Review article

Possible functional scaffolds for periodontal

regeneration
Hidetoshi Shimauchi
a,
*
, Eiji Nemoto
a
, Hiroshi Ishihata
a
,
Masatsugu Shimomura
b
a
Division of Periodontology and Endodontology, Graduate School of Dentistry, Tohoku University, 4-1 Seiryo-machi,
Aoba-ku, Sendai 980-8575, Japan
b
Organized Polymer Material, Institute of Multidisciplinary Research for Advanced Materials Laboratory (IMRAM),
Tohoku University, 2-1-1 Katahira, Aoba-ku, Sendai 980-8577, Japan
Received 9 March 2013; received in revised form 29 April 2013; accepted 22 May 2013
Contents
1. Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119
2. Current strategies for periodontal regeneration. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119
3. Periodontal tissue engineering . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 120
4. The desirable properties of scaffolds designed for periodontal regeneration . . . . . . . . . . . . . . . . . . . . . . . . . . . . 122
4.1. Mimicking the stem cell niche to alter the regulation of stem cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 122
Japanese Dental Science Review (2013) 49, 118130
KEYWORDS
Periodontal regeneration;
Scaffold;
Tissue engineering;
Cellscaffold interaction
Summary Periodontal diseases are the most prevalent infectious diseases with the irreversible
loss of tooth supporting apparatus. To reestablish the stable health of periodontal tissues after
healing, the concept of regenerative surgical procedures has been developed over the past three
decades, including various grafting materials, guided tissue regeneration (GTR), and the use of
enamel matrix derivatives (EMD) in a clinical setting. More recently, tissue engineering strategies
have also been applied and developed for periodontal regeneration: (1) stem cell therapies; (2)
recombinant human growth factor therapies; (3) combined use of cell and growth factors with
matrix-based scaffolds. However, the complete and predictable reconstruction of healthy
periodontal tissues still remains a challenging eld. To overcome therapeutic limitations and
develop stem cell-based strategies, it is necessary to optimize cellscaffold combinations by
understanding the cellular events during periodontal wound healing and regeneration. We
reviewed: (1) the current status and strategies for periodontal regeneration; (2) a possible
biomaterial design for the scaffold used in periodontal tissue engineering; (3) a possible
interaction between scaffold materials and periodontal tissue cells.
#2013 Japanese Association for Dental Science. Published by Elsevier Ltd. All rights reserved.
* Corresponding author. Tel.: +81 22 717 8333; fax: +81 22 717 8339.
E-mail address: simauti@dent.tohoku.ac.jp (H. Shimauchi).
Available online at www.sciencedirect.com
j our nal h o mepage: w ww. el sevi er . co m/ l oc at e/ j dsr
1882-7616/$ see front matter # 2013 Japanese Association for Dental Science. Published by Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.jdsr.2013.05.001
4.2. Cellscaffold interplay and scaffold designing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 124
4.3. Application of a honeycomb microarray lm for PDL cell sheet engineering . . . . . . . . . . . . . . . . . . . . . . . . 124
4.4. Possible induction of cementogenesis by modulation of the extracellular ionic microenvironment
using bioactive ceramic scaffolds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 125
4.5. Application of nanofabricated hydroxyapatite for bone tissue engineering . . . . . . . . . . . . . . . . . . . . . . . . . 127
5. Conclusions and future prospects. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 128
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 128
References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 128
1. Introduction
Periodontal diseases are the most common inammatory
diseases caused by plaque biolm in the oral cavity. During
the progression of periodontal diseases, the pathological
change of gingivitis to periodontitis is characterized by the
irreversible loss of tooth supporting apparatus, ultimately
leading to the loss of the tooth. It includes the progressive
destruction of whole periodontal tissue: the gingiva, period-
ontal ligament (PDL), cementum, and alveolar bone, as well
as the connective tissue attachment between the root surface
and alveolar bone [1]. The concept of conventional period-
ontal treatments is a cause-related therapy aimed at
removing causative agents from the oral cavity, with a focus
on eliminating bacterial plaque and calculus. This therapy
commonly results in successful improvements in the inam-
matory process of periodontal tissue; however, it only induces
the unstable healthy condition of destroyed periodontal tis-
sue in case of progressive periodontitis even after a successful
result. The recovered healthy condition of destroyed tissue is
easily broken by the reaccumulation of bacteria surrounding
the teeth, leading to the recurrence of periodontitis. In the
United States, a recent national survey (20092010 National
Health and Nutrition Examination Survey; NHANES) revealed
that an estimated 47.2% or 64.7 million adults aged 30 and
over suffered from periodontitis, and prevalence rates
increased to 70.1% in adults 65 and older [2]. The prevalence
rates of periodontal diseases in Japan are also similar, indi-
cating that periodontal diseases are one of the most common
infectious diseases in the world. Furthermore, recent clinical
research has indicated the close and bidirectional relation-
ship between periodontitis and systemic disorders, such as
diabetes, cardiovascular disease, and metabolic syndromes.
Thus, it is important to reestablish the stable health of
periodontal tissues after healing, as well as prevent period-
ontal diseases to maintain both systemic and oral health.
