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

Fibroblast phenotype, contractility, and migration are governed by substrate stiffness—even overruling classical TGF-β stimulation. Using human dermal fibroblasts on soft (~9 kPa) and stiff (~30 kPa) polyacrylamide gels, the authors show that stiff microenvironments drive stress-fiber formation, focal-adhesion (FA) maturation, αSMA incorporation, and reduced motility in fully transitioned myofibroblasts. Traction force microscopy revealed ~101 Pa mean tractions on soft vs. ~270 Pa on stiff substrates; TGF-β increased forces on soft gels but slightly decreased them on stiff gels. Single-cell tracking showed higher migration speeds on stiff substrates (e.g., ~0.56 μm/min at 96 h) than on soft (~0.32 μm/min), while TGF-β decreased speed on stiff gels as cells completed FMT. FA morphology depended on stiffness (round on soft, elongated on stiff) and TGF-β further enlarged FAs only on stiff gels. Figures highlight cytoskeletal/FA remodeling (Fig. 1, p. 4), migration trajectories and MSD (Fig. 2, pp. 5–6), traction vector maps (Fig. 3A, p. 6) and FA size/speed distributions (Fig. 4B–C, p. 7). Conclusion: stiffness mechanosensing is a primary regulator of fibroblast-to-myofibroblast transition and phenotype-dependent migration/contractility, with implications for fibrosis and wound repair.