Mesenchymal Stromal Cell-Derived Extracellular Vesicles show Modulatory Potential in an Equine Joint-on-a-Chip Model of Osteoarthritis

Background: Osteoarthritis (OA) is a leading cause of lameness, lost training and premature retirement in horses across all disciplines and breeds. Mesenchymal stromal cell (MSC) therapy is used to treat equine OA with variable clinical efficacy. Extracellular vesicles (EVs) may offer a cell-free alternative by mediating key therapeutic effects of MSCs. Objectives: To develop a three-dimensional (3D) equine joint-on-a-chip model of inflammatory OA-like pathology and investigate cell-specific responses to MSC-derived EVs (MSC-EVs). Study design: Pilot in vitro experimental study. Methods: Equine adipose-derived MSCs (n = 3) were characterised, and MSC-EVs isolated using size exclusion chromatography. EVs were characterised by nanoparticle tracking analysis, cryogenic and transmission electron microscopy and western blotting. Equine chondrocytes (n = 3) and fibroblast-like synoviocytes (FLS) (n = 3 ) were cultured within a 3D hydrogel-based joint-on-a-chip. Inflammatory cytokine stimulation was induced using interleukin 1 beta (IL-1β) and tumor necrosis factor alpha (TNF-α) (10 ng/mL each), followed by treatment with MSC-EVs (3.5 × 108 particles) per synovial-chondral organoid. Targeted gene expression was assessed using the NanoString nCounter analysis platform. Results: MSCs successfully underwent trilineage differentiation while MSC-EV preparations displayed vesicular morphology and expressed CD9. Cytokine stimulation induced inflammatory responses. MSC-EV treatment modulated the expression of interleukin 6 (p = 0.01), C-C motif chemokine ligand 2 (p = 0.03) and NF-kappa-B inhibitor alpha (p = 0.02) towards baseline control levels, with differential (p < 0.05) responses observed between chondrocytes and FLS. Main limitations: The study was limited by the number of matched biological donors, and outcomes were assessed at the gene expression level without functional validation. Conclusion: MSC-EVs modulated inflammatory and catabolic gene expression in a 3D equine joint-on-a-chip model, with cell specific responses. This compartmentalised equine joint-on-a-chip platform provided a promising preclinical tool to explore cell-free regenerative strategies for equine musculoskeletal disease.