Biomaterials 33 (2012) 6974e6986

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Biomaterials
journal homepage: www.elsevier.com/locate/biomaterials

The effect of aging on the pluripotential capacity and regenerative potential

of human periodontal ligament stem cells
Jing Zhang a, b, c,1, Ying An a, b,1, Li-Na Gao a, b, c,1, Yong-Jie Zhang b, c, Yan Jin b, c, **, Fa-Ming Chen a, c, *
a
Department of Periodontology & Oral Medicine, School of Stomatology, Fourth Military Medical University, Xi’an, China
b
Research and Development Center for Tissue Engineering, Fourth Military Medical University, Xi’an, China
c
Translational Research Team, School of Stomatology, Fourth Military Medical University, Xi’an, China

a r t i c l e i n f o a b s t r a c t

Article history: Multipotent postnatal stem cells can be isolated from human periodontal ligaments (PDLs) and have the
Received 27 May 2012 potential for large-scale expansion, offering a reliable cell source for clinical use in periodontal regen-
Accepted 17 June 2012 erative therapies. However, the effects of aging on the mesenchymal stem cell (MSC) properties of these
Available online 11 July 2012
cells remain undefined. The aims of this study were to isolate and characterize the periodontal ligament
stem cells (PDLSCs) derived from human impacted third molars of donors of different ages and to
Keywords:
compare their pluripotential capacity and regenerative potential. PDL tissues were obtained from 90
Aging
surgically extracted third molars and divided into four groups according to the donor’s age. For each
Periodontal ligament stem cells
Cytotherapy
group, the colony-forming ability, proliferative capacity, migratory potential, cell surface antigens,
Periodontal tissue engineering differentiation ability, alkaline phosphatase activity, and gene expression of the PDLSCs were contras-
Regenerative medicine tively evaluated and quantified for statistical analysis. The in vivo tissue regenerative potential of PDLSCs
was assessed by an in vivo ectopic transplantation model. It was found that human PDLSCs were
successfully isolated and characterized as MSCs in all 90 teeth. PDLSCs derived from donors of different
ages were successfully differentiated under an osteogenic and adipogenic microenvironment. The
proliferative and migratory potential and the differentiation capacity of PDLSCs decreased as age
increased (p < 0.05). PDLSCs derived from donors whose age is 62.6
6.8 have a statistically significant
decrease in pluripotential capacity compared with those derived from relatively young donors (p < 0.01).
There is no identified cementum and PDL-like tissue formation in vivo among the two aging groups. We
conclude that human PDLSCs could be successfully isolated from PDL tissue derived from donors of
different ages, but the age-related changes of the MSC properties should be taken into account whenever
they are intended for use in research or cytotherapy.
Ó 2012 Elsevier Ltd. All rights reserved.

1. Introduction involvement of the regeneration of three unique tissue types,

Periodontal-tissue breakdown around natural teeth due to
periodontitis, a worldwide medical concern and a leading cause for
tooth loss, poses a critical challenge for clinical regenerative
dentistry [1]. The reestablishment of damaged tooth-supporting
apparatus is an extremely complex process because of the
* Corresponding author. Department of Periodontology & Oral Medicine, School
of Stomatology, Fourth Military Medical University, 145th West Changle Road, Xi’an,
Shaanxi 710032, China. Tel./fax: þ86 29 84776096.
** Corresponding author. Research and Development Center for Tissue Engi-
neering, School of Stomatology, Fourth Military Medical University, 145th West
Changle Road, Xi’an, Shaanxi 710032, China. Tel.: þ86 29 84776472; fax: þ86 29
83218039.
E-mail addresses: yanjin@fmmu.edu.cn (Y. Jin), cfmsunhh@fmmu.edu.cn
(F.-M. Chen).
1
These authors contributed equally to this manuscript.
namely cementum, periodontal ligament (PDL), and alveolar bone,
and the need to rebuild their intricate structures [2]. It is obvious that
the current treatment strategies for periodontitis fail to completely
and reliably reconstitute all tissues and connections damaged
through periodontal disease [3]. In light of this, cell-based tissue
engineering paradigm is considered as an alternative approach to
the current treatments and may help to alleviate the shortcomings
of more conventional therapeutic options by regenerating living and
functional periodontal structures [4e8]. Among the three essential
components required for tissue engineering design, stem cells are
the most fascinating area of research today and have been used in
the field of pre-clinical and clinical regenerative medicine with the
aim to treat many degradative diseases including periodontitis
[9e11]. Of note, there are substantial data that confirm the presence
of various stem cells in dental tissues, such as PDL, dental pulp,
follicle, and papilla, in and around a tooth; these dental stem cells

