Enhancement of orthodontic tooth movement and root resorption in ovariectomized mice.

Background/purpose: As the number of patients with osteoporosis requiring orthodontic treatment is increasing with the aging of society, it is necessary to evaluate the relations between bone metabolism in old age and orthodontic tooth movement (OTM). However, the effects of changes in bone metabolism due to osteoporosis on OTM and root resorption are still unclear. Therefore, we investigated the effects of OTM and root resorption in a mouse ovariectomy (OVX)-induced osteoporosis model. Materials and methods: Eight-week-old female wild-type mice underwent OVX or sham surgery (Sham) as controls. One month after treatment, a nickel titanium coil spring was used to apply a mesial force to the maxillary left first molars of OVX or Sham mice for 12 days. The distance between the maxillary first molar and the second molar changed due to OTM and osteoclast formation was evaluated. The odontoclast formation and root resorption along the root surface of the distobuccal root of the first molar was also evaluated by histological analysis and scanning electron microscopy. Results: Distance of tooth movement and osteoclast formation were significantly increased in OVX mice compared to Sham controls. Furthermore, root resorption in the mesial surface of the distal molars induced by orthodontic force was significantly increased in OVX mice. Conclusion: The amount of OTM was significantly increased, and the accompanying root resorption was also increased in OVX mice. Therefore, attention should be paid to the risk of root resorption associated with orthodontic treatment in patients with osteoporosis.

Salt-Sensitive Hypertension Induces Osteoclastogenesis and Bone Resorption via Upregulation of Angiotensin II Type 1 Receptor Expression in Osteoblasts.

Hypertension is a chronic-low grade inflammatory disease, which is known to be associated with increased bone loss. Excessive activity of the local renin-angiotensin system (RAS) in bone leads to increased bone resorption. As inflammatory cytokines may activate RAS components, we hypothesized that the elevated proinflammatory cytokine levels in hypertension activate bone RAS and thus lead to increased bone resorption. To investigate whether salt-sensitive hypertension (SSHTN) induces osteoclastogenesis and bone resorption, we generated a model of SSHTN in C57BL/6J mice by post-N ω-nitro-l-arginine methyl ester hydrochloride (l-NAME) high-salt challenge. SSHTN led to the reduction of distal femur trabecular number and bone volume fraction, while trabecular separation of femoral bone showed a significant increase, with no change in cortical thickness. Histomorphometric examination showed a significant reduction in trabecular bone volume fraction with an increased number of multinucleated tartrate-resistant acid phosphatase (TRAP)-positive cells and increased osteoclast surface fraction in the trabecular distal femur of hypertensive mice. Furthermore, analysis of gene expression in bone tissue revealed that TRAP and RANKL/OPG mRNA were highly expressed in hypertensive mice. TNF-α and angiotensin II type 1 receptor (AGTR1) mRNA and protein expression were also upregulated in SSHTN mice. These observations suggested that TNF-α may have an effect on AGTR1 expression leading to osteoclast activation. However, TNF-α stimulation did not promote AGTR1 mRNA expression in osteoclast precursors in culture, while TNF-α increased AGTR1 mRNA expression in osteoblast culture by activation of downstream p38. Angiotensin II was also shown to increase TNF-α-induced RANKL/OPG mRNA expression in primary osteoblast culture and osteoclastogenesis in a TNF-α-primed osteoblast and osteoclast precursor co-culture system. In addition, local injection of lipopolysaccharide into the supracalvariae of SSHTN mice markedly promoted osteoclast and bone resorption. In conclusion, mice with SSHTN show increased osteoclastogenesis and bone resorption due mainly to increased TNF-α and partly to the upregulation of AGTR1 in osteoblasts.

Micro-Osteoperforations Induce TNF-α Expression and Accelerate Orthodontic Tooth Movement via TNF-α-Responsive Stromal Cells.

