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Introduction
Tooth eruption is a complex and tightly regulated
process involved in both odontogenic and osteogenic cells.
The dental follicle, a loose connective tissue sac
surrounding each unerupted tooth, has been demonstrated to play an
important role in this process[1_3]. A major reason for its
requirement is that it provides the eruption molecules, such
as colony-stimulating factor-1 and monocyte chemotactic
protein-1 to initiate and regulate
osteoclastogenesis[4_6].
Vascular endothelial growth factor (VEGF), also known
as vascular permeability factor or vasculotropin, is a main
regulator of blood vessel formation during embryogenesis
and a potent inducer of neovascularization during adult
life[7]. The angiogenic property of VEGF has been attributed to
its several distinct functions. VEGF is an endothelial cell
mitogen and permeability-enhancing factor that influences
the egress of plasma proteins and cells that both directly
and indirectly stimulate
angiogenesis[8,9]. VEGF may also act as a survival factor for endothelial
cells[10,11]. VEGF mediates its activity mainly through specific binding to 2 tyrosine
kinase receptors, the fms-like tyrosine kinase [Flt-1, VEGF
receptor (VEGFR)-1] and the kinase insert
domain-containing receptor (KDR, VEGFR-2). Ligand-receptor interaction
induces the activation of the tyrosine kinase domain of the
VEGFR, which finally leads to the activation of intracellular
signaling transduction pathways that are involved in
regulating cellular proliferation and survival, such as the
Raf/mitogen-activated protein kinase-extracellular
signal-regulated kinase (MEK)/extracellular signal-regulated kinase
(ERK), and the phosphatidylinositol 3' kinase/protein kinase
B pathways[12,13]. Agents directed either against VEGF or
VEGFR have been developed and anti-angiogenic therapies
based on the inhibition of VEGF/VEGFR signalling have been
reported to be powerful clinical strategies in oncology and
ophthalmology[14,15].
The role of VEGF in bone remodeling has also been
extensively studied during the past decade. Many authors
demonstrated that VEGF was implicated in normal
physiological processes such as osteoclastogenesis and bone
resorption[16_19]. VEGF has also been shown to induce osteoblast
chemotaxis, proliferation, differentiation, and 3',5'-cyclic
adenosine monophosphate (cAMP)
production[20_22], indicating that it might be involved in bone formation. Since
substantial evidence indicates that VEGF is involved in bone
remodeling, which is required for tooth eruption, the
question as to whether VEGF is expressed in dental follicle cells is
particularly worthy of attention. Recently, Wise
et al originally reported that VEGF was expressed in cultured rat
dental follicle cells. VEGF was also regarded as one of the
molecules responsible for the minor burst of osteoclastogenesis
at d 10 in the rat first mandibular molar, indicating it might
be an important molecule involved in tooth
eruption[23]. However, whether VEGF can be expressed in human dental
follicle cells (HDFC) is not clear. Moreover, previous
studies have focused on the role of VEGF in a signaling cascade
that initiated tooth eruption. Its biological effect on dental
follicle cells has never been reported. We conducted an
in vitro study to determine whether VEGF is normally expressed in
cultured HDFC and to examine the roles of VEGF in the
proliferation, differentiation, and apoptosis of HDFC.
Materials and methods
Reagents Dulbecco's modified Eagle's medium (DMEM)
was purchased from Hyclone Lab (Logan, UT, USA). Fetal
bovine serum (FBS) and RPMI-1640 medium were purchased
from Gibco BRL (Grand Island, NY, USA). The rabbit
polyclonal antibody against human VEGF and non-immune
normal rabbit IgG were purchased from Santa Cruz
Biotechnology (Santa Cruz, CA, USA). Goat anti-rabbit IgG
conjugated with horseradish peroxidase and
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased
from Sigma-Aldrich (St Louis, MO, USA). The Quantikine
human VEGF ELISA kit was purchased from R&D Systems
(Minneapolis, MN, USA). Trizol was purchased from
Invitrogen (Carlsbad, CA, USA). The First-strand cDNA
synthesis kit was purchased from Toyobo (Osaka, Japan).
Premix Taq (Ex Taq version) was purchased from TaKaRa
(Otsu, Shiga, Japan). The agarose gel was purchased from
Amresco (Solon, OH, USA). VEGF was purchased from
PeproTech (Rocky Hill, NJ, USA).
