Calcified Tissue International

, Volume 72, Issue 5, pp 610–626

Phosphate Ions in Bone: Identification of a Calcium–Organic Phosphate Complex by 31P Solid-State NMR Spectroscopy at Early Stages of Mineralization

  • Y. Wu
  • J. L. Ackerman
  • E. S. Strawich
  • C. Rey
  • H. -M. Kim
  • M. J. Glimcher
Article

Abstract

Previous 31P cross-polarization and differential cross-polarization magic angle spinning (CP/MAS and DCP/MAS) solid-state NMR spectroscopy studies of native bone and of the isolated crystals of the calcified matrix synthesized by osteoblasts in cell culture identified and characterized the major PO4−3 phosphate components of the mineral phase. The isotropic and anisotropic chemical shift parameters of the minor HPO4−2 component in bone mineral and in mineral deposited in osteoblast cell cultures were found to differ significantly from those of brushite, octacalcium phosphate, and other synthetic calcium phosphates. However, because of in vivo and in vitro evidence that phosphoproteins may play a significant role in the nucleation of the solid mineral phase of calcium phosphate in bone and other vertebrate calcified tissues, the focus of the current solid-state 31P NMR experiments was to detect the possible presence of and characterize the phosphoryl groups of phosphoproteins in bone at the very earliest stages of bone mineralization, as well as the possible presence of calcium-phosphoprotein complexes. The present study demonstrates that by far the major phosphate components identified by solid-state 31P NMR in the very earliest stages of mineralization are protein phosphoryl groups which are not complexed with calcium. However, very small amounts of calcium-complexed protein phosphoryl groups as well as even smaller, trace amounts of apatite crystals were also present at the earliest phases of mineralization. These data support the hypothesis that phosphoproteins complexed with calcium play a significant role in the initiation of bone calcification.

Keywords

Phosphoproteins Bone 31P NMR Calcification Apatite 

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. 1.
    Wu, Y, Glimcher, MJ, Rey, C, Ackerman, JL 1994A unique protonated phosphate group in bone mineral not present in synthetic calcium phosphates. Identification by phosphorus-31 solid state NMR spectroscopy.J Mol Biol244423435CrossRefPubMedGoogle Scholar
  2. 2.
    Kuhn, LT, Wu, Y, Rey, C, Gerstenfeld, LC, Grynpas, MD, Ackerman, JL, Kim, HM, Glimcher, MJ 2000Structure, composition, and maturation of newly deposited calcium-phosphate crystals in chicken osteoblast cell cultures.J Bone Miner Res1513011309PubMedGoogle Scholar
  3. 3.
    Glimcher, MJ 1979Phosphopeptides of enamel matrix.J Dent Res58790809PubMedGoogle Scholar
  4. 4.
    Glimcher, MJ 1976

    Composition, structure and organization of bone and other mineralized tissues and the mechanism of calcification.

    Greep, ROAstwood, EB eds. Handbook of Physiology 7: Endocrinology, vol VII.American Physiological SocietyWashington25116
    Google Scholar
  5. 5.
    Glimcher, MJ, Krane, SM 1968

    The organization and structure of bone, and the mechanism of calcification.

    Ramachandran, GNGould, BS eds. Treatise on collagen, vol IIB.Academic PressNew York68251
    Google Scholar
  6. 6.
    Lee, SL, Glonek, T, Glimcher, MJ 198331P nuclear magnetic resonance spectroscopic evidence for ternary complex formation of fetal dentin phosphoprotein with calcium and inorganic orthophosphate ions.Calcif Tissue Int35815818PubMedGoogle Scholar
  7. 7.
    Glimcher, MJ 1984Recent studies of the mineral phase in bone and its possible linkage to the organic matrix by protein-bound phosphate bonds.Philos Trans R Soc Lond B Biol Sci304479508PubMedGoogle Scholar
  8. 8.
    Glimcher, MJ 1989Mechanism of calcification: role of collagen fibrils and collagen–phosphoprotein complexes in vitro and in vivo.Anat Rec244139153Google Scholar
  9. 9.
    Veis, A 1985

    Phosphoroproteins of dentin and bone. Do they have a role in matrix mineralization?

