4.6.2 The effect of printed pressure on cell adhesion and proliferation
In vitro culture of HUVECs in samples: KC-SA-C-15kPa, KC-SA-C-25kPa, and KC-SA-C-35kPa,to assess the effect of printing pressure on the morphology of endothelial cells, the nucleus (blue) and cytoskeleton (green) were stained after endothelial cells laden hydrogels were printed after 24, 48, and 72 h (Figure 13(a–i)).
The proliferation and adhesion of cells increased with an increase in the printing pressure. The cell adhesion in the KC-SA-C-35kPa scaffold was not only more than that in KC-SA-C-15kPa and KC-SA-C-25kPa but also the cell adhesion density and proliferation migration on KC-SA-C-35kPa at the same culture time was significantly better than those under other printing pressures.
Moreover, the diffusion area of cells in the scaffold of other pressures was lower than that in the KC-SA-C-35kPa scaffold. The cells were distributed more uniformly and showed better interaction and fusion with each other in the KC-SA-C-35kPa scaffold. After 24 h of cell culture, the pseudopod spread area of cells in the KC-SA-C-35kPa scaffold was larger, and the interaction among cells was more obvious after 48 h of culture, indicating a tendency of endothelialisation until 72 h of culture.
Cell adhesion, proliferation, and distribution can be attributed to the microstructures and properties of the scaffold.As shown in the microstructure and of KC-SA-C-0.3(Figure 10), most pores were either incompletely formed or had excessively large pore size in the KC-SA-C-15KPA scaffold, which was not conducive to the initial cell adhesion and subsequent growth parameters. Moreover, the cells grew well in the KC-SA-C-35kPa scaffold, possibly because the KC-SA-C-35kPa scaffold had a porous structure with an outer diameter of 3–50 m and a regular and elliptical distribution; this pore size is conducive to cell growth. The orientation growth of cells was observed in the KC-SA-C-35kPa scaffold, possibly caused by the radial orientation of pores in the printed structure, that is, the shear force was distributed along the radial gradient of the printed structure.
In general, the adhesion cells formed effective adhesion 6 h after growth on the material. Therefore, under similar culture conditions, the cell density in the material after 24-h seeding reflected the cell adhesion ability. The cell density of fluorescent images was statistically analysed using the Image-Pro Plus 6.0 software to evaluate the adhesion ability of HUVECs (Figure 14). As shown in Figure 14, the absorbance of cells in the KC-SA-C-35kPa scaffold was the highest, followed by that in the KC-SA-C-25kPa and then in KC-SA-C-15kPa (lowest). Moreover, the initial adhesion of cells in the KC-SA-C-35kPa scaffold was much higher than that in other scaffolds. After 48 h of culture, the number of cells in the KC-SA-C-35kPa scaffold was the highest, almost covering the entire region, while that in the KC-SA-C-15kPa scaffold was the least, with the trend becoming more obvious after 72 h of culture.
The results together demonstrated that a larger printing pressure is conducive to the uniform distribution of cells, with rapid proliferation and promotion of endothelialisation. Low printing pressure leads to uneven cell distribution, local sedimentation, and aggregation, which are not conducive to cell growth and proliferation.