INTRODUCTION

The formation of osteoclasts depends on macrophage colony-stimulating factor (M-CSF) and a ligand for the receptor activator of necrosis factor κB (RANKL). It has been reported recently that administration of an antibody against the M-CSF receptor c-Fms (anti-c-Fms antibody) blocked osteoclastogenesis and orthodontic tooth movement.12

Root resorption often is observed as an undesirable side effect of orthodontic treatment. It is a very serious problem for most clinicians, but it is still not understood. The cause of root resorption is considered to be multifactorial. Several studies have suggested that excessive orthodontic force is a critical factor for the root resorption.34 Associations between root resorption and tooth morphology,5 tooth intrusion,67 periodontal condition,8 or systemic factors such as genetics,9 the immune system,1011 and bone metabolism.1213 have been suggested. The cells responsible for the resorption of tooth roots are called odontoclasts. These cells are considered to be similar to osteoclasts.

Osteoclasts, which differentiate from hematopoietic stem cells, are required for bone resorption and remodeling. Formation of osteoclasts is dependent on M-CSF and a ligand for RANKL. Both M-CSF and RANKL are essential factors for osteoclast differentiation.14 Tumor necrosis factor-α (TNF-α) also was reportedly able to induce osteoclasts from bone marrow macrophages in vitro.15–17 TNF-α–induced osteoclast recruitment is probably central to bone erosive diseases such as rheumatoid arthritis,18 periodontal disease,19 periprosthetic bone loss,20 and postmenopausal osteoporosis.21 Several studies have shown that excessive orthodontic force induces expression of TNF-α and suggests an important role of TNF-α in orthodontic tooth movement.22–24 We also investigated the role of TNF-α in orthodontic tooth movement by using TNFR-deficient mice. Results showed that tooth movement in TNFR-deficient mice was less than that in wild-type mice, and that TNF-α plays an important role in mechanical loading–induced osteoclastogenesis and bone remodeling in orthodontic tooth movement.25

It has been reported recently that administration of an antibody against the M-CSF receptor c-Fms (anti-c-Fms antibody) completely blocked osteoclastogenesis and bone erosion induced by TNF-α administration or inflammatory arthritis.1 Because anti-c-Fms antibody inhibited TNF-α–induced osteoclast recruitment, we investigated whether the anti-c-Fms antibody could inhibit mechanical stress–induced osteoclastogenesis and orthodontic tooth movement in mice. The anti-c-Fms antibody significantly inhibited orthodontic tooth movement, markedly reducing the number of osteoclasts in vivo.2 These findings suggest that M-CSF plays an important role in mechanical loading–induced osteoclastogenesis and bone resorption during orthodontic tooth movement mediated by TNF-α. Because root resorption is understood to be performed by odontoclasts, similarly to osteoclasts in bone resorption, we hypothesized that the anti-c-Fms antibody might inhibit odontoclastogenesis and root resorption during orthodontic tooth movement.

In the present study, we demonstrate the effect of anti-c-Fms antibody on mechanical stress–induced odontoclast and the degree of bone resorption assessed using an orthodontic tooth movement mouse model.

MATERIALS AND METHODS

Experimental Tooth Movement

The mice were anesthetized with an intraperitoneal injection of 50 mg/kg pentobarbital sodium during setting and adjusting of the orthodontic appliance. The orthodontic appliance is composed of an Ni-Ti closed coil spring. The appliance was inserted between the upper incisors and the upper left first molar and was fixed with a 0.1-mm stainless wire around both teeth.25 According to the manufacturer's database, the force level of the coil spring after activation is approximately 10 × g.25 The left maxillary molar in each mouse was used to assess experimental tooth movement (Figure 1A). We evaluated odontoclast area by percentage of odontoclast surface on the pressure side of the distobuccal root of the first molar during orthodontic tooth movement (Figure 1B).

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RESULTS

Mechanical Loading Induces Odontoclastogenesis in an Orthodontic Tooth Movement Model

We assessed mechanical loading odontoclastogenesis during orthodontic tooth movement in a mouse model. We observed histologic pictures of the distobuccal root of the first molar after it had been power loaded for 0, 2, 6, 10, or 12 days. From days 0 to 6, no odontoclasts were observed. On days 10 and 12, odontoclasts were observed on transverse histologic sections of the mesial side of the distobuccal root of the upper first molars (Figure 2).

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Histologic Evaluation of the Inhibitory Effect of the Anti-c-Fms Antibody on Odontoclastogenesis during Orthodontic Tooth Movement

Figure 3 shows the histologic evaluation of odontoclastogenesis after application of mechanical force. We carried out TRAP staining on transverse histologic sections of the distobuccal root. The histology of TRAP staining of control mice, power-loaded mice, and power-loaded mice administered anti-c-Fms antibody on day 12 is shown in Figure 3. The odontoclast area was not observed on the root surface on the mesial side in control mice. Odontoclasts were found on the root surface in the mesial region in power-loaded mice on day 12. On the other hand, significantly fewer odontoclasts were found on the root surface of the mesial region in power-loaded mice administered anti-c-Fms antibody.

