Importance of collagen orientation and depth-dependent fixed charge densities of cartilage on mechanical behavior of chondrocytes

The collagen network and proteoglycan matrix of articular cartilage are thought to play an important role in controlling the stresses and strains in and around chondrocytes, in regulating the biosynthesis of the solid matrix, and consequently in maintaining the health of diarthrodial joints. Understanding the detailed effects of the mechanical environment of chondrocytes on cell behavior is therefore essential for the study of the development, adaptation, and degeneration of articular cartilage. Recent progress in macroscopic models has improved our understanding of depth-dependent properties of cartilage. However, none of the previous works considered the effect of realistic collagen orientation or depth-dependent negative charges in microscopic models of chondrocyte mechanics. The aim of this study was to investigate the effects of the collagen network and fixed charge densities of cartilage on the mechanical environment of the chondrocytes in a depth-dependent manner. We developed an anisotropic, inhomogeneous, microstructural fibril-reinforced finite element model of articular cartilage for application in unconfined compression. The model consisted of the extracellular matrix and chondrocytes located in the superficial, middle, and deep zones. Chondrocytes were surrounded by a pericellular matrix and were assumed spherical prior to tissue swelling and load application. Material properties of the chondrocytes, pericellular matrix, and extracellular matrix were obtained from the literature. The loading protocol included a free swelling step followed by a stress-relaxation step. Results from traditional isotropic and transversely isotropic biphasic models were used for comparison with predictions from the current model. In the superficial zone, cell shapes changed from rounded to elliptic after free swelling. The stresses and strains as well as fluid flow in cells were greatly affected by the modulus of the collagen network. The fixed charge density of the chondrocytes, pericellular matrix, and extracellular matrix primarily affected the aspect ratios (height/width) and the solid matrix stresses of cells. The mechanical responses of the cells were strongly location and time dependent. The current model highlights that the collagen orientation and the depth-dependent negative fixed charge densities of articular cartilage have a great effect in modulating the mechanical environment in the vicinity of chondrocytes, and it provides an important improvement over earlier models in describing the possible pathways from loading of articular cartilage to the mechanical and biological responses of chondrocytes.     

Distribution of newly synthesized aggrecan in explant cultures of bovine cartilage treated with retinoic acid.

This paper describes temporal changes in the metabolism and distribution of newly synthesized aggrecan and the organization of the extracellular matrix when explant cultures of articular cartilage maintained in the presence of fetal calf serum were exposed to retinoic acid for varying periods of time. Explant cultures of articular cartilage were incubated with radiolabeled sulfate prior to exposure to retinoic acid. The radiolabeled and chemical aggrecan present in the tissue and appearing in the culture medium was studied kinetically. Changes in the localization of radiolabeled aggrecan within the extracellular matrix were monitored by autoradiography in relation to type VI collagen distribution in the extracellular matrix. In control cultures where tissue levels of aggrecan remain constant the newly synthesized aggrecan remained closely associated with the territorial matrix surrounding the chondrocytes. Exposure of cultures to retinoic acid for the duration of the experiment, resulted in the extensive loss of aggrecan from the tissue and the redistribution of the remaining radiolabeled aggrecan from the chondron and territorial matrix into the inter-territorial matrix. These changes preceded alterations in the organization of type VI collagen in the extracellular matrix that involved the remodeling of the chondron and the appearance of type VI collagen in the inter-territorial matrix; there was also evidence of chondrocyte proliferation and clustering. In cartilage explant cultures exposed to retinoic acid for 24 h there was no loss of aggrecan from the matrix but there was an extensive redistribution of the radiolabeled aggrecan into the inter-territorial matrix. This work shows that maintenance of the structure and organization of the extracellular matrix that comprises the chondron and pericellular microenvironment of chondrocytes in articular cartilage is important for the regulation of the distribution of newly synthesized aggrecan monomers within the tissue.     

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.     

Role of pericellular matrix in development of a mechanically functional neocartilage

