A fibril reinforced nonhomogeneous poroelastic model for articular cartilage: inhomogeneous response in unconfined compression

The depth dependence of material properties of articular cartilage, known as the zonal differences, is incorporated into a nonlinear fibril-reinforced poroelastic model developed previously in order to explore the significance of material heterogeneity in the mechanical behavior of cartilage. The material variations proposed are based on extensive observations. The collagen fibrils are modeled as a distinct constituent which reinforces the other two constituents representing proteoglycans and water. The Young's modulus and Poisson's ratio of the drained nonfibrillar matrix are so determined that the aggregate compressive modulus for confined geometry fits the experimental data. Three nonlinear factors are considered, i.e. the effect of finite deformation, the dependence of permeability on dilatation and the fibril stiffening with its tensile strain. Solutions are extracted using a finite element procedure to simulate unconfined compression tests. The features of the model are then demonstrated with an emphasis on the results obtainable only with a nonhomogeneous model, showing reasonable agreement with experiments. The model suggests mechanical behaviors significantly different from those revealed by homogeneous models: not only the depth variations of the strains which are expected by qualitative analyses, but also, for instance, the relaxation-time dependence of the axial strain which is normally not expected in a relaxation test. Therefore, such a nonhomogeneous model is necessary for better understanding of the mechanical behavior of cartilage.     

Alterations in mechanical behaviour of articular cartilage due to changes in depth varying material properties--a nonhomogeneous poroelastic model study

The depth dependence of the material properties is present in normal adult cartilage and is believed to have significant implications in its normal mechanical function. Cartilage pathology may alter the depth dependence, e.g. a reduced depth dependence of the fibril stiffness has been observed in osteoarthritic cartilage. The objective of the present study is to investigate the alterations in the mechanical response of articular cartilage when the depth dependence of the material properties is varied to simulate healthy and pathological situations. This study is made possible by a recently developed nonhomogeneous poroelastic model. Depth variations of the strains and stresses for individual material phases (collagen, proteoglycan and fluid) are obtained for cartilage disks in unconfined compression using the finite element method. The mean nominal axial strain considered is up to 15%, while the axial strain at the articular surface can reach 33%. This paper demonstrates how the mechanical behaviours of cartilage are affected by individual depth dependent cartilage properties, while such observations are not fully available in experimental investigations. This study suggests the possibility of diagnosing cartilage health by analysing its mechanical behaviours.     

Extracellular ATP and UTP stimulate cartilage proteoglycan and collagen accumulation in bovine articular chondrocyte pellet cultures

Bovine articular chondrocytes were maintained in high density pellet cultures with and without serum and nucleotide triphosphates for different periods of time. Despite half-lives in culture of about 3 h, adenosine triphosphate and uridine triphosphate in the presence of serum increased sulphated glycosaminoglycan and collagen deposition above control levels. In the presence of serum a single dose of uridine triphosphate on the first day of culture was sufficient to induce significant increases in subsequent proteoglycan and collagen deposition. We conclude that both adenine triphosphate and uridine triphosphate are anabolic for articular chondrocytes, and that this effect on the chondrocyte is long-term.     

Contribution of postnatal collagen reorientation to depth-dependent mechanical properties of articular cartilage

The collagen fibril network is an important factor for the depth-dependent mechanical behaviour of adult articular cartilage (AC). Recent studies show that collagen orientation is parallel to the articular surface throughout the tissue depth in perinatal animals, and that the collagen orientations transform to a depth-dependent arcade-like structure in adult animals. Current understanding on the mechanobiology of postnatal AC development is incomplete. In the current paper, we investigate the contribution of collagen fibril orientation changes to the depth-dependent mechanical properties of AC. We use a composition-based finite element model to simulate in a 1-D confined compression geometry the effects of ten different collagen orientation patterns that were measured in developing sheep. In initial postnatal life, AC is mostly subject to growth and we observe only small changes in depth-dependent mechanical behaviour. Functional adaptation of depth-dependent mechanical behaviour of AC takes place in the second half of life before puberty. Changes in fibril orientation alone increase cartilage stiffness during development through the modulation of swelling strains and osmotic pressures. Changes in stiffness are most pronounced for small stresses and for cartilage adjacent to the bone. We hypothesize that postnatal changes in collagen fibril orientation induce mechanical effects that in turn promote these changes. We further hypothesize that a part of the depth-dependent postnatal increase in collagen content in literature is initiated by the depth-dependent postnatal increase in fibril strain due to collagen fibril reorientation.     

