WorldWideScience

Sample records for human chondrocytes express

  1. Astaxanthin reduces matrix metalloproteinase expression in human chondrocytes.

    Science.gov (United States)

    Chen, Wei-Ping; Xiong, Yan; Shi, Yong-Xiang; Hu, Peng-Fei; Bao, Jia-Peng; Wu, Li-Dong

    2014-03-01

    Astaxanthin is a red carotenoid pigment which exerts multiple biological activities. However, little is known about the effects of astaxanthin on matrix metalloproteinases (MMPs) in OA. The present study investigated the effects of astaxanthin on MMPs in human chondrocytes. Human chondrocytes were pretreated with astaxanthin at 1, 10 or 50μM, then, cells were stimulated with IL-1β (10ng/ml) for 24h. MMP-1, MMP-3 and MMP-13 were observed. We found that astaxanthin reduced the expression of MMP-1, MMP-3 and MMP-13 as well as the phosphorylation of two mitogen-activated protein kinases (MAPK) (p38 and ERK1/2) in IL-1β-stimulated chondrocytes. Astaxanthin also blocked the IκB-α degradation. These results suggest that astaxanthin may be beneficial in the treatment of OA.

  2. Adiponectin and leptin induce VCAM-1 expression in human and murine chondrocytes.

    Directory of Open Access Journals (Sweden)

    Javier Conde

    Full Text Available BACKGROUND: Osteoarthritis (OA and rheumatoid arthritis (RA, the most common rheumatic diseases, are characterized by irreversible degeneration of the joint tissues. There are several factors involved in the pathogenesis of these diseases including pro-inflammatory cytokines, adipokines and adhesion molecules. OBJECTIVE: Up to now, the relationship between adipokines and adhesion molecules at cartilage level was not explored. Thus, the aim of this article was to study the effect of leptin and adiponectin on the expression of VCAM-1 in human and murine chondrocytes. For completeness, intracellular signal transduction pathway was also explored. METHODS: VCAM-1 expression was assessed by quantitative RT-PCR and western blot analysis upon treatment with leptin, adiponectin and other pertinent reagents in cultured human primary chondrocytes. Signal transduction pathways have been explored by using specific pharmacological inhibitors in the adipokine-stimulated human primary chondrocytes and ATDC5 murine chondrocyte cell line. RESULTS: Herein, we demonstrate, for the first time, that leptin and adiponectin increase VCAM-1 expression in human and murine chondrocytes. In addition, both adipokines have additive effect with IL-1β. Finally, we demonstrate that several kinases, including JAK2, PI3K and AMPK are at a play in the intracellular signalling of VCAM-1 induction. CONCLUSIONS: Taken together, our results suggest that leptin and adiponectin could perpetuate cartilage-degrading processes by inducing also factors responsible of leukocyte and monocyte infiltration at inflamed joints.

  3. Differences in Cartilage-Forming Capacity of Expanded Human Chondrocytes From Ear and Nose and Their Gene Expression Profiles

    NARCIS (Netherlands)

    Hellingman, C.A.; Verwiel, E.T.P.; Slagt, I.; Koevoet, W.; Poublon, R.M.L.; Nolst-Trenite, G.J.; de Jong, R.J.B.; Jahr, H.; van Osch, G.J.V.M.

    2011-01-01

    The aim of this study was to evaluate the potential of culture-expanded human auricular and nasoseptal chondrocytes as cell source for regeneration of stable cartilage and to analyze the differences in gene expression profile of expanded chondrocytes from these specific locations. Auricular chondroc

  4. 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

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

    Science.gov (United States)

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

    2013-10-01

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

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

    Directory of Open Access Journals (Sweden)

    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

  7. Mechanical Forces Induce Changes in VEGF and VEGFR-1/sFlt-1 Expression in Human Chondrocytes

    Directory of Open Access Journals (Sweden)

    Rainer Beckmann

    2014-09-01

    Full Text Available Expression of the pro-angiogenic vascular endothelial growth factor (VEGF stimulates angiogenesis and correlates with the progression of osteoarthritis. Mechanical joint loading seems to contribute to this cartilage pathology. Cyclic equibiaxial strains of 1% to 16% for 12 h, respectively, induced expression of VEGF in human chondrocytes dose- and frequency-dependently. Stretch-mediated VEGF induction was more prominent in the human chondrocyte cell line C-28/I2 than in primary articular chondrocytes. Twelve hours of 8% stretch induced VEGF expression to 175% of unstrained controls for at least 24 h post stretching, in promoter reporter and enzyme-linked immunosorbent assay (ELISA studies. High affinity soluble VEGF-receptor, sVEGFR-1/sFlt-1 was less stretch-inducible than its ligand, VEGF-A, in these cells. ELISA assays demonstrated, for the first time, a stretch-mediated suppression of sVEGFR-1 secretion 24 h after stretching. Overall, strained chondrocytes activate their VEGF expression, but in contrast, strain appears to suppress the secretion of the major VEGF decoy receptor (sVEGFR-1/sFlt-1. The latter may deplete a biologically relevant feedback regulation to inhibit destructive angiogenesis in articular cartilage. Our data suggest that mechanical stretch can induce morphological changes in human chondrocytes in vitro. More importantly, it induces disturbed VEGF signaling, providing a molecular mechanism for a stress-induced increase in angiogenesis in cartilage pathologies.

  8. Fetal Mesenchymal Stromal Cells Differentiating towards Chondrocytes Acquire a Gene Expression Profile Resembling Human Growth Plate Cartilage

    NARCIS (Netherlands)

    van Gool, S.A.; Emons, J.A.M.; Leijten, Jeroen Christianus Hermanus; Decker, E.; Sticht, C.; van Houwelingen, J.C.; Goeman, J.J.; Kleijburg, C.; Scherjon, S.; Gretz, N.; Wit, J.M.; Rappold, G.; Post, Janine Nicole; Karperien, Hermanus Bernardus Johannes

    2012-01-01

    Abstract We used human fetal bone marrow-derived mesenchymal stromal cells (hfMSCs) differentiating towards chondrocytes as an alternative model for the human growth plate (GP). Our aims were to study gene expression patterns associated with chondrogenic differentiation to assess whether

  9. JNK phosphorylation promotes degeneration of cervical endplate chondrocytes through down-regulation of the expression of ANK in humans

    Institute of Scientific and Technical Information of China (English)

    XU Hong-guang; SONG Jun-xing; CHENG Jia-feng; ZHANG Ping-Zhi; WANG Hong; LIU Ping; L(U) Kun

    2013-01-01

    Background C-Jun N-terminal kinase (JNK) signaling pathway and ankylosis gene (ANK) play a critical role in endplate chondrocytes degeneration.The purpose of this study was to investigate whether the expression levels of ANK was associated with the activation of JNK.Methods Cartilage endplates of 49 patients were divided into the control group (n=19) and the experimental group (n=30).The patients in the control group were graded 0 and those in the experimental group were graded Ⅰ-Ⅲ according to Miller's classification.Endplate chondrocytes were isolated by enzyme digestion and cultured in vitro.The inverted phase contrast microscope,teluidine blue staining,HE staining,real time RT-PCR,and MTT were used to observe morphological appearances,biological characteristics,and growth curve of endplate chondrocytes from the cartilage endplate of the two groups.Real time RT-PCR and Western blotting were used to analyze the mRNA and protein expression levels of associated factors in the degeneration process in the cultured endplate chondrocytes with or without subjected SP600125.Results The expression levels of type Ⅱ collagen,aggrecan,and ANK in endplate chondrocytes of experimental group were lower than that of control group and phosphorylation level of JNK in the experimental group which was higher than that in the control group.Application of JNK phosphorylation inhibitor to degeneration chondrocytes resulted in a marked decrease in the phosphorylation level of JNK and a significant increase in the expression levels of type Ⅱ collagen,aggrecan,and ANK.Conclusion The degeneration of the human cervical endplate chondrocytes might be promoted by JNK phosphorylation by down-regulating the expression of ANK

  10. Human articular chondrocytes express multiple gap junction proteins: differential expression of connexins in normal and osteoarthritic cartilage.

    Science.gov (United States)

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

    2013-04-01

    Osteoarthritis (OA) is the most common joint disease and involves progressive degeneration of articular cartilage. The aim of this study was to investigate if chondrocytes from human articular cartilage express gap junction proteins called connexins (Cxs). We show that human chondrocytes in tissue express Cx43, Cx45, Cx32, and Cx46. We also find that primary chondrocytes from adults retain the capacity to form functional voltage-dependent gap junctions. Immunohistochemistry experiments in cartilage from OA patients revealed significantly elevated levels of Cx43 and Cx45 in the superficial zone and down through the next approximately 1000 μm of tissue. These zones corresponded with regions damaged in OA that also had high levels of proliferative cell nuclear antigen. An increased number of Cxs may help explain the increased proliferation of cells in clusters that finally lead to tissue homeostasis loss. Conversely, high levels of Cxs in OA cartilage reflect the increased number of adjacent cells in clusters that are able to interact directly by gap junctions as compared with hemichannels on single cells in normal cartilage. Our data provide strong evidence that OA patients have a loss of the usual ordered distribution of Cxs in the damaged zones and that the reductions in Cx43 levels are accompanied by the loss of correct Cx localization in the nondamaged areas. Copyright © 2013 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

  11. Regulation of MMP-3 expression and secretion by the chemokine eotaxin-1 in human chondrocytes

    Directory of Open Access Journals (Sweden)

    Chao Pin-Zhir

    2011-11-01

    Full Text Available Abstract Background Osteoarthritis (OA is characterized by the degradation of articular cartilage, marked by the breakdown of matrix proteins. Studies demonstrated the involvement of chemokines in this process, and some may potentially serve as diagnostic markers and therapeutic targets; however, the underlying signal transductions are not well understood. Methods We investigated the effects of the CC chemokine eotaxin-1 (CCL11 on the matrix metalloproteinase (MMP expression and secretion in the human chondrocyte cell line SW1353 and primary chondrocytes. Results Eotaxin-1 significantly induced MMP-3 mRNA expression in a dose-dependent manner. Inhibitors of extracellular signal-regulated kinase (ERK and p38 kinase were able to repress eotaxin-1-induced MMP-3 expression. On the contrary, Rp-adenosine-3',5'-cyclic monophosphorothioate (Rp-cAMPs, a competitive cAMP antagonist for cAMP receptors, and H-89, a protein kinase A (PKA inhibitor, markedly enhanced eotaxin-1-induced MMP-3 expression. These results suggest that MMP-3 expression is specifically mediated by the G protein-coupled eotaxin-1 receptor activities. Interestingly, little amount of MMP-3 protein was detected in the cell lysates of eotaxin-1-treated SW1353 cells, and most of MMP-3 protein was in the culture media. Furthermore we found that the eotaxin-1-dependent MMP-3 protein secretion was regulated by phospholipase C (PLC-protein kinase C (PKC cascade and c-Jun N-terminal kinase (JNK/mitogen-activated protein (MAP kinase pathways. These data indicate a specific regulation of MMP-3 secretion also by eotaxin-1 receptor activities. Conclusions Eotaxin-1 not only induces MMP-3 gene expression but also promotes MMP-3 protein secretion through G protein-coupled eotaxin-1 receptor activities. Chemokines, such as eotaxin-1, could be a potential candidate in the diagnosis and treatment of arthritis.

  12. ROCK inhibitor prevents the dedifferentiation of human articular chondrocytes

    Energy Technology Data Exchange (ETDEWEB)

    Matsumoto, Emi [Department of Orthopaedic Surgery, Science of Functional Recovery and Reconstruction, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, 2-5-1 Shikatacho, Kitaku, Okayama 700-8558 (Japan); Furumatsu, Takayuki, E-mail: matino@md.okayama-u.ac.jp [Department of Orthopaedic Surgery, Science of Functional Recovery and Reconstruction, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, 2-5-1 Shikatacho, Kitaku, Okayama 700-8558 (Japan); Kanazawa, Tomoko; Tamura, Masanori; Ozaki, Toshifumi [Department of Orthopaedic Surgery, Science of Functional Recovery and Reconstruction, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, 2-5-1 Shikatacho, Kitaku, Okayama 700-8558 (Japan)

    2012-03-30

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

  13. Parathyroid hormone-related protein is induced by hypoxia and promotes expression of the differentiated phenotype of human articular chondrocytes.

    Science.gov (United States)

    Pelosi, Michele; Lazzarano, Stefano; Thoms, Brendan L; Murphy, Chris L

    2013-11-01

    PTHrP (parathyroid hormone-related protein) is crucial for normal cartilage development and long bone growth and acts to delay chondrocyte hypertrophy and terminal differentiation in the growth plate. After growth plate closure adult HACs (human articular chondrocytes) still produce PTHrP, suggesting a possible role for this factor in the permanent articular cartilage. However, the expression regulation and function of PTHrP in the permanent articular cartilage is unknown. Human articular cartilage is an avascular tissue and functions in a hypoxic environment. The resident chondrocytes have adapted to hypoxia and use it to drive their tissue-specific functions. In the present study, we explored directly in normal articular chondrocytes isolated from a range of human donors the effect of hypoxia on PTHrP expression and whether PTHrP can regulate the expression of the permanent articular chondrocyte phenotype. We show that in HACs PTHrP is up-regulated by hypoxia in a HIF (hypoxia-inducible factor)-1α and HIF-2α-dependent manner. Using recombinant PTHrP, siRNA-mediated depletion of endogenous PTHrP and by blocking signalling through its receptor [PTHR1 (PTHrP receptor 1)], we show that hypoxia-induced PTHrP is a positive regulator of the key cartilage transcription factor SOX9 [SRY (sex determining region on the Y chromosome)-box 9], leading to increased COL2A1 (collagen type II, α1) expression. Our findings thus identify PTHrP as a potential factor for cartilage repair therapies through its ability to promote the differentiated HAC phenotype.

  14. Fetal mesenchymal stromal cells differentiating towards chondrocytes acquire a gene expression profile resembling human growth plate cartilage.

    Directory of Open Access Journals (Sweden)

    Sandy A van Gool

    Full Text Available We used human fetal bone marrow-derived mesenchymal stromal cells (hfMSCs differentiating towards chondrocytes as an alternative model for the human growth plate (GP. Our aims were to study gene expression patterns associated with chondrogenic differentiation to assess whether chondrocytes derived from hfMSCs are a suitable model for studying the development and maturation of the GP. hfMSCs efficiently formed hyaline cartilage in a pellet culture in the presence of TGFβ3 and BMP6. Microarray and principal component analysis were applied to study gene expression profiles during chondrogenic differentiation. A set of 232 genes was found to correlate with in vitro cartilage formation. Several identified genes are known to be involved in cartilage formation and validate the robustness of the differentiating hfMSC model. KEGG pathway analysis using the 232 genes revealed 9 significant signaling pathways correlated with cartilage formation. To determine the progression of growth plate cartilage formation, we compared the gene expression profile of differentiating hfMSCs with previously established expression profiles of epiphyseal GP cartilage. As differentiation towards chondrocytes proceeds, hfMSCs gradually obtain a gene expression profile resembling epiphyseal GP cartilage. We visualized the differences in gene expression profiles as protein interaction clusters and identified many protein clusters that are activated during the early chondrogenic differentiation of hfMSCs showing the potential of this system to study GP development.

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

    Science.gov (United States)

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

    2016-04-01

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

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

    Science.gov (United States)

    Lu, Shijin; Xiao, Xungang; Cheng, Minghua

    2015-01-01

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

  17. Regulation of Xylosyltransferase I Gene Expression by Interleukin 1β in Human Primary Chondrocyte Cells

    Science.gov (United States)

    Khair, Mostafa; Bourhim, Mustapha; Barré, Lydia; Li, Dong; Netter, Patrick; Magdalou, Jacques; Fournel-Gigleux, Sylvie; Ouzzine, Mohamed

    2013-01-01

    Xylosyltransferase I (XT-I) is an essential enzyme of proteoglycan (PG) biosynthesis pathway catalyzing the initial and rate-limiting step in glycosaminoglycan chain assembly. It plays a critical role in the regulation of PG synthesis in cartilage; however, little is known about underlying mechanism. Here, we provide evidence that, in human primary chondrocytes, IL-1β regulates XT-I gene expression into an early phase of induction and a late phase of down-regulation. Based on promoter deletions, the region up to −850 bp was defined as a major element of XT-I gene displaying both constitutive and IL-1β-regulated promoter activity. Point mutation and signaling analyses revealed that IL-1β-induced promoter activity is achieved through AP-1 response elements and mediated by SAP/JNK and p38 signaling pathways. Transactivation and chromatin immunoprecipitation assays indicated that AP-1 is a potent transactivator of XT-I promoter and that IL-1β-induced activity is mediated through increased recruitment of AP-1 to the promoter. Finally, we show that Sp3 is a repressor of XT-I promoter and bring evidence that the repressive effect of IL-1β during the late phase is mediated through Sp3 recruitment to the promoter. This suggests that modulation of Sp3 in cartilage could prevent IL-1β inhibition of PG synthesis and limit tissue degradation. PMID:23223231

  18. Upregulation of Bone Morphogenetic Protein-2 Synthesis and Consequent Collagen II Expression in Leptin-stimulated Human Chondrocytes.

    Directory of Open Access Journals (Sweden)

    Shun-Fu Chang

    Full Text Available Bone morphogenetic proteins (BMPs play positive roles in cartilage development, but they can barely be detected in healthy articular cartilage. However, recent evidence has indicated that BMPs could be detected in osteoarthritic and damaged cartilage and their precise roles have not been well defined. Extremely high amounts of leptin have been reported in obese individuals, which can be associated with osteoarthritis (OA development. The aim of this study was to investigate whether BMPs could be induced in human primary chondrocytes during leptin-stimulated OA development and the underlying mechanism. We found that expression of BMP-2 mRNA, but not BMP-4, BMP-6, or BMP-7 mRNA, could be increased in human primary chondrocytes under leptin stimulation. Moreover, this BMP-2 induction was mediated through transcription factor-signal transducer and activator of transcription (STAT 3 activation via JAK2-ERK1/2-induced Ser727-phosphorylation. Of note, histone deacetylases (HDACs 3 and 4 were both involved in modulating leptin-induced BMP-2 mRNA expression through different pathways: HDAC3, but not HDAC4, associated with STAT3 to form a complex. Our results further demonstrated that the role of BMP-2 induction under leptin stimulation is to increase collagen II expression. The findings in this study provide new insights into the regulatory mechanism of BMP-2 induction in leptin-stimulated chondrocytes and suggest that BMP-2 may play a reparative role in regulating leptin-induced OA development.

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

    Science.gov (United States)

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

    2007-01-01

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

  20. Cannabinoid WIN-55,212-2 mesylate inhibits interleukin-1β induced matrix metalloproteinase and tissue inhibitor of matrix metalloproteinase expression in human chondrocytes.

    Science.gov (United States)

    Dunn, S L; Wilkinson, J M; Crawford, A; Le Maitre, C L; Bunning, R A D

    2014-01-01

    Interleukin-1β (IL-1β) is involved in the up-regulation of matrix metalloproteinases (MMPs) leading to cartilage degradation. Cannabinoids are anti-inflammatory and reduce joint damage in animal models of arthritis. This study aimed to determine a mechanism whereby the synthetic cannabinoid WIN-55,212-2 mesylate (WIN-55) may inhibit cartilage degradation. Effects of WIN-55 were studied on IL-1β stimulated production of MMP-3 and -13 and their inhibitors TIMP-1 and -2 in human chondrocytes. Chondrocytes were obtained from articular cartilage of patients undergoing total knee replacement. Chondrocytes were grown in monolayer and 3D alginate bead cultures. Real-time polymerase chain reaction (PCR) was used to determine the gene expression of MMP-3, -13, TIMP-1 and -2 and Enzyme Linked Immunosorbent Assay (ELISA) to measure the amount of MMP-3 and MMP-13 protein released into media. Immunocytochemistry was used to investigate the expression of cannabinoid receptors in chondrocyte cultures. Treatment with WIN-55 alone or in combination with IL-1β, decreased or abolished MMP-3, -13, TIMP-1 and -2 gene expression in human chondrocyte monolayer and alginate bead cultures in both a concentration and time dependent manner. WIN-55 treatment alone, and in combination with IL-1β, reduced MMP-3 and -13 protein production by chondrocytes cultured in alginate beads. Immunocytochemistry demonstrated the expression of cannabinoid receptors in chondrocyte cultures. Cannabinoid WIN-55 can reduce both basal and IL-1β stimulated gene and protein expression of MMP-3 and -13. However WIN-55 also decreased basal levels of TIMP-1 and -2 mRNA. These actions of WIN-55 suggest a mechanism by which cannabinoids may act to prevent cartilage breakdown in arthritis. Copyright © 2013 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.

  1. SERPINE2 Inhibits IL-1α-Induced MMP-13 Expression in Human Chondrocytes: Involvement of ERK/NF-κB/AP-1 Pathways.

    Directory of Open Access Journals (Sweden)

    Anna Santoro

    Full Text Available Osteoarthritis (OA is a chronic joint disease, characterized by a progressive loss of articular cartilage. During OA, proinflammatory cytokines, such as interleukin IL-1, induce the expression of matrix metalloproteinases (MMPs in chondrocytes, contributing thus to the extracellular matrix (ECM degradation. Members of Serpine family, including plasminogen activator inhibitors have been reported to participate in ECM regulation. The aim of this study was to assess the expression of serpin peptidase inhibitor clade E member 2 (SERPINE2, under basal conditions and in response to increasing doses of IL-1α, in human cultured chondrocytes. We also examined the effects of SERPINE2 on IL-1α-induced MMP-13 expression. For completeness, the signaling pathway involved in this process was also explored.SERPINE2 mRNA and protein expression were evaluated by RT-qPCR and western blot analysis in human T/C-28a2 cell line and human primary chondrocytes. These cells were treated with human recombinant SERPINE2, alone or in combination with IL-1α. ERK 1/2, NFκB and AP-1 activation were assessed by western blot analysis.Human cultured chondrocytes express SERPINE2 in basal condition. This expression increased in response to IL-1α stimulation. In addition, recombinant SERPINE2 induced a clear inhibition of MMP-13 expression in IL-1α-stimulated chondrocytes. This inhibitory effect is likely regulated through a pathway involving ERK 1/2, NF-κB and AP-1.Taken together, these data demonstrate that SERPINE2 might prevent cartilage catabolism by inhibiting the expression of MMP-13, one of the most relevant collagenases, involved in cartilage breakdown in OA.

  2. Differential gene expression of human chondrocytes cultured under short-term altered gravity conditions during parabolic flight maneuvers.

    Science.gov (United States)

    Wehland, Markus; Aleshcheva, Ganna; Schulz, Herbert; Saar, Katrin; Hübner, Norbert; Hemmersbach, Ruth; Braun, Markus; Ma, Xiao; Frett, Timo; Warnke, Elisabeth; Riwaldt, Stefan; Pietsch, Jessica; Corydon, Thomas Juhl; Infanger, Manfred; Grimm, Daniela

    2015-03-20

    Chondrocytes are the main cellular component of articular cartilage. In healthy tissue, they are embedded in a strong but elastic extracelluar matrix providing resistance against mechanical forces and friction for the joints. Osteoarthritic cartilage, however, disrupted by heavy strain, has only very limited potential to heal. One future possibility to replace damaged cartilage might be the scaffold-free growth of chondrocytes in microgravity to form 3D aggregates. To prepare for this, we have conducted experiments during the 20th DLR parabolic flight campaign, where we fixed the cells after the first (1P) and the 31st parabola (31P). Furthermore, we subjected chondrocytes to isolated vibration and hypergravity conditions. Microarray and quantitative real time PCR analyses revealed that hypergravity regulated genes connected to cartilage integrity (BMP4, MMP3, MMP10, EDN1, WNT5A, BIRC3). Vibration was clearly detrimental to cartilage (upregulated inflammatory IL6 and IL8, downregulated growth factors EGF, VEGF, FGF17). The viability of the cells was not affected by the parabolic flight, but showed a significantly increased expression of anti-apoptotic genes after 31 parabolas. The IL-6 release of chondrocytes cultured under conditions of vibration was not changed, but hypergravity (1.8 g) induced a clear elevation of IL-6 protein in the supernatant compared with corresponding control samples. Taken together, this study provided new insights into the growth behavior of chondrocytes under short-term microgravity.

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

    Science.gov (United States)

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

    2016-01-01

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

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

    NARCIS (Netherlands)

    Lewallen, E.A.; Bonin, C.A.; Li, Xin; Smith, J.; Karperien, Hermanus Bernardus Johannes; Larson, A.N.; Lewallen, D.G.; Cool, S.M.; Westendorf, J.J.; Krych, A.J.; Leontovich, A.A.; Im, Hee-Jeong; Wijnen, van A.J.

    2016-01-01

    Osteoarthritis (OA) is a disabling degenerative joint disease that prompts pain and has limited treatment options.To permit early diagnosis and treatment of OA, a high resolution mechanistic understanding of human chondrocytes in normal and diseased states is necessary. In this study, we assessed

  5. Lactoferrin from Camelus dromedarius inhibits nuclear transcription Factor-kappa B activation, cyclooxygenase-2 expression and prostaglandin E2 production in stimulated human chondrocytes

    Directory of Open Access Journals (Sweden)

    Naila Rasheed

    2016-01-01

    Full Text Available Background: Osteoarthritis (OA is a progressive joint disorder, which remains the leading cause of chronic disability in aged people. Nuclear factor-kappa B (NF-κB is a major cellular event in OA and its activation by interleukin-1β (IL-1β plays a critical role in cartilage breakdown in these patients. Objective: In this study, we examined the effect of lactoferrin on NF-κB activation, cyclooxygenase-2 (COX-2 expression and prostaglandin E2 (PGE2 production in stimulated human articular chondrocytes. Materials and Methods: Human chondrocytes were derived from OA articular cartilage and treated with camel lactoferrin and then stimulated with IL-1β. Gene expression was determined by TaqMan assays and protein expression was studied by Western immunoblotting. NF-κB activity and PGE2levels were determined by ELISA based assays. NF-κB activity was also determined by treatment of chondrocytes with NF-κB specific inhibitor Bay 11–7082. Results: Lactoferrin inhibited IL-1β-induced activation and nuclear translocation of NF-κB p65 in human OA chondrocytes. Lactoferrin also inhibited mRNA/protein expression of COX-2 and production of PGE2. Moreover, Bay 11–7082 also inhibited IL-1β-induced expression of COX-2 and production of PGE2. The inhibitory effect of lactoferrin on the IL-1β induced expression of COX-2 or production of PGE2was mediated at least in part via suppression of NF-κB activation. Conclusions: Our data determine camel lactoferrin as a novel inhibitor of IL-1β-induced activation of NF-κB signaling events and production of cartilage-degrading molecule PGE2via inhibition of COX-2 expressions. These results may have important implications for the development of novel therapeutic strategies for the prevention/treatment of OA and other degenerative/inflammatory diseases.

  6. 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.

  7. Expression and function of NG2/CSPG4 in human chondrocytes

    OpenAIRE

    Jamil, Nuor Sabah Mohammed

    2013-01-01

    Introduction: NG2/CSPG4 is a unique transmembrane chondroitin sulphate proteoglycan molecule expressed as a core protein and a chondroitin sulphate proteoglycan (CSPG) up to 400kD. NG2/CSPG4 mediates the communication between the extracellular and intracellular compartments through interactions with collagen VI, growth factors and the actin cytoskeleton. NG2/CSPG4 affects cell migration, spreading, apoptosis and proliferation processes. NG2/CSPG4 has been shown to be expressed ...

  8. 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.

  9. Formation of Hyaline Cartilage Tissue by Passaged Human Osteoarthritic Chondrocytes.

    Science.gov (United States)

    Bianchi, Vanessa J; Weber, Joanna F; Waldman, Stephen D; Backstein, David; Kandel, Rita A

    2017-02-01

    When serially passaged in standard monolayer culture to expand cell number, articular chondrocytes lose their phenotype. This results in the formation of fibrocartilage when they are used clinically, thus limiting their use for cartilage repair therapies. Identifying a way to redifferentiate these cells in vitro is critical if they are to be used successfully. Transforming growth factor beta (TGFβ) family members are known to be crucial for regulating differentiation of fetal limb mesenchymal cells and mesenchymal stromal cells to chondrocytes. As passaged chondrocytes acquire a progenitor-like phenotype, the hypothesis of this study was that TGFβ supplementation will stimulate chondrocyte redifferentiation in vitro in serum-free three-dimensional (3D) culture. Human articular chondrocytes were serially passaged twice (P2) in monolayer culture. P2 cells were then placed in high-density (3D) culture on top of membranes (Millipore) and cultured for up to 6 weeks in chemically defined serum-free redifferentiation media (SFRM) in the presence or absence of TGFβ. The tissues were evaluated histologically, biochemically, by immunohistochemical staining, and biomechanically. Passaged human chondrocytes cultured in SFRM supplemented with 10 ng/mL TGFβ3 consistently formed a continuous layer of articular-like cartilage tissue rich in collagen type 2 and aggrecan and lacking collagen type 1 and X in the absence of a scaffold. The tissue developed a superficial zone characterized by expression of lubricin and clusterin with horizontally aligned collagen fibers. This study suggests that passaged human chondrocytes can be used to bioengineer a continuous layer of articular cartilage-like tissue in vitro scaffold free. Further study is required to evaluate their ability to repair cartilage defects in vivo.

  10. MicroRNA-33 suppresses CCL2 expression in chondrocytes.

    Science.gov (United States)

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

    2016-06-01

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

  11. Expression of caspase-3 and -9 relevant to cartilage destruction and chondrocyte apoptosis in human osteoarthritic cartilage.

    Directory of Open Access Journals (Sweden)

    Matsuo M

    2001-12-01

    Full Text Available To clarify the involvement of the caspase family in the pathway of NO-induced chondrocyte apoptosis, osteoarthritis (OA cartilage obtained from 8 patients undergoing total hip arthroplasty were used for histopathological study. Cartilage samples taken from non-fibrillated areas of femoral head resected during surgery for femoral neck fracture were used for comparison. DNA fragmentation of chondrocytes was detected by the nick end-labeling (TUNEL method. Apoptosis was further confirmed by transmission electron microscopy. The distributions of nitrotyrosine (NT, caspase-3, and -9 were examined immunohistochemically. The populations of apoptotic as well as NT-, caspase-3-, and -9-positive cells were quantified by counting the number of cells in the superficial, middle, and deep layers, respectively. The TUNEL-positive cells were observed primarily in superficial proliferating chondrocytes, clustering chondrocytes, and deep-layer chondrocytes of OA cartilage. Few positive cells were seen in the proliferating chondrocytes in the middle layer. Positive reactions for caspase-3 and -9 were observed in chondrocytes in similar areas. Histological OA grade showed significant correlations with the mean populations of apoptotic chondrocytes (% apoptosis over the 3 areas. The populations of NT-positive cells (% NT over the same areas also showed significant correlation with OA grade. Positivity for caspase-3 closely correlated with the OA grade, % apoptosis and %NT. It was concluded that caspase-3 and -9 could play a role in NO-induced chondrocyte apoptosis in OA cartilage.

  12. Human adult chondrocytes express hepatocyte growth factor (HGF) isoforms but not HgF: potential implication of osteoblasts on the presence of HGF in cartilage.

    Science.gov (United States)

    Guévremont, Melanie; Martel-Pelletier, Johanne; Massicotte, Frédéric; Tardif, Ginette; Pelletier, Jean-Pierre; Ranger, Pierre; Lajeunesse, Daniel; Reboul, Pascal

    2003-06-01

    HGF is increased in human OA cartilage, possibly from Ob's. RT-PCR shows HGF isoforms are differently regulated between chondrocytes and Ob. A paracrine cross-talk between subchondral bone and cartilage may occur during OA. Recently, hepatocyte growth factor (HGF) has been identified by immunohistochemistry in cartilage and more particularly in the deep zone of human osteoarthritic (OA) cartilage. By investigating HGF expression in cartilage, we found that chondrocytes did not express HGF; however, they expressed the two truncated isoforms, namely HGF/NK1 and HGF/NK2. Because the only other cells localized near the deep zone are osteoblasts from the subchondral bone plate, we hypothesized that they were expressing HGF. Indeed, we found that HGF was synthesized by osteoblasts from the subchondral bone plate. Moreover, OA osteoblasts produced five times more HGF than normal osteoblasts and almost no HGF/NK1, unlike normal osteoblasts. Because prostaglandin E2 (PGE2) and pro-inflammatory cytokines such as interleukin (IL)-1 and IL-6 are involved in OA progression, we investigated whether these factors impact HGF produced by normal osteoblasts. PGE2 was the only factor tested that was able to stimulate HGF synthesis. However, the addition of NS398, a selective inhibitor of cyclo-oxygenase-2 (COX-2) had no effect on HGF produced by OA osteoblasts. HGF/NK2 had a moderate stimulating effect on HGF production by normal osteoblasts, whereas osteocalcin was not modulated by either HGF or HGF/NK2. When investigating signaling routes that might be implicated in OA osteoblast-produced HGF, we found that protein kinase A was at least partially involved. In summary, this study raises the hypothesis that the HGF found in articular cartilage is produced by osteoblasts, diffuses into the cartilage, and may be implicated in the OA process.

  13. The proinflammatory cytokines interleukin-1α and tumor necrosis factor α promote the expression and secretion of proteolytically active cathepsin S from human chondrocytes.

    Science.gov (United States)

    Caglič, Dejan; Repnik, Urška; Jedeszko, Christopher; Kosec, Gregor; Miniejew, Catherine; Kindermann, Maik; Vasiljeva, Olga; Turk, Vito; Wendt, K Ulrich; Sloane, Bonnie F; Goldring, Mary B; Turk, Boris

    2013-02-01

    Osteoarthritis and rheumatoid arthritis are destructive joint diseases that involve the loss of articular cartilage. Degradation of cartilage extracellular matrix is believed to occur due to imbalance between the catabolic and anabolic processes of resident chondrocytes. Previous work has suggested that various lysosomal cysteine cathepsins participate in cartilage degeneration; however, their exact roles in disease development and progression have not been elucidated. In order to study degradation processes under conditions resembling the in vivo milieu of the cartilage, we cultivated chondrocytes on a type II collagen-containing matrix. Stimulation of the cultivated chondrocytes with interleukin-1α and/or tumor necrosis factor α resulted in a time-dependent increase in cathepsin S expression and induced its secretion into the conditioned media. Using a novel bioluminescent activity-based probe, we were able to demonstrate a significant increase in proteolytic activity of cathepsin S in the conditioned media of proinflammatory cytokine-stimulated chondrocytes. For the first time, cathepsin S was demonstrated to be secreted from chondrocytes upon stimulation with the proinflammatory cytokines, and displayed proteolytic activity in culture supernatants. Its stability at neutral pH and potent proteolytic activity on extracellular matrix components mean that cathepsin S may contribute significantly to cartilage degradation and may thus be considered a potential drug target in joint diseases.

  14. 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

  15. 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.

  16. Regulation of xylosyltransferase I gene expression by interleukin 1β in human primary chondrocyte cells: mechanism and impact on proteoglycan synthesis.

    Science.gov (United States)

    Khair, Mostafa; Bourhim, Mustapha; Barré, Lydia; Li, Dong; Netter, Patrick; Magdalou, Jacques; Fournel-Gigleux, Sylvie; Ouzzine, Mohamed

    2013-01-18

    Xylosyltransferase I (XT-I) is an essential enzyme of proteoglycan (PG) biosynthesis pathway catalyzing the initial and rate-limiting step in glycosaminoglycan chain assembly. It plays a critical role in the regulation of PG synthesis in cartilage; however, little is known about underlying mechanism. Here, we provide evidence that, in human primary chondrocytes, IL-1β regulates XT-I gene expression into an early phase of induction and a late phase of down-regulation. Based on promoter deletions, the region up to -850 bp was defined as a major element of XT-I gene displaying both constitutive and IL-1β-regulated promoter activity. Point mutation and signaling analyses revealed that IL-1β-induced promoter activity is achieved through AP-1 response elements and mediated by SAP/JNK and p38 signaling pathways. Transactivation and chromatin immunoprecipitation assays indicated that AP-1 is a potent transactivator of XT-I promoter and that IL-1β-induced activity is mediated through increased recruitment of AP-1 to the promoter. Finally, we show that Sp3 is a repressor of XT-I promoter and bring evidence that the repressive effect of IL-1β during the late phase is mediated through Sp3 recruitment to the promoter. This suggests that modulation of Sp3 in cartilage could prevent IL-1β inhibition of PG synthesis and limit tissue degradation.

  17. In vitro and in vivo validation of human and goat chondrocyte labeling by green fluorescent protein lentivirus transduction.

    Science.gov (United States)

    Miot, Sylvie; Gianni-Barrera, Roberto; Pelttari, Karoliina; Acharya, Chitrangada; Mainil-Varlet, Pierre; Juelke, Henriette; Jaquiery, Claude; Candrian, Christian; Barbero, Andrea; Martin, Ivan

    2010-02-01

    We investigated whether human articular chondrocytes can be labeled efficiently and for long-term with a green fluorescent protein (GFP) lentivirus and whether the viral transduction would influence cell proliferation and tissue-forming capacity. The method was then applied to track goat articular chondrocytes after autologous implantation in cartilage defects. Expression of GFP in transduced chondrocytes was detected cytofluorimetrically and immunohistochemically. Chondrogenic capacity of chondrocytes was assessed by Safranin-O staining, immunostaining for type II collagen, and glycosaminoglycan content. Human articular chondrocytes were efficiently transduced with GFP lentivirus (73.4 +/- 0.5% at passage 1) and maintained the expression of GFP up to 22 weeks of in vitro culture after transduction. Upon implantation in nude mice, 12 weeks after transduction, the percentage of labeled cells (73.6 +/- 3.3%) was similar to the initial one. Importantly, viral transduction of chondrocytes did not affect the cell proliferation rate, chondrogenic differentiation, or tissue-forming capacity, either in vitro or in vivo. Goat articular chondrocytes were also efficiently transduced with GFP lentivirus (78.3 +/- 3.2%) and maintained the expression of GFP in the reparative tissue after orthotopic implantation. This study demonstrates the feasibility of efficient and relatively long-term labeling of human chondrocytes for co-culture on integration studies, and indicates the potential of this stable labeling technique for tracking animal chondrocytes for in cartilage repair studies.

  18. Cartilage tissue engineering of nasal septal chondrocyte-macroaggregates in human demineralized bone matrix.

    Science.gov (United States)

    Liese, Juliane; Marzahn, Ulrike; El Sayed, Karym; Pruss, Axel; Haisch, Andreas; Stoelzel, Katharina

    2013-06-01

    Tissue Engineering is an important method for generating cartilage tissue with isolated autologous cells and the support of biomaterials. In contrast to various gel-like biomaterials, human demineralized bone matrix (DBM) guarantees some biomechanical stability for an application in biomechanically loaded regions. The present study combined for the first time the method of seeding chondrocyte-macroaggregates in DBM for the purpose of cartilage tissue engineering. After isolating human nasal chondrocytes and creating a three-dimensional macroaggregate arrangement, the DBM was cultivated in vitro with the macroaggregates. The interaction of the cells within the DBM was analyzed with respect to cell differentiation and the inhibitory effects of chondrocyte proliferation. In contrast to chondrocyte-macroaggregates in the cell-DBM constructs, morphologically modified cells expressing type I collagen dominated. The redifferentiation of chondrocytes, characterized by the expression of type II collagen, was only found in low amounts in the cell-DBM constructs. Furthermore, caspase 3, a marker for apoptosis, was detected in the chondrocyte-DBM constructs. In another experimental setting, the vitality of chondrocytes as related to culture time and the amount of DBM was analyzed with the BrdU assay. Higher amounts of DBM tended to result in significantly higher proliferation rates of the cells within the first 48 h. After 96 h, the vitality decreased in a dose-dependent fashion. In conclusion, this study provides the proof of concept of chondrocyte-macroaggregates with DBM as an interesting method for the tissue engineering of cartilage. The as-yet insufficient redifferentiation of the chondrocytes and the sporadic initiation of apoptosis will require further investigations.

  19. Precipitant induced porosity augmentation of polystyrene preserves the chondrogenicity of human chondrocytes.

    Science.gov (United States)

    Joergensen, Natasja L; Foldager, Casper B; Le, Dang Q S; Lind, Martin; Lysdahl, Helle

    2016-12-01

    Cells constantly sense and receive chemical and physical signals from neighboring cells, interstitial fluid, and extracellular matrix, which they integrate and translate into intracellular responses. Thus, the nature of the surface on which cells are cultured in vitro plays an important role for cell adhesion, proliferation, and differentiation. Autologs chondrocyte implantation is considered the treatment of choice for larger cartilage defects in the knee. To obtain a sufficient number of chondrocytes for implantation multiple passaging is often needed, which raises concerns about the changes in the chondrogenic phenotype. In the present study, we analyzed the effect at cellular and molecular level of precipitant induced porosity augmentation (PIPA) of polystyrene surfaces on proliferation and differentiation of human chondrocytes. Human chondrocytes were isolated from healthy patients undergoing anterior cruciate ligament reconstruction and cultured on PIPA modified polystyrene surfaces. Microscopical analysis revealed topographically arranged porosity with micron pores and nanometer pits. Chondrocytes cultured on PIPA surfaces revealed no difference in cell viability and proliferation, but gene- and protein expressions of collagen type II were pronounced in the first passage of chondrocytes when compared to chondrocytes cultured on control surfaces. Additionally, an analysis of 40 kinases revealed that chondrocytes expanded on PIPA caused upregulated PI3K/mTOR pathway activation and inhibition of mTORC1 resulted in reduced sGAG synthesis. These findings indicate that PIPA modified polystyrene preserved the chondrogenicity of expanded human chondrocytes at gene and protein levels, which clinically may be attractive for the next generation of cell-culture surfaces for ex vivo cell growth. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 3073-3081, 2016.

  20. 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.

  1. Human platelet releasates combined with polyglycolic acid scaffold promote chondrocyte differentiation and phenotypic maintenance

    Indian Academy of Sciences (India)

    Giulia Bernardini; Federico Chellini; Bruno Frediani; Adriano Spreafico; Annalisa Santucci

    2015-03-01

    In the present study, we aimed to demonstrate the differentiating properties of platelet-rich plasma releasates (PRPr) on human chondrocytes seeded on a polygtlycolic acid (PGA) 3D scaffold. Gene expression and biochemical analysis were carried out to assess the improved quality of our PGA-based cartilage constructs supplemented with PRPr. We observed that the use of PRPr as cell cultures supplementation to PGA-chondrocyte constructs may promote chondrocyte differentiation, and thus may contribute to maintaining the chondrogenic phenotype longer than conventional supplementation by increasing high levels of important chondrogenic markers (e.g. sox9, aggrecan and type II collagen), without induction of type I collagen. Moreover, our constructs were analysed for the secretion and deposition of important ECM molecules (sGAG, type II collagen, etc.). Our results indicate that PRPr supplementation may synergize with PGA-based scaffolds to stimulate human articular chondrocyte differentiation, maturation and phenotypic maintenance.

  2. bFGF influences human articular chondrocyte differentiation

    DEFF Research Database (Denmark)

    Schmal, H; Zwingmann, J; Fehrenbach, M

    2007-01-01

    FGF concentrations in supernatants of primary human articular chondrocytes peaked immediately after isolation and then declined. In a dose-dependent manner, bFGF enhanced cell amplification and viability. BFGF induced a decrease in the apoptotic cell population, while the number of proliferating cells remained......BACKGROUND: The possible functional role of basic fibroblast growth factor (bFGF) in regulating the mitotic and metabolic activity of primary human articular chondrocytes was investigated. METHODS: [EF1]Chondrocytes were enzymatically isolated from femoral head cartilage, and were cultured in vitro...

  3. Human immunodeficiency virus type 1 enhancer-binding protein 3 is essential for the expression of asparagine-linked glycosylation 2 in the regulation of osteoblast and chondrocyte differentiation.

    Science.gov (United States)

    Imamura, Katsuyuki; Maeda, Shingo; Kawamura, Ichiro; Matsuyama, Kanehiro; Shinohara, Naohiro; Yahiro, Yuhei; Nagano, Satoshi; Setoguchi, Takao; Yokouchi, Masahiro; Ishidou, Yasuhiro; Komiya, Setsuro

    2014-04-01

    Human immunodeficiency virus type 1 enhancer-binding protein 3 (Hivep3) suppresses osteoblast differentiation by inducing proteasomal degradation of the osteogenesis master regulator Runx2. In this study, we tested the possibility of cooperation of Hivep1, Hivep2, and Hivep3 in osteoblast and/or chondrocyte differentiation. Microarray analyses with ST-2 bone stroma cells demonstrated that expression of any known osteochondrogenesis-related genes was not commonly affected by the three Hivep siRNAs. Only Hivep3 siRNA promoted osteoblast differentiation in ST-2 cells, whereas all three siRNAs cooperatively suppressed differentiation in ATDC5 chondrocytes. We further used microarray analysis to identify genes commonly down-regulated in both MC3T3-E1 osteoblasts and ST-2 cells upon knockdown of Hivep3 and identified asparagine-linked glycosylation 2 (Alg2), which encodes a mannosyltransferase residing on the endoplasmic reticulum. The Hivep3 siRNA-mediated promotion of osteoblast differentiation was negated by forced Alg2 expression. Alg2 suppressed osteoblast differentiation and bone formation in cultured calvarial bone. Alg2 was immunoprecipitated with Runx2, whereas the combined transfection of Runx2 and Alg2 interfered with Runx2 nuclear localization, which resulted in suppression of Runx2 activity. Chondrocyte differentiation was promoted by Hivep3 overexpression, in concert with increased expression of Creb3l2, whose gene product is the endoplasmic reticulum stress transducer crucial for chondrogenesis. Alg2 silencing suppressed Creb3l2 expression and chondrogenesis of ATDC5 cells, whereas infection of Alg2-expressing virus promoted chondrocyte maturation in cultured cartilage rudiments. Thus, Alg2, as a downstream mediator of Hivep3, suppresses osteogenesis, whereas it promotes chondrogenesis. To our knowledge, this study is the first to link a mannosyltransferase gene to osteochondrogenesis.

  4. Polyhexanide and hydrogen peroxide inhibit proteoglycan synthesis of human chondrocytes

    OpenAIRE

    Röhner, Eric; Hoff, Paula; Winkler, Tobias; von Roth, Philipp; Seeger, Jörn Bengt; Perka, Carsten; Matziolis, Georg

    2011-01-01

    The use of local antiseptics is a common method in septic joint surgery. We tested polyhexanide and hydrogen peroxide, two of the most frequently used antiseptics with high efficacy and low toxicity. The purpose of this study was to evaluate the effects of both antiseptics on the extracellular cartilaginous matrix synthesis of human chondrocytes. Chondrocytes were isolated from donated human knee joints, embedded in alginate beads, and incubated for 10 and 30 minutes with polyhexanide (0.04%)...

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

    Science.gov (United States)

    Gradišnik, Lidija; Gorenjak, Mario; Vogrin, Matjaž

    2017-01-01

    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 common and very

  6. 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

  7. Human chondrocytes respond discordantly to the protein encoded by the osteoarthritis susceptibility gene GDF5.

    Directory of Open Access Journals (Sweden)

    Madhushika Ratnayake

    Full Text Available A genetic deficit mediated by SNP rs143383 that leads to reduced expression of GDF5 is strongly associated with large-joint osteoarthritis. We speculated that this deficit could be attenuated by the application of exogenous GDF5 protein and as a first step we have assessed what effect such application has on primary osteoarthritis chondrocyte gene expression. Chondrocytes harvested from cartilage of osteoarthritic patients who had undergone joint replacement were cultured with wildtype recombinant mouse and human GDF5 protein. We also studied variants of GDF5, one that has a higher affinity for the receptor BMPR-IA and one that is insensitive to the GDF5 antagonist noggin. As a positive control, chondrocytes were treated with TGF-β1. Chondrocytes were cultured in monolayer and micromass and the expression of genes coding for catabolic and anabolic proteins of cartilage were measured by quantitative PCR. The expression of the GDF5 receptor genes and the presence of their protein products was confirmed and the ability of GDF5 signal to translocate to the nucleus was demonstrated by the activation of a luciferase reporter construct. The capacity of GDF5 to elicit an intracellular signal in chondrocytes was demonstrated by the phosphorylation of intracellular Smads. Chondrocytes cultured with TGF-β1 demonstrated a consistent down regulation of MMP1, MMP13 and a consistent upregulation of TIMP1 and COL2A1 with both culture techniques. In contrast, chondrocytes cultured with wildtype GDF5, or its variants, did not show any consistent response, irrespective of the culture technique used. Our results show that osteoarthritis chondrocytes do not respond in a predictable manner to culture with exogenous GDF5. This may be a cause or a consequence of the osteoarthritis disease process and will need to be surmounted if treatment with exogenous GDF5 is to be advanced as a potential means to overcome the genetic deficit conferring osteoarthritis

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

    Science.gov (United States)

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

    2016-01-01

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

  9. Immortalization of human articular chondrocytes and induction of their phenotype

    Institute of Scientific and Technical Information of China (English)

    何清义; 李起鸿; 杨柳; 许建中

    2003-01-01

    Objective To immortalize human articular chondrocytes (HACs) using gene transfection and to maintain stable phenotype of transformed HACs after induction.Methods HACs were transfected with the retroviral vector pLXSN encoding human papillomavirus 16E7 (HPV16E7), and the transformed clones were sorted and proliferated. Karyotype analysis, clone forming tests and nude mice tumor forming tests were applied to check the characteristics of the transformation. Type Ⅱ collagen of transformed chondrocytes was inducted with free serum medium (FSM) supplemented with nutridoma-sp and ascorbate. Results Immortalized HACs were isolated with fifty passages achieved. The HPV16E7 transformed cells were confirmed to be benign. Induction of FSM with nutridoma-sp and ascorbate promoted type Ⅱ collagen of transformed chondrocytes to the high levels of normal chondrocytes. Conclusion HACs transformed with HPV16E7 survive for long periods in vitro, and type Ⅱ collagen can maintain stability after induction.

  10. Spontaneous Redifferentiation of Dedifferentiated Human Articular Chondrocytes on Hydrogel Surfaces

    OpenAIRE

    2010-01-01

    Chondrocytes rapidly dedifferentiate into a more fibroblastic phenotype on a two-dimensional polystyrene substratum. This impedes fundamental research on these cells as well as their clinical application. This study investigated the redifferentiation behavior of dedifferentiated chondrocytes on a hydrogel substratum. Dedifferentiated normal human articular chondrocyte–knee (NHAC-kn) cells were released from the sixth-passage monolayer cultured on a polystyrene surface. These cells were then s...

  11. Correlation of synovial cytokine expression with quality of cells used for autologous chondrocyte implantation in human knees

    DEFF Research Database (Denmark)

    Schmal, Hagen; Mehlhorn, Alexander T; Dovi-Akue, David

    2014-01-01

    content (TPC) and by ELISA for levels of basic fibroblast growth factor (bFGF), insulin-like growth factor 1, bone morphogenetic proteins 2 and 7 (BMP-2 and BMP-7). Cell quality following amplification for ACI was determined by surface expression of CD44, aggrecan, collagen type II and evaluation of cell...... characteristics. Data of 17 patients were supplemented by epidemiological parameters and clinical scores (IKDC, Lysholm, pain strength, subjective knee function). CD44 expression was positively associated with TPC and bFGF, and negatively linked to BMP-2 levels (p collagen type...... knee function after 1 year was positively linked to intraarticular BMP-2 concentrations (p collagen type II indicated a favorable clinical result reaching statistical significance in case of pain strength (p

  12. Adeno-Associated Vector mediated gene transfer of Transforming Growth Factor-beta1 to normal and osteoarthritic human chondrocytes stimulates cartilage anabolism

    Directory of Open Access Journals (Sweden)

    Ulrich-Vinther M.

    2005-11-01

    Full Text Available The objective of the present study was to investigate whether cartilage anabolism in human primary osteoarthritic chondrocytes could be improved by adeno-associated virus (AAV vector-mediated gene transduction of transforming growth factor TGF-beta1 (TGF-beta1. A bi-cistronic AAV-TGF-beta1-IRES-eGFP (AAV-TGF-beta1 vector was generated and used for transduction of a normal human articular chondrocyte cell line (tsT/AC62 and primary human osteoarthritic articular chondrocytes harvested from 8 patients receiving total knee joint arthroplasty. Transduction efficiency was detected by fluorescent microscopy for gene expression of enhanced green fluorescent protein (eGFP. TGF-beta1 synthesis was determined by ELISA. To assess the influence of TGF-beta1 gene therapy on chondrocyte cartilage metabolism, mRNA expressions of type II collagen, aggrecan, and matrix metalloproteinase 3 (MMP-3 were determined by quantitative real-time PCR. AAV-TGF-beta1 transduction resulted in increased synthesis of TGF-beta1 in both osteoarthritic chondrocytes and the normal articular chondrocyte cell line. The expression levels of the transduced genes were correlated to "multiplicity of infection" (MOI and post-infectious time. In both osteoarthritic chondrocytes and the normal articular chondrocyte cell line, AAV-TGF-beta1 treatment increased mRNA expression of both type II collagen and aggrecan, but decreased MMP-3 mRNA expression. Osteoarthritic chondrocytes and the normal articular chondrocyte cell line could be transduced with equal efficiencies. In conclusion, it was demonstrated that AAV-TGF-beta1 gene transfer stimulates cartilage anabolism and decreases expression of enzymes responsible for cartilage degradation in human osteoarthritic chondrocytes. The results indicate that the AAV vector is an efficient mediator of growth factors to human articular chondrocytes, and that it might be useful in future chondrocyte gene therapy.

  13. Gypenoside inhibits interleukin-1β-induced inflammatory response in human osteoarthritis chondrocytes.

    Science.gov (United States)

    Wan, Zhi-Hong; Zhao, Qing

    2017-09-01

    Gypenoside (GP), the main active ingredient of Gynostemma pentaphyllum, possesses a variety of pharmacological capacities including anti-inflammation, anti-oxidation, and anti-tumor. However, the effects of GP on IL-1β-stimulated human osteoarthritis (OA) chondrocytes are still unknown. Therefore, this study aimed to investigate the anti-inflammatory effects of GP on IL-1β-stimulated human OA chondrocytes and explore the possible mechanism. Our results showed that GP dose-dependently inhibited IL-1β-induced NO and PGE2 production in human OA chondrocytes. In addition, treatment of GP inhibited the expression of MMP3 and MMP13, which was increased by IL-1β. Finally, we found that pretreatment of GP obviously suppressed NF-κB activation in IL-1β-stimulated human OA chondrocytes. Taken together, the results demonstrated that GP has chondro-protective effects, at least in part, through inhibiting the activation of NF-κB signaling pathway in human OA chondrocytes. Thus, these findings suggest that GP may be considered as an alternative therapeutic agent for the management of OA patients. © 2017 Wiley Periodicals, Inc.

  14. 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.

  15. Toxicity of polyhexanide and hydrogen peroxide on human chondrocytes in vitro.

    Science.gov (United States)

    Röhner, Eric; Seeger, Joern B; Hoff, Paula; Dähn-Wollenberg, Stephanie; Perka, Carsten; Matziolis, Georg

    2011-07-07

    The treatment of acute joint infections has an important impact on long-term outcome and remains an unsolved problem. The most frequent bacteria are staphylococci, streptococci, and gram-negative bacteria. In septic surgery, polyhexanide and hydrogen peroxide are the most frequently used local antiseptics. The aim of this study was to examine the hypothesis that antiseptics induce cell death of human chondrocytes after a short incubation time.Human chondrocytes were treated with different concentrations of polyhexanide and hydrogen peroxide. Toxicity analysis was determined by visualization of cell structure using light microscopy, lactate dehydrogenase release, and determination of living and total cell numbers after addition of polyhexanide and hydrogen peroxide. Light microscopic data revealed a defect cell structure after addition of both antiseptics. Lactate dehydrogenase activity showed a significant increase of enzyme expression after a short incubation with polyhexanide. The determination of vital chondrocytes showed a significant decrease of vital and total cell numbers after addition with polyhexanide and hydrogen peroxide.Both antiseptic solutions induce significant cell death of human chondrocytes after a short incubation time. Polyhexanide possibly has more toxic potential than hydrogen peroxide against human chondrocytes after an application >15 minutes. Therefore, both substances should only be applied for a short time (<15 minutes) and the joint irrigated to wash out the antiseptic substance prior to wound closure. Copyright 2011, SLACK Incorporated.

  16. Low oxygen tension stimulates redifferentiation of dedifferentiated adult human nasal chondrocytes

    NARCIS (Netherlands)

    Malda, J.; Blitterswijk, van C.A.; Geffen, van M.; Martens, D.E.; Tramper, J.; Riesle, J.

    2004-01-01

    Objective: To determine the effect of dissolved oxygen tension (DO) on the redifferentiation of dedifferentiated adult human nasal septum chondrocytes cultured as pellets. Design: After isolation, human nasal chondrocytes were expanded in monolayer culture, which resulted in their dedifferentiation.

  17. Low oxygen tension stimulates the redifferentiation of dedifferentiated adult human nasal chondrocytes

    NARCIS (Netherlands)

    Malda, J.; Blitterswijk, van C.A.; Geffen, van M.; Martens, D.E.; Tramper, J.; Riesle, J.

    2004-01-01

    Objective: To determine the effect of dissolved oxygen tension (DO) on the redifferentiation of dedifferentiated adult human nasal septum chondrocytes cultured as pellets. - Design: After isolation, human nasal chondrocytes were expanded in monolayer culture, which resulted in their dedifferentiati

  18. The identification of CD163 expressing phagocytic chondrocytes in joint cartilage and its novel scavenger role in cartilage degradation.

    Directory of Open Access Journals (Sweden)

    Kai Jiao

    Full Text Available BACKGROUND: Cartilage degradation is a typical characteristic of arthritis. This study examined whether there was a subset of phagocytic chondrocytes that expressed the specific macrophage marker, CD163, and investigated their role in cartilage degradation. METHODS: Cartilage from the knee and temporomandibular joints of Sprague-Dawley rats was harvested. Cartilage degradation was experimentally-induced in rat temporomandibular joints, using published biomechanical dental methods. The expression levels of CD163 and inflammatory factors within cartilage, and the ability of CD163(+ chondrocytes to conduct phagocytosis were investigated. Cartilage from the knees of patients with osteoarthritis and normal cartilage from knee amputations was also investigated. RESULTS: In the experimentally-induced degrading cartilage from temporomandibular joints, phagocytes were capable of engulfing neighboring apoptotic and necrotic cells, and the levels of CD163, TNF-α and MMPs were all increased (P0.05. CD163(+ chondrocytes were found in the cartilage mid-zone of temporomandibular joints and knee from healthy, three-week old rats. Furthermore, an increased number of CD163(+ chondrocytes with enhanced phagocytic activity were present in Col-II(+ chondrocytes isolated from the degraded cartilage of temporomandibular joints in the eight-week experimental group compared with their age-matched controls. Increased number with enhanced phagocytic activity of CD163(+ chondrocytes were also found in isolated Col-II(+ chondrocytes stimulated with TNF-α (P<0.05. Mid-zone distribution of CD163(+ cells accompanied with increased expression of CD163 and TNF-α were further confirmed in the isolated Col-II(+ chondrocytes from the knee cartilage of human patients with osteoarthritis, in contrast to the controls (both P<0.05. CONCLUSIONS: An increased number of CD163(+ chondrocytes with enhanced phagocytic activity were discovered within degraded joint cartilage, indicating a

  19. The identification of CD163 expressing phagocytic chondrocytes in joint cartilage and its novel scavenger role in cartilage degradation.

