Dynamic Mechanical Loading Reprograms Meniscus Cell-Derived Extracellular Vesicles to Enhance Their Regenerative Potency

Abstract

The meniscus is a fibrocartilaginous tissue critical for knee stability and load distribution, but its poor healing capacity makes regeneration after injury a major clinical challenge. Extracellular vesicles (EVs) are emerging as promising cell-free therapeutics for tissue regeneration. However, their clinical translation remains limited by low yield and variable bioactivity. Here, we investigated how dynamic mechanical loading regulates the production, composition, and function of meniscus fibrochondrocyte-derived EVs (MFC-EVs). Using a custom bioreactor system, cells were subjected to physiologically relevant cyclic tensile loading. Mechanical stimulation significantly increased EV production and secretion, likely through the ESCRT-independent pathway, without altering vesicle size or tetraspanin expression. Functionally, "mechanically primed" EVs enhanced aggrecan expression in recipient mesenchymal stromal cells (MSCs), mirroring the loading-response phenotype of their source cells. Proteomic profiling identified 380 unique proteins across all EV groups, with loaded EVs enriched in extracellular matrix- and cytoskeleton-associated proteins, as well as pathways related to tissue morphogenesis, cell migration, and cartilage development. Together, these findings demonstrate that physiologic mechanical loading enhances both the yield and regenerative potency of MFC-EVs by enriching their cargo with matrix- and development-associated proteins, providing a scalable and biologically inspired approach for engineering high-efficacy EV therapeutics for meniscus repair and musculoskeletal regeneration.