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Sample records for human chondrocyte gene

  1. Comparing effects of perfusion and hydrostatic pressure on gene profiles of human chondrocyte.

    Science.gov (United States)

    Zhu, Ge; Mayer-Wagner, Susanne; Schröder, Christian; Woiczinski, Matthias; Blum, Helmut; Lavagi, Ilaria; Krebs, Stefan; Redeker, Julia I; Hölzer, Andreas; Jansson, Volkmar; Betz, Oliver; Müller, Peter E

    2015-09-20

    Hydrostatic pressure and perfusion have been shown to regulate the chondrogenic potential of articular chondrocytes. In order to compare the effects of hydrostatic pressure plus perfusion (HPP) and perfusion (P) we investigated the complete gene expression profiles of human chondrocytes under HPP and P. A simplified bioreactor was constructed to apply loading (0.1 MPa for 2 h) and perfusion (2 ml) through the same piping by pressurizing the medium directly. High-density monolayer cultures of human chondrocytes were exposed to HPP or P for 4 days. Controls (C) were maintained in static cultures. Gene expression was evaluated by sequencing (RNAseq) and quantitative real-time PCR analysis. Both treatments changed gene expression levels of human chondrocytes significantly. Specifically, HPP and P increased COL2A1 expression and decreased COL1A1 and MMP-13 expression. Despite of these similarities, RNAseq revealed a list of cartilage genes including ACAN, ITGA10 and TNC, which were differentially expressed by HPP and P. Of these candidates, adhesion related molecules were found to be upregulated in HPP. Both HPP and P treatment had beneficial effects on chondrocyte differentiation and decreased catabolic enzyme expression. The study provides new insight into how hydrostatic pressure and perfusion enhance cartilage differentiation and inhibit catabolic effects. Copyright © 2015 Elsevier B.V. All rights reserved.

  2. 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

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

    NARCIS (Netherlands)

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

    2011-01-01

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

  4. 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

  5. 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

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

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    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.

  7. ROCK inhibitor prevents the dedifferentiation of human articular chondrocytes

    International Nuclear Information System (INIS)

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

    2012-01-01

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

  8. 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

  9. Molecular analysis of expansion, differentiation, and growth factor treatment of human chondrocytes identifies differentiation markers and growth-related genes.

    Science.gov (United States)

    Benz, Karin; Breit, Stephen; Lukoschek, Martin; Mau, Hans; Richter, Wiltrud

    2002-04-26

    This study is intended to optimise expansion and differentiation of cultured human chondrocytes by growth factor application and to identify molecular markers to monitor their differentiation state. We dissected the molecular consequences of matrix release, monolayer, and 3D-alginate culture, growth factor optimised expansion, and re-differentiation protocols by gene expression analysis. Among 19 common cartilage molecules assessed by cDNA array, six proved best to monitor differentiation. Instant down-regulation at release of cells from the matrix was strongest for COL 2A1, fibromodulin, and PRELP while LUM, CHI3L1, and CHI3L2 were expansion-related. Both gene sets reflected the physiologic effects of the most potent growth-inducing (PDGF-BB) and proteoglycan-inducing (BMP-4) factors. Only CRTAC1 expression correlated with 2D/3D switches while the molecular phenotype of native chondrocytes was not restored. The markers and optimised protocols we suggest can help to improve cell therapy of cartilage defects and chondrocyte differentiation from stem cell sources.

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

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    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.

  11. Scaffold-assisted cartilage tissue engineering using infant chondrocytes from human hip cartilage.

    Science.gov (United States)

    Kreuz, P C; Gentili, C; Samans, B; Martinelli, D; Krüger, J P; Mittelmeier, W; Endres, M; Cancedda, R; Kaps, C

    2013-12-01

    Studies about cartilage repair in the hip and infant chondrocytes are rare. The aim of our study was to evaluate the use of infant articular hip chondrocytes for tissue engineering of scaffold-assisted cartilage grafts. Hip cartilage was obtained from five human donors (age 1-10 years). Expanded chondrocytes were cultured in polyglycolic acid (PGA)-fibrin scaffolds. De- and re-differentiation of chondrocytes were assessed by histological staining and gene expression analysis of typical chondrocytic marker genes. In vivo, cartilage matrix formation was assessed by histology after subcutaneous transplantation of chondrocyte-seeded PGA-fibrin scaffolds in immunocompromised mice. The donor tissue was heterogenous showing differentiated articular cartilage and non-differentiated tissue and considerable expression of type I and II collagens. Gene expression analysis showed repression of typical chondrocyte and/or mesenchymal marker genes during cell expansion, while markers were re-induced when expanded cells were cultured in PGA-fibrin scaffolds. Cartilage formation after subcutaneous transplantation of chondrocyte loaded PGA-fibrin scaffolds in nude mice was variable, with grafts showing resorption and host cell infiltration or formation of hyaline cartilage rich in type II collagen. Addition of human platelet rich plasma (PRP) to cartilage grafts resulted robustly in formation of hyaline-like cartilage that showed type II collagen and regions with type X collagen. These results suggest that culture of expanded and/or de-differentiated infant hip cartilage cells in PGA-fibrin scaffolds initiates chondrocyte re-differentiation. The heterogenous donor tissue containing immature chondrocytes bears the risk of cartilage repair failure in vivo, which may be possibly overcome by the addition of PRP. Copyright © 2013 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.

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

    International Nuclear Information System (INIS)

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

    2010-01-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2010-10-22

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

  14. Impact of human platelet lysate on the expansion and chondrogenic capacity of cultured human chondrocytes for cartilage cell therapy.

    Science.gov (United States)

    Sykes, J G; Kuiper, J H; Richardson, J B; Roberts, S; Wright, K T; Kuiper, N J

    2018-05-01

    High hopes have been pinned on regenerative medicine strategies in order to prevent the progression of cartilage damage to osteoarthritis, particularly by autologous chondrocyte implantation (ACI). The loss of chondrocyte phenotype during in vitro monolayer expansion, a necessary step to obtain sufficient cell numbers, may be a key limitation in ACI. In this study, it was determined whether a shorter monolayer expansion approach could improve chondrogenic differentiation. The effects of two supplement types, foetal bovine serum (FBS) and Stemulate™ (a commercial source of human platelet lysate), on the expansion and re-differentiation potential of human chondrocytes, isolated from five individuals, were compared. Chondrocytes were expanded with 10 % FBS or 10 % Stemulate™. Pellets were cultured for 28 d in chondrogenic differentiation medium and assessed for the presence of cartilage matrix molecules and genes associated with chondrogenicity. Stemulate™ significantly enhanced the proliferation rate [average population doubling times: FBS, 25.07 ± 6.98 d (standard error of the mean, SEM) vs. Stemulate™, 13.10 ± 2.57 d (SEM)]. Sulphated glycosaminoglycans (sGAG), total collagen and qRT-PCR analyses of cartilage genes showed that FBS-expanded chondrocytes demonstrated significantly better chondrogenic capacity than Stemulate™-expanded chondrocytes. Histologically, FBS-expanded chondrocyte pellets appeared to be more stable, with a more intense staining for toluidine blue, indicating a greater chondrogenic capacity. Although Stemulate™ positively influenced chondrocyte proliferation, it had a negative effect on chondrogenic differentiation potential. This suggested that, in the treatment of cartilage defects, Stemulate™ might not be the ideal supplement for expanding chondrocytes (which maintained a chondrocyte phenotype) and, hence, for cell therapies (including ACI).

  15. Impact of human platelet lysate on the expansion and chondrogenic capacity of cultured human chondrocytes for cartilage cell therapy

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    JG Sykes

    2018-05-01

    Full Text Available High hopes have been pinned on regenerative medicine strategies in order to prevent the progression of cartilage damage to osteoarthritis, particularly by autologous chondrocyte implantation (ACI. The loss of chondrocyte phenotype during in vitro monolayer expansion, a necessary step to obtain sufficient cell numbers, may be a key limitation in ACI. In this study, it was determined whether a shorter monolayer expansion approach could improve chondrogenic differentiation. The effects of two supplement types, foetal bovine serum (FBS and Stemulate™ (a commercial source of human platelet lysate, on the expansion and re-differentiation potential of human chondrocytes, isolated from five individuals, were compared. Chondrocytes were expanded with 10 % FBS or 10 % Stemulate™. Pellets were cultured for 28 d in chondrogenic differentiation medium and assessed for the presence of cartilage matrix molecules and genes associated with chondrogenicity. Stemulate™ significantly enhanced the proliferation rate [average population doubling times: FBS, 25.07 ± 6.98 d (standard error of the mean, SEM vs. Stemulate™, 13.10 ± 2.57 d (SEM]. Sulphated glycosaminoglycans (sGAG, total collagen and qRT-PCR analyses of cartilage genes showed that FBS-expanded chondrocytes demonstrated significantly better chondrogenic capacity than Stemulate™-expanded chondrocytes. Histologically, FBS-expanded chondrocyte pellets appeared to be more stable, with a more intense staining for toluidine blue, indicating a greater chondrogenic capacity. Although Stemulate™ positively influenced chondrocyte proliferation, it had a negative effect on chondrogenic differentiation potential. This suggested that, in the treatment of cartilage defects, Stemulate™ might not be the ideal supplement for expanding chondrocytes (which maintained a chondrocyte phenotype and, hence, for cell therapies (including ACI.

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

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    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

  17. bFGF influences human articular chondrocyte differentiation

    DEFF Research Database (Denmark)

    Schmal, H; Zwingmann, J; Fehrenbach, M

    2007-01-01

    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......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...... by 53%, which was correlated with diminished mRNA production. Monolayer cultured chondrocytes secreted significant amounts of aggrecan that decreased over time. Secretion of this cartilage-specific marker was further reduced by the addition of bFGF. DISCUSSION: These findings highlight the potential...

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

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    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.

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

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    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.

  20. Low-intensity pulsed ultrasound affects human articular chondrocytes in vitro

    NARCIS (Netherlands)

    Korstjens, C.M.; van der Rijt, R.H.H.; Albers, G.H.; Semeins, C.M.; Klein-Nulend, J.

    2008-01-01

    We investigated whether low-intensity pulsed ultrasound (LIPUS) stimulates chondrocyte proliferation and matrix production in explants of human articular cartilage obtained from donors suffering from unicompartimental osteoarthritis of the knee, as well as in isolated human chondrocytes in vitro.

  1. Activation of Indian Hedgehog Promotes Chondrocyte Hypertrophy and Upregulation of MMP-13 in Human Osteoarthritic Cartilage

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    Wei, Fangyuan; Zhou, Jingming; Wei, Xiaochun; Zhang, Juntao; Fleming, Braden C.; Terek, Richard; Pei, Ming; Chen, Qian; Liu, Tao; Wei, Lei

    2012-01-01

    Objective The objectives of this study were to 1) determine the correlation between osteoarthritis (OA) and Ihh expression, and 2) establish the effects of Ihh on expression of markers of chondrocyte hypertrophy and MMP-13 in human OA cartilage. Design OA cartilage and synovial fluid samples were obtained during total knee arthroplasty. Normal cartilage samples were obtained from intra-articular tumor resections, and normal synovial fluid samples were obtained from healthy volunteers and the contralateral uninjured knee of patients undergoing anterior cruciate ligament reconstruction. OA was graded using the Mankin score. Expression of Ihh in synovial fluid was determined by western blot. Ihh, type X collagen and MMP-13 mRNA were determined by real time PCR. Protein expression of type X collagen and MMP-13 in cartilage samples were analyzed with immunohistochemistry. Chondrocyte size was measured using image analysis. Results Ihh expression was increased 2.6 fold in OA cartilage and 37% in OA synovial fluid when compared to normal control samples. Increased expression of Ihh was associated with the severity of OA and expression of markers of chondrocyte hypertrophy: type X collagen and MMP-13, and chondocyte size. Chondrocytes were more spherical with increasing severity of OA. There was a significant correlation between Mankin score and cell size (r2= 0.80) and Ihh intensity (r2 = 0.89). Exogenous Ihh induced a 6.8 fold increase of type X collagen and 2.8 fold increase of MMP-13 mRNA expression in cultured chondrocytes. Conversely, knockdown of Ihh by siRNA and Hh inhibitor Cyclopamine had the opposite effect. Conclusions Ihh expression correlates with OA progression and changes in chondrocyte morphology and gene expression consistent with chondrocyte hypertrophy and cartilage degradation seen in OA cartilage. Thus, Ihh may be a potential therapeutic target to prevent OA progression. PMID:22469853

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

    Science.gov (United States)

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

    2014-02-01

    To induce human mesenchymal stem cells (hMSCs) to differentiate into chondrocytes in three-dimensional (3D) microenvironments, we developed porous hydrogel scaffolds using the cartilage extracellular matrix (ECM) components of chondroitin sulfate (CS) and collagen (COL). The turbidity and viscosity experiments indicated hydrogel could form through pH-triggered co-precipitation when pH=2-3. Enzyme-linked immunosorbent assay (ELISA) confirmed the hydrogel scaffolds could controllably release growth factors as envisaged. Transforming growth factor-β (TGF-β) was released to stimulate hMSCs differentiation into chondrocytes; and then collagen binding domain-basic fibroblast growth factor (CBD-bFGF) was released to improve the differentiation and preserve the chondrocyte phenotype. In in vitro cell culture experiments, the differentiation processes were compared in different microenvironments: 2D culture in culture plate as control, 3D culture in the fabricated scaffolds without growth factors (CC), the samples with CBD-bFGF (CC-C), the samples with TGF-β (CC-T), the samples with CBD-bFGF/TGF-β (CC-CT). Real-time polymerase chain reaction (RT-PCR) revealed the hMSC marker genes of CD44 and CD105 decreased; at the same time the chondrocyte marker genes of collagen type II and aggrecan increased, especially in the CC-CT sample. Immunostaining results further confirmed the hMSC marker protein of CD 44 disappeared and the chondrocyte marker protein of collagen type II emerged over time in the CC-CT sample. These results imply the ECM-based hydrogel scaffolds with growth factors can supply suitable 3D cell niches for hMSCs differentiation into chondrocytes and the differentiation process can be regulated by the controllably released growth factors. Copyright © 2013 Elsevier B.V. All rights reserved.

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

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    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

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

    Science.gov (United States)

    Ito, Akira; Nagai, Momoko; Tajino, Junichi; Yamaguchi, Shoki; Iijima, Hirotaka; Zhang, Xiangkai; Aoyama, Tomoki; Kuroki, Hiroshi

    2015-01-01

    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 chondrogenesis.

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

    Directory of Open Access Journals (Sweden)

    Christelle Sanchez

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

  6. 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

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

    Science.gov (United States)

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

    2018-01-01

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

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

    International Nuclear Information System (INIS)

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

    2013-01-01

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

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

    Science.gov (United States)

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

    2013-03-01

    Cartilage serves as a low-friction and wear-resistant articulating surface in diarthrodial joints and is also important during early stages of bone remodeling. Recently, regenerative cartilage research has focused on combinations of cells paired with scaffolds. Superhydrophilic vertically aligned carbon nanotubes (VACNTs) are of particular interest in regenerative medicine. The aim of this study is to evaluate cell expansion of human articular chondrocytes on superhydrophilic VACNTs, as well as their morphology and gene expression. VACNT films were produced using a microwave plasma chamber on Ti substrates and submitted to an O2 plasma treatment to make them superhydrophilic. Human chondrocytes were cultivated on superhydrophilic VACNTs up to five days. Quantitative RT-PCR was performed to measure type I and type II Collagen, Sox9, and Aggrecan mRNA expression levels. The morphology was analyzed by scanning electron microscopy (SEM) and confocal microscopy. SEM images demonstrated that superhydrophilic VACNTs permit cell growth and adhesion of human chondrocytes. The chondrocytes had an elongated morphology with some prolongations. Chondrocytes cultivated on superhydrophilic VACNTs maintain the level expression of Aggrecan, Sox9, and Collagen II determined by qPCR. This study was the first to indicate that superhydrophilic VACNTs may be used as an efficient scaffold for cartilage or bone repair. Copyright © 2012 Elsevier B.V. All rights reserved.

  10. 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.

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

    Science.gov (United States)

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

    2016-09-01

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

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

    International Nuclear Information System (INIS)

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

    2008-01-01

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

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

    International Nuclear Information System (INIS)

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

    2013-01-01

    Cartilage serves as a low-friction and wear-resistant articulating surface in diarthrodial joints and is also important during early stages of bone remodeling. Recently, regenerative cartilage research has focused on combinations of cells paired with scaffolds. Superhydrophilic vertically aligned carbon nanotubes (VACNTs) are of particular interest in regenerative medicine. The aim of this study is to evaluate cell expansion of human articular chondrocytes on superhydrophilic VACNTs, as well as their morphology and gene expression. VACNT films were produced using a microwave plasma chamber on Ti substrates and submitted to an O 2 plasma treatment to make them superhydrophilic. Human chondrocytes were cultivated on superhydrophilic VACNTs up to five days. Quantitative RT-PCR was performed to measure type I and type II Collagen, Sox9, and Aggrecan mRNA expression levels. The morphology was analyzed by scanning electron microscopy (SEM) and confocal microscopy. SEM images demonstrated that superhydrophilic VACNTs permit cell growth and adhesion of human chondrocytes. The chondrocytes had an elongated morphology with some prolongations. Chondrocytes cultivated on superhydrophilic VACNTs maintain the level expression of Aggrecan, Sox9, and Collagen II determined by qPCR. This study was the first to indicate that superhydrophilic VACNTs may be used as an efficient scaffold for cartilage or bone repair. Highlights: ► Chondrocytes were cultivated on Superhydrophilic Vertically Aligned Multiwall Carbon Nanotubes (VACNT). ► We have shown a correlation between gene expression and thermodynamics aspects. ► Superhydrhophilic VACNT will be an excellent substrate for cartilage and bone tissue regeneration.

  14. 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.

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

    OpenAIRE

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

    2016-01-01

    Summary Objective To use deep sequencing to identify novel microRNAs (miRNAs) in human osteoarthritic cartilage which have a functional role in chondrocyte phenotype or function. Design A small RNA library was prepared from human osteoarthritic primary chondrocytes using in-house adaptors and analysed by Illumina sequencing. Novel candidate miRNAs were validated by northern blot and qRT-PCR. Expression was measured in cartilage models. Targets of novel candidates were identified by microarray...

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

    OpenAIRE

    1988-01-01

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

  17. Dynamic compression of chondrocyte-agarose constructs reveals new candidate mechanosensitive genes.

    Directory of Open Access Journals (Sweden)

    Carole Bougault

    Full Text Available Articular cartilage is physiologically exposed to repeated loads. The mechanical properties of cartilage are due to its extracellular matrix, and homeostasis is maintained by the sole cell type found in cartilage, the chondrocyte. Although mechanical forces clearly control the functions of articular chondrocytes, the biochemical pathways that mediate cellular responses to mechanical stress have not been fully characterised. The aim of our study was to examine early molecular events triggered by dynamic compression in chondrocytes. We used an experimental system consisting of primary mouse chondrocytes embedded within an agarose hydrogel; embedded cells were pre-cultured for one week and subjected to short-term compression experiments. Using Western blots, we demonstrated that chondrocytes maintain a differentiated phenotype in this model system and reproduce typical chondrocyte-cartilage matrix interactions. We investigated the impact of dynamic compression on the phosphorylation state of signalling molecules and genome-wide gene expression. After 15 min of dynamic compression, we observed transient activation of ERK1/2 and p38 (members of the mitogen-activated protein kinase (MAPK pathways and Smad2/3 (members of the canonical transforming growth factor (TGF-β pathways. A microarray analysis performed on chondrocytes compressed for 30 min revealed that only 20 transcripts were modulated more than 2-fold. A less conservative list of 325 modulated genes included genes related to the MAPK and TGF-β pathways and/or known to be mechanosensitive in other biological contexts. Of these candidate mechanosensitive genes, 85% were down-regulated. Down-regulation may therefore represent a general control mechanism for a rapid response to dynamic compression. Furthermore, modulation of transcripts corresponding to different aspects of cellular physiology was observed, such as non-coding RNAs or primary cilium. This study provides new insight into how

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

    Science.gov (United States)

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

    2007-01-01

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

  19. Human IGF-I propeptide A promotes articular chondrocyte biosynthesis and employs glycosylation-dependent heparin binding.

    Science.gov (United States)

    Shi, Shuiliang; Kelly, Brian J; Wang, Congrong; Klingler, Ken; Chan, Albert; Eckert, George J; Trippel, Stephen B

    2018-03-01

    Insulin-like growth factor I (IGF-I) is a key regulator of chondrogenesis, but its therapeutic application to articular cartilage damage is limited by rapid elimination from the repair site. The human IGF-I gene gives rise to three IGF-I propeptides (proIGF-IA, proIGF-IB and proIGF-IC) that are cleaved to create mature IGF-I. In this study, we elucidate the processing of IGF-I precursors by articular chondrocytes, and test the hypotheses that proIGF-I isoforms bind to heparin and regulate articular chondrocyte biosynthesis. Human IGF-I propeptides and mutants were overexpressed in bovine articular chondrocytes. IGF-I products were characterized by ELISA, western blot and FPLC using a heparin column. The biosynthetic activity of IGF-I products on articular chondrocytes was assayed for DNA and glycosaminoglycan that the cells produced. Secreted IGF-I propeptides stimulated articular chondrocyte biosynthetic activity to the same degree as mature IGF-I. Of the three IGF-I propeptides, only one, proIGF-IA, strongly bound to heparin. Interestingly, heparin binding of proIGF-IA depended on N-glycosylation at Asn92 in the EA peptide. To our knowledge, this is the first demonstration that N-glycosylation determines the binding of a heparin-binding protein to heparin. The biosynthetic and heparin binding abilities of proIGF-IA, coupled with its generation of IGF-I, suggest that proIGF-IA may have therapeutic value for articular cartilage repair. These data identify human pro-insulin-like growth factor IA as a bifunctional protein. Its combined ability to bind heparin and augment chondrocyte biosynthesis makes it a promising therapeutic agent for cartilage damage due to trauma and osteoarthritis. Copyright © 2017 Elsevier B.V. All rights reserved.

  20. Nicotine promotes proliferation and collagen synthesis of chondrocytes isolated from normal human and osteoarthritis patients.

    Science.gov (United States)

    Ying, Xiaozhou; Cheng, Shaowen; Shen, Yue; Cheng, Xiaojie; An Rompis, Ferdinand; Wang, Wei; Lin, Zhongqin; Chen, Qingyu; Zhang, Wei; Kou, Dongquan; Peng, Lei; Tian, Xin Qiao; Lu, Chuan Zhu

    2012-01-01

    The aims of the study were to show the direct effect of nicotine with different concentrations (0, 25, 50, and 100 ng/ml) on chondrocytes isolated from normal human and osteoarthritis patients, respectively. Microscopic observation was performed during the culture with an inverted microscope. Methyl thiazolyl tetrazolium (MTT) assay method was adopted to observe the influence of nicotine on the proliferation of chondrocytes, and real-time PCR and ELISA were used to assay the mRNA and protein expression of type II collagen and aggrecan, respectively. We discovered that the OA chondrocytes were similar to fibroblasts in shape and grow slower than normal chondrocytes. The proliferation of the two kinds of chondrocytes was increased in a concentration-dependent manner and in a time-dependent manner (P<0.05). Also, we found that the mRNA level of type II collagen were upregulated under 25-100 ng/ml nicotine doses both in the two kinds of chondrocytes compared with control. The expression of protein levels of type II collagen were synthesized in line with the increase in mRNA. No effect was observed on aggrecan synthesis with any nicotine dose. We concluded that nicotine has the same effect on both chondrocytes, obtained either from osteoarthritis patients or from normal human, and the positive effect of smoking in OA may relate to the alteration in metabolism of chondrocytes.

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

    NARCIS (Netherlands)

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

    2005-01-01

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

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

    Science.gov (United States)

    Apelgren, Peter; Amoroso, Matteo; Lindahl, Anders; Brantsing, Camilla; Rotter, Nicole; Gatenholm, Paul; Kölby, Lars

    2017-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Peter Apelgren

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

  4. Characterization and chondrocyte differentiation stage-specific expression of KRAB zinc-finger protein gene ZNF470

    International Nuclear Information System (INIS)

    Hering, Thomas M.; Kazmi, Najam H.; Huynh, Tru D.; Kollar, John; Xu, Laura; Hunyady, Aaron B.; Johnstone, Brian

    2004-01-01

    As part of a study to identify novel transcriptional regulators of chondrogenesis-related gene expression, we have cloned and characterized cDNA for zinc-finger protein 470 (ZNF470), the human ortholog of which encodes a 717 amino acid residue protein containing 17 Cys 2 His 2 zinc-finger domains, as well as KRAB-A and KRAB-B motifs. The cDNA library used to isolate the initial ZNF470 clone was prepared from human bone marrow-derived mesenchymal progenitor cells at an intermediate stage of chondrogenic differentiation. We have determined the intron-exon structure of the human ZNF470 gene, which has been mapped to a zinc-finger cluster in a known imprinted region of human chromosome 19q13.4. ZNF470 is expressed at high levels in human testis and is expressed at low or undetectible levels in other adult tissues. Human ZNF470 expressed in mammalian cells as an EGFP fusion protein localizes predominantly to the nucleus, consistent with a role in transcriptional regulation. ZNF470, analyzed by quantitative real time PCR, was transiently expressed before the maximal expression of COL2A1 during chondrogenic differentiation in vitro. We have also characterized the bovine ortholog of human ZNF470, which encodes a 508 amino acid residue protein having 10 zinc-finger domains. A bovine ZNF470 cDNA clone was used to examine expression of ZNF470 in bovine articular chondrocytes treated with retinoic acid to stimulate dedifferentiation. Bovine ZNF470 expression was undetectable in freshly isolated bovine articular chondrocytes, but was dramatically upregulated in dedifferentiated retinoic acid-treated chondrocytes. These results, in two model systems, suggest a possible role for ZNF470 in the regulation of chondrogenesis-specific gene expression

  5. Culture temperature affects redifferentiation and cartilaginous extracellular matrix formation in dedifferentiated human chondrocytes.

    Science.gov (United States)

    Ito, Akira; Aoyama, Tomoki; Iijima, Hirotaka; Tajino, Junichi; Nagai, Momoko; Yamaguchi, Shoki; Zhang, Xiangkai; Kuroki, Hiroshi

    2015-05-01

    To date, there have been few studies on how temperature affects the phenotype and metabolism of human chondrocytes. Thus, the purpose of this study was to elucidate the effects of culture temperature on chondrocyte redifferentiation and extracellular matrix (ECM) formation using dedifferentiated mature human chondrocytes in vitro. Dedifferentiated chondrocytes were cultured in a pellet culture system for up to 21 days. The pellets were randomly divided into three groups with different culture temperature (32, 37, and 41°C). Chondrocyte redifferentiation and ECM formation were evaluated by wet weight, messenger ribonucleic acid (mRNA), histological, and biochemical analyses. The results showed that the wet weight and the mRNA expressions of collagen type II A1 and cartilage oligomeric matrix protein at 37°C were higher than the corresponding values at 32°C. The histological and biochemical analyses revealed that the syntheses of type II collagen and proteoglycan were promoted at 37°C compared to those at 32°C, whereas they were considerably inhibited at 41°C. In conclusion, the results obtained herein indicated that temperature affects chondrocyte redifferentiation and ECM formation, and modulation of temperature might thus represent an advantageous means to regulate the phenotype and biosynthetic activity of chondrocytes. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.

  6. NF-kappaB specifically activates BMP-2 gene expression in growth plate chondrocytes in vivo and in a chondrocyte cell line in vitro.

    Science.gov (United States)

    Feng, Jian Q; Xing, Lianping; Zhang, Jiang-Hong; Zhao, Ming; Horn, Diane; Chan, Jeannie; Boyce, Brendan F; Harris, Stephen E; Mundy, Gregory R; Chen, Di

    2003-08-01

    Bone morphogenetic protein-2 (BMP-2) regulates growth plate chondrogenesis during development and postnatal bone growth, but the control mechanisms of BMP-2 expression in growth plate chondrocytes are unknown. Here we have used both in vitro and in vivo approaches to demonstrate that transcription factor, NF-kappaB, regulates BMP-2 gene expression in chondrocytes. Two putative NF-kappaB response elements were found in the -2712/+165 region of the BMP-2 gene. Cotransfection of mutant I-kappaBalpha expression plasmids with BMP-2 promoter-luciferase reporters into TMC-23 chondrocyte cell line suppressed BMP-2 transcription. Mutations in NF-kappaB response elements in the BMP-2 gene lead to decreases in BMP-2 promoter activity. Electrophoretic mobility shift assay using nuclear extracts from TMC-23 chondrocytic cells revealed that the NF-kappaB subunits p50 and p65 bound to the NF-kappaB response elements of the BMP-2 gene. Thus, NF-kappaB may positively regulate BMP-2 gene transcription. Consistent with these findings, expression of BMP-2 mRNA was significantly reduced in growth plate chondrocytes in NF-kappaB p50/p52 dKO mice, which associated with decreased numbers of 5-bromo-2'-deoxyuridine (BrdUrd)-positive cells in the proliferating zone of growth plate in these mice. Therefore, in postnatal growth plate chondrocytes, expression of BMP-2 is regulated by NF-kappaB, which may play an important role in chondrogenesis.

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

    Directory of Open Access Journals (Sweden)

    L Boo

    2009-11-01

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

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

    International Nuclear Information System (INIS)

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

    2014-01-01

    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

  9. 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.

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

    Directory of Open Access Journals (Sweden)

    Saliya Gurusinghe

    2014-01-01

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

  11. A Tumor Suppressor Gene Product, Platelet-Derived Growth Factor Receptor-Like Protein Controls Chondrocyte Proliferation and Differentiation.

    Science.gov (United States)

    Kawata, Kazumi; Kubota, Satoshi; Eguchi, Takanori; Aoyama, Eriko; Moritani, Norifumi H; Oka, Morihiko; Kawaki, Harumi; Takigawa, Masaharu

    2017-11-01

    The platelet-derived growth factor receptor-like (PDGFRL) gene is regarded as a tumor suppressor gene. However, nothing is known about the molecular function of PDGFRL. In this study, we initially clarified its function in chondrocytes. Among all cell lines examined, the PDGFRL mRNA level was the highest in chondrocytic HCS-2/8 cells. Interestingly, the proliferation of chondrocytic HCS-2/8 cells was promoted by PDGFRL overexpression, whereas that of the breast cancer-derived MDA-MB-231 cells was inhibited. Of note, in PDGFRL-overexpressing HCS-2/8 cells, the expression of chondrocyte differentiation marker genes, SOX9, ACAN, COL2A1, COL10A1, and ALP, was decreased. Moreover, we confirmed the expression of PDGFRL mRNA in normal cartilage tissue and chondrocytes. Eventually, the expression of PDGFRL mRNA in condrocytes except in the case of hypertrophic chondrocytes was demonstrated in vivo and in vitro. These findings suggest that PDGFRL plays the different roles, depending upon cell types. Particularly, in chondrocytes, PDGFRL may play a new and important role which is distinct from the function previously reported. J. Cell. Biochem. 118: 4033-4044, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

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

    Directory of Open Access Journals (Sweden)

    Rajesh Kumar

    2015-04-01

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

  13. Hyperpolarisation of cultured human chondrocytes following cyclical pressure-induced strain: evidence of a role for alpha 5 beta 1 integrin as a chondrocyte mechanoreceptor.

    Science.gov (United States)

    Wright, M O; Nishida, K; Bavington, C; Godolphin, J L; Dunne, E; Walmsley, S; Jobanputra, P; Nuki, G; Salter, D M

    1997-09-01

    Mechanical stimuli influence chondrocyte metabolism, inducing changes in intracellular cyclic adenosine monophosphate and proteoglycan production. We have previously demonstrated that primary monolayer cultures of human chondrocytes have an electrophysiological response after intermittent pressure-induced strain characterised by a membrane hyperpolarisation of approximately 40%. The mechanisms responsible for these changes are not fully understood but potentially involve signalling molecules such as integrins that link extracellular matrix with cytoplasmic components. The results reported in this paper demonstrate that the transduction pathways involved in the hyperpolarisation response of human articular chondrocytes in vitro after cyclical pressure-induced strain involve alpha 5 beta 1 integrin. We have demonstrated, using pharmacological inhibitors of a variety of intracellular signalling pathways, that the actin cytoskeleton, the phospholipase C calmodulin pathway, and both tyrosine protein kinase and protein kinase C activities are important in the transduction of the electrophysiological response. These results suggest that alpha 5 beta 1 is an important chondrocyte mechanoreceptor and a potential regulator of chondrocyte function.

  14. 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.

