The level of OC was measured by using commercially available ELISA kits (Human Osteocalcin Quantikine ELISA Kit, R&D Systems, Inc., Minneapolis, MN, USA) according to the manufacturer’s instructions. A 100 μl volume of Assay Diluent RD1-117 (R&D Systems) was added into each well of the microplate and then 50 μl of standard (0–64 ng/ml) or sample was added to each well. The microplate was incubated for 2 h at room temperature on a horizontal orbital microplate shaker (Labnet International, Inc., Edison, NJ, USA) set at 500 rpm. After that, the solutions were aspirated and each well was washed with 400 μl of Wash Buffer (R&D Systems). This step was repeated three times for a total of four washes. Then, 200 μl of Human Osteocalcin Conjugate were added into each well, and the solutions were incubated for 2 h at room temperature on the shaker. After that, the solutions were aspirated and washed again. A 200 μl volume of substrate solution (tetramethylbenzidine) was added into each well, and the solutions were incubated for 30 min at room temperature on the bench-top. Next, 50 μl of stop solution (2 N sulfuric acid) were added into each well. The color in the wells changed from blue to yellow. After that, the color solution in each well was measured at 450/540 nm within 30 min. The OC level in each sample was calculated from a standard curve and normalized by total protein.

The level of the ALP activity was measured by colorimetric analysis. Briefly, 80 μl of sample solution were added into each well of a 96-well microplate, and then a mixture of 100 μl phosphate substrate solution (p-nitrophenyl phosphate) and 20 μl 1.5 M alkali buffer solution were added to each well. The microplate was incubated at 37 °C for 1 h. Next, 50 μl of stop solution (1 M NaOH) were added into each well. After that, the absorbance was measured at 620 nm. Known concentrations of ALP product (p-nitrophenol) were prepared with the dilutions ranging from 0 to 250 μM. The activity of ALP in the samples were calculated from a standard curve and normalized by total protein.