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Objectives Aging and common diseases alter the stiffness of bone tissue, causing changes to the microenvironment of the mechanosensitive bone cells. Osteoclasts, the sole bone‐resorbing cells, play a vital role in bone remodeling. This study was performed to elucidate the mechanism through which osteoclasts sense and react to substrate stiffness signals. Materials and methods We fabricated polydimethylsiloxane (PDMS) substrates of different stiffness degrees for osteoclast formation progressed from osteoclast precursors including bone marrow‐derived macrophages (BMMs) and RAW264.7 monocytes. Osteoclast differentiation in response to the stiffness signals was determined by examining the cell morphology, fusion/fission activities, transcriptional profile, and resorption function. Cytoskeletal changes and mechanosensitive adhesion molecules were also assessed. Results Stiffer PDMS substrates accelerated osteoclast differentiation, firstly observed by variations in their morphology and fusion/fission activities. Upregulation of canonical osteoclast markers (Nfatc1, Acp5, Ctsk, Camk2a, Mmp9, Rela, and Traf6) and the fusion master regulator DC‐stamp were detected on stiffer substrates, with similar increases in their bone resorption functions. Additionally, the activation of cytoskeleton‐associated adhesion molecules, including fibronectin and integrin αvβ3, followed by biochemical signaling cascades of paxillin, FAK, PKC, and RhoA, was detected on the stiffer substrates. Conclusions This is the first study to provide evidence proving that extracellular substrate stiffness is a strong determinant of osteoclast differentiation and functions. Higher stiffness upregulated the differentiation profile and activity of osteoclasts, revealing the mechanical regulation of osteoclast activity in bone homeostasis and diseases., We fabricated a mechanical model of PDMS substrates that mimic physiologically mechanical properties of extracellular microenvironment, with varied stiffness for cell culture. Osteoclasts respond to stiffness variations in cell morphology, fusion/fission activity, osteoclast differentiation profile (Nfatc1, Acp5, Ctsk, Camk2a, Mmp9, Rela, Traf6, and DC‐stamp) and resorption functions. The potential regulatory mechanism with the involvement of cytoskeletons adhesion molecules: cytoskeletons adhesion molecules, including fibronectin‐integrin αvβ3 activation and following biochemical signaling cascades of paxillin, FAK, PKC, and RhoA were altered in response to stiffness signals. Bioinformatics proposed strong connection between cytoskeleton adhesion and osteoclast differentiation.

Objectives NLRP3 inflammasome is a critical part of the innate immune system and plays an important role in a variety of inflammatory diseases. However, the effects of NLRP3 inflammasome on periodontitis have not been fully studied. Materials and methods We used ligature‐induced periodontitis models of NLRP3 knockout mice (NLRP3KO) and their wildtype (WT) littermates to compare their alveolar bone phenotypes. We further used Lysm‐Cre/RosanTnG mouse to trace the changes of Lysm‐Cre+ osteoclast precursors in ligature‐induced periodontitis with or without MCC950 treatment. At last, we explored MCC950 as a potential drug for the treatment of periodontitis in vivo and in vitro. Results Here, we showed that the number of osteoclast precursors, osteoclast differentiation and alveolar bone loss were reduced in NLRP3KO mice compared with WT littermates, by using ligature‐induced periodontitis model. Next, MCC950, a specific inhibitor of the NLRP3 inflammasome, was used to inhibit osteoclast precursors differentiation into osteoclast. Further, we used Lysm‐Cre/RosanTnG mice to demonstrate that MCC950 decreases the number of Lysm‐Cre+ osteoclast precursors in ligature‐induced periodontitis. At last, treatment with MCC950 significantly suppressed alveolar bone loss with reduced IL‐1β activation and osteoclast differentiation in ligature‐induced periodontitis. Conclusion Our findings reveal that NLRP3 regulates alveolar bone loss in ligature‐induced periodontitis by promoting osteoclastic differentiation., NLRP3 Regulates Alveolar Bone Loss in Ligature‐induced Periodontitis by Promoting Osteoclastic Differentiation. Bacterial infection in periodontal tissues leads to the activation of NLRP3 inflammasome in osteoclast precursor cells, which promotes osteoclast differentiation and alveolar bone resorption. Thus, NLRP3 inflammasome contribute significantly to the pathologic bone loss in periodontitis. MCC950, a specific inhibitor of the NLRP3 inflammasome, inhibits osteoclast differentiation, thereby reduces alveolar bone loss in periodontitis.

Objectives A20 exerts an anti‐osteoclastogenic effect through the inhibition of NF‐κB signalling in periodontitis. It also regulates autophagy via ubiquitin modification. This study was aimed at exploring the relationship between A20 and autophagy in anti‐osteoclastogenesis in human periodontal ligament cells (hPDLCs) under hypoxia. Materials and Methods Real‐time PCR and Western blot were used to detect relative mRNA and protein levels. The formation of autophagosomes was measured by TEM. Osteoclastic differentiation was assessed by TRAP staining and hydroxyapatite resorption assay. The interactions between different proteins were observed by co‐IP. Results Cells cultured under 2% O₂ exhibited decreased A20 expression and increased RANKL/OPG (R/O) ratio. There was a negative correlation between A20 and TRAF6, and similar results were found with autophagic flux. A20 delayed the increase in R/O ratio under hypoxia. Autophagy in hPDLCs and osteoclast differentiation and hydroxyapatite resorption areas in mouse bone marrow mononuclear cells (BMMCs) were inhibited by A20. Moreover, inhibition of autophagy using 3‐MA resulted in increased expression of A20 and decreased number and function of osteoclasts. In addition, A20 inhibited polyubiquitination at K63 and enhanced that at K48 in TRAF6 to suppress autophagy under hypoxic conditions. Conclusions A20 inhibits osteoclastogenesis via inhibition of TRAF6‐dependent autophagy in hPDLCs under hypoxia. These findings suggest that A20 may be a key gene target during bone loss in periodontitis via TRAF6‐mediated inhibition of autophagy.