Dataset Information

Three-dimensional ultrastructural and histomorphological analysis of the periodontal ligament with occlusal hypofunction via focused ion beam/scanning electron microscope tomography.

ABSTRACT: The periodontal ligament (PDL) maintains the environment and function of the periodontium. The PDL has been remodelled in accordance with changes in mechanical loading. Three-dimensional (3D) structural data provide essential information regarding PDL function and dysfunction. However, changes in mechanical loading associated with structural changes in the PDL are poorly understood at the mesoscale. This study aimed to investigate 3D ultrastructural and histomorphometric changes in PDL cells and fibres associated with unloading condition (occlusal hypofunction), using focused ion beam/scanning electron microscope tomography, and to quantitatively analyse the structural properties of PDL cells and fibres. PDL cells formed cellular networks upon morphological changes induced via changes in mechanical loading condition. Drastic changes were observed in a horizontal array of cells, with a sparse and disorganised area of collagen bundles. Furthermore, collagen bundles tended to be thinner than those in the control group. FIB/SEM tomography enables easier acquisition of serial ultrastructural images and quantitative 3D data. This method is powerful for revealing 3D architecture in complex tissues. Our results may help elucidate architectural changes in the PDL microenvironment during changes in mechanical loading condition and regeneration, and advance a wide variety of treatments in dentistry.

SUBMITTER: Hirashima S

PROVIDER: S-EPMC6606634 | biostudies-literature | 2019 Jul

REPOSITORIES: biostudies-literature

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Three-dimensional ultrastructural and histomorphological analysis of the periodontal ligament with occlusal hypofunction via focused ion beam/scanning electron microscope tomography.

Hirashima Shingo S Ohta Keisuke K Kanazawa Tomonoshin T Togo Akinobu A Kakuma Tatsuyuki T Kusukawa Jingo J Nakamura Kei-Ichiro KI

Scientific reports 20190702 1

The periodontal ligament (PDL) maintains the environment and function of the periodontium. The PDL has been remodelled in accordance with changes in mechanical loading. Three-dimensional (3D) structural data provide essential information regarding PDL function and dysfunction. However, changes in mechanical loading associated with structural changes in the PDL are poorly understood at the mesoscale. This study aimed to investigate 3D ultrastructural and histomorphometric changes in PDL cells and ...[more]

PMID: 31266989

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Project description:The pre-occlusal eruption brings the rat molars into functional occlusion, which implicates tensional strains during mastication. We hypothesized that upon establishment of occlusion, the periodontal ligament undergoes cell and extracellular matrix maturation to adopt to this mechanical function. We thus aimed to characterize the protein content and changes in expression levels of tensionally relevant extracellular matrix components in the periodontal ligament between the eruption and newly established occlusion in rat molars. Twelve Wistar male rats were divided into 3 groups based on eruption and occlusal stages: covering the pre-occlusal stages at time of eruption, the first day of occlusion and 1 week after occlusion. We employed laser capture microdissection to obtain samples of the periodontal ligament at three regions, cervical, apical and subapical. The proteome was screened with Tandem Mass Tag 10-plexTM mass spectrometry and the expression of key proteins were confirmed by immunofluorescence. Differential expression of matrisome proteins was seen in the cervical and the apical region. Downregulation of pro-metabolic proteins, such as apolipoproteins implicated in lipid transport was observed. Alpha-fetoprotein, a stem cell marker, was also downregulated indicating cell differentiation and PDL maturation. Upregulated proteins were components of the extracellular matrix, involving several proteoglycans and glycoproteins and the matrisome was thus further analyzed. Periostin appeared around collagen α-1 (III) fibers and its expression was particularly strong on Sharpey’s fibers. This co-localization coincided with organization of collagen fibers in direction of the occlusal forces. Establishment of occlusion coincides with cellular differentiation and the maturation of the extracellular matrix of the periodontal ligament, as seen by downregulation of the stem cell marker Alpha-fetoprotein and apolipoproteins, and the progressive accumulation of collagen type III fibers, proteoglycans, glycoproteins. The increase in collagen fiber associates periostin may reflect a physiological response to enforce cell-ECM contacts during PDL maturation.

Project description:BackgroundCardioprotection by preventing or repairing mitochondrial damage is an unmet therapeutic need. To understand the role of cardiomyocyte mitochondria in physiopathology, the reliable characterization of the mitochondrial morphology and compartment is pivotal. Previous studies mostly relied on two-dimensional (2D) routine transmission electron microscopy (TEM), thereby neglecting the real three-dimensional (3D) mitochondrial organization. This study aimed to determine whether classical 2D TEM analysis of the cardiomyocyte ultrastructure is sufficient to comprehensively describe the mitochondrial compartment and to reflect mitochondrial number, size, dispersion, distribution, and morphology.MethodsSpatial distribution of the complex mitochondrial network and morphology, number, and size heterogeneity of cardiac mitochondria in isolated adult mouse cardiomyocytes and adult wild-type left ventricular tissues (C57BL/6) were assessed using a comparative 3D imaging system based on focused ion beam-scanning electron microscopy (FIB-SEM) nanotomography. For comparison of 2D vs. 3D data sets, analytical strategies and mathematical comparative approaches were performed. To confirm the value of 3D data for mitochondrial changes, we compared the obtained values for number, coverage area, size heterogeneity, and complexity of wild-type cardiomyocyte mitochondria with data sets from mice lacking the cytosolic and mitochondrial protein BNIP3 (BCL-2/adenovirus E1B 19-kDa interacting protein 3; Bnip3-/- ) using FIB-SEM. Mitochondrial respiration was assessed on isolated mitochondria using the Seahorse XF analyser. A cardiac biopsy was obtained from a male patient (48 years) suffering from myocarditis.ResultsThe FIB-SEM nanotomographic analysis revealed that no linear relationship exists for mitochondrial number (r = 0.02; P = 0.9511), dispersion (r = -0.03; P = 0.9188), and shape (roundness: r = 0.15, P = 0.6397; elongation: r = -0.09, P = 0.7804) between 3D and 2D results. Cumulative frequency distribution analysis showed a diverse abundance of mitochondria with different sizes in 3D and 2D. Qualitatively, 2D data could not reflect mitochondrial distribution and dynamics existing in 3D tissue. 3D analyses enabled the discovery that BNIP3 deletion resulted in more smaller, less complex cardiomyocyte mitochondria (number: P < 0.01; heterogeneity: C.V. wild-type 89% vs. Bnip3-/- 68%; complexity: P < 0.001) forming large myofibril-distorting clusters, as seen in human myocarditis with disturbed mitochondrial dynamics. Bnip3-/- mice also show a higher respiration rate (P < 0.01).ConclusionsHere, we demonstrate the need of 3D analyses for the characterization of mitochondrial features in cardiac tissue samples. Hence, we observed that BNIP3 deletion physiologically acts as a molecular brake on mitochondrial number, suggesting a role in mitochondrial fusion/fission processes and thereby regulating the homeostasis of cardiac bioenergetics.

Dataset Information

Three-dimensional ultrastructural and histomorphological analysis of the periodontal ligament with occlusal hypofunction via focused ion beam/scanning electron microscope tomography.

Publications

Three-dimensional ultrastructural and histomorphological analysis of the periodontal ligament with occlusal hypofunction via focused ion beam/scanning electron microscope tomography.

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