Nondestructive Real Time 3-D Imaging and Analysis of cell-ECM in Live Tissue Engi

描述（由申请人提供）：标题：活培养物和组织工程构建物的非破坏性3-D成像PI：托德德林项目摘要组织工程有可能彻底改变临床治疗难题，如修复患病的主动脉瓣或关节软骨。不幸的是，目前的组织工程化构造仍然缺乏功能所需的强度、柔性和长期耐久性。此外，构建体与宿主组织的整合也是一个重要的问题。虽然固定和组织学制备的标本的分子和纤维状微观结构的许多数据是可用的，一个显着的差距仍然在我们的知识的实时细胞形态变化，和沉积和重组的细胞外基质活组织工程文化。这部分是由于缺乏对新鲜标本和活组织构造中的胶原结构进行无损成像和真实的实时分析的工具。最近，我们已经开发出一种新的成像技术，使用椭圆偏振光显微镜，以揭示详细的胶原纤维结构在新鲜组织和培养标本，而不需要破坏性的组织学程序。初步结果提供了引人注目的原位胶原/细胞结构的新图像和活细胞凝胶制剂的延时图像（在这里展示），激发了新提出的实验和开发用于量化3-D纤维尺度组织结构-功能行为的分析工具。本提案的目标是应用这些新的实时成像方法分析一系列的细胞/支架组织工程结构下培养的受控负载条件。广泛影响：利用我们的成像和分析方法以及新的数字成像系统，我们打算为广泛的组织和组织工程构建提供前所未有的组织/胶原蛋白3-D结构和活细胞-基质相互作用的实时可视化和分析。详细了解纤维尺度组织结构和受控载荷下的时间依赖性变化对于研究人员和植入物设计者开发用于患病和受伤软组织的“下一代”组织工程修复选择至关重要。这里开发的工具也可以在理解胶原蛋白组装和细胞介导的重塑/再生的时间序列方面产生广泛的影响。具体目标，假设和可行性的总结：具体目标I（第1年）：适应和应用我们新的无损3-D椭圆偏振光成像和测试系统，以分析培养中的组织结构，并对电纺组织工程结构进行“实时”延时3-D成像。将结果与现有的共聚焦显微镜图像进行比较。假设一：2-D和3-D细胞和基质形态，包括体积和表面拓扑参数，将在统计学上类似于静态共聚焦图像。可验证I：能够进行活细胞/ECM成像的经验证的无损实时系统。特定目标II（2年）：在不同的受控张力-屈曲实验下，细胞-ECM和电纺组织工程构建物的“实时”时间推移变化的成像和分析。假设二：3-D细胞和胶原基质组装/重塑形态将根据支架特性和施加的张力/弯曲载荷而变化。表II：在不同的负载条件下，细胞为一系列组织工程支架组装胶原基质的实时图像（视频）。还将使用共聚焦显微镜进行额外的验证和分析。 公共卫生相关性：组织工程有可能彻底改变临床治疗的难题，如修复病变的主动脉瓣或关节软骨。不幸的是，目前的结构没有足够的生物力学性能或纤维结构。在培养期间监测组织细胞-ECM结构是具有挑战性的。缺乏用于对新鲜标本和活组织构造中的胶原结构进行非破坏性成像和实时分析的工具。最近，我们已经开发出一种新的成像技术，使用椭圆偏振光显微镜，以揭示详细的胶原纤维结构在新鲜组织和培养标本，而不需要破坏性的组织学程序。本提案的目标是应用这些新的实时成像方法分析一系列的细胞/支架组织工程结构下培养的受控负载条件。高分辨率，长时间的延时图像和分析的胶原蛋白组装和纤维化的组织工程构建体可能是非常有价值的开发新的方法，用于改善组织工程构建体的生物力学和生物化学特性，以及改善理解细胞-ECM相互作用。

DESCRIPTION (provided by applicant): Title: Nondestructive 3-D imaging of Live Cultures and Tissue Engineered Constructs PI: Todd Doehring Project Abstract Tissue engineering has the potential to revolutionize the clinical treatment of difficult problems such as the repair of diseased aortic valves or articular cartilage. Unfortunately, current tissue engineered constructs still lack the strength, flexibility, and long term durability required for function. In addition, the integration of construct and host tissue is a significant problem. Although much data on the molecular and fibrillar microstructures of fixed and histologically prepared specimens are available, a significant gap remains in our knowledge of the real-time cell morphology changes, and deposition and reorganization of extracellular matrix in live tissue engineered cultures. This is due in part to a lack of tools for non-destructive imaging and real- time analysis of collagen structure in fresh specimens and live tissue constructs. Recently, we have developed a new imaging technique that uses elliptically polarized light microscopy to reveal detailed collagen fiber structures in fresh tissues and cultured specimens, without the need for destructive histological procedures. Preliminary results have provided striking new images of in-situ collagen/cell structure and time-lapse images of live cell-gel preparations (presented here), motivating the new proposed experiments and development of analytical tools for quantifying 3-D fiber-scale tissue structure-function behavior. The goal of this proposal is to apply these novel real-time imaging methods for analyses of a series of cell/scaffold tissue engineered constructs cultured under controlled loading conditions. Broad Impact: Leveraging