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 J. Zhang et al. / Biomaterials 33 (2012) 6974e6986
have received extensive attention in the field of periodontal regen-
erative medicine due to their accessibility and multilineage differ-
entiation capacity [8,12]. The accumulating knowledge on the cell
biology of dental stem cells, together with recent advances in
materials science and developmental science, have opened new
avenues that aim to use those easily available stem cells in clinical
reconstructive dentistry for the treatment of periodontal disease
[4,6e8]. It is remarkable that, following several feasible/pilot studies
[13e15], several clinical trials (e.g., NCT01357785 ) involving the
transplantation of stem cells (i.e. autologous PDLSCs) into peri-
odontal defects have already begun or are in preparation.
Before the identification of stem cell populations in the PDL
tissue, there has been mounting evidence that PDL cells exhibit
osteoblastic characteristics because they are capable of producing
mineral-like nodules [16] and express bone-associated genes,
osteocalcin (OCN), osteopontin (OPN), bone sialoprotein (BSP), bone
morphogenic protein (BMP)-2, BMP-4, and alkaline phosphatase
(ALP) [17e20]. In 2004, a pioneer study demonstrated that the PDL
contains stem cells, generally termed PDL stem cells or PDLSCs, that
have the potential to differentiate into cementoblast-like cells,
adipocytes, and collagen-forming cells in vitro and to generate new
cementum/PDL-like compartments in vivo [21]. Further research
demonstrated that they exhibit a fibroblast-like morphology and
express CD90, CD29, CD44, CD166, CD105, and CD13 antigens that
have also been identified as stromal precursors of bone marrow [22].
Interestingly, recent findings suggest that PDLSCs could be isolated
from both healthy and inflamed young PDL tissue (16e29 years of
age); the inflamed PDLSCs retain their regenerative potential for
cementum and related PDL tissues, suggesting an alternative rich
source (i.e., teeth extracted for periodontitis reasons) of mesen-
chymal stem cells (MSCs) for cytotherapeutic use due to the large
number of periodontitis patients involved [23]. Clearly, the use of
PDLSCs involves less religious and ethical concerns than using MSCs
derived from bone marrow because they are easily obtainable from
medical waste, i.e., the teeth extracted for orthodontic, impaction, or
irreversible periodontic reasons [8]. Therefore, PDLSCs represent
a unique population of MSCs that may facilitate translational
research and have future clinical application in, but not limited to,
periodontal regenerative medicine [8,24].
The ability to identify and manipulate human PDLSCs has been
a significant advancement in regenerative dentistry and has
contributed to a significant development in the progress toward
predictable periodontal regeneration [21e24]. In general, cell-
based periodontal therapy involves the delivery of ex vivo
expanded cell populations to the periodontal defect [8] or the
mobilization of endogenous progenitor cells capable of prolifer-
ating and differentiating into the required tissues [2]. The latter
strategy that relies on the stimulation of the intrinsic regenerative
potential of host tissues unfortunately does not offer a universal
regenerative solution. Innate powers of regeneration may be
restricted by an age-related decline in progenitor populations that
can be mobilized or by the intrinsically low regenerative potential
of certain tissues [25e27]. In these cases or when the original tissue
has been completely destroyed, biomaterial interventions that
include an external source of stem cells may be required [8]. Today,
the methods to isolate, purify, and characterize multipotent stem
cells in human PDL have been well delineated [21e24], and the
large-scale expansion of stem cells to cater to the need for clinical
quantity without using animal sera as nutritional supplements is
becoming possible [28,29]. Nevertheless, considering the
complexity of the PDL attachment apparatus and the heterogeneity
of its cell populations, the standardization of the appropriate cell
populations from PDL tissues necessary for complete regeneration
remains one of the key factors in implementing optimal approaches
to periodontal regeneration [6e8].
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6975
Despite the presence of a feasible study that uses autologous
PDLSCs in clinical periodontal therapy [14], the influences of aging
on the MSC properties of PDLSCs remain largely unexplored. These
influences may have significant effects on final clinical outcomes
because tissue regenerative potential may be negatively regulated
by aging. Understanding such effects is particularly crucial for
autologous therapy development in older subjects whom degen-
erative diseases typically afflict. In fact, the age-related effects of
stem cells have been noted in different tissue sources [30e36].
Furthermore, the loss of the proliferation and differentiation
capacity of PDLSCs was also found in six aged female donors with
a mean age of 54
3.2 years compared with the capacities of eight
young female donors aged 15
2.4 years [37]. The identification of
the effects of aging on PDLSCs needs further investigation that
involves looking at different groups of people of different ages, each
group including a relative narrow age range of subjects. The aims of
this study were to isolate and characterize the PDLSCs derived from
a wider range of donor ages (from less than 20 years to more than
60 years) and to compare their pluripotential capacity and regen-
erative potential in a relatively large amount of samples.
2. Materials and methods
2.1. Study design
Human third molars without caries, inflammation, or periodontitis were
extracted for impaction reasons from 54 systemically healthy donors between 16
and 75 years of age at the Fourth Military Medical University Dental Hospital, Xi’an,
China. For the aged donors whose third impacted molars were not removed in the
correct stage, the extraction of teeth was based on complete oral health examina-
tions showing that the corresponding teeth were harmful to the donors’ oral health
and must be removed. The age and sex of the donors are shown in Table 1 and their
extracted teeth were divided into A, B, C, and D groups solely based on the donors’
age. If one donor had more than two teeth that could be extracted, two teeth were
randomly included in the present investigation. If a tooth was destroyed or
contaminated during or after the surgery, it was automatically excluded from the
study. However, if the cells failed to grow from tissue samples for nontechnical
reasons, the donor information was included into final statistical analysis. Each tooth
was studied independently, even if two were from the same donor.
2.2. Isolation of PDLSCs
Human PDLSCs were freshly isolated and cultured according to previously
reported protocols with slight modification [24,37]. Briefly, each tooth was rinsed
and then the PDL tissues were separated from the mid-third of the root surface. PDL
tissues were further washed several times and then minced into small tissue cubes
(approximately 1 mm3). Subsequently, the tissue cubes were digested with a solu-
tion of 3 mg/mL collagenase (type I) with 4 mg/mL dispase (both from
SigmaeAldrich, St. Louis, MO, USA) in a-minimum essential medium (a-MEM,
Hyclone, Road Logan, UT, USA) for 15 min at 37  C with vigorous shaking. The tissue
explants were then plated into six-well culture dishes (Costar, Cambridge, MA, USA)
containing a-MEM supplemented with 10% fetal bovine serum (FBS; Hyclone),
0.292 mg/mL glutamine (Hyclone), 100 units/mL penicillin streptomycin (Hyclone),
and 100 mM/L ascorbic acid (SigmaeAldrich) and incubated at 37  C in a humidified
atmosphere containing 5% CO2. Single cell-derived colony cultures were obtained
using the limiting dilution technique and passage 0 (P0) cells were cultured. To
avoid changes in cell behaviors that are associated with prolonged culture, the
cells at passages P3eP5 were used for contrastive investigation in the present study.
Table 1
Isolation of human periodontal ligament stem cells from impacted third molars of
different aged donors.
Groups Age range Average age Donors Sex Teeth (n)
(year) (year) (n)
A 16e30 22.3
7.7 15 M ¼ 7; F ¼ 8 24
B 31e40 35.6
4.3 14 M ¼ 7; F ¼ 7 23
C 41e55 48.2
6.1 12 M ¼ 6; F ¼ 6 21
D 56e75 62.6
6.8 13 M ¼ 6; F ¼ 7 22
A total of 90 healthy teeth from 54 donors were involved in the present study and
divided into A, B, C, and D groups according to the age of the donors. Periodontal
ligament tissues from each tooth were independently collected for cell culture and
subsequent investigations. M, male; F, female.
6976 J. Zhang et al. / Biomaterials 33 (2012) 6974e6986

2.3. Colony-forming assay
The human PDLSCs (P3) were plated at a density of 0.25 to 1 103 cells/well in a six-
well plate (two wells for each cell line) and cultured in complete medium for 14 days for
colony-forming unit-fibroblast (CFU-F) assays. All cultures were fixed with 4% para-
formaldehyde and then stained with 0.1% toluidine blue. The cells were washed twice
with distilled water, and the number of colonies was counted and contrastively
analyzed. For colony number counting, only aggregates of >50 cells were scored as
colonies; colonies < 50 cells in number and/or faintly stained were excluded.
different groups were statistically compared. Also at the day 1, 3, 5, 7, 9, and 12 time
points, cell viability was determined by the trypan blue method. All four fields of the
hemocytometer were used to count cells from each well. Four independent experi-
ments were conducted for each group of cells from a single tooth. For the scratch assay
of cell motility, PDLSCs were seeded in a 6-cm dish (Costar) (0.2  106/dish). When the
cultured cells reached 80% confluence, monolayer cells were scratched using pipette
tips to make a cell-free strip area and washed with phosphate buffered saline (PBS) to
eliminate any floating cells. After 24 and 48 h, the cells were fixed and observed under
a light microscope, and the distance that the cells had migrated was measured for
statistical analysis.