    Butler, WT eds. The Chemistry and Biology of Mineralized Tissues.Ebsco MediaBirmingham, AL170176
    Google Scholar
  10. 10.
    Veis, A 1989Studies of vertebrate tooth mineralization. Insights from studies of dentinogenesis imperfecta type II.Northwest Dent Res135PubMedGoogle Scholar
  11. 11.
    Veis, A, Anesey, J, Mussell, S 1967The phosphoprotein of the dentin matrix.Biochemistry624092416PubMedGoogle Scholar
  12. 12.
    Lee, SL, Veis, A 1980Cooperativity in calcium ion binding to repetitive, carboxylate-serylphosphopolypeptides and the relationship of this property to dentin mineralization.Int J Pept Protein Res16231240PubMedGoogle Scholar
  13. 13.
    George, A, Sabsay, B, Simonian, PAL, Veis, A 1993Characterization of a novel dentin matrix acidic phosphoprotein. Implications for induction of biomineralization.J Biol Chem2681262412630PubMedGoogle Scholar
  14. 14.
    Veis, A 1993Mineral–matrix interactions in bone and dentin.J Bone Miner Res8S493S497PubMedGoogle Scholar
  15. 15.
    Suga, T, Okabe, N 1996Aqua (L-O-serine phosphato)calcium (II).Acta Crystallogr5218941896CrossRefGoogle Scholar
  16. 16.
    Boskey, AL, Posner, AS 1982Optimal conditions for Ca-acidic phospholipid-PO4 formation.Calcif Tisse Int34S17Google Scholar
  17. 17.
    Bradbury, MWB, Kleeman, CR, Bagdoyan, H, Berberian, A 1968The calcium and magnesium content of skeletal muscle, brain, and cerebrospinal fluid as determined by atomic absorption flame photometry.J Lab Clin Med71884PubMedGoogle Scholar
  18. 18.
    Fernandez, FJ, Kahn, HL 1971Clinical methods for atomic absorption spectroscopy.Clin Chem Newslett324Google Scholar
  19. 19.
    Kirkpatrick, D, Bishop, S 1971Simplified wet ash procedure for total phosphorus analysis of organophosphonates in biological samples.Anal Chem4317071709PubMedGoogle Scholar
  20. 20.
    Cohen–Solal, L, Lian, JB, Kossiva, D, Glimcher, MJ 1978The identification of O-phosphothreonine in the soluble non-collagenous phosphoproteins of bone matrix.FEBS Lett89107110CrossRefPubMedGoogle Scholar
  21. 21.
    Cohen–Solal, L, Lian, JB, Kossiva, D, Glimcher, MJ 1979Identification of organic phosphorus covalently bound to collagen and non-collagenous proteins of chicken-bone matrix. The presence of O-phosphoserine and O-phosphothreonine in non-collagenous proteins, and their absence from phosphorylated collagen.Biochem J1778198PubMedGoogle Scholar
