The incretin hormone glucose-dependent insulinotropic polypeptide (GIP) promotes insulin secretion, lowers blood glucose levels, and is increasingly linked to bone remodeling. Native GIP is quickly inactivated by the enzyme dipeptidyl peptidase-4 (DPP-4), whereas (D-Ala2)GIP is a novel GIP analog engineered to resist DPP-4 degradation. Tumor necrosis factor-alpha (TNF-α), a key proinflammatory cytokine, promotes osteoclastogenesis and is notably upregulated during orthodontic tooth movement (OTM). This study aimed to evaluate the effects of (D-Ala2)GIP on TNF-α-induced osteoclast formation and bone resorption in vivo, as well as on OTM and related root resorption. Mice received daily supracalvarial injections of TNF-α with or without (D-Ala2)GIP for 5 days. The (D-Ala2)GIP-treated group showed significantly reduced osteoclast formation, bone resorption, and expression of osteoclastic markers TRAP and cathepsin K, compared to the group that received TNF-α alone. OTM was induced in mice by applying a nickel-titanium closed-coil spring, and mice were treated with either phosphate-buffered saline (PBS) or (D-Ala2)GIP every 2 days. After 12 days, the (D-Ala2)GIP-treated group showed significantly reduced tooth movement and fewer osteoclasts and odontoclasts on the compression side compared to the PBS control. These findings suggest that (D-Ala2)GIP inhibits OTM, potentially by suppressing TNF-α-driven osteoclastogenesis and bone resorption.

Orthodontic tooth movement (OTM) is a biologically orchestrated process involving the dynamic interplay of mechanical force, inflammatory signaling, and bone remodeling. Osteocytes, the most abundant cells within the bone matrix, serve as mechanosensitive regulators that transduce mechanical cues into biochemical signals in response to orthodontic force. This review delineates the multifaceted role of osteocytes in facilitating bone resorption required for OTM. The role of osteocytes is examined in inflammation, mechanical adaptation, and cell death. Additionally, we discuss the evidence on how aging alters osteocyte function, with senescence-associated changes disrupting mechanosensory networks and attenuating bone remodeling. Finally, the possibility that osteocytes themselves undergo morphological adaptation during force application is explored. This structural plasticity may impact individual variability in orthodontic outcomes. Advancing our understanding of osteocyte signaling in OTM holds significant promise for optimizing treatment outcomes across diverse patient populations.

Osteoclastogenesis-the activation and differentiation of osteoclasts-is one of the pivotal processes of bone remodeling and is regulated by RANKL/RANK signaling, the decoy function of osteoprotegerin (OPG), and a cascade of pro- and anti-inflammatory cytokines. The disruption of this balance leads to pathological bone loss in diseases such as osteoporosis and rheumatoid arthritis. FFAR4 (Free Fatty Acid Receptor 4), a G protein-coupled receptor for long-chain omega-3 fatty acids, has been confirmed as a key mediator of metabolic and anti-inflammatory effects. This review focuses on how FFAR4 acts as the selective receptor for the omega-3 fatty acid docosahexaenoic acid (DHA). It activates two divergent signaling pathways. The Gαq-dependent cascade facilitates intracellular calcium mobilization and ERK1/2 activation. Meanwhile, β-arrestin-2 recruitment inhibits NF-κB. These collective actions reshape the cytokine environment. In macrophages, DHA-FFAR4 signaling lowers the levels of TNF-α, interleukin-6 (IL-6), and IL-1β while increasing IL-10 secretion. Consequently, the activation of NFATc1 and NF-κB p65 is profoundly suppressed under TNF-α or RANKL stimulation. Additionally, DHA modulates the RANKL/OPG axis in osteoblastic cells by suppressing RANKL expression, thereby reducing osteoclast differentiation in an inflammatory mouse model.

