Immunogold detection of types I and II chondrocyte collagen fibrils: an in situ atomic force microscopic investigation

Chondrocyte tissue engineering is a major challenge in the field of cartilage repair. The phenotype of chondrocytes consists of cartilage specific proteoglycan and type II collagen. During serial passages, chondrocytes dedifferentiate into cells, presenting a fibroblast-like phenotype consisting predominately of type I collagen synthesis. Observation of native collagen fibers could be visualized by atomic force microscope. Here, we developed an original and useful atomic force microscopy-based immunogold technique allowing biochemical distinction between types I and II collagen fibers. Imaging of 40-nm gold particles staining collagen fibers was performed in tapping mode. Rat 1 fibroblasts and human chondrosarcoma cells were used as positive models for types I and II collagen, respectively. As demonstrated by our data, primary rat chondrocytes adhering for 48 h on a glass substrate synthesize type II collagen native fibers. This technique allows analyses of local areas of the extracellular matrix of fixed cells, providing complementary data about cartilage phenotype. This simple approach could be of major interest for the biologist community in routine laboratory investigations, to localize in situ, macromolecules of the extracellular matrix.     

Microscopical evaluation of extracellular matrix and its relation to the palatopharyngeal muscle in obstructive sleep apnea

Obstructive sleep apnea hypopnea syndrome (SAHS) is a complex disease of the upper respiratory airways. SAHS physiopathology is multifactorial in which airway compliance is a very important component. To evaluate the tissue changes in the palatopharyngeal muscle by morphometric, histochemical, immunohistochemical, and stereological quantification, with special attention to extracellular matrix associated with this muscle at the structural and ultrastructural levels. Thirty patients with SAHS were divided into groups of 10 according to disease severity: mild, moderate, and severe SAHS. In addition, the control group consisted of 10 patients. Fragments of palatopharyngeal muscle removed from patients with SAHS and tonsillectomies from patients in the control group were histopathologically submitted to light microscopy and transmission electron microscopy. Histopathological evaluations by light and transmission electron microscopes showed differences in analyzed groups, such as reduction of the muscle fiber diameter in patients with SAHS, taking disease severity into consideration. In contrast, stereological analysis showed a gradual increase of the collagen and elastic system fibers relative frequencies, proportionally to SAHS seriousness. MMP‐2 and MMP‐9 immunostaining also showed an increased reaction in the muscle fiber cytoplasm and endomisium during SAHS progression. The ultrastructural analysis showed that palatopharyngeal muscle fibers presented cytoplasmic residual corpuscles, a sign of early cell aging. In conclusion, the increase of tissue compliance in individuals with SAHS can be, in addition to other factors, consequence of diminished contractile activity of the muscle fibers, which exhibited clear signs of early senescence. Moreover, extracellular matrix components changes may contribute to muscle myopathy during SAHS progression. Microsc. Res. Tech., 2011. © 2010 Wiley‐Liss, Inc.     

Hybrid Navigation System Based Autonomous Positioning and Path Planning for Mobile Robots

To improve the quality and efficiency of Z-directional 3D preform forming, the Z-yarn frictional force distribution model of the preform and its wear mechanism were investigated. In this study, a tensile force measuring device was designed to measure the force required to replace the guide sleeve, which is equivalent to the Z-yarn frictional forces. The frictional force is proportional to the number of preform layers and is applied to the preform decreased from the corner, edge, sub-edge, and middle in order. A back propagation neural network model was established to predict the friction at different positions of the preform with different layers, and the error was within 1.9%. The wear of Z-yarn was studied at different frictional positions and after different times of successive implantation into the preform. The results showed that with an increase in the number of Z-yarn implantations and frictional forces, the amount of carbon fiber bundle hairiness gradually increased, and the tensile fracture strength damage of the fiber was increasingly affected by the frictional forces. In the corner position of the preform, when the number of implantations was 25, the fiber fracture strength decreased non-linearly and substantially; in order to avoid fiber fracturing in the implantation process, the Z-yarn needs to be replaced in time after 20–25 cycles of continuous implantation. This study solves the problem of difficulty in measuring the force required for individual replacements owing to the excessive number of guide sleeves, puts forward the relationship between fiber wear, preform position, and implantation times, solves the phenomenon of fracture in the preform during Z-direction fiber implantation, and realizes the continuous implantation of fibers.     