  22. 22.
    Gotoh, Y, Salih, E, Glimcher, MJ, Gerstenfeld, LC 1995Characterization of the major non-collagenous proteins of chicken bone: identification of a novel 60 kDa non-collagenous phosphoprotein.Biochem Biophys Res Commun208863870CrossRefPubMedGoogle Scholar
  23. 23.
    Toyosawa, S, Sato, A, O’hUigin, C, Tichy, H, Klein, J 2000Expression of the dentin matrix protein 1 gene in birds.J Mol Evol503138PubMedGoogle Scholar
  24. 24.
    Toyosawa, S, Shintani, S, Fujiwara, T, Ooshima, T, Sato, A, Ijuhin, N, Komori, T 2001Dentin matrix protein 1 is predominantly expressed in chicken and rat osteocytes but not in osteoblasts.J Bone Miner Res1620172026PubMedGoogle Scholar
  25. 25.
    Kim, HM, Rey, C, Glimcher, MJ 1995Isolation of calcium-phosphate crystals of bone by nonaqueous methods at low temperature.J Bone Miner Res1015891601PubMedGoogle Scholar
  26. 26.
    Rothwell, WP, Waugh, JS, Yesinowski, JP 1980High-resolution variable-temperature phosphorus-31 NMR of solid calcium phosphates.J Am Chem Soc10226372643Google Scholar
  27. 27.
    Herzfeld, J, Roufosse, A, Haberkorn, RA, Griffin, RG, Glimcher, MJ 1980Magic angle sample spinning in inhomogeneously broadened systems.Phil Trans R Soc Lond B Biol Sci289459469Google Scholar
  28. 28.
    Yesinowski, JP 1981High-resolution NMR spectroscopy of solids and surface adsorbed species in colloidal suspension: 31P NMR spectra of hydroxyapatite and diphosphonates.J Am Chem Soc1036666267Google Scholar
  29. 29.
    Aue, WP, Rôufosse, AH, Glimcher, MK, Griffin, RG 1984Solid state phosphorus-31 nuclear magnetic resonance studies of synthetic solid phases of calcium phosphate: potential models of bone mineral.Biochemistry2361106114PubMedGoogle Scholar
  30. 30.
    Rôufosse, AH, Aue, WP, Roberts, JE, Glimcher, MJ, Griffin, RG 1984Investigation of the mineral phases of bone by solid-state phosphorus-31 magic angle spinning nuclear magnetic resonance.Biochemistry2361156120PubMedGoogle Scholar
  31. 31.
    Bonar, LC, Shimizu, M, Roberts, JE, Griffin, RG, Glimcher, MJ 1991Structural and composition studies on the mineral of newly formed dental enamel: A chemical, x-ray diffraction, and 31P and proton nuclear magnetic resonance study.J Bone Miner Res611671176PubMedGoogle Scholar
  32. 32.
    Yesinowski, JP 1998

