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BCUR Poster

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BCUR Poster

  1. 1. Aidan Seeley & M. H. Helfrich aidan.seeley.11@aberdeen.ac.uk & m.helfrich@abdn.ac.uk; see http://www.abdn.ac.uk/ims/research/musculoskeletal/ The Musculoskeletal Research Programme, The Institute of Medical Sciences, The University of Aberdeen, Foresterhill, Aberdeen AB25 2ZD Transmission Electron Microscopy (TEM) of Osteocytes Tibia and femur from a three month old rat where high pressure frozen in the Leica EMPACT2. Samples where dehydrated by automatic freeze substitution in the Leica EM AFS2 and embedded in epon resin. Ultrathin sections where cut using a diatome diamond knife and stained with lead citrate and uranyl acetate and viewed using the Jeol 1400 Plus TEM. This method was used to study the pericellular matrix in the osteocyte lacuna. Scanning Electron Microscopy (SEM) of Osteocytes Tibia and fibula taken from a three month old rat where dehydrated and infiltrated with methymethacrylate (MMA) resin in a tissue processor. 9% phosphoric acid was applied to the block surface to dissolve the hydroxyapatite mineral of the bone leaving the MMA resin filled spaces. Samples were then coated with gold and viewed using the Zeiss EVO MA10 SEM. Scanning Electron Microscopy (SEM) of Osteocytes Our studies illustrate that osteocytes reside within lacuna surrounded by a pericellular matrix containing unmineralized type I collagen and matrix vesicles, suggesting osteocytes may have the capacity to synthesize and mineralize collagen matrix in addition to releasing calcium in a process called osteocytic osteolysis. We show that osteocytes make contact with blood vessels and that their processes are longer and more numerous than previously described. This study illustrates the complexity of the osteocyte network in vivo and why it is a major simplification to study individual osteocytes in vitro. We conclude that the methods used here provide a useful tool to obtain detailed anatomical knowledge about the osteocyte network. There are three types of cells in bone tissue; the osteoblast, which is responsible for formation of bone matrix, the osteoclast, responsible for bone-resorption, and the osteocyte that has a role in mechanical sensing and in the maintenance of mineral homeostasis. Osteocytes are found encased in cavities within the bone called lacuna. Osteocytes have many projections emanating from the cell body which travel through the bone in the tunnels called canaliculi as shown in Figure 1. E F G H Image E shows an overview of resin etched cortical bone illustrating the interacting between the osteocyte canalicular system (orange arrow) and an osteonic canal (red arrow). Note how it appears that direct interactions between the two exist with canaliculi containing osteocyte processes touching the osteonic canal containing endothelial cells suggesting direct contact of osteocyte processes with endothelial cells. Image F shows the pericellular space (red arrow) which we known to contain collagen fibers, see images A-D. in which an osteocyte resides (orange arrow) with canaliculi eminating from this space. These contain the osteocyte processes. The osteocyte canaliculi contain the osteocyte processes can be clearly seen by the blue arrow. Image G shows the connections between two osteocytes. Image H shows the connections between osteocytes and the endosteal (END) surface. This project was funded by Medical Research Scotland and the Musculoskeletal Research Programme at the University of Aberdeen. We acknowledge technical support from Mr J. Greenwood, Miss L. Wight, Mr K. MacKenzie and Mrs G. Milne from the Microscopy and Histology Core Facility at the Institute of Medical Sciences, the University of Aberdeen. See http://www.abdn.ac.uk/ims/facilities/microscopy-histology.php Transmission Electron Microscopy (TEM) of Osteocytes A B DC Image A and B show TEM micrographs of an osteocyte located in the femural cortex. The pericellular matrix (PCM) is filled with type I collagen fibers. Red arrows show transverse osteocyte processes and blue arrows shown longitudinal osteocyte processes running in their canaliculi, the spaces within the bone. Images C and D show TEM micrographs of the pericelullar matrix (PCM) surrounding an osteocyte. Red arrows show longitudinal fibers, blue arrows show transverse fibers and orange arrows show matrix vesicles, the start sites of mineralisation. Recent advancements in Electron Microscopy (EM) offer the opportunity for more in-depth anatomical studies of osteocytes. In this project two new methods where used to study bone tissue in the electron microscope: a method to freeze tissue under high pressure and then study very thin sections of that tissue to look inside the bone cells and a method to study the lacuna and canaliculi by embedding bone with resin, cutting the block and etching the surface with acid to create casts. Figure 1. Structure of compact bone. Taken from: Sheir, Butler & Lewis Hole’s Human Anatomy. 10th Edition, McGraw Hill, Boston 2004.

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