Figure 3. Scaffold geometry guides EB tissue formation.

a) Graphical illustrations showing the different scaffold geometry designs investigated (i), bright-field images on day 5 showing EB tissue morphology on the different scaffold geometries (ii) on square grids that create four 90° angles as labeled in the bright-field image, rhombus grids that create two major angles of ≈135°and ≈45°, and triangle grids that create two major angles of ≈90°and ≈45°. b) Curve shortening flow models of EB tissue growth on scaffolds. Tissue growth evolution is modeled on: (i) square, rhombus, and triangle grid scaffolds, with lighter gray representing later time points, and (ii) evolution of normalized line curvature $\tilde{k}$ of the tissue interface. The key represents curvature. c) Characterization of lumen formation on square grid scaffolds at day 2 and day 5 by staining for F-actin (phalloidin), and immunostaining with apical protein marker ZO1 and nuclei (DAPI). A representative example of lumen emergence at the scaffold intersections is shown on day 2 (white line arrowheads) and at the scaffold walls (solid white arrowheads). The bottom row shows the matured tissue comprising the lumen at day 5 (n = minimum of 3 tissue nodes from 1 or 2 independent scaffolds). Scale bar: 200 μm. d) Tiled confocal fluorescence microscopy image of an immunostained scaffold sample with the apical protein marker ZO1, and counterstained with DAPI. Scale bar: 1 mm.