Effects of continuous calcitonin treatment on osteoclasts derived from cocultures of mouse marrow stromal and spleen cells

Background: Continuous calcitonin (CT) treatment for bone diseases associat ed with increased bone resorption may be followed by prolonged depression o f osteoclast response to CT. The mechanisms of this "escape" phenomenon rem ain unclear. Methods: We examined the effects of continuous CT treatment on cell formati on, calcitonin receptor (CTR) expression, response to CT, and bone resorpti on of osteoclasts in a coculture of mouse marrow stromal and spleen cells i n the presence of 1,25-dihydroxyvitamin D-3 [1,25(OH)(2)D-3]. Cells were co cultured and treated with salmon CT (sCT) for 7, 14, or 21 days. The effect s of continuous CT treatment on osteoclast formation was determined by quan titation of tartrate-resistant acid phosphatase (TRAP)-positive multinuclea ted cells (MNCs). CTR expression in osteoclasts was determined by binding o f [I-125]sCT in autoradiography. Bone resorption and CT responsiveness were assessed by examining the formation of resorption pits and by enumerating osteoclast reattachment on dentine slices after sCT rechallenge. Results: TRAP-positive MNCs appeared in cocultures treated with sCT and wer e similar in number and morphology to those in control cultures, regardless of the concentration and duration of sCT treatment. A decrease in CTR expr ession was identified as a loss of silver grains from the TRAP-positive cel ls in cocultures receiving sCT treatment for 14 or 21 days. Partial recover y of CTR expression in TRAP-positive cells was evident in cocultures treate d with sCT for only the first 7 days of coculture. TRAP-positive MNCs in co cultures treated with sCT for 14 or 21 days were resistant to the rechallen ge with sCT. They attached to dentine slices and caused numerous resorption pits compared with control cells and cells treated with sCT for the fir st 7 days of coculture (p < 0.01). Conclusion: These findings suggest that the escape phenomenon that develops after continuous CT treatment may be due, at least in part to: 1) loss of responsiveness to CT in existing osteoclasts; and 2) development of new ost eoclasts that are CTR-deficient and, therefore, refractory to CT rechalleng e.