Extracellular Matrix Of Articular Cartilage Research Articles

Prospects for treating osteoarthritis: enzyme–protein interactions regulating matrix metalloproteinase activity

Primary osteoarthritis (OA) is a musculoskeletal disorder of unknown etiology. OA is characterized by an imbalance between anabolism and catabolism in, and altered homeostasis of articular cartilage. Matrix metalloproteinases (MMPs) and a disintegrin and metalloproteinase with thrombospondin motif are upregulated in OA joints. Extracellular matrix (ECM) proteins are critical for resistance to compressive forces and for maintaining the tensile properties of the tissue. Tissue inhibitor of metalloproteinases (TIMPs) is the endogenous inhibitor of MMPs, but in OA, TIMPs do not effectively neutralize MMP activity. Upregulation of MMP gene expression occurs in OA in a milieu of proinflammatory cytokines such as interleukin (IL)-1, IL-6 and tumor necrosis factor α. Presently, the medical therapy of OA includes mainly nonsteroidal anti-inflammatory drugs and corticosteroids which dampen pain and inflammation but appear to have little effect on restoring joint function. Experimental interventions to restore the imbalance between anabolism and catabolism include small molecule inhibitors of MMP subtypes or inhibitors of the interaction between IL-1 and its receptor. Although these agents have some positive effects on reducing MMP subtype activity they have little efficacy at the clinical level. MMP-9 is one MMP subtype implicated in the degradation of articular cartilage ECM proteins. MMP-9 was found in OA synovial fluid as a complex with neutrophil gelatinase-associated lipocalin (NGAL) which protected MMP-9 from autodegradation. Suppressing NGAL synthesis or promoting NGAL degradation may result in reducing the activity of MMP-9. We also propose initiating a search for enzyme-protein interactions to dampen other MMP subtype activity which could suppress ECM protein breakdown.

Proteoglycan metabolism, cell death and Kashin-Beck Disease

Kashin-Beck Disease (KBD) is an endemic, chronic and degenerative osteoarthropathy principally occurring in children. The characteristic pathological change of KBD is chondrocyte necrosis in hyaline articular cartilage. Proteoglycans are one of the major components in the extracellular matrix of articular cartilage, and disrupted proteoglycan metabolism and loss of proteoglycans in articular cartilage from KBD patients has been observed. In this mini-review, we discuss the close relationship between chondrocyte death including necrosis and loss of proteoglycan, and its potential mechanism during KBD onset and development, which may provide new clues for KBD research.

Endogenous regenerative technology using plasma- and platelet-derived growth factors

Cartilage tissue engineering is becoming increasingly popular for repairing cartilage defects. However, lack of nerves and blood vessels, as well as low cell density cause inadequate cartilage tissue regeneration. As a result, it is critical to create an appropriate microenvironment for chondrocyte proliferation. Hydrogels, as three-dimensional cross-linked polymeric networks, are considered promising alternatives to cartilage tissue due to their high-water content, viscoelasticity, and resemblance to the articular cartilage extracellular matrix. Silk fibroin hydrogels of Bombyx mori, a natural polymer, have recently been widely used to regenerate cartilage due to their ability to mimic the extracellular matrix of cartilage and endurability. For this purpose, silk fibroin is frequently combined with other materials. Composite silk fibroin hydrogels have shown strong potential for cartilage regeneration. This review explains various composite silk fibroin hydrogels studied for cartilage regeneration, as well as their advantages over single SF scaffolds.

Expression of cartilage oligomeric matrix protein in articular cartilage in response to bone morphogenetic protein-7 and tumour necrosis factor : peer reviewed original article

Introduction: The extracellular matrix of articular cartilage consists of collagens, proteoglycans and noncollageneous proteins. Cartilage oligomeric matrix protein (COMP) is a non-collagenous protein isolated from articular cartilage. It is localised in articular cartilage and the proliferative and hypertrophic zones of the epiphyseal growth plate. Aim: The aim of the study was to investigate the role of bone morphogenetic proteins (BMP-7) and tumour necrosis factor (TNF-α) on expression of COMP in articular cartilage. Methods: Stifle joints (n=12) from three month-old calves were obtained from a local abattoir. Articular chondrocytes were isolated from three distinct zones of cartilage and cultured as monolayers in a serum-free chemically defined medium. Results: BMP-7 increased COMP accumulation in dose and time dependent manner. TNF-α was however found to significantly reduce COMP accumulation Conclusion: BMP-7 was shown to be important regulators of the COMP gene in different zones of articular cartilage

