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We have investigated in vitro the effects of the electrical field produced by constant current on freshly isolated rabbit osteoclasts and on well characterized clonal rat osteoblastlike cells. At field strengths of 0.1 and 1 V/mm, the osteoclasts migrated rapidly toward the positive electrode, whereas the osteoblastlike cells migrated in the opposite direction, toward the negative electrode. Thus, different cell types from the same tissue can respond differently to the same electrical signal. These results have important implications for hypotheses concerning the cellular mechanism of galvanotaxis, and may also clarify the cellular basis of the clinical application of electrical stimulation of bone healing.

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It is not clear to what extent the increased bone mass observed in vertebral trabecular bone of fluoride-treated mammals is a consequence of effects of fluoride on the number and activity of osteoclasts or of osteoblasts. In the present communication, we have analyzed the effects of NaF on the activity of isolated rabbit osteoclasts cultured on thin slices of devitalized compact bovine bone. Osteoclastic resorption was quantitated by counting the number of resorption lacunae and measuring their surface area and their depth using scanning electron microscopy. Our results show that NaF in concentrations of 0.5-1.0 mM decreased the number of resorption lacunae made by individual osteoclasts and decreased the resorbed area per osteoclast. We argue that the concentration of fluoride in these experiments may be within the range "seen" by osteoclasts in mammals treated for prolonged periods with approximately 1 mg of NaF/kg body weight (bw) per day.

The ability of osteoclasts (OC) to migrate and resorb bone is thought to be dependent on cytoskeletal function and adhesion. Therefore, we investigated the cytoskeleton and the adhesion patterns of rabbit OC on glass and on devitalized bone slices, using specific antibodies to cytoskeletal elements and fluorescence and interference reflection microscopy. Microtubules (MT) were similar in OC on both substrata, and appeared in a pattern typical of that described for many cells. Multiple centriolar complexes were observed in most OC, either as one large aggregate in the center of the cell or dispersed singly or in small aggregates close to individual nuclei. Staining of microfilaments (MF) was similar on both substrata and appeared primarily as an F-actin network. MF distribution was different in OC associated with resorption lacunae with intense staining over those regions. In the OC on glass, high F-actin staining was detectable at the periphery in dots and rosette-like structures, which also stained for vinculin. The adhesion patterns indicated that OC on glass do not make large focal contacts, but appear to make a few tiny focal contacts that are not associated with the rosette-like structures. Most of the undersurface of the OC appeared either to be involved in close contacts or to be separated by distances of greater than 100 nm from the substratum. These studies indicate that the MF distribution and the adhesion patterns of rabbit OC are typical of motile cells, that the distribution of the cytoskeleton of rabbit OC on glass and on bone slices is similar, and that MF may be involved in the morphological changes associated with resorption.

The behaviour of multinucleated giant cells (GCs), obtained from a giant cell tumour of the tibia and cultured on glass coverslips or on devitalized bone slices, was studied using light and electron microscopy. Monitoring the GCs on bone slices by phase-contrast microscopy revealed that they had removed calcified bone matrix resulting in excavation of lacunae, with subsequent lateral extension and perforation of the bone slices. Electron microscopy demonstrated for the first time that the GCs responsible for exavating lacunae had two specific membrane modifications, ruffled border and clear zone, and showed basically similar cytoplasmic fine structures to those of osteoclasts. Fluorescence images of the GCs on glass and on bone after rhodamine-conjugated phalloidin staining revealed that most of the GCs had an intensely fluorescent peripheral band composed of a number of F-actin dots called podosomes. Some GCs showed unusual arrangements of podosomes suggesting abortive attempts at GC formation. We have demonstrated that the band structure of the GCs cultured on bone is intimately involved in bone resorption. Two stromal cell types could be recognized. The predominant type, which seemed to be the only neoplastic element because of its proliferative capability, showed quite different fine structural and cytoskeletal features from the GCs. The other type, which was much less frequent and seemed not to proliferate, had morphological similarities to the GCs, and seemed to be their precursor. Importantly GCs cultured on bone and the osteoclasts share common structures for adhesion to and resorption of bone, strongly supporting the view that the GCs of the giant cell tumour of bone are potentially active bone resorbers and can be regarded as osteoclasts.

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