Fig. 7.

Density and distribution of cells within the methacryloyl gelatin-alginate (GEAL) sublayers of the fabricated small diameter vascular grafts (SDVGs). a Macroscopic image of a segment of the SDVG (scale bar = 2 mm). b Schematic representation of the SDVG multilayer configuration (see also Fig. 1). c A single isolated graft sublayer from a fabricated cellularized SDVG (scale bar = 200 µm). This particular imaged layer corresponds to a middle graft sublayer (PCL/GEAL sublayer). d Fluorescence microscopy image of a transversal section of an SDVG with cell nucleus staining (Hoechst 33342). e Higher magnification image of the vessel wall in a transversal cut, showing cells present throughout the thickness of the SDVG, from the lumen to the outer layer (right to left). f Confocal image reconstitution of 100 µm z-stacked images showing LIVE/DEAD^® cell staining in the SDVG after 2 weeks of static cell culturing (viable cells = green fluorescence; dead cells = red fluorescence) (scale bar = 100 µm). g LIVE/DEAD^® cell staining after 3 weeks of static cell culturing (confocal image reconstitution of 100 µm z-stacked images). h–j Representative confocal images of LIVE/DEAD^® cell staining of the SDVG after 4 weeks of static cell culturing (scale bar = 100 µm). From the lumen to the outer layer, h is the second, i the sixth and j the eighth individually isolated graft sublayer. k Ki-67 nuclear staining in cells embedded in one graft sublayer demonstrating proliferative state (scale bar = 50 µm). Ki-67 staining is only present in active phases of the cell cycle (G1, S, G2 and mitosis), and absent in quiescent cells