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Abstract There is a tremendous interest in developing hydrogels as tunable in vitro cell culture platforms to study cell response to mechanical cues in a controlled manner. However, little is known about how common cell culture techniques, such as serial expansion on tissue culture plastic, affect subsequent cell behavior when cultured on hydrogels. In this work, a methacrylated hyaluronic acid hydrogel platform is leveraged to study stromal cell mechanotransduction. Hydrogels are first formed through thiol‐Michael addition to model normal soft tissue (e.g., lung) stiffness (E ≈ 1 kPa). Secondary cross‐linking via radical photopolymerization of unconsumed methacrylates allows matching of early‐ (E ≈ 6 kPa) and late‐stage fibrotic tissue (E ≈ 50 kPa). Early passage (P1) human bone marrow mesenchymal stromal cells (hMSCs) display increased spreading, myocardin‐related transcription factor‐A (MRTF‐A) nuclear localization, and focal adhesion size with increasing hydrogel stiffness. However, late passage (P5) hMSCs show reduced sensitivity to substrate mechanics with lower MRTF‐A nuclear translocation and smaller focal adhesions on stiffer hydrogels compared to early passage hMSCs. Similar trends are observed in an immortalized human lung fibroblast line. Overall, this work highlights the implications of standard cell culture practices on investigating cell response to mechanical signals using in vitro hydrogel models. 

We engineered a hydrogel platform matching either normal or diseased lung tissue mechanics and tracked time-dependent changes in fibroblast DNA methylation and chromatin condensation in response to both static and dynamic mechanical cues. 

The advancement of click-functionalized hydrogels in recent years has coincided with rapid growth in the fields of mechanobiology, tissue engineering, and regenerative medicine. Click chemistries represent a group of reactions that possess high reactivity and specificity, are cytocompatible, and generally proceed under physiologic conditions. Most notably, the high level of tunability afforded by these reactions enables the design of user-controlled and tissue-mimicking hydrogels in which the influence of important physical and biochemical cues on normal and aberrant cellular behaviors can be independently assessed. Several critical tissue properties, including stiffness, viscoelasticity, and biomolecule presentation, are known to regulate cell mechanobiology in the context of development, wound repair, and disease. However, many questions still remain about how the individual and combined effects of these instructive properties regulate the cellular and molecular mechanisms governing physiologic and pathologic processes. In this review, we discuss several click chemistries that have been adopted to design dynamic and instructive hydrogels for mechanobiology investigations. We also chart a path forward for how click hydrogels can help reveal important insights about complex tissue microenvironments.