2-(2-amino-3-methoxy-phenyl)-4H-1-benzopyran-4-one (PD98059) and
1,4-diamino-2,3-dicyano-1,4-bis(2-aminophenylthio) butadiene (U0126)
were purchased from Calbiochem (San Diego, CA, USA).
The AnaSpec EnzoLyte pNPP alkaline phosphatase assay
kit was purchased from AnaSpec (San Jose, CA, USA). The
BCA protein assay kit was purchased from Pierce (Rockford,
IL, USA). The Annexin V-FITC apoptosis detection kit was
purchased from Biovision (Mountain View, CA, USA).
Cell culture The human dental follicle was separated
from an impacted mandibular third molar extracted for
orthodontic reasons from a 12-year-old boy suffering class III
malocclusion. Informed written consent was obtained from
the boy and his parents. The study protocol was approved
by the medical ethic committee of Fourth Military Medical
University. The dental follicle samples were placed in the
DMEM supplemented with 100 kU/L penicillin and 100 mg/L
streptomycin. The culture took place within 2 h after the
collection.
The HDFC were cultured as previously described by
Morsczeck et al with minor
modifications[24]. Briefly, after the dental follicle was separated, the surfaces of the follicle
tissues were cleaned and minced by using a sterilized scalpel;
the dental papilla tissue was discarded. Tissues were
digested in a solution of 100 U/L collagenase type I for 1 h at
37 °C. Minced and digested tissues of dental follicle
explants were seeded into 6-well plates in DMEM containing
10% FBS, 0.1 g/L sodium pyruvate, 100 mg/L streptomycin,
and 100 kU/L penicillin at 37 °C in a humidified atmosphere
with 5% CO2. The single cell attached to the plastic surface
remained, while non-adherent cells were removed by
changing the medium every 3 d. Confluent cells were collected by
trypsinization and were subcultured. The HDFC between
the third and seventh passage were used in this study.
Human gastric cancer cell line SGC7901, which is known
to express VEGF, was used in some experiments as a
reference cell[25,26]. SGC7901 cells were obtained from the
Institute of Digestive Diseases of Fourth Military Medical
University. The cells were cultured as previously described
by Meng et al[25].
Immunocytochemistry staining for VEGF The HDFC
and SGC7901 cells (that served as a positive control) were
cultured on coverslips for 2 d and then transferred into
serum-free medium for 5 h. Then the cells were immediately
fixed in 10% neutral-buffered formalin for 5 min and
permeabilized with 0.2% Triton X-100 for 5 min. Thereafter,
immunocytochemistry assay was performed right away. After
rinsing with phosphate buffered saline (PBS), the cells were
incubated in 3% H2O2 for 5 min and then treated with a
blocking agent of 10% goat serum for 30 min. For immunostaining,
the cells were incubated with 5 mg/L rabbit polyclonal
antibody to human VEGF at 4 °C overnight. Goat anti-rabbit IgG
conjugated with horseradish peroxidase was used as a
secondary antibody for 1.5 h, and colour development was
visualized with 3',3'-diaminobenzidine tetrahydrochloride. The
nuclei were slightly counterstained with hematoxylin. The
HDFC, in which the primary antibody was replaced with the
same concentration of non-immune normal rabbit IgG, were
used as a negative control.
VEGF detection using ELISA The HDFC were seeded
into 24-well plates at a density of
2×104/mL in DMEM containing 5% FBS (1 mL/well). After 24 h of incubation, the
medium was replaced by 1 mL fresh DMEM containing 5%
FBS. When the culture reached 80% confluence, the
medium was replaced with 1 mL fresh serum-free DMEM
containing 0.3% bovine serum albumin (BSA) and the cells were
further incubated for 24 h. Then the cell-free medium was
collected and stored at -20 °C until assayed.
VEGF in the culture supernatant was measured by
sandwich ELISA. Briefly, 200 µL of cell culture supernate samples
diluted in 50 µL buffer solution or serially diluted standard
solution (recombinant human VEGF) were added to a 96-well
microtiter plate precoated with a mouse anti-human VEGF
monoclonal antibody and incubated at room temperature for
2 h. After washing, 200 µL of the secondary antibody solution,
a VEGF-specific polyclonal goat antibody, was incubated
for 2 h at room temperature. After 3 washes, 200 µL of
substrate solution was added to each of the wells. After
incubation at room temperature for 20 min (to protect it from light),
50 µL of stop solution was added. The absorbance of each
well was detected at 490 nm with the correction wavelength
set at 570 nm with a microspectrophotometer (Bio-Tek,
Winooski, VT, USA). Recombinant human VEGF diluted in
calibrator diluent was used as a calibration standard,
ranging from 7.8 ng/L to 2000 ng/L. A standard curve was drawn
by plotting absorbance versus the concentration of
recombinant human VEGF. The concentration of the VEGF protein
was calculated according to the standard value.