碳纤维/玻璃纤维/石墨协同改性PTFE复合材料力学性能

通过机械混合、冷压和烧结成型制备了碳纤维、玻璃纤维和石墨填充协同改性聚四氟乙烯(PTFE)复合材料。对比分析了不同样品的拉伸、冲击和压缩等力学性能。结果表明:玻纤和碳纤维使复合材料冲击强度下降;玻纤使复合材料拉伸强度下降,碳纤维则使复合材料拉伸强度稍有增强;玻纤和碳纤维均使复合材料压缩强度增加,但碳纤维的增强效果更为明显;石墨、玻纤和碳纤维协同增强PTFE复合材料的拉伸强度较高,弹性模量较大,断裂伸长率较高,抗压缩性能明显提高,且材料拉伸时呈塑性断裂,是综合力学性能较好的高性能润滑密封材料。     

Cytoskeletal response of microvessel endothelial cells to an applied stress force at the submicrometer scale studied by atomic force microscopy

Cytoskeleton fibers form an intricate three-dimensional network to provide structure and function to microvessel endothelial cells. During accommodation to blood flowing, stress fiber bundles become more prominent and align with the direction of blood flow. This network either mechanically resists the applied shear stress (lateral force) or, if deformed, is dynamically remodeled back to a preferred architecture. However, the detailed response of these stress fiber bundles to applied lateral force at submicrometer scales are as yet poorly understood. In our in vitro study, the tip, topography probe in lateral force microscopy of atomic force microscopy, acted as a tool for exerting quantitative vertical and lateral force on the filaments of the cytoskeleton. Moreover, the authors developed a formula to calculate the value of lateral force exerted on every point of the filaments. The results show that cytoskeleton fibers of healthy tight junctions in rat cerebral microvessel endothelial cells formed a cross-type network, and were reinforced and elongated in the direction of scanning under lateral force of 15-42 nN. Under peroxidation (H(2)O(2) of 300 micromol/L), the cytoskeleton remodeled at intercellular junctions, and changed over the meshwork structures into a dense bundle, that redistributed the stress. Once mechanical forces were exerted on an area, the cells shrank and lost morphologic tight junctions. It would be useful in our understanding of certain pathological processes, such as cerebral ischemia/reperfusion injury, which maybe caused by biomechanical forces and which are overlooked in current disease models.     

A dynamic asymmetrical crack model of bridging fiber pull-out in unidirectional composite materials

As a rule, when a crack happens in composite materials, the fibrous system will generate bridging fibers resulted in the asymmetrical extending of the crack. In this paper, a dynamic asymmetrical crack model of bridging fiber pull-out in unidirectional composite materials is built for analyzing the distributions stress and displacement with the internal asymmetrical crack under the loading conditions of an applied non-stress and the traction forces on crack faces yielded by the bridging fiber pull-out model. Thus the fiber failure is determined by the maximum tensile stress, the fiber ruptures, and hence the crack propagation should also occur in self-similar modality. The formulation involves the development of a Riemann-Hilbert problem. The analytical solution of an asymmetrical extension crack in unidirectional composite materials under the conditions of moving increasing loads Pt²/x² and Px²/t is concluded, respectively. Based on relative material properties, the variable law of dynamic stress intensity factors was depicted perfectly. After the conclusion of analytical solutions with the superposition theorem, the solutions of arbitrary complex problems could be acquired.     

3D-C/SiC复合材料的高温拉伸性能

研究了 3D C/SiC复合材料从室温到 15 0 0℃真空条件的拉伸性能。试验材料用T30 0碳纤维编织为三维四向编织体 ,编织角为 2 2° ,用CVI法在 95 0℃～ 10 0 0℃沉积热解碳界面层、SiC基体。最终得到纤维体积分数约为4 0vol%、热解碳界面层厚度约 0 .2 μm和空隙率为 17vol%的复合材料 ,表面SiC涂层厚度为 5 0 μm。试验在超高温拉伸试验机上进行 ,真空度为 10 -3 Pa ,夹头位移速率为 0 .5 95mm/min。结果表明 ,拉伸应力 应变曲线是非线性的 ,大部分拉伸曲线基本由三段折线组成 ,对应着三段模量。第一阶段的模量和基体裂纹饱和应力对应的应变εsa 基本不随温度的升高而改变 ;第二和第三阶段的模量、损伤开始应力σmc、基体裂纹饱和应力σsa、断裂应力σf 和损伤开始应变εmc随温度有相似的变化规律 ,即随温度升高而增加 ,在 110 0℃ ～ 130 0℃范围内出现最大值 ,尔后随温度增加而下降 ;但是断裂应变的变化规律正好与此相反。试样机械加工后 ,由于残余应力部分得到松弛 ,并去除了表面SiC涂层开裂后引起的应力集中 ,因此材料断裂强度和断裂应变明显升高。高温和室温的拉伸断裂应变小于0 .6 % ,不能有效地松弛材料切口处的应力集中。测量了拉伸过程中试样的电阻相对变化率 ,它与载荷的关系曲线总的走势与拉     

