15502-m-pleumeekers

RESULTS Cell expansion The cell sources showed clear differences in growth rate. NCs proliferated significantly faster than ECs, ACs and AMSCs ( p <0.05). NCs had gone through 8.9 ± 1.7 population doublings (PD) in four passages taking 28 ± 5 days, ECs had gone through 6.8 ± 1.3 in 38 ± 6 days and ACs through 3.9 ± 1.1 PD in 44 ± 13 days. It took 39 ± 8 days for BMSCs and 48 ± 8 days for AMSCs to complete four passages. (Table 3) PD/D Statistically significantly different from EC 0.18 ± 0.04 NC ( p =0.015) ; AC ( p =0.008) NC 0.32 ± 0.07 EC ( p =0.015) ; AC ( p <0.001) ; AMSC ( p =0.013) AC 0.10 ± 0.05 EC ( p =0.008) ; NC ( p <0.001) ; BMSC ( p =0.001) BMSC 0.25 ± 0.09 AC ( p =0.001) AMSC 0.16 ± 0.04 NC ( p =0.013) Table 3. Population Doubling Time of different cell types over four passages. NCs proliferated faster than ECs, ACs and AMSCs. The proliferation rate of BMSCs did not differ from AMSCs. Data are shown as mean ± SD. PD/D = Population Doublings per Day (PD/D = (ln (N2/N1) / ln (2))/D) ; EC = Ear Chondrocytes ( n=3 donors) ; NC = Nasal Chondrocytes ( n=3 donors) ; AC = Articular Chondrocytes ( n=3 donors) ; BMSC = bone-marrow-derived Mesenchymal Stem Cells ( n=3 donors) ; AMSC = adipose-tissue-derived Mesenchymal Stem Cells ( n=3 donors). Chondrogenic differentiation in vitro After cell-expansion, cells were encapsulated in clinical-grade alginate to promote chondrogenesis. Alginate beads cultured without TGFβ1 had maintained their DNA content after five weeks of culture. Addition of TGFβ1 significantly increased the total amount of DNA in alginate beads seeded with ECs and NCs ( p <0.001), which was also significantly higher compared to the other cell sources ( p <0.05), indicating that those cells were able to proliferate after encapsulation in alginate. The other cell conditions remained at a stable cell content. (Figure 1A) Chondrocytes did express low levels of COL2A1 and ACAN without TGFβ1. After chondrogenic induction (with TGFβ1), the COL2A1 and ACAN gene expression levels increased in all cell source used . Both genes were most highly expressed by ACs ( p <0.001), followed by BMSCs. (Figure 1B) This was already seen after two weeks of culture (data not shown), suggesting that chondrogenesis was not only enhanced but also accelerated. Matrix production was quantified by sGAG and collagen content of alginate beads during in- vitro culture. Without TGFβ1 very little sGAG was formed in vitro . Addition of TGFβ1 enhanced sGAG-production and after five weeks of culture ACs deposited significantly more sGAGs (p <0.01). When sGAG content was adjusted to the amount of DNA, similar but more pronounced differences were observed (ACs produced most sGAGs: 60.89 ± 53.04 μg sGAG / μg DNA; p <0.001). sGAG content per alginate bead in constructs containing BMSCs, ECs, NCs 70 CHAPTER 4