS group as well as in IL-4 group when compared with that of
the NS group. The mechanical stress-induced upregulation of the
matrix synthesis was attenuated when IL-4 inhibitor was applied.
Conclusions: The present results show that IL-4 and MS influence
the matrix synthesis of the 3-D embedded chondrocytes. In turn,
the inhibition of the mechanical stress-related enhancement of
matrix synthesis by the IL-4 inhibitor strongly suggests that the
mechanical stress regulates the matrix synthesis via IL-4.
P98
17β-estradiol inhibits the activation of volume-sensitive Cl-
current by doxorubicin in isolated rabbit articular chondrocytes
K. Kumagai, F. Toyoda, S. Imai, N. Okumura, E. Isoya, Y. Matsusue,
H. Matsuura
Otsu/Japan
Purpose: Chondrocyte apoptosis contributes to the disruption of
cartilage integrity in osteoarthritis. It has been suggested that
activation of volume-sensitive Cl- current (ICl,vol) mediates cell
shrinkage triggering apoptosis (apoptotic volume decrease: AVD)
in several cell types. We examined in vitro effects of 17β-estradiol
214 Posters
on the doxorubicin-induced activation of ICl,vol in rabbit articular
chondrocytes using whole-cell patch-clamp technique.
Methods and Materials: Rabbit cartilages were collected from joints of
male animals weighing 2.5 to 3.0 kg. The cartilage was dissected into
slices and cultured in DMEM for 1-3 days. On the day of experiments,
chondrocytes were isolated by enzymatic digestion. Whole-cell membrane
current was recorded under conditions where Na+, K+ and Ca2+ currents
were minimized. Real-time change in cell size was monitored using a CCD
digital camera and the cross-sectional area of cell image was measured.
Results: Exposure of isolated chondrocytes to doxorubicin (1µM)
resulted in an obvious increase in the membrane Cl- conductance
without any appreciable change in cell size. The doxorubicin-evoked
Cl- current exhibited many properties almost identical with ICl,vol
phenotype, including outward rectification, prominent inactivation at
large positive potential, inhibition by hyperosmotic cell shrinkage, and
sensitivity to ICl,vol blockers, arachidonic acid or DCPIB . Pretreatment
of cells with 17β-estradiol inhibited the ICl,vol activation by doxorubicin
as well as subsequent apoptotic events such as AVD and elevation of
caspase 3 activity. It was unlikely that 17β-estradiol produced a direct
action on ICl,vol, because it had little effect on ICl,vol activated by
hyposmotic cell swelling. On the other hand, the effect of 17β-estradiol
was significantly attenuated by an estrogen receptor blocker.
Conclusions: These results suggested that 17β-estradiol may prevent
the doxorubicin-induced apoptosis by interfering the activation of
ICl,vol in rabbit articular chondrocytes.
P100
Chondrocyte micro-aggregates enhances neo-cartilage formation
L. Moreira Teixeira1, J. Leijten1, J. Sobral2, R. Jin1, P. Dijkstra1, J.
Feijen1, C. van Blitterswijk1, M. Karperien1
1
Enschede/Netherlands, 2Cambridge/United Kingdom
Purpose: We recently developed a novel injectable in situ crosslinkable
Dextran-Tyramine 14kDa-DS=15 (Dex-TA) hydrogel, which has shown
high potential for cartilage regeneration. However, cartilage repair
strategies based on autologous chondrocyte implantation still rely on in
vitro expansion to obtain sufficient cells with all inherent drawbacks such
as dedifferentiation. Cartilage repair using mixtures of a limited number
of chondrocytes with Mesenchymal Stem Cells (MSCs) can potentially
overcome this hurdle. It remains unclear whether the hydrogels should
be seeded with single-cell suspension or with preformed micro-
aggregates of defined size. To test, this we have seeded hydrogels
with microaggregates of chondrocytes, MSCs or a mixture of both and
compared their performance with single-cell seeded hydrogels.
Methods and Materials: High throughput formation of micro-
aggregates of 50, 100 and 200 cells was achieved in micromolds. Micro-
aggregates were prepared of chondrocytes, MSCs or a mixture of both.
Morphology, stability and chondrogenic capacity was evaluated.
Aggregates with the optimal cell density of 100 cells were incorporated
into Dex-TA hydrogels, cultured in vitro and in vivo and compared to
single-cell seeded hydrogels.
Results: Micro-aggregates were formed in a very controlled manner
and successfully incorporated into Dex-TA hydrogels. Aggregates
formed by 100 cells showed a superior balance between stability and
gene expression profile, with higher collagen type2 and Aggrecan
expression. After incorporation of micro-aggregates into Dex-TA, long
term stability and survival was observed, as well as enhanced matrix
production, when compared to single-cell seeded hydrogels. More
cartilage was formed in micro-aggregates consisting of a 50%/50%
mixture of chondrocytes and MSCs, compared to both micro-aggregates
of pure cell populations and to single-cell mixtures.
Conclusions: We conclude that neocartilage formation is greatly improved
by seeding hydrogels with micro-aggregates instead of single-cell
suspensions. In addition, this system provided preliminary information
about the effect of micro-cocultures, showing enhancement of neo-
cartilage formation when using 50% chondrocytes mixed with 50% MSCs.
P101
The effects of non-steroidal anti-inflammatory drugs on
chondrocytes
C. Hsieh, J. Tsai-Wu, S. chen, H. Chiang, C. Jiang
Taipei/Taiwan
Purpose: Osteoarthritis (OA) is a painful disease with degenerating
cartilage matrix components. For OA treatment, non-steroidal anti-
inflammatory drugs (NSAIDs) are the most frequently prescribed
medications to relieve pain and reduce inflammation. The effects
of NSAIDs on cartilage matrix remain controversial. To elucidate the
effects of NSAIDs on OA treatment, we evaluated the effect of the
selective cyclooxygenase-2 (COX2) inhibitors aceclofenac, celebrex
and tiaprofenic acid, and non-selective COX inhibitor indomethacin
on extracellular matrix of human chondrocytes in vitro.
Methods and Materials: Human chondrocytes were isolated from
surgical specimen of OA patients. The chondrocytes were cultured
in the normal medium (as a control group) or in the presence of
aceclofenac, celecoxib, tiaprofenic acid and indomethacin for 7
days to examine the change in cell proliferation. The components
of extracellular matrix of chondrocytes such as aggrecan and
collagens were measured after 3-day treatment. The expressions
of type I, II, and X collagens and aggrecan were determined by
quantitative RT-PCR. The amount of total glycosaminoglycan (GAG)
was measured by 1,9-dimethylmethylene blue (DMMB) binding.
Results: There is no significant difference in the cell proliferation
rates in the presence or absence of NSAIDs for 7 days. The
expressions of type I, II, and X collagens are reduced in the presence
of the tested NSAIDs. However, the expression of aggrecan are
significantly increased in the presence of aceclofenac, celecoxib
and tiaprofenic acid. In addition, aceclofenac significantly
increased total GAG production compared to those of celecoxib,
indomethacin, tiaprofenic acid and control.
Conclusions: These results suggest that NSAIDs could affect the
expression and production of the extracellular matrix of human
chondrocytes, including increasing glycosaminoglycan and
aggrecan, and decreasing collagens. Aceclofenac has the most
prominent effect in increasing total GAG production. The effects
of these changes in extracellular matrix components on physical
property of chondrocytes need further evaluation.
P102
TGFβ-1 administration on serum free-expanded chondrocyte
enhances the expression of osteogenic markers and induces
SMADs transcript regulation: in-vitro and in-vivo studies.
R. Narcisi, R. Quarto, P. Giannoni
Genova/Italy
Purpose: Culturing articular chondrocytes is required to perform
cartilage resurfacing in tissue engineering-based approaches
the use of serum-free medium (SF) can opportunely mimic the
physiological (avascular) autocrine environment. Previously
data showed that TGFdministration during the expansion phase
induces loss of matrix components, positive immunodetection for
type-X Collagen and apoptosis once in 3D culture system and, after
osteogenic induction, TGF-expanded cells strongly mineralized
with respect to SF-expanded cells. Thus we evaluated if TGF&beta-1
administration influence the expression of osteogenic markers
and SMADs, signalling proteins involved in the chondrogenic
development and differentiation. Moreover we evaluated the
behaviour of TGF-expanded cells in an in-vivo ectopic model.
Methods and Materials: Human articular chondrocytes were
expanded in SF, with or without TGFβ-1. Subsequently cells were
collected and seeded statically in hydroxyapatite (HA) scaffold
and after 48h in osteogenic induction medium, implanted
subcutaneously in nude mice for the following four weeks.
Decalcified sections were assessed by mean of haematoxylin-eosin
immunocitostaining. Microarray and Real time-PCR analysis were
performed after the expansion phase.
Results: TGF-expanded cells displaying an increased transcripts
levels for Osteopontin (OP; 4.76±2.24, [mean±SD]), Bone
Sialoprotein (BSP; 2.76±0.03) and Collagenase-3 (MMP13;
6.25±3.18), while Osteocalcin (OC) wasn’t influenced by TGFβ-1
(1.02±0.28 fold-increase). Moreover, microarray analysis showed
that TGFβ-1 administration influenced the SMADs expression.
Qualitative-PCR analysis confirmed that treated cells displayed
increased level of SMAD-1 mRNA (1,77±0.26) and down-regulated
SMAD-3 transcript (0.35±0.28). Interestingly, after four weeks in-
vivo TGF-chondrocyte/HA implant showed nascent deposition of
bone matrix and numerous blood-vessels.
Conclusions: TGFβ-1 directs chondrocytes to acquire a different
phenotype, firstly regulating the expression of SMADs signalling
pathways. These cells showed high level of OP, BSP and MMP-13
mRNA indicating the predisposition to undertake the endochondral
ossification-like progression and in-vivo analysis seem to validate
this hypothesis.

P103
Salvage of Contaminated Osteochondral Allografts: The Effects of
Chlorhexidine on Human Articular Chondrocyte Viability
J. Campbell1, G. Filardo2, S. Bajaj1, N.A. Friel1, A.A. Hakimiyan1, R.
Grumet3, S. Chubinskaya1, B.J. Cole1
1
Chicago/United States of America, 2Bologna/Italy, 3Orange/United
States of America
Purpose: Osteochondral allograft (OA) transplantation procedures
carry the risk of graft contamination. Because chondrocyte
viability is imperative for successful transplantation, graft
sterilization techniques must provide antimicrobial effects with
minimal to no cartilage toxicity. Chlorhexidine is an effective
disinfectant, but its use with human articular cartilage requires
further investigation. The purpose of this study was to determine
the maximal chlorhexidine concentration that does not affect
chondrocyte viability in allografts.
Methods and Materials: 6mm osteochondral plugs were
harvested using OATS technique (Arthrex,Inc) from five femoral
condyles obtained from AlloSource. Plugs were subjected to
pulse lavage using 1L solutions of 0.002%, 0.01%, 0.05%, and
0.25% chlorhexidine gluconate (CHG) preceded and followed by
1L saline pulse lavage. Treated plugs were cultured for 0,1,2 and 7
days in media containing 10% fetal-bovine-serum and antibiotics.
Two controls, an osteochondral plug immediately cultured after
harvest and a plug subjected to saline pulse lavage before culture,
were included. Chondrocyte viability was determined using LIVE/
DEAD Assay.
Results: Both controls and 0.002% CHG group showed similar cell
viability ranging from 67±4% to 81±22% at all time points. In 0.01%
CHG group, cell viability was reduced in comparison to control
by 2-fold at Day 2 and remained at this level till Day 7 (p<0.01).
0.05% and 0.25% CHG groups showed a 2-fold reduction in cell
viability already at Day 1 (p<0.01). At Day 7, cell viability was
reduced to 15±18% (4-fold) for the 0.05% CHG group and 10±19%
(6-fold) for the 0.25% CHG group, (p<0.01).
Conclusions: 0.002% CHG pulse lavage does not cause significant
cell death within 7 days following exposure, while CHG at
concentrations greater than 0.002% significantly decreases
chondrocyte viability within 1-2 days after exposure and therefore,
should not be used for disinfection of OA. The antimicrobial
properties of 0.002% CHG and its effect on OA metabolism remain
to be investigated.
P104
Cartilage damage treated with Chondron™ method
R. Benink, C. Pronk-Admiraal
Den Helder/Netherlands
Purpose: Several treatment methods are described for post-
traumatic and chronic degenerative cartilage lesions of the knee,
for example arthroscopic debridement, microfracturing, Becks
drilling, osteochondral transplantation (OATS) and 1st generation
autologous chondrocyte implantation (ACI) and 2nd generation
MACI. For the short term results and safety of the Chondron™
(3rd generation cultivated chondrocyte-gel technology) method a
prospective cohort study was evaluated by ICRS criteria and KOOS
scoring lists were used.
Methods and Materials: Data from the first 160 consecutive
patients were prospectively collected. Among them were 45
patients with a solitary medial condyle lesion(17 men, 28 women).
Average age was 41 years (range: 18-60, SD ± 10), mean BMI was
26 (range: 20-37) and the average size of the chondral defect of the
medial condyle was 6.5 cm2 (range: 2-16cm2; SD ± 3.5). Average
number of cells per vial cultivated was 13.213 x 106 cells. Patients
are under supervision of a physiotherapist for 6 to 12 months. Data
was analyzed with PASW v18.
Results: Clinical evaluations using the IKDC and KOOS score were
processed at preoperative, 3, 6 and 12 months. Table 1 and graphic
1 shows the mean scores of the patients. The Wilcoxon signed
ranks test showed statistical significant improvement at 3, 6 and
12 months. *= statistically significant p <0.05.
Conclusions: These first results of the Chondron™ method for the
treatment of articular cartilage defects of the knee are promising.
The medial condyle lesions showed a significant improvement at 1
year, although we realize that this is only short term results.
Posters 215
P105
Tissue engineering cartilage tissue using elderly human articular
chondrocytes
X. Zhao1, D.A. Bichara1, F. Ballyns2, M.A. Randolph1, L. Bonassar2,
T.J. Gill1
1
Boston/United States of America, 2Ithaca/United States of
America
Purpose: There are many unknown aspects using human
chondrocytes from middle aged or elderly patients for neo-cartilage
formation and lesion repair. We hypothesized that human articular
cartilage can be engineered using human chondrocytes from
these patient populations that would be similar morphologically,
biochemically and biomechanically to juvenile animal neo-
cartilage.
Methods and Materials: Articular chondrocytes from elder healthy
human donors (age range: 50-60 years) and young swine (~3
months) were isolated and expanded in culture. Human and swine
Cells (40-60 x 106 cells/ml) were encapsulated in fibrin gel nodules
(n=30) to determine engineered cartilage formation. To evaluate
cartilage integration, 100ul chondrocyte/fibrin hydrogel mixture
was sandwiched between two 6mm discs of native cartilage (n=36).
All constructs were implanted into nude mice subcutaneously
for 12, 18, and 24 weeks. The specimens were evaluated and
compared histologically, immunohistochemically, biochemically
and biomechanically.
Results: Similar to the swine cell constructs, the nodules with
human cells showed continuous neo-cartilage matrix formation as
evidenced in specimens stained with H&E, Safranin-O, Toluidine
Blue, and immunohistochemical stain (type II collagen). Also neo-
cartilage formed between cartilage discs in the sandwich model
with human articular disks and human chondrocytes showed tight
bonding and equivalent integration strength (stress was 90±26 kPa)
with existing matrix. Biochemical and biomechanical data showed
a similar performance between human and swine chodrocytes in
engineering cartilage tissue.
Conclusions: These results demonstrate that chondrogenesis and
integration using articular chondrocytes from older aged people
can be achieved in a predictable and reliable manner similar to that
from juvenile animals. Since most patients with articular cartilage
lesions are quite often middle aged or older, these results move us
closer to possible clinical application.
P106
Inhibition of TGF-ß prevents dedifferentiation in monolayer
cultured sheep chondrocytes
P. Ripalda Cemboráin, A. Zabalza-Baranguá, M. Vicente-Pascual, J.
Dotor, I. Monreal, F. Borrás-Cuesta, G. Mora, I. Izal Azcárate
Pamplona/Spain
Purpose: Expansion of chondrocytes in vitro can yield a great
number of cells but with a depleted capacity of chondrogenesis.
TGF-beta is a strong inductor of chondrocyte phenotype, but its
role in cartilage is not clear. In this work we analyzed the effect of
two peptides (P17 and P144) with capacity to inhibit the activity of
TGF-beta in cartilage cells.
Methods and Materials: Sheep chondrocytes were cultured in
the presence of peptides for up to 4 passages. Apoptosis and
proliferation rate were assayed after a 24 hours treatment, while
RT-PCR for cartilage markers were assayed at passages 2 and
4. Chondrogenic capacity was determined by placing cells in a 3
dimensional environment for 7 additional days. Pellets formed
were analyzed by immunofluorescence and collagen II content
determined by ELISA.
Results: Results show a significant reduction of the proliferation
rate exerted by P144 but not by P17. None of them results toxic.
Only P144 is able to reduce the TGF-beta induced phosphorilation of
SMAD2, and a better expression of cartilage markers during the first
passages. P144 increased the size of the micromass structure used
for the analysis of chondrogenesis, as well as the type II collagen
content measured by ELISA. The histological characterization of
the pellets showed an increase in the proportion of type II collagen
after the expansion with P17 and an increase in the detection of
type II collagen and aggrecan after the expansion with P144.
Conclusions: Inhibition of TGF-beta using peptides P17 and P144
in the monolayer culture preserves the chondrogenic capacity of
expanded chondrocytes.
216 Posters
P107
Identification of hyaline and fibrous chondrocytes using a novel
genetic marker
A. Campos, I. Garzón, J. Garrido, V. Carriel, C. Martínez, M..S.
Alaminos, M. Alaminos
Granada/Spain
Purpose: Autologous chondrocyte cultures are currently used for
the treatment of several conditions affecting the human cartilage.
It is well known that chondrocytes kept in culture for long periods
of time may tend to lose differentiation capabilities, and no specific
chondrocyte markers have been described to the date. In this work,
we have evaluated primary cultures of hyaline and fibrous human
chondrocytes using genome-wide microarrays to identify the gene
expression of a novel chondrocyte marker in both cell types.
Methods and Materials: Primary cell cultures of human hyaline
chondrocytes were obtained from small biopsies of the knee joint
using collagenase II digestion and of human fibroblasts from oral
mucosa biopsies. Then, RNA was extracted from the cell cultures
and the RNA expression of the gene SUSD2 (sushi domain-
containing 2) was quantified by using Affymetrix Human-Genome
U133 plus 2.0 arrays.
Results: Our analysis revealed that the gene SUSD2 was highly
expressed by both hyaline and fibrous human chondrocytes
cultures, but not by other cells of mesenchymal origin (p<0.001
for both comparisons) (Figure 1). In addition, the expression of
this gene was significantly higher in hyaline chondrocytes than in
fibrous chondrocytes (p<0.001).
Conclusions: These results suggest that expression of the gene
SUSD2 is highly specific of human chondrocytes and could be
used as a novel marker of cartilage cells. Additionally, this marker
allows for an efficient ex vivo distinction of hyaline versus fibrous
chondrocytes to be used in cell and tissue engineering protocols.
Supported by G.I. PAI CTS-115 (Tissue Engineering Group).
P108
Age-related change of collagen in patellar cartilage of normal
rabbits: FTIR and histology
H. Kuroki, M. Kobayashi-Miura, K. Tsuchimoto, A. Ito, H. Inoue,
M. Kobayashi, K. Nishitani, T. Shirai, T. Satake, Y. Nakagawa, T.
Nakamura
Kyoto/Japan
Purpose: Fourier transform infrared spectrometer (FTIR) microscope
has been used for a novel tool to assess cartilage matrix content.
The purpose of this study is to determine whether FTIR could
evaluate age-related change of patellar cartilage from surface to
deep layer, especially more detailed distribution of collagen in
combination with histological information.
Methods and Materials: Five groups of rabbits of various ages
(3-week, 8-week, 6-month, 1-year, 2.5-year) consisting of three
rabbits per group were examined. Patellar cartilage samples were
sectioned and analyzed by FTIR. Collagen (amide-I peak; 1710-1590
cm-1) was evaluated with collagen II immunostaining. The amide-I
peak was measured with five depths of cartilage, from surface to
100-, 200-, 300-, 400-, 500-micrometer, respectively.
Results: Mean intensity of amide-I peak in the depth from surface
to 100-micrometer of 3-week, 8-week, 6-month, 1-year and 2.5-
year samples were 27.5, 45.1, 42.1, 47.0 and 49.5 (arbitrary unit),
respectively. Those were 31.3, 43.0, 40.9, 46.4 and 45.8, respectively,
in the depth from surface to 200-micrometer; 33.6, 43.4, 41.2, 44.1
and 42.6, respectively, in the depth to 300-micrometer; 33.4, 43,4,
40.9, 42.5 and 41.5, respectively, in the depth to 400-micrometer;
and were 30.9, 43.8, 41.4, 42.0 and 41.0, respectively, in the depth
to 500-micrometer. Amide-I peak distribution was consistent with
the localization of collagen-II.
Conclusions: Mean intensity of amide-I peak in the 3-week
specimens differed from those of the others. We suppose that,
combining FTIR with collagen II immunostaining, it is feasible to
determine the age-related change of collagen of patellar cartilage
from surface to deep layer.
P109
TGF-beta protects OA chondrocyte mitochondria from
experimentally induced oxidative stress.
V. Grishko1, A.W. Pearsall, IV2, G.L. Wilson2
1
36688/United States of America, 2Mobile/United States of
America
Purpose: TGF- beta is a powerful anabolic factor for chondrocytes
and plays an important role by enhancing cartilage repair. Oxidative
stress and mitochondrial damage have been found to promote cell
death, functional failure and degeneration. There is a growing body
of evidence that mitochondrial dysfunction is present during the
progression of osteoarthritis. The purpose of the present study
was to evaluate the mitoprotective potential of TGF-beta for human
OA chondrocytes in the conditions of oxidative stress.
Methods and Materials: Primary OA chondrocytes cultures,
generated from cartilage from patients undergoing total knee
replacement, were exposed for 30 min to reactive oxygen species
(ROS) generators xanthine oxidase/ hypoxanthine or peroxynitrite.
Some cells were pre-incubated with 5 ng/ml of TGF-beta for 24 h
prior to treatment. In some experiments cells were incubated for 48
h with IL-1 beta or TNF-alpha alone or in combination with TGF-beta.
Following exposure, cells were immediately lysed for DNA or protein
isolation or ATP analysis or replenished with normal growth media
and left for recovery and then analyzed. Mitochondrial dysfunction
was assessed in terms of mitochondrial DNA damage (Quantitative
Southern blot analysis), ATP content (Bioluminescence kit),
mitochondrial proteins levels (Western blot). Cell viability and
apoptosis were evaluated by flow cytometry.
Results: When OA chondrocytes were exposed to ROS generators,
mitochondrial dysfunction and apoptosis were accumulated as a
consequence. Pretreatment with TGF- beta preserved mitochondrial
DNA integrity and ATP levels, restored drop in mitochondrial protein
levels. Moreover, TGF-beta enhanced chondrocytes viability and
diminished the appearance of apoptosis. The similar results were
obtained for IL-1 beta and TNF-alpha.
Conclusions: The present results demonstrate for the first time the
mitoprotective properties of TGF-beta and offer some explanation
of the mechanisms through which TGF-beta enhances cartilage
repair. More studies required evaluating the precise mechanisms
of observed effects.
P111
The effects of osteogenic protein (OP)-1 on microfracture-treated
cartilage defects in a goat model
L. Jeng1, H. Hsu2, R.F. Padera2, M. Spector2
1
Cambridge/United States of America, 2Boston/United States of
America
Purpose: The benefits of osteogenic protein (OP)-1 in promoting
chondrocyte metabolism in vitro commend it for cartilage repair.
The objective was to evaluate the effects of OP-1 on the amount
and composition of the reparative tissue in microfracture-treated
defects in a goat model.
Methods and Materials: Two 4-mm chondral defects were created in
the trochlear groove of right knees and treated with microfracture,
and immobilized for 8 days. Treated knees (Group II) were injected
with 1 mg of OP-1 in 1 ml of vehicle 3 times: at defect creation, and at
8 and 20-21 days, post-op. Control knees received the vehicle alone
(Group I). After sacrifice, the joints were radiographed. Formalin-fixed,
paraffin-embedded sections were evaluated histomorphometrically
for the percentage fill and types of tissues filling the defect.
Results: Radiographs and histology revealed the presence of
ossicles in the synovial capsules of three of six 16-week Group
II goats as well as in one of four 16-week Group I animals (no
significant difference by Fisher’s Exact test). Histomorphometric
analysis (Table 1) indicated that the percentage of the defect site
that was filled with reparative tissue was much higher for the OP-1
treated group (62 ± 17%, mean ± SEM) than for the control group
(19 ± 5%) 4-5 weeks postoperatively. However, by the 16-week
timepoint, the total fill was similar between the 2 groups (75 ± 6%
for Group I and 80 ± 10% for Group II). Two-factor ANOVA of the
16-week defects indicated that OP-1 treatment had no significant
effect on the percentages of hyaline cartilage, fibrocartilage,
fibrous tissue, bone ingrowth, or total fill.
Conclusions: This study suggested that high dose treatment with
OP-1 may be beneficial during early stages of cartilage repair
but found no significant advantage of the OP-1 treatment on
microfracture-treated cartilage defects in a caprine model by the
16-week timepoint.

P112
IL-1ra solution blocks IL-1β and TNFα-induced MMP-13
production from human articular chondrocytes
J. Woodell-May, A. Matuska, J. Hoeppner
Warsaw/United States of America
Purpose: Inflammatory cytokines such as IL-1β and TNFα can induce
chondrocytes to produce matrix metalloproteinases (MMPs) which are
responsible for degradation of cartilage matrix (1). Autologous protein
solution (APS) rich in IL-1ra can be prepared in less than 30 minutes
from blood collected during a draw (2). The purpose of this study is to
determine if incubation with APS, derived from human blood and rich
in IL-1ra, can decrease the production of MMP-13 from IL-1β and TNFα
stimulated chondrocytes.
Methods and Materials: APS was prepared from 10 consented human
donors. Human knee articular chondrocytes (NHAC, Lonza Inc.) were
seeded in 12 well plates. For the positive controls and treatments, 5ng/
ml rhIL-1β (Sigma) and 100 ng/ml rhTNFα (Prospec) were added to the
wells. APS, rhIL-1ra, or rhsTNF-RI were added 2 hours prior to the addition
of IL-1β and TNFα. Wells without IL-1β, TNFα, or APS were left as negative
controls. After 24 hours, the supernatant was removed and frozen at -50°
C. The supernatant was assayed for MMP-13 by ELISA (R&D Systems).
Results: Chondrocytes stimulated with TNFα, IL-1β, and the combination
of TNFα and IL-1β, produced 104, 403, and 565 fold more MMP-13,
respectively, than negative control chondrocytes. Recombinant IL-1ra
and sTNF-RI blocked MMP-13 production in stimulated cells. Treatment
with APS reduced MMP-13 production of the respective samples by
90.9±9.7%, 92.9±3.3%, and 89.7±4.4%.
Conclusions: Articular chondrocytes dosed with known inflammatory
cytokines and treated with APS demonstrated a reduction in MMP-13
production when compared to untreated samples. The results emphasize
the role of IL-1β and TNFα in the breakdown of cartilage matrix seen in
osteoarthritis and warrant further studies to investigate a potential
treatment for patients suffering from OA. 1.) Goldring MB et al., Arthritus
Rheum, 2000; 43(9); 1916-26. 2.) Vangsness, T. et al., ORS, 2008.
P113
Gene profiles of the regenerated cartilage tissue induced by
implantation of a novel double-network hydrogel
R. Imabuchi, H.J. Kwon, N. Kitamura, T. Kurokawa, J.P. Gong, Y.
Ohmiya, K. Yasuda2
Sapporo/Japan
Purpose: We have developed an innovative method to induce
spontaneous hyaline cartilage regeneration in vivo by implanting a
double-network (DN) hydrogel composed of poly-(2-Acrylamido-2-
methylpropanesulfonic acid) and poly-(N,N´-Dimetyl acrylamide).
The purpose of this study is to investigate the gene expression
profiles in the regenerated cartilage tissue induced by this DN gel
in comparison with normal cartilage.
Methods and Materials: Ten mature Japanese white rabbits
were used. An osteochondral defect having a 4.5-mm diameter
was created in the femoral groove. A cylindrical DN gel plug was
implanted into the defect having 2-mm depth remained after
surgery. Five rabbits were sacrificed at 2 (Group-2W) and 4 (Group-
4W) weeks after surgery, respectively. Regenerated cartilage
in the defect were collected and mRNAs were isolated. The gene
expression profiles were analyzed with use of custom-made DNA
microarray. The normal knee cartilage of the same age of Group-2W
and -4W was used as control.
Results: The number of the probe sets with an expression ratio
greater than 2, 5, 10 between 2W and control was 2,423, 452, and
174, respectively and that between 4W and control was 2,122, 391,
and 164, respectively. We defined a probe with the ratio greater
than 5 as differentially expressed gene for further analysis; with
respected to cartilage-related genes, Col2, Col5, Col10, Agc1, Crtl1,
Dcn, Comp, Prg4, Cilp, Ctgf, Pthr1 were upregulated in both 2W and
4W. The functional classification of the differentially expressed
gene was also performed. A similar distribution of categories was
observed in 2W and 4W when comparing to control (Fig)..
Conclusions: We first reported the gene expression profiles of the
regenerated cartilage tissue using a rabbit model. We conclude that
the gene expression profiles of the regenerated cartilage tissue in
both 2W and 4W were different from control and that of 2W and 4W
were similar to each other.
Posters 217
P114
Osteocyte-derived HB-GAM is associated with bone formation
and mechanical loading
Y. Takemura, S. Imai, Y. Matsusue
Otsu/Japan
Purpose: HB-GAM is a cell matrix-associated protein that is highly
expressed in bone. It affects osteoblast function, and might
therefore play a role in bone development and remodeling. We aimed
to investigate the role of HB-GAM in bone in vivo and in vitro.
Methods and Materials: We used the mice lacking HB-GAM in
C57BL/6 background and the C57BL/6 mice for control. The bones
of HB-GAM deficient mice with an inbred mouse background
were studied by histological, histomorphometrical, radiological,
biomechanical and μ-CT analyses and the effect of immobilization
was evaluated. HB-GAM localization in vivo was studied. MLO-Y4
osteocytes were subjected to fluid shear stress in vitro, and gene
and protein expression were studied by subtractive hybridization,
quantitative PCR and Western blot. Human osteoclasts were
cultured in the presence of rhHB-GAM and their formation and
resorption activities were assayed.
Results: The skeletal structure of the HB-GAM knockout mice
developed normally. However, a growth retardation of the weight-
bearing bones was observed between 3weeks and 2 months
of age. This suggests a link to physical activity. Adult HB-GAM
knockout mice were characterized by low bone formation and
osteopenia, as well as resistance to immobilization-dependent
bone remodeling. HB-GAM was localized around osteocytes and
their processes in vivo. Osteocytic HB-GAM expression was up
regulated by mechanical loading in vitro. HB-GAM did not affect on
human osteoclast formation or resorption in vitro.
Conclusions: Our results suggest that HB-GAM could be involved in
mediating the osteogenic effects of mechanical loading on bone.
P115
Effect of intraarticular pH change on structure and metabolism of
cartilage tissue
S. Ergün, B. Kocaoğlu, U. Akgün, R. Nuran, O. Başçı, M. Karahan
Istanbul/Turkey
Purpose: Hyalin cartilage is an avascular tissue and it is only
nourished from synovial fluid by diffusion. Decrease in pH of
synovial fluid in the presence of osteoarthritis and trauma has been
reported in previous investigations. We hereby aimed to show the
negative effects of low synovial fluid pH on cartilage metabolism
and synthesis of extracellular matrix components by Real Time
Polymerase Chain Reaction method.
Methods and Materials: Cartilage tissue obtained from bovine
femoral condyles were incubated in 3 different medias; acidic (pH
7.2), physiologic (pH 7.4) and basic (pH 7.6), for four days. At the
end of incubation, mRNA isolation and quantitative gene expression
analysis were done. Type 2 collagen, aggrecan and hypoxia
inducible factor 1 alpha were the genes investigated. Normalizing
was done with beta actin housekeeping gene by 2-ddCt method.
Results: According to results, expression levels of all genes in
physiologic pH media was higher compared to acidic and basic
media. Gene expression quantity of type 2 collagen in acidic media
was 0.739, in basic media 0.755 when compared to physiologic pH
media gene expression quantity which is 1. This ratio was 0.615
in acidic and 0.681 in basic media of gene aggrecan and 0.695 in
acidic and 0.652 in basic of gene Hif 1 alpha.
Conclusions: Conclusion, decrease in synovial fluid pH as a
consequence of osteoarthritis, trauma or intraarticular hyaluronic
acid injection, negatively effects chondrocyte metabolism and
production of extracellular matrix components.
P116
Recombinant human fibroblast growth factor-18 combined with a
biphasic collagen/GAG scaffold produces superior articular cartilage
compared with BMP-7 in an ovine osteochondral defect model
A. Getgood1, F.M. Henson1, R. Brooks1, A.K. Lynn1, H. Guehring2, N.
Rushton1
1
Cambridge/United Kingdom, 2Darmstadt/Germany
Purpose: The aim of this study was to investigate the effect of
combining rhFGF18 with a biphasic collagen/GAG osteochondral
scaffold (Chondromimetic), on the treatment of osteochondral
defects in sheep.
218 Posters
Methods and Materials: Osteochondral defects (5.8x6mm) were
created in the medial femoral condyle (MFC) and the lateral trochlea
sulcus (LTS) of the stifle joint of 24 sheep. Sheep were randomly
assigned to four groups in the study (n=6); 1) empty defect, 2)
scaffold only, 3) scaffold + rhFGF-18 (30µg) and 4) scaffld + BMP-7
(100µg). At 6 months the defects underwent mechanical testing,
gross assessment of the repair tissue (ICRS score) and histological
analysis (Modified O’Driscoll score).
Results: ICRS gross repair score (Fig.1): Defects treated with
rhFGF18 (mean 9.83, 95% CI 8.43-11.23) and BMP-7 (10, 9.06-
10.94) in the MFC had significantly improved ICRS repair scores
compared to empty defects (4.2, 0-8.80) (p=0.002). Mechanical
properties: BMP-7 treated defects (mean 64.35, 95% CI 56.88-
71.82) were significantly less stiff than both the rhFGF18 (mean
84.1, 95% CI 76.8-91.4) and empty defects in the LTS, compared
to both contralateral limb (p=0.003), and the perilesional articular
cartilage (p<0.001). Histology (Fig.2): Statistically significant
improvements in the modified O’Driscoll score were observed in the
rhFGF18 treated group (mean 16.83, 95% CI 13.65-20.61) compared
to the empty defects (mean 9, 95% CI 4.88-13.12) (p=0.039).
Excellent tissue fill, lateral integration and proteoglycan staining
was observed. Only the rhFGF18 defects showed pericellular type
VI collagen staining with positive type II collagen and reduced
positive type I collagen staining indicative of hyaline like repair
tissue. The majority of defects in the control and BMP-7 groups’
demonstrated fibrocartilagenous repair tissue.
Conclusions: Statistically significant improvements in gross repair,
mechanical properties and histological score were found over empty
osteochondral defects when Chondromimetic was combined with
rhFGF18. These results suggest that rhFGF18 may play a significant
role in articular cartilage repair applications.
P119
Inhibition of TGFß to repair articular cartilage in a sheep model
R. Escribano, P. Ripalda Cemboráin, P. Sanz-Ramos, A. Zabalza-
Baranguá, J. Dotor, I. Izal Azcárate, G. Mora
Pamplona/Spain
Purpose: Repairing of lesions in cartilage is an issue of interest
for orthopaedic surgeons, as the fully regeneration of the tissue
has not been yet acomplished. TGF-beta is a growth factor with a
well described anabolic activity in cartilage. Interestingly, results
in our laboratory have shown that inhibition of TGF-beta using
peptide P144 brakes the loosing of the chondrogenic capacity of in
vitro expanded chondrocytes. The aim of this study is to evaluate
the effect of the inhibition of TGF-beta has in an in vivo model of
cartilage repairing using a commercially available system.
Methods and Materials: A model of osteochondral lesions was
performed in the load bearing area of internal femoral condyle of
healthy sheep. The peptide was loaded in TruFit cylinders (Smith
& Nephew) by centrifugation, resuspended in PBS and implanted
in the right knee according to the guidelines of the manufacturer.
Left knee was used as a control and implanted with the cylinder
embedded in PBS. After 3 months animals were sacrificed
and samples obtained analyzed by gross appearance and by
histomorphometry.
Results: The gross aspect of the lesioned cartilage show an
improvement of the tissue covering the lesioned area in lesions
treated with the peptide. Histological analysis confirms these
results and showed an improvement of the repairing tissue in knees
that received P144 with respect to untreated knees.
Conclusions: Inhibition of TGF-beta unsing peptide P144 must be
taken into account for the development of new strategies for the
repairing of cartilage lesions.
P120
Collagen type II evaluation of the articular tissue repair treated
with autologous plasma rich in growth factors (PRGF).
C.I. Serra1, C. Soler2, J.I. Redondo1, J.M. Carrillo1, J.J. Sopena1, R.
Cugat2
1
Valencia/Spain, 2Barcelona/Spain
Purpose: To value the autologous plasma rich in platelets
application, in the type II collagen formation of tissue repair in
chondral deffects.
Methods and Materials: 36 Californian rabbits were divided in 3
groups based on the treatment that they would receive (physiologic
saline serum –PCB-, activated plasma rich in growth factors –PRGF-
and healthy cartilage –CTR-). At the same time, they were subdivided
in two subgroups based on the time of study (5 postsurgical weeks
-16s- and 8 postsurgical weeks -19s-). The PRGF and PCB rabbits
were subjected to a complete thickness chondral defect in the
medial femoral condyle of both knees. After this, the therapeutic
infiltrations were begun according to the protocol established for
each group. The presence of type II collagen in the reparative tissues
was examined immunohistochemycally using a mouse monoclonal
antibody against human type II collagen (Fuji Chemical, Takaoda,
Japan) that specifically crossreact with rabbit type II collagen. The
sections were counterstained with hematoxylin. The repaired areas
were microscopically assessed according to a semi-quantitative
scale. Statistical significance among groups was estimated using a
Kruskall-Wallis Test, and a Mann-Whitney’s U test for the difference
between times.
Results: The statistical study showed no significant differences,
either among groups or between the stages of evolution, for the
collagen values.
Conclusions: The application of plasma rich in growth factors has
not produced significant differences in the synthesis of collagen
type II in the articular cartilage tissue repair, comparing with the
healthy cartilage and its physiological repair.
P121
Repeated sub-threshold dosing of TGF-β1 is ineffective in
stimulating human bone marrow cells: implications for cartilage
repair strategies
C. Chu, V. Yao, K.A. Payne
Pittsburgh/United States of America
Purpose: Since the articular cartilage has a limited capability to heal,
improving cartilage repair is an important strategy to reducing pain
and disability. Transforming growth factor-beta 1 (TGF-β1) induces
chondrogenesis of human bone marrow cells (hBMC), an important
cell source for cartilage repair. This study tests the hypothesis that
(1) TGF-β1 signaling can be induced and maintained by either single
or repeated administrations in vitro, and (2) hBMC mediated cartilage
repair cells remain responsive to sustained exposure to TGF-β1 in vivo.
Methods and Materials: To test hypothesis 1, hBMC were stimulated
with single administration of 5, 10 and 15ng/mL of TGF-β1, and the
expression of TβRII and phospho-SMAD3 compared with multiple
administrations of TGF-β1. Protein was isolated at 0, 2h, 4h, 1d, 3d
and 7d after the last administration of TGF-β1. To test hypothesis
2, TβRII expression were assessed in athymic rat osteochondral
defects at 4 weeks following implantation of hBMC transduced with
AAV-GFP or AAV-TGF-β1.
Results: In vitro, TβRII expression increased significantly at 2h
post-stimulation with 15ng/mL of TGF-β1, and at 4h and 1d post-
stimulation with 10ng/mL of TGF-β1. Phospho-SMAD3 expression
increased at 2h post-stimulation with 10ng/mL of TGF-β1. However,
the expression of both proteins declined thereafter. In addition,
multiple administrations of 10ng/mL of TGF-β1 increased TβRII and
phospho-SMAD3 expression up to 7 days post-stimulation.
In vivo, TβRII is strongly expressed throughout the repair tissue of
osteochondral defects receiving hBMC expressing TGF-β1, but not
those receiving hBMC expressing GFP.
Conclusions: The data show that the effects of single
administration of TGF-β1 are transient suggesting that repetitive
administration of 10ng/mL of TGF-β1 may be required to sustain
cellular responsiveness. In addition, cartilage repair cells remain
responsive to repeated exposure to TGF-β1 in vivo. The implication
for in vivo repair strategies are that sustained exposure of hBMC to
TGF-β1 is critical and can be achieved using AAV.
P122
The effect of formalin fixation on Equilibrium Partitioning of
an Ionic Contrast with microcomputed tomography (EPIC-µCT)
imaging of osteochondral samples
K.E.M. Benders1, J. Malda1, D.B. Saris1, W.J.A. Dhert1, R. Steck2, D.W.
Hutmacher3, T.J. Klein2
1
Utrecht/Netherlands, 2Kelvin Grove/Australia, 3Brisbane/Australia
Purpose: EPIC-µCT is a non-invasive technique to quantify and
visualize the three-dimensional distribution of glycosaminoglycans
(GAGs) in cartilage. While this technique provides valuable high-
resolution 3D data for fresh tissues, sample preparation and
imaging are time-intensive and limit the number of samples that can
be analysed at one time-point. Fixation may help overcome these

issues; however, the effects of fixation on EPIC-µCT outcomes are
unknown. This study aimed to determine whether formalin fixation
of bovine cartilage affects x-ray attenuation, and therefore the
interpretation of EPIC-µCT data.
Methods and Materials: Osteochondral samples from bovine
trochlear grooves were incubated with ioxaglate, an anionic
contrast agent, for 22 hours prior to µCT scanning. Samples were
scanned in both fresh and formalin-fixed conditions, and samples
scanned fresh were also re-scanned after fixation (n = 8 per group).
Wet weight, dry weight, and GAG content were determined for fresh
and fixed samples (n = 5). Data were analyzed by ANOVA, with
significance determined by p < 0.05.
Results: The expected zonal distribution of contrast agent/GAGs
was observed for both fixed and fresh cartilage specimens. However,
the output range required for visualization of this distribution varied
between fresh and fixed specimens (A). Correspondingly, average
attenuation levels of formalin-fixed cartilage were significantly
lower (14.3%) than in fresh samples (B, p < 0.001). Despite the
difference in attenuation, there were no significant differences in
wet weight or dry weight (C), or GAG concentration (D,E) between
fixed and fresh samples.
Conclusions: Formalin fixation reduces the attenuation of EPIC-
µCT imaged cartilage. This reduction in attenuation is not due to
changes in volume or GAG concentration following fixation. EPIC-
µCT remains useful for studying GAG distributions in fixed tissues,
such as archival samples, but the reduced attenuation should be
taken into account when quantifying GAG by using standard curves
specific for fixed cartilage.
P124
Omega-3 (n-3) Polyunsaturated Fatty Acids in Regulating Disease
in a Spontaneous Model of Osteoarthritis
J. Tarlton, L. Knott
Bristol/United Kingdom
Purpose: The aim of this study was to examine effects of a
high omega-3 (n-3) poly-unsaturated fatty acid (PUFA) diet on
development of osteoarthritis (OA) in a spontaneous guinea
pig model, and to further characterise the model in terms of its
pathogenesis. Modern diets low in n-3 PUFAs have been linked with
increases in inflammatory disorders. It may be that n-3 is beneficial
in osteoarthritis, as this too has an inflammatory component.
However, n-3 also increases bone density, and this may be a
contributing factor in osteoarthritis. Therefore net effects of n-3 on
OA are not known.
Methods and Materials: The OA-prone Dunkin Hartley (DH)
Guinea pig was compared with the OA-resistant Bristol Strain-2
(BS2), each fed standard or n-3 diets (10-30wks). We examined
parameters of cartilage and subchondral bone pathology and
biochemistry, including histological scoring, collagen crosslinks,
matrix metalloproteinases (MMPs), alkaline phosphatase,
glycosaminoglycan, and total and denatured type II collagen.
Results: Most cartilage parameters in the DH strain were modified
by n-3 diet towards those seen in the non-pathological BS2 strain.
Those reaching significance included cartilage pathology, pro- and
active MMP-2, collagen hydroxylation and lysyl-pyridinoline. The
only exception to this trend was proMMP-9 which increased with n-3
but was lower in the BS2. GAG content was higher in the n-3 group,
but not significantly so in the BS2s. There was no difference in total
or denatured type II collagen. All subchondral bone parameters
in the DH n-3 group also changed towards those seen in the non-
pathological strain, of these alkaline phosphatase, proMMP-9 and
calcium:phosphate ratios were significant.
Conclusions: We confirmed reduced pathology in the BS2 strain
by comparison with DH. Dietary n-3 PUFA reduced pathology in
the OA-prone strain, and changes in biochemical markers were
consistent with those associated with the non-OA strain. Omega-3
did not increase markers of pathology in either strain.
P125
Effects of Na+ Ion channel blocker Lidocaine on metabolism of
cultured rabbit articular chondrocytes
W. Qi1, X. Wei2
1
030001/China, 2Taiyuan/China
Purpose: To study the effect of Na+ Ion channel blocker Lidocaine on
apoptosis and metabolism of cultured rabbit articular chondrocytes.
Methods and Materials: Chondrocytes were isolated from the
knee joints of five two-month New Zealand White Rabbit knees
Posters 219
with sterile operation. Divided into control group and 0.2mmol / L
lidocaine group, Cell density of 2×106/ml,vitro culture. Detect the
apoptosis of chondrocytes at 24h and 48h. The GAG and Collagen type II
concentrations of supernatants were measured at 3th,6th and9th day.
Results: 0.2mmol/L Lidocaine has no effect on apoptosis of
chondrocytes (p>0.05). The secretion of GAG was inhibited by
Lidocaine at the 3th day(p<0.05) but was accelerated at the 9th
day(p<0.05); Compared with the control groupe, the secretion
of Collagen type II of Lidocaine group has reduce trend but no
Statistical differences(p>0.05).
Conclusions: 0.2mmol/L Lidocaine has no effect on apoptosis of
chondrocytes.The secretion of GAG was inhibited by Lidocaine at
Short-term culture,but accelerated at long-term culture; Lidocaine
also has no effect on the secretion of Collagen type II.
P126
The Potential for the End Stage Osteoarthritic Sclerotic Lesion to
Participate in Cartilage Repair
L.L. Johnson1, C. Verioti1, J. Gelber1, D.D. D’Lima1, M. Spector2, A.
Pittsley3
1
La Jolla, CA/United States of America, 2Boston/United States of
America, 3Okemos/United States of America
Purpose: The use of arthroscopic debridement procedures in the
treatment of degenerative arthritis of the knee remains controversial.
Reports in the literature on the end stage osteoarthritic sclerotic
lesion of the knee undergoing cartilage repair following unloading
from high tibial osteotomy indicate an intrinsic potential for
regeneration. The purpose of this study was to examine in depth the
nature of the pathology of such a lesion and explore whether there
was any potential for this lesion to participate in cartilage repair.
Methods and Materials: Specimens harvested following total knee
surgery were examined for gross pathology including staining
with Safranin O. Multiple explants of the lesion were placed in
tissue culture for three and six weeks. Gross examination and
histological examination was made after simulated arthroscopic
surgery and following each interval in culture.
Results: The pathology of the end stage osteoarthritic lesion showed
sclerotic bone, dead osteons, hypervascularity and scattered
cartilaginous aggregates. Cartilaginous aggregates Additional
observations showed multiple pitting on the sclerotic surface which
histologically was related to three events; fragmentation of dead bone,
ruptured blood vessels, and eroded aggregates. Vascular pit There were
no pathological or biological changes following the time in tissue culture,
and no outgrowth of chondrocytes from the cartilaginous aggregates.
Conclusions: The in-depth pathological evaluation showed the
end stage osteoarthritic lesion to have certain features with
potential to facilitate cartilage repair. The cartilaginous aggregates
remained unchanged in tissue culture absent the normal synovial
joint environment and therefore further testing with a different
experimental model would be necessary to establish these aggregates
as a source of cartilage regeneration. The clinical relevance of this
pathological study is that the cartilaginous aggregates remain
following arthroscopic abrasion arthroplasty and microfracture and
therefore may be participants in cartilage repair following those
procedures. The small depressions in the lesion may be a “home” for
various arthroscopic delivered cellular therapeutics.
P127
Interaction Of Human Osteoarthritic Cartilage And Synovium
M. Beekhuizen1, L.B. Creemers1, Y.M. Bastiaansen-Jenniskens2,
D.B. Saris1, W.J.A. Dhert1, G.J.V.M. Van Osch2
1
Utrecht/Netherlands, 2Rotterdam/Netherlands
Purpose: Cartilage and synovium are both affected in osteoarthritis
(OA) and are known to interact. However, a long term co-culture
model with synovium and cartilage explants have not been
described before. Such a co-culture may provide a suitable model
to study these interactions and mimic the OA joint more closely as
well as to screen new therapies for cartilage degeneration in OA.
Therefore, the aim of the study is to develop an in vitro model that
includes both synovium and cartilage.
Methods and Materials: Osteoarthritic cartilage and synovium were
cultured together or alone for 21 days. To assess viability of the synovium
immunohistochemistry, a live/dead assay, and the release of lactate
dehydrogenase (LDH) were used. For functionality of the cultured
synovium a multiplex ELISA was used. Di-methylmethylene-blue assay
was used to determine glycosaminoglycan (GAG) release and content.
220 Posters
Results: Throughout the entire culture synovium showed viable
cells by a LDH assay, a live/dead assay and immunohistochemistry
demonstrated the presence of macrophages and T-cells. Several
cytokines, which were previously demonstrated in synovial fluid
of osteoarthritic patients, were secreted by synovium tissue in
culture during the 21 days of culture. Co-culture of cartilage and
synovium enhanced GAG release and reduced GAG content after
21 days of culture compared to cartilage alone (Fig. 1). Addition of
Triamcinolone to the co-culture inhibited these catabolic effects of
synovium on cartilage.
Conclusions: From the present data, we can conclude that it is
possible to culture synovium explants for at least 21 days. A co-
culture with synovium explants enhances GAG release by the
cartilage explants, indicating the catabolic environment that is
present in OA, may be mimicked more closely in co-culture than
in single explant culture. This study indicates the applicability of
this co-culture model for use in testing new therapies for cartilage
degeneration in OA.
P128
Selection of Chondroprogenitors from Bone Marrow by Adhesion
to Chondroitin Sulfate
J. O’Sullivan1, C. Coleman2, M. Murphy1, F. Barry1
1
Galway/Ireland, 2Galway City/Ireland
Purpose: Osteoarthritis is a disease of the joints characterized
by progressive destruction of articular cartilage. As cartilage is
composed primarily of extracellular matrix (ECM) with limited
capacity for self-repair, the therapeutic application of mesenchymal
stromal cells (MSCs) offers potential to regenerate cartilage. It
was therefore hypothesized that a chondrogenic population of
MSCs within the bone marrow (BM) can be isolated by preferential
adhesion to cartilaginous ECM proteins.
Methods and Materials: Proceeding MSC isolation, culture flasks
were coated with 1mg/ml hyaluronan (HA) or chondrotin sulphate
(CS) at 4°C overnight (Figure 1). BM from the iliac crest of healthy
consenting human donors was exposed to uncoated, HA or CS
coated plates for 24hrs or 5 days before non-adherent cell removal
as per traditional methods. Confluent MSCs were passaged and
expanded on uncoated plastic. At P2, MSCs were induced towards
chondrogenesis, adipogenesis and osteogenesis.1
Results: All isolated cells retained a fibroblastic morphology with
comparable expansion characteristics. Adipogenic differentiation
was significantly increased in all ECM isolated MSCs, especially in
the early adherent HA and CS isolated cells. Osteogenic potential
was enhanced in all ECM selected MSCs especially in early adherent
CS cells. Chondrogenic differentiation was enhanced in all ECM
isolated groups, particularly in CS isolated cells (Figure 2).
Conclusions: Cartilaginous ECM molecules (HA and CS) were utilized
to isolate MSC subpopulations from BM. Cells adhering to CS had the
greatest capacity for chondrogenic differentiation, suggesting cells
expressing receptors for CS in the bone marrow may be more potent
chondroprogenitors. Specific isolation of these subpopulations for
clinical application will enable a reduction in the number of MSCs
required for clinical efficacy and tissue regeneration.
P129
Automated Planning of Computer-Guided Mosaic Arthroplasty
J. Inoue1, M. Kunz1, S. Waldman1, M.B. Hurtig2, J. Stewart1
1
Kingston/Canada, 2Guelph/Canada
Purpose: Computer-guided mosaic arthroplasty requires a time
consuming planningprocess to choose and place several osteochondral
grafts on a computermodel of the joint, in order to reconstruct the
original curvature of the articular surface over a defect. We investigated
whether a computer algorithm can achieve reconstruction plans as
good as those of an expert human.
Methods and Materials: Surface mesh models from 14 sheep knees in
original condition and three months after an impact-induced cartilage
defect were created. For each knee, two plans were made (one by an
expert human, the other by a computer algorithm) to reconstruct the
articular cartilage over the three-month defect. Each plan consisted of
donor and placement sites for three or four osteochondral grafts. The
expert human operator manually selected and placed cartilage grafts
using a computer interface. Grafts could be positioned and oriented
and had their cartilage surface tilted according to the donor site.
With the computer algorithm, a human operator outlined the defect,
outlined potential donor regions, and placed a spline surface over
the defect to represent the desired repair surface. The algorithm then
arranged a set of grafts on the defect site. Each graft was chosen from
the potential donor regions. The algorithm optimized the position and
orientation of the grafts to best fit the desired repair surface. The RMS
errors between the planned repair surfaces and the original, uninjured
surfaces were computed.
Results: The algorithm had mean RMS error of 0.25 mm (95% CI: 0.21-
0.30, min 0.12, max 0.45) and took five minutes. The expert human
achieved mean RMS error of 0.30 mm (95% CI: 0.16-0.44, min 0.08,
max 1.04) and took twenty minutes.
Conclusions: No statistically significant difference in RMS error
between the algorithm and the expert was found. The algorithm was
faster and produced surfaces with less variance.
P130
The degree of osteoarthritic alterations modulates the effects of
drugs on human articular cartilage
J. Steinmeyer, J. Kordelle, H. Stuerz
Giessen/Germany
Purpose: Several studies have implicated that tetracyclines, in addition
to their antimicrobial properties, can slow down the progression of
cartilage damage both in animal models of osteoarthritis (OA) as well as
in humans. In search for the underlying mechansims the purpose of this
in vitro study was to determine systematically (1) whether tetracyclines
influence the synthesis and release of proteoglycans (PGs), MMP-
1, -8, -13 and PGE2 from human OA cartilage, and (2) whether these
pharmacological effects are affected by the degree of OA alterations.
Methods and Materials: Full-thickness cartilage explants of the lateral
compartment of the femoral condyles were taken from OA patients
undergoing knee replacement surgery. Explants from mild (Collins
grade 0-1.5) or moderately (Collins grade >1.5-3) affected human OA
condyles were cultured separately in supplemented Ham´s F12 media
with media changes every 3-4 days. Explants were treated with 1, 10,
50 or 100 µM minocycline, doxycycline or tetracycline in the presence or
absence of rhIL-1ß. PG synthesis was determined by the incorporation
of radioactive sulfate during the final 18 h of the 11 days experiments
whereas the content of PGs were quantitated with the DMMB-assay.
MMP-1, -8, and -13 as well as PGE2 were determined with ELISAs.
Results were compared to untreated explants removed from the same
condyles (N=6).
Results: The degree of OA alterations of explants can significantly
modulate the pharmacological effects of tetracylines on cartilage
metabolism. Furthermore, doxycycline was less effective on cartilage
metabolism than minocycline, whereas tetracycline displayed no effect.
Conclusions: Our study indicate that the pharmacological efficacy of
drugs can be dependent on the clinical stage of OA.
P131
Pleiotropic Mechanism of Action for Flavocoxid: Enzymatic
Inhibition, Antioxidant Activity and Down-regulation of
Inflammatory Gene Expression
B. Burnett, L. Pillai, R. Levy
Scottsdale/United States of America
Purpose: Flavocoxid, a USFDA-regulated, prescription medical
food for the clinical dietary management of osteoarthritis (OA)
under the supervision of a physician, shows equivalent efficacy to
NSAIDs in clinical studies with fewer side effects. Its mechanism of
action, however, for cyclooxygenase-1 (COX-1) and COX-2 as well
as 5-lipoxygenase (5-LOX) inhibition is poorly understood. The
antioxidant capacity of flavocoxid and effect on inducible gene
expression is also not well-defined. Experiments were undertaken
to clarify flavocoxid’s overall mechanism of action.
Methods and Materials: Purified enzymes were used to assess
flavocoxid’s anti-peroxidase and anti-cyclooxygenase activity on COX-
1 and COX-2 as well as inhibitory activity on 5-LOX. Multiple standard
antioxidant assay assays were utilized to judge flavocoxid’s antioxidant
capacity and cell co-culture of LPS-stimulated human peripheral
blood monocytes (PBMCs) with flavocoxid for its effect on inducible
inflammatory gene expression of COX-2, IL-1β, IL-6 and TNFα.
Results: Flavocoxid showed balanced inhibition of COX-1 and COX-2
peroxidase activities with inhibitory concentrations (IC50s) of 12.3
and 11.3µg/ml, respectively, while the 5-LOX IC50 was 110µg/ml.
No detectable 5-LOX inhibition was found for rofecoxib, celecoxib,
valdecoxib, diclofenac, meloxicam, naproxen, ibuprofen or
aspirin. Flavocoxid showed minimal inhibition of cyclooxygenase
activity for COX-1 (IC50=25µg/ml) compared to indomethacin

