Journal of Biological Physics

, Volume 38, Issue 2, pp 279–291 | Cite as

Ca/P concentration ratio at different sites of normal and osteoporotic rabbit bones evaluated by Auger and energy dispersive X-ray spectroscopy

  • Nikolaos Kourkoumelis
  • Ioannis Balatsoukas
  • Margaret Tzaphlidou
Original Paper


Osteoporosis is a systemic skeletal disorder associated with reduced bone mineral density and the consequent high risk of bone fractures. Current practice relates osteoporosis largely with absolute mass loss. The assessment of variations in chemical composition in terms of the main elements comprising the bone mineral and its effect on the bone’s quality is usually neglected. In this study, we evaluate the ratio of the main elements of bone mineral, calcium (Ca), and phosphorus (P), as a suitable in vitro biomarker for induced osteoporosis. The Ca/P concentration ratio was measured at different sites of normal and osteoporotic rabbit bones using two spectroscopic techniques: Auger electron spectroscopy (AES) and energy-dispersive X-ray spectroscopy (EDX). Results showed that there is no significant difference between samples from different genders or among cortical bone sites. On the contrary, we found that the Ca/P ratio of trabecular bone sections is comparable to cortical sections with induced osteoporosis. Ca/P ratio values are positively related to induced bone loss; furthermore, a different degree of correlation between Ca and P in cortical and trabecular bone is evident. This study also discusses the applicability of AES and EDX to the semiquantitative measurements of bone mineral’s main elements along with the critical experimental parameters.


Ca/P ratio Bone mineral Apatite Osteoporosis Auger electron spectroscopy Energy dispersive X-ray spectroscopy EDX Calcium Phosphorus 



The authors thank Assoc. Prof. P. Patsalas for providing the Auger facility and for helpful suggestions, and the Horizontal Laboratory Network, University of Ioannina, for the SEM-EDX.


  1. 1.
    Rachner, T.D., Khosla, S., Hofbauer, L.C.: Osteoporosis: now and the future. Lancet 377, 1276–1287 (2011)CrossRefGoogle Scholar
  2. 2.
    Kanis, J.A., Alexeeva, L., Bonjour, J.P., Burkhardt, P., Christiansen, C., Cooper, C., Delmas, P., Johnell, O., Johnston, C., Kanis, J.A., Khaltaev, N., Lips, P., Mazzuoli, G., Melton, L.J., Meunier, P., Seeman, E., Stepan, J., Tosteson, A.: Assessment of fracture risk and its application to screening for postmenopausal osteoporosis—synopsis of a WHO report. Osteoporos. Int. 4, 368–381 (1994)CrossRefGoogle Scholar
  3. 3.
    DeLaet, C.E.D.H., vanHout, L.B., Burger, H., Hofman, A., Pols, H.A.P.: Bone density and risk of hip fracture in men and women: cross sectional analysis. Br. Med. J. 315, 221–225 (1997)CrossRefGoogle Scholar
  4. 4.
    Schuit, S.C.E., van der Klift, M., Weel, A.E.A.M., de Laet, C.E.D.H., Burger, H., Seeman, E., Hofman, A., Uitterlinden, A.G., van Leeuwen, J.P.T.M., Pols, H.A.P.: Fracture incidence and association with bone mineral density in elderly men and women: the Rotterdam study. Bone 34, 195–202 (2004)CrossRefGoogle Scholar
  5. 5.
    Bouxsein, M.L., Seeman, E.: Quantifying the material and structural determinants of bone strength. Best Pract. Res. Clin. Rheumatol. 23, 741–753 (2009)CrossRefGoogle Scholar
  6. 6.
    Einhorn, T.A.: Bone strength: the bottom line. Calcif. Tissue Int. 51, 333–339 (1992)CrossRefGoogle Scholar
  7. 7.
