archives of oral biology 56 (2011) 1221–1229

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journal homepage: http://www.elsevier.com/locate/aob

Odontoblast-like cell differentiation and dentin formation

induced with TGF-b1

Yucheng Li a,e,*, Xin Lü b,e, Xiang Sun c, Shizhu Bai c, Shibao Li d, Junnan Shi a
a
Department of Endodontics, College of Stomatology, Fourth Military Medical University, Xi’an, Shaanxi 710032, China
b
Department of Microbiology, Fourth Military Medical University, Xi’an, Shaanxi 710032, China
c
Department of Prothodontics, College of Stomatology, Fourth Military Medical University, Xi’an, Shaanxi 710032, China
d
Department of Dental Material, College of Stomatology, Fourth Military Medical University, Xi’an, Shaanxi 710032, China

article info abstract

Article history: Objective: To investigate the inductive potential of scaffold material combing with trans-
Accepted 6 May 2011 forming growth factor-b1 (TGF-b1), and to induce odontoblast differentiation and dentin
formation from dental pulp cells both in vitro and in vivo.
Keywords: Methods: Primarily cultured dental pulp cells were used for MTT, ALP activity assay and
Dentin Alizarin red staining in the presence of TGF-b1. Pelleted cells were put on the filters
Odontoblast combining with or not with TGF-b1 and cultured in vitro or in vivo. The in vitro and
Growth factor in vivo cell response and tissue formation were analysed with Haematoxylin–Eosin (HE),
Tissue engineer transmission electron microscopy (TEM) and immunohistochemical staining.
Scaffold Results: TGF-b1 increased the mineralization and ALP activity of dental pulp cells as
revealed by Alizarin red staining and ALP activity assay. After in vitro culture for 7 days,
cells polarized in the TGF-b1 group and expressed dentin sialoprotein (DSP), osteopotin
(OPN) and type I collagen (Col I). After in vivo transplantation for 7 days, columnar
odontoblast formed on the surface of filter in experimental group, and tubular dentin
expressing DSP formed after 3 months transplantation.
Conclusion: It was concluded that TGF-b1 combining with transfilter could induce odonto-
blast differentiation and dentin formation. Our results implied that suitable substrate for
the progenitors of odontoblast to anchor on and inductive signals to initiate the differentia-
tion of odontoblast should be taken into consideration when designing scaffold material for
inducing dentin tissue engineering.
# 2011 Elsevier Ltd. All rights reserved.

mineralized matrix, which give dentin the tubular character-

1. Introduction
Odontoblasts are cranial neural crest derived cells responsible
for the formation of dentin. Functional mature odontoblasts
organized as a single layer along the interface between dental
pulp and dentin, with long processes extending into the
istics.1
This unique structure and function phenotype of the
dentin pulp complex confers specificity to its tissue engineer-
ing research. Unlike osteoblast, the postmitotic feature of
odontoblast make it impossible to be isolated vital and
expanded in vitro, so how to induce the progenitors of

