Antioxidant functionalized double-net/TA dynamic hydrogel promotes cartilage regeneration through stabilization of chondrocyte phenotype

Cartilage defects, whether congenital or acquired, are highly prevalent in clinical practice. Tissue engineering offers a promising strategy for cartilage regeneration; however, the loss of chondrocyte phenotype during <i>in vitro</i> expansion remains a major barrier to the clinical translation of chondrocyte-based engineered cartilage. Emerging evidence has highlighted that alterations in chondrocyte metabolic states can profoundly impact their phenotypic stability. Nonetheless, how metabolic patterns shift during <i>in vitro</i> expansion, and whether metabolic modulation can stabilize the chondrocyte phenotype, remain insufficiently explored. To address these questions, we first utilized single-cell RNA sequencing combined with bulk transcriptomic analysis to profile the metabolic reprogramming of chondrocytes during <i>in vitro</i> expansion. Our findings revealed a distinct shift from glycolytic metabolism toward oxidative phosphorylation dominance. Based on this insight, we engineered a DN (double-net) hydrogel scaffold composed of collagen, PEG (polyethylene glycol), and CNF (nanocellulose). To endow the scaffold with antioxidant functionality, TA (tannic acid) was incorporated by hydrogen bonding to the CNF network, forming an antioxidant DN-TA hydrogel system. To evaluate whether attenuating aerobic metabolism could preserve chondrocyte phenotype, P3 (passage 3) chondrocytes were cultured within the hydrogel scaffold <i>in vitro</i> and then implanted subcutaneously into nude mice. The DN-TA hydrogel effectively preserved the chondrocyte phenotype by activating HIF-1 signaling pathway and reducing ROS (reactive oxygen species). Furthermore, after 8/12 weeks of subcutaneous implantation, the DN-TA scaffold significantly enhanced <i>in vivo</i> cartilage regeneration, as evidenced by increased extracellular matrix deposition and more mature cartilage formation. Collectively, our study demonstrates that reducing aerobic metabolism helps stabilize the chondrocyte phenotype and promotes functional cartilage regeneration. These findings offer novel insights for optimizing cartilage tissue engineering strategies through metabolic modulation.