Production of an acellular matrix from amniotic membrane for the synthesis of a human skin equivalent

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 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.

Characterization of decellularized ovine small intestine submucosal layer as extracellular matrix‐based scaffold for tissue engineering

“…Owing to the thin thickness of sheep SIS, lower concentrations of reagents could be used which help to avoid the longer exposure time that supposed to have negative impact on the quality of the scaffold. Low concentrations of detergents have been used in a few studies which were mainly related to tissues, such as cornea, the AF vertebral disc, vessels and heart valves, and amniotic membrane with a long 24 and 72 h exposure time. Histological and immunohistological (H&E and DAPI) staining revealed that protocols used for decellularization of OSIS have been effective and a significant percentage of the cells were eliminated compared to the control group.…”

Bovine Decellularized Amniotic Membrane: Extracellular Matrix as Scaffold for Mammalian Skin

“…Four protocols were used to decellularize the BAM (Table 1). A control sample was kept without frozen treatment at −20 • C. For protocols I, II, and III, the separation of two layers, fetal and maternal, was performed as it is reported in most of the investigations [52][53][54][55]. On the other hand, this procedure was not performed in protocol IV, in order to observe variations in the properties of the membrane regarding exposure with the chemical solutions used and in cell culture.…”

Progress in developing decellularized bioscaffolds for enhancing skin construction

“…104 Moreover, 1% trypsin-EDTA has been shown to result in complete elimination of epithelial and mesenchymal cells. 39 After decellularization, the acellular HAM retains all major structural components of the matrix and is biocompatible. There are no significant decreases in ultimate tensile strength, extensibility, and elasticity after decellularization.…”