Load-displacement-time characteristics of articular cartilage

The load-displacement-time characteristics of cartilage are modelled in a simple analytical way, and the behavior under steady and oscillating loads is predicted numerically. Comparison with constant-load creep experiments shows fairly good agreement. The model predicts that under oscillating loads of typical physiological magnitude and frequency, the effect on displacement of fluid transport through the matrix is negligible in any one cycle.     

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.     

Gene therapy for articular cartilage repair

Articular cartilage serves as the gliding surface of joints. It is susceptible to damage from trauma and from degenerative diseases. Restoration of damaged articular cartilage may be achievable through the use of cell-regulatory molecules that augment the reparative activities of the cells, inhibit the cells' degradative activities, or both. A variety of such molecules have been identified. These include insulin-like growth factor I, fibroblast growth factor 2, bone morphogenetic proteins 2, 4, and 7, and interleukin-1 receptor antagonist. It is now possible to transfer the genes encoding such molecules into articular cartilage and synovial lining cells. Although preliminary, data from in-vitro and in-vivo studies suggest that gene therapy can deliver such potentially therapeutic agents to protect existing cartilage and to build new cartilage.     

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.     

The potential of hydrogels as synthetic articular cartilage

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

Stem cells for tissue engineering of articular cartilage

Articular cartilage injuries are one of the most common disorders in the musculo-skeletal system. Injured cartilage tissue cannot spontaneously heal and, if not treated, can lead to osteoarthritis of the affected joints. Although a variety of procedures are being employed to repair cartilage damage, methods that result in consistent durable repair tissue are not yet available. Tissue engineering is a recently developed science that merges the fields of cell biology, engineering, material science, and surgery to regenerate new functional tissue. Three critical components in tissue engineering of cartilage are as follows: first, sufficient cell numbers within the defect, such as chondrocytes or multipotent stem cells capable of differentiating into chondrocytes; second, access to growth and differentiation factors that modulate these cells to differentiate through the chondrogenic lineage; third, a cell carrier or matrix that fills the defect, delivers the appropriate cells, and supports cell proliferation and differentiation. Stem cells that exist in the embyro or in adult somatic tissues are able to renew themselves through cell division without changing their phenotype and are able to differentiate into multiple lineages including the chondrogenic lineage under certain physiological or experimental conditions. Here the application of stem cells as a cell source for cartilage tissue engineering is reviewed.     

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.     

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.     

Automated classification of articular cartilage surfaces based on surface texture

In this study the automated classification system previously developed by the authors was used to classify articular cartilage surfaces with different degrees of wear. This automated system classifies surfaces based on their texture. Plug samples of sheep cartilage (pins) were run on stainless steel discs under various conditions using a pin-on-disc tribometer. Testing conditions were specifically designed to produce different severities of cartilage damage due to wear. Environmental scanning electron microscope (SEM) (ESEM) images of cartilage surfaces, that formed a database for pattern recognition analysis, were acquired. The ESEM images of cartilage were divided into five groups (classes), each class representing different wear conditions or wear severity. Each class was first examined and assessed visually. Next, the automated classification system (pattern recognition) was applied to all classes. The results of the automated surface texture classification were compared to those based on visual assessment of surface morphology. It was shown that the texture-based automated classification system was an efficient and accurate method of distinguishing between various cartilage surfaces generated under different wear conditions. It appears that the texture-based classification method has potential to become a useful tool in medical diagnostics.     

The fate of implanted autologous chondrocytes in regenerated articular cartilage

Autologous chondrocyte implantation (ACI) is used to treat some articular cartilage defects. However, the fate of the cultured chondrocytes after in-vivo transplantation and their role in cartilage regeneration remains unclear. To monitor the survival and fate of such cells in vivo, the chondrocytes were labelled with a lipophilic dye and the resultant regenerated tissue in dogs examined. It was found that, 4 weeks after implantation, the osteochondral defects were filled with regenerative tissue that resembled hyaline cartilage. Fluorescence microscopy of frozen sections of the regenerated tissue revealed that the majority of cells were derived from the DiI-labelled implanted chondrocytes. From these results, it was concluded that a large population of implanted autologous chondrocytes can survive at least 4 weeks after implantation and play a direct role in cartilage regeneration. However, it remains unknown whether other cells, such as periosteal cells or bone marrow stromal stem cells, are involved in the regeneration of cartilage after ACI.     

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 preparation of human articular cartilage for scanning electron microscopy

This paper compares sixteen preparative techniques thought to be of advantage in the study by scanning electron microscopy (SEM) of human articular cartilage surfaces. The adequacy of surface preservation obtained with the techniques, was judged subjectively, first, by the reproducibility of secondary electron images of normal cartilage, and second, by comparing the results with those obtained by reflected light microscopy of the fresh unfixed cartilage surface over a magnification range of × 20 – × 240. Adequate surface preservation was confirmed when cartilage surfaces had been dehydrated through ethanol to propylene oxide and vacuum dried; dehydrated through amyl acetate and quenched in Freon before freeze-drying; dehydrated and passed through amyl acetate at low temperature before freeze drying. Valuable information can be obtained from different specimens by varying the technique of preparation. At different ages, different surface features are best preserved. In a systematic study it has been found essential to adopt a uniform preparative method and to control the results by reflected light microscopy. Even with the most perfect preparation, the surface appearances cannot be identical with those that function under load in vivo.     