* Corresponding author at: Department of Endodontics, College of Stomatology, Fourth Military Medical University, Changle Western Road,
No. 17, Xi’an, Shaanxi 710032, China. Tel.: +86 29 82558692; fax: +86 29 84776476.
E-mail address: liyucheng@fmmu.edu.cn (Y. Li).
e
These authors have equally contributed to the work.
0003–9969/$ – see front matter # 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.archoralbio.2011.05.002
1222 archives of oral biology 56 (2011) 1221–1229
odontoblast into functional ones would be of pivotal impor-
tance for dentin tissue engineering. Though various scaffolds
including hydroxyapatite/tricalcium phosphate (HA/TCP) ce-
ramic powder,2,3 collagraft,4 polyglycolic acid (PGA),5 poly
lactic-co-glycolic acid (PLGA) coated with type I collagen (Col
I)6 have been used for this effort, few of them was specifically
designed for dentin tissue engineering and proved to induce
odontoblast differentiation and maintain their functional
stage in a predictable way.
The elucidation of basic principles of odontoblast differen-
tiation during embryogensis and reparation after trauma
helps to design suitable scaffold for dentin tissue engineering.
Odontoblast differentiation during embryogenesis and tooth
repair undergoes related but different processes,7 and the role
of various growth factors and their presentation pattern have
been emphasized during both processes.8 Odontoblast differ-
entiation during tooth development is controlled by the dental
epithelium–mesenchyme interactions mediated by the base-
ment membrane.9 Under the time and space-specific action of
the inner dental epithelium, the progenitors of odontoblast
undergo recruitment, commitment, cytological differentiation
and functional differentiation, i.e. the formation of the
characteristic tubular dentin. Growth factors such as trans-
forming growth factors (TGFs),10 fibroblast growth factors
(FGFs), and insulin-like growth factors (IGFs)11 might act as
mediators of the epithelial–mesenchyme interactions, which
culminates in the functional differentiation of odontoblast.
New generation of odontoblast-like cells during reparation
occurs in the absence of dental epithelium compared with the
primary dentinogenesis. Various ways have been tried to
induce progenitor cell in the pulp to differentiate into
odontoblast, attempting to elucidate the odontoblast-like cell
differentiation mechanism during repair processes. Tooth
papillae culture in vitro was used successfully in exploring
odontoblast differentiation mechanism,12 and such method
could also possibly be used to explore the inductive potential
of different scaffold for tissue engineering dentin.
Different from porous scaffold like PGA used in bone tissue
engineering, both inductive growth factors and their presen-
tation patterns should be considered during tissue engineered
dentin formation. A suitable substrate should act just like
basement membrane to store and present growth factors
secreted by oral epithelium, and further recruit and anchor
progenitors of odontoblasts and so on.
Evidences showed that constituents present in the EDTA-
soluble fraction of dentin could initiate differentiation of
preodontoblast to functional odontoblast-like cells.13 TGF-b1
or bone morphogenetic protein (BMP)-2 in the presence of
heparin or fibronectin also induced cytological and functional
differentiation of odontoblasts in cultured dental papillae.11
Interestingly, Millipore filters showed effective both in
anchoring progenitor of odontoblast and presentation of
growth factors. When soaked with dentin matrix protein14
or TGF-b1,15 filters consistently induced odontoblast-like cell
differentiation from dog dental pulp in situ. In the previous
study,16 we reported that Millipore filters combining with TGF-
b1 could induce odontoblast differentiation and dentin
formation from rat dental papillae tissue when transplanted
under the renal capsule, which implied the inductive potential
of this combination, while whether it could induce the tissue
engineered dentin formation from cultured dental pulp cells
remains unresolved.
Based on the above discussion, the authors proposed that
Millipore filters combined with growth factors and its alike
might be the suitable substrate for inducing odontoblast
differentiation for dentin tissue engineering. To prove this, we
aimed to induce odontoblast differentiation and dentin
formation from primary cultured dental pulp cells with
Millipore filters combining with TGF-b1 both in vitro and
in vivo.
2. Materials and methods
All procedures were carried out according to the guidelines of
the Animal Care Committee of Fourth Military Medical
University.
2.1. Rat dental pulp cell culture
First molar tooth buds were isolated from 4 days Spraque-
Dawley (SD) rat pups and incubated in 2% trypsin at 4 8C for
20 min, then the papillae were carefully dissected out with
disposable needle under the stereomicroscope (Olympus
Optical Co. Ltd., Tokyo, Japan). The papillae were then further
minced into pieces and then digested in a solution of type I
collagenase (0.66 mg/ml; Sigma, USA) for 40 min at 37 8C. Cell
suspensions were obtained by passing the cells through a 70-
mm strainer and then collected using centrifugation. Cell
suspensions were seeded into 25 cm2 culture flasks (Costar,
Cambridge, MA, USA) in Dulbecco’s modified Eagle medium
(Gibco-BRL, CA, USA) containing 10% fetal bovine serum,
0.292 mg/ml glutamax, 100 U/ml penicillin G, 100 mg/ml
streptomycin and 2.5 mg/ml ascorbic acid and then incubated
at 37 8C in 5% carbon dioxide. Cells were routinely observed
under a phase-contrast inverted microscope (Olympus Optical
Co. Ltd., Tokyo, Japan).
2.2. MTT assay
With the method as previously reported,17 the optimal
concentration of TGF-b1 for the MTT assay were set at 6 ng/
ml. Rat dental pulp cells were seeded onto 96-well plates at
density of 1000 cells per well. The MTT assay was performed at
day 1, 3, 5, 7 and 9. Briefly, 20 ml of MTT (5 mg/ml; Sigma–
Aldrich, St. Louis, MO, USA) was added to each well and
incubated at 37 8C for 4 h, then the supernatant was replaced
with 150 ml of DMSO. Attenuation values for each well were
measured spectrophotometrically at 490 nm and the assay
was repeated five times. Data were analysed statistically.
