Osteoarthritis‐like changes and decreased mechanical function of articular cartilage in the joints of mice with the chondrodysplasia gene (cho)

Objective

To investigate whether heterozygosity for a loss‐of‐function mutation in the gene encoding the α1 chain of type XI collagen (Col11a1) in mice (chondrodysplasia, cho) causes osteoarthritis (OA), and to understand the biochemical and biomechanical effects of this mutation on articular cartilage in knee and temporomandibular (TM) joints.

Methods

Articular cartilage from the knee and TM joints of mice heterozygous for cho (cho/+) and their wild‐type littermates (+/+) was examined. The morphologic properties of cartilage were evaluated, and collagen fibrils were examined by transmission electron microscopy. Immunohistochemical staining was performed to examine the protein expression levels of matrix metalloproteinase 3 (MMP‐3) and MMP‐13 in knee joints. In 6‐month‐old animals, fixed‐charge density was determined using a semiquantitative histochemical method, and tensile stiffness was determined using an osmotic loading technique.

Results

The diameter of collagen fibrils in articular cartilage of knee joints from heterozygous cho/+ mice was increased relative to that in control cartilage, and histologic analysis showed OA‐like degenerative changes in knee and TM joints, starting at age 3 months. The changes became more severe with aging. At 3 months, protein expression for MMP‐3 was increased in knee joints from cho/+ mice. At 6 months, protein expression for MMP‐13 was higher in knee joints from cho/+ mice than in joints from their wild‐type littermates, and negative fixed‐charge density was significantly decreased. Moreover, tensile stiffness in articular cartilage of knee joints from cho/+ mice was moderately reduced and was inversely correlated with the increase in articular cartilage degeneration.

Conclusion

Heterozygosity for a loss‐of‐function mutation in Col11a1 results in the development of OA in the knee and TM joints of cho/+ mice. Morphologic and biochemical evidence of OA appears to precede significant mechanical changes, suggesting that the cho mutation leads to OA through a mechanism that does not initially involve mechanical factors.

     

Pathogenesis of osteoarthritis-like changes in the joints of mice deficient in type IX collagen

OBJECTIVE: To examine the pathogenetic mechanisms of osteoarthritis (OA)-like changes in Col9a1-/- mice, which are deficient in type IX collagen. METHODS: Knee joints and temporomandibular joints (TMJs) from Col9a1-/- mice and their wild-type (Col9a1+/+) littermates were examined by light microscopy. Immunohistochemical staining was performed to examine the expression of matrix metalloproteinase 3 (MMP-3) and MMP-13, degraded type II collagen, and the discoidin domain receptor 2 (DDR-2) in knee joints. Cartilage mechanics were also evaluated for compressive properties by microindentation testing of the tibial plateau and for tensile properties by osmotic loading of the femoral condyle. RESULTS: Histologic analysis showed age-dependent OA-like changes in the knee and TMJs of Col9a1-/- mice starting at the age of 3 months. At the age of 6 months, enhanced proteoglycan degradation was observed in the articular cartilage of the knee and TMJs of the mutant mice. The expression of MMP-13 and DDR-2 protein and the amount of degraded type II collagen were higher in the knee joints of Col9a1-/- mice than in their wild-type littermates at the age of 6 months. Changes in cartilage mechanics were observed in the femoral and tibial plateaus of Col9a1-/- mice at 6 months, including a decrease in the compressive modulus and uniaxial modulus. At 3 and 6 months of age, tibial cartilage in Col9a1-/- mice was found to be more permeable to fluid flow, with an associated compromise in the fluid pressurization mechanism of load support. All of these changes occurred only at medial sites. CONCLUSION: Lack of type IX collagen in Col9a1-/- mice results in age-dependent OA-like changes in the knee joints and TMJs.     

Pathogenesis of osteoarthritis‐like changes in the joints of mice deficient in type IX collagen

Objective

To examine the pathogenetic mechanisms of osteoarthritis (OA)–like changes in Col9a1^−/− mice, which are deficient in type IX collagen.

Methods

Knee joints and temporomandibular joints (TMJs) from Col9a1^−/− mice and their wild‐type (Col9a1^+/+) littermates were examined by light microscopy. Immunohistochemical staining was performed to examine the expression of matrix metalloproteinase 3 (MMP‐3) and MMP‐13, degraded type II collagen, and the discoidin domain receptor 2 (DDR‐2) in knee joints. Cartilage mechanics were also evaluated for compressive properties by microindentation testing of the tibial plateau and for tensile properties by osmotic loading of the femoral condyle.

