Quantitative zonal differentiation of articular cartilage by microscopic magnetic resonance imaging,polarized light microscopy,and Fourier‐transform infrared imaging

This study aimed to synchronize the zonal differentiation of the full‐thickness articular cartilage by three micro‐imaging techniques, namely microscopic magnetic resonance imaging (µMRI), polarized light microscopy (PLM), and Fourier‐transform infrared imaging (FTIRI). Eighteen cartilage‐bone blocks from three canine humeral joints were imaged by: (a) µMRI T₂ relaxation at 0° and 55° orientations in a 7 T magnetic field, (b) PLM optical retardation and azimuthal angle, and (c) FTIRI amide I and amide II anisotropies at 0° and 90° polarizations relative to the articular surface. In addition, µMRI T₁ relaxation was imaged before and after the tissue being immersed in gadolinium (contrast agent) solution, to calculate the proteoglycan concentration. A set of previously established criteria in cartilage imaging was revised. The new criteria could simultaneously correlate the thicknesses of the three consecutive subtissue zones in articular cartilage among these imaging techniques. Microsc. Res. Tech. 76:625–632, 2013. © 2013 Wiley Periodicals, Inc.     

The interface region between articular cartilage and bone by μMRI and PLM at microscopic resolutions

This dual-modality microscopic imaging study quantifies the interface region between the noncalcified cartilage and the subchondral bone plate, which includes the deep portion of the noncalcified articular cartilage and the zone of calcified cartilage (ZCC). This interface region is typically not visible in routine MRI but becomes visible in MRI with the application of an ultra-short echo time (UTE) sequence. A number of cartilage-bone blocks from a well-documented canine humeral head were harvested for imaging by microscopic MRI (μMRI) and PLM (polarized light microscopy). In μMRI, T2 anisotropic images were acquired by 2D gradient-echo, magnetization-prepared spin-echo and UTE sequences at the 0° and 55° (the magic angle) orientations at 11.7 μm/pixel resolution. In PLM, quantitative optical retardation (nm) and collagen orientation (°) were mapped from the thin sections from the same μMRI specimens at 0.5–2 μm pixel resolutions. The orientational and organizational architecture of the collagen matrix in this interface region was quantified and correlated between the complementary imaging. The magic angle effect as seen in the noncalcified cartilage was statistically confirmed in ZCC in μMRI, which was further supported by quantitative PLM. With an enhanced understanding of the tissue properties in this important interface region, it will potentially be possible to monitor the changes of this tissue region which is instrumental to the initiation and development of osteoarthritis and other joint diseases.     

Utilizing confocal microscopy to measure refractive index of articular cartilage

This study proposes a method for measuring the refractive index of articular cartilage within a thin and small specimen slice. The cartilage specimen, with a thickness of about 50 μm, was put next to a thin film of immersion oil of similar thickness. Both the articular cartilage and immersion oil were scanned along the depth direction using a confocal microscope. The refractive index mismatch between the cartilage and the immersion oil induced a slight axial deformation in the confocal images of the cartilage specimen that was accurately measured by a subpixel edge‐detection‐based technique. A theoretical model was built to quantify the focal shift of confocal microscopy caused by the refractive index mismatch. With the quantitative deformations of cartilage images and the quantified function of focal shift, the refractive index of articular cartilage was accurately interpolated. At 561 nm, 0.1 MPa and 20 °C, the overall refractive index of the six cartilage plugs was 1.3975 ± 0.0156. The overall coefficient of variation of all cartilage specimens was 0.68%, which indicated the high repeatability of our method. The verification experiments using distilled water showed a minimal relative error of 0.02%.     

