archives of oral biology 56 (2011) 466–473

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

Immunocompetent cells and cytokine expression in

the rat periodontal ligament at the initial stage of orthodontic
tooth movement

Shunsuke Baba a,*, Noriyuki Kuroda b, Chihiro Arai a, Yoshiki Nakamura a, Tetsuji Sato b
a
Department of Orthodontics, Tsurumi University School of Dental Medicine, 2-1-3 Tsurumi, Tsurumi-ku, Yokohama 230-8501, Japan
b
Department of Anatomy II, Tsurumi University School of Dental Medicine, 2-1-3 Tsurumi, Tsurumi-ku, Yokohama 230-8501, Japan

article info abstract

Article history: Objective: The aim of the present study is to investigate the involvement of immunocom-
Accepted 15 November 2010 petent cells, pro-inflammatory cytokines and HSP, to evaluate a change of periodontal
ligament during the initial stage of orthodontic tooth movement.
Keywords: Design: In the present study, we investigated the distributional density of immunocompe-
Immunocompetent cells tent cells, the localisation of cytokines, and the expression levels of their mRNA in the
Initial stage of Orthodontic tooth periodontal ligament during the initial stage of orthodontic tooth movement, using immu-
movement nohistochemistry and real-time PCR.
OX6 Results: Orthodontic tooth movement led to significant recruitment of OX6+ cells and ED1+
ED1 cells in the rat PDL. Double-immunofluorescence staining showed that some ED1+ cells
IL-1b expressed pro-inflammatory factors of IL-1b and TNF-a in the PDL during orthodontic tooth
TNF-a movement. Real-time PCR analysis revealed that the expression levels of IL-1b (Il1b) and
HSP27 TNF-a (Tnf) mRNA gradually increased following its decrease after 1 h of orthodontic tooth
Rat movement. These findings suggest that ED1+ cells are involved in the expression of TNF-a
and IL-1b and the subsequent regulation of bone resorption on pressure side. HSP27 (Hspb1)
mRNA levels were significantly increased as compared with the control at 1 h of the initial
stage of treatment.
Conclusion: ED1+ cells involved in the expression of TNF-a and IL-1b may play an important
role in the initial reaction of the PDL and in the induction of the osteoclastic bone resorption
during orthodontic tooth movement.
# 2010 Elsevier Ltd. All rights reserved.

1. Introduction PDL cells and consequently histological tissue reactions occur

During orthodontic force application, the thickness of peri-
odontal ligament (PDL) decreases on pressure side and
increases on tension side. A change in the thickness of the
PDL influences the intra- and extracellular environments of
the PDL cells, such as cytoskeleton and their surrounding
tissue pressure.1 Transduction of mechanical forces to the
cells triggers different gene expressions and functions in the
in the PDL, which has been described as an aseptic
inflammation.2,3 A change in the thickness of the PDL is the
critical trigger for its biological reaction causing the synthesis
of prostaglandins, cytokines and growth factors, and is
believed to activate tissue remodelling, characterised by
selective bone resorption or deposition in compressed and
stretched regions of PDL, respectively.4–6 However, orthodon-
tists and biologists have not so far reported the initial change

* Corresponding author. Tel.: +81 45 581 1001.