Results

Histologic analysis showed age‐dependent OA‐like changes in the knee and TMJs of Col9a1^−/− mice starting at the age of 3 months. At the age of 6 months, enhanced proteoglycan degradation was observed in the articular cartilage of the knee and TMJs of the mutant mice. The expression of MMP‐13 and DDR‐2 protein and the amount of degraded type II collagen were higher in the knee joints of Col9a1^−/− mice than in their wild‐type littermates at the age of 6 months. Changes in cartilage mechanics were observed in the femoral and tibial plateaus of Col9a1^−/− mice at 6 months, including a decrease in the compressive modulus and uniaxial modulus. At 3 and 6 months of age, tibial cartilage in Col9a1^−/− mice was found to be more permeable to fluid flow, with an associated compromise in the fluid pressurization mechanism of load support. All of these changes occurred only at medial sites.

Conclusion

Lack of type IX collagen in Col9a1^−/− mice results in age‐dependent OA‐like changes in the knee joints and TMJs.

     

Attenuation of osteoarthritis progression by reduction of discoidin domain receptor 2 in mice

Objective

To investigate whether the reduction of discoidin domain receptor 2 (DDR‐2), a cell membrane tyrosine kinase receptor for native type II collagen, attenuates the progression of articular cartilage degeneration in mouse models of osteoarthritis (OA).

Methods

Double‐heterozygous (type XI collagen–deficient [Col11a1^+/−] and Ddr2‐deficient [Ddr2^+/−]) mutant mice were generated. Knee joints of Ddr2^+/− mice were subjected to microsurgical destabilization of the medial meniscus. Conditions of the articular cartilage from the knee joints of the double‐heterozygous mutant and surgically treated mice were examined by histology, evaluated using a modified Mankin scoring system, and characterized by immunohistochemistry.

Results

The rate of progressive degeneration in knee joints was dramatically reduced in the double‐heterozygous mutant mice compared with that in the type XI collagen–deficient mice. The progression in the double‐heterozygous mutant mice was delayed by ∼6 months. Following surgical destabilization of the medial meniscus, the progressive degeneration toward OA was dramatically delayed in the Ddr2^+/− mice compared with that in their wild‐type littermates. The articular cartilage damage present in the knee joints of the mice was directly correlated with the expression profiles of DDR‐2 and matrix metalloproteinase 13.

Conclusion

Reduction of DDR‐2 expression attenuates the articular cartilage degeneration of knee joints induced either by type XI collagen deficiency or by surgical destabilization of the medial meniscus.

     

Development and characteristics of pannus-like soft tissue in osteoarthritic articular surface in rat osteoarthritis model

OBJECTIVES: Pannus is invasive granulation tissue found on the articular cartilage having rheumatoid arthritis (RA). However, pannus-like tissue has also been found in osteoarthritis (OA). Our previous study showed that pannus-like tissue in OA (OA pannus) was frequently found in human OA samples. The purpose of the study is to investigate the development and the characteristics of OA pannus in a rat OA model. DESIGN: Ligaments of the knee joint were transected in Wister rats to induce OA. The knee joints were removed at weeks 1, 2, 4 and 6, and subjected to histological study. Samples were stained with hematoxylin and eosin (HE), Safranin-O and immuno-stained for vimentin, CD34, type II collagen and MMP-3. The whole knee joint of OA rats was implanted in SCID mice and kept for a further 3 weeks. Then the histological findings were evaluated in HE sections. RESULT: OA pannus appeared at week 2 and extend over the articular surface. OA pannus cells were positive for vimentin and/or CD34. At week 6, a part of articular surface was restored with matrix. OA pannus cells expressed MMP-3 as well as type II collagen. Histological study of rat OA knees implanted in SCID mice showed that OA pannus cells filled the joint space and invaded articular cartilage. CONCLUSIONS: The presence of OA pannus was found in a rat OA model and its features were similar to those in human OA. OA pannus had both catabolic and reparative features, and the latter feature were speculated to be dominant in the later phase of the disease under a certain environmental condition.     

Differential expression patterns of matrix metalloproteinases and their inhibitors during development of osteoarthritis in a transgenic mouse model

Objective: To characterise the expression of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) during degeneration of articular cartilage in a transgenic Del1 mouse model for osteoarthritis.

