Enhancing the Regenerative Potential of MSC-Derived Exosomes via 3D Microenvironment Modulation for Cartilage Repair

Articular cartilage defects remain a major clinical challenge due to their poor self-repair capacity. Mesenchymal stem cell (MSC)-derived exosomes have emerged as promising cell-free therapeutics; however, conventional two-dimensional (2D) cultures yield exosomes with limited bioactivity. Here, we engineered hierarchical macro–microporous scaffolds using gelatin methacryloyl (GelMA) hydrogel and cartilage extracellular matrix (ECM) to establish biomimetic three-dimensional (3D) microenvironments for MSC culture. This approach yielded three distinct exosome types—2D-Exo, GelMA-derived exosomes (G-Exo), and ECM-derived exosomes (E-Exo). Compared with 2D-Exo, 3D-derived exosomes significantly enhanced MSC proliferation, migration, chondrogenic differentiation, immunomodulation, and chondrocyte protection under inflammatory conditions, with E-Exo exhibiting the most potent effects. In vivo, E-Exo combined with a decellularized cartilage ECM (DCM) scaffold promoted robust hyaline cartilage regeneration in a rat model. Mechanistically, we identify a key pathway by which E-Exo drives chondrogenesis: they are enriched in miR-503-5p, which suppresses Smad7 to enhance TGF-β/Smad2/3 signaling. These findings highlight ECM-based 3D culture as an effective strategy to optimize exosome bioactivity and provide a clinically translatable approach for cell-free cartilage regeneration.