J Bone Miner Metab (2006) 24:20–27 DOI 10.1007/s00774-005-0641-4

© Springer-Verlag Tokyo 2006

ORIGINAL ARTICLE Masako Yoshimatsu · Yasuaki Shibata · Hideki Kitaura Xin Chang · Takeshi Moriishi · Fumio Hashimoto Noriaki Yoshida · Akira Yamaguchi

Experimental model of tooth movement by orthodontic force in mice and its application to tumor necrosis factor receptor-deficient mice

Received: April 5, 2005 / Accepted: July 6, 2005

Abstract Orthodontic tooth movement is achieved by mechanical loading; however, the biological mechanism involved in this process is not clearly understood owing to the lack of a suitable experimental model. In the present study, we established an orthodontic tooth movement model in mice using a Ni-Ti closed coil spring that was inserted between the upper incisors and the upper first molar. Histological examination demonstrated that the orthodontic force moved the first upper molar mesially without necrosis of the periodontium during tooth movement. The number of TRAP-positive osteoclasts on the pressure side significantly increased in a time-dependent manner. Quantitative real time-based reverse transcription-polymerase chain reaction analysis demonstrated increased levels of mRNA for cathepsin K. Immunohistochemical staining revealed the expression of tumor necrosis factor-a (TNFa) in periodontium on the pressure side of the first molar during orthodontic tooth movement. When this tooth movement system was applied to TNF type 1 receptor-deficient mice and TNF type 2 receptor-deficient mice, tooth movement observed in TNF type 2 receptor-deficient mice was smaller than that in the wild-type mice and TNF type 1 receptor-deficient mice. The number of TRAP-positive osteoclasts on the pressure

M. Yoshimatsu · Y. Shibata · X. Chang · T. Moriishi Divisions of Oral Pathology and Bone Metabolism, Department of Developmental and Reconstructive Medicine, Nagasaki University, Graduate School of Biomedical Sciences, Nagasaki, Japan M. Yoshimatsu · H. Kitaura · F. Hashimoto · N. Yoshida Division of Orthodontics and Biomedical Engineering, Department of Developmental and Reconstructive Medicine, Nagasaki University, Graduate School of Biomedical Sciences, Nagasaki, Japan X. Chang The Second Affiliated Hospital of Dalian Medical University, Dalian, China A. Yamaguchi (*) Section of Oral Pathology, Department of Oral Restitution, Graduate School of Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8549, Japan Tel. +81-3-5803-5451; Fax +81-3-5803-0188 e-mail: [email protected]

side was significantly small in TNF type 2 receptor-deficient mice compared with that in TNF type 1 receptor-deficient mice on day 6 after application of the appliance. The present study indicates that TNFa signaling plays some important roles in orthodontic tooth movement. Key words orthodontic tooth movement · mechanical loading · bone · bone resorption · TNFa · mouse

Introduction Mechanical loading exerts important effects on the skeleton by controlling bone mass and strength [1]. To investigate the effect of mechanical loading on bone metabolism, several in vivo experimental models, such as jumping [2,3] treadmill running [4,5], squatting [6], and swimming [7] have been reported. In addition to such models, orthodontic tooth movement is a good in vivo model for exploring the mechanism underlying the mechanical loading-induced bone changes [8–11]. In previous reports concerning animal models for orthodontic tooth movement, the most widely used animals were rats, and the use of mice for such model has been limited [12–20]. Recent advances in molecular biology techniques have provided opportunities for the use of various gene-mutated mice, including those with genes that regulate bone metabolism. The suitable application of these mice to tooth movement experiments can be advantageous for exploring the molecular mechanisms underlying not only tooth movement but also mechanical loadinginduced bone changes. This prompted us to assess a mouse model for elucidating the mechanism of orthodontic tooth movement. Orthodontic tooth movement is achieved by the process of repeated alveolar bone resorption on the pressure side and new bone formation on the tension side [8]. In rat tooth movement experiments, excessive orthodontic force induced the expression of tumor necrosis factor-a (TNFa) in periodontal tissues [21]. In addition, orthodontic tooth movement increases the levels of TNFa in the gingival

