Phase-contrast 3D tomography of HeLa cells grown in PLLA polymer electrospun scaffolds using synchrotron X-rays.

Advanced imaging is useful for understanding the three-dimensional (3D) growth of cells. X-ray tomography serves as a powerful noninvasive, nondestructive technique that can fulfill these purposes by providing information about cell growth within 3D platforms. There are a limited number of studies taking advantage of synchrotron X-rays, which provides a large field of view and suitable resolution to image cells within specific biomaterials. In this study, X-ray synchrotron radiation microtomography at Diamond Light Source and advanced image processing were used to investigate cellular infiltration of HeLa cells within poly L-lactide (PLLA) scaffolds. This study demonstrates that synchrotron X-rays using phase contrast is a useful method to understand the 3D growth of cells in PLLA electrospun scaffolds. Two different fiber diameter (2 and 4 µm) scaffolds with different pore sizes, grown over 2, 5 and 8 days in vitro, were examined for infiltration and cell connectivity. After performing visualization by segmentation of the cells from the fibers, the results clearly show deeper cell growth and higher cellular interconnectivity in the 4 µm fiber diameter scaffold. This indicates the potential for using such 3D technology to study cell-scaffold interactions for future medical use.

The oesophageal diverticulum of Dirioxa pornia studied through micro-CT scan, dissection and SEM studies.

BACKGROUND: Dirioxa pornia (Diptera, Tephritidae) (Island fly) is an Australian native species related to a number of pestiferous fruit flies but, unlike many of the pest species, has not been studied extensively due to its non-pest status. However, due to D. pornia's apparent reliance on the bacteria for survival it is an ideal species to undertake studies into interaction between Tephritid species and bacteria associated with the intestinal tract. The oesophageal diverticulum, which is a blind-ended protrusion of the oesophagus, has been studied, described and characterised in many other Tephritid species. Unlike many other species where the oesophageal diverticulum has been observed the organ was only observed in male D. pornia. It is speculated that this sexual dimorphism the organ may be the primary location to host beneficial bacteria in the involved in the production of the nuptial gift and the mating success of this Tephritid species. In case of D. pornia, however, no study on any area of the digestive system has been conducted. This study was conducted to locate and characterize the oesophageal diverticulum in D. pornia. A virtual dissection of the alimentary tract was made through micro-computer tomography studies. These studies were followed by dissection and scanning microscopy studies to elucidate the presence of bacteria. RESULTS: The oesophageal diverticulum of D. pornia is part of the foregut and distends from the oesophagus within the head of the fly. The shape of the oesophageal diverticulum corresponds with the Ceratitis type. Scanning microscopy studies of the oesophageal diverticulum show rod-shaped bacterial cells residing along with yeast cells in the lumen. The organ was only observed in male specimens. CONCLUSIONS: This study classifies the oesophageal diverticulum of D. pornia under the "Ceratitis type" of oesophageal diverticula in Tephritid species. The study also proves that micro-CT scanning is possible to locate soft tissues in Tephritid species and the Avizo® Fire software can be successfully used to visualize 3 dimensional (3D) images from x-rays. The methods used in this experiment can be used in future studies for visualising soft tissues of adult Tephritid species through micro tomography. There is sexual dimorphism with the organ only found in males. Finally this study shows that bacteria are present in the oesophageal diverticulum of D. pornia.

SKYQUANT 3D: Quantifying Vascular Anatomy With an Open-Source Workflow for Comprehensive Analysis of Volumetric Optoacoustic Angiography Data.

Efficient visualization of the vascular system is of key importance in biomedical research into tumor angiogenesis, cerebrovascular alterations, and other angiopathies. Optoacoustic (OA) angiography offers a promising solution combining molecular optical contrast with high resolution and deep penetration of ultrasound. However, its hybrid nature implies complex data collection and processing workflows, with significant variability in methodologies across developers and users. To streamline interoperability, we introduce SKYQUANT 3D, a Python-based set of instructions for the Thermo Fisher Scientific Amira/Avizo 3D Visualization & Analysis Software. Our workflow simplifies the batch processing of volumetric optoacoustic angiography images, extracting meaningful quantitative information while also providing statistical analysis and graphical representation of the results. Quantification performance of SKYQUANT 3D is demonstrated using functional preclinical and clinical in vivo 3D OA angiographic tests involving ambient temperature variations and repositioning of the imaged limb.

