Requirement of mitogen-activated protein kinase for collagenase production by the fibronectin fragment in human articular chondrocytes in culture

Abstract

Fibronectin fragments have been shown to up-regulate matrix metalloproteinase production in chondrocytes. We investigated the roles of mitogen-activated protein kinase (MAPK) pathways activated by the COOH-terminal heparin-binding fibronectin fragment (HBFN-f) in collagenase production by human chondrocytes in culture. In articular cartilage explant culture, HBFN-f stimulated type II collagen cleavage by collagenase in association with increased secretion of MMP-1 and MMP-13. In human articular chondrocytes, HBFN-f induced the collagenases with activation of the extracellular signal-regulated kinase (ERK), p38, and the c-Jun NH₂-terminal kinase (JNK). PD98059 that inhibits the ERK pathway blocked HBFN-f-stimulated production of MMP-1 and MMP-13 in explant culture. SB203580 at 1?µM, the concentration that inhibits p38 only, partially suppressed HBFN-f-induced collagenase production, whereas at 10?µM, the inhibitor that blocks both p38 and JNK almost completely inhibited collagenase induction. PD98059 and SB203580 individually blocked HBFN-f-increased cleavage of type II collagen in the explant culture, although 10?µM SB203580 strongly inhibited the collagen cleavage compared with 1?µM of the inhibitor. These results indicate that collagenase production leading to type II collagen cleavage in cartilage explants requires ERK, p38, and JNK.     

Change in collagen synthesis of human chondrocyte culture

Summary A research system constituted entirely of components of human origin was developed to study conversion of collagen synthesis by human chondrocytes. Type specificity of affinity chromatography-purified antibodies to human type II or type I collagen was proven by ELISA inhibition and immunofluorescence analysis. Human chondrocytes were isolated from articular cartilage and kept in monolayer cultures for eight subpassages. Conversion of type II to type I synthesis by chondrocytes was investigated by immunofluorescence. Staining with anti-type II collagen antibodies could be detected during primary cultures and in the first subpassage, whereas staining with anti-type I collagen antibodies occurred beginning from the end of primary cultures and was present up to the eighth subpassage. Results are compared to observations obtained in animal systems and their relevance to conditions in osteoarthritis is discussed.     

体外重建组织工程关节软骨的实验研究

目的　用胶原蛋白和人血纤维蛋白混合物为载体在体外进行软骨细胞三维立体培养 ,构建人工软骨组织。方法　取 2周龄的新生兔关节软骨 ,经消化 ,将获得的软骨细胞与牛 型胶原、人血冻干纤维蛋白原、凝血酶按一定比例混合 ,制成软骨培养物并在体外培养。培养第 3周时 ,取材进行 HE、甲苯胺蓝染色和透射电镜检查。结果　体外培养 3周 ,培养物内细胞均存活 ,形成软骨陷窝 ,同源性细胞簇出现 ,并分泌软骨基质。透射电镜下可见丰富的粗面内质网和线粒体 ,及少量的高尔基复合体。结论　用胶原蛋白和人血纤维蛋白为载体支架体外培养软骨细胞 ,可构建较大的组织工程软骨     

Analysis for the major contributor of collagenase to the primary cleavage of type II collagens in cartilage degradation

Degradation of type II collagen is a central process in cartilage destruction seen in osteoarthritis and rheumatoid arthritis. Primary cleavage of type II collagen at the collagenase site is rate-limiting and is, therefore, a critical step for its degradation. The major contributor to this cleavage was identified in three isozymes of collagenase in human cartilage. Primary cultured human chondrocytes were used for the study. The production of collagenase-1 was major in total production for three isozymes of collagenase after stimulations with any concentration of tumor necrosis factor-α and/or interleukin-1 at 48 and 72 h, comprising 98% or greater of the total collagenase. When the production of collagenase-1 was specifically suppressed by the transfection with duplexes of 21-nucleotide small interfering ribonucleic acid into the cells, the activity of type II collagen cleavage was linearly decreased at neutral pH after activation. The relative contribution of collagenase-1 to the primary cleavage of type II collagen was determined to be 85%–93%. These findings suggest that collagenase-1 is a major contributor to the primary cleavage of type II collagens in human cartilage and is a potential therapeutic target for osteoarthritis and rheumatoid arthritis.     

