The effect of collagen degradation on chondrocyte volume and morphology in bovine articular cartilage following a hypotonic challenge

Collagen degradation is one of the early signs of osteoarthritis. It is not known how collagen degradation affects chondrocyte volume and morphology. Thus, the aim of this study was to investigate the effect of enzymatically induced collagen degradation on cell volume and shape changes in articular cartilage after a hypotonic challenge. Confocal laser scanning microscopy was used for imaging superficial zone chondrocytes in intact and degraded cartilage exposed to a hypotonic challenge. Fourier transform infrared microspectroscopy, polarized light microscopy, and mechanical testing were used to quantify differences in proteoglycan and collagen content, collagen orientation, and biomechanical properties, respectively, between the intact and degraded cartilage. Collagen content decreased and collagen orientation angle increased significantly (p < 0.05) in the superficial zone cartilage after collagenase treatment, and the instantaneous modulus of the samples was reduced significantly (p < 0.05). Normalized cell volume and height 20 min after the osmotic challenge (with respect to the original volume and height) were significantly (p < 0.001 and p < 0.01, respectively) larger in the intact compared to the degraded cartilage. These findings suggest that the mechanical environment of chondrocytes, specifically collagen content and orientation, affects cell volume and shape changes in the superficial zone articular cartilage when exposed to osmotic loading. This emphasizes the role of collagen in modulating cartilage mechanobiology in diseased tissue.     

The effect of hydrogel injection on cardiac function and myocardial mechanics in a computational post-infarction model

An emerging therapy to limit adverse heart remodelling following myocardial infarction (MI) is the injection of polymers into the infarcted left ventricle (LV). In the few numerical studies carried out in this field, the definition and distribution of the hydrogel in the infarcted myocardium were simplified. In this computational study, a more realistic biomaterial distribution was simulated after which the effect on cardiac function and mechanics was studied. A validated finite element heart model was used in which an antero-apical infarct was defined. Four infarct models were created representing different temporal phases in the progression of a MI. Hydrogel layers were simulated in the infarcted myocardium in each model. Biomechanical and functional improvement of the LV was found after hydrogel inclusion in the ischaemic models representing the early phases of MI. In contrast, only functional but no mechanical restitution was shown in the scar model due to hydrogel presence.     

An in vitro model for the pathological degradation of articular cartilage in osteoarthritis

The objective of this study was to develop an in vitro cartilage degradation model that emulates the damage seen in early-stage osteoarthritis. To this end, cartilage explants were collagenase-treated to induce enzymatic degradation of collagen fibers and proteoglycans at the articular surface. To assess changes in mechanical properties, intact and degraded cartilage explants were subjected to a series of confined compression creep tests. Changes in extracellular matrix structure and composition were determined using biochemical and histological approaches. Our results show that collagenase-induced degradation increased the amount of deformation experienced by the cartilage explants under compression. An increase in apparent permeability as well as a decrease in instantaneous and aggregate moduli was measured following collagenase treatment. Histological analysis of degraded explants revealed the presence of surface fibrillation, proteoglycan depletion in the superficial and intermediate zones and loss of the lamina splendens. Collagen cleavage was confirmed by the Col II–3/4C_short antibody. Degraded specimens experienced a significant decrease in proteoglycan content but maintained total collagen content. Repetitive testing of degraded samples resulted in the gradual collapse of the articular surface and the compaction of the superficial zone. Taken together, our data demonstrates that enzymatic degradation with collagenase can be used to emulate changes seen in early-stage osteoarthritis. Further, our in vitro model provides information on cartilage mechanics and insights on how matrix changes can affect cartilage's functional properties. More importantly, our model can be applied to develop and test treatment options for tissue repair.     

The effect of calcification on the structural mechanics of the costal cartilage

The costal cartilage often undergoes progressive calcification with age. This study sought to investigate the effects of calcification on the structural mechanics of whole costal cartilage segments. Models were developed for five costal cartilage specimens, including representations of the cartilage, the perichondrium, calcification, and segments of the rib and sternum. The material properties of the cartilage were determined through indentation testing; the properties of the perichondrium were determined through optimisation against structural experiments. The calcified regions were then expanded or shrunk to develop five different sensitivity analysis models for each. Increasing the relative volume of calcification from 0% to 24% of the cartilage volume increased the stiffness of the costal cartilage segments by a factor of 2.3–3.8. These results suggest that calcification may have a substantial effect on the stiffness of the costal cartilage which should be considered when modelling the chest, especially if age is a factor.     

