Twisted plywood-like tissue formation in vitro. Does curvature do the twist?

Abstract

Little is known about the contribution of 3D surface geometry on the development of multi-layered tissues containing fibrous extracellular matrix components such as those found in bone. Here we elucidate the role of curvature in the formation of chiral, twisted plywood-like structures. Tissues consisting of murine pre-osteoblast cells (MC3T3-E1) were grown on 3D scaffolds with constant mean curvature and negative Gaussian curvature for up to 32 days. Using 3D fluorescence microscopy, the influence of surface curvature on actin stress-fiber alignment and chirality was investigated. To gain mechanistic insights, also MC3T3-E1 cells deficient in nuclear A-type lamins or treated with drugs targeting cytoskeleton proteins were used in our study. We find that wild type cells grow multilayered tissue with fibers predominantly aligned along directions of negative curvature, but where subsequent layers twist in orientation with respect to older tissues with time. Fiber orientation is conserved below the tissue surface thus creating a twisted plywood like material. We further show that this directional organization strongly depends on structural components of the cells (A-type lamins, actin and myosin). Our data indicate the importance of substrate curvature in the formation of 3D tissues and provides new insights into the emergence of chirality. Significance StatementBiological tissues (like compact bone) often consist of multiple fibrous layers which are staggered with a twisting angle relative to each other, thereby improving mechanical performance. The underlying principles of how such tissues are formed and what determines the fiber direction are still debated. Here we report the formation of a twisted plywood-like tissue grown in vitro on constant mean and negative Gaussian curvature substrates and present evidence that for tissue consisting of pre-osteoblast like cells, surface curvature is a main determinant for fiber orientation.