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Cellular DNA Biomarkers for the Safety of Tissue-Engineered Medical Products Using Artificial Skin as a Model

Published

Author(s)

H Rodriguez, C D. O'Connell, Peter E. Barker, M. Dizdaroglu, Donald H. Atha, Pawel Jaruga, M Birincioglu, M A. Marino, P McAndrew

Abstract

Fundamental to the safety of many tissue-engineered medical products is how cells respond to a given polymer when implanted into the body. Assurance of cellular stability during the manufacturing, storage and shipment of such products is crucial to achieve optimal efficacy. It is therefore important to have cellular biomarkers that measure genetic damage that cells might undergo during tissue engineering. In this study, commercially available tissue-engineered skin (TestSkin II) was obtained and separated into its two cellular layers (epidermal layer consisting of human neonatal keratinocytes and the dermal layer consisting of human neonatal fibroblasts). DNA was extracted following cell layer separation or from material that was not separated into their respective cell layers, and compared to respective neonatal control cells (human neonatal dermal fibroblasts and human neonatal epidermal keratinocytes) and adult fibroblasts obtained from a 55-year-old and 96-year-old human donor. To determine if cells were exposed to inflammatory stress as a result of the manufacturing, storage or shipment of the product, five oxidatively modified DNA bases in cellular DNA were identified and quantified using gas chromatography/isotope-dilution mass spectrometry and liquid chromatography/isotope-dilution mass spectrometry. Endogenous levels of the five modified bases used as biomarkers were not elevated when compared to appropriate cell controls. Furthermore, the TP53 tumor suppressor gene was screened for mutations because TP53 is the most commonly mutated gene in skin cancer. Two measurement technologies that incorporate internal calibration standards were used to ensure the accuracy of the results. TestSkin II was free of TP53 mutations at the level of sensitivity of denaturing high-performance liquid chromatography and capillary electrophoresis-single strand confirmation polymorphism. Furthermore, loss of Y-chromosome, which is associated with excessive cell passage and aging, was investigated as another set of biomarkers. Using fluorescent in-situ hybridization technology, no detectable loss of Y-chromosome was found in the tissue-engineered skin and neonatal controls cells. In contrast, Y-chromosome loss was found in the control fibroblasts from the 96-year-old donor. Biomarkers such as those investigated in this work can provide the basis for an international reference standard of cellular biomarkers to aid in the development and safety of tissue-engineered medical products and to help streamline regulatory matters.
Citation
Journal of Molecular Biology

Keywords

biomarkers, DNA damage, safety, Tissue-Engineered Medical Products

Citation

Rodriguez, H. , O'Connell, C. , Barker, P. , Dizdaroglu, M. , Atha, D. , Jaruga, P. , Birincioglu, M. , Marino, M. and McAndrew, P. (2008), Cellular DNA Biomarkers for the Safety of Tissue-Engineered Medical Products Using Artificial Skin as a Model, Journal of Molecular Biology (Accessed April 24, 2024)
Created October 16, 2008