Reengineering 3D cellular-scale stiffness gradient at the enthesis with biomimetic hydrogels to promote in situ chondrogenesis.

The enthesis links tendon/ligament to bone, forming a soft-hard interface characterized by unique cellular-scale compositional and biomechanical gradients. Enthesis injuries are common and increase with age, yet the lack of biomaterials that faithfully mimic its cellular-scale gradient mechanical environment hinders understanding of the mechanobiological basis of this vulnerability. We showed that the enthesis cellular-scale stiffness gradient progressively flattens with aging. This age-dependent mechanical remodeling diminishes chondrogenesis and may predispose the attachment site to degeneration. To investigate this, we developed a simple, tunable, and cytocompatible three-dimensional hydrogel capable of generating a sharp stiffness gradient (∼1 kPa μm-1) within a physiologically relevant range (2-119 kPa) confined to the cellular scale. Using this platform, we found that the cellular-scale stiffness gradient directs enthesis resident stem cell chondrogenesis. Mechanistically, the adhesion sensor integrin β1/FAK and the mechanosensitive calcium channel TRPV4 converge on the PI3K/AKT pathway to activate the SOX9-driven chondrogenesis. FAK promotes TRPV4 channel opening, while TRPV4-mediated calcium influx amplifies FAK signaling. Furthermore, in an aged rat model of enthesis injury, restoration of young-like stiffness gradient with the cell-laden hydrogel enhanced chondrogenic responses and improved histological, imaging-based, and functional healing outcomes. These findings identify the age-dependent cellular-scale stiffness gradient as an important mechanical feature regulating cellular differentiation and highlight potential strategies for enthesis regeneration.