Tribology-optimised silk protein hydrogels for articular cartilage repair

Porous hydrogels were made from silk fibre as potential materials for cartilage repair. The aim was to develop materials which mimicked the tribological behaviour of cartilage, with controlled pore-sizes and optimised mechanical properties. Mechanical tests showed hydrogels had a comparable compressive modulus to cartilage, with stiffness improved by decreasing pore size. Under static loading and during shear hydrogels demonstrated significant interstitial fluid support. Friction testing showed the hydrogels had a cartilage-like frictional response, dominated by this interstitial fluid support. Silk hydrogels showed little wear, early signs of which were changes in surface morphology that did not correlate with the equilibrium friction coefficient. Consequently both wear and friction should be monitored when assessing the tribological performance of hydrogels.     

Tribological testing of articular cartilage of the medial compartment of the knee using a friction simulator

The aim of this study was to develop a tribological simulation of a unicondylar natural knee, to measure the friction and wear of articular cartilage (AC) against itself (AC-vs-AC) and against stainless steel (SS) simulating a hemiarthroplasty (AC-vs-SS). AC-vs-AC produced low levels of friction and no wear was found. AC-vs-SS showed higher levels of friction and significant AC wear. Using AC-vs-SS with elevated loading, the wear of AC was accelerated and through to bone. This study demonstrated the importance of contact stress in the design of a spacer hemiarthroplasty for the medial compartmental knee. Initial results showed the importance of contact mechanics, stress and biomaterial type in determining short-term tribological function and long-term clinical outcome of hemiarthroplasty.     

Microwave-enhanced fixation of rabbit articular cartilage

Cartilage, being highly aqueous, is difficult to preserve for electron microscopy without artefacts. Microwave-enhanced fixation is suggested as a standard method for block samples of this material, with dimensions of up to 12 × 7 × 3 mm. Cartilage samples from the tibial plateau of adult rabbits were fixed by conventional, cryo- or microwave-enhanced fixation. Constant or cyclical microwave irradiation of samples, immersed in fixatives, was carried out to varying final solution temperatures. Microwave-enhanced fixation and staining is shown to be both rapid and reproducible, giving fine structural preservation. Below 323 K microwave fixation always gave excellent preservation of the fine structure within seconds. At higher temperatures thermal artefacts were introduced. In this study the microwave-enhanced fixation is equal in quality to the best conventional immersion fixation and is nearly as fast as cryo-preservation. It provides a standardized, reproducible fixation for morphological studies on cartilage with good process control.     

Analysis of biphasic lubrication of articular cartilage loaded by cylindrical indenter

Combination of theoretical biphasic analyses and corresponding experimental measurements for articular cartilage has successfully revealed the fundamental material properties and time-depending mechanical behaviors of articular cartilage containing plenty of water. The insight of load partitioning between solid and fluid phases advanced the prediction of the frictional behavior of articular cartilage. One of the recent concerns about biphasic finite element (FE) analysis seems to be a dynamic and physiological condition in terms of mechanical functionality as a load-bearing for articular joint system beyond material testing, which has mainly focused on time-dependent reaction force and deformation in relatively small and low speed compression. Recently, the biphasic FE model for reciprocating sliding motion was applied to confirm the frictional effect on the migrating contact area. The results indicated that the model of a cylindrical indenter sliding over the cartilage surface remarkably sustained the higher proportion of fluid load support than a condition without migrating contact area, but the effectiveness of constitutive material properties has not been sufficiently evaluated for sliding motion. In our present study, at the first stage, the compressive response of the articular cartilage was examined by high precision testing machine. Material properties for the biphasic FE model, which included inhomogeneous apparent Young's modulus of solid phase along depth, strain-dependent permeability and collagen reinforcement in tensile strain, were estimated in cylindrical indentation tests by the curve fitting between the experimental time-dependent behavior and FE model simulation. Then, the biphasic lubrication mechanism of the articular cartilage including migrating contact area was simulated to elucidate functionality as a load-bearing material. The results showed that the compaction effect on permeability of solid phase was functional particularly in the condition without the migrating contact area, whereas in sliding condition the compaction effect did not clearly show its role in terms of the proportion of fluid load support. The reinforcement of solid phase, which represented the collagen network in the tissue, improved the proportion of fluid load support especially in the sliding condition. Thus, a functional integration of constitutive mechanical properties as a load-bearing was evaluated by FE model simulation in this study.     

