Long‐term culture of disaggregated rat osteoclasts: Inhibition of bone resorption and reduction of osteoclast‐like cell number by calcitonin and PTHrP[107‐139]

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Abstract
The islated osteoclast bone resorption assay has proved to be a useful means of examining the response of mammalian and avian osteoclasts to a variety of stimuli. The assay has traditionally been performed over a period of 24 hours. By extending the duration of the osteoclast bone resorption assay, we have been able to assess the long‐term effects of carboxyl‐terminal parathyroid hormone‐related protein (hPTHrP[107‐139]), salmon calcitonin (sCT) and hPTH[1‐34] on bone resorption and TRACP‐positive osteoclast‐like cell number. We found that, in control cultures over a period of up to 144 hours, the osteoclast‐like cells not only remained viable but their numbers also increased. The number of mononucleated and multinucleated osteoclast‐like cells doubled in the first 48 hours before stabilizing over the remainder of the incubation period. Osteoblasts also proliferated, resulting in a resorption response to hPTH [1‐34] being evident from 48 hours onward. hPTHrP[107‐139] persistently inhibited basal and PTH‐stimulated bone resorption for at least 96–144 hours. Where as “escape” from the inhibijtory effect of sCT was seen after 48–72 hours. Decreased numbers of bothe mononucleated and multinucleated TRACP‐positive osteoclast‐like cells were seen by 48 hours in cultures treated with sCT. In contrast, hPTHrP[107‐139] reduced the number of mononuclear TRACP‐positive cells with only a late effect on multinucleated cells. Furthermore, the incsreased number of osteoclast‐like cells seen in response to hPTH[1‐34] was inhibited by carboxyl‐terminal PTHrP. In summary, this study indicates that the extended bone resorption assay system is a complex one where bothe osteoclastic resorption and osteoclast maturation are evident. Using this syste, we have shown that hPTHrP[107‐139] acts as a potent long‐term inhibitor of osteoclastic bone resorption, without evidence of escape from its effect. Its action to reduce the number of mononucleated osteoclst‐like cells suggests that it affects several aspects of osteoclast activity.