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S100 calcium-binding protein P (S100P) is a secretory protein that is expressed in various healthy tissues and tumors. Megakaryocyte-secreted S100P promotes osteoclast differentiation and function; however, its receptor and cellular signaling in osteoclasts remain unclear. Receptor for advanced glycation end products (RAGE), which is the receptor for S100P on cancer cells, was expressed in osteoclast precursors, and S100P-RAGE binding was confirmed through co-immunoprecipitation. Additionally, the phosphorylation of ERK and NF-κB was increased in S100P-stimulated osteoclast precursors but was inhibited by addition of the RAGE antagonistic peptide (RAP). S100P-induced osteoclast differentiation and excessive bone resorption activity were also reduced by the addition of RAP. This study demonstrates that S100P, upon binding with RAGE, activates the ERK and NF-κB signaling pathways in osteoclasts, leading to increased cell differentiation and bone resorption activity., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Obesity and type 2 diabetes mellitus (T2DM) are linked to osteoporosis development, with obesity being a significant risk factor for T2DM. T2DM patients with obesity exhibit a higher fracture rate and often have a poor prognosis post-fracture. To address the urgent need for understanding the mechanisms of diabetic osteoporosis (DOP), research is ongoing to explore how obesity and T2DM impact bone metabolism. The NLRP3 inflammasome has been implicated in the pathogenesis of osteoporosis, and MCC950, an NLRP3 inflammasome inhibitor, has shown promise in various diseases but its role in osteoporosis remains unexplored. In this study, BMMs and BMSCs were isolated and cultured to investigate the effects of MCC950 on bone metabolism, and DOP model was used to evaluate the efficacy of MCC950 in vivo. The study demonstrated that MCC950 treatment inhibited osteoclast differentiation, reduced bone resorption capacity in BMMs without suppression for osteoblast differentiation from BMSCs. Additionally, MCC950 suppressed the activation of the NF-κB signaling pathway and downregulated key factors associated with osteoclast differentiation. Additionally, MCC950 alleviated bone loss in DOP mouse. These findings suggest that MCC950, by targeting the NLRP3 inflammasome, may have a protective role in preventing osteoporosis induced by T2DM with obesity. The study highlights the potential therapeutic implications of MCC950 in managing diabetic osteoporosis and calls for further research to explore its clinical application in high-risk patient populations., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Understanding the intricate cellular interactions involved in bone restoration is crucial for developing effective strategies to promote bone healing and mitigate conditions such as osteoporosis and fractures. Here, we provide compelling evidence supporting the anabolic effects of a pharmacological Pyk2 inhibitor (Pyk2-Inh) in promoting bone restoration. In vitro, Pyk2 signaling inhibition markedly enhances alkaline phosphatase (ALP) activity, a hallmark of osteoblast differentiation, through activation of canonical Wnt/β-catenin signaling. Notably, analysis of human mesenchymal stem cells through RNA-seq revealed a novel candidate, SCARA5, identified through Pyk2-Inh treatment. We demonstrate that Scara5 plays a crucial role in suppressing the differentiation from stromal cells into adipocytes, and accelerates lineage commitment to osteoblasts, establishing Scara5 as a negative regulator of bone formation. Additionally, Pyk2 inhibition significantly impedes osteoclast differentiation and bone resorption. In a co-culture system comprising osteoblasts and osteoclasts, Pyk2-Inh effectively suppressed osteoclast differentiation, accompanied by a substantial increase in the transcriptional expression of Tnfrsf11b and Csf1 in osteoblasts, highlighting a dual regulatory role in osteoblast-osteoclast crosstalk. In an ovariectomized mouse model of osteoporosis, oral administration of Pyk2-Inh significantly increased bone mass by simultaneously reducing bone resorption, promoting bone formation and decreasing bone marrow fat. These results suggest Pyk2 as a potential therapeutic target for both adipogenesis and osteogenesis in bone marrow. Our findings underscore the importance of Pyk2 signaling inhibition as a key regulator of bone remodeling, offering promising prospects for the development of novel osteoporosis therapies., Competing Interests: Declarations. Ethics approval and consent to participate: Experiments using mice were approved by the Institutional animal care and use committee of Hokkaido University, Japan and were performed in accordance with the committee’s guiding principle (approval no.: 21-0018). Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

Background: Epithelial stromal interaction 1 (EPSTI1) plays an important role in M1 macrophages, which induce osteoclastogenesis. One recent genome-wide association study (GWAS) involving 426,824 individuals has shown that EPSTI1 is strongly associated with osteoporosis (P < 5E-8). Therefore, we speculate that EPSTI1 participates in the modulation of osteoporosis through osteoclastogenesis. The roles of EPSTI1 in osteoclastogenesis and bone resorption remain unclear., Methods: Femur specimens were collected from osteoporotic patients and control patients. Immunofluorescence staining was used to detect the expression of EPSTI1 and signaling pathways. The osteoclastic potential of RAW264.7 cells with Sh-EPSTI1 lentivirus infection was tested using tartrate-resistant acid phosphatase (TRAP) staining, western blotting, and quantitative reverse transcription polymerase chain reaction (qRT-PCR). Western blotting was also used to examine signaling pathways., Results: In this study, EPSTI1 was found to be significantly increased in tartrate-resistant acid phosphatase positive (ACP5 + ) osteoclasts of bone sections from osteoporotic patients. Next, we identified EPSTI1 as a positive regulator of osteoclastogenesis and osteoclast differentiation capability. Diminished EPSTI1 expression resulted in reduced osteoclastic resorption. Mechanistically, EPSTI1-driven osteoclastogenesis was regulated by NF-κB pathway, which was mediated by the phosphorylation of protein kinase R (p-PKR). Furthermore, EPSTI1 participating in the modulation of osteoporosis via PKR/NF-κB pathway was also verified in the bone samples of osteoporotic patients., Conclusions: Collectively, our findings suggest that EPSTI1 may regulate osteoclast differentiation and bone resorption through PKR/NF-κB pathway and in vivo experiments are needed to further verify EPSTI1 as the therapy target for osteoporosis., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Postmenopausal osteoporosis (PMOP) is closely related to the pathogenesis of osteoclasts, with the Cathepsin K (CTSK) protein playing a crucial role. Our study aimed to screen small molecule compounds targeting CTSK and evaluate their impact on PMOP. Through molecular docking, we identified NVP-BHG712 as significantly inhibiting osteoclast differentiation and bone resorption. NVP-BHG712 also effectively suppressed CTSK activity and exhibited strong binding affinity to CTSK protein. Furthermore, NVP-BHG712 regulated the expression of inflammatory factors and modulated the balance between M1 and M2 macrophage polarization. In the mouse model of ovariectomy-induced osteoporosis, NVP-BHG712 rescued bone loss by inhibiting excessive osteoclast activation. These findings suggest that NVP-BHG712 may be a promising treatment for pathological osteoporosis by alleviating osteoclast function., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Excessive activity of osteoclasts(OCs) lead to bone resorption in chronic inflammatory conditions. The use of natural compounds to target OCs offers significant promise in the treatment or prevention of OC-associated diseases. Irilin D (IRD), a natural isoflavone derived from Belamcanda chinensis (L.) DC., has potential effects on OC differentiation both in vitro and in vivo that have yet to be thoroughly explored. In our study, we found that IRD inhibited receptor activator of nuclear factor-κB ligand (RANKL)-induced OC differentiation, actin ring formation, and bone resorption in vitro without compromising cell viability. However, IRD did not exhibit anti-inflammatory effects in lipopolysaccharide (LPS)-stimulated macrophages. Furthermore, IRD reduced LPS-induced inflammatory bone loss by blocking osteoclastogenesis in a mouse model. Mechanistically, IRD disrupted RANKL-induced activation of mitogen-activated protein kinases (MAPKs) and nuclear factor-κB (NF-κB), leading to the inhibition of c-Fos and nuclear factor of activated T cells cytoplasmic 1 (NFATc1) activation. We also demonstrated that IRD inhibited RANKL-induced osteoclastic NFATc1 target genes, including DC-STAMP, ACP5, and CtsK. Our results indicate that IRD mitigates LPS-induced inflammatory bone resorption in mice by inhibiting RANKL-activated MAPKs and NF-κB signaling pathways, suggesting its potential as a natural isoflavone for preventing or treating OC-associated diseases., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Aging leads to a reduced anabolic response to mechanical stimuli and a loss of bone mass and structural integrity. Chemotherapy agents such as doxorubicin exacerbate the degeneration of aging skeleton and further subject older cancer patients to a higher fracture risk. To alleviate this clinical problem, we proposed and tested a novel mechanobiology-based therapy. Building upon prior findings that i) Yoda1, the Piezo1 agonist, promoted bone growth in young adult mice and suppressed bone resorption markers in aged mice, and ii) moderate tibial loading protected bone from breast cancer-induced osteolysis, we hypothesized that combined Yoda1 and moderate loading would improve the structural integrity of adult and aged skeletons in vivo and protect bones from deterioration after chemotherapy. We first examined the effects of 4-week Yoda1 (dose 5 mg/kg, 5 times/week) and moderate tibial loading (4.5 N peak load, 4 Hz, 300 cycles for 5 days/week), individually and combined, on mature mice (∼50 weeks of age). Combined Yoda1 and loading was found to mitigate age-associated cortical and trabecular bone loss better than individual interventions. As expected, the non-treated controls experienced an average drop of cortical polar moment of inertia (Ct.pMOI) by -4.3 % over four weeks and the bone deterioration occurred in the majority (64 %) of the samples. Relative to no treatment, loading alone, Yoda1 alone, and combined Yoda1 and loading increased Ct.pMOI by +7.3 %, +9.5 %, +12.0 % and increased the % of samples with positive Ct.pMOI changes by +32 %, +26 %, and +43 %, respectively, suggesting an additive protection of aging-related bone loss for the combined therapy. We further tested if the treatment efficacy was preserved in mature mice following two weeks (six injections) of doxorubicin at the dose of 2.5 or 5 mg/kg. As expected, doxorubicin increased osteocyte apoptosis, altered bone remodeling, and impaired bone structure. However, the effects induced by DOX were too severe to be rescued by Yoda1 and loading, alone or combined, although loading and Yoda1 individually, or combined, increased the number of mice showing positive responsiveness by 0 %, +15 %, and +29 % relative to no intervention after doxorubicin exposure. Overall, this study supported the potentials and challenges of the Yoda1-based strategy in mitigating the detrimental skeletal effects caused by aging and doxorubicin., Competing Interests: Declaration of competing interest The authors have no conflict of interest., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Osteoclasts are bone-resorbing multinucleated giant cells formed by the fusion of monocyte/macrophage lineages. Various small GTPases are involved in the multinucleation and differentiation of osteoclasts. However, the roles of small GTPases regulatory molecules in osteoclast differentiation remain unclear. In the present study, we examined the role of Dennd2c, a putative guanine nucleotide exchange factor for Rab GTPases, in osteoclast differentiation. Knockdown of Dennd2c promoted osteoclast differentiation, resorption, and expression of osteoclast markers. Morphologically, Dennd2c knockdown induced the formation of larger osteoclasts with several protrusions. In contrast, overexpression of Dennd2c inhibited the multinucleation and differentiation of osteoclasts, bone resorption, and the expression of osteoclast markers. Dennd2c -overexpressing macrophages exhibited spindle-shaped mononuclear cells and long thin protrusions. Treatment of Dennd2c -overexpressing cells with the Cdc42 inhibitor ML-141 or the Rac1 inhibitor 6-thio-GTP prevented protrusion formation. Moreover, treatment of Dennd2c -overexpressing cells with the actin polymerization inhibitor latrunculin B restored multinucleated and TRAP-positive osteoclast formation. These results indicate that Dennd2c negatively regulates osteoclast differentiation and multinucleation by modulating protrusion formation in macrophages.