    Science.gov (United States)

    Jiao, Kai; Zhang, Jing; Zhang, Mian; Wei, Yuying; Wu, Yaoping; Qiu, Zhong Ying; He, Jianjun; Cao, Yunxin; Hu, Jintao; Zhu, Han; Niu, Li-Na; Cao, Xu; Yang, Kun; Wang, Mei-Qing

    2013-01-01

    Cartilage degradation is a typical characteristic of arthritis. This study examined whether there was a subset of phagocytic chondrocytes that expressed the specific macrophage marker, CD163, and investigated their role in cartilage degradation. Cartilage from the knee and temporomandibular joints of Sprague-Dawley rats was harvested. Cartilage degradation was experimentally-induced in rat temporomandibular joints, using published biomechanical dental methods. The expression levels of CD163 and inflammatory factors within cartilage, and the ability of CD163(+) chondrocytes to conduct phagocytosis were investigated. Cartilage from the knees of patients with osteoarthritis and normal cartilage from knee amputations was also investigated. In the experimentally-induced degrading cartilage from temporomandibular joints, phagocytes were capable of engulfing neighboring apoptotic and necrotic cells, and the levels of CD163, TNF-α and MMPs were all increased (P0.05). CD163(+) chondrocytes were found in the cartilage mid-zone of temporomandibular joints and knee from healthy, three-week old rats. Furthermore, an increased number of CD163(+) chondrocytes with enhanced phagocytic activity were present in Col-II(+) chondrocytes isolated from the degraded cartilage of temporomandibular joints in the eight-week experimental group compared with their age-matched controls. Increased number with enhanced phagocytic activity of CD163(+) chondrocytes were also found in isolated Col-II(+) chondrocytes stimulated with TNF-α (PCD163(+) cells accompanied with increased expression of CD163 and TNF-α were further confirmed in the isolated Col-II(+) chondrocytes from the knee cartilage of human patients with osteoarthritis, in contrast to the controls (both PCD163(+) chondrocytes with enhanced phagocytic activity were discovered within degraded joint cartilage, indicating a role in eliminating degraded tissues. Targeting these cells provides a new strategy for the treatment of arthritis.

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

    Science.gov (United States)

    Melrose, J.

    2016-01-01

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

  1. Are surface antigens suited to verify the redifferentiation potential and culture purity of human chondrocytes in cell-based implants.

    Science.gov (United States)

    Krüger, M; Krüger, J P; Kinne, R W; Kaps, C; Endres, M

    2015-10-01

    Cell expansion in vitro is a prequisite to obtain a sufficient quantity of cells for cell-based cartilage repair of articular cartilage lesions. During this process verification of redifferentiation potential of highly expanded chondrocytes is required. Furthermore, cellular impurities of chondrocyte cultures have to be excluded. For this purpose, redifferentiation of expanded human chondrocytes in passage 3 or 5 was initiated in bioresorbable polyglycolic acid-fibrin (PGA-fibrin) scaffolds and selected potential markers were analysed during the process of cell expansion and redifferentiation. Chondrocyte expansion was accompanied by a decrease of collagen type II and COMP and an increase of collagen type I expression indicating cell dedifferentiation. Redifferentiation of chondrocytes in PGA-fibrin scaffolds was accompanied by an increase of collagen II/I ratio. Flow cytometric analyses revealed that in contrast to CD44 and CD49e, CD63 and CD166 showed significant changes in the number of positive cells during redifferentiation. CD14 and CD45 are not expressed by chondrocytes and are therefore possible candidates to detect specifically monocytes or haematopoetic cells in chondrocyte cultures. Characterization of surface antigen expression revealed two promising candidates (CD63 and CD166) to describe the process of redifferentiation, while CD14 and CD45 are suitable markers to exclude impurities by monocytes or haematopoetic cells. Copyright © 2015 Elsevier Ltd. All rights reserved.

  2. Cartilage tissue engineering using pre-aggregated human articular chondrocytes

    Directory of Open Access Journals (Sweden)

    F Wolf

    2008-12-01

    Full Text Available In this study, we first aimed at determining whether human articular chondrocytes (HAC proliferate in aggregates in the presence of strong chondrocyte mitogens. We then investigated if the aggregated cells have an enhanced chondrogenic capacity as compared to cells cultured in monolayer. HAC from four donors were cultured in tissue culture dishes either untreated or coated with 1% agarose in the presence of TGFb-1, FGF-2 and PDGF-BB. Proliferation and stage of differentiation were assessed by measuring respectively DNA contents and type II collagen mRNA. Expanded cells were induced to differentiate in pellets or in Hyaff®-11 meshes and the formed tissues were analysed biochemically for glycosaminoglycans (GAG and DNA, and histologically by Safranin O staining. The amount of DNA in aggregate cultures increased significantly from day 2 to day 6 (by 3.2-fold, but did not further increase with additional culture time. Expression of type II collagen mRNA was about two orders of magnitude higher in aggregated HAC as compared to monolayer expanded cells. Pellets generated by aggregated HAC were generally more intensely stained for GAG than those generated by monolayer-expanded cells. Scaffolds seeded with aggregates accumulated more GAG (1.3-fold than scaffolds seeded with monolayer expanded HAC. In conclusion, this study showed that HAC culture in aggregates does not support a relevant degree of expansion. However, aggregation of expanded HAC prior to loading into a porous scaffold enhances the quality of the resulting tissues and could thus be introduced as an intermediate culture phase in the manufacture of engineered cartilage grafts.

  3. Green fluorescent protein as marker in chondrocytes overexpressing human insulin-like growth factor-1 for repair of articular cartilage defects in rabbits

    Institute of Scientific and Technical Information of China (English)

    ZHANG Shao-kun; LIU Yi; SONG Zhi-ming; FU Chang-feng; XU Xin-xiang

    2007-01-01

    Objective:To label the primary articular chondrocytes overexpressing human insulin-like growth factor ( hIGF-1 ) with green fluorescent protein (GFP) for repair of articular cartilage defects in rabbits. Methods:GFP cDNA was inserted into pcDNA3.1-hIGF-1 to label the expression vector.The recombinant vector,pcGI,a mammalian expression vector with multiple cloning sites under two respective cytomegalovirus promoters/enhancers,was transfected into the primary articular chondrocytes with the help of lipofectamine.After the positive cell clones were selected by G418,G418-resistant chondrocytes were cultured in medium for 4 weeks.The stable expression of hIGF-1 in the articular chondrocytes was determined by in situ hybridization and immunocytochemical analysis and the GFP was confirmed under a fluorescence microscope. Methyl thiazolyl tetrazolium (MTT) and flow cytometer methods were employed to determine the effect of transfection on proliferation of chondrocytes. Gray value was used to analyze quantitatively the expression of type Ⅱ collagen. Results:The expression of hIGF-1 and GFP was confirmed in transfected chondrocytes by in situ hybridization, immunocytochemical analysis and fluorescence microscope observation. Green articular chondrocytes overexpressing hIGF-1 could expand and maintain their chondrogenic phenotypes for more than 4 weeks.After the transfection of IGF-1,the proliferation of chondrocytes was enhanced and the chondrocytes could effectively maintain the expression of type Ⅱ collagen. Conclusions:The hIGF-1 eukaryotic expression vector containing GFP marker gene has been successfully constructed.GFP,which can be visualized in real time and in situ, is stably expressed in articular chondrocytes overexpressing hIGF-1.The labeled articular chondrocytes overexpressing hIGF-1 can be applied in cell-mediated gene therapy as well as for other biomedical purposes of transgenic chondrocytes.

  4. Hyaluronic acid abrogates nitric oxide-dependent stimulation of collagen degradation in cultured human chondrocytes.

    Science.gov (United States)

    Surazynski, Arkadiusz; Miltyk, Wojciech; Czarnomysy, Robert; Grabowska, Joanna; Palka, Jerzy

    2009-07-01

    Experimental inflammation induced in cultured chondrocytes by inflammatory cytokine IL-1 beta stimulates collagen degradation by metalloproteinases. We propose that nitric oxide (NO) may represent down stream signaling molecule of IL-1-induced collagen degradation in chondrocytes. It was found that IL-1 beta induced the activity of MMP-2 and MMP-9 during the 48 h time course of the experiment, especially after 24h incubation, while DETA/NO, donor of NO, stimulated the process at 12h incubation. The mechanism of IL-1-dependent stimulation of NO production was found at the level of iNOS expression and activation of NF-kappaB. We found that hyaluronic acid (HA) counteracted IL-induced degradation of collagen in chondrocytes. Although, HA by itself had no effect on the metaloproteinases activity, when added to IL-1 beta or DETA/NO treated chondrocytes it contributed to the restoration of the MMPs activity to the control level. The mechanism of this phenomenon involves inhibition of NF-kappaB activation. The data suggest that NO may represent a target molecule for protective effect of hyaluronic acid on interleukin-1-induced stimulation of metaloproteinases activity in cultured human chondrocytes.

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

    Science.gov (United States)

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

    2015-12-16

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

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

    Directory of Open Access Journals (Sweden)

    G. Musumeci

    2011-09-01

    Full Text Available 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.

  7. Human serum provided additional values in growth factors supplemented medium for human chondrocytes monolayer expansion and engineered cartilage construction.

    Science.gov (United States)

    Chua, K H; Aminuddin, B S; Fuzina, N H; Ruszymah, B H I

    2004-05-01

    We have previously formulated an optimized human chondrocytes growth medium based on 2% fetal bovine serum supplementation. For clinical usage, the animal serum must be replaced by patient own serum. We investigated the effects of human serum concentration for human nasal septum chondrocytes monolayer culture and cartilage reconstruction. Human serum demonstrated a dose dependent manner in promoting chondrocytes growth and cartilage engineering.

  8. Polyhexanide and hydrogen peroxide inhibit proteoglycan synthesis of human chondrocytes.

    Science.gov (United States)

    Röhner, Eric; Hoff, Paula; Winkler, Tobias; von Roth, Philipp; Seeger, Jörn Bengt; Perka, Carsten; Matziolis, Georg

    2011-03-01

    The use of local antiseptics is a common method in septic joint surgery. We tested polyhexanide and hydrogen peroxide, two of the most frequently used antiseptics with high efficacy and low toxicity. The purpose of this study was to evaluate the effects of both antiseptics on the extracellular cartilaginous matrix synthesis of human chondrocytes. Chondrocytes were isolated from donated human knee joints, embedded in alginate beads, and incubated for 10 and 30 minutes with polyhexanide (0.04%), hydrogen peroxide (3%), or phosphate-buffered saline (PBS) for control. Cartilaginous matrix production was quantified through light microscopic analysis of Alcian blue staining. Cell number and morphology were detected by histological analysis. Chondrocytes showed a decreased intensity of blue colouring after antiseptic treatment versus PBS. In contrast to that, neither the cell number per view field nor the cell morphology differed between the groups. Polyhexanide has more toxic potential than hydrogen peroxide. Based on the fact that the cell number and morphology was not altered by the substances at the examined concentrations, the lower intensity of Alcian blue staining of treated chondrocytes indicates a decreased cartilage-specific matrix synthesis by polyhexanide more than by hydrogen peroxide and control.

  9. Conditional expression of constitutively active estrogen receptor {alpha} in chondrocytes impairs longitudinal bone growth in mice

    Energy Technology Data Exchange (ETDEWEB)

    Ikeda, Kazuhiro [Division of Gene Regulation and Signal Transduction, Research Center for Genomic Medicine, Saitama Medical University, Saitama (Japan); Tsukui, Tohru [Experimental Animal Laboratory, Research Center for Genomic Medicine, Saitama Medical University, Saitama (Japan); Imazawa, Yukiko; Horie-Inoue, Kuniko [Division of Gene Regulation and Signal Transduction, Research Center for Genomic Medicine, Saitama Medical University, Saitama (Japan); Inoue, Satoshi, E-mail: INOUE-GER@h.u-tokyo.ac.jp [Division of Gene Regulation and Signal Transduction, Research Center for Genomic Medicine, Saitama Medical University, Saitama (Japan); Department of Geriatric Medicine, Graduate School of Medicine, The University of Tokyo, Tokyo (Japan); Department of Anti-Aging Medicine, Graduate School of Medicine, The University of Tokyo, Tokyo (Japan)

    2012-09-07

    Highlights: Black-Right-Pointing-Pointer Conditional transgenic mice expressing constitutively active estrogen receptor {alpha} (caER{alpha}) in chondrocytes were developed. Black-Right-Pointing-Pointer Expression of caER{alpha} in chondrocytes impaired longitudinal bone growth in mice. Black-Right-Pointing-Pointer caER{alpha} affects chondrocyte proliferation and differentiation. Black-Right-Pointing-Pointer This mouse model is useful for understanding the physiological role of ER{alpha}in 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 caER{alpha}{sup ColII}, expressing constitutively active mutant estrogen receptor (ER) {alpha} in chondrocytes, using the chondrocyte-specific type II collagen promoter-driven Cre transgenic mice. caER{alpha}{sup 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 caER{alpha}{sup ColII} mice. In situ hybridization analysis of type X collagen revealed that the maturation of hypertrophic chondrocytes was impaired in caER{alpha}{sup ColII} mice. These results suggest that ER{alpha} is a critical regulator of chondrocyte proliferation and maturation during skeletal development, mediating longitudinal bone growth in vivo.

  10. 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

  11. Tenascin and aggrecan expression by articular chondrocytes is influenced by interleukin 1ß: a possible explanation for the changes in matrix synthesis during osteoarthritis

    OpenAIRE

    Pfander, D; HEINZ, N.; Rothe, P; Carl, H.; Swoboda, B

    2004-01-01

    Objective: To analyse the distribution patterns of tenascin and proteoglycans in normal and osteoarthritic cartilage, and to determine the effect of interleukin 1ß (IL1ß) on aggrecan and tenascin expression by human articular chondrocytes in vitro.

  12. Induction of vascular endothelial growth factor by nitric oxide in cultured human articular chondrocytes.

    Science.gov (United States)

    Turpaev, K; Litvinov, D; Dubovaya, V; Panasyuk, A; Ivanov, D; Prassolov, V

    2001-06-01

    We investigated the role of nitric oxide (NO) in the control of vascular endothelial growth factor A (VEGF) gene expression in cultured human articular chondrocytes. Cell treatment with the NO-generating compound nitrosoglutathione (GSNO) caused a significant accumulation of 4.4 kb VEGF mRNA, a major VEGF mRNA isoform expressing in chondrocytes. This is the first demonstration that NO can induce VEGF mRNA expression in chondrocytes. VEGF mRNA level was not affected in cells exposed to dibutyryl cGMP, a non-hydrolyzable analog of cGMP, suggesting that the cGMP system is not involved in NO-dependent transcriptional activation of VEGF gene. The GSNO-stimulated induction of VEGF mRNA was slightly attenuated by MAP protein kinase inhibitors PD98058 and SB203580, but was completely blocked in cells incubated with GSNO in the presence of catalase and superoxide dismutase, enzymes scavenging reactive oxygen species (ROS), or in the presence of thiol-containing antioxidants, N-acetyl cysteine and reduced glutathione. These results suggest that in articular chondrocytes the GSNO-induced VEGF gene transcriptional activation is dependent on endogenous ROS production and oxidative thiol modifications.

  13. Hyaline cartilage tissue is formed through the co-culture of passaged human chondrocytes and primary bovine chondrocytes.

    Science.gov (United States)

    Taylor, Drew W; Ahmed, Nazish; Hayes, Anthony J; Ferguson, Peter; Gross, Allan E; Caterson, Bruce; Kandel, Rita A

    2012-08-01

    To circumvent the problem of a sufficient number of cells for cartilage engineering, the authors previously developed a two-stage culture system to redifferentiate monolayer culture-expanded dedifferentiated human articular chondrocytes by co-culture with primary bovine chondrocytes (bP0). The aim of this study was to analyze the composition of the cartilage tissue formed in stage 1 and compare it with bP0 grown alone to determine the optimal length of the co-culture stage of the system. Biochemical data show that extracellular matrix accumulation was evident after 2 weeks of co-culture, which was 1 week behind the bP0 control culture. By 3 to 4 weeks, the amounts of accumulated proteoglycans and collagens were comparable. Expression of chondrogenic genes, Sox 9, aggrecan, and collagen type II, was also at similar levels by week 3 of culture. Immunohistochemical staining of both co-culture and control tissues showed accumulation of type II collagen, aggrecan, biglycan, decorin, and chondroitin sulfate in appropriate zonal distributions. These data indicate that co-cultured cells form cartilaginous tissue that starts to resemble that formed by bP0 after 3 weeks, suggesting that the optimal time to terminate the co-culture stage, isolate the now redifferentiated cells, and start stage 2 is just after 3 weeks.

  14. Reconstruction of Hyaline Cartilage Deep Layer Properties in 3-Dimensional Cultures of Human Articular Chondrocytes.

    Science.gov (United States)

    Nanduri, Vibudha; Tattikota, Surendra Mohan; T, Avinash Raj; Sriramagiri, Vijaya Rama Rao; Kantipudi, Suma; Pande, Gopal

    2014-06-01

    Articular cartilage (AC) injuries and malformations are commonly noticed because of trauma or age-related degeneration. Many methods have been adopted for replacing or repairing the damaged tissue. Currently available AC repair methods, in several cases, fail to yield good-quality long-lasting results, perhaps because the reconstructed tissue lacks the cellular and matrix properties seen in hyaline cartilage (HC). To reconstruct HC tissue from 2-dimensional (2D) and 3-dimensional (3D) cultures of AC-derived human chondrocytes that would specifically exhibit the cellular and biochemical properties of the deep layer of HC. Descriptive laboratory study. Two-dimensional cultures of human AC-derived chondrocytes were established in classical medium (CM) and newly defined medium (NDM) and maintained for a period of 6 weeks. These cells were suspended in 2 mm-thick collagen I gels, placed in 24-well culture inserts, and further cultured up to 30 days. Properties of chondrocytes, grown in 2D cultures and the reconstructed 3D cartilage tissue, were studied by optical and scanning electron microscopic techniques, immunohistochemistry, and cartilage-specific gene expression profiling by reverse transcription polymerase chain reaction and were compared with those of the deep layer of native human AC. Two-dimensional chondrocyte cultures grown in NDM, in comparison with those grown in CM, showed more chondrocyte-specific gene activity and matrix properties. The NDM-grown chondrocytes in 3D cultures also showed better reproduction of deep layer properties of HC, as confirmed by microscopic and gene expression analysis. The method used in this study can yield cartilage tissue up to approximately 1.6 cm in diameter and 2 mm in thickness that satisfies the very low cell density and matrix composition properties present in the deep layer of normal HC. This study presents a novel and reproducible method for long-term culture of AC-derived chondrocytes and reconstruction of cartilage

  15. IGF1 regulates RUNX1 expression via IRS1/2: Implications for antler chondrocyte differentiation.

    Science.gov (United States)

    Yang, Zhan-Qing; Zhang, Hong-Liang; Duan, Cui-Cui; Geng, Shuang; Wang, Kai; Yu, Hai-Fan; Yue, Zhan-Peng; Guo, Bin

    2017-03-19

    Although IGF1 is important for the proliferation and differentiation of chondrocytes, its underlying molecular mechanism is still unknown. Here we addressed the physiologic function of IGF1 in antler cartilage and explored the interplay of IGF1, IRS1/2 and RUNX1 in chondrocyte differentiation. The results showed that IGF1 was highly expressed in antler chondrocytes. Exogenous rIGF1 could increase the proliferation of chondrocytes and cell proportion in the S phase, whereas IGF1R inhibitor PQ401 abrogated the induction by rIGF1. Simultaneously, IGF1 could stimulate the expression of IHH which was a well-known marker for prehypertrophic chondrocytes. Further analysis evidenced that IGF1 regulated the expression of IRS1/2 whose silencing resulted in a rise of IHH mRNA levels, but the regulation was impeded by PQ401. Knockdown of IRS1 or IRS2 with specific siRNA could greatly enhance rIGF1-induced chondrocyte differentiation and reduce the expression of RUNX1. Extraneous rRUNX1 might rescue the effects of IRS1 or IRS2 siRNA on the differentiation. In antler chondrocytes, IGF1 played a role in modulating the expression of RUNX1 through IGF1R. Moreover, attenuation of RUNX1 expression advanced the differentiation elicited by rIGF1, while administration of rRUNX1 to chondrocytes treated with IGF1 siRNA or PQ401 reduced their differentiation. Additionally, siRNA-mediated downregulation of IRS1 or IRS2 in the chondrocytes impaired the interaction between IGF1 and RUNX1. Collectively, IGF1 could promote the proliferation and differentiation of antler chondrocytes. Furthermore, IRS1/2 might act downstream of IGF1 to regulate chondrocyte differentiation through targeting RUNX1.

  16. Expression of type I collagen and tenascin C is regulated by actin polymerization through MRTF in dedifferentiated chondrocytes.

    Science.gov (United States)

    Parreno, Justin; Raju, Sneha; Niaki, Mortah Nabavi; Andrejevic, Katarina; Jiang, Amy; Delve, Elizabeth; Kandel, Rita

    2014-10-16

    This study examined actin regulation of fibroblast matrix genes in dedifferentiated chondrocytes. We demonstrated that dedifferentiated chondrocytes exhibit increased actin polymerization, nuclear localization of myocardin related transcription factor (MRTF), increased type I collagen (col1) and tenascin C (Tnc) gene expression, and decreased Sox9 gene expression. Induction of actin depolymerization by latrunculin treatment or cell rounding, reduced MRTF nuclear localization, repressed col1 and Tnc expression, and increased Sox9 gene expression in dedifferentiated chondrocytes. Treatment of passaged chondrocytes with MRTF inhibitor repressed col1 and Tnc expression, but did not affect Sox9 expression. Our results show that actin polymerization regulates fibroblast matrix gene expression through MRTF in passaged chondrocytes.

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

    Energy Technology Data Exchange (ETDEWEB)

    Antonioli, Eliane, E-mail: eliane.antonioli@einstein.br [Research and Education Institute, Hospital Israelita Albert Einstein, Sao Paulo, SP (Brazil); Lobo, Anderson O., E-mail: aolobo@univap.br [Laboratory of Biomedical Nanotechnology, Universidade do Vale do Paraiba, Sao Jose dos Campos, Sao Paulo (Brazil); Ferretti, Mario, E-mail: ferretti@einstein.br [Research and Education Institute, Hospital Israelita Albert Einstein, Sao Paulo, SP (Brazil); Ortophedic Division, Federal University of Sao Paulo, SP (Brazil); Cohen, Moises, E-mail: m.cohen@uol.com.br [Research and Education Institute, Hospital Israelita Albert Einstein, Sao Paulo, SP (Brazil); Ortophedic Division, Federal University of Sao Paulo, SP (Brazil); Marciano, Fernanda R., E-mail: femarciano@uol.com.br [Laboratory of Biomedical Nanotechnology, Universidade do Vale do Paraiba, Sao Jose dos Campos, Sao Paulo (Brazil); Corat, Evaldo J., E-mail: corat@las.inpe.br [Laboratorio Associado de Sensores e Materiais, Instituto Nacional de Pesquisas Espaciais, Sao Jose dos Campos, Sao Paulo (Brazil); Trava-Airoldi, Vladimir J., E-mail: vladimir@las.inpe.br [Laboratorio Associado de Sensores e Materiais, Instituto Nacional de Pesquisas Espaciais, Sao Jose dos Campos, Sao Paulo (Brazil)

    2013-03-01

    Cartilage serves as a low-friction and wear-resistant articulating surface in diarthrodial joints and is also important during early stages of bone remodeling. Recently, regenerative cartilage research has focused on combinations of cells paired with scaffolds. Superhydrophilic vertically aligned carbon nanotubes (VACNTs) are of particular interest in regenerative medicine. The aim of this study is to evaluate cell expansion of human articular chondrocytes on superhydrophilic VACNTs, as well as their morphology and gene expression. VACNT films were produced using a microwave plasma chamber on Ti substrates and submitted to an O{sub 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: Black-Right-Pointing-Pointer Chondrocytes were cultivated on Superhydrophilic Vertically Aligned Multiwall Carbon Nanotubes (VACNT). Black-Right-Pointing-Pointer We have shown a correlation between gene expression and thermodynamics aspects. Black-Right-Pointing-Pointer Superhydrhophilic VACNT will be an excellent substrate for cartilage and bone tissue regeneration.

  18. Identification of a novel population of human cord blood cells with hematopoietic and chondrocytic potential

    Institute of Scientific and Technical Information of China (English)

    Karen E JAY; Anne ROULEAU; T Michael UNDERHILL; Mickie BHATIA

    2004-01-01

    With the exception of mature erythrocytes, cells within the human hematopoietic system are characterized by the cell surface expression of the pan-leukocyte receptor CD45. Here, we identify a novel subset among mononuclear cord blood cells depleted of lineage commitment markers (Lin-) that are devoid of CD45 expression. Surprisingly, functional examination of Lin-CD45- cells also lacking cell surface CD34 revealed they were capable of multipotential hematopoietic progenitor capacity. Co-culture with mouse embryonic limb bud cells demonstrated that Lin-CD45-CD34- cells were capable of contributing to cartilage nodules and differentiating into human chondrocytes. BMP-4, a mesodermal factor known to promote chondrogenesis, significantly augmented Lin-CD45-CD34- differentiation into chondrocytes.Moreover, unlike CD34+ human hematopoietic stem cells, Lin-CD45-CD34- cells were unable to proliferate or survive in liquid cultures, whereas single Lin-CD45-CD34- cells were able to chimerize the inner cell mass (ICM) of murine blastocysts and proliferate in this embryonic environment. Our study identifies a novel population of Lin-CD45-CD34-cells capable of commitment into both hematopoietic and chondrocytic lineages, suggesting that human cord blood may provide a more ubiquitous source of tissue with broader developmental potential than previously appreciated.

  19. Anti-inflammatory activity of an ethanolic Caesalpinia sappan extract in human chondrocytes and macrophages

    Science.gov (United States)

    Wu, Shengqian Q; Otero, Miguel; Unger, Frank M; Goldring, Mary B; Phrutivorapongkul, Ampai; Chiari, Catharina; Kolb, Alexander; Viernstein, Helmut; Toegel, Stefan

    2012-01-01

    Aim of the study Caesalpinia sappan is a common remedy in Traditional Chinese Medicine and possesses diverse biological activities including anti-inflammatory properties. Osteoarthritis (OA) is a degenerative joint disease with an inflammatory component that drives the degradation of cartilage extracellular matrix. In order to provide a scientific basis for the applicability of Caesalpinia sappan in arthritic diseases, the present study aimed to assess the effects of an ethanolic Caesalpinia sappan extract (CSE) on human chondrocytes and macrophages. Materials and Methods Primary human chondrocytes were isolated from cartilage specimens of OA patients. Primary cells, SW1353 chondrocytes and THP-1 macrophages were serum-starved and pretreated with different concentrations of CSE prior to stimulation with 10 ng/ml of interleukin-1beta (IL-1ß) or lipopolysaccharide (LPS). Following viability tests, nitric oxide (NO) and tumor necrosis factor-alpha (TNF-α) were evaluated by Griess assay and ELISA, respectively. Using validated real-time PCR assays, mRNA levels of IL-1ß, TNF-α, inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2) were quantified. SW1353 cells were cotransfected with a COX-2 luciferase reporter plasmid and nuclear factor-kappa-B (NF-κB) p50 and p65 expression vectors in the presence or absence of CSE. Results CSE dose-dependently inhibited the expression of pro-inflammatory cytokines IL-1ß and TNF-α in IL-1ß-stimulated chondrocytes and LPS-stimulated THP-1 macrophages. CSE further suppressed the synthesis of NO in primary OA chondrocytes by blocking iNOS mRNA expression. The inhibition of COX-2 transcription was found to be related with the CSE inhibition of the p65/p50-driven transactivation of the COX-2 promoter. Conclusions The present report is first to demonstrate the anti-inflammatory activity of CSE in an in vitro cell model of joint inflammation. CSE can effectively abrogate the IL-1ß-induced over-expression of

  20. High glucose mediates endothelial-to-chondrocyte transition in human aortic endothelial cells

    Directory of Open Access Journals (Sweden)

    Tang Rining

    2012-09-01

    Full Text Available Abstract Background Vascular calcification is one of the common complications in diabetes mellitus. Many studies have shown that high glucose (HG caused cardiovascular calcification, but its underlying mechanism is not fully understood. Recently, medial calcification has been most commonly described in the vessels of patients with diabetes. Chondrocytes were involved in the medial calcification. Recent studies have shown that the conversion into mesenchymal stem cells (MSCs via the endothelial-to-mesenchymal transition (EndMT could be triggered in chondrocytes. Our previous research has indicated that HG induced EndMT in human aortic endothelial cells (HAECs. Therefore, we addressed the question of whether HG-induced EndMT could be transitioned into MSCs and differentiated into chondrocytes. Methods HAECs were divided into three groups: a normal glucose (NG group, HG group (30 mmol/L, and mannitol (5.5 mmol/L NG + 24.5 mmol/L group. Pathological changes were investigated using fluorescence microscopy and electron microscopy. Immunofluorescence staining was performed to detect the co-expression of endothelial markers, such as CD31, and fibroblast markers, such as fibroblast-specific protein 1 (FSP-1. The expression of FSP-1 was detected by real time-PCR and western blots. Endothelial-derived MSCs were grown in MSC medium for one week. The expression of the MSCs markers STRO-1, CD44, CD10 and the chondrocyte marker SOX9 was detected by immunofluorescence staining and western blots. Chondrocyte expression was detected by alcian blue staining. Calcium deposits were analyzed by alizarin red staining. Results The incubation of HAECs exposed to HG resulted in a fibroblast-like phenotype. Double staining of the HAECs indicated a co-localization of CD31 and FSP-1. The expression of FSP-1 was significantly increased in the HG group, and the cells undergoing EndMT also expressed STRO-1, CD44 and SOX9 compared with the controls (P  Conclusions Our

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

    Directory of Open Access Journals (Sweden)

    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.

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

    Directory of Open Access Journals (Sweden)

    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. Millicurrent stimulation of human articular chondrocytes cultivated in a collagen type-I gel and of human osteochondral explants

    Directory of Open Access Journals (Sweden)

    Silny Jiri

    2010-08-01

    Full Text Available Abstract Background Here we investigate the effect of millicurrent treatment on human chondrocytes cultivated in a collagen gel matrix and on human osteochondral explants. Methods Human chondrocytes from osteoarthritic knee joints were enzymatically released and transferred into a collagen type-I gel. Osteochondral explants and cell-seeded gel samples were cultivated in-vitro for three weeks. Samples of the verum groups were stimulated every two days by millicurrent treatment (3 mA, sinusoidal signal of 312 Hz amplitude modulated by two super-imposed signals of 0.28 Hz, while control samples remained unaffected. After recovery, collagen type-I, type-II, aggrecan, interleukin-1β, IL-6, TNFα and MMP13 were examined by immunohistochemistry and by real time PCR. Results With regard to the immunostainings 3 D gel samples and osteochondral explants did not show any differences between treatment and control group. The expression of all investigated genes of the 3 D gel samples was elevated following millicurrent treatment. While osteochondral explant gene expression of col-I, col-II and Il-1β was nearly unaffected, aggrecan gene expression was elevated. Following millicurrent treatment, IL-6, TNFα, and MMP13 gene expression decreased. In general, the standard deviations of the gene expression data were high, resulting in rarely significant results. Conclusions We conclude that millicurrent stimulation of human osteoarthritic chondrocytes cultivated in a 3 D collagen gel and of osteochondral explants directly influences cell metabolism.

  4. Cyclic Equibiaxial Tensile Strain Alters Gene Expression of Chondrocytes via Histone Deacetylase 4 Shuttling.

    Directory of Open Access Journals (Sweden)

    Chongwei Chen

    Full Text Available This paper aims to investigate whether equibiaxial tensile strain alters chondrocyte gene expression via controlling subcellular localization of histone deacetylase 4 (HDAC4.Murine chondrocytes transfected with GFP-HDAC4 were subjected to 3 h cyclic equibiaxial tensile strain (CTS, 6% strain at 0.25 Hz by a Flexcell® FX-5000™ Tension System. Fluorescence microscope and western blot were used to observe subcellular location of HDAC4. The gene expression was analyzed by real-time RT-PCR. The concentration of Glycosaminoglycans in culture medium was quantified by bimethylmethylene blue dye; Collagen II protein was evaluated by western blot. Cells phenotype was identified by immunohistochemistry. Cell viability was evaluated by live-dead cell detect kit. Okadaic acid, an inhibitor of HDAC4 nuclear relocation, was used to further validate whether HDAC4 nuclear relocation plays a role in gene expression in response to tension stimulation.87.5% of HDAC4 was located in the cytoplasm in chondrocytes under no loading condition, but it was relocated to the nucleus after CTS. RT-PCR analysis showed that levels of mRNA for aggrecan, collagen II, LK1 and SOX9 were all increased in chondrocytes subjected to CTS as compared to no loading control chondrocytes; in contrast, the levels of type X collagen, MMP-13, IHH and Runx2 gene expression were decreased in the chondrocytes subjected to CTS as compared to control chondrocytes. Meanwhile, CTS contributed to elevation of glycosaminoglycans and collagen II protein, but did not change collagen I production. When Okadaic acid blocked HDAC4 relocation from the cytoplasm to nucleus, the changes of the chondrocytes induced by CTS were abrogated. There was no chondrocyte dead detected in this study in response to CTS.CTS is able to induce HDAC4 relocation from cytoplasm to nucleus. Thus, CTS alters chondrocytes gene expression in association with the relocation of HDAC4 induced by CTS.

  5. Dynamic cyclic compression modulates the chondrogenic phenotype in human chondrocytes from late stage osteoarthritis.

    Science.gov (United States)

    Diao, Hua Jia; Fung, Hon Sing; Yeung, Pan; Lam, K L; Yan, Chun Hoi; Chan, Barbara Pui

    2017-02-16

    Human osteoarthritic chondrocytes (hOACs) are characterized by their "dedifferentiated" and catabolic phenotype and lack the ability for restoring their inherent functions by themselves. Here we investigated whether extrinsically supplemented mechanical signal via compression loading would affect the phenotype of hOACs. Specifically, we applied cyclic compression loading on cultured hOACs-collagen constructs and measured the expression of the major chondrogenic factors, cell-matrix interaction molecules and matrix degradation enzymes. Dynamic compression loading stimulates the expression and nuclear localization of sox9 in hOACs and reduces the catabolic events via downregulated expression of collagenases. These results contribute to better understanding towards mechanoregulation of hOACs.

  6. Mesenchymal stem cells from adipose tissue which have been differentiated into chondrocytes in three-dimensional culture express lubricin.

    Science.gov (United States)

    Musumeci, Giuseppe; Lo Furno, Debora; Loreto, Carla; Giuffrida, Rosario; Caggia, Silvia; Leonardi, Rosalia; Cardile, Venera

    2011-11-01

    The present study focused on the isolation, cultivation and characterization of human mesenchymal stem cells (MSCs) from adipose tissue and on their differentiation into chondrocytes through the NH ChondroDiff medium. The main aim was to investigate some markers of biomechanical quality of cartilage, such as lubricin, and collagen type I and II. Little is known, in fact, about the ability of chondrocytes from human MSCs of adipose tissue to generate lubricin in three-dimensional (3D) culture. Lubricin, a 227.5-kDa mucinous glycoprotein, is known to play an important role in articular joint physiology, and the loss of accumulation of lubricin is thought to play a role in the pathology of osteoarthritis. Adipose tissue is an alternative source for the isolation of multipotent MSCs, which allows them to be obtained by a less invasive method and in larger quantities than from other sources. These cells can be isolated from cosmetic liposuctions in large numbers and easily grown under standard tissue culture conditions. 3D chondrocytes were assessed by histology (hematoxylin and eosin) and histochemistry (Alcian blue and Safranin-O/fast green staining). Collagen type I, II and lubricin expression was determined through immunohistochemistry and Western blot. The results showed that, compared with control cartilage and monolayer chondrocytes showing just collagen type I, chondrocytes from MSCs (CD44-, CD90- and CD105- positive; CD45-, CD14- and CD34-negative) of adipose tissue grown in nodules were able to express lubricin, and collagen type I and II, indicative of hyaline cartilage formation. Based on the function of lubricin in the joint cavity and disease and as a potential therapeutic agent, our results suggest that MSCs from adipose tissue are a promising cell source for tissue engineering of cartilage. Our results suggest that chondrocyte nodules producing lubricin could be a novel biotherapeutic approach for the treatment of cartilage abnormalities.

  7. Salvianolic acid B regulates gene expression and promotes cell viability in chondrocytes.

    Science.gov (United States)

    Yang, Xiaohong; Liu, Shaojie; Li, Siming; Wang, Pengzhen; Zhu, Weicong; Liang, Peihong; Tan, Jianrong; Cui, Shuliang

    2017-02-28

    Articular chondrocytes reside in lacunae distributed in cartilage responsible for the remodelling of the tissue with limited ability of damage repairing. The in vitro expanded chondrocytes enhanced by factors/agents to obtain large numbers of cells with strengthened phenotype are essential for successful repair of cartilage lesions by clinical cell implantation therapies. Because the salvianolic acid B (Sal B), a major hydrophilic therapeutic agent isolated from Salvia miltiorrhiza, has been widely used to treat diseases and able to stimulate activity of cells, this study examines the effects of Sal B on passaged chondrocytes. Chondrocytes were treated with various concentrations of Sal B in monolayer culture, their morphological properties and changes, and mitochondrial membrane potential were analysed using microscopic analyses, including cellular biochemical staining and confocal laser scanning microscopy. The proteins were quantified by BCA and Western blotting, and the transcription of genes was detected by qRT-PCR. The passaged chondrocytes treated with Sal B showed strengthened cellular synthesis and stabilized mitochondrial membrane potential with upregulated expression of the marker genes for chondrocyte phenotype, Col2-α1, Acan and Sox9, the key Wnt signalling molecule β-catenin and paracrine cytokine Cytl-1. The treatments using CYTL-1 protein significantly increased expression of Col2-α1 and Acan with no effect on Sox9, indicating the paracrine cytokine acts on chondrocytes independent of SOX9. Sal B has ultimately promoted cell growth and enhanced chondrocyte phenotype. The chondrocytes treated with pharmaceutical agent and cytokine in the formulated medium for generating large number of differentiated chondrocytes would facilitate the cell-based therapies for cartilage repair.

  8. Association between the chondrocyte phenotype and the expression of adipokines and their receptors: evidence for a role of leptin but not adiponectin in the expression of cartilage-specific markers.

    Science.gov (United States)

    Francin, Pierre-Jean; Guillaume, Cécile; Humbert, Anne-Claude; Pottie, Pascale; Netter, Patrick; Mainard, Didier; Presle, Nathalie

    2011-11-01

    Although extensive evidence support the key role of adipokines in cartilage homeostasis, contradictory data have been found for their expression and their effects in chondrocytes. This study was then undertaken to determine whether a phenotypic modulation may affect the expression of adipokines and their receptors in human chondrocytes. The expression of leptin, adiponectin and their receptors, as well as cartilage-specific genes was examined in chondrocytes obtained from patients with osteoarthritis either directly after cells harvest or after culture in monolayer or in alginate beads. The results showed major changes in the gene expression pattern after culture in monolayer with a shift from the adipokines to their receptors. Interestingly, this downregulation of adipokines was associated with a loss of chondrocyte phenotype, and chondrocytes recovered a cartilage-like expression profile of leptin and adiponectin when cultured in a tridimensional chondrocyte phenotype-inducing system, but ceased expressing their receptors. Further experiments clearly showed that leptin but not adiponectin promoted the expression of cartilage-specific markers through mitogen-activated protein kinase, Janus kinase and phosphatidylinositol-3 kinase signaling pathways. In conclusion, our data indicate that any phenotypic modulation could affect chondrocyte responsiveness to leptin or adiponectin, and provide evidence for an important role for leptin in regulating the expression of cartilage-specific markers.

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

    Science.gov (United States)

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

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

  10. Sesamin inhibits IL-1β-stimulated inflammatory response in human osteoarthritis chondrocytes by activating Nrf2 signaling pathway.

    Science.gov (United States)

    Kong, Pengyu; Chen, Guanghua; Jiang, Anlong; Wang, Yufu; Song, Chengchao; Zhuang, Jinpeng; Xi, Chunyang; Wang, Guangxi; Ji, Ye; Yan, Jinglong

    2016-12-13

    Sesamin, a bioactive component extracted from sesame, has been reported to exert anti-inflammatory and anti-oxidant effects. In this study, we evaluated the anti-inflammatory effects of sesamin on IL-1β-stimulated human osteoarthritis chondrocytes and investigated the possible mechanism. Results demonstrated that sesamin treatment significantly inhibited PGE2 and NO production induced by IL-1β. Sesamin inhibited MMP1, MMP3, and MMP13 production in IL-1β-stimulated chondrocytes. Sesamin also inhibited IL-1β-induced phosphorylation of NF-κB p65 and IκBα. Meanwhile, sesamin was found to up-regulate the expression of Nrf2 and HO-1. However, Nrf2 siRNA reversed the anti-inflammatory effects of sesamin. In conclusion, our results suggested that sesamin showed anti-inflammatory effects in IL-1β-stimulated chondrocytes by activating Nrf2 signaling pathway.

  11. Effect of nitrogen-rich cell culture surfaces on type X collagen expression by bovine growth plate chondrocytes

    Science.gov (United States)

    2011-01-01

    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. PMID:21244651

  12. THE EFFECT ON PROTEOGLYCAN METABOLISM OF DEOXYNIVALENOL AND SELENIUM IN THE CULTURED HUMAN FETAL CHONDROCYTES IN VITRO

    Institute of Scientific and Technical Information of China (English)

    2005-01-01

    Objective To investigate the effect of deoxynivalenol (DON) and selenium (Se) on the morphology of chondrocytes and the metabolism of cartilage matrix, and the expression of aggrecanase-1, 2 mRNA in monolayer cultured chondrocytes in vitro. Methods To plant human fetal chondrocytes on the BMG, the expression of Aggrecanase-1, 2 mRNA were analyzed by RT-PCR, the immunohistochemistry of NITEGE epitope was quantitativly analyzed by the image collection and analysis system. Results With the increase of the concentration of DON, the damage of cultured chondrocytes was more and more severe; the expression of NITEGE epitope showed an increasing trend and the fluorescent bands of aggrecanase-1, 2 mRNA were more and more obvious. After adding Se, the damage was relieved, and there was a decreasing trend of NITEGE epitope expressed in matrix. Conclusion DON can enhance transcription of aggrecanase gene and increase the expression of NITEGE epitope which eventually lead to the metabolic disorder of cartilage proteoglycan. It suggested that Se can partially alleviate the damage of DON on cartilage, but can not completely prevent the occurrence of these changes.

  13. A novel rat tail collagen type-I gel for the cultivation of human articular chondrocytes in low cell density.

    Science.gov (United States)

    Muller-Rath, R; Gavénis, K; Andereya, S; Mumme, T; Schmidt-Rohlfing, B; Schneider, U

    2007-12-01

    Collagen type-I matrix systems have gained growing importance as a cartilage repair device. However, most of the established matrix systems use collagen type-I of bovine origin seeded in high cell densities. Here we present a novel collagen type-I gel system made of rat tail collagen for the cultivation of human chondrocytes in low cell densities. Rat tail collagen type-I gel (CaReS, Arthro Kinetics, Esslingen, Germany) was seeded with human passage 2 chondrocytes in different cell densities to evaluate the optimal cell number. In vitro, the proliferation factor of low density cultures was more than threefold higher compared with high density cultures. After 6 weeks of in vitro cultivation, freshly prepared chondrocytes with an initial cell density of 2x10(5) cells/mL showed a proliferation factor of 33. A cell density of 2x10(5) cells/mL was chosen for in vitro and in vivo cultivation using the common nude mouse model as an in vivo system. Chondrocytes stayed viable as a Live/Dead fluorescence assay and TUNEL staining revealed. During in vitro cultivation, passage 0 cells partly dedifferentiated morphologically. In vivo, passage 0 cells maintained the chondrocyte phenotype and demonstrated an increased synthesis of collagen type-II protein and gene expression compared to passage 2 cells. Passage 2 cells did not redifferentiate in vivo. Cultivating a cell-seeded collagen gel of bovine origin as a control (AtelocollagenTM, Koken, Tokyo, Japan) did not lead to superior results with regard to cell morphology, col-II protein production and col-II gene expression. With the CaReS collagen gel system the best quality of repair tissue was obtained by seeding freshly isolated chondrocytes.

  14. Resveratrol inhibits the IL-1β-induced expression of MMP-13 and IL-6 in human articular chondrocytes via TLR4/MyD88-dependent and -independent signaling cascades.

    Science.gov (United States)

    Gu, Hailun; Jiao, Yongliang; Yu, Xiaolu; Li, Xingyao; Wang, Wei; Ding, Lifeng; Liu, Li

    2017-03-01

    The natural polyphenolic compound, resveratrol, has been shown to exhibit anti-osteoarthritic activity. Therefore it is hypothesized that resveratrol may serve as a nutritional supplement to counteract osteoarthritis (OA). However, the mechanisms responsible for these anti-osteoarthritic effects have not yet been fully elucidated. The aim of this study was to determine whether the biological effects of resveratrol against interleukin (IL)-1β‑induced inflammation in human articular chondrocytes involved both Toll-like receptor 4 (TLR4)/myeloid differentiation factor 88 (MyD88)-dependent and -independent signaling pathways. Human articular chondrocytes derived from patients with OA were stimulated with IL-1β, and then co-treated with resveratrol. Cell viability was subsequently evaluated by MTS assays, and the concentrations of matrix metalloproteinase (MMP)-13 and the pro-inflammatory factor, IL-6, were detected in culture supernatants using ELISA. The mRNA and protein levels of downstream mediators of TLR4/MyD88-dependent and -independent signaling pathways were also assayed by RT-qPCR and western blot analysis, respectively. Our results revealed that resveratrol prevented the IL-1β-induced reduction in cell viability. Furthermore, stimulation of the chondrocytes with IL-1β resulted in a significant upregulation of TLR4 and downstream targets of both TLR4/MyD88-dependent and -independent signaling pathways that are associated with the synthesis of MMP-13 and IL-6. Correspondingly, IL-1β-induced catabolic and inflammatory responses were effectively reversed by resveratrol. Taken together, these data suggest that resveratrol exerted protective effects against matrix degradation and inflammation in OA-affected chondrocytes by inhibiting both TLR4/MyD88-dependent and -independent signaling pathways. Thus, resveratrol represents a potential treatment for OA and warrants further investigation.

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

    Directory of Open Access Journals (Sweden)

    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.

  16. 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

  17. Chondrocyte Culture in Three Dimensional Alginate Sulfate Hydrogels Promotes Proliferation While Maintaining Expression of Chondrogenic Markers

    Science.gov (United States)

    Mhanna, Rami; Kashyap, Aditya; Palazzolo, Gemma; Vallmajo-Martin, Queralt; Becher, Jana; Möller, Stephanie; Schnabelrauch, Matthias

    2014-01-01

    The loss of expression of chondrogenic markers during monolayer expansion remains a stumbling block for cell-based treatment of cartilage lesions. Here, we introduce sulfated alginate hydrogels as a cartilage biomimetic biomaterial that induces cell proliferation while maintaining the chondrogenic phenotype of encapsulated chondrocytes. Hydroxyl groups of alginate were converted to sulfates by incubation with sulfur trioxide–pyridine complex (SO3/pyridine), yielding a sulfated material cross-linkable with calcium chloride. Passage 3 bovine chondrocytes were encapsulated in alginate and alginate sulfate hydrogels for up to 35 days. Cell proliferation was five-fold higher in alginate sulfate compared with alginate (p=0.038). Blocking beta1 integrins in chondrocytes within alginate sulfate hydrogels significantly inhibited proliferation (p=0.002). Sulfated alginate increased the RhoA activity of chondrocytes compared with unmodified alginate, an increase that was blocked by β1 blocking antibodies (p=0.017). Expression and synthesis of type II collagen, type I collagen, and proteoglycan was not significantly affected by the encapsulation material evidenced by quantitative reverse transcription polymerase chain reaction (qRT-PCR) and immunohistochemistry. Alginate sulfate constructs showed an opaque appearance in culture, whereas the unmodified alginate samples remained translucent. In conclusion, alginate sulfate provides a three dimensional microenvironment that promotes both chondrocyte proliferation and maintenance of the chondrogenic phenotype and represents an important advance for chondrocyte-based cartilage repair therapies providing a material in which cell expansion can be done in situ. PMID:24320935

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

    Directory of Open Access Journals (Sweden)

    Koh Terauchi

    2016-06-01

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

  19. Expansion of human nasal chondrocytes on macroporous microcarriers enhances redifferentiation

    NARCIS (Netherlands)

    Malda, J.; Kreijveld, E.; Temenoff, J.S.; Blitterswijk, van C.A.; Riesle, J.

    2003-01-01

    Articular cartilage has a limited capacity for self-repair. To overcome this problem, it is expected that functional cartilage replacements can be created from expanded chondrocytes seeded in biodegradable scaffolds. Expansion of chondrocytes in two-dimensional culture systems often results in dedif

  20. Expansion of human nasal chondrocytes on macroporous macrocarriers enhances redifferentiation

    NARCIS (Netherlands)

    Malda, J.; Kreijveld, E.; Temenoff, J.; Blitterswijk, van C.A.; Riesle, J.

    2003-01-01

    Articular cartilage has a limited capacity for self-repair. To overcome this problem, it is expected that functional cartilage replacements can be created from expanded chondrocytes seeded in biodegradable scaffolds. Expansion of chondrocytes in two-dimensional culture systems often results in dedif

  1. Expansion of human nasal chondrocytes on macroporous microcarriers enhances redifferentiation

    NARCIS (Netherlands)

    Malda, J.; Kreijveld, E.; Temenoff, J.S.; van Blitterswijk, Clemens; Riesle, J.U.

    2003-01-01

    Articular cartilage has a limited capacity for self-repair. To overcome this problem, it is expected that functional cartilage replacements can be created from expanded chondrocytes seeded in biodegradable scaffolds. Expansion of chondrocytes in two-dimensional culture systems often results in dedif

  2. Three-dimensional scaffold-free fusion culture: the way to enhance chondrogenesis of in vitro propagated human articular chondrocytes

    Directory of Open Access Journals (Sweden)

    M. Lehmann

    2013-11-01

    Full Text Available Cartilage regeneration based on isolated and culture-expanded chondrocytes has been studied in various in vitro models, but the quality varies with respect to the morphology and the physiology of the synthesized tissues. The aim of our study was to promote in vitro chondrogenesis of human articular chondrocytes using a novel three-dimensional (3-D cultivation system in combination with the chondrogenic differentiation factors transforming growth factor beta 2 (TGF-b2 and L-ascorbic acid. Articular chondrocytes isolated from six elderly patients were expanded in monolayer culture. A single-cell suspension of the dedifferentiated chondrocytes was then added to agar-coated dishes without using any scaffold material, in the presence, or absence of TGF-b2 and/or L-ascorbic acid. Three-dimensional cartilage-like constructs, called single spheroids, and microtissues consisting of several spheroids fused together, named as fusions, were formed. Generated tissues were mainly characterized using histological and immunohistochemical techniques. The morphology of the in vitro tissues shared some similarities to native hyaline cartilage in regard to differentiated S100-positive chondrocytes within a cartilaginous matrix, with strong collagen type II expression and increased synthesis of proteoglycans. Finally, our innovative scaffold-free fusion culture technique supported enhanced chondrogenesis of human articular chondrocytes in vitro. These 3-D hyaline cartilage-like microtissues will be useful for in vitro studies of cartilage differentiation and regeneration, enabling optimization of functional tissue engineering and possibly contributing to the development of new approaches to treat traumatic cartilage defects or osteoarthritis.

  3. Cartilage homeoprotein 1, a homeoprotein selectively expressed in chondrocytes.

    Science.gov (United States)

    Zhao, G Q; Zhou, X; Eberspaecher, H; Solursh, M; de Crombrugghe, B

    1993-09-15

    We identified a rat cDNA that encodes cartilage homeoprotein 1 (Cart-1). The deduced amino acid sequence of Cart-1 contains a paired-type homeodomain. Northern blot hybridization and RNase protection assay revealed that Cart-1 RNA was present at high levels in a well-differentiated rat chondrosarcoma tumor and in a cell line derived from this tumor. Cart-1 RNA was detected in primary mouse and rat chondrocytes but not in various fibroblasts including mouse 10T1/2 cells, NIH 3T3 cells, BALB 3T3 cells, and rat skin fibroblasts. It was also undetectable in mouse C2 myoblasts, S194 myeloma cells, and embryonic stem cells. Cart-1 RNA was present at a very low level in tested but was not detected in other soft tissues of 8-week-old rats. In situ hybridization of rat embryos between 14.5 and 16.5 days post coitum revealed relatively high levels of Cart-1 RNA in condensed prechondrocytic mesenchymal cells and in early chondrocytes of cartilage primordia. The levels of Cart-1 RNA were lower in mature chondrocytes. No hybridization was observed in brain, spinal cord, heart, spleen, gastrointestinal tract, liver, and muscle. We speculate that Cart-1 has a role in chondrocyte differentiation.

  4. 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.

    Science.gov (United States)

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

    2015-01-01

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

  5. Expression of osteogenic proteins during the intrasplenic transplantation of Meckel's chondrocytes: A histochemical and immunohistochemical study.

    Science.gov (United States)

    Ishizeki, Kiyoto; Kagiya, Tadayoshi; Fujiwara, Naoki; Otsu, Keishi; Harada, Hidemitsu

    2009-03-01

    Meckel's chondrocytes, derived from the ectomesenchyme, have the potential to transform into other phenotypes. In this study, we transplanted cell pellets of Meckel's chondrocytes into isogenic mouse spleens and analyzed their phenotypic transformation into osteogenic cells using histological and immunohistochemical methods. With the increasing duration of transplantation, chondrocytes were incorporated into splenic tissues and formed a von Kossa-positive calcified matrix containing calcium and phosphoric acid, similar to that of intact bone. Type I, II, and X collagens, and the bone-marker proteins osteocalcin, osteopontin, osteonectin, and bone morphogenetic protein-2 (BMP-2) were immunolocalized in the matrix formed by the transplanted chondrocytes. Osteopontin and osteonectin were detected in the calcified matrix at earlier stages than osteocalcin and BMP-2. Type II collagen was expressed during the first week of transplantation, and type X collagen-positive cells appeared scattered during the initial stage of calcification, these collagens being later replaced by type I collagen formed by osteocyte-like cells. Electron microscopic observations revealed that chondrocytes surrounded by the calcified matrix transformed into spindle-shaped osteocytic cells accompanying the formation of bone-type thick-banded collagen fibrils. These results suggest that phenotypic switching of Meckel's chondrocytes can occur under in vivo conditions at a cellular morphological level.

  6. Expression of Transient Receptor Potential Vanilloid (TRPV Channels in Different Passages of Articular Chondrocytes

    Directory of Open Access Journals (Sweden)

    Richard Barrett-Jolley

    2012-04-01

    Full Text Available Ion channels play important roles in chondrocyte mechanotransduction. The transient receptor potential vanilloid (TRPV subfamily of ion channels consists of six members. TRPV1-4 are temperature sensitive calcium-permeable, relatively non-selective cation channels whereas TRPV5 and TRPV6 show high selectivity for calcium over other cations. In this study we investigated the effect of time in culture and passage number on the expression of TRPV4, TRPV5 and TRPV6 in articular chondrocytes isolated from equine metacarpophalangeal joints. Polyclonal antibodies raised against TRPV4, TRPV5 and TRPV6 were used to compare the expression of these channels in lysates from first expansion chondrocytes (P0 and cells from passages 1–3 (P1, P2 and P3 by western blotting. TRPV4, TRPV5 and TRPV6 were expressed in all passages examined. Immunohistochemistry and immunofluorescence confirmed the presence of these channels in sections of formalin fixed articular cartilage and monolayer cultures of methanol fixed P2 chondrocytes. TRPV5 and TRPV6 were upregulated with time and passage in culture suggesting that a shift in the phenotype of the cells in monolayer culture alters the expression of these channels. In conclusion, several TRPV channels are likely to be involved in calcium signaling and homeostasis in chondrocytes.

  7. Piezo1 protein induces the apoptosis of human osteoarthritis-derived chondrocytes by activating caspase-12, the signaling marker of ER stress.

    Science.gov (United States)

    Li, Xiao-Fei; Zhang, Zhao; Chen, Zhu-Ke; Cui, Zhao-Wei; Zhang, Hai-Ning

    2017-09-01

    The present study was carried out to determine whether the mechanically activated cation channel Piezo1 protein plays a role as a signaling pathway which causes the apoptosis of human chondrocytes. The chondrocytes were isolated, cultured, and then subjected to mechanical stretch force for 0, 2, 12, 24 and 48 h, respectively. The expression levels of Piezo1 and the apoptosis-related protein caspase-12 were assessed by reverse transcription-quantitative polymerase chain reaction, as well as the apoptosis-related genes, B cell lymphoma/leukemia-2 (Bcl-2), Bcl-associated X protein (Bax) and Bcl-2-associated death promoter (BAD). Lactate dehydrogenase (LDH) activity was used to discern dead cells. Piezo1 expression was determined by immunofluorescence. In addition, Piezo1 inhibitor, GsMTx4, was used to block the mechanically activated (MA) cation channel Piezo1, and served as a positive control. The results showed that the osteoarthritis (OA)-derived chondrocytes showed a tendency to undergo late-stage apoptosis under compressive loading. Piezo1 and caspase-12 were significantly upregulated under static compressive stimuli and the expression was related to the rate of apoptosis of the OA-derived chondrocytes during compressive loading. The expression of caspase-12 and late-stage apoptosis of the human OA-derived chondrocytes were repressed by GsMTx4, the specific inhibitor of Piezo1, while the expression of Piezo1 and the induction of the apoptosis of the OA-derived chondrocytes during compressive loading was not totally blocked. Thus, we conclude that Piezo1 plays an important role in the apoptosis of human OA-derived chondrocytes through a caspase-12-dependent pathway. The expression of Piezo1 protein was not totally inhibited by GsMTx4.

  8. Immunophenotypic analysis of human articular chondrocytes: changes in surface markers associated with cell expansion in monolayer culture.

    Science.gov (United States)

    Diaz-Romero, Jose; Gaillard, Jean Philippe; Grogan, Shawn Patrick; Nesic, Dobrila; Trub, Thomas; Mainil-Varlet, Pierre

    2005-03-01

    Cartilage tissue engineering relies on in vitro expansion of primary chondrocytes. Monolayer is the chosen culture model for chondrocyte expansion because in this system the proliferative capacity of chondrocytes is substantially higher compared to non-adherent systems. However, human articular chondrocytes (HACs) cultured as monolayers undergo changes in phenotype and gene expression known as "dedifferentiation." To gain a better understanding of the cellular mechanisms involved in the dedifferentiation process, our research focused on the characterization of the surface molecule phenotype of HACs in monolayer culture. Adult HACs were isolated by enzymatic digestion of cartilage samples obtained post-mortem. HACs cultured in monolayer for different time periods were analyzed by flow cytometry for the expression of cell surface markers with a panel of 52 antibodies. Our results show that HACs express surface molecules belonging to different categories: integrins and other adhesion molecules (CD49a, CD49b, CD49c, CD49e, CD49f, CD51/61, CD54, CD106, CD166, CD58, CD44), tetraspanins (CD9, CD63, CD81, CD82, CD151), receptors (CD105, CD119, CD130, CD140a, CD221, CD95, CD120a, CD71, CD14), ectoenzymes (CD10, CD26), and other surface molecules (CD90, CD99). Moreover, differential expression of certain markers in monolayer culture was identified. Up-regulation of markers on HACs regarded as distinctive for mesenchymal stem cells (CD10, CD90, CD105, CD166) during monolayer culture suggested that dedifferentiation leads to reversion to a primitive phenotype. This study contributes to the definition of HAC phenotype, and provides new potential markers to characterize chondrocyte differentiation stage in the context of tissue engineering applications. 2004 Wiley-Liss, Inc.

  9. Effects of sesamin on the biosynthesis of chondroitin sulfate proteoglycans in human articular chondrocytes in primary culture.

    Science.gov (United States)

    Pothacharoen, Peraphan; Najarus, Sumet; Settakorn, Jongkolnee; Mizumoto, Shuji; Sugahara, Kazuyuki; Kongtawelert, Prachya

    2014-04-01

    Osteoarthritis (OA) is a degenerative joint disease that progressively causes a loss of joint functions and the impaired quality of life. The most significant event in OA is a high degree of degradation of articular cartilage accompanied by the loss of chondroitin sulfate-proteoglycans (CS-PGs). Recently, the chondroprotective effects of sesamin, the naturally occurring substance found in sesame seeds, have been proved in a rat model of papain-induced osteoarthritis. We hypothesized that sesamin may be associated with possible promotion of the biosynthesis of CS-PGs in human articular chondrocytes. The aim of the study was to investigate the effects of sesamin on the major CS-PG biosynthesis in primary human chondrocyte. The effects of sesamin on the gene expression of the PG core and the CS biosynthetic enzymes as well as on the secretion of glycosaminoglycans (GAGs) in monolayer and pellet culture systems of articular chondrocytes. Sesamin significantly increased the GAGs content both in culture medium and pellet matrix. Real-time-quantitative PCR showed that sesamin promoted the expression of the genes encoding the core protein (ACAN) of the major CS-PG aggrecan and the biosynthetic enzymes (XYLT1, XYLT2, CHSY1 and CHPF) required for the synthesis of CS-GAG side chains. Safranin-O staining of sesamin treated chondrocyte pellet section confirmed the high degree of GAG accumulation. These results were correlated with an increased level of secreted GAGs in the media of cultured articular chondrocytes in both culture systems. Thus, sesamin would provide a potential therapeutic strategy for treating OA patients.

