Enzyme-Responsive Self-Evolving Hydrogel for Osteochondral Regeneration through Mechanosignaling Pathway

Dynamic and spatially graded mechanical microenvironments are essential for guiding the regeneration of hierarchical osteochondral tissue. Although hydrogels are widely used in stem cells-based tissue regeneration, conventional platforms cannot deliver precisely controlled spatiotemporal mechanical cues required for osteochondral repair. Herein, a self-evolving hydrogel (SE gel) is reported that incorporates a secondary cross-linking network catalyzed by alkaline phosphatase (ALP), formed by the reaction between 2-cyanobenzothiazole (CBT) and cysteine (Cys). This enzymatic cross-linking increases network density and complements the primary photo-cross-linking structure, resulting in a 4-fold increase in the storage modulus from 2.73 to 11.08 kPa. The increased stiffness induces a morphological transition in cell spreading from fusiform to polygonal shapes, promotes a 2.2-fold increase in nuclear localization of yes-associated protein (YAP), and triggers osteogenic differentiation. SE gel exploits the endogenous ALP gradient to form a spatially graded, dual-cross-linked network. In the subchondral bone region, a higher ALP activity (∼294.5 U mg^-1) catalyzes the extensive formation of a dual-cross-linked structure, whereas the articular cartilage region, with a lower ALP activity (∼15.0 U mg^-1), generates a less dense network. This ALP-gradient-driven evolution delivers spatially and temporally dynamic mechanical cues, ranging from soft to stiff, which are transduced through extended integrin-mediated mechanosignaling and subsequently activate the PI3K/AKT/GSK-3β/β-catenin pathway. This cascade regulates key cell functions, such as spreading, migration, and differentiation. The dynamic and gradient-responsive SE gel supports osteochondral regeneration with tissue-specific heterogeneity. To the best of our knowledge, this is the first study to integrate an adaptive hydrogel with an ALP activity gradient, demonstrating its potential in osteochondral regeneration and highlighting the pivotal role of mechanobiology.