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Electron Microscopic Evaluation of the Inhibitory Effect of the Anti-c-Fms Antibody for Root Resorption during Orthodontic Tooth Movement

Electron microscopic photographs of half of the mesial area of the distobuccal root of the first molar are shown in Figure 4. Before tooth movement occurred, most of the roots exhibited areas covered by undamaged cementum with a characteristic smooth surface. By contrast, after tooth movement, root resorption was observed. However, in the anti-c-Fms antibody injection group, root resorption was prevented (Figure 4B).

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DISCUSSION

Since the discovery of the RANK-RANKL signal transduction, RANKL has been thought to be essential for osteoclast differentiation. Recently, it was reported that TNF-α makes osteoclasts independent of RANKL.17 We investigated this using mice whose bone marrow stromal cells and osteoclast precursors are chimeric for the presence of TNF receptors, and found that both cell types mediated the TNF-α–induced osteoclastogenic properties.127 Furthermore, TNF-α induced M-CSF gene expression in stromal cells and increased osteoclast precursor numbers in vivo.1 M-CSF assists in the survival and longevity of osteoclast precursors and organizes the cytoskeletons of osteoclasts.14 Recently, we reported that an anti-c-Fms antibody completely blocked pathologic osteoclastogenesis and bone resorption, whether in inflammatory arthritis or induced by direct injection of TNF-α.1 Therefore, M-CSF and c-Fms are clearly candidate therapeutic targets for TNF-α–related pathologic osteolysis.

It has been reported that orthodontic tooth movement increases the levels of TNF-α in the gingival sulcus in humans.2324 Under pathologic conditions resulting from excessive orthodontic force, TNF-α has been shown to be expressed in rat periodontal tissue, suggesting an important role of TNF-α in orthodontic tooth movement.22 Recently, the anti-c-Fms antibody also inhibited TNF-α–related orthodontic tooth movement and markedly reduced the numbers of osteoclasts in vivo. Findings suggest that M-CSF plays an important role in mechanical loading–induced osteoclastogenesis and bone resorption during orthodontic tooth movement mediated by TNF-α. M-CSF basically is produced by mesenchymal cells, and its regulated secretion has pathologic consequences for osteoclasts. For example, the absence of estrogen in postmenopausal osteoporosis is due to enhanced bone resorption caused by increased production of M-CSF by marrow stromal cells.28 In inflammatory osteolysis, the level of M-CSF is increased in the serum of patients with rheumatoid arthritis who have severe ankylosing spondylitis29 and in synovial fluid around loose joint prostheses.30 These observations suggest that stromal cell– produced M-CSF may be an important mediator of osteoclastogenesis in vivo. In this study, we evaluated the effect of an anti-c-Fms antibody on mechanical stress–induced odontoclastogenesis and root resorption in an orthodontic tooth movement model. We found that the anti-c-Fms antibody inhibited mechanical stress–induced odontoclastogenesis and root resorption. The amount of root resorption strongly differed between control and anti-c-Fms antibody–injected groups on day 12. In our previous study, 200 μg of anti-c-Fms antibody was injected intraperitoneally into an inflammatory arthritis mouse model, and the osteoclast inhibitory effect was determined to be systemic.1 In the present study, a lesser amount of anti-c-Fms antibody (10 μg) was injected into a local site in an orthodontic tooth movement model. Mechanical stress–induced odontoclastogenesis was inhibited by anti-c-Fms antibody at the local site, even though the amount was small.

Our data indicate that M-CSF might be a therapeutic target in mechanical stress–induced root resorption in orthodontic tooth movement. This suggests that localized anti-c-Fms antibody might be useful for the prevention of root resorption during orthodontic tooth movement. However, in our previous study,2 orthodontic tooth movement also was inhibited by same amount of anti-c-Fms antibody. Thus, anti-c-Fms antibody inhibits orthodontic tooth movement at the same time. Additional studies are needed in this area.

M-CSF is concerned with host defense mononuclear phagocytes. Anti-c-Fms antibody prevents pathologic osteoclastogenesis, but the immune-suppressive effects of inhibiting macrophage proliferation and survival coincided. It might be that there is a limitation to M-CSF blockade in clinical applications. In a previous in vitro study, anti-c-Fms antibody prevented RANKL- and M-CSF–induced osteoclast formation at a concentration of at least one order of magnitude less than its capacity to decrease cell number.1 Results suggested that osteoclast formation is more sensitive to M-CSF inhibition than are macrophage proliferation and survival. The dose of antibody might be important when it is applied in clinical applications. The receptor tyrosine kinase inhibitor SU11248, which prevents activation of the receptor for M-CSF, inhibits osteoclast formation and functioning in vitro and in vivo.31 The tyrosine kinase inhibitor imatinib also inhibits c-Fms. It is suggested that there is a possibility of drug treatment for bone destruction due to excessive osteoclast activity.32 However, therapeutic use of M-CSF must be approached with caution, given the significant complications encountered with other forms of anti-cytokine therapy.33 Additional studies are needed to establish the therapeutic use of M-CSF for the treatment of patients.

CONCLUSION

• M-CSF and/or its receptor is a potential therapeutic target for the treatment of mechanical stress–induced odontoclastogenesis, and injection of an anti-c-Fms antibody might be useful for inhibition of mechanical stress–induced root resorption during orthodontic tooth movement.