Mechanical and tribological properties of poly(hydroxyethyl methacrylate) hydrogels as articular cartilage substitutes

Poly(hydroxyethyl methacrylate) (p(HEMA)) hydrogels have been proposed as promising biomaterials to replace damaged articular cartilage. A major obstacle to their use as replacement bearing tissue is their poor mechanical properties in comparison with healthy articular cartilage. The purpose of this study was to obtain p(HEMA) hydrogels with physicochemical and mechanical properties close to healthy articular cartilage, by introducing a hydrophilic monomer, namely acrylic acid (AA). Formulations of hydrogels with different amounts of hydrophilic monomer (acrylic acid, AA) were synthesized and tested: p(HEMA), p(HEMA-co-5%AA), p(HEMA-co-25%AA). The macro-mechanical tests were reproduced at nanoscale in order to verify if the superficial properties of the hydrogels are similar to the bulk ones.     

Helium ion microscopy for high-resolution visualization of the articular cartilage collagen network

The articular cartilage collagen network is an important research focus because network disruption results in cartilage degeneration and patient disability. The recently introduced helium ion microscope (HIM), with its smaller probe size, longer depth of field and charge neutralization, has the potential to overcome the inherent limitations of electron microscopy for visualization of collagen network features, particularly at the nanoscale. In this study, we evaluated the capabilities of the helium ion microscope for high-resolution visualization of the articular cartilage collagen network. Images of rabbit knee cartilage were acquired with a helium ion microscope; comparison images were acquired with a field emission scanning electron microscope (FE-SEM) and a transmission electron microscope (TEM). Sharpness of example high-resolution helium ion microscope and field emission scanning electron microscope images was quantified using the 25-75% rise distance metric. The helium ion microscope was able to acquire high-resolution images with unprecedented clarity, with greater sharpness and three-dimensional-like detail of nanoscale fibril morphologies and fibril connections, in samples without conductive coatings. These nanoscale features could not be resolved by field emission scanning electron microscopy, and three-dimensional network structure could not be visualized with transmission electron microscopy. The nanoscale three-dimensional-like visualization capabilities of the helium ion microscope will enable new avenues of investigation in cartilage collagen network research.     

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%.     

Experimental and numerical investigation of the behaviour of articular cartilage under shear loading—Interstitial fluid pressurisation and lubrication mechanisms

In this paper, the mechanical and frictional responses of articular cartilage when subjected to alternating shearing forces under a constant load were investigated. Shear testing was performed at physiological contact pressures to ascertain the influence of interstitial fluid support on the evolution of frictional forces during cyclic loading.Numerical studies were also performed using the finite element software Abaqus. The tissue was modelled as a biphasic material with strain dependent permeability. The influence of the material characteristics on the lubrication mechanisms occurring when cartilage is subject to compression and shear was studied to corroborate the experimental findings.     

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.     

Freeze-substitution of rabbit tibial articular cartilage reveals that radial zone collagen fibres are tubules

Investigations of the micromorphology of rabbit tibial articular cartilage using scanning and transmission electron microscopy revealed that the collagenous elements in the tissue form fluid-containing tubular structures. The commonly described radial or deep zone longitudinal fibres were found to be tubular structures with internal diameters of 1–2 μm. The walls of the tubules were composed of tightly packed fibrils of collagen. The tangential zone, close to the tibial plateau, was composed mainly of a spongy arrangement of collagen fibrils, containing bunches of tangentially lying small (< 1 μm) diameter tubules. The application of conventional chemical fixation techniques resulted in the fine detail of this tissue being obscured. When the tissue was frozen, followed by cryo-scanning electron microscopy or freeze-drying, prior to observation in the scanning electron microscope the tubule structures were not obviously present. It was only by applying freeze-substitution techniques, followed by critical point drying or resin embedding, that the structure was revealed clearly. Segregation of water into ice crystals did occur during the freezing process, but the formation of those crystals played no part in creating the tubular morphology observed. A similar structure was still revealed following pre-treatment with glycerol, methanol or Triton X-100, provided that concentration of these additives was not too high. The walls of the tubules in the radial region were composed of straight, longitudinally arranged as well as helically arranged, 30 nm diameter fibrils. The lumen of the tubules appears to be lined by a circumferentially arranged array of approximately 10 nm diameter fibres, spaced at regular intervals of 50–70 nm.     

The ultra-low friction of the articular surface is pH-dependent and is built on a hydrophobic underlay including a hypothesis on joint lubrication mechanism

In this study, the influence of pH on interfacial energy distributed over the phospholipids-bilayer surface model and the effect of hydrophobicity on coefficient of friction (f) were investigated by using microelectrophoresis. An important clinical implication of deficiency in hydrophobicity is the loss of phospholipids that is readily observed in osteoarthritis joints. This paper establishes the influence of pH on interfacial energy upon an increase f, which might be associated with a decrease of hydrophobicity of the articular surface.     

Extracellular matrix of ostrich articular cartilage.