The role of the chondrocyte pericellular matrix (PCM) was examined in a three-dimensional chondrocyte culture system to determine whether retention of the native pericellular matrix could stimulate collagen and proteoglycan accumulation and also promote the formation of a mechanically functional hyaline-like neocartilage. Porcine chondrocytes and chondrons, consisting of the chondrocyte with its intact pericellular matrix, were maintained in pellet culture for up to 12 weeks. Sulfated glycosaminoclycans and type II collagen were measured biochemically. Immunocytochemistry was used to examine collagen localization as well as cell distribution within the pellets. In addition, the equilibrium compressive moduli of developing pellets were measured to determine whether matrix deposition contributed to the mechanical stiffness of the cartilage constructs. Pellets increased in size and weight over a 6-week period without apparent cell proliferation. Although chondrocytes quickly rebuilt a PCM rich in type VI collagen, chondron pellets accumulated significantly more proteoglycan and type II collagen than did chondrocyte pellets, indicating a greater positive effect of the native PCM. After 5 weeks in chondron pellets, matrix remodeling was evident by microscopy. Cells that had been uniformly distributed throughout the pellets began to cluster between large areas of interterritorial matrix rich in type II collagen. After 12 weeks, clusters were stacked in columns. A rapid increase in compressive strength was observed between 1 and 3 weeks in culture for both chondron and chondrocyte pellets and, by 6 weeks, both had achieved 25% of the equilibrium compressive stiffness of cartilage explants. Retention of the in vivo PCM during chondrocyte isolation promotes the formation of a mechanically functional neocartilage construct, suitable for modeling the responses of articular cartilage to chemical stimuli or mechanical compression.     

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.     

Retention of the native chondrocyte pericellular matrix results in significantly improved matrix production.

The interaction of the cell with its surrounding extracellular matrix (ECM) has a major effect on cell metabolism. We have previously shown that chondrons, chondrocytes with their in vivo-formed pericellular matrix, can be enzymatically isolated from articular cartilage. To study the effect of the native chondrocyte pericellular matrix on ECM production and assembly, chondrons were compared with chondrocytes isolated without any pericellular matrix. Immediately after isolation from human cartilage, chondrons and chondrocytes were centrifuged into pellets and cultured. Chondron pellets had a greater increase in weight over 8 weeks, were more hyaline appearing, and had more type II collagen deposition and assembly than chondrocyte pellets. Minimal type I procollagen immunofluorescence was detected for both chondron and chondrocyte pellets. Chondron pellets had a 10-fold increase in proteoglycan content compared with a six-fold increase for chondrocyte pellets over 8 weeks (P<0.0001). There was no significant cell division for either chondron or chondrocyte pellets. The majority of cells within both chondron and chondrocyte pellets maintained their polygonal or rounded shape except for a thin, superficial edging of flattened cells. This edging was similar to a perichondrium with abundant type I collagen and fibronectin, and decreased type II collagen and proteoglycan content compared with the remainder of the pellet. This study demonstrates that the native pericellular matrix promotes matrix production and assembly in vitro. Further, the continued matrix production and assembly throughout the 8-week culture period make chondron pellet cultures valuable as a hyaline-like cartilage model in vitro.     

The major basement membrane components localize to the chondrocyte pericellular matrix--a cartilage basement membrane equivalent?

In this study, we demonstrate that articular cartilage chondrocytes are surrounded by the defining basement membrane proteins laminin, collagen type IV, nidogen and perlecan, and suggest that these form the functional equivalent of a basement membrane. We found by real-time PCR that mouse chondrocytes express these four cardinal components of basement membranes and demonstrated by immunohistochemistry that the proteins are present in bovine and mouse cartilage tissues and are deposited in a thin pericellular structure. Immunoelectron microscopy confirmed high laminin concentration in the pericellular matrix. In cartilage from newborn mice, basement membrane components are widespread in the territorial and interterritorial matrix, while in mature cartilage of adult mice the basement membrane components are localized mainly to a narrow pericellular zone. With progression into old age, this layer becomes less distinct, especially in areas of obvious mechanical attrition. Interestingly, individual laminin subunits were located in different zones of the cartilage, with laminin alpha1 showing preferential localization around a select population of superficial layer chondrocytes. We propose that the chondrocyte, like several other cell types of mesenchymal origin, is surrounded by the functional equivalent of a basement membrane. This structure is presumably involved in maintaining chondrocyte phenotype and viability and may well allow a new understanding of cartilage development and provide clues to the progression of degenerative joint disorders.     

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.     

The role of chondrocyte-matrix interactions in maintaining and repairing articular cartilage

