Alteration of viscoelastic properties is associated with a change in cytoskeleton components of ageing chondrocytes from rabbit knee articular cartilage

The cytoskeleton network is believed to play an important role in the biomechanical properties of the chondrocyte. Ours and other laboratories have demonstrated that chondrocytes exhibit a viscoelastic solid creep behavior in vitro and that viscoelastic properties decrease in osteoarthritic chondrocytes. In this study, we aimed to understand whether the alteration of viscoelastic properties is associated with changes in cytoskeleton components of ageing chondrocytes from rabbit knee articular cartilage. Three age groups were used for this study: young (2-months-old, N=23), adult (8-months-old, N=23), and old (31-months-old, N=23) rabbit groups. Cartilage structure and proteoglycan and type II collagen content were determined by H&E and Toluidine Blue staining, and type II collagen antibody. The detailed structure of the chondrocytes in all groups was visualized using transmission electron microscopy (TEM). Chondrocytes were isolated from full-thickness knee cartilage of rabbits from all groups and their viscoelastic properties were quantified within 2 hours of isolation using a micropipette aspiration technique combined with a standard linear viscoelastic solid model. The components and network of the cytoskeleton within the cells were analyzed by laser scanning confocal microscopy (LSCM) with immunofluorescence staining as well as real time PCR and western blotting. With ageing, articular cartilage contained less chondrocytes and less proteoglycans and type II collagen. TEM observations showed that the cell membranes were not clearly defined, organelles were fewer and the nuclei were deformed or shrunk in the old cells compared with the young and adult cells. In suspension, chondrocytes from all three age groups showed significant viscoelastic creep behavior, but the deformation rate and amplitude of old chondrocytes were increased under the same negative pressure when compared to young and adult chondrocytes. Viscoelastic properties of the old cells, including equilibrium modulus (E infinity), instantaneous modulus (E0) and apparent viscosity (mu) were significantly lower than that those of the young and adult ones (P < 0.001). No significant differences were detected between young and adult chondrocytes (P > 0.05). Moreover, we found that the cytoskeletal networks of old cells were sparser, and that the contents of the various components of the intracellular networks were reduced in old cells, compared with adult and young cells. Aged chondrocytes had a different response to mechanical stimulation when compared to young and adult chondrocytes due to alteration of their viscoelastic properties, which was in turn associated with changes in cell structure and cytoskeleton composition.     

Immunohistochemical analysis of ageing and osteoarthritic articular cartilage

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

Alterations in structure and properties of collagen network of osteoarthritic and repaired cartilage modify knee joint stresses

Organization of the collagen network is known to be different in healthy, osteoarthritic and repaired cartilage. The aim of the study was to investigate how the structure and properties of collagen network of cartilage modulate stresses in a knee joint with osteoarthritis or cartilage repair. Magnetic resonance imaging (MRI) at 1.5 T was conducted for a knee joint of a male subject. Articular cartilage and menisci in the knee joint were segmented, and a finite element mesh was constructed based on the two-dimensional section in sagittal projection. Then, the knee joint stresses were simulated under impact loads by implementing the structure and properties of healthy, osteoarthritic and repaired cartilage in the models. During the progression of osteoarthritis, characterized especially by the progressive increase in the collagen fibrillation from the superficial to the deeper layers, the stresses were reduced in the superficial zone of cartilage, while they were increased in and under menisci. Increased fibril network stiffness of repair tissue with randomly organized collagen fibril network reduced the peak stresses in the adjacent tissue and strains at the repair–adjacent cartilage interface. High collagen fibril strains were indicative of stress concentration areas in osteoarthritic and repaired cartilage. The collagen network orientation and stiffness controlled the stress distributions in healthy, osteoarthritic and repaired cartilage. The evaluation of articular cartilage function using clinical MRI and biomechanical modeling could enable noninvasive estimation of osteoarthritis progression and monitoring of cartilage repair. This study presents a step toward those goals.     

Linked decreases in liver kinase B1 and AMP-activated protein kinase activity modulate matrix catabolic responses to biomechanical injury in chondrocytes

Introduction

AMP-activated protein kinase (AMPK) maintains cultured chondrocyte matrix homeostasis in response to inflammatory cytokines. AMPK activity is decreased in human knee osteoarthritis (OA) chondrocytes. Liver kinase B1 (LKB1) is one of the upstream activators of AMPK. Hence, we examined the relationship between LKB1 and AMPK activity in OA and aging cartilages, and in chondrocytes subjected to inflammatory cytokine treatment and biomechanical compression injury, and performed translational studies of AMPK pharmacologic activation.