Micromechanics analysis of progressive failure in cross-ply carbon fiber/epoxy composite under uniaxial loading

Three-dimensional micromechanics models were created for cross-ply carbon fiber/epoxy composite with a layer stacking-sequence arranged in [0/90]_s. Elasto-plastic finite element (FE) analysis was performed to study the effects of thermal residual stress and the stress redistribution as individual fiber fractures. The modified Rice and Tracey (RT) void growth model was used to predict the location of transverse matrix crack. The stress amplification factors (SAF) in intact fibers adjacent to a fractured fiber were calculated and compared with the planar array composite. The FE results show that small defects have already formed in curing process, and ply-delamination is likely to occur near the comer of free-edges. The transverse matrix crack was predicted to occur near the fiber fracture location in the models having little inter-fiber spacing.     

Differential interference contrast and confocal reflectance imaging of collagen organization in three-dimensional matrices

The remodeling of extracellular matrices by cells plays a defining role in developmental morphogenesis and wound healing as well as in tissue engineering. Three-dimensional (3-D) type I collagen matrices have been used extensively as an in vitro model for studying cell-induced matrix reorganization at the macroscopic level. However, few studies have directly assessed the process of 3-D extracellular matrix (ECM) remodeling at the cellular and subcellular level. In this study, we directly compare two imaging modalities for both quantitative and qualitative imaging of 3-D collagen organization in vitro: differential interference contrast (DIC) and confocal reflectance imaging. The results demonstrate that two-dimensional (2-D) DIC images allow visualization of the same population of collagen fibrils as observed in 2-D confocal reflectance images. Thus, DIC can be used for qualitative assessment of fibril organization, as well as tracking of fibril movement in sequential time-lapse 2-D images. However, we also found that quantitative techniques that can be applied to confocal reflectance images, such as Fourier transform analysis, give different results when applied to DIC images. Furthermore, common techniques used for 3-D visualization and reconstruction of confocal reflectance datasets are not generally applicable to DIC. Overall, obtaining a complete understanding of cell-matrix mechanical interactions will likely require a combination of both wide-field DIC imaging to study rapid changes in ECM deformation which can occur within minutes, and confocal reflectance imaging to assess more gradual changes in cell-induced compaction and alignment of ECM which occur over a longer time course.     

Tribological behavior of metal matrix composites

The wear and friction behavior of continuous graphite fiber reinforced metal matrix composites was investigated. Composite materials were tested against 4620 steel at 54 m s^?1 at room temperature in air without lubricant. The graphite fibers studied included rayon-, pitch- and polyacrilonitrile (PAN)-based fibers. Both high modulus and high strength PAN-based fibers were examined. The fibers were incorporated into copper- and silver-based alloys by means of a liquid metal infiltration technique. The results of this study indicate that the type of graphite fiber in the composite is the most significant factor in the wear and friction behavior of metal matrix composites. In some high modulus fiber tin-bronze composites the fiber fraction influences the wear rate but not the coefficient of friction. Neither the matrix alloy nor the composite tensile strength per se correlate with the friction and wear properties; however, there are specific trends for the various matrix alloys.     

Investigate correlation between mechanical property and aging biomarker in passaged human dermal fibroblasts

Skin aging is associated with changes in both the mechanical properties of the skin and extracellular matrix (ECM) components. In this study, we investigated the relationships between mechanical property and aging biomarkers in passaged human dermal fibroblasts (HDFs). The stiffness of HDFs from passages 5–20 was assessed by atomic force microscopy. The ECM components including collagen, elastin, and fibrillin‐1 and that of signaling molecules (SIRTs) were determined from each passage of cells. The stiffness of HDFs increased linearly from passages 5–15 and then became saturated: the average stiffness was 0.356 N/m at passages 5 and 1.186 N/m at passages 15, respectively. Expression of all aging biomarkers, including pro‐collagen I and VII, elastin, fibrillin‐1, and SIRT1 and SIRT6, were down‐regulated by passaging. In particular, a change in the level of procollagen Type I was significantly associated with early aging, while a change in the level of fibrillin‐1 was associated with late aging. All biomarkers except elastin showed a strong correlation with the cellular stiffness of HDFs. Microsc. Res. Tech. 78:277–282, 2015. © 2015 Wiley Periodicals, Inc.     

