Unconfined compression of articular cartilage: nonlinear behavior and comparison with a fibril-reinforced biphasic model

Mechanical behavior of articular cartilage was characterized in unconfined compression to delineate regimes of linear and nonlinear behavior, to investigate the ability of a fibril-reinforced biphasic model to describe measurements, and to test the prediction of biphasic and poroelastic models that tissue dimensions alter tissue stiffness through a specific scaling law for time and frequency. Disks of full-thickness adult articular cartilage from bovine humeral heads were subjected to successive applications of small-amplitude ramp compressions cumulating to a 10 percent compression offset where a series of sinusoidal and ramp compression and ramp release displacements were superposed. We found all equilibrium behavior (up to 10 percent axial compression offset) to be linear, while most nonequilibrium behavior was nonlinear, with the exception of small-amplitude ramp compressions applied from the same compression offset. Observed nonlinear behavior included compression-offset-dependent stiffening of the transient response to ramp compression, nonlinear maintenance of compressive stress during release from a prescribed offset, and a nonlinear reduction in dynamic stiffness with increasing amplitudes of sinusoidal compression. The fibril-reinforced biphasic model was able to describe stress relaxation response to ramp compression, including the high ratio of peak to equilibrium load. However, compression offset-dependent stiffening appeared to suggest strain-dependent parameters involving strain-dependent fibril network stiffness and strain-dependent hydraulic permeability. Finally, testing of disks of different diameters and rescaling of the frequency according to the rule prescribed by current biphasic and poroelastic models (rescaling with respect to the sample's radius squared) reasonably confirmed the validity of that scaling rule. The overall results of this study support several aspects of current theoretical models of articular cartilage mechanical behavior, motivate further experimental characterization, and suggest the inclusion of specific nonlinear behaviors to models.     

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.     

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.     

Investigation of articular cartilage surface morphology with a semiquantitative scanning electron microscopic method

A semiquantitative scanning electron microscopic method for analysis of the articular cartilage surface morphology was developed. The method was based on a survey of large picture montages (ca. 70 X 100 cm) and classification of the cartilage surface changes at three levels. Computer technique was utilized in the analysis. The method ensured numerical expression and statistical treatment of the results. With this method we investigated the effects of physical exercise and immobilization on the articular cartilage of rabbit patella.     

Identification of latexin by a proteomic analysis in rat normal articular cartilage

Background  

Osteoarthritis (OA) is characterized by degeneration of articular cartilage. Animal models of OA induced are a widely used tool in the study of the pathogenesis of disease. Several proteomic techniques for selective extraction of proteins have provided protein profiles of chondrocytes and secretory patterns in normal and osteoarthritic cartilage, including the discovery of new and promising biomarkers. In this proteomic analysis to study several proteins from rat normal articular cartilage, two-dimensional electrophoresis and mass spectrometry (MS) were used. Interestingly, latexin (LXN) was found. Using an immunohistochemical technique, it was possible to determine its localization within the chondrocytes from normal and osteoarthritic articular cartilage.     

Electron microscopic studies of proteoglycan aggregates from bovine articular cartilage.

Proteoglycan aggregates from bovine articular cartilage have been visualized by electron microscopy of mixed proteoglycan-cytochrome c monolayers. The proteoglycan aggregates consist of proteoglycan subunits arising laterally at fairly regular intervals (20 to 30 nm) from the opposite sides of an elongated filamentous structure. The filamentous backbone in individual aggregates varies in length from 400 to 4000 nm. The individual proteoglycan subunits in the aggregate vary in length from 100 to 400 nm. However, there is no difference in the average size of the proteoglycan subunits associated with the largest or smallest aggregates. The sizes of the individual aggregates are determined mainly by the lengths of their filamentous backbones. The stoichiometry of binding of subunits to filament, calculated from the data reported here, is close to that for the binding of subunits to hyaluronic acid reported by others.     

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.     

