Isolation and Osteogenic Differentiation of Rat Bone Marrow Stem Cells

The rats in the study were euthanized by cervical dislocation during the fourth week after injection. Their BMSCs were obtained by flushing the bone marrow from their tibias and femur bones using low-glucose Dulbecco’s modified Eagle’s medium (DMEM; Gibco BRL, Grand Island, USA) containing 10% fetal bovine serum (FBS; Gibco BRL, USA), 100 U/ml streptomycin, 100 U/ml penicillin and 200 U/ml heparin (Sigma-Aldrich, St Louis, MO), according to previously described procedures [29 (link),30 (link)]. The primary cells were cultured in low-glucose DMEM supplemented with 10% FBS, 100 U/ml streptomycin and 100 U/ml penicillin at 37°C in a 5% CO₂ atmosphere. Non-adherent cells were discarded via a change of medium after 24 hours, and the medium was replaced every three days until the cells reached 80–90% confluence (S1 Fig). A Z2 Coulter particle count and size analyzer (Beckman Coulter, USA) was used to evaluate the quantity of cells. The quantities of both normal and diabetic BMSCs were approximately 1.2–1.5×10⁷ cells/dish. Next, 0.25% trypsin (EDTA) was used for cell detachment, and the BMSCs were subcultured at a density of 1×10⁵ cells/ml. Cells at passage 2 or 3 were used for the subsequent experiments. Osteogenic medium (DMEM, 10% FBS, 10 mM dexamethasone, 50 μg/ml L-2-ascorbic acid and 10 mM glycerophosphate) was used to induce the osteogenic differentiation of BMSCs.