Development of an oral mucosa epithelium sheet using amniotic membrane

Objectives: Using the human amniotic membrane (AM), we developed a novel material for cultivated mucosa epithelium cell sheets for clinical applications (oral surgery, periodontal therapy, implant therapy and so on). The objective of this study was to use the AM as an extracellular matrix (ECM) for culturing oral mucosa epithelium cells. Methods: Oral mucosa epithelial cells were obtained from the buccal mucosa of four adult albino rabbits, and were cultivated for 2-3 weeks on an AM carrier with a 3T3 fibroblast co-culture with air-lifting. Samples of the cultivated epithelial cells were examined histomorphologically by hematoxylin and eosin staining, and the immunohistochemical distribution of several keratins (keratins 3, 4, 13, 1 and 10) was investigated by mouse monoclonal anti-keratin antibodies using an indirect fluorescent antibody method. Results: A confluent primary culture of oral epithelial cells was established on AM after 7 days. After 2-3 weeks in culture, the cultivated epithelial cells showed five to seven layers stratification and well-differentiated cells. Immunohistochemistry revealed that the cultivated epithelial cells expressed keratins 3, 4 and 13, but not keratins 1 or 10. The Samples appeared similar to in vivo native oral mucosa epithelium structures. Conclusion: The cultivation of oral mucosa epithelial cells using AM enable mucosa-like differentiation and sheet formation. AM is suitable for ECM since type-…¢ collagen and laminin are in the basement membrane, and for many kinds of growth factors (EGF, KGF, HGF etc.). Furthermore, AM has high biocompatibility, and anti-inflammatory, anti-infection actions. We conclude that this novel material will be a useful new biomaterial for dentistry.