  15. Effects of tofacitinib on nucleic acid metabolism in human articular chondrocytes.

    Science.gov (United States)

    Koizumi, Hideki; Arito, Mitsumi; Endo, Wataru; Kurokawa, Manae S; Okamoto, Kazuki; Omoteyama, Kazuki; Suematsu, Naoya; Beppu, Moroe; Kato, Tomohiro

    2015-07-01

    In our previous screening of chondrocyte protein profiles, the amount of adenosine monophosphate deaminase (AMPD) 2 was found to be decreased by tofacitinib. Extending the study, here we confirmed the decrease of AMPD2 by tofacitinib and further investigated effects of tofacitinib on purine nucleotide metabolism. Human articular chondrocytes and a chondrosarcoma cell line: OUMS-27 were stimulated with tofacitinib. Then the levels of AMPD2 and its related enzymes were investigated by Western blot. The levels of AMP and adenosine were assessed by mass spectrometry. We confirmed the significant decrease of AMPD2 by tofacitinib in chondrocytes (p = 0.025). The levels of adenosine kinase and 5'-nucleotidase were decreased in chondrocytes, although they did not meet statistical significance (p = 0.067 and p = 0.074, respectively). The results from OUMS-27 were similar to those from the chondrocytes. The cellular adenosine levels were significantly decreased by tofacitinib in OUMS-27 (p = 0.014). The cellular AMP levels were increased, although they did not meet statistical significance in OUMS-27 (p = 0.066). Our data indicate that tofacitinib increases the cellular levels of adenosine, which is known to have anti-inflammatory activity, through the downregulation of AMPD2. This would be a novel functional aspect of tofacitinib.

  16. Analysis of human articular chondrocyte CD44 isoform expression and function in health and disease.

    Science.gov (United States)

    Salter, D M; Godolphin, J L; Gourlay, M S; Lawson, M F; Hughes, D E; Dunne, E

    1996-08-01

    Interactions between articular chondrocytes and components of the extracellular matrix are of potential importance in the normal function of cartilage and in the pathophysiology of arthritis. Little is known of the basis of these interactions, but cell adhesive molecules such as CD44 are likely to be involved. Immunohistology using six well-characterized anti-CD44 monoclonal antibodies demonstrated standard CD44 isoform (CD44H) expression by all chondrocytes in normal and osteoarthrotic (OA) cartilage but absence of the CD44E variant. Polymerase chain reaction (PCR) of reverse transcribed mRNA from monolayer cultures of normal and OA chondrocytes using primer sequences which span the region containing variably spliced exons produced a predominant band representing the standard form of CD44, which lacks the variable exons 6-15 (v1-v10). No product was seen at the expected size of the epithelial variant of CD44 (CD44v8-10). Use of exon-specific primers, however, showed expression of variant exons resulting in multiple minor isoforms. Standard CD44 was also shown to be the predominantly expressed isoform identified by immunoprecipitation, but human articular chondrocytes did not adhere to hyaluronan in vitro. Chondrocyte CD44 may function as an adhesion receptor for other matrix molecules such as fibronectin or collagen.

  17. 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

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

  19. Topographic variation in redifferentiation capacity of chondrocytes in the adult human knee joint.

    Science.gov (United States)

    Stenhamre, H; Slynarski, K; Petrén, C; Tallheden, T; Lindahl, A

    2008-11-01

    The aim of this study was to investigate the topographic variation in matrix production and cell density in the adult human knee joint. Additionally, we have examined the redifferentiation potential of chondrocytes expanded in vitro from the different locations. Full thickness cartilage-bone biopsies were harvested from seven separate anatomical locations of healthy knee joints from deceased adult human donors. Chondrocytes were isolated, expanded in vitro and redifferentiated in a pellet mass culture. Biochemical analysis of total collagen, proteoglycans and cellular content as well as histology and immunohistochemistry were performed on biopsies and pellets. In the biochemical analysis of the biopsies, we found lower proteoglycan to collagen (GAG/HP) ratio in the non-weight bearing (NWB) areas compared to the weight bearing (WB) areas. The chondrocytes harvested from different locations in femur showed a significantly better attachment and proliferation ability as well as good post-expansion chondrogenic capacity in pellet mass culture compared with the cells harvested from tibia. These results demonstrate that there are differences in extra cellular content within the adult human knee in respect to GAG/HP ratio. Additionally, the data show that clear differences between chondrocytes harvested from femur and tibia from healthy human knee joints exist and that the differences are not completely abolished during the process of de- and redifferentiation. These findings emphasize the importance of the understanding of topographic variation in articular cartilage biology when approaching new cartilage repair strategies.

  20. 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...

  1. The transcription factor ATF3 is upregulated during chondrocyte differentiation and represses cyclin D1 and A gene transcription

    Directory of Open Access Journals (Sweden)

    James Claudine G

    2006-09-01

    Full Text Available Abstract Background Coordinated chondrocyte proliferation and differentiation are required for normal endochondral bone growth. Transcription factors binding to the cyclicAMP response element (CRE are known to regulate these processes. One member of this family, Activating Tanscription Factor 3 (ATF3, is expressed during skeletogenesis and acts as a transcriptional repressor, but the function of this protein in chondrogenesis is unknown. Results Here we demonstrate that Atf3 mRNA levels increase during mouse chondrocyte differentiation in vitro and in vivo. In addition, Atf3 mRNA levels are increased in response to cytochalasin D treatment, an inducer of chondrocyte maturation. This is accompanied by increased Atf3 promoter activity in cytochalasin D-treated chondrocytes. We had shown earlier that transcription of the cell cycle genes cyclin D1 and cyclin A in chondrocytes is dependent on CREs. Here we demonstrate that overexpression of ATF3 in primary mouse chondrocytes results in reduced transcription of both genes, as well as decreased activity of a CRE reporter plasmid. Repression of cyclin A transcription by ATF3 required the CRE in the cyclin A promoter. In parallel, ATF3 overexpression reduces the activity of a SOX9-dependent promoter and increases the activity of a RUNX2-dependent promoter. Conclusion Our data suggest that transcriptional induction of the Atf3 gene in maturing chondrocytes results in down-regulation of cyclin D1 and cyclin A expression as well as activation of RUNX2-dependent transcription. Therefore, ATF3 induction appears to facilitate cell cycle exit and terminal differentiation of chondrocytes.

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

    OpenAIRE

    Apelgren, Peter; Amoroso, Matteo; Lindahl, Anders; Brantsing, Camilla; Rotter, Nicole; Gatenholm, Paul; Kölby, Lars

    2017-01-01

    Cartilage repair and replacement is a major challenge in plastic reconstructive surgery. The development of a process capable of creating a patient-specific cartilage framework would be a major breakthrough. Here, we described methods for creating human cartilage in vivo and quantitatively assessing the proliferative capacity and cartilage-formation ability in mono- and co-cultures of human chondrocytes and human mesenchymal stem cells in a three-dimensional (3D)-bioprinted hydrogel scaffold....

  3. 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

  4. Influence of bone morphogenetic protein-2 on the extracellular matrix, material properties, and gene expression of long-term articular chondrocyte cultures: loss of chondrocyte stability.

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    Krawczak, David A; Westendorf, Jennifer J; Carlson, Cathy S; Lewis, Jack L

    2009-06-01

    The aim of this study was to determine the effects of bone morphogenetic protein-2 (BMP-2) on articular chondrocyte tissues grown as monolayers in vitro for up to 8 weeks. Articular chondrocytes were isolated from New Zealand White rabbits and plated in monolayer cultures. The cultures were supplemented with 100 ng/mL of BMP-2 for up to 8 weeks and the extracellular matrix (ECM) composition, material properties, and messenger RNA (mRNA) expression were analyzed. mRNA expression of cartilage-specific genes, type II collagen, and aggrecan showed that BMP-2 enhanced chondrocyte stability for up to 3 weeks. After 3 weeks in culture, there was substantially more type I collagen expression and more osteopontin and runt-related transcription factor 2 expression in 5- and 8-week cultures treated with BMP-2 than in controls. Additionally, matrix metalloproteinase-13 and ADAMTS-5 (A disintegrin-like and metalloproteinase with thrombospondin 5) were upregulated in 5- and 8-week cultures treated with BMP-2, coinciding with a loss of ECM density, collagen, and proteoglycan. Eight-week tissue stimulated with BMP-2 was more fragile and tore more easily when removed from the culture dish as compared to controls, suggesting temporal limitations to the effectiveness of BMP-2 in monolayer systems and perhaps other models to enhance the generation of a cartilage-like tissue for tissue engineering purposes.

  5. 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.

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

    Science.gov (United States)

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

    2016-04-01

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

  7. Chondrocyte heterogeneity: immunohistologically defined variation of integrin expression at different sites in human fetal knees.

    Science.gov (United States)

    Salter, D M; Godolphin, J L; Gourlay, M S

    1995-04-01

    During development and at maturity different forms of cartilage vary in morphology and macromolecular content. This reflects heterogeneity of chondrocyte activity, in part involving differential interactions with the adjacent extracellular matrix via specialized cell surface receptors such as integrins. We undertook an immunohistological study on a series of human fetal knee joints to assess variation in the expression of integrins by chondrocytes and potential matrix ligands in articular, epiphyseal, growth plate, and meniscal cartilage. The results show that articular chondrocytes (beta 1+, beta 5 alpha V+, alpha 1+, alpha 2+/-, alpha 5+, weakly alpha 6+, alpha V+) differed from epiphyseal (beta 1+, beta 5 alpha V+, alpha 1+/-, alpha 2+/-, alpha 5+, alpha 6+, alpha V+) growth plate (beta 1+, beta 5 alpha V+, alpha 1-, alpha 2-, alpha 5+, alpha 6+, alpha V+), and meniscal cells (beta 1+, beta 5 alpha V+, alpha 1+, strongly alpha 2+, alpha 5+, alpha 6+, alpha V+ in expression of integrin subunits. There was no expression of beta 3, beta 4, beta 6, or alpha 3 by chondrocytes. These results differ from previous reports on the expression of integrins by adult articular cartilage, where alpha 2 and alpha 6 are not seen. Variation in distribution of matrix ligands was also seen. Fibronectin, laminin and Type VI collagen were expressed in all cartilages but there was restricted expression of tenascin, ED-A and ED-B fibronectin isoforms (articular cartilage and meniscus), and vitronectin (absent from growth plate cartilage). Regulated expression of integrins by chondrocytes, associated with changes in the pericellular matrix composition, is of potential importance in control of cartilage differentiation and function in health and disease.

  8. 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.

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

    Science.gov (United States)

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

    2013-03-15

    Bovine lactoferricin (LfcinB) is a heparan sulfate-binding peptide with multiple bioactivities. In human articular cartilage, LfcinB antagonizes interleukin-1 β (IL-1β) and fibroblast growth factor 2 (FGF-2) in proteoglycan metabolism, catabolic protease expression, and induction of pro-inflammatory mediators. LfcinB specifically activates ERK1/2, p38 and Akt, but whether these signaling pathways control the expression of LfcinB target genes remained unknown. In this report, we characterized a novel aspect of LfcinB-mediated genetic response in human articular chondrocytes, tissue inhibitor of metalloproteinase 3 (TIMP-3) induction. Inhibition of individual signaling pathways revealed that ERK1/2 functions as the major pathway in TIMP-3 expression, whereas Akt plays a minor role. Further investigation identified Sp1 as a critical transcriptional activator in TIMP-3 regulation, and Sp1 activity is modulated by ERK1/2, not Akt. Comparative quantification indicates that significant downregulation of TIMP-3 occurs in OA chondrocytes, suggesting a beneficial role of LfcinB in OA pathogenesis. Our results collectively provide new insights into the mechanism of action of LfcinB, and support the candidacy of LfcinB as a chondroprotective agent. Copyright © 2013 Elsevier B.V. All rights reserved.

  10. 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.

  11. Indian hedgehog signaling promotes chondrocyte differentiation in enchondral ossification in human cervical ossification of the posterior longitudinal ligament.

    Science.gov (United States)

    Sugita, Daisuke; Yayama, Takafumi; Uchida, Kenzo; Kokubo, Yasuo; Nakajima, Hideaki; Yamagishi, Atsushi; Takeura, Naoto; Baba, Hisatoshi

    2013-10-15

    Histological, immunohistochemical, and immunoblot analyses of the expression of Indian hedgehog (Ihh) signaling in human cervical ossification of the posterior longitudinal ligament (OPLL). To examine the hypothesis that Ihh signaling in correlation with Sox9 and parathyroid-related peptide hormone (PTHrP) facilitates chondrocyte differentiation in enchondral ossification process in human cervical OPLL. In enchondral ossification, certain transcriptional factors regulate cell differentiation. OPLL is characterized by overexpression of these factors and disturbance of the normal cell differentiation process. Ihh signaling is essential for enchondral ossification, especially in chondrocyte hypertrophy. Samples of ossified ligaments were harvested from 45 patients who underwent anterior cervical decompressive surgery for symptomatic OPLL, and 6 control samples from patients with cervical spondylotic myelopathy/radiculopathy without OPLL. The harvested sections were stained with hematoxylin-eosin and toluidine blue, examined by transmission electron microscopy, and immunohistochemically stained for Ihh, PTHrP, Sox9, type X, XI collagen, and alkaline phosphatase. Immunoblot analysis was performed in cultured cells derived from the posterior longitudinal ligaments in the vicinity of the ossified plaque and examined for the expression of these factors. The ossification front in OPLL contained chondrocytes at various differentiation stages, including proliferating chondrocytes in fibrocartilaginous area, hypertrophic chondrocytes around the calcification front, and apoptotic chondrocytes near the ossified area. Immunoreactivity for Ihh and Sox9 was evident in proliferating chondrocytes and was strongly positive for PTHrP in hypertrophic chondrocytes. Mesenchymal cells with blood vessel formation were positive for Ihh, PTHrP, and Sox9. Cultured cells from OPLL tissues expressed significantly higher levels of Ihh, PTHrP, and Sox9 than those in non-OPLL cells. Our results

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

    Science.gov (United States)

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

    2016-01-01

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

  13. 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.

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

    Science.gov (United States)

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

    2009-01-01

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

  15. Human articular chondrocytes secrete parathyroid hormone-related protein and inhibit hypertrophy of mesenchymal stem cells in coculture during chondrogenesis.

    Science.gov (United States)

    Fischer, J; Dickhut, A; Rickert, M; Richter, W

    2010-09-01

    The use of bone marrow-derived mesenchymal stem cells (MSCs) has shown promise in cell-based cartilage regeneration. A yet-unsolved problem, however, is the unwanted up-regulation of markers of hypertrophy, such as alkaline phosphatase (AP) and type X collagen, during in vitro chondrogenesis and the formation of unstable calcifying cartilage at heterotopic sites. In contrast, articular chondrocytes produce stable, nonmineralizing cartilage. The aim of this study was to address whether coculture of MSCs with human articular chondrocytes (HACs) can suppress the undesired hypertrophy in differentiating MSCs. MSCs were differentiated in chondrogenic medium that had or had not been conditioned by parallel culture with HAC pellets, or MSCs were mixed in the same pellet with the HACs (1:1 or 1:2 ratio) and cultured for 6 weeks. Following in vitro differentiation, the pellets were transplanted into SCID mice. The gene expression ratio of COL10A1 to COL2A1 and of Indian hedgehog (IHH) to COL2A1 was significantly reduced by differentiation in HAC-conditioned medium, and less type X collagen protein was deposited relative to type II collagen. AP activity was significantly lower (P chondrogenesis. The main inhibitory effects seen with HAC-conditioned medium were reproducible by PTHrP supplementation of unconditioned medium. HAC-derived soluble factors and direct coculture are potent means of improving chondrogenesis and suppressing the hypertrophic development of MSCs. PTHrP is an important candidate soluble factor involved in this effect.

  16. Nitric oxide from both exogenous and endogenous sources activates mitochondria-dependent events and induces insults to human chondrocytes.

    Science.gov (United States)

    Wu, Gong-Jhe; Chen, Tyng-Guey; Chang, Huai-Chia; Chiu, Wen-Ta; Chang, Chia-Chen; Chen, Ruei-Ming

    2007-08-15

    During inflammation, overproduction of nitric oxide (NO) can damage chondrocytes. In this study, we separately evaluated the toxic effects of exogenous and endogenous NO on human chondrocytes and their possible mechanisms. Human chondrocytes were exposed to sodium nitroprusside (SNP), an NO donor, or a combination of lipopolysaccharide (LPS) and interferon-gamma (IFN-gamma) as the exogenous and endogenous sources of NO, respectively. Administration of SNP or a combination of LPS and IFN-gamma in human chondrocytes increased cellular NO levels but decreased cell viability. Exposure to exogenous or endogenous NO significantly induced apoptosis of human chondrocytes. When treated with exogenous or endogenous NO, the mitochondrial membrane potential time-dependently decreased. Exposure to exogenous or endogenous NO significantly enhanced cellular reactive oxygen species (ROS) and cytochrome c (Cyt c) levels. Administration of exogenous or endogenous NO increased caspase-3 activity and consequently induced DNA fragmentation. Suppression of caspase-3 activation by Z-DEVD-FMK decreased NO-induced DNA fragmentation and cell apoptosis. Similar to SNP, exposure of human chondrocytes to S-nitrosoglutathione (GSNO), another NO donor, caused significant increases in Cyt c levels, caspase-3 activity, and DNA fragmentation, and induced cell apoptosis. Pretreatment with N-monomethyl arginine (NMMA), an inhibitor of NO synthase, significantly decreased cellular NO levels, and lowered endogenous NO-induced alterations in cellular Cyt c amounts, caspase-3 activity, DNA fragmentation, and cell apoptosis. Results of this study show that NO from exogenous and endogenous sources can induce apoptotic insults to human chondrocytes via a mitochondria-dependent mechanism.

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

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    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.

  18. Cell expansion of human articular chondrocytes on macroporous gelatine scaffolds-impact of microcarrier selection on cell proliferation

    Energy Technology Data Exchange (ETDEWEB)

    Pettersson, Sofia; Kratz, Gunnar [Laboratory for Reconstructive Plastic Surgery, Department of Clinical and Experimental Medicine, Linkoeping University, SE-581 85 Linkoeping (Sweden); Wetteroe, Jonas [Rheumatology/AIR, Department of Clinical and Experimental Medicine, Linkoeping University, SE-581 85 Linkoeping (Sweden); Tengvall, Pentti, E-mail: sofia.pettersson@liu.se [Institute of Clinical Sciences, Department of Biomaterials, The Sahlgrenska Academy at University of Gothenburg, SE-405 30 Gothenburg (Sweden)

    2011-12-15

    This study investigates human chondrocyte expansion on four macroporous gelatine microcarriers (CultiSpher) differing with respect to two manufacturing processes-the amount of emulsifier used during initial preparation and the gelatine cross-linking medium. Monolayer-expanded articular chondrocytes from three donors were seeded onto the microcarriers and cultured in spinner flask systems for a total of 15 days. Samples were extracted every other day to monitor cell viability and establish cell counts, which were analysed using analysis of variance and piecewise linear regression. Chondrocyte densities increased according to a linear pattern for all microcarriers, indicating an ongoing, though limited, cell proliferation. A strong chondrocyte donor effect was seen during the initial expansion phase. The final cell yield differed significantly between the microcarriers and our results indicate that manufacturing differences affected chondrocyte densities at this point. Remaining cells stained positive for chondrogenic markers SOX-9 and S-100 but extracellular matrix formation was modest to undetectable. In conclusion, the four gelatine microcarriers supported chondrocyte adhesion and proliferation over a two week period. The best yield was observed for microcarriers produced with low emulsifier content and cross-linked in water and acetone. These results add to the identification of optimal biomaterial parameters for specific cellular processes and populations.

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

    Directory of Open Access Journals (Sweden)

    Serena Guidotti

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

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

    Science.gov (United States)

    Guidotti, Serena; Minguzzi, Manuela; Platano, Daniela; Cattini, Luca; Trisolino, Giovanni; Mariani, Erminia; Borzì, Rosa Maria

    2015-01-01

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

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

    NARCIS (Netherlands)

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

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

  2. 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.

  3. Comparison of Four Protocols to Generate Chondrocyte-Like Cells from Human Induced Pluripotent Stem Cells (hiPSCs).

    Science.gov (United States)

    Suchorska, Wiktoria Maria; Augustyniak, Ewelina; Richter, Magdalena; Trzeciak, Tomasz

    2017-04-01

    Stem cells (SCs) are a promising approach to regenerative medicine, with the potential to treat numerous orthopedic disorders, including osteo-degenerative diseases. The development of human-induced pluripotent stem cells (hiPSCs) has increased the potential of SCs for new treatments. However, current methods of differentiating hiPSCs into chondrocyte-like cells are suboptimal and better methods are needed. The aim of the present study was to assess four different chondrogenic differentiation protocols to identify the most efficient method of generating hiPSC-derived chondrocytes. For this study, hiPSCs were obtained from primary human dermal fibroblasts (PHDFs) and differentiated into chondrocyte-like cells using four different protocols: 1) monolayer culture with defined growth factors (GF); 2) embryoid bodies (EBs) in a chondrogenic medium with TGF-β3 cells; 3) EBs in chondrogenic medium conditioned with human chondrocytes (HC-402-05a cell line) and 4) EBs in chondrogenic medium conditioned with human chondrocytes and supplemented with TGF-β3. The cells obtained through these four protocols were evaluated and compared at the mRNA and protein levels. Although chondrogenic differentiation of hiPSCs was successfully achieved with all of these protocols, the two fastest and most cost-effective methods were the monolayer culture with GFs and the medium conditioned with human chondrocytes. Both of these methods are superior to other available techniques. The main advantage of the conditioned medium is that the technique is relatively simple and inexpensive while the directed method (i.e., monolayer culture with GFs) is faster than any protocol described to date because it is does not require additional steps such as EB formation.

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

    Science.gov (United States)

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

    2015-03-01

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

  5. PPAR-δ Agonist With Mesenchymal Stem Cells Induces Type II Collagen-Producing Chondrocytes in Human Arthritic Synovial Fluid.

    Science.gov (United States)

    Heck, Bruce E; Park, Joshua J; Makani, Vishruti; Kim, Eun-Cheol; Kim, Dong Hyun

    2017-08-01

    Osteoarthritis (OA) is an inflammatory joint disease characterized by degeneration of articular cartilage within synovial joints. An estimated 27 million Americans suffer from OA, and the population is expected to reach 67 million in the United States by 2030. Thus, it is urgent to find an effective treatment for OA. Traditional OA treatments have no disease-modifying effect, while regenerative OA therapies such as autologous chondrocyte implantation show some promise. Nonetheless, current regenerative therapies do not overcome synovial inflammation that suppresses the differentiation of mesenchymal stem cells (MSCs) to chondrocytes and the expression of type II collagen, the major constituent of functional cartilage. We discovered a synergistic combination that overcame synovial inflammation to form type II collagen-producing chondrocytes. The combination consists of peroxisome proliferator-activated receptor (PPAR) δ agonist, human bone marrow (hBM)-derived MSCs, and hyaluronic acid (HA) gel. Interestingly, those individual components showed their own strong enhancing effects on chondrogenesis. GW0742, a PPAR-δ agonist, greatly enhanced MSC chondrogenesis and the expression of type II collagen and glycosaminoglycan (GAG) in hBM-MSC-derived chondrocytes. GW0742 also increased the expression of transforming growth factor β that enhances chondrogenesis and suppresses cartilage fibrillation, ossification, and inflammation. HA gel also increased MSC chondrogenesis and GAG production. However, neither GW0742 nor HA gel could enhance the formation of type II collagen-producing chondrocytes from hBM-MSCs within human OA synovial fluid. Our data demonstrated that the combination of hBM-MSCs, PPAR-δ agonist, and HA gel significantly enhanced the formation of type II collagen-producing chondrocytes within OA synovial fluid from 3 different donors. In other words, the novel combination of PPAR-δ agonist, hBM-MSCs, and HA gel can overcome synovial inflammation to form

  6. RHEB: a potential regulator of chondrocyte phenotype for cartilage tissue regeneration.

    Science.gov (United States)

    Ashraf, S; Ahn, J; Cha, B-H; Kim, J-S; Han, I; Park, H; Lee, S-H

    2017-09-01

    As articular cartilage has a limited ability to self-repair, successful cartilage regeneration requires clinical-grade chondrocytes with innate characteristics. However, cartilage regeneration via chondrocyte transplantation is challenging, because chondrocytes lose their innate characteristics during in vitro expansion. Here, we investigated the mechanistic underpinning of the gene Ras homologue enriched in brain (RHEB) in the control of senescence and dedifferentiation through the modulation of oxidative stress in chondrocytes, a hallmark of osteoarthritis. Serial expansion of human chondrocytes led to senescence, dedifferentiation and oxidative stress. RHEB maintained the innate characteristics of chondrocytes by regulating senescence, dedifferentiation and oxidative stress, leading to the upregulation of COL2 expression via SOX9 and the downregulation of p27 expression via MCL1. RHEB also decreased the expression of COL10. RHEB knockdown mimics decreased the expression of SOX9, COL2 and MCL1, while abrogating the suppressive function of RHEB on p27 and COL10 in chondrocytes. RHEB-overexpressing chondrocytes successfully formed cartilage tissue in vitro as well as in vivo, with increased expression of GAG matrix and chondrogenic markers. RHEB induces a distinct gene expression signature that maintained the innate chondrogenic properties over a long period. Therefore, RHEB expression represents a potentially useful mechanism in terms of cartilage tissue regeneration from chondrocytes, by which chondrocyte phenotypic and molecular characteristics can be retained through the modulation of senescence, dedifferentiation and oxidative stress. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

  7. 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.

  8. 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.

  9. NF-κB Mediates the Stimulation of Cytokine and Chemokine Expression by Human Articular Chondrocytes in Response to Fibronectin Fragments1

    Science.gov (United States)

    Pulai, Judit I.; Chen, Hong; Im, Hee-Jeong; Kumar, Sanjay; Hanning, Charles; Hegde, Priti S.; Loeser, Richard F.

    2010-01-01

    Fibronectin fragments (FN-f) that bind to the α5β1 integrin stimulate chondrocyte-mediated cartilage destruction and could play an important role in the progression of arthritis. The objective of this study was to identify potential cytokine mediators of cartilage inflammation and destruction induced by FN-f and to investigate the mechanism of their stimulation. Human articular chondrocytes, isolated from normal ankle cartilage obtained from tissue donors, were treated with a 110-kDa FN-f in serum-free culture, and expression of various cytokine genes was analyzed by cDNA microarray and by a cytokine protein array. Compared with untreated control cultures, stimulation by FN-f resulted in a >2-fold increase in IL-6, IL-8, MCP-1, and growth-related oncogene β (GRO-β). Constitutive and FN-f-inducible expression of GRO-α and GRO-γ were also noted by RT-PCR and confirmed by immunoblotting. Previous reports of IL-1β expression induced by FN-f were also confirmed, while TNF expression was found to be very low. Inhibitor studies revealed that FN-f-induced stimulation of chondrocyte chemokine expression was dependent on NF-κB activity, but independent of IL-1 autocrine signaling. The ability of FN-f to stimulate chondrocyte expression of multiple proinflammatory cytokines and chemokines suggests that damage to the cartilage matrix is capable of inducing a proinflammatory state responsible for further progressive matrix destruction, which also includes the chemoattraction of inflammatory cells. Targeting the signaling pathways activated by FN-f may be an effective means of inhibiting production of multiple mediators of cartilage destruction. PMID:15843581

  10. A standardized extract of Butea monosperma (Lam.) flowers suppresses the IL-1β-induced expression of IL-6 and matrix-metalloproteases by activating autophagy in human osteoarthritis chondrocytes.

    Science.gov (United States)

    Ansari, Mohammad Y; Khan, Nazir M; Haqqi, Tariq M

    2017-12-01

    Osteoarthritis (OA) is a leading cause of joint dysfunction, disability and poor quality of life in the affected population. The underlying mechanism of joint dysfunction involves increased oxidative stress, inflammation, high levels of cartilage extracellular matrix degrading proteases and decline in autophagy-a mechanism of cellular defense. There is no disease modifying therapies currently available for OA. Different parts of the Butea monosperma (Lam.) plant have widely been used in the traditional Indian Ayurvedic medicine system for the treatment of various human diseases including inflammatory conditions. Here we studied the chondroprotective effect of hydromethanolic extract of Butea monosperma (Lam.) flowers (BME) standardized to the concentration of Butein on human OA chondrocytes stimulated with IL-1β. The hydromethanolic extract of Butea monosperma (Lam.) (BME) was prepared with 70% methanol-water mixer using Soxhlet. Chondrocytes viability after BME treatment was measured by MTT assay. Gene expression levels were determined by quantitative polymerase chain reaction (qPCR) using TaqMan assays and immunoblotting with specific antibodies. Autophagy activation was determined by measuring the levels of microtubule associated protein 1 light chain 3-II (LC3-II) by immunoblotting and visualization of autophagosomes by transmission electron and confocal microscopy. BME was non-toxic to the OA chondrocytes at the doses employed and suppressed the IL-1β induced expression of inerleukin-6 (IL-6) and matrix metalloprotease-3 (MMP-3), MMP-9 and MMP-13. BME enhanced autophagy in chondrocytes as determined by measuring the levels of LC3-II by immunoblotting and increased number of autophagosomes in BME treated chondrocytes by transmission electron microscopy and confocal microscopy. BME upregulated the expression of several autophagy related genes and increased the autophagy flux in human OA chondrocytes under pathological conditions. Further analysis revealed that

  11. New bioreactor vessel for tissue engineering of human nasal septal chondrocytes

    Directory of Open Access Journals (Sweden)

    Princz Sascha

    2016-09-01

    Full Text Available Cultivation of human nasal septal chondrocytes in a self-established automated bioreactor system with a new designed reactor glass vessel and the results of a computational fluid dynamics model are presented. The first results show the effect of a homogeneous fluidic condition of the continuous medium flow and the resulting stresses on the scaffolds’ surface and their influence on the migration of the cells into the scaffold matrix under these conditions. For this purpose computational models, generated with the computational fluid dynamics software STAR-CCM+, and the results of alcian blue staining for newly synthesized sulphated glycosaminoglycans have been compared during cultivation in the new and a first version of the glass reactor vessel with inhomogeneous fluidic conditions, with the same automated bioreactor system and under similar cultivation conditions.

  12. [Comparative effects of vitamin C on the effects of local anesthetics ropivacaine, bupivacaine, and lidocaine on human chondrocytes].

    Science.gov (United States)

    Tian, Jun; Li, Yan

    2016-01-01

    Intra-articular injections of local anesthetics are commonly used to enhance post-operative analgesia following orthopedic surgery as arthroscopic surgeries. Nevertheless, recent reports of severe complications due to the use of intra-articular local anesthetic have raised concerns. The study aims to assess use of vitamin C in reducing adverse effects of the most commonly employed anesthetics - ropivacaine, bupivacaine and lidocaine - on human chondrocytes. The chondrocyte viability following exposure to 0.5% bupivacaine or 0.75% ropivacaine or 1.0% lidocaine and/or vitamin C at doses 125, 250 and 500μM was determined by Live/Dead assay and annexin V staining. Expression levels of caspases 3 and 9 were assessed using antibodies by Western blotting. Flow cytometry was performed to analyze the generation of reactive oxygen species. On exposure to the local anesthetics, chondrotoxicity was found in the order ropivacaineC effectively improved the reduced chondrocyte viability and decreased the raised apoptosis levels following exposure to anesthesia. At higher doses, vitamin C was found efficient in reducing the generation of reactive oxygen species and as well down-regulate the expressions of caspases 3 and 9. Vitamin C was observed to effectively protect chondrocytes against the toxic insult of local anesthetics ropivacaine, bupivacaine and lidocaine. Copyright © 2015 Sociedade Brasileira de Anestesiologia. Publicado por Elsevier Editora Ltda. All rights reserved.

  13. Comparative effects of vitamin C on the effects of local anesthetics ropivacaine, bupivacaine, and lidocaine on human chondrocytes.

    Science.gov (United States)

    Tian, Jun; Li, Yan

    2016-01-01

    Intra-articular injections of local anesthetics are commonly used to enhance post-operative analgesia following orthopedic surgery as arthroscopic surgeries. Nevertheless, recent reports of severe complications due to the use of intra-articular local anesthetic have raised concerns. The study aims to assess use of vitamin C in reducing adverse effects of the most commonly employed anesthetics - ropivacaine, bupivacaine and lidocaine - on human chondrocytes. The chondrocyte viability following exposure to 0.5% bupivacaine or 0.75% ropivacaine or 1.0% lidocaine and/or vitamin C at doses 125, 250 and 500 μM was determined by LIVE/DEAD assay and annexin V staining. Expression levels of caspases 3 and 9 were assessed using antibodies by Western blotting. Flow cytometry was performed to analyze the generation of reactive oxygen species. On exposure to the local anesthetics, chondrotoxicity was found in the order ropivacaineC effectively improved the reduced chondrocyte viability and decreased the raised apoptosis levels following exposure to anesthesia. At higher doses, vitamin C was found efficient in reducing the generation of reactive oxygen species and as well down-regulate the expressions of caspases 3 and 9. Vitamin C was observed to effectively protect chondrocytes against the toxic insult of local anesthetics ropivacaine, bupivacaine and lidocaine. Copyright © 2015 Sociedade Brasileira de Anestesiologia. Published by Elsevier Editora Ltda. All rights reserved.

  14. 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...

  15. 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.

