Contribution of tissue composition and structure to mechanical response of articular cartilage under different loading geometries and strain rates

Mechanical function of articular cartilage in joints between articulating bones is dependent on the composition and structure of the tissue. The mechanical properties of articular cartilage are traditionally tested in compression using one of the three loading geometries, i.e., confined compression, unconfined compression or indentation. The aim of this study was to utilize a composition-based finite element model in combination with a fractional factorial design to determine the importance of different cartilage constituents in the mechanical response of the tissue, and to compare the importance of the tissue constituents with different loading geometries and loading rates. The evaluated parameters included water and collagen fraction as well as fixed charge density on cartilage surface and their slope over the tissue thickness. The thicknesses of superficial and middle zones, as based on the collagen orientation, were also included in the evaluated parameters. A three-level resolution V fractional factorial design was used. The model results showed that inhomogeneous composition plays only a minor role in indentation, though that role becomes more significant in confined compression and unconfined compression. In contrast, the collagen architecture and content had a more profound role in indentation than with two other loading geometries. These differences in the mechanical role of composition and structure between the loading geometries were emphasized at higher loading rates. These findings highlight how the results from mechanical tests of articular cartilage under different loading conditions are dependent upon tissue composition and structure.     

The effect of tissue-engineered cartilage biomechanical and biochemical properties on its post-implantation mechanical behavior

The insufficient load-bearing capacity of today’s tissue-engineered (TE) cartilage limits its clinical application. Focus has been on engineering cartilage with enhanced mechanical stiffness by reproducing native biochemical compositions. More recently, depth dependency of the biochemical content and the collagen network architecture has gained interest. However, it is unknown whether the mechanical performance of TE cartilage would benefit more from higher content of biochemical compositions or from achieving an appropriate collagen organization. Furthermore, the relative synthesis rate of collagen and proteoglycans during the TE process may affect implant performance. Such insights would assist tissue engineers to focus on those aspects that are most important. The aim of the present study is therefore to elucidate the relative importance of implant ground substance stiffness, collagen content, and collagen architecture of the implant, as well as the synthesis rate of the biochemical constituents for the post-implantation mechanical behavior of the implant. We approach this by computing the post-implantation mechanical conditions using a composition-based fibril-reinforced poro-viscoelastic swelling model of the medial tibia plateau. Results show that adverse implant composition and ultrastructure may lead to post-implantation excessive mechanical loads, with collagen orientation being the most critical variable. In addition, we predict that a faster synthesis rate of proteoglycans compared to that of collagen during TE culture may result in excessive loads on collagen fibers post-implantation. This indicates that even with similar final contents, constructs may behave differently depending on their development. Considering these aspects may help to engineer TE cartilage implants with improved survival rates.     

On the anisotropy and inhomogeneity of permeability in articular cartilage

Articular cartilage is known to be anisotropic and inhomogeneous because of its microstructure. In particular, its elastic properties are influenced by the arrangement of the collagen fibres, which are orthogonal to the bone-cartilage interface in the deep zone, randomly oriented in the middle zone, and parallel to the surface in the superficial zone. In past studies, cartilage permeability has been related directly to the orientation of the glycosaminoglycan chains attached to the proteoglycans which constitute the tissue matrix. These studies predicted permeability to be isotropic in the undeformed configuration, and anisotropic under compression. They neglected tissue anisotropy caused by the collagen network. However, magnetic resonance studies suggest that fluid flow is "directed" by collagen fibres in biological tissues. Therefore, the aim of this study was to express the permeability of cartilage accounting for the microstructural anisotropy and inhomogeneity caused by the collagen fibres. Permeability is predicted to be anisotropic and inhomogeneous, independent of the state of strain, which is consistent with the morphology of the tissue. Looking at the local anisotropy of permeability, we may infer that the arrangement of the collagen fibre network plays an important role in directing fluid flow to optimise tissue functioning.     

