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.