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Sample records for hypoxia-cultured human chondrocytes

  1. ROCK inhibitor prevents the dedifferentiation of human articular chondrocytes

    SciTech Connect

    Matsumoto, Emi; Furumatsu, Takayuki; Kanazawa, Tomoko; Tamura, Masanori; Ozaki, Toshifumi

    2012-03-30

    Highlights: Black-Right-Pointing-Pointer ROCK inhibitor stimulates chondrogenic gene expression of articular chondrocytes. Black-Right-Pointing-Pointer ROCK inhibitor prevents the dedifferentiation of monolayer-cultured chondrocytes. Black-Right-Pointing-Pointer ROCK inhibitor enhances the redifferentiation of cultured chondrocytes. Black-Right-Pointing-Pointer ROCK inhibitor is useful for preparation of un-dedifferentiated chondrocytes. Black-Right-Pointing-Pointer ROCK inhibitor may be a useful reagent for chondrocyte-based regeneration therapy. -- Abstract: Chondrocytes lose their chondrocytic phenotypes in vitro. The Rho family GTPase ROCK, involved in organizing the actin cytoskeleton, modulates the differentiation status of chondrocytic cells. However, the optimum method to prepare a large number of un-dedifferentiated chondrocytes is still unclear. In this study, we investigated the effect of ROCK inhibitor (ROCKi) on the chondrogenic property of monolayer-cultured articular chondrocytes. Human articular chondrocytes were subcultured in the presence or absence of ROCKi (Y-27632). The expression of chondrocytic marker genes such as SOX9 and COL2A1 was assessed by quantitative real-time PCR analysis. Cellular morphology and viability were evaluated. Chondrogenic redifferentiation potential was examined by a pellet culture procedure. The expression level of SOX9 and COL2A1 was higher in ROCKi-treated chondrocytes than in untreated cells. Chondrocyte morphology varied from a spreading form to a round shape in a ROCKi-dependent manner. In addition, ROCKi treatment stimulated the proliferation of chondrocytes. The deposition of safranin O-stained proteoglycans and type II collagen was highly detected in chondrogenic pellets derived from ROCKi-pretreated chondrocytes. Our results suggest that ROCKi prevents the dedifferentiation of monolayer-cultured chondrocytes, and may be a useful reagent to maintain chondrocytic phenotypes in vitro for chondrocyte

  2. Morphological, genetic and phenotypic comparison between human articular chondrocytes and cultured chondrocytes.

    PubMed

    Mata-Miranda, Mónica Maribel; Martinez-Martinez, Claudia María; Noriega-Gonzalez, Jesús Emmanuel; Paredes-Gonzalez, Luis Enrique; Vázquez-Zapién, Gustavo Jesús

    2016-08-01

    Articular cartilage is an avascular and aneural tissue with limited capacity for regeneration. On large articular lesions, it is recommended to use regenerative medicine strategies, like autologous chondrocyte implantation. There is a concern about morphological changes that chondrocytes suffer once they have been isolated and cultured. Due to the fact that there is little evidence that compares articular cartilage chondrocytes with cultured chondrocytes, in this research we proposed to obtain chondrocytes from human articular cartilage, compare them with themselves once they have been cultured and characterize them through genetic, phenotypic and morphological analysis. Knee articular cartilage samples of 10 mm were obtained, and each sample was divided into two fragments; a portion was used to determine gene expression, and from the other portion, chondrocytes were obtained by enzymatic disaggregation, in order to be cultured and expanded in vitro. Subsequently, morphological, genetic and phenotypic characteristics were compared between in situ (articular cartilage) and cultured chondrocytes. Obtained cultured chondrocytes were rounded in shape, possessing a large nucleus with condensed chromatin and a clear cytoplasm; histological appearance was quite similar to typical chondrocyte. The expression levels of COL2A1 and COL10A1 genes were higher in cultured chondrocytes than in situ chondrocytes; moreover, the expression of COL1A1 was almost undetectable on cultured chondrocytes; likewise, COL2 and SOX9 proteins were detected by immunofluorescence. We concluded that chondrocytes derived from adult human cartilage cultured for 21 days do not tend to dedifferentiate, maintaining their capacity to produce matrix and also retaining their synthesis capacity and morphology. PMID:27094849

  3. Expression of Angiotensin II Receptor-1 in Human Articular Chondrocytes

    PubMed Central

    Kawakami, Yuki; Matsuo, Kosuke; Murata, Minako; Yudoh, Kazuo; Nakamura, Hiroshi; Shimizu, Hiroyuki; Beppu, Moroe; Inaba, Yutaka; Saito, Tomoyuki; Kato, Tomohiro; Masuko, Kayo

    2012-01-01

    Background. Besides its involvement in the cardiovascular system, the renin-angiotensin-aldosterone (RAS) system has also been suggested to play an important role in inflammation. To explore the role of this system in cartilage damage in arthritis, we investigated the expression of angiotensin II receptors in chondrocytes. Methods. Articular cartilage was obtained from patients with osteoarthritis, rheumatoid arthritis, and traumatic fractures who were undergoing arthroplasty. Chondrocytes were isolated and cultured in vitro with or without interleukin (IL-1). The expression of angiotensin II receptor types 1 (AT1R) and 2 (AT2R) mRNA by the chondrocytes was analyzed using reverse transcription-polymerase chain reaction (RT-PCR). AT1R expression in cartilage tissue was analyzed using immunohistochemistry. The effect of IL-1 on AT1R/AT2R expression in the chondrocytes was analyzed by quantitative PCR and flow cytometry. Results. Chondrocytes from all patient types expressed AT1R/AT2R mRNA, though considerable variation was found between samples. Immunohistochemical analysis confirmed AT1R expression at the protein level. Stimulation with IL-1 enhanced the expression of AT1R/AT2R mRNA in OA and RA chondrocytes. Conclusions. Human articular chondrocytes, at least partially, express angiotensin II receptors, and IL-1 stimulation induced AT1R/AT2R mRNA expression significantly. PMID:23346400

  4. Membrane channel gene expression in human costal and articular chondrocytes

    PubMed Central

    Asmar, A.; Barrett-Jolley, R.; Werner, A.; Kelly, R.; Stacey, M.

    2016-01-01

    ABSTRACT Chondrocytes are the uniquely resident cells found in all types of cartilage and key to their function is the ability to respond to mechanical loads with changes of metabolic activity. This mechanotransduction property is, in part, mediated through the activity of a range of expressed transmembrane channels; ion channels, gap junction proteins, and porins. Appropriate expression of ion channels has been shown essential for production of extracellular matrix and differential expression of transmembrane channels is correlated to musculoskeletal diseases such as osteoarthritis and Albers-Schönberg. In this study we analyzed the consistency of gene expression between channelomes of chondrocytes from human articular and costal (teenage and fetal origin) cartilages. Notably, we found 14 ion channel genes commonly expressed between articular and both types of costal cartilage chondrocytes. There were several other ion channel genes expressed only in articular (6 genes) or costal chondrocytes (5 genes). Significant differences in expression of BEST1 and KCNJ2 (Kir2.1) were observed between fetal and teenage costal cartilage. Interestingly, the large Ca2+ activated potassium channel (BKα, or KCNMA1) was very highly expressed in all chondrocytes examined. Expression of the gap junction genes for Panx1, GJA1 (Cx43) and GJC1 (Cx45) was also observed in chondrocytes from all cartilage samples. Together, this data highlights similarities between chondrocyte membrane channel gene expressions in cells derived from different anatomical sites, and may imply that common electrophysiological signaling pathways underlie cellular control. The high expression of a range of mechanically and metabolically sensitive membrane channels suggest that chondrocyte mechanotransduction may be more complex than previously thought. PMID:27116676

  5. Matrix Metalloproteinase-9 Production by Immortalized Human Chondrocyte Lines

    PubMed Central

    Malemud, Charles J.; Meszaros, Evan C.; Wylie, Meredith A.; Dahoud, Wissam; Skomorovska-Prokvolit, Yelenna; Mesiano, Sam

    2016-01-01

    We reported at the Keynote Forum of Immunology Summit-2015 that recombinant human (rh) TNF-α or rhIL-6 stimulated production of matrix metalloproteinase-9 (MMP-9) in the T/C28a2 and C-28/I2 human immortalized chondrocyte cell lines. Furthermore, we reported that tocilizumab (TCZ), a fully humanized monoclonal antibody which neutralizes IL-6-mediated signaling, inhibited the rhIL-6-mediated increase in the production of MMP-9. IL-6 is also a known activator of the JAK/STAT signaling pathway. In that regard, we evaluated the effect of rhIL-6 on total and phosphorylated Signal Transducer and Activator of Transcription by these chondrocyte lines which showed that whereas STAT3 was constitutively phosphorylated in T/C28a2 chondrocytes, rhIL-6 activated STAT3 in C-28/I2 chondrocytes. The finding that rhIL-6 increased the production of MMP-9 by human immortalized chondrocyte cell lines may have important implications with respect to the destruction of articular cartilage in rheumatoid arthritis and osteoarthritis. Thus, the markedly elevated level of IL-6 in rheumatoid arthritis and osteoarthritis sera and synovial fluid would be expected to generate significant MMP-9 to cause the degradation of articular cartilage extracellular matrix proteins. The finding that TCZ suppressed rhIL-6-mediated MMP-9 production suggests that TCZ, currently employed in the medical therapy of rheumatoid arthritis, could be considered as a drug for osteoarthritis.

  6. Telomere erosion and senescence in human articular cartilage chondrocytes.

    PubMed

    Martin, J A; Buckwalter, J A

    2001-04-01

    Aging and the degeneration of articular cartilage in osteoarthritis are distinct processes, but a strong association exists between age and the incidence and prevalence of osteoarthritis. We hypothesized that this association is due to in vivo replicative senescence, which causes age-related declines in the ability of chondrocytes to maintain articular cartilage. For this hypothesis to be tested, senescence-associated markers were measured in human articular chondrocytes from donors ranging in age from 1 to 87 years. These measures included in situ staining for senescence-associated beta-galactosidase activity, (3)H-thymidine incorporation assays for mitotic activity, and Southern blots for telomere length determinations. We found that senescence-associated beta-galactosidase activity increased with age, whereas both mitotic activity and mean telomere length declined. These findings indicate that chondrocyte replicative senescence occurs in vivo and support the hypothesis that the association between osteoarthritis and aging is due in part to replicative senescence. The data also imply that transplantation procedures performed to restore damaged articular surfaces could be limited by the inability of older chondrocytes to form new cartilage after transplantation. PMID:11283188

  7. Human Articular Chondrocytes Express Multiple Gap Junction Proteins

    PubMed Central

    Mayan, Maria D.; Carpintero-Fernandez, Paula; Gago-Fuentes, Raquel; Martinez-de-Ilarduya, Oskar; Wang, Hong-Zhang; Valiunas, Virginijus; Brink, Peter; Blanco, Francisco J.

    2014-01-01

    Osteoarthritis (OA) is the most common joint disease and involves progressive degeneration of articular cartilage. The aim of this study was to investigate if chondrocytes from human articular cartilage express gap junction proteins called connexins (Cxs). We show that human chondrocytes in tissue express Cx43, Cx45, Cx32, and Cx46. We also find that primary chondrocytes from adults retain the capacity to form functional voltage-dependent gap junctions. Immunohistochemistry experiments in cartilage from OA patients revealed significantly elevated levels of Cx43 and Cx45 in the superficial zone and down through the next approximately 1000 μm of tissue. These zones corresponded with regions damaged in OA that also had high levels of proliferative cell nuclear antigen. An increased number of Cxs may help explain the increased proliferation of cells in clusters that finally lead to tissue homeostasis loss. Conversely, high levels of Cxs in OA cartilage reflect the increased number of adjacent cells in clusters that are able to interact directly by gap junctions as compared with hemichannels on single cells in normal cartilage. Our data provide strong evidence that OA patients have a loss of the usual ordered distribution of Cxs in the damaged zones and that the reductions in Cx43 levels are accompanied by the loss of correct Cx localization in the nondamaged areas. PMID:23416160

  8. Comparative study of the chondrogenic potential of human bone marrow stromal cells, neonatal chondrocytes and adult chondrocytes

    SciTech Connect

    Saha, Sushmita; Kirkham, Jennifer; Wood, David; Curran, Stephen; Yang, Xuebin

    2010-10-22

    Research highlights: {yields} This study has characterised three different cell types under conditions similar to those used for autologous chondrocyte implantation (ACI) for applications in cartilage repair/regeneration. {yields} Compared for the first time the chondrogenic potential of neonatal chondrocytes with human bone marrow stromal cells (HBMSCs) and adult chondrocytes. {yields} Demonstrated that adult chondrocytes hold greatest potential for use in ACI based on their higher proliferation rates, lower alkaline phosphatise activity and enhanced expression of chondrogenic genes. {yields} Demonstrated the need for chondroinduction as a necessary pre-requisite to efficient chondrogenesis in vitro and, by extrapolation, for cell based therapy (e.g. ACI or cartilage tissue engineering). -- Abstract: Cartilage tissue engineering is still a major clinical challenge with optimisation of a suitable source of cells for cartilage repair/regeneration not yet fully addressed. The aims of this study were to compare and contrast the differences in chondrogenic behaviour between human bone marrow stromal cells (HBMSCs), human neonatal and adult chondrocytes to further our understanding of chondroinduction relative to cell maturity and to identify factors that promote chondrogenesis and maintain functional homoeostasis. Cells were cultured in monolayer in either chondrogenic or basal medium, recapitulating procedures used in existing clinical procedures for cell-based therapies. Cell doubling time, morphology and alkaline phosphatase specific activity (ALPSA) were determined at different time points. Expression of chondrogenic markers (SOX9, ACAN and COL2A1) was compared via real time polymerase chain reaction. Amongst the three cell types studied, HBMSCs had the highest ALPSA in basal culture and lowest ALPSA in chondrogenic media. Neonatal chondrocytes were the most proliferative and adult chondrocytes had the lowest ALPSA in basal media. Gene expression analysis revealed

  9. Effects of introducing cultured human chondrocytes into a human articular cartilage explant model.

    PubMed

    Secretan, Charles; Bagnall, Keith M; Jomha, Nadr M

    2010-02-01

    Articular cartilage (AC) heals poorly and effective host-tissue integration after reconstruction is a concern. We have investigated the ability of implanted chondrocytes to attach at the site of injury and to be incorporated into the decellularized host matrix adjacent to a defect in an in vitro human explant model. Human osteochondral dowels received a standardized injury, were seeded with passage 3 chondrocytes labelled with PKH 26 and compared with two control groups. All dowels were cultured in vitro, harvested at 0, 7, 14 and 28 days and assessed for chondrocyte adherence and migration into the region of decellularized tissue adjacent to the defects. Additional evaluation included cell viability, general morphology and collagen II production. Seeded chondrocytes adhered to the standardized defect and areas of lamina splendens disruption but did not migrate into the adjacent acellular region. A difference was noted in viable-cell density between the experimental group and one control group. A thin lattice-like network of matrix surrounded the seeded chondrocytes and collagen II was present. The results indicate that cultured human chondrocytes do indeed adhere to regions of AC matrix injury but do not migrate into the host tissue, despite the presence of viable cells. This human explant model is thus an effective tool for studying the interaction of implanted cells and host tissue. PMID:20012649

  10. RECK Is Up-Regulated and Involved in Chondrocyte Cloning in Human Osteoarthritic Cartilage

    PubMed Central

    Kimura, Tokuhiro; Okada, Aiko; Yatabe, Taku; Okubo, Masashi; Toyama, Yoshiaki; Noda, Makoto; Okada, Yasunori

    2010-01-01

    Reversion-inducing cysteine-rich protein with Kazal motifs (RECK) is a membrane-anchored matrix metalloproteinase regulator, but its functions in cartilage are not fully understood. The aim of the present study was to examine the expression and functions of RECK in human osteoarthritic (OA) cartilage. Quantitative RT-PCR indicated that the expression level of RECK is significantly higher in OA cartilage than in normal cartilage. By immunohistochemical analysis, RECK was localized to chondrocytes in OA cartilage, and the immunoreactivity directly correlated with the Mankin score and degree of chondrocyte cloning and proliferation. In cultured OA chondrocytes, RECK was expressed on the cell surface by glycosylphosphatidylinositol anchoring. The expression was stimulated by insulin-like growth factor-1 and suppressed by interleukin-1 and tumor necrosis factor-α. Down-regulation of RECK by small interfering RNA showed reduced spreading and smaller focal adhesions in the chondrocytes. Chondrocyte migration in a monolayer wounding assay was increased by down-regulation of RECK and inhibited by RECK overexpression in an matrix metalloproteinase activity-dependent manner. On the other hand, chondrocyte proliferation was suppressed by RECK silencing, and this was associated with reduced phosphorylation of focal adhesion kinase and extracellular signal-regulated kinase, whereas the proliferation was enhanced by RECK overexpression. These data are the first to demonstrate that RECK is up-regulated in human OA cartilage and suggest that RECK plays a role in chondrocyte cloning probably through suppression and promotion of chondrocyte migration and proliferation, respectively. PMID:20395433

  11. Chondrocyte number and proteoglycan synthesis in the aging and osteoarthritic human articular cartilage

    PubMed Central

    Bobacz, K; Erlacher, L; Smolen, J; Soleiman, A; Graninger, W

    2004-01-01

    Objective: To correlate the number of chondrocytes in healthy and osteoarthritic human articular cartilage with age, and to evaluate the influence of donor age on total proteoglycan synthesis. Methods: Chondrocytes were isolated from human articular cartilage derived from hip joints with and without osteoarthritic lesions. The cell number was normalised to cartilage sample wet weight. In addition, the influence of age on chondrocyte numbers was assessed histomorphometrically. Chondrocytes were grown as monolayer cultures for seven days in a chemically defined serum-free basal medium. Total proteoglycan synthesis was measured by [35S]sulphate incorporation into newly synthesised macromolecules. Results: Chondrocyte numbers in healthy cartilage decreased significantly with advancing age (r = –0.69, p<0.0001). In contrast to healthy specimens, chondrocyte numbers were decreased in osteoarthritic cartilage irrespective of and unrelated to age, and differed markedly, by an average of 38%, from the cell numbers found in healthy individuals (p<0.0001). Regarding synthesis of matrix macromolecules, no dependence on patients' age, either in healthy or in osteoarthritic specimens, could be observed. Conclusions: Under the experimental conditions employed, chondrocytes from healthy and osteoarthritic joints synthesised comparable amounts of cartilage macromolecules, independent of age or underlying osteoarthritic disease. Thus the decrease in chondrocyte number in aging and osteoarthritic joints could be a crucial factor in limiting tissue replenishment. PMID:15547085

  12. Hyaluronan production and chondrogenic properties of primary human chondrocyte on gelatin based hematostatic spongostan scaffold

    PubMed Central

    2012-01-01

    Background Autologous chondrocyte transplantation is a promising technique for treatment of cartilage defects. Three dimensional chondrocyte cultures on a scaffold are widely used to retain the chondrogenic phenotype. Using a biodegradable gelatin scaffold is one option for the cell delivery system, but molecular and histological studies of the method have not yet been done. Methods We evaluated the chondrogenic property of the primary human chondrocyte on a gelatin scaffold as compared to a collagen scaffold over a period of 21 days. We examined the production of glycosaminoglycan by quantitative and histological analysis. Gene expression of cartilage-associated molecules was assessed by quantitative RT-PCR. Results The gelatin scaffold showed the ability to promote chondrocyte expansion, chondrogenic phenotype retention at molecular and mRNA levels. Conclusions This scaffold is thus suitable for use as an in vitro model for chondrocyte 3D culture. PMID:23253362

  13. Effects of telomerase and viral oncogene expression on the in vitro growth of human chondrocytes.

    PubMed

    Martin, James A; Mitchell, Calista J; Klingelhutz, Aloysius J; Buckwalter, Joseph A

    2002-02-01

    Senescent chondrocytes accumulate with aging in articular cartilage, a process that interferes with cartilage homeostasis and increases the risk of cartilage degeneration. We showed previously that chondrocyte telomere length declines with donor age, which suggests that the aging process is telomere dependent. From these results we hypothesized that telomerase should delay the onset of senescence in cultured chondrocytes. Population doubling limits (PDL) were determined for chondrocytes expressing telomerase. We found that telomerase alone did not extend PDL beyond controls that senesced after 25 population doublings. The human papillomavirus 16 oncogenes E6 and E7 were transduced into the same cell population to investigate this telomere-independent form of senescence further. Chondrocytes expressing E6 and E7 grew longer than the telomerase cDNA (hTERT) cells but still senesced at 55 population doublings. In contrast, chondrocytes expressing telomerase with E6 and E7 grew vigorously past 100 population doublings. We conclude that although telomerase is necessary for the indefinite extension of chondrocyte life span, telomere-independent senescence limits PDL in vitro and may play a role in the age-related accumulation of senescent chondrocytes in vivo. PMID:11818423

  14. Effect of Laminin-A4 inhibition on cluster formation of human osteoarthritic chondrocytes.

    PubMed

    Moazedi-Fuerst, Florentine C; Gruber, Gerald; Stradner, Martin H; Guidolin, Diego; Jones, Jonathan C; Bodo, Koppany; Wagner, Karin; Peischler, Daniela; Krischan, Verena; Weber, Jennifer; Sadoghi, Patrick; Glehr, Mathias; Leithner, Andreas; Graninger, Winfried B

    2016-03-01

    Formation of chondrocyte clusters is not only a morphological sign of osteoarthritis but it is also observed in cell culture. Active locomotion of chondrocytes is controlled by integrins in vitro. Integrins bind to Laminin-A4 (LAMA4), a protein that is highly expressed in vivo in clusters of hypertrophic chondrocytes. We tested if LAMA4 is relevant for cluster formation. Human chondrocytes were cultured in a 2D matrigel model and treated with different concentrations of a monoclonal inhibitory anti-LAMA4-antibody. Migration and cluster formation was analysed using live cell imaging technique. Full genome gene expression analysis was performed to assess the effect of LAMA4 inhibition. The data set were screened for genes relevant to cell motility. F-actin staining was performed to document cytoskeletal changes. Anti-LAMA4 treatment significantly reduced the rate of cluster formation in human chondrocytes. Cells changed their surface morphology and exhibited fewer protrusions. Expression of genes associated with cellular motility and migration was affected by anti-LAMA4 treatment. LAMA4-integrin signalling affects chondrocyte morphology and gene expression in vitro, thereby contributing to cluster formation in human osteoarthritic chondrocytes. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:419-426, 2016. PMID:26295200

  15. Comparative potential of juvenile and adult human articular chondrocytes for cartilage tissue formation in three-dimensional biomimetic hydrogels.

    PubMed

    Smeriglio, Piera; Lai, Janice H; Dhulipala, Lakshmi; Behn, Anthony W; Goodman, Stuart B; Smith, Robert L; Maloney, William J; Yang, Fan; Bhutani, Nidhi

    2015-01-01

    Regeneration of human articular cartilage is inherently limited and extensive efforts have focused on engineering the cartilage tissue. Various cellular sources have been studied for cartilage tissue engineering including adult chondrocytes, and embryonic or adult stem cells. Juvenile chondrocytes (from donors below 13 years of age) have recently been reported to be a promising cell source for cartilage regeneration. Previous studies have compared the potential of adult and juvenile chondrocytes or adult and osteoarthritic (OA) chondrocytes. To comprehensively characterize the comparative potential of young, old, and diseased chondrocytes, here we examined cartilage formation by juvenile, adult, and OA chondrocytes in three-dimensional (3D) biomimetic hydrogels composed of poly(ethylene glycol) and chondroitin sulfate. All three human articular chondrocytes were encapsulated in the 3D biomimetic hydrogels and cultured for 3 or 6 weeks to allow maturation and extracellular matrix formation. Outcomes were analyzed using quantitative gene expression, immunofluorescence staining, biochemical assays, and mechanical testing. After 3 and 6 weeks, juvenile chondrocytes showed a greater upregulation of chondrogenic gene expression than adult chondrocytes, while OA chondrocytes showed a downregulation. Aggrecan and type II collagen deposition and glycosaminoglycan accumulation were high for juvenile and adult chondrocytes but not for OA chondrocytes. Similar trend was observed in the compressive moduli of the cartilage constructs generated by the three different chondrocytes. In conclusion, the juvenile, adult and OA chondrocytes showed differential responses in the 3D biomimetic hydrogels. The 3D culture model described here may also provide a useful tool to further study the molecular differences among chondrocytes from different stages, which can help elucidate the mechanisms for age-related decline in the intrinsic capacity for cartilage repair. PMID:25054343

  16. Chitosan Enriched Three-Dimensional Matrix Reduces Inflammatory and Catabolic Mediators Production by Human Chondrocytes

    PubMed Central

    Oprenyeszk, Frederic; Sanchez, Christelle; Dubuc, Jean-Emile; Maquet, Véronique; Henrist, Catherine; Compère, Philippe; Henrotin, Yves

    2015-01-01

    This in vitro study investigated the metabolism of human osteoarthritic (OA) chondrocytes encapsulated in a spherical matrix enriched of chitosan. Human OA chondrocytes were encapsulated and cultured for 28 days either in chitosan-alginate beads or in alginate beads. The beads were formed by slowly passing dropwise either the chitosan 0.6%–alginate 1.2% or the alginate 1.2% solution through a syringe into a 102 mM CaCl2 solution. Beads were analyzed histologically after 28 days. Interleukin (IL)-6 and -8, prostaglandin (PG) E2, matrix metalloproteinases (MMPs), hyaluronan and aggrecan were quantified directly in the culture supernatant by specific ELISA and nitric oxide (NO) by using a colorimetric method based on the Griess reaction. Hematoxylin and eosin staining showed that chitosan was homogeneously distributed through the matrix and was in direct contact with chondrocytes. The production of IL-6, IL-8 and MMP-3 by chondrocytes significantly decreased in chitosan-alginate beads compared to alginate beads. PGE2 and NO decreased also significantly but only during the first three days of culture. Hyaluronan and aggrecan production tended to increase in chitosan-alginate beads after 28 days of culture. Chitosan-alginate beads reduced the production of inflammatory and catabolic mediators by OA chondrocytes and tended to stimulate the synthesis of cartilage matrix components. These particular effects indicate that chitosan-alginate beads are an interesting scaffold for chondrocytes encapsulation before transplantation to repair cartilage defects. PMID:26020773

  17. Detecting new microRNAs in human osteoarthritic chondrocytes identifies miR-3085 as a human, chondrocyte-selective, microRNA

    PubMed Central

    Crowe, N.; Swingler, T.E.; Le, L.T.T.; Barter, M.J.; Wheeler, G.; Pais, H.; Donell, S.T.; Young, D.A.; Dalmay, T.; Clark, I.M.

    2016-01-01

    Summary Objective To use deep sequencing to identify novel microRNAs (miRNAs) in human osteoarthritic cartilage which have a functional role in chondrocyte phenotype or function. Design A small RNA library was prepared from human osteoarthritic primary chondrocytes using in-house adaptors and analysed by Illumina sequencing. Novel candidate miRNAs were validated by northern blot and qRT-PCR. Expression was measured in cartilage models. Targets of novel candidates were identified by microarray and computational analysis, validated using 3′-UTR-luciferase reporter plasmids. Protein levels were assessed by western blot and functional analysis by cell adhesion. Results We identified 990 known miRNAs and 1621 potential novel miRNAs in human osteoarthritic chondrocytes, 60 of the latter were expressed in all samples assayed. MicroRNA-140-3p was the most highly expressed microRNA in osteoarthritic cartilage. Sixteen novel candidate miRNAs were analysed further, of which six remained after northern blot analysis. Three novel miRNAs were regulated across models of chondrogenesis, chondrocyte differentiation or cartilage injury. One sequence (novel #11), annotated in rodents as microRNA-3085-3p, was preferentially expressed in cartilage, dependent on chondrocyte differentiation and, in man, is located in an intron of the cartilage-expressed gene CRTAC-1. This microRNA was shown to target the ITGA5 gene directly (which encodes integrin alpha5) and inhibited adhesion to fibronectin (dependent on alpha5beta1 integrin). Conclusion Deep sequencing has uncovered many potential microRNA candidates expressed in human cartilage. At least three of these show potential functional interest in cartilage homeostasis and osteoarthritis (OA). Particularly, novel #11 (microRNA-3085-3p) which has been identified for the first time in man. PMID:26497608

  18. Expression of mutant cartilage oligomeric matrix protein in human chondrocytes induces the pseudoachondroplasia phenotype.

    PubMed

    Merritt, Thomas M; Alcorn, Joseph L; Haynes, Richard; Hecht, Jacqueline T

    2006-04-01

    Over 70 mutations in the cartilage oligomeric matrix protein (COMP), a large extracellular pentameric glycoprotein synthesized by chondrocytes, have been identified as causing two skeletal dysplasias: multiple epiphyseal dysplasia (MED/EDM1), and a dwarfing condition, pseudoachondroplasia (PSACH). These mutations induce misfolding of intracellular COMP, resulting in retention of the protein in the rough endoplasmic reticulum (rER) of chondrocytes. This accumulation of COMP in the rER creates the phenotypic enlarged rER cisternae in the cells, which is believed to compromise chondrocyte function and eventually cause cell death. To study the molecular mechanisms involved with the disease, we sought to develop an in vitro model that recapitulates the PSACH phenotype. Normal human chondrocytes were transfected with wildtype (wt-) COMP or with mutant COMP (D469del; mt-) recombinant adenoviruses and grown in a nonattachment redifferentiating culture system that provides an environment allowing formation of a differentiated chondrocyte nodule. Visualization of normal cells expressing COMP suggested the hallmarks of the PSACH phenotype. Mutant COMP expressed in normal cells was retained in enlarged rER cisternae, which also retained IX collagen (COL9) and matrilin-3 (MATN3). Although these proteins were secreted normally into the ECM of the wt-COMP nodules, reduced secretion of these proteins was observed in nodules composed of cells transfected with mt-COMP. The findings complement those found in chondrocytes from PSACH patient growth plates. This new model system allows for production of PSACH chondrocyte pathology in normal costochondral chondrocytes and can be used for future mechanistic and potential gene therapy studies. PMID:16514635

  19. Cryopreservation Effect on Proliferative and Chondrogenic Potential of Human Chondrocytes Isolated from Superficial and Deep Cartilage

    PubMed Central

    Muiños-López, Emma; Rendal-Vázquez, Mª Esther; Hermida-Gómez, Tamara; Fuentes-Boquete, Isaac; Díaz-Prado, Silvia; Blanco, Francisco J

    2012-01-01

    Objectives: To compare the proliferative and chondrogenic potential of fresh and frozen chondrocytes isolated from superficial and deep articular cartilage biopsies. Materials and Methodology: The study included 12 samples of fresh and frozen healthy human knee articular cartilage. Cell proliferation was tested at 3, 6 and 9 days. Studies of mRNA quantification, protein expression and immunofluorescence for proliferation and chondrogenic markers were performed. Results: Stimulation of fresh and frozen chondrocytes from both superficial and deep cartilage with fetal bovine serum produced an increase in the proliferative capacity compared to the non-stimulated control group. In the stimulated fresh cells group, the proliferative capacity of cells from the deep biopsy was greater than that from cells from the superficial biopsy (0.046 vs 0.028, respectively, p<0.05). There was also a significant difference between the proliferative capacity of superficial zone fresh (0.028) and frozen (0.051) chondrocytes (p<0.05). CCND1 mRNA and protein expression levels, and immunopositivity for Ki67 revealed a higher proliferative capacity for fresh articular chondrocytes from deep cartilage. Regarding the chondrogenic potential, stimulated fresh cells showed higher SOX9 and Col II expression in chondrocytes from deep than from superficial zone (p<0.05, T student test). Conclusions: The highest rate of cell proliferation and chondrogenic potential of fresh chondrocytes was found in cells obtained from deep cartilage biopsies, whereas there were no statistically significant differences in proliferative and chondrogenic capacity between biopsy origins with frozen chondrocytes. These results indicate that both origin and cryopreservation affect the proliferative and chondrogenic potential of chondrocytes. PMID:22523526

  20. Curcumin synergizes with resveratrol to stimulate the MAPK signaling pathway in human articular chondrocytes in vitro.

    PubMed

    Shakibaei, Mehdi; Mobasheri, Ali; Buhrmann, Constanze

    2011-05-01

    The mitogen-activated protein kinase (MAPK) pathway is stimulated in differentiated chondrocytes and is an important signaling cascade for chondrocyte differentiation and survival. Pro-inflammatory cytokines such as interleukin 1β (IL-1β) play important roles in the pathogenesis of osteoarthritis (OA) and rheumatoid arthritis (RA). In this study, we investigated whether curcumin and resveratrol can synergistically inhibit the catabolic effects of IL-1β, specifically the inhibition of the MAPK and subsequent apoptosis in human articular chondrocytes. Chondrocytes were either left untreated or treated with 10 ng/ml IL-1β or 1 μM U0126, a specific inhibitor of MAPK pathway alone for the indicated time periods or pre-treated with 10 μM curcumin, 10 μM resveratrol or 10 μM resveratrol and 10 μM curcumin for 4 h followed by co-treatment with 10 ng/ml IL-1β or 1 μM U0126 and 10 μM resveratrol, 10 μM curcumin or 10 μM resveratrol and 10 μM curcumin for the indicated time periods. Cultures were evaluated by immunoblotting and transmission electron microscopy. Incubation of chondrocytes with IL-1β resulted in induction of apoptosis, downregulation of β1-integrins and the extracellular signal-regulated kinase 1/2 (Erk1/2). Interestingly, U0126 induced apoptosis and blocked the above-mentioned proteins in a similar way to IL-1β. Furthermore, curcumin and resveratrol inhibited IL-1β- or U0126-induced apoptosis and downregulation of β1-integrins and Erk1/2 in human articular chondrocytes. These results suggest that combining these two natural compounds activates MEK/Erk signaling, a pathway that is involved in the maintenance of chondrocyte differentiation and survival. PMID:21484156

  1. Single cell confocal Raman spectroscopy of human osteoarthritic chondrocytes: a preliminary study.

    PubMed

    Kumar, Rajesh; Singh, Gajendra P; Grønhaug, Kirsten M; Afseth, Nils K; de Lange Davies, Catharina; Drogset, Jon O; Lilledahl, Magnus B

    2015-01-01

    A great deal of effort has been focused on exploring the underlying molecular mechanism of osteoarthritis (OA) especially at the cellular level. We report a confocal Raman spectroscopic investigation on human osteoarthritic chondrocytes. The objective of this investigation is to identify molecular features and the stage of OA based on the spectral signatures corresponding to bio-molecular changes at the cellular level in chondrocytes. In this study, we isolated chondrocytes from human osteoarthritic cartilage and acquired Raman spectra from single cells. Major spectral differences between the cells obtained from different International Cartilage Repair Society (ICRS) grades of osteoarthritic cartilage were identified. During progression of OA, a decrease in protein content and an increase in cell death were observed from the vibrational spectra. Principal component analysis and subsequent cross-validation was able to associate osteoarthritic chondrocytes to ICRS Grade I, II and III with specificity 100.0%, 98.1%, and 90.7% respectively, while, sensitivity was 98.6%, 82.8%, and 97.5% respectively. The overall predictive efficiency was 92.2%. Our pilot study encourages further use of Raman spectroscopy as a noninvasive and label free technique for revealing molecular features associated with osteoarthritic chondrocytes. PMID:25918938

  2. A practical way to prepare primer human chondrocyte culture.

    PubMed

    Isyar, Mehmet; Yilmaz, Ibrahim; Yasar Sirin, Duygu; Yalcin, Sercan; Guler, Olcay; Mahirogullari, Mahir

    2016-09-01

    Biological cartilage repair is one of the most important targets for orthopedic surgeons currently. For this purpose, it is mandatory to know how to prepare a chondrocyte culture. In this study, our purpose was to introduce a method enabling orthopedic surgeons to practice their knowledge and skills on molecular experimental setup at cellular level, based on our experiences from previous pilot studies. Thus, we believe it will encourage orthopedic surgeons. PMID:27408489

  3. Human fetal and adult chondrocytes. Effect of insulinlike growth factors I and II, insulin, and growth hormone on clonal growth.

    PubMed Central

    Vetter, U; Zapf, J; Heit, W; Helbing, G; Heinze, E; Froesch, E R; Teller, W M

    1986-01-01

    Clonal proliferation of freshly isolated human fetal chondrocytes and adult chondrocytes in response to human insulinlike growth factors I and II (IGF I, IGF II), human biosynthetic insulin, and human growth hormone (GH) was assessed. IGF I (25 ng/ml) stimulated clonal growth of fetal chondrocytes (54 +/- 12 colonies/1,000 inserted cells, mean +/- 1 SD), but IGF II (25 ng/ml) was significantly more effective (106 +/- 12 colonies/1,000 inserted cells, P less than 0.05, unstimulated control: 14 +/- 4 colonies/1,000 inserted cells). In contrast, IGF I (25 ng/ml) was more effective in adult chondrocytes (42 +/- 6 colonies/1,000 inserted cells) than IGF II (25 ng/ml) (21 +/- 6 colonies/1,000 inserted cells; P less than 0.05, unstimulated control: 6 +/- 3 colonies/1,000 inserted cells). GH and human biosynthetic insulin did not affect clonal growth of fetal or adult chondrocytes. The clonal growth pattern of IGF-stimulated fetal and adult chondrocytes was not significantly changed when chondrocytes were first grown in monolayer culture, harvested, and then inserted in the clonal culture system. However, the adult chondrocytes showed a time-dependent decrease of stimulation of clonal growth by IGF I and II. This was not true for fetal chondrocytes. The results are compatible with the concept that IGF II is a more potent stimulant of clonal growth of chondrocytes during fetal life, whereas IGF I is more effective in stimulating clonal growth of chondrocytes during postnatal life. Images PMID:3519682

  4. Differences in cartilage-forming capacity of expanded human chondrocytes from ear and nose and their gene expression profiles.

    PubMed

    Hellingman, Catharine A; Verwiel, Eugène T P; Slagt, Inez; Koevoet, Wendy; Poublon, René M L; Nolst-Trenité, Gilbert J; Baatenburg de Jong, Robert J; Jahr, Holger; van Osch, Gerjo J V M

    2011-01-01

    The aim of this study was to evaluate the potential of culture-expanded human auricular and nasoseptal chondrocytes as cell source for regeneration of stable cartilage and to analyze the differences in gene expression profile of expanded chondrocytes from these specific locations. Auricular chondrocytes in monolayer proliferated less and more slowly (two passages took 26.7 ± 2.1 days and were reached in 4.37 ± 0.30 population doublings) than nasoseptal chondrocytes (19.3 ± 2.5 days; 5.45 ± 0.20 population doublings). However, auricular chondrocytes produced larger pellets with more cartilage-like matrix than nasoseptal chondrocytes (2.2 ± 0.71 vs. 1.7 ± 0.13 mm in diameter after 35 days of culture). Although the matrix formed by auricular and nasoseptal chondrocytes contained collagen X, it did not mineralize in an in vitro model or after in vivo subcutaneous implantation. A DNA microarray study on expanded auricular and nasoseptal chondrocytes from the same donors revealed 1,090 differentially expressed genes. No difference was observed in the expression of known markers of chondrogenic capacity (e.g., collagen II, FGFR3, BMP2, and ALK1). The most striking differences were that the auricular chondrocytes had a higher expression of anabolic growth factors BMP5 and IGF1, while matrix-degrading enzymes MMP13 and ADAMTS5 were higher expressed in nasoseptal chondrocytes. This might offer a possible explanation for the observed higher matrix production by auricular chondrocytes. Moreover, chondrocytes isolated from auricular or nasoseptal cartilage had specific gene expression profiles even after expansion. These differently expressed genes were not restricted to known characterization of donor site subtype (e.g., elastic), but were also related to developmental processes. PMID:21054934

  5. Microcontact printing of BMP-2 and its effect on human chondrocytes behavior

    NASA Astrophysics Data System (ADS)

    Pan, Chang-Jiang; Nie, Yu-Dong

    2010-01-01

    The present study is to investigate human chondrocytes behavior on microcontact printed bone morphogenetic protein-2 (BMP-2) lines on polystyrene (PS) surface. It was found that the cells aligned with BMP lines and expressed type II and VI collagen. The chondrocytes in vitro cultured on BMP lines were elongated, which resulted in altered cell morphology. Taking all these results into consideration, BMP-2 lines enhance cell adhesion, restrict spreading, and increase type II and VI collagen expression. The results represented in this study may be an approach to the problem of engineering reparative cartilage in vitro.

  6. The Knee Joint Loose Body as a Source of Viable Autologous Human Chondrocytes

    PubMed Central

    Melrose, J.

    2016-01-01

    Loose bodies are fragments of cartilage or bone present in the synovial fluid. In the present study we assessed if loose bodies could be used as a source of autologous human chondrocytes for experimental purposes. Histochemical examination of loose bodies and differential enzymatic digestions were undertaken, the isolated cells were cultured in alginate bead microspheres and immunolocalisations were undertaken for chondrogenic markers such as aggrecan, and type II collagen. Isolated loose body cells had high viability (≥90% viable), expressed chondrogenic markers (aggrecan, type II collagen) but no type I collagen. Loose bodies may be a useful source of autologous chondrocytes of high viability. PMID:27349321

  7. Diosgenin inhibits IL-1β-induced expression of inflammatory mediators in human osteoarthritis chondrocytes.

    PubMed

    Wang, Leisheng; Ma, Tian; Zheng, Yanpin; Lv, Shiqiao; Li, Yu; Liu, Shaoxian

    2015-01-01

    It is well known that the inflammatory cytokines play important roles in osteoarthritis (OA). Diosgenin is a steroidal saponin found in several plants including Solanum and Dioscorea species and possesses diverse biological activities including anti-inflammatory properties. However, the role of diosgenin in inflammatory responses in OA chondrocytes is still unclear. Therefore, in this study, we investigated the anti-inflammatory properties of diosgenin in human OA chondrocytes. We found that diosgenin inhibited the production of nitric oxide (NO) and prostaglandin E2 (PGE2) induced by interleukin-1-beta (IL-1β). Diosgenin significantly inhibited the IL-1β-stimulated expression of metalloproteinase-3 (MMP-3), MMP-13, inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) in human OA chondrocytes. In addition, diosgenin suppressed the degradation of IκB-α in IL-1β-induced human OA chondrocytes. Taken together, this study showed that diosgenin can effectively inhibit the IL-1β-induced expression of inflammatory mediators, suggesting that diosgenin may be a potential agent in the treatment of OA. PMID:26191174

  8. Diosgenin inhibits IL-1β-induced expression of inflammatory mediators in human osteoarthritis chondrocytes

    PubMed Central

    Wang, Leisheng; Ma, Tian; Zheng, Yanpin; Lv, Shiqiao; Li, Yu; Liu, Shaoxian

    2015-01-01

    It is well known that the inflammatory cytokines play important roles in osteoarthritis (OA). Diosgenin is a steroidal saponin found in several plants including Solanum and Dioscorea species and possesses diverse biological activities including anti-inflammatory properties. However, the role of diosgenin in inflammatory responses in OA chondrocytes is still unclear. Therefore, in this study, we investigated the anti-inflammatory properties of diosgenin in human OA chondrocytes. We found that diosgenin inhibited the production of nitric oxide (NO) and prostaglandin E2 (PGE2) induced by interleukin-1-beta (IL-1β). Diosgenin significantly inhibited the IL-1β-stimulated expression of metalloproteinase-3 (MMP-3), MMP-13, inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) in human OA chondrocytes. In addition, diosgenin suppressed the degradation of IκB-α in IL-1β-induced human OA chondrocytes. Taken together, this study showed that diosgenin can effectively inhibit the IL-1β-induced expression of inflammatory mediators, suggesting that diosgenin may be a potential agent in the treatment of OA. PMID:26191174

  9. Distinct horizontal patterns in the spatial organization of superficial zone chondrocytes of human joints

    PubMed Central

    Rolauffs, Bernd; Williams, James M.; Grodzinsky, Alan J.; Kuettner, Klaus E.; Cole, Ada A.

    2008-01-01

    A better understanding of the unique cellular and functional properties of the superficial zone of articular cartilage may aid current strategies in tissue engineering which attempts a layered design for the repair of cartilage lesions to avert or postpone the onset of osteoarthritis. However, data pertaining to the cellular organization of non-degenerated superficial zone of articular cartilage is not available for most human joints. The present study analyzed the arrangement of chondrocytes of non-degenerated human joints (shoulder, elbow, knee, and ankle) by using fluorescence microscopy of the superficial zone in a top-down view. The resulting horizontal chondrocyte arrangements were tested for randomness, homogeneity or a significant grouping via point pattern analysis and were correlated with the joint type in which they occurred. The present study demonstrated that human superficial chondrocytes occurred in four distinct patterns of strings, clusters, pairs or single chondrocytes. Those patterns represented a significant grouping (p<0.0001) with horizontal alignment. Each articular joint surface was dominated by only one of these four patterns (p<0.001). Specific patterns correlated with specific diarthrodial joint types (p<0.001). Further studies need to establish whether these organizational patterns are a consequence of their surrounding environment or whether they are linked to a functional purpose. PMID:18325787

  10. Distinct horizontal patterns in the spatial organization of superficial zone chondrocytes of human joints.

    PubMed

    Rolauffs, Bernd; Williams, James M; Grodzinsky, Alan J; Kuettner, Klaus E; Cole, Ada A

    2008-05-01

    A better understanding of the unique cellular and functional properties of the superficial zone of articular cartilage may aid current strategies in tissue engineering which attempts a layered design for the repair of cartilage lesions to avert or postpone the onset of osteoarthritis. However, data pertaining to the cellular organization of non-degenerated superficial zone of articular cartilage is not available for most human joints. The present study analyzed the arrangement of chondrocytes of non-degenerated human joints (shoulder, elbow, knee, and ankle) by using fluorescence microscopy of the superficial zone in a top-down view. The resulting horizontal chondrocyte arrangements were tested for randomness, homogeneity or a significant grouping via point pattern analysis and were correlated with the joint type in which they occurred. The present study demonstrated that human superficial chondrocytes occurred in four distinct patterns of strings, clusters, pairs or single chondrocytes. Those patterns represented a significant grouping (p < 0.0001) with horizontal alignment. Each articular joint surface was dominated by only one of these four patterns (p < 0.001). Specific patterns correlated with specific diarthrodial joint types (p < 0.001). Further studies need to establish whether these organizational patterns are a consequence of their surrounding environment or whether they are linked to a functional purpose. PMID:18325787

  11. Regulation of human mesenchymal stem cells differentiation into chondrocytes in extracellular matrix-based hydrogel scaffolds.

    PubMed

    Du, Mingchun; Liang, Hui; Mou, Chenchen; Li, Xiaoran; Sun, Jie; Zhuang, Yan; Xiao, Zhifeng; Chen, Bing; Dai, Jianwu

    2014-02-01

    To induce human mesenchymal stem cells (hMSCs) to differentiate into chondrocytes in three-dimensional (3D) microenvironments, we developed porous hydrogel scaffolds using the cartilage extracellular matrix (ECM) components of chondroitin sulfate (CS) and collagen (COL). The turbidity and viscosity experiments indicated hydrogel could form through pH-triggered co-precipitation when pH=2-3. Enzyme-linked immunosorbent assay (ELISA) confirmed the hydrogel scaffolds could controllably release growth factors as envisaged. Transforming growth factor-β (TGF-β) was released to stimulate hMSCs differentiation into chondrocytes; and then collagen binding domain-basic fibroblast growth factor (CBD-bFGF) was released to improve the differentiation and preserve the chondrocyte phenotype. In in vitro cell culture experiments, the differentiation processes were compared in different microenvironments: 2D culture in culture plate as control, 3D culture in the fabricated scaffolds without growth factors (CC), the samples with CBD-bFGF (CC-C), the samples with TGF-β (CC-T), the samples with CBD-bFGF/TGF-β (CC-CT). Real-time polymerase chain reaction (RT-PCR) revealed the hMSC marker genes of CD44 and CD105 decreased; at the same time the chondrocyte marker genes of collagen type II and aggrecan increased, especially in the CC-CT sample. Immunostaining results further confirmed the hMSC marker protein of CD 44 disappeared and the chondrocyte marker protein of collagen type II emerged over time in the CC-CT sample. These results imply the ECM-based hydrogel scaffolds with growth factors can supply suitable 3D cell niches for hMSCs differentiation into chondrocytes and the differentiation process can be regulated by the controllably released growth factors. PMID:24231133

  12. Mechanotransduction in primary human osteoarthritic chondrocytes is mediated by metabolism of energy, lipids, and amino acids.

    PubMed

    Zignego, Donald L; Hilmer, Jonathan K; June, Ronald K

    2015-12-16

    Chondrocytes are the sole cell type found in articular cartilage and are repeatedly subjected to mechanical loading in vivo. We hypothesized that physiological dynamic compression results in changes in energy metabolism to produce proteins for maintenance of the pericellular and extracellular matrices. The objective of this study was to develop an in-depth understanding for the short term (<30min) chondrocyte response to sub-injurious, physiological compression by analyzing metabolomic profiles for human chondrocytes harvested from femoral heads of osteoarthritic donors. Cell-seeded agarose constructs were randomly assigned to experimental groups, and dynamic compression was applied for 0, 15, or 30min. Following dynamic compression, metabolites were extracted and detected by HPLC-MS. Untargeted analyzes examined changes in global metabolomics profiles and targeted analysis examined the expression of specific metabolites related to central energy metabolism. We identified hundreds of metabolites that were regulated by applied compression, and we report the detection of 16 molecules not found in existing metabolite databases. We observed patient-specific mechanotransduction with aging dependence. Targeted studies found a transient increase in the ratio of NADP+ to NADPH and an initial decrease in the ratio of GDP to GTP, suggesting a flux of energy into the TCA cycle. By characterizing metabolomics profiles of primary chondrocytes in response to applied dynamic compression, this study provides insight into how OA chondrocytes respond to mechanical load. These results are consistent with increases in glycolytic energy utilization by mechanically induced signaling, and add substantial new data to a complex picture of how chondrocytes transduce mechanical loads. PMID:26573901

  13. Carnosol Inhibits Pro-Inflammatory and Catabolic Mediators of Cartilage Breakdown in Human Osteoarthritic Chondrocytes and Mediates Cross-Talk between Subchondral Bone Osteoblasts and Chondrocytes

    PubMed Central

    Sanchez, Christelle; Horcajada, Marie-Noëlle; Membrez Scalfo, Fanny; Ameye, Laurent; Offord, Elizabeth; Henrotin, Yves

    2015-01-01

    Aim The aim of this work was to evaluate the effects of carnosol, a rosemary polyphenol, on pro-inflammatory and catabolic mediators of cartilage breakdown in chondrocytes and via bone-cartilage crosstalk. Materials and Methods Osteoarthritic (OA) human chondrocytes were cultured in alginate beads for 4 days in presence or absence of carnosol (6 nM to 9 μM). The production of aggrecan, matrix metalloproteinase (MMP)-3, tissue inhibitor of metalloproteinase (TIMP)-1, interleukin (IL)-6 and nitric oxide (NO) and the expression of type II collagen and ADAMTS-4 and -5 were analyzed. Human osteoblasts from sclerotic (SC) or non-sclerotic (NSC) subchondral bone were cultured for 3 days in presence or absence of carnosol before co-culture with chondrocytes. Chondrocyte gene expression was analyzed after 4 days of co-culture. Results In chondrocytes, type II collagen expression was significantly enhanced in the presence of 3 μM carnosol (p = 0.008). MMP-3, IL-6, NO production and ADAMTS-4 expression were down-regulated in a concentration-dependent manner by carnosol (p<0.01). TIMP-1 production was slightly increased at 3 μM (p = 0.02) and ADAMTS-5 expression was decreased from 0.2 to 9 μM carnosol (p<0.05). IL-6 and PGE2 production was reduced in the presence of carnosol in both SC and NSC osteoblasts while alkaline phosphatase activity was not changed. In co-culture experiments preincubation of NSC and SC osteoblasts wih carnosol resulted in similar effects to incubation with anti-IL-6 antibody, namely a significant increase in aggrecan and decrease in MMP-3, ADAMTS-4 and -5 gene expression by chondrocytes. Conclusions Carnosol showed potent inhibition of pro-inflammatory and catabolic mediators of cartilage breakdown in chondrocytes. Inhibition of matrix degradation and enhancement of formation was observed in chondrocytes cocultured with subchondral osteoblasts preincubated with carnosol indicating a cross-talk between these two cellular compartments, potentially

  14. Interleukin-1 beta induces synthesis and secretion of interleukin-6 in human chondrocytes.

    PubMed

    Bender, S; Haubeck, H D; Van de Leur, E; Dufhues, G; Schiel, X; Lauwerijns, J; Greiling, H; Heinrich, P C

    1990-04-24

    Increased concentrations of interleukin-6 (IL-6) have been found in the synovial fluid of patients with osteoarthritis, rheumatoid arthritis and crystal-related joint diseases. It is therefore of great interest to identify the cells responsible for the production of IL-6, and to investigate whether IL-6 plays a role in the pathogenesis of degenerative or inflammatory joint diseases. Here we show that human interleukin-1 beta (IL-1 beta) induces IL-6 synthesis and secretion in differentiated human chondrocytes. In organ cultures resembling closely the in vivo system 10(6) chondrocytes incubated with 100 units of interleukin-1 beta per ml of medium led to the release of 6 X 10(3) units of IL-6 within 24 h. Chondrocytes cultured in agarose or as monolayers similarly incubated with IL-1 beta produced even higher amounts of IL-6: 70 X 10(3) units per 10(6) cells within 24 h. The induction of IL-6 synthesis by IL-1 beta was also shown at the mRNA level. IL-6 secreted by stimulated chondrocytes showed heterogeneity upon Western blot analysis. PMID:2335234

  15. Acetylation reduces SOX9 nuclear entry and ACAN gene transactivation in human chondrocytes.

    PubMed

    Bar Oz, Michal; Kumar, Ashok; Elayyan, Jinan; Reich, Eli; Binyamin, Milana; Kandel, Leonid; Liebergall, Meir; Steinmeyer, Juergen; Lefebvre, Veronique; Dvir-Ginzberg, Mona

    2016-06-01

    Changes in the content of aggrecan, an essential proteoglycan of articular cartilage, have been implicated in the pathophysiology of osteoarthritis (OA), a prevalent age-related, degenerative joint disease. Here, we examined the effect of SOX9 acetylation on ACAN transactivation in the context of osteoarthritis. Primary chondrocytes freshly isolated from degenerated OA cartilage displayed lower levels of ACAN mRNA and higher levels of acetylated SOX9 compared with cells from intact regions of OA cartilage. Degenerated OA cartilage presented chondrocyte clusters bearing diffused immunostaining for SOX9 compared with intact cartilage regions. Primary human chondrocytes freshly isolated from OA knee joints were cultured in monolayer or in three-dimensional alginate microbeads (3D). SOX9 was hypo-acetylated in 3D cultures and displayed enhanced binding to a -10 kb ACAN enhancer, a result consistent with higher ACAN mRNA levels than in monolayer cultures. It also co-immunoprecipitated with SIRT1, a major deacetylase responsible for SOX9 deacetylation. Finally, immunofluorescence assays revealed increased nuclear localization of SOX9 in primary chondrocytes treated with the NAD SIRT1 cofactor, than in cells treated with a SIRT1 inhibitor. Inhibition of importin β by importazole maintained SOX9 in the cytoplasm, even in the presence of NAD. Based on these data, we conclude that deacetylation promotes SOX9 nuclear translocation and hence its ability to activate ACAN. PMID:26910618

  16. Acquiring Chondrocyte Phenotype from Human Mesenchymal Stem Cells under Inflammatory Conditions

    PubMed Central

    Kondo, Masahiro; Yamaoka, Kunihiro; Tanaka, Yoshiya

    2014-01-01

    An inflammatory milieu breaks down the cartilage matrix and induces chondrocyte apoptosis, resulting in cartilage destruction in patients with cartilage degenerative diseases, such as rheumatoid arthritis or osteoarthritis. Because of the limited regenerative ability of chondrocytes, defects in cartilage are irreversible and difficult to repair. Mesenchymal stem cells (MSCs) are expected to be a new tool for cartilage repair because they are present in the cartilage and are able to differentiate into multiple lineages of cells, including chondrocytes. Although clinical trials using MSCs for patients with cartilage defects have already begun, its efficacy and repair mechanisms remain unknown. A PubMed search conducted in October 2014 using the following medical subject headings (MeSH) terms: mesenchymal stromal cells, chondrogenesis, and cytokines resulted in 204 articles. The titles and abstracts were screened and nine articles relevant to “inflammatory” cytokines and “human” MSCs were identified. Herein, we review the cell biology and mechanisms of chondrocyte phenotype acquisition from human MSCs in an inflammatory milieu and discuss the clinical potential of MSCs for cartilage repair. PMID:25407530

  17. Biotechnological Chondroitin a Novel Glycosamminoglycan With Remarkable Biological Function on Human Primary Chondrocytes.

