Mar. 2007, Volume 4, No.3 (Serial No.

28) Journal of US-China Medical Science, ISSN1548-6648, USA

Cartilage Regeneration by Delayed Engraftment of an Injectable Fibrin

Clue/Hyaluronate Hybrid Scaffold Mixed with Mesenchymal Stem Cells

YANG Rui-fu1, HU Yun-yu1, WU Yin-song1, ZHANG Yu-fei2, WEI Yi-yong1

(1. Department of Orthopaedics, Xijing Hospital, the Fourth Military Medical University, Xi’an 710032;
2. Department of Respiration, Xijing Hospital, the Fourth Military Medical University, Xi’an 710032)

Abstract: Objective To evaluate the effect of an injectable hybrid fibrin/hyaluronate scaffold in cartilage
defect by delayed engraftment. Methods A cartilage defect model was established by drilling deep into the
femoropatellar grooves of rabbits with a diameter of about 4.5 millimeter. The defects were not treated until 42
days after drilling. An injectable scaffold was developed by combining fibrin glue and hyaluronate to mimic the
structure of cartilage matrix. Autologous mesenchymal stem cells (MSCs) were induced to chondrocytes in vitro,
and mixed with the injectable scaffold and then implanted into the cartilage defects. 30 rabbits (both sides of
knees, n=60) were used in this study and they were randomly divided into three groups: group A (n=20) were
treated by implanting the injectable scaffolds mixed with MSCs; group B (n=20) were treated by drilling the
bottom of defect to reach medullary cavity; group C (n=20) were blank control. The regenerated cartilages were
evaluated by both macroscopic observation and histological scores at 12 and 24 weeks after treatment. Results In
group A, most cartilage defects were well repaired; the histological scores were significantly higher compared
with group B and C (p<0.01). The reconstitution of the cartilage surfaces was also better in group A (p<0.001).
There were no significant difference between 12 and 24 weeks in group A (p=0.917). Conclusion this study
revealed that by using autologous mesenchymal stem cells and an injectable fibrin clue/hyaluronate hybrid
scaffold, defects of articular cartilage can be well repaired in a delayed manner, which has considerable relevance
to the clinical practice.
Key words: cartilage defect; tissue engineering; cartilage regeneration

INTRODUCTION

Defects of the articular cartilage induced by trauma or rheumatism are prevalent in clinical practices;
however, effective therapies that could regenerate the cartilage defects are not available currently. Drilling
therapeutic holes into the subchondral bone-marrow spaces underlying regions of damaged articular cartilage,
used as a simple way of treatment, is unable to reconstruct the intact cartilage surfaces[1]. The joints with untreated
defects can usually developed into osteoarthritis in the long term[2-4]. It can cause more severe cartilage loss, and
ultimately complete erosion of the cartilage surface[5].
Recently, tissue engineering (TE) have brought prospect for cartilage regeneration[6]. To repair articular
cartilage defects, transplantations of various tissues or cells have been investigated in both animal model and
human[6,7]. Previously it was reported that by using autologous bone marrow stromal cells (ABMSCs), fresh

HU Yun-yu (1933- ), female, Ph.D. supervisor, professor of Institute for Department of Orthopaedics, Xijing Hospital, the Fourth
Military Medical University; research field: basic and clinical orthopaedics.

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 Cartilage Regeneration by Delayed Engraftment of an Injectable Fibrin Clue/Hyaluronate Hybrid Scaffold Mixed
with Mesenchymal Stem Cells

defects at the femoropatellar grooves can be repaired to some extent[8,9], but the situation is very different from
those seen clinically, which are often delayed defects with some osteoarthritis characteristics. Until now there are
very few reports describing therapies under these conditions[4]. The reason may be that the disease are more severe,
as well as lack of research on this respect.
The objective of this study was to determine if delivery of MSCs-differentiated chondrocytes, by mixed with
an injectable scaffold mimicking cartilage matrix, to the cartilage defects can be effective and applicable. The
traditional treatment of drilling therapeutic holes was used as a control. The effects of cartilage regeneration were
evaluated at several time points during a 24-week period.

