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Sample records for stimulates chondrocyte biosynthesis

  1. Sphingosine-1-phosphate stimulates rat primary chondrocyte proliferation

    International Nuclear Information System (INIS)

    Kim, Mi-Kyoung; Lee, Ha Young; Kwak, Jong-Young; Park, Joo-In; Yun, Jeanho; Bae, Yoe-Sik

    2006-01-01

    Rat primary chondrocytes express the sphingosine-1-phosphate (S1P) receptor, S1P 2 , S1P 3 , S1P 4 , but not S1P 1 . When chondrocytes were stimulated with S1P or phytosphingosine-1-phosphate (PhS1P, an S1P 1 - and S1P 4 -selective agonist), phospholipase C-mediated cytosolic calcium increase was dramatically induced. S1P and PhS1P also stimulated two kinds of mitogen-activated protein kinases, extracellular signal-regulated kinase (ERK) and p38 kinase in chondrocytes. In terms of the two phospholipids-mediated functional modulation of chondrocytes, S1P and PhS1P stimulated cellular proliferation. The two phospholipids-induced chondrocyte proliferations were almost completely blocked by PD98059 but not by SB203580, suggesting that ERK but not p38 kinase is essentially required for the proliferation. Pertussis toxin almost completely inhibited the two phospholipids-induced cellular proliferation and ERK activation, indicating the crucial role of G i protein. This study demonstrates the physiological role of two important phospholipids (S1P and PhS1P) on the modulation of rat primary chondrocyte proliferation, and the crucial role played by ERK in the process

  2. High throughput generated micro-aggregates of chondrocytes stimulate cartilage formation in vitro and in vivo

    NARCIS (Netherlands)

    Moreira Teixeira, Liliana; Leijten, Jeroen Christianus Hermanus; Sobral, J.; Jin, R.; van Apeldoorn, Aart A.; Feijen, Jan; van Blitterswijk, Clemens; Dijkstra, Pieter J.; Karperien, Hermanus Bernardus Johannes

    2012-01-01

    Cell-based cartilage repair strategies such as matrix-induced autologous chondrocyte implantation (MACI) could be improved by enhancing cell performance. We hypothesised that micro-aggregates of chondrocytes generated in high-throughput prior to implantation in a defect could stimulate cartilaginous

  3. High throughput generated micro-aggregates of chondrocytes stimulate cartilage formation in vitro and in vivo

    Directory of Open Access Journals (Sweden)

    LS Moreira Teixeira

    2012-06-01

    Full Text Available Cell-based cartilage repair strategies such as matrix-induced autologous chondrocyte implantation (MACI could be improved by enhancing cell performance. We hypothesised that micro-aggregates of chondrocytes generated in high-throughput prior to implantation in a defect could stimulate cartilaginous matrix deposition and remodelling. To address this issue, we designed a micro-mould to enable controlled high-throughput formation of micro-aggregates. Morphology, stability, gene expression profiles and chondrogenic potential of micro-aggregates of human and bovine chondrocytes were evaluated and compared to single-cells cultured in micro-wells and in 3D after encapsulation in Dextran-Tyramine (Dex-TA hydrogels in vitro and in vivo. We successfully formed micro-aggregates of human and bovine chondrocytes with highly controlled size, stability and viability within 24 hours. Micro-aggregates of 100 cells presented a superior balance in Collagen type I and Collagen type II gene expression over single cells and micro-aggregates of 50 and 200 cells. Matrix metalloproteinases 1, 9 and 13 mRNA levels were decreased in micro-aggregates compared to single-cells. Histological and biochemical analysis demonstrated enhanced matrix deposition in constructs seeded with micro-aggregates cultured in vitro and in vivo, compared to single-cell seeded constructs. Whole genome microarray analysis and single gene expression profiles using human chondrocytes confirmed increased expression of cartilage-related genes when chondrocytes were cultured in micro-aggregates. In conclusion, we succeeded in controlled high-throughput formation of micro-aggregates of chondrocytes. Compared to single cell-seeded constructs, seeding of constructs with micro-aggregates greatly improved neo-cartilage formation. Therefore, micro-aggregation prior to chondrocyte implantation in current MACI procedures, may effectively accelerate hyaline cartilage formation.

  4. High throughput generated micro-aggregates of chondrocytes stimulate cartilage formation in vitro and in vivo.

    Science.gov (United States)

    Moreira Teixeira, L S; Leijten, J C H; Sobral, J; Jin, R; van Apeldoorn, A A; Feijen, J; van Blitterswijk, C; Dijkstra, P J; Karperien, M

    2012-06-05

    Cell-based cartilage repair strategies such as matrix-induced autologous chondrocyte implantation (MACI) could be improved by enhancing cell performance. We hypothesised that micro-aggregates of chondrocytes generated in high-throughput prior to implantation in a defect could stimulate cartilaginous matrix deposition and remodelling. To address this issue, we designed a micro-mould to enable controlled high-throughput formation of micro-aggregates. Morphology, stability, gene expression profiles and chondrogenic potential of micro-aggregates of human and bovine chondrocytes were evaluated and compared to single-cells cultured in micro-wells and in 3D after encapsulation in Dextran-Tyramine (Dex-TA) hydrogels in vitro and in vivo. We successfully formed micro-aggregates of human and bovine chondrocytes with highly controlled size, stability and viability within 24 hours. Micro-aggregates of 100 cells presented a superior balance in Collagen type I and Collagen type II gene expression over single cells and micro-aggregates of 50 and 200 cells. Matrix metalloproteinases 1, 9 and 13 mRNA levels were decreased in micro-aggregates compared to single-cells. Histological and biochemical analysis demonstrated enhanced matrix deposition in constructs seeded with micro-aggregates cultured in vitro and in vivo, compared to single-cell seeded constructs. Whole genome microarray analysis and single gene expression profiles using human chondrocytes confirmed increased expression of cartilage-related genes when chondrocytes were cultured in micro-aggregates. In conclusion, we succeeded in controlled high-throughput formation of micro-aggregates of chondrocytes. Compared to single cell-seeded constructs, seeding of constructs with micro-aggregates greatly improved neo-cartilage formation. Therefore, micro-aggregation prior to chondrocyte implantation in current MACI procedures, may effectively accelerate hyaline cartilage formation.

  5. Stimulation of artemisinin biosynthesis in Artemisia annua hairy ...

    African Journals Online (AJOL)

    , the OGA-induced reactive oxygen species (ROS) were involved in stimulating the artemisinin biosynthesis in the hairy roots. This is the first report on the stimulation of artemisinin production in hairy roots by an oligogalacturonide elicitor.

  6. Dexamethasone stimulates expression of C-type Natriuretic Peptide in chondrocytes

    Directory of Open Access Journals (Sweden)

    Beier Frank

    2006-11-01

    Full Text Available Abstract Background Growth of endochondral bones is regulated through the activity of cartilaginous growth plates. Disruption of the physiological patterns of chondrocyte proliferation and differentiation – such as in endocrine disorders or in many different genetic diseases (e.g. chondrodysplasias – generally results in dwarfism and skeletal defects. For example, glucocorticoid administration in children inhibits endochondral bone growth, but the molecular targets of these hormones in chondrocytes remain largely unknown. In contrast, recent studies have shown that C-type Natriuretic Peptide (CNP is an important anabolic regulator of cartilage growth, and loss-of-function mutations in the human CNP receptor gene cause dwarfism. We asked whether glucocorticoids could exert their activities by interfering with the expression of CNP or its downstream signaling components. Methods Primary mouse chondrocytes in monolayer where incubated with the synthetic glucocorticoid Dexamethasone (DEX for 12 to 72 hours. Cell numbers were determined by counting, and real-time PCR was performed to examine regulation of genes in the CNP signaling pathway by DEX. Results We show that DEX does influence expression of key genes in the CNP pathway. Most importantly, DEX significantly increases RNA expression of the gene encoding CNP itself (Nppc. In addition, DEX stimulates expression of Prkg2 (encoding cGMP-dependent protein kinase II and Npr3 (natriuretic peptide decoy receptor genes. Conversely, DEX was found to down-regulate the expression of the gene encoding its receptor, Nr3c1 (glucocorticoid receptor, as well as the Npr2 gene (encoding the CNP receptor. Conclusion Our data suggest that the growth-suppressive activities of DEX are not due to blockade of CNP signaling. This study reveals a novel, unanticipated relationship between glucocorticoid and CNP signaling and provides the first evidence that CNP expression in chondrocytes is regulated by endocrine

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

    Energy Technology Data Exchange (ETDEWEB)

    Ryu, Jae-Sung; Jung, Yeon-Hwa; Cho, Mi-Young; Yeo, Jee Eun; Choi, Yun-Jin; Kim, Yong Il; Koh, Yong-Gon, E-mail: yonseranglab@daum.net

    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.

  8. Increase in production of matrix metalloproteinase 13 by human articular chondrocytes due to stimulation with S100A4: Role of the receptor for advanced glycation end products.

    Science.gov (United States)

    Yammani, Raghunatha R; Carlson, Cathy S; Bresnick, Anne R; Loeser, Richard F

    2006-09-01

    S100 proteins have been implicated in various inflammatory conditions, including arthritis. The aims of this study were to determine whether chondrocytes produce S100A4 and whether S100A4 can stimulate the production of matrix metalloproteinase 13 (MMP-13) by articular chondrocytes via receptor for advanced glycation end products (RAGE)-mediated signaling. The expression of chondrocyte S100A4 was analyzed by immunohistochemistry using normal and osteoarthritic (OA) cartilage and by immunoblotting of chondrocyte cell lysates. RAGE signaling was examined by stimulating chondrocytes with S100A4 and monitoring for the activation of MAP kinases and NF-kappaB. Production of MMP-13 was determined in the conditioned medium. A pulldown assay using biotin-labeled S100A4 was used to demonstrate binding to RAGE. S100A4 expression was detected in human articular chondrocytes by immunoblotting and appeared to increase in the cell lysates from OA tissue. Marked positive immunostaining for S100A4 was also noted in sections of human cartilage with changes due to OA. Stimulation of chondrocytes with S100A4 increased the phosphorylation of Pyk-2, MAP kinases, and activated NF-kappaB, followed by increased production of MMP-13 in the conditioned medium. This signaling was inhibited in cells pretreated with soluble RAGE, advanced glycation end product-bovine serum albumin, or the antioxidant Mn(III)tetrakis (4-benzoic acid) porphyrin, or by overexpression of a dominant-negative RAGE construct. A pulldown assay showed that S100A4 binds to RAGE in chondrocytes. This is the first study to demonstrate that S100A4 binds to RAGE and stimulates a RAGE-mediated signaling cascade, leading to increased production of MMP-13. Since both S100A4 and RAGE are up-regulated in OA cartilage, this signaling pathway could contribute to cartilage degradation in OA.

  9. Endogenous versus exogenous growth factor regulation of articular chondrocytes.

    Science.gov (United States)

    Shi, Shuiliang; Chan, Albert G; Mercer, Scott; Eckert, George J; Trippel, Stephen B

    2014-01-01

    Anabolic growth factors that regulate the function of articular chondrocytes are candidates for articular cartilage repair. Such factors may be delivered by pharmacotherapy in the form of exogenous proteins, or by gene therapy as endogenous proteins. It is unknown whether delivery method influences growth factor effectiveness in regulating articular chondrocyte reparative functions. We treated adult bovine articular chondrocytes with exogenous recombinant insulin-like growth factor-I (IGF-I) and transforming growth factor-beta1 (TGF-β1), or with the genes encoding these growth factors for endogenous production. Treatment effects were measured as change in chondrocyte DNA content, glycosaminoglycan production, and aggrecan gene expression. We found that IGF-I stimulated chondrocyte biosynthesis similarly when delivered by either exogenous or endogenous means. In contrast, exogenous TGF-β1 stimulated these reparative functions, while endogenous TGF-β1 had little effect. Endogenous TGF-β1 became more bioactive following activation of the transgene protein product. These data indicate that effective mechanisms of growth factor delivery for articular cartilage repair may differ for different growth factors. In the case of IGF-I, gene therapy or protein therapy appear to be viable options. In contrast, TGF-β1 gene therapy may be constrained by a limited ability of chondrocytes to convert latent complexes to an active form. Published 2013 by Wiley Periodicals, Inc. on behalf of the Orthopaedic Research Society. This article is a U.S. Government work and is in the public domain in the USA.

  10. ADAM12-S stimulates bone growth in transgenic mice by modulating chondrocyte proliferation and maturation

    DEFF Research Database (Denmark)

    Kveiborg, Marie; Albrechtsen, Reidar; Rudkjaer, Lise

    2006-01-01

    ADAM12-S transgenic mice exhibit a pronounced increase in the length of bones, such as femur, tibia, and vertebrae. The effect of ADAM12-S on longitudinal bone growth involves the modulation of chondrocyte proliferation and maturation, likely through proteolytic activities and altered cell......: Transgenic mice expressing the secreted form of human ADAM12, ADAM12-S, or a truncated metalloprotease-deficient form of ADAM12-S in the circulation were used to study the effects of ADAM12 on the skeleton. In addition, murine chondrocyte cultures were used to study the effect of ADAM12-S on cell......-extracellular matrix interactions. RESULTS: ADAM12-S transgenic mice exhibit increased longitudinal bone growth. The increased bone length is progressive and age dependent, with a maximum increase of 17% seen in the femur from 6-month-old transgenic mice. The effect is gene dose dependent, being more pronounced...

  11. Stimulation of reserpine biosynthesis in the callus of Rauvolfia ...

    African Journals Online (AJOL)

    So enhancing this alkaloid in the already available system is a beneficial approach. Tryptophan is the starting material in the biosynthesis of reserpine. Callus was induced from leaf explants of Rauvolfia tetraphylla L. on MS medium supplemented with the combination of 9 μM 2,4-D and 25, 50, 75 and 100 mg/l tryptophan.

  12. Influence of extremely low frequency, low energy electromagnetic fields and combined mechanical stimulation on chondrocytes in 3-D constructs for cartilage tissue engineering.

    Science.gov (United States)

    Hilz, Florian M; Ahrens, Philipp; Grad, Sibylle; Stoddart, Martin J; Dahmani, Chiheb; Wilken, Frauke L; Sauerschnig, Martin; Niemeyer, Philipp; Zwingmann, Jörn; Burgkart, Rainer; von Eisenhart-Rothe, Rüdiger; Südkamp, Norbert P; Weyh, Thomas; Imhoff, Andreas B; Alini, Mauro; Salzmann, Gian M

    2014-02-01

    Articular cartilage, once damaged, has very low regenerative potential. Various experimental approaches have been conducted to enhance chondrogenesis and cartilage maturation. Among those, non-invasive electromagnetic fields have shown their beneficial influence for cartilage regeneration and are widely used for the treatment of non-unions, fractures, avascular necrosis and osteoarthritis. One very well accepted way to promote cartilage maturation is physical stimulation through bioreactors. The aim of this study was the investigation of combined mechanical and electromagnetic stress affecting cartilage cells in vitro. Primary articular chondrocytes from bovine fetlock joints were seeded into three-dimensional (3-D) polyurethane scaffolds and distributed into seven stimulated experimental groups. They either underwent mechanical or electromagnetic stimulation (sinusoidal electromagnetic field of 1 mT, 2 mT, or 3 mT; 60 Hz) or both within a joint-specific bioreactor and a coil system. The scaffold-cell constructs were analyzed for glycosaminoglycan (GAG) and DNA content, histology, and gene expression of collagen-1, collagen-2, aggrecan, cartilage oligomeric matrix protein (COMP), Sox9, proteoglycan-4 (PRG-4), and matrix metalloproteinases (MMP-3 and -13). There were statistically significant differences in GAG/DNA content between the stimulated versus the control group with highest levels in the combined stimulation group. Gene expression was significantly higher for combined stimulation groups versus static control for collagen 2/collagen 1 ratio and lower for MMP-13. Amongst other genes, a more chondrogenic phenotype was noticed in expression patterns for the stimulated groups. To conclude, there is an effect of electromagnetic and mechanical stimulation on chondrocytes seeded in a 3-D scaffold, resulting in improved extracellular matrix production. © 2013 Wiley Periodicals, Inc.

  13. Linkage of chondroitin-sulfate to type I collagen scaffolds stimulates the bioactivity of seeded chondrocytes in vitro.

    NARCIS (Netherlands)

    Susante, J.L.C. van; Pieper, J.S.; Buma, P.; Kuppevelt, A.H.M.S.M. van; Beuningen, H.M. van; Kraan, P.M. van der; Veerkamp, J.H.; Berg, W.B. van den; Veth, R.P.H.

    2001-01-01

    An increasing amount of interest is focused on the potential use of tissue-engineered articular cartilage implants, for repair of defects in the joint surface. In this perspective, various biodegradable scaffolds have been evaluated as a vehicle to deliver chondrocytes into a cartilage defect. This

  14. Biosynthesis of germination stimulants of parasitic weeds Striga and Orobanche

    NARCIS (Netherlands)

    Sun, Z.

    2008-01-01

    My research focused on the biosynthetic origin of germination stimulants of the root parasitic plants, Striga spp. and Orobanche spp., which have an increasing impact on cereal and other economically important crops in many regions of the world. The traditional control methods are not sufficient and

  15. TGF-β1, GDF-5, and BMP-2 stimulation induces chondrogenesis in expanded human articular chondrocytes and marrow-derived stromal cells.

    Science.gov (United States)

    Murphy, Meghan K; Huey, Daniel J; Hu, Jerry C; Athanasiou, Kyriacos A

    2015-03-01

    Replacement of degenerated cartilage with cell-based cartilage products may offer a long-term solution to halt arthritis' degenerative progression. Chondrocytes are frequently used in cell-based FDA-approved cartilage products; yet human marrow-derived stromal cells (hMSCs) show significant translational potential, reducing donor site morbidity and maintaining their undifferentiated phenotype with expansion. This study sought to investigate the effects of transforming growth factor β1 (TGF-β1), growth/differentiation factor 5 (GDF-5), and bone morphogenetic protein 2 (BMP-2) during postexpansion chondrogenesis in human articular chondrocytes (hACs) and to compare chondrogenesis in passaged hACs with that of passaged hMSCs. Through serial expansion, chondrocytes dedifferentiated, decreasing expression of chondrogenic genes while increasing expression of fibroblastic genes. However, following expansion, 10 ng/mL TGF-β1, 100 ng/mL GDF-5, or 100 ng/mL BMP-2 supplementation during three-dimensional aggregate culture each upregulated one or more markers of chondrogenic gene expression in both hACs and hMSCs. Additionally, in both cell types, the combination of TGF-β1, GDF-5, and BMP-2 induced the greatest upregulation of chondrogenic genes, that is, Col2A1, Col2A1/Col1A1 ratio, SOX9, and ACAN, and synthesis of cartilage-specific matrix, that is, glycosaminoglycans (GAGs) and ratio of collagen II/I. Finally, TGF-β1, GDF-5, and BMP-2 stimulation yielded mechanically robust cartilage rich in collagen II and GAGs in both cell types, following 4 weeks maturation. This study illustrates notable success in using the self-assembling method to generate robust, scaffold-free neocartilage constructs using expanded hACs and hMSCs. © 2014 AlphaMed Press.

  16. ROCK inhibitor prevents the dedifferentiation of human articular chondrocytes

    International Nuclear Information System (INIS)

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

    2012-01-01

    Highlights: ► ROCK inhibitor stimulates chondrogenic gene expression of articular chondrocytes. ► ROCK inhibitor prevents the dedifferentiation of monolayer-cultured chondrocytes. ► ROCK inhibitor enhances the redifferentiation of cultured chondrocytes. ► ROCK inhibitor is useful for preparation of un-dedifferentiated chondrocytes. ► 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-based regeneration therapy.

  17. Human growth hormone binding and stimulation of insulin biosynthesis in cloned rat insulinoma cells

    DEFF Research Database (Denmark)

    Billestrup, Nils

    1985-01-01

    Binding of 125I labelled human growth hormone to cloned insulin producing RIN-5AH cells is described. Binding was specific for somatotropic hormones since both human and rat growth hormone could compete for binding sites, whereas much higher concentrations of lactogenic hormones were needed to in...... to inhibit binding. Culture of RIN-5AH cells in the presence of hGH stimulated insulin biosynthesis by 85%....

  18. Stimulation of matrix formation in rabbit chondrocyte cultures by ascorbate. 1. Effect of ascorbate analogs and beta-aminopropionitrile.

    Science.gov (United States)

    Wright, G C; Wei, X Q; McDevitt, C A; Lane, B P; Sokoloff, L

    1988-01-01

    The most consistent effects of 0.2 mM L-ascorbate on monolayer cultures of rabbit articular chondrocytes were a diversion of incorporated radiosulfate into a pericellular matrix and enhancement of cell proliferation. Only with certain batches of fetal bovine serum (FBS) was there a cell-for-cell increase of proteoglycan synthesis. These actions increased as the cell inoculum rose from 0.5 to 2 x 10(5) cells/T25 flask. Maximal effects of ascorbate and D-isoascorbate were found over a range of 0.05-0.2 mM. L-Dehydroascorbic acid was less effective than either, and no stimulatory action was exerted by L-cysteine, glutathione, dithiothreitol, methylene blue, or phenazine methosulfate. Ascorbate increased the hypro:pro ratio of newly synthesized proteins. beta-Aminopropionitrile (1 mM) reduced the proportion of [3H]hydroxyproline and [35S]O4-proteoglycans in the ascorbate-supplemented matrix 31 and 7%, respectively. In corresponding electronmicrographs, the number of pericellular filaments was reduced. We conclude: (a) Ascorbate has a general anabolic effect on chondrocytes in culture and enhances matrix assembly through mechanisms other than its redox function; (b) deposition of proteoglycans in the matrix is not simply the result of mechanical entrapment by allysine- or hydroxyallysine-derived cross-linking of collagen; and (c) contradictory reports on the subject result from variations in the serum employed, inoculum density, and concentration of ascorbate.

  19. Polychlorinated biphenyl 126 stimulates basal and inducible aldosterone biosynthesis of human adrenocortical H295R cells

    International Nuclear Information System (INIS)

    Li, L.-A.; Wang, P.-W.; Chang, Louis W.

    2004-01-01

    To understand the effects of polychlorinated biphenyls (PCBs) on adrenal aldosterone biosynthesis, we have performed a systematical study to characterize the corresponding steroidogenic response of human adrenocortical cell line H295R to PCB126 exposure. We found that PCB126 at high concentrations stimulated basal and inducible aldosterone production. The aldosterone induction occurred concomitantly with activation of the CYP11B2 gene. Despite the fact that PCB126 acted in synergy with both potassium and angiotensin II (Ang II) in activation of aldosterone synthesis, PCB126 only modestly increased CYP11B2 mRNA expression in the presence of Ang II contrary to the synergistic transcriptional induction elicited by PCB126 and potassium. This implicated that PCB126 had differential interactions with the potassium and Ang II signaling systems in the regulation of aldosterone biosynthesis. In addition, high concentrations of PCB126 elevated transcriptional expression of the type I Ang II receptor (AT 1 ) and might thus sensitize the cellular Ang II responsiveness in both basal and inducible aldosterone biosynthesis. SF-1 was not involved in the PCB126-induced transcriptional regulation despite its importance in steroidogenic gene activation

  20. Stimulation of chondrocytes in vitro by gene transfer with plasmids coding for epidermal growth factor (hEGF) and basic fibroblast growth factor (bFGF)

    DEFF Research Database (Denmark)

    Schmal, H; Mehlhorn, A T; Zwingmann, J

    2005-01-01

    Human epidermal growth factor (hEGF) and basic fibroblast growth factor (bFGF) influence critical characteristics of chondrocytes. The effects on metabolism and differentiation were evaluated following transfection using specific plasmids coding for both cytokines. Chondrocytes were isolated from...

  1. Biophysical stimulation improves clinical results of matrix-assisted autologous chondrocyte implantation in the treatment of chondral lesions of the knee.

    Science.gov (United States)

    Collarile, Marco; Sambri, Andrea; Lullini, Giada; Cadossi, Matteo; Zorzi, Claudio

    2018-04-01

    The purpose of the present study was to evaluate the effects of pulsed electromagnetic fields (PEMFs) on clinical outcome in patients who underwent arthroscopic matrix-assisted autologous chondrocyte implantation (MACI) for chondral lesions of the knee. Thirty patients affected by grade III and IV International Cartilage Repair Society chondral lesions of the knee underwent MACI. After surgery, patients were randomly assigned to either experimental group (PEMFs 4 h per day for 60 days) or control group . Clinical outcome was evaluated through International Knee Documentation Committee (IKDC) subjective knee evaluation form, Visual Analog Scale, Short Form-36 (SF-36) and EuroQoL before surgery and 1, 2, 6, and 60 months postoperative. Mean size of chondral lesion was 2.4 ± 0.6 cm 2 in the PEMFs group and 2.5 ± 0.5 cm 2 in the control one. No differences were found between groups at baseline. IKDC score increased in both groups till 6 months, but afterward improvement was observed only in the experimental group with a significant difference between groups at 60 months (p = 0.001). A significant difference between groups was recorded at 60 months for SF-36 (p = 0.006) and EuroQol (p = 0.020). A significant pain reduction was observed in the experimental group at 1-, 2- and 60-month follow-up. Biophysical stimulation with PEMFs improves clinical outcome after arthroscopic MACI for chondral lesions of the knee in the short- and long-term follow-up. Biophysical stimulation should be considered as an effective tool in order to ameliorate clinical results of regenerative medicine. The use of PEMFs represents an innovative therapeutic approach for the survival of cartilage-engineered constructs and consequently the success of orthopaedic surgery. II.

  2. Progesterone-specific stimulation of triglyceride biosynthesis in a breast cancer cell line (T-47D)

    International Nuclear Information System (INIS)

    Judge, S.M.; Chatterton, R.T. Jr.

    1983-01-01

    The purpose of this study was to examine the lactogenic response of human mammary cancer cell lines to hormones in vitro. Progesterone was found to stimulate the incorporation of 14C from [14C]acetate into triglycerides (TG) and to promote accumulation of TG with a fatty acid composition similar to that of human milk fat in T-47D cells. Lipid droplets were observed in larger numbers without concomitant accumulation of casein granules in cells incubated with progesterone, but secretion of lipid into the medium did not occur. An effect of progesterone on TG accumulation was detectable after 12 hr and was maximal at 72 hr. Increasing doses of progesterone (10(-9) to 10(-5) M) caused a progressive increase in TG accumulation. The presence of cortisol and/or prolactin did not alter TG formation nor the dose response of the cells to progesterone. The growth rate of T-47D cells was not altered by the presence of progesterone in the medium. Neither of the human mammary cancer cell lines, MCF-7 and HBL-100, nor the human fibroblast cell lines, 28 and 857, responded to progesterone. The data indicate that, while the normally lactogenic hormones do not stimulate milk product biosynthesis in the cell lines tested, progesterone specifically stimulated synthesis and accumulation of TG in the T-47D cells

  3. Growth factor transgenes interactively regulate articular chondrocytes.

    Science.gov (United States)

    Shi, Shuiliang; Mercer, Scott; Eckert, George J; Trippel, Stephen B

    2013-04-01

    Adult articular chondrocytes lack an effective repair response to correct damage from injury or osteoarthritis. Polypeptide growth factors that stimulate articular chondrocyte proliferation and cartilage matrix synthesis may augment this response. Gene transfer is a promising approach to delivering such factors. Multiple growth factor genes regulate these cell functions, but multiple growth factor gene transfer remains unexplored. We tested the hypothesis that multiple growth factor gene transfer selectively modulates articular chondrocyte proliferation and matrix synthesis. We tested the hypothesis by delivering combinations of the transgenes encoding insulin-like growth factor I (IGF-I), fibroblast growth factor-2 (FGF-2), transforming growth factor beta1 (TGF-β1), bone morphogenetic protein-2 (BMP-2), and bone morphogenetic protien-7 (BMP-7) to articular chondrocytes and measured changes in the production of DNA, glycosaminoglycan, and collagen. The transgenes differentially regulated all these chondrocyte activities. In concert, the transgenes interacted to generate widely divergent responses from the cells. These interactions ranged from inhibitory to synergistic. The transgene pair encoding IGF-I and FGF-2 maximized cell proliferation. The three-transgene group encoding IGF-I, BMP-2, and BMP-7 maximized matrix production and also optimized the balance between cell proliferation and matrix production. These data demonstrate an approach to articular chondrocyte regulation that may be tailored to stimulate specific cell functions, and suggest that certain growth factor gene combinations have potential value for cell-based articular cartilage repair. Copyright © 2012 Wiley Periodicals, Inc.

  4. Acute Exercise Stimulates Carnitine Biosynthesis and OCTN2 Expression in Mouse Kidney

    Directory of Open Access Journals (Sweden)

    Tom L. Broderick

    2017-06-01

    Full Text Available Background/Aims: Carnitine is essential for the transport of long-chain FAs (FA into the mitochondria for energy production. During acute exercise, the increased demand for FAs results in a state of free carnitine deficiency in plasma. The role of kidney in carnitine homeostasis after exercise is not known. Methods: Swiss Webster mice were sacrificed immediately after a 1-hour moderate intensity treadmill run, and at 4-hours and 8-hours into recovery. Non-exercising mice served as controls. Plasma was analyzed for carnitine using acetyltransferase and [14C] acetyl-CoA. Kidney was removed for gene and protein expression of butyrobetaine hydroxylase (γ-BBH, organic cation transporter (OCTN2, and peroxisome proliferator-activated receptor (PPARα, a regulator of fatty acid oxidation activated by FAs. Results: Acute exercise caused a decrease in plasma free carnitine levels. Rapid return of free carnitine to control levels during recovery was associated with increased γ-BBH expression. Both mRNA and protein levels of OCTN2 were detected in kidney after exercise and during recovery, suggesting renal transport mechanisms were stimulated. These changes were accompanied with a reciprocal increase in PPARα protein expression. Conclusions: Our results show that the decrease in free carnitine after exercise rapidly activates carnitine biosynthesis and renal transport mechanism in kidney to establish carnitine homeostasis.

  5. Methamphetamine accelerates cellular senescence through stimulation of de novo ceramide biosynthesis.

    Directory of Open Access Journals (Sweden)

    Giuseppe Astarita

    Full Text Available Methamphetamine is a highly addictive psychostimulant that causes profound damage to the brain and other body organs. Post mortem studies of human tissues have linked the use of this drug to diseases associated with aging, such as coronary atherosclerosis and pulmonary fibrosis, but the molecular mechanism underlying these findings remains unknown. Here we used functional lipidomics and transcriptomics experiments to study abnormalities in lipid metabolism in select regions of the brain and, to a greater extent, peripheral organs and tissues of rats that self-administered methamphetamine. Experiments in various cellular models (primary mouse fibroblasts and myotubes allowed us to investigate the molecular mechanisms of systemic inflammation and cellular aging related to methamphetamine abuse. We report now that methamphetamine accelerates cellular senescence and activates transcription of genes involved in cell-cycle control and inflammation by stimulating production of the sphingolipid messenger ceramide. This pathogenic cascade is triggered by reactive oxygen species, likely generated through methamphetamine metabolism via cytochrome P450, and involves the recruitment of nuclear factor-κB (NF-κB to induce expression of enzymes in the de novo pathway of ceramide biosynthesis. Inhibitors of NF-κB signaling and ceramide formation prevent methamphetamine-induced senescence and systemic inflammation in rats self-administering the drug, attenuating their health deterioration. The results suggest new therapeutic strategies to reduce the adverse consequences of methamphetamine abuse and improve effectiveness of abstinence treatments.

  6. Endurance exercise and conjugated linoleic acid (CLA supplementation up-regulate CYP17A1 and stimulate testosterone biosynthesis.

    Directory of Open Access Journals (Sweden)

    Rosario Barone

    Full Text Available A new role for fat supplements, in particular conjugated linoleic acid (CLA, has been delineated in steroidogenesis, although the underlying molecular mechanisms have not yet been elucidated. The aims of the present study were to identify the pathway stimulated by CLA supplementation using a cell culture model and to determine whether this same pathway is also stimulated in vivo by CLA supplementation associated with exercise. In vitro, Leydig tumour rat cells (R2C supplemented with different concentrations of CLA exhibited increasing testosterone biosynthesis accompanied by increasing levels of CYP17A1 mRNA and protein. In vivo, trained mice showed an increase in free plasma testosterone and an up-regulation of CYP17A1 mRNA and protein. The effect of training on CYP17A1 expression and testosterone biosynthesis was significantly higher in the trained mice supplemented with CLA compared to the placebo. The results of the present study demonstrated that CLA stimulates testosterone biosynthesis via CYP17A1, and endurance training led to the synthesis of testosterone in vivo by inducing the overexpression of CYP17A1 mRNA and protein in the Leydig cells of the testis. This effect was enhanced by CLA supplementation. Therefore, CLA-associated physical activity may be used for its steroidogenic property in different fields, such as alimentary industry, human reproductive medicine, sport science, and anti-muscle wasting.

  7. Stimulating the biosynthesis of antroquinonol by addition of effectors and soybean oil in submerged fermentation of Antrodia camphorata.

    Science.gov (United States)

    Hu, Yong-Dan; Lu, Rui-Qiu; Liao, Xiang-Ru; Zhang, Bo-Bo; Xu, Gan-Rong

    2016-05-01

    Antrodia camphorata is a precious medicinal mushroom that has attracted increasing attentions. Antroquinonol has been considered as being among the most biologically active components of A. camphorata. However, it was hardly biosynthesized via conventional submerged fermentation. Two approaches were applied to stimulate the biosynthesis of antroquinonol in submerged fermentation. On one hand, different kinds of effectors that may involve in the antroquinonol biosynthesis were investigated. Among the tested effectors, camphorwood leach liquor was the most effective for stimulating the antroquinonol production. On the other hand, because of the hydrophobic characteristics of antroquinonol, soybean oil was added to establish an extractive fermentation system for alleviating the product inhibition and resulting in enhanced productivity. The highest antroquinonol concentration could be achieved at 89.06 ± 0.14 mg/L when 10% (v/v) soybean oil was added at the beginning of the fermentation. This study will be of great significance for the study of A. camphorata and the bioprocess regulation of antroquinonol production. © 2015 International Union of Biochemistry and Molecular Biology, Inc.

  8. Biosynthesis of poly(4-hydroxybutyrate) in recombinant Escherichia coli grown on glycerol is stimulated by propionic acid.

    Science.gov (United States)

    Kämpf, Michael M; Thöny-Meyer, Linda; Ren, Qun

    2014-11-01

    One of the most promising polyhydroxyalkanoates (PHAs) for medical applications is poly(4-hydroxybutyrate) (P4HB) due to its biodegradability, biocompatibility and mechanical properties. Currently, the major hurdle for expanding P4HB applications is the production and recovery cost. In this study, we investigated the stimulating factors for P4HB biosynthesis with the ultimate goal of reducing production cost. We found that addition of propionic acid to the culture medium stimulates the P4HB accumulation in recombinant Escherichia coli JM109 grown on glycerol. This stimulating effect was significantly weakened by addition of exogenous methionine, whereas it was not influenced by addition of cysteine. These results suggest that propionic acid enhances P4HB synthesis by reducing the intracellular methionine pool. Utilizing these findings for P4HB production in batch cultures on glycerol, the volumetric yield of P4HB could be improved 4 fold from 0.9g/L to 3.7g/L by adding 2g/L propionic acid into the medium. Copyright © 2014 Elsevier B.V. All rights reserved.

  9. Giant crystals inside mitochondria of equine chondrocytes.

    Science.gov (United States)

    Nürnberger, S; Rentenberger, C; Thiel, K; Schädl, B; Grunwald, I; Ponomarev, I; Marlovits, St; Meyer, Ch; Barnewitz, D

    2017-05-01

    The present study reports for the first time the presence of giant crystals in mitochondria of equine chondrocytes. These structures show dark contrast in TEM images as well as a granular substructure of regularly aligned 1-2 nm small units. Different zone axes of the crystalline structure were analysed by means of Fourier transformation of lattice-resolution TEM images proving the crystalline nature of the structure. Elemental analysis reveals a high content of nitrogen referring to protein. The outer shape of the crystals is geometrical with an up to hexagonal profile in cross sections. It is elongated, spanning a length of several micrometres through the whole cell. In some chondrocytes, several crystals were found, sometimes combined in a single mitochondrion. Crystals were preferentially aligned along the long axis of the cells, thus appearing in the same orientation as the chondrocytes in the tissue. Although no similar structures have been found in the cartilage of any other species investigated, they have been found in cartilage repair tissue formed within a mechanically stimulated equine chondrocyte construct. Crystals were mainly located in superficial regions of cartilage, especially in joint regions of well-developed superficial layers, more often in yearlings than in adult horses. These results indicate that intramitochondrial crystals are related to the high mechanical stress in the horse joint and potentially also to the increased metabolic activity of immature individuals.

  10. Mammalian tyrosinase: biosynthesis, processing, and modulation by melanocyte-stimulating hormone.

    Science.gov (United States)

    Jiménez, M; Kameyama, K; Maloy, W L; Tomita, Y; Hearing, V J

    1988-01-01

    We have examined the rate of synthesis and degradation of tyrosinase (monophenol, 3,4-dihydroxyphenylalanine:oxygen oxidoreductase, EC 1.14.18.1), the critical enzyme involved in mammalian pigmentation, using pulse-chase metabolic labeling of murine melanoma cells and immunoprecipitation of protein extracts with antibodies directed specifically against the enzyme. We have found that tyrosinase is synthesized and glycosylated within melanocytes rapidly, since significant quantities of pulse-labeled enzyme could be detected within 30 min. The maximum amount of enzyme was processed within 4 hr, and the t1/2 of tyrosinase in vivo was 10 hr (compared to 120 hr with purified enzyme), suggesting that tyrosinase activity in melanocytes is at least in part regulated by rapid synthesis and active degradation. We also have examined the melanogenic stimulation caused by melanocyte-stimulating hormone, using metabolic labeling, radiometric assays, and immunofluorescence techniques; responding cells increased their melanogenic potential more than 7-fold within 4 days without increasing their levels of tyrosinase synthesis. The results demonstrate that a pool of inactive tyrosinase exists in melanocytes and that rapid increases in enzyme activity elicited by melanocyte-stimulating hormone reflect an alteration in the activity of a preexisting pool of intracellular tyrosinase. Images PMID:3131764

  11. Methylquercetins stimulate melanin biosynthesis in a three-dimensional skin model.

    Science.gov (United States)

    Yamauchi, Kosei; Mitsunaga, Tohru

    2018-02-13

    In a previous study, we found that both synthetic 3-O-methylquercetin (3MQ) and 3,4',7-O-trimethylquercetin (34'7TMQ) increased extracellular melanin content. 34'7TMQ increased the activity of melanogenic enzymes by stimulating the p38 pathway and the expression of microphthalmia-associated transcription factor (MITF). In contrast, 3MQ increased the activity of melanogenic enzymes without the involvement of MITF, which suggests that 3MQ inhibits the degradation of melanogenic enzymes. In the present study, we investigated the effects of 3MQ and 34'7TMQ on melanogenesis in normal human melanocytes and using a commercial three-dimensional (3D) skin model system. Both 3MQ and 34'7TMQ elongated the dendrites of normal human melanocytes from a Caucasian donor, but did not stimulate melanogenesis in the melanocytes. In the 3D skin model, which included melanocytes from an Asian donor, 3MQ and 34'7TMQ increased and elongated the melanocytes and showed a tendency to stimulate melanogenesis. These results suggest that 3MQ and 34'7TMQ could be put to practical use in skin care products and agents aimed at preventing hair graying.

  12. Biological and Chemical Removal of Primary Cilia Affects Mechanical Activation of Chondrogenesis Markers in Chondroprogenitors and Hypertrophic Chondrocytes.

    Science.gov (United States)

    Deren, Matthew E; Yang, Xu; Guan, Yingjie; Chen, Qian

    2016-02-04

    Chondroprogenitors and hypertrophic chondrocytes, which are the first and last stages of the chondrocyte differentiation process, respectively, are sensitive to mechanical signals. We hypothesize that the mechanical sensitivity of these cells depends on the cell surface primary cilia. To test this hypothesis, we removed the primary cilia by biological means with transfection with intraflagellar transport protein 88 (IFT88) siRNA or by chemical means with chloral hydrate treatment. Transfection of IFT88 siRNA significantly reduced the percentage of ciliated cells in both chondroprogenitor ATDC5 cells as well as primary hypertrophic chondrocytes. Cyclic loading (1 Hz, 10% matrix deformation) of ATDC5 cells in three-dimensional (3D) culture stimulates the mRNA levels of chondrogenesis marker Type II collagen (Col II), hypertrophic chondrocyte marker Type X collagen (Col X), and a molecular regulator of chondrogenesis and chondrocyte hypertrophy bone morphogenetic protein 2 (BMP-2). The reduction of ciliated chondroprogenitors abolishes mechanical stimulation of Col II, Col X, and BMP-2. In contrast, cyclic loading stimulates Col X mRNA levels in hypertrophic chondrocytes, but not those of Col II and BMP-2. Both biological and chemical reduction of ciliated hypertrophic chondrocytes reduced but failed to abolish mechanical stimulation of Col X mRNA levels. Thus, primary cilia play a major role in mechanical stimulation of chondrogenesis and chondrocyte hypertrophy in chondroprogenitor cells and at least a partial role in hypertrophic chondrocytes.

  13. Biological and Chemical Removal of Primary Cilia Affects Mechanical Activation of Chondrogenesis Markers in Chondroprogenitors and Hypertrophic Chondrocytes

    Directory of Open Access Journals (Sweden)

    Matthew E. Deren

    2016-02-01

    Full Text Available Chondroprogenitors and hypertrophic chondrocytes, which are the first and last stages of the chondrocyte differentiation process, respectively, are sensitive to mechanical signals. We hypothesize that the mechanical sensitivity of these cells depends on the cell surface primary cilia. To test this hypothesis, we removed the primary cilia by biological means with transfection with intraflagellar transport protein 88 (IFT88 siRNA or by chemical means with chloral hydrate treatment. Transfection of IFT88 siRNA significantly reduced the percentage of ciliated cells in both chondroprogenitor ATDC5 cells as well as primary hypertrophic chondrocytes. Cyclic loading (1 Hz, 10% matrix deformation of ATDC5 cells in three-dimensional (3D culture stimulates the mRNA levels of chondrogenesis marker Type II collagen (Col II, hypertrophic chondrocyte marker Type X collagen (Col X, and a molecular regulator of chondrogenesis and chondrocyte hypertrophy bone morphogenetic protein 2 (BMP-2. The reduction of ciliated chondroprogenitors abolishes mechanical stimulation of Col II, Col X, and BMP-2. In contrast, cyclic loading stimulates Col X mRNA levels in hypertrophic chondrocytes, but not those of Col II and BMP-2. Both biological and chemical reduction of ciliated hypertrophic chondrocytes reduced but failed to abolish mechanical stimulation of Col X mRNA levels. Thus, primary cilia play a major role in mechanical stimulation of chondrogenesis and chondrocyte hypertrophy in chondroprogenitor cells and at least a partial role in hypertrophic chondrocytes.

  14. On the mechanism of the involvement of monoamine oxidase in catecholamine-stimulated prostaglandin biosynthesis in particulate fraction of rat brain homogenates: role of hydrogen peroxide.

    Science.gov (United States)

    Seregi, A; Serfözö, P; Mergl, Z; Schaefer, A

    1982-01-01

    The mechanism of involvement of monoamine oxidase (MAO) in catecholamine-stimulated prostaglandin (PG) biosynthesis was studied in the particulate fraction of rat brain homogenates. High concentrations of either noradrenaline (NA) or dopamine (DA) stimulated effectively PGF2 alpha formation. The same amount of 2-phenylethylamine (PEA) acted similarly, provided that it was administered together with a catecholamine analogue or metabolite possessing the 3,4-dihydroxyphenyl nucleus--3,4-dihydroxyphenylalanine (DOPA), 3,4-dihydroxyphenylacetic acid (DOPAC), 3,4-dihydroxyphenylglycol (DOPEG), 3,4-dihydroxyphenylacetaldehyde (DOPAL), or alpha-methylnoradrenaline (alpha-met-NA)--or with SnCl2. In the absence of PEA, these compounds were ineffective with regard to stimulation of PGF2 alpha formation. Catalase, pargyline, or indomethacin abolished completely PGF2 alpha formation elicited either by catecholamines or by PEA plus a 3,4-dihydroxyphenyl compound or SnCl2. With regard to the stimulation of PGF2 alpha formation in the presence of alpha-met-NA, PEA could be replaced by H2O2 generated by the glucose oxidase(GOD)-glucose system. The effect of H2O2 was inhibited by indomethacin or catalase, but pargyline was ineffective. It is assumed that catecholamines play a dual role in the activation of PG biosynthesis in brain tissue. During the enzymatic decomposition of catecholamines MAO produces H2O2, which stimulates endoperoxide synthesis. Simultaneously, catecholamines as hydrogen donors promote the nonenzymatic transformation of endoperoxides into PGF2 alpha. The possible physiological importance of these findings is discussed.

  15. Regulative mechanisms of chondrocyte adhesion

    DEFF Research Database (Denmark)

    Schmal, Hagen; Mehlhorn, Alexander T; Fehrenbach, Miriam

    2006-01-01

    Interaction between chondrocytes and extracellular matrix is considered a key factor in the generation of grafts for matrix-associated chondrocyte transplantation. Therefore, our objective was to study the influence of differentiation status on cellular attachment. Adhesion of chondrocytes...... to collagen type II increased after removal from native cartilage up to the third day in monolayer in a dose-dependent manner. Following dedifferentiation after the second passage, adhesion to collagen types I (-84%) and II (-46%) decreased, whereas adhesion to fibrinogen (+59%) and fibronectin (+43......%) increased. A cartilage construct was developed based on a clinically established collagen type I scaffold. In this matrix, more than 80% of the cells could be immobilized by mechanisms of adhesion, filtration, and cell entrapment. Confocal laser microscopy revealed focal adhesion sites as points of cell...

  16. Fibroblast growth factor receptor 3 effects on proliferation and telomerase activity in sheep growth plate chondrocytes

    Directory of Open Access Journals (Sweden)

    Smith Logan B

    2012-12-01

    Full Text Available Abstract Background Fibroblast growth factor receptor 3 (FGFR3 inhibits growth-plate chondrocyte proliferation and limits bone elongation. Gain-of-function FGFR3 mutations cause dwarfism, reduced telomerase activity and shorter telomeres in growth plate chondroyctes suggesting that FGFR3 reduces proliferative capacity, inhibits telomerase, and enhances senescence. Thyroid hormone (T3 plays a role in cellular maturation of growth plate chondrocytes and a known target of T3 is FGFR3. The present study addressed whether reduced FGFR3 expression enhanced telomerase activity, mRNA expression of telomerase reverse transcriptase (TERT and RNA component of telomerase (TR, and chondrocyte proliferation, and whether the stimulation of FGFR3 by T3 evoked the opposite response. Results Sheep growth-plate proliferative zone chondrocytes were cultured and transfected with siRNA to reduce FGFR3 expression; FGFR3 siRNA reduced chondrocyte FGFR3 mRNA and protein resulting in greater proliferation and increased TERT mRNA expression and telomerase activity (p 3 significantly enhanced FGFR3 mRNA and protein expression and reduced telomerase activity (p 3 at the growth plate may be partially mediated through the FGFR3 pathway. Conclusions The results suggest that FGFR3 inhibits chondrocyte proliferation by down-regulating TERT expression and reducing telomerase activity indicating an important role for telomerase in sustaining chondrocyte proliferative capacity during bone elongation.

  17. Comparison between Chondrogenic Markers of Differentiated Chondrocytes from Adipose Derived Stem Cells and Articular Chondrocytes In Vitro

    Directory of Open Access Journals (Sweden)

    Mohmmad Mardani

    2013-06-01

    Full Text Available   Objective(s: Osteoarthritis is one of the most common diseases in middle-aged population in the world. Cartilage tissue engineering (TE has been presented as an effort to introduce the best combination of cells, biomaterial scaffolds and stimulating growth factors to produce a cartilage tissue similar to the natural articular cartilage. In this study, the chondrogenic potential of adipose derived stem cells (ADSCs was compared with natural articular chondrocytes cultured in alginate scaffold.   Materials and Methods: Human ADSCs were obtained from subcutaneous adipose tissue and human articular chondrocytes from non-weight bearing areas of knee joints. Cells were seeded in 1.5% alginate and cultured in chondrogenic media for three weeks with and without TGFβ3. The genes expression of types II and X collagens was assessed by Real Time PCR and the amount of aggrecan (AGC and type I collagen measured by ELISA and the content of glycosaminoglycan evaluated by GAG assay. Results: Our findings showed that type II collagen, GAG and AGC were expressed, in differentiated ADSCs. Meanwhile, they produced a lesser amount of types II and X collagens but more AGC, GAG and type I collagen in comparison with natural chondrocytes (NCs. Conclusion: Further attempt should be carried out to optimize achieving type II collagen in DCs, as much as, natural articular chondrocytes and decline of the production of type I collagen in order to provide efficient hyaline cartilage after chondrogenic induction, prior to the usage of harvested tissues in clinical trials.

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

    Science.gov (United States)

    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

  19. Supplementation with linoleic acid-rich soybean oil stimulates macrophage foam cell formation via increased oxidative stress and diacylglycerol acyltransferase1-mediated triglyceride biosynthesis.

    Science.gov (United States)

    Rom, Oren; Jeries, Helana; Hayek, Tony; Aviram, Michael

    2017-01-02

    During the last decades there has been a staggering rise in human consumption of soybean oil (SO) and its major polyunsaturated fatty acid linoleic acid (LA). The role of SO or LA in cardiovascular diseases is highly controversial, and their impact on macrophage foam cell formation, the hallmark of early atherogenesis, is unclear. To investigate the effects of high SO or LA intake on macrophage lipid metabolism and the related mechanisms of action, C57BL/6 mice were orally supplemented with increasing levels of SO-based emulsion or equivalent levels of purified LA for 1 month, followed by analyses of lipid accumulation and peroxidation in aortas, serum and in peritoneal macrophages (MPM) of the mice. Lipid peroxidation and triglyceride mass in aortas from SO or LA supplemented mice were dose-dependently and significantly increased. In MPM from SO or LA supplemented mice, lipid peroxides were significantly increased and a marked accumulation of cellular triglycerides was found in accordance with enhanced triglyceride biosynthesis rate and overexpression of diacylglycerol acyltransferase1 (DGAT1), the key enzyme in triglyceride biosynthesis. In cultured J774A.1 macrophages treated with SO or LA, triglyceride accumulated via increased oxidative stress and a p38 mitogen-activated protein kinase (MAPK)-mediated overexpression of DGAT1. Accordingly, anti-oxidants (pomegranate polyphenols), inhibition of p38 MAPK (by SB202190) or DGAT1 (by oleanolic acid), all significantly attenuated SO or LA-induced macrophage triglyceride accumulation. These findings reveal novel mechanisms by which supplementation with SO or LA stimulate macrophage foam cell formation, suggesting a pro-atherogenic role for overconsumption of SO or LA. © 2016 BioFactors, 43(1):100-116, 2017. © 2016 International Union of Biochemistry and Molecular Biology.

  20. Hepatic fatty acid biosynthesis is more responsive to protein than carbohydrate in rainbow trout during acute stimulations.

    Science.gov (United States)

    Dai, Weiwei; Panserat, Stéphane; Kaushik, Sadasivam; Terrier, Frédéric; Plagnes-Juan, Elisabeth; Seiliez, Iban; Skiba-Cassy, Sandrine

    2016-01-01

    The link between dietary carbohydrate/protein and de novo lipogenesis (DNL) remains debatable in carnivorous fish. We aimed to evaluate and compare the response of hepatic lipogenic gene expression to dietary carbohydrate intake/glucose and dietary protein intake/amino acids (AAs) during acute stimulations using both in vivo and in vitro approaches. For the in vivo trial, three different diets and a controlled-feeding method were employed to supply fixed amount of dietary protein or carbohydrate in a single meal; for the in vitro trial, primary hepatocytes were stimulated with a low or high level of glucose (3 mM or 20 mM) and a low or high level of AAs (one-fold or four-fold concentrated AAs). In vitro data showed that a high level of AAs upregulated the expression of enzymes involved in DNL [fatty acid synthase (FAS) and ATP citrate lyase (ACLY)], lipid bioconversion [elongation of very long chain fatty acids like-5 (Elovl5), Elovl2, Δ6 fatty acyl desaturase (D6D) and stearoyl-CoA desaturase-1 (SCD1)], NADPH production [glucose-6-phosphate dehydrogenase (G6PDH) and malic enzyme (ME)], and transcriptional factor sterol regulatory element binding protein 1-like, while a high level of glucose only elevated the expression of ME. Data in trout liver also showed that high dietary protein intake induced higher lipogenic gene expression (FAS, ACLY, and Elovl2) regardless of dietary carbohydrate intake, while high carbohydrate intake markedly suppressed the expression of acetyl-CoA carboxylase (ACC) and Elovl5. Overall, we conclude that, unlike rodents or humans, hepatic fatty acid biosynthetic gene expression in rainbow trout is more responsive to dietary protein intake/AAs than dietary carbohydrate intake/glucose during acute stimulations. This discrepancy probably represents one important physiological and metabolic difference between carnivores and omnivores. Copyright © 2016 the American Physiological Society.

  1. Coumestrol Counteracts Interleukin-1β-Induced Catabolic Effects by Suppressing Inflammation in Primary Rat Chondrocytes.

    Science.gov (United States)

    You, Jae-Seek; Cho, In-A; Kang, Kyeong-Rok; Oh, Ji-Su; Yu, Sang-Joun; Lee, Gyeong-Je; Seo, Yo-Seob; Kim, Su-Gwan; Kim, Chun Sung; Kim, Do Kyung; Im, Hee-Jeong; Kim, Jae-Sung

    2017-02-01

    In the present study, we investigated the anti-catabolic effects of coumestrol, a phytoestrogen derived from herbal plants, against interleukin-1β-induced cartilage degeneration in primary rat chondrocytes and articular cartilage. Coumestrol did not affect the viability of human normal oral keratinocytes and primary rat chondrocytes treated for 24 h and 21 days, respectively. Although coumestrol did not significantly increase the proteoglycan contents in long-term culture, it abolished the interleukin-1β-induced loss of proteoglycans in primary rat chondrocytes and knee articular cartilage. Furthermore, coumestrol suppressed the expression of matrix-degrading enzymes such as matrix metalloproteinase-13, -3, and -1 in primary rat chondrocytes stimulated with interleukin-1β. Moreover, the expression of catabolic factors such as nitric oxide synthase, cyclooxygenase-2, prostaglandin E 2 , and inflammatory cytokines in interleukin-1β-stimulated primary rat chondrocytes was suppressed by coumestrol. In summary, these results indicate that coumestrol counteracts the catabolic effects induced by interleukin-1β through the suppression of inflammation. Therefore, based on its biological activity and safety profile, coumestrol could be used as a potential anti-catabolic biomaterial for osteoarthritis.

  2. Articular chondrocyte metabolism and osteoarthritis

    Energy Technology Data Exchange (ETDEWEB)

    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. MicroRNA-29b Contributes to Collagens Imbalance in Human Osteoarthritic and Dedifferentiated Articular Chondrocytes

    Directory of Open Access Journals (Sweden)

    David Moulin

    2017-01-01

    Full Text Available Objective. Decreased expression of collagen type II in favour of collagen type I or X is one hallmark of chondrocyte phenotype changes in osteoarthritic (OA cartilage. MicroRNA- (miR- 29b was previously shown to target collagens in several tissues. We studied whether it could contribute to collagen imbalance in chondrocytes with an impaired phenotype. Methods. After preliminary microarrays screening, miR-29b levels were measured by RT- quantitative PCR in in vitro models of chondrocyte phenotype changes (IL-1β challenge or serial subculturing and in chondrocytes from OA and non-OA patients. Potential miR-29b targets identified in silico in 3′-UTRs of collagens mRNAs were tested with luciferase reporter assays. The impact of premiR-29b overexpression in ATDC5 cells was studied on collagen mRNA levels and synthesis (Sirius red staining during chondrogenesis. Results. MiR-29b level increased significantly in IL-1β-stimulated and weakly in subcultured chondrocytes. A 5.8-fold increase was observed in chondrocytes from OA versus non-OA patients. Reporter assays showed that miR-29b targeted COL2A1 and COL1A2 3′-UTRs although with a variable recovery upon mutation. In ATDC5 cells overexpressing premiR-29b, collagen production was reduced while mRNA levels increased. Conclusions. By acting probably as a posttranscriptional regulator with a different efficacy on COL2A1 and COL1A2 expression, miR-29b can contribute to the collagens imbalance associated with an abnormal chondrocyte phenotype.

  4. Mechanical and hypoxia stress can cause chondrocytes apoptosis through over-activation of endoplasmic reticulum stress.

    Science.gov (United States)

    Huang, Ziwei; Zhou, Min; Wang, Qian; Zhu, Mengjiao; Chen, Sheng; Li, Huang

    2017-12-01

    contributed to the chondrocytes apoptosis. Mechanical stress can cause OA-like pathological change in rat mandibular condylar cartilage via ERS activation and hypoxia existed in the meantime. Both mechanical forces and hypoxia can induce ERS and cause chondrocytes apoptosis only if the stimulate was in higher level. Salubrinal can protect chondrocytes from apoptosis, and relieve OA-liked pathological change on mandibular condylar cartilage under mechanical stress stimulation. Copyright © 2017. Published by Elsevier Ltd.

  5. In-vitro chondrogenic potential of synovial stem cells and chondrocytes allocated for autologous chondrocyte implantation

    DEFF Research Database (Denmark)

    Kubosch, Eva Johanna; Heidt, Emanuel; Niemeyer, Philipp

    2017-01-01

    Purpose: The use of passaged chondrocytes is the current standard for autologous chondrocyte implantation (ACI). De-differentiation due to amplification and donor site morbidity are known drawbacks highlighting the need for alternative cell sources. Methods: Via clinically validated flow cytometry...... analysis, we compared the expression of human stem cell and cartilage markers (collagen type 2 (Col2), aggrecan (ACAN), CD44) of chondrocytes (CHDR), passaged chondrocytes for ACI (CellGenix™), bone marrow derived mesenchymal stem cells (BMSC), and synovial derived stem cells (SDSC). Results: Primary...

  6. Chondrocyte-seeded type I/III collagen membrane for autologous chondrocyte transplantation

    DEFF Research Database (Denmark)

    Niemeyer, Philipp; Lenz, Philipp; Kreuz, Peter C

    2010-01-01

    PURPOSE: We report the 2-year clinical results and identify prognostic factors in patients treated with autologous chondrocyte transplantation by use of a collagen membrane to seed the chondrocytes (ACT-CS). METHODS: This is a prospective study of 59 patients who were treated with ACT-CS and foll......PURPOSE: We report the 2-year clinical results and identify prognostic factors in patients treated with autologous chondrocyte transplantation by use of a collagen membrane to seed the chondrocytes (ACT-CS). METHODS: This is a prospective study of 59 patients who were treated with ACT...

  7. Time-varying magnetic fields: effects of orientation on chondrocyte proliferation

    Energy Technology Data Exchange (ETDEWEB)

    Elliott, J.P.; Smith, R.L.; Block, C.A.

    1988-01-01

    The purpose of this study was to determine the effect of orientation of pulsed electromagnetic fields (PEMFs) on cellular proliferation and extracellular matrix synthesis. Bovine articular chondrocytes were cultured in PEMFs (repetitive pulse at 72 Hz) generated using Helmholtz coils oriented either parallel (horizontal) or perpendicular (vertical) to the plane of cell adhesion. Dissipation of signal energy in the form of heat increased the temperature of the PEMF coils by 2 degrees C and the tissue culture medium by 1 degree C. Therefore, control coils, which emitted no PEMFs, were heated to the temperature of PEMF coils by circulating water. Chondrocytes were cultured in 16-mm-well culture plates, and the data for individual wells were pooled as triplicates. Although not observed by microscopic examination of individual wells, positionally dependent electric field effects may be minimized by this approach. PEMFs generated by coils oriented vertically significantly decreased chondrocyte proliferation. The effect was dependent on the concentration of serum in the culture media. At 3% serum concentration, the total cell number attained after 10 days of culture was reduced by 50% in stimulated cultures when compared with controls. At 5% serum concentration, there was no effect. PEMFs applied by coils oriented horizontally did not alter proliferation of articular chondrocytes. PEMFs had no effect on synthesis of extracellular matrix by chondrocytes plated at high density, irrespective of orientation.

  8. Effect of freezing on rabbit cultured chondrocytes

    Directory of Open Access Journals (Sweden)

    R.R Filgueiras

    2011-02-01

    Full Text Available This work evaluated the effect of freezing on chondrocytes maintained in culture, aiming the establishment of a cell bank for future application as heterologous implant. Chondrocytes extracted from joint cartilage of nine healthy New Zealand White rabbits were cultivated and frozen with the cryoprotector 5% dimethylsulfoxide for six months. Phenotypic and scanning electron microscopy analyses were carried out to identify morphological and functional differences between fresh and thawed cells. After enzymatic digestion, a total of 4.8x10(5cells per rabbit were obtained. Fresh chondrocytes showed a high mitotic rate and abundant matrix was present up to 60 days of culture. Loss of phenotypic stability was notable in the thawed chondrocytes, with a low labeling of proteoglycans and weak immunostaining of type II collagen. The present study showed important loss of chondrocyte viability under the freezing conditions. For future in vivo studies of heterologous implant, these results suggests that a high number of cells should be implanted in the host site in order to achieve an adequate number of viable cells. Furthermore, the chondrocytes should be implanted after two weeks of culture, when the highest viability rate is found

  9. Comparative Analysis of Proteins with Stimulating Activity on Ovarian Estradiol Biosynthesis from Four Different Dioscorea Species in vitro Using Both Phenotypic and Target-based Approaches: Implication for Treating Menopause.

    Science.gov (United States)

    Lu, J; Wong, R N S; Zhang, L; Wong, R Y L; Ng, T B; Lee, K F; Zhang, Y B; Lao, L X; Liu, J Y; Sze, S C W

    2016-09-01

    Rhizomes of Dioscorea species are traditionally used for relieving menopausal syndromes in Chinese medicine. The estrogen-stimulating bioactive principles have been demonstrated in our previous study. In this study, the estrogen-stimulating effects of proteins isolated from four Dioscorea species [D. alata L. (DA), D. zingiberensis C.H. Wright (DH), D. collettii var. hypoglauca (Palib.) S.J. Pei & C.T. Ting (DH), and D. oppositifolia L. (DO)] have been investigated and compared. Microscopic authentication of four Dioscorea species was performed by using paraffin and powder sections of the rhizomes. The potential bioactive proteins of four Dioscorea species have been rapidly isolated by using a DOI-antibody affinity column chromatography on immobilized antibodies against on estradiol-stimulating protein from DO (DOI), and their bioactivity has been rapidly confirmed and compared by phenotypic (i.e., estradiol-stimulating effect) and target-based (i.e., STAR, aromatase, estrogen receptors) screening approaches. The estrogen-stimulating activity of bioactive proteins from DO is the highest. In addition, bioactive proteins from DO upregulated the estradiol-metabolizing enzymes (aromatase and steroidogenic acute regulatory protein). Meanwhile, bioactive proteins from DA, DH and DO upregulated estrogen receptor β (ERβ). All bioactive proteins did not change the expression of estrogen receptor β (ERα). The estrogen-stimulating bioactive proteins isolated from DO increased biosynthesis of estradiol and upregulated the protein expression of aromatase, steroidogenic acute regulatory protein, and ERβ. The results scientifically support the traditional use of DO in Chinese medicine for relieving menopausal syndrome. Besides, proteins from DA and DZ could also upregulate the translational levels of ERβ, and potentially reducing the risk of ovarian cancer, which also support the clinical use of them for treating female aging disorder. Graphical Abstract Comparative

  10. Latexin is involved in bone morphogenetic protein-2-induced chondrocyte differentiation

    International Nuclear Information System (INIS)

    Kadouchi, Ichiro; Sakamoto, Kei; Tangjiao, Liu; Murakami, Takashi; Kobayashi, Eiji; Hoshino, Yuichi; Yamaguchi, Akira

    2009-01-01

    Latexin is the only known carboxypeptidase A inhibitor in mammals. We previously demonstrated that BMP-2 significantly induced latexin expression in Runx2-deficient mesenchymal cells (RD-C6 cells), during chondrocyte and osteoblast differentiation. In this study, we investigated latexin expression in the skeleton and its role in chondrocyte differentiation. Immunohistochemical studies revealed that proliferating and prehypertrophic chondrocytes expressed latexin during skeletogenesis and bone fracture repair. In the early phase of bone fracture, latexin mRNA expression was dramatically upregulated. BMP-2 upregulated the expression of the mRNAs of latexin, Col2a1, and the gene encoding aggrecan (Agc1) in a micromass culture of C3H10T1/2 cells. Overexpression of latexin additively stimulated the BMP-2-induced expression of the mRNAs of Col2a, Agc1, and Col10a1. BMP-2 treatment upregulated Sox9 expression, and Sox9 stimulated the promoter activity of latexin. These results indicate that latexin is involved in BMP-2-induced chondrocyte differentiation and plays an important role in skeletogenesis and skeletal regeneration.

  11. Kaempferol Alleviates the Interleukin-1β-Induced Inflammation in Rat Osteoarthritis Chondrocytes via Suppression of NF-κB.

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    Zhuang, Zhengling; Ye, Guangqun; Huang, Bin

    2017-08-14

    BACKGROUND This study was designed to examine the anti-inflammatory and anti-osteoarthritis (OA) effects of kaempferol in rat articular chondrocytes stimulated with interleukin-1β. MATERIAL AND METHODS Rat articular chondrocytes cultures were treated with interleukin-1β alone or with kaempferol (25, 50, 100, and 200 μM) and interleukin-1β. The effect of kaempferol on chondrocyte cells viability was measured by MTT assay. The effect on prostaglandin E2 (PGE2) and nitric oxide (NO) level were also assessed using the ELISA and Griess reagent, respectively, for kaempferol activity. Moreover, the expression of iNOS, Cox-2 and activation of NF-κB under influence of kaempferol was also assessed by Western blot. RESULTS Kaempferol treatment (up to 100 μM) in a concentration-dependent way caused reduction in the interleukin-1b-stimulated formations of PGE2 and NO. Kaempferol also upregulated the expression of iNOS and Cox-2 in interleukin-1β-stimulated rat OA chondrocytes. Additionally, kaempferol was found to inhibit the IkBa degradation and NF-κB activation in rat chondrocytes stimulated with interleukin-1β. CONCLUSIONS Kaempferol significantly caused reduction in interleukin-1β-stimulated pro-inflammatory mediators in rat OA chondrocytes by inhibiting the NF-κB pathway. These results suggest that kaempferol had significant anti-inflammatory and anti-arthritis effects. Thus, kaempferol, as a novel therapeutic active agent, may prevent, stop, or retard the progression of OA.

  12. Effects of clinically relevant concentrations of glucosamine on equine chondrocytes and synoviocytes in vitro.

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    Byron, Christopher R; Stewart, Matthew C; Stewart, Allison A; Pondenis, Holly C

    2008-09-01

    To evaluate the effects of glucosamine on equine articular chondrocytes and synoviocytes at concentrations clinically relevant to serum and synovial fluid concentrations. Articular cartilage and synovium with normal gross appearance from metacarpophalangeal and metatarsophalangeal joints of 8 horses (1 to 10 years of age). In vitro chondrocyte and synoviocyte cell cultures from 8 horses were treated with glucosamine (0.1 to 20 microg/mL) with or without interleukin-1 (IL-1; 10 ng/mL) for 48 hours. Negative control cultures received no glucosamine or IL-1, and positive control cultures received only IL-1. Cultures were assayed for production of proteoglycan (via media containing sulfur 35 (35S)-labeled sodium sulfate and Alcian blue precipitation), prostaglandin E2 (PGE2; via a colorimetric assay), cyclooxygenase-2 (via real-time reverse-transcriptase PCR assay), microsomal PGE2 synthase (mPGEs; via real-time reverse-transcriptase PCR assay), and matrix metalloproteinase (MMP)-13 (via a colorimetric assay). Glucosamine had no impact on proteoglycan production or MMP-13 production under noninflammatory (no IL-1) or inflammatory (with IL-1) conditions. Glucosamine at 0.1 and 0.5 microg/mL significantly decreased IL-1-stimulated production of mPGEs by chondrocytes, compared with that of positive control chondrocytes. Glucosamine at 0.1 and 5 microg/mL significantly decreased IL-1-stimulated production of mPGEs and PGE2, respectively, compared with that of positive control synoviocytes. Glucosamine had limited effects on chondrocyte and synoviocyte metabolism at clinically relevant concentrations, although it did have some anti-inflammatory activity on IL-1-stimulated articular cells. Glucosamine may have use at clinically relevant concentrations in the treatment of inflammatory joint disease.

  13. Optimization of transport media for human chondrocytes

    International Nuclear Information System (INIS)

    Olender, E.; Uhrynowska-Tyszkiewicz, I.; Gaweda, K.; Kaminski, A.

    2008-01-01

    Full text: Autologous chondrocytes transplantation is a method used in treatment of cartilage defects in joints. Small fragments of patient healthy cartilage are removed and sent to a laboratory or tissue bank for cultivating chondrocytes. Obtained cells are reimplanted into areas of damaged cartilage. Since the transport of cartilage from a recovery site to a cell culture laboratory may be extended, it is very important to optimize the cartilage storage conditions in order to provide specimens with the best cell viability. Fresh human cartilage is stored in Ringer's solution or in normal saline at 4 degree C. Supplements such as hyaluronic acid and glucosamine have been shown to have chondroprotective effects. The aim of this experiment was to evaluate potential new storage media for improving chondrocytes viability. Cartilage fragments were harvested from fresh human femoral condyles. Cartilage samples from each condyle were separately stored at 4 degree C in: normal saline, Ringer solution, normal saline amended with hyaluronic acid and normal saline amended with glucosamine. The cartilage from each donor for each storage method was assayed for viability by MTT reduction assay on the day of recovery and after duration of one, two, three, six, twelve, and twenty-one days. Chondrocytes viability decreased with time in all media except for normal saline amended with glucosamine. The decline in chondrocytes viability was especially distinct for samples maintained in normal saline amended with hyaluronic acid when compared with standard media (normal saline and Ringer solution). In contrast, chondrocytes viability remained high for the whole duration of the experiment in samples maintained in normal saline amended with glucosamine. This finding suggests that the glucosamine supplementation of normal saline reduces the decline in chondrocytes viability and consequently extends the acceptable storage period of cartilage specimens. Further investigations are needed to

  14. Using Costal Chondrocytes to Engineer Articular Cartilage with Applications of Passive Axial Compression and Bioactive Stimuli.

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    Huwe, Le W; Sullan, Gurdeep K; Hu, Jerry C; Athanasiou, Kyriacos A

    2018-03-01

    Generating neocartilage with suitable mechanical integrity from a cell source that can circumvent chondrocyte scarcity is indispensable for articular cartilage regeneration strategies. Costal chondrocytes of the rib eliminate donor site morbidity in the articular joint, but it remains unclear how neocartilage formed from these cells responds to mechanical loading, especially if the intent is to use it in a load-bearing joint. In a series of three experiments, this study sought to determine efficacious parameters of passive axial compressive stimulation that would enable costal chondrocytes to synthesize mechanically robust cartilage. Experiment 1 determined a suitable time window for stimulation by its application during either the matrix synthesis phase, the maturation phase, or during both phases of the self-assembling process. The results showed that compressive stimulation at either time was effective in increasing instantaneous moduli by 92% and 87% in the synthesis and maturation phases, respectively. Compressive stimulation during both phases did not further improve properties beyond a one-time stimulation. The magnitude of passive axial compression was examined in Experiment 2 by applying 0, 3.3, 5.0, or 6.7 kPa stresses to the neocartilage. Unlike 6.7 kPa, both 3.3 and 5.0 kPa significantly increased neocartilage compressive properties by 42% and 48% over untreated controls, respectively. Experiment 3 examined how the passive axial compression regimen developed from the previous phases interacted with a bioactive regimen (transforming growth factor [TGF]-β1, chondroitinase ABC, and lysyl oxidase-like 2). Passive axial compression significantly improved the relaxation modulus compared with bioactive treatment alone. Furthermore, a combined treatment of compressive and bioactive stimulation improved the tensile properties of neocartilage 2.6-fold compared with untreated control. The ability to create robust articular cartilage from passaged costal

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

    Science.gov (United States)

    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. Copyright © 2013 Elsevier B.V. All rights reserved.

  16. Curcumin Inhibits Apoptosis of Chondrocytes through Activation ERK1/2 Signaling Pathways Induced Autophagy

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    Xiaodong Li

    2017-04-01

    Full Text Available Osteoarthritis (OA is an inflammatory disease of load-bearing synovial joints that is currently treated with drugs that exhibit numerous side effects and are only temporarily effective in treating pain, the main symptom of the disease. Consequently, there is an acute need for novel, safe, and more effective chemotherapeutic agents for the treatment of osteoarthritis and related arthritic diseases. Curcumin, the principal curcuminoid and the most active component in turmeric, is a biologically active phytochemical. Evidence from several recent in vitro studies suggests that curcumin may exert a chondroprotective effect through actions such as anti-inflammatory, anti-oxidative stress, and anti-catabolic activity that are critical for mitigating OA disease pathogenesis and symptoms. In the present study, we investigated the protective mechanisms of curcumin on interleukin 1β (IL-1β-stimulated primary chondrocytes in vitro. The treatment of interleukin (IL-1β significantly reduces the cell viability of chondrocytes in dose and time dependent manners. Co-treatment of curcumin with IL-1β significantly decreased the growth inhibition. We observed that curcumin inhibited IL-1β-induced apoptosis and caspase-3 activation in chondrocytes. Curcumin can increase the expression of phosphorylated extracellular signal-regulated kinases 1/2 (ERK1/2, autophagy marker light chain 3 (LC3-II, and Beclin-1 in chondrocytes. The expression of autophagy markers could be decreased when the chondrocytes were incubated with ERK1/2 inhibitor U0126. Our results suggest that curcumin suppresses apoptosis and inflammatory signaling through its actions on the ERK1/2-induced autophagy in chondrocytes. We propose that curcumin should be explored further for the prophylactic treatment of osteoarthritis in humans and companion animals.

  17. A novel compressive stress-based osteoarthritis-like chondrocyte system.

    Science.gov (United States)

    Young, In-Chi; Chuang, Sung-Ting; Gefen, Amit; Kuo, Wei-Ting; Yang, Chun-Ting; Hsu, Chia-Hsien; Lin, Feng-Huei

    2017-05-01

    Mechanical stress damage and insufficient self-repair can contribute to osteoarthritis (OA) in the affected joint. As the effects of stress on chondrocyte metabolism can regulate cartilage homeostasis, the specific stress-response condition is therefore a key to the generation of an OA disease model. We aimed to produce a specific stress- and cell-based OA model after evaluating the metabolic responses of chondrocytes in response to a series of static and cyclic compression stressors. A static load exceeding 40 psi initiated extracellular matrix (ECM) degradation through a decrease in the sulphated-glycosaminoglycan (GAG) content, upregulation of catabolic matrix metalloproteinase (MMP)-13 encoding gene expression, and downregulation of the ECM-related aggrecan and type II collagen encoding genes within 24 h. Indicators of pro-inflammatory events and oxidative stress were found to correlate with increased IL-6 expression and reactive oxygen species (ROS) production, respectively. However, chondrocytes stimulated by moderate cyclic loading (30-40 psi) exhibited increased ECM-related gene expression without significant changes in catabolic and pro-inflammatory gene expression. BMP-7 expression increased at cyclic loading levels above 30-60 psi. These results demonstrated that static compression exceeding 60 psi is sufficient to produce OA-like chondrocytes that exhibit signs of ECM degradation and inflammation. These OA-like chondrocytes could therefore be used as a novel cell-based drug screening system. Impact statement The lack of an effective treatment for osteoarthritis (OA) reflects the great need for alternative therapies and drug discovery. Disease models can be used for early-stage compound screening and disease studies. Chondrocytes are solely responsible for the maintenance of the articular cartilage extracellular matrix. Our strategy involved the generation of a cell-based model of OA, a more readily studied disease. Instead of using animal cartilage

  18. Oxygen tension affects lubricin expression in chondrocytes.

    Science.gov (United States)

    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.