A challenge in orthodontic treatment is the long time taken to move teeth, which extends the long treatment period. Accordingly, various treatment protocols and orthodontic materials have been developed to shorten the orthodontic treatment period. However, controlling biological reactions is considered necessary to further shorten this treatment period. Orthodontic force results in compression of the periodontal ligament in the direction of tooth movement, resulting in various reactions in the periodontal ligament that induce osteoclast development, alveolar bone absorption, and teeth movement. The aforementioned reactions include immune reactions. Cytokines are substances responsible for intercellular communication and are involved in various physiological actions, including immune and inflammatory reactions. They cause various cellular responses, including cell proliferation, differentiation, cell death, and functional expression. Various cytokines are involved in biological reactions during orthodontic tooth movement (OTM). It is important to understand the role of cytokines during OTM in order to elucidate their biological response. This review discusses the role of cytokines during OTM.

BACKGROUND/PURPOSE: Orthodontic treatment is one of the most demanding procedures available for both patients and clinicians. The challenges stem from the extended duration to achieve desired results, often necessitating surgical interventions, such as micro-osteoperforations (MOPs). This study aimed to investigate the biological effects and extent of changes resulting from these interventions. Specifically, we evaluated the degree of root resorption during orthodontic tooth movement accelerated by MOPs. MATERIALS AND METHODS: We assessed the tooth movement rates and root resorption in eight-to ten-week-old male mice. A nickel-titanium (Ni-Ti) closed-coil spring was applied between the maxillary left first molar and maxillary incisors. In the MOPs group, micro-perforations were made on the mesial and palatal surfaces of the left maxillary first molar. Odontoclast formation and root resorption were assessed using histological analysis and scanning electron microscopy. RESULTS: Tooth movement was greater in the MOPs group. Odontoclast formation was remarkably higher in this group than in the orthodontic tooth movement (OTM) group. Additionally, more extensive root resorption was observed on the mesial surface of the distobuccal root of the left maxillary first molar. CONCLUSION: Root resorption significantly increased in mice with MOPs. These findings highlight the need to carefully consider the risk of root resorption in patients undergoing MOPs during orthodontic treatment.

Orthodontic tooth movement (OTM) has been described as a bone remodeling process mediated by the expression of various inflammatory cytokines, including tumor necrosis factor-α (TNF-α). Necroptosis is a form of regulated cell death that is mainly induced by TNF-α, leading to the release of damage-associated molecular patterns (DAMPs) that cause inflammation. However, the role of osteocyte necroptosis in regulating osteoclastogenesis during OTM remains unclear. Here, we investigated the effects of osteocyte necroptosis on osteoclastogenesis in a mouse model of OTM. In wild-type mice, osteocyte death was remarkably increased on day 6 after OTM. Transmission electron microscopy identified apoptotic osteocytes, necrotic osteocytes, and empty lacunae based on morphological characteristics. TNF receptor type 1- and 2-deficient (TNFRsKO) mice showed a reduction in osteocyte death on day 6 after OTM. Immunofluorescence staining detected necroptosis markers in osteocytes on the compression side in wild-type OTM mice, whereas such osteocytes were almost undetectable in TNFRsKO OTM mice. Furthermore, the conditioned medium from primary osteocytes undergoing necroptosis significantly enhanced osteoclastogenesis. These findings suggest that TNF-α-induced osteocyte necroptosis enhances osteoclastogenesis and alveolar bone resorption on the compression side during OTM, involving the release of inflammatory factors including DAMPs.