    PubMed

    Stellavato, Antonietta; Tirino, Virginia; de Novellis, Francesca; Della Vecchia, Antonella; Cinquegrani, Fabio; De Rosa, Mario; Papaccio, Gianpaolo; Schiraldi, Chiara

    2016-09-01

    Cartilage tissue engineering, with in vitro expansion of autologus chondrocytes, is a promising technique for tissue regeneration and is a new potential strategy to prevent and/or treat cartilage damage (e.g., osteoarthritis). The aim of this study was (i) to investigate and compare the effects of new biotechnological chondroitin (BC) and a commercial extractive chondroitin sulfate (CS) on human chondrocytes in vitro culture; (ii) to evaluate the anti-inflammatory effects of the innovative BC compared to extractive CS. A chondrogenic cell population was isolated from human nasoseptal cartilage and in vitro cultures were studied through time-lapse video microscopy (TLVM), immunohistochemical staining and cytometry. In order to investigate the effect of BC and CS on phenotype maintainance, chondrogenic gene expression of aggrecan (AGN), of the transcriptor factor SOX9, of the types I and II collagen (COL1A1 and COL1A2), were quantified through transcriptional and protein evaluation at increasing cultivation time and passages. In addition to resemble the osteoarthritis-like in vitro model, chondrocytes were treated with IL-1β and the anti-inflammatory activity of BC and CS was assessed using cytokines quantification by multiplex array. BC significantly enhances cell proliferation also preserving chondrocyte phenotype increasing type II collagen expression up to 10 days of treatment and reduces inflammatory response in IL-1β treated chondrocytes respect to CS treated cells. Our results, taken together, suggest that this new BC is of foremost importance in translational medicine because it can be applied in novel scaffolds and pharmaceutical preparations aiming at cartilage pathology treatments such as the osteoarthritis. J. Cell. Biochem. 117: 2158-2169, 2016. © 2016 The Authors. Journal of Cellular Biochemistry Published by Wiley Periodicals, Inc. PMID:27018169

  18. Long Intergenic Noncoding RNAs Mediate the Human Chondrocyte Inflammatory Response and Are Differentially Expressed in Osteoarthritis Cartilage

    PubMed Central

    Pearson, Mark J.; Philp, Ashleigh M.; Heward, James A.; Roux, Benoit T.; Walsh, David A.; Davis, Edward T.; Lindsay, Mark A.

    2016-01-01

    Objective To identify long noncoding RNAs (lncRNAs), including long intergenic noncoding RNAs (lincRNAs), antisense RNAs, and pseudogenes, associated with the inflammatory response in human primary osteoarthritis (OA) chondrocytes and to explore their expression and function in OA. Methods OA cartilage was obtained from patients with hip or knee OA following joint replacement surgery. Non‐OA cartilage was obtained from postmortem donors and patients with fracture of the neck of the femur. Primary OA chondrocytes were isolated by collagenase digestion. LncRNA expression analysis was performed by RNA sequencing (RNAseq) and quantitative reverse transcriptase–polymerase chain reaction. Modulation of lncRNA chondrocyte expression was achieved using LNA longRNA GapmeRs (Exiqon). Cytokine production was measured with Luminex. Results RNAseq identified 983 lncRNAs in primary human hip OA chondrocytes, 183 of which had not previously been identified. Following interleukin‐1β (IL‐1β) stimulation, we identified 125 lincRNAs that were differentially expressed. The lincRNA p50‐associated cyclooxygenase 2–extragenic RNA (PACER) and 2 novel chondrocyte inflammation–associated lincRNAs (CILinc01 and CILinc02) were differentially expressed in both knee and hip OA cartilage compared to non‐OA cartilage. In primary OA chondrocytes, these lincRNAs were rapidly and transiently induced in response to multiple proinflammatory cytokines. Knockdown of CILinc01 and CILinc02 expression in human chondrocytes significantly enhanced the IL‐1–stimulated secretion of proinflammatory cytokines. Conclusion The inflammatory response in human OA chondrocytes is associated with widespread changes in the profile of lncRNAs, including PACER, CILinc01, and CILinc02. Differential expression of CILinc01 and CIinc02 in hip and knee OA cartilage, and their role in modulating cytokine production during the chondrocyte inflammatory response, suggest that they may play an important role

  19. Characterization of a type II collagen gene (COL2A1) mutation identified in cultured chondrocytes from human hypochondrogenesis.

    PubMed Central

    Horton, W A; Machado, M A; Ellard, J; Campbell, D; Bartley, J; Ramirez, F; Vitale, E; Lee, B

    1992-01-01

    A subtle mutation in the type II collagen gene COL2A1 was detected in a case of human hypochondrogenesis by using a chondrocyte culture system and PCR-cDNA scanning analysis. Chondrocytes obtained from cartilage biopsies were dedifferentiated and expanded in monolayer culture and then redifferentiated by culture over agarose. Single-strand conformation polymorphism and direct sequencing analysis identified a G----A transition, resulting in a glycine substitution at amino acid 574 of the pro alpha 1(II) collagen triple-helical domain. Morphologic assessment of cartilage-like structures produced in culture and electrophoretic analysis of collagens synthesized by the cultured chondrocytes suggested that the glycine substitution interferes with conversion of type II procollagen to collagen, impairs intracellular transport and secretion of the molecule, and disrupts collagen fibril assembly. This experimental approach has broad implications for the investigation of human chondrodysplasias as well as human chondrocyte biology. Images PMID:1374906

  20. Piperine inhibits IL-β induced expression of inflammatory mediators in human osteoarthritis chondrocyte.

    PubMed

    Ying, Xiaozhou; Chen, Xiaowei; Cheng, Shaowen; Shen, Yue; Peng, Lei; Xu, Hua Zi

    2013-10-01

    Black pepper (Piper nigrum) is a common remedy in Traditional Chinese Medicine and possesses diverse biological activities including anti-inflammatory properties. Osteoarthritis (OA) is a degenerative joint disease with an inflammatory component that drives the degradation of cartilage extracellular matrix. The present study aimed to assess the effects of piperine, the active phenolic component in black pepper extract, on human OA chondrocytes. In this study, human OA chondrocytes were pretreated with piperine at 10, 50 or 100μg/ml and subsequently stimulated with IL-1β (5ng/ml) for 24h. Production of PGE2 and NO was evaluated by the Griess reaction and an ELISA. Gene expression of MMP-3, MMP-13, iNOS and COX-2 was measured by real-time PCR. MMP-3 and MMP-13 proteins in culture medium were determined using cytokine-specific ELISA. Western immunoblotting was used to analyze the iNOS and COX-2 protein production in the culture medium. The regulation of NF-kB activity and the degradation of IkB were explored using luciferase and Western immunoblotting, respectively. We found that piperine inhibited the production of PGE2 and NO induced by IL-1β. Piperine significantly decreased the IL-1β-stimulated gene expression and production of MMP-3, MMP-13, iNOS and COX-2 in human OA chondrocytes. Piperine inhibited the IL-1β-mediated activation of NF-κB by suppressing the degradation of its inhibitory protein IκBα in the cytoplasm. The present report is first to demonstrate the anti-inflammatory activity of piperine in human OA chondrocytes. Piperine can effectively abrogate the IL-1β-induced over-expression of inflammatory mediators; suggesting that piperine may be a potential agent in the treatment of OA. PMID:23838114

  1. The Role of PPARγ in Advanced Glycation End Products-Induced Inflammatory Response in Human Chondrocytes

    PubMed Central

    Li, Yu-qing; Chen, Cheng; Cai, Wei; Zeng, Yue-lin

    2015-01-01

    Objective Advances made in the past ten years highlight the notion that peroxisome proliferator-activated receptors gamma (PPARγ) has protective properties in the pathophysiology of osteoarthritis (OA). The aim of this study was to define the roles of PPARγ in AGEs-induced inflammatory response in human chondrocytes. Methods Primary human chondrocytes were stimulated with AGEs in the presence or absence of neutralizing antibody against RAGE (anti-RAGE), MAPK specific inhibitors and PPARγ agonist pioglitazone. The expression of IL-1, MMP-13, TNF-α, PPARγ, nuclear NF-κB p65 and cytosol IκBα was determined by western blotting and real-time PCR. Results AGEs could enhance the expression of IL-1, TNF-α, and MMP-13, but the level of PPARγ was decreased in a time- and dose-dependent manner, which was inhibited by anti-RAGE, SB203580 (P38 MAPK specific inhibitor) and SP600125 (a selective inhibitor of JNK). PPARγ agonist pioglitazone could inhibit the effects of AGEs-induced inflammatory response and PPARγ down-regulation. In human chondrocytes, AGEs could induce cytosol IκBα degradation and increase the level of nuclear NF-κB p65, which was inhibited by PPARγ agonist pioglitazone. Conclusions In primary human chondrocytes, AGEs could down-regulate PPARγ expression and increase the inflammatory mediators, which could be reversed by PPARγ agonist pioglitazone. Activation of RAGE by AGEs triggers a cascade of downstream signaling, including MAPK JNK/ p38, PPARγ and NF-κB. Taken together, PPARγ could be a potential target for pharmacologic intervention in the treatment of OA. PMID:26024533

  2. Tormentic Acid Inhibits IL-1β-Induced Inflammatory Response in Human Osteoarthritic Chondrocytes.

    PubMed

    Yang, Yang; Wang, Yawei; Wang, Yumin; Zhao, Meng; Jia, Haobo; Li, Bing; Xing, Dan

    2016-06-01

    The pro-inflammatory cytokine interleukin-1beta (IL-1β) plays critical roles in pathogenesis of osteoarthritis (OA). Tormentic acid (TA), a triterpene isolated from Rosa rugosa, has anti-inflammatory activity. However, the anti-inflammatory effect of TA on OA is still unclear. So, in the present study, we examined the effect of TA on IL-1β-induced inflammatory response in primary human OA chondrocytes. Our results demonstrated that TA significantly decreased the IL-1β-stimulated expression of matrix metalloproteinase-3 (MMP-3) and MMP-13. It also inhibited the IL-1β-induced expression of inducible nitric oxide (NO) synthase (iNOS) and cyclooxygenase-2 (COX-2), as well as the production of NO and prostaglandin E2 (PGE2) in human OA chondrocytes. Furthermore, TA greatly inhibited the IL-1β-induced NF-κB activation. In conclusion, this study is the first to demonstrate the anti-inflammatory activity of TA in human OA chondrocytes. TA significantly inhibits the IL-1β-induced inflammatory response by suppressing the NF-κB signaling pathway. Thus, TA may be a potential agent in the treatment of OA. PMID:27102898

  3. Effect of the polyamine analogue N1,N11-diethylnorspermine on cell survival and susceptibility to apoptosis of human chondrocytes.

    PubMed

    Stanic, Ivana; Cetrullo, Silvia; Facchini, Annalisa; Stefanelli, Claudio; Borzì, Rosa Maria; Tantini, Benedetta; Guarnieri, Carlo; Caldarera, Claudio Marcello; Flamigni, Flavio

    2008-07-01

    Chondrocyte survival is closely linked to cartilage integrity, and forms of chondrocyte apoptotic death can contribute to cartilage degeneration in articular diseases. Since growing evidence also implicates polyamines in the control of cell death, we have been investigating the role of polyamine metabolism in chondrocyte survival and apoptosis. Treatment of human C-28/I2 chondrocytes with N(1),N(11)-diethylnorspermine (DENSPM), a polyamine analogue with clinical relevance as an experimental anticancer agent, inhibited polyamine biosynthesis and induced polyamine catabolism, thus rapidly depleting all main polyamines. DENSPM did not increase significantly caspase activity, but provoked a late cell death associated to DNA fragmentation. A short treatment with DENSPM did not reduce cell viability when given alone, but enhanced caspase-3 and -9 activation in chondrocytes exposed to tumor necrosis factor-alpha (TNF) and cycloheximide (CHX). A longer treatment with DENSPM however reduced caspase response to TNF plus CHX. Depletion of all polyamines obtained by specific inhibitors of polyamine biosynthesis did not cause cell death and contrasted apoptosis by decreasing caspase activities. In conclusion, following DENSPM treatment, C-28/I2 chondrocytes are initially sensitized to caspase 9-dependent apoptosis in the presence of TNF and CHX and may eventually undergo a late and mainly caspase-independent cell death in the absence of other stimuli. Moreover, these results indicate that a reduction of polyamine levels not only leads to inhibition of cell proliferation, but also of caspase-mediated pathways of chondrocyte apoptosis. PMID:18231987

  4. Expression and Function of K(ATP) Channels in Normal and Osteoarthritic Human Chondrocytes: Possible Role in Glucose Sensing

    PubMed Central

    Rufino, Ana T; Rosa, Susana C; Judas, Fernando; Mobasheri, Ali; Lopes, M Celeste; Mendes, Alexandrina F

    2013-01-01

    ATP-sensitive potassium [K(ATP)] channels sense intracellular ATP/ADP levels, being essential components of a glucose-sensing apparatus in various cells that couples glucose metabolism, intracellular ATP/ADP levels and membrane potential. These channels are present in human chondrocytes, but their subunit composition and functions are unknown. This study aimed at elucidating the subunit composition of K(ATP) channels expressed in human chondrocytes and determining whether they play a role in regulating the abundance of major glucose transporters, GLUT-1 and GLUT-3, and glucose transport capacity. The results obtained show that human chondrocytes express the pore forming subunits, Kir6.1 and Kir6.2, at the mRNA and protein levels and the regulatory sulfonylurea receptor (SUR) subunits, SUR2A and SUR2B, but not SUR1. The expression of these subunits was no affected by culture under hyperglycemia-like conditions. Functional impairment of the channel activity, using a SUR blocker (glibenclamide 10 or 20 nM), reduced the protein levels of GLUT-1 and GLUT-3 by approximately 30% in normal chondrocytes, while in cells from cartilage with increasing osteoarthritic (OA) grade no changes were observed. Glucose transport capacity, however, was not affected in normal or OA chondrocytes. These results show that K(ATP) channel activity regulates the abundance of GLUT-1 and GLUT-3, although other mechanisms are involved in regulating the overall glucose transport capacity of human chondrocytes. Therefore, K(ATP) channels are potential components of a broad glucose sensing apparatus that modulates glucose transporters and allows human chondrocytes to adjust to varying extracellular glucose concentrations. This function of K(ATP) channels seems to be impaired in OA chondrocytes. J. Cell. Biochem. 114: 1879–1889, 2013. © 2013 Wiley Periodicals, Inc. PMID:23494827

  5. Early induction of a prechondrogenic population allows efficient generation of stable chondrocytes from human induced pluripotent stem cells

    PubMed Central

    Lee, Jieun; Taylor, Sarah E. B.; Smeriglio, Piera; Lai, Janice; Maloney, William J.; Yang, Fan; Bhutani, Nidhi

    2015-01-01

    Regeneration of human cartilage is inherently inefficient; an abundant autologous source, such as human induced pluripotent stem cells (hiPSCs), is therefore attractive for engineering cartilage. We report a growth factor-based protocol for differentiating hiPSCs into articular-like chondrocytes (hiChondrocytes) within 2 weeks, with an overall efficiency >90%. The hiChondrocytes are stable and comparable to adult articular chondrocytes in global gene expression, extracellular matrix production, and ability to generate cartilage tissue in vitro and in immune-deficient mice. Molecular characterization identified an early SRY (sex-determining region Y) box (Sox)9low cluster of differentiation (CD)44lowCD140low prechondrogenic population during hiPSC differentiation. In addition, 2 distinct Sox9-regulated gene networks were identified in the Sox9low and Sox9high populations providing novel molecular insights into chondrogenic fate commitment and differentiation. Our findings present a favorable method for generating hiPSC-derived articular-like chondrocytes. The hiChondrocytes are an attractive cell source for cartilage engineering because of their abundance, autologous nature, and potential to generate articular-like cartilage rather than fibrocartilage. In addition, hiChondrocytes can be excellent tools for modeling human musculoskeletal diseases in a dish and for rapid drug screening.—Lee, J., Taylor, S. E. B., Smeriglio, P., Lai, J., Maloney, W. J., Yang, F., Bhutani, N. Early induction of a prechondrogenic population allows efficient generation of stable chondrocytes from human induced pluripotent stem cells. PMID:25911615

  6. Bioimaging: An Useful Tool to Monitor Differentiation of Human Embryonic Stem Cells into Chondrocytes.

    PubMed

    Suchorska, Wiktoria M; Lach, Michał S; Richter, Magdalena; Kaczmarczyk, Jacek; Trzeciak, Tomasz

    2016-05-01

    To improve the recovery of damaged cartilage tissue, pluripotent stem cell-based therapies are being intensively explored. A number of techniques exist that enable monitoring of stem cell differentiation, including immunofluorescence staining. This simple and fast method enables changes to be observed during the differentiation process. Here, two protocols for the differentiation of human embryonic stem cells into chondrocytes were used (monolayer cell culture and embryoid body formation). Cells were labeled for markers expressed during the differentiation process at different time points (pluripotent: NANOG, SOX2, OCT3/4, E-cadherin; prochondrogenic: SOX6, SOX9, Collagen type II; extracellular matrix components: chondroitin sulfate, heparan sulfate; beta-catenin, CXCR4, and Brachyury). Comparison of the signal intensity of differentiated cells to control cell populations (articular cartilage chondrocytes and human embryonic stem cells) showed decreased signal intensities of pluripotent markers, E-cadherin and beta-catenin. Increased signal intensities of prochondrogenic markers and extracellular matrix components were observed. The changes during chondrogenic differentiation monitored by evaluation of pluripotent and chondrogenic markers signal intensity were described. The changes were similar to several studies over chondrogenesis. These results were confirmed by semi-quantitative analysis of IF signals. In this research we indicate a bioimaging as a useful tool to monitor and semi-quantify the IF pictures during the differentiation of hES into chondrocyte-like. PMID:26354117

  7. Human Umbilical Cord Blood-Derived Mesenchymal Stem Cells Contribute to Chondrogenesis in Coculture with Chondrocytes

    PubMed Central

    Li, Xingfu; Duan, Li; Liang, Yujie; Zhu, Weimin; Xiong, Jianyi; Wang, Daping

    2016-01-01

    Human umbilical cord blood-derived mesenchymal stem cells (hUCB-MSCs) have been shown as the most potential stem cell source for articular cartilage repair. In this study, we aimed to develop a method for long-term coculture of human articular chondrocytes (hACs) and hUCB-MSCs at low density in vitro to determine if the low density of hACs could enhance the hUCB-MSC chondrogenic differentiation as well as to determine the optimal ratio of the two cell types. Also, we compared the difference between direct coculture and indirect coculture at low density. Monolayer cultures of hUCB-MSCs and hACs were investigated at different ratios, at direct cell-cell contact groups for 21 days. Compared to direct coculture, hUCB-MSCs and hACs indirect contact culture significantly increased type II collagen (COL2) and decreased type I collagen (COL1) protein expression levels. SRY-box 9 (SOX9) mRNA levels and protein expression were highest in indirect coculture. Overall, these results indicate that low density direct coculture induces fibrocartilage. However, indirect coculture in conditioned chondrocyte cell culture medium can increase expression of chondrogenic markers and induce hUCB-MSCs differentiation into mature chondrocytes. This work demonstrates that it is possible to promote chondrogenesis of hUCB-MSCs in combination with hACs, further supporting the concept of novel coculture strategies for tissue engineering. PMID:27446948

  8. Low dose short duration pulsed electromagnetic field effects on cultured human chondrocytes: An experimental study

    PubMed Central

    Anbarasan, Selvam; Baraneedharan, Ulaganathan; Paul, Solomon FD; Kaur, Harpreet; Rangaswami, Subramoniam; Bhaskar, Emmanuel

    2016-01-01

    Background: Pulsed electromagnetic field (PEMF) is used to treat bone and joint disorders for over 30 years. Recent studies demonstrate a significant effect of PEMF on bone and cartilage proliferation, differentiation, synthesis of extracellular matrix (ECM) and production of growth factors. The aim of this study is to assess if PEMF of low frequency, ultralow field strength and short time exposure have beneficial effects on in-vitro cultured human chondrocytes. Materials and Methods: Primary human chondrocytes cultures were established using articular cartilage obtained from knee joint during joint replacement surgery. Post characterization, the cells were exposed to PEMF at frequencies ranging from 0.1 to 10 Hz and field intensities ranging from 0.65 to 1.95 μT for 60 min/day for 3 consecutive days to analyze the viability, ECM component synthesis, proliferation and morphology related changes post exposure. Association between exposure doses and cellular effects were analyzed with paired't’ test. Results: In-vitro PEMF exposure of 0.1 Hz frequency, 1.95 μT and duration of 60 min/day for 3 consecutive days produced the most favorable response on chondrocytes viability (P < 0.001), ECM component production (P < 0.001) and multiplication. Exposure of identical chondrocyte cultures to PEMFs of 0.65 μT field intensity at 1 Hz frequency resulted in less significant response. Exposure to 1.3 μT PEMFs at 10 Hz frequency does not show any significant effects in different analytical parameters. Conclusions: Short duration PEMF exposure may represent a new therapy for patients with Osteoarthritis (OA). PMID:26955182

  9. Proliferative re-modeling of the spatial organization of human superficial chondrocytes distant to focal early osteoarthritis (OA)

    PubMed Central

    Rolauffs, Bernd; Williams, James M.; Aurich, Matthias; Grodzinsky, Alan J.; Kuettner, Klaus E.; Cole, Ada A.

    2010-01-01

    Objective Human superficial chondrocytes show distinct spatial organizations whereas they commonly aggregate near osteoarthritic (OA) fissures. It is not known whether remodeling or destruction of the spatial chondrocyte organization may occur distant to focal (early) OA lesions. Methods The intact cartilages (condyles, patellofemoral groove, proximal tibia) distant to focal OA lesions of human grade 2 joints were compared to location-matched non-degenerative (grade 0–1) cartilages. Chondrocyte nuclei were stained with propidium iodide and recorded by fluorescence-microscopy in a top-down view. Chondrocyte arrangements were tested for randomness or significant grouping via point pattern analyses (Clark and Evans Aggregation Index), and were correlated with OA grade and surface cell densities. Results In grade 2 cartilages, superficial chondrocytes were situated in horizontal patterns such as strings, cluster, pairs and singles comparable to non-degenerative cartilage. In the intact cartilages of grade 2 joints, the spatial organization included a novel pattern, consisting of chondrocytes that were aligned in two parallel lines building double strings. These double strings correlated with an increased number of chondrocytes per group (p<0.05), increased corresponding superficial zone cell density (p<0.001), and were observed in all grade 2 condyles (p<0.001), some grade 2 tibiae (p<0.05) but never in grade 0–1 cartilage (p<0.001). Conclusion The present study is the first to identify a distinct spatial re-organization of human superficial chondrocytes in response to distant early OA lesions and suggests that proliferation had occurred distant to focal early OA. This spatial re-organization may serve to recruit metabolically active units as attempt to repair focal damage. PMID:20112377

  10. Taraxasterol inhibits IL-1β-induced inflammatory response in human osteoarthritic chondrocytes.

    PubMed

    Piao, Taikui; Ma, Zhiqiang; Li, Xin; Liu, Jianyu

    2015-06-01

    Osteoarthritis (OA), a chronic degenerative joint disease, is a leading cause of disability among elderly patients. Taraxasterol, a pentacyclic-triterpene isolated from Taraxacum officinale, has been shown to have anti-inflammatory effects. However, the protective effect of taraxasterol on OA remains unclear. In order to provide a scientific basis for the applicability of taraxasterol in OA, the anti-inflammatory effects of taraxasterol on IL-1β-stimulated osteoarthritic chondrocytes were investigated. Chondrocytes were pretreated with taraxasterol 1h before IL-1β treatment. The productions of MMP-1, MMP3, MMP13, PGE2 and NO were measured by ELISA and Griess reaction. The expression of COX-2, iNOS, and NF-κB was detected by western blot analysis. Our results demonstrated that taraxasterol dose-dependently suppressed MMP-1, MMP3, MMP13, PGE2 and NO production induced by IL-1β. The expression of COX-2 and iNOS was also inhibited by taraxasterol. Western blot analysis showed that taraxasterol suppressed IL-1β-induced NF-κB activation in a dose-dependent manner. Taken together, we found that taraxasterol protected human chondrocytes by inhibiting MMPs, NO and PGE2 production. Taraxasterol may be a useful agent for prevention and treatment of OA. PMID:25797286

  11. The effect of piroxicam on the metabolism of isolated human chondrocytes.

    PubMed

    Bulstra, S K; Kuijer, R; Buurman, W A; Terwindt-Rouwenhorst, E; Guelen, P J; van der Linden, A J

    1992-04-01

    The effect of piroxicam on the metabolism of healthy and osteoarthrotic (OA) chondrocytes was studied in vitro. The chondrocytes were obtained from five healthy, five moderately OA, and four severely OA hips or knees. The chondrocytes were cultured in a high-density, short-term in vitro model. In this culture, the healthy chondrocytes as well as the OA chondrocytes retain their metabolic properties. Piroxicam was used in concentrations ranging from 0 to 10 micrograms/ml, which is comparable to the concentrations reached in vivo after oral administration. In cultures of healthy chondrocytes, piroxicam inhibited proliferation and synthesis of proteoglycans. The metabolism of moderately damaged chondrocytes was not influenced by piroxicam. In severely damaged chondrocytes, the proliferation was significantly inhibited by piroxicam. In order to avoid the possible negative side effects of piroxicam on the metabolism of healthy and severely OA chondrocytes, piroxicam treatment of an OA joint with synovitis should be restricted to the period of the effusion. PMID:1555353

  12. Role of miR-146a in human chondrocyte apoptosis in response to mechanical pressure injury in vitro

    PubMed Central

    JIN, LEI; ZHAO, JIAN; JING, WENSEN; YAN, SHIJU; WANG, XIN; XIAO, CHUN; MA, BAOAN

    2014-01-01

    MicroRNA (miR)-146a is known to be overexpressed in osteoarthritis (OA). However, the role of miR-146a in OA has not yet been fully elucidated. In the present study, we applied mechanical pressure of 10 MPa to human chondrocytes for 60 min in order to investigate the expression of miR-146a and apoptosis following the mechanical pressure injury. Normal human chondrocytes were transfected with an miR-146a mimic or an inhibitor to regulate miR-146a expression. Potential target genes of miR-146a were predicted using bioinformatics. Moreover, luciferase reporter assay confirmed that Smad4 was a direct target of miR-146a. The expression levels of miR-146a, Smad4 and vascular endothelial growth factor (VEGF) were quantified by quantitative reverse transcription PCR and/or western blot analysis. The effects of miR-146a on apoptosis were detected by Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) flow cytometry. The results indicated that mechanical pressure affected chondrocyte viability and induced the early apoptosis of chondrocytes. Mechanical pressure injury increased the expression levels of miR-146a and VEGF and decreased the levels of Smad4 in the chondrocytes. In the human chondrocytes, the upregulation of miR-146a induced apoptosis, upregulated VEGF expression and downregulated Smad4 expression. In addition, the knockdown of miR-146a reduced cell apoptosis, upregulated Smad4 expression and downregulated VEGF expression. Smad4 was identified as a direct target of miR-146a by harboring a miR-146a binding sequence in the 3′-untranslated region (3′-UTR) of its mRNA. Furthermore, the upregulation of VEGF induced by miR-146a was mediated by Smad4 in the chondrocytes subjected to mechanical pressure injury. These results demonstrated that miR-146a was overexpressed in our chondrocyte model of experimentally induced human mechanical injury, accompanied by the upregulation of VEGF and the downregulation of Smad4 in vitro. Moreover, our data suggest

  13. Role of miR-146a in human chondrocyte apoptosis in response to mechanical pressure injury in vitro.

    PubMed

    Jin, Lei; Zhao, Jian; Jing, Wensen; Yan, Shiju; Wang, Xin; Xiao, Chun; Ma, Baoan

    2014-08-01

    MicroRNA (miR)-146a is known to be overexpressed in osteoarthritis (OA). However, the role of miR-146a in OA has not yet been fully elucidated. In the present study, we applied mechanical pressure of 10 MPa to human chondrocytes for 60 min in order to investigate the expression of miR-146a and apoptosis following the mechanical pressure injury. Normal human chondrocytes were transfected with an miR-146a mimic or an inhibitor to regulate miR-146a expression. Potential target genes of miR-146a were predicted using bioinformatics. Moreover, luciferase reporter assay confirmed that Smad4 was a direct target of miR-146a. The expression levels of miR-146a, Smad4 and vascular endothelial growth factor (VEGF) were quantified by quantitative reverse transcription PCR and/or western blot analysis. The effects of miR-146a on apoptosis were detected by Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) flow cytometry. The results indicated that mechanical pressure affected chondrocyte viability and induced the early apoptosis of chondrocytes. Mechanical pressure injury increased the expression levels of miR-146a and VEGF and decreased the levels of Smad4 in the chondrocytes. In the human chondrocytes, the upregulation of miR-146a induced apoptosis, upregulated VEGF expression and downregulated Smad4 expression. In addition, the knockdown of miR-146a reduced cell apoptosis, upregulated Smad4 expression and downregulated VEGF expression. Smad4 was identified as a direct target of miR-146a by harboring a miR‑146a binding sequence in the 3'-untranslated region (3'-UTR) of its mRNA. Furthermore, the upregulation of VEGF induced by miR‑146a was mediated by Smad4 in the chondrocytes subjected to mechanical pressure injury. These results demonstrated that miR-146a was overexpressed in our chondrocyte model of experimentally induced human mechanical injury, accompanied by the upregulation of VEGF and the downregulation of Smad4 in vitro. Moreover, our data suggest

  14. The synovial microenvironment of osteoarthritic joints alters RNA-seq expression profiles of human primary articular chondrocytes.

    PubMed

    Lewallen, Eric A; Bonin, Carolina A; Li, Xin; Smith, Jay; Karperien, Marcel; Larson, A Noelle; Lewallen, David G; Cool, Simon M; Westendorf, Jennifer J; Krych, Aaron J; Leontovich, Alexey A; Im, Hee-Jeong; van Wijnen, Andre J

    2016-10-15

    Osteoarthritis (OA) is a disabling degenerative joint disease that prompts pain and has limited treatment options. To permit early diagnosis and treatment of OA, a high resolution mechanistic understanding of human chondrocytes in normal and diseased states is necessary. In this study, we assessed the biological effects of OA-related changes in the synovial microenvironment on chondrocytes embedded within anatomically intact cartilage from joints with different pathological grades by next generation RNA-sequencing (RNA-seq). We determined the transcriptome of primary articular chondrocytes derived from anatomically unaffected knees and ankles, as well as from joints affected by OA. The GALAXY bioinformatics platform was used to facilitate biological interpretations. Comparisons of patient samples by k-means, hierarchical clustering and principal component analyses together reveal that primary chondrocytes exhibit OA grade-related differences in gene expression, including genes involved in cell-adhesion, ECM production and immune response. We conclude that diseased synovial microenvironments in joints with different histopathological OA grades directly alter gene expression in chondrocytes. One ramification of this finding is that anatomically intact cartilage from OA joints is not an ideal source of healthy chondrocytes, nor should these specimens be used to generate a normal baseline for the molecular characterization of diseased joints. PMID:27378743

  15. The effect of oxygen tension on human articular chondrocyte matrix synthesis: Integration of experimental and computational approaches

    PubMed Central

    Li, S; Oreffo, ROC; Sengers, BG; Tare, RS

    2014-01-01

    Significant oxygen gradients occur within tissue engineered cartilaginous constructs. Although oxygen tension is an important limiting parameter in the development of new cartilage matrix, its precise role in matrix formation by chondrocytes remains controversial, primarily due to discrepancies in the experimental setup applied in different studies. In this study, the specific effects of oxygen tension on the synthesis of cartilaginous matrix by human articular chondrocytes were studied using a combined experimental-computational approach in a “scaffold-free” 3D pellet culture model. Key parameters including cellular oxygen uptake rate were determined experimentally and used in conjunction with a mathematical model to estimate oxygen tension profiles in 21-day cartilaginous pellets. A threshold oxygen tension (pO2 ≈ 8% atmospheric pressure) for human articular chondrocytes was estimated from these inferred oxygen profiles and histological analysis of pellet sections. Human articular chondrocytes that experienced oxygen tension below this threshold demonstrated enhanced proteoglycan deposition. Conversely, oxygen tension higher than the threshold favored collagen synthesis. This study has demonstrated a close relationship between oxygen tension and matrix synthesis by human articular chondrocytes in a “scaffold-free” 3D pellet culture model, providing valuable insight into the understanding and optimization of cartilage bioengineering approaches. Biotechnol. Bioeng. 2014;111: 1876–1885. PMID:24668194

  16. Lithium Chloride Dependent Glycogen Synthase Kinase 3 Inactivation Links Oxidative DNA Damage, Hypertrophy and Senescence in Human Articular Chondrocytes and Reproduces Chondrocyte Phenotype of Obese Osteoarthritis Patients

    PubMed Central

    Platano, Daniela; Cattini, Luca; Trisolino, Giovanni; Mariani, Erminia; Borzì, Rosa Maria

    2015-01-01

    Introduction Recent evidence suggests that GSK3 activity is chondroprotective in osteoarthritis (OA), but at the same time, its inactivation has been proposed as an anti-inflammatory therapeutic option. Here we evaluated the extent of GSK3β inactivation in vivo in OA knee cartilage and the molecular events downstream GSK3β inactivation in vitro to assess their contribution to cell senescence and hypertrophy. Methods In vivo level of phosphorylated GSK3β was analyzed in cartilage and oxidative damage was assessed by 8-oxo-deoxyguanosine staining. The in vitro effects of GSK3β inactivation (using either LiCl or SB216763) were evaluated on proliferating primary human chondrocytes by combined confocal microscopy analysis of Mitotracker staining and reactive oxygen species (ROS) production (2',7'-dichlorofluorescin diacetate staining). Downstream effects on DNA damage and senescence were investigated by western blot (γH2AX, GADD45β and p21), flow cytometric analysis of cell cycle and light scattering properties, quantitative assessment of senescence associated β galactosidase activity, and PAS staining. Results In vivo chondrocytes from obese OA patients showed higher levels of phosphorylated GSK3β, oxidative damage and expression of GADD45β and p21, in comparison with chondrocytes of nonobese OA patients. LiCl mediated GSK3β inactivation in vitro resulted in increased mitochondrial ROS production, responsible for reduced cell proliferation, S phase transient arrest, and increase in cell senescence, size and granularity. Collectively, western blot data supported the occurrence of a DNA damage response leading to cellular senescence with increase in γH2AX, GADD45β and p21. Moreover, LiCl boosted 8-oxo-dG staining, expression of IKKα and MMP-10. Conclusions In articular chondrocytes, GSK3β activity is required for the maintenance of proliferative potential and phenotype. Conversely, GSK3β inactivation, although preserving chondrocyte survival, results in

  17. Possible chondroprotective effect of canakinumab: an in vitro study on human osteoarthritic chondrocytes.

    PubMed

    Cheleschi, Sara; Cantarini, Luca; Pascarelli, Nicola Antonio; Collodel, Giulia; Lucherini, Orso Maria; Galeazzi, Mauro; Fioravanti, Antonella

    2015-02-01

    Canakinumab is a human IgGκ monoclonal antibody that neutralizes the activity of interleukin (IL)-1β blocking interaction with IL-1β receptors. Our study aimed to evaluate the in vitro effect of canakinumab on human osteoarthritic (OA) chondrocytes cultivated in the presence or absence of tumor necrosis factor (TNF)-α. Articular cartilage was obtained from the femoral heads of patients with osteoarthritis (OA). Chondrocytes were incubated with two concentrations (1μg/ml and 10μg/ml) of canakinumab alone or with TNF-α (10ng/ml) for 48h. We evaluated cell viability, release of proteoglycans (PG) and nitric oxide (NO) in culture medium, inducible nitric oxide synthase (iNOS) and metalloproteinanes (MMP)-1,3,13 gene expression, apoptosis, necrosis and morphological feature by transmission electron microscopy (TEM). Canakinumab alone did not have cytotoxic effect. Cell viability was reduced significantly (p<0.001) by TNF-α and restored by canakinumab at both concentrations used. TNF-α determined a significant decrease of PG (p<0.001) and an increase of NO (p<0.001) and MMP-1,3,13 gene expression. Canakinumab significantly increased the PG levels and decreased (1μg/ml, p<0.01; 10μg/ml, p<0.01) NO levels in cells cultured with TNF-α. The NO data were confirmed by the immunocytochemistry assay for iNOS. A significant reduction of MMP-1,3,13 gene expression was induced by canakinumab. Our experiments confirmed the pro-apoptotic effect of TNF-α and demonstrated a protective role of canakinumab. The results concerning biochemical data were further confirmed by the morphological findings obtained by TEM. We showed that canakinumab counteracts the negative effects of TNF-α on OA chondrocyte cultures and may have a potential chondroprotective role in OA. PMID:25461395

  18. Human developmental chondrogenesis as a basis for engineering chondrocytes from pluripotent stem cells.

    PubMed

    Wu, Ling; Bluguermann, Carolina; Kyupelyan, Levon; Latour, Brooke; Gonzalez, Stephanie; Shah, Saumya; Galic, Zoran; Ge, Sundi; Zhu, Yuhua; Petrigliano, Frank A; Nsair, Ali; Miriuka, Santiago G; Li, Xinmin; Lyons, Karen M; Crooks, Gay M; McAllister, David R; Van Handel, Ben; Adams, John S; Evseenko, Denis

    2013-01-01

    Joint injury and osteoarthritis affect millions of people worldwide, but attempts to generate articular cartilage using adult stem/progenitor cells have been unsuccessful. We hypothesized that recapitulation of the human developmental chondrogenic program using pluripotent stem cells (PSCs) may represent a superior approach for cartilage restoration. Using laser-capture microdissection followed by microarray analysis, we first defined a surface phenotype (CD166(low/neg)CD146(low/neg)CD73(+)CD44(low)BMPR1B(+)) distinguishing the earliest cartilage committed cells (prechondrocytes) at 5-6 weeks of development. Functional studies confirmed these cells are chondrocyte progenitors. From 12 weeks, only the superficial layers of articular cartilage were enriched in cells with this progenitor phenotype. Isolation of cells with a similar immunophenotype from differentiating human PSCs revealed a population of CD166(low/neg)BMPR1B(+) putative cartilage-committed progenitors. Taken as a whole, these data define a developmental approach for the generation of highly purified functional human chondrocytes from PSCs that could enable substantial progress in cartilage tissue engineering. PMID:24371811

  19. Human Developmental Chondrogenesis as a Basis for Engineering Chondrocytes from Pluripotent Stem Cells

    PubMed Central

    Wu, Ling; Bluguermann, Carolina; Kyupelyan, Levon; Latour, Brooke; Gonzalez, Stephanie; Shah, Saumya; Galic, Zoran; Ge, Sundi; Zhu, Yuhua; Petrigliano, Frank A.; Nsair, Ali; Miriuka, Santiago G.; Li, Xinmin; Lyons, Karen M.; Crooks, Gay M.; McAllister, David R.; Van Handel, Ben; Adams, John S.; Evseenko, Denis

    2013-01-01

    Summary Joint injury and osteoarthritis affect millions of people worldwide, but attempts to generate articular cartilage using adult stem/progenitor cells have been unsuccessful. We hypothesized that recapitulation of the human developmental chondrogenic program using pluripotent stem cells (PSCs) may represent a superior approach for cartilage restoration. Using laser-capture microdissection followed by microarray analysis, we first defined a surface phenotype (CD166low/negCD146low/negCD73+CD44lowBMPR1B+) distinguishing the earliest cartilage committed cells (prechondrocytes) at 5–6 weeks of development. Functional studies confirmed these cells are chondrocyte progenitors. From 12 weeks, only the superficial layers of articular cartilage were enriched in cells with this progenitor phenotype. Isolation of cells with a similar immunophenotype from differentiating human PSCs revealed a population of CD166low/negBMPR1B+ putative cartilage-committed progenitors. Taken as a whole, these data define a developmental approach for the generation of highly purified functional human chondrocytes from PSCs that could enable substantial progress in cartilage tissue engineering. PMID:24371811

  20. Human Bone-Forming Chondrocytes Cultured in the Hydrodynamic Focusing Bioreactor Retain Matrix Proteins: Similarities to Spaceflight Results

    NASA Technical Reports Server (NTRS)

    Duke, P. J.; Hecht, J.; Montufar-Solis, D.

    2006-01-01

    Fracture healing, crucial to a successful Mars mission, involves formation of a cartilaginous fracture callus which differentiates, mineralizes, ossifies and remodels via the endochondral process. Studies of spaceflown and tailsuspended rats found that, without loading, fracture callus formation and cartilage differentiation within the callus were minimal. We found delayed differentiation of chondrocytes within the rat growth plate on Cosmos 1887, 2044, and Spacelab 3. In the current study, differentiation of human bone-forming chondrocytes cultured in the hydrodynamic focusing bioreactor (HFB) was assessed. Human costochondral chondrocytes in suspension were aggregated overnight, then cultured in the HFB for 25 days. Collagen Type II, aggrecan and unsulfated chondroitin were found extracellularly and chondroitin sulfates 4 and 6 within the cell. Lack of secretion was also found in pancreatic cells of spaceflown rats, and in our SL3 studies. The HFB can be used to study cartilage differentiation in simulated microgravity.

  1. High seeding density of human chondrocytes in agarose produces tissue-engineered cartilage approaching native mechanical and biochemical properties.

    PubMed

    Cigan, Alexander D; Roach, Brendan L; Nims, Robert J; Tan, Andrea R; Albro, Michael B; Stoker, Aaron M; Cook, James L; Vunjak-Novakovic, Gordana; Hung, Clark T; Ateshian, Gerard A

    2016-06-14

    Animal cells have served as highly controllable model systems for furthering cartilage tissue engineering practices in pursuit of treating osteoarthritis. Although successful strategies for animal cells must ultimately be adapted to human cells to be clinically relevant, human chondrocytes are rarely employed in such studies. In this study, we evaluated the applicability of culture techniques established for juvenile bovine and adult canine chondrocytes to human chondrocytes obtained from fresh or expired osteochondral allografts. Human chondrocytes were expanded and encapsulated in 2% agarose scaffolds measuring ∅3-4mm×2.3mm, with cell seeding densities ranging from 15 to 90×10(6)cells/mL. Subsets of constructs were subjected to transient or sustained TGF-β treatment, or provided channels to enhance nutrient transport. Human cartilaginous constructs physically resembled native human cartilage, and reached compressive Young's moduli of up to ~250kPa (corresponding to the low end of ranges reported for native knee cartilage), dynamic moduli of ~950kPa (0.01Hz), and contained 5.7% wet weight (%/ww) of glycosaminoglycans (≥ native levels) and 1.5%/ww collagen. We found that the initial seeding density had pronounced effects on tissue outcomes, with high cell seeding densities significantly increasing nearly all measured properties. Transient TGF-β treatment was ineffective for adult human cells, and tissue construct properties plateaued or declined beyond 28 days of culture. Finally, nutrient channels improved construct mechanical properties, presumably due to enhanced rates of mass transport. These results demonstrate that our previously established culture system can be successfully translated to human chondrocytes. PMID:27198889

  2. Demineralized bone promotes chondrocyte or osteoblast differentiation of human marrow stromal cells cultured in collagen sponges.

    PubMed

    Zhou, Shuanhu; Yates, Karen E; Eid, Karim; Glowacki, Julie

    2005-01-01

    Demineralized bone implants have been used for many types of craniomaxillofacial, orthopedic, periodontal, and hand reconstruction procedures. In previous studies, we showed that demineralized bone powder (DBP) induces chondrogenesis of human dermal fibroblasts in a DBP/collagen sponge system that optimized interactions between particles of DBP and target cells in cell culture. In this study, we test the hypothesis that DBP promotes chondrogenesis or osteogenesis of human marrow stromal cells (hMSCs) in 3-D collagen sponge culture, depending upon the culture conditions. We first confirmed that hMSCs have chondrogenic potential when treated with TGF-beta, either in 2-D monolayer cultures or in 3-D porous collagen sponges. Second, we found that DBP markedly enhanced chondrogenesis in hMSCs in 3-D sponges, as assessed by metachromasia and expression of chondrocyte-specific genes AGGRECAN, COL II, and COL X. Human dermal fibroblasts (hDFs) were used to define mechanisms of chondroinduction because unlike hMSCs they have no inherent chondrogenic potential. In situ hybridization revealed that hDFs vicinal to DBPs express chondrocyte-specific genes AGGRECAN or COL II. Macroarray analysis showed that DBP activates TGF-beta/BMP signaling pathway genes in hDFs. Finally, DBP induced hMSCs to express the osteoblast phenotype when cultured with osteogenic supplements. These studies show how culture conditions can influence the differentiation pathway that human marrow stromal cells follow when stimulated by DBP. These results support the potential to engineer cartilage or bone in vitro by using human bone marrow stromal cells and DBP/collagen scaffolds. PMID:15735899

  3. Three-Dimensional Scaffold-Free Fusion Culture: the Way to Enhanced Chondrogenesis of in vitro Propagated Human Articular Chondrocytes

    PubMed Central

    Lehmann, M.; Martin, F.; Mannigel, K.; Kaltschmidt, K.; Sack, U.; Anderer, U.

    2013-01-01

    Cartilage regeneration based on isolated and culture-expanded chondrocytes has been studied in various in vitro models, but the quality varies with respect to the morphology and the physiology of the synthesized tissues. The aim of our study was to promote in vitro chondrogenesis of human articular chondrocytes using a novel three-dimensional (3-D) cultivation system in combination with the chondrogenic differentiation factors transforming growth factor beta 2 (TGF-β2) and L-ascorbic acid. Articular chondrocytes isolated from six elderly patients were expanded in monolayer culture. A single-cell suspension of the dedifferentiated chondrocytes was then added to agar-coated dishes without using any scaffold material, in the presence, or absence of TGF-β2 and/or L-ascorbic acid. Three-dimensional cartilage-like constructs, called single spheroids, and microtissues consisting of several spheroids fused together, named as fusions, were formed. Generated tissues were mainly characterized using histological and immunohistochemical techniques. The morphology of the in vitro tissues shared some similarities to native hyaline cartilage in regard to differentiated S100-positive chondrocytes within a cartilaginous matrix, with strong collagen type II expression and increased synthesis of proteoglycans. Finally, our innovative scaffold-free fusion culture technique supported enhanced chondrogenesis of human articular chondrocytes in vitro. These 3-D hyaline cartilage-like microtissues will be useful for in vitro studies of cartilage differentiation and regeneration, enabling optimization of functional tissue engineering and possibly contributing to the development of new approaches to treat traumatic cartilage defects or osteoarthritis. PMID:24441184

  4. Matrine inhibits IL-1β-induced expression of matrix metalloproteinases by suppressing the activation of MAPK and NF-κB in human chondrocytes in vitro

    PubMed Central

    Lu, Shijin; Xiao, Xungang; Cheng, Minghua

    2015-01-01

    Interleukin (IL)-1β plays an important role in promoting osteoarthritis (OA) lesions by inducing chondrocytes to secrete matrix metalloproteinases (MMPs), which degrade the extracellular matrix and facilitate chondrocyte apoptosis. Matrine was shown to exert anti-inflammatory effects. However, the role of matrine in OA is still unclear. Therefore, in this study, we investigated the effects of matrine on the expression of MMPs in IL-1β-treated human chondrocytes and the underlying mechanism. The cell viability of chondrocytes was detected by MTT assay. The cell apoptosis of chondrocytes was measured by flow cytometric analysis. The protein production of MMPs was determined by ELISA. The protein expression of phosphorylation of mitogen-activated protein kinases (MAPKs) and the inhibitor of kappaB alpha (IκBα) was determined by Western blot. Matrine significantly inhibited the IL-1β-induced apoptosis in chondrocytes. It also significantly inhibited the IL-1β-induced release of MMP-3 and MMP-13, and increased the production of TIMP-1. Furthermore, matrine inhibits the phosphorylation of p-38, extracellular regulated kinase (ERK), c-Jun-N-terminal kinase (JNK) and IκBα degradation induced by IL-1β in chondrocytes. Taken together, our results show that matrine inhibits IL-1β-induced expression of matrix metalloproteinases by suppressing the activation of MAPK and NF-κB in human chondrocytes in vitro. Therefore,-matrine may be beneficial in the treatment of OA. PMID:26191166

  5. C-type natriuretic peptide signalling drives homeostatic effects in human chondrocytes.

    PubMed

    Peake, N J; Bader, D L; Vessillier, S; Ramachandran, M; Salter, D M; Hobbs, A J; Chowdhury, T T

    2015-10-01

    Signals induced by mechanical loading and C-type natriuretic peptide (CNP) represent chondroprotective routes that may potentially prevent osteoarthritis (OA). We examined whether CNP will reduce hyaluronan production and export via members of the multidrug resistance protein (MRP) and diminish pro-inflammatory effects in human chondrocytes. The presence of interleukin-1β (IL-1β) increased HA production and export via MRP5 that was reduced with CNP and/or loading. Treatment with IL-1β conditioned medium increased production of catabolic mediators and the response was reduced with the hyaluronan inhibitor, Pep-1. The induction of pro-inflammatory cytokines by the conditioned medium was reduced by CNP and/or Pep-1, αCD44 or αTLR4 in a cytokine-dependent manner, suggesting that the CNP pathway is protective and should be exploited further. PMID:26307537

  6. Matrilin-3 activates the expression of osteoarthritis-associated genes in primary human chondrocytes.

    PubMed

    Klatt, Andreas R; Klinger, Gabriele; Paul-Klausch, Brigitte; Kühn, Gertrud; Renno, Joerg H; Wagener, Raimund; Paulsson, Mats; Schmidt, Joachim; Malchau, Gebhart; Wielckens, Klaus

    2009-11-19

    Here, we tested the matrilin-3-dependent induction of osteoarthritis-associated genes in primary human chondrocytes. Matrilin stimulation leads to the induction of MMP1, MMP3, MMP13, COX-2, iNOS, IL-1beta, TNFalpha, IL-6 and IL-8. Furthermore, we show the participation of ADAMTS4 and ADAMTS5 in the in vitro degradation of matrilin-3. We provide evidence for a matrilin-3-dependent feed-forward mechanism of matrix degradation, whereby proteolytically-released matrilin-3 induces pro-inflammatory cytokines as well as ADAMTS4 and -5 indirectly via IL-1beta. ADAMTS4 and ADAMTS5, in turn, cleave matrilin-3 and may release more matrilin-3 from the matrix, which could lead to further release of pro-inflammatory cytokines and proteases in cartilage. PMID:19840795

  7. Differential regulation and expression of hyaluronan synthases in human articular chondrocytes, synovial cells and osteosarcoma cells.

