In vitro bone resorption by isolated multinucleated giant cells from giant cell tumour of bone: Light and electron microscopic study

Abstract

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.

Culturing of cells from giant cell tumour of bone on natural and synthetic calcified substrata: the effect of leukaemia inhibitory factor and vitamin D3 on the resorbing activity of osteoclast-like cells

“…The morphology of these lacunae varied from simple small round pits to complex areas reaching 100 pm. Their size and number increased significantly during the following 5 days of culturing (by 5.5-and 4.4-fold, respectively), in agreement with the qualitative observations made by Kanehisa et al [21].…”

“…In agreement with others reporting on osteoclasts from GCT [7,21] we were able to obtain significant number of viable MNC from a tumour specimen by means of mechanical tissue disaggregation and two cycles of trypsinization. Most of the MNC (along with some mononuclear cells) possessed strong TRAP activity and varied in size and nuclear number (from 3 to more than 50).…”

“…The methods used for cell preparation were described elsewhere [21] with some modifications. Tumour specimens were stored for about 1 h in sterile ct-MEM, supplemented with 1% antibiotic mixture (10,000 U/ml penicillin, 10,000 gg/ml streptomycin solution, GIBCO, BRL, Eragny, France) and heparin (50 U/ml).…”

“…Currently, mesenchymal fibroblast-like cells are believed to represent the neoplastic tumour population which produces several factors presumably involved in the recruitment of osteoclasts and their monocyte/macrophage precursors [15,22,36]. MNC from GCT are osteoclasts, as they share with these latter common phenotypic and functional characteristics, such as possession of calcitonin receptors [15], expression of osteoclastic antigens [19] and the ability to excavate resorption lacunae on natural mineralized substrata [7,21]. GCT osteoclasts and their mononuclear precursors thus provide a rich cellular source for the investigation of the cellular mechanisms leading to the formation of MNC, the differentiation pattern of their progenitors, the resorption process and the involvement of hormones and cytokines in these events.…”

The transient appearance of zipper-like actin superstructures during the fusion of osteoclasts

“…In fact, some isolated multinucleated cells obtained from giant cell tumors of bone exhibit characteristics similar to those of hypermultinucleated osteoclasts, i.e. compartmentalization and the zipper-like structure discussed above (Kanehisa et al, 1991).…”