Structural arrangement of the cardiac collagen fibers of healthy and diabetic dogs

The heart is composed by a specialized muscle, whose form and function are essentials for an adequate work and shows an amount of connective tissue which support and provide insertion for this muscle, whose collagen fibers are responsible for determination of tissue feature. Our objective was to identify the structural arrangement of the heart collagen fibers in dogs. The hearts of the dogs were submitted to the process of the controlled digestion with NaOH solution and observed by scanning electron microscope. Our results showed that the collagen fibers of the endomysial wall have structural arrangement composed by an irregular network with one layer in normal dogs but in diabetic dogs the network acquires a greater amount of the fibers and layers, looking like a "rug" of fibers modifying the relationships of the stress/strain of the tissue. Ahead of the observed results we are able to conclude that exist increase in the amount and thickness of cardiac collagen fibers, beyond the increase of layers and architectural disarrangement in the endomysial wall in the diabetic dogs.     

Imaging and 3D morphological analysis of collagen fibrils

The recent booming of multiphoton imaging of collagen fibrils by means of second harmonic generation microscopy generates the need for the development and automation of quantitative methods for image analysis. Standard approaches sequentially analyse two-dimensional (2D) slices to gain knowledge on the spatial arrangement and dimension of the fibrils, whereas the reconstructed three-dimensional (3D) image yields better information about these characteristics. In this work, a 3D analysis method is proposed for second harmonic generation images of collagen fibrils, based on a recently developed 3D fibre quantification method. This analysis uses operators from mathematical morphology. The fibril structure is scanned with a directional distance transform. Inertia moments of the directional distances yield the main fibre orientation, corresponding to the main inertia axis. The collaboration of directional distances and fibre orientation delivers a geometrical estimate of the fibre radius. The results include local maps as well as global distribution of orientation and radius of the fibrils over the 3D image. They also bring a segmentation of the image into foreground and background, as well as a classification of the foreground pixels into the preferred orientations. This accurate determination of the spatial arrangement of the fibrils within a 3D data set will be most relevant in biomedical applications. It brings the possibility to monitor remodelling of collagen tissues upon a variety of injuries and to guide tissues engineering because biomimetic 3D organizations and density are requested for better integration of implants.     

Characterization of ultrastructure and collagen composition of the teratoma membrane: Comparison to the amniotic membrane

The structural and morphological properties of the teratoma membrane were investigated to better understand the pathogenesis of ovarian teratomas. A mature cystic teratoma and amnion were obtained from patients who underwent laparoscopic cystectomy and uncomplicated delivery, respectively. The teratoma membrane was divided into three layers according to the results of the histological analysis. Each layer showed distinct morphological properties, including an outer layer that was uniformly arranged, a middle layer with an irregular pattern of fibers, and an inner layer that was structurally dense with a wavy pattern of fibers. The morphology of the layers of the amniotic membrane was the reverse that of the teratoma membrane. In the teratoma membrane, the outer layer was primarily composed of type III collagen and the inner layer had a large amount of type III and IV collagen. The amniotic membrane showed a small amount of type III collagen in the outer layer, whereas the inner layer had large amounts of type I, III, and IV collagen. In the teratoma membrane, the collagen fibrils were arranged regularly in the outer layer, but irregularly in the inner layer. In the amniotic membrane, the arrangement of collagen fibrils was the reverse that of the teratoma membrane. Additionally, the collagen fibrils in the teratoma membrane were thinner than those of the amniotic membrane and had slightly shorter d‐spacing. Two membranes showed the differences in collagen fibril arrangement, which may caused by the different functional roles. Microsc. Res. Tech. 76:432–441, 2013. © 2013 Wiley Periodicals, Inc.     