Rufy4, a protein belonging to the RUN and FYVE domain-containing protein family, participates in various cellular processes such as autophagy and intracellular trafficking. However, its role in osteoclast-mediated bone resorption remains uncertain. In this study, we investigated the expression and role of the Rufy4 gene in osteoclasts using small interfering RNA (siRNA) transfection and gene overexpression systems. Our findings revealed a significant increase in Rufy4 expression during osteoclast differentiation. Silencing Rufy4 enhanced osteoclast differentiation, intracellular cathepsin K levels, and formation of axial protrusive structures but suppressed bone resorption. Conversely, overexpressing wild-type Rufy4 in osteoclasts hindered differentiation while promoting podosome formation and bone resorption. Similarly, overexpression of a Rufy4 variant lacking the RUN domain mimics the effects of Rufy4 knockdown, significantly increasing intracellular cathepsin K levels, promoting osteoclastogenesis, and elongated axial protrusions formation, yet inhibiting bone resorption. These findings indicate that Rufy4 plays a critical role in osteoclast differentiation and bone resorption by regulating the cytoskeletal organization through its RUN domain. Our study provides new insights into the molecular mechanisms governing osteoclast activity and underscores Rufy4's potential as a novel therapeutic target for bone disorders characterized by excessive bone resorption.

Context: Silicon (Si), which is present in the diet in the bioavailable form of orthosilicic acid (OSA) and is detected as a dissolution product of certain bone-substitute materials, is suggested to promote bone health, and enhance bone healing, respectively. Silicon has been shown to stimulate osteoblastic cell differentiation and function, although the effect of Si on human osteoclasts is unclear., Aim: The present study investigated the direct effects of Si on human osteoclast differentiation, gene expression, and bone resorption., Material & Methods: Human CD14+ monocytes were isolated from buffy coats and cultured with M-CSF and RANKL in medium without or with Si (50 μg/ml; constituting 75% OSA). The effects of Si on osteoclast differentiation were evaluated by TRAP-staining and the expression levels of CtsK, CalcR, TRAP, and DC-STAMP measured by RT-qPCR. The effect of Si on the expression level of AQP9, which encodes a potential Si transporter, was also analyzed. Bone resorption was determined based on the number of resorption pits formed when the RANKL-stimulated monocytes were cultured on bone slices, and by the levels of type I collagen fragments released into the cell culture medium., Results: Silicon significantly inhibited the number of TRAP+ multinucleated cells and the expression of osteoclast related genes but increased the late expression of AQP9. Furthermore, Si significantly inhibited the number of resorption pits and the amount of collagen fragments in the medium when cells were cultured on bone slices., Conclusion: Our results demonstrate that OSA inhibits RANKL-stimulated human osteoclast differentiation, gene expression of osteoclast phenotypic markers, and bone resorption., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Magnusson, Ransjö. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Osteoclasts are the only cells able to resorb all the constituents of the bone matrix. While the modulation of osteoclast activity is well established for preventing bone-related diseases, there is an increasing demand for novel classes of anti-resorption agents. Herein, we investigated non-coding RNA molecules and proposed DLEU1 and miR-16 as potential candidates for modulating osteoclast functions. DLEU1 and miR-16 target cell fusion at both the early and late stages of osteoclastogenesis but operate through independent pathways. DLEU1 silencing hinders the fusion process, leading to abrogation of the phagocytic cup fusion modality and a reduction in the fusion events between mononucleated precursors and multinucleated osteoclasts, while miR-16 influences monocyte-to-osteoclast differentiation, impairing osteoclasts formation but not the number of nuclei at early stages. On the other hand, using these non-coding RNAs to engineer mature osteoclasts has implications for bone resorption. Both DLEU1 and miR-16 influence the speed of resorption in pit-forming osteoclasts, without affecting the resorbed area. However, the impact of increasing miR-16 levels extends more broadly, affecting trench-forming osteoclasts as well, leading to a reduction in their percentage, speed, and resorbed area. These findings offer potential new therapeutic targets to ameliorate bone destruction in skeletal diseases., (© 2024. The Author(s).)

Postmenopausal women experience bone loss and weight gain. To date, crosstalk between estrogen receptor signals and nuclear factor-κB (NF-κB) has been reported, and estrogen depletion enhances bone resorption by osteoclasts via NF-κB activation. However, it is unclear when and in which tissues NF-κB is activated after menopause, and how NF-κB acts as a common signaling molecule for postmenopausal weight gain and bone loss. Therefore, we examined the role of NF-κB in bone and energy metabolism following menopause. NF-κB reporter mice, which can be used to measure NF-κB activation in vivo, were ovariectomized (OVX) and the luminescence intensity after OVX increased in the metaphyses of the long bones and perigonadal white adipose tissue, but not in the other tissues. OVX was performed on wild-type (WT) and p65 mutant knock-in (S534A) mice, whose mutation enhances the transcriptional activity of NF-κB. Weight gain with worsening glucose tolerance was significant in S534A mice after OVX compared with those of WT mice. The bone density of the sham group in WT or S534A mice did not change, whereas in the S534A-OVX group it significantly decreased due to the suppression of bone formation and increase in bone marrow adipocytes. Disulfiram, an anti-alcoholic drug, suppressed OVX-induced activation of NF-κB in the metaphyses of long bones and white adipose tissue (WAT), as well as weight gain and bone loss. Overall, the activation of NF-κB in the metaphyses of long bones and WAT after OVX regulates post-OVX weight gain and bone loss., Competing Interests: Declaration of competing interest The authors have declared no conflict of interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Staphylococcus aureus osteomyelitis leads to extensive bone destruction. Osteoclasts are bone resorbing cells that are often increased in bone infected with S. aureus. The cytokine RANKL is essential for osteoclast formation under physiological conditions but in vitro evidence suggests that inflammatory cytokines may by-pass the requirement for RANKL. The goal of this study was to determine whether RANKL-dependent osteoclast formation is essential for the bone loss that occurs in a murine model of S. aureus osteomyelitis. To this end, humanized-RANKL mice were infected by direct inoculation of S. aureus into a unicortical defect in the femur. Mice were treated with vehicle or denosumab, a human monoclonal antibody that inhibits RANKL, both before and during a 14-day infection period. The severe cortical bone destruction caused by infection was completely prevented by denosumab administration even though the bacterial burden in the femur was not affected. Osteoclasts were abundant near the inoculation site in vehicle-treated mice but absent in denosumab-treated mice. In situ hybridization demonstrated that S. aureus infection potently stimulated RANKL expression in bone marrow stromal cells. The extensive reactive bone formation that occurs in this osteomyelitis model was also reduced by denosumab administration. Lastly, there was a notable lack of osteoblasts near the infection site suggesting that the normal coupling of bone formation to bone resorption was disrupted by S. aureus infection. These results demonstrate that RANKL-mediated osteoclast formation is required for the bone loss that occurs in S. aureus infection and suggest that disruption of the coupling of bone formation to bone resorption may also contribute to bone loss in this condition., Competing Interests: Declaration of competing interest The authors declare that they have no competing interests., (Published by Elsevier Inc.)

