Healthy Human Articular Cartilage Research Articles

The Releasate of Avascular Cartilage Demonstrates Inherent Pro-Angiogenic Properties In Vitro and In Vivo.

ObjectiveCartilage is avascular and numerous studies have identified the presence of single anti- and pro-angiogenic factors in cartilage. To better understand the maintenance hyaline cartilage, we assessed the angiogenic potential of complete cartilage releasate with functional assays in vitro and in vivo.DesignWe evaluated the gene expression profile of angiogenesis-related factors in healthy adult human articular cartilage with a transcriptome-wide analysis generated by next-generation RNAseq. The effect on angiogenesis of the releasate of cartilage tissue was assessed with a chick chorioallantoic membrane (CAM) assay as well as human umbilical vein endothelial cell (HUVEC) migration and proliferation assays using conditioned media generated from tissue-engineered cartilage derived from human articular and nasal septum chondrocytes as well as explants from bovine articular cartilage and human nasal septum. Experiments were done with triplicate samples of cartilage from 3 different donors.ResultsRNAseq data of 3 healthy human articular cartilage donors revealed that the majority of known angiogenesis-related factors expressed in healthy adult articular cartilage are pro-angiogenic. The releasate from generated cartilage as well as from tissue explants, demonstrated at least a 3.1-fold increase in HUVEC proliferation and migration indicating a pro-angiogenic effect of cartilage. Finally, the CAM assay demonstrated that cartilage explants can indeed attract vessels; however, their ingrowth was not observed.ConclusionUsing multiple approaches, we show that cartilage releasate has an inherent pro-angiogenic capacity. It remains vessel free due to anti-invasive properties associated with the tissue itself.

Human Cartilage-Derived Progenitors Resist Terminal Differentiation and Require CXCR4 Activation to Successfully Bridge Meniscus Tissue Tears.

Meniscus injuries are among the most common orthopedic injuries. Tears in the inner one‐third of the meniscus heal poorly and present a significant clinical challenge. In this study, we hypothesized that progenitor cells from healthy human articular cartilage (chondroprogenitor cells [C‐PCs]) may be more suitable than bone‐marrow mesenchymal stem cells (BM‐MSCs) to mediate bridging and reintegration of fibrocartilage tissue tears in meniscus. C‐PCs were isolated from healthy human articular cartilage based on their expression of mesenchymal stem/progenitor marker activated leukocyte cell adhesion molecule (ALCAM) (CD166). Our findings revealed that healthy human C‐PCs are CD166+, CD90+, CD54+, CD106‐ cells with multilineage differentiation potential, and elevated basal expression of chondrogenesis marker SOX‐9. We show that, similar to BM‐MSCs, C‐PCs are responsive to the chemokine stromal cell‐derived factor‐1 (SDF‐1) and they can successfully migrate to the area of meniscal tissue damage promoting collagen bridging across inner meniscal tears. In contrast to BM‐MSCs, C‐PCs maintained reduced expression of cellular hypertrophy marker collagen X in monolayer culture and in an explant organ culture model of meniscus repair. Treatment of C‐PCs with SDF‐1/CXCR4 pathway inhibitor AMD3100 disrupted cell localization to area of injury and prevented meniscus tissue bridging thereby indicating that the SDF‐1/CXCR4 axis is an important mediator of this repair process. This study suggests that C‐PCs from healthy human cartilage may potentially be a useful tool for fibrocartilage tissue repair/regeneration because they resist cellular hypertrophy and mobilize in response to chemokine signaling. stem cells 2019;37:102–114