Mechanical testing of recombinant human bone morphogenetic protein-7 regenerated bone in sheep mandibles

A new method was developed in this study for testing excised sheep mandibles as a cantilever. The method was used to determine the strength and stiffness of sheep hemi-mandibles including a 35 mm defect bridged by regenerated bone. Recombinant human bone morphogenetic protein-7 (rhBMP-7) in a bovine collagen type-I carrier was used for the bone regeneration. Initial tests on ten intact sheep mandibles confirmed that the strength, stiffness and area beneath the load-deformation curves of the right and left hemi-mandibles were not significantly different, confirming the validity of using the contra-lateral hemi-mandible as a control side. Complete bone regeneration occurred in six hemi-mandibles treated with rhBMP, but the quality and mechanical properties of the bone were very variable. The new bone in three samples contained fibrous tissue and was weaker and less stiff than the contra-lateral side (strength, 10-20 per cent; stiffness, 6-15 per cent). The other half had better-quality bone and was significantly stiffer and stronger (p < 0.05), with strength 45-63 per cent and stiffness 35-46 per cent of the contra-lateral side. Hemi-mandibles treated with collagen alone had no regenerated bone bridge suggesting that 35 mm is a critical-size bone defect.     

Sectional differences in tendon response

The objectives of this work here focus on the differences in responses to multiple cyclic tests of different sections along the length of the same tendon. Tendon specimens were obtained from the hindlimbs of canines and frozen to -70°C. After thawing, specimens were mounted in the immersion bath at room temperature (22°C). preloaded to 0.13 N and then subjected to 3% or 4% of the initial length at a strain rate of 5%/sec. It was found that different sections of the same long tendons had different resistances to deformation. In general, the bone end sections were stiffer and carried greater loads for a given strain than the muscle end sections, and the mid-portions were the least stiff and carried the smallest loads for a given strain. The results of this study offer new information about the mechanical responses of collagenous tissues. We know more about their responses to multiple cyclic extensions and how their responses are different from the positions along the length of the tendon specimen. The nature and causes of these differences in the stiffness are not fully known. However, it is clear that differences in the mechanical response of tendons and other connective tissues are significant to musculoskeletal performance.     

分布式能源用新型Senegal式垂直轴风力机尾流分析

针对现有的Jensen尾流模型分析水平轴风力机(HAWTs)的方法,对采用经典Jensen尾流模型时恒定的风速亏损和尾流衰减系数进行改进,提出基于改进型Jensen尾流模型对新型Senegal式垂直轴风力机(VAWTs)尾流分析的新方法。结合流场分析对该新型风力机进行尾流数值模拟,得出了基于改进的Jensen尾流模型的尾流参数二维分布的变化规律。由于尾流分布具有三维性,二维尾流分布不能完全反映尾流特性,因此还得到了尾流参数三维分布。全面地分析了单台VAWT的尾流特性,不仅为进一步的多台VAWTs尾流分析和风电场风力机排布研究打好基础,而且为该类Senegal式VAWT的尾流研究提供了借鉴和参考。     

TOC ‐ Issue Information

Knowledge of the collagen structure of an Achilles tendon is critical to comprehend the physiology, biomechanics, homeostasis and remodelling of the tissue. Despite intensive studies, there are still uncertainties regarding the microstructure. The majority of studies have examined the longitudinally arranged collagen fibrils as they are primarily attributed to the principal tensile strength of the tendon. Few studies have considered the structural integrity of the entire three‐dimensional (3D) collagen meshwork, and how the longitudinal collagen fibrils are integrated as a strong unit in a 3D domain to provide the tendons with the essential tensile properties. Using second harmonic generation imaging, a 3D imaging technique was developed and used to study the 3D collagen matrix in the midportion of Achilles tendons without tissue labelling and dehydration. Therefore, the 3D collagen structure is presented in a condition closely representative of the in vivo status. Atomic force microscopy studies have confirmed that second harmonic generation reveals the internal collagen matrix of tendons in 3D at a fibril level. Achilles tendons primarily contain longitudinal collagen fibrils that braid spatially into a dense rope‐like collagen meshwork and are encapsulated or wound tightly by the oblique collagen fibrils emanating from the epitenon region. The arrangement of the collagen fibrils provides the longitudinal fibrils with essential structural integrity and endows the tendon with the unique mechanical function for withstanding tensile stresses. A novel 3D microscopic method has been developed to examine the 3D collagen microstructure of tendons without tissue dehydrating and labelling. The study also provides new knowledge about the collagen microstructure in an Achilles tendon, which enables understanding of the function of the tissue. The knowledge may be important for applying surgical and tissue engineering techniques to tendon reconstruction.     