Effect of calcipenia on proteoglycan metabolism and aggregation in normal articular cartilage in vitro.

Glycosaminoglycan synthesis in normal adult dog knee cartilage cultured in medium containing 0, 0.3 MM- and 0.9 mM-Ca2+ was 52, 67 and 78%, respectively, of that in cartilage from the same joints cultured in a normal concentration of Ca2+, i.e. 1.8 mM. Pulse-chase experiments indicated that the rate of degradiation of glycosaminoglycans in cartilage cultured in the absence of Ca2+ was similar to that of glycosaminoglycans in cartilage cultured in 1.8 mM-Ca2+. Although [35S]sulphate incorporation into glycosaminoglycans was decreased in the presence of calcipenia, [3H]leucine incorporation into protein was unaffected. The average hydrodynamic size of newly synthesized proteoglycan aggregates and purified disaggregated proteoglycans from cartilage cultured in the absence of Ca2+ was similar to that of aggregates and disaggregated proteoglycans from cartilage cultured in 1.8 mM-Ca2+.     

An immunoelectron microscope study of the organization of proteoglycan monomer, link protein, and collagen in the matrix of articular cartilage

Monospecific antibodies to bovine cartilage proteoglycan monomer (PG) and link protein (LP) have been used with immunoperoxidase electron microscopy to study the distribution and organization of these molecules in bovine articular cartilage. The following observations were made: (a) The interterritorial matrix of the deep zone contained discrete interfibrillar particulate staining for PG and LP. This particulate staining, which was linked by faint bands of staining (for PG) or filaments (for LP), was spaced at 75- to 80-nm intervals. On collagen fibrils PG was also detected as particulate staining spaced at regular intervals (72 nm), corresponding to the periodicity of collagen cross-banding. The interfibrillar PG staining was often linked to the fibrillar PG staining by the same bands or filaments. The latter were cleaved by a proteinase-free Streptomyces hyaluronidase with the removal of much of the interfibrillar lattice. Since this enzyme has a specificity for hyaluronic acid, the observations indicate that the lattice contains a backbone of hyaluronic acid (which appeared as banded or filamentous staining) to which is attached LP and PG, the latter collapsing when the tissue is fixed, reacted with antibodies, and prepared for electron microscopy. Thishyaluronic acid is anchored to collagen fibrils at regular intervals where PG is detected on collagen. PG and LP detected by antibody in the interterritorial zones are essentially fully extractible with 4 M guanidine hydrochloride. These observations indicated that interfibrillar PG and LP is aggregated with HA in this zone. (b) The remainder of the cartilage matrix had a completely different organization of PG and LP. There was no evidence of a similar latticework based on hyaluronic acid. Instead, smaller more closely packed particulate staining for PG was seen everywhere irregularly distributed over and close to collagen fibrils. LP was almost undetectable in the territorial matrix of the deep zone, as observed previously. In the middle and superficial zones, stronger semiparticulate staining for LP was distributed over collagen fibrils. (c) In the superficial zone, reaction product for PG was distributed evenly on collagen fibrils as diffuse staining and also irregularly as particulate staining. LP was observed as semiparticulate staining over collagen fibrils. The diffuse staining for PG remained after extraction with 4 M guanidine hydrochloride. (d) In pericellular matrix, most clearly identified in middle and deep zones, the nature and organization of reaction product for PG and LP were similar to those observed in the territorial matrix, except that LP and PG were more strongly stained and amorphous staining for both components was also observed. (e) This study demonstrates striking regional variations of ultrastructural organization of PG and LP in articular cartilage...     

The inability to prepare high-buoyant-density proteoglycan aggregates from extracts of normal adult human articular cartilage.

