Investigation of micro-cracking behaviors of human femur cortical bone during radial fretting

Radial fretting tests on human femur cortical bone with a ball-on-flat configuration were carried out in vitro under different normal contact loads. The kinetics behaviors and damage characteristics of the cortical bone were analyzed using the load versus displacement (F-D) curves. The fretting scars were examined using laser confocal scanning microscopy (LCSM) and scanning electron microscopy (SEM). A nano-indenter was used to characterize the mechanical property of the osteon of the cortical bone. Morphologies showed that the primary damage form was due to micro-cracking. Around the osteon, three kinds of micro-crack configurations were observed; i.e. annular cracks—located at the concentric lamellae; radial cracks—initiated from the Harversion canal; and interstitial cracks—initiated and propagated between the interstitial lamellae. Plenty of interstitial cracks occurred in the wear scar, which indicated that the interstitial tissue was the weak zone in the structure of cortical bone. In addition, the contact stresses under the radial fretting condition were calculated by the finite element method analysis (FEMs) and the behaviors of cracking were explained. The results demonstrated that the stress concentration that occurred around the Harversian canal and osteon system resulted in a better resistance than that from the interstitial tissue. The concentric lamellae presented a better radial-fretting resistance and contact fatigue resistance than that from the interstitial lamellae.     

The application of heat‐deproteinization to the morphological study of cortical bone: A contribution to the knowledge of the osteonal structure

Observation of heat‐deproteinized cortical bone specimens in incident light enabled the high definition documentation of the osteonal pattern of diaphyseal Haversian bone. This prompted a study to compare these images with those revealed by polarized light microscopy, carried out either on decalcified or thin, undecalcified, resin‐embedded sections. Different bone processing methods can reveal structural aspects of the intercellular matrix, depending on the light diffraction mode: birefringency in decalcified sections can be ascribed to the collagen fibrils orientation alone; in undecalcified sections, to both the ordered layout of collagen and the inorganic phase; in the heat‐deproteinized samples, exclusively to the hydroxyapatite crystals aggregation mode. The elemental chemical analysis documented low content of carbon and hydrogen, no detectable levels of nitrogen and significantly higher content of calcium and phosphorus in heat‐deproteinized samples, as compared with dehydrated controls. In both samples, the X‐ray diffraction (XRD) pattern did not show any significant difference in pattern of hydroxyapatite, with no peaks of any possible decomposition phases. Scanning electron microscopic (SEM) morphology of heat‐deproteinized samples could be documented with the fracturing technique facilitated by the bone brittleness. The structure of crystal aggregates, oriented in parallel and with marks of time periods, was documented. Comparative study of deproteinized and undecalcified samples showed that the matrix inorganic phase did not undergo a coarse grain thermal conversion until it reached 500°C, maintaining the original crystals structure and orientation. Incident light stereomicroscopy, combined with SEM analysis of deproteinized bone fractured surfaces, is a new enforceable technique which can be used in morphometric studies to improve the understanding of the osteonal dynamics. Microsc. Res. Tech. 79:691–699, 2016. © 2016 Wiley Periodicals, Inc.     

High resolution microscopic survey of third metacarpal articular calcified cartilage and subchondral bone in the juvenile horse: possible implications in chondro-osseous disease

The aim was to survey articular calcified cartilage (ACC) and subchondral bone in the palmar and dorsal regions of the condyles of the third metacarpal bone (Mc3) of young horses with minimal or no signs of musculo-skeletal abnormality. Back-scattered electron scanning electron microscopy (BSE SEM) was conducted on polymethyl methacrylate-embedded mediolateral slices and macerated wedges of the right distal Mc3 from seven each of trained and untrained 2-year-old Thoroughbred horses. Furrows or grooves visible to the naked eye in the mineralizing front (MF) of ACC are the commonest "lesion" and are most common in the palmar portions of the medial and lateral condylar grooves. Cracks running predominantly in the parasagittal plane that infill with hypercalcified matrix are found in the same domain. Common to all these defects are deficiencies or absence of the ACC MF. Other anomalies include local excrescences or depressed areas of the MF. More important condylar lesions show displaced fragmented hypermineralized ACC with underlying excess resorption in the bone domain, leaving a thin ACC layer with cavernous space beneath it. The fragments may dislodge and displace to the joint space. Obvious although small lesions are present in horses that have undertaken little or no training. The nature and sites of the lesions indicate that they are possibly the earliest morphological evidence of changes that may lead to specific joint abnormalities. The lesions appear unlikely to be solely due to functional traumatic forces, and developmental influences are likely to be important in their initiation.     