Anisotropic strain transfer through the aortic valve and its relevance to the cellular mechanical environment

Aortic valve interstitial cells are responsible for maintaining the valve in response to their local mechanical environment. However, the complex organization of the extracellular matrix means cell strains cannot be directly derived from gross strains, and knowledge of tissue structure-function correlations is fundamental towards understanding mechanotransduction. This study investigates strain transfer through the valve, hypothesizing that organization of the valve matrix leads to non-homogenous local strains. Radial and circumferential samples were cut from aortic valve leaflets and subjected to quasi-static mechanical characterization. Further samples were imaged using confocal microscopy, to determine local strains in the matrix. Mechanical data demonstrated that the valve was significantly stronger and stiffer when loaded circumferentially, comparable with previous studies. Micromechanical studies demonstrated that strain transfer through the matrix is anisotropic and indirect, with local strains consistently smaller than applied strains in both orientations. Under radial loading, strains were transferred linearly to cells. However, under circumferential loading, strains were only one-third of applied values, with a less direct relationship between applied and local strains. This may result from matrix reorganization, and be important for preventing cellular damage during normal valve function. These findings should be taken into account when investigating interstitial cell behaviours, such as cell metabolism and mechanotransduction.     

The cellular microenvironment and cytoskeletal actin dynamics in liver fibrogenesis

Hepatic stellate cells (HSCs) are the primary effector cells in liver fibrosis. In the normal liver, HSCs serve as the primary vitamin A storage cells in the body and retain a “quiescent” phenotype. However, after liver injury, they transdifferentiate to an “activated” myofibroblast-like phenotype, which is associated with dramatic upregulation of smooth muscle specific actin and extracellular matrix proteins. The result is a fibrotic, stiff, and dysfunctional liver. Therefore, understanding the molecular mechanisms that govern HSC function is essential for the development of anti-fibrotic medications. The actin cytoskeleton has emerged as a key component of the fibrogenic response in wound healing. Recent data indicate that the cytoskeleton receives signals from the cellular microenvironment and translates them to cellular function—in particular, increased type I collagen expression. Dynamic in nature, the actin cytoskeleton continuously polymerizes and depolymerizes in response to changes in the cellular microenvironment. In this viewpoint, we discuss the recent developments underlying cytoskeletal actin dynamics in liver fibrosis, including how the cellular microenvironment affects HSC function and the molecular mechanisms that regulate the actininduced increase in collagen expression typical of activated HSCs.     

Physico-chemical characterization of functional electrospun scaffolds for bone and cartilage tissue engineering