Effects of ageing on the biomechanical properties of rat articular cartilage

Previous studies have demonstrated that male Sprague Dawley (SD) rats experience age-related bone loss with the same characteristics as that in ageing men. As articular cartilage, like bone, is a critical component of the health and function of the musculoskeletal system, the authors hypothesized that articular cartilage in the untreated male SD rats could be a suitable model for studying the age-related deterioration of articular cartilage in men. To test this hypothesis, male SD rats were killed at between 6 and 27 months. The right femur of each rat was removed. The effects of ageing on the structural integrity of the distal femoral articular cartilage were studied by biomechanical testing with a creep indentation apparatus. The aggregate modulus, Poisson's ratio, permeability, thickness, and percentage recovery of articular cartilage were determined using finite element/non-linear optimization modelling. No significant differences were observed in these biomechanical properties of the distal femoral articular cartilage as a function of age. Therefore, untreated male SD rats appear to be unsuitable for studying the age-related changes of articular cartilage as they occur in men. However, and more intriguingly, it is also possible that ageing does not affect the biomechanical properties of articular cartilage in the absence of cartilage pathology.     

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.     

Improved techniques for measuring the indentation and thickness of articular cartilage

A review of the techniques previously employed in the indentation and measurement of the thickness of articular cartilage has led to new and improved techniques for performing both measurements. By utilizing high-speed, microcomputer-controlled data logging techniques, simultaneous monitoring of signals from a dynamic load cell and a displacement transducer could be made throughout an indentation test. The position of the indenter as it touched the articular surface could thus be determined automatically by identifying the moment at which a positive change in the load signal occurred. Less accurate and more time consuming techniques previously required for determining the position of the cartilage surface were hence avoided. The apparatus also included a critically damped dashpot which prevented any transient loads being applied to the cartilage. Depths of indentation could be measured to an accuracy of 0.005 mm with a measurement repeatability of 2.14 per cent. By replacing the indenter with a sharp needle, the apparatus was also capable of measuring the undeformed thickness of cartilage. An accuracy of +/- 0.012 mm could be achieved with a measurement repeatability of 1.2 per cent. The apparatus is particularly suited to survey work where large numbers of indentation tests are to be performed.     

Freeze-drying of articular cartilage: Investigation of rat femoral heads by SEM

The results of comparative investigations of freeze-drying of joint cartilage which had not been separated from the underlying bone are reported. Fixation and ethanol and amyl acetate substitution procedures cause marked loss of ground substance and thus create surface structures which are not present to the same degree in cartilage which is simply freeze-dried. Indeed, it is questionable whether these structures are present in vivo. They were most highly developed in critical-point-dried cartilage. The main difficulty with freeze-drying is the prevention of damage caused by ice crystals. A tissue temperature of ?130°C should always be aimed for and, because of the rapid growth of ice crystals, the temperature should never be allowed to rise above ?90°C. These conditions can be fulfilled if the specimen is in good thermal contact with the cold stage and if the cooling device is equipped with a condenser which is cooled with liquid nitrogen. The better the tissue was processed, the smoother was the resulting cartilage surface and the greater was the degree to which the chondrocytes fitted the walls of their lacunae. At tissue temperatures above ?90°C, the first change which became apparent was destruction of the cell membranes. This was followed by almost complete elution of the ground substance from between the fibers and, finally, the cell nuclei were also destroyed. This damage caused by ice crystals exposed fibrous structures on the surface of the cartilage, and the appearance of these structures was superior to that produced by enzymatic methods of preparation. The disruption caused by freezing uncovered intracellular structures.     

Correlation between friction of articular cartilage and reflectance intensity from superficial images

The lubrication mechanism of articular cartilage is characterized by an efficient performance. In this work, friction of articular cartilage was evaluated with in-site images of articular surface. The images were captured with the laser light reflected at the interface between a prism and articular cartilage. The attenuation of reflectance was associated with the increase of the contact of collagen network of articular cartilage. The light reflectance and friction coefficient for short sliding presented a significant positive correlation. Friction tests were also carried out for short (30 s) and long (300 s) preloading times. The results indicate that depletion of fluid film is responsible for the increase of friction and the recovery of the fluid film was observed for the long preloading after the early stage of sliding.     

Design, validation, and utilization of an articular cartilage impact instrument

This paper describes the development and use of an instrument mechanically to impact bovine articular cartilage and record the event using a piezoelectric accelerometer, as well as to carry out post-impact characterization of the tissue. Two levels of impact (low: 6 cm drop height, 18.4 N tup; high: 10 cm drop height, 27.8 N tup) were chosen such that the former did not show gross damage upon inspection, while the latter showed substantial gross damage. Peak stress, time to peak stress, and impact duration were taken from data recorded by the instrument. Three cartilage biomechanical properties (aggregate modulus, Poisson's ratio, and permeability) were acquired by creep indentation, and tissue morphology rated on a standardized scale was also determined. When subjected to the high level of impact, articular cartilage showed statistically significant (p < 0.05) differences in all three impact metrics and morphology. This high level of impact also resulted in a 37 per cent decrease in the aggregate modulus of the tissue. Lower drop heights resulted in more consistent impact curves, demonstrated less standard deviation, and did not change the biomechanical properties of the tissues. With the instrument and techniques described in this study, articular cartilage can be subjected to specific levels of impact in order to study injury biomechanics of the tissue at specific levels of mechanical damage.