Multienzyme-Mediated Dynamic Cross-Linking of All-Natural Hydrogels with High Adhesion and Redox Modulation for Rapid Tissue Filling and Repair

The in vivo construction of soft-tissue filling hydrogels with cell-adaptive biophysical and biochemical microenvironments is highly desirable for wound healing and tissue regeneration. Herein, inspired by enzyme-mediated cross-linking and metabolic regulation in biological systems, we report a cascade galactose oxidase (GAO)/catalase (CAT)-catalyzed dynamic Schiff-base cross-linking strategy for rapid in situ formation of an all-natural polysaccharide hydrogel under physiological conditions. In this system, natural galactomannan functions simultaneously as the structural component and enzymatic substrate, which can be selectively oxidized by GAO/CAT at C6 hydroxyl groups of galactose into reactive aldehydes, enabling subsequent Schiff-base cross-linking with amino-containing polysaccharides (e.g., glycol chitosan (CHT)) and enzymes, without external initiators, radicals, or chemical premodification. The immobilized enzymes can be repeatedly re-exposed to catalytically oxidize the substrate galactomannan under mechanical stimulation, allowing sustained regulation of network architecture and interfacial reactivity for enhanced tissue adhesion, thereby supporting effective filling of irregular defects. Furthermore, the GAO/CAT-integrated hydrogel demonstrates superoxide dismutase (SOD)-like activity and, together with CAT, enables the synergistic scavenging of reactive oxygen and nitrogen species (ROS/RNS) in human osteoarthritis (OA) synovial fluid, thereby promoting articular cartilage regeneration in an OA mouse model. This enzymatic cross-linking strategy provides a safe and facile approach for dynamic hydrogel construction, allowing direct precursor injection at target sites for in situ gelation with biochemical regulation, thereby demonstrating significant translational potential in biofilling and tissue regeneration.