RNA extraction and RT-PCR The total RNA was
extracted from the cultured HDFC or SGC7901 cells (that
served as a positive control) with Trizol. The pellet of the
total RNA was washed briefly with 75% ethanol, resuspended
in 20 µL diethylpyrocarbonate (DEPC)-treated water, and
stored at -80 °C. The total RNA concentration was
determined by measuring the optical density at 260 nm with a
microspectro-photometer. The ratio of A260/A280 was
greater than 1.8.
For each reverse transcription reaction, 1 µg total RNA
was mixed with first-strand buffer, dNTP (1 mmol/L), oligo
dT (0.5 µmol/L), RNase inhibitor (500 kU/L), ReverTra Ace
reverse-transcriptase (5000 kU/L), and RNase-free water to
make a total reaction volume of 20 µL for the generation of
the first-strand cDNA. The reactions were carried out at
42 °C for 20 min, 99 °C for 5 min, and chilled to 4 °C for 5
min. The PCR primers were designed based on the
published human overall VEGF and GAPDH mRNA
sequences and were commercially synthesized by TaKaRa
(Japan)[27,28]. Specifically, the overall VEGF forward primer was
5'-TCTT-GGGTGCATTGGAGCCTC-3' and the reverse primer was
5'-AGCTCATCTCTCCTATGTGC-3', yielding an amplicon of
349 bp. The GAPDH forward primer was
5'-ACCACAGTCCA-TGCCATCAC-3' and the reverse primer was
5'-TCCACCA-CCCTGTTGCTGTA-3', yielding an amplicon of 452 bp. For
each PCR, 1 uL of template cDNA generated in the above RT
reaction was mixed with 0.5 µmol/L each primer, 25 kU/L
premix Taq mixture, and distilled water to make a total reaction
volume of 20 µL. A total of 35 cycles of PCR was performed with
the amplification protocol of 94 °C for 30 s (denaturation),
55 °C for 30 s (annealing), and 72 °C for 45 s (extension). PCR
was completed by a final extension at 72 °C for 10 min. For
the loading controls, identical amplification procedures were
done with GAPDH primers. Amplified products were
separated by electrophoresis in a 1.5% agarose gel and
visualized with ethidium bromide staining under UV light.
In vitro biological activity detection
Incubation experiments For the cell proliferation and
ALP activity assays, the HDFC were seeded into 96-well
plates at a density of 2×104/mL (unless otherwise stated) in
DMEM containing 5% FBS (100 µL/well). After 24 h
incubation, the medium was replaced by 100 µL serum-free
DMEM containing 0.3% BSA for the designated studies.
For the concentration-dependent studies, the cells were
incubated with the medium plus various concentrations of
VEGF for 3 d. For the time-course studies, the cells were
incubated with the medium in the presence or absence of
100 µg/L VEGF for 0, 1, 3, 5, and 7 d (the medium was changed
every 4 d). To determine whether the mitogen-activated
protein kinase (MAPK) signaling pathway was involved in the
VEGF-mediated HDFC proliferation, the cells were evenly
distributed into seven groups and incubated with the
reagents as follows respectively: 0 µg/L VEGF, 50 µmol/L
PD98059, 50 µmol/L U0126, 100 ug/L VEGF with the
pre-addition of 50 µmol/L PD98059 for 30 min, 100 µg/L VEGF with the
pre-addition of 50 µmol/L U0126 for 30 min, 100 µg/L VEGF
with the pre-addition of both 50 µmol/L PD98059 and 50
µmol/L U0126 for 30 min, and 100 µg/L VEGF alone. Cell
proliferation was measured after 3 d of incubation with these reagents.
In the groups for of the cell proliferation assay, each group
had 8 replications, while each group for the determination of
ALP activity had 5 replications.
