Effect of Sterilization Methods on the Physicochemical Properties of Silk Fibroin Hydrogels

This study investigates the impact of clinically relevant sterilization methods, such as ethylene oxide (EtO), gamma (γ) irradiation, autoclaving, and hydrogen peroxide gas plasma, on the physical, structural, and functional properties of methacrylated silk fibroin hydrogels obtained by photo- and enzymatic crosslinking. EtO, γ irradiation, and hydrogen peroxide gas plasma were applied as terminal sterilization methods to the fabricated hydrogels, whereas autoclaving was performed on the SilMA precursor solution prior to hydrogel formation. Silk fibroin hydrogels at 5 and 7 wt.% concentrations were evaluated for transparency, rheological behavior, water content, secondary structure, chemical composition, thermal stability, microbial growth, and morphology after sterilization and during storage. EtO sterilization effectively maintained high optical transparency (>98%) but compromised the mechanical properties of the hydrogels. In contrast, γ-irradiation and autoclaving promoted greater β-sheet formation, resulting in increased mechanical stiffness and thermal stability but reduced transparency after autoclaving. Plasma sterilization proved unsuitable, as incomplete cycles related to the high-water content of hydrogels. Overall, the results delineate the influence of different sterilization strategies and identify approaches that best preserve or enhance the performance of silk hydrogels, supporting their clinical translation in tissue engineering.