Processing methods for human amniotic membrane as scaffold for tissue engineering with mesenchymal stromal human cells

Tissue engineering is an interdisciplinary field that applies the principles of engineering and life sciences toward the development of biological substitutes that restore, maintain, or improve tissue function. The aims of this work were to compare chemically and physically processed human Amniotic Membranes (hAM) and analyze the cytocompatibility and proliferation rate (PR) of two primary human mesenchymal stromal cell lines, from different sources and donor conditions seeded over these scaffolds. The evaluated hAM processes were: cold shock to obtain a frozen amniotic membrane (FEAM) with remaining dead epithelial cells, denudation of hAM with trypsin for 20/10 min (DEAM20/10) or treatment with sodium dodecyl sulfate to decellularized hAM (DAM). All samples were sterilized with gamma radiation. The selection of the treated hAM to then generate composites was performed by scanning and transmission electron microscopy and characterization by X-ray diffraction, selecting DEAM10 and FEAM as scaffolds for cell seeding. Two sources of primary human stromal cells were used, both developed by our researchers, human Dental Pulp Stem Cells (hDPSC) from living donors and human Mesenchymal Stromal Cells (hMSC) from bone marrow isolated from brain dead donors. This last line of cells conveys a novel source of human cells that, to our knowledge, have not been tested as part of this type of construct. We developed four in vitro constructs without cytotoxicity signs and with different PR depending on the scaffolds and cells. hDPSC and hMSC grew over both FEAM and DEAM10, but DEAM10 allowed higher PR.