The ultrastructure of bone has been widely debated, in part due to limitations in visualizing nanostructural features over relevant micrometer length scales. Here, we employ the high resolving power and compositional contrast of high-angle annular dark-field scanning transmission electron microscopy (HAADF STEM) to investigate new features in human bone with nanometer resolution over microscale areas. Using focused ion beam (FIB)-milled sections that span an area of 50 μm2, we have shown how most of the mineral of cortical human osteonal bone occurs in the form of long, thin polycrystalline plates (mineral lamellae, MLs) which are either flat or curved to wrap closely around collagen fibrils. Close to the collagen fibril (< 20 nm), the radius of curvature matches that of the fibril diameter, while at greater distances, MLs form arcs with much larger radii of curvature. In addition, stacks of closely packed planar (uncurved) MLs occur between fibrils. The curving of mineral lamellae both around and between the fibrils would contribute to the strength of bone. At a larger scale, rosette-like clusters of fibrils are noted for the first time, arranged in quasi-circular arrays that define tube-like structures in alternating osteonal lamellae. At the boundary between adjacent osteonal lamellae, the orientation of fibrils and surrounding mineral lamellae changes abruptly, resembling the “orthogonal” patterns identified by others (Reznikov et al. in Acta Biomater 10:3815–3826, 2014). These features spanning nanometer to micrometer scale have implications for our understanding of bone structure and mechanical integrity.
This is a preview of subscription content, log in to check access.
Buy single article
Instant unlimited access to the full article PDF.
Price includes VAT for USA
Subscribe to journal
Immediate online access to all issues from 2019. Subscription will auto renew annually.
This is the net price. Taxes to be calculated in checkout.
Orgel JPRO, Irving TC, Miller A, Wess TJ (2006) Microfibrillar structure of type I collagen in situ. Proc Natl Acad Sci 103:9001–9005. https://doi.org/10.1073/pnas.0502718103
Rinnerthaler S, Roschger P, Jakob HF et al (1999) Scanning small angle X-ray scattering analysis of human bone sections. Calcif Tissue Int 64:422–429. https://doi.org/10.1007/PL00005824
Weiner S, Price PA (1986) Disaggregation of bone into crystals. Calcif Tissue Int 39:365–375. https://doi.org/10.1007/BF02555173
Kim H-M, Rey C, Glimcher MJ (1995) Isolation of calcium-phosphate crystals of bone by non-aqueous methods at low temperature. J Bone Miner Res 10:1589–1601. https://doi.org/10.1002/jbmr.5650101021
Landis WJ, Song MJ, Leith A et al (1993) Mineral and organic matrix interaction in normally calcifying tendon visualized in three dimensions by high-voltage electron microscopic tomography and graphic image reconstruction. J Struct Biol 110:39–54
Siperko LM, Landis WJ (2001) Aspects of mineral structure in normally calcifying avian tendon. J Struct Biol 135:313–320. https://doi.org/10.1006/jsbi.2001.4414
Hamed E, Novitskaya E, Li J et al (2012) Elastic moduli of untreated, demineralized and deproteinized cortical bone: validation of a theoretical model of bone as an interpenetrating composite material. Acta Biomater 8:1080–1092. https://doi.org/10.1016/j.actbio.2011.11.010
Alexander B, Daulton TL, Genin GM et al (2012) The nanometre-scale physiology of bone: steric modelling and scanning transmission electron microscopy of collagen-mineral structure. J R Soc Interface 9:1774–1786
Su X, Sun K, Cui FZ, Landis WJ (2003) Organization of apatite crystals in human woven bone. Bone 32:150–162
Lees S, Prostak K (1988) The locus of mineral crystallites in bone. Connect Tissue Res 18:41–54. https://doi.org/10.3109/03008208809019071
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. https://doi.org/10.1371/journal.pone.0029258
Fratzl P, Gupta HS, Paschalis EP, Roschger P (2004) Structure and mechanical quality of the collagen–mineral nano-composite in bone. J Mater Chem 14:2115–2123. https://doi.org/10.1039/B402005G
Fratzl P, Gupta HS, Paris O et al (2005) Diffracting “stacks of cards”—some thoughts about small-angle scattering from bone. Prog Colloid Polym Sci 130:33–39. https://doi.org/10.1007/b107343
Schwarcz HP (2015) The ultrastructure of bone as revealed in electron microscopy of ion-milled sections. Semin Cell Dev Biol 46:44–50
Schwarcz HP, Abueidda D, Jasiuk I (2017) The ultrastructure of bone and its relevance to mechanical properties. Front Phys. https://doi.org/10.3389/fphy.2017.00039
