Force-driven evolution of mesoscale structure in engineered 3D microtissues and the modulation of tissue stiffening.

The complex structures of tissues determine their mechanical strength. In engineered tissues formed through self-assembly in a mold, artificially imposed boundary constraints have been found to induce anisotropic clustering of the cells and the extracellular matrix in local regions. To understand how such tissue remodeling at the intermediate length-scale (mesoscale) affects tissue stiffening, we used a novel microtissue mechanical testing system to manipulate the remodeling of the tissue structures and to measure the subsequent changes in tissue stiffness. Microtissues were formed through cell driven self-assembly of collagen matrix in arrays of micro-patterned wells, each containing two flexible micropillars that measured the microtissues' contractile forces and elastic moduli via magnetic actuation. We manipulated tissue remodeling by inducing myofibroblast differentiation with TGF-β1, by varying the micropillar spring constants or by blocking cell contractility with blebbistatin and collagen cross-linking with BAPN. We showed that increased anisotropic compaction of the collagen matrix, caused by increased micropillar spring constant or elevated cell contraction force, contributed to tissue stiffening. Conversely, collagen matrix and tissue stiffness were not affected by inhibition of cell-generated contraction forces. Together, these measurements showed that mesoscale tissue remodeling is an important middle step linking tissue compaction forces and tissue stiffening.