15502-m-pleumeekers

done at 37 ± 1°C for 72 ± 2 hours under orbital motion at 160 rpm. Endotoxin analysis was performed with the PyroGene TM Recombinant Factor C assay by Lonza. This assay has a minimum detection limit of 0.005 Endotoxin Units (EU) per milliliter. According to the USA Food and Drug Administration [352], endotoxin levels in medical devices are not to exceed 0.5 EU/ml or 20 EU/device [352]. Attenuated Total Reflectance Fourier Transform Infrared spectroscopy Removal of EMIMAc residues from the bilayer BNC scaffolds was analyzed with Attenuated Total Reflectance Fourier Transform Infrared (ATR-FTIR) spectroscopy. Samples ( n=2 per group) were freeze-dried after day 1, 7 and 14 of purification. The porous layer was removed from the bilayer BNC scaffolds to expose the interface. This interface, visible on the dense BNC layer, was analyzed with a single reflection ATR accessory fitted with a monolithic diamond crystal (GladiATR™, Pike Technologies, WI, USA). The sample was placed on the small crystal area and a force was applied on the sample to push it onto the diamond surface. ATR-FTIR spectroscopy measurements were made with a System 2000 FT-IR spectrometer (PerkinElmer, MA, USA) in the mid-infrared region, 4000 to 400 cm -1 . 20 scans were taken with a resolution of 4 cm -1 . Pure EMIMAc solution and pure dried BNC films were used as controls. In-vitro cytotoxicity testing Removal of EMIMAc residues from the bilayer BNC scaffolds was also evaluated by in vitro cytotoxicity testing, according to the international standard ISO 10993-5:2009. Bilayer BNC scaffolds ( n=4 per time point) were incubated in growth medium (RPMI 1640 medium supplemented with 1% fetal bovine serum (FBS), and antibiotics (100 U/ml penicillin and 100 µg/ml streptomycin); Biochrom, Germany) for 24 hours to extract potential cytotoxic residues. All incubations were done in standard culture conditions (37°C, 5% CO 2 and 95% relative humidity). Meanwhile, sensitized L929 cells (ACC 2, DSMZ, Germany) were seeded in 96-well cell culture plates (1.0 × 10 4 cells per well) and incubated for 24 hours to allow cell adhesion. The medium was removed and 100 µl of extract or control solutions was added to each well and incubated for 24 hours. Cell culture inserts (ThinCert TM , Greiner BioOne, Germany) incubated in growth medium served as negative control ( n=8 ), while 10% dimethylsulfoxide in growth medium served as positive control ( n=8 ). After 24 hours of incubation in extract or control solutions, the mediumwas removed, 100 µl mediumwere mixed with 20 µl of CellTiter 96 ® AQueous one solution reagent (Promega, WI, USA) MTS and added to each well, followed by incubation for 2 hours at 37°C. Growth mediumwith reagent solution (without cells) served as blank ( n=8 ). After incubation with the reagent, absorbance was measured photometrically (Infinite M200 Pro, Tecan AG, Switzerland) at a wavelength of 490 nm and a reference wavelength of 680 nm. The average absorbance value of the negative control was used to compute the cell viability, where the negative control was regarded as 100% viability. The cytotoxic potential of the test samples was classified as highly cytotoxic when cell viability was below 50%, slightly cytotoxic when it was between 51% and 70% and non-cytotoxic when cell viability was above 71%. 153 NOVEL BILAYER BNC: AN ECM-INSPIRED SCAFFOLD 8