    Nuclear magnetic resonance spectroscopy of calcium phosphates.

    Amjad, Z eds. Calcium phosphates in biological and industrial systems.KluwerAmsterdam103143
    Google Scholar
  33. 33.
    Kaflak, A, Chmielewski, D, Górecki, A, Kolodziejski, W 1998Kinetics of 1H–31P cross-polarization in human trabecular bone.Solid State Nucl Magn Reson10191195CrossRefPubMedGoogle Scholar
  34. 34.
    Kaflak–Hachulska, A, Slosarczyk, A, Kolodziejski, W 2000Kinetics of NMR cross-polarization from protons to phosphorus-31 in natural brushite.Solid-State Nucl Magn Reson15237238CrossRefPubMedGoogle Scholar
  35. 35.
    Wu, Y, Ackerman, JL, Kim, H-M, Rey, C, Barroug, A, Glimcher, MJ 2002Nuclear magnetic resonance spin–spin relaxation of the crystals of bone, dental enamel and synthetic hydroxyapatites.J Bone Miner Res17472480PubMedGoogle Scholar
  36. 36.
    Legrand, AP, Bresson, B, Guidoin, R, Famery, R 2002Mineralization follow-up with the use of NMR spectroscopy and others.J Biomed Mater Res63390395CrossRefPubMedGoogle Scholar
  37. 37.
    Herzfeld, J, Berger, SE 1980Sideband intensities in NMR spectra of sample spinning at the magic angle.J Chem Phys7360216030CrossRefGoogle Scholar
  38. 38.
    Mason, J 1993Conventions for the reporting of nuclear magnetic shielding (or shift) tensors suggested by participants in the NATO ARW on NMR Shielding Constants at the University of Maryland, College Park, July 1992.Solid State Nucl Magn Reson2285288CrossRefPubMedGoogle Scholar
  39. 39.
    Moon, RB, Richards, JH 1973Determination of intracellular pH by 31P magnetic resonance.J Biol Chem24872767278PubMedGoogle Scholar
  40. 40.
    Lian, JB, Roufosse, AH, Reit, B, Glimcher, MJ 1982Concentrations of osteocalcin and phosphoprotein as a function of mineral content and age in cortical bone.Calcif Tissue Int34S82S87PubMedGoogle Scholar
  41. 41.
    Glimcher, MJ 1959Molecular biology of mineralized tissues with particular reference to bone.Rev Mod Phys31359393CrossRefGoogle Scholar
  42. 42.
    Lee, DD, Glimcher, MJ 1991The three-dimensional spatial relationship between the collagen fibrils and the inorganic calcium-phosphate crystals of pickerel (Americanus americanus) and herring (Clupea harengus) bone.J Mol Biol217487501Google Scholar
  43. 43.
    Glimcher, MJ 1968A basic architectural principle in the organization of mineralized tissues.Clin Orthop611636PubMedGoogle Scholar
  44. 44.
    Glimcher, MJ 1984Recent studies of the mineral phase in bone and its possible linkage to the organic matrix by protein-bound phosphate bonds.Philos Trans R Soc Lond B Biol Sci304479508PubMedGoogle Scholar
  45. 45.
    White, SW, Hulmes, DJS, Miller, A, Timmins, PA 1977Collagen-mineral axial relationship in calcified turkey leg tendon by x-ray and neutron diffraction.Nature.266Google Scholar
  46. 46.
    Burger, C, Liu, L-Z, Hsiao, BS, Chu, B, Hanson, J, Hori, T, Glimcher, MJ 2001Synchrotron SAXS/WAXS study of the composite nature of bone.Am Chem Soc PMSE Preprint [abstr]85169170Google Scholar
  47. 47.
    Berthet–Colominas, C, Miller, A, White, SW 1979Structural study of the calcifying collagen in turkey leg tendons.J Mol Biol134431435PubMedGoogle Scholar
  48. 48.
    Katz, EP, Wachtel, E, Yamauchi, M, Mechanic, GL 1989The structure of mineralized collagen fibrils.Connect Tissue Res21149158PubMedGoogle Scholar
  49. 49.
    Katz, EP, Li, S-T 1973Structure and function of bone collagen fibrils.J Mol Biol80115PubMedGoogle Scholar
  50. 50.
    Katz, EP, Li, S-T 1972The molecular packing of collagen in mineralized and non-mineralized tissues.Biochem Biophys Res Commun46115PubMedGoogle Scholar
  51. 51.
    Landis, WJ, Hodgens, KJ, Arena, J 1996Structural relations between collagen and mineral in bone as determined by high voltage electron microscopic tomography.Microsc Res Tech33192202CrossRefPubMedGoogle Scholar
  52. 52.
    Landis, WJ, Song, MJ, Leith, A, McEwen, L, McEwen, B 1992Mineral and organic matrix interaction in normally calcifying tendon visualized in three dimensions by high-voltage electron microscopic tomography and graphic image reconstruction.J Struct Biol1103954CrossRefGoogle Scholar
  53. 53.
    Landis, WJ, Song, MJ 1991Early mineral deposition in calcifying tendon characterized by high voltage electron microscopy and three-dimensional graphic imaging.J Struct Biol107116127PubMedGoogle Scholar
  54. 54.
    Landis, WJ, Moradian–Oldak, J, Weiner, S 1991Topographic imaging of mineral and collagen in the calcifying turkey tendon.Connect Tissue Res25181196PubMedGoogle Scholar
  55. 55.
    Glimcher, MJ 1998

    The nature of the mineral phase in bone: Biological and clinical implications.

