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249
RESEARCH REPORTS
Biological
DOI: 10.1177/0022034509333804
Received April 19, 2008; Last revision January 6, 2009;
Accepted January 30, 2009
A supplemental appendix to this article is published elec-
tronically at http://jdr.sagepub.com/supplemental.
Y. Zheng1, Y. Liu1, C.M. Zhang1, H.Y.
Zhang2, W.H. Li2, S. Shi3, A.D. Le3*,
and S.L. Wang1, 4*
1Salivary Gland Disease Center and the Molecular
Laboratory for Gene Therapy & Tooth Regeneration, Capital
Medical University School of Stomatology, Tian Tan Xi Li
No.4, Beijing 100050, China; 2Department of Cell Biology,
Municipal Laboratory for Liver Protection and Regulation
of Regeneration, Capital Medical University School of
Basic Medical Sciences, You An Men Wai No.10, Beijing
100069, China; 3The Center for Craniofacial Molecular
Biology, University of Southern California School of
Dentistry, 2250 Alcazar Street, CSA 103, Los Angeles, CA
90033, USA; and 4Department of Biochemistry and
Molecular Biology, Capital Medical University School of
Basic Medical Sciences, You An Men Wai No.10, Beijing
100069, China; *corresponding authors, slwang@ccmu.
edu.cn and anhle@usc.edu
J Dent Res 88(3):249-254, 2009
ABSTRACT
Stem cells from human exfoliated deciduous teeth
have been identified as a new post-natal stem cell
population with multipotential differentiation
capabilities, including regeneration of mineralized
tissues in vivo. To examine the efficacy of utilizing
these stem cells in regenerating orofacial bone
defects, we isolated stem cells from miniature pig
deciduous teeth and engrafted the critical-size
bone defects generated in swine mandible models.
Our results indicated that stem cells from minia-
ture pig deciduous teeth, an autologous and easily
accessible stem cell source, were able to engraft
and regenerate bone to repair critical-size man-
dibular defects at 6 months post-surgical recon-
struction. This pre-clinical study in a large-animal
model, specifically swine, allows for testing of a
stem cells/scaffold construct in the restoration of
orofacial skeletal defects and provides rapid trans-
lation of stem-cell-based therapy in orofacial
reconstruction in human clinical trials.
KEY WORDS: deciduous tooth, stem cell,
bone, tissue engineering, miniature pig
Stem Cells from Deciduous
Tooth Repair Mandibular
Defect in Swine
INTRODUCTION
Reconstruction of orofacial defects secondary to tumors and trauma relies on
different sources of bone grafts with inherent morbidity. Stem-cell-based
tissue engineering is a promising alternative for bone regeneration (Petite et al.,
2000; Bianco et al., 2001; Rose and Oreffo, 2002). The stem-cell-based therapeu-
tic approach can restore bone defects without incurring graft donor site morbidity.
Bone marrow mesenchymal stem cells (BMMSCs) have emerged as an important
cell source for bone regeneration (Gronthos et al., 2003; Mankani et al., 2006;
Mastrogiacomo et al., 2007), as has been demonstrated clinically in femur fracture
repair and regeneration (Shao et al., 2006). Previous studies have indicated that
the orofacial bone and dental tissues contain a variety of stem cells, including
dental pulp stem cells (DPSCs), stem cells from human exfoliated deciduous teeth
(SHED), periodontal ligament stem cells (PDLSCs), and orofacial mesenchymal
stem cells (Akintoye et al., 2006). These orofacial stem cells possess a higher
proliferation capacity when compared with BMMSCs, and their origin may be
associated with neural crest cells (Gronthos et al., 2000; Shi et al., 2002; Shi and
Gronthos, 2003; Seo et al., 2004; Akintoye et al., 2006). When transplanted sub-
cutaneously into immunocompromised mice, SHEDs, an easily accessible stem
cell source, are capable of generating robust amounts of bone in vivo (Miura et al.,
2003; Seo et al., 2008), suggesting a potential for bone regeneration. To explore
the feasibility of using SHED-based bone regeneration to treat orofacial bone
defects, we utilized swine as a pre-clinical animal model to test the regeneration
of critical-size mandibular defects using SHEDs. Our previous studies demon-
strated that miniature pigs (minipig) are appropriate large-animal models for oral
and craniofacial tissue engineering and therapies (Shan et al., 2005; Sonoyama
et al., 2006; Wang et al., 2007; Yan et al., 2007; Liu et al., 2008). In this study, we
utilized minipigs as a large-animal model to examine the feasibility of using autol-
ogous stem cells derived from miniature pig deciduous teeth—that is, SPD—to
repair critical-size mandibular bone defects.
