Figure 5. Osteocalcin assay. The graph shows the osteocalcin of the osteoblasts treated once or twice with 0.1 μg/mL neuropeptide Y (NPY), substance P (SP) and human bone morphogenetic protein 2 (hBMP2) alone, as well as their combined applications. The osteoblasts exposed to 0.1 μg/mL NPY had the highest level of osteocalcin. The osteoblasts treated twice with the neuropeptides or hBMP2 displayed higher osteocalcin than those treated only once. Furthermore, the osteocalcin levels of cells exposed to 0.1 μg/mL neuropeptides alone were also significantly higher than those of the osteoblasts exposed to neuropeptides combined with hBMP2 (P<0.05, ANOVA).