M2 macrophage-derived migrasomes orchestrate dual CXCL12/CXCR4 and neutrophil-MMP-9/MSC-EphB2 signaling to enhance fracture healing

Macrophages' promotion of mesenchymal stem cell (MSC) homing is important for fracture healing; but mechanisms are unclear. Migrasomes are newly discovered membrane-bound organelles, which, like exosomes, mediate intercellular communication. This study compare macrophage-derived migrasomes and exosomes in directing MSC homing and enhancing fracture healing. Migrasomes and exosomes isolated from polarized M2 and M1 macrpphages were co-cultured with MSCs to test effects on MSC migration. In addition, M2-migra and M2-exo hydrogel delivery systems, with or without the CXCR4 antagonist AMD3100 or poly(lactic-co-glycolic acid) (PLGA)-encapsulated BMP-2, were injected into mice to evaluate their effects on MSC homing and fracture healing. Single-cell RNA sequencing (scRNA-seq) and microarray analyses were used to define mechanisms. M2-migra, but not M2-exo, expressed abundant CXCL12, which activates the CXCL12/CXCR4 axis to promote MSC migration. AMD3100 (a CXCR4 inhibitor) abolishes this migratory effect, confirming the pathway's specificity. scRNA-seq revealed that M2-migra modulates neutrophil-derived MMP-9 expression, which enhances EphB2 receptor expression on MSCs. This interaction enhances MSC osteogenic differentiation and fracture healing, providing a previously uncharacterized link between migrasomes, immune cells, and MSC function. A PLGA nanoparticle-encapsulated BMP-2 delivery system combined with an M2-migra hydrogel achieved sustained release of bioactive factors, superior MSC homing, and accelerated bone regeneration compared with M2-migra or BMP-2 alone. M2-migra demonstrates superior MSC homing capacity compared with M2-exo through dual mechanisms: CXCL12/CXCR4-mediated recruitment and neutrophil-MMP-9/MSC-EphB2-induced osteogenic differentiation. Moreover, the migrasome-hydrogel system represents a promising tissue-engineering strategy for fracture repair, offering insights into targeted regenerative medicine.