DIMENSIONAL ANALYSIS OF OSTEOCLASTIC BONE-RESORPTION AND THE MEASUREMENT OF BIOLOGICALLY-ACTIVE CALCITONIN

Calcitonin inhibits bone resorption through a direct action on the ost eoclast. We report a quantitative analysis of bone resorption by disag gregated rat osteoclasts. We then used our findings to develop a forma l bioassay for calcitonin. Osteoclasts were mechanically disaggregated from neonatal rat long bones and dispersed at low densities on slices of devitalized bovine cortical bone. The resulting areas of bone exca vation were quantified to micrometric precision by scanning electron m icroscopy together with computer-assisted image analysis. These findin gs were correlated with the volumes of bone resorption in the same sli ces measured by confocal scanning microscopy for the first time. The t otal planar areas of bone resorption per slice correlated linearly (r = 0.78) with the confocal microscopic measurements of total volume res orbed, provided that volume was expressed to its two-thirds power. The latter transformation resulted in representations of the determined a reas ([length](2)) and volumes ([length](3)) which were dimensionally consistent. These findings thus demonstrate that osteoclastic bone exc avations show a consistent relationship between area and volume and th at assessments of the area of excavations accordingly provide an empir ical representation of the volume of bone resorbed. Furthermore, in vi ew of the skewed nature of the distributions of area measurements, we assessed the effect of transforming the response variable to derive a metameter, (planar area of resorption)(1/2). Such transformed data poi nts, which expressed the data in the dimensions of [length], were more normally distributed than the raw data points and had more stable var iances over a wider concentration range. We accordingly determined rel ative potencies using parallel line analyses on the transformed data. The latter offered a consistent correlation to the volume measurements when these were also converted to dimensions of [length] (r = 0.805). It was confirmed that the inhibition of bone resorption by calcitonin s from various species, namely, pig, salmon and eel, was quantitativel y dependent upon concentration of the respective peptides. The resulti ng assay was also found to be sufficiently sensitive to measure picomo lar peptide concentrations with a precision, h (standard deviation/slo pe), ranging between 0.3 and 0.8. Finally, we identified factors affec ting assay precision and sensitivity. INTRODUCTION