    Kanis, J.A., Melton, L.J., Christiansen, C., Johnston, C.C., Khaltaev, N.: The diagnosis of osteoporosis. J. Bone Miner. Res. 9, 1137–1141 (1994)CrossRefGoogle Scholar
  8. 8.
    Cummings, S.R., Cauley, J.A., Palermo, L., Ross, P.D., Wasnich, R.D., Black, D., Faulkner, K.G.: Racial differences in hip axis lengths might explain racial differences in rates of hip fracture. Osteoporos. Int. 4, 226–229 (1994)CrossRefGoogle Scholar
  9. 9.
    Black, D.M., Bouxsein, M.L., Marshall, L.M., Cummings, S.R., Lang, T.F., Cauley, J.A., Ensrud, K.E., Nielson, C.M., Orwoll, E.S.: Proximal femoral structure and the prediction of hip fracture in men: a large prospective study using QCT. J. Bone Miner. Res. 23, 1326–1333 (2008)CrossRefGoogle Scholar
  10. 10.
    Carrey, J.D.: Bones: Structure and Mechanics. Princeton University Press, NJ (2006)Google Scholar
  11. 11.
    van der Harst, M.R., Brama, P.A.J., van de Lest, C.H.A., Kiers, G.H., DeGroot, J., van Weeren, P.R.: An integral biochemical analysis of the main constituents of articular cartilage, subchondral and trabecular bone. Osteoarthr. Cartil. 12, 752–761 (2004)CrossRefGoogle Scholar
  12. 12.
    Hu, Y.Y., Rawal, A., Schmidt-Rohr, K.: Strongly bound citrate stabilizes the apatite nanocrystals in bone. Proc. Natl. Acad. Sci. USA 107, 22425–22429 (2010)ADSCrossRefGoogle Scholar
  13. 13.
    Kahn, A.J., Partridge, N.C.: Bone. Vol. 2: The Osteoclast: Bone Resorption In Vivo. CRC Press, Boca Raton, FL (1991)Google Scholar
  14. 14.
    Peacock, M.: Calcium metabolism in health and disease. Clin. J. Am. Soc. Nephrol. 5, S23–S30 (2010)CrossRefGoogle Scholar
  15. 15.
    Shapiro, R., Heaney, R.P.: Co-dependence of calcium and phosphorus for growth and bone development under conditions of varying deficiency. Bone 32, 532–540 (2003)CrossRefGoogle Scholar
  16. 16.
    Coats, A.M., Zioupos, P., Aspden, R.M.: Material properties of subchondral bone from patients with osteoporosis or osteoarthritis by microindentation testing and electron probe microanalysis. Calcif. Tissue Int. 73, 66–71 (2003)CrossRefGoogle Scholar
  17. 17.
    Oelzner, P., Muller, A., Deschner, F., Huller, M., Abendroth, K., Hein, G., Stein, G.: Relationship between disease activity and serum levels of vitamin D metabolites and PTH in rheumatoid arthritis. Calcif. Tissue Int. 62, 193–198 (1998)CrossRefGoogle Scholar
  18. 18.
    Fountos, G., Yasumura, S., Glaros, D.: The skeletal calcium/phosphorus ratio: a new in vivo method of determination. Med. Phys. 24, 1303–1310 (1997)CrossRefGoogle Scholar
  19. 19.
    Bolotin, H.H., Sievanen, H.: Inaccuracies inherent in dual-energy X-ray absorptiometry in vivo bone mineral density can seriously mislead diagnostic/prognostic interpretations of patient-specific bone fragility. J. Bone Miner. Res. 16, 799–805 (2001)CrossRefGoogle Scholar
  20. 20.
    Tzaphlidou, M., Speller, R., Royle, G., Griffiths, J.: Preliminary estimates of the calcium/phosphorus ratio at different cortical bone sites using synchrotron microCT. Phys. Med. Biol. 51, 1849–1855 (2006)CrossRefGoogle Scholar
  21. 21.
