Fig. 4. Excessive extracellular OPN induced by elevated extracellular Ca²⁺ conditions does not affect cell proliferation and matrix mineralization.

a The effect of recombinant OPN on cell proliferation. C3H10T1/2 cells were incubated in 6 mM Ca²⁺ medium or in the standard medium with the indicated amount of recombinant OPN, and cell proliferation was measured after 48 h treatment. b The effect of elevated extracellular Ca²⁺-induced cell proliferation is not affected by blocking the OPN action. After 48 h of treatment with the OPN neutralizing antibody and elevated extracellular Ca²⁺ medium, a BrdU incorporation assay was performed. Normal mouse IgG antibody was used as a control for antibody treatment. c Elevated extracellular Ca²⁺ increase c-Jun protein level. After 48 h of treatment as indicated, the relative expression of Cyclin D1 and c-Jun was calculated after normalization to β-actin. d The effect of recombinant OPN supplementation on matrix mineralization. C3H10T1/2 cells were incubated in normal growth medium (GM) or osteogenic medium (OM) supplemented with the indicated concentration of CaCl₂ and recombinant OPN. Cells were subjected to AR staining after 16 days of differentiation and staining was quantified. e Supplementation of excess extracellular OPN has no effect on the elevated extracellular Ca²⁺-induced matrix mineralization. C3H10T1/2 cells were incubated in GM supplemented with the indicated concentration of CaCl₂ and recombinant OPN, and were subjected to AR staining at day 12. The bar graph shows the relative intensity of AR staining. *p < 0.05; **p < 0.01; ***p < 0.001 vs. first bar