EX VIVO DEVELOPMENT OF TRANSFECTED ORAL MUCOSAL GRAFTS

点击翻译按钮获取中文摘要

Our long-term goal is to produce a "smart" oral mucosal graft, ex vivo, that will be used for reconstruction of major oral defects that are seen secondary to oncologic resection, traumatic events or developmental disturbances. At present, oral mucosa is in limited supply for use in major reconstructive procedures. Other alternatives such as skin introduce disadvantages, such as the presence of adnexal structures and a different pattern of keratinization that can lead to a compromise in oral function. This proposal plans to harvest human oral keratinocytes from keratinized mucosa, expand them in vitro, and seed them onto a cellular, non- immunogenic dermal equivalent, AllodDerm/TM (already approved for human use), to produce a mucosal equivalent with similar anatomic and handling properties as native oral mucosa. The ex vivo produced oral mucosal composite (EVPOMC) will be developed in an environment free of serum and transformed irradiated feeder cells so that it can be used for grafting back into autogenous human recipients. The oral keratinocytes of the EVPOMC will be transfected with a plasmid, using a novel approach to alter cell membrane viscosity, to cause them to over secrete vascular endothelial growth factor (VEGF), thus making this a "smart" oral mucosal grafting device. We will evaluate vascular ingrowth into the EVPOMC, by histology and immunohistochemistry, to assess the functionality of the inserted plasmid in the device. The safety and efficacy of the device, which is necessary for FDA approval, will be tested in SCID mice and minipigs. Our specific aims are: 1. Characterize the ex vivo produced human oral mucosal composite. 2. Define optimal parameters for transfection of oral keratinocytes with a cDNA plasmid for VEGF. 3. Develop animal models to a) demonstrate the safety of the transfected oral mucosal device (SCID mouse), and b) show that the transfected oral mucosal device can be grafted successfully to an intra-oral site (minipig). The transfection and grafting of oral mucosal device, in this investigation, will allow us to gather the necessary preclinical data, to use for submission to regulatory agencies, for the translation of this technology into early Phase I human clinical trials.