    PubMed Central

    Recklies, A D; White, C; Melching, L; Roughley, P J

    2001-01-01

    Recently three isoforms of hyaluronan synthase (HAS), the enzyme responsible for hyaluronate/hyaluronan (HA) biosynthesis, have been cloned, allowing us to study their expression pattern. Our objective was to determine which of the HAS isoenzymes were expressed in human articular chondrocytes, synovial fibroblasts and osteosarcoma cells, whether their expression could be modulated by growth factors (insulin-like growth factor-1, basic fibroblast growth factor and transforming growth factor (TGF-beta1) and cytokines [interleukin 1beta1 (IL-1beta)], and whether changes in the rate of HA synthesis by the cells correlated with changes in mRNA levels for one or more of the HAS isoforms. All three HAS isoforms were found to be expressed in the cultured cells analysed in this study, although the relative proportions varied for each cell type. HAS2 mRNA was usually predominant in chondrocytes, whereas synovial cells contained increased amounts of HAS1. HAS3 was always the least abundant message. The rapidly growing osteosarcoma cells contained almost exclusively HAS2 message. HAS usage in uncultured cartilage and synovial tissues was similar to that in the cultured cells, with HAS2 message being the predominant species in cartilage and HAS1 usually being the predominant species in synovium. HA synthesis was stimulated by the growth factors, but the extent of the response was cell-type specific. Synovial cells responded particularly well to IL-1beta, and showed a unique synergistic response when IL-1beta was used in combination with TGF-beta1. This response was much reduced in articular chondrocytes and absent in the osteosarcoma cells. Analysis of changes in HAS message levels indicated that there was often no correlation with the changes in HA secretion following exposure to growth factors. Although HAS-1 mRNA was increased in synovial cells after exposure to TGF-beta1/IL-1beta, the magnitude of the change was far less than the effect on HA synthesis. Our data thus

  8. Echinocystic Acid Inhibits IL-1β-Induced COX-2 and iNOS Expression in Human Osteoarthritis Chondrocytes.

    PubMed

    Ma, Zhiqiang; Wang, Yanlong; Piao, Taikui; Liu, Jianyu

    2016-04-01

    Echinocystic acid (EA), a pentacyclic triterpene isolated from the fruits of Gleditsia sinensis Lam, displays a range of pharmacological activities including anti-inflammatory and antioxidant effects. However, the effect of EA on IL-1β-stimulated osteoarthritis chondrocyte has not been reported. The purpose of this study was to assess the effects of EA on IL-1β-stimulated human osteoarthritis chondrocyte. Chondrocytes were stimulated with IL-1β in the absence or presence of EA. NO and PGE2 production were measured by Griess reagent and ELISA. The expression of COX-2, iNOS, nuclear factor-κB (NF-κB), inhibitory kappa B (IκBα), c-Jun N-terminal kinase (JNK), p38, and extracellular signal-regulated kinase (ERK) were detected by Western blot analysis. The results showed that EA suppressed IL-1β-induced collagenase-3 (MMP-13), NO, and PGE2 production in a dose-dependent manner. IL-1β up-regulated the expression of COX-2 and iNOS, and the increase was inhibited by EA. Furthermore, IL-1β-induced NF-κB and mitogen-activated protein kinase (MAPK) activation were inhibited by EA. In conclusion, EA effectively attenuated IL-1β-induced inflammatory response in osteoarthritis chondrocyte which suggesting that EA may be a potential agent in the treatment of osteoarthritis. PMID:26499345

  9. Hydroxytyrosol Prevents Increase of Osteoarthritis Markers in Human Chondrocytes Treated with Hydrogen Peroxide or Growth-Related Oncogene α

    PubMed Central

    Facchini, Annalisa; Cetrullo, Silvia; D'Adamo, Stefania; Guidotti, Serena; Minguzzi, Manuela; Facchini, Andrea; Borzì, Rosa Maria; Flamigni, Flavio

    2014-01-01

    Hydroxytyrosol (HT), a phenolic compound mainly derived from olives, has been proposed as a nutraceutical useful in prevention or treatment of degenerative diseases. In the present study we have evaluated the ability of HT to counteract the appearance of osteoarthritis (OA) features in human chondrocytes. Pre-treatment of monolayer cultures of chondrocytes with HT was effective in preventing accumulation of reactive oxidant species (ROS), DNA damage and cell death induced by H2O2 exposure, as well as the increase in the mRNA level of pro-inflammatory, matrix-degrading and hypertrophy marker genes, such as iNOS, COX-2, MMP-13, RUNX-2 and VEGF. HT alone slightly enhanced ROS production, but did not enhance cell damage and death or the expression of OA-related genes. Moreover HT was tested in an in vitro model of OA, i.e. three-dimensional micromass cultures of chondrocytes stimulated with growth-related oncogene α (GROα), a chemokine involved in OA pathogenesis and known to promote hypertrophy and terminal differentiation of chondrocytes. In micromass constructs, HT pre-treatment inhibited the increases in caspase activity and the level of the messengers for iNOS, COX-2, MMP-13, RUNX-2 and VEGF elicited by GROα. In addition, HT significantly increased the level of SIRT-1 mRNA in the presence of GROα. In conclusion, the present study shows that HT reduces oxidative stress and damage, exerts pro-survival and anti-apoptotic actions and favourably influences the expression of critical OA-related genes in human chondrocytes treated with stressors promoting OA-like features. PMID:25279550

  10. Fibroblast growth factor receptor 1 is principally responsible for fibroblast growth factor 2-induced catabolic activities in human articular chondrocytes

    PubMed Central

    2011-01-01

    Introduction Cartilage degeneration driven by catabolic stimuli is a critical pathophysiological process in osteoarthritis (OA). We have defined fibroblast growth factor 2 (FGF-2) as a degenerative mediator in adult human articular chondrocytes. Biological effects mediated by FGF-2 include inhibition of proteoglycan production, up-regulation of matrix metalloproteinase-13 (MMP-13), and stimulation of other catabolic factors. In this study, we identified the specific receptor responsible for the catabolic functions of FGF-2, and established a pathophysiological connection between the FGF-2 receptor and OA. Methods Primary human articular chondrocytes were cultured in monolayer (24 hours) or alginate beads (21 days), and stimulated with FGF-2 or FGF18, in the presence or absence of FGFR1 (FGF receptor 1) inhibitor. Proteoglycan accumulation and chondrocyte proliferation were assessed by dimethylmethylene blue (DMMB) assay and DNA assay, respectively. Expression of FGFRs (FGFR1 to FGFR4) was assessed by flow cytometry, immunoblotting, and quantitative real-time PCR (qPCR). The distinctive roles of FGFR1 and FGFR3 after stimulation with FGF-2 were evaluated using either pharmacological inhibitors or FGFR small interfering RNA (siRNA). Luciferase reporter gene assays were used to quantify the effects of FGF-2 and FGFR1 inhibitor on MMP-13 promoter activity. Results Chondrocyte proliferation was significantly enhanced in the presence of FGF-2 stimulation, which was inhibited by the pharmacological inhibitor of FGFR1. Proteoglycan accumulation was reduced by 50% in the presence of FGF-2, and this reduction was successfully rescued by FGFR1 inhibitor. FGFR1 inhibitors also fully reversed the up-regulation of MMP-13 expression and promoter activity stimulated by FGF-2. Blockade of FGFR1 signaling by either chemical inhibitors or siRNA targeting FGFR1 rather than FGFR3 abrogated the up-regulation of matrix metalloproteinases 13 (MMP-13) and a disintegrin and

  11. Intracellular Delivery of Poorly Soluble Polyphenols: Elucidating the Interplay of Self-Assembling Nanocarriers and Human Chondrocytes.

    PubMed

    Kann, Birthe; Spengler, Christian; Coradini, Karine; Rigo, Lucas A; Bennink, Martin L; Jacobs, Karin; Offerhaus, Herman L; Beck, Ruy C R; Windbergs, Maike

    2016-07-19

    Increased molecular understanding of multifactorial diseases paves the way for novel therapeutic approaches requiring sophisticated carriers for intracellular delivery of actives. We designed and characterized self-assembling lipid-core nanocapsules for coencapsulation of two poorly soluble natural polyphenols curcumin and resveratrol. The polyphenols were identified as high-potential therapeutic candidates intervening in the intracellular inflammation cascade of chondrocytes during the progress of osteoarthritis. To elucidate the interplay between chondrocytes and nanocapsules and their therapeutic effect, we pursued a complementary analytical approach combining label-free visualization with biological assays. Primary human chondrocytes did not show any adverse effects upon nanocapsule application and coherent anti-Stokes Raman scattering images visualized their intracellular uptake. Further, by systematically blocking different uptake mechanisms, an energy independent uptake into the cells could be identified. Additionally, we tested the therapeutic effect of the polyphenol-loaded carriers on inflamed chondrocytes. Treatment with nanocapsules resulted in a major reduction of nitric oxide levels, a well-known apoptosis trigger during the course of osteoarthritis. For a more profound examination of this protective effect on joint cells, we pursued studies with atomic force microscopy investigations. Significant changes in the cell cytoskeleton as well as prominent dents in the cell membrane upon induced apoptosis were revealed. Interestingly, these effects could not be detected for chondrocytes which were pretreated with the nanocapsules. Overall, besides presenting a sophisticated carrier system for joint application, these results highlight the necessity of establishing combinatorial analytical approaches to elucidate cellular uptake, the interplay of codelivered drugs and their therapeutic effect on the subcellular level. PMID:27329347

  12. The Involvement of Mutual Inhibition of ERK and mTOR in PLCγ1-Mediated MMP-13 Expression in Human Osteoarthritis Chondrocytes

    PubMed Central

    Liu, Zejun; Cai, Heguo; Zheng, Xinpeng; Zhang, Bing; Xia, Chun

    2015-01-01

    The issue of whether ERK activation determines matrix synthesis or degradation in osteoarthritis (OA) pathogenesis currently remains controversial. Our previous study shows that PLCγ1 and mTOR are involved in the matrix metabolism of OA cartilage. Investigating the interplays of PLCγ1, mTOR and ERK in matrix degradation of OA will facilitate future attempts to manipulate ERK in OA prevention and therapy. Here, cultured human normal chondrocytes and OA chondrocytes were treated with different inhibitors or transfected with expression vectors, respectively. The levels of ERK, p-ERK, PLCγ1, p-PLCγ1, mTOR, p-mTOR and MMP-13 were then evaluated by Western blotting analysis. The results manifested that the expression level of ERK in human OA chondrocytes was lower than that in human normal articular chondrocytes, and the up-regulation of ERK could promote matrix synthesis, including the decrease in MMP-13 level and the increase in Aggrecan level in human OA chondrocytes. Furthermore, the PLCγ1/ERK axis and a mutual inhibition of mTOR and ERK were observed in human OA chondrocytes. Interestingly, activated ERK had no inhibitory effect on MMP-13 expression in PLCγ1-transformed OA chondrocytes. Combined with our previous study, the non-effective state of ERK activation by PLCγ1 on MMP-13 may be partly attributed to the inhibition of the PLCγ1/mTOR axis on the PLCγ1/ERK axis. Therefore, the study indicates that the mutual inhibition of ERK and mTOR is involved in PLCγ1-mediated MMP-13 expression in human OA chondrocytes, with important implication for the understanding of OA pathogenesis as well as for its prevention and therapy. PMID:26247939

  13. 3D Bioprinting Human Chondrocytes with Nanocellulose-Alginate Bioink for Cartilage Tissue Engineering Applications.

    PubMed

    Markstedt, Kajsa; Mantas, Athanasios; Tournier, Ivan; Martínez Ávila, Héctor; Hägg, Daniel; Gatenholm, Paul

    2015-05-11

    The introduction of 3D bioprinting is expected to revolutionize the field of tissue engineering and regenerative medicine. The 3D bioprinter is able to dispense materials while moving in X, Y, and Z directions, which enables the engineering of complex structures from the bottom up. In this study, a bioink that combines the outstanding shear thinning properties of nanofibrillated cellulose (NFC) with the fast cross-linking ability of alginate was formulated for the 3D bioprinting of living soft tissue with cells. Printability was evaluated with concern to printer parameters and shape fidelity. The shear thinning behavior of the tested bioinks enabled printing of both 2D gridlike structures as well as 3D constructs. Furthermore, anatomically shaped cartilage structures, such as a human ear and sheep meniscus, were 3D printed using MRI and CT images as blueprints. Human chondrocytes bioprinted in the noncytotoxic, nanocellulose-based bioink exhibited a cell viability of 73% and 86% after 1 and 7 days of 3D culture, respectively. On the basis of these results, we can conclude that the nanocellulose-based bioink is a suitable hydrogel for 3D bioprinting with living cells. This study demonstrates the potential use of nanocellulose for 3D bioprinting of living tissues and organs. PMID:25806996

  14. Changes in Ultrastructure and Cytoskeletal Aspects of Human Normal and Osteoarthritic Chondrocytes Exposed to Interleukin-1β and Cyclical Hydrostatic Pressure

    PubMed Central

    Pascarelli, Nicola Antonio; Collodel, Giulia; Moretti, Elena; Cheleschi, Sara; Fioravanti, Antonella

    2015-01-01

    The aim of this study was to examine the ultrastructure and cytoskeletal organization in human normal and Osteoarhritic (OA) chondrocytes, exposed to interleukin-1β (IL-1β) and cyclic hydrostatic pressure (HP). Morphological examination by transmission electron microscopy (TEM) and scanning electron microscopy (SEM) revealed differences between normal and OA chondrocytes at the nuclear and cytoplasmic level. IL-1β (5 ng/mL) induced a decrease of the number of mitochondria and Golgi bodies and a significant increase on the percentage of cells rich in vacuolization and in marginated chromatin. Cyclical HP (1–5 MPa, 0.25 Hz, for 3 h) did not change the morphology of normal chondrocytes, but had a beneficial effect on OA chondrocytes increasing the number of organelles. Normal and OA cells subjected to IL-1β and HP recovered cytoplasmic ultrastructure. Immunofluorescence (IF) examination of normal chondrocytes showed an actin signal polarized on the apical sides of the cytoplasm, tubulin and vimentin uniformly distributed throughout cytoplasm and vinculin revealed a punctuated pattern under the plasma membrane. In OA chondrocytes, these proteins partially lost their organization. Stimulation with IL-1β caused, in both type of cells, modification in the cytoskeletal organization; HP counteracted the negative effects of IL-1β. Our results showed structural differences at nuclear, cytoplasmic and cytoskeletal level between normal and OA chondrocytes. IL-1β induced ultrastructural and cytoskeletal modifications, counteracted by a cyclical low HP. PMID:26528971

  15. Curcuminoids extract, hydrolyzed collagen and green tea extract synergically inhibit inflammatory and catabolic mediator's synthesis by normal bovine and osteoarthritic human chondrocytes in monolayer.

    PubMed

    Comblain, Fanny; Sanchez, Christelle; Lesponne, Isabelle; Balligand, Marc; Serisier, Samuel; Henrotin, Yves

    2015-01-01

    The main objective of this study was to assess the in vitro effects of curcuminoids extract, hydrolyzed collagen and green tea extract in normal bovine chondrocytes and osteoarthritic human chondrocytes cultured in monolayer. This study also investigated the synergic or additive effects of these compounds. Enzymatically isolated primary bovine or human chondrocytes were cultured in monolayer until confluence and then incubated for 24 hours or 48 hours in the absence or in the presence of interleukin-1β and with or without curcuminoids extract, hydrolyzed collagen or green tea extract, added alone or in combination, at different concentrations. Cell viability was neither affected by these compounds, nor by interleukin 1β. In the absence of interleukin-1β, compounds did not significantly affect bovine chondrocytes metabolism. In human chondrocytes and in the absence of interleukin 1β, curcuminoids extract alone or in combination with hydrolyzed collagen and green tea extract significantly inhibited matrix metalloproteinase-3 production. In interleukin-1β-stimulated bovine chondrocytes, interleukin-6, inducible nitric oxide synthase, cyclooxygenase2, matrix metalloproteinase 3, a disintegrin and metalloproteinase with thrombospondin type I motifs 4 and a disintegrin and metalloproteinase with thrombospondin type I motifs 5 expressions were decreased by curcuminoids extract alone or in combination with hydrolyzed collagen and green tea extract. The combination of the three compounds was significantly more efficient to inhibit interleukin-1β stimulated matrix metalloproteinase-3 expression than curcuminoids extract alone. In interleukin-1β-stimulated human chondrocytes, nitric oxide, interleukin-6 and matrix metalloproteinase 3 productions were significantly reduced by curcuminoids extract alone or in combination with hydrolyzed collagen and green tea extract. These findings indicate that a mixture of curcuminoids extract, hydrolyzed collagen and green tea

  16. Curcuminoids Extract, Hydrolyzed Collagen and Green Tea Extract Synergically Inhibit Inflammatory and Catabolic Mediator’s Synthesis by Normal Bovine and Osteoarthritic Human Chondrocytes in Monolayer

    PubMed Central

    Comblain, Fanny; Sanchez, Christelle; Lesponne, Isabelle; Balligand, Marc; Serisier, Samuel; Henrotin, Yves

    2015-01-01

    The main objective of this study was to assess the in vitro effects of curcuminoids extract, hydrolyzed collagen and green tea extract in normal bovine chondrocytes and osteoarthritic human chondrocytes cultured in monolayer. This study also investigated the synergic or additive effects of these compounds. Enzymatically isolated primary bovine or human chondrocytes were cultured in monolayer until confluence and then incubated for 24 hours or 48 hours in the absence or in the presence of interleukin-1β and with or without curcuminoids extract, hydrolyzed collagen or green tea extract, added alone or in combination, at different concentrations. Cell viability was neither affected by these compounds, nor by interleukin 1β. In the absence of interleukin-1β, compounds did not significantly affect bovine chondrocytes metabolism. In human chondrocytes and in the absence of interleukin 1β, curcuminoids extract alone or in combination with hydrolyzed collagen and green tea extract significantly inhibited matrix metalloproteinase-3 production. In interleukin-1β-stimulated bovine chondrocytes, interleukin-6, inducible nitric oxide synthase, cyclooxygenase2, matrix metalloproteinase 3, a disintegrin and metalloproteinase with thrombospondin type I motifs 4 and a disintegrin and metalloproteinase with thrombospondin type I motifs 5 expressions were decreased by curcuminoids extract alone or in combination with hydrolyzed collagen and green tea extract. The combination of the three compounds was significantly more efficient to inhibit interleukin-1β stimulated matrix metalloproteinase-3 expression than curcuminoids extract alone. In interleukin-1β-stimulated human chondrocytes, nitric oxide, interleukin-6 and matrix metalloproteinase 3 productions were significantly reduced by curcuminoids extract alone or in combination with hydrolyzed collagen and green tea extract. These findings indicate that a mixture of curcuminoids extract, hydrolyzed collagen and green tea

  17. Betulinic acid inhibits IL-1β-induced inflammation by activating PPAR-γ in human osteoarthritis chondrocytes.

    PubMed

    Jingbo, Wang; Aimin, Chen; Qi, Wu; Xin, Li; Huaining, Li

    2015-12-01

    Betulinic acid (BA), a triterpenoid isolated from birch bark, has been reported to have anti-inflammatory effects. In this study, we investigated the anti-osteoarthritic effects of BA in IL-1β-stimulated human osteoarthritis chondrocytes. Human osteoarthritis chondrocytes were pre-incubated with BA (6, 12, 24μM) for 12h and then treated with IL-1β (10ng/ml). The production of PGE2 and NO were detected by ELISA and Griess reagent. The expression of NF-κB, IκB, and PPAR-γ were detected by Western blotting. The results showed that BA dose-dependently inhibited IL-1β-induced MMP-1, MMP-3, MMP-13, PGE2 and NO productions. BA also inhibited IL-1β-induced NF-κB activation. Furthermore, BA was found to activate PPAR-γ and the inhibition of PGE2 and NO by BA can be reversed by PPAR-γ antagonist GW9662. In conclusion, these results suggested that BA inhibited IL-1β-induced inflammation in osteoarthritis chondrocytes by activating PPAR-γ. PMID:26391061

  18. Heparin-based self-assembling peptide scaffold reestablish chondrogenic phenotype of expanded de-differentiated human chondrocytes.

    PubMed

    Recha-Sancho, Lourdes; Semino, Carlos E

    2016-07-01

    The use of chondrocytes in cell-based therapies for cartilage lesions are limited by quantity and, therefore, require an in vitro expansion. As monolayer culture leads to de-differentiation, different culture techniques are currently under development to recover chondrocyte phenotype after cell expansion. In the present work, we studied the capacity of the bimolecular heparin-based self-assembling peptide scaffold (RAD16-I) as a three-dimensional (3D) culture system to foster reestablishment of chondrogenic phenotype of de-differentiated human Articular Chondrocytes (AC). The culture was performed in a serum-free medium under control and chondrogenic induction and good viability results were observed after 4 weeks of culture in both conditions. Cells changed their morphology to a more elongated shape and established a cellular network that induced the condensation of the constructs in the case of chondrogenic medium, leading to a compacted structure with improved mechanical properties. Specific extracellular matrix (ECM) proteins of mature cartilage, such as collagen type II and aggrecan were up-regulated under chondrogenic medium and significantly enhanced with the presence of heparin in the scaffold. 3D constructs became highly stained with toluidine blue dye after 4 weeks of culture, indicating the presence of synthetized proteoglycans (PGs) by the cells. Interestingly, the full viscoelastic behavior was closely related to that found in chicken native cartilage. Altogether, the results suggest that the 3D culture model described can help de-differentiated human chondrocytes to recover its cartilage phenotype. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 1694-1706, 2016. PMID:26939919

  19. Hyaluronan suppresses lidocaine-induced apoptosis of human chondrocytes in vitro by inhibiting the p53-dependent mitochondrial apoptotic pathway

    PubMed Central

    Lee, Yoon-Jin; Kim, Soo A; Lee, Sang-Han

    2016-01-01

    Aim: Intra-articular injection of local anesthetics (LAs) is a common procedure for therapeutic purposes. However, LAs have been found toxic to articular cartilage, and hyaluronan may attenuate this toxicity. In this study we investigated whether hyaluronan attenuated lidocaine-induced chondrotoxicity, and if so, to elucidate the underlying mechanisms. Methods: Human chondrocyte cell line SW1353 and newly isolated murine chondrocytes were incubated in culture medium containing hyaluronan and/or lidocaine for 72 h. Cell viability was evaluated using MTT assay. Cell apoptosis was detected with DAPI staining, caspase 3/7 activity assay and flow cytometry. Cell cycle distributions, ROS levels and mitochondrial membrane potential (ΔΨm) were determined using flow cytometry. The expression of p53 and p53-regulated gene products was measured with Western blotting. Results: Lidocaine (0.005%−0.03%) dose-dependently decreased the viability of SW1353 cells. This local anesthetic (0.015%, 0.025%) induced apoptosis, G2/M phase arrest and loss of ΔΨm, and markedly increased ROS production in SW1353 cells. Hyaluronan (50−800 μg/mL) alone did not affect the cell viability, but co-treatment with hyaluronan (200 μg/mL) significantly attenuated lidocaine-induced apoptosis and other abnormalities in SW1353 cells. Furthermore, co-treatment with lidocaine and hyaluronan significantly decreased the levels of p53 and its transcription targets Bax and p21 in SW1353 cells, although treatment with lidocaine alone did not significantly change these proteins. Similar results were obtained in ex vivo cultured murine chondrocytes. Conclusion: Hyaluronan suppresses lidocaine-induced apoptosis of human chondrocytes in vitro through inhibiting the p53-dependent mitochondrial apoptotic pathway. PMID:27041463

  20. SOCS1 suppresses IL-1β-induced C/EBPβ expression via transcriptional regulation in human chondrocytes

    PubMed Central

    Ha, You-Jung; Choi, Yong Seok; Kang, Eun Ha; Shin, Kichul; Kim, Tae Kyun; Song, Yeong Wook; Lee, Yun Jong

    2016-01-01

    CAAT/enhancer-binding protein-beta (C/EBPβ) is a transcription factor that regulates interleukin-1β (IL-1β)-induced catabolic pathways, including the expression of matrix metalloproteinases (MMPs), in chondrocytes. We previously reported that suppressor of cytokine signaling 1 (SOCS1) inhibits IL-1β signaling in chondrocytes. However, the effect of SOCS1 on C/EBPβ has not been explored. To investigate the interaction between SOCS1 and C/EBPβ, we established human SW1353 cells with overexpression or knockdown of SOCS1 or C/EBPβ. Both SOCS1 and C/EBPβ were involved in transcription of MMP-3 and MMP-13. When stimulated with IL-1β, C/EBPβ levels were significantly increased by SOCS1 knockdown and decreased by SOCS1 overexpression. A similar change in IL-1β-induced C/EBPβ expression was observed in SOCS1-transfected human articular chondrocytes. However, C/EBPβ overexpression or knockdown did not change the levels of IL-1β-induced SOCS1. SOCS1 regulated the levels of C/EBPβ mRNA by ubiquitination of C/EBPβ as well as transcriptional regulation. Furthermore, it suppressed the phosphorylation of cAMP response element-binding protein (CREB), an active transcription factor of C/EBPβ. In addition, p38 mitogen-activated protein kinases, a target of SOCS1, was involved in CREB phosphorylation. The chromatin immunoprecipitation assay confirmed that SOCS1 overexpression led to reduced binding of C/EBPβ to the MMP-13 promoter. Taken together, our results demonstrate that SOCS1 downregulates the p38-CREB-C/EBPβ pathway resulting in increased expression of MMPs in chondrocytes. PMID:27339399

  1. SOCS1 suppresses IL-1β-induced C/EBPβ expression via transcriptional regulation in human chondrocytes.

    PubMed

    Ha, You-Jung; Choi, Yong Seok; Kang, Eun Ha; Shin, Kichul; Kim, Tae Kyun; Song, Yeong Wook; Lee, Yun Jong

    2016-01-01

    CAAT/enhancer-binding protein-beta (C/EBPβ) is a transcription factor that regulates interleukin-1β (IL-1β)-induced catabolic pathways, including the expression of matrix metalloproteinases (MMPs), in chondrocytes. We previously reported that suppressor of cytokine signaling 1 (SOCS1) inhibits IL-1β signaling in chondrocytes. However, the effect of SOCS1 on C/EBPβ has not been explored. To investigate the interaction between SOCS1 and C/EBPβ, we established human SW1353 cells with overexpression or knockdown of SOCS1 or C/EBPβ. Both SOCS1 and C/EBPβ were involved in transcription of MMP-3 and MMP-13. When stimulated with IL-1β, C/EBPβ levels were significantly increased by SOCS1 knockdown and decreased by SOCS1 overexpression. A similar change in IL-1β-induced C/EBPβ expression was observed in SOCS1-transfected human articular chondrocytes. However, C/EBPβ overexpression or knockdown did not change the levels of IL-1β-induced SOCS1. SOCS1 regulated the levels of C/EBPβ mRNA by ubiquitination of C/EBPβ as well as transcriptional regulation. Furthermore, it suppressed the phosphorylation of cAMP response element-binding protein (CREB), an active transcription factor of C/EBPβ. In addition, p38 mitogen-activated protein kinases, a target of SOCS1, was involved in CREB phosphorylation. The chromatin immunoprecipitation assay confirmed that SOCS1 overexpression led to reduced binding of C/EBPβ to the MMP-13 promoter. Taken together, our results demonstrate that SOCS1 downregulates the p38-CREB-C/EBPβ pathway resulting in increased expression of MMPs in chondrocytes. PMID:27339399

  2. Overexpression of microRNA-634 suppresses survival and matrix synthesis of human osteoarthritis chondrocytes by targeting PIK3R1

    PubMed Central

    Cui, Xu; Wang, Shaojie; Cai, Heguo; Lin, Yuan; Zheng, Xinpeng; Zhang, Bing; Xia, Chun

    2016-01-01

    Osteoarthritis (OA) is a degenerative disease characterized by deterioration of articular cartilage. Recent studies have demonstrated the importance of some microRNAs in cartilage damage. The aim of this study was to identify and characterize the expression of microRNA-634 (miR-634) in normal and OA chondrocytes, and to determine its role in OA pathogenesis. Human normal and OA chondrocytes obtained from patients were cultured in vitro. Transfection with miR-634 mimic or inhibitor was employed to investigate the effect of miR-634 on chondrocyte survival and matrix synthesis, and to identify miR-634 target. The results indicated that miR-634 was expressed at lower level in high grade OA chondrocyte compared with normal chondrocytes. Overexpression of miR-634 could inhibit cell survival and matrix synthesis in high grade OA chondrocytes. Furthermore, miR-634 targeted PIK3R1 gene that encodes the regulatory subunit 1 of class I PI3K (p85α) and exerted its inhibitory effect on the phosphorylation of Akt, mTOR, and S6 signal molecules in high grade OA chondrocytes. Therefore, the data suggested that miR-634 could suppress survival and matrix synthesis of high grade OA chondrocytes through targeting PIK3R1 gene to modulate the PI3K/Akt/S6 and PI3K/Akt/mTOR/S6 axes, with important implication for validating miR-634 as a potential target for OA therapy. PMID:26972586

  3. Co-culture with human synovium-derived mesenchymal stem cells inhibits inflammatory activity and increases cell proliferation of sodium nitroprusside-stimulated chondrocytes

    SciTech Connect

    Ryu, Jae-Sung; Jung, Yeon-Hwa; Cho, Mi-Young; Yeo, Jee Eun; Choi, Yun-Jin; Kim, Yong Il; Koh, Yong-Gon

    2014-05-16

    Highlights: • Co-culture of hSDMSCs with SNP-stimulated chondrocytes improves anti-inflammation. • Co-culture system produces IGF-1. • Co-culture system suppresses inflammatory genes expression. • Co-culture system improves cell proliferation. • Exogenous IGF-1 inhibits inflammatory activity in SNP-stimulated chondrocytes. - Abstract: Rheumatoid arthritis (RA) and osteoarthritis (OA) are primarily chronic inflammatory diseases. Mesenchymal stem cells (MSCs) have the ability to differentiate into cells of the mesodermal lineage, and to regulate immunomodulatory activity. Specifically, MSCs have been shown to secrete insulin-like growth factor 1 (IGF-1). The purpose of the present study was to examine the inhibitory effects on inflammatory activity from a co-culture of human synovium-derived mesenchymal stem cells (hSDMSCs) and sodium nitroprusside (SNP)-stimulated chondrocytes. First, chondrocytes were treated with SNP to generate an in vitro model of RA or OA. Next, the co-culture of hSDMSCs with SNP-stimulated chondrocytes reduced inflammatory cytokine secretion, inhibited expression of inflammation activity-related genes, generated IGF-1 secretion, and increased the chondrocyte proliferation rate. To evaluate the effect of IGF-1 on inhibition of inflammation, chondrocytes pre-treated with IGF-1 were treated with SNP, and then the production of inflammatory cytokines was analyzed. Treatment with IGF-1 was shown to significantly reduce inflammatory cytokine secretion in SNP-stimulated chondrocytes. Our results suggest that hSDMSCs offer a new strategy to promote cell-based cartilage regeneration in RA or OA.

  4. The overexpression of SIRT1 inhibited osteoarthritic gene expression changes induced by interleukin-1β in human chondrocytes.

    PubMed

    Matsushita, Takehiko; Sasaki, Hiroshi; Takayama, Koji; Ishida, Kazunari; Matsumoto, Tomoyuki; Kubo, Seiji; Matsuzaki, Tokio; Nishida, Kotaro; Kurosaka, Masahiro; Kuroda, Ryosuke

    2013-04-01

    In this study, we examined the effects of overexpression of SIRT1 on IL-1β-induced gene expression changes in human chondrocytes to explore a protective role of SIRT1 in human chondrocytes. SIRT1 was overexpressed in human chondrocytes by expression plasmid under stimulation with IL-1β. SIRT1 was also inhibited by siRNA under stimulation with IL-1β. Gene expression changes were examined by real-time PCR. The interaction of SIRT1 and p65 (NF-κB) were examined by Western blotting. SIRT1, MMP-13, and ADAMTS-5 expressions in human cartilage were examined by immunohistochemistry. IL-1β stimulation significantly up-regulated MMP-1, 2, 9, and 13 and ADAMTS-5. Overexpression of SIRT1 significantly inhibited the up-regulation of those genes caused by IL-1β while the inhibition of SIRT1 further increased them. In addition, the overexpression of SIRT1 markedly reduced the IL-1β-induced acetylation of p65. SIRT1 expression was clearly detected in the non-OA cartilage while MMP-13 and ADAMTS-5 were undetectable. In contrast, in the OA cartilage, SIRT1 expression was decreased while MMP-13 and ADAMTS-5 were increased. Our observations suggested that SIRT1 can play a protective role by suppressing IL-1β-induced expressions of cartilage-degrading enzymes partially through the modulation of the NF-κB pathway. SIRT1 overexpression might be a new therapeutic approach for OA. PMID:23143889

  5. A hexadecylamide derivative of hyaluronan (HYMOVIS®) has superior beneficial effects on human osteoarthritic chondrocytes and synoviocytes than unmodified hyaluronan

    PubMed Central

    2013-01-01

    Background Intra-articular hyaluronan (HA) injection provides symptomatic benefit in the treatment of osteoarthritis (OA). Previously we found superior beneficial effects in a large animal OA model of a hexadecylamide derivative compared with unmodified HA of the same initial molecular weight. The current study sought to define possible molecular mechanisms whereby this enhanced relief of symptoms was occurring. Methods Chondrocytes and synovial fibroblasts were isolated from tissues of patients undergoing arthroplasty for knee OA. Monolayer cultures of cells were treated with 0, 0.5, 1.0 or 1.5 mg/mL of unmodified HA (500–730 kDa) or a hexadecylamide derivative of HA of the same initial molecular weight (HYADD4®-G; HYMOVIS®) simultaneously or 1 hour before incubation with interleukin (IL)-1beta (2 ng/mL). Cultures were terminated 15 or 30 minutes later (chondrocytes and synovial fibroblasts, respectively) for quantitation of phosphorylated-(p)-JNK, p-NFkappaB, p-p38, or at 24 hours for quantitation of gene expression (MMP1 &13, ADAMTS4 &5, TIMP1 &3, CD44, COL1A1 &2A1, ACAN, PTGS2, IL6, TNF) and matrix metalloproteinase (MMP)-13 activity. Results The hexadecylamide derivative of HA had significantly better amelioration of IL-1beta-induced gene expression of key matrix degrading enzymes (MMP1, MMP13, ADAMTS5), and inflammatory mediators (IL6, PTGS2) by human OA chondrocytes and synovial fibroblasts. Pre-incubation of cells with the derivatized HA for 1 hour prior to IL-1beta exposure significantly augmented the inhibition of MMP1, MMP13, ADAMTS4 and IL6 expression by chondrocytes. The reduction in MMP13 mRNA by the amide derivative of HA was mirrored in reduced MMP-13 protein and enzyme activity in IL-1beta-stimulated chondrocytes. This was associated in part with a greater inhibition of phosphorylation of the cell signalling molecules JNK, p38 and NF-kappaB. Conclusions The present studies have demonstrated several potential key mechanisms whereby the

  6. MicroRNA-411 inhibited matrix metalloproteinase 13 expression in human chondrocytes

    PubMed Central

    Wang, Guodong; Zhang, Yuanmin; Zhao, Xiaowei; Meng, Chunyang; Ma, Longfei; Kong, Ying

    2015-01-01

    Osteoarthritis (OA) is the most common joint degenerative disease affecting the joint structure, leading to loss of joint function and tissue destruction. Recent studies have demonstrated that miRNAs are involved in many pathological conditions, including OA. The study was to investigate the role of miR-411 in the pathogenesis of OA. The expression of miR-411 was downregulated in OA cartilage compared with in normal cartilage. Conversely, the expression of MMP-13 was upregulated in OA cartilage compared with in normal cartilage. IL-1β treatment repressed miR-411 expression in chondrocytes. Moreover, we identified MMP-13 as a direct target gene of miR-411 in chondrocytes and overexpression of miR-411 inhibited the MMP-13 expression. Furthermore, overexpression of miR-411 increased the expression of type II collagen and type IV collagen expression in chondrocytes. MiR-411 is a crucial regulator of MMP-13 in chondrocytes and may response to the development of OA. PMID:26692943

  7. Beneficial effect of a sturgeon-based bioactive compound on gene expression of tumor necrosis factor-alpha, matrix metalloproteinases and type-10 collagen in human chondrocytes.

    PubMed

    Catanzaro, R; Marotta, F; Jain, S; Rastmanesh, R; Allegri, F; Celep, G; Lorenzetti, A; Polimeni, A; Yadav, H

    2012-01-01

    In the present study, we examined the effect of a marine bioactive compound containing high-purity caviar-derived DNA, collagen elastin and protein extracts from sturgeon (LD-1227, Caviarlieri, Laboratoires Dom, Switzerland) on IL-1beta-induced activation and production of TNFalpha and MMP-13 in human osteo-arthritis (OA) chondrocytes and intracellular signaling factors. Human chondrocytes were derived from OA cartilage and stimulated with IL-1beta. Gene expression of TNFalpha, MMP-13, MMP-1 and Col10A1 was measured by quantitative RT-PCR. TNFalpha protein in culture medium was determined using cytokine-specific ELISA. Western immunoblotting was used to analyze the MMP-13 production in the culture medium and the activation of NF-kB. DNA binding activity of NF-kB p65 was determined using a highly sensitive and specific ELISA. MMP-13 activity in the culture medium was assayed by gelatine zymography. LD-1227 significantly decreased IL-1beta-stimulated gene expression and production of TNFalpha, MMP-1, MMP-13 and Col10A1 in human chondrocytes. The inhibitory effect of LD-1227 on the IL-1beta-induced expression of these genes was mediated at least in part via suppression of NF-kB p65. These data show that LD-1227 can inhibit IL-1beta-induced proliferation and inflammatory reactions via inhibited activation of the transcription factor NF-kB pathway in human chondrocytes derived from OA patients. These novel pharmacological actions of LD-1227 on IL-1beta-stimulated human OA chondrocytes provide suggestions that this marine biology compound may inhibit cartilage degradation by suppressing IL-1beta-mediated activation and the catabolic response in human chondrocytes. PMID:23034253

  8. Interleukin-1β induced Stress Granules Sequester COX-2 mRNA and Regulates its Stability and Translation in Human OA Chondrocytes

    PubMed Central

    Ansari, Mohammad Y.; Haqqi, Tariq M.

    2016-01-01

    Enhanced and immediate expression of cyclooxygenase-2 (COX-2) mRNA is observed in IL-1β-stimulated OA chondrocytes but the synthesis of protein found significantly delayed. Here we investigated the role of stress granules (SGs), ribonucleoprotein complexes that regulate mRNA translation, in the delayed translation of COX-2 mRNAs in IL-1β-stimulated OA chondrocytes. Stimulation of human chondrocytes with IL-1β activated the stress response genes and the phosphorylation of eIF2α that triggered the assembly of SGs. Using combined immunofluorescence staining of SGs markers and COX-2 protein, RNA fluorescence in situ hybridization and RNA immunoprecipitation, the COX-2 mRNAs were found sequestered in SGs in IL-1β-stimulated OA chondrocytes. No increase in COX-2 protein expression was observed during the persistence of SGs but enhanced expression of COX-2 protein was noted upon clearance of the SGs. Inhibition of SGs clearance blocked COX-2 mRNA translation whereas blocking the assembly of SGs by TIA-1 depletion resulted in rapid and increased production of COX-2 and PGE2. Our findings show for the first time assembly of SGs and sequestration of COX-2 mRNAs in human OA chondrocytes under pathological conditions. Post-transcriptional regulation of COX-2 mRNAs translation by SGs indicates a role in IL-1β-mediated catabolic response that could be therapeutically targeted in OA. PMID:27271770

  9. Chondroprotective effect of three different classes of anti-inflammatory agents on human osteoarthritic chondrocytes exposed to IL-1β.

    PubMed

    Cheleschi, Sara; Pascarelli, Nicola Antonio; Valacchi, Giuseppe; Di Capua, Angela; Biava, Mariangela; Belmonte, Giuseppe; Giordani, Antonio; Sticozzi, Claudia; Anzini, Maurizio; Fioravanti, Antonella

    2015-09-01

    VA694, a promising cyclooxigenase-2 (COX-2)-inhibiting hybrid drug endowed with nitric oxide (NO) releasing properties (NO-COXIB), showed COX-2-selective inhibitory effects, associated with interesting anti-inflammatory and anti-nociceptive activities. Therefore, we studied the effects of VA694 on cartilage metabolism, in comparison with Naproxcinod, a COX inhibitor and NO donor (CINOD), and Naproxen, a traditional non-steroidal-anti-inflammatory drug (NSAID) on human osteoarthritic chondrocyte cultures. IL-1β-stimulated chondrocytes showed a significant decrease in cell viability (P<0.001). VA694, Naproxcinod and Naproxen alone didn't significantly affect cell viability, while it restored cell viability in cultures stimulated by IL-1β. The presence of IL-1β determined a significant increase (P<0.001) in PGE2 levels measured by an ELISA assay, and in COX-2 and MMP-3, -9, and -13 gene expression analyzed by RT-PCR. VA694, Naproxcinod and Naproxen, at both concentrations analyzed, significantly counteracted the negative effects induced by IL-1β. VA694, Naproxcinod and Naproxen pre-treatment were able to inhibit IL-1β-induced NF-κB activation, when measured as its nuclear translocation (p50 and p65 subunits). Naproxcinod and Naproxen pre-treatment didn't affect cytoplasmic NF-κB levels; VA694 decreased the cytoplasmic levels of both subunits. Our data suggest that VA694, Naproxcinod and Naproxen, exert anti-inflammatory and chondroprotective effects on OA chondrocytes. PMID:26292179

  10. Preventing Friction Induced Chondrocyte Apoptosis: A Comparison of Human Synovial Fluid and Hylan G-F 20

    PubMed Central

    Waller, Kimberly A; Zhang, Ling X; Fleming, Braden C; Jay, Gregory D

    2013-01-01

    Objectives Symptomatic osteoarthritis (OA) is a common painful disease with limited treatment options. A rising number of OA patients have been treated with intraarticular injections of hyaluronic acid, including the high molecular weight hylan G-F 20, which is injected following arthrocentesis. This study investigated the effectiveness of hylan G-F 20 to lower coefficient of friction (COF) and prevent chondrocyte apoptosis in vitro. Methods A disc-on-disc bovine cartilage bearing was used to measure the static and kinetic COF when lubricated with hylan G-F 20, human synovial fluid (HSF) and phosphate buffered saline (PBS). Following friction testing, we stained paraffin embedded sections of these cartilage bearings for activated caspase-3, a marker of apoptosis. Results Bearings lubricated with hylan G-F 20 had kinetic COF values that were similar to bearings lubricated with PBS, but significantly higher than those lubricated with HSF. There were no significant differences in static COF values in bearings lubricated with hylan G-F 20 as compared to PBS or HSF. However, bearings lubricated with HSF had a significantly lower static COF values compared to bearings lubricated with PBS. The mean percentage of caspase-3 positive chondrocytes in the superficial and upper intermediate zones of bearings lubricated with hylan G-F 20 were significantly higher when compared to bearings lubricated with HSF or unloaded controls, but significantly lower than those lubricated with PBS. Conclusion These findings indicate that joint lubrication may prevent chondrocyte apoptosis by lowering the COF. Furthermore, removal of synovial fluid prior to hylan G-F 20 injection may be detrimental to cartilage health. PMID:22660808

  11. Ginkgo biloba Extract Individually Inhibits JNK Activation and Induces c-Jun Degradation in Human Chondrocytes: Potential Therapeutics for Osteoarthritis

    PubMed Central

    Ho, Ling-Jun; Hung, Li-Feng; Liu, Feng-Cheng; Hou, Tsung-Yun; Lin, Leou-Chyr; Huang, Chuan-Yueh; Lai, Jenn-Haung

    2013-01-01

    Osteoarthritis (OA) is a common joint disorder with varying degrees of inflammation. The ideal anti-OA drug should have immunomodulatory effects while at the same time having limited or no toxicity. We examined the anti-inflammatory effects of Ginkgo biloba extract (EGb) in interleukin-1 (IL-1)-stimulated human chondrocytes. Chondrocytes were prepared from cartilage specimens taken from patients with osteoarthritis who had received total hip or total knee replacement. The concentrations of chemokines and the degree of cell migration were determined by ELISA and chemotaxis assays, respectively. The activation of inducible nitric oxide synthase (iNOS), mitogen-activated protein kinases (MAPKs), activator protein-1 (AP-1), and nuclear factor-kappaB (NF-κB) was determined by immunoblotting, immunohistochemistry, and electrophoretic mobility shift assay. We found that EGb inhibited IL-1-induced production of chemokines, which in turn resulted in attenuation of THP-1 cell migration toward EGb-treated cell culture medium. EGb also suppressed IL-1-stimulated iNOS expression and release of nitric oxide (NO). The EGb-mediated suppression of the iNOS-NO pathway correlated with the attenuation of activator protein-1 (AP-1) but not nuclear factor-kappaB (NF-κB) DNA-binding activity. Of the mitogen-activated protein kinases (MAPKs), EGb inhibited only c-Jun N-terminal kinase (JNK). Unexpectedly, EGb selectively caused degradation of c-Jun protein. Further investigation revealed that EGb-mediated c-Jun degradation was preceded by ubiquitination of c-Jun and could be prevented by the proteosome inhibitor MG-132. The results imply that EGb protects against chondrocyte degeneration by inhibiting JNK activation and inducing ubiquitination-dependent c-Jun degradation. Although additional research is needed, our results suggest that EGb is a potential therapeutic agent for the treatment of OA. PMID:24349175

  12. MicroRNA-127-5p regulates osteopontin expression and osteopontin-mediated proliferation of human chondrocytes

    PubMed Central

    Tu, Min; Li, Yusheng; Zeng, Chao; Deng, Zhenhan; Gao, Shuguang; Xiao, Wenfeng; Luo, Wei; Jiang, Wei; Li, Liangjun; Lei, Guanghua

    2016-01-01

    The aim of this study was to determine the specific microRNA (miRNA) that regulates expression of osteopontin (OPN) in osteoarthritis (OA). The potential regulatory miRNAs for OPN messenger RNA (mRNA) were predicted by miRNA prediction programs. Among eight potential regulatory miRNAs, miR-220b, miR-513a-3p and miR-548n increased, while miR-181a, miR-181b, miR-181c, miR-181d and miR-127-5p decreased in OA patients. miRNA-127-5p mimics suppressed OPN production as well as the activity of a reporter construct containing the 3′-UTR of human OPN mRNA. In addition, mutation of miR-127-5p binding site in the 3′-UTR of OPN mRNA abolished miR-127-5p-mediated repression of reporter activity. Conversely, treatment with miR-127-5p inhibitor increased reporter activity and OPN production. Interestingly, miR-127-5p inhibited proliferation of chondrocytes through OPN. In conclusion, miRNA-127-5p is an important regulator of OPN in human chondrocytes and may contribute to the development of OA. PMID:27126955

  13. Proteomic and redox-proteomic evaluation of homogentisic acid and ascorbic acid effects on human articular chondrocytes.

    PubMed

    Braconi, Daniela; Laschi, Marcella; Taylor, Adam M; Bernardini, Giulia; Spreafico, Adriano; Tinti, Laura; Gallagher, James A; Santucci, Annalisa

    2010-11-01

    Alkaptonuria (AKU) is a rare genetic disease associated with the accumulation of homogentisic acid (HGA) and its oxidized/polymerized products in connective tissues up to the deposition of melanin-like pigments (ochronosis). Since little is known on the effects of HGA and its metabolites on articular cells, we carried out a proteomic and redox-proteomic analysis to investigate how HGA and ascorbic acid (ASC) affect the human chondrocytic protein repertoire. We settled up an in vitro model using a human chondrocytic cell line to evaluate the effects of 0.33 mM HGA, alone or combined with ASC. We found that HGA and ASC significantly affect the levels of proteins with specific functions in protein folding, cell organization and, notably, stress response and cell defense. Increased protein carbonyls levels were found either in HGA or ASC treated cells, and evidences produced in this paper support the hypothesis that HGA-induced stress might be mediated by protein oxidation. Our finding can lay the basis towards the settling up of more sophisticated models to study AKU and ochronosis. PMID:20665660

  14. Bovine lactoferricin induces TIMP-3 via the ERK1/2-Sp1 axis in human articular chondrocytes

    PubMed Central

    Yan, Dongyao; Chen, Di; Hawse, John R; van Wijnen, Andre J; Im, Hee-Jeong

    2013-01-01

    Bovine lactoferricin (LfcinB) is a heparan sulfate-binding peptide with multiple bioactivities. In human articular cartilage, LfcinB antagonizes interleukin-1 β (IL-1β) and fibroblast growth factor 2 (FGF-2) in proteoglycan metabolism, catabolic protease expression, and induction of pro-inflammatory mediators. LfcinB specifically activates ERK1/2, p38 and Akt, but whether these signaling pathways control the expression of LfcinB target genes remained unknown. In this report, we characterized a novel aspect of LfcinB-mediated genetic response in human articular chondrocytes, tissue inhibitor of metalloproteinase 3 (TIMP-3) induction. Inhibition of individual signaling pathways revealed that ERK1/2 functions as the major pathway in TIMP-3 expression, whereas Akt plays a minor role. Further investigation identified Sp1 as a critical transcriptional activator in TIMP-3 regulation, and Sp1 activity is modulated by ERK1/2, not Akt. Comparative quantification indicates significant downregulation of TIMP-3 occurs in OA chondrocytes, suggesting a beneficial role of LfcinB in OA pathogenesis. Our results collectively provide new insights into the mechanism of action of LfcinB, and support the candidacy of LfcinB as a chondroprotective agent. PMID:23313877

  15. Human osteoarthritic chondrocytes exposed to extremely low-frequency electromagnetic fields (ELF) and therapeutic application of musically modulated electromagnetic fields (TAMMEF) systems: a comparative study.