Micro-osteoperforations (MOPs) have been reported to accelerate orthodontic tooth movement (OTM), and tumor necrosis factor (TNF)-α has been reported to play a crucial role in OTM. In this report, the influence of MOPs during OTM was analyzed. We evaluated the expression of TNF-α with and without MOPs by RT-PCR analysis. A Ni-Ti closed coil spring was fixed between the maxillary left first molar and the incisors as an OTM mouse model to move the first molar in the mesial direction. MOPs were prepared on the lingual side and mesial side of the upper first molars. Furthermore, to investigate the target cell of TNF-α for osteoclast formation during OTM with MOPs in vivo, we created four types of chimeric mice in which bone marrow of wild-type (WT) or TNF receptor 1- and 2-deficient mice (KO) was transplanted into lethally irradiated WT or KO mice. The results showed that MOPs increased TNF-α expression, the distance of tooth movement and osteoclast formation significantly. Furthermore, mice with TNF-α-responsive stromal cells showed a significant increase in tooth movement and number of osteoclasts by MOPs. We conclude that MOPs increase TNF-α expression, and tooth movement is dependent on TNF-α-responsive stromal cells.

Role of the Interaction of Tumor Necrosis Factor-α and Tumor Necrosis Factor Receptors 1 and 2 in Bone-Related Cells.

Tumor necrosis factor-α (TNF-α) is a pleiotropic cytokine expressed by macrophages, monocytes, and T cells, and its expression is triggered by the immune system in response to pathogens and their products, such as endotoxins. TNF-α plays an important role in host defense by inducing inflammatory reactions such as phagocytes and cytocidal systems activation. TNF-α also plays an important role in bone metabolism and is associated with inflammatory bone diseases. TNF-α binds to two cell surface receptors, the 55kDa TNF receptor-1 (TNFR1) and the 75kDa TNF receptor-2 (TNFR2). Bone is in a constant state of turnover; it is continuously degraded and built via the process of bone remodeling, which results from the regulated balance between bone-resorbing osteoclasts, bone-forming osteoblasts, and the mechanosensory cell type osteocytes. Precise interactions between these cells maintain skeletal homeostasis. Studies have shown that TNF-α affects bone-related cells via TNFRs. Signaling through either receptor results in different outcomes in different cell types as well as in the same cell type. This review summarizes and discusses current research on the TNF-α and TNFR interaction and its role in bone-related cells.

C­X­C receptor 7 agonist acts as a C­X­C motif chemokine ligand 12 inhibitor to ameliorate osteoclastogenesis and bone resorption.

The C­X­C receptor (CXCR) 7 agonist, VUF11207, is a chemical compound that binds specifically to CXCR7, and negatively regulates C­X­C motif chemokine ligand 12 (CXCL12) and CXCR4­induced cellular events. Lipopolysaccharide (LPS) can induce inflammatory cytokines and pathological bone loss. LPS also induces expression of CXCL12, enhancing sensitivity to receptor activator of NF­κB ligand (RANKL) and tumor necrosis factor­α (TNF­α) in vivo. RANKL and TNF­α induce the differentiation of osteoclasts into osteoclast precursors and bone resorption. The current study was performed to examine the effects of a CXCR7 agonist on osteoclastogenesis and bone resorption induced by LPS in vivo. In addition, the mechanisms underlying these in vivo effects were investigated by in vitro experiments. Eight­week­old male C57BL/6J mice were subcutaneously injected over the calvariae with LPS alone or LPS and CXCR7 agonist. After sacrifice, the number of osteoclasts and the bone resorption area were measured. In vitro experiments were performed to investigate the effects of CXCL12 and CXCR7 agonist on osteoclastogenesis induced by RANKL and TNF­α. Mice injected with LPS and CXCR7 agonist showed significantly reduced osteoclastogenesis and bone resorption compared with mice injected with LPS alone. Moreover, the CXCR7 agonist inhibited CXCL12 enhancement of RANKL­ and TNF­α­induced osteoclastogenesis in vitro. Thus, CXCR7 agonist inhibited LPS­induced osteoclast­associated cytokines, such as RANKL and TNF­α, as well as RANKL­ and TNF­α­induced osteoclastogenesis in vitro by modulating CXCL12­mediated enhancement of osteoclastogenesis. In conclusion, CXCR7 agonist reduced CXCL12­mediated osteoclastogenesis and bone resorption.

Tumor necrosis factor-α enhances the expression of vascular endothelial growth factor in a mouse orthodontic tooth movement model.

