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.
Project description:Currently, various tissue engineering strategies have been developed for multiple tissue regeneration and integrative structure formations as well as single tissue formation in musculoskeletal complexes. In particular, the regeneration of periodontal tissues or tooth-supportive structures is still challenging to spatiotemporally compartmentalize PCL (poly-?-caprolactone)-cementum constructs with micron-scaled interfaces, integrative tissue (or cementum) formations with optimal dimensions along the tooth-root surfaces, and specific orientations of engineered periodontal ligaments (PDLs). Here, we discuss current advanced approaches to spatiotemporally control PDL orientations with specific angulations and to regenerate cementum layers on the tooth-root surfaces with Sharpey's fiber anchorages for state-of-the-art periodontal tissue engineering.
Project description:The complex hierarchical structure in biological and synthetic fibrous nanocomposites entails considerable difficulties in the interpretation of the crystallographic texture from diffraction data. Here, we present a novel reconstruction method to obtain the 3D distribution of fibres in such systems. An analytical expression is derived for the diffraction intensity from fibres, explaining the azimuthal intensity distribution in terms of the angles of the three dimensional fibre orientation distributions. The telson of stomatopod (mantis shrimp) serves as an example of natural biological armour whose high impact resistance property is believed to arise from the hierarchical organization of alpha chitin nanofibrils into fibres and twisted plywood (Bouligand) structures at the sub-micron and micron scale. Synchrotron microfocus scanning X-ray diffraction data on stomatopod telson were used as a test case to map the 3D fibre orientation across the entire tissue section. The method is applicable to a range of biological and biomimetic structures with graded 3D fibre texture at the sub-micron and micron length scales.
Project description:Periodontal disease may cause considerable destruction of alveolar bone, periodontal ligaments (PDLs) and cementum and even lead to progressive oral dysfunction. Periodontal tissue regeneration is the ultimate goal of periodontal disease treatment to reconstruct both structures and functions. However, the regenerative efficiency is low, possibly due to the lack of a proper periodontal microenvironment. In this study, we applied an injectable and thermosensitive chitosan/gelatin/glycerol phosphate hydrogel to provide a 3D environment for transplanted stem cells and to enhance stem cell delivery and engraftment. The iPSCs-BMP-6-hydrogel complex promoted osteogenesis and the differentiation of new connective tissue and PDL formation. In animal models of maxillary-molar defects, the iPSCs-BMP-6-hydrogel-treated group showed significant mineralization with increased bone volume, trabecular number and trabecular thickness. Synergistic effects of iPSCs and BMP-6 increased both bone and cementum formation. IPSCs-BMP-6-hydrogel-treated animals showed new bone synthesis (increased ALP- and TRAP-positive cells), new PDL regeneration (shown through Masson's trichrome staining and a qualification assay), and reduced levels of inflammatory cytokines. These findings suggest that hydrogel-encapsulated iPSCs combined with BMP-6 provide a new strategy to enhance periodontal regeneration. This combination not only promoted stem cell-derived graft engraftment but also minimized the progress of inflammation, which resulted in highly possible periodontal regeneration.
Project description:Fibre topography of the extracellular matrix governs local mechanical properties and cellular behaviour including migration and gene expression. While quantifying properties of the fibrous network provides valuable data that could be used across a breadth of biomedical disciplines, most available techniques are limited to two dimensions and, therefore, do not fully capture the architecture of three-dimensional (3D) tissue. The currently available 3D techniques have limited accuracy and applicability and many are restricted to a specific imaging modality. To address this need, we developed a novel fibre analysis algorithm capable of determining fibre orientation, fibre diameter and fibre branching on a voxel-wise basis in image stacks with distinct fibre populations. The accuracy of the technique is demonstrated on computer-generated phantom image stacks spanning a range of features and complexities, as well as on two-photon microscopy image stacks of elastic fibres in bovine tendon and dermis. Additionally, we propose a measure of axial spherical variance which can be used to define the degree of fibre alignment in a distribution of 3D orientations. This method provides a useful tool to quantify orientation distributions and variance on image stacks with distinguishable fibres or fibre-like structures.
Project description: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.
Project description:Tissue paper consumption has been growing for the past years, with a forecasted increase in demand for premium products. Premium tissue paper products are obtained with a balance among softness, strength, and absorption properties, optimized for each kind of tissue paper. These properties are influenced by the three-dimensional structure, made from the spatial distribution of cellulose fibres. To our knowledge, the efforts made to date to improve the softness, strength and absorption properties have overlooked the 3D structure. There is an absence of 3D experimental data in the literature for the simultaneous characterization of individual eucalyptus fibres and the paper structure made from these fibres. The 2D fibre morphology determination, including fibre length and fibre width, was obtained by an image analysis method for pulp fibre suspensions, using the MorFi? equipment. The third fibre dimension, the fibre thickness morphology in the out-of-plane direction, was obtained using SEM images of non-pressed isotropic laboratory-made paper sheets. The effective fibre thickness morphology, consisting of the fibre wall and lumen, was measured in the paper structure, as this is precisely the key fibre parameter, influencing not only the structure-related properties, such as paper thickness, bulk, and porosity, but also the final end-use properties. The paper structures were produced using an ISO standard adapted method, for tissue paper structures, without pressing, with a basis weight range from 20 to 150 g/m2. These data are important, among other possible uses, for paper property optimization and simulation studies with 3D fibre based simulators.