    PubMed

    Corallo, Claudio; Volpi, Nila; Franci, Daniela; Vannoni, Daniela; Leoncini, Roberto; Landi, Giacomo; Guarna, Massimo; Montella, Antonio; Albanese, Antonietta; Battisti, Emilio; Fioravanti, Antonella; Nuti, Ranuccio; Giordano, Nicola

    2013-06-01

    Osteoarthritis (OA) is the most common joint disease, characterized by matrix degradation and changes in chondrocyte morphology and metabolism. Literature reported that electromagnetic fields (EMFs) can produce benefits in OA patients, even if EMFs mechanism of action is debated. Human osteoarthritic chondrocytes isolated from femoral heads were cultured in vitro in bidimensional (2-D) flasks and in three-dimensional (3-D) alginate beads to mimic closely cartilage environment in vivo. Cells were exposed 30 min/day for 2 weeks to extremely low-frequency electromagnetic field (ELF) with fixed frequency (100 Hz) and to therapeutic application of musically modulated electromagnetic field (TAMMEF) with variable frequencies, intensities, and waveforms. Cell viability was measured at days 7 and 14, while healthy-cell density, heavily vacuolized (hv) cell density, and cluster density were measured by light microscopy only for 3-D cultures after treatments. Cell morphology was observed for 2-D and 3-D cultures by transmission electron microscopy (TEM). Chondrocyte exposure to TAMMEF enhances cell viability at days 7 and 14 compared to ELF. Light microscopy analysis showed that TAMMEF enhances healthy-cell density, reduces hv-cell density and clustering, compared to ELF. Furthermore, TEM analysis showed different morphology for 2-D (fibroblast-like) and 3-D (rounded shape) cultures, confirming light microscopy results. In conclusion, EMFs are effective and safe for OA chondrocytes. TAMMEF can positively interfere with OA chondrocytes representing an innovative non-pharmacological approach to treat OA. PMID:23263545

  16. Effects of regenerative radioelectric asymmetric conveyer treatment on human normal and osteoarthritic chondrocytes exposed to IL-1β. A biochemical and morphological study

    PubMed Central

    Collodel, Giulia; Fioravanti, Antonella; Pascarelli, Nicola Antonio; Lamboglia, Antonello; Fontani, Vania; Maioli, Margherita; Santaniello, Sara; Pigliaru, Gianfranco; Castagna, Alessandro; Moretti, Elena; Iacoponi, Francesca; Rinaldi, Salvatore; Ventura, Carlo

    2013-01-01

    Purpose Osteoarthritis (OA) is a degenerative disease characterized by a progressive loss of articular cartilage extracellular matrix and is due to functional impairments occurring in chondrocytes. In previous works, we highlighted that Regenerative Tissue Optimization (TO-RGN) treatment with radioelectric asymmetric conveyer (REAC) technology influenced the gene expression profiles controlling stem cell differentiation and the pluripotency of human skin-derived fibroblasts in vitro. Since interleukin-1 beta signaling has been implicated in the induction and progression of this disease (through metalloproteinase-3 synthesis and nitric oxide production), we investigated whether REAC TO-RGN might influence the biochemical and morphological changes induced by interleukin-1 beta in normal and OA chondrocytes. Methods The induction of metalloproteinase-3 and proteoglycan synthesis was evaluated by a solid-phase enzyme-amplified sensitivity immunoassay, and nitric oxide production was evaluated with the Griess method. Ultrastructural features were observed by transmission electron microscopy. Results REAC TO-RGN treatment decreased nitric oxide and metalloproteinase-3 production in normal and OA chondrocytes, while inducing an increase in proteoglycan synthesis. OA chondrocytes were more affected by REAC TO-RGN treatment than were normal chondrocytes. Ultrastructural changes confirmed that REAC TO-RGN may counteract the negative effects of interleukin-1 beta incubation. Conclusion The results of this in vitro study suggest that REAC TO-RGN treatment may represent a new, promising approach for the management of OA. PMID:23682210

  17. Synoviocyte Derived-Extracellular Matrix Enhances Human Articular Chondrocyte Proliferation and Maintains Re-Differentiation Capacity at Both Low and Atmospheric Oxygen Tensions

    PubMed Central

    Kean, Thomas J.; Dennis, James E.

    2015-01-01

    Background Current tissue engineering methods are insufficient for total joint resurfacing, and chondrocytes undergo de-differentiation when expanded on tissue culture plastic. De-differentiated chondrocytes show poor re-differentiation in culture, giving reduced glycosaminoglycan (GAG) and collagen matrix accumulation. To address this, porcine synoviocyte-derived extracellular matrix and low (5%) oxygen tension were assessed for their ability to enhance human articular chondrocyte expansion and maintain re-differentiation potential. Methods Porcine synoviocyte matrices were devitalized using 3 non-detergent methods. These devitalized synoviocyte matrices were compared against tissue culture plastic for their ability to support human chondrocyte expansion. Expansion was further compared at both low (5%), and atmospheric (20%) oxygen tension on all surfaces. Expanded cells then underwent chondrogenic re-differentiation in aggregate culture at both low and atmospheric oxygen tension. Aggregates were assessed for their GAG and collagen content both biochemically and histologically. Results Human chondrocytes expanded twice as fast on devitalized synoviocyte matrix vs. tissue culture plastic, and cells retained their re-differentiation capacity for twice the number of population doublings. There was no significant difference in growth rate between low and atmospheric oxygen tension. There was significantly less collagen type I, collagen type II, aggrecan and more MMP13 expression in cells expanded on synoviocyte matrix vs. tissue culture plastic. There were also significant effects due to oxygen tension on gene expression, wherein there was greater collagen type I, collagen type II, SOX9 and less MMP13 expression on tissue culture plastic compared to synoviocyte matrix. There was a significant increase in GAG, but not collagen, accumulation in chondrocyte aggregates re-differentiated at low oxygen tension over that achieved in atmospheric oxygen conditions. Conclusions

  18. Viscoelastic properties of human mesenchymally-derived stem cells and primary osteoblasts, chondrocytes, and adipocytes

    PubMed Central

    Darling, Eric M.; Topel, Matthew; Zauscher, Stefan; Vail, Thomas P.; Guilak, Farshid

    2010-01-01

    The mechanical properties of single cells play important roles in regulating cell-matrix interactions, potentially influencing the process of mechanotransduction. Recent studies also suggest that cellular mechanical properties may provide novel biological markers, or “biomarkers,” of cell phenotype, reflecting specific changes that occur with disease, differentiation, or cellular transformation. Of particular interest in recent years has been the identification of such biomarkers that can be used to determine specific phenotypic characteristics of stem cells that separate them from primary, differentiated cells. The goal of this study was to determine the elastic and viscoelastic properties of three primary cell types of mesenchymal lineage (chondrocytes, osteoblasts, and adipocytes) and to test the hypothesis that primary differentiated cells exhibit distinct mechanical properties compared to adult stem cells (adipose-derived or bone marrow-derived mesenchymal stem cells). In an adherent, spread configuration, chondrocytes, osteoblasts, and adipocytes all exhibited significantly different mechanical properties, with osteoblasts being stiffer than chondrocytes and both being stiffer than adipocytes. Adipose-derived and mesenchymal stem cells exhibited similar properties to each other, but were mechanically distinct from primary cells, particularly when comparing a ratio of elastic to relaxed moduli. These findings will help more accurately model the cellular mechanical environment in mesenchymal tissues, which could assist in describing injury thresholds and disease progression or even determining the influence of mechanical loading for tissue engineering efforts. Furthermore, the identification of mechanical properties distinct to stem cells could result in more successful sorting procedures to enrich multipotent progenitor cell populations. PMID:17825308

  19. Cannabinoid WIN‑55,212‑2 mesylate inhibits ADAMTS‑4 activity in human osteoarthritic articular chondrocytes by inhibiting expression of syndecan‑1.

    PubMed

    Kong, Ying; Wang, Wanchun; Zhang, Changjie; Wu, Yi; Liu, Yang; Zhou, Xiaorong

    2016-06-01

    A central feature of osteoarthritis (OA) is the loss of articular cartilage, which is primarily attributed to cartilage breakdown. A group of metalloproteinases termed the A disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) family are reported to be important in cartilage breakdown. Recent studies have suggested that ADAMTS‑4 is a major contributor to the pathogenesis of OA and that syndecan‑1 is closely associated with activation of ADAMTS‑4 in human chondrocytes. Accumulating evidence also suggests that cannabinoids have chondroprotective effects. The current study explored the effects of synthetic cannabinoid WIN‑55,212‑2 mesylate (WIN‑55) on the expression of syndecan‑1 and ADAMTS‑4, as well as ADAMTS‑4 activity, in unstimulated and interleukin (IL)‑1β‑stimulated OA chondrocytes. Primary human OA articular chondrocytes were treated with WIN‑55 in the presence or absence of IL‑1β and cannabinoid receptor antagonists. The results of the present study demonstrated that WIN‑55 inhibited ADAMTS‑4 activity in unstimulated and IL‑1β‑stimulated primary human OA articular chondrocytes in a concentration‑dependent manner. Cannabinoid receptor type 1 (CB1) and 2 (CB2) were constitutively expressed in human OA articular chondrocytes. Furthermore, selective CB2 antagonist, JTE907, but not selective CB1 antagonist, MJ15, abolished the inhibitory effect of WIN‑55 on ADAMTS‑4 activity. WIN55 inhibited the expression of syndecan‑1 but not ADAMTS‑4, and overexpression of syndecan‑1 reversed the inhibitory effect of WIN‑55 on the ADAMTS‑4 activity in unstimulated and IL‑1β‑stimulated human OA articular chondrocytes. Despite having no significant effect on syndecan‑1 gene promoter activity, WIN‑55 markedly decreased the stability of syndecan‑1 mRNA via CB2. In conclusion, to the best of our knowledge, the present study provides the first in vitro evidence supporting that the synthetic

  20. Cannabinoid WIN-55,212-2 mesylate inhibits ADAMTS-4 activity in human osteoarthritic articular chondrocytes by inhibiting expression of syndecan-1

    PubMed Central

    KONG, YING; WANG, WANCHUN; ZHANG, CHANGJIE; WU, YI; LIU, YANG; ZHOU, XIAORONG

    2016-01-01

    A central feature of osteoarthritis (OA) is the loss of articular cartilage, which is primarily attributed to cartilage breakdown. A group of metalloproteinases termed the A disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) family are reported to be important in cartilage breakdown. Recent studies have suggested that ADAMTS-4 is a major contributor to the pathogenesis of OA and that syndecan-1 is closely associated with activation of ADAMTS-4 in human chondrocytes. Accumulating evidence also suggests that cannabinoids have chondroprotective effects. The current study explored the effects of synthetic cannabinoid WIN-55,212-2 mesylate (WIN-55) on the expression of syndecan-1 and ADAMTS-4, as well as ADAMTS-4 activity, in unstimulated and interleukin (IL)-1β-stimulated OA chondrocytes. Primary human OA articular chondrocytes were treated with WIN-55 in the presence or absence of IL-1β and cannabinoid receptor antagonists. The results of the present study demonstrated that WIN-55 inhibited ADAMTS-4 activity in unstimulated and IL-1β-stimulated primary human OA articular chondrocytes in a concentration-dependent manner. Cannabinoid receptor type 1 (CB1) and 2 (CB2) were constitutively expressed in human OA articular chondrocytes. Furthermore, selective CB2 antagonist, JTE907, but not selective CB1 antagonist, MJ15, abolished the inhibitory effect of WIN-55 on ADAMTS-4 activity. WIN55 inhibited the expression of syndecan-1 but not ADAMTS-4, and overexpression of syndecan-1 reversed the inhibitory effect of WIN-55 on the ADAMTS-4 activity in unstimulated and IL-1β-stimulated human OA articular chondrocytes. Despite having no significant effect on syndecan-1 gene promoter activity, WIN-55 markedly decreased the stability of syndecan-1 mRNA via CB2. In conclusion, to the best of our knowledge, the present study provides the first in vitro evidence supporting that the synthetic cannabinoid WIN-55 inhibits ADAMTS-4 activity in unstimulated and IL-1

  1. Tenuigenin Prevents IL-1β-induced Inflammation in Human Osteoarthritis Chondrocytes by Suppressing PI3K/AKT/NF-κB Signaling Pathway.

    PubMed

    Wang, Chunlei; Zeng, Lihong; Zhang, Tao; Liu, Jiakun; Wang, Wenbo

    2016-04-01

    Tenuigenin (TEN), the main active component of Polygala tenuifolia, has been reported to have anti-inflammatory effects. However, the effects of TEN on IL-1β-stimulated osteoarthritis chondrocytes have not been reported. The purpose of this study was to investigate the anti-inflammatory effects and mechanism of TEN on IL-1β-stimulated human osteoarthritis chondrocytes. Human osteoarthritis chondrocytes were pretreated with or without TEN for 1 h and then stimulated with IL-1β. The production of NO and PGE2 were detected by the Griess reagent and ELISA. The expression of NF-κB and MAPKs (p38, JNK, ERK) were measured by Western blot analysis. The production of MMP-1, MMP3, and MMP13 were measured by ELISA. The results showed that treatment of TEN significantly inhibited IL-1β-induced NO and PGE2 production. TEN also suppressed IL-1β-induced MMP-1, MMP3, and MMP13 expression. Furthermore, TEN was found to inhibit IL-1β-induced NF-κB activation, PI3K, and AKT phosphorylation. In conclusion, these results suggest that TEN inhibits IL-1β-induced inflammation in human osteoarthritis chondrocytes by inhibiting PI3K/AKT/NF-κB signaling pathway. PMID:26846886

  2. Up-regulation of the chemo-attractive receptor ChemR23 and occurrence of apoptosis in human chondrocytes isolated from fractured calcaneal osteochondral fragments

    PubMed Central

    Sena, Paola; Manfredini, Giuseppe; Benincasa, Marta; Mariani, Francesco; Smargiassi, Alberto; Catani, Fabio; Palumbo, Carla

    2014-01-01

    To study the expression level of a panel of pro/anti-apoptotic factors and inflammation-related receptors in chondral fragments from patients undergoing surgical treatment for intra-articular calcaneal fractures, cartilage fragments were retrieved from calcaneal fractures of 20 patients subjected to surgical treatment. Primary cultures were performed using chondral fragments from fractured and control patients. Chondrocyte cultures from each patient of the fractured and control groups were subjected to immunofluorescence staining and quantitatively analyzed under confocal microscopy. Proteins extracted from the cultured chondrocytes taken from the fractured and control groups were processed for Western blot experiments and densitometric analysis. The percentage of apoptotic cells was determined using the cleaved PARP-1 antibody. The proportion of labelled cells was 35% for fractured specimens, compared with 7% for control samples. Quantification of caspase-3 active and Bcl-2 proteins in chondrocyte cultures showed a significant increase of the apoptotic process in fractured specimens compared with control ones. Fractured chondrocytes were positively stained for ChemR23 with statistically significant differences with respect to control samples. Densitometric evaluation of the immunoreactive bands confirmed these observations. Human articular chondrocytes obtained from patients with intra-articular calcaneal fractures express higher levels of pivotal pro-apoptotic factors, and of the chemo-attractive receptor ChemR23, compared with control cultures. On the basis of these observations, the authors hypothesize that consistent prolonged chondrocyte death, associated with the persistence of high levels of pro-inflammatory factors, could enhance the deterioration of cartilage tissue with consequent development of post-traumatic arthritis following intra-articular bone fracture. PMID:24689495

  3. Integrin-mediated mechanotransduction pathway of low-intensity continuous ultrasound in human chondrocytes

    PubMed Central

    Whitney, Nicholas P.; Lamb, Allyson C.; Louw, Tobias M.; Subramanian, Anuradha

    2012-01-01

    Chondrocytes are mechanosensitive cells that require mechanical stimulation for proper growth and function in in vitro culture systems. Ultrasound (US) has emerged as a technique to deliver mechanical stress; however, the intracellular signaling components of the mechanotransduction pathways that transmit the extracellular mechanical stimulus to gene regulatory mechanisms are not fully defined. We evaluated a possible integrin/mitogen-activated protein kinase (MAPK) mechanotransduction pathway using Western blotting with antibodies targeting specific phosphorylation sites on intracellular signaling proteins. US stimulation of chondrocytes induced phosphorylation of focal adhesion kinase (FAK), Src, p130 Crk-associated substrate (p130Cas), CrkII, and extracellular-regulated kinase (Erk). Furthermore, pre-incubation with inhibitors of integrin receptors, Src, and MAPK/Erk kinase (MEK) reduced US-induced Erk phosphorylation levels, indicating integrins and Src are upstream of Erk in an US-mediated mechanotransduction pathway. These findings suggest US signals through integrin receptors to the MAPK/Erk pathway via a mechanotransduction pathway involving FAK, Src, p130Cas, and CrkII. PMID:22920546

  4. Association of Reduced Type IX Collagen Gene Expression in Human Osteoarthritic Chondrocytes With Epigenetic Silencing by DNA Hypermethylation

    PubMed Central

    Imagawa, Kei; de Andrés, María C; Hashimoto, Ko; Itoi, Eiji; Otero, Miguel; Roach, Helmtrud I; Goldring, Mary B; Oreffo, Richard O C

    2014-01-01

    Objective To investigate whether the changes in collagen gene expression in osteoarthritic (OA) human chondrocytes are associated with changes in the DNA methylation status in the COL2A1 enhancer and COL9A1 promoter. Methods Expression levels were determined using quantitative reverse transcription–polymerase chain reaction, and the percentage of DNA methylation was quantified by pyrosequencing. The effect of CpG methylation on COL9A1 promoter activity was determined using a CpG-free vector; cotransfections with expression vectors encoding SOX9, hypoxia-inducible factor 1α (HIF-1α), and HIF-2α were carried out to analyze COL9A1 promoter activities in response to changes in the methylation status. Chromatin immunoprecipitation assays were carried out to validate SOX9 binding to the COL9A1 promoter and the influence of DNA methylation. Results Although COL2A1 messenger RNA (mRNA) levels in OA chondrocytes were 19-fold higher than those in the controls, all of the CpG sites in the COL2A1 enhancer were totally demethylated in both samples. The levels of COL9A1 mRNA in OA chondrocytes were 6,000-fold lower than those in controls; 6 CpG sites of the COL9A1 promoter were significantly hypermethylated in OA patients as compared with controls. Treatment with 5-azadeoxycitidine enhanced COL9A1 gene expression and prevented culture-induced hypermethylation. In vitro methylation decreased COL9A1 promoter activity. Mutations in the 5 CpG sites proximal to the transcription start site decreased COL9A1 promoter activity. Cotransfection with SOX9 enhanced COL9A1 promoter activity; CpG methylation attenuated SOX9 binding to the COL9A1 promoter. Conclusion This first demonstration that hypermethylation is associated with down-regulation of COL9A1 expression in OA cartilage highlights the pivotal role of epigenetics in OA, involving not only hypomethylation, but also hypermethylation, with important therapeutic implications for OA treatment. PMID:25048791

  5. Inhibition of Histone Deacetylases Antagonized FGF2 and IL-1β Effects on MMP Expression in Human Articular Chondrocytes

    PubMed Central

    Wang, Xibin; Song, Yingjie; Jacobi, Jennifer L.; Tuan, Rocky S.

    2013-01-01

    Fibroblast growth factor – 2 (FGF2) and interleukin – 1β IL-1β) stimulate the expression of matrix metalloproteinases (MMPs) in articular chondrocytes, which may contribute to cartilage degradation and development of osteoarthritis. Histone deacetylases (HDACs) have recently been implicated in the regulation of MMP gene expression. To investigate the functional involvement of HDACs in the signaling pathway of FGF2 and IL-1β, we examined the effects of HDAC inhibition on activities of FGF2 or IL-1β on gene expression of MMP-1, MMP-3, MMP-13, a disintegrin and metalloproteinase with thrombospondin motifs – 5 (ADAMTS5), collagen type II, and aggrecan. Human articular chondrocyte cultures were treated with FGF2 or IL-1β in the presence or absence of HDAC inhibitor (trichostatin A, TSA). Gene expression levels after treatments were assessed using quantitative real time PCR. Results showed that FGF2 and IL-1β both increased MMP-1 and -13 expression, while IL-1βalso increased MMP-3 mRNA levels. These effects were attenuated in the presence of TSA in a dose dependent manner. In contrast to the effects on MMPs, FGF2 decreased mRNA levels of ADAMTS–5, which was not affected by HDAC inhibition. FGF2, IL-1β, and TSA inhibited expression of aggrecan, while TSA also decreased mRNA levels of collagen type II. These findings showed that HDAC inhibition antagonized FGF2 and IL-1β induced MMP expression. Combination of FGF2 and the HDAC inhibitor decreases both anabolic and catabolic genes, which may slow the cartilage turnover and be beneficial for maintaining cartilage integrity. PMID:19107653

  6. In vitro exposure of human osteoarthritic chondrocytes to ELF fields and new therapeutic application of musically modulated electromagnetic fields: biological evidence.

    PubMed

    Vannoni, D; Albanese, A; Battisti, E; Aceto, E; Giglioni, S; Corallo, C; Carta, S; Ferrata, P; Fioravanti, A; Giordano, N

    2012-01-01

    Osteoarthritis (OA) is the most frequently occurring rheumatic disease, caused by metabolic changes in chondrocytes, the cells that maintain cartilage. Treatment with electromagnetic fields (MF) produces benefits in patients affected by this pathology. Isolated human osteoarthritic (OA) chondrocytes were cultured in vitro under standard conditions or stimulated with IL-1beta or IGF-1, to mimic the imbalance between chondroformation and chondroresorption processes observed in OA cartilage in vivo. The cells were exposed for a specific time to extremely low frequency (ELF; 100-Hz) electromagnetic fields and to the Therapeutic Application of Musically Modulated Electromagnetic Fields (TAMMEF), which are characterized by variable frequencies, intensities, and waveforms. Using flow cytometry, we tested the effects of the different types of exposure on chondrocyte metabolism. The exposure of the cells to both systems enhances cell proliferation, does not generate reactive oxygen species, does not cause glutathione depletion or changes in mitochondrial transmembrane potential and does not induce apoptosis. This study presents scientific support to the fact that MF could influence OA chondrocytes from different points of view (viability, ROS production and apoptosis). We can conclude that both ELF and TAMMEF systems could be recommended for OA therapy and represent a valid non-pharmacological approach to the treatment of this pathology. PMID:22475096

  7. Simvastatin inhibits CD44 fragmentation in chondrocytes.

    PubMed

    Terabe, Kenya; Takahashi, Nobunori; Takemoto, Toki; Knudson, Warren; Ishiguro, Naoki; Kojima, Toshihisa

    2016-08-15

    In human osteoarthritic chondrocytes, the hyaluronan receptor CD44 undergoes proteolytic cleavage at the cell surface. CD44 cleavage is thought to require transit of CD44 into cholesterol-rich lipid rafts. The purpose of this study was to investigate whether statins exert a protective effect on articular chondrocytes due to diminution of cholesterol. Three model systems of chondrocytes were examined including human HCS-2/8 chondrosarcoma cells, human osteoarthritic chondrocytes and normal bovine articular chondrocytes. Treatment with IL-1β + Oncostatin M resulted in a substantial increase in CD44 fragmentation in each of the three chondrocyte models. Pre-incubation with simvastatin prior to treatment with IL-1β + Oncostatin M decreased the level of CD44 fragmentation, decreased the proportion of CD44 that transits into the lipid raft fractions, decreased ADAM10 activity and diminished the interaction between CD44 and ADAM10. In HCS-2/8 cells and bovine articular chondrocytes, fragmentation of CD44 was blocked by the knockdown of ADAM10. Inhibition of CD44 fragmentation by simvastatin also resulted in improved retention of pericellular matrix. Addition of cholesterol and farnesyl-pyrophosphate reversed the protective effects of simvastatin. Thus, the addition of simvastatin exerts positive effects on chondrocytes including reduced CD44 fragmentation and enhanced the retention of pericellular matrix. PMID:27242325

  8. Green tea polyphenol epigallocatechin-3-gallate inhibits advanced glycation end product-induced expression of tumor necrosis factor-α and matrix metalloproteinase-13 in human chondrocytes

    PubMed Central

    Rasheed, Zafar; Anbazhagan, Arivarasu N; Akhtar, Nahid; Ramamurthy, Sangeetha; Voss, Frank R; Haqqi, Tariq M

    2009-01-01

    Introduction The major risk factor for osteoarthritis (OA) is aging, but the mechanisms underlying this risk are only partly understood. Age-related accumulation of advanced glycation end products (AGEs) can activate chondrocytes and induce the production of proinflammatory cytokines and matrix metalloproteinases (MMPs). In the present study, we examined the effect of epigallocatechin-3-gallate (EGCG) on AGE-modified-BSA (AGE-BSA)-induced activation and production of TNFα and MMP-13 in human OA chondrocytes. Methods Human chondrocytes were derived from OA cartilage by enzymatic digestion and stimulated with in vitro-generated AGE-BSA. Gene expression of TNFα and MMP-13 was measured by quantitative RT-PCR. TNFα protein in culture medium was determined using cytokine-specific ELISA. Western immunoblotting was used to analyze the MMP-13 production in the culture medium, phosphorylation of mitogen-activated protein kinases (MAPKs), and the activation of NF-κB. DNA binding activity of NF-κB p65 was determined using a highly sensitive and specific ELISA. IκB kinase (IKK) activity was determined using an in vitro kinase activity assay. MMP-13 activity in the culture medium was assayed by gelatin zymography. Results EGCG significantly decreased AGE-stimulated gene expression and production of TNFα and MMP-13 in human chondrocytes. The inhibitory effect of EGCG on the AGE-BSA-induced expression of TNFα and MMP-13 was mediated at least in part via suppression of p38-MAPK and JNK activation. In addition, EGCG inhibited the phosphorylating activity of IKKβ kinase in an in vitro activity assay and EGCG inhibited the AGE-mediated activation and DNA binding activity of NF-κB by suppressing the degradation of its inhibitory protein IκBα in the cytoplasm. Conclusions These novel pharmacological actions of EGCG on AGE-BSA-stimulated human OA chondrocytes provide new suggestions that EGCG or EGCG-derived compounds may inhibit cartilage degradation by suppressing AGE

  9. MicroRNA-602 and microRNA-608 regulate sonic hedgehog expression via target sites in the coding region in human chondrocytes

    PubMed Central

    Akhtar, Nahid; Makki, Mohammad Shahidul; Haqqi, Tariq M.

    2015-01-01

    Objective Hedgehog(Hh) signaling has recently been associated with cartilage degradation in osteoarthritis(OA). As interleukin-1β(IL-1β) is a critical mediator of OA pathogenesis, here we determined whether IL-1β induces the expression of sonic hedgehog(SHH) and its regulation by microRNAs in human chondrocytes. Methods SHH protein expression in human OA-cartilage and in an animal model of OA was determined by immunohistochemistry and immunofluorescence respectively. Gene and protein expression in IL-1β or SHH-stimulated chondrocytes was determined by TaqMan assays and immunoblotting respectively. Effect of overexpression of miR-602 and miR-608 or their anatgomirs on SHH expression was evaluated by transient transfections of human chondrocytes and HEK-293 cells. Role of signaling pathways was evaluated using small molecule inhibitors. Binding of miRNAs with the putative “seed sequence” in the SHH mRNA was validated with a SHH luciferase reporter assay. Results Expression of SHH, PTCH-1, GLI-1, HHIP, MMP-13, and COL10A1 was high in damaged OAcartilage. Expression of SHH was inversely correlated with the expression of miR-608 in damaged cartilage and in IL-1β-stimulated chondrocytes. Transfection with miR-608 or miR-602 mimics inhibited the reporter activity and mutation of the miRNAs “seed sequences” abolished the repression of reporter activity. Overexpression of miR-602 or miR-608 inhibited the expression of SHH mRNA and protein and this was abrogated by antagomirs. Stimulation with SHH-protein up-regulated the MMP-13 expression and inhibition of Hh signaling blocked MMP-13 expression in OA chondrocytes. Conclusions miR-602 and miR-608 are important regulators of SHH expression in chondrocytes and their suppression by IL-1β may contribute to the enhanced expression of SHH and MMP-13 in OA. PMID:25385442

  10. Effects of weak, low-frequency pulsed electromagnetic fields (BEMER type) on gene expression of human mesenchymal stem cells and chondrocytes: an in vitro study.

    PubMed

    Walther, Markus; Mayer, Florian; Kafka, Wolf; Schütze, Norbert

    2007-01-01

    In vitro effects of electromagnetic fields appear to be related to the type of electromagnetic field applied. Previously, we showed that human osteoblasts display effects of BEMER type electromagnetic field (BTEMF) on gene regulation. Here, we analyze effects of BTEMF on gene expression in human mesenchymal stem cells and chondrocytes. Primary mesenchymal stem cells from bone marrow and the chondrocyte cell line C28I2 were stimulated 5 times at 12-h intervals for 8 min each with BTEMF. RNA from treated and control cells was analyzed for gene expression using the affymetrix chip HG-U133A. A limited number of regulated gene products from both cell types mainly affect cell metabolism and cell matrix structure. There was no increased expression of cancer-related genes. RT-PCR analysis of selected transcripts partly confirmed array data. Results indicate that BTEMF in human mesenchymal stem cells and chondrocytes provide the first indications to understanding therapeutic effects achieved with BTEMF stimulation. PMID:17886005

  11. Primary Human Chondrocyte Extracellular Matrix Formation and Phenotype Maintenance using RGD-derivatized PEGDM Hydrogels Possessing a Continuous Gradient in Modulus

    PubMed Central

    Smith Callahan, Laura A.; Ganios, Anna M.; Childers, Erin P.; Weiner, Scott D.; Becker, Matthew L.

    2013-01-01

    Efficient ex vivo methods for expanding primary human chondrocytes while maintaining phenotype is critical to advancing autologous cell sourcing efforts for tissue engineering applications. While there is significant activity in the literature, systematic approaches are necessary to determine and optimize the chemical and mechanical scaffold properties for hyaline cartilage generation using limited cell numbers. Functionalized hydrogels possessing continuous variations in physico-chemical properties are therefore an efficient three-dimensional platform for studying several properties simultaneously. Herein, we describe a polyethylene glycol dimethacrylate (PEGDM) hydrogel system possessing a gradient in modulus (~27,000 Pa to 3,800 Pa) containing a uniform concentration of arginine–glycine–aspartic acid peptide (RGD) to enhance cellular adhesion for the correlation of primary human osteoarthritic chondrocyte proliferation, phenotype maintenance, and ECM production to the hydrogel properties. Cell number and chondrogenic phenotype (CD14:CD90 ratios) were found to decline in regions with higher storage modulus (>13,100 Pa), while regions with lower storage modulus maintained cell number and phenotype. Over three weeks of culture, hydrogel regions possessing lower storage modulus experience an increase in ECM content (~200%) compared to regions with higher storage modulus. Variations in the amount and organization of cytoskeletal markers actin and vinculin were observed within the modulus gradient which are indicative of the differences in chondrogenic phenotype maintenance and ECM expression. Thus scaffold mechanical properties significantly impact on modulating human osteoarthritic chondrocyte behavior and tissue formation. PMID:23291491

  12. Effect of a Herbal-Leucine mix on the IL-1β-induced cartilage degradation and inflammatory gene expression in human chondrocytes

    PubMed Central

    2011-01-01

    Background Conventional treatments for the articular diseases are often effective for symptom relief, but can also cause significant side effects and do not slow the progression of the disease. Several natural substances have been shown to be effective at relieving the symptoms of osteoarthritis (OA), and preliminary evidence suggests that some of these compounds may exert a favorable influence on the course of the disease. The objective of this study was to investigate the anti-inflammatory/chondroprotective potential of a Herbal and amino acid mixture containing extract of the Uncaria tomentosa, Boswellia spp., Lepidium meyenii and L-Leucine on the IL-1β-induced production of nitric oxide (NO), glycosaminoglycan (GAG), matrix metalloproteinases (MMPs), aggrecan (ACAN) and type II collagen (COL2A1) in human OA chondrocytes and OA cartilage explants. Methods Primary OA chondrocytes or OA cartilage explants were pretreated with Herbal-Leucine mixture (HLM, 1-10 μg/ml) and then stimulated with IL-1β (5 ng/ml). Effect of HLM on IL-1β-induced gene expression of iNOS, MMP-9, MMP-13, ACAN and COL2A1 was verified by real time-PCR. Estimation of NO and GAG release in culture supernatant was done using commercially available kits. Results HLM tested in these in vitro studies was found to be an effective anti-inflammatory agent, as evidenced by strong inhibition of iNOS, MMP-9 and MMP-13 expression and NO production in IL-1β-stimulated OA chondrocytes (p < 0.05). Supporting these gene expression results, IL-1β-induced cartilage matrix breakdown, as evidenced by GAG release from cartilage explants, was also significantly blocked (p < 0.05). Moreover, in the presence of herbal-Leucine mixture (HLM) up-regulation of ACAN and COL2A1 expression in IL-1β-stimulated OA chondrocytes was also noted (p < 0.05). The inhibitory effects of HLM were mediated by inhibiting the activation of nuclear factor (NF)-kB in human OA chondrocytes in presence of IL-1β. Conclusion Our data

  13. Paracrine effects of human adipose-derived mesenchymal stem cells in inflammatory stress-induced senescence features of osteoarthritic chondrocytes.

    PubMed

    Platas, Julia; Guillén, Maria Isabel; Pérez Del Caz, Maria Dolores; Gomar, Francisco; Castejón, Miguel Angel; Mirabet, Vicente; Alcaraz, Maria José

    2016-08-01

    Aging and exposure to stress would determine the chondrocyte phenotype in osteoarthritis (OA). In particular, chronic inflammation may contribute to stress-induced senescence of chondrocytes and cartilage degeneration during OA progression. Recent studies have shown that adipose-derived mesenchymal stem cells exert paracrine effects protecting against degenerative changes in chondrocytes. We have investigated whether the conditioned medium (CM) from adipose-derived mesenchymal stem cells may regulate senescence features induced by inflammatory stress in OA chondrocytes. Our results indicate that CM down-regulated senescence markers induced by interleukin-1β including senescence-associated β-galactosidase activity, accumulation of γH2AX foci and morphological changes with enhanced formation of actin stress fibers. Treatment of chondrocytes with CM also decreased the production of oxidative stress, the activation of mitogen-activated protein kinases, and the expression of caveolin-1 and p21. The effects of CM were related to the reduction in p53 acetylation which would be dependent on the enhancement of Sirtuin 1 expression. Therefore, CM may exert protective effects in degenerative joint conditions by countering the premature senescence of OA chondrocytes induced by inflammatory stress. PMID:27490266

  14. The metabolism of 2-fluoro-2-deoxy-D-glucose in human chondrocytes and its incorporation into keratan sulfate proteoglycans.

    PubMed

    Fedders, G; Kock, R; Van de Leur, E; Greiling, H

    1994-02-01

    The incorporation of 2-fluoro-2-deoxy-D-[14C]glucose in proteoglycans was investigated in a cell culture system, where human articular chondrocytes were cultured in high-cell-density thin-layer soft agarose. The proteoglycans were solubilized from the culture medium and the cell layer fraction by extracting with a guanidine hydrochloride buffer and purified by an ion-exchange-chromatography (DEAE-Sepharose CL-6B). With enzymic decomposition experiments concerning the glycosaminoglycan side-chains it could be shown that 65-69% were digestible by keratanase, whereas 21-29% of the 14C-labeled proteoglycans were digested with chondroitinase AC/ABC. The main constituent of the 2-fluoro-2-deoxy-D-[14C]glucose-metabolites present in the glycosaminoglycan side chains of the proteoglycans was 2-fluoro-2-deoxy-D-[14C]galactose. Therefore, 2-fluoro-2-deoxy-D-glucose was preferentially incorporated into keratan sulfate. We investigated the effect of non-radioactive 2-fluoro-2-deoxy-D-glucose on UDP-sugar and proteoglycan biosynthesis after incubation periods of 1-30 h. A high 2-fluoro-2-deoxy-D-glucose concentration in the culture medium did not influence the pool size of UDP-N-acetylhexosamines, but UDP-D-glucose, UDP-D-galactose, UDP-D-glucuronic acid, UDP-2-fluoro-2-deoxy-D-glucose, UDP-2-fluoro-2-deoxy-D-galactose and UDP-2-fluoro-2-deoxy-D-glucuronic acid accumulated in the chondrocytes time dependently. In a pulse/chase experiment the retarded synthesis of fluorinated UDP-sugars was proved. The half-lives (t1/2) for UDP-2-fluoro-2-deoxy-D-glucose and UDP-2-fluoro-2-deoxy-D-galactose were about 7.7 h and 13.3 h, respectively. UDP-2-fluoro-2-deoxy-D-glucuronic acid could be found with delay. The incubation with 2-fluoro-2-deoxy-D-glucose and [14C]glucosamine resulted in a decreased radioactive labelling of chondroitin sulfate and keratan sulfate. PMID:8112319

  15. Delphinidin inhibits IL-1β-induced activation of NF-κB by modulating the phosphorylation of IRAK-1Ser376 in human articular chondrocytes

    PubMed Central

    Haseeb, Abdul; Chen, Dongxing

    2013-01-01

    Objective. In OA, there is enhanced expression of pro-inflammatory cytokines such as IL-1β in the affected joint. Delphinidin, an anthocyanidin found in pigmented fruits and vegetables, has been shown to possess anti-inflammatory and antioxidant properties. In the present study we determined whether delphinidin would inhibit the IL-1β-induced activation of NF-κB in human chondrocytes and determined the mechanism of its action. Methods. PGE2 levels and activation of NF-κB p65 in human OA chondrocytes were determined by ELISA-based assays. Protein expression of cyclo-oxygenase-2 (COX-2) and phosphorylation of kinases was determined by western immunoblotting. Expression level of mRNAs was determined by TaqMan assays. Results. Delphinidin inhibited IL-1β-induced expression of COX-2 and production of PGE2 in human chondrocytes. Delphinidin also inhibited IL-1β-mediated phosphorylation of IL-1 receptor-associated kinase-1Ser376, phosphorylation of IKKα/β, expression of IKKβ, degradation of IκBα, and activation and nuclear translocation of NF-κB/p65. Phosphorylation of TGF-β-activated kinase 1 was not observed but NF-κB-inducing kinase (NIK) was phosphorylated and phosphorylation of NIK was blocked by delphinidin in IL-1β-treated human chondrocytes. Conclusion. These data identify delphinidin as a novel inhibitor of IL-1β-induced production of cartilage-degrading molecule PGE2 via inhibition of COX-2 expression and provide new insight into the mechanism of its action. Our results also identify inhibition of IRAK1Ser376 phosphorylation by delphinidin in IL-1β-induced activation of NF-κB in human chondrocytes. Given the important role played by IL-1β-induced NF-κB activation, COX-2 expression and PGE2 production in OA, our results may have important implications for the development of novel therapeutic strategies for the prevention/treatment of OA. PMID:23392593

  16. Lactoferrin from Camelus dromedarius Inhibits Nuclear Transcription Factor-kappa B Activation, Cyclooxygenase-2 Expression and Prostaglandin E2 Production in Stimulated Human Chondrocytes

    PubMed Central

    Rasheed, Naila; Alghasham, Abdullah; Rasheed, Zafar

    2016-01-01

    Background: Osteoarthritis (OA) is a progressive joint disorder, which remains the leading cause of chronic disability in aged people. Nuclear factor-kappa B (NF)-κB is a major cellular event in OA and its activation by interleukin-1β (IL-1β) plays a critical role in cartilage breakdown in these patients. Objective: In this study, we examined the effect of lactoferrin on NF-κB activation, cyclooxygenase-2 (COX-2) expression and prostaglandin E2 (PGE2) production in stimulated human articular chondrocytes. Materials and Methods: Human chondrocytes were derived from OA articular cartilage and treated with camel lactoferrin and then stimulated with IL-1β. Gene expression was determined by TaqMan assays and protein expression was studied by Western immunoblotting. NF-κB activity and PGE2 levels were determined by ELISA based assays. NF-κB activity was also determined by treatment of chondrocytes with NF-κB specific inhibitor Bay 11–7082. Results: Lactoferrin inhibited IL-1β-induced activation and nuclear translocation of NF-κB p65 in human OA chondrocytes. Lactoferrin also inhibited mRNA/protein expression of COX-2 and production of PGE2. Moreover, Bay 11–7082 also inhibited IL-1β-induced expression of COX-2 and production of PGE2. The inhibitory effect of lactoferrin on the IL-1β induced expression of COX-2 or production of PGE2 was mediated at least in part via suppression of NF-κB activation. Conclusions: Our data determine camel lactoferrin as a novel inhibitor of IL-1β-induced activation of NF-κB signaling events and production of cartilage-degrading molecule PGE2 via inhibition of COX-2 expressions. These results may have important implications for the development of novel therapeutic strategies for the prevention/treatment of OA and other degenerative/inflammatory diseases. SUMMARY Lactoferrin shows anti-arthritic activity in IL-1β stimulated primary human chondrocytes.Lactoferrin inhibits IL-1β-induced NF-κB activation.Lactoferrin inhibits

  17. PEP-1-FK506BP12 inhibits matrix metalloproteinase expression in human articular chondrocytes and in a mouse carrageenan-induced arthritis model.

    PubMed

    Hwang, Hyun Sook; Park, In Young; Kim, Dae Won; Choi, Soo Young; Jung, Young Ok; Kim, Hyun Ah

    2015-07-01

    The 12 kDa FK506-binding protein (FK506BP12), an immunosuppressor, modulates T cell activation via calcineurin inhibition. In this study, we investigated the ability of PEP-1-FK506BP12, consisting of FK506BP12 fused to the protein transduction domain PEP-1 peptide, to suppress catabolic responses in primary human chondrocytes and in a mouse carrageenan-induced paw arthritis model. Western blotting and immunofluorescence analysis showed that PEP-1-FK506BP12 efficiently penetrated chondrocytes and cartilage explants. In interleukin-1β (IL-1β)-treated chondrocytes, PEP-1-FK506BP12 significantly suppressed the expression of catabolic enzymes, including matrix metalloproteinases (MMPs)-1, -3, and -13 in addition to cyclooxygenase-2, at both the mRNA and protein levels, whereas FK506BP12 alone did not. In addition, PEP-1-FK506BP12 decreased IL-1β-induced phosphorylation of the mitogen-activated protein kinase (MAPK) complex (p38, JNK, and ERK) and the inhibitor kappa B alpha. In the mouse model of carrageenan-induced paw arthritis, PEP-1-FK506BP12 suppressed both carrageenan-induced MMP-13 production and paw inflammation. PEP-1-FK506BP12 may have therapeutic potential in the alleviation of OA progression. PMID:25887750

  18. Thymoquinone Inhibits IL-1β-Induced Inflammation in Human Osteoarthritis Chondrocytes by Suppressing NF-κB and MAPKs Signaling Pathway.

    PubMed

    Wang, Dongyan; Qiao, Jiutao; Zhao, Xin; Chen, Tianxin; Guan, Dehong

    2015-12-01

    Thymoquinone, an active ingredient isolated from Nigella sativa, has been reported to have anti-inflammatory effects. However, the anti-inflammatory effect of thymoquinone on IL-1β-stimulated osteoarthritis chondrocytes remains unclear. In this study, we designed to investigate the anti-inflammatory effects and elucidated the underlying mechanism of thymoquinone on IL-1β-stimulated human osteoarthritis chondrocytes. The effects of thymoquinone on inflammatory mediators COX-2, iNOS, NO, PGE2, as well as MMP-1, MMP3, MMP13 production were detected. The results demonstrated that thymoquinone concentration-dependently inhibited IL-1β-induced COX-2, iNOS, NO, and PGE2 production. Thymoquinone also suppressed IL-1β-induced MMP-1, MMP3, and MMP13 production. We found that thymoquinone significantly inhibited IL-1β-induced NF-κB activation and IκBα degradation. In addition, thymoquinone was found to suppress IL-1β-induced mitogen-activated protein kinases (MAPKs) activation. In conclusion, thymoquinone inhibited IL-1β-induced inflammatory mediator production by inhibition of NF-κB and MAPKs signaling pathways in osteoarthritis chondrocytes. Thymoquinone may be a potential agent in the treatment of osteoarthritis. PMID:26156811

  19. Interstitial Perfusion Culture with Specific Soluble Factors Inhibits Type I Collagen Production from Human Osteoarthritic Chondrocytes in Clinical-Grade Collagen Sponges.

    PubMed

    Mayer, Nathalie; Lopa, Silvia; Talò, Giuseppe; Lovati, Arianna B; Pasdeloup, Marielle; Riboldi, Stefania A; Moretti, Matteo; Mallein-Gerin, Frédéric

    2016-01-01

    Articular cartilage has poor healing ability and cartilage injuries often evolve to osteoarthritis. Cell-based strategies aiming to engineer cartilaginous tissue through the combination of biocompatible scaffolds and articular chondrocytes represent an alternative to standard surgical techniques. In this context, perfusion bioreactors have been introduced to enhance cellular access to oxygen and nutrients, hence overcoming the limitations of static culture and improving matrix deposition. Here, we combined an optimized cocktail of soluble factors, the BIT (BMP-2, Insulin, Thyroxin), and clinical-grade collagen sponges with a bidirectional perfusion bioreactor, namely the oscillating perfusion bioreactor (OPB), to engineer in vitro articular cartilage by human articular chondrocytes (HACs) obtained from osteoarthritic patients. After amplification, HACs were seeded and cultivated in collagen sponges either in static or dynamic conditions. Chondrocyte phenotype and the nature of the matrix synthesized by HACs were assessed using western blotting and immunohistochemistry analyses. Finally, the stability of the cartilaginous tissue produced by HACs was evaluated in vivo by subcutaneous implantation in nude mice. Our results showed that perfusion improved the distribution and quality of cartilaginous matrix deposited within the sponges, compared to static conditions. Specifically, dynamic culture in the OPB, in combination with the BIT cocktail, resulted in the homogeneous production of extracellular matrix rich in type II collagen. Remarkably, the production of type I collagen, a marker of fibrous tissues, was also inhibited, indicating that the association of the OPB with the BIT cocktail limits fibrocartilage formation, favoring the reconstruction of hyaline cartilage. PMID:27584727

  20. PEP-1-FK506BP12 inhibits matrix metalloproteinase expression in human articular chondrocytes and in a mouse carrageenan-induced arthritis model

    PubMed Central

    Hwang, Hyun Sook; Park, In Young; Kim, Dae Won; Choi, Soo Young; Jung, Young Ok; Kim, Hyun Ah

    2015-01-01

    The 12 kDa FK506-binding protein (FK506BP12), an immunosuppressor, modulates T cell activation via calcineurin inhibition. In this study, we investigated the ability of PEP-1-FK506BP12, consisting of FK506BP12 fused to the protein transduction domain PEP-1 peptide, to suppress catabolic responses in primary human chondrocytes and in a mouse carrageenan-induced paw arthritis model. Western blotting and immunofluorescence analysis showed that PEP-1-FK506BP12 efficiently penetrated chondrocytes and cartilage explants. In interleukin-1β (IL-1β)-treated chondrocytes, PEP-1-FK506BP12 significantly suppressed the expression of catabolic enzymes, including matrix metalloproteinases (MMPs)-1, -3, and -13 in addition to cyclooxygenase-2, at both the mRNA and protein levels, whereas FK506BP12 alone did not. In addition, PEP-1-FK506BP12 decreased IL-1β-induced phosphorylation of the mitogen-activated protein kinase (MAPK) complex (p38, JNK, and ERK) and the inhibitor kappa B alpha. In the mouse model of carrageenan-induced paw arthritis, PEP-1-FK506BP12 suppressed both carrageenan-induced MMP-13 production and paw inflammation. PEP-1-FK506BP12 may have therapeutic potential in the alleviation of OA progression. [BMB Reports 2015; 48(7): 407-412] PMID:25887750

  1. miR-139 modulates MCPIP1/IL-6 expression and induces apoptosis in human OA chondrocytes

    PubMed Central

    Makki, Mohammad Shahidul; Haqqi, Tariq M

    2015-01-01

    IL-6 is an inflammatory cytokine and its overexpression plays an important role in osteoarthritis (OA) pathogenesis. Expression of IL-6 is regulated post-transcriptionally by MCPIP1. The 3′ untranslated region (UTR) of MCPIP1 mRNA harbors a miR-139 ‘seed sequence', therefore we examined the post-transcriptional regulation of MCPIP1 by miR-139 and its impact on IL-6 expression in OA chondrocytes. Expression of miR-139 was found to be high in the damaged portion of the OA cartilage compared with unaffected cartilage from the same patient and was also induced by IL-1β in OA chondrocytes. Inhibition of miR-139 decreased the expression of IL-6 mRNA by 38% and of secreted IL-6 protein by 40%. However, overexpression of miR-139 increased the expression of IL-6 mRNA by 36% and of secreted IL-6 protein by 56%. These data correlated with altered expression profile of MCPIP1 in transfected chondrocytes. Studies with a luciferase reporter construct confirmed the interactions of miR-139 with the ‘seed sequence' located in the 3′ UTR of MCPIP mRNA. Furthermore, miR-139 overexpression increased the catabolic gene expression but expression of anabolic markers remained unchanged. Overexpression of miR-139 also induced apoptosis in OA chondrocytes. Importantly, we also discovered that IL-6 is a potent inducer of miR-139 expression in OA chondrocytes. These findings indicate that miR-139 functions as a post-transcriptional regulator of MCPIP1 expression and enhances IL-6 expression, which further upregulates miR-139 expression in OA chondrocytes. These results support our hypothesis that miR-139-mediated downregulation of MCPIP1 promotes IL-6 expression in OA. Therefore, targeting miR-139 could be therapeutically beneficial in the management of OA. PMID:26450708

  2. Chondrocyte Moves: clever strategies?

    PubMed Central

    Morales, Teresa I.

    2007-01-01

    Goals To review the literature on chondrocyte movements and to develop plausible hypothesis for further work. Design Chondrocyte movements are herein defined as translocations of the cell body. To set the stage for a discussion of chondrocyte moves, a brief overview of cell migration in other cell types is presented, including a discussion of the challenges that cells find when moving within tissues. Reports of isolated chondrocyte migration in vitro (isolated cell systems) and ex vivo (cartilage organ cultures) are then summarized, followed by a discussion of recent studies that infer chondrocyte movements in vivo. Results Investigators from different laboratories have observed chondrocyte motility in vitro. I became interested in the question of whether articular chondrocytes retained their phenotype during their migratory excursions. We devised a simple method to separate migratory and stationary chondrocytes and then showed that migratory chondrocytes synthesized collagen II but not I—consistent with a differentiated phenotype. Our time-lapse video microscopy studies showed that the cells displayed appropriate movement kinetics, albeit with low speed and directionality. Similarly, others have presented data consistent with slow movement of chondrocytes out of cartilage explants. It is important to decipher whether these in vitro movements reflect physiological states and if so, which events are simulated. Examples of in vivo studies that have inferred chondrocyte movements include those describing rotational or gliding movements of chondrocytes in the proliferative zone of the growth plate and its importance in the growth process; and the notion that chondrocytes move from the cartilage endplates to the nucleus pulposus in the spine of rabbits and rats during development. Such studies are consistent with the hypothesis that chondrocytes exhibit highly controlled and specialized movements during tissue growth and remodeling in vivo. On the other hand, the

  3. Honokiol, a low molecular weight natural product, prevents inflammatory response and cartilage matrix degradation in human osteoarthritis chondrocytes.

    PubMed

    Chen, Ying Ju; Tsai, Keh Sung; Chan, Ding Cheng; Lan, Kuo Cheng; Chen, Cheng Feng; Yang, Rong Sen; Liu, Shing Hwa

    2014-04-01

    Proinflammatory cytokine interleukin-1β (IL-1β) stimulates several mediators of cartilage degradation and plays an important role in the pathogenesis of osteoarthritis (OA). Honokiol, a low molecular weight natural product isolated from the Magnolia officinalis, has been shown to possess anti-inflammatory effect. Here, we used an in vitro model of cartilage inflammation to investigate the therapeutic potential of honokiol in OA. Human OA chondrocytes were cultured and pretreated with honokiol (2.5-10 µM) with or without IL-1β (10 ng/ml). Nitric oxide (NO) production was quantified by Griess reagent. Prostaglandin (PG)E2 , metalloproteinase-13 (MMP-13), and interleukin-6 (IL-6) productions were quantified by enzyme-linked immunosorbent assay. The expressions of collagen II, cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS), and nuclear factor κB (NF-κB)-related signaling molecules were determined by Western blotting. Our data showed that IL-1β markedly stimulated the expressions of iNOS and COX-2 and the productions of NO, PGE2 , and IL-6, which could be significantly reversed by honokiol. Honokiol could also suppress the IL-1β-triggered activation of IKK/IκBα/NF-κB signaling pathway. Moreover, honokiol significantly inhibited the IL-1β-induced MMP-13 production and collagen II reduction. Taken together, the present study suggests that honokiol may have a chondroprotective effect and may be a potential therapeutic choice in the treatment of OA patients. PMID:24375705

  4. Cordycepin modulates inflammatory and catabolic gene expression in interleukin-1beta-induced human chondrocytes from advanced-stage osteoarthritis: an in vitro study

    PubMed Central

    Hu, Pengfei; Chen, Weiping; Bao, Jiapeng; Jiang, Lifeng; Wu, Lidong

    2014-01-01

    Cordycepin is widely used as for its various pharmacological activities, such as anti-inflammation, anti-angiogenesis, anti-aging, anti-tumor and anti-proliferation. However, the precise role of cordycepin on chondrocytes is not clear. In the present study, we examined the inhibitory effects of cordycepin on interleukin-1 beta (IL-1β)-induced glycosaminoglycan (GAG) release, nitric oxide production as well as gene expressions of inflammatory and catabolic mediators in human cartilage and chondrocytes. Cartilage explants and human chondrocytes were cultured in the absence or in the presence of IL-1β (10 ng/ml) and with or without cordycepin (5-100 μM). GAG content in the cartilage explants was measured by using the dimethylmethylene blue method and Safranin O staining. Nitric oxide level was determined by Griess reaction. Expressions of MMP-1, MMP-13, cathepsin K, cathepsin S, ADAMTS-4 (a disintegrin and metalloproteinase with thrombospondin motifs-4) and ADAMTS-5, inducible nitric oxide synthase (iNOS) and cyclooxgenase-2 (COX-2) were evaluated by real-time quantitative PCR. We found that cordycepin suppressed IL-1β-stimulated GAG release. Gene expressions of catabolic enzymes, including MMP-1, MMP-13, cathepsin K, cathepsin S, ADAMTS-4 and ADAMTS-5, were decreased by cordycepin in a dose-dependent manner. In addition, cordycepin inhibited IL-1β-induced COX-2 and iNOS expression at the transcript level as well as blocked NO production. Our results suggest that cordycepin may possess chondroprotective effect by preventing cartilage denegation and interfering inflammatory response in the pathogenesis of OA. PMID:25400736

  5. A radiochemical high-performance liquid chromatographic method for the analysis of 2-fluoro-2-deoxy-D-glucose-derived metabolites in human chondrocytes.

