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Cartilage Matrix (cartilage + matrix)
Terms modified by Cartilage Matrix Selected AbstractsAnalysis of N-cadherin function in limb mesenchymal chondrogenesis in vitro,DEVELOPMENTAL DYNAMICS, Issue 2 2002Anthony M. Delise Abstract During embryonic limb development, cartilage formation is presaged by a crucial mesenchymal cell condensation phase. N-Cadherin, a Ca2+ -dependent cell,cell adhesion molecule, is expressed in embryonic chick limb buds in a spatiotemporal pattern suggestive of its involvement during cellular condensation; functional blocking of N-cadherin homotypic binding, by using a neutralizing monoclonal antibody, results in perturbed chondrogenesis in vitro and in vivo. In high-density micromass cultures of embryonic limb mesenchymal cells, N-cadherin expression level is high during days 1 and 2, coincident with active cellular condensation, and decreases upon overt chondrogenic differentiation from day 3 on. In this study, we have used a transfection approach to evaluate the effects of gain- and loss-of-function expression of N-cadherin constructs on mesenchymal condensation and chondrogenesis in vitro. Chick limb mesenchymal cells were transfected by electroporation with recombinant expression plasmids encoding wild-type or two mutant extracellular/cytoplasmic deletion forms of N-cadherin. Expression of the transfected N-cadherin forms showed a transient profile, being high on days 1,2 of culture, and decreasing by day 3, fortuitously coincident with the temporal profile of endogenous N-cadherin gene expression. Examined by means of peanut agglutinin (PNA) staining for condensing precartilage mesenchymal cells, cultures overexpressing wild-type N-cadherin showed enhanced cellular condensation on culture days 2 and 3, whereas expression of the deletion mutant forms (extracellular/cytoplasmic) of N-cadherin resulted in a decrease in PNA staining, suggesting that a complete N-cadherin protein is required for normal cellular condensation to occur. Subsequent chondrogenesis was also affected. Cultures overexpressing the wild-type N-cadherin protein showed enhanced chondrogenesis, indicated by increased production of cartilage matrix (sulfated proteoglycans, collagen type II, and cartilage proteoglycan link protein), as well as increased cartilage nodule number and size of individual nodules, compared with control cultures and cultures transfected with either of the two mutant N-cadherin constructs. These results demonstrate that complete N-cadherin function, at the levels of both extracellular homotypic binding and cytoplasmic linkage to the cytoskeleton by means of the catenin complex, is required for chondrogenesis by mediating functional mesenchymal cell condensation. © 2002 Wiley-Liss, Inc. [source] Physical indicators of cartilage health: the relevance of compliance, thickness, swelling and fibrillar textureJOURNAL OF ANATOMY, Issue 6 2003Neil D. Broom Abstract This study uses a bovine patella model to compare the relative merits of on-bone compliance and thickness measurements, free-swelling behaviour, and structural imaging with differential interference contrast (DIC) light microscopy to assess the biomechanical normality of the cartilage matrix. The results demonstrate that across a spectrum of cartilage tissues from immature, mature, through to mildly degenerate, and all with intact articular surfaces, there is a consistent pattern of increased free swelling of the isolated general matrix with age and degeneration. High swelling was always associated with major structural alterations of the general matrix that were readily imaged using DIC light microscopy. Conversely, for all tissue groups, no relationship was observed between thickness vs. compliance and compliance vs. general matrix swelling. Only in the proximal aspects of the normal mature and degenerate tissues was there a correlation between thickness and general matrix swelling. Free-swelling measurements combined with fibrillar texture imaging using DIC light microscopy are therefore recommended as providing a reliable and quick method of assessing the biomechanical condition of the cartilage general matrix. [source] Role of the Latent Transforming Growth Factor ,,Binding Protein 1 in Fibrillin-Containing Microfibrils in Bone Cells In Vitro and In VivoJOURNAL OF BONE AND MINERAL RESEARCH, Issue 1 2000Sarah L. Dallas Abstract Latent transforming growth factor ,,binding proteins (LTBPs) are extracellular matrix (ECM) proteins that bind latent transforming growth factor , (TGF-,) and influence its availability in bone and other connective tissues. LTBPs have homology with fibrillins and may have related functions as microfibrillar