In vitro study on the promotion of osteogenic differentiation by mitochondrial-derived vesicles through activation of inflammation and reprogramming of metabolic pathways.

The secretion of mitochondrial-derived vesicles (MDVs) has been found to increase during osteogenic differentiation, but their role in intercellular communication and osteogenic promotion remains unclear. In this study, we extracted translocase of outer mitochondrial membrane 20 (Tomm20) + MDVs from bone marrow stromal cells (BMSCs) at different osteogenic culture days using differential centrifugation and immunoprecipitation, then co-cultured them with BMSCs to assess osteogenic differentiation, immune response and metabolic levels. The results showed that osteogenic differentiation enhances MDVs' secretion and their mitochondrial DNA (mtDNA) content. In promoting osteogenic differentiation ability, osteogenic-induced MDVs (MDV-OMs, especially MDV-OM14 and MDV-OM21) significantly enhance mineralization with OD values 1.37-fold and 1.32-fold higher than those of MDV-OM7 (p < 0.05) after 21 days, respectively. However, these MDVs containing mtDNA activate immune responses by upregulating cGas, Sting, Caspase-9, Il-6, and Tnf-a mRNA levels, inducing cell apoptosis and oxidative stress. In addition, MDVs containing mitochondrial components also have metabolic regulatory functions. Metabolic level detection revealed that MDV-OMs downregulate lactate, promote tricarboxylic acid cycle (TCA) enzyme expression, and increase mitochondrial membrane potential. Among these MDVs, MDV-OM7, induced for 7 days, shows osteogenic function without strong immune response, possibly related to metabolic reprogramming. This study highlights the potential of osteogenic-induced MDVs for bone regeneration, cGAS-STING activation, and metabolic enhancement, and are expected to be used for the treatment of diseases such as tissue damage.