High-buoyant-density proteoglycan aggregates could not be prepared from extracts of adult human cartilage by associative CsCl-density-gradient centrifugation with a starting density of 1.68 g/ml, even though proteoglycan subunits, hyaluronic acid and link proteins were all present. In contrast, aggregates could be prepared when extracts of neonatal human cartilage or bovine nasal cartilage were subjected to the same procedure. This phenomenon did not appear to be due to a defect within the hyaluronic acid-binding region of the adult proteoglycan subunit, but rather to an interference in the stability of the interaction between the proteoglycan subunit and hyaluronic acid towards centrifugation. The factor responsible for this instability was shown to reside within the low-density cartilage protein preparation obtained by direct dissociative CsCl-density-gradient centrifugation of the adult cartilage extract.     

Age-related changes in the structure of proteoglycan link proteins present in normal human articular cartilage.

Reduced-minus-oxidized difference spectra were recorded on particle preparations of the cyanobacterium Anacystis nidulans. Physiological oxidation of anaerobic membranes was effected either by O2 or by light. In both cases the spectral changes observed in the 550-570nm region were essentially the same. The results were confirmed by dual-wavelength spectrophotometry. It is concluded that a membrane-bound cytochrome f-b complex participates in both respiratory and photosynthetic elevtron transport.     

Effects of copper and zinc on proteoglycan metabolism in articular cartilage

Co-Cultures of porcine articular cartilage and synovium or synovial conditioned medium were used as an in vitro model to mimic inflammatory events at the cartilage/synovial junction in degenerative joint disease. This model provides a useful tool to assess the anti-inflammatory and antiarthritic properties of pharmacological agents. In this study the effects of copper and zinc on (i) PG synthesis by cartilage and (ii) synovial-induced PG depletion have been investigated. Copper sulphate at a concentration of 0.01 mM did not stimulate PG synthesis significantly in cultured cartilage explants but completely abrogated the inhibitory effects of synovial tissue in co-culture experiments. This finding was supported by the histological demonstration of copper-dependent reversal of the PG depletion in cartilage exposed to synovial conditioned medium. Zinc sulphate at 0.01 mM had no effect on PG synthesis and was unable to protect cartilage against synovialinduced PG depletion. These results reveal possible mechanisms by which copper exerts its anti-inflammatory and anti-arthritic actions.     

Differential responses of chondrocytes from normal and osteoarthritic human articular cartilage to mechanical stimulation

Mechanical stimulation is critically important for the maintenance of normal articular cartilage integrity. Molecular events regulating responses of chondrocytes to mechanical forces are beginning to be defined. Chondrocytes from normal human knee joint articular cartilage show increased levels of aggrecan mRNA following 0.33 Hz mechanical stimulation whilst at the same time relative levels of MMP3 mRNA are decreased. This anabolic response, associated with membrane hyperpolarisation, is activated via an integrin-dependent interleukin (IL)-4 autocrine/paracrine loop. Work in our laboratory suggests that this chondroprotective response may be aberrant in osteoarthritis (OA). Chondrocytes from OA cartilage show no changes in aggrecan or MMP3 mRNA following 0.33 Hz mechanical stimulation. alpha5beta1 integrin is the mechanoreceptor in both normal and OA chondrocytes but downstream signalling pathways differ. OA chondrocytes show membrane depolarisation following 0.33 Hz mechanical stimulation consequent to activation of an IL1beta autocrine/paracrine loop. IL4 signalling in OA chondrocytes is preferentially through the type I (IL4alpha/cgamma) receptor rather than via the type II (IL4alpha/IL13R) receptor. Altered mechanotransduction and signalling in OA may contribute to changes in chondrocyte behaviour leading to increased cartilage breakdown and disease progression.     

An elasto-viscoplastic model to describe the ratcheting behavior of articular cartilage

In the present work, a constitutive model for articular cartilage is proposed in finite elasto-viscoplasticity. For simplification, articular cartilage is supposed to be a typical composite composed of a soft basis and a fiber assembly. The stress tensor and free energy function are hence accordingly divided into two components. The high nonlinear stress-strain response is assumed to be mainly related to the fiber assembly and described by an exponential-type hypoelastic relation. Ratcheting is considered according to the viscoplasticity, the evolution rule of which is deduced from the dissipative inequality by the co-directionality hypotheses. Then, the capability of the proposed model is validated by comparing its predictions with related experimental observations. Results show that the ratcheting behavior and stress-strain hysteresis loops are reasonably captured by the proposed model.     