Interleukin-1β Is Essential for Blood-Induced Cartilage Damage In Vitro

Objective: Joint damage due to recurrent joint bleeds remains the most common complication in hemophilia. The combination of cartilage degeneration and synovial inflammation ultimately lead to hemophilic arthropathy. Cartilage destruction is considered to result from both cartilage matrix-degrading proteases as well as cartilage-destructive pro-inflammatory cytokines such as interleukin (IL)-1b. To unravel the role of IL1b in the pathogenesis of blood-induced cartilage damage, we investigated whether direct blocking of IL1b specifically prevents blood-induced cartilage damage in vitro. Moreover, we investigated whether blocking IL1b after the onset of a bleed can still avert cartilage damage.Methods: Full thickness healthy human articular cartilage explants, obtained post-mortem, were cultured for four days in presence or absence of 50% v/v whole blood. A recombinant human IL1b monoclonal antibody (IL1bmAb) was added at the moment of blood exposure in a concentration of 0, 1, 3, 10, 30, or 100ng/mL (n=8). Furthermore, 100ng/mL IL1bmAb was administered directly or after a delay of several hours up to two days (n=7). Cartilage matrix proteoglycan turnover was determined 12 days later to analyse long-term effects.To investigate the direct effects of IL1bmAb on cartilage, explants were cultured for four days in the presence of 10ng/mL IL1bmAb in the absence of blood (n=7).Results: Exposure to blood decreased the proteoglycan synthesis rate and -content, and increased the proteoglycan release (all p<0.05). Adding IL1bmAb resulted in a dose-dependent increase of the proteoglycan synthesis rate leading to normalisation at higher concentrations (see figure A). Proteoglycan release and -content also normalised after addition of IL1bmAb. Its protective effect was most pronounced when IL1bmAb was administered within 4 hours after the bleed, but significant recovery was still achieved by administration within 8-24 hours (see figure B for proteoglycan synthesis). In the absence of blood, IL1bmAb did not have direct effects on cartilage proteoglycan turnover (for all three parameters p>0.50).Conclusions: This study demonstrates that IL1b is a crucial factor in the development of blood-induced cartilage damage in vitro. Blocking this pro-inflammatory cytokine with a monoclonal antibody protects cartilage from the damaging effects of blood exposure in a dose-dependent way. Early administration after blood-exposure is most beneficial. Further research is warranted to investigate the in vivo capacity of IL1bmAb in prevention and its position as a treatment to limit joint damage upon joint bleeding. [Display omitted] Figure -Effect ofIL1bmAbon cartilage proteoglycan synthesis rate is dose-dependent (A) and time-dependent (B). Median values ± IQR of at least 7 individual cartilage and blood donors are shown. Hash tags indicate a statistically significant difference from control values, whereas asterisks indicate a statistically significant difference from blood-exposed cartilage withoutIL1bmAb addition(p<0.05). DisclosuresSchutgens:CSL Behring: Research Funding.

Quantitative proteomic profiling of human articular cartilage degradation in osteoarthritis.

Osteoarthritis (OA) is the most common rheumatic pathology and is characterized primarily by articular cartilage degradation. Despite its high prevalence, there is no effective therapy to slow disease progression or regenerate the damaged tissue. Therefore, new diagnostic and monitoring tests for OA are urgently needed, which would also promote the development of alternative therapeutic strategies. In the present study, we have performed an iTRAQ-based quantitative proteomic analysis of secretomes from healthy human articular cartilage explants, comparing their protein profile to those from unwounded (early disease) and wounded (advanced disease) zones of osteoarthritic tissue. This strategy allowed us to identify a panel of 76 proteins that are distinctively released by the diseased tissue. Clustering analysis allowed the classification of proteins according to their different profile of release from cartilage. Among these proteins, the altered release of osteoprotegerin (decreased in OA) and periostin (increased in OA), both involved in bone remodelling processes, was verified in further analyses. Moreover, periostin was also increased in the synovial fluid of OA patients. Altogether, the present work provides a novel insight into the mechanisms of human cartilage degradation and a number of new cartilage-characteristic proteins with possible biomarker value for early diagnosis and prognosis of OA.