Stresses in the local collagen network of articular cartilage: a poroviscoelastic fibril-reinforced finite element study

Osteoarthritis (OA) is a multifactorial disease, resulting in diarthrodial joint wear and eventually destruction. Swelling of cartilage, which is proportional to the amount of collagen damage, is an initial event of cartilage degeneration, so damage to the collagen fibril network is likely to be one of the earliest signs of OA cartilage degeneration. We propose that the local stresses and strains in the collagen fibrils, which cause the damage, cannot be determined dependably without taking the local arcade-like collagen-fibril structure into account. We investigate this using a poroviscoelastic fibril-reinforced FEA model. The constitutive fibril properties were determined by fitting numerical data to experimental results of unconfined compression and indentation tests on samples of bovine patellar articular cartilage. It was demonstrated that with this model the stresses and strains in the collagen fibrils can be calculated. It was also exhibited that fibrils with different orientations at the same location can be loaded differently, depending on the local architecture of the collagen network. To the best of our knowledge, the present model is the first that can account for these features. We conclude that the local stresses and strains in the articular cartilage are highly influenced by the local morphology of the collagen-fibril network.     

A transversely isotropic, transversely homogeneous microstructural-statistical model of articular cartilage

Articular cartilage is a multi-phasic, composite, fibre-reinforced material. Therefore, its mechanical properties are determined by the tissue microstructure. The presence of cells (chondrocytes) and collagen fibres within the proteoglycan matrix influences, at a local and a global level, the material symmetries. The volumetric concentration and shape of chondrocytes, and the volumetric concentration and spatial arrangement of collagen fibres have been observed to change as a function of depth in articular cartilage. In particular, collagen fibres are perpendicular to the bone-cartilage interface in the deep zone, their orientation is almost random in the middle zone, and they are parallel to the surface in the superficial zone. The aim of this work is to develop a model of elastic properties of articular cartilage based on its microstructure. In previous work, we addressed this problem based on Piola's notation for fourth-order tensors. Here, mathematical tools initially developed for transversely isotropic composite materials comprised of a statistical orientation of spheroidal inclusions are extended to articular cartilage, while taking into account the dependence of the elastic properties on cartilage depth. The resulting model is transversely isotropic and transversely homogeneous (TITH), the transverse plane being parallel to the bone-cartilage interface and the articular surface. Our results demonstrate that the axial elastic modulus decreases from the deep zone to the articular surface, a result that is in good agreement with experimental findings. Finite element simulations were carried out, in order to explore the TITH model's behaviour in articular cartilage compression tests. The force response, fluid flow and displacement fields obtained with the TITH model were compared with the classical linear elastic, isotropic, homogeneous (IH) model, showing that the IH model is unable to predict the non-uniform behaviour of the tissue. Based on considerations that the mechanical stability of the tissue depends on its topological and microstructural properties, our long-term goal is to clearly understand the stability conditions in topological terms, and the relationship with the growth and remodelling mechanisms in the healthy and diseased tissue.     

The Mechanical Behaviour of Chondrocytes Predicted with a Micro-structural Model of Articular Cartilage

The integrity of articular cartilage depends on the proper functioning and mechanical stimulation of chondrocytes, the cells that synthesize extracellular matrix and maintain tissue health. The biosynthetic activity of chondrocytes is influenced by genetic factors, environmental influences, extracellular matrix composition, and mechanical factors. The mechanical environment of chondrocytes is believed to be an important determinant for joint health, and chondrocyte deformation in response to mechanical loading is speculated to be an important regulator of metabolic activity. In previous studies of chondrocyte deformation, articular cartilage was described as a biphasic material consisting of a homogeneous, isotropic, linearly elastic solid phase, and an inviscid fluid phase. However, articular cartilage is known to be anisotropic and inhomogeneous across its depth. Therefore, isotropic and homogeneous models cannot make appropriate predictions for tissue and cell stresses and strains. Here, we modelled articular cartilage as a transversely isotropic, inhomogeneous (TI) material in which the anisotropy and inhomogeneity arose naturally from the microstructure of the depth-dependent collagen fibril orientation and volumetric fraction, as well as the chondrocyte shape and volumetric fraction. The purpose of this study was to analyse the deformation behaviour of chondrocytes using the TI model of articular cartilage. In order to evaluate our model against experimental results, we simulated indentation and unconfined compression tests for nominal compressions of 15%. Chondrocyte deformations were analysed as a function of location within the tissue. The TI model predicted a non-uniform behaviour across tissue depth: in indentation testing, cell height decreased by 43% in the superficial zone and between 11 and 29% in the deep zone. In unconfined compression testing, cell height decreased by 32% in the superficial zone, 25% in the middle, and 18% in the deep zones. This predicted non-uniformity is in agreement with experimental studies. The novelty of this study is the use of a cartilage material model accounting for the intrinsic inhomogeneity and anisotropy of cartilage caused by its microstructure.     

