Extracellular vesicles from SB431542-conditioned human adipose-derived stem cells enhance in vitro and in vivo osteogenesis via a miR-20a-5p- BAMBI -BMP/canonical Wnt signaling axis

BACKGROUND: Critical-sized bone defects resulting from tumors, trauma, or surgery present a significant clinical challenge due to its poor self-regenerating capabilities and limited therapeutic options. Mesenchymal stem cells (MSCs) have shown promise in bone regeneration, possibly mediated through their secretion of bioactive factors rather than direct engraftment and differentiation. MSC-derived extracellular vesicles (EVs) deliver micro-RNAs (miRs), proteins, and lipids that may modulate bone regeneration. METHODS: EVs were isolated by differential ultracentrifugation from osteogenically induced human adipose-derived stem cells (hASCs), treated with or without the small molecule SB431542, an inhibitor of TGFβ signaling (EV-hASCs[-/+]SB). EVs were characterized using western blot and nanoparticle tracking analysis. hASCs were cultured with EV-hASCs[-/+]SBs to assess bone regeneration in vitro. Bone regeneration was assessed in vivo in calvarial defects treated with (EV-hASCs[-/+]SB). EV "cargo" was analyzed for miR-content. RESULTS: Enhanced osteogenesis is shown in vitro and in vivo upon treatment with EV-hASCs[+]SB. EV-hASCs[+]SB-enriched-miR-20a-5p was identified as a modulating factor in osteogenesis, silencing BAMBI and enhancing early downstream phosphorylation of SMAD1/5 and late activation of β-catenin in hASCs undergoing osteogenic differentiation, targets of bone morphogenetic protein (BMP) and canonical Wnt (cWnt) signaling pathways. CONCLUSION: EVs act as carriers for miR-mediated pro-osteogenic signaling, harnessing the therapeutic potential of MSCs in the context of bone tissue repair.