Project description:Tooth-supporting periodontium forms a complex with multiple tissues, including cementum, periodontal ligament (PDL), and alveolar bone. In this study, we developed multiphase region-specific microscaffolds with spatiotemporal delivery of bioactive cues for integrated periodontium regeneration. Polycarprolactione-hydroxylapatite (90:10 wt%) scaffolds were fabricated using three-dimensional printing seamlessly in three phases: 100-?m microchannels in Phase A designed for cementum/dentin interface, 600-?m microchannels in Phase B designed for the PDL, and 300-?m microchannels in Phase C designed for alveolar bone. Recombinant human amelogenin, connective tissue growth factor, and bone morphogenetic protein-2 were spatially delivered and time-released in Phases A, B, and C, respectively. Upon 4-week in vitro incubation separately with dental pulp stem/progenitor cells (DPSCs), PDL stem/progenitor cells (PDLSCs), or alveolar bone stem/progenitor cells (ABSCs), distinctive tissue phenotypes were formed with collagen I-rich fibers especially by PDLSCs and mineralized tissues by DPSCs, PDLSCs, and ABSCs. DPSC-seeded multiphase scaffolds upon in vivo implantation yielded aligned PDL-like collagen fibers that inserted into bone sialoprotein-positive bone-like tissue and putative cementum matrix protein 1-positive/dentin sialophosphoprotein-positive dentin/cementum tissues. These findings illustrate a strategy for the regeneration of multiphase periodontal tissues by spatiotemporal delivery of multiple proteins. A single stem/progenitor cell population appears to differentiate into putative dentin/cementum, PDL, and alveolar bone complex by scaffold's biophysical properties and spatially released bioactive cues.
Project description:Periodontal tissue is a distinctive tissue structure composed three-dimensionally of cementum, periodontal ligament (PDL) and alveolar bone. Severe periodontal diseases cause fundamental problems for oral function and general health, and conventional dental treatments are insufficient for healing to healthy periodontal tissue. Cell sheet technology has been used in many tissue regenerations, including periodontal tissue, to transplant appropriate stem/progenitor cells for tissue regeneration of a target site as a uniform tissue. However, it is still difficult to construct a three-dimensional structure of complex tissue composed of multiple types of cells, and the transplantation of a single cell sheet cannot sufficiently regenerate a large-scale tissue injury. Here, we fabricated a three-dimensional complex cell sheet composed of a bone-ligament structure by layering PDL cells and osteoblast-like cells on a temperature responsive culture dish. Following ectopic and orthotopic transplantation, only the complex cell sheet group was demonstrated to anatomically regenerate the bone-ligament structure along with the functional connection of PDL-like fibers to the tooth root and alveolar bone. This study represents successful three-dimensional tissue regeneration of a large-scale tissue injury using a bioengineered tissue designed to simulate the anatomical structure.
Project description:<h4>Background</h4>Firm attachments binding muscles to skeleton are crucial mechanical components of the vertebrate body. These attachments (entheses) are complex three-dimensional structures, containing distinctive arrangements of cells and fibre systems embedded in the bone, which can be modified during ontogeny. Until recently it has only been possible to obtain 2D surface and thin section images of entheses, leaving their 3D histology largely unstudied except by extrapolation from 2D data. Entheses are frequently preserved in fossil bones, but sectioning is inappropriate for rare or unique fossil material.<h4>Methodology/principal findings</h4>Here we present the first non-destructive 3D investigation, by propagation phase contrast synchrotron microtomography (PPC-SRµCT), of enthesis histology in extant and fossil vertebrates. We are able to identify entheses in the humerus of the salamander Desmognathus from the organization of bone-cell lacunae and extrinsic fibres. Statistical analysis of the lacunae differentiates types of attachments, and the orientation of the fibres, reflect the approximate alignment of the muscle. Similar histological structures, including ontogenetically related pattern changes, are perfectly preserved in two 380 million year old fossil vertebrates, the placoderm Compagopiscis croucheri and the sarcopterygian fish Eusthenopteron foordi.<h4>Conclusions/significance</h4>We are able to determine the position of entheses in fossil vertebrates, the approximate orientation of the attached muscles, and aspects of their ontogenetic histories, from PPC-SRµCT data. Sub-micron microtomography thus provides a powerful tool for studying the structure, development, evolution and palaeobiology of muscle attachments.
Project description:Fibre-shaped materials are useful for creating different functional three-dimensional (3D) structures that could mimic complex tissues. Several methods (e.g. extrusion, laminar flow or electrospinning) have been proposed for building hydrogel microfibres, with distinctive cell types and with different degrees of complexity. However, these methods require numerous protocol adaptations in order to achieve fibre fabricating and lack the ability to control microfibre alignment. Here, we present a simple method for the production of microfibers, based on a core shell approach, composed of calcium alginate and type I collagen. The process presented here allows the removal of the calcium alginate shell, after only 24?hours of culture, leading to stable and reproducible fibre shaped cellular constructs. With time of culture cells show to distribute preferentially to the surface of the fibre and display a uniform cellular orientation. Moreover, when cultured inside the fibres, murine bone marrow mesenchymal stem cells show the capacity to differentiate towards the osteoblastic lineage, under non-osteoinductive culture conditions. This work establishes a novel method for cellular fibre fabrication that due to its inherent simplicity can be easily upscaled and applied to other cell types.