    Avioli, LVKrane, SM eds. Metabolic bone disease and clinically related disorders, 3rd ed.Academic PressSan Diego2350
    Google Scholar
  56. 56.
    Hohling, H, Hall, T, Boothroyd, B, Cooke, C, Duncumb, P, Fitton–Jackson, S 1967Studies on the early stages of bone formation, using ordinary and electron microscopic electron probe x-ray microanalysis.Naturwissenschaften54142143Google Scholar
  57. 57.
    Katz, EP 1969The kinetics of mineralization in vitro.Biochim Biophys Acta194121129CrossRefPubMedGoogle Scholar
  58. 58.
    Glimcher, MJ, Hodge, AJ, Schmitt, FO 1957Macromolecular aggregation states in relation to mineralization: the collagen hydroxyapatite system as studied in vitro.Proc Natl Acad Sci USA43860867Google Scholar
  59. 59.
    Mergenhagen, SE, Martin, GR, Rizzo, AA, Wright, DN, Scott, DB 1960Calcification in vivo of implanted collagens.Biochim Biophys Acta43563565CrossRefPubMedGoogle Scholar
  60. 60.
    Hunter, GK, Goldberg, HA 1993Nucleation of hydroxyapatite by bone sialoprotein.Proc Natl Acad Sci USA9085628565PubMedGoogle Scholar
  61. 61.
    Hunter, GK, Goldberg, HA 1994Modulation of crystal formation by bone phosphoproteins: Role of glutamic acid-rich sequences in the nucleation of hydroxyapatite by bone sialoprotein.Biochem J302175179PubMedGoogle Scholar
  62. 62.
    Hunter, GK, Kyle, CL, Goldberg, HA 1994Modulation of crystal formation by bone phosphoproteins; Structural specificity of the osteopontin-mediated inhibition of hydroxyapatite formation.Biochem J300723728PubMedGoogle Scholar
  63. 63.
    Hunter, GK, Hauschka, PV, Poole, AR, Rosenberg, LC, Goldberg, HA 1996Nucleation and inhibition of hydroxyapatite formation by mineralized tissue proteins.Biochem J3175964PubMedGoogle Scholar
  64. 64.
    Goldberg, HA, Warner, KJ, Stillman, MJ, Hunter, GK 1996Determination of the hydroxyapatite-nucleating region of bone sialoprotein.Connect Tissue Res35385392PubMedGoogle Scholar
  65. 65.
    Goldberg, HA, Hunter, GK 1995The inhibitory activity of osteopontin on hydroxyapatite formation in vitro.Ann NY Acad Sci760305308PubMedGoogle Scholar
  66. 66.
    Stanford, CM, Jacobson, PA, Eanes, ED, Lembke, LA, Midura, RJ 1995Rapidly forming apatitic mineral in an osteoblastic cell line (UMR 106-01 BSP).J Biol Chem27094209428CrossRefPubMedGoogle Scholar
  67. 67.
    Wang, A, Martin, JA, Lemke, LA, Midura, RJ 2000Reversible suppression of in vitro biomineralization by activation of protein kinase A.J Biol Chem2751108211091CrossRefPubMedGoogle Scholar
  68. 68.
    Kohler, SJ, Klein, MP 1977Phosphorus-31 nuclear magnetic resonance chemical shielding tensors of L-O-serine phosphate and 3′-cytidine monophosphate.J Am Chem Soc9982908293PubMedGoogle Scholar
  69. 69.
    Hauser, H, Radloff, C, Ernst, RR, Sundell, S, Pascher, I 1988The 31 P chemical shielding tensor in phospholipids.J Am Chem Soc11010541058Google Scholar

Copyright information

© Springer-Verlag 2003

Authors and Affiliations

  • Y. Wu
    • 1
    • 2
  • J. L. Ackerman
    • 1
    • 2
  • E. S. Strawich
    • 1
  • C. Rey
    • 3
  • H. -M. Kim
    • 4
  • M. J. Glimcher
    • 1
  1. 1.Laboratory for the Study of Skeletal Disorders and Rehabilitation, Department of Orthopaedic SurgeryChildren’s Hospital, Harvard Medical School, Boston, MA 02115USA
  2. 2.Biomaterials Laboratory, NMR Center, Department of RadiologyMassachusetts General Hospital, Harvard Medical School, Charlestown, MA 02129USA
  3. 3.Centre Interuniversitaire de Recherche et d’Ingénierie des Matériaux, INPT, ENSCT, UMR CNRS 5085, 31400 ToulouseFrance
  4. 4.Laboratory of Hard Tissue EngineeringDepartment of Oral Anatomy, College of Dentistry, Seoul National University, SeoulSouth Korea 110-749

Personalised recommendations