Type III collagen is a key regulator of the collagen fibrillar structure and biomechanics of articular cartilage and meniscus

Despite the fact that type III collagen is the second most abundant collagen type in the body, its contribution to the physiologic maintenance and repair of skeletal tissues remains poorly understood. This study queried the role of type III collagen in the structure and biomechanical functions of two structurally distinctive tissues in the knee joint, type II collagen-rich articular cartilage and type I collagen-dominated meniscus. Integrating outcomes from atomic force microscopy-based nanomechanical tests, collagen fibril nanostructural analysis, collagen cross-link analysis and histology, we elucidated the impact of type III collagen haplodeficiency on the morphology, nanostructure and biomechanical properties of articular cartilage and meniscus in Col3a1^+/− mice. Reduction of type III collagen leads to increased heterogeneity and mean thickness of collagen fibril diameter, as well as reduced modulus in both tissues, and these effects became more pronounced with skeletal maturation. These data suggest a crucial role of type III collagen in mediating fibril assembly and biomechanical functions of both articular cartilage and meniscus during post-natal growth. In articular cartilage, type III collagen has a marked contribution to the micromechanics of the pericellular matrix, indicating a potential role in mediating the early stage of type II collagen fibrillogenesis and chondrocyte mechanotransduction. In both tissues, reduction of type III collagen leads to decrease in tissue modulus despite the increase in collagen cross-linking. This suggests that the disruption of matrix structure due to type III collagen deficiency outweighs the stiffening of collagen fibrils by increased cross-linking, leading to a net negative impact on tissue modulus. Collectively, this study is the first to highlight the crucial structural role of type III collagen in both articular cartilage and meniscus extracellular matrices. We expect these results to expand our understanding of type III collagen across various tissue types, and to uncover critical molecular components of the microniche for regenerative strategies targeting articular cartilage and meniscus repair.     

The effect of link peptide on proteoglycan synthesis in equine articular cartilage

The basal rate of in vitro proteoglycan (PG) synthesis in explants of equine articular cartilage was subject to considerable variation in animals of the same age but was greater in younger than older animals. Synthesis of PGs in explant cultures was stimulated by a synthetic link peptide, identical in sequence to the N-terminus of the link protein (LP) of PG aggregates, in a similar manner to that demonstrated previously for human articular cartilage [Biochem. Soc. Trans. 25 (1997) 427; Arthritis Rheum. 41 (1998) 157]. Stimulation occurred in tissue from animals ranging from 1 to 30 years old but older animals required higher concentrations of peptide to produce a measurable response. Synthesis of PGs increased in a concentration-dependent manner and was paralleled by increases in the ability of aggrecan monomers to form aggregates with hyaluronan (HA). In addition to its effect on synthesis of PGs, link peptide also increased synthesis of both aggrecan and LP mRNA. Cartilage explant and chondrocyte cultures secreted small amounts of biologically active interleukin 1 (IL 1) and secretion of this cytokine was reduced considerably by the addition of link peptide. Reduction in the activity of this catabolic cytokine coupled with the increased synthesis of mRNA for aggrecan and link peptide may be the mechanism by which link peptide exerts its positive effect on the rate of PG synthesis in articular cartilage.     

The effect of cyclical compressive loading on gene expression in articular cartilage

Osteoarthritis (OA) develops as a consequence of articular cartilage degeneration possibly initiated by excessive or abnormal loading of the joint, and potentially mediated through a proteinase/proteinase inhibitor imbalance. We have shown previously that physiological loads (0.5 MPa, 1 Hz, 3 hour) elicit increased expression and activation of the matrix metalloproteinases (MMPs) in articular cartilage explants in vitro. The objective of this study was to identify mechanically-regulated genes involved in the observed induction of MMP expression and enhanced activation. Differential RNA Display (DRD) was used to identify mechanically-regulated genes by comparing DRD products derived from loaded and unloaded cartilage. One gene up-regulated in cartilage after 10, 30 and 60 minute loading revealed 83% homology with Mus musculus thymosin beta_4 which is known to induce MMP gene expression. The identification of mechanically regulated genes will greatly enhance our understanding of matrix turnover providing an exciting future in elucidating the role of mechanically-regulated genes in the development of OA.     

