The Effect of Wound Dressings on a Bio-Engineered Human Dermo-Epidermal Skin Substitute in a Rat Model.

Autologous bio-engineered dermo-epidermal skin substitutes are a promising treatment for large skin defects such as burns. For their successful clinical application, the graft dressing must protect and support the keratinocyte layer and, in many cases, possess antimicrobial properties. However, silver in many antimicrobial dressings may inhibit keratinocyte growth and differentiation. The purpose of our study was to evaluate the effect of various wound dressings on the healing of a human hydrogel-based dermo-epidermal skin substitute in preparation for the first-in-human clinical trials. Human dermo-epidermal skin substitutes approved for clinical trials were produced under good manufacturing practice conditions, transplanted onto immuno-incompetent rats, and dressed with either Vaseline Gauze™ (Kendall Medtronic, Minneapolis, USA), Suprathel (PolyMedics Innovations GmbH, Denkendorf, Germany), Urgotul SSD (Urgo Medical, Shepshed, United Kingdom), Mepilex AG (Mölnlycke Health Care, Gothenburg, Sweden), or Acticoat™ (Smith&Nephew, Baar, Switzerland). Grafts were assessed clinically for take, epithelialization, and infection at 10 and 21 days post-transplantation, and histologically at 21 days. There were three subjects each in the Vaseline Gauze™ and Suprathel groups, and four subjects each in the Urgotul SSD, Mepilex AG, and Acticoat™ groups. For all samples, the take rate was 100% and the expected keratinocyte number, epithelialization and epidermal stratification were observed. All of the dressings in the current study were well tolerated by our human dermo-epidermal skin substitute. The tolerance of the silver-based dressings is particularly relevant given the high risk of bacterial contamination with large skin defects, and provides pivotal information as we embark on clinical trials for this novel skin substitute.

Evaluation of cultured human dermal- and dermo-epidermal substitutes focusing on extracellular matrix components: Comparison of protein and RNA analysis.

Treatment of full-thickness skin defects with split-thickness skin grafts is generally associated with contraction and scar formation and cellular skin substitutes have been developed to improve skin regeneration. The evaluation of cultured skin substitutes is generally based on qualitative parameters focusing on histology. In this study we focused on quantitative evaluation to provide a template for comparison of human bio-engineered skin substitutes between clinical and/or research centers, and to supplement histological data. We focused on extracellular matrix proteins since these components play an important role in skin regeneration. As a model we analyzed the human dermal substitute denovoDerm and the dermo-epidermal skin substitute denovoSkin. The quantification of the extracellular matrix proteins type III collagen and laminin 5 in tissue homogenates using western blotting analysis and ELISA was not successful. The same was true for assaying lysyl oxidase, an enzyme involved in crosslinking of matrix molecules. As an alternative, gene expression levels were measured using qPCR. Various RNA isolation procedures were probed. The gene expression profile for specific dermal and epidermal genes could be measured reliably and reproducibly. Differences caused by changes in the cell culture conditions could easily be detected. The number of cells in the skin substitutes was measured using the PicoGreen dsDNA assay, which was found highly quantitative and reproducible. The (dis) advantages of assays used for quantitative evaluation of skin substitutes are discussed.

MAL overexpression leads to disturbed expression of genes that influence cytoskeletal organization and differentiation of Schwann cells.

In the developing peripheral nervous system, a coordinated reciprocal signaling between Schwann cells and axons is crucial for accurate myelination. The myelin and lymphocyte protein MAL is a component of lipid rafts that is important for targeting proteins and lipids to distinct domains. MAL overexpression impedes peripheral myelinogenesis, which is evident by a delayed onset of myelination and reduced expression of the myelin protein zero (Mpz/P0) and the low-affinity neurotrophin receptor p75(NTR). This study shows that MAL overexpression leads to a significant reduction of Mpz and p75(NTR) expression in primary mouse Schwann cell cultures, which was already evident before differentiation, implicating an effect of MAL in early Schwann cell development. Their transcription was robustly reduced, despite normal expression of essential transcription factors and receptors. Further, the cAMP response element-binding protein (CREB) and phosphoinositide 3-kinase signaling pathways important for Schwann cell differentiation were correctly induced, highlighting that other so far unknown rate limiting factors do exist. We identified novel genes expressed by Schwann cells in a MAL-dependent manner in vivo and in vitro. A number of those, including S100a4, RhoU and Krt23, are implicated in cytoskeletal organization and plasma membrane dynamics. We showed that S100a4 is predominantly expressed by nonmyelinating Schwann cells, whereas RhoU was localized within myelin membranes, and Krt23 was detected in nonmyelinating as well as in myelinating Schwann cells. Their differential expression during early peripheral nerve development further underlines their possible role in influencing Schwann cell differentiation and myelination.