Quantikine human comp immunoassay

Release of COMP from OA cartilage was measured in the supernatants of SF cultures over cartilage explants.13 OA joint cartilages from four patients were provided by the Rheumatology Service at Complejo Hospitalario Universitario A Coruña (CHUAC, A Coruña, Spain). Fixed diameter (6 mm) sections were collected from adjacent areas to the injured cartilage. Samples were frozen at −80°C and stored until testing. One explant per well was attached to a 96‐well plate. HD‐ or OA‐SF were added drop‐wise on top of the cartilage surface (2 × 10⁴ SF per explant). After 3 hours of incubation, wells were filled with DMEM in the presence or absence of 10 µM PD98059 or 200 ng/mL DKK1 for 1 hour, followed by treatment with 10 ng/mL IL‐1β or 10 nM 45 kDa Fn‐fs. Cultures were continued for 7 days, and treatments were renewed every two days. Culture supernatants were collected for detection of COMP using a Quantikine® Human COMP Immunoassay (R&D Systems).

Release of glycosaminoglycans (GAGs) and COMP degradation products from cartilage tissue was measured in culture medium from wells containing SF cultured over cartilage explants. Osteoarthritis human cartilages were obtained from three patients undergoing total hip arthroplasty in Hospital del Mar (Barcelona, Spain). Fixed diameter (6 mm) and height (2 mm) sections were collected from cartilage areas without macroscopically signs of OA. The samples were frozen at −80°C and stored until testing. One explant per well was attached to a 24 well plate. Healthy donors‐ or OA‐SF were added drop‐wise on top of the cartilage surface (2 × 10⁴ SF/explant). After 3 hrs of incubation at 37°C, wells were filled with 1 ml DMEM 10% FBS, with the treatments described, and cultures were continued for 14 days. Culture supernatants were collected to detect the release of GAGs and COMP degradation products, 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. Cartilage explants were placed in OCT and frozen in liquid nitrogen. Sections were prepared using a cryostat, placed on slides and stained with Alcian blue and Callejas's tricromic.