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Schweitzer Hospital 2011/7). Cells were cultured at a density of 2300 cells/cm 2 at 37°C and 5% CO 2 in MEM-α, containing 10% FCS, 50 µg/mL gentamicin, 1.5 µg/mL Fungizone, 25 µg/mL L- ascorbic acid 2-phosphate and 1 ng/mL basic Fibroblast Growth Factor 2 (bFGF2; R&D Systems, Minneapolis, USA), from now on referred to as ‘MSC-expansion medium’. For toxicity tests, BMSCs were plated in a 24-well plate at a density of 40,000 cells/cm 2 and after 3 days of culture a decellularized cartilage scaffold was added to each well. Wells containing only medium or only BMSCs were included as controls. After 4 days, the cells and scaffolds were washed with PBS. Next, 5 mg/mL MTT-solution was added and incubated for 3 hours protected from light at 37°C and 5% CO 2 . Finally, the scaffolds were removed from the wells and the MTT-solution was replaced with 100% ethanol (Boom, Meppel, the Netherlands), transferred to a 96-well plate and absorbance was measured at 670 and 570 nm on a VersaMax (Molecular Devices, Sunnyvale, USA). Toxicity experiments were conducted twice with independent BMSC and cartilage donors, with 3 decellularized bovine samples per cartilage type. To further assess the interaction of cells with the decellularized cartilage scaffolds, BMSCs were seeded on the scaffolds by rotation in a tube rotator at 20 rpm (VWR, Radnor, Pennsylvania, USA) in 1.6 mL cell suspension containing 2·10 6 BMSCs/scaffold for 4 hours at 37°C. After seeding, the scaffolds were transferred to a 12-well plate (BD Biosciences) coated with 3% agarose (Eurogentec, Liège, Belgium) to prevent attachment of BMSCs to the culture well and cultured in 2 mL high glucose (4.5 g/L) Dulbecco’s Modified Eagle Medium (DMEM- HG; Gibco) containing 50 µg/mL gentamicin, 1.5 µg/mL Fungizone, Insulin-Transferrin- Selenium (ITS+1, BD Biosciences, New Jersey, USA), 40 µg/mL L-proline, 1mM sodium pyruvate (Gibco), 25 µg/mL L-ascorbic acid 2-phosphate, 10 ng/mL Transforming Growth Factor beta 1 (TGFβ1; R&D Systems, Minneapolis, USA) and 10 -7 M dexamethasone. To confirm the chondrogenic capacity of the seeded BMSCs, pellet cultures of 250,000 BMSCs/pellet were included as positive controls. Therefore, BMSCs were suspended at a density of 5·10 5 cells/mL. Aliquots of 0.5 mL cell-suspension were transferred into polypropylene tubes and pellets were formed by centrifuging at 200 G for 8 minutes. Negative controls included BMSCs cultured in monolayer in DMEM-HG containing 10% FCS, 50 µg/mL gentamicin, 1.5 µg/mL Fungizone and 25 µg/mL L-ascorbic acid 2-phosphate in the absence of TGFβ1. Samples intended for gene- expression analysis and viability analysis were cultured for 21 days at 37°C and 5% CO 2 and medium was refreshed twice a week. After culture, cell viability was evaluated with a LIVE/DEAD® assay (Invitrogen, Carlsbad, USA) according to manufacturer’s instructions. Fluorescent imaging was performed on a Zeiss LSM 510 with the excitation laser set at 488 nm. A 505-530 nm band-pass filter was used to detect living cells and a 650 nm low-pass filter for detecting dead cells. To assess the chondrogenic differentiation of the BMSCs cultured on the scaffolds, gene expression analysis was performed. RNA was isolated from the seeded scaffolds by snap freezing in liquid nitrogen followed by pulverization using a Mikro-Dismembrator (B. Braun Biotech International GmbH, Melsungen, Germany) at 2800 rpm. The tissue was homogenized with 18 µL/mg sample RNA-Bee TM (Tel-Test Inc, Friendswood, USA) and 20% chloroform. RNA was isolated using the RNeasy Micro Kit (Qiagen, Hilden, Germany) according to manufacturer’s instructions. Quantification of total extracted RNA was determined using a 135 DECELLULARIZED CARTILAGE: AN ECM-DERIVED SCAFFOLD 7