2.5. BrdU Assay

The osteoblast proliferation rate was determined by using the BrdU (Bromodeoxyuridine) Cell Proliferation enzyme-linked immunoadsorption tool (Roche Diagnostics, Mannheim, Germany). Similar to the MTT assay, 96-well microtiter plates with 5 × 10³ cells/ well were incubated for 24 h and a sample of 150 μL eluate was obtained. After 48 h, the eluate was changed to a standard nutrient medium and the osteoblasts were incubated for another 72 h. After that, 10 μL of BrdU solution was added to each well and the osteoblasts were incubated for an additional 24 h so that the osteoblasts could incorporate bromodeoxyuridine into their DNA. The optical density of each sample was examined in a microplate reader (Spectra Max plus 384, Molecular Devices, Sunnyvale, CA, USA) at a wavelength of 450 nm.