In this paper we report a fundamental morphological instability of constrained 3D microtissues induced by positive chemomechanical feedback between actomyosin-driven contraction and the mechanical stresses arising from the constraints. Using a 3D model for mechanotransduction we find that perturbations in the shape of contractile tissues grow in an unstable manner leading to formation of "necks" that lead to the failure of the tissue by narrowing and subsequent elongation. The magnitude of the instability is shown to be determined by the level of active contractile strain, the stiffness of the extracellular matrix, and the components of the tissue that act in parallel with the active component and the stiffness of the boundaries that constrain the tissue. A phase diagram that demarcates stable and unstable behavior of 3D tissues as a function of these material parameters is derived. The predictions of our model are verified by analyzing the necking and failure of normal human fibroblast tissue constrained in a loop-ended dog-bone geometry and cardiac microtissues constrained between microcantilevers. By analyzing the time evolution of the morphology of the constrained tissues we have quantitatively determined the chemomechanical coupling parameters that characterize the generation of active stresses in these tissues. More generally, the analytical and numerical methods we have developed provide a quantitative framework to study how contractility can influence tissue morphology in complex 3D environments such as morphogenesis and organogenesis.

The alignment and blend of extracellular matrix (ECM) proteins give a tissue its specific mechanical properties as well as its physiological function. Various tissue engineering methods have taken purified ECM proteins and aligned them into gels, sponges and threads. Although, each of these methods has created aligned ECM, they have had many limitations including loss of hierarchal collagen structure and poor mechanical performance. Here, we have developed a new method to control ECM synthesis using self-assembled cells. Cells were seeded into custom designed, scaffold-free, micro-molds with fixed obstacles that harnessed and directed cell-mediated stresses. Cells within the microtissue reacted to self-generated tension by aligning, elongating, and synthesizing an ECM whose organization was dictated by the strain field that was set by our micro-mold design. We have shown that through cell selection, we can create tissues with aligned collagen II or aligned elastin. We have also demonstrated that these self-assembled microtissues have mechanical properties in the range of natural tissues and that mold design can be used to further tailor these mechanical properties.

When seeded into nonadhesive micro-molds, cells self-assemble three-dimensional (3D) multicellular microtissues via the action of cytoskeletal-mediated contraction and cell-cell adhesion. The size and shape of the tissue is a function of the cell type and the size, shape, and obstacles of the micro-mold. In this article, we used human fibroblasts to investigate some of the elements of mold design and how they can be used to guide the morphological changes that occur as a 3D tissue self-organizes. In a loop-ended dogbone mold with two nonadhesive posts, fibroblasts formed a self-constrained tissue whose tension induced morphological changes that ultimately caused the tissue to thin and rupture. Increasing the width of the dogbone's connecting rod increased the stability, whereas increasing its length decreased the stability. Mapping the rupture points showed that the balance of cell volume between the toroid and connecting rod regions of the dogbone tissue controlled the point of rupture. When cells were treated with transforming growth factor-b1, dogbones ruptured sooner due to increased cell contraction. In mold designs to form tissues with more complex shapes such as three interconnected toroids or a honeycomb, obstacle design controlled tension and tissue morphology. When the vertical posts were changed to cones, they became tension modulators that dictated when and where tension was released in a large self-organizing tissue. By understanding how elements of mold design control morphology, we can produce better models to study organogenesis, examine 3D cell mechanics, and fabricate building parts for tissue engineering.