The effects of proteolytic enzymes on the mechanical properties of adult human articular cartilage.

The effects of the lysosomal proteinase cathepsin D on the mechanical properties of adult human articular cartilage were examined in detail in 7 joints within the age range 21 to 72 years. The results of a preliminary study on the effects of the lysosomal proteinase cathepsin B1 and clostridial collagenase on the mechanical properties of cartilage are also presented. Cartilage which had been incubated with either cathepsin D or cathepsin B1 showed increased deformation in uniaxial compression perpendicular to the articular surface. The enzyme-treated cartilage also showed decreased tensile stiffness at low values of stress. This effect was more pronounced in specimens from the deeper zone of cartilage than in specimens from the superficial zone. It was also more pronounced in specimens which were aligned perpendicular to the predominant alignment of the collagen fibres in the superficial zone than in specimens which were parallel to the collagen fibres. At higher stresses the tensile stiffness of the treated cartilage was not significantly different from that of the untreated tissue. The tensile fracture stress of the cartilage was also not significantly reduced by the action of cathepsin D. In contrast to the effects observed with the cathepsins, the preliminary results obtained by incubating cartilage for 24 h with clostridial collagenase showed that both the tensile stiffness and the fracture stress were considerably lower than the corresponding values for the untreated tissue. Biochemical analysis of the incubation media, and the specimens, revealed that a large proportion of the proteoglycans was released from the cartilage by each of the three enzymes. The proportion of the total collagen which was released from the cartilage was different for each enzyme: cathepsin D released between 0 and 1.5 per cent, cathepsin B1 released between 2.3 and 4.3 per cent and collagenase released between 5.3 and 27.8 per cent of the collagen after 24 h.     

The effects of leucocyte elastase on the mechanical properties of adult human articular cartilage in tension

The effects of leucocyte elastase on the tensile properties of adult human articular cartilage were examined in detail in 99 specimens from hip, knee and ankle joints in the age range 16–83 years. The results showed that elastase reduced the tensile stiffness of cartilage, both at low stress and at fracture. The tensile strength of cartilage was also considerably reduced by the action of elastase. Biochemical analysis of the incubation media, and the specimens, revealed that 90%, or more, of the proteoglycan was released from the cartilage, whilst the release of collagen was negligible. Leucocyte elastase is known to degrade the non-helical terminal peptides of cartilage collagen molecules and thereby disrupt the main intermolecular cross-links in collage fribrils. A previous study (Kempson, G.E., Tuke, M.A., Dingle, J.T., Barrett, A.J. and Horsfield, P.H. (1976) Biochim. Biophys. Acta 428, 741–760) showed the lack of effect of proteoglycan degradation alone on the tensile strength and stiffness of cartilage. The reduction in strength and stiffness recorded in the present study can, therefore, be attributed to the action of elastase on the collagen in cartilage and it emphasises the important of covalent intermolecular cross-links to the mechanical properties of collagen fibrils.     

The effects of selective matrix degradation on the short-term compressive properties of adult human articular cartilage.

The effects of proteoglycan and collagen digestion on the transient response of human articular cartilage when tested in unconfined compression were determined. Small cylindrical specimens of cartilage, isolated from the femoral head of the hip joint and from the femoral condyles of the knee joint, were subjected to a suddenly applied compressive load using a test apparatus designed to yield a transient oscillatory response. From this response values of the elastic stiffness and the viscous damping coefficient were determined. Cathepsin D and cathepsin B1 were used to digest the proteoglycan in some specimens, while in other specimens leukocyte elastase was used to attack the non-helical terminal regions of the Type II tropocollagen molecules and possibly the Type IX collagen molecule and thereby disturb the integrity of the collagen mesh. The results showed that proteoglycan digestion alone reduced the viscous damping coefficient but it did not significantly alter the elastic stiffness as determined from the oscillatory response. In contrast, the action of elastase reduced both the damping coefficient and the elastic stiffness of the cartilage. The results demonstrated the role of proteoglycans in regulating fluid transport in cartilage and hence controlling the time-dependent viscous properties. The elastic stiffness was shown to be dependent on the integrity of the collagen fibre network and not on the proteoglycans.     

