Three-dimensional ultrastructural analysis of cells in the periodontal ligament using focused ion beam/scanning electron microscope tomography

Abstract

The accurate comprehension of normal tissue provides essential data to analyse abnormalities such as disease and regenerative processes. In addition, understanding the proper structure of the target tissue and its microenvironment may facilitate successful novel treatment strategies. Many studies have examined the nature and structure of periodontal ligaments (PDLs); however, the three-dimensional (3D) structure of cells in normal PDLs remains poorly understood. In this study, we used focused ion beam/scanning electron microscope tomography to investigate the whole 3D ultrastructure of PDL cells along with quantitatively analysing their structural properties and ascertaining their orientation to the direction of the collagen fibre. PDL cells were shown to be in contact with each other, forming a widespread mesh-like network between the cementum and the alveolar bone. The volume of the cells in the horizontal fibre area was significantly larger than in other areas, whereas the anisotropy of these cells was lower than in other areas. Furthermore, the orientation of cells to the PDL fibres was not parallel to the PDL fibres in each area. As similar evaluations are recognized as being challenging using conventional two-dimensional methods, these novel 3D findings may contribute necessary knowledge for the comprehensive understanding and analysis of PDLs.

Three‐dimensional ultrastructural analysis and histomorphometry of collagen bundles in the periodontal ligament using focused ion beam/scanning electron microscope tomography

“…In conventional single section analysis, PDL cells are generally oriented with their long axes parallel to the direction of the PDL fibers. We have previously reported that, in 3D images, PDL cells exhibit a flat shape with long processes that are wing‐like, rather than spindle‐shaped. We showed that these PDL cells were in contact with neighboring cells and formed a widespread mesh‐like network between the cementum and alveolar bone.…”

“…Recently, focused ion beam/scanning electron microscope tomography (FIB/SEM tomography) has been developed, 8,9 which facilitates observation of hundreds of serial sections, reconstruction of 3D structures with high resolution and quantitative analysis of structural properties. We have previously reported the 3D ultrastructural properties of PDL cells, 10 and of other tissues [11][12][13][14][15][16][17] and organelles 18,19 after performing a quantitative analysis using FIB/SEM tomography.…”

Three-Dimensional Analysis of Peeled Internal Limiting Membrane Using Focused Ion Beam/Scanning Electron Microscopy

“…The specimens were prepared as previously described. 24 – 26 Briefly, after washing five times with the cacodylate buffer, the specimens were postfixed for 30 minutes in a solution containing 2% osmium tetroxide and 1.5% potassium ferrocyanide in the cacodylate buffer at 4°C. The specimens were then washed five times with distilled water and immersed in 1% thiocarbohydrazide solution for 30 minutes.…”

Effects of an <i>in Vitro</i> Reconstructed Three-dimensional Hematopoietic Microenvironment on Bone Regeneration in a Rat Calvarial Defect Model

“…Specimens were prepared for FIB/SEM tomography as described previously [35][36][37] , with some modifications. On days 7 and 14, the cell cultured membranes were fixed with 2% paraformaldehyde and incubated in 2.5% glutaraldehyde in 0.1 M cacodylate (pH 7.3) buff er for 15 min at room temperature for fi xation.…”