The Annealing Behavior of the Subsurface Zone Induced by Friction in Bismuth Detected by Positron Lifetime Technique

The annealing behavior of the subsurface zone (SZ) in pure bismuth induced by dry sliding was studied using the positron lifetime measurement. This measurement allows us to detect the SZ and its recovery, and recrystallization processes. The comparative measurements of the sample exposed to compression revealed the thermal stability of the SZ. The compressed sample rebuilt its structure due to the recovery and recrystallization processes at the temperature of 60 °C, whereas the sample exposed to dry sliding does it at higher temperature of 260 °C, which is close to the melting point. The isothermal annealing at the temperature of 100 °C confirmed these results. The defect depth profile induced by dry sliding evolves with the annealing temperature in such a way that the concentration of defects at the worn surface gradually decreases, but at the depth between 50 and 170 μm, the generation of new defects takes place at the temperature of 75, 100 and even at 175 °C. At the temperature of 175 °C, the defects still are extended up to the depth of about 60 μm from the worn surface. The results were qualitatively confirmed by the measurements of the Vickers microhardness depth profile. Similar annealing behavior of the SZ was observed in pure magnesium.     

Application of autofluorescence robotic histology for quantitative evaluation of the 3‐dimensional morphology of murine articular cartilage

Murine models of osteoarthritis (OA) are increasingly important for understating pathogenesis and for testing new therapeutic approaches. Their translational potential is, however, limited by the reduced size of mouse limbs which requires a much higher resolution to evaluate their articular cartilage compared to clinical imaging tools. In experimental models, this tissue has been predominantly assessed by time‐consuming histopathology using standardized semi‐quantitative scoring systems. This study aimed to develop a novel imaging method for 3‐dimensional (3D) histology of mouse articular cartilage, using a robotic system—termed here “3D histocutter”—which automatically sections tissue samples and serially acquires fluorescence microscopy images of each section. Tibiae dissected from C57Bl/6 mice, either naïve or OA‐induced by surgical destabilization of the medial meniscus (DMM), were imaged using the 3D histocutter by exploiting tissue autofluorescence. Accuracy of 3D imaging was validated by ex vivo contrast‐enhanced micro‐CT and sensitivity to lesion detection compared with conventional histology. Reconstructions of tibiae obtained from 3D histocutter serial sections showed an excellent agreement with contrast‐enhanced micro‐CT reconstructions. Furthermore, osteoarthritic features, including articular cartilage loss and osteophytes, were also visualized. An in‐house developed software allowed to automatically evaluate articular cartilage morphology, eliminating the subjectivity associated to semi‐quantitative scoring and considerably increasing analysis throughput. The novelty of this methodology is, not only the increased throughput in imaging and evaluating mouse articular cartilage morphology starting from conventionally embedded samples, but also the ability to add the third dimension to conventional histomorphometry which might be useful to improve disease assessment in the model.     

Application of design‐based stereology for estimation of absolute volume and surface area of the articular and calcified cartilage compartments of undecalcified human femoral heads

Design‐based stereological methods using systematic uniform random sampling, the Cavalieri estimator and vertical sections are used to investigate undecalcified human femoral heads. Ten entire human femoral heads, obtained from normal women and normal men, were systematically sampled and thin undecalcified vertical sections were obtained. Absolute volumes and surface areas of the entire femoral head, the articular cartilage and the calcified cartilage compartments were estimated. In addition, the average thickness of the articular cartilage and the calcified cartilage were calculated. The stereological procedures applied to the human femoral heads resulted in average coefficient of errors, which were 0.03–0.06 for the volume estimates and 0.03–0.04 for the surface area estimates. We conclude that design‐based stereology using the Cavalieri estimator and vertical sections can successfully be used in large undecalcified tissue specimens, like the human femoral head, to estimate the absolute volume and surface area of macroscopic as well as of microscopic tissue compartments. The application of well‐known design‐based stereological methods carries potential advantage for investigating the pathology in inflammatory and degenerative joint diseases.     