Background: Ameloblastoma is a locally destructive benign odontogenic tumor. While the neoplastic cells of conventional ameloblastoma can infiltrate the connective tissue and bone, in unicystic ameloblastoma the epithelium is encapsulated. The mechanisms driving ameloblastoma's bone resorption remains unclear., Methods: RNA sequencing (RNA-seq) was performed in a discovery cohort of conventional ameloblastoma, and pathway enrichment analysis was carried out. mRNA levels of MMP13, a gene associated with bone resorption, were assessed using RT-qPCR in a larger cohort of conventional ameloblastoma and in unicystic ameloblastoma. Zymogram gels and the immunoexpression profile of collagenase 3 (encoded by MMP13 gene) were evaluated as well., Results: Enriched pathways related to bone mineralization and upregulation of MMP13 were observed in ameloblastomas. Collagenolytic activity of collagenase 3 was detected in the tumor lysates. Collagenase 3 immunopositivity was observed in ameloblastomatous epithelium infiltrating the fibrous capsule of unicystic ameloblastoma. At the tumor-bone interface, collagenase 3 expression was detected in stromal cells, osteoblasts, and osteocytes., Conclusion: The results indicate a potential involvement of MMP13 in ameloblastoma-related bone resorption and progression., (© 2024 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Abnormalities in osteoclastic generation or activity disrupt bone homeostasis and are highly involved in many pathologic bone-related diseases, including rheumatoid arthritis, osteopetrosis, and osteoporosis. Control of osteoclast-mediated bone resorption is crucial for treating these bone diseases. However, the mechanisms of control of osteoclastogenesis are incompletely understood. In this study, we identified that inosine 5'-monophosphate dehydrogenase type II (Impdh2) positively regulates bone resorption. By histomorphometric analysis, Impdh2 deletion in mouse myeloid lineage cells (Impdh2 LysM-/- mice) showed a high bone mass due to the reduced osteoclast number. qPCR and western blotting results demonstrated that the expression of osteoclast marker genes, including Nfatc1, Ctsk, Calcr, Acp5, Dcstamp, and Atp6v0d2, was significantly decreased in the Impdh2 LysM-/- mice. Furthermore, the Impdh inhibitor MPA treatment inhibited osteoclast differentiation and induced Impdh2-cytoophidia formation. The ability of osteoclast differentiation was recovered after MPA deprivation. Interestingly, genome-wide analysis revealed that the osteoclastic mitochondrial biogenesis and functions, such as oxidative phosphorylation, were impaired in the Impdh2 LysM-/- mice. Moreover, the deletion of Impdh2 alleviated ovariectomy-induced bone loss. In conclusion, our findings revealed a previously unrecognized function of Impdh2, suggesting that Impdh2-mediated mechanisms represent therapeutic targets for osteolytic diseases., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

This study explored the mechanism of curcumin (CUR) suppressing osteoclastogenesis and evaluated its effects on osteoarthritis (OA) mouse. Bone marrow-derived macrophages were isolated as osteoclast precursors. In the presence or absence of CUR, cell proliferation was detected by CCK-8, osteoclastogenesis was detected by tartrate-resistant acid phosphatase (TRAP) staining, F-actin rings formation was detected by immunofluorescence, bone resorption was detected by bone slices, IκBα, nuclear factor kappa-B (NF-κB) and mitogen-activated protein kinase (MAPK) signaling pathways were detected using western blot, osteoclastogenesis-related gens were measured using quantitative polymerase chain reaction. A knee OA mouse model was designed by destabilizing the medial meniscus (DMM). Thirty-six male mice were divided into sham+vehicle, OA+vehicle, and OA+CUR groups. Mice were administered with or without CUR at 25 mg/kg/d from the first post-operative day until sacrifice. After 4 and 8 weeks of OA induction, micro-computed tomography was performed to analyze microstructure changes in subchondral bone, hematoxylin and eosin staining was performed to calculate the thickness of the calcified and hyaline cartilage layers, toluidine blue O staining was performed to assess the degenerated cartilage, TRAP-stained osteoclasts were counted, and NF-κB, phosphorylated Jun N-terminal Kinases (p-JNK), and receptor activator of nuclear factor κB ligand (RANKL) were detected using immunohistochemistry. CUR suppressed osteoclastogenesis and bone resorption without cytotoxicity. CUR restrained RANKL-induced activation of NF-κB, p-JNK and up-regulation of osteoclastogenesis-related genes. CUR delayed cartilage degeneration by suppressing osteoclastogenesis and bone resorption in early OA. The mechanism of CUR inhibiting osteoclastogenesis might be associated with NF-κB/JNK signaling pathway, indicating a novel strategy for OA treatment., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Ding et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Treatments to reduce periodontal inflammation and rescue periodontitis bone resorption have been of interest to researchers. Bone tissue engineering materials have been gradually used in the treatment of bone defects, but periodontal bone tissue regeneration still faces challenges. Considering the biocompatibility factor, constructing bionic scaffolds with natural extracellular matrix properties is an ideal therapeutic pathway. Based on the pathological mechanism of periodontitis, in this study, short peptide and nanometer inorganic particles were comingled to construct NapKFF-nano CaF 2 supramolecular composite hydrogels with different ratios. Material characterization experiments confirmed that the composite hydrogel had suitable mechanical properties and a three-dimensional structure that can function in the resorption region of the alveolar bone and provide spaces for cell proliferation and adhesion. The release of low concentrations of fluoride and calcium ions has been shown to have positive biological effects in both in vivo and in vitro experiments. Vitro experiments confirmed that the composite hydrogel had good biocompatibility and promoted osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs). Microbiological experiments confirmed that the composite hydrogel inhibited the activity of periodontal pathogenic bacteria. In animal studies, composite hydrogel applied to periodontitis rats in vivo can effectively repair alveolar bone resorption. This composite hydrogel has a simple preparation method and is inexpensive to produce, yet it has antibacterial and osteogenesis-promoting incremental effects, which makes it well suited for the treatment of periodontitis bone resorption, providing a new strategy for periodontal bone tissue engineering.

Background: Mechanical unloading-induced bone loss threatens prolonged spaceflight and human health. Recent studies have confirmed that osteoporosis is associated with a significant reduction in bone microvessels, but the relationship between them and the underlying mechanism under mechanical unloading are still unclear., Methods: We established a 2D clinostat and hindlimb-unloaded (HLU) mouse model to simulate unloading in vitro and in vivo. Micro-CT scanning was performed to assess changes in the bone microstructure and mass of the tibia. The levels of CD31, Endomucin (EMCN) and histone deacetylase 6 (HDAC6) in tibial microvessels were detected by immunofluorescence (IF) staining. In addition, we established a coculture system of microvascular endothelial cells (MVECs) and osteoblasts, and qRT‒PCR or western blotting was used to detect RNA and protein expression; cell proliferation was detected by CCK‒8 and EdU assays. ChIP was used to detect whether HDAC6 binds to the miRNA promoter region., Results: Bone mass and bone microvessels were simultaneously significantly reduced in HLU mice. Furthermore, MVECs effectively promoted the proliferation and differentiation of osteoblasts under coculture conditions in vitro. Mechanistically, we found that the HDAC6 content was significantly reduced in the bone microvessels of HLU mice and that HDAC6 inhibited the expression of miR-375-3p by reducing histone acetylation in the miR-375 promoter region in MVECs. miR-375-3p was upregulated under unloading and it could inhibit MVEC proliferation by directly targeting low-density lipoprotein-related receptor 5 (LRP5) expression. In addition, silencing HDAC6 promoted the miR-375-3p/LRP5 pathway to suppress MVEC proliferation under mechanical unloading, and regulation of HDAC6/miR-375-3p axis in MVECs could affect osteoblast proliferation under coculture conditions., Conclusion: Our study revealed that disuse-induced bone loss may be closely related to a reduction in the number of bone microvessels and that the modulation of MVEC function could improve bone loss induced by unloading. Mechanistically, the HDAC6/miR-375-3p/LRP5 pathway in MVECs might be a promising strategy for the clinical treatment of unloading-induced bone loss., (© 2024. The Author(s).)