BACKGROUND/PURPOSE: Orthodontic tooth movement (OTM) is a critical aspect of dental treatment that requires the precise control of bone remodeling processes. Hypertension (HTN) can affect the effectiveness of OTM. Salt-sensitive hypertension (SSHTN) is of particular concern due to its detrimental effects on bone health, potentially altering orthodontic outcomes. This study aimed to investigate the effects of SSHTN on OTM using a mouse model. MATERIALS AND METHODS: Male mice were divided into a normal and an SSHTN group. The SSHTN model was generated by administering N(ω)-nitro-l-arginine methyl ester (l-NAME) followed by a high-salt diet. The OTM was performed using a nickel-titanium (Ni-Ti) closed-coil spring, and the tooth movement was measured after 12 days. Silicone imprinting was used to estimate the OTM distance. Osteoclast activity was assessed using tartrate-resistant acid phosphatase (TRAP) staining of decalcified maxillary sections. RESULTS: SSHTN mice exhibited significantly increased tooth movement compared to normal mice. This enhanced movement was associated with more osteoclasts in the SSHTN group than in the control group. These findings suggest that SSHTN increases OTM levels by promoting bone resorption. CONCLUSION: SSHTN significantly affected OTM by enhancing osteoclast activity and increasing tooth movement. These results underscore the importance of considering hypertensive conditions in orthodontic treatment planning as they may require adjustments in force application to prevent potential adverse effects.

Background/Objective: Osteocytes are the most abundant cell type in the skeleton, with key endocrine functions, particularly in regulating osteoblast and osteoclast activity to maintain bone quality. Angiotensin II (Ang II), a critical component of the renin-angiotensin-aldosterone system, is well-known for its role in vasoconstriction during hypertension. Beyond its cardiovascular functions, Ang II participates in various biological processes, including bone metabolism. While its influence on osteoblast proliferation, differentiation, and osteoclastogenesis has been documented, its effects on osteocytes remain unexplored. This study hypothesized that Ang II enhances the osteoclastogenic activity of osteocytes. Methods: Mouse calvariae were cultured ex vivo in an Ang II-containing medium, analyzed via immunohistochemistry, and evaluated for osteoclastogenic gene expression through real-time PCR. Western blotting was employed to assess protein levels and signaling pathway activation in the MLO-Y4 osteocytic cell line in vitro. Results: Ang II significantly increased the expression of receptor activator of nuclear factor κB ligand (RANKL) and macrophage colony-stimulating factor (M-CSF). These effects were abrogated by azilsartan, a blocker targeting Ang II type 1 receptors (AT1R). p38 and ERK1/2 in the MAPK pathway were also activated by Ang II. Conclusions: Ang II enhances osteocyte-mediated osteoclastogenesis via AT1R activation, highlighting its potential as a therapeutic target for bone diseases.

Tumor necrosis factor-alpha (TNF-α) is a significant cytokine that regulates bone resorption under inflammatory conditions. However, its mechanism of action in osteocytes remains unclear. In this study, highly purified osteocytes were isolated from dentin matrix protein 1 (DMP1)-Topaz mice using cell sorter. RNA sequencing (RNA-seq) revealed that TNF-α stimulation increased C-X-C motif chemokine ligand 10 (CXCL10) gene expression in osteocytes. Although CXCL10 did not affect osteoclast differentiation in vitro, it enhanced the migration of osteoclast precursors. Additionally, in the transwell co-culture system, TNF-α induced the migration of osteoclast precursors. However, this effect was attenuated by a CXCL10-neutralizing antibody. In vivo, mice were administered supracalvarial injections of TNF-α with or without the CXCL10-neutralizing antibody for 5 days. The percentage of CXCL10-positive osteocytes increased after TNF-α administration. Additionally, osteoclast formation and bone resorption were assessed. CXCL10-neutralizing antibody-treated calvariae exhibited a significantly lower number of osteoclasts and bone resorption than those treated with TNF-α alone. These results indicated that TNF-α-induced CXCL10, which affects the migration of osteocyte-derived osteoclast precursors, may enhance TNF-α-triggered osteoclast formation and bone resorption in vivo.