Acoustic properties of articular cartilage under mechanical stress

Mechano-acoustic and elastographic techniques may provide quantitative means for the in vivo diagnostics of articular cartilage. These techniques assume that sound speed does not change during tissue loading. As articular cartilage shows volumetric changes during compression, acoustic properties of cartilage may change affecting the validity of mechano-acoustic measurements. In this study, we examined the ultrasound propagation through human, bovine and porcine articular cartilage during stress-relaxation in unconfined compression. The time of flight (TOF) technique with known cartilage thickness (true sound speed) as well as in situ calibration method [Suh, Youn, Fu, J. Biomech. 34 (2001), 1347-1353] were used for the determination of sound speed. Ultrasound speed and attenuation decreased in articular cartilage during ramp compression, but returned towards the level of original values during relaxation. Variations in ultrasound speed induced an error in strain and compressive moduli provided that constant ultrasound speed and time-of-flight data was used to determine the tissue thickness. Highest errors in strain (-11.8 +/- 12.0%) and dynamic modulus (15.4 +/- 17.9%) were recorded in bovine cartilage. TOF and in situ calibration methods yielded different results for changes in sound speed during compression. We speculate that the variations in acoustic properties in loaded cartilage are related to rearrangement of the interstitial matrix, especially to that of collagen fibers. In human cartilage the changes, are, however relatively small and, according to the numerical simulations, mechano-acoustic techniques that assume constant acoustic properties for the cartilage will not be significantly impaired by this phenomenon.     

Mesenchymal progenitor cells in adult human articular cartilage

The transmembrane receptor Notch-1 regulates cell fate and differentiation and was suggested to identify a cell type with progenitor characteristics in newborn bovine articular cartilage. We show that Notch-1 is expressed on > 70% of BM-MSC in early passage monolayer culture. We also demonstrate that normal articular cartilage contains Notch-1+ cells and that the frequency is increased in OA. Most Notch-1+ cells in OA cartilage are located in the clusters of proliferating cells. These findings indicate that multipotential mesenchymal progenitor cells are present in articular cartilage from adult humans and that their frequency is increased in OA. This observation has implications for understanding the intrinsic repair capacity of articular cartilage and raises the possibility that these progenitor cells might be involved in the pathogenesis of arthritis.     

Delayed aggregation of proteoglycans in adult human articular cartilage

The biosynthesis and macromolecular organization of proteoglycans was studied in explants of adult human articular cartilage. In a series of pulse-chase experiments, labelling with (³⁵S)sulphate, it was shown that the proteoglycan monomer is synthesized as a precursor that has a low affinity for hyaluronic acid. These findings suggest a possible mechanism by which the rate of incorporation of proteoglycans into the extraceHular matrix may be controlled.     

The mechanical environment of chondrocytes in articular cartilage

There is a growing literature concerning chondrocyte responses to mechanical loading, but relatively little is known about the mechanical environment these cells experience in a living joint. Calculations indicate that high forces are applied to limb joints whenever the joints are flexed, because flexion can cause body weight to act on long lever arms compared to the joint centre, whereas the muscles which extend the joint act on much shorter lever arms. As a result, joint reaction forces (which compress the cartilage) can rise to 3-6 times body weight during activities such as stair climbing. Articular cartilage tends to spread this load evenly over the joint surface, but is too thin to do this well, and compressive stresses can rise to 10-20 MPa. Within cartilage, matrix stresses vary locally, possibly as a result of variation in composition or undulations in the subchondral bone, and further modifications of stress occur within each chondron. Articular cartilage is a fibrous solid and cells within it are deformed by mechanical loading rather than subjected to a hydrostatic pressure. The mechanical environment of chondrocytes can best be reproduced in vitro by direct compression of the articular surface of cartilage which is supported naturally by adjacent cartilage and subchondral bone.     