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

    NARCIS (Netherlands)

    Zhong, Leilei; Huang, X; Karperien, Hermanus Bernardus Johannes; Post, Janine Nicole

    2015-01-01

    Hypertrophic differentiation of chondrocytes is a main barrier in application of mesenchymal stem cells (MSCs) for cartilage repair. In addition, hypertrophy occurs occasionally in osteoarthritis (OA). Here we provide a comprehensive review on recent literature describing signal pathways in the

  17. Reprogrammed chondrocytes engineered to produce IL-12 provide novel ex vivo immune-gene therapy for cancer.

    Science.gov (United States)

    Tada, Hiroyuki; Kishida, Tsunao; Fujiwara, Hitoshi; Kosuga, Toshiyuki; Konishi, Hirotaka; Komatsu, Shuhei; Shiozaki, Atsushi; Ichikawa, Daisuke; Okamoto, Kazuma; Otsuji, Eigo; Mazda, Osam

    2017-03-01

    The somatic cell reprogramming technology was applied to a novel and promising ex vivo immune-gene therapy strategy for cancer. To establish a novel ex vivo cytokine gene therapy of cancer using the somatic cell reprogramming procedures. Mouse fibroblasts were converted into chondrocytes and subsequently transduced with IL-12 gene. The resultant IL-12 induced chondrogenic cells were irradiated with x-ray and inoculated into mice bearing CT26 colon cancer. The irradiation at 20 Gy or higher totally eliminated the proliferative potential of the cells, while less significantly influencing the IL-12 production from the cells. An inoculation of the irradiated IL-12 induced chondrogenic cells significantly suppressed tumor by inducing tumor-specific cytotoxic T lymphocytes, enhancing natural killer tumoricidal activity and inhibiting tumor neoangiogenesis in the mice. The somatic cell reprogramming procedures may provide a novel and effective means to treat malignancies.

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

    Directory of Open Access Journals (Sweden)

    Zejun Liu

    2015-08-01

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

  20. Effect of Cell Sheet Manipulation Techniques on the Expression of Collagen Type II and Stress Fiber Formation in Human Chondrocyte Sheets.

    Science.gov (United States)

    Wongin, Sopita; Waikakul, Saranatra; Chotiyarnwong, Pojchong; Siriwatwechakul, Wanwipa; Viravaidya-Pasuwat, Kwanchanok

    2018-03-01

    Cell sheet technology is applied to human articular chondrocytes to construct a tissue-like structure as an alternative treatment for cartilage defect. The effect of a gelatin manipulator, as a cell sheet transfer system, on the quality of the chondrocyte sheets was investigated. The changes of important chondrogenic markers and stress fibers, resulting from the cell sheet manipulation, were also studied. The chondrocyte cell sheets were constructed with patient-derived chondrocytes using a temperature-responsive polymer and a gelatin manipulator as a transfer carrier. The properties of the cell sheets, including sizes, expression levels of collagen type II and I, and the localization of the stress fibers, were assessed and compared with those of the cell sheets harvested without the gelatin manipulator. Using the gelatin manipulator, the original size of the chondrocyte cell sheets was retained with abundant stress fibers, but with a decrease in the expression of collagen type II. Without the gelatin manipulator, although the cell shrinkage occurred, the cell sheet with suppressed stress fiber formation showed significantly higher levels of collagen type II. These results support our observations that stress fiber formation in chondrocyte cell sheets affected the production of chondrogenic markers. These densely packed tissue-like structures possessed a good chondrogenic activity, indicating their potential for use in autologous chondrocyte implantation to treat cartilage defects.

  1. Synergistic chondroprotective effects of curcumin and resveratrol in human articular chondrocytes: inhibition of IL-1beta-induced NF-kappaB-mediated inflammation and apoptosis.

    Science.gov (United States)

    Csaki, Constanze; Mobasheri, Ali; Shakibaei, Mehdi

    2009-01-01

    Currently available treatments for osteoarthritis (OA) are restricted to nonsteroidal anti-inflammatory drugs, which exhibit numerous side effects and are only temporarily effective. Thus novel, safe and more efficacious anti-inflammatory agents are needed for OA. Naturally occurring polyphenolic compounds, such as curcumin and resveratrol, are potent agents for modulating inflammation. Both compounds mediate their effects by targeting the NF-kappaB signalling pathway. We have recently demonstrated that in chondrocytes resveratrol modulates the NF-kappaB pathway by inhibiting the proteasome, while curcumin modulates the activation of NF-kappaB by inhibiting upstream kinases (Akt). However, the combinational effects of these compounds in chondrocytes has not been studied and/or compared with their individual effects. The aim of this study was to investigate the potential synergistic effects of curcumin and resveratrol on IL-1beta-stimulated human chondrocytes in vitro using immunoblotting and electron microscopy. Treatment with curcumin and resveratrol suppressed NF-kappaB-regulated gene products involved in inflammation (cyclooxygenase-2, matrix metalloproteinase (MMP)-3, MMP-9, vascular endothelial growth factor), inhibited apoptosis (Bcl-2, Bcl-xL, and TNF-alpha receptor-associated factor 1) and prevented activation of caspase-3. IL-1beta-induced NF-kappaB activation was suppressed directly by cocktails of curcumin and resveratrol through inhibition of Ikappakappa and proteasome activation, inhibition of IkappaBalpha phosphorylation and degradation, and inhibition of nuclear translocation of NF-kappaB. The modulatory effects of curcumin and resveratrol on IL-1beta-induced expression of cartilage specific matrix and proinflammatory enzymes were mediated in part by the cartilage-specific transcription factor Sox-9. We propose that combining these natural compounds may be a useful strategy in OA therapy as compared with separate treatment with each individual

  2. 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

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    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.

  3. Cellular automata model for human articular chondrocytes migration, proliferation and cell death: An in vitro validation.

    Science.gov (United States)

    Vaca-González, J J; Gutiérrez, M L; Guevara, J M; Garzón-Alvarado, D A

    2017-01-01

    Articular cartilage is characterized by low cell density of only one cell type, chondrocytes, and has limited self-healing properties. When articular cartilage is affected by traumatic injuries, a therapeutic strategy such as autologous chondrocyte implantation is usually proposed for its treatment. This approach requires in vitro chondrocyte expansion to yield high cell number for cell transplantation. To improve the efficiency of this procedure, it is necessary to assess cell dynamics such as migration, proliferation and cell death during culture. Computational models such as cellular automata can be used to simulate cell dynamics in order to enhance the result of cell culture procedures. This methodology has been implemented for several cell types; however, an experimental validation is required for each one. For this reason, in this research a cellular automata model, based on random-walk theory, was devised in order to predict articular chondrocyte behavior in monolayer culture during cell expansion. Results demonstrated that the cellular automata model corresponded to cell dynamics and computed-accurate quantitative results. Moreover, it was possible to observe that cell dynamics depend on weighted probabilities derived from experimental data and cell behavior varies according to the cell culture period. Thus, depending on whether cells were just seeded or proliferated exponentially, culture time probabilities differed in percentages in the CA model. Furthermore, in the experimental assessment a decreased chondrocyte proliferation was observed along with increased passage number. This approach is expected to having other uses as in enhancing articular cartilage therapies based on tissue engineering and regenerative medicine.

  4. 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

    2014-04-04

    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.

  5. Chondroprotective Effects of Ginsenoside Rg1 in  Human Osteoarthritis Chondrocytes and a Rat Model  of Anterior Cruciate Ligament Transection

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    Wendan Cheng

    2017-03-01

    Full Text Available This study aimed to assess whether Ginsenoside Rg1 (Rg1 inhibits inflammatory responses in human chondrocytes and reduces articular cartilage damage in a rat model of osteoarthritis (OA. Gene expression and protein levels of type II collagen, aggrecan, matrix metalloproteinase (MMP‐13 and cyclooxygenase‐2 (COX‐2 were determined in vitro by quantitative real‐time‐polymerase chain reaction and Western blotting. Prostaglandin E2 (PGE2 amounts in the culture medium were determined by enzyme‐linked immunosorbent assay (ELISA. For in vivo assessment, a rat model of OA was generated by anterior cruciate ligament transection (ACLT. Four weeks after ACLT, Rg1 (30 or 60 mg/kg or saline was administered by gavage once a day for eight consecutive weeks. Joint damage was analyzed by histology and immunohistochemistry. Ginsenoside Rg1 inhibited Interleukin (IL‐1β‐induced chondrocyte gene and protein expressions of MMP‐13, COX‐2 and PGE2, and prevented type II collagen and aggrecan degradation, in a dose‐dependent manner. Administration of Ginsenoside Rg1 to OA rats attenuated cartilage degeneration, and reduced type II collagen loss and MMP‐13 levels. These findings demonstrated that Ginsenoside Rg1 can inhibit inflammatory responses in human chondrocytes in vitro and reduce articular cartilage damage in vivo, confirming the potential therapeutic value of Ginsenoside Rg1 in OA.

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

    NARCIS (Netherlands)

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

    2013-01-01

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

  7. Transforming growth factor-beta predominantly stimulates phenotypically changed chondrocytes in osteoarthritic human cartilage

    NARCIS (Netherlands)

    Lafeber, F. P.; van Roy, H. L.; van der Kraan, P. M.; van den Berg, W. B.; Bijlsma, J. W.

    1997-01-01

    One of the most prominent alterations that characterizes osteoarthritic cartilage damage is a reduction of proteoglycan content, reflecting an imbalance between synthesis and release of proteoglycans. Both synthesis and release depend on the activity of cartilage cells. Chondrocytes in the upper

  8. The effects of monosodium urate monohydrate crystals on chondrocyte viability and function: implications for development of cartilage damage in gout.

    Science.gov (United States)

    Chhana, Ashika; Callon, Karen E; Pool, Bregina; Naot, Dorit; Gamble, Gregory D; Dray, Michael; Pitto, Rocco; Bentley, Jarome; McQueen, Fiona M; Cornish, Jillian; Dalbeth, Nicola

    2013-12-01

    Cartilage damage is frequently observed in advanced destructive gout. The aim of our study was to investigate the effects of monosodium urate monohydrate (MSU) crystals on chondrocyte viability and function. The alamarBlue assay and flow cytometry were used to assess the viability of primary human chondrocytes and cartilage explants following culture with MSU crystals. The number of dead chondrocytes in cartilage explants cultured with MSU crystals was quantified. Real-time PCR was used to determine changes in the relative mRNA expression levels of chondrocytic genes. The histological appearance of cartilage in joints affected by gout was also examined. MSU crystals rapidly reduced primary human chondrocyte and cartilage explant viability in a dose-dependent manner (p gout, normal cartilage architecture was lost, with empty chondrocyte lacunae observed. MSU crystals have profound inhibitory effects on chondrocyte viability and function. Interactions between MSU crystals and chondrocytes may contribute to cartilage damage in gout through reduction of chondrocyte viability and promotion of a catabolic state.

  9. Targeted Deletion of Autophagy Genes Atg5 or Atg7 in the Chondrocytes Promotes Caspase-Dependent Cell Death and Leads to Mild Growth Retardation.

    Science.gov (United States)

    Vuppalapati, Karuna K; Bouderlique, Thibault; Newton, Phillip T; Kaminskyy, Vitaliy O; Wehtje, Henrik; Ohlsson, Claes; Zhivotovsky, Boris; Chagin, Andrei S

    2015-12-01

    Longitudinal bone growth takes place in epiphyseal growth plates located in the ends of long bones. The growth plate consists of chondrocytes traversing from the undifferentiated (resting zone) to the terminally differentiated (hypertrophic zone) stage. Autophagy is an intracellular catabolic process of lysosome-dependent recycling of intracellular organelles and protein complexes. Autophagy is activated during nutritionally depleted or hypoxic conditions in order to facilitate cell survival. Chondrocytes in the middle of the growth plate are hypoxic and nutritionally depleted owing to the avascular nature of the growth plate. Accordingly, autophagy may facilitate their survival. To explore the role of autophagy in chondrocyte survival and constitutional bone growth, we generated mice with cartilage-specific ablation of either Atg5 (Atg5cKO) or Atg7 (Atg7cKO) by crossing Atg5 or Atg7 floxed mice with cartilage-specific collagen type 2 promoter-driven Cre. Both Atg5cKO and Atg7cKO mice showed growth retardation associated with enhanced chondrocyte cell death and decreased cell proliferation. Similarly, inhibition of autophagy by Bafilomycin A1 (Baf) or 3-methyladenine (3MA) promoted cell death in cultured slices of human growth plate tissue. To delineate the underlying mechanisms we employed ex vivo cultures of mouse metatarsal bones and RCJ3.IC5.18 rat chondrogenic cell line. Baf or 3MA impaired metatarsal bone growth associated with processing of caspase-3 and massive cell death. Similarly, treatment of RCJ3.IC5.18 chondrogenic cells by Baf also showed massive cell death and caspase-3 cleavage. This was associated with activation of caspase-9 and cytochrome C release. Altogether, our data suggest that autophagy is important for chondrocyte survival, and inhibition of this process leads to stunted growth and caspase-dependent death of chondrocytes. © 2015 American Society for Bone and Mineral Research.

  10. Smad signaling pathway is a pivotal component of tissue inhibitor of metalloproteinases-3 regulation by transforming growth factor beta in human chondrocytes.

    Science.gov (United States)

    Qureshi, Hamid Yaqoob; Ricci, Gemma; Zafarullah, Muhammad

    2008-09-01

    Transforming growth factor beta (TGF-beta1) promotes cartilage matrix synthesis and induces tissue inhibitor of metalloproteinases-3 (TIMP-3), which inhibits matrix metalloproteinases, aggrecanases and TNF-alpha-converting enzyme implicated in articular cartilage degradation and joint inflammation. TGF-beta1 activates Akt, ERK and Smad2 pathways in chondrocytes. Here we investigated previously unexplored roles of specific Smads in TGF-beta1 induction of TIMP-3 gene by pharmacological and genetic knockdown approaches. TGF-beta1-induced Smad2 phosphorylation and TIMP-3 protein expression could be inhibited by the Smad2/3 phosphorylation inhibitors, PD169316 and SB203580 and by Smad2-specific siRNA. Specific inhibitor of Smad3 (SIS3) and Smad3 siRNA abolished TGF-beta induction of TIMP-3. Smad2/3 siRNAs also down regulated TIMP-3 promoter-driven luciferase activities, suggesting transcriptional regulation. SiRNA-driven co-Smad4 knockdown abrogated TIMP-3 augmentation by TGF-beta. TIMP-3 promoter deletion analysis revealed that -828 deletion retains the original promoter activity while -333 and -167 deletions display somewhat reduced activity suggesting that most of the TGF-beta-responsive, cis-acting elements are found in the -333 fragment. Chromatin Immunoprecipitation (ChIP) analysis confirmed binding of Smad2 and Smad4 with the -940 and -333 promoter sequences. These results suggest that receptor-activated Smad2 and Smad3 and co-Smad4 critically mediate TGF-beta-stimulated TIMP-3 expression in human chondrocytes and TIMP-3 gene is a target of Smad signaling pathway.

  11. 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.

  12. The Effect of Soy Isoflavone on the Proliferation and Differentiation of Adipose-Derived Mesenchymal Stem Cells into Chondrocytes and Expression of Collagen II and Aggrecan Genes

    Directory of Open Access Journals (Sweden)

    Fatemeh Bamdadpasand Shekarsarayi

    2017-03-01

    Full Text Available Background and Objectives: Due to the lack of blood vessels in cartilage tissue, its damage is not repairable. This study was conducted to investigate the effect of soy isoflavone on proliferation and differentiation of adipose-derived mesenchymal stem cells into chondrocytes and expression of collagen II and aggrecan genes. Methods: In this experimental study, human subcutaneous fat was obtained during liposuction and incubated with collagenase enzyme (type 1 for the breakdown of collagen, and collagenase was deactivated by DMEM medium, and was cultured in the cell sediment after centrifugation, the cells were isolated after the third passage, were placed in chondrogenic medium for differentiate into the cartilage, and were divided into three groups, including control, treatment with TGF-β1, and treatment with soy isoflavones tablets. The tablets were dissolved in distilled water, sterilized by passing through a 0.2 um filter and were added to the culture medium. After 48 hours, cell viability was determined by MTT assay, and after 14 days, collagen II and aggrecan gene expressions were assessed by real-time PCR technique. Data were statistically analyzed by one-way ANOVA and Tukey's post-hoc test using SPSS 20 and p<0.05. Results: The results of MTT assay showed a significant increase in viability in the TGF-β1 group compared to the control and soy isoflavone groups (p<0.05. The RT-PCR indicated a significant increase in the expression of collagen II and aggrecan genes in isoflavones and TGF-β1 groups compared to the control group, and also, the mean CT associated with collagen II gene had a significant increase in isoflavone and TGF-β1groups compared to the control group (p<0.05. Conclusion: Soy in culture medium increases the expression of collagen II and aggrecan genes and cell proliferation, but this increase is not high compared to the TGF-β1 group.

  13. Adeno-associated virus gene therapy vector scAAVIGF-I for transduction of equine articular chondrocytes and RNA-seq analysis.

    Science.gov (United States)

    Hemphill, D D; McIlwraith, C W; Slayden, R A; Samulski, R J; Goodrich, L R

    2016-05-01

    IGF-I is one of several anabolic factors being investigated for the treatment of osteoarthritis (OA). Due to the short biological half-life, extended administration is required for more robust cartilage healing. Here we create a self-complimentary adeno-associated virus (AAV) gene therapy vector utilizing the transgene for IGF-I. Various biochemical assays were performed to investigate the cellular response to scAAVIGF-I treatment vs an scAAVGFP positive transduction control and a negative for transduction control culture. RNA-sequencing analysis was also performed to establish a differential regulation profile of scAAVIGF-I transduced chondrocytes. Biochemical analyses indicated an average media IGF-I concentration of 608 ng/ml in the scAAVIGF-I transduced chondrocytes. This increase in IGF-I led to increased expression of collagen type II and aggrecan and increased protein concentrations of cellular collagen type II and media glycosaminoglycan vs both controls. RNA-seq revealed a global regulatory pattern consisting of 113 differentially regulated GO categories including those for chondrocyte and cartilage development and regulation of apoptosis. This research substantiates that scAAVIGF-I gene therapy vector increased production of IGF-I to clinically relevant levels with a biological response by chondrocytes conducive to increased cartilage healing. The RNA-seq further established a set of differentially expressed genes and gene ontologies induced by the scAAVIGF-I vector while controlling for AAV infection. This dataset provides a static representation of the cellular transcriptome that, while only consisting of one time point, will allow for further gene expression analyses to compare additional cartilage healing therapeutics or a transient cellular response. Copyright © 2015. Published by Elsevier Ltd.

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

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    LS Moreira Teixeira

    2012-06-01

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

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

    Science.gov (United States)

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

    2012-06-05

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

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

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    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

  17. Mesenchymal stem cells display different gene expression profiles compared to hyaline and elastic chondrocytes

    OpenAIRE

    Zhai, Li-Jie; Zhao, Ke-Qing; Wang, Zhi-Qiang; Feng, Ya; Xing, Shuang-Chun

    2011-01-01

    Cartilage has a poor intrinsic repair capacity, requiring surgical intervention to effect biological repair. Tissue engineering technologies or regenerative medicine strategies are currently being employed to address cartilage repair. Mesenchymal stem cells (MSCs) are considered to be an excellent cell source for this application. However, the different gene expression profiles between the MSCs and differentiated cartilage remain unclear. In this report, we first examined the gene expression ...

  18. 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

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

    International Nuclear Information System (INIS)

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

    2011-01-01

    Research highlights: → Glucosamine and a NF-kB inhibitor reduce inflammation in OA. → Cytokine induced demethylation of CpG site in IL1β promoter prevented by glucosamine. → 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-1β, 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-1β and 2.5 ng/ml oncostatin M (OSM), (iii) cultured with 2 mM N-acetyl GlcN (Sigma

  20. Devitalisation of human cartilage by high hydrostatic pressure treatment: Subsequent cultivation of chondrocytes and mesenchymal stem cells on the devitalised tissue

    Science.gov (United States)

    Hiemer, B.; Genz, B.; Jonitz-Heincke, A.; Pasold, J.; Wree, A.; Dommerich, S.; Bader, R.

    2016-01-01

    The regeneration of cartilage lesions still represents a major challenge. Cartilage has a tissue-specific architecture, complicating recreation by synthetic biomaterials. A novel approach for reconstruction is the use of devitalised cartilage. Treatment with high hydrostatic pressure (HHP) achieves devitalisation while biomechanical properties are remained. Therefore, in the present study, cartilage was devitalised using HHP treatment and the potential for revitalisation with chondrocytes and mesenchymal stem cells (MSCs) was investigated. The devitalisation of cartilage was performed by application of 480 MPa over 10 minutes. Effective cellular inactivation was demonstrated by the trypan blue exclusion test and DNA quantification. Histology and electron microscopy examinations showed undamaged cartilage structure after HHP treatment. For revitalisation chondrocytes and MSCs were cultured on devitalised cartilage without supplementation of chondrogenic growth factors. Both chondrocytes and MSCs significantly increased expression of cartilage-specific genes. ECM stainings showed neocartilage-like structure with positive AZAN staining as well as collagen type II and aggrecan deposition after three weeks of cultivation. Our results showed that HHP treatment caused devitalisation of cartilage tissue. ECM proteins were not influenced, thus, providing a scaffold for chondrogenic differentiation of MSCs and chondrocytes. Therefore, using HHP-treated tissue might be a promising approach for cartilage repair. PMID:27671122

  1. Nitric Oxide Mediates Crosstalk between Interleukin 1β and WNT Signaling in Primary Human Chondrocytes by Reducing DKK1 and FRZB Expression.

    Science.gov (United States)

    Zhong, Leilei; Schivo, Stefano; Huang, Xiaobin; Leijten, Jeroen; Karperien, Marcel; Post, Janine N

    2017-11-22

    Interleukin 1 beta (IL1β) and Wingless-Type MMTV Integration Site Family (WNT) signaling are major players in Osteoarthritis (OA) pathogenesis. Despite having a large functional overlap in OA onset and development, the mechanism of IL1β and WNT crosstalk has remained largely unknown. In this study, we have used a combination of computational modeling and molecular biology to reveal direct or indirect crosstalk between these pathways. Specifically, we revealed a mechanism by which IL1β upregulates WNT signaling via downregulating WNT antagonists, DKK1 and FRZB. In human chondrocytes, IL1β decreased the expression of Dickkopf-1 (DKK1) and Frizzled related protein (FRZB) through upregulation of nitric oxide synthase (iNOS), thereby activating the transcription of WNT target genes. This effect could be reversed by iNOS inhibitor 1400W, which restored DKK1 and FRZB expression and their inhibitory effect on WNT signaling. In addition, 1400W also inhibited both the matrix metalloproteinase (MMP) expression and cytokine-induced apoptosis. We concluded that iNOS/NO play a pivotal role in the inflammatory response of human OA through indirect upregulation of WNT signaling. Blocking NO production may inhibit the loss of the articular phenotype in OA by preventing downregulation of the expression of DKK1 and FRZB.

  2. Mechanical properties and structure-function relationships of human chondrocyte-seeded cartilage constructs after in vitro culture.

    Science.gov (United States)

    Middendorf, Jill M; Griffin, Darvin J; Shortkroff, Sonya; Dugopolski, Caroline; Kennedy, Stephen; Siemiatkoski, Joseph; Cohen, Itai; Bonassar, Lawrence J

    2017-10-01

    Autologous Chondrocyte Implantation (ACI) is a widely recognized method for the repair of focal cartilage defects. Despite the accepted use, problems with this technique still exist, including graft hypertrophy, damage to surrounding tissue by sutures, uneven cell distribution, and delamination. Modified ACI techniques overcome these challenges by seeding autologous chondrocytes onto a 3D scaffold and securing the graft into the defect. Many studies on these tissue engineered grafts have identified the compressive properties, but few have examined frictional and shear properties as suggested by FDA guidance. This study is the first to perform three mechanical tests (compressive, frictional, and shear) on human tissue engineered cartilage. The objective was to understand the complex mechanical behavior, function, and changes that occur with time in these constructs grown in vitro using compression, friction, and shear tests. Safranin-O histology and a DMMB assay both revealed increased sulfated glycosaminoglycan (sGAG) content in the scaffolds with increased maturity. Similarly, immunohistochemistry revealed increased lubricin localization on the construct surface. Confined compression and friction tests both revealed improved properties with increased construct maturity. Compressive properties correlated with the sGAG content, while improved friction coefficients were attributed to increased lubricin localization on the construct surfaces. In contrast, shear properties did not improve with increased culture time. This study suggests the various mechanical and biological properties of tissue engineered cartilage improve at different rates, indicating thorough mechanical evaluation of tissue engineered cartilage is critical to understanding the performance of repaired cartilage. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:2298-2306, 2017. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.

  3. Nasal chondrocyte-based engineered autologous cartilage tissue for repair of articular cartilage defects: an observational first-in-human trial.

    Science.gov (United States)

    Mumme, Marcus; Barbero, Andrea; Miot, Sylvie; Wixmerten, Anke; Feliciano, Sandra; Wolf, Francine; Asnaghi, Adelaide M; Baumhoer, Daniel; Bieri, Oliver; Kretzschmar, Martin; Pagenstert, Geert; Haug, Martin; Schaefer, Dirk J; Martin, Ivan; Jakob, Marcel

    2016-10-22

    Articular cartilage injuries have poor repair capacity, leading to progressive joint damage, and cannot be restored predictably by either conventional treatments or advanced therapies based on implantation of articular chondrocytes. Compared with articular chondrocytes, chondrocytes derived from the nasal septum have superior and more reproducible capacity to generate hyaline-like cartilage tissues, with the plasticity to adapt to a joint environment. We aimed to assess whether engineered autologous nasal chondrocyte-based cartilage grafts allow safe and functional restoration of knee cartilage defects. In a first-in-human trial, ten patients with symptomatic, post-traumatic, full-thickness cartilage lesions (2-6 cm 2 ) on the femoral condyle or trochlea were treated at University Hospital Basel in Switzerland. Chondrocytes isolated from a 6 mm nasal septum biopsy specimen were expanded and cultured onto collagen membranes to engineer cartilage grafts (30 × 40 × 2 mm). The engineered tissues were implanted into the femoral defects via mini-arthrotomy and assessed up to 24 months after surgery. Primary outcomes were feasibility and safety of the procedure. Secondary outcomes included self-assessed clinical scores and MRI-based estimation of morphological and compositional quality of the repair tissue. This study is registered with ClinicalTrials.gov, number NCT01605201. The study is ongoing, with an approved extension to 25 patients. For every patient, it was feasible to manufacture cartilaginous grafts with nasal chondrocytes embedded in an extracellular matrix rich in glycosaminoglycan and type II collagen. Engineered tissues were stable through handling with forceps and could be secured in the injured joints. No adverse reactions were recorded and self-assessed clinical scores for pain, knee function, and quality of life were improved significantly from before surgery to 24 months after surgery. Radiological assessments indicated variable degrees of

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

    DEFF Research Database (Denmark)

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

    2017-01-01

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

  5. 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.

  6. Aging and oxidative stress reduce the response of human articular chondrocytes to insulin-like growth factor 1 and osteogenic protein 1.

    Science.gov (United States)

    Loeser, Richard F; Gandhi, Uma; Long, David L; Yin, Weihong; Chubinskaya, Susan

    2014-08-01

    To determine the effects of aging and oxidative stress on the response of human articular chondrocytes to insulin-like growth factor 1 (IGF-1) and osteogenic protein 1 (OP-1). Chondrocytes isolated from normal articular cartilage obtained from tissue donors were cultured in alginate beads or monolayer. Cells were stimulated with 50-100 ng/ml of IGF-1, OP-1, or both. Oxidative stress was induced using tert-butyl hydroperoxide. Sulfate incorporation was used to measure proteoglycan synthesis, and immunoblotting of cell lysates was performed to analyze cell signaling. Confocal microscopy was performed to measure nuclear translocation of Smad4. Chondrocytes isolated from the articular cartilage of tissue donors ranging in age from 24 years to 81 years demonstrated an age-related decline in proteoglycan synthesis stimulated by IGF-1 and IGF-1 plus OP-1. Induction of oxidative stress inhibited both IGF-1- and OP-1-stimulated proteoglycan synthesis. Signaling studies showed that oxidative stress inhibited IGF-1-stimulated Akt phosphorylation while increasing phosphorylation of ERK, and that these effects were greater in cells from older donors. Oxidative stress also increased p38 phosphorylation, which resulted in phosphorylation of Smad1 at the Ser(206) inhibitory site and reduced nuclear accumulation of Smad1. Oxidative stress also modestly reduced OP-1-stimulated nuclear translocation of Smad4. These results demonstrate an age-related reduction in the response of human chondrocytes to IGF-1 and OP-1, which are 2 important anabolic factors in cartilage, and suggest that oxidative stress may be a contributing factor by altering IGF-1 and OP-1 signaling. Copyright © 2014 by the American College of Rheumatology.

  7. 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.

  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. Human Gene Therapy: Genes without Frontiers?

    Science.gov (United States)

    Simon, Eric J.

    2002-01-01

    Describes the latest advancements and setbacks in human gene therapy to provide reference material for biology teachers to use in their science classes. Focuses on basic concepts such as recombinant DNA technology, and provides examples of human gene therapy such as severe combined immunodeficiency syndrome, familial hypercholesterolemia, and…

  10. 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.

  11. Sorbitol-modified hyaluronic acid reduces oxidative stress, apoptosis and mediators of inflammation and catabolism in human osteoarthritic chondrocytes.

    Science.gov (United States)

    Mongkhon, John-Max; Thach, Maryane; Shi, Qin; Fernandes, Julio C; Fahmi, Hassan; Benderdour, Mohamed

    2014-08-01

    Our study was designed to elucidate the precise molecular mechanisms by which sorbitol-modified hyaluronic acid (HA/sorbitol) exerts beneficial effects in osteoarthritis (OA). Human OA chondrocytes were treated with increasing doses of HA/sorbitol ± anti-CD44 antibody or with sorbitol alone and thereafter with or without interleukin-1beta (IL-1β) or hydrogen peroxide (H2O2). Signal transduction pathways and parameters related to oxidative stress, apoptosis, inflammation, and catabolism were investigated. HA/sorbitol prevented IL-1β-induced oxidative stress, as measured by reactive oxygen species, p47-NADPH oxidase phosphorylation, 4-hydroxynonenal (HNE) production and HNE-metabolizing glutathione-S-transferase A4-4 expression. Moreover, HA/sorbitol stifled IL-1β-induced metalloproteinase-13, nitric oxide (NO) and prostaglandin E2 release as well as inducible NO synthase expression. Study of the apoptosis process revealed that this gel significantly attenuated cell death, caspase-3 activation and DNA fragmentation elicited by exposure to a cytotoxic H2O2 dose. Examination of signaling pathway components disclosed that HA/sorbitol prevented IL-1β-induced p38 mitogen-activated protein kinase and nuclear factor-kappa B activation, but not that of extracellular signal-regulated kinases 1 and 2. Interestingly, the antioxidant as well as the anti-inflammatory and anti-catabolic effects of HA/sorbitol were attributed to sorbitol and HA, respectively. Altogether, our findings support a beneficial effect of HA/sorbitol in OA through the restoration of redox status and reduction of apoptosis, inflammation and catabolism involved in cartilage damage.

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

    Science.gov (United States)

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

    2015-01-01

    To establish a two-dimensional biological printing technique of chondrocytes and compare the difference of related biological characters between printed chondrocytes and unprinted cells so as to control the cell transfer process and keep cell viability after printing. Primary chondrocytes were obtained from human mature and fetal cartilage tissues and then were regularly sub-cultured to harvest cells at passage 2 (P2), which were adjusted to the single cell suspension at a density of 1×10(6)/mL. The experiment was divided into 2 groups: experimental group P2 chondrocytes were transferred by rapid prototype biological printer (driving voltage value 50 V, interval in x-axis 300 μm, interval in y-axis 1500 μm). Afterwards Live/Dead viability Kit and flow cytometry were respectively adopted to detect cell viability; CCK-8 Kit was adopted to detect cell proliferation viability; immunocytochemistry, immunofluorescence and RT-PCR was employed to identify related markers of chondrocytes; control group steps were the same as the printing group except that cell suspension received no printing. Fluorescence microscopy and flow cytometry analyses showed that there was no significant difference between experimental group and control group in terms of cell viability. After 7-day in vitro culture, control group exhibited higher O.D values than experimental group from 2nd day to 7th day but there was no distinct difference between these two groups (P>0.05). Inverted microscope observation demonstrated that the morphology of these two groups had no significant difference either. Similarly, Immunocytochemistry, immunofluorescence and RT-PCR assays also showed that there was no significant difference in the protein and gene expression of type II collagen and aggrecan between these two groups (P>0.05). Conclusion Cell printing has no distinctly negative effect on cell vitality, proliferation and phenotype of chondrocytes. Biological printing technique may provide a novel approach

  13. 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

    NARCIS (Netherlands)

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

    2014-01-01

    Background: There is a need for tools to predict the chondrogenic potency of autologous cells for cartilage repair. Purpose: To evaluate previously proposed chondrogenic biomarkers and to identify new biomarkers in the chondrocyte transcriptome capable of predicting clinical success or failure after

  14. Biocompatibility of Human Auricular Chondrocytes Cultured onto a Chitosan/Polyvynil Alcohol/Epichlorohydrin-Based Hydrogel for Tissue Engineering Application

    OpenAIRE

    Melgarejo-Ramírez, Yaaziel; Sánchez-Sánchez, Roberto; García-Carvajal, Zaira; García-López, Julieta; Gutiérrez-Gómez, Claudia; Luna-Barcenas, Gabriel; Ibarra, Clemente; Velasquillo, Cristina

    2014-01-01

    Tissue engineering (TE) has become an alternative for auricular reconstruction based on the combination of cells, molecular signals and biomaterials. Scaffolds are biomaterials that provide structural support for cell attachment and subsequent tissue development. Ideally, a scaffold should have characteristics such as biocompatibility and bioactivity to adequate support cell functions. Our purpose was to evaluate biocompatibility of microtic auricular chondrocytes seeded onto a chitosan-polyv...

