- Email: [email protected]
Osteocyte bioimaging
Journal of Oral Biosciences 57 (2015) 61–64
Contents lists available at ScienceDirect
Journal of Oral Biosciences journal homepage: www.elsevier.com/locate/job
Review
Osteocyte bioimaging Hiroshi Kamioka n Department of Orthodontics, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, 2-5-1 Shikata, Kita-ku, Okayama 700-8525, Japan
art ic l e i nf o
a b s t r a c t
Article history: Received 20 January 2015 Received in revised form 13 February 2015 Accepted 16 February 2015 Available online 7 April 2015
Background: Newly developed visualization methods often lead to breakthroughs in the bioscience field. In particular, the ability to reveal temporal–spatial responses in cells, while visualizing molecular events through bioimaging techniques is very important. One such event is the regulation of bone remodeling by osteocytes. It is thought that osteocyte processes sense the flow of interstitial fluid that is driven through the osteocyte canaliculi by mechanical stimuli caused in the bone. However, the precise mechanism by which the flow elicits a cellular response is still unknown. Highlight: It is critical to obtain precise morphological and/or morphometrical data from osteocytes and their surrounding microenvironment. In this review, we describe our application of confocal laser scanning microscopy to visualize osteocyte morphology in the bone and the combination of ultra-high voltage electron microscopy (UHVEM) and computer simulation of fluid flow to reveal the mechanosensitivity of osteocytes in the bone. Conclusion: The osteocyte network in the bone as well as the microstructure of osteocyte cell processes and the surrounding bone matrix were visualized. We found fluorescence to be useful for studying the osteocyte network morphology. Additionally, the combination of UHVEM and computer simulation is a powerful tool to study the fluid flow in osteocyte canaliculi. & 2015 Japanese Association for Oral Biology. Published by Elsevier B.V. All rights reserved.
Keywords: 3D image-based model Fluid flow simulation Fluid shear stress Osteocyte Osteocyte canaliculus
Contents 1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Bioimaging of osteocytes for morphological analysis by fluorescence 3. UHVEM to visualize osteocyte morphology . . . . . . . . . . . . . . . . . . . . . 4. UHVEM for analyzing osteocyte canaliculi . . . . . . . . . . . . . . . . . . . . . . 5. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Ethical approval. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conflict of interest. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1. Introduction It is well known that osteocytes are the most predominant cells in the bone and are located inside the mineralized bone matrix. Their cytoplasmic processes form a complex intercellular network via gap junctions. In this network, osteocytes are thought to be the principal cells responsible for sensing mechanical stimuli and transporting
n
Tel.: þ 81 86 2356690; fax: þ81 86 2356694. E-mail address: [email protected]
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signals that coordinate the adaptive bone-remodeling response [1]. Overall, it is thought that mechanical loading-induced matrix strain causes a flow of interstitial fluid around the osteocyte processes [2,3]. However, the mechanism by which this fluid flow excites the osteocytes remains unclear. Therefore, it is very important to obtain precise morphological and/or morphometrical data from osteocytes and their surrounding matrix. In the first chapter, we introduce our application of confocal laser scanning microscopy [4] to characterize the osteocyte network in the bone. In the second chapter, we describe a high-resolution analysis of osteocyte morphology via ultra-high voltage electron microscopy (UHVEM) [5]. In the third chapter, we
http://dx.doi.org/10.1016/j.job.2015.02.002 1349-0079/& 2015 Japanese Association for Oral Biology. Published by Elsevier B.V. All rights reserved.
62
H. Kamioka / Journal of Oral Biosciences 57 (2015) 61–64
Fig. 1. Serial histotomography by CLS microscopy. The fluorescent images show the cells stained with Texas Red-X phalloidin (red) and OB7.3 (green). (A) The vascular-facing surface of the osteoblast layer. Arrow shows the tip of the osteocyte processes. (B) Two and a half micrometers from the first layer. Arrow shows the osteocyte processes. (C) Five micrometers from the first layer. Arrow shows the osteocyte. Bar ¼50 mm. Data are from Kamioka et al. [6], with permission to reprint from Elsevier Science Inc. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
demonstrate how the fluid flow causes fluid shear stress surrounding the osteocyte processes using computer simulation based on a threedimensional nanoscale model of osteocyte canaliculi.