proteins. However, at present little is known about their structural arrangement in the ECM. By using antibodies against purified LTBP1, against a short peptide in LTBP1, and against epitope-tagged LTBP1 constructs, we have shown colocalization of LTBP1 and fibrillin 1 in microfibrillar structures in the ECM of cultured primary osteoblasts. Immunoelectron microscopy confirmed localization of LTBP1 to 10- to 12-nm microfibrils and suggested an ordered aggregation of LTBP1 into these structures. Early colocalization of LTBP1 with fibronectin suggested a role for fibronectin in the initial assembly of LTBP1 into the matrix; however, in more differentiated osteoblast cultures, LTBP1 and fibronectin 1 were found in distinct fibrillar networks. Overexpression of LTBP1 deletion constructs in osteoblast-like cells showed that N-terminal amino acids 67,467 were sufficient for incorporation into fibrillin-containing microfibrils and suggested that LTBP1 can be produced by cells distant from the site of fibril formation. In embryonic long bones in vivo, LTBP1 and fibrillin 1 colocalized at the surface of newly forming osteoid and bone. However, LTBP1-positive fibrils, which did not contain fibrillin 1, were present in cartilage matrix. These studies show that in addition to regulating TGF,1, LTBP1 may function as a structural component of connective tissue microfibrils. LTBP1 may therefore be a candidate gene for Marfan-related connective tissue disorders in which linkage to fibrillins has been excluded. [source] Epidermal growth factor stimulates proton efflux from chondrocytic cellsJOURNAL OF CELLULAR PHYSIOLOGY, Issue 1 2002Kevin E.H. Lui Proton efflux from chondrocytes alters the extracellular pH and ionic composition of cartilage, and influences the synthesis and degradation of extracellular matrix. Epidermal growth factor (EGF) promotes chondrocyte proliferation during skeletal development and accumulates in the synovial fluid in rheumatoid arthritis. The purpose of this study was to investigate the effect of EGF on proton efflux from chondrocytes. When monitored using a Cytosensor microphysiometer, EGF was found to rapidly activate proton efflux from CFK2 chondrocytic cells and rat articular chondrocytes. The actions of EGF were concentration-dependent with half-maximal effects at 0.3,0.7 ng/ml. Partial desensitization and time-dependent recovery of the response were observed following repeated exposures to EGF. EGF-induced proton efflux was dependent on extracellular glucose, and inhibitors of Na+/H+ exchange (NHE) markedly attenuated the initial increase in proton efflux. The response was diminished by inhibitors of phosphatidylinositol 3-kinase and phospholipase C, but not by inhibitors of MEK (MAPK/ERK kinase) or protein kinase A or C. Thus, EGF-induced proton efflux involves glucose metabolism and NHE, and is regulated by a discrete subset of EGF-activated signaling pathways. In vivo, proton efflux induced by EGF may lead to an acidic environment, enhancing turnover of cartilage matrix during development and in rheumatoid arthritis. © 2002 Wiley-Liss, Inc. [source] Smoking delays chondrogenesis in a mouse model of closed tibial fracture healingJOURNAL OF ORTHOPAEDIC RESEARCH, Issue 12 2006Hossam B. El-Zawawy Abstract Smoking delays the healing process and increases morbidity associated with many common musculoskeletal disorders, including long bone fracture. In the current study, a murine model of tibial fracture healing was used to test the hypothesis that smoking delays chondrogenesis after fracture. Mice were divided into two groups, a nonsmoking control group and a group exposed to cigarette smoke for 1 month prior to surgical tibial fracture. Mice were euthanized at 7, 14, and 28 days after surgery. The outcomes measured were immunohistochemical staining for type II collagen protein expression as a marker of cartilage matrix and proliferating cell nuclear antigen (PCNA) staining to measure proliferation at the site of injury. Toluidine blue staining and histomorphometry were used to quantify areas of cartilaginous and noncartilaginous fracture callus. Radiographs were analyzed for evidence of remodeling after injury. At day 7 after injury, mice exposed to cigarette smoke had a smaller fracture callus with less cartilage matrix compared to controls. Proliferation was present at high levels in both groups at this time point, but proliferating cells had a more immature morphology in the smoking group. At day 14, chondrogenesis was more active in smokers compared to controls, while a higher percentage of bone was present in the control animals. At day 28, X-ray analysis revealed a larger fracture callus remaining in the smoking animals. Together, these