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Original Article
Localization of TNF-α and Macrophages in the Periodontal Ligament during
Orthodontic Tooth Movement
Mari Funakoshi1, Masaru Yamaguchi2, Shoji Fujita2, and Kazutaka Kasai2
1Nihon University Graduate School of Dentistry at Matsudo, Orthodontics, Matsudo, Chiba 271-8587, Japan2Department of Orthodontics, Nihon University School of Dentistry at Matsudo, Matsudo, Chiba 271-8587, Japan
Article History
Received 5 October 2012
Accepted 24 October 2012
Keywords :
orthodontic tooth movement, perio-
dontal ligament, TNF-α, RM-4
Abstract
Remodelling of the periodontium after application of mechanical forces constitutes the basis
of clinical orthodontics, and various immunoregulatory molecules are involved in this
process. The present study focused on the localizations of the tumor necrosis factor-α
(TNF-α)and macrophages in periodontal ligament(PDL)during experimental tooth
movement in rats. A total of 15 male 6-weeks-old Wistar rats were subjected to an
orthodontic force of 10g to induce a mesially tipping movement of the upper first molars.
Experimental tooth movement was accomplished for seven days. We determined the
localization of TNF-α and RM-4(an antibody specific for identification of macrophages)in
the PDL during orthodontic tooth movement using immunohistochemistry. Immuno-
reactivity for TNF-α and RM-4 was detected in PDL fibroblasts in the compressive side by
the orthodontic force of 10g. On the first day after tooth movement, the immunoreactivity
of TNF-α and RM-4 was weak. On the third and fifth days, more TNF-α and RM-4 positive
reactions in some nucleuses of fibroblasts were recognized than on the first day.
Furthermore, these positive reactions were decreased after seven days. Therefore, RM-4
(+)cells involved in the expression of TNF-α 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.
Introduction
Orthodontic tooth movement is the result of an organized
remodeling of the periodontal tissues after application of
mechanical forces. At the cellular level, remodeling of the
periodontium consists of bone resorption adjacent to the
periodontal ligament(PDL)in the compression zone, bone
apposition in the tension zone, and degeneration and re-
establishment of the PDL(1). A number of factors have
been recognized as participants in the rather complex
orchestration of tissue remodeling during orthodontic tooth
movement. It has been proposed that chemical mediators,
such as cytokines, play an important role(2). Cytokines are
small protein molecules that regulate cell communication
and function and are actively secreted by different cell types
in response to external stimuli. It has been proposed that
during orthodontic tooth movement, these signaling mole-
cules are produced by inflammatory cells that migrated
from dilated PDL capillaries after orthodontic force
application(3).
Tumor necrosis factor-alpha(TNF-α)are key mediators
in acute-phase inflammatory reactions with overlapping
activities. These pro-inflammatory cytokines are members
of the formerly known osteoclast-activating factor and have
been implicated in the bone remodeling process(4, 5).
At sites of inflammation, TNF-α is expressed in large
quantities by macrophages(6), as well as by many other cell
types, including fibroblasts(7), osteoblasts(8), and osteo-
clasts(9).
Increased levels of TNF-α have been detected in the
gingival crevicular fluid of orthodontic patients(10-14),
speculating that the elevated cytokines observed in gingival
182 Int J Oral-Med Sci 11(3):182-189, 2012
Correspondence to :
Masaru Yamaguchi
E-mail : [email protected]
crevicular fluid(GCF)reflect the biological responses
induced by mechanical stress. The TNF-α synthesis in GCF
during tooth mobilization appeared to level off 24 h after
force application(13, 14), suggesting a central role of these
cytokines in the early phase of orthodontic tooth movement.
Macrophages are widely distributed throughout the body
to play a crucial role in the defense mechanism(15). In
different organs, there exists specifically diffentiated organ-
specific macrophages, such as tangible body macrophages in
the lymphatic follicles, sinus macrophages in the lymphno-
des, red pulp, marginal metallophilic and marginal zone
macrophages in the spleen and Kupffer cells in the liver(16).
Nakamura et al.(17) demonstrated that ED1(anti-mono-
cyte/macrophage-lineage cells and dendritic cells)in the
PDL of the compression side were significantly increased
during the orthodontic tooth movement. Furthermore, a
marked accumulation of ED1-reactive cells was frequently
observed in the area of the hyalinized tissue at 5-7 days
after the start of tooth movement. These results suggest
that after the start of tooth movement ED1-positive cells,
which are mostly exudative macrophages, may be actively
engaged in bone resorption and the remodeling of tissues on
the compression side of the PDL. However, few studies have
attempted to identify the concomitant localization of TNF-α
and macrophages during orthodontic tooth movement on
the compression side.