Ultimate strength prediction of continuous fiber-reinforced brittle matrix composites

A model to predict ultimate strength of continuous fiber-reinforced brittle matirix composites has been developed. A statistical theory for the strength of the uniaxially fiber-reinforced brittle matrix composite is presented. Material of matrix is assumed to be homogeneous and isotropic, so that the strength of material is anywhere constant, whilst that of fiber is considered to show Weibull statistical distribution. The theory may be utilized to optimize the biaxial and multidirectional tensile strength properties of laminated materials. The composite strength is estimated by assuming no interacting matrix cracks. The frictional shear stress caused by bridging fibers is involved in the strength computation. The predicted strength is compared to experimental results with LAS-Glass/Nicalon fiber composite.     

Investigation of nanostructural changes following acute injury using atomic force microscopy in rabbit vocal folds

There continues to be a paucity of data regarding the nanostructural changes of vocal fold (VF) collagen after injury. The aim of this study is to investigate the nanostructural and morphological changes in the rabbit VF lamina propria following acute injury using atomic force microscopy (AFM). Unilateral VF injury was performed on 9 New Zealand breeder rabbits. Sacrifice and laryngeal harvest were performed at three time points: 1 day, 3 days, and 7 days after injury. Histology and immunohistochemistry data were collected to confirm extracellular matrix (ECM) changes in rabbit VF. The progressive changes in thickness and D‐spacing of VF collagen fibrils were investigated over a 7‐day postinjury period using AFM. At post‐injury day 1, a fibrin clot and inflammatory cell infiltration were observed at the injured VF. The inflammatory score at postinjury day 1 was highest in injured VF tissue, with a significant decrease at postinjury day 7. The immunoreactivity of inflammatory proteins (COX‐2, TNF‐α) was observed in VF up to day 7 after injury. AFM investigation showed clustered and disorganized collagen fibrils at the nanoscale resolution at post‐injury day 7. Collagen fibrils in injured VF at postinjury day 7 were significantly thicker than control and postinjury days 1 and 3 (P < 0.001). D‐spacing of collagen at postinjury day 7 was not studied due to loss of distinct edges resulting from immature collagen deposition. AFM investigation of VF could add valuable information to understanding micromechanical changes in VF scar tissue. Microsc. Res. Tech. 78:569–576, 2015. © 2015 Wiley Periodicals, Inc.     

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.     

涂层刀具高速铣削碳纤维复合材料的铣削力研究

由于碳纤维复合材料(CFRP)的各向异性,纤维的铺层方向对其整体性能有重要的影响。本文采用斜角自由切削方法对具有12种不同纤维方向的T800、T700和T300碳纤维复合材料的切削力进行了试验研究,得出了CFRP单向层合板在不同基体类型和不同纤维方向下切削力的变化规律,并分析了纤维结构对切削力的影响机理。结果表明:基体类型对切削力的影响均匀稳定,无方向性;纤维方向对切削力的影响具有显著的方向性,对切削力影响的强弱关系为F_XF_ZF_Y。     

Ultrastructural features of the myotendinous junction of the sternomastoid muscle in Wistar rats: From newborn to aging

The myotendinous junction (MTJ) is a major area for transmitting force from the skeletal muscle system and acts in joint position and stabilization. This study aimed to use transmission electron microscopy to describe the ultrastructural features of the MTJ of the sternomastoid muscle in Wistar rats from newborn to formation during adulthood and possible changes with aging. Ultrastructural features of the MTJ from the newborn group revealed pattern during development with interactions between muscle cells and extracellular matrix elements with thin folds in the sarcolemma and high cellular activity evidenced through numerous oval mitochondria groupings. The adult group had classical morphological features of the MTJ, with folds in the sarcolemma forming long projections called “finger‐like processes” and sarcoplasmic invaginations. Sarcomeres were aligned in series, showing mitochondria near the Z line in groupings between collagen fiber bundles. The old group had altered “finger‐like processes,” thickened in both levels of sarcoplasmic invaginations and in central connections with the lateral junctions. We conclude that the MTJ undergoes intense activity from newborn to its formation during adulthood. With increasing age, changes to the MTJ were observed in the shapes of the invaginations and “finger‐like processes” due to hypoactivity, potentially compromising force transmission and joint stability. Microsc. Res. Tech. 75:1292–1296, 2012. © 2012 Wiley Periodicals, Inc.     