    Neues, F., Epple, M.: X-ray microcomputer tomography for the study of biomineralized endo- and exoskeletons of animals. Chem. Rev. 108, 4734–4741 (2008)CrossRefGoogle Scholar
  22. 22.
    Zaichick, V., Tzaphlidou, M.: Determination of calcium, phosphorus, and the calcium/phosphorus ratio in cortical bone from the human femoral neck by neutron activation analysis. Appl. Radiat. Isotopes 56, 781–786 (2002)CrossRefGoogle Scholar
  23. 23.
    Tzaphlidou, M., Zaichick, V.: Neutron activation analysis of calcium/phosphorus ratio in rib bone of healthy humans. Appl. Radiat. Isotopes 57, 779–783 (2002)CrossRefGoogle Scholar
  24. 24.
    Ishikawa, K., Ducheyne, P., Radin, S.: Determination of the Ca/P ratio in calcium-deficient hydroxyapatite using X-ray diffraction analysis. J. Mater. Sci.: Mater. Med. 4, 165–168 (1993)CrossRefGoogle Scholar
  25. 25.
    Bradley, D.A., Farquharson, M.J., Gundogdu, O., Al-Ebraheem, A., Ismail, E.C., Kaabar, W., Bunk, O., Pfeiffer, F., Falkenberge, G., Bailey, M.: Applications of condensed matter understanding to medical tissues and disease progression: Elemental analysis and structural integrity of tissue scaffolds. Radiat. Phys. Chem. 79, 162–175 (2010)ADSCrossRefGoogle Scholar
  26. 26.
    Bailey, M.J., Coe, S., Grant, D.M., Grime, G.W., Jeynes, C.: Accurate determination of the Ca: P ratio in rough hydroxyapatite samples by SEM-EDS, PIXE and RBS – a comparative study. X-Ray Spectrom. 38, 343–347 (2009)CrossRefGoogle Scholar
  27. 27.
    Milovanovic, P., Potocnik, J., Stoiljkovic, M., Djonic, D., Nikolic, S., Neskovic, O., Djuric, M., Rakocevic, Z.: Nanostructure and mineral composition of trabecular bone in the lateral femoral neck: Implications for bone fragility in elderly women. Acta Biomater. 7, 3446–3451 (2011)CrossRefGoogle Scholar
  28. 28.
    Akesson, K., Grynpas, M.D., Hancock, R.G.V., Odselius, R., Obrant, K.J.: Energy-dispersive X-ray microanalysis of the bone mineral content in human trabecular bone: a comparison with ICPES and neutron activation analysis. Calcif. Tissue Int. 55, 236–239 (1994)CrossRefGoogle Scholar
  29. 29.
    Xu, J.D., Zhu, P.Z., Gan, Z.H., Sahar, N., Tecklenburg, M., Morris, M.D., Kohn, D.H., Ramamoorthy, A.: Natural-abundance Ca-43 solid-state NMR spectroscopy of bone. J. Am. Chem. Soc. 132, 11504–11509 (2010)CrossRefGoogle Scholar
  30. 30.
    Wu, Y., Ackerman, J.L., Strawich, E.S., Rey, C., Kim, H.M., Glimcher, M.J.: Phosphate ions in bone: Identification of a calcium-organic phosphate complex by P-31 solid-state NMR spectroscopy at early stages of mineralization. Calcif. Tissue Int. 72, 610–626 (2003)CrossRefGoogle Scholar
  31. 31.
    Wu, Y.T., Ackerman, J.L., Kim, H.M., Rey, C., Barroug, A., Glimcher, M.J.: Nuclear magnetic resonance spin-spin relaxation of the crystals of bone, dental enamel, and synthetic hydroxyapatites. J. Bone Miner. Res. 17, 472–480 (2002)CrossRefGoogle Scholar
  32. 32.
    Hübler, R., Blando, E., Gaião, L., Kreisner, P.E., Post, L.K., Xavier, C.B., de Oliveira, M.G.: Effects of low-level laser therapy on bone formed after distraction osteogenesis. Laser Med. Sci. 25, 213–219 (2010)CrossRefGoogle Scholar
  33. 33.