Relative contribution of articular cartilage’s constitutive components to load support depending on strain rate

Cartilage is considered a biphasic material in which the solid is composed of proteoglycans and collagen. In biphasic tissue, the hydraulic pressure is believed to bear most of the load under higher strain rates and its dissipation due to fluid flow determines creep and relaxation behavior. In equilibrium, hydraulic pressure is zero and load bearing is transferred to the solid matrix. The viscoelasticity of the collagen network also contributes to its time-dependent behavior, and the osmotic pressure to load bearing in equilibrium. The aim of the present study was to determine the relative contributions of hydraulic pressure, viscoelastic collagen stress, solid matrix stiffness and osmotic pressure to load carriage in cartilage under transient and equilibrium conditions. Unconfined compression experiments were simulated using a fibril-reinforced poroviscoelastic model of articular cartilage, including water, fibrillar viscoelastic collagen and non-fibrillar charged glycosaminoglycans. The relative contributions of hydraulic and osmotic pressures and stresses in the fibrillar and non-fibrillar network were evaluated in the superficial, middle and deep zone of cartilage under five different strain rates and after relaxation. Initially upon loading, the hydraulic pressure carried most of the load in all three zones. The osmotic swelling pressure carried most of the equilibrium load. In the surface zone, where the fibers were loaded in tension, the collagen network carried 20 % of the load for all strain rates. The importance of these fibers was illustrated by artificially modifying the fiber architecture, which reduced the overall stiffness of cartilage in all conditions. In conclusion, although hydraulic pressure dominates the transient behavior during cartilage loading, due to its viscoelastic nature the superficial zone collagen fibers carry a substantial part of the load under transient conditions. This becomes increasingly important with higher strain rates. The interesting and striking new insight from this study suggests that under equilibrium conditions, the swelling pressure generated by the combination of proteoglycans and collagen reinforcement accounts cartilage stiffness for more than 90 % of the loads carried by articular cartilage. This finding is different from the common thought that load is transferred from fluid to solid and is carried by the aggregate modulus of the solid. Rather, it is transformed from hydraulic to osmotic swelling pressure. These results show the importance of considering both (viscoelastic) collagen fibers as well as swelling pressure in studies of the (transient) mechanical behavior of cartilage.     

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 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 articulage were examined in detail in 7 joints within the age rangee 21 to 72 years. The results of 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 unixial 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 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, reveled that a large proportion of the proteoglycans was released from the cartilage by each of the freeze 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 relesed between 5.3 and 27.8 per cent of the collagen after 24 h.     

Dynamic compression augments interstitial transport of a glucose-like solute in articular cartilage

Solute transport through the extracellular matrix is essential for cellular activities in articular cartilage. Increased solute transport via fluid convection may be a mechanism by which dynamic compression stimulates chondrocyte metabolism. However, loading conditions that optimally augment transport likely vary for different solutes. To investigate effects of dynamic loading on transport of a bioactive solute, triangular mechanical loading waveforms were applied to cartilage explants disks while interstitial transport of a fluorescent glucose analog was monitored. Peak-to-peak compression amplitudes varied from 5-50% and frequencies varied from 0.0006-0.1 Hz to alter the spatial distribution and magnitude of oscillatory fluid flow. Solute transport was quantified by monitoring accumulation of fluorescence in a saline bath circulated around the explant. Individual explants were subjected to a series of compression protocols, so that effects of loading on solute desorption could be observed directly. Maximum increases in solute transport were obtained with 10-20% compression amplitudes at 0.1 Hz; similar loading protocols were previously found to stimulate chondrocyte metabolism in vitro. Results therefore support hypotheses relating to increased solute transport as a mediator of the cartilage biological response to dynamic compression, and may have application in mechanical conditioning of cartilage constructs for tissue engineering.     

The importance of superficial collagen fibrils for the function of articular cartilage

It has been proposed that the superficial tangential zone (STZ) of articular cartilage is essential to the tissue’s load-distributing function. However, the exact mechanism by which the STZ fulfills this function has not yet been revealed. Using a channel-indentation experiment, it was recently shown that compared to intact tissue, cartilage without STZ behaves slightly stiffer and deforms significantly different in regions adjacent to mechanically compressed areas (Bevill et al. in Osteoarthr Cartil 18:1310–1318, 2010). We aim to further explore the role of STZ in the load-transfer mechanism of AC by thorough biomechanical analysis of these experiments. Using our previously validated fibril-reinforced swelling model of articular cartilage, which accounts for the depth-dependent collagen structure and biochemical composition of articular cartilage, we simulated the above-mentioned channel-indenter compression experiments for both intact and STZ-removed cartilage. First, we show that the composition of the deep zone in cartilage is most effective in carrying cartilage compression, which explains the apparent tissue stiffening after STZ removal. Second, we show that tangential fibrils in the STZ are responsible for transferring compressive loads from directly loaded regions to adjacent tissue. Cartilage with an intact STZ has superior load-bearing properties compared to cartilage in which the STZ is compromised, because the STZ is able to recruit a larger area of deep zone cartilage to carry compressive loads.     

