New resorption assay with mouse osteoclast-like multinucleated cells formed in vitro

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Abstract
We previously reported a procedure to obtain a preparation containing a large number of mouse osteoclast (OCL)-like multinucleated cells (MNCs) formed in cocultures of mouse osteoblastic and bone marrow cells in the presence of 1α,25-dihydroxyvitamin D3 [1α,25-(OH)2D3]. The MNCs satisfied major criteria of OCLs, such as tartrate-resistant acid phosphatase (TRAP) activity, acid production, calcitonin (CT) receptors, and the ability to form resorption pits on bone slices. In this report, we describe a simple resorption assay system using MNC preparations. After culturing MNC preparations or disaggregated rat OCL preparations on dentin slices, they were stained with Mayer's hematoxylin. The stained area corresponded exactly with the resorption pits visualized by scanning electron microscopy and were measured using an image analysis system attached to a light microscope. Pit formation by MNCs was gradually enhanced by reducing the medium pH (pH 7.5 < 7.2 < 6.9). The plan area resorbed by MNCs increased linearly for up to 72 h. These results are very similar to those obtained with OCL preparations. In multiple standard assays with MNC preparations, more than 250 MNCs could be placed on a dentin slice, and the total area resorbed to a level of up to 9% of the whole surface within 48 h. In contrast, in multiple assays with OCL preparations, it was not easy to place more than 50 OCLs on a slice and the resorbed area was only 0.7% of the surface. The resorbing activity of the MNC preparation expressed by the resorbed area per a TRAP-positive MNC (4.4 ± 0.4 × 10−3 μm2) was greater than that of the OCL preparation (1.9 ± 0.4 × 10−3 μm2). Pit formation by MNCs was dose dependently inhibited by salmon and human CT (ED50: salmon CT, 10−14 M; human CT, 3 × 10−13 M). Bafilomycin A1, a specific inhibitor of vacuolar type H+-ATPase, also inhibited pit formation by MNCs at concentrations of 10−9 M and above. In contrast, estrogen at 10−12–10−6 M had no significant inhibitory effect on pit formation in this assay system. The assay system presented here is sensitive and reproducible and will be useful for examining the effects of various natural factors and synthetic compounds on osteoclastic bone resorption. We have previously established a culture system for examining MNC formation. Therefore, it is now possible to examine the effects of various test compounds on the recruitment of OCLs and pit formation by the same OCLs in a series of experiments.