The composition and organization of the extracellular matrix of ostrich articular cartilage was investigated, using samples from the proximal and distal surfaces of the tarsometatarsus. For morphological analysis, sections were stained with toluidine blue and analyzed by polarized light microscopy. For biochemical analysis, extracellular matrix components were extracted with 4 M guanidinium chloride, fractionated on DEAE-Sephacel and analyzed by SDS-PAGE. Glycosaminoglycans were analyzed by electrophoresis in agarose gels. Structural analysis showed that the fibrils were arranged in different directions, especially on the distal surface. The protein and glycosaminoglycan contents of this region were higher than in the other regions. SDS-PAGE showed the presence of proteins with molecular masses ranging from 17 to 121 kDa and polydisperse components of 67, 80-100, and 250-300 kDa in all regions. The analysis of glycosaminoglycans in agarose-propylene diamine gels revealed the presence of only chondroitin-sulfate. The electrophoretic band corresponding to putative decorin was a small proteoglycan containing chondroitin-sufate and not dermatan-sulfate, unlike other cartilages. The higher amounts of proteins and glycosaminoglycans and the multidirectional arrangement of fibrils seen in the distal region may be correlated with the higher compression normally exerted on this region.     

Vitrification of articular cartilage by high-pressure freezing

For more than 20 years, high-pressure freezing has been used to cryofix bulk biological specimens and reports are available in which the potential and limits of this method have been evaluated mostly based on morphological criteria. By evaluating the presence or absence of segregation patterns, it was postulated that biological samples of up to 600 μm in thickness could be vitrified by high-pressure freezing. The cooling rates necessary to achieve this result under high-pressure conditions were estimated to be of the order of several hundred degrees kelvin per second. Recent results suggest that the thickness of biological samples which can be vitrified may be much less than previously believed. It was the aim of this study to explore the potential and limits of high-pressure freezing using theoretical and experimental methods. A new high-pressure freezing apparatus (Lei?a EM HPF), which can generate higher cooling rates at the sample surface than previously possible, was used. Using bovine articular cartilage as a model tissue system, we were able to vitrify 150-μm-thick tissue samples. Vitrification was proven by subjecting frozen-hydrated cryosections to electron diffraction analysis and was found to be dependent on the proteoglycan concentration and water content of the cartilage. Only the lower radical zone (with a high proteoglycan concentration and a low water content compared to the other zones) could be fully vitrified. Our theoretical calculations indicated that applied surface cooling rates in excess of 5000 K/s can be propagated into specimen centres only if samples are relatively thin (<200 μm). These calculations, taken together with our zone-dependent attainment of vitrification in 150-μm-thick cartilage samples, suggest that the critical cooling rates necessary to achieve vitrification of biological samples under high-pressure freezing conditions are significantly higher (1000–100 000 K/s) than previously proposed, but are reduced by about a factor of 100 when compared to cooling rates necessary to vitrify biological samples at ambient pressure.     

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.     

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.     

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.     

Multi-layer models of friction between solids

Recent simulations of processes in surface layers of rubbing solids have shown the formation of a boundary layer, called quasi-fluid layer. To better understand the physical nature of the quasi-fluid layer, we investigate the processes occurring in the surface layer of rubbing solids in the frame of a simple multi-layer model, which also leads to the formation of a quasi-fluid layer. The multi-layer model can further be used to investigate how the friction force is determined by the material and loading parameters. It has been shown that the friction force and the thickness of the quasi-fluid layer depend on sliding velocity, the viscosity of the material and on a microscopic parameter (layer thickness). Subsequently, two different ways of extending the discussed model are proposed aiming at a model that can also predict the influence of normal pressure and surface topography on the friction force and on the formation of the quasi-fluid layer.     

Investigation of the friction and surface degradation of innovative chondroplasty materials against articular cartilage

Understanding the wear of the biomaterial-cartilage interface is vital for the development of innovative chondroplasty. The aim of this study was to investigate a number of biphasic materials as potential chondroplasty biomaterials. Simple geometry friction and wear studies were conducted using bovine articular cartilage pins loaded against a range of single-phase and biphasic materials. The frictions of each biomaterial was compared within simple and protein-containing lubricants. Longer-term continuous sliding tests within a protein containing lubricant were also conducted at various loading conditions to evaluate the friction and degradation for each surface. All single-phase materials showed a steady rise in friction, which was dependent on the loss of interstitial fluid load support from the opposing cartilage pin. All biphasic materials demonstrated a marked reduction in friction when compared with the single-phase materials. It is postulated that the biphasic nature of each material allowed an element of fluid load support to be maintained by fluid rehydration and expulsion. In the longer-term study, significant differences in the articular cartilage pin (surface damage) between the positive control (stainless steel) and the negative control (articular cartilage) was found. The potential biphasic chondroplasty materials produced a reduction in articular cartilage pin damage when compared with the single-phase materials. The changes in surface topography of the cartilage pin were associated with increased levels of friction achieved during the continuous wear test. The study illustrated the importance of the biphasic properties of potential chondroplasty materials, and future work will focus on the optimization of biphasic properties as well as long-term durability, such that materials will more closely mimic the biotribology of natural articular cartilage.