E-mail address: baba-shunsuke@tsurumi-u.ac.jp (S. Baba).
0003–9969/$ – see front matter # 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.archoralbio.2010.11.010
 archives of oral biology 56 (2011) 466–473
in the thickness of the PDL, because it is difficult to identify the
initial tooth movement. We revealed, in the previous study,7
using three-dimensional image analysis that a slight change in
the thickness of the PDL occurred after 1 h of the orthodontic
force loading, which became significant after 6 h, with the
appearance of pressure-tension regions.
The initial stage of orthodontic tooth movement involves
an acute inflammatory response, characterised by periodontal
vasodilatation and the recruited immunocompetent cells out
of PDL capillaries.8 Pro-inflammatory cytokines are up-
regulated in response to inflammatory triggers and secreted
by different cell populations, e.g. cells of the mononuclear
phagocyte system. There are several immunohistochemical
markers for detection of immunocompetent cells. MHC class II
presenting (OX6-positive) cells have been confirmed in normal
rat PDL9 and in rat PDL induced by mechanical, orthodontic
force.10 Cells immunoreactive to ED1, a general macrophage/
dendritic cell marker, are well known to be broadly distributed
in the PDL.11–13 However, it remains unclear that OX6+ and/or
ED1+ cells are involved in an acute inflammatory response
observed in the PDL at initial stage of orthodontic tooth
movement.
Exposure of cells to stressful stimuli such as mechanical
forces or pro-inflammatory cytokines induces or enhances the
expression of several heat shock proteins (HSPs). One of these
proteins is HSP27 (Hspb1; also known as Hsp25 in the mouse),
a member of the small-HSP family, is highly expressed in
many cell types, at different stages of differentiation/devel-
opment.14,15 HSP27 has been shown to interact with the actin
cytoskeleton, to modulate intracellular reactive oxygen
species content, and to prevent apoptotic cell death triggered
by a variety of stimuli, including TNF-a.16,17 In tissue
remodelling during orthodontic tooth movement, the inter-
leukin-1b (IL-1b) and tumour necrosis factor-a (TNF-a) levels
are found to be up-regulated after orthodontic force applica-
tion and are thought to play important roles in bone
resorption.18,19 Indeed, animal model studies demonstrate
that the absence of IL-1b (Il1b) and/or TNF-a (Tnf) signalling
results in impaired tooth movement,20,21 but the mechanisms
involved in their function remain unknown.
The purpose of the present study is to investigate the total
distribution of the cells immunoreactive to ED1 or OX6
antibodies, the localisation of IL-1b and TNF-a, and mRNA
levels of IL-1b (Il1b), TNF-a (Tnf), and HSP27 (Hspb1) in maxillary
rat first molars during the initial stage of orthodontic tooth
movement, by using immunohistochemistry and real-time
PCR, respectively.
2. Materials and methods
2.1. Experimental design
Male inbred Wistar rats (n = 34) average weighing 264.1 g at 8
weeks of age (CLEA Japan, Inc., Tokyo, Japan), kept in a specific
pathogen-free environment, were divided into the following
four groups and upper first molars (M1) of the three groups
were moved lingually with fixed appliances for 1 h (n = 8), 3 h
(n = 9) and 6 h (n = 8). The appliance with 0.012 in. stainless
steel helical springs is set on the upper incisors with dental
467
[()TD$FIG]
Fig. 1 – Orthodontic appliance used in this study. The
appliance with 0.012 in. stainless steel helical springs is
set on the upper incisors with dental adhesive resin. The
initial force magnitude is about 0.1 N, moving both
maxillary first molars (M1) in the palatal direction
(arrows).
adhesive resin (Fig. 1). The initial force magnitude was about
0.1 N. Nine rats of the other group were used as untreated
controls (0 h). The rats were anesthetised with an intraperito-
neal injection of pentobarbital sodium 1 ml/kg during the
setting and adjusting of the orthodontic appliance. The
handling of animals followed the guidelines for care and
use of experimental animals established by the Ethical
Committee of Animal Experiments of Tsurumi University.
At the end of the experimental periods, the animals were
perfused through the left ventricle of the heart with 0.1 M
phosphate-buffered saline (PBS; pH 7.4) under ether narcosis,
followed by 0.1 M phosphate-buffered (pH 7.4) 4% paraformal-
dehyde. After perfusion fixation, the orthodontic appliances
were removed and tissue blocks containing the maxillary were
immersed in the same fixative solution overnight at room
temperature. The specimens were cut in half along the sagittal
plane, rinsed in 0.1 M PBS, and decalcified in 10% (w/v) EDTA
for approximately 5w. The decalcified specimens were
dehydrated in a graded ethanol series, embedded in paraffin,
and cut into frontal section (6 mm thick). Some sections were
stained with haematoxylin and eosin (HE).
2.2. Immunohistochemical analysis
Immunocompetent cells and cytokines were immunohisto-
chemically labelled with the streptavidin–biotin–peroxidase
complex (SAB) method. Monoclonal antibodies were used that
recognised major histocompatibility complex (MHC) class II
(Ia) antigen (OX6) and macrophages/dendritic cells (ED1),
respectively, and polyclonal/IL1-b and TNF-a. OX6 and ED1
antibodies were purchased from Serotec (Blacktorn, Bicester,
UK), IL1-b from R&D Systems Inc. (Minneapolis, MN) and TNF-
a from U-CyTech biosciences (Utrecht, Netherland). Paraffin
sections were treated with 0.3% hydrogen peroxide (H2O2) in
100% methanol for 20 min and incubated with 20% normal
goat serum (DAKO, Copenhagen, Denmark) in PBS for 30 min
to block non-specific binding. Primary antibodies were applied
at appropriate concentrations (OX6 and ED1, 1:400; IL1-b and
468 archives of oral biology 56 (2011) 466–473