BACKGROUND/PURPOSE: Tooth movement that is achieved using orthodontic mechanical principles relies on bone resorption which takes place on the compression side via osteoclasts. Tumor necrosis factor-α (TNF-α) has been known to affect osteoclast formation in orthodontic tooth movement (OTM). Vascular endothelial growth factor (VEGF), which is one of the mediators of angiogenesis, also plays an important role in OTM by inducing vascular permeability and chemotaxis of osteoclast precursors. Therefore, the purpose of this research was to evaluate the effect of TNF-α on VEGF expression during OTM. MATERIALS AND METHODS: In order to demonstrate the effect of TNF-α on VEGF expression during OTM, a nickel titanium closed coil spring was fixed to the upper left first molar and the alveolar bone beneath the upper incisors of both wild type (WT) and TNF receptors (TNFRs) deficient mice resulting in a mesial movement of the molar for 12 days. The maxilla was removed for histological analysis and real-time RCR analysis of VEGF expression. RESULTS: Immunohistochemical analysis revealed that there were fewer VEGF-positive cells in the periodontal membrane on the mesial side of the distobuccal root in TNFRs-deficient mice than that in WT mice during the OTM for 12 days. Furthermore, expression of VEGF mRNA is lower level in TNFRs-deficient mice than that in WT mice. CONCLUSION: Our results indicate that TNF-α plays an important role in VEGF expression during tooth movement.

Docosahexaenoic acid inhibits TNF-α-induced osteoclast formation and orthodontic tooth movement through GPR120.

Docosahexaenoic acid (DHA) is an omega-3 fatty acid that has a range of positive impacts on human health, including anti-inflammatory effects and inhibition of osteoclast formation via G-protein-coupled receptor 120 (GPR120). Orthodontic force was reported to induce tumor necrosis factor-α (TNF-α) expression, which activates osteoclast differentiation during orthodontic tooth movement (OTM). The aim of this study was to investigate the influence of DHA on TNF-α-induced osteoclast formation and OTM in vivo. We examined osteoclast formation and bone resorption within the calvaria of both wild-type (WT) and GPR120-deficient (GPR120-KO) mice injected with phosphate-buffered saline (PBS), TNF-α, TNF-α and DHA, or DHA. DHA inhibited TNF-α-induced osteoclast formation and bone resorption in WT mice but had no effect in GPR120-KO mice. OTM experiments were performed in mouse strains with or without regular injection of DHA, and the effects of DHA on osteoclast formation in the alveolar bones during OTM were examined. DHA also suppressed OTM in WT but not GPR120-KO mice. Our data showed that DHA suppresses TNF-α-induced osteoclastogenesis and bone resorption via GPR120. TNF-α has considerable significance in OTM, and therefore, DHA may also inhibit TNF-α-induced osteoclast formation and bone resorption in OTM.

Effect of TNF-α on osteocyte RANKL expression during orthodontic tooth movement.

BACKGROUND/PURPOSE: Orthodontic tooth movement (OTM) is facilitated by two events; bone resorption on the compression side and bone formation on the tension side simultaneously termed bone remodeling. Osteocytes play a critical role in bone remodeling during OTM, as they have been described as the critical source of nuclear factor-κB ligand (RANKL) necessary for bone remodeling during OTM. Tumor necrosis factor (TNF)-α is a cytokine that acts by amplifying RANKL expression in osteocytes. In this study, we evaluated the effects of TNF-α on RANKL expression in osteocyte during OTM. MATERIALS AND METHODS: We assessed whether TNF-α influenced RANKL expression in osteocyte during orthodontic tooth movement by using wild-type (WT) and TNF receptor I and II deficient (TNFRsKO) mice. A Nickel-titanium closed coil spring was attached to the maxillary alveolar bone near the incisors and the upper left first molar, and the first molars were moved mesially in WT and TNFRsKO mice. After OTM, the number of RANKL-positive osteocytes in the alveolar bone was evaluated by immunohistochemistry. RESULTS: The number of RANKL-positive osteocyte in the alveolar bone significantly increased in WT mice than in TNFRsKO mice after OTM. CONCLUSION: The results indicate that TNF-α induces the expression of RANKL in osteocyte during OTM.

Effects of Incretin-Related Diabetes Drugs on Bone Formation and Bone Resorption.