2.4. Cell proliferation, viability, and motility assays

The cell proliferative potential was determined by the tetrazolium salt [MTT, 3-
(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay. Briefly, the cells
were plated into 96-multiwell plates (Costar) at a density of 1  104 cells/mL with
200 ml standard culture medium per well and incubated for 24 h at 37  C in
a humidified atmosphere containing 5% CO2 for cell attachment and spreading.
Subsequently, the media was changed to a-MEM containing 10% FBS, and the cells
were cultured for a total of 12 days. Media were changed every 3 days. At time points
of day 1, 3, 5, 7, 9, and 12, 20 ml MTT solution (SigmaeAldrich, 5 mg/m1) was added into
each well and incubated at 37  C for 4 h, allowing viable cells to reduce the yellow
tetrazolium salt (MTT) into dark blue formazan crystals. Finally, the MTT solution and
medium were discarded, and formazan crystals were dissolved with 200 ml of solu-
bilization solution (dimethylsulfoxide). The absorbance in individual wells was
determined at 540 nm by a microplate reader (ELx800, BioTek Instruments Inc., USA)
and expressed as optical density with SoftMax Pro software. The results from the
2.5. Flow cytometric analysis of cell surface markers
The percentages of CD21-, CD29-, CD34-, CD44-, CD90-, CD105-, CD146- and
STRO-1-positive cells were analyzed by flow cytometry. Briefly, the fourth genera-
tion of isolated PDLSC aliquots (>2  105 cells) were washed, fixed by adding 4%
paraformaldehyde for 15 min, and then resuspended in PBS supplemented with 3%
FBS that contained saturating concentrations (1:100 dilution) of primary antibodies
(BD Bioscience, San Jose, CA, USA) raised against CD21, CD29, CD34, CD44, CD90,
CD105, CD146, and STRO-1 for 1 h at 4  C in the dark. The cells were then washed
with buffer, and the secondary antibody (to which fluorescein isothiocyanate FITC
was attached) was added for 45 min at room temperature (20e25  C). Following
buffer washing, the samples were analyzed with a flow cytometry cell sorting
Vantage Cell Sorter (Becton & Dickinson, Mountain View, CA, USA). The data were
analyzed with a Mod-Fit 2.0 cell cycle analysis program (Becton & Dickinson).

Fig. 1. Initial cells growing out from periodontal ligament (PDL) tissues when the tissue explants were plated into six-well culture dishes containing a-minimum essential medium
(a-MEM) supplemented with 10% fetal bovine serum, 0.292 mg/mL glutamine, 100 units/mL penicillin streptomycin, and 100 mM/L ascorbic acid and incubated at 37  C in an
atmosphere containing 5% CO2. Representative figures (a) showing that the rate of initial cells growing was influenced by donor age (14 d, 40). The percentages of cell confluence
at day 14 (b) and the average time to 80% cell confluence (c) showing an age-related reduction in the growth rates of initial cells. The values are the mean
standard deviation
(*p < 0.05; **p < 0.01; ***p < 0.001).

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 J. Zhang et al. / Biomaterials 33 (2012) 6974e6986 6977

a A B C D

b A B c

**
40 *
**
36 * *
32
28

Colony units
24
C D 20
16
12
8
4
0
A group B group C group D group

Fig. 2. Colony-forming unit-fibroblast (CFU-F) assays of periodontal ligament stem cells (PDLSCs). (a) Representative figures showing the proliferation of a single clone of PDLSCs (A,
B, C, and D representing a representative colony from the A, B, C, and D groups, respectively) (14 d, 20); (b) Representative figures showing that the colony number of PDLSCs (A, B,
C, and D representing an investigating plate from the A, B, C, and D groups, respectively) was influenced by donor age (14 d, 20); (c) The graph represents a statistically significant
difference in the total colony number among PDLSCs in different age groups, where values are the mean
standard deviation (*p < 0.05; **p < 0.01).

2.6. ALP activity
3
The cells (P4) were plated on 96-well plates at a density of 2  10 cells/well and
cultured in complete medium for 48 h. Then, the medium was changed to complete
medium with osteoinductive supplements, i.e., 100 mg/mL ascorbic acid, 10 mM b-
glycerophosphate (SigmaeAldrich), and 50 nM dexamethasone (SigmaeAldrich).
After 3, 5, 7, and 10 additional days of culture, the cells were rinsed with PBS
three times and fixed in 70% ethanol. ALP activity was evaluated after incubation
with 10 mM p-nitrophenyl phosphate (3 mM final concentration; Beyotime Institute
of Biotechnology, Nantong, China) as a substrate; the plate was covered with foil and
incubated at 37  C for 30 min. The substrate solution contained the following in
15 mL TriseHCl (pH 9.6): 12 mg Fast Blue BB Salt, 4 mg naphthol AS-TR phosphate,
and 0.15 mL dimethyl formamide (DMF). The naphthol was first dissolved in the
DMF before being mixed with the TriseHCl. The mix was filtered, and 50 mL MgCl2
was added. The addition of NaOH quenched the reaction, and the absorbance was
measured at 520 nm using an enzyme-linked immunosorbent assay reader (EL312,
Bio-Tek Instruments, USA). Thereafter, ALP staining was performed and quantifica-
tion of the staining density was performed with Scion Image software (Scion Corp.,
Frederick, MD, USA).
2.7. Differentiation and gene expression analysis
The cells (P4) were seeded in 60-mm dishes at 6  104 cells/cm2 for gene
expression analysis and in six-well plates at 1  105 cells/well to assess the mineral
nodule and lipid formation in vitro. The cells were cultured in standard medium until
they reached subconfluence. The medium was then switched to a calcification