21 Fig. 1. Orthodontic tooth movement in a mouse using a Ni-Ti closed coil spring. A Intraoral photograph after insertion of the appliance between the upper incisor (white arrow) and the left first molar of mouse (black arrow). B Schema for orthodontic tooth movement of the upper first molar to the mesial side. The open arrow indicates the direction of physiological tooth movement, and the black arrow indicates the direction of orthodontic tooth movement

sulcus in humans [22,23], suggesting an important role for TNFa in orthodontic tooth movement. In contrast, another group using a rat tooth movement model reported that TNFa mRNA expression was not detected during orthodontic tooth movement [24]. Thus, the role of TNFa in orthodontic tooth movement is not clearly understood. Because TNFa induces a number of biological responses via two cell-surface receptors termed TNF receptor type 1 (TNFR1) and TNF receptor type 2 (TNFR2) (also called TNFR p55 and TNFR p75, respectively) [25], the use of TNFR-mutated mice may provide important information for resolving the conflicting role of TNFa in orthodontic tooth movement. In the present study, we applied a tooth movement model to the TNFR-deficient mice to investigate the role of TNF signaling in orthodontic tooth movement. We demonstrate here that TNF signaling plays an important role in orthodontic tooth movement.

Materials and methods

and adjusting of the orthodontic appliance. The orthodontic appliance is composed of a Ni-Ti closed coil spring. The diameter of the Ni-Ti wire, shaped into a coil, is 0.15 mm; and the diameter of the coil is 0.9 mm. The appliance was inserted between the upper incisors and the upper left first molar and fixed with a 0.1 mm stainless wire around both teeth using a dental adhesive agent (Superbond; Sunmedical, Shiga, Japan). During the preliminary experiments, the appliance attached between the incisors often detached from the tooth because of the growing of the incisors during experimental tooth movement. To prevent this detachment of the appliance from the incisor surface, we hooked the stainless wire to a shallow groove that was made approximately 0.5 mm from the gingiva. During the experiments, we changed the position of the groove every 4 days, and rehooked the wire to the new groove. Figure 1 represents the orthodontic appliance used in this study. According to the manufacturer’s database, the force level of the coil spring after activation is approximately 10 g. The left maxillary molar in each mouse was used for the experimental tooth movement, and the right maxillary molar was the control.

Experimental animals Wild-type mice (C57BL6/J), TNFR1-deficient-mice (Tnfrsf1atm1IMak), and TNFR2-deficient mice (Tnfrsf1btm1Mwn) were purchased from the Jackson Laboratory (Bar Harbor, ME, USA). Eight-week-old mice were used in this experiment. The animals were fed a granular diet (Oriental Yeast, Tokyo, Japan) to prevent them from exerting an excessive chewing force. During the experiment, the mice were kept in cages in a room maintained at 25°C with a 12–24 h light/dark cycle, and their body weight and health were checked every day. The protocol for all animal procedures was in accordance with Nagasaki University regulations. Experimental tooth movement The mice were anesthetized with an intraperitoneal injection of pentobarbital sodium 50 mg/kg during the setting

Measurement of tooth movement We measured the distance of tooth movement after application of the orthodontic appliance every other day until day 12, as described later. The mice were perfused with 10 mM phosphate-buffered saline (PBS) to remove their blood and then perfused and immersed in 4% paraformaldehyde for fixation. After removing the maxillae, the individual tray was placed on the maxillary teeth in order to take impressions by dental hydrophilic vinyl polysiloxane impression material (Exafine, injection type; GC Co., Tokyo, Japan) using each individual tray. The amount of tooth movement was evaluated by measuring the closest distance between the first molar and the second molar (1st and 2nd molar distance) in the impression under a stereoscopic microscope (VH-7000; Keyence, Osaka, Japan). To validate the consistency of the measurement, we conducted the preliminary experiment. We measured the distance between the first

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and second molars on day 6 after application of the appliance using the impression three times a day during three consecutive days. There was no significant difference among the values obtained on the three consecutive days, indicating the consistency of our measuring technique. A single examiner (M.Y.) conducted observations blinded to the group status.

of amplification. Each cycle consisted of a denaturation step at 94°C for 45 s, an annealing step at 60°C for 45 s, and an extension step at 72°C for 45 s. The relative expression of cathepsin K mRNA was normalized by GAPDH mRNA.