    PubMed

    Fedders, G; Kock, R; Van de Leur, E; Greiling, H

    1993-05-15

    A high-performance liquid chromatography method with on-line radioactivity monitoring was developed for the measurement of 2-fluoro-2-deoxy-D-[U-14C]-glucose-derived metabolites in a cell culture system of human chondrocytes embedded in soft agarose. To optimize the chromatographic procedure, glucose-analogous substrates derived from 2-fluoro-2-deoxy-D-glucose by enzymatic synthesis in vitro were used. The synthesized metabolites could be separated by anion-exchange chromatography on a Partisil 10 SAX cartridge with a LiChrosorb RP 18-5 guard column eluted with a 35-min ion-strength/pH gradient performed from 15 mM NH4H2PO4, pH 3.8, to 0.75 M NH4H2PO4, pH 4.8, at a flow rate of 2 ml/min. Only by using an on-line radioactivity monitor instead of an off-line counting procedure was the resolution obtained sufficient for the determination of these intermediates. This method was applied to studying the metabolic pathway of 2-fluoro-2-deoxy-D-glucose in human chondrocytes. Due to the resistance of the chondrocytes embedded in soft agarose, the usual cell-lysing methods could not be used; therefore, an extraction procedure for acid-stable glucose metabolites, which may also be applied to other resistant cell lines or critical cell culture systems, was developed. With the procedure presented here, the existence of metabolites of 2-fluoro-2-deoxy-D-glucose resulting from enzymatic reactions following the hexokinase reaction could be proven.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:8323040

  6. Effects of vimentin disruption on the mechanoresponses of articular chondrocyte.

    PubMed

    Chen, Cheng; Yin, Li; Song, Xiongbo; Yang, Hao; Ren, Xiang; Gong, Xiaoyuan; Wang, Fuyou; Yang, Liu

    2016-01-01

    Human articular cartilage is subjected to repetitive mechanical loading during life time. As the only cellular component of articular cartilage, chondrocytes play a key role in the mechanotransduction within this tissue. The mechanoresponses of chondrocytes are largely determined by the cytoskeleton. Vimentin intermediate filaments, one of the major cytoskeletal components, have been shown to regulate chondrocyte phenotype. However, the contribution of vimentin in chondrocyte mechanoresponses remains less studied. In this study, we seeded goat articular chondrocytes on a soft polyacrylamide gel, and disrupted the vimentin cytoskeleton using acrylamide. Then we applied a transient stretch or compression to the cells, and measured the changes of cellular stiffness and traction forces using Optical Magnetic Twisting Cytometry and Traction Force Microscopy, respectively. In addition, to study the effects of vimentin disruption on the intracellular force generation, we treated the cells with a variety of reagents that are known to increase or decrease cytoskeletal tension. We found that, after a compression, the contractile moment and cellular stiffness were not affected in untreated chondrocytes, but were decreased in vimentin-disrupted chondrocytes; after a stretch, vimentin-disrupted chondrocytes showed a lower level of fluidization-resolidification response compared to untreated cells. Moreover, vimentin-disrupted chondrocytes didn't show much difference to control cells in responding to reagents that target actin and ROCK pathway, but showed a weaker response to histamine and isoproterenol. These findings confirmed chondrocyte vimentin as a major contributor in withstanding compressive loading, and its minor role in regulating cytoskeletal tension. PMID:26616052

  7. Selenomethionine Inhibits IL-1β Inducible Nitric Oxide Synthase (iNOS) and Cyclooxygenase 2 (COX2) Expression in Primary Human Chondrocytes

    PubMed Central

    Cheng, Anthony Wai Ming; Stabler, Thomas V.; Bolognesi, Michael; Kraus, Virginia Byers

    2010-01-01

    Objective Several lines of evidence show that selenium has potential protective effects in osteoarthritis (OA), however the exact mechanism is still unclear. As IL-1β is one of the key proinflammatory cytokines contributing to the progression in OA, we investigated the effect of selenium in neutralizing the inflammatory effects of IL-1β on nitric oxide (NO) and prostaglandin E (PGE2) production, and the signaling pathways involved. Methods Isolated primary human chondrocytes were pretreated with selenomethionine (0.5 μM SeMet) for 24 hours then co-treated without or with IL-1β (10 pg/ml or 50 pg/ml) for another 24 hours followed by RNA isolation. Gene expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX2) was determined by quantitative Real Time-Polymerase Chain Reaction. Culture media concentrations of NO and PGE2 were determined by nitrite assay and immunoassay respectively. For analysis of cell signaling pathways, chondrocytes were pretreated with SeMet then stimulated with IL-1β for 0 – 45 minutes. The activity of IL-1β signaling pathways was determined by Western blot screening of phosphorylation states of signal transduction proteins. Results SeMet inhibited chondrocyte gene expression of IL-1β induced iNOS (31–54%, p=0.031) and COX2 (50–65%, p=0.031) with corresponding reductions in both NO (19–47%, p=0.031) and PGE2 (24–32%, p=0.031) production. Pretreatment with SeMet attenuated IL-1β induced activation of p38 MAPK (39%, p=0.039) but not the ERK, JNK or NFkB pathways. Conclusions This study elucidates one potential protective mechanism of selenium, namely through the alteration of cell signaling and downstream transcription of pro-inflammatory effects of IL-1β. PMID:21035557

  8. Astragalin inhibits IL-1β-induced inflammatory mediators production in human osteoarthritis chondrocyte by inhibiting NF-κB and MAPK activation.

    PubMed

    Ma, Zhiqiang; Piao, Taikui; Wang, Yanlong; Liu, Jianyu

    2015-03-01

    Astragalin, a bioactive component isolated from Rosa agrestis, has been described to exhibit anti-inflammatory activity. The aim of this study was to investigate the anti-inflammatory effects and the underlying mechanisms of astragalin on IL-1β-stimulated human osteoarthritis chondrocyte. The production of NO and PGE2 was detected by Griess reaction and ELISA. The expression of iNOS and COX-2 was detected by western blotting. The expression of NF-κB and MAPKs was detected by western blot analysis. We found that astragalin dose-dependently inhibited IL-1β-induced NO and PGE2 production, as well as iNOS and COX-2 expression. Meanwhile, western blot analysis showed that astragalin inhibited IL-1β-induced NF-κB and MAPK activation in human osteoarthritis chondrocyte. In addition, astragalin was found to activate PPAR-γ. The inhibition of astragalin on IL-1β-induced NO and PGE2 production can be reversed by PPAR-γ antagonist GW9662. Astragalin suppressed IL-1β-induced inflammatory mediators via activating PPAR-γ, which subsequently inhibited IL-1β-induced NF-κB and MAPK activation. Astragalin may be a potential agent in the treatment of osteoarthritis. PMID:25637445

  9. PEO-PPO-PEO Carriers for rAAV-Mediated Transduction of Human Articular Chondrocytes in Vitro and in a Human Osteochondral Defect Model.

    PubMed

    Rey-Rico, Ana; Frisch, Janina; Venkatesan, Jagadesh Kumar; Schmitt, Gertrud; Rial-Hermida, Isabel; Taboada, Pablo; Concheiro, Angel; Madry, Henning; Alvarez-Lorenzo, Carmen; Cucchiarini, Magali

    2016-08-17

    Gene therapy is an attractive strategy for the durable treatment of human osteoarthritis (OA), a gradual, irreversible joint disease. Gene carriers based on the small human adeno-associated virus (AAV) exhibit major efficacy in modifying damaged human articular cartilage in situ over extended periods of time. Yet, clinical application of recombinant AAV (rAAV) vectors remains complicated by the presence of neutralizing antibodies against viral capsid elements in a majority of patients. The goal of this study was to evaluate the feasibility of delivering rAAV vectors to human OA chondrocytes in vitro and in an experimental model of osteochondral defect via polymeric micelles to protect gene transfer from experimental neutralization. Interaction of rAAV with micelles of linear (poloxamer PF68) or X-shaped (poloxamine T908) poly(ethylene oxide) (PEO) and poly(propylene oxide) (PPO) copolymers (PEO-PPO-PEO micelles) was characterized by means of isothermal titration calorimetry. Micelle encapsulation allowed an increase in both the stability and bioactivity of rAAV vectors and promoted higher levels of safe transgene (lacZ) expression both in vitro and in experimental osteochondral defects compared with that of free vector treatment without detrimental effects on the biological activity of the cells or their phenotype. Remarkably, protection against antibody neutralization was also afforded when delivering rAAV via PEO-PPO-PEO micelles in all systems evaluated, especially when using T908. Altogether, these findings show the potential of PEO-PPO-PEO micelles as effective tools to improve current gene-based treatments for human OA. PMID:27404480

  10. Chondroprotective effects of a proanthocyanidin rich Amazonian genonutrient reflects direct inhibition of matrix metalloproteinases and upregulation of IGF-1 production by human chondrocytes

    PubMed Central

    Miller, Mark JS; Bobrowski, Paul; Shukla, Meenakshi; Gupta, Kalpana; Haqqi, Tariq M

    2007-01-01

    Background The Amazonian medicinal plant Sangre de grado (Croton palanostigma) has traditional applications for the treatment of wound healing and inflammation. We sought to characterize two extracts (progrado and zangrado) in terms of safety and oligomeric proanthocyanidin chain length. Additionally progrado was evaluated for antioxidant activity and possible chondroprotective actions. Methods Acute oral safety and toxicity was tested in rats according under OECD protocol number 420. The profile of proanthocyanidin oligomers was determined by HPLC and progrado's antioxidant activity quantified by the ORAC, NORAC and HORAC assays. Human cartilage explants, obtained from surgical specimens, were used to assess chondroproteciton with activity related to direct inhibitory effects on human matrix metalloproteinase (MMP, gelatinolytic) activity using synovial fluid and chondrocytes activated with IL-1β (10 ng/ml). Additionally, progrado (2–10 μg/ml) was tested for its ability to maintain optimal IGF-1 transcription and translation in cartilage explants and cultured chondrocytes. Results Both progrado and zangrado at doses up to 2000 mg/kg (po) displayed no evidence of toxicity. Oligomeric proanthocyanidin content was high for both progrado (158 mg/kg) and zangrado (124 mg/kg), with zangrado almost entirely composed of short oligomers (<6 mer), whereas the majority of oligomers in progrado exceeded 10 mers. Progrado was a remarkably potent antioxidant in the standardized tests ORAC, NORAC and HORAC. Progrado was exceptionally effective in reducing both basal and IL-1β induced glycosaminoglycan release from human cartilage explants at concentrations that also directly blocked the gelatinolytic activity of MMP-2 and MMP-9. Progrado prevented IL-1β induced suppression of IGF-1 production from human cartilage explants as well as stimulating basal IGF-1 production (P < 0.05). Comparable changes in IGF-1 gene expression were noted in cultured human chondrocytes

  11. The novel adipokine progranulin counteracts IL-1 and TLR4-driven inflammatory response in human and murine chondrocytes via TNFR1

    PubMed Central

    Abella, Vanessa; Scotece, Morena; Conde, Javier; López, Verónica; Pirozzi, Claudio; Pino, Jesús; Gómez, Rodolfo; Lago, Francisca; González-Gay, Miguel Ángel; Gualillo, Oreste

    2016-01-01

    Progranulin (PGRN) is a recently identified adipokine that is supposed to have anti-inflammatory actions. The proinflammatory cytokine interleukin-1β (IL1β) stimulates several mediators of cartilage degradation. Toll like receptor-4 (TLR4) can bind to various damage-associated molecular patterns, leading to inflammatory condition. So far, no data exist of PGRN effects in inflammatory conditions induced by IL1β or lipopolysaccharide (LPS). Here, we investigated the anti-inflammatory potential of PGRN in IL1β- or LPS-induced inflammatory responses of chondrocytes. Human osteoarthritic chondrocytes and ATDC-5 cells were treated with PGRN in presence or not of IL1β or LPS. First, we showed that recombinant PGRN had no effects on cell viability. We present evidence that PGRN expression was increased during the differentiation of ATDC-5 cell line. Moreover, PGRN mRNA and protein expression is increased in cartilage, synovial and infrapatellar fat pad tissue samples from OA patients. PGRN mRNA levels are upregulated under TNFα and IL1β stimulation. Our data showed that PGRN is able to significantly counteract the IL1β-induced expression of NOS2, COX2, MMP13 and VCAM-1. LPS-induced expression of NOS2 is also decreased by PGRN. These effects are mediated, at least in part, through TNFR1. Taken together, our results suggest that PGRN has a clear anti-inflammatory function. PMID:26853108

  12. Advanced glycation end products induce peroxisome proliferator-activated receptor γ down-regulation-related inflammatory signals in human chondrocytes via Toll-like receptor-4 and receptor for advanced glycation end products.

    PubMed

    Chen, Ying Ju; Sheu, Meei Ling; Tsai, Keh Sung; Yang, Rong Sen; Liu, Shing Hwa

    2013-01-01

    Accumulation of advanced glycation end products (AGEs) in joints is important in the development of cartilage destruction and damage in age-related osteoarthritis (OA). The aim of this study was to investigate the roles of peroxisome proliferator-activated receptor γ (PPARγ), toll-like receptor 4 (TLR4), and receptor for AGEs (RAGE) in AGEs-induced inflammatory signalings in human OA chondrocytes. Human articular chondrocytes were isolated and cultured. The productions of metalloproteinase-13 and interleukin-6 were quantified using the specific ELISA kits. The expressions of related signaling proteins were determined by Western blotting. Our results showed that AGEs enhanced the productions of interleukin-6 and metalloproteinase-13 and the expressions of cyclooxygenase-2 and high-mobility group protein B1 and resulted in the reduction of collagen II expression in human OA chondrocytes. AGEs could also activate nuclear factor (NF)-κB activation. Stimulation of human OA chondrocytes with AGEs significantly induced the up-regulation of TLR4 and RAGE expressions and the down-regulation of PPARγ expression in a time- and concentration-dependent manner. Neutralizing antibodies of TLR4 and RAGE effectively reversed the AGEs-induced inflammatory signalings and PPARγ down-regulation. PPARγ agonist pioglitazone could also reverse the AGEs-increased inflammatory signalings. Specific inhibitors for p38 mitogen-activated protein kinases, c-Jun N-terminal kinase and NF-κB suppressed AGEs-induced PPARγ down-regulation and reduction of collagen II expression. Taken together, these findings suggest that AGEs induce PPARγ down-regulation-mediated inflammatory signalings and reduction of collagen II expression in human OA chondrocytes via TLR4 and RAGE, which may play a crucial role in the development of osteoarthritis pathogenesis induced by AGEs accumulation. PMID:23776688

  13. The Emerging Chondrocyte Channelome

    PubMed Central

    Barrett-Jolley, Richard; Lewis, Rebecca; Fallman, Rebecca; Mobasheri, Ali

    2010-01-01

    Chondrocytes are the resident cells of articular cartilage and are responsible for synthesizing a range of collagenous and non-collagenous extracellular matrix macromolecules. Whilst chondrocytes exist at low densities in the tissue (1–10% of the total tissue volume in mature cartilage) they are extremely active cells and are capable of responding to a range of mechanical and biochemical stimuli. These responses are necessary for the maintenance of viable cartilage and may be compromised in inflammatory diseases such as arthritis. Although chondrocytes are non-excitable cells their plasma membrane contains a rich complement of ion channels. This diverse channelome appears to be as complex as one might expect to find in excitable cells although, in the case of chondrocytes, their functions are far less well understood. The ion channels so far identified in chondrocytes include potassium channels (KATP, BK, Kv, and SK), sodium channels (epithelial sodium channels, voltage activated sodium channels), transient receptor potential calcium or non-selective cation channels and chloride channels. In this review we describe this emerging channelome and discuss the possible functions of a range of chondrocyte ion channels. PMID:21423376

  14. Edible Bird’s nest extract as a chondro-protective agent for human chondrocytes isolated from osteoarthritic knee: in vitro study

    PubMed Central

    2013-01-01

    Background Osteoarthritis (OA) is a degenerative joint disease that results in the destruction of cartilage. Edible Bird’s Nest (EBN) extract contains important components, which can reduce the progression of osteoarthritis and helps in the regeneration of the cartilage. The present study aimed to investigate the effect of EBN extract on the catabolic and anabolic activities of the human articular chondrocytes (HACs) isolated from the knee joint of patients with OA. Methods A single batch of EBN extract was prepared with hot-water extraction and coded as HMG. HACs were isolated from the knee joint cartilage removed during surgery. The optimum concentration of HMG for HAC cultures was determined using MTT assay. The effect of HMG on the catabolic and anabolic genes’ expression in HACs was measured by real-time PCR. The total amount of prostaglandin E2 (PGE2) production was determined by ELISA method, and the total sulphated glycosaminoglycan (GAGs) production was quantified by 1,9-dimethylmethylene blue (DMMB) assay. Results MTT assay showed 0.50% - 1.00% HMG supplementation promoted HACs proliferation. HMG supplementation was able to reduce the catabolic genes’ expression in cultured HACs such as matrix metalloproteinases (MMP1 & MMP3), Interleukin 1, 6 and 8 (IL-1, IL-6 & IL-8), cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS). Prostaglandin E2 (PGE2) production was significantly reduced in HAC cultures supplemented with HMG. With regard to anabolic activity assessment, type II collagen, Aggrecan and SOX-9 gene expression as well as sGAG production was increased in the HMG supplemented groups. Conclusion Edible Bird’s Nest extract coded as HMG demonstrated chondro-protection ability on human articular chondrocytes in vitro. It reduced catabolic activities and increased cartilage extracellular matrix synthesis. It is concluded that HMG is a potential agent in the treatment of osteoarthritis. PMID:23339380

  15. The epigenetic effect of glucosamine and a nuclear factor-kappa B (NF-kB) inhibitor on primary human chondrocytes - Implications for osteoarthritis

    SciTech Connect

    Imagawa, Kei; Andres, MC de; Hashimoto, Ko; Pitt, Dominic; Itoi, Eiji; Goldring, Mary B.; Roach, Helmtrud I.; Oreffo, Richard O.C.

    2011-02-18

    Research highlights: {yields} Glucosamine and a NF-kB inhibitor reduce inflammation in OA. {yields} Cytokine induced demethylation of CpG site in IL1{beta} promoter prevented by glucosamine. {yields} Glucosamine and NF-kB inhibitor have epigenetic effects on human chondrocytes. -- Abstract: Objective: Idiopathic osteoarthritis is the most common form of osteoarthritis (OA) world-wide and remains the leading cause of disability and the associated socio-economic burden in an increasing aging population. Traditionally, OA has been viewed as a degenerative joint disease characterized by progressive destruction of the articular cartilage and changes in the subchondral bone culminating in joint failure. However, the etiology of OA is multifactorial involving genetic, mechanical and environmental factors. Treatment modalities include analgesia, joint injection with steroids or hyaluronic acid, oral supplements including glucosamine and chondroitin sulfate, as well as physiotherapy. Thus, there is significant interest in the discovery of disease modifying agents. One such agent, glucosamine (GlcN) is commonly prescribed even though the therapeutic efficacy and mechanism of action remain controversial. Inflammatory cytokines, including IL-1{beta}, and proteinases such as MMP-13 have been implicated in the pathogenesis and progression of OA together with an associated CpG demethylation in their promoters. We have investigated the potential of GlcN to modulate NF-kB activity and cytokine-induced abnormal gene expression in articular chondrocytes and, critically, whether this is associated with an epigenetic process. Method: Human chondrocytes were isolated from the articular cartilage of femoral heads, obtained with ethical permission, following fractured neck of femur surgery. Chondrocytes were cultured for 5 weeks in six separate groups; (i) control culture, (ii) cultured with a mixture of 2.5 ng/ml IL-1{beta} and 2.5 ng/ml oncostatin M (OSM), (iii) cultured with 2 mM N

  16. Chondrocyte channel transcriptomics

    PubMed Central

    Lewis, Rebecca; May, Hannah; Mobasheri, Ali; Barrett-Jolley, Richard

    2013-01-01

    To date, a range of ion channels have been identified in chondrocytes using a number of different techniques, predominantly electrophysiological and/or biomolecular; each of these has its advantages and disadvantages. Here we aim to compare and contrast the data available from biophysical and microarray experiments. This letter analyses recent transcriptomics datasets from chondrocytes, accessible from the European Bioinformatics Institute (EBI). We discuss whether such bioinformatic analysis of microarray datasets can potentially accelerate identification and discovery of ion channels in chondrocytes. The ion channels which appear most frequently across these microarray datasets are discussed, along with their possible functions. We discuss whether functional or protein data exist which support the microarray data. A microarray experiment comparing gene expression in osteoarthritis and healthy cartilage is also discussed and we verify the differential expression of 2 of these genes, namely the genes encoding large calcium-activated potassium (BK) and aquaporin channels. PMID:23995703

  17. The regulation of human MMP-13 by licofelone, an inhibitor of cyclo-oxygenases and 5-lipoxygenase, in human osteoarthritic chondrocytes is mediated by the inhibition of the p38 MAP kinase signalling pathway

    PubMed Central

    Boileau, C; Pelletier, J; Tardif, G; Fahmi, H; Laufer, S; Lavigne, M; Martel-Pelletier, J

    2005-01-01

    Background: MMP-13 is one of the most important metalloproteases (MMP) involved in osteoarthritis. Licofelone, a novel dual inhibitor of cyclo-oxygenases (COX) and 5-lipoxygenase (5-LOX), can modulate MMP-13 production in human osteoarthritis chondrocytes. Objective: To evaluate the impact of licofelone on MMP-13 expression/production, promoter, and major MAP kinase signalling pathways and transcription factors. Methods: Human osteoarthritis chondrocytes were stimulated by interleukin 1ß (IL1ß) and treated with or without: licofelone (0.3, 1, or 3 µg/ml); NS-398 (10 µM; a specific COX-2 inhibitor); or BayX-1005 (10 µM; a specific 5-LOX inhibitor). MMP-13 synthesis was determined by specific enzyme linked immunosorbent assay, and expression by real time polymerase chain reaction. The effect of licofelone on the MMP-13 promoter was studied through transient transfection; dexamethasone (10–7 M) was used as comparison. The effect on IL1ß induced MMP-13 signalling pathways was determined using specific ELISA for phosphorylated MAP kinases and transcription factors. Results: Licofelone dose dependently inhibited the IL1ß stimulated production and expression of MMP-13. NS-398 and BayX-1005 had very little effect. Licofelone also inhibited MMP-13 transcription on each of the promoter constructs used. The licofelone inhibition was comparable to that obtained with dexamethasone. Licofelone had no effect on phosphorylated p44/42 or JNK1/2; however, it decreased phosphorylated c-jun and inhibited phosphorylated p38, CREB, and AP-1 activity. Conclusions: Licofelone inhibited MMP-13 production under proinflammatory conditions on human osteoarthritis chondrocytes, through inhibition of the p38/AP-1 pathway and the transcription factor CREB. This may explain some of the mechanisms whereby licofelone exerts its positive effect on osteoarthritic changes. PMID:15498796

  18. Insulin-Like Growth Factor (IGF) Binding Protein-3 (IGFBP-3) is Closely Associated with the Chondrocyte Nucleus in Human Articular Cartilage

    PubMed Central

    EB, Hunziker; E, Kapfinger; J, Martin; J, Buckwalter; TI, Morales

    2008-01-01

    Objective The Insulin-like Growth Factor-I (IGF-I) is critically involved in the control of cartilage matrix metabolism. It is well known that its binding protein-3 (IGFBP-3) is increased during osteoarthritis (OA), but its function(s) is not known. In other cells, IGFBP-3 can regulate IGF-I action in the extracellular environment and can also act independently inside the cell; this includes transcriptional gene control in the nucleus. These studies were undertaken to localize IGFBP-3 in human articular cartilage, particularly within cells. Design Cartilage was dissected from human femoral heads derived from arthroplasty for OA, and OA grade assessed by histology. Tissue slices were further characterized by extraction and assay of IGFBPs by IGF Ligand blot (LB) and by ELISA. Immunohistochemistry (IHC) for IGF-I and IGFBP-3 was performed on cartilage from donors with mild, moderate and severe OA. Indirect fluorescence and immunogold labeling IHC studies were included. Results LBs of chondrocyte lysates showed a strong signal for IGFBP-3. IHC of femoral cartilage sections at all OA stages showed IGF-I and IGFBP-3 matrix stain particularly in the top zones, and closely associated with most cells. A prominent perinuclear/nuclear IGFBP-3 signal was seen. Controls using non-immune sera or antigen-blocked antibody showed negative or strongly reduced stain. In frozen sections of human ankle cartilage, immunofluorescent IGFBP-3 stain co-localized with the nuclear DAPI stain in greater than 90% of the cells. Immunogold IHC of thin sections and TEM immunogold microscopy of ultra-thin sections showed distinct intra-nuclear staining. Conclusions IGFBP-3 in human cartilage is located in the matrix and within chondrocytes in the cytoplasm and nuclei. This new data indicates that the range of IGFBP-3 actions in articular cartilage is likely to include IGF independent roles and opens the door to studies of its nuclear actions, including the possible regulation of hormone receptors

  19. Synthesis of classical pathway complement components by chondrocytes.

    PubMed Central

    Bradley, K; North, J; Saunders, D; Schwaeble, W; Jeziorska, M; Woolley, D E; Whaley, K

    1996-01-01

    Using immunohistochemical studies, C1q, C1s, C4 and C2 were detected in chondrocytes in normal human articular cartilage and macroscopically normal articular cartilage from the inferior surfaces of hip joints of patients with osteoarthritis. Using reverse-transcribed polymerase chain reaction (RT-PCR), mRNA for C1q, C1s, C4 and C2 was also detected in RNA extracted from articular cartilage. C1r, C3, C1-inhibitor, C4-binding protein and factor I were not detected by either technique. Articular chondrocytes cultured in vitro synthesized C1r, C1s, C4, C2, C3 and C1-inhibitor but not C1q, C4-binding protein or factor I, as assessed by enzyme-linked immunosorbent assay (ELISA) and Northern blot analysis. Thus cultured articular chondrocytes have a complement profile that is similar to that of cultured human fibroblasts rather than that of articular chondrocytes in vivo. Complement synthesis in cultured chondrocytes was modulated by the cytokines interleukin-1 beta (IL-1 beta), tumour necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma), showing that cytokines can probably regulate complement synthesis in intact cartilage. The possible roles of local synthesis of complement components by chondrocytes in matrix turnover and the regulation chondrocyte function are discussed. Images Figure 1 Figure 2 Figure 4 PMID:8881771

  20. Moderate alterations of the cytoskeleton in human chondrocytes after short-term microgravity produced by parabolic flight maneuvers could be prevented by up-regulation of BMP-2 and SOX-9.

    PubMed

    Aleshcheva, Ganna; Wehland, Markus; Sahana, Jayashree; Bauer, Johann; Corydon, Thomas J; Hemmersbach, Ruth; Frett, Timo; Egli, Marcel; Infanger, Manfred; Grosse, Jirka; Grimm, Daniela

    2015-06-01

    Real and simulated microgravity induce a variety of changes in human cells. Most importantly, changes in the cytoskeleton have been noted, and studies on microtubules have shown that they are gravisensitive. This study focuses on the effects of short-term real microgravity on gene expression, protein content, and cytoskeletal structure of human chondrocytes. We cultivated human chondrocytes, took them along a parabolic flight during the 24th Deutsches Zentrum für Luft- und Raumfahrt Parabolic (DLR) Flight Campaign, and fixed them after the 1st and the 31st parabola. Immunofluorescence microscopy revealed no changes after the 1st parabola, but disruptions of β-tubulin, vimentin, and cytokeratin networks after the 31st parabola. No F-actin stress fibers were detected even after 31 parabolas. Furthermore, mRNA and protein quantifications after the 31st parabola showed a clear up-regulation of cytoskeletal genes and proteins. The mRNAs were significantly up-regulated as follows: TUBB, 2-fold; VIM, 1.3-fold; KRT8, 1.8-fold; ACTB, 1.9-fold; ICAM1, 4.8-fold; OPN, 7-fold; ITGA10, 1.5-fold; ITGB1, 1.2-fold; TGFB1, 1.5-fold; CAV1, 2.6-fold; SOX9, 1.7-fold; BMP-2, 5.3-fold. However, SOX5 (-25%) and SOX6 (-28%) gene expression was decreased. Contrary, no significant changes in gene expression levels were observed during vibration and hypergravity experiments. These data suggest that short-term microgravity affects the gene expression of distinct proteins. In contrast to poorly differentiated follicular thyroid cancer cells or human endothelial cells, chondrocytes only exert moderate cytoskeletal alterations. The up-regulation of BMP-2, TGF-β1, and SOX9 in chondrocytes may play a key role in preventing cytoskeletal alterations. PMID:25681461

  1. Relationship between Initial Telomere Length, Initial Telomerase Activity, Age, and Replicative Capacity of Nucleus Pulposus Chondrocytes in Human Intervertebral Discs: What Is a Predictor of Replicative Potential?

    PubMed Central

    Lee, Jun-Seok; Jeong, Seo-Won; Cho, Sung-Wook; Juhn, Joon-Pyo; Kim, Ki-Won

    2015-01-01

    There is evidence that telomere length (TL), telomerase activity (TA), and age are related to the replicative potential of human nucleus pulposus chondrocytes (NPCs). However, it has not yet been established if any of these factors can serve as predictors of the replicative potential of NPCs. To establish predictors of the replicative potential of NPCs, we evaluated potential relationships between replicative capacity of NPCs, initial TL (telomere length at the first passage), initial TA (telomerase activity at the first passage), and age. Nucleus pulposus specimens were obtained from 14 patients of various ages undergoing discectomy. NPCs were serially cultivated until the end of their replicative lifespans. Relationships among cumulative population doubling level (PDL), initial TL, initial TA, and age were analyzed. Initial TA was negatively correlated with age (r = -0.674, P = 0.008). However, no correlation between initial TL and age was observed. Cumulative PDL was also negatively correlated with age (r = -0.585, P = 0.028). Although the cumulative PDL appeared to increase with initial TL or initial TA, this trend was not statistically significant. In conclusion, age is the sole predictor of the replicative potential of human NPCs, and replicative potential decreases with age. Initial TL and initial TA are not predictors of replicative potential, and can serve only as reference values. PMID:26633809

  2. Effects of sodium selenite on c-Jun N-terminal kinase signalling pathway induced by oxidative stress in human chondrocytes and c-Jun N-terminal kinase expression in patients with Kashin-Beck disease, an endemic osteoarthritis.

    PubMed

    Dai, XiaoXia; Li, YuanYuan; Zhang, RongQiang; Kou, Yan; Mo, XiaoYan; Cao, JunLing; Xiong, YongMin

    2016-05-01

    The c-Jun N-terminal kinases (JNK) are members of the mitogen-activated protein kinase family and are activated by environmental stress. Se plays an important role in the biological pathways by forming selenoprotein. Selenoproteins have been shown to exhibit a variety of biological functions including antioxidant functions and maintaining cellular redox balance, and compromise of such important proteins would lead to oxidative stress and apoptosis. We examined the expression levels of JNK in Kashin-Beck disease (KBD) patients, tested the potential protective effects of sodium selenite on tert-butyl hydroperoxide (tBHP)-induced oxidative injury and apoptosis in human chondrocytes as well as its underlying mechanism in this study. We produced an oxidative damage model induced by tBHP in C28/I2 human chondrocytes to test the essential anti-apoptosis effects of Se in vitro. The results indicated that the expression level of phosphorylated JNK was significantly increased in KBD patients. Cell apoptosis was increased and molecule expressions of the JNK signalling pathway were activated in the tBHP-injured chondrocytes. Na2SeO3 protected against tBHP-induced oxidative stress and apoptosis in cells by increasing cell viability, reducing reactive oxygen species generation, increasing Glutathione peroxidase (GPx) activity and down-regulating the JNK pathway. These results demonstrate that apoptosis induced by tBHP in chondrocytes might be mediated via up-regulation of the JNK pathway; Na2SeO3 has an effect of anti-apoptosis by down-regulating the JNK signalling pathway. PMID:26948765

  3. Extensively Expanded Auricular Chondrocytes Form Neocartilage In Vivo

    PubMed Central

    Tseng, Alan; Pomerantseva, Irina; Cronce, Michael J.; Kimura, Anya M.; Neville, Craig M.; Randolph, Mark A.; Sundback, Cathryn A.

    2014-01-01

    Objective Our goal was to engineer cartilage in vivo using auricular chondrocytes that underwent clinically relevant expansion and using methodologies that could be easily translated into health care practice. Design Sheep and human chondrocytes were isolated from auricular cartilage biopsies and expanded in vitro. To reverse dedifferentiation, expanded cells were either mixed with cryopreserved P0 chondrocytes at the time of seeding onto porous collagen scaffolds or proliferated with basic fibroblast growth factor (bFGF). After 2-week in vitro incubation, seeded scaffolds were implanted subcutaneously in nude mice for 6 weeks. The neocartilage quality was evaluated histologically; DNA and glycosaminoglycans were quantified. Cell proliferation rates and collagen gene expression profiles were assessed. Results Clinically sufficient over 500-fold chondrocyte expansion was achieved at passage 3 (P3); cell dedifferentiation was confirmed by the simultaneous COL1A1/3A1 gene upregulation and COL2A1 downregulation. The chondrogenic phenotype of sheep but not human P3 cells was rescued by addition of cryopreserved P0 chondrocytes. With bFGF supplementation, chondrocytes achieved clinically sufficient expansion at P2; COL2A1 expression was not rescued but COL1A1/3A1genes were downregulated. Although bFGF failed to rescue COL2A1 expression during chondrocyte expansion in vitro, elastic neocartilage with obvious collagen II expression was observed on porous collagen scaffolds after implantation in mice for 6 weeks. Conclusions Both animal and human auricular chondrocytes expanded with low-concentration bFGF supplementation formed high-quality elastic neocartilage on porous collagen scaffolds in vivo. PMID:26069703

  4. Leptin induces ADAMTS-4, ADAMTS-5, and ADAMTS-9 genes expression by mitogen-activated protein kinases and NF-ĸB signaling pathways in human chondrocytes.

    PubMed

    Yaykasli, Kursat Oguz; Hatipoglu, Omer Faruk; Yaykasli, Emine; Yildirim, Kubra; Kaya, Ertugrul; Ozsahin, Mustafa; Uslu, Mustafa; Gunduz, Esra

    2015-01-01

    Elucidation of the causes of inflammation has vital importance in the development of new approaches for the treatment of arthritic diseases. The degradation of aggrecan by upregulated disintegrin and metalloproteinase with trombospondin motifs (ADAMTSs) is the key event in the development of both rheumatoid arthritis (RA) and osteoarthritis (OA). Increased levels of leptin in both RA and OA have been demonstrated, thus linking leptin to arthritic diseases, but the mechanism has not been clarified. This study investigated the putative role of signaling pathways (p38, JNK, MEK1, NF-ĸB, and PI3) involved in leptin-induced cartilage destruction. Normal human articular chondrocytes were cultured with recombinant human leptin at 100, 250, 500, and 1000 ng/mL doses for 6, 12, 24, and 48 h, after which ADAMTS-4, -5, and -9 genes expression were determined by real time-polymerase chain reaction (RT-PCR) and Western Blot methods. The signaling pathways involved in leptin-induced ADAMTSs upregulation were also investigated by using inhibitors of signaling pathways. It was demonstrated that ADAMTSs expression level was peaked at 1000 ng/mL doses for 48 hours, and MAPKs (p38, JNK, and MEK) and NF-ĸB signaling pathways involving in leptin triggered ADAMTSs upregulation. Obesity as a risk for RA and OA may contribute to the inflammation of both RA and OA diseases by secreting adipokines like leptin. We hypothesize that leptin is involved in the development of RA and OA accompanied with obesity by increasing ADAMTS-4, -5, and -9 genes expression via MAPKs and NF-ĸB signaling pathways. PMID:25045124

  5. Equine articular chondrocytes on MACT scaffolds for cartilage defect treatment.

    PubMed

    Nürnberger, S; Meyer, C; Ponomarev, I; Barnewitz, D; Resinger, C; Klepal, W; Albrecht, C; Marlovits, S

    2013-10-01

    Treatment of cartilage defects poses challenging problems in human and veterinary medicine, especially in horses. This study examines the suitability of applying scaffold materials similar to those used for human cartilage regeneration on equine chondrocytes. Chondrocytes gained from biopsies of the talocrural joint of three horses were propagated in 2D culture and grown on two different scaffold materials, hyaluronan (HYAFF®) and collagen (BioGide®), and evaluated by light and electron microscopy. The equine chondrocytes developed well in both types of materials. They were vital and physiologically highly active. On the surface of the scaffolds, they formed cell multilayers. Inside the hyaluronan web, the chondrocytes were regularly distributed and spanned the large scaffold fibre distances by producing their own matrix sheath. Half-circle-like depressions occasionally found in the cell membrane were probably related to movement on the flexible matrix sheath. Inside the dense collagen scaffold, only single cells were found. They passed through the scaffold strands by cell shape adaptation. This study showed that the examined scaffold materials can be used for equine chondrocyte cultivation. Chondrocytes tend to form multilayers on the surface of both, very dense and very porous scaffolds, and have strategies to span between and move in large gaps. PMID:23323689

  6. Choosing the right chondrocyte cell line: Focus on nitric oxide.

    PubMed

    Santoro, Anna; Conde, Javier; Scotece, Morena; Abella, Vanessa; López, Verónica; Pino, Jesús; Gómez, Rodolfo; Gómez-Reino, Juan Jesús; Gualillo, Oreste

    2015-12-01

    Nitric oxide (NO) has been considered a catabolic factor that contributes to OA pathology by inducing chondrocytes apoptosis, matrix metalloproteinases synthesis, and pro-inflammatory cytokines expression. Thus, the research on NO regulation in chondrocytes represents a relevant field which needs to be explored in depth. However, to date, only the murine ATDC-5 cell line and primary chondrocytes are well-established cells to study NO production in cartilage tissues. The goal of this study is to determine whether two commonly used human chondrocytic cell lines: SW-1353 and T/C-28a2 cell lines are good models to examine lipopolysaccharide and/or pro-inflammatory cytokine-driven NO release and iNOS expression. To this aim, we carefully examined NO production and iNOS protein expression in human T/C-28a2 and SW-1353 chondrocytes stimulated with LPS and interleukin (IL)-1 alone or in combination. We also use ATDC-5 cells as a positive control for NO production. NO accumulation has been determined by colorimetric Griess reaction, whereas NOS type II expression was determined by Western Blot analysis. Our results clearly demonstrated that neither human T/C-28a2 nor SW-1353 chondrocytes showed a detectable increase in NO production or iNOS expression after bacterial endotoxin or cytokines challenge with IL-1. Our study demonstrated that T/C-28a2 and SW-1353 human cell lines are not suitable for studying NO release and iNOS expression confirming that ATDC5 and human primary cultured chondrocytes are the best in vitro cell system to study the actions derived from this mediator. PMID:26016689

  7. Effects of Payena dasyphylla (Miq.) on hyaluronidase enzyme activity and metalloproteinases protein expressions in interleukin-1β stimulated human chondrocytes cells

    PubMed Central

    2013-01-01

    Background Hyaluronidases have been found as the target enzymes in the development of osteoarthritis (OA) disease. While there is still no curative treatment for this disease, recent studies on the treatment of OA were focused on the effectiveness of natural products which are expected to improve the symptoms with minimal side effects. The aim of this study was to screen selected Malaysian plants on their anti-hyaluronidase activity as well as to evaluate the active plant and its derived fractions on its potential anti-arthritic and antioxidant activities. Methods A total of 20 methanolic crude extracts (bark and leaf) from ten different plants were screened using a colorimetric hyaluronidase enzymatic assay. The active plant extract (Payena dasyphylla) was then studied for its hyaluronidase inhibitory activity in the interleukin-1β (IL-1β) stimulated human chondrocytes cell line (NHAC-kn) using zymography method. The Payena dasyphylla methanolic bark extract was then fractionated into several fractions in where the ethyl acetate (EA) fraction was evaluated for its inhibitory effects on the HYAL1 and HYAL2 gene expressions using reverse transcription-polymerase chain reaction (RT-PCR) technique. While the MMP-3 and MMP-13 protein expressions were evaluated using western blot method. The phenolic and flavonoid contents of the three fractions as well as the antioxidant property of the EA fraction were also evaluated. Results Bark extract of Payena dasyphylla (100 μg/ml) showed the highest inhibitory activity against bovine testicular hyaluronidase with 91.63%. The plant extract also inhibited hyaluronidase expression in the cultured human chondrocyte cells in response to IL-1β (100 ng/ml). Similarly, treatment with Payena dasyphylla ethyl acetate (EA) fraction (100 μg/ml) inhibited the HYAL1 and HYAL2 mRNA gene expressions as well as MMP-3 and MMP-13 protein expression in a dose dependent manner. Payena dasyphylla EA fraction has demonstrated the highest

  8. Autologous chondrocytes. Autologous chondrocyte implantation: more data needed.

    PubMed

    2011-05-01

    There is no standard surgical treatment for young adults with persistent, incapacitating symptoms of knee cartilage damage. ChondroCelect is the first cell therapy product to be authorised in the European Union. It contains a dense suspension of chondrocytes cultured from a biopsy of the patient's knee cartilage for 4 weeks before being reimplanted. Clinical evaluation of Chondro-Celect only includes one trial, versus subchondral microfracture, in 118 patients. After 3 years of follow-up, there was no difference in the symptom score between the groups. Histological outcome was better after autologous chondrocyte implantation, but methodological problems make it difficult to interpret the observed difference. Long-term functional outcomes remain to be determined. More joint complications occurred after autologous chondrocyte implantation than after subchondral bone microfracture: more frequently symptomatic cartilage hypertrophy (27% versus 13%, possibly related to the implantation technique), joint swelling (22% versus 6.6%), joint effusion (24% versus 9.8%), and joint crepitations (18% versus 6.6%). Autologous chondrocyte implantation was sometimes associated with flu-like syndrome (in 7.8% of patients), which did not occur with the microfracture technique. Autologous chondrocyte implantation is more complex than microfracture. During routine use, there is a risk that one patient will inadvertently receive chondrocytes collected from another patient, leading to a risk of rejection. In practice, this autologous chondrocyte product should only be used by highly specialised teams, and its assessment must continue. PMID:21648176

  9. Monosodium Urate Crystal-Induced Chondrocyte Death via Autophagic Process

    PubMed Central

    Hwang, Hyun Sook; Yang, Chung Mi; Park, Su Jin; Kim, Hyun Ah

    2015-01-01

    Monosodium urate (MSU) crystals, which are highly precipitated in the joint cartilage, increase the production of cartilage-degrading enzymes and pro-inflammatory mediators in cartilage, thereby leading to gouty inflammation and joint damage. In this study, we investigated the effect of MSU crystals on the viability of human articular chondrocytes and the mechanism of MSU crystal-induced chondrocyte death. MSU crystals significantly decreased the viability of primary chondrocytes in a time- and dose-dependent manner. DNA fragmentation was observed in a culture medium of MSU crystal-treated chondrocytes, but not in cell lysates. MSU crystals did not activate caspase-3, a marker of apoptosis, compared with actinomycin D and TNF-α-treated cells. MSU crystals did not directly affect the expression of endoplasmic reticulum (ER) stress markers at the mRNA and protein levels. However, MSU crystals significantly increased the LC3-II level in a time-dependent manner, indicating autophagy activation. Moreover, MSU crystal-induced autophagy and subsequent chondrocyte death were significantly inhibited by 3-methyladenine, a blocker of autophagosomes formation. MSU crystals activated autophagy via inhibition of phosporylation of the Akt/mTOR signaling pathway. These results demonstrate that MSU crystals may cause the death of chondrocytes through the activation of the autophagic process rather than apoptosis or ER stress. PMID:26670233

  10. CCN1 Regulates Chondrocyte Maturation and Cartilage Development

    PubMed Central

    Zhang, Yongchun; Sheu, Tzong-jen; Hoak, Donna; Shen, Jie; Hilton, Matthew J; Zuscik, Michael J; Jonason, Jennifer H; O’Keefe, Regis J

    2016-01-01

    WNT/β-CATENIN signaling is involved in multiple aspects of skeletal development, including chondrocyte differentiation and maturation. Although the functions of β-CATENIN in chondrocytes have been extensively investigated through gain-of-function and loss-of-function mouse models, the precise downstream effectors through which β-CATENIN regulates these processes are not well defined. Here, we report that the matricellular protein, CCN1, is induced by WNT/β-CATENIN signaling in chondrocytes. Specifically, we found that β-CATENIN signaling promotes CCN1 expression in isolated primary sternal chondrocytes and both embryonic and postnatal cartilage. Additionally, we show that, in vitro, CCN1 overexpression promotes chondrocyte maturation, whereas inhibition of endogenous CCN1 function inhibits maturation. To explore the role of CCN1 on cartilage development and homeostasis in vivo, we generated a novel transgenic mouse model for conditional Ccn1 overexpression and show that cartilage-specific CCN1 overexpression leads to chondrodysplasia during development and cartilage degeneration in adult mice. Finally, we demonstrate that CCN1 expression increases in mouse knee joint tissues after meniscal/ligamentous injury (MLI) and in human cartilage after meniscal tear. Collectively, our data suggest that CCN1 is an important regulator of chondrocyte maturation during cartilage development and homeostasis. PMID:26363286

  11. CCN1 Regulates Chondrocyte Maturation and Cartilage Development.

    PubMed

    Zhang, Yongchun; Sheu, Tzong-Jen; Hoak, Donna; Shen, Jie; Hilton, Matthew J; Zuscik, Michael J; Jonason, Jennifer H; O'Keefe, Regis J

    2016-03-01

    WNT/β-CATENIN signaling is involved in multiple aspects of skeletal development, including chondrocyte differentiation and maturation. Although the functions of β-CATENIN in chondrocytes have been extensively investigated through gain-of-function and loss-of-function mouse models, the precise downstream effectors through which β-CATENIN regulates these processes are not well defined. Here, we report that the matricellular protein, CCN1, is induced by WNT/β-CATENIN signaling in chondrocytes. Specifically, we found that β-CATENIN signaling promotes CCN1 expression in isolated primary sternal chondrocytes and both embryonic and postnatal cartilage. Additionally, we show that, in vitro, CCN1 overexpression promotes chondrocyte maturation, whereas inhibition of endogenous CCN1 function inhibits maturation. To explore the role of CCN1 on cartilage development and homeostasis in vivo, we generated a novel transgenic mouse model for conditional Ccn1 overexpression and show that cartilage-specific CCN1 overexpression leads to chondrodysplasia during development and cartilage degeneration in adult mice. Finally, we demonstrate that CCN1 expression increases in mouse knee joint tissues after meniscal/ligamentous injury (MLI) and in human cartilage after meniscal tear. Collectively, our data suggest that CCN1 is an important regulator of chondrocyte maturation during cartilage development and homeostasis. © 2015 American Society for Bone and Mineral Research. PMID:26363286

  12. Bovine achondrogenesis: evidence for defective chondrocyte differentiation.

    PubMed

    Horton, W A; Jayo, M J; Leipold, H W; Machado, M A; Campbell, D; Ahmed, S

    1987-01-01

    A survey study of growth cartilage abnormalities in bovine bone dysplasias revealed that a disorder in Holstein cattle called bulldog calf closely resembles human achondrogenesis Type II. Substantial amounts of Type I collagen and other non Type II collagens were detected in the bulldog cartilage which was comprised primarily of extensive vascular canals and cells having the characteristics of hypertrophic and degenerative chondrocytes normally found in the growth plate. It is proposed that chondrocytes throughout the bulldog growth cartilage prematurely differentiate into hypertrophic cells that degenerate and predispose the cartilage to vascular invasion and the formation of cartilage canals. The presence of these canals probably accounts for most of the observed collagen abnormalities. PMID:3606909

  13. Lectin binding patterns reflect the phenotypic status of in vitro chondrocyte models.

    PubMed

    Toegel, S; Plattner, V E; Wu, S Q; Goldring, M B; Chiari, C; Kolb, A; Unger, F M; Nehrer, S; Gabor, F; Viernstein, H; Wirth, M

    2009-01-01

    In vitro studies using chondrocyte cell cultures have increased our understanding of cartilage physiology and the altered chondrocytic cell phenotype in joint diseases. Beside the use of primary cells isolated from cartilage specimens of donors, immortalized chondrocyte cell lines such as C-28/I2 and T/C-28a2 have facilitated reproducible and standardized experiments. Although carbohydrate structures appear of significance for cartilage function, the contribution of the chondrocyte glycocalyx to matrix assembly and alterations of the chondrocyte phenotype is poorly understood. Therefore, the present study aimed to evaluate the glycoprofile of primary human chondrocytes as well as of C-28/I2 and T/C-28a2 cells in culture. First, the chondrocytic phenotype of primary and immortalized cells was assessed using real-time reverse transcriptase polymerase chain reaction, immunofluorescence, and glycosaminoglycans staining. Then, a panel of lectins was selected to probe for a range of oligosaccharide sequences determining specific products of the O-glycosylation and N-glycosylation pathways. We found that differences in the molecular phenotype between primary chondrocytes and the immortalized chondrocyte cell models C-28/I2 and T/C-28a2 are reflected in the glycoprofile of the cells. In this regard, the glycocalyx of immortalized chondrocytes was characterized by reduced levels of high-mannose type and sialic acid-capped N-glycans as well as increased fucosylated O-glycosylation products. In summary, the present report emphasizes the glycophenotype as an integral part of the chondrocyte phenotype and points at a significant role of the glycophenotype in chondrocyte differentiation. PMID:19263178

  14. Homology of lubricin and superficial zone protein (SZP): products of megakaryocyte stimulating factor (MSF) gene expression by human synovial fibroblasts and articular chondrocytes localized to chromosome 1q25.

    PubMed

    Jay, G D; Tantravahi, U; Britt, D E; Barrach, H J; Cha, C J

    2001-07-01

    We have previously identified megakaryocyte stimulating factor (MSF) gene expression by synovial fibroblasts as the origin of lubricin in the synovial cavity. Lubricin is a mucinous glycoprotein responsible for the boundary lubrication of articular cartilage. MSF has a significant homology to vitronectin and is composed of 12 exons. RNA was purified from human synovial fibroblasts and articular chondrocytes grown in vitro from tissue explants obtained from subjects without degenerative joint disease. RT-PCR was used with multiple complimentary primer pairs spanning the central mucin expressing exon 6 of the MSF gene and individual exons on both the N- and C-terminal sides of exon 6. Exons 2, 4 and 5 appear to be variably expressed by synovial fibroblasts and articular chondrocytes. Lubricating mucin, in the form of MSF, is expressed by both chondrocytes and synovial fibroblasts in vitro. Both lubricin and superficial zone protein (SZP), a related proteoglycan, share a similar primary structure but could differ in post-translational modifications with O-linked oligosaccharides which are predominant in lubricin and with limited amounts chondroitin and keratan sulfate found in SZP. Since most of the MSF exons are involved in the expression of lubricating mucin, a strong homology to vitronectin persists. It is therefore appropriate to consider that both SZP and lubricin occupy a new class of biomolecules termed tribonectins. Screening of a human genome bacterial artificial chromsome (BAC) library with a cDNA primer pair complimentary for exon 6 identified two clones. Both clones were complimentary for chromosome 1q25 by in situ hybridization. This same locus was previously implicated in camptodactyl-arthropathy-pericarditis syndrome (CAP) by genetic mapping. It is hypothesized that CAP, a large joint arthropathy, may be associated with ineffective boundary lubrication provided by synovial fluid. PMID:11518279

  15. IL-1beta, but not BMP-7 leads to a dramatic change in the gene expression pattern of human adult articular chondrocytes--portraying the gene expression pattern in two donors.

