Alterations in the Young's modulus and volumetric properties of chondrocytes isolated from normal and osteoarthritic human cartilage

The mechanical environment of the chondrocyte is an important factor that influences the maintenance of the articular cartilage extracellular matrix. Previous studies have utilized theoretical models of chondrocytes within articular cartilage to predict the stress-strain and fluid flow environments around the cell, but little is currently known regarding the cellular properties which are required for implementation of these models. The objectives of this study were to characterize the mechanical behavior of primary human chondrocytes and to determine the Young's modulus of chondrocytes from non-osteoarthritic ('normal') and osteoarthritic cartilage. A second goal was to quantify changes in the volume of isolated chondrocytes in response to mechanical deformation. The micropipette aspiration technique was used to measure the deformation of a single chondrocyte into a glass micropipette in response to a prescribed pressure. The results of this study indicate that the human chondrocyte behaves as a viscoelastic solid. No differences were found between the Young's moduli of normal (0.65+/-0.63 kPa, n = 44) and osteoarthritic chondrocytes (0.67+/-0.86 kPa, n = 69, p = 0.93). A significant difference in cell volume was observed immediately and 600 s after complete aspiration of the cell into the pipette (p < 0.001), and the magnitude of this volume change between normal (11+/-11%, n = 40) and osteoarthritic (20+/-11%, n = 41) chondroctyes was significantly different at both time points (p < 0.002). This finding suggests that chondrocytes from osteoarthritic cartilage may have altered volume regulation capabilities in response to mechanical deformation. The mechanical and volumetric properties determined in this study will be of use in analytical and finite element models of chondrocyte-matrix interactions in order to better predict the mechanical environment of the cell in vivo.     

Regulatory volume increase (RVI) by in situ and isolated bovine articular chondrocytes

Metabolism of the matrix by chondrocytes is sensitive to alterations in cell volume that occur, for example, during static loading and osteoarthritis. The ability of chondrocytes to respond to changes in volume could be important, and this study was aimed at testing the hypothesis that chondrocytes can regulate their volume following cell shrinking by regulatory volume increase (RVI). We used single cell fluorescence imaging of in situ bovine articular chondrocytes, cells freshly isolated into 280 or 380 mOsm, or 2-D cultured chondrocytes loaded with calcein or fura-2, to investigate RVI and changes to [Ca2+]i during shrinkage. Following a 42% hyperosmotic challenge, chondrocytes rapidly shrunk, however, only approximately 6% of the in situ or freshly isolated chondrocytes demonstrated RVI. This contrasted with 2D-cultured chondrocytes where approximately 54% of the cells exhibited RVI. The rate of RVI was the same for all preparations. During the 'post-RVD/RVI protocol', approximately 60% of the in situ and freshly isolated chondrocytes demonstrated RVD, but only approximately 5% showed RVI. There was no relationship between [Ca2+]i and RVI either during hyperosmotic challenge, or during RVD suggesting that changes to [Ca2+]i were not required for RVI. Depolymerisation of the actin cytoskeleton by latrunculin, increased RVI by freshly isolated chondrocytes, in a bumetanide-sensitive manner. The results showed that in situ and freshly isolated articular chondrocytes have only limited RVI capacity. However, RVI was stimulated by treating freshly isolated chondrocytes with latrunculin B and following 2D culture of chondrocytes, suggesting that cytoskeletal integrity plays a role in regulating RVI activity which appears to be mediated principally by the Na+ - K+ -2Cl- cotransporter.     

Mechanical regulation of proteoglycan synthesis in normal and osteoarthritic human articular chondrocytes--roles for alpha5 and alphaVbeta5 integrins