Methods: Northern analysis was used to measure mRNA levels of MMP-2, -3, -8, -9, -13, and -14, and TIMP-1, -2, and -3 in total RNA extracted from knee joints of transgenic Del1 mice, harbouring a 15 amino acid deletion in the triple helical domain of the α1(II) collagen chain, using their non-transgenic littermates as controls. Immunohistochemistry was used to study the presence of cleavage products (neoepitopes) of type II collagen, and the distribution of MMP-13 and TIMP-1 in degenerating cartilage.

Results: Each of the MMP and TIMP mRNAs analysed exhibited distinct expression patterns during development and osteoarthritic degeneration of the knee joint. The most striking change was up regulation of MMP-13 mRNA expression in the knee joints of Del1 mice at the onset of cartilage degeneration. However, the strongest immunostaining for MMP-13 and its inhibitor TIMP-1 was not seen in the degenerating articular cartilage but in synovial tissue, deep calcified cartilage, and subchondral bone. The localisation of type II collagen neoepitopes in chondrocytes and their pericellular matrix followed a similar pattern; they were not seen in cartilage fibrillations, but in adjacent unaffected cartilage.

Conclusion: The primary localisation of MMP-13 and TIMP-1 in hyperplastic synovial tissue, subchondral bone, and calcified cartilage suggests that up regulation of MMP-13 expression during early degeneration of articular cartilage is a secondary response to cartilage erosion. This interpretation is supported by the distribution of type II collagen neoepitopes. Synovial production of MMP-13 may be related to removal of tissue debris released from articular cartilage. In the deep calcified cartilage and adjacent subchondral bone, MMP-13 probably participates in tissue remodelling.

     

Gene deletion of either interleukin-1beta, interleukin-1beta-converting enzyme, inducible nitric oxide synthase, or stromelysin 1 accelerates the development of knee osteoarthritis in mice after surgical transection of the medial collateral ligament and partial medial meniscectomy

OBJECTIVE: To investigate the development of osteoarthritis (OA) after transection of the medial collateral ligament and partial medial meniscectomy in mice in which genes encoding either interleukin-1beta (IL-1beta), IL-1beta-converting enzyme (ICE), stromelysin 1, or inducible nitric oxide synthase (iNOS) were deleted. METHODS: Sectioning of the medial collateral ligament and partial medial meniscectomy were performed on right knee joints of wild-type and knockout mice. Left joints served as unoperated controls. Serial histologic sections were obtained from throughout the whole joint of both knees 4 days or 1, 2, 3, or 4 weeks after surgery. Sections were graded for OA lesions on a scale of 0-6 and were assessed for breakdown of tibial cartilage matrix proteoglycan (aggrecan) and type II collagen by matrix metalloproteinases (MMPs) and aggrecanases with immunohistochemistry studies using anti-VDIPEN, anti-NITEGE, and Col2-3/4C(short) neoepitope antibodies. Proteoglycan depletion was assessed by Alcian blue staining and chondrocyte cell death, with the TUNEL technique. RESULTS: All knockout mice showed accelerated development of OA lesions in the medial tibial cartilage after surgery, compared with wild-type mice. ICE-, iNOS-, and particularly IL-1beta-knockout mice developed OA lesions in the lateral cartilage of unoperated limbs. Development of focal histopathologic lesions was accompanied by increased levels of MMP-, aggrecanase-, and collagenase-generated cleavage neoepitopes in areas around lesions, while nonlesional areas showed no change in immunostaining. Extensive cell death was also detected by TUNEL staining in focal areas around lesions. CONCLUSION: We postulate that deletion of each of these genes, which encode molecules capable of producing degenerative changes in cartilage, leads to changes in the homeostatic controls regulating the balance between anabolism and catabolism, favoring accelerated cartilage degeneration. These observations suggest that these genes may play important regulatory roles in maintaining normal homeostasis in articular cartilage matrix turnover.     

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.     

