Biological Characteristics Of Human Oral Mucosal Epithelial Cell And Preliminary Study On Tissue Engineered Mucosa

In the oral and maxillofacial surgery, the covering of intraoral epithelial defects is a recurrent issue. Splint thickness skin grafts or mucosal grafts are usually used to reconstruct defect. There has some disadvantages using these methods, including scarcity of donor site, keratinization and hair growth in transplanted skin, donor site morbidity. Tissue engineering offers exciting possibilities for meeting the requirements of oral cavity reconstruction. The study was to investigate human oral mucosal keratinocyte biological charateristics and efficient culture procedure, and reconstitute skin defects in nude mice with tissue engineered mucosa. Part I Study on seeds of tissue engineered mucosa 1.Biological characteristics and the culture of human oral mucosal epithelial cell culture in serum free medium To study the procedure for the culture and amplification of oral mucosal epithelial cells (OMEC) and their biological characteristics. The biological characteristics were observed through phase microscope, cell growth curve, colony forming efficiency. The effect of two kinds of separating methods was compared through cell counting, trypan blue staining. The third generation of OMES maitained a good growth power in K-SFM, and OMEC were induced to differentiate when serum existed in medium. Compared with trypsin group, Dispase group harvested more viable cells and reached confluence earlier. K-SFM demonstrates superior primary and subculture cell growth. A large quality of cells could be gained within a short time using this method. 2.The factors affected primary culture of human oral mucosal epithellal cells To explore an efficient procedure for primary culture of human oral mucosal epithelial cells in serum free keratinocyte medium. Several methods to control microbiological infection were used, and the effects of different enzymes on keratinocytes in the process of separating epithelium from dermis and digesting single cell from epithelium were evaluated. The rate of contamination was reduced 5 dramatically by the storage of the specimen in medium containing a high concentration of antibiotics for 3 days in 4t or incubating the specimen in hibitane solution. At the same time the proportion of vial epithelial cell was not affected. Dispase and Trypsin/EDTA obtained a good effect on separating epithelium from dermis and digesting single cells from epithelium. The high successful rate in primary culture of human oral epithelial cells could be obtained according to above optimal procedure. 3. Effect of retinoic acid on differentiation of human oral mucosal keratinocyte in vitro The aim of this study was to investigate RA effects on oral keratinocyte differentiation induced by high calcium concentration. Oral keratinocytes were cultured in serum free keratinocyte medium containing high calcium (1.2mM) and variable concentration of RA was added in above medium. argyrophilic nucleolar organiser region-associated proteins (AgNORs) in cultured human oral keratinocytes under differential culture conditions were stained with silver staining. Keratinocyte proliferative ability were assessed by comparing the area of intranuclear AgNORs of keratinocytes in diferent groups through image analysis system. The expression of bax in keratinocyte in different groups was also observed by immunohistology. The results showed that the concentration of RA ranging 107M to 1 06M resisted the terminal differentiation caus...