MATERIALS AND METHODS

1. Experimental Design
30 New Zealand white rabbits, 5-8 months old and weighing 2.8-3.5kg, were used in this study. A
full-thickness cartilage defect was formed on the femoropatellar grooves with a bitbrace. The size of the defect
was about 4mm in diameter and 2.5mm in depth. After 42 days, the delayed cartilage defect models were
established. They were divided randomly into three groups: (1) group A was treated by scaffold-MSC composite
engraftment. The scaffold was composed of Fibrin glue (FG, Guangzhou Beixiu, China), Hyaluronate acid (HA,
Chiatia Freda, China), Aminomethylbenzoic acid (AM, Sishui Xierkang, China). Autologous bone marrow
stromal cells (MSCs) were induced to differentiate into chondrocytes in vitro, and then were mixed with the
scaffold and implanted into the cartilage defects. (2) Group B was treated by drilling holes into the subchondral
bone-marrow spaces underlying regions of cartilage defect. (3) Group C served as blank control. The animals
were assigned randomly to sacrifice dates (12 weeks and 24 weeks after operation). This study was approved by
the University Committee for Animal Experimentation.
2. The Chondrogenic Medium
DMEM/F12 medium (Gibco) were supplemented with 20% fetal bovine serum (Hyclone), 5ng/ml
transforming growth factor β1 (TGF-β1, R&D USA), 50ug/ml ascorbate-2 phosphate (Sigma), 50ug/ml
Dexamethasone (R&D), and 1%ITS+TM Premix(B&D, containing 6.25ng/ml insulin/selenious/transferrin,
1.25mg/ml bovine serum albumin, 5.35mg/ml linoleic Acid).
3. Differentiation of MSCs into Chondrocytes
Bone marrow was aspirated from the bilateral femoral trochanters of 14 rabbits in group A. MSCs were
isolated and cultured in vitro, when cells were almost confluent in passage 2, they were treated with 0.05% trypsin
and centrifuged at 1000rpm for 10min. The cell pellet was resuspended in chondrogenic medium and then
incorporated into the hyaluronic acid. The adherent cells were counted in a haemocytometer. The final cell density
was adjusted to 6.0×109cells/ml. They were induced as a pelleted micromass with the chondrogenic medium.
After 10-14 days, six of them were taken out at random and stained for type II collagen (Fig. 1), which showed
most MSCs had transformed into chondrocytes[10].
4. Scaffold/MSCs Composite Preparation
The cell pellets were resuspended in chondrogenic medium containing 5ng/ml TGF-β1 and counted in a
haemocytometer. The cell density was adjusted to 9.0×107cells/ml. Then they were mixed with HA to fabricate
cell-HA gel. The cell density was adjusted to 3.0×107cells/ml.
FG mixing with cell-HA gel was named as solution A. Thrombin, Calcium chloride and 10%
aminomethylbenzoic mixing with cell-HA gel was named as solution B. The final cell density was adjusted to

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 Cartilage Regeneration by Delayed Engraftment of an Injectable Fibrin Clue/Hyaluronate Hybrid Scaffold Mixed
with Mesenchymal Stem Cells

