Extracellular Vesicles Derived from Resveratrol-Treated BMSCs Promote the Repair of Bone Defects in Osteoporotic Rats.

Osteoporotic fracture is the most severe complication of osteoporosis, and impaired bone regeneration and delayed healing after fracture remain major clinical challenges. Extracellular vesicles (EVs) derived from bone marrow mesenchymal stem cells (BMSCs) may play a critical role in promoting bone regeneration after osteoporotic fracture. Enhancing their osteogenic potential and elucidating the underlying mechanisms may contribute to accelerated fracture healing. In this study, resveratrol-stimulated BMSC-derived EVs (Res-EVs) were isolated using differential centrifugation combined with ultracentrifugation. We found that compared with EVs secreted by BMSCs under normal culture conditions (Nor-EVs), Res-EVs significantly enhanced BMSCs osteogenic differentiation capacity. Analysis of miRNA sequencing data identified miR-19a-3p as significantly downregulated and PC-3p-83,608 as significantly upregulated. The reliability of the sequencing data was validated by qPCR results. By transfecting BMSCs with inhibitor and mimic of miR-19a-3p, we found that miR-19a-3p was a negative regulator of osteogenic differentiation. Bioinformatics prediction and dual-luciferase reporter assays confirmed that miR-19a-3p directly targets Raf1. Western blot results indicated that miR-19a-3p regulates osteogenic differentiation of BMSCs by targeting Raf1. In vivo experiments further demonstrated that Res-EVs treatment enhanced repair at osteoporotic fracture sites and increased the expression of RUNX2 and RAF1. Collectively, these findings indicate that Res-EVs promote bone regeneration at osteoporotic bone defect sites in rats, with the miR-19a-3p/Raf1 axis representing a potential underlying mechanism.