Comparison of nonlinear mechanical properties of bovine articular cartilage and meniscus

Nonlinear, linear and failure properties of articular cartilage and meniscus in opposing contact surfaces are poorly known in tension. Relationships between the tensile properties of articular cartilage and meniscus in contact with each other within knee joints are also not known. In the present study, rectangular samples were prepared from the superficial lateral femoral condyle cartilage and lateral meniscus of bovine knee joints. Tensile tests were carried out with a loading rate of 5 mm/min until the tissue rupture. Nonlinear properties of the toe region, linear properties in larger strains, and failure properties of both tissues were analysed. The strain-dependent tensile modulus of the toe region, Young's modulus of the linear region, ultimate tensile stress and toughness were on average 98.2, 8.3, 4.0 and 1.9 times greater (p<0.05) for meniscus than for articular cartilage. In contrast, the toe region strain, yield strain and failure strain were on average 9.4, 3.1 and 2.3 times greater (p<0.05) for cartilage than for meniscus. There was a significant negative correlation between the strain-dependent tensile moduli of meniscus and articular cartilage samples within the same joints (r=−0.690, p=0.014). In conclusion, the meniscus possesses higher nonlinear and linear elastic stiffness and energy absorption capability before rupture than contacting articular cartilage, while cartilage has longer nonlinear region and can withstand greater strains before failure. These findings point out different load carrying demands that both articular cartilage and meniscus have to fulfil during normal physiological loading activities of knee joints.     

Fluid transport and mechanical properties of articular cartilage: a review

This review is aimed at unifying our understanding of cartilage viscoelastic properties in compression, in particular the role of compression-dependent permeability in controlling interstitial fluid flow and its contribution to the observed viscoelastic effects. During the previous decade, it was shown that compression causes the permeability of cartilage to drop in a functional manner described by k = ko exp (epsilon M) where ko and M were defined as intrinsic permeability parameters and epsilon is the dilatation of the solid matrix (epsilon = tr delta u). Since permeability is inversely related to the diffusive drag coefficient of relative fluid motion with respect to the porous solid matrix, the measured load-deformation response of the tissue must therefore also depend on the non-linearly permeable nature of the tissue. We have summarized in this review our understanding of this non-linear phenomenon. This understanding of these flow-dependent viscoelastic effects are put into the historical perspective of a comprehensive literature review of earlier attempts to model the compressive viscoelastic properties of articular cartilage.     

Relationship between triphasic mechanical properties of articular cartilage and osteoarthritic grade

The purpose of this study was to explore the triphasic mechanical properties of osteoarthritic cartilage with different pathological grades. First, samples of cartilage from rabbits with different stages of osteoarthritis (OA) were graded. Following this, the cartilage was strained by a swelling experiment, and changes were measured using a high-frequency ultrasound system. The result, together with fixed charge density and water volume fraction of cartilage samples, was used to estimate the uniaxial modulus of the cartilage tissue, based on a triphasic model. For the control cartilage samples, the uniaxial elastic modulus on the cartilage surface was lower than those in the middle and deep layers. With an increase in the OA grade, the uniaxial elastic modulus of the surface, middle and deep layers decreased. A significant difference was found in the surface elastic modulus of different OA grades (P<0.01), while no significant differences were identified for OA cartilages of Grades 1 and 2 in the middle and deep layers (P<0.01). Compared with Grades 1 and 2, there was a significant reduction in the elastic modulus in the middle and deep layers of Grade 3 OA cartilage (P<0.05). Overall, this study may provide a new quantitative method to evaluate the severity of OA using the mechanical properties of cartilage tissue.     

Biomechanical properties of human articular cartilage under compressive loads

The function of articular cartilage is to support and distribute loads and to provide lubrication in the diarthrodial joints. Cartilage function is described by proper mechanical and rheological properties, strain and depth-dependent, which are not completely assessed. Unconfined and confined compression are commonly used to evaluate the Young's modulus (E) and the aggregate modulus (H(A)), respectively. The Poisson's ratio (nu) can be calculated indirectly from the equilibrium compression data, or using the biphasic indentation technique; it has recently been optically evaluated by using video microscopy during unconfined compression. The transient response of articular cartilage during confined compression depends on its permeability k; a constant value of k can be easily identified by a simple analytical model of confined compression tests, whereas more complex models or direct measurements (permeation tests) are needed to study the permeability dependence on deformation. A poroelastic finite element model of articular cartilage was developed for this purpose. The elastic parameters (E,nu) of the model were evaluated performing unconfined compression creep tests on human articular cartilage disks, whereas k was identified from the confined test response. Our combined experimental and computational method can be used to identify the parameters that define the permeability dependence on deformation, as a function of depth from articular surface.     