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

    Directory of Open Access Journals (Sweden)

    Gupta Kalpana

    2007-08-01

    Full Text Available Abstract Background The Amazonian medicinal plant Sangre de grado (Croton palanostigma has traditional applications for the treatment of wound healing and inflammation. We sought to characterize two extracts (progrado and zangrado in terms of safety and oligomeric proanthocyanidin chain length. Additionally progrado was evaluated for antioxidant activity and possible chondroprotective actions. Methods Acute oral safety and toxicity was tested in rats according under OECD protocol number 420. The profile of proanthocyanidin oligomers was determined by HPLC and progrado's antioxidant activity quantified by the ORAC, NORAC and HORAC assays. Human cartilage explants, obtained from surgical specimens, were used to assess chondroproteciton with activity related to direct inhibitory effects on human matrix metalloproteinase (MMP, gelatinolytic activity using synovial fluid and chondrocytes activated with IL-1β (10 ng/ml. Additionally, progrado (2–10 μg/ml was tested for its ability to maintain optimal IGF-1 transcription and translation in cartilage explants and cultured chondrocytes. Results Both progrado and zangrado at doses up to 2000 mg/kg (po displayed no evidence of toxicity. Oligomeric proanthocyanidin content was high for both progrado (158 mg/kg and zangrado (124 mg/kg, with zangrado almost entirely composed of short oligomers ( Conclusion Progrado has a promising safety profile, significant chondroprotective and antioxidant actions, directly inhibits MMP activity and promotes the production of the cartilage repair factor, IGF-1. This suggests that progrado may offer therapeutic benefits in joint health, wound healing and inflammation.

  16. 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.

  17. 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.

  18. 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

    OBJECTIVE: Maintenance of chondrocyte phenotype is a major issue in prevention of degeneration and repair of articular cartilage. Although the critical pathways in chondrocyte maturation and homeostasis have been revealed, the in-depth understanding is deficient and novel modifying components...... subgroups. Cartilage specific expression was highest in proliferating and prehypertrophic zones during development, and in adult articular cartilage, expression was restricted to the uncalcified zone, including chondrocyte clusters in human osteoarthritic cartilage. Studies with experimental chondrogenesis...... chondrocytes and adult articular chondrocytes with possible functions associated with development and maintenance of chondrocyte phenotype....

  19. Widespread epigenomic, transcriptomic and proteomic differences between hip osteophytic and articular chondrocytes in osteoarthritis.

    Science.gov (United States)

    Steinberg, Julia; Brooks, Roger A; Southam, Lorraine; Bhatnagar, Sahir; Roumeliotis, Theodoros I; Hatzikotoulas, Konstantinos; Zengini, Eleni; Wilkinson, J Mark; Choudhary, Jyoti S; McCaskie, Andrew W; Zeggini, Eleftheria

    2018-05-08

    To identify molecular differences between chondrocytes from osteophytic and articular cartilage tissue from OA patients. We investigated genes and pathways by combining genome-wide DNA methylation, RNA sequencing and quantitative proteomics in isolated primary chondrocytes from the cartilaginous layer of osteophytes and matched areas of low- and high-grade articular cartilage across nine patients with OA undergoing hip replacement surgery. Chondrocytes from osteophytic cartilage showed widespread differences to low-grade articular cartilage chondrocytes. These differences were similar to, but more pronounced than, differences between chondrocytes from osteophytic and high-grade articular cartilage, and more pronounced than differences between high- and low-grade articular cartilage. We identified 56 genes with significant differences between osteophytic chondrocytes and low-grade articular cartilage chondrocytes on all three omics levels. Several of these genes have known roles in OA, including ALDH1A2 and cartilage oligomeric matrix protein, which have functional genetic variants associated with OA from genome-wide association studies. An integrative gene ontology enrichment analysis showed that differences between osteophytic and low-grade articular cartilage chondrocytes are associated with extracellular matrix organization, skeletal system development, platelet aggregation and regulation of ERK1 and ERK2 cascade. We present a first comprehensive view of the molecular landscape of chondrocytes from osteophytic cartilage as compared with articular cartilage chondrocytes from the same joints in OA. We found robust changes at genes relevant to chondrocyte function, providing insight into biological processes involved in osteophyte development and thus OA progression.

  20. Genes, Environment, and Human Behavior.

    Science.gov (United States)

    Bloom, Mark V.; Cutter, Mary Ann; Davidson, Ronald; Dougherty, Michael J.; Drexler, Edward; Gelernter, Joel; McCullough, Laurence B.; McInerney, Joseph D.; Murray, Jeffrey C.; Vogler, George P.; Zola, John

    This curriculum module explores genes, environment, and human behavior. This book provides materials to teach about the nature and methods of studying human behavior, raise some of the ethical and public policy dilemmas emerging from the Human Genome Project, and provide professional development for teachers. An extensive Teacher Background…

  1. 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.

  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. PEDF Is Associated with the Termination of Chondrocyte Phenotype and Catabolism of Cartilage Tissue.

    Science.gov (United States)

    Klinger, P; Lukassen, S; Ferrazzi, F; Ekici, A B; Hotfiel, T; Swoboda, B; Aigner, T; Gelse, K

    2017-01-01

    Objective. To investigate the expression and target genes of pigment epithelium-derived factor (PEDF) in cartilage and chondrocytes, respectively. Methods. We analyzed the expression pattern of PEDF in different human cartilaginous tissues including articular cartilage, osteophytic cartilage, and fetal epiphyseal and growth plate cartilage, by immunohistochemistry and quantitative real-time (qRT) PCR. Transcriptome analysis after stimulation of human articular chondrocytes with rhPEDF was performed by RNA sequencing (RNA-Seq) and confirmed by qRT-PCR. Results. Immunohistochemically, PEDF could be detected in transient cartilaginous tissue that is prone to undergo endochondral ossification, including epiphyseal cartilage, growth plate cartilage, and osteophytic cartilage. In contrast, PEDF was hardly detected in healthy articular cartilage and in the superficial zone of epiphyses, regions that are characterized by a permanent stable chondrocyte phenotype. RNA-Seq analysis and qRT-PCR demonstrated that rhPEDF significantly induced the expression of a number of matrix-degrading factors including SAA1, MMP1, MMP3, and MMP13. Simultaneously, a number of cartilage-specific genes including COL2A1, COL9A2, COMP, and LECT were among the most significantly downregulated genes. Conclusions. PEDF represents a marker for transient cartilage during all neonatal and postnatal developmental stages and promotes the termination of cartilage tissue by upregulation of matrix-degrading factors and downregulation of cartilage-specific genes. These data provide the basis for novel strategies to stabilize the phenotype of articular cartilage and prevent its degradation.

  4. 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

  5. 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....

  6. 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......:3) for identifying designs displaying optimal engagement of OA patient-derived CD44-expressing chondrocytes. Correlation was found between cell binding and CD44 expression, with maximal binding exhibited at a HA/chitosan ratio of 7:3, that was 181% higher than CD44-negative MCF-7 cell control cells. Transfection...... agent-free uptake into OA chondrocytes of fluorescent 13-mer DNA oligonucleotides with a flanked locked nucleic acid (LNA) gapmer design, in contrast to naked siRNA, was demonstrated by confocal and flow cytometric analysis. A sustained and complete release over 5days was found with the 7:3 hydrogel...

  7. Endogenous versus Exogenous Growth Factor Regulation of Articular Chondrocytes

    Science.gov (United States)

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

    2014-01-01

    Anabolic growth factors that regulate the function of articular chondrocytes are candidates for articular cartilage repair. Such factors may be delivered by pharmacotherapy in the form of exogenous proteins, or by gene therapy as endogenous proteins. It is unknown whether delivery method influences growth factor effectiveness in regulating articular chondrocyte reparative functions. We treated adult bovine articular chondrocytes with exogenous recombinant insulin-like growth factor-I (IGF-I) and transforming growth factor-beta1 (TGF-β1), or with the genes encoding these growth factors for endogenous production. Treatment effects were measured as change in chondrocyte DNA content, glycosaminoglycan production, and aggrecan gene expression. We found that IGF-I stimulated chondrocyte biosynthesis similarly when delivered by either exogenous or endogenous means. In contrast, exogenous TGF-ß1 stimulated these reparative functions, while endogenous TGF-ß1 had little effect. Endogenous TGF-ß1 became more bioactive following activation of the transgene protein product. These data indicate that effective mechanisms of growth factor delivery for articular cartilage repair may differ for different growth factors. In the case of IGF-I, gene therapy or protein therapy appear to be viable options. In contrast, TGF-ß1 gene therapy may be constrained by a limited ability of chondrocytes to convert latent complexes to an active form. PMID:24105960

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

    Science.gov (United States)

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

    2014-01-01

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

  9. MSX2 stimulates chondrocyte maturation by controlling Ihh expression.

    Science.gov (United States)

    Amano, Katsuhiko; Ichida, Fumitaka; Sugita, Atsushi; Hata, Kenji; Wada, Masahiro; Takigawa, Yoko; Nakanishi, Masako; Kogo, Mikihiko; Nishimura, Riko; Yoneda, Toshiyuki

    2008-10-24

    Several studies indicated that a homeobox gene, Msx2, is implicated in regulation of skeletal development by controlling enchondral ossification as well as membranous ossification. However, the molecular basis by which Msx2 conducts chondrogenesis is currently unclear. In this study, we examined the role of Msx2 in chondrocyte differentiation using mouse primary chondrocytes and embryonic metatarsal explants. Treatment with BMP2 up-regulated the expression of Msx2 mRNA along with chondrocyte differentiation in murine primary chondrocytes. Overexpression of wild-type Msx2 stimulated calcification of primary chondrocytes in the presence of BMP2. We also found that constitutively active Msx2 (caMsx2) enhanced BMP2-dependent calcification more efficiently than wild-type Msx2. Consistently, caMsx2 overexpression up-regulated the expression of alkaline phosphatase and collagen type X induced by BMP2. Furthermore, organ culture experiments using mouse embryonic metatarsals indicated that caMsx2 clearly stimulated the maturation of chondrocytes into the prehypertrophic and hypertrophic stages in the presence of BMP2. In contrast, knockdown of Msx2 inhibited maturation of primary chondrocytes. The stimulatory effect of Msx2 on chondrocyte maturation was enhanced by overexpression of Smad1 and Smad4 but inhibited by Smad6, an inhibitory Smad for BMP2 signaling. These data suggest that Msx2 requires BMP2/Smad signaling for its chondrogenic action. In addition, caMsx2 overexpression induced Ihh (Indian hedgehog) expression in mouse primary chondrocytes. Importantly, treatment with cyclopamine, a specific inhibitor for hedgehogs, blocked Msx2-induced chondrogenesis. Collectively, our results indicated that Msx2 promotes the maturation of chondrocytes, at least in part, through up-regulating Ihh expression.

  10. Utility of NucleoCounter for the chondrocyte count in the collagenase digest of human native cartilage

    Science.gov (United States)

    Yonenaga, Kazumichi; Nishizawa, Satoru; Akizawa, Miki; Asawa, Yukiyo; Fujihara, Yuko; Takato, Tsuyoshi

    2010-01-01

    In cartilage tissue engineering, viable cell numbers should be correctly counted in the collagenase digest of the biopsied cartilage. However, this is a difficult task due to the presence of matrix debris, cell ghosts and their aggregates. To search for the correct cell counting method in this situation, we evaluated the utility of an automatic cell counting device, the NucleoCounter, and compared it with conventional staining using the LIVE/DEAD® kit. We first measured the cell numbers of a standard chondrocyte sample by the NucleoCounter, which showed a high accuracy (R2 = 0.9999) and reproducibility (%CV: 2.00–8.66). We then calculated the cell numbers and viability in some collagenase digests of native cartilage using either the NucleoCounter or LIVE/DEAD® kit, revealing that the total cell numbers, viable ones and viability were highly correlated between them (R2 = 0.9601, 0.9638 and 0.917, respectively). However, both the intrapersonal and interpersonal variabilities in the NucleoCounter was significantly decreased to about 1/20–1/5, compared to that of the LIVE/DEAD® kit. The NucleoCounter was regarded as a useful tool for simple, rapid, and highly reproducible cell counts, which may not only provide constant experimental data in a certain laboratory, but also contribute to the high reproducibility of the clinical results of cartilage tissue engineering among multiple institutions. PMID:20845070

  11. Unphysiologically high magnesium concentrations support chondrocyte proliferation and redifferentiation.

    Science.gov (United States)

    Feyerabend, Frank; Witte, Frank; Kammal, Michael; Willumeit, Regine

    2006-12-01

    The effect of unphysiologically high extracellular magnesium concentrations on chondrocytes, induced by the supplementation of magnesium sulfate, was studied using a 3-phase tissue engineering model. The experiments showed that chondrocyte proliferation and redifferentiation, on the gene and protein expression level, are enhanced. A negative influence was found during chondrogenesis where an inhibition of extracellular matrix formation was observed. In addition, a direct impact on chondrocyte metabolism, elevated magnesium concentrations also affected growth factor effectiveness by consecutive influences during chondrogenesis. All observations were dosage dependent. The results of this study indicate that magnesium may be a useful tool for cartilage tissue engineering.

  12. Genetic modification of chondrocytes with insulin-like growth factor-1 enhances cartilage healing in an equine model.

    Science.gov (United States)

    Goodrich, L R; Hidaka, C; Robbins, P D; Evans, C H; Nixon, A J

    2007-05-01

    Gene therapy with insulin-like growth factor-1 (IGF-1) increases matrix production and enhances chondrocyte proliferation and survival in vitro. The purpose of this study was to determine whether arthroscopically-grafted chondrocytes genetically modified by an adenovirus vector encoding equine IGF-1 (AdIGF-1) would have a beneficial effect on cartilage healing in an equine femoropatellar joint model. A total of 16 horses underwent arthroscopic repair of a single 15 mm cartilage defect in each femoropatellar joint. One joint received 2 x 10(7) AdIGF-1 modified chondrocytes and the contralateral joint received 2 x 10(7) naive (unmodified) chondrocytes. Repairs were analysed at four weeks, nine weeks and eight months after surgery. Morphological and histological appearance, IGF-1 and collagen type II gene expression (polymerase chain reaction, in situ hybridisation and immunohistochemistry), collagen type II content (cyanogen bromide and sodium dodecyl sulphate-polyacrylamide gel electrophoresis), proteoglycan content (dimethylmethylene blue assay), and gene expression for collagen type I, matrix metalloproteinase (MMP)-1, MMP-3, MMP-13, aggrecanase-1, tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) and TIMP-3 were evaluated. Genetic modification of chondrocytes significantly increased IGF-1 mRNA and ligand production in repair tissue for up to nine weeks following transplantation. The gross and histological appearance of IGF-1 modified repair tissue was improved over control defects. Gross filling of defects was significantly improved at four weeks, and a more hyaline-like tissue covered the lesions at eight months. Histological outcome at four and nine weeks post-transplantation revealed greater tissue filling of defects transplanted with genetically modified chondrocytes, whereas repair tissue in control defects was thin and irregular and more fibrous. Collagen type II expression in IGF-1 gene-transduced defects was increased 100-fold at four weeks and

  13. Gene expression profile in human induced pluripotent stem cells: Chondrogenic differentiation in vitro, part A

    Science.gov (United States)

    Suchorska, Wiktoria Maria; Augustyniak, Ewelina; Richter, Magdalena; Trzeciak, Tomasz

    2017-01-01

    Human induced pluripotent stem cells (hiPSCs) offer promise in regenerative medicine, however more data are required to improve understanding of key aspects of the cell differentiation process, including how specific chondrogenic processes affect the gene expression profile of chondrocyte-like cells and the relative value of cell differentiation markers. The main aims of the present study were as follows: To determine the gene expression profile of chondrogenic-like cells derived from hiPSCs cultured in mediums conditioned with HC-402-05a cells or supplemented with transforming growth factor β3 (TGF-β3), and to assess the relative utility of the most commonly used chondrogenic markers as indicators of cell differentiation. These issues are relevant with regard to the use of human fibroblasts in the reprogramming process to obtain hiPSCs. Human fibroblasts are derived from the mesoderm and thus share a wide range of properties with chondrocytes, which also originate from the mesenchyme. Thus, the exclusion of dedifferentiation instead of chondrogenic differentiation is crucial. The hiPSCs were obtained from human primary dermal fibroblasts during a reprogramming process. Two methods, both involving embryoid bodies (EB), were used to obtain chondrocytes from the hiPSCs: EBs formed in a chondrogenic medium supplemented with TGF-β3 (10 ng/ml) and EBs formed in a medium conditioned with growth factors from HC-402-05a cells. Based on immunofluorescence and reverse transcription-quantiative polymerase chain reaction analysis, the results indicated that hiPSCs have the capacity for effective chondrogenic differentiation, in particular cells differentiated in the HC-402-05a-conditioned medium, which present morphological features and markers that are characteristic of mature human chondrocytes. By contrast, cells differentiated in the presence of TGF-β3 may demonstrate hypertrophic characteristics. Several genes [paired box 9, sex determining region Y-box (SOX) 5, SOX6

  14. Remodelling of human osteoarthritic cartilage by FGF-2, alone or combined with Sox9 via rAAV gene transfer.

    Science.gov (United States)

    Cucchiarini, Magali; Terwilliger, Ernest F; Kohn, Dieter; Madry, Henning

    2009-08-01

    Compensating for the loss of extracellular cartilage matrix, as well as counteracting the alterations of the chondrocyte phenotype in osteoarthritis are of key importance to develop effective therapeutic strategies against this disorder. In the present study, we analysed the benefits of applying a potent gene combination to remodel human osteoarthritic (OA) cartilage. We employed the promising recombinant adeno-associated virus (rAAV) vector to deliver the mitogenic fibroblast growth factor 2 (FGF-2) factor, alone or simultaneously with the transcription factor Sox9 as a key activator of matrix synthesis, to human normal and OA articular chondrocytes. We evaluated the effects of single (FGF-2) or combined (FGF-2/SOX9) transgene expression upon the regenerative activities of chondrocytes in three dimensional cultures in vitro and in cartilage explants in situ. Single overexpression of FGF-2 enhanced the survival and proliferation of both normal and OA chondrocytes, without stimulating the matrix synthetic processes in the increased pools of cells. The mitogenic properties of FGF-2 were maintained when SOX9 was co-overexpressed and concomitant with an increase in the production of proteoglycans and type-II collagen, suggesting that the transcription factor was capable of counterbalancing the effects of FGF-2 on matrix accumulation. Also important, expression of type-X collagen, a marker of hypertrophy strongly decreased following treatment by the candidate vectors. Most remarkably, the levels of activities achieved in co-treated human OA cartilage were similar to or higher than those observed in normal cartilage. The present findings show that combined expression of candidate factors in OA cartilage can re-establish key features of normal cartilage and prevent the pathological shift of metabolic homeostasis. These data provide further motivation to develop coupled gene transfer approaches via rAAV for the treatment of human OA.

  15. 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...

  16. Msx2 Stimulates Chondrocyte Maturation by Controlling Ihh Expression*

    OpenAIRE

    Amano, Katsuhiko; Ichida, Fumitaka; Sugita, Atsushi; Hata, Kenji; Wada, Masahiro; Takigawa, Yoko; Nakanishi, Masako; Kogo, Mikihiko; Nishimura, Riko; Yoneda, Toshiyuki

    2008-01-01

    Several studies indicated that a homeobox gene, Msx2, is implicated in regulation of skeletal development by controlling enchondral ossification as well as membranous ossification. However, the molecular basis by which Msx2 conducts chondrogenesis is currently unclear. In this study, we examined the role of Msx2 in chondrocyte differentiation using mouse primary chondrocytes and embryonic metatarsal explants. Treatment with BMP2 up-regulated the expression of Msx2 mRNA...

  17. Cloning human DNA repair genes

    International Nuclear Information System (INIS)

    Jeggo, P.A.; Carr, A.M.; Lehmann, A.R.

    1994-01-01

    Many human genes involved in the repair of UV damage have been cloned using different procedures and they have been of great value in assisting the understanding of the mechanism of nucleotide excision-repair. Genes involved in repair of ionizing radiation damage have proved more difficult to isolate. Positional cloning has localized the XRCC5 gene to a small region of chromosome 2q33-35, and a series of yeast artificial chromosomes covering this region have been isolated. Very recent work has shown that the XRCC5 gene encodes the 80 kDa subunit of the Ku DNA-binding protein. The Ku80 gene also maps to this region. Studies with fission yeast have shown that radiation sensitivity can result not only from defective DNA repair but also from abnormal cell cycle control following DNA damage. Several genes involved in this 'check-point' control in fission yeast have been isolated and characterized in detail. It is likely that a similar checkpoint control mechanism exists in human cells. (author)

  18. Human platelet lysate successfully promotes proliferation and subsequent chondrogenic differentiation of adipose-derived stem cells: a comparison with articular chondrocytes.

    Science.gov (United States)

    Hildner, F; Eder, M J; Hofer, K; Aberl, J; Redl, H; van Griensven, M; Gabriel, C; Peterbauer-Scherb, A

    2015-07-01

    Fetal calf serum (FCS) bears a potential risk for carrying diseases and eliciting immune reactions. Nevertheless, it still represents the gold standard as medium supplement in cell culture. In the present study, human platelet lysate (PL) was tested as an alternative to FCS for the expansion and subsequent chondrogenic differentiation of human adipose-derived stem cells (ASCs). ASCs were expanded with 10% FCS (group F) or 5% PL (group P). Subsequently, three-dimensional (3D) micromass pellets were created and cultured for 5 weeks in chondrogenic differentiation medium. Additionally, the de- and redifferentiation potential of human articular chondrocytes (HACs) was evaluated and compared to ASCs. Both HACs and ASCs cultured with PL showed strongly enhanced proliferation rates. Redifferentiation of HACs was possible for cells expanded up to 3.3 population doublings (PD). At this stage, PL-expanded HACs demonstrated better redifferentiation potential than FCS-expanded cells. ASCs could also be differentiated following extended passaging. Glycosaminoglycan (GAG) quantification and qRT-PCR of 10 cartilage related markers demonstrated a tendency for increased chondrogenic differentiation of PL-expanded ASCs compared to cells expanded with FCS. Histologically, collagen type II but also collagen type X was mainly present in group P. The present study demonstrates that PL strongly induces proliferation of ASCs, while the chondrogenic differentiation potential is retained. HACs also showed enhanced proliferation and even better redifferentiation when previously expanded with PL. This suggests that PL is superior to FCS as a supplement for the expansion of ASCs and HACs, particularly with regard to chondrogenic (re)differentiation. Copyright © 2013 John Wiley & Sons, Ltd.

  19. Considerations on the use of ear chondrocytes as donor chondrocytes for cartilage tissue engineering

    NARCIS (Netherlands)

    van Osch, Gerjo J. V. M.; Mandl, Erik W.; Jahr, Holger; Koevoet, Wendy; Nolst-Trenité, Gilbert; Verhaar, Jan A. N.

    2004-01-01

    Articular cartilage is often used for research on cartilage tissue engineering. However, ear cartilage is easier to harvest, with less donor-site morbidity. The aim of this study was to evaluate whether adult human ear chondrocytes were capable of producing cartilage after expansion in monolayer

  20. Role of interleukin-1 in antigen presentation by normal articular chondrocytes

    International Nuclear Information System (INIS)

    Tiku, M.L.; Liu, S.; Tiku, K.

    1986-01-01

    Recently the authors have described that normal articular chondrocytes of rabbits present antigen to immune T cells. In the present study the authors investigated the role of interleukin-1 (IL-1) on antigen presentation by chondrocytes. For these experiments the antigen pulsed chondroyctes were either untreated or fixed with paraformaldehyde and then co-cultured with immune T cells. T cell proliferation was measured by 3 H-thymidine incorporation. Pulsed non-fixed chondrocytes presented antigen, as expected, but pulsed and fixed cells failed to present antigen to T cells. The 3 H-TdR incorporation was partially restored by addition of purified human IL-1. Next, IL-1 activity was measured in primary chondrocyte culture supernatants stimulated with or without lipopolysaccharide (LPS) in comitogen thymocyte assay. No activity was detected in chondrocyte supernatants. Propagated chondrocyte culture supernatants also lacked IL-1 activity when stimulated with LPS in the presence of increasing concentration of indomethacin. On the other hand the authors observed that chondrocyte culture supernatants in a dose dependent manner inhibited human IL-1 induced 3 H-TdR incorporation of murine thymocytes. This suggested that these cells may produce an inhibitor of IL-1 and IL-1 production by chondrocytes may be essential for T cell proliferation by these cells. Inability to detect IL-1 in chondrocyte supernatants may be due to the presence of an inhibitor to IL-1. These findings may help in elucidating the immunological mechanisms in situations where chondrocytes and T cell interact, such as in arthritis

  1. 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.

  2. 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.

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

    International Nuclear Information System (INIS)

    Yan, Jidong; Xu, Jing; Fei, Yao; Jiang, Congshan; Zhu, Wenhua; Han, Yan; Lu, Shemin

    2016-01-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2016-05-15

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

  5. Surface modification of cyclic olefin copolymers for osteochondral defect repair can increase pro-destructive potential of human chondrocytes in vitro

    Czech Academy of Sciences Publication Activity Database

    Polanská, M.; Hulejová, H.; Petrtýl, M.; Bastl, Zdeněk; Spirovová, Ilona; Kruliš, Zdeněk; Horák, Zdeněk; Veigl, D.; Šenolt, L.

    2010-01-01

    Roč. 59, č. 2 (2010), s. 247-253 ISSN 0862-8408 R&D Projects: GA ČR GA106/06/0761 Institutional research plan: CEZ:AV0Z40400503; CEZ:AV0Z40500505 Keywords : osteochondral defects * cycloolefin copolymer * chondrocytes * biocompatibility Subject RIV: CF - Physical ; Theoretical Chemistry Impact factor: 1.646, year: 2010

  6. Redifferentiation of in vitro expanded adult articular chondrocytes by combining the hanging-drop cultivation method with hypoxic environment.

    Science.gov (United States)

    Martinez, Inigo; Elvenes, Jan; Olsen, Randi; Bertheussen, Kjell; Johansen, Oddmund

    2008-01-01

    The main purpose of this work has been to establish a new culturing technique to improve the chondrogenic commitment of isolated adult human chondrocytes, with the aim of being used during cell-based therapies or tissue engineering strategies. By using a rather novel technique to generate scaffold-free three-dimensional (3D) structures from in vitro expanded chondrocytes, we have explored the effects of different culture environments on cartilage formation. Three-dimensional chondrospheroids were developed by applying the hanging-drop technique. Cartilage tissue formation was attempted after combining critical factors such as serum-containing or serum-free media and atmospheric (20%) or low (2.5%) oxygen tensions. The quality of the formed microtissues was analyzed by histology, immunohistochemistry, electron microscopy, and real-time PCR, and directly compared with native adult cartilage. Our results revealed highly organized, 3D tissue-like structures developed by the hanging-drop method. All culture conditions allowed formation of 3D spheroids; however, cartilage generated under low oxygen tension had a bigger size, enhanced matrix deposition, and higher quality of cartilage formation. Real-time PCR demonstrated enhanced expression of cartilage-specific genes such us collagen type II and aggrecan in 3D cultures when compared to monolayers. Cartilage-specific matrix proteins and genes expressed in hanging-drop-developed spheroids were comparable to the expression obtained by applying the pellet culture system. In summary, our results indicate that a combination of 3D cultures of chondrocytes in hanging drops and a low oxygen environment represent an easy and convenient way to generate cartilage-like microstructures. We also show that a new specially tailored serum-free medium is suitable for in vitro cartilage tissue formation. This new methodology opens up the possibility of using autogenously produced solid 3D structures with redifferentiated chondrocytes as an

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

    Science.gov (United States)

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

    2015-01-01

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

  8. Patenting human genes: Chinese academic articles' portrayal of gene patents.

    Science.gov (United States)

    Du, Li

    2018-04-24

    The patenting of human genes has been the subject of debate for decades. While China has gradually come to play an important role in the global genomics-based testing and treatment market, little is known about Chinese scholars' perspectives on patent protection for human genes. A content analysis of academic literature was conducted to identify Chinese scholars' concerns regarding gene patents, including benefits and risks of patenting human genes, attitudes that researchers hold towards gene patenting, and any legal and policy recommendations offered for the gene patent regime in China. 57.2% of articles were written by law professors, but scholars from health sciences, liberal arts, and ethics also participated in discussions on gene patent issues. While discussions of benefits and risks were relatively balanced in the articles, 63.5% of the articles favored gene patenting in general and, of the articles (n = 41) that explored gene patents in the Chinese context, 90.2% supported patent protections for human genes in China. The patentability of human genes was discussed in 33 articles, and 75.8% of these articles reached the conclusion that human genes are patentable. Chinese scholars view the patent regime as an important legal tool to protect the interests of inventors and inventions as well as the genetic resources of China. As such, many scholars support a gene patent system in China. These attitudes towards gene patents remain unchanged following the court ruling in the Myriad case in 2013, but arguments have been raised about the scope of gene patents, in particular that the increasing numbers of gene patents may negatively impact public health in China.

  9. 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.

  10. Silencing of microRNA-138-5p promotes IL-1β-induced cartilage degradation in human chondrocytes by targeting FOXC1: miR-138 promotes cartilage degradation.

    Science.gov (United States)

    Yuan, Y; Zhang, G Q; Chai, W; Ni, M; Xu, C; Chen, J Y

    2016-10-01

    Osteoarthritis (OA) is characterised by articular cartilage degradation. MicroRNAs (miRNAs) have been identified in the development of OA. The purpose of our study was to explore the functional role and underlying mechanism of miR-138-5p in interleukin-1 beta (IL-1β)-induced extracellular matrix (ECM) degradation of OA cartilage. Human articular cartilage was obtained from patients with and without OA, and chondrocytes were isolated and stimulated by IL-1β. The expression levels of miR-138-5p in cartilage and chondrocytes were both determined. After transfection with miR-138-5p mimics, allele-specific oligonucleotide (ASO)-miR-138-5p, or their negative controls, the messenger RNA (mRNA) levels of aggrecan (ACAN), collagen type II and alpha 1 (COL2A1), the protein levels of glycosaminoglycans (GAGs), and both the mRNA and protein levels of matrix metalloproteinase (MMP)-13 were evaluated. Luciferase reporter assay, quantitative real-time polymerase chain reaction (qRT-PCR), and Western blot were performed to explore whether Forkhead Box C1 (FOCX1) was a target of miR-138-5p. Further, we co-transfected OA chondrocytes with miR-138-5p mimics and pcDNA3.1 (+)-FOXC1 and then stimulated with IL-1β to determine whether miR-138-5p-mediated IL-1β-induced cartilage matrix degradation resulted from targeting FOXC1. MiR-138-5p was significantly increased in OA cartilage and in chondrocytes in response to IL-1β-stimulation. Overexpression of miR-138-5p significantly increased the IL-1β-induced downregulation of COL2A1, ACAN, and GAGs, and increased the IL-1β-induced over expression of MMP-13.We found that FOXC1 is directly regulated by miR-138-5p. Additionally, co-transfection with miR-138-5p mimics and pcDNA3.1 (+)-FOXC1 resulted in higher levels of COL2A1, ACAN, and GAGs, but lower levels of MMP-13. miR-138-5p promotes IL-1β-induced cartilage degradation in human chondrocytes, possibly by targeting FOXC1.Cite this article: Y. Yuan, G. Q. Zhang, W. Chai,M. Ni, C. Xu, J

  11. Chondrocytic Atf4 regulates osteoblast differentiation and function via Ihh.

    Science.gov (United States)

    Wang, Weiguang; Lian, Na; Ma, Yun; Li, Lingzhen; Gallant, Richard C; Elefteriou, Florent; Yang, Xiangli

    2012-02-01

    Atf4 is a leucine zipper-containing transcription factor that activates osteocalcin (Ocn) in osteoblasts and indian hedgehog (Ihh) in chondrocytes. The relative contribution of Atf4 in chondrocytes and osteoblasts to the regulation of skeletal development and bone formation is poorly understood. Investigations of the Atf4(-/-);Col2a1-Atf4 mouse model, in which Atf4 is selectively overexpressed in chondrocytes in an Atf4-null background, demonstrate that chondrocyte-derived Atf4 regulates osteogenesis during development and bone remodeling postnatally. Atf4 overexpression in chondrocytes of the Atf4(-/-);Col2a1-Atf4 double mutants corrects the reduction in stature and limb in Atf4(-/-) embryos and rectifies the decrease in Ihh expression, Hh signaling, proliferation and accelerated hypertrophy that characterize the Atf4(-/-) developing growth plate cartilages. Unexpectedly, this genetic manipulation also restores the expression of osteoblastic marker genes, namely Ocn and bone sialoprotein, in Atf4(-/-) developing bones. In Atf4(-/-);Col2a1-Atf4 adult mice, all the defective bone parameters found in Atf4(-/-) mice, including bone volume, trabecular number and thickness, and bone formation rate, are rescued. In addition, the conditioned media of ex vivo cultures from wild-type or Atf4(-/-);Col2a1-Atf4, but not Atf4(-/-) cartilage, corrects the differentiation defects of Atf4(-/-) bone marrow stromal cells and Ihh-blocking antibody eliminates this effect. Together, these data indicate that Atf4 in chondrocytes is required for normal Ihh expression and for its paracrine effect on osteoblast differentiation. Therefore, the cell-autonomous role of Atf4 in chondrocytes dominates the role of Atf4 in osteoblasts during development for the control of early osteogenesis and skeletal growth.

