Electron and Ion Beam Imaging of Human Bone Structure Across the Nano- and Mesoscale

Human bone tissue has an inherent hierarchical structure, which is integral to its material properties. It is primarily composed of a collagen fiber matrix that is mineralized with hydroxyapatite. A comprehensive understanding of bone and the linkages between structural and cellular organization is imperative to developing fundamental knowledge that can be applied to better our understanding of bone disease manifestations and its interaction with implant devices. Herein, this thesis investigated non-traditional methods for evaluating bone structure across the nano- and meso-length scales.

Firstly, due to the inhomogeneous organization of collagen fibrils and mineral platelets of bone ultrastructure, a suitable methodology for the investigation of both phases needed to be generated. In this work, focused ion beam (FIB) microscopy was employed to create sitespecific scanning transmission electron microscopy (STEM) lift-outs of human osteonal bone that could be visualized with correlatively with STEM and small angle X-ray scattering (SAXS). Samples were successfully characterized using both techniques, and minimal visual damage was induced during data acquisition. This work is the first to demonstrate the potential for bone to be investigated correlatively using both STEM and SAXS.

Secondly, this work is the first to employ a dual-beam plasma FIB (PFIB) equipped with a scanning electron microscope (SEM), to investigate bone tissue across the mesoscale. This equipment enables large volume three-dimensional (3D) imaging at nanoscale resolution across larger mesoscale volumes. This thesis aimed to reduce ion beam-based artifacts, which presents as curtain-like features by adjusting the composition of protective capping layers. Subsequently, large volume tomograms of bone tissue were acquired, demonstrating the effectiveness of the PFIB to reveal mesoscale features including the cellular network of bone tissue.

Overall, this thesis has developed methods that allow for the application of advanced microscopy techniques to enhance the understanding of bone tissue across the nanoscale and mesoscale.

Year	Entry
2008	Clarke B. Normal bone anatomy and physiology. Clin J Am Soc Nephrol. November 2008;3(suppl 3):S131-S139.
2015	Reznikov N, Chase H, Brumfeld V, Shahar R, Weiner S. The 3D structure of the collagen fibril network in human trabecular bone: relation to trabecular organization. Bone. February 2015;71:189-195.
1999	Weiner S, Traub W, Wagner HD. Lamellar bone: structure–function relations. J Struct Biol. June 1999;126(3):241-255.
2018	Grandfield K, Vuong V, Schwarcz HP. Ultrastructure of bone: hierarchical features from nanometer to micrometer scale revealed in focused ion beam sections in the TEM. Calcif Tiss Int. December 2018;103(6):606-616.
2004	Fratzl P, Gupta HS, Paschalis EP, Roschger P. Structure and mechanical quality of the collagen–mineral nano-composite in bone. J Mater Chem. 2004;14(14):2115-2123.
2012	McNally EA, Schwarcz HP, Botton GA, Arsenault AL. A model for the ultrastructure of bone based on electron microscopy of ion-milled sections. PLoS One. January 17, 2012;7(1):e29258.
1999	Zysset PK, Guo XE, Hoffler CE, Moore KE, Goldstein SA. Elastic modulus and hardness of cortical and trabecular bone lamellae measured by nanoindentation in the human femur. J Biomech. October 1999;32(10):1005-1012.
1997	Weiner S, Arad T, Sabanay I, Traub. W. Rotated plywood structure of primary lamellar bone in the rat: orientations of the collagen fibril arrays. Bone. June 1997;20(6):509-514.
1993	Landis WJ, Song MJ, Leith A, McEwen L, McEwen BF. 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. January 1993;110(1):39-54.
1988	Giraud-Guille MM. Twisted plywood architecture of collagen fibrils in human compact bone osteons. Calcif Tiss Int. May 1988;42(3):167-180.
2012	Grandfield K, Engqvist H. Focused ion beam in the study of biomaterials and biological matter. Adv Mater Sci Eng. 2012;2012:841961.
2014	Reznikov N, Shahar R, Weiner S. Three-dimensional structure of human lamellar bone: the presence of two different materials and new insights into the hierarchical organization. Bone. February 2014;59:93-104.
2017	Wang X, Shah FA, Palmquist A, Grandfield K. 3D characterization of human nano-osseointegration by on-axis electron tomography without the missing wedge. ACS Biomater Sci Eng. 2017;3(1):49-55.
2016	Georgiadis M, Müller R, Schneider P. Techniques to assess bone ultrastructure organization: orientation and arrangement of mineralized collagen fibrils. J R Soc Interface. June 1, 2016;13(119):20160088.
2013	Reznikov N, Almany-Magal R, Shahar R, Weiner S. 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. February 2013;52(2):676-683.
2014	Bellido T. Osteocyte-driven bone remodeling. Calcif Tiss Int. January 2014;94(1):25-34.
2015	Florencio-Silva R, Sasso GRdS, Sasso-Cerri E, Simões MJ, Cerri PS. Biology of bone tissue: structure, function, and factors that influence bone cells. BioMed Res Int. 2015;2015:421746.
2011	Schneider P, Meier M, Wepf R, Müller R. Serial FIB/SEM imaging for quantitative 3D assessment of the osteocyte lacuno-canalicular network. Bone. August 2011;49(2):304-311.
2014	Reznikov N, Shahar R, Weiner S. Bone hierarchical structure in three dimensions. Acta Biomater. September 2014;10(8):3815-3826.
2018	Reznikov N, Bilton M, Lari L, Stevens MM, Kröger R. Fractal-like hierarchical organization of bone begins at the nanoscale. Science. May 4, 2018;360(6388):eaao2189.

Year

Entry

2008

Clarke B. Normal bone anatomy and physiology. Clin J Am Soc Nephrol. November 2008;3(suppl 3):S131-S139.

2015

Reznikov N, Chase H, Brumfeld V, Shahar R, Weiner S. The 3D structure of the collagen fibril network in human trabecular bone: relation to trabecular organization. Bone. February 2015;71:189-195.

1999

Weiner S, Traub W, Wagner HD. Lamellar bone: structure–function relations. J Struct Biol. June 1999;126(3):241-255.

2018

Grandfield K, Vuong V, Schwarcz HP. Ultrastructure of bone: hierarchical features from nanometer to micrometer scale revealed in focused ion beam sections in the TEM. Calcif Tiss Int. December 2018;103(6):606-616.

2004

Fratzl P, Gupta HS, Paschalis EP, Roschger P. Structure and mechanical quality of the collagen–mineral nano-composite in bone. J Mater Chem. 2004;14(14):2115-2123.