Generation of Osteoclastic Function in Mouse Bone Marrow Cultures: Multinuclearity and Tartrate- Resistant Acid Phosphatase Are Unreliable Markers for Osteoclastic Differentiation*

The osteoclast is the cell that resorbs bone. It is known to derive from hemopoietic precursors, and a series of recent experiments has used enumeration of the tartrate-resistant acid phosphatase (TRAP)-positive multinucleate cells that develop in cultures of hemopoietic tissue as a means to analyze the regulation of osteoclast generation. These multinucleate cells have never been definitively characterized as osteoclasts, however, and we elected to assess the relationship among bone resorption (the primary function of the osteoclast), TRAP, and multinuclearity in mouse bone marrow cultures. Mouse bone marrow cells and peritoneal macrophages were incubated on plastic coverslips or bone slices for up to 14 days in the presence or absence of 1 alpha, 25-dihydroxyvitamin D3 [1 alpha,25-(OH)2D3]. Osteoclast generation, as judged by bone resorption, occurred in marrow cell cultures only in the presence of 1 alpha,25-(OH)2D3. However, TRAP-positive multinuclear cells developed both with and without the hormone. The multinuclear cells bound F4/80, a marker for macrophages that does not bind to osteoclasts. Peritoneal macrophages became multinucleate and developed TRAP positivity in culture to levels similar to those in freshly isolated osteoclasts, especially with 1 alpha,25-(OH)2D3, but remained nonresorptive. In cultures of marrow cells incubated with 1 alpha,25-(OH)2D3 bone resorption was more extensive than could readily be accounted for by the number of multinucleate cells present, and the size of excavations and extent of resorption suggested a major contribution by mononuclear cells with osteoclastic function. Thus, while TRAP and multinuclearity are reliable markers for osteoclastic phenotype in bone, they are unreliable markers in culture. Experiments designed to evaluate the regulation of osteoclast generation through enumeration of TRAP-positive multinucleate cells formed in bone marrow cultures will not only overstate, to an unknown and probably variable degree, the number of multinucleate osteoclasts that develop, but will also fail to even identify what may be a considerable and more substantial population of mononuclear cells that possess osteoclastic characteristics.