Preparation for histological observation

We investigated the expression of TNFa by immunohistochemistry. Preliminary experiments using spleen sections obtained from lipopolysaccharide (LPS)-injected mice revealed that only the purified goat anti-TNFa polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) was available for detecting TNFa; however, this antibody could be used only on frozen sections and not on paraffin sections (data not shown). Thus, we conducted immunohistochemical tests for TNFa using undecalcified frozen sections. The small pieces of tissues containing the upper first molar were cryoprotected in 97% hexane with Dry Ice immediately after sacrifice and embedded in 5% carboxymethyl cellulose sodium (Finetec, Tokyo, Japan). They were sectioned to obtain horizontal 4 mm thick sections, which were fixed with 99% acetone at -20°C for 1 min. After blocking the endogenous peroxidase activity in methanol/ H2O2 for 30 min, the sections were preincubated with 1.5% normal rabbit serum (Vector laboratories, Burlingame, CA, USA) for 30 min to avoid nonspecific background staining. Thereafter, the sections were incubated with the above described primary antibody at 1 : 25 dilution with 10 mM PBS at room temperature for 1 h. The sections were incubated with a secondary antibody, biotinylated anti-goat immunoglobulin G (IgG) (Vector), diluted 1 : 500, for 30 min. After washing, the reactions were developed using the VECTASTAIN ABC detection system (Vector) at room temperature, in accordance with the manufacturer’s instructions. The sections were counterstained with methyl green.

After fixation, the maxillae were demineralized in 10% ethylenediaminetetraacetic acid (EDTA) for 10 days at 4°C. Paraffin-embedded samples were sectioned at 4 mm. The vertical and horizontal sections of the first molar region were prepared. The vertical sections were used for general histological examination after staining with hematoxylin and eosin. In mouse first molar, the root length between the bifurcation surface and apical end is about 600 mm. This suggests that about 300 mm of the root from the bifurcation surface at the mesial side is the pressure side during tooth movement. In addition, the alveolar crest is about 80 mm away from the bifurcation surface. Thus, we prepared horizontal sections from five levels of the root: 100, 140, 180, 220, and 260 mm away from the bifurcation surface. These sections were used to count the osteoclast number after staining for tartrate-resistant acid phosphatase (TRAP) activity. For the TRAP staining, the sections were incubated in acetate buffer (pH 5.0) containing naphthol AS-MX phosphate (Sigma Chemical, St. Louis, MO, USA), Fast Red Violet LB salt (Sigma), and 50 mM sodium tartrate. The sections were counterstained with hematoxylin. We expressed the TRAP-positive osteoclast number as a mean of five sections obtained from five levels of the root in each mouse. RNA preparation and real time-based RT-PCR analysis For isolating total RNA, tissues including the first molar root and periodontium on the pressure side were frozen in liquid nitrogen. Subsequently, they were ground using Multi-Beads Shocker (Yasui Kikai, Osaka, Japan) and then centrifuged in 1 ml TRIzol Reagent (Invitrogen, Carlsbad CA, USA). For reverse transcription-polymerase chain reaction (RT-PCR) analysis, cDNA was synthesized from 1 mg of total RNA using reverse transcriptase (Invitrogen) and oligo-dT primers (Invitrogen) in a volume of 25 ml. The expression level of cathepsin K mRNA was quantified by real time-based RT-PCR using the LightCycler System (Roche Diagnostics, Mannheim, Germany). Reactions were performed using a 20 ml volume with 5 ml cDNA, 2 ml FastStart DNA Master SYBR Green I (Roche Diagnostics, Mannheim, Germany), 0.5 mM primers, and 4 mM of MgCl2. The GAPDH primers used were 5¢-GGTGGAGCCAAA AGGGTCA-3¢ and 5¢-GGGGGCTAAGCAGTTGGT-3¢; and for cathepsin K, the primers used were 5¢-GCAGAG GTGTGTACTATGA-3¢ and 5¢-GCAGGCGTTGTTCTT ATT-3¢. The reactions were as follows: incubation at 95°C for 10 min to activate the polymerase followed by 45 cycles

Immunohistochemistry

Statistical analysis The evaluation of each group was expressed as the mean ± SEM. Comparison among the groups was statistically analyzed by one-way analysis of variance (ANOVA) and Fisher’s protected least significant difference (PLSD) or Student’s t-test. P < 0.05 was considered statistically significant.