Glucose-insulinotropic polypeptide (GIP) is an incretin hormone that induces insulin secretion and decreases blood glucose levels. In addition, it has been reported to suppress osteoclast formation. Native GIP is rapidly degraded by dipeptidyl peptidase-4 (DPP-4). (D-Ala2)GIP is a newly developed GIP analog that demonstrates enhanced resistance to DPP-4. This study aimed to evaluate the influence of (D-Ala2)GIP on osteoclast formation and bone resorption during lipopolysaccharide (LPS)-induced inflammation in vivo and in vitro. In vivo, mice received supracalvarial injections of LPS with or without (D-Ala2)GIP for 5 days. Osteoclast formation and bone resorption were evaluated, and TNF-α and RANKL expression were measured. In vitro, the influence of (D-Ala2)GIP on RANKL- and TNF-α-induced osteoclastogenesis, LPS-triggered TNF-α expression in macrophages, and RANKL expression in osteoblasts were examined. Compared to the LPS-only group, calvariae co-administered LPS and (D-Ala2)GIP led to less osteoclast formation, lower bone resorption, and decreased TNF-α and RANKL expression. (D-Ala2)GIP inhibited osteoclastogenesis induced by RANKL and TNF-α and downregulated TNF-α expression in macrophages and RANKL expression in osteoblasts in vitro. Furthermore, (D-Ala2)GIP suppressed the MAPK signaling pathway. The results suggest that (D-Ala2)GIP dampened LPS-triggered osteoclast formation and bone resorption in vivo by reducing TNF-α and RANKL expression and directly inhibiting osteoclastogenesis.

INTRODUCTION: Hypertension is a major risk factor for cardiovascular disease (CVD) and is associated with increased bone loss due to excessive activity of the local renin-angiotensin system (RAS). Angiotensinogen/Angiotensin (ANG) II/Angiotensin II type 1 receptor (AT1R) axis is considered as the core axis regulating RAS activity. Azilsartan is an FDA-approved selective AT1R antagonist that is used to treat hypertension. This study aimed to determine whether azilsartan affects formation of osteoclast, resorption of bone, and the expression of cytokines linked with osteoclastogenesis during lipopolysaccharide (LPS)-triggered inflammation in vivo. METHODS: In vivo, following a 5-day supracalvarial injection of LPS or tumor necrosis factor-alpha (TNF-α) with or without azilsartan, the proportion of bone resorption and the number of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells, which are identified as osteoclasts on mice calvariae were counted. The mRNA expression levels of TRAP, cathepsin K, receptor activator of NF-κB ligand (RANKL), and TNF-α were also evaluated. In vitro, the effect of azilsartan (0, 0.01, 0.1, 1, and 10 μM) on RANKL and TNF-α-triggered osteoclastogenesis were investigated. Also, whether azilsartan restrains LPS-triggered TNF-α mRNA and protein expression in macrophages and RANKL expression in osteoblasts were assessed. Furthermore, western blotting for analysis of mitogen-activated protein kinases (MAPKs) signaling was conducted. RESULTS: Azilsartan-treated calvariae exhibited significantly lower bone resorption and osteoclastogenesis than those treated with LPS alone. In vivo, LPS with azilsartan administration resulted in lower levels of receptor activator of RANKL and TNF-α mRNA expression than LPS administration alone. Nevertheless, azilsartan did not show inhibitory effect on RANKL- and TNF-α-triggered osteoclastogenesis in vitro. Compared to macrophages treated with LPS, TNF-α mRNA and protein levels were lower in macrophages treated by LPS with azilsartan. In contrast, RANKL mRNA and protein expression levels in osteoblasts were the same in cells co-treated with azilsartan and LPS and those exposed to LPS only. Furthermore, azilsartan suppressed LPS-triggered MAPKs signaling pathway in macrophages. After 5-day supracalvarial injection, there is no difference between TNF-α injection group and TNF-α with azilsartan injection group. CONCLUSION: These findings imply that azilsartan prevents LPS-triggered TNF-α production in macrophages, which in turn prevents LPS-Triggered osteoclast formation and bone resorption in vivo.