Ultrastructure of Bone: Hierarchical Features from Nanometer to Micrometer Scale Revealed in Focused Ion Beam Sections in the TEM
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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.
KeywordsBone Ultrastructure Mineral lamellae Collagen Apatite TEM
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.
K.G. and H.P.S designed the experiments. V.V. and K.G. performed electron microscopy. All authors analyzed the data. K.G. and H.P.S prepared the manuscript with contributions, revisions, and figure preparation from V.V.
Compliance with Ethical Standards
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.
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