Joris van Dongen

288 Chapter 12 Fibroblast scratch assay NAMand DAMhydrogels (n=3) were produced as previously described (three technical replicates) (2.6). PK84 fibroblasts were cultured in DMEM (containing 10% FBS, 1% L-Glut, 1% P/S). After confluency, PK84 fibroblasts were seeded into a 24-wells plate with a cell density of 17,500 cells/cm 2 . Then, cells were serum-starved overnight. The next day, a scratch was made with the use of a 1000 µl pipette tip. Subsequently, cells were washed with PBS three times and all the different types of medium were applied. Scratches were analysed after 9h. Fibroblast proliferation assay NAMand DAMhydrogels (n=3) were produced as previously described (three technical replicates) (2.6). PK84 fibroblasts were cultured in DMEM (containing 10% FBS, 1% L-Glut, 1% P/S). After confluency, fibroblasts were seeded into a 96-wells plate with a cell density of 17,500 cells/cm 2 . After 24h, the different types of medium were applied for 24h (2.6). After 24h, 20µl of MTT (5 mg/ml in PBS) was added and incubated for 3h at 37 °C. Next, mediumwas removed and 100 µl of dimethyl sulfoxide was added to dissolve the formazan crystals. Plates were read at 570 nm. The fibroblast proliferation assay was repeated twice. Results were expressed as percentage of cell proliferation compared to normal culture medium (DMEM containing 10% FBS, 1% L-Glut, 1% P/S). Human umbilical vein endothelial cell sprouting assay NAMand DAMhydrogels (n=3) were produced as previously described (three technical replicates) (2.6). Instead of DMEM serum free, RPMI serum free incubated hydrogels were used as a control (2.6). Human umbilical cord vein endothelial cells (HUVEC) were isolated and cultured in RPMI (containing 10% FBS, 1% L-Glut, 1% P/S). µ-Slide Angiogenesis plates (Ibidi GmbH, Germany) were coated with 10 µl of Matrigel TM (BD Bioscience, CA) and incubated at 37 °C for 2h. HUVECwere resuspended in all different types of medium. 60 µl of medium containing 10,000 cells were added per well. The slides were incubated at 37 °C for 6h. The sprouting networks that had formed were photographed with the use of an inverted light microscope (Leica Microsystems, DM IL) and the number of loops was counted manually. Statistics Scratch assay images were analysed using ImageJ, version 1.4.3.67 (NIH). Descriptive statistics were used to evaluate the amount of DNA, sGAG, stiffness and factor release profile of the hydrogels, scratch surface area, fibroblast proliferation as well as the number of loops. Data was expressed as mean with standard error of the mean, except