Positron Studies of Subsurface Zone Created in Sliding Wear in Bismuth

The report about subsurface zone (SZ) created during dry sliding in pure bismuth is presented. The positron lifetime measurements revealed the well defined subsurface zone where the concentration of created during sliding vacancy clusters decreases exponentially with the depth from the worn surface increases. The experimental results are quite well described by the two state trapping model which allows to determine the positron trapping rate and hence deduce about the depth dependency of vacancy voids concentration. Despite of the low recrystallization temperature ca. 115 °C in this semimetal, determined also by the positron lifetime measurements, no significant effect of temperature rise due to sliding on the SZ constitution is observed.     

Design-based estimation of surface area in thick tissue sections of arbitrary orientation using virtual cycloids

Surface area is a first‐order stereological parameter with important biological applications, particularly at the intersection of biological phases. To deal with the inherent anisotropy of biological surfaces, state‐of‐the‐art design‐based methods require tissue rotation around at least one axis prior to sectioning. This paper describes the use of virtual cycloids for surface area estimation of objects and regions in thick, transparent tissue sections cut at any arbitrary (convenient) orientation. Based on the vertical section approach of Baddeley et al., the present approach specifies the vertical axis as the direction of sectioning (i.e. the direction perpendicular to the tissue section), and applies computer‐generated cycloids (virtual cycloids) with their minor axis parallel to the vertical axis. The number of surface‐cycloid intersections counted on focal planes scanned through the z‐axis is proportional to the surface area of interest in the tissue, with no further assumptions about size, shape or orientation. Optimal efficiency at each x–y location can be achieved by three virtual cycloids orientated with their major axes (which are parallel to the observation planes) mutually at an angle of 120°. The major practical advantage of the present approach is that estimates of total surface area (S) and surface density (S_V) can be obtained in tissue sections cut at any convenient orientation through the reference space.     

Mechanical anisotropy of the human knee articular cartilage in compression

Articular cartilage exhibits anisotropic mechanical properties when subjected to tension. However, mechanical anisotropy of mature cartilage in compression is poorly known. In this study, both confined and unconfined compression tests of cylindrical cartilage discs, taken from the adult human patello-femoral groove and cut either perpendicular (normal disc) or parallel (tangential disc) to the articular surface, were utilized to determine possible anisotropy in Young's modulus, E, aggregate modulus, Ha, Poisson's ratio, v and hydraulic permeability, k, of articular cartilage. The results indicated that Ha was significantly higher in the direction parallel to the articular surface as compared with the direction perpendicular to the surface (Ha = 1.237 +/- 0.486 MPa versus Ha = 0.845 +/- 0.383 MPa, p = 0.017, n = 10). The values of Poisson's ratio were similar, 0.158 +/- 0.148 for normal discs compared with 0.180 +/- 0.046 for tangential discs. Analysis using the linear biphasic model revealed that the decrease of permeability during the offset compression of 0-20 per cent was higher (p = 0.015, n = 10) in normal (from 25.5 x 10(-15) to 1.8 x 10(-15) m4/N s) than in tangential (from 12.3 x 10(-15) to 1.3 x 10(-15) m4/N s) discs. Based on the results, it is concluded that the mechanical characteristics of adult femoral groove articular cartilage are anisotropic also during compression. Anisotropy during compression may be essential for normal cartilage function. This property has to be considered when developing advanced theoretical models for cartilage biomechanics.     

Direct visualization of receptor arrays in frozen-hydrated sections and plunge-frozen specimens of E. coli engineered to overproduce the chemotaxis receptor Tsr

