## 3 Results
### Cell Density Can Be Maintained in the Different Zones Overtime In Vitro
Figure 3: Gradient cell density can be maintained in the zonal scaffold over time in in vitro culture; (**a**) fluorescent microscopic images indicate cell viability based on live/dead staining (green: alive; red: dead) and bright-field microscopy images of the H&E staining (matrix: violet; nuclei: dark purple) showing the cell distribution for the three different zones, bottom, middle, and top, of the zonal and homogeneous bioprinted scaffolds at day 0; (**b**) the quantification of the number of the cells from the H&E staining at day 0; (**c**) the live/dead and H&E images of the zonal and homogeneous scaffold at day 25; (**d**) the quantification of the number of cells from the H&E staining at day 28 (\(n=3\); \(p\)-values \(<0.05\) are shown). Scale bar: 100 μm. Figure 4: Bioprinted zonal scaffolds with cell density gradients result in differential cartilage-like tissue deposition; (**a**) histological images of zonal or homogeneous scaffold bioprinted with human chondrocytes and evaluated for sGAG with Alican Blue staining (AB) at day 0; (**b**) the corresponding semiquantitative zonal analysis of the AB staining; (**c**) histological images evaluated for collagen deposition with Picrosirus Red staining (PSR) at day 0; (**d**) the corresponding semiquantitative zonal analysis of the PSR staining; (**e**) the histological images evaluated for sGAG with AB after 25 days in culture; (**f**) the corresponding semiquantitative zonal analysis of the AB staining; (**g**) histological images evaluated for collagen with PSR; (**h**) the corresponding semiquantitative zonal analysis of the PSR staining (\(n=3\); \(p\)-values <0.05 are shown). Scale bar: 100 μm. At day 25 of culture, both types of staining demonstrated that the scaffolds supported the deposition of cartilage-like tissue components. In the zonal scaffold, Alcian Blue staining revealed an increased deposition of sGAG in the top zone of the scaffold at day 25, as indicated by a strong Alcian Blue signal (Figure 4e, zonal, AB, top). The semiquantification of the staining revealed the zonal manner in which sGAG was deposited within the scaffolds over time, displaying a significantly increase from the bottom to the top zone (Figure 4f, left), which was in accordance with the cell density distribution within the scaffolds. The homogeneous scaffolds deposited a similar amount of sGAG in all the different zones at day 25 (Figure 4e, homogeneous, AB and Figure 3f, right). For the collagen deposition, it was observed that chondrocytes appeared to mainly secrete small amounts of pericellular collagen according to the intense orange-red staining observed (which is positive for collagen staining) after 25 days of culture (Figure 4g). Similar to the result of Alcian Blue staining, some samples exhibited a trend toward stronger staining in the top zone of the specimens. By comparison, the homogeneous scaffolds revealed no differences in their staining throughout the section. The statistical analysis of the semiquantitative values corresponding to Picrosirius Red revealed a trend of increasing collagen deposition in the top zone of some of the zonal scaffolds. However, no significant differences were found between the groups after 25 days of culture (Figure 4h).