Substrate viscoelasticity regulates fibroblast adhesion and migration.

Mechanical properties of the extracellular matrix (ECM) modulate cell-substrate interactions and influence cellular behaviors such as contractility, migration, and proliferation. Although the effects of substrate stiffness on mechanobiology have been well studied, the role of ECM viscoelasticity in fibrotic progression remains less understood. To examine how viscoelasticity affects the biophysical properties and regulates signaling of human mammary fibroblasts, we engineered elastic (E) and viscoelastic (VE) polyacrylamide hydrogels with comparable storage moduli (∼14.52 ± 1.03 kPa) but distinctly different loss moduli; mean loss moduli for VE gels was 36.9% higher at 0.05 Hz than E gels. Fibroblasts cultured on E hydrogels spread extensively (2428.93 ± 864.71 μm2), developed prominent stress fibers with higher zyxin intensity, and generated higher traction stresses (2931.57 ± 1732.61 Pa). In contrast, fibroblasts on VE substrates had 54.2% smaller focal adhesion areas, exhibited 51.8% lower critical adhesion strengths, and generated 21% lower traction stresses (p < 0.001). These substrates also promoted migration and showed enhanced proliferation with reduced Yes-associated protein (YAP) activity, suggesting a mechanotransduction shift that may involve alternative signaling pathways. In contrast, E substrates showed YAP nuclear translocation, consistent with greater cytoskeletal tension and contractility. These findings highlight the importance of energy dissipation mechanisms in regulating fibroblast function on substrates mimicking the fibrotic milieu. Our results demonstrate that tuning the ECM viscoelasticity is a useful strategy to regulate cell behaviors in tissue-engineered scaffolds and develop better disease modeling for regenerative medicine.