(IC50=0.012µg/ml) and no cyclooxygenase inhibition of COX-
2 compared to NS-398 (IC50=0.095µg/ml). Flavocoxid also
demonstrated a strong antioxidant capacity against a variety of
reactive oxygen species: ORAChydro=3700 µmolTE/g; ORAClipo=
19µmolTE/g; FRAP=1145µmolTE/g; HORAC= 1326µmolCAE/g;
NORAC=1936µmolTE/g; SORAC= 27kunitSODeq/g;
TEAC=2456µmolTE/g; and DPPH=767µmolTE/g. In LPS-stimulated
PBMCs, flavocoxid strongly reduced gene expression of COX-2
(80-fold), TNFα (11-fold), IL-1β (10-fold) and IL-6 (40-fold) and, to a
lesser extent, COX-1 (2.8-fold). Expression of NF-κB gene was also
reduced (2.2-fold).
Conclusions: These results suggest that the clinically favourable
effects and equivalency to NSAIDs in the management of OA are
achieved by simultaneous modification at multiple points in the
inflammatory process making flavocoxid a unique anti-inflammatory
acting via an antioxidant mechanism.
P132
Joint distraction in canine experimentally induced osteoarthritis
leads to cartilage repair accompanied by sustained relieve of
pain.
S.C. Mastbergen, F. Intema, P.M. van Roermund, H.A.W. Hazewinkel,
F.P.J.G. Lafeber
Utrecht/Netherlands
Purpose: Joint distraction in the treatment of osteoarthritis in
humans results in long-term clinical benefit. The mechanism
responsible for this benefit is unclear. Tissue structure modification
was suggested to be involved. Therefore, joint distraction was
applied in a canine experimental model of osteoarthritis to study
the involvement of cartilage repair.
Methods and Materials: Osteoarthritis was induced in the right
knee joint according to the Groove model (condylar surgically
applied damage) in 16 dogs. Ten weeks post-induction, the right
knee joint was distracted for 3-5 mm by use of a hinged external
fixator for 8 weeks in 9 dogs (distraction group). Seven dogs were
left untreated (OA group). Pain was studied by (un)loading of the
joint using force-plate-analysis every 5-10 weeks. Twenty-five
weeks after removal of the external fixators, cartilage integrity of
the tibial plateau was analysed.
Results: Cartilage showed a decreased PG-content (-18%, p<0.01),
an increased PG-release (+20%, p<0.03), and an increased
histological grade of cartilage damage (+5; p<0.05). This loss of
cartilage integrity was accompanied by decreased loading of the
affected joint. The loss of PG-content was significantly less in the
distraction group (-7%; p<0.02) compared to the OA group. PG-
release (5%) was also less as was the histological grade of cartilage
damage (-3, p<0.05). This relative improvement of cartilage
integrity was accompanied by a persistent increase of loading of
the osteoarthritic joint.
Conclusions: Joint distraction resulted in less cartilage damage
and normalization of loading of the affected knee. This study
corroborates the clinical finding that cartilage repair is induced by
joint distraction in treatment of osteoarthritis and that this may
underlie the clinical benefit.
P133
Surgical preparation for articular cartilage regeneration in
patients with osteoarthritis
J. Mika1, T.O. Clanton2, R.W. Kinne3, C.G. Ambrose2
1
Jena/Germany, 2Houston/United States of America, 3Jena/
Eisenberg/Germany
Purpose: The autologous chondrocyte implantation/transplantation
(ACI/ACT) has been described as a treatment option even in
patients with osteoarthritis (OA). To prevent hemorrhage, fibrin clot
formation, and subsequent activation of the inflammatory response,
surgical preparation for articular cartilage regeneration should
avoid penetration of the subchondral bone plate. We examined
whether current surgical procedures with ring curettes preserve the
integrity of the subchondral bone plate in patients with OA.
Methods and Materials: The subchondral bone plates of femoral
condyles (n = 40) in OA human knees undergoing total knee
arthroplasty (TKA) were prepared in vivo using traditional
debridement for ACI/ACT. The force on the ring curette was 15.2
± 0.4 N. In order to approximate regular wear, only areas with
maximally grade 3A (ICRS-score) cartilage were prepared. Effects
were analyzed by light microscopy.
Posters 221
Results: Standard debridement generally did not violate the tide
mark line in 87.5% of the cases (35/40 specimens), with only
occasional minor openings of approximately 20 µm in diameter with
a smooth edge. Twenty-eight specimens (70%) showed at least
remnants of uncalcified cartilage, 5 samples (12.5%) one large area
with a missing bone plate and an open bone marrow space.
Conclusions: Because of the fine structure of the minor openings,
they are likely due to increased vascular penetration through the
tide mark in the pathologically altered bone-cartilage interface
in OA. Standard debridement only occasionally violates the
subchondral bone plate in OA knee joints under in vivo conditions.
The exact consequences of limited hemorrhage through minor
openings or selected large defects are still unkown. Therefore,
the traditional surgical preparation technique appears suitable for
cartilage regeneration even in cases of osteoarthritic defects.
P134
Therapeutic Effects of Intra-articular Administration of
Ultra-purified Low Endotoxin Alginate on the Progression of
Experimental Osteoarthritis in Rabbits
T. Igarashi, N. Iwasaki, D. Kawamura, N. Tsukuda, Y. Kasahara, A.
Minami
1
Sapporo/Japan
Purpose: We have developed an ultra-purified low-endotoxin
alginate (UPLE-alginate), which can drastically reduce endotoxin
levels. Our hypothesis was that the UPLE-alginate could promote
anti-arthritic activity in experimental osteoarthritis (OA). To
test this hypothesis, we examined the activity of intra-articular
administration of the UPLE-alginate using a rabbit OA model. Our
aims were to clarify the effects of the UPLE-alginate administration
on OA progression, and to determine the adequate molecular
weight of the UPLE-alginate for therapeutic effects.
Methods and Materials: To induce knee OA, 35 Japanese white
rabbits underwent anterior cruciate ligament transection. Intra-
articular injections of a 0.3 ml solution of each material started at
4 weeks postoperatively for a total of 5 weekly injections. Seventy
knees were randomly divided into 5 treatment groups as follows;
AL20 (430 kDa molecular weight UPLE-alginate), AL100 (1,000 kDa),
AL500 (1,700 kDa), HA (hyaluronan), and NS (normal saline). At 9
weeks postoperatively, all knees were assessed macroscopically,
histologically, and mechanically. Mechanical testing determined
the friction coefficient as a joint lubrication.
Results: Gross morphology. The NS and HA groups showed
extensive cartilage erosion. The focal deep erosions were smaller
in the treatment groups than the NS group. The UPLE-alginate
groups exhibited milder cartilage degradation compared to that of
the NS and HA groups. Histological findings. The NS and HA groups
showed loss of the superficial layer and fibrillation. An obvious
reduction in the severerity of OA was found in the UPLE-alginate
groups. The scores described by Kikuchi et al. were superior in
the AL100 group compared to other groups. Mechanical test. The
friction coefficient of samples obtained from the AL20 and AL100
groups was significantly lower than that of the NS group.
Conclusions: The current study indicates that our novel UPLE-
alginates, especially AL100 (1,000 kDa molecular weight), have
promising potential as an effective agent in preventing OA
progression.
P135
Effect of Light-Emitting Diode (LED) Therapy on the Development
of Osteoarthritis (OA) in a Rabbit Model
Y. Oshima1, R.D. Coutts1, N.M. Badlani1, R. Healey1, T. Kubo2, D.
Amiel1
1
La Jolla/United States of America, 2Kyoto/Japan
Purpose: Light-emitting diode (LED) therapy has proven beneficial
for pain relief and wound healing. Our objective was to evaluate the
effect of LED therapy on osteoarthritic (OA) knee joints using the
anterior cruciate ligament transection (ACLT) model in vivo.
Methods and Materials: Four weeks after ACLT of mature New
Zealand rabbits, LED therapy began at intervals of 10 min/day, 5
days/week for 5 weeks (n=7). Therapy on the ACLT knees used
a custom-designed brace that fit over the knee using red and IR
(wavelength: 630 nm and 870 nm, respectively). The amount of
energy delivered to the skin was calculated to be 2.4J/cm2. In
the control group (n=7), the rabbits were sacrificed 9 weeks after
ACLT without treatment. The femoral surface was evaluated with
222 Posters
a modified Outerbridge OA grading: Grades I, II, III and IV. mRNA
expressions of various markers from cartilage of the femoral
condyle and synovial tissue were evaluated by RT-PCR. Statistical
analysis was performed.
Results: The control group showed 4 Grade II, 2 Grade III and 1
Grade IV (average was 2.6) macroscopically. In the experimental
group after the 5-week treatment with the light brace there were
no remarkable side effects seen on the knee joints. There were 2
Grade I and 5 Grade II (average 1.7), however no severe OA joints.
Aggrecan expression in the cartilage showed no differences
between the groups; however, type II collagen expression
increased in the experimental group compared to the control (Table
1). Expression of TNF-α in both cartilage and synovial tissues was
significantly decreased in the experimental group compared to the
control (Table 2).
Conclusions: This is the first trial of LEDs in the orthopaedic
area, and results showed that cartilage was preserved and type
II collagen increased in the experimental group. Inflammation
seemed decreased in both cartilage and synovium.
P136
Effects of potassium and chloride channel blockers on the matrix
anabolism of rabbit articular chondrocytes in vitro
L. Ren, X. Wei
Taiyuan/China
Purpose: To investgate the effects of potassium and chloride
channel blockers on The glycosaminoglycan(GAG) and collagen
type 2 synthesis of rabbit articular chondrocytes in vitro.
Methods and Materials: 2 months New Zealand rabbits were killed
and their knee joint were taken out under aseptic condition. The
chondrocytes were isolated by enzymolysis method and cultivated in
24-well plates (seeded 5×104 cells per well).Then the chondrocytes
were divided into three groups randomly after cultured 2 days when
the cells were adherent. The control group was cultured by DMEM/
F12 while the potassium channel blockon group(4-AP group)was
cultured in the media contained 1mmol/L 4-aminopyridine, which
can lead to the blockon of the potassium channel,and the chloride
channel blockon group(SITS group)was cultured in the media
contained 1mmol/L 4-acetamido-4 -isothiocyano-2,2 -disulfonic
acid stilbene(SITS), which can lead to the blockon of the chloride
channel.The GAG synthesis were measured by Alician Blue method
and collagen type 2 synthesis were estimated by the ELISA in the
media when medium was change after3-, 6-and 9-day.
Results: Compared with the control group,the GAG content of 4-AP group
has significant decrease at 3th day,but has no significant difference
at 6th and 9th day,while the collagen type 2 content has significant
increased at 3th and 6th day, and has decrease tendency at 9th day,
but have no significant difference.The GAG content of SITS group has
significant increased at 3th, 6th and 9th day,while the collagen type 2
content has significant increased at 3th and 6th day,and has increased
tendency at 9th day, but have no significant difference.
Conclusions: The blockon of potassium and chloride channel can
increase the GAG and collagen type2 synthesis of chondrocytes
in vitro.Especially when block chloride channel,the GAG synthesis
was significant.
P137
Prostaglandin E2 prevents the degeneration of articular cartilage
via EP2 receptor in rabbit osteoarthritis model
H. Mitsui1, T. Aoyama2, M. Furu2, K. Ito3, Y. Jin2, T. Kasahara4 , K.
Hayakawa2, K. kobayashi2, T. Maruyama4 , T. Kanaji4 , S. Fujimura4 ,
H. Sugihara4 , T. Otsuka3, T. Nakamura2, J. Toguchida2
1
Kyoto City/Japan, 2Kyoto/Japan, 3Nagoya/Japan, 4Osaka/Japan
Purpose: We have reported that a selective agonist for EP2 receptor
of prostaglandin E2 (EP2 agonist) induces the proliferation of
articular chondrocytes and promotes the regeneration of the
osteochondral defect of rabbits. The purpose of the current
study is to analyze the effect of EP2 agonist on articular cartilage
regeneration in traumatic joint degeneration model of rabbits.
Methods and Materials: Female Japanese white rabbits (3-months-
old) were used for this study. Traumatic degeneration model was
created by transection of anterior cruciate ligament and partial
menisectomy of medial meniscus in both knee joints. Immediately
after the operation, poly lactic-co-glycolic acid gel (PLGA) containing
EP2 agonist (80ug or 400ug) was placed on one side (Treated
groups), and no treatment was performed on contralateral side.
As a control, PLGA gel without EP2 agonist was placed in one side
(Control group). Rabbits were sacrificed either 2 or 12 weeks after
the operation, and articular cartilages were examined histologically
by HE and Safranin O staining and evaluated by Mankin ‘s score.
Results: In the control group, significant cartilage degeneration
was observed in both knee joints at 2 weeks. Among treated
groups, the Mankin’s score of distal femur was significantly lower
in knee joints treated with 80ug of EP2 agonist. At 12 weeks, the
joint degeneration was accelerated in the control group, whereas
no significant effect of EP2 agonist was observed in either 80ug or
400ug-treated group.
Conclusions: We found that the administration of low dose (80ug)
EP2 agonist was effective to prevent joint degeneration for short
term, which may be due to the limitation of current drug delivery
system. Continuous stimulation of EP2 agonist by new drug delivery
system may prevent the degeneration for long term. EP2 agonist
could be a promising drug for osteoarthritis.
P138
Hyperbaric oxygen treatment enhances the expression of heat
shock protein 70 and prevents nitric oxide-induced apoptosis in
chondrocyte
L. Yuan, S. Lin, C. Niu, C. Yang, S.W.N. Ueng, W. Chen
Kweishan, Taoyuan/Taiwan
Purpose: We investigated the effect of hyperbaric oxygen (HBO)
on (1) NO-induced apoptosis of rabbit chondrocytes and (2) the
healing of rabbit articular cartilage defects.
Methods and Materials: In vitro, interleukin-1β treatment induced
NO in chondrocytes, and all hyperoxic cells were exposed to 100%
oxygen at 2.5 atmospheres absolute (ATA) in a hyperbaric chamber.
Reverse transcription polymerase chain reaction (RT-PCR) and
western blotting to detect mRNA and protein expression of HSP
70, inducible NO synthase (iNOS), and caspase 3. In vivo, the HBO
group was exposed to 100% oxygen at 2.5 ATA for 2 h, 5 days a
week for 10 weeks. After sacrifice, specimen sections were sent for
histological and histochemical examination using a standardized
scoring system. In situ analysis of iNOS, HSP 70, and caspase 3
expression were performed by immunohistostaining
Results: Our in vitro study demonstrated that HBO treatment up-
regulated mRNA and protein expression of HSP 70 and suppressed
iNOS and caspase 3 expression. In vivo, the histological and
histochemical scores showed that HBO treatment significantly
enhanced the cartilage repair. Moreover, immunohistostaining
showed that HBO treatment enhanced HSP 70 expression and
suppressed iNOS and caspase 3 expressions in chondrocytes.
Conclusions: HBO treatment enhances the expression of HSP 70
and prevents NO-induced apoptosis in articular cartilage injury.
P139
Tissue Engineering Osteochondral Graft for Cartilage Repair
J. Davies1, S. Wilshaw2, D. Shaw1, Z. Jin2, E. Ingham2, J. Fisher2
1
Bradford/United Kingdom, 2Leeds/United Kingdom
Purpose: A novel acellular xenogenic graft is proposed as a biological
cartilage replacement, for repair of osteochondral defects. Acellular
porcine cartilage has been produced using repeated freeze thaw
cycles and washing using hypotonic buffers and sodium dodecyl
sulphate solution (SDS; Keir, 2008). DNA content was reduced by
93.3% compared to native cartilage, with corresponding reduction
in glycosaminoglycan (GAG) content. It was hypothesised that
penetration of decellularisation solutions into native tissue could
be improved through deformation of cartilage under confined
compression, and then allowing the osteochondral pin to recover in
solution, allowing greater retention of the GAGs
Methods and Materials: A perforated indenter applied a ramped
load in a confined chamber, using a tensile testing machine (Instron).
Osteochondral pins (9.8 mm diameter; n = 3) were loaded to 5 MPa
contact stress for 20 minutes, and then placed in SDS 0.1% (v/v)
in hypotonic Tris buffer; pH 8.0 and then treated according to the
established protocol. For comparison, osteochondral pins (n = 3)
were treated using the unmodified standard protocol. Histological
and biochemical analyses were performed.
Results: There was no difference in the reduction in DNA content
of loading modified versus standard protocol decellularisation
cartilage (0.017 mcg.mg-1 +/- 0.006 mcg.mg-1 (95% CI), loaded,
0.018 mcg.mg-1 +/- 0.011 mcg.mg-1 (95 % CI), standard). There
was no difference in the GAG content (19.769 mcg.mg-1 +/- 33.581

mcg.mg-1 (95% CI), loaded, and 13.530 mcg.mg-1 +/- 21.207 mcg.
mg-1 (95 % CI), standard). DAPI fluorescence demonstrated
residual DNA in deep zones and at the cartilage bone interface in
the standard protocol, which was to a lesser degree with loading
modified cartilage.
Conclusions: Loading modification did not improve DNA reduction
or GAG retention in acellular porcine cartilage.
P140
Repair of rabbit osteochondral defects using a novel
bioabsorbable bone-originated apatite with body fluid
permeabitity
Y. Kasahara1, N. Iwasaki1, M. Murata2, T. Akazawa1, T. Igarashi1, D.
Kawamura1, Y. Tsukuda1, A. Minami1
1
Sapporo/Japan, 2Tobetsu-Chou/Japan
Purpose: We successfully developed a bioabsorbable bone-
originated apatite with body fluid permeability (functionally graded
apatite, fg-HAp). The purpose of this study was to determine the
efficacy of this material for the treatment of osteochondral defects
using a rabbit model.
Methods and Materials: Preparation and physicochemical
characterization of the fg-HAp blocks were described previously.
As a control, commercially manufactured interconnected porous
hydroxyapatite ceramic (IP-CHA; Toshiba Ceramics, Tokyo) was
used in this study. Twenty skeletally mature female rabbits were
used for further analysis. A 4.5 mm diameter osteochondral defect
was created in the femoral trochlea of each rabbit. In both treatment
groups, the HAp blocks were press-fit implanted into the defects
without periosteum coverage. At 4 and 12 weeks postoperatively,
the macroscopic and histological appearance of the samples was
evaluated in each group (n=5) using Wayne and modified O’Driscoll
scoring systems, respectively.
Results: Table 1 summarizes macroscopic and histological
evaluations of both treatment groups. From 4 to 12 weeks
postoperatively, the implanted apatites of the fg-HAp group were
indistinguishable from the reparative tissue and the surface
became smooth. The macroscopic score of the fgHAp group was
statistically higher than that of the IP-CHA group at 4 weeks. The
histological findings of the fg-HAp group demonstrated that the
reparative tissue at the articular surface was complete integration
between native and reparative tissue (Fig. 1). Although the fg-HAp
bulk was stained by HE, the IP-CHA bulk was not stained by HE,
suggesting that body fluid hardly permeated into the bulk structure.
The histological score of the fgHAp group was statistically higher
than that of the IP-CHA group at 12 weeks.
Conclusions: We succeeded in osteochondral repair with the
newly developed fg-HAp alone. Our study indicates the efficacy
of the fg-HAp as a bioabsorbable scaffold for the treatment of
osoteochondral defects without growth factors nor cultured cells.
P142
Chondrogenic media (CM) protects native and engineered
cartilage constructs from cell death after injury
A.R. Tan, J.P. Andry, G.A. Ateshian, C.T. Hung
New York/United States of America
Purpose: In cartilage explant injury models cell death has been
characterized as progressing away from the site of injury and
persisting long after the initial insult. Here, we look at protecting
both native cartilage explants and mature engineered cartilage
after mechanical insult by storage in a serum-free, chondrogenic
media (CM).
Methods and Materials: Explants and chondrocytes were harvested
from juvenile bovine wrists. After 35 days in culture, chondrocyte-
seeded agarose constructs attained native Young’s modulus and
glycosaminoglycan content. Two orthogonal cuts were made in
each construct by pushing a razor blade to 50% of the specimen
thickness. Cuts were similarly made in fresh cartilage explants.
Controls were handled similarly but without insult. Samples
were returned to culture following injury and maintained in CM
(hgDMEM plus 1X PSF, 0.1uM dexamethasone, 50ug/mL ascorbate
2-phosphate, 40ug/mL L-proline, 100ug/mL sodium pyruvate, and
1X ITS+ premix (Becton Dickinson, Franklin Lakes, NJ). Cell viability
was assessed by Live/Dead cytotoxicity assay at days 1, 7 and 21
post-trauma.
Results: Cartilage injury and cell death were mitigated in cartilage
explants and engineered cartilage when cultured in chondrogenic
Posters 223
media. Cell death remained localized to the site of injury immediately
following injury; at 21 days post injury, injured regions were void
of dead cells, suggesting that cell death had not spread through
apoptotic pathways, as previously reported in the literature for in
vitro explant injury models.
Conclusions: Tissue culture storage in CM protects native and
mature engineered cartilage by confining cell death to the
injury site. In engineered cartilage, biochemical and mechanical
properties are maintained at control levels after injury. Future work
will look to utilize CM to enhance explant survival after harvesting.
These findings may lead to development of better strategies for
engineered cartilage as well as osteochondral harvest/storage that
optimize the use of allograft tissues for clinical repair of cartilage.
P143
Arthroscopic Mosaicplasty for Osteochondral Lesions of the
Knee: Computer-Assisted Navigation vs. Freehand Technique
D. Koulalis1, P. Di Benedetto2, M. Citak3, P.F. O’Loughlin4 , C.
Cranchi4 , A. Pearle4 , D. Kendoff4
1
Athens/Greece, 2Udine/Italy, 3Hannover/Germany, 4New York/
United States of America
Purpose: The purpose of this study was to compare a freehand
arthroscopic approach to mosaicplasty for treatment of osteochondral
lesions of the knee with a navigated arthroscopic approach.
Methods and Materials: Four whole cadaveric legs were used.
A conventional navigation system was used in combination with
an autologous ostechondral graft transplantation system. A
customized reference tracker was fixed to both the OATS recipient
and donor guides.
Results: Mean angle of graft harvest was 3.4° (range 0° to 10°, SD
+/- 3.10°) in navigated group versus 14.8° (range 6° to 26°, SD +/-
7.53°) in freehand group (p < 0.0003) Mean angle for recipient
site coring was 1.50° (range 0° to 5°, SD +/- 1.65°) in the navigated
group versus 12.60° (range 4° to 17°, SD +/- 3.98°) in the freehand
group (Fig. 2) (p < 0.0003). Mean angle of graft placement was
2.0° (range 1° to 5°, SD 1.25°) in the navigated group versus 10.8°
(range 5° to 15°, SD +/- 3.23°) in the freehand group (Fig. 3) (p =
0.0002). Mean difference between angle of graft harvest and graft
placement was 2.40° (range 0° to 8°, SD +/- 2.46°) in the navigated
group versus 7° (range 1° to 16°, SD +/- 5.19°) (p = 0.0207) in the
freehand group. Mean difference between the angle of recipient
site coring and graft placement was 0.90° (range 0° to 2°, SD +/-
0.74°) in the navigated group versus 9.2° (range 6° to 14°, SD 2.57°)
in the freehand group (p = 0.002).
Conclusions: This study demonstrates in a cadaveric model
that improved accuracy and reproducibility may be achieved
with a navigated arthroscopic mosaicplasty versus a freehand
arthroscopic technique.
P144
A systematic assessment of hierarchical clustering methods
for flow cytometry data analysis: application to the study of
chondrosarcomas
J. Diaz-Romero1, S. Romeo2, J.V.M.G. Bovée3, P.C.W. Hogendoorn3,
D. Nesic1, P. Mainil-Varlet1
1
Bern/Switzerland, 2Treviso/Italy, 3Leiden/Netherlands
Purpose: We wanted to establish a method to categorize different
cell types of mesenchymal origin based on their pattern of surface
marker expression combining flow cytometry data with hierarchical
clustering. Cell types for which an independent knowledge of cluster
membership was available were used to (a) assess cluster accuracy
and guide the choice of the different clustering approaches tested,
and (b) establish similarities with chondrosarcoma cells that could
provide clues concerning the cellular origin of these tumors.
Methods and Materials: Human primary conventional central
chondrosarcoma cells, articular chondrocytes, mesenchymal stem
cells, fibroblasts, and a panel of tumor cell lines of chondrocytic
or epithelial origin were clustered based on the expression profile
of eleven surface markers, previously shown to be differentially
expressed on the cell types analyzed (Fig.1). Eight hierarchical
clustering algorithms, three distance metrics, as well as several
approaches for data preprocessing, including multivariate outlier
detection, logarithmic transformation, and z-score normalization,
were systematically evaluated.
Results: Careful selection of clustering approaches allowed
separation of chondrocytes, mesenchymal stem cells, fibroblasts,
224 Posters
and tumor cell lines in four well differentiated clusters (Fig.2).
Primary chondrosacoma cells were grouped in two main clusters
with distinctive marker signatures: one group clustered together
with mesenchymal stem cells (CD49b-high/CD10-low/CD221-
high) and a second group formed an independent cluster close
to fibroblasts (CD49b-low/CD10-high/CD221-low). Substantial
differences between primary chondrosarcoma cells and established
chondrosarcoma cell lines were revealed, with the latter not only
segregating apart from primary tumor and normal tissue cells, but
clustering with cell lines from epithelial origin.
Conclusions: Hierarchical clustering of flow cytometry data is a
powerful tool to classify samples according to surface marker
expression patterns. It distinguishes between closely related
cell types, such as fibroblasts from chondrocytes, allows tumor
classification according to similarity with normal cell types, and
could be used to validate the use of cell lines mimicking normal
chondrocytes or primary tumors.
P145
Phenotypic gradient from the avascular to the vascular zone of
the young meniscus
D. Deponti, C. De Palma, A. Pozzi, R. Ballis, A. Di Giancamillo, C.
Domeneghini, L. Mangiavini, G.F. Fraschini, G.M. Peretti
Milan/Italy
Purpose: The meniscus plays an important role in the biomechanics
of the knee joint. This tissue has a poor healing potential, partly due
to the absence of vasculature: blood vessels are present only in the
outer 10-30% of the meniscal body. The term fibrochondrocytes
has been introduced to identify the typical characteristics of the
meniscus cells, but a clear description of the phenotype of these cells
is still missing. This work was aimed to study the characteristics of the
meniscus cells by focusing on three areas of the meniscus, the inner
avascular zone, the intermediate, and the external vascular zone.
Methods and Materials: Meniscus cells proliferation and
differentiation were compared in these three different areas by
FACS analysis, moreover the expression of cartilage specific genes
was confirmed by real time PCR; additionally, cell morphology
was analyzed both in the native meniscus by histology and by
microscopy analysis after enzymatic isolation from the tissue.
Meniscus cells were compared to isolated articular chondrocytes
and tendon fibroblasts.
Results: The data show that the inner region of the meniscus is
composed of cells that are similar to articular chondrocytes in
terms of morphology and proliferation rate, but they express lower
amount of cartilage specific genes, such as collagen type II and
aggrecan; the intermediate and external areas are composed of
cells that showed a gradient in morphology and cartilage specific
gene expression compared to the cells of the inner area; however,
interestingly, they show a lower proliferation rate.
Conclusions: These results suggest the presence of an “intermediate
phenotype” located in the middle and external meniscal areas
having morphological and functional characteristics intermediate
between the fibrochondrocytes of the inner region and the tendon
fibroblasts; these evidences could lead to new options in the choice
of the optimal cell source for meniscus tissue engineering.
P146
Meniscus cells phenotype: new insights in the use of the term
“fibrochondrocytes”
D. Deponti, C. De Palma, A. Pozzi, R. Ballis, M. Melato, A. Di
Giancamillo, C. Domeneghini, G.F. Fraschini, G.M. Peretti
Milan/Italy
Purpose: The meniscus plays an important role in the biomechanics
of the knee joint. This tissue has a poor healing potential, partly due
to the absence of vasculature. Due to the presence of both collagen
I and II, the meniscus has properties of fibrous and cartilaginous
tissue. The term fibrochondrocytes has been introduced to identify
the typical characteristics of the meniscus cells, but a clear
description of the phenotype of these cells is still missing.
Methods and Materials: This work was aimed to study the
characteristics of swine meniscus cells by analyzing three areas of
the meniscus: the inner avascular zone, the intermediate and the
external vascular zone. Meniscus cells morphology, proliferation,
differentiation and gene expression were evaluated.
Results: The data showed that in the young meniscus the inner
region is composed of cells that are similar to chondrocytes in terms
of morphology but not in terms of gene expression; the intermediate
and external areas are composed of cells that present fibroblast-like
morphology and phenotype. Interestingly, there is no expression
of type II collagen throughout the whole young meniscus. In the
adult meniscus, however, the three analyzed areas did not show
the differences in cell morphology noted for the young tissue.
Moreover, all cell populations were positive for type II and type I
collagen presenting a gradient of type II versus type I: from the inner
to the outer area, type II collagen decreases while type I increases.
Conclusions: These results lead to the conclusion that meniscus
maturation, from young to adult, is accompanied by changes in cell
phenotype: in the early stages of life the cells from the intermediate
and outer part are still immature and far from a chondocyte-like
phenotype; in adult life all meniscus cells show an intermediate
phenotype between chondrocytes and fibroblasts.
P147
Integration of Repair Cartilage: an in vitro model
R. Kandel, J.A. DeCroos, J. Theodoropoulos
Toronto/Canada
Purpose: In previous studies, we developed methodology to generate
biphasic constructs composed of hyaline cartilage formed in vitro and
anchored to the top surface of a porous bone-interfacing component.
These constructs are suitable to use for cartilage repair as shown in
an ovine model. In contrast to osteochondral transfer (OATS), the in
vitro formed cartilage layer integrates laterally with the surrounding
native tissue. The aim of this investigation was to develop this as an
in vitro model in order to be able to study the mechanisms regulating
tissue engineered cartilage integration to native cartilage.
Methods and Materials: A single biphasic construct (5.7 mm
diameter) was placed into the center of doughnut-shaped 1cm
diameter osteochondral cylinder, which had been harvested from
bovine joints. These were maintained in culture for up to eight
weeks. Autologous osteochondral plugs served as controls.
Results: Histological evaluation by light and electron microscopy
showed that bioengineered cartilage exhibited increasing
integration over time. Collagen fibers were seen crossing the
interface ultrastructurally in regions of integration. In contrast
autologous osteochondral implants did not integrate. Biochemical
evaluation demonstrated a significant increase in collagen content
in the cartilage of the biphasic implant over time but no change
in proteoglycan content. The strength of repair–native cartilage
integration increased significantly between 4 and 8 weeks. When
carboxy-fluorescein diacetate-labeled chondrocytes were used to
generate cartilage tissue, fluorescent cells were detected in the
adjacent native cartilage up to 1.5mm from the integration site.
Conclusions: This study demonstrates development of an in vitro
model to study repair cartilage integration. The presence of cells
from the repair tissue in the adjacent cartilage suggests that the
integration to the surrounding native tissue may be a result of
these migratory cells. Further study is required to evaluate the role
of these cells in cartilage integration. This work was supported by
a grant from CIHR (RK).
P148
Development of In Vitro Articular Cartilage Injury: Novel Strategy
Utilizing Impaction and Bioreactor to Induce and Treat Lesions
D. Wilensky1, L.D. Kaplan1, C. Wong2, C.C. Huang1
1
Miami/United States of America, 2Coral Gables/United States of
America
Purpose: To induce an articular cartilage injury and recreate the
native environment in vitro to facilitate the assessment of early
hyaluronic acid delivery in a human articular cartilage model.
Methods and Materials: Human full-thickness articular cartilage
plugs were harvested from the femoral condyles of tissue donors
upon cessation. Cartilage injuries were induced with a drop tower
and treated at 1 hr or 24 hrs post impaction. The articular cartilage
plugs were then subjected to displacement-controlled cyclic
compressive loading at a frequency of 1 Hz and magnitude of 10%
strain in the presence of hyaluronic acid. The effectiveness of the
hyaluronic acid treatment was then assessed utilizing an ethidium
homodimer-1 assay for cell viability determination and a TUNEL
assay for apoptotic activity. GAG content was determined using a
DMMB assay while safranin O staining was employed to visualize
proteoglycan distribution in the extracellular matrix. Morphologic
analysis was performed with a Hematoxylin and Eosin stain.

Results: Injured groups subjected to mechanical loading and hyaluronic
acid treatment showed increased viability mainly in the superficial
tangential and middle zones. The injured group with no treatment
revealed lower overall proteoglycan content with reduced distribution
in the more superficial areas. Fluorescent microscopy demonstrated
that there was a strong trend for reduced chondrocyte death in the
mechanically loaded groups challenged with hyaluronic acid.
Conclusions: This study suggested that early hyaluronic acid
delivery coupled with cyclic mechanical loading, to acutely injured
cartilage, reduces cell death and significantly improves cell
metabolism. With further experimentation, the use of hyaluronic
acid shortly following injury may prove to be beneficial for clinical
use in humans to minimize cartilage death and improve healing
after traumatic injury.
P149
T2 relaxation in healthy neutral, varus and valgus knee joints:
does malalignment provoke cartilage deterioration
M. Sauerschnig1, G.M. Salzmann2, L. Kohn1, J.S. Bauer1, K. Wörtler1,
S. Landwehr1, S. Hinterwimmer1, A.B. Imhoff1
1
Munich/Germany, 2Freiburg/Germany
Purpose: We hypothesize that static knee joint malalignment
affects the articular cartilage in asymptomatic subjects who display
evidence of an early chronic joint impairment.
Methods and Materials: The study had institutional review board
approval and was HIPAA compliant; all subjects provided informed
consent prior to testing. In 4 groups of active, age-matched (26.1
± 1.8 years) patients [neutral (0.44° ± 0.29), mild varus (2.95° ±
0.52), severe varus (5.25° ± 1.45), or valgus leg deformity (2.95°
± 0.75) both knee joints were clinically (Lysholm, Tegner) and MR-
radiographically (hip-knee-ankle angle, T2 mapping) evaluated.
Patients were without knee complaints (no history of major
knee trauma, knee surgical intervention) with either to define
4 different groups (n=6 subjects/group; n=12 knees/group).
Regions of interest for T2 assessment were within full-thickness
cartilage across the joint surface and were divided with respect to
compartmental as well as functional joint anatomy.
Results: Leg deformity was significantly different between groups,
while no difference emerged from the clinical measures. When
compared to neutral knee angle subjects, prolonged cartilage T2
was found at the medial tibia plateau and medial posterior femoral
condyle in varus aligned subjects, while no such differences
emerged among valgus knees.
Conclusions: There are early alterations within the articular surface of
asymptomatic subjects that have noteable varus knee malalignment.
These changes can be detected by quantitative MR-imaging.
P150
Single intra-articular injection of AAV results in stable and
controllable in vivo transgene expression
C. Chu1, K.A. Payne1, H.H. Lee1, A.M. Haleem1, C. Martins1, X. Xiao2
1
Pittsburgh/United States of America, 2Chapel Hill/United States of
America
Purpose: This study characterized the localization, persistence, and
ability to externally control transgene expression in the rat knee
joint after a single intra-articular injection of adeno-associated
virus (AAV2).
Methods and Materials: Nine male Sprague-Dawley rats received
AAV2-CMV-GFP and AAV2-CMV-Luciferase into their right and left
knees, respectively. Luciferase expression was evaluated over
a 1-year period using the IVIS® Imaging System (Xenogen). After
sacrifice, tissues within the joint were analyzed by fluorescence
stereomicroscopy to detect GFP+ cells. To study the ability to
control AAV transgene expression in the rat knee joint, five male
Sprague-Dawley rats received AAV2-tetracycline response element
(TRE)-Luciferase and AAV2-CMV-transcriptional transactivator into
the right knee. To induce expression of Luciferase, doxycycline
(Dox; 2 mg/ml) was added to the drinking water, and rats were
imaged twice a week as described above.
Results: Luciferase expression was detectable in all rats by 7
days post-injection, and showed continued and stable expression
through 1 year. GFP+ cells were mainly found in soft tissues
surrounding articular cartilage, with rare GFP+ chondrocytes. With
the use of inducible AAV-TRE-Luciferase, gene expression could
be upregulated with Dox, and could then be downregulated by its
removal from the water. This was repeated in multiple cycles.
Posters 225
Conclusions: Longitudinal in vivo results show persistent and stable
AAV-mediated gene expression following a single intra-articular
injection. The use of inducible AAV-TRE-Luciferase demonstrates
the feasibility to control transgene expression in the rat knee joint.
These results suggest that AAV is a potential therapeutic approach
to deliver growth factors to the knee joint. The rare occurrence of
GFP+ cells in intact articular cartilage indicates that more studies
on AAV delivery to damaged articular cartilage, as seen in cartilage
injuries, are needed. This continued characterization of the length,
location and ability to control transgene expression following intra-
articular injection of AAV will help to better tailor gene therapy for
articular cartilage repair.
P151
Imaging of osteochondral samples using delayed Gadolinium-
Enhanced MR Imaging of Cartilage (dGEMRIC) is not affected by
formalin fixation
K.E.M. Benders, J.E.J. Bekkers, W. Schuurman, L.B. Creemers, W.J.A.
Dhert, D.B. Saris, J. Malda
Utrecht/Netherlands
Purpose: Delayed Gadolinium-Enhanced MR Imaging of Cartilage
(dGEMRIC) is a non-destructive method that could facilitate non-
destructive ex vivo visualization of glycosaminoglycans (GAGs) in
experimental settings. This study aimed at investigating whether
formalin fixation affects dGEMRIC outcomes.
Methods and Materials: Osteochondral plugs (n=72) were harvested
from the medial and lateral condyles of equine stifle joints (n=3).
Samples were divided into three groups: no digestion (samples were
kept in PBS (n=24)), digest 1 (0.25% trypsin for 20 minutes (n=24),
and digest 2 (0.25% trypsin for 40 minutes (n=24). Per group half
of the plugs were fixed using 10% formalin for 22 hours (n=36)
while the other half was kept in PBS. Samples were incubated for
22 hours in 1mM Magnevist (Gadolinium). A 3-dimensional T1 MRI
measurement protocol, at 1.5T, with 5 different inversion times (50,
150, 350, 650 and 1650ms) was used. The T1gd (dGEMRIC index)
was calculated by averaging the T1gd across the region of interest
(ROI) using in-house developed software. The ROI was defined as
the full-thickness cartilage in all the sagittal slices. After scanning,
the cartilage was cut from the samples and GAGs were determined
biochemically using a DMMB assay. Correlations and differences
in GAG/mg wet weight were statistically analyzed using Pearson’s
correlations and one-way ANOVAs with post-hoc LSD tests.
Results: Digestion of the samples resulted in a GAG content
that ranged from approximately 20 to 75 mg GAG per mg tissue.
Good correlations were found between the T1gd and total GAG/
mg cartilage (R=0.763, p<0.01). No significant differences were
found in T1gd and GAG content per mg between fixed and non-fixed
osteochondral samples (p>0.1).
Conclusions: Formalin fixation of osteochondral plugs does not alter
the T1gd-time. Therefore, the results suggest that dGEMRIC can be
used on both fixed and non-fixed osteochondral ex vivo samples.
P152
Construction of a bio-active meniscus via bio-plotting
E. Berneel, J. Schelfhout, H. Declercq, P. Dubruel, M. Cornelissen
Ghent/Belgium
Purpose: Injuries to the meniscus, particularly those in the
a-vascular region, pose a complex problem. Tissue engineering of a
replacement tissue might be the solution. Rapid proto-typing is an
innovating technology that allows to construct tailor-made scaffolds.
Layer-by layer disposition enables to fabricate a 3D-scaffold with a
variable micro-architecture. Another advantage is the possibility to
plot cells together with the material so that plotted scaffolds contain
a homogenous dispersed cell population. Moreover different cell
types can be used during plotting. We started with the construction
of the gelatinous core of the meniscus. A porcine meniscus
functions as a reference because of its analogous structure and
cell distribution as the human meniscus: fibrochondrocytes in
the inner and middle part of the meniscus and fibroblast-like cells
in the outer part of the meniscus. Our aim is to encapsulate and
plot these cells with a suitable material and to evaluate their cell
viability and phenotype. A polyester such a polycaprolactone will
be used to give more strength to the construct.
Methods and Materials: Fibrochondrocytes derived from porcine
menisci were isolated, cultured and identified with following
markers: collagen I, collagen II and aggrecan. MC3T3 cells were
226 Posters
encapsulated in and seeded on methacylamide modified gelatin
(DS 61%). The hydrogels were cross-linked using uv-irradiation in
the presence of a photo-initiator Irgacure® 2959. Sol-gel fractions
of the hydrogels were determined in aqua dest, PBS and culture
medium. The gels (0,4 ml) were casted in a 24 well plate and cross-
linked with uv-irradiation. Cell survival was evaluated with MTT en
live-dead assay. Polycaprolactone scaffolds were plotted with the
3D-bioplotter device and were modified to enhance cell adhesion
and improve biocompatibility of PCL. Cell viability and adhesion of
HFF (human foreskin fibroblast) cells was tested on thin modified
polycaprolactone films (2D) and 3D scaffolds with MTT and live-
dead assay.
Results: First results showed a good cell survival of MC3T3 cells
seeded on the hydrogel constructs but a low cell survival of
encapsulated MC3T3 cells in methacrylamide modified gelatin.
The effect of uv-irradiation on MC3T3 cells was also evaluated and
the preliminary results are promising. This might indicate that the
low cell viability in the hydrogel may be due to radical formation
during uv-irradiation but further research is needed. The HFF cells
demonstrated a good cell survival on the modified 2D PCL films and
3D plotted constructs which indicates that modified PCL is a good
choice of material to construct the outer part of the meniscus.
Conclusions: First results showed a good cell survival of MC3T3
cells seeded on the hydrogel constructs but a low cell survival of
encapsulated MC3T3 cells in methacrylamide modified gelatin.
The effect of uv-irradiation on MC3T3 cells was also evaluated and
the preliminary results are promising. This might indicate that the
low cell viability in the hydrogel may be due to radical formation
during uv-irradiation but further research is needed. The HFF cells
demonstrated a good cell survival on the modified 2D PCL films and
3D plotted constructs which indicates that modified PCL is a good
choice of material to construct the outer part of the meniscus.
P153
Coefficient of Friction Measurement for Early Performance
Screening of Artificial Materials in Articulation Against Joint
Cartilage
N. Stark1, B. Thomas2, R. Klabunde1, M. Bürgi1, T. Schwenke1
1
Winterthur/Switzerland, 2Warsaw/United States of America
Purpose: Cartilage replacement with artificial materials may pose as
a successful early intervention procedure in patients with confined
areas of cartilage lesion or degradation. Cartilage – cartilage
articulations have shown a very low coefficient of friction (COF) in
the range of 0.0181. Therefore we hypothesized that the coefficient
of friction measurement of cartilage articulating against artificial
material can provide early information on the general mechanical
performance of a material for this application.
Methods and Materials: COF testing was performed on a pin-on-
rotating-disk wear testing device (Figure 1). Cartilage pins (ø: 6
mm), harvested from the femoral condyle of bovine calves, were
articulated against flat discs of different artificial materials. Constant
sliding velocity (25 mm / s) and a constant vertical load (5 N ->
0.18 MPa) were applied. The contact was submersed in phosphate-
buffered saline at room temperature (20±2°C). Friction forces were
constantly measured for 60 minutes. The artificial materials were:
cobalt-chrome alloy (CoCr, ISO 5832-12), polyethylene (PE, ISO
5834-2), and PCU (Bionate 55D). Three samples were tested per
material, using a fresh cartilage pin for each test.
Results: Two materials (CoCr, PE) experienced an increasing COF
with increasing testing time (Figure 2). The COF of the PCU discs
in articulation against cartilage pins remained constant at a level
of μ = 0.16.
Conclusions: The three different artificial materials, when tested
against bovine cartilage pins, developed different behavior with
respect to their coefficient of friction over time. Along with our
hypothesis, this testing approach may be useful as an early and
quick assessment parameter for the performance of the artificial
material.
1Schmidt, Sah RL. Effect of synovial fluid on boundary lubrication
of articular cartilage. Osteoarthritis and Cartilage 2007
P154
Manipulative structural re-organization of superficial zone
extracellular matrix: articular chondron density increases in
response to non-ablation radiofrequency energy
K. Ganguly1, I.D. McRury2, P.M. Goodwin1, R.E. Morgan2, W.K. Augé,
II3
1
Los Alamos/United States of America, 2Fall River/United States of
America, 3Santa Fe/United States of America
Purpose: Chondron density within the Superficial Zone has been
shown to decreased with age, disease, injury, and in response
to some interventions and may predispose articular cartilage to
extracellular matrix-based failure through an inability to support
the mechanotransductive demands of physiologic loading (1). Since
chondron shape and orientation reflect inter-territorial extracellular
matrix architecture, chondron density is an important descriptor for
functional cartilage (2, 3). Interventions that alter chondron density
may provide insight into the treatment outcome of focal lesions. This
study evaluates radiofrequency energy effects on native Superficial
Zone chondron density in articular cartilage demonstrating early
lacunar emptying without significant surface fibrillation.
Methods and Materials: Radiofrequency energy was delivered by
two methods to ex-vivo femoral condyle osteochondral specimens
obtained from patients undergoing total joint replacement; Ablation
and Non-ablation. Untreated control and treated specimens were
sectioned, prepared with Live/Dead cell viability stain, and assessed
by confocal laser microscopy. Chondron density was determined by
quantifying live chondrocyte populations per square millimeter in
two-dimensional section images.
Results: The mean total Superficial Zone cell number in control
sections was 1480 per mm2. The Ablation method fully corrupted
the Superficial Zone by volumetric loss or near complete cellular
necrosis. The Non-Ablation method retained the Superficial Zone
with a mean total number of cells of 1468 per mm2 (0.8% difference
from control) with increased live chondron density of 12% over
control. Chondron image character was not altered by treatment;
whereas, the increased live chondron density originated from
preferential extracellular matrix volume contraction.
Conclusions: This report suggests that non-ablative radiofrequency
energy can increase articular cartilage Superficial Zone live
chondron density. The Superficial Zone extracellular matrix,
because of its distinctive composition, is uniquely suited to
manipulative structural reorganization. Resetting functional
chondron density patterns may have the potential to create a
more chondro-supportive environment for articular cartilage as it
inherently responds to focal disease (4).
P156
Mechanical Forces can change the microstructure of cartilage
using a joint mimicking bioreactor system
W.H. Choi1, Y.J. Kim1, B.H. Choi2, S.R. Park2, B. Min1
1
Suwon/Korea, Democratic People’s Republic of, 2Incheon/Korea
Purpose: Articular cartilage consists of 4 horizontal layers separated
by cell arrangement and macromolecular (proteoglycan and
collagen) distribution and organization. The zonation is important
to maintain cartilage function such as lubrication and cushion.
In this study, we hypothesized the complex stimuli (combination
of shear, hydrostatic pressure and compression) during the joint
loading movement effects on zonal differentiation by applying
bioreactor system that imitated human joint loading condition.
We examined the change of cell arrangement and macromolecular
organization on porcine cartilage explants.
Methods and Materials: To mimic the joint loading condition, we
manufactured the bioreactor system that has a device similar to the
shape of a femoral and tibial condyle in human knee. This device is
designed to produce the shear and compression forces. Cylinder
shaped cartilage plugs (D X H = 5mm x 4mm) were taken from a
young porcine (2w) femoral head and were placed between both
condylar mold of the device of bioreactor. The cartilage plugs were
stimulated with bioreactor for 1 hour per day over the course of
4 weeks. Control group was static cultured without stimuli. After
4 weeks, the tissues were cut into 2 pieces (the top and bottom
layers) for chemical analysis (glycosaminoglycan (GAG), collagen,
water). Cell arrangement and macromolecular organization were
analyzed through histology.
Results: All groups have a quantitative difference between the
upper and lower layers on chemical contents. The amount of GAG
and collagen was larger in the lower layer, but water content was
not. In histology data, larger amounts of GAG were expressed

on the bioreactor group, while it degraded on the control group
(static cultivation). In the bioreactor group, the cells were arranged
horizontally paralleled to the surface, but control group was not.
The result of this study suggests that complex stimuli could affect
the cartilage zonal differentiation.
Conclusions: Our bioreactor loading condition that imitated joint
loading movement could be a useful system in manufacturing the
native mimic artificial cartilage using cells/scaffold.
P158
Prostaglandin E2 up-regulation by cyclic compressive loading on
3-D tissue of human synovium-derived stem cells and inhibitory
effects of COX2 selective inhibitor or dexamethasone
K. Shimomura1, T. Kanamoto1, N. Nakamura2, T. Mae1, Y. Take1, H.
Kohda1, S. Kuroda1, Y. Akamine1, K. Ota1, H. Yoshikawa1, K. Nakata1
1
Suita/Japan, 2Osaka/Japan
Purpose: Excessive mechanical stress on synovial joint causes
osteoarthritis along with production of prostaglandin E2 (PGE2), a key
molecule of arthritis, by synovial fibroblasts or chondrocytes. However,
the molecular mechanism of PGE2 production by mechanical stress or
the effects of NSAIDs or steroid is still unclear. The purpose of this
study was to examine the expression of PGE2 by cyclic compressive
loading on human synovium-derived mesenchymal stem cells (MSCs)
and clarify the effects of NSAIDs or steroid.
Methods and Materials: Human synovium-derived MSCs were
cultured onto collagen scaffolds to produce three-dimensional
constructs. Cyclic compressive loading (40kPa, 0.5Hz, 1hr) was
applied to the constructs with or without the administration of COX2
selective inhibitor or dexamethasone. After 6 hours incubation,
the concentrations of PGE2, IL-1beta, TNF-alpha, IL-6, IL-8 in
supernatant were measured by HTRF and the mRNA expressions
for COX2 and mPGES-1 genes were examined by RT-PCR.
Results: The concentrations of PGE2, IL-6, and IL-8 in supernatant were
significantly higher by mechanical stress, but those of IL-1beta and
TNF-alpha unchanged. By administering COX2 selective inhibitor, the
increased concentration of PGE2 by mechanical stress was impeded
but those of IL-6 and IL-8 remained high. mRNA levels of COX2 and
mPGES-1 genes were up-regulated by mechanical stress and this up-
regulation of COX2 was not suppressed by COX2 selective inhibitor,
while that of mPGES-1 was suppressed. With dexamethasone, up-
regulation of PGE2, IL-6, and IL-8 was suppressed, and mRNA levels
of COX2 and mPGES-1 were also suppressed.
Conclusions: Mechanical stress on the constructs up-regulated
PGE2 production, while pro-inflammatory cytokines such as IL-
1beta and TNF-alpha did not change. These results indicate that
PGE2 up-regulation by mechanical stress is controlled not via these
cytokines but via another signal pathway. COX2 selective inhibitor
suppressed PGE2 production without changing COX2 mRNAl level,
while dexamethasone did by suppressing COX2 gene expression.
P159
Train the chondrocytes: Mechanostimulation reverses the
catabolic phenotype of human matrix-embedded chondrocytes -
a preliminary report
F. Halbwirth, E. Niculescu-Morzsa, H. Zwickl, S. Nehrer
1
Krems and der Donau/Austria
Purpose: The goal of this study was to determine the effect of cyclic
mechanical stimulation on matrix-embedded human chondrocytes
derived from osteoarthritic cartilage. For that purpose expression
of genes known to be affected in diseased cartilage (MMP-3,
MMP-13, collagen II, aggrecan) and its alteration upon mechanical
stress was investigated via RT-PCR. In addition, matrix-embedded
chondrocytes were histologically evaluated concerning a potential
effect of mechanostimulation on cell morphology.
Methods and Materials: Human articular cartilage was obtained
from osteoarthritis patients subjected to total knee arthroplasty.
After isolation of the chondrocytes from cartilage tissue and an
expansion under standard cell culture conditions for 14 to 20 days
they were embedded in a collagen I-matrix. Following cultivation
for 12 to 16 days, cell-seeded matrices were either mechanically
stimulated (cyclic sinusoid compression regime for 4 days) or
further cultivated without stimulus. For measurement of gene
expression, chondrocytes were isolated and mRNA levels of the
genes of interest were gathered via RT-PCR. Moreover, morphology
and distribution of chondrocytes in the differentially treated
matrices were histologically determined.
Posters 227
Results: Based on data from literature concerning the preferential
targets of the respective proteinases, results were expressed as
ratios of aggrecan and Col 2 to MMP-3 (MI3) and MMP-13 (MI13),
respectively. Both, MI3 as well as MI13, significantly increased by
mechanostimulation compared to control indicating an “anabolic
shift”. Moreover, chondrocyte morphology proved to be drastically
altered by appearing round rather than spindle-shaped in
histological slices of stimulated compared to control matrices.
Conclusions: We got strong indications of a shift from catabolic
towards anabolic transcription activity. Our findings further
suggest the importance of mechanical stress for metabolism and
function of chondrocytes and indicate that the supposed catabolic
phenotype of matrix-embedded osteoarthritic chondrocytes might
be reversible by mechanostimulation.
P160
Change in cartilage quality following opening wedge valgus
tibial osteotomy, measured using dGEMRIC
M. Rutgers1, W. Bartels1, R.V. Heerwaarden2, L.B. Creemers1, R.
Castelein1, W.J.A. Dhert1, K.L. Vincken1, D.B. Saris1
1
Utrecht/Netherlands, 2Woerden/Netherlands
Purpose: Opening wedge valgus tibial osteotomy (VTO) is clinically
effective for the treatment of knee pain caused by medial compartment
osteoarthritis with varus of the knee. Although its effectiveness in
pain reduction has been repeatedly shown, little is known about the
effects of the change in alignment on knee cartilage quality. This study
aims to evaluate the effects of such an osteotomy on knee cartilage
quality, by means of dGEMRIC non-invasive imaging.
Methods and Materials: 10 patients with unicompartmental
osteoarthritis of the knee were treated with opening wedge VTO.
Before and 9 months after surgery, cartilage quality of medial and
lateral compartments were compared using dGEMRIC (3D TFE T1-
mapping). In order to reduce artifacts, hardware was removed
before the post-operative MRI. T1 measurements were made in
selected anterior and posterior regions of interest (area size: 10
pixels) of medial and lateral condylar cartilage. Changes in clinical
scores (VAS for pain, KOOS, KSCRS) were evaluated using paired
T-testing and related to cartilage quality improvement.
Results: Before osteotomy, average T1 was greater in the lateral
compartment than in the medial compartment (p < 0.01). Clinical scores
of patients undergoing osteotomy improved during follow-up (VAS
p=0.02, KOOS pain p=0.05). dGEMRIC cartilage quality changed in varying
degrees post-operatively, from improvement of medial cartilage quality to
further deterioration of both medial and lateral cartilage quality.
Conclusions: dGEMRIC confirms that patients with unicompartmental
knee osteoarthritis have higher cartilage quality in the lateral
than in the medial compartment. While clinical scores improved
following VTO, changes in medial cartilage were accompanied by
either improvement or worsening of lateral cartilage quality. Axis
alignment alteration alone does not prevent further deterioration
of medial cartilage quality. Moreover, clinical improvement does
not seem to depend on cartilage quality. Although dGEMRIC seems
valuable as research tool, its value for patient selection and clinical
decision making should be further evaluated.
P161
Anti-angiogenic properties of articular cartilage
glycosaminoglycans
J.J. Bara1, W.E.B. Johnson2, B. Caterson3, S. Roberts4
1
Oswestry/United Kingdom, 2Birmingham/United Kingdom, 3Cardiff/
United Kingdom, 4Shropshire/United Kingdom
Purpose: Mature articular cartilage is normally avascular, but
vascularisation can occur in diseased cartilage (e.g. osteoarthritis and
degenerative disc disease). This in-growth of blood vessels has been
associated with a loss of proteoglycan from the tissue. Human aggrecan
is known to inhibit the adhesion and migration of endothelial cells in
monolayer culture depending on the presence of its glycosaminoglycan
(GAG) sidechains. We have developed an in vitro 3D model to
investigate whether GAG depletion of cartilage explants increased the
susceptibility of the tissue to invasion by endothelial cells.
Methods and Materials: Bovine cartilage explants were treated
for 24 hours with hyaluronidase. Explants were seeded with
fluorescently tagged human endothelial cells (HMEC-1). HMEC-1
adherence was quantified 4 hours and 7 days after cell seeding. GAG
content was quantified using the DMMB assay and chondrocyte
viability was assessed using live/dead scoring. Biochemical
228 Posters
composition of the ECM was investigated immunohistochemically
with antibodies against aggrecan core protein, chondroitin-6-
sulphate, chondroitin-4-sulphate, keratan sulphate and lubricin.
Results: Hyaluronidase treatment reduced explant GAG content by
79±3% compared to controls. GAG depletion was associated with
significantly more HMEC-1 adherence (11±1 cells/mm) compared
to controls (3±0.4 cells/mm) at 4 hours (p<0.0001). HMEC-1
cells adhered to the surface and deeper/calcified zones of the
cartilage, but did not appear to migrate into the tissue during
culture. Chondrocyte viability was significantly lower in GAG-
depleted explants compared to controls, i.e. 66±2% vs. 87±1%
respectively (p<0.0001); however further experiments confirmed
that reduced cell viability alone did not increase HMEC-1 adhesion.
Hyaluronidase treatment altered chondroitin sulphate patterning,
but not lubricin.
Conclusions: This work supports the hypothesis that GAGs in
mature articular cartilage have anti-angiogenic properties. Hence,
the loss of proteoglycan that occurs in OA may make the tissue
more susceptible to pathological vascularisation. Furthermore,
manipulating GAG content may be a useful technique to control
vascularisation in osteochondral tissue engineering.
P163
The redifferentiation potential of human articular chondrocytes
can be modulated by the pore size of poly(urethane urea)
scaffolds
H. Stenhamre1, U. Nannmark2, A. Lindahl2, P. Gatenholm2, M.
Brittberg3
1
Gäteborg/Sweden, 2Göteborg/Sweden, 3Kungsbacka/Sweden
Purpose: It is suggested that the chemical and physical properties
of scaffolds affect cellular behavior. The objective of this study
was to assess whether a degradable porous poly(urethane urea)
scaffold (ArtelonÒ) could be a suitable material for cartilage tissue
engineering. In addition we investigated whether the pore sizes
of the scaffold could modulate the redifferentiation capacity of in
vitro expanded articular chondrocytes.
Methods and Materials: Scaffolds with different pore sizes, <150µm,
150-300µm and 300-500µm, were seeded with chondrocytes and
cultured in chondrogenic media up to 28 days in vitro.
Results: The chondrocytes redifferentiated and produced cartilage
specific ECM in the poly(urtehane urea) scaffolds. The scaffolds
with the smaller pore size favoured redifferentiation and thus
enhanced the hyaline-like extracellular matrix production.
Conclusions: In conclusion, our results demonstrate that
poly(urethane urea) may be useful as a scaffold material in
cartilage tissue engineering. The chondrogenic differentiation
capacity of in vitro expanded human articular chondrocytes can be
influenced by the scaffold architecture. By tailoring the pore sizes
the performance of the tissue engineered cartilage constructs is
modulated and thus also the clinical outcome in the long run.
P164
Human stem cells culture into biodegradable collagen scaffolds
for guided tissue regeneration.
R.F.C. Marques1, H. Tavares2, A.F. Fraga3, H.M.G. Tómas4 , J.L.
Santos4
1
Poços de Caldas/Brazil, 2São carlos/Brazil, 3São Carlos/Brazil,
4
Funchal - Ilha da Madeira/Portugal
Purpose: Biomaterials like collagen barriers are current employed
in guided tissue regeneration (GTR) for plastic and reconstructive
surgery, dental implants and bone graft procedures. The actual
challenges of sportive medicine are trauma in muscle, tendon
and cartilage structures where clinical and surgical treatment
shows many controversies. The aim of this preliminary study was
to evaluate the behavior of human stem cells and proliferation
after the initial stage of cell adhesion among collagen matrices,
with different compositions, nanostructures and mechanical
properties.
Methods and Materials: After local ethical committee approbation,
human mesenchimal stem cell (hMSC) from fourth passage with
density of 5x104 cells/sample were seeds onto collagen membranes
manufactured by PROCELL BIOLOGICS LTDA. The experiments were
realized with (n=6) for all experimental groups. The cell adhesion
and proliferation was analyzed by fluorescent method using Alamar
Blue assay. In order to evaluate morphological aspects, fluorescent
labeling by Alexafluor were performed.
Results: The cells were monitored every 24h until day 5, when was
performed the analysis of Alkaline Phosphatase expression and the
quantification of total protein. These evaluations were also realized
at day 10, when was determinate the initial cell differentiation. The
proliferation results shows decrease of RFU (Relative Fluorescence
Units) after 24h due to environment changing and cell adaptation
on the morphologies of each collagen membrane. The results of
resasurin increases after 72h and 96h by the proliferation into the
porous surfaces (see Fluorescence Microscopy day 3) and decrease
after 120h. The ends of these evaluations are the comparison
between Alcaline phosphatase expression and amount of protein
for 5 and 10 days.
Conclusions: The biodegradable collagen membranes Bilayer
and Hydroxyapatite coated showed excellent morphology for cell
growing. These biomaterials have presented good properties
for tissue engineering as scaffold for cells culture from human
mesenchymal stem cells.
The authors are grateful to FAPEMIG for the financial support.
P165
TGF-beta1 release from hyaluronan-coated poly-e-caprolactone
nanofibrous and microfibrous scaffolds promotes expression of
collagens type I and II in human mesenchymal stem cells
J.C. Schagemann1, S. Paul1, M.E. Casper2, J. Rohwedel1, J. Kramer1,
J.S. Fitzsimmons2, C. Kaps3, M. Fehr4 , M. Russlies1, S. O’Driscoll2,
G.G. Reinholz2
1
Luebeck/Germany, 2Rochester/United States of America, 3Berlin/
Germany, 4Hannover/Germany
Purpose: The objective was to develop a scaffold that presents cartilage-
like matrix to bmdMSCs together with a release of growth factors for
cell recruitment, proliferation and chondrogenic differentiation, which
would make supply of inductive factors redundant.
Methods and Materials: Nanofibrous (~400 nm) and microfibrous
(~10 um) PCL scaffolds were combined with 1% HMW sodium
hyaluronate (NHA/MHA), 1% sodium hyaluronate and 200 ng TGF-
beta1 (NTGF/MTGF), or 0.1% BSA (N/M). Scaffolds were seeded
with bmdMSCs and cultured without growth factors in vitro.
Cultures with chondrogenic medium served as controls (ScInd).
Proliferation, migration, and release of TGF-beta1 were investigated.
Cell differentiation was evaluated by rtPCR.
Results: MTGF released TGF-beta1 for 72 hrs, NTGF for 12 hrs.
None of the scaffolds recruited bmdMSCs. Composites containing
HA demonstrated elevated seeding efficiency. TGF-beta1 had no
proliferative effect. There was an increase in COL2 expression with
TGF-beta1 containing scaffolds promoting higher yields. This was
independent of scaffold composition or fiber size. Significantly
higher values were found for ScInd. All scaffolds allowed for COL1
expression with TGF-beta1 containing scaffolds showing highest
yields. COL1 expression decreased in NTGF/MTGF and NHA/MHA after
21 days, whereas scaffolds supplied with TGF-beta1 in the medium
promoted an increase until day 21. PCR identified also bone-specific
markers, yet not as striking as for bmdMSCs supplied with osteogenic
medium. Varying alkaline phosphatase levels were found in N/M and
NHA/MHA, and a decline in NTGF/MTGF after 21 days. Osteopontin
decreased regardless of scaffold composition after 21 days.
Conclusions: Combining HA and TGF-beta1 with PCL scaffolds
fosters cell seeding and proliferation. TGF-beta1 released from
MTGF promotes COL2 expression, although not as effective as
TGF-beta1 supplied with the medium. TGF-beta1 induces also COL1
followed by a decline after 21 days when released from NTGF/
MTGF. The presence of nanofibrous PCL and HA suppresses COL1
compared to a TGF-beta1 containing milieu. No significant impact
of fiber size was seen in our model.
P166
The assembly of platelet lysate-loaded chitosan-chondroitin
sulfate nanoparticles as a three dimensional hydrogel construct
for encapsulation of adipose derived stem cells for cartilage
regeneration
V. Espírito Santo, M.E. Gomes, J.F. Mano, R.L. Reis
Caldas das Taipas, Guimarães/Portugal
Purpose: Platelet lysates (PL) are an outstanding natural source
of growth factors (GFs) that can play an enhancement role over
the proliferation and differentiation ability of mesenchymal stem
cells. Moreover, their autologous nature is an advantage for
regenerative medicine applications as they can be easily obtained

