Hydrogel-Wrapped Calendula officinalis L. extracellular vesicles - A novel approach to enhance fracture healing by Macrophage reprogramming.

Fracture healing is a complex process often complicated by nonunion and delayed healing, with macrophage polarization being a key regulator of inflammation and tissue repair. Single-cell RNA sequencing (scRNA-seq) has identified genes highly expressed in M1 macrophages at fracture sites, which are closely linked to macrophage polarization. Plant-derived extracellular vesicles (EVs) have shown potential as anti-inflammatory agents but are limited by short duration of action and poor targeting. In this study, we used scRNA-seq to elucidate the mechanisms of M1 macrophage polarization during fracture healing and identified MMP12 as a target regulated by Calendula officinalis L.-derived EVs (COEVs) through network pharmacology. To enhance the therapeutic potential of COEVs, we developed a reactive oxygen species (ROS)-responsive hydrogel encapsulating COEVs modified with phosphatidylserine (PS) for macrophage targeting. The hydrogel demonstrated excellent mechanical properties, injectability, self-healing, and ROS responsiveness. Comprehensive in vitro and in vivo experiments confirmed its biocompatibility, ability to regulate target genes, macrophage reprogramming, and osteogenic promotion, offering a novel therapeutic approach for fracture repair.