Maintenance of bone homeostasis and the balance between bone resorption and formation are crucial for maintaining skeletal integrity. This study sought to investigate the role of salt-inducible kinase 3 (SIK3), a key regulator in cellular energy metabolism, during the differentiation of osteoclasts. Despite osteoclasts being high energy-consuming cells essential for breaking down mineralized bone tissue, the specific function of SIK3 in this process remains unclear. To address this issue, we generated osteoclast-specific SIK3 conditional knockout mice and assessed the impact of SIK3 deletion on bone homeostasis. Our findings revealed that SIK3 conditional knockout mice exhibited increased bone mass and an osteopetrosis phenotype, suggesting a pivotal role for SIK3 in bone resorption. Moreover, we assessed the impact of pterosin B, a SIK3 inhibitor, on osteoclast differentiation. The treatment with pterosin B inhibited osteoclast differentiation, reduced the numbers of multinucleated osteoclasts, and suppressed resorption activity in vitro. Gene expression analysis demonstrated that SIK3 deletion and pterosin B treatment influence a common set of genes involved in osteoclast differentiation and bone resorption. Furthermore, pterosin B treatment altered intracellular metabolism, particularly affecting key metabolic pathways, such as the tricarboxylic acid cycle and oxidative phosphorylation. These results provide valuable insights into the involvement of SIK3 in osteoclast differentiation and the molecular mechanisms underlying osteoclast function and bone diseases., (© The Author(s) 2024. Published by Oxford University Press on behalf of the American Society for Bone and Mineral Research. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Developing long bones alter their shape while maintaining uniform cortical thickness via coordinated activity of bone-forming osteoblasts and bone-resorbing osteoclasts at periosteal and endosteal surfaces, a process we designate trans-pairing. Two types of trans-pairing shift cortical bone in opposite orientations: peri-forming trans-pairing (peri-t-p) increases bone marrow space and endo-forming trans-pairing (endo-t-p) decreases it, via paired activity of bone resorption and formation across the cortex. Here, we focused on endo-t-p in growing bones. Analysis of endo-t-p activity in the cortex of mouse fibulae revealed osteoclasts under the periosteum compressed by muscles, and expression of RANKL in periosteal cells of the cambium layer. Furthermore, mature osteoblasts were localized on the endosteum, while preosteoblasts were at the periosteum and within cortical canals. X-ray tomographic microscopy revealed the presence of cortical canals more closely associated with endo- than with peri-t-p. Sciatic nerve transection followed by muscle atrophy and unloading induced circumferential endo-t-p with concomitant spread of cortical canals. Such canals likely supply the endosteum with preosteoblasts from the periosteum under endo-t-p, allowing bone shape to change in response to mechanical stress or nerve injury., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

Osteocytes engage in bone resorption and mineralization surrounding their expansive lacunar-canalicular system (LCS) through peri-LCS turnover. However, fundamental questions persist about where, when, and how often osteocytes engage in peri-LCS turnover and how these processes change with aging. Furthermore, whether peri-LCS turnover is associated with natural variation in cortical tissue strain remains unexplored. To address these questions, we utilized confocal scanning microscopy, immunohistochemistry, and scanning electron microscopy to characterize osteocyte peri-LCS turnover in the cortical (mid-diaphysis) and cancellous (metaphysis) regions of femurs from young adult (5 mo) and early-old-age (22 mo) female C57BL/6JN mice. LCS bone mineralization was measured by the presence of perilacunar fluorochrome labels. LCS bone resorption was measured by immunohistochemical marker of bone resorption. The dynamics of peri-LCS turnover were estimated from serial fluorochrome labeling, where each mouse was administered two labels between 2 and 16 days before euthanasia. Osteocyte participation in mineralizing their surroundings is highly abundant in both cortical and cancellous bone of young adult mice but significantly decreases with aging. LCS bone resorption also decreases with aging. Aging has a greater impact on peri-LCS turnover dynamics in cancellous bone than in cortical bone. Lacunae with recent peri-LCS turnover are larger in both age groups. While peri-LCS turnover is associated with variation in tissue strain between cortical quadrants and intracortical location for 22 mo mice, these associations were not seen for 5 mo mice. The impact of aging on decreasing peri-LCS turnover may have significant implications for bone quality and mechanosensation., Competing Interests: Declaration of competing interest The authors have no conflicts of interest to disclose., (Copyright © 2024 Elsevier Inc. All rights reserved.)

ZC4H2 encodes a C4H2 -type zinc finger protein, mutations of which lead to a spectrum of diseases known as ZC4H2 associated rare disorders (ZARD). In addition to neurological phenotypes, the most typical symptoms of ZARD are multiple joint contractures of varying degrees, accompanied by abnormal development of muscles and bones, and osteoporosis in some cases. The pathogenic mechanisms of such bone related phenotypes, however, remain unclear. Here, we showed that ZC4H2 is expressed in the developing bones in mice. ZC4H2 knockout mice were neonatal-lethal and smaller in size, with reduced calcification of long bones. Upon induced loss of ZC4H2 postnatally, the femoral bones developed an osteoporosis-like phenotype, with reduced bone mineral density, bone-volume fraction, and trabecular bone number. Knockdown of ZC4H2 showed no clear effect on the expression of osteogenic differentiation genes in in vitro models using mesenchymal stem cells. Interestingly, ZC4H2 knockdown significantly enhanced osteoclast differentiation and bone resorption in induced bone marrow-derived macrophages. We further confirmed that the number of osteoclasts in the long bone of ZC4H2 knockout mice was increased, as well as the expression of the serum bone resorption/osteoporosis marker CTX-1. Our study unveils a new role of ZC4H2 in osteoclast differentiation and bone development, providing new clues on the pathology of ZARD.

Osteoclasts are multinucleated bone-resorbing cells, and their formation is tightly regulated to prevent excessive bone loss. However, the mechanisms by which osteoclast formation is restricted remain incompletely determined. Here, we found that sterol regulatory element binding protein 2 (SREBP2) functions as a negative regulator of osteoclast formation and inflammatory bone loss. Cholesterols and SREBP2, a key transcription factor for cholesterol biosynthesis, increased in the late phase of osteoclastogenesis. The ablation of SREBP2 in myeloid cells resulted in increased in vivo and in vitro osteoclastogenesis, leading to low bone mass. Moreover, deletion of SREBP2 accelerated inflammatory bone destruction in murine inflammatory osteolysis and arthritis models. SREBP2-mediated regulation of osteoclastogenesis is independent of its canonical function in cholesterol biosynthesis but is mediated, in part, by its downstream target, interferon regulatory factor 7 (IRF7). Taken together, our study highlights a previously undescribed role of the SREBP2-IRF7 regulatory circuit as a negative feedback loop in osteoclast differentiation and represents a novel mechanism to restrain pathological bone destruction., (© 2024. The Author(s).)

Iron overload is a risk factor for osteoporosis due to its oxidative toxicity. Previous studies have demonstrated that an excessive amount of iron increases osteocyte apoptosis and receptor activator of nuclear factor κ-B ligand (RANKL) production, which stimulates osteoclast differentiation in vitro. However, the effects of exogenous iron supplementation-induced iron overload on osteocytes in vivo and its role in iron overload-induced bone loss are unknown. This work aimed to develop an iron overloaded murine model of C57BL/6 mice by intraperitoneal administration of iron dextran for two months. The iron levels in various organs, bone, and serum, as well as the microstructure and strength of bone, apoptosis of osteocytes, oxidative stress in bone tissue, and bone formation and resorption, were assessed. The results showed that 2 months of exogenous iron supplementation significantly increased iron levels in the liver, spleen, kidney, bone tissue, and serum. Iron overload negatively affected bone microstructure and strength. Osteocyte apoptosis and empty lacunae rate were elevated by exogenous iron. Iron overload upregulated RANKL expression but had no significant impact on osteoprotegerin (OPG) and sclerostin levels. Static and dynamic histologic analyses and serum biochemical assay showed that iron overload increased bone resorption without significantly affecting bone formation. Exogenous iron promoted oxidative stress in osteocytes in vivo and in vitro. Additional supplementation of iron chelator (deferoxamine) or N-acetyl-l-cysteine (NAC) partially alleviated bone loss, osteocyte apoptosis, osteoclast formation, and oxidative stress due to iron overload. These findings, in line with prior in vitro studies, suggest that exogenous iron supplementation induces osteoclastogenesis and osteoporosis by promoting osteocyte apoptosis and RANKL production via oxidative stress., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Type I interferons (IFN-I) are pleiotropic factors endowed with multiple activities that play important roles in innate and adaptive immunity. Although many studies indicate that IFN-I inducers exert favorable effects on broad-spectrum antivirus, immunomodulation, and anti-tumor activities by inducing endogenous IFN-I and IFN-stimulated genes, their function in bone homeostasis still needs further exploration. Here, our study demonstrates 2 distinct IFN-I inducers, diABZI and poly(I:C), as potential therapeutics to alleviate osteolysis and osteoporosis. First, IFN-I inducers suppress the genes that control osteoclast (OC) differentiation and activity in vitro. Moreover, diABZI alleviates bone loss in Ti particle-induced osteolysis and ovariectomized -induced osteoporosis in vivo by inhibiting OC differentiation and function. In addition, the inhibitory effects of IFN-I inducers on OC differentiation are not observed in macrophages derived from Ifnar1-/-mice, which indicate that the suppressive effect of IFN-I inducers on OC is IFNAR-dependent. Mechanistically, RNAi-mediated silencing of IRF7 and IFIT3 in OC precursors impairs the suppressive effect of the IFN-I inducers on OC differentiation. Taken together, these results demonstrate that IFN-I inducers play a protective role in bone turnover by limiting osteoclastogenesis and bone resorption through the induction of OC-specific mediators via the IFN-I signaling pathway., (© The Author(s) 2024. Published by Oxford University Press on behalf of the American Society for Bone and Mineral Research.)

