15502-m-pleumeekers

dissolved in ionic liquid 1-ethyl-3-methylimidazolium acetate (EMIMAc) at a concentration of 10 mg/ml. The cellulose solvent solution was preheated to 80°C and then smeared on the dense BNC layers. Subsequently, the porous layers were aligned on top of the dense layers and the bilayer BNC scaffolds were placed on a heating plate at 80°C for 2 minutes to accelerate the dissolution of nanocellulose at the interface. The bilayer BNC scaffolds were then stabilized in 100 mM CaCl 2 in ethanol to precipitate the dissolved cellulose between the layers (i.e. attach the layers), while simultaneously crosslinking the alginate to bind the BNC in the porous layer. The scaffolds were then rehydrated and washed in non-pyrogenic conical tubes (TPP, Switzerland) with endotoxin-free water (HyClone™ cell culture-grade water, Thermo Fisher Scientific) supplemented with 20 mM CaCl 2 to remove residuals of the ionic liquid EMIMAc and endotoxins. The scaffolds were purified under orbital motion (320 rpm) at 37°C for 14 days, during which the endotoxin-free water and conical tubes were changed every second or third day. Subsequently, the bilayer BNC scaffolds (Ø 8 mm × 3 mm) were steam sterilized (as described above) in endotoxin-free water and stored until use at 4°C. Characterization of bilayer BNC scaffolds The morphology of bilayer BNC scaffolds was characterized by scanning electron microscopy (SEM) and micro–computed tomography (microCT). Moreover, the purity of the bilayer BNC scaffolds was analyzed throughout the purification process by bacterial endotoxin testing, infrared spectroscopy analysis and in vitro cytotoxicity testing. Scanning electron microscopy Samples were lyophilized (as described previously), thereafter sputter coated with a gold film and analyzed using a Leo Ultra 55 field emission gun SEM (Carl Zeiss, Germany). Micro–computed tomography Bilayer BNC scaffolds ( n =3) were incubated in 0.1 M CaCl 2 solution at room temperature overnight and subsequently quenched in liquid nitrogen and lyophilized for 24 hours. The dry scaffolds were scanned with microCT (µCT50, Scanco Medical AG, Switzerland) at 45 kVp and 1 µm nominal resolution. The internal microstructure of the porous layer was then segmented automatically using a constrained Gaussian filter to suppress noise and a global threshold (25% of maximal grayscale value). 3D morphometric parameters such as scaffold porosity (Sc.Po), volume-weighted mean pore size (Pore.Th), scaffold wall thickness (Wall.Th), and scaffold wall number (Wall.N) were calculated using the manufacturer's morphometry software (IPL, Scanco Medical AG) according to the guidelines established for the assessment of bone microstructure. [351] Bacterial endotoxin testing Endotoxin extraction from the bilayer BNC scaffolds was done in accordance to the international standard ISO 10993-12:2009 (Sample preparation and reference materials). After 14 days of purification, bilayer BNC scaffolds ( n=3 ) were weighed and placed in depyrogenated sample containers (Lonza, Belgium). Endotoxin-free water was added to the containers using the ratio of 0.1 grams of BNC/ml of extraction medium. The extraction was 152 CHAPTER 8