Biomarkers Used to Detect Genetic Damage in Tissue Engineered Skin
In this study, tissue-engineered skin (TestSkin II) was obtained, separated into its two cellular layers (epidermis and dermis) and DNA was extracted. The first biomarker tested consisted of screening for DNA point mutations in the p53 gene, the most commonly mutated gene in skin cancer. To ensure the accuracy of the results, two measurement technologies that incorporate internal calibration standards were used. It was shown that tissue-engineered skin did not contain mutations in this gene at the level of sensitivity of capillary electrophoresis-SSCP and Denaturing High Performance Liquid Chromatography. Results were compared to control cells (neonatal fibroblasts and neonatal keratinocytes) and fibroblasts that were obtained from a 55 year-old and 96 year-old human donor. The second set of biomarkers tested looked at the loss of the Y-chromosome. Using Fluorescent In Situ Hybridization technology, Y chromosome loss was examined in the tissue-engineered skin, normal control lymphocytes, and neonatal and aged (55 and 96 year-old) donor control cells. Y-chromosome loss was only detected in the fibroblasts from the 96 year-old donor. Biomarkers such as p53 mutations and chromosome loss can provide the basis for an international reference standard of cellular biomarkers to aid in the development and safety of tissue engineered medical products.
KeywordsChromosome Loss Denature High Performance Liquid Chromatography International Reference Standard Internal Calibration Standard Neonatal Fibroblast
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