Regulation of contractile forces in self-assembling microtissues

细胞外基质（ECM）的结构和机械性质控制成熟组织（如骨、心肌和骨骼肌或皮肤）的功能。这些特性又由ECM分泌细胞，特别是成纤维细胞决定。然而，成纤维细胞本身对它们自己的基质做出反应，产生复杂的反馈回路，该反馈回路被认为在纤维化期间失调，例如在肝脏、肺、气道或心脏中。特别是，我们和其他人以前已经表明，细胞产生的力使周围的基质变硬，并且成纤维细胞和其他细胞在附着到更硬的基质时施加更高的牵引力。本项目将探讨组织形成和纤维化过程中细胞收缩性和基质硬度之间的反馈机制。我们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胶原蛋白或纤维蛋白网络中来实现这一点，并定义了机械边界条件。当细胞伸展和收缩时，网络纤维对齐并将力传递给相邻细胞，最终导致大规模的结构重排和组织形成，这些结构和组织形成可以通过边界条件、细胞密度和ECM组成进行微调。通过在基质网络内放置两个柔性杠杆，基质纤维开始在杠杆之间平行排列，形成与纤维化瘢痕组织非常相似的可收缩组织。该组织的收缩力可以通过柔性杠杆的偏转来测量，并且有效组织刚度可以通过杠杆的弯曲刚度、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，这也允许我们直接测量被动组织刚度及其时间演变。我们将使用光遗传学工程化的成纤维细胞系，其中Rho介导的收缩力可以通过光局部或全局激活。然后，我们将系统地确定复杂的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，然后探索纤维化组织如何形成，这取决于局部基质特性（建筑，机械），全局机械边界条件（刚度，几何形状），光诱导或光诱导收缩激活，细胞密度和细胞排列。我们将使用牵引力显微镜和悬臂弯曲测量由单细胞以及整个组织产生的收缩力;我们将使用循环组织拉伸测量ECM的机械特性;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。这些结果将导致更深入地了解纤维化组织是如何形成的。

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.