Endogenous alkaline phosphatase gradient-driven stiffness-adaptive hydrogel regulates stem cell fate for...

Stiffness-adaptive gradient scaffolds that provide precise mechanobiological cues are highly desirable for coordinated osteochondral repair. However, most existing scaffolds remain mechanically static and spatially homogeneous after implantation, and therefore fail to recapitulate the adaptive stiffening and depth-dependent heterogeneity of native osteochondral interface. Herein, an endogenous alkaline phosphatase (ALP)-responsive hydrogel is developed to translate the native depth-dependent ALP activity of osteochondral tissue into in situ mechanical remodeling. A phosphorylated peptide is covalently grafted onto gelatin methacryloyl and then photo-cross-linked to form a primary network. ALP-mediated dephosphorylation subsequently induces β-sheet-rich assembly of the grafted phosphorylated peptides, establishing a secondary physical network that increases the unconfined compressive modulus from 76.11 to 240.75 kPa and gives rise to a depth-aligned stiffening profile. These evolving mechanical cues regulate stem cell fate through adhesion-associated mechanotransduction, centered on focal adhesion kinase, Rho-associated kinase, and Hippo-Yes-associated protein signaling, with softer regions favoring chondrogenesis and stiffer regions promoting osteogenesis. In a rabbit osteochondral defect model, the stiffness-adaptive hydrogel supports integrated osteochondral regeneration, including nearly continuous cartilage coverage, improved subchondral bone restoration, and superior local mechanical recovery. Overall, this work presents an enzyme-instructed self-strengthening strategy that couples an endogenous biochemical cue with β-sheet-rich secondary physical network formation, thereby providing a versatile stiffness-adaptive platform for stem cell regulation and graded osteochondral regeneration.