Calcified Tissue International

, Volume 98, Issue 2, pp 193–205 | Cite as

The Orientation of Nanoscale Apatite Platelets in Relation to Osteoblastic–Osteocyte Lacunae on Trabecular Bone Surface

  • Furqan A. ShahEmail author
  • Ezio Zanghellini
  • Aleksandar Matic
  • Peter Thomsen
  • Anders Palmquist
Original Research


The orientation of nanoscale mineral platelets was quantitatively evaluated in relation to the shape of lacunae associated with partially embedded osteocytes (osteoblastic–osteocytes) on the surface of deproteinised trabecular bone of adult sheep. By scanning electron microscopy and image analysis, the mean orientation of mineral platelets at the osteoblastic–osteocyte lacuna (Ot.Lc) floor was found to be 19° ± 14° in the tibia and 20° ± 14° in the femur. Further, the mineral platelets showed a high degree of directional coherency: 37 ± 7 % in the tibia and 38 ± 9 % in the femur. The majority of Ot.Lc in the tibia (69.37 %) and the femur (74.77 %) exhibited a mean orientation of mineral platelets between 0° and 25°, with the largest fraction within a 15°–20° range, 17.12 and 19.8 % in the tibia and femur, respectively. Energy dispersive X-ray spectroscopy and Raman spectroscopy were used to characterise the features observed on the anorganic bone surface. The Ca/P (atomic %) ratio was 1.69 ± 0.1 within the Ot.Lc and 1.68 ± 0.1 externally. Raman spectra of NaOCl-treated bone showed peaks associated with carbonated apatite: ν1, ν2 and ν4 PO4 3−, and ν1 CO3 2−, while the collagen amide bands were greatly reduced in intensity compared to untreated bone. The apatite-to-collagen ratio increased considerably after deproteinisation; however, the mineral crystallinity and the carbonate-to-phosphate ratios were unaffected. The ~19°–20° orientation of mineral platelets in at the Ot.Lc floor may be attributable to a gradual rotation of osteoblasts in successive layers relative to the underlying surface, giving rise to the twisted plywood-like pattern of lamellar bone.


Bone Biomineralisation Osteoblastic–osteocyte Lacuna Trabecular bone Electron microscopy Raman spectroscopy 



This study was supported by the Swedish Research Council (Grant K2015-52X-09495-28-4), the BIOMATCELL VINN Excellence Center of Biomaterials and Cell Therapy, the Region Västra Götaland, an ALF/LUA Grant, the IngaBritt and Arne Lundberg Foundation, the Dr. Felix Neubergh Foundation, Promobilia, the Hjalmar Svensson Foundation, and the Materials Science Area of Advance at Chalmers and the Department of Biomaterials, University of Gothenburg.

Compliance with Ethical Standards

Conflict of Interest

Furqan A. Shah, Ezio Zanghellini, Aleksandar Matic, Peter Thomsen, and Anders Palmquist declare that they have no conflict of interest.

Ethical Approval

All applicable international, national, and/or institutional guidelines for the care and use of animals were followed.

Human and Animal Rights and Informed Consent

All procedures performed in studies involving animals were in accordance with the ethical standards of the institution or practice at which the studies were conducted. This article does not contain any studies with human participants performed by any of the authors.

Supplementary material

223_2015_72_MOESM1_ESM.docx (2.1 mb)
Supplementary material 1 (DOCX 2175 kb)