Histomorphometric analysis of adult articular calcified cartilage zone

The aim of this study was to carry out a histomorphometric analysis of calcified cartilage zone (CCZ) and its interfaces between hyaline cartilage and subchondral bone. The study used 40 donated normal human femoral condyles, from which paraffin-embedded sections were prepared after fixation and decalcification. The histomorphology of the CCZ were qualitatively and quantitatively observed by staining, scanning electron microscopy (SEM) and three-dimensional (3D) reconstruction. The hyaline cartilage and CCZ were stained red with Safranin-O, and the subchondral bone was stained blue with Fast green. CCZ was stained black after von Kossa staining. The hyaline cartilage was interlocked tightly in the manner of “ravine-engomphosis” by the CCZ. The surface roughnesses of tidemark and cement line were 1.14 ± 0.04 and 1.99 ± 0.38. The maximum, minimum and mean thicknesses of CCZ were 277.12 ± 8.6, 9.83 ± 6.72 and 104.162 ± 0.87 μm, respectively. The cell density of CCZ (51.25 ± 21.26 cells/mm²) was significantly lower than that of the hyaline cartilage (152.54 ± 35.77 cells/mm²) (P < 0.05). The subchondral bone was anchored tightly in the manner of a “comb-anchor” by the CCZ in our 3D reconstruction model. Thus, we discovered two junctional interfaces of CCZ using different histomorphometric methods. The upper interface of CCZ is a “ravine-engomphosis” shape, while its lower interface is a “comb-anchor” shape.     

The proteoglycans of bovine nasal cartilage and human articular cartilage: a sedimentation equilibrium analysis

Cartilage proteoglycan was isolated from bovine nasal septum and fractionated according to buoyant density after dissociative CsCl density gradient centrifugation. Gel-exclusion chromatography showed that hyaluronic acid was present in fractions of density lower than 1.69 g/mL. The molecular weight, assessed by sedimentation equilibrium analysis, of the proteoglycan present in the fractions with density > 1.69 g/mL, which appeared chromatographically homogeneous and constituted 54% of the preparation, ranged from 1.0 to 2.6 × 10⁶ for v = 0.55 cm³ g^?1. Carbodiimide-induced modification of the carboxyl groups by methylamine resulted in a reduction of the molecular weight to 0.74 – 1.25 × 10⁶. An analogous reduction in molecular weight was obtained after equilibration of this proteoglycan fraction with hyaluronic acid oligomers containing five disaccharide units. Since both procedures are known to cause inhibition of the interaction between proteoglycans and hyaluronic acid, it is suggested that this lower molecular-weight range represents the true degree of polydispersity of the sub-units of hyaline cartilage proteoglycan constituting this fraction, while the higher values obtained for the intact proteoglycan are the result of the presence of hyaluronic acid in the sample. The molecular-weight range of the whole proteoglycan subunit preparation, assessed after carboxyl group modification, was 0.5–1.2 × 10⁶. Apparently normal and abnormal cartilage was excised from single human osteoarthrosic femoral heads. Proteoglycans extracted by 4M guanidine hydrochloride were isolated after dissociative density gradient centrifugation and subjected to carboxyl group modification. Preparations from normal tissue exhibited molecular-weight averages ranging from 5 to 9 × 10⁵. A molecular-weight reduction was observed with proteoglycans isolated from abnormal areas.     

Immunohistochemical analysis of ageing and osteoarthritic articular cartilage

Articular cartilage degeneration seen in osteoarthritis is primarily the consequence of events within the articular cartilage that leads to the production of proteases by chondrocytes. 22 osteoarthritic cartilage specimens were obtained from patients with primary osteoarthritis (46–81 years) undergoing total knee replacement. 12 age-matched (41–86 years) and 16 young (16–40 years) non-osteoarthritic control cartilage specimens were obtained from the cadavers in the department of Anatomy and from patients undergoing lower limb amputation in Trauma center of PGIMER, Chandigarh. 5 μ thick paraffin sections were stained for osteocalcin, osteopontin, osteonectin and alkaline phosphatase to analyze their expression in hypertrophied chondrocytes and osteoarthritic cartilage matrix and to compare the staining intensity with that of normal ageing articular cartilage. Immunohistochemical staining of tissue sections revealed moderate to strong cytoplasmic staining for all four stains in all the specimens of the osteoarthritic group compared to age-matched control. The immunohistochemical scores were significantly higher in the osteoarthritic group for all four stains. The features of the osteoarthritic articular cartilage were markedly different from the non-osteoarthritic age-matched articular cartilage suggesting that osteoarthritis is not an inevitable feature of aging.     

