Y-RBCEVs accelerate fracture repair through osteogenesis and VCAN-mediated CD44/PI3K/AKT-dependent macrophage polarization.

To investigate the effects of young human red blood cell-derived extracellular vesicles (Y-RBCEVs) on fracture healing. Associations of RBC-related indicators with bone mineral density and mortality in fracture populations were analyzed using NHANES data (1999-2020). A murine femoral fracture model was established to investigate RBC transfusion effects. Y-RBCEVs and O-RBCEVs were isolated to examine their effects on osteogenic differentiation and BMM polarization in vitro. In vivo distribution was assessed by IVIS, and therapeutic efficacy was evaluated in fracture mice. Proteomic sequencing identified differentially expressed proteins between Y-RBCEVs and O-RBCEVs, and key protein function was investigated by transcriptomic analysis and in vitro validation. The observational study revealed associations between RBC count and bone mineral density with fracture populations mortality. RBC transfusion modulated the expression of osteogenic and M2 macrophage polarization-related genes. In vitro, RBCEVs were efficiently internalized by BMSCs and BMMs, with Y-RBCEVs enhancing osteogenic activity in BMSCs and promoting M2 macrophage polarization, thereby partially restoring osteogenesis impaired by inflammatory conditions. In vivo, Y-RBCEVs accumulated at fracture sites and enhanced bone regeneration without detectable toxicity, accompanied by increased OCN and CD206 expression. Proteomic analysis identified VCAN as one of the potential functional cargos. Mechanistically, Y-RBCEV-derived VCAN interacted with CD44 on macrophages and was associated with activation of the PI3K/AKT pathway, contributing to M2 polarization. Y-RBCEVs enhance fracture repair by modulating osteogenesis and macrophage polarization, with VCAN-associated CD44/PI3K/AKT signaling contributing to their osteo-immunomodulatory effects. These findings highlight RBCEVs as a potential therapeutic strategy for bone regeneration.