Why is periodontitis at high risk of recurrence? There are
two major reasons: easily occurring bacterial re-accumulation
and the formation of a long junctional epithelium (periodontal
repair) after surgical/non-surgical procedures. Lindhe et al.
[3] investigated the long-term effects of surgical/non-surgical
treatment over a 5-year period, and concluded that the
patients self-plaque control, but not treatment modality,
was the critical determinant of a good prognosis in periodontal
therapy. Both of the procedures induced healing with a long
junctional epithelium that was easily broken by recurrent
inammation due to plaque accumulation [4]. To obtain
good stability and predictability after therapy, periodontal
regeneration of destroyed tissue, which is characterized by de
novo formation of cementum, a functionally organized PDL,
alveolar bone, and gingiva, is desirable. Clinical research has
extensively shown that regeneration remains the favorable
outcome over periodontal repair [5,6].
The desire to induce the complete regeneration of period-
ontal tissue has inspired the introduction of tissue engineer-
ing technology into dental clinics [7,8]. Tissue engineering is
dened as a multi-disciplinary eld of medicine, chemistry,
physics, engineering, and biology [9]. The triad for conven-
tional cell-based tissue engineering involves cells, signaling
molecules, and scaffold/supporting matrices [10]. In this
triad, the role of the scaffold is the niche of cells, and
facilitates the attachment, migration, proliferation, and
three-dimensional (3D) spatial organization of the cell popu-
lation. However, recent progress in material sciences has
provided bioactive properties to scaffold materials for trans-
mitting specic signals to cells that will decode these into
biochemical signals. Topography, chemistry, and physical
properties are considered to be critical parameters for
directing cell fate [11]. This review has focused on the
following topics: (1) the current status of and strategies
for periodontal regeneration; (2) a possible biomaterial
design for the scaffold used in periodontal tissue engineering;
(3) a possible interaction between scaffold materials and
periodontal tissue cells.
2. Current strategies for periodontal
regeneration
For more than three decades, periodontal research has been
attempting to discover clinical treatment regimens that
can regenerate periodontal tissues with good predictability.
These trials successfully developed two types of strategies
with the combined use of biomaterials and grafts shown in
Table 1.
A number of animal and human trials demonstrated that
the combined use of bone grafts/implant materials with ap
surgery successfully stimulated alveolar bone regeneration.
These grafts/materials include: (1) autogenous grafts; (2)
allogeneic graft; (3) xenogeneic grafts, and (4) alloplastic
materials [12]. The use of grafts/biomaterials presumably
served as a scaffold for bone formation and contained the
bone-forming cells and bone-inducting substances that
nally resulted in bone formation. The biological perfor-
mance of bone grafts can be divided into three interrelated,
but not identical rationales: osteogenesis (the formation of
new bone by stem cell lineage derived from graft material);
osteoinduction (bone growth by the surrounding immature
cells recruited by graft material); and osteoconduction
(bone growth on the surface of a material with fabrication)
[13]. Autogenous grafts only contain self-bone forming cells
Possible Functional Scaffolds for Periodontal Regeneration 119
that can induce osteogenesis, and still serves as the golden
standard of bone grafts.
The second strategy aimed at regenerating a 3D arrayed
structure of lost periodontal tissue including root cementum,
alveolar bone, and the PDL with the connective tissue attach-
ment. The biological rationale of this strategy was based on
the Melcher hypothesis [14], which proposed that the
nature of the attachment in periodontal healing depended
on the origin of cells (epithelial, gingival connective, bone,
PDL) repopulating the area adjacent to the root surface. The
hypothesis was successfully demonstrated in a series of
animal experiments, and the principal of Guided Tissue
Regeneration (GTR) was established [15,16]. The cell occlu-
sive membrane of GTR functions to isolate the periradicular
bone and root surface wound area from the rest of the
tissues to maintain a space for the repopulation of cells
originating from the PDL. For this purpose, different barrier
materials have been used, both non-resorbable and resorbable
(biodegradable).
The concept of the GTR technique only awaits the repo-
pulation of PDL cells in the wound healing area and does not
interfere with the growth/differentiation speed of these
cells. Recent biological approaches have attempted to
mimic/enhance the cellular events leading to the normal
development/wound healing process of periodontal tissues
[17]. Enamel Matrix Derivative (EMD), the active component
of Emdogain
1
, was the rst signaling molecule that could
regenerate periodontal tissue. Emdogain
1
is prepared from a
piglets unerupted teeth and has been clinically used
throughout the world after approval for the market in Europe
(1995), the United States (1996), and Japan (1998) [18].
Amelogenin proteins that dominate 90% of EMD are capable
of periodontal regeneration, but the contaminated amelo-
blastin functioned synergistically [19]. Better understanding
of the wound healing/regeneration process has driven
periodontal research to evaluate the role of growth factor,
which is endogenously secreted during the wound healing/
regeneration process. It is also meritorious that advances in
molecular cloning have made unlimited quantities of recom-
binant human proteins available for tissue engineering.
Various recombinant growth factors were studied for their
periodontal regeneration ability in vitro and in vivo. Among
them, platelet derived growth factor (PDGF; GEM21S
1
) and
bone morphogenic protein-2 (BMP-2; Infuse
1
) have already
become commercially available for clinical use in the United
States [20]. Furthermore, broblast growth factor (FGF)-2 is
being investigated in a large clinical trial (Phase III) in Japan,
and its clinical efcacy for periodontal regeneration has
already reported [2123].