T1ρ and T2 mapping reveal the in vivo extracellular matrix of articular cartilage

To determine the relationship between changes in the extracellular matrix (ECM) and T(1ρ) and T(2) values in vivo. The ECM is composed of proteoglycan (PG), collagen, and water. It has been unclear which of the ECM constituents affects T(1ρ) and T(2) mapping in living human cartilage. Sagittal T(1ρ) and T(2) maps were preoperatively obtained from 20 knee osteoarthritis patients. Osteochondral samples harvested from the resected tibial plateaus during total knee arthroplasty were consistent with the MRIs of the patients' knees. Parameters that included histological grading of cartilage degeneration, glycosaminoglycan (GAG) content (which constitutes PG), presence of collagen anisotropy and water content were evaluated along with T(1ρ) and T(2) values, and statistical analysis was performed using multiple regression analysis. T(1ρ) and T(2) values were significantly correlated with the degree of cartilage degeneration (β = 0.397 and 0.357, respectively) and the GAG content (β = -0.340 and -0.244, respectively). The present study demonstrated that T(1ρ) and T(2) values reflect the GAG content of the cartilage and can indicate cartilage degeneration in vivo. Use of these parameters can facilitate the noninvasive diagnosis and evaluation of cartilage degeneration.

Study of the Cytotoxicity of a Composite of Carboxymethylcellulose (CMC) and a BioCeramic (Biphasic Calcium Phosphate-BCP) Injection for Use in Articular Cartilage Repair

The failure of organs and tissues caused by trauma and other injuries is one of the most costly of human health problems. It is estimated that 1.6 million people experience work limitations caused by osteoarthritis and related disorders, representing 8.3% of all main conditions. Joint injuries frequently lead to progressive joint degeneration and post-traumatic osteoarthritis. Articular cartilage has only a limited capacity for self-healing, mainly due to the fact that it is avascular; and once seriously damaged, articular cartilage lesions will not regenerate. There is strong evidence that cartilage lesions may lead to osteoarthritis when left untreated. Numerous animal experiments and clinical studies have shown that early biological reconstruction of circumscribed cartilage defects in the knee is superior to conservative or delayed surgical treatment. Tissue engineering has shown promising therapeutic strategies for repair or regeneration of damaged tissues. Currently, ceramic based and polymeric scaffolds have been developed to bring about the restoration of tissue functions. The bioceramics associated with water-soluble polymers have been developed as substitutes for various orthopedic applications. The objectives of this work are the processing and characterization of a composite of carboxymethylcellulose (CMC) and biphasic calcium phosphate (Biphasic Calcium Phosphate - BCP) in the form of a hydrogel, and a study of its cytotoxicity (in vitro), aimed at its application as an injectable biomaterial in order to repair the extracellular matrix of articular cartilage. The CMC and BCP were characterized by Fourier Transform Infrared Spectrometry (FTIR) and X-Ray Diffraction (XRD), X-ray fluorescence (XRF), respectively, and scanning electron microscopy (SEM) of powders and the composite. To evaluate the biological effect of the composite hydrogel, tests of cytotoxicity (MTT) and rheological tests under real conditions of use were performed. The composite of carboxymethylcellulose (CMC) and bioceramics (biphasic calcium phosphate-BCP) in the form of hydrogel showed an adequate injectability in the conditions studied, and a non-toxic response, presenting potential for use as fillers or to stimulate the healing of cartilage defects in the extracellular matrix of articular cartilage.

Cartilage Intermediate Layer Protein 2 (CILP-2) Is Expressed in Articular and Meniscal Cartilage and Down-regulated in Experimental Osteoarthritis

