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EPIGINGIVAL™ (GIN-100) TISSUE MODELS FOR ORAL IRRITATION STUDIES.
Breyfogle1,B., Kubilus1, J., Dale2, M., Wertz3, P.
1MatTek Corporation, Ashland, MA, USA, 2University of Washington, Seattle, WA, USA, 3University of Iowa, Iowa City, IA, USA.
Presented at 5th World Congress, Berlin, Germany, August, (2005).
Keywords: Air/Liquid interface (ALI), Buccal, Cytokeratin K13, Cytokeratin K14, EpiGingival (GIN-100), EpiOral (ORL-200), Gingival, High performance thin layer chromatography (HPTLC), Human epithelia, Irritation testing, MTT assay, Normal human oral keratinocytes (NHOK), Oral irritation, Surfactant
Endpoints: Immunohistochemistry, Lipid analysis, MTT tissue viability assay, Transepithelial electrical resistance (TEER)
Materials Tested: Triton X-100 (1%)
Summary: Three-dimensional models of the human oral epithelia, exhibiting a buccal or gingival phenotype have been developed using normal human oral epithelial cells cultured in serum free medium. The buccal tissue (ORL-200) contains 8-12 cell layers with cells becoming increasingly squamous toward the apical surface. No evidence of cornification is present in histological slides and immuno-staining shows the expression of cytokeratin K13 human beta-defensins in the suprabasal layers. These features are characteristic of buccal epithelium. The gingival tissue (GIN-100) has 9-13 layers of viable, nucleated cells and is partially cornified at the apical surface. Lipid analysis of ORL-200 revealed that, of the ceramides important in the barrier of epidermis, only ceramide 2 in was present, a result that matches human buccal tissue. GIN-100 showed the presence of the three least polar ceramides, C1, C2, C3, in a ratio of 1: 8.2 : 4.5, respectively. When exposed to the surfactant Triton X-100 (1%), an exposure time of 52 ±20 minutes (n=31) reduces the viability of ORL-200 to 50% as determined by an MTT assay. For GIN-100, an exposure of >8 hours is required to damage the tissue to the same extent. In addition, MTT assay and cytokine release results from ORL-200 tissue have been used to provide a quick, reproducible method for evaluating the irritation potential of oral care excipients and oral care products. The methodology correlates well to human irritation results, and can provide a reliable alternative to animal testing.
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