findings show that the chondrogenic phase of tibial fracture healing is delayed by smoking. This study represents, to our knowledge, the first analysis of molecular and cellular mechanisms of healing in a smoking mouse fracture model. © 2006 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res [source] Membrane type 1 matrix metalloproteinase is a crucial promoter of synovial invasion in human rheumatoid arthritisARTHRITIS & RHEUMATISM, Issue 3 2009Mary-Clare Miller Objective A hallmark of rheumatoid arthritis (RA) is invasion of the synovial pannus into cartilage, and this process requires degradation of the collagen matrix. The aim of this study was to explore the role of one of the collagen-degrading matrix metalloproteinases (MMPs), membrane type 1 MMP (MT1-MMP), in synovial pannus invasiveness. Methods The expression and localization of MT1-MMP in human RA pannus were investigated by Western blot analysis of primary synovial cells and immunohistochemical analysis of RA joint specimens. The functional role of MT1-MMP was analyzed by 3-dimensional (3-D) collagen invasion assays and a cartilage invasion assay in the presence or absence of tissue inhibitor of metalloproteinases 1 (TIMP-1), TIMP-2, or GM6001. The effect of adenoviral expression of a dominant-negative MT1-MMP construct lacking a catalytic domain was also examined. Results MT1-MMP was highly expressed at the pannus,cartilage junction in RA joints. Freshly isolated rheumatoid synovial tissue and isolated RA synovial fibroblasts invaded into a 3-D collagen matrix in an MT1-MMP,dependent manner. Invasion was blocked by TIMP-2 and GM6001 but not by TIMP-1. Invasion was also inhibited by the overexpression of a dominant-negative MT1-MMP, which inhibits collagenolytic activity and proMMP-2 activation by MT1-MMP on the cell surface. Synovial fibroblasts also invaded into cartilage in an MT1-MMP,dependent manner. This process was further enhanced by removing aggrecan from the cartilage matrix. Conclusion MT1-MMP serves as an essential collagen-degrading proteinase during pannus invasion in human RA. Specific inhibition of MT1-MMP,dependent invasion may represent a novel therapeutic strategy for RA. [source] A sodium dodecyl sulfate,polyacrylamide gel electrophoresis,liquid chromatography tandem mass spectrometry analysis of bovine cartilage tissue response to mechanical compression injury and the inflammatory cytokines tumor necrosis factor , and interleukin-1,ARTHRITIS & RHEUMATISM, Issue 2 2008Anna L. Stevens Objective To compare the response of chondrocytes and cartilage matrix to injurious mechanical compression and treatment with interleukin-1, (IL-1,) and tumor necrosis factor , (TNF,), by characterizing proteins lost to the medium from cartilage explant culture. Methods Cartilage explants from young bovine stifle joints were treated with 10 ng/ml of IL-1, or 100 ng/ml of TNF, or were subjected to uniaxial, radially-unconfined injurious compression (50% strain; 100%/second strain rate) and were then cultured for 5 days. Pooled media were subjected to gel-based separation (sodium dodecyl sulfate,polyacrylamide gel electrophoresis) and analysis by liquid chromatography tandem mass spectrometry, and the data were analyzed by Spectrum Mill proteomics software, focusing on protein identification, expression levels, and matrix protein proteolysis. Results More than 250 proteins were detected, including extracellular matrix (ECM) structural proteins, pericellular matrix proteins important in cell,cell interactions, and novel cartilage proteins CD109, platelet-derived growth factor receptor,like, angiopoietin-like 7, and adipocyte enhancer binding protein 1. IL-1, and TNF, caused increased release of chitinase 3,like protein 1 (CHI3L1), CHI3L2, complement factor B, matrix metalloproteinase 3, ECM-1, haptoglobin, serum amyloid A3, and clusterin. Injurious compression caused the release of intracellular proteins, including Grp58, Grp78, ,4-actinin, pyruvate kinase, and vimentin. Injurious compression also caused increased release and evidence of proteolysis of type VI collagen subunits, cartilage oligomeric matrix protein, and fibronectin. Conclusion Overload compression injury caused a loss of cartilage integrity, including matrix damage and cell membrane disruption, which likely occurred through strain-induced mechanical disruption of cells and matrix. IL-1, and TNF, caused the release of proteins associated with an innate immune and stress response by the chondrocytes, which may play a role in host defense against pathogens or may protect cells against stress-induced damage. [source] Mediation of interleukin-1,,induced transforming growth factor ,1 expression by activator protein 4 transcription factor