    PubMed

    Saas, J; Haag, J; Rueger, D; Chubinskaya, S; Sohler, F; Zimmer, R; Bartnik, E; Aigner, T

    2006-10-01

    Anabolic and catabolic cytokines and growth factors such as BMP-7 and IL-1beta play a central role in controlling the balance between degradation and repair of normal and (osteo)arthritic articular cartilage matrix. In this report, we investigated the response of articular chondrocytes to these factors IL-1beta and BMP-7 in terms of changes in gene expression levels. Large scale analysis was performed on primary human adult articular chondrocytes isolated from two human, independent donors cultured in alginate beads (non-stimulated and stimulated with IL-1beta and BMP-7 for 48 h) using Affymetrix gene chips (oligo-arrays). Biostatistical and bioinformatic evaluation of gene expression pattern was performed using the Resolver software (Rosetta). Part of the results were confirmed using real-time PCR. IL-1beta modulated significantly 909 out of 3459 genes detectable, whereas BMP-7 influenced only 36 out of 3440. BMP-7 induced mainly anabolic activation of chondrocytes including classical target genes such as collagen type II and aggrecan, while IL-1beta, both, significantly modulated the gene expression levels of numerous genes; namely, IL-1beta down-regulated the expression of anabolic genes and induced catabolic genes and mediators. Our data indicate that BMP-7 has only a limited effect on differentiated cells, whereas IL-1beta causes a dramatic change in gene expression pattern, i.e. induced or repressed much more genes. This presumably reflects the fact that BMP-7 signaling is effected via one pathway only (i.e. Smad-pathway) whereas IL-1beta is able to signal via a broad variety of intracellular signaling cascades involving the JNK, p38, NFkB and Erk pathways and even influencing BMP signaling. PMID:17161615

  16. Fibroblast Growth Factor–2 Promotes Catabolism Via FGFR1–Ras–Raf–MEK1/2–ERK1/2 Axis That Coordinates With the PKCδ Pathway in Human Articular Chondrocytes

    PubMed Central

    Yan, Dongyao; Chen, Di; Im, Hee-Jeong

    2013-01-01

    Fibroblast growth factor 2 (FGF-2) has been found to play an anti-anabolic and/or a catabolic role in adult human articular cartilage via regulation of multiple signaling pathways. Upon FGF-2 stimulation, a molecular crosstalk between the mitogen activated protein kinase (MAPK) and protein kinase C δ (PKCδ) pathways are initiated, where PKCδ positively regulates downstream MAPK signaling. In this study, we explored the relationship between fibroblast growth factor receptor 1 (FGFR1), Ras, and PKCδ in FGF-2 signaling in human articular chondrocytes. Pathway-specific inhibition using both chemical inhibitors and siRNA targeting FGFR1 demonstrated that, upon FGF-2 stimulation, FGFR1 controlled both Ras and PKCδ activation, which converged on the Raf-MEK1/2-ERK1/2 axis. No crosstalk was observed between Ras and PKCδ. Quantitative PCR analyses revealed that both Ras and PKCδ contributed to FGF-2-mediated upregulation of MMP-13, ADAMTS5, and repression of aggrecan gene. Correspondingly, FGF-2-mediated proteoglycan loss was effectively reversed by individual pathway-specific inhibitor of Ras, PKCδ, and ERK1/2 in both 3-dimensional alginate bead culture and cartilage organ culture systems. Our findings suggest that FGFR1 interacts with FGF-2 and then activates Ras and PKCδ, which concertedly drive MAPK signaling to mediate biological effects of FGF-2. Such an integration of dual inputs constitutes a novel mechanism of FGF-2 signaling cascade in human articular chondrocytes. PMID:22488450

  17. R-spondin 2 facilitates differentiation of proliferating chondrocytes into hypertrophic chondrocytes by enhancing Wnt/β-catenin signaling in endochondral ossification.

    PubMed

    Takegami, Yasuhiko; Ohkawara, Bisei; Ito, Mikako; Masuda, Akio; Nakashima, Hiroaki; Ishiguro, Naoki; Ohno, Kinji

    2016-04-22

    Endochondral ossification is a crucial process for longitudinal growth of bones. Differentiating chondrocytes in growth cartilage form four sequential zones of proliferation, alignment into column, hypertrophy, and substitution of chondrocytes with osteoblasts. Wnt/β-catenin signaling is essential for differentiation of proliferating chondrocytes into hypertrophic chondrocytes in growth cartilage. R-spondin 2 (Rspo2), a member of R-spondin family, is an agonist for Wnt signaling, but its role in chondrocyte differentiation remains unknown. Here we report that growth cartilage of Rspo2-knockout mice shows a decreased amount of β-catenin and increased amounts collagen type II (CII) and Sox9 in the abnormally extended proliferating zone. In contrast, expression of collagen type X (CX) in the hypertrophic zone remains unchanged. Differentiating chondrogenic ATDC5 cells, mimicking proliferating chondrocytes, upregulate Rspo2 and its putative receptor, Lgr5, in parallel. Addition of recombinant human Rspo2 to differentiating ATDC5 cells decreases expressions of Col2a1, Sox9, and Acan, as well as production of proteoglycans. In contrast, lentivirus-mediated knockdown of Rspo2 has the opposite effect. The effect of Rspo2 on chondrogenic differentiation is mediated by Wnt/β-catenin signaling, and not by Wnt/PCP or Wnt/Ca(2+) signaling. We propose that Rspo2 activates Wnt/β-catenin signaling to reduce Col2a1 and Sox9 and to facilitate differentiation of proliferating chondrocytes into hypertrophic chondrocytes in growth cartilage. PMID:27012200

  18. Integrated Study of Globally Expressed microRNAs in IL-1β-stimulated Human Osteoarthritis Chondrocytes and Osteoarthritis Relevant Genes: A Microarray and Bioinformatics Analysis.

    PubMed

    Rasheed, Zafar; Al-Shobaili, Hani A; Rasheed, Naila; Al Salloom, Abdulaziz A M; Al-Shaya, Osama; Mahmood, Amer; Alajez, Nehad M; Alghamdi, Ahmed S S; Mehana, El-Sayed E

    2016-07-01

    This study was undertaken to identify and characterize the globally expressed microRNAs (miRNAs) involved in interleukin-1β (IL-1β)-induced joint damage and to predict whether miRNAs can regulate the catabolic effects in osteoarthritis (OA) chondrocytes. Out of 1347 miRNAs analyzed by microarrays in IL-1β-stimulated OA chondrocytes, 35 miRNAs were down-regulated, 1 miRNA was up-regulated, and the expression of 1311 miRNAs remained unchanged. Bioinformatics analysis showed the key inflammatory mediators and key molecular pathways are targeted by differentially expressed miRNAs. Novel miRNAs identified could have important diagnostic and therapeutic potentials in the development of novel therapeutic strategies for pain managements in OA. PMID:27152662

  19. Circular RNA Related to the Chondrocyte ECM Regulates MMP13 Expression by Functioning as a MiR-136 ‘Sponge’ in Human Cartilage Degradation

    PubMed Central

    Liu, Qiang; Zhang, Xin; Hu, Xiaoqing; Dai, Linghui; Fu, Xin; Zhang, Jiying; Ao, Yingfang

    2016-01-01

    Circular RNAs (circRNAs) are involved in the development of various diseases, but there is little knowledge of circRNAs in osteoarthritis (OA). The aim of study was to identify circRNA expression in articular cartilage and to explore the function of chondrocyte extracellular matrix (ECM)-related circRNAs (circRNA-CER) in cartilage. To identify circRNAs that are specifically expressed in cartilage, we compared the expression of circRNAs in OA cartilage with that in normal cartilage. Bioinformatics was employed to predict the interaction of circRNAs and mRNAs in cartilage. Loss-of-function and rescue experiments for circRNA-CER were performed in vitro. A total of 71 circRNAs were differentially expressed in OA and normal cartilage. CircRNA-CER expression increased with interleukin-1 and tumor necrosis factor levels in chondrocytes. Silencing of circRNA-CER using small interfering RNA suppressed MMP13 expression and increased ECM formation. CircRNA-CER could compete for miR-136 with MMP13. Our results demonstrated that circRNA-CER regulated MMP13 expression by functioning as a competing endogenous RNA (ceRNA) and participated in the process of chondrocyte ECM degradation. We propose that circRNA-CER could be used as a potential target in OA therapy. PMID:26931159

  20. Runx1 Activities in Superficial Zone Chondrocytes, Osteoarthritic Chondrocyte Clones and Response to Mechanical Loading

    PubMed Central

    LeBlanc, Kimberly T.; Walcott, Marie E.; Gaur, Tripti; O’Connell, Shannon L.; Basil, Kirti; Tadiri, Christina P.; Mason-Savas, April; Silva, Jason A.; van Wijnen, Andre J.; Stein, Janet L.; Stein, Gary S; Ayers, David C.; Lian, Jane B.; Fanning, Paul J.

    2015-01-01

    Objective Runx1, the hematopoietic lineage determining transcription factor, is present in perichondrium and chondrocytes. Here we addressed Runx1 functions, by examining expression in cartilage during mouse and human osteoarthritis (OA) progression and in response to mechanical loading. Methods Spared and diseased compartments in knees of OA patients and in mice with surgical destabilization of the medial meniscus were examined for changes in expression of Runx1 mRNA (Q-PCR) and protein (immunoblot, immunohistochemistry). Runx1 levels were quantified in response to static mechanical compression of bovine articular cartilage. Runx1 function was assessed by cell proliferation (Ki67, PCNA) and cell type phenotypic markers. Results Runx1 is enriched in superficial zone (SZ) chondrocytes of normal bovine, mouse, and human tissues. Increasing loading conditions in bovine cartilage revealed a positive correlation with a significant elevation of Runx1. Runx1 becomes highly expressed at the periphery of mouse OA lesions and in human OA chondrocyte ‘clones’ where Runx1 co-localizes with Vcam1, the mesenchymal stem cell (MSC) marker and lubricin (Prg4), a cartilage chondroprotective protein. These OA induced cells represent a proliferative cell population, Runx1 depletion in MPCs decreases cell growth, supporting Runx1 contribution to cell expansion. Conclusion The highest Runx1 levels in SZC of normal cartilage suggest a function that supports the unique phenotype of articular chondrocytes, reflected by upregulation under conditions of compression. We propose Runx1 co-expression with Vcam1 and lubricin in murine cell clusters and human ‘clones’ of OA cartilage, participate in a cooperative mechanism for a compensatory anabolic function. PMID:25078095

  1. The Interplay between Chondrocyte Redifferentiation Pellet Size and Oxygen Concentration

    PubMed Central

    Babur, Betul Kul; Ghanavi, Parisa; Levett, Peter; Lott, William B.; Klein, Travis; Cooper-White, Justin J.; Crawford, Ross; Doran, Michael R.

    2013-01-01

    Chondrocytes dedifferentiate during ex vivo expansion on 2-dimensional surfaces. Aggregation of the expanded cells into 3-dimensional pellets, in the presence of induction factors, facilitates their redifferentiation and restoration of the chondrogenic phenotype. Typically 1×105–5×105 chondrocytes are aggregated, resulting in “macro” pellets having diameters ranging from 1–2 mm. These macropellets are commonly used to study redifferentiation, and recently macropellets of autologous chondrocytes have been implanted directly into articular cartilage defects to facilitate their repair. However, diffusion of metabolites over the 1–2 mm pellet length-scales is inefficient, resulting in radial tissue heterogeneity. Herein we demonstrate that the aggregation of 2×105 human chondrocytes into micropellets of 166 cells each, rather than into larger single macropellets, enhances chondrogenic redifferentiation. In this study, we describe the development of a cost effective fabrication strategy to manufacture a microwell surface for the large-scale production of micropellets. The thousands of micropellets were manufactured using the microwell platform, which is an array of 360×360 µm microwells cast into polydimethylsiloxane (PDMS), that has been surface modified with an electrostatic multilayer of hyaluronic acid and chitosan to enhance micropellet formation. Such surface modification was essential to prevent chondrocyte spreading on the PDMS. Sulfated glycosaminoglycan (sGAG) production and collagen II gene expression in chondrocyte micropellets increased significantly relative to macropellet controls, and redifferentiation was enhanced in both macro and micropellets with the provision of a hypoxic atmosphere (2% O2). Once micropellet formation had been optimized, we demonstrated that micropellets could be assembled into larger cartilage tissues. Our results indicate that micropellet amalgamation efficiency is inversely related to the time cultured as discreet

  2. Autophagy modulates articular cartilage vesicle formation in primary articular chondrocytes.

    PubMed

    Rosenthal, Ann K; Gohr, Claudia M; Mitton-Fitzgerald, Elizabeth; Grewal, Rupinder; Ninomiya, James; Coyne, Carolyn B; Jackson, William T

    2015-05-22

    Chondrocyte-derived extracellular organelles known as articular cartilage vesicles (ACVs) participate in non-classical protein secretion, intercellular communication, and pathologic calcification. Factors affecting ACV formation and release remain poorly characterized; although in some cell types, the generation of extracellular vesicles is associated with up-regulation of autophagy. We sought to determine the role of autophagy in ACV production by primary articular chondrocytes. Using an innovative dynamic model with a light scatter nanoparticle counting apparatus, we determined the effects of autophagy modulators on ACV number and content in conditioned medium from normal adult porcine and human osteoarthritic chondrocytes. Healthy articular chondrocytes release ACVs into conditioned medium and show significant levels of ongoing autophagy. Rapamycin, which promotes autophagy, increased ACV numbers in a dose- and time-dependent manner associated with increased levels of autophagy markers and autophagosome formation. These effects were suppressed by pharmacologic autophagy inhibitors and short interfering RNA for ATG5. Caspase-3 inhibition and a Rho/ROCK inhibitor prevented rapamycin-induced increases in ACV number. Osteoarthritic chondrocytes, which are deficient in autophagy, did not increase ACV number in response to rapamycin. SMER28, which induces autophagy via an mTOR-independent mechanism, also increased ACV number. ACVs induced under all conditions had similar ecto-enzyme specific activities and types of RNA, and all ACVs contained LC3, an autophagosome-resident protein. These findings identify autophagy as a critical participant in ACV formation, and augment our understanding of ACVs in cartilage disease and repair. PMID:25869133

  3. Engineered cartilage using primary chondrocytes cultured in a porous cartilage-derived matrix

    PubMed Central

    Cheng, Nai-Chen; Estes, Bradley T; Young, Tai-Horng; Guilak, Farshid

    2011-01-01

    Aim To investigate the cell growth, matrix accumulation and mechanical properties of neocartilage formed by human or porcine articular chondrocytes on a porous, porcine cartilage-derived matrix (CDM) for use in cartilage tissue engineering. Materials & methods We examined the physical properties, cell infiltration and matrix accumulation in different formulations of CDM and selected a CDM made of homogenized cartilage slurry as an appropriate scaffold for long-term culture of human and porcine articular chondrocytes. Results The CDM scaffold supported growth and proliferation of both human and porcine chondrocytes. Histology and immunohistochemistry showed abundant cartilage-specific macromolecule deposition at day 28. Human chondrocytes migrated throughout the CDM, showing a relatively homogeneous distribution of new tissue accumulation, whereas porcine chondrocytes tended to form a proteoglycan-rich layer primarily on the surfaces of the scaffold. Human chondrocyte-seeded scaffolds had a significantly lower aggregate modulus and hydraulic permeability at day 28. Conclusions These data show that a scaffold derived from native porcine articular cartilage can support neocartilage formation in the absence of exogenous growth factors. The overall characteristics and properties of the constructs depend on factors such as the concentration of CDM used, the porosity of the scaffold, and the species of chondrocytes. PMID:21175289

  4. Chondrocyte Senescence and Telomere Regulation: Implications in Cartilage Aging and Cancer (A Brief Review)

    PubMed Central

    Mollano, Anthony V; Martin, James A; Buckwalter, Joseph A

    2002-01-01

    Recent studies on osteoarthritis and the cartilage aging in our laboratory demonstrate that chronologic age correlates with molecular changes in human chondrocytes that affect cell cycle control and replicative life span. These findings indicate that age-related changes in chondrocytes may explain the heightened risk for development of primary osteoarthritis (OA) with increasing age. Concomitant studies of human chondrosarcoma suggest that these aging mechanisms may also play a role in preventing the malignant transformation of chondrocytes. The convergence at the molecular level of these seemingly dissimilar biologic processes provides an excellent opportunity to deepen our understanding of the fundamental processes underlying cartilage neoplasia, cartilage aging, and osteoarthritis. PMID:12180600

  5. Schisandrae Fructus Inhibits IL-1β-Induced Matrix Metalloproteinases and Inflammatory Mediators Production in SW1353 Human Chondrocytes by Suppressing NF-κB and MAPK Activation.

    PubMed

    Jeong, Jin-Woo; Lee, Hye Hyeon; Choi, Eun-Ok; Lee, Ki Won; Kim, Ki Young; Kim, Sung Goo; Hong, Su Hyun; Kim, Gi-Young; Park, Cheol; Kim, Ho Kyoung; Choi, Young Whan; Choi, Yung Hyun

    2015-12-01

    Proinflammatory cytokine interleukin-1 beta (IL-1β) plays a crucial role in the pathogenesis of osteoarthritis (OA) by stimulating several mediators that contribute to cartilage degradation. Schisandrae Fructus (SF), the dried fruit of Schisandra chinensis (Turcz.) Baill. (Magnoliaceae), is widely used in traditional medicine for the treatment of a number of chronic inflammatory diseases. This study investigated the antiosteoarthritis properties of an ethanol extract of SF on IL-1β-stimulated SW1353 chondrocytes. SF attenuated IL-1β-induced expression and activity of matrix metalloproteinase (MMP)-1, MMP-3, and MMP-13 and also reduced the elevated levels of cyclooxygenase-2 and inducible nitric oxide synthase associated with the inhibition of prostaglandin E2 and nitric oxide production in IL-1β-stimulated SW1353 chondrocytes. In addition, SF markedly suppressed the nuclear translocation of nuclear factor-kappa B (NF-κB) by blocking inhibitor κB-alpha degradation and inhibited the phosphorylation of c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK). These results indicate that the inhibitory effect of SF on IL-1β-stimulated expression of MMPs and inflammatory mediators production in SW1353 cells were associated with the suppression of the NF-κB and JNK/p38 MAPK signaling pathways. The results from this study indicate that SF may have therapeutic potential for the treatment of OA due to its anti-inflammatory and chondroprotective features. PMID:26443270

  6. The effect of resveratrol on normal and osteoarthritic chondrocyte metabolism

    PubMed Central

    Kim, H. J.; Braun, H. J.; Dragoo, J. L.

    2014-01-01

    Background Resveratrol is a polyphenolic compound commonly found in the skins of red grapes. Sirtuin 1 (SIRT1) is a human gene that is activated by resveratrol and has been shown to promote longevity and boost mitochondrial metabolism. We examined the effect of resveratrol on normal and osteoarthritic (OA) human chondrocytes. Methods Normal and OA chondrocytes were incubated with various concentrations of resveratrol (1 µM, 10 µM, 25 µM and 50 µM) and cultured for 24, 48 or 72 hours or for six weeks. Cell proliferation, gene expression, and senescence were evaluated. Results SIRT1 was significantly upregulated in normal chondrocytes with resveratrol concentrations of 25 µM and 50 µM on both two- (2D) (both p = 0.001) and three-dimensional (3D) cultures (p = 0.008 and 0.001, respectively). It was significantly upregulated in OA chondrocytes treated with 10 µM, 25 µM and 50 µM resveratrol on 2D cultures (p = 0.036, 0.002 and 0.001, respectively) and at 50 µM concentration on 3D cultures (p = 0.001). At 72 hours, the expression of collagen (COL)-10, aggrecan (AGG), and runt-related transcription factor 2 (RUNX2) was significantly greater in both 25 µM (p = 0.011, 0.006 and 0.015, respectively) and 50 µM (p = 0.019, 0.004 and 0.002, respectively) resveratrol-treated normal chondrocyte cultures. In OA chondrocytes, expression of COL10 and RUNX2 was significantly greater in 25 µM (p = 0.004 and 0.024) and 50 µM (p = 0.004 and 0.019) cultures at 72 hours on 3D cultures. Conclusions At concentrations of 25 µM and/or 50 µM, resveratrol treatment significantly upregulates SIRT1 gene expression in normal and osteoarthritic chondrocytes. Resveratrol induces chondrocytes into a hypertrophic state through upregulation of COL1, COL10, and RUNX2. Cite this article: Bone Joint Res 2014;3:51–9. PMID:24615710

  7. Protein kinase B and extracellular signal-regulated kinase contribute to the chondroprotective effect of morroniside on osteoarthritis chondrocytes

    PubMed Central

    Cheng, Liang; Zeng, Guoqing; Liu, Zejun; Zhang, Bing; Cui, Xu; Zhao, Honghai; Zheng, Xinpeng; Song, Gang; Kang, Jian; Xia, Chun

    2015-01-01

    Despite extensive studies on the multifaceted roles of morroniside, the main active constituent of iridoid glycoside from Corni Fructus, the effect of morroniside on osteoarthritis (OA) chondrocytes remains poorly understood. Here, we investigated the influence of morroniside on cultured human OA chondrocytes and a rat experimental model of OA. The results showed that morroniside enhanced the cell viability and the levels of proliferating cell nuclear antigen expression (PCNA), type II collagen and aggrecan in human OA chondrocytes, indicating that morroniside promoted chondrocyte survival and matrix synthesis. Furthermore, different doses of morroniside activated protein kinase B (AKT) and extracellular signal-regulated kinase (ERK) in human OA chondrocytes, and in turn, triggered AKT/S6 and ERK/P70S6K/S6 pathway, respectively. The PI3K/AKT inhibitor LY294002 or the MEK/ERK inhibitor U0126 attenuated the effect of morroniside on human OA chondrocytes, indicating that the activation of AKT and ERK contributed to the regulation of morroniside in human OA chondrocytes. In addition, the intra-articular injection of morroniside elevated the level of proteoglycans in cartilage matrix and the thickness of articular cartilage in a rat experimental model of OA, with the increase of AKT and ERK activation. As a consequence, morroniside has chondroprotective effect on OA chondrocytes, and may have the therapeutic potential for OA treatment. PMID:25754021

  8. Protein kinase B and extracellular signal-regulated kinase contribute to the chondroprotective effect of morroniside on osteoarthritis chondrocytes.

    PubMed

    Cheng, Liang; Zeng, Guoqing; Liu, Zejun; Zhang, Bing; Cui, Xu; Zhao, Honghai; Zheng, Xinpeng; Song, Gang; Kang, Jian; Xia, Chun

    2015-08-01

    Despite extensive studies on the multifaceted roles of morroniside, the main active constituent of iridoid glycoside from Corni Fructus, the effect of morroniside on osteoarthritis (OA) chondrocytes remains poorly understood. Here, we investigated the influence of morroniside on cultured human OA chondrocytes and a rat experimental model of OA. The results showed that morroniside enhanced the cell viability and the levels of proliferating cell nuclear antigen expression (PCNA), type II collagen and aggrecan in human OA chondrocytes, indicating that morroniside promoted chondrocyte survival and matrix synthesis. Furthermore, different doses of morroniside activated protein kinase B (AKT) and extracellular signal-regulated kinase (ERK) in human OA chondrocytes, and in turn, triggered AKT/S6 and ERK/P70S6K/S6 pathway, respectively. The PI3K/AKT inhibitor LY294002 or the MEK/ERK inhibitor U0126 attenuated the effect of morroniside on human OA chondrocytes, indicating that the activation of AKT and ERK contributed to the regulation of morroniside in human OA chondrocytes. In addition, the intra-articular injection of morroniside elevated the level of proteoglycans in cartilage matrix and the thickness of articular cartilage in a rat experimental model of OA, with the increase of AKT and ERK activation. As a consequence, morroniside has chondroprotective effect on OA chondrocytes, and may have the therapeutic potential for OA treatment. PMID:25754021

  9. In vitro chondrocyte behavior on porous biodegradable poly(e-caprolactone)/polyglycolic acid scaffolds for articular chondrocyte adhesion and proliferation.

    PubMed

    Jonnalagadda, John B; Rivero, Iris V; Dertien, Janet S

    2015-01-01

    In this study, poly(e-caprolactone)/polyglycolic acid (PCL/PGA) scaffolds for repairing articular cartilage were fabricated via solid-state cryomilling along with compression molding and porogen leaching. Four distinct scaffolds were fabricated using this approach by four independent cryomilling times. These scaffolds were assessed for their suitability to promote articular cartilage regeneration with in vitro chondrocyte cell culture studies. The scaffolds were characterized for pore size, porosity, swelling ratio, compressive, and thermal properties. Cryomilling time proved to significantly affect the physical, mechanical, and morphological properties of the scaffolds. In vitro bovine chondrocyte culture was performed dynamically for 1, 7, 14, 28, and 35 days. Chondrocyte viability and adhesion were tested using MTT assay and scanning electron microscopy micrographs. Glycosaminoglycan (GAG) and DNA assays were performed to investigate the extracellular matrix (ECM) formation and cell proliferation, respectively. PCL/PGA scaffolds demonstrated high porosity for all scaffold types. Morphological analysis and poly(ethylene oxide) continuity demonstrated the existence of a co-continuous network of interconnected pores with pore sizes appropriate for tissue engineering and chondrocyte ingrowth. While mean pore size decreased, water uptake and compressive properties increased with increasing cryomilling times. Compressive modulus of 12, 30, and 60 min scaffolds matched the compressive modulus of human articular cartilage. Viable cells increased besides increase in cell proliferation and ECM formation with progress in culture period. Chondrocytes exhibited spherical morphology on all scaffold types. The pore size of the scaffold affected chondrocyte adhesion, proliferation, and GAG secretion. The results indicated that the 12 min scaffolds delivered promising results for applications in articular cartilage repair. PMID:25671317

  10. Cartilage engineering using chondrocyte cell sheets and its application in reconstruction of microtia

    PubMed Central

    Zhou, Libin; Ding, Ruiying; Li, Baowei; Han, Haolun; Wang, Hongnan; Wang, Gang; Xu, Bingxin; Zhai, Suoqiang; Wu, Wei

    2015-01-01

    The imperfections of scaffold materials have hindered the clinical application of cartilage tissue engineering. The recently developed cell-sheet technique is adopted to engineer tissues without scaffold materials, thus is considered being potentially able to overcome the problems concerning the scaffold imperfections. This study constructed monolayer and bilayer chondrocyte cell sheets and harvested the sheets with cell scraper instead of temperature-responsive culture dishes. The properties of the cultured chondrocyte cell sheets and the feasibility of cartilage engineering using the chondrocyte cell sheets was further investigated via in vitro and in vivo study. Primary extracellular matrix (ECM) formation and type II collagen expression was detected in the cell sheets during in vitro culture. After implanted into nude mice for 8 weeks, mature cartilage discs were harvested. The morphology of newly formed cartilage was similar in the constructs originated from monolayer and bilayer chondrocyte cell sheet. The chondrocytes were located within evenly distributed ovoid lacunae. Robust ECM formation and intense expression of type II collagen was observed surrounding the evenly distributed chondrocytes in the neocartilages. Biochemical analysis showed that the DNA contents of the neocartilages were higher than native human costal cartilage; while the contents of the main component of ECM, glycosaminoglycan and hydroxyproline, were similar to native human costal cartilage. In conclusion, the chondrocyte cell sheet constructed using the simple and low-cost technique is basically the same with the cell sheet cultured and harvested in temperature-responsive culture dishes, and can be used for cartilage tissue engineering. PMID:25755694

  11. Cartilage engineering using chondrocyte cell sheets and its application in reconstruction of microtia.

    PubMed

    Zhou, Libin; Ding, Ruiying; Li, Baowei; Han, Haolun; Wang, Hongnan; Wang, Gang; Xu, Bingxin; Zhai, Suoqiang; Wu, Wei

    2015-01-01

    The imperfections of scaffold materials have hindered the clinical application of cartilage tissue engineering. The recently developed cell-sheet technique is adopted to engineer tissues without scaffold materials, thus is considered being potentially able to overcome the problems concerning the scaffold imperfections. This study constructed monolayer and bilayer chondrocyte cell sheets and harvested the sheets with cell scraper instead of temperature-responsive culture dishes. The properties of the cultured chondrocyte cell sheets and the feasibility of cartilage engineering using the chondrocyte cell sheets was further investigated via in vitro and in vivo study. Primary extracellular matrix (ECM) formation and type II collagen expression was detected in the cell sheets during in vitro culture. After implanted into nude mice for 8 weeks, mature cartilage discs were harvested. The morphology of newly formed cartilage was similar in the constructs originated from monolayer and bilayer chondrocyte cell sheet. The chondrocytes were located within evenly distributed ovoid lacunae. Robust ECM formation and intense expression of type II collagen was observed surrounding the evenly distributed chondrocytes in the neocartilages. Biochemical analysis showed that the DNA contents of the neocartilages were higher than native human costal cartilage; while the contents of the main component of ECM, glycosaminoglycan and hydroxyproline, were similar to native human costal cartilage. In conclusion, the chondrocyte cell sheet constructed using the simple and low-cost technique is basically the same with the cell sheet cultured and harvested in temperature-responsive culture dishes, and can be used for cartilage tissue engineering. PMID:25755694

  12. Pulsed Electromagnetic Fields Increased the Anti-Inflammatory Effect of A2A and A3 Adenosine Receptors in Human T/C-28a2 Chondrocytes and hFOB 1.19 Osteoblasts

    PubMed Central

    Vincenzi, Fabrizio; Targa, Martina; Corciulo, Carmen; Gessi, Stefania; Merighi, Stefania; Setti, Stefania; Cadossi, Ruggero; Goldring, Mary B.; Borea, Pier Andrea; Varani, Katia

    2013-01-01

    Adenosine receptors (ARs) have an important role in the regulation of inflammation and their activation is involved in the inhibition of pro-inflammatory cytokine release. The effects of pulsed electromagnetic fields (PEMFs) on inflammation have been reported and we have demonstrated that PEMFs increased A2A and A3AR density and functionality in different cell lines. Chondrocytes and osteoblasts are two key cell types in the skeletal system that play important role in cartilage and bone metabolism representing an interesting target to study the effect of PEMFs. The primary aim of the present study was to evaluate if PEMF exposure potentiated the anti-inflammatory effect of A2A and/or A3ARs in T/C-28a2 chondrocytes and hFOB 1.19 osteoblasts. Immunofluorescence, mRNA analysis and saturation binding assays revealed that PEMF exposure up-regulated A2A and A3AR expression. A2A and A3ARs were able to modulate cAMP production and cell proliferation. The activation of A2A and A3ARs resulted in the decrease of some of the most relevant pro-inflammatory cytokine release such as interleukin (IL)-6 and IL-8, following the treatment with IL-1β as an inflammatory stimuli. In human chondrocyte and osteoblast cell lines, the inhibitory effect of A2A and A3AR stimulation on the release of prostaglandin E2 (PGE2), an important lipid inflammatory mediator, was observed. In addition, in T/C-28a2 cells, the activation of A2A or A3ARs elicited an inhibition of vascular endothelial growth factor (VEGF) secretion. In hFOB 1.19 osteoblasts, PEMF exposure determined an increase of osteoprotegerin (OPG) production. The effect of the A2A or A3AR agonists in the examined cells was enhanced in the presence of PEMFs and completely blocked by using well-known selective antagonists. These results demonstrated that PEMF exposure significantly increase the anti-inflammatory effect of A2A or A3ARs suggesting their potential therapeutic use in the therapy of inflammatory bone and joint disorders

  13. Viability and Regeneration of Chondrocytes after Laser Cartilage Reshaping Using 1,460 nm Diode Laser

    PubMed Central

    Mo, Ji-Hun; Kim, Ji-Sun; Lee, Jae-Wook; Chung, Phil-Sang

    2013-01-01

    Objectives Cartilage reshaping by laser irradiation is used to correct septal and auricular cartilage deformities. Chondrocyte viability following laser irradiation and reshaping has been well established. However, the regeneration process of chondrocyte after laser irradiation has not been revealed yet. The aims of this study were to determine the mechanism of cartilaginous thermal injury and the regenerative process of damaged cartilage following laser irradiation. Methods Laser irradiation was performed on human septal cartilage and rabbit auricular cartilage using a 1,460-nm diode laser. We observed change in the shape of cartilage and evaluated the extent of cartilage injury using live/dead cell assay via confocal microscopy. Hoechst and propidium iodide (PI) staining was used to evaluate the mechanism of chondrocyte injury after laser irradiation. To evaluate the regeneration of cartilage, laser irradiated cartilages were reimplanted into a subperichondrial pocket and were harvested at 1, 2, and 4 weeks after reimplantation for viability assessment and histologic examination. Results Laser irradiation using a 1,460-nm diode laser produced a marked shape change in both human septal and rabbit auricular cartilages. Thermal damage on cartilage was correlated with the exposure time and the laser power. Hoechst and PI staining showed that chondrocyte death by laser irradiation was due to mainly necrosis, rather than apoptosis. In lower power treatment group (0.3 W and 0.5 W), all the chondrocytes regenerated within 4 weeks, however, in 1 W treatment group, chondrocytes could not regenerate until 4 weeks. Conclusion Reshaping of cartilage using 1,460 nm diode laser was attained concurrently with the thermal injury to the chondrocytes. The extent of thermal damage on chondrocytes was dependent on the exposure time and the laser power and the damaged chondrocytes irradiated with lower level of laser power could be regenerated after reimplantation into

  14. IL-36α: a novel cytokine involved in the catabolic and inflammatory response in chondrocytes

    PubMed Central

    Conde, Javier; Scotece, Morena; Abella, Vanessa; Lois, Ana; López, Verónica; García-Caballero, Tomás; Pino, Jesús; Gómez-Reino, Juan Jesús; Gómez, Rodolfo; Lago, Francisca; Gualillo, Oreste

    2015-01-01

    Recent studies confer to IL-36α pro-inflammatory properties. However, little is known about the expression and function of IL-36α in cartilage. This study sought to analyze the expression of IL-36α in healthy and OA cartilage. Next, we determined the effects of recombinant IL-36α on catabolism and inflammation in chondrocytes. For completeness, part of the signaling pathway elicited by IL-36α was also explored. IL-36α expression was evaluated by immunohistochemistry and RT-qPCR. Expression of MMP-13, NOS2 and COX-2 was also determined in OA articular chondrocytes treated with recombinant IL-36α. IκB-α and P-p38 was explored by western blot. We observed a low constitutive expression of IL-36α in healthy human chondrocytes. However, OA chondrocytes likely expressed more IL-36α than healthy chondrocytes. In addition, immune cells infiltrated into the joint and PBMCs express higher levels of IL-36α in comparison to chondrocytes. OA chondrocytes, treated with IL-36α, showed significant increase in the expression of MMP-13, NOS2 and COX-2. Finally, IL-36α stimulated cells showed NFκB and p38 MAPK activated pathways. IL-36α acts as a pro-inflammatory cytokine at cartilage level, by increasing the expression of markers of inflammation and cartilage catabolism. Like other members of IL-1 family, IL-36α acts through the activation of NFκB and p38 MAPK pathway. PMID:26560022

  15. Implantation of rAAV5-IGF-I transduced autologous chondrocytes improves cartilage repair in full-thickness defects in the equine model.

    PubMed

    Ortved, Kyla F; Begum, Laila; Mohammed, Hussni O; Nixon, Alan J

    2015-02-01

    Cartilage injury often precipitates osteoarthritis which has driven research to bolster repair in cartilage impact damage. Autologous chondrocytes transduced with rAAV5-IGF-I were evaluated in chondral defects in a well-established large animal model. Cartilage was harvested from the talus of 24 horses; chondrocytes were isolated and stored frozen. Twenty million cells were cultured and transduced with 10(5) AAV vg/cell prior to implantation. Chondrocytes from eight horses were transduced with rAAV5-IGF-I, chondrocytes from eight horses with rAAV5-GFP, and chondrocytes from eight horses were not transduced. A 15 mm full-thickness chondral defect was created arthroscopically in the lateral trochlear ridge of the femur in both femoropatellar joints. Treated defects were filled with naive or gene-enhanced chondrocytes, in fibrin vehicle. Control defects in the opposite limb received fibrin alone. rAAV5-IGF-I transduced chondrocytes resulted in significantly better healing at 8 week arthroscopy and 8 month necropsy examination when compared to controls. At 8 months, defects implanted with cells expressing IGF-I had better histological scores compared to control defects and defects repaired with naive chondrocytes. This included increased chondrocyte predominance and collagen type II, both features of hyaline-like repair tissue. The equine model closely approximates human cartilage healing, indicating AAV-mediated genetic modification of chondrocytes may be clinically beneficial to humans. PMID:25311491

  16. Prolonged Application of High Fluid Shear to Chondrocytes Recapitulates Gene Expression Profiles Associated with Osteoarthritis

    PubMed Central

    Zhu, Fei; Wang, Pu; Lee, Norman H.; Goldring, Mary B.; Konstantopoulos, Konstantinos

    2010-01-01

    Background Excessive mechanical loading of articular cartilage producing hydrostatic stress, tensile strain and fluid flow leads to irreversible cartilage erosion and osteoarthritic (OA) disease. Since application of high fluid shear to chondrocytes recapitulates some of the earmarks of OA, we aimed to screen the gene expression profiles of shear-activated chondrocytes and assess potential similarities with OA chondrocytes. Methodology/Principal Findings Using a cDNA microarray technology, we screened the differentially-regulated genes in human T/C-28a2 chondrocytes subjected to high fluid shear (20 dyn/cm2) for 48 h and 72 h relative to static controls. Confirmation of the expression patterns of select genes was obtained by qRT-PCR. Using significance analysis of microarrays with a 5% false discovery rate, 71 and 60 non-redundant transcripts were identified to be ≥2-fold up-regulated and ≤0.6-fold down-regulated, respectively, in sheared chondrocytes. Published data sets indicate that 42 of these genes, which are related to extracellular matrix/degradation, cell proliferation/differentiation, inflammation and cell survival/death, are differentially-regulated in OA chondrocytes. In view of the pivotal role of cyclooxygenase-2 (COX-2) in the pathogenesis and/or progression of OA in vivo and regulation of shear-induced inflammation and apoptosis in vitro, we identified a collection of genes that are either up- or down-regulated by shear-induced COX-2. COX-2 and L-prostaglandin D synthase (L-PGDS) induce reactive oxygen species production, and negatively regulate genes of the histone and cell cycle families, which may play a critical role in chondrocyte death. Conclusions/Significance Prolonged application of high fluid shear stress to chondrocytes recapitulates gene expression profiles associated with osteoarthritis. Our data suggest a potential link between exposure of chondrocytes/cartilage to abnormal mechanical loading and the pathogenesis/progression of OA

  17. Influence of cell printing on biological characters of chondrocytes

    PubMed Central

    Qu, Miao; Gao, Xiaoyan; Hou, Yikang; Shen, Congcong; Xu, Yourong; Zhu, Ming; Wang, Hengjian; Xu, Haisong; Chai, Gang; Zhang, Yan

    2015-01-01

    Objective: To establish a two-dimensional biological printing technique of chondrocytes and compare the difference of related biological characters between printed chondrocytes and unprinted cells so as to control the cell transfer process and keep cell viability after printing. Methods: Primary chondrocytes were obtained from human mature and fetal cartilage tissues and then were regularly sub-cultured to harvest cells at passage 2 (P2), which were adjusted to the single cell suspension at a density of 1×106/mL. The experiment was divided into 2 groups: experimental group P2 chondrocytes were transferred by rapid prototype biological printer (driving voltage value 50 V, interval in x-axis 300 μm, interval in y-axis 1500 μm). Afterwards Live/Dead viability Kit and flow cytometry were respectively adopted to detect cell viability; CCK-8 Kit was adopted to detect cell proliferation viability; immunocytochemistry, immunofluorescence and RT-PCR was employed to identify related markers of chondrocytes; control group steps were the same as the printing group except that cell suspension received no printing. Results: Fluorescence microscopy and flow cytometry analyses showed that there was no significant difference between experimental group and control group in terms of cell viability. After 7-day in vitro culture, control group exhibited higher O.D values than experimental group from 2nd day to 7th day but there was no distinct difference between these two groups (P>0.05). Inverted microscope observation demonstrated that the morphology of these two groups had no significant difference either. Similarly, Immunocytochemistry, immunofluorescence and RT-PCR assays also showed that there was no significant difference in the protein and gene expression of type II collagen and aggrecan between these two groups (P>0.05). Conclusion Cell printing has no distinctly negative effect on cell vitality, proliferation and phenotype of chondrocytes. Biological printing technique may

  18. Evaluating Osteoarthritic Chondrocytes through a Novel 3-Dimensional In Vitro System for Cartilage Tissue Engineering and Regeneration

    PubMed Central

    Li, Hanwei; Davison, Noel; Moroni, Lorenzo; Feng, Felicia; Crist, Joshua; Salter, Erin; Bingham, Clifton O.

    2012-01-01

    Objective: To characterize and evaluate osteoarthritic (OA) chondrocytes, in comparison to normal chondrocytes, through a novel 3-dimensional (3-D) culture system, poly(ethylene-glycol) diacrylate (PEGDA). The cytokine interleukin 1β (IL-1β) was also used to simulate an in vitro OA model. Methods: Normal and OA chondrocytes were cultured in monolayer and analyzed for changes in cartilage-specific gene expressions due to passage number. Then, cells were encapsulated in PEGDA to evaluate phenotype and matrix production capabilities through the in vitro culture system. Characterization was conducted with polymerase chain reaction (PCR), biochemical analyses, and histological staining. 3-D encapsulated chondrocytes (human and bovine) were also treated with IL-1β to characterize how the cytokine affects gene transcription and extracellular matrix (ECM) content. Results: In 2-dimensional monolayer, anabolic genes were down-regulated significantly in both normal and OA chondrocytes. In 3-D culture, OA chondrocytes demonstrated significantly higher expressions of catabolic genes when compared to normal cells. Differentiation medium resulted in significantly more matrix production than growth medium from OA chondrocytes, indicated through histological staining. In addition, normal chondrocytes responded more significantly to exogenous administration of IL-1β than OA chondrocytes. Temporary initial stimulation of IL-1β to OA chondrocytes resulted in comparable gene expressions to untreated cells after 3 weeks of in vitro culture. Conclusions: Our findings demonstrate the use of OA chondrocytes in tissue engineering and their significance for potential future cartilage regeneration research through their matrix production capabilities and the use of a hydrogel culture system. PMID:26069626

  19. Efficient, Low-Cost Nucleofection of Passaged Chondrocytes.

    PubMed

    Parreno, Justin; Delve, Elizabeth; Andrejevic, Katarina; Paez-Parent, Sabrina; Wu, Po-Han; Kandel, Rita

    2016-01-01

    Nucleofection of chondrocytes has been shown to be an adequate method of transfection. Using Amaxa's nucleofection system, transfection efficiencies up to 89% were achievable for vector (pmaxGFP) and 98% for siRNA (siGLO) into passaged chondrocytes. However, such methods rely on costly commercial kits with proprietary reagents limiting its use in basic science labs and in clinical translation. Bovine-passaged chondrocytes were plated in serum reduced media conditionsand then nucleofected using various in laboratory-produced buffers. Cell attachment, confluency, viability, and transfection efficiency was assessed following nucleofection. For each parameter the buffers were scored and a final rank for each buffer was determined. Buffer denoted as 1M resulted in no significant difference for cell attachment, confluency, and viability as compared to non-nucleofected controls. Nucleofection in 1M buffer, in the absence of DNA vectors, resulted in increased col2, ki67, ccnd1 mRNA levels, and decreased col1 mRNA levels at 4 days of culture. Flow cytometry revealed that the transfection efficiency of 1M buffer was comparable to that obtained using the Amaxa commercial kit. siRNA designed against lamin A/C resulted in an average reduction of lamin A and C proteins to 19% and 8% of control levels, respectively. This study identifies a cost-effective, efficient method of nonviral nucleofection of bovine-passaged chondrocytes using known buffer formulations. Human-passaged chondrocytes could also be successfully nucleofected in 1M buffer. Thus this method should facilitate cost-efficient gene targeting of cells used for articular cartilage repair in a research setting. PMID:26958320

  20. Mechanical overloading causes mitochondrial superoxide and SOD2 imbalance in chondrocytes resulting in cartilage degeneration

    PubMed Central

    Koike, Masato; Nojiri, Hidetoshi; Ozawa, Yusuke; Watanabe, Kenji; Muramatsu, Yuta; Kaneko, Haruka; Morikawa, Daichi; Kobayashi, Keiji; Saita, Yoshitomo; Sasho, Takahisa; Shirasawa, Takuji; Yokote, Koutaro; Kaneko, Kazuo; Shimizu, Takahiko

    2015-01-01

    Mechanical stress and aging are major risk factors of cartilage degeneration. Human studies have previously reported that oxidative damage increased, while SOD2 protein was reciprocally downregulated in osteoarthritic degenerated cartilage. However, it remains unclear whether mitochondrial superoxide imbalance in chondrocytes causes cartilage degeneration. We herein demonstrate that mechanical loading promoted mitochondrial superoxide generation and selective Sod2 downregulation in chondrocytes in vivo and that mitochondrial superoxide inducer also downregulated Sod2 expression in chondrocytes in vitro. A genetically manipulated model revealed that Sod2 deficiency in chondrocytes also resulted in mitochondrial superoxide overproduction and dysfunction, thus leading to cartilage degeneration. Intra-articular injection of a permeable antioxidant effectively suppressed the mechanical loading-induced mitochondrial superoxide generation and cartilage degeneration in mice. Our findings demonstrate that mitochondrial superoxide plays a pivotal role in the development and progression of osteoarthritis, and the mitochondrial superoxide balance may therefore be a promising target for the treatment of cartilage degeneration. PMID:26108578

  1. Mechanical overloading causes mitochondrial superoxide and SOD2 imbalance in chondrocytes resulting in cartilage degeneration.

    PubMed

    Koike, Masato; Nojiri, Hidetoshi; Ozawa, Yusuke; Watanabe, Kenji; Muramatsu, Yuta; Kaneko, Haruka; Morikawa, Daichi; Kobayashi, Keiji; Saita, Yoshitomo; Sasho, Takahisa; Shirasawa, Takuji; Yokote, Koutaro; Kaneko, Kazuo; Shimizu, Takahiko

    2015-01-01

    Mechanical stress and aging are major risk factors of cartilage degeneration. Human studies have previously reported that oxidative damage increased, while SOD2 protein was reciprocally downregulated in osteoarthritic degenerated cartilage. However, it remains unclear whether mitochondrial superoxide imbalance in chondrocytes causes cartilage degeneration. We herein demonstrate that mechanical loading promoted mitochondrial superoxide generation and selective Sod2 downregulation in chondrocytes in vivo and that mitochondrial superoxide inducer also downregulated Sod2 expression in chondrocytes in vitro. A genetically manipulated model revealed that Sod2 deficiency in chondrocytes also resulted in mitochondrial superoxide overproduction and dysfunction, thus leading to cartilage degeneration. Intra-articular injection of a permeable antioxidant effectively suppressed the mechanical loading-induced mitochondrial superoxide generation and cartilage degeneration in mice. Our findings demonstrate that mitochondrial superoxide plays a pivotal role in the development and progression of osteoarthritis, and the mitochondrial superoxide balance may therefore be a promising target for the treatment of cartilage degeneration. PMID:26108578

  2. Articular chondrocyte metabolism and osteoarthritis

    SciTech Connect

    Leipold, H.R.

    1989-01-01

    The three main objectives of this study were: (1) to determine if depletion of proteoglycans from the cartilage matrix that occurs during osteoarthritis causes a measurable increase of cartilage proteoglycan components in the synovial fluid and sera, (2) to observe what effect intracellular cAMP has on the expression of matrix components by chondrocytes, and (3) to determine if freshly isolated chondrocytes contain detectable levels of mRNA for fibronectin. Canine serum keratan sulfate and hyaluronate were measured to determine if there was an elevation of these serum glycosaminoglycans in a canine model of osteoarthritis. A single intra-articular injection of chymopapain into a shoulder joint increased serum keratan sulfate 10 fold and hyaluronate less than 2 fold in 24 hours. Keratan sulfate concentrations in synovial fluids of dogs about one year old were unrelated to the presence of spontaneous cartilage degeneration in the joints. High keratan sulfate in synovial fluids correlated with higher keratan sulfate in serum. The mean keratan sulfate concentration in sera of older dogs with osteoarthritis was 37% higher than disease-free controls, but the difference between the groups was not statistically significant. Treatment of chondrocytes with 0.5 millimolar (mM) dibutyryl cAMP (DBcAMP) caused the cells to adopt a more rounded morphology. There was no difference between the amount of proteins synthesized by cultures treated with DBcAMP and controls. The amount of fibronectin (FN) in the media of DBcAMP treated cultures detected by an ELISA was specifically reduced, and the amount of {sup 35}S-FN purified by gelatin affinity chromatography decreased. Moreover, the percentage of FN containing the extra domain. A sequence was reduced. Concomitant with the decrease in FN there was an increase in the concentration of keratan sulfate.

  3. Lysine-specific demethylase 1-mediated demethylation of histone H3 lysine 9 contributes to interleukin 1β-induced microsomal prostaglandin E synthase 1 expression in human osteoarthritic chondrocytes

    PubMed Central

    2014-01-01

    Introduction Microsomal prostaglandin E synthase 1 (mPGES-1) catalyzes the terminal step in the biosynthesis of PGE2, a critical mediator in the pathophysiology of osteoarthritis (OA). Histone methylation plays an important role in epigenetic gene regulation. In this study, we investigated the roles of histone H3 lysine 9 (H3K9) methylation in interleukin 1β (IL-1β)-induced mPGES-1 expression in human chondrocytes. Methods Chondrocytes were stimulated with IL-1β, and the expression of mPGES-1 mRNA was evaluated using real-time RT-PCR. H3K9 methylation and the recruitment of the histone demethylase lysine-specific demethylase 1 (LSD1) to the mPGES-1 promoter were evaluated using chromatin immunoprecipitation assays. The role of LSD1 was further evaluated using the pharmacological inhibitors tranylcypromine and pargyline and small interfering RNA (siRNA)-mediated gene silencing. The LSD1 level in cartilage was determined by RT-PCR and immunohistochemistry. Results The induction of mPGES-1 expression by IL-1β correlated with decreased levels of mono- and dimethylated H3K9 at the mPGES-1 promoter. These changes were concomitant with the recruitment of the histone demethylase LSD1. Treatment with tranylcypromine and pargyline, which are potent inhibitors of LSD1, prevented IL-1β-induced H3K9 demethylation at the mPGES-1 promoter and expression of mPGES-1. Consistently, LSD1 gene silencing with siRNA prevented IL-1β-induced H3K9 demethylation and mPGES-1 expression, suggesting that LSD1 mediates IL-1β-induced mPGES-1 expression via H3K9 demethylation. We show that the level of LSD1 was elevated in OA compared to normal cartilage. Conclusion These results indicate that H3K9 demethylation by LSD1 contributes to IL-1β-induced mPGES-1 expression and suggest that this pathway could be a potential target for pharmacological intervention in the treatment of OA and possibly other arthritic conditions. PMID:24886859

  4. Mori folium inhibits interleukin-1β-induced expression of matrix metalloproteinases and inflammatory mediators by suppressing the activation of NF-κB and p38 MAPK in SW1353 human chondrocytes.

