Cryo-Electrospinning Generates Highly Porous Fiber Scaffolds Which Improves Trabecular Meshwork Cell Infiltration.

Human trabecular meshwork is a sieve-like tissue with large pores, which plays a vital role in aqueous humor outflow. Dysfunction of this tissue can occur, which leads to glaucoma and permanent vision loss. Replacement of trabecular meshwork with a tissue-engineered device is the ultimate objective. This study aimed to create a biomimetic structure of trabecular meshwork using electrospinning. Conventional electrospinning was compared to cryogenic electrospinning, the latter being an adaptation of conventional electrospinning whereby dry ice is incorporated in the fiber collector system. The dry ice causes ice crystals to form in-between the fibers, increasing the inter-fiber spacing, which is retained following sublimation. Structural characterization demonstrated cryo-scaffolds to have closer recapitulation of the trabecular meshwork, in terms of pore size, porosity, and thickness. The attachment of a healthy, human trabecular meshwork cell line (NTM5) to the scaffold was not influenced by the fabrication method. The main objective was to assess cell infiltration. Cryo-scaffolds supported cell penetration deep within their structure after seven days, whereas cells remained on the outer surface for conventional scaffolds. This study demonstrates the suitability of cryogenic electrospinning for the close recapitulation of trabecular meshwork and its potential as a 3D in vitro model and, in time, a tissue-engineered device.

Morphological and biomechanical analyses of the human healthy and glaucomatous aqueous outflow pathway: Imaging-to-modeling.

BACKGROUND AND OBJECTIVE: Intraocular pressure (IOP) is maintained via a dynamic balance between the production of aqueous humor and its drainage through the trabecular meshwork (TM), juxtacanalicular connective tissue (JCT), and Schlemm's canal (SC) endothelium of the conventional outflow pathway. Primary open angle glaucoma (POAG) is often associated with IOP elevation that occurs due to an abnormally high outflow resistance across the outflow pathway. Outflow tissues are viscoelastic and actively interact with aqueous humor dynamics through a two-way fluid-structure interaction coupling. While glaucoma affects the morphology and stiffness of the outflow tissues, their biomechanics and hydrodynamics in glaucoma eyes remain largely unknown. This research aims to develop an image-to-model method allowing the biomechanics and hydrodynamics of the conventional aqueous outflow pathway to be studied. METHODS: We used a combination of X-ray computed tomography and scanning electron microscopy to reconstruct high-fidelity, eye-specific, 3D microstructural finite element models of the healthy and glaucoma outflow tissues in cellularized and decellularized conditions. The viscoelastic TM/JCT/SC complex finite element models with embedded viscoelastic beam elements were subjected to a physiological IOP load boundary; the stresses/strains and the flow state were calculated using fluid-structure interaction and computational fluid dynamics. RESULTS: Based on the resultant hydrodynamics parameters across the outflow pathway, the primary site of outflow resistance in healthy eyes was in the JCT and immediate vicinity of the SC inner wall, while the majority of the outflow resistance in the glaucoma eyes occurred in the TM. The TM and JCT in the glaucoma eyes showed 1.32-fold and 1.13-fold larger beam thickness and smaller trabecular space size (2.24-fold and 1.50-fold) compared to the healthy eyes. CONCLUSIONS: Characterizing the accurate morphology of the outflow tissues may significantly contribute to constructing more accurate, robust, and reliable models, that can eventually help to better understand the dynamic IOP regulation, hydrodynamics of the aqueous humor, and outflow resistance dynamic in the human eyes. This model demonstrates proof of concept for determining changes to outflow resistance in healthy and glaucomatous tissues and thus may be utilized in larger cohorts of donor tissues where disease specificity, race, age, and gender of the eye donors may be accounted for.

A Review of Particle Size Analysis with X-ray CT.

