Oostendorp Corien, Meyer Sarah, Sobrio Monia, van Arendonk Joyce, Reichmann Ernst, Daamen Willeke F, van Kuppevelt Toin H
Department of Biochemistry, Radboud Institute for Molecular Life Sciences, Radboud University Medical Center, Geert Grooteplein 28, 6525 GA, Nijmegen, The Netherlands.
Tissue Biology Research Unit, Department of Surgery, Zurich University Children's Hospital, August Forel Strasse 7, 8008 Zurich, Switzerland.
Burns. 2017 May;43(3):520-530. doi: 10.1016/j.burns.2016.10.002. Epub 2016 Dec 29.
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.
用断层皮片治疗全层皮肤缺损通常会导致收缩和瘢痕形成，因此人们开发了细胞皮肤替代物来促进皮肤再生。对培养的皮肤替代物的评估通常基于关注组织学的定性参数。在本研究中，我们专注于定量评估，以为临床和/或研究中心之间比较人类生物工程皮肤替代物提供一个模板，并补充组织学数据。我们关注细胞外基质蛋白，因为这些成分在皮肤再生中起重要作用。作为模型，我们分析了人真皮替代物denovoDerm和真皮-表皮皮肤替代物denovoSkin。使用蛋白质免疫印迹分析和酶联免疫吸附测定法对组织匀浆中的细胞外基质蛋白III型胶原蛋白和层粘连蛋白5进行定量分析未成功。测定参与基质分子交联的赖氨酰氧化酶也是如此。作为替代方法，使用定量聚合酶链反应测量基因表达水平。探索了各种RNA分离程序。可以可靠且可重复地测量特定真皮和表皮基因的基因表达谱。细胞培养条件变化引起的差异很容易被检测到。使用PicoGreen双链DNA测定法测量皮肤替代物中的细胞数量，发现该方法具有高度的定量性和可重复性。讨论了用于皮肤替代物定量评估的测定方法的（优）缺点。
