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Differentiation versus chondro-induction Using a xenogeneic in-vitro culture system enabled us to determine the contribution of each individual cell type (i.e. h BMSCs, h BMSCs or b ACs) to cartilage matrix production using species- specific gene-expression analyses. First, GAPDH- gene expression was analyzed after 5 weeks of in-vitro culture. h AMSC/ b ACs and h BMSC/ b ACs contained cells from both bovine (AC) and human (AMSC or BMSC) origin. (Figure 5A) Then, chondrogenic gene expression was evaluated by the ACAN and COL2A1 genes. In a growth-factor-free environment, h AMSCs and h BMSCs hardly expressed hsACAN and hsCOL2A1 . Besides, chondrogenic genes were hardly expressed in h AMSC/ b ACs or h BMSC/ b ACs either. Conversely, h AMSC/ b ACs or h BMSC/ b ACs - containing solely 20% bovine articular chondrocytes - expressed as much or even higher levels of bsACAN compared to 100% b ACs ( h AMSC/ b ACs vs b ACs p >0.05 ; h BMSC/ b ACs vs b ACs p <0.001). h AMSC/ b ACs and h BMSC/ b ACs expressed COL2A1 , although gene-expression of hsCOL2A1 was negligible. This means that the COL2A1 expressed was from bovine origin. (Figure 5B) These data indicate that the formed cartilage matrix was from b AC-origin, which suggests a more trophic role for h MSCs herein. Cellular interactions To further understand the complex cellular interaction between h MSCs and b ACs, cells were encapsulated in separate alginate constructs and co-cultured in a Transwell® system as well as in medium conditioned by the other cell type. (Figure 6A and 7A) In addition cell combination were also cultured in pellets, allowing direct cell-cell contact. Alginate constructs containing solely b ACs, h AMSCs or h BMSCs cultured in ‘basic medium’ maintained their DNA content over the 3 weeks of culture. Exposure to paracrine factors of b AC via Transwell® system or b AC-conditioned medium, did not alter the amount of DNA in alginate constructs seeded with either h AMSCs or h BMSCs. (Figure 6B) The presence of factors secreted by h MSC significantly increased the total amount of DNA in constructs containing b ACs ( p <0.01). This effect was independent on the origin of the h MSCs (i.e. h AMSCs, h BMSCs) and co-culture system used (i.e. Transwell® system, h MSC-conditioned medium). (Figure 7B) This suggests MSC have paracrine effects on chondrocytes. Alginate constructs containing h AMSCs or h BMSCs, formed very little sGAG after 3 weeks of culture. sGAG-production remained similarly low when h MSC-constructs were cultured in the presence of paracrine factors of b AC via Transwell® system or b AC-conditioned medium. (Figure 6B) The production of sGAG was higher in constructs containing b ACs. Exposure to paracrine factors of h MSC significantly increased sGAG-production, irrespective to the h MSC-source used (i.e. h AMSCs, h BMSCs, p <0.01). Since the amount of DNA was also enhanced in these constructs, sGAG content was adjusted to the amount of DNA, still showing pronounced differences. sGAG formation was significantly increased in Transwell® system compared to constructs cultured with b AC-conditioned medium ( p <0.01). (Figure 7B) Similar trends were observed at COL2A1 gene-expression level. (Figure 7D) This provides further indications that the effect of the combination of h MSCs and b ACs on chondrogenesis is due to paracrine effect of h MSCs on chondrocytes. 98 CHAPTER 5