Investigation on Three-Dimensional Void Mesostructures and Geometries in Porous Asphalt Mixture Based on Computed Tomography (CT) Images and Avizo.

To investigate the void mesostructure in porous asphalt mixtures (PA), computed tomography (CT) and Avizo were utilized to scan and reconstruct the three-dimensional (3D) void model of PA-16 specimens. The void mesostructure of the specimen was quantitatively characterized through the anisotropy evaluation index. The equivalent pore network model (PNM) was extracted using the medial axis method. Based on the PNM model, the topological structure of the specimen and the morphological characteristics of the connected pores were analyzed. The results showed that the void anisotropy evaluation method can reflect the microscopic morphology of voids in porous asphalt mixtures. The cross-sectional porosity of representative elementary volume (REV) is mainly distributed between 20% and 25%, and about 90% of the macropores have a diameter between 0.5 mm and 3 mm. The distribution of cross-sectional porosity is uneven along the REV height direction. As the smallest cross-section of the seepage path, the equivalent radius of the throat is mainly between 0.1 mm and 1.5 mm, which is much smaller than the equivalent radius of the pore. The topological structure of pores is quite different, and their coordination numbers are mainly concentrated within 18. The pores with coordination numbers 1 to 10 constitute the main body of the pores inside REV, accounting for over 98% of the total number of pores. In addition, the permeability calculation results show that there is a significant difference in the permeability of each axis of REV compared to the total permeability of the superpave gyratory compactor (SGC) specimen, which illustrates that the permeability distribution presents an obvious spatial anisotropy. This study effectively reveals the heterogeneity of the 3D void morphology of porous asphalt mixtures, and it provides a reference for a better understanding of the void flow rules in drainage pavements.

A new straightforward method for semi-automated segmentation of trabecular bone from cortical bone in diverse and challenging morphologies.

Many physiological, biomechanical, evolutionary and clinical studies that explore skeletal structure and function require successful separation of trabecular from cortical compartments of a bone that has been imaged by X-ray micro-computed tomography (micro-CT) prior to analysis. Separation often involves manual subdivision of these two similarly radio-opaque compartments, which can be time-consuming and subjective. We have developed an objective, semi-automated protocol which reduces user bias and enables straightforward, user-friendly segmentation of trabecular from the cortical bone without requiring sophisticated programming expertise. This method can conveniently be used as a 'recipe' in commercial programmes (Avizo herein) and applied to a variety of datasets. Here, we characterize and share this recipe, and demonstrate its application to a range of murine and human bone types, including normal and osteoarthritic specimens, and bones with distinct embryonic origins and spanning a range of ages. We validate the method by testing inter-user bias during the scan preparation steps and confirm utility in the architecturally challenging analysis of growing murine epiphyses. We also report details of the recipe, so that other groups can readily re-create a similar method in open access programmes. Our aim is that this method will be adopted widely to create a reproducible and time-efficient method of segmenting trabecular and cortical bone.

Effect of three-dimensional porosity gradients of biomimetic coatings on their bonding strength and cell behavior.

Surface modification techniques are often used to enhance the properties of Ti-based materials as hard-tissue replacements. While the microstructure of the coating and the quality of the interface between the substrate and coating are essential to evaluate the reliability and applicability of the surface modification. In this study, both a hydroxyapatite (HA) coating and a collagen-hydroxyapatite (Col-HA) composite coating were deposited onto a Ti-6Al-4V substrate using a biomimetic coating process. Importantly, a gradient cross-sectional structure with a porous coating toward the surface, while a dense layer adjacent to the interface between the coating and substrate was observed in three-dimensional (3D) from both the HA and Col-HA coatings via a dual-beam focused ion beam-scanning electron microscope (FIB-SEM). Moreover, the pore distributions within the entire coatings were reconstructed in 3D using Avizo, and the pores size distributions along the coating depth were calculated using RStudio. By evaluating the mechanical property and biocompatibility of these materials and closely observing the cross-sectional cell-coating-substrate interfaces using FIB-SEM, it was revealed that the porous surface created by both coatings well supports osteoblast cell adhesion while the dense inner layer facilitates a good bonding between the coating and the substrate. Although the mechanical property of the coating decreased with the addition of collagen, it is still strong enough for implant handling and the biocompatibility was promoted.