For the Annexin V/propidium iodide (PI) staining assay,
the HDFC were seeded into 75 cm2 T-flasks at a density of
2×104/mL in DMEM containing 5% FBS (12 mL/T-flask). One
day before a given experiment, the medium in each T-flask
was replaced with 12 mL fresh DMEM containing 5% FBS.
On the day of the treatment, the cells were starved in 12 mL
serum-free DMEM containing 0.3% BSA for 5 h. Then the
HDFC were incubated with the medium in the presence of
VEGF (100 µg/L) as the experimental groups or in the
absence of VEGF as the controls for 3 d. The experiments
were replicated 3 times.
MTT assay for cell proliferation determination At the
end of the culture period, 20 µL MTT stock solution (5 g/L)
was added into each well and incubated for a further 4 h.
Then the supernatant was removed and 150 µL DMSO was
added. After shaking at room temperature for 10 min, the
absorbance of each well was detected at 490 nm with a
microspectrophotometer.
Determination of ALP activity At the end of the culture
period, the HDFC were gently washed twice with 1×lysis
buffer and scraped immediately upon the addition of 0.2%
Triton X-100 (50 µL/well). After incubation at 4 °C for 10 min
under agitation, the cell suspension was centrifuged at
2500×g for 10 min at 4 °C. Then the supernatant was
collected for the alkaline phosphatase (ALP) assay according
to the EnzoLyte pNPP alkaline phosphatase assay kit
instructions. 50 µL of the supernatant and 50 µL of pNPP
reaction mixture were mixed and incubated at 37 °C for 30
min. The reaction was arrested by the addition of 50 µL stop
solution. After shaking on a plate shaker for 1 min, the
absorbance of each well was detected at 405 nm with a
microspectrophotometer. The cellular protein concentration
in the supernatant was determined according to BCA
protein assay kit instructions. The absorbance was measured
at 560 nm. The percentages of changes of ALP activity with
respect to the value found in the control were calculated
according to the formula: M=value of absorbance at 405
nm/value of absorbance at 560 nm. The percentage of
change=[(M of the testM of the
control)/M of the
control]×100[29].
Annexin V/PI staining assay After 3 d of incubation,
the cells were harvested. The single cell suspension samples
(approximately 5×105 cells in each sample) were analyzed
within 2 h. The procedure was done according to Annexin
V-FITC apoptosis detection kit protocol. In brief, the HDFC
were washed twice with PBS. Then the cells were
resuspended in binding buffer (500 µL). After that, 5 µL Annexin
V-FITC and 5 µL PI were added to each sample and
incubated at room temperature for 5 min in the dark. Then the
stained cells were analyzed by a flow cytometer
(Beckman-Coulter, Fullerton, CA, USA).
Statistical analysis All the experiments were replicated
at least 3 times. All numerical results were expressed as
mean±SD. Statistical analysis was carried out using SPSS
10.0 software package for windows (SPSS Inc, Chicago, IL,
USA). The statistical significance between the 2 groups
was determined by Student's t-test. The significant level
was set at P<0.05.
Results
Immunostaining, ELISA, and RT-PCR Immunostaining
for VEGF revealed that it was localized in cultured HDFC.
The stain was more evident in the perinuclear region of the
cytoplasm (Figure 1A). The negative control in which the
primary antibody was replaced with non-immune normal
rabbit IgG did not stain (Figure 1B).
After 24 h incubation, the concentration of VEGF protein
in the HDFC culture supernatants was 35.1±4.2 ng/L.
As detected by RT-PCR, the VEGF gene was expressed in
the cultured HDFC (Figure 2).
Effect of VEGF on HDFC proliferation The
growth-stimulative effect of VEGF on cultured HDFC was examined by
MTT assay. In the concentration-dependent pattern,
compared with the control group (VEGF at 0 µg/L), VEGF at 1 µg/L
or 400 µg/L induced a small increase in HDFC proliferation,
but the difference did not reach statistical significance
(P=0.233 and 0.087, respectively). At 10_300 µg/L, VEGF
significantly increased HDFC proliferation
(P<0.01, Figure 3A).
In the time-course pattern, following 1, 3, 5, or 7 d
stimulation, VEGF induced a significant increase in HDFC
proliferation compared with the corresponding control
(P<0.01, Figure 3B).
Before evaluating the effect of the MAPK inhibitors on
the VEGF-mediated HDFC proliferation, we first investigated
the toxic effect of the 2 inhibitors on HDFC proliferation.
