Regulation of contractile forces in self-assembling microtissues

细胞外基质（ECM）的结构和机械特性控制着成熟组织的功能，如骨骼、心脏和骨骼肌或皮肤。这些特性反过来又由分泌ecm的细胞，特别是成纤维细胞决定。然而，成纤维细胞本身对其自身的基质作出反应，产生一个复杂的反馈回路，该回路被认为在纤维化过程中失调，例如在肝、肺、气道或心脏。特别是，我们和其他人之前已经表明，细胞产生的力使周围的基质变硬，当成纤维细胞和其他细胞附着在更硬的基质上时，它们会施加更高的牵引力。本项目将探索组织形成和纤维化过程中细胞收缩性和基质刚度之间的反馈机制。我们的两个小组都开发了模型系统，以前所未有的细节研究成纤维细胞和ECM之间的机械反馈。我们通过将细胞嵌入具有明确机械边界条件的胶原蛋白或纤维蛋白网络来实现这一目标。随着细胞的扩散和收缩，网络纤维排列并将力传递给邻近的细胞，最终导致大规模的结构重排和组织形成，这可以通过边界条件、细胞密度和ECM组成进行微调。通过在基质网络中放置两个柔性悬臂，基质纤维开始在悬臂之间平行排列，形成一个与纤维化疤痕组织非常相似的收缩组织。这种组织的收缩力可以通过柔性悬臂梁的挠度来测量，有效组织刚度可以通过悬臂梁的弯曲刚度、组织形成的高度以及通过对组织施加稳态或循环拉伸来调节，这也使我们能够直接测量被动组织刚度及其时间演变。我们将使用光基因工程成纤维细胞系，其中rho介导的收缩力可以被光局部或全局激活。然后，我们将系统地确定复杂3D组织模型中光诱导收缩扰动的空间传播和时间持久性的特征长度尺度，然后根据局部基质特性（建筑，机械），全局机械边界条件（刚度，几何），药理或光诱导收缩激活，细胞密度和细胞排列，探索纤维化组织如何形成。我们将使用牵引力显微镜和悬臂弯曲来测量单个细胞和整个组织产生的收缩力；我们将使用循环组织拉伸测量机械ECM性能；我们将使用二次谐波成像和免疫荧光显微镜测量ECM的结构和组成。这些结果将导致对纤维化组织如何形成的更深层次的理解。

The architecture and mechanical properties of the extracellular matrix (ECM) control the function of mature tissues, such as bone, cardiac and skeletal muscle, or skin. These properties in turn are determined by ECM-secreting cells, in particular fibroblasts. However, fibroblasts themselves respond to their own matrix, giving rise to a complex feedback loop that is thought to be dysregulated during fibrosis e.g. in the liver, the lungs, the airways, or the heart. In particular, we and others have previously shown that cell-generated forces stiffen the surrounding matrix, and that fibroblasts and other cells exert higher traction forces when they are attached to a stiffer matrix. This project will explore the feedback mechanism between cell contractility and matrix stiffness during tissue formation and fibrosis. Both of our groups have developed model systems to study the mechanical feedback between fibroblasts and the ECM in unprecedented detail. We achieve this by embedding cells in collagen or fibrin networks with defined mechanical boundary conditions. As the cells spread and contract, the network fibers align and transmit forces to neighboring cells, ultimately leading to large-scale architectural rearrangements and tissue formation that can be fine-tuned by the boundary conditions, the cell density, and the ECM composition. By placing two flexible cantilevers within the matrix network, matrix fibers begin to align in parallel between the cantilevers, forming a contractile tissue that closely resembles fibrotic scar tissue. Contractile forces of this tissue can be measured by the deflection of the flexible cantilevers, and the effective tissue stiffness can be modulated by the bending stiffness of the cantilevers, the height at which the tissue forms, and by imposing a steady-state or cyclic stretch onto the tissue, which also allows us to directly measure the passive tissue stiffness and its time evolution. We will use an optogenetically-engineered fibroblast cell line in which Rho-mediated contractile forces can be activated locally or globally by light. We will then systematically determine the characteristic length-scales of spatial propagation and temporal persistence of light-induced contractile perturbations in complex 3D tissue models, before exploring how fibrotic tissue forms, depending on the local matrix properties (architectural, mechanical), global mechanical boundary conditions (stiffness, geometry), pharmacologically- or light-induced contractile activation, cell density, and cell alignment. We will measure contractile forces generated by single cells as well as by the whole tissue using traction force microscopy and cantilever bending; we will measure mechanical ECM properties using cyclic tissue stretching; and we will measure ECM architecture and composition using second harmonic imaging and immunofluorescence microscopy. These results will lead to a deeper understanding of how fibrotic tissue forms.