Long-term cyclical in vivo loading increases cartilage proteoglycan content in a spatially specific manner: an infrared microspectroscopic imaging and polarized light microscopy study

Understanding the changes in collagen and proteoglycan content of cartilage due to physical forces is necessary for progress in treating joint disorders, including those due to overuse. Physical forces in the chondrocyte environment can affect the cellular processes involved in the biosynthesis of extracellular matrix. In turn, the biomechanical properties of cartilage depend on its collagen and proteoglycan content. To understand changes due to physical forces, this study examined the effect of 80 cumulative hours of in vivo cyclical joint loading on the cartilage content of proteoglycan and collagen in the rabbit metacarpophalangeal joint. The forepaw digits of six anesthetized New Zealand White adult female rabbits were repetitively flexed at 1 Hz with an estimated joint contact pressure of 1 to 2 MPa. Joints were collected from loaded and contralateral control specimens, fixed, decalcified, embedded, and thin-sectioned. Sections were examined under polarized light microscopy to identify and measure superficial and mid zone thicknesses of cartilage. Fourier Transform Infrared microspectroscopy was used to measure proteoglycan and collagen contents in the superficial, mid, and deep zones. Loading led to an increase in proteoglycan in the cartilage of all six rabbits. Specifically, there was a 46% increase in the cartilage deep zone (p = 0.003). The collagen content did not change with loading. Joint loading did not change the superficial and mid zone mean thicknesses. We conclude that long-term (80 cumulative hours) cyclical in vivo joint loading stimulates proteoglycan synthesis. Furthermore, stimulation is localized to cartilage regions of high hydrostatic pressure. These data may be useful in developing interventions to prevent overuse injuries or in developing therapies to improve joint function.     

Remodeling of the collagen fiber architecture due to compaction in small vessels under tissue engineered conditions

Mechanical loading protocols in tissue engineering (TE) aim to improve the deposition of a properly organized collagen fiber network. In addition to collagen remodeling, these conditioning protocols can result in tissue compaction. Tissue compaction is beneficial to tissue collagen alignment, yet it may lead to a loss of functionality of the TE construct due to changes in geometry after culture. Here, a mathematical model is presented to relate the changes in collagen architecture to the local compaction within a TE small blood vessel, assuming that under static conditions, compaction is the main factor responsible for collagen fiber organization. An existing structurally based model is extended to incorporate volumetric tissue compaction. Subsequently, the model is applied to describe the collagen architecture of TE constructs under either strain based or stress based stimulus functions. Our computations indicate that stress based simulations result in a helical collagen fiber distribution along the vessel wall. The helix pitch angle increases from a circumferential direction in the inner wall, over about 45 deg in the middle vessel layer, to a longitudinal direction in the outer wall. These results are consistent with experimental data from TE small diameter blood vessels. In addition, our results suggest a stress dependent remodeling of the collagen, suggesting that cell traction is responsible for collagen orientation. These findings may be of value to design improved mechanical conditioning protocols to optimize the collagen architecture in engineered tissues.     

A computational model to describe the collagen orientation in statically cultured engineered tissues

Collagen provides cardiovascular tissues with the ability to withstand haemodynamic loads. A similar network is essential to obtain in tissue-engineered (TE) samples of the same nature. Yet, the mechanism of collagen orientation is not fully understood. Typically collagen remodelling is linked to mechanical loading. However, TE constructs also show an oriented collagen network when developed under static culture. Experiments under these conditions also indicate that the tissue gradually compacts due to contractile stresses developed in the α-actin fibres of the cells. Therefore, it is hypothesised that cellular contractile stresses are responsible for collagen orientation. A model describing the cellular α-actin turnover and the stresses developed by them is integrated in a structural constitutive model describing the mechanical behaviour of collagen fibres. Results show that the model can successfully capture the sample compaction, tissue stress generation and its heterogeneous collagen arrangement.     