Bone homeostasis is a complex process in which some Eph kinase receptors and their ephrin ligands appear to be involved. In the present study, we address this issue by examining, both in vitro and in vivo, the role of EphB2 and EphB3 in mesenchymal stromal/stem cell (MSC) differentiation into bone tissue. This was first evaluated by quantitative reverse transcription PCR (RT-qPCR) and histological staining in MSCs cultured in specific mediums revealing that although EphB2-/- MSCs mainly expressed pro-adipogenic transcription factors, EphB3-/- MSCs showed abundant osteogenic transcripts, such as Runx2, Msx2, and Sp7. To clarify the underlying molecular mechanisms, we found that the lack of EphB3 signaling alters the genetic profile of differentiating MSCs, reducing the expression of many inhibitory molecules and antagonists of the BMP signaling pathway, and increasing Bmp7 expression, a robust bone inductor. Then, to confirm the osteogenic role of EphB3 in vivo, we studied the condition of 2 mouse models of induced bone loss (ovariectomy or long-term glucocorticoid treatment). Interestingly, in both models, both WT and EphB2-/- mice equally developed the disease but EphB3-/- mice did not exhibit the typical bone loss, nor an increase in urine Ca2+ or blood serum CTX-1. This phenotype in EphB3-KO mice could be due to their significantly higher proportions of osteoprogenitor cells and preosteoblasts, and their lower number of osteoclasts, as compared with WT and EphB2-KO mice. Thus, we conclude that EphB3 acts as a negative regulator of the osteogenic differentiation, and its absence prevents bone loss in mice subjected to ovariectomy or dexamethasone treatment., (© The Author(s) 2024. Published by Oxford University Press on behalf of the American Society for Bone and Mineral Research. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Background/aim: Cancer-associated fibroblasts (CAFs) have recently been suggested as critical cellular components of bone invasion in oral squamous cell carcinoma (OSCC). However, the underlying molecular mechanisms and subtypes related to their bone-invasive function are unclear. This study investigated the implications of thymidine phosphorylase (TP)-positive CAFs (TP + CAFs) in OSCC bone invasion., Materials and Methods: TP expression was determined in 116 patients with OSCC using immunohistochemistry. The influence of TP expression on the biological behavior of CAFs was investigated in vitro. The possible impact of TP + CAFs on bone invasion in OSCC was further evaluated using patient-derived xenograft (PDX) mouse models., Results: In bone-invasive OSCC tissues, TP + CAFs were mainly distributed on the surface of resorbed bone tissue rather than on the tumor side. High levels of TP + CAFs were significantly associated with higher T-stage, bone invasion, and worse overall survival and recurrence-free survival in our study cohort. Recombinant human TP promoted the proliferative and invasive abilities of CAFs and increased matrix metalloproteinase-9 mRNA expression in vitro, related to bone resorption. In the PDX mouse models, TP + CAFs were found in early bone resorption on the surface of resorbed bony tissues. Bone resorption occurred more frequently in the PDX models with TP + CAFs than in those without., Conclusion: TP + CAFs were significantly associated with bone invasion and the prognosis of OSCC. This study provides insights into cellular and molecular targets for the early diagnosis and treatment of bone-invasive OSCC., (Copyright © 2024 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Objective: Rheumatoid arthritis (RA), a chronic inflammatory disease, is characterized by joint swelling, cartilage erosion, and bone destruction. This study investigated the therapeutic efficacy of Carnosic acid (CA), a natural compound with anti-inflammatory and antioxidant properties, in an adjuvant-induced arthritis model., Methods: Paw swelling and arthritis index were measured. Oxidative stress markers, including lipid peroxidation and antioxidant enzyme levels, were assessed. Synovial tissue was analyzed for pro-inflammatory markers using real-time Q-PCR and Western blotting. The expression of mPGES-1 was determined by Western blotting. Peripheral neuropathic pain was assessed using cold and mechanical allodynia tests. Bone loss was quantitatively assessed through microcomputed tomography (μCT) scanning of femurs and X-ray radiography. Indomethacin-induced gastric ulcers were evaluated. Molecular docking studies were conducted to analyze the binding affinity of CA to mPGES-1., Results: The CA treatment not only demonstrated a significant reduction in joint inflammation and paw swelling but also mitigated oxidative stress and improved the antioxidant defence system. CA inhibited microsomal prostaglandin E synthase-1 (mPGES-1) expression and the expression of pro-inflammatory molecules such as inducible nitric oxide synthase (iNOS) and cyclooxygenases-2 (COX-2), thus attenuating the arthritis symptoms without severe gastrointestinal side effects. Additionally, it inhibited the expression of pro-inflammatory molecules such as iNOS and COX-2, contributing to the reduction of arthritis symptoms. Notably, CA treatment prevented the common side effects of traditional RA treatments like corticosteroids and non-steroidal anti-inflammatory drugs (NSAIDs), including weight loss, bone degradation, and gastric ulcers., Conclusions: These findings suggest that CA, through specific enzyme inhibition, offers a compelling alternative therapeutic approach for RA. Further research is warranted to explore the potential of CA in other arthritis models and its suitability for human RA treatment.

High cyclic strains induce formation of microcracks in bone, triggering targeted bone remodeling, which entails osteoclastic resorption. Racehorse bone is an ideal model for studying the effects of high-intensity loading, as it is subject to focal formation of microcracks and subsequent bone resorption. The volume of resorption in vitro is considered a direct indicator of osteoclast activity but indirect 2D measurements are used more often. Our objective was to develop an accurate, high-throughput method to quantify equine osteoclast resorption volume in μCT 3D images. Here, equine osteoclasts were cultured on equine bone slices and imaged with μCT pre- and postculture. Individual resorption events were then isolated and analyzed in 3D. Modal volume, maximum depth, and aspect ratio of resorption events were calculated. A convolutional neural network (CNN U-Net-like) was subsequently trained to identify resorption events on post-culture μCT images alone, without the need for pre-culture imaging, using archival bone slices with known resorption areas and paired CTX-I biomarker levels in culture media. 3D resorption volume measurements strongly correlated with both the CTX-I levels (p < 0.001) and area measurements (p < 0.001). Our 3D analysis shows that the shapes of resorption events form a continuous spectrum, rather than previously reported pit and trench categories. With more extensive resorption, shapes of increasing complexity appear, although simpler resorption cavity morphologies (small, rounded) remain most common, in acord with the left-hand limit paradigm. Finally, we show that 2D measurements of in vitro osteoclastic resorption are a robust and reliable proxy., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Efficient cellular fusion of mononuclear precursors is the prerequisite for the generation of fully functional multinucleated bone-resorbing osteoclasts. However, the exact molecular factors and mechanisms controlling osteoclast fusion remain incompletely understood. Here we identify RANKL-mediated activation of caspase-8 as early key event during osteoclast fusion. Single cell RNA sequencing-based analyses suggested that activation of parts of the apoptotic machinery accompanied the differentiation of osteoclast precursors into mature multinucleated osteoclasts. A subsequent characterization of osteoclast precursors confirmed that RANKL-mediated activation of caspase-8 promoted the non-apoptotic cleavage and activation of downstream effector caspases that translocated to the plasma membrane where they triggered activation of the phospholipid scramblase Xkr8. Xkr8-mediated exposure of phosphatidylserine, in turn, aided cellular fusion of osteoclast precursors and thereby allowed generation of functional multinucleated osteoclast syncytia and initiation of bone resorption. Pharmacological blockage or genetic deletion of caspase-8 accordingly interfered with fusion of osteoclasts and bone resorption resulting in increased bone mass in mice carrying a conditional deletion of caspase-8 in mononuclear osteoclast precursors. These data identify a novel pathway controlling osteoclast biology and bone turnover with the potential to serve as target for therapeutic intervention during diseases characterized by pathologic osteoclast-mediated bone loss. Proposed model of osteoclast fusion regulated by caspase-8 activation and PS exposure. RANK/RANK-L interaction. Activation of procaspase-8 into caspase-8. Caspase-8 activates caspase-3. Active capase-3 cleaves Xkr8. Local PS exposure is induced. Exposed PS is recognized by the fusion partner. FUSION. PS is re-internalized., (© 2024. The Author(s).)

Clonal hematopoiesis of indeterminate potential (CHIP) arises from aging-associated acquired mutations in hematopoietic progenitors, which display clonal expansion and produce phenotypically altered leukocytes. We associated CHIP-DNMT3A mutations with a higher prevalence of periodontitis and gingival inflammation among 4,946 community-dwelling adults. To model DNMT3A-driven CHIP, we used mice with the heterozygous loss-of-function mutation R878H, equivalent to the human hotspot mutation R882H. Partial transplantation with Dnmt3a R878H/+ bone marrow (BM) cells resulted in clonal expansion of mutant cells into both myeloid and lymphoid lineages and an elevated abundance of osteoclast precursors in the BM and osteoclastogenic macrophages in the periphery. DNMT3A-driven clonal hematopoiesis in recipient mice promoted naturally occurring periodontitis and aggravated experimentally induced periodontitis and arthritis, associated with enhanced osteoclastogenesis, IL-17-dependent inflammation and neutrophil responses, and impaired regulatory T cell immunosuppressive activity. DNMT3A-driven clonal hematopoiesis and, subsequently, periodontitis were suppressed by rapamycin treatment. DNMT3A-driven CHIP represents a treatable state of maladaptive hematopoiesis promoting inflammatory bone loss., Competing Interests: Declaration of interests P.N. reports research grants from Allelica, Amgen, Apple, Boston Scientific, Genentech/Roche, and Novartis, personal fees from Allelica, Apple, AstraZeneca, Blackstone Life Sciences, Creative Education Concepts, CRISPR Therapeutics, Eli Lilly & Co, Foresite Labs, Genentech/Roche, GV, HeartFlow, Magnet Biomedicine, Merck, and Novartis, scientific advisory board membership of Esperion Therapeutics, Preciseli, and TenSixteen Bio, scientific co-founder of TenSixteen Bio, equity in MyOme, Preciseli, and TenSixteen Bio, and spousal employment at Vertex Pharmaceuticals, all unrelated to the present work. M.G.N. is an advisory board member of Cell and scientific founder of Lemba, TTxD, and Biotrip., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background: Osteoporosis is a disease associated with bone resorption, characterized primarily by the excessive activation of osteoclasts. Ginkgetin is a compound purified from natural ginkgo leaves which has various biological properties, including anti-inflammation, antioxidant, and anti-tumor effects. This study investigated the bone-protective effects of ginkgetin in ovariectomized (OVX) mice and explored their potential signaling pathway in inhibiting osteoclastogenesis in a mouse model of osteoporosis., Methods: Biochemical assays were performed to assess the levels of Ca, ALP, and P in the blood. Micro CT scanning was used to evaluate the impact of ginkgetin on bone loss in mice. RT-PCR was employed to detect the expression of osteoclast-related genes (ctsk, c-fos, trap) in their femoral tissue. Hematoxylin and eosin (H&E) staining was utilized to assess the histopathological changes in femoral tissue due to ginkgetin. The TRAP staining was used to evaluate the impact of ginkgetin osteoclast generation in vivo . Western blot analysis was conducted to investigate the effect of ginkgetin on the expression of p-NF-κB p65 and IκBα proteins in mice., Results: Our findings indicate that ginkgetin may increase the serum levels of ALP and P, while decreasing the serum level of Ca in OVX mice. H&E staining and micro CT scanning results suggest that ginkgetin can inhibit bone loss in OVX mice. The TRAP staining results showed ginkgetin suppresses the generation of osteoclasts in OVX mice. RT-PCR results demonstrate that ginkgetin downregulate the expression of osteoclast-related genes (ctsk, c-fos, trap) in the femoral tissue of mice, and this effect is dose-dependent. Western blot analysis results reveal that ginkgetin can inhibit the expression of p-NF-κB p65 and IκBα proteins in mice., Conclusion: Ginkgetin can impact osteoclast formation and activation in OVX mice by inhibiting the NF-κB/IκBα signaling pathway, thereby attenuating bone loss in mice., Competing Interests: The authors declare that they have no competing interests., (© 2024 Wei et al.)