Influence of Plasma Treatment on Carbon Fabric for Enhancing Abrasive Wear Properties of Polyetherimide Composites

Interfacial adhesion between matrix and fiber plays a crucial role in controlling performance properties of composites. Carbon fibers have major constraint of chemical inertness and hence limited adhesion with the matrix. Surface treatment of fibers is the best solution of the problem. In this work, cold remote nitrogen oxygen plasma (CRNOP) was used for surface treatment. Twill weave carbon fabric (CF) (55–58 vol%) was used with and without plasma treatment with varying content of oxygen (0–1%) in nitrogen plasma to develop composites with Polyetherimide (PEI) matrix. The composites were developed by compression molding and assessed for mechanical and tribological (abrasive wear mode) properties. Improvement in tensile strength, flexural strength, and interlaminar shear strength (ILSS) was observed in composites due to treatment. Similarly, improvement in wear resistance (W _R) and reduction in friction coefficient (μ) were observed in treated fabric composites when slid against silicon carbide (SiC) abrasive paper under varying loads. A correlation between wear resistance and tensile strength was slightly better than that in Lancaster–Ratner plot indicating that ultimate tensile strength (S) and elongation to break (e) were contributing to control the W _R of the composites. It was concluded that enhanced adhesion of fibers with matrix was responsible for improvement in performance properties of composites, as evident from SEM, Fourier Transform Infrared spectroscopy-Attenuated Total Reflectance (FTIR-ATR) technique.     

Diagnostics of Multilayered Composite Structures with the Help of a Fiber-Optic System for Strain Measurements

The paper considers a built-in fiber-optic array system for technical diagnostic of composite structures measuring local strains in hazardous cross sections of these structures. The effect of repeated microscopic bends of optical fibers on the transmitted flux in structures strained by a tensile force of up to 1 kN over a temperature range of 20 to 100°C has been investigated. The increase in the tensile stress in components of a composite structure leads to a growth in the optical losses of up to 20–23% of the initial optical flux. The loss function (F) is linear in the region of mechanical forces of up to 1 kN and temperatures up to 40°C. An explanation has been suggested for the nonlinearity of (F) at temperatures above 40°C. The conclusion is that the developed system enables one to estimate the strength of multilayered composite structures and detect their prefracture states using singular points on opto-mechanical characteristics of optical fibers.     

The process of collagen biomineralization observed by AFM in a model dual membrane diffusion system

Investigation and simulation of naturally occurring mineralization can offer some new ideas in the design and fabrication of new functional materials for bone analogues. In this paper, a model dual membrane diffusion system (DMDS) was used to study the mineralization behaviour of collagen. The process of mineralization was observed by atomic force microscope (AFM). The results showed that the surface roughness and hardness of mineralized collagen fibers increased with time during the process of mineralization. The adhesion force of mineralized collagen fibers decreased with mineralization time. The micromechanical properties and microstructure changes of mineralized collagen fibers suggested that the mineralization was a step-by-step assembling process.     

Biochemical and morphological alterations of the extracellular matrix of chicken calcaneal tendon during maturation

The region in tendons that surrounds bone extremities adapts to compression forces, developing a fibrocartilaginous structure. During maturation, changes occur in the amount and organization of macromolecules of the extracellular matrix of tendons, changing the tissue morphology. To study the effect of maturation on tendons, Pedrês chickens were sacrificed at 1, 5, and 8 months old and had the calcaneal tendon (CT) divided into proximal region, submitted to tension/compression forces ( p ), and distal region submitted to tension force ( d ). Morphological analysis of the p region showed the presence of fibrocartilage in all ages. In the central part of the fibrocartilage, near a diminishment of the metachromasy, there was also a development of a probable fat pad that increased with the maturation. The activity of MMP‐2 and MMP‐9 was higher at 5 and 8 months old, in both regions, compared with 1‐month‐old animals. In SDS‐PAGE analysis, components with electrophoretic migration similar to decorin and fibromodulin increased with maturation, particularly in the d region. The Western blotting confirmed the increased amount of fibromodulin with maturation. In conclusion, our results show that process of maturation leads to the appearance of a probable fat pad in the center of the fibrocartilage of CT, with a reduced amount of glycosaminoglycans and an increased activity of MMPs. Microsc. Res. Tech. 78:949–957, 2015. © 2015 Wiley Periodicals, Inc.