High throughput proteomic analysis of the secretome in an explant model of articular cartilage inflammation

This study employed a targeted high-throughput proteomic approach to identify the major proteins present in the secretome of articular cartilage. Explants from equine metacarpophalangeal joints were incubated alone or with interleukin-1beta (IL-1β, 10ng/ml), with or without carprofen, a non-steroidal anti-inflammatory drug, for six days. After tryptic digestion of culture medium supernatants, resulting peptides were separated by HPLC and detected in a Bruker amaZon ion trap instrument. The five most abundant peptides in each MS scan were fragmented and the fragmentation patterns compared to mammalian entries in the Swiss-Prot database, using the Mascot search engine. Tryptic peptides originating from aggrecan core protein, cartilage oligomeric matrix protein (COMP), fibronectin, fibromodulin, thrombospondin-1 (TSP-1), clusterin (CLU), cartilage intermediate layer protein-1 (CILP-1), chondroadherin (CHAD) and matrix metalloproteinases MMP-1 and MMP-3 were detected. Quantitative western blotting confirmed the presence of CILP-1, CLU, MMP-1, MMP-3 and TSP-1. Treatment with IL-1β increased MMP-1, MMP-3 and TSP-1 and decreased the CLU precursor but did not affect CILP-1 and CLU levels. Many of the proteins identified have well-established extracellular matrix functions and are involved in early repair/stress responses in cartilage. This high throughput approach may be used to study the changes that occur in the early stages of osteoarthritis.     

Effects of introducing cultured human chondrocytes into a human articular cartilage explant model

Articular cartilage (AC) heals poorly and effective host-tissue integration after reconstruction is a concern. We have investigated the ability of implanted chondrocytes to attach at the site of injury and to be incorporated into the decellularized host matrix adjacent to a defect in an in vitro human explant model. Human osteochondral dowels received a standardized injury, were seeded with passage 3 chondrocytes labelled with PKH 26 and compared with two control groups. All dowels were cultured in vitro, harvested at 0, 7, 14 and 28 days and assessed for chondrocyte adherence and migration into the region of decellularized tissue adjacent to the defects. Additional evaluation included cell viability, general morphology and collagen II production. Seeded chondrocytes adhered to the standardized defect and areas of lamina splendens disruption but did not migrate into the adjacent acellular region. A difference was noted in viable-cell density between the experimental group and one control group. A thin lattice-like network of matrix surrounded the seeded chondrocytes and collagen II was present. The results indicate that cultured human chondrocytes do indeed adhere to regions of AC matrix injury but do not migrate into the host tissue, despite the presence of viable cells. This human explant model is thus an effective tool for studying the interaction of implanted cells and host tissue.     

A Conewise Linear Elasticity mixture model for the analysis of tension-compression nonlinearity in articular cartilage