Regional assessment of articular cartilage gene expression and small proteoglycan metabolism in an animal model of osteoarthritis

Osteoarthritis (OA), the commonest form of arthritis and a major cause of morbidity, is characterized by progressive degeneration of the articular cartilage. Along with increased production and activation of degradative enzymes, altered synthesis of cartilage matrix molecules and growth factors by resident chondrocytes is believed to play a central role in this pathological process. We used an ovine meniscectomy model of OA to evaluate changes in chondrocyte expression of types I, II and III collagen; aggrecan; the small leucine-rich proteoglycans (SLRPs) biglycan, decorin, lumican and fibromodulin; transforming growth factor-β; and connective tissue growth factor. Changes were evaluated separately in the medial and lateral tibial plateaux, and were confirmed for selected molecules using immunohistochemistry and Western blotting. Significant changes in mRNA levels were confined to the lateral compartment, where active cartilage degeneration was observed. In this region there was significant upregulation in expession of types I, II and III collagen, aggrecan, biglycan and lumican, concomitant with downregulation of decorin and connective tissue growth factor. The increases in type I and III collagen mRNA were accompanied by increased immunostaining for these proteins in cartilage. The upregulated lumican expression in degenerative cartilage was associated with increased lumican core protein deficient in keratan sulphate side-chains. Furthermore, there was evidence of significant fragmentation of SLRPs in both normal and arthritic tissue, with specific catabolites of biglycan and fibromodulin identified only in the cartilage from meniscectomized joints. This study highlights the focal nature of the degenerative changes that occur in OA cartilage and suggests that altered synthesis and proteolysis of SLRPs may play an important role in cartilage destruction in arthritis.     

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.     

Effects of ceramide on apoptosis, proteoglycan degradation, and matrix metalloproteinase expression in rabbit articular cartilage

Cartilage loss in osteoarthritis is characterized by matrix degradation and chondrocyte death. The lipid messenger ceramide is implicated in signal transduction of the catabolic cytokines tumor necrosis factor (TNF) and interleukin-1 (IL-1), as well as in apoptosis. The aim of this study was to examine the in vitro effects of ceramide on proteoglycan degradation, matrix-metalloproteinase (MMP) expression and activity, and chondrocyte apoptosis in rabbit articular cartilage. Cell-permeant ceramide C(2) stimulated proteoglycan degradation in cartilage explants starting from 3 x 10(-5) M, with 100% increase at the dose of 10(-4) M. This effect was probably due to MMPs since it was blocked by the MMP inhibitor batimastat. Furthermore, in isolated chondrocytes, C(2) stimulated the expression of MMP-1, 3, and 13 at the mRNA level, MMP activity, and MMP-3 production. Ceramide also caused chondrocyte apoptosis at doses ranging from 10(-5) to 10(-4) M. This study supports the hypothesis that ceramide might play a mediatory role in both matrix degradation and apoptosis in processes of cartilage loss such as those observed in osteoarthritis.     

Changes in proteoglycan synthesis of chondrocytes in articular cartilage are associated with the time-dependent changes in their mechanical environment

Explant loading experiments were conducted to investigate the effect of load duration on proteoglycan synthesis. A compressive load of 0.1 MPa applied for 10 min was found to stimulate proteoglycan synthesis, while the same load applied for 20 h suppressed synthesis. This bimodal response suggests that the cells are responding to different mechanical stimuli as time progresses. A theoretical model has therefore been developed to describe the mechanical environment perceived by cells within soft hydrated tissues (e.g. articular cartilage) while the tissue is being loaded. The cells are modeled, using the biphasic theory, as fluid-solid inclusions embedded in and attached to a biphasic extracellular matrix of distinct material properties. A method of solution is developed which is valid for any axisymmetric loading configuration, provided that the cell radius, a, is small relative to the tissue height, h (i.e. h/a 1). A closed-form analytical solution for this inclusion problem is then presented for the confined compression configuration. Results from this model show that the mechanical environment in and around the cells is time dependent and inhomogeneous, and can be significantly influenced by differences in properties between the cell and the extracellular matrix.     

