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来实时评估工程组织的体外活性、结构和代谢活性，以及(2)第四种工具来评估其在SCID小鼠体内原位移植后的活性。作为我们的模型系统，我们将使用一种工程组织，Ex Vivo Produced Oral Mucosal Evalent(EVPOME)，这是一种为口腔内移植程序开发的组织工程化人类口腔粘膜。EVPOME已在一期人体临床试验中成功测试，并提供了一种独特的工程化组织模型，可在其制造过程中遵循(MFG。)在体外过程中。假设：可以开发非侵入性工具，并用于在体外实时评估工程组织EVPOME的细胞成分的活性、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无创性功能和分析测试/方法或工具来验证我们的假设，以评估EVPOME的口腔角质形成细胞层(S)的功能、结构/组成和活性：特定目标：(1)测试芯片上微流控在线电泳法的有效性，以检测在移植功能和生存中至关重要的三种口腔角质形成细胞成分分泌蛋白：人2-防御素1(HBD-1)、(一种天然抗菌剂)和促血管生成细胞因子血管内皮生长因子(VEGF)和白介素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)的氧化还原状态检测定量荧光光谱检测EVPOME细胞成分代谢活性的有效性。(3)测试拉曼光谱对EVPOME构建物化学结构和组成的评估效果。(4)使用特定AIMS 1-3中开发的工具对EVPOME构建物进行体外评估。(5)通过发展反射光谱，结合荧光和拉曼光谱，原位评估移植到SCID小鼠体内的EVPOME的组织灌注量和血管密度。我们的创新工具将被用来以更全面的方式建立工程组织的释放标准，即活性、结构和代谢活性，与目前使用的分析系统(如葡萄糖摄取)相比。实时释放标准的发展将改善MFG过程中工程组织的质量控制。移植到动物体内后，在原位、非侵入性地评估组织活性的过程和能力。 公共卫生相关声明(申请人提供)：体外制造的用于伤口修复、烧伤和其他医疗治疗的组织需要经过质量检测，才能在人体上使用。目前的技术要么破坏了用于这种测试的大部分人造组织，要么依赖于测量一种营养物质的使用，葡萄糖是组织在准备过程中摄取的。我们建议开发、测试和使用三种新的方法来检查体外组织，然后再用于医疗治疗。这些方法使用最先进的技术来测量组织产生的新陈代谢、细胞结构和分子，以确定人造组织在进入人体之前的“健康状况”。使用这些新方法时，人造组织的任何部分都不会被破坏。此外，其中两种方法可以用来跟踪人造组织在放置到体内后的成功生长。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.