  19. Biochemical alterations in inflammatory reactive chondrocytes: evidence for intercellular network communication

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    Eva Skiöldebrand

    2018-01-01

    Full Text Available Chondrocytes are effectively involved in the pathophysiological processes of inflammation in joints. They form cellular processes in the superficial layer of the articular cartilage and form gap junction coupled syncytium to facilitate cell-to-cell communication. However, very little is known about their physiological cellular identity and communication. The aim with the present work is to evaluate the physiological behavior after stimulation with the inflammatory inducers interleukin-1β and lipopolysaccharide. The cytoskeleton integrity and intracellular Ca2+ release were assessed as indicators of inflammatory state. Cytoskeleton integrity was analyzed through cartilage oligomeric matrix protein and actin labeling with an Alexa 488-conjugated phalloidin probe. Ca2+ responses were assessed through the Ca2+ sensitive fluorophore Fura-2/AM. Western blot analyses of several inflammatory markers were performed. The results show reorganization of the actin filaments. Glutamate, 5-hydoxytryptamine, and ATP evoked intracellular Ca2+ release changed from single peaks to oscillations after inflammatory induction in the chondrocytes. The expression of toll-like receptor 4, the glutamate transporters GLAST and GLT-1, and the matrix metalloproteinase-13 increased. This work demonstrates that chondrocytes are a key part in conditions that lead to inflammation in the cartilage. The inflammatory inducers modulate the cytoskeleton, the Ca2+ signaling, and several inflammatory parameters. In conclusion, our data show that the cellular responses to inflammatory insults from healthy and inflammatory chondrocytes resemble those previously observed in astrocyte and cardiac fibroblasts networks.

  20. 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.

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    Fanny Comblain

    Full Text Available 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

  1. Adipose-Derived Stem Cells Cocultured with Chondrocytes Promote the Proliferation of Chondrocytes

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    Jie Shi

    2017-01-01

    Full Text Available Articular cartilage injury and defect caused by trauma and chronic osteoarthritis vascularity are very common, while the repair of injured cartilage remains a great challenge due to its limited healing capacity. Stem cell-based tissue engineering provides a promising treatment option for injured articular cartilage because of the cells potential for multiple differentiations. However, its application has been largely limited by stem cell type, number, source, proliferation, and differentiation. We hypothesized that (1 adipose-derived stem cells are ideal seed cells for articular cartilage repair because of their accessibility and abundance and (2 the microenvironment of articular cartilage could induce adipose-derived stem cells (ADSCs to differentiate into chondrocytes. In order to test our hypotheses, we isolated stem cells from rabbit adipose tissues and cocultured these ADSCs with rabbit articular cartilage chondrocytes. We found that when ADSCs were cocultured with chondrocytes, the proliferation of articular cartilage chondrocytes was promoted, the apoptosis of chondrocytes was inhibited, and the osteogenic and chondrogenic differentiation of ADSCs was enhanced. The study on the mechanism of this coculture system indicated that the role of this coculture system is similar to the function of TGF-β1 in the promotion of chondrocytes.

  2. Cartilage repair: Generations of autologous chondrocyte transplantation

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    Marlovits, Stefan [Department of Traumatology, Center for Joint and Cartilage, Medical University of Vienna, Waehringer Guertel 18-20, A-1090 Vienna (Austria)]. E-mail: stefan.marlovits@meduniwien.ac.at; Zeller, Philip [Department of Traumatology, Center for Joint and Cartilage, Medical University of Vienna, Waehringer Guertel 18-20, A-1090 Vienna (Austria); Singer, Philipp [Department of Traumatology, Center for Joint and Cartilage, Medical University of Vienna, Waehringer Guertel 18-20, A-1090 Vienna (Austria); Resinger, Christoph [Department of Traumatology, Center for Joint and Cartilage, Medical University of Vienna, Waehringer Guertel 18-20, A-1090 Vienna (Austria); Vecsei, Vilmos [Department of Traumatology, Center for Joint and Cartilage, Medical University of Vienna, Waehringer Guertel 18-20, A-1090 Vienna (Austria)

    2006-01-15

    Articular cartilage in adults has a limited capacity for self-repair after a substantial injury. Surgical therapeutic efforts to treat cartilage defects have focused on delivering new cells capable of chondrogenesis into the lesions. Autologous chondrocyte transplantation (ACT) is an advanced cell-based orthobiologic technology used for the treatment of chondral defects of the knee that has been in clinical use since 1987 and has been performed on 12,000 patients internationally. With ACT, good to excellent clinical results are seen in isolated post-traumatic lesions of the knee joint in the younger patient, with the formation of hyaline or hyaline-like repair tissue. In the classic ACT technique, chondrocytes are isolated from small slices of cartilage harvested arthroscopically from a minor weight-bearing area of the injured knee. The extracellular matrix is removed by enzymatic digestion, and the cells are then expanded in monolayer culture. Once a sufficient number of cells has been obtained, the chondrocytes are implanted into the cartilage defect, using a periosteal patch over the defect as a method of cell containment. The major complications are periosteal hypertrophy, delamination of the transplant, arthrofibrosis and transplant failure. Further improvements in tissue engineering have contributed to the next generation of ACT techniques, where cells are combined with resorbable biomaterials, as in matrix-associated autologous chondrocyte transplantation (MACT). These biomaterials secure the cells in the defect area and enhance their proliferation and differentiation.

  3. Enhanced root growth in phosphate-starved Arabidopsis by stimulating de novo phospholipid biosynthesis through the overexpression of LYSOPHOSPHATIDIC ACID ACYLTRANSFERASE 2 (LPAT2).

    Science.gov (United States)

    Angkawijaya, Artik Elisa; Nguyen, Van Cam; Nakamura, Yuki

    2017-09-01

    Upon phosphate starvation, plants retard shoot growth but promote root development presumably to enhance phosphate assimilation from the ground. Membrane lipid remodelling is a metabolic adaptation that replaces membrane phospholipids by non-phosphorous galactolipids, thereby allowing plants to obtain scarce phosphate yet maintain the membrane structure. However, stoichiometry of this phospholipid-to-galactolipid conversion may not account for the massive demand of membrane lipids that enables active growth of roots under phosphate starvation, thereby suggesting the involvement of de novo phospholipid biosynthesis, which is not represented in the current model. We overexpressed an endoplasmic reticulum-localized lysophosphatidic acid acyltransferase, LPAT2, a key enzyme that catalyses the last step of de novo phospholipid biosynthesis. Two independent LPAT2 overexpression lines showed no visible phenotype under normal conditions but showed increased root length under phosphate starvation, with no effect on phosphate starvation response including marker gene expression, root hair development and anthocyanin accumulation. Accompanying membrane glycerolipid profiling of LPAT2-overexpressing plants revealed an increased content of major phospholipid classes and distinct responses to phosphate starvation between shoot and root. The findings propose a revised model of membrane lipid remodelling, in which de novo phospholipid biosynthesis mediated by LPAT2 contributes significantly to root development under phosphate starvation. © 2017 John Wiley & Sons Ltd.

  4. New insight on FGFR3-related chondrodysplasias molecular physiopathology revealed by human chondrocyte gene expression profiling.

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    Laurent Schibler

    Full Text Available Endochondral ossification is the process by which the appendicular skeleton, facial bones, vertebrae and medial clavicles are formed and relies on the tight control of chondrocyte maturation. Fibroblast growth factor receptor (FGFR3 plays a role in bone development and maintenance and belongs to a family of proteins which differ in their ligand affinities and tissue distribution. Activating mutations of the FGFR3 gene lead to craniosynostosis and multiple types of skeletal dysplasia with varying degrees of severity: thanatophoric dysplasia (TD, achondroplasia and hypochondroplasia. Despite progress in the characterization of FGFR3-mediated regulation of cartilage development, many aspects remain unclear. The aim and the novelty of our study was to examine whole gene expression differences occurring in primary human chondrocytes isolated from normal cartilage or pathological cartilage from TD-affected fetuses, using Affymetrix technology. The phenotype of the primary cells was confirmed by the high expression of chondrocytic markers. Altered expression of genes associated with many cellular processes was observed, including cell growth and proliferation, cell cycle, cell adhesion, cell motility, metabolic pathways, signal transduction, cell cycle process and cell signaling. Most of the cell cycle process genes were down-regulated and consisted of genes involved in cell cycle progression, DNA biosynthesis, spindle dynamics and cytokinesis. About eight percent of all modulated genes were found to impact extracellular matrix (ECM structure and turnover, especially glycosaminoglycan (GAG and proteoglycan biosynthesis and sulfation. Altogether, the gene expression analyses provide new insight into the consequences of FGFR3 mutations in cell cycle regulation, onset of pre-hypertrophic differentiation and concomitant metabolism changes. Moreover, impaired motility and ECM properties may also provide clues about growth plate disorganization. These

  5. Sodium Thiosulfate Prevents Chondrocyte Mineralization and Reduces the Severity of Murine Osteoarthritis.

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    Sonia Nasi

    Full Text Available Calcium-containing crystals participate in the pathogenesis of OA. Sodium thiosulfate (STS has been shown to be an effective treatment in calcification disorders such as calciphylaxis and vascular calcification. This study investigated the effects and mechanisms of action of STS in a murine model of OA and in chondrocyte calcification.Hydroxyapatite (HA crystals-stimulated murine chondrocytes and macrophages were treated with STS. Mineralization and cellular production of IL-6, MCP-1 and reactive oxygen species (ROS were assayed. STS's effects on genes involved in calcification, inflammation and cartilage matrix degradation were studied by RT-PCR. STS was administered in the menisectomy model of murine OA, and the effect on periarticular calcific deposits and cartilage degeneration was investigated by micro-CT-scan and histology.In vitro, STS prevented in a dose-dependent manner calcium crystal deposition in chondrocytes and inhibited Annexin V gene expression. In addition, there was a reduction in crystal-induced IL-6 and MCP-1 production. STS also had an antioxidant effect, diminished HA-induced ROS generation and abrogated HA-induced catabolic responses in chondrocytes. In vivo, administration of STS reduced the histological severity of OA, by limiting the size of new periarticular calcific deposits and reducing the severity of cartilage damage.STS reduces the severity of periarticular calcification and cartilage damage in an animal model of OA via its effects on chondrocyte mineralization and its attenuation of crystal-induced inflammation as well as catabolic enzymes and ROS generation. Our study suggests that STS may be a disease-modifying drug in crystal-associated OA.

  6. Effect of hydrostatic pressure of various magnitudes on osteoarthritic chondrocytes exposed to IL-1beta.

    Science.gov (United States)

    Fioravanti, Antonella; Collodel, Giulia; Petraglia, Angela; Nerucci, Fabiola; Moretti, Elena; Galeazzi, Mauro

    2010-08-01

    Several in vitro studies have shown the importance of mechanical compression or hydrostatic pressure (HP) as a modulator of cartilage metabolism. The present study was undertaken to evaluate the in vitro effects of cyclical low HP (1-5 MPa) and continuous high HP (24 MPa) applied in the presence or absence of interleukin (IL)-1beta on human osteoarthritis (OA) chondrocytes. Chondrocytes obtained from OA cartilage were cultivated for 48 h and then exposed to pressurization in the presence or absence of IL-1beta. After pressurization, the culture medium was collected to detect the amount of proteoglycans (PG) and nitric oxide (NO) and the chondrocytes were immediately fixed for transmission electron microscopy (TEM) and processed for immunocytochemistry to localize the inducible nitric oxide synthase (iNOS). A significant increase in the level of PG and a small, non-significant, decrease in NO production were observed upon exposure to cyclical low HP. On the other hand, exposure to continuous high HP resulted in a significant decrease in the PG levels and a significant increase in NO production. The presence of IL-1beta led to a significant decrease in PG levels as well as a significant increase in NO production. The cyclical low HP did not increase the PG levels significantly but caused a statistically significant decrease in NO production in cultures damaged with IL-1beta. The continuous high HP in chondrocyte cultures stimulated with IL-1beta did not significantly decrease PG production, but significantly increased NO production. The results concerning metabolic production were further confirmed by morphological findings obtained by TEM and immunocytochemical studies. The findings of this study confirmed that the response of chondrocytes varies with magnitude and frequency of HP. These findings are important to understand aetiopathogenetic mechanisms of OA and to find out which type of physical activity may be best suited for the prevention and therapy of OA.

  7. Collagen-induced expression of collagenase-3 by primary chondrocytes is mediated by integrin α1 and discoidin domain receptor 2: a protein kinase C-dependent pathway.

    Science.gov (United States)

    Vonk, Lucienne A; Doulabi, Behrouz Z; Huang, ChunLing; Helder, Marco N; Everts, Vincent; Bank, Ruud A

    2011-03-01

    To investigate whether maintaining the chondrocyte's native pericellular matrix prevents collagen-induced up-regulation of collagenase-3 (MMP-13) and whether integrin α1 (ITGα1) and/or discoidin domain receptor 2 (DDR2) modulate MMP-13 expression and which signalling pathway plays a role in collagen-stimulated MMP-13 expression. Goat articular chondrocytes and chondrons were cultured on collagen coatings. Small interfering RNA (siRNA) oligonucleotides targeted against ITGα1 and DDR2 were transfected into primary chondrocytes. Chemical inhibitors for mitogen-activated protein kinase kinase (MEK1) (PD98059), focal adhesion kinase (FAK) (FAK inhibitor 14), mitogen-activated protein kinase 8 (JNK) (SP600125) and protein kinase C (PKC) (PKC412), and a calcium chelator (BAPTA-AM) were used in cell cultures. Real-time PCR was performed to examine gene expression levels of MMP-13, ITGα1 and DDR2 and collagenolytic activity was determined by measuring the amount of hydroxyproline released in the culture medium. Maintaining the chondrocyte's native pericellular matrix prevented MMP-13 up-regulation and collagenolytic activity when the cells were cultured on a collagen coating. Silencing of ITGα1 and DDR2 reduced MMP-13 gene expression and collagenolytic activity by primary chondrocytes cultured on collagen. Incubation with the PKC inhibitor strongly reduced MMP-13 gene expression levels. Gene expression levels of MMP-13 were also decreased by chondrocytes incubated with the MEK, FAK or JNK inhibitor. Maintaining the native pericellular matrix of chondrocytes prevents collagen-induced up-regulation of MMP-13. Both ITGα1 and DDR2 modulate MMP-13 expression after direct contact between chondrocytes and collagen. PKC, FAK, MEK and JNK are involved in collagen-stimulated expression of MMP-13.

  8. A Wnt/β-catenin negative feedback loop inhibits interleukin-1-induced matrix metalloproteinase expression in human articular chondrocytes.

    Science.gov (United States)

    Ma, Bin; van Blitterswijk, Clemens A; Karperien, Marcel

    2012-08-01

    The results of recent animal studies suggest that activation of Wnt/β-catenin signaling in articular chondrocytes might be a driving factor in the pathogenesis of osteoarthritis (OA) by stimulating, for instance, the expression of matrix metalloproteinases (MMPs). The aim of this study was to investigate the role of Wnt/β-catenin signaling in interleukin-1β (IL-1β)-induced MMP expression in human chondrocytes. Primary cultures of human, murine, and bovine articular chondrocytes as well as human mesenchymal stem cells and mouse embryonic fibroblasts were used in the experiments. Multiple strategies for the activation and inhibition of signaling pathways were utilized. Reporter assays and coimmunoprecipitation were performed to study the interaction between β-catenin and NF-κB. In contrast to the role of Wnt/β-catenin in animal chondrocytes, in human chondrocytes it was a potent inhibitor of MMP-1, MMP-3, and MMP-13 expression and generic MMP activity both in basal conditions and after IL-1β stimulation. This effect was independent of the T cell factor/lymphoid enhancer factor family of transcription factors but rather was attributable to an inhibitory protein-protein interaction between β-catenin and NF-κB. IL-1β indirectly activated β-catenin signaling by inducing canonical Wnt-7B expression and by inhibiting the expression of canonical Wnt antagonists. Wnt/β-catenin signaling in human chondrocytes had an unexpected anticatabolic role by counteracting NF-κB-mediated MMP expression induced by IL-1β in a negative feedback loop. Copyright © 2012 by the American College of Rheumatology.

  9. Stable subclones of the chondrogenic murine cell line MC615 mimic distinct stages of chondrocyte differentiation.

    Science.gov (United States)

    Surmann-Schmitt, Cordula; Widmann, Nathalie; Mallein-Gerin, Frédéric; von der Mark, Klaus; Stock, Michael

    2009-10-15

    Fourteen stable subclones derived from the murine chondrogenic cell line MC615 were established and characterised regarding their differentiation stages and responsivity to BMP2. Based on their gene expression profiles which revealed remarkable variances in Col2a1 and Col10a1 expression, subclones could be grouped into at least three distinct categories. Three representative subclones (4C3, 4C6 and 4H4) were further characterised with respect to gene expression pattern and differentiation capacity. These subclones resembled (i) weakly differentiated chondrogenic precursors, strongly responding to BMP2 stimulation (4C3), (ii) collagen II expressing chondrocytes which could be induced to undergo maturation (4C6) and (iii) mature chondrocytes expressing Col10a1 and other markers of hypertrophy (4H4). Interestingly, BMP2 administration caused Smad protein phosphorylation and stimulated Col10a1 expression in all clones, but induced Col2a1 expression only in precursor-like cells. Most remarkably, these clones maintained a stable gene expression profile at least until the 30th passage of subconfluent culture, but revealed reproducible changes in gene expression and differentiation pattern in long term high density cultures. Thus, the newly established MC615 subclones may serve as a potent new tool for investigations on the regulation of chondrocyte differentiation and function. (c) 2009 Wiley-Liss, Inc.

  10. 5-Arylidene-4-thiazolidinone derivatives active as antidegenerative agents on human chondrocyte cultures.

    Science.gov (United States)

    Panico, Annamaria; Maccari, Rosanna; Cardile, Venera; Crasci, Lucia; Ronsisvalle, Simone; Ottanà, Rosaria

    2013-02-01

    5-Arylidene-2-oxo-4-thiazolidinones and 2-phenylimino analogues were evaluated for their antidegenerative activity on human chondrocyte cultures stimulated by IL-1β and for their inhibitory capability against matrix metalloproteinase- 13. Our results indicated that 5-arylidene-4-thiazolidinone derivatives 1-9 exhibit antidegenerative activity and could block multiple cartilage destruction during the osteoarthritic process. Out of the selected compounds, (5-arylidene- 2,4-dioxothiazolidin-3-yl)acetic acids 7-9 showed significant effectiveness in reducing NO release and restoring normal levels of GAGs in chondrocytes treated with IL-1β. Moreover, benzoic acids 1, 5 and 6 proved to be effective MMP-13 inhibitors and were able to restore normal levels of GAGs.

  11. Endogenous versus Exogenous Growth Factor Regulation of Articular Chondrocytes

    OpenAIRE

    Shi, Shuiliang; Chan, Albert G.; Mercer, Scott; Eckert, George J.; Trippel, Stephen B.

    2013-01-01

    Anabolic growth factors that regulate the function of articular chondrocytes are candidates for articular cartilage repair. Such factors may be delivered by pharmacotherapy in the form of exogenous proteins, or by gene therapy as endogenous proteins. It is unknown whether delivery method influences growth factor effectiveness in regulating articular chondrocyte reparative functions. We treated adult bovine articular chondrocytes with exogenous recombinant insulin-like growth factor-I (IGF-I) ...

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

    Directory of Open Access Journals (Sweden)

    Joseph Schwager

    2016-04-01

    Full Text Available 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.

  13. Smad4 regulates growth plate matrix production and chondrocyte polarity.

    Science.gov (United States)

    Whitaker, Amanda T; Berthet, Ellora; Cantu, Andrea; Laird, Diana J; Alliston, Tamara

    2017-03-15

    Smad4 is an intracellular effector of the TGFβ family that has been implicated in Myhre syndrome, a skeletal dysplasia characterized by short stature, brachydactyly and stiff joints. The TGFβ pathway also plays a critical role in the development, organization and proliferation of the growth plate, although the exact mechanisms remain unclear. Skeletal phenotypes in Myhre syndrome overlap with processes regulated by the TGFβ pathway, including organization and proliferation of the growth plate and polarity of the chondrocyte. We used in vitro and in vivo models of Smad4 deficiency in chondrocytes to test the hypothesis that deregulated TGFβ signaling leads to aberrant extracellular matrix production and loss of chondrocyte polarity. Specifically, we evaluated growth plate chondrocyte polarity in tibiae of Col2-Cre +/- ;Smad4 fl/fl mice and in chondrocyte pellet cultures. In vitro and in vivo , Smad4 deficiency decreased aggrecan expression and increased MMP13 expression. Smad4 deficiency disrupted the balance of cartilage matrix synthesis and degradation, even though the sequential expression of growth plate chondrocyte markers was intact. Chondrocytes in Smad4-deficient growth plates also showed evidence of polarity defects, with impaired proliferation and ability to undergo the characteristic changes in shape, size and orientation as they differentiated from resting to hypertrophic chondrocytes. Therefore, we show that Smad4 controls chondrocyte proliferation, orientation, and hypertrophy and is important in regulating the extracellular matrix composition of the growth plate. © 2017. Published by The Company of Biologists Ltd.

  14. Smad4 regulates growth plate matrix production and chondrocyte polarity

    Directory of Open Access Journals (Sweden)

    Amanda T. Whitaker

    2017-03-01

    Full Text Available Smad4 is an intracellular effector of the TGFβ family that has been implicated in Myhre syndrome, a skeletal dysplasia characterized by short stature, brachydactyly and stiff joints. The TGFβ pathway also plays a critical role in the development, organization and proliferation of the growth plate, although the exact mechanisms remain unclear. Skeletal phenotypes in Myhre syndrome overlap with processes regulated by the TGFβ pathway, including organization and proliferation of the growth plate and polarity of the chondrocyte. We used in vitro and in vivo models of Smad4 deficiency in chondrocytes to test the hypothesis that deregulated TGFβ signaling leads to aberrant extracellular matrix production and loss of chondrocyte polarity. Specifically, we evaluated growth plate chondrocyte polarity in tibiae of Col2-Cre+/−;Smad4fl/fl mice and in chondrocyte pellet cultures. In vitro and in vivo, Smad4 deficiency decreased aggrecan expression and increased MMP13 expression. Smad4 deficiency disrupted the balance of cartilage matrix synthesis and degradation, even though the sequential expression of growth plate chondrocyte markers was intact. Chondrocytes in Smad4-deficient growth plates also showed evidence of polarity defects, with impaired proliferation and ability to undergo the characteristic changes in shape, size and orientation as they differentiated from resting to hypertrophic chondrocytes. Therefore, we show that Smad4 controls chondrocyte proliferation, orientation, and hypertrophy and is important in regulating the extracellular matrix composition of the growth plate.

  15. TrxR2 deficiencies promote chondrogenic differentiation and induce apoptosis of chondrocytes through mitochondrial reactive oxygen species

    Energy Technology Data Exchange (ETDEWEB)

    Yan, Jidong [Department of Human Anatomy, Histology and Embryology, School of Basic Medical Sciences, Xi’an Jiaotong University Health Science Center, Xi’an, Shaanxi 710061 (China); Xu, Jing [Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Xi’an Jiaotong University Health Science Center, Xi’an, Shaanxi 710061 (China); Fei, Yao [College of Life Sciences, Northwest University, Xi’an, Shaanxi Province 710069 (China); Jiang, Congshan; Zhu, Wenhua; Han, Yan [Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Xi’an Jiaotong University Health Science Center, Xi’an, Shaanxi 710061 (China); Lu, Shemin, E-mail: lushemin@xjtu.edu.cn [Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Xi’an Jiaotong University Health Science Center, Xi’an, Shaanxi 710061 (China); Key Laboratory of Environment and Genes Related to Diseases, Xi’an Jiaotong University, Ministry of Education of China (China)

    2016-05-15

    Thioredoxin reductase 2 (TrxR2) is a selenium (Se) containing protein. Se deficiency is associated with an endemic osteoarthropathy characterized by impaired cartilage formation. It is unclear whether TrxR2 have roles in cartilage function. We examined the effects of TrxR2 on chondrogenic ATDC5 cells through shRNA-mediated gene silencing of TrxR2. We demonstrated TrxR2 deficiencies could enhance chondrogenic differentiation and apoptosis of ATDC5 cells. TrxR2 deficiencies increased accumulation of cartilage glycosaminoglycans (GAGs) and mineralization. TrxR2 deficiencies also stimulated expression of extracellular (ECM) gene including Collagen II and Aggrecan. The enhanced chondrogenic properties were further confirmed by activation of Akt signaling which are required for chondrogenesis. In addition, TrxR2 deficiencies promoted chondrocyte proliferation through acceleration of cell cycle progression by increase in both S and G2/M phase cell distribution accompanied with induction of parathyroid hormone-related protein (PTHrP). Moreover, TrxR2 deficiencies induced chondrocyte death via apoptosis and increased cell sensitivity to exogenous oxidative stress. Furthermore, TrxR2 deficiencies induced emission of mitochondrial reactive oxygen species (ROS) without alteration of mitochondrial membrane potential and intracellular ATP content. Finally, treatment of TrxR2 deficiency cells with N-acetylcysteine (NAC) inhibited mitochondrial ROS production and chondrocyte apoptosis. NAC also prevented chondrogenic differentiation of TrxR2 deficiency cells by suppression of ECM gene expression, GAGs accumulation and mineralization, as well as attenuation of Akt signaling. Thus, TrxR2-mediated mitochondrial integrity is indispensable for chondrogenic differentiation of ATDC5 cells. TrxR2 deficiency-induced impaired proliferation and death of chondrocytes may be the pathological mechanism of the osteoarthropathy due to Se deficiency. Notably, this study also uncover the roles of

  16. Low nitrogen stress stimulating the indole-3-acetic acid biosynthesis of Serratia sp. ZM is vital for the survival of the bacterium and its plant growth-promoting characteristic.

    Science.gov (United States)

    Ouyang, Liming; Pei, Haiyan; Xu, Zhaohui

    2017-04-01

    Serratia sp. ZM is a plant growth-promoting (PGP) bacterial strain isolated from the rhizospheric soil of Populus euphratica in northwestern China. In this study, low nitrogen supply significantly stimulated the production of indole-3-acetic acid (IAA) in Serratia sp.ZM. The inoculation of the bacterium to wheat seedlings improved plant growth compared with the uninoculated group, and the stimulating effect was more prominent under low nitrogen stress. Inactivation of the predicted key gene in the IAA biosynthesis pathway impaired IAA production and significantly hampered mutant growth in poor medium. Furthermore, the IAA-deficient mutant lost the PGP effect under either normal or low nitrogen conditions in plant experiments. This study revealed the significant impact of environmental nitrogen levels on IAA production in the PGP strain and the vital effect of IAA on resistance physiology of both the bacterium and host plant. The characteristics of Serratia sp. ZM also indicated its application potential as a biofertilizer for plants, especially those suffering from poor nitrogen soil.

  17. Control of chondrocyte gene expression by actin dynamics: a novel role of cholesterol/Ror-alpha signalling in endochondral bone growth.

    Science.gov (United States)

    Woods, Anita; James, Claudine G; Wang, Guoyan; Dupuis, Holly; Beier, Frank

    2009-09-01

    Elucidating the signalling pathways that regulate chondrocyte differentiation, such as the actin cytoskeleton and Rho GTPases, during development is essential for understanding of pathological conditions of cartilage, such as chondrodysplasias and osteoarthritis. Manipulation of actin dynamics in tibia organ cultures isolated from E15.5 mice results in pronounced enhancement of endochondral bone growth and specific changes in growth plate architecture. Global changes in gene expression were examined of primary chondrocytes isolated from embryonic tibia, treated with the compounds cytochalasin D, jasplakinolide (actin modifiers) and the ROCK inhibitor Y27632. Cytochalasin D elicited the most pronounced response and induced many features of hypertrophic chondrocyte differentiation. Bioinformatics analyses of microarray data and expression validation by real-time PCR and immunohistochemistry resulted in the identification of the nuclear receptor retinoid related orphan receptor-alpha (Ror-alpha) as a novel putative regulator of chondrocyte hypertrophy. Expression of Ror-alpha target genes, (Lpl, fatty acid binding protein 4 [Fabp4], Cd36 and kruppel-like factor 5 [Klf15]) were induced during chondrocyte hypertrophy and by cytochalasin D and are cholesterol dependent. Stimulation of Ror-alpha by cholesterol results in increased bone growth and enlarged, rounded cells, a phenotype similar to chondrocyte hypertrophy and to the changes induced by cytochalasin D, while inhibition of cholesterol synthesis by lovastatin inhibits cytochalasin D induced bone growth. Additionally, we show that in a mouse model of cartilage specific (Col2-Cre) Rac1, inactivation results in increased Hif-1alpha (a regulator of Rora gene expression) and Ror-alpha(+) cells within hypertrophic growth plates. We provide evidence that cholesterol signalling through increased Ror-alpha expression stimulates chondrocyte hypertrophy and partially mediates responses of cartilage to actin dynamics.

  18. Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is increased in osteoarthritis and regulates chondrocyte catabolic and anabolic activities

    Science.gov (United States)

    Long, D.L.; Ulici, V.; Chubinskaya, S.; Loeser, R.F.

    2015-01-01

    Objective We determined if the epidermal growth factor receptor ligand HB-EGF is produced in cartilage and if it regulates chondrocyte anabolic or catabolic activity. Methods HB-EGF expression was measured by quantitative PCR using RNA isolated from mouse knee joint tissues and from normal and OA human chondrocytes. Immunohistochemistry was performed on normal and OA human cartilage and meniscus sections. Cultured chondrocytes were treated with fibronectin fragments (FN-f) as a catabolic stimulus and osteogenic protein 1 (OP-1) as an anabolic stimulus. Effects of HB-EGF on cell signaling were analyzed by immunoblotting of selected signaling proteins. MMP-13 was measured in conditioned media, proteoglycan synthesis was measured by sulfate incorporation, and matrix gene expression by quantitative PCR. Results HB-EGF expression was increased in 12-month old mice at 8 weeks after surgery to induce OA and increased amounts of HB-EGF were noted in human articular cartilage from OA knees. FN-f stimulated chondrocyte HB-EGF expression and HB-EGF stimulated chondrocyte MMP-13 production. However, HB-EGF was not required for FN-f stimulation of MMP-13 production. HB-EGF activated the ERK and p38 MAP kinases and stimulated phosphorylation of Smad1 at an inhibitory serine site which was associated with inhibition of OP-1 mediated proteoglycan synthesis and reduced aggrecan (ACAN) but not COL2A1 expression. Conclusion HB-EGF is a new factor identified in OA cartilage that promotes chondrocyte catabolic activity while inhibiting anabolic activity suggesting it could contribute to the catabolic-anabolic imbalance seen in OA cartilage. PMID:25937027

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

    Directory of Open Access Journals (Sweden)

    Abdul-Rehman Phull

    2016-01-01

    Full Text Available 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.

  20. L-Monomethyl-arginine decreases apoptosis of chondrocytes by ...

    African Journals Online (AJOL)

    Yomi

    2012-04-03

    Apr 3, 2012 ... Apoptosis of chondrocytes was detected by terminal deoxynucleotidyl transferase dUTP nick end ... number of degraded chondrocytes to disease severity. A subsequent study of a rabbit knee OA .... (400×) and analyzed with Image-pro Plus6.0 image processing system (Media Cybernetics, Inc. Bethesda, ...

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

    Science.gov (United States)

    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

  2. Evaluation of chondrocyte behavior in a new equine collagen scaffold useful for cartilage repair.

    Science.gov (United States)

    Grigolo, B; Desando, G; Cavallo, C; Zini, N; Ghisu, S; Facchini, A

    2011-01-01

    Association of biomaterials with autologous cells can provide a new generation of implantable devices for cartilage repair. An ideal scaffold should possess a preformed three-dimensional shape, fix the cells to the damaged area and prevent their migration into the articular cavity. Furthermore, the constructs should have sufficient mechanical strength to facilitate handling in a clinical setting and stimulate the uniform spreading of cells and a phenotype re-differentiation process. The aim of this study was to verify the ability of an equine collagen membrane to support the growth of human chondrocytes and to allow the re-expression of their original phenotype. This ability was assessed by the evaluation of collagen type I, II and aggrecan mRNA expression by Real-Time PCR. Immunohistochemical analyses were performed to evaluate collagen type I, II and proteoglycans synthesis. Electron microscopy was utilized to highlight the structure of the biomaterial and its interactions with the cells. Our data indicate that human chondrocytes seeded onto a collagen membrane express and produce collagen type II and aggrecan and downregulate the production of collagen type I during the experimental times analyzed. These results provide an in vitro demonstration for the therapeutic potential of autologous chondrocyte transplantation by an equine collagen membrane as a delivery vehicle in a tissue-engineered approach towards the repair of articular cartilage defects.

  3. Biomarkers of Chondrocyte Apoptosis and Autophagy in Osteoarthritis

    Directory of Open Access Journals (Sweden)

    Giuseppe Musumeci

    2015-08-01

    Full Text Available 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.

  4. Biomarkers of Chondrocyte Apoptosis and Autophagy in Osteoarthritis.

    Science.gov (United States)

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

    2015-08-31

    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.

  5. Arabinogalactan biosynthesis

    DEFF Research Database (Denmark)

    Poulsen, Christian Peter; Dilokpimol, Adiphol; Geshi, Naomi

    2015-01-01

    Arabinogalactan proteins are abundant cell surface proteoglycans in plants and are implicated to act as developmental markers during plant growth. We previously reported that AtGALT31A, AtGALT29A, and AtGLCAT14A-C, which are involved in the biosynthesis of arabinogalactan proteins, localize......GALT29A. Therefore, the electrostatic status of Y144, which is regulated by an unknown kinase/phosphatase system, may regulate AtGALT29A enzyme activity. Moreover, we have identified additional proteins, apyrase 3 (APY3; At1g14240) and UDPglucuronate epimerases 1 and 6 (GAE1, At4g30440; GAE6, At3g23820...

  6. FUNCTIONS OF THE mTOR SIGNALING PATHWAY IN NORMAL ARTICULAR CARTILAGE CHONDROCYTES AND IN OSTEOARTHRITIS

    Directory of Open Access Journals (Sweden)

    E. V. Chetina

    2016-01-01

    Full Text Available Osteoarthritis  (OA is a chronic disease associated with pain, stiffness, limited mobility and joint inflammation, as well as articular cartilage destruction.  Recent studies have shown the importance  of chondrocyte  differentiation (hypertrophy as one of the mechanisms  of cartilage degradation in OA. This suggests that chondrocyte  metabolism undergoes the profound changes during cartilage resorption,  which are due to dysregulation of cell function. One of the major cellular metabolic regulators is the protein mTOR (mechanistic target of rapamycin that controls cell growth, proliferation, protein biosynthesis and integrates extracellular signals from growth factors and hormones with amino acid availability and intracellular energy status. The importance  of mTOR activity for articular cartilage destruction  in OAis confirmed by significant changes in the work of mTOR regulatory network that involves multiple intracellular (growth factors, adenosine triphosphate, oxygen availability, and autophagy and extracellular (glucose, amino acids, lipids, and hexosamine signals. Moreover, the altered expression of the mTOR gene in the blood of patients with OA is associated with either increased pain or synovitis, which indicates that there is a strong metabolic heterogeneity in patients with OA and a need for a differentiated therapeutic  approach. The above problems are discussed in this review.

  7. [Significance and technique of autologous chondrocyte transplantation].

    Science.gov (United States)

    Fritz, J; Gaissmaier, C; Schewe, B; Weise, K

    2005-08-01

    The bad risk for an early onset of osteoarthritis in the knee increases with the size of a cartilage defect. A collateral meniscus- or ligament-tear will enforce this hazard in addition. In order to avoid such a development, relevant full-thickness cartilage defects should be reconstructed biologically and attendant meniscus- or ligament-tears as well as varus- or valgus deformities should be treated. A number of studies, including some prospective-randomized trials, have shown that autologous chondrocyte transplantation (ACT) is the most reliable procedure for a surgical treatment of full-thickness cartilage defects larger than 4 cm (2) in adults. One disadvantage of ACT is the extensive approach to the joint and often a hypertrophy of the repair tissue. To solve these problems, some different biomaterials for a matrix-assisted ACT have been developed. The scaffold we use has a covering membrane upside and a collagen-sponge carrying the chondrocytes. By means of special surgical instruments a minimally invasive implantation is possible, reducing the side-effects of an extensive approach. Animal studies showed the regeneration of a hyaline cartilage using our described system. However, results of current clinical studies with the different scaffolds must be awaited before an universal application of matrix-assisted ACT can be recommended.

  8. Influence of species and anatomical location on chondrocyte expansion

    Directory of Open Access Journals (Sweden)

    Hurtig Mark B

    2005-05-01

    Full Text Available Abstract Background Bovine articular cartilage is often used to study chondrocytes in vitro. It is difficult to correlate in vitro studies using bovine chondrocytes with in vivo studies using other species such as rabbits and sheep. The aim of this investigation was to study the effect of species, anatomical location and exogenous growth factors on chondrocyte proliferation in vitro. Methods Equine (EQ, bovine (BO and ovine (OV articular chondrocytes from metacarpophalangeal (fetlock (F, shoulder (S and knee (K joints were cultured in tissue culture flasks. Growth factors (rh-FGFb: 10 ng/ml; rh-TGFβ: 5 ng/ml were added to the cultures at days 2 and 4. On day 6, cells were counted and flow cytometry analysis was performed to determine cell size and granularity. A three factor ANOVA with paired Tukey's correction was used for statistical analysis. Results After 6 days in culture, cell numbers had increased in control groups of EQ-F, OV-S, OV-F and BO-F chondrocytes. The addition of rh-FGFb led to the highest increase in cell numbers in the BO-F, followed by EQ-F and OV-S chondrocytes. The addition of rh-TGFβ increased cell numbers in EQ-S and EQ-F chondrocytes, but showed nearly no effect on EQ-K, OV-K, OV-S, OV-F and BO-F chondrocytes. There was an overall difference with the addition of growth factors between the different species and joints. Conclusion Different proliferation profiles of chondrocytes from the various joints were found. Therefore, we recommend performing in vitro studies using the species and site where subsequent in vivo studies are planned.

  9. Proteomics of human primary osteoarthritic chondrocytes exposed to extremely low-frequency electromagnetic fields (ELF EMFs) and to therapeutic application of musically modulated electromagnetic fields (TAMMEF).

    Science.gov (United States)

    Corallo, Claudio; Battisti, Emilio; Albanese, Antonietta; Vannoni, Daniela; Leoncini, Roberto; Landi, Giacomo; Gagliardi, Assunta; Landi, Claudia; Carta, Serafino; Nuti, Ranuccio; Giordano, Nicola

    2014-01-01

    Osteoarthritis (OA) is the most frequent joint disease, characterized by degradation of extracellular matrix and alterations in chondrocyte metabolism. Some authors reported that electromagnetic fields (EMFs) can positively interfere with patients affected by OA, even though the nature of the interaction is still debated. Human primary osteoarthritic chondrocytes isolated from the femoral heads of OA-patients undergoing to total hip replacement, were cultured in vitro and exposed 30 min/day for two weeks to extremely-low-frequency electromagnetic field (ELF) with fixed frequency (100 Hz) and to therapeutic application of musically modulated electromagnetic fields (TAMMEF) with variable frequencies, intensities and waveforms. Sham-exposed (S.E.) cells served as control group. Cell viability was measured at days 2, 7 and 14. After two weeks, cell lysates were processed using a proteomic approach. Chondrocyte exposed to ELF and TAMMEF system demonstrated different viability compared to untreated chondrocytes (S.E.). Proteome analysis of 2D-Electrophoresis and protein identification by mass spectrometry showed different expression of proteins derived from nucleus, cytoplasm and organelles. Function analysis of the identified proteins showed changes in related-proteins metabolism (glyceraldeyde-3-phosphate-dehydrogenase), stress response (Mn-superoxide-dismutase, heat-shock proteins), cytoskeletal regulation (actin), proteinase inhibition (cystatin-B) and inflammation regulatory functions (S100-A10, S100-A11) among the experimental groups (ELF, TAMMEF and S.E.). In conclusion, EMFs do not cause damage to chondrocytes, besides stimulate safely OA-chondrocytes and are responsible of different protein expression among the three groups. Furthermore, protein analysis of OA-chondrocytes treated with ELF and the new TAMMEF systems could be useful to clarify the pathogenetic mechanisms of OA by identifying biomarkers of the disease.

  10. Hydrostatic Pressure Regulates MicroRNA Expression Levels in Osteoarthritic Chondrocyte Cultures via the Wnt/β-Catenin Pathway

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    Sara Cheleschi

    2017-01-01

    Full Text Available Mechanical loading and hydrostatic pressure (HP regulate chondrocytes’ metabolism; however, how mechanical stimulation acts remain unclear. MicroRNAs (miRNAs play an important role in cartilage homeostasis, mechanotransduction, and in the pathogenesis of osteoarthritis (OA. This study investigated the effects of a cyclic HP (1–5 MPa, in both normal and OA human chondrocytes, on the expression of miR-27a/b, miR-140, miR-146a/b, and miR-365, and of their target genes (MMP-13, ADAMTS-5, IGFBP-5, and HDAC-4. Furthermore, we assessed the possible involvement of Wnt/β-catenin pathway in response to HP. Chondrocytes were exposed to HP for 3h and the evaluations were performed immediately after pressurization, and following 12, 24, and 48 h. Total RNA was extracted and used for real-time PCR. β-catenin was detected by Western blotting analysis and immunofluorescence. In OA chondrocytes, HP induced a significant increase (p < 0.01 of the expression levels of miR-27a/b, miR-140, and miR-146a, and a significant reduction (p < 0.01 of miR-365 at all analyzed time points. MMP-13, ADAMTS-5, and HDAC-4 were significantly downregulated following HP, while no significant modification was found for IGFBP-5. β-catenin levels were significantly increased (p < 0.001 in OA chondrocytes at basal conditions and significantly reduced (p < 0.01 by HP. Pressurization did not cause any significant modification in normal cells. In conclusion, in OA chondrocytes, HP restores the expression levels of some miRNAs, downregulates MMP-13, ADAMTS-5, and HDAC-4, and modulates the Wnt/β-catenin pathway activation.

  11. Hydrostatic Pressure Regulates MicroRNA Expression Levels in Osteoarthritic Chondrocyte Cultures via the Wnt/β-Catenin Pathway

    Science.gov (United States)

    Cheleschi, Sara; De Palma, Anna; Pecorelli, Alessandra; Pascarelli, Nicola Antonio; Valacchi, Giuseppe; Belmonte, Giuseppe; Carta, Serafino; Galeazzi, Mauro; Fioravanti, Antonella

    2017-01-01

    Mechanical loading and hydrostatic pressure (HP) regulate chondrocytes’ metabolism; however, how mechanical stimulation acts remain unclear. MicroRNAs (miRNAs) play an important role in cartilage homeostasis, mechanotransduction, and in the pathogenesis of osteoarthritis (OA). This study investigated the effects of a cyclic HP (1–5 MPa), in both normal and OA human chondrocytes, on the expression of miR-27a/b, miR-140, miR-146a/b, and miR-365, and of their target genes (MMP-13, ADAMTS-5, IGFBP-5, and HDAC-4). Furthermore, we assessed the possible involvement of Wnt/β-catenin pathway in response to HP. Chondrocytes were exposed to HP for 3h and the evaluations were performed immediately after pressurization, and following 12, 24, and 48 h. Total RNA was extracted and used for real-time PCR. β-catenin was detected by Western blotting analysis and immunofluorescence. In OA chondrocytes, HP induced a significant increase (p < 0.01) of the expression levels of miR-27a/b, miR-140, and miR-146a, and a significant reduction (p < 0.01) of miR-365 at all analyzed time points. MMP-13, ADAMTS-5, and HDAC-4 were significantly downregulated following HP, while no significant modification was found for IGFBP-5. β-catenin levels were significantly increased (p < 0.001) in OA chondrocytes at basal conditions and significantly reduced (p < 0.01) by HP. Pressurization did not cause any significant modification in normal cells. In conclusion, in OA chondrocytes, HP restores the expression levels of some miRNAs, downregulates MMP-13, ADAMTS-5, and HDAC-4, and modulates the Wnt/β-catenin pathway activation. PMID:28085114

  12. Berberine prevents nitric oxide-induced rat chondrocyte apoptosis and cartilage degeneration in a rat osteoarthritis model via AMPK and p38 MAPK signaling.

    Science.gov (United States)

    Zhou, Yan; Liu, Shi-Qing; Yu, Ling; He, Bin; Wu, Shi-Hao; Zhao, Qi; Xia, Shao-Qiang; Mei, Hong-Jun

    2015-09-01

    Chondrocyte apoptosis is an important mechanism involved in osteoarthritis (OA). Berberine (BBR), a plant alkaloid derived from Chinese medicine, is characterized by multiple pharmacological effects, such as anti-inflammatory and anti-apoptotic activities. This study aimed to evaluate the chondroprotective effect and underlying mechanisms of BBR on sodium nitroprusside (SNP)-stimulated chondrocyte apoptosis and surgically-induced rat OA model. The in vitro results revealed that BBR suppressed SNP-stimulated chondrocyte apoptosis as well as cytoskeletal remodeling, down-regulated expressions of inducible nitric oxide synthase (iNOS) and caspase-3, and up-regulated Bcl-2/Bax ratio and Type II collagen (Col II) at protein levels, which were accompanied by increased adenosine monophosphate-activated protein kinase (AMPK) phosphorylation and decreased phosphorylation of p38 mitogen-activated protein kinase (MAPK). Furthermore, the anti-apoptotic effect of BBR was blocked by AMPK inhibitor Compound C (CC) and adenosine-9-β-D-arabino-furanoside (Ara A), and enhanced by p38 MAPK inhibitor SB203580. In vivo experiment suggested that BBR ameliorated cartilage degeneration and exhibited an anti-apoptotic effect on articular cartilage in a rat OA model, as demonstrated by histological analyses, TUNEL assay and immunohistochemical analyses of caspase-3, Bcl-2 and Bax expressions. These findings suggest that BBR suppresses SNP-stimulated chondrocyte apoptosis and ameliorates cartilage degeneration via activating AMPK signaling and suppressing p38 MAPK activity.

  13. Ofloxacin induces apoptosis in microencapsulated juvenile rabbit chondrocytes by caspase-8-dependent mitochondrial pathway

    International Nuclear Information System (INIS)

    Sheng Zhiguo; Cao Xiaojuan; Peng Shuangqing; Wang Changyong; Li Qianqian; Wang Yimei; Liu Mifeng

    2008-01-01

    Quinolones (QNs)-induced arthropathy is an important toxic effect in immature animals leading to restriction of their therapeutic use in pediatrics. However, the exact mechanism still remains unclear. Recently, we have demonstrated that ofloxacin, a typical QN, induces apoptosis of alginate microencapsulated juvenile rabbit joint chondrocytes by disturbing the β 1 integrin functions and inactivating the ERK/MAPK signaling pathway. In this study, we extend our initial observations to further elucidate the mechanism(s) of ofloxacin-induced apoptosis by utilizing specific caspase inhibitors. Pretreatment with both caspase-9-specific inhibitor zLEHD-fmk and caspase-8 inhibitor zIETD-fmk attenuated ofloxacin-induced apoptosis and activation of caspase-3 of chondrocyte in a concentration-dependent manner, as determined by fluorescent dye staining, enzyme activity assay and immunoblotting. Furthermore, the activation of caspase-9, -8 and -3 stimulated by ofloxacin was significantly inhibited in the presence of zIETD-fmk while pretreatment with zLEHD-fmk only blocked the activation of caspase-9 and -3. Ofloxacin also stimulated a concentration-dependent translocation of cytochrome c from mitochondria into the cytosol and a decrease of mitochondrial transmembrane potential, which was completely inhibited by zIETD-fmk. In addition, ofloxacin was found to increase the level of Bax, tBid, p53 in a concentration- and time-dependent manner. Taken together, The current results indicate that the caspase-8-dependent mitochondrial pathway is primarily involved in the ofloxacin-induced apoptosis of microencapsulated juvenile rabbit joint chondrocytes

  14. Divergent responses of chondrocytes and endothelial cells to shear stress: Cross-talk among COX-2, the phase 2 response, and apoptosis

    Science.gov (United States)

    Healy, Zachary R.; Lee, Norman H.; Gao, Xiangqun; Goldring, Mary B.; Talalay, Paul; Kensler, Thomas W.; Konstantopoulos, Konstantinos

    2005-01-01

    Fluid shear exerts anti-inflammatory and anti-apoptotic effects on endothelial cells by inducing the coordinated expression of phase 2 detoxifying and antioxidant genes. In contrast, high shear is pro-apoptotic in chondrocytes and promotes matrix degradation and cartilage destruction. We have analyzed the mechanisms regulating shear-mediated chondrocyte apoptosis by cDNA microarray technology and bioinformatics. We demonstrate that shear-induced cyclooxygenase (COX)-2 suppresses phosphatidylinositol 3-kinase (PI3-K) activity, which represses antioxidant response element (ARE)/NF-E2 related factor 2 (Nrf2)-mediated transcriptional response in human chondrocytes. The resultant decrease in antioxidant capacity of sheared chondrocytes contributes to their apoptosis. Phase 2 inducers, and to a lesser extent COX-2-selective inhibitors, negate the shear-mediated suppression of ARE-driven phase 2 activity and apoptosis. The abrogation of shear-induced COX-2 expression by PI3-K activity and/or stimulation of the Nrf2/ARE pathway suggests the existence of PI3-K/Nrf2/ARE negative feedback loops that potentially interfere with c-Jun N-terminal kinase 2 activity upstream of COX-2. Reconstructing the signaling network regulating shear-induced chondrocyte apoptosis may provide insights to optimize conditions for culturing artificial cartilage in bioreactors and for developing therapeutic strategies for arthritic disorders. PMID:16172382

  15. Phenotyping of chondrocytes from human osteoarthritic cartilage: chondrocyte expression of beta integrins and correlation with anatomic injury

    Directory of Open Access Journals (Sweden)

    G. Lapadula

    2011-09-01

    Full Text Available Chondrocyte-ECM (extracellular matrix interactions are believed to play a pivotal role in the development and metabolic homeostasis of articular cartilage. Cell surface adhesion molecules have been reported to modulate chondrocyte binding to ECM (collagen, fibronectin, laminin and they also act as transducers of critical signals in many biological processes such as growth, differentiation, migration and matrix synthesis. Recently, it has been shown that normal human articular chondrocytes strongly express ß1 integrins, which are constituted by a common chain (ß1 and a variable α chain, but the behaviour of these molecules in human osteoarthritic cartilage has not been extensively investigated. We studied the expression of ß integrins (ß1-5, α1-6, av chains, LFA-1, ICAM-1 and CD44, on freshly isolated chondrocytes obtained from 10 osteoarthritic patients undergoing surgical knee replacement. Chondrocytes were isolated by enzymatic digestion from three zones of each articular cartilage with a differing degree of macroscopic and microscopic damage. Integrin expression and cell cycle analysis were carried out by flowcytometry. Chondrocytes from costal cartilages of 5 human fetuses were also studied. Chondrocytes from osteoarthritic cartilage expressed high levels of ß1 integrin and, at different percantages, all the α chains. The α chain most frequentiy expressed was α1, foilowed by α3, α5, α2, αv. Integrin expression decreased from the least to the most damaged zone of articular cartilage and cell cycle analysis showed that proliferating chondrocytes (S phase were prevalent on the latter zone. ß2, ß3, ß2, ß5, CD44, LFA-1/ICAM-1 complex were very low expressed. Fetal chondrocytes strongly expressed ß1 and ß5 chains. These data provide evidence to show that integrin expression on human chondrocytes changes in osteoarthritis and suggest that perturbations of chondrocyte-ECM signalling occur in the development of the disease. The

  16. Inhibition of T-Type Voltage Sensitive Calcium Channel Reduces Load-Induced OA in Mice and Suppresses the Catabolic Effect of Bone Mechanical Stress on Chondrocytes.

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    Padma P Srinivasan

    Full Text Available Voltage-sensitive calcium channels (VSCC regulate cellular calcium influx, one of the earliest responses to mechanical stimulation in osteoblasts. Here, we postulate that T-type VSCCs play an essential role in bone mechanical response to load and participate in events leading to the pathology of load-induced OA. Repetitive mechanical insult was used to induce OA in Cav3.2 T-VSCC null and wild-type control mouse knees. Osteoblasts (MC3T3-E1 and chondrocytes were treated with a selective T-VSCC inhibitor and subjected to fluid shear stress to determine how blocking of T-VSCCs alters the expression profile of each cell type upon mechanical stimulation. Conditioned-media (CM obtained from static and sheared MC3T3-E1 was used to assess the effect of osteoblast-derived factors on the chondrocyte phenotype. T-VSCC null knees exhibited significantly lower focal articular cartilage damage than age-matched controls. In vitro inhibition of T-VSCC significantly reduced the expression of both early and late mechanoresponsive genes in osteoblasts but had no effect on gene expression in chondrocytes. Furthermore, treatment of chondrocytes with CM obtained from sheared osteoblasts induced expression of markers of hypertrophy in chondrocytes and this was nearly abolished when osteoblasts were pre-treated with the T-VSCC-specific inhibitor. These results indicate that T-VSCC plays a role in signaling events associated with induction of OA and is essential to the release of osteoblast-derived factors that promote an early OA phenotype in chondrocytes. Further, these findings suggest that local inhibition of T-VSCC may serve as a therapy for blocking load-induced bone formation that results in cartilage degeneration.

  17. Altered Chondrocyte Apoptosis Status in Developmental Hip Dysplasia in Rabbits

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    Yi-Shan Wei

    2016-12-01

    Full Text Available Background: Developmental dysplasia of the hip (DDH is an important factor leading to early adult osteoarthritis. Chondrocyte apoptosis has been proven to be an important factor causing osteoarthritis. Aims: The current study aims to explore whether a rabbit model of developmental dysplasia of the hip through cast immobilization in the legs results in chondrocyte apoptosis. Study Design: Animal experimentation. Methods: Thirty-two New Zealand white rabbits were divided in three groups with cast plaster-induced dislocation at 2, 4 and 6 weeks. The contralateral hip joint was utilized as a control group. Ten rabbits in each group were sacrificed, and hip specimens were obtained. Bcl-2/Bax, cleaved caspase-3 and cleaved caspase-8 expression were examined by western blot analysis. Chondrocyte apoptosis was analyzed through transmission electron microscopy (TEM and TUNEL analysis. All experiments were repeated at least three times. Results: In the experimental group, Bcl-2/Bax, cleaved caspase-3 and cleaved caspase-8 expression were significantly altered. The Bcl-2/Bax ratio decreased with time (all p<0.01, whereas levels of cleaved caspase-3 (p<0.01 and p<0.05 and cleaved caspase-8 (all p<0.05 gradually increased. Chondrocyte apoptosis was observed through transmission electron microscopy (TEM and TUNEL analysis (p<0.05 at 4 weeks and p<0.01 at 6 weeks. Conclusion: Prolonged immobilization of rabbit hip caused chondrocyte apoptosis. Reduction of the hip joint may protect chondrocytes from apoptosis, thus preventing secondary osteoarthritis.

  18. The chondrocytic journey in endochondral bone growth and skeletal dysplasia.

    Science.gov (United States)

    Yeung Tsang, Kwok; Wa Tsang, Shun; Chan, Danny; Cheah, Kathryn S E

    2014-03-01

    The endochondral bones of the skeleton develop from a cartilage template and grow via a process involving a cascade of chondrocyte differentiation steps culminating in formation of a growth plate and the replacement of cartilage by bone. This process of endochondral ossification, driven by the generation of chondrocytes and their subsequent proliferation, differentiation, and production of extracellular matrix constitute a journey, deviation from which inevitably disrupts bone growth and development, and is the basis of human skeletal dysplasias with a wide range of phenotypic severity, from perinatal lethality to progressively deforming. This highly coordinated journey of chondrocyte specification and fate determination is controlled by a myriad of intrinsic and extrinsic factors. SOX9 is the master transcription factor that, in concert with varying partners along the way, directs the different phases of the journey from mesenchymal condensation, chondrogenesis, differentiation, proliferation, and maturation. Extracellular signals, including bone morphogenetic proteins, wingless-related MMTV integration site (WNT), fibroblast growth factor, Indian hedgehog, and parathyroid hormone-related peptide, are all indispensable for growth plate chondrocytes to align and organize into the appropriate columnar architecture and controls their maturation and transition to hypertrophy. Chondrocyte hypertrophy, marked by dramatic volume increase in phases, is controlled by transcription factors SOX9, Runt-related transcription factor, and FOXA2. Hypertrophic chondrocytes mediate the cartilage to bone transition and concomitantly face a live-or-die situation, a subject of much debate. We review recent insights into the coordination of the phases of the chondrocyte journey, and highlight the need for a systems level understanding of the regulatory networks that will facilitate the development of therapeutic approaches for skeletal dysplasia. Copyright © 2014 Wiley Periodicals

  19. Blockade of recombinant human IL-6 by tocilizumab suppresses matrix metalloproteinase-9 production in the C28/I2 immortalized human chondrocyte cell line.

    Science.gov (United States)

    Meszaros, Evan C; Dahoud, Wissam; Mesiano, Sam; Malemud, Charles J

    Two immortalized human juvenile chondrocyte cell lines, T/C28a2 and C28/I2, were employed to determine the extent to which recombinant human (rh) IL-6 or rh-TNF-α increased the production of matrix metalloproteinase-9 (MMP-9). The effect of rhIL-6 on neutrophil gelatinase-associated lipocalin (NGAL) was also assessed. Although C28/I2 chondrocytes incubated with rhIL-6 (50 ng/ml) increased MMP-9 production which could not be mimicked by the T/C28a2 chondrocyte line, the effect of rhTNF-α on MMP-9 was more robust than with rhIL-6. The combinations of rhIL-6 and soluble IL-6 receptor-α (sIL-6Rα) or rhIL-6 and tocilizumab (TCZ), a fully-humanized recombinant monoclonal antibody that neutralizes the interaction between IL-6 and IL-6R significantly reduced MMP-9 production by C28/I2 chondrocytes. However, TCZ had no effect on rhTNF-α-induced MMP-9 production. By contrast, rhIL-6 did not increase the production of NGAL by C28/I2 chondrocytes although the number of NGAL-positive cells was significantly reduced by sIL-6R compared to its control group, but not by the combination of rhIL-6 plus TCZ compared to rhIL-6. In summary, these results showed that rhIL-6 stimulated the production of MMP-9, but not NGAL, in the C28/I2 chondrocyte line. TCZ or sIL-6Rα suppressed rhIL-6-induced MMP-9 production.

  20. The influence of “Efial” medicine on the chondrocytes functional state

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    N. А. Volkova

    2014-12-01

    Full Text Available Renewal of articular cartilage is a topical issue of modern orthopedics. High frequency of injuries, complexity of clinical diagnosis and subsequent treatment, and also the delay in recovery lead to the development of osteoarthritis, and in some cases, to disability. Articular cartilage belongs to the highly specialized tissues, which is characterized by the lack of blood supply, the low number of cell elements that are placed in the matrix, include collagen, proteoglycans, non-collagenous proteins and water. For the treatment of articular cartilage lesions the medicine which are tissue specific promoters of regeneration are used. The ability of most reparants to stimulate cartilage regeneration combines with other effects, such as: anti-inflammatory, antioxidant and antibacterial. The purpose of administration of these medicines is to stimulate regeneration of tissue in the area of injury. The aim of research was to investigate the effect of “Efial” medicine on functional state of chondrocytes in cultivation conditions. Materials and methods. The chondrocytes were obtained from articular cartilage of rats by enzymatic disaggregation. In all experiments the seeding concentration of chondrocytes was 1.2 x 104 cells/cm2.The "Efial" medicine in concentration of peptides of 0.137 mg/ml was used. Investigated concentration range was 70; 7.6; 1.5; 0.15µg/ml and 75; 15; 1.5 ng/ml. The medicine was added to the cell culture medium when seeding and on the 3rd cultivation day. The control (comparison group was the cultures of chondrocytes which were cultivated under the same conditions without medicine addition. Functional state of chondrocytes under interaction with investigated "Efial" medicine was evaluated by the presence of glycosaminoglycans after Toluidine blue staining (Fluka, Germany and collagen type II (1:200 and FITC-conjugate, Sigma -Aldrich, USA. For statistical study ANOVA and t-Student tests were used with application of Microsoft

  1. Characterization of collagenase-3 binding and internalization by rabbit chondrocytes

    International Nuclear Information System (INIS)

    Raggatt, L.J.; Choundhury, I.; Williams, S.

    2002-01-01

    Full text: Collagenase-3 (MMP-13) is an extracellular matrix metalloproteinase that cleaves type II collagen, the major protein component of cartilage, with high specificity. Several studies have identified increased levels of MMP-13 in arthritic synovial fluid where it may contribute to matrix destruction in this disease. Our laboratory has previously documented a process where by osteoblastic cells remove MMP-13 from the surrounding milieu by binding the enzyme to a specific receptor. The enzyme is then internalized and degraded through the actions of the endocytotic receptor, the low-density lipoprotein receptor-related protein (LRP). Such a mechanism provides for a controlled elimination of a potentially destructive enzyme from the extracellular environment. This process of MMP-13 internalization also occurs in chondrocytes and is significantly reduced in OA chondrocytes. We are currently characterizing the internalization of MMP-13 in normal rabbit chondrocytes. Primary rabbit chondrocytes were harvested and cultured in monolayers for three passages. Reverse transcription polymerase chain reaction (RT-PCR) was used to asses the cell phenotype during the culture period and the rabbit chondrocytes were found to express the cartilage specific genes aggrecan and type II collagen throughout this time. 125I-MMP-13 was used to assess the ability of the rabbit chondrocytes to bind MMP-13. Appreciable specific cell-association of MMP-13 was detected after 10 mm of exposure to the ligand and equilibrium was obtained after 2 h. After identifying the time to equilibrium we determined whether binding was saturable by incubating the chondrocytes with increasing concentrations of 125I-MMP-13 ranging from 0 to 100 nM at 4 deg C for 2h. The amount of specifically associated MMP-13 approached saturation at 75 nM, allowing assessment of the receptor kinetics. Finally, we have assessed the ability of rabbit chondrocytes to internalize a single cohort of 125I-MMP-13 over time at

  2. Extracellular matrix domain formation as an indicator of chondrocyte dedifferentiation and hypertrophy

    NARCIS (Netherlands)

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

    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

  3. TGF-β2 is involved in the preservation of the chondrocyte phenotype under hypoxic conditions

    NARCIS (Netherlands)

    Das, R.; Timur, U. T.; Edip, S.; Haak, E.; Wruck, C.; Weinans, H.; Jahr, H.

    2015-01-01

    Culturing chondrocytes under oxygen tension closely resembling their in vivo environment has been shown to have positive effects on matrix synthesis. In redifferentiation of expanded chondrocytes, hypoxia increased collagen type II expression. However, the mechanism by which hypoxia enhances

  4. Pronounced biomaterial dependency in cartilage regeneration using nonexpanded compared with expanded chondrocytes

    NARCIS (Netherlands)

    Tsuchida, A.I.; Bekkers, J.E.J.; Beekhuizen, M.; Vonk, L.A.; Dhert, W.J.A.; Saris, Daniël B.F.; Creemers, L.B.

    2013-01-01

    We aimed to investigate freshly isolated compared with culture-expanded chondrocytes with respect to early regenerative response, cytokine production and cartilage formation in response to four commonly used biomaterials. Materials & methods: Chondrocytes were both directly and after expansion to

  5. Roles of Chondrocytes in Endochondral Bone Formation and Fracture Repair.

    Science.gov (United States)

    Hinton, R J; Jing, Y; Jing, J; Feng, J Q

    2017-01-01

    The formation of the mandibular condylar cartilage (MCC) and its subchondral bone is an important but understudied topic in dental research. The current concept regarding endochondral bone formation postulates that most hypertrophic chondrocytes undergo programmed cell death prior to bone formation. Under this paradigm, the MCC and its underlying bone are thought to result from 2 closely linked but separate processes: chondrogenesis and osteogenesis. However, recent investigations using cell lineage tracing techniques have demonstrated that many, perhaps the majority, of bone cells are derived via direct transformation from chondrocytes. In this review, the authors will briefly discuss the history of this idea and describe recent studies that clearly demonstrate that the direct transformation of chondrocytes into bone cells is common in both long bone and mandibular condyle development and during bone fracture repair. The authors will also provide new evidence of a distinct difference in ossification orientation in the condylar ramus (1 ossification center) versus long bone ossification formation (2 ossification centers). Based on our recent findings and those of other laboratories, we propose a new model that contrasts the mode of bone formation in much of the mandibular ramus (chondrocyte-derived) with intramembranous bone formation of the mandibular body (non-chondrocyte-derived).

  6. Altered Chondrocyte Apoptosis Status in Developmental Hip Dysplasia in Rabbits.

    Science.gov (United States)

    Wei, Yi-Shan; Li, Dai-He; Liu, Wan-Lin; Jiang, Dian-Ming

    2016-11-01

    Developmental dysplasia of the hip (DDH) is an important factor leading to early adult osteoarthritis. Chondrocyte apoptosis has been proven to be an important factor causing osteoarthritis. The current study aims to explore whether a rabbit model of developmental dysplasia of the hip through cast immobilization in the legs results in chondrocyte apoptosis. Animal experimentation. Thirty-two New Zealand white rabbits were divided in three groups with cast plaster-induced dislocation at 2, 4 and 6 weeks. The contralateral hip joint was utilized as a control group. Ten rabbits in each group were sacrificed, and hip specimens were obtained. Bcl-2/Bax, cleaved caspase-3 and cleaved caspase-8 expression were examined by western blot analysis. Chondrocyte apoptosis was analyzed through transmission electron microscopy (TEM) and TUNEL analysis. All experiments were repeated at least three times. In the experimental group, Bcl-2/Bax, cleaved caspase-3 and cleaved caspase-8 expression were significantly altered. The Bcl-2/Bax ratio decreased with time (all phip caused chondrocyte apoptosis. Reduction of the hip joint may protect chondrocytes from apoptosis, thus preventing secondary osteoarthritis.

  7. Chondrocyte secreted CRTAC1: a glycosylated extracellular matrix molecule of human articular cartilage.

    Science.gov (United States)

    Steck, Eric; Bräun, Jessica; Pelttari, Karoliina; Kadel, Stephanie; Kalbacher, Hubert; Richter, Wiltrud

    2007-01-01

    Cartilage acidic protein 1 (CRTAC1), a novel human marker which allowed discrimination of human chondrocytes from osteoblasts and mesenchymal stem cells in culture was so far studied only on the RNA-level. We here describe its genomic organisation and detect a new brain expressed (CRTAC1-B) isoform resulting from alternate last exon usage which is highly conserved in vertebrates. In humans, we identify an exon sharing process with the neighbouring tail-to-tail orientated gene leading to CRTAC1-A. This isoform is produced by cultured human chondrocytes, localized in the extracellular matrix of articular cartilage and its secretion can be stimulated by BMP4. Of five putative O-glycosylation motifs in the last exon of CRTAC1-A, the most C-terminal one is modified according to exposure of serial C-terminal deletion mutants to the O-glycosylation inhibitor Benzyl-alpha-GalNAc. Both isoforms contain four FG-GAP repeat domains and an RGD integrin binding motif, suggesting cell-cell or cell-matrix interaction potential. In summary, CRTAC1 acquired an alternate last exon from the tail-to-tail oriented neighbouring gene in humans resulting in the glycosylated isoform CRTAC1-A which represents a new extracellular matrix molecule of articular cartilage.

  8. THE EFFECT OF PIROXICAM ON THE METABOLISM OF ISOLATED HUMAN CHONDROCYTES

    NARCIS (Netherlands)

    BULSTRA, SK; KUIJER, R; BUURMAN, WA; TERWINDTROUWENHORST, E; GUELEN, PJM; VANDERLINDEN, AJ

    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

  9. Spanish Experience in Autologous Chondrocyte Implantation

    Science.gov (United States)

    Pérez-Cachafeiro, Santiago; Ruano-Raviña, Alberto; Couceiro-Follente, José; Benedí-Alcaine, Jose Antonio; Nebot-Sanchis, Ignacio; Casquete-Román, Ciriaco; Bello-Prats, Santiago; Couceiro-Sánchez, Gonzalo; Blanco, Francisco J.

    2010-01-01

    Introduction: The Spanish Ministry of Health commissioned the Galician Agency for Health Technology Assessment to monitor and follow-up Autologous Chondrocyte Implantation (ACI) used to treat chondral lesions of the knee in Spain. The objective of this monitoring was to assess efficacy and safety of the technique. Design: One-hundred and eleven consecutive patients with knee chondral lesions were included in a multi-center study between January 2001 and January 2005. ACI was used in these patients as a second-line treatment option (or a first-line treatment option if the cause was Osteocondritis dissecans). The Cincinnati score and the Short Form 36 (SF-36) questionnaire were used to assess the patients’ self-reported satisfaction with the outcomes of ACI. A descriptive analysis was performed and non-parametric tests were used to establish correlations and compare results among subgroups. A multivariate analysis was also performed to measure the effect of different variables on changes in the condition of the knee. Results: Eighty men (72%) and 31 women (21%) with an age range from 16 to 49 years, underwent ACI surgery. Among these subjects, the most common previous first-line treatment was debridement (64 individuals, 74.4%). The mean size of the lesion treated with ACI was 3.82 cm2, and the most frequent location of the lesion was the inner femoral condyle (53.6%). The patient satisfaction was high or very high in 36 subjects (66.7%). Overall knee joint assessment improved from 4.32 points to 6.78. All SF-36 questionnaire categories improved, notably those related to physical condition. Conclusions: The results of this study indicate that ACI is safe; however, further studies are mandated to assess the efficacy of ACI compared to alternative treatment options. PMID:20148094

  10. Rabbit chondrocytes maintained in serum-free medium. I. Synthesis and secretion of hydrodynamically-small proteoglycans

    International Nuclear Information System (INIS)

    Malemud, C.J.; Papay, R.S.

    1986-01-01

    The biosynthesis of sulfated proteoglycan in vitro by rabbit articular chondrocytes in first passage monolayer culture maintained in fetal bovine serum (FBS) or in serum-free conditions was compared. Neosynthesized proteoglycan in the culture medium in the most dense fraction of an associative CsCl density gradient (fraction dAl) declined with increasing time under serum-free conditions, but not when cells were maintained in the presence of serum. After one day, the major peak of incorporated 35 SO 4 in medium fraction dAl eluted as a retarded peak on Sepharose CL-2B, whether cells were maintained under serum-free or serum-containing conditions. The hydrodynamic size of proteoglycan monomer fraction dAlDl obtained after one day of exposure to serum-free culture media was smaller than dAlDl from serum-containing cultures. The hydrodynamic size of dAlDl obtained from serum-free culture media became even progressively smaller after 2 and 3 days' exposure to these conditions. Hydrodynamically small sulfated proteoglycans were identified in the cell-associated dAlDl fraction as early as one day after switching chondrocytes from serum-containing to serum-free medium. Proteoglycan monomer from serum-containing medium reaggregated more efficiently under both conditions. No change in the size of glycosaminoglycan chains was seen in the smaller proteoglycan subpopulations, nor was there any indication of marked changes in the glycosaminoglycan types

  11. The proteoglycan biglycan mediates inflammatory response by activating TLR-4 in human chondrocytes: Inhibition by specific siRNA and high polymerized Hyaluronan.

    Science.gov (United States)

    Avenoso, Angela; D'Ascola, Angela; Scuruchi, Michele; Mandraffino, Giuseppe; Calatroni, Alberto; Saitta, Antonino; Campo, Salvatore; Campo, Giuseppe M

    2018-02-15

    Cartilage degeneration are hallmarks of wear, tear, mechanical and inflammatory damage of the joint cartilage. Tissue degradation as well as compromising the integrity and function of the organ, produces different intermediates, directly able to stimulate further inflammatory effect, therefore, amplifying the inflammation response. Biglycan is a soluble component of the extracellular matrix that is released during tissue injury. It has been reported that released biglycan is an endogenous ligand for TLR-2/4 in some cell type. We studied the role of biglycan in an experimental model of biglycan-induced inflammatory response in human chondrocytes and the effect of high polymerized HA on reducing its activity. Exposition of chondrocytes to LPS generated cell injury, including high levels of biglycan. Chondrocyte treatment with biglycan produces a high mRNA expression of several detrimental inflammation mediators such as IL-1β, IL-6, MMP-13, and IL-17, as well as NF-kB and TLR-4 activation. Administration of high polymerized HA to chondrocytes exposed to biglycan was able to attenuate the inflammatory response by decreasing the expression of the inflammatory mediators. Involvement of the TLR-4 in the mediation of the biglycan action was confirmed using a specific silent agent (siRNA). Taken together, these data could be used to develop new anti-inflammatory approaches. Copyright © 2018 Elsevier Inc. All rights reserved.

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

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    Saha, Sushmita [Biomaterials and Tissue Engineering Group, Leeds Dental Institute, University of Leeds, LS29LU (United Kingdom); Kirkham, Jennifer [Biomineralisation Group, Leeds Dental Institute, University of Leeds, LS29LU (United Kingdom); NIHR Leeds Musculoskeletal Biomedical Research Unit, University of Leeds, Chapel Allerton Hospital, Leeds LS74SA (United Kingdom); Wood, David [Biomaterials and Tissue Engineering Group, Leeds Dental Institute, University of Leeds, LS29LU (United Kingdom); Curran, Stephen [Smith and Nephew Research Centre, YO105DF (United Kingdom); Yang, Xuebin, E-mail: X.B.Yang@leeds.ac.uk [Biomaterials and Tissue Engineering Group, Leeds Dental Institute, University of Leeds, LS29LU (United Kingdom); NIHR Leeds Musculoskeletal Biomedical Research Unit, University of Leeds, Chapel Allerton Hospital, Leeds LS74SA (United Kingdom)

    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

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

    International Nuclear Information System (INIS)

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

    2010-01-01

    Research highlights: → 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. → Compared for the first time the chondrogenic potential of neonatal chondrocytes with human bone marrow stromal cells (HBMSCs) and adult chondrocytes. → 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. → 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 a difference in the

  14. Clinical Outcomes of Characterized Chondrocyte Implantation

    Science.gov (United States)

    Huylebroek, José; Van Der Bauwhede, Jan; Saris, Daniël; Veeckman, Geert; Bobic, Vladimir; Victor, Jan; Almqvist, Karl Fredrik; Verdonk, Peter; Fortems, Yves; Van Lommel, Nel; Haazen, Ludo

    2012-01-01

    Objective: To assess the clinical outcome of patients treated with autologous chondrocyte implantation using ChondroCelect in daily practice. Methods: The study is a cross-sectional analysis of an open-label, noninterventional cohort. The setting was a compassionate use program, involving 43 orthopaedic centers in 7 European countries. The participants were patients treated with ChondroCelect between October 13, 2004 and July 2, 2008. The measurements used were Clinical Global Impression–Improvement and –Efficacy and solicited adverse event reports. Results: Safety data were collected from 334 patients (90.3%), and effectiveness data were from 282 (76.2%) of the 370 patients treated. Mean age at baseline was 33.6 years (range, 12-57 years), 57% were male, and mean body mass index was 25 kg/m2. Mean follow-up was 2.2 years (range, 0.4-4.1 years). A femoral condyle lesion was reported in 66% (288/379) and a patellar lesion in 19% (84/379). Mean lesion size was 3.5 cm2; a collagen membrane was used in 92.4% (328/355). A therapeutic effect was reported in 89% (234/264) of patients overall and in 87% (40/46) of patellar lesion patients. Rates of much or very much improved patients were similar in patients with short- (18 months: 68% [70/103]) (P = 0.68) and were independent of lesion size (>4 cm2: 75.5% [37/49]; ≤4 cm2: 67.7% [111/164]) (P = 0.38). Adverse events were similar to those reported in the randomized trial with the same product, with more arthrofibrosis, more reduced joint mobility, and more crepitations reported in patellar lesions. Overall, less cartilage hypertrophy was noted, probably due to the use of a biological membrane cover. Conclusions: Implantation of ChondroCelect appeared to result in a positive benefit/risk ratio when used in an unselected heterogenous population, irrespective of the follow-up period, lesion size, and type of lesion treated. PMID:26069630

  15. Comparison of chondrocytes produced from adipose tissue-derived stem cells and cartilage tissue.

    Science.gov (United States)

    Meric, Aysenur; Yenigun, Alper; Yenigun, Vildan Betul; Dogan, Remzi; Ozturan, Orhan

    2013-05-01

    Spontaneous cartilage regeneration is poor after a cartilage defect occurs by trauma, surgical, and other reasons. Importance of producing chondrocytes from stem cells and using tissues to repair a defect is getting popular. The aim of this study was to compare the effects of injectable cartilage produced by chondrocytes differentiated from adipose tissue-derived mesenchymal stem cells and chondrocyte cells isolated directly from cartilage tissue. Mesenchymal stem cells were isolated from rat adipose tissue and characterized by cell-surface markers. Then, they were differentiated to chondrocyte cells. The function of differentiated chondrocyte cells was compared with chondrocyte cells directly isolated from cartilage tissue in terms of collagen and glycosaminoglycan secretion. Then, both chondrocyte cell types were injected to rats' left ears in liquid and gel form, and histologic evaluation was done 3 weeks after the injection. Adipose-derived stem cells were strongly positive for the CD44 and CD73 mesenchymal markers. Differentiated chondrocyte cells and chondrocyte cells directly isolated from cartilage tissue had relative collagen and glycosaminoglycan secretion results. However, histologic evaluations did not show any cartilage formation after both chondrocyte cell types were injected to rats. Strong CD44- and CD73-positive expression indicated that adipose-derived cells had the stem cell characters. Collagen and glycosaminoglycan secretion results demonstrated that adipose-derived stem cells were successfully differentiated to chondrocyte cells.