  1. 1.
    Weiner S, Wagner HD (1998) The material bone: structure-mechanical function relations. Annu Rev Mater Sci 28:271–298CrossRefGoogle Scholar
  2. 2.
    Nudelman F, Pieterse K, George A, Bomans PH, Friedrich H, Brylka LJ, Hilbers PA, de With G, Sommerdijk NA (2010) The role of collagen in bone apatite formation in the presence of hydroxyapatite nucleation inhibitors. Nat Mater 9:1004–1009PubMedCentralCrossRefPubMedGoogle Scholar
  3. 3.
    Wang Y, Azais T, Robin M, Vallee A, Catania C, Legriel P, Pehau-Arnaudet G, Babonneau F, Giraud-Guille MM, Nassif N (2012) The predominant role of collagen in the nucleation, growth, structure and orientation of bone apatite. Nat Mater 11:724–733CrossRefPubMedGoogle Scholar
  4. 4.
    Boskey AL (1996) Matrix proteins and mineralization: an overview. Connect Tissue Res 35:357–363CrossRefPubMedGoogle Scholar
  5. 5.
    Landis WJ, Hodgens KJ, Song MJ, Arena J, Kiyonaga S, Marko M, Owen C, McEwen BF (1996) Mineralization of collagen may occur on fibril surfaces: evidence from conventional and high-voltage electron microscopy and three-dimensional imaging. J Struct Biol 117:24–35CrossRefPubMedGoogle Scholar
  6. 6.
    Weiner S, Traub W (1986) Organization of hydroxyapatite crystals within collagen fibrils. FEBS Lett 206:262–266CrossRefPubMedGoogle Scholar
  7. 7.
    Alexander B, Daulton TL, Genin GM, Lipner J, Pasteris JD, Wopenka B, Thomopoulos S (2012) The nanometre-scale physiology of bone: steric modelling and scanning transmission electron microscopy of collagen–mineral structure. J R Soc Interface 9:1774–1786PubMedCentralCrossRefPubMedGoogle Scholar
  8. 8.
    McNally EA, Schwarcz HP, Botton GA, Arsenault AL (2012) A model for the ultrastructure of bone based on electron microscopy of ion-milled sections. PLoS One 7:e29258PubMedCentralCrossRefPubMedGoogle Scholar
  9. 9.
    Fratzl P, Weinkamer R (2007) Nature’s hierarchical materials. Prog Mater Sci 52:1263–1334CrossRefGoogle Scholar
  10. 10.
    Reznikov N, Shahar R, Weiner S (2014) Bone hierarchical structure in three dimensions. Acta Biomater 10:3815–3826CrossRefPubMedGoogle Scholar
  11. 11.
    Reznikov N, Shahar R, Weiner S (2014) Three-dimensional structure of human lamellar bone: the presence of two different materials and new insights into the hierarchical organization. Bone 59:93–104CrossRefPubMedGoogle Scholar
  12. 12.
    Mann S, Webb J, Williams RJP (1989) Biomineralization: chemical and biochemical perspectives. VCH, WeinheimGoogle Scholar
  13. 13.
    Olszta MJ, Cheng X, Jee SS, Kumar R, Kim Y-Y, Kaufman MJ, Douglas EP, Gower LB (2007) Bone structure and formation: a new perspective. Mater Sci Eng 58:77–116CrossRefGoogle Scholar
  14. 14.
    Franz-Odendaal TA, Hall BK, Witten PE (2006) Buried alive: how osteoblasts become osteocytes. Dev Dyn 235:176–190CrossRefPubMedGoogle Scholar
  15. 15.
    Pazzaglia UE, Congiu T, Sibilia V, Quacci D (2014) Osteoblast-osteocyte transformation. A SEM densitometric analysis of endosteal apposition in rabbit femur. J Anat 224:132–141CrossRefPubMedGoogle Scholar
  16. 16.
    Dallas SL, Bonewald LF (2010) Dynamics of the transition from osteoblast to osteocyte. Ann N Y Acad Sci 1192:437–443PubMedCentralCrossRefPubMedGoogle Scholar
  17. 17.
    Pazzaglia UE, Congiu T, Marchese M, Dell’Orbo C (2010) The shape modulation of osteoblast-osteocyte transformation and its correlation with the fibrillar organization in secondary osteons: a SEM study employing the graded osmic maceration technique. Cell Tissue Res 340:533–540CrossRefPubMedGoogle Scholar
  18. 18.
