Abstract
The aim of this study is to demonstrate the application of focused ion beam-scanning electron microscopy, FIB-SEM for revealing the three-dimensional features of osteocytic cytoplasmic processes in metaphyseal (immature) and diaphyseal (mature) trabeculae. Tibiae of eight-week-old male mice were fixed with aldehyde solution, and treated with block staining prior to FIB-SEM observation. While two-dimensional backscattered SEM images showed osteocytes’ cytoplasmic processes in a fragmented fashion, three-dimensional reconstructions of FIB-SEM images demonstrated that osteocytes in primary metaphyseal trabeculae extended their cytoplasmic processes randomly, thus maintaining contact with neighboring osteocytes and osteoblasts. In contrast, diaphyseal osteocytes extended thin cytoplasmic processes from their cell bodies, which ran perpendicular to the bone surface. In addition, these osteocytes featured thick processes that branched into thinner, transverse cytoplasmic processes; at some point, however, these transverse processes bend at a right angle to run perpendicular to the bone surface. Osteoblasts also possessed thicker cytoplasmic processes that branched off as thinner processes, which then connected with cytoplasmic processes of neighboring osteocytes. Thus, FIB-SEM is a useful technology for visualizing the three-dimensional structures of osteocytes and their cytoplasmic processes.
Similar content being viewed by others
References
Amizuka N, Hasegawa T, Oda K, Luiz de Freitas PH, Hoshi K, Li M, Ozawa H (2012a) Histology of epiphyseal cartilage calcification and endochondral ossification. Front Biosci 4:2085–2100. https://doi.org/10.2741/526
Amizuka N, Hongo H, Sasaki M, Hasegawa T, Suzuki R, Tabata C, Ubaidus S, Masuki H, Guo Y, Freitas PHL, Oda K, Li M (2012b) The distribution of osteocytic lacunar-canalicular system, and immunolocalization of FGF23 and sclerostin in osteocytes. J Oral Biosci 54(1):37–42. https://doi.org/10.1016/j.job.2011.06.002
Burger EH, Klein-Nulend J (1999) Mechanotransduction in bone—role of the lacuno-canalicular network. FASEB J 13:101–112
Burr DB (2002) Targeted and nontargeted remodeling. Bone 30:2–4
De Winter DA, Schneijdenberg CT, Lebbink MN, Lich B, Verkleij AJ, Drury MR, Humbel BM (2009) Tomography of insulating biological and geological materials using focused ion beam (FIB) sectioning and low-kV BSE imaging. J Microsc 233:372–383. https://doi.org/10.1111/j.1365-2818.2009.03139.x
Donahue HJ (2000) Gap junctions and biophysical regulation of bone cell differentiation. Bone 26:417–422. https://doi.org/10.1016/S8756-3282(00)00245-3
Drobne D, Milani M, Ballerini M, Zrimec A, Zrimec MB, Tatti F, Draslar K (2004) Focused ion beam for microscopy and in situ sample preparation: application on a crustacean digestive system. J Biomed Opt 9:1238–1243. https://doi.org/10.1117/1.1803846
Drobne D, Milani M, Zrimec A, Zrimec MB, Tatti F, Draslar K (2005) Focused ion beam/scanning electron microscopy studies of Porcellio scaber (Isopoda, Crustacea) digestive gland epithelium cells. Scanning 27:30–34. https://doi.org/10.1002/sca.4950270106
Drobne D, Milani M, Leser V, Tatti F, Zrimec A, Znidarsic N, Kostanjsek R, Strus J (2008) Imaging of intracellular spherical lamellar structures and tissue gross morphology by a focused ion beam/scanning electron microscope (FIB/SEM). Ultramicroscopy 108:663–670. https://doi.org/10.1016/j.ultramic.2007.10.010