A biphasic mixture model is developed that can account for the observed tension-compression nonlinearity of cartilage by employing the continuum-based Conewise Linear Elasticity (CLE) model of Curnier et al. (J. Elasticity, 37, 1-38, 1995) to describe the solid phase of the mixture. In this first investigation, the orthotropic octantwise linear elasticity model was reduced to the more specialized case of cubic symmetry, to reduce the number of elastic constants from twelve to four. Confined and unconfined compression stress-relaxation, and torsional shear testing were performed on each of nine bovine humeral head articular cartilage cylindrical plugs from 6 month old calves. Using the CLE model with cubic symmetry, the aggregate modulus in compression and axial permeability were obtained from confined compression (H-A = 0.64 +/- 0.22 MPa, k2 = 3.62 +/- 0.97 x 10(-16) m4/N.s, r2 = 0.95 +/- 0.03), the tensile modulus, compressive Poisson ratio, and radial permeability were obtained from unconfined compression (E+Y = 12.75 +/- 1.56 MPa, v- = 0.03 +/- 0.01, kr = 6.06 +/- 2.10 x 10(-16) m4/N.s, r2 = 0.99 +/- 0.00), and the shear modulus was obtained from torsional shear (mu = 0.17 +/- 0.06 MPa). The model was also employed to predict the interstitial fluid pressure successfully at the center of the cartilage plug in unconfined compression (r2 = 0.98 +/- 0.01). The results of this study demonstrate that the integration of the CLE model with the biphasic mixture theory can provide a model of cartilage that can successfully curve-fit three distinct testing configurations while producing material parameters consistent with previous reports in the literature.     

Procyanidin B3 prevents articular cartilage degeneration and heterotopic cartilage formation in a mouse surgical osteoarthritis model

Osteoarthritis (OA) is a common disease in the elderly due to an imbalance in cartilage degradation and synthesis. Heterotopic ossification (HO) occurs when ectopic masses of endochondral bone form within the soft tissues around the joints and is triggered by inflammation of the soft tissues. Procyanidin B3 (B3) is a procyanidin dimer that is widely studied due to its high abundance in the human diet and antioxidant activity. Here, we evaluated the role of B3 isolated from grape seeds in the maintenance of chondrocytes in vitro and in vivo. We observed that B3 inhibited H(2)O(2)-induced apoptosis in primary chondrocytes, suppressed H(2)O(2)- or IL-1?-induced nitric oxide synthase (iNOS) production, and prevented IL-1?-induced suppression of chondrocyte differentiation marker gene expression in primary chondrocytes. Moreover, B3 treatment enhanced the early differentiation of ATDC5 cells. To examine whether B3 prevents cartilage destruction in vivo, OA was surgically induced in C57BL/6J mice followed by oral administration of B3 or vehicle control. Daily oral B3 administration protected articular cartilage from OA and prevented chondrocyte apoptosis in surgically-induced OA joints. Furthermore, B3 administration prevented heterotopic cartilage formation near the surgical region. iNOS protein expression was enhanced in the synovial tissues and the pseudocapsule around the surgical region in OA mice fed a control diet, but was reduced in mice that received B3. Together, these data indicated that in the OA model, B3 prevented OA progression and heterotopic cartilage formation, at least in a part through the suppression of iNOS. These results support the potential therapeutic benefits of B3 for treatment of human OA and heterotopic ossification.     

An articular cartilage contact model based on real surface geometry

Abnormal, excessive stresses acting on articular joint surfaces are speculated to be one of the causes for joint degeneration. However, articular surface stresses have not been studied systematically, since it is technically difficult to measure in vivo contact areas and pressures in dynamic situations. Therefore, we implemented a numerical model of articular surface contact using accurate surface geometries. The model was developed for the cat patellofemoral joint. We demonstrated that small misalignments of the patella relative to the femur change the joint contact mechanics substantially for a given external load. These results suggest that misalignment might be studied as one of the factors causing articular cartilage disorder and joint degeneration.     

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.     

A finite-element model for healing of cutaneous wounds combining contraction, angiogenesis and closure

A simplified finite-element model for wound healing is proposed. The model takes into account the sequential steps of dermal regeneration, wound contraction, angiogenesis and wound closure. An innovation in the present study is the combination of the aforementioned partially overlapping processes, which can be used to deliver novel insights into the process of wound healing, such as geometry related influences, as well as the influence of coupling between the various existing subprocesses on the actual healing behavior. The model confirms the clinical observation that epidermal closure proceeds by a crawling and climbing mechanism at the early stages, and by a stratification process in layers parallel to the skin surface at the later stages. The local epidermal oxygen content may play an important role here. The model can also be used to investigate the influence of local injection of hormones that stimulate partial processes occurring during wound healing. These insights can be used to improve wound healing treatments.