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were decellularized in batches. Samples intended for histological analysis and SEM were stored in 4% formaldehyde and samples for biochemical analysis were stored dry at -80°C. Samples intended for biomechanical analysis were shipped to Eidgenössische Technische Hochschule (ETH; Zurich, Switzerland) in PBS containing protease inhibitors (Roche, Basel, Switzerland) at 4°C. Decellularized b EC scaffolds intended for seeding ( n =1, in 6-fold) were pre-conditioned for at least 2 hours in Minimally Essential Medium Alpha (MEM-α; Gibco, Carlsbad, USA) containing 10% fetal calf serum (FCS; Lonza, Verviers, Belgium), 50 µg/mL gentamicin (Gibco) and 1.5 µg/mL amphotericin B (Fungizone; Gibco) and stored at 4°C until seeding. Biochemical analysis Prior to biochemical analysis, wet weight was determined for all cartilage samples. For DNA, sGAG and collagen analysis, samples were digested overnight at 60°C in a papain solution (0.2 M Na 2 H 2 PO 4 , 0.01 M EDTA.2H 2 O, 250 µg/mL papain, 5 mM L-cystein, pH 6.0). Bovine and human cartilage samples were digested in 400 and 500 µL papain solution, respectively. To assess the removal of nuclear components, the DNA content of the cartilage scaffolds was measured with the CyQUANT® (Invitrogen) proliferation assay. This assay is able to detect low amounts of DNA and has a detection limit of 10 ng per measurement. In short, 250 IU heparin (LEO Pharma, Ballerup, Denmark) and 125 µg RNAse were added to the papain digests and incubated for 30 minutes at 37°C. Finally, 0.375 µL CyQUANT GR dye was added to each papain digested sample and fluorescence was immediately measured (excitation/emission: 480/520 nm) on a SpectraMax Gemini micro plate reader (Molecular Devices, Sunnyvale, USA), using calf thymus DNA as a standard. A 1,9-Dimethylmethylene blue (DMMB; pH 3.0) assay [292] was performed to measure the sulphated GAG content of the cartilage scaffolds. The metachromatic reaction of DMMB was monitored using a VersaMax spectrophotometer at 530 and 590 nm. Shark chondroitin sulphate C was used as a standard. A hydroxyproline assay [213] was performed to measure the total amount of collagen of the cartilage scaffolds. In short, the papain digests were hydrolyzed with equal volumes of 12 M HCl at 108°C for 20 hours, dried (Savant SPD 121P SpeedVac; Thermo Scientific, Massachusetts, USA) and re-dissolved in 1.5 mL Mili-Q water. Hydroxyproline contents were measured using a colorimetric method (extinction 570 nm), with chloramine-T and dimethylaminobenazldehyde as reagents. Hydroxyproline (Merck) was used as a standard to calculate the amount of collagen per sample. Elastin content of the cartilage samples was measured using the Fastin TM Elastin Assay (Biocolor, Carrickfergus, UK) according to manufacturer’s instructions. Briefly, cartilage samples were converted to water soluble α-elastin by 3 overnight heat extraction cycles at 100°C in 0.25M oxalic acid before adding the kit’s dye. Absorption was measured at 513 nm on a VersaMax plate reader. α-elastin from bovine neck ligament (provided by manufacturer) was used as a standard. 133 DECELLULARIZED CARTILAGE: AN ECM-DERIVED SCAFFOLD 7