Objective: Osteoporosis is a global health issue characterized by decreased bone mass and microstructural degradation, leading to an increased risk of fractures. This study aims to explore the molecular mechanism by which P2X7 receptors influence osteoclast formation and bone resorption through the PI3K-Akt-GSK3β signaling pathway., Methods: An osteoporosis mouse model was generated through ovariectomy (OVX) in normal C57BL/6 and P2X7 f/f ; LysM-cre mice. Osteoclasts were isolated for transcriptomic analysis, and differentially expressed genes were selected for functional enrichment analysis. Metabolite analysis was performed using liquid chromatography-tandem mass spectrometry (LC-MS/MS), and multivariate statistical analysis and pattern recognition were used to identify differential lipid metabolism markers and their distribution. Bioinformatics analyses were conducted using the Encyclopedia of Genes and Genomes database and the MetaboAnalyst database to assess potential biomarkers and create a metabolic pathway map. Osteoclast precursor cells were used for in vitro cell experiments, evaluating cell viability and proliferation using the Cell Counting Kit 8 (CCK-8) assay. Osteoclast precursor cells were induced to differentiate into osteoclasts using macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor kappa-beta ligand (RANKL), and tartrate-resistant acid phosphatase (TRAP) staining was performed to compare differentiation morphology, size, and quantity between different groups. Western blot analysis was used to assess the expression of differentiation markers, fusion gene markers, and bone resorption ability markers in osteoclasts. Immunofluorescence staining was employed to examine the spatial distribution and quantity of osteoclast cell skeletons, P2X7 protein, and cell nuclei, while pit assay was used to evaluate osteoclast bone resorption ability. Finally, in vivo animal experiments, including micro computed tomography (micro-CT), hematoxylin and eosin (HE) staining, TRAP staining, and immunohistochemistry, were conducted to observe bone tissue morphology, osteoclast differentiation, and the phosphorylation level of the PI3K-Akt-GSK3β signaling pathway., Results: Transcriptomic and metabolomic data collectively reveal that the P2X7 receptor can impact the pathogenesis of osteoporosis through the PI3K-Akt-GSK3β signaling pathway. Subsequent in vitro experiments showed that cells in the Sh-P2X7 + Recilisib group exhibited increased proliferative activity (1.15 versus 0.59), higher absorbance levels (0.68 versus 0.34), and a significant increase in resorption pit area (13.94 versus 3.50). Expression levels of osteoclast differentiation-related proteins MMP-9, CK, and NFATc1 were markedly elevated (MMP-9: 1.72 versus 0.96; CK: 2.54 versus 0.95; NFATc1: 3.05 versus 0.95), along with increased fluorescent intensity of F-actin rings. In contrast, the OE-P2X7 + LY294002 group showed decreased proliferative activity (0.64 versus 1.29), reduced absorbance (0.34 versus 0.82), and a significant decrease in resorption pit area (5.01 versus 14.96), accompanied by weakened expression of MMP-9, CK, and NFATc1 (MMP-9: 1.14 versus 1.79; CK: 1.26 versus 2.75; NFATc1: 1.17 versus 2.90) and decreased F-actin fluorescent intensity. Furthermore, in vivo animal experiments demonstrated that compared with the wild type (WT) + Sham group, mice in the WT + OVX group exhibited significantly increased levels of CTX and NTX in serum (CTX: 587.17 versus 129.33; NTX: 386.00 versus 98.83), a notable decrease in calcium deposition (19.67 versus 53.83), significant reduction in bone density, increased trabecular separation, and lowered bone mineral density (BMD). When compared with the KO + OVX group, mice in the KO + OVX + recilisib group showed a substantial increase in CTX and NTX levels in serum (CTX: 503.50 versus 209.83; NTX: 339.83 versus 127.00), further reduction in calcium deposition (29.67 versus 45.33), as well as decreased bone density, increased trabecular separation, and reduced BMD., Conclusion: P2X7 receptors positively regulate osteoclast formation and bone resorption by activating the PI3K-Akt-GSK3β signaling pathway., (© 2024. The Author(s).)

Osteoclast (OC) differentiation, vital for bone resorption, depends on osteoclast and precursor fusion. Osteoprotegerin (OPG) inhibits osteoclast differentiation. OPG's influence on fusion and mechanisms is unclear. Osteoclasts and precursors were treated with OPG alone or with ATP. OPG significantly reduced OC number, area and motility and ATP mitigated OPG's inhibition. However, OPG hardly affected the motility of precusors. OPG downregulated fusion-related molecules (CD44, CD47, DC-STAMP, ATP6V0D2) in osteoclasts, reducing only CD47 in precursors. OPG reduced Connexin43 phosphorylated forms (P1 and P2) in osteoclasts, affecting only P2 in precursors. OPG disrupted subcellular localization of CD44, CD47, DC-STAMP, ATP6V0D2, and Connexin43 in both cell types. Findings underscore OPG's multifaceted impact, inhibiting multinucleated osteoclast and mononuclear precursor fusion through distinct molecular mechanisms. Notably, ATP mitigates OPG's inhibitory effect, suggesting a potential regulatory role for the ATP signaling pathway. This study enhances understanding of intricate processes in osteoclast differentiation and fusion, offering insights into potential therapeutic targets for abnormal bone metabolism., Competing Interests: Declaration of competing interest The authors declare that there is no conflict of interest., (Copyright © 2024. Published by Elsevier B.V.)

Aging-related bone loss is driven by various biological factors, such as imbalanced bone metabolism from decreased osteoblast and increased osteoclast activities. Various transcriptional and post-transcriptional factors increase osteoclast activity with aging; however, studies regarding the post-translational regulators of osteoclast activity are still limited. The ubiquitin E3 ligase Pellino-1 is a well-known post-translational regulator of inflammation. However, how Pellino-1 expression regulation affects osteoclast differentiation remains unclear. This study determined that Pellino-1 levels are reduced in bone marrow monocytes (BMMs) from 40-week-old mice compared to 4-week-old mice. Interestingly, conditional Knockout (cKO) of Pellino-1 in 6-week-old mice resulted in decreased bone mass, reduced body size, and lower weight than in Pellino-1 floxed mice; however, these differences are not observed in 20-week-old mice. The increased number of tartrate-resistant acid phosphatase (TRAP)-positive cells and serum levels of C-terminal telopeptides of type I collagen, a marker of bone resorption, in 6-week-old Pellino-1 cKO mice implied a connection between Pellino-1 and the osteoclast population. Enhanced TRAP activity and upregulation of osteoclast genes in BMMs from the cKO mice indicate that Pellino-1 deletion affects osteoclast differentiation, leading to decreased bone mass and heightened osteoclast activity. Thus, targeting Pellino-1 could be a potential gene therapy for managing and preventing osteoporosis., (© 2024 The Authors. Advanced Biology published by Wiley‐VCH GmbH.)