A group ***
**
B group *** **
5.0 ** * * *
C group * **
Absorbance at 540 nm

4.5 *
D group
4.0 *** *
3.5 **
* **
3.0 * *
2.5
2.0 *
1.5
1.0
0.5
0.0
1 3 5 7 9 12
Time cause (day)

Fig. 3. Proliferative activities of human periodontal ligament stem cells (PDLSCs) in different age groups assessed by 3-(4,5-dimethylthazol-2-yl)-2,5-diphenyltetrazolium bromide
(MTT) assay. PDLSCs from all groups showed increased proliferation over the time course: : (A group), ; (B group), C (C group), - (D group) p < 0.05; :: (A group), ;; (B
group) p < 0.01. After 7, 9, and 12 days, PDLSCs from group A showed increased proliferation compared with the other groups. The effect of aging on the cellular proliferation of
PDLSCs showed the most significance at 9 days post-treatment. The values are the mean
standard deviation (*p < 0.05; **p < 0.01; ***p < 0.001).

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6978 J. Zhang et al. / Biomaterials 33 (2012) 6974e6986

b 24 h 48 h A group
B group
C group
D group
***
2.0 **
A group *** * **
1.8 ** * *
Cell migration (mm)

1.6 B group * **
1.4 C group
*
*
1.2 D group
1.0
0.8
0.6
0.4
0.2
0.0
0 hour 24 hour 48 hour

Fig. 4. Migratory activities of human periodontal ligament stem cells (PDLSCs) from donors of different ages. (a) Representative figures showing cell migration of PDLSCs at 24 and
48 h after scratching (20). (b) The distance achieved by cell migration was measured using an image-analysis program, where a statistically significant difference was observed
between different groups after 24e48 h of migration. The values are the mean
standard deviation (*p < 0.05; **p < 0.01; ***p < 0.001).

medium composed of a-MEM containing 10% FBS, 50 mg/mL ascorbic acid, 10 nM
dexamethasone, and 10 mM b-glycerophosphate for osteoblastic differentiation and
gene expression analysis or an adipogenic medium, which consisted of complete
medium supplemented with 100 nM dexamethasone, 0.5 mM 3-Isobutyl-1-Methyl-
xanthine (SigmaeAldrich), and 50 mM indomethacin (Wako Pure Chemical, Osaka,
Japan). The osteogenic/adipogenic inducing medium was refreshed at 3-day inter-
vals. After 2 weeks of adipogenic differentiation, the cells were fixed with 70%
ethanol for 15 min at room temperature and then stained with fresh Oil Red O
solution (SigmaeAldrich) for 15 min at room temperature. After 3 weeks of osteo-
genic differentiation, the cultures were fixed in 70% ethanol for 15 min and stained
with alizarin red (pH 4; SigmaeAldrich) for 15 min. The mineral nodule and lipid
areas were measured quantitatively with an image analysis system (Image-Pro Plus
5.0; Media Cybernetics). In parallel, total cellular RNA was isolated from the main-
tenance cell cultures using a reagent (Gibco) at days 3, 7, 10, and 21 to determine the
expression of OCN, OPN, and BSP by real-time polymerase chain reaction (RT-PCR)
(Takara RNA PCR kit AMV, Takara Shuzo, Tokyo, Japan). The mean fold changes in
gene expression were calculated using the values obtained from PDLSCs without
induction on day 3. The primer sequences for OCN, OPN, or BSP (Sango Biotech,
Shanghai, China) are listed below: ACTIN (forward: 50 -TGGCACCCAGCACAATGAA-30 ,
reverse: 50 -CTAAGTCATAGTCCGCCTAGAAGCA-30 ); BSP (forward: 50 -GGGCAGTAGT-
GACTCATCCGA-30 , reverse: 50 -TCTTCATTGTTTTCTCCTTCATTTG-30 ); OPN (forward:
50 -TCTGGGAGGGCTTGGTTGTC-30 , reverse: 50 -TTTCCTTGGTCGGCGTTTG-30 ); OCN
(forward: 50 -CCCAGGCGCTACCTGTATCAA-30 , reverse: 50 -GGTCAGCCAACTCGTCA
CAGTC-30 ). For each run, water was used as a negative control. The reaction product
was quantified with a relative quantification tool (LightCycler software 4, Roche
Diagnostics) using ACTIN as the reference gene. The reactions were performed under
the following cycling conditions: 95  C for 10 min, 45 cycles of 95  C for 15 s, and
60  C for 1 min.

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 a ZJ-B-29 ZJ-B-31 ZJ-B-34 ZJ-B-44 ZJ-B-90 ZJ-B-105 ZJ-B-146 ZJ-B-STRO-1

50 100 150 200 250 300

50 100 150 200 250 300

50 100 150 200 250 300

50 100 150 200 250

50
100 150 200

A group
150

150

40
Count

Count
Count

Count
Count
Count
Count

30
100

100
Count
P2 P3 P2 P2 P3 P2 P3 P4

20
50

50
50

10
0
0

0
0

0
0

0
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3 3 2 3 4 5 2 2 3 4 5
0 102 10 104 105 0 10
2 103 10
4 10
5 0 10
2 103 104 105 0 102 103 104 105 101 10
2 10 10
4 10
5 0 10 10 10 10 0 10 103 104 105 0 10 10 10 10
-23 -121 -85 -39 0 -57 -93 -166
FITC-A PE-A FITC-A FITC-A PE-A FITC-A PE-A APC-A

ZJ-B-29 ZJ-B-31 ZJ-B-34 ZJ-B-44 ZJ-B-90 ZJ-B-105 ZJ-B-146 ZJ-B-ST1

For personal use only. No other uses without permission. Copyright ©2020. Elsevier Inc. All rights reserved.

