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Production of an artificial skin substitute using tissue engineering principles to be used for toxicological tests in vitro
The EU launched in 2009 an effort to ban animal-tested cosmetics. Since then many models have emerged with the aim of replacing the use of animals in toxicological and cosmetic tests. Several have been pre-validated for this purpose, but they are limited in predicting whether or not a particular molecule is toxic. Brazil follows the same trend; however, we do not have a national in vitro skin substitute technology for those tests. Chitosan, polyhydroxybutyrate (PHB) and gelatin are polymers widely used in tissue engineering due to their biodegradation, biocompatibility and non-toxicity properties.
Objetivos - Metodologia - Resultados - Discussão dos Resultados/Objectives - Methodology - Results - Discussion of Results/Objetivos - Metodología - Resultados - Discusión de los resultados
We set out to produce a bilayer skin substitute - composed of a chitosan and gelatin membrane and a 3D matrix of PHB and chitosan, colonized with human keratinocytes (hKC) and fibroblasts (hFb), respectively - to be used in toxicological tests in vitro. Membranes were produced from the mixture of two 1.5% chitosan and gelatin solutions 2: 1 and left to dry. They were treated with alcohol and glycerin, washed, and then treated with water. The 3D matrix was obtained from the mixture of a 2.5% chitosan and PHB solution. The obtained solution was frozen at -20°C and lyophilized for 24h, followed by crosslinking with 0.005% glutaraldehyde and another lyophilization with the membrane. Subsequently, the materials were cut and sterilized at 15 kGy and used in physical, chemical and biological tests.
We observed that a bilayer structure was formed, consisting of a smooth surface and porous under layer, with pores of about 100 to 500 micrometers. The absorption rate showed that within 15 minutes the bilayers increased their weight by 1000% and thus remained for the rest of the analysis. We also treated the structure with a solution of lipase and lysozyme and after 8 weeks only 20% of the material remained. We tested then the viability of the composite by MTT assay, and we observed a similar viability between the control group and the membrane layer of the bilayer when cultured with keratinocytes. The same was seen when fibroblasts were grown on the porous structure of the bilayer. The viability of those cells was confirmed later by MEV analysis that showed adherence and growth of these primary cells on both layers.
Considerações Finais/Final considerations/Consideraciones finales
We conclude then that the bilayer structure constructed by our group consists of a skin substitute that pending further analysis can be used as an in vitro test platform for chemicals.
Palavras-chave/Key words/Palabras clave
Chitosan, PHB, gelatin, tissue engineering, skin engineering, bilayer skin, human keratinocytes, human fibroblasts.
JOANA LOBATO BARBOSA, ALFREDO MIRANDA DE GOES, DAWIDSON ASSIS GOMES