Macrophages are essential regulators of inflammation and bone loss. Receptor activator of nuclear factor-κβ ligand (RANKL), a pro-inflammatory cytokine, is responsible for macrophage differentiation to osteoclasts and bone loss. We recently showed that 14-3-3ζ-knockout (Ywhaz KO ) rats exhibit increased bone loss in the inflammatory arthritis model. 14-3-3ζ is a cytosolic adaptor protein that actively participates in many signaling transductions. However, the role of 14-3-3ζ in RANKL signaling or bone remodeling is unknown. We investigated how 14-3-3ζ affects osteoclast activity by evaluating its role in RANKL signaling. We utilized 14-3-3ζ-deficient primary bone marrow-derived macrophages obtained from wildtype and Ywhaz KO animals and RAW264.7 cells generated using CRISPR-Cas9. Our results showed that 14-3-3ζ-deficient macrophages, upon RANKL stimulation, have bigger and stronger tartrate-resistant acid phosphatase-positive multinucleated cells and increased bone resorption activity. The presence of 14-3-3ζ suppressed RANKL-induced MAPK and AKT phosphorylation, transcription factors (NFATC1 and p65) nuclear translocation, and subsequently, gene induction (Rank, Acp5, and Ctsk). Mechanistically, 14-3-3ζ interacts with TRAF6, an essential component of the RANKL receptor complex. Upon RANKL stimulation, 14-3-3ζ-TRAF6 interaction was increased, while RANK-TRAF6 interaction was decreased. Importantly, 14-3-3ζ supported TRAF6 ubiquitination and degradation by the proteasomal pathway, thus dampening the downstream RANKL signaling. Together, we show that 14-3-3ζ regulates TRAF6 levels to suppress inflammatory RANKL signaling and osteoclast activity. To the best of our knowledge, this is the first report on 14-3-3ζ regulation of RANKL signaling and osteoclast activation., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Mechanical loading is required for bone homeostasis, but the underlying mechanism is still unclear. Our previous studies revealed that the mechanical protein polycystin-1 (PC1, encoded by Pkd1) is critical for bone formation. However, the role of PC1 in bone resorption is unknown. Here, we found that PC1 directly regulates osteoclastogenesis and bone resorption. The conditional deletion of Pkd1 in the osteoclast lineage resulted in a reduced number of osteoclasts, decreased bone resorption, and increased bone mass. A cohort study of 32,500 patients further revealed that autosomal dominant polycystic kidney disease, which is mainly caused by loss-of-function mutation of the PKD1 gene, is associated with a lower risk of hip fracture than those with other chronic kidney diseases. Moreover, mice with osteoclast-specific knockout of Pkd1 showed complete resistance to unloading-induced bone loss. A mechanistic study revealed that PC1 facilitated TAZ nuclear translocation via the C-terminal tail-TAZ complex and that conditional deletion of Taz in the osteoclast lineage resulted in reduced osteoclastogenesis and increased bone mass. Pharmacological regulation of the PC1-TAZ axis alleviated unloading- and estrogen deficiency- induced bone loss. Thus, the PC1-TAZ axis may be a potential therapeutic target for osteoclast-related osteoporosis., (Copyright © 2024 Science China Press. Published by Elsevier B.V. All rights reserved.)

Condylar resorption occurs in some cases after orthognathic surgery, and the risk factors associated with postoperative condylar head resorption have been extensively described. Nevertheless, even in cases with a combination of risk factors, postoperative condylar resorption may not appear. This study analyzed the microstructure and three-dimensional positional change of the condylar bone via imaging in patients who have undergone bimaxillary orthognathic surgery to determine whether the microstructure or condylar position differs between patients with and without postoperative condylar resorption. Among asymptomatic patients who underwent bimaxillary surgery between April 2021 and March 2022 at our department, 17 patients were analyzed, limited to "female," "skeletal Class II," and "high-angle cases," which are known risk factors for mandibular head resorption. Multidetector computed tomography was performed on these patients before and 6 months after surgery, and the bone microstructure of the condylar head and the three-dimensional positional changes of the condylar bone and the proximal bony fragments were compared with the presence of postoperative condyle resorption using the bone morphology software TRI/3D-BON. Patients with condylar bone abnormalities before surgery and those with high trabecular bone density can develop postoperative resorption if the condyle is misaligned by surgery., (© 2024. The Author(s).)

Interactions between osteolineage cells and myeloid cells play important roles in maintaining skeletal homeostasis. Herein, we find that osteolineage cells transfer mitochondria to myeloid cells. Impairment of the transfer of mitochondria by deleting MIRO1 in osteolineage cells leads to increased myeloid cell commitment toward osteoclastic lineage cells and promotes bone resorption. In detail, impaired mitochondrial transfer from osteolineage cells alters glutathione metabolism and protects osteoclastic lineage cells from ferroptosis, thus promoting osteoclast activities. Furthermore, mitochondrial transfer from osteolineage cells to myeloid cells is involved in the regulation of glucocorticoid-induced osteoporosis, and glutathione depletion alleviates the progression of glucocorticoid-induced osteoporosis. These findings reveal an unappreciated mechanism underlying the interaction between osteolineage cells and myeloid cells to regulate skeletal metabolic homeostasis and provide insights into glucocorticoid-induced osteoporosis progression., (© 2024. The Author(s).)

Osteoporosis is a systemic bone disease characterized by decreased bone mass that is tightly regulated by the coordinated actions of osteoclasts and osteoblasts. Apoptosis as a precise programmed cell death involves a cascade of gene expression events which are mechanistically linked to the regulation of bone metabolism. Nevertheless, the critical biomolecules involved in regulating cell apoptosis in osteoporosis remain unknown. To gain a deeper insight into the relationship between apoptosis and osteoporosis, this study integrated the sequencing results of human samples and using a machine learning workflow to overcome the limitations of a single study. Among all immune cell populations, we assessed the apoptotic level and portrayed the distinct subtypes and lineage differentiation of monocytic cells in osteoporotic tissues. Osteoclasts expressed a higher level of Spermidine/spermine-N1-Acetyltransferase1 (SAT1) during osteoclastogenesis which prevented osteoclasts apoptosis and facilitate osteoporosis progression. In addition, Berenil, one potent SAT1 inhibitor, increased osteoclast apoptosis and reversed the bone loss in the femurs of a murine ovariectomy model. In summary, Berenil promotes osteoclast apoptosis, inhibits the bone resorption and improves the abnormal bone structure in vitro and in vivo models by targeting SAT1, demonstrating its potential as a precise therapeutic strategy for clinical osteoporosis treatment., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Several members of the transforming growth factor beta (TGF-β) superfamily regulate the proliferation, differentiation, and function of bone-forming osteoblasts and bone-resorbing osteoclasts. However, it is still unknown whether Nodal, a member of the TGF-β superfamily, serves a function in bone cells. In this study, we found that Nodal did not have any function in osteoblasts but instead negatively regulated osteoclast differentiation. Nodal inhibited RANKL-induced osteoclast differentiation by downregulating the expression of pro-osteoclastogenic genes, including c-fos, Nfatc1, and Blimp1, and upregulating the expression of antiosteoclastogenic genes, including Bcl6 and Irf8. Nodal activated STAT1 in osteoclast precursor cells, and STAT1 downregulation significantly reduced the inhibitory effect of Nodal on osteoclast differentiation. These findings indicate that Nodal activates STAT1 to downregulate or upregulate the expression of pro-osteoclastogenic or antiosteoclastogenic genes, respectively, leading to the inhibition of osteoclast differentiation. Moreover, the inhibitory effect of Nodal on osteoclast differentiation contributed to the reduction of RANKL-induced bone loss in vivo., (© 2024 Wiley Periodicals LLC.)

Bone diseases are increasing with aging populations and it is important to identify clues to develop innovative treatments. Vasn, which encodes vasorin (Vasn), a transmembrane protein involved in the pathophysiology of several organs, is expressed during the development in intramembranous and endochondral ossification zones. Here, we studied the impact of Vasn deletion on the osteoblast and osteoclast dialog through a cell Coculture model. In addition, we explored the bone phenotype of Vasn KO mice, either constitutive or tamoxifen-inducible, or with an osteoclast-specific deletion. First, we show that both osteoblasts and osteoclasts express Vasn. Second, we report that, in both KO mouse models but not in osteoclast-targeted KO mice, Vasn deficiency was associated with an osteopenic bone phenotype, due to an imbalance in favor of osteoclastic resorption. Finally, through the Coculture experiments, we identify a dysregulation of the Wnt/β-catenin pathway together with an increase in RANKL release by osteoblasts, which led to an enhanced osteoclast activity. This study unravels a direct role of Vasn in bone turnover, introducing a new biomarker or potential therapeutic target for bone pathologies., (© 2024 The Authors. Journal of Cellular Physiology published by Wiley Periodicals LLC.)

The existing suite of therapies for bone diseases largely act to prevent further bone loss but fail to stimulate healthy bone formation and repair. We describe an endogenous osteopeptide (PEPITEM) with anabolic osteogenic activity, regulating bone remodeling in health and disease. PEPITEM acts directly on osteoblasts through NCAM-1 signaling to promote their maturation and formation of new bone, leading to enhanced trabecular bone growth and strength. Simultaneously, PEPITEM stimulates an inhibitory paracrine loop: promoting osteoblast release of the decoy receptor osteoprotegerin, which sequesters RANKL, thereby limiting osteoclast activity and bone resorption. In disease models, PEPITEM therapy halts osteoporosis-induced bone loss and arthritis-induced bone damage in mice and stimulates new bone formation in osteoblasts derived from patient samples. Thus, PEPITEM offers an alternative therapeutic option in the management of diseases with excessive bone loss, promoting an endogenous anabolic pathway to induce bone remodeling and redress the imbalance in bone turnover., Competing Interests: Declaration of interests H.M.M., G.E.R., A.J.I., and M.C. hold patents on the use of PEPITEM in bone diseases and other inflammatory conditions (US9597368B2; US9839671B; EP2802342; JP6055845; CN104168910B; US-2021-0100870-A1; GB2301317.0; PCT/GB2023/052013). H.M.M. and A.J.I. have received funding from Roche., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Mature osteoclasts degrade bone matrix by exocytosis of active proteases from secretory lysosomes through a ruffled border. However, the molecular mechanisms underlying lysosomal trafficking and secretion in osteoclasts remain largely unknown. Here, we show with GeneChip analysis that RUN and FYVE domain-containing protein 4 (RUFY4) is strongly upregulated during osteoclastogenesis. Mice lacking Rufy4 exhibited a high trabecular bone mass phenotype with abnormalities in osteoclast function in vivo. Furthermore, deleting Rufy4 did not affect osteoclast differentiation, but inhibited bone-resorbing activity due to disruption in the acidic maturation of secondary lysosomes, their trafficking to the membrane, and their secretion of cathepsin K into the extracellular space. Mechanistically, RUFY4 promotes late endosome-lysosome fusion by acting as an adaptor protein between Rab7 on late endosomes and LAMP2 on primary lysosomes. Consequently, Rufy4-deficient mice were highly protected from lipopolysaccharide- and ovariectomy-induced bone loss. Thus, RUFY4 plays as a new regulator in osteoclast activity by mediating endo-lysosomal trafficking and have a potential to be specific target for therapies against bone-loss diseases such as osteoporosis., (© 2024. The Author(s).)