  10. Hydroxytyrosol prevents increase of osteoarthritis markers in human chondrocytes treated with hydrogen peroxide or growth-related oncogene α.

    Directory of Open Access Journals (Sweden)

    Annalisa Facchini

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

  11. Human pituitary tissue secretes a potent growth factor for chondrocyte proliferation.

    Science.gov (United States)

    Kasper, S; Friesen, H G

    1986-01-01

    We report the secretion from human pituitary tumor fragments in organ culture of a potent mitogen for chondrocyte proliferation. Primary human pituitary cell and organ cultures were established from pituitary fragments obtained from patients with acromegaly, prolactinomas, and nonfunctional adenomas. The conditioned culture medium contained a mitogenic factor(s) that stimulated rabbit fetal chondrocyte proliferation, causing up to an 8-fold increase in cell number when added to Ham's F-10 medium in the presence of 10% fetal bovine serum. Blood leaking into the surgical field after the adenomectomy is known to contain very high concentrations of pituitary hormones. Serum samples, obtained from this venous "ooze" collected at the site of pituitary surgery, also were found to contain chondrocyte growth-promoting activity. Some venous serum samples stimulated chondrocyte proliferation in a dose-dependent manner down to a 1:10 dilution of 1 microliter serum, indicating that the material being secreted was very potent indeed. Gel filtration on Sephadex G-100 and analytical gel isoelectric focusing of culture media or serum samples from the pituitary fossa demonstrated that the growth factor secreted from the pituitary tumor fragments as well as from the venous serum is similar, if not identical, to chondrocyte growth factor (mol wt, 43,000; pI 7.6-7.9) purified from human pituitaries collected at autopsy. These results suggest that the chondrocyte growth-promoting factor(s) may not only be secreted by pituitary tumor fragments but by normal human pituitary tissue as well.

  12. In vitro isolation and cultivation of human chondrocytes for osteoarthritis renovation.

    Science.gov (United States)

    Xu, Jiaming; Zhang, Changqing

    2014-08-01

    The purpose of this study was to evaluate the repair effects of chondrocytes that were cultured in vitro on osteoarthritis (OA). Chondrocytes were isolated from fetal rabbits and cultured in Biosilon microcarriers. Sixty rabbits were randomly divided into three groups equally (blank group, model group, treatment group). The rabbit knee OA model was established by inducing papain. Rabbits in the treatment group were injected with the chondrocytes that were cultured in vitro. Hematoxylin-eosin (HE) staining and gross morphologic observation were conducted. Expression level of cytokines such as IL-1bβ, IL-6, and TNF-α in cartilage synovial cells was also analyzed by an ELISA assay. The cultured chondrocyte was validated by a positive stain of type II collagen and vimentin by immunofluorescence. Compared to the model group, the articular cartilage of the rabbit knee in the treatment group showed a normal color, smooth surface, and none of malacia and coloboma. HE staining indicated that the articular surface of the treatment group tended to be smooth and flat; the matrix stained tinge and the cartilage destruction and fiber hyperplasia of the synovia were lightened. The expression levels of IL-1bβ, IL-6, and TNF-α also declined in the treatment group. OA symptoms were improved by treating with chondrocytes. In summary, the animal experiment in the present study indicated that chondrocyte injection played an active effect on renovation of OA.

  13. Cartilage homeoprotein 1, a homeoprotein selectively expressed in chondrocytes.

    OpenAIRE

    Zhao, G. Q.; Zhou, X.; Eberspaecher, H; Solursh, M; de Crombrugghe, B

    1993-01-01

    We identified a rat cDNA that encodes cartilage homeoprotein 1 (Cart-1). The deduced amino acid sequence of Cart-1 contains a paired-type homeodomain. Northern blot hybridization and RNase protection assay revealed that Cart-1 RNA was present at high levels in a well-differentiated rat chondrosarcoma tumor and in a cell line derived from this tumor. Cart-1 RNA was detected in primary mouse and rat chondrocytes but not in various fibroblasts including mouse 10T1/2 cells, NIH 3T3 cells, BALB 3T...

  14. 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.

  15. MicroRNA-381 Regulates Chondrocyte Hypertrophy by Inhibiting Histone Deacetylase 4 Expression.

    Science.gov (United States)

    Chen, Weishen; Sheng, Puyi; Huang, Zhiyu; Meng, Fangang; Kang, Yan; Huang, Guangxin; Zhang, Zhiqi; Liao, Weiming; Zhang, Ziji

    2016-08-23

    Chondrocyte hypertrophy, regulated by Runt-related transcription factor 2 (RUNX2) and matrix metalloproteinase 13 (MMP13), is a crucial step in cartilage degeneration and osteoarthritis (OA) pathogenesis. We previously demonstrated that microRNA-381 (miR-381) promotes MMP13 expression during chondrogenesis and contributes to cartilage degeneration; however, the mechanism underlying this process remained unclear. In this study, we observed divergent expression of miR-381 and histone deacetylase 4 (HDAC4), an enzyme that directly inhibits RUNX2 and MMP13 expression, during late-stage chondrogenesis of ATDC5 cells, as well as in prehypertrophic and hypertrophic chondrocytes during long bone development in E16.5 mouse embryos. We therefore investigated whether this miRNA regulates HDAC4 expression during chondrogenesis. Notably, overexpression of miR-381 inhibited HDAC4 expression but promoted RUNX2 expression. Moreover, transfection of SW1353 cells with an miR-381 mimic suppressed the activity of a reporter construct containing the 3'-untranslated region (3'-UTR) of HDAC4. Conversely, treatment with a miR-381 inhibitor yielded increased HDAC4 expression and decreased RUNX2 expression. Lastly, knockdown of HDAC4 expression resulted in increased RUNX2 and MMP13 expression in SW1353 cells. Collectively, our results indicate that miR-381 epigenetically regulates MMP13 and RUNX2 expression via targeting of HDAC4, thereby suggesting the possibilities of inhibiting miR-381 to control chondrocyte hypertrophy and cartilage degeneration.

  16. Mangiferin Inhibits IL-1β-Induced Inflammatory Response by Activating PPAR-γ in Human Osteoarthritis Chondrocytes.

    Science.gov (United States)

    Qu, Yanlong; Zhou, Li; Wang, Chunlei

    2017-02-01

    Inflammation has been reported to play critical roles in the development of osteoarthritis. In the present study, we investigated whether mangiferin (MFN) had anti-inflammatory effects in IL-1β-stimulated human osteoarthritis chondrocytes. The cells were treated with various concentrations of MFN in the presence or absence of IL-1β. The production of MMP-1, MMP-3, PGE2, and NO was measured in this study. The expression of NF-kB and PPAR-γ was detected by western blot analysis. MFN inhibited IL-1β-induced inflammatory mediators PGE2 and NO production. MFN also inhibited IL-1β-induced MMP1 and MMP3 production. IL-1β-induced NF-kB activation was significantly inhibited by MFN. In addition, MFN was found to up-regulate the expression of PPAR-γ in human osteoarthritis chondrocytes. PPAR-γ inhibitor GW9662 significantly reversed the anti-inflammatory effects of MFN. These results suggest that MFN inhibits IL-1β-induced inflammatory response in human osteoarthritis chondrocytes by activating PPAR-γ.

  17. Chondrocyte gene expression is affected by very small iron oxide particles-labeling in long-term in vitro MRI tracking.

    Science.gov (United States)

    Foldager, Casper Bindzus; Pedersen, Michael; Ringgaard, Steffen; Bünger, Cody; Lind, Martin

    2011-03-01

    To investigate the effect and dose response of very small iron oxide particles (VSOP) labeling of human chondrocytes for long-term in vitro MRI tracking. Chondrocytes were isolated from cartilage biopsies from four patients. The cells for the dose-response study were labeled with 25, 50, or 100 μg/mL VSOP. Quantitative gene expression and cellular proliferation were compared with unlabeled controls at day 1, 3, and 7. The cells suited for MRI tracking were labeled with 50 μg/mL VSOP and embedded in alginate beads, followed by MRI (using T2-weighted sequences) at day 0, 1, 3, 7, 14, 21, 28, and histology was performed at each time-point. Histology revealed that VSOP particles were intracellularly confined at all time-points, whereas no extracellular VSOPs were observed. A mean reduction in T2-value of 25.1 ms (±SD 3.5 ms) was found on T2-maps. The chondrocyte-specific genes aggrecan, collagen type 2, and sox9 were all affected by labeling, the two latter in a dose-dependent manner. VSOPs had no effect on proliferation. VSOP labeling of chondrocytes affected gene expression but not proliferation. The labeled chondrocytes could be recognized by MRI for 4 weeks without significant changes in the T2 relaxation time. Copyright © 2011 Wiley-Liss, Inc.

  18. In vitro analysis of integrin expression during chondrogenic differentiation of mesenchymal stem cells and chondrocytes upon dedifferentiation in cell culture.

    Science.gov (United States)

    Goessler, Ulrich Reinhart; Bieback, Karen; Bugert, Peter; Heller, Tobias; Sadick, Haneen; Hörmann, Karl; Riedel, Frank

    2006-02-01

    Tissue engineering represents a promising method for generating chondrogenic grafts for reconstructive surgery. In cultured chondrocytes, the dedifferentiation of cells seems unavoidable for multiplication. Stem cells, however, displaying unlimited self-renewal and the capacity to differentiate towards chondrocytes, might be usable after further characterization. As the interactions between the extracellular matrix and the cellular compartment can alter the cellular behaviour, we investigated the expression of integrins using microarray analysis during chondrogenic differentiation of human mesenchymal stem cells (MSC) in comparison with de-differentiating human chondrocytes (HC) harvested during septoplasty. During chondrogenic differentiation of MSC, the fibronectin-receptor (Integrin beta1alpha5), fibronectin and the GPIIb/IIIa-receptor were downregulated. The components of the vitronectin-receptor (Integrin alphavbeta3) and CD47 were constantly expressed and ILK was downregulated. Vitronectin and osteopontin were not expressed by the cells. In HC, Integrin beta1alpha5 in conjunction with the ligand fibronectin were upregulated during dedifferentiation, Integrin alphavbeta3 as well as the GBIIb/IIIa-receptor were activated on day 21 but neither vitronectin nor osteopontin were expressed by the cells. The integrins, beta2, beta4, beta6, beta8 and alpha2, alpha4, alpha6, alpha7, alpha11, were not expressed at any time. ILK, CD47, and ICAP were activated with ongoing dedifferentiation. In conclusion, a candidate for signal-transmission is the fibronectin receptor (integrin alpha5beta1) in conjunction with its ligand fibronectin. Other receptors, e.g. for vitronectin and osteopontin (alphavbeta3), or their ligands do not seem to be involved in signal transmission for dedifferentiation. The GPIIb/IIIa-receptor might assist the process of dedifferentiation. Intracellularly, ILK, ICAP1 and CD47 might be involved in the transduction of integrin-dependent signals.

  19. 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.

  20. Expression of adhesion molecules and collagen on rat chondrocyte seeded into alginate and hyaluronate based 3D biosystems. Influence of mechanical stresses.

    Science.gov (United States)

    Gigant-Huselstein, C; Hubert, P; Dumas, D; Dellacherie, E; Netter, P; Payan, E; Stoltz, J F

    2004-01-01

    Chondrocytes use mechanical signals, via interactions with their environment, to synthesize an extracellular matrix capable to withstanding high loads. Most chondrocyte-matrix interactions are mediated via transmembrane receptors such as integrins or non-integrins receptors (i.e. annexin V and CD44). The aim of this study was to analyze, by flow cytometry, the adhesion molecules (alpha5/beta1 integrins and CD44) on rat chondrocytes seeded into 3D biosystem made of alginate and hyaluronate. These biosystems were submitted to mechanical stress by knocking the biosystems between them for 48 hours. The expression of type I and type II collagen was also evaluated. The results of the current study showed that mechanical stress induced an increase of type II collagen production and weak variations of alpha5/beta1 receptors expression no matter what biosystems. Moreover, our results indicated that hyaluronan receptor CD44 expression depends on extracellular matrix modifications. Thus, these receptors were activated by signals resulted from cell environment variations (HA addition and modifications owing to mechanical stress). It suggested that this kind of receptor play a crucial role in extracellular matrix homeostasis. Finally, on day 24, no dedifferentiation of chondrocytes was noted either in biosystems or under mechanical stress. For all biosystems, the neosynthesized matrix contained an important level of collagen, which was type II, whatever biosystems. In conclusion, it appeared that the cells, under mechanical stress, maintained their phenotype. In addition, it seems that, on rat chondrocytes, alpha5/beta1 integrins did not act as the main mechanoreceptor (as described for human chondrocytes). In return, hyaluronan receptor CD44 seems to be in relation with matrix composition.

  1. 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.

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

    NARCIS (Netherlands)

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

    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 intracell

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

    Directory of Open Access Journals (Sweden)

    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

  4. Chondroitin sulfate and hyaluronic acid (500-730 kda) inhibit stromelysin-1 synthesis in human osteoarthritic chondrocytes.

    Science.gov (United States)

    Monfort, J; Nacher, M; Montell, E; Vila, J; Verges, J; Benito, P

    2005-01-01

    Chondroitin sulfate (CS) and 500-730 kDa hyaluronic acid (HA) are symptomatic slow-acting drugs for the treatment of osteoarthritis (OA). In addition, a growing body of evidence suggests a role for CS and this specific HA as modifiers of the course of OA. The therapeutic efficacy of CS and HA lies in their different mechanisms of action. Stromelysin-1 (metalloprotease-3 [MMP-3]) is a cartilage proteolytic enzyme, which induces cartilage destruction and acts as a mediator of the inflammatory response. However, there are few studies evaluating the in vitro effect of CS and HA on MMP-3 synthesis in human chondrocyte cultures from OA patients. Thus, the aim of the present study was to analyze the effect of CS and HA (500-730 kDa) on MMP-3 synthesis induced by interleukin-1beta (IL-1beta) in chondrocytes from patients with hip OA. Chondrocyte cultures were incubated for 48 h with IL-1beta (2.5 ng/ml) in the absence or presence of different HA 500-730 kDa (Hyalgan, Bioibérica Farma, Barcelona, Spain) concentrations, or alternatively, CS (Condro.san, Bioibérica Farma) at concentrations of 10, 50, 100, 150, 200 and 1,000 microg/ml. The results revealed that both CS and HA (500-730 kDa) inhibited MMP-3 synthesis induced by IL-1beta in human OA chondrocytes. Specifically, CS and HA (500-730 kDa) reduced MMP-3 expression levels at all tested concentrations. Therefore, our study provides new data on the mechanism of action of these drugs, which could help to explain their clinical efficacy in OA patients.

  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. 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.

  7. 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

  8. 高强度周期性静水压对人软骨细胞超微结构及II 型胶原分泌的影响%Effects of high-intensity cyclical hydrostatic pressures on the ultrastructure and type II collagen expression of chondrocytes of knee joint in human

    Institute of Scientific and Technical Information of China (English)

    王鑫; 白倩; 徐奎; 裘秀春; 范清宇; 马保安

    2016-01-01

    Objective To investigate the effects of high-intensity cyclic hydrostatic pressures on the ultrastructure and type II collagen contents of chondrocytes of the knee joint cultured in vitro in human. Methods The normal chondrocytes of human knee joint were isolated and cultured in vitro. The 3rd generation of chondrocytes were treated with high-intensity cyclical hydrostatic pressures ( 10.0 mPa ) by the multifunctional thermostatic high-insensitive hydrostatic pressure loading device for 2 h per day lasting for 5 days. Toluidine blue staining and immunohistochemical staining of type II collagen were employed to identify the chondrocytes. Cell morphology was observed by light microscopy. The ultrastructure of chondrocytes were observed by transmission electron microscopy ( TEM ). Type II collagen immunohistochemical staining and semi-quantitative analysis were performed to measure contents and expression of type II collagen of chondrocytes in 2 groups. Results Compared with the control group, cell morphology changed from irregular polygon into long spindle, membrane and cytoplasm retracted, the number of cells was reduced significantly and grew sparsely in the 10.0 mPa group. TEM showed that various apoptosis indications such as uneven distribution and margination of chromosomes in the 10.0 mPa group. Collagen type II immunohistochemistry and semi-quantitative analysis showed that the percent of stained area of control group and 10.0 mPa group was ( 61.64 ± 6.19 ) % and ( 43.76 ± 5.61 ) %, compared with the control group, the stained area and extent of chondrocytes were significantly decreased in the 10.0 mPa group ( P < 0.05 ). Conclusions The high-intensity pressure over human physiological range can be regarded as a mechanical injury to result in cell apoptosis, morphology and intracellular ultrastructural changes of human chondrocytes, and also decrease protein expression of human chondrocytes. These data provide the experimental basis for studying the

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

    Directory of Open Access Journals (Sweden)

    Thomas J Kean

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

  10. 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 in...

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

    Science.gov (United States)

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

    2016-04-01

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

  13. Tenascin and aggrecan expression by articular chondrocytes is influenced by interleukin 1ß: a possible explanation for the changes in matrix synthesis during osteoarthritis

    Science.gov (United States)

    Pfander, D; Heinz, N; Rothe, P; Carl, H; Swoboda, B

    2004-01-01

    Objective: To analyse the distribution patterns of tenascin and proteoglycans in normal and osteoarthritic cartilage, and to determine the effect of interleukin 1ß (IL1ß) on aggrecan and tenascin expression by human articular chondrocytes in vitro. Methods: Normal and osteoarthritic cartilage and bone samples were obtained during total knee replacements or necropsies. After fixation and decalcification, paraffin embedded specimens were sectioned perpendicular to the surface. Specimens were graded according to Mankin and subdivided into those with normal, and mild, moderate, and severe osteoarthritic lesions. Serial sections were immunostained for tenascin. Tenascin expression by healthy and osteoarthritic chondrocytes was quantified by real time polymerase chain reaction (PCR). Furthermore, in cell culture experiments, human articular chondrocytes were treated with 0.1 or 10 ng/ml IL1ß. Real time PCR analyses of aggrecan and tenascin transcripts (normalised 18S rRNA) were conducted to determine the effect of IL1ß on later mRNA levels. Results: Tenascin was immunodetected in normal and osteoarthritic cartilage. In osteoarthritic cartilage increased tenascin staining was found. Tenascin was found specifically in upper OA cartilage showing a strong reduction of proteoglycans. Greatly increased tenascin transcript levels were detected in osteoarthritic cartilage compared with healthy articular cartilage. IL1ß treatment of articular chondrocytes in vitro significantly increased tenascin transcripts (~200% of control) and strongly reduced aggrecan mRNA levels (~42% of control). Conclusions: During progression of osteoarthritis the switch in matrix synthesis occurs mainly in upper osteoarthritic cartilage. Furthermore, changes in synthesis patterns of osteoarthritic chondrocytes may be significantly influenced by IL1ß, probably diffusing from the joint cavity within the upper osteoarthritic cartilage. PMID:14962956

  14. Interleukin-1 Acts via the JNK-2 Signaling Pathway to Induce Aggrecan Degradation by Human Chondrocytes.

    Science.gov (United States)

    Ismail, Heba M; Yamamoto, Kazuhiro; Vincent, Tonia L; Nagase, Hideaki; Troeberg, Linda; Saklatvala, Jeremy

    2015-07-01

    Aggrecan enables articular cartilage to bear load and resist compression. Aggrecan loss occurs early in osteoarthritis and rheumatoid arthritis and can be induced by inflammatory cytokines such as interleukin-1 (IL-1). IL-1 induces cleavage of specific aggrecans characteristic of the ADAMTS proteinases. The aim of this study was to identify the intracellular signaling pathways by which IL-1 causes aggrecan degradation by human chondrocytes and to investigate how aggrecanase activity is controlled by chondrocytes. We developed a cell-based assay combining small interfering RNA (siRNA)-induced knockdown with aggrecan degradation assays. Human articular chondrocytes were overlaid with bovine aggrecan after transfection with siRNAs against molecules of the IL-1 signaling pathway. After IL-1 stimulation, released aggrecan fragments were detected with AGEG and ARGS neoepitope antibodies. Aggrecanase activity and tissue inhibitor of metalloproteinases 3 levels were measured by enzyme-linked immunosorbent assay. Low-density lipoprotein receptor-related protein 1 (LRP-1) shedding was analyzed by Western blotting. ADAMTS-5 is a major aggrecanase in human chondrocytes, regulating aggrecan degradation in response to IL-1. The tumor necrosis factor receptor-associated 6 (TRAF-6)/transforming growth factor β-activated kinase 1 (TAK-1)/MKK-4 signaling axis is essential for IL-1-induced aggrecan degradation, while NF-κB is not. Of the 3 MAPKs (ERK, p38, and JNK), only JNK-2 showed a significant role in aggrecan degradation. Chondrocytes constitutively secreted aggrecanase, which was continuously endocytosed by LRP-1, keeping the extracellular level of aggrecanase low. IL-1 induced aggrecanase activity in the medium in a JNK-2-dependent manner, possibly by reducing aggrecanase endocytosis, because IL-1 caused JNK-2-dependent shedding of LRP-1. The signaling axis TRAF-6/TAK-1/MKK-4/JNK-2 mediates IL-1-induced aggrecanolysis. The level of aggrecanase is controlled by its

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

    Science.gov (United States)

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

    2014-06-01

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

  16. Regeneration of human-ear-shaped cartilage by co-culturing human microtia chondrocytes with BMSCs.

    Science.gov (United States)

    Zhang, Lu; He, Aijuan; Yin, Zongqi; Yu, Zheyuan; Luo, Xusong; Liu, Wei; Zhang, Wenjie; Cao, Yilin; Liu, Yu; Zhou, Guangdong

    2014-06-01

    Previously, we had addressed the issues of shape control/maintenance of in vitro engineered human-ear-shaped cartilage. Thus, lack of applicable cell source had become a major concern that blocks clinical translation of this technology. Autologous microtia chondrocytes (MCs) and bone marrow stromal cells (BMSCs) were both promising chondrogenic cells that did not involve obvious donor site morbidity. However, limited cell availability of MCs and ectopic ossification of chondrogenically induced BMSCs in subcutaneous environment greatly restricted their applications in external ear reconstruction. The current study demonstrated that MCs possessed strong proliferation ability but accompanied with rapid loss of chondrogenic ability during passage, indicating a poor feasibility to engineer the entire ear using expanded MCs. Fortunately, the co-transplantation results of MCs and BMSCs (25% MCs and 75% BMSCs) demonstrated a strong chondroinductive ability of MCs to promote stable ectopic chondrogenesis of BMSCs in subcutaneous environment. Moreover, cell labeling demonstrated that BMSCs could transform into chondrocyte-like cells under the chondrogenic niche provided by co-cultured MCs. Most importantly, a human-ear-shaped cartilaginous tissue with delicate structure and proper elasticity was successfully constructed by seeding the mixed cells (MCs and BMSCs) into the pre-shaped biodegradable ear-scaffold followed by 12 weeks of subcutaneous implantation in nude mouse. These results may provide a promising strategy to construct stable ectopic cartilage with MCs and stem cells (BMSCs) for autologous external ear reconstruction.

  17. MiR-15a-5p regulates viability and matrix degradation of human osteoarthritis chondrocytes via targeting VEGFA.

    Science.gov (United States)

    Chen, Hongwei; Tian, Yun

    2017-01-16

    Previous studies demonstrated that miR-15a-5p was probably associated with human hepatocellular carcinoma, while the function of miR-15a-5p in OA (Osteoarthritis) still remains unknown. Here, we uncovered the potential role of miR-15a-5p on OA pathogenesis and confirmed its predicted target VEGFA (Vascular Endothelial Growth Factor A). Measured by RT-PCR, miR-15a-5p expression increased remarkably while VEGFA expression was significantly decreased in OA chondrocytes compared with normal conditions. According to Luciferase activity assay, miR-15a-5p directly targeted the 3'-UTR of VEGFA to inhibit its expression. Functional analysis including CCK-8 assay and flow cytometry revealed that overexpression of VEGFA or inhibition of miR-15a-5p promoted cell proliferation, suppressed cell apoptosis and reduced matrix degradation in OA chondrocytes. Moreover, rescue assays carried out with both expression of VEGFA and miR-15a-5p demonstrated that miR-15a-5p contributes to cell apoptosis and matrix degradation via inhibiting VEGFA. We further provided evidence that multiple proteins related to matrix synthesis were regulated by miR-15a-5p and VEGFA using Western blot and ELISA assays. Taken together, our findings elucidated an underlying mechanism by which miR-15a-5p regulates viability and matrix degradation of OA and indicated a new target for OA diagnosis and therapy.

  18. 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.

  19. 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.

  20. Characterization of human primary chondrocytes of osteoarthritic cartilage at varying severity

    Institute of Scientific and Technical Information of China (English)

    YIN Jing; YANG Zheng; CAO Yong-ping; GE Zi-gang

    2011-01-01

    Background There is a difficulty in evaluating the in vivo functionality of individual chondrocytes,and there is much heterogeneity among cartilage affected by osteoarthritis (OA).In this study,in vitro cultured chondrocytes harvested from varying stages of degeneration were studied as a projective model to further understand the pathogenesis of osteoarthritis.Methods Cartilage of varying degeneration of end-stage OA was harvested,while cell yield and matrix glycosaminoglycan (GAG) content were measured.Cell morphology,proliferation,and gene expression of collagen type Ⅰ,Ⅱ,and Ⅹ,aggrecan,matrix metalloproteinase 13 (MMP-13),and ADAMTS5 of the acquired chondrocytes were measured during subsequent in vitro culture.Results Both the number of cells and the GAG content increased with increasing severity of OA.Cell spreading area increased and gradually showed spindle-like morphology during in vitro culture.Gene expression of collagen type Ⅱ,collagen type X as well as GAG decreased with severity of cartilage degeneration,while expression of collagen type Ⅰ increased.Expression of MMP-13 increased with severity of cartilage degeneration,while expression of ADAMTS-5 remained stable.Expression of collagen type Ⅱ,X,GAG,and MMP-13 substantially decreased with in vitro culture.Expression of collagen type Ⅰ increased with in vitro cultures,while expression of ADAMTS 5 remained stable.Conclusions Expression of functional genes such as collagen type Ⅱ and GAG decreased during severe degeneration of OA cartilage and in vitro dedifferentiation.Gene expression of collagen Ⅰ and MMP-13 increased with severity of cartilage degeneration.

  1. The upregulation of matrix metalloproteinase -2 and -9 genes caused by resistin in human chondrocytes

    Directory of Open Access Journals (Sweden)

    Kürşat Oğuz Yaykaşlı

    2014-02-01

    Full Text Available  OBJECTIVES: The articular cartilage allows movement by absorbing mechanical loading within a physiological range. However, the accumulation of excessive adipose tissue has catabolic effect on extracellular matrix (ECM components in some diseases such as rheumatoid arthritis (RA and osteoarthritis (OA. Resistin, inflammatory adipokines is secreted by adipose tissue, and the elevated serum level was reported in obese subjects and patients with RA and OA. Gelatinases (MMP-2 and MMP-9, a subfamily of matrix metalloproteinases are responsible for destruction of collagen and matrix components. In this study, the effect of resistin on gelatinases genes expression was investigated. METHODS: Human chondrocytes was stimulated by resistin at 100 and 250 ng/ml doses for 3h, 6h, 12h, 24h and 48h. 2 µg RNA was subject to reverse transcription after RNA extraction. Gelatinases expressions were analyzed by quantitative Real-Time Polymerase Chain Reaction method. RESULTS: The expression levels of gelatinases were increased at both 100 and 250 ng/mL and peaked at 250 ng/ml dose for 48 hours. CONCLUSIONS: The clarification of etiology for irreversible destruction of ECM has a vital importance to develop new treatment strategies for RA and OA. In conclusion, increased levels of gelatinases expression caused by resistin were founded. The upregulation of gelatinases caused by resistin is might be a new target for obesity associated patients with RA and OA. However, the molecular pathways of this induction should be investigated in other studies. 

  2. 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.

  3. Na+, K+-ATPase Subunit Composition in a Human Chondrocyte Cell Line; Evidence for the Presence of α1, α3, β1, β2 and β3 Isoforms

    Directory of Open Access Journals (Sweden)

    Ali Mobasheri

    2012-04-01

    Full Text Available Membrane transport systems participate in fundamental activities such as cell cycle control, proliferation, survival, volume regulation, pH maintenance and regulation of extracellular matrix synthesis. Multiple isoforms of Na+, K+-ATPase are expressed in primary chondrocytes. Some of these isoforms have previously been reported to be expressed exclusively in electrically excitable cells (i.e., cardiomyocytes and neurons. Studying the distribution of Na+, K+-ATPase isoforms in chondrocytes makes it possible to document the diversity of isozyme pairing and to clarify issues concerning Na+, K+-ATPase isoform abundance and the physiological relevance of their expression. In this study, we investigated the expression of Na+, K+-ATPase in a human chondrocyte cell line (C-20/A4 using a combination of immunological and biochemical techniques. A panel of well-characterized antibodies revealed abundant expression of the α1, β1 and β2 isoforms. Western blot analysis of plasma membranes confirmed the above findings. Na+, K+-ATPase consists of multiple isozyme variants that endow chondrocytes with additional homeostatic control capabilities. In terms of Na+, K+-ATPase expression, the C-20/A4 cell line is phenotypically similar to primary and in situ chondrocytes. However, unlike freshly isolated chondrocytes, C-20/A4 cells are an easily accessible and convenient in vitro model for the study of Na+, K+-ATPase expression and regulation in chondrocytes.

  4. Hydrostatic pressure decreases membrane fluidity and lipid desaturase expression in chondrocyte progenitor cells.

    Science.gov (United States)

    Montagne, Kevin; Uchiyama, Hiroki; Furukawa, Katsuko S; Ushida, Takashi

    2014-01-22

    Membrane biomechanical properties are critical in modulating nutrient and metabolite exchange as well as signal transduction. Biological membranes are predominantly composed of lipids, cholesterol and proteins, and their fluidity is tightly regulated by cholesterol and lipid desaturases. To determine whether such membrane fluidity regulation occurred in mammalian cells under pressure, we investigated the effects of pressure on membrane lipid order of mouse chondrogenic ATDC5 cells and desaturase gene expression. Hydrostatic pressure linearly increased membrane lipid packing and simultaneously repressed lipid desaturase gene expression. We also showed that cholesterol mimicked and cholesterol depletion reversed those effects, suggesting that desaturase gene expression was controlled by the membrane physical state itself. This study demonstrates a new effect of hydrostatic pressure on mammalian cells and may help to identify the molecular mechanisms involved in hydrostatic pressure sensing in chondrocytes.

  5. Clinical Outcome 3 Years After Autologous Chondrocyte Implantation Does Not Correlate With the Expression of a Predefined Gene Marker Set in Chondrocytes Prior to Implantation but Is Associated With Critical Signaling Pathways.

    Science.gov (United States)

    Stenberg, Johan; de Windt, Tommy S; Synnergren, Jane; Hynsjö, Lars; van der Lee, Josefine; Saris, Daniel B F; Brittberg, Mats; Peterson, Lars; Lindahl, Anders

    2014-09-01

    There is a need for tools to predict the chondrogenic potency of autologous cells for cartilage repair. To evaluate previously proposed chondrogenic biomarkers and to identify new biomarkers in the chondrocyte transcriptome capable of predicting clinical success or failure after autologous chondrocyte implantation. Controlled laboratory study and case-control study; Level of evidence, 3. Five patients with clinical improvement after autologous chondrocyte implantation and 5 patients with graft failures 3 years after implantation were included. Surplus chondrocytes from the transplantation were frozen for each patient. Each chondrocyte sample was subsequently thawed at the same time point and cultured for 1 cell doubling, prior to RNA purification and global microarray analysis. The expression profiles of a set of predefined marker genes (ie, collagen type II α1 [COL2A1], bone morphogenic protein 2 [BMP2], fibroblast growth factor receptor 3 [FGFR3], aggrecan [ACAN], CD44, and activin receptor-like kinase receptor 1 [ACVRL1]) were also evaluated. No significant difference in expression of the predefined marker set was observed between the success and failure groups. Thirty-nine genes were found to be induced, and 38 genes were found to be repressed between the 2 groups prior to autologous chondrocyte implantation, which have implications for cell-regulating pathways (eg, apoptosis, interleukin signaling, and β-catenin regulation). No expressional differences that predict clinical outcome could be found in the present study, which may have implications for quality control assessments of autologous chondrocyte implantation. The subtle difference in gene expression regulation found between the 2 groups may strengthen the basis for further research, aiming at reliable biomarkers and quality control for tissue engineering in cartilage repair. The present study shows the possible limitations of using gene expression before transplantation to predict the chondrogenic and

  6. Upregulation of aggrecan and type II collagen mRNA expression in bovine chondrocytes by the application of hydrostatic pressure.

    Science.gov (United States)

    Toyoda, Takashi; Seedhom, Bahaa B; Kirkham, Jennifer; Bonass, William A

    2003-01-01

    The aim of this study was to investigate the effect of hydrostatic pressure on the expression of messenger ribonucleic acid (mRNA) for specific extracellular matrix proteins in chondrocytes. Chondrocytes obtained from bovine metatarsophalangeal joints were embedded in cylindrical 2% agarose gels. A novel experimental system was used to apply 5 MPa of static hydrostatic pressure to these chondrocytes for 4 hours. The application of hydrostatic pressure caused a significant increase in the level of aggrecan mRNA by almost four fold (papplication of hydrostatic pressure, in the absence of cell deformation, can bring about changes in the matrix components which may play an important role in the homeostasis and mechanical properties of articular cartilage.

  7. 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.

  8. Interplay between cytoskeletal polymerization and the chondrogenic phenotype in chondrocytes passaged in monolayer culture.

    Science.gov (United States)

    Parreno, Justin; Nabavi Niaki, Mortah; Andrejevic, Katarina; Jiang, Amy; Wu, Po-Han; Kandel, Rita A

    2017-02-01

    Tubulin and actin exist as monomeric units that polymerize to form either microtubules or filamentous actin. As the polymerization status (monomeric/polymeric ratio) of tubulin and/or actin have been shown to be important in regulating gene expression and phenotype in non-chondrocyte cells, the objective of this study was to examine the role of cytoskeletal polymerization on the chondrocyte phenotype. We hypothesized that actin and/or tubulin polymerization status modulates the chondrocyte phenotype during monolayer culture as well as in 3D culture during redifferentiation. To test this hypothesis, articular chondrocytes were grown and passaged in 2D monolayer culture. Cell phenotype was investigated by assessing cell morphology (area and circularity), actin/tubulin content, organization and polymerization status, as well as by determination of proliferation, fibroblast and cartilage matrix gene expression with passage number. Bovine chondrocytes became larger, more elongated, and had significantly (P  0.05) modulated, actin polymerization was increased in bovine P2 cells. Actin depolymerization, but not tubulin depolymerization, promoted the chondrocyte phenotype by inducing cell rounding, increasing aggrecan and reducing COL1 expression. Knockdown of actin depolymerization factor, cofilin, in these cells induced further P2 cell actin polymerization and increased COL1 gene expression. To confirm that actin status regulated COL1 gene expression in human P2 chondrocytes, human P2 chondrocytes were exposed to cytochalasin D. Cytochalasin D decreased COL1 gene expression in human passaged chondrocytes. Furthermore, culture of bovine P2 chondrocytes in 3D culture on porous bone substitute resulted in actin depolymerization, which correlated with decreased expression of COL1 and proliferation molecules. In 3D cultures, aggrecan gene expression was increased by cytochalasin D treatment and COL1 was further decreased. These results reveal that actin polymerization

  9. Bone marrow extract as a growth supplement for human iliac apophyseal chondrocyte culture

    Directory of Open Access Journals (Sweden)

    Balasubramanian Balakumar

    2016-01-01

    Full Text Available Background & objectives: Human bone marrow is rich in various growth factors which may support the chondrocyte growth. This study was conducted to compare the culture characteristics of human growth plate chondrocyte in foetal bovine serum (FBS and human autologous bone marrow extract (BME in monolayer culture. Methods: Iliac crest apophyseal cartilage was harvested from four donors, aged between two and nine years, undergoing hip surgery. Chondrocytes were propagated under two culture conditions, with 10 per cent FBS and 10 per cent autologous BME harvested from the same donors. Cells were harvested at 7, 14 and 21 days to assess viability, morphology, cell count and immunocytochemistry. Results: With an initial seeding density of 2500 cells/cm 2 , the average yield in monolayer cultured with FBS was 3.35 × 10 5 , 5.9 × 10 5 , 14.1 × 10 5 and BME was 0.66 × 10 5 , 1.57 × 10 5 and 3.48 × 10 5 at 7, 14 and 21 days, respectively. Viability was 98.21 per cent with FBS and 97.45 per cent with BME at 21 days. In BME supplemented cultures, hyaline phenotype was maintained up to 21 days. The yield was higher in the FBS supplemented group; however, the phenotype could not be maintained by the FBS group as long as BME group. Interpretation & conclusions: Autologous BME was found to be a safer alternative to FBS for human studies. BME could maintain the hyaline phenotype for a longer time. Ways to enhance the cell yield needs to be explored in future studies.

  10. WNT4 acts downstream of BMP2 to mediate the regulation of ATRA signaling on RUNX1 expression: Implications for terminal differentiation of antler chondrocytes.

    Science.gov (United States)

    Zhang, Hong-Liang; Yang, Zhan-Qing; Duan, Cui-Cui; Geng, Shuang; Wang, Kai; Yu, Hai-Fan; Yue, Zhan-Peng; Guo, Bin

    2017-04-24

    Although ATRA is involved in regulating the proliferation and differentiation of chondrocytes, its underlying mechanism remains unknown. Here we showed that ATRA could stimulate the proliferation of antler chondrocytes and expression of COL X and MMP13 which were two well-known markers for hypertrophic chondrocytes. Silencing of CRABP2 prevented the induction of ATRA on chondrocyte terminal differentiation, while overexpression of CRABP2 exhibited the opposite effects. CYP26A1 and CYP26B1 weakened the sensitivity of antler chondrocytes to ATRA. Further analysis evidenced that ATRA might induce chondrocyte terminal differentiation and modulate the expression of BMP2, WNT4, and RUNX1 through RARα/RXRα. Knockdown of BMP2 enhanced the induction of ATRA on the expression of COL X and MMP13, whereas overexpression of BMP2 abrogated this effectiveness. WNT4 might mediate the effects of ATRA and BMP2 on chondrocyte terminal differentiation. Dysregulation of BMP2 impaired the regulation of ATRA on WNT4 expression. Administration of ATRA to antler chondrocytes transfected with RUNX1 siRNA failed to induce the differentiation. Conversely, rRUNX1 strengthened the stimulation of ATRA on the expression of COL X and MMP13. Simultaneously, RUNX1 was a downstream effector of BMP2 and WNT4 in chondrocyte terminal differentiation. Moreover, WNT4 might play an important role in the crosstalk between BMP2 and RUNX1. Attenuation of BMP2 or WNT4 enhanced the interaction between ATRA and RUNX1, while constitutive expression of BMP2 or WNT4 reversed the regulation of ATRA on RUNX1. Collectively, WNT4 may act downstream of BMP2 to mediate the effects of ATRA on the terminal differentiation of antler chondrocytes through targeting RUNX1. © 2017 Wiley Periodicals, Inc.

  11. β-Defensin-4 (HBD-4) is expressed in chondrocytes derived from normal and osteoarthritic cartilage encapsulated in PEGDA scaffold.

    Science.gov (United States)

    Musumeci, Giuseppe; Carnazza, Maria Luisa; Loreto, Corrado; Leonardi, Rosalia; Loreto, Carla

    2012-12-01

    Defensins are antibiotic peptides involved in host defense mechanisms, wound healing and tissue repair. Furthermore, they seem to play an important role in protection mechanisms in articular joints. The aim of this study was to investigate β-defensin-4 expression in chondrocytes taken from articular cartilage of knees of patients with osteoarthritis (OA) compared to normal cartilage, in vivo in explanted tissue, and in vitro in chondrocytes encapsulated in construct PEGDA hydrogels. The present investigation was conducted to try and elucidate the possible use of β-defensin-4 as a relevant marker for the eventual use of successive scaffold allografts, and to provide new insights for hydrogel PEGDA scaffold efficacy in re-differentiation or repair of OA chondrocytes in vitro. Articular cartilage specimens from OA cartilage and normal cartilage were assessed by histology, histochemistry, immunohistochemistry and Western blot analysis. The results showed strong β-defensin-4 immunoexpression in explanted tissue from OA cartilage and weak β-defensin-4 expression in control cartilage. The chondrocytes from OA cartilage after 4 weeks of culture in PEGDA hydrogels showed the formation of new hyaline cartilage and a decreased expression of β-defensin-4 immunostaining comparable to that of control cartilage. Our results suggest the possibility of applying autologous cell transplantation in conjunction with scaffold materials for repair of cartilage lesions in patients with OA using β-defensin-4 as a relevant marker. Copyright © 2012 Elsevier GmbH. All rights reserved.

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

    Science.gov (United States)

    Li, S; Oreffo, R O C; Sengers, B G; Tare, R S

    2014-09-01

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

  13. Regulation of α5 and αV Integrin Expression by GDF-5 and BMP-7 in Chondrocyte Differentiation and Osteoarthritis

    Science.gov (United States)

    Garciadiego-Cázares, David; Aguirre-Sánchez, Hilda I.; Abarca-Buis, René F.; Kouri, Juan B.; Velasquillo, Cristina; Ibarra, Clemente

    2015-01-01

    The Integrin β1 family is the major receptors of the Extracellular matrix (ECM), and the synthesis and degradation balance of ECM is seriously disrupted during Osteoarthritis (OA). In this scenario, integrins modify their pattern expression and regulate chondrocyte differen-tiation in the articular cartilage. Members of the Transforming growth factor beta (Tgf-β) Su-perfamily, such as Growth differentiation factor 5 (Gdf-5) and Bone morphogenetic protein 7 (Bmp-7), play a key role in joint formation and could regulate the integrin expression during chondrocyte differentiation and osteoarthritis progression in an experimental OA rat model. Decrease of α5 integrin expression in articular cartilage was related with chondrocyte dedif-ferentiation during OA progression, while increase of α1, α2, and α3 integrin expression was related with fibrous areas in articular cartilage during OA. Hypertrophic chondrocytes expressedαV integrin and was increased in the articular cartilage of rats with OA. Integrin expression during chondrocyte differentiation was also analyzed in a micromass culture system of mouse embryo mesenchymal cells, micromass cultures was treated with Gdf-5 or Bmp-7 for 4 and 6 days, respectively. Gdf-5 induced the expression of theα5 sub-unit, while Bmp-7 induced the expression of the αV sub-unit. This suggests a switch in signaling for prehypertrophic chondrocyte differentiation towards hypertrophy, where Gdf-5 could maintain the articular chondrocyte phenotype and Bmp-7 would induce hypertrophy. Decrease of Ihh expression during late stages of OA in rat model suggest that the ossification in OA rat knees and endochondral ossification could be activated by Bmp-7 and αV integrin in absence of Ihh. Thus, chondrocyte phenotype in articular cartilage is similar to prehypetrophic chondrocyte in growth plate, and is preserved due to the presence of Indian hedgehog (Ihh), Gdf-5 and α5 integrin to maintain articular cartilage and prevent hy

  14. Human-like collagen/nano-hydroxyapatite scaffolds for the culture of chondrocytes

    Energy Technology Data Exchange (ETDEWEB)

    Jia, Liping; Duan, Zhiguang [Shaanxi Key Laboratory of Degradable Biomedical Materials, Northwest University, 229 Taibai North Road, Xi' an, Shaanxi 710069 (China); Shaanxi R and D Center of Biomaterials and Fermentation Engineering, Northwest University, 229 Taibai North Road, Xi' an, Shaanxi 710069 (China); Fan, Daidi, E-mail: fandaidi@nwu.edu.cn [Shaanxi Key Laboratory of Degradable Biomedical Materials, Northwest University, 229 Taibai North Road, Xi' an, Shaanxi 710069 (China); Shaanxi R and D Center of Biomaterials and Fermentation Engineering, Northwest University, 229 Taibai North Road, Xi' an, Shaanxi 710069 (China); Mi, Yu; Hui, Junfeng [Shaanxi Key Laboratory of Degradable Biomedical Materials, Northwest University, 229 Taibai North Road, Xi' an, Shaanxi 710069 (China); Shaanxi R and D Center of Biomaterials and Fermentation Engineering, Northwest University, 229 Taibai North Road, Xi' an, Shaanxi 710069 (China); Chang, Le [School of Chemical Engineering, Northwest University, Xi' an, Shaanxi 710069 (China)

    2013-03-01

    Three dimensional (3D) biodegradable porous scaffolds play a key role in cartilage tissue repair. Freeze-drying and cross-linking techniques were used to fabricate a 3D composite scaffold that combined the excellent biological characteristics of human-like collagen (HLC) and the outstanding mechanical properties of nano-hydroxyapatite (nHA). The scaffolds were characterized by scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD) and compression tests, using Relive Registered-Sign Artificial Bone (RAB) scaffolds as a control. HLC/nHA scaffolds displayed homogeneous interconnected macroporous structure and could withstand a compression stress of 2.67 {+-} 0.37 MPa, which was higher than that of the control group. Rabbit chondrocytes were seeded on the composite porous scaffolds and cultured for 21 days. Cell/scaffold constructs were examined using SEM, histological procedures, and biochemical assays for cell proliferation and the production of glycosaminoglycans (GAGs). The results indicated that HLC/nHA porous scaffolds were capable of encouraging cell adhesion, homogeneous distribution and abundant GAG synthesis, and maintaining natural chondrocyte morphology compared to RAB scaffolds. In conclusion, the presented data warrants the further exploration of HLC/nHA scaffolds as a potential biomimetic platform for chondrocytes in cartilage tissue engineering. - Highlights: Black-Right-Pointing-Pointer Human-like collagen was first used to prepare cartilage tissue engineering scaffold. Black-Right-Pointing-Pointer Genipin, a natural biological cross-linking agent, was introduced to treat scaffold. Black-Right-Pointing-Pointer We chose market product as a control.

  15. The cytotoxicity of bupivacaine, ropivacaine, and mepivacaine on human chondrocytes and cartilage.

    Science.gov (United States)

    Breu, Anita; Rosenmeier, Katharina; Kujat, Richard; Angele, Peter; Zink, Wolfgang

    2013-08-01

    Intraarticular injections of local anesthetics are frequently used as part of multimodal pain regimens. However, recent data suggest that local anesthetics affect chondrocyte viability. In this study, we assessed the chondrotoxic effects of mepivacaine, ropivacaine, and bupivacaine. We hypothesized that specific cytotoxic potencies directly correlate with analgesic potencies, and that cytotoxic effects in intact cartilage are different than in osteoarthritic tissue. Human articular chondrocytes were exposed to equal and equipotent concentrations of bupivacaine, ropivacaine, and mepivacaine for 1 hour. Cell viability, apoptosis, and necrosis were determined at predefined time points using flow cytometry, live-dead staining, and caspase detection. Intact and osteoarthritic human cartilage explants were treated with equipotent concentrations of named drugs to determine cell viability applying fluorescence microscopy. Chondrotoxic effects increased from ropivacaine to mepivacaine to bupivacaine in a time-dependent and concentration-dependent manner. Compared with control, bupivacaine 0.5% decreased chondrocyte viability to 78% ± 9% (P = 0.0183) 1 hour and 16% ± 10% (P mepivacaine 2%, viable cells were scored 36% ± 6% (P mepivacaine exposure (P = 0.0059). Exposure to concentrations up to 0.25% of bupivacaine, 0.5% of ropivacaine, and 0.5% of mepivacaine did not reveal significant chondrotoxicity in flow cytometry. However, chondrotoxicity did not correlate with potency of local anesthetics. Immediate cell death was mainly due to necrosis followed by apoptosis. Cellular death rates were clearly higher in osteoarthritic compared with intact cartilage after bupivacaine, mepivacaine, and ropivacaine treatment in a decreasing order. Bupivacaine, ropivacaine, and mepivacaine are chondrotoxic in a time-dependent, concentration-dependent, and drug-dependent manner. Chondrotoxic and analgesic potencies do not directly correlate. Cellular death rates were higher in

  16. 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

    1992-01-01

    The effect of piroxicam on the metabolism of healthy and osteoarthrotic (OA) chondrocytes was studied in vitro. The chondrocytes were obtained from five healthy, five moderately OA, and four severely OA hips or knees. The chondrocytes were cultured in a high-density, short-term in vitro model. In th

  17. The effect of scaffold composition on the early structural characteristics of chondrocytes and expression of adhesion molecules.

    Science.gov (United States)

    Schagemann, Jan C; Kurz, Haymo; Casper, Michelle E; Stone, James S; Dadsetan, Mahrokh; Yu-Long, Sun; Mrosek, Eike H; Fitzsimmons, James S; O'Driscoll, Shawn W; Reinholz, Gregory G

    2010-04-01

    Previously we demonstrated that chondrocyte ECM synthesis and mitotic activity was dependent on scaffold composition when cultured on uncoated PCL scaffolds (pPCL) or PCL composites containing hyaluronan (PCL/HA), chitosan (PCL/CS), fibrin (PCL/F), or collagen type I (PCL/COL1). We hypothesized that initial cell contact with these biomaterials results in ultrastructural changes and alters CD44 and integrin beta1 expression. The current study was designed to investigate the early ultrastructural responses of chondrocytes on these scaffolds and expression of CD44 and integrin beta1. A common observation 1 h after seeding was the abundance of cell processes. Different types of cell processes occurred in different areas of the same cell and on different cells within the same composite. Chondrocytes seeded onto PCL/CS had the greatest cell surface enhancement. PCL/HA promoted CD44 expression and almost spherical cells with a low degree of surface enhancement. PCL/COL1 enabled continuing expression of integrin beta1 and CD44. In contrast, cells in PCL/CS, PCL/F and pPCL promoted elliptic cells with a higher degree of surface enhancement and no prolonged CD44 and integrin beta1 expression. A strong variability of cell surface processes indicated either reparative or degenerative adaptation to the artificial environment. Interestingly, we found initial integrin beta1 expression in all composite scaffolds, but not in pPCL although this promoted strong adhesiveness as indicated by the formation of stress fibers. In conclusion, chondrocytes respond to biomaterials early after implantation by altering ultrastructural characteristics and expression of CD44 and integrin beta1.

  18. CD44 knock-down in bovine and human chondrocytes results in release of bound HYAL2

    Science.gov (United States)

    Hida, Daisuke; Danielson, Ben T.; Knudson, Cheryl B.; Knudson, Warren

    2015-01-01

    CD44 shedding occurs in osteoarthritic chondrocytes. Previous work of others has suggested that the hyaluronidase isoform HYAL2 has the capacity to bind to CD44, a binding that may itself induce CD44 cleavage. Experiments were developed to elucidate whether chondrocyte HYAL2: (1) was exposed on the extracellular plasma membrane of chondrocytes, (2) bound to CD44, (3) underwent shedding together with CD44 and lastly, (4) exhibited hyaluronidase activity within a near-neutral pH range. Enhancing CD44 shedding by IL-1β resulted in a proportional increase in HYAL2 released from human and bovine chondrocytes into the medium. CD44 knockdown by siRNA also resulted in increased accumulation of HYAL2 in the media of chondrocytes. By hyaluronan zymography only activity at pH 3.7 was observed and this activity was reduced by pre-treatment of chondrocytes with trypsin. CD44 and HYAL2 were found to co-immunoprecipitate, and to co-localize within intracellular vesicles and at the plasma membrane. Degradation of hyaluronan was visualized by agarose gel electrophoresis. With this approach, hyaluronidase activity could be observed at pH 4.8 under assay conditions in which CD44 and HYAL2 binding remained intact; additionally, weak hyaluronidase activity could be observed at pH 6.8 under these conditions. This study suggests that CD44 and HYAL2 are bound at the surface of chondrocytes. The release of HYAL2 when CD44 is shed could provide a mechanism for weak hyaluronidase activity to occur within the more distant extracellular matrix of cartilage. PMID:25864644

  19. In vitro human chondrocyte culture on plasma-treated poly(glycerol sebacate) scaffolds.

    Science.gov (United States)

    Theerathanagorn, Tharinee; Klangjorhor, Jeerawan; Sakulsombat, Morakot; Pothacharoen, Peraphan; Pruksakorn, Dumnoensun; Kongtawelert, Prachya; Janvikul, Wanida

    2015-01-01

    Porous poly(glycerol sebacate) (PGS) scaffolds were prepared using a salt leaching technique and subsequently surface modified by a low oxygen plasma treatment prior to the use in the in vitro culture of human chondrocytes. Condensation polymerization of glycerol and sebacic acid used at various mole ratios, i.e. 1:1, 1:1.25, and 1:1.5, was initially conducted to prepare PGS prepolymers. Porous elastomeric PGS scaffolds were directly fabricated from the mixtures of each prepolymer and 90% (w/w) NaCl particles and then subjected to the plasma treatment to enhance the surface hydrophilicity of the materials. The properties of both untreated and plasma-treated PGS scaffolds were comparatively evaluated, in terms of surface morphology, surface chemical composition, porosity, and storage modulus using scanning electron microscopy (SEM), X-ray photoelectron spectroscopy, micro-computed tomography, and dynamic mechanical analysis, respectively. The responses of chondrocytes cultured on individual PGS scaffolds were assessed, in terms of cell proliferation and ECM production. The results revealed that average pore sizes and porosity of the scaffolds were increased with an increasing sebacic acid concentration used. The storage moduli of the scaffolds were raised after the plasma treatment, possibly due to the further crosslinking of PGS upon treatment. Moreover, the scaffold prepared with a higher sebacic acid content demonstrated a greater capability of promoting cell infiltration, proliferation, and ECM production, especially when it was plasma-treated; the greatest HA, sGAG, uronic acid, and collagen contents were detected in matrix of this scaffold. The H & E and safranin O staining results also strongly supported this finding. The storage modulus of the scaffold was intensified after incubation with the chondrocytes for 21 days, indicating the accretion and retention of matrix ECM on the cell-cultured scaffold.

  20. Baicalein Inhibits MMPs Expression via a MAPK-Dependent Mechanism in Chondrocytes

    Directory of Open Access Journals (Sweden)

    Wei-Ping Chen

    2015-05-01

    Full Text Available Background: Baicalein is a flavonoid isolated from Scutellaria baicalensis Georgi. Here, we investigated the anti-osteoarthritic effect of baicalein in vitro and in vivo. Methods: Interleukin-1 beta (IL-1β-induced chondrocytes were treated with different concentrations of baicalein, real-time PCR and ELISA were performed to detect the matrix metalloproteinases (MMPs expression. Western blot was used to evaluate the mitogen-activated protein kinase (MAPK expression. In experimental osteoarthritis (OA, rabbits were treated with baicalein, gross morphological and histological assessment was performed to evaluate the cartilage damage. Results: Baicalein significantly reduced the expression of MMPs in vitro and in vivo. Moreover, baicalein significantly reduced the phosphorylation of p38 and extracellular signal regulated kinase (ERK, but not of c-Jun N-terminal kinase (JNK. In addition, intra-articular injection of baicalein ameliorated the cartilage damage in a rabbit model of OA induced by anterior cruciate ligament transection (ACLT. Conclusions: The results indicate that baicalein may be considered as a potential agent for OA treatment.

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

    Science.gov (United States)

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

    2016-06-14

    Animal cells have served as highly controllable model systems for furthering cartilage tissue engineering practices in pursuit of treating osteoarthritis. Although successful strategies for animal cells must ultimately be adapted to human cells to be clinically relevant, human chondrocytes are rarely employed in such studies. In this study, we evaluated the applicability of culture techniques established for juvenile bovine and adult canine chondrocytes to human chondrocytes obtained from fresh or expired osteochondral allografts. Human chondrocytes were expanded and encapsulated in 2% agarose scaffolds measuring ∅3-4mm×2.3mm, with cell seeding densities ranging from 15 to 90×10(6)cells/mL. Subsets of constructs were subjected to transient or sustained TGF-β treatment, or provided channels to enhance nutrient transport. Human cartilaginous constructs physically resembled native human cartilage, and reached compressive Young's moduli of up to ~250kPa (corresponding to the low end of ranges reported for native knee cartilage), dynamic moduli of ~950kPa (0.01Hz), and contained 5.7% wet weight (%/ww) of glycosaminoglycans (≥ native levels) and 1.5%/ww collagen. We found that the initial seeding density had pronounced effects on tissue outcomes, with high cell seeding densities significantly increasing nearly all measured properties. Transient TGF-β treatment was ineffective for adult human cells, and tissue construct properties plateaued or declined beyond 28 days of culture. Finally, nutrient channels improved construct mechanical properties, presumably due to enhanced rates of mass transport. These results demonstrate that our previously established culture system can be successfully translated to human chondrocytes. Copyright © 2016 Elsevier Ltd. All rights reserved.

  2. Matrix stiffness promotes cartilage endplate chondrocyte calcification in disc degeneration via miR-20a targeting ANKH expression.

    Science.gov (United States)

    Liu, Ming-Han; Sun, Chao; Yao, Yuan; Fan, Xin; Liu, Huan; Cui, You-Hong; Bian, Xiu-Wu; Huang, Bo; Zhou, Yue

    2016-05-04

    The mechanical environment is crucial for intervertebral disc degeneration (IDD). However, the mechanisms underlying the regulation of cartilage endplate (CEP) calcification by altered matrix stiffness remain unclear. In this study, we found that matrix stiffness of CEP was positively correlated with the degree of IDD, and stiff matrix, which mimicked the severe degeneration of CEP, promoted inorganic phosphate-induced calcification in CEP chondrocytes. Co-expression analysis of the miRNA and mRNA profiles showed that increasing stiffness resulted in up-regulation of miR-20a and down-regulation of decreased ankylosis protein homolog (ANKH) during inorganic phosphate-induced calcification in CEP chondrocytes. Through a dual luciferase reporter assay, we confirmed that miR-20a directly targets 3'-untranslated regions of ANKH. The inhibition of miR-20a attenuated the calcium deposition and calcification-related gene expression, whereas the overexpression of miR-20a enhanced calcification in CEP chondrocytes on stiff matrix. The rescue of ANKH expression restored the decreased pyrophosphate efflux and inhibited calcification. In clinical samples, the levels of ANKH expression were inversely associated with the degeneration degree of CEP. Thus, our findings demonstrate that the miR-20a/ANKH axis mediates the stiff matrix- promoted CEP calcification, suggesting that miR-20a and ANKH are potential targets in restraining the progression of IDD.