    PubMed

    Jeong, Jin-Woo; Lee, Hye Hyeon; Lee, Ki Won; Kim, Ki Young; Kim, Sung Goo; Hong, Su Hyun; Kim, Gi-Young; Park, Cheol; Kim, Ho Kyoung; Choi, Young Whan; Choi, Yung Hyun

    2016-02-01

    The pro-inflammatory cytokine interleukin-1β (IL-1β) is known to play a crucial role in the pathogenesis of osteoarthritis (OA) by stimulating several mediators that contribute to cartilage degradation. Mori folium, the leaves of Morus alba L., has long been used in traditional medicine to treat diabetes, protect the liver, and lower blood pressure; however, the role of Mori folium in OA is not yet fully understood. Therefore, in the present study, we investigated whether Mori folium water extract (MF) inhibited the catabolic effects of IL-1β in vitro, and also whether it inhibited the matrix metalloproteinases (MMPs), inducible nitric oxide (NO) synthase (iNOS) and cyclooxygenase-2 (COX-2) through the attenuation of nuclear factor-κB (NF-κB) and mitogen activated protein kinase (MAPK) pathways in SW1353 human chondrocytes. MMP proteins in culture medium were determined using a cytokine‑specific enzyme-linked immunosorbent assay (ELISA). The production of NO and prostaglandin E2 (PGE2) were evaluated using Griess reagent and ELISA. Subsequently, the mRNA and protein levels of MMPs, iNOS, COX-2, NF-κB and MAPKs were examined by RT-qPCR and/or western blot analysis. The results indicate that MF significantly reduced the IL-1β‑induced release of MMP-1 and -13 in SW1353 cells, which was associated with the inhibition of MMP-1 and -13 mRNA and protein expression in a concentration‑dependent manner at concentrations with no cytotoxicity. MF also attenuated the IL-1β-induced production of NO and PGE2, and reduced iNOS and COX-2 expression. Furthermore, we noted that MF markedly suppressed the IL-1β‑induced nuclear translocation of NF-κB, which correlated with the inhibitory effects of MF on inhibitor-κB (IκB) degradation, and the phosphorylation of p38 MAPK was selectively restored by MF upon IL-1β stimulation. These results indicate that MF inhibited the production and expression of MMP-1 and -13 and inflammatory mediators, at least in part, through

  5. MiR-34a promotes Fas-mediated cartilage endplate chondrocyte apoptosis by targeting Bcl-2.

    PubMed

    Chen, Huajiang; Wang, Jianxi; Hu, Bo; Wu, Xiaodong; Chen, Yu; Li, Renhu; Yuan, Wen

    2015-08-01

    Apoptosis of cartilage endplate (CEP) chondrocytes is associated with the pathogenesis of intervertebral disk degeneration (IDD). Recent studies have shown that miR-34a is crucially involved in chondrocyte apoptosis during osteoarthritic cartilage. Here, we investigated the involvement of miR-34a in CEP chondrocyte apoptosis in IDD. In human degenerated CEP chondrocytes, miRNA (miR)-34a was markedly elevated in association with increased apoptosis. Bioinformatics target prediction identified Bcl-2 as a putative target of miR-34a. Furthermore, miR-34a inhibited Bcl-2 expression by directly targeting their 3'-untranslated regions, and this inhibition was abolished by mutation of the miR-34a binding sites. In vitro, knockdown of miR-34a in human endplate chondrocytes resulted in overexpression of Bcl-2, whereas upregulation of miR-34a led to repression of Bcl-2. Fas-mediated apoptosis was decreased when antagonizing miR-34a with locked nucleotide analog-miR-34a in human endplate chondrocytes. Taken together, our results demonstrate that upregulated miR-34a potentiates Fas-mediated endplate chondrocyte apoptosis, which is associated with IDD. PMID:25910896

  6. Cytotoxic T lymphocytes recognize and lyse chondrocytes under inflammatory, but not non-inflammatory conditions.

    PubMed

    Cohen, E Suzanne; Bodmer, Helen C

    2003-05-01

    The human major histocompatibility complex (MHC) class I allele HLA-B27 is strongly associated with seronegative spondyloarthropathies including ankylosing spondylitis and reactive arthritis. Although of unknown aetiology, one hypothesis suggests that a cytotoxic T cell (CTL) response against a self-antigen at sites of inflammation, such as entheses or joints may be involved. The chondrocyte is one of the major specialized cell types found both in articular cartilage and cartilaginous entheses and therefore is a possible source of such an antigen. CTL recognition of these cells is a potential mechanism for inflammation and cartilage damage, both through direct lysis of chondrocytes and the secretion of pro-inflammatory cytokines such as tumour necrosis factor and interferon-gamma (IFN-gamma). We test the feasibility of this hypothesis by examining the ability of chondrocytes to present antigen to CTL in vitro. Chondrocytes isolated from the ribcages of mice did not constitutively express detectable levels of MHC class I by fluorescence-activated cell sorting analysis. In addition, they were resistant to lysis by alloreactive and influenza A virus nucleoprotein (NP)-specific CTL. However, treatment of chondrocytes with IFN-gamma up-regulated MHC class I expression and rendered the cells susceptible to lysis by CTL. Similarly, IFN-gamma-treated chondrocytes infected with influenza A virus were recognized by NP-specific CTL, though with variable efficiency. Thus, we suggest that under certain circumstances CTL-mediated lysis of chondrocytes is potentially a potent mechanism for cartilage damage in vivo, but that low levels of MHC class I on healthy chondrocytes protects from immune recognition in health. PMID:12709012

  7. STAT1 and STAT3 do not participate in FGF-mediated growth arrest in chondrocytes.

    PubMed

    Krejci, Pavel; Salazar, Lisa; Goodridge, Helen S; Kashiwada, Tamara A; Schibler, Matthew J; Jelinkova, Petra; Thompson, Leslie Michels; Wilcox, William R

    2008-02-01

    Activating mutations in fibroblast growth factor receptor 3 (FGFR3) cause several human skeletal dysplasias as a result of attenuation of cartilage growth. It is believed that FGFR3 inhibits chondrocyte proliferation via activation of signal transducers and activators of transcription (STAT) proteins, although the exact mechanism of both STAT activation and STAT-mediated inhibition of chondrocyte growth is unclear. We show that FGFR3 interacts with STAT1 in cells and is capable of activating phosphorylation of STAT1 in a kinase assay, thus potentially serving as a STAT1 kinase in chondrocytes. However, as demonstrated by western blotting with phosphorylation-specific antibodies, imaging of STAT nuclear translocation, STAT transcription factor assays and STAT luciferase reporter assays, FGF does not activate STAT1 or STAT3 in RCS chondrocytes, which nevertheless respond to a FGF stimulus with potent growth arrest. Moreover, addition of active STAT1 and STAT3 to the FGF signal, by means of cytokine treatment, SRC-mediated STAT activation or expression of constitutively active STAT mutants does not sensitize RCS chondrocytes to FGF-mediated growth arrest. Since FGF-mediated growth arrest is rescued by siRNA-mediated downregulation of the MAP kinase ERK1/2 but not STAT1 or STAT3, our data support a model whereby the ERK arm but not STAT arm of FGF signaling in chondrocytes accounts for the growth arrest phenotype. PMID:18198189

  8. Identification and characterization of the novel Col10a1 regulatory mechanism during chondrocyte hypertrophic differentiation.

    PubMed

    Gu, J; Lu, Y; Li, F; Qiao, L; Wang, Q; Li, N; Borgia, J A; Deng, Y; Lei, G; Zheng, Q

    2014-01-01

    The majority of human skeleton develops through the endochondral pathway, in which cartilage-forming chondrocytes proliferate and enlarge into hypertrophic chondrocytes that eventually undergo apoptosis and are replaced by bone. Although at a terminal differentiation stage, hypertrophic chondrocytes have been implicated as the principal engine of bone growth. Abnormal chondrocyte hypertrophy has been seen in many skeletal dysplasia and osteoarthritis. Meanwhile, as a specific marker of hypertrophic chondrocytes, the type X collagen gene (COL10A1) is also critical for endochondral bone formation, as mutation and altered COL10A1 expression are often accompanied by abnormal chondrocyte hypertrophy in many skeletal diseases. However, how the type X collagen gene is regulated during chondrocyte hypertrophy has not been fully elucidated. We have recently demonstrated that Runx2 interaction with a 150-bp mouse Col10a1 cis-enhancer is required but not sufficient for its hypertrophic chondrocyte-specific reporter expression in transgenic mice, suggesting requirement of additional Col10a1 regulators. In this study, we report in silico sequence analysis of this 150-bp enhancer and identification of its multiple binding factors, including AP1, MEF2, NFAT, Runx1 and TBX5. Using this enhancer as bait, we performed yeast one-hybrid assay and identified multiple candidate Col10a1-interacting genes, including cyclooxygenase 1 (Cox-1) and Cox-2. We have also performed mass spectrometry analysis and detected EF1-alpha, Fus, GdF7 and Runx3 as components of the specific complex formed by the cis-enhancer and nuclear extracts from hypertrophic MCT (mouse chondrocytes immortalized with large T antigen) cells that express Col10a1 abundantly. Notably, some of the candidate genes are differentially expressed in hypertrophic MCT cells and have been associated with chondrocyte hypertrophy and Runx2, an indispensible Col10a1 regulator. Intriguingly, we detected high-level Cox-2 expression in

  9. Doublecortin May Play a Role in Defining Chondrocyte Phenotype

    PubMed Central

    Ge, Dongxia; Zhang, Qing-Song; Zabaleta, Jovanny; Zhang, Qiuyang; Liu, Sen; Reiser, Brendan; Bunnell, Bruce A.; Braun, Stephen E.; O’Brien, Michael J.; Savoie, Felix H.; You, Zongbing

    2014-01-01

    Embryonic development of articular cartilage has not been well understood and the role of doublecortin (DCX) in determination of chondrocyte phenotype is unknown. Here, we use a DCX promoter-driven eGFP reporter mouse model to study the dynamic gene expression profiles in mouse embryonic handplates at E12.5 to E13.5 when the condensed mesenchymal cells differentiate into either endochondral chondrocytes or joint interzone cells. Illumina microarray analysis identified a variety of genes that were expressed differentially in the different regions of mouse handplate. The unique expression patterns of many genes were revealed. Cytl1 and 3110032G18RIK were highly expressed in the proximal region of E12.5 handplate and the carpal region of E13.5 handplate, whereas Olfr538, Kctd15, and Cited1 were highly expressed in the distal region of E12.5 and the metacarpal region of E13.5 handplates. There was an increasing gradient of Hrc expression in the proximal to distal direction in E13.5 handplate. Furthermore, when human DCX protein was expressed in human adipose stem cells, collagen II was decreased while aggrecan, matrilin 2, and GDF5 were increased during the 14-day pellet culture. These findings suggest that DCX may play a role in defining chondrocyte phenotype. PMID:24758934

  10. Chopping off the chondrocyte proteome

    PubMed Central

    Dvir-Ginzberg, Mona; Reich, Eli

    2015-01-01

    Abstract The progressive nature of osteoarthritis is manifested by the dynamic increase of degenerated articular cartilage, which is one of the major characteristics of this debilitating disease. As articular chondrocytes become exposed to inflammatory stress they enter a pro-catabolic state, which leads to the secretion and activation of a plethora of proteases. In aim to detect the disease before massive areas of cartilage are destroyed, various protein and non-protein biomarkers have been examined in bodily fluids and correlated with disease severity. This review will discuss the widely research extracellular degraded products as well as products generated by affected cellular pathways upon increased protease activity. While extracellular components could be more abundant, cleaved cellular proteins are less abundant and are suggested to possess a significant effect on cell metabolism and cartilage secretome. Subtle changes in cell secretome could potentially act as indicators of the chondrocyte metabolic and biological state. Therefore, it is envisioned that combined biomarkers composed of both cell and extracellular-degraded secretome could provide a valuable platform for testing drug efficacy to halt disease progression at its early stages. PMID:25179281

  11. Coptisine Prevented IL-β-Induced Expression of Inflammatory Mediators in Chondrocytes.

    PubMed

    Zhou, Kai; Hu, Li; Liao, Wenjun; Yin, Defeng; Rui, Feng

    2016-08-01

    Interleukin 1β (IL-1β) is a pleiotropic pro-inflammatory cytokine that plays a critical role in the development of osteoarthritis (OA). Coptisine is an isoquinoline alkaloid extracted from Coptidis rhizome and has been reported to possess anti-inflammatory activity. However, the anti-inflammatory effects of coptisine on interleukin-1 beta (IL-1β)-stimulated chondrocytes have not been reported. Therefore, the aim of this study was to investigate the effects of coptisine on IL-1β-induced inflammation in human articular chondrocytes. Our results showed that coptisine greatly inhibited the production of nitric oxide (NO) and prostaglandin E2 (PGE2), as well as suppressed the expression of inducible NO synthase (iNOS) and cyclooxygenase-2 (COX-2) in human OA chondrocytes induced by IL-1β. It also inhibited the expression of matrix metalloproteinase-3 (MMP-3) and MMP-13 in IL-1β-stimulated human OA chondrocytes. Furthermore, coptisine significantly inhibited the IL-1β-induced NF-kB activation in human OA chondrocytes. Taken together, these data suggest that coptisine inhibits the IL-1β-induced inflammatory response by suppressing the NF-kB signaling pathway. Thus, coptisine may be a potential agent in the treatment of OA. PMID:27294276

  12. Chondrocyte Apoptosis in the Pathogenesis of Osteoarthritis

    PubMed Central

    Hwang, Hyun Sook; Kim, Hyun Ah

    2015-01-01

    Apoptosis is a highly-regulated, active process of cell death involved in development, homeostasis and aging. Dysregulation of apoptosis leads to pathological states, such as cancer, developmental anomalies and degenerative diseases. Osteoarthritis (OA), the most common chronic joint disease in the elderly population, is characterized by progressive destruction of articular cartilage, resulting in significant disability. Because articular cartilage depends solely on its resident cells, the chondrocytes, for the maintenance of extracellular matrix, the compromising of chondrocyte function and survival would lead to the failure of the articular cartilage. The role of subchondral bone in the maintenance of proper cartilage matrix has been suggested as well, and it has been proposed that both articular cartilage and subchondral bone interact with each other in the maintenance of articular integrity and physiology. Some investigators include both articular cartilage and subchondral bone as targets for repairing joint degeneration. In late-stage OA, the cartilage becomes hypocellular, often accompanied by lacunar emptying, which has been considered as evidence that chondrocyte death is a central feature in OA progression. Apoptosis clearly occurs in osteoarthritic cartilage; however, the relative contribution of chondrocyte apoptosis in the pathogenesis of OA is difficult to evaluate, and contradictory reports exist on the rate of apoptotic chondrocytes in osteoarthritic cartilage. It is not clear whether chondrocyte apoptosis is the inducer of cartilage degeneration or a byproduct of cartilage destruction. Chondrocyte death and matrix loss may form a vicious cycle, with the progression of one aggravating the other, and the literature reveals that there is a definite correlation between the degree of cartilage damage and chondrocyte apoptosis. Because current treatments for OA act only on symptoms and do not prevent or cure OA, chondrocyte apoptosis would be a valid

  13. Chondrocyte Apoptosis in the Pathogenesis of Osteoarthritis.

    PubMed

    Hwang, Hyun Sook; Kim, Hyun Ah

    2015-01-01

    Apoptosis is a highly-regulated, active process of cell death involved in development, homeostasis and aging. Dysregulation of apoptosis leads to pathological states, such as cancer, developmental anomalies and degenerative diseases. Osteoarthritis (OA), the most common chronic joint disease in the elderly population, is characterized by progressive destruction of articular cartilage, resulting in significant disability. Because articular cartilage depends solely on its resident cells, the chondrocytes, for the maintenance of extracellular matrix, the compromising of chondrocyte function and survival would lead to the failure of the articular cartilage. The role of subchondral bone in the maintenance of proper cartilage matrix has been suggested as well, and it has been proposed that both articular cartilage and subchondral bone interact with each other in the maintenance of articular integrity and physiology. Some investigators include both articular cartilage and subchondral bone as targets for repairing joint degeneration. In late-stage OA, the cartilage becomes hypocellular, often accompanied by lacunar emptying, which has been considered as evidence that chondrocyte death is a central feature in OA progression. Apoptosis clearly occurs in osteoarthritic cartilage; however, the relative contribution of chondrocyte apoptosis in the pathogenesis of OA is difficult to evaluate, and contradictory reports exist on the rate of apoptotic chondrocytes in osteoarthritic cartilage. It is not clear whether chondrocyte apoptosis is the inducer of cartilage degeneration or a byproduct of cartilage destruction. Chondrocyte death and matrix loss may form a vicious cycle, with the progression of one aggravating the other, and the literature reveals that there is a definite correlation between the degree of cartilage damage and chondrocyte apoptosis. Because current treatments for OA act only on symptoms and do not prevent or cure OA, chondrocyte apoptosis would be a valid

  14. Modeling chondrocyte patterns by elliptical cluster processes.

    PubMed

    Meinhardt, Martin; Lück, Sebastian; Martin, Pascal; Felka, Tino; Aicher, Wilhelm; Rolauffs, Bernd; Schmidt, Volker

    2012-02-01

    Superficial zone chondrocytes (CHs) of human joints are spatially organized in distinct horizontal patterns. Among other factors, the type of spatial CH organization within a given articular surface depends on whether the cartilage has been derived from an intact joint or the joint is affected by osteoarthritis (OA). Furthermore, specific variations of the type of spatial organization are associated with particular states of OA. This association may prove relevant for early disease recognition based on a quantitative structural characterization of CH patterns. Therefore, we present a point process model describing the distinct morphology of CH patterns within the articular surface of intact human cartilage. This reference model for intact CH organization can be seen as a first step towards a model-based statistical diagnostic tool. Model parameters are fitted to fluorescence microscopy data by a novel statistical methodology utilizing tools from cluster and principal component analysis. This way, the complex morphology of surface CH patters is represented by a relatively small number of model parameters. We validate the point process model by comparing biologically relevant structural characteristics between the fitted model and data derived from photomicrographs of the human articular surface using techniques from spatial statistics. PMID:22155191

  15. MicroRNA-33 suppresses CCL2 expression in chondrocytes

    PubMed Central

    Wei, Meng; Xie, Qingyun; Zhu, Jun; Wang, Tao; Zhang, Fan; Cheng, Yue; Guo, Dongyang; Wang, Ying; Mo, Liweng; Wang, Shuai

    2016-01-01

    CCL2-mediated macrophage infiltration in articular tissues plays a pivotal role in the development of the osteoarthritis (OA). miRNAs regulate the onset and progression of diseases via controlling the expression of a series of genes. How the CCL2 gene was regulated by miRNAs was still not fully elucidated. In the present study, we demonstrated that the binding sites of miR-33 in the 3′UTR of CCL2 gene were conserved in human, mouse and rat species. By performing gain- or loss-of-function studies, we verified that miR-33 suppressed CCL2 expression in the mRNA and protein levels. We also found that miR-33 suppressed the CCL2 levels in the supernatant of cultured primary mouse chondrocytes. With reporter gene assay, we demonstrated that miR-33 targeted at AAUGCA in the 3′UTR of CCL2 gene. In transwell migration assays, we demonstrated that the conditional medium (CM) from miR-33 deficient chondrocytes potentiated the monocyte chemotaxis in a CCL2 dependent manner. Finally, we demonstrated that the level of miR-33 was decreased, whereas the CCL2 level was increased in the articular cartilage from the OA patients compared with the control group. In summary, we identified miR-33 as a novel suppressor of CCL2 in chondrocytes. The miR-33/CCL2 axis in chondrocytes regulates monocyte chemotaxis, providing a potential mechanism of macrophage infiltration in OA. PMID:27129293

  16. Sequential differentiation of mesenchymal stem cells in an agarose scaffold promotes a physis-like zonal alignment of chondrocytes.

    PubMed

    Schmitt, Jacqueline Frida; See, Kwee Hua; Hua, See Kwee; Yang, Zheng; Zheng, Yang; Hui, James Hoi Po; Po, James Hui Hoi; Lee, Eng Hin; Hin, Lee Eng

    2012-11-01

    Chondrocytes of the epiphyseal growth plate (physis) differentiate and mature in defined linear zones. The current study examines the differentiation of human bone marrow derived mesenchymal stem cells (hBMSCs) into zonal physeal cartilage. hBMSCs were embedded in an agarose scaffold with only the surface of the scaffold in direct contact with the culture medium. The cells were differentiated using a two-step system involving the sequential addition of TGFβ followed by BMP2. The resultant samples displayed a heterogenic population of physis-like collagen type 2 positive cells including proliferating chondrocytes and mature chondrocytes showing hypertrophy, expression of early bone markers and matrix mineralization. Histological analysis revealed a physis-like linear zonal alignment of chondrocytes in varying stages of differentiation. The less mature chondrocytes were seen at the base of the construct while hypertrophic chondrocytes and matrix mineralization was observed closer to the surface of the construct. The described differentiation protocol using hBMSCs in an agarose scaffold can be used to study the factors and conditions that influence the differentiation, proliferation, maturation, and zonal alignment of physeal chondrocytes. PMID:22517299

  17. Native Chondrocyte Viability during Cartilage Lesion Progression

    PubMed Central

    Ganguly, Kumkum; McRury, Ian D.; Goodwin, Peter M.; Morgan, Roy E.; Augé, Wayne K.

    2010-01-01

    Objective: Early surgical intervention for articular cartilage disease is desirable before full-thickness lesions develop. As early intervention treatments are designed, native chondrocyte viability at the treatment site before intervention becomes an important parameter to consider. The purpose of this study is to evaluate native chondrocyte viability in a series of specimens demonstrating the progression of articular cartilage lesions to determine if the chondrocyte viability profile changes during the evolution of articular cartilage disease to the level of surface fibrillation. Design: Osteochondral specimens demonstrating various degrees of articular cartilage damage were obtained from patients undergoing knee total joint replacement. Three groups were created within a patient harvest based on visual and tactile cues commonly encountered during surgical intervention: group 1, visually and tactilely intact surfaces; group 2, visually intact, tactilely soft surfaces; and group 3, surface fibrillation. Confocal laser microscopy was performed following live/dead cell viability staining. Results: Groups 1 to 3 demonstrated viable chondrocytes in all specimens, even within the fibrillated portions of articular cartilage, with little to no evidence of dead chondrocytes. Chondrocyte viability profile in articular cartilage does not appear to change as disease lesion progresses from normal to surface fibrillation. Conclusions: Fibrillated partial-thickness articular cartilage lesions are a good therapeutic target for early intervention. These lesions retain a high profile of viable chondrocytes and are readily diagnosed by visual and tactile cues during surgery. Early intervention should be based on matrix failure rather than on more aggressive procedures that further corrupt the matrix and contribute to chondrocyte necrosis of contiguous untargeted cartilage. PMID:26069561

  18. Biochemical and proteomic characterization of alkaptonuric chondrocytes.

    PubMed

    Braconi, Daniela; Bernardini, Giulia; Bianchini, Claretta; Laschi, Marcella; Millucci, Lia; Amato, Loredana; Tinti, Laura; Serchi, Tommaso; Chellini, Federico; Spreafico, Adriano; Santucci, Annalisa

    2012-09-01

    Alkaptonuria (AKU) is a rare genetic disease associated with the accumulation of homogentisic acid (HGA) and its oxidized/polymerized products which leads to the deposition of melanin-like pigments (ochronosis) in connective tissues. Although numerous case reports have described ochronosis in joints, little is known on the molecular mechanisms leading to such a phenomenon. For this reason, we characterized biochemically chondrocytes isolated from the ochronotic cartilage of AKU patients. Based on the macroscopic appearance of the ochronotic cartilage, two sub-populations were identified: cells coming from the black portion of the cartilage were referred to as "black" AKU chondrocytes, while those coming from the white portion were referred to as "white" AKU chondrocytes. Notably, both AKU chondrocytic types were characterized by increased apoptosis, NO release, and levels of pro-inflammatory cytokines. Transmission electron microscopy also revealed that intracellular ochronotic pigment deposition was common to both "white" and "black" AKU cells. We then undertook a proteomic and redox-proteomic analysis of AKU chondrocytes which revealed profound alterations in the levels of proteins involved in cell defence, protein folding, and cell organization. An increased post-translational oxidation of proteins, which also involved high molecular weight protein aggregates, was found to be particularly relevant in "black" AKU chondrocytes. PMID:22213341

  19. Biochemical and Proteomic Characterization of Alkaptonuric Chondrocytes

    PubMed Central

    Braconi, Daniela; Bernardini, Giulia; Bianchini, Claretta; Laschi, Marcella; Millucci, Lia; Amato, Loredana; Tinti, Laura; Serchi, Tommaso; Chellini, Federico; Spreafico, Adriano; Santucci, Annalisa

    2012-01-01

    Alkaptonuria (AKU) is a rare genetic disease associated with the accumulation of homogentisic acid (HGA) and its oxidized/polymerized products which leads to the deposition of melanin-like pigments (ochronosis) in connective tissues. Although numerous case reports have described ochronosis in joints, little is known on the molecular mechanisms leading to such a phenomenon. For this reason, we characterized biochemically chondrocytes isolated from the ochronotic cartilage of AKU patients. Based on the macroscopic appearance of the ochronotic cartilage, two sub-populations were identified: cells coming from the black portion of the cartilage were referred to as “black” AKU chondrocytes, while those coming from the white portion were referred to as “white” AKU chondrocytes. Notably, both AKU chondrocytic types were characterized by increased apoptosis, NO release, and levels of pro-inflammatory cytokines. Transmission electron microscopy also revealed that intracellular ochronotic pigment deposition was common to both “white” and “black” AKU cells. We then undertook a proteomic and redox-proteomic analysis of AKU chondrocytes which revealed profound alterations in the levels of proteins involved in cell defence, protein folding, and cell organization. An increased post-translational oxidation of proteins, which also involved high molecular weight protein aggregates, was found to be particularly relevant in “black” AKU chondrocytes. J. Cell. Physiol. 227: 3333–3343, 2012. © 2011 Wiley Periodicals, Inc. PMID:22213341

  20. Lubricin is expressed in chondrocytes derived from osteoarthritic cartilage encapsulated in poly (ethylene glycol) diacrylate scaffold

    PubMed Central

    Musumeci, G.; Loreto, C.; Carnazza, M.L.; Coppolino, F.; Cardile, V.; Leonardi, R.

    2011-01-01

    Osteoarthritis (OA) is characterized by degenerative changes within joints that involved quantitative and/or qualitative alterations of cartilage and synovial fluid lubricin, a mucinous glycoprotein secreted by synovial fibroblasts and chondrocytes. Modern therapeutic methods, including tissue-engineering techniques, have been used to treat mechanical damage of the articular cartilage but to date there is no specific and effective treatment. This study aimed at investigating lubricin immunohistochemical expression in cartilage explant from normal and OA patients and in cartilage constructions formed by Poly (ethylene glycol) (PEG) based hydrogels (PEG-DA) encapsulated OA chondrocytes. The expression levels of lubricin were studied by immunohistochemistry: i) in tissue explanted from OA and normal human cartilage; ii) in chondrocytes encapsulated in hydrogel PEGDA from OA and normal human cartilage. Moreover, immunocytochemical and western blot analysis were performed in monolayer cells from OA and normal cartilage. The results showed an increased expression of lubricin in explanted tissue and in monolayer cells from normal cartilage, and a decreased expression of lubricin in OA cartilage. The chondrocytes from OA cartilage after 5 weeks of culture in hydrogels (PEGDA) showed an increased expression of lubricin compared with the control cartilage. The present study demonstrated that OA chondrocytes encapsulated in PEGDA, grown in the scaffold and were able to restore lubricin biosynthesis. Thus our results suggest the possibility of applying autologous cell transplantation in conjunction with scaffold materials for repairing cartilage lesions in patients with OA to reduce at least the progression of the disease. PMID:22073377

  1. Isolation and characterization of mesenchymal stem cells from human umbilical cord blood: reevaluation of critical factors for successful isolation and high ability to proliferate and differentiate to chondrocytes as compared to mesenchymal stem cells from bone marrow and adipose tissue.

    PubMed

    Zhang, Xiaohong; Hirai, Masako; Cantero, Susana; Ciubotariu, Rodica; Dobrila, Ludy; Hirsh, Allen; Igura, Koichi; Satoh, Hitoshi; Yokomi, Izuru; Nishimura, Toshihide; Yamaguchi, Satoru; Yoshimura, Kotaro; Rubinstein, Pablo; Takahashi, Tsuneo A

    2011-04-01

    Human umbilical cord blood (CB) is a potential source for mesenchymal stem cells (MSC) capable of forming specific tissues, for example, bone, cartilage, or muscle. However, difficulty isolating MSC from CB (CB-MSC) has impeded their clinical application. Using more than 450 CB units donated to two public CB banks, we found that successful cell recovery fits a hyper-exponential function of time since birth with very high fidelity. Additionally, significant improvement in the isolation of CB-MSC was achieved by selecting cord blood units having a volume ≥90  ml and time ≤2  h after donor's birth. This resulted in 90% success in isolation of CB-MSC by density gradient purification and without a requirement for immunoaffinity methods as previously reported. Using MSC isolated from bone marrow (BM-MSC) and adipose tissue (AT-MSC) as reference controls, we observed that CB-MSC exhibited a higher proliferation rate and expanded to the order of the 1 × 10(9)  cells required for cell therapies. CB-MSC showed karyotype stability after prolonged expansion. Functionally, CB-MSC could be more readily induced to differentiate into chondrocytes than could BM-MSC and AT-MSC. CB-MSC showed immunosuppressive activity equal to that of BM-MSC and AT-MSC. Collectively, our data indicate that viable CB-MSC could be obtained consistently and that CB should be reconsidered as a practical source of MSC for cell therapy and regenerative medicine using the well established CB banking system. PMID:21312238

  2. Chondrocytes, Mesenchymal Stem Cells, and Their Combination in Articular Cartilage Regenerative Medicine.

    PubMed

    Nazempour, A; Van Wie, B J

    2016-05-01

    Articular cartilage (AC) is a highly organized connective tissue lining, covering the ends of bones within articulating joints. Its highly ordered structure is essential for stable motion and provides a frictionless surface easing load transfer. AC is vulnerable to lesions and, because it is aneural and avascular, it has limited self-repair potential which often leads to osteoarthritis. To date, no fully successful treatment for osteoarthritis has been reported. Thus, the development of innovative therapeutic approaches is desperately needed. Autologous chondrocyte implantation, the only cell-based surgical intervention approved in the United States for treating cartilage defects, has limitations because of de-differentiation of articular chondrocytes (AChs) upon in vitro expansion. De-differentiation can be abated if initial populations of AChs are co-cultured with mesenchymal stem cells (MSCs), which not only undergo chondrogenesis themselves but also support chondrocyte vitality. In this review we summarize studies utilizing AChs, non-AChs, and MSCs and compare associated outcomes. Moreover, a comprehensive set of recent human studies using chondrocytes to direct MSC differentiation, MSCs to support chondrocyte re-differentiation and proliferation in co-culture environments, and exploratory animal intra- and inter-species studies are systematically reviewed and discussed in an innovative manner allowing side-by-side comparisons of protocols and outcomes. Finally, a comprehensive set of recommendations are made for future studies. PMID:26987846

  3. Extracellular Matrix Domain Formation as an Indicator of Chondrocyte Dedifferentiation and Hypertrophy

    PubMed Central

    Wu, Ling; Gonzalez, Stephanie; Shah, Saumya; Kyupelyan, Levon; Petrigliano, Frank A.; McAllister, David R.; Adams, John S; Karperien, Marcel; Tuan, Tai-Lan; Benya, Paul D.

    2014-01-01

    Cartilage injury represents one of the most significant clinical conditions. Implantation of expanded autologous chondrocytes from noninjured compartments of the joint is a typical strategy for repairing cartilage. However, two-dimensional culture causes dedifferentiation of chondrocytes, making them functionally inferior for cartilage repair. We hypothesized that functional exclusion of dedifferentiated chondrocytes can be achieved by the selective mapping of collagen molecules deposited by chondrogenic cells in a three-dimensional environment. Freshly isolated and in vitro expanded human fetal or adult articular chondrocytes were cultured in a thermoreversible hydrogel at density of 1×107 cells/mL for 24 h. Chondrocytes were released from the gel, stained with antibodies against collagen type 2 (COL II) or COL I or COL X and sorted by fluorescence activated cell sorting. Imaging flow cytometry, immunohistochemistry, quantitative polymerase chain reaction, and glycosaminoglycan (GAG) assays were performed to evaluate the differences between COL II domain forming and COL II domain-negative cells. Freshly dissected periarticular chondrocytes robustly formed domains that consisted of the extracellular matrix surrounding cells in the hydrogel as a capsule clearly detectable by imaging flow cytometry (ImageStream) and confocal microscopy. These domains were almost exclusively formed by COL II. In contrast to that, a significant percentage of freshly isolated growth plate pre-hypertrophic and hyperdrophic chondrocytes deposited matrix domains positive for COL II, COL I, and COL X. The proportion of the cells producing COL II domains decreased with the increased passage of in vitro expanded periarticular fetal or adult articular chondrocytes. Sorted COL II domain forming cells deposited much higher levels of COL II and GAGs in pellet assays than COL II domain-negative cells. COL II domain forming cells expressed chondrogenic genes at higher levels than negative cells

  4. Sodium nitroprusside induces apoptosis of rabbit chondrocytes

    NASA Astrophysics Data System (ADS)

    Liang, Qian; Wang, Xiao-Ping; Chen, Tong-Sheng

    2013-02-01

    Osteoarthritis (OA) is characterized by a slowly progressing degradation of the matrix and destruction of articular cartilage. Apoptosis of chondrocyte is accounted for the mechanism of OA. Nitric oxide (NO), as a stimulus, has been shown to induce chondrocyte apoptosis by activating the matrix metalloproteinases (MMPs), increasing the expression of cyclooxygenase 2 (COX-2) and the level of prostaglandin E2 (PGE2), inhibiting the proteoglycan synthesis and type II collagen expression. In this study, sodium nitroprusside (SNP) was administered to be the NO donor to explore the mechanism of NO-induced apoptosis of rabbit chondrocytes obtained from six weeks old New Zealand rabbits. CCK-8 assay revealed the inhibitory effect of SNP on cell viability. We used flow cytometry (FCM) to assess the form of cell death by Annexin-V/propidium iodide (PI) double staining, and evaluate the change of mitochondrial membrane potential (ΔΨm). We found that the SNP induced chondrocyte apoptosis in a dose- and time-dependent manner and an observable reduction of ΔΨm. In conclusion, our findings indicate that SNP induces apoptosis of rabbit chondrocytes via a mitochondria-mediated pathway.

  5. Oxygen tension affects lubricin expression in chondrocytes.

    PubMed

    Hatta, Taku; Kishimoto, Koshi N; Okuno, Hiroshi; Itoi, Eiji

    2014-10-01

    We assessed the effects of oxygen tension on lubricin expression in bovine chondrocytes and cartilage explants and a role for hypoxia-inducible transcription factor (HIF)-1α in regulating lubricin expression was investigated using a murine chondroprogenitor cell line, ATDC5, and bovine chondrocytes isolated from superficial and middle/deep zones of femoral cartilage. ATDC5 cells and bovine chondrocytes were cultured in micromass under different oxygen tensions (21%, 5%, and 1%). ATDC5 cells and middle/deep zone chondrocytes that initially had low lubricin expression levels were also cultured with or without transforming growth factor (TGF)-β1. Quantitative reverse transcription (RT)-PCR was used to determine lubricin and chondrogenic marker gene mRNA levels and immunohistochemistry was used to assess lubricin protein expression. Explant cartilage plugs cultured under different oxygen tensions were also subjected to immunohistological analysis for lubricin. HIF-1α gene silencing was achieved by electroporatic transfer into ATDC5 cells. A low oxygen tension reduced lubricin gene expression levels in bovine superficial chondrocytes, TGF-β1-treated middle/deep zone chondrocytes, and TGF-β1-treated ATDC5 cells. Lubricin expression in explant cartilage was also suppressed under hypoxia. HIF-1α gene silencing in ATDC5 cells attenuated the lubricin expression response to the oxygen tension. These results corroborate with previous studies that the oxygen tension regulates lubricin gene expression and suggest that HIF-1α plays an important role in this regulation. The normal distribution of lubricin in articular cartilage may be due to the hypoxic oxygen environment of cartilage as it is an avascular tissue. An oxygen tension gradient may be a key factor for engineering cartilage tissue with a layered morphology. PMID:24712343

  6. The TATA-containing core promoter of the type II collagen gene (COL2A1) is the target of interferon-gamma-mediated inhibition in human chondrocytes: requirement for Stat1 alpha, Jak1 and Jak2.

    PubMed Central

    Osaki, Makoto; Tan, Lujian; Choy, Bob K; Yoshida, Yasuhiro; Cheah, Kathryn S E; Auron, Philip E; Goldring, Mary B

    2003-01-01

    Interferon-gamma (IFN-gamma) inhibits the synthesis of the cartilage-specific extracellular matrix protein type II collagen, and suppresses the expression of the type II collagen gene ( COL2A1 ) at the transcriptional level. To further examine this mechanism, the responses of COL2A1 regulatory sequences to IFN-gamma and the role of components of the Janus kinase/signal transducer and activators of transcription (JAK/STAT) pathway were examined in the immortalized human chondrocyte cell line, C-28/I2. IFN-gamma inhibited the mRNA levels of COL2A1 and aggrecan, but not Sox9, L-Sox5 and Sox6, all of which were expressed by these cells as markers of the differentiated phenotype. IFN-gamma suppressed the expression of luciferase reporter constructs containing sequences of the COL2A1 promoter spanning -6368 to +125 bp in the absence and presence of the intronic enhancer and stimulated activity of the gamma-interferon-activated site (GAS) luciferase reporter vector, associated with induction of Stat1 alpha-binding activity in nuclear extracts. These responses to IFN-gamma were blocked by overexpression of the JAK inhibitor, JAK-binding protein (JAB), or reversed by dominant-negative Stat1 alpha Y701F containing a mutation at Tyr-701, the JAK phosphorylation site. IFN-gamma had no effect on COL2A1 promoter expression in Jak1 (U4A)-, Jak2 (gamma 2A)- and Stat1 alpha (U3A)-deficient cell lines. In the U3A cell line, the response to IFN-gamma was rescued by overexpression of Stat1 alpha, but not by either Stat1 alpha Y701F or Stat1 beta. Functional analysis using deletion constructs showed that the IFN-gamma response was retained in the COL2A1 core promoter region spanning -45 to +11 bp, containing the TATA-box and GC-rich sequences but no Stat1-binding elements. Inhibition of COL2A1 promoter activity by IFN-gamma persisted in the presence of multiple deletions within the -45/+11 bp region. Our results indicate that repression of COL2A1 gene transcription by IFN

  7. Processed xenogenic cartilage as innovative biomatrix for cartilage tissue engineering: effects on chondrocyte differentiation and function.

    PubMed

    Schwarz, Silke; Elsaesser, Alexander F; Koerber, Ludwig; Goldberg-Bockhorn, Eva; Seitz, Andreas M; Bermueller, Christian; Dürselen, Lutz; Ignatius, Anita; Breiter, Roman; Rotter, Nicole

    2015-12-01

    One key point in the development of new bioimplant matrices for the reconstruction and replacement of cartilage defects is to provide an adequate microenvironment to ensure chondrocyte migration and de novo synthesis of cartilage-specific extracellular matrix (ECM). A recently developed decellularization and sterilization process maintains the three-dimensional (3D) collagen structure of native septal cartilage while increasing matrix porosity, which is considered to be crucial for cartilage tissue engineering. Human primary nasal septal chondrocytes were amplified in monolayer culture and 3D-cultured on processed porcine nasal septal cartilage scaffolds. The influence of chondrogenic growth factors on neosynthesis of ECM proteins was examined at the protein and gene expression levels. Seeding experiments demonstrated that processed xenogenic cartilage matrices provide excellent environmental properties for human nasal septal chondrocytes with respect to cell adhesion, migration into the matrix and neosynthesis of cartilage-specific ECM proteins, such as collagen type II and aggrecan. Matrix biomechanical stability indicated that the constructs retrieve full stability and function during 3D culture for up to 42 days, proportional to collagen type II and GAG production. Thus, processed xenogenic cartilage offers a suitable environment for human nasal chondrocytes and has promising potential for cartilage tissue engineering in the head and neck region. PMID:23193064

  8. Oxidative Stress Promotes Peroxiredoxin Hyperoxidation and Attenuates Pro-survival Signaling in Aging Chondrocytes.

    PubMed

    Collins, John A; Wood, Scott T; Nelson, Kimberly J; Rowe, Meredith A; Carlson, Cathy S; Chubinskaya, Susan; Poole, Leslie B; Furdui, Cristina M; Loeser, Richard F

    2016-03-25

    Oxidative stress-mediated post-translational modifications of redox-sensitive proteins are postulated as a key mechanism underlying age-related cellular dysfunction and disease progression. Peroxiredoxins (PRX) are critical intracellular antioxidants that also regulate redox signaling events. Age-related osteoarthritis is a common form of arthritis that has been associated with mitochondrial dysfunction and oxidative stress. The objective of this study was to determine the effect of aging and oxidative stress on chondrocyte intracellular signaling, with a specific focus on oxidation of cytosolic PRX2 and mitochondrial PRX3. Menadione was used as a model to induce cellular oxidative stress. Compared with chondrocytes isolated from young adult humans, chondrocytes from older adults exhibited higher levels of PRX1-3 hyperoxidation basally and under conditions of oxidative stress. Peroxiredoxin hyperoxidation was associated with inhibition of pro-survival Akt signaling and stimulation of pro-death p38 signaling. These changes were prevented in cultured human chondrocytes by adenoviral expression of catalase targeted to the mitochondria (MCAT) and in cartilage explants from MCAT transgenic mice. Peroxiredoxin hyperoxidation was observedin situin human cartilage sections from older adults and in osteoarthritic cartilage. MCAT transgenic mice exhibited less age-related osteoarthritis. These findings demonstrate that age-related oxidative stress can disrupt normal physiological signaling and contribute to osteoarthritis and suggest peroxiredoxin hyperoxidation as a potential mechanism. PMID:26797130

  9. Evaluation of thermoreversible polymers containing fibroblast growth factor 9 (FGF-9) for chondrocyte culture

    SciTech Connect

    Au, Angela; Ha, Jinny; Polotsky, Anna; Krzyminski, Karol J.; Gutowska, Anna; Hungerford, Davis S.; Frondoza, Carmelita G.

    2004-05-01

    We have evaluated a biomaterial to serve as a scaffold for the propagation and amplification of chondrocytes that promotes the original cellular phenotype of these cells. The goal of the present study was to investigate the use of thermally reversible polymer gels poly(NiPAAm-co-AAc), as a biocompatible supporting scaffold for the propagation of chondrocytic cells. The polymer gels at temperatures above its lower critical solution temperature (LCST) while liquefying at temperatures below its LCST of 34.5 C. Hence, the polymer, in its gelled form, has the ability to hold cells in situ, forming a matrix similar to the natural cellular environment or the extracellular matrix that comprises cartilage. We tested the hypothesis that the polymer gel promotes cell viability and function. Human osteoblast-like cells, nasal chondrocytes, and articular chondrocytes (1x105/150 ?l) were re-suspended in enriched DMEM media and were plated onto control (without gel) and gel containing 24-well plates. The plates were re-incubated at 37 C, 5% CO2 for the time-point of interest. Additional media was added to the plates and exchanged as needed. Following cell culture, cells were retrieved, enumerated, and cell viability was determined. Other aliquots of the cells were stained for morphological analysis while expression of chondrocyte markers including collagen type II and aggrecan were determined using Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR). The polymer gel was not cytotoxic as the cell number retrieved from three-dimensional culture gel was found to be one to two times higher than that retrieved from monolayer culture. Chondrocytes propagated in the thermo-reversible polymers expressed enhanced or maintained expression of collagen type II and aggrecan. Collagen type I expression was decreased or unaltered. The N-isopropylacrylamide and acrylic acid copolymer gel has potential use as a cell culture substrate and as a cell delivery vehicle.

  10. Mycoplasma synoviae induces upregulation of apoptotic genes, secretion of nitric oxide and appearance of an apoptotic phenotype in infected chicken chondrocytes

    PubMed Central

    2012-01-01

    The role of chondrocytes in the development of infectious arthritis is not well understood. Several examples of mycoplasma-induced arthritis in animals indicate that chondrocytes come into direct contact with bacteria. The objective of this study was to analyze the interaction of an arthrogenic Mycoplasma synoviae strain WVU 1853 with chicken chondrocytes. We found that M. synoviae significantly reduces chondrocyte respiration. This was accompanied by alterations in chondrocyte morphology, namely cell shrinkage and cytoplasm condensation, as well as nuclear condensation and formation of plasma membrane invaginations containing nuclear material, which appeared to cleave off the cell surface. In concordance with these apoptosis-like events in chondrocytes, transcription was increased in several pro-apoptotic genes. Twenty-four hours after infection, strong upregulation was assayed in NOS2, Mapk11, CASP8 and Casp3 genes. Twenty-four and 72 h incubation of chondrocytes with M. synoviae induced upregulation of AIFM1, NFκB1, htrA3 and BCL2. Casp3 and NOS2 remained upregulated, but upregulation ceased for Mapk11 and CASP8 genes. Increased production of nitric oxide was also confirmed in cell supernates. The data suggests that chicken chondrocytes infected with M. synoviae die by apoptosis involving production of nitric oxide, caspase 3 activation and mitochondrial inactivation. The results of this study show for the first time that mycoplasmas could cause chondrocyte apoptosis. This could contribute to tissue destruction and influence the development of arthritic conditions. Hence, the study gives new insights into the role of mycoplasma infection on chondrocyte biology and development of infectious arthritis in chickens and potentially in humans. PMID:22280251

  11. Regulation of osteogenic proteins by chondrocytes.

    PubMed

    Chubinskaya, Susan; Kuettner, Klaus E

    2003-09-01

    The purpose of this review is to summarize the current scientific knowledge of bone morphogenetic proteins (BMPs) in adult articular cartilage. We specifically focus on adult cartilage, since one of the major potential applications of the members of the BMP family may be a repair of adult tissue after trauma and/or disease. After reviewing cartilage physiology and BMPs, we analyze the data on the role of recombinant BMPs as anabolic agents in tissue formation and restoration in different in vitro and in vivo models following with the endogenous expression of BMPs and factors that regulate their expression. We also discuss recent transgenic modifications of BMP genes and subsequent effect on cartilage matrix synthesis. We found that the most studied BMPs in adult articular cartilage are BMP-7 and BMP-2 as well as transforming growth factor-beta (TGF-beta). There are a number of contradicting reports for some of these growth factors, since different models, animals, doses, time points, culture conditions and devices were used. However, regardless of the experimental conditions, only BMP-7 or osteogenic protein-1 (OP-1) exhibits the most convincing effects. It is the only BMP studied thus far in adult cartilage that demonstrates strong anabolic activity in vitro and in vivo with and without serum. OP-1 stimulates the synthesis of the majority of cartilage extracellular matrix proteins in adult articular chondrocytes derived from different species and of different age. OP-1 counteracts the degenerative effect of numerous catabolic mediators; it is also expressed in adult human, bovine, rabbit and goat articular cartilage. This review reveals the importance of the exploration of the BMPs in the cartilage field and highlights their significance for clinical applications in the treatment of cartilage-related diseases. PMID:12798347

  12. Follistatin in chondrocytes: the link between TRPV4 channelopathies and skeletal malformations

    PubMed Central

    Leddy, Holly A.; McNulty, Amy L.; Lee, Suk Hee; Rothfusz, Nicole E.; Gloss, Bernd; Kirby, Margaret L.; Hutson, Mary R.; Cohn, Daniel H.; Guilak, Farshid; Liedtke, Wolfgang

    2014-01-01

    Point mutations in the calcium-permeable TRPV4 ion channel have been identified as the cause of autosomal-dominant human motor neuropathies, arthropathies, and skeletal malformations of varying severity. The objective of this study was to determine the mechanism by which TRPV4 channelopathy mutations cause skeletal dysplasia. The human TRPV4V620I channelopathy mutation was transfected into primary porcine chondrocytes and caused significant (2.6-fold) up-regulation of follistatin (FST) expression levels. Pore altering mutations that prevent calcium influx through the channel prevented significant FST up-regulation (1.1-fold). We generated a mouse model of theTRPV4V620I mutation, and found significant skeletal deformities (e.g., shortening of tibiae and digits, similar to the human disease brachyolmia) and increases in Fst/TRPV4 mRNA levels (2.8-fold). FST was significantly up-regulated in primary chondrocytes transfected with 3 different dysplasia-causing TRPV4 mutations (2- to 2.3-fold), but was not affected by an arthropathy mutation (1.1-fold). Furthermore, FST-loaded microbeads decreased bone ossification in developing chick femora (6%) and tibiae (11%). FST gene and protein levels were also increased 4-fold in human chondrocytes from an individual natively expressing the TRPV4T89I mutation. Taken together, these data strongly support that up-regulation of FST in chondrocytes by skeletal dysplasia-inducing TRPV4 mutations contributes to disease pathogenesis.—Leddy, H. A., McNulty, A. L., Lee, S. H., Rothfusz, N. E., Gloss, B., Kirby, M. L., Hutson, M. R., Cohn, D. H., Guilak, F., Liedtke, W. Follistatin in chondrocytes: the link between TRPV4 channelopathies and skeletal malformations. PMID:24577120

  13. Tissue responses against tissue-engineered cartilage consisting of chondrocytes encapsulated within non-absorbable hydrogel.

    PubMed

    Kanazawa, Sanshiro; Fujihara, Yuko; Sakamoto, Tomoaki; Asawa, Yukiyo; Komura, Makoto; Nagata, Satoru; Takato, Tsuyoshi; Hoshi, Kazuto

    2013-01-01

    To disclose the influence of foreign body responses raised against a non-absorbable hydrogel consisting of tissue-engineered cartilage, we embedded human/canine chondrocytes within agarose and transplanted them into subcutaneous pockets in nude mice and donor beagles. One month after transplantation, cartilage formation was observed in the experiments using human chondrocytes in nude mice. No significant invasion of blood cells was noted in the areas where the cartilage was newly formed. Around the tissue-engineered cartilage, agarose fragments, a dense fibrous connective tissue and many macrophages were observed. On the other hand, no cartilage tissue was detected in the autologous transplantation of canine chondrocytes. Few surviving chondrocytes were observed in the agarose and no accumulation of blood cells was observed in the inner parts of the transplants. Localizations of IgG and complements were noted in areas of agarose, and also in the devitalized cells embedded within the agarose. Even if we had inhibited the proximity of the blood cells to the transplanted cells, the survival of the cells could not be secured. We suggest that these cytotoxic mechanisms seem to be associated not only with macrophages but also with soluble factors, including antibodies and complements. PMID:21916014

  14. Changes in Morphology, Gene Expression and Protein Content in Chondrocytes Cultured on a Random Positioning Machine

    PubMed Central

    Aleshcheva, Ganna; Sahana, Jayashree; Ma, Xiao; Hauslage, Jens; Hemmersbach, Ruth; Egli, Marcel; Infanger, Manfred; Bauer, Johann; Grimm, Daniela

    2013-01-01

    Tissue engineering of chondrocytes on a Random Positioning Machine (RPM) is a new strategy for cartilage regeneration. Using a three-dimensional RPM, a device designed to simulate microgravity on Earth, we investigated the early effects of RPM exposure on human chondrocytes of six different donors after 30 min, 2 h, 4 h, 16 h, and 24 h and compared the results with the corresponding static controls cultured under normal gravity conditions. As little as 30 min of RPM exposure resulted in increased expression of several genes responsible for cell motility, structure and integrity (beta-actin); control of cell growth, cell proliferation, cell differentiation and apoptosis (TGF-β1, osteopontin); and cytoskeletal components such as microtubules (beta-tubulin) and intermediate filaments (vimentin). After 4 hours of RPM exposure disruptions in the vimentin network were detected. These changes were less dramatic after 16 hours on the RPM, when human chondrocytes appeared to reorganize their cytoskeleton. However, the gene expression and protein content of TGF-β1 was enhanced during RPM culture for 24 h. Taking these results together, we suggest that chondrocytes exposed to the RPM seem to change their extracellular matrix production behaviour while they rearrange their cytoskeletal proteins prior to forming three-dimensional aggregates. PMID:24244418

  15. An evaluation of chondrocyte morphology and gene expression on superhydrophilic vertically-aligned multi-walled carbon nanotube films.

    PubMed

    Antonioli, Eliane; Lobo, Anderson O; Ferretti, Mario; Cohen, Moisés; Marciano, Fernanda R; Corat, Evaldo J; Trava-Airoldi, Vladimir J

    2013-03-01

    Cartilage serves as a low-friction and wear-resistant articulating surface in diarthrodial joints and is also important during early stages of bone remodeling. Recently, regenerative cartilage research has focused on combinations of cells paired with scaffolds. Superhydrophilic vertically aligned carbon nanotubes (VACNTs) are of particular interest in regenerative medicine. The aim of this study is to evaluate cell expansion of human articular chondrocytes on superhydrophilic VACNTs, as well as their morphology and gene expression. VACNT films were produced using a microwave plasma chamber on Ti substrates and submitted to an O2 plasma treatment to make them superhydrophilic. Human chondrocytes were cultivated on superhydrophilic VACNTs up to five days. Quantitative RT-PCR was performed to measure type I and type II Collagen, Sox9, and Aggrecan mRNA expression levels. The morphology was analyzed by scanning electron microscopy (SEM) and confocal microscopy. SEM images demonstrated that superhydrophilic VACNTs permit cell growth and adhesion of human chondrocytes. The chondrocytes had an elongated morphology with some prolongations. Chondrocytes cultivated on superhydrophilic VACNTs maintain the level expression of Aggrecan, Sox9, and Collagen II determined by qPCR. This study was the first to indicate that superhydrophilic VACNTs may be used as an efficient scaffold for cartilage or bone repair. PMID:25427468

  16. 3D Hydrogel Scaffolds for Articular Chondrocyte Culture and Cartilage Generation

    PubMed Central

    Yang, Fan; Bhutani, Nidhi

    2015-01-01

    Human articular cartilage is highly susceptible to damage and has limited self-repair and regeneration potential. Cell-based strategies to engineer cartilage tissue offer a promising solution to repair articular cartilage. To select the optimal cell source for tissue repair, it is important to develop an appropriate culture platform to systematically examine the biological and biomechanical differences in the tissue-engineered cartilage by different cell sources. Here we applied a three-dimensional (3D) biomimetic hydrogel culture platform to systematically examine cartilage regeneration potential of juvenile, adult, and osteoarthritic (OA) chondrocytes. The 3D biomimetic hydrogel consisted of synthetic component poly(ethylene glycol) and bioactive component chondroitin sulfate, which provides a physiologically relevant microenvironment for in vitro culture of chondrocytes. In addition, the scaffold may be potentially used for cell delivery for cartilage repair in vivo. Cartilage tissue engineered in the scaffold can be evaluated using quantitative gene expression, immunofluorescence staining, biochemical assays, and mechanical testing. Utilizing these outcomes, we were able to characterize the differential regenerative potential of chondrocytes of varying age, both at the gene expression level and in the biochemical and biomechanical properties of the engineered cartilage tissue. The 3D culture model could be applied to investigate the molecular and functional differences among chondrocytes and progenitor cells from different stages of normal or aberrant development. PMID:26484414

  17. Role of lubricin and boundary lubrication in the prevention of chondrocyte apoptosis.

    PubMed

    Waller, Kimberly A; Zhang, Ling X; Elsaid, Khaled A; Fleming, Braden C; Warman, Matthew L; Jay, Gregory D

    2013-04-01

    Osteoarthritis is a complex disease involving the mechanical breakdown of articular cartilage in the presence of altered joint mechanics and chondrocyte death, but the connection between these factors is not well established. Lubricin, a mucinous glycoprotein encoded by the PRG4 gene, provides boundary lubrication in articular joints. Joint friction is elevated and accompanied by accelerated cartilage damage in humans and mice that have genetic deficiency of lubricin. Here, we investigated the relationship between coefficient of friction and chondrocyte death using ex vivo and in vitro measurements of friction and apoptosis. We observed increases in whole-joint friction and cellular apoptosis in lubricin knockout mice compared with wild-type mice. When we used an in vitro bovine explant cartilage-on-cartilage bearing system, we observed a direct correlation between coefficient of friction and chondrocyte apoptosis in the superficial layers of cartilage. In the bovine explant system, the addition of lubricin as a test lubricant significantly lowered the static coefficient of friction and number of apoptotic chondrocytes. These results demonstrate a direct connection between lubricin, boundary lubrication, and cell survival and suggest that supplementation of synovial fluid with lubricin may be an effective treatment to prevent cartilage deterioration in patients with genetic or acquired deficiency of lubricin. PMID:23530215

  18. 3D Hydrogel Scaffolds for Articular Chondrocyte Culture and Cartilage Generation.