6.0×106cells/ml in solution A and B respectively. Solution A and B were drawn into the chambers of a syn-push
needle respectively.
5. Implant Procedures:
The operations were performed under general anaesthesia induced by an intramuscular injection of
pentobarbital sodium (Somnopentyl). A lateral parapatellar anteriomedialis incision was made and the patella
everted dislocate. After the cartilage defects of femoropatellar grooves were debrided, their sizes were more than
4.5mm in diameter and 3.5mm in depth.
In group A, solution A and B were immediately injected into the defects after debridement. In group B, the
defect bottom was drilled about 6-8 times to be spongia-like with needles of 0.5mm in diameter to reach
medullary cavity. Then the knee joint capsule, patellar ligament and skin were carefully sutured separately.
Animals in group C were not treated. All rabbits were allowed free movement in standard rearing cages after
operation.
6. Macroscopic and Histological Assessments
Rabbits were sacrificed by injecting a lethal dose of pentobarbital sodium respectively at 12w and 24w
post-operation, according to their preassigned designations. The specimens containing the original defects in the
femoropatellar grooves were harvested and photographed. They were then prepared for histologic analysis.
Histological sections with the regenerated tissue were assessed and scored blindly, using the International
Cartilage Repair Society (ICRS) visual histological assessment scale[11]. The ICRS criteria evaluates regularity of
the cartilage surface, matrix morphology, cell distribution, cell population viability, subchondral bone,
mineralisation of cartilage and staining for type II collagen.
7. Statistical Analyses
All data were expressed as mean±standard deviation (SD), and a three-way classification analysis of variance
was analyzed. We considered a P value of less than 0.05 to be significant. SPSS10.0 software was used in this
study.

RESULTS

1. Gross Morphological Finding

In most of the 30 rabbits, when the joints were exposed, the synovial tissue and cartilage appeared to be
degenerated to some extent. With the lapse of times, degeneration was more severe and extended from
femoropatellar grooves to total knee. In 12w and 24w, cartilage degeneration of group A was significantly slighter
than that of group B and C. Typically, at 24 weeks, 17 out of 30 cartilages of femoropatellar grooves had
characteristics of degeneration. There were 1 out of 10 knees in group A, 8 out of 10 knees in group B, and all of
10 knees degenerated obviously in group C.
As shown in figure 2, the surface of femoropatellar groove in group A appeared smooth and tenacious
compared with groups B and C. In group A, the defect was completely filled with white translucent tissue which
was similar to the surrounding cartilage in color and luster, and the conjunction between neogenetic cartilage and
host cartilage was compact and ill-demarcated at both 12 and 24 weeks (Fig. 2a, b). In particular at 24 weeks, the
neogenetic cartilage in 5 joints was hardly discriminated from the surrounding host-cartilage. Although the defect
was partially filled with cartilage-like tissues in group B, the surface of femoropatellar groove was uneven (Fig.
2c, d). In group C, the boundary of defect was blunt, and the bottom was only covered with little connective tissue

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 Cartilage Regeneration by Delayed Engraftment of an Injectable Fibrin Clue/Hyaluronate Hybrid Scaffold Mixed
with Mesenchymal Stem Cells

and island-like neocartilage (Fig. 2e, f).

2. Histological Findings
When we stained with HE staining at both 12 and 24 weeks, We found that neogenetic cartilage were thicker,
the neogenetic chondrocytes were mature and distributed more orderly, and cartilage degenerated more slightly in
group A than those in other two control groups(Fig. 3a), though tidemark was less distinctive (seemingly because
the ossification front was still in evolution). However, cartilage layer was thin and depressed in group B (Fig. 3b).
As for group C, HE staining showed that there were obvious proliferation of chondrocytes in the border and some
fibrous tissue on the bottom (Fig. 3c). safranin-O staining showed that the neogenetic cartilages in group A
contained more proteoglycan than those of groups B and C (Fig. 3d, e, f).
At no time period was there any infection in knees joints, nor did there appear to be any adverse reaction by
the surrounding or replacing tissues to the presence of the scaffold. The scaffold was absorbed by the sacrifice time.
3. Histological and Histochemical Assessments
The mean scores are shown in Table 1. The scores of group A were significantly better than those of group B
and C in terms of their surface morphology (p<0.001), matrix morphology (p=0.003 and p<0.001, respectively),
cell distribution (p=0.014 and p<0.001, respectively), cell population viability (p=0.016 and p<0.001,
respectively), subchondral bone (p<0.001 respectively) and Type II collagen staining of the matrix (p=0.01 and
p<0.001, respectively). There was no significant difference between group A and B in terms of their cartilage
mineralisation (p=0.307). The total histological scores in group A were significantly higher than those in group B
and C (p<0.01). There was also significant difference between group B and C (p<0.01). But there was no
significant difference between the results of 12 weeks and 24weeks in group A (p＝0.917).

