15502-m-pleumeekers

DISCUSSION A novel bilayer BNC scaffold was successfully evaluated for auricular cartilage TE. This study demonstrates that non-pyrogenic and non-cytotoxic bilayer BNC scaffolds offer a good mechanical stability and maintain a structural integrity while providing a porous architecture that supports cell ingrowth. Moreover, bilayer BNC scaffolds, together with alginate, provide a suitable environment for human nasoseptal chondrocytes to form cartilage. As shown by the endotoxin analysis, the purification process reduced the endotoxins in the bilayer BNC scaffolds to a level well below the endotoxin limit set by the FDA for medical devices [352]. This low endotoxin content (0.15 ± 0.09 EU/ml) found in the bilayer BNC scaffolds is in good agreement with our previous results (0.10 EU/ml, [316]), where densified BNC hydrogel disks (i.e. dense layer) of similar dimensions were considered non-pyrogenic after purification with endotoxin-free water for 14 days. The ionic liquid EMIMAc offers a novel cellulose solvent system to achieve a strong interfacial molecular bonding between the cellulosic dense and porous layers. We are not aware of any other methods that can achieve such result. On the other hand, using EMIMAc increases the risk of having cytotoxic compounds in the bilayer BNC scaffolds, if these residues are not properly removed during the purification process. Since the toxicity of ionic liquids is not well understood, the use of EMIMAc to fabricate the bilayer BNC scaffolds was investigated with precaution. The cytotoxicity of imidazole ionic liquids has been studied in a human lung carcinoma epithelial cell line model, and it was found that the alkyl-chain length of the ionic liquid has an influence on cytotoxicity [357]. However, the cytotoxicity of EMIMAc, in particular, has not been studied in eukaryotes. Consequently, we analyzed the removal of EMIMAc from the bilayer BNC scaffolds with ATR-FTIR spectroscopy, followed by in-vitro cytotoxicity testing with sensitized L929 cells. The strong peak at wavenumber 1566 cm -1 was used to detect EMIMAc residues in the ATR spectra of bilayer BNC scaffolds, as it has been shown that this peak is composed of two overlapped peaks that correspond to the carboxyl group of the acetate and an underlying ring mode of the cation [355, 356]. Since the peak at 1566 cm -1 was found in the ATR spectra of bilayer BNC scaffolds that had been washed for 1 and 7 days, it was considered necessary to continue washing the scaffolds in endotoxin-free water. The washing process proved to be successful in removing the EMIMAc residues, as first observed in the ATR spectra of samples that were washed for 14 days. The absence of this peak confirmed the removal of EMIMAc from the BNC bilayer scaffolds. In-vitro cytotoxicity testing supported our findings from ATR-FTIR. Bilayer BNC scaffolds washed for 7 days still had residues of EMIMAc that were highly cytotoxic to L929 cells (cell viability: 18.4 ± 3.6%). However, these residues were further reduced after the purification process with endotoxin-free water; yielding non-cytotoxic bilayer BNC scaffolds. These results are in good agreement with our previous study which evaluated the cytotoxic potential of pure densified BNC hydrogel disks (i.e. dense layer) and found the material to be non-cytotoxic [316]. Altogether, the results from ATR-FTIR and in vitro cytotoxicity testing demonstrated that EMIMAc residues were successfully removed from the bilayer BNC scaffolds after the purification process with endotoxin-free water; whereat no peak at 1566 cm -1 and no cytotoxic effects were observed. 167 NOVEL BILAYER BNC: AN ECM-INSPIRED SCAFFOLD 8