2.3. ALP activity assay
Rat dental pulp cells were seeded at density of 103 cells per well
onto 96-well plate in the presence of 6 ng/ml TGF-b1. The ALP
assay was performed at day 1, 3, 5, 7 and 9. Briefly, cells were
washed three times with 0.01 M PBS, then incubated in 50 ml of
0.1% Triton X-100 at 4 8C overnight. 100 ml ALP substrate (2 mM
MgCl2 and 16 mM p-nitrophenyl phosphate) was added to each
sample and incubated at 37 8C for 30 min. Reaction was
 archives of oral biology 56 (2011) 1221–1229
stopped by adding 50 ml of 0.2 M NaOH and measured
spectrophotometrically at 410 nm. Each assay was repeated
5 times and analysed statistically.
2.4. Alizarin red staining
For Alizarin red staining, rat dental pulp cells were seeded into
24-well plate and induced in the presence of 6 ng/ml TGF-b1
for mineralization for 4 weeks. Cells were fixed with 4%
paraformaldehyde, and stained with 2% Alizarin red.
2.5. Preparation of the specimens
Filters binding with TGF-b1 were prepared in the same way as
previously reported.16 Briefly, small pieces of Millipore filters
(pore size, 0.45 mm) not larger than 2 mm  4 mm were
soaked in 2 ml of TGF-b1 containing PBS for 24 h at room
temperature, and the mean amount of TGF-b1 (Sigma, USA)
bound per filter was roughly calculated to be 24  4 ng. In
control group, pieces of filters sized about 2 mm  4 mm were
soaked in PBS.
About 1  107 dental pulp cells were centrifuged to pellet
and put onto the prepared filters and used for in vitro or in vivo
culture.
2.6. In vitro culture and examination
Prepared specimen was cultured as above at 37 8C in 5% carbon
dioxide in Dulbecco’s modified Eagle medium (Gibco-BRL, CA,
USA) containing 10% fetal bovine serum, 0.292 mg/ml gluta-
max, 100 U/ml penicillin G, 100 mg/ml streptomycin and
2.5 mg/ml ascorbic acid. The culture medium was changed
each day, and 7 days later the specimen were harvested for
examination.
Fifteen specimens in each group were fixed in buffered
formalin solution. Paraffin-embedded specimens were cut in
5 mm sections and stained with HE for examination under an
Olympus compound microscope (Olympus Optical Co. Ltd.,
Tokyo, Japan). The induced odontoblast-like cells in experi-
mental group and control group were immunohistochemically
characterized with odontoblast-identifying protein markers.
Antibodies included: anti-DSP antibody (Santa Cruz Biotech-
nology, Santa Cruz, CA, USA), anti-ColIantibody (Santa Cruz
Biotechnology, Santa Cruz, CA, USA), and anti-OPN antibody
(Santa Cruz Biotechnology, Santa Cruz, CA, USA). The
procedures were carried out according to the protocol
recommended by the manufacture. Stained specimens were
examined with an Olympus compound microscope (Olympus
Optical Co. Ltd., Tokyo, Japan). All samples were counter-
stained with hematoxylin. Stained sections were examined
under an Olympus compound microscope (Olympus Optical
Co. Ltd., Tokyo, Japan).
Five specimens in each group were fixed for 10 min in 3%
glutaraldehyde buffered with 0.1 M cacodylate, pH 7.3. They
were then rinsed in 0.1 M cacodylate buffer, fixed in 1%
osmium tetroxide in the same buffer and embedded in Epon
812. Semithin sections were stained with toluidine blue and
ultrathin sections with uranyl acetate and lead citrate for
examination with a transmission electron microscope (Japan
Electron-optics Laboratory, Japan).
1223
Specimens in experimental group and control group were
serially sectioned every 5 mm, and randomly selected 10
section of each specimen were then stained with HE. The
average number of polarized cells per 100 mm length on the
surface of transfilter in each group, with the height more than
4.0 mm, was calculated under the microscope. Difference
between groups was compared using the parametric Student’s
t-test, with statistical significance evaluated at P < 0.05.
2.7. In vivo examination
For in vivo transplantation, adult rats aged 6 months were
used as hosts. The prepared implants were transplanted under
the renal capsule.
The host rats were raised for 7 days or 3 months, and then
15 implants were dissected at each time point. The specimens
were fixed for 24 h in buffered formalin solution (pH 7.2), and
demineralized in 10% formic acid for 1 week. Sections 5 mm
thick were stained with HE and examined with a microscope
(Olympus Optical Co. Ltd., Tokyo, Japan). The 3 months
specimens were characterized using odontoblast-identifying
protein markers anti-DSP antibody (Santa Cruz Biotechnology,
Santa Cruz, CA, USA), and examined using an Olympus
compound microscope.
3. Results
3.1. TGF-b1’s effect on cultured rat dental pulp cells
After culture for 7 days, the rat dental pulp cells reached
confluence (Fig. 1A) and were used for further experiments. To
determine whether TGF-b1 could affect the differentiation of
dental pulp cells, cells were cultured in media containing 6 ng/
ml TGF-b1 for 4 weeks, Alizarin red staining showed that
calcification nodule formation in experimental group was
significantly increased compared to control cells (Fig. 1B). MTT
results showed that TGF-b1 could not affect the proliferation
rate of dental pulp cells significantly (Fig. 1C), and TGF-b1
increased ALP activity in dental pulp cell significantly (Fig. 1D).
3.2. Examination of in vitro cultured specimens
After in vitro culture for 7 days, the specimen of both groups
shrank to about half of its original size.
As shown in Fig. 2A and B, all specimens in experimental
group observed polarized cells perpendicular to the filters with
processes invading into the pores of the filter. The nuclei of the
elongated cells took up basal positions, which appeared as
odontoblast-like cell. One sample in the experimental group
saw layers of accumulated cell along the transfilter (Fig. 2C), 2
specimens in experimental group saw obviously elongated
cells with secretion of matrix as shown in Fig. 2D. Only cells in
contact with the filter gained polarization in all specimens of
experimental group.
In control group, elongated cells were observed along the
interface between filter and papillae too, but not as obvious as
experimental group. Polarized secreted matrix was not
observed between cells and filter in control groups (Fig. 2E
and F).
1224 archives of oral biology 56 (2011) 1221–1229