  12. Comprehensive high-resolution genomic profiling and cytogenetics of human chondrocyte cultures by GTG-banding, locus-specific FISH, SKY and SNP array.

    Science.gov (United States)

    Wallenborn, M; Petters, O; Rudolf, D; Hantmann, H; Richter, M; Ahnert, P; Rohani, L; Smink, J J; Bulwin, G C; Krupp, W; Schulz, R M; Holland, H

    2018-04-23

    In the development of cell-based medicinal products, it is crucial to guarantee that the application of such an advanced therapy medicinal product (ATMP) is safe for the patients. The consensus of the European regulatory authorities is: "In conclusion, on the basis of the state of art, conventional karyotyping can be considered a valuable and useful technique to analyse chromosomal stability during preclinical studies". 408 chondrocyte samples (84 monolayers and 324 spheroids) from six patients were analysed using trypsin-Giemsa staining, spectral karyotyping and fluorescence in situ hybridisation, to evaluate the genetic stability of an ATMP named Spherox®. Single nucleotide polymorphism (SNP) array analysis was performed on chondrocyte spheroids from five of the six donors. Applying this combination of techniques, the genetic analyses performed revealed no significant genetic instability until passage 3 in monolayer cells and interphase cells from spheroid cultures at different time points. Clonal occurrence of polyploid metaphases and endoreduplications were identified associated with prolonged cultivation time. Also, gonosomal losses were observed in chondrocyte spheroids, with increasing passage and duration of the differentiation phase. Interestingly, in one of the donors, chromosomal aberrations that are also described in extraskeletal myxoid chondrosarcoma were identified. The SNP array analysis exhibited chromosomal aberrations in two donors and copy neutral losses of heterozygosity regions in four donors. This study showed the necessity of combined genetic analyses at defined cultivation time points in quality studies within the field of cell therapy.

  13. 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

    of dlk1/FA1 as a novel surface marker for chondroprogenitor cells during hESC differentiation. We found that, Dlk1/FA1 is expressed specifically in cells undergoing transition from proliferating to prehypertrophic chondrocytes during endochondral ossification of the mouse limb. In hESC cells, dlk1/FA1...... 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......, a member of TGF-ss family, markedly increased Dlk1 expression in association with up-regulation of the mesoderm-specific markers (e.g. FOXF1, KDR and VE-cadherin) and SOX9. dlk1/FA1(+) cells isolated by fluorescence activated cell sorting (FACS) were capable of differentiating into chondrocytic cells when...

  14. 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.

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

    Directory of Open Access Journals (Sweden)

    Elena Vasil'evna Chetina

    2010-01-01

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

  16. The Signaling Pathways Involved in Chondrocyte Differentiation and Hypertrophic Differentiation

    Directory of Open Access Journals (Sweden)

    Jianmei Li

    2016-01-01

    Full Text Available Chondrocytes communicate with each other mainly via diffusible signals rather than direct cell-to-cell contact. The chondrogenic differentiation of mesenchymal stem cells (MSCs is well regulated by the interactions of varieties of growth factors, cytokines, and signaling molecules. A number of critical signaling molecules have been identified to regulate the differentiation of chondrocyte from mesenchymal progenitor cells to their terminal maturation of hypertrophic chondrocytes, including bone morphogenetic proteins (BMPs, SRY-related high-mobility group-box gene 9 (Sox9, parathyroid hormone-related peptide (PTHrP, Indian hedgehog (Ihh, fibroblast growth factor receptor 3 (FGFR3, and β-catenin. Except for these molecules, other factors such as adenosine, O2 tension, and reactive oxygen species (ROS also have a vital role in cartilage formation and chondrocyte maturation. Here, we outlined the complex transcriptional network and the function of key factors in this network that determine and regulate the genetic program of chondrogenesis and chondrocyte differentiation.

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

    Directory of Open Access Journals (Sweden)

    Margot E Bowen

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

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

    Science.gov (United States)

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

    2014-03-01

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

  19. Human proton/oligopeptide transporter (POT) genes

    DEFF Research Database (Denmark)

    Botka, C. W.; Wittig, T. W.; Graul, R. C.

    2000-01-01

    The proton-dependent oligopeptide transporters (POT) gene family currently consists of approximately 70 cloned cDNAs derived from diverse organisms. In mammals, two genes encoding peptide transporters, PepT1 and PepT2 have been cloned in several species including humans, in addition to a rat...... histidine/peptide transporter (rPHT1). Because the Candida elegans genome contains five putative POT genes, we searched the available protein and nucleic acid databases for additional mammalian/human POT genes, using iterative BLAST runs and the human expressed sequence tags (EST) database. The apparent...... and introns of the likely human orthologue (termed hPHT2). Northern analyses with EST clones indicated that hPHT1 is primarily expressed in skeletal muscle and spleen, whereas hPHT2 is found in spleen, placenta, lung, leukocytes, and heart. These results suggest considerable complexity of the human POT gene...

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

    International Nuclear Information System (INIS)

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

    2002-01-01

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

  1. Atf4 regulates chondrocyte proliferation and differentiation during endochondral ossification by activating Ihh transcription.

    Science.gov (United States)

    Wang, Weiguang; Lian, Na; Li, Lingzhen; Moss, Heather E; Wang, Weixi; Perrien, Daniel S; Elefteriou, Florent; Yang, Xiangli

    2009-12-01

    Activating transcription factor 4 (Atf4) is a leucine-zipper-containing protein of the cAMP response element-binding protein (CREB) family. Ablation of Atf4 (Atf4(-/-)) in mice leads to severe skeletal defects, including delayed ossification and low bone mass, short stature and short limbs. Atf4 is expressed in proliferative and prehypertrophic growth plate chondrocytes, suggesting an autonomous function of Atf4 in chondrocytes during endochondral ossification. In Atf4(-/-) growth plate, the typical columnar structure of proliferative chondrocytes is disturbed. The proliferative zone is shortened, whereas the hypertrophic zone is transiently expanded. The expression of Indian hedgehog (Ihh) is markedly decreased, whereas the expression of other chondrocyte marker genes, such as type II collagen (Col2a1), PTH/PTHrP receptor (Pth1r) and type X collagen (Col10a1), is normal. Furthermore, forced expression of Atf4 in chondrocytes induces endogenous Ihh mRNA, and Atf4 directly binds to the Ihh promoter and activates its transcription. Supporting these findings, reactivation of Hh signaling pharmacologically in mouse limb explants corrects the Atf4(-/-) chondrocyte proliferation and short limb phenotypes. This study thus identifies Atf4 as a novel transcriptional activator of Ihh in chondrocytes that paces longitudinal bone growth by controlling growth plate chondrocyte proliferation and differentiation.

  2. 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.

  3. Regulative mechanisms of chondrocyte adhesion

    DEFF Research Database (Denmark)

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

    2006-01-01

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

  4. Culture of chondrocytes in alginate surrounded by fibrin gel: characteristics of the cells over a period of eight weeks

    NARCIS (Netherlands)

    Almqvist, K. F.; Wang, L.; Wang, J.; Baeten, D.; Cornelissen, M.; Verdonk, R.; Veys, E. M.; Verbruggen, G.

    2001-01-01

    OBJECTIVE: To produce tissue engineered cartilage by human articular chondrocytes in vitro for further use in in vivo manipulations for the treatment of cartilage defects. METHODS: Human articular chondrocytes were cultured in 0.5%, 1.0%, and 2.0% of alginate for up to four weeks. The optimal

  5. Comparison of Efficacy of Endogenous and Exogenous IGF-I in Stimulating Matrix Production in Neonatal and Mature Chondrocytes.

    Science.gov (United States)

    Aguilar, Izath N; Trippel, Stephen B; Shi, Shuiliang; Bonassar, Lawrence J

    2015-10-01

    The goal of this study was to compare the efficacy of endogenous upregulation of IGF-I by gene therapy and exogenous addition of insulin-like growth factor I (IGF-I) in enhancing proteoglycan synthesis by skeletally mature and neonatal chondrocytes. Chondrocyte transplantation therapy is a common treatment for focal cartilage lesions, with both mature and neonatal chondrocytes used as a cell source. Additionally, gene therapy strategies to upregulate growth factors such as IGF-I have been proposed to augment chondrocyte transplantation therapies. Both skeletally mature and neonatal chondrocytes were exposed to either an adeno-associated virus-based plasmid containing the IGF-I gene or exogenous IGF-I. Analysis of IGF-I and glycosaminoglycan production using a 4-parameter dose-response model established a clear connection between the amount of IGF-I produced by cells and their biosynthetic response. Both neonatal and mature chondrocytes showed this relationship, but the sensitivities were quite different, with EC50 of 0.57 ng/mL for neonatal chondrocytes and EC50 of 8.70 ng/mL IGF-I for skeletally mature chondrocytes. These data suggest that IGF-I gene therapy may be more effective with younger cell sources. Both cell types were less sensitive to exogenous IGF-I than endogenous IGF-I.

  6. The role of imprinted genes in humans

    OpenAIRE

    Ishida, Miho; Moore, Gudrun E.

    2013-01-01

    Detailed comprehensive molecular analysis using families and multiple matched tissues is essential to determine whether imprinted genes have a functional role in humans. See research article: http://genomebiology.com/2011/12/3/R25

  7. 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.

  8. Effect of donor age on DNA repair by articular chondrocytes

    International Nuclear Information System (INIS)

    Lipman, J.M.

    1986-01-01

    The hypothesis that aging of articular chondrocytes at a cellular level results from loss of DNA repair capability was studied by two different measures: unscheduled DNA synthesis (UDS) and O 6 -methylguanine acceptor protein (MGAP) activity. UDS following damage by 254 nm ultraviolet irradiation (20J/m 2 ) was examined in intact articular cartilage from rabbits of different ages. Semiconservative DNA synthesis was suppressed with hydroxurea and repair followed by the incorporation of [ 3 H]-thymidine ([ 3 H]-dThd). After repair the cartilage was digested in proteinase K (0.5mg/ml) with dodecyl sodium sulfate (0.2%) and DNA determined with Hoechst 33258 dye. UDS (dpm [ 3 H]-dThd/μg DNA) was greater in articular cartilage from 3- than 39-month-old rabbits. MGAP was studied in cell extracts of cultured human and rabbit chondrocytes by transfer of [ 3 H] O 6 -methyl groups from exogenous DNA to protein. It was significantly less in rabbit than in human cells on a per protein or DNA basis. There was no decline in this activity in human chondrocytes from newborn to 60 years of age; and rabbits from 3- to 36-months-old. The data indicate that in the two different repair mechanisms, age differences are found with resting but not dividing chondrocytes

  9. Inflammation induction of Dickkopf-1 mediates chondrocyte apoptosis in osteoarthritic joint.

    Science.gov (United States)

    Weng, L-H; Wang, C-J; Ko, J-Y; Sun, Y-C; Su, Y-S; Wang, F-S

    2009-07-01

    Dysregulated Wnt signaling appears to modulate chondrocyte fate and joint disorders. Dickkopf-1 (DKK1) regulates the pathogenesis of skeletal tissue by inhibiting Wnt actions. This study examined whether DKK1 expression is linked to chondrocyte fate in osteoarthritis (OA). Articular cartilage specimens harvested from nine patients with knee OA and from six controls with femoral neck fracture were assessed for DKK1, interleukin-1beta (IL-1beta), tumor necrosis factor-alpha (TNF-alpha), Bad, Bax, Bcl2 and caspase-3 expression by real time-polymerase chain reaction (RT-PCR) and immunohistochemistry. Apoptotic chondrocytes were detected by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end-labelling (TUNEL) and 4', 6-dianidino-2-phenylindole dihydrochloride (DAPI) staining. Human chondrocyte cultures were treated with recombinant IL-1beta and monoclonal DKK1 antibody to determine whether DKK1 impairs chondrocyte survival. Expression of DKK1 correlated with inflammatory cytokine levels (IL-1beta and TNF-alpha expressions), proapoptosis regulators (Bad and caspase-3 expressions) and TUNEL staining in OA cartilage tissues. The IL-1beta induced expressions of DKK1, Bax, Bad and caspase-3-dependent apoptosis of chondrocyte cultures. Neutralization of DKK1 by monoclonal DKK1 antibody significantly abrogated IL-1beta-mediated caspase-3 cleavage and apoptosis and reversed chondrocyte proliferation. Recombinant DKK1 treatment impaired chondrocyte growth and promoted apoptosis. By suppressing nuclear beta-catenin accumulation and Akt phosphorylation, DKK1 mediated IL-1beta promotion of chondrocyte apoptosis. Chondrocyte apoptosis correlates with joint OA. Expression of DKK1 contributes to cartilage deterioration and is a potent factor in OA pathogenesis. Attenuating DKK1 may reduce cartilage deterioration in OA.

  10. 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

  11. Hydrostatic Pressure Influences HIF-2 Alpha Expression in Chondrocytes

    Directory of Open Access Journals (Sweden)

    Hiroaki Inoue

    2015-01-01

    Full Text Available Hypoxia-inducible factor (HIF-2α is considered to play a major role in the progression of osteoarthritis. Recently, it was reported that pressure amplitude influences HIF-2α expression in murine endothelial cells. We examined whether hydrostatic pressure is involved in expression of HIF-2α in articular chondrocytes. Chondrocytes were cultured and stimulated by inflammation or hydrostatic pressure of 0, 5, 10, or 50 MPa. After stimulation, heat shock protein (HSP 70, HIF-2α, nuclear factor kappa B (NF-κB, matrix metalloproteinase (MMP-13, MMP-3, and vascular endothelial growth factor (VEGF gene expression were evaluated. The levels of all gene expression were increased by inflammatory stress. When chondrocytes were exposed to a hydrostatic pressure of 5 MPa, HIF-2α, MMP-13, and MMP-3 gene expression increased significantly although those of HSP70 and NF-κB were not significantly different from the control group. In contrast, HIF-2α gene expression did not increase under a hydrostatic pressure of 50 MPa although HSP70 and NF-κB expression increased significantly compared to control. We considered that hydrostatic pressure of 5 MPa could regulate HIF-2α independent of NF-κB, because the level of HIF-2α gene expression increased significantly without upregulation of NF-κB expression at 5 MPa. Hydrostatic pressure may influence cartilage degeneration, inducing MMP-13 and MMP-3 expression through HIF-2α.

  12. Hydrostatic pressure influences HIF-2 alpha expression in chondrocytes.

    Science.gov (United States)

    Inoue, Hiroaki; Arai, Yuji; Kishida, Tsunao; Terauchi, Ryu; Honjo, Kuniaki; Nakagawa, Shuji; Tsuchida, Shinji; Matsuki, Tomohiro; Ueshima, Keiichirou; Fujiwara, Hiroyoshi; Mazda, Osam; Kubo, Toshikazu

    2015-01-05

    Hypoxia-inducible factor (HIF)-2α is considered to play a major role in the progression of osteoarthritis. Recently, it was reported that pressure amplitude influences HIF-2α expression in murine endothelial cells. We examined whether hydrostatic pressure is involved in expression of HIF-2α in articular chondrocytes. Chondrocytes were cultured and stimulated by inflammation or hydrostatic pressure of 0, 5, 10, or 50 MPa. After stimulation, heat shock protein (HSP) 70, HIF-2α, nuclear factor kappa B (NF-κB), matrix metalloproteinase (MMP)-13, MMP-3, and vascular endothelial growth factor (VEGF) gene expression were evaluated. The levels of all gene expression were increased by inflammatory stress. When chondrocytes were exposed to a hydrostatic pressure of 5 MPa, HIF-2α, MMP-13, and MMP-3 gene expression increased significantly although those of HSP70 and NF-κB were not significantly different from the control group. In contrast, HIF-2α gene expression did not increase under a hydrostatic pressure of 50 MPa although HSP70 and NF-κB expression increased significantly compared to control. We considered that hydrostatic pressure of 5 MPa could regulate HIF-2α independent of NF-κB, because the level of HIF-2α gene expression increased significantly without upregulation of NF-κB expression at 5 MPa. Hydrostatic pressure may influence cartilage degeneration, inducing MMP-13 and MMP-3 expression through HIF-2α.

  13. Duplicability of self-interacting human genes.

    LENUS (Irish Health Repository)

    Pérez-Bercoff, Asa

    2010-01-01

    BACKGROUND: There is increasing interest in the evolution of protein-protein interactions because this should ultimately be informative of the patterns of evolution of new protein functions within the cell. One model proposes that the evolution of new protein-protein interactions and protein complexes proceeds through the duplication of self-interacting genes. This model is supported by data from yeast. We examined the relationship between gene duplication and self-interaction in the human genome. RESULTS: We investigated the patterns of self-interaction and duplication among 34808 interactions encoded by 8881 human genes, and show that self-interacting proteins are encoded by genes with higher duplicability than genes whose proteins lack this type of interaction. We show that this result is robust against the system used to define duplicate genes. Finally we compared the presence of self-interactions amongst proteins whose genes have duplicated either through whole-genome duplication (WGD) or small-scale duplication (SSD), and show that the former tend to have more interactions in general. After controlling for age differences between the two sets of duplicates this result can be explained by the time since the gene duplication. CONCLUSIONS: Genes encoding self-interacting proteins tend to have higher duplicability than proteins lacking self-interactions. Moreover these duplicate genes have more often arisen through whole-genome rather than small-scale duplication. Finally, self-interacting WGD genes tend to have more interaction partners in general in the PIN, which can be explained by their overall greater age. This work adds to our growing knowledge of the importance of contextual factors in gene duplicability.

  14. Mechanical confinement regulates cartilage matrix formation by chondrocytes

    Science.gov (United States)

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

    2017-12-01

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

  15. Human DNA repair and recombination genes

    International Nuclear Information System (INIS)

    Thompson, L.H.; Weber, C.A.; Jones, N.J.

    1988-09-01

    Several genes involved in mammalian DNA repair pathways were identified by complementation analysis and chromosomal mapping based on hybrid cells. Eight complementation groups of rodent mutants defective in the repair of uv radiation damage are now identified. At least seven of these genes are probably essential for repair and at least six of them control the incision step. The many genes required for repair of DNA cross-linking damage show overlap with those involved in the repair of uv damage, but some of these genes appear to be unique for cross-link repair. Two genes residing on human chromosome 19 were cloned from genomic transformants using a cosmid vector, and near full-length cDNA clones of each gene were isolated and sequenced. Gene ERCC2 efficiently corrects the defect in CHO UV5, a nucleotide excision repair mutant. Gene XRCC1 normalizes repair of strand breaks and the excessive sister chromatid exchange in CHO mutant EM9. ERCC2 shows a remarkable /approximately/52% overall homology at both the amino acid and nucleotide levels with the yeast RAD3 gene. Evidence based on mutation induction frequencies suggests that ERCC2, like RAD3, might also be an essential gene for viability. 100 refs., 4 tabs

  16. Human gene therapy and imaging: cardiology

    International Nuclear Information System (INIS)

    Wu, Joseph C.; Yla-Herttuala, Seppo

    2005-01-01

    This review discusses the basics of cardiovascular gene therapy, the results of recent human clinical trials, and the rapid progress in imaging techniques in cardiology. Improved understanding of the molecular and genetic basis of coronary heart disease has made gene therapy a potential new alternative for the treatment of cardiovascular diseases. Experimental studies have established the proof-of-principle that gene transfer to the cardiovascular system can achieve therapeutic effects. First human clinical trials provided initial evidence of feasibility and safety of cardiovascular gene therapy. However, phase II/III clinical trials have so far been rather disappointing and one of the major problems in cardiovascular gene therapy has been the inability to verify gene expression in the target tissue. New imaging techniques could significantly contribute to the development of better gene therapeutic approaches. Although the exact choice of imaging modality will depend on the biological question asked, further improvement in image resolution and detection sensitivity will be needed for all modalities as we move from imaging of organs and tissues to imaging of cells and genes. (orig.)

  17. Patenting Human Genes in Europe

    DEFF Research Database (Denmark)

    Minssen, Timo

    2017-01-01

    In accordance with the concept of the book and the assigned scope of the contribution, this chapter describes the European law with respect to the patent-eligibility of isolated DNA sequences. This chapter will further include a brief comparison with recent developments from the US and Australia....... It will, however, not focus on the important debates regarding the patent-eligibility of other biological material, diagnostic methods patents (as data aggregators) or abstract ideas which will be addressed by other contributions. Moreover, the analysis will merely concentrate on patent-eligibility. Other...... patentability requirement will only be briefly touched upon in the discussion part. The paper starts out in section 1.5.2 by discussing the patent-eligibility of isolated human DNA sequences on the European national level and under the Biotechnology Directive. Then the patent-eligibility of isolated human DNA...

  18. Tamoxifen-inducible gene deletion reveals a distinct cell type associated with trabecular bone, and direct regulation of PTHrP expression and chondrocyte morphology by Ihh in growth region cartilage.

    Science.gov (United States)

    Hilton, Matthew J; Tu, Xiaolin; Long, Fanxin

    2007-08-01

    Indian hedgehog (Ihh) controls multiple aspects of endochondral skeletal development by signaling to both chondrocytes and perichondrial cells. Previous efforts to delineate direct effects of Ihh on chondrocytes by Col2-Cre-mediated ablation of Smoothened (Smo, encoding a transmembrane protein indispensable for Ihh signaling) has been only partially successful, due to the inability to discriminate between chondrocytes and perichondrial cells. Here we report a transgenic line (Col2-Cre) expressing under the control of the Colalpha1(II) promoter an inert form of Cre that is activatable by exogenous tamoxifen (TM); TM administration at proper times during embryogenesis induced Cre activity in chondrocytes but not in the perichondrium. By using this mouse line, we deleted Smo within subsets of chondrocytes without affecting the perichondrium and found that Smo removal led to localized disruption of the expression of parathyroid hormone-related protein (PTHrP) and the morphology of chondrocytes. Unexpectedly, TM invariably induced Cre activity in a subset of cells associated with the trabecular bone surface of long bones. These cells, when genetically marked and cultured in vitro, were capable of producing bone nodules. Expression of the Col2-Cre transgene in these cells likely reflected the endogenous Colalpha1(II) promoter activity as similar cells were found to express the IIA isoform of Colalpha1(II) mRNA endogenously. In summary, the present study has not only provided evidence that Ihh signaling directly controls PTHrP expression and chondrocyte morphology in the growth region cartilage, but has also uncovered a distinct cell type associated with the trabecular bone that appears to possess osteogenic potential.

  19. CARTILAGE CONSTRUCTS ENGINEERED FROM CHONDROCYTES OVEREXPRESSING IGF-I IMPROVE THE REPAIR OF OSTEOCHONDRAL DEFECTS IN A RABBIT MODEL

    Science.gov (United States)

    Madry, Henning; Kaul, Gunter; Zurakowski, David; Vunjak-Novakovic, Gordana; Cucchiarini, Magali

    2015-01-01

    Tissue engineering combined with gene therapy is a promising approach for promoting articular cartilage repair. Here, we tested the hypothesis that engineered cartilage with chondrocytes over expressing a human insulin-like growth factor I (IGF-I) gene can enhance the repair of osteochondral defects, in a manner dependent on the duration of cultivation. Genetically modified chondrocytes were cultured on biodegradable polyglycolic acid scaffolds in dynamic flow rotating bioreactors for either 10 or 28 d. The resulting cartilaginous constructs were implanted into osteochondral defects in rabbit knee joints. After 28 weeks of in vivo implantation, immunoreactivity to ß-gal was detectable in the repair tissue of defects that received lacZ constructs. Engineered cartilaginous constructs based on IGF-I-over expressing chondrocytes markedly improved osteochondral repair compared with control (lacZ) constructs. Moreover, IGF-I constructs cultivated for 28 d in vitro significantly promoted osteochondral repair vis-à-vis similar constructs cultivated for 10 d, leading to significantly decreased osteoarthritic changes in the cartilage adjacent to the defects. Hence, the combination of spatially defined overexpression of human IGF-I within a tissue-engineered construct and prolonged bioreactor cultivation resulted in most enhanced articular cartilage repair and reduction of osteoarthritic changes in the cartilage adjacent to the defect. Such genetically enhanced tissue engineering provides a versatile tool to evaluate potential therapeutic genes in vivo and to improve our comprehension of the development of the repair tissue within articular cartilage defects. Insights gained with additional exploration using this model may lead to more effective treatment options for acute cartilage defects. PMID:23588785

  20. Cartilage constructs engineered from chondrocytes overexpressing IGF-I improve the repair of osteochondral defects in a rabbit model

    Directory of Open Access Journals (Sweden)

    H Madry

    2013-04-01

    Full Text Available Tissue engineering combined with gene therapy is a promising approach for promoting articular cartilage repair. Here, we tested the hypothesis that engineered cartilage with chondrocytes overexpressing a human insulin-like growth factor I (IGF-I gene can enhance the repair of osteochondral defects, in a manner dependent on the duration of cultivation. Genetically modified chondrocytes were cultured on biodegradable polyglycolic acid scaffolds in dynamic flow rotating bioreactors for either 10 or 28 d. The resulting cartilaginous constructs were implanted into osteochondral defects in rabbit knee joints. After 28 weeks of in vivo implantation, immunoreactivity to ß-gal was detectable in the repair tissue of defects that received lacZ constructs. Engineered cartilaginous constructs based on IGF-I-overexpressing chondrocytes markedly improved osteochondral repair compared with control (lacZ constructs. Moreover, IGF-I constructs cultivated for 28 d in vitro significantly promoted osteochondral repair vis-à-vis similar constructs cultivated for 10 d, leading to significantly decreased osteoarthritic changes in the cartilage adjacent to the defects. Hence, the combination of spatially defined overexpression of human IGF-I within a tissue-engineered construct and prolonged bioreactor cultivation resulted in most enhanced articular cartilage repair and reduction of osteoarthritic changes in the cartilage adjacent to the defect. Such genetically enhanced tissue engineering provides a versatile tool to evaluate potential therapeutic genes in vivo and to improve our comprehension of the development of the repair tissue within articular cartilage defects. Insights gained with additional exploration using this model may lead to more effective treatment options for acute cartilage defects.

  1. Beta1 integrins regulate chondrocyte rotation, G1 progression, and cytokinesis

    DEFF Research Database (Denmark)

    Aszodi, Attila; Hunziker, Ernst B; Brakebusch, Cord

    2003-01-01

    Beta1 integrins are highly expressed on chondrocytes, where they mediate adhesion to cartilage matrix proteins. To assess the functions of beta1 integrin during skeletogenesis, we inactivated the beta1 integrin gene in chondrocytes. We show here that these mutant mice develop a chondrodysplasia...... of various severity. beta1-deficient chondrocytes had an abnormal shape and failed to arrange into columns in the growth plate. This is caused by a lack of motility, which is in turn caused by a loss of adhesion to collagen type II, reduced binding to and impaired spreading on fibronectin, and an abnormal F......-actin organization. In addition, mutant chondrocytes show decreased proliferation caused by a defect in G1/S transition and cytokinesis. The G1/S defect is, at least partially, caused by overexpression of Fgfr3, nuclear translocation of Stat1/Stat5a, and up-regulation of the cell cycle inhibitors p16 and p21...

  2. Good genes, complementary genes and human mate preferences.

    Science.gov (United States)

    Roberts, S Craig; Little, Anthony C

    2008-09-01

    The past decade has witnessed a rapidly growing interest in the biological basis of human mate choice. Here we review recent studies that demonstrate preferences for traits which might reveal genetic quality to prospective mates, with potential but still largely unknown influence on offspring fitness. These include studies assessing visual, olfactory and auditory preferences for potential good-gene indicator traits, such as dominance or bilateral symmetry. Individual differences in these robust preferences mainly arise through within and between individual variation in condition and reproductive status. Another set of studies have revealed preferences for traits indicating complementary genes, focussing on discrimination of dissimilarity at genes in the major histocompatibility complex (MHC). As in animal studies, we are only just beginning to understand how preferences for specific traits vary and inter-relate, how consideration of good and compatible genes can lead to substantial variability in individual mate choice decisions and how preferences expressed in one sensory modality may reflect those in another. Humans may be an ideal model species in which to explore these interesting complexities.

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

    International Nuclear Information System (INIS)

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

    2016-01-01

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

  4. 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

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

    International Nuclear Information System (INIS)

    Zeng, Lei; Yao, Yongchang; Wang, Dong-an; Chen, Xiaofeng

    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

  6. 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.

  7. 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.

  8. Ethical issues of perinatal human gene therapy.

    Science.gov (United States)

    Fletcher, J C; Richter, G

    1996-01-01

    This paper examines some key ethical issues raised by trials of human gene therapy in the perinatal period--i.e., in infants, young children, and the human fetus. It describes five resources in ethics for researchers' considerations prior to such trials: (1) the history of ethical debate about gene therapy, (2) a literature on the relevance of major ethical principles for clinical research, (3) a body of widely accepted norms and practices, (4) knowledge of paradigm cases, and (5) researchers' own professional integrity. The paper also examines ethical concerns that must be met prior to any trial: benefits to and safety of subjects, informed assent of children and informed parental permission, informed consent of pregnant women in fetal gene therapy, protection of privacy, and concerns about fairness in the selection of subjects. The paper criticizes the position that cases of fetal gene therapy should be restricted only to those where the pregnant woman has explicitly refused abortion. Additional topics include concerns about genetic enhancement and germ-line gene therapy.

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

    DEFF Research Database (Denmark)

    Kveiborg, Marie; Albrechtsen, Reidar; Rudkjaer, Lise

    2006-01-01

    ADAM12-S transgenic mice exhibit a pronounced increase in the length of bones, such as femur, tibia, and vertebrae. The effect of ADAM12-S on longitudinal bone growth involves the modulation of chondrocyte proliferation and maturation, likely through proteolytic activities and altered cell......: Transgenic mice expressing the secreted form of human ADAM12, ADAM12-S, or a truncated metalloprotease-deficient form of ADAM12-S in the circulation were used to study the effects of ADAM12 on the skeleton. In addition, murine chondrocyte cultures were used to study the effect of ADAM12-S on cell...... studies showed that ADAM12-S inhibits chondrocyte adhesion to fibronectin and collagen type II. CONCLUSIONS: ADAM12-S stimulates bone growth in mice by modulating chondrocyte proliferation and maturation through mechanisms probably involving both metalloprotease and adhesion activities....

  10. 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.

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

    Science.gov (United States)

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

    2004-01-01

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

  12. Histone Deacetylase 3 Suppresses Erk Phosphorylation and Matrix Metalloproteinase (Mmp)-13 Activity in Chondrocytes

    Science.gov (United States)

    Carpio, Lomeli R.; Bradley, Elizabeth W.; Westendorf, Jennifer J.

    2017-01-01

    Histone deacetylase inhibitors are emerging therapies for many diseases including cancers and neurological disorders; however, these drugs are teratogens to the developing skeleton. Hdac3 is essential for proper endochondral ossification as its deletion in chondrocytes increases cytokine signaling and the expression of matrix remodeling enzymes. Here we explored the mechanism by which Hdac3 controls Mmp13 expression in chondrocytes. In Hdac3-depleted chondrocytes, Erk1/2 as well as its downstream substrate, Runx2, were hyperphosphorylated as a result of decreased expression and activity of the Erk1/2 specific phosphatase, Dusp6. Erk1/2 kinase inhibitors and Dusp6 adenoviruses reduced Mmp13 expression and partially rescued matrix production in Hdac3-deficient chondrocytes. Postnatal chondrocyte-specific deletion of Hdac3 with an inducible Col2a1-Cre caused premature production of pErk1/2 and Mmp13 in the growth plate. Thus, Hdac3 controls the temporal and spatial expression of tissue-remodeling genes in chondrocytes to ensure proper endochondral ossification during development. PMID:27662443

  13. Secondary chondrocyte-derived Ihh stimulates proliferation of periosteal cells during chick development.

    Science.gov (United States)

    Buxton, Paul G; Hall, Brian; Archer, Charles W; Francis-West, Philippa

    2003-10-01

    The development of the skull is characterised by its dependence upon epigenetic influences. One of the most important of these is secondary chondrogenesis, which occurs following ossification within certain membrane bone periostea, as a result of biomechanical articulation. We have studied the genesis, character and function of the secondary chondrocytes of the quadratojugal of the chick between embryonic days 11 and 14. Analysis of gene expression revealed that secondary chondrocytes formed coincident with Sox9 upregulation from a precursor population expressing Cbfa1/Runx2: a reversal of the normal sequence. Such secondary chondrocytes rapidly acquired a phenotype that is a compound of prehypertrophic and hypertrophic chondrocytes, exited from the cell cycle and upregulated Ihh. Pulse and pulse/chase experiments with BrdU confirmed the germinal region as the highly proliferative source of the secondary chondrocytes, which formed by division of chondrocyte-committed precursors. By blocking Hh signalling in explant cultures we show that the enhanced proliferation of the germinal region surrounding the secondary chondrocytes derives from this Ihh source. Additionally, in vitro studies on membrane bone periosteal cells (non-germinal region) demonstrated that these cells can also respond to Ihh, and do so both by enhanced proliferation and precocious osteogenesis. Despite the pro-osteogenic effects of Ihh on periosteal cell differentiation, mechanical articulation of the quadratojugal/quadrate joint in explant culture revealed a negative role for articulation in the regulation of osteocalcin by germinal region descendants. Thus, the mechanical stimulus that is the spur to secondary chondrocyte formation appears able to override the osteogenic influence of Ihh on the periosteum, but does not interfere with the cell cycle-promoting component of Hh signalling.