Particle size and morphology analysis is a problem common to a wide range of applications, including additive manufacturing, geological and agricultural materials' characterisation, food manufacturing and pharmaceuticals. Here, we review the use of microfocus X-ray computed tomography (X-ray CT) for particle analysis. We give an overview of different sample preparation methods, image processing protocols, the morphology parameters that can be determined, and types of materials that are suitable for analysis of particle sizes using X-ray CT. The main conclusion is that size and shape parameters can be determined for particles larger than approximately 2 to 3 µm, given adequate resolution of the X-ray CT setup. Particles composed of high atomic number materials (Z > 40) require careful sample preparation to ensure X-ray transmission. Problems occur when particles with a broad range of sizes are closely packed together, or when particles are fused (sintered or cemented). The use of X-ray CT for particle size analysis promises to become increasingly widespread, offering measurements of size, shape, and porosity of large numbers of particles within one X-ray CT scan.

An Optimized Method to Decellularize Human Trabecular Meshwork.

Glaucoma is linked to raised intraocular pressure (IOP). The trabecular meshwork (TM) plays a major role in regulating IOP by enabling outflow of aqueous humor from the eye through its complex 3D structure. A lack of therapies targeting the dysfunctional TM highlights the need to develop biomimetic scaffolds that provide 3D in vitro models for glaucoma research or as implantable devices to regenerate TM tissue. To artificially mimic the TM's structure, we assessed methods for its decellularization and outline an optimized protocol for cell removal and structural retention. Using bovine TM, we trialed 2 lysing agents-Trypsin (0.05% v/v) and Ammonium Hydroxide (NH4OH; 2% v/v). Twenty-four hours in Trypsin caused significant structural changes. Shorter exposure (2 h) reduced this disruption whilst decellularizing the tissue (dsDNA 26 ± 14 ng/mL (control 1970 ± 146 ng/mL)). In contrast, NH4OH lysed all cells (dsDNA 25 ± 21 ng/mL), and the TM structure remained intact. For human TM, 2% v/v NH4OH similarly removed cells (dsDNA 52 ± 4 ng/mL (control 1965 ± 233 ng/mL)), and light microscopy and SEM presented no structural damage. X-ray computed tomography enabled a novel 3D reconstruction of decellularized human TM and observation of the tissue's intricate architecture. This study provides a new, validated method using NH4OH to decellularize delicate human TM without compromising tissue structure.

Olfactory flow in the sea catfish, Ariopsis felis (L.): Origin, regulation, and resampling.

The olfactory epithelium of the sea catfish, Ariopsis felis, is found on a pinnate array of lamellae (the olfactory rosette) housed within a nasal chamber. The nasal anatomy of A. felis suggests an ability to capture external water currents. We prepared models from X-ray micro-computed tomography scans of two preserved specimens of A. felis. We then used dye visualisation and computational fluid dynamics to show that an external current induced a flow of water through a) the nasal chamber and b) the sensory channels of the olfactory rosette. The factors responsible for inducing flow through the nasal chamber are common to fishes from two other orders. The dye visualisation experiments, together with observations of sea catfishes in vivo, indicate that flow through the nasal chamber is regulated by a mobile nasal flap. The position of the nasal flap - elevated (significant flow) or depressed (reduced flow) - is controlled by the sea catfish's movements. Flow in the sensory channels of the olfactory rosette can pass through either a single channel or, via multiple pathways, up to four consecutive channels. Flow through consecutive sensory channels (olfactory resampling) is more extensive at lower Reynolds numbers (200 and 300, equivalent to swimming speeds of 0.5-1.0 total lengths s-1), coinciding with the mean swimming speed of the sea catfishes observed in vivo (0.6 total lengths s-1). Olfactory resampling may also occur, via a vortex, within single sensory channels. In conclusion, olfactory flow in the sea catfish is regulated and thoroughly sampled by novel mechanisms.

Postcopulatory sexual selection and the evolution of shape complexity in the carnivoran baculum.

