Measurement of DNA biomarkers for the safety of tissue-engineered medical products, using artificial skin as a model.
To test the hypothesis that the process of tissue engineering introduces genetic damage to tissue-engineered medical products, we employed the use of five state-of-the-art measurement technologies to measure a series of DNA biomarkers in commercially available tissue-engineered skin as a model. DNA was extracted from the skin and compared with DNA from cultured human neonatal control cells (dermal fibroblasts and epidermal keratinocytes) and adult human fibroblasts from a 55-year-old donor and a 96-year-old donor. To determine whether tissue engineering caused oxidative DNA damage, gas chromatography/isotope-dilution mass spectrometry and liquid chromatography/isotope-dilution mass spectrometry were used to measure six oxidatively modified DNA bases as biomarkers. Normal endogenous levels of the modified DNA biomarkers were not elevated in tissue-engineered skin when compared with control cells. Next, denaturing high-performance liquid chromatography and capillary electrophoresis-single strand conformation polymorphism were used to measure genetic mutations. Specifically, the TP53 tumor suppressor gene was screened for mutations, because it is the most commonly mutated gene in skin cancer. The tissue-engineered skin was found to be free of TP53 mutations at the level of sensitivity of these measurement technologies. Lastly, fluorescence in situ hybridization was employed to measure the loss of Y chromosome, which is associated with excessive cell passage and aging. Loss of Y chromosome was not detected in the tissue-engineered skin and cultured neonatal cells used as controls. In this study, we have demonstrated that tissue engineering (for TestSkin II) does not introduce genetic damage above the limits of detection of the state-of-the-art technologies used. This work explores the standard for measuring genetic damage that could be introduced during production of novel tissue-engineered products. More importantly, this exploratory work addresses technological considerations that need to be addressed in order to expedite accurate and useful international reference standards for the emerging tissue-engineering industry.
Rodriguez
Henry
H
Chemical Science and Technology Laboratory, National Institute of Standards and Technology, Gaithersburg, MD 20899, USA. henry.rodriguez@nist.gov
O'Connell
Catherine
C
Barker
Peter E
PE
Atha
Donald H
DH
Jaruga
Pawel
P
Birincioglu
Mustafa
M
Marino
Michael
M
McAndrew
Patricia
P
Dizdaroglu
Miral
M
D015415:Biomarkers / Q000032:analysis; D004249:DNA Damage / Q000235:genetics; D004252:DNA Mutational Analysis / Q000379:methods*; D019544:Equipment Failure Analysis / Q000379:methods; D004869:Equipment Safety / Q000379:methods*; D020869:Gene Expression Profiling / Q000379:methods; D005819:Genetic Markers / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D011785:Quality Assurance, Health Care / Q000379:methods; D012867:Skin / Q000378:metabolism*; D017768:Skin, Artificial / Q000009:adverse effects*; D023822:Tissue Engineering / Q000295:instrumentation* / Q000379:methods*; D016159:Tumor Suppressor Protein p53 / Q000032:analysis / Q000235:genetics* / Q000378:metabolism
D015415:Biomarkers; D005819:Genetic Markers; D016159:Tumor Suppressor Protein p53