Earl JS, Leary RK, Perrin JS, Brydson R, Harrington JP, Markowitz K, Milne SJ (2010) Characterization of dentine structure in three dimensions using FIB-SEM. J Microsc 240:1–5. https://doi.org/10.1111/j.1365-2818.2010.03396.x
Frost HM (1960) Presence of microscopic cracks in vivo in bone. Bull Henry Ford Hosp 8:25–35
Hasegawa T, Li M, Hara K, Sasaki M, Tabata C, de Freitas PH, Hongo H, Suzuki R, Kobayashi M, Inoue K, Yamamoto T, Oohata N, Oda K, Akiyama Y, Amizuka N (2011) Morphological assessment of bone mineralization in tibial metaphyses of ascorbic acid-deficient ODS rats. Biomed Res 32:259–269. https://doi.org/10.2220/biomedres.32.259
Hasegawa T, Amizuka N, Yamada T, Liu Z, Miyamoto Y, Yamamoto T, Sasaki M, Hongo H, Suzuki R, Freitus de PHL, Yamamoto T, Oda K, Li M (2013) Sclerostin is differently immunolocalized in metaphyseal trabecules and cortical bones of mouse tibiae. Biomed Res 34(3):153–159. https://doi.org/10.2220/biomedres.34.153
Hasegawa T, Endo T, Tsuchiya E, Kudo A, Shen Z, Moritani Y, Abe M, Yamamoto T, Hongo H, Tsuboi K, Yoshida T, Nagai T, Khadiza N, Yokoyama A, Freitas de PHL, Li M, Amizuka N (2017a) Biological application of focus ion beam-scanning electron microscopy (FIB-SEM) to the imaging of cartilaginous fibrils and osteoblastic cytoplasmic processes. J Oral Biosci 59:55–62. https://doi.org/10.1016/j.job.2016.11.004
Hasegawa T, Yamamoto T, Tsuchiya E, Hongo H, Tsuboi K, Kudo A, Abe M, Yoshida T, Nagai T, Khadiza N, Yokoyama A, Oda K, Ozawa H, Freitas de PHL, Li M, Amizuka N (2017b) Ultrastructural and biochemical aspects of matrix vesicle-mediated mineralization. Japanese Dent Sci Review 53:34–45. https://doi.org/10.1369/0022155416665577
Hazenberg JG, Freeley M, Foran E, Lee TC, Taylor D (2006) Microdamage: a cell transducing mechanism based on ruptured osteocyte processes. J Biomech 39:2096–2103. https://doi.org/10.1016/j.jbiomech.2005.06.006
Heymann JA, Shi D, Kim S, Bliss D, Milne JL, Subramaniam S (2009) 3D imaging of mammalian cells with ion-abrasion scanning electron microscopy. J Struct Biol 166:1–7. https://doi.org/10.1016/j.jsb.2008.11.005
Hirose S, Li M, Kojima T, de Freitas PH, Ubaidus S, Oda K, Saito C, Amizuka N (2007) A histological assessment on the distribution of the osteocytic lacunar canalicular system using silver staining. J Bone Miner Metab 25:374–382. https://doi.org/10.1007/s00774-007-0764-x
Hongo H, Hasegawa T, Sasaki M, Suzuki R, Yamada T, Shimoji S, Yamamoto T, Amizuka N (2012) Bone-Orchestrating Cells, Osteocytes. Hokkaido J Dent Sci 32:93–103
Huiskes R, Ruimerman R, van Lenthe GH, Janssen JD (2000) Effects of mechanical forces on maintenance and adaptation of form in trabecular bone. Nature 405:704–706. https://doi.org/10.1038/35015116
Kizilyaprak C, Longo G, Daraspe J, Humbel BM (2015) Investigation of resins suitable for the preparation of biological sample for 3-D electron microscopy. J Struct Biol 189:135–146. https://doi.org/10.1016/j.jsb.2014.10.009
Knothe Tate ML, Niederer P, Knothe U (1998) In vivo tracer transport through the lacunocanalicular system of rat bone in an environment devoid of mechanical loading. Bone 22:107–117
Knothe Tate ML, Adamson JR, Tami AE, Bauer TW (2004) The osteocyte. Int J Biochem Cell Biol 36(1):1–8
Knott G, Marchman H, Wall D, Lich B (2008) Serial section scanning electron microscopy of adult brain tissue using focused ion beam milling. J Neurosci 28:2959–2964. https://doi.org/10.1523/JNEUROSCI.3189-07.2008