Long non-coding rnas in osteoarthritis and chondrogenesis

Long non-coding rnas in osteoarthritis and chondrogenesis

Interleukin-1B IS essential for blood-induced cartilage damage in vitro

Purpose: Exposure of joint cartilage to blood can occur after joint trauma, during or after major joint surgery, or due to hemophilia. This ultimately leads to joint damage, both by direct effects of blood on cartilage and via synovial inflammation. Cartilage destruction is considered to result from both cartilage matrix-degrading proteases as well as cartilage-destructive pro-inflammatory cytokines such as IL1β. To unravel the role of IL1β in the pathogenesis of blood-induced cartilage damage, we investigated whether direct blocking of IL1β specifically prevents blood-induced cartilage damage in vitro. Methods: Full thickness healthy human articular cartilage explants, obtained post-mortem, were cultured for 4 days in presence or absence of 50% v/v whole blood. A recombinant human IL1β monoclonal antibody (IL1β mAb) was only added during blood exposure in a concentration of 0, 1, 2, 10, 30, or 100 ng/mL. Cartilage matrix proteoglycan turnover was determined 12 days later to analyse long-term effects. Moreover, to investigate the direct effects of IL1β mAb on cartilage, explants were cultured for 4 days in the presence of 10 ng/mL IL1β mAb in the absence of blood. Results: Exposure to blood decreased the proteoglycan-synthesis rate (-62%) and proteoglycan content (-19%), and increased the proteoglycan release (+191%, all p < 0.05). Adding IL1βmAb resulted in a dose-dependent increase of the proteoglycan synthesis rate leading to normalisation at higher concentrations (see figure A). Moreover, proteoglycan release was statistically significantly decreased from a concentration of 3ng/mL and above (see figure B). Similar, the proteoglycan content increased statistically significantly upon addition of the IL1βmAb (3 ng/mL and above; all p < 0.05). In the absence of blood, IL1βmAb did not have direct effects on cartilage proteoglycan turnover (p = 0.51). Conclusions: This study demonstrates that IL1β is a crucial factor in the development of blood-induced cartilage damage in vitro. Blocking this pro-inflammatory cytokine with a monoclonal antibody protects cartilage from the damaging effects of blood exposure. Further research is warranted to investigate the in vivo capacity of IL1β mAb in prevention and its position as a treatment of hemophilic arthropathy.

MiR-1247 Functions by Targeting Cartilage Transcription Factor SOX9

microRNAs are a large and essential class of gene regulators that play key roles in development, homeostasis, and disease. They are necessary for normal skeletal development, and their expression is altered in arthritis. However, the specific role of individual microRNAs is only beginning to be unraveled. Using microRNA expression profiling in healthy human articular cartilage cells (chondrocytes), we identified miR-1247 expression as highly correlated with that of the differentiated cell phenotype. Transcribed from the DLK1-DIO3 locus, the function of miR-1247 is completely unknown. In mice its expression level was relatively high in cartilage tissue, and correlated with cartilage-associated microRNA miR-675 across a range of 15 different mouse tissues. To further probe miR-1247 function, overexpression and inhibition studies were performed in isolated human chondrocytes. Modulation of miR-1247 was found to exert profound phenotypic effects altering expression levels of cartilage master regulator transcription factor SOX9. SOX9 is essential for cartilage development and subsequent function throughout life, and mutations in this gene result in severe dwarfism. Putative miR-1247 binding sites were further investigated using luciferase reporter assays, which indicated binding of miR-1247 to a highly conserved region in the coding sequence of SOX9 but not in its 3'-UTR. Interestingly, depletion of SOX9 in human chondrocytes resulted in increased levels of the mature, processed microRNA, suggesting a negative feedback loop between miR-1247 and its target SOX9.

A comparison of healthy human and swine articular cartilage dynamic indentation mechanics

Articular cartilage is a multicomponent, poroviscoelastic tissue with nonlinear mechanical properties vital to its function. A consequent goal of repair or replacement of injured cartilage is to achieve mechanical properties in the repair tissue similar to healthy native cartilage. Since fresh healthy human articular cartilage (HC) is not readily available, we tested whether swine cartilage (SC) could serve as a suitable substitute for mechanical comparisons. To a first approximation, cartilage tissue and surgical substitutes can be evaluated mechanically as viscoelastic materials. Stiffness measurements (dynamic modulus, loss angle) are vital to function and are also a non-destructive means of evaluation. Since viscoelastic material stiffness is strongly strain rate dependent, stiffness was tested under different loading conditions related to function. Stiffness of healthy HC and SC specimens was determined and compared using two non-destructive, mm-scale indentation test modes: fast impact and slow sinusoidal deformation. Deformation resistance (dynamic modulus) and energy handling (loss angle) were determined. For equivalent anatomic locations, there was no difference in dynamic modulus. However, the HC loss angle was ~35% lower in fast impact and ~12% higher in slow sinusoidal mode. Differences seem attributable to age (young SC, older HC) but also to species anatomy and biology. Test mode-related differences in human-swine loss angle support use of multiple function-related test modes. Keeping loss angle differences in mind, swine specimens could serve as a standard of comparison for mechanical evaluation of e.g. engineered cartilage or synthetic repair materials.