Using transcriptome profiling to determine differential gene expression between the permanent mouse articular cartilage and the transient growth plate cartilage, we identified a highly expressed gene, Cilp2, which is expressed differentially by articular chondrocytes. CILP-2 is highly homologous to CILP-1 (cartilage intermediate layer protein 1), which is expressed in the intermediate zone of articular cartilage and has been linked to cartilage degenerative diseases. We demonstrated that Cilp2 has a restricted mRNA distribution at the surface of the mouse articular cartilage during development, becoming localized to the intermediate zone of articular cartilage and meniscal cartilage with maturity. Although the extracellular CILP-2 protein localization is broadly similar to CILP-1, CILP-2 appears to be more localized in the deeper intermediate zone of the articular cartilage extracellular matrix at maturity. CILP-2 was shown to be proteolytically processed, N-glycosylated, and present in human articular cartilage. In surgically induced osteoarthritis in mice, Cilp1 and Cilp2 gene expression was dysregulated. However, whereas Cilp1 expression was increased, Cilp2 gene expression was down-regulated demonstrating a differential response to mechanically induced joint destabilization. CILP-2 protein was reduced in the mouse osteoarthritic cartilage. Ultrastructural analysis also suggested that CILP-2 may be associated with collagen VI microfibrils and thus may mediate interactions between matrix components in the territorial and inter-territorial articular cartilage matrix. mRNA expression analysis indicated that whereas Cilp1 and Cilp2 are expressed most abundantly in cartilaginous tissues, expression can be detected in muscle and heart.

Arthrodiatasis for Management of Knee Osteoarthritis

Osteoarthritic disease is the result of mechanical and biological events that destabilize the normal processes of degradation and synthesis of articular cartilage chondrocytes, extracellular matrix, and subchondral bone. Osteoarthritis of the knee can cause symptoms ranging from mild to disabling. Initial management of most patients should be nonoperative, but because of the progressive nature of the disease, many patients with osteoarthritis of the knee eventually benefit from operative treatment. Various procedures have been described for treatment of the osteoarthritic knee, ranging from arthroscopic lavage and debridement to total knee arthroplasty. The aim of this study was to evaluate the clinical results of distraction arthroplasty combined with arthroscopic lavage and drilling of cartilage defects for treatment of osteoarthritis of the knee. Nineteen patients (15 women and 4 men; age range, 39-65 years) were operated on. Pre- and postoperative findings were compared. A control group comprising 42 patients treated with only arthroscopic procedures was evaluated for comparison. Follow-up ranged from 3 to 5 years. Results were evaluated both clinically and radiologically postoperatively and throughout the follow-up period. Clinically, pain and walking capacity improved in most patients. Radiologically, joint space widening and improvement of the tibiofemoral angle was noted in nearly all patients.

Chondrocyte apoptosis determined by caspase-3 expression varies with fibronectin distribution in equine articular cartilage

The purpose of this study was to investigate associations between the extent of chondrocyte apoptosis and expression of the articular cartilage (AC) extracellular matrix (ECM) molecules, cartilage oligomeric matrix protein (COMP) and fibronectin. Cartilage from four sites (when available) on equine left middle carpal joints (n = 12) were used. Expression of COMP and fibronectin was determined using specific polyclonal antibodies and a biotin-streptavidin/peroxidase method. The intensity of staining for matrix molecules was graded (none, mild, moderate, strong) in each cartilage zone. Apoptosis of chondrocytes in AC sections was assessed by their expression of active caspase-3 using immunohistochemistry. The intensity of fibronectin expression varied significantly according to cartilage depth, with greater expression in the deep zone than in either the superficial or middle layers (P < 0.001). A significant positive association was found overall between intensity of fibronectin expression and chondrocyte apoptosis (r = 0.44, P = 0.0187). The data were also significant for superficial and deep zones (r = 0.44, P = 0.0239 and r = 0.42, P = 0.0279 respectively). Conversely, intensity of COMP expression did not show zonal differences and was un-associated with degree of apoptosis. However, COMP expression was significantly more intense in cartilage than fibronectin (P = 0.0007), and the correlation between overall intensity of COMP and fibronectin was statistically significant (r = 0.56, P = 0.0018). The positive correlation between the incidence of apoptosis and expression of fibronectin, a key ECM molecule involved in communication between the chondrocyte and surrounding matrix, suggests that chondrocyte death by apoptosis may alter cartilage metabolism, supporting the role of this process in the pathogenesis of osteoarthritis.