The importance of biomechanical forces in regulating normal chondrocyte metabolism is well established and the mechanisms whereby mechanical forces are transduced into biochemical responses by chondrocytes are beginning to be understood. Previous studies have indicated that cyclical mechanical stimulation induces increased aggrecan gene expression in normal but not osteoarthritic chondrocytes in monolayer. It remains unclear, however, whether these effects on gene expression are associated with changes in proteoglycan production and whether any changes in proteoglycan expression is dependent on integrins or integrin associated proteins. Normal and osteoarthritic articular chondrocytes in monolayer were exposed to 0.33 Hz mechanical stimulation for 20 min in the absence or presence of function modifying anti-integrin antibodies. Following stimulation GAG and proteoglycan (PG) synthesis was assessed by DMMB assay and western blotting. Mechanical stimulation of normal chondrocytes resulted in increased GAG synthesis that was blocked by the presence of antibodies to alpha5 and alphaVbeta5 integrins and CD47. Electrophoretic patterns of PGs released from normal chondrocytes following mechanical stimulation showed an increase in newly-synthesized aggrecan that was not fragmented or degraded. Chondrocytes from osteoarthritic cartilage showed lower levels of GAG production when compared to normal chondrocytes and synthesis was not influenced by mechanical stimulation. These studies show that chondrocytes derived from normal and OA cartilage have different matrix production responses to mechanical stimulation and suggest previously unrecognised roles for alphaVbeta5 integrin in regulation of chondrocyte responses to biomechanical stimulation.     

Regulatory volume decrease (RVD) by isolated and in situ bovine articular chondrocytes

Articular chondrocytes in vivo are exposed to a changing osmotic environment under both physiological (static load) and pathological (osteoarthritis) conditions. Such changes to matrix hydration could alter cell volume in situ and influence matrix metabolism. However the ability of chondrocytes to regulate their volume in the face of osmotic perturbations have not been studied in detail. We have investigated the regulatory volume decrease (RVD) capacity of bovine articular chondrocytes within, and isolated from the matrix, before and following acute hypotonic challenge. Cell volumes were determined by visualising fluorescently-labelled chondrocytes using confocal laser scanning microscopy (CLSM) at 21 degrees C. Chondrocytes in situ were grouped into superficial (SZ), mid (MZ), and deep zones (DZ). When exposed to 180mOsm or 250mOsm hypotonic challenge, cells in situ swelled rapidly (within approximately 90 sec). Chondrocytes then exhibited rapid RVD (t(1/2) approximately 8 min), with cells from all zones returning to approximately 3% of their initial volume after 20 min. There was no significant difference in the rates of RVD between chondrocytes in the three zones. Similarly, no difference in the rate of RVD was observed for an osmotic shock from 280 to 250 or 180mOsm. Chondrocytes isolated from the matrix into medium of 380mOsm and then exposed to 280mOsm showed an identical RVD response to that of in situ cells. The RVD response of in situ cells was inhibited by REV 5901. The results suggested that the signalling pathways involved in RVD remained intact after chondrocyte isolation from cartilage and thus it was likely that there was no role for cell-matrix interactions in mediating RVD.     

NMDA receptor expression and roles in human articular chondrocyte mechanotransduction

Mechanical forces influence articular cartilage structure by regulating chondrocyte activity. Mechanical stimulation results in activation of an alpha5beta1 integrin dependent intracellular signal cascade involving focal adhesion kinase and protein kinase C, triggering the release of interleukin-4 from the cell. In normal HAC the response to physiological mechanical stimulation is characterised by increased levels of aggrecan mRNA and a decrease in levels of mRNA for matrix metalloproteinase 3 (MMP-3), the net result of which would be to maintain and optimise cartilage structure and function. This protective/anabolic response is not seen when chondrocytes from osteoarthritic cartilage are subjected to an identical mechanical stimulation regime. Following the observation that the neurotransmitter substance P is involved in chondrocyte mechanotransduction the present study was undertaken to establish potential roles for glutamate receptors in the control of chondrocyte mechanical responses. Using immunohistochemistry and RTPCR normal and OA chondrocytes are shown to express NR1 and NR2a subunits of the NMDA receptor. Addition of NMDA receptor agonists to chondrocytes in primary culture resulted in changes in membrane potential consistent with expression of functional receptors. NMDA receptor antagonists inhibited the hyperpolarisation response of normal chondrocytes to mechanical stimulation but had no effect on the depolarisation response of osteoarthritic chondrocytes to mechanical stimulation. These studies indicate that at least one subset of the NMDA receptor family of molecules is expressed in cartilage and may have important modulatory effects on mechanotransduction and cellular responses following mechanical stimulation. Indeed the results suggest that there is an alteration of NMDA receptor signalling in OA chondrocytes, which may be critical in the abnormal response of OA chondrocytes to mechanical stimulation. Thus NMDA receptors appear to be involved in the regulation of human articular chondrocyte responses to mechanical stimulation, and in OA, mechanotransduction pathways may be modified as a result of altered activation and function of these receptors.     

