Substrate Stress Relaxation Regulates Cell‐Mediated Assembly of Extracellular Matrix

Abstract The viscoelasticity of the extracellular matrix (ECM) regulates diverse cellular functions, yet its influence in guiding ECM assembly and organization under physiologically relevant stiffness remains poorly defined. In this study, silicone‐based substrates with comparable stiffness (≈80 kPa) but distinct stress relaxation profiles are used to investigate how matrix viscoelasticity affects cellular mechanosensing and cell‐mediated ECM remodeling in the stiff regime. Increased substrate stress relaxation enhances fibronectin reorganization, focal adhesion maturation, and traction force generation for similar fibronectin surface density. Cells on viscoelastic substrates exhibit increased nuclear localization of YAP and form β1 integrin‐enriched adhesions, correlating with localized ECM reorganization. These findings reveal that mechanical properties alone, decoupled from biochemical cues, are sufficient to direct ECM reorganization. This platform allows dissecting mechano‐regulated tissue remodeling and designing mechanically tunable biomaterials for regenerative medicine.