  3. Expression of hypoxia-inducible factor 2 alpha and vascular endothelial growth factor in chondrocytes of articular cartilages in human osteoarthritis%骨关节炎关节软骨细胞中低氧诱导因子2α及血管内皮生长因子的表达

    Institute of Scientific and Technical Information of China (English)

    刘丰; 彭昊; 周建林; 方洪松; 邓爽; 杨骁; 翁金清

    2015-01-01

    BACKGROUND:Studies have found that vascular endothelial growth factor and hypoxia inducible factor are involved in the development process of osteoarthritis, but their correlation is rarely reported. OBJECTIVE:To observe the expression and correlation of hypoxia inducible factor-2α and vascular endothelial growth factor in chondrocytes of articular cartilages in human osteoarthritis. METHODS: Articular cartilage specimens were colected from 50 patients with knee osteoarthritis undergoing total knee joint replacement. According to the joint Kelgren-Lawrance (K-L) X-ray grouping classification standard, there were 18 cases of K-LIII level and 32 cases of K-LIV level. Besides, articular cartilage specimens from 10 patients undergoing amputation for legs tumor or traffic accident served as control group. Hematoxylin-eosin staining, Safranin O-Fast Green staining and Mankin scoring were performed to observe and evaluate the histological characteristics of articular cartilages of each group, immunohistochemical staining was conducted to detect the expression of hypoxia inducible factor-2α and vascular endothelial growth factor in chondrocytes of articular cartilages, and their correlations were analyzed. RESULTS AND CONCLUSION:The Mankin score of K-LIV group was significantly higher than those of K-LIII group and control group. Immunohistochemical staining revealed that the number of chondrocytes with positive expression of hypoxia inducible factor-2α or vascular endothelial growth factor in K-LIV group was significantly higher than that in K-LIII group and control group (P < 0.05). The expression of hypoxia inducible factor-2α and vascular endothelial growth factor increased in chondrocytes of articular cartilages of osteoarthritis patients, and to up-regulate the expression of vascular endothelial growth factor may be the regulatory mechanism of hypoxia inducible factor-2αinthe pathogenesis of osteoarthritis.%背景:已有研究发现血管内皮生长因子

  4. Meniscal repair in vivo using human chondrocyte-seeded PLGA mesh scaffold pretreated with platelet-rich plasma.

    Science.gov (United States)

    Kwak, Hong Suk; Nam, Jinwoo; Lee, Ji-Hye; Kim, Hee Joong; Yoo, Jeong Joon

    2017-02-01

    The objective of this study was to test the hypothesis that platelet-rich plasma (PRP) pretreatment on a poly-lactic-co-glycolic acid (PLGA) mesh scaffold enhances the healing capacity of the meniscus with human chondrocyte-seeded scaffolds in vivo, even when the seeded number of cells was reduced from 10 million to one million. A flexible PLGA mesh scaffold was pretreated with PRP using a centrifugal technique. One million human articular chondrocytes were seeded onto the scaffold by dynamic oscillation. After 7 days, scaffolds were placed between human meniscal discs and were implanted subcutaneously in nude mice for 6 weeks (n = 16/group). Fluorescence microscopy demonstrated uniform attachment of the chondrocytes throughout the scaffolds 24 h following seeding. Cell attachment analysis revealed a significantly increased number of chondrocytes on PRP-pretreated than non-treated scaffolds (p network at 24 h and day 7 of culture. Of the 16 constructs containing PRP-pretreated scaffolds implanted in mice, six menisci healed completely, nine healed incompletely and one did not heal. Histological results from the 16 control constructs containing non-treated scaffolds revealed that none had healed completely, four healed incompletely and 12 did not heal. The histological outcome between the groups was significantly different (p mesh scaffolds demonstrate increased cell attachment and enhance the healing capacity of meniscus with a reduced number of seeding cells in a meniscal repair mouse model. Copyright © 2014 John Wiley & Sons, Ltd.

  5. Proteome analysis during chondrocyte differentiation in a new chondrogenesis model using human umbilical cord stroma mesenchymal stem cells.

    Science.gov (United States)

    De la Fuente, Alexandre; Mateos, Jesús; Lesende-Rodríguez, Iván; Calamia, Valentina; Fuentes-Boquete, Isaac; de Toro, Francisco J; Arufe, Maria C; Blanco, Francisco J

    2012-02-01

    Umbilical cord stroma mesenchymal stem cells were differentiated toward chondrocyte-like cells using a new in vitro model that consists of the random formation of spheroids in a medium supplemented with fetal bovine serum on a nonadherent surface. The medium was changed after 2 days to one specific for the induction of chondrocyte differentiation. We assessed this model using reverse transcriptase-polymerase chain reaction, flow cytometry, immunohistochemistry, and secretome analyses. The purpose of this study was to determine which proteins were differentially expressed during chondrogenesis. Differential gel electrophoresis analysis was performed, followed by matrix-assisted laser desorption/ionization mass spectrometry protein identification. A total of 97 spots were modulated during the chondrogenesis process, 54 of these spots were identified as 39 different proteins and 15 were isoforms. Of the 39 different proteins identified 15 were down-regulated, 21 were up-regulated, and 3 were up- and down-regulated during the chondrogenesis process. Using Pathway Studio 7.0 software, our results showed that the major cell functions modulated during chondrogenesis were cellular differentiation, proliferation, and migration. Five proteins involved in cartilage extracellular matrix metabolism found during the differential gel electrophoresis study were confirmed using Western blot. The results indicate that our in vitro chondrogenesis model is an efficient and rapid technique for obtaining cells similar to chondrocytes that express proteins characteristic of the cartilage extracellular matrix. These chondrocyte-like cells could prove useful for future cell therapy treatment of cartilage pathologies.

  6. Proteome Analysis During Chondrocyte Differentiation in a New Chondrogenesis Model Using Human Umbilical Cord Stroma Mesenchymal Stem Cells*

    Science.gov (United States)

    De la Fuente, Alexandre; Mateos, Jesús; Lesende-Rodríguez, Iván; Calamia, Valentina; Fuentes-Boquete, Isaac; de Toro, Francisco J.; Arufe, Maria C.; Blanco, Francisco J.

    2012-01-01

    Umbilical cord stroma mesenchymal stem cells were differentiated toward chondrocyte-like cells using a new in vitro model that consists of the random formation of spheroids in a medium supplemented with fetal bovine serum on a nonadherent surface. The medium was changed after 2 days to one specific for the induction of chondrocyte differentiation. We assessed this model using reverse transcriptase-polymerase chain reaction, flow cytometry, immunohistochemistry, and secretome analyses. The purpose of this study was to determine which proteins were differentially expressed during chondrogenesis. Differential gel electrophoresis analysis was performed, followed by matrix-assisted laser desorption/ionization mass spectrometry protein identification. A total of 97 spots were modulated during the chondrogenesis process, 54 of these spots were identified as 39 different proteins and 15 were isoforms. Of the 39 different proteins identified 15 were down-regulated, 21 were up-regulated, and 3 were up- and down-regulated during the chondrogenesis process. Using Pathway Studio 7.0 software, our results showed that the major cell functions modulated during chondrogenesis were cellular differentiation, proliferation, and migration. Five proteins involved in cartilage extracellular matrix metabolism found during the differential gel electrophoresis study were confirmed using Western blot. The results indicate that our in vitro chondrogenesis model is an efficient and rapid technique for obtaining cells similar to chondrocytes that express proteins characteristic of the cartilage extracellular matrix. These chondrocyte-like cells could prove useful for future cell therapy treatment of cartilage pathologies. PMID:22008206

  7. 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......, 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...... 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...

  8. Micromass co-culture of human articular chondrocytes and human bone marrow mesenchymal stem cells to investigate stable neocartilage tissue formation in vitro

    Directory of Open Access Journals (Sweden)

    S Giovannini

    2010-10-01

    Full Text Available Cell therapies for articular cartilage defects rely on expanded chondrocytes. Mesenchymal stem cells (MSC represent an alternative cell source should their hypertrophic differentiation pathway be prevented. Possible cellular instruction between human articular chondrocytes (HAC and human bone marrow MSC was investigated in micromass pellets. HAC and MSC were mixed in different percentages or incubated individually in pellets for 3 or 6 weeks with and without TGF-beta1 and dexamethasone (±T±D as chondrogenic factors. Collagen II, collagen X and S100 protein expression were assessed using immunohistochemistry. Proteoglycan synthesis was evaluated applying the Bern score and quantified using dimethylmethylene blue dye binding assay. Alkaline phosphatase activity (ALP was detected on cryosections and soluble ALP measured in pellet supernatants. HAC alone generated hyaline-like discs, while MSC formed spheroid pellets in ±T±D. Co-cultured pellets changed from disc to spheroid shape with decreasing number of HAC, and displayed random cell distribution. In -T-D, HAC expressed S100, produced GAG and collagen II, and formed lacunae, while MSC did not produce any cartilage-specific proteins. Based on GAG, collagen type II and S100 expression chondrogenic differentiation occurred in -T-D MSC co-cultures. However, quantitative experimental GAG and DNA values did not differ from predicted values, suggesting only HAC contribution to GAG production. MSC produced cartilage-specific matrix only in +T+D but underwent hypertrophy in all pellet cultures. In summary, influence of HAC on MSC was restricted to early signs of neochondrogenesis. However, MSC did not contribute to the proteoglycan deposition, and HAC could not prevent hypertrophy of MSC induced by chondrogenic stimuli.

  9. Serum-free media for articular chondrocytes in vitro expansion

    Institute of Scientific and Technical Information of China (English)

    SHAO Xin-xin; Neil A.Duncan; LIN Lin; FU Xin; ZHANG Ji-ying; YU Chang-long

    2013-01-01

    Background In vitro chondrocyte expansion is a major challenge in cell-based therapy for human articular cartilage repair.Classical culture conditions usually use animal serum as a medium supplement,which raises a number of undesirable questions.In the present study,two kinds of defined,serum-free media were developed to expand chondrocytes in monolayer culture for the purpose of cartilage tissue engineering.Methods Bovine chondrocytes were expanded in serum-free media supplemented with fibroblast growth factor-2 and platelet-derived growth factor or fibroblast growth factor-2 and insulin-like growth factor.Expansion culture in a conventional 10% fetal bovine serum (FBS) medium served as control.Fibronectin coating was used to help cell adhesion in serum-free medium.Next,in vitro three-dimensional pellet culture was used to evaluate the chondrocyte capacity.Cell pellets were expanded in different media to re-express the differentiated phenotype (re-differentiation) and to form cartilaginous tissue.The pellets were assessed by glycosaminoglycans contents,collagen II,collagen I and collagen X immunohistological staining.Results Chondrocytes cultured in serum-free media showed no proliferation difference than cells grown with 10% FBS medium.In addition,chondrocytes expanded in both serum-free media expressed more differentiated phenotypes at the end of monolayer culture,as indicated by higher gene expression ratios of collagen type Ⅱ to collagen type Ⅰ.Pellets derived from chondrocytes cultured in both serum-free media displayed comparable chondrogenic capacities to pellets from cells expanded in 10% FBS medium.Conclusion These findings provide alternative culture approaches for chondrocytes in vitro expansion,which may benefit the clinical use of autologous chondrocytes implantation.

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

    Science.gov (United States)

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

    2016-01-01

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

  11. Cell-engineered human elastic chondrocytes regenerate natural scaffold in vitro and neocartilage with neoperichondrium in the human body post-transplantation.

    Science.gov (United States)

    Yanaga, Hiroko; Imai, Keisuke; Koga, Mika; Yanaga, Katsu

    2012-10-01

    We have developed a unique method that allows us to culture large volumes of chondrocyte expansion from a small piece of human elastic cartilage. The characteristic features of our culturing method are that fibroblast growth factor-2 (FGF2), which promotes proliferation of elastic chondrocytes, is added to a culture medium, and that cell-engineering techniques are adopted in the multilayered culture system that we have developed. We have subsequently discovered that once multilayered chondrocytes are transplanted into a human body, differentiation induction that makes use of surrounding tissue occurs in situ, and a large cartilage block is obtained through cartinogenesis and matrix formation. We have named this method two-stage transplantation. We have clinically applied this transplantation method to the congenital ear defect, microtia, and reported successful ear reconstruction. In our present study, we demonstrated that when FGF2 was added to elastic chondrocytes, the cell count increased and the level of hyaluronic acid, which is a major extracellular matrix (ECM) component, increased. We also demonstrated that these biochemical changes are reflected in the morphology, with the elastic chondrocytes themselves producing a matrix and fibers in vitro to form a natural scaffold. We then demonstrated that inside the natural scaffold thus formed, the cells overlap, connect intercellularly to each other, and reconstruct a cartilage-like three-dimensional structure in vitro. We further demonstrated by immunohistochemical analysis and electron microscopic analysis that when the multilayered chondrocytes are subsequently transplanted into a living body (abdominal subcutaneous region) in the two-stage transplantation process, neocartilage and neoperichondrium of elastic cartilage origin are regenerated 6 months after transplantation. Further, evaluation by dynamic mechanical analysis showed the regenerated neocartilage to have the same viscoelasticity as normal auricular

  12. In vitro study of intracellular IL-1beta production and beta1 integrins expression in stimulated chondrocytes--effect of rhein.

    Science.gov (United States)

    Gigant-Huselstein, C; Dumas, D; Payan, E; Muller, S; Bensoussan, D; Netter, P; Stoltz, J F

    2002-01-01

    The purpose of the present study was to investigate the intracellular IL-1beta production and beta1 integrins (alpha4/beta1 and alpha5/beta1) expression on chondrocytes. Chondroytes monolayer (human chondrosarcoma cell line HEM-C55) were incubated for 12, 24 and 48 hours in the presence of Tumor Necrosis Factor-alpha (TNF-alpha, Sigma, France) or recombinant human IL-1alpha (rh-IL1alpha, Becton Dickinson, France). After direct immunolabelling, cells were either analyzed on FACScan flow cytometer (Becton Dickinson, France), or observed under an epi-fluorescence inverted microscope equipped with the CellScan EPR optical scanning acquisition system (IPLab-Scanalytics, USA). We found that the IL-1beta mean fluorescence intensity in flow cytometry and in 3D microscopy was increased in the presence of TNF-alpha or rh-IL-1alpha, and alpha4/beta1 or alpha5/beta1 expression was higher on stimulated cells than on control cells. On the other hand, we have evaluated the in vitro effects of rhein (10(-5) M, Negma, France), an active metabolite of diacerein, on the intracellular IL-1beta and beta1 integrins expressed by stimulated or no-stimulated chondrocytes. The results indicated that rhein leads to a reduction of IL-1beta synthesis whereas a weak decrease of beta1 integrins receptors expression is observed. From this study, it seems that rhein partially reduce cytokine-induced intracellular IL-1beta production, and it has a weak action on alpha4/beta1 or alpha5/beta1 receptors.

  13. Salvianolic acid B inhibits IL-1β-induced inflammatory cytokine production in human osteoarthritis chondrocytes and has a protective effect in a mouse osteoarthritis model.

    Science.gov (United States)

    Lou, Yiting; Wang, Chenggui; Zheng, Wenhao; Tang, Qian; Chen, Yu; Zhang, Xiaolei; Guo, Xiaoshan; Wang, Jianshun

    2017-02-27

    Osteoarthritis (OA) is a chronic progressive disease that has complicated mechanisms that involve inflammation and cartilage degradation. In this study, we investigated the anti-inflammatory action of Salvianolic acid B (Sal B) in both human OA chondrocytes and a mouse OA model that was induced by destabilization of the medial meniscus. In vitro, chondrocytes were pretreated with Sal B (0, 25, 50, 100μM) for 2h, then incubated with IL-1β (10ng/mL) for 24h. NO production was determined by Griess method and PGE2 was assessed by ELISA. The expression of INOS, COX-2, MMP-13, ADAMTS-5 and NF-κB-related signaling molecules were tested by Western blotting. Immunofluorescence staining was used to detect P65 nuclear translocation. In vivo, the mouse OA model received intraperitoneal-injection of either Sal B (25mg/kg) or saline every other day. Hematoxylin and Eosin, as well as Safranin-O-Fast green staining, were utilized to evaluate the severity of cartilage lesions up to 8weeks following the surgery. Sal B inhibited the over-production of NO and PGE2, while the elevated expression of INOS, COX-2, MMP-13 and ADAMTS-5 were reversed by Sal B in IL-1β-induced chondrocytes. In addition, IL-1β significantly induced phosphorylation of NF-κB signaling, and this phosphorylation response was blocked by Sal B. Immunofluorescence staining demonstrated that Sal B could suppress IL-1β-induced p65 nuclear translocation. In vivo, the cartilage in Sal B-treated mice exhibited less cartilage degradation and lower OARSI scores. Taken together, Sal B possesses great potential value as a therapeutic agent for OA treatment.

  14. Changes in chondrocyte gene expression following in vitro impaction of porcine articular cartilage in an impact injury model.

    Science.gov (United States)

    Ashwell, Melissa S; Gonda, Michael G; Gray, Kent; Maltecca, Christian; O'Nan, Audrey T; Cassady, Joseph P; Mente, Peter L

    2013-03-01

    Our objective was to monitor chondrocyte gene expression at 0, 3, 7, and 14 days following in vitro impaction to the articular surface of porcine patellae. Patellar facets were either axially impacted with a cylindrical impactor (25 mm/s loading rate) to a load level of 2,000 N or not impacted to serve as controls. After being placed in organ culture for 0, 3, 7, or 14 days, total RNA was isolated from full thickness cartilage slices and gene expression measured for 17 genes by quantitative real-time RT-PCR. Targeted genes included those encoding proteins involved with biological stress, inflammation, or anabolism and catabolism of cartilage extracellular matrix. Some gene expression changes were detected on the day of impaction, but most significant changes occurred at 14 days in culture. At 14 days in culture, 10 of the 17 genes were differentially expressed with col1a1 most significantly up-regulated in the impacted samples, suggesting impacted chondrocytes may have reverted to a fibroblast-like phenotype.

  15. Identification of the calcitonin receptor in osteoarthritic chondrocytes

    Directory of Open Access Journals (Sweden)

    Christensen Tjorbjoern

    2011-10-01

    Full Text Available Abstract Background Preclinical and clinical studies have shown that salmon calcitonin has cartilage protective effects in joint degenerative diseases, such as osteoarthritis (OA. However, the presence of the calcitonin receptor (CTR in articular cartilage chondrocytes is yet to be identified. In this study, we sought to further investigate the expression of the CTR in naïve human OA articular chondrocytes to gain further confirmation of the existents of the CTR in articular cartilage. Methods Total RNA was purified from primary chondrocytes from articular cartilage biopsies from four OA patients undergoing total knee replacement. High quality cDNA was produced using a dedicated reverse transcription polymerase chain reaction (RT-PCR protocol. From this a nested PCR assay amplifying the full coding region of the CTR mRNA was completed. Western blotting and immunohistochemistry were used to characterize CTR protein on protein level in chondrocytes. Results The full coding transcript of the CTR isoform 2 was identified in all four individuals. DNA sequencing revealed a number of allelic variants of the gene including two potentially novel polymorphisms: a frame shift mutation, +473del, producing a shorter form of the receptor protein, and a single nucleotide polymorphism in the 3' non coding region of the transcript, +1443 C>T. A 53 kDa protein band, consistent with non-glycosylated CTR isoform 2, was detected in chondrocytes with a similar size to that expressed in osteoclasts. Moreover the CTR was identified in the plasma membrane and the chondrocyte lacuna of both primary chondrocytes and OA cartilage section. Conclusions Human OA articular cartilage chondrocytes do indeed express the CTR, which makes the articular a pharmacological target of salmon calcitonin. In addition, the results support previous findings suggesting that calcitonin has a direct anabolic effect on articular cartilage.

  16. Co-culture of dedifferentiated and primary human chondrocytes obtained from cadaveric donor enhance the histological quality of repair tissue: an in-vivo animal study.

    Science.gov (United States)

    Olivos-Meza, Anell; Velasquillo Martínez, Cristina; Olivos Díaz, Brenda; Landa-Solís, Carlos; Brittberg, Mats; Pichardo Bahena, Raul; Ortega Sanchez, Carmina; Martínez, Valentin; Alvarez Lara, Enrique; Ibarra-Ponce de León, José Clemente

    2017-06-05

    To compare the quality of the repair tissue in three-dimensional co-culture of human chondrocytes implanted in an in vivo model. Six cadaveric and five live human donors were included. Osteochondral biopsies from the donor knees were harvested for chondrocyte isolation. Fifty percent of cadaveric chondrocytes were expanded until passage-2 (P2) while the remaining cells were cryopreserved in passage-0 (P0). Fresh primary chondrocytes (P0f) obtained from live human donors were co-cultured. Three-dimensional constructs were prepared with a monolayer of passage-2 chondrocytes, collagen membrane (Geistlich Bio-Gide(®)), and pellet of non-co-cultured (P2) or co-cultured chondrocytes (P2 + P0c, P2 + P0f). Constructs were implanted in the subcutaneous tissue of athymic mice and left for 3 months growth. Safranin-O and Alcian blue staining were used to glycosaminoglycan content assessment. Aggrecan and type-II collagen were evaluated by immunohistochemistry. New-formed tissue quality was evaluated with an adaptation of the modified O'Driscoll score. Histological quality of non-co-cultured group was 4.37 (SD ±4.71), while co-cultured groups had a mean score of 8.71 (SD ±3.98) for the fresh primary chondrocytes and 9.57 (SD ±1.27) in the cryopreserved chondrocytes. In immunohistochemistry, Co-culture groups were strongly stained for type-II and aggrecan not seen in the non-co-cultured group. It is possible to isolate viable chondrocytes from cadaveric human donors in samples processed in the first 48-h of dead. There is non-significant difference between the numbers of chondrocytes isolated from live or cadaveric donors. Cryopreservation of cadaveric primary chondrocytes does not alter the capability to form cartilage like tissue. Co-culture of primary and passaged chondrocytes enhances the histological quality of new-formed tissue compared to non-co-cultured cells.

  17. The chrondoprotective actions of a natural product are associated with the activation of IGF-1 production by human chondrocytes despite the presence of IL-1β

    Directory of Open Access Journals (Sweden)

    Bobrowski Paul

    2006-04-01

    Full Text Available Abstract Background Cartilage loss is a hallmark of arthritis and follows activation of catabolic processes concomitant with a disruption of anabolic pathways like insulin-like growth factor 1 (IGF-1. We hypothesized that two natural products of South American origin, would limit cartilage degradation by respectively suppressing catabolism and activating local IGF-1 anabolic pathways. One extract, derived from cat's claw (Uncaria guianensis, vincaria®, is a well-described inhibitor of NF-κB. The other extract, derived from the vegetable Lepidium meyenii (RNI 249, possessed an uncertain mechanism of action but with defined ethnomedical applications for fertility and vitality. Methods Human cartilage samples were procured from surgical specimens with consent, and were evaluated either as explants or as primary chondrocytes prepared after enzymatic digestion of cartilage matrix. Assessments included IGF-1 gene expression, IGF-1 production (ELISA, cartilage matrix degradation and nitric oxide (NO production, under basal conditions and in the presence of IL-1β. Results RNI 249 enhanced basal IGF-1 mRNA levels in human chondrocytes by 2.7 fold, an effect that was further enhanced to 3.8 fold by co-administration with vincaria. Enhanced basal IGF-1 production by RNI 249 alone and together with vincaria, was confirmed in both explants and in primary chondrocytes (P Conclusion The identification of agents that activate the autocrine production of IGF-1 in cartilage, even in the face of suppressive pro-inflammatory, catabolic cytokines like IL-1β, represents a novel therapeutic approach to cartilage biology. Chondroprotection associated with prevention of the catabolic events and the potential for sustained anabolic activity with this natural product suggests that it holds significant promise in the treatment of debilitating joint diseases.

  18. 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.

  19. Molecular hydrogen protects chondrocytes from oxidative stress and indirectly alters gene expressions through reducing peroxynitrite derived from nitric oxide

    Directory of Open Access Journals (Sweden)

    Hanaoka Teruyasu

    2011-08-01

    Full Text Available Abstract Background Molecular hydrogen (H2 functions as an extensive protector against oxidative stress, inflammation and allergic reaction in various biological models and clinical tests; however, its essential mechanisms remain unknown. H2 directly reacts with the strong reactive nitrogen species peroxynitrite (ONOO- as well as hydroxyl radicals (•OH, but not with nitric oxide radical (NO•. We hypothesized that one of the H2 functions is caused by reducing cellular ONOO-, which is generated by the rapid reaction of NO• with superoxides (•O2-. To verify this hypothesis, we examined whether H2 could restore cytotoxicity and transcriptional alterations induced by ONOO- derived from NO• in chondrocytes. Methods We treated cultured chondrocytes from porcine hindlimb cartilage or from rat meniscus fibrecartilage with a donor of NO•, S-nitroso-N-acetylpenicillamine (SNAP in the presence or absence of H2. Chondrocyte viability was determined using a LIVE/DEAD Viability/Cytotoxicity Kit. Gene expressions of the matrix proteins of cartilage and the matrix metalloproteinases were analyzed by reverse transcriptase-coupled real-time PCR method. Results SNAP treatment increased the levels of nitrated proteins. H2 decreased the levels of the nitrated proteins, and suppressed chondrocyte death. It is known that the matrix proteins of cartilage (including aggrecan and type II collagen and matrix metalloproteinases (such as MMP3 and MMP13 are down- and up-regulated by ONOO-, respectively. H2 restoratively increased the gene expressions of aggrecan and type II collagen in the presence of H2. Conversely, the gene expressions of MMP3 and MMP13 were restoratively down-regulated with H2. Thus, H2 acted to restore transcriptional alterations induced by ONOO-. Conclusions These results imply that one of the functions of H2 exhibits cytoprotective effects and transcriptional alterations through reducing ONOO-. Moreover, novel pharmacological strategies

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

    Science.gov (United States)

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

    2015-06-01

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

  1. Standardized butanol fraction of WIN-34B suppresses cartilage destruction via inhibited production of matrix metalloproteinase and inflammatory mediator in osteoarthritis human cartilage explants culture and chondrocytes

    Directory of Open Access Journals (Sweden)

    Huh Jeong-Eun

    2012-12-01

    Full Text Available Abstract Background WIN-34B is a novel Oriental medicine, which represents the n-butanol fraction prepared from dried flowers of Lonicera japonica Thunb and dried roots of Anemarrhena asphodeloides BUNGE. The component herb of WIN-34B is used for arthritis treatment in East Asian countries. The aim of this study was to determine the cartilage-protective effects and mechanisms of WIN-34B and its major phenolic compounds, chlorogenic acid and mangiferin, in osteoarthritis (OA human cartilage explants culture and chondrocytes. Methods The investigation focused on whether WIN-34B and its standard compounds protected cartilage in interleukin (IL-1β-stimulated cartilage explants culture and chondrocytes derived from OA patients. Also, the mechanisms of WIN-34B on matrix metalloproteinases (MMPs, tissue inhibitor of matrix metalloproteinases (TIMPs, inflammatory mediators, and mitogen-activated protein kinases (MAPKs pathways were assessed. Results WIN-34B was not cytotoxic to cultured cartilage explants or chondrocytes. WIN-34B dose-dependently inhibited the release of glycosaminoglycan and type II collagen, increased the mRNA expression of aggrecan and type II collagen, and recovered the intensity of proteoglycan and collagen by histological analysis in IL-1β-stimulated human cartilage explants culture. The cartilage protective effect of WIN-34B was similar to or better than that of chlorogenic acid and mangiferin. Compared to chlorogenic acid and mangiferin, WIN-34B displayed equal or greater decreases in the levels of MMP-1, MMP-3, MMP-13, ADAMTS-4, and ADAMTS-5, and markedly up-regulated TIMP-1 and TIMP-3. WIN-34B inhibited inflammatory mediators involved in cartilage destruction, such as prostaglandin E2, nitric oxide, tumor necrosis factor-alpha, and IL-1β. The phosphorylation of extracellular signal-regulated kinase, c-Jun N-terminal kinase (JNK, and p38 was significantly reduced by WIN-34B treatment, while phosphorylation of JNK was only

  2. Standardized butanol fraction of WIN-34B suppresses cartilage destruction via inhibited production of matrix metalloproteinase and inflammatory mediator in osteoarthritis human cartilage explants culture and chondrocytes

    Science.gov (United States)

    2012-01-01

    Background WIN-34B is a novel Oriental medicine, which represents the n-butanol fraction prepared from dried flowers of Lonicera japonica Thunb and dried roots of Anemarrhena asphodeloides BUNGE. The component herb of WIN-34B is used for arthritis treatment in East Asian countries. The aim of this study was to determine the cartilage-protective effects and mechanisms of WIN-34B and its major phenolic compounds, chlorogenic acid and mangiferin, in osteoarthritis (OA) human cartilage explants culture and chondrocytes. Methods The investigation focused on whether WIN-34B and its standard compounds protected cartilage in interleukin (IL)-1β-stimulated cartilage explants culture and chondrocytes derived from OA patients. Also, the mechanisms of WIN-34B on matrix metalloproteinases (MMPs), tissue inhibitor of matrix metalloproteinases (TIMPs), inflammatory mediators, and mitogen-activated protein kinases (MAPKs) pathways were assessed. Results WIN-34B was not cytotoxic to cultured cartilage explants or chondrocytes. WIN-34B dose-dependently inhibited the release of glycosaminoglycan and type II collagen, increased the mRNA expression of aggrecan and type II collagen, and recovered the intensity of proteoglycan and collagen by histological analysis in IL-1β-stimulated human cartilage explants culture. The cartilage protective effect of WIN-34B was similar to or better than that of chlorogenic acid and mangiferin. Compared to chlorogenic acid and mangiferin, WIN-34B displayed equal or greater decreases in the levels of MMP-1, MMP-3, MMP-13, ADAMTS-4, and ADAMTS-5, and markedly up-regulated TIMP-1 and TIMP-3. WIN-34B inhibited inflammatory mediators involved in cartilage destruction, such as prostaglandin E2, nitric oxide, tumor necrosis factor-alpha, and IL-1β. The phosphorylation of extracellular signal-regulated kinase, c-Jun N-terminal kinase (JNK), and p38 was significantly reduced by WIN-34B treatment, while phosphorylation of JNK was only inhibited by chlorogenic

  3. Quantitative proteomics reveals regulatory differences in the chondrocyte secretome from human medial and lateral femoral condyles in osteoarthritic patients.

    Science.gov (United States)

    Stenberg, Johan; Rüetschi, Ulla; Skiöldebrand, Eva; Kärrholm, Johan; Lindahl, Anders

    2013-10-04

    Osteoarthritis (OA) is a destructive joint disease and there are no known biomarkers available for an early diagnosis. To identify potential disease biomarkers and gain further insight into the disease mechanisms of OA we applied quantitative proteomics with SILAC technology on the secretomes from chondrocytes of OA knees, designated as high Mankin (HM) scored secretome. A quantitative comparison was made between the secretomes of the medial and lateral femur condyle chondrocytes in the same knee since the medial femur condyle is usually more affected in OA than the lateral condyle, which was confirmed by Mankin scoring. The medial/lateral comparison was also made on the secretomes from chondrocytes taken from one individual with no clinically apparent joint-disease, designated as low Mankin (LM) scored secretome. We identified 825 proteins in the HM secretome and 69 of these showed differential expression when comparing the medial and lateral femoral compartment. The LM scored femoral condyle showed early signs of OA in the medial compartment as assessed by Mankin score. We here report the identification and relative quantification of several proteins of interest for the OA disease mechanism e.g. CYTL1, DMD and STAB1 together with putative early disease markers e.g. TIMP1, PPP2CA and B2M. The present study reveals differences in protein abundance between medial/lateral femur condyles in OA patients. These regulatory differences expand the knowledge regarding OA disease markers and mechanisms.

  4. Knockdown of the intraflagellar transport protein IFT46 stimulates selective gene expression in mouse chondrocytes and affects early development in zebrafish.

    Science.gov (United States)

    Gouttenoire, Jérôme; Valcourt, Ulrich; Bougault, Carole; Aubert-Foucher, Elisabeth; Arnaud, Estelle; Giraud, Lionel; Mallein-Gerin, Frédéric

    2007-10-19

    Bone morphogenetic proteins (BMPs) act as multifunctional regulators in morphogenesis during development. In particular they play a determinant role in the formation of cartilage molds and their replacement by bone during endochondral ossification. In cell culture, BMP-2 favors chondrogenic expression and promotes hypertrophic maturation of chondrocytes. In mouse chondrocytes we have identified a BMP-2-sensitive gene encoding a protein of 301 amino acids. This protein, named mIFT46, is the mouse ortholog of recently identified Caenorhabditis elegans and Chlamydomonas reinhardtii intraflagellar transport (IFT) proteins. After generation of a polyclonal antibody against mIFT46, we showed for the first time that the endogenous protein is located in the primary cilium of chondrocytes. We also found that mIFT46 is preferentially expressed in early hypertrophic chondrocytes located in the growth plate. Additionally, mIFT46 knockdown by small interfering RNA oligonucleotides in cultured chondrocytes specifically stimulated the expression of several genes related to skeletogenesis. Furthermore, Northern blotting analysis indicated that mIFT46 is also expressed before chondrogenesis in embryonic mouse development, suggesting that the role of mIFT46 might not be restricted to cartilage. To explore the role of IFT46 during early development, we injected antisense morpholino oligonucleotides in Danio rerio embryos to reduce zebrafish IFT46 protein (zIFT46) synthesis. Dramatic defects in embryonic development such as a dorsalization and a tail duplication were observed. Thus our results taken together indicate that the ciliary protein IFT46 has a specific function in chondrocytes and is also essential for normal development of vertebrates.

  5. An RGD-restricted substrate interface is sufficient for the adhesion, growth and cartilage forming capacity of human chondrocytes

    Directory of Open Access Journals (Sweden)

    D Vonwil

    2010-11-01

    Full Text Available This study aimed at testing whether an RGD-restricted substrate interface is sufficient for adhesion and growth of human articular chondrocytes (HAC, and whether it enhances their post expansion chondrogenic capacity. HAC/substrate interaction was restricted to RGD by modifying tissue culture polystyrene (TCPS with a poly(ethylene glycol (PEG based copolymer system that renders the surface resistant to protein adsorption while at the same time presenting the bioactive RGD-containing peptide GCRGYGRGDSPG (RGD. As compared to TCPS, HAC cultured on RGD spread faster (1.9-fold, maintained higher type II collagen mRNA expression (4.9-fold and displayed a 19% lower spreading area. On RGD, HAC attachment efficiency (66±10% and proliferation rate (0.56±0.04 doublings/day, as well as type II collagen mRNA expression in the subsequent chondrogenic differentiation phase, were similar to those of cells cultured on TCPS. In contrast, cartilaginous matrix deposition by HAC expanded on RGD was slightly but consistently higher (15% higher glycosaminoglycan-to-DNA ratio. RDG (bioinactive peptide and PEG (no peptide ligand controls yielded drastically reduced attachment efficiency (lower than 11% and proliferation (lower than 0.20 doublings/day. Collectively, these data indicate that restriction of HAC interaction with a substrate through RGD peptides is sufficient to support their adhesion, growth and maintenance of cartilage forming capacity. The concept could thus be implemented in materials for cartilage repair, whereby in situ recruited/infiltrated chondroprogenitor cells would proliferate while maintaining their ability to differentiate and generate cartilage tissue.

  6. Isolation and differentiation of chondrocytic cells derived from human embryonic stem cells using dlk1/FA1 as a novel surface marker

    DEFF Research Database (Denmark)

    Harkness, Linda; Taipaleenmaki, Hanna; Mahmood, Amer

    2009-01-01

    was not expressed by undifferentiated hESC, but expressed during in vitro embryoid bodies (hEBs) formation upon down-regulation of undifferentiated markers e.g. Oct 3/4. Similarly, dlk1/FA1 was expressed in chondrocytic cells during in vivo teratoma formation. Interestingly, treatment of hEBs with Activin B...

  7. Synergistic Effects of FGF-2 with Insulin or IGF-I on the Proliferation of Human Auricular Chondrocytes.

    Science.gov (United States)

    Takahashi, Tsuguharu; Ogasawara, Toru; Kishimoto, Junji; Liu, Guangyao; Asato, Hirotaka; Nakatsuka, Takashi; Uchinuma, Eijyu; Nakamura, Kozo; Kawaguchi, Hiroshi; Takato, Tsuyoshi; Hoshi, Kazuto

    2005-10-01

    Chondrocyte preparation with the safety and efficiency is the first step in cartilage regenerative medicine. To prepare a chondrocyte proliferation medium that does not contain fetal bovine serum (FBS) and that provides more than a 1000-fold increase in cell numbers within approximately 1 month, we attempted to use the medium containing 5% human serum (HS), but it exerted no more than twofold increase in 2 weeks. To compensate for the limited proliferation ability in HS, we investigated the combinational effects of 12 factors [i.e., fibroblast growth factor(FGF)-2, insulin-like growth factor(IGF)-I, insulin, bone morphogenetic protein-2, parathyroid hormone, growth hormone, dexamethasone, 1α25-dihydroxy vitamin D3, L-3,3',5'-triodothyronine, interleukine-1 receptor antagonist, 17β-estradiol, and testosterone] on the proliferation of human auricular chondrocytes by analysis of variance in fractional factorial design. As a result, FGF-2, dexamethasone, insulin, and IGF-I possessed promotional effects on proliferation, while the combination of FGF-2 with insulin or IGF-I synergistically enhanced the proliferation. Actually, the chondrocytes increased 7.5-fold in number in 2 weeks in a medium containing 5% HS with 10 ng/ml FGF-2, while the cell number synergistically gained a 10-12-fold increase with 5 μg/ml insulin or 100 ng/ml IGF-I in the same period. The proliferation effects were more enhanced at a concentration of 100 ng/ml for FGF-2, and especially for the combination of 100 ng/ml FGF-2 and 5 μg/ml insulin (approximately 16-fold within 2 weeks). In the long-term culture with repeated passaging, this combination provided more than 10,000-fold within 8 weeks (i.e., passage 4). Thus, we concluded that such a combination of FGF-2 with insulin or IGF-I may be useful for promotion of auricular chondrocyte proliferation in a clinical application for cartilage regeneration.

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

    Directory of Open Access Journals (Sweden)

    Chua Kien-Hui

    2013-01-01

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

  9. 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,

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

    NARCIS (Netherlands)

    Rutgers, M.; Saris, D.B.F.; Vonk, L.A.; Rijen, van M.H.P.; Akrum, V.; Langeveld, D.; Boxtel, van 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, ho

  11. Cryptotanshinone protects against IL-1β-induced inflammation in human osteoarthritis chondrocytes and ameliorates the progression of osteoarthritis in mice.

    Science.gov (United States)

    Feng, Zhenhua; Zheng, Wenhao; Li, Xiaobin; Lin, Jian; Xie, Chenglong; Li, Hang; Cheng, Liang; Wu, Aimin; Ni, Wenfei

    2017-09-01

    Osteoarthritis (OA) is a common degenerative disease characterized by progressive erosion of articular cartilage, subchondral bone sclerosis and synovitis. Cryptotanshinone (CTS), an active component extracted from the root of Salvia miltiorrhiza Bunge, has been shown to have potent anti-inflammatory effects. However, its effects on OA have not been clearly elucidated. This study aimed to assess the effect of CTS on human OA chondrocytes and mice OA models. Human OA chondrocytes were pretreated with CTS (5, 10 and 20μM) for 2h and subsequently stimulated with IL-1β for 24h. Production of NO, PGE2, IL-6, TNF-α was evaluated by the Griess reaction and ELISA. The protein expression of COX-2, iNOs, MMP-3, MMP13, COX-2, ADAMTS-5, JNK, p-JNK, ERK, p-ERK, p38, p-p38, p-IKKα/β, p65, p-p65, IκB-α, and p-IκB-α was tested by Western blot. In vivo, the severity of OA was determined by histological analysis. We found that CTS significantly inhibited the IL-1β-induced production of NO and PGE2; expression of COX-2, iNOS, MMP-3, MMP-13, and ADAMTS-5. Furthermore, CTS in dramatically suppressed IL-1β-stimulated NF-κB and MAPK activation. Immunofluorescence staining demonstrated that CTS could suppress IL-1β-induced phosphorylation of p65 nuclear translocation. In vivo, treatment of CTS prevented the destruction of cartilage and the thickening of subchondral bone in mice OA models. These results indicate that the therapeutic effect of CTS on OA is accomplished through the inhibition of both NF-κB and MAPK signaling pathways. Our findings provide the evidence to develop CTS as a potential therapeutic agent f or patients with OA. Copyright © 2017 Elsevier B.V. All rights reserved.

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

    Science.gov (United States)

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

    2016-08-17

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

  13. Study on human chondrocyte culture viability for autologous transplantation in clinical application

    Directory of Open Access Journals (Sweden)

    Christiane Lombello

    2003-06-01

    Full Text Available Objective: The limited regenerative capacity of the cartilage tissuemakes the treatment of chondral lesions difficult. The techniquescurrently available to treat cartilage lesions may relieve symptoms,but do not regenerate the injured tissue. Autologous chondrocytetransplantation uses cell biology and cell culture techniques toregenerate the hyaline cartilage. Methods: In this study, we analyzechondrocyte biopsy collection and culture for autologoustransplantation. Ultrastructural analyses of hyaline cartilage biopsieswere performed 0, 6, 24 and 48 hours after collection. The tissue evenafter 48 hours. Eleven cell culture assays were performed to evaluateisolation, viability, morphology, proliferation and absence ofcontaminants. Results: The cell culture techniques used allowedchondrocyte proliferation. Rates on cell viability were maintained abovethe acceptable patterns (above 90. Control of cell culture laboratoryconditions showed absence of contaminants, assuring safety of theprocess. The chondrocytes obtained presented the morphology typicalof cultured cell monolayers. Conclusion: The results indicate viabilityof chondrocyte culture technique for clinical application in autologoustransplantation.

  14. In-vitro interactions of human chondrocytes and mesenchymal stem cells, and of mouse macrophages with phospholipid-covered metallic implant materials

    Directory of Open Access Journals (Sweden)

    R Willumeit

    2007-03-01

    Full Text Available Phospholipid-coatings on metallic implant surfaces were evaluated in terms of adhesion, proliferation and matrix production of skeletal cells, and of macrophage stimulation. The working hypothesis is that mimicking a model biomembrane by phospholipids on surfaces to which cells adhere, the surface recognition by surrounding cells is altered. In this study, 1 mirror-like polished Ti-6Al-7Nb and 2 porous Ti-6Al-4V specimens were covered with the phospholipids POPE (palmitoyl-oleoyl phosphatidyl-ethanolamine and POPC (palmitoyl-oleoyl phosphatidyl-choline, and the interactions of a human articular chondrocytes (HAC, b human mesenchymal stem cells (HMSC, and c mouse macrophages (RAW 264.7 were tested in vitro. On POPE-covered polished surfaces adherence of HAC (42% of seeded cells after 2 hrs and metabolic activity (MTT after 3 days were reduced, while on porous surfaces 99% HAC adhered, and metabolic activity was significantly increased, compared to respective native surfaces. On both POPE-covered surfaces the chondrocyte phenotype was present. After 3 weeks of chondrogenic differentiation, cartilage matrix production (measuring chondroitin sulphate per HAC number was significantly increased by about 30% on both POPE-covered metallic surfaces. On both POPC-covered surfaces nearly no adhering and surviving HAC were found. HMSC grown on POPE-covered porous substrates showed osteogenic differentiation by improved osteopontin and collagen I expression in RT-PCR, and osteocalcin fluorescence and bone nodule formation was only detectable on POPE-covered porous surfaces. In contrast to POPC and other phospholipids used as positive controls, POPE did not stimulate the NO production in mouse macrophage cultures. We therefore conclude that a phospholipid coating by POPE shows potential as surface modification for metallic implant materials.

  15. Interleukin-6 synthesis in human chondrocytes is regulated via the antagonistic actions of prostaglandin (PGE2 and 15-deoxy-Δ(12,14-PGJ2.

    Directory of Open Access Journals (Sweden)

    Pu Wang

    Full Text Available BACKGROUND: Elevated levels of interleukin-6 (IL-6, prostaglandin (PGE(2, PGD(2 and its dehydration end product 15-deoxy-Δ(12,14-PGJ(2 (15d-PGJ(2 have been detected in joint synovial fluids from patients with rheumatoid arthritis (RA. PGE(2 directly stimulates IL-6 production in human articular chondrocytes. However, the effects of PGD(2 and 15d-PGJ(2 in the absence or presence of PGE(2 on IL-6 synthesis in human chondrocytes have yet to be determined. It is believed that dysregulated overproduction of IL-6 is responsible for the systemic inflammatory manifestations and abnormal laboratory findings in RA patients. METHODOLOGY/PRINCIPAL FINDINGS: Using the T/C-28a2 chondrocyte cell line as a model system, we report that exogenous PGE(2 and PGD(2/15d-PGJ(2 exert antagonistic effects on IL-6 synthesis in human T/C-28a2 chondrocytes. Using a synthesis of sophisticated molecular biology techniques, we determined that PGE(2 stimulates Toll-like receptor 4 (TLR4 synthesis, which is in turn responsible for the activation of the ERK1/2, PI3K/Akt and PKA/CREB pathways that phosphorylate the NF-κB p65 subunit leading to NF-κB activation. Binding of the activated NF-κB p65 subunit to IL-6 promoter induces IL-6 synthesis in human T/C28a2 chondrocytes. PGD(2 or 15d-PGJ(2 concurrently downregulates TLR4 and upregulates caveolin-1, which in turn inhibit the PGE(2-dependent ERK1/2, PI3-K and PKA activation, and ultimately with NF-κB-dependent IL-6 synthesis in chondrocytes. CONCLUSIONS/SIGNIFICANCE: We have delineated the signaling cascade by which PGE(2 and PGD(2/15d-PGJ(2 exert opposing effects on IL-6 synthesis in human chondrocytes. Elucidation of the molecular pathway of IL-6 synthesis and secretion by chondrocytes will provide insights for developing strategies to reduce inflammation and pain in RA patients.

  16. Endoplasmic reticulum stress participates in the progress of senescence and apoptosis of osteoarthritis chondrocytes.

    Science.gov (United States)

    Liu, Yake; Zhu, Hai; Yan, Xin; Gu, Haoye; Gu, Zhifeng; Liu, Fan

    2017-09-16

    Endoplasmic reticulum stress (ERS) has been shown to participate in many disease pathologies. Recent reports have reported that ERS exists in human osteoarthritis (OA) chondrocytes. During OA, chondrocytes exhibit increased level of some senescence marker, such as senescence-associated β-galactosidase (SA β-gal) activity. However, the persistence and accumulation of senescent cells in various tissues can also impair function and have been involved in the pathogenesis of many age-related diseases, including OA. In this present study, we used IL-1β (10 ng/ml) to mimic OA chondrocytes and we found that IL-1β stimulated chondrocytes caused the increasing expression of ADAMTS5 and MMP13, decreasing COL2A1 expression, which were in accord with OA chondrocytes changes. Our data also showed that ERS is involved in the OA chondrocytes, SA β-gal activity significantly increases and inhibition of ERS can decrease the SA β-gal activity, apoptosis of OA chondrocytes and increase cell viability. These results help us to open new perspectives for the development of molecular-targeted treatment approaches and thus present an effective novel therapeutic approach for OA. Copyright © 2017 Elsevier Inc. All rights reserved.

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

    Energy Technology Data Exchange (ETDEWEB)

    Imagawa, Kei, E-mail: k.Imagawa@soton.ac.uk [University of Southampton Medical School, Bone and Joint Research Group, Southampton (United Kingdom); Tohoku University School of Medicine, Sendai (Japan); Andres, MC de [University of Southampton Medical School, Bone and Joint Research Group, Southampton (United Kingdom); Hashimoto, Ko [Hospital for Special Surgery, NY (United States); Pitt, Dominic [University of Southampton Medical School, Bone and Joint Research Group, Southampton (United Kingdom); Itoi, Eiji [Tohoku University School of Medicine, Sendai (Japan); Goldring, Mary B. [Hospital for Special Surgery, NY (United States); Roach, Helmtrud I.; Oreffo, Richard O.C. [University of Southampton Medical School, Bone and Joint Research Group, Southampton (United Kingdom)

    2011-02-18

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

  18. Pannexin 3 is a novel target for Runx2, expressed by osteoblasts and mature growth plate chondrocytes.

    Science.gov (United States)

    Bond, Stephen R; Lau, Alice; Penuela, Silvia; Sampaio, Arthur V; Underhill, T Michael; Laird, Dale W; Naus, Christian C

    2011-12-01

    Pannexins are a class of chordate channel proteins identified by their homology to insect gap junction proteins. The pannexin family consists of three members, Panx1, Panx2, and Panx3, and the role each of these proteins plays in cellular processes is still under investigation. Previous reports of Panx3 expression indicate enrichment in skeletal tissues, so we have further investigated this distribution by surveying the developing mouse embryo with immunofluorescence. High levels of Panx3 were detected in intramembranous craniofacial flat bones, as well as long bones of the appendicular and axial skeleton. This distribution is the result of expression in both osteoblasts and hypertrophic chondrocytes. Furthermore, the Panx3 promoter contains putative binding sites for transcription factors involved in bone formation, and we show that the sequence between bases -275 and -283 is responsive to Runx2 activation. Taken together, our data suggests that Panx3 may serve an important role in bone development, and is a novel target for Runx2-dependent signaling.

  19. 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

  20. Short waves-induced enhancement of proliferation of human chondrocytes: involvement of extracellular signal-regulated map-kinase (erk).

    Science.gov (United States)

    Wang, Jue-Long; Chan, Rai-Chi; Cheng, He-Hsiung; Huang, Chun-Jen; Lu, Yih-Chau; Chen, I-Shu; Liu, Shiuh-Inn; Hsu, Shu-Shong; Chang, Hong-Tai; Huang, Jong-Khing; Chen, Jin-Shyr; Ho, Chin-Man; Jan, Chung-Ren

    2007-07-01

    1. Short-wave diathermy (SWD) is a form of radiofrequency radiation that is used therapeutically by physiotherapists. The cellular mechanisms of SWD are unclear. The present study was performed to explore the effect of different conditions of short-wave exposure on the proliferation of cultured human chondrocytes. 2. Cells exposed to short waves once per day for seven consecutive days exhibited a significant increase in proliferation by 42% compared with the control cells. In cells that were treated with short waves twice per day for seven consecutive days, or only once on Day 1 and then examined for proliferation on Day 7, cell proliferation was greater than the control cells by 40% and 30%, respectively. 3. Given the importance of mitogen-activated protein kinases (MAPK) in the proliferation of different cell types, efforts were extended to explore the role of three major types of MAPK; that is, extracellular signal-regulated kinase (ERK), c-Jun NH(2)-terminal protein kinase (JNK) and p38. 4. It was found that the level of phosphorylated ERK (phospho-ERK 1 and ERK 2) increased significantly within 5-120 min following consecutive exposure to short waves for 7 days. Exposure to short waves failed to alter the intensity of phosphorylated JNK and p38 within 0-240 min. 5. Cells were exposed to short waves once for seven consecutive days in the presence of 0, 10 micromol/L, 20 micromol/L or 50 micromol/L PD98059 (an ERK inhibitor). PD98059 totally inhibited short waves-induced enhancement of proliferation without altering normal control viability. In the presence of short waves and PD98059, the cell viability was lower than the normal control. Together, the data suggest that short waves could increase proliferation in human chondrocytes through activation of the ERK pathway, which is also involved in maintaining normal cell proliferation under physiological conditions.

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

    Science.gov (United States)

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

    2015-06-25

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

  2. EFFECT OF LOW SELENIUM ON CHONDROCYTE DIFFERENTIATION AND DIFFERENTIAL EXPRESSION OF COLLAGEN TYPES Ⅰ , Ⅱ AND X IN ARTICULAR CARTILAGE FROM MINI-PIGS

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Objective According to the distribution of low selenium areas, low nutrition state of the residents and the affecting cartilage growth and articular cartilage of Kashin-Beck Disease(KBD),the chondrocyte differentia- tion and differential expression of collagen types Ⅰ , Ⅱ and Ⅹ in articular cartilage from Chinese mini-pigs treated with low selenium were investigated in order to gain insight into the effects of these conditions on chondrocyte differ- entiation in KBD cartilage. Mothods Eleven male juvenile mini-pigs, aged from 4 weeks to 6 weeks after birth, were divided into 3 groups. The Se content in the diet of the “low Se” group was 0. 035mg/kg diet, and 0. 175 mg/kg diet in the control. For Se-supplemented group 0. 390mg /kg diet was added. The content of Se in blood was assayed at the beginning and at the end of each experiment. Samples of articular cartilage were taken from the right femur condylus, and collagen types Ⅰ , Ⅱ and Ⅹ in articular cartilage were analyzed by immunohistochemistry and in situ hybridization. Results ①All cartilage samples from juvenile mini-pigs fed with low selenium diet revealed a re- duction in type Ⅹ collagen mRNA expression in the hypertrophic chondrocytes as shown by in situ hybridization, and reduced type Ⅹ collagen deposition in the lower hypertrophic zone as shown by immunohistochemistry. ②Addition of selenium to the diet restored the type Ⅹ collagen to normal level. ③Type Ⅱ collagen was evenly distributed over the entire articular cartilage in all experimental and control groups. Type Ⅱ collagen mRNA signals were most prominent in the upper articular layer as well as in the hypertrophic zone in all groups. Type Ⅱ collagen expression was restrict- ed to the zone of endochondral ossification in all experimental groups and the control. Conclusion Low selenium has an down-regulatory role on the synthesis and deposition of collagen type Ⅹ in hypertrophic chondrocytes in articular cartilage of

  3. Chitosan-plasmid DNA nanoparticles encoding small hairpin RNA targeting MMP-3 and -13 to inhibit the expression of dedifferentiation related genes in expanded chondrocytes.

    Science.gov (United States)

    Zhao, Jingxin; Fan, Xiangli; Zhang, Qiang; Sun, Fangfei; Li, Xiaojian; Xiong, Chuan; Zhang, Chunli; Fan, Hongbin

    2014-02-01

    Overexpression of matrix metalloproteinase (MMP)-3 and -13 can lead to the dedifferentiation of expanded chondrocytes. After implanting dedifferentiated cells for cartilage defect repair, graft failure may occur. Short hairpin RNA (shRNA) is a powerful genetic tool to reduce the expression of target genes. This study investigated the effects of chitosan-plasmid DNA (pDNA) nanoparticles encoding shRNA targeting MMP-3 and -13 on the dedifferentiation of expanded chondrocytes. The objective was to optimize the parameters of chitosan-pDNA formulation for achieving higher efficiency of pDNA delivery and gene silencing. The chitosan-pDNA nanoparticles were prepared using a complex coacervation process. Then the characteristics including size, shape, stability, and transfection efficiency were compared in different groups. The results indicated that chitosan of 800 kDa at N/P ratio of 4 and pH 7.0 was optimal to prepare chitosan-pDNA nanoparticles. These nanoparticles showed high DNA loading efficiency (95.8 ± 1.5%) and high gene transfection efficiency (24.5 ± 1.6%). After the expanded chondrocytes were transfected by chitosan-pDNA nanoparticles, MMP-3-610 and MMP-13-2024 groups showed greater suppression in mRNA and protein levels. The results indicated that chitosan-pDNA nanoparticles encoding shRNA targeting MMP-3 and -13 had great potential in silencing the dedifferentiation-related genes for regenerating prolonged and endurable cartilage.

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

    Energy Technology Data Exchange (ETDEWEB)

    Oh, Jung-Hoon [Department of Molecular Medicine, Cell and Matrix Research Institute, BK21 Medical Education Program for Human Resources, Kyungpook National University School of Medicine, Daegu (Korea, Republic of); Park, Seung-Yoon [Department of Biochemistry, School of Medicine, Dongguk University, Gyeongju 780-714 (Korea, Republic of); Crombrugghe, Benoit de [Department of Genetics, University of Texas, M.D. Anderson Cancer Center, Houston (United States); Kim, Jung-Eun, E-mail: kjeun@knu.ac.kr [Department of Molecular Medicine, Cell and Matrix Research Institute, BK21 Medical Education Program for Human Resources, Kyungpook National University School of Medicine, Daegu (Korea, Republic of)

    2012-02-24

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

  5. Simvastatin inhibits CD44 fragmentation in chondrocytes.

    Science.gov (United States)

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

    2016-08-15

    In human osteoarthritic chondrocytes, the hyaluronan receptor CD44 undergoes proteolytic cleavage at the cell surface. CD44 cleavage is thought to require transit of CD44 into cholesterol-rich lipid rafts. The purpose of this study was to investigate whether statins exert a protective effect on articular chondrocytes due to diminution of cholesterol. Three model systems of chondrocytes were examined including human HCS-2/8 chondrosarcoma cells, human osteoarthritic chondrocytes and normal bovine articular chondrocytes. Treatment with IL-1β + Oncostatin M resulted in a substantial increase in CD44 fragmentation in each of the three chondrocyte models. Pre-incubation with simvastatin prior to treatment with IL-1β + Oncostatin M decreased the level of CD44 fragmentation, decreased the proportion of CD44 that transits into the lipid raft fractions, decreased ADAM10 activity and diminished the interaction between CD44 and ADAM10. In HCS-2/8 cells and bovine articular chondrocytes, fragmentation of CD44 was blocked by the knockdown of ADAM10. Inhibition of CD44 fragmentation by simvastatin also resulted in improved retention of pericellular matrix. Addition of cholesterol and farnesyl-pyrophosphate reversed the protective effects of simvastatin. Thus, the addition of simvastatin exerts positive effects on chondrocytes including reduced CD44 fragmentation and enhanced the retention of pericellular matrix. Copyright © 2016 Elsevier Inc. All rights reserved.

  6. 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.

  7. Autoimmune regulator, Aire, is a novel regulator of chondrocyte differentiation.

    Science.gov (United States)

    Si, Yuan; Inoue, Kazuki; Igarashi, Katsuhide; Kanno, Jun; Imai, Yuuki

    2013-08-09

    Chondrocyte differentiation is controlled by various regulators, such as Sox9 and Runx2, but the process is complex. To further understand the precise underlying molecular mechanisms of chondrocyte differentiation, we aimed to identify a novel regulatory factor of chondrocyte differentiation using gene expression profiles of micromass-cultured chondrocytes at different differentiation stages. From the results of microarray analysis, the autoimmune regulator, Aire, was identified as a novel regulator. Aire stable knockdown cells, and primary cultured chondrocytes obtained from Aire(-/-) mice, showed reduced mRNA expression levels of chondrocyte-related genes. Over-expression of Aire induced the early stages of chondrocyte differentiation by facilitating expression of Bmp2. A ChIP assay revealed that Aire was recruited on an Airebinding site (T box) in the Bmp2 promoter region in the early stages of chondrocyte differentiation and histone methylation was modified. These results suggest that Aire can facilitate early chondrocyte differentiation by expression of Bmp2 through altering the histone modification status of the promoter region of Bmp2. Taken together, Aire might play a role as an active regulator of chondrocyte differentiation, which leads to new insights into the regulatory mechanisms of chondrocyte differentiation. Copyright © 2013 Elsevier Inc. All rights reserved.

  8. 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.

  9. 15-Deoxy-delta12,14-PGJ2, but not troglitazone, modulates IL-1beta effects in human chondrocytes by inhibiting NF-kappaB and AP-1 activation pathways.

    Science.gov (United States)

    Boyault, S; Simonin, M A; Bianchi, A; Compe, E; Liagre, B; Mainard, D; Bécuwe, P; Dauça, M; Netter, P; Terlain, B; Bordji, K

    2001-07-13

    The activation of peroxisome proliferator-activated receptor gamma (PPARgamma) has been shown to inhibit the production and the effects of proinflammatory cytokines. Since interleukin-1beta (IL-1beta) directly mediates cartilage degradation in osteoarthritis, we investigated the capability of PPARgamma ligands to modulate IL-1beta effects on human chondrocytes. RT-PCR and Western blot analysis revealed that PPARgamma expression was decreased by IL-1beta. 15-deoxy-Delta12,14-prostaglandin J2 (15d-PGJ2), in contrast to troglitazone, was highly potent to counteract IL-1beta-induced cyclooxygenase-2 and inductible nitric oxide synthase expression, NO production and the decrease in proteoglycan synthesis. Western blot and gel-shift analyses demonstrated that 15d-PGJ2 inhibited NF-kappaB activation, while troglitazone was ineffective. Although 15d-PGJ2 attenuated activator protein-1 binding on the DNA, it potentiated c-jun migration in the nucleus. The absence or the low effect of troglitazone suggests that 15d-PGJ2 action in human chondrocytes is mainly PPARgamma-independent.

  10. The trans-well coculture of human synovial mesenchymal stem cells with chondrocytes leads to self-organization, chondrogenic differentiation, and secretion of TGFβ

    DEFF Research Database (Denmark)

    Kubosch, Eva Johanna; Heidt, Emanuel; Bernstein, Anke

    2016-01-01

    BACKGROUND: Synovial mesenchymal stem cells (SMSC) possess a high chondrogenic differentiation potential, which possibly supports natural and surgically induced healing of cartilage lesions. We hypothesized enhanced chondrogenesis of SMSC caused by the vicinity of chondrocytes (CHDR). METHODS....... RESULTS: After 7 days, phase-contrast microscopy revealed cell aggregation of SMSC in coculture with CHDR. Afterwards, cells formed spheres and lost adherence. However, this phenomenon was not observed when culturing SMSC alone. Fluorescence labeling showed concurrent collagen type II expression. Addition...