Alterations in the mechanical properties of the human chondrocyte pericellular matrix with osteoarthritis

In articular cartilage, chondrocytes are surrounded by a pericellular matrix (PCM), which together with the chondrocyte have been termed the "chondron." While the precise function of the PCM is not know there has been considerable speculation that it plays a role in regulating the biomechanical environment of the chondrocyte. In this study, we measured the Young's modulus of the PCM from normal and osteoarthritic cartilage using the micropipette aspiration technique, coupled with a newly developed axisymmetric elastic layered half-space model of the experimental configuration. Viable, intact chondrons were extracted from human articular cartilage using a new microaspiration-based isolation technique. In normal cartilage, the Young's modulus of the PCM was similar in chondrons isolated from the surface zone (68.9 +/- 18.9 kPa) as compared to the middle and deep layers (62.0 +/- 30.5 kPa). However, the mean Young's modulus of the PCM (pooled for the two zones) was significantly decreased in osteoarthritic cartilage (66.5 +/- 23.3 kPa versus 41.3 +/- 21.1 kPa, p < 0.001). In combination with previous theoretical models of cell-matrix interactions in cartilage, these findings suggest that the PCM has an important influence on the stress-strain environment of the chondrocyte that potentially varies with depth from the cartilage surface. Furthermore, the significant loss of PCM stiffness that was observed in osteoarthritic cartilage may affect the magnitude and distribution of biomechanical signals perceived by the chondrocytes.     

Determination of the Poisson's ratio of the cell: recovery properties of chondrocytes after release from complete micropipette aspiration

Chondrocytes in articular cartilage are regularly subjected to compression and recovery due to dynamic loading of the joint. Previous studies have investigated the elastic and viscoelastic properties of chondrocytes using micropipette aspiration techniques, but in order to calculate cell properties, these studies have generally assumed that cells are incompressible with a Poisson's ratio of 0.5. The goal of this study was to measure the Poisson's ratio and recovery properties of the chondrocyte by combining theoretical modeling with experimental measures of complete cellular aspiration and release from a micropipette. Chondrocytes isolated from non-osteoarthritic and osteoarthritic cartilage were fully aspirated into a micropipette and allowed to reach mechanical equilibrium. Cells were then extruded from the micropipette and cell volume and morphology were measured throughout the experiment. This experimental procedure was simulated with finite element analysis, modeling the chondrocyte as either a compressible two-mode viscoelastic solid, or as a biphasic viscoelastic material. By fitting the experimental data to the theoretically predicted cell response, the Poisson's ratio and the viscoelastic recovery properties of the cell were determined. The Poisson's ratio of chondrocytes was found to be 0.38 for non-osteoarthritic cartilage and 0.36 for osteoarthritic chondrocytes (no significant difference). Osteoarthritic chondrocytes showed an increased recovery time following full aspiration. In contrast to previous assumptions, these findings suggest that chondrocytes are compressible, consistent with previous studies showing cell volume changes with compression of the extracellular matrix.     

Stimulation of regulatory volume decrease (RVD) by isolated bovine articular chondrocytes following F-actin disruption using latrunculin B

Articular chondrocytes are exposed to significant changes in extracellular osmolarity during normal joint activity, which can lead to changes in cell volume and metabolism of the extracellular matrix (ECM). Chondrocytes can respond to cell swelling/shrinking by volume regulatory pathways, but the signalling pathways are poorly understood although a role for the cytoskeleton is frequently implicated. Here, we have investigated the effects of disruption of the chondrocyte F-actin cytoskeleton on the recovery of cell volume by RVD. The cytoskeleton was perturbed using the relatively specific agent latrunculin B (5 microM; 30 min) and loss of F-actin integrity quantified using fluorescent phalloidin-labelling and confocal laser scanning microscopy (CLSM). Imaging of isolated chondrocytes labelled with Fura-2 to measure the fluorescence associated with cell volume changes, showed that the extent of hypo-osmotic swelling was unaffected by latrunculin B treatment. Two categories of the chondrocyte RVD response were observed: 'fast' RVD where at 3 min post-osmotic challenge there was a recovery in cell fluorescence of >or=80%, whereas other cells exhibited 'slow' RVD. Latrunculin B increased the proportion of chondrocytes demonstrating 'fast' RVD by approximately 10 fold and reduced those cells showing 'slow' RVD. An inhibitor of chondrocyte RVD (REV 5901) had no significant effect on the integrity of the cytoskeleton showing that the RVD response could be inhibited independent of the state of the F-actin cytoskeleton. These results suggest that the intact cortical F-actin cytoskeleton has a restraining effect on the RVD response of isolated bovine articular chondrocytes.     

