Mechanotransduction-driven macrophage polarization via Integrin-SRC-STAT6 pathway in distraction osteogenesis.

BACKGROUND: Mechanical stimuli are indispensable for bone regeneration. Distraction osteogenesis (DO) is a widely used clinical technique for limb lengthening and bone defect repair; however, its specific mechanobiological mechanisms remain unclear. Macrophages play crucial regulatory roles throughout bone fracture healing. Recent studies indicate that macrophages are mechanosensitive and can modulate the local immune microenvironment in response to mechanical cues. This study aims to investigate how macrophages respond to mechanical stimulation and regulate bone regeneration during DO. METHODS: Animal models of DO (with external fixation) and fracture healing (with internal fixation) were established to compare bone regeneration under different mechanical conditions. Immunohistochemistry (IHC) was used to quantify M1 and M2 macrophage infiltration. An in vitro model of cyclic mechanical stretch (10 %, 0.5 Hz, 12 h) was applied to RAW264.7 cells to study macrophage polarization. Flow cytometry, PCR, and western blot were used to assess macrophage phenotypes. An indirect co-culture system was employed to evaluate the effect of mechanically stimulated M2 macrophages on osteogenic differentiation. Single-cell RNA sequencing analysis of public data was performed to identify key biological processes in macrophage subpopulations during DO. Western blot and immunofluorescence were used to measure expression and phosphorylation levels of SRC and STAT-6. Pathway inhibitors were applied to elucidate regulatory mechanisms. In vivo, Saracatinib and TGF-β were administered locally in DO models. Bone regeneration was evaluated using micro-CT, mechanical testing, and histology. RESULTS: DO significantly enhanced M2 macrophage polarization at 1st, 2nd, and 4th week post-surgery compared to controls. Cyclic stretch promoted M2 polarization in vitro and increased secretion of TGF-β and IL-10. Mechanically induced macrophages enhanced osteoblast differentiation in co-culture. Mechanical activation of the Integrin-SRC-STAT6 pathway drove M2 polarization. Local SRC inhibition suppressed M2 polarization and impaired bone regeneration in DO, which was partially rescued by TGF-β supplementation. CONCLUSION: Mechanical stimulation during DO promotes M2 macrophage polarization via the Integrin-SRC-STAT6 pathway. TGF-β appears to be a key cytokine secreted by mechanically induced M2 macrophages that facilitates osteogenesis. These findings reveal a novel mechano-immune regulatory axis that supports bone regeneration in DO. THE TRANSLATIONAL POTENTIAL OF THIS ARTICLE: This research confirms the core concept of "mechano-immunoregulation" and identifies actionable therapeutic targets, enabling the development of targeted therapies for refractory bone defects by modulating the integrin-β1/SRC/STAT6 pathway and TGF-β1 to enhance bone regeneration.