The chondrocyte cytoskeleton in mature articular cartilage: structure and distribution of actin, tubulin, and vimentin filaments.

We investigated the structure of the chondrocyte cytoskeleton in intact tissue sections of mature bovine articular cartilage using confocal fluorescence microscopy complemented by protein extraction and immunoblotting analysis. Actin microfilaments were present inside the cell membrane as a predominantly cortical structure. Vimentin and tubulin spanned the cytoplasm from cell to nuclear membrane, the vimentin network appearing finer compared to tubulin. These cytoskeletal structures were present in chondrocytes from all depth zones of the articular cartilage. However, staining intensity varied from zone to zone, usually showing more intense staining for the filament systems at the articular surface compared to the deeper zones. These results obtained on fluorescently labeled sections were also corroborated by protein contents extracted and observed by immunoblotting. The observed cytoskeletal structures are compatible with some of the proposed cellular functions of these systems and support possible microenvironmental regulation of the cytoskeleton, including that due to physical forces from load-bearing, which are known to vary through the depth layers of articular cartilage.     

Hypoxia. HIF-mediated articular chondrocyte function: prospects for cartilage repair

In a chronically hypoxic tissue such as cartilage, adaptations to hypoxia do not merely include cell survival responses, but also promotion of its specific function. This review will focus on describing such hypoxia-mediated chondrocyte function, in particular in the permanent articular cartilage. The molecular details of how chondrocytes sense and respond to hypoxia and how this promotes matrix synthesis have recently been examined, and specific manipulation of hypoxia-induced pathways is now considered to have potential therapeutic application to maintenance and repair of articular cartilage.     

Integrins and chondrocyte–matrix interactions in articular cartilage

The integrin family of cell adhesion receptors plays a major role in mediating interactions between cells and the extracellular matrix. Normal adult articular chondrocytes express α1β1, α3β1, α5β1, α10β1, αVβ1, αVβ3, and αVβ5 integrins, while chondrocytes from osteoarthritic tissue also express α2β1, α4β1, α6β1. These integrins bind a host of cartilage extracellular matrix (ECM) proteins, most notably fibronectin and collagen types II and VI, which provide signals that regulate cell proliferation, survival, differentiation, and matrix remodeling. By initiating signals in response to mechanical forces, chondrocyte integrins also serve as mechanotransducers. When the cartilage matrix is damaged in osteoarthritis, fragments of fibronectin are generated that signal through the α5β1 integrin to activate a pro-inflammatory and pro-catabolic response which, if left unchecked, could contribute to progressive matrix degradation. The cell signaling pathways activated in response to excessive mechanical signals and to fibronectin fragments are being unraveled and may represent useful therapeutic targets for slowing or stopping progressive matrix destruction in arthritis.     

RNA extraction from human articular cartilage by chondrocyte isolation

We report an optimized method for RNA extraction from human articular cartilage that does not require the use of specialized equipment or column purification. To maximize RNA yield while minimizing degradation and contamination, chondrocytes are isolated from the extracellular matrix and the traditional TRIzol protocol is modified to include two RNA-DNA-protein phase separations. We compared RNA extracted using this modified method with the traditional TRIzol method by spectrophotometry, Bioanalyzer, and real-time polymerase chain reaction (PCR). With the modified method, RNA recovery is increased by nearly 1μg per 100mg of cartilage, and RNA integrity number (RIN) is improved from 2.0 to 7.5.     

