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 osteocla st-like multinucleated cells (OCLs) induced by 1 alpha,25-dihydroxyvit amin D-3 in cocultures of mouse osteoblastic cells and bone marrow cel ls. OCLs obtained from cocultures on collagen gel-coated dishes, treat ed with tiludronate, formed as many resorption pits on dentine slices as those obtained from the control cocultures. However, pit formation by OCLs was dose-dependently inhibited when tiludronate was added dire ctly to the pit formation assay. Other bisphosphonates such as alendro nate and etidronate 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 recrui tment of OCLs induced by 1 alpha,25-dihydroxyvitamin D-3 in the cocult ures. When OCLs were placed on dentine slices, they formed the ringed structure of F-actin-containing podosomes and ruffled borders (polariz ed 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 for med neither actin rings nor ruffled borders (nonpolarized OCLs), and s howed no response to tiludronate. OCLs formed from the spleen cells of osteosclerotic (oc/oc) mice developed the ringed structure of podosom es, but not ruffled borders, on dentine slices. The actin ring in the oc/oc spleen cell-derived OCLs placed on dentine slices was not disrup ted 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 inc orporated into polarized OCLs that have a ruffled border, suggesting t hat the bisphosphonate is a specific inhibitor of functioning osteocla sts.