We have recently reported electron tomographic studies of sections obtained from chemically fixed E. coli cells overproducing the 60‐kDa chemotaxis receptor Tsr. Membrane extracts from these cells prepared in the presence of Tween‐80 display hexagonally close‐packed microcrystalline assemblies of Tsr, with a repeating unit large enough to accommodate six Tsr molecules arranged as trimers of receptor dimers. Here, we report the direct visualization of the Tsr receptor clusters in (i) vitrified cell suspensions of cells overproducing Tsr, prepared by rapid plunge‐freezing, and (ii) frozen‐hydrated sections obtained from cells frozen under high pressure. The frozen‐hydrated sections were generated by sectioning at ?150 °C using a diamond knife with a 25° knife angle, with nominal thicknesses ranging from 20 to 60 nm. There is excellent correspondence between the spatial arrangement of receptors in thin frozen‐hydrated sections and the arrangements found in negatively stained membrane extracts and plunge‐frozen cells, highlighting the potential of using frozen‐hydrated sections for the study of macromolecular assemblies within cells under near‐native conditions.     

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.     

Non‐Contact Evaluation of Osmosis‐Induced Shrinkage and Swelling Behavior of Articular Cartilage In‐Situ Using High‐Frequency Ultrasound

Abstract

The aim of the present study was to use high‐frequency ultrasound for the investigation of the transient osmosis‐induced free shrinkage‐swelling behaviors of normal articular cartilage in situ. Full‐thickness cartilage‐bone specimens were prepared from normal bovine patellae. The transient shrinkage and swelling strains of the cartilage induced by changing the bathing solution between physiological saline (0.15 M) and hypertonic saline (2 M) were monitored using a 50 MHz focused ultrasound beam. Both shrinkage and swelling strains showed temporary overshoots, followed by relaxation phases. The absolute peak value of the shrinkage strain (1.01%±0.62%) was significantly larger (p<0.05) than that of the swelling strain (0.40%±0.33%). It was found that the change of the mean ultrasound speed in cartilage could be approximately represented by an exponential function of time after the change of saline concentration.

This study successfully demonstrated that a high‐frequency focused ultrasound beam could be used to monitor the transient osmosis‐induced deformation of articular cartilage in a non‐contact way. Since the osmosis‐swelling behavior of cartilage relates to its compositional and structural characteristics and degeneration status, the reported ultrasound method may have potential for the characterization of cartilage degeneration, such as osteoarthritis.     

Alteration of cartilage matrix morphology with histological processing

An interlacunar network in the extracellular matrix of femoral head articular cartilage of neontal rats was seen by light microscopy to: (1) consist of elements, 0·5 μm thick, which occurred as individual elements, as bundles of elements, and as fused elements, (2) stain intensely with toluidine blue, methylene blue, and safranin O, and (3) connect chondrocytes by inserting on the chondrocyte capsules which were composed of morphologically and cytochemically similar material. By electron microscopy, the single elements were seen to be composed of thicker, denser staining areas of the honeycomb appearing matrix and the fused elements appeared as non-membrane bound channels containing granular material. Articular cartilage was processed using combinations of fixatives, dehydrating agents, and embedding media. Regardless of fixation, demineralization, or embedding, the network was not seen after dehydration of the cartilage with methanol, ethanol, acetone or tert-butanol but was seen after dehydration with aqueous solutions of glycol methacrylate, propylene oxide, 2-propanol or 2,2-dimethoxypropane. Network visualization following a variety of methods demonstrated that no single fixative, dehydrating agent, or embedding medium caused its formation. The presence of the network in different cartilage zones, its consistent morphology by light and electron microscopy, the uniformity of the elements in their connection with the chondrocytes, and presence in fresh-frozen sections suggest the network may be real, but rigorous evidence for its existence in vivo is still required. Since cartilage morphology was altered by histological methods, especially dehydration, common methods used in studying connective tissue matrix should be evaluated to determine their effect on matrix morphology.     