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.     

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.     

Vitreous preservation of articular cartilage from cryoinjury in rabbits

Frozen osteoarticular grafts treated with liquid nitrogen are utilized for joint reconstruction after tumor resection, but the joints may subsequently develop osteoarthritic changes. To preserve articular cartilage from cryoinjury, we modified a vitrification method utilized for embryo cryopreservation and demonstrated in vitro that our vitrification protocol was effective for protecting cartilage from cryoinjury. In this study, we investigated in vivo whether this vitrification method could protect against osteoarthritic changes in articular cartilage. Osteochondral plugs were obtained from the distal femur of rabbits. These grafts were divided into 3 groups: Fresh group (F-group), non-vitrification group (N-group), and vitrification group (V-group). After treatment, the plugs were re-implanted as autografts. Histological findings, chondrocyte viability, and ultrastructural examinations were examined 6, 12, and 24weeks after implantation. Histological findings of chondrocytes for the V-group showed no significant difference from those of the F-group at any time point except at 24weeks postimplantation at the non-weight bearing site (p<0.05). Viability of chondrocyte showed no significant difference from those of the F-group except at 12weeks postimplantation at the bearing site (p<0.05). In contrast, viable cells disappeared from the N-group and histology and viability significantly differed between the N-group and the V-group. Transmission electron microscopy demonstrated preservation of chondrocyte structure in the V-group and the F-group, but chondrocytes of the N-group were abnormally electron dense. Our vitrification method was effective in protecting chondrocytes from cryoinjury that might lead to cartilage degeneration. Reconstructing joints with osteoarticular grafts containing living cartilage may help to avert osteoarthritic changes. Our vitrification method could prove useful for reconstruction with frozen tumor-containing autografts and for long-term storage of living cartilage for allografts.     

Soluble recombinant neprilysin induces aggrecanase-mediated cleavage of aggrecan in cartilage explant cultures.

Neprilysin (neutral endopeptidase, enkephalinase, CALLA, CD10, NEP) is a regulatory Zn metallopeptidase expressed in the brush border membranes of the kidney and has been found in porcine chondrocytes and rat articular cartilage as well as other cell types and tissues. Although its function in cartilage is not currently known, previous observations of high levels of NEP enzymatic activity in the synovial fluid of arthritic patients and on the chondrocyte membranes of human osteoarthritic cartilage have led to the hypothesis that NEP is involved in the inflammation or degradation pathways in articular cartilage. Our study localized endogenous NEP to the membranes of mature bovine articular chondrocytes in a tissue explant model and demonstrated that the addition of soluble recombinant NEP (sNEP) to the culture medium of bovine cartilage explants leads to the degradation of aggrecan through the action of aggrecanase. A 6-day exposure to sNEP was necessary to initiate the degradation, suggesting that the chondrocytes were responding in a delayed manner to an altered composition of regulatory peptides. This NEP-induced degradation was completely inhibited by the NEP inhibitors thiorphan and phosphoramidon. These results suggest that NEP is present as a transmembrane enzyme on articular chondrocytes where it can cleave regulatory peptides and lead to the induction of aggrecanase.     