AFM study for morphological characteristics and biomechanical properties of human cataract anterior lens capsules

The aim of this study was to quantitatively investigate the morphologies (surface roughness) and biomechanical properties (Young's modulus) of human anterior lens capsules (ALCs) for noncataract and cataract groups using atomic force microscopy. Eight human ALCs obtained during phacoemulsification from patients with senile cataracts (72 ± 13 years) were investigated in both the hydrated and dehydrated conditions. The cataract group showed clearly the proliferated lens epithelial cells (LECs) with a monomorphic cell structure, a diameter of 12.54 ± 4.31 μm, and a height of 0.23 ± 0.04 μm, whereas the control group showed no LECs. A substantial amount of false-positive calcification was observed caused by the deposition of remnants of dried salt solution. Cataract group showed significantly higher surface roughness (382.06 nm, p ≤ 0.001) than control group in the anterior side of ALCs, whereas cataract group showed significantly lower surface roughness (353.79 nm, p ≤ 0.001) than control group in their posterior side. Cataract group showed significantly higher Young's modulus (69.52 kPa, p ≤ 0.001) compared to the control group, regardless of the ALC side. Therefore, it is significant that this study provides a new method to examine the nanostructural characteristic and biomechanical property of human ALCs through a nanometer-scale resolution microscopy technique.     

冷轧钢压印接头拉伸-剪切和疲劳性能研究

对冷轧钢圆形连接点和矩形连接点的压印接头进行拉伸-剪切试验,研究连接点形状对接头拉伸-剪切性能的影响.并对圆形连接点压印接头进行疲劳试验.试验结果表明,两种接头拉伸-剪切强度和刚度相当.圆形连接点拉伸-剪切过程中的能量吸收值大于矩形连接点,矩形连接点的失效形式为上板拉脱失效.应力比R=0.5,当最大疲劳载荷为接头强度的95％时,接头循环寿命可以达到137万次,为80％时,可以达到疲劳极限500万次.疲劳失效形式为上板接头处产生裂纹,裂纹方向与加载方向大致呈90°.提出了圆形点压印接头的拉伸-剪切强度预测公式和疲劳寿命计算公式,拉伸-剪切强度公式的误差为6.9％.     

Viscometric properties of a multigrade motor oil in relation to its bright-stock content

Viscometric properties for a multigrade motor oil with SAE 20W/50 viscosity grade were studied in the presence of different viscosity index improvers. The neat base blends contained increasing percentages of a bright-stock. Measured viscometric properties for both the neat and VI improved blends included the following: kinematic viscosity, viscosity increase by incorporation of a VI improver, viscosity index, specific viscosity and thickening tendency. These measurements were carried out over a wide temperature range. Increase of the bright-stock content showed that most of the VI improvers behave as thickeners. Polyalkylmethacrylate and styrene-isoprene copolymers were found to be the only exception from such behaviour for the VI improvers.     

Development of preparation methods for and insights obtained from atomic force microscopy of fluid spaces in cortical bone

Several preparation methods were developed to investigate the dimensions and surface structure of fluid spaces within cortical bone, using atomic force microscopy (AFM). Of special interest was the morphology of the lacunocanalicular system, which serves as a conduit between osteocytes encased in bone tissue, the intramedullary cavity, blood vessels running through the bone, and the periosteal surface of bone. Fracture and the removal of either the mineral or the organic component is a method by which each component can be investigated at a very high resolution in situ. Although fractured bone was too rough to image details of the lacunocanalicular system, post-treatment with ethylene diamine tetraacetic acid (EDTA) or papain allowed for investigation of the collagen matrix or the mineral crystals of bone, respectively. Cut and polished bone was smooth enough for identifying the lacunae of bone using AFM, but unambiguous differentiation between the canaliculi and cracks in the bone surface was not possible. However, when the lacunocanalicular system was filled with polymethylmethacrylate (PMMA), it was possible to image casts of the lacunocanalicular system by selectively etching away the surrounding bone matrix. Using this method, we identified individual canaliculi and measured their dimensions. Furthermore, by carefully etching away the bone matrix in successive etches, it was shown that the wall structure of the canaliculus is dominated by collagen fibrils. These observations have important implications for fluid flow in bone.     