Bonding of articular cartilage using a combination of biochemical degradation and surface cross-linking

After trauma, articular cartilage often does not heal due to incomplete bonding of the fractured surfaces. In this study we investigated the ability of chemical cross-linkers to facilitate bonding of articular cartilage, either alone or in combination with a pre-treatment with surface-degrading agents. Articular cartilage blocks were harvested from the femoropatellar groove of bovine calves. Two cartilage blocks, either after pre-treatment or without, were assembled in a custom-designed chamber in partial apposition and subjected to cross-linking treatment. Subsequently, bonding of cartilage was measured as adhesive strength, that is, the maximum force at rupture of bonded cartilage blocks divided by the overlap area. In a first approach, bonding was investigated after treatment with cross-linking reagents only, employing glutaraldehyde, 1-ethyl-3-diaminopropyl-carbodiimide (EDC)/N-hydroxysuccinimide (NHS), genipin, or transglutaminase. Experiments were conducted with or without compression of the opposing surfaces. Compression during cross-linking strongly enhanced bonding, especially when applying EDC/NHS and glutaraldehyde. Therefore, all further experiments were performed under compressive conditions. Combinations of each of the four cross-linking agents with the degrading pre-treatments, pepsin, trypsin, and guanidine, led to distinct improvements in bonding compared to the use of cross-linkers alone. The highest values of adhesive strength were achieved employing combinations of pepsin or guanidine with EDC/NHS, and guanidine with glutaraldehyde. The release of extracellular matrix components, that is, glycosaminoglycans and total collagen, from cartilage blocks after pre-treatment was measured, but could not be directly correlated to the determined adhesive strength. Cytotoxicity was determined for all substances employed, that is, surface degrading agents and cross-linkers, using the resazurin assay. Taking the favourable cell vitality after treatment with pepsin and EDC/NHS and the cytotoxic effects of guanidine and glutaraldehyde into account, the combination of pepsin and EDC/NHS appeared to be the most advantageous treatment in this study. In conclusion, bonding of articular cartilage blocks was achieved by chemical fixation of their surface components using cross-linking reagents. Application of compressive forces and prior modulation of surface structures enhanced cartilage bonding significantly. Enzymatic treatment in combination with cross-linkers may represent a promising addition to current techniques for articular cartilage repair.     

The effect of perfusion on soft tissue mechanical properties: a computational model

Some simple finite element models were constructed to investigate the magnitude and character of changes in mechanical properties of very soft tissues due to the loss of perfusion. Changes in the apparent stress-strain curve were used to characterise the effect of simulated blood perfusion pressure on the engineering stress-strain curve. The results indicated that the blood to tissue volume ratio and the perfusion pressure have the strongest effect on the effective stress-strain response of a representative tissue cell. Tissue viscoelasticity increased the sensitivity of the system to perfusion pressure changes.     

Material and functional properties of articular cartilage and patellofemoral contact mechanics in an experimental model of osteoarthritis

The purposes of this study were to determine the in situ functional and material properties of articular cartilage in an experimental model of joint injury, and to quantify the corresponding in situ joint contact mechanics. Experiments were performed in the anterior cruciate ligament (ACL) transected knee of the cat and the corresponding, intact contralateral knee, 16 weeks following intervention. Cartilage thickness, stiffness, effective Young’s modulus, and permeability were measured and derived from six locations of the knee. The total contact area and peak pressures in the patellofemoral joint were obtained in situ using Fuji Pressensor film, and comparisons between experimental and contralateral joint were made for corresponding loading conditions. Total joint contact area and peak pressure were increased and decreased significantly (=0.01), respectively, in the experimental compared to the contralateral joint. Articular cartilage thickness and stiffness were increased and decreased significantly (=0.01), respectively, in the experimental compared to the contralateral joint in the four femoral and patellar test locations. Articular cartilage material properties (effective Young’s modulus and permeability) were the same in the ACL-transected and intact joints. These results demonstrate for the first time the effect of changes in articular cartilage properties on the load transmission across a joint. They further demonstrate a substantial change in the joint contact mechanics within 16 weeks of ACL transection. The results were corroborated by theoretical analysis of the contact mechanics in the intact and ACL-transected knee using biphasic contact analysis and direct input of cartilage properties and joint surface geometry from the experimental animals. We conclude that the joint contact mechanics in the ACL-transected cat change within 16 weeks of experimental intervention.     