Throughout life chondrocytes maintain the articular cartilage matrix by replacing degraded macromolecules and respond to focal cartilage injury or degeneration by increasing local synthesis activity. These observations suggest that mechanisms exist within articular cartilage that stimulate chondrocyte anabolic activity in response to matrix degradation or damage. An important cartilage anabolic factor, insulin-like growth factor I (IGF-I), appears to have a role in stimulating chondrocyte anabolic activity. Although IGF-I is ubiquitous, its bioavailability is controlled by a class of secreted proteins, IGF binding proteins (IGFPBs). Of the six known IGFPBs, IGFBP-3 is the most abundant in human articular cartilage. We recently found that with increasing age, articular chondrocytes increase their expression of IGFBP-3. This observation led us to investigate the potential role of IGFBP-3 in chondrocyte-matrix interactions. Using immunofluorescent staining and confocal microscopy we found that IGFBP-3 accumulates with increasing age in the chondrocyte territorial matrix where it co-localizes with fibronectin, but not with tenascin-C or type VI collagen. Using purified proteins we demonstrated that IGFBP-3 binds to fibronectin in a dose dependent manner, but not to tenascin-C. In vitro studies showed that IGFBP-3 alone inhibited chondrocyte synthetic activity while intact fibronectin alone significantly stimulated activity. When fibronectin and IGFBP-3 were combined we found that the inhibitory activity of low concentrations of IGFPB-3 was enhanced. These observations indicate that in mature articular cartilage IGF-I is stored in the chondrocyte territorial matrix through binding to a complex of IGFPB-3 and intact fibronectin. Storage of IGF-I of the territorial matrix may help maintain a relatively constant level of available IGF-I and the local increase in matrix synthesis following matrix damage may result from release of IGF-I. This mechanism may have an important role in maintaining and repairing articular cartilage and failure of this mechanism may lead to progressive articular cartilage degeneration.     

Fibril reinforced poroelastic model predicts specifically mechanical behavior of normal,proteoglycan depleted and collagen degraded articular cartilage

Degradation of collagen network and proteoglycan (PG) macromolecules are signs of articular cartilage degeneration. These changes impair cartilage mechanical function. Effects of collagen degradation and PG depletion on the time-dependent mechanical behavior of cartilage are different. In this study, numerical analyses, which take the compression-tension nonlinearity of the tissue into account, were carried out using a fibril reinforced poroelastic finite element model. The study aimed at improving our understanding of the stress-relaxation behavior of normal and degenerated cartilage in unconfined compression. PG and collagen degradations were simulated by decreasing the Young's modulus of the drained porous (nonfibrillar) matrix and the fibril network, respectively. Numerical analyses were compared to results from experimental tests with chondroitinase ABC (PG depletion) or collagenase (collagen degradation) digested samples. Fibril reinforced poroelastic model predicted the experimental behavior of cartilage after chondroitinase ABC digestion by a major decrease of the drained porous matrix modulus (-64+/-28%) and a minor decrease of the fibril network modulus (-11+/-9%). After collagenase digestion, in contrast, the numerical analyses predicted the experimental behavior of cartilage by a major decrease of the fibril network modulus (-69+/-5%) and a decrease of the drained porous matrix modulus (-44+/-18%). The reduction of the drained porous matrix modulus after collagenase digestion was consistent with the microscopically observed secondary PG loss from the tissue. The present results indicate that the fibril reinforced poroelastic model is able to predict specifically characteristic alterations in the stress-relaxation behavior of cartilage after enzymatic modifications of the tissue. We conclude that the compression-tension nonlinearity of the tissue is needed to capture realistically the mechanical behavior of normal and degenerated articular cartilage.     

Confocal analysis of the molecular heterogeneity in the pericellular microenvironment produced by adult canine chondrocytes cultured in agarose gel

Adult articular chondrocytes are each surrounded by a heterogeneous microenvironment and together form the chondron. Since little is known of chondron development, agarose gel culture, confocal immunohistochemistry and image analysis have been used to characterize the molecular anatomy and temporal development of the chondrocyte pericellular microenvironment in vitro. Two structurally distinct domains were identified during the 12-week culture period. The first comprised a narrow glycocalyx, 1–3 ·m in width, which consolidated over time and was rich in collagen types II, VI, IX and XI, fibronectin, decorin and the aggrecan epitopes, 5D4 and HABR. The second region emerged after 4–6 weeks in culture and progressively developed a broad territorial region up to 12 ·m wide around the chondrocyte and pericellular glycocalyx. Co-localization studies confirmed the dominance of aggrecan epitopes 2B6, EFG-4, 5D4 and HABR in the territorial domain, whereas surface density mapping with NIH image revealed two patterns of staining, one punctate and stippled, the other more uniform in distribution. The pericellular differentiation identified appeared analogous to the chondrons of adult articular cartilage, and provides an appropriate in vitro model for further studies of cell surface receptor function in the orchestration of pericellular matrix assembly This revised version was published online in November 2006 with corrections to the Cover Date.     