Mimicking the zonal organization of the bone-cartilage interface will aid the production of functional osteochondral grafts for regeneration of skeletal joint defects. This study investigates the potential of the electrospinning technique to build a three-dimensional construct recapitulating the zonal matrix of this interface. Poly(lactic-co-glycolic acid) (PLGA) and PLGA-collagen solutions containing different concentrations of hydroxyapatite nanoparticles (nHAp) were electrospun on a thin layer of phosphate buffer saline solution spread on the collector in order to facilitate membrane detachment and recovery. Incorporation of increasing amounts of nHAp in PLGA solutions did not affect significantly the average diameter of the fibres, which was about 700 nm. However, in the presence of collagen, fibres with diameters below 100 nm were generally observed and the number of these fibres was inversely proportional to the ratio PLGA:collagen and proportional to the content of nHAp. PLGA membranes were rather hydrophobic, although the aqueous drop contact angles progressively fell from 125 degrees to 110 degrees when the content of nHAp was increased from 0 per cent to 50 per cent (w/v). PLGA-collagen membranes were more hydrophilic with contact angles between 60 degrees and 110 degrees; the values being proportional to the ratio PLGA:collagen and the content of nHAp. Also, the addition of nHAp from 0 per cent to 50 per cent (w/v) in the absence of collagen resulted in decreasing dramatically both the Young's modulus (Ym), from 34.3 +/- 1.8 MPa to 0.10 +/- 0.06 MPa, and the ultimate tensile strain (epsilon max), from a value higher than 40 per cent to 5 per cent. However, the presence of collagen together with nHAp allowed the creation of membranes much stiffer, although more brittle, as shown for membranes made with a ratio 8:2 and 10 per cent of nHAp, for which Ym = 70.0 +/- 6.6 MPa and epsilon max = 7 per cent.     

Fabrication of two-layered Al-B4C composites by conventional hot pressing uuder nitrogen atmosphere and their characterization

In this study, we describe the conventional hot pressing (CHP) of layered Al-B₄C composites and their characterization. The matrix alloy A1-5 wt.%Cu was prepared from elemental powder mixtures. The metal and B₄C powders were mixed to produce either Al-Cu-10vol.%B₄C or Al-Cu-30vol.%B₄C combinations. Then, these powder mixtures were stacked as layers in the hot pressing die to form a two-layered composite. Hot pressing was carried out under nitrogen atmosphere to produce 30×40×5 mm specimens. Microstructural features and age hardening characteristics of composites were determined by specimens cut longitudinally. The flexural strength of both layered composites and their monolithic counterparts were investigated via three point bending tests. In the case of layered specimens of both 10vol.%B₄C and 30vol.%B₄C containing layers were loaded for three-point test. The results show that a homogeneous distribution of B₄C particles in the matrix alloy which is free of pores, can be obtained by CHP method. The ageing behavior of the composites was found to be influenced by the reinforced materials, i.e. higher hardness values were reached in 8 hrs for the composites than that for the matrix alloy. Flexural strength test showed that two-layered composites exhibited improved damage tolerance depending on layer arrangement. Microstructural investigation of the fracture surfaces of the bending specimens was performed by means of scanning electron microscope (SEM). While layer with lower reinforcement content exhibited large plastic deformation under loading, the other with higher reinforcement content exhibited less plastic deformation.     

The potential of hydrogels as synthetic articular cartilage

The relatively poor mechanical properties of conventional synthetic hydrogels are illustrated and compared with those of articular cartilage. By using the composite structure of the natural material as a model a new family of hydrogels, based on interpenetrating polymer network (IPN) technology, has been developed. The underlying synthetic strategies are discussed and the properties of a novel representative network presented. IPN formation produces networks that are stiffer and stronger than the hydrogel copolymers of similar water content. In this behaviour these simple IPNs begin to mimic the properties of biological hydrogel composites. Thus, these materials have exciting potential for demanding in vivo applications.     

Morphological and ultrastructural evaluation of the golden retriever muscular dystrophy trachea,lungs, and diaphragm muscle