2. Bioimaging of osteocytes for morphological analysis by fluorescence To visualize the osteocyte network, we first employed a combination of confocal laser scanning (CLS) microscopy and differential interference contrast (DIC) microscopy for the observation of osteocytes in mineralized embryonic chicken calvariae [6]. The CLS microscopic images identified whole cells stained with Texas-Red-X phalloidin as well as osteocytes immunofluorescently labeled by the monoclonal antibody OB7.3, which specifically identifies chicken osteocytes [7]. The complementation of CLS microscopy with DIC microscopy in the same confocal layer revealed the 3D organization of the cells as well as the lacunar and canalicular walls. In this study, we revealed for the first time that osteocytes elongate their processes to cells on the bone surface and that their dendritic processes run through the osteoblast layer, implying that there is a possibility for a direct contact of osteocytes with the marrow-residing cells (Fig. 1). The distribution of osteocyte processes between the osteocytes and osteoblasts was also analyzed. The ratio of the processes through the osteoblast layer was 2.6374.17% [6]. Therefore, there is a possibility that stress-sensing osteocytes propagate the information to surrounding cells in the bone.
3. UHVEM to visualize osteocyte morphology Although the fluorescent analysis of the osteocyte network is useful for examining a large sample volume, a high-resolution analysis was necessary for the precise histomorphological analysis of the osteocyte cell processes and osteocyte canaliculi. The diameter of the cell processes and osteocyte canaliculi is typically less than 250 nm, which is the theoretical resolution of the fluorescent analysis. Thus, we employed electron tomography to reveal the 3D morphology of the osteocytes at the nanometer level. UHVEM (3 MeV) [5] was utilized to reconstruct the osteocyte network in 3-mm-thick sections. We succeeded in acquiring transmission electron micrographs from 3-mm-thick sections of the bone at a resolution of 8 nm/pixel and reconstructed the three-dimensional morphology of the osteocytes [8] (Fig. 2). We focused on the young osteocytes in the modeling site of chick calvaria. Reconstructed young osteocytes clearly showed that the surfaces of the cell body and processes were not smooth, but irregular.
Fig. 2. Three-dimensional reconstruction of young chick osteoid-osteocytes. The reconstructed images were rotated and are shown at 01 and 361 in the horizontal plane. Data are from Kamioka et al. [8].
H. Kamioka / Journal of Oral Biosciences 57 (2015) 61–64
In addition, variations in size and shape were prominent in single young osteocyte processes.
4. UHVEM for analyzing osteocyte canaliculi We made a three-dimensional reconstruction of a single canaliculus containing an osteocyte cell process (Fig. 3) using the serial
63
tomographic images obtained by UHVEM [9]. The three-dimensional reconstruction clearly illustrates that the osteocyte cell process runs in the center of its canaliculus without direct attachment to the canalicular wall (Fig. 3A–C and supplemental movie). Furthermore, a threedimensional distribution of collagen fibrils was apparent (Fig. 3D, E). The contour of the canalicular wall was determined by the linear collagen fibrils running parallel to the cell process [green] and in a direction perpendicular to the cell process (red; Fig. 3D–I).
Fig. 3. Three-dimensional reconstruction of a canaliculus containing an osteocyte process. Row A shows an osteocyte process (orange) and a translucent canalicular wall (blue). Row B shows the same structures observed in Row A with collagen fibrils that run along the cell process (green) and in a direction perpendicular to the cell process (red). Row C shows the same structures observed in Row B with a translucent canalicular wall. Data are from Kamioka et al. [8]. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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H. Kamioka / Journal of Oral Biosciences 57 (2015) 61–64
Fig. 4. Microscale fluid flow analysis in an almost straight osteocyte canaliculus. (a) A three-dimensional model of canaliculi with an osteocyte process. (b) Distribution of the absolute value of fluid velocity on the stream lines. The fluid flow in the pericellular space is generated by a uniform body force directed from minus to plus along the direction of the z-axis. Data are from Kamioka et al. [9].