  16. Role of G-proteins in the differentiation of epiphyseal chondrocytes.

    Science.gov (United States)

    Chagin, Andrei S; Kronenberg, Henry M

    2014-10-01

    Herein, we review the regulation of differentiation of the growth plate chondrocytes by G-proteins. In connection with this, we summarize the current knowledge regarding each family of G-protein α subunit, specifically, Gα(s), Gα(q/11), Gα(12/13), and Gα(i/o). We discuss different mechanisms involved in chondrocyte differentiation downstream of G-proteins and different G-protein-coupled receptors (GPCRs) activating G-proteins in the epiphyseal chondrocytes. We conclude that among all G-proteins and GPCRs expressed by chondrocytes, Gα(s) has the most important role and prevents premature chondrocyte differentiation. Receptor for parathyroid hormone (PTHR1) appears to be the major activator of Gα(s) in chondrocytes and ablation of either one leads to accelerated chondrocyte differentiation, premature fusion of the postnatal growth plate, and ultimately short stature. © 2014 Society for Endocrinology.

  17. Mechanical confinement regulates cartilage matrix formation by chondrocytes

    Science.gov (United States)

    Lee, Hong-Pyo; Gu, Luo; Mooney, David J.; Levenston, Marc E.; Chaudhuri, Ovijit

    2017-12-01

    Cartilage tissue equivalents formed from hydrogels containing chondrocytes could provide a solution for replacing damaged cartilage. Previous approaches have often utilized elastic hydrogels. However, elastic stresses may restrict cartilage matrix formation and alter the chondrocyte phenotype. Here we investigated the use of viscoelastic hydrogels, in which stresses are relaxed over time and which exhibit creep, for three-dimensional (3D) culture of chondrocytes. We found that faster relaxation promoted a striking increase in the volume of interconnected cartilage matrix formed by chondrocytes. In slower relaxing gels, restriction of cell volume expansion by elastic stresses led to increased secretion of IL-1β, which in turn drove strong up-regulation of genes associated with cartilage degradation and cell death. As no cell-adhesion ligands are presented by the hydrogels, these results reveal cell sensing of cell volume confinement as an adhesion-independent mechanism of mechanotransduction in 3D culture, and highlight stress relaxation as a key design parameter for cartilage tissue engineering.

  18. In vitro exposure of human chondrocytes to pulsed electromagnetic fields

    Directory of Open Access Journals (Sweden)

    V Nicolin

    2009-08-01

    Full Text Available The effect of pulsed electromagnetic fields (PEMFs on the proliferation and survival of matrix-induced autologous chondrocyte implantation (MACI®-derived cells was studied to ascertain the healing potential of PEMFs. MACI-derived cells were taken from cartilage biopsies 6 months after surgery and cultured. No dedifferentiation towards the fibroblastic phenotype occurred, indicating the success of the surgical implantation. The MACI-derived cultured chondrocytes were exposed to 12 h/day (short term or 4 h/day (long term PEMFs exposure (magnetic field intensity, 2 mT; frequency, 75 Hz and proliferation rate determined by flow cytometric analysis. The PEMFs exposure elicited a significant increase of cell number in the SG2M cell cycle phase. Moreover, cells isolated from MACI® scaffolds showed the presence of collagen type II, a typical marker of chondrocyte functionality. The results show that MACI® membranes represent an optimal bioengineering device to support chondrocyte growth and proliferation in surgical implants. The surgical implant of MACI® combined with physiotherapy is suggested as a promising approach for a faster and safer treatment of cartilage traumatic lesions.

  19. Influence of cell printing on biological characters of chondrocytes.

    Science.gov (United States)

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

    2015-01-01

    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. 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 1×10(6)/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. 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 provide a novel approach

  20. RAGE, Receptor of Advanced Glycation Endoproducts, Negatively Regulates Chondrocytes Differentiation

    Science.gov (United States)

    Kurosaka, Yuko; Nishimura, Haruka; Tanabe, Motoki; Takakura, Yuuki; Iwai, Keisuke; Waki, Takuya; Fujita, Takashi

    2014-01-01

    RAGE, receptor for advanced glycation endoproducts (AGE), has been characterized as an activator of osteoclastgenesis. However, whether RAGE directly regulates chondrocyte proliferation and differentiation is unclear. Here, we show that RAGE has an inhibitory role in chondrocyte differentiation. RAGE expression was observed in chondrocytes from the prehypertrophic to hypertrophic regions. In cultured cells, overexpression of RAGE or dominant-negative-RAGE (DN-RAGE) demonstrated that RAGE inhibited cartilaginous matrix production, while DN-RAGE promoted production. Additionally, RAGE regulated Ihh and Col10a1 negatively but upregulated PTHrP receptor. Ihh promoter analysis and real-time PCR analysis suggested that downregulation of Cdxs was the key for RAGE-induced inhibition of chondrocyte differentiation. Overexpression of the NF-κB inhibitor I-κB-SR inhibited RAGE-induced NF-κB activation, but did not influence inhibition of cartilaginous matrix production by RAGE. The inhibitory action of RAGE was restored by the Rho family GTPases inhibitor Toxin B. Furthermore, inhibitory action on Ihh, Col10a1 and Cdxs was reproduced by constitutively active forms, L63RhoA, L61Rac, and L61Cdc42, but not by I-κB-SR. Cdx1 induced Ihh and Col10a1 expressions and directly interacted with Ihh promoter. Retinoic acid (RA) partially rescued the inhibitory action of RAGE. These data combined suggests that RAGE negatively regulates chondrocyte differentiation at the prehypertrophic stage by modulating NF-κB-independent and Rho family GTPases-dependent mechanisms. PMID:25275461

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

    Science.gov (United States)

    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. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  2. Nanosized fibers' effect on adult human articular chondrocytes behavior

    International Nuclear Information System (INIS)

    Stenhamre, Hanna; Thorvaldsson, Anna; Enochson, Lars; Walkenström, Pernilla; Lindahl, Anders; Brittberg, Mats; Gatenholm, Paul

    2013-01-01

    Tissue engineering with chondrogenic cell based therapies is an expanding field with the intention of treating cartilage defects. It has been suggested that scaffolds used in cartilage tissue engineering influence cellular behavior and thus the long-term clinical outcome. The objective of this study was to assess whether chondrocyte attachment, proliferation and post-expansion re-differentiation could be influenced by the size of the fibers presented to the cells in a scaffold. Polylactic acid (PLA) scaffolds with different fiber morphologies were produced, i.e. microfiber (MS) scaffolds as well as nanofiber-coated microfiber scaffold (NMS). Adult human articular chondrocytes were cultured in the scaffolds in vitro up to 28 days, and the resulting constructs were assessed histologically, immunohistochemically, and biochemically. Attachment of cells and serum proteins to the scaffolds was affected by the architecture. The results point toward nano-patterning onto the microfibers influencing proliferation of the chondrocytes, and the overall 3D environment having a greater influence on the re-differentiation. In the efforts of finding the optimal scaffold for cartilage tissue engineering, studies as the current contribute to the knowledge of how to affect and control chondrocytes behavior. - Highlights: ► Chondrocyte behavior in nanofiber-coated microfiber versus microfiber scaffolds ► High porosity (> 90%) and large pore sizes (a few hundred μm) of nanofibrous scaffolds ► Proliferation enhanced by presence of nanofibers ► Differentiation not significantly affected ► Cell attachment improved in presence of both nanofibers and serum

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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

  5. Controlled Release of Interleukin-1 Receptor Antagonist from Hyaluronic Acid-Chitosan Microspheres Attenuates Interleukin-1β-Induced Inflammation and Apoptosis in Chondrocytes

    Directory of Open Access Journals (Sweden)

    Bo Qiu

    2016-01-01

    Full Text Available This paper investigates the protective effect of interleukin-1 receptor antagonist (IL-1Ra released from hyaluronic acid chitosan (HA-CS microspheres in a controlled manner on IL-1β-induced inflammation and apoptosis in chondrocytes. The IL-1Ra release kinetics was characterized by an initial burst release, which was reduced to a linear release over eight days. Chondrocytes were stimulated with 10 ng/ml IL-1β and subsequently incubated with HA-CS-IL-1Ra microspheres. The cell viability was decreased by IL-1β, which was attenuated by HA-CS-IL-1Ra microspheres as indicated by an MTT assay. ELISA showed that HA-CS-IL-1Ra microspheres inhibited IL-1β-induced inflammation by attenuating increases in NO2- and prostaglandin E2 levels as well as increase in glycosaminoglycan release. A terminal deoxyribonucleotide transferase deoxyuridine triphosphate nick-end labeling assay revealed that the IL-1β-induced chondrocyte apoptosis was decreased by HA-CS-IL-1Ra microspheres. Moreover, HA-CS-IL-1Ra microspheres blocked IL-1β-induced chondrocyte apoptosis by increasing B-cell lymphoma 2 (Bcl-2 and decreasing Bcl-2-associated X protein and caspase-3 expressions at mRNA and protein levels, as indicated by reverse-transcription quantitative polymerase chain reaction and western blot analysis, respectively. The results of the present study indicated that HA-CS-IL-1Ra microspheres as a controlled release system of IL-1Ra possess potential anti-inflammatory and antiapoptotic properties in rat chondrocytes due to their ability to regulate inflammatory factors and apoptosis associated genes.

  6. Viability of chondrocytes seeded onto a collagen I/III membrane for matrix-induced autologous chondrocyte implantation.

    Science.gov (United States)

    Hindle, Paul; Hall, Andrew C; Biant, Leela C

    2014-11-01

    Cell viability is crucial for effective cell-based cartilage repair. The aim of this study was to determine the effect of handling the membrane during matrix-induced autologous chondrocyte implantation surgery on the viability of implanted chondrocytes. Images were acquired under five conditions: (i) Pre-operative; (ii) Handled during surgery; (iii) Cut edge; (iv) Thumb pressure applied; (v) Heavily grasped with forceps. Live and dead cell stains were used. Images were obtained for cell counting and morphology. Mean cell density was 6.60 × 10(5) cells/cm(2) (5.74-7.11 × 10(5) ) in specimens that did not have significant trauma decreasing significantly in specimens that had been grasped with forceps (p < 0.001) or cut (p = 0.004). Cell viability on delivery grade membrane was 75.1%(72.4-77.8%). This dropped to 67.4%(64.1-69.7%) after handling (p = 0.002), 56.3%(51.5-61.6%) after being thumbed (p < 0.001) and 28.8%(24.7-31.2%) after crushing with forceps (p < 0.001). When cut with scissors there was a band of cell death approximately 275 µm in width where cell viability decreased to 13.7%(10.2-18.2%, p < 0.001). Higher magnification revealed cells without the typical rounded appearance of chondrocytes. We found that confocal laser-scanning microscope (CLSM) can be used to quantify and image the fine morphology of cells on a matrix-induced autologous chondrocyte implantation (MACI) membrane. Careful handling of the membrane is essential to minimise chondrocyte death during surgery. © 2014 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.

  7. Conditional expression of constitutively active estrogen receptor α in chondrocytes impairs longitudinal bone growth in mice

    International Nuclear Information System (INIS)

    Ikeda, Kazuhiro; Tsukui, Tohru; Imazawa, Yukiko; Horie-Inoue, Kuniko; Inoue, Satoshi

    2012-01-01

    Highlights: ► Conditional transgenic mice expressing constitutively active estrogen receptor α (caERα) in chondrocytes were developed. ► Expression of caERα in chondrocytes impaired longitudinal bone growth in mice. ► caERα affects chondrocyte proliferation and differentiation. ► This mouse model is useful for understanding the physiological role of ERαin vivo. -- Abstract: Estrogen plays important roles in the regulation of chondrocyte proliferation and differentiation, which are essential steps for longitudinal bone growth; however, the mechanisms of estrogen action on chondrocytes have not been fully elucidated. In the present study, we generated conditional transgenic mice, designated as caERα ColII , expressing constitutively active mutant estrogen receptor (ER) α in chondrocytes, using the chondrocyte-specific type II collagen promoter-driven Cre transgenic mice. caERα ColII mice showed retardation in longitudinal growth, with short bone lengths. BrdU labeling showed reduced proliferation of hypertrophic chondrocytes in the proliferating layer of the growth plate of tibia in caERα ColII mice. In situ hybridization analysis of type X collagen revealed that the maturation of hypertrophic chondrocytes was impaired in caERα ColII mice. These results suggest that ERα is a critical regulator of chondrocyte proliferation and maturation during skeletal development, mediating longitudinal bone growth in vivo.

  8. Ant trail pheromone biosynthesis is triggered by a neuropeptide hormone.

    Directory of Open Access Journals (Sweden)

    Man-Yeon Choi

    Full Text Available Our understanding of insect chemical communication including pheromone identification, synthesis, and their role in behavior has advanced tremendously over the last half-century. However, endocrine regulation of pheromone biosynthesis has progressed slowly due to the complexity of direct and/or indirect hormonal activation of the biosynthetic cascades resulting in insect pheromones. Over 20 years ago, a neurohormone, pheromone biosynthesis activating neuropeptide (PBAN was identified that stimulated sex pheromone biosynthesis in a lepidopteran moth. Since then, the physiological role, target site, and signal transduction of PBAN has become well understood for sex pheromone biosynthesis in moths. Despite that PBAN-like peptides (∼200 have been identified from various insect Orders, their role in pheromone regulation had not expanded to the other insect groups except for Lepidoptera. Here, we report that trail pheromone biosynthesis in the Dufour's gland (DG of the fire ant, Solenopsis invicta, is regulated by PBAN. RNAi knock down of PBAN gene (in subesophageal ganglia or PBAN receptor gene (in DG expression inhibited trail pheromone biosynthesis. Reduced trail pheromone was documented analytically and through a behavioral bioassay. Extension of PBAN's role in pheromone biosynthesis to a new target insect, mode of action, and behavioral function will renew research efforts on the involvement of PBAN in pheromone biosynthesis in Insecta.

  9. The effect of a slightly acidic somatomedin peptide (ILAs) on the sulphation of proteoglycans from articular and growth plate chondrocytes in culture

    International Nuclear Information System (INIS)

    Corvol, M.-T.; Dumontier, M.-F.; Rappaport, R.; Guyda, H.; Posner, B.I.

    1978-01-01

    Chondrocyte cultures were prepared from rabbit growth plate (GPC) and articular (ARC) chondrocytes. These two cell types have distinct morphological characteristics. The cells reached maximum numbers by days 10 and 21 for ARC and GPC, respectively. The proteoglycans (PG) contained in the cellular pool were extracted and purified by DEAE cellulose chromatography. The effect of a partially purified somatomedin peptide with insulin-like activity on [ 35 S]sulphate incorporation into PG was evaluated. In both ARC and GPC a significant stimulation of [ 35 S]sulphate uptake into PG subunits was obtained with 1 ng Eq./ml of somatomedin peptide. In order to obtain the same stimulatory effect with porcine insulin, a 1000-fold greater concentration was required. The electrophoretic patterns of the PG subunits on acrylamide-agarose electrophoresis were identical on control incubations and after stimulation with the somatomedin peptide. These data demonstrate in vitro biological activity of this peptide on well differentiated articular and epiphyseal growth plate chondrocytes in culture. These cultures appear to provide a sensitive biological assay for somatomedin peptides. (author)

  10. Conditioned Media from Adipose-Tissue-Derived Mesenchymal Stem Cells Downregulate Degradative Mediators Induced by Interleukin-1β in Osteoarthritic Chondrocytes

    Directory of Open Access Journals (Sweden)

    Julia Platas

    2013-01-01

    Full Text Available Osteoarthritis (OA is the most frequent joint disorder and an important cause of disability. Recent studies have shown the potential of adipose-tissue-derived mesenchymal stem cells (AD-MSC for cartilage repair. We have investigated whether conditioned medium from AD-MSC (CM may regulate in OA chondrocytes a number of key mediators involved in cartilage degeneration. CM enhanced type II collagen expression in OA chondrocytes while decreasing matrix metalloproteinase (MMP activity in cell supernatants as well as the levels of MMP-3 and MMP-13 proteins and mRNA in OA chondrocytes stimulated with interleukin- (IL- 1β. In addition, CM increased IL-10 levels and counteracted the stimulating effects of IL-1β on the production of tumor necrosis factor-α, IL-6, prostaglandin E2, and NO measured as nitrite and the mRNA expression of these cytokines, CCL-2, CCL-3, CCL-4, CCL-5, CCL-8, CCL-19, CCL-20, CXCL-1, CXCL-2, CXCL-3, CXCL-5, CXCL-8, cyclooxygenase-2, microsomal prostaglandin E synthase-1, and inducible NO synthase. These effects may be dependent on the inhibition of nuclear factor-κB activation by CM. Our data demonstrate the chondroprotective actions of CM and provide support for further studies of this approach in joint disease.

  11. MR imaging of autologous chondrocyte implantation of the knee

    Energy Technology Data Exchange (ETDEWEB)

    James, S.L.J.; Connell, D.A.; Saifuddin, A.; Skinner, J.A.; Briggs, T.W.R. [RNOH Stanmore, Department of Radiology, Stanmore, Middlesex (United Kingdom)

    2006-05-15

    Autologous chondrocyte implantation (ACI) is a surgical technique that is increasingly being used in the treatment of full-thickness defects of articular cartilage in the knee. It involves the arthroscopic harvesting and in vitro culture of chondrocytes that are subsequently implanted into a previously identified chondral defect. The aim is to produce a repair tissue that closely resembles hyaline articular cartilage that gradually becomes incorporated, restoring joint congruity. Over the long term, it is hoped that this will prevent the progression of full-thickness articular cartilage defects to osteoarthritis. This article reviews the indications and operative procedure performed in ACI. Magnetic resonance imaging (MRI) sequences that provide optimal visualization of articular cartilage in the post-operative period are discussed. Normal appearances of ACI on MRI are presented along with common complications that are encountered with this technique. (orig.)

  12. Triterpenoid biosynthesis in Euphorbia lathyris latex

    International Nuclear Information System (INIS)

    Hawkins, D.R.

    1987-11-01

    The structures of triterpenols, not previously been known, from Euphorbia lathyris latex are reported. A method for quantifying very small amounts of these compounds was developed. Concerning the biochemistry of the latex, no exogenous cofactors were required for the biosynthesis and the addition of compounds such as NADPAH and ATP do not stimulate the biosynthesis. The addition of DTE or a similar anti-oxidant was found to help reduce the oxidation of the latex, thus increasing the length of time that the latex remains active. The requirement of a divalent cation and the preference for Mn in the pellet was observed. The effect of several inhibitors on the biosynthesis of the triterpenoids was examined. Mevinolin was found to inhibit the biosynthesis of the triterpenoids from acetate, but not mevalonate. A dixon plot of the inhibition of acetate incorporation showed an I 50 concentration of 3.2 μM. Fenpropimorph was found to have little or no effect on the biosynthesis. Tridemorph was found to inhibit the biosynthesis of all of the triterpenoids with an I 50 of 4 μM. It was also observed that the cyclopropyl containing triterpenols, cycloartenol and 24-methylenecycloartenol were inhibited much more strongly than those containing an 8-9 double bond, lanosterol and 24-methylenelanosterol. The evidence indicates, but does not definetely prove, that lanosterol and 24-methylenelanosterol are not made from cycloartenol and 24-methylenecycloartenol via a ring-opening enzyme such as cycloeucalenol-obtusifoliol isomerase. The possibilty that cycloartenol is made via lanosterol was investigated by synthesizing 4-R-4- 3 H-mevalonic acid and incubating latex with a mixture of this and 14 C-mevalonic acid. From the 3 H/ 14 C ratio it was shown that cycloartenol and 24-methylenecycloartenol are not made via an intermediate containing as 8-9 double bond. 88 refs., 15 figs., 30 tabs

  13. Statins do not inhibit the FGFR signaling in chondrocytes

    Czech Academy of Sciences Publication Activity Database

    Fafílek, B.; Hampl, Marek; Říčánková, N.; Veselá, Iva; Bálek, L.; Kunová Bosáková, M.; Gudernová, I.; Vařecha, M.; Buchtová, Marcela; Krejčí, P.

    2017-01-01

    Roč. 25, č. 9 (2017), s. 1522-1530 ISSN 1063-4584 R&D Projects: GA ČR(CZ) GA14-31540S Grant - others:GA MŠk(CZ) LH12004 Institutional support: RVO:67985904 Keywords : statins * FGF signaling * chondrocytes Subject RIV: EB - Genetics ; Molecular Biology OBOR OECD: Developmental biology Impact factor: 4.742, year: 2016

  14. Inhibition of Chondrocyte Hypertrophy of Osteoarthritis by Disruptor Peptide

    Science.gov (United States)

    2017-07-01

    generated the disruptor peptides conjugated with green fluorescent protein (GFP) and/or Pen. Primary chondrocytes were treated with 10 M of disruptor...were stained with Safranin-O and Fast green . Arrow indicates cartilage loss. 7 PTHrP-induced PTHR coupling to G protein subunits will be...vitro and protect cartilage damage in a mouse OA model. 15. SUBJECT TERMS Osteoarthritis; Parathyroid hormone-related protein ; PTH receptor; Beta

  15. RAGE and activation of chondrocytes and fibroblast-like synoviocytes in joint diseases

    OpenAIRE

    Steenvoorden, Marjan Maria Claziena

    2007-01-01

    This dissertation describes a new model in which cartilage degradation can be studied. New cartilage is formed by bovine chondrocytes obtained from the slaughterhouse and cocultured with synovial cells from rheumatoid arthritis (RA) patients to study the interaction between the chondrocytes and synoviocytes.The results of our study show that the role of synoviocytes in cartilage degradation is dependent on the presence of live chondrocytes. In osteoarthritis (OA) patients an increased level o...

  16. Doublecortin May Play a Role in Defining Chondrocyte Phenotype

    Directory of Open Access Journals (Sweden)

    Dongxia Ge

    2014-04-01

    Full Text Available 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.

  17. Quantitative Proteomic Analysis of Rat Condylar Chondrocytes during Postnatal Development.

    Science.gov (United States)

    Jiang, Li Ting; Xie, Yin Yin; Wei, Li; Zhou, Qi; Shen, Xing; Gao, Yi Ming; Jiang, Xin Quan

    To investigate differentially expressed proteins in rat mandibular condylar cartilage (MCC) chondrocytes caused by initial mastication for short postnatal periods. Four groups of protein samples were extracted from primary cultured rat MCC chondrocytes, harvested from eigthy postnatal SD rats aged 1,7,14 and 28 days, with twenty in each group. Total proteins were labelled with isobaric tags for relative and absolute quantification (iTRAQ) reagents. Two-dimensional nano-high-performance liquid chromatography (HPLC) and matrix-assisted laser desorption ionization-time-of-flight/ time-of-flight (MALDI-TOF/TOF) mass spectrometry analysis with iTRAQ technique were performed. All data were analysed by MASCOT software with the SWISSPROT protein database. Furthermore, bioinformatics and statistical analysis were performed to classify their cellular components, biological processes, molecular functions and metabolic pathway by the PANTHER database. In total, 137 differentially expressed proteins were identified during MCC growth and were assigned to one or more cellular components. According to the PANTHER analysis, a significant proportion of proteins are involved in the metabolic process, cellular process, biological regulation, developmental process and response to stimulus. The most extensive molecular function was 43% in catalytic activity. In addition, it was found that proteins in MCC chondrocytes change markedly on the growth stage of eruption of the teeth. This study provides an integrated perspective of molecular mechanisms regulating early normal postnatal growth and development of rat MCC at the protein level.

  18. Gamma-aminobutyric acid mediates nicotine biosynthesis in tobacco under flooding stress

    Directory of Open Access Journals (Sweden)

    Xiaoming Zhang

    2016-02-01

    Full Text Available Gamma-aminobutyric acid (GABA is a four-carbon non-protein amino acid conserved from bacteria to plants and vertebrates. Increasing evidence supports a regulatory role for GABA in plant development and the plant's response to environmental stress. The biosynthesis of nicotine, the main economically important metabolite in tobacco, is tightly regulated. GABA has not hitherto been reported to function in nicotine biosynthesis. Here we report that water flooding treatment (hypoxia markedly induced the accumulation of GABA and stimulated nicotine biosynthesis. Suppressing GABA accumulation by treatment with glutamate decarboxylase inhibitor impaired flooding-induced nicotine biosynthesis, while exogenous GABA application directly induced nicotine biosynthesis. Based on these results, we propose that GABA triggers nicotine biosynthesis in tobacco seedlings subjected to flooding. Our results provide insight into the molecular mechanism of nicotine biosynthesis in tobacco plants exposed to environmental stress.

  19. Biosynthesis of tylophora alkaloids

    International Nuclear Information System (INIS)

    Mulchandani, N.B.; Iyer, S.S.; Badheka, L.P.

    1974-01-01

    Using labelled precursors, biosynthesis of the tylophora alkaloids, tylophorine, tylophorinidine and tylophorinide has been investigated in Tylophora asthmatica plants. The radioactive precursors, phenylalanine-2- 14 C, benzoic acid-1- 14 C, benzoic acid-ring 14 C, acetate-2- 14 C, ornithine-5- 14 C, acetate-2- 14 C, ornithine-5- 14 C and cinnamic acid-2- 14 C were administered to the plants individually by wick technique. Tylophorine was isolated in each case and assayed for its radioactivity to find out the incorporation of the label into it. The results indicate that: (1) phenylalanine via cinnamic acid is an important precursor in the biosynthesis of tylophorine (2) orinithine participates in tylophorine biosynthesis via pyrroline and (3) tylophorinidine may be a direct precursor of tylophorine. (M.G.B.)

  20. Aflatoxin biosynthesis: current frontiers.

    Science.gov (United States)

    Roze, Ludmila V; Hong, Sung-Yong; Linz, John E

    2013-01-01

    Aflatoxins are among the principal mycotoxins that contaminate economically important food and feed crops. Aflatoxin B1 is the most potent naturally occurring carcinogen known and is also an immunosuppressant. Occurrence of aflatoxins in crops has vast economic and human health impacts worldwide. Thus, the study of aflatoxin biosynthesis has become a focal point in attempts to reduce human exposure to aflatoxins. This review highlights recent advances in the field of aflatoxin biosynthesis and explores the functional connection between aflatoxin biosynthesis, endomembrane trafficking, and response to oxidative stress. Dissection of the regulatory mechanisms involves a complete comprehension of the aflatoxin biosynthetic process and the dynamic network of transcription factors that orchestrates coordinated expression of the target genes. Despite advancements in the field, development of a safe and effective multifaceted approach to solve the aflatoxin food contamination problem is still required.

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

    Directory of Open Access Journals (Sweden)

    Haqqi Tariq M

    2011-08-01

    Full Text Available Abstract 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 Leucine mixture (HLM up-regulation of ACAN and COL2A1 expression in IL-1β-stimulated OA chondrocytes was also noted (p Conclusion Our data suggests that HLM could be chondroprotective and anti-inflammatory agent in arthritis, switching chondrocyte gene expression from catabolic direction towards anabolic and regenerative, and consequently this approach may be potentially useful as a new adjunct therapeutic/preventive agent for OA or injury recovery.

  2. Reduction of Environmental Temperature Mitigates Local Anesthetic Cytotoxicity in Bovine Articular Chondrocytes

    Directory of Open Access Journals (Sweden)

    Tarik Onur, Alexis Dang

    2014-09-01

    Full Text Available The purpose of this study was to assess whether reducing environmental temperature will lead to increased chondrocyte viability following injury from a single-dose of local anesthetic treatment. Bovine articular chondrocytes from weight bearing portions of femoral condyles were harvested and cultured. 96-well plates were seeded with 15,000 chondrocytes per well. Chondrocytes were treated with one of the following conditions: ITS Media, 1x PBS, 2% lidocaine, 0.5% bupivacaine, or 0.5% ropivacaine. Each plate was then incubated at 37°C, 23°C, or 4°C for one hour and then returned to media at 37°C. Chondrocyte viability was assessed 24 hours after treatment. Chondrocyte viability is presented as a ratio of the fluorescence of the treatment group over the average of the media group at that temperature (ratio ± SEM. At 37°C, lidocaine (0.35 ± 0.04 and bupivacaine (0.30 ± 0.05 treated chondrocytes show low cell viability when compared to the media (1.00 ± 0.03 control group (p < 0.001. Lidocaine treated chondrocytes were significantly more viable at 23°C (0.84 ± 0.08 and 4°C (0.86±0.085 than at 37°C (p < 0.001. Bupivacaine treated chondrocytes were significantly more viable at 4°C (0.660 ± 0.073 than at 37°C or 23°C (0.330 ± 0.069 (p < 0.001 and p = 0.002 respectively. Reducing the temperature from 37°C to 23°C during treatment with lidocaine increases chondrocyte viability following injury. Chondrocytes treated with bupivacaine can be rescued by reducing the temperature to 4°C.

  3. SHP2 regulates chondrocyte terminal differentiation, growth plate architecture and skeletal cell fates.

    Directory of Open Access Journals (Sweden)

    Margot E Bowen

    Full Text Available Loss of PTPN11/SHP2 in mice or in human metachondromatosis (MC patients causes benign cartilage tumors on the bone surface (exostoses and within bones (enchondromas. To elucidate the mechanisms underlying cartilage tumor formation, we investigated the role of SHP2 in the specification, maturation and organization of chondrocytes. Firstly, we studied chondrocyte maturation by performing RNA-seq on primary chondrocyte pellet cultures. We found that SHP2 depletion, or inhibition of the ERK1/2 pathway, delays the terminal differentiation of chondrocytes from the early-hypertrophic to the late-hypertrophic stage. Secondly, we studied chondrocyte maturation and organization in mice with a mosaic postnatal inactivation of Ptpn11 in chondrocytes. We found that the vertebral growth plates of these mice have expanded domains of early-hypertrophic chondrocytes that have not yet terminally differentiated, and their enchondroma-like lesions arise from chondrocytes displaced from the growth plate due to a disruption in the organization of maturation and ossification zones. Furthermore, we observed that lesions from human MC patients also display disorganized chondrocyte maturation zones. Next, we found that inactivation of Ptpn11 in Fsp1-Cre-expressing fibroblasts induces exostosis-like outgrowths, suggesting that loss of SHP2 in cells on the bone surface and at bone-ligament attachment sites induces ectopic chondrogenesis. Finally, we performed lineage tracing to show that exostoses and enchondromas in mice likely contain mixtures of wild-type and SHP2-deficient chondrocytes. Together, these data indicate that in patients with MC, who are heterozygous for inherited PTPN11 loss-of-function mutations, second-hit mutations in PTPN11 can induce enchondromas by disrupting the organization and delaying the terminal differentiation of growth plate chondrocytes, and can induce exostoses by causing ectopic chondrogenesis of cells on the bone surface. Furthermore, the

  4. Ofloxacin induces apoptosis via β1 integrin-EGFR-Rac1-Nox2 pathway in microencapsulated chondrocytes

    International Nuclear Information System (INIS)

    Sheng, Zhi-Guo; Huang, Wei; Liu, Yu-Xiang; Yuan, Ye; Zhu, Ben-Zhan

    2013-01-01

    Quinolones (QNs)-induced arthropathy is an important toxic side-effect in immature animals leading to the restriction of their therapeutic use in pediatrics. Ofloxacin, a typical QN, was found to induce the chondrocytes apoptosis in the early phase (12–48 h) of arthropathy in our previous study. However, the exact mechanism(s) is unclear. Microencapsulated juvenile rabbit joint chondrocytes, a three-dimensional culture system, is utilized to perform the present study. Ofloxacin, at a therapeutically relevant concentration (10 μg/ml), disturbs the interaction between β1 integrin and activated intracellular signaling proteins at 12 h, which is inhibited when supplementing Mg 2+ . Intracellular reactive oxygen species (ROS) significantly increases in a time-dependent manner after exposure to ofloxacin for 12–48 h. Furthermore, ofloxacin markedly enhances the level of activated Rac1 and epidermal growth factor receptor (EGFR) phosphorylation, and its inhibition in turn reduces the ROS production, apoptosis and Rac1 activation. Silencing Nox2, Rac1 or supplementing Mg 2+ inhibits ROS accumulation, apoptosis occurrence and EGFR phosphorylation induced by ofloxacin. However, depletion of Nox2, Rac1 and inhibition of EGFR do not affect ofloxacin-mediated loss of interaction between β1 integrin and activated intracellular signaling proteins. In addition, ofloxacin also induces Vav2 phosphorylation, which is markedly suppressed after inactivating EGFR or supplementing Mg 2+ . These results suggest that ofloxacin causes Nox2-mediated intracellular ROS production by disrupting the β1 integrin function and then activating the EGFR-Vav2-Rac1 pathway, finally resulting in apoptosis within 12–48 h exposure. The present study provides a novel insight regarding the potential role of Nox-driven ROS in QNs-induced arthropathy. - Highlights: ► Ofloxacin induces Nox2-driven ROS in encapsulated chondrocyte at 12–48 h. ► Ofloxacin stimulates ROS production via the β1

  5. Ofloxacin induces apoptosis via β1 integrin-EGFR-Rac1-Nox2 pathway in microencapsulated chondrocytes

    Energy Technology Data Exchange (ETDEWEB)

    Sheng, Zhi-Guo [State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-Environmental Science, Chinese Academy of Sciences, 18 Shuangqing Road, Beijing 100085 (China); Huang, Wei [Department of Chemical Biology, School of Pharmaceutical Sciences, Peking University Health Science Center, Beijing 1000191 (China); Liu, Yu-Xiang [State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-Environmental Science, Chinese Academy of Sciences, 18 Shuangqing Road, Beijing 100085 (China); Yuan, Ye [Beijing Institute of Pharmacology and Toxicology, 27 Taiping Road, Beijing 100850 (China); Zhu, Ben-Zhan, E-mail: bzhu@rcees.ac.cn [State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-Environmental Science, Chinese Academy of Sciences, 18 Shuangqing Road, Beijing 100085 (China); Linus Pauling Institute, Oregon State University, Corvallis, OR 97331 (United States)

    2013-02-15

    Quinolones (QNs)-induced arthropathy is an important toxic side-effect in immature animals leading to the restriction of their therapeutic use in pediatrics. Ofloxacin, a typical QN, was found to induce the chondrocytes apoptosis in the early phase (12–48 h) of arthropathy in our previous study. However, the exact mechanism(s) is unclear. Microencapsulated juvenile rabbit joint chondrocytes, a three-dimensional culture system, is utilized to perform the present study. Ofloxacin, at a therapeutically relevant concentration (10 μg/ml), disturbs the interaction between β1 integrin and activated intracellular signaling proteins at 12 h, which is inhibited when supplementing Mg{sup 2+}. Intracellular reactive oxygen species (ROS) significantly increases in a time-dependent manner after exposure to ofloxacin for 12–48 h. Furthermore, ofloxacin markedly enhances the level of activated Rac1 and epidermal growth factor receptor (EGFR) phosphorylation, and its inhibition in turn reduces the ROS production, apoptosis and Rac1 activation. Silencing Nox2, Rac1 or supplementing Mg{sup 2+} inhibits ROS accumulation, apoptosis occurrence and EGFR phosphorylation induced by ofloxacin. However, depletion of Nox2, Rac1 and inhibition of EGFR do not affect ofloxacin-mediated loss of interaction between β1 integrin and activated intracellular signaling proteins. In addition, ofloxacin also induces Vav2 phosphorylation, which is markedly suppressed after inactivating EGFR or supplementing Mg{sup 2+}. These results suggest that ofloxacin causes Nox2-mediated intracellular ROS production by disrupting the β1 integrin function and then activating the EGFR-Vav2-Rac1 pathway, finally resulting in apoptosis within 12–48 h exposure. The present study provides a novel insight regarding the potential role of Nox-driven ROS in QNs-induced arthropathy. - Highlights: ► Ofloxacin induces Nox2-driven ROS in encapsulated chondrocyte at 12–48 h. ► Ofloxacin stimulates ROS production via

  6. Modulation of Hyaluronan Synthesis by the Interaction between Mesenchymal Stem Cells and Osteoarthritic Chondrocytes

    Directory of Open Access Journals (Sweden)

    Eliane Antonioli

    2015-01-01

    Full Text Available Bone marrow mesenchymal stem cells (BM-MSCs are considered a good source for cellular therapy in cartilage repair. But, their potential to repair the extracellular matrix, in an osteoarthritic environment, is still controversial. In osteoarthritis (OA, anti-inflammatory action and extracellular matrix production are important steps for cartilage healing. This study examined the interaction of BM-MSC and OA-chondrocyte on the production of hyaluronan and inflammatory cytokines in a Transwell system. We compared cocultured BM-MSCs and OA-chondrocytes with the individually cultured controls (monocultures. There was a decrease in BM-MSCs cell count in coculture with OA-chondrocytes when compared to BM-MSCs alone. In monoculture, BM-MSCs produced higher amounts of hyaluronan than OA-chondrocytes and coculture of BM-MSCs with OA-chondrocytes increased hyaluronan production per cell. Hyaluronan synthase-1 mRNA expression was upregulated in BM-MSCs after coculture with OA-chondrocytes, whereas hyaluronidase-1 was downregulated. After coculture, lower IL-6 levels were detected in BM-MSCs compared with OA-chondrocytes. These results indicate that, in response to coculture with OA-chondrocytes, BM-MSCs change their behavior by increasing production of hyaluronan and decreasing inflammatory cytokines. Our results indicate that BM-MSCs per se could be a potential tool for OA regenerative therapy, exerting short-term effects on the local microenvironment even when cell:cell contact is not occurring.

  7. Modelling and Simulating the Adhesion and Detachment of Chondrocytes in Shear Flow

    Science.gov (United States)

    Hao, Jian; Pan, Tsorng-Whay; Rosenstrauch, Doreen

    Chondrocytes are typically studied in the environment where they normally reside such as the joints in hips, intervertebral disks or the ear. For example, in [SKE+99], the effect of seeding duration on the strength of chondrocyte adhesion to articulate cartilage has been studied in shear flow chamber since such adhesion may play an important role in the repair of articular defects by maintaining cells in positions where their biosynthetic products can contribute to the repair process. However, in this investigation, we focus mainly on the use of auricular chondrocytes in cardiovascular implants. They are abundant, easily and efficiently harvested by a minimally invasive technique. Auricular chondrocytes have ability to produce collagen type-II and other important extracellular matrix constituents; this allows them to adhere strongly to the artificial surfaces. They can be genetically engineered to act like endothelial cells so that the biocompatibility of cardiovascular prothesis can be improved. Actually in [SBBR+02], genetically engineered auricular chondrocytes can be used to line blood-contacting luminal surfaces of left ventricular assist device (LVAD) and a chondrocyte-lined LVAD has been planted into the tissue-donor calf and the results in vivo have proved the feasibility of using autologous auricular chondrocytes to improve the biocompatibility of the blood-biomaterial interface in LVADs and cardiovascular prothesis. Therefore, cultured chondrocytes may offer a more efficient and less invasive means of covering artificial surface with a viable and adherent cell layer.

  8. RAGE and activation of chondrocytes and fibroblast-like synoviocytes in joint diseases

    NARCIS (Netherlands)

    Steenvoorden, Marjan Maria Claziena

    2007-01-01

    This dissertation describes a new model in which cartilage degradation can be studied. New cartilage is formed by bovine chondrocytes obtained from the slaughterhouse and cocultured with synovial cells from rheumatoid arthritis (RA) patients to study the interaction between the chondrocytes and

  9. Adipose mesenchymal stem cells protect chondrocytes from degeneration associated with osteoarthritis.

    Science.gov (United States)

    Maumus, Marie; Manferdini, Cristina; Toupet, Karine; Peyrafitte, Julie-Anne; Ferreira, Rosanna; Facchini, Andrea; Gabusi, Elena; Bourin, Philippe; Jorgensen, Christian; Lisignoli, Gina; Noël, Danièle

    2013-09-01

    Our work aimed at evaluating the role of adipose stem cells (ASC) on chondrocytes from osteoarthritic (OA) patients and identifying the mediators involved. We used primary chondrocytes, ASCs from different sources and bone marrow mesenchymal stromal cells (MSC) from OA donors. ASCs or MSCs were co-cultured with chondrocytes in a minimal medium and using cell culture inserts. Under these conditions, ASCs did not affect the proliferation of chondrocytes but significantly decreased camptothecin-induced apoptosis. Both MSCs and ASCs from different sources allowed chondrocytes in the cocultures maintaining a stable expression of markers specific for a mature phenotype, while expression of hypertrophic and fibrotic markers was decreased. A number of factors known to regulate the chondrocyte phenotype (IL-1β, IL-1RA, TNF-α) and matrix remodeling (TIMP-1 and -2, MMP-1 and -9, TSP-1) were not affected. However, a significant decrease of TGF-β1 secretion by chondrocytes and induction of HGF secretion by ASCs was observed. Addition of a neutralizing anti-HGF antibody reversed the anti-fibrotic effect of ASCs whereas hypertrophic markers were not modulated. In summary, ASCs are an interesting source of stem cells for efficiently reducing hypertrophy and dedifferentiation of chondrocytes, at least partly via the secretion of HGF. This supports the interest of using these cells in therapies for osteo-articular diseases. Copyright © 2013 Elsevier B.V. All rights reserved.

  10. Curcumin Inhibits Chondrocyte Hypertrophy of Mesenchymal Stem Cells through IHH and Notch Signaling Pathways.

    Science.gov (United States)

    Cao, Zhen; Dou, Ce; Dong, Shiwu

    2017-01-01

    Using tissue engineering technique to repair cartilage damage caused by osteoarthritis is a promising strategy. However, the regenerated tissue usually is fibrous cartilage, which has poor mechanical characteristics compared to hyaline cartilage. Chondrocyte hypertrophy plays an important role in this process. Thus, it is very important to find out a suitable way to maintain the phenotype of chondrocytes and inhibit chondrocyte hypertrophy. Curcumin deriving from turmeric was reported with anti-inflammatory and anti-tumor pharmacological effects. However, the role of curcumin in metabolism of chondrocytes, especially in the chondrocyte hypertrophy remains unclear. Mesenchymal stem cells (MSCs) are widely used in cartilage tissue engineering as seed cells. So we investigated the effect of curcumin on chondrogenesis and chondrocyte hypertrophy in MSCs through examination of cell viability, glycosaminoglycan synthesis and specific gene expression. We found curcumin had no effect on expression of chondrogenic markers including Sox9 and Col2a1 while hypertrophic markers including Runx2 and Col10a1 were down-regulated. Further exploration showed that curcumin inhibited chondrocyte hypertrophy through Indian hedgehog homolog (IHH) and Notch signalings. Our results indicated curcumin was a potential agent in modulating cartilage homeostasis and maintaining chondrocyte phenotype.

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

    Energy Technology Data Exchange (ETDEWEB)

    Takegami, Yasuhiko [Division of Neurogenetics, Center of Neurological Disease and Cancer, Nagoya University Graduate School of Medicine, Nagoya (Japan); Department of Orthopaedic Surgery, Nagoya University School of Medicine, Nagoya (Japan); Ohkawara, Bisei; Ito, Mikako; Masuda, Akio [Division of Neurogenetics, Center of Neurological Disease and Cancer, Nagoya University Graduate School of Medicine, Nagoya (Japan); Nakashima, Hiroaki; Ishiguro, Naoki [Department of Orthopaedic Surgery, Nagoya University School of Medicine, Nagoya (Japan); Ohno, Kinji, E-mail: ohnok@med.nagoya-u.ac.jp [Division of Neurogenetics, Center of Neurological Disease and Cancer, Nagoya University Graduate School of Medicine, Nagoya (Japan)

    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{sup 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. - Highlights: • Rspo2 is a secreted activator of Wnt, and its knockout shows extended proliferating chondrocytes in endochondral ossification. • In proliferating chondrocytes of Rspo2-knockout mice, Sox9 and collagen type 2 are increased and β-catenin is decreased. • Rspo2 and its receptor Lgr5, as well as Sox9 and collagen type 2, are expressed in differentiating ATDC5 chondrogenic cells. • In ATDC5 cells, Rspo2 decreases

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

    International Nuclear Information System (INIS)

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

    2016-01-01

    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. - Highlights: • Rspo2 is a secreted activator of Wnt, and its knockout shows extended proliferating chondrocytes in endochondral ossification. • In proliferating chondrocytes of Rspo2-knockout mice, Sox9 and collagen type 2 are increased and β-catenin is decreased. • Rspo2 and its receptor Lgr5, as well as Sox9 and collagen type 2, are expressed in differentiating ATDC5 chondrogenic cells. • In ATDC5 cells, Rspo2 decreases expressions

  13. Lactoferrin inhibits dexamethasone-induced chondrocyte impairment from osteoarthritic cartilage through up-regulation of extracellular signal-regulated kinase 1/2 and suppression of FASL, FAS, and Caspase 3

    International Nuclear Information System (INIS)

    Tu, Yihui; Xue, Huaming; Francis, Wendy; Davies, Andrew P.; Pallister, Ian; Kanamarlapudi, Venkateswarlu; Xia, Zhidao

    2013-01-01

    Highlights: •Dex exerts dose-dependant inhibition of HACs viability and induction of apoptosis. •Dex-induced impairment of chondrocytes was attenuated by rhLF. •ERK and FASL/FAS signaling are involved in the effects of rhLF. •OA patients with glucocorticoid-induced cartilage damage may benefit from treatment with rhLF. -- Abstract: Dexamethasone (Dex) is commonly used for osteoarthritis (OA) with excellent anti-inflammatory and analgesic effect. However, Dex also has many side effects following repeated use over prolonged periods mainly through increasing apoptosis and inhibiting proliferation. Lactoferrin (LF) exerts significantly anabolic effect on many cells and little is known about its effect on OA chondrocytes. Therefore, the aim of this study is to investigate whether LF can inhibit Dex-induced OA chondrocytes apoptosis and explore its possible molecular mechanism involved in. MTT assay was used to determine the optimal concentration of Dex and recombinant human LF (rhLF) on chondrocytes at different time and dose points. Chondrocytes were then stimulated with Dex in the absence or presence of optimal concentration of rhLF. Cell proliferation and viability were evaluated using MTT and LIVE/DEAD assay, respectively. Cell apoptosis was evaluated by multi-parameter apoptosis assay kit using both confocal microscopy and flow cytometry, respectively. The expression of extracellular signal-regulated kinase (ERK), FAS, FASL, and Caspase-3 (CASP3) at the mRNA and protein levels were examined by real-time polymerase chain reaction (PCR) and immunocytochemistry, respectively. The optimal concentration of Dex (25 μg/ml) and rhLF (200 μg/ml) were chosen for the following experiments. rhLF significantly reversed the detrimental effect of Dex on chondrocytes proliferation, viability, and apoptosis. In addition, rhLF significantly prevented Dex-induced down-regulation of ERK and up-regulation of FAS, FASL, and CASP3. These findings demonstrated that rhLF acts as

  14. Lactoferrin inhibits dexamethasone-induced chondrocyte impairment from osteoarthritic cartilage through up-regulation of extracellular signal-regulated kinase 1/2 and suppression of FASL, FAS, and Caspase 3

    Energy Technology Data Exchange (ETDEWEB)

    Tu, Yihui [Department of Orthopaedics, Yangpu District Central Hospital Affiliated to Tongji University School of Medicine, 450 Tengyue Road, Shanghai (China); Xue, Huaming [Department of Orthopaedics, Yangpu District Central Hospital Affiliated to Tongji University School of Medicine, 450 Tengyue Road, Shanghai (China); Institute of Life Science, College of Medicine, Swansea University, Singleton Park (United Kingdom); Francis, Wendy [Institute of Life Science, College of Medicine, Swansea University, Singleton Park (United Kingdom); Davies, Andrew P. [Department of Orthopaedics and Trauma, Moriston Hospital, Swansea (United Kingdom); Pallister, Ian; Kanamarlapudi, Venkateswarlu [Institute of Life Science, College of Medicine, Swansea University, Singleton Park (United Kingdom); Xia, Zhidao, E-mail: zhidao.xia@gmail.com [Institute of Life Science, College of Medicine, Swansea University, Singleton Park (United Kingdom)

    2013-11-08

    Highlights: •Dex exerts dose-dependant inhibition of HACs viability and induction of apoptosis. •Dex-induced impairment of chondrocytes was attenuated by rhLF. •ERK and FASL/FAS signaling are involved in the effects of rhLF. •OA patients with glucocorticoid-induced cartilage damage may benefit from treatment with rhLF. -- Abstract: Dexamethasone (Dex) is commonly used for osteoarthritis (OA) with excellent anti-inflammatory and analgesic effect. However, Dex also has many side effects following repeated use over prolonged periods mainly through increasing apoptosis and inhibiting proliferation. Lactoferrin (LF) exerts significantly anabolic effect on many cells and little is known about its effect on OA chondrocytes. Therefore, the aim of this study is to investigate whether LF can inhibit Dex-induced OA chondrocytes apoptosis and explore its possible molecular mechanism involved in. MTT assay was used to determine the optimal concentration of Dex and recombinant human LF (rhLF) on chondrocytes at different time and dose points. Chondrocytes were then stimulated with Dex in the absence or presence of optimal concentration of rhLF. Cell proliferation and viability were evaluated using MTT and LIVE/DEAD assay, respectively. Cell apoptosis was evaluated by multi-parameter apoptosis assay kit using both confocal microscopy and flow cytometry, respectively. The expression of extracellular signal-regulated kinase (ERK), FAS, FASL, and Caspase-3 (CASP3) at the mRNA and protein levels were examined by real-time polymerase chain reaction (PCR) and immunocytochemistry, respectively. The optimal concentration of Dex (25 μg/ml) and rhLF (200 μg/ml) were chosen for the following experiments. rhLF significantly reversed the detrimental effect of Dex on chondrocytes proliferation, viability, and apoptosis. In addition, rhLF significantly prevented Dex-induced down-regulation of ERK and up-regulation of FAS, FASL, and CASP3. These findings demonstrated that rhLF acts as

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

    OpenAIRE

    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...

  16. Adipose-Derived Stem Cells Suppress Inflammation Induced by IL-1β through Down-Regulation of P2X7R Mediated by miR-373 in Chondrocytes of Osteoarthritis.

    Science.gov (United States)

    Jin, Rilong; Shen, Miaoda; Yu, Liedao; Wang, Xuanwei; Lin, Xiangjin

    2017-03-01

    Adipose-derived stem cells (ADSCs) were previously considered to have an anti-inflammatory effect, and Interleukin-1β (IL-1β) was found to be a pro-inflammatory factor in chondrocytes, but the mechanism underlying ADSCs and IL-1β is unclear. In this study, we investigate whether P2X7 receptor (P2X7R) signalling, regulated by microRNA 373 (miR-373), was involved in the ADSCs and IL-1β mediated inflammation in osteoarthritis (OA). Chondrocytes were collected from 20 OA patients and 20 control participants, and ADSCs were collected from patients who had undergone abdominal surgery. The typical surface molecules of ASDCs were detected by flow cytometry. The level of nitric oxide (NO) was determined by Griess reagent. Concentrations of prostaglandin E2 (PGE2), interleukin 6 (IL-6), matrix metallopeptidase 3 (MMP-3) were detected by enzyme-linked immunosorbent assay (ELISA). The expressions of IL-6, MMP-3, miR-373 and P2X7R were determined by real-time polymerase chain reaction (PCR), and Western blot was used to detect the protein expression of P2X7R. The typical potential characters of ADSCs were verified. In chondrocytes or OA tissues, the miR-373 expression level was decreased, but the P2X7R expression was increased. IL-1β stimulation increased the level of inflammatory factors in OA chondrocytes, and ADSCs co-cultured with IL-1β-stimulated chondrocytes decreased the inflammation. OA chondrocytes transfected with the miR-373 inhibitor increased the inflammation level. The miR-373 mimic suppressed the inflammation by targeting P2X7R and regulated its expression, while its effect was reversed by overexpression of P2X7R. IL-1β induced inflammation in OA chondrocytes, while ADSCs seemed to inhibit the expression of P2X7R that was regulated by miR-373 and involved in the anti-inflammatory process in OA.

  17. Proteoglycon synthesis by articular chondrocytes in agarose culture

    International Nuclear Information System (INIS)

    Sweet, M.B.E.; Grisillo, A.; Coehlo, A.; Schnitzler, C.M.

    1987-01-01

    Articular chondrocytes were isolated from knee joints of full-term bovine foetuses and grown in long-term agarose cultures. At intervals, cultures were labelled with 35 S-[sulphate] or D[6- 3 H] glucosamine. Newly synthesized proteoglycans were extracted with 4 M guanidine HCl and purified by isopycnic density gradient centrifugation or on DEAE cellulose in the presence of 8 M urea. Characterization of the proteoglycans revealed them to be identical in size to those present in the tissue and to be similarly capable of aggregation with hyaluronate. Newly synthesized chondroitin sulphate chains were identical in size, but newly synthesized keratan sulphate chains were somewhat larger than those present in the tissue. The newly synthesized proteoglycans were shown to contain the same range of O-linked oligosaccharides identified in proteoglycans of the Swarm rat chondrosarcoma. Cartilage-specific proteoglycan continued to be synthesized by the chondrocytes for up to 60 days; however, with time, proportionately more of a small non-aggregating proteoglycan appeared

  18. MR imaging of osteochondral grafts and autologous chondrocyte implantation

    Energy Technology Data Exchange (ETDEWEB)

    Trattnig, S. [Medical University of Vienna, MR Centre of Excellence, Department of Radiology, Vienna (Austria); University Hospital of Vienna, MR-Center, Department of Radiology, Vienna (Austria); Millington, S.A. [Medical University of Vienna, MR Centre of Excellence, Department of Radiology, Vienna (Austria); Medical University of Vienna, Department of Trauma Surgery, Center for Joints and Cartilage, Vienna (Austria); Szomolanyi, P. [Medical University of Vienna, MR Centre of Excellence, Department of Radiology, Vienna (Austria); Marlovits, S. [Medical University of Vienna, Department of Trauma Surgery, Center for Joints and Cartilage, Vienna (Austria)

    2007-01-15

    Surgical articular cartilage repair therapies for cartilage defects such as osteochondral autograft transfer, autologous chondrocyte implantation (ACI) or matrix associated autologous chondrocyte transplantation (MACT) are becoming more common. MRI has become the method of choice for non-invasive follow-up of patients after cartilage repair surgery. It should be performed with cartilage sensitive sequences, including fat-suppressed proton density-weighted T2 fast spin-echo (PD/T2-FSE) and three-dimensional gradient-echo (3D GRE) sequences, which provide good signal-to-noise and contrast-to-noise ratios. A thorough magnetic resonance (MR)-based assessment of cartilage repair tissue includes evaluations of defect filling, the surface and structure of repair tissue, the signal intensity of repair tissue and the subchondral bone status. Furthermore, in osteochondral autografts surface congruity, osseous incorporation and the donor site should be assessed. High spatial resolution is mandatory and can be achieved either by using a surface coil with a 1.5-T scanner or with a knee coil at 3 T; it is particularly important for assessing graft morphology and integration. Moreover, MR imaging facilitates assessment of complications including periosteal hypertrophy, delamination, adhesions, surface incongruence and reactive changes such as effusions and synovitis. Ongoing developments include isotropic 3D sequences, for improved morphological analysis, and in vivo biochemical imaging such as dGEMRIC, T2 mapping and diffusion-weighted imaging, which make functional analysis of cartilage possible. (orig.)

  19. L-carnitine enhances extracellular matrix synthesis in human primary chondrocytes.

    Science.gov (United States)

    Stoppoloni, Daniela; Politi, Laura; Dalla Vedova, Pietro; Messano, Masa; Koverech, Aleardo; Scandurra, Roberto; Scotto d'Abusco, Anna

    2013-09-01

    Osteoarthritis (OA) is one of the most common degenerative joint disease for which there is no cure. It is treated mainly with non-steroidal anti-inflammatory drugs to control the symptoms and some supplements, such as glucosamine and chondroitin sulphate in order to obtain structure-modifying effects. Aim of this study is to investigate the effects of L-carnitine, a molecule with a role in cellular energy metabolism, on extracellular matrix synthesis in human primary chondrocytes (HPCs). Dose-dependent effect of L-carnitine on cartilage matrix production, cell proliferation and ATP synthesis was examined by incubating HPCs with various amounts of molecule in monolayer (2D) and in hydromatrix scaffold (3D). L-Carnitine affected extracellular matrix synthesis in 3D in a dose-dependent manner; moreover, L-carnitine was very effective to stimulate cell proliferation and to induce ATP synthesis, mainly in 3D culture condition. In conclusion, L-carnitine enhances cartilage matrix glycosaminoglycan component production and cell proliferation, suggesting that this molecule could be useful in the treatment of pathologies where extracellular matrix is degraded, such as OA. To our knowledge, this is the first study where the effects of L-carnitine are evaluated in HPCs.

  20. Chondrocyte survival in osteochondral transplant cylinders depends on the harvesting technique.

    Science.gov (United States)

    Hafke, Benedikt; Petri, Maximilian; Suero, Eduardo; Neunaber, Claudia; Kwisda, Sebastian; Krettek, Christian; Jagodzinski, Michael; Omar, Mohamed

    2016-07-01

    In autologous osteochondral transplantation, the edges of the harvested plug are particularly susceptible to mechanical or thermal damage to the chondrocytes. We hypothesised that the applied harvesting device has an impact on chondrocyte vitality. Both knees of five blackhead sheep (ten knees) underwent open osteochondral plug harvesting with three different circular harvesting devices (osteoarticular transfer system harvester [OATS; diameter 8 mm; Arthrex, Munich, Germany], diamond cutter [DC; diameter 8.35 mm; Karl Storz, Tuttlingen, Germany] and hollow reamer with cutting crown [HRCC; diameter 7 mm; Dannoritzer, Tuttlingen, Germany]) from distinctly assigned anatomical sites of the knee joint. The rotary cutters (DC and HRCC) were either used with (+) or without cooling (-). Surgical cuts of the cartilage with a scalpel blade were chosen as control method. After cryotomy cutting, chondrocyte vitality was assessed using fluorescence microscopy and a Live/Dead assay. There were distinct patterns of chondrocyte vitality, with reproducible accumulations of dead chondrocytes along the harvesting edge. No statistical difference in chondrocyte survivorship was seen between the OATS technique and the control method, or between the HRCC+ technique and the control method (P > 0.05). The DC+, HRCC- and DC- techniques yielded significantly lower chondrocyte survival rates compared with the control method (P vitality.

  1. Crucial Role of Elovl6 in Chondrocyte Growth and Differentiation during Growth Plate Development in Mice.

    Directory of Open Access Journals (Sweden)

    Manami Kikuchi

    Full Text Available ELOVL family member 6, elongation of very long chain fatty acids (Elovl6 is a microsomal enzyme, which regulates the elongation of C12-16 saturated and monounsaturated fatty acids. Elovl6 has been shown to be associated with various pathophysiologies including insulin resistance, atherosclerosis, and non-alcoholic steatohepatitis. To investigate a potential role of Elovl6 during bone development, we here examined a skeletal phenotype of Elovl6 knockout (Elovl6-/- mice. The Elovl6-/- skeleton was smaller than that of controls, but exhibited no obvious patterning defects. Histological analysis revealed a reduced length of proliferating and an elongated length of hypertrophic chondrocyte layer, and decreased trabecular bone in Elovl6-/- mice compared with controls. These results were presumably due to a modest decrease in chondrocyte proliferation and accelerated differentiation of cells of the chondrocyte lineage. Consistent with the increased length of the hypertrophic chondrocyte layer in Elovl6-/- mice, Collagen10α1 was identified as one of the most affected genes by ablation of Elovl6 in chondrocytes. Furthermore, this elevated expression of Collagen10α1 of Elovl6-null chondrocytes was likely associated with increased levels of Foxa2/a3 and Mef2c mRNA expression. Relative increases in protein levels of nuclear Foxa2 and cytoplasmic histone deacethylase 4/5/7 were also observed in Elovl6 knockdown cells of the chondrocyte lineage. Collectively, our data suggest that Elovl6 plays a critical role for proper development of embryonic growth plate.

  2. Prolonged application of high fluid shear to chondrocytes recapitulates gene expression profiles associated with osteoarthritis.

    Directory of Open Access Journals (Sweden)

    Fei Zhu

    Full Text Available 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/cm(2 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

  3. Effects of intermittent versus continuous parathyroid hormone administration on condylar chondrocyte proliferation and differentiation

    International Nuclear Information System (INIS)

    Liu, Qi; Wan, Qilong; Yang, Rongtao; Zhou, Haihua; Li, Zubing

    2012-01-01

    Highlights: ► Different PTH administration exerts different effects on condylar chondrocyte. ► Intermittent PTH administration suppresses condylar chondrocyte proliferation. ► Continuous PTH administration maintains condylar chondrocyte proliferating. ► Intermittent PTH administration enhances condylar chondrocyte differentiation. -- Abstract: Endochondral ossification is a complex process involving chondrogenesis and osteogenesis regulated by many hormones and growth factors. Parathyroid hormone (PTH), one of the key hormones regulating bone metabolism, promotes osteoblast differentiation and osteogenesis by intermittent administration, whereas continuous PTH administration inhibits bone formation. However, the effects of PTH on chondrocyte proliferation and differentiation are still unclear. In this study, intermittent PTH administration presented enhanced effects on condylar chondrocyte differentiation and bone formation, as demonstrated by increased mineral nodule formation and alkaline phosphatase (ALP) activity, up-regulated runt-related transcription factor 2 (RUNX2), ALP, collagen type X (COL10a1), collagen type I (COL1a1), osteocalcin (OCN), bone sialoprotein (BSP), bone morphogenetic protein 2 (BMP2) and osterix (OSX) mRNA and/or protein expression. On the contrary, continuous PTH administration promoted condylar chondrocyte proliferation and suppressed its differentiation, as demonstrated by up-regulated collagen type II (COL2a1) mRNA expression, reduced mineral nodule formation and down-regulated expression of the mRNAs and/or proteins mentioned above. Our data suggest that PTH can regulate condylar chondrocyte proliferation and differentiation, depending on the type of PTH administration. These results provide new insight into the effects of PTH on condylar chondrocytes and new evidence for using local PTH administration to cure mandibular asymmetry.

  4. Effects of intermittent versus continuous parathyroid hormone administration on condylar chondrocyte proliferation and differentiation

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Qi; Wan, Qilong; Yang, Rongtao; Zhou, Haihua [The State Key Laboratory Breeding Base of Basic Science of Stomatology (Hubei-MOST) and Key Laboratory of Oral Biomedicine Ministry of Education, School and Hospital of Stomatology, Wuhan University, 237 Luoyu Road, Wuhan 430079 (China); Li, Zubing, E-mail: lizubing0827@163.com [The State Key Laboratory Breeding Base of Basic Science of Stomatology (Hubei-MOST) and Key Laboratory of Oral Biomedicine Ministry of Education, School and Hospital of Stomatology, Wuhan University, 237 Luoyu Road, Wuhan 430079 (China); Department of Oral and Maxillofacial Surgery, School and Hospital of Stomatology, Wuhan University, 237 Luoyu Road, Wuhan 430079 (China)

    2012-07-20

    Highlights: Black-Right-Pointing-Pointer Different PTH administration exerts different effects on condylar chondrocyte. Black-Right-Pointing-Pointer Intermittent PTH administration suppresses condylar chondrocyte proliferation. Black-Right-Pointing-Pointer Continuous PTH administration maintains condylar chondrocyte proliferating. Black-Right-Pointing-Pointer Intermittent PTH administration enhances condylar chondrocyte differentiation. -- Abstract: Endochondral ossification is a complex process involving chondrogenesis and osteogenesis regulated by many hormones and growth factors. Parathyroid hormone (PTH), one of the key hormones regulating bone metabolism, promotes osteoblast differentiation and osteogenesis by intermittent administration, whereas continuous PTH administration inhibits bone formation. However, the effects of PTH on chondrocyte proliferation and differentiation are still unclear. In this study, intermittent PTH administration presented enhanced effects on condylar chondrocyte differentiation and bone formation, as demonstrated by increased mineral nodule formation and alkaline phosphatase (ALP) activity, up-regulated runt-related transcription factor 2 (RUNX2), ALP, collagen type X (COL10a1), collagen type I (COL1a1), osteocalcin (OCN), bone sialoprotein (BSP), bone morphogenetic protein 2 (BMP2) and osterix (OSX) mRNA and/or protein expression. On the contrary, continuous PTH administration promoted condylar chondrocyte proliferation and suppressed its differentiation, as demonstrated by up-regulated collagen type II (COL2a1) mRNA expression, reduced mineral nodule formation and down-regulated expression of the mRNAs and/or proteins mentioned above. Our data suggest that PTH can regulate condylar chondrocyte proliferation and differentiation, depending on the type of PTH administration. These results provide new insight into the effects of PTH on condylar chondrocytes and new evidence for using local PTH administration to cure mandibular

  5. Primary Culture of Canine Growth Plate Chondrocytes as a Model of Biomineralization

    OpenAIRE

    Ryuji, HOSOKAWA; Kenji, KIKUZAKI; Daisuke, CHIBA; Yasumasa, AKAGAWA; Department of Removable Prosthodontics, Hiroshima University School of Dentistry; Department of Removable Prosthodontics, Hiroshima University School of Dentistry; Department of Removable Prosthodontics, Hiroshima University School of Dentistry; Department of Removable Prosthodontics, Hiroshima University School of Dentistry

    1999-01-01

    This study investigated the mineralization process in primary cultures of dog growth plate chondrocytes as a model of biomineralization. Chondrocytes were isolated from the growth plates of ribs of 1-week-old dogs. The chondrocytes were maintained at extremely high density (5x10^4 cells/well) in collagen-coated 96-well dishes in a-MEM supplemented with 10% fetal bovine serum and 50 μg/ml ascorbic acid. Mineralization was initiated between days 20 and 24; however, the addition of fibroblast gr...

  6. Nuclear deformation and expression change of cartilaginous genes during in vitro expansion of chondrocytes

    International Nuclear Information System (INIS)

    Hoshiba, Takashi; Yamada, Tomoe; Lu, Hongxu; Kawazoe, Naoki; Tateishi, Tetsuya; Chen, Guoping

    2008-01-01

    Cartilaginous gene expression decreased when chondrocytes were expanded on cell-culture plates. Understanding the dedifferentiation mechanism may provide valuable insight into cartilage tissue engineering. Here, we demonstrated the relationship between the nuclear shape and gene expression during in vitro expansion culture of chondrocytes. Specifically, the projected nuclear area increased and cartilaginous gene expressions decreased during in vitro expansion culture. When the nuclear deformation was recovered by cytochalasin D treatment, aggrecan expression was up-regulated and type I collagen (Col1a2) expression was down-regulated. These results suggest that nuclear deformation may be one of the mechanisms for chondrocyte dedifferentiation during in vitro expansion culture

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

    International Nuclear Information System (INIS)

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

    2008-01-01

    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

  8. Chondrocyte activity is increased in psoriatic arthritis and axial spondyloarthritis

    DEFF Research Database (Denmark)

    Gudmann, Natasja Stæhr; Munk, Heidi Lausten; Christensen, Anne Friesgaard

    2016-01-01

    BACKGROUND: Psoriatic arthritis (PsA) and axial spondyloarthritis (axSpA) are chronic inflammatory rheumatic diseases with complex origins. Both are characterized by altered extracellular matrix remodeling in joints and entheses that results in destructive and osteochondral proliferative lesions.......30 ng/ml, 0.16-0.41) (p treatment. C-Col10 was slightly but equally elevated in the PsA and axSpA groups vs. the control group, but it was significantly lower in patients with axSpA undergoing tumor necrosis factor-α inhibitor (TNFi) treatment. ROC curve......SpA undergoing TNFi treatment may reflect that hypertrophic chondrocytes in axSpA are targeted by TNFi. ROC curve analysis showed a diagnostic potential for Pro-C2 in axSpA and PsA....

  9. Multiphoton microscope measurement-based biphasic multiscale analyses of knee joint articular cartilage and chondrocyte by using visco-anisotropic hyperelastic finite element method and smoothed particle hydrodynamics method.

    Science.gov (United States)

    Nakamachi, Eiji; Noma, Tomohiro; Nakahara, Kaito; Tomita, Yoshihiro; Morita, Yusuke

    2017-11-01

    The articular cartilage of a knee joint has a variety of functions including dispersing stress and absorbing shock in the tissue and lubricating the surface region of cartilage. The metabolic activity of chondrocytes under the cyclic mechanical stimulations regenerates the morphology and function of tissues. Hence, the stress evaluation of the chondrocyte is a vital subject to assess the regeneration cycle in the normal walking condition and predict the injury occurrence in the accidents. Further, the threshold determination of stress for the chondrocytes activation is valuable for development of regenerative bioreactor of articular cartilage. In this study, in both macroscale and microscale analyses, the dynamic explicit finite element (FE) method was used for the solid phase and the smoothed particle hydrodynamics (SPH) method was used for the fluid phase. In the homogenization procedure, the representative volume element for the microscale finite element model was derived by using the multiphoton microscope measured 3D structure comprising 3 different layers: surface, middle, and deep layers. The layers had different anisotropic structural and rigidity characteristics because of the collagen fiber orientation. In both macroscale and microscale FE analyses, the visco-anisotropic hyperelastic constitutive law was used. Material properties were identified by experimentally determined stress-strain relationships of 3 layers. With respect to the macroscale and microscale SPH models for non-Newtonian viscous fluid, the previous observation results of interstitial fluid and proteoglycan were used to perform parameter identifications. Biphasic multiscale FE and SPH analyses were conducted under normal walking conditions. Therefore, the hydrostatic and shear stresses occurring in the chondrocytes caused by the compressive load and shear viscous flow were evaluated. These stresses will be used to design an ex-vivo bioreactor to regenerate the damaged articular cartilage

  10. [Optimization of oxytetracycline biosynthesis].

    Science.gov (United States)

    Maksimova, E A; Falkov, N N; Izmaĭlov, N N; Romanchuk, N N

    1988-06-01

    It was shown that rising of temperature up to 30 degrees C at the stage of the oxytetracycline-producing organism growth promoted acceleration of the culture growth rate and increasing of the antibiotic concentration by the 114th hour of the biosynthetic process. For the apparatus used in the study optimal aeration and agitation conditions were developed. To provide optimal parameters during biosynthesis of oxytetracycline, it was recommended to use the aeration rate of 1 v/v.min and the specific mechanical power for mixing of not less than 1 kW/m3.

  11. Biosynthesis of Rishirilide B

    Directory of Open Access Journals (Sweden)

    Philipp Schwarzer

    2018-03-01

    Full Text Available Rishirilide B was isolated from Streptomyces rishiriensis and Streptomyces bottropensis on the basis of its inhibitory activity towards alpha-2-macroglobulin. The biosynthesis of rishirilide B was investigated by feeding experiments with different 13C labelled precursors using the heterologous host Streptomyces albus J1074::cos4 containing a cosmid encoding of the gene cluster responsible for rishirilide B production. NMR spectroscopic analysis of labelled compounds demonstrate that the tricyclic backbone of rishirilide B is a polyketide synthesized from nine acetate units. One of the acetate units is decarboxylated to give a methyl group. The origin of the starter unit was determined to be isobutyrate.

  12. Synthesis of collagen by bovine chondrocytes cultured in alginate; posttranslational modifications and cell-matrix interaction

    NARCIS (Netherlands)

    Beekman, B.; Verzijl, N.; Bank, R.A.; Von Der Mark, K.; TeKoppele, J.M.

    1997-01-01

    The extracellular matrix synthesized by articular chondrocytes cultured in alginate beads was investigated. Collagen levels increased sigmoidally with time and remained constant after 2 weeks of culture. The presence of cartilage-specific type II collagen was confirmed immunohistochemically.

  13. Exploration of mechanisms underlying the strain-rate-dependent mechanical property of single chondrocytes

    Energy Technology Data Exchange (ETDEWEB)

    Nguyen, Trung Dung; Gu, YuanTong, E-mail: yuantong.gu@qut.edu.au [School of Chemistry, Physics and Mechanical Engineering, Queensland University of Technology, Brisbane, Queensland (Australia)

    2014-05-05

    Based on the characterization by Atomic Force Microscopy, we report that the mechanical property of single chondrocytes has dependency on the strain-rates. By comparing the mechanical deformation responses and the Young's moduli of living and fixed chondrocytes at four different strain-rates, we explore the deformation mechanisms underlying this dependency property. We found that the strain-rate-dependent mechanical property of living cells is governed by both of the cellular cytoskeleton and the intracellular fluid when the fixed chondrocytes are mainly governed by their intracellular fluid, which is called the consolidation-dependent deformation behavior. Finally, we report that the porohyperelastic constitutive material model which can capture the consolidation-dependent behavior of both living and fixed chondrocytes is a potential candidature to study living cell biomechanics.

  14. Fluoroquinolone's effect on growth of human chondrocytes and chondrosarcomas. In vitro and in vivo correlation

    DEFF Research Database (Denmark)

    Multhaupt, H A; Alvarez, J C; Rafferty, P A

    2001-01-01

    Clinical and in vitro studies have demonstrated that fluoroquinolones are toxic to chondrocytes; however, the exact mechanism of fluoroquinolone arthropathy is unknown. We investigated the toxicity of ciprofloxacin on normal cartilage and on cartilaginous tumors. Normal human cartilage, enchondroma...

  15. A Preliminary Study of Human Amniotic Membrane as a Potential Chondrocyte Carrier

    Directory of Open Access Journals (Sweden)

    L Boo

    2009-11-01

    Full Text Available PURPOSE: To investigate the feasibility of using processed human amniotic membrane (HAM to support the attachment and proliferation of chondrocytes in vitro which in turn can be utilised as a cell delivery vehicle in tissue engineering applications. METHODS: Fresh HAM obtained from patients undergoing routine elective caesarean sections was harvested, processed and dried using either freeze drying (FD or air drying (AD methods prior to sterilisation by gamma irradiation. Isolated, processed and characterised rabbit autologous chondrocytes were seeded on processed HAM and cultured for up to three weeks. Cell attachment and proliferation were examined qualitatively using inverted brightfield microscopy. RESULTS: Processed HAM appeared to allow cell attachment when implanted with chondrocytes. Although cells seeded on AD and FD HAM did not appear to attach as strongly as those seeded on glycerol preserved intact human amniotic membrane, these cells to be proliferated in cell culture conditions. CONCLUSION: Preliminary results show that processed HAM promotes chondrocyte attachment and proliferation.