from a simple blood sample from the own patient in treatment.
Natural based chitosan/chondroitin sulfate nanoparticles (CH/CS
NPs) were developed and characterized with the ultimate goal of
encapsulating bioactive agents to promote and enhance cartilage
regeneration. Previous studies performed in our group reported
the successful incorporation of PL in these NPs, which were then
released in a controlled manner in two and three dimensional
(2D and 3D) in vitro cultures of human adipose derived stem cells
(hASCs), enhancing the proliferation rate of hASCs while they are
differentiating into the chondrogenic phenotype. When used at
determined concentrations, the PL-releasing NPs can assemble
in simple and quick mode and form a 3D stable hydrogel while in
suspension with hASCs, following a mild centrifugation, allowing
the cells to be entrapped in this enriched 3D environment.
Methods and Materials: The hydrogels were cultured in vitro in
chondrogenic medium and basal medium up to 28 days and were
characterized for cell viability, proliferation, glycosaminoglycans
production, histology, immunohistochemistry and gene expression
(by polymerase chain reaction).
Results: The presence of high levels of GFs such as Transforming
Growth Factor β1 (TGF-β1) influence the biological response of
the entrapped cells, stimulating their viability, proliferation and
the production of a cartilage extracellular matrix throughout the
culture time.
Conclusions: PL is a strengthening factor in the morphological
stability of the assembled NPs as they enhance the interactions
between the individual natural-based complexes and, in
combination with ASCs, it is obtained an innovative system with a
high potential of application in cartilage tissue regeneration.
P167
Long-term clinical and radiological follow-up of collagen
meniscal implants.
P.C. Verdonk1, J. van der Maas2, T. Tampere2, K.F. Almqvist2, R.
Verdonk2
1
Gent-Zwijnaarde/Belgium, 2Gent/Belgium
Purpose: There is growing evidence in literature that meniscal
substitution in the post-meniscectomized knee results in significant
pain relief and functional improvement of the involved joint. The
collagen matrix implant (CMI) is a bioabsorbable scaffold to substitute
a partial medial meniscus defect. Long-term data on clinical and
radiological outcome of this CMI implant are however scarce.
Methods and Materials: We evaluated 15 transplants with a mean
time of follow-up of 9,9 ± 1,9 years, mean age at surgery was 31,6
years (range 16-49). Clinically, the patients were evaluated using
a KOOS, SF-36, HSS, VAS, Tegner and Lysholm score. Each patient
received radiographs (AP, profile and Rosenberg view). Radiological
outcome parameters were joint space width narrowing and Fairbank
changes and were scored according to IKDC. Failures were defined
as graft removal or conversion to an arthroplasty.
Results: HSS-scores at long-term follow-up were 184 ± 21. Lysholm-
score was 84 ± 19, which was defined as a good result. Mean
VAS-score was 1,7 ± 2,7, mean Tegner was 4 ± 2. There were no
significant differences between following subgroups: left or right
knee and male or female. Five (33%) of the fifteen implants failed
after a mean of 3,8 years.
Conclusions: Transplantation of a collagen matrix implant scaffold
can significantly relieve pain and improve function of the knee
joint. Survival analysis showed that this beneficial clinical effect
remained in approximately only 70% of the patients at ten years.
P168
Chondrocyte viability is affected by PCL molecular weight in
structurally graded polycaprolactone (SG-PCL) scaffolds
C.B. Foldager, B.B. Christensen, A. Kristiansen, D. Le, J.V. Nygaard,
C. Bünger, M. Lind
1
Aarhus/Denmark
Purpose: The aim of the study is to investigate cellular effects
of the physical structure in a novel SG-PCL scaffold on human
chondrocytes, when the scaffold is produced with different
molecular weights (MW) and water-dioxane ratios.
Methods and Materials: Rapid prototyping was used to make
a porous PCL backbone macro-structure (Ø=4mm, height=2,
MW=50kDa). Micro and nano-structure in the scaffold porosity
was made by lyophilization of a PCL, water and dioxane mixture.
Two different lyophilized structures was made using PCL with a
Posters 229
MW of either 25kDa or 50kDa. Based on a large sweep, two water-
dioxane (0.0105 and 0.0405) ratios were used producing four
different scaffolds. The cells were cultured for 1, 3 and 6 days.
Using quantitative RT-PCR expression of chodrogenic markers
sox9, collagen type 1 and 2 and aggrecan as well as cytoskeletal-
and adhesion markes beta-actin, fibronectin, vinculin and integrin
10aplha were investigated. Reference genes were B2M and RPL13A.
Possible differences were investigated using three-way ANOVA
measurements.
Results: Scaffolds with a lyophilized PCL compound of 25kDa
resulted in a significantly higher expression of collagen type 2,
aggrecan and vinculin (p
Conclusions: We successfully constructed a structurally graded
scaffold for cartilage repair. There was a significant positive effect
of the structure made by 25kDa-lyophilized material compared
to 50kDa. The lower MW might lead to a combination of faster
degradation time and increased chondrogenic viability for scaffold-
aided cartilage repair techniques.
P169
In situ gelling Tetronic depots for sustained delivery of growth
factors
J. Couceiro, A. Rey-Rico, M. Silva, A. Concheiro, C. Alvarez-Lorenzo
Santiago de Compostela/Spain
Purpose: Local delivery of growth factors using minimally invasive
in situ gelling solutions is an efficient and patient-friendly
alternative to surgical grafts. The aim of this work was to evaluate
the potential of X-shaped poly(ethylene oxide)-poly(propylene
oxide) block copolymers, named as Tetronics, as components of
liquid and easily syringeable solutions that undergo a transition to
the gel state at the body temperature
Methods and Materials: A cytoxicity screening of a wide number
of Tetronic varieties, the in situ gelling capability of Tetronic 908,
1107, 1301 and 1307 solutions was rheologically characterized. The
release profile of BSA and BMP-2 was recorded and the ALP activity
measured using mesenchymal stem cells.
Results: The gelling temperature of 20% w/w solutions of 908
was 33ºC and of 1107, 1301 and 1307 solutions around 25ºC; being
highly viscoelastic at 37ºC. Such solutions evidenced a remarkable
capability to sustain the delivery of proteins under physiological-
like conditions. Particularly, formulation of BMP-2 led to relevant
differentiation of mesenchymal cells to osteoblasts, quantified as
alkaline phosphatase activity.
Conclusions: Tetronic varieties (908, 1107, 1301 and 1307)
combine good cytocompatibility with the ability to undergo sol
to gel transitions at body temperature. Autoclaving does not
alter the rheological properties, which is an important aspect
for the development of injectable formulations. Tetronic gels can
modulate the release of proteins depending on hydrophilicity and
molecular weight of the copolymer. Initially rapid delivery of an
important amount of BMP-2 followed by slower release enables
the Tetronic gels to exhibit a marked ALP activity at day 7 and
matrix mineralization at day 14. Thus, Tetronic is a very promising
material for syringeable in situ gelling formulations with osteogenic
performance, acting as a suitable BMP carrier. Similar application
could be used for cartilage cells differentiation by means of delivery
of proper growth factors.
P170
Development of a structurally graded polycaprolactone (SG-PCL)
scaffolds for hyaline cartilage repair
B.B. Christensen, C.B. Foldager, A. Kristiansen, D. Le, J.V. Nygaard,
C. Bünger, M. Lind
Aarhus/Denmark
Purpose: Rapid prototyping is a very precise scaffold manufacturing
technique where scaffolds can be constructed from MRI or CT scans
to fit into the individual tissue defects. The aim of this study is to
develop a novel Structurally Graded Poly-Capro-Lactone scaffold
using rapid prototyping for cartilage tissue engineering.
Methods and Materials: A novel SG-PCL scaffold was constructed
using rapid prototyping (Ø 4 mm, height 2 mm). PCL fibers (MW
50 kDa) with a diameter of 120 µm were plotted producing a
3-dimensional web. The scaffold was subsequently submerged
into a mixture of dioxane, PCL and water, and lyophilized at -32°C,
creating an extremely porous graded structure. By shifting the
water-dioxane ratio, 16 batches of scaffolds with different pore
230 Posters
sizes were made. Using scanning electron microscopy, two scaffolds
were selected, based on porous structure. The two scaffolds were
then constructed with a graded structure of either 25 kDa or 50 kDa,
giving a total of four different scaffolds. They were cultured with
human chondrocytes and the viability was analyzed using confocal
microscopy after 1, 3 and 6 days. The scaffolds were rated based on
cell migration, cell shape and distribution of viable cells.
Results: The scaffolds contained macro-, micro-, and nano-pores.
A large difference in investigated parameters was observed and a
water-dioxane ratio of 0.0415 provided the most viable environment
for chondrocytes.
Conclusions: We successfully constructed a SG-PCL scaffold that
can be used in future in vivo experiments, and has the potential of
subsequent functionalization with nanoparticles and growth- and
differentiation factors.
P171
A Hybrid Scaffold For The Functional Repair Of Articular Cartilage
Defects
K.W. Ng1, H. Hsu2, K. Joh1, P.A. Torzilli1, R.F. Warren1, S.A. Maher1
1
New York/United States of America, 2Taichung/Taiwan
Purpose: Clinical success in the use of scaffolds to treat focal
cartilage defects depends on the scaffold’s ability to bear
mechanical loads and integrate with surrounding tissue. In this
study, we hypothesized that a hybrid scaffold with a solid polymer
core and a porous periphery would improve the load-bearing
ability of the scaffold while maintaining the ability to facilitate
chondrocyte migration.
Methods and Materials: Scaffold Fabrication: Cylindrical gelatin
sponges (Ø7x2mm) were impregnated with 10% polyvinyl-alcohol
(PVA) solution and freeze-thawed 6 times. In half of the sponges, a
hybrid scaffold was creating by excising a Ø5mm central core and
filling the hole with 10% PVA solution. All sponges were freeze-
thawed an additional 6 times and digested with collagenase.
Scaffold morphology was evaluated using environmental scanning
electron microscopy. Mechanical properties were determined using
unconfined compression tests. In vitro cartilage defect model:
Disks of middle-zone, calf cartilage were cored to create annuli.
These annuli were treated briefly with collagenase and then cell-
free porous scaffolds were press-fit to fill the central defects of the
annuli. These scaffold-cartilage constructs were then cultured and
histologically analyzed.
Results: Macropores were present in the porous scaffolds and
the porous periphery of the hybrid scaffolds (Figure 1), with
no significant differences in average pore diameter (~15μm) or
apparently porosity (~80%). Compressive modulus was ~12kPa
for porous scaffolds and ~55kPa for hybrid scaffolds. In the
defect model (Figure 2), chondrocytes infiltrated the scaffolds
and produced a proteoglycan rich matrix, with cell number and
migration distance increasing significantly over time to ~500 μm
after 28 days.
Conclusions: This hybrid scaffold presents a modular design where
the compressive modulus of the polymer core can be controlled and
the porous periphery can facilitate cell infiltration towards tissue
integration. Varying the polymer concentration of the solid core to
improve load bearing of the scaffold will be explored in future work.
P172
Localization of VEGF in cartilage repair using bioabsorbable
synthetic polymer scaffold
R. Sakata1, H. Fujioka2, K. Makino2, T. kokubu1, I. Nagura1, N.
Toyokawa1, A. Inui1, M. Satake3, H. Kaneko3, M. Kurosaka1
1
Kobe/Japan, 2Nishinomiya/Japan, 3Tokyo/Japan
Purpose: The purpose of the present study is to investigate the
localization of VEGF at the early stage of cartilage repair using
bioabsorbable synthetic scaffold in a rabbit model.
Methods and Materials: Forty-eight female Japanese white rabbits
were used in this study. Osteochondral defect (5 mm diameter, 5 mm
depth) was created on the patellar groove of the right knee under
general anesthesia using the Osteochondral Autograft Transfer
System. Then, rabbits were divided into two groups. The defect was
treated with PLG scaffold in the scaffold group and the defect was
left untreated in the control group. At postoperative 1, 3, 5 days
and 1, 2, 4 weeks, the specimens are examined macroscopically
and histologically. Immunolocalization of VEGF and type II collagen
were also analyzed.
Results: By postoperative week 1, the osteochondral defects
of both groups were filled with hematoma. At week 2, fibrous
membrane covered the surface and the border between the defect
and surrounding bone became unclear at the deep area in both
groups. At week 4, cartilage regeneration was found at the whole
surface of the scaffold. In the control group, the regenerated tissue
was concaved and cartilage regeneration was observed only at the
peripheral area of the defect even at week 4. VEGF was detected
in the whole area of defect by week 2. At week 4, in the scaffold
group, localization of VEGF decreased and type II collagen was
observed at the surface of the scaffold. VEGF was still remained at
the concaved surface in the control group.
Conclusions: In the present study, VEGF was localized at the whole
osteochondral defect and decreased time dependently at the
surface of regenerated tissue, and type II collagen was observed
at week 4. The angiogenesis during chondrogenic differentiation is
one of the important issues affecting the application of stem cells
for cartilage regeneration.
P173
Osteochondral defect repair using a bilayer scaffold implant
containing BMP-7 and IGF-1
M. Ast1, P. Razzano2, D. Grande2
1
New Hyde Park/United States of America, 2Manhasset/United
States of America
Purpose: Articular cartilage injuries in the knee are painful and
disabling, predisposing the patient to early arthritis. In this study
we use a bilayer scaffold implanted with cells engineered to
overexpress BMP-7 and IGF-1 in an attempt to improve the quality
of the repair tissue. It is our hypothesis that gene-modified tissue
engineering using this bilayer implant will regenerate a durable,
physiologic articular surface.
Methods and Materials: BMP-7 and IGF-1 were isolated from
human cells using RT-PCR and retroviral expression plasmids were
generated for each based on the LNCX series of vectors. Northern
blot analysis was then used to demonstrate effective gene transfer
and ELISA was performed to confirm BMP-7 and IGF-1 secretion from
primary rabbit periosteal cells. 4 x 106 gene-enhanced periosteal
cells were seeded onto PGA scaffolds prior to implantation. Animals
were randomly assigned to one of four experimental groups which
included: 1) Empty Defect, 2) PGA Alone, 3) LNCX/PGA, 4) BMP-1/
IGF-1. The BMP-7 cell laden scaffold was placed in the bony defect
and the IGF-1 containing scaffold layer was placed congruent with
the cartilage layer. Animals were then sacrificed at 6, 10, 12, and 24
post operative weeks. The rabbit knees were fixed and evaluated
for histology. Histological sections were graded using a modified
O’Driscoll scoring system.
Results: The experimental defects treated with a bilayer BMP-7 and
IGF-1 scaffold showed histologically superior repair tissue when
compared to each of the other groups.
Conclusions: Gene-modified tissue engineering using a bilayer implant
containing both BMP-7 and IGF-1 to repair an osteochondral defect
can regenerate a histologically superior repair tissue when compared
to no intervention, or implantation of an PGA scaffold alone.
P174
Growth Factor Release from Platelet-rich Plasma (PRP) Loaded
TRUFIT(TM) Scaffolds without the Use of Thrombin
A. Au
Andover/United States of America
Purpose: To assess the loading of PRP in TRUFIT scaffolds,
determine the in vitro growth factor (GF) release kinetics from
thrombin-free PRP-loaded TRUFIT scaffolds, and the activation
state of the platelets.
Methods and Materials: PRP was produced using Caption PRC and
loaded into TRUFIT scaffolds. No thrombin was used in any part of
the process. To measure GF release, PRP-loaded scaffolds (n=3)
were placed in DMEM media at 37ºC. During the course of 10 days,
media was collected at each time point and replenished with fresh
DMEM. GF concentration in the collected media was measured by
ELISA. Platelet activation in PRP and whole blood was quantified by
microparticle count, percentage of P-selectin+ platelets and PAC-1+
platelets using flow cytometry (n=3).
Results: PRP filled up (95.0±7.3)% of the voids in TRUFIT scaffolds
after loading. PRP gelled in TRUFIT scaffolds in ~5 minutes, faster
than PRP alone and likely owing to the calcium sulphate in the

implant. The significantly higher percentage of microparticles,
P-selectin positive platelets and PAC-1 positive platelets in PRP than
whole blood indicates the platelets in PRP were activated, even
without thrombin. SEM showed activated platelets of spiculated
shape and mesh-like fibrin network within the scaffold. The gradual
release of GFs from PRP-loaded TRUFIT scaffolds continued out to
10 days in vitro. The 10-day cumulative amount of VEGF, TGF-β1 and
PDGF-AB released was (2.7±0.9), (4.0±1.8) and (0.8±0.0) times the
GF content of lysed whole blood of the same volume respectively.
The low enrichment of PDGF-AB is expected as the concentration of
PDGF-AB in PRP generated by Caption was less than 1.5 times the
concentration in whole blood.
Conclusions: PRP-loaded TRUFIT scaffolds released GFs gradually
for up to 10 days without thrombin. TRUFIT scaffolds with PRP can
be used to deliver autologous GFs to repair sites.
P175
In vitro 3-D scaffold-based cultures for potential human meniscus
repair
U. Freymann, D. Paape, L. Litau, L. Morawietz, K. Neumann, M.
Endres, C. Kaps
Berlin/Germany
Purpose: Treatment options for meniscal lesions are rarely developed.
Recent approaches use scaffold-based techniques based on the
long experience with matrix-induced treatment options for hyaline
cartilage.The aim of the study was the investigation of meniscal matrix
formation of in vitro expanded human meniscal cells in a 3-dimensional
bioresorbable scaffold for potential meniscal repair approaches.
Methods and Materials: Cultivation of the human meniscus
cells was performed in a resorbable scaffold material made of
polyglycolic acid (PGA) with the application of hyaluronic acid (HA).
To investigate the impact of human serum (HS) and HA as potential
inducing factors for meniscus matrix formation, high-density
micromass cultures supplemented with human serum (HS) or HA
were additionally examined in comparison to an non-stimulated
control. Biocompatibility and cell vitality of human meniscus cell
seeded PGA scaffolds were evaluated by fluorescein diacetate (FDA)
and propidium iodide (PI) staining. Verification of typical meniscal
markers like type I collagen and other extracellular matrix molecules
was performed histologically, immunohistochemically and by gen
expression analysis in meniscus cell seeded PGA scaffolds and in
high-density micromass cultures.
Results: In results, 3-D scaffold-based meniscus cultures showed an
excellent cell vitality and cell expansion over an observational period of
21 days. On the protein level type I collagen and proteoglycan expression
was documented. Gen expression analysis confirmed the expression of
meniscus-specific markers. Supplementation of HS or HA in high-density
cultures was shown to stimulate meniscal matrix formation.
Conclusions: In this study we demonstrated, that in vitro expanded
human meniscus cells allow for formation of meniscal matrix
components when cultured in PGA scaffolds supplemented with HS
and HA. This results encourage scaffold-based approaches for the
treatment of meniscal lesions.
P176
Porous Poly(vinyl Alcohol) Hydrogel Matrix-Engineered Bio-
Synthetic Cartilage
D.A. Bichara1, X. Zhao1, H. Bodugoz-Senturk1, F. Ballyns2, L.
Bonasser2, M.A. Randolph1, O.K. Muratoglu1
1
Boston/United States of America, 2Ithaca/United States of
America
Purpose: Poly(vinyl alcohol) (PVA) gel is a non-degradable material
that can be tailored to be viscoelastic and have a high water
content. We hypothesized that porous PVA combined with articular
chondrocytes suspended in fibrin gel (FG) creating a PVA-FG hybrid
construct could mimic the properties of native cartilage.
Methods and Materials: PVA hydrogel was prepared by theta
gelation by dissolving PVA, polyacrylamide-co-acrylic acid and
poly(ethylene glycol) in 90°C deionized water. The solution was
molded and cooled down for gelation. Hydrogel was imaged by SEM
(Fig. 1A). Chondrocytes from swine knees were isolated, expanded
and mixed with thrombin and fibrinogen to create FG that was injected
into PVA discs (6mm x 2.1mm) and a three layered construct model
(Peretti, Tissue Eng. 1999). Constructs were implanted for 12w into
nude mice and subjected to histological and immunohistochemical
analysis. PVA discs underwent creep strain by applying a 0.5MPa
Posters 231
compressive stress for 10h followed by a 10h relaxation period under
0.05MPa. Integration strength testing was performed on the three
layered constructs using plexiglass rods mounted in a mechanical
test frame and pulled to failure in tension at a displacement rate of
10 µm/s, with resultant loads recorded at 5Hz.
Results: All constructs had a similar appearance to native cartilage
shown by Safranin-O (Fig. 1B) and toluidine blue stains (Fig. 1C).
Specimens stained positive for COL II (Fig. 1D). Creep strain (%) after
20 h of loading showed the PVA-FG chondrocyte group performing
similarly to native cartilage (Fig. 1E). The presence of cells and FG
significantly enhanced the integration strength of layered constructs
(p<0.05 for both parameters by 2 way ANOVA). The combination of
cells and fibrin glue increased integration strength more than 11 fold
from 12 kPa to 133 kPa (Fig. 1F).
Conclusions: Bio-synthetic cartilage was engineered using articular
chondrocytes in vivo with a novel porous PVA-gel hybrid.
P177
Mechanical properties of PLLA scaffolds for cartilage tissue
engineering depending on their micro and macrostructure
V. Acosta1, H. Deplaine2, I. Ochoa1, G. Gallego Ferrer2, M. Doblaré1,
J.L. Gómez-Ribelles2, J.M. García-Aznar1
1
Zaragoza/Spain, 2Valencia/Spain
Purpose: Actually, the application of biodegradable polymeric
materials with three-dimensional structure to facilitate
the adhesion, diffusion and proliferation of cells for tissue
regeneration represents an important field of investigation. The
structural scaffolds not only permit passive mechanical support
but also perform an interactive physical-chemical role in the tissue
regeneration. This work presents a characterization of Poly,L-
lactic acid (PLLA) scaffolds, comparing experimental mechanical
properties (Young and Aggregate Modulus), permeability, porosity
and pore size. This information allows a better understanding of
how the mechanical properties of PLLA scaffolds change depending
on the micro and macrostructure. Different concentrations of PLLA/
Dioxan and PLLA/Porogen could be used in order to achieve a
correct structural scaffold’s design.
Methods and Materials: Uniaxial Compression (Unconfined (UC)
and Confined (CC)) static tests were performed to characterize the
mechanical properties of the scaffolds. To determine a relation between
interconnected porosity and pore size, a permeability test was carried
out.The microstruture was obtained by micro CT and SEM.
Results: Regarding the PLLA/Porogen concentrations, a higher
amount of Porogen causes lost of stiffness and raise of the
permeability and porosity values (figure 1). macropore distribution
and micropore sizes are associated with an augment of structure
stiffness (figure 2). The results show that an increase of the wt%
PLLA augments the uniformity and the distribution of scaffold’s
pores and trabeculae, granting higher mechanical properties
illustrated by the Modulus values. When the wt% PLLA increases
the permeability value decreases. All this results are independent
from the changes in porogen relation.
Conclusions: It’s important to underline that if scaffolds are
designed for confined application, Aggregate Modulus should be
taken as critical factor, whereas for scaffolds subject to a high
lateral deformation, the Young Modulus represents the most
relevant design parameter. A correct scaffold design should take
into account the microstrututre as a key factor to control the
mechanical properties.
P178
Ultrasonic Noninvasive Evaluation of Preconditioned Scaffolds in
Articular Cartilage
S.R. Ghorayeb1, T. Awad1, P. Razzano2, D. Grande2
1
Hempstead/United States of America, 2Manhasset/United States
of America
Purpose: While several imaging modalities have been utilized to
observe articular cartilage injuries in the knee, ultrasonography
has not played a major role in this area. This study demonstrates
that ultrasound may be used to non-invasively monitor the
healing process of osteochondral defects that are implanted with
preconditioned bioactive scaffolds.
Methods and Materials: Rabbit marrow stromal cells were
retrovirally transduced with either BMP-7 or IGF-1 genes and
altered for 9 weeks in non-woven PLLA scaffolds, then frozen and
lyophilized. A total of 16 adult male White New Zealand rabbits
232 Posters
underwent bilateral knee arthrotomies to create 3.7 mm diameter
osteochondral defects. 24 defects were randomly implanted with
preconditioned PLLA scaffolds and 8 defects were left empty as
controls. The knees were then harvested at 3, 6, and 12 weeks
post-surgery and evaluated using scanning acoustic microscopy.
B-scans were obtained and compared to histological results.
Results: The osteochondral defects were clearly observed in each
ultrasound signature. Images containing an empty defect were
characterized as having a gap between the grooves of the femur,
while those containing a scaffold displayed a filling within the area.
There were no significant differences between images of scaffolds
treated with IGF-1 or BMP-7. A defect depth of 4 mm and scaffold
thickness of 2.5 mm were measured. Also a gradual increase of
healing bordering the defects for the 3, 6 and 12 week samples was
observed. Histological results revealed similar outcomes where the
scaffold was still intact along with the cartilage and minimal bone
growth within the defect.
Conclusions: Ultrasound can aid in monitoring implantation of
preconditioned scaffolds in osteochondral defects and thus assessing
the healing process and cartilage/bone quality. This method was
successful at distinguishing the physical border between the defect
and the scaffold. Lateral edge integration was observed in the B-scans
and correlates well with histological results.
P179
Cell proliferation and differentiation capacity of magnetically
labeled mesenchymal stem cells – the effect of an external
magnetic force
G. Kamei, N. Adachi, H. Shibuya, Y. Nagata, S. Ohkawa, M. Ochi
Hiroshima/Japan
Purpose: Many studies on regenerative medicine are currently
focused on tissue engineering. Tissue engineering usually requires
technically demanding procedures with proper scaffolds or growth
factors. Therefore, intravenous or intraarticular cell transplantation
without scaffolds and growth factors is a more attractive option. We
developed a novel stem cell delivery system for regenerated medicine
using MSCs with superparamagnetic iron oxide (magnetically labeled
MSCs:m-MSCs) and an external magnetic device to accumulate a
relatively small number of MSCs to a desired area. But, it had not been
approved whether m-MSCs that were exposed to external magnetic
force could proliferate and differentiate. The purpose of this study
was to evaluate cell proliferation, differentiation into chondrogenesis,
osteogenesis and adipogenesis of m- MSCs that was exposed to
external magnetic force, and the effect of the magnetic force strength
and exposure time.
Methods and Materials: MSCs were labeled overnight with
ferucarbotran and protamine as a transfection agent. We
evaluated cell proliferation using cell-counting kit and compared
the absorbance of day 0 with that of day 7 about non-magnetically
labeled MSCs (non m-MSCs), m-MSCs and m-MSCs that were
exposed to magnetic force with various condition (Magnetic force:
0.6T, 1.5T, and 3.0T. Exposure time: 10 minutes, 30minutes and 60
minutes). Also we evaluated chondrogenesis, osteogenesis and
adipogenesis in their conditions.
Results: Cell proliferation of m- MSCs that were exposed to
magnetic force was significantly larger than that of m-MSCs and non
m-MSCs. And magnetic labeling and exposure to magnetic force do
not have any adverse effects to chondrogenesis, osteogenesis and
adipogenesis.
Conclusions: This study showed that magnetically labeling of MSCs
had no advertise effect on cell proliferation and differentiation, and
we could use them safety. We believe that we can apply this novel
stem cell delivery system using m-MSCs and an external magnetic
device for regenerated medicine.
P180
Cartilage tissue engineering by human embryonic stem cells
K. Ridgway1, H. Jia1, S.C. Dickinson1, T.J. Sims1, S. Minger2, A.P.
Hollander1, W. Kafienah1
1
Bristol/United Kingdom, 2London/United Kingdom
Purpose: The use of human embryonic stem cells (hESCs) as a
source for chondrogenic stem cells remains to be established.
Previous attempts were qualitative and limited to using H1 and H9
cell lines in pellet cultures or hydrogels. Our aim was to investigate
quantitatively the optimal factors that drive cartilage tissue
engineering by hESCs using therapeutic biodegradable scaffolds.
Methods and Materials: Multipotent mesenchyaml stem cell-like
cells (MSC-hESCs) were derived from the hESC line, KCL-2, by plating
7-day embryoid bodies (EBs) in serum containing medium with FGF-2.
The cells were characterized for surface markers by FACS and were
seeded onto PGA or HYAFF-11 scaffolds with or without fibronectin
coating. The constructs were incubated with different combinations
of growth factors for 35 days on a rotating platform and markers of
chondrogenic differentiation were analyzed by quantitative RT-PCR
and immunoassays.
Results: MSC-hESCs had fibroblastic morphology and expanded
for more than 60 days. They expressed several makers found on
adult MSCs including CD105, CD73, CD117, BMPR1 and VCAM and
were multipotent upon adipogenic, osteogenic and chondrogenic
stimulation in monolayer. The best chondrogenic stimulation on 3D
scaffolds was found with fibronectin-coated scaffolds in the presence
of TGF-β1 and BMP-7 but not with BMP-2, BMP-7 or TGF-β1 alone.
Quantitative analysis of dry weight, aggrecan and type II collagen
mRNA and protein in cartilage engineered constructs revealed
levels that were 10-30% of constructs engineered using adults MSCs
stimulated with TGF-β3. The MSC-hESC constructs had double the
hydroxyproline content when compared to adult MSC constructs.
Conclusions: The data highlights the importance for quantitative
analysis in assessing the quality of cartilage tissue engineering
using hESCs and the need for optimal matrix and soluble signals to
drive their chondrogenic differentiation.
P182
The influence of culture media and supplements on phenotype
and differentiation properties of human bone-marrow derived
mesenchymal stromal cells
S. Hagmann, B. Moradi, W. Richter, T. Gotterbarm
Heidelberg/Germany
Purpose: Chondrogenic differentiation of bone marrow-derived
mesenchymal stromal cells (BM-MSCs) is of major importance
in modern tissue engineering, including novel cartilage repair
procedures. Different cell isolation and expansion conditions
have been described, with distinct variations of culture media,
growth factors and other supplements. The aim of this study was
to analyse the influence of culture media and supplements on
immunophenotype and chondrogenic differentiation potential of
human BM-MSCs.
Methods and Materials: Bone marrow derived mononuclear
cells (BM-MNCs) were isolated with gradient centrifugation and
expanded in different cell culture media (alphaMEM, DMEM-
LG, DMEM-HG, DMEM-F12, n=6), with different supplements
described earlier. Additionally, cells from 6 donors were expanded
in alpha-MEM and DMEM with or without addition of bFGF. In
P4, immunophenotyping was performed by FACS analysis for
fluorochrome labelled CD10, CD13, CD14, CD34, CD44, CD45,
CD49, CD73, CD90, CD105, CD117, CD133, CD 140b, CD 166, CD271,
CD340, Stro-1 and HLA-ABC. Also, chondrogenic and osteogenic
differentiation were induced using standard protocols.
Results: Cell proliferation differed substantially between the
media with significantly lower growth index values for DMEM-LG
with 10% FCS. “Negative markers” for MSCs (e.g. CD14, CD34,
CD45) showed a high reliability in all donors and media, while
expression of certain surface markers (e.g. CD10, CD49) was
strongly donor-dependent. The expression of CD10 and CD146
showed significant differences depending on the media used for
expansion. Supplementing bFGF caused significant changes of
CD10, CD49 (Fig.1) and CD146 expression while the expression
of CD13, CD73 and CD133 appeared to be independent of bFGF
addition. “Negative” surface markers showed to be independent
of bFGF addition. The different cell culture media had a significant
influence on the differentiation ability of MSCs, with the highest
chondrogenic potential obtained after expansion in DMEM-LG
with 10% FCS.
Conclusions: Our results show that distinct culture media
have a determining influence on both immunophenotype and
therapeutic potential of MSCs.

P183
Bone marrow-derived culture-expanded mesenchymal stem cells
to augment healing of chondral lesions treated with microfracture
W. McIlwraith1, D.D. Frisbie1, W.G. Rodkey2, J.R. Steadman2, J.
Kisiday3, C. Kawcak1
1
Fort Collins/United States of America, 2Vail/United States of America
Purpose: We tested bone marrow-derived culture-expanded
mesenchymal stem cells (BMSC) to augment healing after full-thickness
cartilage defects were microfractured. We hypothesized that intraarticular
injection of autogenous BMSC with microfracture would enhance healing
in full-thickness defects compared to microfracture alone.
Methods and Materials: At index surgery, ten skeletally mature
horses had 1cm2 defects arthroscopically made on medial femoral
condyles of both stifles (femorotibial joints). Defects were debrided
to subchondral bone followed by routine microfracture. One month
later, one medial femorotibial joint in each horse randomly received
intraarticular injection of 20x106 BMSC with hyaluronan or hyaluronan
alone. A routine strenuous postoperative rehabilitation protocol
was followed. Throughout the study, horses underwent routine
musculoskeletal and radiographic examinations bimonthly and re-
look arthroscopy at six months post index surgery. All examinations
were done with evaluators unaware of treatment. Horses were
euthanized 12 months post index surgery, and study joints underwent
MRI. Gross, histologic, histomorphometric, immunohistochemical
and biochemical examinations were performed.
Results: Subjective arthroscopic and gross evaluation of study
joints confirmed significantly increased repair tissue firmness
in BMSC-treated joints. There also was a trend for better overall
repair tissue quality (cumulative score of all arthroscopic and gross
grading criteria) in BMSC-treated joints. Immunohistochemical
analysis demonstrated significantly greater levels of aggrecan
in repair tissue associated with BMSC treatment although no
difference in type II collagen was noted. There were no significant
effects between treatment groups on clinical (musculoskeletal,
radiographic or MRI) parameters. Likewise, histologic analysis of
synovial membrane and articular cartilage including repair tissue
showed no differences between treatments.
Conclusions: Long-term improvement was enhanced in repair
tissue firmness and aggrecan staining following intraarticular
administration of BMSC. These findings persisted even with
strenuous exercise and may be of long-term clinical benefits in
other species. These positive findings support further in vivo
research, including human clinical trials to use BMSC to enhance
outcomes of microfracture.
P184
The chondrogenic potential of human synovial tissue derived
from young adults with femoral acetabular impingement and
from elderly osteoarthritic patients
Y. Himeda, N. Shintani, E. Hunziker
Bern/Switzerland
Purpose: The synovium – like other mesenchymal tissues – contains
sub-populations of multipotent stem cells that are capable of
undergoing chondrogenesis, as well as osteogenesis, adipogenesis and
myogenesis. We wished to ascertain whether synovial explants that
were derived from young adults with femoral acetabular impingement
(FAI) and from elderly osteoarthritic patients could be induced by
exogenous stimulation to undergo chondrogenesis in vitro.
Methods and Materials: Synovial biopsies were derived from the hip
joints of five 20- to 28-year-old individuals with FAI and from the knee
joints of six 67- to 81-year-old osteoarthritic patients. Agarose-cultured
synovial explants were exposed for two weeks under serum-free
conditions to a combination of BMP-2 (2000ng/ml) and TGF-β1 (10ng/
ml). The gene-expression levels of a panel of cartilaginous markers and
of catabolic ones were quantified by the real-time PCR-method.
Results: In both groups of patients, stimulation with BMP-2/TGF-β1
induced the most profound increases (>10-fold) in the gene-
expression levels of the chondrogenic markers collagen types II,
IX, X and XI, aggrecan, COMP and alkaline phosphatase; those of
lubricin and type-I collagen were lower (<10-fold increases), and
that of osteocalcin remained unchanged. Amongst the catabolic
markers, the levels of MMP-13, Cox-2 and iNOS were elevated
(<10-fold), those of IL-1, IL-6 and ADAMTS-4 remained unchanged,
and those of IL-4, matrilin-1 and TNF-α lay below the detection
level. Although the trends were similar for the two groups of
patients, the magnitude of the increases for collagen types II and
XI, aggrecan, COMP, Cox-2 and iNOS were greater in the FAI- than in
the osteoarthritic patients.
Posters 233
Conclusions: Our data reveal that in young and elderly patients alike,
synovial explants can be induced to undergo chondrogenesis in
vitro, even if the tissue is derived from differently diseased joints.
P185
Tendon cells from rotator cuff differentiate into chondrogenic
lineage
I. Nagura, T. kokubu, T. Makino, H. Nishimoto, R. Sakata, A. Inui,
M. Kurosaka
Kobe/Japan
Purpose: Entheses serve to decrease stress between the bone
and the tendon and reconstruction of these interfaces is an issue
of considerable importance. We targeted degenerated cells from
human rotator cuff and investigated whether human rotator cuff
derived cells have the capacity for chondrogenic differentiation.
Methods and Materials: The edges of the rotator cuff were harvested
from patients who had sustained a rotator cuff tear and underwent
arthroscopic rotator cuff repair. Cells were cultured in monolayer
culture with α-MEM containing 10%FBS, 2mM L-glutamine and
antibiotics on 100 mm diameter culture dish. The cultures were
incubated at 37oC with 5% humidified CO2. Flow cytometric
analyses have also performed using monoclonal antibodies for the
following antigens: CD14, CD29, CD34, CD44, CD45, CD105, CD133,
CD166. To induce chondrogenesis, a pellet culture was performed
for three-dimensional culture. About 2.5x105 cells were spun in
serum free ITS-medium containing dexamethasone, ascorbate,
proline, recombinant BMP-6 and recombinant TGF-β3.
Results: Flow cytometric analyses showed positive
immunoreactivities for CD29, 44, 105, 166. The other tested
markers were negative. After 21 days of differentiation culture the
cells showed a chondrogenic differentiation potential as evidenced
by the histological immunohistochemistryies and RT-PCR analyses.
In contrast, the tissue specific markers of the control cells without
supplements did not increased.
Conclusions: The results of this study showed that degenerated cells
derived from human rotator cuff have the capacity for chondrogenic
differentiation. The ability to generate bone-ligament interface is
specifically of great importance, as it can be useful in healing torn
tendons that require reattachment to bone. This study suggests that
degenerated human rotator cuff derived cells have a chondrogenic
differentiation potential. Further investigation was needs to
analysis the best condition to chodrogenic differentiation.
P186
Enhanced in vitro cartilage formation by co-culture of human
primary chondrocytes and mesenchymal stromal cells
A. Barbero1, C. Acharya1, A. Adesida2, P. Zajac1, S. Schaeren1, M.
Jakob1, J. Riesle3, I. Martin1
1
Basel/Switzerland, 2Basel/Canada, 3Bilthoven/Netherlands
Purpose: Co-culture of mesenchymal stromal cells (MSC) with
chondrocytes (Ch) has been reported to improve cartilaginous
matrix accumulation (phenomenon here named chondro-induction,
CI). In this study, we investigate the type(s) of communication
between the two cell types responsible for CI.
Methods and Materials: Expanded bone marrow-MSC or fibroblast
(as control cells) and freshly isolated Ch were cultured in pellets
alone (pure pellets) or after being mixed (co-culture pellets, Ch:MSC
ratio 25%:75%). Selected pellets were generated combining:
human MSC with bovine Ch, and MSC from HLA-A2+ with Ch from
HLA-A2- donors. MSC and Ch were also cultured in transwells, with
the two cell types physically separated. Pellets were assessed
biochemically [to quantify CI as a ratio GAGmeasured/GAGexpected
(GAGexpected=75%GAGpure_MSC+25%GAGpure_Ch)], by RT-PCR
using human and bovine specific primers and probes for collagen-
II, and cytofluorimetrically. Tissues formed in the inserts of the
transwells were assessed histologically and biochemically.
Results: CI was higher when Ch were co-cultured with MSC (1.6±0.1)
than with fibroblasts (1.3±0.1). RT-PCR of pellets generated by
bovine Ch and human MSC showed an increase in the expression
of human collagen-II following co-culture. FACS quantification with
antibodies specific for HLA-A2 indicated that: Ch number increased
(4.2-fold) in the co-culture pellets while remaining constant in pure
pellets, MSC number decreased in the co-culture and pure pellets to
a similar extent (5.0-fold). GAG content of tissues formed by MSC or
Ch in the inserts of transwells were not modulated by the presence
of either cell types in the bottom layer of the same transwells.
234 Posters
Conclusions: Mutual communication between Ch and MSC occurs
in co-culture: Ch stimulate MSC for higher collagen-II expression
and, in turn, MSC stimulate Ch to proliferate. These effects are not
mediated by soluble factors alone but require cell-cell contacts. In
vivo studies are necessary to assess the clinical relevance of our
findings in the context of cartilage repair.
P187
A Strategy of Using Cartilage Fragments for Chondrogenesis of
MSC.
C.C. Chen, C.H. Chang, H.W. Fang, C.H. Liao
Taipei/Taiwan
Purpose: Human mesenchymal stem cells (hMSC) can differentiate
into cells of connective tissue lineages, including cartilage. To
overcome the limiting autogenous chondrocyte populations available
in cartilage repair, various methods have been developed to maximize
chondrogenesis of hMSCs in vitro. In this study, we investigated the
effects of cartilage fragments for chondrogenesis of MSCs in fibrin
glue and developed a favor method for cartilage repair.
Methods and Materials: Human cartilage fragments obtained from
TKR surgery and immortalized human bone marrow-derived MSC
were mixed well and embedded into fibrin glue. The cartilage-glue-
BMMSC constructs were implanted into subcutaneous tissue of
nude mice. After 2 and 4 weeks, gene expression, histological and
immunohistochemical stain of the constructs were analyzed. The
fibrin glue-MSC constructs without minced cartilage fragments
were as a control group.
Results: The results of the cartilage fragments-fibrin glue-MSC
constructs presented round and elongated chondrocyte-like
appearance with positive stain of GAG which inducted into cartilage
tissue. Moreover, RT-PCR analysis showed the gene expression of
aggrecan, type II, X collagen, and sox-9 increased over time in the
cartilage fragments-fibrin glue-MSC constructs. However, there
was no gene expression of type II collagen in control group, and
suggesting the MSCs in fibrin glue without cartilage fragments did
not differentiate into chondrocytes.
Conclusions: According to our results in this study, we have
suggested that cartilage fragments own the potential to help
chondrogenic induction of MSCs and develop a favor method for
cartilage repair.
P188
Relative percentage and zonal distribution of mesenchymal
progenitor cells in adult human articular cartilage
D. Pretzel, S. Linss, S. Rochler, R. Kinne
Eisenberg/Germany
Purpose: Combined expression of the surface molecules CD105
and CD166 unequivocally identifies mesenchymal progenitor cells
(MPC) in adult human articular cartilage. Therefore, these marker
molecules were employed to quantify and analyze the zonal
distribution of resident MPC in cartilage.
Methods and Materials: Specimens of human osteoarthritic cartilage
(n = 8) were either used for cell isolation or for immunohistochemistry
in paraffin sections. Isolated cells were analyzed by flow cytometry
(FACS) or immunofluorescence in chamber slides for the expression
of CD105 and CD166. Following separation of CD166+/- cells with
magnetobead-coupledantibodies,multi-lineagedifferentiationassays
were performed. In addition, the zonal distribution of CD166+ cells
within the cartilage matrix was analyzed by immunohistochemistry in
paraffin-embedded tissue sections.
Results: FACS analysis showed that 16.2 ± 2.1% (mean ± SEM)
of the chondrocytes were CD105+/CD166+ and thus carried the
established MPC marker combination. Similar results (approx.
12% CD105+/CD166+ cells) were observed by immunofluorescence
in adherent cells. The CD166+-enriched cell population showed a
stronger induction of the chondrogenic phenotype in differentiation
assays than the CD166--enriched cell population and thereby
underlined the chondrogenic potential of the MPC. Interestingly,
the CD166+ cells were mainly located in the superficial and middle
zone of articular cartilage and only sporadically in the deep zone.
Conclusions: CD 166 appears to be a novel suitable biomarker to
characterize the zonal distribution of resident MPC with a high
chondrogenic potential in human articular cartilage. Strikingly,
the percentage of MPC in osteoarthritic cartilage was substantially
higher than previously reported, supporting the concept of a yet
unexplored reserve capacity for regeneration.
P189
Cartilage tissue engineering with acellular cartilage matrix and
synovial stem cells
Y. Hsu, C.H. Liao, H.W. Fang, C.H. Chang
Taipei/Taiwan
Purpose: Cartilage tissue engineering has been a new strategy for
treatment of cartilage injury. However, there are some limitations
from the clinical perspective, such as cell source or type, ideal
scaffold, and optimal culture conditions. The aim of this study was
to investigate the potential of the discard cartilage and synovium
from total knee replacement (TKR) surgery to develop decellularized
cartilage ECM powder as chondrogenic scaffold and to isolate
synovial stem cells for replacement of autogenous chondrocytes.
Methods and Materials: The cartilage slices were harvested from
the patients who underwent TKR surgery. After decellularization
process, the DNA, GAG, and collagen contents of acellular
cartilage matrix (ACM) were determined and its morphology was
observed. The cytotoxicity of ACM was analyzed as well. Finally,
the chondrogenic properties of isolated synovial stem cells in cell-
ACM construct were characterized.
Results: The DNA assay showed that the amount of DNA of
the cartilage powder after the decellularization procedure was
decreased, and the GAG and collagen assays showed that the
amounts of GAG and collagen were retained from the untreated
powder. The cytotoxicity assay indicated that the ACM was toxic
free for cell adhesion and growth. After induction, the results from
qPCR of synovial stem cell and ACM co-culture showed that the
chondrogenesis was slightly promoted with ACM existence.
Conclusions: In this study, we utilized human, natural, and discard
materials from the TKR surgery to develop a method for cartilage
tissue engineering with ACM and synovial stem cells. The results of
this study may provide an alternative option for clinical treatment
on articular cartilage injury.
P190
Iron oxide labeling does not affect the chondrogenic
differentiation ‎capacity of mesenchymal stem cells
H. Nejadnik1, S. Wang2, J.H.P. Hui1
1
Singapore/Singapore, 2Sydney/Australia
Purpose: Cell based therapy is one of the most promising treatments
for cartilage injuries. Monitoring cells’ fate by live tracking of the
cells is essential to understand the homing mechanism and for the
improvement of cell therapy. Magnetic resonance imaging (MRI) is a
repeatable, non invasive way to track the cells in the living animal;
by labeling the stem cells with super para magnetic iron oxide (SPIO)
nanoparticle, monitoring of the cells with MRI will be possible.
Methods and Materials: Bone marrow derived mesenchymal stem
cells (BMSCs) were extracted and expanded till passage 2 from iliac
crest of 3 different porcines. Labeling was done using five different
concentration of Resovist® (Ferucarbotran) (25, 50, 75, 100, 125
µg/ml in culture media). To optimize labeling concentration and to
evaluate effects of labeling on the cells, proliferation study (MTS
assay), atomic absorption spectrometry (AAS), Prussian blue
staining, transmission electron microscopy (TEM), adipogenic,
osteogenic, and chondrogenic differentiation studies were done.
Results: Prussian blue staining showed that the efficiency of
labeling was more than 90 percent of the cells. Transmission
electron microscopy confirmed that the endocytosed particles
were in the cytoplasm. The average iron content of the BMSCs was
increased according to the increase of the particle concentration
in the culture media. Labeling of the BMSCs with different
concentration of Resovist did not affect the proliferation rate of the
cells. The detection capability of the labeled cells was assessed
with clinical MR imaging at 1.5T and 3T. Moreover, labeling of the
BMSCs did not affect the adipogenic, osteogenic and especially the
chondrogenic differentiation capacity of the cells.
Conclusions: This study showed that labeling of BMSCs by SPIO
does not affect cellular function of the BMSCs especially the
chondrogenic differentiation capacity. Therefore, it can be used for
tracking the cells and to study their homing in the articular cartilage
defect.

P191
Comparative labelling of mesenchymal stem cells with magnetic
iron oxide nanoparticles for MR imaging in vitro
H. Jülke, C. Geißler, I. Ribitsch, W. Brehm, E. Ludewig, U. Delling
Leipzig/Germany
Purpose: In vitro and magnetic resonance imaging (MRI) trials
were employed to investigate 3 commercial supraparamagnetic
iron oxide nanoparticles (SPIO) products for labelling bone marrow
derived mesenchymal stem cells (MSC). Labelling efficiency,
longitudinal cellular detectability by MRI and biologic effects on
MSC were evaluated. Results of this study will help to define the
most appropriate SPIO for in vivo cell tracking of locally applied
MSC in degenerative joint disease.
Methods and Materials: Ovine and equine MSC were routinely
obtained from 5 donors, expanded until p4 and labelled with one
of the following SPIO products: Molday ION Rhodamine B (Biopal,
USA), Endorem (Guerbet, France), or Resovist (Bayer HealthCare,
Germany). Labelling efficiency and cellular SPIO retention until
p7 were graded by a semiquantitative histological scoring system
based on Prussian blue staining. Cellular detection was evaluated
by a 0.5T MRI system using GRE 3DT2*w sequences for up to 3
weeks after labelling (p4 to p7). Proliferation and differentiation
capacities were assessed by in vitro assays.
Results: MSC were successfully labelled by all 3 SPIO products. High
iron uptake and selective intracellular iron presence was achieved by
Molday ION Rhodamine B, only. Labelling with Resovist led to prominent
extracellular iron presence, labelling with Endorem was less intense.
In MRI, all labelled cells showed strong hypointense signals contrary
to unlabelled controls. Resovist caused most hypointense signals,
followed by Molday ION Rhodamine B and Endorem. MRI detectability
decreased proportionally from p4 to p6. In p7, 3 weeks after labelling,
only Resovist labelled cells were detectable. Proliferation, adipogenic
and osteogenic differentiation were similar between labelled and
unlabelled cells. Chondrogenic differentiation decreased proportional
to increasing intracellular iron quantities.
Conclusions: Out of the 3 products, Molday ION Rhodamine B is the
most promising labelling agent for in vivo cell tracking. Advantages
are reliable intracellular iron uptake without extracellular SPIO and
consistent hypointense signals in MRI.
P192
Heterogeneity in the chondrogenic capacity of bone marrow
stromal cells
S.C. Dickinson, C.A. Sutton, W. Kafienah, A.P. Hollander
Bristol/United Kingdom
Purpose: There is great potential for the use of cell-based therapies
for the treatment of cartilage lesions caused by osteoarthritis (OA).
Bone marrow stromal cells (BMSCs) are a heterogeneous population
of multipotent stem cells which can differentiate into chondrocytes
and produce three-dimensional hyaline cartilage. The aim of our
study is to determine whether sub-populations of BMSCs are able
to create cartilage of superior quality to the whole population and
to identify markers of the most chondrogenic cells.
Methods and Materials: BMSCs were isolated from patients with
OA. Stem cell clones were prepared by using a sterile cell sorter to
deliver individual cells into wells of a 96-well plate. Each clone was
proliferated in the presence of FGF-2 to generate a sufficient number
of cells to assess multipotential. Chondrogenesis was assessed by
TGF-β3 stimulated cartilage tissue engineering. The quality of the
cartilage produced by each individual clone was measured using
specific biochemical assays.
Results: A total of 21 individual stem cell clones were proliferated,
with negligible signs of senescence, to generate sufficient cells
to perform cartilage tissue engineering. Both the macroscopic
appearance (Figures 1A&B) and the extracellular matrix protein
content of the resulting cartilage were highly variable between
clones. Five stem cell clones were highly chondrogenic, generating
cartilage containing more type II collagen than cartilage produced
from the whole BMSC population (Figure 1C). There was no
correlation between the chondrogenic capacity of different clones
and their adipogenic and osteogenic potential.
Conclusions: We have demonstrated that the BMSC population
is highly heterogeneous in terms of chondrogenic capacity. This
highlights the importance of identifying and isolating the most
chondrogenic cells for use in clinical therapies. We are currently
performing a gene array analysis to compare gene expression in
highly and poorly chondrogenic clones to identify unique markers
for the isolation of the most effective cells for therapeutic use.
Posters 235
P193
Correlation of human bone marrow mesenchymal stem cell
characteristics with donor age
V. Dexheimer, S. Müller, F. Braatz, W. Richter
Heidelberg/Germany
Purpose: Due to their self-renewing and multilineage differentiation
capacity, mesenchymal stem cells (MSC) are an attractive source for cell-
based cartilage repair strategies. Therefore the question arises if donor
age has an influence on MSC properties, in particular their number, clonal
expandability and chondrogenic differentiation capacity.
Methods and Materials: MSC from 28 donors of three age groups
were analyzed for their ability to form Colony-forming unit
fibroblasts (CFU-F). During expansion single cell cloning efficiency
(SSCE) was assessed at passage 0 (P0) and generation determined
at P0-P4. Chondrogenic differentiation was induced in a high
density pellet culture at P3 for 6 weeks in chondrogenic induction
medium with TGF-β1 and pellets were analyzed for proteoglycan
and collagen type II content.
Results: The number of MSC obtained per 106 mononuclear cells
was highly variable between donors but independent of donor age.
Clonal expandability was reduced in the old age group compared to
the young and middle aged groups. Cells proliferated slower with
increasing passages. Generation time in passage two and three
was however significantly higher in the old compared to the young
and middle aged group. After chondrogenesis the collagen type II
and proteoglycan content of pellets was similar in all age groups.
Generation time correlated with collagen type II deposition in every
age group with the fastest cells producing the best differentiating
results in each age group.
Conclusions: The cells from aged donors show a reduced speed of
proliferation. The in vitro chondrogenic differentiation potential
is highly variable between donors but donor age would not be a
criterion to exclude patients from future applications of MSC. In
the context of cartilage repair similar numbers of MSC are obtained
from bone marrow of young and old donors.
P194
PTHrP - Indian Hedgehog autoregulation during chondrogenic
differentiation of human mesenchymal stem cells – a PTH-
receptor-independent mechanism
A. Brauckhoff, J. Fischer, E. Steck, W. Richter
Heidelberg/Germany
Purpose: Bone marrow-derived mesenchymal stem cells (BMSC)
are an attractive alternative for cartilage tissue engineering.
However, long-term phenotypic stability along with functional
suitability in-vivo and the adoption of a non-hypertrophic
chondrocyte phenotype are imperative for cartilage repair and still
represent a challenge for current in vitro chondrogenic protocols.
Parathyroid hormone-like peptide (PTHrP) is a negative regulator
of chondrocyte differentiation and MSC chondrogenesis, and a
known inhibitor of chondrocyte hypertrophy. The aim of this study
was to evaluate whether modulation of PTHrP-signalling is a means
to improve chondrogenic in vitro differentiation of human MSC.
Methods and Materials: Chondrogenic differentiation of human
BMSC was induced in pellet culture for 6 weeks in chondrogenic
medium containing TGFß. Expression of PTHrP, PTH-receptor 1
(PTHR1) and Indian Hedgehog (IHH) mRNA was assessed over
time. Part of the cultures were supplemented with PTHrP(1-34), its
antagonist PTHrP(7-34) or a combination of both from day 0 or 21
on. Differentiation was assessed by histology, real-time PCR and
detection of ALP activity.
Results: PTHrP was expressed up to 2-3 weeks and downregulated
in favour of IHH and PTH1R up-regulation. PTHrP(1-34) inhibited the
TGF-β-driven collagen type II and collagen type X expression and
alkaline phosphatase (ALP) induction, while the antagonist alone
showed no evident effects. PTHrP action could surprisingly not be
antagonised by PTHrP(7-34).
Conclusions: Similar to observations in immature chondrocytes in
the growth plate, early chondrogenesis of MSC was characterized
by PTHrP expression which declined spontaneously in favour of
IHH up-regulation allowing further maturation of cells towards
hypertrophy. PTHrP exposure from day 21 on suppressed IHH up-
regulation and reduced hypertrophy and surprisingly, this occurred
independent of PTH1R action. Future steps will be to develop
alternate differentiation protocols in which the here discovered
PTHrP/IHH autoregulation is modulated in favour of a sustained
nonhypertrophic chondrocyte phenotype desired for articular
cartilage repair studies.
236 Posters
P195
Effects of coculture conditions on chondrogenic commitment of
human wharton jelly stem cells
R.C. Pereira, A.R. Pinto, A.M. Frias, N.M. Neves, H.S. Azevedo, R.L.
Reis
Caldas das Taipas – Guimarães/Portugal
Purpose: Cell-based therapies have shown great promise for
the repair of articular cartilage lesions.Autologous chondrocyte
implantation(ACI)proposed by Britteberg is a clear example of
a cell-based therapy with excellent clinical results.The use of
ACI is associated with several limitations involving lengthy and
costly cell isolation and expansion steps of human articular
chondrocytes(hACs)which have low cellular mitosis and are prone
to dedifferentiation.Potential improvement in ACI procedure is the
application of mesenchymal stem cells(MSCs),recognized for the
potential to differentiate into the chondrogenic lineage,instead of
hACs.Coculture systems using relevant cells can be used as valuable
tools to offer therapeutic possibilities in cartilage regeneration.
Realizing that MSCs present a limited degree of proliferation with
maintenance of their multipotency capacity of differentiation,we
used human wharton jelly stem cells(hWJSCs)to evaluate their
chondrogenic differentiation capability.This study was undertaken
to test if hWJSCs,in non direct coculture with hACs,would be able
to present a chondrogenic commitment compared to those not
exposed to chondrocytes metabolic products.
Methods and Materials: Cell differential potential was assessed by
qRT-PCR,and by micromass pellet culture.Immunohistochemistry
was also performed.
Results: Immunolocalization of collagen type I and II of hWJSCs
cocultured with hACs in micromass pellets shows a promising trend
towards chondrogenic phenotype.Moreover,hWJSCs in coculture
presented a homogeneous cell distribution and, for at least 20
cell duplications, higher levels of collagen type II, aggrecan, Sox9,
TGF-β and COMP expression compared to those cultured alone,
suggesting a potential commitment toward the chondrogenic
lineage.
Conclusions: These findings suggest that coculture of hWJSCs with
hACs may provide a suitable environment for their chondrogenic
commitment and could significantly improve the development of
cell-based therapies for treating cartilage lesions.
P196
Articular chondrocytes secrete PTHrP and inhibit hypertrophy of
mesenchymal stem cells in coculture during chondrogenesis
J. Fischer, A. Dickhut, M. Rickert, W. Richter
Heidelberg/Germany
Purpose: Bone marrow-derived mesenchymal stem cells (MSC)
are promising for cell-based cartilage regeneration. A yet unsolved
problem is, however, the unwanted upregulation of hypertrophic
markers like alkaline phosphatase (ALP) and collagen-type X
during in vitro chondrogenesis and formation of instable calcifying
cartilage at heterotopic sites. In contrast, stable non-mineralizing
cartilage is obtained from articular chondrocytes. Aim of this
study was to address whether coculture with human articular
chondrocytes (HAC) has the capacity to suppress undesired
hypertrophy in differentiating MSC.
Methods and Materials: MSC were differentiated in chondrogenic
medium which had or had not been conditioned by parallel
chondrocyte pellet cultures, or were mixed in the same pellet with
chondrocytes (1:1, 1:2) and cultured for six weeks. Following in vitro
differentiation, pellets were transplanted into SCID mice.
Results: The gene expression ratio of COL10A1/COL2A1 and IHH/
COL2A1 was significantly reduced by HAC-conditioned medium and
less collagen-type X protein was deposited relative to collagen-type
II. ALP-activity was significantly lower (p<0.05) in the conditioned-
medium-group and transplants showed significantly reduced
calcification in vivo. In mixed HAC/MSC pellets, suppression of ALP
was dose-dependent and in vivo calcification was fully inhibited.
Chondrocytes secreted PTHrP throughout culture while PTHrP
was downregulated in favour of IHH upregulation in control MSC
after 2-3 weeks of chondrogenesis. Main inhibitory effects seen
with HAC-conditioned medium could be reproduced by PTHrP
supplementation of unconditioned medium.
Conclusions: HAC-derived soluble factors and direct coculture
are potent means to improve chondrogenesis and suppress
hypertrophic development of MSC. PTHrP is one important soluble
candidate factor involved in this effect.
P197
Retinoic acid signalling is important for chondrogenic
differentiation of human mesenchymal stem cells
S. Diederichs, W. Richter
Heidelberg/Germany
Purpose: Retinoid signalling has long been known to play a major role
in early skeletal development. On the one hand, retinoic acid (RA) – the
active vitamin A metabolite – inhibited in vitro chondrogenesis of primary
mouse and chick limb bud mesenchymal cultures and stimulated
hypertrophy of chicken sternal chondrocytes. On the other hand, RA
receptor antagonists promoted chondrogenic differentiation of mouse
limbbudmesenchymalculturesandpreventedchondrocytehypertrophy
in developing chick limbs, suggesting that they are attractive factors
to improve chondrogenic differentiation of mesenchymal stromal cell
(MSC) cultures. Indeed, the RA receptor-β selective antagonist (LE135)
was recently reported to drive chondrogenesis of human MSC in PGA
scaffolds. Aim of this study was to unravel whether LE135 can drive and
improve in vitro chondrogenesis of human MSCs in pellet culture in the
presence or absence of TGF-β.
Methods and Materials: Expanded MSCs from five human donors
were cultured as micromasses (500,000 cells each) in serum free
chondrogenic medium supplemented either with TGF-β (positive
control), LE135 (0.1 µM / 1 µM) or a combination of both. Chondrogenic
differentiation was assessed after six weeks based on proteoglycan
and collagen II detection (histology, quantitative PCR).
Results: While TGF-β treated MSCs were strongly positive for
proteoglycans and collagen-type II, LE135 was not sufficient to promote
chondrogenesis of human MSCs. In combination with TGF-β, LE135
even inhibited chondrogenesis.
Conclusions:WhilepreventionofRAsignallingbyRAreceptor-βinhibition
improved chondrogenesis of in vivo committed chondroprogenitors,
such an inhibition did not create a sufficiently positive signal to drive
non-committed human MSCs into the chondrogenic lineage. In the
presence of the potent inducer TGF-β, RA receptor-β inhibition blocked
differentiation suggesting that RA signalling is, however, a crucial part
also of in vitro chondrogenesis of human MSCs. LE135 is, thus, currently
not attractive to improve chondrogenesis in this model.
P198
Inhibition of chondrogenesis by sex hormones
P. Angele, Z. Jenei-Lanzl, T. Dienstknecht, R. Kujat
Regensburg/Germany
Purpose: Objective Articular cartilage is a sex-hormone sensitive
tissue. estrogens have been shown to affect cartilage under
physiological and pathological conditions. Estrogens have
different mechanisms of action via classical (ER α/β) or membrane
receptors such as GPR30. The purpose of the present study was
to investigate the effect and the possible mechanism of estradiol
treatment during chondrogenesis of bone marrow derived MSCs
with respect to repair of chondral defects and for the prevention of
trauma-dependent osteoarthritis.
Methods and Materials: Methods Bone marrow was obtained
from the iliac crest of young male humans. Ficoll-separated
hMSCs proliferated as a monolayer in serum-containing medium.
After achieving confluence, aggregates were created and
cultured in a serum-free differentiation medium. 17β-estradiol
(E2) with or without the specific estrogen receptor inhibitor ICI
182.780, membrane impermeable E2-BSA, ICI 182.780 alone, a
GPR30 agonist G-1, and a GPR30 antagonist G15, were added.
After 21 days, the aggregates were analyzed histologically and
immunohistochemically; synthesized type II collagen, DNA content,
glycosaminoglycan concentrations, and type X collagen and MMP-
13 expression were quantified.
Results: Results The existence of intracellular and membrane-
associated E2 receptors was shown at various stages of
chondrogenesis. Smaller aggregates, significant lower type II
collagen, and sGAG content but enhanced type X collagen and
MMP-13 expression were detected after E2 and E2-BSA treatment
in a dose-dependent manner. The co-incubation of ICI 182.780
with estradiol enhanced suppression of chondrogenesis compared
to estradiol alone. The treatment with specific GPR30 agonists
alone (G-1 and ICI 182.780) resulted in a considerable inhibition of
chondrogenesis and enhancement of hypertrophy.
Conclusions: Conclusion The experiments revealed a suppression
of chondrogenesis by estradiol via membrane receptors (GPR30).
The study opens new perspectives to influence chondrogenesis on
the basis of classical and non-classical estradiol signalling.

