Abstract Biomechanical contributions of the extracellular matrix underpin cell growth and proliferation, differentiation, signal transduction, and other fate decisions. As such, biomaterials whose mechanics can be spatiotemporally altered‐ particularly in a reversible manner‐ are extremely valuable for studying these mechanobiological phenomena. Herein, a poly(ethylene glycol) (PEG)‐based hydrogel model consisting of two interpenetrating step‐growth networks is introduced that are independently formed via largely orthogonal bioorthogonal chemistries and sequentially degraded with distinct recombinant sortases, affording reversibly tunable stiffness ranges that span healthy and diseased soft tissues (e.g., 500 Pa–6 kPa) alongside terminal cell recovery for pooled and/or single‐cell analysis in a near “biologically invisible” manner. Spatiotemporal control of gelation within the primary supporting network is achieved via mask‐based and two‐photon lithography; these stiffened patterned regions can be subsequently returned to the original soft state following sortase‐based secondary network degradation. Using this approach, the effects of 4D‐triggered network mechanical changes on human mesenchymal stem cell morphology and Hippo signaling, as well as Caco‐2 colorectal cancer cell mechanomemory using transcriptomics and metabolic assays are investigated. This platform is expected to be of broad utility for studying and directing mechanobiological phenomena, patterned cell fate, and disease resolution in softer matrices.

Stepwise Stiffening/Softening of and Cell Recovery from Reversibly Formulated Hydrogel Interpenetrating Networks