Results Histological changes in the periodontium during orthodontic tooth movement Figure 2 summarizes the histological changes in the periodontium at the distobuccal root of the upper first molar before and after application of orthodontic force. Prior to

23 Fig. 2. Vertical sections of the distobuccal root of the first molar and alveolar bone on day 2 (B), day 4 (C), day 6 (D), day 8 (E), and day 10 (F) after application of the orthodontic appliance. A Histology before application of the appliance. a, alveolar bone; p, periodontal space; r, root. The direction of tooth movement caused by orthodontic force is toward the mesial side (open arrow). Bone resorption lacunae are shown by arrowheads

application of the orthodontic force, the first molar retained the well-reserved periodontal space between the tooth root and alveolar bone. Because the mouse upper molars move to the distal portion of the maxilla in the control teeth, the distal sides of the roots undergo mechanical loading, and the mesial sides of the roots undergo tension. In the control teeth, the surface of the alveolar bone was smooth at the mesial side and irregular on the distal side due to bone resorption. There was no apparent resorption lacuna on the surfaces of any roots in the control teeth. On day 2 after application of orthodontic force, the periodontal space on the pressure side (mesial side) had narrowowed and that on the tension side (distal side) became wider. The alveolar bone surface on the pressure side showed a few resorption lacunae. In some cases, the root surface at the apical region on the pressure side showed an irregular shape due to resorption of the cementum; however, no such changes were observed on the root surface of the tension side. On day 4, bone resorption lacunae became more apparent at the surface of the alveolar edge on the pressure side. Other histological findings were almost identical to those observed on day 2. On day 6, the resorption lacunae on the alveolar bone surface extended to the apical side from the alveolar ridge. The range of the bone resorption lacunae extended to further apical regions on day 10. Changes in TRAP-positive osteoclasts during orthodontic tooth movement

Fig. 3. Tartrate-resistant acid phosphatase (TRAP)-stained horizontal section before (A) and after tooth movement (B–D). TRAP activity is shown in red. Open arrow indicates the direction of physiological tooth movement for control teeth. TRAP activity on day 2 (B), day 6 (C), and day 10 (D) after application of the orthodontic appliance. Black arrow indicates the direction of orthodontic force. M and D, mesial side and distal side, respectively; a, alveolar bone; p, periodontal space; r, root

To identify osteoclasts histologically, we performed tartrate-resistant acid phosphatase (TRAP) staining on the transverse histological sections of the distobuccal root. Figure 3 summarizes the histology of TRAP staining before (day 0) and after (days 2, 6, and 10) the application of orthodontic force. On day 0, TRAP activity was observed

on the alveolar bone surface and in osteoclasts at the distal region of the periodontium, and no activity was observed in the mesial region of the periodontium (Fig. 3). In contrast, on day 2, TRAP activity appeared on the limited surface of the alveolar bone and in a few osteoclasts in the mesial

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region of the periodontium, on which pressure was exerted due to the application of orthodontic force. On day 6, strong TRAP activity was found on the resorption surface as well as in the osteoclasts at the mesial region of the periodontium, and its activity increased more extensively on the bone surface and osteoclasts on day 10. The black bars in Fig. 6B (see later) summarizes the changes in TRAPpositive osteoclast number in wild-type mice. The number of TRAP-positive osteoclasts increased on day 2, compared with that on day 0; however, there was no significant difference between these two groups. The numbers of TRAPpositive osteoclasts significantly increased on days 6 and 10 in the wild-type mice, and there was a significant increase relative to day 0.

Immunohistochemistry of TNFa during orthodontic tooth movement Because TNFa is one of the candidate cytokines involved in orthodontic tooth movement, we investigated the expression of TNFa during tooth movement. Figure 5 shows the results of immunohistochemistry experiments for TNFa on the pressure side of the distobuccal root of the first molar. On day 0, no expression of TNFa was observed on the

Expression of cathepsin K during orthodontic tooth movement Because cathepsin K is a marker for identifying osteoclasts, we investigated the changes in the expression levels of cathepsin K mRNA using real-time-based RT-PCR during orthodontic tooth movement. As shown in Fig. 4, there was no significant difference between the expression levels of cathepsin K mRNA from samples obtained on day 0 and day 2. On day 6, the expression of cathepsin K mRNA had increased; however, no significant increase was observed relative to day 0. Finally, on day 10, the expression level was 2.8-fold in the tissues obtained from the pressure side of the root of the first molar, and there was a significant increase relative to day 0.