Compared with the control group, PD98059 or U0126 could
slightly inhibit HDFC proliferation, but the difference did
not reach statistical significance (P=0.227 and 0.083,
respectively).
The effect of the MAPK inhibitors on the
VEGF-mediated HDFC proliferation was then evaluated. PD98059
and/or U0126 could significantly inhibit the VEGF-mediated HDFC
proliferation when compared to the treatment group with
VEGF alone (P<0.01). However, the proliferative levels of
HDFC of the groups with the inhibitors plus 100 µg/L VEGF
were still higher than those of the control group
(P<0.01, Figure 3C).
Effect of VEGF on ALP activity in cultured
HDFC The effect of VEGF on ALP activity in cultured HDFC was
determined by colorimetric ALP assay kit. In a
concentration-dependent pattern, compared with the control group (VEGF
at 0 µg/L), VEGF at a concentration of 10_300 µg/L
significantly increased ALP activity (P<0.01, Figure 4A).
In a time-dependent pattern, following 3, 5, or 7 d
stimula-tion, VEGF induced a significant increase in ALP activity
when compared to the corresponding control
(P<0.01, Figure 4B).
Effect of VEGF on cell apoptosis The results from the
Annexin V/PI staining assay showed that fewer apoptotic
cells were observed in the experimental groups after an
incubation period of 3 d (Figure 5), but there was no significant
difference in the apoptosis rate between the experimental
groups and the control groups (P=0.057).
Discussion
Tooth eruption depends on the local changes in bone
metabolism and these changes in turn depend upon the
dental follicle[30]. The dental follicle cells, as the basic structure
and functional unit of the dental follicle, have been the aim
of intensive research for fully understanding the precise role
of the dental follicles. It is not clear whether the conclusions
obtained from rodentine dental follicle are applicable to the
human dental follicle. Recently, Morsczeck et
al reported the culturing and identification of dental follicle cells
obtained from human wisdom teeth[24]. Their substantial report
provides excellent support for HDFC culture.
In the present study, the RT-PCR result demonstrated
that the VEGF gene was transcribed in cultured HDFC.
Moreover, as shown by immunostaining, the VEGF gene was
translated. The VEGF protein was also detected in the HDFC
culture supernatant by ELISA. Thus, the dental follicle
served as an endogenous source for the VEGF protein.
Considering its function in bone remodeling, the presence of
VEGF in cultured HDFC strongly suggests that VEGF played
a role in tooth eruption.
Among the numerous studies on the dental follicle, only
a few have investigated whether the proliferation of HDFC is
under the regulation of endogenous factors that are secreted
by HDFC or that of the surrounding cells. The incubation of
cultured rat dental follicle cells with the epidermal growth
factor (EGF) over 6 d increased their growth 2-fold compared
to the controls[31], while the platelet-derived growth factor
(PDGF) and basic fibroblast growth factor seemed to
stimulate mouse dental follicle cell DNA
synthesis[32]. Since VEGF (a member of the PDGF family) was found to be expressed in
cultured HDFC (in the first part of the study), the present
study also explored the roles of VEGF in the proliferation,
differentiation, and apoptosis of cultured HDFC. In our
observation, VEGF at 10_100 µg/L could obviously promote
HDFC proliferation in a concentration-dependent manner.
At higher concentrations over 100 µg/L, the
growth-stimulative effect of VEGF on cultured HDFC diminished
proportionally to the increase in concentration. Thus, it seemed
that 100 µg/L was the optimal concentration to stimulate
HDFC proliferation. Moreover, VEGF at 100 µg/L could
stimulate HDFC proliferation in a time-dependent manner. This
suggests that VEGF at a proper concentration range might
act as a mitogen for HDFC, just as it does for human dental
pulp cells[33]. The results also indicate that HDFC has a
sensitive response to the change of VEGF concentration.
Annexin V/PI reagents were widely used to measure
apoptosis. Fluorescence marked Annexin V can bind to the
phosphatidyl serine in the outer membrane of apoptotic cells,
and thus the cells can be detected through a flow
cytometer[34]. The results from the Annexin V/PI staining assay showed
that VEGF could protect the HDFC from apoptosis. Coupled
with the results of the MTT assay, it seemed that the effect
of VEGF on the HDFC viability was mainly through
promoting HDFC proliferation, but not inhibiting HDFC apoptosis.