Injurious mechanical compression of bovine articular cartilage induces chondrocyte apoptosis

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

Depletion of chondrocyte primary cilia reduces the compressive modulus of articular cartilage

Primary cilia are slender, microtubule based structures found in the majority of cell types with one cilium per cell. In articular cartilage, primary cilia are required for chondrocyte mechanotransduction and the development of healthy tissue. Loss of primary cilia in Col2aCre;ift88^fl/fl transgenic mice results in up-regulation of osteoarthritic (OA) markers and development of OA like cartilage with greater thickness and reduced mechanical stiffness. However no previous studies have examined whether loss of primary cilia influences the intrinsic mechanical properties of articular cartilage matrix in the form of the modulus or just the structural properties of the tissue. The present study describes a modified analytical model to derive the viscoelastic moduli based on previous experimental indentation data. Results show that the increased thickness of the articular cartilage in the Col2aCre;ift88^fl/fl transgenic mice is associated with a reduction in both the instantaneous and equilibrium moduli at indentation strains of greater than 20%. This reveals that the loss of primary cilia causes a significant reduction in the mechanical properties of cartilage particularly in the deeper zones and possibly the underlying bone. This is consistent with histological analysis and confirms the importance of primary cilia in the development of a mechanically functional articular cartilage.     

A method for determining DNA and chondrocyte content of articular cartilage

A novel and precise method was devised to study the DNA and chondrocyte content of articular cartilage. It involved the sequential digestion of cartilage matrix with hyaluronidase, trypsin, and collagenase to release the chondrocytes. A direct cell count and DNA assays were then performed on the cells. The concentration of cells was the quotient of the total number of cells and the weight of cartilage used. The DNA content of cartilage is identical to the amount of DNA in the chondrocytes. Our data also confirmed the earlier findings that cell density and DNA content of articular cartilage decreased gradually to a relatively constant level as animals matured to adulthood.     

Human chondrocyte cell lines from articular cartilage of metatarsal phalangeal joints

Chondrocytes can be isolated from human adult cartilage from metatarsal phalangeal joints. After enzymatic digestion to isolate viable cells, confluent monolayers were obtained 2-4 weeks after the start of cell division. Chondrocytes cultures, initiated and maintained in HAM's F12 with bovine fetal serum without the addition of other growth factors, produced in vitro a matrix rich in collagen and proteoglycans. Although several studies reported phenotypic instability, our results showed that the cell retain for more than 5 months in culture their differentiated characteristics, including the ability to produce cartilage-specific molecules. Chondrocyte cell lines should be useful in studying the functions of these cells from normal and abnormal tissue and for pharmacological studies in vitro.     

A novel method for determining articular cartilage chondrocyte mechanics in vivo

Work relating the mechanical states of articular cartilage chondrocytes to their biosynthetic responses is based on measurements in isolated cells or cells in explant samples removed from their natural in situ environment. Neither the mechanics nor the associated biological responses of chondrocytes have ever been studied in cartilage within a joint of a live animal, and no such measurements have ever been performed using physiologically relevant joint loading through muscular contractions. The purpose of this study was to design and apply a method to study the mechanics of chondrocytes in the exposed but fully intact knee of live animals, which was loaded near-physiologically through muscular contraction. In order to achieve this purpose, we developed an accurate and reliable method based on two-photon laser excitation microscopy. Near-physiological knee joint loading was achieved through controlled electrical activation of the knee extensor muscles that compress the articulating surfaces of the femur, tibia and patella. Accuracy of the system was assessed by inserting micro-beads of known dimensions into the articular cartilage of the mouse knee and comparing the measured volumes and diameters in the principal directions with known values of the beads. Accuracy was best in the plane perpendicular to the optical axis (average error = 1%) while it was slightly worse, but still excellent, along the optical axis (average error = 3%). Reliability of cell volume and shape measurements was 0.5% on average, and 2.9% in the worst-case-scenario. Pilot measurements of chondrocyte deformations upon sub-maximal muscular loading causing a mean articular contact pressure of 1.9 ± 0.2 MPa showed an "instantaneous" decrease in cell height (17 ± 4.5%) and loss of cell volume (22.3 ± 2.4%) that took minutes to recover upon deactivation of the knee extensor muscles.     

