Cell transplantation for nucleus pulposus (NP) regeneration represents a promising strategy for intervertebral disc degeneration (IVDD). Nonetheless, the hostile microenvironment within the degenerated intervertebral discs, characterized by redox imbalance and elevated mechanical pressure, poses risks of low cell survival and inadequate cell colonization for efficient NP regeneration. To address these challenges, we developed a biomimetic, esterase-responsive composite hydrogel microsphere (GHKM) for cell delivery, consisting of gelatin methacrylate (GelMA) mixed with HAMA-KGN, a conjugate of hyaluronic acid methacrylate (HAMA) and the small heterocyclic molecule kartogenin (KGN) via ester bonds. GHKM mimic the NP extracellular matrix (ECM), providing essential adhesion and mechanical support for cell proliferation, while facilitating cellular adaptation to the adverse microenvironment through the esterase-responsive release of KGN. Furthermore, GHKM exhibit favorable biocompatibility and promote or protect ECM synthesis by nucleus pulposus cells (NPCs) under both normal and inflammatory conditions. Transcriptomic sequencing analysis indicates a correlation between enhanced ECM synthesis and enrichment of antioxidant-related pathways. Subsequent cellular biological studies reveal that GHKM can also reduce reactive oxygen species production within the inflammatory milieu. The underlying mechanism of its protective effect on matrix metabolism may involve the activation of nuclear factor erythroid 2-related factor 2 (NRF2) and the upregulation of downstream antioxidant enzymes. In vivo implantation of NPCs-laden GHKM into rat tail nuclectomy models for 4 and 8 weeks preserved disc height, structure, and biological function, with histological analysis confirming NP regeneration. These findings present GHKM as a promising, synergistic transplantation strategy for NP regeneration in IVDD. STATEMENT OF SIGNIFICANCE: This study introduces an esterase-responsive gelatin methacrylate/hyaluronic acid methacrylate-kartogenin composite hydrogel microsphere (GHKM) system, aimed at mimicing the extracellular matrix (ECM) of the nucleus pulposus (NP) to address the pressing challenge of intervertebral disc degeneration (IVDD). These microspheres offer an innovative solution for cell transplantation therapy by simultaneously addressing two critical barriers: the harsh microenvironment of the degenerated disc and the need for sustained therapeutic effects. GHKM provide mechanical support, enhance cell survival, and adapt dynamically to adverse conditions through esterase-responsive release of kartogenin (KGN), a multifunctional molecule with chondrogenic, anti-inflammatory, and antioxidative properties. This study will not only interest researchers focused on regenerative medicine and biomaterials but also inspire new directions for tackling complex degenerative diseases.