  11. Implantation of juvenile human chondrocytes demonstrates no adverse effect on spinal nerve tissue in rats.

    Science.gov (United States)

    Külling, Fabrice A; Liu, Jane J; Liebenberg, Ellen; Lotz, Jeffrey C

    2016-09-01

    Degenerative disc disease (DDD) is a common disabling condition for millions of individuals. Injection of xenogenic juvenile chondrocytes (XJC) into the disc space has been shown to have a therapeutic potential for disc repair. In the current study, XJC were injected extra-discally on neural structures in an in vivo rat hemilaminectomy model to compare the histological and behavioral effects on XJC and fibrin glue carrier. Twenty-four rats were assigned to four groups: cells plus carrier, carrier alone, sham hemi-laminectomy, and a positive control (nerve root ligation). A right-sided hemilaminectomy was performed and the study material was placed on and around the exposed L4 nerve root and the spinal cord. Pre- and postoperatively mechanical allodynia was tested on the ipsilateral hind paw using the von Frey up-down method. The lumbar spines were harvested after 6 and 12 weeks for nerve histology and TNF-α quantification. After a brief period of hyperalgesia, the von Frey data indicate there are no adverse effects of placing XJC on spinal nerve roots in rats. However ligation of nerve root showed significant allodynia compared to the other groups. These behavioral data were supported by histological analyses. While these results need to be confirmed over a larger period of time, they suggest that XJC transplantation into the disc space shows no adverse effect on nerve tissue.

  12. 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

    -tag was expressed in Cos7 cells, and the cell lysate was studied for putative glycosaminoglycan attachment by digestion with chondroitinase ABC and Western blotting. RESULTS: The predicted molecule is a small, 121 amino acids long type I single-pass transmembrane chondroitin sulfate proteoglycan, that contains ER...

  13. Differentiation of synovial CD-105(+) human mesenchymal stem cells into chondrocyte-like cells through spheroid formation.

    Science.gov (United States)

    Arufe, M C; De la Fuente, A; Fuentes-Boquete, I; De Toro, Francisco J; Blanco, Francisco J

    2009-09-01

    Mesenchymal stem cells (MSCs) have the capacity to differentiate into several cell lineages, some of which can generate bone, cartilage, or adipose tissue. The presence of MSCs in the synovial membrane was recently reported. Data from comparative studies of MSCs derived from various mesenchymal tissues suggest that MSCs from synovial membranes have a superior chondrogenesis capacity. Previous chondrogenic differentiation studies have used the total population of MSCs, including cells with several MSC markers, such as CD44, CD90, CD105, or CD73. However the chondrogenic capacity of an individual population of MSCs has not been examined. Our aim was to study the chondrogenic capacity of the cellular MSC subset, CD105(+), derived from synovial membrane tissues of patients with osteoarthritis (OA) and normal donors. The tissues were digested with a cocktail of collagenase/dispase and the isolated MSCs were seeded into plates. The subpopulation of CD105(+)-MSCs was separated using a magnetic separator. The MSCs were then differentiated towards chondrocyte-like cells using a specific medium to promote spheroid formation. Spheroids were collected after 14, 28, and 46 days in chondrogenic medium and stained with hematoxylin, eosin, Safranin O or Alcian blue to evaluate the extracellular matrix. Immunohistochemistry was performed to study collagen types I (COLI) and II (COLII) and aggrecan expression. Phenotypic characterization of the isolated CD105(+)-MSCs shows that these cells are also positive for CD90 and CD44, but negatives for CD34 and CD45. In addition, this cellular subset expressed Sox-9. Spheroids appeared after 7 days in culture in the presence of chondrogenic medium. Our studies show no differences between MSCs obtained from OA and normal synovial membranes during chondrogenesis. The morphological analysis of spheroids revealed characteristics typical of chondrocyte cells. The intensity of Safranin O, Alcian blue and aggrecan staining was positive and constant

  14. Human YKL39 (chitinase 3-like protein 2), an osteoarthritis-associated gene, enhances proliferation and type II collagen expression in ATDC5 cells

    Energy Technology Data Exchange (ETDEWEB)

    Miyatake, Kazumasa [Department of Joint Surgery and Sports Medicine, Tokyo Medical and Dental University, Tokyo (Japan); Tsuji, Kunikazu, E-mail: ktsuji.gcoe@tmd.ac.jp [International Research Center for Molecular Science in Tooth and Bone Diseases (Global Center of Excellence Program), Tokyo Medical and Dental University, Tokyo (Japan); Yamaga, Mika; Yamada, Jun; Matsukura, Yu; Abula, Kahaer [Department of Joint Surgery and Sports Medicine, Tokyo Medical and Dental University, Tokyo (Japan); Sekiya, Ichiro [Section of Cartilage Regeneration, Tokyo Medical and Dental University, Tokyo (Japan); Muneta, Takeshi [Department of Joint Surgery and Sports Medicine, Tokyo Medical and Dental University, Tokyo (Japan); International Research Center for Molecular Science in Tooth and Bone Diseases (Global Center of Excellence Program), Tokyo Medical and Dental University, Tokyo (Japan)

    2013-02-01

    Highlights: ► hYKL-39 expression is increased in osteoarthritic articular chondrocytes. ► To examine the molecular functions of hYKL-39 in chondrocytes, we overexpressed hYKL-39 in chondrocytic ATDC5 cells. ► hYKL-39 enhanced proliferation and colony formation in ATDC5 cells. ► hYKL-39 increased type II collagen expression in ATDC5 cells treated with chondrogenic medium. -- Abstract: Human YKL39 (chitinase 3-like protein 2/CHI3L2) is a secreted 39 kDa protein produced by articular chondrocytes and synoviocytes. Recent studies showed that hYKL-39 expression is increased in osteoarthritic articular chondrocytes suggesting the involvement of hYKL-39 in the progression of osteoarthritis (OA). However little is known regarding the molecular function of hYKL-39 in joint homeostasis. Sequence analyses indicated that hYKL-39 has significant identity with the human chitotorisidase family molecules, although it is considered that hYKL-39 has no enzymatic activity since it lacks putative chitinase catalytic motif. In this study, to examine the molecular function of hYKL-39 in chondrocytes, we overexpressed hYKL-39 in ATDC5 cells. Here we report that hYKL-39 enhances colony forming activity, cell proliferation, and type II collagen expression in these cells. These data suggest that hYKL-39 is a novel growth and differentiation factor involved in cartilage homeostasis.

  15. Decrease in the expression of the type 1 PTH/PTHrP receptor (PTH1R on chondrocytes in animals with osteoarthritis

    Directory of Open Access Journals (Sweden)

    Skwara Adrian

    2010-04-01

    Full Text Available Abstract Background To evaluate the expression of the type 1 PTH/PTHrP receptor (PTH1R on chondrocytes from hyaline cartilage over the course of osteoarthritis (OA. Methods In 12 NZW rabbits, the anterior cruciate ligament (ACL was resected to create anterior instability of the knee. In 12 control rabbits, only a sham operation, without resection of the ACL, was performed. Four animals from each group were killed at 3, 6, and 12 weeks. After opening the knee joint, OA was macroscopically graded and hyaline cartilage of the load-bearing area was evaluated histologically according to the Mankin scale and by immunostaining for PTH1R. Results There was a positive linear correlation between the time after surgery and the macroscopic and histologic OA scores. The scores in the control group were constant over the time course. Immunostaining showed significantly less expression of PTH1R in the experimental compared to the control group after 6 (P Conclusions The results show an in vivo decrease in the expression of PTH1R on chondrocytes over the time course of OA. Further studies are needed to evaluate whether new treatment approaches could evolve from this knowledge.

  16. 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.

  17. ATRA Signaling Regulates the Expression of COL9A1 through BMP2-WNT4-RUNX1 Pathway in Antler Chondrocytes.

    Science.gov (United States)

    Zhang, Hong-Liang; Guo, Bin; Yang, Zhan-Qing; Duan, Cui-Cui; Geng, Shuang; Wang, Kai; Yu, Hai-Fan; Yue, Zhan-Peng

    2017-09-01

    Although all-trans retinoic acid (ATRA) is involved in the regulation of cartilage growth and development, its regulatory mechanisms remain unknown. Here, we showed that ATRA could induce the expression of COL9A1 in antler chondrocytes. Silencing of cellular retinoic acid binding protein 2 (CRABP2) could impede the ATRA-induced upregulation of COL9A1, whereas overexpression of CRABP2 presented the opposite effect. RARα agonist Am80 induced the expression of COL9A1, whereas treatment with RARα antagonist Ro 41-5253 or RXRα small-interfering RNA (siRNA) caused an obvious blockage of ATRA on COL9A1. In antler chondrocytes, CYP26A1 and CYP26B1 weakened the sensitivity of ATRA to COL9A1. Simultaneously, Bone morphogenetic protein 2 (BMP2) and WNT4 mediated the regulation of ATRA on COL9A1 expression. Knockdown of WNT4 could abrogate the inhibitory effect of BMP2 overexpression on COL9A1. Conversely, constitutive expression of WNT4 reversed the upregulation of COL9A1 elicited by BMP2 siRNA. Together these data indicated that WNT4 might act downstream of BMP2 to mediate the effect of ATRA on COL9A1 expression. Further analysis evidenced that attenuation of runt-related transcription factor 1 (RUNX1) could prevent the stimulation of ATRA on COL9A1 expression, while exogenous rRUNX1 further enhanced this effectiveness. Moreover, RUNX1 might serve as an intermediate to mediate the regulation of BMP2 and WNT4 on COL9A1 expression. Collectively, ATRA signaling might regulate the expression of COL9A1 through BMP2-WNT4-RUNX1 pathway. © 2017 Wiley Periodicals, Inc.

  18. Induced superficial chondrocyte death reduces catabolic cartilage damage in murine posttraumatic osteoarthritis.

    Science.gov (United States)

    Zhang, Minjie; Mani, Sriniwasan B; He, Yao; Hall, Amber M; Xu, Lin; Li, Yefu; Zurakowski, David; Jay, Gregory D; Warman, Matthew L

    2016-08-01

    Joints that have degenerated as a result of aging or injury contain dead chondrocytes and damaged cartilage. Some studies have suggested that chondrocyte death precedes cartilage damage, but how the loss of chondrocytes affects cartilage integrity is not clear. In this study, we examined whether chondrocyte death undermines cartilage integrity in aging and injury using a rapid 3D confocal cartilage imaging technique coupled with standard histology. We induced autonomous expression of diphtheria toxin to kill articular surface chondrocytes in mice and determined that chondrocyte death did not lead to cartilage damage. Moreover, cartilage damage after surgical destabilization of the medial meniscus of the knee was increased in mice with intact chondrocytes compared with animals whose chondrocytes had been killed, suggesting that chondrocyte death does not drive cartilage damage in response to injury. These data imply that chondrocyte catabolism, not death, contributes to articular cartilage damage following injury. Therefore, therapies targeted at reducing the catabolic phenotype may protect against degenerative joint disease.

  19. 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

    chain reaction (RT-PCR). In bovine articular cartilage explants, cartilage degradation was investigated by release of C-terminal telopeptides of collagen type II (CTX-II), induced by tumor necrosis factor-alpha (TNF-alpha) [20 ng/ml] and oncostatin M (OSM) [10 ng/ml], with salmon calcitonin [0.......0001-1 microM]. In vivo, cartilage degradation was investigated in ovariectomized (OVX) rats administered with oral calcitonin [2 mg/kg calcitonin] for 9 weeks. RESULTS: The calcitonin receptor was identified in articular chondrocytes by immunohistochemistry and RT-PCR. Calcitonin concentration......-dependently increased cAMP levels in isolated chondrocytes. Explants cultured with TNF-alpha and OSM showed a 100-fold increase in CTX-II release compared to vehicle-treated controls (Pprotection...

  20. Prolactin inhibits the apoptosis of chondrocytes induced by serum starvation.

    Science.gov (United States)

    Zermeño, C; Guzmán-Morales, J; Macotela, Y; Nava, G; López-Barrera, F; Kouri, J B; Lavalle, C; de la Escalera, G Martínez; Clapp, C

    2006-05-01

    The apoptosis of chondrocytes plays an important role in endochondral bone formation and in cartilage degradation during aging and disease. Prolactin (PRL) is produced in chondrocytes and is known to promote the survival of various cell types. Here we show that articular chondrocytes from rat postpubescent and adult cartilage express the long form of the PRL receptor as revealed by immunohistochemistry of cartilage sections and by RT-PCR and Western blot analyses of the isolated chondrocytes. Furthermore, we demonstrate that PRL inhibits the apoptosis of these same chondrocytes cultured in low-serum. Chondrocyte apoptosis was measured by hypodiploid DNA content determined by flow cytometry and by DNA fragmentation evaluated by the ELISA and the TUNEL methods. The anti-apoptotic effect of PRL was dose-dependent and was prevented by heat inactivation. These data demonstrate that PRL can act as a survival factor for chondrocytes and that it has potential preventive and therapeutic value in arthropathies characterized by cartilage degradation.

  1. Serum-free medium supplemented with high-concentration FGF2 for cell expansion culture of human ear chondrocytes promotes redifferentiation capacity.

    Science.gov (United States)

    Mandl, Erik W; van der Veen, Simone W; Verhaar, Jan A N; van Osch, Gerjo J V M

    2002-08-01

    For tissue engineering of autologous cartilage, cell expansion is needed to obtain the cell numbers required. Standard expansion media contain bovine serum. This has several disadvantages, that is, the risk of transmitting diseases and serum-batch variations. The aim of this study was to find a serum-free medium with at least the same potential to expand cell numbers as serum-containing media. Ear chondrocytes of three young children were expanded in either serum-containing medium (SCM; DMEM with 10% fetal calf serum) or serum-free medium (SFM; DMEM with ITS+) supplemented with 5 or 100 ng/mL fibroblast growth factor-2 (FGF2). To promote cell adherence onto the culture flask, the serum-free conditions were cultured with 10% serum for 1 day after each trypsinization. After the fourth passage, the chondrocytes were encapsuled in alginate beads and redifferentiated in a SFM (DMEM with ITS+, hydrocortisone, and L-ascorbic acid) supplemented with 10 ng/mL IGF-I and 10 ng/mL TGFbeta-2. Results showed that expansion in SFM with 100 ng/mL FGF2 was comparable to expansion in SCM. Redifferentiation with SFM with IGF-I and TGFbeta-2 showed high collagen type II expression and high GAG/DNA production regardless of which expansion medium had been used. However, chondrocytes expanded in SFM with 100 ng/mL FGF2 resulted in less positive cells for collagen type I and 11-fibrau (a fibroblast membrane marker). The present study shows that it is possible to use serum-free medium for tissue engineering of cartilage. Expansion of immature ear chondrocytes in SFM supplemented with high-concentration FGF2 resulted in high cell numbers, which in addition had better redifferentiation capacity than cells expanded in medium with 10% serum.

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

    Science.gov (United States)

    Talò, Giuseppe; Lovati, Arianna B.; Pasdeloup, Marielle; Riboldi, Stefania A.; Moretti, Matteo; Mallein-Gerin, Frédéric

    2016-01-01

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

  3. PGE2 signal through EP2 promotes the growth of articular chondrocytes.

    Science.gov (United States)

    Aoyama, Tomoki; Liang, Bojian; Okamoto, Takeshi; Matsusaki, Takashi; Nishijo, Koichi; Ishibe, Tatsuya; Yasura, Ko; Nagayama, Satoshi; Nakayama, Tomitaka; Nakamura, Takashi; Toguchida, Junya

    2005-03-01

    EP2 was identified as the major PGE2 receptor expressed in articular cartilage. An EP2 agonist increased intracellular cAMP in articular chondrocytes, stimulating DNA synthesis in both monolayer and 3D cultures. Hence, the EP2 agonist may be a potent therapeutic agent for degenerative cartilage diseases. Prostaglandin E2 (PGE2) exhibits pleiotropic effects in various types of tissue through four types of receptors, EP1-4. We examined the expression of EPs and effects of agonists for each EP on articular chondrocytes. The expression of each EP in articular chondrocytes was examined by immunohistochemistry and RT-PCR. A chondrocyte cell line, MMA2, was established from articular cartilage of p53(-/-) mice and used to analyze the effects of agonists for each EP. A search for molecules downstream of the PGE2 signal through the EP2 agonist was made by cDNA microarray analysis. The growth-promoting effect of the EP2 agonist on chondrocytes surrounded by cartilage matrix was examined in an organ culture of rat femora. EP2 was identified as the major EP expressed in articular cartilage. Treatment of MMA2 cells with specific agonists for each EP showed that only the EP2 agonist significantly increased intracellular cAMP levels in a dose-dependent manner. Gene expression profiling of MMA2 revealed a set of genes upregulated by the EP2 agonist, including several growth-promoting and apoptosis-protecting genes such as the cyclin D1, fibronectin, integrin alpha5, AP2alpha, and 14-3-3gamma genes. The upregulation of these genes by the EP2 agonist was confirmed in human articular chondrocytes by quantitative mRNA analysis. On treatment with the EP2 agonist, human articular chondrocytes showed an increase in the incorporation of 5-bromo-2-deoxyuracil (BrdU), and the organ culture of rat femora showed an increase of proliferating cell nuclear antigen (PCNA) staining in articular chondrocytes surrounded by cartilage matrix, suggesting growth-promoting effects of the PGE2 signal

  4. BMP-2, hypoxia, and COL1A1/HtrA1 siRNAs favor neo-cartilage hyaline matrix formation in chondrocytes.

    Science.gov (United States)

    Ollitrault, David; Legendre, Florence; Drougard, Carole; Briand, Mélanie; Benateau, Hervé; Goux, Didier; Chajra, Hanane; Poulain, Laurent; Hartmann, Daniel; Vivien, Denis; Shridhar, Vijayalakshmi; Baldi, Alfonso; Mallein-Gerin, Frédéric; Boumediene, Karim; Demoor, Magali; Galera, Philippe

    2015-02-01

    Osteoarthritis (OA) is an irreversible pathology that causes a decrease in articular cartilage thickness, leading finally to the complete degradation of the affected joint. The low spontaneous repair capacity of cartilage prevents any restoration of the joint surface, making OA a major public health issue. Here, we developed an innovative combination of treatment conditions to improve the human chondrocyte phenotype before autologous chondrocyte implantation. First, we seeded human dedifferentiated chondrocytes into a collagen sponge as a scaffold, cultured them in hypoxia in the presence of a bone morphogenetic protein (BMP), BMP-2, and transfected them with small interfering RNAs targeting two markers overexpressed in OA dedifferentiated chondrocytes, that is, type I collagen and/or HtrA1 serine protease. This strategy significantly decreased mRNA and protein expression of type I collagen and HtrA1, and led to an improvement in the chondrocyte phenotype index of differentiation. The effectiveness of our in vitro culture process was also demonstrated in the nude mouse model in vivo after subcutaneous implantation. We, thus, provide here a new protocol able to favor human hyaline chondrocyte phenotype in primarily dedifferentiated cells, both in vitro and in vivo. Our study also offers an innovative strategy for chondrocyte redifferentiation and opens new opportunities for developing therapeutic targets.

  5. Effects of extracellular matrix proteins in chondrocyte-derived matrices on chondrocyte functions.

    Science.gov (United States)

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

    2013-01-01

    Loss of cartilaginous phenotype during in vitro expansion culture of chondrocytes is a major barrier to the application of chondrocytes for tissue engineering. In previous study, we showed that dedifferentiation of chondrocytes during the passage culture was delayed by matrices formed by primary chondrocytes (P0-ECM). In this study, we investigated bovine chondrocyte functions when being cultured on isolated extracellular matrix (ECM) protein-coated substrata and P0-ECM. Low chondrocyte attachment was observed on aggrecan-coated substratum and P0-ECM. Cell proliferation on aggrecan- and type II collagen/aggrecan-coated substrata and P0-ECM was lower than that on the other ECM protein (type I collagen and type II collagen)-coated substrata. When chondrocytes were subcultured on aggrecan-coated substratum, decline of cartilaginous gene expression was delayed, which was similar to the cells subcultured on P0-ECM. These results indicate that aggrecan plays an important role in the regulation of chondrocyte functions and P0-ECM may be a good experimental control for investigating the role of each ECM protein in cartilage ECM.

  6. The roles of canonical and non-canonical Wnt signaling in human de-differentiated articular chondrocytes

    NARCIS (Netherlands)

    Sassi, N.; Laadhar, L.; Allouche, M.; Zandieh-Doulabi, B.; Hamdoun, M.; Klein-Nulend, J.; Makni, S.; Sellami, S.

    2014-01-01

    Osteoarthritis is the most prevalent form of arthritis in the world and it is becoming a major public health problem. Osteoarthritic chondrocytes undergo morphological and biochemical changes that lead to de-differentiation. The involvement of signaling pathways, such as the Wnt pathway, during cart

  7. 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 E2, 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.

  8. Indian hedgehog signals independently of PTHrP to promote chondrocyte hypertrophy

    National Research Council Canada - National Science Library

    Kinglun Kingston Mak; Henry M. Kronenberg; Pao-Tien Chuang; Susan Mackem; Yingzi Yang

    2008-01-01

    .... Indian hedgehog (Ihh) and PTHrP signaling play crucial roles in regulating the onset of chondrocyte hypertrophy by forming a negative feedback loop, in which Ihh signaling regulates chondrocyte hypertrophy by controlling PTHrP expression...

  9. Resisin stimulates the expression of CCL3 and CCL4 in chondrocytes%抵抗素刺激软骨细胞趋化因子CCL3及CCL4基因表达及机制

    Institute of Scientific and Technical Information of China (English)

    张紫机; 廖威明; 张志奇; 康焱; 杨子波; 侯昌禾; 黄广鑫; 陈蔚深; 盛璞义; 何爱珊; 傅明

    2015-01-01

    BACKGROUND:Previous studies have indicated that resistin stimulates a large set of chemokines in chondrocytes that are known to be important in inflammatory joint lesions. OBJECTIVE:To further investigate the mechanism of co-regulation roles of transcription and post-transcription in the up-regulation of two chemokine genes CCL3 and CCL4 in chondrocytes in response to resistin. METHODS:Human chondrocytes, T/C-28a2 and ATDC5 cels were cultured. The function of resistin on the chemokine genes, and the expression of C/EBPβ, nuclear factor-κB isoforms and chondrogenic specific miRNAs were tested by qPCR. The co-regulation of C/EBPβ and nuclear factor-κB was investigated by nuclear factor-κB inhibitor (IKK-NBD) and C/EBPβ inhibitor (SB303580) treatments, and subcelular localization was detected with or without resistin stimulation. RESULTS AND CONCLUSION:Resistin could increase the expression of chemokine genes independently. Chondrocytes reacted in a non-restrictedly cel-specific manner to resistin; C/EBPβ inhibitor, nuclear factor-κB and some chondrogenic specific miRNAs in a combinatorial manner regulated chemokine gene expression. The activity of C/EBPβ was augmented by a transient increase in activity of nuclear factor-κB, and both transcription factors acted independently on the chemokine genes, CCL3 and CCL4.%背景:既往研究表明抵抗素可刺激软骨细胞产生大量趋化因子,在炎症性关节病变中具重要作用,但具体作用机制未明。  目的:进一步探讨抵抗素刺激软骨细胞趋化因子CCL3及CCL4基因表达上调的机制。  方法:培养人源性软骨细胞,T/C-28a2细胞及ATDC5细胞,采用qPCR检测抵抗素刺激趋化因子基因的作用,C/EBPβ表达,核因子κB亚型及软骨特异性miRNAs。给予核因子κB抑制剂(IKK-NBD)和C/EBPβ抑制剂(SB303580),对C/EBPβ及核因子κB的共同调节作用进行检测。在给予抵抗素刺激或无抵抗素刺激时分别进

  10. 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

    2014-06-01

    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 TRPV4(V620I) 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 the TRPV4(V620I) 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 TRPV4(T89I) mutation. Taken together, these data strongly support that up-regulation of FST in chondrocytes by skeletal dysplasia-inducing TRPV4 mutations contributes to disease pathogenesis. © FASEB.

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

    Directory of Open Access Journals (Sweden)

    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

  12. Collagen-induced expression of collagenase-3 by primary chondrocytes is mediated by integrin alpha 1 and discoidin domain receptor 2 : a protein kinase C-dependent pathway

    NARCIS (Netherlands)

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

    2011-01-01

    Methods. Goat articular chondrocytes and chondrons were cultured on collagen coatings. Small interfering RNA (siRNA) oligonucleotides targeted against ITG alpha 1 and DDR2 were transfected into primary chondrocytes. Chemical inhibitors for mitogen-activated protein kinase kinase (MEK1) (PD98059), fo

  13. Enhanced hyaline cartilage matrix synthesis in collagen sponge scaffolds by using siRNA to stabilize chondrocytes phenotype cultured with bone morphogenetic protein-2 under hypoxia.

    Science.gov (United States)

    Legendre, Florence; Ollitrault, David; Hervieu, Magalie; Baugé, Catherine; Maneix, Laure; Goux, Didier; Chajra, Hanane; Mallein-Gerin, Frédéric; Boumediene, Karim; Galera, Philippe; Demoor, Magali

    2013-07-01

    Cartilage healing by tissue engineering is an alternative strategy to reconstitute functional tissue after trauma or age-related degeneration. However, chondrocytes, the major player in cartilage homeostasis, do not self-regenerate efficiently and lose their phenotype during osteoarthritis. This process is called dedifferentiation and also occurs during the first expansion step of autologous chondrocyte implantation (ACI). To ensure successful ACI therapy, chondrocytes must be differentiated and capable of synthesizing hyaline cartilage matrix molecules. We therefore developed a safe procedure for redifferentiating human chondrocytes by combining appropriate physicochemical factors: hypoxic conditions, collagen scaffolds, chondrogenic factors (bone morphogenetic protein-2 [BMP-2], and insulin-like growth factor I [IGF-I]) and RNA interference targeting the COL1A1 gene. Redifferentiation of dedifferentiated chondrocytes was evaluated using gene/protein analyses to identify the chondrocyte phenotypic profile. In our conditions, under BMP-2 treatment, redifferentiated and metabolically active chondrocytes synthesized a hyaline-like cartilage matrix characterized by type IIB collagen and aggrecan molecules without any sign of hypertrophy or osteogenesis. In contrast, IGF-I increased both specific and noncharacteristic markers (collagens I and X) of chondrocytes. The specific increase in COL2A1 gene expression observed in the BMP-2 treatment was shown to involve the specific enhancer region of COL2A1 that binds the trans-activators Sox9/L-Sox5/Sox6 and Sp1, which are associated with a decrease in the trans-inhibitors of COL2A1, c-Krox, and p65 subunit of NF-kappaB. Our procedure in which BMP-2 treatment under hypoxia is associated with a COL1A1 siRNA, significantly increased the differentiation index of chondrocytes, and should offer the opportunity to develop new ACI-based therapies in humans.

  14. Chondrocyte physiopathology and drug efficacy.

    Science.gov (United States)

    Serni, U; Mannoni, A

    1991-01-01

    After a brief exposition on the physiopathology of cartilage, and characteristic features of chondrocytes and proteoglycans (PGs) in osteoarthritis (OA), it is underlined how different molecules of GAGs and aggregated PGs added to the culture media can prevent damage and reduction of GAGs and fibril production in chondrocytes cultured with NSAIDs and corticosteroids. In animal models of OA, the local or general administration of GAGPS reduces the proteinase activity, the level of uronic acid in synovial fluid and the number of inflammatory cells in synovia. In the Pond-Nuki dog, GAGPS improves the cartilage surface. These favourable events can also occur in human OA, where it is, moreover, difficult to monitor the patients. For this purpose, patients must be selected in the first two stages of primary OA, and followed using NMR, the only device able to scan cartilage and subchondral bone, to determine their consistency and thickness, and to provide information on water content.

  15. Chondrocytes from congenital microtia possess an inferior capacity for in vivo cartilage regeneration to healthy ear chondrocytes.

    Science.gov (United States)

    Gu, Yunpeng; Kang, Ning; Dong, Ping; Liu, Xia; Wang, Qian; Fu, Xin; Yan, Li; Jiang, Haiyue; Cao, Yilin; Xiao, Ran

    2016-11-15

    The remnant auricular cartilage from microtia has become a valuable cell source for ear regeneration. It is important to clarify the issue of whether the genetically defective microtia chondrocytes could engineer cartilage tissue comparable to healthy ear chondrocytes. In the current study, the histology and cell yield of native microtia and normal ear cartilage were investigated, and the biological characteristics of derived chondrocytes examined, including proliferation, chondrogenic phenotype and cell migration. Furthermore, the in vivo cartilage-forming capacity of passaged microtia and normal auricular chondrocytes were systematically compared by seeding them onto polyglycolic acid/polylactic acid scaffold to generate tissue engineered cartilage in nude mice. Through histological examinations and quantitative analysis of glycosaminoglycan, Young's modulus, and the expression of cartilage-related genes, it was found that microtia chondrocytes had a slower dedifferentiation rate with the decreased expression of stemness-related genes, and weaker migration ability than normal ear chondrocytes, and the microtia chondrocytes-engineered cartilage was biochemically and biomechanically inferior to that constructed using normal ear chondrocytes. This study provides valuable information for the clinical application of the chondrocytes derived from congenital microtia to engineer cartilage. Copyright © 2016 John Wiley & Sons, Ltd.

  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. 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...... of recombinant hEGF and bFGF resulted in a significant increase in cell proliferation and glucosaminoglycan production. Chondrocytes were transfected with vectors coding for either hEGF or bFGF and the production of these proteins was measured in supernatants by ELISA. Expression kinetics showed different...... patterns: hEGF was detectable 2.5 days following transfection and peaked at day 5.5, whereas bFGF-production reached its maximum 1.5 days after transfection, declining thereafter. Chondrocytes endogenously produced significant amounts of bFGF within 5 days following isolation. Proliferation of h...

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

    Directory of Open Access Journals (Sweden)

    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.

  19. Basic fibroblast growth factor induces matrix metalloproteinase-13 via ERK MAP kinase-altered phosphorylation and sumoylation of Elk-1 in human adult articular chondrocytes

    Directory of Open Access Journals (Sweden)

    Hee-Jeong Im

    2009-10-01

    Full Text Available Hee-Jeong Im,1–4 Andrew D Sharrocks,5 Xia Lin,6 Dongyao Yan,1 Jaesung Kim,1 Andre J van Wijnen,7 Robert A Hipskind81Departments of Biochemistry, 2Internal Medicine, 3Section of Rheumatology, Orthopedic Surgery, 4Rush University Medical Center, and Department of Bioengineering; University of Illinois at Chicago, IL USA; 5Faculty of Life Sciences, University of Manchester, Oxford Rd, Manchester, UK; 6Michael D DeBakey Department of Surgery, Baylor College of Medicine, Houston, Texas, USA; 7Department of Cell Biology, University of Massachusetts Medical School, Worcester, MA, USA; 8Institute De Genetique Moleculaire de Montpellier, FranceAbstract: Degradation of the extracellular matrix (ECM by matrix metalloproteinases (MMPs and release of basic fibroblast growth factor (bFGF are principal aspects of the pathology of osteoarthritis (OA. ECM disruption leads to bFGF release, which activates the extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK/MAPK pathway and its downstream target the Ets-like transcription factor Elk-1. Previously we demonstrated that the bFGF-ERK-Elk-1 signaling axis is responsible for the potent induction of MMP-13 in human primary articular chondrocytes. Here we report that, in addition to phosphorylation of Elk-1, dynamic posttranslational modification of Elk-1 by small ubiquitin-related modifier (SUMO serves as an important mechanism through which MMP-13 gene expression is regulated. We show that bFGF activates Elk-1 mainly through the ERK pathway and that increased phosphorylation of Elk-1 is accompanied by decreased conjugation of SUMO to Elk-1. Reporter gene assays reveal that phosphorylation renders Elk-1 competent for induction of MMP-13 gene transcription, while sumoylation has the opposite effect. Furthermore, we demonstrate that the SUMO-conjugase Ubc9 acts as a key mediator for Elk-1 sumoylation. Taken together, our results suggest that sumoylation antagonizes the phosphorylation

  20. Sprifermin (rhFGF18) enables proliferation of chondrocytes producing a hyaline cartilage matrix.

    Science.gov (United States)

    Gigout, A; Guehring, H; Froemel, D; Meurer, A; Ladel, C; Reker, D; Bay-Jensen, A C; Karsdal, M A; Lindemann, S

    2017-08-18

    Fibroblast growth factor (FGF) 18 has been shown to increase cartilage volume when injected intra-articularly in animal models of osteoarthritis (OA) and in patients with knee OA (during clinical development of the recombinant human FGF18, sprifermin). However, the exact nature of this effect is still unknown. In this study, we aimed to investigate the effects of sprifermin at the cellular level. A combination of different chondrocyte culture systems was used and the effects of sprifermin on proliferation, the phenotype and matrix production were evaluated. The involvement of MAPKs in sprifermin signalling was also studied. In monolayer, we observed that sprifermin promoted a round cell morphology and stimulated both cellular proliferation and Sox9 expression while strongly decreasing type I collagen expression. In 3D culture, sprifermin increased the number of matrix-producing chondrocytes, improved the type II:I collagen ratio and enabled human OA chondrocytes to produce a hyaline extracellular matrix (ECM). Furthermore, we found that sprifermin displayed a 'hit and run' mode of action, with intermittent exposure required for the compound to fully exert its anabolic effect. Finally, sprifermin appeared to signal through activation of ERK. Our results indicate that intermittent exposure to sprifermin leads to expansion of hyaline cartilage-producing chondrocytes. These in vitro findings are consistent with the increased cartilage volume observed in the knees of OA patients after intra-articular injection with sprifermin in clinical studies. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

  1. Sodium nitroprusside induces apoptosis of rabbit chondrocytes

    Science.gov (United States)

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

    2013-02-01

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

  2. Chondrocyte behavior on nanostructured micropillar polypropylene and polystyrene surfaces

    Energy Technology Data Exchange (ETDEWEB)

    Prittinen, Juha [Department of Applied Physics, University of Eastern Finland, Kuopio (Finland); Jiang, Yu [Department of Chemistry, University of Eastern Finland, Joensuu (Finland); Ylärinne, Janne H. [Department of Applied Physics, University of Eastern Finland, Kuopio (Finland); Pakkanen, Tapani A. [Department of Chemistry, University of Eastern Finland, Joensuu (Finland); Lammi, Mikko J., E-mail: mikko.lammi@uef.fi [Department of Applied Physics, University of Eastern Finland, Kuopio (Finland); Qu, Chengjuan [Department of Applied Physics, University of Eastern Finland, Kuopio (Finland)

    2014-10-01

    This study was aimed to investigate whether patterned polypropylene (PP) or polystyrene (PS) could enhance the chondrocytes' extracellular matrix (ECM) production and phenotype maintenance. Bovine primary chondrocytes were cultured on smooth PP and PS, as well as on nanostructured micropillar PP (patterned PP) and PS (patterned PS) for 2 weeks. Subsequently, the samples were collected for fluorescein diacetate-based cell viability tests, for immunocytochemical assays of types I and II collagen, actin and vinculin, for scanning electronic microscopic analysis of cell morphology and distribution, and for gene expression assays of Sox9, aggrecan, procollagen α{sub 1}(II), procollagen α{sub 1}(X), and procollagen α{sub 2}(I) using quantitative RT-PCR assays. After two weeks of culture, the bovine primary chondrocytes had attached on both patterned PP and PS, while practically no adhesion was observed on smooth PP. However, the best adhesion of the cells was on smooth PS. The cells, which attached on patterned PP and PS surfaces synthesized types I and II collagen. The chondrocytes' morphology was extended, and an abundant ECM network formed around the attached chondrocytes on both patterned PP and PS. Upon passaging, no significant differences on the chondrocyte-specific gene expression were observed, although the highest expression level of aggrecan was observed on the patterned PS in passage 1 chondrocytes, and the expression level of procollagen α{sub 1}(II) appeared to decrease in passaged chondrocytes. However, the expressions of procollagen α{sub 2}(I) were increased in all passaged cell cultures. In conclusion, the bovine primary chondrocytes could be grown on patterned PS and PP surfaces, and they produced extracellular matrix network around the adhered cells. However, neither the patterned PS nor PP could prevent the dedifferentiation of chondrocytes. - Highlights: • Methods to avoid chondrocyte dedifferentiation would be useful for cartilage

  3. 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

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

    Science.gov (United States)

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

    2016-03-25

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

  5. Dicer-dependent pathways regulate chondrocyte proliferation and differentiation.

    Science.gov (United States)

    Kobayashi, Tatsuya; Lu, Jun; Cobb, Bradley S; Rodda, Stephen J; McMahon, Andrew P; Schipani, Ernestina; Merkenschlager, Matthias; Kronenberg, Henry M

    2008-02-12

    Small noncoding RNAs, microRNAs (miRNAs), bind to messenger RNAs through base pairing to suppress gene expression. Despite accumulating evidence that miRNAs play critical roles in various biological processes across diverse organisms, their roles in mammalian skeletal development have not been demonstrated. Here, we show that Dicer, an essential component for biogenesis of miRNAs, is essential for normal skeletal development. Dicer-null growth plates show a progressive reduction in the proliferating pool of chondrocytes, leading to severe skeletal growth defects and premature death of mice. The reduction of proliferating chondrocytes in Dicer-null growth plates is caused by two distinct mechanisms: decreased chondrocyte proliferation and accelerated differentiation into postmitotic hypertrophic chondrocytes. These defects appear to be caused by mechanisms downstream or independent of the Ihh-PTHrP signaling pathway, a pivotal signaling system that regulates chondrocyte proliferation and differentiation. Microarray analysis of Dicer-null chondrocytes showed limited expression changes in miRNA-target genes, suggesting that, in the majority of cases, chondrocytic miRNAs do not directly regulate target RNA abundance. Our results demonstrate the critical role of the Dicer-dependent pathway in the regulation of chondrocyte proliferation and differentiation during skeletal development.

  6. Chondrocytes transdifferentiate into osteoblasts in endochondral bone during development, postnatal growth and fracture healing in mice.

    Science.gov (United States)

    Zhou, Xin; von der Mark, Klaus; Henry, Stephen; Norton, William; Adams, Henry; de Crombrugghe, Benoit

    2014-12-01

    One of the crucial steps in endochondral bone formation is the replacement of a cartilage matrix produced by chondrocytes with bone trabeculae made by osteoblasts. However, the precise sources of osteoblasts responsible for trabecular bone formation have not been fully defined. To investigate whether cells derived from hypertrophic chondrocytes contribute to the osteoblast pool in trabecular bones, we genetically labeled either hypertrophic chondrocytes by Col10a1-Cre or chondrocytes by tamoxifen-induced Agc1-CreERT2 using EGFP, LacZ or Tomato expression. Both Cre drivers were specifically active in chondrocytic cells and not in perichondrium, in periosteum or in any of the osteoblast lineage cells. These in vivo experiments allowed us to follow the fate of cells labeled in Col10a1-Cre or Agc1-CreERT2 -expressing chondrocytes. After the labeling of chondrocytes, both during prenatal development and after birth, abundant labeled non-chondrocytic cells were present in the primary spongiosa. These cells were distributed throughout trabeculae surfaces and later were present in the endosteum, and embedded within the bone matrix. Co-expression studies using osteoblast markers indicated that a proportion of the non-chondrocytic cells derived from chondrocytes labeled by Col10a1-Cre or by Agc1-CreERT2 were functional osteoblasts. Hence, our results show that both chondrocytes prior to initial ossification and growth plate chondrocytes before or after birth have the capacity to undergo transdifferentiation to become osteoblasts. The osteoblasts derived from Col10a1-expressing hypertrophic chondrocytes represent about sixty percent of all mature osteoblasts in endochondral bones of one month old mice. A similar process of chondrocyte to osteoblast transdifferentiation was involved during bone fracture healing in adult mice. Thus, in addition to cells in the periosteum chondrocytes represent a major source of osteoblasts contributing to endochondral bone formation in vivo.

  7. 骨髓源性肥大细胞对软骨细胞表达Ⅱ型胶原及糖胺多糖的影响%Effects of bone marrow- derived mast cells on expressions of type II collagen and glycosaminoglycan in co-cultured chondrocytes

    Institute of Scientific and Technical Information of China (English)

    欧阳晴晴; 赵进军; 杨敏

    2014-01-01

    Objective To investigate the influence of the bone marrow-derived mast cells (BMMCs) on the expression of type II collagen and glycosaminoglycan (GAG) in chondrocytes co-cultured with BMMCs. Methods Primarily cultured mouse BMMCs at 4 weeks and the second passage of chondrocytes were plated in a Transwell co-cultured system at a ratio of 1∶10 in the presence or absence of sodium cromoglycate (DSCG) or compound 48/80 (C48/80). The chondrocytes were harvested and lysed for detecting type II collagen expression with ELISA and Western blotting and GAG expression using 1,9 dimethylmethylene blue (DBM). Results After a 24-hour culture, the chondrocytes co-cultured with BMMCs showed similar expression levels of type II collagen and GAG to the control group regardless of the presence of DSCG (P>0.05). Compared with chondrocytes cultured alone or with BMMCs, the co- cultured chondrocytes in the presence of C48/80 showed significantly lower expressions of type II collagen and GAG (P0.05),C48/80组Ⅱ型胶原与GAG含量相对于对照组和BMMCs组显著降低(P0.05)。结论C48/80激活的BMMCs可降低软骨细胞Ⅱ型胶原以及GAG表达。

  8. Altered mRNA Splicing, Chondrocyte Gene Expression and Abnormal Skeletal Development due to SF3B4 Mutations in Rodriguez Acrofacial Dysostosis

    Science.gov (United States)

    Nevarez, Lisette; Pogue, Robert; Krakow, Deborah; Cohn, Daniel H.

    2016-01-01

    The acrofacial dysostoses (AFD) are a genetically heterogeneous group of inherited disorders with craniofacial and limb abnormalities. Rodriguez syndrome is a severe, usually perinatal lethal AFD, characterized by severe retrognathia, oligodactyly and lower limb abnormalities. Rodriguez syndrome has been proposed to be a severe form of Nager syndrome, a non-lethal AFD that results from mutations in SF3B4, a component of the U2 small nuclear ribonucleoprotein particle (U2 snRNP). Furthermore, a case with a phenotype intermediate between Rodriguez and Nager syndromes has been shown to have an SF3B4 mutation. We identified heterozygosity for SF3B4 mutations in Rodriguez syndrome, confirming that the phenotype is a dominant disorder that is allelic with Nager syndrome. The mutations led to reduced SF3B4 synthesis and defects in mRNA splicing, primarily exon skipping. The mutations also led to reduced expression in growth plate chondrocytes of target genes, including the DLX5, DLX6, SOX9, and SOX6 transcription factor genes, which are known to be important for skeletal development. These data provide mechanistic insight toward understanding how SF3B4 mutations lead to the skeletal abnormalities observed in the acrofacial dysostoses. PMID:27622494

  9. Altered mRNA Splicing, Chondrocyte Gene Expression and Abnormal Skeletal Development due to SF3B4 Mutations in Rodriguez Acrofacial Dysostosis.

    Directory of Open Access Journals (Sweden)

    Felipe Marques

    2016-09-01

    Full Text Available The acrofacial dysostoses (AFD are a genetically heterogeneous group of inherited disorders with craniofacial and limb abnormalities. Rodriguez syndrome is a severe, usually perinatal lethal AFD, characterized by severe retrognathia, oligodactyly and lower limb abnormalities. Rodriguez syndrome has been proposed to be a severe form of Nager syndrome, a non-lethal AFD that results from mutations in SF3B4, a component of the U2 small nuclear ribonucleoprotein particle (U2 snRNP. Furthermore, a case with a phenotype intermediate between Rodriguez and Nager syndromes has been shown to have an SF3B4 mutation. We identified heterozygosity for SF3B4 mutations in Rodriguez syndrome, confirming that the phenotype is a dominant disorder that is allelic with Nager syndrome. The mutations led to reduced SF3B4 synthesis and defects in mRNA splicing, primarily exon skipping. The mutations also led to reduced expression in growth plate chondrocytes of target genes, including the DLX5, DLX6, SOX9, and SOX6 transcription factor genes, which are known to be important for skeletal development. These data provide mechanistic insight toward understanding how SF3B4 mutations lead to the skeletal abnormalities observed in the acrofacial dysostoses.

  10. Expression of NGF, Trka and p75 in human cartilage

    Directory of Open Access Journals (Sweden)

    A Gigante

    2009-06-01

    Full Text Available Nerve growth factor (NGF exerts its action through two types of receptor: high-affinity tyrosine kinase A receptor (trkA and low-affinity p75 receptor. NGF has a neurotrophic role in central and peripheral nervous system development, but there is also clear evidence of its involvement in the developing skeleton. The aim of the present immunohistochemical study was to investigate the expression and distribution of NGF, trkA, and p75 in normal cartilaginous tissues from adult subjects: articular and meniscal cartilage of the knee, cartilage from the epiglottis, and intervertebral disc tissue. Detection of NGF mRNA was also performed by in situ hybridization. Immunoreaction for NGF and the two receptors in articular chondrocytes, chondrocyte-like cells of meniscus and annulus fibrosus, and chondrocytes of the epiglottis demonstrated that they are all expressed in hyaline, fibrous and elastic cartilaginous tissues, suggesting that they could be involved in cartilage physio-pathology.

  11. Pilose antler polypeptides promote chondrocyte proliferation via the tyrosine kinase signaling pathway

    Directory of Open Access Journals (Sweden)

    Lin Jian-Hua

    2011-11-01

    Full Text Available Abstract Background Pilose antler polypeptides (PAP have been reported to promote chondrocyte proliferation. However, the underlying mechanism remains unclear. The present study was to investigate the effects of PAP on the proliferation of chondrocytes and its underlying mechanism. Methods Chondrocytes isolated from the knee of Zealand white rabbits were cultured. The second generation chondrocytes were collected and identified using safranin-O staining. The chondrocytes were divided into the following 4 groups including serum-free, PAP, genistein (an inhibitor of tyrosine kinases, and PAP plus genistein group. Cell viability was analyzed using the MTT assay. The cell cycle distribution of the chondrocytes was analyzed by flow cytometry. The expression levels of cyclin A was detected using immunocytochemical staining. Results No significant difference was observed between serum-free and genistein group. Treatment of the cultures with PAP produced a significant dose-dependent increase in cell viability, the percentage proportion of chondrocytes in the S phase and Cyclin A expression as well. However, the promoting effect of PAP on chondrocyte proliferation were dose-dependently inhibited by genistein, whereas genistein alone had no effect on proliferation of isolated chondrocytes. Conclusions The data demonstrate that PAP promotes chondrocyte proliferation with the increased cell number, percentage proportion of chondrocytes in S phase and expression of protein cyclin A via the TK signaling pathway.

  12. Toxicity of antiseptics against chondrocytes: What is best for the cartilage in septic joint surgery?

    OpenAIRE

    Röhner, Eric; Kolar, Paula; Seeger, Joern B.; Arnholdt, Joerg; Thiele, Kathi; Perka, Carsten; Matziolis, Georg

    2011-01-01

    In septic joint surgery, the most frequently used antiseptics are polyhexanide, hydrogen peroxide and taurolidine. The aim of this study was to examine the effects of these antiseptics on viability of human chondrocytes. Our hypothesis was that antiseptics and supplemental irrigation with sodium chloride lavage are less toxic on human chondrocytes than treatment with antiseptics only. Primary human chondrocytes were isolated and cultured from six donated human knee joints. Polyhexanide, hydro...

  13. 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.

  14. 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 col...

  15. Vascular endothelial growth factor activities on osteoarthritic chondrocytes.

    Science.gov (United States)

    Pulsatelli, L; Dolzani, P; Silvestri, T; Frizziero, L; Facchini, A; Meliconi, R

    2005-01-01

    Evaluation of the role of VEGF in cartilage pathophysiology. VEGF release from chondrocytes in the presence of IL-1beta, TGFbeta and IL-10 was detected by immunoassay. VEGF receptor -1 and -2 expression and VEGF ability to modulate caspase -3 and cathepsin B expression were detected by immunohistochemistry on cartilage biopsies and cartilage explants. VEGF effects on chondrocyte proliferation was analysed by a fluorescent dye that binds nucleic acids. VEGF production by osteoartritis (OA) chondrocytes was significantly reduced by IL-1beta while it was increased in the presence of TGFbeta. Cartilage VEGFR-1 immunostaining was significantly downregulated in 'early' OA patients compared to normal controls (NC). VEGFR-2 expression was negligible both in OA and in NC. VEGF decreased the expression of caspase-3 and cathepsin B, whereas it did not affect proliferation. VEGF is able to down-modulate chondrocyte activities related to catabolic events involved in OA cartilage degradation.

  16. Stromal cell-derived factor 1 regulates the actin organization of chondrocytes and chondrocyte hypertrophy.

    Directory of Open Access Journals (Sweden)

    Koichi Murata

    Full Text Available Stromal cell-derived factor 1 (SDF-1/CXCL12/PBSF plays important roles in the biological and physiological functions of haematopoietic and mesenchymal stem cells. This chemokine regulates the formation of multiple organ systems during embryogenesis. However, its roles in skeletal development remain unclear. Here we investigated the roles of SDF-1 in chondrocyte differentiation. We demonstrated that SDF-1 protein was expressed at pre-hypertrophic and hypertrophic chondrocytes in the newly formed endochondral callus of rib fracture as well as in the growth plate of normal mouse tibia by immunohistochemical analysis. Using SDF-1(-/- mouse embryo, we histologically showed that the total length of the whole humeri of SDF-1(-/- mice was significantly shorter than that of wild-type mice, which was contributed mainly by shorter hypertrophic and calcified zones in SDF-1(-/- mice. Actin cytoskeleton of hypertrophic chondrocytes in SDF-1(-/- mouse humeri showed less F-actin and rounder shape than that of wild-type mice. Primary chondrocytes from SDF-1(-/- mice showed the enhanced formation of philopodia and loss of F-actin. The administration of SDF-1 to primary chondrocytes of wild-type mice and SDF-1(-/- mice promoted the formation of actin stress fibers. Organ culture of embryonic metatarsals from SDF-1(-/- mice showed the growth delay, which was recovered by an exogenous administration of SDF-1. mRNA expression of type X collagen in metatarsals and in primary chondrocytes of SDF-1(-/- mouse embryo was down-regulated while the administration of SDF-1 to metatarsals recovered. These data suggests that SDF-1 regulates the actin organization and stimulates bone growth by mediating chondrocyte hypertrophy.

  17. Paracrine effect of chondrocytes on gene expression of matrix metalloproteinases and tissue inhibitors of metalloproteinases in osteoblasts%成骨细胞旁分泌影响软骨细胞中MMPs与TIMPs的表达

    Institute of Scientific and Technical Information of China (English)

    关鹏; 赵伟; 张全有; 谢静; 尹利军; 赵虎成; 许建文

    2015-01-01

    metaloproteinase inhibitors (TIMPs), resulting in extracelular matrix degradation of the articular cartilage, functional disorders of chondrocytes and articular cartilage degeneration. OBJECTIVE:To study the effect of interleukin-1β on migration, MMP and TIMP expression of chondrocytes co-cultured with osteoblast supernatantin vitro. METHODS:There were three groups: chondrocyte monoculture group, osteoblast+chondrocyte group (co-culture group), osteoblast+chondrocyte+interleukin-1β group (interleukin-1β group). Cel scratch test was conducted to observe the migration of chondrocytes within 24 hours. Semi-quantitative PCR test was used to detect the changes in expressions of MMP-1, MMP-2, MMP-3, MMP-9, TIMP-1, TIMP-2, TIMP-3, TIMP-9 in chondrocytes within 24 hours. RESULTS AND CONCLUSION:Compared with the monoculture group, cel migration rate of the other two groups were increased significantly (P< 0.01). Compared with the monoculture group, the gene expressions of MMP-1, MMP-2, MMP-3 and MMP-9 were increased significantly in the coculture group (P < 0. 05); the gene expressions of MMP-1, MMP-3, MMP-9 were increased significantly in the interleukin-1β group (P< 0. 01). Compared with monoculture group, the gene expression of TIMP-1 was increased significantly (P < 0. 01), but the gene expressions of TIMP-3 and TIMP-4 were declined significantly (P < 0. 05) in the other two groups. These findings indicate that co-culture of chondrocytes with osteoblasts can promote chondrocytes migration, enhance gene expression of chondrocytes MMP-1, MMP-2, MMP-3, MMP-9 and regulate gene expression of TIMPs family. Interleukin-1β inhibitsthe migration of chondrocytes co-cultured with osteoblasts and gene expression of TIMPs family.

  18. 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.

  19. Chondrocalcin is internalized by chondrocytes and triggers cartilage destruction via an interleukin-1β-dependent pathway.

    Science.gov (United States)

    Bantsimba-Malanda, Claudie; Cottet, Justine; Netter, Patrick; Dumas, Dominique; Mainard, Didier; Magdalou, Jacques; Vincourt, Jean-Baptiste

    2013-01-01

    Chondrocalcin is among the most highly synthesized polypeptides in cartilage. This protein is released from its parent molecule, type II pro-collagen, after secretion by chondrocytes. A participation of extracellular, isolated chondrocalcin in mineralization was proposed more than 25 years ago, but never demonstrated. Here, exogenous chondrocalcin was found to trigger MMP13 secretion and cartilage destruction ex vivo in human cartilage explants and did so by modulating the expression of interleukin-1β in primary chondrocyte cultures in vitro. Chondrocalcin was found internalized by chondrocytes. Uptake was found mediated by a single 18-mer peptide of chondrocalcin, which does not exhibit homology to any known cell-penetrating peptide. The isolated peptide, when artificially linked as a tetramer, inhibited gene expression regulation by chondrocalcin, suggesting a functional link between uptake and gene expression regulation. At the same time, the tetrameric peptide potentiated chondrocalcin uptake by chondrocytes, suggesting a cooperative mechanism of entry. The corresponding peptide from type I pro-collagen supported identical cell-penetration, suggesting that this property may be conserved among C-propeptides of fibrillar pro-collagens. Structural modeling localized this peptide to the tips of procollagen C-propeptide trimers. Our findings shed light on unexpected function and mechanism of action of these highly expressed proteins from vertebrates.

  20. A hyaluronic acid-based hydrogel enabling CD44-mediated chondrocyte binding and gapmer oligonucleotide release for modulation of gene expression in osteoarthritis

    DEFF Research Database (Denmark)

    Cai, Yunpeng; López-Ruiz, Elena; Wengel, Jesper

    2017-01-01

    Hyaluronic acid (HA) is an attractive biomaterial for osteoarthritis (OA) treatment due to inherent functional and compatibility properties as an endogenous knee joint component. In this work, we describe a HA-based hydrogel with the dual functionality of increased CD44-dependent chondrocyte......, in contrast, the 5:5 and 3:7 hydrogel released 60% and 43% of loaded gapmers, respectively over the same period. A COX-2-specific gapmer designed with maximal chondrocyte gene silencing (~70% silencing efficiency at 500nM compared with a mismatch gapmer sequence) resulted in effective COX-2 silencing over 14...

  1. Elucidation of IL-1/TGF-beta interactions in mouse chondrocyte cell line by genome-wide gene expression

    DEFF Research Database (Denmark)

    Takahashi, N; Rieneck, K; van der Kraan, P M;

    2005-01-01

    To elucidate the antagonism between interleukin-1 (IL-1) and transforming growth factor-beta (TGF-beta) at the gene expression level, as IL-1 and TGF-beta are postulated to be critical mediators of cartilage degeneration/protection in rheumatic diseases.......To elucidate the antagonism between interleukin-1 (IL-1) and transforming growth factor-beta (TGF-beta) at the gene expression level, as IL-1 and TGF-beta are postulated to be critical mediators of cartilage degeneration/protection in rheumatic diseases....

  2. Elucidation of IL-1/TGF-beta interactions in mouse chondrocyte cell line by genome-wide gene expression

    DEFF Research Database (Denmark)

    Takahashi, N; Rieneck, K; van der Kraan, P M;

    2005-01-01

    To elucidate the antagonism between interleukin-1 (IL-1) and transforming growth factor-beta (TGF-beta) at the gene expression level, as IL-1 and TGF-beta are postulated to be critical mediators of cartilage degeneration/protection in rheumatic diseases....

  3. The species-specific regenerative effects of notochordal cell-conditioned medium on chondrocyte-like cells derived from degenerated human intervertebral discs

    NARCIS (Netherlands)

    Bach, F C; de Vries, S A; Krouwels, A; Creemers, L B; Ito, K; Meij, B P; Tryfonidou, M A

    2015-01-01

    During intervertebral disc (IVD) maturation, the main cell type shifts from notochordal cells (NCs) to chondrocyte-like cells (CLCs). NCs secrete factors with regenerative potential, making them an interesting focus for regenerative treatments. During initial development, these strategies preferably

  4. The species-specific regenerative effects of notochordal cell-conditioned medium on chondrocyte-like cells derived from degenerated human intervertebral discs.

    NARCIS (Netherlands)

    Bach, FC; de Vries, S A H; Krouwels, A; Creemers, L B; Ito, K; Meij, B P; Tryfonidou, M A

    2015-01-01

    During intervertebral disc (IVD) maturation, the main cell type shifts from notochordal cells (NCs) to chondrocyte-like cells (CLCs). NCs secrete factors with regenerative potential, making them an interesting focus for regenerative treatments. During initial development, these strategies preferably

  5. Contraction-induced Mmp13 and-14 expression by goat articular chondrocytes in collagen type I but not type II gels

    NARCIS (Netherlands)

    Berendsen, Agnes D.; Vonk, Lucienne A.; Zandieh-Doulabi, Behrouz; Everts, Vincent; Bank, Ruud A.

    2012-01-01

    Collagen gels are promising scaffolds to prepare an implant for cartilage repair but several parameters, such as collagen concentration and composition as well as cell density, should be carefully considered, as they are reported to affect phenotypic aspects of chondrocytes. In this study we

  6. Contraction-induced Mmp13 and-14 expression by goat articular chondrocytes in collagen type I but not type II gels

    NARCIS (Netherlands)

    Berendsen, Agnes D.; Vonk, Lucienne A.; Zandieh-Doulabi, Behrouz; Everts, Vincent; Bank, Ruud A.

    2012-01-01

    Collagen gels are promising scaffolds to prepare an implant for cartilage repair but several parameters, such as collagen concentration and composition as well as cell density, should be carefully considered, as they are reported to affect phenotypic aspects of chondrocytes. In this study we investi

  7. Up-regulated expression of cartilage intermediate-layer protein and ANK in articular hyaline cartilage from patients with calcium pyrophosphate dihydrate crystal deposition disease.

    Science.gov (United States)

    Hirose, Jun; Ryan, Lawrence M; Masuda, Ikuko

    2002-12-01

    Excess accumulation of extracellular inorganic pyrophosphate (ePPi) in aged human cartilage is crucial in calcium pyrophosphate dihydrate (CPPD) crystal formation in cartilage matrix. Two sources of ePPi are ePPi-generating ectoenzymes (NTPPPH) and extracellular transport of intracellular PPi by ANK. This study was undertaken to evaluate the role of NTPPPH and ANK in ePPi elaboration, by investigating expression of NTPPPH enzymes (cartilage intermediate-layer protein [CILP] and plasma cell membrane glycoprotein 1 [PC-1]) and ANK in human chondrocytes from osteoarthritic (OA) articular cartilage containing CPPD crystals and without crystals. Chondrocytes were harvested from knee cartilage at the time of arthroplasty (OA with CPPD crystals [CPPD], n = 8; OA without crystals [OA], n = 10). Normal adult human chondrocytes (n = 1) were used as a control. Chondrocytes were cultured with transforming growth factor beta1 (TGFbeta1), which stimulates ePPi elaboration, and/or insulin-like growth factor 1 (IGF-1), which inhibits ePPi elaboration. NTPPPH and ePPi were measured in the media at 48 hours. Media CILP, PC-1, and ANK were determined by dot-immunoblot analysis. Chondrocyte messenger RNA (mRNA) was extracted for reverse transcriptase-polymerase chain reaction to study expression of mRNA for CILP, PC-1, and ANK. NTPPPH and ANK mRNA and protein were also studied in fresh frozen cartilage. Basal ePPi elaboration and NTPPPH activity in conditioned media from CPPD chondrocytes were elevated compared with normal chondrocytes, and tended to be higher compared with OA chondrocytes. Basal expression of mRNA for CILP (chondrocytes) and ANK (cartilage) was higher in both CPPD chondrocytes and CPPD cartilage extract than in OA or normal samples. PC-1 mRNA was less abundant in CPPD chondrocytes and cartilage extract than in OA chondrocytes and extract, although the difference was not significant. CILP, PC-1, and ANK protein levels were similar in CPPD, OA, and normal chondrocytes

  8. Fisetin inhibits IL-1β-induced inflammatory response in human osteoarthritis chondrocytes through activating SIRT1 and attenuates the progression of osteoarthritis in mice.