Prevention of cartilage destruction with intraarticular osteoclastogenesis inhibitory factor/osteoprotegerin in a murine model of osteoarthritis

OBJECTIVE: To investigate the effect of osteoclastogenesis inhibitory factor/osteoprotegerin (OPG) on chondrocytes in the development of osteoarthritis (OA) in vivo. METHODS: To determine the role of endogenous OPG in the progression of OA, OA was surgically induced in OPG+/- mice and their wild-type (WT) littermates. To determine the role of exogenous OPG, knee joints of C57BL/6J mice with surgically induced OA were injected intraarticularly with recombinant human OPG (rHuOPG) or vehicle 5 times a week. All mice were euthanized 4 weeks after OA induction; joints were harvested and evaluated immunohistochemically. RESULTS: Although OA changes were induced in both WT and OPG+/- mice, the degenerative changes in the articular cartilage were significantly enhanced in OPG+/- mice. In C57BL/6J mice with surgically induced OA, intraarticular OPG administration protected the articular cartilage from the progression of OA. The Mankin and cartilage destruction scores in OPG-treated animals were approximately 50% of those seen in the control group. Furthermore, OPG administration significantly protected articular cartilage thickness. Findings of the TUNEL assay indicated that rHuOPG prevented chondrocyte apoptosis in joints with surgically induced OA. Results of immunostaining indicated that OPG protein was detected in the synovium and in resident chondrocytes at higher levels in the OPG-treated group than in the control group. CONCLUSION: These data indicate that endogenous OPG had a protective effect against the cartilage destruction that occurs during OA progression. Furthermore, direct administration of rHuOPG to articular chondrocytes prevented cartilage destruction in an experimental murine model of OA via prevention of chondrocyte apoptosis.     

Increased expression of the collagen receptor discoidin domain receptor 2 in articular cartilage as a key event in the pathogenesis of osteoarthritis

OBJECTIVE: To investigate the role of the collagen receptor discoidin domain receptor 2 (DDR-2) in the pathogenesis of osteoarthritis (OA). METHODS: Histologic and immunohistochemical analyses were performed to characterize femoral head cartilage from 7 patients with OA and 4 patients with fracture, as well as articular cartilage from the knee joints of mice with surgically induced OA. Gene constructs encoding human Raf kinase inhibitor protein (RKIP), DDR-2 lacking the discoidin (DS) domain (DeltaDS-DDR-2) or the protein tyrosine kinase (PTK) core (DeltaPTK-DDR-2), DDR-2 containing a substitution of tyrosine for alanine at position 740 (Y740A), and luciferase driven by the matrix metalloproteinase 13 (MMP-13) promoter were transfected into human chondrocyte cell lines. Activated and neutralized alpha2beta1 integrin polyclonal antibodies, interleukin-1 receptor antagonist, and the chemical inhibitors SB203580, for p38, and SP600125, for JNKs, were used in cell cultures. Real-time polymerase chain reaction was performed to examine MMP-13 and DDR-2 messenger RNA (mRNA). RESULTS: Increased immunostaining for DDR-2, MMP-13, and MMP-derived type II collagen fragments was detected in cartilage from patients with OA and from mice with surgically induced OA. The discoidin domain and PTK core of DDR-2 were essential for signal transmission and the resulting increased expression of MMP-13 in chondrocytes. Y740A mutation of DDR-2 reduced levels of mRNA for MMP-13 and endogenous DDR-2. The overexpression of RKIP or preincubation with the p38 inhibitor reduced MMP-13 mRNA levels. DDR-2 signaling was independent of the alpha2beta1 integrin and the interleukin-1-induced signaling pathways in chondrocytes. CONCLUSION: These findings suggest that increased expression of DDR-2, resulting in the elevated expression of MMP-13, may be one of the common events in OA progression.     

Inactivation of one allele of the type II collagen gene alters the collagen network in murine articular cartilage and makes cartilage softer

OBJECTIVE: To evaluate the influence of inactivation of one allele ("heterozygous knockout" or "heterozygous inactivation") of the type II procollagen gene (Col2a1) on the biomechanical properties and structure of the articular cartilage and subchondral bone in 15 month old mice. METHODS: Indentation stiffness of the humerus head articular cartilage was measured by a microindentation method. Cartilage and subchondral bone were prepared for digital densitometry of proteoglycans (PGs), polarised light microscopy (PLM) of collagen, and osteoarthrosis (OA) grading. RESULTS: Heterozygous inactivation of the Col2a1 gene softened articular cartilage (p=0.002) as measured by indentation stiffness ((mean (SEM) 0.50 (0.07) MPa v 0.94 (0.13) MPa in controls). Fibrillar collagen network exhibited lower birefringence in the intermediate (p=0.04) and deep zones (p=0.01) of cartilage by PLM, indicating either decreased collagen content or a lower degree of fibril parallelism in the knockout mice. The total and zonal thicknesses of articular cartilage were unchanged. Zonal PG contents did not differ significantly. In knockout mice, the prevalence of superficial fibrillation-that is, a sign of OA, was higher than in controls (73% v 21%, p=0.002). The collagen induced birefringence of the superficial zone was not reduced. The subchondral bone volume fraction was lower in knockout mice than in controls, 31% v 43% (p=0.01), and optical retardation values in PLM of bone collagen were slightly but significantly lower (p=0.01). CONCLUSION: Heterozygous inactivation of the Col2a1 gene made articular cartilage softer, altered the collagenous network, reduced subchondral bone volume, and altered its microstructure. Changes in the cartilage collagen network probably contributed to increased susceptibility to OA.     

