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

描述(由申请人提供)：组织工程学有可能彻底改变疑难问题的临床治疗，如病变的主动脉瓣或关节软骨的修复。不幸的是，目前的组织工程构建仍然缺乏功能所需的强度、灵活性和长期耐用性。此外，构建体和宿主组织的整合是一个重要的问题。尽管已有大量关于固定标本和组织学标本的分子和纤维微结构的数据，但我们对活组织工程培养中细胞形态的实时变化以及细胞外基质的沉积和重组的了解仍然存在着显著的差距。这在一定程度上是由于缺乏对新鲜标本和活组织结构进行非破坏性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样本中详细的胶原纤维结构，而不需要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/细胞结构图像和活细胞凝胶制剂的时间推移图像(此处列出)，推动了新提出的实验和分析工具的开发，以量化三维纤维尺度的组织结构-功能行为。该方案的目标是将这些新的实时成像方法应用于在受控加载条件下培养的一系列细胞/支架组织工程构建物的分析。广泛的影响：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，我们打算为各种组织和组织工程结构提供前所未有的组织/胶原三维结构和活细胞-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随时间变化的情况，对于研究人员和植入物设计者开发用于疾病和受伤软组织的“下一代”组织工程修复方案至关重要。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、假设和交付成果摘要：具体目标I(1年级)：调整和应用我们新的非破坏性3-D椭圆偏振光成像和测试系统，利用电纺组织工程构建物的‘实时’延时3-D成像分析培养中的组织构建物。用现有的共聚焦显微镜图像验证我们的结果。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，将在统计上类似于静态共焦图像。交付件I：能够进行活细胞/细胞外基质成像的经过验证的无损实时系统。特定目标II(第2年)：在不同的受控拉伸-屈曲实验下，对细胞-ECM和电纺组织工程构建物的‘活体’时间推移变化进行成像和分析。假设II：3-D细胞和胶原基质组装/重塑的形态，将根据支架的性质和施加的拉伸/弯曲载荷而变化。交付II：细胞在不同加载条件下为一系列组织工程支架组装胶原基质的实时实时图像(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.