Osteoblast Proliferation on Ceramic Composites

The human osteoblast cell lines (hFOB 1.19 SV40 transfected osteoblasts, ATCC, Rockville, MD, USA) were seeded on the sterilized pure CS and GNP/CS composites samples at a density of 1×10⁴ cells ml⁻¹ in 96-well culture plates. Cells were maintained and propagated in DME/F-12 (HyClone, UT) cell culture medium supplemented with 10% fetal bovine serum (Gibco, NY), 100 U/mL penicillin and 100 µg/mL streptomycin at 37°C in a humidified atmosphere with 5% CO₂ and cultured for 1,3 and 5 days. Proliferation of the cells cultured on the sterilized pellets (5 mm in diameter) was analysed using the methyl thiazole tetrazodium (MTT) assay. An MTT stock solution of 5 mg ml⁻¹ (Sigma, St. Louis, MO, USA) was prepared by dissolving MTT in PBS, filtered through a 0.2 µm filter and stored at 4°C. Then the 96-well plate was removed from the incubator and 20 µl MTT stock solutions were added to each well. Cells were incubated for 4 h at 37°C in an atmosphere of 100% humidity and 5% CO₂. After the incubation, the MTT solution was removed and replaced with 100 µl DMSO. At each culture period (1, 3 and 5 days), the samples were taken out and removed to new 24-well tissue culture plates. After being washed three times with PBS solution, cells were detached with trypsin/EDTA and stained with trypan blue, after which the living cells were counted with a hemocytometer (Becton Dickinson, Germany). Five samples of each composite were tested, and each test was carried out in triplicate.