Bone Research Protocols pp 103-117

Part of the Methods in Molecular Biology book series (MIMB, volume 816)

Rodent Osteoclast Cultures



This chapter describes quantitative methods for isolating and culturing rodent osteoclasts on dentine, a bone-like, resorbable substrate. These techniques generate relatively large numbers of osteoclasts and allow the key processes of osteoclast formation and activation to be studied independently. A special focus will be on the role of extracellular pH, a critical factor in the control of osteoclast function.

Key words

Osteoclast Resorption pH Bone Dentine 


  1. 1.
    Boyde, A., Ali, N. N., and Jones, S. J. (1984) Resorption of dentine by isolated osteoclasts in vitro. Br. Dent. J. 156, 216–220.PubMedCrossRefGoogle Scholar
  2. 2.
    Chambers, T. J., Revell, P. A., Fuller, K., and Athanasou, N. A. (1984) Resorption of bone by isolated rabbit osteoclasts. J. Cell Sci. 66, 383–399.PubMedGoogle Scholar
  3. 3.
    Arnett, T. R., and Dempster, D. W. (1986) Effect of pH on bone resorption by rat osteoclasts in vitro. Endocrinology 119, 119–124.PubMedCrossRefGoogle Scholar
  4. 4.
    McSheehy, P. M., and Chambers, T. J. (1986) Osteoblast-like cells in the presence of parathyroid hormone release soluble factor that stimulates osteoclastic bone resorption. Endocrinology 119, 1654–1659.PubMedCrossRefGoogle Scholar
  5. 5.
    Arnett, T. R., and Dempster, D. W. (1987) A comparative study of disaggregated chick and rat osteoclasts in vitro: effects of calcitonin and prostaglandins. Endocrinology 120, 602–608.PubMedCrossRefGoogle Scholar
  6. 6.
    Takahashi, N., Yamana, H., Yoshiki, S., Roodman, G. D., Mundy, G. R., Jones, S. J., Boyde, A., and Suda, T. (1988) Osteoclast-like cell formation and its regulation by osteotropic hormones in mouse bone marrow cultures. Endocrinology 122, 1373–1382.PubMedCrossRefGoogle Scholar
  7. 7.
    Suda, T., Takahashi, N., and Martin, T. J. (1992) Modulation of osteoclast differentiation. Endocr. Rev. 13, 66–80.PubMedGoogle Scholar
  8. 8.
    Takahashi, N., Akatsu, T., Udagawa, N., Sasaki, T., Yamaguchi, A., Moseley, J. M., Martin, T. J., and Suda, T. (1988) Osteoblastic cells are involved in osteoclast formation. Endocrinology 123, 2600–2602.PubMedCrossRefGoogle Scholar
  9. 9.
    Yoshida, H., Hayashi, S., Kunisada, T., Ogawa, M., Nishikawa, S., Okamura, H., Sudo, T., Shultz, L. D., and Nishikawa, S. (1990) The murine mutation osteopetrosis is in the coding region of the macrophage colony stimulating factor gene. Nature 345, 442–444.PubMedCrossRefGoogle Scholar
  10. 10.
    Lacey, D. L., Timms, E., Tan, H. L., Kelley, M. J., Dunstan, C. R., Burgess, T., Elliott, R., Colombero, A., Elliott, G., Scully, S., Hsu, H., Sullivan, J., Hawkins, N., Davy, E., Capparelli, C., Eli, A., Qian, Y. X., Kaufman, S., Sarosi, I., Shalhoub, V., Senaldi, G., Guo, J., Delaney, J., and Boyle, W. J. (1998) Osteoprotegerin ligand is a cytokine that regulates osteoclast differentiation and activation. Cell 93, 165–176.PubMedCrossRefGoogle Scholar
  11. 11.
    Yasuda, H., Shima, N., Nakagawa, N., Yamaguchi, K., Kinosaki, M., Mochizuki, S., Tomoyasu, A., Yano, K., Goto, M., Murakami, A., Tsuda, E., Morinaga, T., Higashio, K., Udagawa, N., Takahashi, N., and Suda, T. (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.PubMedCrossRefGoogle Scholar
  12. 12.
    Goldhaber, P. and Rabadjija, L. (1987) H+ stimulation of cell-mediated bone resorption in tissue culture. Am. J. Physiol 253, E90–E98.PubMedGoogle Scholar
  13. 13.
    Walsh, C. A., Beresford, J. N., Birch, M. A., Boothroyd, B., and Gallagher, J. A. (1991) Application of reflected light microscopy to identify and quantitate resorption by isolated osteoclasts. J. Bone Miner. Res. 6, 661–671.PubMedCrossRefGoogle Scholar
  14. 14.
    Arnett, T. R., and Spowage, M. (1996) Modulation of the resorptive activity of rat osteoclasts by small changes in extracellular pH near the physiological range. Bone 18, 277–279.PubMedCrossRefGoogle Scholar
  15. 15.
    Arnett, T. R., Gibbons, D. C., Utting, J. C., Orriss, I. R., Hoebertz, A., Rosendaal, M., and Meghji, S. (2003) Hypoxia is a major stimulator of osteoclast formation and bone resorption. J. Cell Physiol. 196, 2–8.PubMedCrossRefGoogle Scholar
  16. 16.
    Meghji, S., Morrison, M. S., Henderson, B., and Arnett, T. R. (2001) pH dependence of bone resorption: mouse calvarial osteoclasts are activated by acidosis. Am. J. Physiol. Endocrinol. Metab. 280, E112–E119.PubMedGoogle Scholar
  17. 17.
    Morrison, M. S. and Arnett, T. R. (1997) Effect of extracellular pH on resorption pit formation by chick osteoclasts. J. Bone Miner. Res. 12, S290–S290.CrossRefGoogle Scholar
  18. 18.
    Bushinsky, D. A. (1987) Net calcium influx from live bone during chronic metabolic, but not respiratory, acidosis. Am. J. Physiol. 256, F836–F842.Google Scholar
  19. 19.
    Morrison, M. S., Turin, L., King, B. F., Burnstock, G., and Arnett, T. R. (1998) ATP is a potent stimulator of the activation and formation of rodent osteoclasts. J. Physiol. 511 (Pt 2), 495–500.PubMedCrossRefGoogle Scholar
  20. 20.
    Hoebertz, A., Meghji, S., Burnstock, G., and Arnett, T. R. (2001) Extracellular ADP is a powerful osteolytic agent: evidence for signaling through the P2Y1 receptor on bone cells. FASEB J. 15, 1139–1148.PubMedCrossRefGoogle Scholar
  21. 21.
    Lees, R. L., Sabharwal, V. K., and Heersche, J. N. (2001) Resorptive state and cell size influence intracellular pH regulation in rabbit osteoclasts cultured on collagen-hydroxyapatite films. Bone 28, 187–194.PubMedCrossRefGoogle Scholar
  22. 22.
    Walsh, N. C., Cahill, M., Carninci, P., Kawai, J., Okazaki, Y., Hayashizaki, Y., Hume, D. A., and Cassady, A. I. (2003) Multiple tissue-specific promoters control expression of the murine tartrate-resistant acid phosphatase gene. Gene 307, 111–123.PubMedCrossRefGoogle Scholar
  23. 23.
    Boyde, A., and Jones, S. J. (1991) Pitfalls in pit measurement. Calcif. Tissue Int. 49, 65–70.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  1. 1.Department of Cell and Developmental BiologyUniversity College LondonLondonUK

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