Analysis for the major contributor of collagenase to the primary cleavage of type II collagens in cartilage degradation

Abstract

Degradation of type II collagen is a central process in cartilage destruction seen in osteoarthritis and rheumatoid arthritis. Primary cleavage of type II collagen at the collagenase site is rate-limiting and is, therefore, a critical step for its degradation. The major contributor to this cleavage was identified in three isozymes of collagenase in human cartilage. Primary cultured human chondrocytes were used for the study. The production of collagenase-1 was major in total production for three isozymes of collagenase after stimulations with any concentration of tumor necrosis factor-α and/or interleukin-1 at 48 and 72?h, comprising 98% or greater of the total collagenase. When the production of collagenase-1 was specifically suppressed by the transfection with duplexes of 21-nucleotide small interfering ribonucleic acid into the cells, the activity of type II collagen cleavage was linearly decreased at neutral pH after activation. The relative contribution of collagenase-1 to the primary cleavage of type II collagen was determined to be 85%–93%. These findings suggest that collagenase-1 is a major contributor to the primary cleavage of type II collagens in human cartilage and is a potential therapeutic target for osteoarthritis and rheumatoid arthritis.     

Type X collagen synthesis in human osteoarthritic cartilage. Indication of chondrocyte hypertrophy.

OBJECTIVE: To investigate the appearance of hypertrophic chondrocytes in osteoarthritic (OA) cartilage, using type X collagen as a specific marker. METHODS. The biosynthesis of type X collagen was examined by metabolic labeling of freshly isolated articular chondrocytes with 3H-proline, immunoprecipitation, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the synthesized collagens. Extracellular deposition of types X and II collagen was analyzed immunohistochemically. RESULTS. Immunostaining revealed an irregular distribution of type X collagen, which was localized around chondrocyte clusters in fibrillated OA cartilage, but was absent from the noncalcified region of normal articular cartilage. Freshly isolated OA chondrocytes synthesized predominantly type X collagen, while control chondrocytes synthesized mostly type II collagen. CONCLUSION. Our findings indicate focal premature chondrocyte differentiation to hypertrophic cells in OA cartilage.     

Requirement of mitogen-activated protein kinase for collagenase production by the fibronectin fragment in human articular chondrocytes in culture

Fibronectin fragments have been shown to up-regulate matrix metalloproteinase production in chondrocytes. We investigated the roles of mitogen-activated protein kinase (MAPK) pathways activated by the COOH-terminal heparin-binding fibronectin fragment (HBFN-f) in collagenase production by human chondrocytes in culture. In articular cartilage explant culture, HBFN-f stimulated type II collagen cleavage by collagenase in association with increased secretion of MMP-1 and MMP-13. In human articular chondrocytes, HBFN-f induced the collagenases with activation of the extracellular signal-regulated kinase (ERK), p38, and the c-Jun NH₂-terminal kinase (JNK). PD98059 that inhibits the ERK pathway blocked HBFN-f-stimulated production of MMP-1 and MMP-13 in explant culture. SB203580 at 1µM, the concentration that inhibits p38 only, partially suppressed HBFN-f-induced collagenase production, whereas at 10µM, the inhibitor that blocks both p38 and JNK almost completely inhibited collagenase induction. PD98059 and SB203580 individually blocked HBFN-f-increased cleavage of type II collagen in the explant culture, although 10µM SB203580 strongly inhibited the collagen cleavage compared with 1µM of the inhibitor. These results indicate that collagenase production leading to type II collagen cleavage in cartilage explants requires ERK, p38, and JNK.     