The baculum is an enigmatic bone within the mammalian glans penis, and the driving forces behind its often bizarre shape have captivated evolutionary biologists for over a century. Hypotheses for the function of the baculum include aiding in intromission, stimulating females and assisting with prolonged mating. Previous attempts to test these hypotheses have focused on the gross size of the baculum and have failed to reach a consensus. We conducted three-dimensional imaging and apply a new method to quantify three-dimensional shape complexity in the carnivoran baculum. We show that socially monogamous species are evolving towards complex-shaped bacula, whereas group-living species are evolving towards simple bacula. Overall three-dimensional baculum shape complexity is not related to relative testes mass, but tip complexity is higher in induced ovulators and species engaging in prolonged copulation. Our study provides evidence of postcopulatory sexual selection pressures driving three-dimensional shape complexity in the carnivore baculum.

X-ray Micro-Computed Tomography: An Emerging Technology to Analyze Vascular Calcification in Animal Models.

Vascular calcification describes the formation of mineralized tissue within the blood vessel wall, and it is highly associated with increased cardiovascular morbidity and mortality in patients with chronic kidney disease, diabetes, and atherosclerosis. In this article, we briefly review different rodent models used to study vascular calcification in vivo, and critically assess the strengths and weaknesses of the current techniques used to analyze and quantify calcification in these models, namely 2-D histology and the o-cresolphthalein assay. In light of this, we examine X-ray micro-computed tomography (µCT) as an emerging complementary tool for the analysis of vascular calcification in animal models. We demonstrate that this non-destructive technique allows us to simultaneously quantify and localize calcification in an intact vessel in 3-D, and we consider recent advances in µCT sample preparation techniques. This review also discusses the potential to combine 3-D µCT analyses with subsequent 2-D histological, immunohistochemical, and proteomic approaches in correlative microscopy workflows to obtain rich, multifaceted information on calcification volume, calcification load, and signaling mechanisms from within the same arterial segment. In conclusion we briefly discuss the potential use of µCT to visualize and measure vascular calcification in vivo in real-time.

The functional nasal anatomy of the pike, Esox lucius L.

Olfactory flow in fishes is a little-explored area of fundamental and applied importance. We investigated olfactory flow in the pike, Esox lucius, because it has an apparently simple and rigid nasal region. We characterised olfactory flow by dye visualisation and computational fluid dynamics, using models derived from X-ray micro-computed tomography scans of two preserved specimens. An external current induced a flow of water through the nasal chamber at physiologically relevant Reynolds numbers (200-300). We attribute this externally-induced flow to: the location of the incurrent nostril in a region of high static pressure; the nasal bridge deflecting external flow into the nasal chamber; an excurrent nostril normal to external flow; and viscous entrainment. A vortex in the incurrent nostril may be instrumental in viscous entrainment. Flow was dispersed over the olfactory sensory surface when it impacted on the floor of the nasal chamber. Dispersal may be assisted by: the radial array of nasal folds; a complementary interaction between a posterior nasal fold and the ventral surface of the nasal bridge; and the incurrent vortex. The boundary layer could delay considerably (up to ~ 3 s) odorant transport from the external environment to the nasal region. The drag incurred by olfactory flow was almost the same as the drag incurred by models in which the nasal region had been replaced by a smooth surface. The boundary layer does not detach from the nasal region. We conclude that the nasal bridge and the incurrent vortex are pivotal to olfaction in the pike.

Olfactory flow in the sturgeon is externally driven.

Fluid dynamics plays an important part in olfaction. Using the complementary techniques of dye visualisation and computational fluid dynamics (CFD), we investigated the hydrodynamics of the nasal region of the sturgeon Huso dauricus. H. dauricus offers several experimental advantages, including a well-developed, well-supported, radial array (rosette) of visible-by-eye olfactory sensory channels. We represented these features in an anatomically accurate rigid model derived from an X-ray scan of the head of a preserved museum specimen. We validated the results from the CFD simulation by comparing them with data from the dye visualisation experiments. We found that flow through both the nasal chamber and, crucially, the sensory channels could be induced by an external flow (caused by swimming in vivo) at a physiologically relevant Reynolds number. Flow through the nasal chamber arises from the anatomical arrangement of the incurrent and excurrent nostrils, and is assisted by the broad, cartilage-supported, inner wall of the incurrent nostril. Flow through the sensory channels arises when relatively high speed flow passing through the incurrent nostril encounters the circular central support of the olfactory rosette, decelerates, and is dispersed amongst the sensory channels. Vortices within the olfactory flow may assist odorant transport to the sensory surfaces. We conclude that swimming alone is sufficient to drive olfactory flow in H. dauricus, and consider the implications of our results for the three other extant genera of sturgeons (Acipenser, Pseudoscaphirhynchus and Scaphirhynchus), and for other fishes with olfactory rosettes.