Expression of the cartilage derived anti-angiogenic factor chondromodulin-I decreases in the early stage of experimental osteoarthritis

OBJECTIVE: Chondromodulin-I (ChM-I), a cartilage derived anti-angiogenic factor, has been shown to regulate the vascular invasion during endochondral bone formation. We evaluated the expression and localization of ChM-I in articular cartilage during the progression of osteoarthritis (OA) in the rat, and correlated ChM-I expression with the increase in vascular invasion into OA articular cartilage. METHODS: Expression of ChM-I, type II collagen, basic fibroblast growth factor, vascular endothelial growth factor (VEGF), and matrix metalloproteinases MMP-9 and MMP-13 were examined in articular cartilage of intact growing and adult rats and in the surgically induced OA model using in situ hybridization, Western blot analysis, and immunohistochemistry. Co-immunostaining for ChM-I and CD-31 was performed to localize ChM-I and neovascularization in articular cartilage at advanced stage of OA. RESULTS: Abundant expression of ChM-I protein was detected in avascular regions of the developing and adult healthy articular cartilage. In early OA, ChM-I expression decreased in the superficial zone of articular cartilage, while levels of proteoglycan and type II collagen were comparable to control. In advanced OA, ChM-I expression was reduced in all zones of articular cartilage, and the number of VEGF-expressing cells was increased. Immunohistochemical studies showed that vascular invasion occurred in proximity to chondrocytes with high expression of pro-angiogenic markers, and decreased expression of ChM-I. CONCLUSION: High expression of ChM-I was detected in articular cartilage of growing and normal adult joints, implicating its role in the maintenance of avascularity of intact articular cartilage. Expression of ChM-I decreased, while expression of VEGF and other pro-angiogenic factors increased, in OA cartilage. These findings suggest the loss of ChM-I from articular cartilage might be responsible in part for promoting blood vessel invasion into the cartilage during progression of OA.     

A type I collagen defect leads to rapidly progressive osteoarthritis in a mouse model

OBJECTIVE: This study was undertaken to test the hypothesis that abnormalities of the subchondral bone can result in osteoarthritis (OA). METHODS: We used a knockin model of human osteogenesis imperfecta, the Brittle IV (Brtl) mouse, in which defective type I collagen is expressed in bone. OA in individual mice was documented by micro-magnetic resonance imaging (micro-MRI) and micro-computed tomography (micro-CT). Alterations in the knee joints were confirmed by histopathologic and immunohistochemical analysis. In addition, atomic force microscopy (AFM) was used to assess the ultrastructure of the articular cartilage and subchondral bone matrix. RESULTS: Brtl mice had decreased integrity of bone but initially normal articular cartilage. However, by the second month of life, Brtl mice developed alterations of the cartilage that were characteristic of OA, as documented by micro-CT, micro-MRI, and histologic evaluation. In addition, chondrocyte loss and breakdown of the collagen matrix in the residual cartilage were demonstrated using AFM. CONCLUSION: The Brtl mouse model demonstrates that progressive destruction of articular cartilage characteristic of OA may be secondary to altered architecture of the underlying subchondral bone.     

Developmental and osteoarthritic changes in Col6a1‐knockout mice: Biomechanics of type VI collagen in the cartilage pericellular matrix

Objective

Chondrocytes, the sole cell type in articular cartilage, maintain the extracellular matrix (ECM) through a homeostatic balance of anabolic and catabolic activities that are influenced by genetic factors, soluble mediators, and biophysical factors such as mechanical stress. Chondrocytes are encapsulated by a narrow tissue region termed the “pericellular matrix” (PCM), which in normal cartilage is defined by the exclusive presence of type VI collagen. Because the PCM completely surrounds each cell, it has been hypothesized that it serves as a filter or transducer for biochemical and/or biomechanical signals from the cartilage ECM. The present study was undertaken to investigate whether lack of type VI collagen may affect the development and biomechanical function of the PCM and alter the mechanical environment of chondrocytes during joint loading.

