Substrate stiffness regulates the proliferation and inflammation of chondrocytes and macrophages through exosomes.

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Osteoarthritis (OA) progression is characterized by decreased cartilage stiffness and degradation of the extracellular matrix (ECM), which significantly influence cartilage behavior and fate. In contrast, processes such as chondrocyte calcification and aging often result in increased stiffness. Despite extensive studies on how ECM stiffness regulates cellular functions, the impact of substrate stiffness on the cartilage microenvironment and intercellular communications remains not well understood. Using tunable stiffness Gelatin methacryloyl (GelMA) hydrogel, we demonstrated that a potential optimal substrate stiffness can promote maximal chondrocyte proliferation and exosome secretion. The exogenous addition of stiffness-tuned exosomes induced significant changes in chondrocyte morphology, proliferation, migration, and inflammation. Notably, blocking Yes-associated protein (YAP) synthesis negated the proliferation enhancement induced by exosomes from chondrocytes cultured on medium stiffness substrates (ExoMedium), confirming that substrate stiffness regulates cell proliferation through exosomes by modulating YAP expression and its nuclear localization. Moreover, our study revealed that exosomes from medium stiffness substrates-mimicking normal cartilage stiffness-not only reduce inflammation in chondrocytes but also shift macrophage polarization from M1 to M2. Conversely, exosomes from soft stiffness substrates, akin to osteoarthritic tissue, exacerbate chondrocytes inflammation and M1 macrophage polarization. These findings highlight the crucial role of stiffness-tuned exosomes in OA progressing, affecting chondrocyte proliferation, migration, inflammation and macrophage polarization, and provide new insights into the potential novel treatment strategies using engineered scaffolds and exosomes. STATEMENT OF SIGNIFICANCE: Osteoarthritis (OA) is a prevalent degenerative joint disease characterized by decreased cartilage stiffness and degradation of the extracellular matrix (ECM). While some studies suggest that increased substrate stiffness enhances cell proliferation, others have reported the opposite effect. Whether there exists an optimal matrix stiffness that promotes chondrocytes anabolism and how matrix stiffness regulates the cartilage microenvironment and intercellular communications remain unclear. Utilizing tunable stiffness Gelatin methacryloyl (GelMA) hydrogel, this study demonstrated that a potential optimal substrate stiffness can maximize chondrocyte proliferation and exosome secretion. The introduction of stiffness-tuned exosomes induced significant changes in chondrocyte and macrophage proliferation, migration, and inflammation, offering new insights into OA progression and highlighting their potential as a promising therapeutic strategy for osteoarthritis treatment and tissue regeneration.