Trefoil factor 3 is induced during degenerative and inflammatory joint disease, activates matrix metalloproteinases, and enhances apoptosis of articular cartilage chondrocytes

Trefoil factor 3 (TFF3, also known as intestinal trefoil factor) is a member of a family of protease-resistant peptides containing a highly conserved motif with 6 cysteine residues. Recent studies have shown that TFF3 is expressed in injured cornea, where it plays a role in corneal wound healing, but not in healthy cornea. Since cartilage and cornea have similar matrix properties, we undertook the present study to investigate whether TFF3 could induce anabolic functions in diseased articular cartilage. We used reverse transcriptase-polymerase chain reaction, Western blot analysis, and immunohistochemistry to measure the expression of TFF3 in healthy articular cartilage, osteoarthritis (OA)-affected articular cartilage, and septic arthritis-affected articular cartilage and to assess the effects of cytokines, bacterial products, and bacterial supernatants on TFF3 production. The effects of TFF3 on matrix metalloproteinase (MMP) production were measured by enzyme-linked immunosorbent assay, and effects on chondrocyte apoptosis were studied by caspase assay and annexin V assay. Trefoil factors were not expressed in healthy human articular cartilage, but expression of TFF3 was highly up-regulated in the cartilage of patients with OA. These findings were confirmed in animal models of OA and septic arthritis, as well as in tumor necrosis factor alpha- and interleukin-1beta-treated primary human articular chondrocytes, revealing induction of Tff3/TFF3 under inflammatory conditions. Application of the recombinant TFF3 protein to cultured chondrocytes resulted in increased production of cartilage-degrading MMPs and increased chondrocyte apoptosis. In this study using articular cartilage as a model, we demonstrated that TFF3 supports catabolic functions in diseased articular cartilage. These findings widen our knowledge of the functional spectrum of TFF peptides and demonstrate that TFF3 is a multifunctional trefoil factor with the ability to link inflammation with tissue remodeling processes in articular cartilage. Moreover, our data suggest that TFF3 is a factor in the pathogenesis of OA and septic arthritis.

Spatial variation inT1of healthy human articular cartilage of the knee joint

The longitudinal relaxation time T(1) of native cartilage is frequently assumed to be constant. To redress this, the spatial variation of T(1) in unenhanced healthy human knee cartilage in different compartments and cartilage layers was investigated. Knees of 25 volunteers were examined on a 1.5 T MRI system. A three-dimensional gradient-echo sequence with a variable flip angle, in combination with parallel imaging, was used for rapid T(1) mapping of the whole knee. Regions of interest (ROIs) were defined in five different cartilage segments (medial and lateral femoral cartilage, medial and lateral tibial cartilage and patellar cartilage). Pooled histograms and averaged profiles across the cartilage thickness were generated. The mean values were compared for global variance using the Kruskal-Wallis test and pairwise using the Mann-Whitney U-test. Mean T(1) decreased from 900-1100 ms in superficial cartilage to 400-500 ms in deep cartilage. The averaged T(1) value of the medial femoral cartilage was 702+/-68 ms, of the lateral femoral cartilage 630+/-75 ms, of the medial tibial cartilage 700+/-87 ms, of the lateral tibial cartilage 594+/-74 ms and of the patellar cartilage 666+/-78 ms. There were significant differences between the medial and lateral compartment (p<0.01). In each cartilage segment, T(1) decreased considerably from superficial to deep cartilage. Only small variations of T(1) between different cartilage segments were found but with a significant difference between the medial and lateral compartments.