Effect of hyaluronic acid on human chondrocyte cell lines from articular cartilage

The effects of hyaluronic acid (HA) derivative on the proliferation and metabolism of human chondrocytes were examined. Cells were obtained from cartilage from metatarsal phalangeal joints of 20 adult humans (aged 22-63) and from femoral knee condyles of 10 subjects (aged 22-77). Chondrocytes isolated by collagenase/Dnase digestion were cultured with addition of different doses of HA for 4 weeks. Morphological studies demonstrated that HA enhanced the adhesion of cells to substrate; HA-treated chondrocytes proliferated better than chondrocytes cultured in HA-free medium. This study shows that HA improves in vitro substrate adhesion ability and proliferative activity of human cartilage cells and that the response to the treatment varies on an individual basis.     

Effects of Wnt3A and mechanical load on cartilage chondrocyte homeostasis

Introduction  

Articular cartilage functions in withstanding mechanical loads and provides a lubricating surface for frictionless movement of joints. Osteoarthritis, characterised by cartilage degeneration, develops due to the progressive erosion of structural integrity and eventual loss of functional performance. Osteoarthritis is a multi-factorial disorder; two important risk factors are abnormal mechanical load and genetic predisposition. A single nucleotide polymorphism analysis demonstrated an association of hip osteoarthritis with an Arg324Gly substitution mutation in FrzB, a Wnt antagonist. The purpose of this study was two-fold: to assess whether mechanical stimulation modulates β-catenin signalling and catabolic gene expression in articular chondrocytes, and further to investigate whether there is an interplay of mechanical load and Wnt signalling in mediating a catabolic response.     

A second-generation autologous chondrocyte implantation approach to the treatment of focal articular cartilage defects

Autologous chondrocyte implantation (ACI) is the most widely used cell-based surgical procedure for the repair of articular cartilage defects. Challenges to successful ACI outcomes include limitation in defect size and geometry as well as inefficient cell retention. Second-generation ACI procedures have thus focused on developing three-dimensional constructs using native and synthetic biomaterials. Clinically significant and satisfactory results from applying autologous chondrocytes seeded in fibrin within a biodegradable polymeric material were recently reported. In the future, third-generation cell-based articular cartilage repair should focus on the use of chondroprogenitor cells and biofunctionalized biomaterials for more extensive and permanent repair.     

The prostaglandins of articular cartilage I. Correlates of prostaglandin activity in a chondrocyte culture system

Suspensions of aggregated chondrocytes display active prostaglandin (PG) production. Radioimmunoassay of culture media and thin layer chromatographic analysis suggests that PGE₂ is the primary PG synthesized. In order of decreasing concentration, the following PG were tentatively identified; PGE> PGI> PGA + PGB PGF₁₊₂ > T×B. An inverse logarithmic relationship was identified between PG synthesis and cells cultured at densities of 1.5 to 7.5 × 10⁶ cells/ml. Little or no change in the PG distribution profile was seen at these high cell densities. Maximum PG synthesis was attained after 36 hours of incubation with persistence of high synthetic levels up to 48 hours. PGE₂ production measured at various post-isolation intervals indicated an initial high rate of synthesis during the first 4 hours which decreased with time up to 24 hours. Cartilage explant organ cultures demonstrated a similar level of PG synthesis suggesting minimal effect of matrix on cellular PG production. Indomethacin (5 μg/ml) inhibited PG synthesis by 70% within 4 hours and 85% after 24 hours of exposure. Arachidonic acid supplementation (10 μM) stimulated PG synthesis by 300%.     

