Blyscan sulfated glycosaminoglycan assay

Release of GAG and COMP from cartilage was measured in culture supernatants from wells containing co-cultures of SF over cartilage explants. 20 OA human cartilages were obtained from three patients undergoing total hip arthroplasty from Hospital del Mar (Barcelona, Spain). Fixed diameter (6 mm) and height (2 mm) sections were collected from cartilage areas without macroscopic signs of OA. Samples were frozen at À80 C and stored until testing. One explant per well was attached to a 24-well plate. HD-or OA-SF were added drop-wise on top of the cartilage surface (2 Â 10 4 SF per explant). After 3 hours of incubation, wells were filled with DMEM in the absence or presence of 10 ng/ mL IL-1b or 10 nmol/L Fn-fs 45 kDa, and cultures were continued for 14 days. Culture supernatants were collected for detection of GAG and COMP, using a Blyscan Sulfated Glycosaminoglycan Assay (Biocolor Ltd County Antrim, Ireland, UK), and a Quantikine Human COMP Immunoassay (R&D Systems, Abingdon, OX, UK), respectively. Frozen sections were prepared using a cryostat and stained with Alcian blue and Callejas's tricromic. Sections were observed using an Olympus BX51 microscope with DP72 camera model (objective 20Â).

Midstream morning urine samples were collected into sterile urine collection tubes and each urine sample was centrifuged at 5000 g for 10 minutes, the supernatant was aliquoted in 1.5-mL microcentrifuge tubes and stored at -800C until further analysis. Total GAG levels in supernatants were determined using the Blyscan Sulfated Glycosaminoglycan assay (Blyscan Assay, Biocolor Ltd., Northern Ireland, UK) according to the manufacturer' s instructions (14) . The Blyscan Assay as a direct colorimetric method quantifying urinary glycosaminoglycan excretion exploits the specific binding of 1,9-dimethylmethylene blue that provides a specific label for the sulfated polysaccharide component of proteoglycans or the protein-free sulfated glycosaminoglycan chains (15) . The detection limit of assay was 2.5 µg/mL. Urinary creatinine (Cr) concentration was determined on each sample using the kinetic Jaffe method in the AU5800 clinical chemistry system (Beckman Coulter Inc., CA, USA). The urinary concentrations of GAG were normalized to the concentration of urinary Cr and results were expressed as, milligram per gram of Cr.

Sulfated Glycosaminoglycan (sGAG) was measured by a Blyscan Sulfated Glycosaminoglycan assay (Biocolor, B3000). Cartilage Oligomeric Matrix Protein (COMP) was measured by a Cartilage Oligomeric Matrix Protein Human ELISA kit (BioVender, RD194080200). MMP-1, MMP-3, and MMP-9 were measured by a Human MMP 3-Plex Ultra-Sensitive Kit (MSD, K15034C). Results of soluble CD163, CD14, and elastase, IL-6, IL-8 and IL-10 were previously measured and published (10, 16 ) (see Supplementary Methods).