Human bone marrow mesenchymal stem cell–derived small extracellular vesicle preparations combined with salvianolic acid B enhance early bone-defect repair

This study evaluated whether small extracellular vesicle (sEV)-enriched preparations derived from human bone marrow mesenchymal stem cells (hBMSCs) enhance the osteogenic effects of salvianolic acid B (Sal B) during early bone-defect repair. hBMSCs were treated with hBMSC-derived sEV preparations (5–25 μg/mL), Sal B (50 μM), or their combination. Early ALP responses, mineralization, Transwell cell passage, osteogenic gene/protein expression, and Type I collagen were assessed in vitro. In vivo, a 2 mm cranial defect was created in 32 male C57BL/6 mice assigned to Control, sEV, Sal B, or sEV + Sal B groups ( n = 8/group). Animals were evaluated at 2 weeks using H&E, Masson staining, immunohistochemistry, and Western blotting. The hBMSC-derived vesicle preparation showed approximately 100 nm particles by the reported size analysis and was positive for CD63 and CD9. The 25 μg/mL sEV condition increased the early ALP-associated signal and calcium deposition ( P < 0.001). The sEV + Sal B group showed a 3.57-fold increase in cells traversing the Transwell membrane and higher RUNX2 and OCN expression than control ( P < 0.001). At 2 weeks in vivo, the combined group showed more prominent repair-associated tissue formation, collagen deposition, and OCN/BSP staining. BMP-2, Smad1, HIF-1α, and VEGF protein abundance was also increased; these expression changes are associative and do not establish pathway causality. hBMSC-derived sEV preparations combined with Sal B enhanced osteogenic responses in vitro and were associated with improved early-repair features in a mouse cranial-defect model. The observed changes in BMP/Smad- and HIF-1α/VEGF-related proteins support candidate pathways for future testing but do not demonstrate causal pathway activation or functional angiogenesis.