Osteogenic differentiation is a critical biological process for bone formation and regeneration. Exosomes, as extracellular vesicles, play vital roles in intercellular communication by transferring bioactive molecules. This study aimed to investigate the role and mechanism of exosomal miRNAs derived from human bone mesenchymal stem cells (BMSCs) in regulating osteogenic differentiation of human osteoblast cells (hFOB1.19). BMSCs were characterized by assessing multipotential differentiation capacity. Exosomes from BMSCs were isolated and identified. miRNA qPCR array analysis was used to screen differentially expressed miRNAs in BMSCs-derived exosomes. Alkaline phosphatase (ALP) and Alizarin Red S (ARS) staining were performed to evaluate osteogenic differentiation. Reverse transcription quantitative polymerase chain reaction was utilized to detect mRNA expression levels. Dual-luciferase reporter assay, RNA pull-down, and RNA fluorescence in situ hybridization were employed to verify the interaction between miR-509-5p and secreted frizzled-related protein 1 (SFRP1) in hFOB1.19 cells. The activation of the Wnt/β-catenin pathway was examined through Western blot. An ovariectomized (OVX) rat model was used to evaluate the therapeutic effects of BMSC-exosomes and miR-509-5p mimics in vivo. BMSCs-derived exosomes promoted osteogenic differentiation of hFOB1.19 cells, as evidenced by enhanced ALP and ARS staining and upregulated expression of osteogenic markers. This effect was mediated through activation of the Wnt/β-catenin pathway. Additionally, miR-509-5p was upregulated in BMSCs-derived exosomes. Both inhibition and overexpression experiments confirmed that miR-509-5p regulates osteogenic differentiation via the Wnt/β-catenin pathway. SFRP1 was identified as a direct target of miR-509-5p, and its inhibition suppressed osteogenic differentiation. Conversely, SFRP1 overexpression reversed the pro-osteogenic effect of miR-509-5p mimics. In vivo, administration of BMSC-exosomes or miR-509-5p mimics attenuated OVX-induced bone loss, restoring bone microarchitecture parameters. BMSCs-derived exosomal miR-509-5p promoted osteogenic differentiation of hFOB1.19 cells by targeting SFRP1 and activating the Wnt/β-catenin signaling pathway, providing a potential therapeutic target for bone-related diseases.