Effects of enzymatic degradation on the frictional response of articular cartilage in stress relaxation

It was recently shown experimentally that the friction coefficient of articular cartilage correlates with the interstitial fluid pressurization, supporting the hypothesis that interstitial water pressurization plays a fundamental role in the frictional response by supporting most of the load during the early time response. A recent study showed that enzymatic treatment with chondroitinase ABC causes a decrease in the maximum fluid load support of bovine articular cartilage in unconfined compression. The hypothesis of this study is that treatment with chondroitinase ABC will increase the friction coefficient of articular cartilage in stress relaxation. Articular cartilage samples (n = 34) harvested from the femoral condyles of five bovine knee joints (1-3 months old) were tested in unconfined compression with simultaneous continuous sliding (+/-1.5 mm at 1 mm/s) under stress relaxation. Results showed a significantly higher minimum friction coefficient in specimens treated with 0.1 micro/ml of chondroitinase ABC for 24 h (micro(min) = 0.082+/-0.024) compared to control specimens (micro(min) = 0.047+/-0.014). Treated samples also exhibited higher equilibrium friction coefficient (micro(eq) = 0.232+/-0.049) than control samples (micro(eq) = 0.184+/-0.036), which suggest that the frictional response is greatly influenced by the degree of tissue degradation. The fluid load support was predicted from theory, and the maximum value (as a percentage of the total applied load) was lower in treated specimens (77+/-12%) than in control specimens (85+/-6%). Based on earlier findings, the increase in the ratio micro(min)/micro(eq) may be attributed to the decrease in fluid load support.     

The effect of loading rate on the development of early damage in articular cartilage

Experimental reports suggest that cartilage damage depends on strain magnitude. Additionally, because of its poro-viscoelastic nature, strain magnitude in cartilage can depend on strain rate. The present study explores whether cartilage damage may develop dependent on strain rate, even when the presented damage numerical model is strain-dependent but not strain-rate-dependent. So far no experiments have been distinguished whether rate-dependent cartilage damage occurs in the collagen or in the non-fibrillar network. Thus, this research presents a finite element analysis model where, among others, collagen and non-fibrillar matrix are incorporated as well as a strain-dependent damage mechanism for these components. Collagen and non-fibrillar matrix stiffness decrease when a given strain is reached until complete failure upon reaching a maximum strain. With such model, indentation experiments at increasing strain rates were simulated on cartilage plugs and damage development was monitored over time. Collagen damage increased with increasing strain rate from 21 to 42 %. In contrast, damage in the non-fibrillar matrix decreased with increasing strain rates from 72 to 34 %. Damage started to develop at a depth of approximately 20 % of the sample height, and this was more pronounced for the slow and modest loading rates. However, the most severe damage at the end of the compression step occurred at the surface for the plugs subjected to 120 mm/min strain rate. In conclusion, the present study confirms that the location and magnitude of damage in cartilage may be strongly dependent on strain rate, even when damage occurs solely through a strain-dependent damage mechanism.     

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.     

The mechanical environment of the chondrocyte: a biphasic finite element model of cell-matrix interactions in articular cartilage

Mechanical compression of the cartilage extracellular matrix has a significant effect on the metabolic activity of the chondrocytes. However, the relationship between the stress–strain and fluid-flow fields at the macroscopic “tissue” level and those at the microscopic “cellular” level are not fully understood. Based on the existing experimental data on the deformation behavior and biomechanical properties of articular cartilage and chondrocytes, a multi-scale biphasic finite element model was developed of the chondrocyte as a spheroidal inclusion embedded within the extracellular matrix of a cartilage explant. The mechanical environment at the cellular level was found to be time-varying and inhomogeneous, and the large difference (3 orders of magnitude) in the elastic properties of the chondrocyte and those of the extracellular matrix results in stress concentrations at the cell–matrix border and a nearly two-fold increase in strain and dilatation (volume change) at the cellular level, as compared to the macroscopic level. The presence of a narrow “pericellular matrix” with different properties than that of the chondrocyte or extracellular matrix significantly altered the principal stress and strain magnitudes within the chondrocyte, suggesting a functional biomechanical role for the pericellular matrix. These findings suggest that even under simple compressive loading conditions, chondrocytes are subjected to a complex local mechanical environment consisting of tension, compression, shear, and fluid pressure. Knowledge of the local stress and strain fields in the extracellular matrix is an important step in the interpretation of studies of mechanical signal transduction in cartilage explant culture models.     

Diffusion tensor of water in model articular cartilage

We used Monte Carlo simulations of Brownian dynamics of water to study anisotropic water diffusion in an idealised model of articular cartilage. The main aim was to use the simulations as a tool for translation of the fractional anisotropy of the water diffusion tensor in cartilage into quantitative characteristics of its collagen fibre network. The key finding was a linear empirical relationship between the collagen volume fraction and the fractional anisotropy of the diffusion tensor. Fractional anisotropy of the diffusion tensor is potentially a robust indicator of the microstructure of the tissue because, to a first approximation, it is invariant to inclusion of proteoglycans or chemical exchange between free and collagen-bound water in the model. We discuss potential applications of Monte Carlo diffusion-tensor simulations for quantitative biophysical interpretation of magnetic resonance diffusion-tensor images of cartilage. Extension of the model to include collagen fibre disorder is also discussed.     

