Abstract
Osteoclasts are bone-resorbing multinucleated cells derived from the monocytemacrophage 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 1α,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 κB ligand (RANKL) and macrophage colony-stimulating factor (M-CSF). Using RANKL and M-CSF, we can induce osteoclasts from monocytemacrophage 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.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Chambers, T. J. (2000) Regulation of the differentiation and function of osteoclasts. J. Pathol. 192, 4–13.
Roodman, G. D. (1996) Advances in bone biology: the osteoclast. Endocr. Rev. 17, 308–332.
Teitelbaum, S. L. and Ross, F. P. (2003) Genetic regulation of osteoclast development and function. Nat. Rev. Genet. 4, 638–649.
Takahashi, N., Yamana, H., Yoshiki, S., et al. (1988) Osteoclast-like cell formation and its regulation by osteotropic hormones in mouse bone marrow cultures. Endocrinology 122, 1373–1382.
Suda, T., Takahashi, N., and Martin, T. J. (1992) Modulation of osteoclast differentiation. Endocr. Rev. 13, 66–80.
Takahashi, N., Akatsu, T., Udagawa, N., et al. (1988) Osteoblastic cells are involved in osteoclast formation. Endocrinology 123, 2600–2602.
Yoshida, H., Hayashi, S., Kunisada, T., et al. (1990) The murine mutation osteopetrosis is in the coding region of the macrophage colony stimulating factor gene. Nature 345, 442–444.
Felix, R., Cecchini, M. G., and Fleisch, H. (1990) Macrophage colony stimulating factor restores in vivo bone resorption in the op/op osteopetrotic mouse. Endocrinology 127, 2592–2594.
Takahashi, N., Udagawa, N., Akatsu, T., Tanaka, H., Isogai, Y., and Suda, T. (1991) Deficiency of osteoclasts in osteopetrotic mice is due to a defect in the local microenvironment provided by osteoblastic cells. Endocrinology 128, 1792–1796.
Yasuda, H., Shima, N., Nakagawa, N., et al. (1998) Osteoclast differentiation factor is a ligand for osteoprotegerin/osteoclastogenesis-inhibitory factor and is identical to TRANCE/RANKL. Proc. Natl. Acad. Sci. USA 95, 3597–3602.
Lacey, D. L., Timms, E., Tan, H. L., et al. (1998) Osteoprotegerin ligand is a cytokine that regulates osteoclast differentiation and activation. Cell 93, 165–176.
Suda, T., Takahashi, N., Udagawa, N., Jimi, E., Gillespie, M. T., and Martin, T. J. (1999) Modulation of osteoclast differentiation and function by the new members of the tumor necrosis factor receptor and ligand families. Endocr. Rev. 20, 345–357.
Boyle, W. J., Simonet, W. S., and Lacey, D. L. (2003) Osteoclast differentiation and activation. Nature 423, 337–342.
Hsu, H., Lacey, D. L., Dunstan, C. R., et al. (1999) Tumor necrosis factor receptor family member RANK mediates osteoclast differentiation and activation induced by osteoprotegerin ligand. Proc. Natl. Acad. Sci. USA 96, 3540–3545.
Akatsu, T., Tamura, T., Takahashi, N et al. (1992) Preparation and characterization of a mouse multinucleated cell population. J. Bone Miner. Res. 7, 1297–1306.
Tamura, T., Takahashi, N., Akatsu, T., et al. (1993) A new resorption assay with mouse osteoclast-like multinucleated cells formed in vitro. J. Bone Miner. Res. 8, 953–960.
Udagawa, N., Takahashi, N., Akatsu, T., et al. (1990) Origin of osteoclasts: Mature monocytes and macrophages are capable of differentiating into osteoclasts under a suitable microenvironment prepared by bone marrow-derived stromal cells. Proc. Natl. Acad. Sci. USA 87, 7260–7264.
Kobayashi, K., Takahashi, N., Jimi, E., et al. (2000) Tumor necrosis factor a stimulates osteoclast differentiation by a mechanism independent of the ODF/RANKLRANK interaction. J. Exp. Med. 191, 275–286.
Fuller, K., Murphy, C., Kirstein, B., Fox, S. W., and Chambers, T. J. (2002) TNFα potently activates osteoclasts, through a direct action independent of and strongly synergistic with RANKL. Endocrinology 143, 1108–1118.