In the present study, we investigated the localization of
TNF-α and RM-4(an antibody specific for identification of
macrophages)in PDL of the compressive side during the
experimental tooth movement in rats using immunohisto-
chemical analysis.
Materials and Methods
Animals
The animal experimental protocol in this study was
approved by the Ethics Committee for Animal Experiments
at Nihon University School of Dentistry at Matsudo(appro-
val No. ECA-08-0039). A total of 15 male 6-weeks-old Wistar
strain rats(Sankyo Labo Service Co., Tokyo, Japan)
weighting 180±10g were used for the experiment. They
were kept in the animal center of Nihon University School of
Dentistry at Matsudo in separate cages, in a 12-hour light/
dark environment at a constant temperature of 23ºC and
provided with food and water ad libitum. The health status
of each rat was evaluated by daily body weight monitoring
for 1 week before the start of the experiments.
Application of orthodontic devices and tissue harvesting
Fifteen male 6-weeks-old Wistar rats with an average
body weight of 180±10g were used. Animals were
anaesthetized with pentobarbital sodium(40mg/kg body
weight)for application of orthodontic devices. Experimental
tooth movement was performed using the method of Fujita
et al.(18), with a closed-coil spring(wire size: 0.005 inch,
diameter: 1/12 inch, Accurate Sales Co., Chiba. Japan)
ligated to the maxillary first molar cleat by a 0.008 inch
stainless steel ligature wire(Tomy International Inc., Tokyo,
Japan). The other side of the coil spring was also ligated,
with the holes in the maxillary incisors drilled laterally just
above the gingival papilla with a #1/4 round bur, using the
same ligature wire. The upper first molar was moved
mesially by the closed coil spring with a force of 10g(Fig. 1).
The period of experiment was performed for 7 days.
Tissue preparation
The experimental periods were set at 1, 3, 5 and 7 days
after tooth movement. Animals were deeply anesthetized by
pentobarbital sodium, and perfused with 4% paraformalde-
hyde in 0.1 M phosphate buffer(PB)in a trans-cardial
manner, after which the maxilla was immediately dissected
and immersed in the same fixative overnight at 4 ˚C. The
specimens were decalcified in 10% disodium ethylenedi-
amine tetraacetic acid(EDTA, pH 7.4)solution for 4 weeks,
and then decalcified specimens were dehydrated through an
ethanol series and embedded in paraffin. Each sample was
sliced into 6μm continuous sections in the horizontal
direction, and prepared for Hematoxylin and Eosin(H.E.)
and also for immunohistochemistry staining for TNF-α and
RM-4. The periodontal tissues in the distal part of the distal
buccal root of a first upper molar(M1)was observed. The
one that was not moved was defined as the control group.
Measurement of tooth movement
Measurement of tooth movement was performed accord-
ing to the method of Fujita et al.(18). To determine the
amount of tooth movement, plaster models of the maxillae
were made using silicone impression material(Dent
Silicone-V, Shofu Inc. Kyoto, Japan)before(day 0)and after
initiating tooth movement(days 1, 3, 5, and 7). The plaster
models were scanned using a contact-type three-
dimensional measurement apparatus(3D-picza, Roland Co.)
by setting the plane to pass through three points, which
were the bilateral interpapillary crests between the first and
Int J Oral-Med Sci 11(3):182-189, 2012 183
second molars, and the interpapillary crest between the
second and third molars. Using three-dimensional morpho-
logical analysis software(3D-RUGLE, Medic Engineering
Inc.), we measured the distance between the first molar
central fossa and second molar mesial surface to determine
tooth movement.
Immunohistochemistry
Immunohistochemical staining was performed as follows:
The sections were deparaffinized and the endogenous
peroxidase activities were quenched by incubation in a
Peroxidase-blocking solution(DAKO, Japan)for 10 mi-
nutes at room temperature(RT). After washing in PBS, the
sections were incubated with polyclonal anti-rabbit TNF-
alpha(IHCWORLD, ready to use), and polyclonal anti-
rabbit RM-4(Trans Genic, working dilution, 1:200)
antibodies for 18 hours at 4 ºC. TNF-α and RM-4 were
stained using the histofine simple stain MAX-Po(multi)kit
(Nichirei, Co., Tokyo, Japan)according to the manufacturerʼ
s protocol.
