The mechanical properties of the native extracellular matrix play a key role in regulating cell behavior during developmental, healing and homeostatic processes. Since these properties change over time, it may be valuable to have the capacity to dynamically vary the mechanical properties of engineered hydrogels used in tissue engineering strategies to better mimic the dynamic mechanical behavior of native extracellular matrix. However, in situ repeatedly reversible dynamic tuning of hydrogel mechanics is still limited. In this study, we have engineered a hydrogel system with reversible dynamic mechanics using a dual-crosslinkable alginate hydrogel. The effect of reversible mechanical signals on encapsulated stem cells in dynamically tunable hydrogels has been demonstrated. In situ stiffening of hydrogels decreases cell spreading and proliferation, and subsequent softening of hydrogels gives way to an increase in cell spreading and proliferation. The hydrogel stiffening and softening, and resulting cellular responses are repeatedly reversible. This hydrogel system provides a promising platform for investigating the effect of repeatedly reversible changes in extracellular matrix mechanics on cell behaviors. Statement of significance Since the mechanical properties of native extracellular matrix (ECM) change over time during development, healing and homeostatic processes, it may be valuable to have the capacity to dynamically control the mechanics of biomaterials used in tissue engineering and regenerative medicine applications to better mimic this behavior. Unlike previously reported biomaterials whose mechanical properties can be changed by the user only a limited number of times, this system provides the capacity to induce unlimited alterations to the mechanical properties of an engineered ECM for 3D cell culture. This study presents a strategy for on-demand dynamic and reversible control of materials’ mechanics by single and dual-crosslinking mechanisms using oxidized and methacrylated alginates. By demonstrating direct changes in encapsulated human mesenchymal stem cell morphology, proliferation and chondrogenic differentiation in response to multiple different dynamic changes in hydrogel mechanics, we have established a repeatedly reversible 3D cellular mechanosensing system. This system provides a powerful platform tool with a wide range of stiffness tunability to investigate the role of dynamic mechanics on cellular mechanosensing and behavioral responses.