Table 1 The modified ICRS visual histological assessment scale and results (Mean ± SD)
cell Cell population Subchondral Cartilage Type II collagen
weeks group n surface matrix Total
distribution viability bone mineralisation staining
CFH 8 2.25±1.38 0.88±.64 1.75± 0.71 2.75±0.71 1.87±0.35 2.63±1.06 1.50± 0.75 1.95±1.01△¥
6 D 8 0.00±0.00 0.63±0.51 1.37±0.51 2.25±1.03 1.37±0.51 2.25±1.38 1.00±0.75 1.27±1.07
FH 8 0.00±0.00 0.25±0.46 0.25±0.46 0.25±0.46 0.25±0.46 0.75±1.38 0.25±0.46 0.29±0.65
CFH 10 2.40±1.26 1.80±0.63 2.50±0.71 2.80±0.63 2.00±0.00 2.70±0.94 2.10±0.73 2.33±0.83☆
12 D 10 0.00±0.00 1.10±0.73 2.10±0.74 2.40±0.96 1.70±0.48 2.40±1.26 1.30±0.67 1.57±1.09
FH 10 0.00±0.00 0.20±0.42 0.20±0.42 0.20±0.42 0.20±0.42 0.60±1.26 0.20±0.42 0.23±0.59
CFH 10 2.70±0.94 2.10±0.56 2.80±0.42 2.60±0.84 2.00±0.47 2.70±0.94 1.60±0.69 2.36±0.82#$
24 D 10 0.00±0.00 1.60±0.84 2.40±0.69 2.00±1.05 1.30±0.67 2.40±1.26 1.20±0.78 1.56±1.12
FH 10 0.00±0.00 0.20±0.42 0.20±0.42 0.10±0.31 0.10±0.31 0.30±0.94 0.10±0.31 0.14±0.45
* The scale has seven categories assigning a total score ranging from 0 (worst) to 21 (best). I-VII: graded from 0 to 3. Those
calculations were based on observed means and the mean difference is significant at the 0.05 level. △p<0.05 (CFH group vs the D
group and FH group at 6w). ☆p<0.05 (CFH group vs the D group and FH group at 12w). #p<0.05 (CFH group vs the D group and FH
group at 24w). ¥p<0.05 (sig. 0.025 and 0.032) (the 6w vs the 12w and 24w in CFH group). $p>0.05 (sig. 0.917) (24w vs the 12w in
CFH group).

DISCUSSION

Cartilage defects due to trauma or rheumatism can not only damage the cartilage surface, but also can be the
prelude to the development of osteoarthritis, because the stimuli include mechanical stress and degradation
products of ECM components including fibronectin fragments can stimulate production of matrix-degrading
proteases[12]. Those potential stimuli and cartilage defect in the local environment catalyze a cascade of events

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 Cartilage Regeneration by Delayed Engraftment of an Injectable Fibrin Clue/Hyaluronate Hybrid Scaffold Mixed
with Mesenchymal Stem Cells