Formation of nodular structures resembling mature articular cartilage in long–term primary cultures of human fetal epiphyseal chondrocytes on a hydrogel substrate

Objective. To establish long–term cultures of human fetal epiphyseal chondrocytes under conditions that allow the preservation of a cartilage–specific phenotype. Methods. Chondrocytes isolated from 20—24–week human fetal epiphyseal cartilage were cultured for up to 180 days on plastic dishes previously coated with the hydrogel, poly–(2–hydroxyethyl methacrylate). Morphologic, ultrastructural, and biochemical characteristics of the cultures were examined at various intervals, and the expression of genes encoding types I, II, and IX collagen and aggrecan core protein was determined by Northern hybridizations of total cellular RNA with human–specific complementary DNAs. Results. Human fetal epiphyseal chondrocytes cultured for 180 days under conditions that prevented their attachment to the underlying substratum formed nodular structures with morphologic and structural characteristics resembling mature articular cartilage. The cells in the center of the nodules remained spherical and were surrounded by an abundant cartilaginous extracellular matrix, as evidenced by histochemical and ultrastructural examinations. The cells in the periphery of the nodules acquired a discoid morphology and were surrounded by a sparse extracellular matrix. Biosyn–thetic studies demonstrated the maintenance of a cartilagespecific phenotype throughout the 180 days of culture, with the production of aggrecan and types II, IX, and XI collagens but not type I collagen. Northern hybridizations showed high levels of messenger RNAs (mRNAs) for aggrecan core protein, type II procollagen, and type IX collagen, but type I procollagen mRNA was not detectable even at 180 days of culture. Conclusion. The human chondrocyte culture system described here allows the maintenance of a chondrocyte–specific phenotype for prolonged periods (up to 180 days). The long–term chondrocyte cultures formed nodular structures that resemble mature articular cartilage morphologically, ultrastructurally, biosynthetically, and in the pattern of cartilage–specific gene expression.     

Type x collagen synthesis in human osteoarthritic cartilage. indication of chondrocyte hypertrophy

Objective. To investigate the appearance of hypertrophic chondrocytes in osteoarthritic (OA) cartilage, using type X collagen as a specific marker. Methods. The biosynthesis of type X collagen was examined by metabolic labeling of freshly isolated articular chondrocytes with ³H-proline, immunoprecipitation, and sodium dodecyl sulfate–polyacrylamide gel electrophoresis of the synthesized collagens. Extracellular deposition of types X and II collagen was analyzed immunohistochemically. Results. Immunostaining revealed an irregular distribution of type X collagen, which was localized around chondrocyte clusters in fibrillated OA cartilage, but was absent from the noncalcified region of normal articular cartilage. Freshly isolated OA chondrocytes synthesized predominantly type X collagen, while control chondrocytes synthesized mostly type II collagen. Conclusion. Our findings indicate focal premature chondrocyte differentiation to hypertrophic cells in OA cartilage.     

Restoration of the extracellular matrix in human osteoarthritic articular cartilage by overexpression of the transcription factor SOX9

OBJECTIVE: Human osteoarthritis (OA) is characterized by a pathologic shift in articular cartilage homeostasis toward the progressive loss of extracellular matrix (ECM). The purpose of this study was to investigate the ability of rAAV-mediated SOX9 overexpression to restore major ECM components in human OA articular cartilage. METHODS: We monitored the synthesis and content of proteoglycans and type II collagen in 3-dimensional cultures of human normal and OA articular chondrocytes and in explant cultures of human normal and OA articular cartilage following direct application of a recombinant adeno-associated virus (rAAV) SOX9 vector in vitro and in situ. We also analyzed the effects of this treatment on cell proliferation in these systems. RESULTS: Following SOX9 gene transfer, expression levels of proteoglycans and type II collagen increased over time in normal and OA articular chondrocytes in vitro. In situ, overexpression of SOX9 in normal and OA articular cartilage stimulated proteoglycan and type II collagen synthesis in a dose-dependent manner. These effects were not associated with changes in chondrocyte proliferation. Notably, expression of the 2 principal matrix components could be restored in OA articular cartilage to levels similar to those in normal cartilage. CONCLUSION: These data support the concept of using direct, rAAV-mediated transfer of chondrogenic genes to articular cartilage for the treatment of OA in humans.     

