Search tips
Search criteria 


Logo of jcinvestThe Journal of Clinical Investigation
J Clin Invest. 1990 March; 85(3): 632–639.
PMCID: PMC296476

Oxygen-derived free radicals stimulate osteoclastic bone resorption in rodent bone in vitro and in vivo.


The mechanisms by which bone resorbing osteoclasts form and are activated by hormones are poorly understood. We show here that the generation of oxygen-derived free radicals in cultured bone is associated with the formation of new osteoclasts and enhanced bone resorption, identical to the effects seen when bones are treated with hormones such as parathyroid hormone (PTH) and interleukin 1 (IL-1). When free oxygen radicals were generated adjacent to bone surfaces in vivo, osteoclasts were also formed. PTH and IL-1-stimulated bone resorption was inhibited by both natural and recombinant superoxide dismutase, an enzyme that depletes tissues of superoxide anions. We used the marker nitroblue tetrazolium (NBT) to identify the cells that were responsible for free radical production in resorbing bones. NBT staining was detected only in osteoclasts in cultures of resorbing bones. NBT staining in osteoclasts was decreased in bones coincubated with calcitonin, an inhibitor of bone resorption. We also found that isolated avian osteoclasts stained positively for NBT. NBT staining in isolated osteoclasts was increased when the cells were incubated with bone particles, to which they attach. We confirmed the formation of superoxide anion in isolated avian osteoclasts using ferricytochrome c reduction as a method of detection. The reduction of ferricytochrome c in isolated osteoclasts was inhibited by superoxide dismutase. Our results suggest that oxygen-derived free radicals, and particularly the superoxide anion, are intermediaries in the formation and activation of osteoclasts.

Full text

Full text is available as a scanned copy of the original print version. Get a printable copy (PDF file) of the complete article (2.6M), or click on a page image below to browse page by page. Links to PubMed are also available for Selected References.

Images in this article

Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Nathan CF. Secretory products of macrophages. J Clin Invest. 1987 Feb;79(2):319–326. [PMC free article] [PubMed]
  • Gowen M, Mundy GR. Actions of recombinant interleukin 1, interleukin 2, and interferon-gamma on bone resorption in vitro. J Immunol. 1986 Apr 1;136(7):2478–2482. [PubMed]
  • Teti A, Oreffo RO, Zambonin Zallone A, Triffitt JT, Francis MJ. Acid phosphatase activity is stimulated in isolated osteoclasts by vitamin A. Boll Soc Ital Biol Sper. 1986 Oct 30;62(10):1311–1314. [PubMed]
  • Zambonin Zallone A, Teti A, Primavera MV. Isolated osteoclasts in primary culture: first observations on structure and survival in culture media. Anat Embryol (Berl) 1982 Dec;165(3):405–413. [PubMed]
  • Zambonin Zallone A, Teti A. The osteoclasts of hen medullary bone under hypocalcaemic conditions. Anat Embryol (Berl) 1981;162(4):379–392. [PubMed]
  • Fridovich I. Quantitative aspects of the production of superoxide anion radical by milk xanthine oxidase. J Biol Chem. 1970 Aug 25;245(16):4053–4057. [PubMed]
  • McCord JM, Fridovich I. The reduction of cytochrome c by milk xanthine oxidase. J Biol Chem. 1968 Nov 10;243(21):5753–5760. [PubMed]
  • Yates AJ, Gutierrez GE, Smolens P, Travis PS, Katz MS, Aufdemorte TB, Boyce BF, Hymer TK, Poser JW, Mundy GR. Effects of a synthetic peptide of a parathyroid hormone-related protein on calcium homeostasis, renal tubular calcium reabsorption, and bone metabolism in vivo and in vitro in rodents. J Clin Invest. 1988 Mar;81(3):932–938. [PMC free article] [PubMed]
  • Sabatini M, Boyce B, Aufdemorte T, Bonewald L, Mundy GR. Infusions of recombinant human interleukins 1 alpha and 1 beta cause hypercalcemia in normal mice. Proc Natl Acad Sci U S A. 1988 Jul;85(14):5235–5239. [PubMed]
  • Rook GA, Steele J, Umar S, Dockrell HM. A simple method for the solubilisation of reduced NBT, and its use as a colorimetric assay for activation of human macrophages by gamma-interferon. J Immunol Methods. 1985 Sep 3;82(1):161–167. [PubMed]
  • Murray HW, Cohn ZA. Macrophage oxygen-dependent antimicrobial activity. III. Enhanced oxidative metabolism as an expression of macrophage activation. J Exp Med. 1980 Dec 1;152(6):1596–1609. [PMC free article] [PubMed]
  • Murray HW, Cohn ZA. Mononuclear phagocyte antimicrobial and antitumor activity: the role of oxygen intermediates. J Invest Dermatol. 1980 May;74(5):285–288. [PubMed]
  • Babior BM. Oxidants from phagocytes: agents of defense and destruction. Blood. 1984 Nov;64(5):959–966. [PubMed]
  • Stock JL, Coderre JA, Levine PH. Effects of calcium-regulating hormones and drugs on human monocyte chemiluminescence. J Clin Endocrinol Metab. 1982 Nov;55(5):956–960. [PubMed]
  • Freund M, Pick E. The mechanism of action of lymphokines. IX. The enzymatic basis of hydrogen peroxide production by lymphokine-activated macrophages. J Immunol. 1986 Aug 15;137(4):1312–1318. [PubMed]
  • Clement LT, Lehmeyer JE. Regulation of the growth and differentiation of a human monocytic cell line by lymphokines. I. Induction of superoxide anion production and chemiluminescence. J Immunol. 1983 Jun;130(6):2763–2766. [PubMed]
  • Friedman J, Au WY, Raisz LG. Responses of fetal rat bone to thyrocalcitonin in tissue culture. Endocrinology. 1968 Jan;82(1):149–156. [PubMed]
  • Weiss SJ, LoBuglio AF. Phagocyte-generated oxygen metabolites and cellular injury. Lab Invest. 1982 Jul;47(1):5–18. [PubMed]
  • Babior BM. The respiratory burst of phagocytes. J Clin Invest. 1984 Mar;73(3):599–601. [PMC free article] [PubMed]
  • Takahashi N, MacDonald BR, Hon J, Winkler ME, Derynck R, Mundy GR, Roodman GD. Recombinant human transforming growth factor-alpha stimulates the formation of osteoclast-like cells in long-term human marrow cultures. J Clin Invest. 1986 Oct;78(4):894–898. [PMC free article] [PubMed]
  • Thomson BM, Mundy GR, Chambers TJ. Tumor necrosis factors alpha and beta induce osteoblastic cells to stimulate osteoclastic bone resorption. J Immunol. 1987 Feb 1;138(3):775–779. [PubMed]

Articles from The Journal of Clinical Investigation are provided here courtesy of American Society for Clinical Investigation