Application of combined EBSD and 3D‐SEM technique on crystallographic facet analysis of steel at low temperature

Electron backscatter diffraction has been increasingly used to identify the crystallographic planes and orientation of cleavage facets with respect to the rolling direction in fracture surfaces. The crystallographic indices of cleavage planes can be determined either directly from the fracture surface or indirectly from metallographic sections perpendicular to the plane of the fracture surface. A combination of electron backscatter diffraction and 3D scanning electron microscopy imaging technique has been modified to determine crystallographic facet orientations. The main purpose of this work has been to identify the macroscopic crystallographic orientations of cleavage facets in the fracture surfaces of weld heat affected zones in a well‐known steel fractured at low temperatures. The material used for the work was an American Petroleum Institute (API) X80 grade steel developed for applications at low temperatures, and typical heat affected zone microstructures were obtained by carrying out weld thermal simulation. The fracture toughness was measured at different temperatures (0°C, ?30°C, ?60°C and ?90°C) by using Crack Tip Opening Displacement testing. Fracture surfaces and changes in microstructure were analyzed by scanning electron microscopy and light microscopy. Crystallographic orientations were identified by electron backscatter diffraction, indirectly from a polished section perpendicular to the major fracture surface of the samples. Computer assisted 3D imaging was used to measure the angles between the cleavage facets and the adjacent polished surface, and then these angles were combined with electron backscatter diffraction measurements to determine the macroscopic crystallographic planes of the facets. The crystallographic indices of the macroscopic cleavage facet planes were identified to be {100}, {110}, {211} and {310} at all temperatures.     

A novel double‐image Fizeau system for accurate investigation of anisotropic polymer fibres

This paper introduces a double‐image multiple‐beam Fizeau fringes system. The introduced system can dynamically determine the variations of the refractive indices for both parallel and perpendicular polarization simultaneously. This is achieved by the simultaneous capturing of two multiple‐beam interference patterns during the mechanical processing of isotactic polypropylene fibre. This parallel determination of the refractive indices of both polarization directions allowed us to determine the full‐field distribution of the stress vector, S . To accomplish this, a mathematical model was deduced to calculate the components of the stress vector, S , i.e. parallel stress component, S₁, and perpendicular stress component, S₂. Double‐image Fizeau fringes system and the deduced mathematical model were used to investigate the variation of the refractive index and stress components of the fibre during the stretching process and propagation of necked regions.     

Glycol methacrylate embedding of bone and cartilage for light microscopic staining

A method is described for embedding bone and cartilage in glycol methacrylate (GMA) for light microscopy. Dehydration-infiltration of the hard tissue is with aqueous GMA solutions minimizing solvent and dehydration artefact, and polymerization is by UV light in the cold to minimize thermal damage. Over fifty stains, enzyme localizations and related histochemical methods for 0·5–3·0 μm thick sections of GMA embedded tissue are listed. The increased resolution plus the localization of cellular and extracellular chemical moieties is now easier and more accurate providing an improved method for the study of the musculo-skeletal system by light microscopic histochemistry.     

Studies on insect fibrous proteins: the structural protein of the ootheca in the praying mantis, Sphodromatis centralis Rehn

Histological studies have been made on the left colleterial gland which secretes the α-type structural protein in the ootheca of Sphodromantis centralis. Three distinct types of secretory cell are present in the gland wall, and each has a complex apical ‘end-apparatus’ composed of closely packed canalicules radiating from a central cavity. Structural protein is synthesized in the cytoplasm, passed through the canalicules into the cavity of the end-apparatus, and extruded into the lumen as strongly birefringent, apparently membrane-free globules. At this stage the globules consist of numerous concentric layers of long, parallel fibrils, 0·05–0·1 μm in diameter. In each successive layer the fibril direction is rotated through an angle of about 18° about an axis perpendicular to the layers, in the manner of cholesteric phase systems. When sectioned, the globules reveal a regular ‘lamellated’ structure similar to that apparent in sections of some insect and crustacean cuticles. A three-dimensional model has been constructed to illustrate this phenomenon. During ootheca formation fibrils become transformed into thin ‘crystalline’ ribbons, about 15 μm long, 1–2 μm wide and 200–300 Å thick. Very regular diamond-shaped ribbons may be obtained in vitro by mixing structural protein globules from dissected left glands with optimum concentrations (0·005–·025 m ) of calcium chloride in unbuffered solution. Experiments suggest that calcium ions play an important part in the natural process of ribbon formation.     

