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

Abstract

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.