15502-m-pleumeekers

RESULTS Cartilage regeneration in co-cultures In vitro outcomes After 3 weeks, DNA content of alginate constructs containing either co-cultures of h MSCs and b ACs or single-cell-type populations, did not change in relation to their initial DNA content. (Figure 2A) Because the amount of DNA had not changed significantly in any of the conditions, matrix deposition was expressed per construct and per initially seeded primary ACs. After 5 weeks, DNA content did significantly decrease in constructs containing h BMSCs only ( p <0.001), but remained unchanged in the remaining culture conditions. (Supplementary figure 1) Since constructs were cultured in the absence of chondrogenic factors, constructs containing solely h AMSCs or h BMSCs produced very little sGAG (Figure 2B) and collagen (Figure 2C). To demonstrate the additional effect of h MSCs in mixed-cell-type populations, a control condition - containing similar numbers of b ACs (0.8*10 6 nc/mL) without the supplementation of h MSCs - was evaluated (Figure 2 dotted lines). The addition of either h AMSCs or h BMSCs to b ACs demonstrated a significant increase in the production of sGAG over their controls ( h AMSC/ b ACs p =0.018 ; h BMSC/ b ACs p <0.001). Compared to constructs containing single-cell-type populations, the deposition of sGAG was most evidently enhanced in co-cultures combining h BMSCs and b ACs ( p <0.001). Constructs containing h AMSC/ b ACs deposited significantly less sGAG compared to h BMSC/ b ACs ( p <0.001) and equal amounts compared to constructs containing b ACs only. (Figure 2B) The production of collagen was enhanced in co-cultures of both h AMSC/ b ACs and h BMSC/ b ACs compared to single-cell-type populations ( h AMSC/ b ACs p =0.002 ; h BMSC/ b ACs p <0.001). (Figure 2C) Normalization of the total sGAG content to the initially seeded primary ACs revealed even more distinct differences between co-cultures and single-cell-type populations: h BMSC/ b ACs produced significantly more sGAG compared to b ACs only and co-cultures of h AMSC/ b ACs (both p <0.001) ; collagen production was significantly enhanced in both co-cultures ( h AMSC/ b ACs p =0.013 ; h BMSC/ b ACs p <0.001). (Figure 2B and 2C) Similar results were obtained after 5 weeks of culture. (Data not shown) These results demonstrate that co-cultures of h MSCs and b ACs improve cartilage formation in vitro , depending on the h MSC-source used ( h BMSC ≥ h AMSC). 94 CHAPTER 5