Confocal analysis of the molecular heterogeneity in the pericellular microenvironment produced by adult canine chondrocytes cultured in agarose gel

Adult articular chondrocytes are each surrounded by a heterogeneous microenvironment and together form the chondron. Since little is known of chondron development, agarose gel culture, confocal immunohistochemistry and image analysis have been used to characterize the molecular anatomy and temporal development of the chondrocyte pericellular microenvironment in vitro. Two structurally distinct domains were identified during the 12-week culture period. The first comprised a narrow glycocalyx, 1–3 ·m in width, which consolidated over time and was rich in collagen types II, VI, IX and XI, fibronectin, decorin and the aggrecan epitopes, 5D4 and HABR. The second region emerged after 4–6 weeks in culture and progressively developed a broad territorial region up to 12 ·m wide around the chondrocyte and pericellular glycocalyx. Co-localization studies confirmed the dominance of aggrecan epitopes 2B6, EFG-4, 5D4 and HABR in the territorial domain, whereas surface density mapping with NIH image revealed two patterns of staining, one punctate and stippled, the other more uniform in distribution. The pericellular differentiation identified appeared analogous to the chondrons of adult articular cartilage, and provides an appropriate in vitro model for further studies of cell surface receptor function in the orchestration of pericellular matrix assembly This revised version was published online in November 2006 with corrections to the Cover Date.     

Latrunculin and cytochalasin decrease chondrocyte matrix retention.

The proteoglycan-rich extracellular matrix (ECM) directly associated with the cells of articular cartilage is anchored to the chondrocyte plasma membrane via interaction with the hyaluronan receptor CD44. The cytoplasmic tail of CD44 interacts with the cortical cytoskeleton. The objective of this study was to determine the role of the actin cytoskeleton in CD44-mediated matrix assembly by chondrocytes and cartilage matrix retention and homeostasis. Adult bovine articular cartilage tissue slices and isolated chondrocytes were treated with latrunculin or cytochalasin. Tissues were processed for histology and chondrocytes were examined for CD44 expression and pericellular matrix assembly. Treatments that disrupt the actin cytoskeleton reduced chondrocyte pericellular matrix assembly and the retention of proteoglycan within cartilage explants. There was enhanced detection of a neoepitope resulting from proteolysis of aggrecan. Cytoskeletal disruption did not reduce CD44 expression, as monitored by flow cytometry, but detergent extraction of CD44 was enhanced and hyaluronan binding was decreased. Thus, disruption of the cytoskeleton reduces the anchorage of CD44 in the chondrocyte membrane and the capacity of CD44 to bind its ligand. The results suggest that cytoskeletal disruption within cartilage uncouples chondrocytes from the matrix, resulting in altered metabolism and deleterious changes in matrix structure.     

The C5 domain of Col6A3 is cleaved off from the Col6 fibrils immediately after secretion.

In articular cartilage, type VI collagen is concentrated in the pericellular matrix compartment. During protein synthesis and processing at least the alpha3(VI) chain undergoes significant posttranslational modification and cleavage. In this study, we investigated the processing of type VI collagen in articular cartilage. Immunostaining with a specific polyclonal antiserum against the C5 domain of alpha3(VI) showed strong cellular staining seen in nearly all chondrocytes of articular cartilage. Confocal laser-scanning microscopy and immunoelectron microscopy allowed localization of this staining mainly to the cytoplasm and the immediate pericellular matrix. Double-labeling experiments showed a narrow overlap of the C5 domain and the pericellular mature type VI collagen. Our results suggest that at least in human adult articular cartilage the C5 domain of alpha3(VI) collagen is synthesized and initially incorporated into the newly formed type VI collagen fibrils, but immediately after secretion is cut off and is not present in the mature pericellular type VI matrix of articular cartilage.     

The distribution of type VI collagen in the developing tissues of the bovine femoral head

Type VI collagen appears central to the maintenance of tissue integrity. In adult articular cartilage, type VI collagen is preferentially localised in the chondron where it may be involved in cell attachment. In actively remodelling developing cartilage, the distribution is less certain. We have used confocal immunohistochemistry and in situ hybridisation to investigate type VI collagen distribution in third trimester bovine proximal femoral epiphyses. In general, type VI collagen immunofluorescence was concentrated in the chondrocyte pericellular matrix, with staining intensity strongest in regions which persist to maturity and weakest in regions that remodel during development. Type VI collagen was also present in cartilage canals. In the growth plate and around the secondary centre of ossification, the intensity of type VI collagen stain rapidly decreased with chondrocyte maturation and was absent at hypertrophy, except where canal branches penetrated the growth plate and stain was retained around the adjacent chondrocytes. In situ hybridisation confirmed the presence of type VI collagen mRNA in cartilage canal mesenchymal cells but the signal was low in chondrocytes, suggesting minimal levels of synthesis and turnover. The results are consistent with a role for type VI collagen in stabilising the extracellular matrix during development.     