Morphological analysis of pulps from orange tree trimmings and its relation to mechanical properties

To optimize the pulping and refining processes of new alternative raw materials for papermaking, researchers generally perform tests that consume considerable time and large amounts of sample. We propose measuring the morphological properties of pulps from orange tree trimmings by image analysis systems, which are fast and user-friendly, to develop models relating key mechanical properties to the dimensions, the deformation and the population of fibers. Data modeling involves multiple linear regression, as used in other studies, and support vector regression, not used before for this purpose, achieving higher R² values (up to 0.90). Although tensile, tear and burst tests are still required to obtain accurate values, a quick morphological characterization allows for a rough but satisfactory prediction of paper strength. In this case, chemical pulping and moderate refining are shown to be necessary to obtain pulps of acceptable quality from orange tree trimmings.     

An investigation of segmentation methods and texture analysis applied to tomographic images of human vertebral cancellous bone

The goal of this study is to determine architectural and textural parameters on computed tomographic (CT) images, allowing us to explain the mechanical compressive properties of bone. Although the resolution (150 μm) is of the same order of magnitude as the trabecular thickness, this method enables the possibility of perfecting an in vivo peripheral CT system with an acceptable radiation dose for the patient. This study was performed on L2 vertebrae cancellous bone specimens taken after necropsy in 22 subjects aged 47–95 years (mean: 79 years). The segmentation process is a crucial point in the determination of accurate architectural parameters. In this paper the use of two different segmentation methods is investigated, based on an edge enhancement and a region growing approach. The images are compared and the architectural parameters extracted from the images segmented by both methods lead to a quantitative evaluation. The parameters are found to be globally robust towards the segmentation process, although some of them are much more sensitive to the approach used. Highly significant correlations ( P  < 0.0005) have been obtained between the two segmentation methods for all the parameters, with ρ ranging from 0.70 to 0.93. In order to improve the assessment of bone architecture, texture analysis (run length method) was investigated. New features are obtained from an image reduced to 16 grey-levels. Textural parameters in addition to architectural parameters in a multivariate regression model increase significantly ( P  = 0.01) the prediction of the maximum compressive strength (variation of r ² from 0.75 up to 0.89).     

Ultrastructural analysis of human bone marrow mesenchymal stem cells during in vitro osteogenesis and chondrogenesis

The main purpose of this article was to describe the morphology of mesenchymal stem cells (MSCs) differentiated in vitro towards osteogenic and chondrogenic lineages and to focus on the ultrastructural features associated with these processes. Human mononuclear cells (hMNC) were isolated, expanded, and analyzed for the expression of specific cell surface markers to demonstrate their stem cell characteristics. Human mononuclear cells were differentiated in vitro in an osteogenic and in a chondrogenic sense for 7, 14, 21, and 28 days. Subsequently, they were processed using electron microscopic analysis (FEISEM). Alizarin red and alcian blue staining were carried out to demonstrate the deposition of mineral salts and proteoglycans in the extracellular matrix. Undifferentiated MSCs showed a cell surface covered by filopodia and ondulopodia. During differentiation, the MSCs changed their shape from a round to a fibroblastic-like shape. At the end of the differentiation, several filaments with a parallel orientation in the osteogenic samples as well as a network organization in the chondrogenic samples were detected in the extracellular spaces. This study demonstrated that there are morphological features associated with the undifferentiated and differentiated states of the MSCs, which could be utilized as new parameters for identifying and classifying these cells.     

Spatial and temporal changes of subchondral bone proceed to articular cartilage degeneration in rats subjected to knee immobilization

This study was aimed to investigate the spatial and temporal changes of subchondral bone and its overlying articular cartilage in rats following knee immobilization. A total of 36 male Wistar rats (11–13 months old) were assigned randomly and evenly into 3 groups. For each group, knee joints in 6 rats were immobilized unilaterally for 1, 4, or 8 weeks, respectively, while the remaining rats were allowed free activity and served as external control groups. For each animal, femurs at both sides were dissected after sacrificed. The distal part of femur was examined by micro‐CT. Subsequently, femoral condyles were collected for further histological observation and analysis. For articular cartilage, significant changes were observed only at 4 and 8 weeks of immobilization. The thickness of articular cartilage and chondrocytes numbers decreased with time. However, significant changes in subchondral bone were defined by micro‐CT following immobilization in a time‐dependent manner. Immobilization led to a thinner and more porous subchondral bone plate, as well as a reduction in trabecular thickness and separation with a more rod‐like architecture. Changes in subchondral bone occurred earlier than in articular cartilage. More importantly, immobilization‐induced changes in subchondral bone may contribute, at least partially, to changes in its overlying articular cartilage. Microsc. Res. Tech. 79:209–218, 2016. © 2016 Wiley Periodicals, Inc.     