On the Structural Peculiarities of Self-Reinforced Composite Materials Based on UHMWPE Fibers.

The structure of self-reinforced composites (SRCs) based on ultra-high molecular weight polyethylene (UHMWPE) was studied by means of Wide-Angle X-ray Scattering (WAXS), X-ray tomography, Raman spectroscopy, Scanning Electron Microscopy (SEM) and in situ tensile testing in combination with advanced processing tools to determine the correlation between the processing conditions, on one hand, and the molecular structure and mechanical properties, on the other. SRCs were fabricated by hot compaction of UHMWPE fibers at different pressure and temperature combinations without addition of polymer matrix or softener. It was found by WAXS that higher compaction temperatures led to more extensive melting of fibers with the corresponding reduction of the Herman's factor reflecting the degree of molecular orientation, while the increase of hot compaction pressure suppressed the melting of fibers within SRCs at a given temperature. X-ray tomography proved the absence of porosity while polarized light Raman spectroscopy measurements for both longitudinal and perpendicular fiber orientations showed qualitatively the anisotropy of SRC samples. SEM revealed that the matrix was formed by interlayers of molten polymer entrapped between fibers in SRCs. Moreover, in situ tensile tests demonstrated the increase of Young's modulus and tensile strength with increasing temperature.

Multi-Scale Digital Image Correlation Analysis of In Situ Deformation of Open-Cell Porous Ultra-High Molecular Weight Polyethylene Foam.

Porous ultra-high molecular weight polyethylene (UHMWPE) is a high-performance bioinert polymer used in cranio-facial reconstructive surgery in procedures where relatively low mechanical stresses arise. As an alternative to much stiffer and more costly polyether-ether-ketone (PEEK) polymer, UHMWPE is finding further wide applications in hierarchically structured hybrids for advanced implants mimicking cartilage, cortical and trabecular bone tissues within a single component. The mechanical behaviour of open-cell UHMWPE sponges obtained through sacrificial desalination of hot compression-moulded UHMWPE-NaCl powder mixtures shows a complex dependence on the fabrication parameters and microstructural features. In particular, similarly to other porous media, it displays significant inhomogeneity of strain that readily localises within deformation bands that govern the overall response. In this article, we report advances in the development of accurate experimental techniques for operando studies of the structure-performance relationship applied to the porous UHMWPE medium with pore sizes of about 250 µm that are most well-suited for live cell proliferation and fast vascularization of implants. Samples of UHMWPE sponges were subjected to in situ compression using a micromechanical testing device within Scanning Electron Microscope (SEM) chamber, allowing the acquisition of high-resolution image sequences for Digital Image Correlation (DIC) analysis. Special masking and image processing algorithms were developed and applied to reveal the evolution of pore size and aspect ratio. Key structural evolution and deformation localisation phenomena were identified at both macro- and micro-structural levels in the elastic and plastic regimes. The motion of pore walls was quantitatively described, and the presence and influence of strain localisation zones were revealed and analysed using DIC technique.

Union operation image processing of data cubes separately processed by different objective filters and its application to void analysis in an all-solid-state lithium-ion battery.

In this article, we propose a smart image-analysis method suitable for extracting target features with hierarchical dimension from original data. The method was applied to three-dimensional volume data of an all-solid lithium-ion battery obtained by the automated sequential sample milling and imaging process using a focused ion beam/scanning electron microscope to investigate the spatial configuration of voids inside the battery. To automatically fully extract the shape and location of the voids, three types of filters were consecutively applied: a median blur filter to extract relatively larger voids, a morphological opening operation filter for small dot-shaped voids and a morphological closing operation filter for small voids with concave contrasts. Three data cubes separately processed by the above-mentioned filters were integrated by a union operation to the final unified volume data, which confirmed the correct extraction of the voids over the entire dimension contained in the original data.