A biphasic multiscale study of the mechanical microenvironment of chondrocytes within articular cartilage under unconfined compression

Computational analyses have been used to study the biomechanical microenvironment of the chondrocyte that cannot be assessed by in vitro experimental studies; yet all computational studies thus far have focused on the effect of zonal location (superficial, middle, and deep) on the mechanical microenvironment of chondrocytes. The aim of this paper was to study the effect of both zonal and radial locations on the biomechanical microenvironment of chondrocytes in inhomogeneous cartilage under unconfined stress relaxation. A biphasic multiscale approach was employed and nine chondrocytes in different locations were studied. Hyperelastic biphasic theory and depth-dependent aggregate modulus and permeability of articular cartilage were included in the models. It was found that both zonal and radial locations affected the biomechanical stresses and strains of the chondrocytes. Chondrocytes in the mid-radial location had increased volume during the early stage of the loading process. Maximum principal shear stress at the interface between the chondrocyte and the extracellular matrix (ECM) increased with depth, yet that at the ECM–pericellular matrix (PCM) interface had an inverse trend. Fluid pressure decreased with depth, while the fluid pressure difference between the top and bottom boundaries of the microscale model increased with depth. Regardless of location, fluid was exchanged between the chondrocyte, PCM, and ECM. These findings suggested that even under simple compressive loading conditions, the biomechanical microenvironment of the chondrocytes, PCM and ECM was spatially dependent. The current study provides new insight on chondrocyte biomechanics.     

A proteomic approach for identification and localization of the pericellular components of chondrocytes

Although the pericellular matrix (PCM) plays a central role in the communication between chondrocytes and extracellular matrix, its composition is largely unknown. In this study, the PCM was investigated with a proteomic approach using chondrons, which are enzymatically isolated constructs including the chondrocyte and its surrounding PCM. Chondrons and chondrocytes alone were isolated from human articular cartilage. Proteins extracted from chondrons and chondrocytes were used for two-dimensional electrophoresis. Protein spots were quantitatively compared between chondron and chondrocyte gels. Cellular proteins, which had similar density between chondron and chondrocyte gels, did not proceed for analysis. Since chondrons only differ from chondrocytes in association of the PCM, protein spots in the chondron gels that had higher quantity than that in the chondrocyte gels were selected as candidates of the PCM components and processed for mass spectrometry. Among 15 identified peptides, several were fragments of the three type VI collagen chains (α-1, α-2, and α-3). Other identified PCM proteins included triosephosphate isomerase, transforming growth factor-β induced protein, peroxiredoxin-4, ADAM (A disintegrin and metalloproteinases) 28, and latent-transforming growth factor beta-binding protein-2. These PCM components were verified with immunohisto(cyto)chemistry for localization in the PCM region of articular cartilage. The abundance of type VI collagen in the PCM emphasizes its importance to the microenvironment of chondrocytes. Several proteins were localized in the PCM of chondrocytes for the first time and that warrants further investigation for their functions in cartilage biology.     

The deformation behavior and viscoelastic properties of chondrocytes in articular cartilage

Chondrocytes in articular cartilage utilize mechanical signals in conjunction with other environmental factors to regulate their metabolic activity. However, the sequence of biomechanical and biochemical events involved in the process of mechanical signal transduction has not been fully deciphered. A fundamental step in determining the role of various factors in regulating chondrocyte activity is to characterize accurately the biophysical environment within the tissue under physiological conditions of mechanical loading. Microscopic imaging studies have revealed that chondrocytes as well as their nuclei undergo shape and volume changes in a coordinated manner with deformation of the tissue matrix. Through micromechanical experiments, it has been shown that the chondrocyte behaves as a viscoelastic solid material with a mechanical stiffness that is several orders of magnitude lower than that of the cartilage extracellular matrix. These properties seem to be due to the structure of the chondrocyte cytoskeleton, and in part, the viscoelastic properties of the cell nucleus. The mechanical properties of the pericellular matrix that immediately surrounds the chondrocyte significantly differ from those of the chondrocyte and the extracellular matrix, suggesting that the pericellular matrix plays an important role in defining the mechanical environment of the chondrocyte. These experimentally measured values for chondrocyte and cartilage mechanical properties have been used in combination with theoretical constitutive modeling of the chondrocyte within articular cartilage to predict the non-uniform and time-varying stress-strain and fluid flow environment of the cell. The ultimate goal of these studies has been to elucidate the sequence of biomechanical and biochemical events through which mechanical stress influences chondrocyte activity in both health and in disease.     

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.