Methods

We assessed activity (phosphorylation) of LKB1 and AMPKα in mouse knee OA cartilage, in aging mouse cartilage (6 to 24 months), and in chondrocytes after mechanical injury by dynamic compression, via immunohistochemistry or western blot. We knocked down LKB1 by siRNA transfection. Nitric oxide, matrix metalloproteinase (MMP)-3, and MMP-13 release were measured by Griess reaction and ELISA, respectively.

Results

Knockdown of LKB1 attenuated chondrocyte AMPK activity, and increased nitric oxide, MMP-3 and MMP-13 release (P <0.05) in response to IL-1β and TNFα. Both LKB1 and AMPK activity were decreased in mouse knee OA and aged knee cartilage, and in bovine chondrocytes after biomechanical injury. Pretreatment of bovine chondrocytes with AMPK activators AICAR and A-769662 inhibited both AMPKα dephosphorylation and catabolic responses after biomechanical injury.

Conclusion

LKB1 is required for chondrocyte AMPK activity, thereby inhibiting matrix catabolic responses to inflammatory cytokines. Concurrent loss of LKB1 and AMPK activity in articular chondrocytes is associated with OA, aging and biomechanical injury. Conversely, pharmacologic AMPK activation attenuates catabolic responses to biomechanical injury, suggesting a potentially novel approach to inhibit OA development and progression.     

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.     

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.     

3D Hydrogel Scaffolds for Articular Chondrocyte Culture and Cartilage Generation

Human articular cartilage is highly susceptible to damage and has limited self-repair and regeneration potential. Cell-based strategies to engineer cartilage tissue offer a promising solution to repair articular cartilage. To select the optimal cell source for tissue repair, it is important to develop an appropriate culture platform to systematically examine the biological and biomechanical differences in the tissue-engineered cartilage by different cell sources. Here we applied a three-dimensional (3D) biomimetic hydrogel culture platform to systematically examine cartilage regeneration potential of juvenile, adult, and osteoarthritic (OA) chondrocytes. The 3D biomimetic hydrogel consisted of synthetic component poly(ethylene glycol) and bioactive component chondroitin sulfate, which provides a physiologically relevant microenvironment for in vitro culture of chondrocytes. In addition, the scaffold may be potentially used for cell delivery for cartilage repair in vivo. Cartilage tissue engineered in the scaffold can be evaluated using quantitative gene expression, immunofluorescence staining, biochemical assays, and mechanical testing. Utilizing these outcomes, we were able to characterize the differential regenerative potential of chondrocytes of varying age, both at the gene expression level and in the biochemical and biomechanical properties of the engineered cartilage tissue. The 3D culture model could be applied to investigate the molecular and functional differences among chondrocytes and progenitor cells from different stages of normal or aberrant development.     

Effect of Age and Cytoskeletal Elements on the Indentation-Dependent Mechanical Properties of Chondrocytes

Articular cartilage chondrocytes are responsible for the synthesis, maintenance, and turnover of the extracellular matrix, metabolic processes that contribute to the mechanical properties of these cells. Here, we systematically evaluated the effect of age and cytoskeletal disruptors on the mechanical properties of chondrocytes as a function of deformation. We quantified the indentation-dependent mechanical properties of chondrocytes isolated from neonatal (1-day), adult (5-year) and geriatric (12-year) bovine knees using atomic force microscopy (AFM). We also measured the contribution of the actin and intermediate filaments to the indentation-dependent mechanical properties of chondrocytes. By integrating AFM with confocal fluorescent microscopy, we monitored cytoskeletal and biomechanical deformation in transgenic cells (GFP-vimentin and mCherry-actin) under compression. We found that the elastic modulus of chondrocytes in all age groups decreased with increased indentation (15–2000 nm). The elastic modulus of adult chondrocytes was significantly greater than neonatal cells at indentations greater than 500 nm. Viscoelastic moduli (instantaneous and equilibrium) were comparable in all age groups examined; however, the intrinsic viscosity was lower in geriatric chondrocytes than neonatal. Disrupting the actin or the intermediate filament structures altered the mechanical properties of chondrocytes by decreasing the elastic modulus and viscoelastic properties, resulting in a dramatic loss of indentation-dependent response with treatment. Actin and vimentin cytoskeletal structures were monitored using confocal fluorescent microscopy in transgenic cells treated with disruptors, and both treatments had a profound disruptive effect on the actin filaments. Here we show that disrupting the structure of intermediate filaments indirectly altered the configuration of the actin cytoskeleton. These findings underscore the importance of the cytoskeletal elements in the overall mechanical response of chondrocytes, indicating that intermediate filament integrity is key to the non-linear elastic properties of chondrocytes. This study improves our understanding of the mechanical properties of articular cartilage at the single cell level.     