An Atypical Degenerative Osteoarthropathy in Hyp Mice is Characterized by a Loss in the Mineralized Zone of Articular Cartilage

Patients with X-linked hypophosphatemia (XLH) develop enthesophytes and osteophytes secondary to articular cartilage degeneration and together are the primary cause of morbidity in adult patients so afflicted. We have previously characterized the enthesopathy in Hyp mice, a murine model of XLH. We now extend these studies to the synovial joint in order to characterize potential cellular changes in articular cartilage that may predispose patients to the osteoarthropathy of XLH. We report that, despite highly elevated levels of alkaline phosphatase activity throughout articular cartilage, there is a complete loss in the mineralized zone of articular cartilage as assessed by von Kossa staining of mineral and as quantified by EPIC-microCT analysis and evidence of vascular invasion. We also identify the downregulation of extracellular matrix (ECM) factors identified as regulators of terminally differentiated mineralizing articular chondrocytes. There is also a striking increase in the histochemical staining of sulfated proteoglycans, a change that may reflect the loss of a transitional tissue that reduces mechanical stress at the interface between cartilage and subchondral bone. The failure of mineralizing articular chondrocytes to develop in the hypophosphatemic state suggests that phosphate may be a key regulator of chondrocyte mineralization. Accordingly, we find that the appropriate zonal arrangement and phenotypic markers of articular cartilage are significantly reestablished by phosphate-replacement therapy. Given the turnover and maintenance of articular cartilage ECM, the identification of early and abnormal cellular changes unique to XLH will undoubtedly aid in a more effective management of this disease to minimize the onset of degenerative osteoarthropathy.

Mesenchymal stem cells on a decellularized cartilage matrix for cartilage tissue engineering

An ideal scaffold for cartilage tissue engineering should be biomimetic in not only its biochemical composition, but also in the morphological structure of the scaffold. In this study, we fabricated a scaffold with an oriented structure using a nanofibrous articular cartilage extracellular matrix (ACECM), in which the ACECM was used to mimic the biochemical composition and oriented structure of articular cartilage. Histology analysis showed that the scaffold contained cartilage ECM (GAGs and collagen II). Scanning electron microscopy (SEM) indicated that the scaffolds were composed of nanofibers and possessed vertical microtubules. Chondrogenic differentiation-induced mesenchymal stem cells (MSCs) were seeded on the scaffold in vitro. SEM showed that MSCs proliferated well and aligned along the vertical microtubules, which mimicked the orientation of deep zone articular cartilage. A cell proliferation assay and live/dead cell staining demonstrated that the ACECM possessed good cell affinity, which favored cell adherence and proliferation. The MSCs that had been labeled with the fluorescent dye PKH26 and seeded on scaffolds were implanted into nude mice. The differentiated cells/ACECM implants formed cartilage-like tissue 4 weeks after implantation, and stained positive for collagen type II and toluidine blue. In addition, the in vivo fluorescent images verified that the MSCs in the implants were the labeled MSCs. These results demonstrated that the oriented ACECM scaffolds hold great promise for use in cartilage tissue engineering applications.

Fabrication and cell affinity of biomimetic structured PLGA/articular cartilage ECM composite scaffold

An ideal scaffold for cartilage tissue engineering should be biomimetic in not only mechanical property and biochemical composition, but also the morphological structure. In this research, we fabricated a composite scaffold with oriented structure to mimic cartilage physiological morphology, where natural nanofibrous articular cartilage extracellular matrix (ACECM) was used to mimic the biochemical composition, and synthetic PLGA was used to enhance the mechanical strength of ACECM. The composite scaffold has well oriented structure and more than 89% of porosity as well as about 107 μm of average pore diameter. The composite scaffold was compared with ACECM and PLGA scaffolds. Cell proliferation test showed that the number of MSCs in ACECM and composite scaffolds was noticeably bigger than that in PLGA scaffold, which was coincident with results of SEM observation and cell viability staining. The water absorption of ACECM and composite scaffolds were 22.1 and 10.2 times respectively, which was much higher than that of PLGA scaffolds (3.8 times). The compressive modulus of composite scaffold in hydrous status was 1.03 MPa, which was near 10 times higher than that of hydrous ACECM scaffold. The aforementioned results suggested that the composite scaffold has the potential for application in cartilage tissue engineering.

Matrix metalloproteinase 17 is necessary for cartilage aggrecan degradation in an inflammatory environment

ObjectiveAggrecan is a critical component of cartilage extracellular matrix. Several members of the ‘a disintegrin and metalloproteinase with thrombospondin motifs’ (ADAMTS) family have been characterised as aggrecanases by their ability...

