Hypoxia-programmed apoptotic-mimetic M2 macrophage-derived small extracellular vesicles for hydrogel-mediated immunoregenerative bone repair

Abstract

Effective regeneration of critical-sized bone defects requires the coordinated reconstruction of immune homeostasis, vascular networks, and osteogenic matrix formation. However, conventional bone-regenerative materials frequently provide structural support or single-pathway osteoinduction, while failing to actively orchestrate the multicellular repair microenvironment. Here, we develop a hydrogel-assisted immunoregenerative strategy based on hypoxia-programmed apoptotic-mimetic small extracellular vesicles derived from M2-like macrophages. Interleukin-4-polarized macrophages were subjected to hypoxic preconditioning to generate M2-HP-Apo-sEVs with increased phosphatidylserine exposure, enhanced cellular internalization, and amplified immunomodulatory potency. Compared with normoxic M2-sEVs, M2-HP-Apo-sEVs more effectively reprogrammed inflammatory macrophages toward a pro-resolving phenotype, suppressed inflammatory cytokine production, and enhanced reparative cytokine secretion. The vesicles further promoted osteogenic differentiation of bone marrow mesenchymal stem cells and stimulated endothelial migration and capillary-like network formation, demonstrating coordinated osteo–immuno–angiogenic activity. To enable sustained local delivery, M2-HP-Apo-sEVs were incorporated into photocrosslinkable GelMA hydrogels and implanted into critical-sized calvarial defects. The GelMA–M2-HP-Apo-sEV system substantially enhanced bone regeneration, vascularization, and osteogenic matrix deposition while attenuating local inflammation. This work establishes a cell-free vesicle–hydrogel platform that integrates donor-cell hypoxic programming, apoptotic-mimetic membrane signaling, and local biomaterial delivery for immunoregenerative bone repair.