Duchenne muscular dystrophy (DMD) is a genetic disease, characterized by atrophy and muscle weakness. The respiratory failure is a common cause of early death in patients with DMD. Golden retriever muscular dystrophy (GRMD) is a canine model which has been extensively used for many advances in therapeutics applications. As the patients with DMD, the GRMD frequently died from cardiac and respiratory failure. Observing the respiratory failure in DMD is one of the major causes of mortality we aimed to describe the morphological and ultrastructural data of trachea, lungs (conductive and respiratory portion of the system), and diaphragm muscle using histological and ultrastructural analysis. The diaphragm muscle showed discontinuous fibers architecture, with different diameter; a robust perimysium inflammatory infiltrate and some muscle cells displayed central nuclei. GRMD trachea and lungs presented collagen fibers and in addition, the GRMD lungs showed higher of levels collagen fibers that could limit the alveolar ducts and alveoli distension. Therefore, the most features observed were the collagen areas and fibrosis. We suggested in this study that the collagen remodeling in the trachea, lungs, and diaphragm muscle may increase fibrosis and affect the trachea, lungs, and diaphragm muscle function that can be a major cause of respiratory failure that occur in patients with DMD. Microsc. Res. Tech. 77:857–861, 2014. © 2014 Wiley Periodicals, Inc.     

Application of Fourier transform‐second‐harmonic generation imaging to the rat cervix

We present the application of Fourier transform‐second‐harmonic generation (FT‐SHG) imaging to evaluate the arrangement of collagen fibers in five nonpregnant rat cervices. Tissue slices from the mid‐cervix and near the external orifice of the cervix were analyzed in both two‐dimensions (2D) and three‐dimensions (3D). We validate that the cervical microstructure can be quantitatively assessed in three dimensions using FT‐SHG imaging and observe collagen fibers oriented both in and out‐of‐plane in the outermost and the innermost layers, which cannot be observed using 2D FT‐SHG analysis alone. This approach has the potential to be a clinically applicable method for measuring progressive changes in collagen organization during cervical remodeling in humans.     

Technique to quantify local clustering of synaptic vesicles using single section data

Synaptic vesicles are organelles that specialize in the storage of a neurotransmitter that continuously undergo an exo-endocytotic cycle. During this cycle vesicles change their positions within a presynaptic terminal and their numbers as well as spatial arrangement can provide insight into a neurotransmitter turnover. This article introduces a technique based on the nearest-neighbor formalism to quantify the proximity of vesicles to active zones and vesicle clustering in different regions of a terminal. The technique, implemented in a software package, uses the two-dimensional coordinates of features identified in digitized electron micrographs as an input. It has been validated in the analysis of asymmetric synapses of the rat hippocampal CA1 stratum radiatum affected by transient cerebral ischemia. It was shown that a 15-minute-long ischemic episode influenced the spatial arrangement of vesicles that were more distant from active zones and had larger intervesicle spacings with respect to the control. The latter effect was apparently stronger within 200 nm distance of active zones.     

Extracellular breakdown of collagen by mice decidual cells. A cytochemical and ultrastructural study.

The interaction of antimesometrial decidual cells and collagen fibrils was studied by light microscopy and ultrastructural cytochemistry in fed and acutely fasted mice on days 9-11 of pregnancy. Fibrillar elements in the extracellular space consisted of collagen fibrils and filamentous aggregates (disintegrating collagen fibrils). Intracellular vacuoles exhibited typical collagen immersed in electron-translucent material (clear vacuoles) and faint cross-banded collagen immersed in electron-opaque material (dark vacuoles). Fibrillar elements showed extracellular acid phosphatase activity which was stronger in the region of mature decidua than in predecidual cells region in all animals; it was conspicuous in mature decidua of fasted animals. Intracellular acid phosphatase activity was observed in dark vacuoles and lysosomes, and was absent in clear vacuoles in all cells studied. Since acid phosphatase activity reflects the presence of lysosomal hydrolases in general, the results indicate that breakdown of extracellular collagen occurs by release of lysosomal enzymes by decidual cells and also by internalization of collagen for intracellular degradation in fed and fasted mice. Collagen breakdown may be part of the process of tissue remodeling in mature and predecidual regions, however, in mature decidua, collagen breakdown is enhanced and may therefore contribute to nutrition of the fetus, specially in acutely fasted mice.     