P199
Clinically applicable cell tracking in cartilage repair using MRI
G.M. van Buul, G. Kotek, P.A. Wielopolski, E. Farrell, P.K. Bos, H.
Weinans, J.A.N. Verhaar, G.P. Krestin, M.R. Bernsen, G.J.V.M. Van
Osch
Rotterdam/Netherlands
Purpose: Cell tracking is a useful tool to optimize the use of human bone
marrow stromal cells (hBMSCs) for cartilage repair and to elucidate the
relevant repair mechanisms. Cell labeling using superparamagnetic
iron oxides (SPIOs) provides the possibility for non-invasive in vivo
cell tracking using MRI. We investigated the safety, intra-articular MRI
traceability and SPIO re-uptake of this cell tracking technique.
Methods and Materials: Part 1: hBMSCs were labeled with SPIO
(ferumoxides-protamine sulphate), dose range 0 - 250 μg/ml. Cell
viability was assessed and cell activity was quantified up to 7 days.
Part 2: SPIO-labeled hBMSCs (100,000 – 5,000,000 cells) were injected
in pig knees post mortem. Furthermore, SPIO-labeled cells (30,000 -
100,000 per 75 μl) were seeded in cartilage defects in vitro. Scanning
was performed on a clinical 3.0 T MRI scanner. Part 3: To show possible
SPIO re-uptake by synovial cells, viable and dead GFP-SPIO double-
labeled chondrocytes were co-cultured on human synovium explants
for five days. Samples were analyzed using fluorescence- and light
microscopy.
Results: Part 1: SPIO labeling resulted in labeling efficiencies of ± 95%
and did not impair cell viability or -activity at any dose. Part 2: Intra-
articular injected SPIO-labeled hBMSCs were visualized by MRI in a
dose dependent manner, and could be differentiated from articular
anatomical structures. SPIO-labeled cells seeded in cartilage defects
were visualized and quantified using a T2* mapping MRI technique
Part 3: GFP+-SPIO+ cells, indicating originally seeded cells, were seen
in samples containing live cells. GFP‾-SPIO+ cells, indicating SPIO re-
uptake by synovial cells, were found in samples containing dead cells.
Conclusions: Although possible SPIO re-uptake by host cells has to be
considered, we showed promising results for the use of SPIO labeling
for cell tracking in clinical cartilage repair. This approach provides the
extra advantage to simultaneously track cells and evaluate cartilage
repair in one MRI session.
P201
Efficacy of Adeno-associated gene therapy in equine bone
marrow derived mesenchymal stem cells for cartilage resurfacing
applications
L. Goodrich1, A. Mandel1, V. Choi2, J. Kisiday1, W. McIlwraith1, J.
Samulski3
1
Fort Collins/United States of America, 2Cambridge/United States of
America, 3Chapel Hill/United States of America
Purpose: Gene therapy for joint disease relies on a non-immunogenic
gene delivery vector that efficiently and persistently transduces joint
specific tissues. Recombinant self-complimentary adenoassociated
virus (scAAV) is a promising vector due to its potential to efficiently
express therapeutic genes for long periods of time. Our goal was to
investigate which serotypes of AAV would be best suited to deliver
the gene encoding green fluorescent protein (GFP) into bone marrow
derived mesenchymal stem cells (BMDMSC) for future gene transfer
applications in the joint.
Methods and Materials: Bone marrow derived mesenchymal stem cells
were harvested from adult horses and humans. Cell monolayers were
cultured in a mesenchymogenic media. Two days after seeding, the
scAAV vector serotypes including S1-6 and S8 carrying a GFP expression
cassette were used to transduce cells. The cells were suspended into
1.6% alginate for 6 weeks. Flow cytometry was performed every two
weeks to measure efficacy of gene modification of these cells and
realtime PCR was performed to detect any increases in inflammatory
molecules as a result of gene modification.
Results: Excellent gene modification of these cells was accomplished
for scAAV serotype 2 and 3. Moderate transduction was achieved for
scAAV5 and 6. Intense fluorescence was achieved for a period of at
least 6 weeks for these cells when transduced by scAAV 2 and 3 as
measured by flow cytometry. No significant elevations were noted in
inflammatory molecules as measured by PCR.
Conclusions: We established the use of specific AAV serotypes
for efficient gene modification of BMDMSC. Transgene expression
continued past 60 days and no apparent inflammation or reduction in
cell viability appears to be associated with gene modification of these
cells. Gene therapy using BMDMSC for gene therapy in joint resurfacing
applications appear to be clinically applicable with scAAV serotype 2
and 3 being the most efficacious means of vector transfer.
Posters 237
P202
Repair of chondral defects and meniscus using autologous
mesenchymal stem cells: a preliminary study in sheep
J. Vives1, F. García Arnás1, J. Barrachina2, J. García2, R. Soler Rich2,
L. Orozco2
1
Bellaterra/Spain, 2Barcelona/Spain
Purpose: We herein present our recent findings, in an ovine model,
on the use of autologous mesenchymal cells expanded from bone
marrow aspirates for the repair of damaged cartilage, as a model,
of degenerative effects of osteoarthritis or osteochondral lesions.
Such animal model also permitted us to study the regeneration of
meniscal lesions in the same knee.
Methods and Materials: Ten 2-year old ewes were divided in two
groups (for analysis at 6 and 12 month). Arthroscopically guided
chondral lesions of approximately 60 mm2 were created in the
femoral medial condyles and the anterior horn of the medial
meniscus of the two posterior legs. Cellular treatments were
applied at a max dosage of 50x106 MSCs 30 days later and the
animals were monitored by MRI, echographic and X ray at 6 and 12
months. A full necropsy was performed at 12 months to evaluate
possible adverse effects of the treatment.
Results: Although MRI, ecographic and X ray control were not
extremely informative to assess the progression of the reparative
process, the histological and immunohystochemical analysis (at 6
and 12 months) confirmed the hyaline quality of the regenerated
cartilage of the condyles of the knee of treated animals. Meniscal
lesions were also partially repaired in some cases. Most importantly,
the intra-joint application of autologous mesenchymal stem cells
was demonstrated to be safe at 12 months post-treatment.
Conclusions: We demonstrated that autologous mesenchymal
stem cells are safe and capable to regenerate both hyaline
cartilage and meniscal fibrocartilage with the advantage of using
a straightforward infiltration approach.
P204
Microarray analysis reveals signature clusters of gene
expression during in vitro chondrogenesis of human bone
marrow-derived mesenchymal stem cells
S.R. Herlofsen, A.M. Küchler, J.E. Brinchmann
Oslo/Norway
Purpose: Chondrogenic differentiation of hBM-MSCs in alginate
discs was performed to develop clinically useful cartilage implants,
and to facilitate the kinetic analysis of gene and protein expression.
Methods and Materials: After monolayer expansion MSCs were
set into a selfgelling alginate system forming a 1-2 mm thick disc
and differentiated in chondrogenic medium containing TGF-β1 and
BMP2. Samples were collected at day 0, 7, 14 and 21. Chondrogenesis
was evaluated by realtime PCR and immunofluorescence
histochemistry. Matrix production was additionally quantified by
GAG concentration measurement. Finally, microarray technology
and similarity search statistics were used to identify signature
clusters of gene expression patterns.
Results: COL2, COL10 and SOX5, 6 and 9 mRNA were greatly elevated
already at day 7, while COL1 and versican were gradually reduced.
COL2 and aggrecan were seen throughout the entire ECM. COL1
seemed to be produced by only some of the cells. COL10 was found
predominantly in the cytoplasm while SOX proteins were localized in
the nuclei. We detected increasing levels of GAGs in the supernatant.
By similarity search of differentially expressed genes (p<0.05,
>2-fold change) we identified a signature cluster of ECM genes
upregulated similar to COL2 by differentiation medium, and clusters
of genes involved in blood vessel development and immune
responses downregulated similar to the chemokine CXCL12.
Conclusions: We show that hBM-MSCs differentiated in alginate
discs express genes and secrete proteins with kinetics and
distribution similar to hyaline cartilage. At the same time they
downregulate genes involved in non-chondrogenic functions of BM-
MSCs. Analysis of the signature chondrogenic clusters identified
here may provide a better understanding of how the stem cell fate
could be directed to produce perfect chondrocytes for celltherapy.
238 Posters
P206
Stem cell- based augmentation of meniscus repair
E. Chen1, M. Ast1, M. Goldstein1, N.V. Shah2, N. Chahine2, P. Razzano2,
D. Grande2
1
New Hyde Park/United States of America, 2Manhasset/United
States of America
Purpose: Meniscal injuries represent one of the most common
musculoskeletal complaints, with partial meniscectomy being
the most frequently performed orthopaedic procedure in the US.
The importance of the meniscus as a weight-bearing structure
has been established, with meniscectomy shown to predispose
to earlier onset of articular cartilage degeneration. Yet meniscal
repair is infrequently performed, in part from concerns for healing
due to limited vascular supply. In this study, we attempt to enhance
the meniscus repair process. It is our hypothesis that cellular
augmentation with either meniscal fibrochondrocytes or stem cells
will enhance the histological repair quality of meniscus tissue.
Methods and Materials: Experimental defects were created in fresh
bovine menisci in a radial orientation. Defects were treated with
either meniscal fibrochondrocytes, adipose-derived stem cells,
synovial-derived stem cells, or without cells. Cells were delivered
either alone in a collagen gel, scaffold, or with hyalurinoc acid. Each
of these cell lines were derived from bovine tissue, cultured and
expanded, and labeled with BrdU for later tracking. The repaired
meniscal samples were then implanted subcutaneously into the
dorsum of nude rats and the animals sacrificed at 3, 6, and 9 weeks.
The samples were then be prepared for histologic examination and
stained with H & E, Mallory Trichrome, and DAPI.
Results: Meniscal defects treated with any of these cell types showed a
histologically superior repair when compared to controls. Cells implanted
in the absence of scaffold showed a higher density of cellular repair
tissue, although it is unclear whether this reflects a more structurally
sound repair. Synovial-derived cells resulted in significantly higher levels
of collagen matrix synthesis and better overall integrative repair.
Conclusions: The addition of meniscal fibrochondrocytes, adipose-,
or synovial- derived stem cells to a bovine meniscus injury model
results in a histologically superior repair tissue when compared to
repair without cellular augmentation.
P208
GDF-5 Supplementation Enhances MSC Chondrogenesis
C. Coleman1, C. Curtin2, F. Barry3
1
Galway City/Ireland, 2Dublin/Ireland, 3Galway/Ireland
Purpose: Deficient secretion of growth/differentiation factor
5 (GDF-5) contributes to the induction of arthritis. As arthritic
cartilage fails to self-repair, treatment with mesenchymal stem
cells (MSCs) may contribute to the repair of fibrillated cartilage.
The aim of this project was to assess the effects of MSC treatment
with GDF-5, focusing on the deposition of matrix proteins.
Methods and Materials: Bone marrow (BM) was harvested from
healthy, consenting donors. MSCs were isolated and expanded
from the BM by direct plating using traditional methods, followed by
induction towards chondrogenesis by pellet culture in incomplete
chondrogenic medium (ICM), ICM supplemented with TGF-beta3
(CCM) or CCM with 100, 150 or 200ng/ml GDF-5. Glycosaminoglycan
(GAG) content was determined histologically by Safranin-O staining,
and quantitatively by DMMB assay. DNA content was determined
by Pico Green analysis. Immunohistological (IHC) analysis following
paraffin embedding included blocking in 2.5% BSA, incubation with
anti-collagen II or X antibodies and DAB development.
Results: MSCs in pellet culture initiated the deposition of a collagen
and GAG containing ECM. As assessed by Safranin-O and DMMB
analysis, pellets treated with GDF-5 contained significantly greater
quantities of GAG as compared to CCM controls (Figure 1). Although
treatment with GDF-5 resulted in enhanced GAG deposition, IHC
analysis for collagen II illustrated the presence of similar quantities of
protein in pellets treated with CCM or GDF-5. Collagen X expression,
a marker of hypertrophy, was eliminated in GDF-5 treated pellets
when expression was observed in CCM pellets (Figure 2).
Conclusions: Treatment of MSC chondrogenic pellets with 150ng/
ml GDF-5 resulted specifically in significant enhancement of
GAG deposition, but no obvious change in collagen II secretion.
The presence of GDF-5, however, eliminated collagen X from the
ECM, an indication of undesirable hypertrophy. Therefore, the co-
administration of GDF-5 with MSCs as a therapeutic for arthritis
may result in enhanced chondrogenesis as well as the prevention
of hypertrophy.
P209
Development of the scaffold free cell processing robot for
osteochodral regeneration
K. Nakayama, T. Shimoto, S. Akieda, X.Y. Zhang, S. Matsuda, Y.
Iwamoto
Fukuoka/Japan
Purpose: We’ve been working for developing scaffold free
cell delivery system, and show good regeneration of rabbit
osteochondral defect until more than three years by implantation
of molded mesenchymal stem cells (MSC) construct without use
of exogenous factors. The entire procedure is simple, yet, it takes
time and labor to build a single construct even experienced hands.
In this study, we developed a cell processing robot for building
Scaffold free HD MACs (High-density mesenchymal stem cell
autologus constructs).
Methods and Materials: We customized ready-made-scalar robot
and the electric cylinder for cell handling under sterilized and GMP-
ready condition. The entire system are placed in clean bench. The
system has automatic 96-well plate changer. When the isolated
cells and empty 96-well plates in a magazine rack were set, the
system automatically dispense the cell suspension into each well.
After 24 hours in normal incubator with the magazine rack,the
system will also automatically collect cell aggregate into the HD-
MACs molding chamber. Another 2 to 3 days in the molding chamber
under incubator, the HD-MACs were ready to implant to the knee.
Results: The obtained scaffold free HD MACs as same as manual
procedure without contamination. The processing times are almost
same compared with experienced human,while the robot system
may able to work much faster with some optimization.
Conclusions: In this study, we developed the cell processing robot for
cartilage regeneration. The entire system is almost GMP ready. We are
preparing clinical trials in Kyushu university hospital for autologous
MSC implantation to cartilage defect with this robot system.
P210
hMSC TNAP Expression is a Marker of Differentiation Potential
S. D’Arcy1, C. Coleman2, M. Murphy1, F. Barry1
1
Galway/Ireland, 2Galway City/Ireland
Purpose: Due to their regenerative, tri-lineage potential,
mesenchymal stromal cells (hMSC) offer new prospects for clinical
treatments over current therapies. Although hMSCs are CD90+,
CD105+, CD73+, CD44+ and CD45-, no standardized marker has
been identified. Recently, expression of the cell surface protein
Tissue Non-specific Alkaline Phosphatase (TNAP) has been used
to directly isolate hMSC from bone marrow (1, 2). We hypothesized
that expression of TNAP may offer a methodology to isolate hMSC
subpopulations with varying differentiation potential.
Methods and Materials: hMSC subpopulations were isolated from
whole BM using anti-TNAP microbeads according to manufacturer’s
instructions (3). TNAP+, TNAP-, and parent (TNAP+ and TNAP-)
populations were expanded in alpha-MEM with 10% FBS and 5ng/
ml FGF-2. All populations were assessed for surface phenotype
(flow cytometry), expansion potential, morphology, and tri-lineage
differentiation potential using standard methods (4).
Results: TNAP+ hMSC, unlike parent and TNAP- fractions, initiated
as distinct colonies and showed enhanced proliferative ability (Fig
1). Expression of traditional hMSC markers (CD105, CD73, CD44)
was maintained in all populations, however TNAP- hMSC also
showed CD271 expression was increased significantly, indicative
of osteogenic potential (5). Osteogenic differentiation confirmed
this when TNAP- hMSC showed significantly enhanced calcium
deposition compared to parent and TNAP+ populations (Fig 2a).
TNAP+ hMSC demonstrated enhanced adipogenic potential,
which was completely depleted in the TNAP- fraction (Fig 2c).
Chondrogenic assessment (GAG deposition) indicated complete
inhibition of TNAP- hMSC potential, accompanied by significant
reduction of TNAP+ hMSC, to chondrify (Fig 2b).
Conclusions: TNAP expression was found to be related to both
hMSC proliferation and differentiation capability. TNAP absence
on hMSC was indicative of osteogenic potential, and presence of
TNAP suggested a more proliferative cell with enhanced adipogenic
potential. Interestingly, for chondrogenic differentiation to arise,
the combination of TNAP expressing and non-expressing cells was
required, indicating a necessity for subpopulations to communicate
for hMSC chondrogenesis.

P212
A novel cell therapy for cartilage regeneration based on adipose
mesenchymal stem cells and a new biomaterial of natural origin.
B. Castro1, V. Soto-Cerrato2, M. Nicolàs2, A. Herrero1, I. Gartzia1, M.
Montes1, M. del Olmo1, A.G. Fernández2, A. Guglietta2
1
Derio/Spain, 2Barcelona/Spain
Purpose: Articular cartilage damage is a common disease that may
lead to severe osteoarthritis of the joint. Spontaneous healing of
cartilage is difficult due to the avascular nature and low cell density
of this tissue. Our purpose is to develop a new tissue engineering
strategy to optimize cartilage repair by integrating cells in a scaffold
material that functions as an extracellular matrix.
Methods and Materials: We evaluated cell viability using live/dead
and MTT assays. Gene expression profile was analyzed by qRT-PCR.
Results: We first describe Histogel, a novel biomaterial of
natural origin with optimal properties for its use in cartilage
regeneration. Histogel behaves as a hydrogel, with viscoelastic
properties yielding high adhesiveness in the injury and fluidity to
be used through arthroscopic implantation. We have identified
the expression profile of several key genes to comparatively study
the chondrogenesis process in human adipose mesenchimal stem
cells (aMSC) either conventionally cultured or introduced into the
biomaterial. In the same conditions, cell viability was evaluated
showing cell biocompatibility up to 96 hours in the biomaterial.
Furthermore, when human cartilage explants were incubated
together with this new biomaterial, chondrocytes seemed to be
attracted by the hydrogel in a higher extent than when incubated
with hyaluronic acid.
Conclusions: These results show the potential of Histogel to be
a very advantageous scaffold for cartilage regeneration owing
to its biological safety and its ability to promote cell adhesion,
proliferation and chondrocytic differentiation. Additionally,
Histogel has chemotactic trails that will be relevant to recruit cells
to the chondral defect.
P214
Effects of combining chondrogenic and anti-angiogenic factors on
a one-step cartilage repair approach
K. Gelse, J. Gusinde, M. Blanke, A. Olk, F. Hennig
Erlangen/Germany
Purpose: Microfracture (Mfx) is a simple, minimally-invasive
technique that is frequently applied for the induction of cartilage
repair tissue, however, this approach mostly generates inferior
fibrocartilage. Furthermore, an unphysiological hypertrophy of the
subchondral bone plate with vascular invasion and subsequent
endochondral ossification within the repair tissue is a frequently
observed phenomenon that may interfere with the long-term
outcome. Therefore, this study followed a strategy to combine Mfx
with the application of recombinant osteogenic protein-1 (OP1) to
promote chondrogenesis, and with the application of recombinant
Thrombospondin-1 (TSP-1), a protein with anti-angiogenic
properties, to prevent excessive endochondral ossification within
the repair matrix.
Methods and Materials: In miniature pigs, cartilage defects in a
total of 12 knee joints were treated by the microfracture technique
(Mfx). The Mfx-treated defects either received no further treatment
(Mfx), or were filled with collagen-bound OP1 (Mfx+OP1), collagen-
bound TSP-1 (Mfx+TSP-1), or with a combination of the two factors
(Mfx+TSP-1+OP1), respectively. Six or 26 weeks later, the outcome
was assessed by the ICRS histological assessment scale.
Results: Defects treated by Mfx were typically characterized
by fibrocartilaginous repair tissue and hypertrophic reactions
of subchondral bone tissue. The application of OP1 (Mfx+OP)
significantly improved the quality of the repair tissue with a hyaline-
like, proteoglycan-rich matrix, but was also associated with excessive
endochondral ossification. In contrast, the application of TSP-1
(Mfx+TSP) completely prevented subchondral bone hypertrophy
and matrix calcification, but did not support chondrogenesis within
the repair tissue. Only the combined treatment with Mfx+TSP-1+OP1
could achieve both generation of a hyaline-like repair matrix and
prevention of endochondral ossification.
Conclusions: These data indicate that the induction of chondrogenesis
and the prevention of terminal chondrocyte differentiation have
to be addressed by different factors. Future studies may further
optimize this “factor-cocktail” and release kinetics in order to yield
the unique articular chondrocyte phenotype within the cartilage
repair tissue.
Posters 239
P215
Gene expression in subchondral bone osteoblasts from hip
osteoarthritis, rapidly destructive coxarthrosis and rheumatoid
arthritis
K. Honjo1, Y. Arai1, K. Takahashi1, R. Terauchi1, S. Nakagawa1, N.
Hiraoka1, H. Inoue1, M. Saito1, S. Tsuchida1, T. Kubo2
1
Kyoto/Japan, 2Kyoto City/Japan
Purpose: The representative hip joint diseases include osteoarthritis
(OA), rapidly destructive coxarthrosis (RDC) and rheumatoid arthritis
(RA). OA progresses slowly, whereas RDC and RA result in rapid
joint deterioration. Recent studies suggested that an abnormal
subchondral bone metabolism might be involved in the progression
of joint destruction. In this study, we examined the gene expression
of inflammatory cytokines, proteases and factors related to bone
metabolism in the subchondral bone of these hip joint diseases.
Methods and Materials: The subjects were 7 OA, 3 RDC and 3 RA
patients who underwent total hip arthroplasty. Four patients with
femoral neck fracture (FNF) were used as controls. Subchondral
bone tissues of femoral head were harvested. The subchondral bone
osteoblasts (SBOs) were isolated. The mRNA expression of IL-1β,
IL-6, IL-8, TNF-α, MMP-13, ADAMTS-5, RANKL, RANK and OPG were
analyzed by real time RT-PCR.
Results: The expression levels of IL-β, IL-6, ADAMTS-5 and MMP-13 in
the SBOs from OA patients were significantly higher than those from
FNF. RANK/ RANKL/ OPG among these two groups had no significant
difference. Comparing RDC with OA, IL-8 gene expression and RANKL/
OPG ratio in the RDC were higher. The ADAMTS-5 mRNA in the RA was
significantly higher than OA, but the level of OPG was lower. RANKL/
OPG ratio in the RA was upregulated.
Conclusions: In relation to the joint destruction, vulnerability of
subchondral bone and activities of osteoclasts have been discussed.
In the present study, highly expressed IL-β, IL-6, ADAMTS-5 and MMP-
13 around the subchondral bone area might have an influence on the
bone turnover, which contribute to the progression of OA. In addition
to the highly expression of inflammatory cytokines and proteases,
upregulated osteoclastic activities might accelerate the joint
destruction and result in the rapid clinical course of RDC and RA.
P216
Bipolar fresh osteochondral allograft in the shoulder: a possible
alternative to traditional arthroplasty?
S. Giannini, R. Buda, A. Ruffilli, M. Cavallo, B. Grigolo, M. Nanni, F.
Vannini
Bologna/Italy
Purpose: Surgical treatment of severe post-traumatic shoulder arthritis
in young active patients is a challenge. Bipolar fresh osteochondral
allograft (BFOA) is a fascinating option for patients without severe cuff
tear arthropaty. The purpose of this study is to report the clinical and
histological results of 2 cases of BFOA in the shoulder.
Methods and Materials: 2 patients, mean age 48.5 ± 0.7 years,
affected by post-traumatic unilateral arthritis of shoulder,
received BFOA. Patients evaluation was carried out clinically and
radiographically by X-Rays, CT scans and MRI. Immunosuppressive
therapy was used for 6 months. Bioptic specimens were harvested
during hardware removal at 12 months follow-up in both
patients. Bioptic specimens were examined histochemically and
immunohistochemically and by immunohistochemistry to analyze
specific cellular markers. ICRS II scoring system was used to
evaluate the grafted tissue.
Results: At 24 months both patients obtained good clinical result
with pain relief and ROM comparable with that of the controlateral
healthy joint. Good radiographic consolidation of the graft was
observed at 5 months. Radiographic arthritis of the BFOA was
nevertheless evident at follow-up. Bioptic specimens showed
cartilaginous structure with an high proteoglycan content and a
normal cellular distribution, collagen Type II expression. Analysis
of synovial biopsies demonstrated limited number of macrophages,
without prominent perivascular inflammatory cell infiltrates or
lymphoid aggregates.
Conclusions: For properly selected patients without symptomatic and
disabling rotator cuff deficiency, BFOA can result in improvements in
pain, motion, function, and patient satisfaction. Furthermore, unknown
factors still have an influence on allografts outcome. Radiographic
arthritis at follow-up, was evident although immunosuppressive
therapy, correct positioning and size of the graft. This finding, even
if uncorrelated to the clinical results, still remain cause of concern.
Further research regarding the immunological behavior of transplanted
cartilage are needed in order to improve the results.
240 Posters
P217
Isolated Osteochondral Allografting of the Patella
W. Bugbee, A. DeYoung, S. Görtz
La Jolla/United States of America
Purpose: Cartilage disease of the patellofemoral joint represents
a difficult clinical challenge. Although osteochondral allografting
has been shown to be effective in the treatment of chondral and
osteochondral lesions of the femoral condyle and tibial plateau,
there are few clinical studies on osteochondral allografting of the
patellofemoral joint. In this study, we report on the outcome of
isolated osteochondral allografting of the patella.
Methods and Materials: Twenty patients (22 knees) underwent
isolated osteochondral allografting (OCA) of the patella. Patients
with bipolar or multifocal allografts were excluded. The mean age
was 35 years (range, 17–64 years) and 60% were female. 91% had
previous surgery (range, 1–10 procedures), including seven patients
who had surgery for realignment of the extensor mechanism. The
majority of patients had traumatic or degenerative chondral disease
or fracture malunion. Mean allograft area was 11.1 cm2 (range, 4.0–
17.8 cm2) and 86% (19/22) had total patellar resurfacing.
Results: Thirty-two percent had failure of the allograft (defined
as revision, patellectomy, or conversion to TKA) at a mean of 19.6
months (range, 6–40 months). Seven of 22 (32%) had further
surgery with retention of the allograft. Mean follow-up of the 15 non-
failures was 102 months (range, 24–310 months). Mean IKDC pain
scores improved from 6.8 to 3.2 (p< 0.01). Mean IKDC function
scores improved from 4.3 to 6.6 (p= 0.06). 93% reported less pain,
80% reported better function, and 93% reported improvement.
All patients were satisfied with their results and would choose
allograft surgery again under similar circumstances.
Conclusions: Patella allografting resulted in a high (64%)
reoperation rate. However, patients that retained their allografts
had significant improvement in pain, better function, and were
satisfied with their treatment. We conclude that patella allografting
is a useful salvage treatment option in carefully selected patients.
P218
Fresh total shell osteochondral allograft of the knee: surgical
technique, clinical and histological results
S. Giannini, R. Buda, A. Ruffilli, M. Cavallo, G. Desando, S. Neri, B.
Grigolo, F. Vannini
Bologna/Italy
Purpose: Post-traumatic arthritis of the knee poses a
reconstructive challenge for the young active patient. Fresh total
shell osteochondral allograft (FTSOA) is a fascinating option since
chondrocytes showed to be viable many years after transplantation.
Nevertheless, the source of cells in transplanted cartilage has not
been confirmed as being from the donor or the recipient. Purpose
of this study was to examine the effect of FTSOA as an alternative
to prosthesis in post-traumatic arthritis of the knee and to provide
a characterization of chondrocytes DNA.
Methods and Materials: 8 patients, age 42±12 years, affected
by pst-traumatic knee arthritis, received FTSOA. Patients were
evaluated clinically and by X-Rays, CT scans and MRI. The technique
provided a complete total joint resurfacing with a continuous 1.5cm
thick FTSOA, including the resurfacing of the patella and menisci.
Biopsies, including cartilage, ligament, synovia and menisci
were obtained during revision and evaluated by histological,
immunohistochemical analyses and by allograft genetic typing by
microsatellite analysis in respect to recipient and donor DNA.
Results: Satisfactory allograft consolidation, ROM and weight-
bearing were resumed at 6-8 months in all the cases. A severe
joint laxity developed at 10-16 months in 6 patients requiring
arthroplasty. Two patients are satisfied with the result.
Histological evaluation showed cartilage degenerative changes
with hypocellularity. Fibrocartilaginous aspect and positivity to
extracellular matrix degrading enzymes were evident in the soft
tissues. The genetic typing showed a mixed recipient/donor DNA
profile in all the cases.
Conclusions: Knee FTSOA demonstrated high rate of failure,
although specifically designed jigs, excellent positioning and
good consolidation rates. The integration of recipient cells into
transplanted knee allograft investigated by DNA typing, resulted
in persistence of donor cells in the graft together with presence
of cells from the host, thus suggesting partial in growth of the
transplanted allograft by host cells at different degrees.
P219
Mid-Term Functional and Radiological Outcomes of Allograft
Meniscal Transplantation
J.C. Monllau, G. Gonzalez-Lucena, P.E. Gelber, X. Pelfort, M. Tey
Barcelona/Spain
Purpose: The purpose of this study was to evaluate the functional and
radiographic results on a mid term basis as well as the complications
in a first series of meniscal allograft transplantation (MAT) performed
by means of suture fixation without any bone block.
Methods and Materials: A series of 33 meniscal allograft
transplantation were operated on in our institution from January
2001 to October 2003. Inclusion criteria was patients with
compartmental joint line pain due to a previous meniscectomy
in an otherwise well aligned knee. There were 24 males and 9
females with a mean age of 38.8 years (range, 21 to 54 years). The
functional outcomes were evaluated by use of Lysholm and Tegner
scores at a mean follow-up of 6.5 years. A Visual Analogical Scale
(VAS) for pain was also used. Radiographic assessment included
joint space narrowing in Rosenberg view and MRI evaluation.
Results: The Lysholm and Tegner scores significantly improved
from 65.4 to 88.6 (p < 0.001) and from 3.1 to 5.5 (p < 0.001)
respectively, after index surgery. Similarly, the VAS score
significantly dropped from 6.4 to 1.5 (p < 0.001). The radiographic
evaluation did not show any joint space narrowing (p = 0.38).
Meniscal extrusion was a constant finding, averaging 36.3% of
total meniscal size. According to the Van Arkel criteria, the survival
rate was 87.8% at 6.5 years. The rate of complications was 33%.
Conclusions: This study suggests that MAT provides significant pain
relief and functional improvement in selected symptomatic individuals
on a mid term basis. with a non-negligible reoperation rate.
P220
Matrix associated and Autologous Chondrocyte Implantation in
the Ankle: Clinical and MRI Follow-up after 2 to 14 Years
C. Hirschfeld, S. Domayer, D. Stelzeneder, R. Dorotka
Vienna/Austria
Purpose: The aim of this study was to assess the clinical mid- to
long-term results of autologous condrocyte transplantation in
the ankle in 19 cases and to evaluate repair tissue quality with
T2 mapping at 3T.
Methods and Materials: 19 cases with talar cartilage defects were
assessed with the AOFAS Score, a modified Cincinnati Score and
a subjective Ankle-Hindfoot Score at mean 4.5 ( 2 to 14) years
after surgery. Nine patients consented to an additional MRI exam
including T2 mapping at 3T. ACI was carried out with a periosteal
flap (4 cases), with a matrix assisted ACI technique (Hyalograft
C, 13 cases) or with a matrix assisted collagen-I-gel condrocyte
transplantation system (CaReS, 2 cases). At the time of surgery,
the patients had a mean age of 27,8 ± 7,5 years (21-42) and the
defect size measured during surgery was 1.48 ± 0,64 cm² (0.5-3).
Results: Significant improvement was found in all cases. The
AOFAS improved from 52.2 to 87.0 , the AHS from 45.8 to 81.4 ,
and the modified Cincinnati Score from 3.0 to 6.9. All cases that
underwent MRI had an intact interface of the repair tissue and
the adjacent talar cartilage. T2 mapping results indicated that the
collagen and water content of the repair tissue was comparable
to adjacent cartilage. Repair tissue (RT) T2 was 30.9 ± 7.9 (22 –
43) ms and reference cartilage (RC) T2 was 37.3 ± 7.5 (27 – 47)
ms. The unpaired double tailed student t-test demonstrated no
significant difference between RT and RC T2 (P=0.095).
Conclusions: ACI is a valuable treatment for talar cartilage lesions
that can provide excellent outcome in the long term. Matrix
assisted implantation allows for a less invasive procedure and can
apparently provide good defect filling as well as repair tissue with
a water and collagen content similar to the adjacent cartilage.
P221
Autologous Chondrocyte Implantation of the Ankle
R. Ferkel, S.K. Kwak
Van Nuys/United States of America
Purpose: To report long-term outcomes on patients who have had
autologous chondrocyte implantation of the talus
Methods and Materials: Thirty-two patients (16 male, 16 female;
mean age 34) underwent ACI of the talus after previous failed

surgical management for osteochondral lesions. There were 24
medial and 8 lateral lesions, with an average size of 18x11 mm (198
mm2). Twenty-three patients underwent ACI of the talus alone;
nine had ACI with a “sandwich procedure”. Mean follow-up was 66
months (range 24-113 months). 25 of 32 patients underwent second
look arthroscopy during hardware removal at 1 year.
Results: Preoperatively, 25 patients rated their ankles as poor and
3 as fair, using the simplified symptomatology evaluation. At last
follow-up, 8 were classified as excellent, 12 as good, 5 as fair and 1 as
poor. One patient qualified as a failure having had a fusion at 4 years
post-op. Tegner activity scale improved from 1.6 to 4.4. The Finsen
score (modified Weber score) showed significant improvement in
total score. There was also overall agreement between the Finsen
score and the American Orthopaedic Foot and Ankle Society ankle
hindfoot score, with significant improvement from 50.2 to 86.4.
Nine of eleven previously reported patients showed continued
maintanence of improvement in Tegner, Finsen and AOFAS scores.
Conclusions: Autologous chondrocyte implantation of the talus
yields significant functional improvement with enduring long-term
results in patients who have failed previous surgery..
P222
Synovial fibrinolysis activity in foals with septic joint disease:
Ribera T, Monreal L, Armengou L & Prades M : Departament de
Medicina i Cirurgia Animals, Facultat de Veterinària, Universitat
Autònoma de Barcelona, Barcelona, Spain.
M. Prades1, T. Ribera2
1
Cerdanyola/Spain, 2Barcelona/Spain
Purpose: The objective of this study was to assess synovial
fibrinolysis activity in foals with septic arthritis
Methods and Materials: Prospective observational clinical study.
Foals admitted for septic joint disease (neonates or not) were
included. Synovial D-dimer concentration was determined together
with routine analysis. Septic joint was diagnosed when it resulted
consistent with septic disease (synovial TNCC> 30,000 cells/ïL,
total protein> 4 g/dL and percentage of neutrophils> 80%, or a
positive synovial culture)
Results: The study included synovial fluid samples from 18 septic
newborn foals with polyarthritis, 9 septic newborn foals without
polyarthritis, 9 older foals with a septic joint, and 4 control foals.
Synovial D-dimer concentration was markedly higher in septic/
polyarthritic foals (median, interquartile range 299,104 ng/mL,
122,960-679,390) and septic joint foals (400,384 ng/mL, 256,768-
632,064) when compared to septic/non-polyarthritis foals (40,640
ng/mL, 21,240-89,426) and controls (36,753 ng/mL, 30,760-43,562).
A decrease towards normalization in synovial D-dimer concentration
was seen in 10 septic foals after receiving treatment. A positive
correlation between an increased synovial D-dimer concentration
and other synovial fluid analysis variables was observed. Discussion:
Septic joint disorders in foals are associated with a marked synovial
fibrinolysis activation to subsequently destroy fibrin formed. This
has been studied in degenerative joint diseases in humans and
experimental animals, and more recently in horses, but no information
is available in patients with acute septic arthritis
Conclusions: Synovial fibrinolysis activity was higher in foals with
septic joint disease, and may be used as a marker of inflammatory
joint disease for diagnostic and follow up purposes.
P223
Local PRP therapy induces fluctuations on plasmatic and synovial
fluid biomarker concentrations
M. Prades1, I. Abellanet2
1
Cerdanyola/Spain, 2Barcelona/Spain
Purpose: To report the effect of intralesional or synovial injection of
PRP on plasmatic and synovial fluid concentrations of 2 biomarkers
(COMP and GAGS) and hyaluronan.
Methods and Materials: Seven horses with DJD, 8 horses with
tendonitis, 4 patients with tenosynovitis, seven horses free of
musculoskeletal disease (negative controls) and 2 horses with
lesions and no treatment (2 positive controls) were included in the
study. Baseline COMP, GAGs and HA plasmatic and sinovial fluid
concentrations (in DJD affected horses) were measured initially and
then after treatment with PRP and normal saline in some horses. A non
parametric test was used for statystical analysis (Wilcoxon p<0.05).
Biomarker concentrations and HA were measured by ELISA at the IBB
(Biomedicine and Biotechnology Institute at the UAB).
Posters 241
Results: PRP administration induced a significant decrease
P<0,005 in COMP concentrations both in plasma ( tendinitis:
11,06 U/L±1,55SD); tenosynovitis 14,15 U/L±7,32SD; OA:19,98
U/L ±7,4SD), and increases in GAG concentrations although not
statistically significant in the samples tested. Fluctuations in HA
concentrations were seen but were not significant. . Discussion: The
significant decrease in COMP concentrations may be interpreted as
a sign of decrease in degradation and the increase in GAGs although
not statistically significant, as a sign of synthesis.
Conclusions: From the 3 mollecules tested in this study COMP
was the marker that reflected both baseline lesional status and
response to PRP treatment.
P224
Synovial fibrinolysis activity as a potential early marker in horses
with inflammatory joint disease.Thais Ribera, Luis Monreal, M.
Ángeles Delgado, José Ríos, M Prades Departament de Medicina i
Cirurgia Animal , Universitat Autònoma de Barcelona
M. Prades1, T. Ribera2
1
Cerdanyola/Spain, 2Barcelona/Spain
Purpose: To assess synovial fibrinolysis activity in horses with joint
inflammatory disease.
Methods and Materials: Study Design: Prospective observational
clinical study and pilot experimental study. Sample Population: 34
synovial fluid samples from 24 horses with joint disease and 35
controls. Additionally, 5 synovial fluid samples collected before
and after induction of chondral lesion from 3 experimental horses.
Methods: Horses with synovial disease had a diagnosis consistent
with osteoarthritis (OA) or osteochondritis dissecans (OCD).
D-dimer concentration was measured in synovial fluid samples.
Synovial fluid also was routinely analyzed.
Results: In the clinical study, synovial D-dimer concentration was
significantly (P= 0.002) higher in the inflammatory group (median,
interquartile range; 25,024 ng/mL, 7,914-55,104) compared to
controls (10,832 ng/mL, 4,808-16,652). The logistic regression
analysis showed that horses with synovial D-dimer concentration
above 20,000 ng/mL were statistically (P<0.001) more consistent
with inflammatory joint disease, resulting in an OR= 9.8 (95% CI=
2.9 to 33.8) for inflammatory joint disease when synovial D-dimer
concentration was above 20,000 ng/mL.
Conclusions: Inflammatory joint disorders in horses are associated
with an increased fibrinolysis activity in synovial fluid.
P225
Clinical and radiological evaluation of matrix-induced autologous
chondrocyte implantation (MACI) at five years.
J. Ebert, M. Fallon, W.B. Robertson, T.R. Ackland, M.H. Zheng, D.J. Wood
Perth/Australia
Purpose: This study presents radiological and clinical outcome
to five years post surgery, for a consecutive series of patients
following matrix-induced autologous chondrocyte implantations
(MACI), to evaluate whether MACI provides a suitable mid-term
treatment option for articular cartilage defects in the knee.
Methods and Materials: An uncontrolled, prospective study design
was used to assess clinical and radiological outcome in 41 patients
(44 knees; 53 grafts) to five years following MACI. Following surgery,
patients underwent a structured, supervised rehabilitation program
of progressive exercise and graduated load bearing to protect, and
then stimulate the healing process. Clinical outcomes were measured
using the Knee Injury and Osteoarthritis Outcome Score (KOOS), the
Short-Form Health Survey (SF-36), the six-minute walk test and knee
range of motion. High resolution magnetic resonance imaging (MRI)
was undertaken at 3, 12 and 24 months, as well as at five years post-
surgery, to describe the quality and quantity of repair tissue.
Results: Patients demonstrated a significant improvement (p<0.05)
throughout the post-operative timeline for all five subscales of the
KOOS, both subscales of the SF-36, the six-minute walk test and
active knee extension. Patients also demonstrated an increased MRI
composite score over time that improved significantly (p<0.05)
from three months to five years post-surgery. Post-hoc analysis
demonstrated the improvement occurred predominantly in the first
12 months post-operatively, though was maintained to five years.
Conclusions: Both clinical and radiological outcome improved up until two
years post-surgery, and were maintained to five years. Based on these
data, it would appear that MACI provides a suitable mid-term treatment
option for full thickness, localised articular cartilage defects in the knee.
242 Posters
P226
Open Autologous Osteochondral Transplantation on Posterior
Aspect of Medial Femoral Chondyle with Medial Epicondylectomy
Y.G. Koh, S. Kim, S. Jo
Seoul/Korea
Purpose: The purpose of this presentation is to introduce a novel
approaching method of autologous osteochondral transplantation
for treatment of chondral and osteochondral lesions on posterior
aspect of the medial femoral chondyle(MFC) of the knee.
Methods and Materials: Operative technique : After the arthroscopic
procedure, about 8cm skin incision is made on the midline of the
knee joint and arthrotomy is performed with midvastus approach.
MFC is exposed and the medial epicondyle is confirmed. Medial
epicondylectomy is done with including about 2cm diameter bone
fragment and attached medial collateral ligament. Subsequently was
carried out with the OATS technique, which allows for press-fit graft
implantation. Without medial epicondylectomy, OATS devices cannot
be applied perpendicularly on the osteochondral lesion of posterior
aspect of the MFC then, it is impossible to achieve perpendicular
graft insertion. After autologous osteochondral transplantation is
performed, reattachment of the medial epicondyle is done with one
washer and one 5.0 mm diameter cannulated screw.
Results: Case: Patient was a 48-year old male complained pain
on the right knee when he standed up from sitting or squating
position, climbed a mountain and went up the stairs. There
was an osteochondral lesion on posterior aspect of the MFC on
the preoperatively evaluated MRI. Autologous osteochondral
Transplantation was performed with medial epicondylectomy
approach. Postoperative evaluation was performed when
one year passed after surgery. Lysholm score improved from
56 preoperatively to 89 postoperatively. IKDC assessment
was improved from abnormal(C) preoperatively to normal(A)
postoperatively. VAS score were improved 8 to 2 on standing-up
motion and 8 to 3 on going-up stairs motion. Congruency of the
articular surface was restored on postoperatively evaluated MRI.
Conclusions: An approach with medial epicondylectomy including
attached medial collateral ligament is one of the recommendable
alternative for treatment of chondral and osteochondral lesions on
posterior aspect of the MFC of the knee with OATS technique.
P227
Combined autologous chondrocyte implantation (ACI) with supra-
condylar femoral varus osteotomy, following lateral growth-plate
damage in the knee of an adolescent
S. Vijayan1, G. Bentley2, R. Carrington1
1
Stanmore/United Kingdom, 2London/United Kingdom
Purpose: We report the 8-year clinical and radiographic outcome
of an adolescent patient with a large osteochondral defect of the
lateral femoral condyle, and ipsilateral genu valgum secondary to
an epiphyseal injury.
Methods and Materials: This patient was successfully managed
with Autologous Chondrocyte Implantation (ACI) and supracondylar
re-alignment femoral osteotomy.
Results: Long-term clinical success was achieved using this method,
with the patient reporting significant improvements in Modified
cincinnati, Bentley and Visual analogue scores post-operatively
when compared to their pre-opartive state. Since treatment, the
patient has resumed semi-professional sporting activities which
was was not possible before.
Conclusions: We conclude that this technique illustrates the
effective use of re-alignment osteotomy in correcting mal-alignment
of the knee, protecting the ACI graft site and providing the optimum
environment for cartilage repair and regeneration. This is the first
report of combined ACI and femoral osteotomy for such a case.
P228
Xenograft chondrocytes for cartilage repair– A unique source for
genuine hyaline cartilage implant
G. Maor, R. Goldberg, S. Bar-Zvi, G. Nierenberg
Haifa/Israel
Purpose: Since hyaline cartilage does not regenerate, biological
long term solutions are based on tissue regenerative therapies.
The current available cell base therapy is autologous chondrocyte
implantation (ACI), which results invariably in mixed fibrous /
hyaline-like tissue in various proportions. Chondrocytes separated
from articular cartilage inevitably dedifferente into fibroblast-
like cells. Spontaneous generation of hyaline cartilage - is
physiologically present only in skeletal growth centers. A unique
accessible site of hyaline producing cells was found in the neonatal
porcine mandibular condyle with the capacity to differentiate into
stable hyaline cartilage spontaneously.
Methods and Materials: Mandibular condyle (MC)-derived
chondrocytes (MCDC), harvested from neonatal SPF porcine
were cultured in vitro. The cultured cells create a continuous
implantable cartilage film (Cartimove™), offering the possibility of
simple mechanical handling. Re-culturing/implantation preserves
its proliferating and differentiating activities as demonstrated by
immunohistochemistry and RT-PCR. In the pre-clinical studies,
Cartimove was implanted in lesions created in the medial femoral
condyle of 8 goats. Three, 6 and 13 months implants were retrieved
and analyzed morphologically, biochemically and biomechanically
Results: In vitro, at 10 days, the expression of type II collagen is amplified
by about 50 folds (RT-PCR), concomitantly, type I collagen expression is
reducing to negligible values. Co-culturing with human cells, showed no
transmittable pathogen. The preclinical studies, demonstrated tissue
filling, environmental integration and hyaline characteristics. Local as well
as lymphatic organ analysis, showed no detectable immune response.
Conclusions: We propose that Cartimove cultured from porcine-
derived MCDC cells, is capable of replenishing cartilage lesions
with genuine hyaline cartilage.
P229
Correlation between localisation of 4° chondral leasions in
knees and the clinical result after autologous chondrocyte
transplantation
B. Böttenberg, P. Schaeferhoff
Köln/Germany
Purpose: The method of the ACT demands to repair deep and huge
size cartilage defects with hyalin respectively hyalin like cartilage.
Previous studys describe only rare the effect of the ACT in knees
regarding to their chondral defect region.
Methods and Materials: This monocenter study presents the
correlation between the localisation of 4° chondral leasions in
knees and the clinical outcome after ACT treatment. The clinical
evaluation was performed using the subjective and the objective
part of the IKDC2000 score for 52 patients before and after treated
with ACT (meanfollowup 38,6 months).
Results: 52(35m/17f) patients in the age between 18 and 45 years
(mean age 38 years) participate in this study. The defect size
average is 6,13cm². Concerning to the cartilage defect localisation
we develop five different cartilage defect region groups.
Group 1 (medial femoral, n=15), Group 2 (lateral femoral, n=10),
Group 3 (trochlear, n=10 ), Group 4 (retropatellar, n=8) and Group 5
(combinied defect regions, n=9). The follow up examination shows
in all five groups a signifivant rise concerning to the subjective und
objective IKDC2000 score after ACT treatment ( p<0,001). The
subjective part of the IKDC2000 score shows significant poorly
post-operatively results for the the patients in the groups 4 and 5
than in the groups 1,2,3 (p<0,001), where as there is no significant
difference betwenn the 5 groups pre-operatively. Regarding to the
objective part of the IKDC2000 score with a view to the chondral
leasion area there are no significant differences between the pre-
and postoperative evaluated IKDC2000 scoure values (p<0,001).
Conclusions: The presented data indicate autologous chondrocyte
transplantation as an effective and safe option for the treatment of
large full thickness cartilage defects in knee joints with significant
better subjective results for patients with cartilage defects in the
medial or femoral condyle and trochlear region than in retropatellar
or combinied cartilage leasion regions.
P230
Can Joint Preservation be possible for Osteonecrosis of the Knee
by Autogenous Osteochondral Graft Transplantation? -Middle-
term Results-
Y. Matsusue1, M. Kubo1, Y. Nakagawa2
1
Otsu/Japan, 2Kyoto/Japan
Purpose: Repair of the osteochondral lesion of osteonecrosis of the
knee is a difficult and controversial issue. In this paper, we present
the middle-term clinical results of autogenous osteochondral
grafting for osteonecrosis of the knee including steroid-induced
osteonecrosis.

Methods and Materials: Thirty-two patients (36 knees) with at least
24-months follow-up periods were included in this study. The age
ranged from 21 to 76 years of age, with a mean of 48. The follow-up
period ranged from 24 to 165 months with a mean of 52. The cause
of osteonecrosis was steroid-induced in 10 and idiopathic in 26.
There was one knee with osteonecrosis of the medial tibial plateau.
Correction osteotomy was performed in 22 knees at the same time
of osteochondral grafting. The size of the osteochondral defect
ranged from 156 to 1080 mm2 with a mean of 475. The number of
transplanted grafts ranged from 1 to 6 with a mean of 2.94.
Results: The clinical results by ICRS cartilage evaluation form were
normal in 14, nearly normal in 20, and abnormal in 2 knees. The
range of motion was normal except in one knee with a flexion of 135
degrees. Second-look arthroscopy revealed complete integration
in 59% of the cases and the others showed partially incomplete
integration between the grafts. MRI showed that complete
healing of the necrotic lesion was observed in 14 knees (41%) and
a significant lesion was remained in one knee. In steroid induced
osteonecrosis with unstable but intact articular cartilage on the
necrotic lesion, the lesion is treated by multiple drilling and bone
grafting through cartilage windows created OATS instruments; then
followed by two large osteochondral plugs transplantation into the
windows (eyeplass-plasty). Abnormal results were obtained in one
patient who had non-union of osteotomy and another patient with
steroid-induced osteonecrosis who had avulsion of the remaining
cartilage after transplantation. When a proper alignment was
obtained, autogenous osteochondral grafting for osteonecrosis of
the knee can provide a good knee function including full flexion
and could avoid total knee arthroplasty.
Conclusions: Autogenous osteochondral grafting can give
satisfactory middle-term results for the osteonecrosis of the knee
including steroid-induced osteonecrosis.
P231
Arthroscopic matrix-induced autologous chondrocyte
implantation (MACI) in combination with accelerated
rehabilitation
J. Ebert1, R. Carey-Smith2, M. Fallon1, H. Davies1, S. Garett1, D.J.
Wood1, G. Janes1
1
Perth/Australia, 2Coventry/United Kingdom
Purpose: This study presents the clinical and radiological outcome
in patients treated with arthroscopically performed matrix-induced
autologous chondrocyte implantation (MACI), combined with an
‘accelerated’ return to full weight bearing (WB).
Methods and Materials: Fifteen patients with full-thickness defects
to the femoral or tibial condyles were treated with MACI performed
arthroscopically. Following surgery, patients underwent a graduated
rehabilitation program that aimed to protect the implant initially,
then incrementally increase the load until full WB was attained
at 8-weeks post-surgery. Clinical outcomes were measured pre-
surgery and at 3, 6 and 12 months post-surgery using the KOOS,
VAS, the six-minute walk test and knee range of motion. Clinical
outcomes were compared with previously established clinical
scores from patients who had undergone MACI performed through
open arthrotomy, in combination with an identical, accelerated
rehabilitation protocol. High resolution MRI was undertaken at 3
and 12 months post-surgery.
Results: From pre-surgery to 12 months post-surgery, KOOS and VAS
scores demonstrated significant improvement (p<0.05), though
there was no difference (p>0.05) to patients who underwent
open arthrotomic MACI. There was no difference (p>0.05) in six-
minute walk distance, though significantly better (p<0.05) active
knee flexion was observed at 3 and 6 months in the arthroscopic
MACI group. Patients demonstrated an increased MRI composite
score over time that improved significantly (p<0.05) from three to
12 months post-surgery, but did not differ (p>0.05) from patients
following open MACI.
Conclusions: Arthroscopic MACI in combination with ‘accelerated’
rehabilitation has shown encouraging early results, with good
early graft infill and no graft complications. Arthroscopically
performed MACI may reduce the co-morbidity associated with
open arthrotomy, such as adhesions, decreased range of motion,
pain and impressive scars, whilst providing faster post-operative
rehabilitation due to reduced pain and muscular deficits.
Posters 243
P232
Matrix guided Autologous Chondrocyte Transplantation (MACI) is
effective as second line treatment of large cartilage defects in the
knee: 2-year clinical results
P.E. Müller, M.F. Pietschmann, A. Horng, T. Niethammer, S.
Lehmann, M. Feist, I. Pagenstert, C. Glaser, V. Jansson
Munich/Germany
Purpose: Over the last years Matrix guided Autologous Chondrocyte
Implantation (MACI) has become an important surgical technique
for treating large cartilage defects. The aim of our prospective study
was to evaluate the effectiveness of MACI as first and second line
treatment option for cartilage repair of large defects in the knee.
Methods and Materials: 43 pts. were treated with MACI for cartilage
defects of the knee between 2004 and 2008. We assessed clinical
situation pre- and postoperatively using the IKDC Knee Examination
Form, VAS for “pain at rest” and “pain at weight bearing”. MRI
scans for evaluation of cartilage regeneration were obtained
postoperatively. The modified MOCART score and T2 relaxation
time were applied to assess cartilage regeneration.
Results: One third of the cartilage defects were localized retropatellar
the remaining on the femoral condyles. The average defect size
was 5,7 cm2. After one year the clinical assessment showed a
significant improvement across all patients. After two years we
found an improvement as well. With regard to first or second line
therapy MACI showed similar results in both groups. Etiology and
age of the cartilage lesion are important factors that influence the
postoperative clinical outcome. The MRI showed an significant
improvement of the implanted scaffold over time as well.
Conclusions: The present study confirms the benefits of MACI in
young pts. with large cartilage defects of the knee. Patients with
MACI as second treatment option achieved similar results as patient
which had MACI done as first therapy. We expect a good long-term
outcome of MACI comparable to that of classic ACI.
P233
Patellar dislocation causing severe patellar-trochlear cartilage
damage, treated by BioCart™II implantation. A case study
A. Friedman1, G. Chaimsky1, J. Lowe1, H. Barkay2, A. Yayon2
1
Jerusalem/Israel, 2Ness Ziona/Israel
Purpose: A major challenge for matrix-assisted autologous
chondrocyte implantation is the susceptibility of implants to
mechanical loads and shear forces in the joint during neocartilage
maturation, integration and remodeling, particularly in multiple
lesions where rapid implant maturation is critical for rapid
mobilization and rehabilitation. Here we describe treatment with
BioCart™II, a novel fibrin-HA porous mechano-transductive
scaffold, supporting autologous chondrocyte implantation in both
opposing lesions in a patella-trochlear kissing lesion
Methods and Materials: Right knee arthroscopy in a 24-year
old policeman injured playing football revealed severe chondral
damage to the medial patellar facet (1x0.8cm) and lateral trochlea
(2x2cm), both ICRS grade IIIc, compatible with lateral patellar
dislocation. Several loose cartilagenous bodies were removed.
Pre-operative MRI revealed a “kissing” lesion. Five weeks later, the
patient underwent arthrotomy. BioCart™II was implanted in both
the patella and trochlea using autologous chondrocytes cultured
in ProChon. A protective osteotomy (Fulkerson) with anterio-
medialization of the tibial tuberosity was performed. Post-surgery,
a knee immobilizer was applied and weight bearing in extension
was allowed with CPM 0-40 degrees then intensive physiotherapy
rehabilitation for 4 months. .
Results: MRI 8 months post-surgery showed almost normal
appearance of the implanted cartilage. Two years after implantation
of two opposing but overlapping BioCart™II implants it the patella
and trochlea, the patient is back to full service as a policeman,
including sport activities. The physical examination of the knee is
normal with only 10 degrees of reduction in knee flexion and very
mild reduction in quadriceps muscle tone.
Conclusions: BioCart™II has been scientifically designed to mimic
the topography of natural hyaline cartilage. Together with the
growth factor-directed culture of the chondrocytes which preserves
the chondrogenic potential of the cells an accelerated rehabilitation
schedule can be followed which facilitates the return to normal
physical activity. There is a need for more experience before
extensively prescribing BioCart™II for such complex injuries.
244 Posters
P234
Osteochondral transplantation in the therapy of osteochondral
lesions in the elbow
S. Vogt, A.B. Imhoff
München/Germany
Purpose: Effective treatment of osteochondral lesions in the elbow
is difficult. Débridement and microfracture or drilling techniques
are often insufficient and provide only temporary symptomatic
relief. The purpose of this study was to evaluate the treatment of
these lesions with osteochondral autografts.
Methods and Materials: From 1996 to 2009, 19 patients with
osteochondral lesions (n=20) of the capitellum humeri, trochlea,
or radial head were treated with osteochondral grafts, which were
harvested from the proximal aspect of the lateral femoral condyle.
The patients were evaluated preoperatively and postoperatively,
with an average follow-up of 6.6 years. The Broberg/Morrey
score was chosen for functional evaluation of the elbow, and
the ASES score was used for the analysis of pain. All patients
had imaging studies done preoperatively to evaluate the defect,
postoperatively to assess the ingrowth and viability of the graft
and at the final follow-up. The ipsilateral knee was examined for
donor-site morbidity.
Results: The Broberg and Morrey score improved significantly from
preoperatively to postoperatively, and pain scores were significantly
reduced (p < 0.05). At the time of the final follow-up, flexion and
extension were equal bilaterally in all patients. The postoperative
magnetic resonance imaging scans showed graft viability and a
congruent chondral surface in all patients. At the last follow-up
there were only minor degenerative changes in some patients. No
donor-site morbidity was noted at one year postoperatively and at
the final follow-up.
Conclusions: The osteochondral autograft procedure described in
the present study provides the opportunity to retain viable hyaline
cartilage for the repair of osteochondral lesions in the elbow while
restoring joint congruity and function. These results suggest that
the risks of a two joint procedure are modest and justifiable.
P235
Phenotypic modulation of chondrocytes from human knee joints
with osteochondritis dissecans: Implications for chondrocyte
implantation procedures.
M. Aurich1, G.O. Hofmann1, B. Rolauffs2, J. Mollenhauer3
1
Jena/Germany, 2Tuebingen/Germany, 3Reutlingen/Germany
Purpose: To characterize the biological quality and potential of
chondrocytes from the dissected fragment.
Methods and Materials: 20 patients with cartilage lesions in the
knee were involved. Dissected fragments from 10 patients with
typical OCD were harvested at arthroscopy. A biopsy of cartilage
from the intercondylar notch was taken from the same joint and
from 10 patients with a traumatic full thickness cartilage defect as
part of the autologous chondrocyte implantation (ACI) procedure.
Chondrocytes were isolated by enzymatic digestion and expanded
by subsequent primary cell culture. Before and after cultivation,
mRNA levels of collagen types I, -II, and -X were determined by semi
quantitative RT-PCR. In two patients, a biopsy of the repair tissue
was taken 6 and 18 months after ACI.
Results: In total, 0.81 ± 0.33 x 106 cells per gram tissue could be
recovered with no difference between dissecate and notch cartilages
(cell viability ≥ 90 %). Compared with the notch chondrocytes, cells
from the dissecate expressed similar levels of collagen type I and -II
mRNA including a 100,000fold relative increase of col1 over col2 after
cell expansion. Expression of collagen type X mRNA is significantly
less in trauma joints compared to OCD cartilages before and after
cell culture. The level of collagen type X message is approx. 50fold
decreased after cell culture, indicating a loss of hypertrophic
cells or expression of hypertrophic genes in chondrocytes. Post-
implantation biopsies show features of hyaline-like cartilage
without signs of hypertrophy or mineralization (Figure 1).
Conclusions: The high viability, quality and activity of the extracted
cells suggest a still preserved intrinsic repair capacity of the
dissecates. The molecular analysis indicates phenotypic modulation
of the isolated chondrocytes during cell culture. The similar quality
of the cells from both dissecate and notch after cell culture suggests
the use of either cartilages as a cell source for ACI.
P236
Therapeutic response to Autologous Cartilage Tissue Implant
(ACTI), Neocart is significantly greater in comparison to
microfracture (MF) arthroplasty surgery following treatment of
distal femoral cartilage lesions
D.C. Crawford1, R.J. Williams, III2
1
Portland/United States of America, 2New York/United States of
America
Purpose: This exploratory multi-site (FDA and IRB monitored) phase II
prospective randomized clinical trial evaluates the efficacy of a tissue-
engineered bio-implant, Neocart, for treatment of hyaline cartilage
knee injury in direct comparison to microfracture arthroplasty.
Methods and Materials: Thirty patients were randomized
(2:1;ACTI:MF) at arthroscopic confirmation of grade III ICRS
lesion(s). Lesional microfracture arthroplasty or hyaline cartilage
biopsy was performed. The ACTI (Neocart) produced by seeding
collagen I matrix scaffold with autogenous chondrocytes and
bioreactor treatment, was implanted 6 weeks post-arthroscopic
biopsy. Standard interval evaluations applied multiple validated
clinical outcomes measures. Responder analysis was applied
using a dual threshold criteria based on previously reported MPCI
(minimal perceptible clinical improvement) thresholds for both the
KOOS pain and IKDC outcomes measures.
Results: Twelve month data for all enrolled (21ACTI:9MF) and for
the first ten (7ACTI:3MF) reaching 24 months is reported. Mean
age (40+/-9yrs), BMI (28+/-4), injury acuity (3+/-5yrs) and lesion
size (ACTI 287+/-138mm2 v. MF 247+/-127mm2) is similar. Clinical
outcomes demonstrated by IKDC, KOOS, SF-36, and VAS Pain scores
are shown in Table 1. In both groups SF-36 physical and IKDC improved
from baseline (p<0.025) at one year. Improvement for ACTI v.
baseline was significant (p<0.025) for all additional measures at
twelve months and all but KOOS Sports at six months. ACTI showed
greater change from baseline than MF in IKDC (p<0.025) and KOOS
pain (p<0.025) at one and two years. Using ANOVA, the difference
in both IKDC and KOOS Pain change from baseline between MF
and ACTI was also significant (p=0.028) and (p=0.016) for each
measure. Similarly, more patients (P=0.0125, Fischer’s exact test)
in the ACTI arm were therapeutic responders at 6 months (48% v.
25%, data not shown) and 12 months (76% v. 22%) (Fig. 1).
Conclusions: This initial prospective randomized study indicates
ACTI, using the NeoCart implant treatment has greater clinical
efficacy than microfracture.
P237
A Novel technique for fully arthroscopically performed
3-dimensional autologous chondrocyte implantation (ACT-3D )
for retropatellar lesions. Preliminary results.
S. Alevrogiannis, G. Skarpas, A. Triantafyllopoulos
Athens/Greece
Purpose: To present our experience in using a novel technique for
autologous 3D chondrocyte implantation (ACT-3D), performed in fully
arthroscopical manner, for treatment of retropatellar cartilage defects.
Methods and Materials: We treated operatively in our Dept., 5
symptomatic patients with retropatellar lesions. All patients were
recreational athletes and the mean age was 32 years old (17-54 y.o.).
The mean area of cartilage defect was 3.8 cm² and all the cases were
classified as grade III(3) and IV(2) according to Outerbrigde scale. All
of them were treated fully arthroscopically. 3 patients were male and
4 patients had the operation at the right knee. In 2 cases the defect
was due to trauma, while 3 were due to osteochondritis dissecans. 3
of them had MFx procedure performed elsewhere, more than 5 years
ago. We performed 5 applications of ACT3D as single procedure, with
the patient in prone position. Preop. and postoperative evaluation
of patients was done using the Modified Cincinatti (MC) Rating
System(0-100), the VAS (visual analogue pain score) (0-10), IKDC
Knee examination score and Patient Outcome Function score.
Results: All the procedures progressed uneventfully. A specialized
rehabilitation protocol was followed. We assessed the patient at six months
and one year post-operatively; using Lysholm & Gillquist Score, VAS pain
score and the Patient Outcome Function score. The follow-up using MRI
showed adequate filling of the defect without significant graft-associated
complications for the same period. The clinical outcome was excellent .
Conclusions: Our preliminary results of this novel technique for autologous-
3D chondrocyte implantation for the treatment of retropatellar cartilage
defects, seems to be more than encouraging.Rehabilitation protocol is
quicker due to minimal trauma. As far as we know this is the first publication
in the literature regarding a novel technique for 3rd generation ACI fully
arthroscopically performed, concerning retropatellar lesions.

