Thioketal-functionalized gelatin methacryloyl hydrogel enables ROS-responsive dencichine delivery for osteoporotic...

Osteoporotic bone defects are difficult to repair because excessive oxidative stress, heightened osteoclast activity, and impaired osteogenic differentiation act concurrently within the defect microenvironment. Here, a reactive oxygen species (ROS)-responsive hydrogel was developed by conjugating thioketal dicarboxylic acid (TK-DA) to gelatin methacryloyl (GelMA) through amide coupling and physically encapsulating dencichine (Denc), yielding G-TK@Denc. Denc showed a concentration-dependent biological response in vitro, with 10 μM providing the most favorable balance between cytocompatibility, osteogenic stimulation, and inhibition of osteoclast differentiation and resorptive function. The G-TK@Denc hydrogel retained an interconnected porous architecture and underwent rapid photocrosslinking. Under H2O2-containing conditions, the hydrogel exhibited reduced mechanical integrity, accelerated degradation, and enhanced Denc release, consistent with ROS-mediated cleavage of thioketal linkages. G-TK@Denc extracts inhibited RANKL-induced osteoclast formation, disrupted F-actin sealing rings, and reduced osteoclast-associated gene and protein expression. The formulation also decreased intracellular ROS accumulation, restored superoxide dismutase and catalase activity, and was associated with an Nrf2/Sirt1-related antioxidant response. In parallel, G-TK@Denc enhanced alkaline phosphatase activity, matrix mineralization, and osteogenic marker expression in bone marrow mesenchymal stem cells. In an ovariectomized mouse femoral condyle defect model, G-TK@Denc increased new bone formation and improved trabecular microarchitecture. Transcriptomic analysis further suggested coordinated regulation of osteoclastogenic, antioxidant, and osteogenic programs. These findings support thioketal-functionalized GelMA as a local, microenvironment-responsive platform for Denc delivery and osteoporotic bone regeneration.