Transglutaminase 2 as a biomarker of osteoarthritis: an update

Osteoarthritis is a progressive joint disease characterized by cartilage degradation and bone remodelling. Under physiologic conditions, articular cartilage displays a stable chondrocyte phenotype, whereas in osteoarthritis a chondrocyte hypertrophy develops near the sites of cartilage surface damage and associates to the pathologic expression of type X collagen. Transglutaminases (TGs) include a family of Ca²⁺-dependent enzymes that catalyze the formation of γ-glutamyl cross-links. Their substrates include a variety of intracellular and extracellular macromolecular components. TGs are ubiquitously and abundantly expressed and implicated in a variety of physiopathological processes. TGs activity is modulated by inflammatory cytokines. TG2 (also known as tissue transglutaminase) mediates the hypertrophic differentiation of joint chondrocytes and interleukin-1-induced calcification. Histomorphometrical and biomolecular investigations document increased TG2 expression in human and experimental osteoarthritis. Consequently, the level of TG2 expression may represent an adjuvant additional marker to monitor tissue remodelling occurring in osteoarthritic joint tissue. Experimental induction of osteoarthritis in TG2 knockout mice is followed from reduced cartilage destruction and increased osteophyte formation compared to wild-type mice, suggesting a different influence on joint bone and cartilage remodelling. The capacity of transamidation by TG2 to regulate activation of latent TGF-β seems to have a potential impact on the regulation of inflammatory response in osteoarthritic tissues. Additional studies are needed to define TG2-regulated pathways that are differently modulated in osteoblasts and chondrocytes during osteoarthritis.     

Effects of shear stress on articular chondrocyte metabolism

The articular cartilage of diarthrodial joints experiences a variety of stresses, strains and pressures that result from normal activities of daily living. In normal cartilage, the extracellular matrix exists as a highly organized composite of specialized macromolecules that distributes loads at the bony ends. The chondrocyte response to mechanical loading is recognized as an integral component in the maintenance of articular cartilage matrix homeostasis. With inappropriate mechanical loading of the joint, as occurs with traumatic injury, ligament instability, bony malalignment or excessive weight bearing, the cartilage exhibits manifestations characteristic of osteoarthritis. Breakdown of cartilage in osteoarthritis involves degradation of the extracellular matrix macromolecules and decreased expression of chondrocyte proteins necessary for normal joint function. Osteoarthritic cartilage often exhibits increased amounts of type I collagen and synthesis of proteoglycans characteristic of immature cartilage. The shift in cartilage phenotype in response to altered load yields a matrix that fails to support normal joint function. Mathematical modeling and experimental studies in animal models confirm an association between altered loading of diarthrotic joints and arthritic changes. Both types of studies implicate shear forces as a critical component in the destructive profile. The severity of cartilage destruction in response to altered loads appears linked to expression of biological factors influencing matrix integrity and cellular metabolism. Determining how shear stress alters chondrocyte metabolism is fundamental to understanding how to limit matrix destruction and stimulate cartilage repair and regeneration. At present, the precise biochemical and molecular mechanisms by which shear forces alter chondrocyte metabolism from a normal to a degenerative phenotype remain unclear. The results presented here address the hypothesis that articular chondrocyte metabolism is modulated by direct effects of shear forces that act on the cell through mechanotransduction processes. The purpose of this work is to develop critical knowledge regarding the basic mechanisms by which mechanical loading modulates cartilage metabolism in health and disease. This presentation will describe the effects of using fluid induced shear stress as a model system for stimulation of articular chondrocytes in vitro. The fluid induced shear stress was applied using a cone viscometer system to stimulate all the cells uniformly under conditions of minimal turbulence. The experiments were carried using high-density primary monolayer cultures of normal and osteoarthritic human and normal bovine articular chondrocytes. The analysis of the cellular response included quantification of cytokine release, matrix metalloproteinase expression and activation of intracellular signaling pathways. The data presented here show that articular chondrocytes exhibit a dose- and time-dependent response to shear stress that results in the release of soluble mediators and extracellular matrix macromolecules. The data suggest that the chondrocyte response to mechanical stimulation contributes to the maintenance of articular cartilage homeostasis in vivo.     

Osteopontin is expressed by adult human osteoarthritic chondrocytes: protein and mRNA analysis of normal and osteoarthritic cartilage.