  14. 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.

  15. 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.

  16. Human serum amyloid genes--molecular characterization

    International Nuclear Information System (INIS)

    Sack, G.H.; Lease, J.J.

    1986-01-01

    Three clones containing human genes for serum amyloid A protein (SAA) have been isolated and characterized. Each of two clones, GSAA 1 and 2 (of 12.8 and 15.9 kilobases, respectively), contains two exons, accouting for amino acids 12-58 and 58-103 of mature SAA; the extreme 5' termini and 5' untranslated regions have not yet been defined but are anticipated to be close based on studies of murine SAA genes. Initial amino acid sequence comparisons show 78/89 identical residues. At 4 of the 11 discrepant residues, the amino acid specified by the codon is the same as the corresponding residue in murine SAA. Identification of regions containing coding regions has permitted use of selected subclones for blot hybridization studies of larger human SAA chromosomal gene organization. The third clone, GSAA 3 also contains SAA coding information by DNA sequence analysis but has a different organization which has not yet been fully described. We have reported the isolation of clones of human DNA hybridizing with pRS48 - a plasmid containing a complementary DNA (cDNA) clone for murine serum amyloid A (SAA; 1, 2). We now present more detailed data confirming the identity and defining some of the organizational features of these clones

  17. Gli3 acts as a repressor downstream of Ihh in regulating two distinct steps of chondrocyte differentiation.

    Science.gov (United States)

    Koziel, Lydia; Wuelling, Manuela; Schneider, Sabine; Vortkamp, Andrea

    2005-12-01

    During endochondral ossification, the secreted growth factor Indian hedgehog (Ihh) regulates several differentiation steps. It interacts with a second secreted factor, parathyroid hormone-related protein (PTHrP), to regulate the onset of hypertrophic differentiation, and it regulates chondrocyte proliferation and ossification of the perichondrium independently of PTHrP. To investigate how the Ihh signal is translated in the different target tissues, we analyzed the role of the zinc-finger transcription factor Gli3, which acts downstream of hedgehog signals in other organs. Loss of Gli3 in Ihh mutants restores chondrocyte proliferation and delays the accelerated onset of hypertrophic differentiation observed in Ihh-/- mutants. Furthermore the expression of the Ihh target genes patched (Ptch) and PTHrP is reactivated in Ihh-/-;Gli3-/- mutants. Gli3 seems thus to act as a strong repressor of Ihh signals in regulating chondrocyte differentiation. In addition, loss of Gli3 in mice that overexpress Ihh in chondrocytes accelerates the onset of hypertrophic differentiation by reducing the domain and possibly the level of PTHrP expression. Careful analysis of chondrocyte differentiation in Gli3-/- mutants revealed that Gli3 negatively regulates the differentiation of distal, low proliferating chondrocytes into columnar, high proliferating cells. Our results suggest a model in which the Ihh/Gli3 system regulates two distinct steps of chondrocyte differentiation: (1) the switch from distal into columnar chondrocytes is repressed by Gli3 in a PTHrP-independent mechanism; (2) the transition from proliferating into hypertrophic chondrocytes is regulated by Gli3-dependent expression of PTHrP. Furthermore, by regulating distal chondrocyte differentiation, Gli3 seems to position the domain of PTHrP expression.

  18. Trophic effects of adipose-tissue-derived and bone-marrow-derived mesenchymal stem cells enhance cartilage generation by chondrocytes in co-culture.

    Science.gov (United States)

    Pleumeekers, M M; Nimeskern, L; Koevoet, J L M; Karperien, M; Stok, K S; van Osch, G J V M

    2018-01-01

    Combining mesenchymal stem cells (MSCs) and chondrocytes has great potential for cell-based cartilage repair. However, there is much debate regarding the mechanisms behind this concept. We aimed to clarify the mechanisms that lead to chondrogenesis (chondrocyte driven MSC-differentiation versus MSC driven chondroinduction) and whether their effect was dependent on MSC-origin. Therefore, chondrogenesis of human adipose-tissue-derived MSCs (hAMSCs) and bone-marrow-derived MSCs (hBMSCs) combined with bovine articular chondrocytes (bACs) was compared. hAMSCs or hBMSCs were combined with bACs in alginate and cultured in vitro or implanted subcutaneously in mice. Cartilage formation was evaluated with biochemical, histological and biomechanical analyses. To further investigate the interactions between bACs and hMSCs, (1) co-culture, (2) pellet, (3) Transwell® and (4) conditioned media studies were conducted. The presence of hMSCs-either hAMSCs or hBMSCs-increased chondrogenesis in culture; deposition of GAG was most evidently enhanced in hBMSC/bACs. This effect was similar when hMSCs and bAC were combined in pellet culture, in alginate culture or when conditioned media of hMSCs were used on bAC. Species-specific gene-expression analyses demonstrated that aggrecan was expressed by bACs only, indicating a predominantly trophic role for hMSCs. Collagen-10-gene expression of bACs was not affected by hBMSCs, but slightly enhanced by hAMSCs. After in-vivo implantation, hAMSC/bACs and hBMSC/bACs had similar cartilage matrix production, both appeared stable and did not calcify. This study demonstrates that replacing 80% of bACs by either hAMSCs or hBMSCs does not influence cartilage matrix production or stability. The remaining chondrocytes produce more matrix due to trophic factors produced by hMSCs.

  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. 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.

  1. IGF-1 Gene Transfer to Human Synovial MSCs Promotes Their Chondrogenic Differentiation Potential without Induction of the Hypertrophic Phenotype

    Directory of Open Access Journals (Sweden)

    Yasutoshi Ikeda

    2017-01-01

    Full Text Available Mesenchymal stem cell- (MSC- based therapy is a promising treatment for cartilage. However, repair tissue in general fails to regenerate an original hyaline-like tissue. In this study, we focused on increasing the expression levels for insulin-like growth factor-1 (IGF-1 to improve repair tissue quality. The IGF-1 gene was introduced into human synovial MSCs with a lentiviral vector and examined the levels of gene expression and morphological status of MSCs under chondrogenic differentiation condition using pellet cultures. The size of the pellets derived from IGF-1-MSCs were significantly larger than those of the control group. The abundance of glycosaminoglycan (GAG was also significantly higher in the IGF-1-MSC group. The histology of the IGF-1-induced pellets demonstrated similarities to hyaline cartilage without exhibiting features of a hypertrophic chondrocyte phenotype. Expression levels for the Col2A1 gene and protein were significantly higher in the IGF-1 pellets than in the control pellets, but expression levels for Col10, MMP-13, ALP, and Osterix were not higher. Thus, IGF-1 gene transfer to human synovial MSCs led to an improved chondrogenic differentiation capacity without the detectable induction of a hypertrophic or osteogenic phenotype.

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

    International Nuclear Information System (INIS)

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

    2014-01-01

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

  3. Hepatocyte specific expression of human cloned genes

    Energy Technology Data Exchange (ETDEWEB)

    Cortese, R

    1986-01-01

    A large number of proteins are specifically synthesized in the hepatocyte. Only the adult liver expresses the complete repertoire of functions which are required at various stages during development. There is therefore a complex series of regulatory mechanisms responsible for the maintenance of the differentiated state and for the developmental and physiological variations in the pattern of gene expression. Human hepatoma cell lines HepG2 and Hep3B display a pattern of gene expression similar to adult and fetal liver, respectively; in contrast, cultured fibroblasts or HeLa cells do not express most of the liver specific genes. They have used these cell lines for transfection experiments with cloned human liver specific genes. DNA segments coding for alpha1-antitrypsin and retinol binding protein (two proteins synthesized both in fetal and adult liver) are expressed in the hepatoma cell lines HepG2 and Hep3B, but not in HeLa cells or fibroblasts. A DNA segment coding for haptoglobin (a protein synthesized only after birth) is only expressed in the hepatoma cell line HepG2 but not in Hep3B nor in non hepatic cell lines. The information for tissue specific expression is located in the 5' flanking region of all three genes. In vivo competition experiments show that these DNA segments bind to a common, apparently limiting, transacting factor. Conventional techniques (Bal deletions, site directed mutagenesis, etc.) have been used to precisely identify the DNA sequences responsible for these effects. The emerging picture is complex: they have identified multiple, separate transcriptional signals, essential for maximal promoter activation and tissue specific expression. Some of these signals show a negative effect on transcription in fibroblast cell lines.

  4. [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.

  5. Dietary methanol regulates human gene activity.

    Directory of Open Access Journals (Sweden)

    Anastasia V Shindyapina

    Full Text Available Methanol (MeOH is considered to be a poison in humans because of the alcohol dehydrogenase (ADH-mediated conversion of MeOH to formaldehyde (FA, which is toxic. Our recent genome-wide analysis of the mouse brain demonstrated that an increase in endogenous MeOH after ADH inhibition led to a significant increase in the plasma MeOH concentration and a modification of mRNA synthesis. These findings suggest endogenous MeOH involvement in homeostasis regulation by controlling mRNA levels. Here, we demonstrate directly that study volunteers displayed increasing concentrations of MeOH and FA in their blood plasma when consuming citrus pectin, ethanol and red wine. A microarray analysis of white blood cells (WBC from volunteers after pectin intake showed various responses for 30 significantly differentially regulated mRNAs, most of which were somehow involved in the pathogenesis of Alzheimer's disease (AD. There was also a decreased synthesis of hemoglobin mRNA, HBA and HBB, the presence of which in WBC RNA was not a result of red blood cells contamination because erythrocyte-specific marker genes were not significantly expressed. A qRT-PCR analysis of volunteer WBCs after pectin and red wine intake confirmed the complicated relationship between the plasma MeOH content and the mRNA accumulation of both genes that were previously identified, namely, GAPDH and SNX27, and genes revealed in this study, including MME, SORL1, DDIT4, HBA and HBB. We hypothesized that human plasma MeOH has an impact on the WBC mRNA levels of genes involved in cell signaling.

  6. Normal proliferation and differentiation of Hoxc-8 transgenic chondrocytes in vitro

    Directory of Open Access Journals (Sweden)

    Mello Maria

    2003-04-01

    Full Text Available Abstract Background Hox genes encode transcription factors that are involved in pattern formation in the skeleton, and recent evidence suggests that they also play a role in the regulation of endochondral ossification. To analyze the role of Hoxc-8 in this process in more detail, we applied in vitro culture systems, using high density cultures of primary chondrocytes from neonatal mouse ribs. Results Cultured cells were characterized on the basis of morphology (light microscopy and production of cartilage-specific extracellular matrix (sulfated proteoglycans and type II Collagen. Hypertrophy was demonstrated by increase in cell size, alkaline phosphatase activity and type X Collagen immunohistochemistry. Proliferation was assessed by BrdU uptake and flow cytometry. Unexpectedly, chondrocytes from Hoxc-8 transgenic mice, which exhibit delayed cartilage maturation in vivo 1, were able to proliferate and differentiate normally in our culture systems. This was the case even though freshly isolated Hoxc-8 transgenic chondrocytes exhibited significant molecular differences as measured by real-time quantitative PCR. Conclusions The results demonstrate that primary rib chondrocytes behave similar to published reports for chondrocytes from other sources, validating in vitro approaches for studies of Hox genes in the regulation of endochondral ossification. Our analysis of cartilage-producing cells from Hoxc-8 transgenic mice provides evidence that the cellular phenotype induced by Hoxc-8 overexpression in vivo is reversible in vitro.

  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. Lipofection of rabbit chondrocytes and long lasting expression of a lacZ reporter system in alginate beads.

    Science.gov (United States)

    Stöve, J; Fiedler, J; Huch, K; Günther, K-P; Puhl, W; Brenner, R

    2002-03-01

    Our aim was to investigate the maintenance of the transfection status of non-viral transfected chondrocytes in an alginate culture system. Chondrocytes harvested from rabbit knees were isolated by sequential digestion and cultivated in monolayer culture. At 60-70% cell density, chondrocytes were transfected with different transfection systems (FuGENE6, CaCl2, Lipofectin). A lac Z expression vector (pcDNA 3.1/Myc-His+ lacZ) was used as a reporter system. In order to improve transfection rates, hyaluronidase (4 U/ml) was used prior and during the transfection procedure. Thereafter, transfected cells were either kept in monolayer culture or embedded in alginate beads and kept in culture for up to the next 30 weeks. Transfection efficiency was maximal using FuGENE6TM/DNA at a ratio of 3:2 and hyaluronidase (4 U/ml). Transfection efficiency reached up to 40.8% (+/- 3.2%) after 36 h. In alginate beads lac Z positive cells declined to 8.5% +/- 3.3% after 4 weeks and to 4.6% +/- 3.2% after 12 weeks of culturing. After 30 weeks 3% of chondrocytes still expressed lac Z. In contrast, during culturing in monolayer, no lac Z expression was detectable after 4 weeks. Differentiation status of the chondrocytes was confirmed by histology and immunohistochemistry methods. After successful gene transfer to rabbit chondrocytes the alginate system made it possible to culture lipofected chondrocytes phenotypically stable. Genetically engineered chondrocytes express the lac Z reporter gene over a period of at least 30 weeks. This transfection and culture system provides a promising tool to further investigate the over-expression of growth factors and enzyme inhibitors. Copyright 2002 OsteoArthritis Research Society International.

  9. 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.

  10. CCN2/CTGF is required for matrix organization and to protect growth plate chondrocytes from cellular stress.

    Science.gov (United States)

    Hall-Glenn, Faith; Aivazi, Armen; Akopyan, Lusi; Ong, Jessica R; Baxter, Ruth R; Benya, Paul D; Goldschmeding, Roel; van Nieuwenhoven, Frans A; Hunziker, Ernst B; Lyons, Karen M

    2013-08-01

    CCN2 (connective tissue growth factor (CTGF/CCN2)) is a matricellular protein that utilizes integrins to regulate cell proliferation, migration and survival. The loss of CCN2 leads to perinatal lethality resulting from a severe chondrodysplasia. Upon closer inspection of Ccn2 mutant mice, we observed defects in extracellular matrix (ECM) organization and hypothesized that the severe chondrodysplasia caused by loss of CCN2 might be associated with defective chondrocyte survival. Ccn2 mutant growth plate chondrocytes exhibited enlarged endoplasmic reticula (ER), suggesting cellular stress. Immunofluorescence analysis confirmed elevated stress in Ccn2 mutants, with reduced stress observed in Ccn2 overexpressing transgenic mice. In vitro studies revealed that Ccn2 is a stress responsive gene in chondrocytes. The elevated stress observed in Ccn2-/- chondrocytes is direct and mediated in part through integrin α5. The expression of the survival marker NFκB and components of the autophagy pathway were decreased in Ccn2 mutant growth plates, suggesting that CCN2 may be involved in mediating chondrocyte survival. These data demonstrate that absence of a matricellular protein can result in increased cellular stress and highlight a novel protective role for CCN2 in chondrocyte survival. The severe chondrodysplasia caused by the loss of CCN2 may be due to increased chondrocyte stress and defective activation of autophagy pathways, leading to decreased cellular survival. These effects may be mediated through nuclear factor κB (NFκB) as part of a CCN2/integrin/NFκB signaling cascade.

  11. Regulation of collagenase inhibitor production in chondrosarcoma chondrocytes

    International Nuclear Information System (INIS)

    Harper, J.; Harper, E.

    1987-01-01

    Swarm rat chondrosarcoma chondrocytes produce an inhibitor of collagenase. This inhibitor is similar to those isolated from normal cartilage tissues. These cells will synthesize proteins in the absence of serum. Since serum contains inhibitors of collagenase, it is necessary to culture cells without serum in order to obtain accurate measurements of enzyme and inhibitor levels. They examined the effect of insulin on inhibitor secretion by cultures of Swarm rat chondrosarcoma chondrocytes. They observed a 2.5 to 3.5 fold stimulation of inhibitory activity in the presence of as little as 10 ng/ml insulin as compared to controls in serum free Dulbecco's modified Eagle's medium supplemented with 4.5 g/l glucose. The units of inhibitor were determined over a 7 day culture period. Medium was harvested daily and assayed for collagenase activity and for inhibition of a known collagenase from rabbit skin or human skin, using the 14 C-glycine peptide release assay. The amount of inhibitor obtained from days 2 through 7 were: 1.4 unit (control), 3.8 units (10 ng/ml insulin), 5.2 units (1 μg/ml insulin). The addition of 1 mM dibutyryl cyclic AMP to these chondrocytes in the presence of 1 μg/ml insulin caused a decrease in the level of inhibitor, suggesting that a dephosphorylation event may be necessary for this stimulation by insulin to occur

  12. Targeting the human lysozyme gene on bovine αs1- casein gene ...

    African Journals Online (AJOL)

    Targeting an exogenous gene into a favorable gene locus and for expression under endogenous regulators is an ideal method in mammary gland bioreactor research. For this purpose, a gene targeting vector was constructed to targeting the human lysozyme gene on bovine αs1-casein gene locus. In this case, the ...

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

    Science.gov (United States)

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

    2018-03-15

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

  14. Injury, inflammation and the emergence of human specific genes

    Science.gov (United States)

    2016-07-12

    genes in circulating and resident human immune cells can be studied in mice after the transplantation and engraft- ment of human hemato- lymphoid immune...Martinek J, Strowig T, Gearty SV, Teichmann LL, et al. Development and function of human innate immune cells in a humanized mouse model. Nat Bio...normal wound repair and regeneration, we hypothesize that the preponderance of human-specific genes expressed in human inflammatory cells is commensurate

  15. 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.

  16. 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.

  17. Structure and chromosomal localization of the human lymphotoxin gene

    International Nuclear Information System (INIS)

    Nedwin, G.E.; Jarrett-Nedwin, J.; Smith, D.H.; Naylor, S.L.; Sakaguchi, A.Y.; Goeddel, D.V.; Gray, P.W.

    1987-01-01

    The authors have isolated, sequenced, and determined the chromosomal localization of the gene encoding human lymphotoxin (LT). The single copy gene was isolated from a human genomic library using a /sup 32/P-labeled 116 bp synthetic DNA fragment whose sequence was based on the NH/sub 2/-terminal amino acid sequence of LT. The gene spans 3 kb of DNA and is interrupted by three intervening sequences. The LT gene is located on human chromosome 6, as determined by Southern blot analysis of human-murine hybrid DNA. Putative transcriptional control regions and areas of homology with the promoters of interferon and other genes are identified

  18. Primary chondrocytes enhance cartilage tissue formation upon co-culture with expanded chondrocytes, dermal fibroblasts, 3T3 feeder cells and embryonic stem cells

    NARCIS (Netherlands)

    Hendriks, J.A.A.; Miclea, Razvan L.; Schotel, Roka; de Bruijn, Ewart; Moroni, Lorenzo; Karperien, Hermanus Bernardus Johannes; Riesle, J.U.; van Blitterswijk, Clemens

    2010-01-01

    Co-culture models have been increasingly used in tissue engineering applications to understand cell–cell interactions and consequently improve regenerative medicine strategies. Aiming at further elucidating cartilage tissue formation, we co-cultured bovine primary chondrocytes (BPCs) with human

  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. 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.

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

    Directory of Open Access Journals (Sweden)

    Eleonora Olivotto

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

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

    Science.gov (United States)

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

    2015-06-24

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

  3. 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

  4. Gene expression studies on human keratinocytes transduced with human growth hormone gene for a possible utilization in gene therapy

    International Nuclear Information System (INIS)

    Mathor, Monica Beatriz.

    1994-01-01

    Taking advantage of the recent progress in the DNA-recombinant techniques and of the potentiality of normal human keratinocytes primary culture to reconstitute the epidermis, it was decided to genetically transform these keratinocytes to produce human growth hormone under controllable conditions that would be used in gene therapy at this hormone deficient patients. The first step to achieve this goal was to standardize infection of keratinocytes with retrovirus producer cells containing a construct which included the gene of bacterial b-galactosidase. The best result was obtained cultivating the keratinocytes for 3 days in a 2:1 mixture of retrovirus producer cells and 3T3-J2 fibroblasts irradiated with 60 Gy, and splitting these infected keratinocytes on 3T3-J2 fibroblasts feeder layer. Another preliminary experiment was to infect normal human keratinocytes with interleukin-6 gene (hIL-6) that, in pathologic conditions, could be reproduced by keratinocytes and secreted to the blood stream. Thus, we verify that infected keratinocytes secrete an average amount of 500 ng/10 6 cell/day of cytokin during the in vitro life time, that certify the stable character of the injection. These keratinocytes, when grafted in mice, secrete hIL-6 to the blood stream reaching levels of 40 pg/ml of serum. After these preliminary experiments, we construct a retroviral vector with the human growth hormone gene (h GH) driven by human metallothionein promoter (h PMT), designated DChPMTGH. Normal human keratinocytes were infected with DChPMTGH producer cells, following previously standardized protocol, obtaining infected keratinocytes secreting to the culture media 340 ng h GH/10 6 cell/day without promoter activation. This is the highest level of h GH secreted in human keratinocytes primary culture described in literature. The h GH value increases approximately 10 times after activation with 100 μM Zn +2 for 8-12 hours. (author). 158 refs., 42 figs., 6 tabs

  5. Widespread of horizontal gene transfer in the human genome.

    Science.gov (United States)

    Huang, Wenze; Tsai, Lillian; Li, Yulong; Hua, Nan; Sun, Chen; Wei, Chaochun

    2017-04-04

    A fundamental concept in biology is that heritable material is passed from parents to offspring, a process called vertical gene transfer. An alternative mechanism of gene acquisition is through horizontal gene transfer (HGT), which involves movement of genetic materials between different species. Horizontal gene transfer has been found prevalent in prokaryotes but very rare in eukaryote. In this paper, we investigate horizontal gene transfer in the human genome. From the pair-wise alignments between human genome and 53 vertebrate genomes, 1,467 human genome regions (2.6 M bases) from all chromosomes were found to be more conserved with non-mammals than with most mammals. These human genome regions involve 642 known genes, which are enriched with ion binding. Compared to known horizontal gene transfer regions in the human genome, there were few overlapping regions, which indicated horizontal gene transfer is more common than we expected in the human genome. Horizontal gene transfer impacts hundreds of human genes and this study provided insight into potential mechanisms of HGT in the human genome.

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

    Science.gov (United States)

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

    2016-10-01

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

  7. Widespread of horizontal gene transfer in the human genome

    OpenAIRE

    Huang, Wenze; Tsai, Lillian; Li, Yulong; Hua, Nan; Sun, Chen; Wei, Chaochun

    2017-01-01

    Background A fundamental concept in biology is that heritable material is passed from parents to offspring, a process called vertical gene transfer. An alternative mechanism of gene acquisition is through horizontal gene transfer (HGT), which involves movement of genetic materials between different species. Horizontal gene transfer has been found prevalent in prokaryotes but very rare in eukaryote. In this paper, we investigate horizontal gene transfer in the human genome. Results From the pa...

  8. 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

    2017-12-01

    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.

  9. The formation of human auricular cartilage from microtic tissue: An in vivo study.

    Science.gov (United States)

    Ishak, Mohamad Fikeri bin; See, Goh Bee; Hui, Chua Kien; Abdullah, Asma bt; Saim, Lokman bin; Saim, Aminuddin bin; Idrus, Ruszymah bt Haji

    2015-10-01

    This study aimed to isolate, culture-expand and characterize the chondrocytes isolated from microtic cartilage and evaluate its potential as a cell source for ear cartilage reconstruction. Specific attention was to construct the auricular cartilage tissue by using fibrin as scaffold. Cell culture experiment with the use of microtic chondrocytes. Cell culture experiment with the use of microtic chondrocytes. After ear reconstructive surgery at the Universiti Kebangsaan Malaysia Medical Center, chondrocytes were isolated from microtic cartilage. Chondrocytes isolated from the tissue were cultured expanded until passage 4 (P4). Upon confluency at P4, chondrocytes were harvested and tissue engineered constructs were made with human plasma polymerized to fibrin. Constructs formed later is implanted at the dorsal part of nude mice for 8 weeks, followed by post-implantation evaluation with histology staining (Hematoxylin and Eosin (H&E) and Safranin O), immunohistochemistry and RT-PCR for chondrogenic associated genes expression level. Under gross assessment, the construct after 8 weeks of implantation showed similar physical characteristics that of cartilage. Histological staining showed abundant lacunae cells embedded in extracellular matrix similar to that of native cartilage. Safranin O staining showed positive staining which indicates the presence of proteoglycan-rich matrix. Immunohistochemistry analysis showed the strong positive staining for collagen type II, the specific collagen type in the cartilage. Gene expression quantification showed no significant differences in the expression of chondrogenic gene used which is collagen type I, collagen type II, aggrecan core protein (ACP), elastin and sox9 genes when compared to construct formed from normal auricular tissue. Chondrocytes isolated from microtia cartilage has the potential to be used as an alternative cell source for external ear reconstruction in future clinical application. Copyright © 2015 Elsevier

  10. High fat-diet and saturated fatty acid palmitate inhibits IGF-1 function in chondrocytes.

    Science.gov (United States)

    Nazli, S A; Loeser, R F; Chubinskaya, S; Willey, J S; Yammani, R R

    2017-09-01

    Insulin-like growth factor-1 (IGF-1) promotes matrix synthesis and cell survival in cartilage. Chondrocytes from aged and osteoarthritic cartilage have a reduced response to IGF-1. The purpose of this study was to determine the effect of free fatty acids (FFA) present in a high-fat diet on IGF-1 function in cartilage and the role of endoplasmic reticulum (ER) stress. C57BL/6 male mice were maintained on either a high-fat (60% kcal from fat) or a low-fat (10% kcal from fat) diet for 4 months. Mice were then sacrificed; femoral head cartilage caps were collected and treated with IGF-1 to measure proteoglycan (PG) synthesis. Cultured human chondrocytes were treated with 500 μM FFA palmitate or oleate, followed by stimulation with (100 ng/ml) IGF-1 overnight to measure CHOP (a protein marker for ER stress) and PG synthesis. Human chondrocytes were pre-treated with palmitate or 1 mM 4-phenyl butyric acid (PBA) or 1 μM C-Jun N terminal Kinase (JNK) inhibitor, and IGF-1 function (PG synthesis and signaling) was measured. Cartilage explants from mice on the high fat-diet showed reduced IGF-1 mediated PG synthesis compared to a low-fat group. Treatment of human chondrocytes with palmitate induced expression of CHOP, activated JNK and inhibited IGF-1 function. PBA, a small molecule chemical chaperone that alleviates ER stress rescued IGF-1 function and a JNK inhibitor rescued IGF-1 signaling. Palmitate-induced ER stress inhibited IGF-1 function in chondrocytes/cartilage via activating the mitogen-activated protein (MAP) kinase JNK. This is the first study to demonstrate that ER stress is metabolic factor that regulates IGF-1 function in chondrocytes. Copyright © 2017 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.

  11. Mutation analysis of the MCHR1 gene in human obesity

    DEFF Research Database (Denmark)

    Wermter, Anne-Kathrin; Reichwald, Kathrin; Büch, Thomas

    2005-01-01

    The importance of the melanin-concentrating hormone (MCH) system for regulation of energy homeostasis and body weight has been demonstrated in rodents. We analysed the human MCH receptor 1 gene (MCHR1) with respect to human obesity....

  12. Bioinformatic prediction and functional characterization of human KIAA0100 gene

    Directory of Open Access Journals (Sweden)

    He Cui

    2017-02-01

    Full Text Available Our previous study demonstrated that human KIAA0100 gene was a novel acute monocytic leukemia-associated antigen (MLAA gene. But the functional characterization of human KIAA0100 gene has remained unknown to date. Here, firstly, bioinformatic prediction of human KIAA0100 gene was carried out using online softwares; Secondly, Human KIAA0100 gene expression was downregulated by the clustered regularly interspaced short palindromic repeats (CRISPR/CRISPR-associated (Cas 9 system in U937 cells. Cell proliferation and apoptosis were next evaluated in KIAA0100-knockdown U937 cells. The bioinformatic prediction showed that human KIAA0100 gene was located on 17q11.2, and human KIAA0100 protein was located in the secretory pathway. Besides, human KIAA0100 protein contained a signalpeptide, a transmembrane region, three types of secondary structures (alpha helix, extended strand, and random coil , and four domains from mitochondrial protein 27 (FMP27. The observation on functional characterization of human KIAA0100 gene revealed that its downregulation inhibited cell proliferation, and promoted cell apoptosis in U937 cells. To summarize, these results suggest human KIAA0100 gene possibly comes within mitochondrial genome; moreover, it is a novel anti-apoptotic factor related to carcinogenesis or progression in acute monocytic leukemia, and may be a potential target for immunotherapy against acute monocytic leukemia.

  13. Identification and characterization of human GUKH2 gene in silico.

    Science.gov (United States)

    Katoh, Masuko; Katoh, Masaru

    2004-04-01

    Drosophila Guanylate-kinase holder (Gukh) is an adaptor molecule bridging Discs large (Dlg) and Scribble (Scrib), which are implicated in the establishment and maintenance of epithelial polarity. Here, we searched for human homologs of Drosophila gukh by using bioinformatics, and identified GUKH1 and GUKH2 genes. GUKH1 was identical to Nance-Horan syndrome (NHS) gene, while GUKH2 was a novel gene. FLJ35425 (AK092744.1), DKFZp686P1949 (BX647246.1) and KIAA1357 (AB037778.1) cDNAs were derived from human GUKH2 gene. Nucleotide sequence of GUKH2 cDNA was determined by assembling 5'-part of FLJ35425 cDNA and entire region of DKFZp686P1949 cDNA. Human GUKH2 gene consists of 8 exons. Exon 5 (132 bp) of GUKH2 gene was spliced out in GUKH2 cDNA due to alternative splicing. GUKH2-REPS1 locus at human chromosome 6q24.1 and GUKH1-REPS2 locus at human chromosome Xp22.22-p22.13 are paralogous regions within the human genome. Mouse Gukh2 and zebrafish gukh2 genes were also identified. N-terminal part of human GUKH2, mouse Gukh2 and zebrafish gukh2 proteins were completely divergent from human GUKH1 protein. Human GUKH2 and GUKH1, consisting of eight GUKH homology (GKH1-GKH8) domains and Proline-rich domain, showed 28.5% total-amino-acid identity. GKH1, GKH4, GKH5, GKH7 and GKH8 domains were conserved among human GUKH1, human GUKH2 and Drosophila Gukh. Because human homologs of Drosophila dlg (DLG1-DLG7) as well as human homologs of Drosophila scrib (SCRIB, ERBB2IP and Densin-180) are cancer-associated genes, human homologs of Drosophila gukh (GUKH1 and GUKH2) are predicted cancer-associated genes.

  14. ICAM-1 expression on chondrocytes in rheumatoid arthritis: induction by synovial cytokines

    Directory of Open Access Journals (Sweden)

    M. E. Davies

    1992-01-01

    Full Text Available The intercellular adhesion molecule-1 (ICAM-1 was found by immunostaining chondrocytes in cartilage from three patients with rheumatoid arthritis. Expression of ICAM-1 was restricted to chondrocytes in areas of erodedcartilage adjacent to the invading synovial tissue. Toluidine blue staining of these areas demonstrated severe depletion of the cartilage extracellular matrix. In areas of undamaged cartilage there was no ICAM-1 expression. Since ICAM-1 is not constitutively expressed on normal human articular cartilage, but could be induced in vitro by exogenous IL-1α, TNFα and IFNγ or by co-culturing cartilage with inflammatory rheumatoid synovium, we conclude that the induction of ICAM-1 on rheumatoid chondrocytes results from the synergistic action of a variety of cytokines produced by the inflammatory cells of the invading pannus.

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

    International Nuclear Information System (INIS)

    Miyatake, Kazumasa; Tsuji, Kunikazu; Yamaga, Mika; Yamada, Jun; Matsukura, Yu; Abula, Kahaer; Sekiya, Ichiro; Muneta, Takeshi

    2013-01-01

    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

  16. 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.

  17. Electroporation-mediated transfer of SOX trio genes (SOX-5, SOX-6, and SOX-9) to enhance the chondrogenesis of mesenchymal stem cells.

    Science.gov (United States)

    Kim, Hye-Joung; Im, Gun-Il

    2011-12-01

    The purpose of this study was to test the hypothesis that the SOX trio genes (SOX-5, SOX-6, and SOX-9) have a lower level of expression during the chondrogenic differentiation of mesenchymal stem cells (MSCs) compared with chondrocytes and that the electroporation-mediated gene transfer of SOX trio promotes chondrogenesis from human MSCs. An in vitro pellet culture was carried out using MSCs or chondrocytes at passage 3 and analyzed after 7 and 21 days. Then, MSCs were transfected with SOX trio genes and analyzed for the expression of chondrogenic markers after 21 days of in vitro culture. Without transforming growth factor-β1, the untransfected MSCs had a lower level of SOX trio gene and protein expression than chondrocytes. However, the level of SOX-9 gene expression increased in MSCs when treated with transforming growth factor-β1. GAG level significantly increased 7-fold in MSCs co-transfected with SOX trio, which was corroborated by Safranin-O staining. SOX trio co-transfection significantly increased COL2A1 gene and protein and decreased COL10A1 protein in MSCs. It is concluded that the SOX trio have a significantly lower expression in human MSCs than in chondrocytes and that the electroporation-mediated co-transfection of SOX trio enhances chondrogenesis and suppresses hypertrophy of human MSCs.