MAPK are widespread and important signal
transduction enzymes in eukaryotes. ERK, a key molecule in the
MAPK pathway, is involved in several cellular processes
such as proliferation, cell survival, differentiation, and
cytokine-activated migration[35]. As demonstrated in other
cell types, VEGF produced a time- and dose-dependent
activation of ERK1/2. The specific
ERK1/2 inhibitor PD98059 could abolish
ERK1/2 activation and endothelial cell proliferation in
a dose-dependent manner[36]. The present study showed
that PD98059 attenuated the VEGF-mediated HDFC
proli-feration. To confirm that the inhibitory effect of PD98059
was not due to its toxic effect, we investigated the toxic
effect of PD98059 on HDFC proliferation. The data indicated
that PD98059 could slightly inhibit HDFC proliferation
compared with the control group, but the difference was not
statistically significant. It suggested that the inhibitory
effect of PD98059 on the VEGF-mediated HDFC proliferation
was induced by blocking the MAPK signaling pathway and
not by its toxic effect. However, the data also showed that
PD98059 failed to completely block the VEGF-mediated HDFC
proliferation. To exclude the possibility that PD98059 tested
was not strong enough to completely inhibit
ERK1/2, we tested U0126, another MEK-ERK specific inhibitor. U0126 inhibits
both active and inactive MEK1/2 (the upstream protein of
ERK1/2), unlike PD98059 which only inhibits activation of
inactive MEK1/2. However, U0126 or U0126 plus PD98059 did
not completely block cell proliferation induced by VEGF
either. We suggest 2 possible explanations for the
pheno-menon. One is that the MAPK signaling pathway is not the
sole mechanism responsible for VEGF-mediated HDFC proliferation; there are other signaling pathways involved.
The other is that VEGF might enhance the expression of other
growth factors or cytokines in cultured HDFC, such as EGF.
These growth factors or cytokines further stimulate HDFC
proliferation. Thus, it is an indirect effect of VEGF on HDFC
proliferation. Regardless, a more detailed study is required
to elucidate the precise mechanism of VEGF on HDFC
proliferation.
Previous studies based on the rodentine dental follicle
show that the dental follicle cells could exhibit partial
characteristics of a mineralized tissue-forming
phenotype[37]. Recently, HDFC have been reported to share some of the
biochemical features of osteoblastic phenotypes. Moreover,
dexamethasone and insulin could upregulate the expression
of some typical osteoblast- or cementoblast-related genes
such as osteocalcin and nestin in cultured HDFC. This could
indicate that dexamethasone and insulin can promote
differentiation of HDFC into cementoblasts and
osteoblasts[38]. ALP is an indication of early osteoblast differentiation. It is
one of the enzymes necessary for bone formation and its
secretion indicates that bone formation is occurring and
differentiation is beginning. However, the change of the ALP
level and activity in cultured HDFC induced by cytokines
has not been investigated. The present study suggests that
ALP was expressed in cultured HDFC. Moreover, VEGF
could enhance ALP activity in a dose- and time-dependent
manner. This suggests that VEGF also has the ability to
promote HDFC differentiation along a
"cementoblast/osteoblast" pathway. However, since the dental follicle cells, which
are progenitors for cementoblasts, PDL fibroblasts and
alveolar osteoblasts, have multipotential differentiation
ability, we do not know whether all of the follicle cells
respond to VEGF treatment or only a selected population
(the progenitors for cementoblasts and alveolar osteoblasts)
respond. Thus, these results need further detailed
investi-gation.
In summary, the present study demonstrates that both
the VEGF gene and protein were expressed in cultured HDFC.
The present study also provides evidence that VEGF at a
proper concentration range could stimulate HDFC
prolifera-tion, promote HDFC to differentiate along a
"cementoblast/osteoblast" pathway, and protect HDFC from apoptosis. The
MAPK signaling pathway might be involved in the
VEGF-mediated HDFC proliferation, but the precise signal
regulation mechanism remains to be further investigated in the
future.
Acknowledgements
Sincere thanks are expressed to Prof Yan JIN (Tissue
Engineering Center, Qindu Stomatological College, Fourth
Military Medical University, Xi'an, China) for technical
assistance and Dr Ye-fei ZHU (Department of Veterinary and
Biomedical Sciences, University of Nebraska-Lincoln, Lincoln,
USA) for critical reading of the manuscript.
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