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

活组织工程中细胞 ECM 的无损实时 3D 成像和分析

• 批准号: 7776563
• 负责人: Todd Doehring
• 金额: $ 7.68万
• 依托单位: DREXEL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-01-01 至 2011-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7776563
• 关键词: 3-Dimensional; Actins; Affect; Behavior; Biochemical; Biomechanics; Cells; Cellular Morphology; Cellular Structures; Clinical Treatment; Collagen; Collagen Fiber; Computers; Confocal Microscopy; D Cells; Data; Deposition; Development; Disease; Employee Strikes; Extracellular Matrix; Fiber; Fresh Tissue; Gel; Goals; Histologic; Image; Image Analysis; Imagery; Imaging Techniques; Implant; In Situ; Incubators; Knowledge; Life; Mediating; Methods; Microscope; Molecular; Monitor; Morphology; Motor; Natural regeneration; Polarization Microscopy; Preparation; Procedures; Property; Real-Time Systems; Regimen; Research Personnel; Resolution; Series; Specimen; Structure; Surface; System; Technology; Testing; Three-Dimensional Imaging; Time; Tissue Engineering; Tissues; Validation; abstracting; analytical tool; aortic valve; articular cartilage; cell assembly; cellular imaging; digital imaging; fibrogenesis; flexibility; imaging modality; improved; injured; insight; next generation; novel; polarized light; polydimethylsiloxane; prototype; public health relevance; repaired; research study; sample fixation; scaffold; soft tissue; three dimensional structure; time use; tissue culture; tool

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.

描述（由申请人提供）： 标题：活体培养物和组织工程结构的无损 3D 成像 PI：Todd Doehring 项目 摘要 组织工程有可能彻底改变诸如修复患病主动脉瓣或关节软骨等难题的临床治疗。不幸的是，当前的组织工程结构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。尽管有大量关于固定和组织学制备的样本的分子和纤维微结构的数据，但我们对实时细胞形态变化以及活体组织工程培养物中细胞外基质的沉积和重组的了解仍然存在重大差距。部分原因是缺乏对新鲜标本和活体组织结构中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揭示新鲜组织和培养标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/细胞结构新图像和活细胞凝胶制剂的延时图像（此处介绍），激发了新提出的实验和开发用于量化 3D 纤维尺度组织结构功能行为的分析工具。该提案的目标是应用这些新颖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/支架组织工程构建体。广泛影响：利用我们的成像和分析方法以及新推出的数字成像系统，我们打算为各种组织和组织工程构建体提供前所未有的组织/胶原 3D 结构和活细胞-基质相互作用的实时可视化和分析。详细了解受控负载下的纤维尺度组织结构和时间依赖性变化对于研究人员和植入物设计者为患病和受伤的软组织开发“下一代”组织工程修复方案至关重要。这里开发的工具还可能对理解胶原蛋白组装和细胞介导的重塑/再生的时间顺序产生广泛影响。具体目标、假设和可交付成果摘要： 具体目标 I（第一年）：调整并应用我们新的无损 3-D 椭圆偏振光成像和测试系统，通过对电纺组织工程构建体进行“实时”延时 3-D 成像来分析培养物中的组织构建体。根据现有的共焦显微镜图像验证我们的结果。假设 I：2-D 和 3-D 细胞和矩阵形态（包括体积和表面拓扑参数）在统计上与静态共焦图像相似。可交付成果 I：经过验证的非破坏性实时系统，能够进行活细胞/ECM 成像。具体目标 II（第二年）：在不同的受控张力-弯曲实验下，对细胞 ECM 和电纺组织工程结构的“实时”延时变化进行成像和分析。假设 II：3-D 细胞和胶原蛋白基质组装/重塑形态将根据支架特性和施加的张力/弯曲载荷而变化。可交付成果 II：细胞在不同负载条件下为一系列组织工程支架组装胶原基质的实时图像（视频）。还将使用共焦显微镜进行额外的验证和分析。 公共健康相关性：项目叙述组织工程有可能彻底改变疑难问题的临床治疗，例如修复患病主动脉瓣或关节软骨。不幸的是，当前的构造不具有足够的生物力学特性或纤维结构。在培养过程中监测组织细胞-ECM 结构具有挑战性。缺乏对新鲜标本和活组织结构中的胶原结构进行无损成像和实时分析的工具。最近，我们开发了一种新颖的成像技术，使用椭圆偏振光显微镜来揭示新鲜组织和培养标本中详细的胶原纤维结构，而不需要破坏性的组织学程序。该提案的目标是应用这些新颖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/支架组织工程构建体。组织工程结构中胶原蛋白组装和纤维发生的高分辨率、长期延时图像和分析对于开发改善组织工程结构生物力学和生化特性的新方法以及增进对细胞-ECM相互作用的理解非常有价值。