PURPOSE: The purpose of this study was to investigate the effects of EMD on the growth and differentiation of osteoblastic cells (MC3T3-E1) and on the expression of osteoprotegerin (OPG), a key cytokine that inhibits osteoclastogenesis and osteoclast function. STUDY DESIGN: MC3T3-E1 cells were treated with 100 microg/mL EMD in serum-free medium for 1, 2, 3, 5, and 7 days, or in 2% fetal bovine serum (FBS) for 3 weeks. Cells incubated without EMD served as negative control. At the end of each incubation period, cell numbers were counted and total cellular mRNA was extracted. Northern blot analysis and RT-PCR were performed to determine the mRNA levels of core binding factor alpha (Cbfa1), collagen alpha1 (I), bone sialoprotein (BSP), osteocalcin (OC), insulin-like growth factor I (IGF-I), and OPG. Alkaline phosphatase (ALP) activity was also determined and compared between treatment and control groups. RESULTS: A marked increase in cell numbers was observed in EMD-treated groups from day 2 to day 7 (P < .01). mRNA expression of collagen alpha1 (I), BSP, OC, OPG, and IGF-I were up-regulated in cells treated with EMD. ALP activity was significantly increased by EMD treatment after 3-week culture under differentiating conditions (P < .05). The expression of Cbfa1 was not affected by EMD treatment from day 1 to day 5; the levels were elevated after culturing for 3 weeks in EMD-treated cells. CONCLUSIONS: EMD promotes both proliferation and differentiation of MC3T3-E1 cells and indirectly inhibits osteoclastogenesis and osteoclast function by stimulating the expression of OPG.
PURPOSE: The purpose of this study was to investigate the effects of EMD on the growth and differentiation of osteoblastic cells (MC3T3-E1) and on the expression of osteoprotegerin (OPG), a key cytokine that inhibits osteoclastogenesis and osteoclast function. STUDY DESIGN: MC3T3-E1 cells were treated with 100 microg/mL EMD in serum-free medium for 1, 2, 3, 5, and 7 days, or in 2% fetal bovine serum (FBS) for 3 weeks. Cells incubated without EMD served as negative control. At the end of each incubation period, cell numbers were counted and total cellular mRNA was extracted. Northern blot analysis and RT-PCR were performed to determine the mRNA levels of core binding factor alpha (Cbfa1), collagen alpha1 (I), bone sialoprotein (BSP), osteocalcin (OC), insulin-like growth factor I (IGF-I), and OPG. Alkaline phosphatase (ALP) activity was also determined and compared between treatment and control groups. RESULTS: A marked increase in cell numbers was observed in EMD-treated groups from day 2 to day 7 (P < .01). mRNA expression of collagen alpha1 (I), BSP, OC, OPG, and IGF-I were up-regulated in cells treated with EMD. ALP activity was significantly increased by EMD treatment after 3-week culture under differentiating conditions (P < .05). The expression of Cbfa1 was not affected by EMD treatment from day 1 to day 5; the levels were elevated after culturing for 3 weeks in EMD-treated cells. CONCLUSIONS: EMD promotes both proliferation and differentiation of MC3T3-E1 cells and indirectly inhibits osteoclastogenesis and osteoclast function by stimulating the expression of OPG.
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