which then alters joint homeostasis[13]. In delayed cartilage defects, not only the original autologous tissues are
damaged, but also the transplanted and newly formed tissues in the construct are affected. So the neogenetic
cartilage is difficult to grow and fill the defect.
In this study we formed cartilage defects in the non-weight-bearing area of the femoropatellar grooves of the
knee joints[14]. This method were adopted to reduce contact pressure on the regenerated area and could result in
the formation of a space for cell proliferation and differentiation[2,3,15]. Compared with former researches that used
cartilage tissue engineering technique which was limited to fresh defect of normal cartilage[8,9], this model imitate
microenvironment and cartilage defect that are similar to that of human seen in clinical practice. Also in the
present study, the 4.5mm diameter×3.5mm depth defect in rabbit femoropatellar groove might be so massive
injury that the spontaneous repair of the experimental model would be reduced greatly. The 4.5mm diameter ×
3.5mm depth cylindrical defect (which encompasses a large volume and surface area of the rabbit femoropatellar
groove) may be repaired successfully by our promising therapeutic interventions.
In this study, we used a new injectable scaffold as ABMSCs delivery system which was different from the
previous scaffolds. The advantages of previous biomaterials scaffolds such as hyroxyapatite or Poly (a-hydroxy
esters) are their mechanical strength and absorbability[16]. However, it is clear that most those resorbable
biomaterials are resorbed over 1-2 years and the liberated crystallites could induce an inflammatory response and
the typical pH drop[17]. Our new scaffold was composed of FG, HA and AM, and possessed most advantages as
previous scaffolds. Both FG and HA have good biodegradability and biocompatibility, and HA was reported even
to be able to upregulate chondrocyte metabolism[18,19]. AM, which has similar chemical constituents to lysine,
could competitively block the lysine binding site of fibrinolysin and delay the formative fibrin clot to be degraded
because of fibrinolysis, and has no harmful effect on cell proliferation and can maintain phenotype and matrix
secretion[20,21]. After HA, FG and AM were mixed, the degradation rates of HA and FG were dramatically
decreased in vitro, especially the HA-fibrin mixture was put at rest and not exposed to a mechanical stress[22]. Our
study demonstrated that HA and FG degraded during a period 6 weeks, which contributed to the growth of
neogenetic cartilage. In addition, our scaffold was more convenient for its injectability, which could eliminate the
complex operation procedures performed using those previous scaffolds, and in turn could reduce the incidence of
infection caused by operation. Although mechanical strength of our scaffold is somewhat weak, we did not find
obvious influence of this scaffold on the cartilage repair by reducing contact pressure on the regenerated areas.
Using the injectable scaffold, we delivered ABMSCs differentiated chondrocytes to the old defects. We
observed that the defects in group A had been filled with cartilage–like tissue and the cartilage-like tissues showed
a chondrocytes morphology at 12 week. The degree of damage in femoropatellar groove was significantly
mitigated at 24 week compared with that at 12 week. In group B, the defect was partly repaired. This traditional
repair approach that is based on the rationale of drilling holes into the subchondral bone-marrow space underlying
regions of damaged articular cartilage can stimulate a spontaneous repair reaction, but could not get satisfactory
results as that of our approach in group A for large defects as shown in our study. However, in group C, the
cartilage-like tissues were hardly observed at the bottom of defects even at 24 week. The reason was that FG
could interrupt the ingression of ABMSCs and some growth factors.

CONCLUSION

In conclusion, our study has considerable clinical relevance to the treatment of large defects of articular

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 Cartilage Regeneration by Delayed Engraftment of an Injectable Fibrin Clue/Hyaluronate Hybrid Scaffold Mixed
with Mesenchymal Stem Cells

cartilage, and provides a simple but effective repair technique for the regeneration of large cartilage defect.

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(Edited by Jane Chen)

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 Cartilage Regeneration by Delayed Engraftment of an Injectable Fibrin Clue/Hyaluronate Hybrid Scaffold Mixed
with Mesenchymal Stem Cells

Fig. 1 Photomicrographs showed immunohistochemical staining of type II collagen after Pelleted micromass had been induced
for 10-14 days. The intensity of staining of type II collagen showed most MSCs had transformed into chondrocytes. (×100).

Fig. 2 Gross morphological findings at 12 (a, b, c) and 24 weeks (d, e, f) showed the neogenetic cartilages in group A (a, d)
was better than that in group B (b, e) and group C (c, f). At 24 weeks, the defect in group A was totally repaired with
cartilage-like tissues and it integrated smoothly with the surrounding host tissues. (×100).

Fig. 3 The 24-week histological Photomicrographs of HE staining (a, b, c) and Safranin-O staining (d, e, f) showed the
neogenetic cartilages in group A (a, d) was better than that in group B (b, e) and group C (c, f). Good integration between
neo-cartilage and the surrounding cartilage in group A was observed at 24 weeks. Arrows showed areas of neogenetic
cartilage. (×100).

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