Evaluation of cultured human dermal- and dermo-epidermal substitutes focusing on extracellular matrix components: Comparison of protein and RNA analysis
Authors
Corien Oostendorp, Sarah Meyer, Monia Sobrio , Joyce van Arendonk, Ernst Reichmann, Willeke F. Daamen, Toin H. van Kuppevelt
Institution
Radboud University Medical Center
Country
Netherlands
Year
2017
Journal
ScienceDirect
Abstract
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 usingwestern blotting analysis andELISAwas not successful. Thesamewas 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.