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As such, biomaterials whose mechanics can be spatiotemporally altered\u2010 particularly in a reversible manner\u2010 are extremely valuable for studying these mechanobiological phenomena. Herein, a poly(ethylene glycol) (PEG)\u2010based hydrogel model consisting of two interpenetrating step\u2010growth 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\u00a0Pa\u20136\u00a0kPa) alongside terminal cell recovery for pooled and\/or single\u2010cell analysis in a near \u201cbiologically invisible\u201d manner. Spatiotemporal control of gelation within the primary supporting network is achieved via mask\u2010based and two\u2010photon lithography; these stiffened patterned regions can be subsequently returned to the original soft state following sortase\u2010based secondary network degradation. Using this approach, the effects of 4D\u2010triggered network mechanical changes on human mesenchymal stem cell morphology and Hippo signaling, as well as Caco\u20102 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.<\/jats:p>","DOI":"10.1002\/adma.202404880","type":"journal-article","created":{"date-parts":[[2024,9,6]],"date-time":"2024-09-06T09:37:40Z","timestamp":1725615460000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":32,"title":["Stepwise Stiffening\/Softening of and Cell Recovery from Reversibly Formulated Hydrogel Interpenetrating Networks"],"prefix":"10.1002","volume":"36","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-3740-6971","authenticated-orcid":false,"given":"Irina","family":"Kopyeva","sequence":"first","affiliation":[{"name":"Department of Bioengineering University of Washington  Seattle WA 98105 USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0000-6347-2353","authenticated-orcid":false,"given":"Ethan 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