Surface strength of silicon nitride in relation to rolling contact performance measured on ball-on-rod and modified four-ball tests

Silicon nitride (Si₃N₄) has been used in various rolling contact applications in turbomachinery, automotive and power industry. It is favoured to replace conventional steel due to its low density, low friction, corrosion resistance and good performance under extreme condition. However, a major limitation of its wider application is its high material and machining cost, especially the cost associated with the finishing process. In the present study, a low cost sintered and reaction bonded silicon nitride (SRBSN) is used to study the surface machining effects on its rolling contact performance. Attempt has been made to link the surface strengths of Si₃N₄ derived from half-rod and C-sphere flexure strength specimens to the rolling contact lifetimes of Si₃N₄ rod and ball specimens. The rolling contact fatigue tests are carried out on ball-on-rod and modified four ball machines. Three types of surfaces with coarse, fine and conventional finishing conditions are examined. Flexure strength tests on half-rod and C-sphere show an increasing surface strength from specimens with coarse, fine to conventionally machined conditions. During rolling contact fatigue test of as-machined specimens, there are no failures observed on both ball-on-rod and four ball tests after 100 million stress cycles. However, there is a trend of decreasing wear volumes measured on the contact path of rods and balls with coarse, fine and conventional conditions. In four ball test, spall failures are observed on pre-crack specimens. There is a trend of increasing rolling contact fatigue lifetime from pre-cracked specimens with coarse, fine to conventional machining conditions.     

SNOM and AFM microscopy techniques to study the effect of non-ionizing radiation on the morphological and biochemical properties of human keratinocytes cell line (HaCaT)

In this study we have employed atomic force microscopy (AFM) and scanning near‐field optical microscopy (SNOM) techniques to study the effect of the interaction between human keratinocytes (HaCaT) and electromagnetic fields at low frequency. HaCaT cells were exposed to a sinusoidal magnetic field at a density of 50 Hz, 1 mT. AFM analysis revealed modification in shape and morphology in exposed cells with an increase in the areas of adhesion between cells. This latter finding was confirmed by SNOM indirect immunofluorescence analysis performed with a fluorescent antibody against the adhesion marker β4 integrin, which revealed an increase of β4 integrin segregation in the cell membrane of 50‐Hz exposed cells, suggesting that a higher percentage of these cells shows a modified pattern of this adhesion marker.     

Application of the EBSD technique to describe the initiation and growth behaviour of microstructurally short fatigue cracks in a duplex steel

Up to 90% of the fatigue life of engineering alloys results from the initiation and propagation of microstructurally short cracks. Owing to their strong interactions with microstructural features, e.g. grain and phase boundaries, they exhibit substantially non‐uniform propagation kinetics as compared with the growth rate of long cracks, which can be well described using a power‐law function of the range of the stress‐intensity factor ΔK. In the present paper interactions between the crystallographic misorientation of grain and phase boundaries and microcracks in an austenitic/ferritic stainless steel are discussed and quantified by means of fatigue experiments in combination with the electron backscattered diffraction technique. In the second part a numerical model for the simulation of microcracks is introduced, which is capable of taking real microstructural arrangements into consideration.     

Influence of low-level laser associated with osteogenic proteins recombinant human BMP-2 and Hevea brasiliensis on bone repair in Wistar rats

This study analyzed the newly formed bone tissue after application of recombinant human BMP-2 (rhBMP-2) and P-1 (extracted from Hevea brasiliensis) proteins, 2 weeks after the creation of a critical bone defect in male Wistar rats treated or not with a low-intensity laser (GaAlAs 780 nm, 60 mW of power, and energy density dose of 30 J/cm(2)). The animals were divided into two major groups: (1) bone defect plus low-intensity laser treatment and (2) bone defect without laser irradiation. The following subgroups were also analyzed: (a) 5 μg of pure rhBMP-2; (b) 5 μg of pure P-1 fraction; (c) 5 μg of rhBMP-2/monoolein gel; (d) 5 μg of P-1 fraction/monoolein gel; (e) pure monoolein gel. Comparisons of the groups receiving laser treatment with those that did not receive laser irradiation show differences in the areas of new bone tissue. The group treated with 5 μg of rhBMP-2 and laser irradiation was not significantly different (P >0.05) than the nonirradiated group that received the same treatment. The irradiated, rhBMP-2/monoolein gel treatment group showed a lower area of bone formation than the nonirradiated, rhBMP-2/gel monoolein treatment group (P < 0.001). The area of new bone tissue in the other nonirradiated and irradiated groups was not significantly different (P > 0.05). Furthermore, the group that received the 5 μg of rhBMP-2 application showed the greatest bone formation. We conclude that the laser treatment did not interfere with the area of new bone tissue growth and that the greatest stimulus for bone formation involved application of the rhBMP-2 protein.     

