Extracellular Vesicle-Mediated Communication Between Anterior Cruciate Ligament and Bone Marrow Cells Modulates...

Following anterior cruciate ligament (ACL) reconstruction, enhancing hamstring tenocyte activity and minimizing apoptosis are critical for preventing graft failure and promoting ligamentization. This study investigated the therapeutic potential of extracellular vesicles (EVs) derived from a coculture of ACL remnant cells and bone marrow stromal cells (BMSCs), termed coculture-EV, in comparison with EVs from BMSC monoculture (BMSC-EV). We hypothesized that coculture-EVs could enhance tenocyte activity and reduce apoptosis, with greater effects than BMSC-EVs. ACL remnants, bone marrow, and hamstring tendons were harvested from rabbits 4 weeks post-ACL transection, and the cultured cells were used for coculture and subsequent experiments. EVs were isolated by ultracentrifugation and characterized by nanoparticle size analysis, electron microscopy, and EV-specific markers. Hamstring tenocytes treated with coculture-EVs exhibited significantly improved viability, proliferation (EdU assay), and migration (scratch and transwell assays), along with increased expression of genes related to collagen synthesis, transforming growth factor beta (TGF-β), and vascular endothelial growth factor (VEGF). Importantly, coculture-EV treatment more effectively suppressed both intrinsic and extrinsic apoptotic pathways. Coculture-EV treatment reduced early apoptosis in tenocytes by 54.5%, whereas BMSC-EV produced a 21.1% reduction. These findings suggest that EVs from ACL/BMSC coculture possess superior bioactivity compared with BMSC-derived EVs alone. Coculture-EV enhances tenocyte function and survival, indicating its potential as a cell-free therapeutic strategy to promote hamstring graft maturation and improve outcomes after ACL reconstruction.