  16. Effects of scaffold composition and architecture on human nasal chondrocyte redifferentiation and cartilaginous matrix deposition

    NARCIS (Netherlands)

    Miot, Sylvie; Woodfield, T.B.F.; Daniels, Alma U.; Suetterlin, Rosemarie; Peterschmitt, Iman; Heberer, Michael; van Blitterswijk, Clemens; Riesle, J.U.; Martin, Ivan

    2005-01-01

    We investigated whether the post-expansion redifferentiation and cartilage tissue formation capacity of adult human nasal chondrocytes can be regulated by controlled modifications of scaffold composition and architecture. As a model system, we used poly(ethylene

  17. Hyaluronan Protects Bovine Articular Chondrocytes against Cell Death Induced by Bupivacaine under Supraphysiologic Temperatures

    Science.gov (United States)

    Liu, Sen; Zhang, Qing-Song; Hester, William; O’Brien, Michael J.; Savoie, Felix H.; You, Zongbing

    2013-01-01

    Background Bupivacaine and supraphysiologic temperature can independently reduce cell viability of articular chondrocytes. In combination these two deleterious factors could further impair cell viability. Hypothesis Hyaluronan may protect chondrocytes from death induced by bupivacaine at supraphysiologic temperatures. Study Design Controlled laboratory study. Methods Bovine articular chondrocytes were treated with hyaluronan at physiologic (37°C) and supraphysiologic temperatures (45°C and 50°C) for one hour, and then exposed to bupivacaine for one hour at room temperature. Cell viability was assessed at three time points: immediately after treatment, six hours later, and twenty-four hours later using flow cytometry and fluorescence microscopy. The effects of hyaluronan on the levels of sulfated glycosaminoglycan in the chondrocytes were determined using Alcian blue staining. Results (1) Bupivacaine alone did not induce noticeable chondrocyte death at 37°C; (2) bupivacaine and temperature synergistically increased chondrocyte death, that is, when the chondrocytes were conditioned to 45°C and 50°C, 0.25% and 0.5% bupivacaine increased the cell death rate by 131% to 383% in comparison to the phosphate-buffered saline control group; and, (3) addition of hyaluronan reduced chondrocyte death rates to approximately 14% and 25% at 45°C and 50°C, respectively. Hyaluronan’s protective effects were still observed at six and twenty-four hours after bupivacaine treatment at 45°C. However, at 50°C, hyaluronan delayed but did not prevent the cell death caused by bupivacaine. One-hour treatment with hyaluronan significantly increased sulfated glycosaminoglycan levels in the chondrocytes. Conclusions Bupivacaine and supraphysiologic temperature synergistically increase chondrocyte death and hyaluronan may protect articular chondrocytes from death caused by bupivacaine. Clinical Relevance This study provides a rationale to perform pre-clinical and clinical studies to

  18. Isolation and characterization of human articular chondrocytes from surgical waste after total knee arthroplasty (TKA

    Directory of Open Access Journals (Sweden)

    Jakob Naranda

    2017-03-01

    Full Text Available Background Cartilage tissue engineering is a fast-evolving field of biomedical engineering, in which the chondrocytes represent the most commonly used cell type. Since research in tissue engineering always consumes a lot of cells, simple and cheap isolation methods could form a powerful basis to boost such studies and enable their faster progress to the clinics. Isolated chondrocytes can be used for autologous chondrocyte implantation in cartilage repair, and are the base for valuable models to investigate cartilage phenotype preservation, as well as enable studies of molecular features, nature and scales of cellular responses to alterations in the cartilage tissue. Methods Isolation and consequent cultivation of primary human adult articular chondrocytes from the surgical waste obtained during total knee arthroplasty (TKA was performed. To evaluate the chondrogenic potential of the isolated cells, gene expression of collagen type 2 (COL2, collagen 1 (COL1 and aggrecan (ACAN was evaluated. Immunocytochemical staining of all mentioned proteins was performed to evaluate chondrocyte specific production. Results Cartilage specific gene expression of COL2 and ACAN has been shown that the proposed protocol leads to isolation of cells with a high chondrogenic potential, possibly even specific phenotype preservation up to the second passage. COL1 expression has confirmed the tendency of the isolated cells dedifferentiation into a fibroblast-like phenotype already in the second passage, which confirms previous findings that higher passages should be used with care in cartilage tissue engineering. To evaluate the effectiveness of our approach, immunocytochemical staining of the evaluated chondrocyte specific products was performed as well. Discussion In this study, we developed a protocol for isolation and consequent cultivation of primary human adult articular chondrocytes with the desired phenotype from the surgical waste obtained during TKA. TKA is a

  19. Role of Insulin-Transferrin-Selenium in Auricular Chondrocyte Proliferation and Engineered Cartilage Formation in Vitro

    Directory of Open Access Journals (Sweden)

    Xia Liu

    2014-01-01

    Full Text Available The goal of this study is to determine the effects of Insulin-Transferrin-Selenium (ITS on proliferation of auricular chondrocytes and formation of engineered cartilage in vitro. Pig auricular monolayer chondrocytes and chondrocyte pellets were cultured in media containing 1% ITS at different concentrations of fetal bovine serum (FBS, 10%, 6%, 2%, 0%, or 10% FBS alone as a control for four weeks. Parameters including cell proliferation in monolayer, wet weight, collagen type I/II/X (Col I, II, X and glycosaminoglycan (GAG expression, GAG content of pellets and gene expression associated with cartilage formation/dedifferentiation (lost cartilage phenotype/hypertrophy within the chondrocyte pellets were assessed. The results showed that chondrocytes proliferation rates increased when FBS concentrations increased (2%, 6%, 10% FBS in ITS supplemented groups. In addition, 1% ITS plus 10% FBS significantly promoted cell proliferation than 10% FBS alone. No chondrocytes grew in ITS alone medium. 1% ITS plus 10% FBS enhanced cartilage formation in terms of size, wet weight, cartilage specific matrices, and homogeneity, compared to 10% FBS alone group. Furthermore, ITS prevented engineered cartilage from dedifferentiation (i.e., higher index of Col II/Col I mRNA expression and expression of aggrecan and hypertrophy (i.e., lower mRNA expression of Col X and MMP13. In conclusion, our results indicated that ITS efficiently enhanced auricular chondrocytes proliferation, retained chondrogenic phenotypes, and promoted engineered cartilage formation when combined with FBS, which is potentially used as key supplementation in auricular chondrocytes and engineered cartilage culture.

  20. Changes in the expression of collagen genes show two stages in chondrocyte differentiation in vitro

    OpenAIRE

    1988-01-01

    This report deals with the quantitation of both mRNA and transcription activity of type I collagen gene and of three cartilage-specific collagens (types II, IX, and X) during in vitro differentiation of chick chondrocytes. Differentiation was obtained by transferal to suspension culture of dedifferentiated cells passaged for 3 wk as adherent cells. The type I collagen mRNA, highly represented in the dedifferentiated cells, rapidly decreased during chondrocyte differentiation. On the contrary,...

  1. (+)-Germacrene A Biosynthesis

    Science.gov (United States)

    de Kraker, Jan-Willem; Franssen, Maurice C.R.; de Groot, Aede; König, Wilfried A.; Bouwmeester, Harro J.

    1998-01-01

    The leaves and especially the roots of chicory (Cichorium intybus L.) contain high concentrations of bitter sesquiterpene lactones such as the guianolides lactupicrin, lactucin, and 8-deoxylactucin. Eudesmanolides and germacranolides are present in smaller amounts. Their postulated biosynthesis through the mevalonate-farnesyl diphosphate-germacradiene pathway has now been confirmed by the isolation of a (+)-germacrene A synthase from chicory roots. This sesquiterpene cyclase was purified 200-fold using a combination of anion-exchange and dye-ligand chromatography. It has a Km value of 6.6 μm, an estimated molecular mass of 54 kD, and a (broad) pH optimum around 6.7. Germacrene A, the enzymatic product, proved to be much more stable than reported in literature. Its heat-induced Cope rearrangement into (−)-β-elemene was utilized to determine its absolute configuration on an enantioselective gas chromatography column. To our knowledge, until now in sesquiterpene biosynthesis, germacrene A has only been reported as an (postulated) enzyme-bound intermediate, which, instead of being released, is subjected to additional cyclization(s) by the same enzyme that generated it from farnesyl diphosphate. However, in chicory germacrene A is released from the sesquiterpene cyclase. Apparently, subsequent oxidations and/or glucosylation of the germacrane skeleton, together with a germacrene cyclase, determine whether guaiane- or eudesmane-type sesquiterpene lactones are produced. PMID:9701594

  2. Incorporation of biomimetic matrix molecules in PEG hydrogels enhances matrix deposition and reduces load-induced loss of chondrocyte-secreted matrix.

    Science.gov (United States)

    Roberts, Justine J; Nicodemus, Garret D; Giunta, Suzanne; Bryant, Stephanie J

    2011-06-01

    Poly(ethylene glycol) (PEG) hydrogels offer numerous advantages in designing controlled 3D environments for cartilage regeneration, but offer little biorecognition for the cells. Incorporating molecules that more closely mimic the native tissue may provide key signals for matrix synthesis and may also help in the retention of neotissue, particularly when mechanical stimulation is employed. Therefore, this research tested the hypothesis that exogenous hyaluronan encapsulated within PEG hydrogels improves tissue deposition by chondrocytes, while the incorporation of Link-N (DHLSDNYTLDHDRAIH), a fragment of link protein that is involved in stabilizing hyaluronan and aggrecan in cartilage, aids in the retention of the entrapped hyaluronan as well as cell-secreted glycosaminoglycans (GAGs), particularly when dynamic loading is employed. The incorporation of Link-N as covalent tethers resulted in a significant reduction, ~60%, in the loss of entrapped exogenous hyaluronan under dynamic stimulation. When chondrocytes were encapsulated in PEG hydrogels containing exogenous hyaluronan and/or Link-N, the extracellular matrix (ECM) analogs aided in the retention of cell-secreted GAGs under loading. The presence of hyaluronan led to enhanced deposition of collagen type II and aggrecan. In conclusion, our results highlight the importance of ECM analogs, specifically hyaluronan and Link-N, in matrix retention and matrix development and offer new strategies for designing scaffolds for cartilage regeneration. Copyright © 2011 Wiley Periodicals, Inc.

  3. Non-woven PGA/PVA fibrous mesh as an appropriate scaffold for chondrocyte proliferation.

    Science.gov (United States)

    Rampichová, M; Koštáková, E; Filová, E; Prosecká, E; Plencner, M; Ocheretná, L; Lytvynets, A; Lukáš, D; Amler, E

    2010-01-01

    Non-woven textile mesh from polyglycolic acid (PGA) was found as a proper material for chondrocyte adhesion but worse for their proliferation. Neither hyaluronic acid nor chitosan nor polyvinyl alcohol (PVA) increased chondrocyte adhesion. However, chondrocyte proliferation suffered from acidic byproducts of PGA degradation. However, the addition of PVA and/or chitosan into a wet-laid non-woven textile mesh from PGA improved chondrocyte proliferation seeded in vitro on the PGA-based composite scaffold namely due to a diminished acidification of their microenvironment. This PVA/PGA composite mesh used in combination with a proper hydrogel minimized the negative effect of PGA degradation without dropping positive parameters of the PGA wet-laid non-woven textile mesh. In fact, presence of PVA and/or chitosan in the PGA-based wet-laid non-woven textile mesh even advanced the PGA-based wet-laid non-woven textile mesh for chondrocyte seeding and artificial cartilage production due to a positive effect of PVA in such a scaffold on chondrocyte proliferation.

  4. Characterization of pediatric microtia cartilage: a reservoir of chondrocytes for auricular reconstruction using tissue engineering strategies.

    Science.gov (United States)

    Melgarejo-Ramírez, Y; Sánchez-Sánchez, R; García-López, J; Brena-Molina, A M; Gutiérrez-Gómez, C; Ibarra, C; Velasquillo, C

    2016-09-01

    The external ear is composed of elastic cartilage. Microtia is a congenital malformation of the external ear that involves a small reduction in size or a complete absence. The aim of tissue engineering is to regenerate tissues and organs clinically implantable based on the utilization of cells and biomaterials. Remnants from microtia represent a source of cells for auricular reconstruction using tissue engineering. To examine the macromolecular architecture of microtia cartilage and behavior of chondrocytes, in order to enrich the knowledge of this type of cartilage as a cell reservoir. Auricular cartilage remnants were obtained from pediatric patients with microtia undergoing reconstructive procedures. Extracellular matrix composition was characterized using immunofluorescence and histological staining methods. Chondrocytes were isolated and expanded in vitro using a mechanical-enzymatic protocol. Chondrocyte phenotype was analyzed using qualitative PCR. Microtia cartilage preserves structural organization similar to healthy elastic cartilage. Extracellular matrix is composed of typical cartilage proteins such as type II collagen, elastin and proteoglycans. Chondrocytes displayed morphological features similar to chondrocytes derived from healthy cartilage, expressing SOX9, COL2 and ELN, thus preserving chondral phenotype. Cell viability was 94.6 % during in vitro expansion. Elastic cartilage from microtia has similar characteristics, both architectural and biochemical to healthy cartilage. We confirmed the suitability of microtia remnant as a reservoir of chondrocytes with potential to be expanded in vitro, maintaining phenotypical features and viability. Microtia remnants are an accessible source of autologous cells for auricular reconstruction using tissue engineering strategies.

  5. Uhrf1 is indispensable for normal limb growth by regulating chondrocyte differentiation through specific gene expression.

    Science.gov (United States)

    Yamashita, Michiko; Inoue, Kazuki; Saeki, Noritaka; Ideta-Otsuka, Maky; Yanagihara, Yuta; Sawada, Yuichiro; Sakakibara, Iori; Lee, Jiwon; Ichikawa, Koichi; Kamei, Yoshiaki; Iimura, Tadahiro; Igarashi, Katsuhide; Takada, Yasutsugu; Imai, Yuuki

    2018-01-08

    Transcriptional regulation can be tightly orchestrated by epigenetic regulators. Among these, ubiquitin-like with PHD and RING finger domains 1 (Uhrf1) is reported to have diverse epigenetic functions, including regulation of DNA methylation. However, the physiological functions of Uhrf1 in skeletal tissues remain unclear. Here, we show that limb mesenchymal cell-specific Uhrf1 conditional knockout mice ( Uhrf1 Δ Limb/ Δ Limb ) exhibit remarkably shortened long bones that have morphological deformities due to dysregulated chondrocyte differentiation and proliferation. RNA-seq performed on primary cultured chondrocytes obtained from Uhrf1 Δ Limb/ Δ Limb mice showed abnormal chondrocyte differentiation. In addition, integrative analyses using RNA-seq and MBD-seq revealed that Uhrf1 deficiency decreased genome-wide DNA methylation and increased gene expression through reduced DNA methylation in the promoter regions of 28 genes, including Hspb1 , which is reported to be an IL1-related gene and to affect chondrocyte differentiation. Hspb1 knockdown in cKO chondrocytes can normalize abnormal expression of genes involved in chondrocyte differentiation, such as Mmp13 These results indicate that Uhrf1 governs cell type-specific transcriptional regulation by controlling the genome-wide DNA methylation status and regulating consequent cell differentiation and skeletal maturation. © 2018. Published by The Company of Biologists Ltd.

  6. Distinct Transcriptional Programs Underlie Sox9 Regulation of the Mammalian Chondrocyte

    Directory of Open Access Journals (Sweden)

    Shinsuke Ohba

    2015-07-01

    Full Text Available Sox9 encodes an essential transcriptional regulator of chondrocyte specification and differentiation. When Sox9 nuclear activity was compared with markers of chromatin organization and transcriptional activity in primary chondrocytes, we identified two distinct categories of target association. Class I sites cluster around the transcriptional start sites of highly expressed genes with no chondrocyte-specific signature. Here, Sox9 association reflects protein-protein association with basal transcriptional components. Class II sites highlight evolutionarily conserved active enhancers that direct chondrocyte-related gene activity through the direct binding of Sox9 dimer complexes to DNA. Sox9 binds through sites with sub-optimal binding affinity; the number and grouping of enhancers into super-enhancer clusters likely determines the levels of target gene expression. Interestingly, comparison of Sox9 action in distinct chondrocyte lineages points to similar regulatory strategies. In addition to providing insights into Sox family action, our comprehensive identification of the chondrocyte regulatory genome will facilitate the study of skeletal development and human disease.

  7. Chondrocytes and stem cells in 3D-bioprinted structures create human cartilage in vivo.

    Directory of Open Access Journals (Sweden)

    Peter Apelgren

    Full Text Available Cartilage repair and replacement is a major challenge in plastic reconstructive surgery. The development of a process capable of creating a patient-specific cartilage framework would be a major breakthrough. Here, we described methods for creating human cartilage in vivo and quantitatively assessing the proliferative capacity and cartilage-formation ability in mono- and co-cultures of human chondrocytes and human mesenchymal stem cells in a three-dimensional (3D-bioprinted hydrogel scaffold. The 3D-bioprinted constructs (5 × 5 × 1.2 mm were produced using nanofibrillated cellulose and alginate in combination with human chondrocytes and human mesenchymal stem cells using a 3D-extrusion bioprinter. Immediately following bioprinting, the constructs were implanted subcutaneously on the back of 48 nude mice and explanted after 30 and 60 days, respectively, for morphological and immunohistochemical examination. During explantation, the constructs were easy to handle, and the majority had retained their macroscopic grid appearance. Constructs consisting of human nasal chondrocytes showed good proliferation ability, with 17.2% of the surface areas covered with proliferating chondrocytes after 60 days. In constructs comprising a mixture of chondrocytes and stem cells, an additional proliferative effect was observed involving chondrocyte production of glycosaminoglycans and type 2 collagen. This clinically highly relevant study revealed 3D bioprinting as a promising technology for the creation of human cartilage.

  8. The Ihh signal is essential for regulating proliferation and hypertrophy of cultured chicken chondrocytes.

    Science.gov (United States)

    Ma, R S; Zhou, Z L; Luo, J W; Zhang, H; Hou, J F

    2013-10-01

    The Indian hedgehog (Ihh) signal plays a vital role in regulating proliferation and hypertrophy of chondrocytes. To investigate its function in postnatal chicken (Gallus gallus) chondrocytes, cyclopamine was used to inhibit Ihh signaling. The MTT and ALP assays revealed the downgrade-proliferation and upgrade-differentiation of chondrocytes. To further elucidate the mechanism, the mRNA expression levels of Ihh, parathyroid hormone related protein (PTHrP), Gli-2, Bcl-2, Bone Morphogenetic Protein 6 (BMP-6), type X collagen (Col X) and type II collagen (Col II) were detected by quantitative real-time RT-PCR analysis, and the protein expressions of Ihh, Col X, and Col II were determined using Western blot analysis. After the Ihh signal was blocked, chondrocytes demonstrated high expression levels of PTHrP and Col X and low levels of Gli-2, BMP-6, Bcl-2 and Col II although Ihh expression was increased. Based on these results, the Ihh signal is essential for balancing chicken chondrocyte proliferation and hypertrophy, and the regulatory function of PTHrP acts in an Ihh-dependent manner. Furthermore, BMP-6 and Bcl-2 played roles in maintaining the development of chondrocytes and may be downstream regulatory factors of Ihh signaling. © 2013.

  9. Effects of manganese deficiency on chondrocyte development in tibia growth plate of Arbor Acres chicks.

    Science.gov (United States)

    Wang, Jian; Wang, Zhen Yong; Wang, Zhao Jun; Liu, Ran; Liu, Shao Qiong; Wang, Lin

    2015-01-01

    The aim of this study was to investigate the effects of manganese (Mn) deficiency on chondrocyte development in tibia growth plate. Ninety 1-day-old Arbor Acres chicks were randomly divided into three groups and fed on control diet (60 mg Mn/kg diet) and manganese deficient diets (40 mg Mn/kg diet, manganese deficiency group I; 8.7 mg Mn/kg diet, manganese deficiency group II), respectively. The width of the proliferative zone of growth plate was measured by the microscope graticule. Chondrocyte apoptosis was estimated by TUNEL staining. Gene expression of p21 and Bcl-2, and expression of related proteins were analyzed by quantitative real time reverse transcription polymerase chain reaction and immunohistochemistry, respectively. Compared with the control group, manganese deficiency significantly decreased the proliferative zone width and Bcl-2 mRNA expression level, while significantly increased the apoptotic rates and the expression level of p21 gene in chondrocytes. The results indicate that manganese deficiency had a negative effect on chondrocyte development, which was mediated by the inhibition of chondrocyte proliferation and promotion of chondrocyte apoptosis.

  10. Oxidative stress-induced apoptosis and matrix loss of chondrocytes is inhibited by eicosapentaenoic acid.

    Science.gov (United States)

    Sakata, Shuhei; Hayashi, Shinya; Fujishiro, Takaaki; Kawakita, Kohei; Kanzaki, Noriyuki; Hashimoto, Shingo; Iwasa, Kenjiro; Chinzei, Nobuaki; Kihara, Shinsuke; Haneda, Masahiko; Ueha, Takeshi; Nishiyama, Takayuki; Kuroda, Ryosuke; Kurosaka, Masahiro

    2015-03-01

    Eicosapentaenoic acid (EPA) is an antioxidant and n-3 polyunsaturated fatty acid that reduces the production of inflammatory cytokines. We evaluated the role of EPA in chondrocyte apoptosis and degeneration. Normal human chondrocytes were treated with EPA and sodium nitroprusside (SNP). Expression of metalloproteinases (MMPs) was detected by real-time polymerase chain reaction (PCR) and that of apoptosis-related proteins was detected by western blotting. Chondrocyte apoptosis was detected by flow cytometry. C57BL/6J mice were used for the detection of MMP expression by immunohistochemistry and for investigation of chondrocyte apoptosis. EPA inhibited SNP-induced chondrocyte apoptosis, caspase 3 and poly(ADP-ribose) polymerase cleavage, phosphorylation of p38 MAPK and p53, and expression of MMP3 and MMP13. Intra-articular injection of EPA prevented the progression of osteoarthritis (OA) by inhibiting MMP13 expression and chondrocyte apoptosis. EPA treatment can control oxidative stress-induced OA progression, and thus may be a new approach for OA therapy. © 2014 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.

  11. Efficiency of Human Epiphyseal Chondrocytes with Differential Replication Numbers for Cellular Therapy Products

    Directory of Open Access Journals (Sweden)

    Michiyo Nasu

    2016-01-01

    Full Text Available The cell-based therapy for cartilage or bone requires a large number of cells; serial passages of chondrocytes are, therefore, needed. However, fates of expanded chondrocytes from extra fingers remain unclarified. The chondrocytes from human epiphyses morphologically changed from small polygonal cells to bipolar elongated spindle cells and to large polygonal cells with degeneration at early passages. Gene of type II collagen was expressed in the cells only at a primary culture (Passage 0 and Passage 1 (P1 cells. The nodules by implantation of P0 to P8 cells were composed of cartilage and perichondrium. The cartilage consisted of chondrocytes with round nuclei and type II collagen-positive matrix, and the perichondrium consisted of spindle cells with type I collage-positive matrix. The cartilage and perichondrium developed to bone with marrow cavity through enchondral ossification. Chondrogenesis and osteogenesis by epiphyseal chondrocytes depended on replication number in culture. It is noteworthy to take population doubling level in correlation with pharmaceutical efficacy into consideration when we use chondrocytes for cell-based therapies.

  12. Leptin Antagonizes Peroxisome Proliferator-Activated Receptor-γ Signaling in Growth Plate Chondrocytes

    Directory of Open Access Journals (Sweden)

    Lai Wang

    2012-01-01

    Full Text Available Leptin is an obesity-associated cytokine-like hormone encoded by the ob gene. Recent studies reveal that leptin promotes proliferation and differentiation of chondrocytes, suggesting a peripheral role of leptin in regulating growth plate function. Peroxisome proliferator-activated receptor-γ (PPARγ is a transcriptional regulator of adipogenesis. Locally, PPARγ negatively regulates chondrogenic differentiation and terminal differentiation in the growth plate. The aim of this study was to test the hypothesis that leptin may suppress the inhibitory effects of PPARγ on growth plate chondrocytes. Chondrocytes were collected from distal femoral growth plates of newborn rats and were cultured in monolayer or cell pellets in the presence or absence of leptin and the PPARγ agonist ciglitazone. The results show that leptin attenuates the suppressive effects of PPARγ on chondrogenic differentiation and T3-mediated chondrocyte hypertrophy. Leptin treatment also leads to a mild downregulation of PPAR mRNA expression and a significant MAPK/ERK-dependent PPARγ phosphorylation at serine 112/82. Blocking MAPK/ERK function with PD98059 confirmed that leptin antagonizes PPARγ function in growth plate chondrocytes through the MAPK/ERK signaling pathway. Furthermore, leptin signaling in growth plate cells is also negatively modulated by activation of PPARγ, implying that these two signaling pathways are mutually regulated in growth plate chondrocytes.

  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.

    Directory of Open Access Journals (Sweden)

    Christelle Sanchez

    Full Text Available 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.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.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.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 mediated via inhibition of IL-6 in

  14. A network of transcriptional and signaling events is activated by FGF to induce chondrocyte growth arrest and differentiation

    OpenAIRE

    Dailey, Lisa; Laplantine, Emmanuel; Priore, Riccardo; Basilico, Claudio

    2003-01-01

    Activating mutations in FGF receptor 3 (FGFR3) cause several human dwarfism syndromes by affecting both chondrocyte proliferation and differentiation. Using microarray and biochemical analyses of FGF-treated rat chondrosarcoma chondrocytes, we show that FGF inhibits chondrocyte proliferation by initiating multiple pathways that result in the induction of antiproliferative functions and the down-regulation of growth-promoting molecules. The initiation of growth arrest is characterized by the r...

  15. Type II collagen peptide is able to accelerate embryonic chondrocyte differentiation: an association with articular cartilage matrix resorption in osteoarthrosis

    Directory of Open Access Journals (Sweden)

    Elena Vasil'evna Chetina

    2010-01-01

    Conclusion. The effect of CP on gene expression and collagen decomposition activity depends on the morphotype of embryonic chondrocytes. Lack of effect of CP on collagen decomposition activity in both the embryonic hypertrophic chondrocytes and the cartilage explants from OA patients supports the hypothesis that the hypertrophic morphotype is a dominant morphotype of articular chondrocytes in OA. Moreover, collagen decomposition products can be involved in the resorption of matrix in OA and in the maintenance of chronic nature of the pathology.

  16. [Bio-modification of polyhydroxyalkanoates and its biocompatibility with chondrocytes].

    Science.gov (United States)

    Gao, Tianxi; Chang, Huimin; Fan, Minjie; Lu, Xiaoyun; Wang, Zhenghui; Zhang, Xianghong; Jing, Xiaohong; Shi, Yanxia; Li, Zhihui

    2014-08-01

    To study the hydrophilicity and the cell biocompatibility of the poly(3-hydroxybutyrate-co- 3-hydroxyvalerate) (PHBV) and poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx) coated with a fusion protein polyhydroxyalkanoates granule binding protein (PhaP) fused with Arg-Gly-Asp (RGD) peptide (PhaP-RGD). PHBV and PHBHHx films were fabricated by solvent evaporation. Scanning electronic microscope (SEM) was used to study the morphology of the films. PhaP-RGD fusion proteins were expressed and purified by the technology of protein engineering; PHBV and PHBHHx films were immersed in the PhaP-RGD with an amount of 3.5 mg/mL protein/per sample respectively. The hydrophilicity of the surface were detected by the contact angle measurements. Septal cartilage cells obtained from human septal cartilage were cultured in vitro. The 2nd passage chondrocytes were incubated on PHBV unmodified with PhaP-RGD in group A1, PHBV modified with PhaP-RGD in group A2, PHBHHx unmodified with PhaP-RGD in group Bl, PHBHHx modified with PhaP-RGD in group B2, and on the cell culture plates in group C. After cultured for 3 days, the proliferation of cells was detected by the DAPI staining; the proliferation viability of cells was detected by the MTT assay after cultured for 3 and 7 days; after cultured for 7 days, the adhesion and morphology of the cells on the surface of the biomaterial films were observed by SEM and the matrix of the cells was detected through the toluidine blue staining. SEM observation showed that PHBV and PHBHHx films had porous structures. The contact angle of the surface of the PHBV and PHBHHx films modified with PhaP-RGD fusion proteins were significantly reduced when compared with the films unmodified with PhaP-RGD fusion proteins (P films could grow in all groups. After 3 days of cultivation in vitro, the cell proliferation and viability of group B2 were the strongest among all groups (P films of PHBHHx modified with PhaP-RGD fusion protein can promote its

  17. Gold Nanoparticles of Diameter 13 nm Induce Apoptosis in Rabbit Articular Chondrocytes

    Science.gov (United States)

    Huang, Hao; Quan, Ying-yao; Wang, Xiao-ping; Chen, Tong-sheng

    2016-05-01

    Gold nanoparticles (AuNPs) have been widely used in biomedical science including antiarthritic agents, drug loading, and photothermal therapy. In this report, we studied the effects of AuNPs with diameters of 3, 13, and 45 nm, respectively, on rabbit articular chondrocytes. AuNPs were capped with citrate and their diameter and zeta potential were measured by dynamic light scattering (DLS). Cell viability was evaluated by Cell Counting Kit-8 (CCK-8) assay after the rabbit articular chondrocytes were pre-incubated with 3, 13, and 45 nm AuNPs, respectively, for 24 h. Flow cytometry (FCM) analysis with annexin V/propidium iodide (PI) double staining and fluorescence imaging with Hoechst 33258 staining were used to determine the fashion of AuNPs-induced chondrocyte death. Further, 13 nm AuNPs (2 nM) significantly induced chondrocyte death accompanying apoptotic characteristics including mitochondrial damage, externalization of phosphatidylserine and nuclear concentration. However, 3 nm AuNPs (2 nM) and 45 nm (0.02 nM) AuNPs did not induce cytotoxicity in chondrocytes. Although 13 nm AuNPs (2 nM) increased the intracellular reactive oxygen species (ROS) level, pretreatment with Nacetyl cysteine (NAC), a ROS scavenger, did not prevent the cytotoxicity induced by 13 nm AuNPs, indicating that 13 nm AuNPs (2 nM) induced ROS-independent apoptosis in chondrocytes. These results demonstrate the size-dependent cytotoxicity of AuNPs in chondrocytes, which must be seriously considered when using AuNPs for treatment of osteoarthritis (OA).

  18. Investigation of the Effects of Extracellular Osmotic Pressure on Morphology and Mechanical Properties of Individual Chondrocyte.

    Science.gov (United States)

    Nguyen, Trung Dung; Oloyede, Adekunle; Singh, Sanjleena; Gu, YuanTong

    2016-06-01

    It has been demonstrated that most cells of the body respond to osmotic pressure in a systematic manner. The disruption of the collagen network in the early stages of osteoarthritis causes an increase in water content of cartilage which leads to a reduction of pericellular osmolality in chondrocytes distributed within the extracellular environment. It is therefore arguable that an insight into the mechanical properties of chondrocytes under varying osmotic pressure would provide a better understanding of chondrocyte mechanotransduction and potentially contribute to knowledge on cartilage degeneration. In this present study, the chondrocyte cells were exposed to solutions with different osmolality. Changes in their dimensions and mechanical properties were measured over time. Atomic force microscopy (AFM) was used to apply load at various strain-rates and the force-time curves were logged. The thin-layer elastic model was used to extract the elastic stiffness of chondrocytes at different strain-rates and at different solution osmolality. In addition, the porohyperelastic (PHE) model was used to investigate the strain-rate-dependent responses under the loading and osmotic pressure conditions. The results revealed that the hypo-osmotic external environment increased chondrocyte dimensions and reduced Young's modulus of the cells at all strain-rates tested. In contrast, the hyper-osmotic external environment reduced dimensions and increased Young's modulus. Moreover, using the PHE model coupled with inverse FEA simulation, we established that the hydraulic permeability of chondrocytes increased with decreasing extracellular osmolality which is consistent with previous work in the literature. This could be due to a higher intracellular fluid volume fraction with lower osmolality.

  19. Deconvoluting heme biosynthesis to target blood-stage malaria parasites

    Science.gov (United States)

    Sigala, Paul A; Crowley, Jan R; Henderson, Jeffrey P; Goldberg, Daniel E

    2015-01-01

    Heme metabolism is central to blood-stage infection by the malaria parasite Plasmodium falciparum. Parasites retain a heme biosynthesis pathway but do not require its activity during infection of heme-rich erythrocytes, where they can scavenge host heme to meet metabolic needs. Nevertheless, heme biosynthesis in parasite-infected erythrocytes can be potently stimulated by exogenous 5-aminolevulinic acid (ALA), resulting in accumulation of the phototoxic intermediate protoporphyrin IX (PPIX). Here we use photodynamic imaging, mass spectrometry, parasite gene disruption, and chemical probes to reveal that vestigial host enzymes in the cytoplasm of Plasmodium-infected erythrocytes contribute to ALA-stimulated heme biosynthesis and that ALA uptake depends on parasite-established permeability pathways. We show that PPIX accumulation in infected erythrocytes can be harnessed for antimalarial chemotherapy using luminol-based chemiluminescence and combinatorial stimulation by low-dose artemisinin to photoactivate PPIX to produce cytotoxic reactive oxygen. This photodynamic strategy has the advantage of exploiting host enzymes refractory to resistance-conferring mutations. DOI: http://dx.doi.org/10.7554/eLife.09143.001 PMID:26173178

  20. High throughput transcriptome profiling of lithium stimulated human mesenchymal stem cells reveals priming towards osteoblastic lineage.

    Directory of Open Access Journals (Sweden)

    Neeraj Kumar Satija

    Full Text Available Human mesenchymal stem cells (hMSCs present in the bone marrow are the precursors of osteoblasts, chondrocytes and adipocytes, and hold tremendous potential for osteoregenerative therapy. However, achieving directed differentiation into osteoblasts has been a major concern. The use of lithium for enhancing osteogenic differentiation has been documented in animal models but its effect in humans is not clear. We, therefore, performed high throughput transcriptome analysis of lithium-treated hMSCs to identify altered gene expression and its relevance to osteogenic differentiation. Our results show suppression of proliferation and enhancement of alkaline phosphatase (ALP activity upon lithium treatment of hMSCs under non-osteogenic conditions. Microarray profiling of lithium-stimulated hMSC revealed decreased expression of adipogenic genes (CEBPA, CMKLR1, HSD11B1 and genes involved in lipid biosynthesis. Interestingly, osteoclastogenic factors and immune responsive genes (IL7, IL8, CXCL1, CXCL12, CCL20 were also downregulated. Negative transcriptional regulators of the osteogenic program (TWIST1 and PBX1 were suppressed while genes involved in mineralization like CLEC3B and ATF4 were induced. Gene ontology analysis revealed enrichment of upregulated genes related to mesenchymal cell differentiation and signal transduction. Lithium priming led to enhanced collagen 1 synthesis and osteogenic induction of lithium pretreated MSCs resulted in enhanced expression of Runx2, ALP and bone sialoprotein. However, siRNA-mediated knockdown of RRAD, CLEC3B and ATF4 attenuated lithium-induced osteogenic priming, identifying a role for RRAD, a member of small GTP binding protein family, in osteoblast differentiation. In conclusion, our data highlight the transcriptome reprogramming potential of lithium resulting in higher propensity of lithium "primed" MSCs for osteoblastic differentiation.

  1. High throughput transcriptome profiling of lithium stimulated human mesenchymal stem cells reveals priming towards osteoblastic lineage.

    Science.gov (United States)

    Satija, Neeraj Kumar; Sharma, Deepa; Afrin, Farhat; Tripathi, Rajendra P; Gangenahalli, Gurudutta

    2013-01-01

    Human mesenchymal stem cells (hMSCs) present in the bone marrow are the precursors of osteoblasts, chondrocytes and adipocytes, and hold tremendous potential for osteoregenerative therapy. However, achieving directed differentiation into osteoblasts has been a major concern. The use of lithium for enhancing osteogenic differentiation has been documented in animal models but its effect in humans is not clear. We, therefore, performed high throughput transcriptome analysis of lithium-treated hMSCs to identify altered gene expression and its relevance to osteogenic differentiation. Our results show suppression of proliferation and enhancement of alkaline phosphatase (ALP) activity upon lithium treatment of hMSCs under non-osteogenic conditions. Microarray profiling of lithium-stimulated hMSC revealed decreased expression of adipogenic genes (CEBPA, CMKLR1, HSD11B1) and genes involved in lipid biosynthesis. Interestingly, osteoclastogenic factors and immune responsive genes (IL7, IL8, CXCL1, CXCL12, CCL20) were also downregulated. Negative transcriptional regulators of the osteogenic program (TWIST1 and PBX1) were suppressed while genes involved in mineralization like CLEC3B and ATF4 were induced. Gene ontology analysis revealed enrichment of upregulated genes related to mesenchymal cell differentiation and signal transduction. Lithium priming led to enhanced collagen 1 synthesis and osteogenic induction of lithium pretreated MSCs resulted in enhanced expression of Runx2, ALP and bone sialoprotein. However, siRNA-mediated knockdown of RRAD, CLEC3B and ATF4 attenuated lithium-induced osteogenic priming, identifying a role for RRAD, a member of small GTP binding protein family, in osteoblast differentiation. In conclusion, our data highlight the transcriptome reprogramming potential of lithium resulting in higher propensity of lithium "primed" MSCs for osteoblastic differentiation.

  2. Localization and biosynthesis of polyamines in insulin-producing cells

    DEFF Research Database (Denmark)

    Hougaard, D M; Larsson, L I; Nielsen, Jens Høiriis

    1986-01-01

    determinations carried out on isolated rat and mouse pancreatic islets revealed large amounts of polyamines. Compared with extracts of whole pancreas, the islets contained very high concentrations of spermine relative to spermidine. Biosynthesis of polyamines from [3H]ornithine or from [3H]putrescine in isolated...... islets was significantly stimulated at high glucose concentrations. Moreover, significant incorporation of label from [3H]putrescine was also detected in gamma-aminobutyric acid. This incorporation, however, was not stimulated by high glucose. Possible roles for polyamines associated with the secretory...

  3. Serine biosynthesis and transport defects.

    Science.gov (United States)

    El-Hattab, Ayman W

    2016-07-01

    l-serine is a non-essential amino acid that is biosynthesized via the enzymes phosphoglycerate dehydrogenase (PGDH), phosphoserine aminotransferase (PSAT), and phosphoserine phosphatase (PSP). Besides its role in protein synthesis, l-serine is a potent neurotrophic factor and a precursor of a number of essential compounds including phosphatidylserine, sphingomyelin, glycine, and d-serine. Serine biosynthesis defects result from impairments of PGDH, PSAT, or PSP leading to systemic serine deficiency. Serine biosynthesis defects present in a broad phenotypic spectrum that includes, at the severe end, Neu-Laxova syndrome, a lethal multiple congenital anomaly disease, intermediately, infantile serine biosynthesis defects with severe neurological manifestations and growth deficiency, and at the mild end, the childhood disease with intellectual disability. A serine transport defect resulting from deficiency of the ASCT1, the main transporter for serine in the central nervous system, has been recently described in children with neurological manifestations that overlap with those observed in serine biosynthesis defects. l-serine therapy may be beneficial in preventing or ameliorating symptoms in serine biosynthesis and transport defects, if started before neurological damage occurs. Herein, we review serine metabolism and transport, the clinical, biochemical, and molecular aspects of serine biosynthesis and transport defects, the mechanisms of these diseases, and the potential role of serine therapy. Copyright © 2016 Elsevier Inc. All rights reserved.

  4. RhoA activation and nuclearization marks loss of chondrocyte phenotype in crosstalk with Wnt pathway.

    Science.gov (United States)

    Öztürk, Ece; Despot-Slade, Evelin; Pichler, Michael; Zenobi-Wong, Marcy

    2017-11-15

    De-differentiation comprises a major drawback for the use of autologous chondrocytes in cartilage repair. Here, we investigate the role of RhoA and canonical Wnt signaling in chondrocyte phenotype. Chondrocyte de-differentiation is accompanied by an upregulation and nuclear localization of RhoA. Effectors of canonical Wnt signaling including β-catenin and YAP/TAZ are upregulated in de-differentiating chondrocytes in a Rho-dependent manner. Inhibition of Rho activation with C3 transferase inhibits nuclear localization of RhoA, induces expression of chondrogenic markers on 2D and enhances the chondrogenic effect of 3D culturing. Upregulation of chondrogenic markers by Rho inhibition is accompanied by loss of canonical Wnt signaling markers in 3D or on 2D whereas treatment of chondrocytes with Wnt-3a abrogates this effect. However, induction of canonical Wnt signaling inhibits chondrogenic markers on 2D but enhances chondrogenic re-differentiation on 2D with C3 transferase or in 3D. These data provide insights on the context-dependent role of RhoA and Wnt signaling in de-differentiation and on mechanisms to induce chondrogenic markers for therapeutic approaches. Copyright © 2017 Elsevier Inc. All rights reserved.

  5. The Regulatory Role of Signaling Crosstalk in Hypertrophy of MSCs and Human Articular Chondrocytes.

    Science.gov (United States)

    Zhong, Leilei; Huang, Xiaobin; Karperien, Marcel; Post, Janine N

    2015-08-14

    Hypertrophic differentiation of chondrocytes is a main barrier in application of mesenchymal stem cells (MSCs) for cartilage repair. In addition, hypertrophy occurs occasionally in osteoarthritis (OA). Here we provide a comprehensive review on recent literature describing signal pathways in the hypertrophy of MSCs-derived in vitro differentiated chondrocytes and chondrocytes, with an emphasis on the crosstalk between these pathways. Insight into the exact regulation of hypertrophy by the signaling network is necessary for the efficient application of MSCs for articular cartilage repair and for developing novel strategies for curing OA. We focus on articles describing the role of the main signaling pathways in regulating chondrocyte hypertrophy-like changes. Most studies report hypertrophic differentiation in chondrogenesis of MSCs, in both human OA and experimental OA. Chondrocyte hypertrophy is not under the strict control of a single pathway but appears to be regulated by an intricately regulated network of multiple signaling pathways, such as WNT, Bone morphogenetic protein (BMP)/Transforming growth factor-β (TGFβ), Parathyroid hormone-related peptide (PTHrP), Indian hedgehog (IHH), Fibroblast growth factor (FGF), Insulin like growth factor (IGF) and Hypoxia-inducible factor (HIF). This comprehensive review describes how this intricate signaling network influences tissue-engineering applications of MSCs in articular cartilage (AC) repair, and improves understanding of the disease stages and cellular responses within an OA articular joint.

  6. Mefloquine inhibits chondrocytic proliferation by arresting cell cycle in G2/M phase.

    Science.gov (United States)

    Li, Qiong; Chen, Zeng-Gan; Xia, Qing; Lin, Jian-Ping; Yan, Zuo-Qin; Yao, Zheng-Jun; Dong, Jian

    2015-01-01

    Mefloquine (MQ), an analog of chloroquine, exhibits a promising cytotoxic activity against carcinoma cell lines and for the treatment of glioblastoma patients. The present study demonstrates the effect of mefloquine on proliferation and cell cycle in chondrocytes. MTT assay and propidium iodide staining were used for the analysis of proliferation and cell cycle distribution, respectively. Western blot analysis was used to examine the expression levels of cyclin B1/cdc2, cdc25c, p21WAF1/CIP1 and p53. The results revealed that mefloquine inhibited the proliferation of chondrocytes and caused cell cycle arrests in the G2/M phase. The proliferation of chondrocytes was reduced to 27% at 40 μM concentration of mefloquine after 48 h. The population of chondrocytes in G2/M phase was found to be 15.7 and 48.4%, respectively at 10 and 40 μM concentration of mefloquine at 48 h following treatment. The expression of the cell cycle regulatory proteins including, cyclin B1/cdc2 and cdc25c was inhibited. On the other hand, mefloquine treatment promoted the expression of p21WAF1/CIP1 and p53 at 40 μM concentration after 48 h. Therefore, mefloquine inhibits proliferation and induces cell cycle arrest in chondrocytes.

  7. Effect of bone marrow-derived stem cells on chondrocytes from patients with osteoarthritis.

    Science.gov (United States)

    Zhang, Qiangzhi; Chen, Yong; Wang, Qiang; Fang, Chaoyong; Sun, Yu; Yuan, Tao; Wang, Yuebei; Bao, Rongni; Zhao, Ningjian

    2016-02-01

    Increasing numbers of individuals are suffering from osteoarthritis every year, and the directed intra-articular injection of bone marrow stem cells has provided a promising treatment strategy for osteoarthritis. Although a number of studies have demonstrated that intra-articular injection of bone marrow stem cells produced desirable results, the mechanism underlying this effect has not been elucidated. In the current study, the effect of bone marrow stem cells on chondrocytes from patients with osteoarthritis was observed in a co-culture system. Human chondrocytes were obtained from patients with osteoarthritis who underwent surgical procedures and bone marrow stem cells were obtained from bone marrow aspirates, and then the chondrocytes were then cultured alone or cocultured with bone marrow stem cells in 0.4-µm Transwell inserts. The differentiation and biological activity of chondrocytes in the culture system were measured, and the inflammatory factors and OA-associated markers were also measured. The results indicated that coculture with human bone marrow stem cells increases cell proliferation of chondrocytes and inhibits inflammatory activity in osteoarthritis.

  8. Osteoarthritis-derived chondrocytes are a potential source of multipotent progenitor cells for cartilage tissue engineering.

    Science.gov (United States)

    Oda, Tomoyuki; Sakai, Tadahiro; Hiraiwa, Hideki; Hamada, Takashi; Ono, Yohei; Nakashima, Motoshige; Ishizuka, Shinya; Matsukawa, Tetsuya; Yamashita, Satoshi; Tsuchiya, Saho; Ishiguro, Naoki

    2016-10-21

    The natural healing capacity of damaged articular cartilage is poor, rendering joint surface injuries a prime target for regenerative medicine. While autologous chondrocyte or mesenchymal stem cell (MSC) implantation can be applied to repair cartilage defects in young patients, no appropriate long-lasting treatment alternative is available for elderly patients with osteoarthritis (OA). Multipotent progenitor cells are reported to present in adult human articular cartilage, with a preponderance in OA cartilage. These facts led us to hypothesize the possible use of osteoarthritis-derived chondrocytes as a cell source for cartilage tissue engineering. We therefore analyzed chondrocyte- and stem cell-related markers, cell growth rate, and multipotency in OA chondrocytes (OACs) and bone marrow-derived MSCs, along with normal articular chondrocytes (ACs) as a control. OACs demonstrated similar phenotype and proliferation rate to MSCs. Furthermore, OACs exhibited multilineage differentiation ability with a greater chondrogenic differentiation ability than MSCs, which was equivalent to ACs. We conclude that chondrogenic capacity is not significantly affected by OA, and OACs could be a potential source of multipotent progenitor cells for cartilage tissue engineering. Copyright © 2016 Elsevier Inc. All rights reserved.

  9. The Regulatory Role of Signaling Crosstalk in Hypertrophy of MSCs and Human Articular Chondrocytes

    Science.gov (United States)

    Zhong, Leilei; Huang, Xiaobin; Karperien, Marcel; Post, Janine N.

    2015-01-01

    Hypertrophic differentiation of chondrocytes is a main barrier in application of mesenchymal stem cells (MSCs) for cartilage repair. In addition, hypertrophy occurs occasionally in osteoarthritis (OA). Here we provide a comprehensive review on recent literature describing signal pathways in the hypertrophy of MSCs-derived in vitro differentiated chondrocytes and chondrocytes, with an emphasis on the crosstalk between these pathways. Insight into the exact regulation of hypertrophy by the signaling network is necessary for the efficient application of MSCs for articular cartilage repair and for developing novel strategies for curing OA. We focus on articles describing the role of the main signaling pathways in regulating chondrocyte hypertrophy-like changes. Most studies report hypertrophic differentiation in chondrogenesis of MSCs, in both human OA and experimental OA. Chondrocyte hypertrophy is not under the strict control of a single pathway but appears to be regulated by an intricately regulated network of multiple signaling pathways, such as WNT, Bone morphogenetic protein (BMP)/Transforming growth factor-β (TGFβ), Parathyroid hormone-related peptide (PTHrP), Indian hedgehog (IHH), Fibroblast growth factor (FGF), Insulin like growth factor (IGF) and Hypoxia-inducible factor (HIF). This comprehensive review describes how this intricate signaling network influences tissue-engineering applications of MSCs in articular cartilage (AC) repair, and improves understanding of the disease stages and cellular responses within an OA articular joint. PMID:26287176

  10. Treatment of osteoarthritis using a helper-dependent adenoviral vector retargeted to chondrocytes

    Directory of Open Access Journals (Sweden)

    Merry ZC Ruan

    2016-01-01

    Full Text Available Osteoarthritis (OA is a joint disease characterized by degeneration of the articular cartilage, subchondral bone remodeling, and secondary inflammation. It is among the top three causes of chronic disability, and currently there are no treatment options to prevent disease progression. The localized nature of OA makes it an ideal candidate for gene and cell therapy. However, gene and cell therapy of OA is impeded by inefficient gene transduction of chondrocytes. In this study, we developed a broadly applicable system that retargets cell surface receptors by conjugating antibodies to the capsid of helper-dependent adenoviral vectors (HDVs. Specifically, we applied this system to retarget chondrocytes by conjugating an HDV to an α-10 integrin monoclonal antibody (a10mab. We show that a10mab-conjugated HDV (a10mabHDV-infected chondrocytes efficiently in vitro and in vivo while detargeting other cell types. The therapeutic index of an intra-articular injection of 10mabHDV-expressing proteoglycan 4 (PRG4 into a murine model of post-traumatic OA was 10-fold higher than with standard HDV. Moreover, we show that PRG4 overexpression from articular, superficial zone chondrocytes is effective for chondroprotection in postinjury OA and that α-10 integrin is an effective protein for chondrocyte targeting.

  11. The Regulatory Role of Signaling Crosstalk in Hypertrophy of MSCs and Human Articular Chondrocytes

    Directory of Open Access Journals (Sweden)

    Leilei Zhong

    2015-08-01

    Full Text Available Hypertrophic differentiation of chondrocytes is a main barrier in application of mesenchymal stem cells (MSCs for cartilage repair. In addition, hypertrophy occurs occasionally in osteoarthritis (OA. Here we provide a comprehensive review on recent literature describing signal pathways in the hypertrophy of MSCs-derived in vitro differentiated chondrocytes and chondrocytes, with an emphasis on the crosstalk between these pathways. Insight into the exact regulation of hypertrophy by the signaling network is necessary for the efficient application of MSCs for articular cartilage repair and for developing novel strategies for curing OA. We focus on articles describing the role of the main signaling pathways in regulating chondrocyte hypertrophy-like changes. Most studies report hypertrophic differentiation in chondrogenesis of MSCs, in both human OA and experimental OA. Chondrocyte hypertrophy is not under the strict control of a single pathway but appears to be regulated by an intricately regulated network of multiple signaling pathways, such as WNT, Bone morphogenetic protein (BMP/Transforming growth factor-β (TGFβ, Parathyroid hormone-related peptide (PTHrP, Indian hedgehog (IHH, Fibroblast growth factor (FGF, Insulin like growth factor (IGF and Hypoxia-inducible factor (HIF. This comprehensive review describes how this intricate signaling network influences tissue-engineering applications of MSCs in articular cartilage (AC repair, and improves understanding of the disease stages and cellular responses within an OA articular joint.

  12. Single Cell Confocal Raman Spectroscopy of Human Osteoarthritic Chondrocytes: A Preliminary Study

    Directory of Open Access Journals (Sweden)

    Rajesh Kumar

    2015-04-01

    Full Text Available 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.

  13. Carotenoid Biosynthesis in Fusarium

    Directory of Open Access Journals (Sweden)

    Javier Avalos

    2017-07-01

    Full Text Available Many fungi of the genus Fusarium stand out for the complexity of their secondary metabolism. Individual species may differ in their metabolic capacities, but they usually share the ability to synthesize carotenoids, a family of hydrophobic terpenoid pigments widely distributed in nature. Early studies on carotenoid biosynthesis in Fusarium aquaeductuum have been recently extended in Fusarium fujikuroi and Fusarium oxysporum, well-known biotechnological and phytopathogenic models, respectively. The major Fusarium carotenoid is neurosporaxanthin, a carboxylic xanthophyll synthesized from geranylgeranyl pyrophosphate through the activity of four enzymes, encoded by the genes carRA, carB, carT and carD. These fungi produce also minor amounts of β-carotene, which may be cleaved by the CarX oxygenase to produce retinal, the rhodopsin’s chromophore. The genes needed to produce retinal are organized in a gene cluster with a rhodopsin gene, while other carotenoid genes are not linked. In the investigated Fusarium species, the synthesis of carotenoids is induced by light through the transcriptional induction of the structural genes. In some species, deep-pigmented mutants with up-regulated expression of these genes are affected in the regulatory gene carS. The molecular mechanisms underlying the control by light and by the CarS protein are currently under investigation.

  14. Biosynthesis of Tetrahydroisoquinoline Antibiotics.

    Science.gov (United States)

    Tang, Gong-Li; Tang, Man-Cheng; Song, Li-Qiang; Zhang, Yue

    2016-01-01

    The tetrahydroisoquinoline (THIQ) alkaloids are naturally occurring antibiotics isolated from a variety of microorganisms and marine invertebrates. This family of natural products exhibit broad spectrum antimicrobial and strong antitumor activities, and the potency of clinical application has been validated by the marketing of ecteinascidin 743 (ET-743) as anticancer drug. In the past 20 years, the biosynthetic gene cluster of six THIQ antibiotics has been characterized including saframycin Mx1 from Myxococcus xanthus, safracin-B from Pseudomonas fluorescens, saframycin A, naphthyridinomycin, and quinocarcin from Streptomyces, as well as ET-743 from Ecteinascidia turbinata. This review gives a brief summary of the current status in understanding the molecular logic for the biosynthesis of these natural products, which provides new insights on the biosynthetic machinery involved in the nonribosomal peptide synthetase system. The proposal of the THIQ biosynthetic pathway not only shows nature's route to generate such complex molecules, but also set the stage to develop a different process for production of ET-743 by synthetic biology.

  15. Stereoselectivity in Polyphenol Biosynthesis

    Science.gov (United States)

    Lewis, Norman G.; Davin, Laurence B.

    1992-01-01

    Stereoselectivity plays an important role in the late stages of phenyl-propanoid metabolism, affording lignins, lignans, and neolignans. Stereoselectivity is manifested during monolignol (glucoside) synthesis, e.g., where the geometry (E or Z) of the pendant double bond affects the specificity of UDPG:coniferyl alcohol glucosyltransferases in different species. Such findings are viewed to have important ramifications in monolignol transport and storage processes, with roles for both E- and Z-monolignols and their glucosides in lignin/lignan biosynthesis being envisaged. Stereoselectivity is also of great importance in enantiose-lective enzymatic processes affording optically active lignans. Thus, cell-free extracts from Forsythia species were demonstrated to synthesize the enantiomerically pure lignans, (-)-secoisolariciresinol, and (-)-pinoresinol, when NAD(P)H, H2O2 and E-coniferyl alcohol were added. Progress toward elucidating the enzymatic steps involved in such highly stereoselective processes is discussed. Also described are preliminary studies aimed at developing methodologies to determine the subcellular location of late-stage phenylpropanoid metabolites (e.g., coniferyl alcohol) and key enzymes thereof, in intact tissue or cells. This knowledge is essential if questions regarding lignin and lignan tissue specificity and regulation of these processes are to be deciphered.

  16. Glycolipid biosynthesis in cyanobacteria

    International Nuclear Information System (INIS)

    Van Dusen, W.J.; Jaworski, J.G.

    1987-01-01

    The biosynthesis of monogalactosyldiacyl-glycerol (MGDG) was studied in five different cyanobacteria. Previous work has shown Anabaena variabilis to synthesize both MGDG and monoglucosyl-diacylglycerol (MG1cDG) with MG1cDG being the precursor of MGDG. They have examined four other cyanobacteria to determine if a similar relationship exists. The cyanobacteria studied were Anabaena variabilis, Chlorogloeopsis sp., Schizothrix calcicola, Anacystis nidulans, and Anacystis marina. Each were grown in liquid culture and lipids were labeled with 14 C]CO 2 for 20 min., 1.0 hr, 1.0 hr + 10 hr chase. Glycolipids were analyzed by initial separation of MGDG and MG1cDG by TLC followed by further analysis by HPLC. Complete separation of molecular species was obtained isocratically on an ODS column. All of the cyanobacteria labeled 16-C and 18-C fatty acids except for A. marina which labeled only 14-C and 16-C fatty acids. Desaturation of the fatty acids could be observed in the 1.0 hr and chase experiments. All were capable of labeling both MG1cDG and MGDG with the precursor-product relationship being observed. There does not appear to be a direct relationship between the epimerization of the sugar moiety and fatty acid desaturation

  17. Biosynthesis of silver nanoparticles.

    Science.gov (United States)

    Poulose, Subin; Panda, Tapobrata; Nair, Praseetha P; Théodore, Thomas

    2014-02-01

    Metal nanoparticles have unique optical, electronic, and catalytic properties. There exist well-defined physical and chemical processes for their preparation. Those processes often yield small quantities of nanoparticles having undesired morphology, and involve high temperatures for the reaction and the use of hazardous chemicals. Relatively, the older technique of bioremediation of metals uses either microorganisms or their components for the production of nanoparticles. The nanoparticles obtained from bacteria, fungi, algae, plants and their components, etc. appear environment-friendly, as toxic chemicals are not used in the processes. In addition to this, the formation of nanoparticles takes place at almost normal temperature and pressure. Control of the shape and size of the nanoparticles is possible by appropriate selection of the pH and temperature. Three important steps are the bioconversion of Ag+ ions, conversion of desired crystals to nanoparticles, and nanoparticle stability. Generally, nanoparticles are characterized by the UV-visible spectroscopy and use of the electron microscope. Silver nanoparticles are used as antimicrobial agents and they possess antifungal, anti-inflammatory, and anti-angiogenic properties. This review highlights the biosynthesis of silver nanoparticles by various organisms, possible mechanisms of their synthesis, their characterization, and applications of silver nanoparticles.

  18. Adhesion-mediated signal transduction in human articular chondrocytes: the influence of biomaterial chemistry and tenascin-C

    NARCIS (Netherlands)

    Mahmood, Tahir A.; de Jong, Ruben; Riesle, J.U.; Langer, Robert; van Blitterswijk, Clemens

    2004-01-01

    Chondrocyte ‘dedifferentiation’ involves the switching of the cell phenotype to one that no longer secretes extracellular matrix found in normal cartilage and occurs frequently during chondrocyte expansion in culture. It is also characterized by the differential expression of receptors and

  19. Botanical Extracts from Rosehip (Rosa canina, Willow Bark (Salix alba, and Nettle Leaf (Urtica dioica Suppress IL-1β-Induced NF-κB Activation in Canine Articular Chondrocytes

    Directory of Open Access Journals (Sweden)

    Mehdi Shakibaei

    2012-01-01

    Full Text Available The aim of this study was to characterize the anti-inflammatory mode of action of botanical extracts from rosehip (Rosa canina, willow bark (Salix alba, and nettle leaf (Urtica dioica in an in vitro model of primary canine articular chondrocytes. Methods. The biological effects of the botanical extracts were studied in chondrocytes treated with IL-1β for up to 72 h. Expression of collagen type II, cartilage-specific proteoglycan (CSPG, β1-integrin, SOX-9, COX-2, and MMP-9 and MMP-13 was examined by western blotting. Results. The botanical extracts suppressed IL-1β-induced NF-κB activation by inhibition of IκBα phosphorylation, IκBα degradation, p65 phosphorylation, and p65 nuclear translocation. These events correlated with downregulation of NF-κB targets including COX-2 and MMPs. The extracts also reversed the IL-1β-induced downregulation of collagen type II, CSPG, β1-integrin, and cartilage-specific transcription factor SOX-9 protein expression. In high-density cultures botanical extracts stimulated new cartilage formation even in the presence of IL-1β. Conclusions. Botanical extracts exerted anti-inflammatory and anabolic effects on chondrocytes. The observed reduction of IL-1β-induced NF-κB activation suggests that further studies are warranted to demonstrate the effectiveness of plant extracts in the treatment of OA and other conditions in which NF-κB plays pathophysiological roles.

  20. Botanical Extracts from Rosehip (Rosa canina), Willow Bark (Salix alba), and Nettle Leaf (Urtica dioica) Suppress IL-1β-Induced NF-κB Activation in Canine Articular Chondrocytes

    Science.gov (United States)

    Shakibaei, Mehdi; Allaway, David; Nebrich, Simone; Mobasheri, Ali

    2012-01-01

    The aim of this study was to characterize the anti-inflammatory mode of action of botanical extracts from rosehip (Rosa canina), willow bark (Salix alba), and nettle leaf (Urtica dioica) in an in vitro model of primary canine articular chondrocytes. Methods. The biological effects of the botanical extracts were studied in chondrocytes treated with IL-1β for up to 72 h. Expression of collagen type II, cartilage-specific proteoglycan (CSPG), β1-integrin, SOX-9, COX-2, and MMP-9 and MMP-13 was examined by western blotting. Results. The botanical extracts suppressed IL-1β-induced NF-κB activation by inhibition of IκBα phosphorylation, IκBα degradation, p65 phosphorylation, and p65 nuclear translocation. These events correlated with downregulation of NF-κB targets including COX-2 and MMPs. The extracts also reversed the IL-1β-induced downregulation of collagen type II, CSPG, β1-integrin, and cartilage-specific transcription factor SOX-9 protein expression. In high-density cultures botanical extracts stimulated new cartilage formation even in the presence of IL-1β. Conclusions. Botanical extracts exerted anti-inflammatory and anabolic effects on chondrocytes. The observed reduction of IL-1β-induced NF-κB activation suggests that further studies are warranted to demonstrate the effectiveness of plant extracts in the treatment of OA and other conditions in which NF-κB plays pathophysiological roles. PMID:22474508

  1. Botanical Extracts from Rosehip (Rosa canina), Willow Bark (Salix alba), and Nettle Leaf (Urtica dioica) Suppress IL-1β-Induced NF-κB Activation in Canine Articular Chondrocytes.

    Science.gov (United States)

    Shakibaei, Mehdi; Allaway, David; Nebrich, Simone; Mobasheri, Ali

    2012-01-01

    The aim of this study was to characterize the anti-inflammatory mode of action of botanical extracts from rosehip (Rosa canina), willow bark (Salix alba), and nettle leaf (Urtica dioica) in an in vitro model of primary canine articular chondrocytes. Methods. The biological effects of the botanical extracts were studied in chondrocytes treated with IL-1β for up to 72 h. Expression of collagen type II, cartilage-specific proteoglycan (CSPG), β1-integrin, SOX-9, COX-2, and MMP-9 and MMP-13 was examined by western blotting. Results. The botanical extracts suppressed IL-1β-induced NF-κB activation by inhibition of IκBα phosphorylation, IκBα degradation, p65 phosphorylation, and p65 nuclear translocation. These events correlated with downregulation of NF-κB targets including COX-2 and MMPs. The extracts also reversed the IL-1β-induced downregulation of collagen type II, CSPG, β1-integrin, and cartilage-specific transcription factor SOX-9 protein expression. In high-density cultures botanical extracts stimulated new cartilage formation even in the presence of IL-1β. Conclusions. Botanical extracts exerted anti-inflammatory and anabolic effects on chondrocytes. The observed reduction of IL-1β-induced NF-κB activation suggests that further studies are warranted to demonstrate the effectiveness of plant extracts in the treatment of OA and other conditions in which NF-κB plays pathophysiological roles.

  2. Chondrocytes co-cultured with Stromal Vascular Fraction of adipose tissue present more intense chondrogenic characteristics than with Adipose Stem Cells

    NARCIS (Netherlands)

    Wu, Ling; Prins, H.J.; Leijten, Jeroen Christianus Hermanus; Helder, M.; Evseenko, D.; Moroni, L; van Blitterswijk, Clemens; Lin, Y.; Karperien, Hermanus Bernardus Johannes

    2016-01-01

    Partly replacement of chondrocytes by stem cells has been proposed to improve the performance of autologous chondrocytes implantation (ACI). Our previous studies showed that the increased cartilage production in pellet co-cultures of chondrocytes and mesenchymal stem cells (MSCs) is due to a trophic

  3. Andrographolide Enhances Proliferation and Prevents Dedifferentiation of Rabbit Articular Chondrocytes: An In Vitro Study

    Directory of Open Access Journals (Sweden)

    Li-ke Luo

    2015-01-01

    Full Text Available As the main active constituent of Andrographis paniculata that was applied in treatment of many diseases including inflammation in ancient China, andrographolide (ANDRO was found to facilitate reduction of edema and analgesia in arthritis. This suggested that ANDRO may be promising anti-inflammatory agent to relieve destruction and degeneration of cartilage after inflammation. In this study, the effect of ANDRO on rabbit articular chondrocytes in vitro was investigated. Results showed that not more than 8 μM ANDRO did no harm to chondrocytes (P0.05. The viability assay, hematoxylin-eosin, safranin O, and immunohistochemical staining also showed better performances in ANDRO groups. As to the doses, 3 μM ANDRO showed the best performance. The results indicate that ANDRO can accelerate proliferation of rabbit articular chondrocytes in vitro and meanwhile maintain the phenotype, which may provide valuable references for further exploration on arthritis.

  4. Pterosin B prevents chondrocyte hypertrophy and osteoarthritis in mice by inhibiting Sik3

    Science.gov (United States)

    Yahara, Yasuhito; Takemori, Hiroshi; Okada, Minoru; Kosai, Azuma; Yamashita, Akihiro; Kobayashi, Tomohito; Fujita, Kaori; Itoh, Yumi; Nakamura, Masahiro; Fuchino, Hiroyuki; Kawahara, Nobuo; Fukui, Naoshi; Watanabe, Akira; Kimura, Tomoatsu; Tsumaki, Noriyuki

    2016-01-01

    Osteoarthritis is a common debilitating joint disorder. Risk factors for osteoarthritis include age, which is associated with thinning of articular cartilage. Here we generate chondrocyte-specific salt-inducible kinase 3 (Sik3) conditional knockout mice that are resistant to osteoarthritis with thickened articular cartilage owing to a larger chondrocyte population. We also identify an edible Pteridium aquilinum compound, pterosin B, as a Sik3 pathway inhibitor. We show that either Sik3 deletion or intraarticular injection of mice with pterosin B inhibits chondrocyte hypertrophy and protects cartilage from osteoarthritis. Collectively, our results suggest Sik3 regulates the homeostasis of articular cartilage and is a target for the treatment of osteoarthritis, with pterosin B as a candidate therapeutic. PMID:27009967

  5. Stress relaxation analysis of single chondrocytes using porohyperelastic model based on AFM experiments

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    Trung Dung Nguyen

    2014-01-01

    Full Text Available Based on atomic force microscopytechnique, we found that the chondrocytes exhibits stress relaxation behavior. We explored the mechanism of this stress relaxation behavior and concluded that the intracellular fluid exuding out from the cells during deformation plays the most important role in the stress relaxation. We applied the inverse finite element analysis technique to determine necessary material parameters for porohyperelastic (PHE model to simulate stress relaxation behavior as this model is proven capable of capturing the non-linear behavior and the fluid-solid interaction during the stress relaxation of the single chondrocytes. It is observed that PHE model can precisely capture the stress relaxation behavior of single chondrocytes and would be a suitable model for cell biomechanics.

  6. Arthroscopic autologous chondrocyte implantation in the ankle joint.

    Science.gov (United States)

    Giannini, Sandro; Buda, Roberto; Ruffilli, Alberto; Cavallo, Marco; Pagliazzi, Gherardo; Bulzamini, Maria Chiara; Desando, Giovanna; Luciani, Deianira; Vannini, Francesca

    2014-06-01

    Autologous chondrocyte implantation (ACI) is an established procedure in the ankle providing satisfactory results. The development of a completely arthroscopic ACI procedure in the ankle joint made the technique easier and reduced the morbidity. The purpose of this investigation was to report the clinical results of a series of patients who underwent arthroscopic ACI of the talus at a mean of 7 ± 1.2-year follow-up. Forty-six patients (mean age 31.4 ± 7.6) affected by osteochondral lesions of the talar dome (OLT) received arthroscopic ACI between 2001 and 2006. Patients were clinically evaluated using AOFAS score pre-operatively and at 12, 36 months and at final follow-up of 87.2 ± 14.5 months. The mean pre-operative AOFAS score was 57.2 ± 14.3. At the 12-month follow-up, the mean AOFAS score was 86.8 ± 13.4 (p = 0.0005); at 36 months after surgery, the mean score was 89.5 ± 13.4 (p = 0.0005); whereas at final follow-up of 87.2 ± 14.5 months it was 92.0 ± 11.2 (p = 0.0005). There were three failures. Histological and immunohistochemical evaluations of specimens harvested from failed implants generally showed several aspects of a fibro-cartilaginous tissue associated with some aspects of cartilage tissue remodelling as indicated by the presence of type II collagen expression. This study confirmed the ability of arthroscopic ACI to repair osteochondral lesions in the ankle joint with satisfactory clinical results after mid-term follow-up. IV, retrospective case series.

  7. Gentiopicroside prevents interleukin-1 beta induced inflammation response in rat articular chondrocyte.

    Science.gov (United States)

    Zhao, Lei; Ye, Juan; Wu, Guo-Tai; Peng, Xue-Jing; Xia, Peng-Fei; Ren, Yuan

    2015-08-22

    In traditional Chinese medicine, Gentiana macrophylla Pall have been prescribed for the treatment of pain and inflammatory conditions. In addition, it is a common Tibetan medicinal herb used for the treatment of tonsillitis, urticaria, and rheumatoid arthritis (RA), while the flowers of G. macrophylla Pall have been traditionally treated as an anti-inflammatory agent to clear heat in Mongolian medicine. The secoiridoid glycosides and their derivatives are the primary active components of G. macrophylla and have been demonstrated to be effective as anti-inflammatory agents. Solvent extraction and D101 macroporous resin columns were employed to concentratethe gentiopicroside. Gentiopicroside cytotoxicity was assessed by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay; the toxicity of gentiopicroside in chondrocytes was reconfirmed using Hoechst staining. Western blotting, reverse transcriptase-polymerase chain reaction (RT-PCR) and immunohistochemistry were utilized to explore the protective effects and mechanisms of gentiopicroside prevents interleukin-1 beta induced inflammation response in rat articular chondrocyte. The MTT assay demonstrated that 50, 500, and 1,500 μg/mL of gentiopicroside exhibited no significant toxicity to chondrocytes (P>0.05) after 24h. Using immunohistochemistry, ELISA, RT-PCR, Western blot method to explore the protective effect and mechanism of gentiopicroside on chondrocytes induced by IL-1β. The results showed some pathways of IL-1β signal transduction were inhibited by gentiopicroside in rat chondrocytes: p38, ERK and JNK. Meanwhile, gentiopicroside showed inhibition in the IL-1β-induced release of MMPs while increasing Collagen type II expression. The current study demonstrated that gentiopicroside exhibited a potent protective effect on IL-1β induced inflammation response in rat articular chondrocyte. Thus, gentiopicroside could be a potential therapeutic strategy for treatment of OA. Copyright © 2015

  8. Biomechanical properties of single chondrocytes and chondrons determined by micromanipulation and finite-element modelling

    Science.gov (United States)

    Nguyen, Bac V.; Wang, Qi Guang; Kuiper, Nicola J.; El Haj, Alicia J.; Thomas, Colin R.; Zhang, Zhibing

    2010-01-01

    A chondrocyte and its surrounding pericellular matrix (PCM) are defined as a chondron. Single chondrocytes and chondrons isolated from bovine articular cartilage were compressed by micromanipulation between two parallel surfaces in order to investigate their biomechanical properties and to discover the mechanical significance of the PCM. The force imposed on the cells was measured directly during compression to various deformations and then holding. When the nominal strain at the end of compression was 50 per cent, force relaxation showed that the cells were viscoelastic, but this viscoelasticity was generally insignificant when the nominal strain was 30 per cent or lower. The viscoelastic behaviour might be due to the mechanical response of the cell cytoskeleton and/or nucleus at higher deformations. A finite-element analysis was applied to simulate the experimental force-displacement/time data and to obtain mechanical property parameters of the chondrocytes and chondrons. Because of the large strains in the cells, a nonlinear elastic model was used for simulations of compression to 30 per cent nominal strain and a nonlinear viscoelastic model for 50 per cent. The elastic model yielded a Young's modulus of 14 ± 1 kPa (mean ± s.e.) for chondrocytes and 19 ± 2 kPa for chondrons, respectively. The viscoelastic model generated an instantaneous elastic modulus of 21 ± 3 and 27 ± 4 kPa, a long-term modulus of 9.3 ± 0.8 and 12 ± 1 kPa and an apparent viscosity of 2.8 ± 0.5 and 3.4 ± 0.6 kPa s for chondrocytes and chondrons, respectively. It was concluded that chondrons were generally stiffer and showed less viscoelastic behaviour than chondrocytes, and that the PCM significantly influenced the mechanical properties of the cells. PMID:20519215

  9. Autologous chondrocytes as a novel source for neo-chondrogenesis in haemophiliacs.

    Science.gov (United States)

    Stocco, Elena; Barbon, Silvia; Radossi, Paolo; Rajendran, Senthilkumar; Dalzoppo, Daniele; Bortolami, Marina; Bagno, Andrea; Grandi, Francesca; Gamba, Pier Giorgio; Parnigotto, Pier Paolo; Tagariello, Giuseppe; Grandi, Claudio

    2016-10-01

    Haemophilic arthropathy is the major cause of disability in patients with haemophilia and, despite prophylaxis with coagulation factor concentrates, some patients still develop articular complications. We evaluate the feasibility of a tissue engineering approach to improve current clinical strategies for cartilage regeneration in haemophiliacs by using autologous chondrocytes (haemophilic chondrocytes; HaeCs). Little is known about articular chondrocytes from haemophilic patients and no characterisation has as yet been performed. An investigation into whether blood exposure alters HaeCs should be interesting from the perspective of autologous implants. The typical morphology and expression of specific target genes and surface markers were therefore assessed by optical microscopy, reverse transcription plus the polymerase chain reaction (PCR), real-time PCR and flow-cytometry. We then considered chondrocyte behaviour on a bio-hybrid scaffold (based on polyvinyl alcohol/Wharton's jelly) as an in vitro model of articular cartilage prosthesis. Articular chondrocytes from non-haemophilic donors were used as controls. HaeC morphology and the resulting immunophenotype CD44(+)/CD49c(+)/CD49e(+)/CD151(+)/CD73(+)/CD49f(-)/CD26(-) resembled those of healthy donors. Moreover, HaeCs were active in the transcription of genes involved in the synthesis of the extracellular matrix proteins of the articular cartilage (ACAN, COL1A, COL2A, COL10A, COL9A, COMP, HAS1, SOX9), although the over-expression of COL1A1, COL10A1, COMP and HAS was observed. In parallel, the composite scaffold showed adequate mechanical and biological properties for cartilage tissue engineering, promoting chondrocyte proliferation. Our preliminary evidence contributes to the characterisation of HaeCs, highlighting the opportunity of using them for autologous cartilage implants in patients with haemophilia.