In either approach, there is a therapeutic limitation due to
the morphology of alveolar resorption to obtain a predictable
outcome. Both strategies are only applicable to angular bony
defects, and not horizontal resorption. The classication of
infrabony defects that referred to the number of surrounding
bony walls also exerted a signicant inuence on the success
and failure of regeneration [24,25]. To overcome these lim-
itations and achieve complete regeneration, the introduction
of modern tissue engineering technologies is anticipated in
this eld.
3. Periodontal tissue engineering
Periodontal regeneration is one of the earliest clinical dis-
ciplines that has achieved the therapeutic application of
tissue engineering-based technologies. As well as the clinical
application of growth factors (signaling molecules), precli-
nical studies of cell therapy targeted for periodontal regen-
eration have been carried out using various sources of
somatic stem cells (Table 2) [9]. Somatic stem cells were
obtained from both dental and non-dental tissues: period-
ontal ligament-derived stem cells (PDLSC), dental follicular
cells (FDC), bone marrow stem cells (BMSC), and adipose
stem cells (ASC). These stem cells have the capability of
periodontal tissue formation as well as bone formation
[26,27]. In addition to adult/somatic stem cells, induced
pluripotent stem (iPS) cells were successfully generated
from gingival broblasts by the introduction of four factors
(OcT3/4, Sox2, Klf4, and c-Myc), suggesting the potential for
its clinical application in regenerative dentistry [28]. It is also
Table 1 Proposed strategies clinically available or tested for periodontal tissue reconstruction.
1. Targeted for alveolar bone repair 2. Targeted for periodontal tissue regeneration
(1) Autologous bone grafts (autografts) (1) Non-resorbable cell occlusive barrier membranes
Intra-oral grafts Polytetrauorethylene (ePTFE)
Extra-oral grafts (2) Resorbable cell occlusive barrier membranes
(2) Allogenic bone grafts (allografts) polygolicol acid/polylactic acid
Freeze-dried bone grafts Collagen membrane
Demineralized bone grafts (3) Growth factors (bio-regeneration)
(3) Xenogenic bone grafts (xenografts) Enamel matrix derivative (EMD)
Bovine mineral matrix Platelet-derived growth factor (PDGF)
(4) Alloplastic bone grafts Bone morphogenic protein-2 (BMP-2)
Hydroxiapatite Fibroblast growth factor-2 (FGF-2)
b-Tricalcium phosphate Platelet-rich plasma (PRP)
Bioactive glass (4) Cell therapy
Coral-derived calcium carbonate Autologous somatic stem cells
(5) Polymer and collagen sponges Allogenic stem cells
Collagen
Hard-tissue replacement polymer
Modied from Ramseier CA et al., Periodontology 2000, 38:185202, 2012 [12].
120 H. Shimauchi et al.
important to provide a large amount of cells at the site of
regeneration. For this purpose, PDL cell sheet technology has
been developed and successfully regenerated both new bone
and cementum connecting with collagen bers in canine
periodontal defect models [29]. However, none of these cell
transplantation models showed the complete regeneration of
horizontally lost periodontal tissues.
Therefore, periodontal tissue still remains one of the most
difcult tissues to achieve complete regeneration because
many clinical and biological variables may affect the regen-
eration process. Firstly, this tissue is always in contact with
the outside environment of the oral cavity, and is at risk of
infection during the regeneration process. Secondly, several
types of mechanical stresses are also loaded onto periodontal
tissue: one is from occlusal forces and the other is the tension
stress from gingiva and mucosal membranes, which disrupts
horizontally resorbed periodontal tissue. As shown in Fig. 1, it
may be difcult to overcome these environmental factors
and induce complete periodontal regeneration without the
golden triad of cell/signaling molecule/scaffold for con-
ventional regenerative medicine. It is important to maintain
the space of horizontally lost tissues and protect regenerat-
ing tissues from infection and mechanical stresses.
The last component of the tissue engineering triad is the
scaffold. Table 3 shows the scaffold materials used for growth
factor therapy aimed at periodontal regeneration [30]. Poly-
meric scaffolds commonly have porous and biodegradable
structures fabricated from natural/synthetic materials, and
Table 2 Preclinical application of cell therapies for peridontal regeneration.
Cell type Cell origin Experimental model Reference
Animal Defect type
BMSC Auto Dog Class III defects Hasegawa N et al. (2006), Li H et al. (2009)
Auto Dog Fenestration Yamada Y et al. (2004a)
Auto Dog, Rat Osteotomy Yamada Y et al. (2004b, 2004c), Tobita M et al. (2008)
ASC Auto Dog Palatal defects Dogan A et al. (2003)
PDL cells Auto Rat Fenestration Lekic PC et al. (2001)
Auto/Xeno Human Fenestration Seo BM et al. (2005)
PDL cell sheet Auto Dog Class II defects Akizuki T et al. (2005)
Auto Dog 1-Wall defects Tsumanuma Y et al. (2011)
PDLSC Auto Nude rat Ectopic Chang J et al. (2007)
Auto Dog, Rat Fenestration Akizuki T et al. (2005), Majewski M et al. (2008)
Auto Rat Periodontal defects Jin QM et al. (2003)
CM Allo Rat Ectopic Zhao M et al. (2004)
DFC Allo Rat Ectopic Zhao M et al. (2004)
Modied from Rios HF et al., J Periodontol 82:12231227, 2011 [9].