in primary cultures of bovine articular chondrocytes: Possible cooperation with activator protein 1ARTHRITIS & RHEUMATISM, Issue 6 2003R. Andriamanalijaona Objective Interleukin-1 (IL-1) and transforming growth factor ,1 (TGF,1) play major roles in osteoarticular diseases, exerting opposite effects on both the catabolism and anabolism of cartilage matrix. Previous findings suggest that IL-1 and TGF,1 could function in a feedback interaction. However, the effect exerted by IL-1 on expression of TGF, by articular chondrocytes is, so far, poorly understood. The present study was carried out to determine the influence of IL-1, on the expression of TGF,1 by bovine articular chondrocytes (BACs) in primary culture. Methods BAC primary cultures were treated with IL-1,, and TGF,1 messenger RNA (mRNA) steady-state levels and protein expression were measured by real-time reverse transcription,polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. Transient transfection of TGF,1 gene promoter constructs was performed to delineate the DNA sequences that mediate the IL-1, effect. Electrophoretic mobility shift assays (EMSAs) and supershift analysis were used to characterize the transcription factors binding to these sequences. Results Cultured BACs responded to IL-1, exposure by exhibiting an increase of TGF,1 expression at both the mRNA and protein levels. The effect was found to be mediated by a major 80-bp sequence located between ,732 and ,652 upstream of the transcription initiation site. EMSA and supershift analysis revealed that the transcription factors activator protein 4 (AP-4) and AP-1 specifically bound to the ,720/,696 part of this sequence under IL-1, treatment. Overexpression of AP-4 in the BAC cultures resulted in stimulation of the transcriptional activity of the ,732/+11 TGF,1 promoter construct through the same IL-1,,responsive element. Conclusion IL-1, induces an increase of TGF,1 in articular chondrocytes through activation of AP-4 and AP-1 binding to the TGF,1 gene promoter. These findings may help us understand the role of IL-1, in the disease process. Notwithstanding its deleterious effect on cartilage, IL-1 could initiate the repair response displayed by injured cartilage in the early stages of osteoarthritis through its ability to enhance TGF,1 expression by local chondrocytes. [source] Kinetics of bone protection by recombinant osteoprotegerin therapy in Lewis rats with adjuvant arthritisARTHRITIS & RHEUMATISM, Issue 7 2002Giuseppe Campagnuolo Objective To assess the effect of different dosages and treatment schedules of osteoprotegerin (OPG) on joint preservation in an experimental model of adjuvant-induced arthritis (AIA). Methods Male Lewis rats with AIA (6,8 per group) were treated with a subcutaneous bolus of recombinant human OPG according to one of the following schedules: daily OPG (an efficacious regimen) starting at disease onset (days 9,15), early intervention (days 9,11), delayed intervention (days 13,15), and extended therapy (days 9,22). Inflammation (hind paw swelling) was quantified throughout the clinical course; osteoporosis (bone mineral density [BMD], by quantitative dual x-ray absorptiometry) and morphologic appraisals of inflammation, bone damage, intralesional osteoclasts (by semiquantitative histopathologic scoring), and integrity of the articular cartilage matrix (by retention of toluidine blue stain) were determined in histology sections of arthritic hind paws. Results OPG provided dose- and schedule-dependent preservation of BMD and periarticular bone while essentially eliminating intralesional osteoclasts. Dosages ,2.5 mg/kg/day preserved or enhanced BMD and prevented essentially all erosions. A dosage of 4 mg/kg/day protected joint integrity to a comparable degree when given for 7 (days 9,15) or 14 (days 9,22) consecutive days. At this dosage, early intervention (days 9,11) was twice as effective as delayed intervention (days 13,15) at preventing joint dissolution. Erosions and osteoclast scores were greatly decreased for 26 days (measured from the first treatment) after 7 or 14 daily doses of OPG (4 mg/kg/day). OPG treatment also prevented loss of cartilage matrix proteoglycans, an indirect consequence of protecting the subchondral bone. No OPG dosage or regimen alleviated weight loss, inflammation, or periosteal osteophyte production. Conclusion These data indicate that OPG preserves articular bone and (indirectly) articular cartilage in arthritic joints in a dose- and schedule-dependent manner, halts bone erosion when given at any point during the course of arthritis, produces sustained antierosive activity after a short course, and is most effective when initiated early in the disease. [source] | |||||||||||||