Avian myelocytomatosis virus immortalizes differentiated quail chondrocytes.

Quail embryo chondrocytes in culture display two morphological phenotypes: polygonal epithelial-like and floating cells. Both cell populations synthesize cartilage extracellular matrix proteins (type II collagen and specific proteoglycans), whereas type X collagen, which appears to be a marker of later stages of chondrocyte differentiation, is expressed only by the epithelial-like cells. Avian myelocytomatosis virus strain MC29 does not induce morphological transformation in quail embryo chondrocytes but stimulates these cells to proliferate with a progressively reduced doubling time. MC29-infected chondrocytes can be established in culture as a continuous cell line, whereas control (uninfected) cultures only survive a few months. Rapidly dividing MC29-infected chondrocytes still express type II collagen and cartilage proteoglycans but do not synthesize type X collagen.     

Morphology and phenotype expression of types I, II, III, and X collagen and MMP-13 of chondrocytes cultured from articular cartilage of Kashin-Beck Disease

OBJECTIVE: We investigated the characteristics of cell morphology and expression of types I, II, III, and X collagen and matrix metalloproteinase-13 (MMP-13) of chondrocytes from articular cartilage of adult patients with Kashin-Beck Disease (KBD) in vitro to understand the pathogenesis in chondrocytes. METHODS: Samples of articular cartilage were divided into 2 groups: KBD group (8 samples, 8 cases) and the control (8 samples, 8 cases). KBD patients were diagnosed according to "Pathological Criteria to Diagnose KBD in China." Hyaline cartilage was digested with collagenase into cell suspensions and cultured in monolayers. Chondrocyte ultrastructure was observed by electron microscope at 10th day in vitro. Primary articular chondrocytes were seeded on microscope slides and immunostained on 12th day of cultivation for types I, II, III, and X collagens and MMP-13. Positive findings were counted by light microscopy and confirmed by flow cytometric analyses. RESULTS: Considerable amounts of vacuoles and distorted nuclei, as well as thickening and irregular arrangement of collagen fibrils, were seen in the KBD samples by electron microscopy. Types I, III, and X collagen were stained in the KBD, but not in the control cultures. The percentages of positive staining for type II collagen were significantly lower in KBD than those in controls (t col II = -5.54, p < 0.001), and for MMP-13 in the KBD group were significantly higher (t MMP-13 = 3.70, p < 0.01). CONCLUSION:Phenotype expressions of types I, II, III, and X collagen and MMP-13 in chondrocytes cultured in vitro were significantly different between the KBD and control cultures, indicating degenerative and hypertrophic changes in chondrocytes of KBD articular cartilage.     

Increased expression of discoidin domain receptor 2 is linked to the degree of cartilage damage in human knee joints: a potential role in osteoarthritis pathogenesis

OBJECTIVE: To investigate the relationship between increased discoidin domain receptor 2 (DDR-2) expression and cartilage damage in osteoarthritis (OA). METHODS: Full-thickness cartilage tissue samples from 16 human knee joints were obtained and the grade of cartilage damage was evaluated according to the Mankin scale. Expression of DDR-2, matrix metalloproteinase 13 (MMP-13), and MMP-derived type II collagen fragments was visualized immunohistochemically. Moreover, upon stimulation with either type II collagen or gelatin, levels of DDR-2 and MMP-13 messenger RNA (mRNA) in primary human articular chondrocytes were assessed by real-time polymerase chain reaction. RESULTS: Immunohistochemical analysis showed an increase in DDR-2 expression in human articular cartilage, which was correlated with the degree of tissue damage. In parallel, the extent of MMP-13 and type II collagen breakdown products was elevated as a function of increased DDR-2 expression and cartilage damage. Furthermore, in vitro experiments revealed an up-regulation of both DDR-2 and MMP-13 mRNA in human articular chondrocytes after stimulation with type II collagen. CONCLUSION: Our data indicate that 3 factors, DDR-2 expression, MMP-13 expression, and the degree of cartilage damage, are linked, such that DDR-2 promotes tissue catabolism, and tissue degradation promotes DDR-2 up-regulation and activation. Thus, the perpetuation of DDR-2 expression and activation can be seen as a vicious circle that ultimately leads to cartilage destruction in OA.     