Collagen arrangement in space of Disse correlates with fluid flow in normal and cirrhotic rat livers

Little is known about collagen arrangement in the space of Disse was related to the fluid flow both in normal and cirrhotic liver. We examined the changes in the arrangement of type‐I collagen in thioacetamide‐induced cirrhotic rat livers with immunohistochemistry and SEM after maceration of the noncollagenous tissues with NaOH. The sparse bundles of collagen fibers in the spaces of Disse were mostly elongated fibers with a disorganized arrangement in each nodule. They connected with the broad fibrous septa. Based on a comparison of the architecture of the collagen fibers and the established flow of fluid in the space of Disse, we hypothesize that the fluid in the space of Disse streams along collagen fibers in all directions to broad fibrous septa. The appearance of perinodular plexus in cirrhotic rat livers probably helps to reduce portal hypertension. Microsc. Res. Tech. 78:187–193, 2015. © 2014 Wiley Periodicals, Inc.     

Automated jet polishing of transmission electron microscope specimens for in situ straining

A jet-polishing technique has been developed for use in the preparation of microtensile specimens for HVEM examination. The technique requires the use of a pair of Teflon sheet inserts with rectangular openings in a conventional specimen holder. When inserts with optimum opening dimensions are used, specimens having elliptical holes close to the center of the gauge section are produced with large electron-transparent areas at both ends of the long axis. Annealed metal specimens, such as brass or aluminum, prepared by this method are stronger, and can be handled more easily, than those prepared by conventional methods. An advantage of the technique is that reproducible electropolishing conditions and the automatic detection of perforation by a photocell can be used in the normal way.     

High voltage microscopy of biological specimens: some practical considerations

Techniques are now available for the preparation of specimens giving excellent image contrast at high voltages, either by block-staining, staining an already embedded block or by post-staining. Further techniques are being developed for selectively staining certain features of the specimen thereby reducing the stain content of the specimen and permitting the examination of very thick specimens at adequate resolution. Specimens having inadequate contrast, particularly preparations of macro-molecules, may be imaged in one of the various dark-field modes, giving a considerable enhancement in image contrast on the viewing screen. Further enhancement of the recorded image contrast can be achieved by exposing the photographic plate to a much higher density than is normal. The complexity of image detail which occurs when the many features of a thick specimen are contracted into a two-dimensional micrograph may be elucidated by stereo-imaging techniques. Care must be taken to optimize the stereo-tilt angles, the orientation of the tilt axis and the register of the two micrographs in the stereo-pair, for the maximum depth in the specimen to be observed.     

A 360° single-axis tilt stage for the high-voltage electron microscope

A new type of specimen stage that permits more than 180° of tilting about the axis of a side-entry rod has been developed for a high-voltage electron microscope (HVEM). Roughly cylindrical specimens, with radial dimensions of less than a few micrometres, that can be mounted on the tip of a microneedle or micropipette are applicable. For glass micropipettes, the energy of the 1-MeV beam of the HVEM is sufficient to image specimens through both walls. The stage employs a spindle mechanism that holds these needles or micropipettes coaxial with the tilt axis, allowing the specimen to be rotated without restriction. This arrangement, along with the cylindrical form of the specimen, is an important development for single-axis tomography, because it permits a complete 180° set of projections to be recorded. The angular accuracy of the stage was demonstrated to be within ±0.20°, with a cumulative error of less than 1.0° over a 180° span. The new stage was tested using puffball spores mounted on a micropipette. A 180° tilt series was recorded and processed to yield a tomographic three-dimensional reconstruction which was displayed both as a cross-sectional view perpendicular to the tilt axis, and as a shaded surface viewed from different directions. The same computations were repeated using subsets of the tilt series to assess the effect of various amounts of missing information. Visual inspection of a selected cross-section from these reconstructions indicated that limiting the angular range to 160° produced results nearly as good as the full data set. Limiting the range to 140°, however, produced a noticeable geometric distortion, which became increasingly severe with ranges of 120° and 100°.