    Cassella, J.P., Garrington, N., Stamp, T.C.B., Ali, S.Y.: An electron-probe X-ray microanalytical study of bone mineral in osteogenesis imperfecta. Calcif. Tissue Int. 56, 118–122 (1995)CrossRefGoogle Scholar
  34. 34.
    Benhayoune, H., Charlier, D., Jallot, E., Laquerriere, P., Balossier, G., Bonhomme, P.: Evaluation of the Ca/P concentration ratio in hydroxyapatite by STEM-EDXS: influence of the electron irradiation dose and temperature processing. J. Phys. D Appl. Phys. 34, 141–147 (2001)ADSCrossRefGoogle Scholar
  35. 35.
    Kourkoumelis, N., Tzaphlidou, M.: Spectroscopic assessment of normal cortical bone: differences in relation to bone site and sex. TheScientificWorldJOURNAL 10, 402–412 (2010). doi: 10.1100/tsw.2010.43 CrossRefGoogle Scholar
  36. 36.
    Watanabe, K., Okawa, S., Kanatani, M., Homma, K.: Surface analysis of commercially pure titanium implant retrieved from rat bone. Part 1: Initial biological response of sandblasted surface. Dent. Mater. J. 28, 178–184 (2009)CrossRefGoogle Scholar
  37. 37.
    Zoehrer, R., Perilli, E., Kuliwaba, J., Shapter, J., Fazzalari, N., Voelcker, N.: Human bone material characterization: Integrated imaging surface investigation of male fragility fractures. Osteoporos. Int. 1–13 (2011). doi: 10.1007/s00198-011-1688-9 Google Scholar
  38. 38.
    Balatsoukas, I., Kourkoumelis, N., Tzaphlidou, M.: Auger electron spectroscopy for the determination of sex and age related Ca/P ratio at different bone sites. J. Appl. Phys. 108, 074701 (2010). doi: 10.1063/1.3490118 CrossRefGoogle Scholar
  39. 39.
    Miller, R.G., Bowles, C.Q., Eick, J.D., Gutshall, P.L.: Auger electron spectroscopy of dentin: Elemental quantification and the effects of electron and ion bombardment. Dent. Mater. 9, 280–285 (1993)CrossRefGoogle Scholar
  40. 40.
    Wieliczka, D.M., Spencer, P., Kruger, M.B., Eick, J.D.: Spectroscopic characterization of the dentin/adhesive interface. J. Dent. Res. 75, 1758–1758 (1996)Google Scholar
  41. 41.
    Korn, D., Soyez, G., Elssner, G., Petzow, G., Bres, E.F., d’Hoedt, B., Schulte, W.: Study of interface phenomena between bone and titanium and alumina surfaces in the case of monolithic and composite dental implants. J. Mater. Sci.: Mater. Med. 8, 613–620 (1997)CrossRefGoogle Scholar
  42. 42.
    Kang, B.S., Sul, Y.T., Oh, S.J., Lee, H.J., Albrektsson, T.: XPS, AES and SEM analysis of recent dental implants. Acta. Biomater. 5, 2222–2229 (2009)CrossRefGoogle Scholar
  43. 43.
    Jones, F.H.: Teeth and bones: applications of surface science to dental materials and related biomaterials. Surf. Sci. Rep. 42 75–205 (2001)ADSCrossRefGoogle Scholar
  44. 44.
    Obrant, K.J., Odselius, R.: Electron-microprobe investigation of calcium and phosphorus concentration in human-bone trabeculae—both normal and in posttraumatic osteopenia. Calcif. Tissue Int. 37, 117–120 (1985)CrossRefGoogle Scholar
  45. 45.
    Rai, D.V., Darbari, R., Aggarwal, L.M.: Age-related changes in the elemental constituents and molecular behaviour of bone. Indian J. Biochem. Biophys. 42, 127–130 (2005)Google Scholar
  46. 46.