Fig. 1 – Culture of rat dental pulp cells and TGF-b1’s effect on its proliferation and mineralization. (A) Rat dental pulp cells
reached confluence after culture for 7 days; (B) calcification nodule formation in experimental group and control group; (C)
TGF-b1 could not affect the proliferation rate of dental pulp cells significantly; (D) TGF-b1 increased ALP activity in dental
pulp cell significantly.

3.3. Immunohistochemistry analysis
For the nature of the Millipore filter, almost all of them
exfoliated during the immunohistochemical staining. The
polarized cells in experimental group stained positively for
DSP (Fig. 3A), OPN (Fig. 3B) and Col I (Fig. 3C). The cells in the
peripheral layer of both control and experimental group,
though did not gain polarization, still expressed DSP, OPN and
Col I.
ratio was high, and the nucleus located eccentrically apart
from the filter. The zone between the nucleus and the
transfilter contained endoplasmic reticulum and mitochon-
drion (Fig. 3E). Intercellular junctions between adjacent
polarized cells were generally absent. Immature desmo-
some-like structures were observed occasionally between
the elongated cells along the filter. Cells in the control group
attached to the filter via processes, but cell morphology was
much more squamous (Fig. 3F).

3.4. TEM examination 3.5. Statistical analysis

Transmission electron microscopy examination results were
shown in Fig. 3D–F. Polarized cells with the height of about
5.0 mm were observed attaching to the filter in experimental
group, with the cytoplasmic processes extending about 20–
30 mm into the pores of filter (Fig. 3D). The nucleus/cytoplasm
The frequency of polarized cells showed relevance with the
growth factors combining with the filter. After 7 days of
culture, all samples in the experimental group showed
polarized cells lining along the filter, while 3 out of 15 in the
control group saw similar results. The average number of

Table 1 – Comparison of the average number of polarized odontoblast-like cells per 100 mm length of transfilter after
culture for 7 days between experimental group and control group.
Groups Number of dental papillae with Average number of polarized
polarized cell/total number of cultured tooth papille odontoblast-like cells per 100 mm length of filter
Experimental 15/15* 25*
Control 3/15 10
*
Significant difference between experimental and control group (P < 0.01).
 archives of oral biology 56 (2011) 1221–1229 1225

