M2 macrophage-derived extracellular vesicles regulate mitochondrial dynamics to enhance osteogenesis of human periodontal ligament stem cells during orthodontic tooth movement

Orthodontic tooth movement (OTM) induces sterile inflammation and metabolic disturbances in periodontal tissues, which may impair the osteogenic capacity of human periodontal ligament stem cells (hPDLSCs), disrupt periodontal homeostasis, and contribute to clinically relevant complications associated with alveolar bone resorption, including orthodontic-related bone dehiscence, fenestration, and tooth mobility. Mitochondrial dysfunction has emerged as an important regulator of stem cell fate under inflammatory and mechanical stress conditions; however, whether immune cell-derived extracellular vesicles (EVs) can ameliorate the inflammatory microenvironment induced by orthodontic force and simultaneously regulate mitochondrial homeostasis to enhance hPDLSC osteogenesis remains unclear. Extracellular vesicles derived from M2 macrophages (M2-EVs) were isolated and characterized. Their effects on hPDLSC proliferation, osteogenic differentiation, mitochondrial function, and macrophage polarization were evaluated in vitro. Mechanistic studies focused on dynamin-related protein 1 (DRP1)-mediated mitochondrial dynamics. A rat OTM model was established to assess the in vivo therapeutic effects of M2-EVs on alveolar bone remodeling. In vitro experiments demonstrated that M2-EVs were efficiently internalized by hPDLSCs and significantly enhanced their proliferation and osteogenic differentiation. Mechanistically, M2-EVs inhibited DRP1-mediated mitochondrial fission, reduced intracellular reactive oxygen species levels, and restored mitochondrial homeostasis. Inhibition of DRP1 recapitulated the pro-osteogenic effects of M2-EVs, confirming that this regulatory mechanism is mitochondria-dependent. In vivo, local administration of M2-EVs reduced DRP1 expression by approximately 50% compared with the control group and increased tension-side BV/TV by 20%, while no significant difference in tooth movement distance was observed among the groups. In addition, M2-EVs promoted macrophage polarization toward an anti-inflammatory M2 phenotype, thereby contributing to the establishment of a pro-regenerative microenvironment. M2 macrophage-derived EVs promote hPDLSC osteogenesis through coordinated regulation of mitochondrial dynamics and the immune microenvironment. These findings reveal a mitochondrial–immune coupling mechanism underlying stem cell-mediated periodontal regeneration and highlight M2-EVs as a potential therapeutic strategy for improving orthodontic outcomes.