Supplementary material related to this article can be found online at http://dx.doi.org/10.1016/j.job.2015.02.002. To investigate the effect of the microscopic surface roughness of the canalicular wall, which is created by collagen fibrils, on the flow profiles in the pericellular space, we conducted a computational fluid dynamics analysis using the Lattice Boltzmann method. The fluid flow profile in an almost straight canaliculus is shown in Fig. 4 [9]. We found that the surface roughness of the canalicular wall induces a complex distribution of fluid velocities. Inhomogeneous flow patterns may induce the deformation of cytoskeletal elements in the osteocyte process, thereby amplifying the mechanical signals. 5. Conclusions In summary, we were able to visualize the entire osteocyte network in the bone as well as the microstructure of osteocyte cell processes and the surrounding bone matrix. We found the fluorescent analysis to be useful for studying the osteocyte network morphology. However, it was not sufficient to analyze the spatial relationship between osteocytes and the canalicular wall, where the mechanical response in osteocytes is dynamic. Using a three-dimensional imagebased model of two distinct canaliculi, we found that the microscopic surface roughness of the canalicular wall strongly influenced the profile of the mechanical loading-induced flow of interstitial fluid, whereby highly inhomogeneous flow patterns emerged. These inhomogeneous flow patterns may induce the deformation of cytoskeletal elements in the osteocyte process, thereby amplifying mechanical signals. Ethical approval The study protocol was approved by the Animal Ethics Review Committee of Okayama University (OKU-2013260). All animal care and procedures were performed in accordance with the Guidelines for Animal Experimentation at Okayama University, the Japanese Government Animal Protection and Management Law, and the Japanese Government Notification on Feeding and Safekeeping of Animals.
Conflict of interest The authors declare no conflict of interest.
Acknowledgments Part of this work was supported by the Nanotechnology Network Project of the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan, at the Research Center for Ultrahigh Voltage Electron Microscopy, Osaka University (Handai Multifunctional Nano-Foundry), and the Japan Society for the Promotion of Science in the form of a Grants-in-Aid for Scientific Research (#25293419).
References [1] Klein-Nulend J, Nijweide PJ, Burger EH. Osteocyte and bone structure. Curr Osteoporos Rep 2003;1:5–10. [2] Weinbaum S, Cowin SC, Zeng Y. A model for the excitation of osteocytes by mechanical loading-induced bone fluid shear stresses. J Biomech 1994;27:339–60. [3] Burger EH, Klein-Nulend J. Mechanotransduction in bone – role of the lacunocanalicular network. FASEB J 1999;13(Suppl):S101–12. [4] Boyde A. Stereoscopic images in confocal (tandem scanning) microscopy. Science 1985;230:1270–2. [5] Takaoka A, Hasegawa T, Yoshida K, Mori H. Microscopic tomography with ultraHVEM and applications. Ultramicroscopy 2008;108:230–8. [6] Kamioka H, Honjo T, Takano-Yamamoto T. A three-dimensional distribution of osteocyte processes revealed by the combination of confocal laser scanning microscopy and differential interference contrast microscopy. Bone 2001;28:145–9. [7] Nijweide PJ, Mulder RJ. Identification of osteocytes in osteoblast-like cell cultures using a monoclonal antibody specifically directed against osteocytes. Histochemistry 1986;84:342–7. [8] Kamioka H, Murshid SA, Ishihara Y, Kajimura N, Hasegawa T, Ando R, Sugawara Y, Yamashiro T, Takaoka A, Takano-Yamamoto T. A method for observing silverstained osteocytes in situ in 3-mm sections using ultra-high voltage electron microscopy tomography. Microsc Microanal 2009;15:377–83. [9] Kamioka H, Kameo Y, Imai Y, Bakker AD, Bacabac RG, Yamada N, Takaoka A, Yamashiro T, Adachi T, Klein-Nulend J. Microscale fluid flow analysis in a human osteocyte canaliculus using a realistic high-resolution image-based three-dimensional model. Integr Biol (Camb) 2012;4:1198–206.