Culture of chondrocytes in alginate surrounded by fibrin gel: characteristics of the cells over a period of eight weeks

OBJECTIVE: To produce tissue engineered cartilage by human articular chondrocytes in vitro for further use in in vivo manipulations for the treatment of cartilage defects. METHODS: Human articular chondrocytes were cultured in 0.5%, 1.0%, and 2.0% of alginate for up to four weeks. The optimal concentration of an alginate matrix for cell replication and for aggrecan synthesis by chondrocytes was determined. DNA content in the different culture conditions was measured after two and four weeks. Aggrecan synthesis rates and accumulation in the surrounding extracellular matrix were assessed by [(35)S]sulphate incorporation after the same periods of culture. To follow the outgrowth of chondrocytes from the alginate beads, chondrocytes were cultured for four weeks in 0.5 or 1.0% alginate surrounded by 0.25 or 0.5% fibrin gel. DNA content of each culture was measured after different culture periods. Finally, human chondrocytes in 1.0% alginate beads were embedded in 0.5% fibrin gel for eight weeks. Immunohistochemical analysis for aggrecan, type I and II collagen was performed weekly. RESULTS: At two weeks the DNA content in each culture significantly increased in 0.5 and 1.0% alginate cultures in comparison with baseline values. This increase continued until week 4 at the three alginate concentrations. Aggrecan synthesis at two weeks was highest in 0.5 and 1.0% alginate cell cultures. At four weeks aggrecan synthesis rates decreased independently of the alginate concentrations. Aggrecan mainly accumulated in the interterritorial matrix. Proliferation of chondrocytes in alginate and outgrowth of these cells in the surrounding fibrin gel were evident throughout the culture period. The accumulation of aggrecan and type II collagen around the cells, in alginate as well as in fibrin gel, gradually increased over the culture period. Type I collagen appeared after six weeks in alginate and in the surrounding fibrin. CONCLUSION: Human chondrocytes proliferate in this culture system, show an outgrowth into the surrounding fibrin, and synthesise a cartilage-like matrix for up to eight weeks.     

Bone morphogenetic protein and transforming growth factor beta inhibitory Smads 6 and 7 are expressed in human adult normal and osteoarthritic cartilage in vivo and are differentially regulated in vitro by interleukin-1beta

OBJECTIVE: Bone morphogenetic protein (BMP) and transforming growth factor beta (TGFbeta) are potent anabolic factors in adult articular chondrocytes. In this study, we investigated whether intracellular inhibitors of BMP and TGFbeta signaling, inhibitory Smad6 (I-Smad6) and I-Smad7, are expressed in articular chondrocytes in normal and osteoarthritic (OA) cartilage, and whether their expression shows a correlation with the anabolic activity of OA chondrocytes in vivo and after interleukin-1beta (IL-1beta) stimulation in vitro. METHODS: RNA isolated directly from normal and OA human knee cartilage as well as from cultured articular chondrocytes was analyzed by (quantitative) polymerase chain reaction technology. Immunolocalization of the I-Smads was performed on tissue sections and compared with the anabolic cellular activity as documented by in situ hybridization experiments for aggrecan and type II collagen. RESULTS: Both Smad6 and Smad7 were expressed in all samples of normal and OA cartilage. Immunostaining (including confocal microscopy) confirmed the presence of Smad6 and Smad7 in the majority of normal and degenerated articular chondrocytes; localization was mostly cytoplasmic. No correlation between expression of the main anabolic genes and expression of the I-Smads was found. In cultured articular chondrocytes, stimulation with IL-1beta showed up-regulation of Smad7, whereas Smad6 was down-regulated. CONCLUSION: Both Smad6 and Smad7 are expressed in adult human articular chondrocytes. The primarily cytoplasmic localization suggests permanent activation of the I-Smads in articular cartilage in vivo. No evidence was found that up-regulation or down-regulation of I-Smads in OA cartilage correlates directly with the anabolic (or catabolic) activity of articular chondrocytes. The regulation in chondrocytes of Smad6 and Smad7 expression by IL-1beta suggests a potentially important role of IL-1beta signaling in chondrocytes, via indirect influencing of the BMP/TGFbeta signaling cascade.     

