Non-invasive Assessment of Tissue Engineered Human Oral Mucosa

组织工程人体口腔粘膜的无创评估

• 批准号: 8074510
• 负责人: STEPHEN Elliott FEINBERG
• 金额: $ 79.25万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-25 至 2013-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8074510
• 关键词: Animal Model; Animals; Biological Assay; Biological Models; Burn injury; Cell Survival; Cells; Cellular Structures; Chemical Structure; Clinical Trials; Defensins; Development; Devices; Electrophoresis; Engraftment; Flavin-Adenine Dinucleotide; Fluorescence; Fluorescence Spectroscopy; Glucose; Graft Survival; Growth; Health; Human; Immunoassay; In Situ; In Vitro; Interleukin-8; Measures; Medical; Metabolic; Metabolism; Methods; Microfluidics; Molecular; Monitor; NADH; Nicotinamide adenine dinucleotide; Nutrient; Optics; Oral; Oral cavity; Oral mucous membrane structure; Organ Transplantation; Oxidation-Reduction; Performance; Perfusion; Phase; Process; Proteins; Punch Biopsy; Qualifying; Quality Control; Raman Spectrum Analysis; Request for Applications; Research Personnel; SCID Mice; Spectrum Analysis; Structure; System; Techniques; Technology; Testing; Time; Tissue Engineering; Tissue Model; Tissue Viability; Tissues; Vascular Endothelial Growth Factors; Wound Healing; antimicrobial; base; cell behavior; cytokine; efficacy testing; fluorophore; glucose metabolism; glucose uptake; graft function; improved; innovation; keratinocyte; manufacturing process; novel; public health relevance; response; tool

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.

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力、结构和代谢活性，以及​​（2）第四种工具来评估其在 SCID 小鼠中原位移植后的活力。作为我们的模型系统，我们将使用工程组织，体外生产的口腔粘膜等效物（EVPOME），这是一种为口腔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测试，并提供了独特的工程组织模型，可在体外制造 (mfg.) 过程中遵循。 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 的细胞成分的活力、结构、功能和代谢活动。其中一些工具还可用于原位评估 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 口腔角质形成细胞层的功能、结构/组成和活力：具体目标：（1）测试芯片上微流体在线电泳免疫测定的功效，以测定口腔的三种组成型分泌蛋白 对移植物功能和存活很重要的角质形成细胞：人 2-防御素 1 (hBD-1)（一种先天抗菌剂）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) 测试定量荧光光谱通过内源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) 的氧化还原状态检查 EVPOME 细胞成分代谢活性的功效。 (3) 测试拉曼光谱评估 EVPOME 构建体的化学结构和组成的功效。 (4) 使用具体目标 1-3 中开发的工具体外评估 EVPOME 构建体。 (5) 通过开发与荧光和拉曼光谱结合使用的反射光谱，在移植到 SCID 小鼠后原位评估 EVPOME 的组织灌注和血管分布。 与目前使用的测定系统（例如葡萄糖摄取）相比，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。实时发布标准的制定将改善工程组织在制造过程中的质量控制。移植到动物体内后，以非侵入方式原位评估组织活力的过程和能力。 公共卫生相关性声明（由申请人提供）：用于伤口修复、烧伤和其他医学治疗的体外制造的组织需要经过质量测试才能用于人体。目前的技术要么破坏了大部分制造的组织来进行这种测试，要么依赖于测量组织在准备过程中摄取的一种营养物质葡萄糖的使用情况。我们建议开发、测试和使用三种新方法来在用于医学治疗之前检查体外制造的组织。这些方法使用最先进的技术来测量新陈代谢、细胞结构和组织产生的分子，以确定制造的组织在放入体内之前的“健康状况”。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入体内后的成功生长。我们将在动物模型中测试这些方法。