Matrix-Bound and Media-Derived Extracellular Vesicles from Mineralized Osteoblasts Exhibit Distinct Osteogenic Activities

Extracellular vesicles (EVs) derived from osteoblasts are emerging as key regulators of bone formation, yet functional differences between vesicles from distinct extracellular compartments remain unclear. In this study, we compared media-derived extracellular vesicles (MEs) and matrix-bound extracellular vesicle-enriched fractions (MBEs) isolated from mineralized osteoblasts (MOBs) and evaluated their effects on human bone marrow-derived mesenchymal stromal cells (hBMSCs). Both preparations, characterized by nanoparticle tracking analysis and transmission electron microscopy, displayed similar size distributions (30–200 nm) and concentrations. Moreover, both preparations showed enrichment of the EV-associated marker CD63, with no detectable GAPDH and only minimal Grp94 signals in a subset of samples. mRNA profiling revealed that MBE-enriched fractions were selectively enriched in RUNX2, whereas other osteogenesis-related transcripts were comparable between them. Functional analyses demonstrated that both EV populations promoted osteogenic differentiation of hBMSCs, although with distinct biological profiles. MBE-enriched fractions were associated with higher alkaline phosphatase activity under control conditions, whereas MEs induced greater osteocalcin expression and showed a numerical tendency toward increased matrix mineralization, particularly under osteogenic conditions. These findings suggest that extracellular vesicles associated with different extracellular compartments exhibit distinct osteogenic activities rather than a uniform biological effect. Although the matrix-bound preparation likely contains extracellular matrix-associated components co-isolated during the extraction procedure, the present study highlights the importance of extracellular compartmentalization in shaping EV-associated bioactivity and provides a foundation for future studies aimed at optimizing EV-based strategies for bone regeneration.