TNF-a, 1:50) in PBS with 1% bovine serum albumin (BSA;
Sigma–Aldrich, St. Louis, MO, USA) and 0.03% Triton X-100
(Wako, Tokyo, Japan) overnight at 37 8C. Sections were rinsed
with PBS without Triton X-100 (20 min 3) between each step.
Immunoreactions were visualised using 0.025% 3,30 -diamino-
benzidine tetrahydrochloride (DAB; Sigma–Aldrich) and 0.01%
hydrogen peroxide in 0.05 M Tris–HCl buffer (pH 7.3) for 5–
10 min. After being counterstained with haematoxylin, they
were dehydrated and mounted with Permount (Fisher
Scientific, Fair Lawn, NJ, USA). As the secondary antibodies
for immunofluorescence-labelling, Alexa Fluor 488 (green)-
labelled donkey anti-goat IgG (Invitrogen, Carlsbad, CA, USA)
for anti-IL-1b antibody, Alexa Fluor 594 (red)-conjugated goat
anti-rabbit IgG (Invitrogen) for anti-TNF-a antibody, and Alexa
Fluor 546 (red)-labelled goat anti-mouse IgG (Invitrogen) or
Alexa Fluor 488 (green)-labelled goat anti-mouse IgG (Invitro-
gen) for ED1 and OX6 antibodies were used and finally the
sections were counterstained with 40 ,6-diamidino-2-pheny-
lindole (DAPI 1:1000; Roche Diagnostics, Mannheim,
Germany). Stained sections were mounted with crystalmount
(Biomeda, Foster city, CA, USA) and observed with a fluores-
cence microscope (BZ9000-Analyzer, Keyence, Tokyo, Japan).
As immunohistochemical controls, sections were pro-
cessed by replacing the primary antibodies with normal
non-immune serum. The immunoreactions were completely
absent in control sections.
2.3. Observed area
As IL-1b, TNF-a and HSP27 probe, Probe#119, #78 and #22
were used (Universal Probe Library; Roche Diagnostics,
Indianapolis, IN, USA) and Fast Start Universal Probe Master
(ROX) (Roche Diagnostics) as PCR reagent. Actb was used as an
internal control (Universal Probe Library Rat ACTB Gene Assay;
Roche Diagnostics).
2.5. Statistical study
All values are indicated as mean  SEM. The unpaired ANOVA
and Tukey’s HDS tests were used for evaluating statistical
significance. A value of p < 0.05 was considered statistically
significant.
3. Results
In the present study, immunohistochemistry for OX6 and ED1
revealed OX6- and ED1-positive cells were distributed in the
PDL (Figs. 3a–f and 4a–f). OX6 caused membrane staining,
whereas ED1 caused patchy, granular intracytoplasmic stain-
ing. OX6 recognised macrophage-like cells of predominantly
irregular or dendritic appearance. ED1 was reactive to large
number of macrophages that had various morphological
features, i.e. oval, irregular, spindle and dendritic. In the
control rats, cells immunostained with OX6 or ED1 were
scatteringly distributed throughout the PDL. Negative control
[()TD$FIG]
staining occasionally showed a non-specific reaction on a few

The areas observed in the rat PDL were as follows: 300 mm (A)
lower from the palatal alveolar crest, (B) upper from the palatal
root apex, (C) upper from the interradicular septal root apex,
and (D) lower from the interradicular septal alveolar crest,
respectively (Fig. 2). The volume of each observed area (only
the PDL of whole visual field under microscope) was measured
at a magnification level of 400 by use of imaging analyser
(TRY/3D-SRFII, RATOC System Engineering Co., Ltd., Tokyo,
Japan). Three representative sections were selected in the
identical sample of each group and the number of OX6 and ED1
positive cells was determined in each area. The distributional
density of labelled cells was expressed as cell number per
square millimetre.