Rebound bone loss following denosumab discontinuation is an important clinical challenge. Current treatment strategies to prevent this fail to suppress the rise and overshoot in osteoclast-mediated bone resorption. In this study, we use a murine model of denosumab treatment and discontinuation to show the temporal changes in osteoclast formation and activity during RANKL inhibition and withdrawal. We show that the cellular processes that drive the formation of osteoclasts and subsequent bone resorption following withdrawal of RANKL inhibition precede the rebound bone loss. Furthermore, a rise in serum TRAP and RANKL levels is detected before markers of bone turnover used in current clinical practice. These mechanistic advances may provide insight into a more defined window of opportunity to intervene with sequential therapy following denosumab discontinuation., (© The Author(s) 2024. Published by Oxford University Press on behalf of the American Society for Bone and Mineral Research.)

Absent, small, or homeotic1-like (ASH1L) is a histone lysine methyltransferase that generally functions as a transcriptional activator in controlling cell fate. So far, its physiological relevance in bone homeostasis and osteoclast differentiation remains elusive. Here, by conditional deleting Ash1l in osteoclast progenitors of mice, we found ASH1L deficiency resulted in osteoporosis and potentiation of osteoclastogenesis in vivo and in vitro. Mechanistically, ASH1L binds the promoter of the Src homology 3 and cysteine-rich domain 2 (Stac2) and increases the gene's transcription via histone 3 lysine 4 (H3K4) trimethylation modification, thus augmenting the STAC2's protection against receptor activator of nuclear factor kB ligand (RANKL)-initiated inflammation during osteoclast formation. Collectively, we demonstrate the first piece of evidence to prove ASH1L as a critical checkpoint during osteoclastogenesis. The work sheds new light on our understanding about the biological function of ASH1L in bone homeostasis, therefore providing a valuable therapeutic target for the treatment of osteoporosis or inflammatory bone diseases., (© 2024. The Author(s), under exclusive licence to ADMC Associazione Differenziamento e Morte Cellulare.)

Bacterial infection remains a significant problem associated with orthopaedic surgeries leading to surgical site infection (SSI). This unmet medical need can become an even greater complication when surgery is due to malignant bone tumor. In the present study, we evaluated in vitro titanium (Ti) implants subjected to gallium (Ga) and silver (Ag)-doped thermochemical treatment as strategy to prevent SSI and improve osteointegration in bone defects caused by diseases such as osteoporosis, bone tumor, or bone metastasis. Firstly, as Ga has been reported to be an osteoinductive and anti-resorptive agent, its performance in the mixture was proved by studying human mesenchymal stem cells (hMSC) and pre-osteoclasts (RAW264.7) behaviour. Then, the antibacterial potential provided by Ag was assessed by resembling "The Race for the Surface" between hMSC and Pseudomonas aeruginosa in two co-culture methods. Moreover, the presence of quorum sensing molecules in the co-culture was evaluated. The results highlighted the suitability of the mixture to induce osteodifferentiation and reduce osteoclastogenesis in vitro. Furthermore, the GaAg surface promoted strong survival rate and retained osteoinduction potential of hMSCs even after bacterial inoculation. Therefore, GaAg-modified titanium may be an ideal candidate to repair bone defects caused by excessive bone resorption, in addition to preventing SSI. STATEMENT OF SIGNIFICANCE: This article provides important insights into titanium for fractures caused by osteoporosis or bone metastases with high incidence in surgical site infection (SSI) because in this situation bacterial infection can become a major disaster. In order to solve this unmet medical need, we propose a titanium implant modified with gallium and silver to improve osteointegration, reduce bone resorption and avoid bacterial infection. For that aim, we study osteoblast and osteoclast behavior with the main novelty focused on the antibacterial evaluation. In this work, we recreate "the race for the surface" in long-term experiments and study bacterial virulence factors (quorum sensing). Therefore, we believe that our article could be of great interest, providing a great impact on future orthopedic applications., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Inflammatory diseases, such as rheumatoid arthritis, periodontitis, chronic obstructive pulmonary disease, and celiac disease, disrupt the delicate balance between bone resorption and formation, leading to inflammatory bone loss. Conventional approaches to tackle this issue encompass pharmaceutical interventions and surgical procedures. Nevertheless, pharmaceutical interventions exhibit limited efficacy, while surgical treatments impose trauma and significant financial burden upon patients. Biomaterials show outstanding spatiotemporal controllability, possess a remarkable specific surface area, and demonstrate exceptional reactivity. In the present era, the advancement of emerging biomaterials has bestowed upon more efficacious solutions for combatting the detrimental consequences of inflammatory bone loss. In this review, the advances of biomaterials for ameliorating inflammatory bone loss are listed. Additionally, the advantages and disadvantages of various biomaterials-mediated strategies are summarized. Finally, the challenges and perspectives of biomaterials are analyzed. This review aims to provide new possibilities for developing more advanced biomaterials toward inflammatory bone loss., (© 2024 Wiley‐VCH GmbH.)

The overactivation of the osteoclasts is a crucial pathological factor in the development of osteoporosis. MZF1, belonging to the scan-zinc finger family, plays a significant role in various processes associated with tumor malignant progression and acts as an essential transcription factor regulating osteoblast expression. However, the exact role of MZF1 in osteoclasts has not been determined. In this study, the purpose of our study was to elucidate the role of MZF1 in osteoclastogenesis. First, we established MZF1-deficient female mice and evaluated the femur bone phenotype by micro-computed tomography and histological staining. Our findings indicate that MZF1-/- mice exhibited a low bone mass osteoporosis phenotype. RANKL could independently induce the differentiation of RAW264.7 cells into osteoclasts, and we found that the expression level of MZF1 protein decreased gradually. Then, the CRISPR/Cas 9 gene-editing technique was used to build a RAW264.7 cell model with MZF1 knockout, and RANKL was used to independently induce MZF1-/- and wild-type cells to differentiate into mature osteoclasts. Tartrate-resistant acid phosphatase staining and F-actin fluorescence results showed that the MZF1-/- group produced more tartrate-resistant acid phosphatase-positive mature osteoclasts and larger actin rings. The expression of osteoclast-associated genes (including tartrate-resistant acid phosphatase, CTSK, c-Fos, and NFATc1) was evaluated by reverse transcription quantitative polymerase chain reaction and Western blot. The expression of key genes of osteoclast differentiation in the MZF1-/- group was significantly increased. Furthermore, we found that cell viability was increased in the early stages of RANKL-induced cell differentiation in the MZF1-/- group cells. We examined some prevalent ferroptosis markers, including malondialdehyde, glutathione, and intracellular Fe, the active form of iron in the cytoplasm during the early stages of osteoclastogenesis. The results suggest that MZF1 may be involved in osteoclast differentiation by regulating RANKL-induced ferroptosis of osteoclasts. Collectively, our findings shed light on the essential involvement of MZF1 in the regulation of osteoclastogenesis in osteoporosis and provide insights into its potential underlying mechanism., Competing Interests: Conflict of interest: The authors declare no conflict of interest., (© The Author(s) 2024. Published by Oxford University Press on behalf of Society for Leukocyte Biology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Introduction: This study evaluated the effects of cigarette smoke inhalation (CSI) on apical periodontitis (AP) induced in rats by histometric, immunohistochemical, and microtomographic analysis., Methods: A total of 32 male Wistar rats were divided into 4 experimental groups (n = 8): control, CSI, AP, and CSI + AP. Rats in the CSI and CSI + AP groups inhaled cigarette smoke by remaining inside a smoking chamber for 8 minutes 3 times a day for 50 days. After 20 days of smoke inhalation, rats in the AP and CSI + AP groups had the pulp of their first right lower molar exposed to induce AP. Blood was collected on day 50 to evaluate nicotine and serum cotinine levels. The animals' mandibles were removed for histologic processing to evaluate bone resorption by histometric, immunohistochemical (receptor activator of nuclear factor kappa B ligand/osteoprotegerin), and microtomographic analysis. The Student t test was applied., Results: Histometric analysis showed a larger area of bone resorption (P < .05) and microtomographic analysis found greater resorption volume (P < .001) for the CSI + AP group compared with the AP group. The CSI + AP group presented a high RANKL immunostaining pattern compared with the AP group (P < .001)., Conclusions: CSI increased bone resorption caused by AP., (Copyright © 2024 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.)