Experimental steering of electron microscopy studies using prior X-ray computed tomography.

Transmission electron microscopy (TEM) and scanning electron microscopy (SEM) can provide unrivalled high-resolution images of specific features and volumes of interest. However, the regions interrogated are typically very small, and sample preparation is both time-consuming and destructive. Here we consider how prior X-ray micro-computed tomography (microCT) presents an opportunity to increase the efficiency of electron microscopy in biology. We demonstrate how it can be used to; select the most promising samples and target site-specific locations; provide a wider context of the location being interrogated (multiscale correlative imaging); guide sample preparation and 3D imaging schemes; as well as quantify the effects of destructive sample preparation and staining procedures. We present a workflow utilising open source software in which microCT can be used either broadly, or precisely, to experimentally steer and inform subsequent electron microscopy studies. As automated sample registration procedures are developed to enable correlative microscopy, experimental steering by prior CT could be beneficially routinely incorporated into many experimental workflows.

Alpha shapes: determining 3D shape complexity across morphologically diverse structures.

BACKGROUND: Following recent advances in bioimaging, high-resolution 3D models of biological structures are now generated rapidly and at low-cost. To use this data to address evolutionary and ecological questions, an array of tools has been developed to conduct shape analysis and quantify topographic complexity. Here we focus particularly on shape techniques applied to irregular-shaped objects lacking clear homologous landmarks, and propose a new 'alpha-shapes' method for quantifying 3D shape complexity. METHODS: We apply alpha-shapes to quantify shape complexity in the mammalian baculum as an example of a morphologically disparate structure. Micro- computed-tomography (µCT) scans of bacula were conducted. Bacula were binarised and converted into point clouds. Following application of a scaling factor to account for absolute size differences, a suite of alpha-shapes was fitted per specimen. An alpha shape is formed from a subcomplex of the Delaunay triangulation of a given set of points, and ranges in refinement from a very coarse mesh (approximating convex hulls) to a very fine fit. 'Optimal' alpha was defined as the refinement necessary in order for alpha-shape volume to equal CT voxel volume, and was taken as a metric of overall 'complexity'. RESULTS: Our results show that alpha-shapes can be used to quantify interspecific variation in shape 'complexity' within biological structures of disparate geometry. The 'stepped' nature of alpha curves is informative with regards to the contribution of specific morphological features to overall 'complexity'. Alpha-shapes agrees with other measures of complexity (dissection index, Dirichlet normal energy) in identifying ursid bacula as having low shape complexity. However, alpha-shapes estimates mustelid bacula as being most complex, contrasting with other shape metrics. 3D fractal dimension is identified as an inappropriate metric of complexity when applied to bacula. CONCLUSIONS: Alpha-shapes is used to calculate 'optimal' alpha refinement as a proxy for shape 'complexity' without identifying landmarks. The implementation of alpha-shapes is straightforward, and is automated to process large datasets quickly. We interpret alpha-shapes as being particularly sensitive to concavities in surface topology, potentially distinguishing it from other shape complexity metrics. Beyond genital shape, the alpha-shapes technique holds considerable promise for new applications across evolutionary, ecological and palaeoecological disciplines.

Abrogation of TGF-beta signalling in TAGLN expressing cells recapitulates Pentalogy of Cantrell in the mouse.