    PubMed

    Smeriglio, Piera; Lai, Janice H; Yang, Fan; Bhutani, Nidhi

    2015-01-01

    Human articular cartilage is highly susceptible to damage and has limited self-repair and regeneration potential. Cell-based strategies to engineer cartilage tissue offer a promising solution to repair articular cartilage. To select the optimal cell source for tissue repair, it is important to develop an appropriate culture platform to systematically examine the biological and biomechanical differences in the tissue-engineered cartilage by different cell sources. Here we applied a three-dimensional (3D) biomimetic hydrogel culture platform to systematically examine cartilage regeneration potential of juvenile, adult, and osteoarthritic (OA) chondrocytes. The 3D biomimetic hydrogel consisted of synthetic component poly(ethylene glycol) and bioactive component chondroitin sulfate, which provides a physiologically relevant microenvironment for in vitro culture of chondrocytes. In addition, the scaffold may be potentially used for cell delivery for cartilage repair in vivo. Cartilage tissue engineered in the scaffold can be evaluated using quantitative gene expression, immunofluorescence staining, biochemical assays, and mechanical testing. Utilizing these outcomes, we were able to characterize the differential regenerative potential of chondrocytes of varying age, both at the gene expression level and in the biochemical and biomechanical properties of the engineered cartilage tissue. The 3D culture model could be applied to investigate the molecular and functional differences among chondrocytes and progenitor cells from different stages of normal or aberrant development. PMID:26484414

  19. NF-{kappa}B regulates Lef1 gene expression in chondrocytes

    SciTech Connect

    Yun, Kangsun; Choi, Yoo Duk; Nam, Jong Hee; Park, Zeeyoung; Im, Sin-Hyeog . E-mail: imsh@gist.ac.kr

    2007-06-08

    The relation of Wnt/{beta}-catenin signaling to osteoarthritis progression has been revealed with little information on the underlying molecular mechanism. In this study we found overexpression of Lef1 in cartilage tissue of osteoarthritic patients and elucidated molecular mechanism of NF-{kappa}B-mediated Lef1 gene regulation in chondrocytes. Treatment of IL-1{beta} augmented Lef1 upregulation and nuclear translocation of NF-{kappa}B in chondrocytes. Under IL-1{beta} signaling, treatment of NF-{kappa}B nuclear translocation inhibitor SN-50 reduced Lef1 expression. A conserved NF-{kappa}B-binding site between mouse and human was selected through bioinformatic analysis and mapped at the 14 kb upstream of Lef1 transcription initiation site. NF-{kappa}B binding to the site was confirmed by chromatin immunoprecipitation assay. Lef1 expression was synergistically upregulated by interactions of NF-{kappa}B with Lef1/{beta}-catenin in chondrocytes. Our results suggest a pivotal role of NF-{kappa}B in Lef1 expression in arthritic chondrocytes or cartilage degeneration.

  20. [Growth behavior of chondrocytes on various biomaterials].

    PubMed

    Rudert, M; Hirschmann, F; Wirth, C J

    1999-01-01

    Chondrocytes can be cultured on different three-dimensional culture systems suitable for transplantation to enhance the repair of localized cartilage defects. Articular cartilage chondrocytes from adult rabbit knees and from bovine calf metacarpophalangeal joints were isolated by enzymatic digestion and cultured in a monolayer system to amplify cell count. After amplification the cells were seeded on different biocompatible materials. We investigated two types of bioresorbable polymer fleece matrices (a composite fleece of polydioxanon and polyglactin and a resorbable poly-L-lactic acid fleece) and lyophilized dura as a biological carrier. On all three types of transport media the phenotypic and morphological appearance of cultured chondrocytes could be observed. The production of glycosaminoglycans was revealed by Alcian blue staining and immunohistochemical detection of Chondroitin-4 and 6-sulfate in the created constructs. The material properties of the carriers allow for transplantation of the artificial cartilage-like products into full thickness articular cartilage defects and could therefore improve the minor intrinsic healing capacity of cartilage tissue. Bioartificial cartilage may become a future perspective in the treatment options of orthopaedic and plastic surgery. PMID:10081046

  1. Changes in gene expression, protein content and morphology of chondrocytes cultured on a 3D Random Positioning Machine and 2D rotating clinostat

    NASA Astrophysics Data System (ADS)

    Aleshcheva, Ganna; Hauslage, Jens; Hemmersbach, Ruth; Infanger, Manfred; Bauer, Johann; Grimm, Daniela; Sahana, Jayashree

    Chondrocytes are the only cell type found in human cartilage consisting of proteoglycans and type II collagen. Several studies on chondrocytes cultured either in Space or on a ground-based facility for simulation of microgravity revealed that these cells are very resistant to adverse effects and stress induced by altered gravity. Tissue engineering of chondrocytes is a new strategy for cartilage regeneration. Using a three-dimensional Random Positioning Machine and a 2D rotating clinostat, devices designed to simulate microgravity on Earth, we investigated the early effects of microgravity exposure on human chondrocytes of six different donors after 30 min, 2 h, 4 h, 16 h, and 24 h and compared the results with the corresponding static controls cultured under normal gravity conditions. As little as 30 min of exposure resulted in increased expression of several genes responsible for cell motility, structure and integrity (beta-actin); control of cell growth, cell proliferation, cell differentiation and apoptosis; and cytoskeletal components such as microtubules (beta-tubulin) and intermediate filaments (vimentin). After 4 hours disruptions in the vimentin network were detected. These changes were less dramatic after 16 hours, when human chondrocytes appeared to reorganize their cytoskeleton. However, the gene expression and protein content of TGF-β1 was enhanced for 24 h. Based on the results achieved, we suggest that chondrocytes exposed to simulated microgravity seem to change their extracellular matrix production behavior while they rearrange their cytoskeletal proteins prior to forming three-dimensional aggregates.

  2. Changes in the Chondrocyte and Extracellular Matrix Proteome during Post-natal Mouse Cartilage Development*

    PubMed Central

    Wilson, Richard; Norris, Emma L.; Brachvogel, Bent; Angelucci, Constanza; Zivkovic, Snezana; Gordon, Lavinia; Bernardo, Bianca C.; Stermann, Jacek; Sekiguchi, Kiyotoshi; Gorman, Jeffrey J.; Bateman, John F.

    2012-01-01

    Skeletal growth by endochondral ossification involves tightly coordinated chondrocyte differentiation that creates reserve, proliferating, prehypertrophic, and hypertrophic cartilage zones in the growth plate. Many human skeletal disorders result from mutations in cartilage extracellular matrix (ECM) components that compromise both ECM architecture and chondrocyte function. Understanding normal cartilage development, composition, and structure is therefore vital to unravel these disease mechanisms. To study this intricate process in vivo by proteomics, we analyzed mouse femoral head cartilage at developmental stages enriched in either immature chondrocytes or maturing/hypertrophic chondrocytes (post-natal days 3 and 21, respectively). Using LTQ-Orbitrap tandem mass spectrometry, we identified 703 cartilage proteins. Differentially abundant proteins (q < 0.01) included prototypic markers for both early and late chondrocyte differentiation (epiphycan and collagen X, respectively) and novel ECM and cell adhesion proteins with no previously described roles in cartilage development (tenascin X, vitrin, Urb, emilin-1, and the sushi repeat-containing proteins SRPX and SRPX2). Meta-analysis of cartilage development in vivo and an in vitro chondrocyte culture model (Wilson, R., Diseberg, A. F., Gordon, L., Zivkovic, S., Tatarczuch, L., Mackie, E. J., Gorman, J. J., and Bateman, J. F. (2010) Comprehensive profiling of cartilage extracellular matrix formation and maturation using sequential extraction and label-free quantitative proteomics. Mol. Cell. Proteomics 9, 1296–1313) identified components involved in both systems, such as Urb, and components with specific roles in vivo, including vitrin and CILP-2 (cartilage intermediate layer protein-2). Immunolocalization of Urb, vitrin, and CILP-2 indicated specific roles at different maturation stages. In addition to ECM-related changes, we provide the first biochemical evidence of changing endoplasmic reticulum function during

  3. Streptococcus pyogenes degrades extracellular matrix in chondrocytes via MMP-13

    SciTech Connect

    Sakurai, Atsuo; Okahashi, Nobuo; Maruyama, Fumito; Ooshima, Takashi; Hamada, Shigeyuki; Nakagawa, Ichiro

    2008-08-29

    Group A streptococcus (GAS) causes a wide range of human diseases, including bacterial arthritis. The pathogenesis of arthritis is characterized by synovial proliferation and the destruction of cartilage and subchondral bone in joints. We report here that GAS strain JRS4 invaded a chondrogenic cell line ATDC5 and induced the degradation of the extracellular matrix (ECM), whereas an isogenic mutant of JRS4 lacking a fibronectin-binding protein, SAM1, failed to invade the chondrocytes or degrade the ECM. Reverse transcription-PCR and Western blot analysis revealed that the expression of matrix metalloproteinase (MMP)-13 was strongly elevated during the infection with GAS. A reporter assay revealed that the activation of the AP-1 transcription factor and the phosphorylation of c-Jun terminal kinase participated in MMP-13 expression. These results suggest that MMP-13 plays an important role in the destruction of infected joints during the development of septic arthritis.

  4. Genome-wide mapping of DNA hydroxymethylation in osteoarthritic chondrocytes

    PubMed Central

    Taylor, Sarah E. B.; Li, Ye Henry; Wong, Wing H.; Bhutani, Nidhi

    2015-01-01

    Objective To examine genome-wide 5hmC distribution in osteoarthritic (OA) and normal chondrocytes to investigate the effect on OA-specific gene expression. Methods Cartilage was obtained from OA patients undergoing total knee arthroplasty or control patients undergoing anterior cruciate ligament reconstruction. Genome-wide sequencing of 5hmC-enriched DNA (5hmC-seq) was performed for a small cohort of normal and OA chondrocytes to identify differentially hydroxymethylated regions (DhMRs) in OA chondrocytes. 5hmC-seq data was intersected with global OA gene expression data to define subsets of genes and pathways potentially affected by increased 5hmC levels in OA chondrocytes. Results 70591 DhMRs were identified in OA chondrocytes compared to normal chondrocytes, 44288 (63%) of which were increased in OA chondrocytes. The majority of DhMRs (66%) were gained in gene bodies. Increased DhMRs were observed in ~50% of genes previously implicated in OA pathology including MMP3, LRP5, GDF5 and COL11A1. Furthermore, analyses of gene expression data revealed gene body gain of 5hmC appears to be preferentially associated with activated but not repressed genes in OA chondrocytes. Conclusion This study provides the first genome-wide profiling of 5hmC distribution in OA chondrocytes. We had previously reported a global increase in 5hmC levels in OA chondrocytes. Gain of 5hmC in the gene body is found to be characteristic of activated genes in OA chondrocytes, highlighting the influence of 5hmC as an epigenetic mark in OA. In addition, this study identifies multiple OA-associated genes that are potentially regulated either singularly by gain of DNA hydroxymethylation or in combination with loss of DNA methylation. PMID:25940674

  5. Purification of a peptide from seahorse, that inhibits TPA-induced MMP, iNOS and COX-2 expression through MAPK and NF-kappaB activation, and induces human osteoblastic and chondrocytic differentiation.

    PubMed

    Ryu, BoMi; Qian, Zhong-Ji; Kim, Se-Kwon

    2010-03-30

    Ongoing efforts to search for naturally occurring, bioactive substances for the amelioration of arthritis have led to the discovery of natural products with substantial bioactive properties. The seahorse (Hippocampus kuda Bleeler), a telelost fish, is one source of known beneficial products, yet has not been utilized for arthritis research. In the present work, we have purified and characterized a bioactive peptide from seahorse hydrolysis. Among the hydrolysates tested, pronase E-derived hydrolysate exhibited the highest alkaline phosphatase (ALP) activity, a phenotype marker of osteoblast and chondrocyte differentiation. After its separation from the hydrolysate by several purification steps, the peptide responsible for the ALP activity was isolated and its sequence was identified as LEDPFDKDDWDNWK (1821Da). We have shown that the isolated peptide induces differentiation of osteoblastic MG-63 and chondrocytic SW-1353 cells by measuring ALP activity, mineralization and collagen synthesis. Our results indicate that the peptide acts during early to late stages of differentiation in MG-63 and SW-1353 cells. We also assessed the concentration dependence of the peptide's inhibition of MMP (-1, -3 and -13), iNOS and COX-2 expression after treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA), a common form of phorbol ester. The peptide also inhibited NO production in MG-63 and SW-1353 cells. To elucidate the mechanisms by which the peptide acted, we examined its effects on TPA-induced MAPKs/NF-kappaB activation and determined that the peptide treatment significantly reduced p38 kinase/NF-kappaB in MG-63 cells and MAPKs/NF-kappaB in SW-1353 cells. PMID:20004183

  6. Chondrocyte-specific ablation of Osterix leads to impaired endochondral ossification

    SciTech Connect

    Oh, Jung-Hoon; Park, Seung-Yoon; Crombrugghe, Benoit de; Kim, Jung-Eun

    2012-02-24

    Highlights: Black-Right-Pointing-Pointer Conditional ablation of Osterix (Osx) in chondrocytes leads to skeletal defects. Black-Right-Pointing-Pointer Osx regulates chondrocyte differentiation and bone growth in growth plate chondrocytes. Black-Right-Pointing-Pointer Osx has an autonomous function in chondrocytes during endochondral ossification. -- Abstract: Osterix (Osx) is an essential transcription factor required for osteoblast differentiation during both intramembranous and endochondral ossification. Endochondral ossification, a process in which bone formation initiates from a cartilage intermediate, is crucial for skeletal development and growth. Osx is expressed in differentiating chondrocytes as well as osteoblasts during mouse development, but its role in chondrocytes has not been well studied. Here, the in vivo function of Osx in chondrocytes was examined in a chondrocyte-specific Osx conditional knockout model using Col2a1-Cre. Chondrocyte-specific Osx deficiency resulted in a weak and bent skeleton which was evident in newborn by radiographic analysis and skeletal preparation. To further understand the skeletal deformity of the chondrocyte-specific Osx conditional knockout, histological analysis was performed on developing long bones during embryogenesis. Hypertrophic chondrocytes were expanded, the formation of bone trabeculae and marrow cavities was remarkably delayed, and subsequent skeletal growth was reduced. The expression of several chondrocyte differentiation markers was reduced, indicating the impairment of chondrocyte differentiation and endochondral ossification in the chondrocyte-specific Osx conditional knockout. Taken together, Osx regulates chondrocyte differentiation and bone growth in growth plate chondrocytes, suggesting an autonomous function of Osx in chondrocytes during endochondral ossification.

  7. Applications of Chondrocyte-Based Cartilage Engineering: An Overview

    PubMed Central

    Eo, Seong-Hui; Abbas, Qamar; Ahmed, Madiha

    2016-01-01

    Chondrocytes are the exclusive cells residing in cartilage and maintain the functionality of cartilage tissue. Series of biocomponents such as different growth factors, cytokines, and transcriptional factors regulate the mesenchymal stem cells (MSCs) differentiation to chondrocytes. The number of chondrocytes and dedifferentiation are the key limitations in subsequent clinical application of the chondrocytes. Different culture methods are being developed to overcome such issues. Using tissue engineering and cell based approaches, chondrocytes offer prominent therapeutic option specifically in orthopedics for cartilage repair and to treat ailments such as tracheal defects, facial reconstruction, and urinary incontinence. Matrix-assisted autologous chondrocyte transplantation/implantation is an improved version of traditional autologous chondrocyte transplantation (ACT) method. An increasing number of studies show the clinical significance of this technique for the chondral lesions treatment. Literature survey was carried out to address clinical and functional findings by using various ACT procedures. The current study was conducted to study the pharmacological significance and biomedical application of chondrocytes. Furthermore, it is inferred from the present study that long term follow-up studies are required to evaluate the potential of these methods and specific positive outcomes.

  8. Proliferation and differentiation potential of chondrocytes from osteoarthritic patients

    PubMed Central

    Tallheden, Tommi; Bengtsson, Catherine; Brantsing, Camilla; Sjögren-Jansson, Eva; Carlsson, Lars; Peterson, Lars; Brittberg, Mats; Lindahl, Anders

    2005-01-01

    Autologous chondrocyte transplantation (ACT) has been shown, in long-term follow-up studies, to be a promising treatment for the repair of isolated cartilage lesions. The method is based on an implantation of in vitro expanded chondrocytes originating from a small cartilage biopsy harvested from a non-weight-bearing area within the joint. In patients with osteoarthritis (OA), there is a need for the resurfacing of large areas, which could potentially be made by using a scaffold in combination with culture-expanded cells. As a first step towards a cell-based therapy for OA, we therefore investigated the expansion and redifferentiation potential in vitro of chondrocytes isolated from patients undergoing total knee replacement. The results demonstrate that OA chondrocytes have a good proliferation potential and are able to redifferentiate in a three-dimensional pellet model. During the redifferentiation, the OA cells expressed increasing amounts of DNA and proteoglycans, and at day 14 the cells from all donors contained type II collagen-rich matrix. The accumulation of proteoglycans was in comparable amounts to those from ACT donors, whereas total collagen was significantly lower in all of the redifferentiated OA chondrocytes. When the OA chondrocytes were loaded into a scaffold based on hyaluronic acid, they bound to the scaffold and produced cartilage-specific matrix proteins. Thus, autologous chondrocytes are a potential source for the biological treatment of OA patients but the limited collagen synthesis of the OA chondrocytes needs to be further explained. PMID:15899043

  9. The NAD-Dependent Deacetylase Sirtuin-1 Regulates the Expression of Osteogenic Transcriptional Activator Runt-Related Transcription Factor 2 (Runx2) and Production of Matrix Metalloproteinase (MMP)-13 in Chondrocytes in Osteoarthritis

    PubMed Central

    Terauchi, Koh; Kobayashi, Hajime; Yatabe, Kanaka; Yui, Naoko; Fujiya, Hiroto; Niki, Hisateru; Musha, Haruki; Yudoh, Kazuo

    2016-01-01

    Aging is one of the major pathologic factors associated with osteoarthritis (OA). Recently, numerous reports have demonstrated the impact of sirtuin-1 (Sirt1), which is the NAD-dependent deacetylase, on human aging. It has been demonstrated that Sirt1 induces osteogenic and chondrogenic differentiation of mesenchymal stem cells. However, the role of Sirt1 in the OA chondrocytes still remains unknown. We postulated that Sirt1 regulates a hypertrophic chondrocyte lineage and degeneration of articular cartilage through the activation of osteogenic transcriptional activator Runx2 and matrix metalloproteinase (MMP)-13 in OA chondrocytes. To verify whether sirtuin-1 (Sirt1) regulates chondrocyte activity in OA, we studied expressions of Sirt1, Runx2 and production of MMP-13, and their associations in human OA chondrocytes. The expression of Sirt1 was ubiquitously observed in osteoarthritic chondrocytes; in contrast, Runx2 expressed in the osteophyte region in patients with OA and OA model mice. OA relating catabolic factor IL-1βincreased the expression of Runx2 in OA chondrocytes. OA chondrocytes, which were pretreated with Sirt1 inhibitor, inhibited the IL-1β-induced expression of Runx2 compared to the control. Since the Runx2 is a promotor of MMP-13 expression, Sirt1 inactivation may inhibit the Runx2 expression and the resultant down-regulation of MMP-13 production in chondrocytes. Our findings suggest thatSirt1 may regulate the expression of Runx2, which is the osteogenic transcription factor, and the production of MMP-13 from chondrocytes in OA. Since Sirt1 activity is known to be affected by several stresses, including inflammation and oxidative stress, as well as aging, SIRT may be involved in the development of OA. PMID:27367673

  10. The oncofetal gene survivin is re-expressed in osteoarthritis and is required for chondrocyte proliferation in vitro

    PubMed Central

    2011-01-01

    Background Regulation of cell death and cell division are key processes during chondrogenesis and in cartilage homeostasis and pathology. The oncogene survivin is considered to be critical for the coordination of mitosis and maintenance of cell viability during embryonic development and in cancer, and is not detectable in most adult differentiated tissues and cells. We analyzed survivin expression in osteoarthritic cartilage and its function in primary human chondrocytes in vitro. Methods Survivin expression was analyzed by immunoblotting and quantitative real-time PCR. The localization was visualized by immunofluorescence. Survivin functions in vitro were investigated by transfection of a specific siRNA. Results Survivin was expressed in human osteoarthritic cartilage, but was not detectable in macroscopically and microscopically unaffected cartilage of osteoarthritic knee joints. In primary human chondrocyte cultures, survivin was localized to heterogeneous subcellular compartments. Suppression of survivin resulted in inhibition of cell cycle progression and sensitization toward apoptotic stimuli in vitro. Conclusions The present study indicates a role for survivin in osteoarthritic cartilage and human chondrocytes. In vitro experiments indicated its involvement in cellular division and viability. Learning more about the functions of survivin in chondrocyte biology might further help toward understanding and modulating the complex processes of cartilage pathology and regeneration. PMID:21729321

  11. Triptolide suppresses proinflammatory cytokine-induced matrix metalloproteinase and aggrecanase-1 gene expression in chondrocytes.

    PubMed

    Liacini, Abdelhamid; Sylvester, Judith; Zafarullah, Muhammad

    2005-02-01

    A hallmark of rheumatoid- and osteoarthritis (OA) is proinflammatory cytokine-induced degeneration of cartilage collagen and aggrecan by matrix metalloproteinases (MMPs) and aggrecanases (ADAMTS). Effects of the Chinese herb, Tripterygium wilfordii Hook F (TWHF), on cartilage and its anti-arthritic mechanisms are poorly understood. This study investigated the impact of a purified derivative of TWHF, PG490 (triptolide), on cytokine-stimulated expression of the major cartilage damaging proteases, MMP-3, MMP-13, and ADAMTS4. PG490 inhibited cytokine-induced MMP-3, MMP-13 gene expression in primary human OA chondrocytes, bovine chondrocytes, SW1353 cells, and human synovial fibroblasts. Triptolide was effective at low doses and blocked the induction of MMP-13 by IL-1 in human and bovine cartilage explants. TWHF extract and PG490 also suppressed IL-1-, IL-17-, and TNF-alpha-induced expression of ADAMTS-4 in bovine chondrocytes. Thus, PG490 could protect cartilage from MMP- and aggrecanase-driven breakdown. The immunosuppressive, cartilage protective, and anti-inflammatory properties could make PG490 potentially a new therapeutic agent for arthritis. PMID:15629465

  12. Monolayer expansion induces an oxidative metabolism and ROS in chondrocytes

    SciTech Connect

    Heywood, H.K. Lee, D.A.

    2008-08-22

    This study tests the hypothesis that articular chondrocytes shift from a characteristically glycolytic to an oxidative energy metabolism during population expansion in monolayer. Bovine articular chondrocytes were cultured in monolayer under standard incubator conditions for up to 14 days. Cellular proliferation, oxygen consumption, lactate production, protein content, ROS generation and mitochondrial morphology were examined. Lactate release increased {approx}5-fold within 1 week, but this was limited to {approx}2-fold increase when normalized to cellular protein content. By contrast, per cell oxidative phosphorylation increased 98-fold in 1 week. The increase in oxidative phosphorylation was evident within 24 h, preceding cell proliferation and was associated with augmented reactive oxygen species generation. The autologous chondrocyte implantation procedure requires 14-21 days for population expansion. The alterations in metabolic phenotype we report within 7 days in vitro are thus pertinent to autologous chondrocyte implantation with significant implications for the chondrocyte functionality.

  13. Biomarkers of Chondrocyte Apoptosis and Autophagy in Osteoarthritis

    PubMed Central

    Musumeci, Giuseppe; Castrogiovanni, Paola; Trovato, Francesca Maria; Weinberg, Annelie Martina; Al-Wasiyah, Mohammad K.; Alqahtani, Mohammed H.; Mobasheri, Ali

    2015-01-01

    Cell death with morphological and molecular features of apoptosis has been detected in osteoarthritic (OA) cartilage, which suggests a key role for chondrocyte death/survival in the pathogenesis of OA. Identification of biomarkers of chondrocyte apoptosis may facilitate the development of novel therapies that may eliminate the cause or, at least, slow down the degenerative processes in OA. The aim of this review was to explore the molecular markers and signals that induce chondrocyte apoptosis in OA. A literature search was conducted in PubMed, Scopus, Web of Science and Google Scholar using the keywords chondrocyte death, apoptosis, osteoarthritis, autophagy and biomarker. Several molecules considered to be markers of chondrocyte apoptosis will be discussed in this brief review. Molecular markers and signalling pathways associated with chondroycte apoptosis may turn out to be therapeutic targets in OA and approaches aimed at neutralizing apoptosis-inducing molecules may at least delay the progression of cartilage degeneration in OA. PMID:26334269

  14. Celecoxib can suppress expression of genes associated with PGE2 pathway in chondrocytes under inflammatory conditions

    PubMed Central

    Sun, Tian-Wen; Wu, Zhi-Hong; Weng, Xi-Sheng

    2015-01-01

    This study aimed to investigate the effect of a selective cyclooxygenase-2 (COX-2) inhibitor (celecoxib) on the expression of arachidonate-associated inflammatory genes in cultured human normal chondrocytes. Normal chondrocytes were obtained from the cartilage of three different amputated patients without osteoarthritis (OA). Affymetrix Human microarray was used to assess the alterations in gene expression in three groups of cells: untreated cells (negative control group), cells treated with interleukin-1β (IL-1β) (positive control group), and cells treated with IL-1β and celecoxib. The patterns of up-regulation and down-regulation of gene expression were further validated by real-time PCR. A total of 1091 up-regulated genes and 1252 down-regulated genes were identified in the positive control group compared with the negative control group. Among them, PTGS2, ADAMTS5, PTGER2, mPTGES and PTGER4 are known to be involved in chondrocyte inflammation, while VEGFA, BCL2, TRAF1, CYR61, BMP6, DAPK1, DUSP7, IL1RN, MMP13 and TNFSF10 were reported being associated with cytokine and chemokine signaling. 189 up-regulated genes and 177 down-regulated genes were identified in the positive control group compared with intervention group. PTGS1, PTGS2, ADAMTS5, PTGER2, mPTGES and PTGER4 were among the genes down-regulated upon the treatment with celecoxib. Our results demonstrated that the OA chondrocytes are the site of active eicosanoid production. IL-1β can activate inflammation in chondrocytes and trigger the production of various proteins involved in cyclooxygenase pathway. The expression of genes corresponding to these proteins can be down-regulated by celecoxib. The findings indicate that the therapy with prostaglandin E2 (PGE2)-blocking agents may decrease the PGE2 production not only by direct inhibition of COX-2 activity, but also by down-regulating the expression of genes encoding for COX-2, microsomal prostaglandin-endoperoxide synthase 1 (mPGES-1) and prostaglandin

  15. 5-Aza-2'-deoxycytidine acts as a modulator of chondrocyte hypertrophy and maturation in chick caudal region chondrocytes in culture.

    PubMed

    Haq, Samina Hyder

    2016-06-01

    This study was carried out to explore the effect of DNA hypomethylation on chondrocytes phenotype, in particular the effect on chondrocyte hypertrophy, maturation, and apoptosis. Chondrocytes derived from caudal region of day 17 embryonic chick sterna were pretreated with hypomethylating drug 5-aza-2'-deoxycytidine for 48 hours and then maintained in the normal culture medium for up to 14 days. Histological studies showed distinct morphological changes occurred in the pretreated cultures when compared to the control cultures. The pretreated chondrocytes after 7 days in culture became bigger in size and acquired more flattened fibroblastic phenotype as well as a loss of cartilage specific extracellular matrix. Scanning electron microscopy at day 7 showed chondrocytes to have increased in cell volume and at day 14 in culture the extracellular matrix of the pretreated cultures showed regular fibrillar structure heavily embedded with matrix vesicles, which is the characteristic feature of chondrocyte hypertrophy. Transmission electron microscopic studies indicated the terminal fate of the hypertrophic cells in culture. The pretreated chondrocytes grown for 14 days in culture showed two types of cells: dark cells which had condense chromatin in dark patches and dark cytoplasm. The other light chondrocytes appeared to be heavily loaded with endoplasmic reticulum indicative of very active protein and secretory activity; their cytoplasm had large vacuoles and disintegrating cytoplasm. The biosynthetic profile showed that the pretreated cultures were actively synthesizing and secreting type X collagen and alkaline phosphatase as a major biosynthetic product. PMID:27382512

  16. 5-Aza-2'-deoxycytidine acts as a modulator of chondrocyte hypertrophy and maturation in chick caudal region chondrocytes in culture

    PubMed Central

    2016-01-01

    This study was carried out to explore the effect of DNA hypomethylation on chondrocytes phenotype, in particular the effect on chondrocyte hypertrophy, maturation, and apoptosis. Chondrocytes derived from caudal region of day 17 embryonic chick sterna were pretreated with hypomethylating drug 5-aza-2'-deoxycytidine for 48 hours and then maintained in the normal culture medium for up to 14 days. Histological studies showed distinct morphological changes occurred in the pretreated cultures when compared to the control cultures. The pretreated chondrocytes after 7 days in culture became bigger in size and acquired more flattened fibroblastic phenotype as well as a loss of cartilage specific extracellular matrix. Scanning electron microscopy at day 7 showed chondrocytes to have increased in cell volume and at day 14 in culture the extracellular matrix of the pretreated cultures showed regular fibrillar structure heavily embedded with matrix vesicles, which is the characteristic feature of chondrocyte hypertrophy. Transmission electron microscopic studies indicated the terminal fate of the hypertrophic cells in culture. The pretreated chondrocytes grown for 14 days in culture showed two types of cells: dark cells which had condense chromatin in dark patches and dark cytoplasm. The other light chondrocytes appeared to be heavily loaded with endoplasmic reticulum indicative of very active protein and secretory activity; their cytoplasm had large vacuoles and disintegrating cytoplasm. The biosynthetic profile showed that the pretreated cultures were actively synthesizing and secreting type X collagen and alkaline phosphatase as a major biosynthetic product. PMID:27382512

  17. Joint aging and chondrocyte cell death

    PubMed Central

    Grogan, Shawn P; D’Lima, Darryl D

    2010-01-01

    Articular cartilage extracellular matrix and cell function change with age and are considered to be the most important factors in the development and progression of osteoarthritis. The multifaceted nature of joint disease indicates that the contribution of cell death can be an important factor at early and late stages of osteoarthritis. Therefore, the pharmacologic inhibition of cell death is likely to be clinically valuable at any stage of the disease. In this article, we will discuss the close association between diverse changes in cartilage aging, how altered conditions influence chondrocyte death, and the implications of preventing cell loss to retard osteoarthritis progression and preserve tissue homeostasis. PMID:20671988

  18. The impact of polyphenols on chondrocyte growth and survival: a preliminary report

    PubMed Central

    Fernández-Arroyo, Salvador; Huete-Toral, Fernando; Jesús Pérez de Lara, María; de la Luz Cádiz-Gurrea, María; Legeai-Mallet, Laurence; Micol, Vicente; Segura-Carretero, Antonio; Joven, Jorge; Pintor, Jesús

    2015-01-01

    Background Imbalances in the functional binding of fibroblast growth factors (FGFs) to their receptors (FGFRs) have consequences for cell proliferation and differentiation that in chondrocytes may lead to degraded cartilage. The toxic, proinflammatory, and oxidative response of cytokines and FGFs can be mitigated by dietary polyphenols. Objective We explored the possible effects of polyphenols in the management of osteoarticular diseases using a model based on the transduction of a mutated human FGFR3 (G380R) in murine chondrocytes. This mutation is present in most cases of skeletal dysplasia and is responsible for the overexpression of FGFR3 that, in the presence of its ligand, FGF9, results in toxic effects leading to altered cellular growth. Design Different combinations of dietary polyphenols derived from plant extracts were assayed in FGFR3 (G380R) mutated murine chondrocytes, exploring cell survival, chloride efflux, extracellular matrix (ECM) generation, and grade of activation of mitogen-activated protein kinases. Results Bioactive compounds from Hibiscus sabdariffa reversed the toxic effects of FGF9 and restored normal growth, suggesting a probable translation to clinical requests in humans. Indeed, these compounds activated the intracellular chloride efflux, increased ECM generation, and stimulated cell proliferation. The inhibition of mitogen-activated protein kinase phosphorylation was interpreted as the main mechanism governing these beneficial effects. Conclusions These findings support the rationale behind the encouragement of the development of drugs that repress the overexpression of FGFRs and suggest the dietary incorporation of supplementary nutrients in the management of degraded cartilage. PMID:26445212

  19. Carnosol and Related Substances Modulate Chemokine and Cytokine Production in Macrophages and Chondrocytes.

    PubMed

    Schwager, Joseph; Richard, Nathalie; Fowler, Ann; Seifert, Nicole; Raederstorff, Daniel

    2016-01-01

    Phenolic diterpenes present in Rosmarinus officinalis and Salvia officinalis have anti-inflammatory and chemoprotective effects. We investigated the in vitro effects of carnosol (CL), carnosic acid (CA), carnosic acid-12-methylether (CAME), 20-deoxocarnosol and abieta-8,11,13-triene-11,12,20-triol (ABTT) in murine macrophages (RAW264.7 cells) and human chondrocytes. The substances concentration-dependently reduced nitric oxide (NO) and prostaglandin E₂ (PGE₂) production in LPS-stimulated macrophages (i.e., acute inflammation). They significantly blunted gene expression levels of iNOS, cytokines/interleukins (IL-1α, IL-6) and chemokines including CCL5/RANTES, CXCL10/IP-10. The substances modulated the expression of catabolic and anabolic genes in chondrosarcoma cell line SW1353 and in primary human chondrocytes that were stimulated by IL-1β (i.e., chronic inflammation In SW1353, catabolic genes like MMP-13 and ADAMTS-4 that contribute to cartilage erosion were down-regulated, while expression of anabolic genes including Col2A1 and aggrecan were shifted towards pre-pathophysiological homeostasis. CL had the strongest overall effect on inflammatory mediators, as well as on macrophage and chondrocyte gene expression. Conversely, CAME mainly affected catabolic gene expression, whereas ABTT had a more selectively altered interleukin and chemokine gene exprssion. CL inhibited the IL-1β induced nuclear translocation of NF-κBp65, suggesting that it primarily regulated via the NF-κB signalling pathway. Collectively, CL had the strongest effects on inflammatory mediators and chondrocyte gene expression. The data show that the phenolic diterpenes altered activity pattern of genes that regulate acute and chronic inflammatory processes. Since the substances affected catabolic and anabolic gene expression in cartilage cells in vitro, they may beneficially act on the aetiology of osteoarthritis. PMID:27070563

  20. The Circadian Clock in Murine Chondrocytes Regulates Genes Controlling Key Aspects of Cartilage Homeostasis

    PubMed Central

    Gossan, Nicole; Zeef, Leo; Hensman, James; Hughes, Alun; Bateman, John F; Rowley, Lynn; Little, Christopher B; Piggins, Hugh D; Rattray, Magnus; Boot-Handford, Raymond P; Meng, Qing-Jun

    2013-01-01

    ObjectiveTo characterize the circadian clock in murine cartilage tissue and identify tissue-specific clock target genes, and to investigate whether the circadian clock changes during aging or during cartilage degeneration using an experimental mouse model of osteoarthritis (OA). MethodsCartilage explants were obtained from aged and young adult mice after transduction with the circadian clock fusion protein reporter PER2::luc, and real-time bioluminescence recordings were used to characterize the properties of the clock. Time-series microarrays were performed on mouse cartilage tissue to identify genes expressed in a circadian manner. Rhythmic genes were confirmed by quantitative reverse transcription–polymerase chain reaction using mouse tissue, primary chondrocytes, and a human chondrocyte cell line. Experimental OA was induced in mice by destabilization of the medial meniscus (DMM), and articular cartilage samples were microdissected and subjected to microarray analysis. ResultsMouse cartilage tissue and a human chondrocyte cell line were found to contain intrinsic molecular circadian clocks. The cartilage clock could be reset by temperature signals, while the circadian period was temperature compensated. PER2::luc bioluminescence demonstrated that circadian oscillations were significantly lower in amplitude in cartilage from aged mice. Time-series microarray analyses of the mouse tissue identified the first circadian transcriptome in cartilage, revealing that 615 genes (∼3.9% of the expressed genes) displayed a circadian pattern of expression. This included genes involved in cartilage homeostasis and survival, as well as genes with potential importance in the pathogenesis of OA. Several clock genes were disrupted in the early stages of cartilage degeneration in the DMM mouse model of OA. ConclusionThese results reveal an autonomous circadian clock in chondrocytes that can be implicated in key aspects of cartilage biology and pathology. Consequently

  1. Effect of hyaluronic acid on chondrocyte apoptosis

    PubMed Central

    Barreto, Ronald Bispo; Sadigursky, David; de Rezende, Marcia Uchoa; Hernandez, Arnaldo José

    2015-01-01

    OBJECTIVE: To determine the percentage of apoptotic cells in a contusion model of osteoarthritis (OA) and to assess whether intra-articular injection of high doses of hyaluronic acid (HA) immediately after trauma reduces chondrocyte apoptosis. METHODS: Forty knees from adult rabbits were impacted thrice with a 1 kg block released through a 1 meter tall cylinder (29.4 Joules). Subsequently, 2 mL of HA was injected in one knee and 2 mL saline in the contra-lateral knee. Medication were administered twice a week for 30 days, when animals were sacrificed. Specimens were prepared for optical microscopy exam and terminal deoxynucleotidyl transferase end labeling assay (TUNEL). RESULTS: The apoptosis rate in the contusion model was 68.01% (± 19.73%), a higher rate than previously described. HA significantly reduced the rate of apoptosis to 53.52% (± 18.09) (p <0.001). CONCLUSION: Intra-articular HA administration started immediately after trauma reduces impact-induced chondrocyte apoptosis rates in rabbits. Level of Evidence I, Experimental Study. PMID:27069407

  2. Fibroblast growth factor is an inhibitor of chondrocyte terminal differentiation

    SciTech Connect

    Kato, Y.; Iwamoto, M. )

    1990-04-05

    The effects of basic fibroblast growth factor (bFGF) on terminal differentiation of chondrocytes and cartilage-matrix calcification were investigated. Rabbit growth-plate chondrocytes maintained as a pelleted mass in a centrifuge tube produced an abundant proteoglycan matrix during the matrix-maturation stage, yielding a cartilage-like tissue. Thereafter, they terminally differentiated to hypertrophic chondrocytes which produced high levels of alkaline phosphatase. These cells induced extensive calcification of the matrix in the absence of additional phosphate. Addition of bFGF to the chondrocyte cultures abolished the increases in alkaline phosphatase activity, {sup 45}Ca deposition, and the calcium content. These effects were dose-dependent, reversible, and observed in the presence of cytosine arabinoside, an inhibitor of DNA synthesis. The inhibitory effects could be observed only when chondrocytes were exposed to bFGF in a transition period between the matrix-maturation and hypertrophic stages. As chondrocytes differentiated to hypertrophic cells, bFGF became less effective in inhibiting the expression of the mineralization-related phenotypes. The present study also shows that although the rate of ({sup 35}S)sulfate incorporation into large, chondroitin sulfate proteoglycan in the cell-matrix fraction is very high during the matrix-maturation stage, it abruptly decreases by 90% after terminal differentiation. Furthermore, the terminal differentiation-associated decrease in proteoglycan synthesis was delayed by bFGF. These results provide evidence that bFGF inhibits terminal differentiation of chondrocytes and calcification.

  3. A Qualitative Model of the Differentiation Network in Chondrocyte Maturation: A Holistic View of Chondrocyte Hypertrophy.

    PubMed

    Kerkhofs, Johan; Leijten, Jeroen; Bolander, Johanna; Luyten, Frank P; Post, Janine N; Geris, Liesbet

    2016-01-01

    Differentiation of chondrocytes towards hypertrophy is a natural process whose control is essential in endochondral bone formation. It is additionally thought to play a role in several pathophysiological processes, with osteoarthritis being a prominent example. We perform a dynamic analysis of a qualitative mathematical model of the regulatory network that directs this phenotypic switch to investigate the influence of the individual factors holistically. To estimate the stability of a SOX9 positive state (associated with resting/proliferation chondrocytes) versus a RUNX2 positive one (associated with hypertrophy) we employ two measures. The robustness of the state in canalisation (size of the attractor basin) is assessed by a Monte Carlo analysis and the sensitivity to perturbations is assessed by a perturbational analysis of the attractor. Through qualitative predictions, these measures allow for an in silico screening of the effect of the modelled factors on chondrocyte maintenance and hypertrophy. We show how discrepancies between experimental data and the model's results can be resolved by evaluating the dynamic plausibility of alternative network topologies. The findings are further supported by a literature study of proposed therapeutic targets in the case of osteoarthritis. PMID:27579819

  4. Production of three-dimensional tissue-engineered cartilage through mutual fusion of chondrocyte pellets.

    PubMed

    Hoshi, K; Fujihara, Y; Mori, Y; Asawa, Y; Kanazawa, S; Nishizawa, S; Misawa, M; Numano, T; Inoue, H; Sakamoto, T; Watanabe, M; Komura, M; Takato, T

    2016-09-01

    In this study, the mutual fusion of chondrocyte pellets was promoted in order to produce large-sized tissue-engineered cartilage with a three-dimensional (3D) shape. Five pellets of human auricular chondrocytes were first prepared, which were then incubated in an agarose mold. After 3 weeks of culture in matrix production-promoting medium under 5.78g/cm(2) compression, the tissue-engineered cartilage showed a sufficient mechanical strength. To confirm the usefulness of these methods, a transplantation experiment was performed using beagles. Tissue-engineered cartilage prepared with 50 pellets of beagle chondrocytes was transplanted subcutaneously into the cell-donor dog for 2 months. The tissue-engineered cartilage of the beagles maintained a rod-like shape, even after harvest. Histology showed fair cartilage regeneration. Furthermore, 20 pellets were made and placed on a beta-tricalcium phosphate prism, and this was then incubated within the agarose mold for 3 weeks. The construct was transplanted into a bone/cartilage defect in the cell-donor beagle. After 2 months, bone and cartilage regeneration was identified on micro-computed tomography and magnetic resonance imaging. This approach involving the fusion of small pellets into a large structure enabled the production of 3D tissue-engineered cartilage that was close to physiological cartilage tissue in property, without conventional polyper scaffolds. PMID:27173826

  5. Rutin protects rat articular chondrocytes against oxidative stress induced by hydrogen peroxide through SIRT1 activation.

    PubMed

    Na, Ji-Young; Song, Kibbeum; Kim, Sokho; Kwon, Jungkee

    2016-05-13

    The progressive degeneration and ossification of articular chondrocytes are main symptoms in the pathogenesis of osteoarthritis (OA). Several flavonoids may provide an adjunctive alternative for the management of moderate OA in humans. Rutin, a natural flavone derivative (quercetin-3-rhamnosylglucoside), is well known for its potent anti-inflammatory and anti-oxidant properties against oxidative stress. However, the protective function of rutin related to OA, which is characterized by deterioration of articular cartilage, remains unclear. The present study investigated the protective effects of rutin, an activator of silent information regulator 1 (SIRT1), involved in the inhibition of NF-κB/MAPK signaling pathway in hydrogen peroxide (H2O2)-induced oxidative stress in rat chondrocytes. SIRT1 activation by rutin attenuated levels of inflammatory cytokines and NF-κB/MAPK signaling, whereas the inhibition of SIRT1 by sirtinol counteracted the beneficial effects of rutin in H2O2-treated chondrocytes. The findings of these studies suggested the potential involvement of SIRT1 in the pathogenesis of OA, and indicated that rutin is a possible therapeutic option for OA. PMID:27086847

  6. Increased Production of Clusterin in Biopsies of Repair Tissue following Autologous Chondrocyte Implantation

    PubMed Central

    Malda, Jos; Richardson, James B.; Roberts, Sally

    2013-01-01

    Objective. To characterize the immunolocalization of clusterin in the repair cartilage of patients having undergone autologous chondrocyte implantation (ACI) and evaluate correlation to clinical outcome. Design. Full-depth core biopsies of repair tissue were obtained from 38 patients who had undergone ACI at an average of 18 ± 13 months previously (range 8-67 months). The biopsies were snap frozen, cryosectioned, and clusterin production immunolocalized using a specific monoclonal clusterin antibody and compared with normal and osteoarthritic cartilage. Clinical outcome was assessed from patients preoperatively, at the time of biopsy, and annually postoperatively. Results. Intensity of immunostaining for clusterin decreased with age in healthy cartilage tissue. Clusterin was detected to a variable degree in 37 of the 38 ACI cartilage biopsies, in single and clustered chondrocytes, in the pericellular capsule and the cartilage extracellular matrix, as well as the osteocytes and osteoid within the bone. Chondrocytes in hyaline repair tissue were significantly more immunopositive than those in fibrocartilage repair tissue. Clinical outcome improved significantly post-ACI, but did not correlate with the presence of clusterin in the repair tissue. Conclusions. These results demonstrate the presence of clusterin in actively repairing human cartilage and indicate a different distribution of clusterin in this tissue compared to normal cartilage. Variability in clusterin staining in the repair tissue could indicate different states of chondrogenic differentiation. The clinical significance of clusterin within repair tissue is difficult to assess, although the ideal functioning repair tissue morphology should resemble that of healthy adult cartilage. PMID:26069669

  7. Chondrocyte-Specific Pathology During Skeletal Growth and Therapeutics in a Murine Model of Pseudoachondroplasia

    PubMed Central

    Posey, Karen L; Coustry, Francoise; Veerisetty, Alka C; Liu, Peiman; Alcorn, Joseph L; Hecht, Jacqueline T

    2014-01-01

    Mutations in the gene encoding cartilage oligomeric matrix protein (COMP) cause pseudoachondroplasia (PSACH), a severe dwarfing condition. Pain, a significant complication, has generally been attributed to joint abnormalities and erosion and early onset osteoarthritis. Previously, we found that the Inflammatory-related transcripts were elevated In growth plate and articular cartilages, Indicating that Inflammation plays an important role in the chondrocyte disease pathology and may contribute to the overall pain sequelae. Here, we describe the effects of D469-delCOMP expression on the skeleton and growth plate chondrocytes with the aim to define a treatment window and thereby reduce pain. Consistent with the human PSACH phenotype, skeletal development of D469del-COMP mice was normal and similar to controls at birth. By postnatal day 7 (P7), the D469del-COMP skeleton, limbs, skull and snout were reduced and this reduction was progressive during postnatal growth, resulting in a short-limbed dwarfed mouse. Modulation of prenatal and postnatal expression of D469del-COMP showed minimal retention/cell death at P7 with some retention/cell death by P14, suggesting that earlier treatment intervention at the time of PSACH diagnosis may produce optimal results. Important and novel findings were an increase In Inflammatory proteins generally starting at P21 and that exercise exacerbates Inflammation. These observations suggest that pain in PSACH may be related to an intrinsic inflammatory process that can be treated symptomatically and is not related to early joint erosion. We also show that genetic ablation of CHOP dampens the inflammatory response. observed in mice expressing D469del-COMP. Toward identifying potential treatments, drugs known to decrease cellular stress (lithlum, phenyl butyric add, and valproate) were assessed. Interestingly, all diminished the chondrocyte pathology but had untoward outcomes on mouse growth, development, and longevity. Collectively, these

  8. Biomechanical signals exert sustained attenuation of proinflammatory gene induction in articular chondrocytes

    PubMed Central

    Madhavan, S.; Anghelina, M.; Rath-Deschner, B.; Wypasek, E.; John, A.; Deschner, J.; Piesco, N.; Agarwal, S.

    2016-01-01

    Objectives Physical therapies are commonly used for limiting joint inflammation. To gain insight into their mechanisms of actions for optimal usage, we examined persistence of mechanical signals generated by cyclic tensile strain (CTS) in chondrocytes, in vitro. We hypothesized that mechanical signals induce anti-inflammatory and anabolic responses that are sustained over extended periods. Methods Articular chondrocytes obtained from rats were subjected to CTS for various time intervals followed by a period of rest, in the presence of interleukin-1β (IL-1β). The induction for cyclooxygenase (COX-2), inducible nitric oxide synthase (iNOS), matrix metalloproteinase (MMP)-9, MMP-13 and aggrecan was analyzed by real-time polymerase chain reaction (PCR), Western blot analysis and immunofluorescence. Results Exposure of chondrocytes to constant CTS (3% CTS at 0.25 Hz) for 4e24 h blocked more than 90% (P < 0.05) of the IL-1β-induced transcriptional activation of proinflammatory genes, like iNOS, COX-2, MMP-9 and MMP-13, and abrogated inhibition of aggrecan synthesis. CTS exposure for 4, 8, 12, 16, or 20 h followed by a rest for 20, 16, 12, 8 or 4 h, respectively, revealed that 8 h of CTS optimally blocked (P < 0.05) IL-1β-induced proinflammatory gene induction for ensuing 16 h. However, CTS for 8 h was not sufficient to inhibit iNOS expression for ensuing 28 or 40 h. Conclusions Data suggest that constant application of CTS blocks IL-1β-induced proinflammatory genes at transcriptional level. The signals generated by CTS are sustained after its removal, and their persistence depends upon the length of CTS exposure. Furthermore, the sustained effects of mechanical signals are also reflected in their ability to induce aggrecan synthesis. These findings, once extrapolated to human chondrocytes, may provide insight in obtaining optimal sustained effects of physical therapies in the management of arthritic joints. PMID:16731008

  9. Catabolic effects of muramyl dipeptide on rabbit chondrocytes

    SciTech Connect

    Ikebe, T.; Iribe, H.; Hirata, M.; Yanaga, F.; Koga, T. )

    1990-12-01

    Muramyl dipeptide, an essential structure for the diverse biologic activities of bacterial cell wall peptidoglycan, inhibited the synthesis of glycosaminoglycan/proteoglycan in cultured rabbit costal chondrocytes in a dose-dependent manner. Muramyl dipeptide, as well as lipopolysaccharide and interleukin-1 alpha, also enhanced the release of 35S-sulfate-prelabeled glycosaminoglycan/proteoglycan from the cell layer, which seems to reflect, at least partially, the increasing degradation of glycosaminoglycan/proteoglycan. Five synthetic analogs of muramyl dipeptide known to be adjuvant active or adjuvant inactive were tested for their potential to inhibit synthesis of glycosaminoglycan/proteoglycan and to enhance the release of glycosaminoglycan/proteoglycan in chondrocytes. The structural dependence of these synthetic analogs on chondrocytes was found to parallel that of immunoadjuvant activity. These results suggest that muramyl dipeptide is a potent mediator of catabolism in chondrocytes.

  10. Resveratrol Interferes with IL1-β-Induced Pro-Inflammatory Paracrine Interaction between Primary Chondrocytes and Macrophages.

    PubMed

    Limagne, Emeric; Lançon, Allan; Delmas, Dominique; Cherkaoui-Malki, Mustapha; Latruffe, Norbert

    2016-01-01

    State of the art. Osteoarthritis (OA) is a chronic articular disease characterized by cartilage degradation and osteophyte formation. OA physiopathology is multifactorial and involves mechanical and hereditary factors. So far, there is neither preventive medicine to delay cartilage breakdown nor curative treatment. Objectives. To investigate pro-inflammatory paracrine interactions between human primary chondrocytes and macrophages following interleukin-1-β (IL-1β) treatment; to evaluate the molecular mechanism responsible for the inhibitory effect of resveratrol. Results. The activation of NF-κB in chondrocytes by IL-1β induced IL-6 secretion. The latter will then activate STAT3 protein in macrophages. Moreover, STAT3 was able to positively regulate IL-6 secretion, as confirmed by the doubling level of IL-6 in the coculture compared to macrophage monoculture. These experiments confirm the usefulness of the coculture model in the inflammatory arthritis-linked process as a closer biological situation to the synovial joint than separated chondrocytes and macrophages. Il also demonstrated the presence of an inflammatory amplification loop induced by IL-1β. Resveratrol showed a strong inhibitory effect on the pro-inflammatory marker secretion. The decrease of IL-6 secretion is dependent on the NFκB inhibition in the chondrocytes. Such reduction of the IL-6 level can limit STAT3 activation in the macrophages, leading to the interruption of the inflammatory amplification loop. Conclusion. These results increase our understanding of the anti-inflammatory actions of resveratrol and open new potential approaches to prevent and treat osteoarthritis. PMID:27187448