Influence of tissue- and cell-scale extracellular matrix distribution on the mechanical properties of tissue-engineered cartilage

The insufficient load-bearing capacity of today’s tissue- engineered (TE) cartilage limits its clinical application. Generally, cartilage TE studies aim to increase the extracellular matrix (ECM) content, as this is thought to determine the load-bearing properties of the cartilage. However, there are apparent inconsistencies in the literature regarding the correlation between ECM content and mechanical properties of TE constructs. In addition to the amount of ECM, the spatial inhomogeneities in ECM distribution at the tissue scale as well as at the cell scale may affect the mechanical properties of TE cartilage. The relative importance of such structural inhomogeneities on mechanical behavior of TE cartilage is unknown. The aim of the present study was, therefore, to theoretically elucidate the influence of these inhomogeneities on the mechanical behavior of chondrocyte-agarose TE constructs. A validated non-linear fiber-reinforced poro-elastic swelling cartilage model that can accommodate for effects of collagen reinforcement and swelling by proteoglycans was used. At the tissue scale, ECM was gradually varied from predominantly localized in the periphery of the TE construct toward an ECM-rich inner core. The effect of these inhomogeneities in relation to the total amount of ECM was also evaluated. At the cell scale, ECM was gradually varied from localized in the pericellular area, toward equally distributed throughout the interterritorial area. Results from the tissue-scale model indicated that localization of ECM in either the construct periphery or in the inner core may reduce construct stiffness compared with that of constructs with homogeneous ECM. Such effects are more significant at high ECM amounts. At the cell scale, localization of ECM around the cells significantly reduced the overall stiffness, even at low ECM amounts. The compressive stiffness gradually increased when ECM distribution became more homogeneous and the osmotic swelling pressure in the interterritorial area increased. We conclude that for the same amount of ECM content in TE cartilage constructs, superior mechanical properties can be achieved with more homogeneous ECM distribution at both tissue and cell scale. Inhomogeneities at the cell scale are more important than those at the tissue scale.     

The generalized triphasic correspondence principle for simultaneous determination of the mechanical properties and proteoglycan content of articular cartilage by indentation

The triphasic mixture theory has been used to describe the mechanical and physicochemical behaviors of articular cartilage under some specialized loading conditions. However, the mathematical complexities of this theory have limited its applications for theoretical analyses of experimental studies and models for predicting cartilage and other biological tissues' deformational behaviors. A generalized correspondence principle has been established in the present study, and this principle shows that the equilibrium deformational behavior of a charged-hydrated material under loading is identical to that of an elastic medium without charge. A set of explicit formulas has been derived to correlate the mechanical properties of an equivalent material with the intrinsic elastic moduli, fixed charge density and free-ion concentration within the cartilage tissue. The validity of these formulas is independent of the deformation state of the elastic solid matrix under an infinitesimal strain. Therefore they can be employed for any loading conditions, such as confined or unconfined compression, tension, and indentation tests, etc. In the current study, the fixed charge density of bovine cartilage is determined from the indentation creep data using this generalized correspondence principle. The proteoglycan content results were then compared with those from biochemical assay, yielding a linear regression slope of 1.034. Additionally a correspondence principle within a framework of cubic symmetry and a bilinear response in tension-compression (the conewise linear elasticity model) has also been developed to demonstrate the potential application of current methodology for inhomogeneous, anisotropic and nonlinear situations.     

How preconditioning affects the measurement of poro-viscoelastic mechanical properties in biological tissues

It is known that initial loading curves of soft biological tissues are substantially different from subsequent loadings. The later loading curves are generally used for assessing the mechanical properties of a tissue, and the first loading cycles, referred to as preconditioning, are omitted. However, slow viscoelastic phenomena related to fluid flow or collagen viscoelasticity are initiated during these first preconditioning loading cycles and may persist during the actual data collection. When these data are subsequently used for fitting of material properties, the viscoelastic phenomena that occurred during the initial cycles are not accounted for. The aim of the present study is to explore whether the above phenomena are significant for articular cartilage, by evaluating the effect of such time-dependent phenomena by means of computational modeling. Results show that under indentation, collagen viscoelasticity dominates the time-dependent behavior. Under UC, fluid-dependent effects are more important. Interestingly, viscoelastic and poroelastic effects may act in opposite directions and may cancel each other out in a stress–strain curve. Therefore, equilibrium may be apparent in a stress–strain relationship, even though internally the tissue is not in equilibrium. Also, the time-dependent effects of viscoelasticity and poroelasticity may reinforce each other, resulting in a sustained effect that lasts longer than suggested by their individual effects. Finally, the results illustrate that data collected from a mechanical test may depend on the preconditioning protocol. In conclusion, preconditioning influences the mechanical response of articular cartilage significantly and therefore cannot be neglected when determining the mechanical properties. To determine the full viscoelastic and poroelastic properties of articular cartilage requires fitting to both preconditioning and post-preconditioned loading cycles.     