Interaction between proteoglycan subunit and type II collagen from bovine nasal cartilage, and the preferential binding of proteoglycan subunit to type I collagen.

We studied the interaction of proteoglycan subunit with both types I and II collagen. All three molecular species were isolated from the ox. Type II collagen, prepared from papain-digested bovine nasal cartilage, was characterized by gel electrophoresis, amino acid analysis and CM-cellulose chromatography. By comparison of type I collagen, prepared from papain-digested calf skin, with native calf skin acid-soluble tropocollagen, we concluded that the papain treatment left the collagen molecules intact. Interactions were carried out at 4 degrees C in 0.06 M-sodium acetate, pH 4.8, and the results were studied by two slightly different methods involving CM-cellulose chromatography and polyacrylamide-gel electrophoresis. It was demonstrated that proteoglycan subunit, from bovine nasal cartilage, bound to cartilage collagen. Competitive-interaction experiments showed that, in the presence of equal amounts of calf skin acid-soluble tropocollagen (type I) and bovine nasal cartilage collagen (type II), proteoglycan subunit bound preferentially to the type I collagen. We suggest from these results that, although not measured under physiological conditions, it is unlikely that the binding in vivo between type II collagen and proteoglycan is appreciably stronger than that between type I collagen and proteoglycan.     

Synthesis of fibronectin in normal and osteoarthritic articular cartilage

The content and the biosynthesis of fibronectin was examined in disease-free articular cartilage and in articular cartilage from osteoarthritic canine joints. Fibronectin content was increased in extracts of cartilage from osteoarthritic joints. Incubation of cartilage in vitro with [³H]phenylalanine and subsequent isolation of [³H]fibronectin from a gelatin affinity column and characterization by SDS-polyacrylamide gel electrophoresis and by immunoprecipitation indicated that disease-free and osteoarthritic cartilage explants synthesized fibronectin. About 50% of the [³H]fibronectin was recovered in the incubation medium. The osteoarthritic cartilage synthesized and accumulated up to 5-fold more [³H]fibronectin than disease-free cartilage.     

Comparison of single-phase isotropic elastic and fibril-reinforced poroelastic models for indentation of rabbit articular cartilage

Classically, single-phase isotropic elastic (IE) model has been used for in situ or in vivo indentation analysis of articular cartilage. The model significantly simplifies cartilage structure and properties. In this study, we apply a fibril-reinforced poroelastic (FRPE) model for indentation to extract more detailed information on cartilage properties. Specifically, we compare the information from short-term (instantaneous) and long-term (equilibrium) indentations, as described here by IE and FRPE models. Femoral and tibial cartilage from rabbit (age 0–18 months) knees (n=14) were tested using a plane-ended indenter (diameter=0.544 mm). Stepwise creep tests were conducted to equilibrium. Single-phase IE solution for indentation was used to derive instantaneous modulus and equilibrium (Young's) modulus for the samples. The classical and modified Hayes’ solutions were used to derive values for the indentation moduli. In the FRPE model, the indentation behavior was sample-specifically described with three material parameters, i.e. fibril network modulus, non-fibrillar matrix modulus and permeability. The instantaneous and fibril network modulus, and the equilibrium Young's modulus and non-fibrillar matrix modulus showed significant (p<0.01) linear correlations of R²=0.516 and 0.940, respectively (Hayes’ solution) and R²=0.531 and 0.960, respectively (the modified Hayes’ solution). No significant correlations were found between the non-fibrillar matrix modulus and instantaneous moduli or between the fibril network modulus and the equilibrium moduli. These results indicate that the instantaneous indentation modulus (IE model) provides information on tensile stiffness of collagen fibrils in cartilage while the equilibrium modulus (IE model) is a significant measure for stiffness of PG matrix. Thereby, this study highlights the feasibility of a simple indentation analysis.