EX VIVO DEVELOPMENT OF TRANSFECTED ORAL MUCOSAL GRAFTS

我们的长期目标是生产一种“智能”的口腔黏膜移植物，体外，将用于重建主要的口腔缺损，被认为是继发于肿瘤切除，创伤事件或发育障碍。目前，口腔粘膜在主要重建手术中的使用是有限的。其他的替代品，如皮肤，会带来缺点，如附件结构的存在和不同的角化模式，这可能导致口腔功能的损害。这项计划计划从角化的口腔粘膜中获取人类口腔角质形成细胞，在体外进行扩增，并将它们种植到细胞、非免疫原性真皮仿真皮上(已被批准用于人类使用)，以生产出与天然口腔粘膜具有相似解剖和操控特性的粘膜仿真皮。体外制备的口腔黏膜复合材料(EVPOMC)将在无血清和转化的辐照饲养层细胞的环境中开发，以便用于移植回自体人类受体。EVPOMC的口腔角质形成细胞将被导入一种新的方法来改变细胞膜的粘度，使其过度分泌血管内皮生长因子(VEGF)，从而使其成为一种“智能”的口腔粘膜移植装置。我们将通过组织学和免疫组织化学的方法评估血管在EVPOMC中的生长，以评估插入到装置中的质粒的功能。该设备的安全性和有效性是FDA批准所必需的，将在SCID小鼠和小型猪身上进行测试。我们的具体目标是：1.对体外制备的人口腔黏膜复合材料进行表征。2.确定血管内皮生长因子基因表达载体转染口腔角质形成细胞的最佳条件。3.建立动物模型，a)证明转基因口腔黏膜装置(SCID小鼠)的安全性，b)证明转基因口腔黏膜装置可以成功地移植到口腔内部位(小型猪)。在这项研究中，口腔粘膜装置的转基因和移植将使我们能够收集必要的临床前数据，用于提交给监管机构，用于将这项技术转化为早期的人类临床试验。

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.