BMSC = Bone marrow stem cells; ASC = Adipose stromal cells; PDL cells = Periodontal ligament cells; PDLSC = Periodontal ligament stem
cells; CM = Cementoblasts; DFC = Dental follicle cell; Auto = Autograft; Allo = Allograft; Xeno = Xenograft.
Figure 1 Required pieces for complete periodontal regeneration.
Possible Functional Scaffolds for Periodontal Regeneration 121
have been tailored to lms, bers, sheets, gels, and sponges
[31]. Inorganic materials also have been used for periodontal
regeneration to specically regenerate alveolar bone
defects, including hydroxyapatite (HA) and calcium phos-
phates (CaP). CaP materials such as b-tricalcium phosphate
(b-TCP) have excellent properties including: (1) a similar
composition to bone minerals; (2) the ability to form bone
apatite-like materials or carbonate HA; (3) the ability to
stimulate cells, leading to the formation of bone-CaP; and
(4) osteoconductivity [32]. HA is the major mineral compo-
nent (approx. 60%) of human hard tissues, and is often
clinically applied to ll alveolar bone defects (shown in
Table 1). HA can eliminate donor site morbidity, but can lead
to granular migration and incomplete resorption, resulting in
long-term difculties [33]. b-TCP has been shown to be an
osteoconductive material, allowing for bone growth onto the
scaffold adjacent to bony surfaces in both animal and human
studies [34,35], and has been clinically applied in combina-
tion with PDGF [36].
Even if the material is a polymer or inorganic, it is
expected to ll a specic anatomic defect to allow the
localization of transplanted cells; to serve as scaffolding for
the formation of new tissue; to provide a niche maintaining
stem cell viability and proliferation; and to recruit host
stem cells for subsequent homing to the site of injury [37].
The rst aim of the scaffold should be to contribute to the
maintenance of space for horizontally lost periodontal
tissues; however, no material was found to fulll this
requirement and induce complete periodontal regenera-
tion. Another point for successful periodontal regeneration
is how to protect the regenerating tissue from infection by
oral microorganisms. Periodontal tissue is the only appara-
tus in which soft and hard tissue attachments are exposed
to the outside environment. In skin tissue engineering,
the same property is also required; nanobrous polymer
materials were developed to release low molecular weight
antibacterial agents during degradation [38,39]. GTR/
guided bone regeneration (GBR) procedures have been
shown to fail when membranes were exposed to the oral
cavity and became infected [40]. To avoid contamination,
multiple studies were conducted to incorporate antibac-
terial agents such as tetracycline hydroxide and metroni-
dazole [41]. However, no such trial has been carried out
to develop an anti-infective scaffold specially designed
for periodontal regeneration. Further exploration of scaf-
fold-based infection control methods may contribute to the
elimination of bacteria, resulting in the successful genera-
tion of new tissue.
4. The desirable properties of scaffolds
designed for periodontal regeneration
4.1. Mimicking the stem cell niche to alter the
regulation of stem cells
The goal of tissue engineering and its application in genera-
tive medicine is the creation of functional tissues or organs.
Therefore, it is important not only to generate hard and soft
tissues in periodontal regeneration, but also to mimic the
attachment of both tissues. Cell-based tissue engineering
requires both cells from an appropriate origin to create the
target organ and a 3-dimensionally designed scaffold that
promotes cellcell proximity and enhances self-assembly
and tissue functions [42]. The ideal combination of cell
and scaffold sources varies in relation to the target tissue
to be created. Furthermore, recent advances in material
science developed new feasible biomaterials and fabrication
techniques, resulting in the creation of functional/bioactive
scaffolds that can activate cellular functions.
Table 3 Scaffold materials applied with/without signaling molecules for periodontal tissue regeneration.
Scaffold Growth factor (combination) Experimental model Reference
1. Naturally occurring polymers
Porous chitosan/coral composites PDGF or BMP-7gene Mouse Zhang et al. (2007)
Methylcellulose gel rhPDGF-BB/rhIGF-1 Human Lynch et al. (1997)
Hydroxypropyl cellurose FGF-2 Human Kitamura et al. (2008, 2011)
2. Synthetic polymers
PLA-PGLA copolymer + gelatin sponge rhBMP-2 Cat Takahashi et al. (2007)
Pluronic F127 TGF-b1 Sheep Mohammed et al. (1998)
3. Hydrogels
Dex-GMA gelatin gel rhBMP-2 Dog Chen et al. (2005)
Bovine insoluble collageous matrix - Monkey Ripamonti et al. (1996)
Collagen gel FGF-2 Dog Sato et al. (2004)
Gelatinous carrier FGF-2 Monkey Takayama et al. (2001)
4. Bioactive ceramics
Calcium phosphate cement - Dog Shirakata et al. (2002)
Calcium carbonate career TGF-b1 Dog Tatakis et al. (2000)
b-Tricalcium phosphate rhPDGF-BB Human Giannobile et al. (2006)
rhGDF-5 Monkey Emerton KB et al. (2012)
Hydroxyapatite (HA) rhBMP-2 Dog Wiskesjo et al. (2000, 2002)
5. Bone grafts
Bone allograft rhPDGF-BB Human Lynch et al. (2003)
Modied from Cheng F-M and Jin Y, Tissue Eng Part B 16:219255, 2010 [28].