Contents lists available at ScienceDirect
Journal of Oral Biosciences journal homepage: www.elsevier.com/locate/job
Review
Osteocyte bioimaging Hiroshi Kamioka n Department of Orthodontics, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, 2-5-1 Shikata, Kita-ku, Okayama 700-8525, Japan
art ic l e i nf o
a b s t r a c t
Article history: Received 20 January 2015 Received in revised form 13 February 2015 Accepted 16 February 2015 Available online 7 April 2015
Background: Newly developed visualization methods often lead to breakthroughs in the bioscience field. In particular, the ability to reveal temporal–spatial responses in cells, while visualizing molecular events through bioimaging techniques is very important. One such event is the regulation of bone remodeling by osteocytes. It is thought that osteocyte processes sense the flow of interstitial fluid that is driven through the osteocyte canaliculi by mechanical stimuli caused in the bone. However, the precise mechanism by which the flow elicits a cellular response is still unknown. Highlight: It is critical to obtain precise morphological and/or morphometrical data from osteocytes and their surrounding microenvironment. In this review, we describe our application of confocal laser scanning microscopy to visualize osteocyte morphology in the bone and the combination of ultra-high voltage electron microscopy (UHVEM) and computer simulation of fluid flow to reveal the mechanosensitivity of osteocytes in the bone. Conclusion: The osteocyte network in the bone as well as the microstructure of osteocyte cell processes and the surrounding bone matrix were visualized. We found fluorescence to be useful for studying the osteocyte network morphology. Additionally, the combination of UHVEM and computer simulation is a powerful tool to study the fluid flow in osteocyte canaliculi. & 2015 Japanese Association for Oral Biology. Published by Elsevier B.V. All rights reserved.
Keywords: 3D image-based model Fluid flow simulation Fluid shear stress Osteocyte Osteocyte canaliculus
Contents 1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Bioimaging of osteocytes for morphological analysis by fluorescence 3. UHVEM to visualize osteocyte morphology . . . . . . . . . . . . . . . . . . . . . 4. UHVEM for analyzing osteocyte canaliculi . . . . . . . . . . . . . . . . . . . . . . 5. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Ethical approval. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conflict of interest. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1. Introduction It is well known that osteocytes are the most predominant cells in the bone and are located inside the mineralized bone matrix. Their cytoplasmic processes form a complex intercellular network via gap junctions. In this network, osteocytes are thought to be the principal cells responsible for sensing mechanical stimuli and transporting
n
Tel.: þ 81 86 2356690; fax: þ81 86 2356694. E-mail address: [email protected]
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61 62 62 63 64 64 64 64 64
signals that coordinate the adaptive bone-remodeling response [1]. Overall, it is thought that mechanical loading-induced matrix strain causes a flow of interstitial fluid around the osteocyte processes [2,3]. However, the mechanism by which this fluid flow excites the osteocytes remains unclear. Therefore, it is very important to obtain precise morphological and/or morphometrical data from osteocytes and their surrounding matrix. In the first chapter, we introduce our application of confocal laser scanning microscopy [4] to characterize the osteocyte network in the bone. In the second chapter, we describe a high-resolution analysis of osteocyte morphology via ultra-high voltage electron microscopy (UHVEM) [5]. In the third chapter, we
http://dx.doi.org/10.1016/j.job.2015.02.002 1349-0079/& 2015 Japanese Association for Oral Biology. Published by Elsevier B.V. All rights reserved.
62
H. Kamioka / Journal of Oral Biosciences 57 (2015) 61–64
Fig. 1. Serial histotomography by CLS microscopy. The fluorescent images show the cells stained with Texas Red-X phalloidin (red) and OB7.3 (green). (A) The vascular-facing surface of the osteoblast layer. Arrow shows the tip of the osteocyte processes. (B) Two and a half micrometers from the first layer. Arrow shows the osteocyte processes. (C) Five micrometers from the first layer. Arrow shows the osteocyte. Bar ¼50 mm. Data are from Kamioka et al. [6], with permission to reprint from Elsevier Science Inc. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
demonstrate how the fluid flow causes fluid shear stress surrounding the osteocyte processes using computer simulation based on a threedimensional nanoscale model of osteocyte canaliculi.