MATERIALS & METHODS
Animals
Sixteen inbred female minipigs (4–6 mos old, weighing 20–30 kg each) were obtained
from the Institute of Animal Science of the Chinese Agriculture University. Minipigs
were kept under conventional conditions, with free access to water and a regular supply
of soft food diet. The study was performed in accordance with a protocol approved by
the Animal Care and Use Committees of Capital Medical University.
Cell Culture
Deciduous incisor pulp tissues from first and second deciduous incisors were harvested
from 16 inbred female minipigs. SPDs were isolated and cloned following established
250 Zheng et al. J Dent Res 88(3) 2009
protocols (Gronthos et al., 2000; Miura et al., 2003; Seo et al., 2008;
Appendix).
Immunocytochemistry
SPDs were subcultured in 24-chamber slides. Cells were fixed in 4%
paraformaldehyde for 15 min, blocked with non-specific antibodies,
and incubated with either anti-STRO-1 (R&D, Minneapolis, MN, USA)
at dilutions of 1:200 to 1:500 or anti-vimentin (Chemicon, Temecula,
CA, USA) at a dilution of 1:500 for 1 hr according to the manufactur-
er’s protocol. To test mouse anti-human STRO-1 antibody cross-
reactivity with pig tissues, we performed immunocytochemical stain on
human SHEDs, minipig SPDs, and lymphocytes, and found that only
human SHEDs and pig SPDs were specifically stained with human
STRO-1 antibody. Based on these findings, we utilized mouse anti-
human monoclonal antibody to STRO-1 in this study. Samples were
subsequently incubated with goat secondary antibodies for 45 min, and
observed by fluorescence microscopy. Non-immune serum served as
negative control. Subsequently, sections were counterstained with
DAPI. We used an alkaline phosphatase detection kit (Chemicon) to
examine the expression of ALP according to the manufacturer’s proto-
col, and the result was observed by light microscopy.
Flow Cytometric Analysis
Detached cells were permeabilized with PBS containing 0.1% (wt/v)
saponin at room temperature for 20 min. After being blocked with nor-
mal serum, the cells were incubated with fluorescein isothiocyanate
(FITC)-conjugated STRO-1 antibodies (R&D Systems; clone STRO-1)
or phycoerythrin (PE)-conjugated ALP antibodies (R&D systems; clone
B4-78) for 30 min at room temperature. After 3 washes with PBS con-
taining 0.1% saponin, fluorescence was analyzed by a FACSCalibur
flow cytometer with CellQuest software (BD Bioscience, Palo Alto,
CA, USA). Positive cells were identified by comparison with the cor-
responding isotype controls (FITC- or PE-conjugated IgG) in which a
false-positive rate of less than 2% was accepted.
Transfection of eGFP Genes
Conditional retroviral supernatants were produced by the stable retrovirus-
producing cell lines PT67/eGFP as described previously (Brazelton and
Blau, 2005; Zhang et al., 2005). For transfection, about 1 x 105 SPD
grown in 6-well plates were incubated for approximately 20 hrs with a
mixture of 1 vol of viral supernatant and growth medium at equal vols
and in the presence of 8 μg/mL polybrene (Sigma, St. Louis, MO,
USA). A repeated transfection was performed in a period of 72 hrs, and
the transfected cultures were selected with G418 (100 μg/mL, Sigma).
The transfection efficiency of the cells was 80%.
Scanning Electron Microscopy
GFP-positive SPDs were grown on β-TCP carrier (Biomedical Materials
and Engineering Center of Wuhan University of Technology, China) for
7 days and fixed with 2.5% glutaraldehyde in 0.1 M sodium cacodylate
buffer (pH 7.2) for 2 hrs at 4˚C. The samples were examined under a
Hitachi S-520 scanning electron microscope (Hitachi, Tokyo, Japan;
http://www.hitachi.com/).
Reconstruction of Mandibular Bone Defects
in Swine Model with SPDs
A critical-size defect of 2.5 x 1.5 x 1.5 cm3 was surgically created in the
parasymphyseal region of the mandible in 10 minipigs for a long-term
(24 wks) follow-up so that we could evaluate bone regeneration (Henkel
et al., 2005). In another 6 minipigs, we created 2 smaller defects of 1.0
x 1.0 x 0.5 cm3 bilaterally in the parasymphysis of the mandible and
evaluated short-term post-surgical follow-ups, at 2 wks (n = 3) and 4
wks (n = 3) (Appendix Fig. 1).