197 INTERLEUKIN-10 PROTECTS AGAINST BLOOD-INDUCED JOINT DAMAGE

Purpose: Joint bleeds occur frequently in patients suffering from haemophilia or can occur as a consequence of joint trauma or major joint surgery. Even a limited number of bleeds has been demonstrated to lead to severe joint damage in time. Small amounts of IL-1 that are produced by activated monocytes/macrophages as present within blood increase the production of hydrogen peroxide (H2O2) by chondrocytes. The hydrogen peroxide reacts with haemoglobin-derived iron from damaged and phagocytosed red blood cells in the joint cavity, which results in the formation of hydroxyl radicals in the vicinity of chondrocytes. This leads to chondrocyte apoptosis and with that the irreversible inhibition of cartilage matrix synthesis. This primary cartilage-driven joint damage is followed by synovial inflammation adding to joint damage. In search of possible interventions to prevent or limit the cartilage damaging effects of joint bleeds, we evaluated the potential of IL-10. Methods: Healthy human articular cartilage tissue explants were cultured in the presence or absence of 50% v/v blood for 4 days, followed by a recovery period of 12 days. IL-10 was added in 0.1, 1 or 10 ng/ml. Also cartilage and synovium in case of haemophilic arthropathy, obtained at joint replacement surgery, were cultured in the presence of IL-10 (10 ng/ml). The effect on cartilage matrix proteoglycan synthesis, -release, and -content were determined as well as the IL-1β and TNFα production by the synovium. Results: IL-10 was able to prevent dose dependently the decrease in proteoglycan synthesis and the increase in proteoglycan release of cartilage exposed to blood (p<0.05). As a consequence, the decrease in proteoglycan content after blood exposure could be prevented dose dependently (p<0.05). With 10 ng/ml IL-10, which is still a low dose for local therapeutic treatment, at least 50% reduction in adverse effects was observed (p<0.05). The matrix turnover of haemophilic cartilage improved upon addition of IL-10 and also the production of IL-1β and TNFα by haemophilic synovium decreased in the presence of IL-10 (all p<0.05). Conclusions: The present results show that interleukin-10 prevents the direct harmful effects of blood on articular cartilage and has a beneficial effect on the matrix turnover of haemophilic cartilage and pro-inflammatory cytokine production by haemophilic synovium. Therefore, although treatment studies in addition to prevention studies have to be performed; IL-10 might be worthwhile to test in clinical practice for local treatment of joint haemorrhages, to prevent joint damage in time.

Protective Abilities of Interleukin-10 in Blood-Induced Joint Damage.

PURPOSE: Joint bleeds occur frequently in patients suffering from haemophilia or can occur as a consequence of joint trauma. Even a limited number of bleeds has been demonstrated to lead to severe joint damage in time (1,2). With respect to the mechanisms of blood induced joint damage, we have demonstrated that monocytes/macrophages within the mononuclear cell population together with the red blood cells as present in blood are responsible for the irreversible inhibition of cartilage matrix synthesis. Small amounts of IL-1 that are produced by activated monocytes/macrophages increase the production of hydrogen peroxide (H2O2) by chondrocytes. The hydrogen peroxide reacts with haemoglobin-derived iron from damaged and phagocytosed red blood cells, which results in the formation of hydroxyl radicals in the vicinity of chondrocytes. This leads to chondrocyte apoptosis and with that the irreversible inhibition of cartilage matrix synthesis (2). In search of possible interventions to prevent or limit the cartilage damaging effects of joint bleeds, we tested interleukin-10 (IL-10) as an inflammation controlling cytokine on blood-induced cartilage damage.METHODS: Healthy human articular cartilage tissue explants were cultured in the presence or absence of 50% v/v blood for 4 days, followed by a recovery period of 12 days. IL-10 was added in 0.1, 1 or 10 ng/ml. Also cartilage and synovium in case of haemophilic arthropathy, obtained at joint replacement surgery, was cultured in the presence of IL-10 (10 ng/ml). The effect on cartilage matrix proteoglycan synthesis, -release, and -content were determined as well as the IL-1β and TNFα production by the synovium.RESULTS: IL-10 was able to prevent the decrease in proteoglycan synthesis and the increase in proteoglycan release of cartilage exposed to blood dose dependently (p<0.05). As a consequence, the decrease in proteoglycan content after blood exposure could be prevented dose dependently (p<0.05). With 10 ng/ml IL-10, which is still a low dose for local therapeutic treatment, at least 50% reduction in adverse effects was observed (p<0.05). The matrix turnover of haemophilic cartilage improved upon addition of IL-10 and also the production of IL-1β and TNFα by haemophilic synovium decreased in the presence of IL-10 (all p<0.05).CONCLUSIONS: The present results show that interleukin-10 prevents the direct harmful effects of blood on articular cartilage and has a beneficial effect on the matrix turnover of haemophilic cartilage and pro-inflammatory cytokine production by haemophilic synovium. Therefore, although treatment studies in addition to prevention studies have to be performed; IL-10 might be worthwhile to test in clinical practice for local treatment of joint haemorrhages, to prevent joint damage in time.

