## 3 Results
### Cell Density Can Be Maintained in the Different Zones Overtime In Vitro  
To investigate the maintenance of the zonal distribution of the cells over time, we cultured human chondrocytes in the hydrogel for 25 days. We compared the scaffolds with different cell densities, herein called the zonal scaffolds, with the scaffolds in which the cell density (10E6 cells/mL) was constant throughout the entire scaffold. At day 0, right after the bioprinting process took place, a high cell viability was detected for the embedded cells (Figure 3a). In addition, the quantification of the H&E staining at day 0 showed significant differences between the bottom/middle, middle/top, and bottom/top zones of the graded scaffolds (Figure 3b, left), whereas no difference in the cell density was observed throughout the homogeneous scaffolds (Figure 3b, right). After 25 days of culture, cell viability remained high for all the specimens (Figure 3c). In addition, a decrease in the cell density of the middle zone was observed, rendering the cell density between the middle and the bottom zones as not significantly different, while the cell density in the top zone remained significantly higher than the other zones (Figure 3d, left). The cell density of the homogeneous scaffolds remained constant throughout the scaffold thickness for all the timepoints (Figure 3d, right). Figure 2: PCL-reinforced alginate scaffolds with different cell density zones can be bioprinted as a single unit. (**a**) The solid structure created in Solidworks corresponding to the outer PCL frame; (**b**) the solid structure corresponding to the hydrogel part; (**c**) a macroscopic image of the top view of a 3D printed scaffold after ionic crosslinking; (**d**) the different solid structures combined by Slic3r software, showing the PCL in yellow and the hydrogel in different shades of red for the different cell density zones; (**e**) Slic3r design of the printing pattern showing a 100% infill for the PCL and a 10% infill for the hydrogel; (**f**) a bar chart showing the mechanical properties (stiffness) of the outer PCL frame, the combined PCL frame + hydrogel, and the hydrogel alone printed scaffolds (\(n=3\)); (**g**) a fluorescent microscopic image of cell viability with live/dead staining (green: live; red: dead) showing the cell distribution in the scaffold bioprinted with three different cell densities for each of the zones: bottom (5E6 cell/mL), middle (10E6 cells/mL), and top (20E cells/mL) at day 0. Lines are drawn on the image to illustrate the approximate division of the zones. Scale bar = 200 μm; (**h**) the corresponding quantification of the overall live and dead cells (\(n=4\)). Bioprinted Zonal Scaffold with Different Cell Densities Leads to a Gradient Cell-Derived ECM Deposition  
The evaluation of the sGAG and collagen deposition in the bioprinted scaffolds was performed on the basis of the analysis of Alcian Blue and Picrosirius Red histological staining of the scaffold cross-sections. At day 0, the histological analysis showed the background staining for the basal biobank material (alginate and methylcellulose) with a weak staining for Alcian Blue (Figure 4a) and a strong pink (negative for collagen) staining for Picrosirius Red (Figure 4c). A semiquantitative analysis of the stained samples was performed using an optimized threshold to subtract the background staining from positively stained areas (Figure 4b,d).