Computed Tomography (CT) Assessment
of Bone Formation
CT imaging of the minipig’s mandible (Siemens Company, Bensheim,
Germany) was carried out at 2, 4, 12, and 24 wks after transplantation
at a scanning length of 0.75 mm.
Quantitative and Histological Evaluation
of Regenerated Bone
Bone specimens were fixed in 10% buffered formalin. Half of the
specimens were decalcified and embedded in paraffin, while the other
half were processed as non-decalcified. Sections of 5- to 6-μm thickness
from the embedded specimen were stained with H&E. The extent of
ossification within each section was analyzed semi-quantitatively by
histomorphometric techniques (Appendix). The non-decalcified sample
was sectioned into a 400-μm series of slices (Donath and Breuner,
1982), and GFP-labeled SPDs were observed by fluorescence micros-
copy (Olympus BX/TF, U-LH100HG, Tokyo, Japan). For pair-wise
comparison, data were analyzed by one-way ANOVA and the Bonferroni
method.
RESULTS
Isolation and Characterization of SPDs
In culture, SPDs form adherent clonogenic cell clusters of
fibroblast-like cells, similar to the morphology described in
SHEDs; approximately 67 single colonies were generated
from 105 single cells cultured at low cell density. These
colony-forming cells were spindle-shaped (Fig. 1A) and posi-
tively stained for STRO-1, vimentin, and ALP (Figs. 1B-1D).
Flow cytometric analysis showed an average of 10.9% of
SPDs positive for STRO-1 and 21.1% positive for ALP (Figs.
1G, 1H), suggesting a heterogeneic population of cells, as
previously reported in other post-natal mesenchymal stem
cells, including bone marrow (Shi and Gronthos, 2003), dental
pulp (Gronthos et al., 2000), and periodontal ligament stem
cells (Seo et al., 2004).
Expression of eGFP Gene and Fabrication
of an SPD-Scaffold Construct
The retroviral transfection was carried out on day 2 in culture,
followed by a repeated transfection on day 3. Subsequently,
transfected SPDs were selected with G418 (100 μg/mL), and
colonies expressing green fluorescence were expanded in growth
medium (Fig. 1E). After several passages, the progenies of GFP-
positive SPDs continued to express eGFP.
For fabrication of the cell-scaffold construct, approximately
2 x 107-4 x 108 GFP-positive SPDs at third and fourth passages
were seeded onto β-TCP scaffolds and cultured in growth
medium for 7 days in vitro. SEM studies showed that GFP+-
SPDs were able to grow on β-TCP scaffolds (Fig. 1F).
J Dent Res 88(3) 2009 Regeneration of Bone Tissues 251
SPD-mediated Bone
Regeneration in the
Reconstructed Mandibular
Defect
At 24 wks, animals were
killed, and the mandibles were
harvested for CT, gross mor-
phology, and histological anal-
yses (Fig. 2, Appendix Fig. 2).
CT scan analyses showed
nearly complete regeneration
of initial defect sites, with new
bone formation in the SPD/β-
TCP treatment group as shown
in coronal (Fig. 2A) and axial
sections (Appendix Fig.
2A), three-dimensional view
(Appendix Fig. 2B), as well as
gross bone morphology of a
sagittal bone section (Appendix
Fig. 2C). Histological sections
of the SPD/β-TCP grafted site
revealed newly formed lamel-
lar bone and degraded β-TCP
scaffold (Fig. 2B). In contrast,
in the β-TCP group, the defect
site was only partially restored,
with significant remaining
defect in the lateral cortex
of the mandible (Fig. 2C,
Appendix Figs. 2D, 2E, and
2F). Histologically, the scaf-
fold-treated defect was par-
tially filled with some new
bone formation, connective
tissues, and degraded β-TCP
(Fig. 2D). In the control, or
untreated, group, a marked
Figure 1. Characterization of stem cells derived from miniature pig deciduous teeth (SPD). (A) Single
colonies of SPD showed typical fibroblast-like morphology under light microscopy. (B) SPDs expressed
STRO-1 by immunohistochemical staining with anti-STRO-1 antibody and (C) ALP-positive staining with the
alkaline phosphatase detection kit under light microscopy. (D) Immunohistochemical staining with anti-
vimentin antibody showed 99.5% positive staining. (E) GFP-expressed SPDs showed fibroblast-like
morphology. (F) Scanning electron microscopy revealed GFP-labeled SPDs grown on β-TCP scaffold. (G-H)
An average of 10.9% of the 3rd-passage SPDs were positively stained for STRO-1 (G) and 21.1% for ALP
by flow cytometric analysis (H). Abbreviations: SPD, stem cells from pig deciduous teeth; ALP, alkaline
phosphatase. Scale bars: 100 µm in A; 30 µm in B, C, D, E; 4 µm in F.
bone defect remained with predominantly connective tissues
(Figs. 2E, 2F). The regenerated defect site displayed a mean of
83.1% of mineralized matrix in the SPD/β-TCP-treated group,
significantly higher than the 52.2% in the scaffold group (P <
0.01), and 28.4% higher than in the control group (P < 0.01)
(Fig. 2G).