    Jones SJ, Boyde A, Pawley JB (1975) Osteoblasts and collagen orientation. Cell Tissue Res 159:73–80CrossRefPubMedGoogle Scholar
  19. 19.
    Marotti G (1979) Osteocyte orientation in human lamellar bone and its relevance to the morphometry of periosteocytic lacunae. Metab Bone Dis Relat Res 1:325–333CrossRefGoogle Scholar
  20. 20.
    Kerschnitzki M, Wagermaier W, Roschger P, Seto J, Shahar R, Duda GN, Mundlos S, Fratzl P (2011) The organization of the osteocyte network mirrors the extracellular matrix orientation in bone. J Struct Biol 173:303–311CrossRefPubMedGoogle Scholar
  21. 21.
    Boyde A (1972) Scanning electron microscope studies of bone. In: Bourne GH (ed) The biochemistry and physiology of bone. Academic Press Inc, New YorkGoogle Scholar
  22. 22.
    Rezakhaniha R, Agianniotis A, Schrauwen JTC, Griffa A, Sage D, Bouten CVC, van de Vosse FN, Unser M, Stergiopulos N (2012) Experimental investigation of collagen waviness and orientation in the arterial adventitia using confocal laser scanning microscopy. Biomech Model Mechanobiol 11:461–473CrossRefPubMedGoogle Scholar
  23. 23.
    Mazet V, Carteret C, Brie D, Idier J, Humbert B (2005) Background removal from spectra by designing and minimising a non-quadratic cost function. Chemom Intell Lab Syst 76:121–133CrossRefGoogle Scholar
  24. 24.
    Morris MD, Mandair GS (2011) Raman assessment of bone quality. Clin Orthop Relat Res 469:2160–2169PubMedCentralCrossRefPubMedGoogle Scholar
  25. 25.
    Dooley KA (2011) Raman spectroscopic studies of bone biomechanical function and development in animal models. University of MichiganGoogle Scholar
  26. 26.
    Kazanci M, Wagner HD, Manjubala NI, Gupta HS, Paschalis E, Roschger P, Fratzl P (2007) Raman imaging of two orthogonal planes within cortical bone. Bone 41:456–461CrossRefPubMedGoogle Scholar
  27. 27.
    Chen P-Y, Toroian D, Price PA, McKittrick J (2011) Minerals form a continuum phase in mature cancellous bone. Calcif Tissue Int 88:351–361PubMedCentralCrossRefPubMedGoogle Scholar
  28. 28.
    Midura RJ, Vasanji A, Su X, Midura SB, Gorski JP (2009) Isolation of calcospherulites from the mineralization front of bone. Cells Tissues Organs 189:75–79CrossRefPubMedGoogle Scholar
  29. 29.
    Kerschnitzki M, Kollmannsberger P, Burghammer M, Duda GN, Weinkamer R, Wagermaier W, Fratzl P (2013) Architecture of the osteocyte network correlates with bone material quality. J Bone Miner Res 28:1837–1845CrossRefPubMedGoogle Scholar
  30. 30.
    Pritchard JJ (1972) The Osteoblast. In: Bourne GH (ed) The biochemistry and physiology of bone. Academic Press Inc, New YorkGoogle Scholar
  31. 31.
    Shah FA, Johansson BR, Thomsen P, Palmquist A (2015) Ultrastructural evaluation of shrinkage artefacts induced by fixatives and embedding resins on osteocyte processes and pericellular space dimensions. J Biomed Mater Res A 103:1565–1576CrossRefPubMedGoogle Scholar
  32. 32.
    Janko M, Davydovskaya P, Bauer M, Zink A, Stark RW (2010) Anisotropic Raman scattering in collagen bundles. Opt Lett 35:2765–2767CrossRefPubMedGoogle Scholar
  33. 33.
    Kazanci M, Roschger P, Paschalis EP, Klaushofer K, Fratzl P (2006) Bone osteonal tissues by Raman spectral mapping: orientation-composition. J Struct Biol 156:489–496CrossRefPubMedGoogle Scholar
  34. 34.
    Bonifacio A, Sergo V (2010) Effects of sample orientation in Raman microspectroscopy of collagen fibers and their impact on the interpretation of the amide III band. Vib Spectrosc 53:314–317CrossRefGoogle Scholar
  35. 35.
