Potent ability of CME to promote the osteogenic differentiation of MG-63 cells.

<p>(A) ALP activity of MG-63 cells cultured in OIM with or without AME or CME (200–400 μg/mL) for 3 or 7 days was measured. *<i>p</i>< 0.05 and ^#<i>p</i>< 0.001 versus OIM of day 7. (B) MG-63 cells were cultured in OIM, and AME or CME was added in a dose-dependent manner. Calcium concentration after 7 days was measured. *<i>p</i>< 0.05 and **<i>p</i>< 0.01 versus OIM. (C) MG-63 cells were cultured in OIM with or without 200 μg/mL of AME or CME to evaluate the expression of osteogenic marker genes by quantitative real-time RT-PCR. The mRNA levels of 3 genes encoding <i>RUNX2</i>, <i>OSTERIX</i>, and <i>IBSP</i> were determined on days 3 and 7. Expression of the gene encoding <i>OCN</i>, which is expressed by osteoblasts in the late stage of osteogenic differentiation, was analyzed on day 24. Results of gene expression were normalized with those obtained for <i>GAPDH</i> and were presented as fold change. *<i>p</i>< 0.05, **<i>p</i>< 0.01, and ^#<i>p</i>< 0.001, Student’s t-test. (D) MG-63 cells grown in OIM and treated with 400 μg/mL CME were cultured for 9 days and were stained with Alizarin red S. Control cells were cultured in only OIM. Scale bars represent 500 μm. Data are presented as the mean ± SD of multiple repeated experiments.