2.4. Real-time polymerase chain reaction (PCR)

For quantitative analysis of IL-1b, TNF-a, and HSP27 mRNA, real-

time polymerase chain reaction (PCR) was performed with the
PDL obtained from the maxillary bilateral M1 extraction whose
gingival tissue of both control group and three experimental
groups (n = 6 for each group) must not be attached to these PDL.
Total RNA was extracted from the rat M1 with TRIzol Reagent
(Invitrogen) and synthesised into cDNA with Super Script III
First-Strand Synthesis System for RT-PCR (Invitrogen) accord- Fig. 2 – Showing the direction (arrow) of orthodontic force
ing to the manufacturer’s instructions. and the areas (A–D) of the rat periodontal ligament
Primer sequences of IL-1b, TNF-a and HSP27 are as follows observed in the present study. The dotted black frame of
(forward, reverse): 50 -TGTGATGAAAGACGGCACAC-30 and 50 - (A) shows 300 mm lower from the palatal alveolar crest, (B)
CTTCTTCTTTCGGGTATTGTTTGG-30 (IL-1b), 50 -GCCCAGACCC 300 mm upper from the palatal root apex, (C) 300 mm upper
TCACACTC-30 and 50 -CCACTCCAGCTGCTCCTCT-30 (TNF-a), 50 - from the interradicular septal root apex, and (D) 300 mm
GGAGCTCACAGTGAAGACCA-30 and 50 -TTCATCCTGCCTTTCT lower from the interradicular septal alveolar crest,
TCGT-30 (HSP27). respectively.
 archives of oral biology 56 (2011) 466–473 469
[()TD$FIG]

Fig. 3 – Immunocytochemical staining to OX6 and time-lapse analysis for the number of OX6+ cells distributed in the PDL (A–
D) during the initial stage (0–6 h) of orthodontic tooth movement. Cells immunoreactive to OX6 antibody (arrows) are widely
distributed throughout the PDL. In control group (0 h), OX6+ cells are scatteringly observed in the PDL of each area (a and d).
In 1 h group, positive cells (arrows) are often distributed in the perivascular tissue (b and e). At this stage, OX6+ cells with
irregular or dendritic appearance predominantly found. In 6 h group, a few OX6+ cells with oval or spindle appearance are
found and immonopositive reaction is similar to that of control group (c and f). The number of these positive cells is
significantly decreased in the areas of A, B, C as compared with 1 h group after 6 h of treatment (g). Bo, bone; De, dentine.
Scale bars = 100 mm for (a–f). Error bars indicate the SEM for (g). Significance *p < 0.05 for (g).

erythrocytes, whilst these cells were easily identified by their
morphology and location. These immunocompetent cells
represented fluctuations in their number distributed in the
PDL during the initial stage of orthodontic tooth movement
(Figs. 3g and 4g). The formation of cell-free hyaline zone was
not found in the PDL of the pressure side on the sections
stained with HE. Furthermore, double-immunofluorescence
for OX6/IL-1b, OX6/TNF-a, ED1/IL-1b, or ED1/TNF-a demon-
strated that IL-1b and TNF-a were detected on the ED1-
immunolabelled cells (Fig. 5g–l), but not on the OX6-immuno-
reactive cells (Fig. 5a–f). Real-time PCR analysis showed time-
lapse changes in the mRNA expression levels of IL-1b, TNF-a
and HSP27 in the PDL during the initial stage of orthodontic
tooth movement (Fig. 6).
3.1. OX6- or ED1-immunoreactive cells in the PDL during
the initial stage of orthodontic tooth movement
The number of OX6+ cells was significantly increased in the
PDL of the pressure side (Fig. 2A and C) and that of the tension
side located in the apical region of the palatal root (Figs. 2B and
3g) after 1 h of orthodontic tooth movement, thereafter were
470 archives of oral biology 56 (2011) 466–473
[()TD$FIG]

Fig. 4 – Immunocytochemical staining to ED1 and time-lapse analysis for the number of ED1+ cells distributed in the PDL (A–
D) during the initial stage (0–6 h) of orthodontic tooth movement. In control (0 h) group, cells immunoreactive to ED1
antibody (arrows) are widely distributed throughout the PDL and sometimes located along the surface of alveolar bone (a
and d). In 1 h groups, intensely labelled ED1+ cells (arrows) with oval, irregular and spindle figures are often in close vicinity
to blood vessels (b and e). In 6 h group, numbers of ED1+ cells with irregular or dendritic appearance are observed in the PDL
especially on pressure side (c and f). Positive cells are often located along the surface of alveolar bone. ED1+ cells in the PDL
are gradually increased in number during orthodontic tooth movement and their number is significantly increased in the
PDL of pressure side (A and C) as compared with controls after 6 h of orthodontic force application (g). Bo, bone; De, dentine.
Scale bars = 100 mm for (a–f). Error bars indicate the SEM for (g). Significance *p < 0.05 for (g).

