Inhibitory effect of ipriflavone on osteoclast-mediated bone resorption and new osteoclast formation in long-term cultures of mouse unfractionated bone cells

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Abstract
To study the effect of ipriflavone on osteoclast-mediated bone resorption and new osteoclast formation, we used an unfractionated bone cell culture system containing mature osteoclasts from femur and tibia of newborn mice. Ipriflavone (10−5 M) inhibited pit formation on dentin slices and caused a decrease in the number of tartrate-resistant acid phosphatase (TRAP)-positive (+) multinucleate cells (MNCs) in a 4-day culture period in which no increase in the number of TRAP(+)-MNCs was observed in the presence of 5% fetal bovine serum (FBS) and 10−8 M 1α,25-dihydroxy-vitamin D3 (1α,25(OH)2D3). During the following 12 days, both the total area of the pits and the number of TRAP(+)-MNCs increased in the control. Continuous treatment with ipriflavone also inhibited the increase in pit area during this period. These effects of ipriflavone were reversible. Furthermore, the differentiation of osteoclasts was examined when preexisting TRAP(+)-MNCs were removed by incubation in the absence of 1α,25(OH)2D3 for the initial 4 days in culture dishes without dentin slices. When 1α,25(OH)2D3 and ipriflavone were added to the medium on the 4th day, ipriflavone inhibited new TRAP(+)-MNC formation stimulated by 1α,25(OH)2D3 in a dose-dependent manner. However, pretreatment of the cells with ipriflavone before the addition of 1α,25(OH)2D3 did not inhibit TRAP(+)-MNC formation. These results indicate that ipriflavone inhibits both the activation of mature osteoclasts and the formation of new osteoclasts without affecting growth of TRAP-negative progenitor cells.