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It has been established, that the osteoclast is derived from the haemopoietic stem cell, but its exact lineage is still controversial. It is sometimes suggested, that osteoclasts and monocytes/macrophages are related cells. It has also been suggested that osteoclast differentiation is regulated by osteoblasts and 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3). In the present paper we addressed the question whether avian monocytes can differentiate into osteoclasts in vitro, using an array of immunocytochemical, enzyme cytochemical and function markers. We have also determined the effects of osteoblasts, osteoblast conditioned medium and 1,25-(OH)2D3 on the expression of osteoclastic features on monocytes during culture. Monocytes developed tartrate resistant acid phosphatase (TRAcP) enzyme activity and antigens for all anti-osteoclast antibodies tested, during culture. However, they did not acquire the ability to resorb dentine and still showed phagocytosis of latex spheres. This indicates that the monocytes developed into cells resembling osteoclasts but lacking their function while retaining the function of macrophages. Osteoblast conditioned medium stimulated TRAcP enzyme activity and proliferation of monocytes in cultures. Addition of osteoblasts or osteoblast conditioned medium to monocyte cultures on dentine in the presence or absence of 1,25-(OH)2D3 did not result in the generation of genuine osteoclasts, nor in pit formation. 1,25-(OH)2D3 appeared to be cytotoxic to the avian monocytes in concentrations considered optimal for mouse osteoclast formation. These results suggest that avian monocytes do not readily differentiate into osteoclasts under in vitro conditions that stimulate osteoclast differentiation from bone marrow derived haemopoietic cells. Furthermore, labelling with anti-osteoclast antibodies and TRAcP as osteoclast-markers should be used only with great caution in the identification of osteoclasts formed in vitro.

Six murine macrophage precursor cell lines, thought to be arrested around the CFU-GM stage of the myeloid differentiation and shown to be negative for acid phosphatase, F4/80 antigen expression and phagocytosis capacity, were tested for their ability to differentiate into osteoclasts. Their differentiation potential was compared with that of the haemopoietic stem cell line FDCP-mix C2GM. None of the macrophage precursor cell lines could be induced to differentiate into osteoclasts when the cells were cocultured with either periosteum-free metatarsal bones of fetal mice, or monolayers of osteoblast-like cells. In contrast, when the haemopoietic stem cell line FDCP-mix C2GM, murine fetal liver cells or murine spleen cells were used as a source of haemopoietic precursor cells, numerous osteoclasts were formed in both culture systems. During cell culture a small percentage of the macrophage precursor cells attached to the bottom of the culture well. These firmly attached cells acquired acid phosphatase activity, F4/80 antigen expression and phagocytosis capacity. Furthermore, when the cell lines were cultured for 2 or 4 days with 1% DMSO, up to 30% of the precursor cells differentiated into metamyelocytes. These results suggest that the macrophage precursor cell lines are able to acquire macrophage and granulocyte characteristics, but are unable to differentiate into osteoclasts. In contrast, the haemopoietic stem cell line FDCP-mix C2GM is able to differentiate into both macrophages and osteoclasts. We therefore suggest that the osteoclast lineage branches off at an early stage of the myeloid differentiation pathway.

The fully differentiated osteoblast may be easily recognized in bone tissue. Its cuboidal shape, its position directly opposed to the bone surface and its capacity to produce calcified bone matrix are characteristic. Three other differentiation stages are also reasonably well defined--the preosteoblast, the osteocyte and the lining cell. These differentiation stages are preceded by an unknown number of precursor, progenitor and stem cell stages. Little is known about the regulation of the transitions between the various osteogenic phenotypes and their reversibility or irreversibility. One of the reasons for this is the lack of adequate tools with which to recognize the various differentiation stages. We have developed a number of monoclonal antibodies (in bone) specifically directed against osteocytes, osteoblasts and as yet unidentified cells in the periosteum. The anti-osteocyte monoclonals were used to recognize osteocytes in bone cell cultures and we obtained purified osteocyte populations for metabolic studies. Osteocytes were shown to have binding sites for parathyroid hormone. The antibodies directed against osteoblasts showed that at present our culture conditions are inadequate to allow osteoblast differentiation in vitro.