Calcified Tissue International

, Volume 79, Issue 5, pp 354–359 | Cite as

Complementary Information on In Vitro Conversion of Amorphous (Precursor) Calcium Phosphate to Hydroxyapatite from Raman Microspectroscopy and Wide-Angle X-Ray Scattering

  • M. Kazanci
  • P. Fratzl
  • K. Klaushofer
  • E. P. Paschalis


In addition to mechanical functions, bones have an essential role in metabolic activity as mineral reservoirs that are able to absorb and release ions. Bioapatite, considered the major component in the mineralized part of mammalian bones, is a calcium phosphate mineral with a structure that closely resembles hydroxyapatite (HA, Ca10[PO4]6[OH]2) with variable chemical substitutions. It is important to note that it continues to be chemically active long after it has been initially deposited. Detailed understanding of changes in the mineral phase as HA matures is essential for understanding how normal bone achieves its remarkable mechanical performance, how it is altered in disease, as well as the effects of therapeutic interventions. A model system for investigation of the in vivo maturation of HA is available, namely, the in vitro conversion of amorphous calcium phosphate (ACP) to HA in a supersaturated solution of calcium and phosphate ions. In the present study, this system was employed to correlate with the changes in chemistry and poorly crystalline HAP crystal size, shape, and habit. The results of the X-ray diffraction as well as Raman analyses showed that as the crystallites mature in the 002 and 310 directions both the full width at half-height and wavelength at maximum of the Raman peaks change as a function of reaction extent and crystallite maturation, size, and shape. Moreover, such analyses can be performed in intact bone specimens through Raman microspectroscopic and imaging analyses with a spatial resolution of 0.6–1 μ, by far superior to the one offered by other microspectroscopic techniques, thus potentially yielding important new information on the organization and mineral quality of normal and fragile bone.


Amorphous calcium phosphate Hydroxyapatite Raman microspectroscopy Wide-angle X-ray scattering 