Patients with type 2 diabetes have an increased risk of fracture compared to the general population. Glucose absorption is accelerated by incretin hormones, which induce insulin secretion from the pancreas. The level of the incretin hormone, glucagon-like peptide-1 (GLP-1), shows an immediate postprandial increase, and the circulating level of intact GLP-1 is reduced rapidly by dipeptidyl peptidase-4 (DPP-4)-mediated inactivation. Therefore, GLP-1 receptor agonists and DPP-4 inhibitors are effective in the treatment of type 2 diabetes. However, these incretin-related diabetic agents have been reported to affect bone metabolism, including bone formation and resorption. These agents enhance the expression of bone markers, and have been applied to improve bone quality and bone density. In addition, they have been reported to suppress chronic inflammation and reduce the levels of inflammatory cytokine expression. Previously, we reported that these incretin-related agents inhibited both the expression of inflammatory cytokines and inflammation-induced bone resorption. This review presents an overview of current knowledge regarding the effects of incretin-related diabetes drugs on osteoblast differentiation and bone formation as well as osteoclast differentiation and bone resorption. The mechanisms by which incretin-related diabetes drugs regulate bone formation and bone resorption are also discussed.

Local administration of high-dose diabetes medicine exendin-4 inhibits orthodontic tooth movement in mice.

OBJECTIVES: To investigate the effects of exendin-4 on orthodontic tooth movement distance, root resorption, and expression levels of osteoclast-related cytokines in a mouse model. MATERIALS AND METHODS: A 10-g NiTi coil spring was placed between the anterior alveolar bone and upper left first molar of 8-week-old male C57BL/6 mice. Twenty microliters of exendin-4 solution (containing 0.2 µg, 4 µg, or 20 µg exendin-4) or phosphate-buffered saline (PBS) were injected on the buccal side of the upper left first molar at 2-day intervals (4 mice per group). Mice were sacrificed on day 12; silicone impressions were taken to record tooth movement distance. The left maxillae of the PBS and 20 µg exendin-4 groups were also excised for histological analysis and quantitative reverse transcription polymerase chain reaction analysis. RESULTS: Orthodontic tooth movement distance was smaller in the 20 µg exendin-4 group than in the PBS group (P < .01). Compared with the PBS group, the 20 µg exendin-4 group showed lower osteoclast number (P < .05), odontoclast number (P < .05), and root resorption surface percentage (P < .05). Relative to maxillae with PBS injections, maxillae with 20 µg exendin-4 injections had lower receptor activator of nuclear factor kappa-B ligand (RANKL) mRNA expression (P < .05), TNF-α mRNA expression (P < .05), and RANKL/osteoprotegerin (OPG) ratio (P < .01). There were no differences in the expression of OPG mRNA. CONCLUSIONS: Exendin-4 inhibits orthodontic tooth movement. Therefore, additional attention is needed for orthodontic patients who receive exendin-4 for diabetes treatment. GLP-1 receptor may be a treatment target for patients with severe root resorption.

Effect of a DPP-4 Inhibitor on Orthodontic Tooth Movement and Associated Root Resorption.

OBJECTIVES: Dipeptidyl peptidase-4 (DPP-4) inhibitors are used as a treatment for type 2 diabetes mellitus and have also recently been applied to enhance bone quality and density, and increase the expression of bone markers. This study aimed to investigate the effect of a DPP-4 inhibitor on orthodontic tooth movement (OTM) and related root resorption in a mouse model. MATERIALS AND METHODS: Mice were randomly divided into three groups: those undergoing OTM with the addition of a DPP-4 inhibitor (30 µg), those undergoing OTM and receiving phosphate-buffered saline (PBS), and those without force loading (control group). OTM was achieved by means of a nickel-titanium closed coil spring that moved the first molar in a mesial direction for 12 days. The distance of OTM was measured using silicone impression. Maxillae were removed for histological analysis or real-time PCR analysis. RESULTS: The distance of OTM and the number of osteoclasts were significantly decreased after administration of the DPP-4 inhibitor, which also significantly suppressed the number of odontoclasts and root resorption after OTM. Furthermore, the mRNA expression of tumour necrosis factor-α (TNF-α) and the receptor activator of nuclear factor kappa-B ligand (RANKL) were decreased in DPP-4 inhibitor-treated mice compared with those receiving PBS and control animals. CONCLUSION: The DPP-4 inhibitor inhibited tooth movement and associated root resorption by blocking the formation of osteoclasts and odontoclasts, respectively. It also appeared to inhibit osteoclastogenesis and odontoclastogenesis by suppressing the expression of TNF-α and/or RANKL.

Anti-c-fms Antibody Prevents Osteoclast Formation and Bone Resorption in Co-Culture of Osteoblasts and Osteoclast Precursors In Vitro and in Ovariectomized Mice.

