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and AMSCs underwent chondrogenic differentiation in vitro and in vivo , although matrix production was less than in constructs containing chondrocytes (ACs, NCs or ECs). Constructs containing BMSCs had a higher chondrogenic potential than AMSCs, demonstrated by an increased ACAN and COL2A1 gene-expression and an improved sGAG-deposition. With the exception of a few studies [223-226], this confirms other studies [55, 207, 227-235]. Nevertheless, the assumptions that MSCs are fundamentally less chondrogenic than chondrocytes and that BMSCs are more in favor for cartilage regeneration than AMSCs, seems unjustified. It appears that cell culture conditions for both BMSCs and AMSCs remain to be improved. For instance, it was found that another member of the TGFβ-superfamily, Bone Morphogenetic Protein 6, is obligatory to improve chondrogenic differentiation in AMSCs. [236] Finally, in order to understand how the distribution and composition of matrix components resulted in a mechanically functional cartilage matrix, the compositional biomechanical relationship of the cartilage constructs was evaluated. After in-vivo implantation, mechanical properties increased in constructs containing ECs and NCs. Only for constructs containing NCs, matrix components were significantly correlated to their biomechanical functionality. It is already known that sGAGs and collagens, the main components of the ECM, are both associated with the biomechanical properties of native cartilage: (1) the negatively charged sGAGs provide an osmotic pressure within the tissue ; (2) the architecture of the collagen network capture the sGAGs and prevent them from leaking out of the tissue. [237] In contrast, the elastic fiber network in constructs containing ECs might have influenced the biomechanical properties in vivo as well, although the exact contribution of elastin to mechanical functionality is not yet fully understood. Besides the existence of matrix components, the quality of the matrix is not only determined by the amount of matrix components deposited, but also influenced by the number of cross-links between matrix molecules (i.e. collagen cross links). [238] The distribution of matrix components in the ECM was clearly different between cell sources: ECs deposited most matrix components pericellularly, whereas NCs deposited these matrix components homogenously throughout their matrix, which was clearly visible on the immunohistochemical collagen type II staining. It is well-known that a heterogeneous distributed matrix alters the biomechanical properties of the matrix, as the physical properties are determined by the weakest point in the matrix [140]. The present study has certain limitations. Firstly, cell density plays a critical role in functional and stable cartilage formation. Others have demonstrated that cell densities greater than 20x10 6 cells per milliliter are desirable, while low cell densities resulted in decreased cartilage formation. [239] Therefore, a potential drawback of our study is that we only could use a cell-seeding density of 4x10 6 cells per milliliter of alginate, since the size of our experimental set-up did not enable higher densities. Secondly, as mentioned before, we have culture-expanded all cells until passage four to obtain a sufficient number of cells. In order to be able to compare all different cell sources, we have used standardized protocols for the culture-expansion of both chondrocytes and MSCs. Differences in expansion rates between the different cells were obvious. While culture-expansion is associated with chondrocyte dedifferentiation and replicative cell senescence, the enforcement of population 80 CHAPTER 4