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

    DEFF Research Database (Denmark)

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

    2010-01-01

    PURPOSE: We report the 2-year clinical results and identify prognostic factors in patients treated with autologous chondrocyte transplantation by use of a collagen membrane to seed the chondrocytes (ACT-CS). METHODS: This is a prospective study of 59 patients who were treated with ACT......-CS represents a technical modification of membrane-associated autologous chondrocyte transplantation that combines easy handling and attractive application properties with reliable clinical results 24 months after surgery, especially in patients with isolated cartilage defects. Even though the failure rate...

  19. In-silico human genomics with GeneCards

    Directory of Open Access Journals (Sweden)

    Stelzer Gil

    2011-10-01

    Full Text Available Abstract Since 1998, the bioinformatics, systems biology, genomics and medical communities have enjoyed a synergistic relationship with the GeneCards database of human genes (http://www.genecards.org. This human gene compendium was created to help to introduce order into the increasing chaos of information flow. As a consequence of viewing details and deep links related to specific genes, users have often requested enhanced capabilities, such that, over time, GeneCards has blossomed into a suite of tools (including GeneDecks, GeneALaCart, GeneLoc, GeneNote and GeneAnnot for a variety of analyses of both single human genes and sets thereof. In this paper, we focus on inhouse and external research activities which have been enabled, enhanced, complemented and, in some cases, motivated by GeneCards. In turn, such interactions have often inspired and propelled improvements in GeneCards. We describe here the evolution and architecture of this project, including examples of synergistic applications in diverse areas such as synthetic lethality in cancer, the annotation of genetic variations in disease, omics integration in a systems biology approach to kidney disease, and bioinformatics tools.

  20. Autologous Chondrocyte Implantation in Osteoarthritic Surroundings

    DEFF Research Database (Denmark)

    Ossendorff, Robert; Grad, Sibylle; Stoddart, Martin J

    2018-01-01

    BACKGROUND: Autologous chondrocyte implantation (ACI) fails in up to 20% of cases. Advanced intra-articular degeneration paired with an inflammatory environment may be closely related to implantation failure. Certain cytokines have been identified to play a major role during early osteoarthritis....... PURPOSE: To investigate the effects of tumor necrosis factor α (TNFα) and its potential inhibition by adalimumab on cartilage regeneration in an in vitro model of ACI. STUDY DESIGN: Controlled laboratory study. METHODS: Bovine articular chondrocytes were cultivated and transferred at passage 3 to fibrin...

  1. Human reporter genes: potential use in clinical studies

    Energy Technology Data Exchange (ETDEWEB)

    Serganova, Inna [Department of Neurology, Memorial Sloan-Kettering Cancer Center, New York, NY 10021 (United States); Ponomarev, Vladimir [Department of Radiology, Memorial Sloan-Kettering Cancer Center, New York, NY 10021 (United States); Blasberg, Ronald [Department of Neurology, Memorial Sloan-Kettering Cancer Center, New York, NY 10021 (United States); Department of Radiology, Memorial Sloan-Kettering Cancer Center, New York, NY 10021 (United States)], E-mail: blasberg@neuro1.mskcc.org

    2007-10-15

    The clinical application of positron-emission-tomography-based reporter gene imaging will expand over the next several years. The translation of reporter gene imaging technology into clinical applications is the focus of this review, with emphasis on the development and use of human reporter genes. Human reporter genes will play an increasingly more important role in this development, and it is likely that one or more reporter systems (human gene and complimentary radiopharmaceutical) will take leading roles. Three classes of human reporter genes are discussed and compared: receptors, transporters and enzymes. Examples of highly expressed cell membrane receptors include specific membrane somatostatin receptors (hSSTrs). The transporter group includes the sodium iodide symporter (hNIS) and the norepinephrine transporter (hNET). The endogenous enzyme classification includes human mitochondrial thymidine kinase 2 (hTK2). In addition, we also discuss the nonhuman dopamine 2 receptor and two viral reporter genes, the wild-type herpes simplex virus 1 thymidine kinase (HSV1-tk) gene and the HSV1-tk mutant (HSV1-sr39tk). Initial applications of reporter gene imaging in patients will be developed within two different clinical disciplines: (a) gene therapy and (b) adoptive cell-based therapies. These studies will benefit from the availability of efficient human reporter systems that can provide critical monitoring information for adenoviral-based, retroviral-based and lenteviral-based gene therapies, oncolytic bacterial and viral therapies, and adoptive cell-based therapies. Translational applications of noninvasive in vivo reporter gene imaging are likely to include: (a) quantitative monitoring of gene therapy vectors for targeting and transduction efficacy in clinical protocols by imaging the location, extent and duration of transgene expression; (b) monitoring of cell trafficking, targeting, replication and activation in adoptive T-cell and stem/progenitor cell therapies

  2. Human reporter genes: potential use in clinical studies

    International Nuclear Information System (INIS)

    Serganova, Inna; Ponomarev, Vladimir; Blasberg, Ronald

    2007-01-01

    The clinical application of positron-emission-tomography-based reporter gene imaging will expand over the next several years. The translation of reporter gene imaging technology into clinical applications is the focus of this review, with emphasis on the development and use of human reporter genes. Human reporter genes will play an increasingly more important role in this development, and it is likely that one or more reporter systems (human gene and complimentary radiopharmaceutical) will take leading roles. Three classes of human reporter genes are discussed and compared: receptors, transporters and enzymes. Examples of highly expressed cell membrane receptors include specific membrane somatostatin receptors (hSSTrs). The transporter group includes the sodium iodide symporter (hNIS) and the norepinephrine transporter (hNET). The endogenous enzyme classification includes human mitochondrial thymidine kinase 2 (hTK2). In addition, we also discuss the nonhuman dopamine 2 receptor and two viral reporter genes, the wild-type herpes simplex virus 1 thymidine kinase (HSV1-tk) gene and the HSV1-tk mutant (HSV1-sr39tk). Initial applications of reporter gene imaging in patients will be developed within two different clinical disciplines: (a) gene therapy and (b) adoptive cell-based therapies. These studies will benefit from the availability of efficient human reporter systems that can provide critical monitoring information for adenoviral-based, retroviral-based and lenteviral-based gene therapies, oncolytic bacterial and viral therapies, and adoptive cell-based therapies. Translational applications of noninvasive in vivo reporter gene imaging are likely to include: (a) quantitative monitoring of gene therapy vectors for targeting and transduction efficacy in clinical protocols by imaging the location, extent and duration of transgene expression; (b) monitoring of cell trafficking, targeting, replication and activation in adoptive T-cell and stem/progenitor cell therapies

  3. Determinants of human adipose tissue gene expression

    DEFF Research Database (Denmark)

    Viguerie, Nathalie; Montastier, Emilie; Maoret, Jean-José

    2012-01-01

    weight maintenance diets. For 175 genes, opposite regulation was observed during calorie restriction and weight maintenance phases, independently of variations in body weight. Metabolism and immunity genes showed inverse profiles. During the dietary intervention, network-based analyses revealed strong...... interconnection between expression of genes involved in de novo lipogenesis and components of the metabolic syndrome. Sex had a marked influence on AT expression of 88 transcripts, which persisted during the entire dietary intervention and after control for fat mass. In women, the influence of body mass index...... on expression of a subset of genes persisted during the dietary intervention. Twenty-two genes revealed a metabolic syndrome signature common to men and women. Genetic control of AT gene expression by cis signals was observed for 46 genes. Dietary intervention, sex, and cis genetic variants independently...

  4. 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.

  5. HIF-1α-induced HSP70 regulates anabolic responses in articular chondrocytes under hypoxic conditions.

    Science.gov (United States)

    Tsuchida, Shinji; Arai, Yuji; Takahashi, Kenji A; Kishida, Tsunao; Terauchi, Ryu; Honjo, Kuniaki; Nakagawa, Shuji; Inoue, Hiroaki; Ikoma, Kazuya; Ueshima, Keiichiro; Matsuki, Tomohiro; Mazda, Osam; Kubo, Toshikazu

    2014-08-01

    We assessed whether heat shock protein 70 (HSP70) is involved in hypoxia inducible factor 1 alpha (HIF-1α)-dependent anabolic pathways in articular chondrocytes under hypoxic conditions. Primary rabbit chondrocytes were cultured under normoxia (20% oxygen condition) or hypoxia (1% oxygen condition). Alternatively, cells cultured under normoxia were treated with CoCl2 , which induces HIF-1α, to simulate hypoxia, or transfected with siRNAs targeting HIF-1α (si-HIF-1α) and HSP70 (si-HSP70) under hypoxia. HSP70 expression was enhanced by the increased expression of HIF-1α under hypoxia or simulated hypoxia, but not in the presence of si-HIF-1α. Hypoxia-induced overexpression of ECM genes was significantly suppressed by si-HIF-1α or si-HSP70. Cell viability positively correlated with hypoxia, but transfection with si-HIF-1α or si-HSP70 abrogated the chondroprotective effects of hypoxia. Although LDH release from sodium nitroprusside-treated cells and the proportion of TUNEL positive cells were decreased under hypoxia, transfection with si-HIF-1α or si-HSP70 almost completely blocked these effects. These findings indicated that HIF-1α-induced HSP70 overexpression increased the expression levels of ECM genes and cell viability, and protected chondrocytes from apoptosis. HIF-1α may regulate the anabolic effects of chondrocytes under hypoxic conditions by regulating HSP70 expression. © 2014 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.

  6. 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

  7. Targeting the human lysozyme gene on bovine αs1- casein gene ...

    African Journals Online (AJOL)

    ajl yemi

    2011-11-28

    Nov 28, 2011 ... Targeting an exogenous gene into a favorable gene locus and for expression under endogenous regulators is ... case, the expression of human lysozyme could be regulated by the endogenous cis-element of αs1- casein gene in .... Mouse mammary epithelial C127 cells (Cell Bank, Chinese. Academy of ...

  8. Reduced primary cilia length and altered Arl13b expression are associated with deregulated chondrocyte Hedgehog signaling in alkaptonuria.

    Science.gov (United States)

    Thorpe, Stephen D; Gambassi, Silvia; Thompson, Clare L; Chandrakumar, Charmilie; Santucci, Annalisa; Knight, Martin M

    2017-09-01

    Alkaptonuria (AKU) is a rare inherited disease resulting from a deficiency of the enzyme homogentisate 1,2-dioxygenase which leads to the accumulation of homogentisic acid (HGA). AKU is characterized by severe cartilage degeneration, similar to that observed in osteoarthritis. Previous studies suggest that AKU is associated with alterations in cytoskeletal organization which could modulate primary cilia structure/function. This study investigated whether AKU is associated with changes in chondrocyte primary cilia and associated Hedgehog signaling which mediates cartilage degradation in osteoarthritis. Human articular chondrocytes were obtained from healthy and AKU donors. Additionally, healthy chondrocytes were treated with HGA to replicate AKU pathology (+HGA). Diseased cells exhibited shorter cilia with length reductions of 36% and 16% in AKU and +HGA chondrocytes respectively, when compared to healthy controls. Both AKU and +HGA chondrocytes demonstrated disruption of the usual cilia length regulation by actin contractility. Furthermore, the proportion of cilia with axoneme breaks and bulbous tips was increased in AKU chondrocytes consistent with defective regulation of ciliary trafficking. Distribution of the Hedgehog-related protein Arl13b along the ciliary axoneme was altered such that its localization was increased at the distal tip in AKU and +HGA chondrocytes. These changes in cilia structure/trafficking in AKU and +HGA chondrocytes were associated with a complete inability to activate Hedgehog signaling in response to exogenous ligand. Thus, we suggest that altered responsiveness to Hedgehog, as a consequence of cilia dysfunction, may be a contributing factor in the development of arthropathy highlighting the cilium as a novel target in AKU. © 2017 The Authors. Journal of Cellular Physiology Published by Wiley Periodicals Inc.

  9. Human gene therapy: novel approaches to improve the current gene delivery systems.

    Science.gov (United States)

    Cucchiarini, Magali

    2016-06-01

    Even though gene therapy made its way through the clinics to treat a number of human pathologies since the early years of experimental research and despite the recent approval of the first gene-based product (Glybera) in Europe, the safe and effective use of gene transfer vectors remains a challenge in human gene therapy due to the existence of barriers in the host organism. While work is under active investigation to improve the gene transfer systems themselves, the use of controlled release approaches may offer alternative, convenient tools of vector delivery to achieve a performant gene transfer in vivo while overcoming the various physiological barriers that preclude its wide use in patients. This article provides an overview of the most significant contributions showing how the principles of controlled release strategies may be adapted for human gene therapy.

  10. 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.

    2016-01-01

    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 observed in situ in human cartilage sections from older adults and in osteoarthritic cartilage. MCAT transgenic mice exhibited less age-related osteoarthritis. These findings demonstrate that age-related oxidative stress can disrupt normal physiological signaling and contribute to osteoarthritis and suggest peroxiredoxin hyperoxidation as a potential mechanism. PMID:26797130

  11. 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.

  12. Different level of population differentiation among human genes

    Directory of Open Access Journals (Sweden)

    Zhang Ya-Ping

    2011-01-01

    Full Text Available Abstract Background During the colonization of the world, after dispersal out of African, modern humans encountered changeable environments and substantial phenotypic variations that involve diverse behaviors, lifestyles and cultures, were generated among the different modern human populations. Results Here, we study the level of population differentiation among different populations of human genes. Intriguingly, genes involved in osteoblast development were identified as being enriched with higher FST SNPs, a result consistent with the proposed role of the skeletal system in accounting for variation among human populations. Genes involved in the development of hair follicles, where hair is produced, were also found to have higher levels of population differentiation, consistent with hair morphology being a distinctive trait among human populations. Other genes that showed higher levels of population differentiation include those involved in pigmentation, spermatid, nervous system and organ development, and some metabolic pathways, but few involved with the immune system. Disease-related genes demonstrate excessive SNPs with lower levels of population differentiation, probably due to purifying selection. Surprisingly, we find that Mendelian-disease genes appear to have a significant excessive of SNPs with high levels of population differentiation, possibly because the incidence and susceptibility of these diseases show differences among populations. As expected, microRNA regulated genes show lower levels of population differentiation due to purifying selection. Conclusion Our analysis demonstrates different level of population differentiation among human populations for different gene groups.

  13. Different level of population differentiation among human genes.

    Science.gov (United States)

    Wu, Dong-Dong; Zhang, Ya-Ping

    2011-01-14

    During the colonization of the world, after dispersal out of African, modern humans encountered changeable environments and substantial phenotypic variations that involve diverse behaviors, lifestyles and cultures, were generated among the different modern human populations. Here, we study the level of population differentiation among different populations of human genes. Intriguingly, genes involved in osteoblast development were identified as being enriched with higher FST SNPs, a result consistent with the proposed role of the skeletal system in accounting for variation among human populations. Genes involved in the development of hair follicles, where hair is produced, were also found to have higher levels of population differentiation, consistent with hair morphology being a distinctive trait among human populations. Other genes that showed higher levels of population differentiation include those involved in pigmentation, spermatid, nervous system and organ development, and some metabolic pathways, but few involved with the immune system. Disease-related genes demonstrate excessive SNPs with lower levels of population differentiation, probably due to purifying selection. Surprisingly, we find that Mendelian-disease genes appear to have a significant excessive of SNPs with high levels of population differentiation, possibly because the incidence and susceptibility of these diseases show differences among populations. As expected, microRNA regulated genes show lower levels of population differentiation due to purifying selection. Our analysis demonstrates different level of population differentiation among human populations for different gene groups.

  14. 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.

  15. Cartilage repair: Generations of autologous chondrocyte transplantation

    International Nuclear Information System (INIS)

    Marlovits, Stefan; Zeller, Philip; Singer, Philipp; Resinger, Christoph; Vecsei, Vilmos

    2006-01-01

    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

  16. 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.

  17. De novo origin of human protein-coding genes.

    Directory of Open Access Journals (Sweden)

    Dong-Dong Wu

    2011-11-01

    Full Text Available The de novo origin of a new protein-coding gene from non-coding DNA is considered to be a very rare occurrence in genomes. Here we identify 60 new protein-coding genes that originated de novo on the human lineage since divergence from the chimpanzee. The functionality of these genes is supported by both transcriptional and proteomic evidence. RNA-seq data indicate that these genes have their highest expression levels in the cerebral cortex and testes, which might suggest that these genes contribute to phenotypic traits that are unique to humans, such as improved cognitive ability. Our results are inconsistent with the traditional view that the de novo origin of new genes is very rare, thus there should be greater appreciation of the importance of the de novo origination of genes.

  18. Structure and chromosomal localization of the human renal kallikrein gene

    International Nuclear Information System (INIS)

    Evans, B.A.; Yun, Z.X.; Close, J.A.

    1988-01-01

    Glandular kallikreins are a family of proteases encoded by a variable number of genes in different mammalian species. In all species examined, however, one particular kallikrein is functionally conserved in its capacity to release the vasoactive peptide, Lys-bradykinin, from low molecular weight kininogen. This kallikrein is found in the kidney, pancreas, and salivary gland, showing a unique pattern of tissue-specific expression relative to other members of the family. The authors have isolated a genomic clone carrying the human renal kallikrein gene and compared the nucleotide sequence of its promoter region with those of the mouse renal kallikrein gene and another mouse kallikrein gene expressed in a distinct cell type. They find four sequence elements conserved between renal kallikrein genes from the two species. They have also shown that the human gene is localized to 19q13, a position analogous to that of the kallikrein gene family on mouse chromosome 7

  19. De Novo Origin of Human Protein-Coding Genes

    Science.gov (United States)

    Wu, Dong-Dong; Irwin, David M.; Zhang, Ya-Ping

    2011-01-01

    The de novo origin of a new protein-coding gene from non-coding DNA is considered to be a very rare occurrence in genomes. Here we identify 60 new protein-coding genes that originated de novo on the human lineage since divergence from the chimpanzee. The functionality of these genes is supported by both transcriptional and proteomic evidence. RNA–seq data indicate that these genes have their highest expression levels in the cerebral cortex and testes, which might suggest that these genes contribute to phenotypic traits that are unique to humans, such as improved cognitive ability. Our results are inconsistent with the traditional view that the de novo origin of new genes is very rare, thus there should be greater appreciation of the importance of the de novo origination of genes. PMID:22102831

  20. Chromosomal localization of the human and mouse hyaluronan synthase genes

    Energy Technology Data Exchange (ETDEWEB)

    Spicer, A.P.; McDonald, J.A. [Mayo Clinic Scottsdale, AZ (United States); Seldin, M.F. [Univ. of California Davis, CA (United States)] [and others

    1997-05-01

    We have recently identified a new vertebrate gene family encoding putative hyaluronan (HA) synthases. Three highly conserved related genes have been identified, designated HAS1, HAS2, and HAS3 in humans and Has1, Has2, and Has3 in the mouse. All three genes encode predicted plasma membrane proteins with multiple transmembrane domains and approximately 25% amino acid sequence identity to the Streptococcus pyogenes HA synthase, HasA. Furthermore, expression of any one HAS gene in transfected mammalian cells leads to high levels of HA biosynthesis. We now report the chromosomal localization of the three HAS genes in human and in mouse. The genes localized to three different positions within both the human and the mouse genomes. HAS1 was localized to the human chromosome 19q13.3-q13.4 boundary and Has1 to mouse Chr 17. HAS2 was localized to human chromosome 8q24.12 and Has2 to mouse Chr 15. HAS3 was localized to human chromosome 16q22.1 and Has3 to mouse Chr 8. The map position for HAS1 reinforces the recently reported relationship between a small region of human chromosome 19q and proximal mouse chromosome 17. HAS2 mapped outside the predicted critical region delineated for the Langer-Giedion syndrome and can thus be excluded as a candidate gene for this genetic syndrome. 33 refs., 2 figs.

  1. Identification and validation of suitable endogenous reference genes for gene expression studies in human peripheral blood

    Directory of Open Access Journals (Sweden)

    Turner Renee J

    2009-08-01

    Full Text Available Abstract Background Gene expression studies require appropriate normalization methods. One such method uses stably expressed reference genes. Since suitable reference genes appear to be unique for each tissue, we have identified an optimal set of the most stably expressed genes in human blood that can be used for normalization. Methods Whole-genome Affymetrix Human 2.0 Plus arrays were examined from 526 samples of males and females ages 2 to 78, including control subjects and patients with Tourette syndrome, stroke, migraine, muscular dystrophy, and autism. The top 100 most stably expressed genes with a broad range of expression levels were identified. To validate the best candidate genes, we performed quantitative RT-PCR on a subset of 10 genes (TRAP1, DECR1, FPGS, FARP1, MAPRE2, PEX16, GINS2, CRY2, CSNK1G2 and A4GALT, 4 commonly employed reference genes (GAPDH, ACTB, B2M and HMBS and PPIB, previously reported to be stably expressed in blood. Expression stability and ranking analysis were performed using GeNorm and NormFinder algorithms. Results Reference genes were ranked based on their expression stability and the minimum number of genes needed for nomalization as calculated using GeNorm showed that the fewest, most stably expressed genes needed for acurate normalization in RNA expression studies of human whole blood is a combination of TRAP1, FPGS, DECR1 and PPIB. We confirmed the ranking of the best candidate control genes by using an alternative algorithm (NormFinder. Conclusion The reference genes identified in this study are stably expressed in whole blood of humans of both genders with multiple disease conditions and ages 2 to 78. Importantly, they also have different functions within cells and thus should be expressed independently of each other. These genes should be useful as normalization genes for microarray and RT-PCR whole blood studies of human physiology, metabolism and disease.

  2. Chromosomal localization of the human diazepam binding inhibitor gene

    International Nuclear Information System (INIS)

    DeBernardi, M.A.; Crowe, R.R.; Mocchetti, I.; Shows, T.B.; Eddy, R.L.; Costa, E.

    1988-01-01

    The authors have used in situ chromosome hybridization and human-mouse somatic cell hybrids to map the gene(s) for human diazepam binding inhibitor (DBI), an endogenous putative modulator of the γ-aminobutyric acid receptor acting at the allosteric regulatory center of this receptor that includes the benzodiazepine recognition site. In 784 chromosome spreads hybridized with human DBI cDNA, the distribution of 1,476 labeled sites revealed a significant clustering of autoradiographic grains (11.3% of total label) on the long arm of chromosome 2 (2q). Furthermore, 63.5% of the grains found on 2q were located on 2q12-21, suggesting regional mapping of DBI gene(s) to this segment. Secondary hybridization signals were frequently observed on other chromosomes and they were statistically significant mainly for chromosomes 5, 6, 11, and 14. In addition, DNA from 32 human-mouse cell hybrids was digested with BamHI and probed with human DBI cDNA. A 3.5-kilobase band, which probably represents the human DBI gene, was assigned to chromosome 2. Four higher molecular weight bands, also detected in BamHI digests, could not be unequivocally assigned. A chromosome 2 location was excluded for the 27-, 13-, and 10-kilobase bands. These results assign a human DBI gene to chromosome 2 (2q12-21) and indicate that three of the four homologous sequences detected by the human DBI probe are located on three other chromosomes

  3. Protective Effect of Ginger (Zingiber officinale Roscoe) Extract against Oxidative Stress and Mitochondrial Apoptosis Induced by Interleukin-1β in Cultured Chondrocytes.

    Science.gov (United States)

    Hosseinzadeh, Azam; Bahrampour Juybari, Kobra; Fatemi, Mohammad Javad; Kamarul, Tunku; Bagheri, Aboulfazl; Tekiyehmaroof, Neda; Sharifi, Ali Mohammad

    2017-01-01

    The protective effects of ginger (Zingiber officinale Roscoe) extract on IL-1β-mediated oxidative stress and mitochondrial apoptosis were investigated in C28I2 human chondrocytes. The effects of various concentrations of ginger extract on C28I2 human chondrocyte viability were evaluated in order to obtain noncytotoxic concentrations of the drug by methylthiotetrazole assay. The cells were pretreated with 5 and 25 μg/mL ginger extract for 24 h, followed by incubation with IL-1β (10 ng/mL) for 24 h. The effects of ginger extract on IL-1β-induced intracellular reactive oxygen species (ROS) production and lipid peroxidation were examined. The mRNA expressions of antioxidant enzymes including catalase, superoxide dismutase-1, glutathione peroxidase-1, glutathione peroxidase-3, and glutathione peroxidase-4 were evaluated by reverse transcription polymerase chain reaction. The protein expressions of Bax, Bcl-2, and caspase-3 were analyzed by Western blotting. No cytotoxicity was observed at any concentration of ginger extract in C28I2 cells. Ginger extract pretreatment remarkably increased the gene expression of antioxidant enzymes and reduced the IL-1β-induced elevation of ROS, lipid peroxidation, the Bax/Bcl-2 ratio, and caspase-3 activity. Ginger extract could considerably reduce IL-1β-induced oxidative stress and consequent mitochondrial apoptosis as the major mechanisms of chondrocyte cell death. These beneficial effects of ginger extract may be due to its antioxidant properties. It may be considered as a natural herbal product to prevent OA-induced cartilage destruction in the clinical setting. © 2017 S. Karger AG, Basel.

  4. Effect of Cyclic Dynamic Compressive Loading on Chondrocytes and Adipose-Derived Stem Cells Co-Cultured in Highly Elastic Cryogel Scaffolds

    Directory of Open Access Journals (Sweden)

    Chih-Hao Chen

    2018-01-01

    Full Text Available In this study, we first used gelatin/chondroitin-6-sulfate/hyaluronan/chitosan highly elastic cryogels, which showed total recovery from large strains during repeated compression cycles, as 3D scaffolds to study the effects of cyclic dynamic compressive loading on chondrocyte gene expression and extracellular matrix (ECM production. Dynamic culture of porcine chondrocytes was studied at 1 Hz, 10% to 40% strain and 1 to 9 h/day stimulation duration, in a mechanical-driven multi-chamber bioreactor for 14 days. From the experimental results, we could identify the optimum dynamic culture condition (20% and 3 h/day to enhance the chondrocytic phenotype of chondrocytes from the expression of marker (Col I, Col II, Col X, TNF-α, TGF-β1 and IGF-1 genes by quantitative real-time polymerase chain reactions (qRT-PCR and production of ECM (GAGs and Col II by biochemical analysis and immunofluorescence staining. With up-regulated growth factor (TGF-β1 and IGF-1 genes, co-culture of chondrocytes with porcine adipose-derived stem cells (ASCs was employed to facilitate chondrogenic differentiation of ASCs during dynamic culture in cryogel scaffolds. By replacing half of the chondrocytes with ASCs during co-culture, we could obtain similar production of ECM (GAGs and Col II and expression of Col II, but reduced expression of Col I, Col X and TNF-α. Subcutaneous implantation of cells/scaffold constructs in nude mice after mono-culture (chondrocytes or ASCs or co-culture (chondrocytes + ASCs and subject to static or dynamic culture condition in vitro for 14 days was tested for tissue-engineering applications. The constructs were retrieved 8 weeks post-implantation for histological analysis by Alcian blue, Safranin O and Col II immunohistochemical staining. The most abundant ectopic cartilage tissue was found for the chondrocytes and chondrocytes + ASCs groups using dynamic culture, which showed similar neo-cartilage formation capability with half of the

  5. Identification of Human HK Genes and Gene Expression Regulation Study in Cancer from Transcriptomics Data Analysis

    Science.gov (United States)

    Zhang, Zhang; Liu, Jingxing; Wu, Jiayan; Yu, Jun

    2013-01-01

    The regulation of gene expression is essential for eukaryotes, as it drives the processes of cellular differentiation and morphogenesis, leading to the creation of different cell types in multicellular organisms. RNA-Sequencing (RNA-Seq) provides researchers with a powerful toolbox for characterization and quantification of transcriptome. Many different human tissue/cell transcriptome datasets coming from RNA-Seq technology are available on public data resource. The fundamental issue here is how to develop an effective analysis method to estimate expression pattern similarities between different tumor tissues and their corresponding normal tissues. We define the gene expression pattern from three directions: 1) expression breadth, which reflects gene expression on/off status, and mainly concerns ubiquitously expressed genes; 2) low/high or constant/variable expression genes, based on gene expression level and variation; and 3) the regulation of gene expression at the gene structure level. The cluster analysis indicates that gene expression pattern is higher related to physiological condition rather than tissue spatial distance. Two sets of human housekeeping (HK) genes are defined according to cell/tissue types, respectively. To characterize the gene expression pattern in gene expression level and variation, we firstly apply improved K-means algorithm and a gene expression variance model. We find that cancer-associated HK genes (a HK gene is specific in cancer group, while not in normal group) are expressed higher and more variable in cancer condition than in normal condition. Cancer-associated HK genes prefer to AT-rich genes, and they are enriched in cell cycle regulation related functions and constitute some cancer signatures. The expression of large genes is also avoided in cancer group. These studies will help us understand which cell type-specific patterns of gene expression differ among different cell types, and particularly for cancer. PMID:23382867

  6. The mechanism of gene targeting in human somatic cells.

    Directory of Open Access Journals (Sweden)

    Yinan Kan

    2014-04-01

    Full Text Available Gene targeting in human somatic cells is of importance because it can be used to either delineate the loss-of-function phenotype of a gene or correct a mutated gene back to wild-type. Both of these outcomes require a form of DNA double-strand break (DSB repair known as homologous recombination (HR. The mechanism of HR leading to gene targeting, however, is not well understood in human cells. Here, we demonstrate that a two-end, ends-out HR intermediate is valid for human gene targeting. Furthermore, the resolution step of this intermediate occurs via the classic DSB repair model of HR while synthesis-dependent strand annealing and Holliday Junction dissolution are, at best, minor pathways. Moreover, and in contrast to other systems, the positions of Holliday Junction resolution are evenly distributed along the homology arms of the targeting vector. Most unexpectedly, we demonstrate that when a meganuclease is used to introduce a chromosomal DSB to augment gene targeting, the mechanism of gene targeting is inverted to an ends-in process. Finally, we demonstrate that the anti-recombination activity of mismatch repair is a significant impediment to gene targeting. These observations significantly advance our understanding of HR and gene targeting in human cells.

  7. Radioactive probes for human gene localisation by in situ hybridisation

    International Nuclear Information System (INIS)

    Fennell, S.J.

    1980-07-01

    Radioactive probes of high specific activity have been used for human gene localisation on metaphase chromosome preparations. Human 5S ribosomal RNA was used as a model system, as a probe for the localisation of human 5S ribosomal genes. 125 I-labelled mouse 5S ribosomal RNA was used to study the 5S ribosomal gene content and arrangement in families with translocations on the long arm of chromosome 1 close to or containing the 5S ribosomal RNA locus, by in situ hybridisation to human metaphase chromosomes from peripheral blood cultures. This confirmed the chromosomal assignment of 5S ribosomal genes to 1q 42-43. In situ hybridisation probes were also prepared from recombinant plasmids containing Xenopus laevis oocyte 5S or 28S/18S gene sequences to give [ 3 H]-labelled cRNA and [ 3 H]-labelled nick-translated plasmid DNA. Studies on the kinetics of hybridisation of plasmid probes with and without ribosomal gene sequences questioned the role of plasmid DNA for amplification of signal during gene localisation. Gene localisation was obtained with nick-translated plasmid DNA containing the 28S/18S ribosomal DNA insert after short exposure times, but poor results were obtained using a [ 3 H]-labelled cRNA probe transcribed from the plasmid with the 5S gene insert. (author)

  8. 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

  9. Exertional Heat Illness and Human Gene Expression

    National Research Council Canada - National Science Library

    Sonna, L.A; Sawka, M. N; Lilly, C. M

    2007-01-01

    Microarray analysis of gene expression at the level of RNA has generated new insights into the relationship between cellular responses to acute heat shock in vitro, exercise, and exertional heat illness...

  10. More than 9,000,000 unique genes in human gut bacterial community: estimating gene numbers inside a human body.

    Science.gov (United States)

    Yang, Xing; Xie, Lu; Li, Yixue; Wei, Chaochun

    2009-06-29

    Estimating the number of genes in human genome has been long an important problem in computational biology. With the new conception of considering human as a super-organism, it is also interesting to estimate the number of genes in this human super-organism. We presented our estimation of gene numbers in the human gut bacterial community, the largest microbial community inside the human super-organism. We got 552,700 unique genes from 202 complete human gut bacteria genomes. Then, a novel gene counting model was built to check the total number of genes by combining culture-independent sequence data and those complete genomes. 16S rRNAs were used to construct a three-level tree and different counting methods were introduced for the three levels: strain-to-species, species-to-genus, and genus-and-up. The model estimates that the total number of genes is about 9,000,000 after those with identity percentage of 97% or up were merged. By combining completed genomes currently available and culture-independent sequencing data, we built a model to estimate the number of genes in human gut bacterial community. The total number of genes is estimated to be about 9 million. Although this number is huge, we believe it is underestimated. This is an initial step to tackle this gene counting problem for the human super-organism. It will still be an open problem in the near future. The list of genomes used in this paper can be found in the supplementary table.