Human chondrocyte senescence and osteoarthritis

Although osteoarthritis (OA) is not an inevitable consequence of aging, a strong association exists between age and increasing incidence of OA. We hypothesized that this association is due to in vivo articular cartilage chondrocyte senescence which causes an age-related decline in the ability of the cells to maintain articular cartilage, that is, increasing age increases the risk of OA because chondrocytes lose their ability to replace their extracellular matrix. To test this hypothesis, we measured senescence markers in human articular cartilage chondrocytes from 27 donors ranging in age from one to 87 years. The markers included expression of the senescence-associated enzyme beta-galactosidase, mitotic activity measured by 3H-thymidine incorporation, and telomere length. beta-galactosidase expression increased with age (r=0.84, p=0.0001) while mitotic activity and mean telomere length declined (r=-0.774, p=0.001 and r=-0.71, p=0.0004, respectively). Decreasing telomere length was strongly correlated with increasing expression of beta-galactosidase and decreasing mitotic activity. These findings help explain the previously reported age related declines in chondrocyte synthetic activity and responsiveness to anabolic growth factors and indicate that in vivo articular cartilage chondrocyte senescence is responsible, at least in part, for the age related increased incidence of OA. The data also imply that people vary in their risk of developing OA because of differences in onset of chondrocyte senescence; and, the success of chondrocyte transplantation procedures performed to restore damaged articular surfaces in older patients could be limited by the inability of older chondrocytes to form new cartilage. New efforts to prevent the development or progression of OA might include strategies that delay the onset of chondrocyte senescence or replace senescent cells.     

Cytoskeletal architecture and cathepsin B trafficking in human articular chondrocytes

In the differentiated state, human articular chondrocytes exhibited modestly developed cytoskeletal components, while cells dedifferentiated by serial subcultures in vitro displayed a prominent cytoskeleton. Morphological changes, a well-developed actin cytoskeleton, and the presence of numerous intracellular organelles were characteristic features of the dedifferentiated chondrocyte phenotype. These properties correlated with the expression, biosynthesis, storage, and secretion of the cysteine peptidase, cathepsin B, a marker of the dedifferentiated chondrocyte phenotype and a potent mediator of cartilage catabolism in osteoarthritis. Both the actin cytoskeleton and microtubules were responsible for trafficking of cathepsin B between cellular compartments in chondrocytes. Despite the endosomes and lysosomes storing high amounts of mature cathepsin B, this enzyme could not be visualized in its active form within these organelles. However, enzymatically active cathepsin B was associated with polymerized tubulin, and was no longer detectable after disruption of the microtubules. This enzyme species possibly represents the mature cathepsin B form in transport vesicles, after cleavage of the inhibitory propeptide, on the way to a final target. These results suggest noteworthy parallels between osteoarthritic articular cartilage and the artificially dedifferentiated cell phenotype, including the expression of type I collagen, the expression of cathepsin B, a significant modification of the cytoskeleton, and the formation of abundant secretory vesicles. These similarities justify the use of chondrocyte cultures as models of the behavior of cartilage cells in osteoarthritis.     

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.     

Effects of osmotic challenges on membrane potential in human articular chondrocytes from healthy and osteoarthritic cartilage

Changes in external osmolarity arise from variations in mechanical loads on joints and may affect the homeostasis of chondrocytes, which are the only cell type responsible for matrix turnover. Accordingly, variations in membrane potential may affect cartilage production. The present study assessed the effects of variations in external osmolarity on membrane potential and the possible mechanisms responsible for this response. Membrane potential was measured by the patch clamp whole-cell technique using human articular chondrocytes freshly isolated from healthy and osteoarthritic cartilage. The membrane potential was -39±4 mV in articular human chondrocytes from healthy cartilage and -26±4 mV in those from osteoarthritic cartilage. Increasing the osmolarity produced a reversible hyperpolarization mediated by K+ efflux through BKCa channels in both groups of chondrocytes, but the response in osteoarthritic cells was significantly reduced; no other K+ pathways were involved in this effect. Alternatively, decreasing the osmolarity elicited depolarization in healthy chondrocytes but did not produce any response in chondrocytes from osteoarthritic cartilage. The depolarization was dependent on Na+ influx through Gd3+-sensitive stretch-activated cation channels and was independent of external Ca2+. The differential responses observed in chondrocytes from osteoarthritic cartilage suggest that disregulation on the responses to external osmolarity may be involved in the process that leads to the alterations in the cartilage structure observed in osteoarthritis.     