122 H. Shimauchi et al.
Recently, the term biomimeticism has been introduced
in tissue engineering and refers to the creative initiation of
various specic biological systems gaining inspiration from
nature [4345]. It is also the case for adult stem cell therapy
because these cells reside in specialized niches that coordi-
nate self-renewal versus differentiation in vivo [46,47].
Thus, the mimicking stem cell niche is considered to facil-
itate self-renewal (proliferation) and differentiation both ex
vivo and in vivo after transplantation.
The microenvironment or niche surrounding stem cells is
shown in Fig. 2. Cytokine/growth factor signaling regulates
the proliferation and differentiation of stem cells as pre-
viously described. Cellcell and cellmatrix interactions
also transmit signals into stem cells, controlling stem cell
functions. Cellcell interactions occurred not only between
stem cells, but also between stem cells and supporting cells
that modulate stem cell retention and regulation. Several
cell surface ligands are known for their association with stem
cell activation, including cadherins and the Notch ligand
[48,49].
The third pathway is cellextracellular matrix (ECM)
interactions, including matrix composition, stiffness, and
topography. The ECM contains various proteins such as bro-
nectin and laminin, as well as proteoglycans (GAG), hyaluro-
nic acid, and bers (collagens and elastins) [50]. These
components can regulate cell behavior as well as support
cell growth because stem cells also have cell adhesion mole-
cules, including integrins and CD44, initiate intracellular
signaling, and associate with the cellular cytoskeleton
[51]. Differences in the composition and crosslink density
of the ECM in each organ and tissue have also been adapted
for their mechanical properties of stiffness and topography.
Cells can sense and respond to various signals, consisting
of biochemical and biophysical cues provided by the ECM.
In combination with the mechanical properties of cell
membrane, matrix stiffness affects the proliferation and
differentiation of stem cells [5254]. Cells are exposed to
a diverse topography including brous ECM and mineralized
bone with a rough surface. The ECM presents various geome-
trically dened and 3D physical cues in the order of a micron
and sub-micron scale, known as topographies [55]. Physical
cues in a cells surrounding environment are integrated and
converted to biochemical, intracellular signaling responses,
leading to the modication of cell function through a process
of mechanotransduction [56].
Oxygen gradients in the niche also affect stem cell func-
tion. Stem cell niches are known to be located in low oxygen
tension and low pO
2
regions, where the rate of cell differ-
entiation is decreased and proliferative potential is increased
[57]. Furthermore, oxidative stress was found to suppress
the E-cadherin-mediated cellcell adhesion of hematopoie-
tic stem cells (HSC) to osteoblasts, inducing the exit of HSC
from the niche [58].
Additionally, another cue should be added when we
consider effective bone regeneration strategies. Bone is a
biocompatible and self-remodeling tissue consisting of an
organic phase (mainly collagen type I, 20%) and an inorganic
phase (mainly carbonated hydroxyapatite, 60%) [32]. Dur-
ing bone metabolism, osteoclasts release Ca
2+
and PO
4
2
(Pi)
derived from the matrix, causing a local increase in ion
concentrations in the microenvironment, which plays a
role in osteoblast proliferation and differentiation, as well
as on subsequent bone formation. Increased extracellular
Ca
2+
concentrations are potent chemical signals for cell
migration and directed growth [59,60], as well as homing
signals that bring together the different cell types required
for the initiation of bone remodeling [61]. Pi is also a reg-
ulator of osteoblast proliferation and differentiation [62],
and the high concentration of Pi in the microenvironment
induced osteoblast apoptosis in vitro [63] and the in vivo
mineralization of the bone matrix [64]. Moreover, specic
Ca
2+
and Pi concentrations have been suggested to induce the
higher proliferation and osteogenic differentiation of MSC
[65]. CaP bioceramics appear to be candidates for scaffold-
aimed bone tissue engineering because they can release
inorganic ions during dissolution.
Figure 2 Interactions of stem cells with microenvironmental factors within the niche Ca: Calcium ion, Pi: Phosphate ion.
Possible Functional Scaffolds for Periodontal Regeneration 123
4.2. Cellscaffold interplay and scaffold
designing
As discussed above, many factors are associated with mimick-
ing of the cell niche/microenvironment to enforce regenera-
tion by the application of tissue engineering. Not only
scaffold properties affect stem cell functions; cells can also
induce the deformation and degradation of scaffolds during
the process of regeneration, leading to the altered mechan-
ical properties of scaffolds (Fig. 3). Thus, the cellscaffold
interplay is bidirectional and involves a feedback loop of cells
and scaffolds. Forces are generated in the context of cell
adhesion to ECM/scaffolds, and mechanotransduction occurs
to transduce them into intracellular signals that drive func-
tional modulations in cells: proliferation, differentiation,
migration, and apoptosis [55]. The actin cytoskeleton plays
the most prominent role in these events [66]. Human MSC
also express specic transcription factors of mechanotrans-
duction and undergo tissue-specic cell fate switches when
cultured on ECMs with mechanical stiffness mirroring the
physical properties of respective specic tissue [67].