  1. 1.
    Rey C, Shimizu M, Collins B, Glimcher MJ (1991) Resolution enhanced Fourier transform infrared spectroscopy study of the environment of phosphate ion in the early deposits of a solid phase calcium phosphate in bone and enamel and their evolution with age: investigation in the ν3 PO4 domain. Calcif Tissue Int 49:383–388PubMedGoogle Scholar
  2. 2.
    Pleshko NL, Boskey AL, Mendelsohn R (1991) Novel infrared spectroscopic method for the determination of crystallinity of hydroxyapatite minerals. Biophys J 60:786–793PubMedCrossRefGoogle Scholar
  3. 3.
    Ou-Yang H, Paschalis EP, Boskey AL, Mendelsohn R (1999) Two-dimensional vibrational correlation spectroscopy of in vitro hydroxyapatite maturation. Biopolymers 57:129–139CrossRefGoogle Scholar
  4. 4.
    Gadaleta SJ, Paschalis EP, Betts F, Mendelsohn R, Boskey AL (1996) Fourier transform infrared spectroscopy of the solution mediated conversion of amorphous calcium phosphate to hydroxyapatite: new correlations between X-ray diffraction and infrared data. Calcif Tissue Int 58:9–16PubMedGoogle Scholar
  5. 5.
    Sauer GR, Zunic WB, During JR, Wuthier RE (1994) Fourier transform Raman spectroscopy of synthetic and biological calcium phosphate. Calcif Tissue Int 54:414–420PubMedCrossRefGoogle Scholar
  6. 6.
    Penel G, Leroy G, Rey C, Bres E (1998) MicroRaman spectral study of the PO4 and CO3 vibrational modes in synthetic and biological apatites. Calcif Tissue Int 63:475–481PubMedCrossRefGoogle Scholar
  7. 7.
    Walters MA, Leung YC, Blumenthal NC, LeGeros RZ, Konsker KA (1990) A Raman and infrared spectroscopic investigation of biological hydroxyapatite. J Inorg Biochem 39:193–200PubMedCrossRefGoogle Scholar
  8. 8.
    Tarnowski CP, Ignelzi MA Jr, Wang W, Taboas JM, Goldstein SA, Morris MD (2004) Earliest mineral and matrix changes in force induced musculoskeletal disease as revealed by raman microspectroscopic imaging. J Bone Miner Res 19:64–70PubMedCrossRefGoogle Scholar
  9. 9.
    Timlin JA, Carden A, Morris MD (1999) Chemical microstructure of cortical bone probed by Raman transects. Appl Spectrosc 53:1429–1435CrossRefGoogle Scholar
  10. 10.
    Timlin JA, Carden A, Morris MD, Bonadio JF, Hoffler CE II, Kozloff KM, Goldstein SA (1999) Spatial distribution of phosphate species in mature and newly generated mammalian bone by hyperspectral Raman imaging. J Biomed Optics 4:28–34CrossRefGoogle Scholar
  11. 11.
    Akkus O, Adar F, Schaffler MB (2004) Age-related changes in physicochemical properties of mineral crystals are related to impaired mechanical function of cortical bone Bone 34:443–453PubMedCrossRefGoogle Scholar
  12. 12.
    Tarnowski CP, Ignelzi MA, Morris MD (2002) Mineralization of developing mouse calvaria as revealed by Raman microspectroscopy. J Bone Miner Res 17:1118–1126PubMedCrossRefGoogle Scholar
  13. 13.
    Tsuda H, Arends J (1994) Orientational micro-Raman spectroscopy on hydroxyapatite single crystals and human enamel crystallites. J Dent Res 73:1703–1710PubMedGoogle Scholar
  14. 14.
    Kazanci M, Roschger P, Paschalis EP, Klaushofer K, Fratzl P (2006) Bone osteonal tissues by Raman spectral mapping: orientation-composition. J Struct Biol (in press)Google Scholar
  15. 15.
    Stewart S, Shea DA, Tarnowski CP, Morris MD, Wang D, Francheschi R, Lin DL, Keller E (2002) Trends in early mineralization of murine calvarial osteoblastic cultures: a Raman microscopic study. J Raman Spectrosc 33:536–543CrossRefGoogle Scholar
  16. 16.
    Carden A, Morris MD (2000) Application of vibrational spectroscopy to the study of mineralized tissue. J Biomed Optics 5:259–268CrossRefGoogle Scholar
  17. 17.
    Danilchenko SN, Kukharenko OG, Moseke C, Protsenko IY, Sukhodub LF, Sulkio-Cleff B (2002) Determination of the bone mineral crystallite size and lattice strain from diffraction line broadening. Cryst Res Technol 37:1234–1240CrossRefGoogle Scholar
  18. 18.
    Rusu VM, Ng C-H, Wilke M, Tiersch B, Fratzl P, Peter MG (2005) Size-controlled hydroxyapatite nanoparticles as self-organized organic-inorganic composite materials. Biomaterials 26:5414–5426PubMedCrossRefGoogle Scholar
  19. 19.
    Fratzl P, Gupta HS, Paschalis EP, Roschger P (2004) Structure and mechanical quality of the collagen-mineral nano-composite in bone. J Mater Chem 14:2115–2123CrossRefGoogle Scholar
  20. 20.
    Urist MR (1980) Fundamental and Clinical Bone Physiology. Lippincott: PhiladelphiaGoogle Scholar
  21. 21.
    Currey JD (2002) Bones – Structure and Mechanics. Princeton, NJ: Princeton University PressGoogle Scholar
  22. 22.
    Roschger P, Fratzl P, Klaushofer K, Rodan G (1997) Mineralization of cancellous bone after alendronate and sodium fluoride treatment: a quantitative backscattered electron imaging study on minipig ribs. Bone 20:393–397PubMedCrossRefGoogle Scholar
  23. 23.
    Termine JD, Posner AS (1967) Amorphous/crystalline interrelationships in bone mineral. Calcif Tissue Res 1:8–23PubMedCrossRefGoogle Scholar
  24. 24.
    Brecevic LJ, Furedi-Milhofer H (1972) Precipitation of calcium phosphates from electrolyte solutions. II. The formation and transformation of precipitates. Calcif Tissue Res 10:82–90PubMedCrossRefGoogle Scholar
  25. 25.
    Crane NJ, Popescu V, Morris MD, Steenhuis P, Ignelzi MA (2006) Raman spectroscopic evidence for octacalcium phosphate and other transient mineral species deposited during intramembranous mineralization. Bone 39:434–442PubMedCrossRefGoogle Scholar
  26. 26.
    Crane NJ, Morris MD, Ignelzi MA, Yu GG (2005) Raman imaging demonstrates FGF2-induced craniosynostosis in mouse calvaria. J Biomed Optics 10: art. no 031119: 1–8Google Scholar

Copyright information

© Springer Science+Business Media, Inc. 2006

Authors and Affiliations

  • M. Kazanci
    • 1
  • P. Fratzl
    • 1
  • K. Klaushofer
    • 2
  • E. P. Paschalis
    • 2
  1. 1.Department of BiomaterialsMax Planck Institute of Colloids and InterfacesPotsdamGermany
  2. 2.Ludwig Boltzmann Institute of Osteology at the Hanusch Hospital, 4th Medical DepartmentHanusch HospitalViennaAustria

Personalised recommendations