ESTABLISHMENT OF A RAPID BONE-RESORPTION IN-VITRO ASSAY USING PREVIOUSLY FROZEN MOUSE UNFRACTIONATED BONE-CELLS PRETREATED WITH PARATHYROID-HORMONE

We established a useful assay system for evaluating osteoclast-mediate d bone resorption based on the use of unfractionated bone cells obtain ed from 10- to 11-day-old mice. When cells from 10 to 11 mice were tre ated for 7 days with rat parathyroid hormone (rPTH, 10(-8)M), a total of 4 to 5 x 10(7) cells could be obtained from the culture by treatmen t with 0.05% trypsin and 0.02% EDTA in PBS. These harvested cells cont ained about 20% tartrate-resistant acid phosphatase (TRAP)-positive mo nonuclear cells. When the harvested cells were cultured on dentine sli ces without rPTH, after 1 day, they formed TRAP-positive multinucleate cells that were active in bone resorption. Eel calcitonin (eCT) decre ased the number of pits in a dose-dependent manner, and its half maxim al inhibition dose (ID50) was 1.08 x 10(-11)M. Even after having been frozen in liquid nitrogen for 5 months, upon thawing, these cells were capable of forming pits; and this pit formation was inhibited by eCT. Since no appropriate osteoclastic cell line for evaluating bone resor ption is available at present, this system can provide a useful, pract ical means for assaying osteoclastic bone-resorbing activity.