Osteoporosis morphology is characterized by bone resorption and decreases in micro-architecture parameters. Anti-osteoporosis therapy targets osteoclasts because bone resorption is a unique function of osteoclasts. Anti-c-fms antibodies against the receptor for macrophage colony-stimulating factor (M-CSF) inhibit osteoclast formation and bone resorption in vitro and in vivo. However, the effect of anti-c-fms antibodies on bone resorption in ovariectomized (OVX) mice is unknown. In this study, we evaluated the effect of anti-c-fms antibodies on osteoclast formation and bone resorption in osteoblast-osteoclast precursor co-culture in vitro and in OVX mice. Osteoblast and osteoclast precursor co-cultures treated with anti-c-fms antibodies showed significantly inhibited osteoclast formation, while cultures without anti-c-fms antibody treatment showed osteoclast formation. However, anti-c-fms antibodies did not change the receptor activator of nuclear factor kappa-B ligand (RANKL) or osteoprotegrin (OPG) expression during osteoblast and osteoclast differentiation in vitro. These results indicate that anti-c-fms antibodies directly affected osteoclast formation from osteoclast precursors in co-culture. OVX mice were treated with intraperitoneal injections of anti-c-fms antibody. The trabecular bone structure of the femur was assessed by micro-computer tomography. The anti-c-fms antibody inhibited osteoclast formation and bone loss compared with PBS-treated OVX mice. These results indicate potential for the therapeutic application of anti-c-fms antibodies for postmenopausal osteoporosis.

Osteocyte-Related Cytokines Regulate Osteoclast Formation and Bone Resorption.

The process of bone remodeling is the result of the regulated balance between bone cell populations, namely bone-forming osteoblasts, bone-resorbing osteoclasts, and the osteocyte, the mechanosensory cell type. Osteoclasts derived from the hematopoietic stem cell lineage are the principal cells involved in bone resorption. In osteolytic diseases such as rheumatoid arthritis, periodontitis, and osteoporosis, the balance is lost and changes in favor of bone resorption. Therefore, it is vital to elucidate the mechanisms of osteoclast formation and bone resorption. It has been reported that osteocytes express Receptor activator of nuclear factor κΒ ligand (RANKL), an essential factor for osteoclast formation. RANKL secreted by osteocytes is the most important factor for physiologically supported osteoclast formation in the developing skeleton and in pathological bone resorption such as experimental periodontal bone loss. TNF-α directly enhances RANKL expression in osteocytes and promotes osteoclast formation. Moreover, TNF-α enhances sclerostin expression in osteocytes, which also increases osteoclast formation. These findings suggest that osteocyte-related cytokines act directly to enhance osteoclast formation and bone resorption. In this review, we outline the most recent knowledge concerning bone resorption-related cytokines and discuss the osteocyte as the master regulator of bone resorption and effector in osteoclast formation.

Obtaining Primary Osteocytes through Murine Calvarial Fractionation of GFP-Expressing Osteocytes.

The osteocyte, once thought to be a passive resident of the bone given the backstage function of sensing mechanical loading, is now brought to the spotlight and has been shown to have multiple major functions like actively modifying the extracellular matrix and forming an endocrine organ with the lacunocanalicular system that encloses it sending messages to distant sites. Owing to the methods that made it possible to test the osteocyte in vitro from isolating primary osteocytes to osteocyte-like cell lines, osteocytes are now experiencing a resounding interest and a surge of knowledge on structure and function. Many aspects of the osteocyte biology and interaction with other molecular components are yet to be discovered. In this protocol, we describe in detail the efficient isolation of primary osteocytes from dmp1-topaz neonatal mouse calvaria, which express the green fluorescent protein in osteocytes, through cell fractionation and subsequently acquiring cultures of primary osteocytes by FACS.

TNF-α stimulates the expression of RANK during orthodontic tooth movement.

OBJECTIVE: This study aimed to evaluate the effects of tumor necrosis factor (TNF)-α on receptor activator of nuclear factor-κB (RANK) expression in osteoclast precursors in vitro and during orthodontic tooth movement (OTM) in vivo. DESIGN: We assessed whether TNF-α influenced RANK expression levels in osteoclast precursors in vitro by real-time PCR and western blotting. For in vivo experiments, TNF-α was subcutaneously injected into mouse calvariae daily for 5 days. Mice were sacrificed and RANK expression was evaluated by real-time PCR and immunohistochemistry. For OTM, a nickel-titanium closed-coil spring was fixed between the upper incisors and upper-left first molar to move the first molar in the mesial direction in wild-type (WT) and TNFR1/TNFR2-deficient (TNFRsKO) mice. After OTM, the number of RANK-positive cells on the compression side was evaluated by immunohistochemistry. RESULTS: RANK levels were enhanced in TNF-α-treated osteoclast precursors in vitro. RANK mRNA expression levels and the number of RANK-positive cells were higher in TNF-α-injected mice than in phosphate-buffered saline-injected mice. RANK-positive cells increased on the compression side of the alveolar bone in WT mice because of the mechanical loading. In addition, the number of RANK-positive cells on the compression side was significantly higher in WT mice than in TNFRsKO mice after OTM. CONCLUSION: These results suggest that TNF-α induces RANK expression in vitro and at baseline in vivo, as well as on the compression side during OTM.

