Elucidating the signal for contact guidance in aligned fibrils to improve tissue engineering

主要研究者：Tranquillo，Robert T.Proposal #：1606008接触引导，即细胞响应于地形特征（如平行凹槽）而定向和迁移的趋势，是许多生理过程和组织工程策略的基本细胞行为关键。本项目的目标是确定细胞在与纤维蛋白凝胶中对齐的原纤维相互作用时感受到的信号，并表现出接触指导。该项目将专门测试的假设，取向的原纤维网络的刚度和粘附力的方向依赖性是主要的贡献信号。这将通过仅系统地改变纤维蛋白网络的刚度方向组分并观察纤维蛋白凝胶中成纤维细胞的接触引导响应是否改变，然后用阻断抗体进行类似的研究以调节粘附，从而调节潜在的粘附方向依赖性信号来实现。 除了回答关于细胞在检测对齐的原纤维时感测到什么信号的问题的基本价值之外，该研究还应该通过为设计产生所需细胞对齐和相关组织机械功能的原纤维支架提供理论基础来改进工程组织。接触引导是多种生理过程和组织工程策略的基本细胞行为关键。虽然它已经在平面基质上进行了大量研究，甚至与某些几何形状的细胞骨架有关，但在对齐的纤维状网络（如天然组织或用作组织模型的重建胶原蛋白和纤维蛋白凝胶）中的研究很少。 尽管其重要性，在对齐的纤维状网络中的细胞接触引导的机制由于这样的网络呈现给细胞的多个相互依赖的信号，包括粘附的各向异性、孔隙率和刚度而难以阐明。 拟议的研究结合了几个关键技术，沿着与适当的实验设计，以测试的假设，即接触指导主要是由各向异性刚度或粘附在当地的纤维网络的传感驱动。 通过形成具有相同对齐强度但交联程度不同的磁性对齐纤维蛋白凝胶，将测试接触导引的各向异性刚度和各向异性粘附假设。将评价接种在凝胶后交联上的成纤维细胞的细胞取向行为和细胞突起的动态模式，初步数据显示刚度各向异性增加，并产生更强的接触引导。使用在富含酪氨酸的纤维蛋白原纤维中形成二酪氨酸键的氯铵催化的光交联方法实现交联。将进行实验以确认初步结果，即交联仅影响刚度各向异性，而不影响粘附力或孔隙率的各向异性，这是迄今为止报告的接触指南研究中的混杂效应。在这些接触引导实验中，将类似地评价使用针对纤连蛋白和同源b1整联蛋白上结合位点的抗体修饰细胞粘附的影响，这可能与各向异性粘附假设相关。将通过接种在纤维蛋白凝胶上的成纤维细胞的离心测定来量化粘附，并且将通过分析来自共聚焦图像堆栈的3D重建来量化孔隙率（更一般地，网络微观结构）。 除了所提出的研究在回答这个问题上的基本价值--细胞在检测排列的原纤维时感受到什么信号？该研究通过为纤维状支架的设计提供基本原理，从而产生所需的细胞排列强度和相关的组织机械功能，从而导致改进的工程化组织。 教育影响是通过增强课程，获得共享的成像系统，这将促进学生和工程师之间的互动，从当地行业和合作预期与细胞生物学家。

PI: Tranquillo, Robert T.Proposal #: 1606008Contact guidance, the tendency of a cell to orient and migrate in response to topographical features such as parallel grooves, is a fundamental cell behavior key to many physiological processes and tissue engineering strategies. The goal of this project is to identify the signal that cells sense when interacting with aligned fibrils in a fibrin gel and exhibit contact guidance. The project will specifically test the hypotheses that the directional dependences of stiffness and adhesion of the network of aligned fibrils are the major contributing signals. This will be accomplished by systematically changing only the stiffness directional components of the fibrin network and observing whether or not the contact guidance response of fibroblasts in the fibrin gel changes, and then conducting similar studies with blocking antibodies to modulate adhesion and thereby modulate a potential adhesion directional dependent signal. Beyond the fundamental value in answering the question regarding what signal cells sense when detecting aligned fibrils, the research should lead to improved engineered tissues by providing a rationale for designing fibrillar scaffolds that yield the desired cell alignment and the associated tissue mechanical function. Contact guidance is a fundamental cell behavior key to multiple physiological processes and tissue engineering strategies. While it has been highly studied on planar substrata, and even related to the cell cytoskeleton for certain geometries, it has been little studied in aligned fibrillar networks such as native tissues or the reconstituted collagen and fibrin gels that are used as tissue models. Despite its importance, the mechanism underlying cell contact guidance in an aligned fibrillar network has defied elucidation due to multiple interdependent signals that such a network presents to cells, including anisotropy of adhesion, porosity, and stiffness. The proposed research combines several key technologies along with appropriate experimental design to test the hypothesis that contact guidance is primarily driven by sensing of anisotropic stiffness or adhesion in the local fibrillar network. By forming magnetically-aligned fibrin gels with the same alignment strength, but crosslinked to different extents, the anisotropic stiffness and anisotropic adhesion hypotheses of contact guidance will be tested. Cell orientation behavior and the dynamic pattern of cell protrusions will be evaluated for fibroblasts seeded on the gel post-crosslinking, which preliminary data show increases stiffness anisotropy and yields stronger contact guidance. Crosslinking is achieved using a ruthenium-catalyzed photocrosslinking method that forms dityrosine bonds in tyrosine-rich fibrin fibrils. Experiments will be conducted to confirm preliminary findings that the crosslinking only affects stiffness anisotropy and does not affect anisotropy of adhesion or porosity, which are confounding effects in reported contact guidance studies to date. The effects of modifying cell adhesion using antibodies to binding sites on fibronectin and cognate b1 integrins will similarly be evaluated in these contact guidance experiments, which can be related to the anisotropic adhesion hypothesis. Adhesion will be quantified with a centrifugation assay of fibroblasts seeded on fibrin gels and porosity (more generally network microstructure) will be quantified from analysis of 3D reconstructions from confocal image stacks. Beyond the fundamental value of the proposed studies in answering the question--What signal do cells sense when detecting aligned fibrils?-- the research should lead to improved engineered tissues by providing a rationale for design of fibrillar scaffolds so as to yield the desired strength of cell alignment and the associated tissue mechanical function. Educational impact is achieved through enhanced courses, access to a shared imaging system that will facilitate interactions between students and engineers from local industries and collaborations expected with cell biologists.