Chondrocyte Culture, its Maintenance, and Ethics

The human chondrocytes cultures were obtained from patients who underwent joint replacement therapy. The osteoarthritic cartilage harvested from patients was turned into pieces and digested with an enzymatic solution [8 mg/ml hyaluronidase (Sigma-Aldrich, St. Louis, MO, USA), 8 mg/ml collagenase (Sigma-Aldrich), and 2.5 mg/ml trypsin (Sigma-Aldrich)] for 6 h at 37°C. The cellular suspension was centrifuged at 1,500 rpm for 5 min and resuspended into Dulbecco’s modified Eagle’s medium (DMEM)/F12 (Gibco BRL, New York, NY, USA) with 10% fetal bovine serum (FBS; Gibco BRL), and 1% Penicillin-Streptomycin-Amphotericin B (PSA) (Biological Industries, Beit Haemek, Israel) in a humidified atmosphere containing 5% CO₂. Thereafter, the primary OA chondrocytes were cultured, passaged, and maintained in DMEM/F12 medium.

Based on previous reports showing various inhibitory activities of nicotine against bone density¹⁹, human renal proximal tubular epithelial cells²⁰, and myoblast differentiation²¹, we firstly established in vitro model of nicotine (Sigma 36733) OA, by treatment of various doses of nicotine (100, 500, and 1,000 µM) to chondrocytes (passage 2) in the culture medium. After validating OA properties in nicotine-treated chondrocytes, we further treated them with PDB for 7 days.