Advanced Osteoarthritis in Humans Is Associated With Altered Collagen VI Expression and Upregulation of ER-stress Markers Grp78 and Bag-1

To test the hypothesis that a perturbation of endoplasmic reticulum (ER) function is involved in the pathogenesis of osteoarthritis (OA), articular cartilage was isolated from non-OA patients secondary to resection of osteo- or chondrosarcomas. Intra-joint samples of minimal and advanced osteoarthritic cartilage were isolated from patients undergoing total knee arthroplasty and scored for disease severity. Glucose-regulated protein-78 (grp78) and bcl-2–associated athanogene-1 (bag-1) were detected via immunofluorescence as markers of non-homeostatic ER function. Additionally, the expression of type VI collagen and its integrin receptor, NG2, was determined to examine cartilage matrix health and turnover. There was an upregulation of grp78 in advanced OA, and variable expression in minimal OA. Non-OA cartilage was consistently grp78 negative. The downstream regulator bag-1 was also upregulated in OA compared with normal cartilage. Collagen VI was mainly cell-associated in non-OA cartilage, with a more widespread distribution observed in OA cartilage along with increased intracellular staining intensity. The collagen VI integral membrane proteoglycan receptor NG2 was downregulated in advanced OA compared with its patient-matched minimally involved cartilage sample. These results suggest that chondrocytes exhibit ER stress during OA, in association with upregulation of a large secreted molecule, type VI collagen. (J Histochem Cytochem 57:923–931, 2009)     

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.     

Relationship between T1rho magnetic resonance imaging,synovial fluid biomarkers,and the biochemical and biomechanical properties of cartilage

Non-invasive techniques for quantifying early biochemical and biomechanical changes in articular cartilage may provide a means of more precisely assessing osteoarthritis (OA) progression. The goals of this study were to determine the relationship between T1rho magnetic resonance (MR) imaging relaxation times and changes in cartilage composition, cartilage mechanical properties, and synovial fluid biomarker levels and to demonstrate the application of T1rho imaging to evaluate cartilage composition in human subjects in vivo. Femoral condyles and synovial fluid were harvested from healthy and OA porcine knee joints. Sagittal T1rho relaxation MR images of the condyles were acquired. OA regions of OA joints exhibited an increase in T1rho relaxation times as compared to non-OA regions. Furthermore in these regions, cartilage sGAG content and aggregate modulus decreased, while percent degraded collagen and water content increased. In OA joints, synovial fluid concentrations of sGAG decreased and C2C concentrations increased compared to healthy joints. T1rho relaxation times were negatively correlated with cartilage and synovial fluid sGAG concentrations and aggregate modulus and positively correlated with water content and permeability. Additionally, we demonstrated the application of these in vitro findings to the study of human subjects. Specifically, we demonstrated that walking results in decreased T1rho relaxation times, consistent with water exudation and an increase in proteoglycan concentration with in vivo loading. Together, these findings demonstrate that cartilage MR imaging and synovial fluid biomarkers provide powerful non-invasive tools for characterizing changes in the biochemical and biomechanical environments of the joint.     

Do chondrocytes undergo "activation" and "transdifferentiation" during the pathogenesis of osteoarthritis? A review of the ultrastructural and immunohistochemical evidence

Chondrocytes, which are the only cell type in the articular cartilage, show substantial morphological and functional differences, depending on their location within the tissue. In OA cartilage, outstanding modifications have been reported concerning their structure and functions. Based on the principle that both structure and function run in a parallel manner, new concepts are arising related to morphological observations. Observations on OA chondrocytes, such as cytoskeleton disruption, development of the secretory machinery (rough endoplasmic reticulum and Golgi complex), and cell death by apoptosis, among others, certainly must be related to the role of chondrocytes in OA pathogenesis. In this degradative process, it has been acknowledged that cell death, matrix degradation and subchondral bone remodelling are the main causes of cartilage breakdown in osteoarthritis. The aim of this review was to correlate and integrate in a logical manner the modifications of chondrocytes with cartilage breakdown during osteoarthritis pathogenesis. Furthermore, we intend to open a debate on cell cycle and mitosis, as well as on signalling molecules that might be involved in the morphofunctional changes in OA chondrocytes, which we propose to name "activation" and "transdifferentiation" of chondrocytes. We expect this analysis to be useful for studying OA pathogenesis in depth, with the aim of finding new strategies for the early diagnosis and therapeutic procedures for this invalidating disease, which is already an important public health problem.     

