Fig. 3: Comparative study of polaprezinc and zinc-sulfate differentiation into osteoblast and osteoclast lineages.

a Alizarin red S staining was performed to detect mineral deposition on day 14 in differentiated hBMSCs treated with vehicle, polaprezinc (50 μM), or zinc sulfate (50 μM). b For quantitative analysis of alizarin red S staining, absorbance was measured at 595 nm following destaining with 10% cetylpyridinium for 30 min. c The protein level for RUNX2 was analyzed in hBMSCs using western blotting for cells treated with vehicle, polaprezinc (50 μM), or zinc sulfate (50 μM). The band intensity was quantified using ImageJ software (n = 3, in triplicate) and each RUNX2 protein level was normalized to the HSP90 protein level. *P < 0.05; **P < 0.01 compared with vehicle-treated hBMSCs. d Representative images of osteoclast differentiation. RAW264.7 treated with vehicle, polaprezinc (50 μM), or zinc sulfate (50 μM) were seeded into 12-well culture plates and treated for 5 days with osteoclastogenesis-related reagents. TRAP staining was performed to visualize TRAP-positive RAW264.7. Scale bar = 200 μm. e TRAP activity was measured at 405 nm, and the data are expressed as the mean ± S.D. (n = 3) from each independent experiment. f The number of TRAP-positive multinucleated osteoclasts (5 ≥ nuclei) was counted. g The protein level for NFATc1 was analyzed in RAW264.7 using western blotting for cells treated with vehicle, polaprezinc (50 μM) or zinc sulfate (50 μM). The band intensity was quantified using ImageJ software (n = 3, in triplicate) and each Nfatc1 protein level was normalized to the β-Actin protein level. **P < 0.01 compared with vehicle-treated RAW264.7. h Cells treated with vehicle, polaprezinc (50 μM), or zinc sulfate (50 μM) were incubated with 25 μM of Zinquin for 30 min at 37 °C. The fluorescence intensity was measured at excitation 368 nm and emission 490 nm using a fluorometer.