A comparison of healthy human and swine articular cartilage dynamic indentation mechanics

Articular cartilage is a multicomponent, poroviscoelastic tissue with nonlinear mechanical properties vital to its function. A consequent goal of repair or replacement of injured cartilage is to achieve mechanical properties in the repair tissue similar to healthy native cartilage. Since fresh healthy human articular cartilage (HC) is not readily available, we tested whether swine cartilage (SC) could serve as a suitable substitute for mechanical comparisons. To a first approximation, cartilage tissue and surgical substitutes can be evaluated mechanically as viscoelastic materials. Stiffness measurements (dynamic modulus, loss angle) are vital to function and are also a non-destructive means of evaluation. Since viscoelastic material stiffness is strongly strain rate dependent, stiffness was tested under different loading conditions related to function. Stiffness of healthy HC and SC specimens was determined and compared using two non-destructive, mm-scale indentation test modes: fast impact and slow sinusoidal deformation. Deformation resistance (dynamic modulus) and energy handling (loss angle) were determined. For equivalent anatomic locations, there was no difference in dynamic modulus. However, the HC loss angle was ~35% lower in fast impact and ~12% higher in slow sinusoidal mode. Differences seem attributable to age (young SC, older HC) but also to species anatomy and biology. Test mode-related differences in human-swine loss angle support use of multiple function-related test modes. Keeping loss angle differences in mind, swine specimens could serve as a standard of comparison for mechanical evaluation of e.g. engineered cartilage or synthetic repair materials.     

The effect of retinoic acid on proteoglycan biosynthesis in bovine articular cartilage cultures

The addition of retinoic acid to adult bovine articular cartilage cultures produces a concentration-dependent decrease in both proteoglycan synthesis and the proteoglycan content of the tissue. Total protein synthesis was not affected by the presence of retinoic acid, indicating that the inhibition of proteoglycan synthesis was not due to cytotoxicity. The proteoglycans synthesized in the presence of retinoic acid were similar in hydrodynamic size, ability to form aggregates with hyaluronate, and glycosaminoglycan composition to those of control cultures. However, the presence of larger glycosaminoglycan chains suggests that the core protein was substituted with fewer but longer glycosaminoglycan chains. In cultures maintained with retinoic acid, a decreased ratio of the large proteoglycan was synthesized relative to the small proteoglycan compared to that measured in control cultures. In cultures maintained with retinoic acid for 1 day and then switched to medium with 20% (v/v) fetal calf serum, the rate of proteoglycan synthesis and hexuronate contents increased within 5 days to levels near those of control cultures. Within 2 days of switching to medium with 20% (v/v) fetal calf serum, the relative proportions of the proteoglycan species were similar to those produced in cultures maintained in medium with 20% (v/v) fetal calf serum throughout. The rate of proteoglycan synthesis by bovine articular cartilage cultures exhibited an exponential decay following exposure to retinoic acid, with estimated half-lives of 11.5 and 5.3 h for tissue previously maintained in medium alone or containing 20% (v/v) fetal calf serum, respectively. The addition of 1 mM benzyl beta-D-xyloside only partially reversed the retinoic acid-mediated inhibition of proteoglycan synthesis. This indicates that the inhibition of proteoglycan synthesis by retinoic acid was due to both a decreased availability of xylosylated core protein and a decreased capacity of the chondrocytes to synthesize chondroitin sulfate chains.     

The effect of benzyl beta-D-xyloside on keratan sulphate synthesis in ox articular cartilage

Cartilage from adult bovine hock joints was incubated with [3H]galactose or [35S]sulphate in the presence of benzyl beta-D-xyloside. Radioisotope incorporation into proteoglycan was inhibited by the xyloside; the magnitude of this inhibition depended on the concentration of xyloside used. With 0.2mM xyloside radioisotope incorporation into keratan sulphate was not altered but inhibition was observed at xyloside concentrations of 1.0mM or higher. The decrease in radioisotope incorporation into keratan sulphate in the presence of 1.0mM benzyl beta-xyloside was directly related to a reduction in the average length of the keratan sulphate chains. This effect of beta-xyloside on keratan sulphate biosynthesis was markedly different from its effect on chondroitin sulphate biosynthesis.