To instruct stem cells and modify their fates, scaffold
biomaterial should provide informative microenvironments
mimicking a physiological niche of the target tissue. Bioma-
terials can have a suitable design to transmit specic signals
to cells that can be decoded into biochemical signals depend-
ing on its composition and processing methods. Both biophy-
sical and biochemical cues have been involved in cellECM
interactions in nature. Hence, topography, chemistry and
physical properties are involved and are critical for deter-
mining cell fate [68]. To accomplish the desirable interplay
between cells, biomaterial designing should include para-
meters within (a) surface, (b) mechanical, (c) morphological,
and (d) electrical properties (Fig. 3). Surface topography and
chemical composition were able to drive cell adhesion, pro-
liferation, migration, and differentiation [55,69,70]. Stem
cells can also respond to the mechanical properties of the
substrate, which were enhanced by the combination of
polymers and organic/inorganic llers [33,71], and osteoin-
duction without the supplementation of growth factors has
recently been reported [72]. Conductive nanostructures,
such as metal nanoparticles and carbon nanostructures,
may modulate the electrical properties of biomaterials,
affecting stem cell functions [73,74]. Scaffold morphology,
e.g., pore size and shape, is critical for cellbiomaterial
interactions in terms of mimicking the morphology of sur-
rounding tissue. High porosity and an adequate pore size in
particular are key conditions for increasing the surface area
available for cell attachment and growth. In the following
sections, the interactions between PDL cells and each para-
meter of the scaffold design discussed above will be covered
in detail using our ndings.
4.3. Application of a honeycomb microarray lm
for PDL cell sheet engineering
Cell sheet technology is a concept of tissue engineering that
provides a mass of cells to the site of regeneration by the
transplantation of an in vitro cultured multilayered cell
sheet. This technology has already been applied to the
regeneration of many organs in clinical settings, including
corneal surface reconstruction, endoscopic treatment of
esophageal ulceration, and myocardial tissue reconstruction
[75]. The cell sheet was successfully prepared from PDL cells,
and scaffold-free sheet transplantation could regenerate a
complete periodontium in a canine model [29]. Cell sheet
engineering is also applicable to 3D-tissue reconstruction of
tissue by applying an appropriate scaffold material to
increase the number of cell layers [76]. Skin reconstruction
is another medical eld applicable to cell sheet-based tissue
engineering. The honeycomb biodegradable collagen scaf-
fold has been shown to be suitable for 3D cell cultures [77]
because the honeycomb structure has many advantages, such
as mechanical stability under various physical conditions, the
capability to exchange nutrients and waste products through
the honeycomb pores, and the ability to retain its structure
without a deformity or collapse until its biodegradation [78].
However, the pore size of natural polymers such as collagen is
difcult to control.
The honeycomb pattern was also prepared on various
types of polymers by a simple casting method [7981].
Briey, the hydrophobic polymer solution was casted on a
basal plate, water droplets self-assembled on the polymer
solution with the addition of humid air, and the honeycomb
Figure 3 Cellscaffold interplay and the components of scaffold designing.
124 H. Shimauchi et al.
pattern was observed on the polymer lm after drying. We
could control the pore size of the honeycomb structure and
fabricate uniform pores using the casting method. This cast-
ing method is applicable for biodegradable polyester lms
made from poly(lactic acid) and poly(e-caprolactone) (PCL)
[82], which is useful as a cell culture substrate.
We cultured PDL cells on the honeycomb-patterned poly-
mer lms fabricated by the casting method with different
pore sizes. PDL cells were obtained from extracted molars
with a healthy periodontium, and subjected to experiments
during 35 passages. PDL cells were cultured on at (control)
and honeycomb PCL lms with 5- and 10-mm pores for 72 h.
Scanning electron microscopy (SEM) images were shown in
Fig. 4. PDL cells seemed to be spread on at lms (Fig. 4A),
although they rmly caught the pillar structure of the hon-
eycomb on the 5 mm-pored lm (Fig. 4B). Interestingly, PDL
cells migrated through the pores of the honeycomb structure
of the 10 mm lm (Fig. 4C). The schematic illustrations of PDL
cell behavior cultured on 5 and 10 mm-pored honeycomb
lms were given in Fig. 4D and E, respectively.
The 3D orientations of PDL cells in the honeycomb lms
were further observed using confocal laser scanning micro-
scopy after a long-term culture (28 days; Fig. 5). Fig. 5A
shows the 3D-constructed image of PDL cells cultured on the
10 mm-pored lm. PDL cells were seen inside the lm and
spread their bodies horizontally into the contiguously lined
pores. PDL cells constructed multi-layered cell sheet-like
structures after 28 days, and the shapes of cells on the upper
cell layer, on the surface, and inside of the lm were sepa-
rately presented in Fig. 5BD. The shapes and forms of the
cells were markedly exchanged by moving between the out-
side and inside of the honeycomb lms. PDL cells seemed to
be desperate to move through the honeycomb lumens and
showed a dendrite-like morphology form. Our results clearly
indicated that the pore size of articial substrates has a
marked effect on cell behavior, and the honeycomb structure
is suitable for the construction of a multi-layered cell sheet.
