Extracellular matrix stiffness facilitates neurite outgrowth by reprogramming the fatty acid oxidation-dependent macrophage polarization

The extracellular matrix (ECM) is involved in various of pathophysiology processes, such as wound healing and neurogenesis. During tissue injury, the recruited bone marrow-derived monocytes in the impaired site undergo functional and phenotypic changes and participate in the initiation, maintenance, and resolution phases of tissue repair. However, the effects of ECM stiffness on monocyte differentiation and function remain largely unknown. Herein, we developed a gelatin-hydroxyphenylpropionic acid-based hydrogel with different substrate stiffnesses by varying hydrogen peroxide concentrations, which demonstrated good biocompatibility. Furthermore, the high substrate stiffness hydrogel could polarize macrophage into immunosuppressive phenotype with increased expression of interleukin 10, transforming growth factor β, CD206, and CD163. Twenty three differentially expressed metabolites were identified in stiff hydrogel-cultured macrophages in comparison with soft hydrogel cultured macrophages via metabolite analysis. In addition, 4-hydroxybenzoic acid was the most upregulated metabolite, which could confer protection against neuronal and acute inflammation. Mechanistically, the high substrate stiffness induced macrophage immunosuppressive differentiation by upregulating the expression of the fatty acid oxidation (FAO)-related proteins peroxisome proliferator-activated receptor (PPAR)-γ and PPAR-δ. Consistently, the FAO inhibitor etomoxir reversed the high substrate stiffness mediated macrophage immunosuppressive polarization and neurite outgrowth. Therefore, the alteration in macrophage phenotype induced by increased substrate stiffness can promote tissue repair in clinical applications.