Effects of doxycycline on articular cartilage GAG release and mechanical properties following impact

The effects of doxycycline were examined on articular cartilage glycosaminoglycan (GAG) release and biphasic mechanical properties following two levels of impact loading at 1 and 2 weeks post-injury. Further, treatment for two continuous weeks was compared to treatment for only the 1st week of a 2-week culture period. Following impact at two levels, articular cartilage explants were cultured for 1 or 2 weeks with 0, 50, or 100 microM doxycycline. Histology, GAG release to the media, and creep indentation biomechanical properties were examined. The "High" (2.8 J) impact level had gross surface damage, whereas "Low" (1.1 J) impact was indiscernible from non-impacted controls. GAG staining decreased after High impact, but doxycycline did not visibly affect staining. High impact resulted in decreased aggregate moduli at both 1 and 2 weeks and increased permeability at 2 weeks, but tissue mechanical properties were not affected by doxycycline treatment. At 1 week, High impact resulted in more GAG release compared to non-impacted controls. However, following High impact, 100 microM doxycycline reduced cumulative GAG release at 1 and 2 weeks by 30% and 38%, respectively, compared to no treatment. Interestingly, there was no difference in GAG release comparing 2 weeks continuous treatment with 1 week on, 1 week off. These results support the hypothesis that doxycycline can mitigate GAG release from articular cartilage following impact loads. However, doxycycline was unable to prevent the loss of tissue stiffness observed post-impact, presumably due to initial matrix damage resulting solely from mechanical trauma.     

Expression of the ED B fibronectin isoform in adult human articular cartilage

The most recently described region of alternate splicing of fibronectin mRNA results in expression of an isoform which includes the type III repeat termed ED B (EIII B). To date this isoform has been detected in transformed cells in culture, in the synovial membrane and ovary but in no other adult tissue, and in embryonic chick cartilage to a much greater extent than in other cells of mesenchymal origin. Monoclonal antibody BC-1, which recognizes an epitope within the ED B segment, was used in two different assays and provided evidence that adult human articular cartilage contains ED B fibronectin. The extent of expression of this isoform, however, was variable and less than that found in fibronectin produced by the transformed fibroblast cell line WI-38VA13.     

Effect of a single impact loading on the structure and mechanical properties of articular cartilage

AIM: To study the effect of a single impact on the structure and mechanical properties of cartilage. MATERIALS AND METHOD: Osteochondral plugs harvested from bovine femora were subjected each to a single impact using an in-house designed drop-tower. Impact masses of different values were released from different drop heights in selected combinations to apply stresses at strain rates and impact energies within specific ranges. Changes in the storage and loss moduli were estimated from cyclic compressive loading test undertaken before and after impact. The conditions of these tests were set to those occurring during walking and running. The extent of the damage on cartilage surface and depth was assessed using optical and confocal microscopy. RESULTS: The storage modulus varied slightly at level walking and running after performing impact tests up to the impact energy of 0.736 J. However, the decrease in the storage modulus was considerable at the impact energy of 1.962 J for test conditions representing both walking and running. This impact energy resulted in strain rate of 1500 s(-1), stress of 25 MPa and energy absorbed per unit volume of 12.79 mJ/mm(3). After impact the loss modulus increased over the loading cycles at all energies. Severe fissures were observed across the cartilage surface and through its thickness at levels of impact energy equal or greater than 1.472 J. CONCLUSIONS: This study identified thresholds for the strain rate, impact stress and impact energy, which caused permanent changes in the mechanical properties and damage to structure of cartilage.