Non-invasive Assessment of Tissue Engineered Human Oral Mucosa

描述（由申请人提供）： 我们建议（1）开发和测试三种非侵入性工具，以评估体外活力，结构和代谢活性的工程组织在真实的时间和（2）第四个评估其生存能力后，原位移植在SCID小鼠。作为我们的模型系统，我们将使用一种工程组织，离体产生的口腔粘膜等效物（EVPOME），一种为口内移植手术开发的组织工程人类口腔粘膜。EVPOME已在I期人体临床试验中成功进行了测试，并提供了一种独特的工程组织模型，可在其制造过程中进行跟踪。体外过程。 假设：可以开发非侵入性工具并用于体外真实的评估工程组织EVPOME的细胞组分的活力、结构、功能和代谢活性。这些工具中的几种也可用于原位评估EVPOME植入后的组织性能。 我们将通过开发三种非侵入性功能和分析测定/方法或工具来测试我们的假设，以评估EVPOME的口腔角质形成细胞层的功能、结构/组成和活力：具体目的：（1）测试芯片上的微流体在线电泳免疫测定的功效，以测定在移植物功能和存活中重要的口腔角质形成细胞的三种组成型分泌蛋白：人2-防御素1（hBD-1）（一种先天性抗微生物剂）和促血管生成细胞因子血管内皮生长因子（VEGF）和白细胞介素8（IL-8）。(2)通过内源性荧光团黄素腺嘌呤二核苷酸（FAD）和烟酰胺腺嘌呤二核苷酸（NADH）的氧化还原状态，测试定量荧光光谱法检查EVPOME细胞组分代谢活性的有效性。(3)测试拉曼光谱法评估EVPOME结构的化学结构和组成的有效性。(4)使用特定目标1-3中开发的工具在体外评估EVPOME构建体。(5)通过开发反射光谱法，结合荧光和拉曼光谱法，评估移植到SCID小鼠体内后EVPOME的原位组织灌注和血管分布。 我们的创新工具将用于以更全面的方式建立工程组织的释放标准，即，活力、结构和代谢活性，与目前使用的测定系统相比，如葡萄糖摄取。真实的释放标准的发展将改善工程组织在释放过程中的质量控制。过程和能力，以评估组织活力原位，非侵入性，移植到动物。 公共卫生相关性声明（申请人提供）：体外制造的用于伤口修复、烧伤和其他医学治疗的组织在用于人体之前需要经过质量检测。目前的技术要么破坏了大部分用于该测试的制造组织，要么取决于测量一种营养物葡萄糖的使用，该营养物是组织在准备时摄取的。我们建议开发、测试和使用三种新方法，在用于医学治疗之前检查体外组织。这些方法使用最先进的技术来测量新陈代谢、细胞结构和由组织制造的分子，以在被放入体内之前确定所制造的组织的“健康”。当使用这些新方法时，制造的组织的任何部分都不会被破坏。此外，其中两种方法可用于跟踪制造的组织放置在体内后的成功生长。我们将在动物模型中测试这些方法。

DESCRIPTION (provided by applicant): We propose to (1) develop and test three non-invasive Tools to assess the in vitro viability, structure, and metabolic activity of an engineered tissue in real time and (2) a fourth to assess its viability after grafting in situ in SCID mice. As our model system we will use an engineered tissue, Ex Vivo Produced Oral Mucosal Equivalent (EVPOME), a tissue engineered human oral mucosa developed for intraoral grafting procedures. The EVPOME has been successfully tested in a Phase I human clinical trial and offers a unique engineered tissue model that can be followed during its manufacturing (mfg.) process in vitro. HYPOTHESIS: Non-invasive Tools can be developed and used to assess viability, structure, function and metabolic activity of the cellular component of an engineered tissue, EVPOME, in real time in vitro. Several of these Tools can also be used to assess, in situ, tissue performance of the EVPOME after its engraftment. We will test our hypothesis by developing three non-invasive functional and analytical assays/methods or Tools to assess function, structure/composition and viability of the oral keratinocyte layer(s) of the EVPOME: SPECIFIC AIMS: (1) Test the efficacy of a microfluidic on-line electrophoresis immunoassay on a chip to assay three constitutively secreted proteins of oral keratinocytes important in graft function and survival: human 2-defensin 1 (hBD-1), (an innate antimicrobial), and pro-angiogenic cytokines vascular endothelial growth factor (VEGF) and interleukin 8 (IL-8). (2) Test the efficacy of Quantitative Fluorescence Spectroscopy to examine the metabolic activity of the cellular component of the EVPOME via the redox state of endogenous fluorophores flavin adenine dinucleotide (FAD) and nicotinamide adenine dinucleotide (NADH). (3) Test the efficacy of Raman Spectroscopy to assess the chemical structure and composition of the EVPOME construct. (4) To assess EVPOME constructs in vitro using Tools developed in Specific Aims 1-3. (5) To assess tissue perfusion and vascularity of the EVPOME, in situ, after grafting into SCID mice, by developing Reflectance Spectroscopy, to be used in conjunction with Fluorescence and Raman Spectroscopy. Our innovative Tools will be used to establish release criteria of an engineered tissue in a more comprehensive fashion, i.e., viability, structure and metabolic activity, compared with presently used assay systems, such as glucose uptake. The development of release criteria in real time will improve the quality control of engineered tissues during the mfg. process and the ability to assess tissue viability in situ, non- invasively, after grafting into animals. Public Health Relevance Statement (provided by applicant): Tissue made outside the body for use in wound repair, burn and other medical therapies needs to go through quality testing before it can be used in human beings. The present day technology either destroys a large part of the manufactured tissue for this testing, or depends on measuring the use of one nutrient, glucose, which the tissue ingests while it is being prepared. We propose to develop, test and use three new methods for checking tissues made outside the body before use in medical therapies. These methods use state of the art technologies to measure metabolism, cell structure and molecules made by the tissue to determine the "health" of the manufactured tissue before being put into the body. No part of the manufactured tissue will be destroyed when using these new methods. In addition, two of the methods can be used to follow the successful growth of the manufactured tissue after it is placed in the body. We will test the methods in an animal model.