Muscle derived,cell based ex vivo gene therapy for treatment of full thickness articular cartilage defects

OBJECTIVE: To evaluate the effectiveness of transplanted allogeneic muscle derived cells (MDC) embedded in collagen gels for the treatment of full thickness articular cartilage defects, to compare the results to those from chondrocyte transplantation, and to evaluate the feasibility of MDC based ex vivo gene therapy for cartilage repair. METHODS: Rabbit MDC and chondrocytes were transduced with a retrovirus encoding for the beta-galactosidase gene (LacZ). The cells were embedded in type I collagen gels, and the cell proliferation and transgene expression were investigated in vitro. In vivo, collagen gels containing transduced cells were grafted to the experimental full thickness osteochondral defects. The repaired tissues were evaluated histologically and histochemically, and collagen typing of the tissue was performed. RESULTS: The MDC and chondrocyte cell numbers at 4 weeks of culture were 305 +/- 25% and 199 +/- 25% of the initial cell number, respectively. The initial percentages of LacZ positive cells in the MDC and chondrocyte groups were 95.4 +/- 1.9% and 93.4 +/- 3.4%, and after 4 weeks of culture they were 84.2 +/- 3.9% and 76.9 +/- 4.3%, respectively. In vivo, although grafted cells were found in the defects only up to 4 weeks after transplantation, the repaired tissues in the MDC and chondrocyte groups were similarly better histologically than control groups. Repaired tissues in the MDC group were mainly composed of type II collagen, as in the chondrocyte group. CONCLUSION: Allogeneic MDC could be used for full thickness articular cartilage repair as both a gene delivery vehicle and a cell source for tissue repair.     

Institute of Bone and Joint Research Symposium Regeneration and repair of connective tissues: Fact or fiction? 24 November 2001, University of Sydney,Australia

