3.5 Cellular vessel structures
SMCs were added into GelMA which was acting as bioink to form the wall of vessel structure; while HUVECs were perfused into the inner space of vessel lumen to form a biomimetic barrier between blood and vessel wall.
The viabilities of SMCs and HUVECs in the vascular structure were analyzed by live/dead staining. The fluorescence images of 1, 4 and 7 days were obtained by confocal microscope. The cells were in granular state at the first day after printing. On the fourth day, the number of cells began to increase, and some cells began to expand in their shape. Most cells expanded and SMCs showed the long spindle shape in 7 days (Figure 5A). The cell survival rates were 93.64 ± 1.455 (day 1), 91.62 ± 2.070 (day 4), and 95.62 ± 1.312 (day 7). All the survival rates in the structures were more than 90% (Figure 5B). CCK-8 was used to analyze cell proliferation. The CCK-8 absorption values of HUVECs and SMCs on day 0, 1, 4 and 7 were 0.110 ± 0.0138, 0.110 ± 0.0068, 0.209 ± 0.0121 and 0.315 ± 0.0179, respectively (Figure 5C). Cells significantly proliferated in the vascular structures.
H&E and Masson’s trichrome staining were used for morphological detection, and immunofluorescence was used for functional assessment. After one week of culture, spreading out SMCs were well distributed in the outer layer. HUVECs formed a monolayer cell coverage on the inner lumen surface (Figure 5D-5E). CD31 and α-SMA immunofluorescence confirmed an inner endothelial layer and the outer smooth muscle layer from the vascular structure.
The morphology of the outer, middle, and inner layers of GelMA vascular construct in were further observed by SEM. Interestingly, the SMCs in the outer layer and middle layer were spreading out and arranged linearly along the vertical direction of printing. The HUVECs in the inner layers attached tightly to the surface of GelMA lumen surface and expanded to form a smooth barrier evenly.