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

描述（由申请人提供）：组织工程有可能彻底改变临床治疗难题，如修复病变主动脉瓣或关节软骨。不幸的是，目前的组织工程结构仍然缺乏功能所需的强度、柔韧性和长期耐久性。此外，构建体与宿主组织的整合也是一个重要的问题。虽然有很多关于固定和组织学制备标本的分子和纤维微观结构的数据，但我们对活组织工程培养中实时细胞形态变化以及细胞外基质沉积和重组的了解仍然存在重大差距。这部分是由于缺乏对新鲜标本和活体组织结构的胶原蛋白结构进行无损成像和实时分析的工具。最近，我们开发了一种新的成像技术，使用椭圆偏振光显微镜来显示新鲜组织和培养标本中详细的胶原纤维结构，而无需破坏性的组织学程序。初步结果提供了令人震惊的原位胶原/细胞结构的新图像和活细胞凝胶制剂的延时图像（如图所示），推动了新的实验和定量三维纤维尺度组织结构-功能行为的分析工具的开发。本提案的目标是应用这些新的实时成像方法来分析在受控负载条件下培养的一系列细胞/支架组织工程构建物。广泛影响：利用我们的成像和分析方法以及最新可用的数字成像系统，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3d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控制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“下一代”组织工程修复病变和损伤软组织的选择至关重要。这里开发的工具也可以在理解胶原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，假设和交付成果总结：具体目标I（一年级）：适应并应用我们新的无损三维椭圆偏振光成像和测试系统，以“实时”延时三维成像的静电纺组织工程结构来分析培养中的组织结构。验证我们的结果对现有的共聚焦显微镜图像。假设一：二维和三维的细胞和矩阵形态，包括体积和表面拓扑参数，在统计上与静态共聚焦图像相似。可交付成果1：一个经过验证的非破坏性实时系统，能够进行活细胞/ECM成像。特定目标II（二年级）：在不同控制的张力弯曲实验下，细胞- ecm和电纺组织工程构建物的“实时”延时变化的成像和分析。假设二：三维细胞和胶原基质组装/重塑形态，将根据支架性能和施加的张力/弯曲载荷而变化。交付II：在不同的加载条件下，为一系列组织工程支架组装胶原基质的细胞的实时图像（视频）。还将使用共聚焦显微镜进行额外的验证和分析。

DESCRIPTION (provided by applicant): 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.