    Glimcher MJ (2006) Bone: nature of the calcium phosphate crystals and cellular, structural, and physical chemical mechanisms in their formation. Rev Mineral Geochem 64:223–282CrossRefGoogle Scholar
  36. 36.
    Wopenka B, Pasteris JD (2005) A mineralogical perspective on the apatite in bone. Mater Sci Eng C 25:131–143CrossRefGoogle Scholar
  37. 37.
    Legeros RZ, Trautz OR, Legeros JP, Klein E, Shirra WP (1967) Apatite crystallites: effects of carbonate on morphology. Science 155:1409–1411CrossRefPubMedGoogle Scholar
  38. 38.
    Davies E, Müller KH, Wong WC, Pickard CJ, Reid DG, Skepper JN, Duer MJ (2014) Citrate bridges between mineral platelets in bone. Proc Natl Acad Sci 111:E1354–E1363PubMedCentralCrossRefPubMedGoogle Scholar
  39. 39.
    Shah FA, Nilson B, Brånemark R, Thomsen P, Palmquist A (2014) The bone-implant interface—nanoscale analysis of clinically retrieved dental implants. Nanomed Nanotechnol Biol Med 10:1729–1737CrossRefGoogle Scholar
  40. 40.
    Midura RJ, Vasanji A, Su X, Wang A, Midura SB, Gorski JP (2007) Calcospherulites isolated from the mineralization front of bone induce the mineralization of type I collagen. Bone 41:1005–1016PubMedCentralCrossRefPubMedGoogle Scholar
  41. 41.
    Mahamid J, Aichmayer B, Shimoni E, Ziblat R, Li C, Siegel S, Paris O, Fratzl P, Weiner S, Addadi L (2010) Mapping amorphous calcium phosphate transformation into crystalline mineral from the cell to the bone in zebrafish fin rays. Proc Natl Acad Sci USA 107:6316–6321PubMedCentralCrossRefPubMedGoogle Scholar
  42. 42.
    Yamamoto T, Hasegawa T, Sasaki M, Hongo H, Tabata C, Liu Z, Li M, Amizuka N (2012) Structure and formation of the twisted plywood pattern of collagen fibrils in rat lamellar bone. J Electron Microsc 61:113–121CrossRefGoogle Scholar
  43. 43.
    Lees S, Heeley JD, Cleary PF (1979) A study of some properties of a sample of bovine cortical bone using ultrasound. Calcif Tissue Int 29:107–117CrossRefPubMedGoogle Scholar
  44. 44.
    Mahamid J, Sharir A, Addadi L, Weiner S (2008) Amorphous calcium phosphate is a major component of the forming fin bones of zebrafish: indications for an amorphous precursor phase. Proc Natl Acad Sci USA 105:12748–12753PubMedCentralCrossRefPubMedGoogle Scholar
  45. 45.
    Rehman I, Smith R, Hench LL, Bonfield W (1995) Structural evaluation of human and sheep bone and comparison with synthetic hydroxyapatite by FT-Raman spectroscopy. J Biomed Mater Res 29:1287–1294CrossRefPubMedGoogle Scholar
  46. 46.
    Boyde A, Sela J (1978) Scanning electron microscope study of separated calcospherites from the matrices of different mineralizing systems. Calcif Tissue Res 26:47–49CrossRefPubMedGoogle Scholar
  47. 47.
    Broz JJ, Simske SJ, Corley WD, Greenberg AR (1997) Effects of deproteinization and ashing on site-specific properties of cortical bone. J Mater Sci Mater Med 8:395–401CrossRefPubMedGoogle Scholar
  48. 48.
    Bi L, Li DC, Huang ZS, Yuan Z (2013) Effects of sodium hydroxide, sodium hypochlorite, and gaseous hydrogen peroxide on the natural properties of cancellous bone. Artif Organs 37:629–636CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  • Furqan A. Shah
    • 1
    • 2
    Email author
  • Ezio Zanghellini
    • 3
  • Aleksandar Matic
    • 3
  • Peter Thomsen
    • 1
    • 2
  • Anders Palmquist
    • 1
    • 2
  1. 1.Department of Biomaterials, Institute of Clinical SciencesSahlgrenska Academy at University of GothenburgGöteborgSweden
  2. 2.BIOMATCELL VINN Excellence Center of Biomaterials and Cell TherapyGöteborgSweden
  3. 3.Department of Applied PhysicsChalmers University of TechnologyGöteborgSweden

Personalised recommendations