Methods

Col6a1^−/− mice, which lack type VI collagen in their organs, were generated for use in these studies. At ages 1, 3, 6, and 11 months, bone mineral density (BMD) was measured, and osteoarthritic (OA) and developmental changes in the femoral head were evaluated histomorphometrically. Mechanical properties of articular cartilage from the hip joints of 1‐month‐old Col6a1^−/−, Col6a1^+/−, and Col6a1^+/+ mice were assessed using an electromechanical test system, and mechanical properties of the PCM were measured using the micropipette aspiration technique.

Results

In Col6a1^−/− and Col6a1^+/− mice the PCM was structurally intact, but exhibited significantly reduced mechanical properties as compared with wild‐type controls. With age, Col6a1^−/− mice showed accelerated development of OA joint degeneration, as well as other musculoskeletal abnormalities such as delayed secondary ossification and reduced BMD.

Conclusion

These findings suggest that type VI collagen has an important role in regulating the physiology of the synovial joint and provide indirect evidence that alterations in the mechanical environment of chondrocytes, due to either loss of PCM properties or Col6a1^−/−‐derived joint laxity, can lead to progression of OA.

     

Selective enhancement of collagenase-mediated cleavage of resident type II collagen in cultured osteoarthritic cartilage and arrest with a synthetic inhibitor that spares collagenase 1 (matrix metalloproteinase 1)

OBJECTIVE: To examine whether type II collagen cleavage by collagenase and loss of proteoglycan are excessive in human osteoarthritic (OA) articular cartilage compared with nonarthritic articular cartilage, and whether this can be inhibited by a selective synthetic inhibitor that spares collagenase 1 (matrix metalloproteinase 1 [MMP-1]). METHODS: Articular cartilage samples were obtained during surgery from 11 patients with OA and at autopsy from 5 adults without arthritis. The articular cartilage samples were cultured in serum-free medium. A collagenase-generated neoepitope, which reflects cleavage of type II collagen, and proteoglycan glycosaminoglycan (GAG), which predominantly reflects aggrecan release, were assayed in culture media. In addition, cultures were performed using either of 2 synthetic MMP inhibitors, both of which inhibited collagenase 2 (MMP-8) and collagenase 3 (MMP-13), but one of which spared collagenase 1. Cultures were also biolabeled with 3H-proline in the presence and absence of these inhibitors to measure collagen synthesis (as tritiated hydroxyproline) and incorporation in articular cartilage. RESULTS: As a group, cleavage of type II collagen by collagenase was significantly increased in OA cartilage samples. In contrast, proteoglycan (GAG) release was not increased. This release of a collagenase-generated epitope was inhibited by both MMP inhibitors in 2 of 5 nonarthritic samples and in 9 of 11 OA cartilage samples. The inhibitor that spared collagenase 1 was generally more effective and inhibited release from 4 of 5 nonarthritic cartilage samples and the same OA cartilage samples. Group analyses revealed that the inhibition of collagenase neoepitope release by both inhibitors was significant in the OA patient cartilage, but not in the nonarthritic cartilage. Proteoglycan loss was unaffected by either inhibitor. Newly synthesized collagen (predominantly, type II) exhibited increased incorporation in OA cartilage, but only in the presence of the inhibitor that arrested collagenase 1 activity. CONCLUSION: These results further indicate that the digestion of type II collagen by collagenase is selectively increased in OA cartilage, and that this can be inhibited in the majority of cases by a synthetic inhibitor that can inhibit collagenases 2 and 3, but not collagenase 1. The results also suggest that in OA, newly synthesized collagen is digested, but in a different manner than that of resident molecules. Proteoglycan release was not increased in OA cartilage and was unaffected by these inhibitors. Inhibitors of this kind may be of value in preventing damage to type II collagen in human arthritic articular cartilage.     

