Jiajia Lu, Sheng Wang, Rui Wu, Xiaojian Shi, Demeng Xia, Nan Lu, Lei Zhu
Abstract This research focuses on the development and application of a novel enzyme‐responsive hydrogel, isoquercitrin‐loaded MMP‐responsive PEG–peptide hydrogel (PEG‐pp‐Iso), for osteochondral defect repair. The hydrogel is synthesized by conjugating isoquercitrin to poly (ethylene glycol) (PEG) and is further enhanced with hydroxyapatite (HAp) to facilitate cartilage and subchondral bone regeneration. To understand the molecular mechanisms behind its regenerative potential, transcriptomic analysis, including Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment, least absolute shrinkage and selection operator (LASSO) regression, and support vector machine‐recursive feature elimination (SVM‐RFE) algorithms, reveals that the CXCL12/CXCR4‐CDC42 signaling axis plays a crucial role in regulating bone marrow mesenchymal stem cells (BMSCs) migration, chondrogenic differentiation, and immune modulation. In vitro, PEG‐pp‐Iso effectively enhances the antioxidant activity, proliferation, migration, and chondrogenic differentiation of BMSCs, as evidenced by increased SOX9 and COL II expression. The inhibition of the CXCL12/CXCR4‐CDC42 axis significantly reduces these effects, confirming its importance in promoting cartilage repair. In vivo, PEG‐pp‐Iso hydrogels promote the migration of BMSCs to osteochondral defect sites, enhances cartilage matrix formation, and improve immune microenvironment regulation by increasing anti‐inflammatory macrophages and reducing pro‐inflammatory ones. These results highlight the potential of PEG‐pp‐Iso in promoting osteochondral regeneration and offer a novel approach to osteochondral defect repair through modulation of immune responses and stem cell activity.