Osteoblast-derived Extracellular Vesicles Exert Bone Formation Effects by WIF1-mediated Regulation of Mitophagy

Osteoporosis arises mainly from an imbalance in bone remodeling, characterized by the impaired ability of osteoblasts (OBs) to form new bone. A significant challenge remains in identifying and validating key genes involved in osteogenic differentiation to develop effective treatments. The regulatory role of WIF1 in osteogenic differentiation stages was investigated using Western blot and quantitative polymerase chain reaction assays to measure the expression levels of osteogenic-related genes in MC3T3-E1 cells. Mineralization of OBs was assessed through alizarin red S (ARS) and alkaline phosphatase (ALP) staining assays. To explore the relationship with mitophagy, RNA sequencing was performed to examine the effects of Wif1 overexpression on genes related to osteogenic differentiation and mitophagy processes. Additionally, histological staining and micro-computed tomography were conducted on ovariectomized (OVX) mice treated with Wif1-overexpressing OB-derived extracellular vesicles (EVs) to evaluate their impact on bone loss and osteogenic differentiation in bone marrow stromal cells (BMSCs). Wif1 was identified as a crucial marker for late-stage osteogenic differentiation, playing a role in regulating mitophagy to release functional EVs. In vitro analyses showed that Wif1 overexpression accelerated osteogenic differentiation by activating genes related to late-stage osteogenic differentiation and mitophagy processes. In vivo experiments demonstrated that administering Wif1-overexpressing OB-derived EVs to OVX mice partially reversed bone loss and countered the suppression of osteogenic differentiation in BMSCs. Our findings shed light on the molecular mechanism of Wif1 in osteogenic differentiation and suggested the therapeutic efficacy of Wif1-overexpressing OB-derived EVs in osteoporosis.