Exposure of human cartilage tissue to low concentrations of blood for a short period of time leads to prolonged cartilage damage: An in vitro study

Joint bleeding, or hemarthrosis, leads in time to severe joint damage. This study was carried out to test the in vitro thresholds of exposure time and concentration that lead to irreversible joint damage, to add to the discussion on the usefulness of aspiration of the joint after a hemorrhage. Explants of healthy human articular cartilage tissue were cultured in the presence or absence of 50% (volume/volume) blood for 1, 2, 3, or 4 days or in the presence of 0%, 5%, 10%, 20%, 30%, or 50% (v/v) blood for 4 days, followed by a 12-day period of recovery after withdrawal of blood. The effect of blood exposure on cartilage was determined by measuring the rate of proteoglycan synthesis as well as the release and content of cartilage matrix proteoglycans and the activity of matrix metalloproteinases. Exposure of cartilage to 50% (v/v) blood led to adverse changes that were largely independent of the exposure time. The adverse effects persisted after an initial exposure of up to or exceeding 2 days. Exposure of cartilage to increasing concentrations of blood for 4 days led to concentration-dependent adverse changes. These effects persisted when the concentration equaled or exceeded 10% (v/v) blood. Moreover, after 2 days of exposure to a blood load of 10% (v/v), the adverse effects on cartilage were not reversible. A 2-day exposure of cartilage in vitro to 10% (v/v) blood leads to prolonged impairment of joint cartilage. This suggests that aspiration of blood from the joint within 2 days after hemarthrosis should be considered to prevent blood-induced joint damage in the long term.

Selective COX-2 inhibition is favorable to human early and late-stage osteoarthritic cartilage: a human in vitro study

Nonsteroidal anti-inflammatory drugs (NSAIDs) are widely used in the treatment of osteoarthritis (OA). For the outcome of treatment the direct effects of NSAIDs on cartilage may be more important than indirect effects on inflammation, considered being secondary in OA. For clinical practice, it is relevant to study effects of NSAIDs on early stages of OA. Therefore we studied the direct effects of celecoxib on human degenerated OA cartilage and compared the effects with those on human healthy cartilage and human end-stage OA cartilage. Degenerated, late-stage OA, and healthy human articular cartilage were exposed (7 days of culture) to celecoxib (0.1-10 microM). Changes in cartilage proteoglycan turnover (synthesis, retention, and release), proteoglycan content, prostaglandin E2 (PGE2) and nitric oxide (NO) production were determined. Both degenerated and established OA cartilage showed its characteristic changes in proteoglycan turnover (all P<0.05). Celecoxib at 1 microM was able to increase synthesis of degenerated cartilage and normalize both releases of newly formed and resident proteoglycans. Importantly, 1 microM celecoxib influenced matrix integrity by enhancing proteoglycan content. Similar results were found for end-stage OA cartilage. Enhanced PGE2 production in degenerative and OA cartilage could be decreased by celecoxib, whereas no effect on enhanced NO production was found. No significant effects of celecoxib on normal cartilage were found. Celecoxib, in a clinical relevant concentration, showed in vitro a significant beneficial effect, not only on late-stage OA but also on more early stages of OA, whereas healthy cartilage remained unaffected, suggesting chondroprotective properties of celecoxib in the treatment of degenerative joint disorders.

Selective COX-2 inhibition prevents proinflammatory cytokine-induced cartilage damage.

