Three-dimensional ultrastructure of osteocytes assessed by focused ion beam-scanning electron microscopy (FIB-SEM)
- 205 Downloads
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.
KeywordsFIB-SEM Osteocyte Osteoblast Three-dimensional structure Bone
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).
Compliance with ethical standards
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.
Supplementary material 1 (AVI 38299 KB)
Supplementary material 2 (MP4 5633 KB)
- 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 CrossRefGoogle Scholar
- 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 CrossRefPubMedGoogle Scholar
- 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 CrossRefPubMedGoogle Scholar
- Frost HM (1960) Presence of microscopic cracks in vivo in bone. Bull Henry Ford Hosp 8:25–35Google Scholar
- 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 CrossRefPubMedGoogle Scholar
- 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 CrossRefPubMedGoogle Scholar
- 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 CrossRefGoogle Scholar
- 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 CrossRefGoogle Scholar
- 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 CrossRefPubMedGoogle Scholar
- 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 32:93–103Google Scholar
- 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 CrossRefPubMedGoogle Scholar
- 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 CrossRefGoogle Scholar
- 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 CrossRefPubMedGoogle Scholar