    Science.gov (United States)

    Zheng, Wenhao; Feng, Zhenhua; You, Shengban; Zhang, Hui; Tao, Zhenyu; Wang, Quan; Chen, Hua; Wu, Yaosen

    2017-04-01

    Osteoarthritis (OA) is a degenerative joint disease characterized by cartilage degradation and inflammation. Fisetin, a polyphenol extracted from fruits and vegetables, has been reported to have anti-inflammatory effects. Our study aimed to investigate the effect of fisetin on OA both in vitro and in vivo. In vitro, chondrocytes were pretreated with fisetin alone or fisetin combined with sirtinol (an inhibitor of SIRT1) for 2h before IL-1β stimulation. Production of NO, PGE2, TNF-α and IL-6 were evaluated by the Griess reaction and ELISAs. The mRNA (COX-2, iNOS, MMP-3, MMP-13, ADAMTS-5, Sox-9, aggrecan and collagen-II) and protein expression (COX-2, iNOS, MMP-3, MMP-13, ADAMTS-5 and SIRT1) were measured by qRT-PCR and Western blot respectively. Immunofluorescence was used to assess the expression of collagen-II and SIRT1. SIRT1 activity was quantified with SIRT1 fluorometric assay kit. The in vivo effect of fisetin was evaluated by gavage in mice OA models induced by destabilization of the medial meniscus (DMM). We found that fisetin inhibited IL-1β-induced expression of NO, PGE2, TNF-α, IL-6, COX-2, iNOS, MMP-3, MMP-13, ADAMTS-5. Besides, fisetin remarkably decreased IL-1β-induced degradation of Sox-9, aggrecan and collagen-II. Furthermore, fisetin significantly inhibited IL-1β-induced SIRT1 decrease and inactivation. However, the inhibitory effect of fisetin was obvious abolished by sirtinol, suggesting that fisetin exerts anti-inflammatory effects through activating SIRT1. In vivo, fisetin-treated mice exhibited less cartilage destruction and lower OARSI scores. Moreover, fisetin reduced subchondral bone plate thickness and alleviated synovitis. Taken together, these findings indicate that fisetin may be a potential agent in the treatment of OA. Copyright © 2017 Elsevier B.V. All rights reserved.

  9. Human Lacrimal Gland Gene Expression

    Science.gov (United States)

    Aakalu, Vinay Kumar; Parameswaran, Sowmya; Maienschein-Cline, Mark; Bahroos, Neil; Shah, Dhara; Ali, Marwan; Krishnakumar, Subramanian

    2017-01-01

    Background The study of human lacrimal gland biology and development is limited. Lacrimal gland tissue is damaged or poorly functional in a number of disease states including dry eye disease. Development of cell based therapies for lacrimal gland diseases requires a better understanding of the gene expression and signaling pathways in lacrimal gland. Differential gene expression analysis between lacrimal gland and other embryologically similar tissues may be helpful in furthering our understanding of lacrimal gland development. Methods We performed global gene expression analysis of human lacrimal gland tissue using Affymetrix ® gene expression arrays. Primary data from our laboratory was compared with datasets available in the NLM GEO database for other surface ectodermal tissues including salivary gland, skin, conjunctiva and corneal epithelium. Results The analysis revealed statistically significant difference in the gene expression of lacrimal gland tissue compared to other ectodermal tissues. The lacrimal gland specific, cell surface secretory protein encoding genes and critical signaling pathways which distinguish lacrimal gland from other ectodermal tissues are described. Conclusions Differential gene expression in human lacrimal gland compared with other ectodermal tissue types revealed interesting patterns which may serve as the basis for future studies in directed differentiation among other areas. PMID:28081151

  10. Effect of autophagy induced by dexamethasone on senescence in chondrocytes

    Science.gov (United States)

    Xue, Enxing; Zhang, Yu; Song, Bing; Xiao, Jun; Shi, Zhanjun

    2016-01-01

    The aim of the current study was to explore the effects of dexamethasone (DXM) on autophagy and senescence in chondrocytes. Collagen II and aggrecan were examined in normal chondrocytes isolated from Sprague-Dawley rats. Following stimulation with DXM, LysoTracker Red staining, monodansylcadaverine (MDC) staining, green fluorescent protein-red fluorescent protein-light chain 3 (LC3) and western blotting were used to detect autophagy levels in the chondrocytes. Mechanistic target of rapamycin (mTOR) pathway-associated molecules were investigated by western blotting. Cell senescence was analyzed by senescence-associated (SA)-β-galactosidase (β-gal) staining. A dose-dependent increase in the number of autophagic vacuoles was observed in the DXM-treated chondrocytes, as demonstrated by LysoTracker Red and MDC staining. A dose-dependent increase in autophagosome formation was observed in the DXM-treated chondrocytes. Expression of LC3-II and beclin-1 was increased by DXM, in particular in the cells treated with DXM for 4 days. However, P62 expression was reduced as a result of treatment. SA-β-gal staining indicated that DXM increased cell senescence. Notably, DXM-induced cell senescence was exacerbated by the autophagic inhibitor 3-MA. Autophagy induced by DXM protected chondrocytes from senescence, and it is suggested that the mTOR pathway may be involved in the activation of DXM-induced autophagy. PMID:27572674

  11. Functional expression of smooth muscle-specific ion channels in TGF-β1-treated human adipose-derived mesenchymal stem cells

    OpenAIRE

    Park, Won Sun; Heo, Soon Chul; Jeon, Eun Su; Hong, Da Hye; Son, Youn Kyoung; Ko, Jae-Hong; Kim, Hyoung Kyu; Lee, Sun Young; Kim, Jae Ho; Han, Jin

    2013-01-01

    Human adipose tissue-derived mesenchymal stem cells (hASCs) have the power to differentiate into various cell types including chondrocytes, osteocytes, adipocytes, neurons, cardiomyocytes, and smooth muscle cells. We characterized the functional expression of ion channels after transforming growth factor-β1 (TGF-β1)-induced differentiation of hASCs, providing insights into the differentiation of vascular smooth muscle cells. The treatment of hASCs with TGF-β1 dramatically increased the contra...

  12. Catabolic effects of FGF-1 on chondrocytes and its possible role in osteoarthritis.

    Science.gov (United States)

    El-Seoudi, Abdellatif; El Kader, Tarek Abd; Nishida, Takashi; Eguchi, Takanori; Aoyama, Eriko; Takigawa, Masaharu; Kubota, Satoshi

    2017-03-25

    Fibroblast growth factor 1 (FGF-1) is a classical member of the FGF family and is produced by chondrocytes cultured from osteoarthritic patients. Also, this growth factor was shown to bind to CCN family protein 2 (CCN2), which regenerates damaged articular cartilage and counteracts osteoarthritis (OA) in an animal model. However, the pathophysiological role of FGF-1 in cartilage has not been well investigated. In this study, we evaluated the effects of FGF-1 in vitro and its production in vivo by use of an OA model. Treatment of human chondrocytic cells with FGF-1 resulted in marked repression of genes for cartilaginous extracellular matrix components, whereas it strongly induced matrix metalloproteinase 13 (MMP-13), representing its catabolic effects on cartilage. Interestingly, expression of the CCN2 gene was dramatically repressed by FGF-1, which repression eventually caused the reduced production of CCN2 protein from the chondrocytic cells. The results of a reporter gene assay revealed that this repression could be ascribed, at least in part, to transcriptional regulation. In contrast, the gene expression of FGF-1 was enhanced by exogenous FGF-1, indicating a positive feedback system in these cells. Of note, induction of FGF-1 was observed in the articular cartilage of a rat OA model. These results collectively indicate a pathological role of FGF-1 in OA development, which includes an insufficient cartilage regeneration response caused by CCN2 down regulation.

  13. Growth differentiation factor-5 stimulates the growth and anabolic metabolism of articular chondrocytes

    Institute of Scientific and Technical Information of China (English)

    Xu Peng; Guo Xiong; Yao Jianfeng; Zhang Yingang; Klaus von der Mark

    2005-01-01

    Objective: To observe the effect of growth differentiation factor-5 (GDF-5) on the growth and anabolic metabolism of articular chondrocytes. Methods: The articular chondrocytes isolated from rats were treated with various concentrations of rmGDF-5, and the growth of chondrocytes measured by MTT assay, the cellular cartilage matrices formation detected sulfated glycosaminoglycan by Alcian blue staining and type Ⅱ collagen by RT-PCR,the collagen phenotypic expression of chondrocytes detected by immunofluorescence. Results: After 7 days culture,MTT assay showed that GDF-5 enhanced the growth of chondrocytes in a dose-dependent manner, RT-PCR showed that GDF-5 clearly induced the synthesis of type Ⅱ collagen because of the col2a1 mRNA band more and more strong in a dose-dependent. Chondrocytes were cultured with GDF-5 for 14 days, the intensity of Alcian blue staining was greatly enhanced, especially, at a high concentration of 1000ng/ml, and GDF-5 enhanced the accumulation of the Alcian blue-stainable material in a concentration-dependent manner and in a does-dependent manner. Chondrocytes were cultured with GDF-5 for 21 days, immunofluorescent staining of type Ⅱ collagen was clear, the type Ⅰ and X collagen were negative. Conclusion: GDF-5 enhanced the growth of mature articular chondrocytes, and stimulated the cellular cartilage matrices formation, but did not change the collagen phenotypic expression of chondrocytes in mono-layer culture.

  14. The properties of bioengineered chondrocyte sheets for cartilage regeneration

    Directory of Open Access Journals (Sweden)

    Ota Naoshi

    2009-03-01

    Full Text Available Abstract Background Although the clinical results of autologous chondrocyte implantation for articular cartilage defects have recently improved as a result of advanced techniques based on tissue engineering procedures, problems with cell handling and scaffold imperfections remain to be solved. A new cell-sheet technique has been developed, and is potentially able to overcome these obstacles. Chondrocyte sheets applicable to cartilage regeneration can be prepared with this cell-sheet technique using temperature-responsive culture dishes. However, for clinical application, it is necessary to evaluate the characteristics of the cells in these sheets and to identify their similarities to naive cartilage. Results The expression of SOX 9, collagen type 2, 27, integrin α10, and fibronectin genes in triple-layered chondrocyte sheets was significantly increased in comparison to those in conventional monolayer culture and in a single chondrocyte sheet, implying a nature similar to ordinary cartilage. In addition, immunohistochemistry demonstrated that collagen type II, fibronectin, and integrin α10 were present in the triple-layered chondrocyte sheets. Conclusion The results of this study indicate that these chondrocyte sheets with a consistent cartilaginous phenotype and adhesive properties may lead to a new strategy for cartilage regeneration.

  15. Tissue engineering applications: cartilage lesions repair by the use of autologous chondrocytes

    Directory of Open Access Journals (Sweden)

    L. De Franceschi

    2011-09-01

    Full Text Available Promising new therapies based on tissue engineering have been recently developed for cartilage repair. The association of biomaterials with autologous chondrocytes expanded in vitro can represent a useful tool to regenerate this tissue. The scaffolds utilised in such therapeutical applications should provide a pre-formed three-dimensional shape, prevent cells from floating out of the defect, have sufficient mechanical strength, facilitate uniform spread of cells and stimulate the phenotype of transplanted cells. Hyaff®-11 is a hyaluronic-acid based biodegradable polymer, that has been shown to provide successful cell carrier for tissue-engineered repair. From our findings we can state that human chondrocytes seeded on Hyaff®-11 are able to maintain in vitro the characteristic of differentiated cells, expressing and producing collagen type II and aggrecan which are the main markers of cartilage phenotype, down-regulating collagen type I. Moreover, it seems to be a useful scaffold for cartilage repair both in animal models and clinical trials in humans, favouring the formation of a hyaline-like tissue. In the light of these data, we can hypothesise, for the future, the use of autologous chondrocyte transplantation together with gene therapy as a treatment for rheumatic diseases such as osteoarthritis.

  16. OA cartilage derived chondrocytes encapsulated in poly(ethylene glycol) diacrylate (PEGDA) for the evaluation of cartilage restoration and apoptosis in an in vitro model.

    Science.gov (United States)

    Musumeci, G; Loreto, C; Carnazza, M L; Strehin, I; Elisseeff, J

    2011-10-01

    Osteoarthritis (OA) is characterized by cartilage attrition, subchondral bone remodeling, osteophyte formation and synovial inflammation. Perturbed homeostasis caused by inflammation, oxidative stress, mitochondrial dysfunction and proapoptotic/antiapoptotic dysregulation is known to impair chondrocyte survival in joint microenvironments and contribute to OA pathogenesis. However, the molecular mechanisms underlying the programmed cell death (apoptosis) of chondral cells are not yet well defined. The present study was conducted to evaluate apoptosis of chondrocytes from knee articular cartilage of patients with OA. The aim of this study was to investigate and compare the apoptosis through the expression of caspase-3 in tissue explants, in cells cultured in monolayer, and in cells encapsulated in a hydrogel (PEGDA) scaffold. Chondrocytes were also studied following cell isolation and encapsulation in poly(ethylene glycol) diacrylate (PEGDA) hydrogels. Specifically, articular cartilage specimens were assessed by histology (Hematoxlyn and Eosin) and histochemistry (Safranin-O and Alcian Blue). The effector of apoptosis caspase-3 was studied through immunohistochemistry, immunocytochemistry and immunofluorescence. DNA strand breaks were evaluated in freshly isolated chondrocytes from human OA cartilage using the TUNEL assay, and changes in nuclear morphology of apoptotic cells were detected by staining with Hoechst 33258. The results showed an increased expression of caspase-3 in tissue explants, in pre-confluent cells and after four passages in culture, and a decreased expression of caspase-3 comparable to control cartilage in cells encapsulated in hydrogels (PEGDA) after 5 weeks in culture. The freshly isolated chondrocytes were TUNEL positive. The chondrocytes after 5 weeks of culture in hydrogels (PEGDA) showed the formation of new hyaline cartilage with increased cell growth, cellular aggregations and extracellular matrix (ECM) production. This is of particular

  17. MicroRNA-34a affects chondrocyte apoptosis and proliferation by targeting the SIRT1/p53 signaling pathway during the pathogenesis of osteoarthritis.

    Science.gov (United States)

    Yan, Shiju; Wang, Meng; Zhao, Jian; Zhang, Hongtao; Zhou, Chengpei; Jin, Lei; Zhang, Yinglong; Qiu, Xiuchun; Ma, Baoan; Fan, Qingyu

    2016-07-01

    Osteoarthritis (OA) is the most prevalent degenerative joint disease with multifactorial etiology caused by risk factors such as ageing, obesity and trauma. Previously, it was reported that the inhibition of microRNA-34a (miR-34a) may reduce rat chondrocyte apoptosis induced by IL-1β, whereas the molecular mechanism and the role of miR-34a in human chondrocyte as well as in OA progression remains to be determined. In the current study, using MTT, luciferase reporter assays and western blot analysis we identified that miR-34a was upregulated while silent information regulator 1 (SIRT1) was inhibited in chondrocytes from 12 OA patients compared with healthy chondrocytes from 10 trauma amputees. Overexpression of miR-34a promoted apoptosis and inhibited cell proliferation in human chondrocytes. Transfection with miR-34a mimic inhibited SIRT1 expression, which attenuated the deacetylation of p53, leading to the upregulation of Bax and downregulation of Bcl-2. Furthermore, results from the western blot analysis and luciferase reporter assay demonstrated that SIRT1 was directly regulated by miR-34a in human chondrocytes. A rat model of OA was induced through anterior cruciate ligament transection and medial meniscus resection (ACLT+MMx). The results showed that the intra‑articular injection of lentiviral vector encoding anti-miR‑34a sequence effectively ameliorated the progression of OA. The results suggest that miR-34a has a crucial role in the pathogenesis of OA through direct regulation of the SIRT1/p53 signaling pathway and serves as a potential therapeutic target of OA.

  18. The Wnt/beta-catenin pathway interacts differentially with PTHrP signaling to control chondrocyte hypertrophy and final maturation.

    Directory of Open Access Journals (Sweden)

    Xizhi Guo

    Full Text Available Sequential proliferation, hypertrophy and maturation of chondrocytes are required for proper endochondral bone development and tightly regulated by cell signaling. The canonical Wnt signaling pathway acts through beta-catenin to promote chondrocyte hypertrophy whereas PTHrP signaling inhibits it by holding chondrocytes in proliferating states. Here we show by genetic approaches that chondrocyte hypertrophy and final maturation are two distinct developmental processes that are differentially regulated by Wnt/beta-catenin and PTHrP signaling. Wnt/beta-catenin signaling regulates initiation of chondrocyte hypertrophy by inhibiting PTHrP signaling activity, but it does not regulate PTHrP expression. In addition, Wnt/beta-catenin signaling regulates chondrocyte hypertrophy in a non-cell autonomous manner and Gdf5/Bmp signaling may be one of the downstream pathways. Furthermore, Wnt/beta-catenin signaling also controls final maturation of hypertrophic chondrocytes, but such regulation is PTHrP signaling-independent.

  19. Elastic cartilage reconstruction by transplantation of cultured hyaline cartilage-derived chondrocytes.

    Science.gov (United States)

    Mizuno, M; Takebe, T; Kobayashi, S; Kimura, S; Masutani, M; Lee, S; Jo, Y H; Lee, J I; Taniguchi, H

    2014-05-01

    Current surgical intervention of craniofacial defects caused by injuries or abnormalities uses reconstructive materials, such as autologous cartilage grafts. Transplantation of autologous tissues, however, places a significant invasiveness on patients, and many efforts have been made for establishing an alternative graft. Recently, we and others have shown the potential use of reconstructed elastic cartilage from ear-derived chondrocytes or progenitors with the unique elastic properties. Here, we examined the differentiation potential of canine joint cartilage-derived chondrocytes into elastic cartilage for expanding the cell sources, such as hyaline cartilage. Articular chondrocytes are isolated from canine joint, cultivated, and compared regarding characteristic differences with auricular chondrocytes, including proliferation rates, gene expression, extracellular matrix production, and cartilage reconstruction capability after transplantation. Canine articular chondrocytes proliferated less robustly than auricular chondrocytes, but there was no significant difference in the amount of sulfated glycosaminoglycan produced from redifferentiated chondrocytes. Furthermore, in vitro expanded and redifferentiated articular chondrocytes have been shown to reconstruct elastic cartilage on transplantation that has histologic characteristics distinct from hyaline cartilage. Taken together, cultured hyaline cartilage-derived chondrocytes are a possible cell source for elastic cartilage reconstruction. Crown Copyright © 2014. Published by Elsevier Inc. All rights reserved.

  20. Enrichment of committed human nucleus pulposus cells expressing chondroitin sulfate proteoglycans under alginate encapsulation.

    Science.gov (United States)

    Sun, Y; Lv, M; Zhou, L; Tam, V; Lv, F; Chan, D; Wang, H; Zheng, Z; Cheung, K M C; Leung, V Y L

    2015-07-01

    Intervertebral disc (IVD) degeneration is associated with a malfunction of the nucleus pulposus (NP). Alginate culturing provides a favorable microenvironment for the phenotypic maintenance of chondrocyte-like NP cells. However, NP cells are recently evidenced to present heterogeneous populations, including progenitors, fibroblastic cells and primitive NP cells. The aim of this study is to profile the phenotypic changes of distinct human NP cells populations and describe the dynamic expression of chondroitin sulfate glycosaminoglycans (CS-GAGs) in extended alginate encapsulation. Non-degenerated (ND-NPC) and degenerated (D-NPC) NP cells were expanded in monolayers, and subject to 28-day culture in alginate after serial passaging. CS-GAG compositional expression in monolayer-/alginate-cultured NP cells was evaluated by carbohydrate electrophoresis. Cellular phenotypic changes were assessed by immunologic detection and gene expression analysis. Relative to D-NPC, ND-NPC displayed remarkably higher expression levels of chondroitin-4-sulfate GAGs over the 28-day culture. Compared with monolayer culture, ND-NPC showed increased NP marker expression of KRT18, KRT19, and CDH2, as well as chondrocyte markers SOX9 and MIA in alginate culture. In contrast, expression of fibroblastic marker COL1A1, COL3A1, and FN1 were reduced. Interestingly, ND-NPC showed a loss of Tie2+ but gain in KRT19+/CD24+ population during alginate culture. In contrast, D-NPC showed more consistent expression levels of NP surface markers during culture. We demonstrate for the first time that extended alginate culture selectively enriches the committed NP cells and favors chondroitin-4-sulfate proteoglycan production. These findings suggest its validity as a model to investigate IVD cell function. Copyright © 2015 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.

  1. Fibroblast growth factor-1 is a mesenchymal stromal cell-secreted factor stimulating proliferation of osteoarthritic chondrocytes in co-culture

    NARCIS (Netherlands)

    Wu, Ling; Leijten, Jeroen; Blitterswijk, van Clemens A.; Karperien, Marcel

    2013-01-01

    Previously, we showed that mesenchymal stromal cells (MSCs) in co-culture with primary chondrocytes secrete soluble factors that increase chondrocyte proliferation. The objective of this study is to identify these factors. Human primary chondrocytes (hPCs) isolated from late-stage osteoarthritis pat

  2. Autologous chondrocyte implantation in children and adolescents

    DEFF Research Database (Denmark)

    Schmal, H; Pestka, J M; Salzmann, G

    2013-01-01

    PURPOSE: Autologous chondrocyte implantation (ACI) is a well-established treatment method for cartilage defects in knees. Age-related grouping was based on expression data of cartilage-specific markers. Specificities of ACI in the different populations were analysed. METHODS: Two hundred and sixt...... months after the operation. CONCLUSIONS: Age-related expression of cartilage-specific markers allows definition of adolescents in cartilage regenerating surgery. Chondromalacia in these patients is mainly caused by OCD or trauma. LEVEL OF EVIDENCE: Case series, Level IV....

  3. Chondrocyte hypertrophy and osteoarthritis: role in initiation and progression of cartilage degeneration?

    NARCIS (Netherlands)

    Kraan, P.M. van der; Berg, W.B. van den

    2012-01-01

    OBJECTIVE: To review the literature on the role and regulation of chondrocyte terminal differentiation (hypertrophy-like changes) in osteoarthritis (OA) and to integrate this in a conceptual model of primary OA development. METHODS: Papers investigating chondrocyte terminal differentiation in human

  4. Chondrocyte hypertrophy and osteoarthritis: role in initiation and progression of cartilage degeneration?

    NARCIS (Netherlands)

    Kraan, P.M. van der; Berg, W.B. van den

    2012-01-01

    OBJECTIVE: To review the literature on the role and regulation of chondrocyte terminal differentiation (hypertrophy-like changes) in osteoarthritis (OA) and to integrate this in a conceptual model of primary OA development. METHODS: Papers investigating chondrocyte terminal differentiation in human

  5. Autophagy protects end plate chondrocytes from intermittent cyclic mechanical tension induced calcification.

    Science.gov (United States)

    Xu, Hong-guang; Yu, Yun-fei; Zheng, Quan; Zhang, Wei; Wang, Chuang-dong; Zhao, Xiao-yn; Tong, Wen-xue; Wang, Hong; Liu, Ping; Zhang, Xiao-ling

    2014-09-01

    Calcification of end plate chondrocytes is a major cause of intervertebral disc (IVD) degeneration. However, the underlying molecular mechanism of end plate chondrocyte calcification is still unclear. The aim of this study was to clarify whether autophagy in end plate chondrocytes could protect the calcification of end plate chondrocytes. Previous studies showed that intermittent cyclic mechanical tension (ICMT) contributes to the calcification of end plate chondrocytes in vitro. While autophagy serves as a cell survival mechanism, the relationship of autophagy and induced end plate chondrocyte calcification by mechanical tension in vitro is unknown. Thus, we investigated autophagy, the expression of the autophagy genes, Beclin-1 and LC3, and rat end plate chondrocyte calcification by ICMT. The viability of end plate chondrocytes was examined using the LIVE/DEAD viability/cytotoxicity kit. The reverse transcription-polymerase chain reaction and western blotting were used to detect the expression of Beclin-1; LC3; type I, II and X collagen; aggrecan; and Sox-9 genes. Immunofluorescent and fluorescent microscopy showed decreased autophagy in the 10- and 20-day groups loaded with ICMT. Additionally, Alizarin red and alkaline phosphatase staining detected the palpable calcification of end plate chondrocytes after ICMT treatment. We found that increased autophagy induced by short-term ICMT treatment was accompanied by an insignificant calcification of end plate chondrocytes. To the contrary, the suppressive autophagy inhibited by long-term ICMT was accompanied by a more significant calcification. The process of calcification induced by ICMT was partially resisted by increased autophagy activity induced by rapamycin, implicating that autophagy may prevent end plate chondrocyte calcification.

  6. Use of non-degenerate human osteochondral tissue and confocal laser scanning microscopy for the study of chondrocyte death at cartilage surgery

    Directory of Open Access Journals (Sweden)

    Huntley J. S.

    2005-02-01

    Full Text Available Although autologous osteochondral grafting has been widely applied in humans, most in vitro work has been on animal models. The aims of this study were to: (i elaborate a full thickness human femoral condylar model using discard material from knee arthroplasty resections, and (ii use this model to assess chondrocyte viability in response to surgical trauma. Homogeneous regions of human lateral femoral condyle bone-cartilage were procured from knee arthroplasty resections. These were graded prospectively, firstly by visual inspection, and then by confocal laser scanning microscopy (CLSM. Samples were subjected to tests of tissue hydration, including analysis of water content and swelling after excision from underlying bone. Surgical cuts were made in explants that were macroscopically and microscopically normal (i.e. Grade 0. Associated margins of death were assessed from both transverse and surface perspectives. Thirty-nine samples were obtained from anterior and distal femoral cuts (16 knees from 13 patients for (1 macroscopic grading, (2 microscopic analysis, (3 analyses of water content as cut and on re-equilibration after excision from bone. Thirteen were Grade 0 on macroscopic viewing - however one showed fibrillation on microscopy and was therefore reassigned Grade 1. Grade 0 tissue had a water content of 73.8±0.38%, in agreement with control values from the literature. Tissues of Grades 2 and 3 were significantly (P=0.03, and P=0.004 more hydrated (76.0±0.59%, 76.7±0.99% than Grade 0 tissue. Grade 0 tissue from the anterior cut did not swell significantly following excision from subchondral bone. However Grade 0 tissue from the distal cut showed a small but statistically significant (P=0.019 increase in water content (1.68±0.39% following excision. With increasing grade there was increased tendency to swell off the bone (P<0.0001. Transverse imaging showed that the Acufex MP surgical harvester caused a greater margin of cell death

  7. 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.

  8. Growth Differentiation Factor-5 Stimulates the Growth and Anabolic Metabolism of Articular Chondrocytes

    Institute of Scientific and Technical Information of China (English)

    Xu Peng; Yao Jianfeng; Guo Xiong; Zhang Yingang; Klaus von der Mark

    2009-01-01

    Objective: To observe the effect of growth differentiation factor-5 (GDF-5) on the growth and anabolic metabolism of articular chondrocytes. Methods: The articular chondrocytes isolated from rats were treated with various concentrations of rmGDF-5, and the growth of chondrocytes measured by MTr assay, the cellular cartilage matrices formation detected sulfated glycosaminoglycan by Alcian blue staining and type 11 collagen by RT-PCR, the collagen phenotypic expression of chondrocytes detected by immunofluorescence. Results: After 7 days culture, MTF assay showed that GDF-5 enhanced the growth of ehondrocytes in a dose-dependent manner, RT-PCR showed that GDF-5 clearly induced the synthesis of type Ⅱ collagen because of the colal mRNA band more and more strong in a dose-dependent. Chondrocytes were cultured with GDF-5 for 14 days, the intensity of Alcian blue staining was gready enhanced, especially, at a high concentration of 1000ng/ml, and GDF-5 enhanced the accumulation of the Alcian blue-stainable material in a concentration-dependent manner and in a does-dependent manner. Chondrocytes were cultured with GDF-5 for 21 days, immunofluorescent staining of type Ⅱ collagen was clear, the type Ⅰ and Ⅹ collagen were negative. Conclusion: GDF-5 enhanced the growth of mature articular chon-drocytes, and stimulated the cellular cartilage matrices formation, but did not change the collagen phenotypic ex-pression of chondrocytes in mono-layer culture.

  9. 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

  10. 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.

  11. Epigenetic Regulation of Chondrocyte Catabolism and Anabolism in Osteoarthritis.

    Science.gov (United States)

    Kim, Hyeonkyeong; Kang, Donghyun; Cho, Yongsik; Kim, Jin-Hong

    2015-08-01

    Osteoarthritis (OA) is one of the most prevalent forms of joint disorder, associated with a tremendous socioeconomic burden worldwide. Various non-genetic and lifestyle-related factors such as aging and obesity have been recognized as major risk factors for OA, underscoring the potential role for epigenetic regulation in the pathogenesis of the disease. OA-associated epigenetic aberrations have been noted at the level of DNA methylation and histone modification in chondrocytes. These epigenetic regulations are implicated in driving an imbalance between the expression of catabolic and anabolic factors, leading eventually to osteoarthritic cartilage destruction. Cellular senescence and metabolic abnormalities driven by OA-associated risk factors appear to accompany epigenetic drifts in chondrocytes. Notably, molecular events associated with metabolic disorders influence epigenetic regulation in chondrocytes, supporting the notion that OA is a metabolic disease. Here, we review accumulating evidence supporting a role for epigenetics in the regulation of cartilage homeostasis and OA pathogenesis.

  12. 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.

  13. The Biological Effects of Sex Hormones on Rabbit Articular Chondrocytes from Different Genders

    Directory of Open Access Journals (Sweden)

    Shwu Jen Chang

    2014-01-01

    Full Text Available The aim of this study was to investigate the biological effects of sex hormones (17β-estradiol and testosterone on rabbit articular chondrocytes from different genders. We cultured primary rabbit articular chondrocytes from both genders with varying concentration of sex hormones. We evaluate cell proliferation and biochemical functions by MTT and GAG assay. The chondrocyte function and phenotypes were analyzed by mRNA level using RT-PCR. Immunocytochemical staining was also used to evaluate the generation of collagen-II. This study demonstrated that 17β-estradiol had greater positive regulation on the biological function and gene expressions of articular chondrocytes than testosterone, with the optimal concentrations of 10−6 and 10−7 M, particularly for female chondrocytes.

  14. Deciphering chondrocyte behaviour in matrix-induced autologous chondrocyte implantation to undergo accurate cartilage repair with hyaline matrix.

    Science.gov (United States)

    Demoor, M; Maneix, L; Ollitrault, D; Legendre, F; Duval, E; Claus, S; Mallein-Gerin, F; Moslemi, S; Boumediene, K; Galera, P

    2012-06-01

    Since the emergence in the 1990s of the autologous chondrocytes transplantation (ACT) in the treatment of cartilage defects, the technique, corresponding initially to implantation of chondrocytes, previously isolated and amplified in vitro, under a periosteal membrane, has greatly evolved. Indeed, the first generations of ACT showed their limits, with in particular the dedifferentiation of chondrocytes during the monolayer culture, inducing the synthesis of fibroblastic collagens, notably type I collagen to the detriment of type II collagen. Beyond the clinical aspect with its encouraging results, new biological substitutes must be tested to obtain a hyaline neocartilage. Therefore, the use of differentiated chondrocytes phenotypically stabilized is essential for the success of ACT at medium and long-term. That is why researchers try now to develop more reliable culture techniques, using among others, new types of biomaterials and molecules known for their chondrogenic activity, giving rise to the 4th generation of ACT. Other sources of cells, being able to follow chondrogenesis program, are also studied. The success of the cartilage regenerative medicine is based on the phenotypic status of the chondrocyte and on one of its essential component of the cartilage, type II collagen, the expression of which should be supported without induction of type I collagen. The knowledge accumulated by the scientific community and the experience of the clinicians will certainly allow to relief this technological challenge, which influence besides, the validation of such biological substitutes by the sanitary authorities. Copyright © 2012 Elsevier Masson SAS. All rights reserved.

  15. 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.

  16. Cell manipulation in autologous chondrocyte implantation: from research to cleanroom.

    Science.gov (United States)

    Roseti, Livia; Serra, Marta; Tigani, Domenico; Brognara, Irene; Lopriore, Annamaria; Bassi, Alessandra; Fornasari, Pier Maria

    2008-04-01

    In the field of orthopaedics, autologous chondrocyte implantation is a technique currently used for the regeneration of damaged articular cartilage. There is evidence of the neo-formation of tissue displaying characteristics similar to hyaline cartilage. In vitro chondrocyte manipulation is a crucial phase of this therapeutic treatment consisting of different steps: cell isolation from a cartilage biopsy, expansion in monolayer culture and growth onto a three-dimensional biomaterial to implant in the damaged area. To minimise the risk of in vitro cell contamination, the manipulation must be performed in a controlled environment such as a cleanroom. Moreover, the choice of reagents and raw material suitable for clinical use in humans and the translation of research protocols into standardised production processes are important. In this study we describe the preliminary results obtained by the development of chondrocyte manipulation protocols (isolation and monolayer expansion) in cleanrooms for the application of autologous implantation.

  17. Cultured articular chondrocytes sheets for partial thickness cartilage defects utilizing temperature-responsive culture dishes

    Directory of Open Access Journals (Sweden)

    N Kaneshiro

    2007-05-01

    Full Text Available The extracellular matrix (ECM of articular cartilage has several functions that are unique to joints. Although a technique for transplanting cultured chondrocytes has already been introduced, it is difficult to collect intact ECM when using enzymes to harvest samples. Temperature-responsive culture dishes have already been clinically applied in the fields of myocardial and corneal transplantation. Earlier studies have shown that a sheet of cultured cells with intact ECM and adhesive factors can be harvested using such culture dishes, which allow the surface properties of the dish to be reversibly altered by changing the temperature. Human chondrocytes were subjected to enzymatic digestion and then were seeded in temperature-responsive culture dishes. A sheet of chondrocytes was harvested by only reducing the temperature after the cultured cells reached confluency. A real-time PCR analysis of the chondrocyte sheets confirmed that type II collagen, aggrecan, and fibronectin were present. These results suggested that, although chondrocytes undergo dedifferentiation in a monolayer culture, multilayer chondrocyte sheets grown in a similar environment to that of three-dimensional culture may be able to maintain a normal phenotype. A histological examination suggested that multilayer chondrocyte sheets could thus prevent the loss of proteoglycans because the area covered by the sheets was well stained by safranin-O. The present experiments suggested that temperature-responsive culture dishes are useful for obtaining cultured chondrocytes, which may then be clinically employed as a substitute for periosteal patches because such sheets can be applied without a scaffold.

  18. Effects of Zinc on the Expression of ZnT-7 in Growth Plate Chondrocytes%锌对大鼠生长板软骨细胞ZnT-7表达的影响

    Institute of Scientific and Technical Information of China (English)

    初立伟; 杨茂伟; 于江; 王铜浩; 李亚伦

    2009-01-01

    目的 研究锌对大鼠生长板软骨细胞锌转移体-7(ZnT-7)表达的影响及其规律.方法 原代培养Wistar大鼠肋生长板软骨细胞,不同浓度的锌螯合剂TPEN分别处理培养生长板软骨细胞12 h.免疫组化检测生长板软骨细胞特异性Ⅱ型胶原表达.免疫荧光技术对ZnT-7进行定位研究,实时RT-PCR和Western blot检测znT-7表达.结果 Ⅱ型胶原免疫组化检测阳性.ZnT-7在生长板软骨细胞中定位于高尔基体上;Western blot和RT-PCR结果显示;5 μmol/L(TPEN)组与对照组相比略有增高,而10 μmol/L、20 μmol/L组与对照组相比逐渐降低.结论 ZnT-7定位于高尔基体上,同时在缺锌的条件下其对锌离子稳定有一定的代偿作用.%Objective To study the effects of zinc on the expression of zinc transporter-7( ZnT-7) in the proliferation of the rat growth plate chondrocytes. Methods Growth plate chondrocytes were isolated from rih cartilage of Wistar rat. The cell3 were treated with zinc chelating agent N, N, N', N'-tetrakis (2-pyridylmethyl )ethylene-diamine (TPEN) of different concentration (0,5,10 and 20 μmol/L) for 12 horns. The expression of rat growth plate chondrocytes specificity collagen type Ⅱ was detected by immunohisloehemistiy. The localization of ZnT-7 was checked by immunofluorescent staining. Real-time RT-PCR and Western blot were used to measure the expression level of ZNT-7 in the cell. Results The result of the immunofluorescence showed that ZnT-7 located in the Golgi apparatus. The expression level of ZnT-7 was slightly higher in the cells treated with 5 μ-mol/L TPEN than the control group, while it was lower in the cells treated with 10 or 20 μmol/L TPEN than the control group. Conclusion ZnT-7 locates in Golgi apparatus and maintains the zinc ion stabilization in the condition of the zinc depletion.

  19. Suitability of porcine chondrocyte micromass culture to model osteoarthritis in vitro.

    Science.gov (United States)

    Schlichting, Niels; Dehne, Tilo; Mans, Karsten; Endres, Michaela; Stuhlmüller, Bruno; Sittinger, Michael; Kaps, Christian; Ringe, Jochen

    2014-07-07

    In vitro tissue models are useful tools for the development of novel therapy strategies in cartilage repair and care. The limited availability of human primary tissue and high costs of animal models hamper preclinical tests of innovative substances and techniques. In this study we tested the potential of porcine chondrocyte micromass cultures to mimic human articular cartilage and essential aspects of osteoarthritis (OA) in vitro. Primary chondrocytes were enzymatically isolated from porcine femoral condyles and were maintained in 96-multiwell format to establish micromass cultures in a high-throughput scale. Recombinant porcine tumor necrosis factor alpha (TNF-α) was used to induce OA-like changes documented on histological (Safranin O, collagen type II staining), biochemical (hydroxyproline assay, dimethylmethylene blue method), and gene expression level (Affymetrix porcine microarray, real time PCR) and were compared with published data from human articular cartilage and human micromass cultures. After 14 days in micromass culture, porcine primary chondrocytes produced ECM rich in proteoglycans and collagens. On gene expression level, significant correlations of detected genes with porcine cartilage (r = 0.90), human cartilage (r = 0.71), and human micromass culture (r = 0.75) were observed including 34 cartilage markers such as COL2A1, COMP, and aggrecan. TNF-α stimulation led to significant proteoglycan (-75%) and collagen depletion (-50%). Comparative expression pattern analysis revealed the involvement of catabolic enzymes (MMP1, -2, -13, ADAM10), chemokines (IL8, CCL2, CXCL2, CXCL12, CCXL14), and genes associated with cell death (TNFSF10, PMAIPI, AHR) and skeletal development (GPNMB, FRZB) including transcription factors (WIF1, DLX5, TWIST1) and growth factors (IGFBP1, -3, TGFB1) consistent with published data from human OA cartilage. Expression of genes related to cartilage ECM formation (COL2A1, COL9A1, COMP, aggrecan) as well as hypertrophic bone

  20. Indian hedgehog signals independently of PTHrP to promote chondrocyte hypertrophy.

    Science.gov (United States)

    Mak, Kinglun Kingston; Kronenberg, Henry M; Chuang, Pao-Tien; Mackem, Susan; Yang, Yingzi

    2008-06-01

    Chondrocyte hypertrophy is an essential process required for endochondral bone formation. Proper regulation of chondrocyte hypertrophy is also required in postnatal cartilage homeostasis. Indian hedgehog (Ihh) and PTHrP signaling play crucial roles in regulating the onset of chondrocyte hypertrophy by forming a negative feedback loop, in which Ihh signaling regulates chondrocyte hypertrophy by controlling PTHrP expression. To understand whether there is a PTHrP-independent role of Ihh signaling in regulating chondrocyte hypertrophy, we have both activated and inactivated Ihh signaling in the absence of PTHrP during endochondral skeletal development. We found that upregulating Ihh signaling in the developing cartilage by treating PTHrP(-/-) limb explants with sonic hedgehog (Shh) protein in vitro, or overexpressing Ihh in the cartilage of PTHrP(-/-) embryos or inactivating patched 1 (Ptch1), a negative regulator of hedgehog (Hh) signaling, accelerated chondrocyte hypertrophy in the PTHrP(-/-) embryos. Conversely, when Hh signaling was blocked by cyclopamine or by removing Smoothened (Smo), a positive regulator of Hh signaling, chondrocyte hypertrophy was delayed in the PTHrP(-/-) embryo. Furthermore, we show that upregulated Hh signaling in the postnatal cartilage led to accelerated chondrocyte hypertrophy during secondary ossification, which in turn caused reduction of joint cartilage. Our results revealed a novel role of Ihh signaling in promoting chondrocyte hypertrophy independently of PTHrP, which is particularly important in postnatal cartilage development and homeostasis. In addition, we found that bone morphogenetic protein (Bmp) and Wnt/beta-catenin signaling in the cartilage may both mediate the effect of upregulated Ihh signaling in promoting chondrocyte hypertrophy.

  1. Effects of adenovirus-mediated basic fibroblast growth factor and the related cytokines gene transfection on human osteoarthritis chondrocytes in vitro%碱性成纤维细胞生长因子及相关细胞因子转染对人骨关节炎软骨细胞的作用

    Institute of Scientific and Technical Information of China (English)

    陈彪; 陈廖斌; 秦俊; Jaques Magdalou; 汪晖

    2010-01-01

    目的 探讨腺病毒介导的人碱性成纤维细胞生长因子(bFGF)单独及与白细胞介素-1受体拮抗蛋白(IL-1Ra)和(或)胰岛素样生长因子(IGF)-1共同转染人骨关节炎(OA)软骨细胞后对软骨细胞的影响.方法 采用单独编码人bFGF的重组腺病毒载体或多重组合的重组腺病毒载体转染单层培养的人OA软骨细胞.6 d后分别检测培养上清液中目的 基因表达和糖胺聚糖(GAG)含量.四甲基偶氮唑蓝(MTT)法及流式细胞术分析软骨细胞的增殖及凋亡.甲苯胺蓝染色及Ⅱ型胶原免疫组织化学染色观察软骨细胞基质的合成.免疫印迹法检测Ⅱ型胶原、基质金属蛋白酶(MMP)-3及其抑制剂-1(TIMP-1)的表达.采用单因素方差分析,并进行组间两两比较.结果 各基因转染后,细胞上清液日的基因表达与OA对照组相比明显增高(P<0.05 ). bFGF单独转染可促进软骨细胞增殖,增加Ⅱ型胶原和蛋白多糖的合成(P<0.05).与bFGF单独转染相比,联合IL-1Ra和(或)IGF-1共同转染后,可降低软骨细胞的凋亡率[分别为:(26.1±1.6)%、(19.4±1.0)%、(18.4±1.1)%、(13.9±1.8)%,P<0.05],进一步增加了软骨基质的生物合成(P<0.05).同时,抑制了MMP-3的表达,增加了TIMP-1的表达.结论 腺病毒介导的bFGF转染入OA软骨细胞可促进细胞增殖,增加基质的合成.与IL-1Ra和IGF-1共转染后可发挥协同作用,进一步增加基质合成;同时,抑制了基质的降解.%Objective To investigate the effect of recombinant adenovirus-mediated basic fibroblast growth factor (bFGF),interleukin-1 receptor antagonist protein (IL-Ra) and insulin-like growth factor(IGF)-1 gene transfection on human osteoarthritis chondrocytes.Methods Monolayer cultures of human osteoarthritis chondrocytes were transfected with recombinant adenovirus carrying genes encoding the following cytokines: human bFGF,IL-1Ra and IGF-1.Six days later,levels of gene expression and glycosaminoglycan (GAG) in culture

  2. Antiangiogenic treatment delays chondrocyte maturation and bone formation during limb skeletogenesis.

    Science.gov (United States)

    Yin, Melinda; Gentili, Chiara; Koyama, Eiki; Zasloff, Michael; Pacifici, Maurizio

    2002-01-01

    Hypertrophic chondrocytes have important roles in promoting invasion of cartilage by blood vessels and its replacement with bone. However, it is unclear whether blood vessels exert reciprocal positive influences on chondrocyte maturation and function. Therefore, we implanted beads containing the antiangiogenic molecule squalamine around humeral anlagen in chick embryo wing buds and monitored the effects over time. Fluorescence microscopy showed that the drug diffused from the beads and accumulated in humeral perichondrial tissues, indicating that these tissues were the predominant targets of drug action. Diaphyseal chondrocyte maturation was indeed delayed in squalamine-treated humeri, as indicated by reduced cell hypertrophy and expression of type X collagen, transferrin, and Indian hedgehog (Ihh). Although reduced in amount, Ihh maintained a striking distribution in treated and control humeri, being associated with diaphyseal chondrocytes as well as inner perichondrial layer. These decreases were accompanied by lack of cartilage invasion and tartrate-resistant acid phosphatase-positive (TRAP+) cells and a significant longitudinal growth retardation. Recovery occurred at later developmental times, when in fact expression in treated humeri of markers such as matrix metalloproteinase 9 (MMP-9) and connective tissue growth factor (CTGF) appeared to exceed that in controls. Treating primary cultures of hypertrophic chondrocytes and osteoblasts with squalamine revealed no obvious changes in cell phenotype. These data provide evidence that perichondrial tissues and blood vessels in particular influence chondrocyte maturation in a positive manner and may cooperate with hypertrophic chondrocytes in dictating the normal pace and location of the transition from cartilage to bone.

  3. 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

  4. Deferoxamine Suppresses Collagen Cleavage and Protease, Cytokine, and COL10A1 Expression and Upregulates AMPK and Krebs Cycle Genes in Human Osteoarthritic Cartilage

    Directory of Open Access Journals (Sweden)

    Elena V. Tchetina

    2016-01-01

    Full Text Available This study reports the effects of the iron chelator deferoxamine (DFO on collagen cleavage, inflammation, and chondrocyte hypertrophy in relation to energy metabolism-related gene expression in osteoarthritic (OA articular cartilage. Full-depth explants of human OA knee articular cartilage from arthroplasty were cultured with exogenous DFO (1–50 μM. Type II collagen cleavage and phospho-adenosine monophosphate-activated protein kinase (pAMPK concentrations were measured using ELISAs. Gene expression studies employed real-time PCR and included AMPK analyses in PBMCs. In OA explants collagen cleavage was frequently downregulated by 10–50 μM DFO. PCR analysis of 7 OA patient cartilages revealed that 10 μM DFO suppressed expression of MMP-1, MMP-13, IL-1β, and TNFα and a marker of chondrocyte hypertrophy, COL10A1. No changes were observed in the expression of glycolysis-related genes. In contrast, expressions of genes associated with the mitochondrial Krebs cycle (TCA, AMPK, HIF1α, and COL2A1 were upregulated. AMPK gene expression was reduced in OA cartilage and increased in PBMCs from the same patients compared to healthy controls. Our studies demonstrate that DFO is capable of suppressing excessive collagenase-mediated type II collagen cleavage in OA cartilage and reversing phenotypic changes. The concomitant upregulation of proanabolic TCA-related gene expressions points to a potential for availability of energy generating substrates required for matrix repair by end-stage OA chondrocytes. This might normally be prevented by high whole-body energy requirements indicated by elevated AMPK expression in PBMCs of OA patients.

  5. Deferoxamine Suppresses Collagen Cleavage and Protease, Cytokine, and COL10A1 Expression and Upregulates AMPK and Krebs Cycle Genes in Human Osteoarthritic Cartilage.

    Science.gov (United States)

    Tchetina, Elena V; Markova, Galina A; Poole, A Robin; Zukor, David J; Antoniou, John; Makarov, Sergey A; Kuzin, Aleksandr N

    2016-01-01

    This study reports the effects of the iron chelator deferoxamine (DFO) on collagen cleavage, inflammation, and chondrocyte hypertrophy in relation to energy metabolism-related gene expression in osteoarthritic (OA) articular cartilage. Full-depth explants of human OA knee articular cartilage from arthroplasty were cultured with exogenous DFO (1-50 μM). Type II collagen cleavage and phospho-adenosine monophosphate-activated protein kinase (pAMPK) concentrations were measured using ELISAs. Gene expression studies employed real-time PCR and included AMPK analyses in PBMCs. In OA explants collagen cleavage was frequently downregulated by 10-50 μM DFO. PCR analysis of 7 OA patient cartilages revealed that 10 μM DFO suppressed expression of MMP-1, MMP-13, IL-1β, and TNFα and a marker of chondrocyte hypertrophy, COL10A1. No changes were observed in the expression of glycolysis-related genes. In contrast, expressions of genes associated with the mitochondrial Krebs cycle (TCA), AMPK, HIF1α, and COL2A1 were upregulated. AMPK gene expression was reduced in OA cartilage and increased in PBMCs from the same patients compared to healthy controls. Our studies demonstrate that DFO is capable of suppressing excessive collagenase-mediated type II collagen cleavage in OA cartilage and reversing phenotypic changes. The concomitant upregulation of proanabolic TCA-related gene expressions points to a potential for availability of energy generating substrates required for matrix repair by end-stage OA chondrocytes. This might normally be prevented by high whole-body energy requirements indicated by elevated AMPK expression in PBMCs of OA patients.

  6. Overexpression of hsa-miR-148a promotes cartilage production and inhibits cartilage degradation by osteoarthritic chondrocytes

    NARCIS (Netherlands)

    Vonk, Lucienne A.; Kragten, Angela H.M.; Dhert, Wouter J.; Saris, Daniël B.F.; Creemers, Laura B.

    2014-01-01

    Objective Hsa-miR-148a expression is decreased in OA cartilage, but its functional role in cartilage has never been studied. Therefore, our aim was to investigate the effects of overexpressing hsa-miR-148a on cartilage metabolism of OA chondrocytes. Design OA chondrocytes were transfected with a

  7. Overexpression of hsa-miR-148a promotes cartilage production and inhibits cartilage degradation by osteoarthritic chondrocytes

    NARCIS (Netherlands)

    Vonk, L A; Kragten, A H M; Dhert, W J A|info:eu-repo/dai/nl/10261847X; Saris, D B F; Creemers, L B

    OBJECTIVE: Hsa-miR-148a expression is decreased in Osteoarthritis (OA) cartilage, but its functional role in cartilage has never been studied. Therefore, our aim was to investigate the effects of overexpressing hsa-miR-148a on cartilage metabolism of OA chondrocytes. DESIGN: OA chondrocytes were

  8. Ontogeny of rat chondrocyte proliferation: studies in embryo, adult and osteoarthritic (OA) cartilage

    Institute of Scientific and Technical Information of China (English)

    Madaí A GóMEZ-CAMARILLO; Juan B.KOURI

    2005-01-01

    The aim of this work was to study the ontogeny of chondrocyte cell division using embryo, adult and osteoarthritic (OA) cartilage. We searched for mitosis phases and performed a comparative evaluation of mitotic index, basic fibroblast growth factor b (FGFb), transforming growth factor β1 (TGF-β1) receptors, cyclin dependent kinase (CDK1)and Cyclin-B expression in fetal, neonate, 3, 5, 8 weeks old rats and experimental OA. Our results showed that mitosis phases were observed in all normal cartilage studied, although, we found a decrease in mitotic index in relation to tissue development. No mitosis was detected in OA cartilage. We also found a statistical significant reduction in cell number in OA cartilage, compared with the normal tissue. Furthermore, FGFb and TGF-β1 receptors diminished in relation to tissue development, and were very scarce in experimental OA. Western blot assays showed CDK-1 expression in all cases, including human-OA cartilage. Similar results were observed for Cyclin-B, except for 8 weeks, when it was not expressed. Our results suggest that cell division seems to be scarce, if not absent within the OA cartilage studied.Nevertheless, the existence of factors essential for cell division leaves open the question concerning chondrocyte proliferation in OA cartilage, which is likely to be present in the early stages of the disease.

  9. Human Neuroepithelial Cells Express NMDA Receptors

    Directory of Open Access Journals (Sweden)

    Cappell B

    2003-11-01

    Full Text Available Abstract L-glutamate, an excitatory neurotransmitter, binds to both ionotropic and metabotropic glutamate receptors. In certain parts of the brain the BBB contains two normally impermeable barriers: 1 cerebral endothelial barrier and 2 cerebral epithelial barrier. Human cerebral endothelial cells express NMDA receptors; however, to date, human cerebral epithelial cells (neuroepithelial cells have not been shown to express NMDA receptor message or protein. In this study, human hypothalamic sections were examined for NMDA receptors (NMDAR expression via immunohistochemistry and murine neuroepithelial cell line (V1 were examined for NMDAR via RT-PCR and Western analysis. We found that human cerebral epithelium express protein and cultured mouse neuroepithelial cells express both mRNA and protein for the NMDA receptor. These findings may have important consequences for neuroepithelial responses during excitotoxicity and in disease.

  10. 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.

  11. The Effect of Salvia miltiorrhiza Injection on phenotype Expression of Collagen and Proteoglycan of Chondrocyte%丹参注射液对大鼠关节软骨细胞合成胶原和蛋白多糖的影响

    Institute of Scientific and Technical Information of China (English)

    赵忠; 刘德春; 宋阳春

    2013-01-01

    Purpose Chondrocytes in vitro experimental study , to explore the impact of Danshen injection on the cartilage cells to synthesize collagen and proteoglycan. Methods On cartilage cells and passage of culture through on morphology of cultured cells and toluidine blue staining and immunocytochemical identification of articular cartilage cells, select the appropriate passage of chondrocytes, TNF-alpha compare danshen injection after intervention on chondrocytes in vitro expression of type II collagen and proteoglycan change. Results 2.5% , 5% , 10% Danshen injection can increase the expression of TNF-α -induced articular chondrocytes collagen type Ⅱ and proteoglycans. Conclusion Danshen injection to promote extracellular matrix synthesis in chondrocytes , reducing the catabolism of chondrocytes , play a protective effect on articular cartilage.%目的 通过对体外培养软骨细胞的实验研究,探寻丹参注射液对软骨细胞合成胶原和蛋白多糖的影响.方法 对软骨细胞进行原代及传代培养,通过对培养细胞形态学及甲苯胺蓝染色和免疫细胞化学方法鉴定关节软骨细胞,选择适合传代软骨细胞,应用TNF-α干预后比较丹参注射液干预对体外培养软骨细胞Ⅱ型胶原和蛋白多糖的表达变化.结果 2.5%、5%、10%丹参注射液可以增加TNF-α诱导的关节软骨细胞Ⅱ型胶原及蛋白多糖的表达.结论 丹参注射液有促进软骨细胞外基质合成的作用,减少软骨细胞分解代谢,对关节软骨起到保护作用.

  12. The critical role of UDP-galactose-4-epimerase in osteoarthritis: modulating proteoglycans synthesis of the articular chondrocytes.

    Science.gov (United States)

    Wen, Yinxian; Qin, Jun; Deng, Yu; Wang, Hui; Magdalou, Jacques; Chen, Liaobin

    2014-10-03

    UDP-galactose-4-epimerase (GALE) is a key enzyme catalyzing the interconversion of UDP-glucose and UDP-galactose, as well as UDP-N-acetylglucosamine and UDP-N-acetylgalactosamine, which are all precursors for the proteoglycans (PGs) synthesis. However, whether GALE is essential in cartilage homeostasis remains unknown. Therefore, we investigated the role of GALE in PGs synthesis of human articular chondrocytes, the GALE expression in OA, and the regulation of GALE expression by interleukin-1beta (IL-1β). Silencing GALE gene with specific siRNAs resulted in a markedly inhibition of PGs synthesis in human articular chondrocytes. GALE protein levels were also decreased in both human and rat OA cartilage, thus leading to losses of PGs contents. Moreover, GALE mRNA expression was stimulated by IL-1β in early phase, but suppressed in late phase, while the suppression of GALE expression induced by IL-1β was mainly mediated by stress-activated protein kinase/c-Jun N-terminal kinase pathway. These data indicated a critical role of GALE in maintaining cartilage homeostasis, and suggested that GALE inhibition might contribute to OA progress.

  13. Expression of immunoreactive urocortin in human tissue

    Institute of Scientific and Technical Information of China (English)

    GU Qing; Vicki L Clifton; CUI Ying; HUI Ning; ZHOU Xiao-ning; HE Qian; HAN Qing-feng; SHA Jin-yan; Roger Smith

    2001-01-01

    To localize where urocortin is expressed in human tissue in an attempt to study its physiological functions. Methods: Expression of immunoreactive urocortin in different human tissue was examined using a specific urocortin antibody and the immunoperoxidase staining method. Results: Immunoreactive urocortin was observed in the anterior pituitary cells, decidual stromal cells, syncytiotrophoblasts, amnion epithelium, the vascular smooth muscles of myometrium, fallopian tube and small intestine. Conclusion: The study indicates that urocortin is expressed in some specific areas of human tissue. The data are consistent with the hypothesis that urocortin is produced locally as an endocrine factor, which may act as a neural regulator and a regulator of local blood flow.

  14. 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.

  15. Chondrogenic potential of subpopulations of cells expressing mesenchymal stem cell markers derived from human synovial membranes.

    Science.gov (United States)

    Arufe, M C; De la Fuente, A; Fuentes, I; de Toro, F J; Blanco, F J

    2010-11-01

    In this study we analyzed the chondrogenic potential of subpopulations of mesenchymal stem cells (MSCs) derived from human synovial membranes enriched for CD73, CD106, and CD271 markers. Subpopulations of human synovial membrane MSCs enriched for CD73, CD106, and CD271 markers were isolated using a cytometry sorter and characterized by flow cytometry for MSC markers. The expression of Sox9, Nanog, and Runx2 genes by these cells was measured by reverse transcriptase-polymerase chain reaction. The chondrogenesis of each subpopulation was assessed by culturing the cells in a defined medium to produce spontaneous spheroid formation and differentiation towards chondrocyte-like cells. The examination of the spheroids by histological and immunohistochemical analyses for collagen type II (COL2), aggrecan, collagen type I (COL1), metalloprotease 13 (MMP13), and collagen type X (COLX) levels were performed to assess their chondrogenesis capacity. The adipogenesis and osteogenesis potential of each subpopulation was determined using commercial media; the resulting cells were stained with oil red O or red alizarin to test the degree of differentiation. The subpopulations had different profiles of cells positive for the MSC markers CD44, CD69, CD73, CD90, and CD105 and showed different expression levels of the genes Sox9, Nanog, and Runx2 involved in chondrogenesis, undifferentiation, and osteoblastogenesis, respectively. Immunohistochemical analysis demonstrated that COL1, COL2, COLX, MMP13, and aggrecan were expressed in the spheroids as soon as 14 days of culture. The CD271(+) subpopulation expressed the highest levels of COL2 staining compared to the other subpopulations. CD105 and Runx2 were shown by immunohistochemistry and genetic analysis to have significantly higher expression CD271(+) subpopulation than the other subpopulations. Spheroids formed from CD271-enriched and CD73-enriched MSCs from normal human synovial membranes mimic the native cartilage extracellular

  16. Softening Substrates Promote Chondrocytes Phenotype via RhoA/ROCK Pathway.

    Science.gov (United States)

    Zhang, Tao; Gong, Tao; Xie, Jing; Lin, Shiyu; Liu, Yao; Zhou, Tengfei; Lin, Yunfeng

    2016-09-01

    Due to its evascular, aneural, and alymphatic conditions, articular cartilage shows extremely poor regenerative ability. Thus, directing chondrocyte toward a desired location and function by utilizing the mechanical cues of biomaterials is a promising approach for effective tissue regeneration. However, chondrocytes cultured on Petri dish will lose their typical phenotype which may lead to compromised results. Therefore, we fabricated polydimethylsiloxane (PDMS) materials with various stiffness as culture substrates. Cell morphology and focal adhesion of chondrocytes displayed significant changes. The cytoskeletal tension of the adherent cells observed by average myosin IIA fluorescent intensity increased as stiffness of the underlying substrates decreased, consistent with the alteration of chondrocyte phenotype in our study. Immunofluorescent images and q-PCR results revealed that chondrocyte cultured on soft substrates showed better chondrocyte functionalization by more type II collagen and aggrecan expression, related to the lowest mRNA level of Rac-1, RhoA, ROCK-1, and ROCK-2. Taken together, this work not only points out that matrix elasticity can regulate chondrocyte functionalization via RhoA/ROCK pathway, but also provides new prospect for biomechanical control of cell behavior in cell-based cartilage regeneration.

