15502-m-pleumeekers

weeks. ACAN and COL2A1 expression after 6 weeks was 3.4- and 4.9-fold higher, respectively, compared to gene expression levels at week 2. Expression of COL1A1 was also upregulated during 3D culture, where after 6 weeks was 1.7-fold higher compared to gene expression levels at week 2. On the other hand, expression of VCAN remained relatively close to zero during the 6 weeks of 3D culture. (Figure 4E, F) The upregulation of the chondrogenic markers ACAN and COL2A1 was clearly enhanced compared to the expression of dedifferentiation markers, revealing the chondrogenic potential of the NCs in the bilayer BNC scaffolds. Cell ingrowth and cell distribution in the bilayer BNC scaffolds were also assessed by histological analysis. As demonstrated in figure 4A, the porous layer supported the ingrowth of NCs and facilitated a homogeneous cell distribution. However, it took 4 weeks of in-vitro culture to get a dense and homogenous cell distribution since there was a substantial loss of cells after seeding in medium. As a means to increase the number of cells retained in the scaffolds, cells were seeded in alginate solution. This significantly improved cell retention in the scaffolds, even after 1 day of seeding. (Data not shown) Performance of bilayer BNC scaffolds and neocartilage formation in vivo The stability and neocartilage formation in MNC/NC-seeded and cell-free bilayer BNC scaffolds were evaluated after 8 weeks of subcutaneous implantation in nude mice. The mice survived until the end of the study period, during which no extrusion of constructs was observed. At 8 weeks post-implantation, a thin fibrous capsule surrounded all MNC/NC-seeded and cell-free bilayer BNC scaffolds - considered a normal non-pathological foreign body reaction. Macroscopic examination of the explants revealed that the shape and size of the bilayer BNC scaffolds remained stable and no delamination of the dense and porous layers was observed in any of the constructs. Furthermore, bilayer BNC scaffolds seeded with MNCs and NCs encapsulated in alginate had a macroscopically cartilage-like appearance. These MNC/NC- seeded constructs were stiffer and more stable upon handling, compared to bilayer BNC scaffolds seeded with cell-free alginate solution. The cells encapsulated in alginate were homogeneously distributed in the porous layer of the scaffolds at 8 weeks post-implantation, as observed by the histology images. (Figure 5B) Proteoglycan synthesis was examined using a Safranin-O staining. As expected, no positive stain for Safranin-O was found in the cell-free bilayer BNC scaffolds. Depositions of proteoglycans were observed in the MNC/NC-seeded bilayer BNC scaffolds after 8 weeks of subcutaneous implantation, as shown by the strong Safranin-O stain surrounding the cells. (Figure 5B) The results pointing towards chondrogenic ECMproduced by the cells in the bilayer BNC scaffolds were confirmed by the positive immunostaining of type II collagen, which was intensely stained in areas of the construct. (Figure 5B) Moreover, a Kolmogorov-Smirnov test indicated a significant difference ( p<0.05 ) between mean sGAG content for MNC/NC-seeded (0.87 ± 0.65 μg sGAG/mg wet weight) and cell-free bilayer BNC scaffolds (0.07 ± 0.11 μg sGAG/mg wet weight). sGAG-production in the MNC/NC group was almost 12-fold higher compared to the control condition. (Figure 5C) 163 NOVEL BILAYER BNC: AN ECM-INSPIRED SCAFFOLD 8