50 100 150 200 250

75 100 125
200
50 100 150 200 250 300

0 10 20 30 40 50 60 70
200

75 100 125

B group
150
150
150

Count

Count
Count

Count
Count

Count
Count

Count

100
100
P4 P3 P4
100

P4 P3 P4 P3 P2

50

50
50

50
50

25

25
0

0
0

0
0
101 102 103 104 105 0 102 103 104 105 0 10
2 103 104 105 102 103 104 105 101 10
2 103 104 105 2
0 -79 -46 -31
0
0 0 10 103 104 105 0 10
2
103 104 105 0 103 104 105
FITC-A PE-A FITC-A FITC-A PE-A -43 -57 -275
FITC-A PE-A APC-A

ZJ-A-29 ZJ-A-31 ZJ-A-34 ZJ-A-44 ZJ-A-90 ZJ-A-105 ZJ-A-146 ZJ-A-STRO-1

200
200
50 100 150 200 250

50 100 150 200 250 300

50 100 150 200 250 300

150

C group
50 100 150 200 250

J. Zhang et al. / Biomaterials 33 (2012) 6974e6986

150

150
150

100
Count

Count

Count
Count

Count

Count

Count
Count
100

100
100

P4 P3 P4 P4 P3 P4 P3 P2

50
50

50
50

0
0
0
0

0
0

2 3 4 5 2 4 5
101 102 103 104 105 0 102 103 104 105 0 10
2 103 104 105 0 102 103 104 105
1
10 10
2 10
3 10
4 10
5
0 10 10 10 10 0 10 103 10 10 0 103 104 105
-6 -113 -51 -23 0 -23 -52 -291
FITC-A PE-A FITC-A FITC-A PE-A FITC-A PE-A APC-A

ZJ-C-29 ZJ-C-31 ZJ-C-34 ZJ-C-44 ZJ-C-90 ZJ-C-105 ZJ-C-146 ZJ-C-STRO-1

D group
150
50 100 150 200 250

50 100 150 200 250

150

0 50 100 150 200 250 300

100 150 200

50 100 150 200 250
150

100
100
Count

Count
Count

Count

Count
Count

Count
Count
100
P2 P3 P2 P2 P3 P2 P3 P4

50
50

50

50
0

0
0

0
0

0
0 102 103 104 105 0 102 103 104 105 0 102 103 104 105 0 102 103 104 105 101 10
2 103 104 105 0 10
2 103 104 105 0 10
2
103 104 105 0 102 103 104 105
-62 -85 -82 -62 0 -48 -108 -132
FITC-A PE-A FITC-A FITC-A PE-A FITC-A PE-A APC-A

CD29 CD31 CD34 CD44 CD90 CD105 CD146 STRO-1

b

A
99.3 2.2 0.8 99.9 100.0 93.5 27.2 14.4

B
99.8 2.3 1.6 100.0 100.0 90.5 21.6 12.2

C
99.3 1.9 3.7 99.7 100.0 91.3 19.5 11.0

D
97.6 2.3 1.8 100.0 99.9 96.6 15.3 6.9

Fig. 5. Representative figures of cytometric flow test (a) and mean data (b) obtained from all testing periodontal ligament stem cell (PDLSC) samples. Cytometric flow analysis indicated that PDLSCs derived from the A, B, C, and D
groups have similar surface molecule phenotype including testing negative for CD31 and CD34, and positive for CD29, CD44, CD90, CD105, CD146, and STRO-1. However, the expression levels of CD146 and STRO-1 in PDLSCs of the D
q
group appeared significantly lower than that in PDLSCs of the other 3 groups ( p < 0.05, compared with A group; +p < 0.05, compared with B group; :p < 0.05, compared with C group). The experiments were repeated three times for
each cell source.

6979
6980 J. Zhang et al. / Biomaterials 33 (2012) 6974e6986

a A B

C D

b **
1.8 *
1.6 *
Nodule Area (%)

1.4
1.2
1.0
0.8
0.6
0.4
0.2
0.0
A group B group C group D group

Fig. 6. Osteogenic differentiation of human periodontal ligament stem cells (PDLSCs) from donors of different ages. (a) Representative figures (A, B, C, and D representing
a representative region from the A, B, C, and D groups, respectively) of mineralized nodules formed after 3 weeks of osteogenic induction (stained with Alizarin Red, 40). (b) The
total areas of mineralized nodules in different groups were compared and showed a statistically significant decrease in the total area of mineralized nodules from groups A to D
(p < 0.05). The values are the mean
standard deviation (*p < 0.05; **p < 0.01; ***p < 0.001).

2.8. Transplantation of PDLSCs into the ectopic transplantation model
The cell carrier used for ectopic transplantation was 80 mg of Geistlich Bio-OssÒ
bone substitute (free products obtained from Geistlich Biomaterials, Wolhusen,
Switzerland). PDLSCs at P5 were used to prepare multilayered-cell sheets for
implantation according to the methods described in the literature [38].
Multilayered-cell sheets material constructs were transplanted subcutaneously into
the dorsal region of immunodeficient mice (4e6 week-old males; Fourth Military
Medical University Animal Center, Xi’an, China) that were anesthetized with 2%
inhaled isoflurane and ventilated using a rodent mechanical ventilator. Eight cell
samples were randomly selected for investigation from each testing group. Incisions
were made subcutaneously on the dorsa of the rats, and the incisions were closed
with 5-0 silk threads (Jinhuan Medical Supplies Company, Shanghai, China). The
animals were allowed to heal for 8 weeks and were then sacrificed for analysis. The
specimens were fixed with 4% paraformaldehyde and routinely processed into 7-
mm-thick paraffin-embedded sections. The most central sections of 5 mm in
thickness were cut at approximately 50-mm intervals and were stained with
hematoxylineeosin (HE) or Masson’s trichrome stain. Histological analyses were
performed using light and polarized microscopy (BX50, Olympus Optical). The
formation and organization of the mineralized and related tissues were observed
following Picrosirius staining with polarized light microscopy. For histometric
analysis of PDL-like and cementum-like tissue formation, computer-assisted histo-
metric measurements were acquired with an automated image analysis.
2.9. Statistical analysis
All experiments were performed in triplicate and repeated on at least three
separate occasions. Continuous variables with normal distribution were expressed
as the average
standard deviation (SD) and compared using Student’s t-test or
One-way analysis of variance (ANOVA). Post-hoc analysis using Tukey’s test was
applied, when appropriate. A level of p < 0.05 was accepted to be statistically
significant. The analyses were performed using SAS for Windows statistical package
(version 8.0).
3. Results
3.1. Extraction of human PDLSCs
A total of 90 wisdom teeth (Table 1) were obtained in the
present study, and cells successfully grew out from PDL tissue
pieces for all 90 teeth after 3e14 days of culture (Fig. 1a). At day 14,
the cells reached 97.6
2.2%, 78.6
8.3%, 77.3.6
9.7%, and
42.8
12.5% confluence of the A, B, C, and D groups, respectively,
showing that the rates of initial cells growing were significantly
influenced by donor age (p < 0.05) (Fig. 1b). We used a modified
tissue cube method for primary cell cultureeculture of small tissue
cubes following 15 min of digestion by collagenase to allow cells to
grow out from tissue cubes more easily. The average time for cells
to reach 80% confluence of the A, B, C, and D groups was 7
3 days,
9
2 days, 12
4 days, and 15
3 days, respectively, showing
a significant difference between either two groups of the testing 4

