4.1. Sample Collection and Cell Cultures

Human OA articular cartilage was collected from femoral heads of five non-obese (body mass index ranging from 20 to 23 kg/m²) and non-diabetic patients (two men and three women, age ranging from 65 to 75) with coxarthrosis according to ACR criteria [61], undergoing hip replacement surgery.

OA grade ranged from moderate to severe, and cartilage showed typical disease changes, as the presence of chondrocyte clusters, fibrillation, and loss of metachromasia (Mankin degree 3–7) [62]. The femoral heads were supplied by the Orthopaedic Surgery, University of Siena, Italy. The use of human articular samples was authorized by the Ethic Committee of Azienda Ospedaliera Universitaria Senese/Siena University Hospital (decision no. 13931/18) and the informed consent of the donor.

After surgery, cartilage fragments were aseptically dissected and processed by an enzymatic digestion, as previously described [63]. For growth and expansion, cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) (Euroclone, Milan, Italy) with phenol red and 4 mM L-glutamine, supplemented with 10% fetal bovine serum (FBS) (Euroclone, Milan, Italy), 200 U/mL penicillin, and 200 µg/mL streptomycin (P/S) (Sigma-Aldrich, Milan, Italy). The medium was changed twice a week, and the cell morphology was examined daily with an inverted microscope (Olympus IMT-2, Tokyo, Japan). OA primary chondrocytes at the first passage were employed for the experiments [64]. For each single experiment a cell culture from a unique donor was used.