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Figure 5. Performance of bilayer BNC scaffolds and neocartilage formation in vivo . (A) Photographs of cell-free and MNC/NC-seeded bilayer BNC scaffolds after 8 weeks of subcutaneous implantation. (B) Histological evaluation of ECM in bilayer BNC scaffolds seeded with a combination of freshly isolated NCs and MNCs in alginate after 8 weeks of subcutaneous implantation. Safranin-O stain was used to examine proteoglycans present in the newly synthesized ECM, whereas immunohistochemical analysis was used to detect type II collagen. (C) Cell-free and MNC/NC-seeded bilayer BNC scaffolds were analysed for sGAG content after 8 weeks of subcutaneous implantation ( n =4 per group). A two-sample Kolmogorov-Smirnov test indicated a significant difference between mean sGAG content for MNC/NC-seeded and cell-free bilayer BNC scaffolds. (D) Biomechanical evaluation of cell-free and MNC/NC-seeded constructs after 8 weeks of subcutaneous implantation ( n =4 per group), and non-implanted bilayer BNC scaffolds with cell-free alginate solution ( n =5). Post hoc comparisons using the Tukey HSD test indicated that the mean Ein for the MNC/NC condition was significantly higher than the non-implanted and cell-free conditions. Moreover, post hoc comparisons using the Mann Whitney tests indicated that the median t1/2 for the MNC/NC condition was significantly higher than the non-implanted and cell-free conditions. Error bars represent the standard deviation of the mean. *, ** or *** indicates p-values less than 0.05, 0.01 or 0.001, respectively. The scale bars indicate 4 mm (A) and 1 mm (B) . 166 CHAPTER 8