Generation of osteoclasts in vitro, and assay of osteoclast activity

Osteoclasts are bone-resorbing multinucleated cells derived from the monocyte-macrophage lineage. The authors have developed a mouse marrow culture system and a coculture system of mouse osteoblasts and hemopoietic cells, in which osteoclasts are formed in response to various osteotropic factors such as 1alpha,25-dihydroxyvitamin D3, parathyroid hormone, prostaglandin E2, and interleukin-11. Recent studies have revealed that osteoblasts express two cytokines essential for osteoclastogenesis: receptor activator of nuclear factor kappaB ligand (RANKL) and macrophage colony-stimulating factor (M-CSF). Using RANKL and M-CSF, we can induce osteoclasts from monocyte-macrophage lineage cells even in the absence of osteoblasts. This chapter describes the methods for osteoclast formation in vitro in the presence and absence of osteoblasts, and for pit-formation assay using dentine slices and osteoclasts formed in vitro. These culture systems have made it possible to investigate each step of osteoclast development and function separately.