Benezra Rosen V, Hobbs LW, Spector M (2002) The ultrastructure of anorganic bovine bone and selected synthetic hyroxyapatites used as bone graft substitute materials. Biomaterials 23:921–928. https://doi.org/10.1016/S0142-9612(01)00204-6
Nalla RK, Porter AE, Daraio C et al (2005) Ultrastructural examination of dentin using focused ion-beam cross-sectioning and transmission electron microscopy. Micron 36:672–680
Jantou-Morris V, Horton MA, McComb DW (2010) The nano-morphological relationships between apatite crystals and collagen fibrils in ivory dentine. Biomaterials 31:5275–5286. https://doi.org/10.1016/j.biomaterials.2010.03.025
Langelier B, Wang X, Grandfield K (2017) Atomic scale chemical tomography of human bone. Sci Rep 7:39958
Grandfield K, Engqvist H (2012) Focused ion beam in the study of biomaterials and biological matter. Adv Mater Sci Eng 2012:1–6
Grandfield K (2015) Bone, implants, and their interfaces. Phys Today 68:40–45. https://doi.org/10.1063/PT.3.2748
Reznikov N, Shahar R, Weiner S (2014) Bone hierarchical structure in 3 dimensions. Acta Biomater 10:3815–3826. https://doi.org/10.1016/j.actbio.2014.05.024
Giraud-Guille MM (1988) Twisted plywood architecture of collagen fibrils in human compact bone osteons. Calcif Tissue Int 42:167–180. https://doi.org/10.1007/BF02556330
Schindelin J, Arganda-Carreras I, Frise E et al (2012) Fiji: an open-source platform for biological-image analysis. Nat Methods 9:676–682. https://doi.org/10.1038/nmeth.2019
Weiner S, Wagner HD (1998) The material bone: structure-mechanical function relations. Annu Rev Mater Sci 28:271–298. https://doi.org/10.1146/annurev.matsci.28.1.271
Raspanti M, Viola M, Forlino A et al (2008) Glycosaminoglycans show a specific periodic interaction with type I collagen fibrils. J Struct Biol 164:134–139. https://doi.org/10.1016/j.jsb.2008.07.001
Orgel JPRO, Eid A, Antipova O et al (2009) Decorin core protein (decoron) shape complements collagen fibril surface structure and mediates its binding. PLoS ONE 4:e7028
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 59:93–104
Reznikov N, Almany-Magal R, Shahar R, Weiner S (2013) Three-dimensional imaging of collagen fibril organization in rat circumferential lamellar bone using a dual beam electron microscope reveals ordered and disordered sub-lamellar structures. Bone 52:676–683
Gebhardt W (1906) Ueber funktionell wichtige Anordnungsweisen der groberen und feineren Bauelemente des Wirbeltierknochens. Arch Entw Mech 20:187–322
Katz JLL, Ukraincik K (1971) On the anisotropic elastic properties of hydroxyapatite. J Biomech 4:221–227. https://doi.org/10.1016/0021-9290(71)90007-8
Schwarcz HP, McNally EA, Botton GA (2014) Dark-field transmission electron microscopy of cortical bone reveals details of extrafibrillar crystals. J Struct Biol 188:240–248. https://doi.org/10.1016/j.jsb.2014.10.005
McKee MD, Cole WG (2012) Bone matrix and mineralization. In: Pediatric bone. pp 9–37
Roschger P, Paschalis EP, Fratzl P, Klaushofer K (2008) Bone mineralization density distribution in health and disease. Bone 42:456–466. https://doi.org/10.1016/j.bone.2007.10.021
Shahar R, Weiner S (2017) Open questions on the 3D structures of collagen containing vertebrate mineralized tissues: A perspective. J Struct Biol https://doi.org/10.1016/j.jsb.2017.11.008
The work was supported by the Discovery Grant program from the Natural Sciences and Engineering Research Council of Canada (NSERC) to HS and KG. Microscopy was performed at the Canadian Centre for Electron Microscopy at McMaster University, a facility supported by NSERC and other government agencies. The authors acknowledge Dakota Binkley and Xiaoyue Wang for assistance with electron tomography videos. We are also grateful to one of the referees for pointing out the possibility of higher hierarchical levels of organization visible in Figs. 5 and 7.
Kathryn Grandfield, Vicky Vuong and Henry P. Schwarcz declare no conflict of interest.
Human and Animal Rights and Informed Consent
This study was obtained with ethical approval as a by-product of restorative surgery. Informed consent was obtained from all subjects and tissues were collected under ethical approval from the institutional human ethics review board.
About this article
Cite this article
Grandfield, K., Vuong, V. & Schwarcz, H.P. Ultrastructure of Bone: Hierarchical Features from Nanometer to Micrometer Scale Revealed in Focused Ion Beam Sections in the TEM. Calcif Tissue Int 103, 606–616 (2018) doi:10.1007/s00223-018-0454-9
- Mineral lamellae