This study evaluated the in vitro effect of the selective cyclooxygenase-2 (COX) inhibitor celecoxib on cartilage matrix turnover under normal and inflammatory conditions. Healthy human articular cartilage tissue alone, in co-culture with peripheral blood mononuclear cells (PBMC) or in the presence of interleukin 1 (IL-1beta) plus tumour necrosis factor alpha (TNF-alpha) was cultured for 7 days in the presence of celecoxib. Changes in cartilage matrix turnover were measured. No direct effects of celecoxib on healthy normal cartilage were found. Both PBMC and IL-1beta plus TNF-alpha induced strong inhibition of cartilage proteoglycan synthesis and significant enhancement of the release of proteoglycans, diminishing proteoglycan content. Celecoxib was able to reverse these adverse effects up to complete normalization. The results suggest that, under the influence of inflammation, COX-2 is up-regulated, which results in disturbance of cartilage matrix turnover. Celecoxib, by diminishing COX-2 activity, prevents these adverse effects without having a direct effect on healthy cartilage.

Activation of annexin II and V expression, terminal differentiation, mineralization and apoptosis in human osteoarthritic cartilage

Objective To test the hypothesis that terminal differentiation of chondrocytes in human osteoarthritic cartilage might lead to the failure of repair mechanisms and might cause progressive loss of structure and function of articular cartilage.Design Markers for terminally differentiated chondrocytes, such as alkaline phosphatase, annexin II, annexin V and type X collagen, were detected by immunohistochemical analysis of human normal and osteoarthritic knee cartilage from medial and lateral femoral condyles. Apoptosis in these specimens was detected using the TUNEL labeling. Mineralization and matrix vesicles were detected by alizarin red S staining and electron microscopic analysis.Results Alkaline phosphatase, annexin II, annexin V and type X collagen were expressed by chondrocytes in the upper zone of early stage and late stage human osteoarthritic cartilage. However, these proteins, which are typically expressed in hypertrophic and calcifying growth plate cartilage, were not detectable in the upper, middle and deep zones of healthy human articular cartilage. TUNEL labeling of normal and osteoarthritic human cartilage sections provided evidence that chondrocytes in the upper zone of late stage osteoarthritic cartilage undergo apoptotic changes. In addition, mineral deposits were detected in the upper zone of late stage osteoarthritic cartilage. Needle-like mineral crystals were often associated with matrix vesicles in these areas, as seen in calcifying growth plate cartilage.Conclusion Human osteoarthritic chondrocytes adjacent to the joint space undergo terminal differentiation, release alkaline phosphatase-, annexin II- and annexin V-containing matrix vesicles, which initiate mineral formation, and eventually die by apoptosis. Thus, these cells resume phenotypic changes similar to terminal differentiation of chondrocytes in growth plate cartilage culminating in the destruction of articular cartilage in osteoarthritis.

Ageing and zonal variation in post-translational modification of collagen in normal human articular cartilage: The age-related increase in non-enzymatic glycation affects biomechanical properties of cartilage

A biomechanical failure of the collagen network is postulated in many hypotheses of the development of osteoarthritis with advancing age. Here we investigate the accumulation of non-enzymatic glycation (NEG) products in healthy human articular cartilage, its relation to tissue remodelling and its role in tissue stiffening. Pentosidine levels were low up to age 20 years, and increased linearly after this age. This indicates extensive tissue remodelling at young age, and slow turnover of collagen after maturity has been reached. The slow remodelling is supported by the finding that enzymatic modifications of collagen (hydroxylysine, hydroxylysylpyridinoline, and lysylpyridinoline) were not related to age. The high remodelling is supported by levels of the crosslink lysylpyridinoline (LP) as a function of distance from the articular surface. LP was highest at the surface in mature cartilage (>20 years), whereas in young cartilage (<10 years) the opposite was seen; highest levels were close to the bone. LP levels in cartilage sections at age 14 years are high at the surface and close to the bone, but they are low in the middle region. This indicates that maturation of cartilage in the second decade of life starts in the upper half of the tissue, and occurs last in the tissue close to the bone. The effect of NEG products on instantaneous deformation of cartilage was investigated as a functional of topographical variations in pentosidine levels in vivo and in relation to in vitro induced NEG. Consistently, higher pentosidine levels were associated with a stiffer collagen network. A stiffer and more crosslinked collagen network may become more brittle and more prone to fatigue.