Cationic contrast agents improve quantification of glycosaminoglycan (GAG) content by contrast enhanced CT imaging of cartilage

Minimally invasive and non-destructive methods to quantify glycosaminoglycans (GAGs) in articular cartilage extracellular matrix are of significant interest for the biochemical analysis of cartilage and diagnosis and tracking of osteoarthritis in vivo. Here, we report the use of cationic iodinated contrast agents in comparison to conventional anionic contrast agents for the quantitative monitoring of GAG concentrations with peripheral quantitative computed tomography. Using an ex vivo bovine osteochondral plug model, the cationic contrast agents were evaluated for their ability to distribute into articular cartilage and generate a positive relationship with GAG content. The cationic agents resulted in much higher equilibrium X-ray attenuations in cartilage extracellular matrix (ECM) than anionic agents. Experiments with samples subjected to enzymatic GAG degradation demonstrated that the cationic agents were up to five times more sensitive (p = 0.0001) to changes in GAG content and had a 24% higher correlation (p = 0.002) compared to the anionic agent (R(2) = 0.86, p < 0.0001 compared with R(2) = 0.62, p = 0.004). The natural inhomogeneous distribution of GAGs in the ECM could clearly be identified in undegraded samples.

Effects of TGF-β1 on alternative splicing of Superficial Zone Protein in articular cartilage cultures

Superficial Zone Protein (SZP) is expressed by the superficial zone chondrocytes and is involved in boundary lubrication of the articular cartilage surface. SZP protein expression is dependent on anatomical location and is regulated by the transforming growth factor-β (TGF-β) pathway. The hypothesis of this study was that between load-bearing, and non-load-bearing locations, of the femoral medial condyle alternative splice isoforms of SZP are different, and regulated by TGF-β1. Using reverse transcription-polymerase chain reaction (RT-PCR) we identified differentially expressed SZP alternative splicing. Using recombinant proteins of the N-terminal region produced from these isoforms, we identified differences in binding to heparin and the extracellular matrix. We identified a novel splice form of SZP (isoform E), lacking exons 2-5. Differences in alternative splicing were observed between anterior load-bearing locations of the femoral medial condyle (M1) compared to the posterior non-load-bearing location (M4). TGF-β1 increased splicing out of exons 4 and 5 encoding a heparin binding domain. The minimal induction time for changes in splicing by TGF-β1 at the M1 location was 1h, although this did change total SZP mRNA levels. Inhibition of Smad3 phosphorylation inhibited TGF-β1 induced splicing, and SZP protein expression. Recombinant proteins corresponding to isoforms upregulated by TGF-β1 had reduced binding. The SZP dimerization domain is located within exon 3. In conclusion, alternative splicing of SZP is regulated by TGF-β1 signaling and may regulate SZP interaction with heparin/heparan sulfate or other components in the extracellular matrix of articular cartilage by splicing out of the heparin binding domain.

Joint aging and chondrocyte cell death

Articular cartilage extracellular matrix and cell function change with age and are considered to be the most important factors in the development and progression of osteoarthritis. The multifaceted nature of joint disease indicates that the contribution of cell death can be an important factor at early and late stages of osteoarthritis. Therefore, the pharmacologic inhibition of cell death is likely to be clinically valuable at any stage of the disease. In this article, we will discuss the close association between diverse changes in cartilage aging, how altered conditions influence chondrocyte death, and the implications of preventing cell loss to retard osteoarthritis progression and preserve tissue homeostasis.