Pentalogy of Cantrell (PC) is a rare multi-organ congenital anomaly that impedes ventral body wall closure and results in diaphragmatic hernia, intra- and pericardial defects. The underlying cellular and molecular changes that lead to these severe developmental defects have remained unknown largely due to the lack of representative animal models. Here we provide in depth characterization of a mouse model with conditional ablation of TGFßRII in Transgelin (Tagln) expressing cells. We show that Tagln is transiently expressed in a variety of cells that participate in the embryonic development and patterning of ventral structures. Genetic ablation of TGFßRII in these cells leads to ventral midline closure defect, diaphragmatic hernia, dilated cardiac outflow tract and aberrant cardiac septation, providing a reliable model to study the morphological changes leading to PC. We show that myogenisis in the diaphragm is independent of TGFß and the diaphragmatic hernia arises from fibroblast-specific migration defect. In the dorsal body wall Tagln expression is initiated after the closure process, revealing a remarkable difference between ventral and dorsal body walls development. Our study demonstrates the use of micro-CT scanning to obtain a 3-dimensional high-resolution overview of embryonic anomalies and provides the first mechanistic insight into the development of PC.

X-ray micro-computed tomography (µCT): an emerging opportunity in parasite imaging.

X-ray micro-computed tomography (µCT) is a technique which can obtain three-dimensional images of a sample, including its internal structure, without the need for destructive sectioning. Here, we review the capability of the technique and examine its potential to provide novel insights into the lifestyles of parasites embedded within host tissue. The current capabilities and limitations of the technology in producing contrast in soft tissues are discussed, as well as the potential solutions for parasitologists looking to apply this technique. We present example images of the mouse whipworm Trichuris muris and discuss the application of µCT to provide unique insights into parasite behaviour and pathology, which are inaccessible to other imaging modalities.

Residual stress distribution analysis of heat treated APS TBC using image based modelling.

We carried out a residual stress distribution analysis in a APS TBC throughout the depth of the coatings. The samples were heat treated at 1150 °C for 190 h and the data analysis used image based modelling based on the real 3D images measured by Computed Tomography (CT). The stress distribution in several 2D slices from the 3D model is included in this paper as well as the stress distribution along several paths shown on the slices. Our analysis can explain the occurrence of the "jump" features near the interface between the top coat and the bond coat. These features in the residual stress distribution trend were measured (as a function of depth) by high-energy synchrotron XRD (as shown in our related research article entitled 'Understanding the Residual Stress Distribution through the Thickness of Atmosphere Plasma Sprayed (APS) Thermal Barrier Coatings (TBCs) by high energy Synchrotron XRD; Digital Image Correlation (DIC) and Image Based Modelling') (Li et al., 2017) [1].

Geochemical Evidence of the Seasonality, Affinity and Pigmenation of Solenopora jurassica.

Solenopora jurassica is a fossil calcareous alga that functioned as an important reef-building organism during the Palaeozoic. It is of significant palaeobiological interest due to its distinctive but poorly understood pink and white banding. Though widely accepted as an alga there is still debate over its taxonomic affinity, with recent work arguing that it should be reclassified as a chaetetid sponge. The banding is thought to be seasonal, but there is no conclusive evidence for this. Other recent work has, however demonstrated the presence of a unique organic boron-containing pink/red pigment in the pink bands of S. jurassica. We present new geochemical evidence concerning the seasonality and pigmentation of S. jurassica. Seasonal growth cycles are demonstrated by X-ray radiography, which shows differences in calcite density, and by varying δ13C composition of the bands. Temperature variation in the bands is difficult to constrain accurately due to conflicting patterns arising from Mg/Ca molar ratios and δ18O data. Fluctuating chlorine levels indicate increased salinity in the white bands, when combined with the isotope data this suggests more suggestive of marine conditions during formation of the white band and a greater freshwater component (lower chlorinity) during pink band precipitation (δ18O). Increased photosynthesis is inferred within the pink bands in comparison to the white, based on δ13C. Pyrolysis Gas Chromatography Mass Spectrometry (Py-GCMS) and Fourier Transform Infrared Spectroscopy (FTIR) show the presence of tetramethyl pyrrole, protein moieties and carboxylic acid groups, suggestive of the presence of the red algal pigment phycoerythrin. This is consistent with the pink colour of S. jurassica. As phycoerythrin is only known to occur in algae and cyanobacteria, and no biomarker evidence of bacteria or sponges was detected we conclude S. jurassica is most likely an alga. Pigment analysis may be a reliable classification method for fossil algae.