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Introduction:Hydrogels are biomaterials that possess appropriate physicochemical and mechanical properties enabling the formation of a three-dimensional, stable structure used in tissue engineering, providing an environment conducive to the survival and proliferation of cells. The cytotoxicity test is performed to assess the overall toxicity of developed materials. Evaluation of the cytotoxicity of the tested materials is quantified by the MTT test. This test uses the fact that the dehydrogenase found in living cells reduces the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazoline bromide, known as MTT reagent, to purple formazan. Enzymes from the dehydrogenases group perform the reaction in which the tetrazole ring opens. Thus the cytotoxicity effect is based on the assumption that only living cells reduce MTT to formazan. Biocompatibility tests of the gelatin-alginate hydrogels cross-linked covalently by squaric acid – SQ, dialdehyde starch – DAS, and ionically by calcium chloride – CaCl2 (5%, 2,5%, 1%) treatment were tested because of their significance for regenerative medicine applications. Extracts cytotoxicity was tested on Mouse embryonic fibroblasts - 3T3 and Keratinocytes - KerCT 4048 cell lines.
Methodology:The experiment consisted of 6% gelatin and 2% sodium alginate based hydrogels, DMEM / F12K medium containing 10% FBS, and 1% mixture of antibiotics (identical to the 2D culture medium), and the addition of cross-linkers. Appropriate amounts of polymers and cross-linker were dissolved in the medium, then 30,000 cells / 1.5 ml of final gel were added, and the finished mixtures were poured, onto 6-well plates to form a thin layer. The gels were left for a few minutes to stabilize the shape of the gels and then cross-linked with a CaCl2 solution at room temperature. After 10 min, gels were washed with PBS, and 2 ml of culture medium was added, to each well. The MTT test was performed based on ISO 10993 standard.
Results:The cell viability was reduced after 24h of incubation. An unfavourable effect of a high concentration of Ca2+ ions on cell survival was observed, especially in the case of the KerCT 4048 cell culture. The survival of 3T3 cells grown in an extract of chemically non-cross-linked and cross-linked hydrogel oscillates around 70%. Reducing the concentration of CaCl2 had a positive effect on 3T3 cells. These cells exposed to extracts from the base samples G6_A2_CaCl2 1%, and 2.5% revealed stable and high survivability comparable with control. The addition of chemical cross-linking agents only slightly affected the viability of 3T3 cells. The KerCT 4048 exposed to extracts from G6_A2_SQ1 CaCl2 1% presented the highest viability for this cell line.
Conclusion:Based on the conducted research, gels containing 2% sodium alginate and 6% gelatin presented promising results for future medical applications. Lowering the concentration of CaCl2 solution from 5% to 1% reduced the toxicity of the final material. All significant values in the MTT test remained above the 70% viability established by the ISO 10993 standard. Therefore, all tested gelatin-alginate hydrogels can be treated as biocompatible.
Acknowledgments:Grant NCBiR no:TECHMATSTRATEG2/407770/2/NCBR/2020
References:1.Tyliszczak, B. et al., J Polym Res. 24,153 (2017)
2.Rodríguez‐Rodríguez, R. et al., J Biomed Mater Res A. 108,81-93 (2020)
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