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contribution of each individual cell type to cartilage matrix production using species-specific gene-expression analyses. In this way, we proved that cartilage matrix formation originated from bovine chondrocytes and that h BMSCs fulfilled a trophic role herein. Although numerous cellular communication pathways have been hypothesized in order to explain beneficial effect of mixed cultures [73], this outcome was in accordance with previous studies, investigating the effect of MSCs on articular chondrocytes. [74, 263, 275, 280] We found no evidence that paracrine soluble factors released by chondrocytes enhanced the chondrogenic differentiation of h BMSCs, as stated by others. [255-259] Although the importance of juxtacrine or gap-junctional signalling is still unclear in literature [263], our mixed cells encapsulated in alginate hydrogels implicated that such signaling pathways are of less importance than paracrine signaling pathways, since the alginate hydrogel hinders direct cell- cell contact. Besides the trophic effect of h BMSCs on chondrocytes we demonstrated that this effect was also depended on the chondrocyte source used. The differences between the chondrocyte sources was most obvious in the in-vitro experiments: b NCs were clearly stimulated by h BMSCs, while b ECs were not at all influenced by them. Although the in-vivo experiments showed a positive effect of h BMSCs on both b ECs and b NCs, it was obvious that the use of b NCs lead to constructs with a higher amount of sGAG and collagen and higher equilibrium modulus than b ECs. Clear subtype-specific differences in cartilage forming potential is in accordance with our previously published work, confirming that ECs and NCs have unique gene-expression profiles inducing dissimilar proliferation capacity, cartilage matrix formation and elastin fiber deposition. [40, 56] Before this method can be successfully applied as a one-step clinical application, there are some limitations to overcome. First, the elastic modulus after 8 weeks of subcutaneous implantation was low and approximately 1% of that of native human ear or nasal cartilage. [281] Although the biomechanical properties of the constructs were rather low, alginate enabled a homogeneous cell distribution and prevented cells from floating out while permitting nutrient diffusion and oxygen transfer to the cells in order to create an environment to form new cartilage matrix with sufficient properties. [89] Therefore, injected into a mechanical stable scaffold, alginate could be an excellent cell-carrying gel for future cell-based cartilage repair. Secondary, the cell density used in this study might not be optimal to obtain engineered tissue that are clinical applicable. Our experimental set-up did not allow us to further increase cell density due to limitations in the number of cells available. Nevertheless it allowed us to study the interactions between the cell types. For clinical application, it would be ideal to only use low numbers of human primary chondrocytes supplemented with h BMSCs. We have combined h BMSCs and chondrocytes at a 4:1 ratio, as the effect of h BMSCs on articular chondrocytes was already studied by us at such ratio. Although others have used a 4:1 ratio for their research as well [74, 253], no consensus on optimal BMSCs-to-chondrocytes ratios have been established for ECs and NCs. Future research needs to clarify if we could further reduce the amount of primary chondrocytes without inhibiting cartilage matrix production. Finally, for future clinical application the use of allogeneic h BMSCs can be considered as MSCs have been demonstrated to be immune 122 CHAPTER 6