Ccm3 Mechanosignaling Enhances Macrophage-Driven Osteogenesis Via Chromatin Remodeling

Macrophages critically regulate bone immune microenvironment homeostasis and are mechanosensitive during bone repair. Understanding how mechanical signals drive macrophage reprogramming is essential to control excessive inflammation. Macrophages were 3D-cultured in soft(S) /hard(H) hydrogels. Macrophage polarization markers were analyzed by flow cytometry and qPCR. A femoral defect model in mice was established, and cell-laden hydrogels were implanted. Bone regeneration was evaluated using microCT and immunohistochemistry. Gene expression and protein affinity of CCM3, FAK, and Paxillin in S and H matrices were examined via immunofluorescence colocalization and molecular dynamics simulation. SiRNA interference was performed to confirm their interactions. ATAC-seq was conducted to analyze chromatin accessibility changes post-CCM3 inhibition, followed by motif and pathway enrichment analysis. Compared with soft (H) matrix, the hard matrix (H) promoted M2 polarization of macrophages, and macrophage-laden H hydrogels significantly enhanced bone defect repair in mice, as demonstrated by increased bone density, bone volume fraction, and denser trabeculae. In the H group, CCM3 expression was reduced, and FAK-Paxillin affinity was higher compared to CCM3-Paxillin. CCM3 inhibition further upregulated FAK expression, enhancing M2 polarization, cytoskeletal reorganization, and chromatin accessibility. Reduced CCM3 expression resulted in the enrichment of transcription factors (e.g., JunB, Fra1, Fra2) and signaling pathways such as TGF-beta and calcium signaling in differentially open chromatin regions related to tissue repair and mechanotransduction. CCM3 depletion strengthens mechanotransduction via FAK/Paxillin pathway, enhancing cytoskeletal reorganization and chromatin accessibility of repair genes to direct macrophage reprogramming for bone regeneration.