The sections were rinsed with Tris-buffered saline(TBS)
and final color reactions were performed using the substrate
reagent 3, 3ʼ-diaminobenzidine tetra-hydrochloride and
aminoethylcarbazole, then they were counter-stained with
hematoxylin. As the immunohistochemical controls, some
sections were incubated in the same way, and then
incubated with either non-immune rabbit IgG or 0.01 M
phosphate buffered saline(PBS)alone, instead of the
primary antibody. Negative reactivity was observed for the
controls.
Statistical analysis
The values in each figure represent the mean±standard
deviation(SD)for each group. Intergroup comparisons of
average values were evaluated by one-way analysis of
variance(ANOVA), followed by a Tukey test, with values
of p<0.01 considered to indicate a significant difference
(Fig. 2).
Results
The body weights and tooth movement during experimental
period
The body weights of the rats in the application of
orthodontic devices(10g)decreased transiently on day one
and then recovered(data not shown). The amount of tooth
movement increased from zero to seven days during
experimental period(p<0.01, Tukey test; p<0.01, by one-
way ANOVA)(Fig. 2).
Histological changes in periodontal tissues during tooth
movement(H.E. staining)
Fig. 3a showed histological changes in periodontal tissues
during tooth movement. In control(day 0), rat PDL
specimens were composed of relatively dense connective
tissue fibers and fibroblasts that regularly ran in a horizontal
direction from the root cementum toward the alveolar bone.
On the first day after tooth movement, blood capillaries
were mainly recognized near the alveolar bone in the PDL.
Only a few mononuclear and multinucleate osteoclasts were
rarely observed on the alveolar bone surface(Fig. 3b). The
arrangement of the fibers and fibroblasts become coarse and
irregular, and blood capillaries were pressured on the third
and fifth days(Figs. 3c, d). On the surface of the alveolar
bone, bone resorption lacunae with multinucleate osteo-
clasts were recognized. Osteoclasts on the alveolar bone
were increased in comparison with these after 3 days. On
the seventh day, the PDL was recomposed of the coarse
arrangement of fibers and expanded blood capillaries. The
resorption lacunae with multinucleate osteoclasts decreased
on the alveolar bone compared to the third day(Fig. 3e).
184 Int J Oral-Med Sci 11(3):182-189, 2012
Fig. 1 Experimental tooth movement was performed with aclosed-coil spring(wire size: 0.005 inch, diameter: 1/12inch)ligated to the maxillary first molar cleat by a 0.008-inch stainless steel ligature wire. The other side of thecoil spring was also ligated, with the holes in themaxillary incisors drilled laterally just above the gingivalpapilla with a #1/4 round bur, using the same ligaturewire. The upper first molar was moved mesially by theclosed coil spring at 10g. The period of experiment wasfor 7 days.
Int J Oral-Med Sci 11(3):182-189, 2012 185
Fig. 2 Tooth movement by the orthodontic force of 10 g for seven days. The amount of tooth movement increased from 0 to 7 daysduring the experimental period (p<0.01, Tukey test; p<0.01, by one-way ANOVA).
Fig. 3 Effect of orthodontic forces on the multinucleate osteoclasts by light microscopic images(H.E.). After 7 days, the resorptionlacunae with multinucleate osteoclasts decreased on the alveolar bone compared with after 3 days.AB: alveolar bone, PDL: periodontal ligament, C: cementum, D: dentin. Bar=50μm.
Protein localizations of TNF-α and RM-4
The immunoreactivity of TNF-α and RM-4 was per-
formed 7 days after tooth movement(Figs. 4 and 5). The
positive reaction of TNF-α and RM-4 recognized in the
fibroblastic PDL cells. On the first day after tooth
movement, some of TNF-α and RM-4 was localized in
fibroblastic PDL cells and pericytes near the alveolar bone
surface(Figs. 4c and 5c). On the third day, more TNF-α and
RM-4 positive reactions in fibroblastic PDL cells were
recognized than on the first day in the compressed PDL
(Figs. 4d and 5d). TNF-α and RM-4 positive reactions in
fibroblastic PDL cells were increased after five days(Figs.
4e and 5e). Furthermore, these positive reactions were
decreased after 7 days(Figs. 4f and 5f).