  17. Toxicity of antiseptics against chondrocytes: what is best for the cartilage in septic joint surgery?

    Science.gov (United States)

    Röhner, Eric; Kolar, Paula; Seeger, Joern B; Arnholdt, Joerg; Thiele, Kathi; Perka, Carsten; Matziolis, Georg

    2011-11-01

    In septic joint surgery, the most frequently used antiseptics are polyhexanide, hydrogen peroxide and taurolidine. The aim of this study was to examine the effects of these antiseptics on viability of human chondrocytes. Our hypothesis was that antiseptics and supplemental irrigation with sodium chloride lavage are less toxic on human chondrocytes than treatment with antiseptics only. Primary human chondrocytes were isolated and cultured from six donated human knee joints. Polyhexanide, hydrogen peroxide or taurolidine were added to the cultures. Toxicity analysis was performed by visualisation of cell structure using light microscopy and LDH activity. The determination of vital cells and total cell numbers of chondrocytes treated with antiseptics partly followed by irrigation with sodium chloride solution was performed by using Casy Cell-Counter. Light microscopic data revealed a defect in cell structure after addition of antiseptics. We showed a significant increase of LDH enzyme activity after the treatment with polyhexanide or taurolidine. After treatment with antiseptics followed by sodium chloride solution a significant increase of vital and total cell numbers resulted in comparison with the chondrocytes that were only treated with antiseptics. The data show that treatment with polyhexanid, hydrogen peroxide or taurolidine induces cell death of human chondroctes in vitro. The application of sodium chloride solution after the treatment with polyhexanide and hydrogen peroxide possibly has a protective effect on chondrocyte viability.

  18. RAGE, receptor of advanced glycation endoproducts, negatively regulates chondrocytes differentiation.

    Directory of Open Access Journals (Sweden)

    Tatsuya Kosaka

    Full Text Available 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.

  19. 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

  20. Neuropharmacology of Human Appetite Expression

    Science.gov (United States)

    Halford, Jason C. G.; Harrold, Joanne A.

    2008-01-01

    The regulation of appetite relies on the integration of numerous episodic (meal) and tonic (energy storage) generated signals in energy regulatory centres within the central nervous system (CNS). These centers provide the pharmacological potential to modify human appetite (hunger and satiety) to increase or decrease caloric intake, or to normalize…

  1. Construction and expression of recombined human AFP eukaryotic expression vector

    Institute of Scientific and Technical Information of China (English)

    Li-Wang Zhang; Yang-Lin Pan; Stephen M Festein; Jun Ren; Liang Zhang; Hong-Mei Zhang; Bin Jin; Bo-Rong Pan; Xiao-Ming Si; Yan-Jun Zhang; Zhong-Hua Wang

    2003-01-01

    AIM: To construct a recombined human AFP eukaryotic expression vector for the purpose of gene therapy and target therapy of hepatocellular carcinoma (HCC).METHODS: The full length AFP-cDNA of prokaryotic vector was digested, and subcloned to the multi-clony sites of the eukaryotic vector. The constructed vector was confirmed by enzymes digestion and electrophoresis, and the product expressed was detected by electrochemiluminescence and immunofluorescence methods.RESULTS: The full length AFP-cDNA successfully cloned to the eukaryotic vector through electrophoresis, 0.9723 IU/ml AFP antigen was detected in the supernatant of AFPCHO by electrochemiluminescence method. Compared with the control groups, the differences were significant (P<0.05).AFP antigen molecule was observed in the plasma of AFPCHO by immunofluorescence staining.CONCLUSION: The recombined human AFP eukaryotic expression vector can express in CHO cell line. It provides experimental data for gene therapy and target therapy of hepatocellular carcinoma.

  2. 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.

  3. 人真皮成纤维细胞体外构建组织工程化软骨的初步探索%Preliminary study on tissue-engineered cartilage with human dermal fibroblasts co-cultured with porcine chondrocytes in vitro

    Institute of Scientific and Technical Information of China (English)

    刘霞; 周广东; 刘伟; 曹谊林

    2009-01-01

    Objective To explore the feasibility of constructing tissue-engineered cartilage with human dermal fibroblasts(HDFs)in vitro. Methods Porcine articular chondrocytes and HDFs were isolated and in vitro expanded respectively. Then they were mixed at the ratio of 1:1 (chondrocytes: fibroblasts). The mixed cells were seeded onto polyglycolic acid(PGA)scaffold at the ultimate concentration of 5.0×10~7/ml as co-culture group.Chondrocytes and HDFs at the same ultimate concentration were seeded respectively onto the scaffold as chondrocyte group (positive control group)and fibroblast group(negative control group). The specimens were collected after in vitro culture for 8 weeks. Gross observation, histology and immunohistochemistry were used to evaluate the results. Results In chondrocyte group, the cell-scaffold constructs could maintain the original size and shape during in vitro culture. The new formed cartilage-like tissue had typical histological structure and extracellular matrix staining similar to normal cartilage. In co-culture group the constructs shrunk slightly at 8 weeks, cartilage-like tissue formed and GAG could be detected for strong expression by Safranin O staining.Furthermore, using the specific identification, a few HDFs derived cells were found to form lacuna structure at the peripheral area of cartilage-like tissue. In fibroblast group, the constructs deformed and shrunk gradually without mature cartilage lacuna in histology. Conclusion The 3D-co-culture system can effectively induce the differenciation of HDFs to chondrocytes. The tissue-engineered cartilage can be constructed in vitro with the 3D-co-culture system.%目的 利用软骨细胞提供的体外软骨诱导微环境,探讨人真皮成纤维细胞在体外构建软骨的可行性.方法 分别培养猪的软骨细胞与人真皮成纤维细胞,将2种细胞按1:1(软骨细胞:成纤维细胞)比例混匀,以5.0×10~7/ml的终浓度接种于聚羟基乙酸支架(PGA,直径9 mm,高2mm)作为

  4. Effect of microcavitary alginate hydrogel with different pore sizes on chondrocyte culture for cartilage tissue engineering

    Energy Technology Data Exchange (ETDEWEB)

    Zeng, Lei; Yao, Yongchang [School of Materials Science and Engineering, South China University of Technology, Guangzhou 510641 (China); National Engineering Research Center for Tissue Restoration and Reconstruction, Guangzhou 510006 (China); Wang, Dong-an, E-mail: DAWang@ntu.edu.sg [National Engineering Research Center for Tissue Restoration and Reconstruction, Guangzhou 510006 (China); Division of Bioengineering, School of Chemical and Biomedical Engineering, Nanyang Technological University, Singapore 637457 (Singapore); Chen, Xiaofeng, E-mail: chenxf@scut.edu.cn [School of Materials Science and Engineering, South China University of Technology, Guangzhou 510641 (China); National Engineering Research Center for Tissue Restoration and Reconstruction, Guangzhou 510006 (China)

    2014-01-01

    In our previous work, a novel microcavitary hydrogel was proven to be effective for proliferation of chondrocytes and maintenance of chondrocytic phenotype. In present work, we further investigated whether the size of microcavity would affect the growth and the function of chondrocytes. By changing the stirring rate, gelatin microspheres in different sizes including small size (80–120 μm), middle size (150–200 μm) and large size (250–300 μm) were prepared. And then porcine chondrocytes were encapsulated into alginate hydrogel with various sizes of gelatin microspheres. Cell Counting Kit-8 (CCK-8), Live/dead staining and real-time PCR were used to analyze the effect of the pore size on cell proliferation and expression of specific chondrocytic genes. According to all the data, cells cultivated in microcavitary hydrogel, especially in small size, had preferable abilities of proliferation and higher expression of cartilaginous markers including type II collagen, aggrecan and cartilage oligomeric matrix protein (COMP). Furthermore, it was shown by western blot assay that the culture of chondrocytes in microcavitary hydrogel could improve the proliferation of cells potentially by inducing the Erk1/2-MAPK pathway. Taken together, this study demonstrated that chondrocytes favored microcavitary alginate hydrogel with pore size within the range of 80–120 μm for better growth and ECM synthesis, in which Erk1/2 pathway was involved. This culture system would be promising for cartilage tissue engineering. - Highlights: • A novel model with microcavitary structure was set up to study the interaction between cells and materials. • Microcavitary alginate hydrogel could enhance the proliferation of chondrocytes and promote the expression of cartilaginous genes as compared with plain alginate hydrogel. • Cells in microcavitary alginate hydrogel with pore size within the range of 80–120 μm were capable of better growth and ECM synthesis.

  5. Collagen type XII and versican are present in the early stages of cartilage tissue formation by both redifferentating passaged and primary chondrocytes.

    Science.gov (United States)

    Taylor, Drew W; Ahmed, Nazish; Parreno, Justin; Lunstrum, Gregory P; Gross, Allan E; Diamandis, Eleftherios P; Kandel, Rita A

    2015-02-01

    Current approaches to cartilage tissue engineering require a large number of chondrocytes. Although chondrocyte numbers can be expanded in monolayer culture, the cells dedifferentiate and unless they can be redifferentiated are not optimal to use for cartilage repair. We took advantage of the differential effect of culture conditions on the ability of passaged and primary chondrocytes to form cartilage tissue to dissect out the extracellular matrix (ECM) molecules produced and accumulated in the early stages of passaged cell cartilage tissue formation as we hypothesized that passaged bovine cells that form cartilage accumulate a pericellular matrix that differs from cells that do not form cartilage. Twice passaged bovine chondrocytes (P2) (cartilage forming), or as a control primary chondrocytes (P0) (which do not generate cartilage), were cultured on three-dimensional membrane inserts in serum-free media. P2 redifferentiation was occurring during the first 8 days as indicated by increased expression of the chondrogenic genes Sox9, collagen type II, aggrecan, and COMP, suggesting that this is an appropriate time period to examine the ECM. Mass spectrometry showed that the P2 secretome (molecules released into the media) at 1 week had higher levels of collagen types I, III, and XII, and versican while type II collagen and COMP were found at higher levels in the P0 secretome. There was increased collagen synthesis and retention by P2 cells compared to P0 cells as early as 3 days of culture. Confocal microscopy showed that types XII, III, and II collagen, aggrecan, versican, and decorin were present in the ECM of P2 cells. In contrast, collagen types I, II, and III, aggrecan, and decorin were present in the ECM of P0 cells. As primary chondrocytes grown in serum-containing media, a condition that allows for the generation of cartilage tissue in vitro, also accumulate versican and collagen XII, this study suggests that these molecules may be necessary to provide a

  6. Losartan increases bone mass and accelerates chondrocyte hypertrophy in developing skeleton.

    Science.gov (United States)

    Chen, Shan; Grover, Monica; Sibai, Tarek; Black, Jennifer; Rianon, Nahid; Rajagopal, Abbhirami; Munivez, Elda; Bertin, Terry; Dawson, Brian; Chen, Yuqing; Jiang, Ming-Ming; Lee, Brendan; Yang, Tao; Bae, Yangjin

    2015-05-01

    Angiotensin receptor blockers (ARBs) are a group of anti-hypertensive drugs that are widely used to treat pediatric hypertension. Recent application of ARBs to treat diseases such as Marfan syndrome or Alport syndrome has shown positive outcomes in animal and human studies, suggesting a broader therapeutic potential for this class of drugs. Multiple studies have reported a benefit of ARBs on adult bone homeostasis; however, its effect on the growing skeleton in children is unknown. We investigated the effect of Losartan, an ARB, in regulating bone mass and cartilage during development in mice. Wild type mice were treated with Losartan from birth until 6 weeks of age, after which bones were collected for microCT and histomorphometric analyses. Losartan increased trabecular bone volume vs. tissue volume (a 98% increase) and cortical thickness (a 9% increase) in 6-weeks old wild type mice. The bone changes were attributed to decreased osteoclastogenesis as demonstrated by reduced osteoclast number per bone surface in vivo and suppressed osteoclast differentiation in vitro. At the molecular level, Angiotensin II-induced ERK1/2 phosphorylation in RAW cells was attenuated by Losartan. Similarly, RANKL-induced ERK1/2 phosphorylation was suppressed by Losartan, suggesting a convergence of RANKL and angiotensin signaling at the level of ERK1/2 regulation. To assess the effect of Losartan on cartilage development, we examined the cartilage phenotype of wild type mice treated with Losartan in utero from conception to 1 day of age. Growth plates of these mice showed an elongated hypertrophic chondrocyte zone and increased Col10a1 expression level, with minimal changes in chondrocyte proliferation. Altogether, inhibition of the angiotensin pathway by Losartan increases bone mass and accelerates chondrocyte hypertrophy in growth plate during skeletal development.

  7. Expression of polarity genes in human cancer.

    Science.gov (United States)

    Lin, Wan-Hsin; Asmann, Yan W; Anastasiadis, Panos Z

    2015-01-01

    Polarity protein complexes are crucial for epithelial apical-basal polarity and directed cell migration. Since alterations of these processes are common in cancer, polarity proteins have been proposed to function as tumor suppressors or oncogenic promoters. Here, we review the current understanding of polarity protein functions in epithelial homeostasis, as well as tumor formation and progression. As most previous studies focused on the function of single polarity proteins in simplified model systems, we used a genomics approach to systematically examine and identify the expression profiles of polarity genes in human cancer. The expression profiles of polarity genes were distinct in different human tissues and classified cancer types. Additionally, polarity expression profiles correlated with disease progression and aggressiveness, as well as with identified cancer types, where specific polarity genes were commonly altered. In the case of Scribble, gene expression analysis indicated its common amplification and upregulation in human cancer, suggesting a tumor promoting function.

  8. Clusterin expression and human testicular seminoma.

    Science.gov (United States)

    Tang, Min; Li, Jie; Liu, Bianjiang; Song, Ninghong; Wang, Zengjun; Yin, Changjun

    2013-10-01

    Clusterin expression has a positive correlation with the occurrence and progression of various types of tumors from different genetic backgrounds. Clusterin overexpression may protect tumor cells from apoptosis and damage caused by autoimmunity or anti-tumor therapy. Using immunohistochemisty, one previous study showed that clusterin protein expression is downregulated in human testicular seminoma, which is highly sensitive to radiotherapy and chemotherapy. We thus postulate that clusterin expression in human testicular seminoma differs from clusterin expression in other tumors. It may be the cause of the treatable characteristics of testicular seminoma. In the present preliminary study, we detected the abundance of clusterin mRNA in human testicular seminoma and normal testis. The results showed decreased clusterin expression in seminoma at the gene transcription level. Our primary data and summarized previous literature suggest that the downregulation of clusterin expression may be the cause of the high sensitivity of testicular seminoma to radiotherapy and chemotherapy. It may be that the scarcity of clusterin leaves tumor cells with insufficient protection from treatment. This is the first study to focus on the relationship between clusterin expression and human testicular cancer. Copyright © 2013 Elsevier Ltd. All rights reserved.

  9. Inducible chondrocyte-specific overexpression of BMP2 in young mice results in severe aggravation of osteophyte formation in experimental OA without altering cartilage damage

    NARCIS (Netherlands)

    Davidson, E.N.; Vitters, E.L.; Bennink, M.B.; Lent, P.L. van; Caam, A.P. van; Blom, A.B.; Berg, W.B. van den; Loo, F.A.J. van de; Kraan, P.M. van der

    2015-01-01

    OBJECTIVES: In osteoarthritis (OA) chondrocytes surrounding lesions express elevated bone morphogenetic protein 2 (BMP2) levels. To investigate the functional consequence of chondrocyte-specific BMP2 expression, we made a collagen type II dependent, doxycycline (dox)-inducible BMP2 transgenic mouse

  10. Effects of Selenium on Fusarium Growth and Associated Fermentation Products and the Relationship with Chondrocyte Viability.

    Science.gov (United States)

    Yin, Hong; Zhang, Yan; Zhang, Feng; Hu, Jin Tao; Zhao, Yu Meng; Cheng, Bo Lun

    2017-02-01

    This study determined the effects of selenium on the growth of Fusarium strains and the effects of products extracted from the fungal cultures on relevant indicators of chondrocytes injury. The results showed that selenium supplementation resulted in differential effects on the mycelial growth of the strains. Levels of the chondrocyte injury indicators, including cell viability, proteoglycan and type II collagen contents and their mRNA expressions, were all reduced to varying degrees when the chondrocytes were incubated with fermentation extracts, the inhibitory effect varied depending on selenium content supplemented to fungal culture media. The results indicated that certain chain relations existed between the content of selenium in the environment, the production of some metabolites by fungi, and the occurrence of chondrocyte damage. The extent of this relationship and the role it plays in Kaschin-Beck disease pathogenesis merit further study. Copyright © 2017 The Editorial Board of Biomedical and Environmental Sciences. Published by China CDC. All rights reserved.

  11. Downregulation of protein kinase CK2 activity facilitates tumor necrosis factor-α-mediated chondrocyte death through apoptosis and autophagy.

    Directory of Open Access Journals (Sweden)

    Sung Won Lee

    Full Text Available Despite the numerous studies of protein kinase CK2, little progress has been made in understanding its function in chondrocyte death. Our previous study first demonstrated that CK2 is involved in apoptosis of rat articular chondrocytes. Recent studies have suggested that CK2 downregulation is associated with aging. Thus examining the involvement of CK2 downregulation in chondrocyte death is an urgently required task. We undertook this study to examine whether CK2 downregulation modulates chondrocyte death. We first measured CK2 activity in articular chondrocytes of 6-, 21- and 30-month-old rats. Noticeably, CK2 activity was downregulated in chondrocytes with advancing age. To build an in vitro experimental system for simulating tumor necrosis factor (TNF-α-induced cell death in aged chondrocytes with decreased CK2 activity, chondrocytes were co-treated with CK2 inhibitors and TNF-α. Viability assay demonstrated that CK2 inhibitors facilitated TNF-α-mediated chondrocyte death. Pulsed-field gel electrophoresis, nuclear staining, flow cytometry, TUNEL staining, confocal microscopy, western blot and transmission electron microscopy were conducted to assess cell death modes. The results of multiple assays showed that this cell death was mediated by apoptosis. Importantly, autophagy was also involved in this process, as supported by the appearance of a punctuate LC3 pattern and autophagic vacuoles. The inhibition of autophagy by silencing of autophage-related genes 5 and 7 as well as by 3-methyladenine treatment protected chondrocytes against cell death and caspase activation, indicating that autophagy led to the induction of apoptosis. Autophagic cells were observed in cartilage obtained from osteoarthritis (OA model rats and human OA patients. Our findings indicate that CK2 down regulation facilitates TNF-α-mediated chondrocyte death through apoptosis and autophagy. It should be clarified in the future if autophagy observed is a consequence

  12. Zfp521 is a target gene and key effector of parathyroid hormone-related peptide signaling in growth plate chondrocytes.

    Science.gov (United States)

    Correa, Diego; Hesse, Eric; Seriwatanachai, Dutmanee; Kiviranta, Riku; Saito, Hiroaki; Yamana, Kei; Neff, Lynn; Atfi, Azeddine; Coillard, Lucie; Sitara, Despina; Maeda, Yukiko; Warming, Soren; Jenkins, Nancy A; Copeland, Neal G; Horne, William C; Lanske, Beate; Baron, Roland

    2010-10-19

    In the growth plate, the interplay between parathyroid hormone-related peptide (PTHrP) and Indian hedgehog (Ihh) signaling tightly regulates chondrocyte proliferation and differentiation during longitudinal bone growth. We found that PTHrP increases the expression of Zfp521, a zinc finger transcriptional coregulator, in prehypertrophic chondrocytes. Mice with chondrocyte-targeted deletion of Zfp521 resembled PTHrP(-/-) and chondrocyte-specific PTHR1(-/-) mice, with decreased chondrocyte proliferation, early hypertrophic transition, and reduced growth plate thickness. Deleting Zfp521 increased expression of Runx2 and Runx2 target genes, and decreased Cyclin D1 and Bcl-2 expression while increasing Caspase-3 activation and apoptosis. Zfp521 associated with Runx2 in chondrocytes, antagonizing its activity via an HDAC4-dependent mechanism. PTHrP failed to upregulate Cyclin D1 and to antagonize Runx2, Ihh, and collagen X expression when Zfp521 was absent. Thus, Zfp521 is an important PTHrP target gene that regulates growth plate chondrocyte proliferation and differentiation.

  13. IL-21 Receptor Expression in Human Tendinopathy

    Science.gov (United States)

    Campbell, Abigail L.; Smith, Nicola C.; Reilly, James H.; Kerr, Shauna C.; Leach, William J.; Fazzi, Umberto G.; Rooney, Brian P.; Murrell, George A. C.; Millar, Neal L.

    2014-01-01

    The pathogenetic mechanisms underlying tendinopathy remain unclear, with much debate as to whether inflammation or degradation has the prominent role. Increasing evidence points toward an early inflammatory infiltrate and associated inflammatory cytokine production in human and animal models of tendon disease. The IL-21/IL-21R axis is a proinflammatory cytokine complex that has been associated with chronic inflammatory diseases including rheumatoid arthritis and inflammatory bowel disease. This project aimed to investigate the role and expression of the cytokine/receptor pair IL-21/IL-21R in human tendinopathy. We found significantly elevated expression of IL-21 receptor message and protein in human tendon samples but found no convincing evidence of the presence of IL-21 at message or protein level. The level of expression of IL-21R message/protein in human tenocytes was significantly upregulated by proinflammatory cytokines (TNFα/IL-1β) in vitro. These findings demonstrate that IL-21R is present in early human tendinopathy mainly expressed by tenocytes and macrophages. Despite a lack of IL-21 expression, these data again suggest that early tendinopathy has an inflammatory/cytokine phenotype, which may provide novel translational targets in the treatment of tendinopathy. PMID:24757284

  14. IL-21 Receptor Expression in Human Tendinopathy

    Directory of Open Access Journals (Sweden)

    Abigail L. Campbell

    2014-01-01

    Full Text Available The pathogenetic mechanisms underlying tendinopathy remain unclear, with much debate as to whether inflammation or degradation has the prominent role. Increasing evidence points toward an early inflammatory infiltrate and associated inflammatory cytokine production in human and animal models of tendon disease. The IL-21/IL-21R axis is a proinflammatory cytokine complex that has been associated with chronic inflammatory diseases including rheumatoid arthritis and inflammatory bowel disease. This project aimed to investigate the role and expression of the cytokine/receptor pair IL-21/IL-21R in human tendinopathy. We found significantly elevated expression of IL-21 receptor message and protein in human tendon samples but found no convincing evidence of the presence of IL-21 at message or protein level. The level of expression of IL-21R message/protein in human tenocytes was significantly upregulated by proinflammatory cytokines (TNFα/IL-1β in vitro. These findings demonstrate that IL-21R is present in early human tendinopathy mainly expressed by tenocytes and macrophages. Despite a lack of IL-21 expression, these data again suggest that early tendinopathy has an inflammatory/cytokine phenotype, which may provide novel translational targets in the treatment of tendinopathy.

  15. Fabrication of cartilage in predetermined shapes from human nasoseptal chondrocytes with tissue engineering method%鼻中隔软骨细胞组织工程法构建预定形态软骨

    Institute of Scientific and Technical Information of China (English)

    崔鹏程; 陈文弦; 罗家胜

    2001-01-01

    目的探讨利用人鼻中隔软骨细胞组织工程方法构建预定形态软骨的可能性。方法将人鼻中隔软骨细胞播种在聚乙醇酸(polyglycolic acid, PGA)无纺网支架材料上,制成片状和管状结构,埋入裸鼠体内,经4、6、8周后取材作大体及组织学观察。结果大体观察见裸鼠体内形成了预定的片状和管状软骨。组织学观察:6周时软骨细胞基本成熟, Masson 三色染色显示胶原形成,番红花-“O”染色证实其基质中存在糖氨多糖。对照组于6周时PGA纤维基本消失。结论人鼻中隔软骨细胞与PGA无纺网复合可在裸鼠体内形成预定形态软骨。%Objective To investigate the feasibility of fabricating a new cartilage in predetermined shapes with tissue engineering methods. Methods Human nasoseptal chondrocytes were seeded onto a nonwoven mesh of polyglycolic acid(PGA) to form a cell-PGA construct. The construction was then configured in sheet and tube shapes, and implanted subcutaneously into the dorsa of 11 athymic mice. The specimens were harvested 4,6,8 weeks after implantation and subjected to gross morphologic and histologic analysis. Results Gross observation showed that the predetermined sheet and tube shapes of new cartilage were formed. Histological observation demonstrated that new mature cartilages were formed in 6-week. A Masson's trichrome stain showed the interwining bands of collagen at the periphery of the cartilage. Staining of Safranin O evaluated that the new cartilage was bound of glycosaminoglycan. In the control group, the PGA of the specimens were completely absorbed at 6 weeks. Conclusion Human nasoseptal chondrocytes-PGA construct could develop into a new cartilage in predetermined shapes in athymic mice.

  16. 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.

  17. Human embryonic stem cell derived mesenchymal progenitors express cardiac markers but do not form contractile cardiomyocytes.

    Directory of Open Access Journals (Sweden)

    Christophe M Raynaud

    Full Text Available Mesenchymal progenitors or stromal cells have shown promise as a therapeutic strategy for a range of diseases including heart failure. In this context, we explored the growth and differentiation potential of mesenchymal progenitors (MPs derived in vitro from human embryonic stem cells (hESCs. Similar to MPs isolated from bone marrow, hESC derived MPs (hESC-MPs efficiently differentiated into archetypical mesenchymal derivatives such as chondrocytes and adipocytes. Upon treatment with 5-Azacytidine or TGF-β1, hESC-MPs modified their morphology and up-regulated expression of key cardiac transcription factors such as NKX2-5, MEF2C, HAND2 and MYOCD. Nevertheless, NKX2-5+ hESC-MP derivatives did not form contractile cardiomyocytes, raising questions concerning the suitability of these cells as a platform for cardiomyocyte replacement therapy. Gene profiling experiments revealed that, although hESC-MP derived cells expressed a suite of cardiac related genes, they lacked the complete repertoire of genes associated with bona fide cardiomyocytes. Our results suggest that whilst agents such as TGF-β1 and 5-Azacytidine can induce expression of cardiac related genes, but treated cells retain a mesenchymal like phenotype.

  18. Collagen-induced expression of collagenase-3 by primary chondrocytes is mediated by integrin a1 and discoidin domain receptor 2: a protein kinase C-dependent pathway

    NARCIS (Netherlands)

    Vonk, L.A.; Doulabi, B.Z.; Huang, C.L.; Helder, M.N.; Everts, V.; Bank, R.A.

    2011-01-01

    Objectives. 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

  19. Gene Expression in the Human Endolymphatic Sac

    DEFF Research Database (Denmark)

    Møller, Martin Nue; Kirkeby, Svend; Vikeså, Jonas

    2015-01-01

    OBJECTIVES/HYPOTHESIS: The purpose of the present study is to explore, demonstrate, and describe the expression of genes related to the solute carrier (SLC) molecules of ion transporters in the human endolymphatic sac. STUDY DESIGN: cDNA microarrays and immunohistochemistry were used for analyses...... of fresh human endolymphatic sac tissue samples. METHODS: Twelve tissue samples of the human endolymphatic sac were obtained during translabyrinthine surgery for vestibular schwannoma. Microarray technology was used to investigate tissue sample expression of solute carrier family genes, using adjacent dura...... mater as control. Immunohistochemistry was used for verification of translation of selected genes, as well as localization of the specific protein within the sac. RESULTS: An extensive representation of the SLC family genes were upregulated in the human endolymphatic sac, including SLC26a4 Pendrin, SLC4...

  20. 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

  1. Expression of RHOGTPase regulators in human myometrium

    Directory of Open Access Journals (Sweden)

    Morrison John J

    2008-01-01

    Full Text Available Abstract Background RHOGTPases play a significant role in modulating myometrial contractility in uterine smooth muscle. They are regulated by at least three families of proteins, RHO guanine nucleotide exchange factors (RHOGEFs, RHOGTPase-activating proteins (RHOGAPs and RHO guanine nucleotide inhibitors (RHOGDIs. RHOGEFs activate RHOGTPases from the inactive GDP-bound to the active GTP-bound form. RHOGAPs deactivate RHOGTPases by accelerating the intrinsic GTPase activity of the RHOGTPases, converting them from the active to the inactive form. RHOGDIs bind to GDP-bound RHOGTPases and sequester them in the cytosol, thereby inhibiting their activity. Ezrin-Radixin-Moesin (ERM proteins regulate the cortical actin cytoskeleton, and an ERM protein, moesin (MSN, is activated by and can also activate RHOGTPases. Methods We therefore investigated the expression of various RHOGEFs, RHOGAPs, a RHOGDI and MSN in human myometrium, by semi-quantitative reverse transcription PCR, real-time fluorescence RT-PCR, western blotting and immunofluorescence microscopy. Expression of these molecules was also examined in myometrial smooth muscle cells. Results ARHGEF1, ARHGEF11, ARHGEF12, ARHGAP5, ARHGAP24, ARHGDIA and MSN mRNA and protein expression was confirmed in human myometrium at term pregnancy, at labour and in the non-pregnant state. Furthermore, their expression was detected in myometrial smooth muscle cells. It was determined that ARHGAP24 mRNA expression significantly increased at labour in comparison to the non-labour state. Conclusion This study demonstrated for the first time the expression of the RHOGTPase regulators ARHGEF1, ARHGEF11, ARHGEF12, ARHGAP5, ARHGAP24, ARHGDIA and MSN in human myometrium, at term pregnancy, at labour, in the non-pregnant state and also in myometrial smooth muscle cells. ARHGAP24 mRNA expression significantly increased at labour in comparison to the non-labouring state. Further investigation of these molecules may enable us

  2. 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.

  3. 白藜芦醇干预对已退变椎间盘终板软骨细胞SIRT1表达的影响%Duration-and concentration-dependent induction of SIRT1 on human degenerative endplate chondrocytes by Resveratrol

    Institute of Scientific and Technical Information of China (English)

    钟小明; 胡侦明; 沈皆亮; 甘强; 郝杰

    2013-01-01

    目的 探讨不同浓度及不同时间的白藜芦醇干预对退变椎间盘终板软骨细胞沉默信息调节因子2相关酶1(SIRT1)表达的影响.方法 老年患者的终板软骨细胞培养、传代,P2代细胞随机分组,分别用不同浓度及不同作用时间的白藜芦醇干预,终止培养后检测SIRT1蛋白及mRNA表达水平.结果 与空白对照组相比较,DMSO组SIRT1蛋白及mRNA表达无显著差异(P>0.05);白藜芦醇干预的浓度在12.5 μmol/L、作用24 h,以及浓度在50 μmol/L、作用12h以上时能够显著促进已退变椎间盘终板软骨细胞表达SIRT1蛋白及mRNA(P <0.05).结论 白藜芦醇干预可促进退变椎间盘终板软骨细胞表达SIRT1蛋白及mRNA,且该作用呈现浓度、时间依赖关系;0.1% DMSO对细胞表达SIRT1蛋白无抑制作用.%Objective To explore the effect of different concentrations and time of Resveratrol intervention on the expression of SIRT1 in human degenerative endplate chondrocytes. Methods Degenerative disc endplate chondrocytes ( DEC) were harvested from elder patient with lumbar disc hernia, and then cultured. Passage 2 DEC were stimulated by Resveratrol of different concentrations and duration re spectively , and then expression levels of SIRT1 protein and mRNA were detected by the end of stimulation. Results Compared with the ex pression levels of SIRT1 and of SIRT1 mRNA in control group, there was no significant difference in DMSO group (P>0. 05). Resveratrol of 12. 5 μmol/L and 24 hours, Resveratrol of 50 μmol/L and 12 hours could stimulate the expressions of SIRT1 protein and mRNA (P < 0. 05). Conclusions Resveratrol intervention can significantly stimulate the expressions of SIRT1 protein and mRNA in human DEC in a concentration- and time-dependent manner. 0. 1 % DMSO has no inhibiting effect of SIRT1 protein expression.

  4. Access to Chondrocyte Culture, with Alginate, In Iran

    Directory of Open Access Journals (Sweden)

    Ebrahim Esfandiary

    2008-01-01

    Full Text Available In this study, chondrocyte culture was established for the first time in Iran,and calcium alginate was used for longer culture of chondrocyte in vitro. Thestudy was programmed in order to be used for future human chondrocytetransplantation. The cartilage specimen obtained from 50 patients whounderwent total knee and hip operations in Isfahan University of MedicalSciences. Cartilage specimens were used for monolayer as well as suspensionculture in alginate beads. Approximately 12±1 millions cells were harvestedfrom the 3rd passage. The cells were round with large euchromatic nucleusand several nucleoli and small vacuoles. The cells derived from passages 1to 4, which were grown up then, in alginate beads, showed higher stainingwith alcian blue. The harvested cells in some patients were immediately andsuccessfully used for autologus transplantation. This later work will be reportedseparately.

  5. Study of differential properties of fibrochondrocytes and hyaline chondrocytes in growing rabbits.

    Science.gov (United States)

    Huang, L; Li, M; Li, H; Yang, C; Cai, X

    2015-02-01

    We aimed to build a culture model of chondrocytes in vitro, and to study the differential properties between fibrochondrocytes and hyaline chondrocytes. Histological sections were stained with haematoxylin and eosin so that we could analyse the histological structure of the fibrocartilage and hyaline cartilage. Condylar fibrochondrocytes and femoral hyaline chondrocytes were cultured from four, 4-week-old, New Zealand white rabbits. The production of COL2A1, COL1OA1, SOX9 and aggrecan was detected by real time-q polymerase chain reaction (RT-qPCR) and immunoblotting and the differences between them were compared statistically. Histological structures obviously differed between fibrocartilage and hyaline cartilage. COL2A1 and SOX9 were highly expressed within cell passage 2 (P2) of both fibrochondrocytes and hyaline chondrocytes, and reduced significantly after cell passage 4 (P4). The mRNA expressions of COL2A1 (p=0.05), COL10A1 (p=0.04), SOX9 (p=0.03), and aggrecan (p=0.04) were significantly higher in hyaline chondrocytes than in fibrochondrocytes, whereas the expression of COL1A1 (p=0.02) was the opposite. Immunoblotting showed similar results. We have built a simple and effective culture model of chondrocytes in vitro, and the P2 of chondrocytes is recommended for further studies. Condylar fibrocartilage and femoral hyaline cartilage have unique biological properties, and the regulatory mechanisms of endochondral ossification for the condyle should be studied independently in the future. Copyright © 2014 The British Association of Oral and Maxillofacial Surgeons. Published by Elsevier Ltd. All rights reserved.

  6. Hypoxia induces apelin expression in human adipocytes.

    Science.gov (United States)

    Geiger, K; Muendlein, A; Stark, N; Saely, C H; Wabitsch, M; Fraunberger, P; Drexel, H

    2011-06-01

    Adipokines play a central role in the development of diseases associated with insulin resistance and obesity. Hypoxia in adipose tissue leads to a dysregulation of the expression of adipokines. The effect of hypoxia on the more recently identified adipokine apelin in human adipocytes is unclear. Therefore, we aimed at investigating the role of hypoxia on the expression of the adipokine apelin. Differentiated human Simpson-Golabi-Behmel syndrome (SGBS) adipocytes were cultured under hypoxic conditions for varying time periods. A modular incubator chamber was used to create a hypoxic tissue culture environment (defined as 1% O(2), 94% N, and 5% CO(2)). In addition, hypoxic conditions were mimicked by using CoCl(2). The effect of hypoxia on the expression of the investigated adipokines was measured by real-time PCR and the secretion of apelin was quantified by ELISA. Induction of hypoxia significantly induced mRNA expression of leptin and apelin in differentiated SGBS adipocytes compared with the normoxic control condition. Expression of adiponectin was significantly decreased by hypoxia. In addition, the amount of secreted apelin protein in response to hypoxia was elevated compared to untreated cells. Furthermore, we could demonstrate that the observed hypoxia-induced induction of apelin mRNA expression is in the first phase dependent on HIF-1α. In our study, we could demonstrate for the first time that apelin expression and secretion by human adipocytes are strongly induced under hypoxic conditions and that the early response on hypoxia with apelin induction is dependent on HIF-1α. © Georg Thieme Verlag KG Stuttgart · New York.

  7. Regulation of gene expression in human tendinopathy

    Science.gov (United States)

    2011-01-01

    Background Chronic tendon injuries, also known as tendinopathies, are common among professional and recreational athletes. These injuries result in a significant amount of morbidity and health care expenditure, yet little is known about the molecular mechanisms leading to tendinopathy. Methods We have used histological evaluation and molecular profiling to determine gene expression changes in 23 human patients undergoing surgical procedures for the treatment of chronic tendinopathy. Results Diseased tendons exhibit altered extracellular matrix, fiber disorientation, increased cellular content and vasculature, and the absence of inflammatory cells. Global gene expression profiling identified 983 transcripts with significantly different expression patterns in the diseased tendons. Global pathway analysis further suggested altered expression of extracellular matrix proteins and the lack of an appreciable inflammatory response. Conclusions Identification of the pathways and genes that are differentially regulated in tendinopathy samples will contribute to our understanding of the disease and the development of novel therapeutics. PMID:21539748

  8. Human plasma cells express granzyme B.

    Science.gov (United States)

    Xu, Wei; Narayanan, Priya; Kang, Ning; Clayton, Sandra; Ohne, Yoichiro; Shi, Peiqing; Herve, Marie-Cecile; Balderas, Robert; Picard, Capucine; Casanova, Jean-Laurent; Gorvel, Jean-Pierre; Oh, Sangkon; Pascual, Virginia; Banchereau, Jacques

    2014-01-01

    While studying the plasma cell (PC) compartment in human tonsils, we identified that immunoglobulin kappa or lambda chain-expressing PCs are the main cells expressing granzyme B (GrzB). In vitro studies revealed that activated B cells differentiated into GrzB-expressing PCs when co-cultured with macrophages and follicular helper T cells. This effect could be reproduced on combined stimulation of IL-15 (produced by macrophages) and IL-21 (produced by T follicular helper cells) in a STAT3-dependent manner. Whereas IL-21 triggers the transcription of mRNA of GrzB, IL-15 synergizes the translation of GrzB proteins. The precise role of GrzB in PC biology remains to be understood and studies in mice will not help as their PCs do not express GrzB.

  9. Posttraumatic Chondrocyte Apoptosis in the Murine Xiphoid

    Science.gov (United States)

    Davis, Christopher G.; Eisner, Eric; McGlynn, Margaret; Shelton, John M.; Richardson, James

    2013-01-01

    Objective. To demonstrate posttraumatic chondrocyte apoptosis in the murine xiphoid after a crush-type injury and to ultimately determine the pathway (i.e., intrinsic or extrinsic) by which chondrocytes undergo apoptosis in response to mechanical injury. Design. The xiphoids of adult female wild-type mice were injured with the use of a modified Kelly clamp. Postinjury xiphoid cartilage was analyzed via 3 well-described independent means of assessing apoptosis in chondrocytes: hematoxylin and eosin staining, terminal deoxynucleotidyl transferase dUTP nick end labeling assay, and activated caspase-3 staining. Results. Injured specimens contained many chondrocytes with evidence of apoptosis, which is characterized by cell shrinkage, chromatin condensation, nuclear fragmentation, and the liberation of apoptotic bodies. There was a statistically significant increase in the number of chondrocytes undergoing apoptosis in the injured specimens as compared with the uninjured specimens. Conclusions. Chondrocytes can be stimulated to undergo apoptosis as a result of mechanical injury. These experiments involving predominantly cartilaginous murine xiphoid in vivo establish a baseline for future investigations that employ the genetic and therapeutic modulation of chondrocyte apoptosis in response to mechanical injury. PMID:26069679

  10. Evc is a positive mediator of Ihh-regulated bone growth that localises at the base of chondrocyte cilia.

    Science.gov (United States)

    Ruiz-Perez, Victor L; Blair, Helen J; Rodriguez-Andres, M Elena; Blanco, Maria Jose; Wilson, Amy; Liu, Yu-Ning; Miles, Colin; Peters, Heiko; Goodship, Judith A

    2007-08-01

    EVC is a novel protein mutated in the human chondroectodermal dysplasia Ellis-van Creveld syndrome (EvC; OMIM: 225500). We have inactivated Evc in the mouse and show that Evc(-/-) mice develop an EvC-like syndrome, including short ribs, short limbs and dental abnormalities. lacZ driven by the Evc promoter revealed that Evc is expressed in the developing bones and the orofacial region. Antibodies developed against Evc locate the protein at the base of the primary cilium. The growth plate of Evc(-/-) mice shows delayed bone collar formation and advanced maturation of chondrocytes. Indian hedgehog (Ihh) is expressed normally in the growth plates of Evc(-/-) mice, but expression of the Ihh downstream genes Ptch1 and Gli1 was markedly decreased. Recent studies have shown that Smo localises to primary cilia and that Gli3 processing is defective in intraflagellar transport mutants. In vitro studies using Evc(-/-) cells demonstrate that the defect lies downstream of Smo. Chondrocyte cilia are present in Evc(-/-) mice and Gli3 processing appears normal by western blot analysis. We conclude that Evc is an intracellular component of the hedgehog signal transduction pathway that is required for normal transcriptional activation of Ihh target genes.

  11. 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.

  12. Activation of α2A-adrenergic signal transduction in chondrocytes promotes degenerative remodelling of temporomandibular joint

    Science.gov (United States)

    Jiao, Kai; Zeng, Guang; Niu, Li-Na; Yang, Hong-xu; Ren, Gao-tong; Xu, Xin-yue; Li, Fei-fei; Tay, Franklin R.; Wang, Mei-qing

    2016-01-01

    This study tested whether activation of adrenoreceptors in chondrocytes has roles in degenerative remodelling of temporomandibular joint (TMJ) and to determine associated mechanisms. Unilateral anterior crossbite (UAC) was established to induce TMJ degeneration in rats. Saline vehicle, α2- and β-adrenoreceptor antagonists or agonists were injected locally into the TMJ area of UAC rats. Cartilage degeneration, subchondral bone microarchitecture and the expression of adrenoreceptors, aggrecans, matrix metalloproteinases (MMPs) and RANKL by chondrocytes were evaluated. Chondrocytes were stimulated by norepinephrine to investigate signal transduction of adrenoreceptors. Increased α2A-adrenoreceptor expression was observed in condylar cartilage of UAC rats, together with cartilage degeneration and subchondral bone loss. Norepinephrine depresses aggrecans expression but stimulates MMP-3, MMP-13 and RANKL production by chondrocytes through ERK1/2 and PKA pathway; these effects were abolished by an α2A-adrenoreceptor antagonist. Furthermore, inhibition of α2A-adrenoreceptor attenuated degenerative remodelling in the condylar cartilage and subchondral bone, as revealed by increased cartilage thickness, proteoglycans and aggrecan expression, and decreased MMP-3, MMP-13 and RANKL expressions in cartilage, increased BMD, BV/TV, and decreased Tb.Sp in subchondral bone. Conversely, activation of α2A-adrenoreceptor intensified aforementioned degenerative changes in UAC rats. It is concluded that activation of α2A-adrenergic signal in chondrocytes promotes TMJ degenerative remodelling by chondrocyte-mediated pro-catabolic activities. PMID:27452863

  13. Chondrocyte Apoptosis in the Pathogenesis of Osteoarthritis

    Science.gov (United States)

    Hwang, Hyun Sook; Kim, Hyun Ah

    2015-01-01

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

  14. Fibulin-5 expression in the human placenta.

    Science.gov (United States)

    Gauster, Martin; Berghold, Veronika M; Moser, Gerit; Orendi, Kristina; Siwetz, Monika; Huppertz, Berthold

    2011-02-01

    Fibulin-5 is a secreted extracellular matrix glycoprotein and displays a diverse panel of biological functions, which can be segregated into elastogenic as well as extra-elastogenic functions. While elastogenic functions of fibulin-5 include essential roles in early steps of elastic fibre assembly, extra-elastogenic functions are widespread. Depending on the cell type used, fibulin-5 mediates cell adherence via a subset of integrins, antagonizes angiogenesis and inhibits migration as well as proliferation of endothelial and smooth muscle cells. In this study, we focused on the spatiotemporal expression of fibulin-5 in the human placenta. With progressing gestation, placental fibulin-5 expression increased from first trimester towards term. At term, placental fibulin-5 mRNA expression is lower when compared with other well-vascularized organs such as lung, kidney, heart, uterus and testis. In first trimester, placenta immunohistochemistry localized fibulin-5 in villous cytotrophoblasts and extravillous cytotrophoblasts of the proximal cell column. In term placenta, fibulin-5 was detected in the endothelial basement membrane and adventitia-like regions of vessels in the chorionic plate and stem villi. Cell culture experiments with the villous trophoblast-derived cell line BeWo showed that fibulin-5 expression was downregulated during functional differentiation and intercellular fusion. Moreover, cultivation of BeWo cells under low oxygen conditions impaired intercellular fusion and upregulated fibulin-5 expression. The spatiotemporal shift from the trophoblast compartment in first trimester to the villous vasculature at term suggests a dual role of fibulin-5 in human placental development.

  15. Circadian Kisspeptin expression in human term placenta.

    Science.gov (United States)

    de Pedro, M A; Morán, J; Díaz, I; Murias, L; Fernández-Plaza, C; González, C; Díaz, E

    2015-11-01

    Kisspeptin is an essential gatekeeper of reproductive function. During pregnancy high circulating levels of kisspeptin have been described, however the clear role of this neuropeptide in pregnancy remains unknown. We tested the existence of rhythmic kisspeptin expression in human full-term placenta from healthy pregnant women at six different time points during the day. The data obtained by Western blotting were fitted to a mathematical model (Fourier series), demonstrating, for the first time, the existence of a circadian rhythm in placental kisspeptin expression.

  16. [Chondrocyte mecanobiology. Application in cartilage tissue engineering].

    Science.gov (United States)

    Stoltz, Jean François; Netter, Patrick; Huselstein, Céline; de Isla, Natalia; Wei Yang, Jing; Muller, Sylvaine

    2005-11-01

    Cartilage is a hydrated connective tissue that withstands and distributes mechanical forces within joints. Chondrocytes utilize mechanical signals to maintain cartilaginous tissue homeostasis. They regulate their metabolic activity through complex biological and biophysical interactions with the extracellular matrix (ECM). Some mechanotransduction mechanisms are known, while many others no doubt remain to be discovered. Various aspects of chondrocyte mechanobiology have been applied to tissue engineering, with the creation of replacement tissue in vitro from bioresorbable or non-bioresorbable scaffolds and harvested cells. The tissues are maintained in a near-physiologic mechanical and biochemical environment. This paper is an overview of both chondrocyte mechanobiology and cartilage tissue engineering

  17. Functional Characterization of TRPV4 As an Osmotically Sensitive Ion Channel in Articular Chondrocytes

    Science.gov (United States)

    Phan, Mimi N.; Leddy, Holly A.; Votta, Bartholomew J.; Kumar, Sanjay; Levy, Dana S.; Lipshutz, David B.; Lee, Sukhee; Liedtke, Wolfgang; Guilak, Farshid

    2010-01-01

    Objective Transient receptor potential vanilloid 4 (TRPV4) is a Ca2+ permeable channel that can be gated by tonicity (osmolarity) and mechanical stimuli. Chondrocytes, the cells in cartilage, respond to their osmotic and mechanical environments; however, the molecular basis of this signal transduction is not fully understood. The objective of this study was to demonstrate the presence and functionality of TRPV4 in chondrocytes. Methods TRPV4 protein expression was measured by immunolabeling and Western blotting. In response to TRPV4 agonist/antagonists, osmotic stress, and interleukin-1 (IL-1), changes in Ca2+ signaling, cell volume, and prostaglandin E2 (PGE2) production were measured in porcine chondrocytes using fluorescence microscopy, light microscopy, or immunoassay, respectively. Results TRPV4 was expressed abundantly at the RNA and protein level. Exposure to 4αPDD, a TRPV4 activator, caused Ca2+ signaling in chondrocytes, which was blocked by the selective TRPV4 antagonist, GSK205. Blocking TRPV4 diminished the chondrocytes' response to hypo-osmotic stress, reducing the fraction of Ca2+ responsive cells, regulatory volume decrease (RVD), and PGE2 production. Ca2+ signaling was inhibited by removal of extracellular Ca2+ or depletion of intracellular stores. Specific activation of TRPV4 restored defective RVD caused by IL-1. Chemical disruption of the primary cilium eliminated Ca2+ signaling in response to either 4αPDD or hypo-osmotic stress. Conclusion TRPV4 is present in articular chondrocytes, and chondrocyte response to hypo-osmotic stress is mediated by this channel, which involves both an extracellular Ca2+ and intracellular Ca2+ release. TRPV4 may also be involved in modulating the production or influence of pro-inflammatory molecules in response to osmotic stress. PMID:19790068

  18. A-raf and B-raf are dispensable for normal endochondral bone development, and parathyroid hormone-related peptide suppresses extracellular signal-regulated kinase activation in hypertrophic chondrocytes.

    Science.gov (United States)

    Provot, Sylvain; Nachtrab, Gregory; Paruch, Jennifer; Chen, Adele Pin; Silva, Alcino; Kronenberg, Henry M

    2008-01-01

    Parathyroid hormone-related peptide (PTHrP) and the parathyroid hormone-PTHrP receptor increase chondrocyte proliferation and delay chondrocyte maturation in endochondral bone development at least partly through cyclic AMP (cAMP)-dependent signaling pathways. Because data suggest that the ability of cAMP to stimulate cell proliferation involves the mitogen-activated protein kinase kinase kinase B-Raf, we hypothesized that B-Raf might mediate the proliferative action of PTHrP in chondrocytes. Though B-Raf is expressed in proliferative chondrocytes, its conditional removal from cartilage did not affect chondrocyte proliferation and maturation or PTHrP-induced chondrocyte proliferation and PTHrP-delayed maturation. Similar results were obtained by conditionally removing B-Raf from osteoblasts. Because A-raf and B-raf are expressed similarly in cartilage, we speculated that they may fulfill redundant functions in this tissue. Surprisingly, mice with chondrocytes deficient in both A-Raf and B-Raf exhibited normal endochondral bone development. Activated extracellular signal-regulated kinase (ERK) was detected primarily in hypertrophic chondrocytes, where C-raf is expressed, and the suppression of ERK activation in these cells by PTHrP or a MEK inhibitor coincided with a delay in chondrocyte maturation. Taken together, these results demonstrate that B-Raf and A-Raf are dispensable for endochondral bone development and they indicate that the main role of ERK in cartilage is to stimulate not cell proliferation, but rather chondrocyte maturation.

  19. Epiphyseal abnormalities, trabecular bone loss and articular chondrocyte hypertrophy develop in the long bones of postnatal Ext1-deficient mice.

    Science.gov (United States)

    Sgariglia, Federica; Candela, Maria Elena; Huegel, Julianne; Jacenko, Olena; Koyama, Eiki; Yamaguchi, Yu; Pacifici, Maurizio; Enomoto-Iwamoto, Motomi

    2013-11-01

    Long bones are integral components of the limb skeleton. Recent studies have indicated that embryonic long bone development is altered by mutations in Ext genes and consequent heparan sulfate (HS) deficiency, possibly due to changes in activity and distribution of HS-binding/growth plate-associated signaling proteins. Here we asked whether Ext function is continuously required after birth to sustain growth plate function and long bone growth and organization. Compound transgenic Ext1(f/f);Col2CreERT mice were injected with tamoxifen at postnatal day 5 (P5) to ablate Ext1 in cartilage and monitored over time. The Ext1-deficient mice exhibited growth retardation already by 2weeks post-injection, as did their long bones. Mutant growth plates displayed a severe disorganization of chondrocyte columnar organization, a shortened hypertrophic zone with low expression of collagen X and MMP-13, and reduced primary spongiosa accompanied, however, by increased numbers of TRAP-positive osteoclasts at the chondro-osseous border. The mutant epiphyses were abnormal as well. Formation of a secondary ossification center was significantly delayed but interestingly, hypertrophic-like chondrocytes emerged within articular cartilage, similar to those often seen in osteoarthritic joints. Indeed, the cells displayed a large size and round shape, expressed collagen X and MMP-13 and were surrounded by an abundant Perlecan-rich pericellular matrix not seen in control articular chondrocytes. In addition, ectopic cartilaginous outgrowths developed on the lateral side of mutant growth plates over time that resembled exostotic characteristic of children with Hereditary Multiple Exostoses, a syndrome caused by Ext mutations and HS deficiency. In sum, the data do show that Ext1 is continuously required for postnatal growth and organization of long bones as well as their adjacent joints. Ext1 deficiency elicits defects that can occur in human skeletal conditions including trabecular bone loss

  20. The human endolymphatic sac expresses natriuretic peptides

    DEFF Research Database (Denmark)

    Møller, Martin Nue; Kirkeby, Svend; Vikeså, Jonas

    2017-01-01

    in intracranial pressure homeostasis. However, no direct evidence of such capacity exists. This study aims to explore and identify the hypothesized endocrine capacity of the human ES. STUDY DESIGN: DNA microarrays and immunohistochemistry were used for analyses of fresh human ES tissue samples. METHODS: Twelve...... vasopressin receptors and aquaporin-2 channels in the inner ear via OXT expression. We hypothesize that the ES is likely to regulate inner ear endolymphatic homeostasis, possibly through secretion of several peptides, but it may also influence systemic and/or intracranial blood pressure through direct...

  1. Rebamipide attenuates pain severity and cartilage degeneration in a rat model of osteoarthritis by downregulating oxidative damage and catabolic activity in chondrocytes.

    Science.gov (United States)

    Moon, S-J; Woo, Y-J; Jeong, J-H; Park, M-K; Oh, H-J; Park, J-S; Kim, E-K; Cho, M-L; Park, S-H; Kim, H-Y; Min, J-K

    2012-11-01

    The objectives were to investigate the in vivo effects of treatment with rebamipide on pain severity and cartilage degeneration in an experimental model of rat osteoarthritis (OA) and to explore its mode of action. OA was induced in rats by intra-articular injection of monosodium iodoacetate (MIA). Oral administration of rebamipide was initiated on the day of MIA injection, 3 or 7 days after. Limb nociception was assessed by measuring the paw withdrawal latency and threshold. We analyzed the samples macroscopically and histomorphologically, and used immunohistochemistry to investigate the expression of matrix metalloproteinase-13 (MMP-13), interleukin-1β (IL-1β), hypoxia-inducible factor-2α (HIF-2α), inducible nitric oxide synthase (iNOS), and nitrotyrosine in knee joints. Real-time quantitative reverse transcription-polymerase chain reaction was used to quantify the mRNA for catabolic and anticatabolic factors in human OA chondrocytes. Rebamipide showed an antinociceptive property and attenuated cartilage degeneration. Rebamipide reduced the expression of MMP-13, IL-1β, HIF-2α, iNOS, and nitrotyrosine in OA cartilage in a dose-dependent manner. Nitrotyrosine expression in the subchondral bone region was decreased in the rebamipide-treated joints. mRNA expression of MMP-1, -3, and -13, and ADAMTS5 was attenuated in IL-1β-stimulated human OA chondrocytes. By contrast, rebamipide induced the mRNA expression of tissue inhibitor of metalloproteinase-1 and -3. The results show the inhibitory effects of rebamipide on pain production and cartilage degeneration in experimentally induced OA. The suppression of oxidative damage and the restoration of extracellular matrix homeostasis of articular chondrocyte suggest that rebamipide is a potential therapeutic strategy for OA. Crown Copyright © 2012. Published by Elsevier Ltd. All rights reserved.

  2. THE ACTIVATION OF MATRIX METALLOPROTEINASES AND CHONDROCYTE DIFFERENTIATION, WHICH ACCOMPANIES THE INDUCTION OF COLLAGEN DECOMPOSITION UNDER THE ACTION OF COLLAGEN PEPTIDE IN THE CARTILAGE OFHEALTHY INDIVIDUALS

    Directory of Open Access Journals (Sweden)

    Elena Vasil'evna Chetina

    2010-01-01

    Conclusion. This study has shown that the induction of collagenase activity by CB12-2 in the human articular cartilage chondrocytes is attended by terminal differentiation/hypertrophy of these cells. The terminal differentiation of chondrocytes may be one of the mechanisms of chondrolysis in osteoarthrosis since it naturally occurs not only in endochondrial ossification, but also in the development of pathology.

  3. Genes of periodontopathogens expressed during human disease.

    Science.gov (United States)

    Song, Yo-Han; Kozarov, Emil V; Walters, Sheila M; Cao, Sam Linsen; Handfield, Martin; Hillman, Jeffrey D; Progulske-Fox, Ann

    2002-12-01

    Since many bacterial genes are environmentally regulated, the screening for virulence-associated factors using classical genetic and molecular biology approaches can be biased under laboratory growth conditions of a given pathogen, because the required conditions for expression of many virulence factors may not occur during in vitro growth. Thus, technologies have been developed during the past several years to identify genes that are expressed during disease using animal models of human disease. However, animal models are not always truly representative of human disease, and with many pathogens, there is no appropriate animal model. A new technology, in vivo-induced antigen technology (IVIAT) was thus engineered and tested in our laboratory to screen for genes of pathogenic organisms induced specifically in humans, without the use of animal or artificial models of infection. This technology uses pooled sera from patients to probe for genes expressed exclusively in vivo (or ivi, in vivo-induced genes). IVIAT was originally designed for the study of Actinobacillus actinomycetemcomitans pathogenesis, but we have now extended it to other oral pathogens including Porphyromonas gingivalis. One hundred seventy-one thousand (171,000) clones from P. gingivalis strain W83 were screened and 144 were confirmed positive. Over 300,000 A. actinomycetemcomitans clones were probed, and 116 were confirmed positive using a quantitative blot assay. MAT has proven useful in identifying previously unknown in vivo-induced genes that are likely involved in virulence and are thus excellent candidates for use in diagnostic : and therapeutic strategies, including vaccine design.

  4. 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.

  5. A Comparison of Artificial Subtle Expressions with Human-like Expressions on Expressing Confidence Level

    Science.gov (United States)

    Komatsu, Takanori; Kobayashi, Kazuki; Yamada, Seiji; Funakoshi, Kotaro; Nakano, Mikio

    Expressing the confidence level of a system's suggestions by using speech sounds is an important cue to users of the system for perceiving how likely it is for the suggestions to be correct. We assume that expressing confidence levels by using human-like expressions would cause users to have a poorer impression of the systems than if artificial subtle expressions (ASEs) were used when the quality of the presented information does not match the expressed confidence level. We confirmed that this assumption was correct by conducting a psychological experiment.

  6. The Effect of the Human Peptide GHK on Gene Expression Relevant to Nervous System Function and Cognitive Decline.

    Science.gov (United States)

    Pickart, Loren; Vasquez-Soltero, Jessica Michelle; Margolina, Anna

    2017-02-15

    Neurodegeneration, the progressive death of neurons, loss of brain function, and cognitive decline is an increasing problem for senior populations. Its causes are poorly understood and therapies are largely ineffective. Neurons, with high energy and oxygen requirements, are especially vulnerable to detrimental factors, including age-related dysregulation of biochemical pathways caused by altered expression of multiple genes. GHK (glycyl-l-histidyl-l-lysine) is a human copper-binding peptide with biological actions that appear to counter aging-associated diseases and conditions. GHK, which declines with age, has health promoting effects on many tissues such as chondrocytes, liver cells and human fibroblasts, improves wound healing and tissue regeneration (skin, hair follicles, stomach and intestinal linings, boney tissue), increases collagen, decorin, angiogenesis, and nerve outgrowth, possesses anti-oxidant, anti-inflammatory, anti-pain and anti-anxiety effects, increases cellular stemness and the secretion of trophic factors by mesenchymal stem cells. Studies using the Broad Institute Connectivity Map show that GHK peptide modulates expression of multiple genes, resetting pathological gene expression patterns back to health. GHK has been recommended as a treatment for metastatic cancer, Chronic Obstructive Lung Disease, inflammation, acute lung injury, activating stem cells, pain, and anxiety. Here, we present GHK's effects on gene expression relevant to the nervous system health and function.