2. Bioimaging of osteocytes for morphological analysis by fluorescence To visualize the osteocyte network, we first employed a combination of confocal laser scanning (CLS) microscopy and differential interference contrast (DIC) microscopy for the observation of osteocytes in mineralized embryonic chicken calvariae [6]. The CLS microscopic images identified whole cells stained with Texas-Red-X phalloidin as well as osteocytes immunofluorescently labeled by the monoclonal antibody OB7.3, which specifically identifies chicken osteocytes [7]. The complementation of CLS microscopy with DIC microscopy in the same confocal layer revealed the 3D organization of the cells as well as the lacunar and canalicular walls. In this study, we revealed for the first time that osteocytes elongate their processes to cells on the bone surface and that their dendritic processes run through the osteoblast layer, implying that there is a possibility for a direct contact of osteocytes with the marrow-residing cells (Fig. 1). The distribution of osteocyte processes between the osteocytes and osteoblasts was also analyzed. The ratio of the processes through the osteoblast layer was 2.6374.17% [6]. Therefore, there is a possibility that stress-sensing osteocytes propagate the information to surrounding cells in the bone.
3. UHVEM to visualize osteocyte morphology Although the fluorescent analysis of the osteocyte network is useful for examining a large sample volume, a high-resolution analysis was necessary for the precise histomorphological analysis of the osteocyte cell processes and osteocyte canaliculi. The diameter of the cell processes and osteocyte canaliculi is typically less than 250 nm, which is the theoretical resolution of the fluorescent analysis. Thus, we employed electron tomography to reveal the 3D morphology of the osteocytes at the nanometer level. UHVEM (3 MeV) [5] was utilized to reconstruct the osteocyte network in 3-mm-thick sections. We succeeded in acquiring transmission electron micrographs from 3-mm-thick sections of the bone at a resolution of 8 nm/pixel and reconstructed the three-dimensional morphology of the osteocytes [8] (Fig. 2). We focused on the young osteocytes in the modeling site of chick calvaria. Reconstructed young osteocytes clearly showed that the surfaces of the cell body and processes were not smooth, but irregular.
Fig. 2. Three-dimensional reconstruction of young chick osteoid-osteocytes. The reconstructed images were rotated and are shown at 01 and 361 in the horizontal plane. Data are from Kamioka et al. [8].
H. Kamioka / Journal of Oral Biosciences 57 (2015) 61–64
In addition, variations in size and shape were prominent in single young osteocyte processes.
4. UHVEM for analyzing osteocyte canaliculi We made a three-dimensional reconstruction of a single canaliculus containing an osteocyte cell process (Fig. 3) using the serial
63
tomographic images obtained by UHVEM [9]. The three-dimensional reconstruction clearly illustrates that the osteocyte cell process runs in the center of its canaliculus without direct attachment to the canalicular wall (Fig. 3A–C and supplemental movie). Furthermore, a threedimensional distribution of collagen fibrils was apparent (Fig. 3D, E). The contour of the canalicular wall was determined by the linear collagen fibrils running parallel to the cell process [green] and in a direction perpendicular to the cell process (red; Fig. 3D–I).
Fig. 3. Three-dimensional reconstruction of a canaliculus containing an osteocyte process. Row A shows an osteocyte process (orange) and a translucent canalicular wall (blue). Row B shows the same structures observed in Row A with collagen fibrils that run along the cell process (green) and in a direction perpendicular to the cell process (red). Row C shows the same structures observed in Row B with a translucent canalicular wall. Data are from Kamioka et al. [8]. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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H. Kamioka / Journal of Oral Biosciences 57 (2015) 61–64
Fig. 4. Microscale fluid flow analysis in an almost straight osteocyte canaliculus. (a) A three-dimensional model of canaliculi with an osteocyte process. (b) Distribution of the absolute value of fluid velocity on the stream lines. The fluid flow in the pericellular space is generated by a uniform body force directed from minus to plus along the direction of the z-axis. Data are from Kamioka et al. [9].
