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

接触指导，即细胞根据诸如平行沟槽等地形特征进行定向和迁移的趋势，是许多生理过程和组织工程策略的关键。该项目的目标是识别细胞在与纤维蛋白凝胶中排列的纤维相互作用时感受到的信号，并展示接触指导。该项目将专门测试这样的假设，即定向纤维网络的硬度和粘附性的方向相关性是主要的贡献信号。这将通过系统地只改变纤维蛋白网络的刚性方向成分并观察纤维蛋白凝胶中成纤维细胞的接触引导反应是否改变来实现，然后利用阻断抗体进行类似的研究以调节黏附，从而调节潜在的黏附方向依赖的信号。除了在回答有关细胞在检测排列的纤维时感觉到什么信号的问题上的基本价值之外，这项研究还应该通过为设计产生所需的细胞排列和相关的组织机械功能的纤维支架提供理论基础来改进工程化组织。接触指导是多种生理过程和组织工程策略的关键。虽然它在平面基质上已经得到了高度的研究，甚至与某些几何形状的细胞骨架有关，但在排列的纤维网络中，如天然组织或用作组织模型的重组胶原和纤维蛋白凝胶中，它的研究还很少。尽管它很重要，但由于这种网络呈现给细胞的多个相互依赖的信号，包括黏附、孔隙率和硬度的各向异性，因此在排列的纤维网络中细胞接触指导的机制仍然无法阐明。这项拟议的研究结合了几项关键技术和适当的实验设计，以验证接触制导主要由局部纤维网络中各向异性刚度或粘连的感知驱动的假设。通过形成具有相同排列强度、但交联度不同的磁性排列的纤维蛋白凝胶，将检验接触制导的各向异性刚性和各向异性粘附性假设。将评估种植在凝胶上的成纤维细胞在交联后的细胞取向行为和细胞突起的动态模式，初步数据显示，这增加了刚性各向异性，并产生了更强的接触指导。交联是使用Ru催化的光交联法实现的，这种方法在富含酪氨酸的纤维蛋白纤维中形成双酪氨酸键。将进行实验以证实初步发现，交联剂只影响硬度各向异性，而不影响附着力或孔隙率的各向异性，这是迄今报道的接触指导研究中的混淆效应。在这些接触指导实验中，将同样评估使用针对纤维连接蛋白和同源b1整合素结合部位的抗体来改变细胞黏附的效果，这可能与各向异性黏附假说有关。粘附性将通过种植在纤维蛋白凝胶上的成纤维细胞的离心法进行量化，孔隙率(更一般的网络微结构)将通过分析来自共聚焦图像堆栈的3D重建来定量。除了拟议中的研究在回答这个问题--细胞在检测到排列的纤维时感觉到什么信号？--方面的基本价值之外，这项研究应该通过为纤维支架的设计提供理论基础来改进工程化组织，从而产生所需的细胞排列强度和相关的组织机械功能。教育的影响是通过加强课程，使用共享成像系统，促进学生和来自当地行业的工程师之间的互动，以及预期与细胞生物学家的合作来实现的。

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.