15502-m-pleumeekers

Figure 3. Purification and cytotoxicity testing of bilayer BNC scaffold. (A) ATR spectra of (1) 1-Ethyl-3-methylimidazolium acetate (EMIMAc) ; bilayer BNC scaffolds after (2) 1 day, (3) 7 days and (4) 14 days of purification in endotoxin-free water ; and (5) pure BNC. (B) In vitro cytotoxicity testing of bilayer BNC scaffolds after 7 and 14 days of purification in endotoxin-free water ( n =4 per time point). Post hoc comparisons using the Tukey HSD test indicated that the mean cell viability for the 14-day condition (97.8 ± 4.7%) was significantly higher than the 7-day (18.4 ± 3.6%) and positive control conditions (25.6 ± 5.5%) at the * p < 0.05 level. Furthermore, there was no significant difference between the negative control and 14-day conditions. Thus, the cytotoxic potential of bilayer BNC scaffolds after 14 days of purification was classified as non-cytotoxic (cell viability >71%). Error bars represent the standard deviation of the mean. Performance of bilayer BNC scaffolds and neocartilage formation in vitro Gross examination of the cell-seeded constructs throughout the 6 weeks of cell culture revealed that the adhesion between the dense support layer and porous layer remained good. Moreover, the size and shape of the bilayer BNC scaffolds remained stable during the cell culture. Deposition of ECM by the NCs seeded in bilayer BNC scaffolds was assessed qualitatively by immunohistological staining. During 3D culture, NCs produced and accumulated cartilage-specific ECM components in the bilayer BNC scaffolds. A positive staining for sGAGs was found around clusters of chondrocytes in the porous layer after 2 weeks of culture, as shown by the Alcian blue staining. (Figure 4A) Moreover, synthesis and accumulation of sGAGs, aggrecan as well as type II collagen increased visibly during 3D culture. (Figure 4A-C) After 6 weeks of 3D culture, a homogeneous production of chondrogenic ECM was observed throughout the porous layer, even at the center. However, fibrocartilage ECM was also synthesized by the NCs in the bilayer BNC scaffolds, as demonstrated by the positive immunostaining of type I collagen. (Figure 4D) The capacity of NCs to synthesize cartilage-specific ECM components when seeded in the BNC scaffold was also investigated on the basis of the expression of the chondrogenic marker genes ACAN and COL2A1 . To assess whether the NCs were able to redifferentiate and maintain their chondrogenic phenotype, the expression not only of the chondrogenic markers but also of the dedifferentiation markers, VCAN and COL1A1 , was determined. Gene expression analyses confirmed the positive immunostains of aggrecan, type II and type I collagen. NCs cultured in bilayer BNC scaffolds were able to express ACAN and COL2A1 . The expression of both chondrogenic markers increased clearly during 3D culture for up to 6 162 CHAPTER 8