Impact of Sterilization Techniques on the Physicochemical and Biological Properties, and 3D Printability of Thermoresponsive Hydrogels

Abstract Three-dimensional bioprinting offers transformative potential for functional disease modeling and tissue engineering. These applications demand effective biomaterial sterilization to ensure reproducibility and clinical translation. This study evaluated pharmacopeia-standard disinfection and sterilization methods, including autoclaving, dry heat treatment (aerobic and anaerobic), ethanol washing (70%), UV irradiation, and filtration on three thermoresponsive hydrogels. The investigated hydrogels consist of Pluronic F127 (poloxamer 407) or two poly(2-oxazolines)/poly(2-oxazine) based block copolymers, namely poly(2-methyl-2-oxazoline)-b-poly(2-n-propyl-2-oxazine) (pMeOx-b-pnPrOzi), or poly(2-methyl-2-oxazoline)-b-poly(2-phenyl-2-oxazine)-b-poly(2-methyl-2-oxazoline) (pMeOx-b-pPheOzi-b-pMeOx). Results showed that ethanol washing and UV irradiation sterilized inefficiently, while dry heat treatment caused partial thermal degradation (all polymers), compromised 3D-printability, and increased cytotoxicity (Pluronic F127), likely due to the formation of oxidative degradation products. Filtration effectively sterilized both pMeOx-based block copolymers, while Pluronic F127 required a combined approach of autoclaving and triple filtration to achieve complete sterility. Overall, filtration and autoclaving emerged as the most suitable sterilization method, balancing chemical stability, mechanical integrity, printability, and cytocompatibility.