Volumes of chick and rat osteoclasts cultured on glass

Abstract

We have examined the relationship between the number of nuclei of an osteoclast and its volume. Chick and rat cells were released from long bones by chopping the shafts and flushing the fragments in Eagle's Minimum Essential Medium with added 10% fetal calf serum. The bone cell suspension was seeded onto glass coverslips. In Experiment 1, rat and chick cells were allowed to settle for 15 minutes, more medium was then added, and the cells were cultured in 5% CO2 at 37 degrees C for 4 hours. In Experiment 2, only rat cells were used, and the cells were cultured in the presence or absence of 10(-6) M 3-amino-1-hydroxypropylidene-1,1-bisphosphonate (APD) in the medium for 4 or 6 hours. The coverslips were washed in 37 degrees C phosphate-buffered saline and fixed for 24 hours in 2.5% glutaraldehyde in isotonic cacodylate buffer (initially 37 degrees C). The chick cells were critical point dried (CPD) or freeze dried (FD); all rat cells were FD. After drying, cells were coated with gold by vacuum evaporation. The volumes and areas of osteoclasts were measured using a video-rate, line-confocal reflection laser scanning microscope and the number of nuclei in each cell was counted. The volumes and volumes per nucleus of the FD cells were larger than those of the CPD cells but there was no significant difference in plan-areas. Rat osteoclasts were larger than chick cells in all the measured parameters except the mean number of nuclei/cell. The correlation coefficients for the areas, volumes, and the numbers of nuclei for rat and chick cells were all high (r > 0.725). The volumes and volumes per nucleus, but not the areas or areas per nucleus, of the osteoclasts cultured with APD were significantly smaller than control cells. We conclude that FD causes less shrinkage than CPD; chick osteoclasts are about two-thirds the size of rat osteoclasts; and 10(-6) M APD caused a reduction of rat osteoclast volume and volume per nucleus of 21%.