Microstructural evolution of Cu-1 at% Ti alloy aged in a hydrogen atmosphere and its relation with the electrical conductivity

Copper alloys with titanium additions between 1 and 6 at% Ti emerge currently as attractive conductive materials for electrical and electronic commercial products, since they exhibit superior mechanical and electrical properties. However, their electrical conductivity is reduced owing to the residual amount of Ti solutes in the Cu solid solution (Cu_ss) phase. Since Cu shows only poor reactivity with hydrogen (H), while Ti exhibits high affinity to it, we were inspired by the idea that hydrogenation of Cu–Ti alloys would influence their microstructure, resulting in a significant change of their properties. In this contribution, the influence of aging under a deuterium (D₂) atmosphere of Cu-1 at% Ti alloys on their microstructure is investigated to explore the effects on the electrical conductivity. The specimens were investigated by means of transmission electron microscopy (TEM), field ion microscopy (FIM), computer-aided field ion image tomography (cFIIT), and atom probe tomography (APT).     

Trabecular bone microarchitecture is related to the number of risk factors and etiology in osteoporotic men

Microarchitecture of trabecular bone is a very important component of bone quality in osteoporosis and a determinant of vertebral fracture in men with low bone mineral density (BMD). In contrast to women, male osteoporosis is, in most cases, secondary. The relationships between microarchitecture and different risk factors have never been evaluated in men. About 152 men with low BMD at the lumbar spine or hip (BMD, T-score < -2.5) were included in this study. Risk factors were: age, BMI, alcohol intake, corticosteroid therapy, hypogonadism, and chronic diseases. Transiliac bone biopsies were obtained and histomorphometry was done on an image analyzer; the following parameters were measured: cortical thickness (Ct.Th), trabecular bone volume (BV/TV), trabecular thickness (Tb.Th), separation (Tb.Sp) and number (Tb.N), interconnectivity Index (ICI), star volume of the bone marrow, and strut analysis with node and free-end count. The 50 men with two risk factors had a lower BMD, lower Ct.Th and a significant higher star volume than those with one factor or idiopathic osteoporosis. The 26 men with at least three risk factors, had a lower BMD, a reduction of BV/TV and Ct.Th and a marked disorganization of the trabecular network (increased Tb.Sp, ICI, star volume, and free-end to free-end struts). The prevalence of vertebral fractures was higher in these patients. When the main risk factor was considered, a marked decrease in trabecular bone connectivity was observed in hypogonadic men. In osteoporotic men, higher the number of risk factors, lower the connectivity of trabecular network and higher the vertebral fracture risk.     

Long bone human anlage longitudinal and circumferential growth in the fetal period and comparison with the growth plate cartilage of the postnatal age

The patterns of longitudinal and peripheral growth were analyzed in human autopod cartilage anlagen (fetal developmental stage 20th–22nd week) through morphometric assessment of chondrocyte parameter size, shape, alignment and orientation between peripheral and central sectors of the anlage transition zone defined by primary ossification center and the epiphyseal basis. The aim was to correlate the chondrocyte dynamics with the longitudinal and peripheral growth. A further comparison was carried out between the corresponding sectors of the postnatal (3–5 months old) growth plate cartilage documenting: (1) the different chondrocyte framework and the new peripheral mechanism; (2) the opposite direction of fetal periosteal ossification versus the Lacroix bone bark. Measurement of multiple parameters (% lac area, % total matrix area, total lac density and mean single lac area), which characterize the cartilage Anlage growth, suggested the following correlations with chondrocyte duplication rate: (a) slow duplication rate ≈ coupled, intralacunar chondrocytes (in central epiphysis); (b) repeated/frequent cell duplications ≈ clusters (in the basal epiphyseal layer); (c) clusters of chondrocytes before becoming hypertrophic were stacked up on the top of each other (both in the Anlage transition zone or in the columns of metaphyseal growth plate); (d) enhanced osteoclastic resorption of the Lacroix bone bark lower end, extended to the more external metaphyseal trabeculae counterbalancing the discrepancy between the epiphyseal and the diaphyseal circumferential growth.     

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.