Regulation of contractile forces in self-assembling microtissues

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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.