  10. Ski inhibits TGF-β/phospho-Smad3 signaling and accelerates hypertrophic differentiation in chondrocytes.

    Science.gov (United States)

    Kim, Kyung-Ok; Sampson, Erik R; Maynard, Robert D; O'Keefe, Regis J; Chen, Di; Drissi, Hicham; Rosier, Randy N; Hilton, Matthew J; Zuscik, Michael J

    2012-06-01

    Since transforming growing factor-β (TGF-β)/Smad signaling inhibits chondrocyte maturation, endogenous negative regulators of TGF-β signaling are likely also important regulators of the chondrocyte differentiation process. One such negative regulator, Ski, is an oncoprotein that is known to inhibit TGF-β/Smad3 signaling via its interaction with phospho-Smad3 and recruitment of histone deacetylases (HDACs) to the DNA binding complex. Based on this, we hypothesized that Ski inhibits TGF-β signaling and accelerates maturation in chondrocytes via recruitment of HDACs to transcriptional complexes containing Smads. We tested this hypothesis in chick upper sternal chondrocytes (USCs), where gain and loss of Ski expression experiments were performed. Over-expression of Ski not only reversed the inhibitory effect of TGF-β on the expression of hypertrophic marker genes such as type X collagen (colX) and osteocalcin, it induced these genes basally as well. Conversely, knockdown of Ski by RNA interference led to a reduction of colX and osteocalcin expression under basal conditions. Furthermore, Ski blocked TGF-β induction of cyclinD1 and caused a basal up-regulation of Runx2, consistent with the observed acceleration of hypertrophy. Regarding mechanism, not only does Ski associate with phospho-Smad2 and 3, but its association with phospho-Smad3 is required for recruitment of HDAC4 and 5. Implicating this recruitment of HDACs in the phenotypic effects of Ski in chondrocytes, the HDAC inhibitor SAHA reversed the up-regulation of colX and osteocalcin in Ski over-expressing cells. These results suggest that inhibition of TGF-β signaling by Ski, which involves its association with phospho-Smad3 and recruitment of HDAC4 and 5, leads to accelerated chondrocyte differentiation. Copyright © 2012 Wiley Periodicals, Inc.

  11. Alpha-Mangostin suppresses interleukin-1β-induced apoptosis in rat chondrocytes by inhibiting the NF-κB signaling pathway and delays the progression of osteoarthritis in a rat model.

    Science.gov (United States)

    Pan, Tianlong; Chen, Rong; Wu, Dengying; Cai, Ningyu; Shi, Xuchao; Li, Bin; Pan, Jun

    2017-11-01

    Osteoarthritis (OA) is a chronic degenerative joint disease that is characterized by progressive joint dysfunction and pain. Apoptosis and catabolism in chondrocytes play critical roles in the development of OA. Alpha-Mangostin (α-MG), one of the main components of the mangosteen, has been reported to have anti-apoptotic, anti-inflammatory and antioxidant effects. We investigated the therapeutic effects of α-MG on OA through experiments on rat chondrocytes in vitro and in a rat model of OA induced by destabilization of the medial meniscus (DMM). In vitro, we provided experimental evidence that α-MG inhibits the expression of MMP-13 and ADAMTs-5, and promotes the expression of SOX-9 in rat chondrocytes stimulated with interleukin-1β (IL-1β). In addition, we also found that α-MG can inhibit the expression of pro-apoptotic proteins such as Bax, Cyto-c, and C-caspase3, and increase the expression of the anti-apoptotic protein Bcl-2. These changes may be related to an α-MG induced inhibition of the IL-1β-induced activation of the NF-kB signaling pathway. In vivo, we also found that α-MG can limit the development of OA in rat models. The above results show that α-MG has a potential therapeutic effect on OA, and that this effect may be achieved by inhibiting the mitochondrial apoptosis of chondrocytes induced by an activation of the NF-kB pathway. Copyright © 2017 Elsevier B.V. All rights reserved.

  12. Regulation of hypoxia-inducible factor-1α (HIF-1α expression by interleukin-1β (IL-1 β, insulin-like growth factors I (IGF-I and II (IGF-II in human osteoarthritic chondrocytes

    Directory of Open Access Journals (Sweden)

    Angelica Rossi Sartori-Cintra

    2012-01-01

    Full Text Available OBJECTIVE: Hypoxia-inducible factor 1 alpha regulates genes related to cellular survival under hypoxia. This factor is present in osteroarthritic chondrocytes, and cytokines, such as interleukin-1 beta, participate in the pathogenesis of osteoarthritis, thereby increasing the activities of proteolytic enzymes, such as matrix metalloproteinases, and accelerating cartilage destruction. We hypothesize that Hypoxia Inducible Factor-1 alpha (HIF-1α can regulate cytokines (catabolic action and/or growth factors (anabolic action in osteoarthritis. The purpose of this study was to investigate the modulation of HIF-1α in human osteoarthritic chondrocytes by interleukin-1 beta (IL-1β and insulin-like growth factors I (IGF-I and II (IGF-II and to determine the involvement of the phosphatidylinositol-3kinase (PI-3K pathway in this process. METHODS: Human osteroarthritic chondrocytes were stimulated with IL-1β, IGF-I and IGF-II and LY294002, a specific inhibitor of PI-3K. Nuclear protein levels and gene expression were analyzed by western blot and quantitative reverse transcription-polymerase chain reaction analyses, respectively. RESULTS: HIF-1α expression was upregulated by IL-1β at the protein level but not at the gene level. IGF-I treatment resulted in increases in both the protein and mRNA levels of HIF-1α , whereas IGF-II had no effect on its expression. However, all of these stimuli exploited the PI-3K pathway. CONCLUSION: IL-1β upregulated the levels of HIF-1α protein post-transcriptionally, whereas IGF-I increased HIF-1α at the transcript level. In contrast, IGF-II did not affect the protein or gene expression levels of HIF-1α . Furthermore, all of the tested stimuli exploited the PI-3K pathway to some degree. Based on these findings, we are able to suggest that Hypoxia inducible Factor-1 exhibits protective activity in chondrocytes during osteoarthritis.

  13. Fluoroquinolone's effect on growth of human chondrocytes and chondrosarcomas. In vitro and in vivo correlation

    DEFF Research Database (Denmark)

    Multhaupt, H A; Alvarez, J C; Rafferty, P A

    2001-01-01

    with use of conventional light microscopy, electron microscopy, and immunohistochemistry to identify extracellular matrix, cell proliferation, and apoptosis. Cultures of normal chondrocytes expressed type-II collagen. Electron microscopy revealed a large amount of glycogen in the cells; the presence of fat...... of vimentin filaments. The treated chondrocytes showed a decrease in cell proliferation, but there was no induction of apoptosis or effect on the expression of extracellular matrix proteins. Ciprofloxacin-treated chondrosarcoma cultures and tissue samples showed changes in cartilage matrix composition...

  14. The Results of Fetal Chondrocytes Transplantation in Patients with Rheumatoid Arthritis

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    Natalya Krivoruchko

    2014-12-01

    Full Text Available Introduction. Nowadays anti-inflammatory and immunosuppressive therapy has significantly improved the quality of life and prognosis of rheumatoid arthritis (RA. Nevertheless, there are still many patients with progressive rheumatoid inflammation, resulting in the destruction of joints. Cell therapy seems like a promising direction in rheumatology. The aim of our research was to evaluate the efficacy of fetal chondrocyte transplantation in patients with RA.Methods. We examined 60 patients with rheumatoid arthritis (I - III stages between 20 and 63 years of age. They were divided into 2 groups: the first group underwent the fetal chondrocytes transplantation (n = 40, and the second was a control group who got conservative therapy (n = 20. Donor cells were taken from the chondrogenic layer of the humerus or femur heads and hip condyles of human embryos in gestation for 17-20 weeks. A suspension of fetal chondrocytes injected into affected areas of the articular surfaces under X-ray control. Cell viability was determined before the injection. Efficacy of the therapy was assessed by clinical, instrumental, and laboratory tests. This clinical trial was allowed by The Ministry of Public Health and Ethics Committee. All of our patients gave informed consent for the fetal chondrocytes transplantation.Results. Evaluation of the clinical manifestations of RA in the first group of patients showed 3.7 times decrease in pain and 1.6 times relief of synovitis. Complete reduction of contracture was observed in 82% of patients in the first group. Morphometric changes in X-ray demonstrated inhibition of the destruction in articular cartilage and surfaces of bones after transplantation of fetal chondrocytes. The dynamics of morphological changes in synovium showed 2.5 times reduction of the inflammatory reaction. Transplantation of fetal chondrocytes led to a significant reduction in ESR, CRP, fibrinogen , γ-globulin after a period of 12 months (p < 0

  15. Activation of PPARs α, β/δ, and γ Impairs TGF-β1-Induced Collagens' Production and Modulates the TIMP-1/MMPs Balance in Three-Dimensional Cultured Chondrocytes

    Directory of Open Access Journals (Sweden)

    Paul-Emile Poleni

    2010-01-01

    Full Text Available Background and Purpose. We investigated the potency of Peroxisome Proliferators-Activated Receptors (PPARs α, β/δ, and γ agonists to modulate Transforming Growth Factor-β1 (TGF-β1- induced collagen production or changes in Tissue Inhibitor of Matrix Metalloproteinase- (TIMP- 1/Matrix Metalloproteinase (MMP balance in rat chondrocytes embedded in alginate beads. Experimental Approach. Collagen production was evaluated by quantitative Sirius red staining, while TIMP-1 protein levels and global MMP (-1, -2, -3, -7, and -9 or specific MMP-13 activities were measured by ELISA and fluorigenic assays in culture media, respectively. Levels of mRNA for type II collagen, TIMP-1, and MMP-3 & 13 were quantified by real-time PCR. Key Results. TGF-β1 increased collagen deposition and type II collagen mRNA levels, while inducing TIMP-1 mRNA and protein expression. In contrast, it decreased global MMP or specific MMP-13 activities, while decreasing MMP-3 or MMP-13 mRNA levels. PPAR agonists reduced most of the effects of TGF-β1 on changes in collagen metabolism and TIMP-1/MMP balance in rat in a PPAR-dependent manner, excepted for Wy14643 on MMP activities. Conclusions and Implications. PPAR agonists reduce TGF-β1-modulated ECM turnover and inhibit chondrocyte activities crucial for collagen biosynthesis, and display a different inhibitory profile depending on selectivity for PPAR isotypes.

  16. Lipophagy Contributes to Testosterone Biosynthesis in Male Rat Leydig Cells.

    Science.gov (United States)

    Ma, Yi; Zhou, Yan; Zhu, Yin-Ci; Wang, Si-Qi; Ping, Ping; Chen, Xiang-Feng

    2018-02-01

    In recent years, autophagy was found to regulate lipid metabolism through a process termed lipophagy. Lipophagy modulates the degradation of cholesteryl esters to free cholesterol (FC), which is the substrate of testosterone biosynthesis. However, the role of lipophagy in testosterone production is unknown. To investigate this, primary rat Leydig cells and varicocele rat models were administered to inhibit or promote autophagy, and testosterone, lipid droplets (LDs), total cholesterol (TC), and FC were evaluated. The results demonstrated that inhibiting autophagy in primary rat Leydig cells reduced testosterone production. Further studies demonstrated that inhibiting autophagy increased the number and size of LDs and the level of TC, but decreased the level of FC. Furthermore, hypoxia promoted autophagy in Leydig cells. We found that short-term hypoxia stimulated testosterone secretion; however, the inhibition of autophagy abolished stimulated testosterone release. Hypoxia decreased the number and size of LDs in Leydig cells, but the changes could be largely rescued by blocking autophagy. In experimental varicocele rat models, the administration of autophagy inhibitors substantially reduced serum testosterone. These data demonstrate that autophagy contributes to testosterone biosynthesis at least partially through degrading intracellular LDs/TC. Our observations might reveal an autophagic regulatory mode regarding testosterone biosynthesis. Copyright © 2018 Endocrine Society.

  17. Shape of chondrocytes within articular cartilage affects the solid but not the fluid microenvironment under unconfined compression.

    Science.gov (United States)

    Guo, Hongqiang; Torzilli, Peter A

    2016-01-01

    Metabolic activity of the chondrocytes in articular cartilage is strongly related to their zone-specific shape and the composition and mechanical properties of their surrounding extracellular matrix (ECM). However the mechanisms by which cell shape influences the response of the ECM microenvironment to mechanical loading is yet to be elucidated. This relationship was studied using a biphasic multiscale finite element model of different shaped chondrocytes in the superficial and deep zones of the ECM during unconfined stress relaxation. For chondrocytes in the superficial zone, increasing the cell's initial aspect ratio (length/height) increased the deformation and solid stresses of the chondrocyte and pericellular matrix (PCM) during the loading phase; for chondrocytes in the deep zone the effect of the cell shape on the solid microenvironment was time and variable dependent. However, for superficial and deep zone chondrocytes the cell shape did not affect the fluid pressure and fluid shear stress. These results suggest that mechanotransduction of chondrocytes in articular cartilage may be regulated through the solid phase rather than the fluid phase, and that high stresses and deformations in the solid microenvironment in the superficial zone may be essential for the zone-specific biosynthetic activity of the chondrocyte. The biphasic multiscale computational analysis suggests that maintaining the cell shape is critical for regulating the microenvironment and metabolic activity of the chondrocyte in tissue engineering constructs. We investigated the effect of chondrocyte shape on the cellular microenvironment using a biphasic multiscale finite element analysis. Our study showed that cell shapes affects the solid but not the fluid microenvironment of the chondrocyte, and that maintaining the cell shape is critical for regulating the microenvironment and metabolic activity of the chondrocyte in native cartilage and tissue engineering constructs. As far as we know

  18. TNF/TNFR1 pathway and endoplasmic reticulum stress are involved in ofloxacin-induced apoptosis of juvenile canine chondrocytes

    International Nuclear Information System (INIS)

    Zhang, Fu-Tao; Ding, Yi; Shah, Zahir; Xing, Dan; Gao, Yuan; Liu, Dong Ming; Ding, Ming-Xing

    2014-01-01

    Background and purpose: Quinolones cause obvious cartilaginous lesions in juvenile animals by chondrocyte apoptosis, which results in the restriction of their use in pediatric and adolescent patients. Studies showed that chondrocytes can be induced to produce TNFα, and the cisternae of the endoplasmic reticulum in quinolone-treated chondrocytes become dilated. We investigated whether TNF/TNFR 1 pathway and endoplasmic reticulum stress (ERs) are involved in ofloxacin (a typical quinolone)-induced apoptosis of juvenile canine chondrocytes. Experimental approach: Canine juvenile chondrocytes were treated with ofloxacin. Cell survival and apoptosis rates were determined with MTT method and flow cytometry, respectively. The gene expression levels of the related signaling molecules (TNFα, TNFR 1 , TRADD, FADD and caspase-8) in death receptor pathways and main apoptosis-related molecules (calpain, caspase-12, GADD153 and GRP78) in ERs were measured by qRT-PCR. The gene expression of TNFR 1 was suppressed with its siRNA. The protein levels of TNFα, TNFR 1 and caspase-12 were assayed using Western blotting. Key results: The survival rates decreased while apoptosis rates increased after the chondrocytes were treated with ofloxacin. The mRNA levels of the measured apoptosis-related molecules in death receptor pathways and ERs, and the protein levels of TNFα, TNFR 1 and caspase-12 increased after the chondrocytes were exposed to ofloxacin. The downregulated mRNA expressions of TNFR 1 , Caspase-8 and TRADD, and the decreased apoptosis rates of the ofloxacin-treated chondrocytes occurred after TNFR 1 –siRNA interference. Conclusions and implications: Ofloxacin-induced chondrocyte apoptosis in a time- and concentration-dependent fashion. TNF/TNFR 1 pathway and ERs are involved in ofloxacin-induced apoptosis of juvenile canine chondrocytes in the early stage. - Highlights: • Chondrocyte apoptosis is induced by ofloxacin in a time- and concentration-dependent manners.

  19. Stimulation of artemisinin biosynthesis in Artemisia annua hairy ...

    African Journals Online (AJOL)

    STORAGESEVER

    2010-06-07

    . Hairy root (HR3) of A. annua was used in this study, which was routinely ... H2O2 generation. H2O2 production from elicited hairy roots was measured by chemiluminescence method (Hu et al., 2003). The luminescence.

  20. Auxin biosynthesis and storage forms

    Science.gov (United States)

    Strader, Lucia C.

    2013-01-01

    The plant hormone auxin drives plant growth and morphogenesis. The levels and distribution of the active auxin indole-3-acetic acid (IAA) are tightly controlled through synthesis, inactivation, and transport. Many auxin precursors and modified auxin forms, used to regulate auxin homeostasis, have been identified; however, very little is known about the integration of multiple auxin biosynthesis and inactivation pathways. This review discusses the many ways auxin levels are regulated through biosynthesis, storage forms, and inactivation, and the potential roles modified auxins play in regulating the bioactive pool of auxin to affect plant growth and development. PMID:23580748

  1. Hypericin: chemical synthesis and biosynthesis.

    Science.gov (United States)

    Huang, Lin-Fang; Wang, Zeng-Hui; Chen, Shi-Lin

    2014-02-01

    Hypericin is one of the most important phenanthoperylene quinones extracted mainly from plants of the genus Hypericum belonging to the sections Euhypericum and Campylosporus of Keller's classification. Widespread attention to the antiviral and anti-tumor properties of hypericin has spurred investigations of the chemical synthesis and biosynthesis of this unique compound. However, the synthetic strategies are challenging for organic and biological chemists. In this review, specific significant advances in total synthesis, semi-synthesis, and biosynthesis in the past decades are summarized. Copyright © 2014 China Pharmaceutical University. Published by Elsevier B.V. All rights reserved.

  2. Design of group IIA secreted/synovial phospholipase A(2 inhibitors: an oxadiazolone derivative suppresses chondrocyte prostaglandin E(2 secretion.

    Directory of Open Access Journals (Sweden)

    Jean-Edouard Ombetta

    Full Text Available Group IIA secreted/synovial phospholipase A(2 (GIIAPLA(2 is an enzyme involved in the synthesis of eicosanoids such as prostaglandin E(2 (PGE(2, the main eicosanoid contributing to pain and inflammation in rheumatic diseases. We designed, by molecular modeling, 7 novel analogs of 3-{4-[5(indol-1-ylpentoxy]benzyl}-4H-1,2,4-oxadiazol-5-one, denoted C1, an inhibitor of the GIIAPLA(2 enzyme. We report the results of molecular dynamics studies of the complexes between these derivatives and GIIAPLA(2, along with their chemical synthesis and results from PLA(2 inhibition tests. Modeling predicted some derivatives to display greater GIIAPLA(2 affinities than did C1, and such predictions were confirmed by in vitro PLA(2 enzymatic tests. Compound C8, endowed with the most favorable energy balance, was shown experimentally to be the strongest GIIAPLA(2 inhibitor. Moreover, it displayed an anti-inflammatory activity on rabbit articular chondrocytes, as shown by its capacity to inhibit IL-1beta-stimulated PGE(2 secretion in these cells. Interestingly, it did not modify the COX-1 to COX-2 ratio. C8 is therefore a potential candidate for anti-inflammatory therapy in joints.

  3. Effect of hyaluronic acid and polysaccharides from Opuntia ficus indica (L.) cladodes on the metabolism of human chondrocyte cultures.

    Science.gov (United States)

    Panico, A M; Cardile, V; Garufi, F; Puglia, C; Bonina, F; Ronsisvalle, S

    2007-05-04

    Conventional medications in articular disease are often effective for symptom relief, but they can also cause significant side effects and do not slow the progression of the disease. Several natural substances have been shown to be effective as non-steroidal anti-inflammatory drugs at relieving the symptoms of osteoarthritis (OA), and preliminary evidence suggests that some of these compounds may exert a favourable influence on the course of the disease. In this study, we assay the anti-inflammatory/chondroprotective effect of some lyophilised extracts obtained from Opuntia ficus indica (L.) cladodes and of hyaluronic acid (HA) on the production of key molecules released during chronic inflammatory events such as nitric oxide (NO), glycosaminoglycans (GAGs), prostaglandins (PGE(2)) and reactive oxygen species (ROS) in human chondrocyte culture, stimulated with proinflammatory cytokine interleukin-1 beta (IL-1 beta). Further the antioxidant effect of these extracts was evaluated in vitro employing the bleaching of the stable 1,1-diphenyl-2-picrylhydrazyl radical (DPPH test). All the extracts tested in this study showed an interesting profile in active compounds. Particularly some of these extracts were characterized by polyphenolic and polysaccharidic species. In vitro results pointed out that the extracts of Opuntia ficus indica cladodes were able to contrast the harmful effects of IL-1 beta. Our data showed the protective effect of the extracts of Opuntia ficus indica cladodes in cartilage alteration, which appears greater than that elicited by hyaluronic acid (HA) commonly employed as visco-supplementation in the treatment of joint diseases.

  4. Upregulation of matrix synthesis in chondrocyte-seeded agarose following sustained bi-axial cyclic loading

    Directory of Open Access Journals (Sweden)

    Belinda Pingguan-Murphy

    2012-08-01

    Full Text Available OBJECTIVES: The promotion of extracellular matrix synthesis by chondrocytes is a requisite part of an effective cartilage tissue engineering strategy. The aim of this in vitro study was to determine the effect of bi-axial cyclic mechanical loading on cell proliferation and the synthesis of glycosaminoglycans by chondrocytes in threedimensional cultures. METHOD: A strain comprising 10% direct compression and 1% compressive shear was applied to bovine chondrocytes seeded in an agarose gel during two 12-hour conditioning periods separated by a 12-hour resting period. RESULTS: The bi-axial-loaded chondrocytes demonstrated a significant increase in glycosaminoglycan synthesis compared with samples exposed to uni-axial or no loading over the same period (p<0.05. The use of a free-swelling recovery period prior to the loading regime resulted in additional glycosaminoglycan production and a significant increase in DNA content (p<0.05, indicating cell proliferation. CONCLUSIONS: These results demonstrate that the use of a bi-axial loading regime results in increased matrix production compared with uni-axial loading.

  5. Melatonin protects chondrocytes from impairment induced by glucocorticoids via NAD+-dependent SIRT1.

    Science.gov (United States)

    Yang, Wei; Kang, Xiaomin; Qin, Na; Li, Feng; Jin, Xinxin; Ma, Zhengmin; Qian, Zhuang; Wu, Shufang

    2017-10-01

    Intra-articular injection of glucocorticoids is used to relieve pain and inflammation in osteoarthritis patients, which is occasionally accompanied with the serious side effects of glucocorticoids in collagen-producing tissue. Melatonin is the major hormone released from the pineal gland and its beneficial effects on cartilage has been suggested. In the present study, we investigated the protective role of melatonin on matrix degeneration in chondrocytes induced by dexamethasone (Dex). The chondrocytes isolated from mice knee joint were treated with Dex, melatonin, EX527 and siRNA targeted for SIRT6, respectively. Dex treatment induced the loss of the extracellular matrix, NAD + /NADH ratio and NADPH concentration in chondrocytes. Melatonin alone have no effect on the quantity of proteoglycans and collagen type IIa1, however, the pretreatment of melatonin reversed the negative effects induced by Dex. Meanwhile, the significant decrease in NAD + /NADH ratio and NADPH concentration in Dex group were up-regulated by pretreatment of melatonin. Furthermore, it was revealed that inhibition of SIRT1 blocked the protective effects of melatonin. The enhancement of NAD + -dependent SIRT1 activity contributes to the chondroprotecfive effects of melatonin, which has a great benefit to prevent dexamethasone-induced chondrocytes impairment. Copyright © 2017 Elsevier Inc. All rights reserved.

  6. Effect of Collagen Type I or Type II on Chondrogenesis by Cultured Human Articular Chondrocytes

    NARCIS (Netherlands)

    Rutgers, M.; Saris, Daniël B.F.; Vonk, L.A.; van Rijen, M.H.P.; Akrum, V.; Langeveld, D.; van Boxtel, A.; Dhert, W.J.A.; Creemers, L.B.

    2013-01-01

    Introduction: Current cartilage repair procedures using autologous chondrocytes rely on a variety of carriers for implantation. Collagen types I and II are frequently used and valuable properties of both were shown earlier in vitro, although a preference for either was not demonstrated. Recently,

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

    Directory of Open Access Journals (Sweden)

    Masahiro Kondo

    2014-11-01

    Full Text Available 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.

  8. Evolution of Autologous Chondrocyte Repair and Comparison to Other Cartilage Repair Techniques

    Directory of Open Access Journals (Sweden)

    Ashvin K. Dewan

    2014-01-01

    Full Text Available Articular cartilage defects have been addressed using microfracture, abrasion chondroplasty, or osteochondral grafting, but these strategies do not generate tissue that adequately recapitulates native cartilage. During the past 25 years, promising new strategies using assorted scaffolds and cell sources to induce chondrocyte expansion have emerged. We reviewed the evolution of autologous chondrocyte implantation and compared it to other cartilage repair techniques. Methods. We searched PubMed from 1949 to 2014 for the keywords “autologous chondrocyte implantation” (ACI and “cartilage repair” in clinical trials, meta-analyses, and review articles. We analyzed these articles, their bibliographies, our experience, and cartilage regeneration textbooks. Results. Microfracture, abrasion chondroplasty, osteochondral grafting, ACI, and autologous matrix-induced chondrogenesis are distinguishable by cell source (including chondrocytes and stem cells and associated scaffolds (natural or synthetic, hydrogels or membranes. ACI seems to be as good as, if not better than, microfracture for repairing large chondral defects in a young patient’s knee as evaluated by multiple clinical indices and the quality of regenerated tissue. Conclusion. Although there is not enough evidence to determine the best repair technique, ACI is the most established cell-based treatment for full-thickness chondral defects in young patients.

  9. Role of Chondrocytes in Cartilage Formation, Progression of Osteoarthritis and Cartilage Regeneration

    Directory of Open Access Journals (Sweden)

    Hemanth Akkiraju

    2015-12-01

    Full Text Available Articular cartilage (AC covers the diarthrodial joints and is responsible for the mechanical distribution of loads across the joints. The majority of its structure and function is controlled by chondrocytes that regulate Extracellular Matrix (ECM turnover and maintain tissue homeostasis. Imbalance in their function leads to degenerative diseases like Osteoarthritis (OA. OA is characterized by cartilage degradation, osteophyte formation and stiffening of joints. Cartilage degeneration is a consequence of chondrocyte hypertrophy along with the expression of proteolytic enzymes. Matrix Metalloproteinases (MMPs and A Disintegrin and Metalloproteinase with Thrombospondin Motifs (ADAMTS are an example of these enzymes that degrade the ECM. Signaling cascades involved in limb patterning and cartilage repair play a role in OA progression. However, the regulation of these remains to be elucidated. Further the role of stem cells and mature chondrocytes in OA progression is unclear. The progress in cell based therapies that utilize Mesenchymal Stem Cell (MSC infusion for cartilage repair may lead to new therapeutics in the long term. However, many questions are unanswered such as the efficacy of MSCs usage in therapy. This review focuses on the role of chondrocytes in cartilage formation and the progression of OA. Moreover, it summarizes possible alternative therapeutic approaches using MSC infusion for cartilage restoration.

  10. Platelet rich plasma associated with heterologous fresh and thawed chondrocytes on osteochondral lesions of rabbits

    Directory of Open Access Journals (Sweden)

    R.R. Filgueiras

    2014-02-01

    Full Text Available Chondrocytes obtained from stifle joint of New Zealand White rabbits were cultivated. Half of cells were maintained in culture for later implantation and the others frozen during six months to evaluate viability. A circular osteochondral defect was created in the right stifle of other twenty seven rabbits. The control group (CG received no treatment. The thawed (TH and fresh (FH heterologous groups received, respectively, an implant of cultivated thawed or fresh heterologous chondrocytes associated with platelet rich plasma (PRP. The CG group showed greatest pain and lameness compared to the other groups seven days after the implantation. Microscopically, at 45 and 90 days, the TH and FH groups showed filling with cartilaginous tissue containing chondrocytes surrounded by a dense matrix of glycosaminoglycans. In the CG group, healing occurred with vascularized fibrous connective tissue without integration to the subchondral bone. Cryopreserved heterologous chondrocytes were viable for implantation and healing of osteochondral lesions; the association with PRP allows the fixation of cells in the lesion and offers growth factors which accelerates repair with tissue similar to articular hyaline cartilage.

  11. Scaffold-free cartilage tissue engineering with a small population of human nasoseptal chondrocytes.

    Science.gov (United States)

    Chiu, Loraine L Y; To, William T H; Lee, John M; Waldman, Stephen D

    2017-03-01

    Cartilage tissue engineering is a promising approach to provide suitable materials for nasal reconstruction; however, it typically requires large numbers of cells. We have previously shown that a small number of chondrocytes cultivated within a continuous flow bioreactor can elicit substantial tissue growth, but translation to human chondrocytes is not trivial. Here, we aimed to demonstrate the application of the bioreactor to generate large-sized tissues from a small population of primary human nasoseptal chondrocytes. Experimental study. Chondrocytes were cultured in the bioreactor using different medium compositions, with varying amounts of serum and with or without growth factors. Resulting engineered tissues were analyzed for physical properties, biochemical composition, tissue microstructure, and protein localization. Bioreactor-cultivated constructs grown with serum and growth factors (basic fibroblast growth factor and transforming growth factor beta 2) had greater thickness, as well as DNA and glycosaminoglycan (GAG) contents, compared to low serum and no growth factor controls. These constructs also showed the most intense proteoglycan and collagen II staining. The combination of bioreactor conditions, serum, and growth factors allowed the generation of large, thick scaffold-free human cartilaginous tissues that resembled the native nasoseptal cartilage. There also may be implications for patient selection in future clinical applications of these engineered tissues because their GAG content decreased with donor age. NA. Laryngoscope, 127:E91-E99, 2017. © 2016 The American Laryngological, Rhinological and Otological Society, Inc.

  12. [Pulsed electromagnetic field therapy inhibits chondrocyte apoptosis in rabbits with osteoarthritis].

    Science.gov (United States)

    Xie, Wei; Zhou, Jun; Luo, Qing-Lua; Liu, Hui-Fang; He, Cheng-Qi

    2014-01-01

    To determine the effect of pulsed electromagnetic field (PEMF) treatment on chondrocyte morphology, chondrocyte apoptosis, and the expression of apoptosis related proteins in rabbits. 24 white New Zealand rabbits were randomly divided into three groups: normal control group (NC group), anterior cruciate ligament transection without treatment (ACLT group), and anterior cruciate ligament transection with pulsed electromagnetic field treatment (PEMF group). Six weeks after anterior cruciate ligament transection, the rabbits in the PEMF group were given 2 weeks of pulsed electromagnetic field treatment. Rabbits in the PEMF group had significantly lower Mankin scores than those in the ACLT group, although the scores were higher than that of the NC group. The rabbits in the PEMF groups also had significantly lower levels of apoptosis index of chondrocytes and expression of caspase-3 compared with those in the ACLT group. The expression of caspase-8 in the rabbits in the PEMF group was higher compared to the NC group, but no significant difference compared with that of the ACLT group. Pulsed electromagnetic field treatment has therapeutic effect on the experimental osteoarthritis, which is likely a result of inhibition of apoptosis in chondrocytes.

  13. Surviving endoplasmic reticulum stress is coupled to altered chondrocyte differentiation and function.

    Directory of Open Access Journals (Sweden)

    Kwok Yeung Tsang

    2007-03-01

    Full Text Available In protein folding and secretion disorders, activation of endoplasmic reticulum (ER stress signaling (ERSS protects cells, alleviating stress that would otherwise trigger apoptosis. Whether the stress-surviving cells resume normal function is not known. We studied the in vivo impact of ER stress in terminally differentiating hypertrophic chondrocytes (HCs during endochondral bone formation. In transgenic mice expressing mutant collagen X as a consequence of a 13-base pair deletion in Col10a1 (13del, misfolded alpha1(X chains accumulate in HCs and elicit ERSS. Histological and gene expression analyses showed that these chondrocytes survived ER stress, but terminal differentiation is interrupted, and endochondral bone formation is delayed, producing a chondrodysplasia phenotype. This altered differentiation involves cell-cycle re-entry, the re-expression of genes characteristic of a prehypertrophic-like state, and is cell-autonomous. Concomitantly, expression of Col10a1 and 13del mRNAs are reduced, and ER stress is alleviated. ERSS, abnormal chondrocyte differentiation, and altered growth plate architecture also occur in mice expressing mutant collagen II and aggrecan. Alteration of the differentiation program in chondrocytes expressing unfolded or misfolded proteins may be part of an adaptive response that facilitates survival and recovery from the ensuing ER stress. However, the altered differentiation disrupts the highly coordinated events of endochondral ossification culminating in chondrodysplasia.

  14. Chondrogenic differentiation of human articular chondrocytes differs in biodegradable PGA/PLA scaffolds

    DEFF Research Database (Denmark)

    Zwingmann, Joern; Mehlhorn, Alexander T; Südkamp, Norbert

    2007-01-01

    Cartilage tissue engineering is applied clinically to cover and regenerate articular cartilage defects. Two bioresorbable nonwoven scaffolds, polyglycolic acid (PGA) and poly(lactic-co-glycolic acid) (PLGA) (90/10 copolymer of L-lactide and glycolide), were seeded with human chondrocytes after...

  15. Ovarian ecdysteroid biosynthesis and female germline stem cells.

    Science.gov (United States)

    Ameku, Tomotsune; Yoshinari, Yuto; Fukuda, Ruriko; Niwa, Ryusuke

    2017-07-03

    The germline stem cells (GSCs) are critical for gametogenesis throughout the adult life. Stem cell identity is maintained by local signals from a specialized microenvironment called the niche. However, it is unclear how systemic signals regulate stem cell activity in response to environmental cues. In our previous article, we reported that mating stimulates GSC proliferation in female Drosophila. The mating-induced GSC proliferation is mediated by ovarian ecdysteroids, whose biosynthesis is positively controlled by Sex peptide signaling. Here, we characterized the post-eclosion and post-mating expression pattern of the genes encoding the ecdysteroidogenic enzymes in the ovary. We further investigated the biosynthetic functions of the ovarian ecdysteroid in GSC maintenance in the mated females. We also briefly discuss the regulation of the ecdysteroidogenic enzyme-encoding genes and the subsequent ecdysteroid biosynthesis in the ovary of the adult Drosophila.

  16. Effects of limited exposure of rabbit chondrocyte cultures to parathyroid hormone and dibutyryl adenosine 3',5'-monophosphate on cartilage-characteristic proteoglycan synthesis

    International Nuclear Information System (INIS)

    Kato, Y.; Koike, T.; Iwamoto, M.; Kinoshita, M.; Sato, K.; Hiraki, Y.; Suzuki, F.

    1988-01-01

    Treatment of rabbit chondrocyte cultures with PTH or (Bu)2cAMP for 30 h increased by 2- to 3-fold the incorporation of [35S]sulfate and 3H radioactivity with glucosamine as the precursor into large chondroitin sulfate proteoglycans characteristically found in cartilage matrix. However, PTH and (Bu)2cAMP did not increase either [35S]sulfate incorporation into small proteoglycans or the incorporation of 3H radioactivity into hyaluronic acid and other glycosaminoglycans. PTH and (Bu)2cAMP also increased the incorporation of [3H] serine into both proteoglycans and total protein. In all cultures described above, the stimulation of [3H]serine incorporation into proteoglycans exceeded that of [3H]serine incorporation into total protein. These data indicate that PTH and (Bu)2cAMP selectively stimulate cartilage proteoglycan synthesis while they increase total protein synthesis. Since cAMP seems to play a mediatory role in the action of PTH, we elected to examine the effects of a limited exposure of chondrocytes to PTH or (Bu)2cAMP on the synthesis of proteoglycans. Treatment with PTH or (Bu)2cAMP for only the initial 2-7 h did not increase the rates of incorporation of [35S]sulfate, the 3H radioactivity with glucosamine, and [3H]serine into proteoglycans, as measured at 30 h, despite the fact that this treatment brought about a rapid and transient rise in the cAMP level. Furthermore, the application of prostaglandin I2 at concentrations that increased cAMP levels in a similar fashion as did PTH did not affect [35S] sulfate incorporation into proteoglycans

  17. 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.

    Directory of Open Access Journals (Sweden)

    Serena Guidotti

    Full Text Available 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.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.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.In articular chondrocytes, GSK3β activity is required for the maintenance of proliferative potential and phenotype. Conversely, GSK3β inactivation, although preserving chondrocyte survival, results in functional impairment via

  18. Culture temperature affects human chondrocyte messenger RNA expression in monolayer and pellet culture systems.

    Directory of Open Access Journals (Sweden)

    Akira Ito

    Full Text Available Cell-based therapy has been explored for articular cartilage regeneration. Autologous chondrocyte implantation is a promising cell-based technique for repairing articular cartilage defects. However, there are several issues such as chondrocyte de-differentiation. While numerous studies have been designed to overcome some of these issues, only a few have focused on the thermal environment that can affect chondrocyte metabolism and phenotype. In this study, the effects of different culture temperatures on human chondrocyte metabolism- and phenotype-related gene expression were investigated in 2D and 3D environments. Human chondrocytes were cultured in a monolayer or in a pellet culture system at three different culture temperatures (32°C, 37°C, and 41°C for 3 days. The results showed that the total RNA level, normalized to the threshold cycle value of internal reference genes, was higher at lower temperatures in both culture systems. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH and citrate synthase (CS, which are involved in glycolysis and the citric acid cycle, respectively, were expressed at similar levels at 32°C and 37°C in pellet cultures, but the levels were significantly lower at 41°C. Expression of the chondrogenic markers, collagen type IIA1 (COL2A1 and aggrecan (ACAN, was higher at 37°C than at 32°C and 41°C in both culture systems. However, this phenomenon did not coincide with SRY (sex-determining region Y-box 9 (SOX9, which is a fundamental transcription factor for chondrogenesis, indicating that a SOX9-independent pathway might be involved in this phenomenon. In conclusion, the expression of chondrocyte metabolism-related genes at 32°C was maintained or enhanced compared to that at 37°C. However, chondrogenesis-related genes were further induced at 37°C in both culture systems. Therefore, manipulating the culture temperature may be an advantageous approach for regulating human chondrocyte metabolic activity and

  19. Molecular analysis of chondrocytes cultured in agarose in response to dynamic compression

    Directory of Open Access Journals (Sweden)

    Mallein-Gerin Frédéric

    2008-09-01

    Full Text Available Abstract Background Articular cartilage is exposed to high mechanical loads under normal physiological conditions and articular chondrocytes regulate the composition of cartilaginous matrix, in response to mechanical signals. However, the intracellular pathways involved in mechanotransduction are still being defined. Using the well-characterized chondrocyte/agarose model system and dynamic compression, we report protocols for preparing and characterizing constructs of murine chondrocytes and agarose, and analyzing the effect of compression on steady-state level of mRNA by RT-PCR, gene transcription by gene reporter assay, and phosphorylation state of signalling molecules by Western-blotting. The mouse model is of particular interest because of the availability of a large choice of bio-molecular tools suitable to study it, as well as genetically modified mice. Results Chondrocytes cultured in agarose for one week were surrounded by a newly synthesized pericellular matrix, as revealed by immunohistochemistry prior to compression experiments. This observation indicates that this model system is suitable to study the role of matrix molecules and trans-membrane receptors in cellular responsiveness to mechanical stress. The chondrocyte/agarose constructs were then submitted to dynamic compression with FX-4000C™ Flexercell® Compression Plus™ System (Flexcell. After clearing proteins off agarose, Western-blotting analysis showed transient activation of Mitogen-activated protein kinases (MAPK in response to dynamic compression. After assessment by capillary electrophoresis of the quality of RNA extracted from agarose, steady-state levels of mRNA expression was measured by real time PCR. We observed an up-regulation of cFos and cJun mRNA levels as a response to compression, in accordance with the mechanosensitive character observed for these two genes in other studies using cartilage explants submitted to compression. To explore further the

  20. Increased adipogenesis in cultured embryonic chondrocytes and in adult bone marrow of dominant negative Erg transgenic mice.

    Directory of Open Access Journals (Sweden)

    Sébastien Flajollet

    Full Text Available In monolayer culture, primary articular chondrocytes have an intrinsic tendency to lose their phenotype during expansion. The molecular events underlying this chondrocyte dedifferentiation are still largely unknown. Several transcription factors are important for chondrocyte differentiation. The Ets transcription factor family may be involved in skeletal development. One family member, the Erg gene, is mainly expressed during cartilage formation. To further investigate the potential role of Erg in the maintenance of the chondrocyte phenotype, we isolated and cultured chondrocytes from the rib cartilage of embryos of transgenic mice that express a dominant negative form of Erg (DN-Erg during cartilage formation. DN-Erg expression in chondrocytes cultured for up to 20 days did not affect the early dedifferentiation usually observed in cultured chondrocytes. However, lipid droplets accumulated in DN-Erg chondrocytes, suggesting adipocyte emergence. Transcriptomic analysis using a DNA microarray, validated by quantitative RT-PCR, revealed strong differential gene expression, with a decrease in chondrogenesis-related markers and an increase in adipogenesis-related gene expression in cultured DN-Erg chondrocytes. These results indicate that Erg is involved in either maintaining the chondrogenic phenotype in vitro or in cell fate orientation. Along with the in vitro studies, we compared adipocyte presence in wild-type and transgenic mice skeletons. Histological investigations revealed an increase in the number of adipocytes in the bone marrow of adult DN-Erg mice even though no adipocytes were detected in embryonic cartilage or bone. These findings suggest that the Ets transcription factor family may contribute to the homeostatic balance in skeleton cell plasticity.

  1. Effect of chondrocyte-derived early extracellular matrix on chondrogenesis of placenta-derived mesenchymal stem cells.

    Science.gov (United States)

    Park, Yong-Beom; Seo, Sinji; Kim, Jin-A; Heo, Jin-Chul; Lim, Young-Cheol; Ha, Chul-Won

    2015-06-24

    The extracellular matrix (ECM) surrounding cells contains a variety of proteins that provide structural support and regulate cellular functions. Previous studies have shown that decellularized ECM isolated from tissues or cultured cells can be used to improve cell differentiation in tissue engineering applications. In this study we evaluated the effect of decellularized chondrocyte-derived ECM (CDECM) on the chondrogenesis of human placenta-derived mesenchymal stem cells (hPDMSCs) in a pellet culture system. After incubation with or without chondrocyte-derived ECM in chondrogenic medium for 1 or 3 weeks, the sizes and wet masses of the cell pellets were compared with untreated controls (hPDMSCs incubated in chondrogenic medium without chondrocyte-derived ECM). In addition, histologic analysis of the cell pellets (Safranin O and collagen type II staining) and quantitative reverse transcription-PCR analysis of chondrogenic markers (aggrecan, collagen type II, and SOX9) were carried out. Our results showed that the sizes and masses of hPDMSC pellets incubated with chondrocyte-derived ECM were significantly higher than those of untreated controls. Differentiation of hPDMSCs (both with and without chondrocyte-derived ECM) was confirmed by Safranin O and collagen type II staining. Chondrogenic marker expression and glycosaminoglycan (GAG) levels were significantly higher in hPDMSC pellets incubated with chondrocyte-derived ECM compared with untreated controls, especially in cells precultured with chondrocyte-derived ECM for 7 d. Taken together, these results demonstrate that chondrocyte-derived ECM enhances the chondrogenesis of hPDMSCs, and this effect is further increased by preculture with chondrocyte-derived ECM. This preculture method for hPDMSC chondrogenesis represents a promising approach for cartilage tissue engineering.

  2. (vitamin B1) biosynthesis genes

    African Journals Online (AJOL)

    In this study, the gene transcripts of first two enzymes in thiamine biosynthesis pathway, THIC and THI1/THI4 were identified and amplified from oil palm tissues. Primers were designed based on sequence comparison of the genes from Arabidopsis thaliana, Zea mays, Oryza sativa and Alnus glutinosa. Oil palm's responses ...

  3. Articular chondrocyte network mediated by gap junctions: role in metabolic cartilage homeostasis

    Science.gov (United States)

    Mayan, Maria D; Gago-Fuentes, Raquel; Carpintero-Fernandez, Paula; Fernandez-Puente, Patricia; Filgueira-Fernandez, Purificacion; Goyanes, Noa; Valiunas, Virginijus; Brink, Peter R; Goldberg, Gary S; Blanco, Francisco J

    2017-01-01

    Objective This study investigated whether chondrocytes within the cartilage matrix have the capacity to communicate through intercellular connections mediated by voltage-gated gap junction (GJ) channels. Methods Frozen cartilage samples were used for immunofluorescence and immunohistochemistry assays. Samples were embedded in cacodylate buffer before dehydration for scanning electron microscopy. Co-immunoprecipitation experiments and mass spectrometry (MS) were performed to identify proteins that interact with the C-terminal end of Cx43. GJ communication was studied through in situ electroporation, electrophysiology and dye injection experiments. A transwell layered culture system and MS were used to identify and quantify transferred amino acids. Results Microscopic images revealed the presence of multiple cellular projections connecting chondrocytes within the matrix. These projections were between 5 and 150 μm in length. MS data analysis indicated that the C-terminus of Cx43 interacts with several cytoskeletal proteins implicated in Cx trafficking and GJ assembly, including α-tubulin and β-tubulin, actin, and vinculin. Electrophysiology experiments demonstrated that 12-mer oligonucleotides could be transferred between chondrocytes within 12 min after injection. Glucose was homogeneously distributed within 22 and 35 min. No transfer was detected when glucose was electroporated into A549 cells, which have no GJs. Transwell layered culture systems coupled with MS analysis revealed connexins can mediate the transfer of L-lysine and L-arginine between chondrocytes. Conclusions This study reveals that intercellular connections between chondrocytes contain GJs that play a key role in cell-cell communication and a metabolic function by exchange of nutrients including glucose and essential amino acids. A three-dimensional cellular network mediated through GJs might mediate metabolic and physiological homeostasis to maintain cartilage tissue. PMID:24225059

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

    Science.gov (United States)

    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

  5. Low oxygen reduces the modulation to an oxidative phenotype in monolayer-expanded chondrocytes.

    Science.gov (United States)

    Heywood, Hannah K; Lee, David A

    2010-01-01

    Autologous chondrocyte implantation requires a phase of in vitro cell expansion, achieved by monolayer culture under atmospheric oxygen levels. Chondrocytes reside under low oxygen conditions in situ and exhibit a glycolytic metabolism. However, oxidative phosphorylation rises progressively during culture, with concomitant reactive oxygen species production. We determine if the high oxygen environment in vitro provides the transformation stimulus. Articular chondrocytes were cultured in monolayer for up to 14 days under 2%, 5%, or 20% oxygen. Expansion under 2% and 5% oxygen reduced the rate at which the cells developed an oxidative phenotype compared to 20% oxygen. However, at 40 +/- 4 fmol cell(-1) h(-1) the oxygen consumption by chondrocytes expanded under 2% oxygen for 14 days was still 14 times the value observed for freshly isolated cells. Seventy-five to 78% of the increased oxygen consumption was accounted for by oxidative phosphorylation (oligomycin sensitive). Expansion under low oxygen also reduced cellular proliferation and 8-hydroxyguanosine release, a marker of oxidative DNA damage. However, these parameters remained elevated compared to freshly isolated cells. Thus, expansion under physiological oxygen levels reduces, but does not abolish, the induction of an oxidative energy metabolism. We conclude that simply transferring chondrocytes to low oxygen is not sufficient to either maintain or re-establish a normal energy metabolism. Furthermore, a hydrophobic polystyrene culture surface which promotes rounded cell morphology had no effect on the development of an oxidative metabolism. Although the shift towards an oxidative energy metabolism is often accompanied by morphological changes, this study does not support the hypothesis that it is driven by them.

  6. Nanosized fibers' effect on adult human articular chondrocytes behavior

    Energy Technology Data Exchange (ETDEWEB)

    Stenhamre, Hanna [Biopolymer Technology, Department of Chemical and Biological Engineering, Chalmers University of Technology, Gothenburg (Sweden); Department of Clinical Chemistry and Transfusion Medicine, Sahlgrenska University Hospital, Gothenburg (Sweden); Thorvaldsson, Anna, E-mail: anna.thorvaldsson@swerea.se [Biopolymer Technology, Department of Chemical and Biological Engineering, Chalmers University of Technology, Gothenburg (Sweden); Swerea IVF, Mölndal (Sweden); Enochson, Lars [Department of Clinical Chemistry and Transfusion Medicine, Sahlgrenska University Hospital, Gothenburg (Sweden); Walkenström, Pernilla [Swerea IVF, Mölndal (Sweden); Lindahl, Anders [Department of Clinical Chemistry and Transfusion Medicine, Sahlgrenska University Hospital, Gothenburg (Sweden); Brittberg, Mats [Cartilage Research Unit, University of Gothenburg, Department Orthopaedics, Kungsbacka Hospital, Kungsbacka (Sweden); Gatenholm, Paul [Biopolymer Technology, Department of Chemical and Biological Engineering, Chalmers University of Technology, Gothenburg (Sweden)

    2013-04-01

    Tissue engineering with chondrogenic cell based therapies is an expanding field with the intention of treating cartilage defects. It has been suggested that scaffolds used in cartilage tissue engineering influence cellular behavior and thus the long-term clinical outcome. The objective of this study was to assess whether chondrocyte attachment, proliferation and post-expansion re-differentiation could be influenced by the size of the fibers presented to the cells in a scaffold. Polylactic acid (PLA) scaffolds with different fiber morphologies were produced, i.e. microfiber (MS) scaffolds as well as nanofiber-coated microfiber scaffold (NMS). Adult human articular chondrocytes were cultured in the scaffolds in vitro up to 28 days, and the resulting constructs were assessed histologically, immunohistochemically, and biochemically. Attachment of cells and serum proteins to the scaffolds was affected by the architecture. The results point toward nano-patterning onto the microfibers influencing proliferation of the chondrocytes, and the overall 3D environment having a greater influence on the re-differentiation. In the efforts of finding the optimal scaffold for cartilage tissue engineering, studies as the current contribute to the knowledge of how to affect and control chondrocytes behavior. - Highlights: ► Chondrocyte behavior in nanofiber-coated microfiber versus microfiber scaffolds ► High porosity (> 90%) and large pore sizes (a few hundred μm) of nanofibrous scaffolds ► Proliferation enhanced by presence of nanofibers ► Differentiation not significantly affected ► Cell attachment improved in presence of both nanofibers and serum.

  7. Increased rate of chondrocyte aggregation in a wavy-walled bioreactor.

    Science.gov (United States)

    Bueno, Ericka M; Bilgen, Bahar; Carrier, Rebecca L; Barabino, Gilda A

    2004-12-20

    A novel wavy-walled bioreactor designed to enhance mixing at controlled shear stress levels was used to culture chondrocytes in suspension. Chondrocyte aggregation in suspensions mixed at 30, 50, and 80 rpm was characterized in the wavy-walled bioreactor and compared with that in conventional smooth-walled and baffled-walled spinner flask bioreactors. Aggregation was characterized in terms of the percentage of cells that aggregated over time, and aggregate size changes over time. The kinetics of chondrocyte aggregation observed in the bioreactors was composed of two phases: early aggregation between 0 and 2 h of culture, and late aggregation between 3 and 24 h of culture. At 50 rpm, the kinetics of early aggregation in the wavy-walled bioreactor was approximately 25% and 65% faster, respectively, than those in the smooth-walled and baffled-walled spinner flask bioreactors. During the late aggregation phase, the kinetics of aggregation in the wavy-walled bioreactor were approximately 45% and 65% faster, respectively, than in the smooth-walled and baffled-walled spinner flasks. The observed improved kinetics of chondrocyte aggregation was obtained at no cost to the cell survival rate. Results of computerized image analysis suggest that chondrocyte aggregation occurred initially by the formation of new aggregates via cell-cell interactions and later by the joining of small aggregates into larger cell clumps. Aggregates appeared to grow for only a couple of hours in culture before reaching a steady size, possibly determined by limitations imposed by the hydrodynamic environment. These results suggest that the novel geometry of the wavy-walled bioreactor generates a hydrodynamic environment distinct from those traditionally used to culture engineered cartilage. Such differences may be useful in studies aimed at distinguishing the effects of the hydrodynamic environment on tissue-engineered cartilage. Characterizing the wavy-walled bioreactor's hydrodynamic environment

  8. [Study on the method of two dimensional polycrylamide gel electrophoresis on rat condylar chondrocyte].

    Science.gov (United States)

    Wu, Tuo-jiang; Li, Huang; Ma, Qiao-lin; Wang, Wen-mei

    2010-08-01

    To investigate the protein profile by two dimensional polycrylamide gel electrophoresis on the rat condylar chondrocyte in vitro. The third-passage chondrocytes were harvested from the mandibular condyles of 2-day-old rats in this study. The protein profile of the rat mandibular condylar chondrocytes was examined by two dimensional polycrylamide gel electrophoresis (2-DE-PAGE). The 2-DE gel maps on different pH gradients were obtained. The result of modified coomassi blue-sliver staining and sliver staining was compared using Pdquest 7.1 image analysis software. The results showed that the good protein profile of the condylar chondrocytes was obtained by standard Bio-Rad manual. The protein was mainly in the field from pH4 to pH7. The 1203±86 protein points were examined on 2-DE gel map by modified coomassi blue-sliver staining, and 1769±97 protein points was examined by sliver staining. The silver staining map showed more distinctly but higher background than modified coomassi blue-sliver staining. The protein profile of the condylar chondrocytes enriches the proteomic database and gives evidence to further proteomic research. The 2-DE map obtained by modified coomassi blue-sliver staining is more suitable for MALDI-TOF mass identification. Supported by National Natural Science Foundation of China (Grant No. C30700963), China Postdoctoral Science Foundation(Grant No.20090461088), Jiangsu Provincial Postdoctoral Science Foundation (Grant No.0802003C) and Nanjing City's Science and Technology Foundation (Grant No.200905011).

  9. IKKα/CHUK regulates extracellular matrix remodeling independent of its kinase activity to facilitate articular chondrocyte differentiation.

    Directory of Open Access Journals (Sweden)

    Eleonora Olivotto

    Full Text Available BACKGROUND: The non-canonical NF-κB activating kinase IKKα, encoded by CHUK (conserved-helix-loop-helix-ubiquitous-kinase, has been reported to modulate pro- or anti- inflammatory responses, cellular survival and cellular differentiation. Here, we have investigated the mechanism of action of IKKα as a novel effector of human and murine chondrocyte extracellular matrix (ECM homeostasis and differentiation towards hypertrophy. METHODOLOGY/PRINCIPAL FINDINGS: IKKα expression was ablated in primary human osteoarthritic (OA chondrocytes and in immature murine articular chondrocytes (iMACs derived from IKKα(f/f:CreERT2 mice by retroviral-mediated stable shRNA transduction and Cre recombinase-dependent Lox P site recombination, respectively. MMP-10 was identified as a major target of IKKα in chondrocytes by mRNA profiling, quantitative RT-PCR analysis, immunohistochemistry and immunoblotting. ECM integrity, as assessed by type II collagen (COL2 deposition and the lack of MMP-dependent COL2 degradation products, was enhanced by IKKα ablation in mice. MMP-13 and total collagenase activities were significantly reduced, while TIMP-3 (tissue inhibitor of metalloproteinase-3 protein levels were enhanced in IKKα-deficient chondrocytes. IKKα deficiency suppressed chondrocyte differentiation, as shown by the quantitative inhibition of.Alizarin red staining and the reduced expression of multiple chondrocyte differentiation effectors, including Runx2, Col10a1 and Vegfa,. Importantly, the differentiation of IKKα-deficient chondrocytes was rescued by a kinase-dead IKKα protein mutant. CONCLUSIONS/SIGNIFICANCE: IKKα acts independent of its kinase activity to help drive chondrocyte differentiation towards a hypertrophic-like state. IKKα positively modulates ECM remodeling via multiple downstream targets (including MMP-10 and TIMP-3 at the mRNA and post-transcriptional levels, respectively to maintain maximal MMP-13 activity, which is required for ECM

  10. (-)-Menthol biosynthesis and molecular genetics

    Science.gov (United States)

    Croteau, Rodney B.; Davis, Edward M.; Ringer, Kerry L.; Wildung, Mark R.

    2005-12-01

    (-)-Menthol is the most familiar of the monoterpenes as both a pure natural product and as the principal and characteristic constituent of the essential oil of peppermint ( Mentha x piperita). In this paper, we review the biosynthesis and molecular genetics of (-)-menthol production in peppermint. In Mentha species, essential oil biosynthesis and storage is restricted to the peltate glandular trichomes (oil glands) on the aerial surfaces of the plant. A mechanical method for the isolation of metabolically functional oil glands, has provided a system for precursor feeding studies to elucidate pathway steps, as well as a highly enriched source of the relevant biosynthetic enzymes and of their corresponding transcripts with which cDNA libraries have been constructed to permit cloning and characterization of key structural genes. The biosynthesis of (-)-menthol from primary metabolism requires eight enzymatic steps, and involves the formation and subsequent cyclization of the universal monoterpene precursor geranyl diphosphate to the parent olefin (-)-(4 S)-limonene as the first committed reaction of the sequence. Following hydroxylation at C3, a series of four redox transformations and an isomerization occur in a general “allylic oxidation-conjugate reduction” scheme that installs three chiral centers on the substituted cyclohexanoid ring to yield (-)-(1 R, 3 R, 4 S)-menthol. The properties of each enzyme and gene of menthol biosynthesis are described, as are their probable evolutionary origins in primary metabolism. The organization of menthol biosynthesis is complex in involving four subcellular compartments, and regulation of the pathway appears to reside largely at the level of gene expression. Genetic engineering to up-regulate a flux-limiting step and down-regulate a side route reaction has led to improvement in the composition and yield of peppermint oil.

  11. Synergistic Effects of a Mixture of Glycosaminoglycans to Inhibit Adipogenesis and Enhance Chondrocyte Features in Multipotent Cells

    Directory of Open Access Journals (Sweden)

    Petar D. Petrov

    2015-11-01

    Full Text Available Background/Aims: Multipotent mesenchymal stem cells affect homeostasis of adipose and joint tissues. Factors influencing their differentiation fate are of interest for both obesity and joint problems. We studied the impact of a mixture of glycosaminoglycans (GAGs (hyaluronic acid: dermatan sulfate 1:0.25, w/w used in an oral supplement for joint discomfort (Oralvisc™ on the differentiation fate of multipotent cells. Methods: Primary mouse embryo fibroblasts (MEFs were used as a model system. Post-confluent monolayer MEF cultures non-stimulated or hormonally stimulated to adipogenesis were chronically exposed to the GAGs mixture, its individual components or vehicle. The appearance of lipid laden cells, lipid accumulation and expression of selected genes at the mRNA and protein level was assessed. Results: Exposure to the GAGs mixture synergistically suppressed spontaneous adipogenesis and induced the expression of cartilage extracellular matrix proteins, aggrecan core protein, decorin and cartilage oligomeric matrix protein. Hormonally-induced adipogenesis in the presence of the GAGs mixture resulted in decreased adipogenic differentiation, down-regulation of adipogenic/lipogenic factors and genes for insulin resistance-related adipokines (resistin and retinol binding protein 4, and up-regulation of oxidative metabolism-related genes. Adipogenesis in the presence of dermatan sulfate, the minor component of the mixture, was not impaired but resulted in smaller lipid droplets and the induction of a more complete brown adipocyte-related transcriptional program in the cells in the adipose state. Conclusions: The Oralvisc™ GAGs mixture can tip the adipogenic/chondrogenic fate balance of multipotent cells away from adipogenesis while favoring chondrocyte related gene expression. The mixture and its dermatan sulfate component also have modulatory effects of interest on hormonally-induced adipogenesis and on metabolic and secretory capabilities of

  12. Constitutive activation of MEK1 in chondrocytes causes Stat1-independent achondroplasia-like dwarfism and rescues the Fgfr3-deficient mouse phenotype

    Science.gov (United States)

    Murakami, Shunichi; Balmes, Gener; McKinney, Sandra; Zhang, Zhaoping; Givol, David; de Crombrugghe, Benoit

    2004-01-01

    We generated transgenic mice that express a constitutively active mutant of MEK1 in chondrocytes. These mice showed a dwarf phenotype similar to achondroplasia, the most common human dwarfism, caused by activating mutations in FGFR3. These mice displayed incomplete hypertrophy of chondrocytes in the growth plates and a general delay in endochondral ossification, whereas chondrocyte proliferation was unaffected. Immunohistochemical analysis of the cranial base in transgenic embryos showed reduced staining for collagen type X and persistent expression of Sox9 in chondrocytes. These observations indicate that the MAPK pathway inhibits hypertrophic differentiation of chondrocytes and negatively regulates bone growth without inhibiting chondrocyte proliferation. Expression of a constitutively active mutant of MEK1 in chondrocytes of Fgfr3-deficient mice inhibited skeletal overgrowth, strongly suggesting that regulation of bone growth by FGFR3 is mediated at least in part by the MAPK pathway. Although loss of Stat1 restored the reduced chondrocyte proliferation in mice expressing an achondroplasia mutant of Fgfr3, it did not rescue the reduced hypertrophic zone, the delay in formation of secondary ossification centers, and the achondroplasia-like phenotype. These observations suggest a model in which Fgfr3 signaling inhibits bone growth by inhibiting chondrocyte differentiation through the MAPK pathway and by inhibiting chondrocyte proliferation through Stat1. PMID:14871928

  13. Calcitonin directly attenuates collagen type II degradation by inhibition of matrix metalloproteinase expression and activity in articular chondrocytes

    DEFF Research Database (Denmark)

    Sondergaard, B C; Wulf, H; Henriksen, K

    2006-01-01

    OBJECTIVE: Calcitonin was recently reported to counter progression of cartilage degradation in an experimental model of osteoarthritis, and the effects were primarily suggested to be mediated by inhibition of subchondral bone resorption. We investigated direct effects of calcitonin on chondrocytes...

  14. Role of Map Kinase in Mediating the Effects of Vitamin D3 Metabolites on Growth Plate Chondrocytes

    National Research Council Canada - National Science Library

    Haris, Ehland

    2003-01-01

    .... The process is required for normal long bone growth and in certain kinds of bone repair. Vitamin D3 plays an important regulatory role in chondrocyte differentiation and maturation and therefore is essential for proper endochondral ossification...

  15. Incorporation of hyaluronic acid into collagen scaffolds for the control of chondrocyte-mediated contraction and chondrogenesis

    Energy Technology Data Exchange (ETDEWEB)

    Tang Shunqing [Department of Biomedical Engineering, Jinan University, Guangzhou 510632 (China); Spector, Myron [Tissue Engineering, VA Boston Healthcare System, Boston, MA 02130 (United States)

    2007-09-15

    Hyaluronic acid (HA), a principal matrix molecule in many tissues, is present in high amounts in articular cartilage. HA contributes in unique ways to the physical behavior of the tissue, and has been shown to have beneficial effects on chondrocyte activity. The goal of this study was to incorporate graduated amounts of HA into type I collagen scaffolds for the control of chondrocyte-mediated contraction and chondrogenesis in vitro. The results demonstrated that the amount of contraction of HA/collagen scaffolds by adult canine articular chondrocytes increased with the HA content of the scaffolds. The greatest amount of chondrogenesis after two weeks was found in the scaffolds which had undergone the most contraction. HA can play a useful role in adjusting the mechanical behavior of tissue engineering scaffolds and chondrogenesis in chondrocyte-seeded scaffolds.

  16. Long-term and real-time monitoring of chondrocyte behavior synthesizing extracellular matrix with biologically coupled field effect transistor

    Science.gov (United States)

    Satake, Hiroto; Saito, Akiko; Mizuno, Shuichi; Kajisa, Taira; Sakata, Toshiya

    2017-04-01

    In this study, we report the differential measurement method of accurately monitoring cellular metabolism with a semiconductor-based field effect transistor (FET), focusing on the proliferation potency of chondrocytes utilized in the field of orthopedics. By adding growth factors to chondrocytes on the gate, cellular activity was induced and continuously monitored as a change in pH during a cellular respiration for ten days using the FET biosensor. Moreover, the electrical signal of the FET device reflected the reproduction property of chondrocytes to synthesize extracellular matrix (ECM). A platform based on the FET device is suitable as a noninvasive, real-time and long-term monitoring system for cellular functions; it will contribute to the elucidation of the mechanism of ECM synthesis by chondrocytes.

  17. Prostaglandin E2 Regulation of Chondrocyte Proliferation and Differentiation

    Science.gov (United States)

    1994-05-01

    increased bone resorption in osteolytic lesions(3 ,31), periodontal disease (32-34), osteomyelitis (35), and rheumatoid arthritis (36). Stimulation of...a, and IL-18 have been shown to increase production of E-series prostaglandins in osteoblasts(4 -42), periodontal ligament fibroblasts(43,44), and...enhanced bone formation during regenerative and augmentation procedures. LITERATURE CITED 1. Raisz, L.G. and B.E. Kream. 1983. Regulation of Bone

  18. SOX9 governs differentiation stage-specific gene expression in growth plate chondrocytes via direct concomitant transactivation and repression.

    Directory of Open Access Journals (Sweden)

    Victor Y L Leung

    2011-11-01

    Full Text Available Cartilage and endochondral bone development require SOX9 activity to regulate chondrogenesis, chondrocyte proliferation, and transition to a non-mitotic hypertrophic state. The restricted and reciprocal expression of the collagen X gene, Col10a1, in hypertrophic chondrocytes and Sox9 in immature chondrocytes epitomise the precise spatiotemporal control of gene expression as chondrocytes progress through phases of differentiation, but how this is achieved is not clear. Here, we have identified a regulatory element upstream of Col10a1 that enhances its expression in hypertrophic chondrocytes in vivo. In immature chondrocytes, where Col10a1 is not expressed, SOX9 interacts with a conserved sequence within this element that is analogous to that within the intronic enhancer of the collagen II gene Col2a1, the known transactivation target of SOX9. By analysing a series of Col10a1 reporter genes in transgenic mice, we show that the SOX9 binding consensus in this element is required to repress expression of the transgene in non-hypertrophic chondrocytes. Forced ectopic Sox9 expression in hypertrophic chondrocytes in vitro and in mice resulted in down-regulation of Col10a1. Mutation of a binding consensus motif for GLI transcription factors, which are the effectors of Indian hedgehog signaling, close to the SOX9 site in the Col10a1 regulatory element, also derepressed transgene expression in non-hypertrophic chondrocytes. GLI2 and GLI3 bound to the Col10a1 regulatory element but not to the enhancer of Col2a1. In addition to Col10a1, paired SOX9-GLI binding motifs are present in the conserved non-coding regions of several genes that are preferentially expressed in hypertrophic chondrocytes and the occurrence of pairing is unlikely to be by chance. We propose a regulatory paradigm whereby direct concomitant positive and negative transcriptional control by SOX9 ensures differentiation phase-specific gene expression in chondrocytes. Discrimination between

  19. Early-term effect of adult chondrocyte transplantation in an osteoarthritis animal model.

    Science.gov (United States)

    Desando, Giovanna; Cavallo, Carola; Tschon, Matilde; Giavaresi, Gianluca; Martini, Lucia; Fini, Milena; Giardino, Roberto; Facchini, Andrea; Grigolo, Brunella

    2012-08-01

    In this study, we investigated the efficacy of the transplantation of autologous articular chondrocytes seeded onto a hyaluronan-based scaffold, known as Hyaff(®)-11, on the treatment of early cartilage lesions in a rabbit osteoarthritis (OA) model. The hypothesis of the study was that this treatment could enhance cartilage repair after OA induction. OA was surgically induced by Anterior Cruciate Ligament Transection (ACLT) in thirty rabbits. Animals were divided into three groups, according to treatment: group 1: ACLT; group 2: Chondrocytes+Hyaff-11; and group 3: Hyaff-11 alone. The animals were euthanized, respectively, at 3 and 6 months after the treatment. Histomorphometrical analyses were performed by means of fibrillation index, cartilage thickness, and subchondral bone thickness evaluations. Histological appearance was scored according to Modified Kraus' Mankin and Osteoarthritis Research Society International (OARSI) scores. Immunohistochemical analyses were carried out for type I and II collagens, MMP-1, and MMP-3. A comparison between groups and follow-up for each outcome was performed with the general linear model with Sidak correction. Histomorphometrical evaluations at 3 and 6 months demonstrated that OA lesions became significantly worse followed by Hyaff-11 treatment, whereas Chondrocytes+Hyaff-11 treatment had the best overall subjective grade. Overall raw histological scores demonstrated a significant improvement with chondro-hyaluronic acid (HA) treatment at 3 months compared with HA in cartilage repair processes. Immunohistochemical analyses displayed a strong positivity for type II collagen in the Chondrocytes+Hyaff-11group at 3 months compared with the HA group. No staining was observed in MMP-3 expression in this group at any experimental point. The use of Chondrocytes+Hyaff-11 for the treatment of early OA lesions produced, already at 3 months, a repair tissue showing better macroscopic, histological, and immunohistochemical results than

  20. The effect of dexamethasone and triiodothyronine on terminal differentiation of primary bovine chondrocytes and chondrogenically differentiated mesenchymal stem cells.

    Directory of Open Access Journals (Sweden)

    Thomas M Randau

    Full Text Available The newly evolved field of regenerative medicine is offering solutions in the treatment of bone or cartilage loss and deficiency. Mesenchymal stem cells, as well as articular chondrocytes, are potential cells for the generation of bone or cartilage. The natural mechanism of bone formation is that of endochondral ossification, regulated, among other factors, through the hormones dexamethasone and triiodothyronine. We investigated the effects of these hormones on articular chondrocytes and chondrogenically differentiated mesenchymal stem cells, hypothesizing that these hormones would induce terminal differentiation, with chondrocytes and differentiated stem cells being similar in their response. Using a 3D-alginate cell culture model, bovine chondrocytes and chondrogenically differentiated stem cells were cultured in presence of triiodothyronine or dexamethasone, and cell proliferation and extracellular matrix production were investigated. Collagen mRNA expression was measured by real-time PCR. Col X mRNA and alkaline phosphatase were monitored as markers of terminal differentiation, a prerequisite of endochondral ossification. The alginate culture system worked well, both for the culture of chondrocytes and for the chondrogenic differentiation of mesenchymal stem cells. Dexamethasone led to an increase in glycosaminoglycan production. Triiodothyronine increased the total collagen production only in chondrocytes, where it also induced signs of terminal differentiation, increasing both collagen X mRNA and alkaline phosphatase activity. Dexamethasone induced terminal differentiation in the differentiated stem cells. The immature articular chondrocytes used in this study seem to be able to undergo terminal differentiation, pointing to their possible role in the onset of degenerative osteoarthritis, as well as their potential for a cell source in bone tissue engineering. When chondrocyte-like cells, after their differentiation, can indeed be moved on

  1. Bone morphogenetic protein (BMP)-2 enhances the expression of type II collagen and aggrecan in chondrocytes embedded in alginate beads.

    Science.gov (United States)

    Gründer, Tatiana; Gaissmaier, Christoph; Fritz, Jürgen; Stoop, Reinout; Hortschansky, Peter; Mollenhauer, Jürgen; Aicher, Wilhelm K

    2004-07-01

    For autologous chondrocyte transplantation (ACT) chondrocytes are expanded in vitro. During expansion these cells may dedifferentiate. This change in phenotype is characterized by a raised expression of type I collagen and a decrease in type II collagen expression. Since high expression of type II collagen is of central importance for the properties of hyaline cartilage, we investigated if the growth factor bone morphogenetic protein-2 (BMP-2) may modulate the chondrogenic phenotype in monolayer cell cultures and in three-dimensional culture systems. Chondrocytes from articular knee cartilage of 11 individuals (average age: 39.8 years) with no history of joint disease were isolated and seeded either in monolayer cultures or embedded in alginate beads in presence or absence of human recombinant BMP-2 (hr-BMP-2). Then, cells were harvested and analysis of the chondrogenic phenotype was performed using quantitative RT-PCR, immunocytochemistry and ELISA. Addition of BMP-2 to chondrocytes expanded in two-dimensional (2D) cultures during the first subculture (P1) had no effect on mRNA amounts encoding type II collagen and interleukin-1beta (IL-1beta). In contrast, seeding chondrocytes in three-dimensional (3D) alginate cultures raised type II collagen expression significantly and addition of BMP-2 enhanced this effect. We conclude that chondrocytes during expansion for ACT may benefit from BMP-2 activation only when seeded in an appropriate 3D culture system. Copyright 2004 OsteoArthritis Research Society International

  2. The Effect of Chondroitin Sulphate and Hyaluronic Acid on Chondrocytes Cultured within a Fibrin-Alginate Hydrogel

    Directory of Open Access Journals (Sweden)

    Christopher J. Little

    2014-09-01

    Full Text Available Osteoarthritis is a painful degenerative joint disease that could be better managed if tissue engineers can develop methods to create long-term engineered articular cartilage tissue substitutes. Many of the tissue engineered cartilage constructs currently available lack the chemical stimuli and cell-friendly environment that promote the matrix accumulation and cell proliferation needed for use in joint cartilage repair. The goal of this research was to test the efficacy of using a fibrin-alginate hydrogel containing hyaluronic acid (HA and/or chondroitin sulphate (CS supplements for chondrocyte culture. Neonatal porcine chondrocytes cultured in fibrin-alginate hydrogels retained their phenotype better than chondrocytes cultured in monolayer, as evidenced by analysis of their relative expression of type II versus type I collagen mRNA transcripts. HA or CS supplementation of the hydrogels increased matrix glycosaminoglycan (GAG production during the first week of culture. However, the effects of these supplements on matrix accumulation were not additive and were no longer observed after two weeks of culture. Supplementation of the hydrogels with CS or a combination of both CS and HA increased the chondrocyte cell population after two weeks of culture. Statistical analysis indicated that the HA and CS treatment effects on chondrocyte numbers may be additive. This research suggests that supplementation with CS and/or HA has positive effects on cartilage matrix production and chondrocyte proliferation in three-dimensional (3D fibrin-alginate hydrogels.

  3. The rate of hypo-osmotic challenge influences regulatory volume decrease (RVD) and mechanical properties of articular chondrocytes.

    Science.gov (United States)

    Wang, Z; Irianto, J; Kazun, S; Wang, W; Knight, M M

    2015-02-01

    Osteoarthritis (OA) is associated with a gradual reduction in the interstitial osmotic pressure within articular cartilage. The aim of this study was to compare the effects of sudden and gradual hypo-osmotic challenge on chondrocyte morphology and biomechanics. Bovine articular chondrocytes were exposed to a reduction in extracellular osmolality from 327 to 153 mOsmol/kg applied either suddenly (osmotic stress, 66% of chondrocytes exhibited an increase in diameter followed by RVD, whilst 25% showed no RVD. By contrast, cells exposed to gradual hypo-osmotic stress exhibited reduced cell swelling without subsequent RVD. There was an increase in the equilibrium modulus for cells exposed to sudden hypo-osmotic stress. However, gradual hypo-osmotic challenge had no effect on cell mechanical properties. This cell stiffening response to sudden hypo-osmotic challenge was abolished when actin organization was disrupted with cytochalasin D or RVD inhibited with REV5901. Both sudden and gradual hypo-osmotic challenge reduced cortical F-actin distribution and caused chromatin decondensation. Sudden hypo-osmotic challenge increases chondrocyte mechanics by activation of RVD and interaction with the actin cytoskeleton. Moreover, the rate of hypo-osmotic challenge is shown to have a profound effect on chondrocyte morphology and biomechanics. This important phenomenon needs to be considered when studying the response of chondrocytes to pathological hypo-osmotic stress. Copyright © 2014 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.

  4. The effects of photosensitizing antibiotics and ultraviolet irradiation on the biosynthesis of prostaglandins

    International Nuclear Information System (INIS)

    Lord, J.T.; Ziboh, V.A.; Blick, G.; Poitier, J.; Kursunoglu, I.; Penneys, N.S.

