15502-m-pleumeekers

Figure 4. Performance of bilayer BNC scaffolds and neocartilage formation in vitro . Histological and immunohistochemical analysis of human NCs after 2, 4 and 6 weeks of culture in vitro on bilayer BNC scaffolds. Samples were stained with (A) Alcian blue to detect deposition of sGAG ( PL = porous layer). Immunohistochemical staining was used to detect cartilage-specific proteins such as (B) aggrecan, (C) collagen type II and (D) the dedifferentiation marker collagen type I. (E and F) Gene-expression analysis of human NCs seeded in bilayer BNC scaffolds and cultured in vitro for 2, 4 and 6 weeks ( n =4 per time point). Gene-expression levels of ACAN , COL2A1 , COL1A1 and VCAN relative to the housekeeping gene glyceraldehyde 3-phosphate dehydrogenase ( GAPDH ). Error bars represent the standard deviation of the mean. The scale bar indicates 1 mm. Biomechanical analysis A typical stress relaxation behavior was observed in all bilayer BNC scaffolds (MNC/NC-seeded and cell-free controls), and the following measurements were determined for the MNC/NC- seeded constructs at 8 weeks post-implantation; 0.76 ± 0.19 MPa for Ein, 0.19 ± 0.08 MPa for Eeq, 0.16 ± 0.07 MPa for σ max and 7.1 ± 3.4 seconds for t 1/2 . A one-way ANOVA was conducted to compare the effect of implantation and seeding of MNC/NCs on initial matrix stiffness (i.e. Ein) of the constructs at 8 weeks post-implantation. There was a significant effect of 164 CHAPTER 8