Mechanical Loading Reshapes Mesenchymal Stem/Stromal Cell-Derived Extracellular Vesicle microRNA Cargo Toward Predicted Immunomodulatory and EV Cargo-Trafficking Pathways: An In Vitro Mechanobiology Study

Mesenchymal stem/stromal cell-derived extracellular vesicles (MSC-EVs) are emerging as cell-free therapeutic candidates for osteoarthritis, but most approaches treat the secretome as a passively collected product rather than a tunable biologic. Because joint loading regulates cartilage and synovial biology, we investigated whether biomechanical priming in a three-dimensional culture system could alter extracellular vesicle microRNA cargo toward cartilage- and immune-relevant regulatory programs. Bone marrow MSCs were seeded into macroporous polyurethane scaffolds within fibrinogen/thrombin hydrogels and cultured under four conditions: chondropermissive static (CP), chondropermissive uniaxial load (CPL), chondropermissive load plus oscillatory shear (CPLS), and chondrogenic static (CH). After 3 days, conditioned media were collected for extracellular vesicles isolation, particle concentration comparisons, RNA extraction, 166-microRNA profiling, and target/pathway analysis. EV preparations across all conditions demonstrated nanoscale particle distributions and strong CD63+/CD9+ positivity. CPLS-derived preparations achieved significantly higher particle abundance (2.9 × 10⁸ particles/mL) than all other conditions (p < 0.05). MicroRNA profiling identified a conserved 59-microRNA core shared across groups, with superimposed condition-specific remodeling. CH produced the broadest repertoire (138 microRNAs) with the strongest cell-cycle, senescence, and stress-regulatory signatures. Conversely, CPLS generated a highly focused profile (107 microRNAs) with a distinct membrane-directed cargo transport signature and the greatest overlap with the CH condition. Mechanically stimulated groups prominently engaged load-responsive MAPK and Wnt pathways, with CPLS demonstrating exceptionally strong predicted transforming growth factor-β (TGF-β) pathway engagement. Both CH and CPLS generated the strongest predicted M2 macrophage-associated regulatory signatures. Parental-cell validation confirmed that mechanical priming selectively modulated EV cargo sorting rather than passively mirroring intracellular abundance. Biochemical chondrogenic induction and mechanical priming generate distinct, non-interchangeable EV microRNA profiles. Combined compression and oscillatory shear increased particle yield and produced a focused cargo signature associated with predicted immunomodulatory, macrophage-related, and membrane-directed cargo-sorting programs. These findings support mechanobiological conditioning as a controllable upstream biomanufacturing variable for shaping MSC-EV miRNA cargo, while direct functional validation is required to determine therapeutic relevance in OA models.