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a
A B

C D

b ***
1.4 **
* **
1.2 * *
Lipid Area (%)

1.0
0.8
0.6
0.4
0.2
0.0
A group B group C group D group

Fig. 7. Adipogenic differentiation of human periodontal ligament stem cells (PDLSCs) from donors of different ages. (a) Representative figures (A, B, C, and D representing
a representative region from the A, B, C, and D groups, respectively) of PDLSCs after 2 weeks of adipogenic induction (stained with Oil Red O, 40) are shown. (b) The total areas of
lipid vacuole formations were compared between the different groups. The values are the mean
standard deviation (*p < 0.05; **p < 0.01; ***p < 0.001).

groups (p < 0.05, 0.01, or 0.001) (Fig. 1c). PDLSCs proliferated
clonally, and the population doubling time in all groups was <24 h
for 7 weeks, without significant differences between the 4 groups
(data not shown). The functional capacity of stem cells in the PDL
was determined by measuring the colony-forming activity.
Different profiles of colony-forming capability were observed in
different groups. The cellular proliferation in each colony varied
(Fig. 2a), and the number of colony units significantly decreased
from the A to D groups, showing an age-related reduction in
colony-forming ability (Fig. 2b,c).
3.2. Proliferative and migratory activities
In culture, the cell populations isolated from all teeth were
capable of forming adherent colonies, a characteristic of other
stromal stem cell populations, and retained their fibroblastic
spindle shape (data not shown). The proliferative capacities of all
cells were assessed at passage two at the following time points: 1, 3,
5, 7, 9, and 12 days. The PDLSCs from the A group demonstrated
a higher proliferative rate at day 7, 9, and 12 (p < 0.01) compared
with the other groups. After 7 days, the number of young PDLSCs (A
group) was almost 2 times more than the number of elderly PDLSCs
(D group) (Fig. 3). However, there were no statistically significant
differences (p > 0.05) in cell viability among groups A, B, C, and D at
1 to 10 days (data not shown). Whether aging affects the migratory
potential of the PDLSCs was investigated by seeding and culturing
cells separately onto dishes, marking their initial position with
a scratch and then by measuring the distance the cells migrated
every 24 h using an image-analysis program (Fig.4a). By 48 h after
scratching, the PDLSCs in group A (1.42
0.34 mm) had migrated
significantly further than the PDLSCs from group D
(0.81
0.24 mm; p < 0.01). Cell migration assays revealed that the
migratory activity of the PDLSCs significantly decreased as the
donor ages increased (Fig. 4b).
3.3. Flow cytometry results
The expression of putative MSC markers was observed in all
types of cells in each group by flow cytometric analysis. The surface
molecule expression detected in single cell suspension via a specific
antibody binding to the antigen indicated that PDLSCs were anal-
ogous to MSCs; the PDLSCs were negative to hematopoietic
markers to CD31 and CD34, but highly expressed positive mesen-
chymal associated markers CD29, CD44, CD90, and CD105. In
addition, PDLSCs were found to express the cell surface molecules
STRO-1 and CD146, two early MSC markers previously found to be

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6982 J. Zhang et al. / Biomaterials 33 (2012) 6974e6986

a A group B group C group D group

3d

5d

7d

10 d

b A group B group ***

C group D group **
2.0
ALP activity (OD, 520 nm)

1.8
* **
***
1.6 *
1.4 ** **
* *
1.2 *
1.0 *
0.8 * *
*
0.6 *
0.4
0.2
0.0
3 days 5 days 7 days 10 days

Fig. 8. The alkaline phosphatase (ALP) activity of human periodontal ligament stem cells (PDLSCs) in groups of cells from donors of different ages during a 10-day culture period
(complete medium containing 100 mg/mL ascorbic acid, 10 mM b-glycerophosphate, and 50 nM dexamethasone). (a) Representative images of ALP staining for human PDLSCs in
different groups at different time intervals. (b) Data analysis of ALP activity. *p < 0.05, **p < 0.01, ***p < 0.001; significant differences by a one-way analysis of variance, as indicated
by brackets at the same time point; : (A group),; (B group), C (C group), - (D group) p < 0.05; :: (A group), ;; (B group) p < 0.01; the significant value increases along with
the culture time (time-dependent effects) in the same group. The values are expressed as the mean
standard deviation.

present on bone marrow MSCs, but the expression levels of CD146
(15.3%) and STRO-1 (6.9%) in PDLSCs of the D group appeared
significantly lower than that in the PDLSCs of the A, B, or C groups
(Fig.5). Particularly, STRO-1, an epitope originally suggested as
a marker for MSCs, was expressed in <15% of cells for all types of
cells.
3.4. Differentiation potential of PDLSCs
In addition to the colony-forming ability and the expression of
specific antigens on the cell surface of PDLSCs, the capacity for
differentiation into different mesenchymal tissues is one of the key
properties of any MSC. The differentiation potential of PDLSCs was
evaluated by culturing them in osteogenic and adipogenic media to
stimulate osteogenic differentiation (Fig. 6) and adipogenic (Fig. 7)
differentiation. After 3 weeks of osteogenic induction in vitro, the
PDLSCs from all groups exhibited osteogenic potential, as deter-
mined by the presence of Alizarin-Red-positive mineral deposits
(indicating calcium accumulation) (Fig. 6a). The total area of
mineralized nodules was significantly decreased along with the
donor aging; the PDLSCs from the A, B, and C groups showed
significant osteogenic potential compared with that of the D group