P238
Simultaneous autologous chondrocyte implantation (ACI) and
mosaicplasty for “Kissing” osteochondral defects of the patello-
femoral joint : A 10-year follow-up of 2 cases
S. Vijayan1, G. Bentley2, J. Skinner1, R. Carrington1
1
Stanmore/United Kingdom, 2London/United Kingdom
Purpose: Bi-polar lesions, especially those involving the patello-
femoral joint, are difficult-to-treat. The ideal approach to
management of “kissing” osteochondral lesions of the patello-
femoral joint is a topic of much debate.
Methods and Materials: We report the 10-year clinical outcome of
two patients with “kissing” osteochondral defects of the patella
and trochlear groove of the knee, managed by simultaneous patellar
autologous chondrocyte implantation (ACI) with type I/III collagen
(chondrogide) membrane (ACI-C) and trochlear mosaicplasty. This
is unreported in the Orthopaedic literature.
Results: At 10 year follow-up, long-term clinical success was
achieved using this method. Significant improvements in the
Modified cincinnati, Bentley and Visual analogue scores post-
operatively when compared with pre-operative scores were
reported. Clinical and arthroscopic assessment of both patients
showed excellent outcome, with adequate cartilage regeneration
being shown.
Conclusions: This case illustrates the effective use of trochlear
mosaicplasty for re-creating the anatomical contour of the joint
and therefore reducing the likelihood of disrupting the opposing
patellar ACI. We conclude that such a technique, allows for the
successfull treatment of “kissing” defects of the patello-femoral
joint in an otherwise diffcult to treat group of patients.
P239
Change over time in cartilage repair after chondrocyte
transplantation with a fibrin-hyaluronan matrix - correlation
of morphological MRI, biochemical T2 mapping and clinical
outcome.
I. Eshed1, S. Trattnig2, M. Sharon1, R. Arbel3, G. Nierenberg4 , E.
Konen1, A. Yayon5
1
Ramat Gan/Israel, 2Vienna/Austria, 3Hod Hasharon/Israel, 4Haifa/
Israel, 5Rehovot/Israel
Purpose: BioCart™II is a second generation matrix-assisted
implantation system composed of autologous chondrocytes
cultured in vitro, seeded on a fibrin/ hyaluronic acid based
scaffold and implanted by miniarthrotomy. We aimed to evaluate
change over time after implantation with BioCart™II of clinical
scores, morphological MRI and quantitative T2 values, novel MR
sequences that permit the assessment of ultrastructural elements
in the neocartilage.
Methods and Materials: Thirty-one patients (18-55 years) including
those with previous knee operations (e.g. ACL, meniscal repair),
were recruited 6-49 months post surgery with BioCart™II to correct
a single full-thickness cartilage defect in the femoral condyle.
Each subject underwent MRI (morphological (Figures 1 and 2)and
T2-mapping sequences) and completed a clinical questionnaire
(International Knee Documentation Committee (IKDC) knee
evaluation form). MRI scans were scored using the Magnetic
resonance Observation of Cartilage Repair Tissue (MOCART)
system and a T2-mapping evaluation of the zones in the repair
and non-operated cartilage. Analysis included correlation of IKDC
scores, MOCART and T2 evaluation with each other, with implant
age and with previous intervention history.
Results: The IKDC score correlated significantly with implant
age (r=0.35 p=0.056) and MOCART score (r=-0.39 p=0.031) and
inversely correlated with previous interventions (r=-0.39 p=0.034).
A slight, but not significant higher MOCART score was seen in
patients with longer duration implants (p=0.199). Zonal cartilage
T2 values, indicative of collagen concentration and orientation
were significantly lower in longer duration implants (p<0.002) in
patients without previous intervention. This trend was also seen
in patients with previous interventions although absolute values
were higher.
Conclusions: Significant improvement with time from BioCart™II
implantation was seen by IKDC scoring and MRI T2-mapping values.
Patients with previous knee operations also improved, though to a
lesser extent, and can benefit from this procedure.
Posters 245
P240
Characterized Chondrocyte Implantation in the Patellofemoral
Joint: a 1 to 3 Year Follow-Up
J. Vanlauwe, T. Claes
Leuven/Belgium
Purpose: To assess clinical outcome in patients treated with
Characterized Chondrocyte Implantation (CCI) for full thickness
lesions in the patellofemoral joint up to 36 months post surgery.
Methods and Materials: Patients with symptomatic patellofemoral full
thickness cartilage lesions were treated with ACI using characterized
chondrocytes (ChondroCelect®) covered with a Chondro-gide®
membrane. clinical outcome was assessed using the Knee Injury and
Osteoarthritis Outcome score (KOOS) and a Visual Analogue Scale
(VAS) for pain. responders were defined using 5 categories (>10
points and >20, 30, 50, 70%) based on KOOS and VAS.
Results: Thirty-six patients, with a mean defect size of 4.89 cm²
(1.5 to 11 cm²), were treated in the patella (n=25), trochlea (n=8) or
kissing lesion (trochlea and patella; n=3). The mean follow-up period
was 24.67 months . Treated patients showed statistically significant
improvements in KOOS (at 6, 12, 18, 24 and 36 months) and VAS (at
12 and 24 months). Responder analysis identified approximately
75% of patients with a clinically relevant improvement greater than
10 points. Treatment failure was observed in 4 patients. The most
commonly reported adverse events were joint crepitation (n=17)
and arthrofibrosis (n=7).
Conclusions: CCI resulted in statistically significant and clinically relevant
improvement over time. these results add to the evidence demonstrating
that ACI is a useful technique for the repair of patellofemoral cartilage
lesions and illustrate the potential of CCI in the context.
P241
Autologous chondrocyte implantation does not prevent the need
for arthroplasty in patients with pre-existing osteoarthritis
S. Nawaz1, P.K. Jaiswal2, K. Gallagher2, G. Bentley3, D. Park3, R.
Carrington3, J. Skinner3, T. Briggs3
1
4LP/United Kingdom, 2Stanmore/United Kingdom, 3London/United
Kingdom
Purpose: The rate of arthroplasty or osteotomy in patients who
had undergone autologous chondrocyte implantation (ACI) for
osteochondral defects in the knee was determined. Furthermore, we
investigated whether any radiographic evidence of osteoarthritis (OA)
prior to ACI was associated with poorer outcome following surgery.
Methods and Materials: We retrospectively reviewed the medical
notes and radiographs of 144 patients (mean age 34.9) who
underwent ACI from 1998 to 2005 at our institution. Knee function
was assessed according to the Modified Cincinnati Score (MCS)
pre-operatively and at a mean of 64.3 months postoperatively
(range 12 – 130). Radiographic changes were graded according to
the Stanmore grading system.
Results: Patients were divided into 2 groups; Group A were patients
with no evidence of OA (n=72) and Group B were patients with OA
(n=72). In group A, two patients required total knee replacement
(TKR) or unicondylar knee replacement (UKR) and 3 required high
tibial osteotomy (overall revision rate 6.9%). In group B, 14 patients
required patello-femoral replacement, or UKR or TKR and 7 patients
required osteotomy (overall revision rate 29.2%). This difference
was significant (p < 0.01). At latest follow up, the mean MCS was
significantly higher in Group A (72.5 versus 51.8, p < 0.01).
Conclusions: Patients with early radiographic of evidence of OA are
unlikely to gain maximum benefit from ACI. The results suggest that
ACI does not prevent patients from progressing in their arthritic
process and hence requiring joint replacement.
P242
Autologous chondrocyte implantation for degenerative cartilage
defects of patello - femoral joint.
K.P. Slynarski, E. Kurowska
Warsaw/Poland
Purpose: Autologous Chondrocyte Implantation (ACI) is typically
indicated for focal lesions contained within boundaries of healthy
tissue of femoral condyles. Our hypothesis was that population of
patients with degenerative changes of patello-femoral joint could
also benefit from this procedure and this could reverse or stop the
course of osteoarthritis.
246 Posters
Methods and Materials: We evaluated 11 patients, aged between
36 to 55 years, with extensive degenerative changes located on
patella or patellar facet of femur. Follow up ranged from 4 years
to 6 months. All patients underwent chondrocyte implantation
on collagen membrane and same rehabilitation process. Weight-
bearing status, range of motion restrictions, muscle strengthening,
proprioceptive retraining and soft tissue conditioning were the
main goals of rehabilitation in those patients.
All patients were prospectively clinically evaluated using the KOOS
score, SF-36 score, analogue pain score and patient’s subjective
assessment and MRI.
Results: The KOOS score increased significantly during the
observation period. For pain the mean preoperative score was 42
and the score at follow-up was 84. The corresponding scores for
symptoms were 36 and 81 for activity of daily life (ADL) 38 and 85
for sport and recreation 26 and 64 and quality of life 19 and 63. The
MRI evaluation well correlated with clinical outcome.
Conclusions: ACI for degenerative changes of patello-femoral joint
is off-label indication however our results suggest that covering
exposed degenerative bone with implanted cells on collagen
membrane, leading to cartilage-like tissue regeneration as
confirmed on MRI, will reduce the pain, improve joint function and
possibly protect from further joint destruction.
P243
Treatment of cartilage defects in the knee using alginate beads
containing human mature allogenic chondrocytes: Clinical
results at 3 years of follow-up.
A.A.M. Dhollander1, P.C. Verdonk2, R. Verdonk1, G. Verbruggen1,
K.F. Almqvist1
1
Ghent/Belgium, 2Gent-Zwijnaarde/Belgium
Purpose: The present study was designed to evaluate the implantation
of alginate beads containing human mature allogenic chondrocytes
for the treatment of symptomatic cartilage defects in the knee.
MethodsandMaterials:Abiodegradable,alginate-basedbiocompatible
scaffold containing human mature allogenic chondrocytes was used
for the treatment of chondral and osteochondral lesions in the knee.
Twenty-one patients were clinically prospectively evaluated with use
of the Western Ontario and McMaster Universities Osteoarthritis Index
(WOMAC) and a Visual Analogue Scale (VAS) for pain preoperatively
and at 3, 6, 9, 12, 24 and 36 months of follow-up.
Results: A statistically significant clinical improvement became
apparent after 6 months and patients continued to improve during
the 36 months of follow-up. Adverse reactions to the alginate/fibrin
matrix seeded with the allogenic cartilage cells were not observed.
Two of the procedures failed. One of the patients had loosening of
the periosteal flap, which was attributed to a failure of the surgical
procedure. The other failure case was the result of the poor quality
and quantity of the repair tissue itself.
Conclusions: The results of this pilot study show that the alginate-
based scaffold containing human mature allogenic chondrocytes is
feasible for the treatment of symptomatic cartilage defects in the
knee. The described technique provides clinical outcomes equal to
those of other cartilage repair techniques.
P244
Scaffold ACI and cancellous bone grafting for osteochondritis
dissecans in the knee – mid-term results
S. Anders, J. Schaumburger, O. Wiech, J. Beckmann, J. Grifka
Bad Abbach/Germany
Purpose: Osteochondritis dissecans (OD) of the femoral condyle
in the knee joint needs extensive osseous debridement for
revitalization as well as reconstruction of the joint surface. This
can be achieved by autologous cancellous bone grafting combined
with scaffold autologous chondrocyte implantation (ACI). Our mid-
term results with this technique are presented.
Methods and Materials: 20 patients (mean age 23.7 (14-42) years,
mean defect size 4.4 (1.8-8.0) cm²) with 21 cases of osteochondritis
dissecans of the femoral condyle (ICRS III-IV°, 19 medial, 2 lateral)
were treated by scaffold ACI (MACI®) and autologous cancellous
bone grafting. 14 patients have had a at least one (1-3) previous
OD treatments before (6x drilling, 4x refixation, 2x cancellous bone
grafting, 2x retrogarde drilling, 1x shaving, 1x removal only). 7 cases
were treated primarily. All results were evaluated prospectively by
functional outcome scores, subjective clinical ratings and MRI with
an average follow-up of 30.2 (6-62) months.
Results: Significant improvements were found in the Lysholm score
(64.1 (±8.7) to 91.4 (±8.3) points) as well as in the Tegner´s activity level
(2.7 (±1.2) to 4.5 (±0.7) points, both p<0.001). On a visual analogous
10-point scale (VAS) pain decreased significantly from 5.9 (±2.1) to
1.7 (±2.0) while subjective knee function improved from 4.9 (±1.9) to
7.5 (±1.9). There were two arthroscopic revisions after 8/18 months.
Both repair tissues revealed a nearly normal result with regards to
surface formation, filling and integration according to Brittberg´s
classification (each 11 out of 12 points). MRI follow-ups showed an
adequate filling of the defects, no prolonged effusion occurred. In
three cases some residual bone marrow edema was detectable. There
were no complications like loss of the scaffold or the bone graft.
Conclusions: Combination of scaffold ACI and autologous cancellous
bone grafting was shown to be an efficient therapeutic option for
osteochondritis dissecans in the knee. Performed as an one step
procedure invasivity can be reduced and rehabilitation fastened. Primary
and secondary interventions did not show a different outcome.
P245
Early results of Autologous Chondrocyte Implantation in the Hip
K.S. Kumar1, H.S. McCarthy1, S. Roberts1, J.C. Parker1, I.W. McCall2,
V. Murakibhavi3, E. Robinson2, P. Harrison2, J. Richardson2
1
Shropshire/United Kingdom, 2
Oswestry/United Kingdom,
3
Belgaum/India
Purpose: There are few surgical options available to treat symptomatic
chondral or osteochondral lesions of the hip. Autologous chondrocyte
implantation (ACI) is most commonly used to treat cartilage defects in
the knee with good results and a long term durable outcome. There
are only a few studies of its use in other joints, mainly the ankle. The
aim of this study was to assess whether ACI could be used in the
treatment of osteochondral lesions in the hip.
Methods and Materials: We describe a consecutive series of 16
patients with chondral or osteochondral lesions of the femoral head
that underwent ACI. Pre-operative hip function and structure was
assessed by the patient-completed Harris hip score (HHS) and where
possible, magnetic resonance imaging (MRI). ACI procedures were
performed via an antero-lateral or posterior approach, covering the
defect with collagen patches (Chondro-Gide®) in twelve patients
and periosteum in four. MRI was performed pre-operatively (n=11),
at 1 year (n=7) and at 2 years or later (n=8) and HHS was collected
annually. Failure was defined as receiving hip arthroplasty.
Results: Patients were on average 34 years old (range 14-52) when
treated. They improved clinically with time with HHS improving from
55±15 (range 31-78) pre-operatively to 74±17 (range 46-100) at two
years and 96±7 (range 86-100) at five years post-operatively. However,
7 patients progressed to hip arthroplasty (mean 30 months, range
13-47 months); of these 6 had cyst formation, 5 had osteophytes, 5
oedema and 5 avascular necrosis (AVN).
Conclusions: These results suggest that ACI can be a viable option
for treatment of either chondral or osteochondral lesions of the hip
but only with careful patient selection. Young patients may benefit, at
least in the short term, delaying the need for arthroplasty. However,
the presence of cysts, osteophytes, oedema or the development of
AVN may indicate a shorter period of benefit.
P248
matrix-based autologous chondrocyte implantation for cartilage
repair of knee: two-year follow-up in 15 patients
Z. Zhang, S. Hou, Z. Tang, Z. Yang, X. Zhong
Beijing/China
Purpose: To evaluate the safety and efficacy of matrix-induced
autologous chondrocyte implantation treatment (MACI, Genzyme
America) for patients suffering from cartilage lesions of knee in two
years follow-up study.
Methods and Materials: MACI operation were performed in a total of
15 patients (16 knees), with a median age of 33.7 years and average
defect size of 8.54 cm2 from 2004 to 2009. The procedure of MACI began
with harvesting of cartilage tissue which obtained from autologous
non-weight-bearing region for in vitro proliferation. The cultivated
chrondrocytes were seeded onto type â… /type â…¢ collagen bilayer
membrane which was glued by fibrin sealant to cartilage defect
area after suitable shaped. Knee injury and Osteoarthritis Outcome
Score（KOOS）and Magnetic resonance imaging (MRI) were
evaluated for clinical rehabilitation at postoperative 3,6,12 and
24months. In addition, 4 arthroscopic biopsies and 2 histological
examinations were performed post-surgery.

Results: There were no postoperative complications in 15 patients
and no adverse events related with MACI operation. In postoperative
3 months, evaluation of KOOS in pain showed significantly improved
compared with baseline(p<0.05). In postoperative 6 months,
KOOS results demonstrated significantly improved (p<0.05) in
pain, ymptoms, ADL, sport and recreation function and knee-related
QoL. MRI score in postoperative 3 months showed the regenerated
cartilage filled the defect areas nearly completely. In postoperative 6
months, MRI score exhibited that neo generated cartilage completely
filled defect regions. MRI score in postoperative 12 and 24 months
presented significantly improved in defect filling, integration,
signal intensity and subchondral bone. Histological evaluation
of postoperative 15 and 24 months presented the predominated
hyaline cartilage in new generated tissue. MACI operation time was
controlled in 2.0 hours with less than 100mL bleeding.
Conclusions: Our clinical results of two-year follow-up study affirmed
that MACI technique was a safe, reliable and valid treatment with
characteristics with non-complicated technique, short operating
time and small amount of surgical bleeding.
P249
Clinical and radiological results in patients operated on the
femoral condyles with the classical ACI after 10 years
M. Drobnič, D. Martinčič, B. Koritnik, M. Gorensek, D.
Radosavljevič
Ljubljana/Slovenia
Purpose: A prospective non-randomized clinical study analyzed
long-term clinical and radiological results in a cohort of patients
treated (1996-2000) with the classical ACI technique due to the
cartilage lesions on femoral condyles.
Methods and Materials: The initial study group comprised of 30
patients with the average age of 28.6 years. 2 patients did not
respond to follow-up, 1 sustained another injury of the operated
knee, and 1 suffered from another handicapping illness. 3 patients
had to undergo additional joint resurfacing procedure (1 osteotomy,
2 arthroplasties). Therefore only 23 patients were available for
clinical and radiological examination (Lysholm-Tegner, IKDC
subjective and examination, and Kellgren-Lawrence radiologic
scores) at 10 years. The acquired results were compared to their
follow-up scores at 2 and 5 years.
Results: The average scores for the cohort of patients were:
Lysholm 50 pre-operatively, 76 at 2 years, 72 at 5 years, 73 at 10
years; Tegner 2.9 pre-operatively, 4.0 at 2 years, 4.1 at 5 years, 3.8
at 10 years; IKDC 36 pre-operatively, 61 at 2 years, 65 at 5 years,
59 at 10 years. 23 operated knees were at 10 years examination
classified as: normal 9, nearly normal 8, abnormal 5, and severely
abnormal 1. The average Kellgren-Lawrence score at 10 years
was 1.9. Kellgren-Lawrence scores of 2 or more were found in the
subgroups of patients as follows: solitary lesions 28%, OCD 15%,
lesion with an ACL reconstruction 67%.
Conclusions: The clinical results demonstrated stability between
2 and 10 years of follow-up. Patients with low clinical scores at the
2nd post-operative year did not improve later. High radiological
evidence of OA in ACL reconstructed subgroup and low OA in OCD
patients indicate that classical ACI worked best for localized low-
impact cartilage lesions.
P250
Impact of patient’s age, etiology and site of cartilage defect
in the knee on clinical outcome of autologous matrix-based
chondrocyte transplantation
J. Fritz1, C. Gaissmaier2, J. Volck2, B. Schewe3
1
Dusslingen/Germany, 2Reutlingen/Germany, 3Rottenburg/Germany
Purpose: The main indications for autologous chondrocyte
transplantation (ACT) are circumscribed full-thickness defects in
younger patients, located mainly at the femoral condyles, caused
by trauma or OD. Little is known about the performance on ACT in
degenerative defects or in patellar lesions. We analyzed our results
of a retrospective survey and a prospective clinical observation to
learn more about the impact of defect-site, etiology of defects and
patients age on the clinical outcome of matrix-based ACT.
Methods and Materials: 1. From 433 patients, who underwent
matrix-ACT (NOVOCART 3D, TETEC AG, Germany), 422 (=97,4%)
have been followed-up after 7 months (2-30) with means of a
standardized questionnaire. This survey was focusing on early basic
clinical outcome and mainly on adverse events. 2. A prospective
Posters 247
examination was performed in 149 patients between 06/03 and
12/07 by capturing the IKDC score pre-operatively, after 6 months,
12 months and then yearly. This investigation was focusing on
clinical improvement performing covariance calculations to learn
about the impact of defect size, patients age, etiology, cell-viability
and more. The average follow-up here is 3 years (0,5-5).
Results: The IKDC-score increased for 18 points on an average and
reached a plateau after 2 years. Graft-related adverse events have
been observed in 6% of all patients (CI: 2,6- 12,4%). Patients age
just had impact on the postoperative joint function (p=0,004). The
patella showed highly significant (p<0,001)worse results in terms
of graft failure and revision surgeries.
Regarding etiology, degenerative defects showed significantly
(p=0,005) worse results compared to traumatic defects or OD.
Whereas in the latter in IKDC-increase for 18,4 points can be
expected, improvement in degenerative defects will be just 10,8
points (p=0,07) on average.
Conclusions: The performed covariance-analyses allow a pretty
detailed chance- and risk-profile for ACT-patients that can be used for
a more sophisticated pre-operative interview and informed consent.
P251
The harvest site in ACI treatment is not associated with long term
joint morbidity: a study of ACI treated hips with knee harvest site
H.S. McCarthy1, J. Richardson2, E. Robinson2, J.C. Parker1, S.
Roberts1
1
Shropshire/United Kingdom, 2Oswestry/United Kingdom
Purpose: Autologous chondrocyte implantation (ACI) is commonly
used for the treatment of cartilage defects in the knee but more
recently has been used to treat similar defects in the hip. Patients
who have had ACI of the hip with chondrocytes harvested from the
knee provide a unique opportunity to assess the contribution of
the harvest site to joint morbidity up to 7 years post-treatment.
Methods and Materials: In a study of 12 patients receiving ACI
treatment of the hip, chondrocytes were harvested from the knee
(11 ipsilateral, 1 contralateral). Clinical knee scores (Lysholm)
were obtained pre-harvest, post-harvest (range 2-12 weeks) and
annually thereafter. Statistical significance was determined using
the Kruskal-Wallis analysis of ranks and the Mann-Whitney U test.
Results: Pre-harvest, mean Lysholm scores were 87±18 (range 59-
100) indicating that the joint was generally in good health prior to
cell harvest. Initially, the Lysholm scores post-harvest were reduced
(but not significantly) to 72±17 (range 61-97), with an increase in
pain, locking sensation and limping, a decrease in movement and an
impaired ability to climb stairs. Cell harvest did not cause swelling.
By 12 months post-ACI however, mean Lysholm scores had increased
to just below pre-harvest scores (mean 82±25, range 44-100) with
all normal functioning of the knee restored. Scores had returned to
pre-harvest levels or greater from 2 years through to 7 years.
Conclusions: These results indicate that that there is an initial
decrease in joint function post-harvest for ACI. However, by 2 years
post-harvest, joint function had returned to pre-harvest levels. Hence,
there is no long term harvest site morbidity of the knee associated
with harvesting cells for use in ACI treatment of the hip. Thus, the
harvest site is unlikely to have contributed to the demise of the joint
for those patients for whom ACI treatment of the knee has failed.
P253
Novel arthroscopic matrix-encapsulated autologous chondrocyte
implantation: clinical, ultrasonographic, MRI, T2-mapping, and
arthroscopic results. Pilot study.
E. Villalobos Jr, C. Ibarra, C. Velasquillo, V. Martinez, F. Pérez, A.
Izaguirre, S. Cortes, G. Franco, D. Chavez, C. Pineda, V. Guevara,
L.G. Ibarra
Mexico City/Mexico
Purpose: The purpose of this study is to evaluate the efficacy
and biosafety of a novel autologous chondrocyte implantation
technique at 6 to18 months follow up.
Methods and Materials: We selected patients younger than 50
years old with localized 1-2 cm² ICRS grade IV femoral condyles
chondral lesions for arthroscopic chondrocyte implantation. We
treated concomitant pathology of patients and took a chondral
biopsy of non-weight bearing site near the femoral notch. After
cell culture and proliferation, we implanted matrix encapsulated
autologous chondrocytes arthroscopically and fixed the construct
with a bioabsorbable mini-anchor. All the patients followed the
same rehabilitation protocol with supervised physical therapy, 8
248 Posters
weeks of non-weight bearing, 6 weeks of CPM, and an isokinetic
strengthening program at the 4th month after the surgery. We
assessed prospectively clinical and structural outcomes at 3, 6,
and 12 mo follow-up. Second look evaluation was done in patients
at one year f/u. We conducted non parametric hypothesis tests
with 2-tailed significance set at (p<0.05).
Results: Mean age was 32.3 years old (23-46). All patients had
clinical improvement after surgery, with mean Lysholm preop
results 51.82±22.12 and postop 72.86 ± 18 .7 (p=0.028); Tegner
activity scale preinjury was 7.58±1.4 and 7.63± 1.2 (p=0.61) at final
f/u. Safety evaluation with ultrasonographic assessment through
the study was done and permitted identify that the implant was
placed correctly. A slight increase in MOCART evaluation and a
decrease in relaxation time with T2-mapping was observed. Second
look evaluation showed a repaired area with cartilage like tissue.
No serious adverse events were observed in this study.
Conclusions:Matrixencapsulatedautologouschondrocyteimplantation
with a novel arthroscopic technique is efficacious and safe supported
by clinical, macroscopic, and imaging studies at the short term.
P254
Matrix associated autologous chondrocyte transplantation in the
knee: Clinical results at 3 to 8 years follow-up
D. Stelzeneder1, S. Domayer1, R. Dorotka1, M. Brix1, C. Chiari1, S.
Nehrer2
1
Vienna/Austria, 2Krems/Austria
Purpose: The aim of this study was to prospectively assess
clinical outcome after matrix associated autologous chondrocyte
transplantation (MACT) in the knee using Hyalograft C.
Methods and Materials: Fifty-three patients were treated with
Hyalograft C in the knee. Patient characteristics at baseline were as
follows (mean±standard deviation): 22 female, 31 male, age 32±12
years, body mass index (BMI) 24.5±3.8 kg/m2 and defect size 4.4±1.9
cm2. Fifty patients had single defects and 3 had multiple defects (41
medial femoral condyle, 6 lateral femoral condyle, 2 patella, 1 tibia).
All defects were rated Outerbridge grade III or IV. Primary inclusion
criteria (stable ligaments, regular knee alignment (±3°), singular defect
within otherwise healthy adjacent cartilage, age <55 years) were
met by 42 patients. However 11 patients with secondary indication,
not fulfilling all criteria, were included. Instability or malalignment
were treated before or at the time of graft implantation. For patients
with small defects (<2 cm2) microfracturing was used as first-line
treatment. Full weight bearing was allowed 7-12 weeks after graft
implantation. Clinical assessment was performed once a year with the
following scoring systems: Subjective IKDC knee form, Lysholm score,
a modified Cincinnati Knee Rating System and objective IKDC knee
form. Descriptive statistics are given.
Results: Clinical parameters at baseline were as follows (median
and interquartile range): Lysholm 59 (47;69), subjective IKDC 40.0
(27.1;46.5), Cincinnati 3 (1;4), objective IKDC C (B;D) Results at 8 years
were: Lysholm 92 (77;95), subjective IKDC 76.3 (61.1;91.7), Cincinnati 8.5
(6;10), objective IKDC B (A;B). Twelve cases (9 secondary indications)
needed further surgical intervention (graft failure). Graphical results
are shown in Figure 1 and 2.
Conclusions: Our findings suggest that MACT with Hyalograft C is able to
provide satisfactory clinical results at 8 years of follow-up. In particular
young patients with singular defects benefit from this treatment.
P255
Treatment of chondral defects with autologous chondrocytes
on type I/III collagen membranes: comparison between 1 and 5
million cells in the sheep animal model
P. Guillen, E. Rodriguez-Iñigo, I. Guillen-Vicente, R. Caballero-Santos,
M. Guillen-Vicente, F. Garcia-Gomez, E. Santos, T. Fernandez-Jaen,
J.M. Lopez-Alcorocho
Madrid/Spain
Purpose: Autologous chondrocyte implantation (ACI) combined
with a periosteal flap was first performed in the human knee in
1987. The success of this technique as an alternative treatment
for symptomatic chondral defects has motivated a continuous
effort to improve it and today many modifications, that involve
tissue engineering, coexist. In MACI , chondrocytes are seeded in
a collagen I/III membrane functioning as cell carrier. The density
in MACI is around 106 cells/ cm2. We have evaluated in an ovine
model if increasing five times the cell density in a type I/III collagen
membrane improves tissue repair.
Methods and Materials: Ten 2-3 years-old female sheep were
included. A full 10 x 10 mm incision was made in the articular cartilage
of the medial femoral condyle. After the induction of a chondral
defect in the femoral condyle the size of the defect was measured,
and a fragment of the same size was cut from the membrane. All the
cell culture (1 million or 5 million cells) was deposited on the fragment
and after waiting for 15 minutes it was implanted to the animal. After
12 weeks the animal were sacrificed and the implantation area was
compared between both groups and also compared with a control
healthy sample taken near the implantation area.
Results: Control samples showed the highest score of the histological
parameters recommended by the International Cartilage Repair
Society to evaluate cartilage reparation. Although without statistical
differences, we have found that the histological parameters scores
were highest in the samples taken from the 5 million cells implantation.
We have also found that the expression of agrecan and type II collagen
was highest in the samples from the 5 million cells implantation.
Conclusions: The implantation of 5 million of cultured autologous
chondrocytes on I/III collagen membranes seems to give better
histological and molecular results than 1 million cells.
P256
Tissue engineering in the treatment of 41 patients with
osteochondritis dissecans: results at mean 6 years follow-up
G. Filardo, E. Kon, M. Berruto, A. Di Martino, S. Patella, G. Altadonna,
M. Marcacci
Bologna/Italy
Purpose: Osteochondritis Dissecans (OCD) is a focal osteochondral
separation of the weight-bearing portion of the articular surface.
Autologous tissue engineered cartilage (Hyalograft C) using a
biodegradable, biocompatible hyaluronian-based scaffold for cell
proliferation was developed for the treatment of chondral knee
lesions. The use of autologous chondrocyte transplantation in
OCD presents a problem to promote only the cartilaginous but not
the bone regeneration. For this reason we utilized the two-step
technique: autologous bone grafting followed by autologouos
condrocyte Hyalograft C transplantation. The aim of this study is to
evaluate Hyalograft C transplantation with or without bone grafting
for the treatment of OCD at mean 6 years follow-up.
Methods and Materials: Autologous chondrocytes transplantation
was performed from 1997 in 41 patients. Two-step technique
consists in the first arthroscopic surgical step autologous bone
harvested from omolateral tibia is utilized to fill the bone defect.
In the same surgical procedure healthy cartilage is harvested from
intercondylar notch in order to expand autologous chondrocytes.
The second surgical procedure is performed 4/6 months later,
when the integration of the autologous bone graft is achieved and
consists of arthroscopic autologous chondrocyte transplantation.
All the patients were clinically evaluated and analyzed according to
the International Repair Cartilage Society score.
Results: No complications related to the Hyalograft C implant and
no serious adverse events were observed during the treatment
and follow-up period. IKDC subjective score showed satisfactory
clinical results, with 79/100 at the latest follow-up.
Conclusions: The autologous chondrocyte transplantation provides
satisfactory clinical results. The second generation of autologous
tissue engineered cartilage transplantation avoids the use of
periosteal flap, simplify the surgical procedure and permit to perform
the arthroscopic implant. Thus, recovery time for the patient are
reduced. The association of the autologous bone grafting permits
to restore also a bone loss in order to recreate the articular surface.
P257
Autologous chondrocyte implantation for treatment of
symptomatic deep cartilage defects in the knee. 3-year follow-up
study comparing clinical and MRI evaluation.
W. Widuchowski1, H. Bursig1, A. Wysocka1, W. Wawrzynek2, R.
Faltus2, B. Koczy2, J. Widuchowski2
1
Katowice/Poland, 2Piekary Śląskie/Poland
Purpose: This prospective cohort study reviews and compares
clinical and morphologic results of ACI in the treatment of deep
cartilage defects in the knee.
Methods and Materials: Forty patients with symptomatic single
ICRS grade IV lesion localized within medial or lateral femoral
condyle participated in that prospective controlled trial. Average
size of defect was 3.5 cm2 (range; 2-9 cm2). Mean follow-up was

26.2 months (range; 12-38 months). Clinical and MRI evaluation
were performed preoperatively and 6, 12, 24 and 36 months
postoperatively. Functional outcome scores were collected using
Lysholm, Tegner and ICRS scores.
Results: All clinical rating scores showed significant improvements
(p<0.05) compared to pre-operative levels with ongoing
sequential improvement. MRI showed overall good healing
process of cartilage defects; 70-90% of defect filling, osseous or
cartilagenous hypertrophy in 32% of cases, subchondral bone
marrow edema in 11% of cases. There was no statistically significant
correlation between clinical and MRI scores.
Conclusions: In short-term observation ACI produces significant
improvement in knee function in patients affected by symptomatic
full-thickness isolated focal cartilage defect. There are likely to be
factors other than morphology of repaired tissue that may influence
the overall outcome of ACI.
P258
ACI or AMIC for osteochondritis dissecans of the knee? A 3-year
follow-up study.
W. Widuchowski1, P. Łukasik2, H. Bursig1, W. Wawrzynek2, M.
Szczęśniak2, J. Widuchowski2
1
Katowice/Poland, 2Piekary Śląskie/Poland
Purpose: The aim of the study was to compare the results of Autologous
Chondrocyte Implantation (ACI) and Autologous Matrix-Induced
Chondrogenesis for osteochondritis dissecans treatment of the knee joint.
Methods and Materials: Clinical evaluation of the results are based on
established scores (Lysholm, Tegner, ICRS). MRI was performed for each
patient; preoperatively and 6, 12, 24 and 36 months postoperatively.
The study comprised of 31 patients (21 male, 10 female, average age
at surgery 28; range 15-32) affected by single grade III and IV isolated
focal lesion (3-8 cm2; average 4,1 cm2). 17 patients were treated with
ACI and 14 with AMIC. Both groups were evaluated prospectively.
Results: Significant (p<0.05) differences between pre- and post-
operative values have been observed for all patients within both groups,
but without distinction between groups. MRI evaluation revealed
better defect filling in ACI patients, but difference was not statistically
significant. Osseous or cartilagenous hypertrophy was observed in
30% (ACI) of cases in ACI group and in 40% of cases (AMIC).
Conclusions: Clinical and MRI scores demonstrate that both ACI
and AMIC techniques allow to obtain good short-term results in the
treatment of OD of the knee.
P259
ACT3D fully arthroscopically performed for multiple chondral
defects, at the knee. Preliminary results.
S. Alevrogiannis, G. Skarpas, A. Triantafyllopoulos, G. Kakavas
Athens/Greece
Purpose: To present our experience in using 3rd generation autologous
cartilage transplantation with 3-dimensional chondrospheres for
treatment of multiple cartilage defects, at the knee.
Methods and Materials: 12 symptomatic patients with multifocal cartilage
lesions, were treated operatively in our Dept., between March 2007 and
June 2009. All patients were recreational athletes and the mean age was
32 years old. The mean area of cartilage defect was 6.75cm² and were
classified as Outerbridge grade III(7) and IV(5). All of them were treated
fully arthroscopically. 8 patients were male; 6 pts had the operation
at the right knee and the remaining 6 at their left knee. The cartilage
lesions were located in the weight-bearing surface of the MFC+LFC in
7 patients, 2 in LFC+FT, 2 in the MFC together with retropatellar lesions
and 1 in MFC+LFC+LTP. In most of the cases (9) the defect was due to
trauma, while 3 of them were caused due to OCD. 3 patients had failed
MFx performed elsewhere in the past. We performed 12 applications of
ACT3D as a single procedure. Preop. and postoperative evaluation of
patients was done using the Modified Cincinatti Rating System, the VAS,
IKDC score and Patient Outcome Function score.
Results: All cases followed a specialized rehabilitation protocol. We
assessed the patient at six months and 1 year post-operatively using the
MCK score , the VAS and the IKDC scores. No significant graft-associated
complications were observed in MRI scans.Second look arthroscopy,
showed hyaline like cartilage formation immunohistologically.
Conclusions: Our preliminary results of biological resurfacing
techniques at the knee, for multiple chondral defects, seems to
be more than encouraging. A greater number of cases and further
mid and long term follow-up has to be studied in order to prove the
efficacy of the method.
Posters 249
P260
Our experience in the treatment of chondral defects with
autologous chondrocyte implantation, chondrotissue and other
scaffolds
E. Somma, G. La Cava
Roma/Italy
Purpose: Introduction.We report our 3 year experience in the
treatment of chondral defects caused by trauma or degeneration,
where we implanted autologous chondrocytes on a 3D scaffold
(BioSeed-C), chondrotissue and other scaffolds.
Methods and Materials: We treated 197 cases of focal cartilage
defects, single and multiple, with a 1 to 3-4 cm. surface, situated in
the femoral chondilars, tibial plate, patella, femoral and astragalus
trochlea in III and IV stage (Outerbridge classification); in some cases
we associated a reconstruction of the cruciate ligaments of the knee.
BioSeed-C was used in 150 cases, and it consisted in drawing a small
sample of cartilage, through arthroscopy, from a healthy area of the
knee without loading and a 120 ml. blood sample for culture of the
chondrocytes in laboratory. The cells were then cultivated in vitro
for approximately 3 weeks and spread out on a 3D reabsorbable
polymeric scaffold. During the 2nd phase of treatment the matrix
with the chondrocytes was implanted with an open technique (mini
open), fixing the matrix with a reabsorbable pin or reabsorbable
suture passing through the trans-bone tunnel with a Kirschner suture
according to the technique of Erggelet. Particular post operative
attention is required to deal with the active and passive flex-
extension. Complete loading is not allowed for 3 weeks. After the
3rd week we allow partial loading of 15% with progressive loading to
reach complete loading after the 6th week. br>
Results: Our follow-up is presently 3 years; our French-Swiss
colleagues (Prof. Rhenter, Prof. Erggelet) have an 8 year follow up with
very satisfactory clinical radiographic results. As regards the use of
Chondrotissue and other scaffolds this has been used in 47 cases and
requires the application of a reabsorbable scaffold of 2 cm x 3 cm x 1
mm of polyclicolic acid (marked CE class 3) and ialuronic acid (marked
CE class 3) in the seat of the cartilage damage prior to courettage
of the lesion, Pridie perforations by means of reabasorbable pins
or reabsorbable suture passed through the trans bone tunnel with
Kirschner suture according to Erggelet’s technique or, in minor
lesions, and particularly in the astragalar and tibial region, with fibrin
glue. After the operation we used the same protocol utilized for the
previous technique but we loaded earlier in percentage. Our follow-
up with this technique is 1 year and 6 months with RMN controls after
3, 6 and 9 months. br>
Conclusions: Considering the simple surgical procedure which
foresees only one operation and very encouraging results, we are
of the opinion that Chondrotissue and other scaffold can be utilized
more frequently in this type of lesion especially in the over 50s. br>
P261
Arthroscopic Cartilage Regeneration Facilitating Procedure
(ACRFP) for OA Knee
S. Lyu
Chiayi/Taiwan
Purpose: A novel concept of arthroscopic procedure for the treatment
of osteoarthritic (OA) knee has been developed based on the
conceptualization of a possible pathogenesis process for OA knee
featured by focal abrasion phenomenon and soft tissue imbalance.
Methods and Materials: From January to December of 2005, 571 knees
of 367 patients with OA knee have received this procedure. We used
Kallgren-Lawrence classification for pre-operative staging. Pain domain
of Knee Society Score (KSS-P) and Knee Injury and Osteoarthritis
Outcome Score (KOOS) were used for subjective outcome study.
Measurement for the roentgenographic changes of femoral-tibial angle
(FTA) and joint space width (JSW) was evaluated for objective outcome
study. The mean follow-up period was 43 months (range, 41 to 54).
Results: There were 70 (19%) male and 297 (81%) female and the mean
age was 60 years (range, 29 to 82). One hundred and thirty-four knees
retured and completed the thorough outcome evaluation. Another 381
knees completed the subjective telephone questionnaires outcome
evaluation. The total follow-up rate was 90.2%. The satisfactory rate for
the whole series was 85.5%. FTA improved from 1.52 (95% confidence
interval, 0.84~2.19) to 1.93 (1.21~2.64)(p=0.03). JSW increased from
2.03 (2.81~3.24) to 2.18 (2.97~3.38)(p=0.01).
Conclusions: ACRFP is a good option of treatment for OA knee. The
beneficial effects of this procedure might be the improvement of the
general environment of the knee joint for cartilaginous regeneration
after eradication of the possible etiologic factors.
250 Posters
P262
Intra-articular Ultrasonic Assessment of Cartilage Integrity
E. Kaleva1, T. Viren1, S. Saarakkala1, J. Sahlman1, J. Sirola1, J. Puhakka2,
T. Paatela2, I. Kiviranta2, H. Kröger1, J. Jurvelin1, J. Töyräs1
1
Kuopio/Finland, 2Helsinki/Finland
Purpose: We tested whether a minimally invasive intra-articular
ultrasound (IAUS) method could be used to evaluate structural
integrity of articular cartilage in human knee joints in vivo.
Methods and Materials: Five patients (2 female, 3 male, age
22-69 years) coming to arthroscopic surgery of the knee were
enrolled in the study. The study plan was approved by the local
ethics committee. During arthroscopy, ultrasonic measurements
of cartilage surfaces were conducted using a miniature rotating
unfocused high-frequency (40 MHz) ultrasound transducer
inside a catheter (dia.=1mm, Boston Scientific). The catheter
was inserted into the knee joint though the normal arthroscopic
portals and the ultrasound transducer was positioned on the
desired joint location under arthroscopic control. In addition
to collecting ultrasound backscatter data, an IAUS video
and arthroscopic video were recorded. Cartilage surfaces at
the measurement sites were classified according to the ICRS
macroscopic score.
Results: The IAUS assessment was able to distinguish between
different characteristics of the articular surfaces (e.g., intact
surface, surface fibrillation and lesions of varying depth) of
human articular cartilage. In addition abnormal internal cartilage
structure and subchondral bone were visible in some cases in the
IAUS images.
Conclusions: The IAUS method served as a minimally invasive
diagnostic in vivo tool for evaluation of articular cartilage. The
method provided quantitative information on cartilage integrity
and thickness not available in conventional arthroscopy. The
ultrasound equipment is FDA approved for intravascular use,
making the use of the instrument easier for intra-articular
applications. The present results suggest that also the integrity
of the articular cartilage repair may be quantitatively monitored
with the IAUS approach in vivo.
P263
Role of Compound Real Time Sonography in Evaluation of
Meniscal Injuries
M. Sandhu, K. Shanbhogue, P. Singh, K. Sodhi, R. Sen
Chandigarh/India
Purpose: This study was designed to assess the utility of Real
Time Spatial compound sonography ( CS ) in diagnosis of
meniscal injuries of the knee.
Methods and Materials: 24 patients presenting with a history of
meniscal injury participated in this study. All patients underwent
conventional sonography ( CNS ) and CS examination followed by
MRI. Only unilateral injuries were included, contra lateral knee
was used as a control.
Results: CS was significantly superior with respect to the clarity of
tissue plane definition, excellent contrast resolution, reduction of
artifacts like speckle and refractive shadows resulting in reduced
posterior shadowing from the bones. Overall, the sensitivity,
specificity and accuracy was 80%, 89.3%, 85.4% for CNS,and
90%, 85.7% and 87.5% for CS respectively.Negative predictive
values were 86.2% for CNS and 92.3% for CS.
Conclusions: We feel that CS should be routinely used for
evaluation of meniscal injuries as it has better sensitivity, making
it an attractive screening tool. It has the potential to become
a gatekeeper to MR especially when the clinical suspicion of a
meniscal tear is not very high. Unnecessary MRI examination
can safely be avoided with 90% accuracy when the clinical
suspicion is not very high and when USG is normal. Although it
cannot replace MRI , CS is an excellent low cost alternative when
MR is not available or when waiting period for MRI can cause
unnecessary delay in management.
P264
Satisfaction after arthroscopic femoral osteoplasty
J. Miquel, J.I. Erquicia, M. Tey Pons, X. Pelfort, J.C. Monllau, E.
Cáceres Palou
Barcelona/Spain
Purpose: Femoracetabular impingment is a well-know predisposal
factor to hip cartilage damage. The objective of the present
paper is to evaluate the satisfaction degree of patients in whom
arthroscopic femoral osteoplasty was performed and its possible
correlation with alpha angle correction and postoperative pain.
Methods and Materials: A series of 20 patients operated on 2009
were prospectively studied. Inclusion criteria were patients in whom
arthroscopic femoral osteoplasty was performed. The patients were
clinically assessed by the Satisfaction Cincinatti Score (SCS) and
Visual Analogical Scale (VAS) pre and postoperatively. The degree
of alpha angle correction was radiologically calculated by means
of a Dünn axial view. Femoral head correction was correlated to
VAS and SCS. Spearman’s-rho test was used to statistically study
correlations among variables, using the programme SPSS 15.0. The
level of significance was set at p< 0.05.
Results: At a mean postoperative follow-up of 9.9 months, femoral
head correction averaged 21.9º ± 11.9 (range 2 to 41º), while VAS
averaged 2.8 ± 2.4 (6.2 ± 2 preoperatively). Patients rated 6.9 out of
10 points in the average activity score (SCS). Most of the patients
(80%) showed a moderate to excellent satisfaction level according
to SCS. A significant correlation was found between patient
satisfaction and alpha angle correction, as well as postoperative
pain (p<0,05).
Conclusions: Arthroscopic femoroacetabular osteoplasty reduces
pain with a high percentatge of patient satisfaction at a short term
follow-up. The procedure satisfaction seems to correlate to the
alpha angle correction.
P265
Further clinical improvement in a cohort of 528 patients affected
by symptomatic hip osteoarthritis undergoing to repeated
intra-articular ultrasound-guided administrations of hylan g-f20
(Synvisc®) during second year of followup.
A. Migliore1, E. Bizzi1, U. Massafra1, S. Martin2, M. Granata1, F.
Vacca1, S. Tormenta1
1
Rome/Italy, 2Albano Laziale/Italy
Purpose: No data are currently available in the scientific literature
reporting long followup times on patients affected by hip OA
undergoing ultrasound-guided intra-articular injection of the
hip with hylan G-F 20. Aim of the Study is to evaluate variability
and duration of long-term benefit obtained by the ultrasound-
guided intra-articular injection of the hip with hylan G-F 20 in hip
OA patients and investigate if further clinical improvements are
achieved during second year of therapy.
Methods and Materials: Adult patients suffering from hip OA
grade 1-3 according to Kellgren-Lawrence grading were considered
eligible for the study. Patients were injected with one syringe (2
ml) of hylanG-F20 every 3-6 months under ultrasound guidance
following Migliore-Tormenta technique. The efficacy was assessed
by using the Lequesne index and VAS pain score at baseline and,
then, every three months after the first injection of hylan G-F 20.
NSAIDs consumption was also evaluated.
Results: 528 patients were enrolled in the study; 851 injections
were performed in 24 months. Scores obtained at each visit after
baseline show an improvement when compared with baseline with
a statistically significant difference, p<0,001 for every time point.
No infectious complications were reported.
Conclusions: Our data suggest that beneficial effects obtained
by the ultrasound-guided intra-articular injection of the hip are
present after the first injection and are maintained over two years
by repeat injections. Furthermore, when comparing data obtained
at 12 months followup whit data obtained at 24 months followup,
a statistically significative reduction was observed in NSAID
consumption (p=0.01) and mean Lequesne index value (p=0.01)
(Fig.1 and Fig.2).

P266
PRP intra-articular ultrasound-guided injections for the treatment
of hip osteoarthritis
A. Timoncini, M. Battaglia, R. Buda, G. Filardo, T. Buscio, S.
Giannini
Bologna/Italy
Purpose: Platelet Rich Plasma (PRP) is a natural concentrate of
autologous growth factors from the blood. Currently PRP is widely
experimented in orthopaedic practice in order to test its potential
in tissue regeneration, especially its benefits on articular cartilage
degeneration of the knee. The aim of our study is to explore this
novel approach to treat osteoarthritis of the hip.
Methods and Materials: Nineteen patients affected by unilateral
symptomatic osteoarthritis of the hip were treated with 3 PRP intra-
articular ultrasound-guided injections. The procedure consisted of
150 ml of venous blood collected and twice centrifuged to produce
3 units of 5 ml each of PRP. Three ml PRP added of 0,3 ml Calcium
Gluconate were biweekly injected in the hip joint under ultrasound-
guide using Convex probe (1-4 Mhz) aligned with the anterior long
axis of the femoral neck. Median value of follow-up was 8 months
(range 6-12): patients were clinically evaluated at enrolment, and
at 1, 3, 6 months and at final follow-up according to HHS and VAS.
Statistical analysis (Non-parametric Mann Whitney test, Paired
t-test, Pearson and Spearman correlations) was performed in order
to evaluate the final outcome and find its correlation with sex, age,
grade of OA, BMI and femoral-acetabular impingement.
Results: A statistically significant improvement of all clinical scores
was obtained from basal evaluation to 1 and 3 months follow-up
(p<0.0005). The results decreased with statistical significance
(p=0,016) from 3 to 6 months follow up, and remained stable
(p=0,5) until final follow-up even if still significantly higher with
respect to the basal level (p<0,005). OA grade and femoral-
acetabular impingement showed statistical correlation with the
clinical improvement at 1 and 3 months follow-up (p<0,023,
p=0,049). One patient stopped the treatment at first injection.
Conclusions: PRP intra-articular ultrasound-guided injections
showed to reduce pain and improve articular function in hip
osteoarthritis without femoral-acetabular impingement at short
follow-up.
P267
Primary stability in the hip of a membrane used for autologous
chondrocyte transplantation. A cadaveric study
A. Fontana
Monza/Italy
Purpose: The treatment of chondral defects is a challenging issue in
joint surgery. Tissue engineering and chondrocyte transplantation
are actually giving new prospectives to this field, especially in the
knee joint. Nevertheless the use of membranes as scaffolds for
chondrocytes implantation in the hip is scarcely investigated. The
aim of this study is to evaluate the primary stability of a membrane
used for autologous chondrocyte transplantation.
Methods and Materials: The membrane was a polyglactin 910 and
poly-p-dioxanone reabsorbable, which favors the three-dimensional
growth of the cellular culture. The stability of the membrane was
evaluated after implantation in 12 hips in 6 cadavers on a cartilage
defect specifically formed on the anterior part of the acetabulum
and on a cartilage defect specifically formed on the polar sector of
the femoral head. All the samples were subjected to 50 complete
movement cycles including coxo-femoral movements, for the
acetabulum and for the femoral head, respectively. Fifty additional
cycles were evaluated for the implants on the acetabulum which
showed stability at the first evaluation.
Results: The implanted membranes showed stability in 83.3% of
the acetabular defects and in 33.3% of the defects of the femoral
head. The additional 50 movement cycles carried out for evaluating
the acetabulum showed stability of the implant in 80% of the cases.
The results suggest the specific use of this tissue, therefore, in the
treatment of cartilage defects of the acetabulum.
Conclusions: The hypothesis to use membranes as scaffolds for
chondrocytes implantation in the hip and their stability without
addictive fixation is extremely attractive for hip surgery and hip
arthroscopy.
Posters 251
P268
Evaluation of post-ACI cartilage repair tissue using FT-IR imaging
spectroscopy
A. Hanifi1, S. Roberts2, J.B. Richardson2, N. Pleshko1
1
Philadelphia/United States of America, 2Shropshire/United Kingdom
Purpose: There has been limited success in correlating the clinical
outcome of cartilage repair procedures with quality of the repair
tissue formed. Techniques that can assess the molecular features
of repair tissue could aid in evaluating the tissue response to such
therapeutic interventions. Fourier transform infrared imaging
spectroscopy (FT-IRIS) is a modality based on molecular vibrations
that has been used to evaluate articular cartilage degradation and
repair. The aim of the current study was to investigate molecular
properties of cartilage repair tissue after autologous chondrocyte
implantation (ACI) procedures and to assess whether there was
a correlation between the FT-IRIS derived properties and the
immunohistochemical and clinical outcomes.
Methods and Materials: Osteochondral biopsies were obtained
from knees of ACI treated patients from 1 to 5 years post-ACI
procedure (N=10). Thin sections of the biopsies were evaluated
for type I and II collagen immunostaining and by FT-IRIS. Clinical
outcome was assessed by Lysholm score.
Results: Improvement in Lysholm score occurred in 9 out of 10
samples and correlated with the FT-IRIS determined proteoglycan
(PG) content of the tissue. Increase in years post-ACI correlated
with an increase in FT-IRIS determined PG content, collagen helical
integrity, and collagen maturity based on crosslinking (ratio of
mature: immature crosslinks). Immunohistochemical staining
showed a greater amount of collagen type II near the calcified
cartilage and bone, which was inversely related to the distribution
of the collagen crosslinking parameter.
Conclusions: We conclude that FT-IRIS derived parameters could
be useful for predicting cartilage repair success.
P269
Clinical utility of T2 relaxation time at proximal tibiofibular
cartilage in osteoarthritis MR imaging
K. Kwack, B. Min, S. Yoon, Y.M. Lee
Suwon/Korea, Democratic People’s Republic of
Purpose: The proximal tibiofibular joint (PTFJ) can be considered
the fourth compartment of the knee joint. However, there have been
no studies of the T2 values (T2 relaxation time) of PTFJ cartilage.
Purpose: To assess the T2 values of PTFJ cartilage at 3T magnetic
resonance imaging (MRI), and to show the clinical utility of T2 values
of PTFJ cartilage for the diagnosis of osteoarthritis (OA).
Methods and Materials: 118 patients who had knee MR imaging
and knee radiography were enrolled. MRI was performed using a
3T MRI scanner, and T2 maps were calculated from a sagittal multi-
echo acquisition. Two regions of interest (ROIs) were positioned
within PTFJ cartilage and medial femoral condyle (MFC) cartilage.
The average T2 value and standard deviation (SD) of each ROI were
recorded. Using PTFJ cartilage as a standard reference, the T2 index
((MFC/PTFJ)x100) and T2(SD) index ((MFC(SD)/PTFJ(SD))x100) were
calculated. A paired t test was performed to compare the mean
and SD of ROIs within PTFJ and MFC cartilage. Correlation analyses
were performed among the parameters age, Kellgren-Lawrence
(KL) score, means and SDs of ROIs within PTFJ and MFC cartilage,
T2 index, and T2(SD) index.
Results: PTFJ cartilage had a significantly shorter T2 value than
did MFC cartilage (P<0.0001). ROIs within PTFJ cartilage showed
significantly smaller SDs than did those within MFC cartilage
(P<0.0001). The average T2 value and SD of MFC and the T2(SD)
index showed a positive correlation to the KL score (P<0.05).
The correlation coefficients for the average T2 value, SD, and
T2(SD) index of MFC were R=0.203, 0.254, and 0.268, respectively.
However, there was no significant correlation between T2 values of
PTFJ cartilage and KL score (P=0.643).
Conclusions: PTFJ cartilage may be a useful internal standard
reference to diagnose OA and would be a good candidate as a
standard reference for cartilage imaging.
252 Posters
P270
Sequentially Programmed Magnetic Field (SPMF) Therapy as an
effective Treatment for Osteoarthritis, our Experience
V.G. Vasishta
Bangalore/India
Purpose: Sequentially Programmed Electromagnetic Field (SPMF)
Therapy utilizes Magnetic Field Generators (MFG) that can be
precisely controlled and focused onto the affected tissues. The
normal physiological remodeling of cartilage occurs due to
piezoelectric stimuli which are lost in osteoarthritic patients. By
utilizing specific frequencies in the range of 6-30Hz based on tissue
type and grade of osteoarthritis this stimulus is recreated by SPMF
therapy leading to cartilage regeneration.
Methods and Materials: Methods: 195 patients with bilateral
osteoarthritis (OA) of knees were assessed by internationally
recognized Knee Society clinical rating system; the scores
computed prior to treatment, after 21 days of therapy, and at 3
months. In addition, MRI of the treated knees was done using
standard protocol, before treatment and at three months, to
measure objective changes in cartilage thickness.
Results: Results: The results showed statistically highly significant
improvement in pain scores, total knee scores, total functional
scores and the range of motion, immediately after the treatment
vis-à-vis pre-treatment values, and this improvement persisted
when re-evaluated at three months. There was also a significant
increase in cartilage thickness at three months, from 0.64mm
(±0.02) baseline to 0.88 mm (±0.07) in left knee, and 0.65mm
(±0.02) to 0.89mm (±0.05) in the right knee joint (p<0.001). The
9 month follow up results showed a significant improvement in
the cartilage thickness, of 1.26mm (±0.02mm)in the left knee and
1.23mm (±0.03mm) in the right knee
Conclusions: Conclusion: Therapeutic exposure to SPMF therapy
is effective in ameliorating the signs and symptoms of OA, and
inducing regeneration of chondrocytes as evidenced by increase
in cartilage thickness. SPMF therapy is an effective treatment
modality for osteoarthritis.
P271
Anteriomedialization of tibial tubercle for patellofemoral
disorders
M.I. Iosifidis, D. Neophytou, I. Melas, T. Liakos, D. Alvanos, A.
Kyriakidis
Thessaloniki/Greece
Purpose: Patella recurrent dislocation and patellofemoral pain
syndrome affect many young people. In the present study we
present the results of the extension mechanism realignment
through the oblique osteotomy of the tibial tubercle
Methods and Materials: During the last three years 18 patients
(12 men, 6 women, mean age 30.3/ range 20-41) were treated
operatively for recurrent patella dislocation (12 patients) and
patellofemoral pain syndrome without patella dislocation episodes
(6 patients). All patients underwent knee arthroscopy for the
treatment of possible chondral lesions or loose bodies removal and
for lateral retinaculum release. After that, we performed oblique
tibial tubercle osteotomy, which is fixed with 2 cortical screws.
This oblique osteotomy allows the concomitant anteriorization
and medialization of tibial tubercle. In addition, we performed also
medial patella retinaculum plication. All the patients followed the
same rehabilitation program. We followed up the patients a mean
period of 14 months (range 9-24 months). Tegner activity and
Lysholm scores were recorded pre- and postoperatively at 3, 6, 9,
12 months and 2 years. We also evaluated the patellofemoral pain
through the visual analogue scale (VAS).
Results: There was no new patella dislocation episode and
any other early or long term complication. The improvement of
both functional scores after the 6th p.o. month was statistically
significant (p<0.001). There was also statistically significant
improvement of pain (p<0.001). Overall, all patients during their
last follow up examination had a painless knee with almost full
ROM and marked improvement of the patella tracking.
Conclusions: Our results support that the oblique osteotomy of
tibila tubercle along with lateral release and medial retinaculum
plication, is an excellent option for the treatment of recurrent
patella instability and relief of the patellofemoral pain.
P274
Autologous Matrix Induced Chondrogenesis (AMIC plus) for the
treatment of patellar cartilage defects in the knee
A.A.M. Dhollander1, F. De Neve1, K.F. Almqvist1, R. Verdonk1, G.
Verbruggen1, P.C. Verdonk2
1
Gent/Belgium, 2Gent-Zwijnaarde/Belgium
Purpose: The present study was designed to evaluate the AMIC
plus technique for the treatment of symptomatic patellar cartilage
defects in the knee. MRI was used for the morphological analysis
of cartilage repair.
Methods and Materials: The AMIC plus technique (combination of
the original AMIC technique and the use of platelet rich plasma) was
used for the treatment of symptomatic chondral and osteochondral
patellar lesions in the knee. Five patients were clinically prospectively
evaluated with use of the Knee Injury and Osteoarthritis Outcome
Score (KOOS) , a Visual Analogue Scale (VAS) for pain, the Tegner
activity level scale and Kujala scale preoperatively and at 12 and
24 months of follow-up. All 5 patients had consented to follow the
postoperative MRI evaluation protocol. MRI data were analyzed
based on the original MOCART (Magnetic Resonance Observation
of Cartilage Repair Tissue) scoring system.
Results: A statistically significant clinical improvement became
apparent after 24 months of follow-up. The MOCART scoring system
revealed no significant deterioration or improvement of the repair
tissue between one and two years of follow-up. Twenty-four months
after the operation hypertrophy was found in 40 %. Subchondral
bone changes and intralesional osteophytes were seen in all cases
(100%). Synovitis and adhesions were not observed in the study
patients at that time of follow-up.
Conclusions: AMIC plus resulted in clinically significant improvement
in all patients. The favourable clinical outcome of the AMIC plus
technique was not confirmed by the MRI findings as determined by
the MOCART score. More specifically, all cases showed subchondral
lamina and bone changes, including intralesional osteophytes,
were observed.
P275
T2 relaxation time mapping of human cartilage with variable
kinds of MR pulse sequences: correlation analysis with
biochemical assay
K. Kwack1, B. Min1, S. Yoon1, L.H. Jin1, X. Cui2, S. Hong2, H. Kim2,
Y.M. Lee1
1
Suwon/Korea, Democratic People’s Republic of, 2Incheon/Korea
Purpose: T2 relaxation time mapping has been universally used
to evaluate the cartilage of knee joint. Variable kinds of pulse
sequences have been used to do T2 relaxation time mapping.
However, there has been no study that compares variable kinds
of T2 relaxation time mapping with variable MR pulse sequences.
Our study aim is to compare variable kinds of T2 relaxation time
mapping with Turbo Spin Echo (TSE), GRASE (hybrid echo) and Fast
Field Echo (FFE for T2 star mapping).
Methods and Materials: In this study, 11 osteochondral specimens
were taken from TKR samples. We performed 3 different T2
relaxation time mappings with 3 kinds of pulse sequences (TSE,
GRASE and FFE for T2 star mapping). After 3 kinds of MR imaging
all the osteochondral specimens were analyzed with biochemical
assay (glycosaminoglycans (GAG), water content and hydroxy
proline (HP) for collagen content). T2 relaxation time (T2 msec) of
the cartilage in the same ROI (region of interest) from 3 different
pulse sequences were calculated. Correlation analyses were
performed between T2 msec, GAG, HP and water contents.
Results: The mean values of ROIs with each pulse sequences
(TSE, GRASE and FFE) were 58.2 msec, 70.4 msec and 20.8 msec
respectively. Correlation analyses revealed significant correlation
between T2 relaxation values with FFE and HP (r=0.782, p < 0.01).
Statistically, no other significant correlation was proven between
T2 relaxation values with TSE and GRASE and biochemical contents
in cartilage.
Conclusions: T2 msec in T2 star mapping using FFE correlated well
with HP content in the cartilage of human knee. T2 star mapping
with FFE is a promising MR imaging method to detect collagen
depletion in cartilage.

