The extracellular matrix (ECM) is a complex, temporally-regulated environment, and engineered hydrogels are increasingly used as ECM surrogates for probing related cell functions. ECM remodeling events related to injury or disease result in local ECM softening and stiffening (e.g., degradation followed by deposition/crosslinking). Inspired by these events, this work establishes an approach for pseudo-reversible mechanical property modulation in synthetic hydrogels by integrating orthogonal, enzymatically-triggered crosslink degradation and light-triggered photopolymerization stiffening. Hydrogels are formed by a photo-initiated thiol-ene reaction between multi-arm polyethylene glycol (PEG) and a dually enzymatically degradable peptide linker, which incorporates a thrombin-degradable sequence for triggered softening and a matrix metalloproteinase (MMP)-degradable sequence for cell-driven remodeling. Hydrogels are stiffened by photopolymerization using a flexible, MMP-degradable polymer-peptide conjugate and multi-arm macromers, increasing the synthetic matrix crosslink density while retaining degradability. Integration of these tools enables sequential softening and stiffening inspired by matrix remodeling events within loose connective tissues (Young's modulus (E)∼5 to 1.5 to 6 kPa with >3xDE). The cytocompatibility and utility of this approach is examined with breast cancer cells, where cell proliferation showed a dependence on the timing of triggered softening. This work provides innovative tools for 3D dynamic property modulation that are synthetically accessible and cell compatible.

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