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(Fig. 6b). Furthermore, the PDLSCs from all testing donors were
capable of undergoing adipogenic differentiation, developing into
Oil-Red-O-positive cells after 2 weeks of induction (Fig. 7a). The
decrease correlated to aging of total area of lipid vacuoles were
similar to what was observed in mineralized nodules (Fig. 7b).
Furthermore, the ALP activity of PDLSCs increased from the D group
to A group in a time-dependent manner (Fig. 8). Real-time PCR
analyses showed the expression levels of OCN, OPN, and BSP in
PDLSCs on Days 14 and 21 had significant differences between the
different testing groups (Fig. 9). However, the expression levels of
all three genes were decreased on Day 21. Taken together, these
results suggest potential age-related changes of cell differentiation
capability in human PDLSCs.
3.5. Transplantation
To confirm the osteogenic potential in vivo, multilayered PDLSC
cell sheets with adjuvant use of Bio-OssÒ bone materials were
transplanted into the backs of athymic rats. After being cultured
with an osteoinductive medium for 14 days, the cells were
a OCN ***
**
40 A group * **
Relatave expression
multilayered with Bio-OssÒ bone materials and transplanted into
the backs of athymic rats [22,24,38]. Because PDLSCs might
represent a potential source of cells for periodontal tissue engi-
neering, the regenerative potential of PDLSCs for cementum and
related PDL tissue formation is of critical importance. In all animals
tested, clinical healing was generally uneventful, and material
exposure or complications at the surgical site were not observed.
The newly formed cementum-like tissue comprised mineralized
tissue that was in direct contact with Bio-OssÒ was found very
frequently in the A and B groups, where cementum was formed
along the surface of the carrier, and there was reliable evidence of
PDL-like tissue formation. However, there is less cementum- and
PDL-like tissue formation in the C and D groups (Fig. 10). Interest-
ingly, when analyzed with light and polarized microscopy, the total
area of the newly formed cementum and PDL-like tissue was
significantly smaller in the B group compared with the A group
(Fig. 11), which was consistent with in vitro results of osteogenic
differentiation (Fig. 6). PDLSCs from both the A and B groups were
able to form a dense PDL tissue around the newly formed
cementum. Dense collagen fibers that were organized in parallel
and with a high degree of cellular invasion were also associated
with newly formed cementum (as evidenced by Masson’s tri-
chrome stain). In groups C and D, the histological results demon-
strated that the PDLSCs had very little ability to regenerate new
cementum-like tissue and PDL fibers (Fig. 11), showing that

36 PDLSCs isolated from an aging population may lose their regener-

level (fold change)

B group * *
32 ative potential for cementum-like tissue and PDL fibers.
28 C group **
24 D group *
* * 4. Discussion
20
16
12 The PDL tissue appears to possess the ability to reestablish lost
8 periodontal tissues throughout adulthood, to different degrees,
4 with the ability decreasing along with age. This regenerative
0 capacity of the PDL is attributed to a few progenitor cells main-
Day 3 Day 7 Day 14 Day 21
taining their proliferation and differentiation potential in the
*** periodontium [39,40]. It is now well recognized that these cell
b OPN ** populations have many osteoblast- and cementoblast-like proper-
* **
800 A group * * ties, including the capacity to form mineralized nodules in vitro and
Relatave expression
level (fold change)

700 B group expression of the bone/cementum-associated markers [16e24]. It is

600 C group therefore possible that the remaining healthy PDL plays a key role
500 D group ** in the regeneration of new compartments during both tissue
* turnover and periodontal regenerative procedures.
400 * Current evidence suggests that stem cells derived from PDL tissue
300
(i.e., PDLSCs) may prevent ankylosis and root resorption and may
200
possibly produce new PDL inserted into the tooth and adjacent bone
100 [7]. Thus, PDL-derived cell sources are one of the most promising
0 candidates for cell-based therapies and tissue engineering-associated
Day 3 Day 7 Day 14 Day 21
periodontal regeneration [9e11,14,23,24,41e43]. Although mounting
c BSP
*** evidence recommends autologous PDLSCs for clinical use, the data
20 A group ** related to the formation of new bone and cementum vary from
Relatave expression

18 * ** **
level (fold change)

B group * * a complete absence to coverage of parts of the debrided root surfaces,

16 *
14 C group * mainly at the borders of the defects, to a thin layer of newly formed
12 D group cementum with complete new bone formation covering the entire
10 previously denuded root surface (reviewed in Refs. [6e8]). The
8 heterogeneity of the results obtained may be caused in part by
6 * increasing age and might hamper MSC suitability for periodontal
4 regeneration in elderly animals or patients [37]. These findings taken
2
together, it is logical to conclude that the therapeutic potential of cell
0
Day 3 Day 7 Day 14 Day 21 populations must be well defined before they are employed in future
clinical trials. Given that large inter-donor variations may exist in the
Fig. 9. Effects of osteoinductive supplements on the expression of osteocalcin (OCN, a), pluripotential capacity and growth of PDLSCs [37,44], it is indis-
osteopontin (OPN, b) and bone sialoprotein (BSP, c) during osteoinduction in human pensable to identify that whether autologous stem cells obtained
periodontal ligament stem cells (PDLSCs); the gene expression at various time points
(3e21 days) was analyzed by real-time polymerase chain reaction (RT-PCP). Each cell
from elderly patients have the same proliferative kinetics and
source was examined in triplicate, and fold changes are expressed as the phenotype plasticity as those from younger donors. Furthermore,
mean
standard deviation (*p < 0.05; **p < 0.01; ***p < 0.001). whether aging causes a decrease in the number of PDLSCs and

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6984 J. Zhang et al. / Biomaterials 33 (2012) 6974e6986

a A B

C D

b A B

C D

Fig. 10. Histological analysis of in vivo ectopic transplantation of human periodontal ligament (PDL) stem cells (PDLSCs) obtained from populations of different ages (A, B, C and D
groups). (a) Hematoxylin and eosin (HE) staining; (b) Masson’s trichrome staining. In the A and B groups, the cementum-like structures formed (Ce) with PDL-like tissue incor-
porated inside the newly formed mineralized tissue (arrows) after 8 weeks of transplantation. In the C group, new bone (NB) tissue formed instead of cementum-like structures.
There was no obvious PDL-like tissue formation in either groups C or D.

modifies their MSC properties, as notified in other stem cell pop-
ulations [30e36].
It was reported recently that age influences the proliferation and
differentiation potential of human PDLSCs, but those data were
obtained from only eight young female donors with a mean age of
15
2.4 years and six older female donors with a mean age of
54
3.2 years [37]. In another study, how aging affects the
phenotypic characteristics of human PDL cells and their response to
hormonal stimulation were evaluated, where cells were from
subjects aged 12e14 (group 1), 41e55 (group 2), and 61e70 years
(group 3) [44]. Ideally, the characteristics of PDLSCs should be
compared between different age groups of a wider range of donor
ages. However, there are practical difficulties to compare these
groups in advanced countries because chronic periodontitis is
dominantly observed in the elderly, while orthodontic treatment
and extraction of wisdom teeth are common in the young. We note
that there are aged populations whose third impacted molars were
not removed in the correct stage, especially in suburban regions of
developing countries, such as in the west region of China. Thus, the
hypothesis that aging could curtail the ability of PDLSCs to differ-
entiate into adipogenic and osteogenic lineages was tested in the
present study.
To determine whether there are intrinsic, age-related changes
of PDLSCs, we evaluated freshly cultured cells at very early
passage. This approach was used to avoid changes in cell behaviors
that are associated with prolonged culture, such as in vitro
senescence or culture stress. Analysis of the data obtained from
the present study suggests that CD105þ PDLSCs are capable of