Udagawa, N., Takahashi, N., Akatsu, T., et al. (1989) The bone marrow-derived stromal cell lines MC3T3-G2/PA6 and ST2 support osteoclast-like cell differentiation in cocultures with mouse spleen cells. Endocrinology 125, 1805–1813.
Suda, T., Jimi, E., Nakamura, I., and Takahashi, N. (1997) Role of 1α, 25-dihydroxyvitamin D3 in osteoclast differentiation and function. Methods Enzymol. 282, 223–235.
Jimi, E., Ikebe, T., Takahashi, N., Hirata, N., Suda, T., and Koga, T. (1996) Interleukin-1b activates an NF-κB-like factor in osteoclast-like cells. J. Biol. Chem. 271, 4605–4608.
Nakamura, I., Jimi, E., Duong, L. T., et al. (1998), Tyrosine phosphorylation of p130Cas is involved in actin organization in osteoclasts. J. Biol. Chem. 273, 11,144–11,149.
Suda, T., Nakamura, I., Jimi, E., and Takahashi, N. (1997) Regulation of osteoclast function. J. Bone Miner. Res. 12, 869–879.
Udagawa, N., Takahashi, N., Sasaki, T., et al. (1992) Failure of bone resorption in osteosclerotic (oc/oc) mice is due to a microenvironment. In: Calcium Regulating Hormones and Bone Metabolism, Cohn, D. V., Gennari C, Tashjian, A. H. Jr., eds., Elsevier Science Publishers, pp. 151–156.
Zambonin-Zallone, A., Teti, A., Carano, A., and Marchisio, P. C. (1988) The distribution of podosomes in osteoclasts cultured on bone laminae, effect of retinol. J. Bone Miner. Res. 3, 517–523.
Chellaiah, M. A., Soga, N., Swanson, S., et al. (2000) Rho-A is critical for osteoclast podosome organization, motility, and bone resorption. J. Biol. Chem. 275, 11,993–12,002.
Nakamura, I., Takahashi, N., Sasaki, T., Jimi, E., Kurokawa, T., and Suda, T. (1996) Chemical and physical properties of the extracellular matrix are required for the actin ring formation in osteoclasts. J. Bone Miner. Res. 11, 1873–1879.
Suzuki, H., Nakamura, I., Takahashi, N., et al. (1996) Calcitonin-induced changes in cytoskeleton are mediated by a signal pathway associated with protein kinase A in osteoclasts. Endocrinology 137, 4685–4690.
Murakami, H., Takahashi, N., Sasaki, T., et al. (2995) A possible mechanism of the specific action of bisophosphonates on osteoclasts: Tiludronate preferentially affects polarized osteoclasts having ruffled borders. Bone 17, 137–144.
Simonet, W. S., Lacey, D. L., Dunstan, C. R., et al. (1997) Osteoprotegerin, a novel secreted protein involved in the regulation of bone density. Cell 89, 309–319.
Tsuda, E., Goto, M., Mochizuki, S., et al. (1997) Isolation of a novel cytokine from human fibroblasts that specifically inhibits osteoclastogenesis. Biochem. Biophys. Res. Commun. 234, 137–142.
Wong, B. R., Rho, J., Arron, J., et al. (1997) TRANCE is a novel ligand of the tumor necrosis factor receptor family that activates c-Jun N-terminal kinase in T cells. J. Biol. Chem. 272, 25,190–25,194.
Anderson, D. M., Maraskovsky, E., Billingsley, W. L., et al. (1997) A homologue of the TNF receptor and its ligand enhance T-cell growth and dendritic-cell function. Nature 390, 175–179.
The American Society for Bone and Mineral Research President’s Committee on Nomenclature (2000) Proposed standard nomenclature for new tumor necrosis factor family members involved in the regulation of bone resorption. J. Bone Miner. Res. 15, 2293–2296.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2007 Humana Press Inc., Totowa, NJ
About this protocol
Cite this protocol
Takahashi, N., Udagawa, N., Kobayashi, Y., Suda, T. (2007). Generation of Osteoclasts In Vitro, and Assay of Osteoclast Activity. In: Cope, A.P. (eds) Arthritis Research. Methods in Molecular Medicine, vol 135. Humana Press. https://doi.org/10.1007/978-1-59745-401-8_18
Download citation
DOI: https://doi.org/10.1007/978-1-59745-401-8_18
Publisher Name: Humana Press
Print ISBN: 978-1-58829-344-2
Online ISBN: 978-1-59745-401-8
eBook Packages: Springer Protocols