15502-m-pleumeekers

MATERIALS AND METHODS Chemicals were obtained from Sigma-Aldrich, USA unless stated otherwise. Fabrication and purification of bilayer BNC scaffolds Production of dense and porous scaffold layers BNC hydrogel disks with increased cellulose content (i.e. dense layer) were produced and purified as described elsewhere [316]. Briefly, a suspension of Gluconacetobacter xylinus (ATCC ® 700178, LGC Standards, Sweden) was inoculated in 250 ml conical flasks containing sterile culture medium (described by Matsuoka et al. [349]) and cultured at 30°C for 18 days, until large BNC cylinders (Ø 48 mm × 20 mm) were biosynthesized. The BNC cylinders were purified in a built-in-house perfusion system and compressed to 1 mm in height to increase the cellulose content. The compressed BNC pellicles were frozen to -80°C overnight and lyophilized (Heto PowerDry PL3000, Thermo Fisher Scientific, MA, USA) for 3 days. Dense BNC disks (Ø 8 mm × 1 mm) were then cut with a sterile biopsy punch (Miltex GmbH, Germany). The criterion for selecting the thickness of the dense BNC layer is based on morphometric analysis from MRI scans of human auricular cartilage, where Nimeskern et al. reported a cartilage thickness of 1.15 ± 0.10 mm [350]. BNC/alginate composite scaffolds (i.e. porous layer) were fabricated by a freeze-drying process. First, purified BNC pellicles were homogenized with a blender, until a pulp consistency was obtained, and then with a dispersing element (S25N-18G, IKA, Germany) at 25,000 rpm for 20 minutes. Afterwards, the homogenized BNC suspension was steam sterilized (100 kPa, 121°C for 20 minutes) and the cellulose content was determined using a halogen moisture analyzer (HB43, Mettler-Toledo, OH, USA). The following steps were carried out in sterile conditions. The BNC suspension was mixed with 1.1% w/w clinical grade alginate dissolved in 0.9% NaCl (CellMed AG, Germany) to get a final composition of 90% dry weight BNC and 10% dry weight alginate compared to the total dry weight. The weight of alginate solution (W Alg ) added to a known weight of BNC suspension (W BNC ) was calculated by using the formula: W Alg = W BNC × (%DW Alg ÷ %DW BNC ) × (%CC i ÷ %AC i ). Where %DW Alg and %DW BNC are the targeted percent dry weight of alginate (10%) and BNC (90%) compared to the total dry weight; and %CC i and %AC i are the initial cellulose and alginate concentrations. The BNC/alginate mixture was then dispersed at 25,000 rpm for 15 minutes, transferred to sterile containers (TP52, Gosselin, France) and degased in a vacuum desiccator. The containers were then placed inside Nalgene ® cryo freezing containers (Thermo Fisher Scientific) and frozen to -80°C overnight at a rate of 1°C/min. The frozen BNC/alginate mixtures were lyophilized for 5 days to sublimate the ice crystals, creating a macroporous architecture. The dry BNC/alginate sponges were then sliced to 2 mm-thick slices and porous BNC/alginate composite scaffolds (Ø 8 mm × 2 mm) were cut with a sterile biopsy punch (Miltex GmbH). Fabrication of bilayer BNC scaffolds A novel cellulose solvent system (i.e. ionic liquid EMIMAc) was used to attach the dense and porous layers and achieve a strong interfacial molecular bonding between the layers. The following steps were carried out in sterile conditions. First, dry homogenized BNC was 151 NOVEL BILAYER BNC: AN ECM-INSPIRED SCAFFOLD 8