Discussion
Considering the method of tooth movement, 10g of light
force application produced tooth movement without root
resorption over a period of 7 days in rats. The resorption
lacunae with multinucleate osteoclasts appeared on the
alveolar bone on the third, fifth, and seventh days after tooth
movement(Fig. 2 and 3). Gonzales et al.(19)showed that 10
g of light force application produced significantly larger
tooth movement with significantly less root resorption over
a period of 28 days in relation to a heavier force application
in rats. The optimum force for the movement of the ratsʼ
upper molars may be less than 10g as previously suggested
(20). Therefore, the model in this study was supported as
the method of efficient tooth movement.
Fig. 4 and 5 demonstrated that after the start of expe-
rimental tooth movement, significant changes in TNF-α and
RM-4 positive reaction found in the compression side of PDL
on the fifth day. TNF-α is a candidate cytokine involved in
orthodontic tooth movement(14,21,22). Yoshimatsu et al.
(23)demonstrated that on days 2,6,and 10 after applica-
tion of the orthodontic force, expression of TNF-α was
identified in the osteoclasts and mononuclear cells located on
the alveolar bone surface as well as in fibroblastic cells in the
PDL on the compression side. Mitsuhashi et al.(24)reported
that the mRNA expression of TNF-α in compressed PDL
186 Int J Oral-Med Sci 11(3):182-189, 2012
Fig. 4 Effect of orthodontic force on TNF-α positive fibroblastic PDL cells by immunohistochemistry. The immunoreactivity of TNF-αpositive was observed in the fibroblastic PDL cells on the alveolar bone surface. TNF-α positive fibroblasts increased after 5days.AB: alveolar bone, PDL: periodontal ligament, C: cementum, D: dentin. Bar=50μm.
cells was found to be increased in a time and magnitude-
dependent manner, detectable after nine hours, and TNF-α
expression gradually decreased after 12 hours. In this study,
TNF-α were detected during tooth movement by immuno-
histochemistry.
Osteoclasts are derived from macrophage-lineages pre-
cursors. RM-4 is an antibody that can recognize this lineage
of cells. Xie et al.(25) reported that the number of ED-1
positive cells, an antibody for macrophage-lineages precur-
sors, increased in the bone marrow on 5 days after
application of orthodontic force. Thereafter, these cells
decreased. Among the anti-rat macrophage monoclonal
antibody(mAB), ED-1 reacts with monocyte/macrophages
and dendritic cells. However, ED-1 mAB react with
monocytes and with some cell types other than macro-
phages and dendritic cells(26). Iyonaga et al.(27)reported
that RM-4 did not label monocytes, granulocytes or
fibroblasts, and concluded that RM-4 is considered to be a
useful tool for identifying macrophage/dendritic cells.
Therefore, the macrophages were detected specifically
during tooth movement in this study.
Considering the relationship between TNF-α and macro-
phages during orthodontic tooth movement, Baba et al.(28)
demonstrated that macrophages expressed TNF-α in the
PDL during orthodontic tooth movement by double-
immunofluorescence staining. These findings suggested that
macrophages involved in the localization of TNF-α may play
an important role in the initial reaction of the PDL. Previous
studies reported that activated macrophages, monocytes,
lymphoid cells, and fibroblasts produce TNF-α(29, 30). Kook
et al.(31)suggest that PDL fibroblasts secreted relatively
higher levels of TNF-α at the compression side than at the
tension side, and this imbalance leads to RANKL expression
by activating CD4+Tcells, thereby facilitating bone resorp-
tion during orthodontic tooth movement. Previous in vitro
studies suggest that macrophages in the PDL may be closely
associated with bone resorption during tooth movement by
the production of TNF-α(32, 33). Therefore, TNF-α may
play an important role in osteoclastic recruitment and
activation. However, more research is required to under-
Int J Oral-Med Sci 11(3):182-189, 2012 187
Fig. 5 Effect of orthodontic forces on RM-4 positive PDL fibroblasts by immunohistochemistry. The immunoreactivity of RM-4positive was observed in the fibroblastic PDL cells on the alveolar bone surface. RM-4 positive fibroblasts increased after 5 days.AB: alveolar bone, PDL: periodontal ligament, C: cementum, D: dentin. Bar=50μm.
stand the effects of macrophage-derived cytokines such as
interleukin (IL) -1β, IL-6, which are known to be very
important stimulators of osteoclastic bone resorption on
bone remodeling during tooth movement.
Conclusion
Macrophages involved in the localization of TNF-α 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.
Acknowledgments
This research was supported, in part, by a Grant-in-Aid
for Scientific Research from the Japan Society for the
Promotion of Science(22592297, 23792449, 23593044).
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