    Kolroser, G., Kasimir, M.T., Eichmair, E., Nigisch, A., Simon, P., Weigel, G.: Scanning electron microscopy and energy-dispersive X-ray microanalysis: a valuable tool for studying cell surface antigen expression on tissue-engineered scaffolds. Tissue Eng. Pt. C: Meth. 15, 257–263 (2009)CrossRefGoogle Scholar
  47. 47.
    Roschger, P., Fratzl, P., Klaushofer, K., Rodan, G.: Mineralization of cancellous bone after alendronate and sodium fluoride treatment: a quantitative backscattered electron imaging study on minipig ribs. Bone 20, 393–397 (1997)CrossRefGoogle Scholar
  48. 48.
    Kanaya, K., Okayama, S.: Penetration and energy-loss theory of electrons in solid targets. J. Phys. D Appl. Phys. 5, 43–58 (1972)ADSCrossRefGoogle Scholar
  49. 49.
    Krause, M.O., Oliver, J.H.: Natural widths of atomic K and L levels, Kα X-ray lines and several KLL Auger lines. J. Phys. Chem. Ref. Data. 8, 329–338 (1979)ADSCrossRefGoogle Scholar
  50. 50.
    Armour, K.J., Armour, K.E.: Methods in Molecular Medicine, Vol. 80: Bone Research Protocols: Inflammation-Induced Osteoporosis, The IMO Model. Humana Press Inc., Totowa, NJ (2003)Google Scholar
  51. 51.
    Speller, R., Pani, S., Tzaphlidou, M., Horrocks, J.: MicroCT analysis of calcium/phosphorus ratio maps at different bone sites. Nucl. Instrum. Meth. A 548, 269–273 (2004)ADSCrossRefGoogle Scholar
  52. 52.
    Turner, A.S.: Animal models of osteoporosis—necessity and limitations. Eur. Cells Mater. 1, 66–81 (2001)Google Scholar
  53. 53.
    Pignatel, G.U., Queirolo, G.: Electron and ion beam effects in Auger electron spectroscopy on insulating materials. Radiat. Eff. Defects Solids 79, 291–303 (1983)CrossRefGoogle Scholar
  54. 54.
    Vanraemdonck, W., Ducheyne, P., Demeester, P.: Auger-electron spectroscopic analysis of hydroxylapatite coatings on titanium. J. Am. Ceram. Soc. 67, 381–384 (1984)CrossRefGoogle Scholar
  55. 55.
    Bloebaum, R.D., Holmes, J.L., Skedros, J.G.: Mineral content changes in bone associated with damage induced by the electron beam. Scanning 27, 240–248 (2005)CrossRefGoogle Scholar
  56. 56.
    Egerton, R.F., Li, P., Malac, M.: Radiation damage in the TEM and SEM. Micron 35, 399–409 (2004)CrossRefGoogle Scholar
  57. 57.
    Emfietzoglou, D., Kyriakou, I., Garcia-Molina, R., Abril, I., Kostarelos, K.: Analytic expressions for the inelastic scattering and energy loss of electron and proton beams in carbon nanotubes. J. Appl. Phys. 108, 054312 (2010). doi: 10.1063/1.3463405 ADSCrossRefGoogle Scholar
  58. 58.
    Holmes, J.L., Bachus, K.N., Bloebaum, R.D.: Thermal effects of the electron beam and implications of surface damage in the analysis of bone tissue. Scanning 22, 243–248 (2000)CrossRefGoogle Scholar
  59. 59.
    Walther, P., Wehrli, E., Hermann, R., Müller, M.: Double-layer coating for high-resolution low-temperature scanning electron microscopy. J. Microsc. 179, 229–237 (1995)CrossRefGoogle Scholar
  60. 60.
    Vajda, E.G., Skedros, J.G., Bloebaum, R.D.: Errors in quantitative backscattered electron analysis of bone standardized by energy-dispersive X-ray spectrometry. Scanning 20, 527–535 (1998)CrossRefGoogle Scholar
  61. 61.