IL-33 Inhibits TNF-α-Induced Osteoclastogenesis and Bone Resorption.

Interleukin (IL)-33 is a member of the IL-1 family, which acts as an alarmin. Several studies suggested that IL-33 inhibited osteoclastogenesis and bone resorption. Tumor necrosis factor-α (TNF-α) is considered a direct inducer of osteoclastogenesis. However, there has been no report regarding the effect of IL-33 on TNF-α-induced osteoclastogenesis and bone resorption. The objective of this study is to investigate the role of IL-33 on TNF-α-induced osteoclastogenesis and bone resorption. In an in vitro analysis of osteoclastogenesis, osteoclast precursors, which were derived from bone marrow cells, were treated with or without IL-33 in the presence of TNF-α. Tartrate-resistant acid phosphatase (TRAP) staining solution was used to assess osteoclast formation. In an in vivo analysis of mouse calvariae, TNF-α with or without IL-33 was subcutaneously administrated into the supracalvarial region of mice daily for 5 days. Histological sections were stained for TRAP, and osteoclast numbers were determined. Using micro-CT reconstruction images, the ratio of bone destruction area on the calvariae was evaluated. The number of TRAP-positive cells induced by TNF-α was significantly decreased with IL-33 in vitro and in vivo. Bone resorption was also reduced. IL-33 inhibited IκB phosphorylation and NF-κB nuclear translocation. These results suggest that IL-33 inhibited TNF-α-induced osteoclastogenesis and bone resorption.

TNF-α is responsible for the contribution of stromal cells to osteoclast and odontoclast formation during orthodontic tooth movement.

Compressive force during orthodontic tooth movement induces osteoclast formation in vivo. TNF-α plays an important role in mouse osteoclast formation and bone resorption induced by compressive force during orthodontic tooth movement. Stromal cells, macrophages and T cells take part in TNF-α-induced osteoclast formation in vitro. Root resorption caused by odontoclasts is a major clinical problem during orthodontic tooth movement. In this study, we determined the cell type targeted by TNF-α during compressive-force-induced osteoclast and odontoclast formation to elucidate the mechanism of bone and root resorption in vivo. An orthodontic tooth movement mouse model was prepared with a nickel-titanium closed coil spring inserted between the maxillary incisors and the first molar. Using TNF receptor 1- and 2-deficient (KO) mice, we found that osteoclast and odontoclast formation was mediated by TNF-α in orthodontic tooth movement. We generated four types of chimeric mice: wild-type (WT) bone marrow cells transplanted into lethally irradiated WT mice (WT>WT), KO bone marrow cells transplanted into lethally irradiated WT mice (KO>WT), WT bone marrow cells transplanted into lethally irradiated KO mice (WT>KO), and KO marrow cells transplanted into lethally irradiated KO mice (KO>KO). Using anti-CD4 and anti-CD8 antibodies, T cells were eliminated from these mice. We subjected these chimeric mice to orthodontic tooth movement. Orthodontic tooth movement was evaluated and tartrate-resistant acid phosphatase-positive cells along the alveolar bone (osteoclasts) and along the tooth root (odontoclasts) were counted after 12 days of tooth movement. The amount of orthodontic tooth movement, and the number of osteoclasts and odontoclasts on the compression side were significantly lower in WT>KO and KO>KO mice than in WT>WT and KO>WT mice. According to these results, we concluded that TNF-α-responsive stromal cells are important for osteoclast and odontoclast formation during orthodontic tooth movement.

Effect of TNF-α-Induced Sclerostin on Osteocytes during Orthodontic Tooth Movement.

