Production of an acellular matrix from amniotic membrane for the synthesis of a human skin equivalent
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Production of an acellular matrix from amniotic membrane for the synthesis of a human skin equivalent Anahı´ Sanluis-Verdes • Ma Teresa Yebra-Pimentel Vilar • Juan Javier Garcı´a-Barreiro • Marta Garcı´a-Camba • Jacinto Sa´nchez Iba´n˜ez • Nieves Dome´nech • Ma Esther Rendal-Va´zquez
Received: 19 June 2014 / Accepted: 29 November 2014 Ó Springer Science+Business Media Dordrecht 2015
Abstract Human amniotic membrane (HAM) has useful properties as a dermal matrix substitute. The objective of our work was to obtain, using different enzymatic or chemical treatments to eliminate cells, a scaffold of acellular HAM for later use as a support for the development of a skin equivalent. The HAM was separated from the chorion, incubated and cryopreserved. The membrane underwent different enzymatic and chemical treatments to eliminate the cells. Fibroblasts and keratinocytes were separately obtained from skin biopsies of patients following a sequential double digestion with first collagenase and then trypsin–EDTA (T/E). A skin equivalent was then constructed by A. Sanluis-Verdes J. S. Iba´n˜ez M. E. Rendal-Va´zquez (&) Unidad de Criobiologı´a-Banco de Tejidos, Instituto de Investigacio´n Biome´dica de A Corun˜a (INIBIC), Complexo Hospitalario Universitario de A Corun˜a (CHUAC), Sergas, As Xubias, s/n, 15006 A Corun˜a, Spain e-mail: [email protected] A. Sanluis-Verdes e-mail: [email protected] J. S. Iba´n˜ez e-mail: [email protected] M. T. Yebra-Pimentel Vilar Servicio de Anatomı´a Patolo´gica, Instituto de Investigacio´n Biome´dica de A Corun˜a (INIBIC), Complexo Hospitalario Universitario de A Corun˜a (CHUAC), Sergas, As Xubias, s/n, 15006 A Corun˜a, Spain e-mail: [email protected]
seeding keratinocytes on the epithelial side and fibroblasts on the chorionic side of the decellularizated HAM. Histological, immunohistochemical, inmunofluorescent and molecular biology studies were performed. Treatment with 1 % T/E at 37 °C for 30 min totally removed epithelial and mesenchymal cells. The HAM thus treated proved to be a good matrix to support adherence of cells and allowed the achievement of an integral and intact scaffold for development of a skin equivalent, which could be useful as a skin substitute for clinical use. Keywords Skin equivalent Human amniotic membrane Keratinocytes Biomaterial Trypsin J. J. Garcı´a-Barreiro Unidad de Quemados, Instituto de Investigacio´n Biome´dica de A Corun˜a (INIBIC), Complexo Hospitalario Universitario de A Corun˜a (CHUAC), Sergas, As Xubias, s/n, 15006 A Corun˜a, Spain e-mail: [email protected] M. Garcı´a-Camba N. Dome´nech (&) Biobanco A Corun˜a, Instituto de Investigacio´n Biome´dica de A Corun˜a (INIBIC), Complexo Hospitalario Universitario de A Corun˜a (CHUAC), Sergas, As Xubias, s/n, 15006 A Corun˜a, Spain e-mail: [email protected] M. Garcı´a-Camba e-mail: [email protected]
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