Crosslinking strategies govern the morphology and biological performance of decellularized extracellular matrix particle–tyramine-modified hyaluronic acid hydrogels
Decellularized extracellular matrix (dECM) particle-based hydrogels hold promise for cartilage repair. Due to its chondropermissive properties, tuneable viscoelastic profile, and the potential to form covalent bonds with ECM proteins, tyramine-modified hyaluronic acid (THA) is an ideal candidate as a binder for dECM particles. However, the incorporation of the dECM particles into a viscoelastic hydrogel may interfere with its crosslinking. The goal of this study was to compare two crosslinking methods: (1) horseradish peroxidase/Eosin (HRP/Eo) with H2O2 and (2) sodium persulfate/ruthenium (SPS/Ru) for fabricating THA hydrogels containing dECM particles, and to investigate their biological and biomechanical effect for cartilage tissue engineering. Incorporation of dECM markedly enhanced the mechanical properties of the composites in both crosslinking methods, increasing the storage modulus by 10-fold, while leaving the linear viscoelastic region unchanged. Chondrocyte-laden dECM hydrogels demonstrated sustained cytocompatibility over 14 days, evidenced by stable DNA content throughout the culture period. Compared with SPS/Ru, hydrogels showed more effective incorporation of dECM particles into the HRP/Eo-crosslinked network, as indicated by a lower I∼1630/I∼1030 ratio from FTIR analysis, clear morphological changes in confocal microscopy, higher glycosaminoglycan (GAG) and collagen retention, as well as slower GAG and collagen release. HRP/Eo-dECM hydrogels promoted stronger gene expression of type II collagen (COL2) and higher compressive modulus compared with SPS/Ru-dECM hydrogels. In summary, HRP/Eo crosslinked dECM hydrogels demonstrated improved mechanical performance and chondrocyte redifferentiation effect compared to SPS/Ru, attributed to the difference in hydrogel morphology. These findings identify the HRP/Eo crosslinking method as a favorable strategy for engineering dECM particle–THA hydrogels for cartilage regeneration.