P276
Functional and radiographic results after fixation of the unstable
oeteochondritis dissecans lesions using PLLA pins
N. Adachi, M. Ochi, M. Deie, A. Okuhara
Hiroshima/Japan
Purpose: There are several surgical treatments for unstable
osteochondritis dissecans (OCD) lesions of the knee. If the
unstable OCD lesions are left in the joint with the adequate
conditions for fixation, internal fixation should be the preferred
method of treatment, since it preserves the natural contour of
the distal femur. The purpose of this study was to evaluate the
functional and radiographic outcome of fixation of the unstable
OCD lesions of the knee after minimum 2 years follow-up.
Methods and Materials: 30 unstable OCD lesions in 28 patients
who were treated with fixation of the OCD lesions and followed
up for more than 2 years were included in this study. They
were 22 males and 6 females with an average age of 14.8 years
(range:11-22). Fixation of the OCD lesions was performed through
arthrotomy in 22 knees and under arthroscopy in 8 knees using
PLLA pins. The functional outcomes were evaluated using the
Lysholm score at an average follow-up of 3.2 years after the
surgery and healing of the lesions were confirmed using plain
radiographs and MRI.
Results: The average Lysholm score significantly improved
postoperatively (from 78.4 to 96.8). 28 of 30 lesions healed
after fixation of the lesion. Healing was achieved at an average
of 2.4 months on plain radiographs and 4.2 months on MRI. Two
lesions which did not heal required additional surgery. 22 of 24
patients who had been involved in sports activities returned to
their previous activities without reduction of their activity levels
within 6 months after the operation.
Conclusions: This study clearly demonstrated that fixation of
the lesions was an effective treatment option for patients with
unstable OCD of the knee, as proved by their functional and
radiographic improvement.
P277
Arthroscopic pullout repair of posterior root tear of the medial
meniscus: The anterior approach using medial collateral
ligament partial release
Y.G. Koh
Seoul/Korea
Purpose: The purpose of this study was to report the clinical outcome
after arthroscopic double transosseous pullout repair of posterior
root tears of the medial meniscus through the anterior approach
using partial controlled medial collateral ligament release.
Methods and Materials: Patients who underwent arthroscopic
repair of posterior root tears of the medial meniscus between
May 2008 and March 2009 were reviewed retrospectively. Fifty
four cases (8 men and 46 women) were available with a mean
follow-up of 15 months (range, 12 to 22 months). The age at
surgery ranged from 36 to 75 years of age (average: 57 years).
The posterior part of deep medial collateral ligament was
partially released to expand the medial joint space with an 18
gauge needle. Two anteromedial transtibial tunnels were made
into the foot print. Two PDS sutures were passed in the red-red
zone approximately 5mm medial to the torn margin and pulled
out to the anteromedial tibial cortex and secured by a sliding
knot. After surgery, a cylinder splint was applied for 4 weeks
with the knee in extension. Clinical outcome was evaluated with
Lysholm score preoperatively and at the latest follow-up.
Results: The mean preoperative Lysholm score significantly
improved from 43 before surgery to 79 after surgery. In most
patients, the pain and the tenderness on the MCL disappeared
by 3 months after surgery and no clinical medial instability or
stiffness of the knee joint occurred.
Conclusions: This technique using partial MCL release can be a
safe and easy alternative in arthroscopic repair of the posterior
root tears of the medial meniscus.
Posters 253
P278
Correlation between the three-dimensional deformation of
meniscus with horizontal tear during knee flexion and patients’
Lysholm score
H. Amano1, K. Nakata2, T. Mae2, K. Shino2, H. Yoshikawa2
1
Toyonaka/Japan, 2Suita/Japan
Purpose: The purpose of this study was to quantitatively evaluate
the three-dimensional deformation of menisci with horizontal tear
during knee flexion using MRI and compare the deformation of
horizontal tear with patients’ Lysholm score.
Methods and Materials: Fifteen patients (10 male, 5 female, mean
age: 32 y/o,) with symptomatic horizontal tear in the posterior
segment of medial meniscus underwent 3-D MRI evaluation at 0
and 60 degrees of knee flexion. Volume of bone, cartilage, and
meniscus was semi-automatically extracted from the 3-D images
by intensity threshold techniques, and 3-D models were created.
The percent of circumferential length of horizontal tear against
the whole circumferential length of meniscus in the 3-D meniscus
model was defined as %tear. The deformation and opening of
horizontal tear during knee flexion were calculated in the para-
sagittal plane in the 3-D meniscus model. Patients’ Lysholm scores
were compared with the degree of opening of meniscus tear with
knee flexion.
Results: The mean value of %tear was 36.0%. The gap of tear
became wide by knee flexion and the mean vertical gap statistically
significantly increased from 1.7mm at 0 degree to 3.2mm at 60
degree of knee flexion (p=0.004). There was a positive correlation
(r=0.60, p<0.05) between the %tear and the opening of vertical
gap. The patient with wide open tear of meniscus by knee flexion
showed lower Lysholm, score. There was an inverse correlations
between patients’ Lysholm score and the opening of meniscus tear
with knee flexion (r= -0.7, p<0.01).
Conclusions: Three-dimensional dynamic MRI evaluation of meniscus
with horizontal tear has revealed that the gap of tear opened with
knee flexion in accordance with the size of circumferential length
of horizontal tear, and patients’ Lysholm score correlated with the
opening of meniscus tear by knee flexion.
P279
Use of Magnetic Resonance Imaging to Assess Tissue
Regeneration Following Implantation of a Novel Meniscus
Scaffold
P.C. Verdonk1, W.C.J. Huysse2, R. Verdonk2
1
Gent-Zwijnaarde/Belgium, 2Gent/Belgium
Purpose: To assess tissue ingrowth, meniscal defect filling and
cartilage status using dynamic contrast-enhanced magnetic
resonance imaging (DCE-MRI) and anatomic magnetic resonance
imaging (MRI) up to 24 months after implantation of a novel
resorbable meniscal scaffold for the treatment of partial meniscal
tears or meniscal loss.
Methods and Materials: This was a prospective, non-randomised,
single-arm, multicentre clinical investigation in patients with an
irreparable medial or lateral meniscal tear or partial meniscus
loss, with an intact rim and the presence of both horns and an
International Cartilage Repair Society (ICRS) classification ≤2, and
≤3 previous surgeries on the index knee. Following implantation,
DCE-MRI was performed to assess tissue ingrowth at 3 months,
and anatomic MRI was performed to assess meniscal defect filling
and cartilage scores at 1 week, and 3, 12 and 24 months.
Results: Overall, 52 patients (34 medial: 18 lateral) were treated
with the meniscal scaffold. Subjects were predominantly Caucasian
(51/52; 98%), male (75%) and had previously undergone one (65%)
or two (23%) surgeries on the treated meniscus. Subjects had a
mean age of 30.8±9.4 years and a mean longitudinal defect length
of 47.1±10.0 mm. DCE-MRI performed 3 months post-implantation
revealed tissue ingrowth in 37/43 (86%) subjects. Anatomic MRI
scans indicated that articular cartilage grading remained stable or
improved in the majority of patients (43/46; 93%) and there was
no indication that the scaffold caused cartilage damage. Filling
of the meniscal defect was observed in all subjects who had data
available at 3 and 12 months. Data obtained at 24 months post-
implantation will also be presented.
Conclusions: Tissue ingrowth and stable or improved cartilage
scores were demonstrated by MRI assessments performed up
to 12 months after implantation of a novel meniscus scaffold for
the treatment of meniscal tears or loss. Data at 24 months post-
implantation will be presented.
254 Posters
P280
The short term clinical and MRI results after meniscus
transplantation ; ninety nine cases
Y.G. Koh
Seoul/Korea
Purpose: The purpose of this study was to compare the allograft
extrusion using MRI in short term period after the medial and lateral
meniscal allograft transplantation and correlate the extrusion with
clinical outcome.
Methods and Materials: Ninety nine cases (67 men and 32 women)
were available for MRI evaluations. The age at surgery ranged from
21 to 52 years of age (average: 35 years). Seventy three lateral and
26 medial meniscus allografts were evaluated with a mean follow-
up of 22 months (range, 12 to 53 months). The absolute value and
relative percentage of the width of extruded menisci was measured
in the coronal image that showed maximal extrusion. Clinical
outcome was evaluated with Lysholm score.
Results: The mean extrusion was 4.5mm in lateral meniscus versus
2.7mm in medial meniscus (p=0.000) and the relative percentage
of extrusion was 50.0% versus 30.8% (p=0.000). Lysholm
score significantly increased from 49.0 preoperatively to 87.3
postoperatively in lateral meniscus and from 50.9 to 89.7 in medial
meniscus. The difference of final score between the groups was not
significant (p=0.381). The overall Lysholm score was not correlated
with the degree of extrusion (p=0.202)
Conclusions: The presented data shows that the transplanted
lateral meniscus extrudes in the lateral direction significantly
more than the medial meniscus. However, the clinical outcome
after meniscus transplantation was not adversely affected by the
allograft extrusion.
P281
Twenty-six years of Meniscal Allograft Transplantation (MAT): Is
it still experimental? Meta-analysis of 44 trials.
P.C. Verdonk1, M. ElAttar2, K.F. Almqvist2, R. Verdonk2
1
Gent-Zwijnaarde/Belgium, 2Gent/Belgium
Purpose: Since the first MAT procedure in 1984, thousands of
patients with post-meniscectomy symptoms have been treated
through allograft replacement. Nevertheless, MAT is still considered
experimental surgery. AIM OF THE WORK: Collection, presentation
and meta-analysis of published trials presenting outcomes of
meniscal transplantation to establish safety and reliability of the
MAT procedure.
Methods and Materials: A pubmed search was conducted using
different combinations of keywords with reviewing of the abstracts
excluding all but English-language trials that presented more than
6 months clinical, radiological and/or histological outcome in
human subjects. We analyzed 44 trials presenting 1136 grafts in
1068 patients.
Results: The literature presented the outcomes of 678 medial and
458 lateral grafts in 613 male, 265 female and 190 non-defined
patients with an average age of 34.8 years. Sixty-four percent
of MATs were parts of combined procedures while only 36%
were isolated. The outcomes were presented through 12 scoring
systems, 4 radiographic modalities, 2nd look arthroscopy plus
histological analysis. Whatever the follow-up period and the scoring
system used, patients continuously showed clinical improvement.
Failure rate averaged 10.6% and a total of 128 (21.3%) non-major
complications, mainly observed in combined procedures, was
reported.
Conclusions: Continuous satisfactory outcomes with restoration
of working ability in this active patients group were observed
in all studies. The complication and failure rate are considered
acceptable by all authors. Salvage procedures included osteotomy
and arthroplasty without secondary difficulties. MAT can be
considered safe and reliable for the treatment of refractory post-
meniscectomy symptoms in selected patients.
P282
Clinical outcome scores following implantation of a novel meniscal
scaffold for the treatment of irreparable partial meniscus tears
and/or partial meniscal tissue loss, 2 year follow up.
R. Verdonk1, P. beaufils2, J. Bellemans3, P. colombet4 , R. Cugat5, P.
djian6, H. Laprell7, P. Neyret8, H. paessler9, P.C. Verdonk10
1
Gent/Belgium, 2Versailles/France, 3Leuven/Belgium, 4Bordeaux/
France, 5Barcelona/Spain, 6Paris/France, 7Kiel/Germany, 8Lyon/
France, 9Heidelberg/Germany, 10Gent-Zwijnaarde/Belgium
Purpose: To assess the clinical efficacy of a novel, biodegradable,
polyurethane meniscal scaffold designed for the treatment of
irreparable partial meniscus tears and/or partial meniscal tissue loss.
Methods and Materials: This was a prospective, non-randomised,
single-arm, multicentre clinical investigation in patients with an
irreparable medial or lateral meniscal tear or partial meniscus loss, with
an intact rim and the presence of both horns. The scaffold is designed
to support the body’s own physiological pathways of tissue repair
by providing a temporary three-dimensional matrix for cellular and
vascular ingrowth. Clinical efficacy was assessed at up to 24 months
post-surgery using the following scores: Visual Analog Scale (VAS), the
International Knee Documentation Committee (IKDC), the Knee and
Osteoarthritis Outcome Score (KOOS) and the Lysholm score.
Results: Overall, 52 patients (34 medial: 18 lateral) were treated
with the meniscal scaffold. The population recruited into the study
was young (mean age, 30.8±9.4 years), predominately male (75%)
and the majority (88%) had undergone 1 or 2 prior surgeries on the
involved meniscus. The mean longitudinal length of the meniscal
defect after surgical debridement was 47.1±10.0 mm. Clinical
efficacy of the meniscal scaffold was demonstrated at 12 months
by a statistically and clinically significant mean reduction in knee
pain on VAS (p<0.0001), and statistically and clinically significant
improvements in IKDC and Lysholm scores, and all outcome
categories of the KOOS questionnaire (p≤0.0001). It is anticipated
that the 24 month data will be presented here.
Conclusions:Implantationofthemeniscalscaffoldresultedinstatistically
significant and clinically relevant improvements in all subjective clinical
outcome scores at 12 months post-surgery demonstrating the efficacy
of the scaffold in the treatment of irreparable meniscal tears. It is
envisaged that 24 month data will further support the efficacy of this
novel meniscal scaffold and will be presented.
P283
Treatment with mesenquimal cells derived from the Hoffa’s fat pad
for the reparation of chondral defects in a ovine animal model
P. Guillen, E. Rodriguez-Iñigo, I. Guillen-Vicente, R. Caballero-Santos,
M. Guillen-Vicente, E. Santos, F. Garcia-Gomez, T. Fernandez-Jaen,
J.M. Lopez-Alcorocho
Madrid/Spain
Purpose: Current therapies, such as transplantation of healthy cartilage,
mosaicplasty, microfracture of the subchondral bone and implantation
of artificial polymers or metal prostheses, have many limitations for
cartilage reparation. Some research have been focused in developing
techniques based on cell therapy using autologous chondrocytes and
mesenchymal cells (MSC). We have used an ovine model to evaluate
from the histological and molecular point of view the use of Hoffa’s fat
pad derived MSC for the reparation of chondral defects.
Methods and Materials: Five 2-3 years-old female sheep were
included. A full 10 x 10 mm incision was made in the articular cartilage
of the medial femoral condyle. A second lesion of the same size was
done at the trochlea. In this lesion, microperforations were done. A
sample of adipose tissue from the Hoffa’s fat pad was taken to isolate
MSC and 5 million of cultured MSC seeded on a collagen I/III membrane
were implanted. After 12 weeks the animals were sacrificed and tissue
samples in the following areas were taken: a) MSC implant area, b)
perforations area, and c) healthy tissue near of perforation area.
Histological study was carried-out made by hematoxilin-eosin and
safranin-O staining. Relative expression of agrecan and types I and II
collagens was determined by real-time polymerase chain-reaction.
Results: Among the parameters recommended by International
Cartilage Repair Society to study cartilage reparation we have
found that most of them were similar when the samples taken
from the implant area were compared to those of the perforated
ones, being different to controls. The differences were statistically
significant in the case of surface and cell distribution. Expression
of agrecan and type II was similar in perforations and implanted
samples and lower than that found in control samples.
Conclusions: Microperforations and Hoffa’s fat pad derived MSC
seem to have no role in the reparation of damaged cartilage.

P284
The Efficacy of Combination of Microfracture and ArtifilmTM Covering
for the Treatment of Cartilage Defects : Comparison with Conventional
Microfracture Technique in a Prospective Randomized Trial
B. Min, K. Oh, Y. Lee, J. Kim
Suwon/Korea, Democratic People’s Republic of
Purpose: Microfracture has been used as a first-line treatment to repair
symptomatic articular cartilage defects. In this study, a new technique
using an extracelluar matrix biomembrane (ArtifilmTM) to cover cartilage
lesions during microfracture healing was compared with conventional
microfracture technique in a prospective randomized trial.
Methods and Materials: A total of 35 patients (37 cases) without
general osteoarthritis or ligament instability who had symptomatic
articular cartilage lesions were randomly assigned in each group.
Fourteen patients (15 cases) underwent conventional microfracture
procedure (group M) and 21 patients (22 cases) received
microfracture and ArtifilmTM covering concomitantly (group MA).
Clinical assessment was done through 1 year postoperatively
using the subjective International Knee Documentation Committee
IKDC questionnaire, and visual analog scale (VAS) for pain and
satisfaction. Magnetic resonance imaging (MRI) was performed at
1 year after the operation in all patients.
Results: In clinical outcomes, no significant differences were
observed between the groups at each visit (final outcomes of VAS
for pain, p=0.413; VAS for satisfaction, p=0.579; IKDC, p=0.365).
The MRI showed good to complete defect fill (67 to 100%) in 6 (group
M) and 17 (group MA), respectively. In group M, 6 patients with
poor defect fill (less than 33%) was observed, whereas 3 in group
MA (p=0.067). Assessment of peripheral integration revealed no
gap formation in 5 patients (group M) and 14 patients (group MA).
While, 6 patients in group M showed fissures greater than 2mm
and 4 patients in group MA (p=0.112). No serious complications or
adverse effects related to the ArtifilmTM were reported.
Conclusions: Although not statistically significant, the patients
undergoing microfracture only had less defect fill on MRI scan.
These results indicated a trend toward increased incomplete
cartilage healing in patients undergoing conventional microfracture
compared with those undergoing combined procedure,
microfracture and ArtifilmTM covering.
P285
Effect of microfracture and jamshidi needle biopsy on ex vivo
symptomatic OA knee condyles
C.D. Hoemann1, J. Sun2, Y. Gosselin1, M.B. Hurtig3, A. Carli1, H. Chen1,
W.D. Stanish4
1
Montreal/Canada, 2Laval/Canada, 3Guelph/Canada, 4Halifax/
Canada
Purpose: The aim of this study was to elucidate practical aspects
of microfracture and Jamshidi osteochondral biopsy of debrided
symptomatic condyles and resulting subchondral bone hole structure.
Methods and Materials: Lateral and medial femoral condyle (LFC
and MFC) surgical waste was obtained through IRB-approved
protocols from total knee arthroplasty (N=4) in consented patients
(49 to 73 years, BMI 30.2 – 34.2, 8-10 pain score with 0=none to
10=extreme). 2 subjects had prior steroid treatment. Condyles were
warmed to 37°C for 1 hour, biopsied in the middle with a Jamshidi
11G bone biopsy needle; the proximal region was debrided of the
calcified layer then microfractured 4mm deep with 3 awls (Arthrex,
Linvatec, Smith & Nephew). Samples were fixed and scanned with
a SkyScan 1172 at 15 µm resolution to measure bone hole depth
and diameter by 3-D Micro-computed tomography. Agarose gels
impregnated with calcium-phosphate were used as tool phantoms.
Results: The subchondral plate was very hard below full-thickness
grade IV MFC lesions compared to LFC specimens debrided of 2.5±0.9
mm thickness cartilage. Bone was difficult to microfracture in samples
from subjects with prior intra-articular steroid treatment. Arthrex,
Linvatec and Smith & Nephew awls formed 2.9-3.9 mm deep holes
with a 1.8, 1.3 and 1.6 mm superficial hole diameter: 0.16-0.3 mm
smaller than the respective hole diameter cast in agarose. 7mm deep
Jamshidi holes were 2.8 mm diameter in both agarose and bone.
Conclusions: MFX holes did not always attain the 4mm target
depth, potentially due to non-elastic bone material properties that
preserved the contours specific to each microfracture awl. Jamshidi
holes were less affected by condyle hardness than the MFX tools,
probably because that the biopsy removed osseous tissue instead
of compacting bone into a conical hole. Patient subchondral bone
has a stiffness spectrum that tends to extreme hardness as lesions
become chronically degraded.
Posters 255
P286
Distraction arthroplasty for severe osteoarthritis of the ankle
joint
H. Shibuya, N. Adachi, T. Nakasa, K. Fukuhara, M. Ochi
Hiroshima/Japan
Purpose: There are many reports for the treatment for osteoarthritis
(OA)and rheumatoid arthritis(RA) of ankle joint. However, we have
no consensus on treatment of severe stage of arthritis. Distraction
arthroplasty has been proposed as one of these options for the
patients in whom fusion or joint replacement is not appropriate.
Distraction arthroplasty and arthroscopic bone marrow stimulating
technique can be a new treatment strategy for severe OA of the
ankle joint.The purpose of this study was to evaluate the results of
distraction arthroplasty and arthroscopic bone marrow stimulating
technique for severe OA of ankle joint.
Methods and Materials: We performed drilling or microfracture
extensively for cartilage defect of tibial plafound and tatar dome.
Then we applied an external fixtator to distract the joint. This
device allow the patients to more joint actively. arthroplasty system
(Kobayashi MedicalTM). Patients began ROM exercise as soon as
possible after the surgery. Weight bearing was permitted 2ï½Å¾3
months postoperatively.
Between April 2002 and March 2010, 5 patients underwent
distraction arthroplasty at our hospital. Of these, OA was 4 cases
and RA was one case. 3 cases were male, 2 were female. The mean
age was 48.6 years (range, 38 to 65) at operation time.
Results: Mean AOSAF score improved significantly postoperatively
in 4 patients. The other patient underwent arthrodesis 24 months
after the initial operation due to residual ankle pain. Representative
case: 44 years old male. He was amateur high level soccer player. He
suffered from left ankle joint pain from 40 years old. After operation,
his symptom was disappeared. He finally returned to soccer.
Conclusions: This report indicated that this procedure is one
option for patients with severe OA of the ankle joint replacement
is not appropriate.
P287
A Retrospective Functional and T2 MRI Comparison Study of
Arthroscopic Bone Marrow Stimulation for Osteochondral Lesions
of the Talus – With and Without BMAC
C.D. Murawski, J.G. Kennedy
New York/United States of America
Purpose: Osteochondral lesions of the talus are increasingly
recognized injuries and may occur in up to 50% of acute and chronic
ankle sprains. Arthroscopic marrow stimulation (i.e., microfracture,
drilling) has provided good results in the short to medium-term
as a means of treatment for lesions up to 15 millimeters in size.
Nevertheless, fibrocartilage is biomechanically inferior to native
hyaline cartilage and is subject to degradation over time. In an
effort to address these concerns, the current authors reserve the
marrow stimulation technique for smaller sized lesions and utilize
an autologous bone marrow aspirate concentrate (BMAC). The
current authors hypothesize that the BMAC in conjunction with
smaller sized lesions will provide durable functional outcomes
and improved appearance of quantitative T2 mapping MRI by
comparison to the control group.
Methods and Materials: Between January 2005 and February
2009, 76 patients underwent arthroscopic microfracture surgery
under the care of the senior author. Thirty-five patients underwent
microfracture without BMAC injection while 31 patients did have
the BMAC injection. The Harvest SmartPReP 2 BMAC device was
utilized to prepare the bone marrow aspirate. No lesion treated was
larger than 8 millimeters in diameter. All patients had functional
AOFAS outcome scores at 1 year post-operatively in addition to
quantitative T2 mapping MRI.
Results: The average size lesion was 6.22 mm (range 1.52 - 8.04
mm) in the anterior to posterior direction and 6.04 mm (range 1.64
- 7.87 mm) in the medial to lateral direction. The average patient
age at the time of surgery was 35.55 years (range 13-66 years). The
mean AOFAS functional outcome score at 1 year post-operatively in
the group not treated with BMAC was 86.51. The group treated with
BMAC had a mean AOFAS score at 1 year of 88.12. These differences
were not statistically significant (p=.127). Quantitative T2 mapping
MRI in the group not treated with BMAC demonstrated prolongation
of T2 relaxation times in 32/35 patients and poor collagen fiber
architecture in 35/35 patients. By comparison, the group treated
with BMAC demonstrated improved fiber orientation in 21/31
patients and good overlying repair cartilage infill in 23/31 patients.
256 Posters
Conclusions: The functional outcome scores of both groups (with/
without BMAC) had good AOFAS functional outcome scores at
1 year post-operatively. Nevertheless, quantitative mapping T2
MRI demonstrated improved repair cartilage in the BMAC group.
These differences may be indicative of more rapid deterioration
in the group not treated with BMAC. While further investigation is
warranted with longer-term follow-up, BMAC is a beneficial adjunct
in cartilage repair.
P288
Cartilage repair by a cell-free polymer-based cartilage implant
immersed with platelet-rich plasma: histological and functional
results of 52 patients
A. Siclari1, G. Mascaro1, C. Gentili2, R. Cancedda2, E. Boux1
1
Biella/Italy, 2Genova/Italy
Purpose: In 52 patients, cartilage repair by a cell-free cartilage
implant (chondrotissue®) immersed with autologous platelet-rich
plasma was evaluated clinically.
Methods and Materials: During arthroscopy, a chondrotissue®
matrix and platelet-rich plasma were implanted after Pride drilling
into articular cartilage defects. At 9 months after surgery, clinical
outcome was assessed by second-look arthroscopy, histological
staining and by evaluation of the Knee injury and Osteoarthritis
Outcome Score (KOOS) compared to the pre-operative situation.
Results: In results, second-look arthroscopy showed an excellent
defect filling with cartilaginous repair tissue, firmly integrated to
the adjacent tissue. Histologically, the formation of an homogenous
matrix with hyaline-like repair tissue was evident. KOOS evaluation at
9 months after surgery showed significant improvement (P< 0,05)
of the patients´ situation with increased subscores for pain (55 to
90; P = 2.75E-033), symptom (57 to 86.5; P = 2.40E-033), activities
of daily living (69 to 85; P = 2.64E-033), sports and recreation (36
to 69.5; P = 1.78E-033) and quality of life (38 to 71; P = 2.39E-033).
In the follow-up period, no revision surgery was indicated. No
serious side effects like infection, inflammation, allergic reaction,
thrombosis or symptomatic hypertrophy occurred.
Conclusions: In conclusion, the chondrotissue® implant immersed
with platelet-rich plasma supports cartilage repair after bone-
marrow stimulation and showed significant clinical improvement
in the patients´ situation and mobility. This advanced technique is
suggested to be a safe and effective approach for cartilage repair.
P289
Repair of full-thickness cartilage defects with human umbilical
cord blood-derived mesenchymal stem cells and hyaluronate gel
composite in a rabbit model
C. Ha
Seoul/Korea, Democratic People’s Republic of
Purpose: This study investigated the cartilage regeneration effects
of human umbilical cord blood derived mesenchymal stem cells
(hUCB-MSCs) plus sodium hyaluronate using an articular cartilage
defect model in rabbits.
Methods and Materials: Full-thickness chondral defects, 3 mm
wide × 3 mm deep, were created in the trochlear groove of the right
femur in 53 healthy New Zealand White (NZW) rabbits. The control
group had only a chondral defect in the trochlear groove and 4
study groups administered the composite directly into the defect
(0.2 ml/cm2). The MSCs concentration ranged between 0.1 and 1.5
x 107 cells/mL were applied. At 4, 8, and 16 weeks after surgery,
cartilage repair was evaluated macroscopically and histologically
using a semiquantitative grading scale.
Results: The mean scores of the gross and histological evaluation
grade in the experimental groups were significantly superior to
those in the control group at 8 and 16 weeks (P <0.05).
Conclusions: These findings suggested that human umbilical cord
blood derived mesenchymal stem cells (hUCB-MSCs) plus sodium
hyaluronate can be used for the regenerative treatment of full
thickness articular cartilage defect.
P291
Evaluation of the structure-modifying effect of avocado-soybean
unsaponifiables (ASU) in hip osteoarthritis (OA): results of the
ERADIAS study, a 3-year prospective, randomized, double-blind,
placebo controlled trial.
E. Maheu1, C. Cadet1, M. Marty2, D. Moyse3, I. Kerloch4 , P. Coste4 ,
M. Dougados1, B. Mazières5, T. Spector6, E. Vignon7, J. Grouin8, M.
Lequesne1
1
Paris/France, 2Créteil/France, 3Tours/France, 4Courbevoie/France,
5
Toulouse/France, 6London/United Kingdom, 7Lyon/France,
8
Rouen/France
Purpose: To assess the ability of ASU to slow radiographic
progression in hip OA
Methods and Materials: Randomized, double-blind, placebo-
controlled 3-year trial. Patients with hip OA (ACR), ≥ 45 years,
symptomatic (pain ≥ 1 year, Lequesne ≥ 3) with a minimum joint space
width (JSW) between 1-4 mm on a pelvic radiograph were randomly
assigned to ASU 300 mg or placebo. 3 standing radiographs were
taken annually: pelvis, target hip, and oblique views. JSW was
measured at the narrowest point on pelvic or target hip view by
manual measurement (0.1 mm-graduated magnifying glass) by the
best of 2 readers. The primary outcome was based on the 3-year
loss in the narrowest JSW. Patients with at least 2 radiographs of
the same view composed the Full Analysis dataSet (FAS). Minimum
JSW change was compared by a continuous approach (mean JS
loss) and a more robust analysis: comparison of the percentage of
“progressors” (loss ≥ 0.5 mm, SDD of the reader).
Results: 399 patients randomized, 345 in the FAS. Demographic
and hip OA characteristics were similar in both groups: age 62, 54%
women, BMI= 27, symptom duration 4 years, 0-100 normalised
Lequesne index 30, pain VAS 37 mm. Baseline JSW was 2.8 (0.9)
mm. Mean JSW loss and % of progressors at year 3
% Mean JSW loss
progressors (sem)
ASU n = 166 Placebo n = 179 Cochrane ASU Placebo P
Mantel
Haenzel P
adjusted
o n
stratus
Total 40% 50% 0.039 -0.64 (0.07) - 0.67
(0.06)
There was no significant intergroup difference on the mean JSW
loss, but the number of progressors was 20% lower in the ASU
group (p = 0.039).
Conclusions: A 3-year treatment by ASU appears to reduce the %
of JSW deteriorating patients compared to placebo, indicating a
structure-modifying effect in hip OA. The clinical relevance of these
results requires further assessment.
P292
Efficacy of chondroitin sulfate on synovitis in patients with knee
osteoarthritis: an ultrasound study
I. Möller
Barcelona/Spain
Purpose: To assess the efficacy of chondroitin sulfate (CS) alone
or combined with glucosamine sulfate (GS) on synovitis in patients
with knee osteoarthritis.
Methods and Materials: Retrospective study in 115 patients who
received CS 800mg (N=36), CS800mg +SG1500mg (N=41) or
acetaminophen (ACET) 500mg (N=38) daily for 6 months. Synovitis was
measured in the suprapatellar recess using ultrasonography with high
frequency linear array. Synovitis was defined as present in measures ≥
4mm. Huskisson’s VAS, extra acetaminophen consumption, meniscal
extrusions and Baker’s cysts were also assessed. Patient’s follow-up
was performed after 1, 2, 3 and 6 months.
Results: Among all subjects, a correlation between BMI and size of
synovitis (r=0,138;p=0,002) and knee pain (r=0,136;p=0,003) was
noted. A correlation between synovitis and pain (r=0,385;p=0,000)
was also detected.
Regarding ultrasonography assessments, all groups experienced
a decrease in synovitis. Patients treated with SYSADOAs reached
physiological values after 2 months. This improvement was
maintained in the course of treatment with a mean synovitis (SD)
of 3,2 (2,7) mm in CS group and of 2,9 (2,6) mm in CS+SG group.

A significant decrease in synovitis after SYSADOA therapy vs ACET
treatment after 1 month of follow-up was observed.
Statistical analysis demonstrated a faster decrease in pain with CS
vs ACET (p=0,000) and in the CS+SG vs ACET (p=0,000). Analysis at
each visit for pain decrease showed that differences between CS+SG
vs ACET were significant after 1 month of treatment (p=0,037) and
between CS vs ACET after 2 months of treatment (p=0,017).
Conclusions: This study suggests that CS alone or in combination with
GS decrease significantly synovitis measured by ultrasonography.
This decrease after SYSADOA treatment has correlated with an
improvement in knee pain. The decrease in synovitis elicited by
SYSADOA is in accordance with former reduction of synovitis signs
shown by CS and could contribute to explain its symptomatic and
structure disease modifying effects.
P293
Clinical utility of (99m)Tc-methylene diphosphonate (MDP)
bone scan in osteoarthritis of knee with concomitant analysis of
subchondral bone change
L.H. Jin, Y.J. Kim, K. Kwack, Y. An, B. Min
Suwon/Korea, Democratic People’s Republic of
Purpose: Bone scan grade system has been used to evaluate
alterations of knee cartilage in osteoarthritis (OA). The aims of this
study were to evaluate the correlations between bone scan score,
subchondral bone change and Osteoarthritis Research Society
International (OARSI) score, and to show the clinical usefulness of
the bone scan score to determine the severity of osteoarthritis.
Methods and Materials: In this study, 54 human osteochondral
specimens from total knee replacement (TKR) samples of 38 patients
were taken after all patients were examined with 99mTc-methylene
diphosphonate (MDP) bone scan. The intensity of MDP isotope
uptakes was graded 0 to 3 (0, normal; 1, mild; 2, moderate; 3, severe)
by 3 specialists of nuclear medicine. We measured mean values
and standard deviations of subchondral Hounsfield Units (HU, CT
number) values of the osteochondral specimens with a Multidetector
Computed Tomography (MDCT). The histological grading by OARSI
system was performed after staining with hematoxylin & eosin and
Safranin-O stains. The correlations between bone scan scores, two
HU values and OARSI scores were statistically analyzed.
Results: Correlation analysis revealed significant correlations
between bone scan and OARSI score (r=0.574, P<0.01). There were
also significant positive correlations between bone scan score,
OARSI score and subchondral MDCT HU (P<0.01). Interestingly,
the standard deviation of subchondral bone MDCT HU correlated
well with the bone scan score and OARSI score.
Conclusions: In this study, we found statistically significant
correlation between the bone scan score, subchondral bone MDCT
HU and OARSI score. Our results suggest that the bone scan is a
valuable tool in clinical research to evaluate OA of knee.
P294
Correlation between the presence of cytokines in the synovial
fluid and postural control in early degrees of knee osteoarthritis
S.M. Mattielo-Rosa, K. Gramani-Say, P.R. Serrão, F.A. Vasilceac,
G.C. Lessi, A.I. Medeiros, R. Reiff, J.A. Barela
São Carlos/Brazil
Purpose: The aim of this study was to verify the correlation between
cytokines detectable in synovial fluid (SF) and functional tests in
patients with OA.
Methods and Materials: 31 male subjects (40 to 65 years) with OA
of the knee, grades I or II, was enrolled. All subjects were selected
by clinical and radiographic evaluation (Kellgren & Lawrence
scale). The SF knee was aspirated for analysis of cytokines (TNF-a,
IL-1β, IL-6, IL-8, IL-12, IL-10, TGF-b) by ELISA method (Enzyme
Linked Immuno Sorbent Assay). Later, the subjects performed one
leg stance postural control on the force platform with eyes open
and closed. Center of pressure (COP) variables were analyzed
(area, amplitude, speed and frequency in anteroposterior and
mediolateral) in anteroposterior and mediolateral directions.
Pearson test correlation was used (R> 0.7 strong; p ≤ 0.05).
Results: There was detected in the SF cytokines IL6, IL8 and
IL12, which related to inflammatory process. According to Table
1, we found a positive correlation between cytokines detected
and postural control variables. Table 1: Correlation between SF
cytokines and postural control variables
Posters 257
Correlation R S
IL-8 OLS open eyes/Area 0,44 0,04
IL-8 OLS open eyes/Amplitude ML 0,54 0,01
IL-12 OLS open eyes/Velocity ML 0,56 0,028
IL-12 OLS open eyes/Velocity AP 0,57 0,027
IL-12 OLS close eyes/Velocity AP 0,50 0,05
IL-6 OLS open eyes/Amplitude ML 0,43 0,04
IL-6 OLS open eyes/Area 0,55 0,009
IlL-6 OLS close eyes/Amplitude ML 0,52 0,015
IL-6 OLS close eyes/Amplitude AP 0,52 0,015
OLS = One leg stance ML = Mediolateral AP= Anteroposterior
Conclusions: The biological markers were detected in SF from
patients with OA in the early grades and were correlated to the
functional activities that influence postural control, thus, these
finds could be used as a way to measure the evolution of the
functional capacity of patients with OA.
P295
A Novel Implantable Load Absorber for the Treatment of Medial
Compartment Knee OA
N.J. London1, C.S. Waller2, D.A. Hayes3, A.G. Clifford4
1
Harrogate/United Kingdom, 2Sidney/Australia, 3Brisbane/
Australia, 4Hayward/United States of America
Purpose: Knee overload correlates with OA incidence, symptoms,
radiographic, morphologic and biological changes. An implantable
load absorber has been developed to treat the medial OA knee. The
implant resides in the extra-capsular tissue of the medial knee and
provides compartment unloading whilst preserving joint integrity.
This study reports on the basic science of, and our early clinical
observations with, this novel device.
Methods and Materials: Changes in intra-articular loads provided
by the implant were determined through dynamic gait simulations
on cadaver knees with early OA. Preclinical safety was determined
using a chronic ovine model (n=15) sacrificed at 4, 12, 26 and 52
weeks. The surgical approach used in our early clinical experience
was similar to that described in preclinical studies. Post operative
recovery and soft tissue healing in these patients were monitored
as part of standard clinical follow-up.
Results: Dynamic gait simulations in cadaveric specimens
demonstrated a statistically significant reduction in medial
compartment loads. Medial compartment loads were reduced by
129 ± 64 N, comparable to results reported for high tibial osteotomy.
Wound healing in the chronic ovine model followed a normal
course. Post implantation, a fibrous pseudocapsule, non adherent
to the implant, formed around the device and matured between 12
and 26 weeks. In our clinical experience post operative recovery
was rapid and medial knee tissues remodeled to accommodate the
subcutaneous device. Patient’s exhibited excellent tolerance for
the implant through a broad range of knee motions and activities.
Conclusions: The load absorber preserves the structural tissues of
the knee whilst providing clinically meaningful joint unloading. This
unique surgical solution may be adopted as a primary or adjunctive
surgical treatment to address medial knee degeneration. Our
clinical experience suggests a major role for this treatment would
be for the active patient, for whom preservation of both joint
structure and active lifestyle are primary objectives.
P296
A comparison of patellofemoral alignment between individuals
with and without patellofemoral cartilage lesions
S. Duncan1, B. Noehren2, C. Lattermann2
1
0200/United States of America, 2Lexington/United States of
America
Purpose: Patellofemoral malaligment is often implicated as a
important factor in the development of patellofemoral osteoarthritis
(PFOA). However, to date no study has directly compared
patellofemoral alignment between those with symptomatic PFOA
vs those without. Therefore, the purpose of this study was to
determine the differences in patellofemoral alignment between
those with grade IV lateral patellofemoral articular cartilage lesions
versus normal controls.
258 Posters
Methods and Materials: 9 subjects (average age 33) diagnosed with
lateral PFOA participated in this study. They were compared to 11
subjects (average age 34) with no patellofemoral lesions documented
during an anterior cruciate ligament repair. Radiological measurements
of interest included, the Insall-Salvati ratio, Trochlear angle, lateral
patellofemoral angle and the tibial tuberosity trochlear grove distance
(TTTG). Groups were compared using a two tail, independent samples,
t-test. Additionally, means and effect sizes were calculated.
Results: The PFOA group had a significantly higher Insall Salvati
ratio (1.13 vs 0.94, p= 0.004, ES= 1.6), Troclear angle (142.2 vs 136,
p= 0.04, ES= 1.8), and TTTG (15 vs 9, p= 0.02, ES= 1.81). Although
there was no significant difference in lateral patellfemoral angle (12
vs 16, p= 0.09, ES= 0.8) it was associated with a large effect size.
Conclusions: The results of this study clearly show that those
with PFOA have alterations in their patellofemoral alignment
as compared to controls.The greater patellar height, shallower
trochlea, and higher Q angle seen in this group will increase shear
and compression on the lateral aspect of the patella.This may over
time result in the cartilage lesions seen in PFOA. These variables
are easily measured with x-ray, or MRI and should be considered
in surgical planning. Lastly, in light of these findings, physicians
should consider concomitant realignment procedures in addition
to cartilage restoration to promote an optimal outcome.
P297
Intrarticular injections of hyalouronans for knee osteoarthritis
C. Zidrou, M.I. Iosifidis, I. Melas, D. Neophytou, T. Liakos, N.
Valanos, D. Alvanos, A. Kyriakidis
Thessaloniki/Greece
Purpose: The aim of this study is to investigate the safety and
efficacy of intrarticular injections with hyalouronic acid, in patients
with knee osteoarthritis.
Methods and Materials: Our study performed from June 2008 until
December 2009 and was carried out on 412 patients (114 men and
298 women) with osteoarthritis of the knee, suffering from painful
joint symptoms. The mean age of the patients was 62,5±8,4 years.
Osteoarthritis was diagnosed on the basis of clinical and radiological
examinations. Three intrarticular injections of hyalouronic acid, once
per week, were carried out in all patients. They were excluded from the
trial those patients with severe systematic disease (e.g. rheumatoid
arthritis), suspected joint infection and those receiving concomitant
treatments likely to interfere with the results (e.g. corticosteroids).
All patients answered a questionnaire which contained questions on
basic demographic information; pre- and post injection pain, mobility
and the number of nights per week the patient was awakened from
sleep by knee discomfort; the occurrence of side effects (e.g. pain,
allergy); whether the injection allowed them to postpone or prevent
joint replacement; and willingness to repeat the treatment if required.
Participants were asked to rate pre-and post- therapy pain (both at
rest and with weight-bearing) using a 10-point scale (1=no pain;
10=worst possible pain). A similar scale was used to access mobility
(1=patient unable to move; 10=no restriction of movement).
Results: The average scores for pre- and post-therapy pain were
significantly improved (rest pain: before injection 5,9±2,4; after
injection 3,8±2,3 – pain with weight-bearing: before injection
8,1±2,3; after injection 4,2±2,5). Average scores for mobility (before
injection 4,2±2,5; after injection 6,8±2,6) and frequency of night-
time awakenings (before injection 3,9±2,6; after injection 1,5±2,2)
were also significantly improved. Knee replacement surgery was
temporary avoided in 49% of the patients while 51% were willing to
repeat the treatment. Side effects were not recorded.
Conclusions: Intrarticular injections of hyalouronans are a safe
and effective treatment for knee osteoarthritis and can be used to
postpone or prevent joint replacement.
P298
A combination of surgical and conservative treatment for knee
articular cartilage degeneration
M.I. Iosifidis, I. Melas, D. Neophytou, T. Liakos, E. Papantoniou, D.
Metaxiotis, D. Alvanos, C. Zidrou, A. Kyriakidis
Thessaloniki/Greece
Purpose: Articular cartilage degeneration is a common finding
during arthroscopy. The aim of this early report is to present our
results of a combination between arthroscopic debridement and
postoperative use of intrarticular injections of hyaluronans and
glucosamine sulfate per os.
Methods and Materials: During the last two years we performed 67
knee arthroscopies (67 patients, 25 men and 42 women, mean age
51,7 years), in which we found cartilage lesions alone or along with
intrarticular soft tissue injuries. We classified the lesions according
to Outerbridge classification (grades I through IV). During the
arthroscopy chondral lesions were shaved. We classified our
patients in two groups taking care each of them to be consisted
of similar cartilage lesions according to size and gradinfg. Group
A (30 patients) were not treated in any additionally way. Group B
(37 patients) were treated postoperatively by intrarticular injection
of hyaluronan in 3-time course (after the 1st p.o. month, one per
week), and took from the early p.o. period glucosamine sulfate per
os, at least for 6 months.
Results: We followed up all our patients using Lysholm score the
1st p.o. month, the 3rd, and the 6th and after 1 year (mean 11.1
months). There was a prevalence of group B scores when there was
I to III grade of cartilage defect. For the grade IV lesions there was
no significant deference between the two groups.
Conclusions: We believe that the combination of arthroscopic
debridment and postoperative use of intrarticular injection of
hyaluronan and glucosamine sulfate per os, offers better functional
results and pain relief in cartilage degenerative lesions.
P299
Distraction arthroplasty for treatment of the osteoarthritic knee
A. Nakamae, M. Deie, N. Adachi, T. Nakasa, H. Shibuya, T. Niimoto,
A. Okuhara, M. Ochi
Hiroshima/Japan
Purpose: Joint distraction is a relatively new approach in treatment
of osteoarthritis. We developed a new articulated distraction
arthroplasty device for treatment of the osteoarthritic knee
joint. This device allowed widening of the joint spaces and the
continuation of ROM exercises. The purpose of this study was
to evaluate the clinical results of a new distraction arthroplasty
device when used in conjunction with a bone marrow stimulating
technique for the treatment of osteoarthritis of the knee.
Methods and Materials: We followed up 7 patients (age range,
40 to 63 years) who underwent distraction arthroplasty using our
device with an average follow-up of 39 months (8 to 67 months).
Distraction was carried out for 10 weeks (7 to 13 weeks) during
which full weight bearing was allowed. Assessment included the
Japanese Orthopaedic Association (JOA) knee score, range of
motion, pain scale, and radiographic evaluation.
Results: The JOA knee scores significantly improved from a
mean of 54 points before treatment to a mean of 78 points after
treatment. Scores on a visual analog pain scale and joint space
values, as measured by the Rosenberg radiographic view, were
also significantly improved at the latest follow-up.
Conclusions: We conclude that the treatment using this new
device in combination with a bone marrow stimulating method
was effective for osteoarthritic knees in middle-aged patients.
Until now, there has been no useful treatment for middle-aged
osteoarthritis. We hope that this procedure not only will delay the
requirement for total knee arthroplasty but will also encourage a
change in strategy for treatment of osteoarthritic knee.
P301
Autologous Matrix-Induced Chondrogenesis (AMIC) on the
patella plus periosteal coverage on the trochlea combined
with mechanical realignment- A New Treatment Option in
Symptomatic Isolated Femoropatellar Osteoarthritis due to
Subluxation of the Patella
R. Jakob, M. Jacobi
Fribourg/Switzerland
Purpose: Symptomatic isolated femoropatellar osteoarthritis is
reported for 8% of women and 2% of men over the age of 55 years.
Surgical options include osteotomy of the tibial tubercle with
patellar debridement and realignment (1), patellar hemiarthroplasty
and femoropatellar arthroplasty and patellectomy. We present
our results of a new biological treatment option, in which (1) is
combined with the AMIC procedure (autologous matrix-induced
chondrogenesis) and periosteal resurfacing of the lateral trochlea
femoris.
Methods and Materials: Only symptomatic isolated lateral
femoropatellar osteoarthritis with an unsuccessful conservative
treatment was included in the study. The surgery consisted of

an AMIC procedure described by Behrens on the retropatellar
cartilage defect and a periosteal coverage of the trochlear cartilage
defect, both naked surfaces prepared by 1, 1 mm drillings combined
with resection of the lateral pole of the patella with lateral release,
medial reefing and a tibial tubercle medialisation and advancement.
Lack of improvement and need for reoperation were defined as
endpoints.
Results: From 2003 to 2009 a total number of 21 patients underwent
this described new procedure of which two were operated bilaterally.
1/4 of patients had a lack of improvement, thus 3/4 were satisfied.
One patient who underwent bilateral surgery was satisfied at the
last follow-up but died later of an unrelated cause. The other patient
with bilateral surgery had persistent anterior knee pain on the one
knee and a good result on the other. One patient had a reoperation
with the AMIC procedure and is now satisfied. Another patient
underwent partial meniscectomy. His arthroscopy showed fibrous
cartilage coverage. One patient had total knee replacement. The
oldest patient in this series showed radiographic signs of patellar
osteolysis. Patients over the age of 75 years all were failures.
Conclusions: The proposed combination of mechanical and
biologic treatment modalities has proven to be a valid alternative
to debridement and alignment alone or to arthroplasty in patients
under the age of 75 years. It shows that there is a chondroplastic
potential in osteoarthritis and advanced age.
P302
MRI Analysis of the Progression of Articular Cartilage Loss in
Knee Osteoarthritis Patients
G. Yildirim, S. Arno, E. Khelmenstkaya, R. Regatte, P. Walker
New York/United States of America
Purpose: Magnetic Resonance Imaging (MRI) has been used to
characterize the properties of articular cartilage, as well as local
thickness. Medical and engineering software is now available
whereby three dimensional models can be made from MRI
scans, together with analyses of thickness and volume. These
techniques can have a useful application in studying the progress of
Osteoarthritis (OA) and in planning and designing Early Intervention
(EI) treatments. While many studies have been carried out on the
end-stage of OA, there have been few reports of the progress of the
cartilage loss from the earliest stages. The goal of this study was
to determine the locations and magnitudes of the cartilage loss of
the knee joint, and the progression of OA in order to understand the
mechanical factors involved. Further, from this data, our second
goal was to determine what types of EI treatments, such as small
implant components, could be employed to arrest the progress of
the OA process.
Methods and Materials: The MRI scans (3T Siemens, Malvern, PA)
from 18 patients with symptomatic early OA were analyzed. The
cases were taken at random from a group of 150 patients enrolled
in the NIH study titled Leukocyte Gene Expression in Osteoarthritis
by PI Steven B Abramson at the Department of Rheumatology,
NYU-HJD. The Kellgren-Lawrence (K-L) scale was the biomarker
used to classify the severity of the OA. 3D Doctor Software (Able
Software Corp., Lexington, MA) was used to contour the articular
cartilage layers at 0.6mm resolution. 3D models of the cartilage
layers were then rendered in RapidForm software by lofting the
contour slices (INUS Technology Inc., Seoul, Korea). The femoral
and tibial cartilage models were subdivided into 6 regions, as
follows: anterior lateral (AL), distal lateral (DL), posterior lateral
(PL), anterior medial (AM), distal medial (DM), and posterior medial
(PM). The anterior femoral region was defined as the anterior-most
point of the cartilage to 2-3mm above the trochlear groove. The
posterior region began 10mm posterior to the center of the circle
formed by the condylar curvature to the posterior-most point of
the femoral cartilage. Finally, the distal section lay between the
anterior and posterior regions. The tibia was subdivided into 3 equal
regions for each condyle. Each region then underwent thickness
analysis displayed with a color-coded thickness map in RapidForm.
From the 18 cases, 4 representative examples with increasing K-L
grades were selected to show the progression of the cartilage
loss (Figure 1). The overall cartilage volume was measured and
the thickness at each point was divided by this overall volume to
give a ratio for each region. This data was used to construct Table 1
showing the variations between the regions across the 18 patients,
for increasing K-L grades.
Results: For the majority of the 18 knees analyzed, the distal
femoral regions showed the most cartilage loss; ranging between
0 and 1.5mm of remaining thickness. As expected, the medial distal
femoral region showed greater thinning than the lateral region
Posters 259
(Table 1). The AMF and lateral compartments consistently showed
the greatest thickness (red areas on figure 1), corresponding with
the thick cartilage commonly found in the PF joint. Some wear of
the PF joint was seen in the knees with more advanced OA. The
posterior regions of the lateral and medial condyles were intact
in most cases, with relatively minor cartilage loss in some of
the severe cases. The medial femoral and tibial lesions mirrored
each other (Figure 1), starting with small areas in the centers of
the condyles, then extending both A-P and medially as the OA
progressed. On the tibia, anterior medial and posterior medial
cartilage showed most loss for the majority of the patients. The
medial side consistently showed more wear than the lateral side
but there was no single region on the medial side which showed
consistent wear for all patients.
Conclusions: Our results showed that most early OA in the knee
presented in the distal region of the medial femoral articular
cartilage, leaving the posterior condyles intact. The medial tibia
also matched the wear shown on the femoral condyle but did not
present a consistent region of wear, which may be explained by
the variations in ligament laxity and the condition of the meniscus.
These patterns of cartilage loss are consistent with the impact
forces generated at the heel strike and the toe off phases of
walking. The information from this study is crucial for the design
of future EI implants, as it points to the opportunity of preserving
maximal viable cartilage while resurfacing only the damaged areas.
Such early intervention techniques could significantly delay the
progression of knee osteoarthritis and thus improve the quality of
life of many patients.
P303
Secondary Osteoarthritis in the Pediatric Hip Joint: Underlying
Conditions
H.K. Gahunia, A.S. Doria, P.S. Babyn
Toronto/Canada
Purpose: The aim of this study was to investigate the incidence of
secondary OA in the pediatric hip joint.
Methods and Materials: We retrospectively reviewed the imaging
data of all identified pediatric patients with secondary OA of the hip
(N=18, age range 8-17 years, 1:1 male:female ratio) obtained from
our computerized database (1996 to 2009). Study cohort inclusion
criteria for hip OA were based on two or more radiographically
characteristic features of OA: joint space narrowing, subchondral
bone sclerosis, cyst, and osteophyte formation. Primary diagnoses
included: Dysplasia (spondyloepiphyseal-SED; Developmental
Dysplasia of the Hip-DDH), rheumatologic diseases (Systemic
juvenile rheumatoid arthritis-SJRA, Systemic Lupus Erythematosus-
SLE), infections (Septic Arthritis-SA) as well as Slipped Capital
Femoral Epiphysis (SCFE) and Legg-Calve-Perthes Disease (LCPD).
Results: Unilateral hip OA (N = 12) presented more commonly
in the left hip (67%) compared to the right (33%), with the OA
lesions predominantly found in the acetabulum and femoral head.
Unilateral hip OA was more commonly seen in patients with SCFE
(25%) and LCPD (25%), followed by children with DDH (16%), SA
(16%), SED (8%) and SLE (8%). In contrast, bilateral hip OA (N=6)
lesions were identified in SJRA (83%) and one case of SED. In
addition to the narrowing of the hip joint space, the radiographic
presentation of OA lesions included subchondral sclerosis, cyst
formation of the acetabulum and/or femoral head, osteophyte
formation at the acetabular margins and irregular outline of the
acetabulum. The magnetic resonance imaging of subchondral
sclerosis showed low signal intensity on both T1- and T2-weighted
images whereas subchondral cyst showed high signal intensity on
T2-weighted images.
Conclusions: Our series of cases suggests that pediatric
secondary OA of the hip predominantly affects the left hip joint
with similar incidence amongst females and males. Patients
diagnosed with inflammatory arthritis, particularly systemic
juvenile rheumatoid arthritis, appear to have an increased risk of
developing secondary OA.
260 Posters
P305
Use of autologous grafts for reconstruction of osteochondral
defects of the knee: 18 years follow-up.
E. Kon, A. Di Martino, G. Filardo, S. Patella, B. Di Matteo, S.
Zaffagnini, M. Marcacci
Bologna/Italy
Purpose: One of the most important problems in orthopaedic
surgery is reconstruction of damaged articular surface because
of its limited regeneration capacity. An osteochondral defect is
defined as a full-thickness, localized loss of bone and cartilage
tissue of the articular surface with no spontaneous healing
capacity. This study evaluates the very long results of a surgical
technique for repairing osteochondral defects with autologous
osteochondral grafts in young and middle-aged adult patients and
evaluates the osteoarthritis progression of the treated knee with
respect to the contralateral.
Methods and Materials: Thirteen patients with femoral
condylar osteochondral defects were treated with autologous
osteochondral grafting. Mean patient age was 31 years (range:
16-52) at the time of surgery and mean follow-up was 18 years.
The male:female ratio was 7:6. Defects were located on the medial
condyle in 11 cases, on the lateral condyle in 1 case and on the
lateral condyle associated with a tibial plate defect 1 case. For the
clinical evaluation the Lysholm, International Knee Documentation
Committee (IKDC) and Tegner scales were used. X-rays were used
for radiological evaluation.
Results: The results at long-term follow-up are very interesting
with an high percentage of subjective satisfaction. Nine of thirteen
patients are satisfied with their postoperative outcomes. The mean
value of IKDC was 68 (range: 23-98.9). The Tegner mean value was
4.2. Knee degenerative progression was documented by X-ray.
Conclusions: Autologous osteochondral transplantation is a valid
therapeutic solution for treatment of deep osteochondral defects of the
knee. At long follow-up evaluation the results are still good. Clinical and
radiographic examinations confirmed good integrative capacity of the
autologous graft and also survival of the chondral transplant.
P306
Osteochondral autogenous transfer to the patello-femoral joint
T. Satake1, M. Kobayashi2, S. Nakamura2, R. Arai2, Y. Nakagawa2, T.
Nakamura2
1
Otsu City/Japan, 2Kyoto/Japan
Purpose: The purpose of this study is to evaluate the result of
treatment of chondral or osteochondral lesions of knee joints
especially at the patello-femoral joint with osteochondral
autogenous transfer (OAT).
Methods and Materials: Seven knees of five patients were
included in this study. Six knees of four patients were diagnosed
as osteoarthritis, and one patient was diagnosed as osteonecrosis.
Chondral or osteochondral lesions between 48 to 120 mm
diameters were located at patellar surface in one knee and at
patellar groove in six knees, and were treated with OAT. One or
two grafts between 7 and 10 mm in diameter were harvested from
non-weight bearing area of the femoral condyle and press-ï¬t into
holes drilled into the defect. All patients were evaluated both
preoperatively and postoperatively with the Lysholm knee score
and Kellgren-Lawrence grade by sky-line view of plain radiographs
of knee joint.
Results: 5 patients of a mean age of 63 years old (55-74 years
old) were followed-up for mean period of 39.1 months (24-57
months). The mean Lysholm score improved significantly from
64.57 preoperatively to 95.00 postoperatively (p<0.01). With
radiographic evaluation, five knees in four patients did not be
changed and two knees in one patient were improved.
Conclusions: The results suggest that OAT is effective and safe
method to treat chondral or osteochondral lesions of the patello-
femoral joint. However, further long-term study is necessary
to determine improvement of symptoms and the restoration of
structural and functional integrity of graft over time.
P307
Osteochondral juvenile allografting: Is juvenile tissue a way to
improve osteochondral defect repair?: A pilot horse study.
S.M. Cokelaere, H. Brommer, J. Malda, P.R. van Weeren
Utrecht/Netherlands
Purpose: Mature articular cartilage has developed definitive
topographical biomechanical and biochemical heterogeneity and
lacks adaptability and regenerative capacity. In contrast, fetal
cartilage is still “blank” with regard to biomechanical and biochemical
properties and has the ability to functionally adapt until maturity.
We therefore explored a new approach in osteochondral repair by
transplanting juvenile osteochondral plugs into freshly made defects
in an equine carpal model.
Methods and Materials: By means of arthroscopy / mini-arthrotomy,
osteochondral plugs (6-mm diameter, 8-10 mm deep) were drilled out
of the third carpal bone of a 5-month-old foal and directly press-fit
implanted in a freshly made defect (6-mm diameter, 8-10 mm deep)
in the proximal articular surface of the third carpal bone in 2 adult
horses. At 2, 4 and 6 months arthrocenthesis, radiography, CT-
scan and follow-up arthroscopy were performed. Sacrifices were
at 6 months, with assessments by MRI (+ contrast), macroscopic
inspection and histology.
Results: Defects treated with osteochondral juvenile plugs showed
better repair than the untreated donor sites. Follow-up arthroscopy
showed a fibrocartilaginous surface with moderate integration
into the surrounding cartilage. Indentation tests showed that the
subchondral area was soft at 2 months, but became considerably
firmer at 4 and 6 months. On CT and MRI, a gradual increase in
subchondral bone density of the plug together with surrounding
sclerosis was visualized. At the level of the plug, the articular surface
was not exactly flush with the adjacent tissue on contrast MRI.
Macroscopically and histologically, good integration at the bone-to-
bone interface and minor cleft formation (filled with fibrous tissue)
at the fibrocartilaginous articular surface was seen. Histologically,
presence of type II collagen was evident and slight Safranin-O staining
showed the (reduced) presence of glycosaminoglycans in the matrix.
Conclusions: This equine pilot study shows that juvenile osteochondral
allografting is an interesting concept that may be potentially useful in
osteochondral defect repair.
P308
Treatment of Osteochondral Lesions of the Talus
A. Dalmau, D. Codina, J. Vega, F. Alvarez, R. Viladot
Sant Cugat del Vallés/Spain
Purpose: The treatment of the osteochondral lesions in the
weithgbearing articular surfaces is a common orthopaedic
problem. Osteochondral ankle defects cause various symptoms
including pain, swelling, and limited range of motion. Several
surgical procedures, open or arthroscopic, attempt to resolve
osteochondral lesions in the ankle: debridement and microfractures,
osteochondral graft, or chondrocyte transplantation. The aim of
this study is show our results of osteochondral lesion in the talar
dome treated with osteochondral autograft technique (OATS), and
present a guideline for its treatment.
Methods and Materials: 34 patients with osteochondral lesions
of talar dome, 5 to 27 mm in diameter, were treated using the
osteochondral autograft transfer system (OATS). Osteochondral
cylindrical grafts from the ipsilateral knee were delivered into the
talar defect. These procedures were done by arthrotomy
Results: The results have been evaluated by a clinical hindfoot score
(AOFAS), radiographs, CT and MR images. An histological study has
been made in any patients. Postoperative plain radiographs and CT
images have been used to evaluate the congruence and integration
of the graft. The AOFAS Score showed good to excellent results in
most of the case, with a mean of 89 points, and all the patients
returned to their activities and work.
Conclusions: Restoring the normal convexity of the articular talar
surface is a perplexing problem. To performance the graft correctly,
malleolar osteotomy is necessary. Hystological exams demostrates
that characteristics of hyaline cartilage is a normal hyaline cartilage
with collagen type II, better with OATS than other techniques.
Although OATS is more aggressive than arthroscopic procedures,
no complications at the ankle or morbidity at the donnor site
has been observed. Autogenous osteochondral mosaicoplasty
is an alternative to treat a localized talar dome lesion with good
functional results.