Osteoarthritic changes in the biphasic mechanical properties of the chondrocyte pericellular matrix in articular cartilage

The pericellular matrix (PCM) is a narrow region of cartilaginous tissue that surrounds chondrocytes in articular cartilage. Previous modeling studies indicate that the mechanical properties of the PCM relative to those of the extracellular matrix (ECM) can significantly affect the stress-strain, fluid flow, and physicochemical environments of the chondrocyte, suggesting that the PCM plays a biomechanical role in articular cartilage. The goals of this study were to measure the mechanical properties of the PCM using micropipette aspiration coupled with a linear biphasic finite element model, and to determine the alterations in the mechanical properties of the PCM with osteoarthritis (OA). Using a recently developed isolation technique, chondrons (the chondrocyte and its PCM) were mechanically extracted from non-degenerate and osteoarthritic human cartilage. The transient mechanical behavior of the PCM was well-described by a biphasic model, suggesting that the viscoelastic response of the PCM is attributable to flow-dependent effects, similar to that of the ECM. With OA, the mean Young's modulus of the PCM was significantly decreased (38.7+/-16.2 kPa vs. 23.5+/-12.9 kPa, p < 0.001), and the permeability was significantly elevated (4.19+/-3.78 x10(-17) m(4)/Ns vs. 10.2+/-9.38 x 10(-17) m(4)/Ns, p < 0.01). The Poisson's ratio was similar for both non-degenerate and OA PCM (0.044+/-0.063 vs. 0.030+/-0.068, p > 0.6). These findings suggest that the PCM may undergo degenerative processes with OA, similar to those occurring in the ECM. In combination with previous theoretical models of cell-matrix interactions in cartilage, our findings suggest that changes in the properties of the PCM with OA may have an important influence on the biomechanical environment of the chondrocyte.     

Functional adaptation of the articular cartilage.

The effect of prolonged sparing and prolonged loading of the knee-joint of dogs on the glucosamine, sialic acid, sulphate and hydroxyproline contents of the articular cartilage was investigated. (a) In the articular cartilage of the spared leg the amount of sulphate decreased by 24.7%, while the sialic acid content remained unchanged. Hydroxyproline showed a slight decrease. (b) On increased loading, glucosamine augmented by 53% and sialic acid by 32.5%. No appreciable changes occurred in sulphate and hydroxyproline. It is concluded that an increased loading brings about accumulation of glycoproteins while the amount of sulphated glycosaminoglycans does not change appreciably; the glycoprotein content of the spared articular cartilage remains unchanged, whereas the chondroitin sulphate content decreases considerably.     

Influence of stress magnitude on water loss and chondrocyte viability in impacted articular cartilage

The objective of this study was to assess mechano-biological response of articular cartilage when subjected to a single impact stress. Mature bovine cartilage explants were impacted with peak stresses ranging from 10 to 60 MPa at a stress rate of 350 MPa/s. Water loss, matrix axial deformation, dynamic impact modulus (DIM), and cell viability were measured immediately after impaction. The water loss through the articular surface (AS) was small and ranged from 1% to 6% with increasing peak stress. The corresponding axial strains ranged from 2.5% to 25%, respectively, while the DIM was 455.9 +/- 111.9 MPa. Chondrocyte death started at the articular surface and increased in depth to a maximum of 6% (70 microns) of the cartilage thickness at the highest stress. We found that the volumetric (axial) strain was more than twice the amount of water loss at the highest peak stress. Furthermore, specimens impacted such that the interstitial water was forced through the deep zone (DZ) had less water loss, a higher DIM, and no cell death. These findings appear to be due to matrix compaction in the superficial region causing higher compressive strains to occur at the surface rather than in the deeper zones.     

Static compression of articular cartilage can reduce solute diffusivity and partitioning: implications for the chondrocyte biological response.

Chondrocytes depend upon solute transport within the avascular extracellular matrix of adult articular cartilage for many of their biological activities. Alterations to bioactive solute transport may, therefore, represent a mechanism by which cartilage compression is transduced into cellular metabolic responses. We investigated the effects of cartilage static compression on diffusivity and partitioning of a range of model solutes including dextrans of molecular weights 3 and 40 kDa, and tetramethylrhodamine (a 430 Da fluorophore). New fluorescence methods were developed for real-time visualization and measurement of transport within compressed cartilage explants. Experimental design allowed for multiple measurements on individual explants at different compression levels in order to minimize confounding influences of compositional variations. Results demonstrate that physiological levels of static compression may significantly decrease solute diffusivity and partitioning in cartilage. Effects of compression were most dramatic for the relatively high molecular weight solutes. For 40 kDa dextran, diffusivity decreased significantly (p<0.01) between 8% and 23% compression, while partitioning of 3 and 40 kDa dextran decreased significantly (p<0.01) between free-swelling conditions and 8% compression. Since diffusivity and partitioning can influence pericellular concentrations of bioactive solutes, these observations support a role for perturbations to solute transport in mediating the cartilage biological response to compression.     

