15502-m-pleumeekers

Prior to implantation, constructs pre-cultured without TGFβ1 produced very little sGAG in vitro , being on average 1,10 ± 1,20 µg sGAG per construct. After in vivo implantation, these constructs greatly increased their production of matrix components, although they did not reach levels which equaled the matrix content found in constructs cultured with TGFβ1. (Figure 2B) After subcutaneous implantation preceded by chondrogenic culture (with TGFβ1), ACs, BMSCs and AMSCs retained their sGAG content but did not further increase it. On the contrary, ECs and NCs significantly enhanced matrix formation in vivo (EC 7.26-fold and NC 2.86-fold; both p <0.001) leading to a superior sGAG-deposition after implantation compared to the other cell sources (both p <0.001). These results were further confirmed by a Thionin- staining (data not shown). Total collagen deposition was hugely increased after implantation and no significant differences could be detected between the different cell sources. (Figure 3B) Constructs containing ACs, BMSCs or AMSCs exhibited a very weak staining for cartilage-specific collagen type II (Figure 3C), which was in contrast to the overall production of collagens (Figure 3B), thus indicating that other collagens were also produced (e.g. collagen type I or type III). The cartilage matrix of constructs containing ECs and NCs showed a strong staining for collagen type II, although the dissimilar distribution of collagen-type-II fibers within the cartilage matrices were apparent. The semi-quantitative histological scores of constructs containing ECs or NCs were significantly better than the scores of the other cell sources. (Figure 3C) The presence of elastin was determined to evaluate differentiation into elastic cartilage. There was no elastin detectable in any of the constructs with an elastin immunostaining after five weeks of in-vitro cell culture. (Data not shown) After subcutaneous implantation, elastin was only present in alginate constructs containing ECs, and predominantly found in constructs which were pre-cultured with TGFβ1. Most elastin was located around the cell. (Figure 3D) To ensure that these cartilage constructs were from human origin, a human-specific vimentin stain was used on histological sections. It confirmed that the cartilage constructs were indeed of human origin (Figure 3E), while the surrounding fibrous tissue was not (data not shown). 73 CARTILAGE-FORMING CAPACITY OF SEVERAL CELL SOURCES 4