P309
Concurrent meniscal allograft transplatation and autologous
osteochondral transplantation in meniscectomized Patient
Y.G. Koh, S. Jo
Seoul/Korea
Purpose: The purpose of this study was to report the short -term clinical
outcome performed concomitantly, AOTs(autologous osteochondral
transplantation) and MAT(meniscal allograft transplantation)
Methods and Materials: Patients who underwent MAT and AOTs
between July 2008 and November 2008 were reviewed retrospectively
. 8 cases were available with a mean follow-up 18 months(range 16
-21 months) The age at surgery ranged from 36 to 55 years of age
(average : 42 years)
Results: As a combined group, statistically significant improvements
were observed in all standardized outcome scores at a mean
18months follow-up. Lysholm score significantly increased from 41.4
preoperatively to 82.0. Overall 87% of patients were classified as
normal or nearly normal at their recent follow-up using Interanational
Knee Documatation Committee examination score.
Conclusions: Combined meniscal allofraft transplantation and
autologous osteochondral transplanatation offers a safe alternative
for patients with persistent symptoms after meniscetomy and
focal cartilage injury. Long-term follow-up is needed to define the
survivorship of these procedures.
P310
2 year experience of Trufit osteochondral plugs for articular
cartilage defects of the knee
N. Verghese1, S. Joshy2, M. Cronin2, M. Forster2, A. Robertson2
1
Bridgend/United Kingdom, 2Cardiff/United Kingdom
Purpose: Recently biodegradable synthetic scaffolds (Trufit plug)
have provided a novel approach to the management of chondral
and osteochondral lesions. The aim of this study was to assess our
2 year experience with the Trufit plug system.
Methods and Materials: 22 patients aged 20 to 50 years old all
presenting with knee pain over a 2 year period were diagnosed
either by MRI or arthroscopically with an isolated chondral or
osteochondral lesion and proceeded to either arthroscopic or
mini arthrotomy Trufit plug implantation. In 5 patients plug
implantation was undertaken along with ACL reconstruction (3),
medial meniscal repair (1) and contralateral knee OCD screw
fixation (1). Pre and post operative IKDC scores were obtained to
assess change in knee symptoms and function.
Results: At a mean follow up of 15 months (range 2 – 24 months)
improved IKDC scores were achieved with the scores improving
with time. 2 patients had a poor result and have had further
surgery for their chondral lesions. One patient had failure of
graft incorporation at second look arthroscopy and went onto to
have a good result after ACI. The second patient had good graft
incorporation on second look but had progression of osteoarthritic
degeneration throughout the other compartments of the knee
which were not initially identified at the time of Trufit plugging.
Conclusions: We conclude that Trufit plug is a viable alternative
method for managing isolated chondral and osteochondral lesions
of the knee which avoids harvest site morbidity or the need for
staged surgery.
P311
An allograft composed of cancellous and demineralized bone to
manage osteochondral defects
J. Farr1, A. Nawab2, S. Weinerman3, P. Stull3, D.C. Flanigan4 , J. Dugas5,
T.R. Carter6, D. May7, P. Bursac8
1
Indianapolis/United States of America, 2Louisville/United States
of America, 3Englewood/United States of America, 4Columbus/
United States of America, 5Birmingham/United States of America,
6
Phoenix/United States of America, 7Mechanicsville/United States
of America, 8Alachua/United States of America
Purpose: This prospective multi-center study was designed to evaluate
the use of a novel scaffold (CR-plug) composed of allograft cancellous
bone and demineralized cortical bone for filling osteochondral defects
(Fig.1). The implant was designed to a) closely biomechanically match
the surrounding tissue, and b) utilize natural allograft material with a
long history of safety and proven ability to induce cartilage and bone
formation in animal models (e.g. Xang et al., 2008 v29(35) p4616-29).
Posters 261
Methods and Materials: In study group 1, the CR-plugs were used
to repair the femoral condylar or trochlear defects in a group of ten
patients. The patients had an isolated grade III or IV articular cartilage
lesion < 2.5 cm2. MRI scans as well as KOOS, Lysholm, IKDC subjective
and SF-36 were collected pre-op and 6 months post-op with pending
follow-up at 12 and 24 months. In study group 2, the CR-plugs were
used as backfill in ten patients qualified by surgeon for OATS procedure.
MRI scans at 6 months were collected with pending 12 and 24 months
follow-up. Adverse events (AEs) were recorded.
Results: One AE (tingling in the foot) possibly related to the study
product was recorded and resolved. No other unexpected AEs related
to the CR-plug were reported. At the 6 month post-op follow-up, the
analyzed patients from study group 1 demonstrated an increase in
KOOS, Lysholm, IKDC subjective and SF-36 when compared to baseline
(Fig. 2C). The MRI scans at 6 months demonstrated initial remodeling of
both cancellous and demineralized bone portion of the CR-plugs (Fig.
2A&B).
Conclusions: We report the preliminary results from the 6 month follow-
up of an ongoing clinical study using a novel allograft implant to manage
small articular joint lesions. The up-to-date results demonstrate good
record of safety, early signs of product incorporation and remodeling,
and improvements in patient well-being.
P312
Second-look arthroscopic and clinical evaluation of
osteochondral autograft transplantation in osteoarthritic patients
older than fifty years old
H.S. Seo, S.C. Lee
Seoul/Korea, Democratic People’s Republic of
Purpose: The aim of this study is to assess second-look arthroscopic
findings and clinical outcomes following osteochondral autograft
transplantation in early osteoarthritic patients older than fifty
years old.
Methods and Materials: From June 2006 to October 2007, 23
consecutive patients (23 knees) underwent arthroscopic or
mini-open osteochondral autograft transplantation (OATS) for
osteoarthritic lesions of the knee joint. Inclusion criteria were: 1)
age older than 50 years old; 2) persistent knee pain unresponsive
to at least 6 months of conservative treatment; 3) radiologic grade
1 or 2 arthritis by Kellgren and Lawrence; 4) varus alignment less
than 3 degrees; 5) Outerbridge grade 4 chondral lesion on weight-
bearing area of the femoral condyle; 6) lesion size ranged from
1~4cm2. Among them, 20 patients (20 knees) received second-
look arthroscopy at a mean of 12.7 months (range, 6-19 months)
and were available for clinical evaluation with a minimum of 2
years’ follow-up. We evaluated the chondral lesion on second-look
arthroscopy and assessed clinical outcomes using the WOMAC and
IKDC scores, both preoperatively and at final follow-up.
Results: On second-look arthroscopy, recipient sites of
osteochondral autograft transplantation had healed completely
showing normal cartilage (ICRS grade 0) in all knees. A radiographic
evaluation at final follow-up revealed an increase by 1 grade
according to Kellgren-Lawrence in only 1 case. The mean WOMAC
score improved from 56.1 (range, 41 to 71) preoperatively to 73.0
(range, 59 to 81) at final follow-up (P = .033), and the average IKDC
score also significantly increased from 54.1 (range, 40 to 66) to
77.4 (range, 67 to 85) (P = .028).
Conclusions: Osteochondral autograft transplantation may be
effective in treating single, localized osteoarthritic lesions in
patients over 50 years old if the alignment of the knee is within the
normal range.
P313
Axial knee realignment technique for medial opening wedge
osteotomy of the tibia with concomittant bioresorbable medial
meniscus implant.
K.P. Slynarski, E. Kurowska, T. Scinski
Warsaw/Poland
Purpose: Main principle of medial open wedge high tibial
osteotomies (MOWHTO) is to achieve a transfer of loading from
medial - diseased, arthritic areas of the joint to lateral compartment
with healthy cartilage. However, MOWHTO correct only knee
alignment, but does not correct intraarticular pathologies of medial
compartment. Our hypothesis was that patients with malalignment
and medial meniscus loss would benefit with concomittant MOWTO
and meniscus repair with bioresorbable meniscus implants.
262 Posters
Methods and Materials: Eight patients underwent medial opening
wedge osteotomies using the AKRFX Surgical Instrument System
and the iFX PEEK implant with concomittant arthroscopic medial
meniscus repair with collagen (four patients) and polyurethane
implants (four patients). Each of the eight patients was case-
matched to a control patient who had undergone MOWHTO using the
same AKRFX system with concomittant debridement arthroscopy.
Patients were evaluated with specific quality of life (KOOS), SF
36 and radiographic assessments of union and maintenance of
correction. Clinical union was measured by the patient’s ability to
full weight bear and walk without the use of crutches.
Results: At six months all 16 patients met the clinical criteria for
osteotomy healing and showed statistically significant improvement
in symptoms and in function. All KOOS score subscales showed
statistically significant higher increase from baseline to 6 months
in meniscus repair group, than in debridement alone arthroscopy.
Statistically significant improvement in the SF-36 physical
component score at 6 months in both groups was also observed,
without statistically significant differences between them. There
was no statistically significant differences with regard to type of
meniscus implant used.
Conclusions: The AKRFX osteotomy system achieved perfect rates of
union and maintenance of correction. Clinical improvement in patients
treated with concomittant medial meniscus repair were superior
to group treated with osteotomy and debridement alone without
statistical differences between types meniscus implants used.
P314
The response of articular cartilage to periacetabular osteotomy
as measured using the 3D dGEMRIC technique
J. Chan1, T.C. Mamisch2, Y. Kim1
1
Boston/United States of America, 2Berne/Switzerland
Purpose: Periacetabular osteotomy (PAO) is a reconstructive
surgery designed to treat symptoms and possibly delay the onset of
osteoarthritis in patients with developmental dysplasia of the hip. The
purpose of our study was to evaluate the effect of this procedure on
the articular cartilage using a three-dimensional dGEMRIC sequence.
Methods and Materials: Ten hips in ten patients treated with PAO for
dysplasia were analyzed retrospectively. All patients were imaged
pre- and post-PAO using a 1.5T MRI with three-dimensional isotropic
fast T1 mapping dGEMRIC sequence (TR 15 msec, TE 3.27 msec, flip
angles of 5 and 23 deg., matrix size 192/192, 16 cm FoV, voxel size
0.8 x 0.8 x 0.8 mm, acquisition 30 min post intravenous injection of
contrast agent). The 3D data was reconstructed into seven radial
slices of 2 mm thickness spaced 30 deg. apart (anterior - posterior)
and oriented orthogonal to the acetabulum or parallel to the femoral
neck axis. The mean T1 relaxation time was calculated separately for
acetabular and femoral cartilage.
Results: The mean age of the population was 22 ± 5.7 years and the
follow-up scan was performed 12 ± 3.8 months after surgery. The mean
overall dGEMRIC index (average of 7 slices) was 559 ± 109 ms (mean ±
SD) pre-operatively and 519 ± 90 ms post-operatively, and the WOMAC
pain score decreased by 4 ± 7.4. A global decrease in average T1 values
was observed overall in both acetabular and femoral cartilage, with
the largest decrease occurring in the superior-posterior region of the
acetabulum and posterior-superior region of the femoral head.
Conclusions: The three-dimensional assessment of cartilage in DDH
patients suggests that the PAO has a minimal impact on the overall
T1 average of cartilage. While the osteotomy appears to help relieve
symptoms for patients, its short-term effect on the integrity of
cartilage is not statistically significant.
P315
Autologous Chondrocyte Implantation for the treatment of
cartilage lesions of the knee. A systematic review or Randomized
Control Trials
H.S. Vasiliadis1, J. Wasiak2, G. Salanti1, A. Georgoulis1
1
Ioannina/Greece, 2Melburne/Australia,
Purpose: Autologous Chondrocyte Implantation (ACI) techniques
are becoming more popular for the treatment of full thickness
cartilage lesions of the knee joint. However, there is no systematic
information for the efficacy of the new generation ACI techniques
comparing to other treatment options. A systematic review of the
existing evidence from randomized clinical trials of ACI treatment
would contribute to understanding the advantages and limitations
of this method and would inform the planning of future studies.
Methods and Materials: Using pre-defined criteria, we searched a
number of electronic databases such as MEDLINE, EMBASE, The
Cochrane Library to identify all the existing randomised control
trials of any type of ACI treatment. Risk of bias was assessed
according to the adequacy of sequence generation, concealment of
allocation, blinding, completeness of follow-up, selective reporting
and differences at baseline between the randomized groups. An
analysis of the reported outcomes was performed. Information on
the clinical efficacy and safety of ACI compared to other interventions
was collected and presented.
Results: Nine trials were identified with 626 patients. Patients ranged
from 15 to 52 years and the size of treated lesions was between 1 and
22 cm2. ACI was associated with improvement in clinical outcomes
compared to baseline. However, the body of evidence did not suggest
any superiority of ACI over other treatments. Complications rates
were comparable between interventions except from an increased
rate of graft hypertrophies after ACI with periosteum.
Conclusions: ACI is an effective treatment for full thickness chondral
defects of the knee, providing an improvement of clinical outcomes.
However, there is insufficient data to say whether ACI is superior
to other treatment strategies. More, high quality, studies and
harmonisation in the reported outcomes are needed before specific
suggestions for practice can be made.
P316
Measuring Pain and Function in Patients with Articular Cartilage
Repair
S. Lewis1, C. DeMuro1, M.M. Mordin1, J. Farr2, B.J. Cole3, K. Mithoefer4 ,
L. Engelhart5
1
Research Triangle Park/United States of America, 2Indianapolis/United
States of America, 3Chicago/United States of America, 4Cambridge/
United States of America, 5Milford/United States of America
Purpose: The US FDA draft guidance for industry entitled
“Preparation of IDEs and INDs for Products Intended to Repair or
Replace Knee Cartilage” recommends including improvement in
pain and physical function as co-primary endpoints for confirmatory
clinical studies. Insights into how patients assess pain intensity
(PI) and the impact of pain on function are crucial to the proper
selection of these outcomes for use in clinical trials. Therefore,
we conducted in-depth individual interviews to assess the content
validity of a single item of PI (Numeric Rating Scale - NRS) and the
Knee injury and Osteoarthritis Outcome Score (KOOS) in patients
with articular cartilage injury.
Methods and Materials: Inclusion and exclusion criteria and a semi-
structured interview guide were developed. Participants aged 18 to
65 who were either surgical candidates or at least 2 months post-
op were identified through clinical sites. Participants were asked
to describe the history of their knee injury and discuss ways that
their lives had been impacted, followed by cognitive debriefing of
the KOOS and NRS.
Results: Fifteen patients aged 25-52 with mixed educational and
ethnic background participated. NRS scores collected at screening
ranged from 1-7 on affected knees and 0- 2 on unaffected knees.
Most patients (13/15) indicated the NRS as an appropriate method
to assess PI, noting its simplicity and patient familiarity with the
scale. All participants stated that the KOOS was comprehensive
and appropriate. Particularly, activities on the “Function, sports
and recreational activities” subscale were often precipitating
factors leading to injury and reflected the level of function to which
participants wished to return.
Conclusions: Results of this qualitative study support the use of
the NRS and the KOOS subscales in assessing PI and function in
patients with articular cartilage injury.
P317
Biomechanical evaluation of the knee after autologous
chondrocyte implantation
P.C. Kreuz1, S. Müller2, A. Hirschmüller2, J. Mika3, C. Erggelet4
1
Munich/Germany, 2Freiburg/Germany, 3Staufen/Germany, 4Zurich/
Switzerland
Purpose: Autologous chondrocyte implantation is an effective
clinical procedure for the regeneration of articular cartilage defects.
However biomechanical loading and muscle strength tests are still
missing in the postoperative follow-up. The present study presents
a biomechanical evaluation of patients after second generation ACI
in the knee.

Methods and Materials: Biomechanical evaluation was performed
on 44 patients 4 years after ACI with a second generation cell loaded
scaffold. Isokinetic single-joint maximum strength measurements
were investigated in different concentric and eccentric test modes
with an isokinetic dynamometer. Reciprocal movements of the knee
were performed for the flexion and extension of the knee. Statistical
evaluation was performed in different defect locations of the knee
using the Wilcoxon-and Mann Whitney U tests. Differences were
considered significant at a p < 0.05.
Results: The biomechanical evaluation revealed significant inferior
maximum strength measurements for the extensor and flexor
muscles in all defect locations of the knee after ACI compared to
the untreated healthy knee (p < 0.05). Maximum strength values
of patients with patellofemoral defects were significantly inferior
compared to patients with defects on the femoral condyles (p <
0.05). Sportive patients with a higher KOOS-score and a continuous
postoperative training revealed significant higher maximum
strength measurements and a better balancing between extensor
and flexor muscles of the thigh (p < 0.05).
Conclusions: Biomechanical evaluation after ACI of the knee reveals
reduced strength measurements compared to the healthy contralateral
knee joint. This deficiency is often associated with a dysbalance
to the disadvantage of the extensor muscles. The results have an
outstanding importance in the postoperative treatment of ACI and
underline the demand for a specific training of the operated extremity
to avoid biomechanical deficiencies and muscle dysbalance.
P318
Would you grow your own joint? An exploratory study into
perceptions of endocultivation.
R.G. Geddes1, J. Richardson2, P.H. Warnke3, A. Kerr2, S. Sivananthan2,
R. Richardson1
1
DE22 3HS/United Kingdom, 2Oswestry/United Kingdom,
3
Queensland/Australia
Purpose: Regenerative medicine as an alternative to conventional
orthopaedic surgery is gaining momentum in medicine through its
promise of using your own body to heal your body. Endocultivation
is a specific and innovative type of regenerative medicine, and
has been used successfully to cultivate a mandible (Warnke et al
2004). It involves using autologous stem cells and culturing them
in vivo, thus using the patient as the ‘bioreactor’ Our research
aims to assess the public understanding and concerns regarding
endocultivation and represents the first study in this area. We can
summarise the aim of our study by asking: do people understand
and want this kind of therapy?
Methods and Materials: A survey instrument developed to assess
attitudes towards different sources of stem cells, regenerative
medicine and willingness to grow your own body part was distributed
to members of the public in several European countries. Educational
material on sources of stem cells was supplied to all participants.
The process of endocultivation did always require an explanation.
Results: Respondents (N=160) had a median age of 43 years and
were from a variety of socio-cultural backgrounds. Support for
a variety of sources of stem cells was higher than has been seen
previously. Nearly 80% of participants would consider becoming
a bioreactor and ‘growing their own joint’. In addition, people are
willing to accept stem cell therapies in certain contexts.
Conclusions: Acceptance of stem cell therapy in Europe is high. The use
of autologous stem cells to regenerate an organ had the highest level
of support, suggesting acceptance of stem cell therapy is dependent
on personal context. We suggest more research is carried out in this
area to inform ethics committees, policy makers and clinicians.
P320
The influence of a physical training in the motor control
-quadriceps steadiness - of patients with osteoarthritis grades I
or II
S.M. Mattielo-Rosa, K. Gramani-Say, P.R. Serrão, Y.M. Kawaguchi,
G.C. Lessi
São Carlos/Brazil
Purpose: Osteoarthritis (OA) is a chronic and progressive
degenerative disease, characterized by gradual loss of articular
cartilage. A subject with OA exhibited substantially impaired
motor control, balance instability and quadriceps weakness. All
these factors come to a motor control deficit in the quadriceps
steadiness. The aim of this study is to compare the average of
Posters 263
the maximum isometric extensor peak torque (MIEPT) and the
quadriceps steadiness (QS) between subjects of the control
group (CG) and Osteoarthritis Group (OAG) and also verify the
effectiveness of 11 weeks treatment protocol applied to the OAG
subjects.
Methods and Materials: This has been checked analyzing the
average of the MIEPT and the QS for these subjects before, after
5 and 11 weeks of treatment. The sample was composed with 20
sedentary men, (45 to 65 years old), separated equally in 2 groups:
CG (53,8 ± 7,7 years) without OA or knee injury and the OAG (53,1
± 6,9 years) with knee OA grade I or II with diagnosis made by the
Kellgreen & Lawrence radiological assessment. All volunteers
were submitted to Isokinetic dynamometer evaluations of knee
extension to verify their MIEPT and QS. The statistical tests used
were ANOVA and Kruskal- Wallis to analyze the difference inter
group. And Shapiro-Wilk, t-student and U Mann-Whitney test to
analyze the OAG data before and after the treatment (α≤0,05).
Results: At the variables analyzed peak torque (PT) and coefficient
of variance (CV) any significant statistics difference was shown in
both analyses, between the groups and inside the OAG before the
5 and 11 week of treatment.
Conclusions: Despite of the results which didn’t show significant
statistics values to prove your effectiveness, the importance to
the clinic practice was notice since we checked that subjects with
the initial grades of OA didn’t exhibit alterations in the maximum
and steadiness force (PT and CV).
P321
Case Study: Rehabilitation of an elite rugby player following
osteochondral lesions in the knee. Train smart not hard!
J.M. Stephen
6SX/United Kingdom
Purpose: A number of published case series have presented
chondrolysis of the knee and its devastating consequences
once it has occurred in young, male, elite sporting populations.
This presentation outlines a successful, structured, progressive
rehabilitation programme for returning an elite sportsman back to
international rugby.
Methods and Materials: A 23 year old male international rugby
player presented with progressively worsening right knee pain
and swelling on returning to play following a partial lateral
meniscectomy 9 months previously. Clinical examination revealed
a moderate knee joint effusion with loss of hyperextension and
lateral joint line tenderness. Magnetic resonance imaging showed
femoral and tibial bone oedema. Nine months following primary
surgery, second look arthroscopy identified grade 3 and grade
2 chondral lesions of the lateral femur and tibia respectively,
alongside a complex degenerative lateral meniscal tear (Figure
1). Surgical treatment involved a wash out and clean up of joint
surfaces, with a meniscal repair undertaken to the remaining
meniscal segment. The player in question underwent a lengthy
rehabilitation period. A strong emphasis was placed on aquatic
rehabilitation to offload the joint and encourage resumption of
normal lower limb movement mechanics.
Results: The player successfully returned to professional level
rugby, however experienced ongoing symptoms of swelling and
pain. He therefore continues to be carefully managed in terms of
his training and playing. He presently plays one match in 2 weeks
and undertakes team training every other day. From experience
load managing these compromised athletes represents the most
crucial aspect of enabling them to successfully return to sport with
some career longevity.
Conclusions: Articular surface compromised athletes need
managed carefully in terms of weekly volume and load. They
require career long modification of typical training patterns, but
with correct management can demonstrate sustainability in the
sport despite compromised joint integrity.
264 Posters
P322
Treatment of osteochondral defects using poly-lactic-glycolic
acid PLGA scaffold and mesenchymal stem cells (MSCs)
K. Uematsu1, Y. Ishimoto1, Y. Inagaki1, M. Ogawa1, Y. Tanaka1, K.
Hattori2, H. Ohgushi2
1
Kashihara/Japan, 2Amagasaki site/Japan
Purpose: The successful cartilage regeneration need a scaffold
that can keep the stem cells in the defect and support to
differentiate to hyaline cartilage. We have developed originally
the biodegradable scaffold and that feature is that the cells can
infiltrate into the scaffold without cell loss and lay in an uniform
array at palisade. We have already reported the good results of
the cartilage regeneration in an animal study. The aim of this study
was to investigate the treatment of the symptomatic knee cartilage
defects by the implantation of PLGA/MSCs composite.
Methods and Materials: The patients were four men (age: 23,
25, 29 and 40 years) and one woman (age: 56 years), and three
posttraumatic cartilage defects and osteonecrosis of the femur
were treated. The average size of defect was about 1.7×1.8 cm.
MSCs were harvested from in each ilium and incubated by standard
method. After confluence, the cultured cells were seeded into
PLGA scaffold, and the density of MSCs in the scaffold were 1×107
cells/cm3. We implanted PLGA/MSCs composite onto the cartilage
defect by press-fit. Magnetic resonance imaging (MRI) and relook
by the arthroscopy were performed in all patients at a mean of
36 months postoperatively. We evaluated the regenerated tissue
endoscopically and conducted histologic assessment by a biopsy.
Results: The inflammatory reaction was not recognized after one
week postoperatively. The histologic evaluation by the biopsies
showed the formation of hyaline-like cartilage.
Conclusions: We conclude this scaffold was useful in the cartilage
regeneration in human.
P323
Articular cartilage repair using tru-fit plugs
S. Tafazal, R. Shahid, R. Mansouri, M. Maqsood
Lincoln/United Kingdom
Purpose: Articular cartilage damage is known to predispose to
osteoarthritis. Many different techniques have been used to
attempt repair of articular cartilage defects. Synthetic scaffolds can
support cartilage formation in areas of cartilage defects. Study: We
present our experience with a synthetic scaffold (Tru-Fit) which we
are using to treat patients with chondral defects in knee.
Methods and Materials: 15 patients with full thickness cartilage
defects affecting the medial femoral condyle were treated with Tru-
Fit plugs. They were followed up at a mean of 9.6 months (0.5-21)
after surgery. At a minimum of 6 months follow-up MRI scans were
obtained in all cases to asses healing of defect. International Knee
Documentation Committee (IKDC) scores and Lysholm scores were
also obtained.
Results: The mean IKDC score was 55 (20-90) and the mean
Lysholm score was 63 (20-100). Patients with isolated cartilage
defects and no other knee pathology had a much better outcome
with a mean IKDC score of 76 and a mean Lysholm score of 86. MRI
scans showed good cartilage cover of the defects.
Conclusions: Our early results of using Tru-Fit plugs to treat articular
cartilage defects in knee are promising. Patients with isolated
chondral defects in the knee show the most favourable outcomes.
P324
Trufit bioresorbable scaffolds: clinical good results associated to
delayed biological incorporation
F.V. Sciarretta, C. Ascani
Rome/Italy
Purpose: Trufit resorbable scaffolds, made of semiporous
copolymer, are press-fit introduced in chondral defects of articular
surfaces in order to promote filling and regeneration of damaged
bone and cartilage tissues.
Methods and Materials: In another previous work, we have
presented our good and promising results obtanied at 1 yy follow-
up. Then, we have had the chance to go on on follow-ups and check
patients through second-look arthroscopies and serial MRI’s: IKDC
score showed 38 points improvement. WOMAC score showed
statistically significant pain improvement in 89% of cases and
function improvement in 86% of cases. Serial MRI’s of the knees
showed progressive incorporation of the synthetic plugs and no
adverse inflammatory reaction. Second-look arthroscopies showed
complete and flush fill of the defects and their resurfacing with
hyaline-like tissue under different stages of maturation. Recently,
we have been able to check, clinically and by serial MRI’s, the first
patients operated 24 months ago.
Results: Despite the mantainance of clinical very good results, as
showed by other authors, MRI images showed a delayed biologic
process of incorporation of the plugs.
Conclusions: This finding has not to be misinterpreted as an implant
failure and the post-op rehabilitation has to be continued in order
to give regenerating cartilage time to complete the maturation
process.
P325
The clinical safety and utility of an osteoconductive,
bioabsorbable, scaffold for the use in treatment of full thickness
chondral and osteochondral defects of the distal femur
R.C. Lehman1, P.A. Davidson2
1
Kirkwood/United States of America, 2Park City/United States of
America
Purpose: This report describes the clinical safety and utility of
an osteoconductive, bioabsorbable, scaffold consisting of bTCP,
PLA and Type I collagen for the use in treatment of full thickness
chondral and osteochondral defects (< 2cm²) of the distal femur.
Methods and Materials: Patients enrolled in the study received 1
or 2 OsseoFit™ Porous Tissue Matrix™ implants on the femoral
condyles (MFC or LFC) or trochlea. The study received IRB approval.
Study endpoints are rate of complications related to the implant,
change in KOOS scores and MRI findings with the MOCART Scale.
The study was retrospective, conducted at a single center. MRI at
approximately 6, 18 and 36 months post-operatively. 22 subjects
with a mean age 37.7 ± 11.2 years were enrolled. 16 subjects
received 1 implant and 6 subjects received 2 implants. Cartilage
lesions were 1.2 ± 0.6 cm2 (0.64–2.64). 28 OsseoFit devices were
implanted as follows: MFC=14, LFC=7 and trochlea=7. Concurrent
surgical procedures included: meniscectomy in 13 subjects, ACL
reconstruction in 6 subjects, debridement of other chondral
surfaces in 16 subjects and other cartilage reparative techniques
in 4 subjects.
Results: At an average follow-up of 17.3 ± 4.5 months, post-
operative KOOS Pain and ADL subscale scores increased by 33.8
± 24.9 points (p<0.001) and 34.8 ± 25.3 points (p<0.001),
respectively. There was no evidence of implant failure, implant
delamination, hypertrophy, osseous necrosis, or significant
reactive marrow edema. MRI evaluation was performed at 159 ±
80.5 days post-operative. Complete defect filling was seen in 19/28
implants. Complete integration to the surrounding cartilage was
seen in 27/28 implants. Complications associated with the knee
surgeries were minimal.
Conclusions: At early follow-up, the OsseoFit device appears to be
safe and contributed to improved clinical outcomes in the treatment
of full thickness cartilage lesions (< 2cm²) of the knee.
P326
Optimization of autologous fibrin carrier for chondrocyte
transplantation.
A. Wysocka1, H. Bursig1, P. Malinowska1, F. Kepski1, J. Dec2, T.S.
Gaździk2
1
Katowice/Poland, 2Sosnowiec/Poland
Purpose: There is an increasing number of publications concerning
applications of cells in fibrin scaffold. Our Regional Blood Center
has developed a method for obtaining fibrinogen from a single
donor, which allows accurate identification and eliminates the risk
of transmitting viral diseases.
The objective of our work was to develop a method for autologous
fibrin – chondrocyte graft preparation stabilized by fibrynolytic
inhibitirs for transplantation into a cartilage defect.
Methods and Materials: Cartilage was taken from the patient’s
femoral condyle, non-load bearing area of knee joint, as well as
blood, for the purpose of preparing autologous serum. Chondrocyte
were cultured about 3 weeks. 450 ml patient own blood was
collected prior to transplantation to produce autologous fibrinogen.
Before surgery the chondrocyte suspension was mixed with fibrin,
glue and applied into special form to prepare gel-like fibrograft.

Results: Within 2 years 32 cultures of autologous chondrocytes were
completed. The mean age of patients was 34 years. The average
defect size 6,1cm2. The average concentration of autologous
fibrinogen was 45 mg/ml. Stabilization of chondrocyte-fibrin
construct was achieved by high doses of antifibrinolitic agent.
Conclusions: Chondrocyte implantation in fibrin glue is a promising
method for treatment of cartilage defect. Fibrinogen prepared
at Regional Blood Center in combination with cells constituted
highly plastic and adhesive grafts. This facilitates and accelerates
implantation - arthroscopy can be applied. It was possible to
set disintegration of fibrograft, by different concentration of
antifibrinolitic agent. A novelty is the application of autologous
fibrinogen in combination with autologous chondrocytes.
Application of autologous fibrinogen is safer for the patient, as
compared with allogenic fibrinogen, since it does not carry the risk
of transmitting infectious diseases. The study was supported by
grant NN403184034
P327
New biodegradable and biocompatible synthetic scaffold for
meniscal regeneration: preliminary clinical experience
S. Patella, E. Kon, A. Di Martino, G. Filardo, S. Zaffagnini, B. Di
Matteo, M. Marcacci2
Bologna/Italy
Purpose: Either a lesion or the complete absence of the menisci can
invalidate the physiological function of the knee causing important
damages, even at long term. Meniscal tears are often found during
the ordinary orthopaedic practice while menisci regenerative
potential is very low and limited to its peripheral-vascularized part;
this is why the surgeon is often almost forced to perform a partial,
subtotal or even total meniscectomy, regardless of the well-known
consequences of this kind of surgery.
Methods and Materials: Recently a porous, biodegradable scaffold
made of an aliphatic polyurethane (Actifit™,Orteq Ltd) has been
developed for the arthroscopic treatment of partial and irreparable
meniscal tears; this scaffold facilitates menisci regeneration
preventing the potential cartilage damage due to its complete or
partial lack. We treated 17 patients affected by a massive loss of
meniscal substance with intraarticular or global knee pain and/or
swelling. We analyzed the patients either clinically and by using the
International Knee Document Committee’s (IKDC) Subjective and
Objective Knee Evaluation Form and the Tegner score at the time
of the very first visit with the presurgery and prelesional ones. A
control MRI was done at 6 and 12 months after surgery.
Results: Apparently, the properties of this scaffold help in vessels
formation and tissue regeneration potentially allowing the
restoration of the surgically removed portion and preventing, or
delaying at least, both chondral and articular degeneration. We also
performed some biopsy associated arthroscopic “second-looks”
that reinforced the already good clinical results and confirmed the
new tissue ingrowth into the biomaterial.
Conclusions: Preliminary results suggest that this surgical
procedure can be considered a really promising method for the
treatment of both inveterate and symptomatic meniscal tears;
however, other randomized studies with a longer follow-up should
be done to confirm its reliability and potentialities.
P328
Modified Mocart scoring system and three year MRI followup of
Chondral repair using TruFit plugs
M. Dhillon1, G. Collin1, R. Wellings1, P. Thompson1, T. Crane1, T.
Spalding2
1
Coventry/United Kingdom, 2Leamington Spa/United Kingdom
Purpose: TruFit plugs (a biodegradeable synthetic scaffold - Smith
and Nephew) are a novel treatment of chondral damage in the knee.
The normal MRI findings following TruFit repair have only a limited
description in the literature. We present a qualitative longitudinal study
of the TruFit plugs MRI changes in our cohort over a 3 year period with a
qunatitative assessment with a modified Mocart scoring system.
Methods and Materials: Twenty four patients with osteochondral
defects in the knee have undergone TruFit plug synthetic bilayer
scaffold repair. These patients have been followed up with MRI at
six monthly intervals where possible. MRI findings of: effusion;
synovitis; bone oedema; TruFit plug resorption; subchondral
lamina formation; cartilage integration and surface contour were
assessed and scored.
Posters 265
Results: Five patients had small effusions, three of which resolved.
One of the patients had a sizeable effusion with evidence of synovitis
which persisted. The plugs show evidence of bone ingrowth in all
patients by one year, which progressed on later scans. Similarly
cartilage integration and subchondral lamina formation improve
over time. Bone oedema is variable on early scans but improves on
later scans. A good surface contour is demonstrated in all patients
on early scanning, although it does deteriorate in a minority of
patients over time.
Conclusions: We demonstrate that the MRI appearances of TruFit
plug repair follow a graded differentiation in to the respective
layers over time - bone, subchondral lamina and articular like
cartilage which appears to correspond with clinical improvement.
These findings cannot be scored under the current Mocart scoring
system and we present a robust modified Mocart scoring system to
monitor the progress of this novel form of cartilage repair.
P329
Ultrasound-guided corticosteroid injection in subacromial
bursa for painful shoulder: a randomized, double-blind, placebo
controlled, comparative trial of three dose regimens
S. Yoon, J. Hong, K. Kwack, B. Min
Suwon/Korea, Democratic People’s Republic of
Purpose: To determine whether ultrasound-guided subacromial
bursa injection with high dose corticosteroid (4cc of 40mg
triamcinolone acetonide and 1cc of 1% lidocaine, group 1, n=20),
in patients with periarticular shoulder disorders, is better than low
dose (2cc of 20mg triamcinolone acetonide and 3cc of 1% lidocaine,
group 2, n=20) or placebo (5cc of 1% lidocaine, group 3, n=20) in
improving pain, function, and active range of motion (AROM) at 2,
4, and 8 weeks.
Methods and Materials: A randomized, placebo controlled,
comparative trial with participant, injection operator, and outcome
assessor blinding in which subacromial bursa injection with high
dose regimen was compared with low dose and placebo. Outcome
measures, assessed at 2, 4, and 8 weeks, included a visual analog
scale (VAS) of the previous week’s average pain intensity, an AROM
(includes flexion, extension, external rotation, internal rotation,
and abduction angles) of shoulder, and a Shoulder Disability
Questionnaire (SDQ).
Results: (1) VAS: At 2 and 4 weeks, significantly greater
improvements were found in participants in group 1 and 2 than in
group 3. At 8 weeks, significantly greater improvement was found
in group 1 than in group 2 and 3. (2) SDQ: At 8 weeks, significantly
greater improvement was found in participants in group 1 than in
group 2 and 3. (3) AROM: At 2, 4, and 8 weeks, significantly greater
improvement was found in group 1 than in group 2 and 3. No
improvement was found in other cases.
Conclusions: Dose dependent efficacy of ultrasound-guided
subacromial bursa injection with corticosteroid was demonstrated
in patients with painful shoulder.
P330
Quantification of intervertebral disc degeneration by parametric
T2 and T2* mapping in patients with low back pain - initial
results on the clinical use with 3.0 Tesla MRI
G.H. Welsch1, D. Stelzeneder1, T. Paternostro-Sluga1, S. Goed1, D.
Hornung2, S. Trattnig1, T.C. Mamisch3
1
Vienna/Austria, 2Erlangen/Germany, 3Berne/Switzerland
Purpose: Quantitative T2 and T2* provide information about the
interaction of water molecules and the collagen-network within the
intervertebral disc (IVD). Aim of the study was to assess, compare
and correlate quantitative T2 and T2* relaxation time measurements
of IVDs in patients suffering from low back pain, with respect to
the IVD degeneration as assessed by the morphological Pfirrmann
Score. Special focus was set on the spatial variation of T2 and T2* in
between the annulus fibrosus (AF) and the nucleus pulposus (NP).
Methods and Materials: Thirty patients (38.1±9.1 years) suffering
from low back pain were enclosed. Morphological (sagittal T1-FSE,
sagittal and axial T2-FSE) and biochemical (sagittal T2- and T2*-
mapping) MRI was performed at 3T covering IVDs L1-L2 to L5-S1.
All IVDs were morphologically classified using the Pfirrmann score.
Region-of-interest (ROI) analysis was performed on midsagittal T2/
T2* maps at 5 ROIs from anterior to posterior to obtain information
on spatial variation between the AF and the NP. Statistical analysis-
of-variance and Pearson correlation was performed.
266 Posters
Results: Both, T2 and T2* were able to clearly differentiate
between all grades of IVD degeneration according to the Pfirrmann
score (p<0.05). The spatial variation, as an increase in T2 and T2*
values from the AF to the NP, was highest at Pfirmann grade I and
declined at higher Pfirmann grades II-IV (p<0.05). Where T2 was
more sensitive in the description of the NP, T2* was more sensitive
in the depiction of the AP. Correlation between T2 and T2* revealed
a medium Pearson correlation (0.210 to 0.356 (p<0.001)).
Conclusions: T2 mapping as well as the newer T2* mapping
provide a fast and stable tool in the evaluation of IVDs. The clear
differentiation of IVD degeneration and the possible quantification
by means of T2 and fast T2* mapping may provide a new tool to
follow-up therapy protocols in patients with low back pain.
P331
T2 mapping of facet joints and intervertebral discs: Biochemical
MRI at 3.0 Tesla in patients with low back pain
D. Stelzeneder1, G.H. Welsch1, S. Goed1, A. Messner1, T. Paternostro-
Sluga1, V. Pflueger1, G. Scheurecker2, K. Friedrich1, S. Trattnig1
1
Vienna/Austria, 2Linz/Austria
Purpose: Degenerative changes of the facet joints
represent a possible source of pain. Our objective was to
assess lumbar facet joint cartilage in comparison to the
intervertebral discs by means of quantitative T2-mapping.
Methods and Materials: Forty-nine lumbar spine segments from
25 patients (mean age 36 years) suffering from low back pain
were examined by axial T2-mapping and morphological sequences
at 3.0 Tesla. Regions of interest were drawn on a single slice
across the facet joints, the nucleus pulposus and in both anterior
and posterior annulus fibrosus. The Pfirrmann-score was used
for morphological grading (“normal” vs. “abnormal” discs). A
Pearson-correlation analysis was performed to compare disc and
facet joint T2 values.
Results: Twenty-six discs were graded as normal (Pfirrmann 2)
and 23 as abnormal (Pfirrmann 3+4). Mean (±standard deviation)
T2-relaxation-time-values (in ms) were: facet joints: 57.4±12.4,
nucleus: 97.3±25.9, anterior annulus: 49.0±11.0, posterior annulus
61.6±11.5. There was no difference between facet joint mean T2-
values in normal and abnormal discs: 57.1 vs 57.7 ms. Correlation
analysis showed no association between nucleus and facet joint
T2-values. Facet joint T2-values of all analyzed segments showed
a weak correlation with the anterior annulus (r=0.31;p=0.03)
and a moderate correlation with the posterior annulus fibrosus
T2-values (r=0.53;p<0.001). These associations were not seen
in the subgroup of normal discs. The correlation of facet joints
with the posterior annulus was stronger in abnormal discs
(r=0.66;p<0.001). For the facet joints and the anterior annulus
only a trend was seen (r=0.41;p=0.053) in abnormal discs.
Conclusions: Our report represents the first attempt of a
quantitative analysis of lumbar facet joint cartilage via T2 mapping
in comparison to morphological grading. Our evaluation may
suggest that the biochemical structure of the posterior part of
annulus fibrosus affects biochemical properties of facet joint
cartilage in particular in degenerated discs. T2 mapping may help
to detect early degeneration of facet joints.
P332
Osteochondral Injuries: Application of Bone Marrow-Derived
Mesenchymal Stem Cell Assisted by Arthroscopy.
J.F. Urraza1, P. Vidal2
1
Sant Pere de Ribes/Spain, 2Barcelona/Spain
Purpose: The successful regeneration of the “Hyaline cartilage” can
be considered as one of the more important challenges in the field
of Orthopaedic surgery. The purpose is to present a preliminary
study using autologous bone marrow-derived mesenchymal stem
cell in the treatment of osteochondral injuries, analyzing the
feasibility of the process.
Methods and Materials: We report the preliminary data of a
prospective clinical study with five patients . The ICRS classification
was used (grade IV B and C). The average patient age was 21 years
old (17-30). All patients had received surgical treatment prior to
this study and they had had the lesion for a year or more (1-11).
The feasibility of the method was appraised with Rx., M.R.I.,
functional clinical assessment, and histological studies. Puncture
of posterior iliac crest for bone marrow aspiration was made.
The identification and isolation of mesenchimals cells was made
by tissue engineering technique and with rich platelets growth
factor, spongy bone chips or calcium triphosphate forming a
biological structure. The affected joint was under arthroscopic
assessment and we prepared the damaged area with all affected
tissue resection. We finished the procedure with the application of
biological structure in the bone defect.
Results: In all cases we found improvement in the articular R.O.M.
The Rx and M.R.I. showed bone or substitute bone integrated and
soft tissue covered the defect in all cases. Histologically we found
in all the biopsies performed the morphology of the “articular
cartilage Hyaline”, homogeneous tissue with typical round
cells forming lagoons and collagen type II with a normal basal
integration that represent a “Tide-Mark” regeneration.
Conclusions: The autologous bone marrow-derived Mesenchymal
stem cell, associated with spongy bone chips or calcium
triphosphate, show histological data of hyaline cartilage articular
regeneration with very good mechanical behavior and clinical
results after more than 13 months of follow up.
P333
Depth-dependent healing strength of cartilage-like tissue to host
cartilage: Application of a stem cell-based tissue engineered
construct
R. Nansai1, K. Adachi1, N. Nakamura2, H. Fujie1
1
Tokyo/Japan, 2Osaka/Japan
Purpose: We have been developing a new tissue engineering
technique for cartilage repair using a scaffold-free, tissue
engineered construct (TEC) bio-synthesized from synovium-
derived mesenchymal stem cells (MSCs). The present study was
performed to determine the depth-dependent healing strength
between the repaired cartilage-like tissue and surrounding host
cartilage.
Methods and Materials: Synovium-derived MSCs obtained from
immature porcine knee joints were cultured in a monolayer in
DMEM. After ascorbic acid 2-phosphate was added, the cells were
allowed to undergo active contraction for 8 hours to develop a TEC.
The TEC mass was allografted to a cylindrically shaped, chondro-
defect created in the medial condyle of 3 month-old porcine femur.
Six months after surgery, a dumbbell-shaped specimen including
the boundary between the repaired tissue and host cartilage was
extracted. The tensile test was performed for the specimen at a
rate of 0.01 mm/s.
Results: The healing strength of the TEC-treated repaired tissue
was higher than that of the TEC-untreated repaired tissue, although
it was lower than the tensile strength of the normal cartilage. The
strength was almost negligible at the surface layer but was 1.7 and
2.8 MPa at the middle and deep layers, respectively. Histological
examination indicated that, at the intermediate-to-deep area, the
TEC-treated repaired tissue was hyaline cartilage-like while the
TEC-untreated repaired tissue was fibrous cartilage-like.
Conclusions: The present study indicated that the healing strength
of cartilage-like repaired tissue to surrounding host cartilage
was increased with the treatment of TEC although the strength
remained lower than the strength of host cartilage. In addition,
the healing strength was depth-dependently increased in the
TEC-treated cartilage-like tissue in the same way as shown in
normal cartilage. These results suggest that the healing process
of cartilage-like tissue is promoted by the TEC in conjunction with
an extrinsic factor possibly permeated from the bottom of the
chondro-defect.
P334
Regeneration of lumbar intervertebral disc using MSVs*.
*Autologous Bone marrow expanded MSCs under GMP
guidelines from IBGM
C. Morera1, R. Soler Rich1, J.J. Velázquez1, A. Sánchez2, M. Alberca2,
J. García Sancho2, L. Orozco1
1
Barcelona/Spain, 2Valladolid/Spain
Purpose: We report the methodology and outcomes of the
clinical trial phase I-II, Eudra-NCT:2008-001191-68, assessing the
feasibility, safety and efficacy findings of regeneration after the
percutaneous intradiscal infiltration of autologous bone marrow
mesenchimal cells expanded under GMP. This study is carried
out under the framework of the “Plan for Advanced Therapy and
Regenerative Medicine” (Consejo de Ministros of the Spanish
Government, 2007).

Methods and Materials: 10 patients with degenerative disc
disease were recruited . One of the inclusion criteria was proven
competence of the annulus fibrosus by discography. The procedure
starts harvesting bone marrow aspirate (80 ml) from iliac crest
under sedation and local anesthesia. Collected samples are sent
to the Cell Therapy Unit of the IBGM, where they carry out the cell
extraction and expansion process for three weeks. The product
generated (MSVs) is held to the same standard as medication
under investigation. It was liberated after undergoing the bio-
safety controls (sterilization, viability, phenotype and cariotype).
MSVs are percutaneously infiltrated under RX guidance at a dosage
of 15±5x106 MSCs per disc (max 2 discs, max dose 40x106 MSVs).
Results: At the moment (6 months follow-up) the procedure has
proven to be feasible and we have registered no adverse effects
or toxicity signs. We are assessing the efficacy findings of the
regenerative therapy evaluated through changes in density at the
nucleous pulposus (re-hydration) assessed by semi quantitative
MRI and the clinical outcomes using the Visual Analogue Score
for pain, Oswestry Disability Index and the SF-36 Questionnaire
for quality of life, all showing positive statistically significant
difference.
Conclusions: Infiltration of autologous bone marrow MSCs into the
degenerated intervertebral nucleous pulposus showing clinical
signs may improve symptoms and even achieve regression of the
degenerative process. The final outcomes shall be communicated
at the ICRS Congress 2010, upon completion of the one year follow-
up period.
P335
Chondrocytic differentiation of bone marrow-derived equine
mesenchymal stem cells for future therapeutic applications in
horses
A. Pineda1, M. Masri2, R. Gomez2, C. Landa2, C. Velasquillo2, C.
Ibarra2
1
México/Mexico, 2Mexico City/Mexico
Purpose: The objective of this study was to isolate and
characterized chondrocytic differentiation of bone marrow-derived
equine mesenchymal stem cells for possible future therapeutic
applications in horses. We characterized using , CD45, CD34 and
CD14 typical hematopoietic antigens.
Methods and Materials: Bone marrow aspirates were obtained from
sternebra of 7 horses, 3-5 years old. The aspirates were obtained
by Jamshidi bone marrow aspirate needles and were collected into
syringes containing heparin to a final concentration of 1,000 UI/
ml. The fraction of mononuclear cells was separated with Ficoll
(Amersham Bioscience Ficoll- Paquete Plus) and washed with PBS.
The pelleted stromal cells were resuspended in DMEM F12 medium
containing 10% fetal bovine serum and 100 UI/ml of penicillin and
100 µg/ml of streptomycin and cells were incubated. Part of the
sample was utilized for flow cytometry test with human antibodies
(CD34, CD14, and CD45). To induce chondrogenic differentiation
the original media was replaced with an induction medium
containing transforming growth factor β1 and bone morphogenetic
protein-2 (100ng/ml). The chondrogenic differentiation potential
was measure by alcian blue and safranin-O stains.
Results: We found that cells from equine bone marrow stem cell
markers negative for CD14, CD34 and CD 45. Under tridimensional
culture conditions to promote chondrogenic differentiation, cell
aggregates were detected positive for alcian blue and safranin-O.
Conclusions: Our data indicate that bone marrow-derived stromal
cells of horses can be characterized as MSC’s, have the capacity
to undergo chondrogenic differentiation and may be a clinically
plausible source of cells for neo-chondrogenesis at damaged
articular sites. As an autologous cell population, equine MSC’s can
be regarded as a promising cell population for tissue engineering
in lesions of the musculoskeletal system in horses.
Posters 267
P336
Pre-differentiation of mesenchymal stem cells improves cartilage
regeneration for treatment of chronic osteochondral lesions
J.S. Somerson1, B. Marquass2, P. Hepp2, R. Richter2, T. Aigner2, C.
Josten2, A. Bader2, M. Zscharnack2, R.M. Schulz2
1
Toledo/United States of America, 2Leipzig/Germany
Purpose: Large osteochondral lesions are a challenge to treat due
to the lack of spontaneous healing. This project evaluated the use
of marrow-derived autologous mesenchymal stem cells (MSC) in
hydrogel constructs to treat osteochondral lesions. Repair quality
in vivo with MSCs that had undergone in vitro chondrogenic pre-
differentiation was compared with undifferentiated MSCs and cell-
free constructs.
Methods and Materials: An initial surgery was performed to create
two osteochondral lesions (7mm diameter) on the medial femoral
condyle of each hind leg in 19 merino sheep. Bone marrow aspirate
was taken from the iliac crest at the time of surgery. The defects
were allowed to degenerate in natural fashion for 6 weeks. After
isolation of MSCs, 5 x 10^5 cells/ml were seeded into a collagen-I
matrix. One group of the MSC-gels underwent chondrogenic pre-
differentiation while the other MSC-gel group was cultivated using
DMEM and 10% autologous serum. After 6 weeks in vitro, the
constructs were implanted. Untreated lesions, cell-free constructs
and chondrocyte-based gels were used in control groups. The
animals were sacrificed after 6 or 12 months and the constructs
were evaluated using histology and micro-MRI.
Results: Quantitative assessment confirmed chondrogenic in
vitro differentiation of the ovine MSCs in the collagen gels. After 6
months, the pre-differentiated MSC-gels showed the best results
(ICRS Visual Histological Score (VHS):13.3±2.6; O’Driscoll score:
17.6±2.5). The cell-free gels showed a VHS of 8.2±4.3 points and an
O’Driscoll score of 11.3±6.8. After 12 months, the pre-differentiated
MSC-gels showed superior histological properties (O’Driscoll:
18.2±2.5) to undifferentiated MSC-gels (10.5±5.9). No significant
difference was noted between the results for repair using pre-
differentiated MSCs at 6 and 12 months.
Conclusions: Implantation of collagen gels seeded with autologous
MSCs results in regeneration with a hyaline-like structure.
Repair using MSC-seeded gels appears to produce better results
when chondrogenic pre-differentiation is performed prior to
implantation.
P337
AMIC technique enhanced by the use of concentrated bone
marrow for the treatment of chondral lesion.
L. de Girolamo1, P. Adravanti2, H. Schoenhuber1, P. Volpi1
1
Milan/Italy, 2Parma/Italy
Purpose: AMICÒ (autologous matrix induced chondrogenesis)
combines microfracturing with the application of Chondro-Gide®,
a porcine collagen type I/III bilayer matrix. However, all marrow
stimulation techniques collect only a poor number of mesenchymal
stem cells and this number is variable among patients. For this
reason it could be useful to enhance these techniques with
progenitor cells harvested from iliac crest bone marrow, in order
to provide a better and faster defect resurfarcing. Here we present
the clinical outcome of 30 patients treated either with the standard
AMICÒ technique or combined with the use of concentrated bone
marrow (cBM) from a minimum follow up of 6 months up to 24
months.
Methods and Materials: 30 patients (range 18-50 y/o) presenting
focal cartilage lesions of the knee were treated either with the
standard (n=11) or the modified AMICÒ technique (n=19). Clinical
evaluation was based on Lysholm Knee Score, IKDC and VAS pain
scale. For each patient MRI was performed preoperatively and 6,
12 month and 24 months postoperatively.
Results: Significant differences between pre- and post-operative
score values have been observed for all patients starting from 6
months. After 12 months significant best results were found in
patients treated with the modified AMIC® technique (n=11) respect
to those treated with standard AMICÒ (n=8), in particular in term
of pain reduction (2.5±0.7 and 0.8±0.4, respectively, p<.05) and
Lysholm score (84±1.4 and 95.4±4.4, p<.05). MRI showed good
healing processes of the cartilage defects already after 6 months
in both groups of patients.
Conclusions: Enhancing the AMICÒ technique with concentrated
bone marrow seems to be an effective and safe method. However,
longer follow-up times are necessary to evaluate the long-term
regeneration success.
268 Posters

P338
State of the art on cartilage tissue regeneration
P. Vidal1, J.F. Urraza2
1
Barcelona/Spain, 2Sant Pere de Ribes/Spain
Purpose: Successfully hyaline cartilage regeneration is one of the
most significant challenges in the orthopedic field. Unfortunately
cartilage injured doesn’t naturally grow back, and treatment
options remain limited even innovative therapies which improved
substantially over the past few years.
Methods and Materials: The tissue engineering discipline pursues
basic knowledge in tissue development and self regeneration
instead of repair. It is critical to choose suitably cells, appropriate
signaling proteins and biomaterials to mimic the natural
physiological process of tissue regeneration. Although mostly
composed by water any regenerated cartilage needs to contain a
structure capable of bearing weight and being strong but flexible,
definitely the main challenge to achieve. The cell-based therapies
strategy fall into matrices where is depending the body natural
capability to regenerate and conditioned by biomaterials applied,
some tend to slowly degrade and other takes too long time. Thus,
there is dissociation between cells growing to become specific
local cell phenotype and matrix degradation, too short or long
term degradation implies an incomplete chondrogenesis.
Results: Our approach has been focused on structured autologous
multilevel matrix supplying proteins and cytokines to the implanted
cells in a proper environment niche that promote the ingrowing cells
and support their sequential function of protein secretions, cellular
metabolism, local cells commitment and differentiation.
Conclusions: Bone marrow-derived mesenchymal cells are our
preferred cells by their multipotentiality and progenity which
correlated directly with regenerative capability. Cartilage tissue
engineering incorporates cell transplantation, materials , and new
surgical cell delivery, personnel who have mastered the procedures
of cell harvest, manipulation, and graft design are essential for the
successful application of this technology.

