Osteogenic priming of canine perivascular mesenchymal stromal cells enhances extracellular vesicle yield and in vitro activity for bone regeneration.

Abstract

The therapeutic efficacy of mesenchymal stromal cells (MSCs) is intrinsically linked to their response to the host microenvironment by the interplay with resident cells. While MSC-derived extracellular vesicles (EVs) offer a promising cell-free alternative, optimizing their production and cargo for specific clinical applications remains a challenge. We investigated the impact of in vitro osteogenic priming on the yield, molecular profile, and functional activity of EVs derived from canine umbilical cord perivascular MSCs (cPMSCs). The EVs secreted by osteogenic primed and naïve cPMSC were isolated by ultracentrifugation and characterized by nanoparticle tracking analysis, western blotting, and electronic transmission microscopy. In vitro functional assays of cellular uptake, proliferation, migration, and mineralization were performed. MicroRNAs known to participate in osteogenesis (cfa-miR-21, cfa-miR26a, cfa-miR-27a and cfa-miR-29a) were evaluated in EV samples of both groups through RT-qPCR. Notably, osteogenic priming led to a 1.5-fold increase in vesicle yield (5.6 ± 0.5 ×1010 particles/ml) compared to naïve MSCs (3.7 ± 0.7 ×1010 particles/ml), without altering vesicle distribution nor TSG101 expression (similar expression for both groups). Functional assays revealed that while both EV populations were efficiently internalized, osteogenic priming significantly improved extracellular matrix mineralization (P=0.0003) and enhanced EV-mediated cell migration (P=0.005). Molecular analysis confirmed the enrichment of key osteogenic microRNAs, including cfa-miR-21, cfa-miR-26a, cfa-miR-27a, and cfa-miR-29a, in both groups. These findings demonstrate that osteogenic priming is a robust strategy to improve EV production efficiency and migratory potential, providing a molecularly characterized foundation for advanced cell-free orthopedic therapies in veterinary medicine.