Fig. 5. Immunohistochemistry for tumor necrosis factor-a (TNFa) in horizontal sections before (A) and after (B–D) application of orthodontic appliance. B Day 2 after application of the appliance. C Day 6. D Day 10. White arrow indicates the direction of the physiological tooth movement in control teeth. Black arrow indicates the direction of the orthodontic force. Arrowheads indicate immunopositive cells. a, aloveolar bone; p, periodontal space; r, root

Fig. 4. Changes in expression level of cathepsin K mRNA during orthodontic tooth movement assessed by real-time-based reverse transcription-polymerase chain reaction (RT-PCR), as described in the text. There is no significant difference in expression levels on days 0, 2, and 6. The expression level on day 10 is significantly different from that on day 2. Four mice were used in each group. The data were expressed as the mean ± SEM. *P < 0.05 by one-way analysis of variance (ANOVA) and Fisher’s protested least significant difference (PLSD)

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pressure side; however, on days 2, 6, and 10 after application of the orthodontic force, expression of TNFa was identified in the osteoclasts and mononuclear cells located on the alveolar bone surface as well as in fibroblastic cells in the periodontium on the pressure side. Application of tooth movement technique to TNFR-deficient mice Figure 6A demonstrates the cumulative 1st and 2nd molar distance during the experimental period in the wild-type mice, TNFR1-deficient mice, and TNFR2-deficient mice. In all groups, tooth movement occurred on day 2. From day 4 to day 8, large tooth movement was not observed in either group; however 1st and 2nd molar distance in TNFR2deficient mice was significantly lower than that in wildtype mice on days 6 and 8. On day 10, the distance of tooth movement increased in all groups, but the increase in TNFR2-deficient mice was significantly lower than that in wild-type mice and TNFR1-deficient mice. Figure 6B summarizes the changes in osteoclast number during tooth movement in wild-type mice, TNFR1-deficient mice, and TNFR2-deficient mice. Although the number of TRAP-positive osteoclasts increased gradually depending on the days after application of the appliance in all of these mice, TNFR2-deficient mice showed significantly lower values than those in TNFR1-deficient mice on day 6.

Discussion There have been a limited number of reports on mice used for tooth movement experiments. The studies employed an elastic band [15,19], stainless steel coil spring [13], stainless steel round wire [14], and a Ni-Ti coil spring [18] to move the teeth. These experiments demonstrated the essential events that occurred during the tooth movement process, including increases in osteoclast number and the expression level of cathepsin K mRNA in mice and in rodent models. We applied a Ni-Ti coil spring to obtain a continuous force for tooth movement of the mouse upper molars, giving an initial force of 10 g. This material is more suitable for exerting continuous orthodontic force than elastic bands and stainless appliances. Chung et al. [18] also used the same material successfully to analyze tooth movement of the mouse upper molars. They extensively examined tooth movement by soft X-ray and micro-computed tomography analyses at 7 and 21 days after the application of the appliance and demonstrated the validity of this model for mouse tooth movement. This report provided important information about tooth movement in the mouse model; however, it did not include any histological analysis during tooth movement. Therefore, we analyzed the histological changes during tooth movement to evaluate the biological validity of this model. To assess the effect of our orthodontic appliance on tooth movement, we measured the distance of the movement. This experiment verified that our orthodontic appli-

Fig. 6. A Time course of changes in first and second molar distance in wild-type mice, TNF receptor-1 (TNFR1)-deficient mice, and TNFR2deficient mice. The first and second molar distance was measured as described in the text. Filled squares, wild-type mice; open circles, TNFR1-deficient mice; open triangles, TNFR2-deficient mice. There were 10–15 wild-type mice in each group and 6–11 TNFR-mutated mice. The data were expressed as the mean ± SEM. *Significantly different from the corresponding control group on each day. *P < 0.05 by Student’s t-test. B The number of TRAP-positive cells on the pressure side of the distobuccal root of the first molar during orthodontic tooth movement in wild-type mice (black bars), TNFR1-deficient mice (hatched bars), and TNFR2-deficient mice (dotted bars). The number of TRAP-positive multinuclear cells on the pressure side per section was quantified as described in the text. There were six mice in each group. The data were expressed as the mean ± SEM. *Significantly different from the value on day 0 in wild-type mice. #Significantly different from the value of day 6 in TNFR1-deficient mice. P < 0.05 by one-way ANOVA and Fisher’s PLSD

ance induced two phases of tooth movement; the first phase occurred by day 2, and the second phase occurred between days 8 and 10. The first phase of tooth movement might be related to the findings that narrowing of the periodontium at the mesial side occurred from day 2, and that TRAPpositive osteoclasts appeared on the pressure side of the

