Biocompatibility of gelatins A and B after sterilization by different methods

Introduction: The reliable sterilization of gelatin-based hydrogels is of critical importance for the clinical application of a multitude of biomanufactured functional constructs for cardiac surgery. However, the sterilization methods employed can have deleterious effects on the properties of natural hydrogels, which are utilised as vascular sealants, bioinks in three-dimensional bioprinting and scaffolds in tissue engineering. Objective: The aim of the study was to investigate the impact of sterilization methods on the biocompatibility and structural organization of gelatins A and B. Methods: Gelatins A and B were sterilized by ultraviolet irradiation, autoclaving and ethylene oxide treating. After sterilization, the following indicators were assessed: the cytotoxicity of the gelatin hydrogels, proliferation and adhesion of endothelial EA.hy926 cells, as well as the expression level of adhesion molecules on the cell surface. Raman scattering and infrared absorption spectroscopy were used to evaluate the structure of the hydrogels; the denaturation process of the gelatins was examined by differential scanning calorimetry. Results: The studied sterilization methods did not induce the cytotoxicity of gelatins A and B. Sterilization of gelatin B1 by ultraviolet irradiation and ethylene oxide treating caused a decrease in the proliferation level of endothelial EA.hy926 cells after 168 hours of cultivation. All types of sterilization, except of ultraviolet irradiation of gelatins A1 and B1, caused a decrease in endothelial cell adhesion at the early stages compared to the control. The augmentation of the number of adherent cells on all gelatins compared to the control after 168 hours of cultivation was stipulated by increased expression of adhesion molecules on endothelial cells. The types of sterilization studied did not cause significant chemical changes in the molecules of gelatine A and B. However, autoclave sterilization reduced the denaturation transition temperature in gelatines of all types. Conclusion: Sterilizations by ultraviolet irradiation and ethylene oxide treatment did not significantly affect the chemical structure and biocompatibility of gelatin A1 but reduced the biocompatibility of other sterilized gelatin types. Sterilization of gelatins A and B by autoclaving was accompanied by a change in the conformational state of the sterilized gelatins. Received 30 October 2024. Revised 29 November 2024. Accepted 2 December 2024. FundingThis research was funded by the state assignment of the Ministry of Health of the Russian Federation No. 124022500251-0, state assignment of the Russian Federation No. FWNR-2025-0016, No. FWNG-2024-0023. Conflict of interestThe authors declare no conflict of interest. Contribution of the authorsConception and study design: I.I. Kim, M.A. Surovtseva, N.A. Bondarenko, E.V. Chepeleva, N.V. Surovtsev, I.Yu. Zhuravleva, O.V. PoveshchenkoData collection and analysis: I.I. Kim, M.A. Surovtseva, N.A. Bondarenko, E.V. Chepeleva, I.V. Zaytseva, Yu.V. Zaytseva, N.V. SurovtsevStatistical analysis: I.I. Kim, M.A. Surovtseva, N.A. Bondarenko, E.V. Chepeleva, I.V. Zaytseva, Yu.V. Zaytseva, N.V. SurovtsevDrafting the article: I.I. Kim, M.A. Surovtseva, N.V. SurovtsevCritical revision of the article: I.I. Kim, M.A. Surovtseva, O.V. PoveshchenkoFinal approval of the version to be published: I.I. Kim, M.A. Surovtseva, I.Yu. Zhuravleva, N.A. Bondarenko, E.V. Chepeleva, I.V. Zaytseva, Yu.V. Zaytseva, N.V. Surovtsev, O.V. Poveshchenko