A possible mechanism of the specific action of bisphosphonates on osteoclasts: Tiludronate preferentially affects polarized osteoclasts having ruffled borders

The mechanism of action of tiludronate [(4-chlorophenyl)-thiomethylene bisphosphonate] on osteoclastic bone resorption was examined in mouse culture systems. Tiludronate did not inhibit the formation of osteoclast-like multinucleated cells (OCLs) induced by 1α,25-dihydroxyvitamin D3 in co-cultures of mouse osteoblastic cells and bone marrow cells. OCLs obtained from cocultures on collagen gel-coated dishes, treated with tiludronate, formed as many resorption pits on dentine slices as those obtained from the control co-cultures. However, pit formation by OCLs was dose-dependently inhibited when tiludronate was added directly to the pit formation assay. Other bisphosphonates such as alendronate and endronate dose-dependently inhibited pit formation according to the in vivo potencies of the respective bisphosphonates to inhibit bone resorption. However, they had no inhibitory effect on the recruitment of OCLs induced by 1α,25-dihydroxyvitamin D3 in the cocultures. When OCLs were placed on dentine slices, they formed the ringed structure of F-actin-containing podosomes and ruffled borders (polarized OCLs) even in the presence of tiludronate. However, the actin rings in OCLs were disrupted by the addition of tiludronate soon after they began to resorb dentine. In contrast, OCLs placed on collagen gel formed neither actin rings nor ruffled borders (nonpolarized OCLs), and showed no response to tiludronate. OCLs formed from the spleen cells of osteosclerotic (oc/oc) mice developed the ringed structure of podosomes, but not ruffled borders, on dentine slices. The actin ring in the oc/oc spleen cell-derived OCLs placed on dentine slices was not disrupted by the addition of tiludronate. However, the actin rings formed in normal OCLs and in oc/oc OCLs placed on plastic dishes were similarly and rapidly disrupted by the microinjection of tiludronate into their cytoplasm. These results indicate that tiludronate is selectively incorporated into polarized OCLs that have a ruffled border, suggesting that the bisphosphonate is a specific inhibitor of functioning osteoclasts.