## 4 Discussion  
In this study, we aimed to generate a cartilage scaffold of a clinically relevant size that mimics the gradient distribution of cells that is observed across the three distinct zones of the native tissue in hyaline articular cartilage of the human condyle and to analyze the cell viability and cell-derived matrix deposition. Over time in culture, the generated zonal scaffolds led to a gradient in cartilage-like matrix deposition. For printing each zonal scaffold, four different inks (one ink and three bioinks) were used to print different parts of the scaffold. Every printing part used its own printhead except for the middle and top zones where the bioink-containing cartridge had to be interchanged for every printed scaffold. This allowed us to simultaneously 3D print a PCL frame and the different chondrocyte-embedded zones with clinically relevant thicknesses and zone-specific cell densities. Few papers have investigated the 3D printing of more than one bioink for a single construct [10; 19; 24; 25; 26], probably due to the lack of several printheads or impracticality due to the constant changing of the printing cartridges that results in a time-consuming process. Recent studies suggest the use of coaxial needles [27] or continuous chaotic printing [28] as alternatives to generate multicellular heterogeneous systems. Several studies have combined PCL with bioinks to enhance the compressive stiffness of hydrogels [29; 30; 31]. In our study, the PCL frame helped the maintenance of the structural integrity of the scaffolds during bioprinting and subsequent in vitro culture. In addition, a PCL frame can be useful for future in vivo implantation [32; 33]. Previous studies have shown that different cell densities can lead to different mechanical properties which are correlated with the amount of sGAG produced. In this study, we did not perform mechanical characterization of the hydrogel overtime. Therefore, future studies should also focus on the effect of different cell densities on the mechanical properties of the scaffold overtime. Regarding the PCL reinforcement, in most studies, this reinforcement is done by printing a mesh of PCL fibers in between hydrogel fibers, where the fiber orientation alternates orthogonally after every layer. In this study, however, it was determined that passing the hot (210 \({}^{\circ}\)C) thermoplastic nozzle right next to the cell-embedded hydrogel fibers negatively affects the chondrocyte viability, which has been also noted in other studies [34]. Therefore, another design approach was chosen in which the PCL would act as an outer skeleton for the hydrogel, concentrating the cell-embedded volume of the bioink at the center and maintaining its shape throughout its thickness. This approach was able to significantly increase the bulk mechanical properties compared to the bioink alone, as others have demonstrated [29; 30; 31]. Other studies also explored the pre-printing of PCL structures and then either cast or \(z\)-printed the bioink in the channels of the PCL mesh [35]. Despitethis option giving more freedom, as it decouples the fabrication process of the outer frame and the bioprinting of the bioink, \(z\)-print is not yet an option available in all 3D printing systems. After fabrication of such complex structures, live/dead and histological analysis demonstrated that human chondrocytes still retained high viability and zone-specific cell densities. In addition, this cell density gradient was shown to be maintained during in vitro culture for 25 days, resulting in a gradient in tissue deposition as compared to homogeneous scaffolds. Previous studies aiming to fabricate similar constructs with different cell density layers through bioprinting have not shown such clear cell distribution or its maintenance over time [19]. For example, it has been reported that cell gradient in scaffolds of 3 mm thickness fail due to the fusion of the zones during bioprinting. Moreover, the analysis of that study was focused on the overall scaffold rather than on the individual zones [19].