Osteopontin, a sulfated phosphoprotein with cell binding and matrix binding properties, is expressed in a variety of tissues. In the embryonic growth plate, osteopontin expression was found in bone-forming cells and in hypertrophic chondrocytes. In this study, the expression of osteopontin was analyzed in normal and osteoarthritic human knee cartilage. Immunohistochemistry, using a monoclonal anti-osteopontin antibody was negative on normal cartilage. These results were confirmed in Western blot experiments, using partially purified extracts of normal knee cartilage. No osteopontin gene expression was observed in chondrocytes of adult healthy cartilage, however, in the subchondral bone plate, expression of osteopontin mRNA was detected in the osteoblasts. In cartilage from patients with osteoarthritis, osteopontin could be detected by immunohistochemistry, Western blot analysis, in situ hybridization, and Northern blot analysis. A qualitative analysis indicated that osteopontin protein deposition and mRNA expression increase with the severity of the osteoarthritic lesions and the disintegration of the cartilaginous matrix. Osteopontin expression in the cartilage was limited to the chondrocytes of the upper deep zone, showing cellular and territorial deposition. The strongest osteopontin detection was found in deep zone chondrocytes and in clusters of proliferating chondrocytes from samples with severe osteoarthritic lesions. These data show the expression of osteopontin in adult human osteoarthritic chondrocytes, suggesting that chondrocyte differentiation and the expression of differentiation markers in osteoarthritic cartilage resembles that of epiphyseal growth plate chondrocytes.     

In situ chondrocyte deformation with physiological compression of the feline patellofemoral joint

The mechanical environment is an important factor affecting the maintenance and adaptation of articular cartilage, and thus the function of the joint and the progression of joint degeneration. Recent evidence suggests that cartilage deformation caused by mechanical loading is directly associated with deformation and volume changes of chondrocytes. Furthermore, in vitro experiments have shown that these changes in the mechanical states of chondrocytes correlate with a change in the biosynthetic activity of cartilage cells. The purpose of this study was to apply our knowledge of contact forces within the feline patellofemoral joint to quantify chondrocyte deformation in situ under loads of physiological magnitude. A uniform, static load of physiological magnitude was applied to healthy articular cartilage still fully intact and attached to its native bone. The compressed cartilage was then chemically fixed to enable the evaluation of cartilage strain, chondrocyte deformation and chondrocyte volumetric fraction. Patella and femoral groove articular cartilages differ in thickness, chondrocyte aspect ratio, and chondrocyte volumetric fraction in both magnitude and depth distribution. Furthermore, when subjected to the same compressive loads, changes to all of these parameters differ in magnitude and depth distribution between patellar and femoral groove articular cartilage. This evidence suggests that significant chondrocyte deformation likely occurs during in vivo joint loading, and may influence chondrocyte biosynthetic activity. Furthermore, we hypothesise that the contrasts between patella and femoral groove cartilages may explain, in part, the site-specific progression of osteoarthritis in the patellofemoral joint of the feline anterior cruciate ligament transected knee.     

Hypertrophic differentiation of chondrocytes in osteoarthritis: the developmental aspect of degenerative joint disorders

Osteoarthritis is characterized by a progressive degradation of articular cartilage leading to loss of joint function. The molecular mechanisms regulating pathogenesis and progression of osteoarthritis are poorly understood. Remarkably, some characteristics of this joint disease resemble chondrocyte differentiation processes during skeletal development by endochondral ossification. In healthy articular cartilage, chondrocytes resist proliferation and terminal differentiation. By contrast, chondrocytes in diseased cartilage progressively proliferate and develop hypertrophy. Moreover, vascularization and focal calcification of joint cartilage are initiated. Signaling molecules that regulate chondrocyte activities in both growth cartilage and permanent articular cartilage during osteoarthritis are thus interesting targets for disease-modifying osteoarthritis therapies.     

Articular cartilage vesicles contain RNA

Small membrane-bound extracellular organelles known as articular cartilage matrix vesicles (ACVs) participate in pathologic mineralization in osteoarthritic articular cartilage. ACVs are also present in normal cartilage, although they have no known functions other than mineralization. Recently, RNA was identified in extracellular vesicles derived from mast cells, suggesting that such vesicles might carry coding information from cell to cell. We found that ACVs from normal porcine and human articular cartilage and primary chondrocyte conditioned media contained 1 μg RNA/80 μg ACV protein. No DNA could be detected. RT-PCR of ACV RNA demonstrated the presence of full length mRNAs for factor XIIIA, type II transglutaminase, collagen II, aggrecan, ANKH and GAPDH. RNA in intact ACVs was resistant to RNase, despite the fact that ACV preparations contained measurable levels of active RNases. Significantly, radiolabeled RNA in ACVs could be transferred to unlabeled chondrocytes by co-incubation and produced changes in levels of chondrocyte enzymes and proteins. The demonstration that ACVs contain mRNAs suggests that they may function to shuttle genetic information between articular cells and indicate novel functions for these structures in articular cartilage.