The extracellular matrix (ECM) of tissues progressively changes its mechanical properties in processes such as tissue development, repair, and disease progression. While stiffness has become a key design parameter of biomaterials, most synthetic biomaterials employed in cell culture or tissue regeneration do not display these gradual changes in mechanical properties. Here, we report on a hydrogel platform with the capacity to exhibit progressive stiffening from 0.8 to 7.4 kPa within a ∼48 h time period. The material integrates the tyramine derivative of hyaluronic acid (HAT) and Laponite® (Lap) and harnesses the diffusion of cations from culture media to trigger gradual secondary Lap-HAT cross-linking, resulting in the progressive stiffening of the hydrogel. We assessed the applicability of the hydrogel by first using it as a substrate for in vitro culture to investigate cross-talk between human bone marrow stromal cells (HBMSCs) and human umbilical vein endothelial cells (HUVECs). The progressively stiffening hydrogel led to changes in cell morphology and enhanced differentiation and communication compared to control substrates. In addition, we also tested the potential of the progressively stiffening hydrogels for bone regeneration using a critical-size rat cranial defect model and found that the hydrogel construct promoted vascularized bone regeneration. The current study introduces a hydrogel material that offers a more physiologically relevant environment for in vitro and in vivo applications and provides insight into the mechanical complexity of the ECM and its role in tissue physiology. STATEMENT OF SIGNIFICANCE: This study presents a dynamic hydrogel platform that imitates the progressive mechanical changes of the native extracellular matrix (ECM), transitioning from soft (0.8 kPa) to stiff (7.4 kPa) over 48 h. By co-assembling HAT and Lap, the hydrogel achieves gradual stiffening through cation diffusion - mediated gradual secondary Lap-HAT cross-linking, offering a physiologically relevant microenvironment. In vitro, it enhances HBMSC and HUVEC cross-talk, improving differentiation and morphology. In vivo, it promotes vascularized bone regeneration in a critical-size cranial defect model. This innovation bridges the gap between static synthetic biomaterials and dynamic ECM mechanics, advancing applications in tissue engineering, disease modeling, and regenerative medicine.