This author index lists the names of all
authors and co-authors of the all congress
abstracts (Podium Presentations, Poster
Presentations & submitted Extended
Abstracts by the invited faculty). Requests
for changes have been considered until
June 30, 2010. The numbers in the index
refer to the final programme number and
the letter “P” before the final programme
number refers to the poster section.
A Azevedo, H. S. P195
Alaminos, M. P93, P93, P107
Abe, S. P87
Abellanet, I. 12.1.7, P223
Abelow, S. P. 20.1.1
Aberman, H. 25.3.4
Acharya, C. P186
Ackland, T. R. 12.2.2, P225
Acosta, V. P48, P177
Adachi, K. P333
Adachi, N. 22.0.2, P23, P74,
P179, P276, P286,
P299
Adelson, W. 9.1.4
Adesida, A. P186
Adravanti, P. P337
Aicher, W. K. P83
Aigner, T. P336
Akamine, Y. P158
Akazawa, T. P140
Akgün, U. P115
Akieda, S. P209
Alberca, M. P334
Alblas, J. 25.1.3
Albrecht, C. 17.1.6
Alcaine, C. P91
Alevrogiannis, S. P237, P259
Alibegovic, A. 9.1.3
Alini, M. 9.4.2
Allendorf, S. P58, P60
Almqvist, K. F. 12.1.2, 12.4.2,
17.3.4, P167,
P243, P274, P281
Altadonna, G. 17.1.5, P256
Alvanos, D. P271, P297, P298
Alvarez-Lorenzo, C. P169
Alvarez, F. P308
Alves da Silva, M. P37
Amano, H. P278
Ambrose, C. G. P133
Amiel, D. 25.1.2, P1, P135
Amler, E. 12.3.4
An, Y. P293
Anders, S. 9.2.8, P244
Anderson, D. G. 12.3.2
Ando, K. P97
Ando, W. 3.2.3, 9.1.9, 24.3.3
Andreas, K. P56
Andry, J. P. P142
Angele, P. P198
Anitua, E. 20.1.2, P20
Aoyama, T. P137
Apostolidis, K. 17.4.6
Apprich, S. 17.2.4, 25.2.2
Arai, R. 25.1.7, P306
Arai, Y. P215
Arakaki, K. 9.4.5, P7, P31, P35
Araki, S. P12, P17, P59,
P68, P97
Arbel, R. 2.1.1, 9.4.9, P239
Archer, C. 9.2.1
Arno, S. P49, P50, P302
Authors‘ Index
Arnold, M. P38
Årøen, A. 3.1.1
Ascani, C. P324
Ast, M. P173, P206
Aszodi, A. 9.2.9, 17.4.3
Ateshian, G. A. P142
Athanasiou, K. 2.3.1
Au, A. P174
Augé, II, W. K. P154
Aurich, M. P235
Awad, T. P178
Azofra, J. P20
B
Babyn, P. S. P303
Bach, Jr, B. R. P2
Bader, A. P336
Badke, A. P83
Badlani, N. M. P135
Bajaj, S. 12.2.7, 17.1.7, P103
Balakrishnan, S. 9.2.8
Balboni, F. 25.4.4
Ball, S. T. 25.1.2, P1
Ballis, R. P145, P146
Ballyns, F. P105, P176
Bar-Zvi, S. P228
Bara, J. P161
Barbero, A. 19.3.3, P30, P186
Bardana, D. 25.4.6
Barela, J. A. P294
Barkay, H. P233
Barnouin, L. 17.3.5, 17.3.8, 25.4.3
Barr, L. 9.2.6
Barrachina, J. P15, P202
Barreto, R. B. P6
Barry, F. 19.3.1, P128, P208,
P210
Bartels, W. P160
Bartl, C. 9.3.7
Bartl, R. 9.3.7
Basad, E. 25.1.4
Bassett, E. 12.3.2
Bassit, A. F. P6
Bastiaansen-Jenniskens, Y. M. P127
Bateman, J. F. 11.2.1
Battacharya, M. P37
Battaglia, M. 25.2.6, P266
Bauer, J. S. P149
Baum, P. 12.4.3
Bascı, O. P115
Beaufils, P. P282
Beaulieu, A. 17.4.5
Becher, C. 10.2.2
Beckmann, J. P244
Bédouet, L. P27
Beekhuizen, M. P127
Behrens, P. 17.1.3
Bekkers, J. E. 12.2.8, 17.3.7, P70,
P84, P151
Bell, A. 9.4.4, P58, P60
Bellemans, J. 12.2.4, 12.4.2, P282
Benders, K. E. 25.3.5, P122, P151
Benetti, D. P9
Benetti, V. P9
Benink, R. P92, P94, P104
Bentley, G. 12.2.6, P227, P238,
P241
Berbig, R. 17.3.6
Berger, M. 12.1.3
Berneel, E. P152
Berninger, M. 9.3.2, 12.1.5
269
Bernsen, M. R. P199
Berruto, M. P256
Bessette, L. 17.4.5
Bhullar, T. P65
Biant, L. C. 12.2.6
Bichara, D. A. 12.3.2, P75, P105,
P176
Bielecki, T. 25.4.7
Bijlsma, J. W. 9.3.6
Bird, J. 8.1.2, 17.1.4
Bittencourt, C. 25.4.5
Bittencourt, P. 25.4.5
Bizzi, E. P9, P265
Bizzini, M. 1.0.1
Blagus, R. 9.1.3
Blanke, M. P214
Blasiak, A. 25.4.7
Boatwright, P. P55
Bobic, V. 22.0.1
Bodugoz-Senturk, H. P176
Bonasia, D. 12.3.5, P64
Bonassar, L. 2.3.2, P105, P176
Bonneau, M. P27
Borrás-Cuesta, F. P106
Bos, P. P199
Boux, E. P288
Bovée, J. P144
Braatz, F. P193
Bradica, G. 25.1.6
Brantsing, C. 9.2.7, P21
Brauckhoff, A. P194
Brehm, W. P11, P191
Brenner, O. 12.2.9
Briggs, T. P241
Brinchmann, J. E. P204
Brinkerhuff, H. P34
Brittberg, M. 3.1.2, 9.2.7, 10.4.1,
17.2.2, 18.4.1, P21,
P163
Brix, M. P254
Brochhausen, C. 16.2.1
Brommer, H. P307
Brooks, R. P116
Brune, T. 17.3.5, 17.3.8, 25.4.3
Bruzzone, M. 12.3.5, P64
Brzoska, R. 25.4.7
Buda, R. 9.1.5, 9.3.4, 17.2.8,
25.2.6, 25.2.9, P66,
P216, P218, P266
Bugbee, W. 9.1.2, 9.1.6, 25.1.2,
P1, P217
Bulgheroni, P. 17.3.6
Burnett, B. P131
Bursac, P. P60, P311
Bursig, H. P257, P258, P326
Buschmann, M. D. 9.4.3, 12.4.7, 12.4.8,
15.0.1, 17.1.2, 25.3.8,
25.4.1
Buscio, T. P266
Bussiere, C. 12.4.9
Byers - Kraus, V. 8.2.2
Böttenberg, B. P229
Bünger, C. 9.2.2, 9.4.8, P168,
P170
Bürgi, M. P153
C
Carriel, V. P93, P107
Caballero-Santos, R. 17.3.3, P255, P283
Caborn, D. 9.2.6
Cadet, C. P291
Cadossi, M. 11.1.1
Cairó, J. J. P15
270
Calabrese, G. 17.4.7
Caminal, M. P15
Campbell, J. P103
Campos, A. P93, P107
Cancedda, R. P288
Carey-Smith, R. P231
Carli, A. P54, P285
Carnelli, D. 9.4.6
Carrillo, J. M. P120
Carrington, R. P227, P238, P241
Carter, T. R. P311
Casino, D. P43
Casper, M. E. P165
Castelein, R. P160
Castiglione, E. 25.1.6
Castoldi, F. 12.3.5, P64
Castro, B. P212
Caterson, B. P161
Catt, C. J. 9.2.3
Cavallo, C. 17.2.8, P66
Cavallo, M. 9.1.5, 17.2.8, 25.2.6,
25.2.9, P66, P216,
P218
Chabane, N. P78, P79, P82
Chahine, N. P206
Chaimsky, G. P233
Chan, E. F. 25.3.4
Chan, J. P314
Chang, C. H. P187, P189
Changoor, A. 12.4.8
Chase, D. P1
Chaudhary, M. P49
Chavez, D. P253
Chen, A. C. 25.1.2, 25.3.4, P1
Chen, C. C. P187
Chen, E. P206
Chen, G. 12.4.7
Chen, H. 25.3.8, P285
Chen, S. P101
Chen, W. P62, P138
Cheney, M. 12.3.2
Cheverud, J. 11.2.3
Chevrier, A. 9.4.3, 25.3.8
Chiang, H. 17.2.7, P86, P101
Chiari, C. 25.3.1, P13, P254
Choi, B. P16, P156
Choi, V. P201
Choi, W. P156
Christensen, B. B. P168, P170
Chu, C. 24.2.1, P121, P150
Chubinskaya, S. 25.1.9, P103
Citak, M. P143
Claassen, B. 17.3.7
Claes, T. P240
Clanton, T. O. P133
Clifford, A. G. P295
Codina, D. P15, P308
Cokelaere, S. M. P307
Cole, B. J. 12.2.7, 17.1.7, 17.2.5,
25.1.9, P46, P103,
P316
Coleman, C. P128, P208, P210
Collett, A. 25.3.7
Collin, G. P328
Collo, G. P64
Colombet, P. P282
Concaro, C. P21
Concaro, S. 9.2.7, 10.4.3, P61
Concheiro, A. P169
Condello, V. 2.1.1
Cornelissen, M. P152
Correlo, V. M. P37
Cortes, S. P253
Authors‘ Index
Costa-Pinto, A. P37
Coste, P. P291
Cottino, U. 12.3.5
Couceiro, J. 19.2.1, P169
Coutts, R. D. P135
Cranchi, C. P143
Crane, T. P328
Crawford, D. C. P236
Crawford, R. 9.2.5
Creemers, L. 12.2.8, 17.3.7, P70,
P84, P127, P151, P160
Crespo, R. 17.3.6
Cronin, M. P310
Cugat, R. P120, P282
Cui, X. P275
Curtin, C. P208
Cáceres Palou, E. P3, P264
D Domayer, S. 12.3-9, 12.4.4, 25.2.4,
D’Arcy, S. P210
D’Lima, D. D. P126
D’Orazio, L. 17.1.5, 19.0.2, 25.1.8,
25.4.4
Da Fonseca, C. F. P15
Dalemans, W. 8.3.1
Daley, E. 12.2.7, 17.1.7
Dalmau, A. P308
Daniels, D. P38
Danielson, B. 25.2.7
Davidson, P. A. 18.3.2, P325
Davies, H. P231
Davies, J. P139
De Biase, C. F. 12.3.8
De Boer, T. N. 9.3.6
DeCroos, J. A. P147
DeMuro, C. P316
De Neve, F. P274
De Palma, C. P145, P146
DeYoung, A. 9.1.2, 9.1.6, P217
Dec, J. P326
Declercq, H. P152
Dediu, V. P43
Deforce, D. 12.1.2
Dehne, T. P21
Deie, M. P23, P74, P276, P299
Dekker, E. 12.1.6
Delattre, O. P26
Delcogliano, A. 12.3.8
Delcogliano, M. 9.3.4, 12.3.8
Della Villa, S. 1.0.1
Delling, U. P191
Deplaine, H. P177
Deponti, D. P145, P146
DesJardins, J. P55
Desando, G. P66, P218
Desnoyers, J. 12.4.7, 12.4.8, 17.1.2,
25.4.1
Devlin, S. 25.4.6
Devreese, K. P76
Dexheimer, V. P193
de Girolamo, L. P337
de Grauw, J. C. 25.3.5
del Olmo, M. P212
Dhert, W. 9.2.3, 12.2.8, 16.2.3,
17.3.7, 25.1.3, 25.3.5,
P70, P84, P122, P127,
P151, P160
Dhillon, M. 10.1.3, 17.1.4, P328
Dhollander, A. A. P76, P243, P274
Di Benedetto, P. P143
Di Caprio, F. 25.2.9
Di Giancamillo, A. P145, P146
Di Martino, A. 9.3.4, 17.1.5, 19.0.2,
25.1.8, 25.4.4, P256,
P305, P327
Di Matteo, B. 19.0.2, 25.1.8, P305,
P327
DiMicco, M. P63
Diaz-Romero, J. P144
Dickhut, A. P196
Dickinson, S. C. P72, P180, P192
Diederichs, S. P197
Dienstknecht, T. P198
Djian, P. P282
Dijkstra, P. P100
Dionigi, C. P43
Dirhold, B. P96
Dishkin-Paset, J. P46
Doblaré, M. P48, P177
Docheva, D. P69
P220, P254
Domeneghini, C. P145, P146
Dono, D. P77
Dorais, M. 17.4.5
Doria, A. S. P303
Dorotka, R. 12.3-9, 25.2.4, P13,
P14, P220, P254
Dos Santos, G. B. P6
Dotor, J. P106, P119
Dougados, M. P291
Doukas, S. 9.2.8
Dragomir, L. 25.3.8
Drobnic, M. 9.1.3, 19.1.1, P249
Drosse, I. 9.2.9
Dubrana, F. 12.4.9
Dubruel, P. P152
Dueren, F. 17.2.9
Dugas, J. P311
Duguay, S. J. P63, P77
Duncan, S. P296
Dvorak, J. 1.0.1, 5.0.2
E
Ebert, J. 12.2.2, P225, P231
El Attar, M. P281
El Mansouri, F. P78, P79, P82
Elewaut, D. 12.1.2, P76
Eliaz, N. P41
Elisseeff, J. 9.4.7, 12.3.1, 16.2.2,
P95
Eliyahu, E. P88
Ellermeijer, B. P92, P94
Ellä, V. P25
Elsner, J. 2.1.1, 9.4.9, 12.2.9,
P41
Emons, J. 12.1.6
Endres, M. P56, P175
Engelhart, L. 17.2.5, P316
Erggelet, C. 3.1.3, 15.0.2, P317
Ergün, S. P115
Erquicia, J. P3, P264
Escribano, R. P119
Eshed, I. P239
Esparza, R. 12.1.9
Espírito Santo, V. P166
Eyre, D. R. 8.2.1
F
Facchini, A. P66
Fahmi, H. P78, P79, P82
Fallon, M. 12.2.2, P225, P231
Faltus, R. 12.4.6, P257
Fang, H. W. P187, P189

Faria, S. P37
Farr, J. 9.1.7, 9.1.8, 16.1.1,
17.2.5, P311, P316
Farrell, E. P199
Fedorovich, N. E. 25.1.3
Fehr, M. P165
Feijen, J. P100
Feist, M. P232
Feller, J. 17.2.3
Ferkel, R. P221
Fernandes Dias, M. 25.4.5
Fernandez-Jaen, T. 17.3.3, P255, P283
Fernández, A. G. P212
Filardo, G. 9.3.4, 17.1.5, 19.0.2,
25.1.8, 25.1.9, 25.4.4,
P103, P256, P266,
P305, P327
Filova, E. 12.3.4
Fischer, J. P194, P196
Fisher, J. P44, P52, P139
Fitzsimmons, J. S. P165
Fiz, N. P20
Flanigan, D. C. P311
Flannery, C. R. 3.2.1
Flory, C. 25.3.7
Foldager, C. 9.2.2, 9.4.8, P168,
P170
Fontana, A. 24.1.1, P267
Foo, L. F. 12.3.7
Forman, R. P49
Forriol Campos, F. 12.1.9, 19.2.4
Forsten, M. P25
Forster, M. P310
Forsyth, R. 25.4.9
Fortier, L. A. 11.2.2, 18.3.1, 23.0.1,
25.1.6
Fraga, A. F. P164
Francin, P. 17.4.4
Franco, G. P253
Frankewycz, B. P69
Fraschini, G. P145, P146
Freire, M. F. 12.3.3
Freymann, U. P175
Frias, A. M. P195
Friederich, N. F. P38
Friedman, A. P233
Friedrich, K. P331
Friel, N. A. P103
Frisbie, D. D. P183
Fritz, J. P250
Fujie, H. 9.1.9, 24.3.3, P333
Fujimura, S. P137
Fujioka, H. P172
Fujita, N. 9.3.8, P89
Fukuhara, K. P286
Furu, M. P137
G Henson, F. M. 9.2.6, P65, P116
Garrido, J. P107
Garzón, I. P93, P107
Gahunia, H. K. P303
Gaissmaier, C. P250
Gallagher, K. P241
Gallego Ferrer, G. P177
Ganey, T. P19
Ganguly, K. P154
Garcia-Campillo, H. P5
Garcia-Gomez, F. 17.3.3, P255, P283
Garcia, Z. P5, P28
García-Aznar, J. P48, P177
García Arnás, F. P202
García, F. P15
Authors‘ Index
García, J. P15, P202
García Sancho, J. P334
Garett, S. P231
Garnica, M. I. P28
Gartzia, I. P212
Gatenholm, P. P163
Gavenis, K. P45
Gaździk, T. S. P326
Ge, Y. P88
Geddes, R. G. P318
Geißler, C. P191
Gelber, J. P126
Gelber, P. E. P219
Gellissen, J. 17.1.3
Gelse, K. P214
Gentili, C. P288
Georgi, N. P81
Georgoulis, A. P315
Gerner, C. 12.1.3
Gersoff, W. 20.2.1
Getgood, A. 9.2.6, 25.1.1, P65,
P116
Ghermandi, R. 9.1.5, 17.2.8
Ghodadra, N. P2
Ghorayeb, S. R. P178
Giacomi, R. 12.3.8
Giannini, S. 9.1.5, 9.3.4, 17.2.8,
25.2.6, 25.2.9, P66,
P216, P218, P266
Giannoni, P. P102
Gibson, M. P95
Gigante, A. 18.1.3
Gill, T. J. P75, P105
Gille, J. 17.1.3
Gillet, P. P26
Giza, E. 18.1.2
Glaser, C. P232
Glassner, P. P49
Goad, E. 25.3.7
Gobbi, A. W. 16.1.2, 18.4.2
Goed, S. 12.3-9, P330, P331
Goldberg, R. P228
Goldring, M. 9.3.1
Goldstein, M. J. 9.3.5, P206
Golub, L. 9.3.5
Gomes, M. E. P166
Gomez-Garcia, R. P5
Gomez, R. P335
Gong, J. P. 9.4.5, P7, P31, P35,
P38, P113
Gonzalez-Lucena, G. P219
Gonzalez, G. P5
Goodrich, L. P201
Goodwin, P. M. P154
Goranov, V. P43
Gorensek, M. P249
Gosselin, Y. P285
Gottardi, R. 9.4.6
Gotterbarm, T. P182
Goyal, A. D. 25.4.8
Goyal, D. 12.4.1, 25.4.8
Grad, S. 9.4.2
Gramani-Say, K. P294, P320
Granata, M. P265
Grande, D. 9.3.5, P173, P178,
P206
Greenwald, R. 9.3.5
Griffin, D. R. 24.1.2
Grifka, J. 9.2.8, P244
Grigolo, B. 9.1.5, 17.2.8, 25.2.9,
P66, P216, P218
Grishko, V. 17.4.2, P109
Grodzinsky, A. 16.3.1
271
Groeneboer, S. P76
Grouin, J. P291
Gruber, M. P13, P14
Grumet, R. P103
Guadilla, J. P20
Guehring, H. P116
Guevara, V. P253
Guglietta, A. P212
Guilak, F. 2.1.1, 9.4.9, 12.2.9,
P51
Guillaume, C. 17.4.4
Guillen-Vicente, I. 17.3.3, 20.1.1, P255,
P283
Guillen-Vicente, M. 17.3.3, 20.1.1, 20.1.3,
P255, P283
Guillen, P. 17.3.3, 20.1.1, P255,
P283
Gusinde, J. P214
Guzman-Morales, J. P54, P57
Gòdia, F. P15
Gómez-Ribelles, J. L. P177
Görtz, S. 9.1.2, 9.1.6, 17.3.1,
19.1.2, 25.1.2, P1,
P217
H
Ha, C. 9.3.9, 25.4.2, P289
Hadlock, T. 12.3.2
Haeupl, T. P56
Hagmann, S. P182
Hakimiyan, A. A. 25.1.9, P103
Halbwirth, F. 12.1.3, 15.0.3, P159
Haleem, A. M. P150
Hambly, K. 12.2.1
Hangody, L. 10.3.1
Hanifi, A. P268
Hanypsiak, B. T. 17.4.7
Harris, A. P54
Harrison, P. P245
Hart, D. A. 9.1.9
Hashimoto, S. 11.2.3
Haskell, M. 25.3.7
Haspl, M. P72
Hattori, K. P322
Haudek, V. 12.1.3
Haupt, O. P30
Hayakawa, K. P137
Hayes, D. A. P295
Hazewinkel, H. A. P132
He, X. P88
Healey, R. P1, P135
Hedrick, M. P19
Heerwaarden, R. V. P160
Helder, M. 17.3.9, P53
Henderson, J. P54
Hennig, F. 12.4.4, P214
Hepp, P. P336
Herlofsen, S. R. P204
Hermida, E. 17.2.6
Hermida, L. F. 17.2.6
Herrenbruck, T. 17.4.7
Herrero, A. P212
Hershman, E. 2.1.1, 12.2.9
Himeda, Y. P184
Hinterwimmer, S. P149
Hirakura, Y. P12
Hiraoka, N. P215
Hirschfeld, C. P220
Hirschmüller, A. P317
Hoch, J. M. 12.1.8, 12.2.3, 12.2.5,
12.4.5
272
Hoechsmann, N. 17.2.9
Hoemann, C. D. 9.4.1, 9.4.3, 9.4.4,
12.4.7, 16.1.3, 17.1.2,
25.3.8, P54, P57,
P60, P285
Hoeppner, J. P112
Hofmann, G. O. P235
Hogendoorn, P. P144
Hollander, A. P. 5.0.1, 10.4.4, 17.3.7,
P180, P192
Holsten, D. G. 17.3.6
Hong, J. P329
Hong, S. P275
Honjo, K. P215
Honold, S. P39
Horibe, S. 25.2.8
Horng, A. P232
Hornung, D. P330
Hoshi, K. 7.2, P80
Hosiner, S. 17.1.6
Hou, S. P248
Howard, J. S. 12.1.8, 12.2.3, 12.2.5,
12.4.5
Hoyle, M. P55
Hsieh, C. P86, P101
Hsu, H. P111, P171
Hsu, Y. P189
Hu, J. 2.3.1
Huang, C. C. P148
Huang, Y. 17.2.7
Hudetz, D. P72
Hui, J. H. 7.1, 25.3.9, P190
Huisman, A. M. 9.3.6
Hung, C. T. P142
Hunziker, E. 9.1.1, 9.2.9, P184
Hurtig, M. B. 9.4.4, 24.3.1, 25.3.3,
25.3.8, 25.4.6, P58,
P60, P129, P285
Hussam, A. 18.5.2
Hutmacher, D. 9.2.5, 25.3.5, P122
Hutton, W. P19
Huysse, W. C. 25.4.9, P279
Hwang, J. H. P71
Hwang, N. 12.3.2
I Kramer, J. P165
Ibarra, C. P5, P28, P253, P335
Ibarra, L. P253
Igarashi, T. P134, P140
Imabuchi, R. P113
Imai, S. P12, P17, P59, P67,
P68, P97, P98, P114
Imhoff, A. 9.3.2, 9.3.7, 12.1.5,
P149, P234
Inagaki, Y. P322
Ingham, E. P44, P139
Inoue, H. P108, P215
Inoue, J. P129
Intema, F. P132
Inui, A. P172, P185
Iosifidis, M. I. 17.4.6, P271, P297,
P298
Ishaque, B. 25.1.4
Ishida, K. 9.3.8, P89
Ishii, T. 11.3.1
Ishimoto, Y. P322
Isoya, E. P98
Ito, A. P108
Ito, K. P137
Itoman, M. 25.3.6, P10
Ivkovic, A. P72
Iwamoto, Y. P209
Iwasaki, N. P134, P140
Authors‘ Index
Izaguirre, A. P5, P253
Izaguirre, F. P5
Izal Azcárate, I. P91, P106, P119
J
Jacobi, M. 17.3.2, 17.4.8, P301
Jacques, P. P76
Jaiswal, P. P241
Jakob, M. P186
Jakob, R. 17.3.2, 17.4.8, P301
Janes, G. P231
Jansen, N. W. 9.3.3
Jansson, V. P69, P232
Jarry, C. 12.4.7
Jelic, M. 19.1.3, P72
Jenei-Lanzl, Z. P198
Jeng, L. P111
Jia, H. P180
Jiang, C. 17.2.7, P86, P101
Jin, L. P275, P293
Jin, R. P100
Jin, Y. P137
Jin, Z. P44, P52, P139
Jo, S. P226, P309
Joh, K. P171
John, D. P50
Johnson, L. L. P126
Johnson, W. E. P161
Johnstone, B. 19.3.2
Joshy, S. P310
Josten, C. P336
Junge, A. 1.0.1
Juras, V. 25.2.4
Jurvelin, J. 25.3.2, P262
Järvinen, E. P25
Jülke, H. P191
K Konen, E. P239
Kafienah, W. P180, P192
Kakavas, G. P259
Kaleva, E. P262
Kalwitz, G. P56
Kamei, G. P74, P179
Kanaji, T. P137
Kanamoto, T. 9.1.9, P158
Kanaya, F. P31, P35
Kanazawa, S. P80
Kandel, R. 16.3.2, P147
Kaneko, H. P172
Kannan, K. 25.3.9
Kaplan, L. D. P148
Kaps, C. P56, P165, P175
Karahan, M. P115
Karperien, M. 12.1.6, P81, P100
Kasahara, T. P137
Kasahara, Y. P134, P140
Kautzky-Willer, A. 12.4.4
Kawaguchi, Y. M. P320
Kawamura, D. P134, P140
Kawcak, C. P183
Kellomäki, M. P25
Kendoff, D. P143
Kennedy, J. G. P287
Kepski, F. P326
Kercher, J. 9.1.7, 12.2.7, 17.1.7,
P46
Kerloch, I. P291
Kerr, A. P318
Khelmenstkaya, E. P302
Kik, M. 25.3.5
Kili, S. P65
Kim, B. P16
Kim, H. P275
Kim, J. P284
Kim, M. P29
Kim, S. P226
Kim, Y. 24.1.3, P29, P156,
P293, P314
Kinne, R. W. P133, P188
Kirk, S. 25.1.9
Kirkpatrick, C. J. 16.2.1
Kisiday, J. P183, P201
Kita, K. 3.2.3, 24.3.3
Kitamura, H. P12
Kitamura, N. 9.4.5, P7, P31, P35,
P113
Kiviranta, I. 19.0.1, 25.3.2, P25,
P262
Klabunde, R. P153
Klein, T. 9.2.5, 25.3.5, P122
Kleinschmidt, K. P11
Knott, L. P124
Knutsen, G. 3.3.1
Kobayashi-Miura, M. P108
Kobayashi, M. 25.1.7, P108, P306
Kobayashi, T. P74
Kobayashi, K. P137
Kokubu, T. P172, P185
Kocaoğlu, B. P115
Koch, C. P24
Koczy, B. P257
Koh, Y. P226, P277, P280,
P309
Kohda, H. P158
Kohn, L. P149
Kon, E. 9.3.4, 10.4.2, 17.1.5,
18.2.5, 18.4.3, 19.0.2,
25.1.8, 25.4.4, P256,
P305, P327
Kondo, E. P7
Kordelle, J. P130
Koritnik, B. P249
Koroglu, A. 25.1.5
Kotek, G. P199
Koulalis, D. P143
Krestin, G. P. P199
Kreuz, P. P317
Kristiansen, A. P168, P170
Kröger, H. P262
Kubo, M. P12, P59, P68, P230
Kubo, S. 9.3.8, P89
Kubo, T. P135, P215
Kujat, R. P198
Kulig, K. M. 12.3.2
Kumagai, K. P98
Kumar, K. P245
Kunz, M. 25.4.6, P129
Kuo, L. P50
Kuroda, R. 9.3.8, P89
Kuroda, S. P158
Kurokawa, T. 9.4.5, P7, P31, P35,
P38, P113
Kuroki, H. P108
Kurosaka, M. 9.3.8, P89, P172,
P185
Kurowska, E. P242, P313
Kurz, A. 25.1.9
Kusano, T. 17.4.8
Kwack, K. P269, P275, P293,
P329
Kwak, S. K. P221
Kwiatkowski, G. 12.4.6
Kwon, H. P113

Kyriakidis, A. 17.4.6, P271, P297,
P298
Kyriakidis, T. 17.4.6
Küchler, A. M. P204
L Luginbuehl, R. P39
Lobo, M. P93
La Cava, G. P260
Labarre, D. P27
Ladenburger, A. P45
Lafantaisie Favreau, C. P54, P57
Lafeber, F. 9.3.3, 9.3.6, 11.3.2,
P8, P132
Lagae, K. 17.3.6
Laganier, L. 25.4.3
Lambrecht, S. 12.1.2, P76
Lammi, M. 25.3.2
Landa, C. P335
Landini, E. 9.4.6
Landwehr, S. P149
Langer, R. 12.3.2
Laprell, H. 18.2.4, P282
Lascau-Coman, V. 9.4.4, 12.4.7, 25.3.8
Lattermann, C. 12.1.8, 12.2.3, 12.2.5,
12.4.5, P296
Laurent, A. P27
Le, D. P168, P170
Leander, M. P21
Lechler, P. 9.2.8
Lecona, H. P5
Lee, D. 11.1.2
Lee, E. H. 25.3.9, P71
Lee, H. H. P150
Lee, J. P71, P71
Lee, S. P312
Lee, W. P86
Lee, Y. P269, P275, P284
Lehman, R. C. 25.1.6, P325
Lehmann, S. P232
Leijten, J. 12.1.6, P81, P100
Lequesne, M. P291
Lessi, G. C. P294, P320
Levy, R. P131
Lewis, P. B. 12.2.7
Lewis, S. P316
Li, K. 17.4.9, P4
Liakos, T. 17.4.6, P271, P297,
P298
Liao, C. 17.2.7, P187, P189
Lichtenberg, D. P11
Lickorish, D. 25.3.7
Lin, S. P62, P138
Lin, Z. P90
Lind, M. 9.2.2, 9.4.8, 17.2.3,
P168, P170
Lindahl, A. 9.2.7, 17.2.2, 25.2.7,
P21, P61, P163
Linder-Ganz, E. 2.1.1, 9.4.9, 12.2.9,
P41, P51
Linss, S. P188
Litau, L. P175
Little, C. B. 24.3.2
Liu, I. 25.3.4
Lizhang, J. P52
Ljungberg, M. 25.2.7
Lloyd, D. G. 12.2.2
Lohmander, S. L. 8.2.3
London, N. J. P295
Loosli, Y. P39
Loparic, M. P72
Lopez-Alcorocho, J. 17.3.3, 19.2.3, P255,
P283
Lopez-Olivia, F. 12.4.7, 12.4.8, 17.1.2,
Authors‘ Index 273
25.4.1 Martins, A. P37
Lopez, E. P26 Martins, C. P150
Lowe, J. P233 Martinčič, D. P249
Lowerison, M. 9.4.4, P58, P60 Marty, M. P291
Ludewig, E. P191 Maruyama, T. P137
Mascaro, G. P288
Lukasik, P. P258 Masri, M. P335
Luksch, T. 12.3-9 Massafra, U. P9, P265
Luna-Barcenas, G. P5, P28 Mastbergen, S. 9.3.6, P8, P132
Luyten, F. 12.2.4, 12.4.2 Maticic, D. P72
Lynn, A. 10.3.1, P116 Matsuda, S. P209
Lytvynets, A. 12.3.4 Matsumoto, T. 9.3.8, P89
Lyu, S. P261 Matsuno, T. P87
Matsushita, T. 9.3.8, P89
M Matsusue, Y. P12, P17, P59, P67,
P68, P97, P98, P114,
Martínez, C. P107 P230
Morales, A. P93 Matsuura, H. P98
MacDonald, P. 12.4.7, 12.4.8, 17.1.2, Mattacola, C. G. 12.1.8, 12.2.3, 12.2.5,
25.4.1 12.4.5
MacMull, S. 12.2.6 Matthews, G. P63
Macule, F. 12.4.7, 12.4.8, 17.1.2, Mattielo-Rosa, S. M. P294, P320
25.4.1 Matuska, A. P112
Madry, H. 18.4.4, 22.0.3 Mauerer, A. 25.2.2, 25.2.3
Mae, T. 9.1.9, P158, P278 Mauthe von Degerfeld, M. 12.3.5
Maher, S. A. P171 May, D. P311
Maheu, E. P291 Mayer, S. 17.2.9
Maiello, A. P64 Mazières, B. P291
Mainard, D. 17.4.4 McArthur, S. P46
Mainil-Varlet, P. 17.3.2, P144 McCall, I. W. P245
Makino, K. P172 McCarrel, T. M. 25.1.6
Makino, T. P185 McCarthy, H. S. P245, P251
Malda, J. 9.2.3, 9.2.4, 16.2.3, McCormack, R. 12.4.7, 12.4.8, 17.1.2,
25.1.3, 25.3.5, P122, 25.4.1
P151, P307 McDonough, M. P55
Malinowska, P. P326 McGill, K. P2
Malo, M. 12.4.7, 12.4.8, 17.1.2, McIlwraith, W. 12.1.1, P183, P201
25.4.1 McKee, M. D. 25.3.8
Mamisch, T. C. 12.3-9, 12.4.4, 25.2.2, McKeon, B. 25.2.7
25.2.3, 25.2.4, P314,
P330 McLeod, L. D. 17.2.5
Mandel, A. P201 McRury, I. D. P154
Mandelbaum, B. 1.0.2, 9.3.4 Mcclelland, J. 17.2.3
Mangiavini, L. P145 Mcnamara, I. P65
Manili, M. 12.3.8 Medeiros, A. I. P294
Mano, J. F. P166 Mehlhorn, A. T. P96
Mansouri, R. P323 Meininger, A. 25.1.9
Maor, G. P228 Meisel, H. P19
Maqsood, M. P323 Melas, I. 17.4.6, P271, P297,
P298
Marcacci, M. 9.3.4, 17.1.5, 17.3.6,
19.0.2, 25.1.8, 25.4.4, Melato, M. P146
P43, P256, P305, Melcher, C. 17.2.9
P327 Memisoglu, K. 25.1.5
Marchand, C. 12.4.7 Messner, A. 17.2.4, P331
Mardones, R. M. 17.2.1 Metaxiotis, D. P298
Marks, P. 12.4.7, 12.4.8, 17.1.2, Methot, S. 12.4.7, 12.4.8
25.3.3, 25.4.1, P58 Mezape, Y. P41, P51
Marlovits, S. 12.4.4, 17.1.6, 18.1.1, Midura, R. 12.3.3
25.2.1, 25.2.2, 25.2.3, Migliore, A. P9, P265
25.2.5 Mika, J. P133, P317
Marmotti, A. 12.3.5, P64 Mimnaugh, B. P34
Marotta, D. 12.3.8 Mimura, T. P12, P59, P68
Marquass, B. P336 Min, B. P16, P29, P156, P269,
Marques, R. F. P164 P275, P284, P293, P329
Martel-Pelletier, J. 17.4.5, P78, P79, P82 Minami, A. P134, P140
Marti, C. 17.4.8 Minas, T. 17.1.1, 20.2.2
Martin, C. 17.3.5, 17.3.8 Minger, S. P180
Martin, I. 19.3.3, P30, P186 Minh, D. P16
Martin, S. P265 Miquel, J. P3, P264
Martinez-Lopez, V. P28 Mithoefer, K. 1.0.3, P316
Martinez, I. 12.1.4 Mitsui, H. P137
Martinez, V. P253 Mohd Hassan, A. 25.3.9
Martinez de Albornoz, P. 12.1.9 Mohtadi, N. 12.4.7, 12.4.8, 17.1.2,
Martinova, L. 12.3.4 25.4.1
274
Moine, L. P27
Moll, X. P15
Mollenhauer, J. P235
Monllau, J. C. 17.3.6, P3, P219,
P264
Monreal, I. P106
Montaperto, C. 25.4.4
Montes, M. P212
Monti, C. 25.2.6
Mora, G. P91, P106, P119
Moradi, B. P182
Morawietz, L. P175
Mordin, M. M. 17.2.5, P316
Moreira Teixeira, L. P100
Morera, C. P334
Morgan, R. E. P154
Mori, K. P97
Morin, F. 17.4.5
Morist, A. P15
Moseley, B. 3.3.2
Motlík, J. 12.3.4
Moya-Angeler, J. 12.1.9
Moyse, D. P291
Mroczka, A. 12.4.6
Muezzinoglu, B. 25.1.5
Muezzinoglu, S. U. 25.1.5
Muhonen, V. P25
Mukai, S. 25.1.7
Mullender, M. 17.3.9, P53
Munir, S. 9.4.8
Murakibhavi, V. P245
Murata, M. P140
Muratoglu, O. K. P176
Murawski, C. D. P287
Murphy, M. P128, P210
Muruzabal, F. P20
Méthot, S. 17.1.2
Míčková, A. 12.3.4
Möller, I. P292
Müller-Rath, R. P45
Müller, P. E. 17.2.9, P69, P232
Müller, S. P193, P317
N Ohgushi, H. P322
Nagata, Y. P179
Nagura, I. P172, P185
Nakagawa, S. P215
Nakagawa, Y. 25.1.7, P108, P230,
P306
Nakamae, A. P299
Nakamura, N. 3.2.3, 9.1.9, 24.3.3,
P158, P333
Nakamura, S. P306
Nakamura, T. 25.1.7, P108, P137,
P306
Nakasa, T. P23, P286, P299
Nakata, K. 3.2.3, 9.1.9, 24.3.3,
P158, P278
Nakayama, K. P209
Nanni, M. P216
Nannmark, U. P163
Nansai, R. 9.1.9, P333
Narcisi, R. P102
Naruse, K. 25.3.6, P10
Nawab, A. P311
Nawaz, S. P241
Nebelung, S. P45
Negrin, L. 25.2.4
Nehrer, S. 12.1.3, 12.3-9, 15.0.3,
P13, P14, P159, P254
Nejadnik, H. P190
Nelea, M. 12.4.8
Authors‘ Index
Nelson, L. 17.2.5
Neophytou, D. 17.4.6, P271, P297,
P298
Neri, S. 9.1.5, P218
Nesic, D. 17.3.2, P144
Netter, P. P26
Neumann, K. P56, P175
Neves, N. M. P37, P195
Neyret, P. P282
Ng, K. W. P171
Nguyen, V. P27
Nicolàs, M. P212
Niculescu-Morzsa, E. 12.1.3, P159
Nielsen, A. 9.4.8
Niemeyer, P. P96
Nierenberg, G. P228, P239
Niethammer, T. P232
Niimoto, T. P23, P299
Nishimoto, H. P185
Nishitani, K. P108
Nishizawa, K. P17, P59, P68, P97
Nishizawa, S. P80
Nishuzawa, K. P12
Niu, C. P62, P138
Nochi, H. P87
Noda, K. P12
Noehren, B. P296
Notbohm, H. P24
Nuran, R. P115
Nygaard, J. V. P168, P170
Nöth, U. 17.1.8
O Pittsley, A. P126
O’Driscoll, S. P165
O’Loughlin, P. F. P143
O’Sullivan, J. P128
Ochi, M. 11.3.3, P23, P74,
P179, P276, P286,
P299
Ochoa, I. P48, P91, P177
Ode, G. E. P46
Ogawa, M. 9.4.5, P322
Oh, K. P284
Ohkawa, S. P74, P179
Ohmiya, Y. P113
Okuhara, A. P23, P276, P299
Okumura, N. P98
Olk, A. P214
Onodera, S. 9.4.5, P7
Orive, G. P20
Orozco, L. P202, P334
Oshima, Y. P135
Ota, K. P158
Otsuka, T. P137
Ouyang, W. 25.3.8
P Pérez, F. P253
Paape, D. P175
Paatela, T. P262
Padera, R. F. P111
Paessler, H. 12.4.3, P282
Pagenstert, I. P232
Pallante, A. L. 25.1.2, P1
Pallu, S. 17.4.4
Panseri, S. P43
Papantoniou, E. P298
Park, D. P241
Park, S. P16, P29, P156
Parker, J. C. P245, P251
Parker, R. 17.4.7
Pascale, F. P27
Pascher, A. P72
Patella, S. 9.3.4, 17.1.5, 19.0.2,
25.1.8, 25.4.4, P256,
P305, P327
Paternostro-Sluga, T. P330, P331
Patsch, I. 24.2.2
Paul, C. 17.3.9, P53
Paul, S. P165
Payne, K. A. P121, P150
Pearle, A. P143
Pearsall, IV, A. W. 17.4.2, P109
Pecina, M. P72
Pedersen, M. 9.2.2
Pelet, S. 12.4.7, 12.4.8, 17.1.2,
25.4.1
Pelfort, X. P3, P219, P264
Pelletier, J. 17.4.5, P78, P79, P82
Pelsis, J. R. 9.1.4
Pereira, R. C. P195
Peretti, G. M. P145, P146
Peris, D. P15
Peterson, L. 17.2.2, 25.2.7, P61
Petrigliano, F. A. 12.3.7
Peyrone, B. 12.3.5
Pezzillo, F. 12.3.8
Pflueger, V. P331
Pietschmann, M. F. P69, P232
Pillai, L. P131
Pineda, A. P335
Pineda, C. P5, P253
Pinto, A. R. P195
Pla, A. P15
Please, C. P. 9.2.3
Pleshko, N. P268
Plummer, O. 18.5.1
Polacek, M. 12.1.4
Polak, A. A. 9.3.6
Polzella, M. P9
Pomerantseva, I. 12.3.2
Portnoy, S. P51
Potel, J. 12.4.9
Potter, H. G. 12.3.7
Pottie, P. 17.4.4
Pozzi, A. P145, P146
Prades, M. 12.1.7, P222, P223,
P224
Prado, R. P20
Prendergast, P. 11.1.3
Presle, N. 17.4.4
Pretzel, D. P188
Prins, H. 25.1.3
Pronk-Admiraal, C. P92, P94, P104
Prosecká, E. 12.3.4
Provencher, M. T. P2
Puhakka, J. P262
Pulkkinen, H. J. 25.3.2
Q
Qi, W. P125
Quarto, R. P102
R
Rabanal, R. P15
Radosavljevič, D. P249
Raducanu, A. 9.2.9
Rai, M. 11.2.3
Raiteri, R. 9.4.6
Rampichová, M. 12.3.4

Randolph, M. A. 12.3.2, P75, P105,
P176
Rapko, S. P77
Rappold, G. 12.1.6
Rappoport, L. 25.1.9
Raynauld, J. 17.4.5
Razzano, P. 9.3.5, P173, P178,
P206
Realmuto, C. 12.3.5, P64
Reddi, H. A. 2.2.1
Redondo, J. I. P120
Regatte, R. P49, P302
Reiff, R. P294
Reinholz, G. G. P165
Reis, R. L. P37, P166, P195
Ren, L. P136
Renger, J. P24
Renkawitz, T. 9.2.8
Responte, D. 2.3.1
Restrepo, A. 12.4.7, 12.4.8, 17.1.2,
25.4.1
Rey-Rico, A. P169
Rezende, M. U. P6
Ribera, T. P222, P224
Ribitsch, I. P191
Richardson, J. P245, P251, P268,
P318
Richardson, R. P318
Richmond, B. 17.4.7
Richter, R. P336
Richter, W. 7.3, 10.2.1, P11, P182,
P193, P194, P196,
P197
Rickert, M. P196
Ridgway, K. P180
Riek, J. K. P22
Riesle, J. P186
Rijen, M. P70
Ringe, J. P21
Ringgard, S. 9.2.2
Ripalda Cemboráin, P. P91, P106, P119
Ristiluoma, M. 17.4.3
Rivard, G. P57
Robert, H. 12.4.9
Roberts, S. P161, P245, P251,
P268
Robertson, A. P310
Robertson, W. B. 12.2.2, P225
Robinson, E. P245, P251
Rochler, S. P188
Rodeo, S. 12.3.7
Rodkey, W. G. P183
Rodrigo, J. P55
Rodriguez-Iñigo, E. 17.3.3, P255, P283
Roessing, S. 12.4.3
Roessler, N. P14
Rohwedel, J. P165
Rolauffs, B. P83, P235
Romeo, A. A. P2
Romeo, S. P144
Ronken, S. P38
Roosendaal, G. 9.3.3
Rossi, P. 12.3.5, P64
Rossi, R. 12.3.5, P64
Rossomacha, E. 12.4.7, 17.1.2
Rothdiener, M. P83
Rothrock, C. 17.4.7
Rousseau, M. P26
Royen van, B. 17.3.9, P53
Rudan, J. 25.4.6
Ruffilli, A. 9.1.5, 17.2.8, 25.2.6,
25.2.9, P216, P218
Ruike, T. P87
Authors‘ Index 275
Rushton, N. 9.2.6, P116 Semler, E. 25.3.4
Russlies, M. P165 Sen, R. P263
Russo, A. P43 Sengers, B. 9.2.3
Ruta, D. 25.1.9 Seo, H. P312
Rutgers, M. P160 Serra, C. P120
Serrão, P. R. P294, P320
S Servien, E. 12.4.9
Sgaglione, N. A. 2.1.2
Saarakkala, S. 25.3.2, P262 Shah, N. V. 9.3.5, P206
Sachot, S. P88 Shahid, R. P323
Sadigursky, D. P6 Shanbhogue, K. P263
Sadlik, B. 25.4.7 Shani, J. 12.2.9
Sague Doimeadios, J. L. P39 Sharon, M. P239
Sah, R. L. 2.3.3, 19.0.3, 25.1.2, Shasha, N. P51
25.3.4, P1 Shaw, D. P139
Sahlman, J. P262 Shelyakova, T. P43
Saito, M. P215 Shen, C. 17.2.7
Sakamoto, F. A. 12.3.3 Shewman, E. P46
Sakata, R. P172, P185 Shibuya, H. P23, P74, P179, P286,
Salanti, G. P315 P299
Salata, M. 9.1.7, 17.1.7 Shieh, C. 17.2.7
Salzmann, G. M. 9.3.2, 9.3.7, 9.4.2, Shikano, M. 8.3.2
12.1.5, P149 Shimizu, T. 25.2.8
Samulski, J. P201 Shimomura, K. 9.1.9, 24.3.3, P158
Samuoh, L. P13 Shimoto, T. P209
Sanchez Hidalgo, R. 12.1.9 Shino, K. 9.1.9, 24.3.3, P278
Sanchez, M. 19.2.2, P20 Shintani, N. P184
Sandell, L. J. 11.2.3 Shioji, S. P59, P97
Sandhu, M. P263 Shiozaki, Y. 25.2.8
Sandri, M. P43 Shirai, T. P108
Santander, S. P91 Shive, M. S. 12.4.7, 12.4.8, 17.1.2,
Santoro, R. 19.3.3 25.3.8, 25.4.1
Santos, E. 17.3.3, P255, P283 Shterling, A. 2.1.1, 9.4.9, 12.2.9,
Santos, J. L. P164 P41, P51
Sanz-Ramos, P. P91, P119 Siclari, A. P288
Saris, D. B. 3.3.3, 10.1.1, 10.3.2, Sievers, B. 17.2.9
12.2.4, 12.2.8, 12.4.2, Silva, M. P169
17.3.7, 20.3.2, 23.0.2, Simon, T. 25.3.4
25.3.5, P70, P84,
P122, P127, P151, Simonaro, C. M. P88
Sims, T. J. P180
P160
Sasaki, H. 9.3.8, P89 Simson, J. A. 9.4.7
Singh, P. P263
Saska, R. 25.1.6
Satake, M. P172 Singleton, S. B. P55
Siren, E. P25
Satake, T. P108, P306
Sauerschnig, M. 9.3.2, 12.1.5, P149 Sirola, J. P262
Schaeferhoff, P. P229 Sittinger, M. P56
Schaeren, S. P186 Sivananthan, S. P318
Schagemann, J. C. P165 Skarpas, G. P237, P259
Schaumburger, J. 9.2.8, P244 Skinner, J. P238, P241
Schelfhout, J. P152 Slynarski, K. P. 18.2.2, P242, P313
Scheurecker, G. 17.2.4, P331 Smit, T. 17.3.9, P53
Schewe, B. P250 Smith, R. 16.3.3
Schieker, M. P69 Smith, T. D. P54
Schinhan, M. P13, P14 Sobral, J. P100
Schirlin, A. 17.3.8 Sodhi, K. P263
Schmal, H. P96 Sol, P. P37
Schmitz, P. P69 Solecki, A. 25.4.7
Schneider, E. 12.3.3 Soler, C. P120
Schoenhuber, H. P337 Soler Rich, R. P202, P334
Schreyer, T. 12.4.3, 17.1.9 Solis-Arrieta, L. P5
Schrier, D. 25.3.7 Solsky, I. 12.3.7
Schrobback, K. 9.2.5 Somerson, J. S. P336
Schuchman, E. H. P88 Somma, E. P260
Schulz, R. M. P336 Son, Y. P71
Schuseil, E. 17.1.3 Song, B. P29
Schuurman, W. 9.2.3, 16.2.3, 25.1.3, Sopena, J. J. P120
P151 Soto-Cerrato, V. P212
Schwenke, T. P153 Spalding, T. 8.1.2, 10.1.2, 17.1.4,
18.2.3, P328
Schönfelder, M. 9.3.2, 12.1.5
Schöttle, P. B. 9.3.2, 12.1.5 Spector, M. P111, P126
Spector, T. P291
Sciarretta, F. P324
Scinski, T. P313 Spindler, K. 17.4.7
Scopp, J. M. 8.1.1 Spruijt, S. 20.2.3
276
Staes, F. 12.2.4
Stanish, W. D. 12.4.7, 12.4.8, 17.1.2,
25.4.1, P54, P285
Stark, N. P153
Steadman, J. P183
Steck, E. P11, P194
Steck, R. P122
Steinmeyer, J. 3.2.2, P130
Steinwachs, M. 17.4.1
Stelzeneder, D. 12.3-9, 12.4.4, 17.2.4,
25.2.4, P220, P254,
P330, P331
Stenhamre, H. 9.2.7, P61, P163
Stephen, J. M. P321
Sterner, H. P21
Stewart, J. 25.4.6, P129
Sticht, C. 12.1.6
Stindel, E. 12.4.9
Stoddart, M. 9.4.2
Stoffel, M. P45
Stolz, M. 9.2.9
Stone, K. R. 9.1.4
Straub, R. H. 9.2.8
Strauss, E. 12.2.7
Strehin, I. 9.4.7
Strem, B. P19
Stroebel, S. 9.2.4
Stuerz, H. P130
Stull, P. P311
Stürz, H. 25.1.4
Su, H. 12.3.7
Suedkamp, N. P. P96
Sugihara, H. P137
Sun, J. 9.4.3, 9.4.4, 25.3.8,
P54, P57, P285
Sundback, C. A. 12.3.2
Suri, M. P55
Sutton, C. A. P192
Szczęśniak, M. P258
Szomolanyi, P. 25.2.4
Sánchez, A. P334
Søballe, K. 9.4.8
Südkamp, N. P. 9.4.2
Sütfels, T. 9.3.2, 12.1.5
T Urraza, J. P332, P338
Tabet, S. K. 12.3.6
Tafazal, S. P323
Taffetani, M. 9.4.6
Takahashi, K. P215
Takakura, Y. 9.4.5
Takato, T. P80
Takayama, K. 9.3.8, P89
Take, Y. P158
Takemura, Y. P59, P97, P114
Tampere, T. 17.3.4, P167
Tampieri, A. P43
Tan, A. R. P142
Tan, N. 25.4.3
Tanaka, Y. 9.4.5, 25.2.8, P322
Tang, Z. P248
Tarella, C. P64
Tarlton, J. P124
Tateishi, K. 3.2.3, 9.1.9
Tavares, H. P164
Taylor, S. D. P44
Terauchi, R. P215
Tey, M. P219
Tey Pons, M. P3, P264
Thakkar, V. 25.4.8
Theodoropoulos, J. P147
Thermann, H. 12.4.3
Authors‘ Index
Thomas, B. P34, P153
Thompson, P. 17.1.4, P328
Tichy, B. 17.1.6
Tiitu, V. 25.3.2
Timoncini, A. 9.3.4, P266
Toguchida, J. P137
Tonnarelli, B. 19.3.3
Tormenta, S. P265
Torzilli, P. A. P171
Toyama, M. 25.3.6
Toyoda, F. P98
Toyokawa, N. P172
Tran-Khanh, N. 12.4.7, 12.4.8, 25.3.8
Trattnig, S. 12.3-9, 12.4.4, 17.2.4,
24.2.3, 25.2.2,
25.2.3, 25.2.4,
25.2.5, P239, P330,
P331
Triantafyllopoulos, A. P237, P259
Trice, M. P95
Truncale, K. 25.3.4
Trüssel, A. P30
Tsai-Wu, J. P86, P101
Tsiridis, E. P44
Tsuchida, A. I. P84
Tsuchida, S. P215
Tsuchimoto, K. P108
Tsukuda, N. P134
Tsukuda, Y. P140
Tuan, R. 2.2.2
Turek, T. 9.1.4
Tómas, H. M. P164
Töyräs, J. 25.3.2, P262
U Villegas, H. P5
Uchida, K. 25.3.6, P10
Uddin, S. 17.2.5
Ueba, H. P17, P68
Uematsu, K. P322
Uenaka, K. P67, P97
Ueng, S. P138
Uhlík, J. 12.3.4
Ulrich-Vinther, M. 9.4.8
Urabe, K. 25.3.6, P10
Usvald, D. 12.3.4
V Waldman, S. 25.4.6, P129
Vidal, P. P332, P338
Vacanti, J. 12.3.2
Vacca, F. P265
Vajner, L. 12.3.4
Valanos, N. P297
Valderrabano, V. 10.2.3
Valonen, P. 25.3.2
Van Assche, D. 12.2.4
Van Blitterswijk, C. 12.1.6, P81, P100
Van Caspel, D. 12.2.4
Van Osch, G. J. 2.2.3, P127, P199
Van Thiel, G. S. P46
van Buul, G. M. P199
van Meegeren, M. E. 9.3.3
van Roermund, P. M. P132
van Weeren, P. 16.2.3, 25.1.3, 25.3.5,
P307
van der Lee, J. P21
van der Maas, J. 17.3.4, P167
Vandercruyssen, B. P76
Vanlauwe, J. 12.2.4, 12.4.2, P240
Vannini, F. 9.1.5, 17.2.8, 25.2.6,
25.2.9, P66, P216,
P218
Vaquero, J. 12.4.7, 12.4.8, 17.1.2,
19.2.5, 25.4.1
Vasanji, A. 12.3.3
Vasilceac, F. A. P294
Vasiliadis, H. S. 17.2.2, 25.2.7, P315
Vasishta, V. G. P270
Vavken, P. P13
Vecsei, V. 17.1.6
Veen van der, A. 17.3.9, P53
Vega, J. P308
Velasquillo, C. P5, P28, P253, P335
Velázquez, J. P334
Vena, P. 9.4.6
Verbruggen, G. 12.1.2, P76, P243,
P274
Verdonk, P. C. 2.1.3, 12.1.2, 17.3.4,
18.2.1, 25.4.9, P167,
P243, P274, P279,
P281, P282
Verdonk, R. 12.4.2, 17.3.4, 18.2.1,
25.4.9, P167, P243,
P274, P279, P281,
P282
Verghese, N. P310
Verhaar, J. A. P199
Verioti, C. P126
Verma, N. N. P2
Verra, W. P84
Vicente-Pascual, M. P106
Victor, J. 12.4.2
Vignon, E. P291
Vijayan, S. 12.2.6, P227, P238
Viladot, R. P308
Villalobos Cordova, F. P28
Villalobos Jr, E. P253
Vincken, K. L. P160
Viren, T. 25.3.2, P262
Vives, J. P15, P202
Vlychou, M. 17.2.4
Vogt, J. P39
Vogt, S. 9.3.2, 18.4.5, P234
Volck, J. P250
Volpi, P. P337
Vonwil, D. P30
W
Wakitani, S. 25.2.8
Walgenbach, A. 9.1.4
Walker, P. P49, P50, P302
Waller, C. S. P295
Wallstabe, S. 17.1.3
Wang, N. 9.4.2
Wang, Q. P83
Wang, S. P190
Wang, V. M. P46
Warnke, P. H. P318
Warren, R. F. 12.3.7, P171
Wasiak, J. P315
Wassef, M. P27
Watzer, B. 16.2.1
Wawrzynek, W. P257, P258
Webster, K. 17.2.3
Wei, L. 17.4.9
Wei, X. 17.4.9, P4, P73, P125,
P136
Weinans, H. P199
Weinerman, S. P311
Wellings, R. P328
Welsch, G. H. 12.3-9, 12.4.4, 17.2.4,
25.2.2, 25.2.3,
25.2.4, 25.2.5, P330,
 Authors‘ Index 277

P331 Zaslav, K. R. 8.3.3, 20.3.1

Wendlandt, R. P24 Zayed, N. P78, P79, P82
Wendt, D. 19.3.3 Zbyn, S. 25.2.4
Werner, S. 12.4.4 Zehbe, R. 16.2.1
White, L. M. P58 Zhang, Z. P248
Whitehead, T. 17.2.3 Zhang, X. P209
Whiteside, R. 25.3.3, P58 Zhao, X. 12.3.2, P75, P105,
Wickiewicz, T. L. 12.3.7 P176
Wicks, J. P. P63 Zheng, M. H. 12.2.2, P90, P225
Widuchowski, J. 12.4.6, P257, P258 Zheng, Q. P90
Widuchowski, W. 12.4.6, P257, P258 Zhong, X. P248
Wiech, O. P244 Zhou, L. 12.3.2
Wielopolski, P. A. P199 Zidrou, C. P297, P298
Wiewiorski, M. 8.1.3 Zivkovic, S. 11.2.1
Wijnberg, H. M. 25.1.3 Zorzi, C. 2.1.1
Wildi, L. 17.4.5 Zscharnack, M. P336
Wilensky, D. P148 Zuiderbaan, H. 17.3.9, P53
Williams, III, R. J. 12.3.7, P236 Zur, G. 2.1.1, 9.4.9, 12.2.9,
Williams, S. P44, P52 P41, P51
Wilshaw, S. P139 Zwickl, H. 12.1.3, P159
Wilson, G. L. 17.4.2, P109
Wilson, R. 11.2.1
Wimmer, J. 17.1.3
Winalski, C. S. 12.3.3
Windhager, R. P72
Wirz, D. P38
Wit, J. 12.1.6
Wittwer, J. 17.2.3
Wong, C. P148
Wood, D. J. 12.2.2, P225, P231
Wood, S. 25.3.7
Woodell-May, J. P112
Woodfield, T. B. 9.2.4, 9.2.5
Wu, L. P81
Wyland, D. P55
Wysocka, A. P257, P326
Wörtler, K. P149

X
Xiafei, R. 25.3.9
Xiao, X. P150
Xu, J. P90

Y
Yoo, J. P75
Yang, C. P62, P138
Yang, Z. P73, P248
Yao, J. 9.1.7, 9.1.8
Yao, V. P121
Yasuda, K. 9.4.5, P7, P31, P35,
P113
Yayon, A. P233, P239
Yildirim, G. P302
Yokota, M. P7
Yonetani, Y. 25.2.8
Yoon, J. P29
Yoon, S. P269, P275, P329
Yoshikawa, H. 3.2.3, 9.1.9, 24.3.3,
P158, P278
Yuan, L. P62, P138

Z
Zabalza-Baranguá, A. P91, P106, P119
Zaffagnini, S. 17.1.5, 17.3.6, P305,
P327
Zajac, P. P186
Zak, L. 12.4.4, 17.1.6, 25.2.2,
25.2.3, 25.2.5
Zandieh Doulabi, B. 17.3.9, P53
Zapero, I. 12.1.9