decreased in number, and after 6 h of treatment their number
was almost same as controls (Fig. 3g). At this stage, some
positive cells without any apparent relationship to the
vasculature were found in the center portion of the PDL
(Fig. 3b and e). The number of OX6+ cells was not significantly
increased in the PDL of the furcal area on tension side (Figs. 2D
and 3g).
Cells immunoreactive to ED1 were gradually increased in
number during the initial stage of orthodontic tooth
movement (Fig. 4g). Their number displayed a significant
increase in A region of pressure side after 3 h and 6 h of
orthodontic tooth movement, and in C region of pressure
side after 6 h of orthodontic force application (Fig. 4g). In
contrast, their number displayed no significant increase in B
and D regions of tension side (Fig. 4g). ED1+ cells were often
distributed close to the bone surface after 6 h of orthodontic
tooth movement (Fig. 4c and f). Some of OX6+ and ED1+ cells
were located in the vicinity of the vasculature during the
 archives of oral biology 56 (2011) 466–473 471
[()TD$FIG]

Fig. 5 – Double-immunofluorescenc staining for OX6/IL-1b, OX6/TNF-a, ED1/IL-1b, and ED1/TNF-a in the PDL on pressure
side after 3 h (a–f) or 6 h (g–l) of orthodontic force application. Two red-labelled OX6+ cells (white arrows; a) and a green-
labelled IL-1b+ cells (white open arrow; b) are found, but both cells are not merged (c). Fluorescence reaction for OX6 (green;
white arrow) is detected in the PDL (d). (e) for TNF-a (red; white open arrow). From merged image of (d) and (e), TNF-a is not
expressed on OX6+ cells (f). (g) Fluorescence for ED1+ cells (red; white arrows), which is marker of macrophages/dendritic
cells. (i) Merged image of (g) and (h). Coexpression of ED1 and IL-1b (green; white open arrows) is shown as yellow
(arrowheads). Two green-labelled ED1+ (white arrows; j) and red-labelled TNF-a+ cells (white open arrows; k) are detected in
the PDL and coexpression of ED1 and TNF-a is shown as yellow (arrowheads; l). Nuclei are stained with DAPI. Scale
bars = 50 mm for (a–l).

