Glycosaminoglycan-functionalized hydrogels for sustained delivery of tissue inhibitor of metalloproteinase-3 mediating matrix metalloprotease inhibition and extracellular matrix stabilization

Excessive protease activity and impaired tissue regeneration are hallmarks of many disease states. Elevated matrix metalloproteinase-9 (MMP-9) plays a key role in adverse tissue remodeling by excessively degrading extracellular matrix (ECM) components and growth factors. Tissue inhibitor of metalloproteinase-3 (TIMP-3) regulates ECM turnover, and its bioavailability is influenced by glycosaminoglycans (GAGs). This study aimed to develop a methacrylated gelatin (GelMA)-based hydrogel functionalized with acrylated sulfated hyaluronan (sHA_c) as a TIMP-3 delivery system to decrease ECM degradation under pathophysiological conditions. sHA_c incorporation enhanced hydrogel stiffness, reduced degradation rates and yielded sustained TIMP-3 release for up to 28 days. Molecular modeling and surface plasmon resonance demonstrated preferential binding of TIMP-3 to sHA_c over hyaluronan methacrylates, together providing a molecular rationale for the reduced and sustained release of TIMP-3 from sHA_c-containing hydrogels. Angiogenesis-related functional assays, supported by molecular modeling studies, indicate that sHA_c modulates the anti-angiogenic activity of TIMP-3 by altering vascular endothelial growth factor receptor-associated signaling, while preserving metalloproteinase inhibition. Released TIMP-3 from GelMA/sHA_c hydrogels retained bioactivity, effectively inhibiting MMP-9 activity and mitigating ECM degradation in-vitro and in human ex-vivo models. In a murine subcutaneous implantation model, sHA_c-functionalized TIMP-3-loaded hydrogels were associated with reduced inflammatory cell presence and altered vascular- and matrix-related tissue signatures compared with GelMA controls. These findings underscore the potential of sHA_c-functionalized GelMA hydrogels as biomaterials for therapeutics delivery, offering controlled TIMP-3 release and sustained bioactivity to promote ECM stability and on-demand MMP inhibition. This system represents a promising strategy for addressing the challenges of excessive MMP activity.