Supplementary material related to this article can be found online at http://dx.doi.org/10.1016/j.job.2015.02.002. To investigate the effect of the microscopic surface roughness of the canalicular wall, which is created by collagen fibrils, on the flow profiles in the pericellular space, we conducted a computational fluid dynamics analysis using the Lattice Boltzmann method. The fluid flow profile in an almost straight canaliculus is shown in Fig. 4 [9]. We found that the surface roughness of the canalicular wall induces a complex distribution of fluid velocities. Inhomogeneous flow patterns may induce the deformation of cytoskeletal elements in the osteocyte process, thereby amplifying the mechanical signals. 5. Conclusions In summary, we were able to visualize the entire osteocyte network in the bone as well as the microstructure of osteocyte cell processes and the surrounding bone matrix. We found the fluorescent analysis to be useful for studying the osteocyte network morphology. However, it was not sufficient to analyze the spatial relationship between osteocytes and the canalicular wall, where the mechanical response in osteocytes is dynamic. Using a three-dimensional imagebased model of two distinct canaliculi, we found that the microscopic surface roughness of the canalicular wall strongly influenced the profile of the mechanical loading-induced flow of interstitial fluid, whereby highly inhomogeneous flow patterns emerged. These inhomogeneous flow patterns may induce the deformation of cytoskeletal elements in the osteocyte process, thereby amplifying mechanical signals. Ethical approval The study protocol was approved by the Animal Ethics Review Committee of Okayama University (OKU-2013260). All animal care and procedures were performed in accordance with the Guidelines for Animal Experimentation at Okayama University, the Japanese Government Animal Protection and Management Law, and the Japanese Government Notification on Feeding and Safekeeping of Animals.
Conflict of interest The authors declare no conflict of interest.
Acknowledgments Part of this work was supported by the Nanotechnology Network Project of the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan, at the Research Center for Ultrahigh Voltage Electron Microscopy, Osaka University (Handai Multifunctional Nano-Foundry), and the Japan Society for the Promotion of Science in the form of a Grants-in-Aid for Scientific Research (#25293419).
References [1] Klein-Nulend J, Nijweide PJ, Burger EH. Osteocyte and bone structure. Curr Osteoporos Rep 2003;1:5–10. [2] Weinbaum S, Cowin SC, Zeng Y. A model for the excitation of osteocytes by mechanical loading-induced bone fluid shear stresses. J Biomech 1994;27:339–60. [3] Burger EH, Klein-Nulend J. Mechanotransduction in bone – role of the lacunocanalicular network. FASEB J 1999;13(Suppl):S101–12. [4] Boyde A. Stereoscopic images in confocal (tandem scanning) microscopy. Science 1985;230:1270–2. [5] Takaoka A, Hasegawa T, Yoshida K, Mori H. Microscopic tomography with ultraHVEM and applications. Ultramicroscopy 2008;108:230–8. [6] Kamioka H, Honjo T, Takano-Yamamoto T. A three-dimensional distribution of osteocyte processes revealed by the combination of confocal laser scanning microscopy and differential interference contrast microscopy. Bone 2001;28:145–9. [7] Nijweide PJ, Mulder RJ. Identification of osteocytes in osteoblast-like cell cultures using a monoclonal antibody specifically directed against osteocytes. Histochemistry 1986;84:342–7. [8] Kamioka H, Murshid SA, Ishihara Y, Kajimura N, Hasegawa T, Ando R, Sugawara Y, Yamashiro T, Takaoka A, Takano-Yamamoto T. A method for observing silverstained osteocytes in situ in 3-mm sections using ultra-high voltage electron microscopy tomography. Microsc Microanal 2009;15:377–83. [9] Kamioka H, Kameo Y, Imai Y, Bakker AD, Bacabac RG, Yamada N, Takaoka A, Yamashiro T, Adachi T, Klein-Nulend J. Microscale fluid flow analysis in a human osteocyte canaliculus using a realistic high-resolution image-based three-dimensional model. Integr Biol (Camb) 2012;4:1198–206.