    Boyce, T.M., Bloebaum, R.D., Bachus, K.N., Skedros, J.G.: Reproducible method for calibrating the backscattered electron signal for quantitative assessment of mineral-content in bone. Scanning Microsc. 4, 591–603 (1990)Google Scholar
  62. 62.
    Suetsugu, Y., Hirota, K., Fujii, K., Tanaka, J.: Compositional distribution of hydroxyapatite surface and interface observed by electron spectroscopy. J. Mater. Sci. 31, 4541–4544 (1996)ADSCrossRefGoogle Scholar
  63. 63.
    Tanuma, S., Powell, C.J., Penn, D.R.: Calculations of electron inelastic mean free paths. VIII. Data for 15 elemental solids over the 50–2000 eV range. Surf. Interface Anal. 37, 1–14 (2005)CrossRefGoogle Scholar
  64. 64.
    Smekal, W., Werner, W.S.M., Powell, C.J.: Simulation of electron spectra for surface analysis (SESSA): a novel software tool for quantitative Auger-electron spectroscopy and X-ray photoelectron spectroscopy. Surf. Interface Anal. 37, 1059–1067 (2005)CrossRefGoogle Scholar
  65. 65.
    Tzaphlidou, M., Zaichick, V.: Sex and age related Ca/P ratio in cortical bone of iliac crest of healthy humans. J. Radioanal. Nucl. Chem. 259, 347–349 (2004)CrossRefGoogle Scholar
  66. 66.
    Kuhn, L.T., Grynpas, M.D., Rey, C.C., Wu, Y., Ackerman, J.L., Glimcher, M.J.: A comparison of the physical and chemical differences between cancellous and cortical bovine bone mineral at two ages. Calcif. Tissue Int. 83, 146–154 (2008)CrossRefGoogle Scholar
  67. 67.
    Bigi, A., Cojazzi, G., Panzavolta, S., Ripamonti, A., Roveri, N., Romanello, M., Suarez, K.N., Moro, L.: Chemical and structural characterization of the mineral phase from cortical and trabecular bone. J. Inorg. Biochem. 68, 45–51 (1997)CrossRefGoogle Scholar
  68. 68.
    Lipschitz, S.: Advances in understanding bone physiology: influences of treatment. Menopause Update 12, 2–5 (2009)Google Scholar
  69. 69.
    Ammann, P., Rizzoli, R.: Bone strength and its determinants. Osteoporos. Int. 14, S13–S18 (2003)CrossRefGoogle Scholar
  70. 70.
    Fei, Y.R., Zhang, M., Li, M., Huang, Y.Y., He, W., Ding, W.J., Yang, J.H.: Element analysis in femur of diabetic osteoporosis model by SRXRF microprobe. Micron 38, 637–642 (2007)CrossRefGoogle Scholar
  71. 71.
    Tzaphlidou, M., Speller, R., Royle, G., Griffiths, J., Olivo, A., Pani, S., Longo, R.: High resolution Ca/P maps of bone architecture in 3D synchrotron radiation microtomographic images. Appl. Radiat. Isotopes 62, 569–575 (2005)CrossRefGoogle Scholar
  72. 72.
    Cummings, S.R., Palermo, L., Browner, W., Marcus, R., Wallace, R., Pearson, J., Blackwell, T., Eckert, S., Black, D., Gr, F.I.T.R.: Monitoring osteoporosis therapy with bone densitometry—Misleading changes and regression to the man. J. Am. Med. Assoc. 283, 1318–1321 (2000)CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2011

Authors and Affiliations

  • Nikolaos Kourkoumelis
    • 1
  • Ioannis Balatsoukas
    • 1
  • Margaret Tzaphlidou
    • 1
  1. 1.Department of Medical Physics, Medical SchoolUniversity of IoanninaIoanninaGreece

Personalised recommendations