    1978-01-01

    Oxygenation of arachidonic acid in vitro by calf skin microsomal acetone powder was enhanced by UV-irradiation at wavelengths of 254 and 360 nm. Further enhancement of the oxygenation reaction was observed in the presence of two photosensitizing cyclic antibiotics, tetracycline and demethylchlortetracycline. To test whether or not the oxygenation of arachidonic acid was related to the biosynthesis of prostaglandins, [I- 14 C]-arachidonic acid was incubated with calf skin acetone powder in the presence of UV-irradiation and the cyclic antibiotics. Prostaglandin biosynthesis from arachidonic acid by the calf skin microsomal acetone powder was enhanced after exposure to UV-irradiation at 254 nm and moderately at 360 nm. Incubation in the presence of demethylchlortetra-cycline (0.2 mM) increased prostaglandin biosynthesis approximately 95% over control by UV-irradiation at 254 nm. No significant stimulation of prostaglandin biosynthesis was observed at 360 nm. Non-photosensitizing antibiotics had no effect either on the oxygenation of arachidonic acid or on the biosynthesis of prostaglandin with or without UV-irradiation. It is suggested that the inflammatory reactions associated with these photo-reactive antibiotics may in part, be via the biosynthesis and release of the prostaglandins which are known to produce cutaneous inflammatory reactions. (author)

  5. Estrogen biosynthesis in human uterine adenomyosis

    International Nuclear Information System (INIS)

    Urabe, Mamoru; Yamamoto, Takara; Kitawaki, Jo; Honjo, Hideo; Okada, Hiroji

    1989-01-01

    Estrogen biosynthesis (aromatiase activity) was investigated in human adenomyosis tissue and compared with that of the normal myometrium, endometrium, and endometrical cancer tissues. Homogenates were incubated with [1,2,6,7- 3 H]androstenedione and NADPH at 37 deg. C for 1 h. After stopping the enzymatic reaction with ethyl acetate, [4- 14 C]estrone and [4- 14 C]estradiol-17β were added to the incubated sample. Estrone and estradiol were purified and identified by Bio-Rad AG1-X2 column chromatography, thin-layer chromatography and co-crystallization. Estrogen formed in the incubated sample was calculated from the 3 H/ 14 C ratio of the final crystal. The value for estrone formed from androstenedione was 52-132 fmol . h -1. g -1 wet weight. Aromatase activity in the adenomyosis tissues was higher than that in normal endometrial or myometrial tissues, but lower than that found in myometrial or endometrial tumour tissue. Furthermore, we investigated the effect of danazol, progresterone, and medroxyprogesterone acetate on adenomyosis cells in primary cultures. Aromatase activity in adenomyosis was blocked by danazol, but stimulated by progesterone and MPA. These results indicate that aromatase activity in adenomyosis may contribute to the growth of the ectopic endometrial tissue which occurs in this disease. (author)

  6. Gene Modification of Mesenchymal Stem Cells and Articular Chondrocytes to Enhance Chondrogenesis

    Directory of Open Access Journals (Sweden)

    Saliya Gurusinghe

    2014-01-01

    Full Text Available Current cell based treatment for articular cartilage and osteochondral defects are hampered by issues such as cellular dedifferentiation and hypertrophy of the resident or transplanted cells. The reduced expression of chondrogenic signalling molecules and transcription factors is a major contributing factor to changes in cell phenotype. Gene modification of chondrocytes may be one approach to redirect cells to their primary phenotype and recent advances in nonviral and viral gene delivery technologies have enabled the expression of these lost factors at high efficiency and specificity to regain chondrocyte function. This review focuses on the various candidate genes that encode signalling molecules and transcription factors that are specific for the enhancement of the chondrogenic phenotype and also how epigenetic regulators of chondrogenesis in the form of microRNA may also play an important role.

  7. Stem cells catalyze cartilage formation by neonatal articular chondrocytes in 3D biomimetic hydrogels

    Science.gov (United States)

    Lai, Janice H.; Kajiyama, Glen; Smith, Robert Lane; Maloney, William; Yang, Fan

    2013-12-01

    Cartilage loss is a leading cause of disability among adults and effective therapy remains elusive. Neonatal chondrocytes (NChons) are an attractive allogeneic cell source for cartilage repair, but their clinical translation has been hindered by scarce donor availability. Here we examine the potential for catalyzing cartilage tissue formation using a minimal number of NChons by co-culturing them with adipose-derived stem cells (ADSCs) in 3D hydrogels. Using three different co-culture models, we demonstrated that the effects of co-culture on cartilage tissue formation are dependent on the intercellular distance and cell distribution in 3D. Unexpectedly, increasing ADSC ratio in mixed co-culture led to increased synergy between NChons and ADSCs, and resulted in the formation of large neocartilage nodules. This work raises the potential of utilizing stem cells to catalyze tissue formation by neonatal chondrocytes via paracrine signaling, and highlights the importance of controlling cell distribution in 3D matrices to achieve optimal synergy.

  8. A programmable ramp waveform generator for PEMF exposure studies on chondrocytes.

    Science.gov (United States)

    Jahns, M; Durdle, N; Lou, E; Raso, V J

    2006-01-01

    Osteoarthritis is a debilitating joint disease where the surface of articular cartilage degrades and is unable to repair itself through natural processes. Controlling the migration of transplanted chondrocytes to the defective cartilage non-invasively could be a novel treatment for osteoarthritis. Our research group has performed an in-vitro investigation into the response of cultured human chondrocytes to pulsed electromagnetic fields (PEMF). Development of a treatment for osteoarthritis patients will require the use of a programmable waveform generator to generate the PEMF. This paper discusses the design and testing of a programmable ramp waveform generator for such purpose. When this ramp waveform generator is connected to the PEMF coil driver circuitry, it will be able to produce linearly ramping magnetic fields ranging in strength from 0.5 mT to 4.5 mT. It also has an attainable pulse width ranging from 6 ms to 100 ms, with a selectable duty cycle from 1% to 99%

  9. Could Oxidative Stress Regulate the Expression of MicroRNA-146a and MicroRNA-34a in Human Osteoarthritic Chondrocyte Cultures?

    Science.gov (United States)

    Cheleschi, Sara; De Palma, Anna; Pascarelli, Nicola Antonio; Giordano, Nicola; Galeazzi, Mauro; Tenti, Sara

    2017-01-01

    Oxidative stress and the overproduction of reactive oxygen species (ROS) play an important role in the pathogenesis of osteoarthritis (OA). Accumulating evidence has demonstrated the involvement of microRNAs (miRNAs) dysregulation in disease development and progression. In this study, we evaluated the effect of oxidative stress on miR-146a and miR-34a expression levels in human OA chondrocytes cultures stimulated by H2O2. Mitochondrial ROS production and cell apoptosis were detected by flow cytometry. The antioxidant enzymes SOD-2, CAT, GPx, the transcriptional factor NRF2 and the selected miRNAs were analyzed by qRT-PCR. The H2O2-induced oxidative stress was confirmed by a significant increase in superoxide anion production and of the apoptotic ratio. Furthermore, H2O2 significantly up-regulated the expression levels of SOD-2, CAT, GPx and NRF2, and modulated miR-146a and miR-34a gene expression. The same analyses were carried out after pre-treatment with taurine, a known antioxidant substance, which, in our experience, counteracted the H2O2-induced effect. In conclusion, the induction of oxidative stress affected cell apoptosis and the expression of the enzymes involved in the oxidant/antioxidant balance. Moreover, we demonstrated for the first time the modification of miR-146a and miR-34a in OA chondrocytes subjected to H2O2 stimulus and we confirmed the antioxidant effect of taurine. PMID:29292727

  10. Could Oxidative Stress Regulate the Expression of MicroRNA-146a and MicroRNA-34a in Human Osteoarthritic Chondrocyte Cultures?

    Directory of Open Access Journals (Sweden)

    Sara Cheleschi

    2017-12-01

    Full Text Available Oxidative stress and the overproduction of reactive oxygen species (ROS play an important role in the pathogenesis of osteoarthritis (OA. Accumulating evidence has demonstrated the involvement of microRNAs (miRNAs dysregulation in disease development and progression. In this study, we evaluated the effect of oxidative stress on miR-146a and miR-34a expression levels in human OA chondrocytes cultures stimulated by H2O2. Mitochondrial ROS production and cell apoptosis were detected by flow cytometry. The antioxidant enzymes SOD-2, CAT, GPx, the transcriptional factor NRF2 and the selected miRNAs were analyzed by qRT-PCR. The H2O2-induced oxidative stress was confirmed by a significant increase in superoxide anion production and of the apoptotic ratio. Furthermore, H2O2 significantly up-regulated the expression levels of SOD-2, CAT, GPx and NRF2, and modulated miR-146a and miR-34a gene expression. The same analyses were carried out after pre-treatment with taurine, a known antioxidant substance, which, in our experience, counteracted the H2O2-induced effect. In conclusion, the induction of oxidative stress affected cell apoptosis and the expression of the enzymes involved in the oxidant/antioxidant balance. Moreover, we demonstrated for the first time the modification of miR-146a and miR-34a in OA chondrocytes subjected to H2O2 stimulus and we confirmed the antioxidant effect of taurine.

  11. Microscale consolidation analysis of relaxation behavior of single living chondrocytes subjected to varying strain-rates.

    Science.gov (United States)

    Nguyen, Trung Dung; Oloyede, Adekunle; Singh, Sanjleena; Gu, YuanTong

    2015-09-01

    Besides the elastic stiffness, the relaxation behavior of single living cells is also of interest of various researchers when studying cell mechanics. It is hypothesized that the relaxation response of the cells is governed by both intrinsic viscoelasticity of the solid phase and fluid-solid interactions mechanisms. There are a number of mechanical models have been developed to investigate the relaxation behavior of single cells. However, there is lack of model enable to accurately capture both of the mechanisms. Therefore, in this study, the porohyperelastic (PHE) model, which is an extension of the consolidation theory, combined with inverse Finite Element Analysis (FEA) technique was used at the first time to investigate the relaxation response of living chondrocytes. This model was also utilized to study the dependence of relaxation behavior of the cells on strain-rates. The stress-relaxation experiments under the various strain-rates were conducted with the Atomic Force Microscopy (AFM). The results have demonstrated that the PHE model could effectively capture the stress-relaxation behavior of the living chondrocytes, especially at intermediate to high strain-rates. Although this model gave some errors at lower strain-rates, its performance was acceptable. Therefore, the PHE model is properly a promising model for single cell mechanics studies. Moreover, it has been found that the hydraulic permeability of living chondrocytes reduced with decreasing of strain-rates. It might be due to the intracellular fluid volume fraction and the fluid pore pressure gradients of chondrocytes were higher when higher strain-rates applied. Copyright © 2015 Elsevier Ltd. All rights reserved.

  12. Effects of Extracellular Matrix on the Morphology and Behaviour of Rabbit Auricular Chondrocytes in Culture

    OpenAIRE

    Villar-Suárez, Vega; Colaço, B.; Calles-Venal, I.; Bravo, I. G.; Fernández-Álvarez, J. G.; Fernández-Caso, M.; Villar-Lacilla, J. M.

    2005-01-01

    Isolated chondrocytes dedifferentiate to a fibroblast-like shape on plastic substrata and proliferate extensively, but rarely form nodules. However, when dissociation is not complete and some cartilage remnants are included in the culture, proliferation decreases and cells grow in a reticular pattern with numerous nodules, which occasionally form small cartilage-like fragments. In an attempt to reproduce this stable chondrogenic state, we added a cartilage protein extract, ...

  13. Inhibition of apoptosis signal-regulating kinase 1 enhances endochondral bone formation by increasing chondrocyte survival

    Science.gov (United States)

    Eaton, G J; Zhang, Q-S; Diallo, C; Matsuzawa, A; Ichijo, H; Steinbeck, M J; Freeman, T A

    2014-01-01

    Endochondral ossification is the result of chondrocyte differentiation, hypertrophy, death and replacement by bone. The careful timing and progression of this process is important for normal skeletal bone growth and development, as well as fracture repair. Apoptosis Signal-Regulating Kinase 1 (ASK1) is a mitogen-activated protein kinase (MAPK), which is activated by reactive oxygen species and other cellular stress events. Activation of ASK1 initiates a signaling cascade known to regulate diverse cellular events including cytokine and growth factor signaling, cell cycle regulation, cellular differentiation, hypertrophy, survival and apoptosis. ASK1 is highly expressed in hypertrophic chondrocytes, but the role of ASK1 in skeletal tissues has not been investigated. Herein, we report that ASK1 knockout (KO) mice display alterations in normal growth plate morphology, which include a shorter proliferative zone and a lengthened hypertrophic zone. These changes in growth plate dynamics result in accelerated long bone mineralization and an increased formation of trabecular bone, which can be attributed to an increased resistance of terminally differentiated chondrocytes to undergo cell death. Interestingly, under normal cell culture conditions, mouse embryonic fibroblasts (MEFs) derived from ASK1 KO mice show no differences in either MAPK signaling or osteogenic or chondrogenic differentiation when compared with wild-type (WT) MEFs. However, when cultured with stress activators, H2O2 or staurosporine, the KO cells show enhanced survival, an associated decrease in the activation of proteins involved in death signaling pathways and a reduction in markers of terminal differentiation. Furthermore, in both WT mice treated with the ASK1 inhibitor, NQDI-1, and ASK1 KO mice endochondral bone formation was increased in an ectopic ossification model. These findings highlight a previously unrealized role for ASK1 in regulating endochondral bone formation. Inhibition of ASK1 has

  14. Fluoroquinolone's effect on growth of human chondrocytes and chondrosarcomas. In vitro and in vivo correlation.

    Science.gov (United States)

    Multhaupt, H A; Alvarez, J C; Rafferty, P A; Warhol, M J; Lackman, R D

    2001-01-01

    Clinical and in vitro studies have demonstrated that fluoroquinolones are toxic to chondrocytes; however, the exact mechanism of fluoroquinolone arthropathy is unknown. We investigated the toxicity of ciprofloxacin on normal cartilage and on cartilaginous tumors. Normal human cartilage, enchondroma, and chondrosarcoma explants were cultured either alone or with the addition of ciprofloxacin at 1, 10, or 20 mg/L of medium. Samples were collected up to twenty-one days after treatment and were processed for electron microscopy and conventional light microscopy. The specimens were characterized morphologically with use of conventional light microscopy, electron microscopy, and immunohistochemistry to identify extracellular matrix, cell proliferation, and apoptosis. Cultures of normal chondrocytes expressed type-II collagen. Electron microscopy revealed a large amount of glycogen in the cells; the presence of fat droplets, rough endoplasmic reticulum, and prominent Golgi apparatus; and a proteoglycan layer surrounding the cells. With prolonged ciprofloxacin treatment and with increased doses, there was an increase in dilated rough endoplasmic reticulum, the appearance of phagosomes, and disintegrated bundles of vimentin filaments. The treated chondrocytes showed a decrease in cell proliferation, but there was no induction of apoptosis or effect on the expression of extracellular matrix proteins. Ciprofloxacin-treated chondrosarcoma cultures and tissue samples showed changes in cartilage matrix composition. Ultrastructural analysis demonstrated clumped glycogen, dilation of endoplasmic reticulum, numerous abnormal lysosomes containing degeneration products, and a decreased proteoglycan deposit surrounding the tumor cells. Treated chondrosarcoma cells and tissue specimens did not proliferate, and apoptosis was induced. In contrast, the in vitro growth of other noncartilaginous malignant tumors like osteosarcoma and liposarcoma was unaffected by ciprofloxacin. Our results

  15. Substrate Stiffness Controls Osteoblastic and Chondrocytic Differentiation of Mesenchymal Stem Cells without Exogenous Stimuli.

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    Rene Olivares-Navarrete

    Full Text Available Stem cell fate has been linked to the mechanical properties of their underlying substrate, affecting mechanoreceptors and ultimately leading to downstream biological response. Studies have used polymers to mimic the stiffness of extracellular matrix as well as of individual tissues and shown mesenchymal stem cells (MSCs could be directed along specific lineages. In this study, we examined the role of stiffness in MSC differentiation to two closely related cell phenotypes: osteoblast and chondrocyte. We prepared four methyl acrylate/methyl methacrylate (MA/MMA polymer surfaces with elastic moduli ranging from 0.1 MPa to 310 MPa by altering monomer concentration. MSCs were cultured in media without exogenous growth factors and their biological responses were compared to committed chondrocytes and osteoblasts. Both chondrogenic and osteogenic markers were elevated when MSCs were grown on substrates with stiffness <10 MPa. Like chondrocytes, MSCs on lower stiffness substrates showed elevated expression of ACAN, SOX9, and COL2 and proteoglycan content; COMP was elevated in MSCs but reduced in chondrocytes. Substrate stiffness altered levels of RUNX2 mRNA, alkaline phosphatase specific activity, osteocalcin, and osteoprotegerin in osteoblasts, decreasing levels on the least stiff substrate. Expression of integrin subunits α1, α2, α5, αv, β1, and β3 changed in a stiffness- and cell type-dependent manner. Silencing of integrin subunit beta 1 (ITGB1 in MSCs abolished both osteoblastic and chondrogenic differentiation in response to substrate stiffness. Our results suggest that substrate stiffness is an important mediator of osteoblastic and chondrogenic differentiation, and integrin β1 plays a pivotal role in this process.

  16. Biomechanics of Meniscus Cells: Regional Variation and Comparison to Articular Chondrocytes and Ligament Cells

    OpenAIRE

    Sanchez-Adams, Johannah; Athanasiou, Kyriacos A.

    2012-01-01

    Central to understanding mechanotransduction in the knee meniscus is the characterization of meniscus cell mechanics. In addition to biochemical and geometric differences, the inner and outer regions of the meniscus contain cells that are distinct in morphology and phenotype. This study investigated the regional variation in meniscus cell mechanics in comparison to articular chondrocytes and ligament cells. It was found that the meniscus contains two biomechanically distinct cell populations,...

  17. Study on the effects of gradient mechanical pressures on the proliferation, apoptosis, chondrogenesis and hypertrophy of mandibular condylar chondrocytes in vitro.

    Science.gov (United States)

    Li, Hui; Huang, Linjian; Xie, Qianyang; Cai, Xieyi; Yang, Chi; Wang, Shaoyi; Zhang, Min

    2017-01-01

    To investigate the effects of gradient mechanical pressure on chondrocyte proliferation, apoptosis, and the expression of markers of chondrogenesis and chondrocyte hypertrophy. Mandibular condylar chondrocytes from 5 rabbits were cultured in vitro, and pressed with static pressures of 50kPa, 100kPa, 150kPa and 200kPa for 3h, respectively. The chondrocytes cultured without pressure (0kPa) were used as control. Cell proliferation, apoptosis, and the expression of aggrecan (AGG), collagen II (COL2), collagen X (COL10), alkaline phosphatase (ALP) were investigated. Ultrastructures of the pressurized chondrocytes under transmission electron microscopy (TEM) were observed. Chondrocyte proliferation increased at 100kPa and decreased at 200kPa. Chondrocyte apoptosis increased with peak pressure at 200kPa in a dose-dependent manner. Chondrocyte necrosis increased at 200kPa. The expression of AGG increased at 200kPa. The expression of COL2 decreased at 50kPa and increased at 150kPa. The expression of COL10 and ALP increased at 150kPa. Ultrastructure of the pressurized chondrocytes under TEM showed: at 100kPa, cells were enlarged with less cellular microvillus and a bigger nucleus; at 200kPa, cells shrank with the sign of apoptosis, and apoptosis cells were found. The mechanical loading of 150kPa is the moderate pressure for chondrocyte: cell proliferation and apoptosis is balanced, necrosis is reduced, and chondrogenesis and chondrocyte hypertrophy are promoted. When the pressure is lower, chondrogenesis and chondrocyte hypertrophy are inhibited. At 200kPa, degeneration of cartilage is implied. Copyright © 2016 Elsevier Ltd. All rights reserved.

  18. Effect of polystyrene and polyether imide cell culture inserts with different roughness on chondrocyte metabolic activity and gene expression profiles of aggrecan and collagen.

    Science.gov (United States)

    König, Josephine; Kohl, Benjamin; Kratz, Karl; Jung, Friedrich; Lendlein, Andreas; Ertel, Wolfgang; Schulze-Tanzil, Gundula

    2013-01-01

    In vitro cultured autologous chondrocytes can be used for implantation to support cartilage repair. For this purpose, a very small number of autologous cells harvested from a biopsy have to be expanded in monolayer culture. Commercially available polymer surfaces lead to chondrocyte dedifferentiation. Hence, the demanding need for optimized polymers and surface topologies supporting chondrocytes' differentiated phenotypes in vitro arises. In this study we explored the effect of tailored cell culture plate inserts prepared from polystyrene (PS) and polyether imide (PEI) exhibiting three different roughness levels (R0, RI, RII) on chondrocyte morphology, metabolism and gene expression profile. As a control, commercially available tissue culture plastic (TCP) dishes were included. Primary porcine articular chondrocytes were seeded on tailored PS and PEI inserts with three different roughness levels. The metabolic activity of the chondrocytes was determined after 24 hours using alamar blue assay. Chondrocyte gene expression profiles (aggrecan, type I and type II collagen) were monitored after 48 hours using Real Time Detection (RTD)-PCR. Chondrocytes cultured on PS and PEI surfaces formed cell clusters after 24 and 48 hours, which was not observed on TCP. The metabolic activity of chondrocytes cultured on PS was lower than of chondrocytes cultured on PEI, but also lower than on TCP. Gene expression analyses revealed an elevated expression of cartilage-specific aggrecan and an impaired expression of both collagen types by chondrocytes on PS and PEI compared with TCP. In summary, PEI is a biocompatible biomaterial suitable for chondrocyte culturing, which can be further chemically functionalized for generating specific surface interactions or covalent binding of biomolecules.

  19. Regulation of neurosteroid biosynthesis by neurotransmitters and neuropeptides

    Directory of Open Access Journals (Sweden)

    Jean-Luc eDo-Rego

    2012-01-01

    Full Text Available The enzymatic pathways leading to the synthesis of bioactive steroids in the brain are now almost completely elucidated in various groups of vertebrates and, during the last decade, the neuronal mechanisms involved in the regulation of neurosteroid production have received increasing attention. This report reviews the current knowledge concerning the effects of neurotransmitters, peptide hormones and neuropeptides on the biosynthesis of neurosteroids. Anatomical studies have been carried out to visualize the neurotransmitter- or neuropeptide-containing fibers contacting steroid-synthesizing neurons as well as the neurotransmitter, peptide hormones or neuropeptide receptors expressed in these neurons. Biochemical experiments have been conducted to investigate the effects of neurotransmitters, peptide hormones or neuropeptides on neurosteroid biosynthesis, and to characterize the type of receptors involved. Thus, it has been found that glutamate, acting through kainate and/or AMPA receptors, rapidly inactivates P450arom, and that melatonin produced by the pineal gland and eye inhibits the biosynthesis of 7-hydroxypregnenolone (7-OH-5P, while prolactin produced by the adenohypophysis enhances the formation of 7-OH-5P. It has also been demonstrated that the biosynthesis of neurosteroids is inhibited by GABA, acting through GABAA receptors, and neuropeptide Y, acting through Y1 receptors. In contrast, it has been shown that the octadecaneuropetide ODN, acting through central-type benzodiazepine receptors, the triakontatetraneuropeptide TTN, acting though peripheral-type benzodiazepine receptors, and vasotocine, acting through V1a-like receptors, stimulate the production of neurosteroids. Since neurosteroids are implicated in the control of various neurophysiological and behavioral processes, these data suggest that some of the neurophysiological effects exerted by neurotransmitters and neuropeptides may be mediated via the regulation

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

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    Xingfu Li

    2016-01-01

    Full Text Available 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.

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

    Science.gov (United States)

    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.

  2. Investigation of the direct effects of salmon calcitonin on human osteoarthritic chondrocytes

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    Pedersen Christian

    2010-04-01

    Full Text Available Abstract Background Calcitonin has been demonstrated to have chondroprotective effects under pre-clinical settings. It is debated whether this effect is mediated through subchondral-bone, directly on cartilage or both in combination. We investigated possible direct effects of salmon calcitonin on proteoglycans and collagen-type-II synthesis in osteoarthritic (OA cartilage. Methods Human OA cartilage explants were cultured with salmon calcitonin [100 pM-100 nM]. Direct effects of calcitonin on articular cartilage were evaluated by 1 measurement of proteoglycan synthesis by incorporation of radioactive labeled 35SO4 [5 μCi] 2 quantification of collagen-type-II formation by pro-peptides of collagen type II (PIINP ELISA, 3 QPCR expression of the calcitonin receptor in OA chondrocytes using four individual primer pairs, 4 activation of the cAMP signaling pathway by EIA and, 5 investigations of metabolic activity by AlamarBlue. Results QPCR analysis and subsequent sequencing confirmed expression of the calcitonin receptor in human chondrocytes. All doses of salmon calcitonin significantly elevated cAMP levels (P 35SO4 incorporation, with a 96% maximal induction at 10 nM (P Conclusion Calcitonin treatment increased proteoglycan and collagen synthesis in human OA cartilage. In addition to its well-established effect on subchondral bone, calcitonin may prove beneficial to the management of joint diseases through direct effects on chondrocytes.

  3. Visualization of living terminal hypertrophic chondrocytes of growth plate cartilage in situ by differential interference contrast microscopy and time-lapse cinematography.

    Science.gov (United States)

    Farnum, C E; Turgai, J; Wilsman, N J

    1990-09-01

    The functional unit within the growth plate consists of a column of chondrocytes that passes through a sequence of phases including proliferation, hypertrophy, and death. It is important to our understanding of the biology of the growth plate to determine if distal hypertrophic cells are viable, highly differentiated cells with the potential of actively controlling terminal events of endochondral ossification prior to their death at the chondro-osseous junction. This study for the first time reports on the visualization of living hypertrophic chondrocytes in situ, including the terminal hypertrophic chondrocyte. Chondrocytes in growth plate explants are visualized using rectified differential interference contrast microscopy. We record and measure, using time-lapse cinematography, the rate of movement of subcellular organelles at the limit of resolution of this light microscopy system. Control experiments to assess viability of hypertrophic chondrocytes include coincubating organ cultures with the intravital dye fluorescein diacetate to assess the integrity of the plasma membrane and cytoplasmic esterases. In this system, all hypertrophic chondrocytes, including the very terminal chondrocyte, exist as rounded, fully hydrated cells. By the criteria of intravital dye staining and organelle movement, distal hypertrophic chondrocytes are identical to chondrocytes in the proliferative and early hypertrophic cell zones.

  4. Mesenchymal stem cell-derived extracellular matrix enhances chondrogenic phenotype of and cartilage formation by encapsulated chondrocytes in vitro and in vivo.

    Science.gov (United States)

    Yang, Yuanheng; Lin, Hang; Shen, He; Wang, Bing; Lei, Guanghua; Tuan, Rocky S

    2018-03-15

    Mesenchymal stem cell derived extracellular matrix (MSC-ECM) is a natural biomaterial with robust bioactivity and good biocompatibility, and has been studied as a scaffold for tissue engineering. In this investigation, we tested the applicability of using decellularized human bone marrow derived MSC-ECM (hBMSC-ECM) as a culture substrate for chondrocyte expansion in vitro, as well as a scaffold for chondrocyte-based cartilage repair. hBMSC-ECM deposited by hBMSCs cultured on tissue culture plastic (TCP) was harvested, and then subjected to a decellularization process to remove hBMSCs. Compared with chondrocytes grown on TCP, chondrocytes seeded onto hBMSC-ECM exhibited significantly increased proliferation rate, and maintained better chondrocytic phenotype than TCP group. After being expanded to the same cell number and placed in high-density micromass cultures, chondrocytes from the ECM group showed better chondrogenic differentiation profile than those from the TCP group. To test cartilage formation ability, composites of hBMSC-ECM impregnated with chondrocytes were subjected to brief trypsin treatment to allow cell-mediated contraction, and folded to form 3-dimensional chondrocyte-impregnated hBMSC-ECM (Cell/ECM constructs). Upon culture in vitro in chondrogenic medium for 21 days, robust cartilage formation was observed in the Cell/ECM constructs. Similarly prepared Cell/ECM constructs were tested in vivo by subcutaneous implantation into SCID mice. Prominent cartilage formation was observed in the implanted Cell/ECM constructs 14 days post-implantation, with higher sGAG deposition compared to controls consisting of chondrocyte cell sheets. Taken together, these findings demonstrate that hBMSC-ECM is a superior culture substrate for chondrocyte expansion and a bioactive matrix potentially applicable for cartilage regeneration in vivo. Current cell-based treatments for focal cartilage defects face challenges, including chondrocyte dedifferentiation, need for

  5. Evidence for regulated interleukin-4 expression in chondrocyte-scaffolds under in vitro inflammatory conditions.

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    Muhammad Farooq Rai

    Full Text Available OBJECTIVE: To elucidate the anti-inflammatory and anabolic effects of regulated expression of IL-4 in chondrocyte-scaffolds under in vitro inflammatory conditions. METHODS: Mature articular chondrocytes from dogs (n = 3 were conditioned through transient transfection using pcDNA3.1.cIL-4 (constitutive or pCOX-2.cIL-4 (cytokine-responsive plasmids. Conditioned cells were seeded in alginate microspheres and rat-tail collagen type I matrix (CaReS® to generate two types of tissue-engineered 3-dimensional scaffolds. Inflammatory arthritis was simulated in the packed chondrocytes through exogenous addition of recombinant canine (rc IL-1β (100 ng/ml plus rcTNFα (50 ng/ml in culture media for 96 hours. Harvested cells and culture media were analyzed by various assays to monitor the anti-inflammatory and regenerative (anabolic properties of cIL-4. RESULTS: cIL-4 was expressed from COX-2 promoter exclusively on the addition of rcIL-1β and rcTNFα while its expression from CMV promoter was constitutive. The expressed cIL-4 downregulated the mRNA expression of IL-1β, TNFα, IL-6, iNOS and COX-2 in the cells and inhibited the production of NO and PGE(2 in culture media. At the same time, it up-regulated the expression of IGF-1, IL-1ra, COL2a1 and aggrecan in conditioned chondrocytes in both scaffolds along with a diminished release of total collagen and sGAG into the culture media. An increased amount of cIL-4 protein was detected both in chondrocyte cell lysate and in concentrated culture media. Neutralizing anti-cIL-4 antibody assay confirmed that the anti-inflammatory and regenerative effects seen are exclusively driven by cIL-4. There was a restricted expression of IL-4 under COX-2 promoter possibly due to negative feedback loop while it was over-expressed under CMV promoter (undesirable. Furthermore, the anti-inflammatory /anabolic outcomes from both scaffolds were reproducible and the therapeutic effects of cIL-4 were both scaffold- and

  6. Experimental study of tissue-engineered cartilage allograft with RNAi chondrocytes in vivo

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    Wang ZH

    2014-05-01

    Full Text Available Zhenghui Wang,1 Xiaoli Li,2 Xi-Jing He,3 Xianghong Zhang,1 Zhuangqun Yang,4 Min Xu,1 Baojun Wu,1 Junbo Tu,5 Huanan Luo,1 Jing Yan11Department of Otolaryngology – Head and Neck Surgery, 2Department of Dermatology, 3Department of Orthopedics, The Second Hospital, Xi’an Jiaotong University, 4Department of Plastic and Burns Surgery, The First Hospital, Xi’an Jiaotong University, 5Department of Oral and Maxillofacial Plastic Surgery, The Stomatological Hospital, Xi’an Jiaotong University, Xi’an, People’s Republic of ChinaPurpose: To determine the effects of RNA interference (RNAi on chondrocyte proliferation, function, and immunological rejection after allogenic tissue-engineered cartilage transplantation within bone matrix gelatin scaffolds.Methods: Seven million rat normal and RNAi chondrocytes were harvested and separately composited with fibrin glue to make the cell suspension, and then transplanted subcutaneously into the back of Sprague Dawley rats after being cultured for 10 days in vitro. Untransplanted animals served as the control group. The allograft and immunological response were examined at 1, 2, 4, 8, and 12 months postoperatively with hematoxylin and eosin histochemical staining, immunohistochemical staining (aggrecan, type II collagen, class I and II major histocompatibility complex, and flow cytometry for peripheral blood cluster of differentiation 4+ (CD4+ and CD8+ T-cells.Results: There was no infection or death in the rats except one, which died in the first week. Compared to the control group, the RNAi group had fewer eukomonocytes infiltrated, which were only distributed around the graft. The ratio of CD4+/CD8+ T-cells in the RNAi group was significantly lower than the normal one (P<0.05. There were many more positively stained chondrocytes and positively stained areas around the cells in the RNAi group, which were not found in the control group.Conclusion: The aggrecanase-1 and aggrecanase-2 RNAi for chondrocytes

  7. Stable subcutaneous cartilage regeneration of bone marrow stromal cells directed by chondrocyte sheet.

    Science.gov (United States)

    Li, Dan; Zhu, Lian; Liu, Yu; Yin, Zongqi; Liu, Yi; Liu, Fangjun; He, Aijuan; Feng, Shaoqing; Zhang, Yixin; Zhang, Zhiyong; Zhang, Wenjie; Liu, Wei; Cao, Yilin; Zhou, Guangdong

    2017-05-01

    In vivo niche plays an important role in regulating differentiation fate of stem cells. Due to lack of proper chondrogenic niche, stable cartilage regeneration of bone marrow stromal cells (BMSCs) in subcutaneous environments is always a great challenge. This study explored the feasibility that chondrocyte sheet created chondrogenic niche retained chondrogenic phenotype of BMSC engineered cartilage (BEC) in subcutaneous environments. Porcine BMSCs were seeded into biodegradable scaffolds followed by 4weeks of chondrogenic induction in vitro to form BEC, which were wrapped with chondrocyte sheets (Sheet group), acellular small intestinal submucosa (SIS, SIS group), or nothing (Blank group) respectively and then implanted subcutaneously into nude mice to trace the maintenance of chondrogenic phenotype. The results showed that all the constructs in Sheet group displayed typical cartilaginous features with abundant lacunae and cartilage specific matrices deposition. These samples became more mature with prolonged in vivo implantation, and few signs of ossification were observed at all time points except for one sample that had not been wrapped completely. Cell labeling results in Sheet group further revealed that the implanted BEC directly participated in cartilage formation. Samples in both SIS and Blank groups mainly showed ossified tissue at all time points with partial fibrogenesis in a few samples. These results suggested that chondrocyte sheet could create a chondrogenic niche for retaining chondrogenic phenotype of BEC in subcutaneous environment and thus provide a novel research model for stable ectopic cartilage regeneration based on stem cells. In vivo niche plays an important role in directing differentiation fate of stem cells. Due to lack of proper chondrogenic niche, stable cartilage regeneration of bone marrow stromal cells (BMSCs) in subcutaneous environments is always a great challenge. The current study demonstrated that chondrocyte sheet generated by

  8. Evidence that TNF-β (lymphotoxin α) can activate the inflammatory environment in human chondrocytes

    Science.gov (United States)

    2013-01-01

    Introduction Inflammatory cytokines play a key role in the pathogenesis of joint diseases such as rheumatoid arthritis (RA). Current therapies target mainly tumor necrosis factor α (TNF-α) as this has proven benefits. However, a large number of patients do not respond to or become resistant to anti-TNF-α therapy. While the role of TNF-α in RA is quite evident, the role of TNF-β, also called lymphotoxin-α (LT-α), is unclear. In this study we investigated whether TNF-β and its receptor play a role in chondrocytes in the inflammatory environment. Methods An in vitro model of primary human chondrocytes was used to study TNF-β-mediated inflammatory signaling. Results Cytokine-induced inflammation enhances TNF-β and TNF-β-receptor expression in primary human chondrocytes accompanied by the up-regulation of inflammatory (cyclooxygenase-2), matrix degrading (matrix metalloproteinase-9 and -13) and apoptotic (p53, cleaved caspase-3) signaling pathways, all known to be regulated by NF-κB. In contrast, anti-TNF-β, similar to the natural NF-κB inhibitor (curcumin, diferuloylmethane) or the knockdown of NF-κB by using antisense oligonucleotides (ASO), suppressed IL-1β-induced NF-κB activation and its translocation to the nucleus, and abolished the pro-inflammatory and apoptotic effects of IL-1β. This highlights, at least in part, the crucial role of NF-κB in TNF-β-induced-inflammation in cartilage, similar to that expected for TNF-α. Finally, the adhesiveness between TNF-β-expressing T-lymphocytes and the responding chondrocytes was significantly enhanced through a TNF-β-induced inflammatory microenvironment. Conclusions These results suggest for the first time that TNF-β is involved in microenvironment inflammation in chondrocytes during RA parallel to TNF-α, resulting in the up-regulation of NF-κB signaling and activation of pro-inflammatory activity. PMID:24283517

  9. Vitamin B biosynthesis in plants.

    Science.gov (United States)

    Roje, Sanja

    2007-07-01

    The vitamin B complex comprises water-soluble enzyme cofactors and their derivatives that are essential contributors to diverse metabolic processes in plants as well as in animals and microorganisms. Seven vitamins form this complex: B1 (thiamin (1)), B2 (riboflavin (2)), B3 (niacin (3)), B5 (pantothenic acid (4)), B6 (pyridoxine, pyridoxal (5), and pyridoxamine), B8 (biotin (6)), and B9 (folate (7)). All seven B vitamins are required in the human diet for proper nutrition because humans lack enzymes to synthesize these compounds de novo. This review aims to summarize the present knowledge of vitamin B biosynthesis in plants.

  10. Early resumption of physical activities leads to inferior clinical outcomes after matrix-based autologous chondrocyte implantation in the knee.

    Science.gov (United States)

    Niethammer, Thomas R; Müller, Peter E; Safi, Elem; Ficklscherer, Andreas; Roßbach, Björn P; Jansson, Volkmar; Pietschmann, Matthias F

    2014-06-01

    Matrix-based autologous chondrocyte implantation is a well-established operation procedure for full cartilage defects. When to resume physical activity after matrix-based autologous chondrocyte implantation is controversial. Our hypothesis was that early resumption of physical activity leads to a worse clinical outcome after matrix-based autologous chondrocyte implantation in the knee two years post-operatively. Physical activity is defined as any kind of impact sport. Forty-four patients with cartilage defects of the knee were treated with matrix-based autologous chondrocyte implantation (Novocart3D). All patients were assessed preoperatively and after a period of 24 months with the University of California Los Angeles (UCLA) Activity score. The return to physical activities or sports after matrix-based autologous chondrocyte implantation was documented. Patients were evaluated using the International Knee Documentation Committee Knee Examination Form and visual analogue scale for pain after 6, 12 and 24 months. Fifty-five percent showed an unchanged level of physical activity in the UCLA Activity score post-operatively. About 35% showed a lower level and 10% a higher level of physical activity. The average return to physical activities or sports after matrix-based autologous chondrocyte implantation procedure was 10.2 months. Patients with a later return of sports after 12 months showed significantly better clinical results after two years. In particular, patients who started practicing impact sport after 12 months post-operatively showed significantly better results. Resuming physical activity including impact sports without waiting at least 12 months after the operation leads to inferior outcomes up to 24 months after matrix-based autologous chondrocyte implantation. Level IV.

  11. Similar properties of chondrocytes from osteoarthritis joints and mesenchymal stem cells from healthy donors for tissue engineering of articular cartilage.

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    Amilton M Fernandes

    Full Text Available Lesions of hyaline cartilage do not heal spontaneously, and represent a therapeutic challenge. In vitro engineering of articular cartilage using cells and biomaterials may prove to be the best solution. Patients with osteoarthritis (OA may require tissue engineered cartilage therapy. Chondrocytes obtained from OA joints are thought to be involved in the disease process, and thus to be of insufficient quality to be used for repair strategies. Bone marrow (BM derived mesenchymal stem cells (MSCs from healthy donors may represent an alternative cell source. We have isolated chondrocytes from OA joints, performed cell culture expansion and tissue engineering of cartilage using a disc-shaped alginate scaffold and chondrogenic differentiation medium. We performed real-time reverse transcriptase quantitative PCR and fluorescence immunohistochemistry to evaluate mRNA and protein expression for a range of molecules involved in chondrogenesis and OA pathogenesis. Results were compared with those obtained by using BM-MSCs in an identical tissue engineering strategy. Finally the two populations were compared using genome-wide mRNA arrays. At three weeks of chondrogenic differentiation we found high and similar levels of hyaline cartilage-specific type II collagen and fibrocartilage-specific type I collagen mRNA and protein in discs containing OA and BM-MSC derived chondrocytes. Aggrecan, the dominant proteoglycan in hyaline cartilage, was more abundantly distributed in the OA chondrocyte extracellular matrix. OA chondrocytes expressed higher mRNA levels also of other hyaline extracellular matrix components. Surprisingly BM-MSC derived chondrocytes expressed higher mRNA levels of OA markers such as COL10A1, SSP1 (osteopontin, ALPL, BMP2, VEGFA, PTGES, IHH, and WNT genes, but lower levels of MMP3 and S100A4. Based on the results presented here, OA chondrocytes may be suitable for tissue engineering of articular cartilage.

  12. Similar properties of chondrocytes from osteoarthritis joints and mesenchymal stem cells from healthy donors for tissue engineering of articular cartilage.

    Science.gov (United States)

    Fernandes, Amilton M; Herlofsen, Sarah R; Karlsen, Tommy A; Küchler, Axel M; Fløisand, Yngvar; Brinchmann, Jan E

    2013-01-01

    Lesions of hyaline cartilage do not heal spontaneously, and represent a therapeutic challenge. In vitro engineering of articular cartilage using cells and biomaterials may prove to be the best solution. Patients with osteoarthritis (OA) may require tissue engineered cartilage therapy. Chondrocytes obtained from OA joints are thought to be involved in the disease process, and thus to be of insufficient quality to be used for repair strategies. Bone marrow (BM) derived mesenchymal stem cells (MSCs) from healthy donors may represent an alternative cell source. We have isolated chondrocytes from OA joints, performed cell culture expansion and tissue engineering of cartilage using a disc-shaped alginate scaffold and chondrogenic differentiation medium. We performed real-time reverse transcriptase quantitative PCR and fluorescence immunohistochemistry to evaluate mRNA and protein expression for a range of molecules involved in chondrogenesis and OA pathogenesis. Results were compared with those obtained by using BM-MSCs in an identical tissue engineering strategy. Finally the two populations were compared using genome-wide mRNA arrays. At three weeks of chondrogenic differentiation we found high and similar levels of hyaline cartilage-specific type II collagen and fibrocartilage-specific type I collagen mRNA and protein in discs containing OA and BM-MSC derived chondrocytes. Aggrecan, the dominant proteoglycan in hyaline cartilage, was more abundantly distributed in the OA chondrocyte extracellular matrix. OA chondrocytes expressed higher mRNA levels also of other hyaline extracellular matrix components. Surprisingly BM-MSC derived chondrocytes expressed higher mRNA levels of OA markers such as COL10A1, SSP1 (osteopontin), ALPL, BMP2, VEGFA, PTGES, IHH, and WNT genes, but lower levels of MMP3 and S100A4. Based on the results presented here, OA chondrocytes may be suitable for tissue engineering of articular cartilage.

  13. Nicotine acts on growth plate chondrocytes to delay skeletal growth through the alpha7 neuronal nicotinic acetylcholine receptor.

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    Atsuo Kawakita

    Full Text Available BACKGROUND: Cigarette smoking adversely affects endochondral ossification during the course of skeletal growth. Among a plethora of cigarette chemicals, nicotine is one of the primary candidate compounds responsible for the cause of smoking-induced delayed skeletal growth. However, the possible mechanism of delayed skeletal growth caused by nicotine remains unclarified. In the last decade, localization of neuronal nicotinic acetylcholine receptor (nAChR, a specific receptor of nicotine, has been widely detected in non-excitable cells. Therefore, we hypothesized that nicotine affect growth plate chondrocytes directly and specifically through nAChR to delay skeletal growth. METHODOLOGY/PRINCIPAL FINDINGS: We investigated the effect of nicotine on human growth plate chondrocytes, a major component of endochondral ossification. The chondrocytes were derived from extra human fingers. Nicotine inhibited matrix synthesis and hypertrophic differentiation in human growth plate chondrocytes in suspension culture in a concentration-dependent manner. Both human and murine growth plate chondrocytes expressed alpha7 nAChR, which constitutes functional homopentameric receptors. Methyllycaconitine (MLA, a specific antagonist of alpha7 nAChR, reversed the inhibition of matrix synthesis and functional calcium signal by nicotine in human growth plate chondrocytes in vitro. To study the effect of nicotine on growth plate in vivo, ovulation-controlled pregnant alpha7 nAChR +/- mice were given drinking water with or without nicotine during pregnancy, and skeletal growth of their fetuses was observed. Maternal nicotine exposure resulted in delayed skeletal growth of alpha7 nAChR +/+ fetuses but not in alpha7 nAChR -/- fetuses, implying that skeletal growth retardation by nicotine is specifically mediated via fetal alpha7 nAChR. CONCLUSIONS/SIGNIFICANCE: These results suggest that nicotine, from cigarette smoking, acts directly on growth plate chondrocytes to decrease

  14. Electrospun gelatin/polycaprolactone nanofibrous membranes combined with a coculture of bone marrow stromal cells and chondrocytes for cartilage engineering

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    He X

    2015-03-01

    Full Text Available Xiaomin He,1,* Bei Feng,1,2,* Chuanpei Huang,1 Hao Wang,1 Yang Ge,1 Renjie Hu,1 Meng Yin,1 Zhiwei Xu,1 Wei Wang,1 Wei Fu,1,2 Jinghao Zheng1 1Department of Pediatric Cardiothoracic Surgery, 2Institute of Pediatric Translational Medicine, Shanghai Children’s Medical Center School of Medicine, Shanghai Jiao Tong University, Shanghai, People’s Republic of China *These authors contributed equally to this work Abstract: Electrospinning has recently received considerable attention, showing notable potential as a novel method of scaffold fabrication for cartilage engineering. The aim of this study was to use a coculture strategy of chondrocytes combined with electrospun gelatin/polycaprolactone (GT/PCL membranes, instead of pure chondrocytes, to evaluate the formation of cartilaginous tissue. We prepared the GT/PCL membranes, seeded bone marrow stromal cell (BMSC/chondrocyte cocultures (75% BMSCs and 25% chondrocytes in a sandwich model in vitro, and then implanted the constructs subcutaneously into nude mice for 12 weeks. Gross observation, histological and immunohistological evaluation, glycosaminoglycan analyses, Young’s modulus measurement, and immunofluorescence staining were performed postimplantation. We found that the coculture group formed mature cartilage-like tissue, with no statistically significant difference from the chondrocyte group, and labeled BMSCs could differentiate into chondrocyte-like cells under the chondrogenic niche of chondrocytes. This entire strategy indicates that GT/PCL membranes are also a suitable scaffold for stem cell-based cartilage engineering and may provide a potentially clinically feasible approach for cartilage repairs. Keywords: electrospinning, nanocomposite, cartilage tissue engineering, nanomaterials, stem cells

  15. Defective postnatal endochondral bone development by chondrocyte-specific targeted expression of parathyroid hormone type 2 receptor.

    Science.gov (United States)

    Panda, Dibyendu Kumar; Goltzman, David; Karaplis, Andrew C

    2012-12-15

    The human parathyroid hormone type 2 receptor (PTH2R) is activated by PTH and by tuberoinfundibular peptide of 39 residues (TIP39), the latter likely acting as its natural ligand. Although the receptor is expressed at highest levels in the nervous system, we have observed that both PTH2R and TIP39 are expressed in the newborn mouse growth plate, with the receptor localizing in the resting zone and the ligand TIP39 localizing exclusively in prehypertrophic and hypertrophic chondrocytes. To address the role of PTH2R in postnatal skeletal growth and development, Col2a1-hPTH2R (PTH2R-Tg) transgenic mice were generated. The mice were viable and of nearly normal size at birth. Expression of the transgene in the growth plate was limited to chondrocytes. We found that chondrocyte proliferation was decreased, as determined by in vivo BrdU labeling of proliferating chondrocytes and CDK4 and p21 expression in the growth plate of Col2a1-hPTH2R transgenic mice. Similarly, the differentiation and maturation of chondrocytes was delayed, as characterized by decreased Sox9 expression and weaker immunostaining for the chondrocyte differentiation markers collagen type II and type X and proteoglycans. As well, there was altered expression of Gdf5, Wdr5, and β-catenin, factors implicated in chondrocyte maturation, proliferation, and differentiation.These effects impacted on the process of endochondral ossification, resulting in delayed formation of the secondary ossification center, and diminished trabecular bone volume. The findings substantiate a role for PTH2R signaling in postnatal growth plate development and subsequent bone mass acquisition.

  16. Co-Compartmentation of Terpene Biosynthesis and Storage via Synthetic Droplet

    Energy Technology Data Exchange (ETDEWEB)

    Zhao, Cheng [Texas; amp,M Agrilife Synthetic and Systems Biology Innovation Hub, Texas A& amp,M University, College Station, Texas 77843, United States; Department; amp,M University, College Station, Texas 77843, United States; Institute; amp,M University, College Station, Texas 77843, United States; Kim, YongKyoung [Texas; amp,M Agrilife Synthetic and Systems Biology Innovation Hub, Texas A& amp,M University, College Station, Texas 77843, United States; Department; amp,M University, College Station, Texas 77843, United States; Institute; amp,M University, College Station, Texas 77843, United States; Zeng, Yining [Biosciences; Li, Man [Texas; amp,M Agrilife Synthetic and Systems Biology Innovation Hub, Texas A& amp,M University, College Station, Texas 77843, United States; Department; amp,M University, College Station, Texas 77843, United States; Institute; amp,M University, College Station, Texas 77843, United States; Wang, Xin [Texas; amp,M Agrilife Synthetic and Systems Biology Innovation Hub, Texas A& amp,M University, College Station, Texas 77843, United States; Department; amp,M University, College Station, Texas 77843, United States; Institute; amp,M University, College Station, Texas 77843, United States; Hu, Cheng [Texas; amp,M Agrilife Synthetic and Systems Biology Innovation Hub, Texas A& amp,M University, College Station, Texas 77843, United States; Department; amp,M University, College Station, Texas 77843, United States; Institute; amp,M University, College Station, Texas 77843, United States; Gorman, Connor [Texas; amp,M Agrilife Synthetic and Systems Biology Innovation Hub, Texas A& amp,M University, College Station, Texas 77843, United States; Department; amp,M University, College Station, Texas 77843, United States; Institute; amp,M University, College Station, Texas 77843, United States; Dai, Susie Y. [State; Ding, Shi-You [Department; Yuan, Joshua S. [Texas; amp,M Agrilife Synthetic and Systems Biology Innovation Hub, Texas A& amp,M University, College Station, Texas 77843, United States; Department; amp,M University, College Station, Texas 77843, United States; Institute; amp,M University, College Station, Texas 77843, United States

    2018-02-16

    Traditional bioproduct engineering focuses on pathway optimization, yet is often complicated by product inhibition, downstream consumption, and the toxicity of certain products. Here, we present the co-compartmentation of biosynthesis and storage via a synthetic droplet as an effective new strategy to improve the bioproduct yield, with squalene as a model compound. A hydrophobic protein was designed and introduced into the tobacco chloroplast to generate a synthetic droplet for terpene storage. Simultaneously, squalene biosynthesis enzymes were introduced to chloroplasts together with the droplet-forming protein to co-compartmentalize the biosynthesis and storage of squalene. The strategy has enabled a record yield of squalene at 2.6 mg/g fresh weight without compromising plant growth. Confocal fluorescent microscopy imaging, stimulated Raman scattering microscopy, and droplet composition analysis confirmed the formation of synthetic storage droplet in chloroplast. The co-compartmentation of synthetic storage droplet with a targeted metabolic pathway engineering represents a new strategy for enhancing bioproduct yield.

  17. Biosynthesis of bacterial aromatic polyketides.

    Science.gov (United States)

    Zhan, Jixun

    2009-01-01

    Aromatic polyketides represent important members of the family of polyketides, which have displayed a wide assortment of bioactive properties, such as antibacterial, antitumor, and antiviral activities. Bacterial aromatic polyketides are mainly synthesized by type II polyketide synthases (PKSs). Whereas malonyl-CoA is exclusively used as the extender unit, starter units can vary in different aromatic polyketide biosynthetic pathways, leading to a variety of polyketide backbones. Once the polyketide chains are elongated by the minimal PKSs to the full length, the immediate tailoring enzymes including ketoreductases, oxygenases and cyclases will work on the nascent chains to form aromatic structures, which will be further decorated by those late tailoring enzymes such as methyltransferases and glycosyltransferases. The mechanistic studies on the biosynthetic pathways of aromatic polyketides such as oxytetracycline and pradimicin A have been extensively carried out in recent years. Engineered biosynthesis of novel "unnatural" polyketides has been achieved in heterologous hosts such as Streptomyces coelicolor and Escherichia coli. This review covers the most recent advances in aromatic polyketide biosynthesis, which provide new enzymes or methods for building novel polyketide biosynthetic machinery.

  18. Fatty acid biosynthesis in actinomycetes

    Science.gov (United States)

    Gago, Gabriela; Diacovich, Lautaro; Arabolaza, Ana; Tsai, Shiou-Chuan; Gramajo, Hugo

    2011-01-01

    All organisms that produce fatty acids do so via a repeated cycle of reactions. In mammals and other animals, these reactions are catalyzed by a type I fatty acid synthase (FAS), a large multifunctional protein to which the growing chain is covalently attached. In contrast, most bacteria (and plants) contain a type II system in which each reaction is catalyzed by a discrete protein. The pathway of fatty acid biosynthesis in Escherichia coli is well established and has provided a foundation for elucidating the type II FAS pathways in other bacteria (White et al., 2005). However, fatty acid biosynthesis is more diverse in the phylum Actinobacteria: Mycobacterium, possess both FAS systems while Streptomyces species have only the multi-enzyme FAS II system and Corynebacterium species exclusively FAS I. In this review we present an overview of the genome organization, biochemical properties and physiological relevance of the two FAS systems in the three genera of actinomycetes mentioned above. We also address in detail the biochemical and structural properties of the acyl-CoA carboxylases (ACCases) that catalyzes the first committed step of fatty acid synthesis in actinomycetes, and discuss the molecular bases of their substrate specificity and the structure-based identification of new ACCase inhibitors with anti-mycobacterial properties. PMID:21204864

  19. Longitudinal bone growth is impaired by direct involvement of caffeine with chondrocyte differentiation in the growth plate.

    Science.gov (United States)

    Choi, Hyeonhae; Choi, Yuri; Kim, Jisook; Bae, Jaeman; Roh, Jaesook

    2017-01-01

    We showed previously that caffeine adversely affects longitudinal bone growth and disrupts the histomorphometry of the growth plate during the pubertal growth spurt. However, little attention has been paid to the direct effects of caffeine on chondrocytes. Here, we investigated the direct effects of caffeine on chondrocytes of the growth plate in vivo and in vitro using a rapidly growing young rat model, and determined whether they were related to the adenosine receptor signaling pathway. A total of 15 male rats (21 days old) were divided randomly into three groups: a control group and two groups fed caffeine via gavage with 120 and 180 mg kg -1  day -1 for 4 weeks. After sacrifice, the tibia processed for the analysis of the long bone growth and proliferation of chondrocytes using tetracycline and BrdU incorporation, respectively. Caffeine-fed animals showed decreases in matrix mineralization and proliferation rate of growth plate chondrocytes compared with the control. To evaluate whether caffeine directly affects chondrocyte proliferation and chondrogenic differentiation, primary rat chondrocytes were isolated from the growth plates and cultured in either the presence or absence of caffeine at concentrations of 0.1-1 mm, followed by determination of the cellular proliferation or expression profiles of cellular differentiation markers. Caffeine caused significant decreases in extracellular matrix production, mineralization, and alkaline phosphatase activity, accompanied with decreases in gene expression of the cartilage-specific matrix proteins such as aggrecan, type II collagen and type X. Our results clearly demonstrate that caffeine is capable of interfering with cartilage induction by directly inhibiting the synthetic activity and orderly expression of marker genes relevant to chondrocyte maturation. In addition, we found that the adenosine type 1 receptor signaling pathway may be partly involved in the detrimental effects of caffeine on chondrogenic

  20. Hydroxylation of methylated DNA by TET1 in chondrocyte differentiation of C3H10T1/2 cells

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    Ryo Ito

    2016-03-01

    Full Text Available DNA methylation is closely involved in the regulation of cellular differentiation, including chondrogenic differentiation of mesenchymal stem cells. Recent studies showed that Ten–eleven translocation (TET family proteins converted 5-methylcytosine (5mC to 5-hydroxymethylcytosine, 5-formylcytosine and 5carboxylcytosine by oxidation. These reactions constitute potential mechanisms for active demethylation of methylated DNA. However, the relationship between the DNA methylation patterns and the effects of TET family proteins in chondrocyte differentiation is still unclear. In this study, we showed that DNA hydroxylation of 5mC was increased during chondrocytic differentiation of C3H10T1/2 cells and that the expression of Tet1 was particularly enhanced. Moreover, knockdown experiments revealed that the downregulation of Tet1 expression caused decreases in chondrogenesis markers such as type 2 and type 10 collagens. Furthermore, we found that TET proteins had a site preference for hydroxylation of 5mC on the Insulin-like growth factor 1 (Igf1 promoter in chondrocytes. Taken together, we showed that the expression of Tet1 was specifically facilitated in chondrocyte differentiation and Tet1 can regulate chondrocyte marker gene expression presumably through its hydroxylation activity for DNA.

  1. Suture compression induced midpalatal suture chondrocyte apoptosis with increased caspase-3, caspase-9, Bad, Bak, Bax and Bid expression.

    Science.gov (United States)

    Lan, Tingting; Zhao, Hanchi; Xiang, Bilu; Wang, Jun; Liu, Yang

    2017-07-22

    Previous studies found bone resorption and chondrocytes loss in mouse models of mid-palatal suture when given continuous compressive force, although chondrocytes response remained unknown. Herein, we design this study to determine how continuous compression force induces chondrocytes apoptosis. Thirty C57BL/6 male mice (aged 6 weeks) were randomly assigned into controls (not ligated to a spring), blank controls (ligated with no compression) and the compression group (ligated with 20-g compression). After 4 d, palatal tissues were sampled and stained by TB and safranin-O. Tunel staining measured the percentage of apoptotic chondrocytes, and immunohistochemistry was performed to label apoptosis-associated proteins (e.g., Bcl-2, Bcl-xl, Bax, Bak, Bid, Bad, caspase-3, caspase-8 and caspase-9). Intergroup comparison was made by the rank sum test, and P compression group was significantly decreased, while the control group remained largely unaltered. Tunel staining showed that apoptotic cell numbers in the mid-palatal suture were significantly higher than the control group. Immunohistochemistry showed that mice in the compression group had significantly increased expression of caspase-3, caspase-9, Bad, Bak, Bax and Bid; However, caspase-8 remained unaltered. No expression of Bcl-2 and Bcl-xl was detected. Continuous compression force induces chondrocytes apoptosis in the mid-palatal suture. This process might be associated with the mitochondrial pathway. Copyright © 2017 Elsevier Inc. All rights reserved.

  2. Engineered cartilage regeneration from adipose tissue derived-mesenchymal stem cells: A morphomolecular study on osteoblast, chondrocyte and apoptosis evaluation.

    Science.gov (United States)

    Szychlinska, Marta Anna; Castrogiovanni, Paola; Nsir, Houda; Di Rosa, Michelino; Guglielmino, Claudia; Parenti, Rosalba; Calabrese, Giovanna; Pricoco, Elisabetta; Salvatorelli, Lucia; Magro, Gaetano; Imbesi, Rosa; Mobasheri, Ali; Musumeci, Giuseppe

    2017-08-15

    The poor self-repair capacity of cartilage tissue in degenerative conditions, such as osteoarthritis (OA), has prompted the development of a variety of therapeutic approaches, such as cellular therapies and tissue engineering based on the use of mesenchymal stem cells (MSCs). The aim of this study is to demonstrate, for the first time, that the chondrocytes differentiated from rat adipose tissue derived-MSCs (AMSCs), are able to constitute a morphologically and biochemically healthy hyaline cartilage after 6 weeks of culture on a Collagen Cell Carrier (CCC) scaffold. In this study we evaluated the expression of some osteoblasts (Runt-related transcription factor 2 (RUNX2) and osteocalcin), chondrocytes (collagen I, II and lubricin) and apoptosis (caspase-3) biomarkers in undifferentiated AMSCs, differentiated AMSCs in chondrocytes cultured in monolayer and AMSCs-derived chondrocytes seeded on CCC scaffolds, by different techniques such as immunohistochemistry, ELISA, Western blot and gene expression analyses. Our results showed the increased expression of collagen II and lubricin in AMSCs-derived chondrocytes cultured on CCC scaffolds, whereas the expression of collagen I, RUNX2, osteocalcin and caspase-3 resulted decreased, when compared to the controls. In conclusion, this innovative basic study could be a possible key for future therapeutic strategies for articular cartilage restoration through the use of CCC scaffolds, to reduce the morbidity from acute cartilage injuries and degenerative joint diseases. Copyright © 2017 Elsevier Inc. All rights reserved.

  3. Expression Profiling and Functional Implications of a Set of Zinc Finger Proteins, ZNF423, ZNF470, ZNF521, and ZNF780B, in Primary Osteoarthritic Articular Chondrocytes

    Directory of Open Access Journals (Sweden)

    Maria Mesuraca

    2014-01-01

    Full Text Available Articular chondrocytes are responsible for the maintenance of healthy articulations; indeed, dysregulation of their functions, including the production of matrix proteins and matrix-remodeling proteases, may result in fraying of the tissue and development of osteoarthritis (OA. To explore transcriptional mechanisms that contribute to the regulation of chondrocyte homeostasis and may be implicated in OA development, we compared the gene expression profile of a set of zinc finger proteins potentially linked to the control of chondrocyte differentiation and/or functions (ZNF423, ZNF470, ZNF521, and ZNF780B in chondrocytes from patients affected by OA and from subjects not affected by OA. This analysis highlighted a significantly lower expression of the transcript encoding ZNF423 in chondrocytes from OA, particularly in elderly patients. Interestingly, this decrease was mirrored by the similarly reduced expression of PPARγ, a known target of ZNF423 with anti-inflammatory and chondroprotective properties. The ZNF521 mRNA instead was abundant in all primary chondrocytes studied; the RNAi-mediated silencing of this gene significantly altered the COL2A/COL1 expression ratio, associated with the maintenance of the differentiated phenotype, in chondrocytes cultivated in alginate beads. These results suggest a role for ZNF423 and ZNF521 in the regulation of chondrocyte homeostasis and warrant further investigations to elucidate their mechanism of action.

  4. Regulatory variability of camalexin biosynthesis.

    Science.gov (United States)

    Schuhegger, Regina; Rauhut, Thomas; Glawischnig, Erich

    2007-05-01

    The anthranilate synthase ASA1, CYP79B2 and CYP71B15 (PAD3) are biosynthetic genes of the Arabidopsis phytoalexin camalexin, which are induced after pathogen infection and abiotic treatments like silver nitrate spraying. The natural variation of camalexin biosynthesis in response to Pseudomonas syringae infection was determined in several ecotypes, and differential CYP71B15 regulation as a potential basis for this variation was investigated. The expression of camalexin biosynthetic genes was restricted to the tissue undergoing cell death. After droplet infection with Alternaria alternata, a potent camalexin inducer in the Col-0 ecotype, camalexin formation and the induction of ASA1, CYP79B2 and CYP71B15 were strictly co-localized with the infection site.

  5. ATX-LPA1 axis contributes to proliferation of chondrocytes by regulating fibronectin assembly leading to proper cartilage formation.

    Science.gov (United States)

    Nishioka, Tatsuji; Arima, Naoaki; Kano, Kuniyuki; Hama, Kotaro; Itai, Eriko; Yukiura, Hiroshi; Kise, Ryoji; Inoue, Asuka; Kim, Seok-Hyung; Solnica-Krezel, Lilianna; Moolenaar, Wouter H; Chun, Jerold; Aoki, Junken

    2016-03-23

    The lipid mediator lysophosphatidic acid (LPA) signals via six distinct G protein-coupled receptors to mediate both unique and overlapping biological effects, including cell migration, proliferation and survival. LPA is produced extracellularly by autotaxin (ATX), a secreted lysophospholipase D, from lysophosphatidylcholine. ATX-LPA receptor signaling is essential for normal development and implicated in various (patho)physiological processes, but underlying mechanisms remain incompletely understood. Through gene targeting approaches in zebrafish and mice, we show here that loss of ATX-LPA1 signaling leads to disorganization of chondrocytes, causing severe defects in cartilage formation. Mechanistically, ATX-LPA1 signaling acts by promoting S-phase entry and cell proliferation of chondrocytes both in vitro and in vivo, at least in part through β1-integrin translocation leading to fibronectin assembly and further extracellular matrix deposition; this in turn promotes chondrocyte-matrix adhesion and cell proliferation. Thus, the ATX-LPA1 axis is a key regulator of cartilage formation.

  6. Relationship of chondrocyte apoptosis to matrix degradation and swelling potential of osteoarthritic cartilage.

    Science.gov (United States)

    Chen, Min-Huey; Wang, Jue-Long; Wong, Chi-Yin; Yao, Chung-Chen; Chen, Yi-Jane; Jiang, Ching-Chuan

    2005-04-01

    Softening of cartilage is the initial degenerative step of osteoarthritic cartilage by matrix degradation and corruption of interconnection of the collagen fibrillar network. The purpose of this study was to investigate the correlation of chondrocyte apoptosis, matrix degradation, and the corruption of collagen architecture in the development of severe swelling of osteoarthritic cartilage. Twenty osteoarthritic and 7 normal femoral neck fractured cartilage samples were obtained from patients with knee osteoarthritis and normal patients with femoral neck fracture at the time of total hip joint replacement surgery. Apoptosis was verified by TUNEL (terminal deoxynucleotidyl transferase-mediated deoxyuridine 5-triphosphate nick end-labeling) staining and structural changes were observed under phase-contrast microscopy. Matrix degradation was evaluated by histochemical analysis of proteoglycans. Swelling tests were performed by immersing the cartilage slices in hypotonic solution. The results of ultrastructural study of collagen architecture of osteoarthritic cartilage performed by scanning electron microscopy before and after swelling were compared. Matrix degradation was most prominent in the middle zone of osteoarthritic cartilage. The percentage of chondrocytes in osteoarthritic cartilage showing apoptosis ranged from 15 to 20% (average, 18%; standard deviation (SD) = 3.2%) and was correlated with the extent of structural changes and matrix degradation. The swelling strain of the osteoarthritic cartilage varied from 120 to 200% (average, 160%; SD = 40%) depending on the degree of matrix degradation and structural changes. The loss of interconnectivity of collagen fibrillar architecture was correlated with the increased swelling potential of osteoarthritic cartilage. This study demonstrated that chondrocyte apoptosis was correlated with matrix degradation and the corruption of fibrillar architecture and that the extent of these manifestations correlated with the

  7. Revision surgery after third generation autologous chondrocyte implantation in the knee.

    Science.gov (United States)

    Niethammer, Thomas R; Niethammer, Thomas; Valentin, Siegfried; Ficklscherer, Andreas; Gülecyüz, Mehmet F; Gülecyüz, Mehmet; Pietschmann, Matthias F; Pietschmann, Matthias; Müller, Peter E; Müller, Peter

    2015-08-01

    Third generation autologous chondrocyte implantation (ACI) is an established treatment for full thickness cartilage defects in the knee joint. However, little is known about cases when revision surgery is needed. The aim of the present study is to investigate the complication rates and the main reasons for revision surgery after third generation autologous chondrocyte implantation in the knee joint. It is of particular interest to examine in which cases revision surgery is needed and in which cases a "wait and see" strategy should be used. A total of 143 consecutive patients with 171 cartilage defects were included in this study with a minimum follow-up of two years. All defects were treated with third generation ACI (NOVACART®3D). Clinical evaluation was carried out after six months, followed by an annual evaluation using the International Knee Documentation Committee (IKDC) subjective score and the visual analogue scale (VAS) for rest and during activity. Revision surgery was documented. The revision rate was 23.4 % (n = 36). The following major reasons for revision surgery were found in our study: symptomatic bone marrow edema (8.3 %, n = 3), arthrofibrosis (22.2 %, n = 8) and partial graft cartilage deficiency (47.2 %, n = 17). The following revision surgery was performed: retrograde drilling combined with Iloprost infusion therapy for bone marrow oedema (8.4 %, n = 3), arthroscopic arthrolysis of the suprapatellar recess (22.2 %, n = 8) and microfracturing/antegrade drilling (47.3 %, n = 17). Significant improvements of clinical scores after revision surgery were observed. Revision surgery after third generation autologous chondrocyte implantation is common and is needed primarily in cases with arthrofibrosis, partial graft cartilage deficiency and symptomatic bone marrow oedema resulting in a significantly better clinical outcome.

  8. Adipose stem cells differentiated chondrocytes regenerate damaged cartilage in rat model of osteoarthritis.

    Science.gov (United States)

    Latief, Noreen; Raza, Fahad Ali; Bhatti, Fazal-Ur-Rehman; Tarar, Moazzam Nazir; Khan, Shaheen N; Riazuddin, Sheikh

    2016-05-01

    Transplantation of mesenchymal stem cells (MSCs) or autologous chondrocytes has been shown to repair damages to articular cartilage due to osteoarthritis (OA). However, survival of transplanted cells is considerably reduced in the osteoarthritic environment and it affects successful outcome of the transplantation of the cells. Differentiated chrondroytes derived from adipose stem cells have been proposed as an alternative source and our study investigated this possibility in rats. We investigated the regenerative potential of ADSCs and DCs in osteoarthritic environment in the repair of cartilage in rats. We found that ADSCs maintained fibroblast morphology in vitro and also expressed CD90 and CD29. Furthermore, ADSCs differentiated into chondrocytes, accompanied by increased level of proteoglycans and expression of chondrocytes specific genes, such as, Acan, and Col2a1. Histological examination of transplanted knee joints showed regeneration of cartilage tissue compared to control OA knee joints. Increase in gene expression for Acan, Col2a1 with concomitant decrease in the expression of Col1a1 suggested formation of hyaline like cartilage. A significant increase in differentiation index was observed in DCs and ADSCs transplanted knee joints (P = 0.0110 vs. P = 0.0429) when compared to that in OA control knee joints. Furthermore, transplanted DCs showed increased proliferation along with reduction in apoptosis as compared to untreated control. In conclusion, DCs showed better survival and regeneration potential as compared with ADSCs in rat model of OA and thus may serve a better option for regeneration of osteoarthritic cartilage. © 2016 International Federation for Cell Biology.

  9. Characteristic complications after autologous chondrocyte implantation for cartilage defects of the knee joint

    DEFF Research Database (Denmark)

    Niemeyer, Philipp; Pestka, Jan M; Kreuz, Peter C

    2008-01-01

    BACKGROUND: Although autologous chondrocyte implantation (ACI) is a well-established therapy for the treatment of isolated cartilage defects of the knee joint, little is known about typical complications and their treatment after ACI. HYPOTHESIS: Unsatisfactory outcome after ACI is associated...... with technique-related typical complications. STUDY DESIGN: Case series; Level of evidence, 4. METHODS: A total of 309 consecutive patients with 349 ACI procedures of the knee joint were analyzed. Three different ACI techniques were used: periosteum-covered ACI in 52 cases (14.9%), Chondrogide (Geistlich...

  10. Non-woven PGA/PVA Fibrous Mesh as an Appropriate Scaffold for Chondrocyte Proliferation

    Czech Academy of Sciences Publication Activity Database

    Rampichová, Michala; Košťáková, E.; Filová, Eva; Prosecká, Eva; Plencner, Martin; Ocheretná, L.; Lytvynets, Andriy; Lukáš, D.; Amler, Evžen

    2010-01-01

    Roč. 59, č. 5 (2010), s. 773-781 ISSN 0862-8408 R&D Projects: GA AV ČR IAA500390702; GA ČR GAP304/10/1307 Grant - others:GA UK(CZ) 119209; EU(XE) BIOSCENT ID 214539; GA MŠk(CZ) 1M0510; GA ČR(CZ) GA202/09/1151; GA MŠk(CZ) 2B06130 Program:1M; GA; 2B Institutional research plan: CEZ:AV0Z50390703; CEZ:AV0Z50390512 Keywords : PGA * PVA * chondrocyte Subject RIV: BO - Biophysics Impact factor: 1.646, year: 2010

  11. Optimized alkylated cyclodextrin polysulphates with reduced risks on thromboembolic accidents improve osteoarthritic chondrocyte metabolism.

    Science.gov (United States)

    Groeneboer, Sara; Lambrecht, Stijn; Dhollander, Aad; Jacques, Peggy; Vander Cruyssen, Bert; Lories, Rik J; Devreese, Katrien; Chiers, Koen; Elewaut, Dirk; Verbruggen, Gust

    2011-07-01

    To compare the ability of different cyclodextrin polysulphate (CDPS) derivatives to affect human articular cartilage cell metabolism in vitro. OA chondrocytes were cultured in alginate and exposed to 5 µg/ml of 2,3,6-tri-O-methyl-β-cyclodextrin (ME-CD), 2,3-di-O-methyl-6-sulphate-β-cyclodextrin (ME-CD-6-S), 2,6-di-O-methyl-3-sulphate-β-cyclodextrin (ME-CD-3-S), (2-carboxyethyl)-β-CDPS (CE-CDPS), (2-hydroxypropyl)-β-CDPS (HP-CDPS), 6-monoamino-6-monodeoxy-β-CDPS (MA-CDPS) or β-CDPS for 5 days. Effects on IL-1-driven chondrocyte extracellular matrix (ECM) metabolism were assayed by analysis of the accumulation of aggrecan in the interterritorial matrix, IL-6 secretion and qPCR. MA-CDPS, HP-CDPS, CE-CDPS and CDPS were analysed for their in vitro effect on coagulation and their ability to activate platelets in an in vitro assay to detect possible cross-reactivity with heparin-induced thrombocytopenia (HIT) antibodies. The monosulphated cyclodextrins ME-CD-6-S and -3-S failed to affect aggrecan synthesis and IL-6 secretion by the OA chondrocytes. Polysulphated cyclodextrins MA-CDPS, HP-CDPS, CE-CDPS and CDPS at 5 µg/ml concentrations, on the other hand, significantly induced aggrecan production and repressed IL-6 release by the chondrocytes in culture. aPTT and PT for all derivatives were lengthened for polysaccharide concentrations >50 µg/ml. Five micrograms per millilitre of β-CDPS concentrations that significantly modulated ECM ground substance production in vitro did not affect aPTT or PT. Furthermore, CE-CDPS, in contrast to MA-CDPS, HP-CDPS and CDPS, did not significantly activate platelets, suggesting a minimal potential to induce HIT thromboembolic accidents in vivo. CE-CDPS is a new, structurally adjusted, sulphated β-cyclodextrin derivative with preserved chondroprotective capacity and a promising safety profile.

  12. Expression profiling of Dexamethasone-treated primary chondrocytes identifies targets of glucocorticoid signalling in endochondral bone development

    Directory of Open Access Journals (Sweden)

    Beier Frank

    2007-07-01

    Full Text Available Abstract Background Glucocorticoids (GCs are widely used anti-inflammatory drugs. While useful in clinical practice, patients taking GCs often suffer from skeletal side effects including growth retardation in children and adolescents, and decreased bone quality in adults. On a physiological level, GCs have been implicated in the regulation of chondrogenesis and osteoblast differentiation, as well as maintaining homeostasis in cartilage and bone. We identified the glucocorticoid receptor (GR as a potential regulator of chondrocyte hypertrophy in a microarray screen of primary limb bud mesenchyme micromass cultures. Some targets of GC regulation in chondrogenesis are known, but the global effects of pharmacological GC doses on chondrocyte gene expression have not been comprehensively evaluated. Results This study systematically identifies a spectrum of GC target genes in embryonic growth plate chondrocytes treated with a synthetic GR agonist, dexamethasone (DEX, at 6 and 24 hrs. Conventional analysis of this data set and gene set enrichment analysis (GSEA was performed. Transcripts associated with metabolism were enriched in the DEX condition along with extracellular matrix genes. In contrast, a subset of growth factors and cytokines were negatively correlated with DEX treatment. Comparing DEX-induced gene expression data to developmental changes in gene expression in micromass cultures revealed an additional layer of complexity in which DEX maintains the expression of certain chondrocyte marker genes while inhibiting factors that promote vascularization and ultimately ossification of the cartilaginous template. Conclusion Together, these results provide insight into the mechanisms and major molecular classes functioning downstream of DEX in primary chondrocytes. In addition, comparison of our data with microarray studies of DEX treatment in other cell types demonstrated that the majority of DEX effects are tissue-specific. This study provides

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

    International Nuclear Information System (INIS)

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

    2013-01-01

    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 O 2 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. Highlights: ► Chondrocytes were cultivated on Superhydrophilic Vertically Aligned Multiwall Carbon Nanotubes (VACNT). ► We have shown a correlation between gene expression and thermodynamics aspects. ► Superhydrhophilic VACNT will be an excellent substrate for cartilage and bone tissue regeneration.

  14. Glucocorticoids induce apoptosis and matrix metalloproteinase-13 expression in chondrocytes through the NOX4/ROS/p38 MAPK pathway.