our imaging and analysis methods and newly available digital imaging systems we intend to provide unprecedented real-time visualizations and analyses of tissue/collagen 3-D structure and live cell-matrix interactions for a wide range of tissues and tissue engineered constructs. Detailed understanding of fiber scale tissue structure and time- dependent changes under controlled loads is critical for researchers and implant designers developing "next generation" tissue engineered repair options for diseased and injured soft tissues. The tools developed here could also have wide impact in understanding the temporal sequence of collagen assembly and cell mediated remodeling/regeneration. Summary of Specific Aims, Hypotheses, and Deliverables: Specific Aim I (Year 1): Adapt and apply our new nondestructive 3-D elliptically polarized light imaging and testing system to analyses of tissue constructs in culture with 'live' time-lapse 3-D imaging of electrospun tissue engineered constructs. Validate our results against existing confocal microscope images. Hypothesis I: 2-D and 3-D cell and matrix morphologies, including volumetric and surface topology parameters, will be statistically similar to static confocal images. Deliverable I: A validated nondestructive real-time system capable of live cell/ECM imaging. Specific Aim II (Year 2): Imaging and analysis of 'live' time-lapse changes in cell-ECM and electrospun tissue engineered constructs under varying controlled tension-flexion experiments. Hypothesis II: 3-D cell and collagen matrix assembly/remodeling morphologies, will vary depending on the scaffold properties and applied tension/flexion loads. Deliverable II: Live, real-time images (video) of cells assembling collagen matrix for a series of tissue engineered scaffolds and during varying loading conditions. Additional validation and analyses using confocal microscopy will also be performed. PUBLIC HEALTH RELEVANCE: Project Narrative Tissue engineering has the potential to revolutionize the clinical treatment of difficult problems such as the repair of diseased aortic valves or articular cartilage. Unfortunately, current constructs do not have adequate biomechanical properties or fiber structure. Monitoring tissue cell-ECM structure during culture is challenging. There is a lack of tools for non-destructive imaging and real-time analysis of collagen structure in fresh specimens and live tissue constructs. Recently, we have developed a novel imaging technique that uses elliptically polarized light microscopy to reveal detailed collagen fiber structures in fresh tissues and cultured specimens, without the need for destructive histological procedures. The goal of this proposal is to apply these novel real-time imaging methods for analyses of a series of cell/scaffold tissue engineered constructs cultured under controlled loading conditions. High resolution, long term time-lapse images and analysis of collagen assembly and fibrogenesis in tissue engineered constructs could be highly valuable for development of new methods for improving tissue engineered construct biomechanical and biochemical properties, as well as an improved understanding of cell-ECM interactions.