Fig. 2 – Transfilter combining with TGF-b1 could induce odontoblast differentiation in vitro. (A and B) Polarized cells
perpendicular to the filters with processes extending into the pores of the filter in experimental group; nuclei of the
elongated cells took up basal positions; (C) accumulated cell lined along the transfilter; (D) elongated cells with polarized
secretion of matrix differentiated along the transfilter; (E and F) sparse elongated cells formed along the interface between
filter and papillae in control groups.

polarized odontoblast-like cells per 100 mm length of transfil-
ter was much higher in the experimental group than that in
the control group with the significance less than 0.01 (Table 1).
3.6. In vivo examination
OPN (B) and (C). In the control group, after 7 days transplanta-
tion, or 3 months transplantation, no obvious mineralized
tubular dentin formed around the transfilter (Fig. 4G and H),
while sparse elongated cell could be observed along the
interface.

Thirteen specimens were available in each group for the

in vivo analysis at each time point. After 7 days of implanta-
tion, odontoblast differentiated on the surface of Millipore
transfilter in the experimental group (Fig. 4A and B). Three
months later, on 8 specimens in the experimental group,
tubular dentin formed on the surface of the transfilter with
columnar odontoblast lying between the interface of dentin
and dental pulp. A layer of fibrodentin between the transfilter
and the tubular dentin characterized this mineralized matrix
(Fig. 4C and D). Odontoblasts expressing dentin sialoprotein
(Fig. 4E) and Col I (Fig. 4F) were located along the tubular
dentin, with processes extending perpendicular into it;
interestingly though some cells on the surface of dentin
was squamous like, they also stained positively for DSP (A),
4. Discussion
Here we provided evidences that filters combining with TGF-
b1 could induce cytological differentiation of odontoblast from
dental pulp cell in vitro and tissue engineered dentin
formation in vivo. Dental pulp cells could be induced to
express odontoblast specific DSP and acquired the columnar
shape when cultured in vitro. When transplanted in vivo,
tubular dentin formed along the transfilter in the experimen-
tal group. Our data implied that inductive growth factors and
their presentation patterns, a suitable substrate for anchoring
the progenitors of odontoblasts should all be taken into
consideration during dentin tissue engineering. The filter
1226 archives of oral biology 56 (2011) 1221–1229

Fig. 3 – Immunohistochemical and TEM results. (A–C) The polarized cells in experimental group stained positively for DSP
(A), OPN (B) and Col I (C); (D) polarized cells with the height of about 5.0 mm were observed attaching to the filter in
experimental group, and cytoplasmic processes extended into the pores of filter; (E) the area between the nuclear and
transfilter contained many cytological organelles; (F) squamous-like cells formed along the surface of filter in the control
group.

combining with growth factors and its alike should be good
reference for designing the scaffold for dentin tissue engi-
neering.
Growth factors and their unique expression patterns play
important roles in odontoblast differentiation during tooth
development and tooth pulp repair.8 Odontoblast differentia-
tion is under matrix-mediated control by the basement
membrane during development. The basement membrane
acts as a reservoir of the growth factors secreted by the inner
dental epithelium, and presents them to the progenitor cell of
odontoblast.18
Following the last mitosis and prior to odontoblast
differentiation, the pre-odontoblast aligns perpendicular to
the basement membrane, and only the daughter cells adjacent
to the basement membrane differentiate into functional
odontoblast.19 The basement membrane confers spatial
configuration characteristics to the growth factor presentation
during the whole process. Growth factors secreted by
odontoblasts after differentiation may be sequestrated within
the dentin matrix, where they may be released following
injury to the tooth. They may be able to signal reparative
processes leading to odontoblast-like cell differentiation
during repair process after dental pulp trauma.
A suitable insoluble substrate to which pulp cells could
attach and acquire the phenotype of odontoblast-like cells
seems to be of critical importance during reparative dentino-
genesis. The presence of fibrodentin matrix at the sites of
repair, with its endogenous bio-active molecular constituents,
may take the role of the basement membrane during tooth
development to present inductive signals.13 Cell–matrix
interaction plays important roles in the initiation of differen-
tiation and maintenance of the functional stage of the
odontoblast.18,20 Though the exact role the basement mem-
brane and fibrodentin remains unclear, the adhesion molecule
fibronectin on their surfaces seems to mediate interactions
between this substrate and the progenitor cell of the
odontoblast.
How to properly apply the exogenous bioactive molecules
to induce odontoblast differentiation and maintain their
functional secreting stage will be pivotal to dentin tissue
 archives of oral biology 56 (2011) 1221–1229 1227