The topographical effects of the honeycomb lm also have a
signicant impact on PDL cell differentiation. We measured
the mRNA expression levels of the osteoblastic markers of
PDL cells cultured for 4 weeks on the honeycomb lm [83].
Osteopontin (OPN) and osteocalcin (OCN) expression levels
were higher than those on at lms, suggesting differentia-
tion into osteoblastic cells. This result was further conrmed
by the formation of calcied nodules on 10 mm-pored hon-
eycomb lms (data not shown).
4.4. Possible induction of cementogenesis
by modulation of the extracellular ionic
microenvironment using bioactive ceramic
scaffolds
To accomplish the restoration of the original architecture of
the periodontal apparatus, it is important to promote cemen-
togenesis rapidly on the root surface after root planning/
conditioning because the cementum is the only hard tissue
that can insert PDLs and assists in anchoring the tooth to the
surrounding alveolar bone [84]. Cementoblasts express alka-
line phosphatase (ALP), runt-related gene 2, type I collagen,
noncollagenous proteins, bone sialoprotein (BSP), and OCN in
a similar manner to osteoblasts [84,85]. According to the
anatomical location, which is in proximity to osteoblasts/
alveolar bone, but is separated by a PDL, cementoblasts may
be under a specic microenvironment resembling bone with
higher extracellular Ca
2+
and Pi concentrations in part
related to osteoclast-mediated bone resorption during alveo-
lar bone remodeling. Thus, these cells are physiologically
and/or pathologically confronted with alterations in the
concentrations of extracellular inorganic ions. Calcium
phosphate ceramics are widely used as bone substitutes
or scaffolds in dental elds because of their excellent
Figure 4 SEM images of PDL cells cultured on honeycomb (HF) lms. Periodontal ligament (PDL) cells were cultured for 72 h on (A): a
at PCL lm, (B): a 5 mm-pored honeycomb lm (HF), and (C): a 10 mm-pored HF. D and E show the schematic illustration of PDL cells on
5 mm- and 10 mm-HF, respectively.
Possible Functional Scaffolds for Periodontal Regeneration 125
osteoconductivity [86]. These ceramics are classied as non-
resorbable and resorbable, and can release or exchange Ca
2+
and Pi ions into their surroundings after implantation [87] as
discussed in the previous section. Whether cementoblasts
could sense extracellular Ca
2+
and Pi ionic concentrations and
altered cell functions such as cementogenesis and cytokine
production remained unclear [88]. An immortalized murine
cementoblast cell line (OCCM-30), established by the isola-
tion of tooth root-surface cells from transgenic mice contain-
ing a SV40 large T-antigen under the control of the OCN
promoter [89], was used for the following studies. OCCM-
30 cells were stimulated with 10 mM CaCl
2
for 24 h because
basal [Ca
2+
] was 1.8 mM. The expression of COX-2 and PGE
2
was signicantly increased in a time-dependent manner and
subsequently increased Fgf-2 mRNA (Fig. 6). OCCM-30
expressed all EP1, EP2, EP3, and EP4 receptors to PGE
2
. Only
an EP4 receptor agonist synergistically enhanced CaCl
2
-
induced Fgf-2 gene expression (data not shown). We nally
concluded that the exposure of cementoblasts to CaCl
2
activated NF-kB signaling and induced the expression of
Cox-2 as well as Ep4, which led to the sequential activation
of PGE
2
/EP4 signaling and increase in Fgf-2 expression levels
(Fig. 7). COX-2/PGE
2
/EP4 signaling may function as a positive
regulator for FGF-2 induction in cementoblasts. We pre-
viously reported that increased extracellular Ca
2+
increased
BMP-2 mRNA expression in human PDL cells as well as in the
dental pulp (DP) cells [90]. Furthermore, hDP and PDL cells
expressed Na-dependent Pi transporters (Pit-1, Pit-2) and
Figure 5 Confocal laser microscopy images of PDL cells cultured for 28 days on a 10 mm-pored honeycomb lm. (A) Three-
dimensional reconstructed image of vertical slices, (BD) PDL cells on the top of multilayered cells (B), center of cell layers (C), and
inside of the honeycomb lm (D), respectively.
Figure 6 Elevated concentrations of extracellular Ca
2+
induced Cox-2 mRNA expression and PGE
2
production, and subsequently up-
regulated the expression of Fgf-2 mRNA.
126 H. Shimauchi et al.
sensed extracellular Pi, resulting in the up-regulation of BMP-
2 mRNA [91]. Taken together, periodontal and dental pulp
cells can respond to changes in extracellular inorganic ion
concentrations, resulting in the signal transduction that leads
to proliferation/differentiation.