At 4 wks post-transplantation, CT scan revealed partial bridg-
ing of the lateral cortical continuity defect and moderate bone
regeneration in the SPD/β-TCP-treated group, in conjunction with
early scaffold degradation (Fig. 3A). The gross view of the recon-
structed defect site displayed marked absorption of scaffold at the
junction of native bone and graft in the SPD/β-TCP-treated group
at the early stage of bone healing (Appendix Fig. 3B). Relatively
intact scaffold was observed in the β-TCP group (Appendix Fig.
3C), and a large bony defect remained in the untreated group
(Appendix Fig. 3D). Histological sections of decalcified speci-
mens showed abundant new islands of bone and blood vessels
amid degraded scaffold in the SPD-treated group at 4 wks post-
reconstruction (Figs. 3B, 3C). In contrast, in the β-TCP group, the
lateral cortical rim defect remained, with minimal β-TCP absorp-
tion (Fig. 3D). The bone void at the defect site remained unfilled
in the control group (Fig. 3G, Appendix Fig. 3D). Histological
sections showed a lack of new bone formation at the junction of
β-TCP scaffold (Figs. 3E, 3F) and abundant connective tissue
proper in the untreated group (Figs. 3H, 3I). These findings dem-
onstrated that bone regeneration was significantly higher in the
SPD/β-TCP-treated group as compared with the β-TCP group. In
the blank (control) group, limited bone regeneration was observed
in the defect area, which was filled predominantly with connec-
tive tissue proper (Figs. 3G, 3H, 3I, Appendix Fig. 4D).
Detection of Transplanted GFP-positive SPD
in Regenerative Bone
To identify if the GFP-positive SPDs engrafted at the transplanted
bone defect sites had differentiated into osteoblasts, we first
screened non-decalcified sections using light microscopy for loca-
tion of osteoblasts and bone lacunae in the new bone at 2 wks and
252 Zheng et al. J Dent Res 88(3) 2009
4 wks post-operatively (Figs. 4A-4D).
Using the same visual field, we captured
GFP expressing SPDs under a fluorescence
microscope (Figs. 4B, 4D). When both
images in Figs. 4C and 4D were overlaid,
the locations of some GFP-positive SPDs
were superimposed with the osteoblasts
and bone lacunae (Fig. 4E, yellow arrows
showing GFP-negative osteoblasts, red
arrows showing GFP-positive osteoblasts),
suggesting that GFP-positive SPD cells
might have differentiated to osteoblasts. As
expected, the normal bone tissue section
showed a uniformly distributed green fluo-
rescence signal without local accumulation
of GFP-positive cells under immunofluo-
rescence microscopy (Fig. 4F).
DISCUSSION
Craniofacial tissue engineering by stem
cells is a fast-moving field with consider-
able potential clinical applications (Mao
et al., 2006; Kaigler et al., 2006; Zhao
et al., 2007). Deciduous tooth stem cells are
an easily accessible stem cell source and
capable of robust ex vivo expansion for
several potential clinical applications
(Miura et al., 2003; Mao et al., 2006; Seo
et al., 2008). Embryo logically derived from
the neural crest cell, SHEDs and SPDs may
share similar tissue origin with the man-
dibular bone cells, and therefore, may serve
as a better cell source for the regeneration
of alveolar and orofacial bone defects.
A previous study showed that the bone-
regenerative capacity of SHEDs was simi-
lar to that of bone marrow mesenchymal
stem cells (BMMSC) when transplanted
into immunocompromised mice at 8 wks
post-transplantation (33% mineralized
matrix/area in the SHED group vs. 31%
mineralized matrix/area in the BMMSC
group) (Seo et al., 2008). Similar to SHEDs
and other post-natal mesenchymal stem
cells derived from bone marrow (Shi and
Gronthos, 2003), dental pulp (Gronthos
et al., 2000), and periodontal ligament stem
cells (Seo et al., 2004), it is not unexpected
that the SPDs are a heterogeneic population
of cells which, following ex vivo cultures,
may display different percentages of cells
positive for STRO-1, or form mineralized
nodules, or be positive for Oil red O under
differentiation inductive conditions.
