Development of a tissue-engineered skin substitute on a base of human amniotic membrane

Abstract

Allogenic graft material and tissue engineering have recently shown promising results for the improvement of both esthetic and functional outcomes in the treatment of large skin defects. We chose human amniotic membrane as a cellular scaffold in order to develop a skin substitute for later in vivo uses. Various methods of de-epithelialization of the human amniotic membrane were evaluated by histological analysis including hematoxylin–eosin and laminin staining, optic coherence tomography, and scanning electron microscopy with 0.25/0.02% trypsin/ethylenediaminetetraacetic acid treatment and mechanical cell removal showing an almost complete loss of the epithelium and a mainly intact basement membrane. Novel examination of human amniotic membrane by optic coherence tomography was feasible, but difficulties were experienced in handling and interpretation of the tissue as no comparable data exist. Subsequently, we developed an air–liquid interface cell culture to cultivate keratinocytes and fibroblasts on the de-epithelialized human amniotic membrane. We achieved a mostly keratinized surface on the epidermal side with a confluent fibroblast network on the chorion side.

Nanofibrous Scaffolds for Skin Tissue Engineering and Wound Healing Based on Synthetic Polymers

“…At the air-liquid interface, the keratinocytes or cells differentiating towards keratinocytes have been cultured on various substrates, e.g., acellular dermis [133], porous sponge-like gelatin scaffolds incorporated with chrondroitin-6-sulfate and hyaluronic acid [134], de-epithelialized human amniotic membrane [135], collagen IV and fibronectin [132] and fibrin in the form of layer [131], hydrogel or clot [136]. On the mentioned substrates, keratinocytes were grown either alone or in combination with fibroblasts submerged in the culture medium.…”

“…In a study by Wang et al [134], fibroblasts were grown inside the porous gelatin-based scaffolds submerged in the medium, while keratinocytes were grown on the top of the scaffolds, exposed to the air-liquid interface. Similarly, on the de-epithelialized amniotic membrane, fibroblasts were cultured on the lower side of the membrane, submerged in the culture medium, while keratinocytes were grown on the upper side at the air-liquid interface [135]. In a study by Keck et al [136], even a three-layered skin substitute was created.…”

Acellular Amniotic Membrane Patch Regeneration for Burn Injury: The Future for Wound Healing

“…Human amniotic membrane is composed of 5 layers containing various types of cells, but the three are basal, mesenchymal and epithelial and basal layer critical to the healing process because it is rich in type IV collagen and laminin, cell-signaling proteins (cytokines), and growth factors which are important components during the inflammatory process the wound. The epithelial layer is basically composed of cuboidal epithelium that supports the membrane and the mesenchymal layer which is composed of connective tissue and is not vascularized [11].…”

“…It can then be used as a structure for recellularization. The decellularization process may involve physical and chemical methods such as deformation, pressurization, agitation, sonication or freezing-defrosting cycles; chemical methods such as acid, base, alcohol or exposure to detergents, chelation treatments or use of osmotic stress; or enzymatic methods [3][4][5][6][7][8][9][10][11].…”

“…Acellular amniotic membrane (AM) patch has been used in various surgeries, because it contains essential proangiogenic growth factors, including fibroblast growth factor-2 (FGF-2) and vascular endothelial growth factor (VEGF) that also promote endogenous mechanisms of repair and regeneration, such as angiogenesis or vasculogenesis [4][5][6][7][8][9][10][11][12][13][14][15][16][17][18].…”