4.5. Application of nanofabricated
hydroxyapatite for bone tissue engineering
Hydroxyapatite (HA) is the prevalent form of CaP found in the
bone; therefore, it has been used as the stable alloplast and
scaffold for bone regeneration. However, HA has a lower
dissolution rate at physiological pH (7.27.6) than the other
types of CaP such as octacalcium phosphate (OCP) and
tricalcium phosphate (TCP), resulting in poor biological
responses. As this dissolution behavior has been associated
with osteoinductivity, previous studies attempted to engi-
neer CaP with an appropriately high solubility [87]. Combin-
ing the different scaffold fabrication technologies and
different biomaterials can provide cells with mechanical,
physicochemical, and biological cues at the macro- and
micro- scale, as well as at the nano-scale. Due to size effects
and surface phenomena at the nanoscale, nanosize HA (nano-
HA) possessed unique properties over its bulk-phase counter-
part. The high surface-to-volume ratio, reactivities, and
biomimetic morphologies may make nano-HA more favorable
in applications for bone tissue engineering [92,93]. We inves-
tigated the effect of nano-HA on BMP-2 expression in human
PDL cells [94]. Nano-HA selectively increased the expression
of BMP-2 in dose- and time-dependent manners (Fig. 8A and
B) at mRNA and protein levels, but not of BMP-4, -7, or -9
(Fig. 8C). However, concentrations of Ca
2+
as well as Pi were
not changed in culture supernatants (Fig. 9), suggesting that
nano-HA functioned as a nanoparticle rather than as a pos-
sible source of Ca
2
+
and/or Pi extracellularly, which were
Figure 7 Increased extracellular Ca
2+
synergistically up-regu-
lated FGF-2 expression via COX-2/PGE
2
/EP4 signaling, COX-2:
cyclooxygenase-2, EP4: EP4 receptor.
Figure 8 Nano-HA stimulated BMP-2 expression at the gene level in human PDL cells. (A) Cells were stimulated with the indicated
concentration of nano-HA for 72 h. (B) Cells were stimulated with 200 mg/ml nano-HA for the indicated times with a medium change
every 3 days. (D) Cells were stimulated with 200 mg/ml nano-HA for 72 h. Total cellular RNA was extracted and transcripts were
analyzed by real-time PCR.
Figure 9 Nano-HA mediated increases in BMP-2 were not caused by extracellular Ca
2+
or Pi. (A) Cells were stimulated with/without
200 mg/ml nano-HA, 10 mM CaCl2, or 3 mM Pi (as a mixture of NaH
2
PO
4
and Na
2
HPO
4
, pH 7.4) for the indicated times. Total cellular RNA
was extracted and transcripts were analyzed by real-time PCR. (B and C) Conuent cells were stimulated with/without 200 mg/ml
nano-HA for the indicated times. The concentrations of Ca
2+
and Pi in the supernatant were measured.
Possible Functional Scaffolds for Periodontal Regeneration 127
shown to also enhance the expression of BMP-2 in PDL cells.
We further revealed that nano-HA-dependent BMP-2 expres-
sion was dependent on p38 MAP kinase, but not on ERK1/2
MAP kinase (data not shown). Thus, nano-fabricated HA may
regulate the differentiation of hPDL cells via a mechanosen-
sitive signaling pathway. This novel mechanism of the action
of nano-HA may offer the promise of new strategies for bone
and periodontal tissue engineering.
5. Conclusions and future prospects
This review focused on the cellscaffold interaction possibly
encountered when tissue-engineering approaches are
applied to periodontal regeneration. Recent progresses in
periodontal regeneration technology allowed the clinical
application of cytokine therapies in dental clinics. However,
as discussed in the previous sections, these cytokine thera-
pies still have therapeutic limitations similar to those of
Emdogain and the GTR technique. To overcome the limita-
tions, researchers have extensively studied the application of
stem cell therapy in this eld, including autologous somatic
stem cells from a dental origin and iPS cells. Current progress
in tissue engineering approaches for periodontal regenera-
tion has been summarized in Fig. 10. The biocompatible
scaffold is another important strategy in tissue engineering
technology that has been adopted for the creation of new
tissue, whether stem cells are utilized or not. Scaffolds not
only can contain stem cells and mimic the microenvironment
suitable for these cells, but also can provide the controlled
release of growth factors including gene delivery [95].
Furthermore, the scaffold should ll a specic anatomic
space of the lost periodontal tissue including horizontally
resorbed alveolar bone. Thus, a 3D version of biomimicry is
the key for complete periodontal regeneration, although this
research eld is currently under intense exploration.
We discussed the possible periodontal cellscaffold inter-
actions that may be a powerful tool for developing the most
suitable scaffold. To date, many cellular and molecular
events involved in periodontal tissue repair/regeneration
have been revealed. These advances in understanding may
serve as the driving force toward a breakthrough for cell-
based regeneration strategies in our research eld. The rapid
progress in material sciences and micro/nano-fabrication
technology also pave the way for the development of new
tissue engineering techniques for complete periodontal
regeneration.
Conict of interest
None of authors have a conict of interest related to this
review.
Acknowledgements
Portions of the authors research discussed in this review
were supported by A Grant-in Aid for Scientic Research
(23390745 and 23659910 to H.S.). The authors would like
to thank Drs. Sousuke Kanaya, Nagayoshi Iwama, and Mizuki
Suto for their contributions in the research results shown
here.
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