The effect of glycosaminoglycan depletion on the friction and deformation of articular cartilage

Glycosaminoglycans (GAGs) have been shown to be responsible for the interstitial fluid pressurization of articular cartilage and hence its compressive stiffness and load-bearing properties. Contradictory evidence has been presented in the literature on the effect of depleting GAGs on the friction properties of articular cartilage. The aim of this study was to investigate the effect of depleting GAGs on the friction and deformation characteristics of articular cartilage under different tribological conditions. A pin-on-plate machine was utilized to measure the coefficient of friction of native and chondroitinase ABC (CaseABC)-treated articular cartilage under two different models: static (4 mm/s start-up velocity) and dynamic (4 mm/s sliding velocity; 4 mm stroke length) under a load of 25 N (0.4 MPa contact stress) and with phosphate-buffered saline as the lubricant. Indentation tests were carried out at 1 N and 2 N loads (0.14 MPa and 0.28 MPa contact stress levels) to study the deformation characteristics of both native and GAG-depleted cartilage samples. CaseABC treatment rendered the cartilage tissue soft owing to the loss of compressive stiffness and a sulphated-sugar assay confirmed the loss of GAGs from the cartilage samples. CaseABC treatment significantly increased (by more than 50 per cent) the friction levels in the dynamic model (p < 0.05) at higher loading times owing to the loss of biphasic lubrication. CaseABC treatment had no effect on friction in the static model in which the cartilage surfaces did not have an opportunity to recover fluid because of static loading unlike the cartilage tissue in the dynamic model, in which translation of the cartilage surfaces was involved, ensuring effective biphasic lubrication. Therefore the depletion of GAGs had a smaller effect on the coefficient of friction for the static model. Indentation tests showed that GAG-depleted cartilage samples had a lower elastic modulus and higher permeability than native tissue. These results corroborate the role of GAGs in the compressive and friction properties of articular cartilage and emphasize the need for developing strategies to control GAG loss from diseased articular cartilage tissue.     

Anisotropic properties of bovine nasal cartilage

To investigate the structural anisotropy in bovine septal cartilage, quantitative procedures in microscopic magnetic resonance imaging (μMRI), polarized light microscopy (PLM), and mechanical indentation were used to measure the tissue in three orthogonal planes: vertical, medial, and caudocephalic. The quantitative T2 imaging experiments in μMRI found strong anisotropy in the images of both vertical and caudocephalic planes but little anisotropy in the images from the medial plane. The PLM birefringent experiments found that the retardation values in the medial section were only about 10% of these in the vertical and caudocephalic sections and that the angle values in all three sections followed the rotation of the tissue section in the microscope stage. The stress relaxation experiments in mechanical indentation showed reduced stiffness in the medial plane compared to stiffness in either the vertical or caudocephalic planes. Collectively, the results in this project coherently indicate a marked structural anisotropy in cartilage from the nasal septum, where the long axis of the collagen fibrils is oriented in parallel with the medial axis.     

Positron Studies of Subsurface Zone in Pure Iron Induced by Sliding

Positron annihilation spectroscopy and microhardness measurements were performed for pure iron samples after dry sliding. Well-defined depth profiles of edge dislocations decorated by vacancies and vacancy clusters were observed in the subsurface zone (SZ) of the samples studied. The vacancy clusters contained more than ten vacancies and their concentration decreased with the depth from the worn surface. Like in other metals studied, the depth profile of the defects detected exhibited the exponential decay with the depth. The total depth of the SZ was correlated with the load applied during sliding but did not exceed 220 μm. The defect profile in the SZ was revealed also in raw iron samples after dry sliding and blasted iron samples. In the case of raw iron, the profile induced by sliding overlaps defect structure which already exists in a raw material.