Osteoclast development in the coculture system of periostless metatarsal bones and hemopoietic cells studied by in situ hybridization with a probe for Y chromosomes

In the coculture system of periostless metatarsal bones of 17-day-old fetal mice and osteoclast progenitors, osteoclasts will develop. Our goal in the present report was to provide further evidence that in the coculture system of fetal metatarsal bone rudiments with hemopoietic cells, the osteoclasts developing inside the bone rudiments are exclusively derived from the cells suspended in the plasma clot and not from endogenous precursor cells of the bone explants themselves, by using the technique of in situ hybridization with a probe for the mouse Y chromosome. Osteoclast formation in unstripped male metatarsal rudiments, occurring after 3–4 days of culture, was compared with osteoclast formation in cocultures of female metatarsal rudiments and male bone marrow cells, occurring after 5–6 days of culture. Osteoclasts were recognized by their tartrate-resistant acid phosphatase activity. In paraffin sections of cultured male metatarsals, the mean percentage of microscopically identifiable osteoclast nuclei, in which the Y chromosome could be detected, was 43.1±4.2% (n=12). For cocultures of female metatarsal bones and male bone marrow cells this mean percentage was 40.9±5.7% (n=17). Statistical comparison by means of the two sample t-test indicated no significant difference in the percentages of osteoclast nuclei containing the Y chromosome for both groups. We concluded that the osteoclasts do derive from cocultured cells and not from precursor cells in the bone explant itself. Therefore, the coculture system is a reliable in vitro system for studying osteoclast formation from progenitor/precursor cells.