Inhibitory function of parathyroid hormone-related protein on chondrocyte hypertrophy: the implication for articular cartilage repair

Cartilage repair tissue is usually accompanied by chondrocyte hypertrophy and osseous overgrowths, and a role for parathyroid hormone-related protein (PTHrP) in inhibiting chondrocytes from hypertrophic differentiation during the process of endochondral ossification has been demonstrated. However, application of PTHrP in cartilage repair has not been extensively considered. This review systemically summarizes for the first time the inhibitory function of PTHrP on chondrocyte hypertrophy in articular cartilage and during the process of endochondral ossification, as well as the process of mesenchymal stem cell chondrogenic differentiation. Based on the literature review, the strategy of using PTHrP for articular cartilage repair is suggested, which is instructive for clinical treatment of cartilage injuries as well as osteoarthritis.     

The effect of finite compressive strain on chondrocyte viability in statically loaded bovine articular cartilage

Recent studies have reported that certain regimes of compressive loading of articular cartilage result in increased cell death in the superficial tangential zone (STZ). The objectives of this study were (1) to test the prevalent hypothesis that preferential cell death in the STZ results from excessive compressive strain in that zone, relative to the middle and deep zones, by determining whether cell death correlates with the magnitude of compressive strain and (2) to test the corollary hypothesis that the viability response of cells is uniform through the thickness of the articular layer when exposed to the same loading environment. Live cartilage explants were statically compressed by approximately 65% of their original thickness, either normal to the articular surface (axial loading) or parallel to it (transverse loading). Cell viability after 12 h was compared to the local strain distribution measured by digital image correlation. Results showed that the strain distribution in the axially loaded samples was highest in the STZ (77%) and lowest in the deep zone (55%), whereas the strain was uniformly distributed in the transversely loaded samples (64%). In contrast, axially and transversely loaded samples exhibited very similar profiles of cell death through the depth, with a preferential distribution in the STZ. Unloaded control samples showed negligible cell death. Thus, under prolonged static loading, depth-dependent variations in chondrocyte death did not correlate with the local depth-dependent compressive strain, and the prevalent hypothesis must be rejected. An alternative hypothesis, suggested by these results, is that superficial zone chondrocytes are more vulnerable to prolonged static loading than chondrocytes in the middle and deep zones.     

Caspase cleavage of vimentin disrupts intermediate filaments and promotes apoptosis.

Caspases are key mediators of apoptosis. Using a novel expression cloning strategy we recently developed to identify cDNAs encoding caspase substrates, we isolated the intermediate filament protein vimentin as a caspase substrate. Vimentin is preferentially cleaved by multiple caspases at distinct sites in vitro, including Asp85 by caspases-3 and -7 and Asp259 by caspase-6, to yield multiple proteolytic fragments. Vimentin is rapidly proteolyzed by multiple caspases into similar sized fragments during apoptosis induced by many stimuli. Caspase cleavage of vimentin disrupts its cytoplasmic network of intermediate filaments and coincides temporally with nuclear fragmentation. Moreover, caspase proteolysis of vimentin at Asp85 generates a pro-apoptotic amino-terminal fragment whose ability to induce apoptosis is dependent on caspases. Taken together, our findings suggest that caspase proteolysis of vimentin promotes apoptosis by dismantling intermediate filaments and by amplifying the cell death signal via a pro-apoptotic cleavage product.