Luiz de Freitas PH, Li M, Ninomiya T, Nakamura M, Ubaidus S, Oda K, Udagawa N, Maeda T, Takagi R, Amizuka N (2009) Intermittent PTH administration stimulates pre-osteoblastic proliferation without leading to enhanced bone formation in osteoclast-less c-fos(−/−) mice. J Bone Miner Res 24(9):1586–1597. https://doi.org/10.1359/jbmr.090413
Mori S, Burr DB (1993) Increased intracortical remodeling following fatigue damage. Bone 14:103–109
Ohta K, Sadayama S, Togo A, Higashi R, Tanoue R, Nakamura K (2012) Beam deceleration for block-face scanning electron microscopy of embedded biological tissue. Micron 43:612–620. https://doi.org/10.1016/j.micron.2011.11.001
Ozawa H, Hoshi K, Amizuka N (2008) Current concepts of bone biomineralization. J Oral Biosci 50:1–14. https://doi.org/10.1016/S1349-0079(08)80014-X
Sasaki T, Yamaguchi A, Higashi S, Yoshiki S (1985) Uptake of horseradish peroxidase by bone cells during endochondral bone development. Cell Tissue Res 239:547–553
Sasaki M, Hongo H, Hasegawa T, Suzuki R, Liu Z, Freitas PHL, Yamada T, Oda K, Yamamoto T, Li M, Totsuka Y, Amizuka N (2012) Morphological aspects of the biological function of the osteocytic lacunar canalicular system and of osteocyte-derived factors. Oral Sci Int 9:1–8
Shapiro F (1997) Variable conformation of GAP junctions linking bone cells: a transmission electron microscopic study of linear stacked linear, curvilinear, oval, and annular junctions. Calcif Tissue Int 61:285–293
Thomas JD, Eric AB, Andrea T, Mark HE. NCMIR METHODS FOR 3D EM: a new protocol for preparation of biological specomens for serial block face scanning electron microscopy. NCMIR 2010 Available at: http://www.ncmir.ucsd.edu/sbem-protocol/
Ubaidus S, Li M, Sultana S, de Freitas PH, Oda K, Maeda T, Takagi R, Amizuka N (2009) FGF23 is mainly synthesized by osteocytes in the regularly distributed osteocytic lacunar canalicular system established after physiological bone remodeling. J Electron Microsc (Tokyo) 58:381–392. https://doi.org/10.1093/jmicro/dfp032
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 (Tokyo) 61:113–121. https://doi.org/10.1093/jmicro/dfs033
Yamamoto T, Hasegawa T, Sasaki M, Hongo H, Tsuboi K, Shimizu T, Ota M, Haraguchi M, Takahata M, Oda K, Luiz de Freitas PH, Takakura A, Takao-Kawabata R, Isogai Y, Amizuka N (2016) Frequency of teriparatide administration affects the histological pattern of bone formation in young adult male mice. Endocrinology 157:2604–2620. https://doi.org/10.1210/en.2015-2028
Acknowledgements
This study was partially supported by the Grants-in Aid for Scientific Research of JSPS (Hasegawa T and Amizuka N) and the Nanotechnology Platform Program of the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan (Hasegawa T).
Author information
Authors and Affiliations
Corresponding author
Ethics declarations
Ethical approval
The animal protocols used in this study were approved by the Hokkaido University Guidelines for Animal Experimentation (approval No. 15-0041).
Conflict of interest
No potential conflicts of interest exist.
Electronic supplementary material
Below is the link to the electronic supplementary material.
Supplementary material 1 (AVI 38299 KB)
Supplementary material 2 (MP4 5633 KB)
Rights and permissions
About this article
Cite this article
Hasegawa, T., Yamamoto, T., Hongo, H. et al. Three-dimensional ultrastructure of osteocytes assessed by focused ion beam-scanning electron microscopy (FIB-SEM). Histochem Cell Biol 149, 423–432 (2018). https://doi.org/10.1007/s00418-018-1645-1
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00418-018-1645-1