Substrate stiffness modulates phenotype-dependent fibroblast contractility and migration independent of TGF-β stimulation

During wound healing, fibroblasts undergo radical processes that impact their phenotype and behavior. They are activated, recruited to the injury site, assume a contractile phenotype, and secrete extracellular matrix proteins to orchestrate tissue repair. Thus, fibroblast responses require dynamic changes in cytoskeleton assembly and organization, adhesion morphology, and force generation. At the same time, fibroblasts experience changes in environmental stiffness during tissue wounding and healing. Although cells are generally known to use their adhesion-contraction machinery to sense microenvironmental stiffness, little is known about how stiffness affects the fibroblast phenotypical transition and behavior in wound healing. Here we demonstrate that stiffness plays a deterministic role in determining fibroblast phenotype, surprisingly even overruling the classical TGF-<i>β</i>-mediated stimulation. By combining morphometric analysis, traction force microscopy, and single-cell migration analysis, we show that environmental stiffness primes the cytoskeletal and mechanical responses of fibroblasts, strongly modulating their morphology, force generation, and migration behavior. Our study, therefore, points to the importance of tissue stiffness as a key mechanobiological regulator of fibroblast behavior, thus serving as a potential target for controlling tissue repair.