3.9 In Vitro Cell Studies
To evaluate the cell compatibility and vascular potential of the 3D
printed A-SA-Gel hydrogel scaffold, HUVECs were cultured as a model cell
type. The SA/Gel hydrogel scaffold served as a control group. The
morphology of cultured cells and detection of live/dead cells were
performed to analyze the viability of HUVECs in 3D printed constructs
after 4 days of culture (Figure 7). The microscopic images of cultured
cells (Figure 7A, 7D, and 7G) showed that the cells were evenly
distributed and adhered well to the scaffolds. The cells were maintained
their normal cellular phenotypes. The cell density of the A-SA-Gel
hydrogel scaffold (Figure 7D) was higher than that of SA/Gel scaffold
(Figure 7A). The live/dead cell staining images showed that the majority
of HUVECs in all groups were stained green (Figure 7B, 7E, and 7H),
indicating the high cellular viability. However, there few dead cells
were also observed (Figure 7C, 7F, and 7I). Importantly, the endothelial
sprout formation and primitive microvascular networks were observed for
the A-SA-Gel hydrogel scaffold (Figure 7E and 7H) as compared to the
SA/Gel scaffold (Figure 7B). To this end, more representative
fluorescence images of endothelial sprouts, vessel networks, and
neovessels were captured at different A-SA-Gel hydrogel scaffold areas,
as shown in Figure 8, at higher magnification. The endothelial vascular
network formation due to endothelial cellular function was captured in
Figure 8A, along with lacunae (micro-rings) microstructures within the
network. The diameter of the lacunae (micro-rings) of endothelial
network was measured (Figure 8G), and the average was found to be
31.25±0.58μm. Figure 8B presented a microvessel of endothelial cells
like a bridge, interconnected between adjacent sides. Exhilaratingly, a
branched microvessel network was observed like a tree branch in Figure
8C. Moreover, several large neovessels with obvious open lumen were
captured in Figure 8D-8F, and the average diameter was more than 100μm.
To further analyze vasculogenesis, the diameter of endothelial sprouting
and sprouting length was measured (Figure 8H and 8I). The minimum
diameter of endothelial sprouting was 8μm, and the maximum diameter was
about 112μm. For the sprouting length, the minimum sprouting length was
118μm, the maximum length was 476μm, and the average length was 289 μm.
Additionally, fluorescent staining of the cell-cultured A-SA-Gel
hydrogel scaffold was carried out to visualize the cell nucleus and
cytoskeleton after 4 days of culturing (Figure 9A-A2, 9B-B2, and 9C-C2).
A homogeneous distribution of cells on the surface of the scaffold was
observed in the DAPI (cell nucleus) and TRITC-phalloidin (cytoskeleton)
stained images
(Figure
9 A-A2). Similarly, the representative fluorescence images of the
endothelial sprouts and neovessels were also presented (Figure 9B-B2 and
9C-C2). Moreover, the SEM photographs of HUVECs on the scaffold were
shown in Figure 9D-9F. The SEM examination of the cells confirmed good
attachment and spreading of cells on the surface of the scaffold. These
cells were observed to be flat-shaped and stretch on the whole surface
of scaffold structures.