Anticytokine Therapy for Osteoarthritis

Several recent in vitro investigations and experimental studies performed in animal models of osteoarthritis (OA) sustained the previously held view that interleukin (IL)-1 or tumour necrosis factor-alpha (TNFalpha) disrupt the metabolism of synovial joint tissues. The evidence to date indicates that, in addition to IL-1 and TNFalpha, other pro-inflammatory cytokines, including IL-6, members of the IL-6 protein superfamily, IL-7, IL-17 and IL-18, can also promote articular cartilage extracellular matrix protein degradation or synergize with other cytokines to amplify and accelerate cartilage destruction. Most importantly, many of these cytokines have been implicated in causing synovial tissue activation and damage to subchondral bone as well as altering cartilage homeostasis in spontaneously occurring or surgically induced animal models of OA and in transgenic mice genetically primed to develop OA. In this regard, these pro-inflammatory cytokines may also play a significant role in the pathogenesis of human OA. However, attempts to modify the progression of human OA in well designed, controlled clinical trials with an IL-1 receptor antagonist protein (IRAP) have not been successful. Several anabolic cytokines (also termed growth factors), including transforming growth factor-beta (TGF-beta), insulin-like growth factor-1 (IGF-1), fibroblast growth factor-2 (FGF-2), platelet-derived growth factor (PDGF) and connective tissue growth factor (CTGF), have also been proposed as regulators of skeletal long bone growth and development as well as cartilage and bone homeostasis. TGF-beta, IGF-1 and FGF-2, in particular, have been characterized as potential chondroprotective agents. Thus, enzymatic disruption and removal of these growth factors from cartilage extracellular matrix proteins, as in the case of TGF-beta and FGF-2, or disruption of their function, as in the case of the enhanced binding of free IGF-1 with IGF binding proteins in OA joint synovial fluid, may compromise and ultimately be responsible for the inadequate repair of articular cartilage in OA. An improved understanding of the cellular and molecular mechanisms by which pro-inflammatory and/or anabolic cytokines alter both the structure and function of synovial joints may eventually result in the commercial development of disease-modifying OA drugs (DMOADs). Since the prevalence of OA is high in the elderly population, future development of DMOADs must also take into account potential differences in the way DMOADs would be metabolized in the older individual compared with younger people.

Regeneration of static-load-degenerated articular cartilage extracellular matrix by vitamin C supplementation

Regeneration of static-load-degenerated articular cartilage extracellular matrix by vitamin C supplementation

Chondrogenic Differentiation of Adipose-Derived Adult Stem Cells by a Porous Scaffold Derived from Native Articular Cartilage Extracellular Matrix

Adipose-derived adult stem cells (ASCs) have the ability to differentiate into a chondrogenic phenotype in response to specific environmental signals such as growth factors or artificial biomaterial scaffolds. In this study, we examined the hypothesis that a porous scaffold derived exclusively from articular cartilage can induce chondrogenesis of ASCs. Human ASCs were seeded on porous scaffolds derived from adult porcine articular cartilage and cultured in standard medium without exogenous growth factors. Chondrogenesis of ASCs seeded within the scaffold was evident by quantitative RT-PCR analysis for cartilage-specific extracellular matrix (ECM) genes. Histological and immunohistochemical examination showed abundant production of cartilage-specific ECM components-particularly, type II collagen-after 4 or 6 weeks of culture. After 6 weeks of culture, the cellular morphology in the ASC-seeded constructs resembled those in native articular cartilage tissue, with rounded cells residing in the glycosaminoglycan-rich regions of the scaffolds. Biphasic mechanical testing showed that the aggregate modulus of the ASC-seeded constructs increased over time, reaching 150 kPa by day 42, more than threefold higher than that of the unseeded controls. These results suggest that a porous scaffold derived from articular cartilage has the ability to induce chondrogenic differentiation of ASCs without exogenous growth factors, with significant synthesis and accumulation of ECM macromolecules, and the development of mechanical properties approaching those of native cartilage. These findings support the potential for a processed cartilage ECM as a biomaterial scaffold for cartilage tissue engineering. Additional in vivo evaluation is necessary to fully recognize the clinical implication of these observations.

Multiple matrix metalloproteinases in type II collagen induced arthritis

To study the relative contribution of various matrix degrading enzymes in the pathogenesis of arthritis, changes in the levels of various matrix metalloprtoteinases (MMPs) during the progression of collagen induced arthritis was studied in experimental animals. Arthritis was induced in male wistar rats by injecting an emulsion containing collagen type II and Freund's complete adjuvant. The duration of the experiment was 35 days. Synovial effusate was collected at regular intervals after induction. At the end of the experiment serum and cartilage were collected and analysed. Synovial fluid of osteoarthritic patients was also analyzed. Levels of MMP-2, MMP-3, MMP-9 and MT1-MMP were found to be high in synovial effusate and cartilage of experimental animals. In synovial effusate of arthritic animals the expression of MMP-3 was found to be high during the early stages while increase in MMP-2 and MMP-9 occurred at later stages. Synovial fluid of osteoarthritic patients also showed elevated levels of MMP-2, MMP-3 and MMP-9. Our results indicated that sequential action of MMPs such as MMP-3, MMP-2 and MMP-9 can cause degradation of articular cartilage extracellular matrix.