Depth-dependent analysis of the role of collagen fibrils, fixed charges and fluid in the pericellular matrix of articular cartilage on chondrocyte mechanics

The pericellular matrix of articular cartilage has been shown to regulate the mechanical environment of chondrocytes. However, little is known about the mechanical role of collagen fibrils in the pericellular matrix, and how fibrils might help modulate strains acting on chondrocytes when cartilage is loaded. The primary objective was to clarify the effect of pericellular collagen fibrils on cell volume changes and strains during cartilage loading. Secondary objectives were to investigate the effects of pericellular fixed charges and fluid on cell responses. A microstructural model of articular cartilage, in which chondrocytes and pericellular matrices were represented with depth-dependent structural and morphological properties, was created. The extracellular matrix and pericellular matrices were modeled as fibril-reinforced, biphasic materials with swelling capabilities, while chondrocytes were assumed to be isotropic and biphasic with swelling properties. Collagen fibrils in the extracellular matrix were represented with an arcade-like architecture, whereas pericellular fibrils were assumed to run tangential to the cell surface. In the early stages of a stress-relaxation test, pericellular fibrils were found to sensitively affect cell volume changes, even producing a reversal from increasing to decreasing cell volume with increasing fibril stiffness in the superficial zone. Consequently, steady-state volume of the superficial zone cell decreased with increasing pericellular fibril stiffness. Volume changes in the middle and deep zone chondrocytes were smaller and opposite to those observed in the superficial zone chondrocyte. An increase in the pericellular fixed charge density reduced cell volumes substantially in every zone. The sensitivity of cell volume changes to pericellular fibril stiffness suggests that pericellular fibrils play an important, and as of yet largely neglected, role in regulating the mechanical environment of chondrocytes, possibly affecting matrix synthesis during cartilage development and degeneration, and affecting biosynthetic responses associated with articular cartilage loading.     

Injurious mechanical compression of bovine articular cartilage induces chondrocyte apoptosis

A bovine cartilage explant system was used to evaluate the effects of injurious compression on chondrocyte apoptosis and matrix biochemical and biomechanical properties within intact cartilage. Disks of newborn bovine articular cartilage were compressed in vitro to various peak stress levels and chondrocyte apoptotic cell death, tissue biomechanical properties, tissue swelling, glycosaminoglycan loss, and nitrite levels were quantified. Chondrocyte apoptosis occurred at peak stresses as low as 4.5 MPa and increased with peak stress in a dose-dependent manner. This increase in apoptosis was maximal by 24 h after the termination of the loading protocol. At high peak stresses (>20 MPa), greater than 50% of cells apoptosed. When measured in uniaxial confined compression, the equilibrium and dynamic stiffness of explants decreased with the severity of injurious load, although this trend was not significant until 24-MPa peak stress. In contrast, the equilibrium and dynamic stiffness measured in radially unconfined compression decreased significantly after injurious stresses of 12 and 7 MPa, respectively. Together, these results suggested that injurious compression caused a degradation of the collagen fibril network in the 7- to 12-MPa range. Consistent with this hypothesis, injurious compression caused a dose-dependent increase in tissue swelling, significant by 13-MPa peak stress. Glycosaminoglycans were also released from the cartilage in a dose-dependent manner, significant by 6- to 13-MPa peak stress. Nitrite levels were significantly increased above controls at 20-MPa peak stress. Together, these data suggest that injurious compression can stimulate cell death as well as a range of biomechanical and biochemical alterations to the matrix and, possibly, chondrocyte nitric oxide expression. Interestingly, chondrocyte programmed cell death appears to take place at stresses lower than those required to stimulate cartilage matrix degradation and biomechanical changes. While chondrocyte apoptosis may therefore be one of the earliest responses to tissue injury, it is currently unclear whether this initial cellular response subsequently drives cartilage matrix degradation and changes in the biomechanical properties of the tissue.