Female patients with OA (n=7; 50-74 years) and healthy volunteers (n=4; 43-48 years) were enrolled between May 2019 and December 2019 during routine medical check-ups at Centro di Medicina (Ferrara, Italy) after obtaining written informed consent; the study was approved by the Centro di Medicina's research committee (approval no. 172201). Briefly, peripheral blood mononuclear cells (PBMCs) were obtained from 20 ml peripheral blood and separated using Histopaque-1077 as previously described (16). hMCs were purified from PBMCs via adhesion selection on polystyrene plates. PBMCs (1×106/cm2) were plated and allowed to settle for 4 h at 37°C, and flasks were then rinsed to remove non-adherent cells. In order to confirm the ability of isolated hMCs to differentiate into mature osteoclasts (hOCs), macrophage colony-stimulating factor (25 ng/ml) and receptor activator of NF-κB ligand (RANKL; 30 ng/ml; PeproTech EC Ltd.) were added to the culture medium; after 14 days, TRAP staining was performed. The expression levels of the osteoclast-specific markers cathepsin K and NFATc1 were assessed via immunocytochemistry.

Human osteoblasts (hOBs) were obtained from vertebral laminae discarded during spinal surgery to remove lumbar herniated discs (Pfirrmann grade 2). Bone fragments were obtained between September 2019 and December 2019, after obtaining written informed consent from 4 donors with no comorbidity (43-48 years; 2 males and 2 females) using research protocols approved by the Ethics Committee of the University of Ferrara and St. Anna Hospital (approved on November 17, 2016). Briefly, bone fragments were placed in sterile PBS at 4°C and dissected within 16 h after removal. Bone chips were minced into smaller pieces as previously reported (17), washed twice with PBS, plated in T-25 culture flasks (Sarstedt, Inc.) and cultured in high-glucose DMEM/Ham's F12 (1:1) supplemented with 10% FCS, 1 mM L-glutamine and antibiotics [(penicillin (100 µg/ml) and streptomycin, (10 µg/ml)]. From each patient, a primary cell culture was obtained. Upon detection of a cell colony from the bone fragments (after 7 days), the cells were expanded until confluent [passage (P)0)]. The cells were then harvested after treatment with 0.05% trypsin EDTA for 2 min at 37°C (Sigma-Aldrich; Merck KGaA), washed, counted via hemocytometric analysis and used for further experiments (P1-3). During the culture period, cells were incubated at 37°C in a humidified atmosphere of 5% CO2, and the medium was changed every 3 days. hOBs (P0) were characterized for the presence of OPN, Runx2 and COL1a1 via immunostaining.

Based on previous studies, osteogenic differentiation was performed by culturing hOBs for up to 14 days in osteogenic medium (OM) (18,19) consisting of high-glucose DMEM, 10% FCS, 10 mM β-glycerophosphate, 100 nM dexamethasone and 100 µM ascorbic acid-2-phosphate.

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