The proposed studies will test the hypothesis that a full-thickness oral mucosal equivalent (FTOME) can be produced in vitro that has physical and biochemical characteristics similar to oral mucosa in vivo. The FTOME will be composed of two components; a collagenous dermal substratum and a stratified squamous epithelial surface.. The FTOME will be evaluated to determine the optimal culture conditions, the expression of molecular markers of epithelial differentiation and the expression of transplantation antigens. FTOME prepared in vitro will be evaluated in vivo in a nude mouse xenograft model to determine whether these conditions alter either the pattern of epithelial differentiation or antigenicity. Canine FTOME will be produced in vitro and transplanted to intraoral sites to determine the suitability jof the material as a graft alternative and the longterm outcome of its use intraorally. Procedures will also be developed to permit longterm cryopreservation of tissue- typed FTOME> IN the present study we will continue to develop the procedures necessary to produce a FTOME in vitro that has the characteristics of oral mucosa and to examine the features of this material after transplantation in vivo. We will define the growth parameters of human oral epithelial cells in vitro to determine the time required to produce adequate quantities of FTOME. The pattern of expression of markers of epithelial differentiation, keratin, filaggrin and involucrin will be examined in FTOME both in vitro and in vivo. The development of basement membrane specific elements, laminin and collagen Type IV will be examined both immunohistochemically and by electron microscopy. The expression of transplantation antigens will be characterized for FTOME in vitro, in vivo and after cryopreservation. The longterm survival of transplanted FTOME epithelium will be examined by detecting the presence of DNA markers which were transfected into the cells in culture. The ingrowth of connective tissue elements in the DE portion will be examined by characterizing the synthesis of Type I and Tjype III collagne and the numbers of vimention positive fibroblasts. The procedures for cryopreservation of tissue-typed FTOME will be developed and the viability and retention of markers of differentiation and transplantation antigens examined. These characterizations of FTOME will precede the use of the material in human patients to replace oral mucosa, and no human trials are included in this proposal.
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