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alveolar bone on day 2. The vertical histological sections revealed that extensive alveolar bone resorption occurred in further apical regions on day 10, compared with day 6. This finding may be closely related to the second phase of tooth movement that occurred between days 8 and 10. However, bone morphometric analysis revealed that the number of TRAP-positive osteoclasts increased gradually from day 2 to day 10 depending on the numbers of days after application of the orthodontic appliance; this result was not in agreement with the results of measuring the amount of tooth movement (1st and 2nd molar distance). We counted osteoclast number using horizontal sections of the teeth. We first tried to count osteoclasts using vertical sections, but there was a large variation in each experimental group. This seemed to be due to the difficulty of obtaining the same level of sections along the long axis of the tooth root because mouse molar roots are very thin compared with those in rats. Thus, we counted the osteoclast number using horizontal sections. This measurement generated less variation in the osteoclast number in each group compared with that measured using vertical sections (data not shown). However, more extensive, careful studies on the distribution of osteoclasts using vertical sections may provide the exact correlation between the number of osteoclasts and the tooth movement distance. The expression level of cathepsin K mRNA greatly increased on day 10 compared with that on day 6. This may be related to the second phase of tooth movement in our model. We applied approximately 10 g of continuous force in mice based on the previous reports that described mouse tooth movement [13]. Although there was no apparent necrosis or hyaline degeneration in the periodontium on the pressure side, we found root resorption at the periapical region of the pressure side from day 2 after application of the orthodontic force. These results suggest that 10 g of continuous force might be too strong to induce tooth movement without root resorption because a strong force magnitude is one of the causes of root resorption during orthodontic treatment [26]. Further experiments are required to identify the optimal force magnitude for ideal tooth movement in a mouse model. Ren et al. [27] failed to find evidence concerning the optimal force level for orthodontic tooth movement in animal experiments through a systemic literature review and suggested that more standardized mouse tooth movement models are necessary to provide more insight into the relation between the applied force and the rate of tooth movement. Because TNFa is a candidate cytokine involved in orthodontic tooth movement [21–23], we investigated the expression of TNFa in the periodontium during tooth movement by immunohistochemistry. These cells were osteoclasts and mononuclear cells located on the alveolar bone surface and fibroblastic cells in the periodontium. Because activated macrophages, monocytes, lymphoid cells, and fibroblasts produce TNFa [28,29], there is a possibility that several kinds of cell are concerned with the production of TNFa during orthodontic tooth movement. In the present study, one antibody against TNFa was available for

immunohistochemical investigations, and it could be used only on frozen sections, not on paraffin sections. Resolution of hard tissue frozen sections is not sufficient to observe the details of the cells reacting to the antibody. To identify more exact cell types producing TNFa, further studies using an antibody that reacts on paraffin sections are required. In preliminary experiments using real-time-based RT-PCR analysis, we found that the expression levels of TNFa mRNA slightly increased on days 2 and 6 after application of the appliance, and it had strongly increased on day 10 (data not shown). To confirm a more precise expression profile of TNFa mRNA during tooth movement, further molecular analyses including in situ hybridization should be performed in the future. To explore the role of TNFa signaling during orthodontic tooth movement, we applied our tooth movement model to TNFR-mutated mice. We examined which receptors were involved in tooth movement using both TNFR1deficient mice and TNFR2-deficient mice. This experiment demonstrated that tooth movement was delayed beginning 6 days after application of the appliance, and that there was no significant difference in tooth movement on days 2 and 4 after application of the appliance. This suggests that TNFa plays some important role in tooth movement at a later stage, but not at an early stage. Considered together, our experiments using TNFR-mutated mice revealed certain roles of TNF signaling in orthodontic tooth movement; however, other cytokines may be concerned with bone resorption during orthodontic tooth movement because the tooth movement could not be blocked completely in these mice. Our experimental model provides great opportunities for the use of various gene mutated mice for exploring the roles of other cytokines in the mechanical loading induced by orthodontic tooth movement. Acknowledgments We thank Atsuko Yamaguchi for providing technical assistance in the preparation of histological sections. This work was supported by a Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science and a Grant-in Aid for Scientific Research on Priority Areas from the Ministry of Education, Culture, Sports, Science, and Technology of Japan.

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