    Science.gov (United States)

    Huang, Ying; Cai, Gui-Quan; Peng, Jian-Ping; Shen, Chao

    2018-03-08

    Based on the results from our previous study, dexamethasone (Dex) increases reactive oxygen species (ROS) levels and subsequently induces cell death and matrix catabolism in chondrocytes. Nevertheless, the mechanism underlying this phenomenon remains unclear. Nicotinamide adenine dinucleotide (phosphate) (NADPH) oxidase 4 (NOX4) is one of the major enzymes responsible for intracellular ROS production during the inflammatory process. The objective of the current study was to investigate the role of NOX4 in Dex-induced ROS over-production. Healthy chondrocytes were harvested from the cartilage debris from 6 female patients. NOX4 and p38 mitogen-activated protein kinase (MAPK) expression levels in these cells were evaluated in the presence of Dex. Changes in the number of apoptotic and viable Dex-treated chondrocytes were recorded after the cells were treated with NOX and p38 MAPK inhibitors. Changes in matrix metalloproteinase 13 (MMP-13) expression levels in Dex-treated chondrocytes were also investigated. The Dex treatment increased NOX4 expression via the glucocorticoid receptor (GR). Treatment of cells with apocynin, a NOX inhibitor, decreased intracellular ROS levels and inhibited p38 MAPK activation. Treatment of cells with a ROS scavenger also reduced p38 MAPK expression. Treatment of cells with a NOX inhibitor, ROS scavenger and p38 MAPK inhibitor rescued chondrocytes from Dex-induced apoptosis. Moreover, treatment of cells with these agents blocked MMP-13 expression in Dex-treated chondrocytes. NOX4 silencing also suppressed p38 MAPK and MMP-13 expression. Dex triggered apoptosis and MMP-13 expression through the NOX4/ROS/p38 MAPK signaling pathway. NOX4 may be a therapeutic target in the management of Dex-induced complications. Copyright © 2018 Elsevier Ltd. All rights reserved.

  15. Transplantation of allogenic chondrocytes with chitosan hydrogel-demineralized bone matrix hybrid scaffold to repair rabbit cartilage injury.

    Science.gov (United States)

    Man, Zhentao; Hu, Xiaoqing; Liu, Zhenlong; Huang, Hongjie; Meng, Qingyang; Zhang, Xin; Dai, Linghui; Zhang, Jiying; Fu, Xin; Duan, Xiaoning; Zhou, Chunyan; Ao, Yingfang

    2016-11-01

    Cartilage tissue engineering is the hotspot of cartilage repair. The allogenic chondrocytes appear to be a promising source of seed cells in cartilage tissue engineering. In this study, we aimed to transplant allogenic chondrocytes with chitosan hydrogel (CS)-demineralized bone matrix (DBM) hybrid scaffold (CS/DBM) to repair rabbit cartilage injury with one-step operation. After the CS/DBM scaffold was successfully fabricated, it showed that the porous CS filled the large pores of DBM, which improved the distribution of seed cells in the CS/DBM scaffold. The allogenic chondrocytes at second passage were transplanted with different scaffolds to repair rabbit cartilage injury. Twenty-four weeks after surgery, the cartilage defect in the CS/DBM group was successfully filled as shown by MRI. Moreover, the histological score of CS/DBM group was significantly higher than that of the other groups. On the aspect of biomechanical property, the regenerated cartilage in the CS/DBM group were superior to those in the other groups as determined by nanoindentation. Meanwhile, no obvious inflammatory response was observed after the transplantation of allogenic chondrocytes at 24 weeks post-surgery. Furtherly, gene expression profile for cells within the repair tissue was compared with the allogenic chondrocytes before transplantation using Agilent microarray and RT-qPCR. The results showed that some genes beneficial to cartilage regeneration, such as BMP-7, HGF, and IGF-1, were upregulated one month after transplantation. Consequently, our study demonstrated that the transplantation of allogenic chondrocytes with CS/DBM scaffold successfully repaired rabbit cartilage injury with only one-step operation, thereby providing new insights into cartilage tissue engineering. Copyright © 2016 Elsevier Ltd. All rights reserved.

  16. Nano-hydroxyapatite/collagen film as a favorable substrate to maintain the phenotype and promote the growth of chondrocytes cultured in vitro.

    Science.gov (United States)

    Jiang, Xianfang; Zhong, Yanping; Zheng, Li; Zhao, Jinmin

    2018-04-01

    Autologous chondrocyte implantation (ACI) has emerged as a novel approach to cartilage repair through the use of harvested chondrocytes. However, the expansion of the chondrocytes from the donor tissue in vitro is restricted by the limited cell numbers and the dedifferentiation of the chondrocytes. The present study investigated the effect of collagen-based films, including collagen, hydroxyapatite (HA)/collagen (HC) and in situ synthesis of nano‑HC (nHC), on monolayer cultures of chondrocytes. As a substrate for the chondrocytes monolayer culture in vitro, nHC was able to restrain the dedifferentiation of chondrocytes and facilitate cell expansion, which was detected by methyl thiazolyl tetrazolium assay, scanning electron microscopy, calcein‑acetoxymethyl/propidium iodide staining, hematoxylin and eosin staining, Safranin O staining, immunohistochemical staining and reverse transcription‑quantitative polymerase chain reaction. Furthermore, the nHC films significantly facilitated cell growth and enhanced the expression of cartilage‑specific extracellular matrix (ECM) components, including aggrecan and type II collagen. In addition, nHC films markedly downregulated the expression of collagen type I, an indicator of dedifferentiation. The results indicated that nHC, a collagen‑based substrate optimized by nanoparticles, was able to better support cell growth and preserve cell phenotype compared with collagen alone or HC. The nHC film, which favors cell growth and prevents the dedifferentiation of chondrocytes, may therefore serve as a useful cartilage‑like ECM for chondrocytes. In conclusion, nHC film is a promising substrate for the culture of chondrocytes in cell-based therapy.

  17. Inhibition of somatotroph growth and growth hormone biosynthesis by activin in vitro

    DEFF Research Database (Denmark)

    Billestrup, Nils; González-Manchón, C; Potter, E

    1990-01-01

    Activin-A, a homodimeric protein composed of two inhibin beta A-subunits, was first isolated from gonadal fluids based upon its ability to stimulate FSH secretion and biosynthesis, but was also observed to suppress GH secretion. The present report describes the effects of activin on the biosynthe......Activin-A, a homodimeric protein composed of two inhibin beta A-subunits, was first isolated from gonadal fluids based upon its ability to stimulate FSH secretion and biosynthesis, but was also observed to suppress GH secretion. The present report describes the effects of activin...... the effect of activin on FSH secretion, did not reverse the effect of activin on GH biosynthesis. Treatment of somatotrophs with activin for 3 days completely inhibited the growth-promoting effect of GRF on somatotrophs. However, no effect of activin on GRF-stimulated expression of the c-fos protooncogene...... was observed. These data demonstrate that activin, in addition to its stimulatory effect on FSH secretion, is able to inhibit both expression of GH and growth of somatotropic cells....

  18. Monoterpene biosynthesis potential of plant subcellular compartments

    NARCIS (Netherlands)

    Dong, L.; Jongedijk, E.J.; Bouwmeester, H.J.; Krol, van der A.R.

    2016-01-01

    Subcellular monoterpene biosynthesis capacity based on local geranyl diphosphate (GDP) availability or locally boosted GDP production was determined for plastids, cytosol and mitochondria. A geraniol synthase (GES) was targeted to plastids, cytosol, or mitochondria. Transient expression in Nicotiana

  19. Antibacterial Targets in Fatty Acid Biosynthesis

    Science.gov (United States)

    Wright, H. Tonie; Reynolds, Kevin A.

    2008-01-01

    Summary The fatty acid biosynthesis pathway is an attractive but still largely unexploited target for development of new anti-bacterial agents. The extended use of the anti-tuberculosis drug isoniazid and the antiseptic triclosan, which are inhibitors of fatty acid biosynthesis, validates this pathway as a target for anti-bacterial development. Differences in subcellular organization of the bacterial and eukaryotic multi-enzyme fatty acid synthase systems offer the prospect of inhibitors with host vs. target specificity. Platensimycin, platencin, and phomallenic acids, newly discovered natural product inhibitors of the condensation steps in fatty acid biosynthesis, represent new classes of compounds with antibiotic potential. An almost complete catalogue of crystal structures for the enzymes of the type II fatty acid biosynthesis pathway can now be exploited in the rational design of new inhibitors, as well as the recently published crystal structures of type I FAS complexes. PMID:17707686

  20. Lincomycin, cultivation of producing strains and biosynthesis

    Czech Academy of Sciences Publication Activity Database

    Spížek, Jaroslav; Řezanka, Tomáš

    2004-01-01

    Roč. 63, - (2004), s. 510-519 ISSN 0175-7598 Institutional research plan: CEZ:AV0Z5020903 Keywords : lincomycin * cultivation * biosynthesis Subject RIV: EE - Microbiology, Virology Impact factor: 2.358, year: 2004

  1. Role of glutathione biosynthesis in endothelial dysfunction and fibrosis

    Directory of Open Access Journals (Sweden)

    Cristina Espinosa-Díez

    2018-04-01

    Full Text Available Glutathione (GSH biosynthesis is essential for cellular redox homeostasis and antioxidant defense. The rate-limiting step requires glutamate-cysteine ligase (GCL, which is composed of the catalytic (GCLc and the modulatory (GCLm subunits. To evaluate the contribution of GCLc to endothelial function we generated an endothelial-specific Gclc haplo-insufficient mouse model (Gclc e/+ mice. In murine lung endothelial cells (MLEC derived from these mice we observed a 50% reduction in GCLc levels compared to lung fibroblasts from the same mice. MLEC obtained from haplo-insufficient mice showed significant reduction in GSH levels as well as increased basal and stimulated ROS levels, reduced phosphorylation of eNOS (Ser 1177 and increased eNOS S-glutathionylation, compared to MLEC from wild type (WT mice. Studies in mesenteric arteries demonstrated impaired endothelium-dependent vasodilation in Gclc(e/+ male mice, which was corrected by pre-incubation with GSH-ethyl-ester and BH4. To study the contribution of endothelial GSH synthesis to renal fibrosis we employed the unilateral ureteral obstruction model in WT and Gclc(e/+ mice. We observed that obstructed kidneys from Gclc(e/+ mice exhibited increased deposition of fibrotic markers and reduced Nrf2 levels. We conclude that the preservation of endothelial GSH biosynthesis is not only critical for endothelial function but also in anti-fibrotic responses. Keywords: Glutamate-cysteine ligase, ROS, Glutathione, Endothelial dysfunction, Kidney Fibrosis

  2. TNF/TNFR{sub 1} pathway and endoplasmic reticulum stress are involved in ofloxacin-induced apoptosis of juvenile canine chondrocytes

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Fu-Tao; Ding, Yi; Shah, Zahir; Xing, Dan; Gao, Yuan; Liu, Dong Ming; Ding, Ming-Xing, E-mail: dmx@mail.hzau.edu.cn

    2014-04-15

    Background and purpose: Quinolones cause obvious cartilaginous lesions in juvenile animals by chondrocyte apoptosis, which results in the restriction of their use in pediatric and adolescent patients. Studies showed that chondrocytes can be induced to produce TNFα, and the cisternae of the endoplasmic reticulum in quinolone-treated chondrocytes become dilated. We investigated whether TNF/TNFR{sub 1} pathway and endoplasmic reticulum stress (ERs) are involved in ofloxacin (a typical quinolone)-induced apoptosis of juvenile canine chondrocytes. Experimental approach: Canine juvenile chondrocytes were treated with ofloxacin. Cell survival and apoptosis rates were determined with MTT method and flow cytometry, respectively. The gene expression levels of the related signaling molecules (TNFα, TNFR{sub 1}, TRADD, FADD and caspase-8) in death receptor pathways and main apoptosis-related molecules (calpain, caspase-12, GADD153 and GRP78) in ERs were measured by qRT-PCR. The gene expression of TNFR{sub 1} was suppressed with its siRNA. The protein levels of TNFα, TNFR{sub 1} and caspase-12 were assayed using Western blotting. Key results: The survival rates decreased while apoptosis rates increased after the chondrocytes were treated with ofloxacin. The mRNA levels of the measured apoptosis-related molecules in death receptor pathways and ERs, and the protein levels of TNFα, TNFR{sub 1} and caspase-12 increased after the chondrocytes were exposed to ofloxacin. The downregulated mRNA expressions of TNFR{sub 1}, Caspase-8 and TRADD, and the decreased apoptosis rates of the ofloxacin-treated chondrocytes occurred after TNFR{sub 1}–siRNA interference. Conclusions and implications: Ofloxacin-induced chondrocyte apoptosis in a time- and concentration-dependent fashion. TNF/TNFR{sub 1} pathway and ERs are involved in ofloxacin-induced apoptosis of juvenile canine chondrocytes in the early stage. - Highlights: • Chondrocyte apoptosis is induced by ofloxacin in a time- and

  3. In vitro effects of cisplatin-functionalized silica nanoparticles on chondrocytes

    Energy Technology Data Exchange (ETDEWEB)

    Bhowmick, Tridib Kumar; Yoon, Diana [University of Maryland, Department of Chemical and Biomolecular Engineering (United States); Patel, Minal; Fisher, John [University of Maryland, Fischell Department of Bioengineering (United States); Ehrman, Sheryl, E-mail: sehrman@umd.ed [University of Maryland, Department of Chemical and Biomolecular Engineering (United States)

    2010-10-15

    In this study, we evaluated the combined effect of a known toxic molecule, cisplatin, in combination with relatively nontoxic nanoparticles, amorphous fumed silica, on chondrocyte cells. Cisplatin was attached to silica nanoparticles using aminopropyltriethoxy silane as a linker molecule, and characterized in terms of size, shape, specific surface area, as well as the dissolution of cisplatin from the silica surface. The primary particle diameter of the as-received silica nanoparticles ranged from 7.1 to 61 nm, estimated from measurements of specific surface area, and the primary particles were aggregated. The effects of cisplatin-functionalized silica particles with different specific surface areas (41, 85, 202, 237, and 297 m{sup 2}/g) were compared in vitro on chondrocytes, the parenchymal cell of hyaline cartilage. The results show that adverse effects on cell function, as evidenced by reduced metabolic activity measured by the MTT assay and increased membrane permeability observed using the Live/Dead stain, can be correlated with specific surface area of the silica. Cisplatin-functionalized silica nanoparticles with the highest specific surface area incited the greatest response, which was almost equivalent to that induced by free cisplatin. This result suggests the importance of particle specific surface area in interactions between cells and surface-functionalized nanomaterials.

  4. In vitro effects of cisplatin-functionalized silica nanoparticles on chondrocytes

    International Nuclear Information System (INIS)

    Bhowmick, Tridib Kumar; Yoon, Diana; Patel, Minal; Fisher, John; Ehrman, Sheryl

    2010-01-01

    In this study, we evaluated the combined effect of a known toxic molecule, cisplatin, in combination with relatively nontoxic nanoparticles, amorphous fumed silica, on chondrocyte cells. Cisplatin was attached to silica nanoparticles using aminopropyltriethoxy silane as a linker molecule, and characterized in terms of size, shape, specific surface area, as well as the dissolution of cisplatin from the silica surface. The primary particle diameter of the as-received silica nanoparticles ranged from 7.1 to 61 nm, estimated from measurements of specific surface area, and the primary particles were aggregated. The effects of cisplatin-functionalized silica particles with different specific surface areas (41, 85, 202, 237, and 297 m 2 /g) were compared in vitro on chondrocytes, the parenchymal cell of hyaline cartilage. The results show that adverse effects on cell function, as evidenced by reduced metabolic activity measured by the MTT assay and increased membrane permeability observed using the Live/Dead stain, can be correlated with specific surface area of the silica. Cisplatin-functionalized silica nanoparticles with the highest specific surface area incited the greatest response, which was almost equivalent to that induced by free cisplatin. This result suggests the importance of particle specific surface area in interactions between cells and surface-functionalized nanomaterials.

  5. The Importance of the mTOR Regulatory Network in Chondrocyte Biology and Osteoarthritis

    Directory of Open Access Journals (Sweden)

    Elena V. Tchetina

    2014-07-01

    Full Text Available Osteoarthritis (OA is a chronic disorder associated mainly with pain, limited range of motion, stiffness, joint inflammation, and articular cartilage (AC destruction. Recent studies demonstrated the involvement of chondrocyte differentiation (hypertrophy as one of the mechanisms of cartilage degradation in OA. This indicates the involvement of profound alterations in chondrocyte metabolism in the course of cartilage resorption orchestrated by principal changes in the regulation of cellular function. Mammalian target of rapamycin (mTOR controls critical cellular processes such as growth, proliferation, and protein synthesis, and integrates extracellular signals from growth factors and hormones with amino acid availability and intracellular energy status. The importance of mTOR activity during AC destruction in OA is supported by considerable alterations in the mTOR regulatory network, involving multiple intracellular (availability of growth factors, adenosine triphosphate [ATP], and oxygen as well as autophagy and extracellular (glucose, amino acid, lipid, and hexosamine signals. Moreover, variable mTOR gene expression in the peripheral blood of OA patients is associated with increases in pain or synovitis, and indicates a profound metabolic dissimilarity among patients that might require differential approaches to treatment. These issues are discussed in the present review article.

  6. Dual mechanism for cAMP-dependent modulation of Ca2+ signalling in articular chondrocytes.

    Science.gov (United States)

    D'Andrea, P; Paschini, V; Vittur, F

    1996-09-01

    The ability of cAMP to modulate the actions of Ca(2+)-mobilizing agonists was studied in single Fura-2-loaded pig articular chondrocytes in primary culture. Forskolin and 8-Br-cAMP increased both the frequency and amplitude of Ca2+ oscillations induced by ATP, and, in unstimulated cells, induced single Ca2+ transients or even Ca2+ oscillations. The cAMP-dependent protein kinase inhibitor H89 totally prevented the effect of cAMP-elevating agents on Ca2+ signalling. Forskolin and 8-Br-cAMP promptly increased the rate of Mn2+ quenching, when administered in the presence of ATP, suggesting a potentiation of receptor-mediated Ca2+ influx. In Ca(2+)-free medium, ATP-induced Ca2+ oscillations decreased and stopped after a few cycles: subsequent ATP additions temporarily resumed the activity, an effect that could be mimicked by forskolin. The same agent induced single Ca2+ transients in 42% of the cell population maintained in Ca(2+)-free medium. Thapsigargin prevented Ca2+ responses to both ATP and forskolin. The results indicate a dual mechanism for cAMP-induced potentiation of Ca2+ signalling in articular chondrocytes: an increase of receptor-mediated Ca2+ influx and a positive modulation of intracellular Ca2+ release.

  7. Chitosan-Coated Collagen Membranes Promote Chondrocyte Adhesion, Growth, and Interleukin-6 Secretion

    Directory of Open Access Journals (Sweden)

    Nabila Mighri

    2015-11-01

    Full Text Available Designing scaffolds made from natural polymers may be highly attractive for tissue engineering strategies. We sought to produce and characterize chitosan-coated collagen membranes and to assess their efficacy in promoting chondrocyte adhesion, growth, and cytokine secretion. Porous collagen membranes were placed in chitosan solutions then crosslinked with glutaraldehyde vapor. Fourier transform infrared (FTIR analyses showed elevated absorption at 1655 cm-1 of the carbon–nitrogen (N=C bonds formed by the reaction between the (NH2 of the chitosan and the (C=O of the glutaraldehyde. A significant peak in the amide II region revealed a significant deacetylation of the chitosan. Scanning electron microscopy (SEM images of the chitosan-coated membranes exhibited surface variations, with pore size ranging from 20 to 50 µm. X-ray photoelectron spectroscopy (XPS revealed a decreased C–C groups and an increased C–N/C–O groups due to the reaction between the carbon from the collagen and the NH2 from the chitosan. Increased rigidity of these membranes was also observed when comparing the chitosan-coated and uncoated membranes at dried conditions. However, under wet conditions, the chitosan coated collagen membranes showed lower rigidity as compared to dried conditions. Of great interest, the glutaraldehyde-crosslinked chitosan-coated collagen membranes promoted chondrocyte adhesion, growth, and interleukin (IL-6 secretion. Overall results confirm the feasibility of using designed chitosan-coated collagen membranes in future applications, such as cartilage repair.

  8. Effect of extracellular fatty acids on lipid metabolism in cultured rabbit articular chondrocytes

    International Nuclear Information System (INIS)

    Nagao, M.; Ishii, S.; Murata, Y.; Akino, T.

    1991-01-01

    Rabbit articular chondrocytes were cultured for 8 h in the presence of various concentrations (5-500 microM) of 14 C oleic, 14 C linoleic, and 3H arachidonic acids. The radioactive unsaturated fatty acids were incorporated into triacylglycerol (TG) and phosphatidylcholine (PC) in a concentration-dependent manner; more fatty acids were incorporated into TG than into PC, at higher concentrations of extracellular fatty acids. Among these fatty acids, arachidonic acid was incorporated into TG much more than into PC, in spite of a very low concentration of arachidonic acid in TG. After transfer of the labeled cells to maintenance medium, the radioactivity in TG declined rapidly and 3 H arachidonic acid radioactivity in PC increased continuously during the chase time periods. Palmitoyl-unsaturated species were mainly formed in PC when cultured at a concentration of 5 microM of each fatty acid. However, when cultured at 500 microM, unsaturated-unsaturated species, specific for each unsaturated fatty acid were actively formed. These findings indicate that (1) fatty acid composition of TG and PC in articular chondrocytes is influenced by the degree of fatty acid supply, (2) formation and turnover of TG plays a role in fatty acid metabolism of cells, and (3) fatty acid pairing in PC is modulated by extracellular fatty acid concentrations

  9. Natural polysaccharides promote chondrocyte adhesion and proliferation on magnetic nanoparticle/PVA composite hydrogels.

    Science.gov (United States)

    Hou, Ruixia; Nie, Lei; Du, Gaolai; Xiong, Xiaopeng; Fu, Jun

    2015-08-01

    This paper aims to investigate the synergistic effects of natural polysaccharides and inorganic nanoparticles on cell adhesion and growth on intrinsically cell non-adhesive polyvinyl alcohol (PVA) hydrogels. Previously, we have demonstrated that Fe2O3 and hydroxyapatite (nHAP) nanoparticles are effective in increasing osteoblast growth on PVA hydrogels. Herein, we blended hyaluronic acid (HA) and chondroitin sulfate (CS), two important components of cartilage extracellular matrix (ECM), with Fe2O3/nHAP/PVA hydrogels. The presence of these natural polyelectrolytes dramatically increased the pore size and the equilibrium swelling ratio (ESR) while maintaining excellent compressive strength of hydrogels. Chondrocytes were seeded and cultured on composite PVA hydrogels containing Fe2O3, nHAP and Fe2O3/nHAP hybrids and Fe2O3/nHAP with HA or CS. Confocal laser scanning microscopy (CLSM) and cell counting kit-8 (CCK-8) assay consistently confirmed that the addition of HA or CS promotes chondrocyte adhesion and growth on PVA and composite hydrogels. Particularly, the combination of HA and CS exhibited further promotion to cell adhesion and proliferation compared with any single polysaccharide. The results demonstrated that the magnetic composite nanoparticles and polysaccharides provided synergistic promotion to cell adhesion and growth. Such polysaccharide-augmented composite hydrogels may have potentials in biomedical applications. Copyright © 2015 Elsevier B.V. All rights reserved.

  10. Beclin 1 overexpression inhibits chondrocyte apoptosis and downregulates extracellular matrix metabolism in osteoarthritis.

    Science.gov (United States)

    Song, Bin; Song, Hong; Wang, Weiguo; Wang, Hongru; Peng, Hanyuan; Cui, Jing; Wang, Rong; Huang, Hua; Wang, Wei; Wang, Lili

    2017-10-01

    In the present study, the expression of Beclin 1 in osteoarthritis (OA) cartilage tissue was investigated, and also its role in proliferation, apoptosis and expression of matrix metalloproteinases (MMPs) in chondrocytes obtained from patients with OA. Beclin 1 expression in cartilage tissue from OA patients, and in the age- and sex-matched controls, was detected by immunohistochemistry, semi-quantitative polymerase chain reaction and western blotting. Chondrocytes were divided into control and Beclin 1-overexpressed groups. After transfection for 48, 72 and 96 h, cell viability, apoptosis, the phosphatidylinositol-3-kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) signaling pathway and MMPs were examined. The mRNA and protein expression levels of Beclin 1 were significantly decreased in cartilage tissue from OA patients compared with the sex- and age-matched controls (Poverexpression significantly increased cell viability (Poverexpression additionally decreased the degree of apoptosis, as demonstrated by Hoechst staining and flow cytometric analysis. B-cell lymphoma-2 (Bcl-2) was upregulated, and Bcl-2 associated X was downregulated, following Beclin 1 overexpression (Poverexpression (Poverexpression (Poverexpression increased cell viability, inhibited apoptosis and MMPs, likely via the PI3K/Akt/mTOR signaling pathway.

  11. Introduction of exogenous growth hormone receptors augments growth hormone-responsive insulin biosynthesis in rat insulinoma cells

    International Nuclear Information System (INIS)

    Billestrup, N.; Moeldrup, A.; Serup, P.; Nielsen, J.H.; Mathews, L.S.; Norstedt, G.

    1990-01-01

    The stimulation of insulin biosynthesis in the pancreatic insulinoma cell line RIN5-AH by growth hormone (GH) is initiated by GH binding to specific receptors. To determine whether the recently cloned rat hepatic GH receptor is able to mediate the insulinotropic effect of GH, the authors have transfected a GH receptor cDNA under the transcriptional control of the human metallothionein promoter into RIN5-AH cells. The transfected cells were found to exhibit an increased expression of GH receptors and to contain a specific GH receptor mRNA that was not expressed in the parent cell line. The expression of GH receptors in one clone (1.24) selected for detailed analysis was increased 2.6-fold compared to untransfected cells. The increased GH receptor expression was accompanied by an increased responsiveness to GH. Thus, the maximal GH-stimulated increase of insulin biosynthesis was 4.1-fold in 1.24 cells compared to 1.9-fold in the nontransfected RIN5-AH cells. The expression of the transfected receptor was stimulated 1.6- and 2.3-fold when cells were cultured in the presence of 25 or 50 μM Zn 2+ was associated with an increased magnitude of GH-stimulated insulin biosynthesis. A close stoichiometric relationship between the level of receptor expression and the level of GH-stimulated insulin biosynthesis was observed. They conclude from these results that the hepatic GH receptor is able to mediate the effect of GH on insulin biosynthesis in RIN5-AH cells

  12. Control of tylosin biosynthesis in Streptomyces fradiae.

    Science.gov (United States)

    Cundliffe, Eric

    2008-09-01

    Tylosin biosynthesis is controlled in cascade fashion by multiple transcriptional regulators, acting positively or negatively, in conjunction with a signalling ligand that acts as a classical inducer. The roles of regulatory gene products have been characterized by a combination of gene expression analysis and fermentation studies, using engineered strains of S. fradiae in which specific genes were inactivated or overexpressed. Among various novel features of the regulatory model, involvement of the signalling ligand is not essential for tylosin biosynthesis.

  13. Brain Stimulation Therapies

    Science.gov (United States)

    ... Magnetic Seizure Therapy Deep Brain Stimulation Additional Resources Brain Stimulation Therapies Overview Brain stimulation therapies can play ... for a shorter recovery time than ECT Deep Brain Stimulation Deep brain stimulation (DBS) was first developed ...

  14. Biosynthesis of antibiotic chuangxinmycin from Actinoplanes tsinanensis

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    Yuanyuan Shi

    2018-03-01

    Full Text Available Chuangxinmycin is an antibiotic isolated from Actinoplanes tsinanensis CPCC 200056 in the 1970s with a novel indole-dihydrothiopyran heterocyclic skeleton. Chuangxinmycin showed in vitro antibacterial activity and in vivo efficacy in mouse infection models as well as preliminary clinical trials. But the biosynthetic pathway of chuangxinmycin has been obscure since its discovery. Herein, we report the identification of a stretch of DNA from the genome of A. tsinanensis CPCC 200056 that encodes genes for biosynthesis of chuangxinmycin by bioinformatics analysis. The designated cxn cluster was then confirmed to be responsible for chuangxinmycin biosynthesis by direct cloning and heterologous expressing in Streptomyces coelicolor M1146. The cytochrome P450 CxnD was verified to be involved in the dihydrothiopyran ring closure reaction by the identification of seco-chuangxinmycin in S. coelicolor M1146 harboring the cxn gene cluster with an inactivated cxnD. Based on these results, a plausible biosynthetic pathway for chuangxinmycin biosynthesis was proposed, by hijacking the primary sulfur transfer system for sulfur incorporation. The identification of the biosynthetic gene cluster of chuangxinmycin paves the way for elucidating the detail biochemical machinery for chuangxinmycin biosynthesis, and provides the basis for the generation of novel chuangxinmycin derivatives by means of combinatorial biosynthesis and synthetic biology. KEY WORDS: Chuangxinmycin, Actinoplanes tsinanensis, Biosynthesis gene cluster, Heterologous expression, Cytochrome P450, Seco-chuangxinmycin, C–S bond formation, Sulfur incorporation

  15. Brain modulation of Dufour's gland ester biosynthesis in vitro in the honeybee ( Apis mellifera)

    Science.gov (United States)

    Katzav-Gozansky, Tamar; Hefetz, Abraham; Soroker, Victoria

    2007-05-01

    Caste-specific pheromone biosynthesis is a prerequisite for reproductive skew in the honeybee. Nonetheless, this process is not hardwired but plastic, in that egg-laying workers produce a queen-like pheromone. Studies with Dufour’s gland pheromone revealed that, in vivo, workers’ gland biosynthesis matches the social status of the worker, i.e., sterile workers showed a worker-like pattern whereas fertile workers showed a queen-like pattern (production of the queen-specific esters). However, when incubated in vitro, the gland spontaneously exhibits the queen-like pattern, irrespective of its original worker type, prompting the notion that ester production in workers is under inhibitory control that is queen-dependent. We tested this hypothesis by exposing queen or worker Dufour’s glands in vitro to brain extracts of queens, queenright (sterile) workers and males. Unexpectedly, worker brain extracts activated the queen-like esters biosynthesis in workers’ Dufour’s gland. This stimulation was gender-specific; queen or worker brains demonstrated a stimulatory activity, but male brains did not. Queen gland could not be further stimulated. Bioassays with heated and filtered extracts indicate that the stimulatory brain factor is below 3,000 Da. We suggest that pheromone production in Dufour’s gland is under dual, negative positive control. Under queenright conditions, the inhibitor is released and blocks ester biosynthesis, whereas under queenless conditions, the activator is released, activating ester biosynthesis in the gland. This is consistent with the hypothesis that queenright workers are unequivocally recognized as non-fertile, whereas queenless workers try to become “false queens” as part of the reproductive competition.

  16. Biosynthesis of promatrix metalloproteinase-9/chondroitin sulphate proteoglycan heteromer involves a Rottlerin-sensitive pathway.

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    Nabin Malla

    Full Text Available BACKGROUND: Previously we have shown that a fraction of the matrix metalloproteinase-9 (MMP-9 synthesized by the macrophage cell line THP-1 was bound to a chondroitin sulphate proteoglycan (CSPG core protein as a reduction sensitive heteromer. Several biochemical properties of the enzyme were changed when it was bound to the CSPG. METHODOLOGY/PRINCIPAL FINDINGS: By use of affinity chromatography, zymography, and radioactive labelling, various macrophage stimulators were tested for their effect on the synthesis of the proMMP-9/CSPG heteromer and its components by THP-1 cells. Of the stimulators, only PMA largely increased the biosynthesis of the heteromer. As PMA is an activator of PKC, we determined which PKC isoenzymes were expressed by performing RT-PCR and Western Blotting. Subsequently specific inhibitors were used to investigate their involvement in the biosynthesis of the heteromer. Of the inhibitors, only Rottlerin repressed the biosynthesis of proMMP-9/CSPG and its two components. Much lower concentrations of Rottlerin were needed to reduce the amount of CSPG than what was needed to repress the synthesis of the heteromer and MMP-9. Furthermore, Rottlerin caused a minor reduction in the activation of the PKC isoenzymes δ, ε, θ and υ (PKD3 in both control and PMA exposed cells. CONCLUSIONS/SIGNIFICANCE: The biosynthesis of the proMMP-9/CSPG heteromer and proMMP-9 in THP-1 cells involves a Rottlerin-sensitive pathway that is different from the Rottlerin sensitive pathway involved in the CSPG biosynthesis. MMP-9 and CSPGs are known to be involved in various physiological and pathological processes. Formation of complexes may influence both the specificity and localization of the enzyme. Therefore, knowledge about biosynthetic pathways and factors involved in the formation of the MMP-9/CSPG heteromer may contribute to insight in the heteromers biological function as well as pointing to future targets for therapeutic agents.

  17. Does Platelet-Rich Plasma Freeze-Thawing Influence Growth Factor Release and Their Effects on Chondrocytes and Synoviocytes?

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    Alice Roffi

    2014-01-01

    Full Text Available PRP cryopreservation remains a controversial point. Our purpose was to investigate the effect of freezing/thawing on PRP molecule release, and its effects on the metabolism of chondrocytes and synoviocytes. PRP was prepared from 10 volunteers, and a half volume underwent one freezing/thawing cycle. IL-1β, HGF, PDGF AB/BB, TGF-β1, and VEGF were assayed 1 hour and 7 days after activation. Culture media of chondrocytes and synoviocytes were supplemented with fresh or frozen PRP, and, at 7 days, proliferation, gene expression, and secreted proteins levels were evaluated. Results showed that in the freeze-thawed PRP the immediate and delayed molecule releases were similar or slightly lower than those in fresh PRP. TGF-β1 and PDGF AB/BB concentrations were significantly reduced after freezing both at 1 hour and at 7 days, whereas HGF concentration was significantly lower in frozen PRP at 7 days. In fresh PRP IL-1β and HGF concentrations underwent a significant further increase after 7 days. Similar gene expression was found in chondrocytes cultured with both PRPs, whereas in synoviocytes HGF gene expression was higher in frozen PRP. PRP cryopreservation is a safe procedure, which sufficiently preserves PRP quality and its ability to induce proliferation and the production of ECM components in chondrocytes and synoviocytes.

  18. In vitro cell quality of articular chondrocytes assigned for autologous implantation in dependence of specific patient characteristics

    DEFF Research Database (Denmark)

    Pestka, Jan M; Schmal, Hagen; Salzmann, Gian

    2011-01-01

    OBJECTIVE: Autologous chondrocyte implantation (ACI) is a well-established therapeutic option for the treatment of cartilage defects of the knee joint. Since information concerning the cellular aspects of ACI is still limited, the aim of the present study was to investigate relevant differences b...

  19. A network of transcriptional and signaling events is activated by FGF to induce chondrocyte growth arrest and differentiation.

    Science.gov (United States)

    Dailey, Lisa; Laplantine, Emmanuel; Priore, Riccardo; Basilico, Claudio

    2003-06-23

    Activating mutations in FGF receptor 3 (FGFR3) cause several human dwarfism syndromes by affecting both chondrocyte proliferation and differentiation. Using microarray and biochemical analyses of FGF-treated rat chondrosarcoma chondrocytes, we show that FGF inhibits chondrocyte proliferation by initiating multiple pathways that result in the induction of antiproliferative functions and the down-regulation of growth-promoting molecules. The initiation of growth arrest is characterized by the rapid dephosphorylation of the retinoblastoma protein (pRb) p107 and repression of a subset of E2F target genes by a mechanism that is independent of cyclin E-Cdk inhibition. In contrast, hypophosphorylation of pRb and p130 occur after growth arrest is first detected, and may contribute to its maintenance. Importantly, we also find a number of gene expression changes indicating that FGF promotes many aspects of hypertrophic differentiation, a notion supported by in situ analysis of developing growth plates from mice expressing an activated form of FGFR3. Thus, FGF may coordinate the onset of differentiation with chondrocyte growth arrest in the developing growth plate.

  20. Iterative design of peptide-based hydrogels and the effect of network electrostatics on primary chondrocyte behavior.

    Science.gov (United States)

    Sinthuvanich, Chomdao; Haines-Butterick, Lisa A; Nagy, Katelyn J; Schneider, Joel P

    2012-10-01

    Iterative peptide design was used to generate two peptide-based hydrogels to study the effect of network electrostatics on primary chondrocyte behavior. MAX8 and HLT2 peptides have formal charge states of +7 and +5 per monomer, respectively. These peptides undergo triggered folding and self-assembly to afford hydrogel networks having similar rheological behavior and local network morphologies, yet different electrostatic character. Each gel can be used to directly encapsulate and syringe-deliver cells. The influence of network electrostatics on cell viability after encapsulation and delivery, extracellular matrix deposition, gene expression, and the bulk mechanical properties of the gel-cell constructs as a function of culture time was assessed. The less electropositive HLT2 gel provides a microenvironment more conducive to chondrocyte encapsulation, delivery, and phenotype maintenance. Cell viability was higher for this gel and although a moderate number of cells dedifferentiated to a fibroblast-like phenotype, many retained their chondrocytic behavior. As a result, gel-cell constructs prepared with HLT2, cultured under static in vitro conditions, contained more GAG and type II collagen resulting in mechanically superior constructs. Chondrocytes delivered in the more electropositive MAX8 gel experienced a greater degree of cell death during encapsulation and delivery and the remaining viable cells were less prone to maintain their phenotype. As a result, MAX8 gel-cell constructs had fewer cells, of which a limited number were capable of laying down cartilage-specific ECM. Published by Elsevier Ltd.

  1. The major basement membrane components localize to the chondrocyte pericellular matrix--a cartilage basement membrane equivalent?

    DEFF Research Database (Denmark)

    Kvist, Alexander J.; Nyström, Alexander; Hultenby, Kjell

    2007-01-01

    In this study, we demonstrate that articular cartilage chondrocytes are surrounded by the defining basement membrane proteins laminin, collagen type IV, nidogen and perlecan, and suggest that these form the functional equivalent of a basement membrane. We found by real-time PCR that mouse...... to the progression of degenerative joint disorders....

  2. Age-related degeneration of articular cartilage in the pathogenesis of osteoarthritis: molecular markers of senescent chondrocytes.

    Science.gov (United States)

    Musumeci, Giuseppe; Szychlinska, Marta Anna; Mobasheri, Ali

    2015-01-01

    Aging is a natural process by which every single living organism approaches its twilight of existence in a natural way. However, aging is also linked to the pathogenesis of a number of complex diseases. This is the case for osteoarthritis (OA), where age is considered to be a major risk factor of this important and increasingly common joint disorder. Half of the world's population, aged 65 and older, suffers from OA. Although the relationship between the development of OA and aging has not yet been completely understood, it is thought that age-related changes correlate with other risk factors. The most prominent hypothesis linking aging and OA is that chondrocytes undergo premature aging due to several factors, such as excessive mechanical load or oxidative stress, which induce the so called "stress-induced senescent state", which is ultimately responsible for the onset of OA. This review focuses on molecular markers and mechanisms implicated in chondrocyte aging and the pathogenesis of OA. We discuss the most important age-related morphological and biological changes that affect articular cartilage and chondrocytes. We also identify the main senescence markers that may be used to recognize molecular alterations in the extracellular matrix of cartilage as related to senescence. Since the aging process is strongly associated with the onset of osteoarthritis, we believe that strategies aimed at preventing chondrocyte senescence, as well as the identification of new increasingly sensitive senescent markers, could have a positive impact on the development of new therapies for this severe disease.

  3. Comparative effects of vitamin C on the effects of local anesthetics ropivacaine, bupivacaine, and lidocaine on human chondrocytes.

    Science.gov (United States)

    Tian, Jun; Li, Yan

    2016-01-01

    Intra-articular injections of local anesthetics are commonly used to enhance post-operative analgesia following orthopedic surgery as arthroscopic surgeries. Nevertheless, recent reports of severe complications due to the use of intra-articular local anesthetic have raised concerns. The study aims to assess use of vitamin C in reducing adverse effects of the most commonly employed anesthetics - ropivacaine, bupivacaine and lidocaine - on human chondrocytes. The chondrocyte viability following exposure to 0.5% bupivacaine or 0.75% ropivacaine or 1.0% lidocaine and/or vitamin C at doses 125, 250 and 500 μM was determined by LIVE/DEAD assay and annexin V staining. Expression levels of caspases 3 and 9 were assessed using antibodies by Western blotting. Flow cytometry was performed to analyze the generation of reactive oxygen species. On exposure to the local anesthetics, chondrotoxicity was found in the order ropivacaineC effectively improved the reduced chondrocyte viability and decreased the raised apoptosis levels following exposure to anesthesia. At higher doses, vitamin C was found efficient in reducing the generation of reactive oxygen species and as well down-regulate the expressions of caspases 3 and 9. Vitamin C was observed to effectively protect chondrocytes against the toxic insult of local anesthetics ropivacaine, bupivacaine and lidocaine. Copyright © 2015 Sociedade Brasileira de Anestesiologia. Published by Elsevier Editora Ltda. All rights reserved.

  4. [Comparative effects of vitamin C on the effects of local anesthetics ropivacaine, bupivacaine, and lidocaine on human chondrocytes].

    Science.gov (United States)

    Tian, Jun; Li, Yan

    2016-01-01

    Intra-articular injections of local anesthetics are commonly used to enhance post-operative analgesia following orthopedic surgery as arthroscopic surgeries. Nevertheless, recent reports of severe complications due to the use of intra-articular local anesthetic have raised concerns. The study aims to assess use of vitamin C in reducing adverse effects of the most commonly employed anesthetics - ropivacaine, bupivacaine and lidocaine - on human chondrocytes. The chondrocyte viability following exposure to 0.5% bupivacaine or 0.75% ropivacaine or 1.0% lidocaine and/or vitamin C at doses 125, 250 and 500μM was determined by Live/Dead assay and annexin V staining. Expression levels of caspases 3 and 9 were assessed using antibodies by Western blotting. Flow cytometry was performed to analyze the generation of reactive oxygen species. On exposure to the local anesthetics, chondrotoxicity was found in the order ropivacaineC effectively improved the reduced chondrocyte viability and decreased the raised apoptosis levels following exposure to anesthesia. At higher doses, vitamin C was found efficient in reducing the generation of reactive oxygen species and as well down-regulate the expressions of caspases 3 and 9. Vitamin C was observed to effectively protect chondrocytes against the toxic insult of local anesthetics ropivacaine, bupivacaine and lidocaine. Copyright © 2015 Sociedade Brasileira de Anestesiologia. Publicado por Elsevier Editora Ltda. All rights reserved.

  5. XBP1-Independent UPR Pathways Suppress C/EBP-β Mediated Chondrocyte Differentiation in ER-Stress Related Skeletal Disease.

    Directory of Open Access Journals (Sweden)

    Trevor L Cameron

    2015-09-01

    Full Text Available Schmid metaphyseal chondrodysplasia (MCDS involves dwarfism and growth plate cartilage hypertrophic zone expansion resulting from dominant mutations in the hypertrophic zone collagen, Col10a1. Mouse models phenocopying MCDS through the expression of an exogenous misfolding protein in the endoplasmic reticulum (ER in hypertrophic chondrocytes have demonstrated the central importance of ER stress in the pathology of MCDS. The resultant unfolded protein response (UPR in affected chondrocytes involved activation of canonical ER stress sensors, IRE1, ATF6, and PERK with the downstream effect of disrupted chondrocyte differentiation. Here, we investigated the role of the highly conserved IRE1/XBP1 pathway in the pathology of MCDS. Mice with a MCDS collagen X p.N617K knock-in mutation (ColXN617K were crossed with mice in which Xbp1 was inactivated specifically in cartilage (Xbp1CartΔEx2, generating the compound mutant, C/X. The severity of dwarfism and hypertrophic zone expansion in C/X did not differ significantly from ColXN617K, revealing surprising redundancy for the IRE1/XBP1 UPR pathway in the pathology of MCDS. Transcriptomic analyses of hypertrophic zone cartilage identified differentially expressed gene cohorts in MCDS that are pathologically relevant (XBP1-independent or pathologically redundant (XBP1-dependent. XBP1-independent gene expression changes included large-scale transcriptional attenuation of genes encoding secreted proteins and disrupted differentiation from proliferative to hypertrophic chondrocytes. Moreover, these changes were consistent with disruption of C/EBP-β, a master regulator of chondrocyte differentiation, by CHOP, a transcription factor downstream of PERK that inhibits C/EBP proteins, and down-regulation of C/EBP-β transcriptional co-factors, GADD45-β and RUNX2. Thus we propose that the pathology of MCDS is underpinned by XBP1 independent UPR-induced dysregulation of C/EBP-β-mediated chondrocyte differentiation

  6. MicroRNA-195 induced apoptosis in hypoxic chondrocytes by targeting hypoxia-inducible factor 1 alpha.

    Science.gov (United States)

    Bai, R; Zhao, A-Q; Zhao, Z-Q; Liu, W-L; Jian, D-M

    2015-02-01

    The chondrocytes, the resident cells of cartilage, are maintained and take effects in the whole life upon chronic hypoxic exposure, which hypoxia-inducible factor 1 alpha (HIF-1α) play pivotal roles in response to. Dysregulation of some microRNA (miRNAs) have also been identified to be involved in hypoxia-related physiologic and pathophysiologic responses in some tissues or cell lines. However, the mechanism of miRNAs reponse to hypoxia remain largely unknown in chondrocytes, including the microRNA-195 (miR-195). AIM To investigate the effects of microRNAs (miRNAs) and hypoxia-inducible factor 1 alpha (HIF-1α) on chondrocytes in physiologic environment. We compared the expression of miR-195 and HIF-1α mRNA on hypoxia with that on normoxia in ATDC 5 cells by qRT-PCR. Further experiments was performed to confirmed the relationships of miR-195 and HIF-1α by bioinformatics analysis and dual reporter gene assay. we also assessed the effect of miR-195 on apoptosis in hypoxic ATDC 5 cells by transfect with miR-195 mimics. It was found the downregulated miR-195 and upregulated HIF-1α were present in hypoxic ATDC 5 cells. miR-195 negatively regulated HIF-1α by targeting its 3'-untranslated region. Moreover, the founding indicated miR-195 greatly increased apoptosis and downregulated HIF-1α mRNA occurred simultaneously in hypoxic chondrocytes. We concluded that miR-195 induced apoptosis in hypoxic chondrocytes by directly targeting HIF-1α.

  7. Ginsenoside Ro suppresses interleukin-1β-induced apoptosis and inflammation in rat chondrocytes by inhibiting NF-κB.

    Science.gov (United States)

    Zhang, Xiao-Hong; Xu, Xian-Xiang; Xu, Tao

    2015-04-01

    This study investigated effects of Ginsenoside Ro (Ro) on interleukin-1β (IL-1β)-induced apoptosis and inflammation in rat chondrocytes. The rat chondrocytes were co-treated with IL-1β (10 ng·kg(-1)) and Ro (50, 100 and 200 μmol·L(-1)) for 48 h. Chondrocytes viability was detected by the MTT assay and Annexin V-FITC/PI dual staining assay. Caspase 3 activity was measured by using caspase 3 colorimetric assay kit. Apoptosis related proteins Bax, Bad, Bcl-xL, PCNA, p53 and phospho-p53, along with inflammation related protein MMP 3, MMP 9 and COX-2, and the expression of phospho-NF-κB p65 were assayed by western blotting analyses. Ro could improve IL-1β-induced chondrocytes viability. Ro could suppress IL-1β-induced apoptosis by inhibiting levels of Bax and Bad, decreasing p53 phosphorylation and promoting the expression of Bcl-xL and PCNA. Ro inhibited caspase 3 activity. IL-1β-induced inflammation and matrix degration were also alleviated by Ro with down-regulating the expression of MMP 3, MMP 9 and COX-2. Moreover, Ro inhibited NF-κB p65 phosphorylation induced by IL-1β. In conclusion, these results suggested Ro exerted anti-apoptosis and anti-inflammation in IL-1β-induced rat chondrocytes, which might be related to NF-κB signal pathway. Therefore, we propose that Ro might be a potential novel drug for the treatment of osteoarthritis. Copyright © 2015 China Pharmaceutical University. Published by Elsevier B.V. All rights reserved.

  8. Effect of nitrogen-rich cell culture surfaces on type X collagen expression by bovine growth plate chondrocytes

    Directory of Open Access Journals (Sweden)

    Wertheimer Michael R

    2011-01-01

    Full Text Available Abstract Background Recent evidence indicates that osteoarthritis (OA may be a systemic disease since mesenchymal stem cells (MSCs from OA patients express type X collagen, a marker of late stage chondrocyte hypertrophy (associated with endochondral ossification. We recently showed that the expression of type X collagen was suppressed when MSCs from OA patients were cultured on nitrogen (N-rich plasma polymer layers, which we call "PPE:N" (N-doped plasma-polymerized ethylene, containing up to 36 atomic percentage (at.% of N. Methods In the present study, we examined the expression of type X collagen in fetal bovine growth plate chondrocytes (containing hypertrophic chondrocytes cultured on PPE:N. We also studied the effect of PPE:N on the expression of matrix molecules such as type II collagen and aggrecan, as well as on proteases (matrix metalloproteinase-13 (MMP-13 and molecules implicated in cell division (cyclin B2. Two other culture surfaces, "hydrophilic" polystyrene (PS, regular culture dishes and nitrogen-containing cation polystyrene (Primaria®, were also investigated for comparison. Results Results showed that type X collagen mRNA levels were suppressed when cultured for 4 days on PPE:N, suggesting that type X collagen is regulated similarly in hypertrophic chondrocytes and in human MSCs from OA patients. However, the levels of type X collagen mRNA almost returned to control value after 20 days in culture on these surfaces. Culture on the various surfaces had no significant effects on type II collagen, aggrecan, MMP-13, and cyclin B2 mRNA levels. Conclusion Hypertrophy is diminished by culturing growth plate chondrocytes on nitrogen-rich surfaces, a mechanism that is beneficial for MSC chondrogenesis. Furthermore, one major advantage of such "intelligent surfaces" over recombinant growth factors for tissue engineering and cartilage repair is potentially large cost-saving.

  9. Chondrocyte and mesenchymal stem cell-based therapies for cartilage repair in osteoarthritis and related orthopaedic conditions.

    Science.gov (United States)

    Mobasheri, Ali; Kalamegam, Gauthaman; Musumeci, Giuseppe; Batt, Mark E

    2014-07-01

    Osteoarthritis (OA) represents a final and common pathway for all major traumatic insults to synovial joints. OA is the most common form of degenerative joint disease and a major cause of pain and disability. Despite the global increase in the incidence of OA, there are no effective pharmacotherapies capable of restoring the original structure and function of damaged articular cartilage. Consequently cell-based and biological therapies for osteoarthritis (OA) and related orthopaedic disorders have become thriving areas of research and development. Autologous chondrocyte implantation (ACI) has been used for treatment of osteoarticular lesions for over two decades. Although chondrocyte-based therapy has the capacity to slow down the progression of OA and delay partial or total joint replacement surgery, currently used procedures are associated with the risk of serious adverse events. Complications of ACI include hypertrophy, disturbed fusion, delamination, and graft failure. Therefore there is significant interest in improving the success rate of ACI by improving surgical techniques and preserving the phenotype of the primary chondrocytes used in the procedure. Future tissue-engineering approaches for cartilage repair will also benefit from advances in chondrocyte-based repair strategies. This review article focuses on the structure and function of articular cartilage and the pathogenesis of OA in the context of the rising global burden of musculoskeletal disease. We explore the challenges associated with cartilage repair and regeneration using cell-based therapies that use chondrocytes and mesenchymal stem cells (MSCs). This paper also explores common misconceptions associated with cell-based therapy and highlights a few areas for future investigation. Copyright © 2014 The Authors. Published by Elsevier Ireland Ltd.. All rights reserved.

  10. A cell shrinkage artefact in growth plate chondrocytes with common fixative solutions: importance of fixative osmolarity for maintaining morphology

    Directory of Open Access Journals (Sweden)

    MY Loqman

    2010-05-01

    Full Text Available The remarkable increase in chondrocyte volume is a major determinant in the longitudinal growth of mammalian bones. To permit a detailed morphological study of hypertrophic chondrocytes using standard histological techniques, the preservation of normal chondrocyte morphology is essential. We noticed that during fixation of growth plates with conventional fixative solutions, there was a marked morphological (shrinkage artifact, and we postulated that this arose from the hyper-osmotic nature of these solutions. To test this, we fixed proximal tibia growth plates of 7-day-old rat bones in either (a paraformaldehyde (PFA; 4%, (b glutaraldehyde (GA; 2% with PFA (2% with ruthenium hexamine trichloride (RHT; 0.7%, (c GA (2% with RHT (0.7%, or (d GA (1.3% with RHT (0.5% and osmolarity adjusted to a ‘physiological’ level of ~280mOsm. Using conventional histological methods, confocal microscopy, and image analysis on fluorescently-labelled fixed and living chondrocytes, we then quantified the extent of cell shrinkage and volume change. Our data showed that the high osmolarity of conventional fixatives caused a shrinkage artefact to chondrocytes. This was particularly evident when whole bones were fixed, but could be markedly reduced if bones were sagittally bisected prior to fixation. The shrinkage artefact could be avoided by adjusting the osmolarity of the fixatives to the osmotic pressure of normal extracellular fluids (~280mOsm. These results emphasize the importance of fixative osmolarity, in order to accurately preserve the normal volume/morphology of cells within tissues.

  11. hWJECM-Derived Oriented Scaffolds with Autologous Chondrocytes for Rabbit Cartilage Defect Repairing.

    Science.gov (United States)

    Zhao, Peng; Liu, Shuyun; Bai, Yuhe; Lu, Shibi; Peng, Jiang; Zhang, Li; Huang, Jingxiang; Zhao, Bin; Xu, Wenjing; Guo, Quanyi

    2018-02-02

    Previously, we synthesized an articular cartilage extracellular matrix (ECM)-derived oriented scaffold for cartilage tissue engineering, which was biomimetic in terms of structure and biochemical composition. However, the limit resource of the cartilage-derived ECM is a hindrance for its application. In this study, we developed a new material for cartilage tissue engineering-human umbilical cord Wharton's jelly-derived ECM (hWJECM). The hWJECM has an abundant resource and similar biochemistry with cartilage ECM, and the use of it is not associated with ethical controversy. We adopted the method previously used in cartilage ECM-derived oriented scaffold preparation to generate the oriented hWJECM-derived scaffold, and the scaffold properties were tested in vitro and in vivo. The three-dimensional scaffold has a porous and well-oriented structure, with a mean pore diameter of ∼104 μm. Scanning electron microscopy and cell viability staining results demonstrated that the oriented scaffold has good biocompatibility and cell alignment. In addition, we used functional autologous chondrocytes to seed the hWJECM-derived oriented scaffold and tested the efficacy of the cell-scaffold constructs to repair the full-thickness articular cartilage defect in a rabbit model. Defects of 4 mm diameter were generated in the patellar grooves of the femurs of both knees and were implanted with chondrocyte-scaffold constructs (group A) or scaffolds alone (group B); rabbits with untreated defects were used as a control (group C). Six months after surgery, all defects in group A were filled completely with repaired tissue, and most of which were hyaline cartilage. In contrast, the defects in group B were filled partially with repaired tissue, and approximately half of these repaired tissues were hyaline cartilage. The defects in group C were only filled with fibrotic tissue. Histological grading score of group A was lower than those of groups B and C. Quantification of

  12. Biodistribution and Immunogenicity of Allogeneic Mesenchymal Stem Cells in a Rat Model of Intraarticular Chondrocyte Xenotransplantation

    Directory of Open Access Journals (Sweden)

    Maribel Marquina

    2017-11-01

    Full Text Available Xenogeneic chondrocytes and allogeneic mesenchymal stem cells (MSC are considered a potential source of cells for articular cartilage repair. We here assessed the immune response triggered by xenogeneic chondrocytes when injected intraarticularly, as well as the immunoregulatory effect of allogeneic bone marrow-derived MSC after systemic administration. To this end, a discordant xenotransplantation model was established by injecting three million porcine articular chondrocytes (PAC into the femorotibial joint of Lewis rats and monitoring the immune response. First, the fate of MSC injected using various routes was monitored in an in vivo imaging system. The biodistribution revealed a dependency on the injection route with MSC injected intravenously (i.v. succumbing early after 24 h and MSC injected intraperitoneally (i.p. lasting locally for at least 5 days. Importantly, no migration of MSC to the joint was detected in rats previously injected with PAC. MSC were then administered either i.v. 1 week before PAC injection or i.p. 3 weeks after to assess their immunomodulatory function on humoral and adaptive immune parameters. Anti-PAC IgM and IgG responses were detected in all PAC-injected rats with a peak at week 2 postinjection and reactivity remaining above baseline levels by week 18. IgG2a and IgG2b were the predominant and long-lasting IgG subtypes. By contrast, no anti-MSC antibody response was detected in the cohort injected with MSC only, but infusion of MSC before PAC injection temporarily augmented the anti-PAC antibody response. Consistent with a cellular immune response to PAC in PAC-injected rats, cytokine/chemokine profiling in serum by antibody array revealed a distinct pattern relative to controls characterized by elevation of multiple markers at week 2, as well as increases in proliferation in draining lymph nodes. Notably, systemic administration of allogeneic MSC under the described conditions did not diminish the immune

  13. Dynamic Mechanical Compression of Chondrocytes for Tissue Engineering: A Critical Review.

    Science.gov (United States)

    Anderson, Devon E; Johnstone, Brian

    2017-01-01

    Articular cartilage functions to transmit and translate loads. In a classical structure-function relationship, the tissue resides in a dynamic mechanical environment that drives the formation of a highly organized tissue architecture suited to its biomechanical role. The dynamic mechanical environment includes multiaxial compressive and shear strains as well as hydrostatic and osmotic pressures. As the mechanical environment is known to modulate cell fate and influence tissue development toward a defined architecture in situ , dynamic mechanical loading has been hypothesized to induce the structure-function relationship during attempts at in vitro regeneration of articular cartilage. Researchers have designed increasingly sophisticated bioreactors with dynamic mechanical regimes, but the response of chondrocytes to dynamic compression and shear loading remains poorly characterized due to wide variation in study design, system variables, and outcome measurements. We assessed the literature pertaining to the use of dynamic compressive bioreactors for in vitro generation of cartilaginous tissue from primary and expanded chondrocytes. We used specific search terms to identify relevant publications from the PubMed database and manually sorted the data. It was very challenging to find consensus between studies because of species, age, cell source, and culture differences, coupled with the many loading regimes and the types of analyses used. Early studies that evaluated the response of primary bovine chondrocytes within hydrogels, and that employed dynamic single-axis compression with physiologic loading parameters, reported consistently favorable responses at the tissue level, with upregulation of biochemical synthesis and biomechanical properties. However, they rarely assessed the cellular response with gene expression or mechanotransduction pathway analyses. Later studies that employed increasingly sophisticated biomaterial-based systems, cells derived from different

  14. MRI evaluation of a new scaffold-based allogenic chondrocyte implantation for cartilage repair

    Energy Technology Data Exchange (ETDEWEB)

    Dhollander, A.A.M., E-mail: Aad.Dhollander@Ugent.b [Department of Orthopaedic Surgery and Traumatology, Ghent University Hospital, De Pintelaan 185, 1P5, B9000 Gent (Belgium); Huysse, W.C.J., E-mail: Wouter.Huysse@Ugent.b [Department of Radiology, Ghent University Hospital, De Pintelaan 185, -1K12 IB, B9000 Gent (Belgium); Verdonk, P.C.M., E-mail: pverdonk@yahoo.co [Department of Orthopaedic Surgery and Traumatology, Ghent University Hospital, De Pintelaan 185, 1P5, B9000 Gent (Belgium); Verstraete, K.L., E-mail: Koenraad.Verstraete@Ugent.b [Department of Radiology, Ghent University Hospital, De Pintelaan 185, -1K12 IB, B9000 Gent (Belgium); Verdonk, R., E-mail: Rene.Verdonk@Ugent.b [Department of Orthopaedic Surgery and Traumatology, Ghent University Hospital, De Pintelaan 185, 1P5, B9000 Gent (Belgium); Verbruggen, G., E-mail: Gust.Verbruggen@Ugent.b [Laboratory of Connective Tissue Biology, Department of Rheumatology, Ghent University Hospital, De Pintelaan 185, Ghent (Belgium); Almqvist, K.F., E-mail: Fredrik.Almqvist@Ugent.b [Department of Orthopaedic Surgery and Traumatology, Ghent University Hospital, De Pintelaan 185, 1P5, B9000 Gent (Belgium)

    2010-07-15

    Aim: The present study was designed to evaluate the implantation of alginate beads containing human mature allogenic chondrocytes for the treatment of symptomatic cartilage defects of the knee. MRI was used for the morphological analysis of cartilage repair. The correlation between MRI findings and clinical outcome was also studied. Methods: A biodegradable, alginate-based biocompatible scaffold containing human mature allogenic chondrocytes was used for the treatment of symptomatic chondral and osteochondral lesions in the knee. Twenty-one patients were prospectively evaluated with use of the Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) and the Visual Analogue Scale (VAS) for pain preoperatively and at 3, 6, 9 and 12 months of follow-up. Of the 21 patients, 12 had consented to follow the postoperative MRI evaluation protocol. MRI data were analyzed based on the original MOCART (Magnetic Resonance Observation of Cartilage Repair Tissue) and modified MOCART scoring system. The correlation between the clinical outcome and MRI findings was evaluated. Results: A statistically significant clinical improvement became apparent after 6 months and patients continued to improve during the 12 months of follow-up. One of the two MRI scoring systems that were used, showed a statistically significant deterioration of the repair tissue at 1 year of follow-up. Twelve months after the operation complete filling or hypertrophy was found in 41.6%. Bone-marrow edema and effusion were seen in 41.7% and 25% of the study patients, respectively. We did not find a consistent correlation between the MRI criteria and the clinical results. Discussion: The present study confirmed the primary role of MRI in the evaluation of cartilage repair. Two MOCART-based scoring systems were used in a longitudinal fashion and allowed a practical and morphological evaluation of the repair tissue. However, the correlation between clinical outcome and MRI findings was poor. Further

  15. MRI evaluation of a new scaffold-based allogenic chondrocyte implantation for cartilage repair

    International Nuclear Information System (INIS)

    Dhollander, A.A.M.; Huysse, W.C.J.; Verdonk, P.C.M.; Verstraete, K.L.; Verdonk, R.; Verbruggen, G.; Almqvist, K.F.

    2010-01-01

    Aim: The present study was designed to evaluate the implantation of alginate beads containing human mature allogenic chondrocytes for the treatment of symptomatic cartilage defects of the knee. MRI was used for the morphological analysis of cartilage repair. The correlation between MRI findings and clinical outcome was also studied. Methods: A biodegradable, alginate-based biocompatible scaffold containing human mature allogenic chondrocytes was used for the treatment of symptomatic chondral and osteochondral lesions in the knee. Twenty-one patients were prospectively evaluated with use of the Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) and the Visual Analogue Scale (VAS) for pain preoperatively and at 3, 6, 9 and 12 months of follow-up. Of the 21 patients, 12 had consented to follow the postoperative MRI evaluation protocol. MRI data were analyzed based on the original MOCART (Magnetic Resonance Observation of Cartilage Repair Tissue) and modified MOCART scoring system. The correlation between the clinical outcome and MRI findings was evaluated. Results: A statistically significant clinical improvement became apparent after 6 months and patients continued to improve during the 12 months of follow-up. One of the two MRI scoring systems that were used, showed a statistically significant deterioration of the repair tissue at 1 year of follow-up. Twelve months after the operation complete filling or hypertrophy was found in 41.6%. Bone-marrow edema and effusion were seen in 41.7% and 25% of the study patients, respectively. We did not find a consistent correlation between the MRI criteria and the clinical results. Discussion: The present study confirmed the primary role of MRI in the evaluation of cartilage repair. Two MOCART-based scoring systems were used in a longitudinal fashion and allowed a practical and morphological evaluation of the repair tissue. However, the correlation between clinical outcome and MRI findings was poor. Further

  16. Snorc is a novel cartilage specific small membrane proteoglycan expressed in differentiating and articular chondrocytes

    DEFF Research Database (Denmark)

    Heinonen, J; Taipaleenmäki, H; Roering, P

    2011-01-01

    and interaction partners are still likely to be discovered. Our focus in this study was to characterize a novel cartilage specific gene that was identified in mouse limb cartilage during embryonic development. METHODS: Open access bioinformatics tools were used to characterize the gene, predicted protein...... subgroups. Cartilage specific expression was highest in proliferating and prehypertrophic zones during development, and in adult articular cartilage, expression was restricted to the uncalcified zone, including chondrocyte clusters in human osteoarthritic cartilage. Studies with experimental chondrogenesis...... and orthologs in vertebrate species. Immunohistochemistry and mRNA expression methodology were used to study tissue specific expression. Fracture callus and limb bud micromass culture were utilized to study the effects of BMP-2 during experimental chondrogenesis. Fusion protein with C-terminal HA...

  17. Repair of cartilage defects in osteoarthritis rats with induced pluripotent stem cell derived chondrocytes.

    Science.gov (United States)

    Zhu, Yanxia; Wu, Xiaomin; Liang, Yuhong; Gu, Hongsheng; Song, Kedong; Zou, Xuenong; Zhou, Guangqian

    2016-11-09

    The incapacity of articular cartilage (AC) for self-repair after damage ultimately leads to the development of osteoarthritis. Stem cell-based therapy has been proposed for the treatment of osteoarthritis (OA) and induced pluripotent stem cells (iPSCs) are becoming a promising stem cell source. Three steps were developed to differentiate human iPSCs into chondrocytes which were transplanted into rat OA models induced by monosodium iodoacetate (MIA). After 6 days embryonic body (EB) formation and 2 weeks differentiation, the gene and protein expression of Col2A1, GAG and Sox9 has significantly increased compare to undifferentiated hiPSCs. After 15 weeks transplantation, no immune responses were observed, micro-CT showed gradual engraftment and the improvement of subchondrol plate integrity, and histological examinations demonstrated articular cartilage matrix production. hiPSC could be an efficient and clinically translatable approach for cartilage tissue regeneration in OA cartilages.

  18. Use of fibroblast growth factor 2 for expansion of chondrocytes and tissue engineering

    Science.gov (United States)

    Martin, Ivan (Inventor); Freed, Lisa E. (Inventor); Langer, Robert (Inventor); Vunjak-Novakovic, Gordana (Inventor)

    2003-01-01

    The present invention provides an improved method for expanding cells for use in tissue engineering. In particular the method provides specific biochemical factors to supplement cell culture medium during the expansion process in order to reproduce events occurring during embryonic development with the goal of regenerating tissue equivalents that resemble natural tissues both structurally and functionally. These specific biochemical factors improve proliferation of the cells and are capable of de-differentiation mature cells isolated from tissue so that the differentiation potential of the cells is preserved. The bioactive molecules also maintain the responsiveness of the cells to other bioactive molecules. Specifically, the invention provides methods for expanding chondrocytes in the presence of fibroblast growth factor 2 for use in regeneration of cartilage tissue.

  19. Treatment of full thickness cartilage defects in human knees with Autologous Chondrocyte Transplantation

    Directory of Open Access Journals (Sweden)

    Khalilallah Nazem

    2011-01-01

    Full Text Available Background: Although a variety of strategies have been employed for managing articular cartilage defects in the knee, overall outcomes have not been satisfactory. An alternative option may be autologous chondrocyte transplantation (ACT. However, as this method is still under investigation, here we assessed the efficacy of ACT for human knee defect cartilage repair. Methods: In a randomized clinical trial study, eleven patients (mean age 31.09 years were enrolled in the study with full thickness cartilage defects in the knee. Arthroscopically, healthy cartilage was obtained, chondrocytes expanded for 2-3 weeks and ACT performed. Clinical status was evaluated before ACT, 6 and 12 months after ACT using the Brittberg-Peterson functional assessment and modified Cincinnati rating score. Magnetic resonance imaging (MRI findings were evaluated based on the scoring systems used by Sally Roberts and by Henderson. Results: Modified Cincinnati rating indicated significant improvement of clinical score before ACT compared to 6 (p = 0.000 and 12 (p = 0.000 months after ACT (from 2.73 before ACT to 7.27, 8.36 and 9.5 at 6, 12, and 48 months after ACT, respectively. Brittberg-Peterson functional assessment indicated a decline from 79.27 to 25.82 and 19.27 at 6 and 12 months post ACT. Further, statistical test demonstrated significant differences 6, 12 and 48 months post ACT (p = 0.007. Evaluation of MRI revealed a score of 6.5 for Henderson criteria and a score of 2.5 for Robert criteria. Conclusions: Our study demonstrated that ACT of the knee provides an excellent treatment for full thickness cartilage defects with outstanding clinical and radiological outcomes.

  20. Enhanced Chondrocyte Proliferation in a Prototyped Culture System with Wave-Induced Agitation

    Directory of Open Access Journals (Sweden)

    Pilarek Maciej

    2017-06-01

    Full Text Available One of the actual challenges in tissue engineering applications is to efficiently produce as high of number of cells as it is only possible, in the shortest time. In static cultures, the production of animal cell biomass in integrated forms (i.e. aggregates, inoculated scaffolds is limited due to inefficient diffusion of culture medium components observed in such non-mixed culture systems, especially in the case of cell-inoculated fiber-based dense 3D scaffolds, inside which the intensification of mass transfer is particularly important. The applicability of a prototyped, small-scale, continuously wave-induced agitated system for intensification of anchorage-dependent CP5 chondrocytes proliferation outside and inside three-dimensional poly(lactic acid (PLA scaffolds has been discussed. Fibrous PLA-based constructs have been inoculated with CP5 cells and then maintained in two independent incubation systems: (i non-agitated conditions and (ii culture with wave-induced agitation. Significantly higher values of the volumetric glucose consumption rate have been noted for the system with the wave-induced agitation. The advantage of the presented wave-induced agitation culture system has been confirmed by lower activity of lactate dehydrogenase (LDH released from the cells in the samples of culture medium harvested from the agitated cultures, in contrast to rather high values of LDH activity measured for static conditions. Results of the proceeded experiments and their analysis clearly exhibited the feasibility of the culture system supported with continuously wave-induced agitation for robust proliferation of the CP5 chondrocytes on PLA-based structures. Aside from the practicability of the prototyped system, we believe that it could also be applied as a standard method offering advantages for all types of the daily routine laboratory-scale animal cell cultures utilizing various fiber-based biomaterials, with the use of only regular laboratory

  1. The Spatial Organization of Glucosinolate Biosynthesis

    DEFF Research Database (Denmark)

    Nintemann, Sebastian

    between the individual classes of glucosinolates under constitutive and induced conditions and identified the source tissues of these defense compounds. Protein-protein interaction studies were carried out to investigate the subcellular organization of glucosinolate biosynthesis. We identified a family...... resistance and nutritional value and many plant specialized metabolites are of high value due to their health promoting characteristics. Glucosinolates are defense compounds found in many crops from the Brassicaceae family and are of high interest because of their nutritional and antinutritional properties...... cells is an open question. Likewise, it is not known how glucosinolate biosynthesis is orchestrated at the subcellular level. These open questions were addressed with several approaches in this project, with the aim of shedding light on the spatial organization of glucosinolate biosynthesis from...

  2. Functional and Evolutionary Relationship between Arginine Biosynthesis and Prokaryotic Lysine Biosynthesis through α-Aminoadipate

    Science.gov (United States)

    Miyazaki, Junichi; Kobashi, Nobuyuki; Nishiyama, Makoto; Yamane, Hisakazu

    2001-01-01

    Our previous studies revealed that lysine is synthesized through α-aminoadipate in an extremely thermophilic bacterium, Thermus thermophilus HB27. Sequence analysis of a gene cluster involved in the lysine biosynthesis of this microorganism suggested that the conversion from α-aminoadipate to lysine proceeds in a way similar to that of arginine biosynthesis. In the present study, we cloned an argD homolog of T. thermophilus HB27 which was not included in the previously cloned lysine biosynthetic gene cluster and determined the nucleotide sequence. A knockout of the argD-like gene, now termed lysJ, in T. thermophilus HB27 showed that this gene is essential for lysine biosynthesis in this bacterium. The lysJ gene was cloned into a plasmid and overexpressed in Escherichia coli, and the LysJ protein was purified to homogeneity. When the catalytic activity of LysJ was analyzed in a reverse reaction in the putative pathway, LysJ was found to transfer the ɛ-amino group of N2-acetyllysine, a putative intermediate in lysine biosynthesis, to 2-oxoglutarate. When N2-acetylornithine, a substrate for arginine biosynthesis, was used as the substrate for the reaction, LysJ transferred the δ-amino group of N2-acetylornithine to 2-oxoglutarate 16 times more efficiently than when N2-acetyllysine was the amino donor. All these results suggest that lysine biosynthesis in T. thermophilus HB27 is functionally and evolutionarily related to arginine biosynthesis. PMID:11489859

  3. In vitro evaluation of chondrosarcoma cells and canine chondrocytes on layer-by-layer (LbL) self-assembled multilayer nanofilms

    International Nuclear Information System (INIS)

    Shaik, J; Mohammed, J Shaikh; McShane, M J; Mills, D K

    2013-01-01

    Short-term cell–substrate interactions of two secondary chondrocyte cell lines (human chondrosarcoma cells, canine chondrocytes) with layer-by-layer self-assembled multilayer nanofilms were investigated for a better understanding of cellular-behaviour dependence on a number of nanofilm layers. Cell–substrate interactions were studied on polyelectrolyte multilayer nanofilms (PMNs) of eleven different biomaterials. Surface characterization of PMNs performed using AFM showed increasing surface roughness with increasing number of layers for most of the biomaterials. LDH-L and MTT assays were performed on chondrosarcoma cells and canine chondrocytes, respectively. A major observation was that 10-bilayer nanofilms exhibited lesser cytotoxicity towards human chondrosarcoma cells than their 5-bilayer counterparts. In the case of canine chondrocytes, BSA enhanced cell metabolic activity with increasing number of layers, underscoring the importance of the multilayer nanofilm architecture on cellular behaviour. (paper)

  4. Hydrostatic compress force enhances the viability and decreases the apoptosis of condylar chondrocytes through integrin-FAK-ERK/PI3K pathway

    NARCIS (Netherlands)

    Ma, D.; Kou, X.; Jin, J.; Xu, T.; Wu, M.; Deng, L.; Fu, L.; Liu, Y.; Wu, G.; Lu, H.

    2016-01-01

    Reduced mechanical stimuli in many pathological cases, such as hemimastication and limited masticatory movements, can significantly affect the metabolic activity of mandibular condylar chondrocytes and the growth of mandibles. However, the molecular mechanisms for these phenomena remain unclear. In

  5. Nucleoside antibiotics: biosynthesis, regulation, and biotechnology.

    Science.gov (United States)

    Niu, Guoqing; Tan, Huarong

    2015-02-01

    The alarming rise in antibiotic-resistant pathogens has coincided with a decline in the supply of new antibiotics. It is therefore of great importance to find and create new antibiotics. Nucleoside antibiotics are a large family of natural products with diverse biological functions. Their biosynthesis is a complex process through multistep enzymatic reactions and is subject to hierarchical regulation. Genetic and biochemical studies of the biosynthetic machinery have provided the basis for pathway engineering and combinatorial biosynthesis to create new or hybrid nucleoside antibiotics. Dissection of regulatory mechanisms is leading to strategies to increase the titer of bioactive nucleoside antibiotics. Copyright © 2014. Published by Elsevier Ltd.

  6. Combinatorial Biosynthesis of Polyketides – A Perspective

    Science.gov (United States)

    Wong, Fong T.; Khosla, Chaitan

    2012-01-01

    Since their discovery, polyketide synthases have been attractive targets of biosynthetic engineering to make “unnatural” natural products. Although combinatorial biosynthesis has made encouraging advances over the past two decades, the field remains in its infancy. In this enzyme-centric perspective, we discuss the scientific and technological challenges that could accelerate the adoption of combinatorial biosynthesis as a method of choice for the preparation of encoded libraries of bioactive small molecules. Borrowing a page from the protein structure prediction community, we propose a periodic challenge program to vet the most promising methods in the field, and to foster the collective development of useful tools and algorithms. PMID:22342766

  7. Method for determining heterologous biosynthesis pathways

    KAUST Repository

    Gao, Xin

    2017-08-10

    The present invention relates to a method and system for dynamically analyzing, determining, predicting and displaying ranked suitable heterologous biosynthesis pathways for a specified host. The present invention addresses the problem of finding suitable pathways for the endogenous metabolism of a host organism because the efficacy of heterologous biosynthesis is affected by competing endogenous pathways. The present invention is called MRE (Metabolic Route Explorer), and it was conceived and developed to systematically and dynamically search for, determine, analyze, and display promising heterologous pathways while considering competing endogenous reactions in a given host organism.

