Matrix degradation promotes fibronectin deposition and spatial remodeling in 3D.

The extracellular matrix (ECM) coordinates the interplay between biochemical and biophysical cues to regulate cell behavior, yet isolating their individual contributions remains difficult as they are interdependent in native tissues. Here, we present a modular hydrogel platform that independently controls growth factor delivery and user-defined enzymatic matrix degradation. We show that matrix degradation alone, in the absence of exogenous growth factors, is sufficient to drive cell spreading, migration, and fibronectin deposition and assembly. Growth factor stimulation enhanced spheroid expansion but did not induce organized fibronectin remodeling unless combined with matrix degradation. Mechanistic inhibitor studies revealed that degradation-enabled fibronectin remodeling is primarily contractility dependent and MMP independent. These results support a feedback model wherein matrix degradation creates a permissive microenvironment for cell migration and fibronectin deposition, reinforcing focal adhesion formation, cell contractility, and further ECM assembly. This work identifies matrix degradation as an instructive regulator of fibronectin deposition, assembly, and remodeling, offering a framework for designing dynamic hydrogels that emulate tissue remodeling during development and healing.