## 4 Discussion
It is noteworthy that the difference in cell number between the middle and the bottom zones in the gradient scaffold is less apparent after culture in differentiation medium. Although cells do not tend to migrate in alginate, this particular bioink formulation containing methylcellulose may allow for some cellular migration [36] from the middle to the bottom layer, perhaps in the search for available nutrients and oxygen. This potential migration may have been caused by the cell gradient itself, as no changes in cell number were observed in the homogeneous scaffold. Further research is needed to verify this hypothesis. Despite the presence of the tissue deposition gradients, low amounts of glycosaminoglycan and collagen were seen in all the groups. This may be due to the alginate-based bioinks that offer good printability [37; 38; 39; 40; 25; 30; 35; 36; 37; 38; 39; 41] but do not offer enzymatic-derived tissue remodeling [42], which have been suggested to potentially act as a physical barrier for tissue secretion in vitro, as also observed by others [43; 44; 45]. On the other hand, alginate-based biomaterials have been shown to be capable of generating cartilage when implanted in vivo [46; 47], suggesting that other factors may also be playing a role in this regard. For instance, chondrocytes from only one donor were used in our study, and it is known that there can be some variability between donors or even between chondrocytes from different zones [45]. Regarding tissue deposition, it is interesting to point out that the superficial zone of native mature articular cartilage is characterized by a lower sGAG presence than the other zones. Our scaffold demonstrated that having a higher cell density in the superficial zone will result in higher sGAG. However, the tissue formed in our scaffold is still in development and yet far from what is seen in articular cartilage tissue. It will be necessary to evaluate how applications of these different zones would influence eventual tissue that is formed using in vivo models. Our study is in agreement with the previous studies showing that constructs with a higher cell density result in higher cartilage-like matrix deposition [48; 49] and enhanced gene expression of cartilage markers [19]. This study focused on the recapitulation of the cell gradient observed in native hyaline articular cartilage. However, there are other gradients that could be interesting to investigate or even to combine. For instance, AC has been shown to have a gradient in stiffness [50; 51; 52; 53]. Previous research has aimed to combine a stiffness gradient achieved by photo-crosslinking with a growth factor gradient achieved by mixing both TGF-\(\beta\)1 and BMP-2 in the hydrogel (before crosslinking). These gradients proved capable of regulating the number of embedded hMSCs and their differentiation toward bone- or cartilage-like matrix-producing cells [18]. In addition, from a developmental biology perspective, cartilage starts as a soft template. Therefore, it may be interesting to explore the generation of the stiffness gradients that occur in time. This points to the need for the development of new 4D bioprinting approaches that investigate the use of stimulus-responsive biomaterials to answer such complex questions [54].