  8. Convergent Evolution of Ergothioneine Biosynthesis in Cyanobacteria.

    Science.gov (United States)

    Liao, Cangsong; Seebeck, Florian P

    2017-11-02

    Biosynthesis of N-α-trimethyl-2-thiohistidine (ergothioneine) is a frequent trait in cyanobacteria. This sulfur compound may provide essential relief from oxidative stress related to oxygenic photosynthesis. The central steps in ergothioneine biosynthesis are catalyzed by a histidine methyltransferase and an iron-dependent sulfoxide synthase. In this report, we present evidence that some cyanobacteria recruited and adapted a sulfoxide synthase from a different biosynthetic pathway to make ergothioneine. The discovery of a second origin of ergothioneine production underscores the physiological importance of this metabolite and highlights the evolutionary malleability of the thiohistidine biosynthetic machinery. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  9. Chondrocyte viability is higher after prolonged storage at 37 degrees C than at 4 degrees C for osteochondral grafts.

    Science.gov (United States)

    Pallante, Andrea L; Bae, Won C; Chen, Albert C; Görtz, Simon; Bugbee, William D; Sah, Robert L

    2009-11-01

    Osteochondral allografts are currently stored at 4 degrees C for 2 to 6 weeks before implantation. At 4 degrees C, chondrocyte viability, especially in the superficial zone, deteriorates starting at 2 weeks. Alternative storage conditions could maintain chondrocyte viability beyond 2 weeks, and thereby facilitate increased graft availability and enhanced graft quality. The objective of the study was to determine the effects of prolonged 37 degrees C storage compared with traditional 4 degrees C storage on chondrocyte viability and cartilage matrix content. Controlled laboratory study. Osteochondral samples from humeral heads of adult goats were analyzed (i) fresh, or after storage in medium for (ii) 14 days at 4 degrees C including 10% fetal bovine serum, (iii) 28 days at 4 degrees C including 10% fetal bovine serum, (iv) 28 days at 37 degrees C without fetal bovine serum, (v) 28 days at 37 degrees C including 2% fetal bovine serum, or (vi) 28 days at 37 degrees C including 10% fetal bovine serum. Portions of samples were analyzed by microscopy after LIVE/DEAD staining to determine chondrocyte viability and density, both en face (to visualize the articular surface) and vertically (overall and in superficial, middle, and deep zones). The remaining cartilage was analyzed for sulfated glycosaminoglycan and collagen. The 37 degrees C storage maintained high chondrocyte viability compared with 4 degrees C storage. Viability of samples after 28 days at 37 degrees C was approximately 80% at the cartilage surface en face, approximately 65% in the superficial zone, and approximately 70% in the middle zone, which was much higher than approximately 45%, approximately 20%, and approximately 35%, respectively, in 4 degrees C samples after 28 days, and slightly decreased from approximately 100%, approximately 85%, and approximately 95%, respectively, in fresh controls. Cartilage thickness, glycosaminoglycan content, and collagen content were maintained for 37 degrees C and 4

  10. Synergistic interactions of bradykinin, thrombin, interleukin 1 and tumor necrosis factor on prostanoid biosynthesis in human periodontal-ligament cells.

    Science.gov (United States)

    Ransjö, M; Marklund, M; Persson, M; Lerner, U H

    1998-04-01

    Prostaglandins are involved in force-induced orthodontic tooth movement. Bradykinin (BK) and thrombin are known to cause a significant time- and concentration-dependent burst of prostanoid biosynthesis in cultured human periodontal-ligament (PDL) cells. The aim now was to investigate interactive effects between interleukin 1 alpha, -beta (IL-1 alpha, -1 beta), tumour necrosis factor-alpha,-beta (TNF-alpha, -beta) and BK or thrombin on prostaglandin biosynthesis in human PDL cells. IL-1 alpha and -1 beta produced time- and concentration-dependent stimulation of prostanoid biosynthesis [prostaglandin (PG)E2 and 6-keto-PGF1alpha]. Synergistic stimulation of prostanoid biosynthesis was demonstrated when BK or thrombin were added together with IL-1 alpha or -1 beta. BK and IL-1 beta both significantly stimulated the release of [3H]arachidonic acid. No synergistic effect on [3H]arachidonic acid release was seen when BK and IL-1 beta were added simultaneously. These data suggest that the synergistic effect of BK and IL-1 beta on prostanoid biosynthesis is not due to interactions at the receptor level nor to enhanced release of arachidonic acid, but may be due to increased activity of cyclo-oxygenase. Also, TNF-alpha and -beta produced a concentration-dependent stimulation of PGE2 formation in cultured human PDL cells. Synergistic effects of BK and thrombin were demonstrated when PGE2 production was stimulated in combination with TNF-beta. In addition, a synergistic effect on the PGE2 response to IL-1 alpha or -1 beta was demonstrated when added in combination with TNF-alpha. These experiments demonstrate synergistic interactions between BK, thrombin, IL-1 and TNF on prostaglandin biosynthesis in cultured human PDL cells. The findings suggest that inflammatory mediators may act in concert in stimulating prostanoid production in response to pro-inflammatory stimuli. As an inflammatory reaction is seen in the periodontal ligament when teeth are orthodontically treated, this

  11. Biosynthesis of silver nanoparticles by Aspergillus niger , Fusarium ...

    African Journals Online (AJOL)

    ... scanning electron microscope (SEM). Results indicate the synthesis of silver nanoparticles in the reaction mixture. The synthesis of nanoparticles would be suitable for developing a microbial nanotechnology biosynthesis process for mass scale production. Keywords: Silver nanoparticles, biosynthesis, fungi, Aspergillus.

  12. Effects of non-steroidal anti-inflammatory drugs on cell proliferation and death in cultured epiphyseal-articular chondrocytes of fetal rats

    International Nuclear Information System (INIS)

    Chang, J.-K.; Wu, S.-C.; Wang, G.-J.

    2006-01-01

    Previous reports indicated that non-steroidal anti-inflammatory drugs (NSAIDs) suppress bone repair. Our previous study further found that ketorolac delayed the endochondral bone formation, and the critical effective timing was at the early stage of repair. Furthermore, we found that NSAIDs suppressed proliferation and induced cell death of cultured osteoblasts. In this study, we hypothesized that chondrocytic proliferation and death, which plays an important role at the early stage of endochondral bone formation, might be affected by NSAIDs. Non-selective NSAIDs, indomethacin, ketorolac, diclofenac and piroxicam; cyclooxygenase-2 (COX-2) selective NSAIDs, celecoxib and DFU (an analog of rofecoxib); prostaglandins (PGs), PGE1, PGE2 and PGF2α; and each NSAID plus each PG were tested. The effects of NSAIDs on proliferation, cell cycle kinetics, cytotoxicity and cell death of epiphyseal-articular chondrocytes of fetal rats were examined. The results showed that all the tested NSAIDs, except DFU, inhibited thymidine incorporation of chondrocytes at a concentration range (10 -8 to 10 -4 M) covering the theoretic therapeutic concentrations. Cell cycle was arrested by NSAIDs at the G /G 1 phase. Upon a 24 h treatment, LDH leakage and cell death (both apoptosis and necrosis) were significantly induced by the four non-selective NSAIDs in chondrocyte cultures. However, COX-2 inhibitors revealed non-significant effects on cytotoxicity of chondrocytes except higher concentration of celecoxib (10 -4 M). Replenishments of PGE1, PGE2 or PGF2α could not reverse the effects of NSAIDs on chondrocytic proliferation and cytotoxicity. In this study, we found that therapeutic concentrations of non-selective NSAIDs caused proliferation suppression and cell death of chondrocytes, suggesting these adverse effects may be one of the reasons that NSAIDs delay the endochondral ossification during bone repair found in previous studies. Furthermore, these effects of NSAIDs may act via PG

  13. Evaluation of nonbiomedical and biomedical grade alginates for the transplantation of genetically modified articular chondrocytes to cartilage defects in a large animal model in vivo.

    Science.gov (United States)

    Heiligenstein, Susanne; Cucchiarini, Magali; Laschke, Matthias W; Bohle, Rainer M; Kohn, Dieter; Menger, Michael D; Madry, Henning

    2011-04-01

    Genetically modified chondrocytes embedded in alginate improve cartilage repair in experimental models, and alginates are clinically used for articular chondrocyte transplantation. In the present study, we tested the hypothesis that the alginate system allows for sustained transgene expression in cartilage defects in a preclinical large animal model in vivo. Primary cultures of ovine articular chondrocytes were transfected with the Photinus pyralis luc or the Escherichia coli lacZ genes in monolayer culture in vitro using eight different nonviral compounds. Optimally transfected chondrocytes were encapsulated in spheres composed of nonbiomedical or biomedical grade alginates for evaluation of luciferase expression, cell numbers and viabilities in vitro. Transfected chondrocytes encapsulated in spheres comprised of the different alginates were then implanted into osteochondral defects in the knee joints of sheep to examine the profiles of transgene expression in vivo. Ovine articular chondrocytes were efficiently transfected with FuGENE 6. Transgene expression was detectable after encapsulation in the alginates over 21 days in vitro. Transplantation of genetically modified chondrocytes to cartilage defects in vivo resulted in maximal transgene expression on day 1 after transfection, with a decrease by day 21, the longest time point evaluated. Remarkably, the reduction in luciferase activity was less pronounced when biomedical grade alginates were employed, compared to nonbiomedical grade alginates, suggesting that such alginates might be better suited to support elevated transgene expression after transplantation of genetically modified chondrocytes. This approach may be of value to study the effects of potential therapeutic genes upon cartilage repair in a clinically relevant setting. Copyright © 2011 John Wiley & Sons, Ltd.

  14. Mutations in fam20b and xylt1 Reveal That Cartilage Matrix Controls Timing of Endochondral Ossification by Inhibiting Chondrocyte Maturation

    Science.gov (United States)

    Eames, B. Frank; Yan, Yi-Lin; Swartz, Mary E.; Levic, Daniel S.; Knapik, Ela W.; Postlethwait, John H.; Kimmel, Charles B.

    2011-01-01

    Differentiating cells interact with their extracellular environment over time. Chondrocytes embed themselves in a proteoglycan (PG)-rich matrix, then undergo a developmental transition, termed “maturation,” when they express ihh to induce bone in the overlying tissue, the perichondrium. Here, we ask whether PGs regulate interactions between chondrocytes and perichondrium, using zebrafish mutants to reveal that cartilage PGs inhibit chondrocyte maturation, which ultimately dictates the timing of perichondral bone development. In a mutagenesis screen, we isolated a class of mutants with decreased cartilage matrix and increased perichondral bone. Positional cloning identified lesions in two genes, fam20b and xylosyltransferase1 (xylt1), both of which encode PG synthesis enzymes. Mutants failed to produce wild-type levels of chondroitin sulfate PGs, which are normally abundant in cartilage matrix, and initiated perichondral bone formation earlier than their wild-type siblings. Primary chondrocyte defects might induce the bone phenotype secondarily, because mutant chondrocytes precociously initiated maturation, showing increased and early expression of such markers as runx2b, collagen type 10a1, and ihh co-orthologs, and ihha mutation suppressed early perichondral bone in PG mutants. Ultrastructural analyses demonstrated aberrant matrix organization and also early cellular features of chondrocyte hypertrophy in mutants. Refining previous in vitro reports, which demonstrated that fam20b and xylt1 were involved in PG synthesis, our in vivo analyses reveal that these genes function in cartilage matrix production and ultimately regulate the timing of skeletal development. PMID:21901110

  15. Mutations in fam20b and xylt1 reveal that cartilage matrix controls timing of endochondral ossification by inhibiting chondrocyte maturation.

    Directory of Open Access Journals (Sweden)

    B Frank Eames

    2011-08-01

    Full Text Available Differentiating cells interact with their extracellular environment over time. Chondrocytes embed themselves in a proteoglycan (PG-rich matrix, then undergo a developmental transition, termed "maturation," when they express ihh to induce bone in the overlying tissue, the perichondrium. Here, we ask whether PGs regulate interactions between chondrocytes and perichondrium, using zebrafish mutants to reveal that cartilage PGs inhibit chondrocyte maturation, which ultimately dictates the timing of perichondral bone development. In a mutagenesis screen, we isolated a class of mutants with decreased cartilage matrix and increased perichondral bone. Positional cloning identified lesions in two genes, fam20b and xylosyltransferase1 (xylt1, both of which encode PG synthesis enzymes. Mutants failed to produce wild-type levels of chondroitin sulfate PGs, which are normally abundant in cartilage matrix, and initiated perichondral bone formation earlier than their wild-type siblings. Primary chondrocyte defects might induce the bone phenotype secondarily, because mutant chondrocytes precociously initiated maturation, showing increased and early expression of such markers as runx2b, collagen type 10a1, and ihh co-orthologs, and ihha mutation suppressed early perichondral bone in PG mutants. Ultrastructural analyses demonstrated aberrant matrix organization and also early cellular features of chondrocyte hypertrophy in mutants. Refining previous in vitro reports, which demonstrated that fam20b and xylt1 were involved in PG synthesis, our in vivo analyses reveal that these genes function in cartilage matrix production and ultimately regulate the timing of skeletal development.

  16. 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.

    Directory of Open Access Journals (Sweden)

    Fabrizio Vincenzi

    Full Text Available 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

  17. 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

    Science.gov (United States)

    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

  18. Biosynthesis of NAD from nicotinic acid and nicotinamide by resting cells of Arthrobacter globiformis

    International Nuclear Information System (INIS)

    Kuwahara, Masaaki

    1978-01-01

    Isotopically labeled nicotinic acid and nicotinamide were incorporated into the metabolites of nicotinic acid-dependent pathway (Preiss-Handler pathway) of the NAD biosynthesis by resting cells of Arthrobacter globiformis. Azaserine and adenosine markedly stimulated the accumulation of NAD in the cells. Radioactive nicotinic acid and nicotinamide were also incorporated into an unknown compound when the cells were incubated in the presence of azaserine. Cell-free extract of the organism showed the NAD synthetase activity, which required ammonium ion and ATP for the amidation of deamido-NAD. Adenosine inhibited the enzyme activity. The organism possessed nicotinamidase, suggesting deamidation is the first step in the biosynthesis of NAD from nicotinamide. The activity was inhibited by NAD, NADP and NMN. (auth.)

  19. How peroxisomes affect aflatoxin biosynthesis in Aspergillus flavus.

    Directory of Open Access Journals (Sweden)

    Massimo Reverberi

    Full Text Available In filamentous fungi, peroxisomes are crucial for the primary metabolism and play a pivotal role in the formation of some secondary metabolites. Further, peroxisomes are important site for fatty acids β-oxidation, the formation of reactive oxygen species and for their scavenging through a complex of antioxidant activities. Oxidative stress is involved in different metabolic events in all organisms and it occurs during oxidative processes within the cell, including peroxisomal β-oxidation of fatty acids. In Aspergillus flavus, an unbalance towards an hyper-oxidant status into the cell is a prerequisite for the onset of aflatoxin biosynthesis. In our preliminary results, the use of bezafibrate, inducer of both peroxisomal β-oxidation and peroxisome proliferation in mammals, significantly enhanced the expression of pex11 and foxA and stimulated aflatoxin synthesis in A. flavus. This suggests the existence of a correlation among peroxisome proliferation, fatty acids β-oxidation and aflatoxin biosynthesis. To investigate this correlation, A. flavus was transformed with a vector containing P33, a gene from Cymbidium ringspot virus able to induce peroxisome proliferation, under the control of the promoter of the Cu,Zn-sod gene of A. flavus. This transcriptional control closely relates the onset of the antioxidant response to ROS increase, with the proliferation of peroxisomes in A. flavus. The AfP33 transformant strain show an up-regulation of lipid metabolism and an higher content of both intracellular ROS and some oxylipins. The combined presence of a higher amount of substrates (fatty acids-derived, an hyper-oxidant cell environment and of hormone-like signals (oxylipins enhances the synthesis of aflatoxins in the AfP33 strain. The results obtained demonstrated a close link between peroxisome metabolism and aflatoxin synthesis.

  20. Biosynthesis and metabolic pathways of pivalic acid

    Czech Academy of Sciences Publication Activity Database

    Řezanka, Tomáš; Kolouchová, I.; Čejková, A.; Sigler, Karel

    2012-01-01

    Roč. 95, č. 6 (2012), s. 1371-1376 ISSN 0175-7598 R&D Projects: GA ČR(CZ) GAP503/11/0215 Institutional support: RVO:61388971 Keywords : Pivalic acid * Isooctane * Biosynthesis Subject RIV: EE - Microbiology, Virology Impact factor: 3.689, year: 2012

  1. Combinatorial biosynthesis of medicinal plant secondary metabolites

    NARCIS (Netherlands)

    Julsing, Mattijs K.; Koulman, Albert; Woerdenbag, Herman J.; Quax, Wim J.; Kayser, Oliver

    2006-01-01

    Combinatorial biosynthesis is a new tool in the generation of novel natural products and for the production of rare and expensive natural products. The basic concept is combining metabolic pathways in different organisms on a genetic level. As a consequence heterologous organisms provide precursors

  2. Biosynthesis of silver nanoparticles synthesized by Aspergillus

    Indian Academy of Sciences (India)

    In the present study, biosynthesis of silver nanoparticles and its antioxidant, antimicrobial and cytotoxic activities were investigated. Silver nanoparticles were extracellularly synthesized using Aspergillus flavus and the formation of nanoparticles was observed after 72 h of incubation. The results recorded from colour ...

  3. Bile acid biosynthesis and its regulation

    Directory of Open Access Journals (Sweden)

    Areta Hebanowska

    2010-10-01

    Full Text Available Bile acid biosynthesis is the main pathway of cholesterol catabolism. Bile acids are more soluble than cholesterol so are easier to excrete. As amphipathic molecules they participate in lipid digestion and absorption in the intestine and they help to excrete free cholesterol with bile. They are also ligands for nuclear receptors regulating the expression of genes involved in cholesterol metabolism. Interconversion of cholesterol into bile acids is an important point of its homeostasis. Seventeen enzymes are engaged in this process and many of them are cytochromes P450. Bile acid synthesis initiation may proceed with the “classical” pathway (starting with cholesterol hydroxylation at the C7α position or the “alternative” pathway (starting with cholesterol hydroxylation at the C27 position. Two additional pathways are possible, though their quantitative significance is small (initiated with cholesterol hydroxylations of C24 and C25 positions. Oxysterols produced are not only intermediates of bile acid biosynthesis but also important regulators of metabolism. Bile acid biosynthesis takes place in the liver, but some enzymes are also present in other organs, where they participate in regulation of cholesterol metabolism. Those enzymes are potential targets for new drugs against cholesterol metabolism disturbances. This article is a brief description of the bile acid biosynthesis pathway and participating enzymes.

  4. Biosynthesis of polyhydroxyalkanotes in wildtype yeasts | Desuoky ...

    African Journals Online (AJOL)

    Biosynthesis of the biodegradable polymers polyhydroxyalkanotes (PHAs) are studied extensively in wild type and genetically modified prokaryotic cells, however the content and structure of PHA in wild type yeasts are not well documented. The purpose of this study was to screen forty yeast isolates collected from different ...

  5. Unedoside derivatives in Nuxia and their biosynthesis

    DEFF Research Database (Denmark)

    Jensen, Søren Rosendal; Ravnkilde, Lene; Schripsema, Jan

    1998-01-01

    isolated, while from N. oppositifolia 2 "-acetyl-3 "-benzoyl-nuxioside was obtained. Both plants contained verbascoside. The biosynthesis of unedoside in N. floribunda was investigated and deoxyloganic acid was found to be a precursor, similar to wh;lt was found for the eight-carbon iridoids in Thunbergia...

  6. Biosynthesis of furanochromones in Pimpinella monoica

    Indian Academy of Sciences (India)

    polyketide origin of their aromatic and pyrone rings while the furan ring originates via an acetate-mevalonate pathway. The plant also utilises glycine and leucine as substrate via acetate. Biotransformation of 3-H-visnagin to (6) but not to (2) was also observed. Keywords. Biosynthesis; furochromones; polyketide origin; ...

  7. Biosynthesis of silver nanoparticles synthesized by Aspergillus ...

    Indian Academy of Sciences (India)

    In the present study, biosynthesis of silver nanoparticles and its antioxidant, antimicrobial and cytotoxic activities were investigated. Silver nanoparticles were extracellularly synthesized using Aspergillus flavus and the formation of nanoparticles was observed after 72 h of incubation. The results recorded from colour ...

  8. Temperature changes and chondrocyte death during drilling in a bovine cartilage model and chondroprotection by modified irrigation solutions.

    Science.gov (United States)

    Farhan-Alanie, Muhamed M H; Hall, Andrew C

    2014-11-01

    Drilling into cartilage/bone is often required for orthopaedic surgery. While drilling into bone has been studied, the response of cartilage has received little attention. We have measured cartilage and drill bit temperatures during drilling and quantified the zone of chondrocyte death (ZCD) around the hole in the presence/absence of irrigation solutions. Drilling was performed using a 1.5-mm orthopaedic drill bit applied to bovine metatarsophalangeal joints and temperatures recorded by infrared camera. Osteochondral explants were then incubated with 5-chloromethylfluorescein diacetate (CMFDA) and propidium iodide (PI) to label living/dead chondrocytes respectively. The width of the ZCD was quantified by confocal laser scanning microscopy (CLSM) and image analysis. Without irrigation, the ZCD following drilling for two seconds was 135 ± 15 μm and this increased (>fourfold, P Irrigation reduced the ZCD following drilling for both two and five seconds (P irrigation, drill bit and cartilage temperature increased rapidly to >265 and 119 °C respectively, whereas the camera saturated at >282 °C during drilling for five seconds. With irrigation, the drill bit temperature was significantly reduced during drilling for two and five seconds (approx. 90 °C) with negligible change in cartilage temperature. Drilling while irrigating with hyperosmotic saline (600 mOsm) reduced (P irrigation markedly suppressed, but did not abolish chondrocyte death. Optimising the irrigation solution by raising osmolarity and reducing Ca(2+) content significantly reduced chondrocyte death during drilling and may be clinically beneficial.

  9. Effects of platelet rich plasma and chondrocyte co-culture on MSC chondrogenesis, hypertrophy and pathological responses

    OpenAIRE

    Ramezanifard, Rouhallah; Kabiri, Mahboubeh; Hanaee Ahvaz, Hana

    2017-01-01

    Regarding the inadequate healing capability of cartilage tissue, cell-based therapy is making the future of cartilage repair and regeneration. Mesenchymal stem cells (MSC) have shown great promise in cartilage regeneration. However, a yet-unresolved issue is the emergence of hypertrophic and pathologic markers during in vitro MSC chondrogenesis. Articular chondrocytes (AC) can suppress the undesired hypertrophy when co-cultured with MSC. On the other hand, platelet rich plasma (PRP), is consi...

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

    Science.gov (United States)

    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

  11. Conditional inactivation of TNFα-converting enzyme in chondrocytes results in an elongated growth plate and shorter long bones.

    Directory of Open Access Journals (Sweden)

    Kenta Saito

    Full Text Available TNFα-converting enzyme (TACE is a membrane-bound proteolytic enzyme with essential roles in the functional regulation of TNFα and epidermal growth factor receptor (EGFR ligands. Previous studies have demonstrated critical roles for TACE in vivo, including epidermal development, immune response, and pathological neoangiogenesis, among others. However, the potential contribution of TACE to skeletal development is still unclear. In the present study, we generated a Tace mutant mouse in which Tace is conditionally disrupted in chondrocytes under the control of the Col2a1 promoter. These mutant mice were fertile and viable but all exhibited long bones that were approximately 10% shorter compared to those of wild-type animals. Histological analyses revealed that Tace mutant mice exhibited a longer hypertrophic zone in the growth plate, and there were fewer osteoclasts at the chondro-osseous junction in the Tace mutant mice than in their wild-type littermates. Of note, we found an increase in osteoprotegerin transcripts and a reduction in Rankl and Mmp-13 transcripts in the TACE-deficient cartilage, indicating that dysregulation of these genes is causally related to the skeletal defects in the Tace mutant mice. Furthermore, we also found that phosphorylation of EGFR was significantly reduced in the cartilage tissue lacking TACE, and that suppression of EGFR signaling increases osteoprotegerin transcripts and reduces Rankl and Mmp-13 transcripts in primary chondrocytes. In accordance, chondrocyte-specific abrogation of Egfr in vivo resulted in skeletal defects nearly identical to those observed in the Tace mutant mice. Taken together, these data suggest that TACE-EGFR signaling in chondrocytes is involved in the turnover of the growth plate during postnatal development via the transcriptional regulation of osteoprotegerin, Rankl, and Mmp-13.

  12. Molecular analysis of expansion, differentiation, and growth factor treatment of human chondrocytes identifies differentiation markers and growth-related genes.

    Science.gov (United States)

    Benz, Karin; Breit, Stephen; Lukoschek, Martin; Mau, Hans; Richter, Wiltrud

    2002-04-26

    This study is intended to optimise expansion and differentiation of cultured human chondrocytes by growth factor application and to identify molecular markers to monitor their differentiation state. We dissected the molecular consequences of matrix release, monolayer, and 3D-alginate culture, growth factor optimised expansion, and re-differentiation protocols by gene expression analysis. Among 19 common cartilage molecules assessed by cDNA array, six proved best to monitor differentiation. Instant down-regulation at release of cells from the matrix was strongest for COL 2A1, fibromodulin, and PRELP while LUM, CHI3L1, and CHI3L2 were expansion-related. Both gene sets reflected the physiologic effects of the most potent growth-inducing (PDGF-BB) and proteoglycan-inducing (BMP-4) factors. Only CRTAC1 expression correlated with 2D/3D switches while the molecular phenotype of native chondrocytes was not restored. The markers and optimised protocols we suggest can help to improve cell therapy of cartilage defects and chondrocyte differentiation from stem cell sources.

  13. H2O2 INDUCES APOPTOSIS OF RABBIT CHONDROCYTES VIA BOTH THE EXTRINSIC AND THE CASPASE-INDEPENDENT INTRINSIC PATHWAYS

    Directory of Open Access Journals (Sweden)

    CAIPING ZHUANG

    2013-07-01

    Full Text Available Osteoarthritis (OA, one of the most common joint diseases with unknown etiology, is characterized by the progressive destruction of articular cartilage and the apoptosis of chondrocytes. The purpose of this study is to elucidate the molecular mechanisms of H2O2-mediated rabbit chondrocytes apoptosis. CCK-8 assay showed that H2O2 treatment induced a remarkable reduction of cell viability, which was further verified by the remarkable phosphatidylserine externalization after H2O2 treatment for 1 h, the typical characteristics of apoptosis. H2O2 treatment induced a significant dysfunction of mitochondrial membrane potential (ΔΨm, but did not induce casapse-9 activation, indicating that H2O2 treatment induced caspase-independent intrinsic apoptosis that was further verified by the fact that silencing of AIF but not inhibiting caspase-9 potently prevented H2O2-induced apoptosis. H2O2 treatment induced a significant increase of caspase-8 and -3 activation, and inhibition of caspase-8 or -3 significantly prevented H2O2-induced apoptosis, suggesting that the extrinsic pathway played an important role. Collectively, our findings demonstrate that H2O2 induces apoptosis via both the casapse-8-mediated extrinsic and the caspase-independent intrinsic apoptosis pathways in rabbit chondrocytes.

  14. Extraneuronal monoamine transporter mediates the permissive action of cortisol in the Guinea pig trachea: possible involvement of tracheal chondrocytes.

    Directory of Open Access Journals (Sweden)

    Chen Wang

    Full Text Available Cortisol, a member of glucocorticoids, could potentiate the action of catecholamine by a non-genomic mechanism. Although this permissive effect has been well appreciated in the anti-asthmatic medication, the underlying signaling pathway has remained mysterious. Here, we show that extraneuronal monoamine transporter (EMT, a membraneous reuptake transporter for circulating catecholamine clearance, is the direct target of cortisol in its permissive effect. We found that BSA-conjugated cortisol, which functions as a cortisol but cannot penetrate cell membrane, enhanced the spasmolytic effect of β-adrenoceptor agonist (isoprenaline in histamine-sensitized tracheal spirals of guinea pigs, and pharmacological inhibition of EMT with famotidine was powerful enough to imitate the permissive action of cortisol. To our surprise, EMT protein expression was high in the chondrocytes of tracheal cartilage, but was undetectable in tracheal smooth muscle cells. The functionality of EMT was further confirmed with measurement of catecholamine uptake by tracheal chondrocytes. Moreover, cortisol-initiated membrane signaling could activate protein kinase C (PKC, which phosphorylates EMT and induces its internalization via a lipid raft-dependent pathway. Both of the mechanisms slow down the reuptake process by chondrocytes, leading to extracellular catecholamine accumulation and results in a more profound adrenergic signaling activation in tracheal smooth muscle cells. Thus, an EMT-centered pathway was proposed to explain the permissive action of cortisol. Collectively, our results highlight the role of EMT in the crosstalk between glucocorticoid and catecholamine. EMT may represent a promising target for adrenergic signaling modulation.

  15. Discrimination of micromass-induced chondrocytes from human mesenchymal stem cells by focal plane array-Fourier transform infrared microspectroscopy.

    Science.gov (United States)

    Chonanant, Chirapond; Bambery, Keith R; Jearanaikoon, Nichada; Chio-Srichan, Sirinart; Limpaiboon, Temduang; Tobin, Mark J; Heraud, Philip; Jearanaikoon, Patcharee

    2014-12-01

    Rapid and sensitive methods for identifying stem cell differentiation state are required for facilitating future stem cell therapies. We aimed to evaluate the capability of focal plane array-Fourier transform infrared (FPA-FTIR) microspectroscopy for characterising the differentiation of chondrocytes from human mesenchymal stem cells (hMSCs). Successful induction was validated by reverse transcription polymerase chain reaction (RT-PCR) and Western blot analysis for collagen and aggrecan expression as chondrocyte markers in parallel with the spectroscopy. Spectra derived from chondrocyte-induced cells revealed strong IR absorbance bands attributed to collagen near 1338 and 1234 cm(-1) and proteoglycan at 1245 and 1175-960 cm(-1) compared to the non-induced cells. In addition, spectra from control and induced cells are segregated into separate clusters in partial least squares discriminant analysis score plots at the very early stages of induction and discrimination of an independent set of validation spectra with 100% accuracy. The predominant bands responsible for this discrimination were associated with collagen and aggrecan protein concordant with those obtained from RT-PCR and Western blot techniques. Our findings support the capability of FPA-FTIR microspectroscopy as a label-free tool for stem cell characterization allowing rapid and sensitive detection of macromolecular changes during chondrogenic differentiation. Copyright © 2014 Elsevier B.V. All rights reserved.

  16. Treatment of osteochondritis dissecans of the femoral condyle with autologous bone grafts and matrix-supported autologous chondrocytes

    Science.gov (United States)

    Bruns, Juergen; Deuretzbacher, Georg; Ruether, Wolfgang; Fuerst, Martin; Niggemeyer, Oliver

    2009-01-01

    The objective of this study was to determine the clinical outcome of combined bone grafting and matrix-supported autologous chondrocyte transplantation in patients with osteochondritis dissecans of the knee. Between January 2003 and March 2005, 21 patients (mean age 29.33 years) with symptomatic osteochondritis dissecans (OCD) of the medial or lateral condyle (grade III or IV) of the knee underwent reconstruction of the joint surface by autologous bone grafts and matrix-supported autologous chondrocyte transplantation. Patients were followed up at three, six, 12 and 36 months to determine outcomes by clinical evaluation based on Lysholm score, IKDC and ICRS score. Clinical results showed a significant improvement of Lysholm-score and IKDC score. With respect to clinical assessment, 18 of 21 patients showed good or excellent results 36 months postoperatively. Our study suggests that treatment of OCD with autologous bone grafts and matrix-supported autologous chondrocytes is a possible alternative to osteochondral cylinder transfer or conventional ACT. PMID:19626325

  17. Different biosynthesis patterns among flavonoid 3-glycosides with distinct effects on accumulation of other flavonoid metabolites in pears (Pyrus bretschneideri Rehd..

    Directory of Open Access Journals (Sweden)

    Rui Zhai

    Full Text Available Flavonoid biosynthesis profile was clarified by fruit bagging and re-exposure treatments in the green Chinese pear 'Zaosu' (Pyrus bretschneideri Rehd. and its red mutant 'Red Zaosu'. Two distinct biosynthesis patterns of flavonoid 3-glycosides were found in 'Zaosu' pear. By comparison with 'Red Zaosu', the biosynthesis of flavonoid 3-galactosides and flavonoid 3-arabinosides were inhibited by bagging and these compounds only re-accumulated to a small degree in the fruit peel of 'Zaosu' after the bags were removed. In contrast, the biosynthesis of flavonoid 3-gluctosides and flavonoid 3-rutinosides was reduced by bagging and then increased when the fruits were re-exposed to sunlight. A combination of correlation, multicollinearity test and partial-correlation analyses among major flavonoid metabolites indicated that biosynthesis of each phenolic compound was independent in 'Zaosu' pear, except for the positive correlation between flavonoid 3-rutincosides and flavanols. In contrast with the green pear cultivar, almost all phenolic compounds in the red mutant had similar biosynthesis patterns except for arbutin. However, only the biosynthesis of flavonoid 3-galactosides was relatively independent and strongly affected the synthesis of the other phenolic compounds. Therefore, we propose a hypothesis that the strong accumulation of flavonoid 3-galactosides stimulated the biosynthesis of other flavonoid compounds in the red mutant and, therefore, caused systemic variation of flavonoid biosynthesis profiles between 'Zaosu' and its red mutant. This hypothesis had been further demonstrated by the enzyme activity of UFGT, and transcript levels of flavonoid biosynthetic genes and been well tested by a stepwise linear regression forecasting model. The gene that encodes flavonoid 3-galacosyltransferase was also identified and isolated from the pear genome.

  18. Atg12 Maintains Skeletal Integrity by Modulating Pro-Osteoclastogenic Signals and Chondrocyte Differentiation

    Science.gov (United States)

    Tahimic, Candice; Bahl, Disha; Shirazi-Fard, Yasaman; Marsh, Timothy; Schreurs, Anne-Sofie; Rael, Victoria E.; Glikbarg, Chloe; Debnath, Jayantha; Globus, Ruth K.

    2016-01-01

    thickness and periosteal perimeter consistent with bone loss; and a longer primary spongiosa in male Atg12 iKOs display compared to male controls. These decrements were less pronounced in the female Atg12 iKOs. Cancellous bone structure was not significantly different between iKOs and controls in both genders. Histological analysis also revealed that compared to male controls, male iKOs showed a profound increase in chondrocyte column length of the growth plate with hyper-expansion of both proliferating and hypertrophic zones. Taken together, these findings indicate that autophagy plays an important role in the maintenance of bone structural integrity by mediating the production of proosteoclastogenic signals and regulating chondrocyte proliferation and differentiation.

  19. Fuyuan Decoction Enhances SOX9 and COL2A1 Expression and Smad2/3 Phosphorylation in IL-1β-Activated Chondrocytes

    Directory of Open Access Journals (Sweden)

    Yudi Zhang

    2015-01-01

    Full Text Available Fuyuan Decoction (FYD, a herbal formula in China, has been widely used for osteoarthritis (OA treatment. Herein, we determined the effects of FYD on the expression of transcription factor SOX9 and its target gene collagen type II, alpha 1 (COL2A1 as well as the activation of Smad2/3 in interleukin- (IL- 1β-stimulated SW1353 chondrosarcoma cells. Serum-derived FYD (FYD-CS was prepared to treat SW1353 cells with or without SB431542, a TGF-β1 receptor inhibitor. Cell cycle progression was tested by flow cytometry. The expression of SOX9 and COL2A1 and the activation of Smad2/3 (p-Smad2/3 were analyzed by quantitative reverse transcription polymerase chain reaction (qRT-PCR and/or western blot. The results showed that, after treatment, FYD-CS, while inducing S-phase cell cycle arrest, enhanced cell proliferation and protected the cells against IL-1β- and/or SB431542-induced cell growth inhibition. Furthermore, FYD-CS reversed the decreased expression of COL2A1 and SOX9 induced by IL-1β and SB431542 and blocked the decreased phosphorylation of Smad2/3 induced by IL-1β alone or in combination with SB431542. Our results suggest that FYD promotes COL2A1 and SOX9 expression as well as Smad2/3 activation in IL-1β-induced chondrocytes, thus benefiting cell survival.

  20. Fuyuan Decoction Enhances SOX9 and COL2A1 Expression and Smad2/3 Phosphorylation in IL-1β-Activated Chondrocytes

    Science.gov (United States)

    Zhang, Yudi; Li, Rongheng; Zhong, Yu; Zhang, Sihan; Zhou, Lingyun; Shang, Shike

    2015-01-01

    Fuyuan Decoction (FYD), a herbal formula in China, has been widely used for osteoarthritis (OA) treatment. Herein, we determined the effects of FYD on the expression of transcription factor SOX9 and its target gene collagen type II, alpha 1 (COL2A1) as well as the activation of Smad2/3 in interleukin- (IL-) 1β-stimulated SW1353 chondrosarcoma cells. Serum-derived FYD (FYD-CS) was prepared to treat SW1353 cells with or without SB431542, a TGF-β1 receptor inhibitor. Cell cycle progression was tested by flow cytometry. The expression of SOX9 and COL2A1 and the activation of Smad2/3 (p-Smad2/3) were analyzed by quantitative reverse transcription polymerase chain reaction (qRT-PCR) and/or western blot. The results showed that, after treatment, FYD-CS, while inducing S-phase cell cycle arrest, enhanced cell proliferation and protected the cells against IL-1β- and/or SB431542-induced cell growth inhibition. Furthermore, FYD-CS reversed the decreased expression of COL2A1 and SOX9 induced by IL-1β and SB431542 and blocked the decreased phosphorylation of Smad2/3 induced by IL-1β alone or in combination with SB431542. Our results suggest that FYD promotes COL2A1 and SOX9 expression as well as Smad2/3 activation in IL-1β-induced chondrocytes, thus benefiting cell survival. PMID:26770254

  1. Culture of bovine articular chondrocytes in funnel-like collagen-PLGA hybrid sponges.

    Science.gov (United States)

    Lu, Hongxu; Ko, Young-Gwang; Kawazoe, Naoki; Chen, Guoping

    2011-08-01

    Three-dimensional porous scaffolds play an important role in tissue engineering and regenerative medicine. Structurally, these porous scaffolds should have an open and interconnected porous architecture to facilitate a homogeneous cell distribution. Moreover, the scaffolds should be mechanically strong to support new tissue formation. We developed a novel type of funnel-like collagen sponge using embossing ice particulates as a template. The funnel-like collagen sponges could promote the homogeneous cell distribution, ECM production and chondrogenesis. However, the funnel-like collagen sponges deformed during cell culture due to their weak mechanical strength. To solve this problem, we reinforced the funnel-like collagen sponges with a knitted poly(D,L-lactic-co-glycolic acid) (PLGA) mesh by hybridizing these two types of materials. The hybrid scaffolds were used to culture bovine articular chondrocytes. The cell adhesion, distribution, proliferation and chondrogenesis were investigated. The funnel-like structure promoted the even cell distribution and homogeneous ECM production. The PLGA knitted mesh protected the scaffold from deformation during cell culture. Histological and immunohistochemical staining and cartilaginous gene expression analyses revealed the cartilage-like properties of the cell/scaffold constructs after in vivo implantation. The hybrid scaffold, composed of a funnel-like collagen sponge and PLGA mesh, would be a useful tool for cartilage tissue engineering. © 2011 IOP Publishing Ltd

  2. An additive manufacturing-based PCL-alginate-chondrocyte bioprinted scaffold for cartilage tissue engineering.

    Science.gov (United States)

    Kundu, Joydip; Shim, Jin-Hyung; Jang, Jinah; Kim, Sung-Won; Cho, Dong-Woo

    2015-11-01

    Regenerative medicine is targeted to improve, restore or replace damaged tissues or organs using a combination of cells, materials and growth factors. Both tissue engineering and developmental biology currently deal with the process of tissue self-assembly and extracellular matrix (ECM) deposition. In this investigation, additive manufacturing (AM) with a multihead deposition system (MHDS) was used to fabricate three-dimensional (3D) cell-printed scaffolds using layer-by-layer (LBL) deposition of polycaprolactone (PCL) and chondrocyte cell-encapsulated alginate hydrogel. Appropriate cell dispensing conditions and optimum alginate concentrations for maintaining cell viability were determined. In vitro cell-based biochemical assays were performed to determine glycosaminoglycans (GAGs), DNA and total collagen contents from different PCL-alginate gel constructs. PCL-alginate gels containing transforming growth factor-β (TGFβ) showed higher ECM formation. The 3D cell-printed scaffolds of PCL-alginate gel were implanted in the dorsal subcutaneous spaces of female nude mice. Histochemical [Alcian blue and haematoxylin and eosin (H&E) staining] and immunohistochemical (type II collagen) analyses of the retrieved implants after 4 weeks revealed enhanced cartilage tissue and type II collagen fibril formation in the PCL-alginate gel (+TGFβ) hybrid scaffold. In conclusion, we present an innovative cell-printed scaffold for cartilage regeneration fabricated by an advanced bioprinting technology. Copyright © 2013 John Wiley & Sons, Ltd.

  3. Superficial Collagen Fibril Modulus and Pericellular Fixed Charge Density Modulate Chondrocyte Volumetric Behaviour in Early Osteoarthritis

    Directory of Open Access Journals (Sweden)

    Petri Tanska

    2013-01-01

    Full Text Available The aim of this study was to investigate if the experimentally detected altered chondrocyte volumetric behavior in early osteoarthritis can be explained by changes in the extracellular and pericellular matrix properties of cartilage. Based on our own experimental tests and the literature, the structural and mechanical parameters for normal and osteoarthritic cartilage were implemented into a multiscale fibril-reinforced poroelastic swelling model. Model simulations were compared with experimentally observed cell volume changes in mechanically loaded cartilage, obtained from anterior cruciate ligament transected rabbit knees. We found that the cell volume increased by 7% in the osteoarthritic cartilage model following mechanical loading of the tissue. In contrast, the cell volume decreased by 4% in normal cartilage model. These findings were consistent with the experimental results. Increased local transversal tissue strain due to the reduced collagen fibril stiffness accompanied with the reduced fixed charge density of the pericellular matrix could increase the cell volume up to 12%. These findings suggest that the increase in the cell volume in mechanically loaded osteoarthritic cartilage is primarily explained by the reduction in the pericellular fixed charge density, while the superficial collagen fibril stiffness is suggested to contribute secondarily to the cell volume behavior.

  4. Role of interleukin-17 in chondrocytes of herniated intervertebral lumbar discs

    Science.gov (United States)

    TIAN, PENG; LI, ZHI-JUN; FU, XIN; MA, XIN-LONG

    2015-01-01

    Lumbar disc herniation (LDH) is a common cause of lumbosacral radiculopathy. An autoimmune response to a herniated nucleus pulposus (NP) has been suggested to play an important role in the initiation of radiculopathy. Interleukin-17 (IL-17) is a cytokine associated with inflammation and autoimmunity. The presence of IL-17 has been studied in patients with LDH; however, extensive investigation into the expression of IL-17 in different disc pathologies of LDH has not yet been conducted. The aim of the present study was to investigate the role of neovascularization and hypertrophic chondrocytes in herniated intervertebral lumbar discs. Fifty-two intervertebral lumbar disc specimens were extracted from 46 patients with LDH and were subsequently classified as either contained or non-contained disc herniation (CDH and NCDH, respectively). The specimens were stained with hematoxylin and eosin or toluidine blue, or were immunostained with polyclonal antibodies to IL-17 using the streptavidin-peroxidase method. The neovascular tissue and staining results were graded to establish the histological differences between the two herniation types. The intervertebral discs (IVDs) obtained from patients with NCDH showed significantly more neovascularization and granulation tissue than the discs obtained from patients with CDH (Pherniated IVDs. PMID:26170916

  5. 3D Bioprinting Human Chondrocytes with Nanocellulose-Alginate Bioink for Cartilage Tissue Engineering Applications.

    Science.gov (United States)

    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.

  6. Chondrocyte Behavior on Micropatterns Fabricated Using Layer-by-Layer Lift-Off: Morphological Analysis

    Directory of Open Access Journals (Sweden)

    Jameel Shaik

    2013-01-01

    Full Text Available Cell patterning has emerged as an elegant tool in developing cellular arrays, bioreactors, biosensors, and lab-on-chip devices and for use in engineering neotissue for repair or regeneration. In this study, micropatterned surfaces were created using the layer-by-layer lift-off (LbL-LO method for analyzing canine chondrocytes response to patterned substrates. Five materials were chosen based on our previous studies. These included: poly(dimethyldiallylammonium chloride (PDDA, poly(ethyleneimine (PEI, poly(styrene sulfonate (PSS, collagen, and chondroitin sulfate (CS. The substrates were patterned with these five different materials, in five and ten bilayers, resulting in the following multilayer nanofilm architectures: (PSS/PDDA5, (PSS/PDDA10; (CS/PEI4/CS, (CS/PEI9/CS; (PSS/PEI5, (PSS/PEI10; (PSS/Collagen5, (PSS/Collagen10; (PSS/PEI4/PSS, (PSS/PEI9/PSS. Cell characterization studies were used to assess the viability, longevity, and cellular response to the configured patterned multilayer architectures. The cumulative cell characterization data suggests that cell viability, longevity, and functionality were enhanced on micropatterned PEI, PSS, collagen, and CS multilayer nanofilms suggesting their possible use in biomedical applications.

  7. Combinatorial Biosynthesis – Potential and Problems

    Science.gov (United States)

    Floss, Heinz G.

    2007-01-01

    Because of their ecological functions, natural products have been optimized in evolution for interaction with biological systems and receptors. However, they have not necessarily been optimized for other desirable drug properties and thus can often be improved by structural modification. Using examples from the literature, this paper reviews the opportunities for increasing structural diversity among natural products by combinatorial biosynthesis, i.e., the genetic manipulation of biosynthetic pathways. It distinguishes between combinatorial biosynthesis in a narrower sense to generate libraries of modified structures, and metabolic engineering for the targeted formation of specific structural analogs. Some of the problems and limitations encountered with these approaches are also discussed. Work from the author’s laboratory on ansamycin antibiotics is presented which illustrates some of the opportunities and limitations. PMID:16414140

  8. Combinatorial biosynthesis of polyketides--a perspective.

    Science.gov (United States)

    Wong, Fong T; Khosla, Chaitan

    2012-04-01

    Since their discovery, polyketide synthases have been attractive targets of biosynthetic engineering to make 'unnatural' natural products. Although combinatorial biosynthesis has made encouraging advances over the past two decades, the field remains in its infancy. In this enzyme-centric perspective, we discuss the scientific and technological challenges that could accelerate the adoption of combinatorial biosynthesis as a method of choice for the preparation of encoded libraries of bioactive small molecules. Borrowing a page from the protein structure prediction community, we propose a periodic challenge program to vet the most promising methods in the field, and to foster the collective development of useful tools and algorithms. Copyright © 2012 Elsevier Ltd. All rights reserved.

  9. Structural basis for phosphatidylinositol-phosphate biosynthesis

    Science.gov (United States)

    Clarke, Oliver B.; Tomasek, David; Jorge, Carla D.; Dufrisne, Meagan Belcher; Kim, Minah; Banerjee, Surajit; Rajashankar, Kanagalaghatta R.; Shapiro, Lawrence; Hendrickson, Wayne A.; Santos, Helena; Mancia, Filippo

    2015-10-01

    Phosphatidylinositol is critical for intracellular signalling and anchoring of carbohydrates and proteins to outer cellular membranes. The defining step in phosphatidylinositol biosynthesis is catalysed by CDP-alcohol phosphotransferases, transmembrane enzymes that use CDP-diacylglycerol as donor substrate for this reaction, and either inositol in eukaryotes or inositol phosphate in prokaryotes as the acceptor alcohol. Here we report the structures of a related enzyme, the phosphatidylinositol-phosphate synthase from Renibacterium salmoninarum, with and without bound CDP-diacylglycerol to 3.6 and 2.5 Å resolution, respectively. These structures reveal the location of the acceptor site, and the molecular determinants of substrate specificity and catalysis. Functional characterization of the 40%-identical ortholog from Mycobacterium tuberculosis, a potential target for the development of novel anti-tuberculosis drugs, supports the proposed mechanism of substrate binding and catalysis. This work therefore provides a structural and functional framework to understand the mechanism of phosphatidylinositol-phosphate biosynthesis.

  10. Occurrence and biosynthesis of carotenoids in phytoplankton.

    Science.gov (United States)

    Huang, Jim Junhui; Lin, Shaoling; Xu, Wenwen; Cheung, Peter Chi Keung

    2017-09-01

    Naturally occurring carotenoids are important sources of antioxidants, anti-cancer compounds and anti-inflammatory agents and there is thus considerable market demand for their pharmaceutical applications. Carotenoids are widely distributed in marine and freshwater organisms including microalgae, phytoplankton, crustaceans and fish, as well as in terrestrial plants and birds. Recently, phytoplankton-derived carotenoids have received much attention due to their abundance, rapid rate of biosynthesis and unique composition. The carotenoids that accumulate in particular phytoplankton phyla are synthesized by specific enzymes and play unique physiological roles. This review focuses on studies related to the occurrence of carotenoids in different phytoplankton phyla and the molecular aspects of their biosynthesis. Recent biotechnological advances in the isolation and characterization of some representative carotenoid synthases in phytoplankton are also discussed. Copyright © 2017 Elsevier Inc. All rights reserved.

  11. Chemical genetics to examine cellulose biosynthesis

    Directory of Open Access Journals (Sweden)

    Seth eDebolt

    2013-01-01

    Full Text Available Long-term efforts to decode plant cellulose biosynthesis via molecular genetics and biochemical strategies are being enhanced by the ever-expanding scale of omics technologies. An alternative approach to consider are the prospects for inducing change in plant metabolism using exogenously supplied chemical ligands. Cellulose biosynthesis inhibitors (CBI have been identified among known herbicides, during diverse combinatorial chemical libraries screens, and natural chemical screens from microbial agents. In this review, we summarize the current knowledge of the inhibitory effects of CBIs and further group them by how they influence fluorescently tagged cellulose synthase A (CESA proteins. Additional attention is paid to the continuing development of the CBI toolbox to explore the cell biology and genetic mechanisms underpinning effector molecule activity.

  12. Microbial biosynthesis of nontoxic gold nanoparticles

    Energy Technology Data Exchange (ETDEWEB)

    Roy, Swarup, E-mail: swaruproy@klyuniv.ac.in [Department of Biochemistry and Biophysics, University of Kalyani, Kalyani 741235, West Bengal (India); Das, Tapan Kumar [Department of Biochemistry and Biophysics, University of Kalyani, Kalyani 741235, West Bengal (India); Maiti, Guru Prasad [Department of Molecular Biology and Biotechnology, University of Kalyani, Kalyani 741235, West Bengal (India); Department of Anesthesiology, Texas Tech University Health science Center, 3601 4th Street, Lubbock, TX 79430 (United States); Basu, Utpal [Department of Molecular Biology and Biotechnology, University of Kalyani, Kalyani 741235, West Bengal (India)

    2016-01-15

    Graphical abstract: The manuscript deals with the fungus mediated optimized biologically synthesized GNPs using Aspergillus foetidus and characterization of biosynthesized GNPs using various physico-chemical methods. The fairly stable synthesized nanoparticles have size in the range of 10–40 nm. Cytotoxicity study of biosynthesized GNPs on Human lung cancer cell line A549 showed no significant toxicity of GNPs. - Highlights: • A novel biosynthesis process of GNPs using Aspergillus foetidus. • Biosynthesized GNPs are in the range of 10–40 nm as observed from TEM. • This process of synthesis is an optimized biosynthesis process of GNPs. • Biosynthesized GNPs are noncytotoxic against A549 cell line. - Abstract: We study the extracellular biosynthesis of gold nanoparticles (GNPs) using the fungal species Aspergillus foetidus. The formation of GNPs were initially monitored by visual observation and then characterized with the help of various characterization techniques. X-ray diffraction (XRD) results revealed distinctive formation of face centered cubic crystalline GNPs. From field emission scanning electron microscopy (FESEM) the morphology of the nanoparticles were found to be roughly spherical and within the size range of 30–50 nm. The spherical and polydispersed GNPs in the range of 10–40 nm were observed by transmission electron microscopy (TEM) analysis. It was established that alkaline pH, 1 mM gold salt concentration and 75 °C temperature were the respective optimum parameter for biosynthesis of GNPs. Cell cytotoxicity of GNP was compared with that of normal gold salt solution on A549 cell. The A549 cell growth in presence of GNPs was found to be comparatively less toxic than the gold ion.

  13. Microbial biosynthesis of nontoxic gold nanoparticles

    International Nuclear Information System (INIS)

    Roy, Swarup; Das, Tapan Kumar; Maiti, Guru Prasad; Basu, Utpal

    2016-01-01

    Graphical abstract: The manuscript deals with the fungus mediated optimized biologically synthesized GNPs using Aspergillus foetidus and characterization of biosynthesized GNPs using various physico-chemical methods. The fairly stable synthesized nanoparticles have size in the range of 10–40 nm. Cytotoxicity study of biosynthesized GNPs on Human lung cancer cell line A549 showed no significant toxicity of GNPs. - Highlights: • A novel biosynthesis process of GNPs using Aspergillus foetidus. • Biosynthesized GNPs are in the range of 10–40 nm as observed from TEM. • This process of synthesis is an optimized biosynthesis process of GNPs. • Biosynthesized GNPs are noncytotoxic against A549 cell line. - Abstract: We study the extracellular biosynthesis of gold nanoparticles (GNPs) using the fungal species Aspergillus foetidus. The formation of GNPs were initially monitored by visual observation and then characterized with the help of various characterization techniques. X-ray diffraction (XRD) results revealed distinctive formation of face centered cubic crystalline GNPs. From field emission scanning electron microscopy (FESEM) the morphology of the nanoparticles were found to be roughly spherical and within the size range of 30–50 nm. The spherical and polydispersed GNPs in the range of 10–40 nm were observed by transmission electron microscopy (TEM) analysis. It was established that alkaline pH, 1 mM gold salt concentration and 75 °C temperature were the respective optimum parameter for biosynthesis of GNPs. Cell cytotoxicity of GNP was compared with that of normal gold salt solution on A549 cell. The A549 cell growth in presence of GNPs was found to be comparatively less toxic than the gold ion.

  14. Intracellular salicylic acid is involved in signal cascade regulating low ammonium-induced taxoid biosynthesis in suspension cultures of Taxus chinensis.

    Science.gov (United States)

    Zhou, Xin; Zhong, Jian-Jiang

    2011-05-01

    It was previously reported that low initial ammonium (2 mM) in medium had significant stimulating effects on the biosynthesis of taxuyunnanine C (Tc) by Taxus chinensis cells. However, the secondary metabolism induction mechanism of the low initial ammonium is yet unknown in plant cells. To provide an insight into the defense signals response to the low initial ammonium, oxidative burst and intracellular salicylic acid (SA) were detected, and their influences on the expression of important genes in taxoid biosynthetic pathway were examined in the cell cultures of T. chinensis. Induced H(2)O(2) production, elevated phenylalanine ammonia-lyase (PAL) activity, and enhanced SA biosynthesis were observed. Interestingly, inhibition of SA biosynthesis by paclobutrazol and (BOC-aminooxy) acetic acid significantly depressed the Tc stimulation and up-regulation of Tc biosynthetic genes of geranylgeranyl diphosphate synthase and taxadiene synthase. The role of intracellular SA in regulating Tc biosynthesis was further confirmed by applying exogenous SA in normal ammonium (20 mM) medium. The results indicated that SA acted as a signal in low initial ammonium-induced Tc biosynthesis. A signal transduction cascade from defense signal response to activated transcription of taxoid biosynthetic genes and enhanced Tc production is proposed.

  15. Tetrahydrobiopterin biosynthesis, utilization and pharmacological effects.

    Science.gov (United States)

    Werner-Felmayer, G; Golderer, G; Werner, E R

    2002-04-01

    Tetrahydrobiopterin (H4-biopterin) is an essential cofactor of a set of enzymes that are of central metabolic importance, i.e. the hydroxylases of the three aromatic amino acids phenylalanine, tyrosine, and tryptophan, of ether lipid oxidase, and of the three nitric oxide synthase (NOS) isoenzymes. As a consequence, H4-biopterin plays a key role in a vast number of biological processes and pathological states associated with neurotransmitter formation, vasorelaxation, and immune response. In mammals, its biosynthesis is controlled by hormones, cytokines and certain immune stimuli. This review aims to summarize recent developments concerning regulation of H4-biopterin biosynthetic and regulatory enzymes and pharmacological effects of H4-biopterin in various conditions, e.g. endothelial dysfunction or apoptosis of neuronal cells. Also, approaches towards gene therapy of diseases like the different forms of phenylketonuria or of Parkinson's disease are reviewed. Additional emphasis is given to H4-biopterin biosynthesis and function in non-mammalian species such as fruit fly, zebra fish, fungi, slime molds, the bacterium Nocardia as well as to the parasitic protozoan genus of Leishmania that is not capable of pteridine biosynthesis but has evolved a sophisticated salvage network for scavenging various pteridine compounds, notably folate and biopterin.

  16. Exopolysaccharide biosynthesis by Lactobacillus helveticus ATCC 15807.

    Science.gov (United States)

    Torino, M I; Mozzi, F; Font de Valdez, G

    2005-08-01

    Exopolysaccharide (EPS) production and the activities of the enzymes involved in sugar nucleotide biosynthesis in Lactobacillus helveticus ATCC 15807 under controlled pH conditions were investigated. Batch fermentations using lactose as energy source showed higher EPS synthesis by L. helveticus ATCC 15807 at pH 4.5 with respect to pH 6.2, the enzyme alpha-phosphoglucomutase (alpha-PGM) being correlated with both total and specific EPS production. When glucose was used as carbon source instead of lactose, the lower EPS synthesis obtained was linked to a decrease in alpha-PGM and galactose 1-phosphate-uridyltransferase (GalT) activities, the reduction of the latter being more pronounced. Higher EPS production by L. helveticus ATCC 15807 at the acidic constant pH of 4.5 requires that both alpha-PGM and GalT activities are high. These enzymes are needed to synthesize UDP-glucose and UDP-galactose for supplying the corresponding monomers for EPS biosynthesis. Although differences are observed in EPS production by this strain regarding the energy source (lactose or glucose), the monomeric composition of the polymers produced is independent of the carbohydrate used. The obtained results contribute to a better understanding of the physiological factors that affect EPS biosynthesis by lactobacilli, which could help in the correct handling of the fermentation parameters within the fermented dairy industry.

  17. Lipopolysaccharide Structure and Biosynthesis in Helicobacter pylori.

    Science.gov (United States)

    Li, Hong; Liao, Tingting; Debowski, Aleksandra W; Tang, Hong; Nilsson, Hans-Olof; Stubbs, Keith A; Marshall, Barry J; Benghezal, Mohammed

    2016-12-01

    This review covers the current knowledge and gaps in Helicobacter pylori lipopolysaccharide (LPS) structure and biosynthesis. H. pylori is a Gram-negative bacterium which colonizes the luminal surface of the human gastric epithelium. Both a constitutive alteration of the lipid A preventing TLR4 elicitation and host mimicry of the Lewis antigen decorated O-antigen of H. pylori LPS promote immune escape and chronic infection. To date, the complete structure of H. pylori LPS is not available, and the proposed model is a linear arrangement composed of the inner core defined as the hexa-saccharide (Kdo-LD-Hep-LD-Hep-DD-Hep-Gal-Glc), the outer core composed of a conserved trisaccharide (-GlcNAc-Fuc-DD-Hep-) linked to the third heptose of the inner core, the glucan, the heptan and a variable O-antigen, generally consisting of a poly-LacNAc decorated with Lewis antigens. Although the glycosyltransferases (GTs) responsible for the biosynthesis of the H. pylori O-antigen chains have been identified and characterized, there are many gaps in regard to the biosynthesis of the core LPS. These limitations warrant additional mutagenesis and structural studies to obtain the complete LPS structure and corresponding biosynthetic pathway of this important gastric bacterium. © 2016 John Wiley & Sons Ltd.

  18. Evaluation of Magnetic Nanoparticle-Labeled Chondrocytes Cultivated on a Type II Collagen–Chitosan/Poly(Lactic-co-Glycolic Acid Biphasic Scaffold

    Directory of Open Access Journals (Sweden)

    Juin-Yih Su

    2017-01-01

    Full Text Available Chondral or osteochondral defects are still controversial problems in orthopedics. Here, chondrocytes labeled with magnetic nanoparticles were cultivated on a biphasic, type II collagen–chitosan/poly(lactic-co-glycolic acid scaffold in an attempt to develop cultures with trackable cells exhibiting growth, differentiation, and regeneration. Rabbit chondrocytes were labeled with magnetic nanoparticles and characterized by scanning electron microscopy (SEM, transmission electron (TEM microscopy, and gene and protein expression analyses. The experimental results showed that the magnetic nanoparticles did not affect the phenotype of chondrocytes after cell labeling, nor were protein and gene expression affected. The biphasic type II collagen–chitosan/poly(lactic-co-glycolic acid scaffold was characterized by SEM, and labeled chondrocytes showed a homogeneous distribution throughout the scaffold after cultivation onto the polymer. Cellular phenotype remained unaltered but with increased gene expression of type II collagen and aggrecan, as indicated by cell staining, indicating chondrogenesis. Decreased SRY-related high mobility group-box gene (Sox-9 levels of cultured chondrocytes indicated that differentiation was associated with osteogenesis. These results are encouraging for the development of techniques for trackable cartilage regeneration and osteochondral defect repair which may be applied in vivo and, eventually, in clinical trials.

  19. Effects of low oxygen tension on gene profile of soluble growth factors in co-cultured adipose-derived stromal cells and chondrocytes.

    Science.gov (United States)

    Shi, Sirong; Xie, Jing; Zhong, Juan; Lin, Shiyu; Zhang, Tao; Sun, Ke; Fu, Na; Shao, Xiaoru; Lin, Yunfeng

    2016-06-01

    Moving towards development of optimized cartilage regeneration with adipose-derived stromal cells (ASCs), the focus of this study was on investigating the influence of hypoxia on soluble factors secreted by ASCs and chondrocytes after crosstalk. We established direct contact co-culture and non-contact co-culture systems by using red or green fluorescent protein (R/GFP)-labelled mice and SD rats respectively. Gene variation of growth factors of the two cell types, in both hypoxic and normoxic conditions, were screened using semi-quantitative polymerase chain reaction (PCR). Co-culture with ASCs and chondrocytes under hypoxia was shown to successfully induce or enhance ASC to chondrogenic differentiation. To be specific, chondrogenic maker genes: AGC, COL II and SOX9 were remarkably enhanced in both ASCs and chondrocytes after crosstalk under low oxygen tension. Subsequently, screening growth factors in ASCs and chondrocytes under hypoxia showed that HIF-1α, VEGF-A/B, BMP-2/-4/-6, FGF-2 and IGF-1 were significantly increased, but not TGF-β1. These results revealed that both hypoxia and co-culture systems can notably enhance chondrogenesis of ASCs as well as increase proliferation of ASCs and chondrocytes. © 2016 John Wiley & Sons Ltd.

  20. Autophagy Is a Protective Response to the Oxidative Damage to Endplate Chondrocytes in Intervertebral Disc: Implications for the Treatment of Degenerative Lumbar Disc

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    Ke Chen

    2017-01-01

    Full Text Available Low back pain (LBP is the leading cause of disability in the elderly. Intervertebral disc degeneration (IDD was considered as the main cause for LBP. Degeneration of cartilaginous endplate was a crucial harmful factor during the initiation and development of IDD. Oxidative stress was implicated in IDD. However, the underlying molecular mechanism for the degeneration of cartilaginous endplate remains elusive. Herein, we found that oxidative stress could induce apoptosis and autophagy in endplate chondrocytes evidenced by western blot analysis, flow cytometry, immunofluorescence staining, GFP-LC3B transfection, and MDC staining. In addition, we also found that the apoptosis of endplate chondrocytes was significantly increased after the inhibition of autophagy by bafilomycin A1 shown by flow cytometry. Furthermore, mTOR pathway upstream autophagy was greatly suppressed suggested by western blot assay. In conclusion, our study strongly revealed that oxidative stress could increase autophagy and apoptosis of endplate chondrocytes in intervertebral disc. The increase of autophagy activity could prevent endplate chondrocytes from apoptosis. The autophagy in endplate chondrocytes induced by oxidative stress was mTOR dependent. These findings might shed some new lights on the mechanism for IDD and provide new strategies for the treatments of IDD.

  1. Conditioned Medium of Wharton's Jelly Derived Stem Cells Can Enhance the Cartilage Specific Genes Expression by Chondrocytes in Monolayer and Mass Culture Systems

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    Maryam Hassan Famian

    2017-04-01

    Full Text Available Purpose: Mesenchymal stem cells (MSCs have been introduced for cell therapy strategies in osteoarthritis (OA. Despite of their capacity for differentiation into chondrocyte, there are some evidences about their life-threatening problem after transplantation. So, some researchers shifted on the application of stem cells conditioned medium. The goal of this study is to evaluate whether Wharton's jelly derived stem cell conditioned medium (WJSCs-CM can enhance the gene expression profile by chondrocytes in monolayer and mass culture systems. Methods: Conditioned medium was obtained from WJSCs at fourth passage. Isolated chondrocytes were plated at density of 1×106 for both monolayer and high density culture. Then cells in both groups were divided into control (received medium and experiment group treated with WJ-CM for 3 and 6 days. Samples were prepared to evaluate gene expression profile of collagen II, aggrecan, cartilage oligomeric matrix protein (COMP and sox-9 using real-time RT-PCR. Results: After 3 days, Chondrocytes treated with WJSCs-CM expressed significantly higher level of genes compared to the control group in both culture systems. After 6 days, the expression of genes in monolayer cultivated chondrocytes was decreased but that of the mass culture were up-regulated significantly. Conclusion: WJ-SCs-CM can increase the expression of cartilage-specific genes and can be introduced as a promoting factor for cartilage regeneration.

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

    Science.gov (United States)

    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.

  3. Chondrocytes from patients with osteoarthritis express typical extracellular matrix molecules once grown onto a three-dimensional hyaluronan-based scaffold.

    Science.gov (United States)

    Cavallo, Carola; Desando, Giovanna; Facchini, Andrea; Grigolo, Brunella

    2010-04-01

    The opportunity to apply autologous chondrocyte transplantation in repairing cartilage lesions in osteoarthritis (OA) is of great interest. To this end, chondrocytes from cartilage of these patients and from healthy donors were used to evaluate the expression of some extracellular matrix molecules once these cells were grown onto a hyaluronan-based scaffold already used in clinical practice. Constructs were analyzed by immunohistochemical and real-time PCR analyses. Chondrocytes from control and patients with OA cartilages expressed the same extracellular matrix molecules even if at different amount. These differences, which were appreciable both at protein and molecular levels, were not evident once the cells were grown onto Hyaff-11 scaffold. In this experimental culture condition, the cells derived from control and patients with OA showed a significant increase of collagen type II, Sox-9, and aggrecan and a decrease of collagen type I compared with chondrocytes grown in monolayer. On the other hand, MMPs were downregulated in both the cell types evaluated by the specific action of TIMP-1 which was highly expressed at molecular and protein levels in the two groups. The growth of chondrocytes onto Hyaff-11 membrane seems to erase the differences between the cells derived from normal and OA cartilages. The cells seem to benefit of the "hyaluronan" presence which is able to create an ideal environment for the expression of cartilage genes even in absence of specific growth factors. This is of particular relevance hypothesizing the use of tissue engineering therapeutical approach also in patients with OA.

  4. Advanced oxidation protein products induce chondrocyte apoptosis via receptor for advanced glycation end products-mediated, redox-dependent intrinsic apoptosis pathway.

    Science.gov (United States)

    Wu, Qian; Zhong, Zhao-Ming; Zhu, Si-Yuan; Liao, Cong-Rui; Pan, Ying; Zeng, Ji-Huan; Zheng, Shuai; Ding, Ruo-Ting; Lin, Qing-Song; Ye, Qing; Ye, Wen-Bin; Li, Wei; Chen, Jian-Ting

    2016-01-01

    Pro-inflammatory cytokine-induced chondrocyte apoptosis is a primary cause of cartilage destruction in the progression of rheumatoid arthritis (RA). Advanced oxidation protein products (AOPPs), a novel pro-inflammatory mediator, have been confirmed to accumulate in patients with RA. However, the effect of AOPPs accumulation on chondrocyte apoptosis and the associated cellular mechanisms remains unclear. The present study demonstrated that the plasma formation of AOPPs was enhanced in RA rats compared with normal. Then, chondrocyte were treated with AOPPs-modified rat serum albumin (AOPPs-RSA) in vitro. Exposure of chondrocyte to AOPPs activated nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and increased expression of NADPH oxidase subunits, which was mediated by receptor for advanced glycation end products (RAGE), but not scavenger receptor CD36. Moreover, AOPPs challenge triggered NADPH oxidase-dependent ROS generation which induced mitochondrial dysfunction and endoplasmic reticulum stress resulted in activation of caspase family that eventually lead to apoptosis. Lastly, blockade of RAGE, instead of CD36, largely attenuated these signals. Our study demonstrated first time that AOPPs induce chondrocyte apoptosis via RAGE-mediated and redox-dependent intrinsic apoptosis pathway in vitro. These data implicates that AOPPs may represent a novel pathogenic factor that contributes to RA progression. Targeting AOPPs-triggered cellular mechanisms might emerge as a promising therapeutic option for patients with RA.

  5. Biosynthesis and biotransformation of bile acids

    Directory of Open Access Journals (Sweden)

    Šarenac Tanja M.

    2017-01-01

    Full Text Available Bile acids are steroidal compounds, which contain 24 carbon atoms. They can be classified into two major groups: primary and secondary. The most abundant bile acids: The primary bile acids include cholic acid and chenodeoxycholic acid, while the major secondary bile acids are deoxycholic acid and litocholic acid. Bile acids are important physiological agents for intestinal absorption of nutrients and are used for biliary lipid secretion, toxic metabolites and xenobiotics. The aim of this paper is to analyze biosynthesis and biotransformation of bile acids, as preparation for practical usage in laboratory and clinical conditions. Topic: Biosynthesis and biotransformation of bile acids: The biosynthesis of bile acids is the dominant metabolic pathway for catabolism of cholesterol in humans. The classical route of biosynthesis of bile acids is embarking on the conversion of cholesterol into 7α-hydroxycholesterol using enzyme 7α-cholesterol hydroxylase (CYP7A1. This enzyme is one of the microsomal cytochrome P450 enzyme is localized exclusively in the liver. Classical road is the main road in the biosynthesis of bile acids, and its total contribution amounts to 90% for people, and 75% in mice. CYP 7A1 enzyme is considered to be sensitive to the inhibition of carbon monoxide, and the condition for the effect of NADPH, the oxygen, lecithin, and the NADPH-cytochrome P450 reductase. Bile acids are important signaling molecules and metabolic controls which activate the nuclear receptor and the G protein-coupled receptors (GPCR, a signaling lipid regulation of the liver, glucose and energy homeostasis. Also, bile acids maintain metabolic homeostasis. Biotransformation of bile acids: The conversion of cholesterol into bile acids just important for maintenance of cholesterol homeostasis, but also to prevent the accumulation of cholesterol, triglycerides and toxic metabolites as well as violations of the liver and other organs. Enterohepatic circulation of

  6. Biosynthesis of Alkyl Lysophosphatidic Acid by Diacylglycerol Kinases

    Science.gov (United States)

    Gellett, Amanda M.; Kharel, Yugesh; Sunkara, Manjula; Morris, Andrew J.; Lynch, Kevin R.

    2012-01-01

    Lysophosphatidic acid (LPA) designates a family of bioactive phosphoglycerides that differ in the length and degree of saturation of their radyl chain. Additional diversity is provided by the linkage of the radyl chain to glycerol: acyl, alkyl, or alk-1-enyl. Acyl-LPAs are the predominate species in tissues and biological fluids. Alkyl-LPAs exhibit distinct pharmacodynamics at LPA receptors, potently drive platelet aggregation, and contribute to ovarian cancer aggressiveness. Multiple biosynthetic pathways exist for alkyl-LPA production. Herein we report that diacylglycerol kinases (DGKs) contribute to cell-associated alkyl-LPA production involving phosphorylation of 1-alkyl-2-acetyl glycerol and document the biosynthesis of alkyl-LPA by DGKs in SKOV-3 ovarian cancer cells, specifically identifying the contribution of DGKα. Concurrently, we discovered that treating SKOV-3 ovarian cancer cell with a sphingosine analog stimulates conversion of exogenous 1-alkyl-2-acetyl glycerol to alkyl-LPA, indicating that DGKα contributes significantly to the production of alkyl-LPA in SKOV-3 cells and identifying cross-talk between the sphingolipid and glycerol lipid pathways. PMID:22627129

  7. Effects of whole-body γ-irradiation on the biosynthesis of certain serum proteins. Final report, November 29, 1967--June 30, 1976

    International Nuclear Information System (INIS)

    Neuhaus, O.W.

    1976-01-01

    Whole-body exposure of rats to ionizing radiations yielded an increased incorporation of labeled amino acids into serum albumin in in vivo studies suggesting a stimulation of biosynthesis. Actually this may have been caused by an elevated hepatic transport of labeled amino acids (see below). A suppressed biosynthesis of albumin was observed when the experiments were performed in vitro using liver microsomes. Impaired biosynthesis appeared to be caused by a reduced mRNA production. Irradiation stimulated the biosynthesis of acute-phase plasma proteins (stress response) and inhibited the excretion of α/sub 2u/-globulin, the sex-dependent protein of the adult male rat. Exposure of rats to γ-rays stimulated amino acid transport into the liver. This process which is Na + and energy-dependent was studied with α-aminoisobutyric acid, cycloleucine, and L-methionine among others. After irradiation the serum glucagon and insulin, as well as hepatic cAMP levels, were elevated. Amino acid transport may be an important factor in controlling the increased gluconeogenesis and glycogenesis observed in rats following whole-body irradiation

  8. Expression of TGF-betas and their receptors is differentially modulated by reactive oxygen species and nitric oxide in human articular chondrocytes.

    Science.gov (United States)

    Ayache, N; Boumediene, K; Mathy-Hartert, M; Reginster, J-Y; Henrotin, Y; Pujol, J-P

    2002-05-01

    To study the effects exerted by two antioxidants, N-monomethyl-L-arginine (L-NMMA), as an inhibitor of nitric oxide (NO) synthesis, and N-acetylcysteine (NAC), a reactive oxygen species (ROS) scavenger, on the expression of the major growth factor involved in cartilage repair, TGF-beta, under the three isoforms beta1, beta2 and beta3, and the receptors I and II of this factor, using lipopolysaccharide (LPS)-treated human chondrocytes in culture. Suspension cultures of human chondrocytes derived from the knee of osteoarthritic patients were treated for 48 h with lipopolysaccharide (LPS) (10 microg/ml), L-NMMA (0.5 mM) or NAC (1 mM). Nitrite levels were assayed on the culture media using the Griess spectrophotometric method. After total RNA extraction, the expression of inducible NO synthase (iNOS), TGF-beta1, TGF-beta2, TGF-beta3, TGF-beta receptors I and II, was determined by semi-quantitative polymerase chain-reaction (RT-PCR). LPS induced a dramatic increase of both NO production and iNOS mRNA level. The addition of L-NMMA (0.5 mM) abolished NO production without affecting iNOS mRNA levels. In contrast NAC (1 mM) strongly synergized with LPS to stimu