The quantitation of a native chondroitin sulfate epitope in synovial fluid lavages and articular cartilage from canine experimental osteoarthritis and disuse atrophy

Objective. Previous studies have shown the presence of a native chondroitin sulfate epitope in articular cartilage proteoglycans from canine knee joints with experimental early osteoarthritis (OA), but not in normal cartilage. The objective of this study was to quantitate the native epitope recognized by monoclonal antibody 3-B-3 in synovial fluids and articular cartilage of diseased joints. Methods. An immunoassay with monoclonal antibody 3-B-3, which recognizes a native chondroitin-6-sulfate structure, was developed and used to analyze synovial fluid lavage material and extracts of articular cartilage from canine knee joints with early experimental OA or with mild disuse atrophy, and from control animals. Results. The concentration of epitope in the OA fluids was elevated 33–35-fold, and in the OA articular cartilage extracts it was elevated > 200-fold, compared with samples from the control group. No significant difference was detected in the levels of 3-B-3 epitope in the synovial fluid lavage material or cartilage extracts from the joints of the disuse group versus the control group. Conclusion. The native 3-B-3 epitope in articular cartilage and synovial fluids may be a specific marker of ongoing anabolic events in early degenerative joint disease.     

Accelerated, aging-dependent development of osteoarthritis in alpha1 integrin-deficient mice

OBJECTIVE: Cell-matrix interactions regulate chondrocyte differentiation and survival. The alpha1beta1 integrin is a major collagen receptor that is expressed on chondrocytes. Mice with targeted inactivation of the integrin alpha1 gene (alpha1-KO mice) provide a model that can be used to address the role of cell-matrix interactions in cartilage homeostasis and osteoarthritis (OA) pathogenesis. METHODS: Knee joints from alpha1-KO and wild-type (WT) BALB/c mice were harvested at ages 4-15 months. Knee joint sections were examined for inflammation, cartilage degradation, and loss of glycosaminoglycans (by Safranin O staining). Immunohistochemistry was performed to detect the distribution of alpha1 integrin, matrix metalloproteinases (MMPs), and chondrocyte apoptosis. RESULTS: In WT mice, the alpha1 integrin subunit was detected in hypertrophic chondrocytes in the growth plate and in a subpopulation of cells in the deep zone of articular cartilage. There was a marked increase in alpha1-positive chondrocytes in the superficial and upper mid-zones in OA-affected areas in joints from old WT mice. The alpha1-KO mice showed more severe cartilage degradation, glycosaminoglycan depletion, and synovial hyperplasia as compared with the WT mice. MMP-2 and MMP-3 expression was increased in the OA-affected areas. In cartilage from alpha1-KO mice, the cellularity was reduced and the frequency of apoptotic cells was increased. These results suggest that the alpha1 integrin subunit is involved in the early remodeling process in OA cartilage. CONCLUSION: Deficiency in the alpha1 integrin subunit is associated with an earlier deregulation of cartilage homeostasis and an accelerated, aging-dependent development of OA.     

Lifelong voluntary joint loading increases osteoarthritis in mice housing a deletion mutation in type II procollagen gene,and slightly also in non-transgenic mice

Objectives: To investigate the effects of voluntary running on the incidence and severity of osteoarthritis (OA) and associated changes in cartilage matrix and subchondral bone in a transgenic Del1 mouse model for OA.

Methods: Del1 mice and their non-transgenic littermate controls were housed from the age of 5–6 weeks to 15 months in individual cages with running wheels. The running activity of each mouse was monitored for the entire 12 month period. Additional Del1 and control mice were housed in individual cages without running wheels. At the end of the experiment the severity of OA was evaluated by light microscopy, and the articular cartilage matrix changes by digital densitometry and quantitative polarised light microscopy.

Results: Lifelong voluntary running increased the incidence and severity of OA significantly in Del1 mice (transgenic runners), and slightly also in non-transgenic runners. Severe OA changes increased from 39% in transgenic non-runners to 90% in transgenic runners (p=0.006) in lateral tibial condyles, and from 24% to 80% (p=0.013) in lateral femoral condyles, respectively. The proteoglycan content of articular cartilage was reduced in transgenic runners in comparison with transgenic non-runners (p=0.0167), but a similar effect was not seen in non-transgenic runners compared with non-transgenic non-runners. No attributable differences were seen in the collagen network of articular cartilage or in the subchondral bone between any of the groups.

Conclusion: The Del1 mutation has earlier been shown to disturb the assembly of the cartilage collagen network and thereby increase the incidence and severity of OA with age. In this study, voluntary running was shown to increase further cartilage damage in the lateral compartments of the knee. This suggests that articular cartilage in Del1 mice is less resistant to physical loading than in control mice. Despite severe OA lesions in the knee joint at the age of 15 months, Del1 mice continued to run voluntarily 2–3 km every night.