M2 macrophage-derived migrasomes orchestrate dual CXCL12/CXCR4 and neutrophil-MMP-9/MSC-EphB2 signaling to enhance fracture healing
Siyu Zhang, Abudurexiti Kutibiding, Mengci Wang, Xiaodan Zhou, Xiaohan Tian, Dandan Liu, Tuersunnayi Manafu, Ruize Li, Hailong Guo, Wen Zhao, Yi Yang
Abstract
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.