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Öğe Biomarkers used to detect genetic damage in tissue engineered skin(Springer Science and Business Media Deutschland GmbH, 2003) O'Connell C.; Barker P.E.; Marino M.; McAndrew P.; Atha D.H.; Jaruga P.; Birincio?lu M.[No abstract available]Öğe A comparative study of biomarkers of oxidative DNA damage used to detect free radical damage in tissue-engineered skin(ASTM International, 2004) Rodriguez H.; Jaruga P.; Birincioglu M.; Barker P.E.; O'Connell C.; Dizdaroglu M.The process of tissue engineering often involves the mixing of cells with polymers that may elevate the level of endogenous free radicals and thus cause genetic damage to the cell. In order to assure that such composite materials are free of genetic damage, our laboratory is responding to the need for test methods used to assess the safety and performance of tissue-engineered materials. Specifically, we are identifying cellular biomarkers that could be used to ensure that the cells of tissue-engineered materials have not undergone any oxidative DNA damage from the production of free radicals by oxidative stress during the development, storage or shipment of the product. Using the technique of gas chromatography/mass spectrometry, we have screened for the oxidatively modified DNA base 8-hydroxyguanine in tissue-engineered skin and compared the levels to those in control cells, neonatal fibroblasts and neonatal keratinocytes. No significant level of damage was detected compared to control cells. The technique of liquid chromatography/mass spectrometry was also used in the validation of this biomarker by measuring its nucleoside form. The results obtained with this technique were nearly identical to those obtained with gas chromatography/mass spectrometry. Biomarker programs such as this can provide the basis for an international reference standard of cellular biomarkers that can aid in the development and safety of tissue-engineered medical products.Öğe Molecular biomarkers used to detect cellular/genetic damage in tissue-engineered skin(ASTM International, 2004) O'Connell C.; Barker P.E.; Marino M.; McAndrew P.; Atha D.H.; Jaruga P.; Birincioglu M.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 exons 5 and 6 of the TP53 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 SSCP-capillary electrophoresis 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, no detectable loss of Y-chromosome was found in the tissue-engineered skin and neonatal control cells. Y-chromosome loss was found in the fibroblasts from the 96-year-old donor. Biomarkers such as TP53 mutations and chromosome loss can provide the basis for an international reference standard of cellular biomarkers that can aid in the development and safety of tissue engineered medical products.Öğe Oxidative DNA damage biomarkers used in tissue engineered skin(Springer Science and Business Media Deutschland GmbH, 2003) Rodriguez H.; Jaruga P.; Birincio?lu M.; Barker P.E.; O'Connell C.; Dizdaro?lu M.[No abstract available]
