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Co-culture of mechanically injured cartilage with joint capsule tissue alters chondrocyte expression patterns and increases ADAMTS5 production.

ABSTRACT: We studied changes in chondrocyte gene expression, aggrecan degradation, and aggrecanase production and activity in normal and mechanically injured cartilage co-cultured with joint capsule tissue. Chondrocyte expression of 21 genes was measured at 1, 2, 4, 6, 12, and 24h after treatment; clustering analysis enabled identification of co-expression profiles. Aggrecan fragments retained in cartilage and released to medium and loss of cartilage sGAG were quantified. Increased expression of MMP-13 and ADAMTS4 clustered with effects of co-culture, while increased expression of ADAMTS5, MMP-3, TGF-beta, c-fos, c-jun clustered with cartilage injury. ADAMTS5 protein within cartilage (immunohistochemistry) increased following injury and with co-culture. Cartilage sGAG decreased over 16-days, most severely following injury plus co-culture. Cartilage aggrecan was cleaved at aggrecanase sites in the interglobular and C-terminal domains, resulting in loss of the G3 domain, especially after injury plus co-culture. Together, these results support the hypothesis that interactions between injured cartilage and other joint tissues are important in matrix catabolism after joint injury.

SUBMITTER: Lee JH

PROVIDER: S-EPMC2752630 | biostudies-literature | 2009 Sep

REPOSITORIES: biostudies-literature

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Co-culture of mechanically injured cartilage with joint capsule tissue alters chondrocyte expression patterns and increases ADAMTS5 production.

Lee J H JH Fitzgerald J B JB DiMicco M A MA Cheng D M DM Flannery C R CR Sandy J D JD Plaas A H AH Grodzinsky A J AJ

Archives of biochemistry and biophysics 20090714 1-2

We studied changes in chondrocyte gene expression, aggrecan degradation, and aggrecanase production and activity in normal and mechanically injured cartilage co-cultured with joint capsule tissue. Chondrocyte expression of 21 genes was measured at 1, 2, 4, 6, 12, and 24h after treatment; clustering analysis enabled identification of co-expression profiles. Aggrecan fragments retained in cartilage and released to medium and loss of cartilage sGAG were quantified. Increased expression of MMP-13 an ...[more]

PMID: 19607802

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Project description:BackgroundsAbnormal cartilage calcification is one of the pathological changes of temporomandibular joint (TMJ) osteoarthritis (OA). Recent studies have reported that exosomes can regulate the formation of abnormal calcified nodules in diseases including atherosclerosis and chronic kidney disease. However, the influences of chondrocyte-derived exosomes on abnormal cartilage calcification in TMJ OA are still unclear.MethodsTMJ OA was induced by unilateral anterior crossbite (UAC) for 4, 8, or 12 weeks in rats to observe abnormal calcification in TMJ condylar cartilage and exosome formation. Concomitantly, GW4869, the inhibitor of exosome formation, was locally injected to the TMJ of rats under stimulation of UAC, while the exosomes extracted from primary condylar chondrocytes stimulated with fluid flow shear stress (FFSS) were locally injected to rats TMJ.ResultsAbnormal calcification was enhanced in the degenerative cartilage of TMJ OA in UAC rats, and a large number of exosome-like structures with diameters of 50-150 nm were found in the calcified cartilage together with decreased expression of matrix Gla protein (MGP) and increased expression of CD63, tissue-nonspecific alkaline phosphatase (TNAP) and nucleotide pyrophosphatase/phosphodiesterase-1 (NPP1). After FFSS stimulation, the number of exosomes secreted by chondrocytes and the numbers of calcified nodules were increased in cultured cells, and the protein levels of MGP, TNAP, and NPP1 in exosomes were changed. Inhibition of exosome formation, TNAP, and NPP1 or supplementation with exogenous MGP effectively alleviated FFSS-induced chondrocyte calcification. Local injection of GW4869, the exosome inhibitor, alleviated TMJ OA-related cartilage degeneration and calcification in UAC rats. Local injection of exosomes obtained from chondrocytes stimulated by FFSS to the TMJs of normal rats induced cartilage degeneration and calcification similar to that in TMJ OA.ConclusionsAbnormal biomechanical loading leads to enhanced formation of chondrocyte-derived exosomes, in which promoters of calcification increased and inhibitors decreased, resulting in accelerating abnormal cartilage calcification in TMJ OA. The inhibition of degenerative chondrocyte-derived exosomes is expected to be a new way to prevent and treat TMJ OA.

Project description:Culture conditions that preserve a stable chondrocyte phenotype are desirable in cell-based cartilage repair to maximize efficacy and clinical outcome. This study investigates whether low-glucose conditions will preserve the chondrocyte phenotype during culture expansion. Articular chondrocytes were culture-expanded in media supplemented with either low (1 mM) or high (10 mM) glucose. The metabolic phenotype, reactive oxygen species generation, and mRNA expression of markers of differentiation or catabolism were assessed by reverse-transcription quantitative polymerase chain reaction after four population doublings (PDs) and subsequent tissue formation capacity determined using pellet cultures. Continuous monolayer culture was used to determine the population doubling limit. After expansion in monolayer for four PDs, chondrocytes expanded in low-glucose conditions exhibited higher expression of the differentiation markers SOX9 and COL2A1 and reduced expression of the catabolic metalloproteinase matrix metallopeptidase 13. When chondrocytes expanded in low glucose were cultured in micropellets, they consistently generated more cartilaginous extracellular matrix than those expanded in high glucose, as evaluated by wet weight, sulfated glycosaminoglycan content, and hydroxyproline assay for collagen content. The same pattern was observed whether high or low glucose was used during the pellet culture. During expansion, chondrocytes in high-glucose generated 50% more reactive oxygen species than low-glucose conditions, despite a lower dependence on oxidative phosphorylation for energy. Furthermore low-glucose cells exhibited >30% increased population doubling limit. These data suggests that low-glucose expansion conditions better preserve the expression of differentiation markers by chondrocytes and enhance their subsequent capacity to form cartilage in vitro. Therefore, low glucose levels should be considered for the expansion of chondrocytes intended for tissue engineering applications.

Dataset Information

Co-culture of mechanically injured cartilage with joint capsule tissue alters chondrocyte expression patterns and increases ADAMTS5 production.

Publications

Co-culture of mechanically injured cartilage with joint capsule tissue alters chondrocyte expression patterns and increases ADAMTS5 production.

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