In this study, to investigate SPD-
mediated bone formation in vivo, we labeled
SPDs with GFP and used SPDs to repair
critical-size bone defects in the mandibles in
swine. At 2 and 4 wks after transplantation,
Figure 3. SPD-mediated bone regeneration of small-size mandibular defect at the short-term (wks)
follow-up. (A, B, C) SPD/β-TCP-treated group: CT axial image showing replacement of β-TCP scaffold
with new bone restoration (red arrow), and bridging of the lateral cortical defect with new bone
formation (blue arrow). H&E stain of the regenerated defect site at 10X (B) and 40X (C) revealed
several new islands of bones and blood vessels amid degraded scaffold. (D, E, F) β-TCP-treated
group: CT axial scan showing a partial bridging of the cortical defect and no visible bone formation;
H&E stains (E, F) showing sparse new bone formation at the junction of β-TCP scaffold and partially
degraded β-TCP. (G, H, I) Untreated, or control, group: CT axial scan showing a continuity defect in
the lateral cortex and lack of bone regeneration. Histological sections show primarily connective tissues
filling the defect of the control group (H and I). Scale bars: 1cm in A, D, G; 1 mm in B, E, H; 100 µm
in C, F, I. BT, bone tissue; BV, blood vessel; CT, connective tissue.
Figure 2. SPD-mediated bone regeneration of critical-size mandibular defect at long-term follow-up. (A,
B) SPD/β-TCP-treated group: CT coronal image showed a large quantity of new bone formation filling
the bone defect at 24 wks post-transplantation; yellow dot area indicates the original site of the bone
defect (A); the histological section shows that the defect was filled with new bone (B). (C, D) β-TCP-treated
group: CT coronal image shows remaining smaller bone defect at the reconstructed defect site;
histological section shows that the defect was partially filled with connective tissues, β-TCP scaffold, and
new bone formation (D). (E, F) Untreated, or control, group: CT coronal image shows limited bone
regeneration and large bone defect remaining; histological section shows that the defect was filled
primarily with connective tissues. (G) Semi-quantitative analysis of bone formation showed a statistically
significant increase in mineralized matrix formation at the regenerated defect site, 83.1 ± 5.75% (mean
± SD, n = 4) in the SPD/β-TCP treated group, compared with 52.2 ± 4.54% (n = 3) in the scaffold group
(P < 0.01), and 28.4 ± 2.79% (n = 3) in the control group (P < 0.01). Scale bars: 1 cm in A, C, E; 50
µm in B, F; 30 µm in D. BT, bone tissue; BV, blood vessel; CT, connective tissue.
J Dent Res 88(3) 2009 Regeneration of Bone Tissues 253
green fluorescence signals were
detected by fluorescence microscopy
within newly formed woven bone.
Photomicrographs of the same visual
fields confirmed that GFP-labeled
SPDs had differentiated directly into
new bone, while the normal bone tis-
sue section was non-specifically labeled
with green fluorescence. These find-
ings suggested that SPDs were
engrafted to some extent at the treated
site and contributed to new bone regen-
eration in the restoration of the bone
defect in the swine mandible model.
The results of CT scan, gross view, and
histological analyses consistently
showed that the SPD group had the
earliest and strongest capacity of bone
regeneration compared with other
groups. At 4 wks post-transplantation,
β-TCP was partly degraded and was
replaced with a large quantity of new
bone formation. At 6 mos post-recon-
struction, the defects in the SPD group
were markedly restored with new bone,
while in the β-TCP group and control
group, much less bone regeneration
and predominantly connective tissue
granulation were evident at the defect
sites. Further studies to regulate bone
regeneration are under way to optimize
the transplanted stem cell numbers,
scaffolds, and their immediate niche
component.
Overall, our study provides the
first evidence that SPDs are capable of
regenerating critical-size defects in
the orofacial bone in a large-animal
model, specifically swine, and may
potentially serve as an alternative
stem-cell-based approach in the recon-
struction of alveolar and orofacial
bone defects.
ACKNOWLEDGMENTS
This work was supported by
grants from the National Basic
Research Program of China (No.
2007CB947304), the Beijing Major
Scientific Program (D090600700
0091), the National Natural Science
Foundation of China (Grants 3042
8009 and 30801297), and the US National Institutes of Health
(NIDCR R01DE17449 to S.S.).
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