Fig. 4 – Transfilter combining with TGF-b1 could induce odontoblast differentiation in vivo. (A and B) After 7 days of
implantation, cells polarized on the surface of filter in the experimental group; (C and D) 3 months later, large amounts of
tubular dentin matrix deposited along the filter in experimental group; (E and F) the tissue engineered tubular dentin
expressed dentin sialoprotein (E) and Col I (F); (G) in the control group, after 7 days transplantation, sparse elongated cells
formed along the transfilter; (H) after 3 months transplantation, there was no tubular dentin formed around the transfilter.

engineering. Though lack of evidence which growth factors
are responsible for signalling odontoblast differentiation
in vivo, TGF-b1 and b3, BMP-2 and IGF-1 appeared capable
of signalling odontoblast differentiation in vitro.10,12 TGF-b
superfamily had been shown to have wide ranging effects on
the mesenchymal cells of many connective tissues. TGF-b1
had been shown to stimulate differentiation of functional
odontoblasts and maintain their physiological gradients of
differentiation within the tooth pulp.21,22 Expression of TGF-bs
receptors in odontoblasts and other cells of rat dental pulp
imply possible roles for these growth factors both in the
induction of odontoblast differentiation and subsequent
control of odontoblast secretory behaviour.23 TGF-bs have
been reported to influence the synthesis of several extracellu-
lar matrix components in mesenchymal cells including
collagen and osteoadherin.24,25 The transcriptional phenotype
of odontoblasts induced by TGF-bs or BMP was very similar to
that of in vivo differentiating odontoblasts. In this study, TGF-
b1 on Millipore transfilter present the growth factors neces-
sary for odontoblast differentiation with defined spatial
pattern, for the pores within the transfilter does not allowing
the infilteration of the whole cell. The mechanical support of
1228 archives of oral biology 56 (2011) 1221–1229
the transfilter also contributes to the odontoblast-like cell
polarization and thus its later secretion.
The cytological polarization of the odontoblast-like cell
implies changes in the organization of the cytoskeleton, and
cytoskeleton–plasma membrane–extracellular matrix inter-
actions might take part in. It has been proved that fibronectin
is re-distributed during odontoblast polarization and interacts
with cell-surface molecules, and a non-integrin 165 kDa
fibronectin-binding protein, transiently expressed by odonto-
blasts is involved in microfilaments reorganization.26–28
Dentin matrix could induce odontoblast-like cell differentia-
tion when implanted in the pulp tissue. Contact between the
odontoblast and the dentin matrix is required to maintain
their functional differentiation stage.29 Thus a suitable
substrate with which the odontoblast could interact is of
fundamental importance.
The phenotype of the odontoblasts may be defined by the
morphology of the cell, the matrix secreted as well as by its
gene expression pattern. All these processes seemed to be
relatively independent, for IGF could only cause the
cytological differentiation,11 but not the functional differ-
entiation. As revealed in the results, under the induction of
the growth factors in the serum of the culturing medium,
the cell not in contact with the transfilter expressed
odontoblastic gene phenotype like DSP and OPN, while no
polarization and polarized secretion of these cells were
observed in these cells. Only those contacting to the
transfilter in experimental group showed polarized mor-
phological changes, and polarized secretion in the 7 days
culturing period. It seems that growth factors alone are not
enough to induce the odontoblast to differentiate into
functional polarized secretion stage.
5. Conclusion
In summary, the dental pulp cell culture method described
here provides a valuable approach to examine the inductive
ability of scaffold material for dentin engineering. TGF-bs
combining with Millipore transfilter was proved to be a good
substrate for inducing odontoblast differentiation and dentin
formation both in vitro and in vivo. Specific consideration
should be taken according to the anatomic, postmitotic and
differentiation feature of odontoblast for dentin tissue
engineering. The inductive growth factors involved in odon-
toblast differentiation and their presentation patterns, suit-
able substrate for anchoring the progenitors of odontoblasts
all should be considered when designing scaffold for dentin
tissue engineering.
Acknowledgements
Funding: This work was supported by grants from the Nature
Science Foundation of China (No. 30801309).
Competing interests: There is no conflict of interests.
Ethical approval: The study protocol was approved by the
guidelines of the Animal Care Committee of Fourth Military
Medical University.
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