Articular cartilage is an avascular, aneural and alymphatic tissue that covers the ends of bones and is responsible for the distribution of mechanical loads over the surface area of the subchondral bone as well as giving a near frictionless surface. Cartilage is made up of chondrocytes embedded in an extracellular matrix and it is the presence of aggrecan complexes trapped within the insoluble collagen network that gives articular cartilage its unique biomechanical properties. Articular cartilage obtains nutrients from the synovial fluid; joint movement and compression are important in facilitating the movement of nutrients into, and waste molecules out of, the extracellular matrix. During growth, chondrocytes are responsible for the expansion of the extracellular matrix that is part of endochrondral bone growth. In diseases such as osteoarthritis, chondrocytes have the capacity to degrade the extracellular matrix of cartilage that can result in the complete loss of cartilage from the surface of bones. Studies have revealed that articular cartilage is an extremely complex structure at the cellular and molecular level. Articular cartilage has been shown to be made up of a number of distinct layers that contain chondrocytes that have distinctive morphologies and metabolism. It has also been shown that chondrocytes from weight‐bearing and non‐weight‐bearing regions of articular cartilage have different metabolic characteristics, which suggest that chondrocytes are sensitive to their biomechanical environment. Studies have shown that the extracellular matrix is made up by the interaction of many individual molecular components and is a complex structure that shows heterogeneity in its organization. The collagen network is made up of type II/XI collagen fibres that are crossed‐linked by type IX collagen. Associated with the collagen network are small proteoglycans and noncollagenous proteins that appear to be involved in the stabilization and organization of the extracellular matrix through specific matrix–matrix and matrix–cell interactions. The extracellular matrix surrounding each chondrocyte can be divided into three distinct regions. The pericellular matrix that surrounds each chondrocyte is rich in aggrecan complexes and contains a paucity of collagen fibres; encapsulating the pericellular matrix is the territorial matrix defined by a basket arrangement of collagen fibres. Joining the territorial matrix surrounding each chondrocyte is the interterritorial matrix that makes up the bulk of the extracellular matrix of articular cartilage and contains aggrecan complexes. The collagen fibres in the interterritorial region are either arranged in large bundles that form arcade‐like structures that are continuous through all the layers of the tissue or they are arranged in a random manner in association with the large collagen bundles. Articular cartilage has the ability to replace aggrecan complexes that have been lost from the extracellular matrix, but it is evident that if the collagen network is damaged then cartilage is unable to achieve a long‐term repair of the damaged network. The ability of articular cartilage to repair is compromised by the lack of an inflammatory wound‐repair response and this is evidenced by the inability of partial‐thickness injuries of the tissue to repair. Full‐thickness injuries that result in bleeding from the bone surface show such an inflammatory wound‐repair response that gives rise to the formation of a clot that is then infiltrated by cells that synthesize an extracellular matrix. In this situation only partial repair of the injured area is usually achieved and in the long term the repaired matrix will degenerate. It has been suggested that the reason for this degeneration is that there is a lack of biomechanical integrity between the original and restored matrix. The degeneration of the repaired matrix occurs as a combination of the metabolic activity of the cells within the repair and by mechanical failure of the restored matrix. This work shows that cartilage has a potential to repair but in order to elicit a mechanically stable repair, consideration needs to be given to obtaining a mechanical continuum between the original and restored matrix. Indeed, the challenge appears to be in the present inability to manipulate the interactive processes that are involved in the formation of the extracellular matrix and reflects the complexity of the cellular and molecular organization of articular cartilage.     

Increased collagen and aggrecan degradation with age in the joints of Timp3(-/-) mice

OBJECTIVE: To investigate the in vivo effect of an imbalance between metalloproteinases and their inhibitors, tissue inhibitors of metalloproteinases (TIMPs), in mouse articular cartilage. METHODS: Hind joints of Timp3(-/-) and wild-type mice were examined by routine staining and by immunohistochemical analysis using antibodies specific for type X collagen and for the neoepitopes produced on proteolytic cleavage of aggrecan (... VDIPEN and ... NVTEGE) and type II collagen. The neoepitope generated on cleavage of type II collagen by collagenases was quantitated in sera by enzyme-linked immunosorbent assay. RESULTS: Articular cartilage from Timp3-knockout animals (ages > or =6 months) showed reduced Safranin O staining and an increase in ...VDIPEN content compared with cartilage from heterozygous and wild-type animals. There was also a slight increase in ... NVTEGE content in articular cartilage and menisci of Timp3(-/-) animals. Chondrocytes showed strong pericellular staining for type II collagen cleavage neoepitopes, particularly in the superficial layer, in knockout mice. Also, there was more type X collagen expression in the superficial zone of articular cartilage, especially around clusters of proliferating chondrocytes, in the knockout mice. More type II collagen cleavage product was found in the serum of Timp3(-/-) mice compared with wild-type animals. This increase was significant in 15-month-old animals. CONCLUSION: These results indicate that TIMP-3 deficiency results in mild cartilage degradation similar to changes seen in patients with osteoarthritis, suggesting that an imbalance between metalloproteinases and TIMP-3 may play a pathophysiologic role in the development of this disease.