Exploring the molecular cargos associated with extracellular vesicles extracted from bone tissue to identify novel players in osteoblast function.

Extracellular vesicles (EVs), composed of proteins, lipids, and nucleic acids, are key mediators of intercellular communication across various tissues. However, research on EVs isolated directly from bone tissue remains limited. Here we identified for the first time and characterized two EV subpopulations extracted from bone tissue and enhanced our understanding of their roles in bone physiology. These two EV subpopulations were reproducibly isolated, a large EV population (lEV; 237.7 ± 8.8 nm) and a small EV population (sEV; 109.2 ± 8.3 nm), both exhibiting the expected shape and presence of EV and bone cell markers. Comparative analysis of their cargos revealed unique or enriched proteins and miRNAs profile for each, suggesting shared functional characteristics with bone cells, including osteocytes, osteoblasts, and osteoclasts. Notably, lEVs contained proteins such as FHL2 and pleiotrophin, along with miRNAs including miR-15b-5p, miR-29a-3p, and miR-128-3p, all of which are involved in early osteogenic signaling pathways such as Hippo, TGF-β, and Wnt. Furthermore, sEVs contained ALP, PAPSS2 and miR-125b, both known regulators of matrix mineralization. Both EV subpopulations were internalized by stromal ST2 and pre-osteoblastic MC3T3-E1 cells. We present evidence that lEVs significantly enhanced ALP activity in ST2 cells, indicating early osteogenic stimulation, while sEVs partially promoted matrix mineralization in primary osteoblasts. Our findings provide novel insights into the role of bone-derived EVs as possible complementary mediators of osteogenesis in vivo and highlight the potential of their cargos in advancing bone regeneration strategies.