Sequential gelation of tyramine-substituted hyaluronic acid hydrogels enhances mechanical integrity and cell...

Tyramine-substituted hyaluronic acid (HA-Tyr) hydrogels formed by the oxidative coupling reaction of hydrogen peroxide (H₂O₂) and horseradish peroxidase (HRP) have been used for cellular encapsulation and protein delivery. Crosslinking density and gelation time can be tuned by altering the H₂O₂ and HRP concentrations. Previous studies using HA-Tyr constructs report significant mechanical degradation after 21 days of culture. In this work, exogenous supplementation of HRP after initial gelation resulted in superior mechanical properties in acellular hydrogels and improved viability and proliferation in cell-laden constructs. Swelling of the acellular hydrogels was prevented in the samples receiving exogenous HRP. Monolayer studies showed no negative effect of relevant HRP concentrations on the viability of human adipose-derived stem cells (hASCs) and improved the viability of hASCs cultured with HRP and H₂O₂ compared to H₂O₂ alone. Taken together, this study demonstrates that HA-Tyr hydrogel properties could be modified by exogenous supplementation of HRP to tune scaffold degradation and improve cell viability by mitigating the negative effects of oxidative stress.