Osteocytes are abundant cells in bone, which contribute to bone maintenance. Osteocytes express receptor activator of nuclear factor kappa-B ligand (RANKL) and regulate osteoclast formation. Orthodontic tooth movement (OTM) occurs by osteoclast resorption of alveolar bone. Osteocyte-derived RANKL is critical in bone resorption during OTM. Additionally, tumor necrosis factor-α (TNF-α) is important in osteoclastogenesis during OTM. Sclerostin has been reported to enhance RANKL expression in the MLO-Y4 osteocyte-like cell line. This study investigated the effect of TNF-α on sclerostin expression in osteocytes during OTM. In vitro analysis of primary osteocytes, which were isolated from DMP1-Topaz mice by sorting the Topaz variant of GFP-positive cells, revealed that SOST mRNA expression was increased when osteocytes were cultured with TNF-α and that RANKL mRNA expression was increased when osteocytes were cultured with sclerostin. Moreover, the number of TRAP-positive cells was increased in osteocytes and osteoclast precursors cocultured with sclerostin. In vivo analysis of mouse calvariae that had been subcutaneously injected with phosphate-buffered saline (PBS) or TNF-α revealed that the number of TRAP-positive cells and the percentage of sclerostin-positive osteocytes were higher in the TNF-α group than in the PBS group. Furthermore, the level of SOST mRNA was increased by TNF-α. As an OTM model, a Ni-Ti closed-coil spring connecting the upper incisors and upper-left first molar was placed to move the first molar to the mesial direction in wild-type (WT) mice and TNF receptor 1- and 2-deficient (TNFRsKO) mice. After 6 days of OTM, the percentage of sclerostin-positive osteocytes on the compression side of the first molar in TNFRsKO mice was lower than that in WT mice. In this study, TNF-α increased sclerostin expression in osteocytes, and sclerostin enhanced RANKL expression in osteocytes. Thus, TNF-α may play an important role in sclerostin expression in osteocytes and enhance osteoclast formation during OTM.

Establishment of an orthodontic retention mouse model and the effect of anti-c-Fms antibody on orthodontic relapse.

Orthodontic relapse after orthodontic treatment is a major clinical issue in the dental field. However, the biological mechanism of orthodontic relapse is still unclear. This study aimed to establish a mouse model of orthodontic retention to examine how retention affects the rate and the amount of orthodontic relapse. We also sought to examine the role of osteoclastogenesis in relapse using an antibody to block the activity of M-CSF, an essential factor of osteoclast formation. Mice were treated with a nickel-titanium closed-coil spring that was fixed between the upper incisors and the upper-left first molar to move the first molar in a mesial direction over 12 days. Mice were randomly divided into three groups: group 1, no retention (G1); group 2, retention for 2 weeks (G2); and group 3, retention for 4 weeks (G3). In G2 and G3, a light-cured resin was placed in the space between the first and second molars as a model of retention. Orthodontic relapse was assessed by measuring changes in the dimensions of the gap created between the first and second molars. To assess the activity and role of osteoclasts, mice in G3 were injected with anti-c-Fms antibody or PBS, and assessed for changes in relapse distance and rate. Overall, we found that a longer retention period was associated with a slower rate of relapse and a shorter overall amount of relapse. In addition, inhibiting osteoclast formation using the anti-c-Fms antibody also reduced orthodontic relapse. These results suggest that M-CSF and/or its receptor could be potential therapeutic targets in the prevention and treatment of orthodontic relapse.

Effect of Anti-c-fms Antibody on Osteoclast Formation and Proliferation of Osteoclast Precursor In Vitro.

Bone remodeling is a complex process and it involves periods of deposition and resorption. Bone resorption is a process by which bone is broken down by osteoclasts in response to different stimuli. Osteoclast precursors differentiate into multinuclear osteoclasts in response to macrophage colony stimulating factor (M-CSF) and receptor activator of nuclear factor Kappa-B ligand (RANKL). Under pathologic conditions, the cytokine profile is different and involves a mixture of inflammatory cytokines. Tumor necrosis factor alpha (TNF-α) is one of the most important cytokines as it is found in large amounts in areas involved with inflammatory osteolysis. The purpose of this protocol is to provide a method by which murine bone marrow is isolated to generate osteoclasts through induction with M-CSF and either RANKL or TNF-α which will be subsequently inhibited by increasing doses of anti-c-fms antibody, the receptor for M-CSF. This experiment highlights the therapeutic value of anti-c-fms antibody in diseases of inflammatory bone resorption.