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 J. Zhang et al. / Biomaterials 33 (2012) 6974e6986 6985

Cementum Formation PDL-like Tissue Formation

a b
** ***
8 * 40 ***
Average area (mm2)

36 * ***

Average area (mm2)

7 **
6 32 **
28
5 24
4
*
20
3 16
12
2
8
1 4
0 0
A group B group C group D group A group B group C group D group

Fig. 11. Measurement of the area of the newly formed cementum and PDL-like tissue. The total area of the newly formed cementum (a) and PDL-like tissue (b) significantly
decreased from groups A to C (*p < 0.05; **p < 0.01; ***p < 0.001). No mineralized tissue was observed in the D group. The values are expressed as the mean
standard deviation,
n ¼ 8.

differentiating toward two main mesenchymal pathways, i.e.,
adipogenic and osteogenic lineages, irrespective of cell-donor age.
However, there are significant differences in the adipogenic and
osteogenic activity observed in cells from donors of different ages
(Fig. 4). Therefore, the results obtained herein should reflect the
effects of aging because cells from subjects of different ages were
treated the same way and evaluated upon expansion at early
passage. The age-related reduction of STRO-1 and CD146 in the
present study indicated that the number of PDLSCs significantly
decreases with age. Similar age-related changes occur in other
type of stem cells [30,36]. The high proliferation potential of
PDLSCs from juvenile donors, as documented in our proliferation
assays, was confirmed by clonogenic capabilities and a higher
percentage of S and G2/M-phase cells (data not shown). Therefore,
we found clear differences in the growth pattern of PDLSCs
obtained from young donors and those from aged donors with
a decrease in the proliferation rate with donor age. These results
suggested a significant impact of aging on PDLSCs, in terms of
proliferative activity. Our results also corroborated the results
obtained in other cell types (e.g., human fibroblasts, bone marrow
stromal cells, and human arterial smooth muscle cells) that
demonstrated a negative correlation between donor age and the
proliferative potential of the cells [45].
In addition to the effect of donor age on PDLSC number and
proliferation, we investigated its effects on cell functionality. A
reduction was found in the osteogenic and adipogenic activity
observed in aged donors. These results are supported by the gene
expression profiles, as determined by RT-PCR analyses in a previous
study [37], which revealed age-related declines in the expression of
Runx2, the earliest differentiation marker for committed osteoge-
netic precursor cells, and the osteoblast markers collagen I and
OCN, as described in the literature [30]. This finding suggested that
the impact of aging on the commitment of PDLSCs toward an
osteoblastic/cementoblastic lineage. These results indicated that
the age-related reduction of PDLSC number might also be accom-
panied by a loss of functionality, implying that for a given number
of PDLSCs, there are fewer stem cells with osteogenic and adipo-
genic potential in aged donors than in younger donors. Whether
this reduced MSC osteogenic activity may be related to in vivo
cementum and PDL-related tissue formation was further investi-
gated by an in vivo ectopic transplantation model. Not surprisingly,
there is less cementum- and PDL-like tissue formation in the C and
D groups compared with the A and B groups (Figs. 10,11), suggesting
that advanced donor age ameliorated the ability of the PDLSCs to
form periodontal tissues. Thus, defects in the number and
proliferative potential of PDLSCs may underlie age-related defects
in the functions. The current data have demonstrated in vivo that
aging may impair the healing process of periodontal tissues [46].
The present findings suggest that this impairment might be due to
qualitative and quantitative stem cell defects. However, as sug-
gested by a recent study, the impaired regenerative potential of
aged PDLSCs may be modulated by the extrinsic cell culture
microenvironment. For example, when cultured in a young PDL-
derived cell-conditioned medium, PDLSCs from aged donors
showed an enhanced proliferation and differentiation potential.
Meanwhile, the tissue-regenerative capacity to produce
cementum/PDL-like structures was also largely improved [37].
Thus can be seen, both the age-related changes of the PDLSCs and
the effects of local cell culture microenvironment on cell activity
should be taken into account whenever they are intended to be
used for therapeutic applications.
Stem cells combat the aging process by replenishing old or
damaged cells, particularly in the skin, the gut, and the hemato-
poietic system, with a fresh supply to maintain and repair tissue. In
general, they also have slow turnover and reside in specialized
niches, protected from the environment such that only a few are
activated at a time [33]. If stem cells are protected from aging, as
has been proposed, then perhaps scientists could find a way to
bolster their ability to rejuvenate aging tissue. Unfortunately, new
evidence suggests that there is an age-related decline in function of
many stem cell types and that their regenerative capacity also
declines with age. To gain further insight into the molecular
mechanisms that underlie these deficits, additional studies need to
be performed. In particular, gene expression in PDLSCs as a function
of age on a genome-wide scale must be addressed to provide
a comprehensive molecular portrait of aging in PDLSCs.
5. Conclusion
In summary, we have shown that the aging process encom-
passes a decline in PDLSC number, proliferation, and differentia-
tion. If PDLSCs emerge as a candidate cell source for periodontal
engineering, they should be harvested from younger patients to
ensure the highest amounts of stem cells because the contribution
of PDLSCs to tissue repair in the aged will be greatly restricted due
to the conserved inhibitory influence of aged differentiated niches.
The senescence data observed in human PDLSCs should be taken
into consideration when evaluating the potential clinical use of
these cells due to the inevitable in vitro expansion required prior to
delivery into damaged tissue.

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6986 J. Zhang et al. / Biomaterials 33 (2012) 6974e6986
Acknowledgments
This research was supported by the National Natural Science
Foundation of China (81071253, 31170912, and 31030033) and the
National Basic Research Program (973 Program) (2010CB944800).
The authors declare that this manuscript has been presented in the
7th Congress of the European Federation of Periodontology (poster
presentation, the Abstract of this article appears in Journal of Clinical
Periodontology, Volume 39, Issue Supplement s13). The authors state
that they have obtained appropriate institutional review board
approval or have followed the principles outlined in the Declaration of
Helsinki for all human or animal experimental investigations. In
addition, for investigations involving the use of human teeth,
informed consent has been obtained from the participants involved.
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