Do changes in the mechanical properties of articular cartilage promote catabolic destruction of cartilage and osteoarthritis?

Osteoarthritis (OA) is a joint disease characterized by cartilage degeneration, a thickening of subchondral bone, and formation of marginal osteophytes. Previous mechanical characterization of cartilage in our laboratory suggests that energy storage and dissipation is reduced in osteoarthritis as the extent of fibrillation and fissure formation increases. It is not clear whether the loss of energy storage and dissipation characteristics is a result of biochemical and/or biophysical changes that occur to hyaline cartilage in joints. The purpose of this study is to present data, on the strain rate dependence of the elastic and viscous behaviors of cartilage, in order to further characterize changes that occur in the mechanical properties that are associated with OA. We have previously hypothesized that the changes seen in the mechanical properties of cartilage may be due to altered mechanochemical transduction by chondrocytes. Results of incremental tensile stress-strain tests at strain rates between 100%/min and 10,000%/min conducted on OA cartilage indicate that the slope of the elastic stress-strain curve increases with increasing strain rate, unlike the reported behavior of skin and self-assembled collagen fibers. It is suggested that the strain-rate dependence of the elastic stress-strain curve is due to the presence of large quantities of proteoglycans (PGs), which protect articular cartilage by increasing the apparent stiffness. The increased apparent stiffness of articular cartilage at high strain rates may limit the stresses borne and prolong the onset of OA. It is further hypothesized that increased compressive loading of chondrocytes in the intermediate zone of articular cartilage occurs as a result of normal wear to the superficial zone or from excessive impact loading. Once the superficial zone of articular cartilage is worn away, the tension is decreased throughout all cartilage zones leading to increased chondrocyte compressive loading and up-regulation of mechanochemical transduction processes that elaborate catabolic enzymes.     

Effect of eldecalcitol on articular cartilage through the regulation of transcription factor Erg in a murine model of knee osteoarthritis

Clinical studies have reported an association between low blood levels of 25-hydroxyvitamin D and the progression of osteoarthritis (OA), but the mechanism and effects of vitamin D signaling on articular chondrocytes and cartilage remains unclear. The purpose of this study was to investigate the effects of vitamin D on articular cartilage degeneration using eldecalcitol (ED-71), which is an active vitamin D₃ analog. Eight-week old male C57BL/6NCrSlc mice were subjected to experimental surgery to induce OA and local treatments with 10 μL ED-71 (0.5 μg/mL) were administered weekly. Four and 12 weeks after surgery, joints were evaluated using histological scoring systems. In addition, gene expression was analyzed in chondrocytes that were isolated from wildtype neonatal mice, cultured, and treated with ED-71 (10^?8 M). Joints treated with ED-71 demonstrated slowed progression of OA at 4 weeks after surgery, but few effects were observed at 12 weeks after surgery. Ets-related gene (Erg) expression was upregulated in OA articular cartilage, and further increased by ED-71 treatment. In primary chondrocytes cultured with ED-71, the gene expression of Erg and lubricin/proteoglycan 4 significantly increased, as compared to that of cells cultured without ED-71. Local treatment with ED-71 reduced degenerative changes to the articular cartilage during the early phase of experimental OA. Regulation of Erg by ED-71 in articular cartilage could confer resistance to early osteoarthritic changes.     

Immunohistochemical demonstration of -(carboxymethyl)lysine protein adducts in normal and osteoarthritic cartilage

N(epsilon)-(carboxymethyl)lysine (CML) is an advanced glycation end product formed by non-enzymatic glycation and oxidation of proteins. The distribution pattern of CML-modified proteins in normal and osteoarthritic (OA) cartilage was investigated using specific antibodies. In healthy articular cartilage, immunoreactivity for CML was preferably found in the extracellular matrix (ECM) of the superficial layer. In OA samples, CML immunoreactivity was not restricted to the ECM of the superficial layer. Interestingly, OA chondrocytes showed a remarkable cytoplasmic immunoreactivity for CML. With the help of a western blot analysis CML-modified proteins between 68 and 39 kDa could be demonstrated in OA cartilage samples. These results suggest that the accumulation of CML adducts contributes to the matrix damage in osteoarthritis. Therefore, the inhibition of CML accumulation may represent an effective therapeutic strategy to prevent severe OA cartilage injury.