initial stage of orthodontic tooth movement (Figs. 3e
and 4c, f).
3.2. Expression of pro-inflammatory cytokine in OX6+ and
ED1+ cells
We examined immunoreactive expression of pro-inflamma-
tory cytokines in OX6+ and ED1+ cells using double-immuno-
fluorescence analysis for IL-1b/TNF-a and OX6/ED1. Double-
immunofluorescence analysis for OX6/IL-1b or OX6/TNF-a did
not detect any immunoreactive expression of IL-1b and TNF-a
on OX6+ cells in the PDL (Fig. 5a–f). On the contrary, double-
immunofluorescence staining for ED1/IL-1b or ED1/TNF-a
revealed that some ED1+ cells expressed pro-inflammatory
factors of IL-1b and TNF-a in the PDL (Fig. 5g–l).
3.3. Expression levels of IL-1b, TNF-a and HSP27 mRNA
In the present study, we analysed the relative concentration of
IL-1b, TNF-a and HSP27 mRNA in the PDL during the initial
stage of orthodontic tooth movement by using real-time PCR
(Fig. 6). The expression levels of IL-1b and TNF-a mRNA were
472 archives of oral biology 56 (2011) 466–473
[()TD$FIG]
Fig. 6 – The graphs show time-lapse changes in the relative
concentration of HSP27 mRNA expression levels during
orthodontic tooth movement using real-time PCR.
Expression levels of HSP27 mRNA are significantly
increased as compared with the control after 1 h of
orthodontic force application and thereafter gradually
decreased. *p < 0.05.
decreased as compared with controls after 1 h of orthodontic
tooth movement and thereafter gradually increased, but not
significant at every stage. IL-1b mRNA levels were higher than
those of TNF-a 3 h and 6 h after orthodontic tooth movement,
but not significant (data not shown). On the contrary, HSP27
mRNA levels were significantly increased as compared with
the control after 1 h of orthodontic tooth movement and then
gradually decreased after 3 h and 6 h of orthodontic tooth
movement (Fig. 6).
4. Discussion
Orthodontic tooth movement is achieved by the remodelling
of periodontal tissue including bone resorption on pressure
side and bone formation on tension side in response to
mechanical loading.1,4 A number of studies have reported the
presence of dendritic cells (DCs) in the gingiva,22,23 the PDL,13
and the lingual tissue.24 These may act as antigen-presenting
cells (APCs) to induce T lymphocyte-mediated immunity
during the pathological conditions of periodontium. Light-
and electron-microscopic analyses have revealed the diverse
distributinal patterns and densities of nonlymphoid cells such
as DC-like cells and macrophages in various (furcal, mesial,
distal, and periapical) regions of PDL.12 In response to
experimental tooth movement, the distribution and number
of macrophage-like cells show temporary changes together
with the remodelling of the PDL.10,25 In the present study,
orthodontic tooth movement led to significant recruitment of
OX6+ cells and ED1+ cells in the rat PDL.
Several studies have provided the experimental evidence to
support the suggestion that cytokines regulate some remodel-
ling processes during orthodontic tooth movement.8,15
Amongst the cytokines, interleukin-1 (IL-1) and TNF-a are
thought to play a prominent role.26–29 Various studies have
shown that IL-1 stimulates bone resorption and inhibits bone
formation in vivo and that it is secreted in response to a variety
of stimuli, be they mechanical, hormonal or inflammatory.30,31
The observation that transient IL-1 elevation in alveolar bone
precedes the increase in osteoclasts population in several days
suggests that recruitment of new preosteoclasts may be
important in orthodontic tooth movement.32 According to the
report of Wellington et al.,33 osteoclasts in orthodontically
treated sites originate by the fusion of recently recruited
preosteoclasts from the marrow instead of from local PDL
cells.
In the present study, the number of ED1+ cells was
significantly increased in the PDL of pressure side as compared
with controls after 3 h and 6 h of orthodontic tooth movement.
Furthermore, double-immunofluorescence staining showed
that some ED1+ cells expressed pro-inflammatory factors of IL-
1b and TNF-a in the PDL during orthodontic tooth movement.
The present results suggest that monocyte-derived ED1+
macrophages are involved in the remodelling of the PDL.
In our experimental system, HSP27 mRNA levels were
significantly increased as compared with the control after
1 h of orthodontic tooth movement, whereas the expression
levels of IL-1b and TNF-a mRNA were decreased at the same
stage. The expression of HSP27 mRNA in the PDL, when
induced in response to orthodontic force, possibly has the
protective effect of the protein to maintain the cytoskeleton.
In the present study, the number of OX6+ (a marker of MHC
class II (Ia) antigen) cells was significantly increased in the
PDL as compared with the control as well as HSP27 mRNA
expression levels after 1 h of orthodontic tooth movement.
However, we could not evaluate whether OX6+ cells are
involved in the expression of HSP27 mRNA. To our
knowledge, the present study is the first report that showed
a change in the distributional density of OX6+ and ED1+ cells
in the PDL at the initial stage of orthodontic tooth movement.
Our results revealed that the time-lapse change in the
number of OX6+ and ED1+ cells and in the expression levels
of IL-1b, TNF-a and HSP27 mRNA, within 6 h after orthodontic
force loading, might contribute to the structural changes of
the PDL observed during the initial tooth movement.7 These
findings suggest that immune system is involved in the
tissue reaction of the PDL during tooth movement. Further
investigations will be required to clarify the physiological
response of the periodontal tissue caused by orthodontic
tooth movement.
Funding
This work was partly supported by the Academic Frontier
Project for Private Universities (a matching fund subsidy) from
the Ministry of Education, Cultures, Sports, Sciences, and
Technology (to T.S.).
Ethical approval
Our research conformed to the guidelines for care and use of
experimental animals established by the Ethical Committee of
Animal Experiments of Tsurumi University. We do not have
any judgement’s reference number.
 archives of oral biology 56 (2011) 466–473
Conflict of interests
None declared.
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