  7. The Effect of the Human Peptide GHK on Gene Expression Relevant to Nervous System Function and Cognitive Decline

    Science.gov (United States)

    Pickart, Loren; Vasquez-Soltero, Jessica Michelle; Margolina, Anna

    2017-01-01

    Neurodegeneration, the progressive death of neurons, loss of brain function, and cognitive decline is an increasing problem for senior populations. Its causes are poorly understood and therapies are largely ineffective. Neurons, with high energy and oxygen requirements, are especially vulnerable to detrimental factors, including age-related dysregulation of biochemical pathways caused by altered expression of multiple genes. GHK (glycyl-l-histidyl-l-lysine) is a human copper-binding peptide with biological actions that appear to counter aging-associated diseases and conditions. GHK, which declines with age, has health promoting effects on many tissues such as chondrocytes, liver cells and human fibroblasts, improves wound healing and tissue regeneration (skin, hair follicles, stomach and intestinal linings, boney tissue), increases collagen, decorin, angiogenesis, and nerve outgrowth, possesses anti-oxidant, anti-inflammatory, anti-pain and anti-anxiety effects, increases cellular stemness and the secretion of trophic factors by mesenchymal stem cells. Studies using the Broad Institute Connectivity Map show that GHK peptide modulates expression of multiple genes, resetting pathological gene expression patterns back to health. GHK has been recommended as a treatment for metastatic cancer, Chronic Obstructive Lung Disease, inflammation, acute lung injury, activating stem cells, pain, and anxiety. Here, we present GHK’s effects on gene expression relevant to the nervous system health and function. PMID:28212278

  8. The Effect of the Human Peptide GHK on Gene Expression Relevant to Nervous System Function and Cognitive Decline

    Directory of Open Access Journals (Sweden)

    Loren Pickart

    2017-02-01

    Full Text Available Neurodegeneration, the progressive death of neurons, loss of brain function, and cognitive decline is an increasing problem for senior populations. Its causes are poorly understood and therapies are largely ineffective. Neurons, with high energy and oxygen requirements, are especially vulnerable to detrimental factors, including age-related dysregulation of biochemical pathways caused by altered expression of multiple genes. GHK (glycyl-l-histidyl-l-lysine is a human copper-binding peptide with biological actions that appear to counter aging-associated diseases and conditions. GHK, which declines with age, has health promoting effects on many tissues such as chondrocytes, liver cells and human fibroblasts, improves wound healing and tissue regeneration (skin, hair follicles, stomach and intestinal linings, boney tissue, increases collagen, decorin, angiogenesis, and nerve outgrowth, possesses anti-oxidant, anti-inflammatory, anti-pain and anti-anxiety effects, increases cellular stemness and the secretion of trophic factors by mesenchymal stem cells. Studies using the Broad Institute Connectivity Map show that GHK peptide modulates expression of multiple genes, resetting pathological gene expression patterns back to health. GHK has been recommended as a treatment for metastatic cancer, Chronic Obstructive Lung Disease, inflammation, acute lung injury, activating stem cells, pain, and anxiety. Here, we present GHK’s effects on gene expression relevant to the nervous system health and function.

  9. Human basophils express interleukin-4 receptors

    Energy Technology Data Exchange (ETDEWEB)

    Valent, P.; Besemer, J.; Kishi, K.; Di Padova, F.; Geissler, K.; Lechner, K.; Bettelheim, P. (Univ. of Vienna (Austria))

    1990-11-01

    Interleukin-4 (IL-4), a multipotential lymphokine reputed to play an important role in the regulation of immune responses, interacts with a variety of hemopoietic target cells through specific cell surface membrane receptors. The present study was designed to investigate whether human basophils express IL-4 binding sites. For this purpose, basophils were enriched to homogeneity (93% and 98% purity, respectively) from the peripheral blood of two chronic granulocytic leukemia (CGL) donors using a cocktail of monoclonal antibodies (MoAbs) and complement. Purified basophils bound 125I-radiolabeled recombinant human (rh) IL-4 in a specific manner. Quantitative binding studies and Scatchard plot analysis revealed the presence of a single class of high affinity IL-4 binding sites (280 +/- 40 sites per cell in donor 1 and 640 +/- 45 sites per cell in donor 2) with an apparent dissociation constant, kd, of 7.12 x 10(-11) +/- 2.29 x 10(-11) and 9.55 +/- 3.5 x 10(-11) mol/L, respectively. KU812-F, a human basophil precursor cell line, was found to express a single class of 810 to 1,500 high affinity IL-4 binding sites with a kd of 2.63 to 5.54 x 10(-10) mol/L. No change in the numbers or binding constants of IL-4 receptors was found after exposure of KU812-F cells to rhIL-3 (a potent activator of basophils) for 60 minutes. No effect of rhIL-4 on 3H-thymidine uptake, release or synthesis of histamine, or expression of basophil differentiation antigens (Bsp-1, CD11b, CD25, CD40, CD54) on primary human CGL basophils or KU812-F cells was observed.

  10. 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

  11. Combining Targeted Metabolomic Data with a Model of Glucose Metabolism: Toward Progress in Chondrocyte Mechanotransduction

    Science.gov (United States)

    Salinas, Daniel; Carlson, Ross P.; McCutchen, Carley N.

    2017-01-01

    Osteoarthritis is a debilitating disease likely involving altered metabolism of the chondrocytes in articular cartilage. Chondrocytes can respond metabolically to mechanical loads via cellular mechanotransduction, and metabolic changes are significant because they produce the precursors to the tissue matrix necessary for cartilage health. However, a comprehensive understanding of how energy metabolism changes with loading remains elusive. To improve our understanding of chondrocyte mechanotransduction, we developed a computational model to calculate the rate of reactions (i.e. flux) across multiple components of central energy metabolism based on experimental data. We calculated average reaction flux profiles of central metabolism for SW1353 human chondrocytes subjected to dynamic compression for 30 minutes. The profiles were obtained solving a bounded variable linear least squares problem, representing the stoichiometry of human central energy metabolism. Compression synchronized chondrocyte energy metabolism. These data are consistent with dynamic compression inducing early time changes in central energy metabolism geared towards more active protein synthesis. Furthermore, this analysis demonstrates the utility of combining targeted metabolomic data with a computational model to enable rapid analysis of cellular energy utilization. PMID:28056047

  12. 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.

  13. Prolyl Hydroxylase Domain-Containing Protein 2 (Phd2) Regulates Chondrocyte Differentiation and Secondary Ossification in Mice

    Science.gov (United States)

    Cheng, Shaohong; Aghajanian, Patrick; Pourteymoor, Sheila; Alarcon, Catrina; Mohan, Subburaman

    2016-01-01

    Endochondral ossification plays an important role in the formation of the primary ossification centers (POCs) and secondary ossification centers (SOCs) of mammalian long bones. However, the molecular mechanisms that regulate POC and SOC formation are different. We recently demonstrated that Prolyl Hydroxylase Domain-containing Protein 2 (Phd2) is a key mediator of vitamin C effects on bone. We investigated the role of Phd2 on endochondral ossification of the epiphyses by conditionally deleting the Phd2 gene in osteoblasts and chondrocytes. We found that the deletion of Phd2 in osteoblasts did not cause changes in bone parameters in the proximal tibial epiphyses in 5 week old mice. In contrast, deletion of Phd2 in chondrocytes resulted in increased bone mass and bone formation rate (normalized to tissue volume) in long bone epiphyses, indicating that Phd2 expressed in chondrocytes, but not osteoblasts, negatively regulates secondary ossification of epiphyses. Phd2 deletion in chondrocytes elevated mRNA expression of hypoxia-inducible factor (HIF) signaling molecules including Hif-1α, Hif-2α, Vegfa, Vegfb, and Epo, as well as markers for chondrocyte hypertrophy and mineralization such as Col10, osterix, alkaline phosphatase, and bone sialoprotein. These data suggest that Phd2 expressed in chondrocytes inhibits endochondral ossification at the epiphysis by suppressing HIF signaling pathways. PMID:27775044

  14. Aquaporin 1 contributes to chondrocyte apoptosis in a rat model of osteoarthritis.

    Science.gov (United States)

    Gao, Hangfei; Gui, Jiancao; Wang, Liming; Xu, Yan; Jiang, Yiqiu; Xiong, Mingyue; Cui, Yongguang

    2016-12-01

    Aquaporins (AQPs) have been found to be associated with a number of diseases. However, the role of AQP‑1 in the pathogenesis of osteoarthritis remains unclear. We previously found that AQP‑1 expression was upregulated in osteoarthritic cartilage and strongly correlated with caspase‑3 expression and activity. The aim of this study was to further investigate the association of AQP‑1 expression with chondrocyte apoptosis in a rat model of osteoarthritis, using RNA interference to knock down AQP‑1. For this purspose, 72 male Sprague‑Dawley rats were randomly assigned to 3 groups as follows: the control group not treated surgically (n=24), the sham‑operated group (n=24), and the osteoarthritis group (n=24). Osteoarthritis was induced by amputating the anterior cruciate ligament and medial collateral ligament and partially excising the medial meniscus. Chondrocytes from the rats with osteoarthritis were isolated and cultured. shRNAs were used to knock down AQP‑1 expression in the cultured chondrocytes. The expression of AQP‑1 and caspase‑3 was determined by reverse transcription-quantitative polymerase chain reaction. Caspase‑3 activity was measured using a caspase‑3 colorimetric assay. The rats in our model of osteoarthritis exhibited severe cartilage damage. The knockdown of AQP‑1 decreased caspase‑3 expression and activity in the cultured chondrocytes. In addition, the expression of AQP‑1 positively correlated with caspase‑3 expression and activity. Thus, the findings of our study, suggest that AQP‑1 promotes caspase‑3 activation and thereby contributes to chondrocyte apoptosis and to the development of osteoarthritis.

  15. Overexpression of Galnt3 in chondrocytes resulted in dwarfism due to the increase of mucin-type O-glycans and reduction of glycosaminoglycans.

    Science.gov (United States)

    Yoshida, Carolina Andrea; Kawane, Tetsuya; Moriishi, Takeshi; Purushothaman, Anurag; Miyazaki, Toshihiro; Komori, Hisato; Mori, Masako; Qin, Xin; Hashimoto, Ayako; Sugahara, Kazuyuki; Yamana, Kei; Takada, Kenji; Komori, Toshihisa

    2014-09-19

    Galnt3, UDP-N-acetyl-α-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase 3, transfers N-acetyl-D-galactosamine to serine and threonine residues, initiating mucin type O-glycosylation of proteins. We searched the target genes of Runx2, which is an essential transcription factor for chondrocyte maturation, in chondrocytes and found that Galnt3 expression was up-regulated by Runx2 and severely reduced in Runx2(-/-) cartilaginous skeletons. To investigate the function of Galnt3 in chondrocytes, we generated Galnt3(-/-) mice and chondrocyte-specific Galnt3 transgenic mice under the control of the Col2a1 promoter-enhancer. Galnt3(-/-) mice showed a delay in endochondral ossification and shortened limbs at embryonic day 16.5, suggesting that Galnt3 is involved in chondrocyte maturation. Galnt3 transgenic mice presented dwarfism, the chondrocyte maturation was retarded, the cell cycle in chondrocytes was accelerated, premature chondrocyte apoptosis occurred, and the growth plates were disorganized. The binding of Vicia villosa agglutinin, which recognizes the Tn antigen (GalNAc-O-Ser/Thr), was drastically increased in chondrocytes, and aggrecan (Acan) was highly enriched with Tn antigen. However, safranin O staining, which recognizes glycosaminoglycans (GAGs), and Acan were severely reduced. Chondroitin sulfate was reduced in amount, but the elongation of chondroitin sulfate chains had not been severely disturbed in the isolated GAGs. These findings indicate that overexpression of Galnt3 in chondrocytes caused dwarfism due to the increase of mucin-type O-glycans and the reduction of GAGs, probably through competition with xylosyltransferases, which initiate GAG chains by attaching O-linked xylose to serine residues, suggesting a negative effect of Galnt family proteins on Acan deposition in addition to the positive effect of Galnt3 on chondrocyte maturation. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  16. Hepatocyte growth factor/scatter factor modulates cell motility, proliferation, and proteoglycan synthesis of chondrocytes.

    Science.gov (United States)

    Takebayashi, T; Iwamoto, M; Jikko, A; Matsumura, T; Enomoto-Iwamoto, M; Myoukai, F; Koyama, E; Yamaai, T; Matsumoto, K; Nakamura, T

    1995-06-01

    Hepatocyte growth factor/scatter factor (HGF/SF) is a multifunctional growth factor that promotes proliferation, motility, and morphogenesis in epithelial cells. Recently the HGF receptor, c-met protooncogene product, has been shown to be expressed in developing limb buds (Sonnenberg, E., D. Meyer, M. Weidner, and C. Birchmeiyer, 1993. J. Cell Biol. 123: 223-235), suggesting that some populations of mesenchymal cells in limb buds respond to HGF/SF. To test the possibility that HGF/SF is involved in regulation of cartilage development, we isolated chondrocytes from knee joints and costal cartilages of 23-d embryonic and 4-wk-old rabbits, and analyzed the effects of HGF/SF on migration and proliferation of these cells. We found that HGF/SF stimulated migration of cultured articular chondrocytes but did not scatter limb mesenchymal fibroblasts or synovial fibroblasts in culture. HGF/SF also stimulated proliferation of chondrocytes; a maximum three-fold stimulation in DNA synthesis was observed at the concentration of 3 ng/ml of HGF/SF. Moreover, HGF/SF had the ability to enhance proteoglycan synthesis in chondrocytes. The responsiveness of chondrocytes to HGF/SF was also supported by the observation that they expressed the HGF/SF receptor. Addition of the neutralizing antibody to rat HGF/SF affected neither DNA synthesis nor proteoglycan synthesis in rat chondrocytes, suggesting a paracine mechanism of action of HGF/SF on these cells. In situ hybridization analysis showed that HGF/SF mRNA was restrictively expressed in the areas of future joint regions in developing limb buds and in the intercostal spaces of developing costal cartilages. These findings suggest that HGF/SF plays important roles in cartilage development through its multiple activities.

  17. Hepatocyte growth factor/scatter factor modulates cell motility, proliferation, and proteoglycan synthesis of chondrocytes

    Science.gov (United States)

    1995-01-01

    Hepatocyte growth factor/scatter factor (HGF/SF) is a multifunctional growth factor that promotes proliferation, motility, and morphogenesis in epithelial cells. Recently the HGF receptor, c-met protooncogene product, has been shown to be expressed in developing limb buds (Sonnenberg, E., D. Meyer, M. Weidner, and C. Birchmeiyer, 1993. J. Cell Biol. 123: 223-235), suggesting that some populations of mesenchymal cells in limb buds respond to HGF/SF. To test the possibility that HGF/SF is involved in regulation of cartilage development, we isolated chondrocytes from knee joints and costal cartilages of 23-d embryonic and 4-wk-old rabbits, and analyzed the effects of HGF/SF on migration and proliferation of these cells. We found that HGF/SF stimulated migration of cultured articular chondrocytes but did not scatter limb mesenchymal fibroblasts or synovial fibroblasts in culture. HGF/SF also stimulated proliferation of chondrocytes; a maximum three-fold stimulation in DNA synthesis was observed at the concentration of 3 ng/ml of HGF/SF. Moreover, HGF/SF had the ability to enhance proteoglycan synthesis in chondrocytes. The responsiveness of chondrocytes to HGF/SF was also supported by the observation that they expressed the HGF/SF receptor. Addition of the neutralizing antibody to rat HGF/SF affected neither DNA synthesis nor proteoglycan synthesis in rat chondrocytes, suggesting a paracine mechanism of action of HGF/SF on these cells. In situ hybridization analysis showed that HGF/SF mRNA was restrictively expressed in the areas of future joint regions in developing limb buds and in the intercostal spaces of developing costal cartilages. These findings suggest that HGF/SF plays important roles in cartilage development through its multiple activities. PMID:7775584

  18. Mechanical vibrations increase the proliferation of articular chondrocytes in high-density culture.

    Science.gov (United States)

    Kaupp, J A; Waldman, S D

    2008-07-01

    Tissue engineering is a promising approach for articular cartilage repair; however, it still has proven a challenge to produce tissue from the limited number of cells that can be extracted from a single individual. Relatively few cell expansion methods exist without the problems of dedifferentiation and/or loss of potency. Previously, it has been shown that mechanical vibrations can enhance chondrocyte proliferation in monolayer culture. Thus, it was hypothesized that chondrocytes grown in high-density culture would respond in a similar fashion while maintaining phenotypic stability. Isolated bovine articular chondrocytes were seeded in high-density culture on Millicell filters and subjected to mechanical vibrations 48 h after seeding. Mechanical vibrations enhanced chondrocyte proliferation at frequencies above 350 Hz, with the peak response occurring at a 1g amplitude for a duration of 30 min. Under these conditions, the gene expression of cartilage-specific and dedifferentiation markers (collagen II, collagen I, and aggrecan) were unchanged by the imposed stimulus. To determine the effect of accumulated extracellular matrix (ECM) on this proliferative response, selected cultures were stimulated under the same conditions after varying lengths of preculture. The amount of accumulated ECM (collagen and proteoglycans) decreased this proliferative response, with the cultures becoming insensitive to the stimulus after 1 week of preculture. Thus, mechanical vibration can serve as an effective means preferentially to stimulate the proliferation of chondrocytes during culture, but its effects appear to be limited to the early stages where ECM accumulation is at a minimum.

  19. 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.

  20. Ear-Shaped Stable Auricular Cartilage Engineered from Extensively Expanded Chondrocytes in an Immunocompetent Experimental Animal Model.

    Science.gov (United States)

    Pomerantseva, Irina; Bichara, David A; Tseng, Alan; Cronce, Michael J; Cervantes, Thomas M; Kimura, Anya M; Neville, Craig M; Roscioli, Nick; Vacanti, Joseph P; Randolph, Mark A; Sundback, Cathryn A

    2016-02-01

    Advancement of engineered ear in clinical practice is limited by several challenges. The complex, largely unsupported, three-dimensional auricular neocartilage structure is difficult to maintain. Neocartilage formation is challenging in an immunocompetent host due to active inflammatory and immunological responses. The large number of autologous chondrogenic cells required for engineering an adult human-sized ear presents an additional challenge because primary chondrocytes rapidly dedifferentiate during in vitro culture. The objective of this study was to engineer a stable, human ear-shaped cartilage in an immunocompetent animal model using expanded chondrocytes. The impact of basic fibroblast growth factor (bFGF) supplementation on achieving clinically relevant expansion of primary sheep chondrocytes by in vitro culture was determined. Chondrocytes expanded in standard medium were either combined with cryopreserved, primary passage 0 chondrocytes at the time of scaffold seeding or used alone as control. Disk and human ear-shaped scaffolds were made from porous collagen; ear scaffolds had an embedded, supporting titanium wire framework. Autologous chondrocyte-seeded scaffolds were implanted subcutaneously in sheep after 2 weeks of in vitro incubation. The quality of the resulting neocartilage and its stability and retention of the original ear size and shape were evaluated at 6, 12, and 20 weeks postimplantation. Neocartilage produced from chondrocytes that were expanded in the presence of bFGF was superior, and its quality improved with increased implantation time. In addition to characteristic morphological cartilage features, its glycosaminoglycan content was high and marked elastin fiber formation was present. The overall shape of engineered ears was preserved at 20 weeks postimplantation, and the dimensional changes did not exceed 10%. The wire frame within the engineered ear was able to withstand mechanical forces during wound healing and neocartilage

  1. The Effects of Vitamin D3 on Proliferation and Apoptosis of Primary Cultured Chondrocytes from Human Articular Cartilage%活性维生素D3对体外原代培养人骨关节炎软骨细胞的影响

    Institute of Scientific and Technical Information of China (English)

    张良; 郭艾

    2012-01-01

    Objective To obtain articular chondrocyte from patients with osteoarthritis by primary culture,to explore the relationship between different concentrate [1, 25-(OH)2D3]and proliferation, apoptosis of chondrocytes. Methods Human articular chondrocytes were obtained by modified digestive method with enzyme in vitro and inden-tified by alkaline phosphatase (AKP) staining. OA Chondrocytes were cultured in medium with different concentrate [1,25-(OH)2D3]. MMT method was used to assay proliferation of chondrocytes. Optical density(OD) at 490nm was determined by ELISA. Flow cytometry was used to assay the apoptosis ratio at different time point. Results 10~5 u-mol/L[l ,25-(OH)2D3]can promote the proliferation of chondrocytes obviously,and the proper react time point is 48 hour(P<0. 01). At the same time 10-5 umol/L[1, 25-(OH)2D3]can inhibit the apoptosis ratio obviously at 48 hour time point. Conclusion To promote the proliferation and inhibit the apoptosis of OA chondrocytes in vitro,the better concentration of [1,25-(OH)2D3]is 1×10-5 umol/L. 48 hour after[1,25-(OH)2D3]added is the best time for the experiment objective.%目的 探讨不同浓度的1α,25二羟基维生素D3[1,25 -(OH)2D3]对体外培养的人骨关节炎患者关节软骨细胞的增殖及凋亡率的影响.方法 酶二步消化法体外分离培养人软骨细胞,以碱性磷酸酶染色法鉴定.加入不同剂量的[1,25 (OH)2D3],通过噻唑蓝比色试验检测细胞存活和增殖情况,以及用流式细胞仪检测软骨细胞在不同药物浓度、不同作用时间的凋亡率.结论 [1,25 (OH)2D3]作用于入骨关节炎软骨细胞的最佳作用浓度为1×10-5umol/L,最佳作用时间点为48h.高浓度的[1,25-(OH)2D3]能明显促进细胞坏死,极低浓度的[1,25 -(OH)2D3]对软骨细胞增殖、凋亡无明显影响.

  2. [The synergistic effect of amygdalin and HSYA on the IL-1beta induced endplate chondrocytes of rat intervertebral discs].

    Science.gov (United States)

    Niu, Kai; Zhao, Yong-Jian; Zhang, Lei; Li, Chen-Guang; Wang, Yong-Jun; Zheng, Wei-Chao

    2014-08-01

    The effect of amygdalin joint hydroxysafflor yellow A (HSYA) on the endplate chondrocytes derived from intervertebral discs of rats induced by IL-1beta and the possible mechanism were studied and explored. Chondrocytes were obtained from endplate of one-month SD rat intervertebral discs and cultured primary endplate chondrocytes. After identification, they were divided into normal group, induced group, amygdalin group, HSYA group and combined group. CCK-8 kit was adopted to detect the proliferation of the endplate chondrocytes. FCM was measured to detect the apoptosis. Real-time PCR method was adopted to observe the mRNA expression of Aggrecan, Col 2 alpha1, Col 10 alpha1, MMP-13 and the inflammatory cytokines IL-1beta. The protein expression of Col II, Col X was tested through immunofluorescence. Compared with the normal group, the proliferation of the endplate chondrocytes decreased while the apoptosis increased (P amygdalin group, HSYA group, the combined group could inhibit the apoptosis and promote the proliferation (P amygdalin and HSYA. Amygdalin joint HSYA could inhibit the degeneration of the endplate chondrocytes derived from intervertebral discs of rats induced by IL-1beta and better than the single use of amygdalin or HSYA.

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

    Science.gov (United States)

    Nazempour, A; Van Wie, B J

    2016-05-01

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

  4. 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.

  5. Expression of BMP-receptor type 1A correlates with progress of osteoarthritis in human knee joints with focal cartilage lesions

    DEFF Research Database (Denmark)

    Schmal, Hagen; Pilz, Ingo H; Mehlhorn, Alexander T;

    2012-01-01

    was significantly higher in cartilage biopsies taken in type 3 lesions with intact subchondral layer compared with type 4 defects (P ...BACKGROUND AIMS: Bone morphogenetic protein-2 (BMP-2) and its receptor type 1A (BMPR-1A) play significant roles in cartilage metabolism. The aim of this study was to evaluate a possible correlation between intra-articular expression of these proteins and the degree of osteoarthritis (OA) in human...... knees. METHODS: Biopsies of synovia and debrided cartilage were taken in 15 patients undergoing autologous chondrocyte implantation. Expression of BMP-2 and BMPR-1A was evaluated semi-quantitatively by immunohistologic staining. These data were complemented by grading of cartilage lesions according...

  6. Potential of Raloxifene in reversing osteoarthritis-like alterations in rat chondrocytes: An in vitro model study

    Indian Academy of Sciences (India)

    Aysegul Kavas; Seda Tuncay Cagatay; Sreeparna Banerjee; Dilek Keskin; Aysen Tezcaner

    2013-03-01

    The aim of this study was to investigate the effects of Raloxifene (Ral) on degeneration-related changes in osteoarthritis (OA)-like chondrocytes using two- and three-dimensional models. Five-azacytidine (Aza-C) was used to induce OA-like alterations in rat articular chondrocytes and the model was verified at molecular and macrolevels. Chondrocytes were treated with Ral (1, 5 and 10 M) for 10 days. Caspase-3 activity, gene expressions of aggrecan, collagen II, alkaline phosphatase (ALP), collagen X, matrix metalloproteinases (MMP-13, MMP-3 and MMP-2), and MMP-13, MMP-3 and MMP-2 protein expressions were studied in two-dimensional model. Matrix deposition and mechanical properties of agarose-chondrocyte discs were evaluated in three-dimensional model. One M Ral reduced expression of OA-related genes, decreased apoptosis, and MMP-13 and MMP-3 protein expressions. It also increased aggrecan and collagen II gene expressions relative to untreated OA-like chondrocytes. In three-dimensional model, 1 M Ral treatment resulted in increased collagen deposition and improved mechanical properties, although a significant increase for sGAG was not observed. In summation, 1 M Ral improved matrix-related activities, whereas dose increment reversed these effects except ALP gene expression and sGAG deposition. These results provide evidence that low-dose Ral has the potential to cease or reduce the matrix degeneration in OA.

  7. Effects of RNAi-mediated inhibition of aggrecanase-1 and aggrecanase-2 on rat costochondral chondrocytes in vitro

    Institute of Scientific and Technical Information of China (English)

    Zheng-hui WANG; Zhuang-qun YANG; Xi-jing HE; Li WANG; Li-xia LI; Jun-bo TU

    2008-01-01

    Aim:Failure of transplanted cartilage or allogenic chondrocytes is attributed mainly to immunological rejection and cartilage degradation.A major feature is the loss of aggrecan from the cartilage matrix,primarily due to the action of the specific proteinases aggrecanase-1 and aggrecanase-2.The aim of this in vitro study was to determine whether the specific inhibition of aggrecanase-1 and aggrecanase-2 by RNAi would mitigate aggrecan loss from cultured chondrocytes.Methods:Expression plasmid vectors of shRNA targeting aggrecanase-1 and aggrecanase-2 were constructed and transfected into cultured rattus costochondral chondrocytes.The transfected cells were induced with interleukin-1 β (IL-1β).Gene mRNA levels were analyzed by RT-PCR.Aggrecan and collagen Ⅱ content were measured by immunohistochemistry and Western blotting.Results:As the chondrocytes underwent dedifferentiation,agggrecanase-1 increased significantly.The specific inhibition of aggrecanase-1 and aggrecanase-2 by RNAi had no negative effect on the morphology and growth velocity of the chondrocytes.The mRNA of aggrecanase-1 and aggrecanase-2 decreased significantly.The α-2-macroglobulin expression level was increased by the shRNA specific for aggrecanase-1.Other genes of the chondrocytic extracellular matrix were not affected.RNAi significantly increased the aggrecan and collagen Ⅱ content of chondrocytes treated with IL-1β.Conclusion:The results suggest that inhibition of aggrecanase-1 and aggrecanase-2 by RNAi can mitigate aggrecan degradation,without interfering with chondrocytic gene phenotype recovery.RNAi technology can be a useful tool for studying degenerative processes in cartilage.

  8. Brown Fat Expresses Adiponectin in Humans

    Directory of Open Access Journals (Sweden)

    Gianluca Iacobellis

    2013-01-01

    Full Text Available The presence of brown adipose tissue (BAT in humans is unclear. Pheochromocytomas (PHEO are rare tumors of neuroectodermal origin which occur in 0.1-0.2% of patients with hypertension. We sought to evaluate the presence and activity of BAT surrounding adrenal PHEO in a well-studied sample of 11 patients who were diagnosed with PHEO and then underwent adrenalectomy. Areas of white fat (WAT and BAT surrounding PHEO were obtained by Laser Capture Microdissection for analysis of uncoupling protein (UCP-1 and adiponectin mRNA expression. Adiponectin and UCP-1 mRNA levels were significantly higher in BAT than in WAT (0.62 versus 0.15 and 362.4 versus 22.1, resp., for both. Adiponectin mRNA levels significantly correlated with urinary metanephrines (, , vanilly mandelic acid (VMA (, , and serum adiponectin levels (, . Serum adiponectin levels significantly decreased ( μg/mL versus  μg/mL, after adrenalectomy in PHEO subjects. This study provides the following findings: (1 BAT surrounding PHEO expresses adiponectin and UCP-1 mRNA, (2 expression of adiponectin mRNA is significantly higher in BAT than in WAT surrounding PHEO, and (3 catecholamines and serum adiponectin levels significantly correlate with BAT UCP-1 and adiponectin mRNA.

  9. [Effects of in vitro continuous passaging on the phenotype of mouse hyaline chondrocytes and the balance of the extra- cellular matrix].

    Science.gov (United States)

    Linyi, Cai; Xiangli, Kong; Jing, Xie

    2016-06-01

    This study aimed to investigate the effects of in vitro continuous passaging on the morphological phenotype and differentiation characteristics of mouse hyaline chondrocytes, as well as on the balance of the extracellular matrix (ECM). Enzymatic digestion was conducted to isolate mouse hyaline chondrocytes, which expanded over five passages in vitro. Hematoxylin-eosin stain was used to show the changes in chondrocyte morphology. Semi-quantitative polymerase chain reaction was performed to analyze the mRNA changes in the marker genes, routine genes, matrix metalloproteinases (MMPs), and tissue inhibitors of MMPs (TIMPs) in chondrocytes. Zymography was carried out to elucidate changes in gelatinase activities. After continuous expansion in vitro, the morphology of round or polygonal chondrocytes changed to elongated and spindled shape. The expression of marker genes significantly decreased (P 0.05). Meanwhile, the ratio of MMPs/TIMPs was altered. At the protein level, the activities of gelatinases decreased after passaging, especially for P4 and P5 chondrocytes (P cartilage ECM became uncontrollable and led to the imbalance of ECM homeostasis. When hyaline chondrocytes are applied in research on relevant diseases or cartilage tissue engineering, P0-P2 chondrocytes should be used.

  10. The in vitro biocompatibility of d-(+) raffinose modified chitosan: Two-dimensional and three-dimensional systems for culturing of horse articular chondrocytes.

    Science.gov (United States)

    De Angelis, Elena; Ravanetti, Francesca; Martelli, Paolo; Cacchioli, Antonio; Ivanovska, Ana; Corradi, Attilio; Nasi, Sonia; Bianchera, Annalisa; Passeri, Benedetta; Canelli, Elena; Bettini, Ruggero; Borghetti, Paolo

    2017-06-15

    The present study investigated the biocompatibility of chitosan films and scaffolds modified with d-(+)raffinose and their capability to support the growth and maintenance of the differentiation of articular chondrocytes in vitro. Primary equine articular chondrocytes were cultured on films and scaffolds of modified d-(+) raffinose chitosan. Their behavior was compared to that of chondrocytes grown in conventional bi- and three-dimensional culture systems, such as micromasses and alginate beads. Chitosan films maintained the phenotype of differentiated chondrocytes (typical round morphology) and sustained the synthesis of cartilaginous extracellular matrix (ECM), even at 4weeks of culture. Indeed, starting from 2weeks of culture, chondrocytes seeded on chitosan scaffolds were able to penetrate the surface pores and to colonize the internal matrix. Moreover they produced ECM expressing the genes of typical chondrocytes differentiation markers such as collagen II and aggrecan. In conclusion, chitosan modified with d-raffinose represents an ideal support for chondrocyte adhesion, proliferation and for the maintenance of cellular phenotypic and genotypic differentiation. This novel biomaterial could potentially be a reliable support for the re-differentiation of dedifferentiated chondrocytes. Copyright © 2017 Elsevier Ltd. All rights reserved.

  11. PTHrP regulates chondrocyte maturation in condylar cartilage.

    Science.gov (United States)

    Rabie, A B M; Tang, G H; Xiong, H; Hägg, U

    2003-08-01

    PTHrP is a key factor regulating the pace of endochondral ossification during skeletal development. Mandibular advancement solicits a cascade of molecular responses in condylar cartilage. However, the pace of cellular maturation and its effects on condylar growth are still unknown. The purpose of this study was to evaluate the pattern of expression of PTHrP and correlate it to cellular dynamics of chondrocytes in condylar cartilage during natural growth and mandibular advancement. We fitted 35-day-old Sprague-Dawley rats with functional appliances. Experimental animals with matched controls were labeled with bromodeoxyuridine 3 days before their death, so that mesenchymal cell differentiation could be traced. Mandibular advancement increased the number of differentiated chondroblasts and subsequently increased the cartilage volume. Higher levels of PTHrP expression in experimental animals coincided with the slowing of chondrocyte hypertrophy. Thus, mandibular advancement promoted mesenchymal cell differentiation and triggered PTHrP expression, which retarded their further maturation to allow for more growth.

  12. YKL-39 (chitinase 3-like protein 2), but not YKL-40 (chitinase 3-like protein 1), is up regulated in osteoarthritic chondrocytes

    OpenAIRE

    Knorr, T; Obermayr, F; Bartnik, E.; A. Zien; Aigner, T

    2003-01-01

    Methods: cDNA array and online quantitative polymerase chain reaction (PCR) were used to measure mRNA expression levels of YKL-39 and YKL-40 in chondrocytes in normal, early degenerative, and late stage osteoarthritic cartilage samples.

  13. TNF Accelerates Death of Mandibular Condyle Chondrocytes in Rats with Biomechanical Stimulation-Induced Temporomandibular Joint Disease.

    Directory of Open Access Journals (Sweden)

    Hongxu Yang

    Full Text Available To determine if temporomandibular joint chondrocyte apoptosis is induced in rats with dental biomechanical stimulation and what a role TNF takes.Thirty-two rats were divided into 4 groups (n = 8/group and exposed to incisor mal-occlusion induced by unilateral anterior crossbite biomechanical stimulation. Two groups were sampled at 2 or 4 weeks. The other two groups were treated with local injections of a TNF inhibitor or PBS into the temporomandibular joints area at 2 weeks and then sampled at 4 weeks. Twenty-four rats either served as unilateral anterior crossbite mock operation controls (n = 8/group with sampling at 2 or 4 weeks or received a local injection of the TNF inhibitor at 2 weeks with sampling at 4 weeks. Chondrocytes were isolated from the temporomandibular joints of 6 additional rats and treated with TNF in vitro. Joint samples were assessed using Hematoxylin&eosin, Safranin O, TUNEL and immunohistochemistry staining, real-time PCR, fluorogenic activity assays and Western blot analyses. The isolated chondrocytes were also analyzed by flow cytometry.Unilateral anterior crossbite stimulation led to temporomandibular joint cartilage degradation, associated with an increase in TUNEL-positive chondrocytes number, caspase-9 expression levels, and the release of cytochrome c from mitochondria at 2 weeks without changes in TNF and caspase-8 levels until after 4 weeks. TNF stimulated apoptosis of the isolated chondrocytes and up-regulated caspase-8 expression, but did not change caspase-9 expression levels. Local injection of TNF inhibitor down-regulated caspase-8 expression and reduced TUNEL-positive cell number, but did not reverse cartilage thickness reduction, caspase-9 up-regulation or cytochrome c release.Unilateral anterior crossbite stimulation induces mitochondrion-mediated apoptosis of articular chondrocytes. TNF accelerated the unilateral anterior crossbite induced chondrocytes apoptosis via death-receptor pathway. However, anti

  14. TNF Accelerates Death of Mandibular Condyle Chondrocytes in Rats with Biomechanical Stimulation-Induced Temporomandibular Joint Disease.

    Science.gov (United States)

    Yang, Hongxu; Zhang, Mian; Wang, Xin; Zhang, Hongyun; Zhang, Jing; Jing, Lei; Liao, Lifan; Wang, Meiqing

    2015-01-01

    To determine if temporomandibular joint chondrocyte apoptosis is induced in rats with dental biomechanical stimulation and what a role TNF takes. Thirty-two rats were divided into 4 groups (n = 8/group) and exposed to incisor mal-occlusion induced by unilateral anterior crossbite biomechanical stimulation. Two groups were sampled at 2 or 4 weeks. The other two groups were treated with local injections of a TNF inhibitor or PBS into the temporomandibular joints area at 2 weeks and then sampled at 4 weeks. Twenty-four rats either served as unilateral anterior crossbite mock operation controls (n = 8/group) with sampling at 2 or 4 weeks or received a local injection of the TNF inhibitor at 2 weeks with sampling at 4 weeks. Chondrocytes were isolated from the temporomandibular joints of 6 additional rats and treated with TNF in vitro. Joint samples were assessed using Hematoxylin&eosin, Safranin O, TUNEL and immunohistochemistry staining, real-time PCR, fluorogenic activity assays and Western blot analyses. The isolated chondrocytes were also analyzed by flow cytometry. Unilateral anterior crossbite stimulation led to temporomandibular joint cartilage degradation, associated with an increase in TUNEL-positive chondrocytes number, caspase-9 expression levels, and the release of cytochrome c from mitochondria at 2 weeks without changes in TNF and caspase-8 levels until after 4 weeks. TNF stimulated apoptosis of the isolated chondrocytes and up-regulated caspase-8 expression, but did not change caspase-9 expression levels. Local injection of TNF inhibitor down-regulated caspase-8 expression and reduced TUNEL-positive cell number, but did not reverse cartilage thickness reduction, caspase-9 up-regulation or cytochrome c release. Unilateral anterior crossbite stimulation induces mitochondrion-mediated apoptosis of articular chondrocytes. TNF accelerated the unilateral anterior crossbite induced chondrocytes apoptosis via death-receptor pathway. However, anti-TNF therapy

  15. TNF Accelerates Death of Mandibular Condyle Chondrocytes in Rats with Biomechanical Stimulation-Induced Temporomandibular Joint Disease

    Science.gov (United States)

    Zhang, Hongyun; Zhang, Jing; Jing, Lei; Liao, Lifan; Wang, Meiqing

    2015-01-01

    Objective To determine if temporomandibular joint chondrocyte apoptosis is induced in rats with dental biomechanical stimulation and what a role TNF takes. Methods Thirty-two rats were divided into 4 groups (n = 8/group) and exposed to incisor mal-occlusion induced by unilateral anterior crossbite biomechanical stimulation. Two groups were sampled at 2 or 4 weeks. The other two groups were treated with local injections of a TNF inhibitor or PBS into the temporomandibular joints area at 2 weeks and then sampled at 4 weeks. Twenty-four rats either served as unilateral anterior crossbite mock operation controls (n = 8/group) with sampling at 2 or 4 weeks or received a local injection of the TNF inhibitor at 2 weeks with sampling at 4 weeks. Chondrocytes were isolated from the temporomandibular joints of 6 additional rats and treated with TNF in vitro. Joint samples were assessed using Hematoxylin&eosin, Safranin O, TUNEL and immunohistochemistry staining, real-time PCR, fluorogenic activity assays and Western blot analyses. The isolated chondrocytes were also analyzed by flow cytometry. Results Unilateral anterior crossbite stimulation led to temporomandibular joint cartilage degradation, associated with an increase in TUNEL-positive chondrocytes number, caspase-9 expression levels, and the release of cytochrome c from mitochondria at 2 weeks without changes in TNF and caspase-8 levels until after 4 weeks. TNF stimulated apoptosis of the isolated chondrocytes and up-regulated caspase-8 expression, but did not change caspase-9 expression levels. Local injection of TNF inhibitor down-regulated caspase-8 expression and reduced TUNEL-positive cell number, but did not reverse cartilage thickness reduction, caspase-9 up-regulation or cytochrome c release. Conclusions Unilateral anterior crossbite stimulation induces mitochondrion-mediated apoptosis of articular chondrocytes. TNF accelerated the unilateral anterior crossbite induced chondrocytes apoptosis via death

  16. Regulation of chondrocyte functions by transient receptor potential cation channel V6 in osteoarthritis.

    Science.gov (United States)

    Song, Tengfei; Ma, Jun; Guo, Lei; Yang, Peng; Zhou, Xuhui; Ye, Tianwen

    2017-11-01

    Transient receptor potential vanilloid (TRPV) channels function to maintain the dynamic balance of calcium signaling and calcium metabolism in bones. The goal of this study was to determine the potential role of TRPV6 in regulation of chondrocytes. The level of TRPV6 expression was analyzed by western blot in articular cartilage derived from the knee joints of osteoarthritis (OA) rat models and OA patients. Bone structure and osteoarthritic changes in the knee joints of TRPV6 knockout mice were examined using micro-computed and histological analysis at the age of 6 and 12 months old. Furthermore, to investigate the effects of TRPV6 on chondrocyte extracellular matrix secretion, the release of matrix degrading enzymes, cell proliferation, and apoptosis, we decreased and increased TRPV6 expression in chondrocytes with lentiviral constructs encoding shRNA targeting TRPV6 and encoding TRPV6, respectively. The results showed that the level of TRPV6 expression in an OA rat model was markedly down-regulated. TRPV6 knockout mice showed severe osteoarthritis changes, including cartilage fibrillation, eburnation, and loss of proteoglycans. In addition, deficiency of TRPV6 clearly affected chondrocyte function, such as extracellular matrix secretion, the release of matrix degrading enzymes, cell proliferation, and apoptosis. Taken together, our results implicated that TRPV6 channel, as a chondro-protective factor, was involved in the pathogenesis of OA. © 2017 Wiley Periodicals, Inc.

  17. Chondrocyte BMP2 signaling plays an essential role in bone fracture healing.

    Science.gov (United States)

    Mi, Meng; Jin, Hongting; Wang, Baoli; Yukata, Kiminori; Sheu, Tzong-Jen; Ke, Qiao Han; Tong, Peijian; Im, Hee-Jeong; Xiao, Guozhi; Chen, Di

    2013-01-10

    The specific role of endogenous Bmp2 gene in chondrocytes and in osteoblasts in fracture healing was investigated by generation and analysis of chondrocyte- and osteoblast-specific Bmp2 conditional knockout (cKO) mice. The unilateral open transverse tibial fractures were created in these Bmp2 cKO mice. Bone fracture callus samples were collected and analyzed by X-ray, micro-CT, histology analyses, biomechanical testing and gene expression assays. The results demonstrated that the lack of Bmp2 expression in chondrocytes leads to a prolonged cartilage callus formation and a delayed osteogenesis initiation and progression into mineralization phase with lower biomechanical properties. In contrast, when the Bmp2 gene was deleted in osteoblasts, the mice showed no significant difference in the fracture healing process compared to control mice. These findings suggest that endogenous BMP2 expression in chondrocytes may play an essential role in cartilage callus maturation at an early stage of fracture healing. Our studies may provide important information for clinical application of BMP2.

  18. MRTF-A signaling regulates the acquisition of the contractile phenotype in dedifferentiated chondrocytes.

    Science.gov (United States)

    Parreno, Justin; Raju, Sneha; Wu, Po-Han; Kandel, Rita A

    2016-10-14

    Chondrocyte culture as a monolayer for cell number expansion results in dedifferentiation whereby expanded cells acquire contractile features and increased actin polymerization status. This study determined whether the actin polymerization based signaling pathway, myocardin-related transcription factor-a (MRTF-A) is involved in regulating this contractile phenotype. Serial passaging of chondrocytes in monolayer culture to passage 2 resulted in increased gene and protein expression of the contractile molecules alpha-smooth muscle actin, transgelin and vinculin compared to non-passaged, primary cells. This resulted in a functional change as passaged 2, but not primary, chondrocytes were capable of contracting type I collagen gels in a stress-relaxed contraction assay. These changes were associated with increased actin polymerization and MRTF-A nuclear localization. The involvement of actin was demonstrated by latrunculin B depolymerization of actin which reversed these changes. Alternatively cytochalasin D which activates MRTF-A increased gene and protein expression of α-smooth muscle actin, transgelin and vinculin, whereas CCG1423 which deactivates MRTF-A decreased these molecules. The involvement of MRTF-A signaling was confirmed by gene silencing of MRTF or its co-factor serum response factor. Knockdown experiments revealed downregulation of α-smooth muscle actin and transgelin gene and protein expression, and inhibition of gel contraction. These findings demonstrate that passaged chondrocytes acquire a contractile phenotype and that this change is modulated by the actin-MRTF-A-serum response factor signaling pathway.

  19. 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.

  20. GLUT-3 expression in human skeletal muscle

    Science.gov (United States)

    Stuart, C. A.; Wen, G.; Peng, B. H.; Popov, V. L.; Hudnall, S. D.; Campbell, G. A.

    2000-01-01

    Muscle biopsy homogenates contain GLUT-3 mRNA and protein. Before these studies, it was unclear where GLUT-3 was located in muscle tissue. In situ hybridization using a midmolecule probe demonstrated GLUT-3 within all muscle fibers. Fluorescent-tagged antibody reacting with affinity-purified antibody directed at the carboxy-terminus demonstrated GLUT-3 protein in all fibers. Slow-twitch muscle fibers, identified by NADH-tetrazolium reductase staining, possessed more GLUT-3 protein than fast-twitch fibers. Electron microscopy using affinity-purified primary antibody and gold particle-tagged second antibody showed that the majority of GLUT-3 was in association with triads and transverse tubules inside the fiber. Strong GLUT-3 signals were seen in association with the few nerves that traversed muscle sections. Electron microscopic evaluation of human peripheral nerve demonstrated GLUT-3 within the axon, with many of the particles related to mitochondria. GLUT-3 protein was found in myelin but not in Schwann cells. GLUT-1 protein was not present in nerve cells, axons, myelin, or Schwann cells but was seen at the surface of the peripheral nerve in the perineurium. These studies demonstrated that GLUT-3 mRNA and protein are expressed throughout normal human skeletal muscle, but the protein is predominantly found in the triads of slow-twitch muscle fibers.

  1. The pharmacology of human appetite expression.

    Science.gov (United States)

    Halford, Jason C G; Cooper, Gillian D; Dovey, Terence M

    2004-04-01

    The discovery of the adiposity signal leptin a decade ago revolutionised our understanding of the hypothalamic mechanisms underpinning the central control of ingestive behaviour. Subsequently, the structure and function of various hypothalamic peptide systems (Neuropeptide Y (NPY), Orexins, Melanocortins, Cocaine and Amphetamine Regulating Transcript (CART), Galanin/Galanin Like Peptides (GALP) and endocannabinoids) have been characterised in detail in rodent models. The therapeutic benefit of targeting these systems remains to be discovered. More is becoming known about the pharmacological potential of peripheral, meal-induced, episodic endogenous peptides. Hormones such as Cholecystokinin (CCK), Gastrin Releasing Peptides (GRP), Glucagon-Like Peptide I (GLP-1) Enterostatin, Amylin, Peptide YY (PYY) and Ghrelin are released prior to, during and/or after a meal, controlling intake and subjective feelings of appetite (hunger and satiety). In addition, there is an expanding body of literature detailing the effects of a wide variety of drugs on human appetite and food intake. Some of these drugs act upon CNS monoamine systems such as Serotonin (5-HT). Dopamine (DA) and Noradrenaline (NA), have long been implicated in appetite regulation. Detailed examination of both the effect of agonising endogenous gut peptide systems and the effect of various monoaminergic drugs on the expression of human appetite can provide a greater understanding of mechanisms underpinning normal appetite regulation. However, such an understanding must be based on knowledge of the effect of the treatment on meal size, eating rate, meal pattern, food choice and the subjective experience of appetite flux (hunger and satiety), and notjust food intake.

  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. Study of cryopreservation of articular chondrocytes using the Taguchi method.

    Science.gov (United States)

    Lyu, Shaw-Ruey; Wu, Wei Te; Hou, Chien Chih; Hsieh, Wen-Hsin

    2010-04-01

    This study evaluates the effect of control factors on cryopreservation of articular cartilage chondrocytes using the Taguchi method. Freeze-thaw experiments based on the L(8)(2(7)) two-level orthogonal array of the Taguchi method are conducted, and ANOVA (analysis of variables) is adopted to determine the statistically significant control factors that affect the viability of the cell. Results show that the type of cryoprotectant, freezing rate, thawing rate, and concentration of cryoprotectant (listed in the order of influence) are the statistically significant control factors that affect the post-thaw viability. The end temperature and durations of the first and second stages of freezing do not affect the post-thaw viability. Within the ranges of the control factors studied in this work, the optimal test condition is found to be a freezing rate of 0.61+/-0.03 degrees C/min, a thawing rate of 126.84+/-5.57 degrees C/min, Me(2)SO cryoprotectant, and a cryoprotectant concentration of 10% (v/v) for maximum cell viability. In addition, this study also explores the effect of cryopreservation on the expression of type II collagen using immunocytochemical staining and digital image processing. The results show that the ability of cryopreserved chondrocytes to express type II collagen is reduced within the first five days of monolayer culture.

  4. CKbeta-8 [CCL23], a novel CC chemokine, is chemotactic for human osteoclast precursors and is expressed in bone tissues.

    Science.gov (United States)

    Votta, B J; White, J R; Dodds, R A; James, I E; Connor, J R; Lee-Rykaczewski, E; Eichman, C F; Kumar, S; Lark, M W; Gowen, M

    2000-05-01

    We have previously demonstrated that a tartrate-resistant acid phosphatase (TRAP)-positive subpopulation of mononuclear cells isolated from collagenase digests of human osteoclastoma tissue exhibits an osteoclast phenotype and can be induced to resorb bone. Using these osteoclast precursors as a model system, we have assessed the chemotactic potential of 16 chemokines. Three CC chemokines, the recently described CKbeta-8, RANTES, and MIP-1alpha elicited significant chemotactic responses. In contrast, 10 other CC chemokines (MIP-1beta, MCP-1, MCP-2, MCP-3, MCP-4, HCC-1, eotaxin-2, PARC, SLC, ELC) and 3 CXC chemokines (IL-8, GROalpha, SDF-1) were inactive. None of these chemokines showed any chemotactic activity for either primary osteoblasts derived from human bone explants or the osteoblastic MG-63 cell line. The identity of the osteoclast receptor that mediates the chemotactic response remains to be established. However, all three active chemokines have been reported to bind to CCR1 and cross-desensitization studies demonstrate that RANTES and MIP-1alpha can partially inhibit the chemotactic response elicited by CKbeta-8. CKbeta-8, the most potent of the active CC chemokines (EC(max) 0.1-0.3 nM), was further characterized with regard to expression in human bone and cartilage. Although expression is not restricted to these tissues, CKbeta-8 mRNA was shown to be highly expressed in osteoblasts and chondrocytes in human fetal bone by in situ hybridization. In addition, CKbeta-8 protein was shown to be present in human osteophytic tissue by immunolocalization. These observations suggest that CKbeta-8, and perhaps other chemokines, may play a role in the recruitment of osteoclast precursors to sites of bone resorption.

  5. Expression of adipokines in osteoarthritis osteophytes and their effect on osteoblasts.

    Science.gov (United States)

    Junker, Susann; Frommer, Klaus W; Krumbholz, Grit; Tsiklauri, Lali; Gerstberger, Rüdiger; Rehart, Stefan; Steinmeyer, Jürgen; Rickert, Markus; Wenisch, Sabine; Schett, Georg; Müller-Ladner, Ulf; Neumann, Elena

    2017-10-01

    Osteophyte formation in osteoarthritis (OA) is mediated by increased osteoblast activity, which is -in turn- regulated by the Wnt signaling pathway. Obesity is regarded a risk factor in OA, yet little is known about the interaction between adipose tissue-derived factors, the adipokines, and bone formation, although adipokines are associated with the pathogenesis of OA. Therefore, the effect of adipokines on bone and cartilage forming cells and osteophyte development was analyzed. Human OA osteophytes were histologically characterized and adipokine expression was evaluated by immunohistochemistry. Osteoblasts and chondrocytes were isolated from OA tissue and stimulated with adiponectin, resistin, or visfatin. Cytokine and osteoblast/chondrocyte markers were quantified and activation of Wnt and p38 MAPK signaling was analyzed. Adiponectin, resistin, and visfatin were expressed in OA osteophytes by various articular cell types. Stimulation of OA osteoblasts with adiponectin and of OA chondrocytes with visfatin led to an increased release of proinflammatory mediators but not to osteoblast differentiation or activation. Additionally, visfatin increased matrix degrading factors in chondrocytes. Wnt signaling was not altered by adipokines, but adiponectin induced p38 MAPK signaling in osteoblasts. Adipokines are present in OA osteophytes, and adiponectin and visfatin increase the release of proinflammatory mediators by osteoblasts and chondrocytes. The effects of adiponectin were mediated by p38 MAPK but not Wnt signaling in osteoblasts. Therefore, the results support the idea that adipokines do not directly influence osteophyte development but the proinflammatory conditions in OA. Copyright © 2016 Elsevier B.V. All rights reserved.

  6. Focal adhesion kinase and mitogen-activated protein kinases are involved in chondrocyte activation by the 29-kDa amino-terminal fibronectin fragment.

    Science.gov (United States)

    Gemba, Takefumi; Valbracht, Jean; Alsalameh, Saifeddin; Lotz, Martin

    2002-01-11

    The 29-kDa amino-terminal fibronectin fragment (FN-f) has a potent chondrolytic effect and is thought to be involved in cartilage degradation in arthritis. However, little is known about signal transduction pathways that are activated by FN-f. Here we demonstrated that FN-f induced nitric oxide (NO) production from human articular chondrocytes. Expression of inducible nitric-oxide synthase (iNOS) mRNA and NO production were observed at 6 and 48 h after FN-f treatment, respectively. Interleukin-1beta (IL-1beta) mRNA up-regulation was stimulated by FN-f in human chondrocytes. To address the possibility that FN-f-induced NO release is mediated by IL-1beta production, the effect of IL-1 receptor antagonist (IL-1ra) was determined. IL-1ra partially inhibited FN-f-induced NO release although it almost completely inhibited IL-1beta-induced NO release. Tyrosine phosphorylation of focal adhesion kinase was induced transiently by FN-f treatment. Blocking antibodies to alpha(5) or beta(1) integrin and Arg-Gly-Asp-containing peptides did not inhibit FN-f-induced NO production. PP2, a Src family kinase inhibitor, or cytochalasin D, which selectively disrupts the network of actin filaments, inhibited both FAK phosphorylation and NO production induced by FN-f, but the phosphatidylinositol 3-kinase inhibitor wortmannin had no effect. Analysis of mitogen-activated protein kinases (MAPK) showed activation of extracellular signal-regulated kinase (ERK), c-Jun NH(2)-terminal kinase, and p38 MAPK. High concentrations of SB203580, which inhibit both JNK and p38 MAPK, and PD98059 a selective inhibitor of MEK1/2 that blocks ERK activation, inhibited FN-f induced NO production. These data suggest that focal adhesion kinase and MAPK mediate FN-f induced activation of human articular chondrocytes.

  7. Positive selection on gene expression in the human brain

    DEFF Research Database (Denmark)

    Khaitovich, Philipp; Tang, Kun; Franz, Henriette

    2006-01-01

    Recent work has shown that the expression levels of genes transcribed in the brains of humans and chimpanzees have changed less than those of genes transcribed in other tissues [1] . However, when gene expression changes are mapped onto the evolutionary lineage in which they occurred, the brain...... shows more changes than other tissues in the human lineage compared to the chimpanzee lineage [1] , [2] and [3] . There are two possible explanations for this: either positive selection drove more gene expression changes to fixation in the human brain than in the chimpanzee brain, or genes expressed...... in the brain experienced less purifying selection in humans than in chimpanzees, i.e. gene expression in the human brain is functionally less constrained. The first scenario would be supported if genes that changed their expression in the brain in the human lineage showed more selective sweeps than other genes...

  8. PTHrP, PTHr, and FGFR3 are involved in the process of endochondral ossification in human osteophytes.

    Science.gov (United States)

    Huch, Klaus; Kleffner, Sandra; Stöve, Johannes; Puhl, Wolfhart; Günther, Klaus-Peter; Brenner, Rolf Erwin

    2003-04-01

    To elucidate the process of endochondral ossification in human osteophytes we have studied the expression of parathyroid hormone-related protein (PTHrP), its receptor (PTHr), and fibroblast growth factor receptor 3 (FGFR3). Osteophytes from patients undergoing total knee replacement ( n=13), and fetal growth plate cartilages ( n=4) were processed for safranin O staining and immunohistochemistry. Chondrocytes and their matrix were preferentially stained for PTHrP in the middle and deep zones of the osteophytes examined. Ossified areas did not show a positive staining. In fetal joints the cartilaginous surface and the perichondrium as well as the osteoblasts in the trabecular bone were positive. PTHr was expressed at large in chondrocytes and osteoblasts of all osteophytes and fetal joints. Cells of the perichondrium were also positive. The FGFR3 antibody stained only single chondrocytes in some osteophytes, and groups of cells in others. In fetal samples, chondrocytes of the proliferating and the hypertrophic zone showed staining for FGFR3. This is the first report on the expression of PTHrP, PTHr, and FGFR3 in human osteophytes. As in fetal joints these mediators might regulate proliferation and differentiation of chondrocytes playing an important role in osteo(chondro)phyte growth.

  9. EXPRESSION OF Fas LIGAND IN HUMAN COLON CANCER CELL LINES

    Institute of Scientific and Technical Information of China (English)

    张建军; 丁尔迅; 王强; 陈学云; 付志仁

    2001-01-01

    To investigate the expression of Fas ligand in human colon carcinoma cell lines. Methods: A total of six human colon cancer cell lines were examined for the expression of Fas ligand mRNA and cell surface protein by using RT-PCR and flow cytometry respectively. Results: The results showed that Fas ligand mRNA was expressed in all of the six cancer cell lines and Fas ligand cell surface protein was expressed in part of them. Conclusion: These data suggest that Fas ligand was expressed, at least in part, in human colon cancer cell lines and might facilitate to escape from immune surveillance of the host.

  10. Increased chondrocyte adhesion on nanotubular anodized titanium.

    Science.gov (United States)

    Burns, Kevin; Yao, Chang; Webster, Thomas J

    2009-03-01

    Previous studies have demonstrated increased osteoblast (bone-forming cells) functions (including adhesion, synthesis of intracellular collagen, alkaline phosphatase activity, and deposition of calcium-containing minerals) on titanium anodized to possess nanometer features compared with their unanodized counterparts. Such titanium materials were anodized to possess novel nanotubes also capable of drug delivery. Since titanium has not only experienced wide spread commercial use in orthopedic but also in cartilage applications, the objective of the present in vitro study was for the first time to investigate chondrocyte (cartilage synthesizing cells) functions on titanium anodized to possess nanotubes. For this purpose, titanium was anodized in dilute hydrofluoric acid at 20 V for 20 min. Results showed increased chondrocyte adhesion on anodized titanium with nanotube structures compared with unanodized titanium. Importantly, the present study also provided evidence why. Since material characterization studies revealed significantly greater nanometer roughness and similar chemistry as well as crystallinity between nanotubular anodized and unanodized titanium, the results of the present study highlight the importance of the nanometer roughness provided by anodized nanotubes on titanium for enhancing chondrocyte adhesion. In this manner, the results of the present in vitro study indicated that anodization might be a promising quick and inexpensive method to modify the surface of titanium-based implants to induce better chondrocyte adhesion for cartilage applications.

  11. 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.

  12. Cartilage repair: Generations of autologous chondrocyte transplantation

    Energy Technology Data Exchange (ETDEWEB)

    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.

  13. Expression and immunohistochemical localization of leptin in human periapical granulomas

    OpenAIRE

    Martín González, Jenifer; Carmona Fernández, Antonio; Pérez Pérez, Antonio; Sánchez Jiménez, Flora; Sánchez-Margalet, Víctor; Segura-Egea, Juan J.

    2015-01-01

    Background Leptin, initially described as an adipocyte-derived hormone to regulate weight control, is expressed in normal and inflamed human dental pulp, being up-regulated during pulp experimental inflammation. Leptin receptor (LER) has been identified in human periapical granulomas. The aim of this study was to analyze and characterize the expression of leptin in human periapical granulomas. Material and Methods Fifteen periapical inflammatory lesions were obtained from extracted human teet...

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

    Directory of Open Access Journals (Sweden)

    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