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 : yamaguchi.masaru@nihon-u.ac.jp

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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