  11. 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

    2017-11-01

    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.

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

    International Nuclear Information System (INIS)

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

    2009-01-01

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

  13. Dlx5 Is a cell autonomous regulator of chondrocyte hypertrophy in mice and functionally substitutes for Dlx6 during endochondral ossification.

    Directory of Open Access Journals (Sweden)

    Hui Zhu

    Full Text Available The axial and appendicular skeleton of vertebrates develops by endochondral ossification, in which skeletogenic tissue is initially cartilaginous and the differentiation of chondrocytes via the hypertrophic pathway precedes the differentiation of osteoblasts and the deposition of a definitive bone matrix. Results from both loss-of-function and misexpression studies have implicated the related homeobox genes Dlx5 and Dlx6 as partially redundant positive regulators of chondrocyte hypertrophy. However, experimental perturbations of Dlx expression have either not been cell type specific or have been done in the context of endogenous Dlx5 expression. Thus, it has not been possible to conclude whether the effects on chondrocyte differentiation are cell autonomous or whether they are mediated by Dlx expression in adjacent tissues, notably the perichondrium. To address this question we first engineered transgenic mice in which Dlx5 expression was specifically restricted to immature and differentiating chondrocytes and not the perichondrium. Col2a1-Dlx5 transgenic embryos and neonates displayed accelerated chondrocyte hypertrophy and mineralization throughout the endochondral skeleton. Furthermore, this transgene specifically rescued defects of chondrocyte differentiation characteristic of the Dlx5/6 null phenotype. Based on these results, we conclude that the role of Dlx5 in the hypertrophic pathway is cell autonomous. We further conclude that Dlx5 and Dlx6 are functionally equivalent in the endochondral skeleton, in that the requirement for Dlx5 and Dlx6 function during chondrocyte hypertrophy can be satisfied with Dlx5 alone.

  14. 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.

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

    Science.gov (United States)

    Murata, Koichi; Kitaori, Toshiyuki; Oishi, Shinya; Watanabe, Naoki; Yoshitomi, Hiroyuki; Tanida, Shimei; Ishikawa, Masahiro; Kasahara, Takashi; Shibuya, Hideyuki; Fujii, Nobutaka; Nagasawa, Takashi; Nakamura, Takashi; Ito, Hiromu

    2012-01-01

    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.

  16. Production of Recombinant Adenovirus Containing Human Interlukin-4 Gene

    OpenAIRE

    Mojarrad, Majid; Abdolazimi, Yassan; Hajati, Jamshid; Modarressi, Mohammad Hossein

    2011-01-01

    Objective(s) Recombinant adenoviruses are currently used for a variety of purposes, including in vitro gene transfer, in vivo vaccination, and gene therapy. Ability to infect many cell types, high efficiency in gene transfer, entering both dividing and non dividing cells, and growing to high titers make this virus a good choice for using in various experiments. In the present experiment, a recombinant adenovirus containing human IL-4 coding sequence was made. IL-4 has several characteristics ...

  17. Characteristics of functional enrichment and gene expression level of human putative transcriptional target genes.

    Science.gov (United States)

    Osato, Naoki

    2018-01-19

    Transcriptional target genes show functional enrichment of genes. However, how many and how significantly transcriptional target genes include functional enrichments are still unclear. To address these issues, I predicted human transcriptional target genes using open chromatin regions, ChIP-seq data and DNA binding sequences of transcription factors in databases, and examined functional enrichment and gene expression level of putative transcriptional target genes. Gene Ontology annotations showed four times larger numbers of functional enrichments in putative transcriptional target genes than gene expression information alone, independent of transcriptional target genes. To compare the number of functional enrichments of putative transcriptional target genes between cells or search conditions, I normalized the number of functional enrichment by calculating its ratios in the total number of transcriptional target genes. With this analysis, native putative transcriptional target genes showed the largest normalized number of functional enrichments, compared with target genes including 5-60% of randomly selected genes. The normalized number of functional enrichments was changed according to the criteria of enhancer-promoter interactions such as distance from transcriptional start sites and orientation of CTCF-binding sites. Forward-reverse orientation of CTCF-binding sites showed significantly higher normalized number of functional enrichments than the other orientations. Journal papers showed that the top five frequent functional enrichments were related to the cellular functions in the three cell types. The median expression level of transcriptional target genes changed according to the criteria of enhancer-promoter assignments (i.e. interactions) and was correlated with the changes of the normalized number of functional enrichments of transcriptional target genes. Human putative transcriptional target genes showed significant functional enrichments. Functional

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

    Directory of Open Access Journals (Sweden)

    Amilton M Fernandes

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

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

    Science.gov (United States)

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

    2017-01-01

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

  20. Are mice pigmentary genes throwing light on humans?

    Directory of Open Access Journals (Sweden)

    Bose S

    1993-01-01

    Full Text Available In this article the rapid advances made in the molecular genetics of inherited disorders of hypo and hyperpigmentation during the past three years are reviewed. The main focus is on studies in mice as compared to homologues in humans. The main hypomelanotic diseases included are, piebaldism (white spotting due to mutations of c-KIT, PDGF and MGF genes; vitiligo (microphathalmia mice mutations of c-Kit and c-fms genes; Waardenburg syndrome (splotch locus mutations of mice PAX-3 or human Hup-2 genes; albinism (mutations of tyrosinase genes, Menkes disease (Mottled mouse, premature graying (mutations in light/brown locus/gp75/ TRP-1; Griscelli disease (mutations in TRP-1 and steel; Prader-willi and Angelman syndromes, tyrosinase-positive oculocutaneous albinism and hypomelanosis of lto (mutations of pink-eyed dilution gene/mapping to human chromosomes 15 q 11.2 - q12; and human platelet storage pool deficiency diseases due to defects in pallidin, an erythrocyte membrane protein (pallid mouse / mapping to 4.2 pallidin gene. The genetic characterization of hypermelanosis includes, neurofibromatosis 1 (Café-au-lait spots and McCune-Albright Syndrome. Rapid evolving knowledge about pigmentary genes will increase further the knowledge about these hypo and hyperpigmentary disorders.

  1. Translational selection in human: More pronounced in housekeeping genes

    KAUST Repository

    Ma, Lina

    2014-07-10

    Background: Translational selection is a ubiquitous and significant mechanism to regulate protein expression in prokaryotes and unicellular eukaryotes. Recent evidence has shown that translational selection is weakly operative in highly expressed genes in human and other vertebrates. However, it remains unclear whether translational selection acts differentially on human genes depending on their expression patterns.Results: Here we report that human housekeeping (HK) genes that are strictly defined as genes that are expressed ubiquitously and consistently in most or all tissues, are under stronger translational selection.Conclusions: These observations clearly show that translational selection is also closely associated with expression pattern. Our results suggest that human HK genes are more efficiently and/or accurately translated into proteins, which will inevitably open up a new understanding of HK genes and the regulation of gene expression.Reviewers: This article was reviewed by Yuan Yuan, Baylor College of Medicine; Han Liang, University of Texas MD Anderson Cancer Center (nominated by Dr Laura Landweber) Eugene Koonin, NCBI, NLM, NIH, United States of America Sandor Pongor, International Centre for Genetic Engineering and biotechnology (ICGEB), Italy. © 2014 Ma et al.; licensee BioMed Central Ltd.

  2. Cloning and characterization of human DNA repair genes

    International Nuclear Information System (INIS)

    Thompson, L.H.; Brookman, K.W.; Weber, C.A.; Salazar, E.P.; Stewart, S.A.; Carrano, A.V.

    1987-01-01

    The isolation of two addition human genes that give efficient restoration of the repair defects in other CHO mutant lines is reported. The gene designated ERCC2 (Excision Repair Complementing Chinese hamster) corrects mutant UV5 from complementation group 1. They recently cloned this gene by first constructing a secondary transformant in which the human gene was shown to have become physically linked to the bacterial gpt dominant-marker gene by cotransfer in calcium phosphate precipitates in the primary transfection. Transformants expressing both genes were recovered by selecting for resistance to both UV radiation and mycophenolic acid. Using similar methods, the human gene that corrects CHO mutant EM9 was isolated in cosmids and named XRCC1 (X-ray Repair Complementing Chinese hamster). In this case, transformants were recovered by selecting for resistance to CldUrd, which kills EM9 very efficiently. In both genomic and cosmid transformants, the XRCC1 gene restored resistance to the normal range. DNA repair was studied using the kinetics of strand-break rejoining, which was measured after exposure to 137 Cs γ-rays

  3. Identification of DNA repair genes in the human genome

    International Nuclear Information System (INIS)

    Hoeijmakers, J.H.J.; van Duin, M.; Westerveld, A.; Yasui, A.; Bootsma, D.

    1986-01-01

    To identify human DNA repair genes we have transfected human genomic DNA ligated to a dominant marker to excision repair deficient xeroderma pigmentosum (XP) and CHO cells. This resulted in the cloning of a human gene, ERCC-1, that complements the defect of a UV- and mitomycin-C sensitive CHO mutant 43-3B. The ERCC-1 gene has a size of 15 kb, consists of 10 exons and is located in the region 19q13.2-q13.3. Its primary transcript is processed into two mRNAs by alternative splicing of an internal coding exon. One of these transcripts encodes a polypeptide of 297 aminoacids. A putative DNA binding protein domain and nuclear location signal could be identified. Significant AA-homology is found between ERCC-1 and the yeast excision repair gene RAD10. 58 references, 6 figures, 1 table

  4. Promoted Chondrogenesis of Cocultured Chondrocytes and Mesenchymal Stem Cells under Hypoxia Using In-situ Forming Degradable Hydrogel Scaffolds

    NARCIS (Netherlands)

    Huang, Xiaobin; Hou, Yong; Zhong, Leilei; Huang, Dechun; Qian, Hongliang; Karperien, Marcel; Chen, Wei

    2018-01-01

    We investigated the effects of different oxygen tension (21% and 2.5% O2) on the chondrogenesis of different cell systems cultured in pH-degradable PVA hydrogels, including human articular chondrocytes (hACs), human mesenchymal stem cells (hMSCs), and their cocultures with a hAC/hMSC ratio of 20/80.

  5. Automated discovery of functional generality of human gene expression programs.

    Directory of Open Access Journals (Sweden)

    Georg K Gerber

    2007-08-01

    Full Text Available An important research problem in computational biology is the identification of expression programs, sets of co-expressed genes orchestrating normal or pathological processes, and the characterization of the functional breadth of these programs. The use of human expression data compendia for discovery of such programs presents several challenges including cellular inhomogeneity within samples, genetic and environmental variation across samples, uncertainty in the numbers of programs and sample populations, and temporal behavior. We developed GeneProgram, a new unsupervised computational framework based on Hierarchical Dirichlet Processes that addresses each of the above challenges. GeneProgram uses expression data to simultaneously organize tissues into groups and genes into overlapping programs with consistent temporal behavior, to produce maps of expression programs, which are sorted by generality scores that exploit the automatically learned groupings. Using synthetic and real gene expression data, we showed that GeneProgram outperformed several popular expression analysis methods. We applied GeneProgram to a compendium of 62 short time-series gene expression datasets exploring the responses of human cells to infectious agents and immune-modulating molecules. GeneProgram produced a map of 104 expression programs, a substantial number of which were significantly enriched for genes involved in key signaling pathways and/or bound by NF-kappaB transcription factors in genome-wide experiments. Further, GeneProgram discovered expression programs that appear to implicate surprising signaling pathways or receptor types in the response to infection, including Wnt signaling and neurotransmitter receptors. We believe the discovered map of expression programs involved in the response to infection will be useful for guiding future biological experiments; genes from programs with low generality scores might serve as new drug targets that exhibit minimal

  6. Genetic effects on gene expression across human tissues

    NARCIS (Netherlands)

    Battle, Alexis; Brown, Christopher D.; Engelhardt, Barbara E.; Montgomery, Stephen B.; Aguet, François; Ardlie, Kristin G.; Cummings, Beryl B.; Gelfand, Ellen T.; Getz, Gad; Hadley, Kane; Handsaker, Robert E.; Huang, Katherine H.; Kashin, Seva; Karczewski, Konrad J.; Lek, Monkol; Li, Xiao; MacArthur, Daniel G.; Nedzel, Jared L.; Nguyen, Duyen T.; Noble, Michael S.; Segrè, Ayellet V.; Trowbridge, Casandra A.; Tukiainen, Taru; Abell, Nathan S.; Balliu, Brunilda; Barshir, Ruth; Basha, Omer; Bogu, Gireesh K.; Brown, Andrew; Castel, Stephane E.; Chen, Lin S.; Chiang, Colby; Conrad, Donald F.; Cox, Nancy J.; Damani, Farhan N.; Davis, Joe R.; Delaneau, Olivier; Dermitzakis, Emmanouil T.; Eskin, Eleazar; Ferreira, Pedro G.; Frésard, Laure; Gamazon, Eric R.; Garrido-Martín, Diego; Gewirtz, Ariel D. H.; Gliner, Genna; Gloudemans, Michael J.; Guigo, Roderic; Hall, Ira M.; Han, Buhm; He, Yuan

    2017-01-01

    Characterization of the molecular function of the human genome and its variation across individuals is essential for identifying the cellular mechanisms that underlie human genetic traits and diseases. The Genotype-Tissue Expression (GTEx) project aims to characterize variation in gene expression

  7. Expression of a partially deleted gene of human type II procollagen (COL2A1) in transgenic mice produces a chondrodysplasia

    Energy Technology Data Exchange (ETDEWEB)

    Vandenberg, P.; Khillan, J.S.; Prockop, D.J.; Helminen, H.; Kontusaari, S.; Ala-Kokko, L. (Thomas Jefferson Univ., Philadelphia, PA (United States))

    1991-09-01

    A minigene version of the human gene for type II procollagen (COL2AI) was prepared that lacked a large central region containing 12 of the 52 exons and therefore 291 of the 1523 codons of the gene. The construct was modeled after sporadic in-frame deletions of collagen genes that cause synthesis of shortened pro{alpha} chains that associate with normal pro{alpha} chains and thereby cause degradation of the shortened and normal pro{alpha} chains through a process called procollagen suicide. The gene construct was used to prepare five lines of transgenic mice expressing the minigene. A large proportion of the mice expressing the minigene developed a phenotype of a chondrodysplasia with dwarfism, short and thick limbs, a short snout, a cranial bulge, a cleft palate, and delayed mineralization of bone. A number of mice died shortly after birth. Microscopic examination of cartilage revealed decreased density and organization of collagen fibrils. In cultured chondrocytes from the transgenic mice, the minigene was expressed as shortened pro{alpha}1(II) chains that were disulfide-linked to normal mouse pro{alpha}1(II) chains. Therefore, the phenotype is probably explained by depletion of the endogenous mouse type II procollagen through the phenomenon of procollagen suicide.

  8. In vitro evaluation of chondrosarcoma cells and canine chondrocytes on layer-by-layer (LbL) self-assembled multilayer nanofilms

    International Nuclear Information System (INIS)

    Shaik, J; Mohammed, J Shaikh; McShane, M J; Mills, D K

    2013-01-01

    Short-term cell–substrate interactions of two secondary chondrocyte cell lines (human chondrosarcoma cells, canine chondrocytes) with layer-by-layer self-assembled multilayer nanofilms were investigated for a better understanding of cellular-behaviour dependence on a number of nanofilm layers. Cell–substrate interactions were studied on polyelectrolyte multilayer nanofilms (PMNs) of eleven different biomaterials. Surface characterization of PMNs performed using AFM showed increasing surface roughness with increasing number of layers for most of the biomaterials. LDH-L and MTT assays were performed on chondrosarcoma cells and canine chondrocytes, respectively. A major observation was that 10-bilayer nanofilms exhibited lesser cytotoxicity towards human chondrosarcoma cells than their 5-bilayer counterparts. In the case of canine chondrocytes, BSA enhanced cell metabolic activity with increasing number of layers, underscoring the importance of the multilayer nanofilm architecture on cellular behaviour. (paper)

  9. Human γ-globin genes silenced independently of other genes in the β-globin locus.

    NARCIS (Netherlands)

    N.O. Dillon (Niall); F.G. Grosveld (Frank)

    1991-01-01

    textabstractErythropoiesis during human development is characterized by switches in expression of beta-like globin genes during the transition from the embryonic through fetal to adult stages. Activation and high-level expression of the genes is directed by the locus control region (LCR), located 5'

  10. Gene × Smoking Interactions on Human Brain Gene Expression: Finding Common Mechanisms in Adolescents and Adults

    Science.gov (United States)

    Wolock, Samuel L.; Yates, Andrew; Petrill, Stephen A.; Bohland, Jason W.; Blair, Clancy; Li, Ning; Machiraju, Raghu; Huang, Kun; Bartlett, Christopher W.

    2013-01-01

    Background: Numerous studies have examined gene × environment interactions (G × E) in cognitive and behavioral domains. However, these studies have been limited in that they have not been able to directly assess differential patterns of gene expression in the human brain. Here, we assessed G × E interactions using two publically available datasets…

  11. LINE FUSION GENES: a database of LINE expression in human genes

    Directory of Open Access Journals (Sweden)

    Park Hong-Seog

    2006-06-01

    Full Text Available Abstract Background Long Interspersed Nuclear Elements (LINEs are the most abundant retrotransposons in humans. About 79% of human genes are estimated to contain at least one segment of LINE per transcription unit. Recent studies have shown that LINE elements can affect protein sequences, splicing patterns and expression of human genes. Description We have developed a database, LINE FUSION GENES, for elucidating LINE expression throughout the human gene database. We searched the 28,171 genes listed in the NCBI database for LINE elements and analyzed their structures and expression patterns. The results show that the mRNA sequences of 1,329 genes were affected by LINE expression. The LINE expression types were classified on the basis of LINEs in the 5' UTR, exon or 3' UTR sequences of the mRNAs. Our database provides further information, such as the tissue distribution and chromosomal location of the genes, and the domain structure that is changed by LINE integration. We have linked all the accession numbers to the NCBI data bank to provide mRNA sequences for subsequent users. Conclusion We believe that our work will interest genome scientists and might help them to gain insight into the implications of LINE expression for human evolution and disease. Availability http://www.primate.or.kr/line

  12. Isolating human DNA repair genes using rodent-cell mutants

    International Nuclear Information System (INIS)

    Thompson, L.H.; Weber, C.A.; Brookman, K.W.; Salazar, E.P.; Stewart, S.A.; Mitchell, D.L.

    1987-01-01

    The DNA repair systems of rodent and human cells appear to be at least as complex genetically as those in lower eukaryotes and bacteria. The use of mutant lines of rodent cells as a means of identifying human repair genes by functional complementation offers a new approach toward studying the role of repair in mutagenesis and carcinogenesis. In each of six cases examined using hybrid cells, specific human chromosomes have been identified that correct CHO cell mutations affecting repair of damage from uv or ionizing radiations. This finding suggests that both the repair genes and proteins may be virtually interchangeable between rodent and human cells. Using cosmid vectors, human repair genes that map to chromosome 19 have cloned as functional sequences: ERCC2 and XRCC1. ERCC1 was found to have homology with the yeast excision repair gene RAD10. Transformants of repair-deficient cell lines carrying the corresponding human gene show efficient correction of repair capacity by all criteria examined. 39 refs., 1 fig., 1 tab

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

    Directory of Open Access Journals (Sweden)

    Ryo Ito

    2016-03-01

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

  14. Effects of low molecular weight hyaluronan combined with carprofen on canine osteoarthritis articular chondrocytes and cartilage explants in vitro.

    Science.gov (United States)

    Euppayo, Thippaporn; Siengdee, Puntita; Buddhachat, Kittisak; Pradit, Waranee; Viriyakhasem, Nawarat; Chomdej, Siriwadee; Ongchai, Siriwan; Harada, Yasuji; Nganvongpanit, Korakot

    2015-09-01

    Intra-articular injection with non-steroidal anti-inflammatory drugs (NSAIDs) is used to treat inflammatory joint disease, but the side effects of NSAIDs include chondrotoxicity. Hyaluronan has shown positive effects on chondrocytes by reducing apoptosis and increasing proteoglycan synthesis. The purposes of this study were to evaluate the effects of low molecular weight hyaluronan (low MW HA), carprofen 25 mg/ml, carprofen 12.5 mg/ml, and a combination of HA and carprofen on canine osteoarthritis (OA) articular chondrocytes and a cartilage explant model in terms of cell viability, extracellular matrix remaining, and gene expression after exposure. In chondrocyte culture, MTT assay was used to evaluate the chondrotoxicity of IC50 and IC80 of carprofen with HA. In cartilage explant culture, two kinds of extracellular matrix (uronic acid and collagen) remaining in cartilage were used to evaluate cartilage damage for 14 d after treatment. Expression of COL2A1, AGG, and MMP3 was used to evaluate the synthesis and degradation of the matrix for 7 d after treatment. In chondrocyte culture, low MW HA could preserve OA chondrocyte viability but could not reduce the chondrotoxicity level of carprofen (P carprofen caused less destruction of uronic acid and collagen structure when compared with the control (P carprofen resulted in higher COL2A1 and AGG expression levels than carprofen alone.

  15. Eco RI RFLP in the human IGF II gene

    Energy Technology Data Exchange (ETDEWEB)

    Cocozza, S; Garofalo, S; Robledo, R; Monticelli, A; Conti, A; Chiarotti, L; Frunzio, R; Bruni, C B; Varrone, S

    1988-03-25

    The probe was a 500 bp cDNA containing exons 2-3 and 4 of the human IGF II gene. The clone was isolated by screening a human liver cDNA library with synthetic oligonucleotides. Eco RI digestion of genomic DNA and hybridization with the IGF II probe detects a two allele polymorphism with allelic fragments of 13.5 kb and 10.5 kb. The frequency was studied 38 unrelated Caucasians: Human IGF II gene was localized on the short arm of chromosome 11 (p15) by in situ hybridization. Codominant segregation was observed in 2 Caucasian families (10 individuals).

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

    Science.gov (United States)

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

    2012-12-15

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

  17. Mutations in fam20b and xylt1 reveal that cartilage matrix controls timing of endochondral ossification by inhibiting chondrocyte maturation.

    Directory of Open Access Journals (Sweden)

    B Frank Eames

    2011-08-01

    Full Text Available Differentiating cells interact with their extracellular environment over time. Chondrocytes embed themselves in a proteoglycan (PG-rich matrix, then undergo a developmental transition, termed "maturation," when they express ihh to induce bone in the overlying tissue, the perichondrium. Here, we ask whether PGs regulate interactions between chondrocytes and perichondrium, using zebrafish mutants to reveal that cartilage PGs inhibit chondrocyte maturation, which ultimately dictates the timing of perichondral bone development. In a mutagenesis screen, we isolated a class of mutants with decreased cartilage matrix and increased perichondral bone. Positional cloning identified lesions in two genes, fam20b and xylosyltransferase1 (xylt1, both of which encode PG synthesis enzymes. Mutants failed to produce wild-type levels of chondroitin sulfate PGs, which are normally abundant in cartilage matrix, and initiated perichondral bone formation earlier than their wild-type siblings. Primary chondrocyte defects might induce the bone phenotype secondarily, because mutant chondrocytes precociously initiated maturation, showing increased and early expression of such markers as runx2b, collagen type 10a1, and ihh co-orthologs, and ihha mutation suppressed early perichondral bone in PG mutants. Ultrastructural analyses demonstrated aberrant matrix organization and also early cellular features of chondrocyte hypertrophy in mutants. Refining previous in vitro reports, which demonstrated that fam20b and xylt1 were involved in PG synthesis, our in vivo analyses reveal that these genes function in cartilage matrix production and ultimately regulate the timing of skeletal development.

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

    Science.gov (United States)

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

    2016-03-23

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

  19. The progress of radiosensitive genes of human brain glioma

    International Nuclear Information System (INIS)

    Wang Xi; Liu Qiang

    2008-01-01

    Human gliomas are one of the most aggressive tumors in brain which grow infiltrativly. Surgery is the mainstay of treatment. But as the tumor could not be entirely cut off, it is easy to relapse. Radiotherapy plays an important role for patients with gliomas after surgery. The efficacy of radiotherapy is associated with radio sensitivity of human gliomas. This paper makes a summary of current situation and progress for radiosensitive genes of human brain gliomas. (authors)

  20. Isolation and characterization of the human uracil DNA glycosylase gene

    International Nuclear Information System (INIS)

    Vollberg, T.M.; Siegler, K.M.; Cool, B.L.; Sirover, M.A.

    1989-01-01

    A series of anti-human placental uracil DNA glycosylase monoclonal antibodies was used to screen a human placental cDNA library in phage λgt11. Twenty-seven immunopositive plaques were detected and purified. One clone containing a 1.2-kilobase (kb) human cDNA insert was chosen for further study by insertion into pUC8. The resultant recombinant plasmid selected by hybridization a human placental mRNA that encoded a 37-kDa polypeptide. This protein was immunoprecipitated specifically by an anti-human placenta uracil DNA glycosylase monoclonal antibody. RNA blot-hybridization (Northern) analysis using placental poly(A) + RNA or total RNA from four different human fibroblast cell strains revealed a single 1.6-kb transcript. Genomic blots using DNA from each cell strain digested with either EcoRI or PstI revealed a complex pattern of cDNA-hydridizing restriction fragments. The genomic analysis for each enzyme was highly similar in all four human cell strains. In contrast, a single band was observed when genomic analysis was performed with the identical DNA digests with an actin gene probe. During cell proliferation there was an increase in the level of glycosylase mRNA that paralleled the increase in uracil DNA glycosylase enzyme activity. The isolation of the human uracil DNA glycosylase gene permits an examination of the structure, organization, and expression of a human DNA repair gene

  1. ANALYSES ON DIFFERENTIALLY EXPRESSED GENES ASSOCIATED WITH HUMAN BREAST CANCER

    Institute of Scientific and Technical Information of China (English)

    MENG Xu-li; DING Xiao-wen; XU Xiao-hong

    2006-01-01

    Objective: To investigate the molecular etiology of breast cancer by way of studying the differential expression and initial function of the related genes in the occurrence and development of breast cancer. Methods: Two hundred and eighty-eight human tumor related genes were chosen for preparation of the oligochips probe. mRNA was extracted from 16 breast cancer tissues and the corresponding normal breast tissues, and cDNA probe was prepared through reverse-transcription and hybridized with the gene chip. A laser focused fluorescent scanner was used to scan the chip. The different gene expressions were thereafter automatically compared and analyzed between the two sample groups. Cy3/Cy5>3.5 meant significant up-regulation. Cy3/Cy5<0.25 meant significant down-regulation. Results: The comparison between the breast cancer tissues and their corresponding normal tissues showed that 84 genes had differential expression in the Chip. Among the differently expressed genes, there were 4 genes with significant down-regulation and 6 with significant up-regulation. Compared with normal breast tissues, differentially expressed genes did partially exist in the breast cancer tissues. Conclusion: Changes in multi-gene expression regulations take place during the occurrence and development of breast cancer; and the research on related genes can help understanding the mechanism of tumor occurrence.

  2. Gene therapy, human nature and the churches.

    Science.gov (United States)

    Dunstan, G R

    1991-12-01

    Moral analysis must begin with respect for the empirical features, the "facts of the case". Major advances in genetic knowledge and technology -- as in other sciences -- inevitably change mental attitudes. But they could not change human nature, a product of the distinctively human cerebral cortex. Human capacities like compassion and justice are our own and for us to guard. To ask (as some do) about a "right" to inherit a non-manipulated genome is to ask an unanswerable question: the language of rights is inappropriate in this context. Parents have a duty to safeguard and to serve the interests of their potential child. The medical duty is to help in that task in ways which they have limited freedom to choose. The role of churches is to be faithful to their deposit of faith and their theological principles, including that of freedom of conscience. Churches are too easily led in practice to over-rule conscience on grounds of authority, ecclesiastical or biblical, not sustained by convincing reason. This is most evident in some declarations concerning human reproduction. Better were it for them to help their faithful in moral reasoning, the ethics of choice; to keep consciences tender.

  3. Hidden Markov Models for Human Genes

    DEFF Research Database (Denmark)

    Baldi, Pierre; Brunak, Søren; Chauvin, Yves

    1997-01-01

    We analyse the sequential structure of human genomic DNA by hidden Markov models. We apply models of widely different design: conventional left-right constructs and models with a built-in periodic architecture. The models are trained on segments of DNA sequences extracted such that they cover com...

  4. Gene Expression in the Human Endolymphatic Sac

    DEFF Research Database (Denmark)

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

    2015-01-01

    a1 sodium-bicarbonate transporter, SLC9a2 sodium-hydrogen transporter, SLC12a3 thiazide-sensitive Na-Cl transporter, and SLC34a2 sodium-phosphate transporter. CONCLUSIONS: Several important ion transporters of the SLC family are expressed in the human endolymphatic sac, including Pendrin...

  5. Systematic identification of human housekeeping genes possibly useful as references in gene expression studies.

    Science.gov (United States)

    Caracausi, Maria; Piovesan, Allison; Antonaros, Francesca; Strippoli, Pierluigi; Vitale, Lorenza; Pelleri, Maria Chiara

    2017-09-01

    The ideal reference, or control, gene for the study of gene expression in a given organism should be expressed at a medium‑high level for easy detection, should be expressed at a constant/stable level throughout different cell types and within the same cell type undergoing different treatments, and should maintain these features through as many different tissues of the organism. From a biological point of view, these theoretical requirements of an ideal reference gene appear to be best suited to housekeeping (HK) genes. Recent advancements in the quality and completeness of human expression microarray data and in their statistical analysis may provide new clues toward the quantitative standardization of human gene expression studies in biology and medicine, both cross‑ and within‑tissue. The systematic approach used by the present study is based on the Transcriptome Mapper tool and exploits the automated reassignment of probes to corresponding genes, intra‑ and inter‑sample normalization, elaboration and representation of gene expression values in linear form within an indexed and searchable database with a graphical interface recording quantitative levels of expression, expression variability and cross‑tissue width of expression for more than 31,000 transcripts. The present study conducted a meta‑analysis of a pool of 646 expression profile data sets from 54 different human tissues and identified actin γ 1 as the HK gene that best fits the combination of all the traditional criteria to be used as a reference gene for general use; two ribosomal protein genes, RPS18 and RPS27, and one aquaporin gene, POM121 transmembrane nucleporin C, were also identified. The present study provided a list of tissue‑ and organ‑specific genes that may be most suited for the following individual tissues/organs: Adipose tissue, bone marrow, brain, heart, kidney, liver, lung, ovary, skeletal muscle and testis; and also provides in these cases a representative

  6. Loss of ATRX in chondrocytes has minimal effects on skeletal development.

    Directory of Open Access Journals (Sweden)

    Lauren A Solomon

    Full Text Available BACKGROUND: Mutations in the human ATRX gene cause developmental defects, including skeletal deformities and dwarfism. ATRX encodes a chromatin remodeling protein, however the role of ATRX in skeletal development is currently unknown. METHODOLOGY/PRINCIPAL FINDINGS: We induced Atrx deletion in mouse cartilage using the Cre-loxP system, with Cre expression driven by the collagen II (Col2a1 promoter. Growth rate, body size and weight, and long bone length did not differ in Atrx(Col2cre mice compared to control littermates. Histological analyses of the growth plate did not reveal any differences between control and mutant mice. Expression patterns of Sox9, a transcription factor required for cartilage morphogenesis, and p57, a marker of cell cycle arrest and hypertrophic chondrocyte differentiation, was unaffected. However, loss of ATRX in cartilage led to a delay in the ossification of the hips in some mice. We also observed hindlimb polydactily in one out of 61 mutants. CONCLUSIONS/SIGNIFICANCE: These findings indicate that ATRX is not directly required for development or growth of cartilage in the mouse, suggesting that the short stature in ATR-X patients is caused by defects in cartilage-extrinsic mechanisms.

  7. Characterization of human cardiac myosin heavy chain genes

    International Nuclear Information System (INIS)

    Yamauchi-Takihara, K.; Sole, M.J.; Liew, J.; Ing, D.; Liew, C.C.

    1989-01-01

    The authors have isolated and analyzed the structure of the genes coding for the α and β forms of the human cardiac myosin heavy chain (MYHC). Detailed analysis of four overlapping MYHC genomic clones shows that the α-MYHC and β-MYHC genes constitute a total length of 51 kilobases and are tandemly linked. The β-MYHC-encoding gene, predominantly expressed in the normal human ventricle and also in slow-twitch skeletal muscle, is located 4.5 kilobases upstream of the α-MYHC-encoding gene, which is predominantly expressed in normal human atrium. The authors have determined the nucleotide sequences of the β form of the MYHC gene, which is 100% homologous to the cardiac MYHC cDNA clone (pHMC3). It is unlikely that the divergence of a few nucleotide sequences from the cardiac β-MYHC cDNA clone (pHMC3) reported in a MYHC cDNA clone (PSMHCZ) from skeletal muscle is due to a splicing mechanism. This finding suggests that the same β form of the cardiac MYHC gene is expressed in both ventricular and slow-twitch skeletal muscle. The promoter regions of both α- and β-MYHC genes, as well as the first four coding regions in the respective genes, have also been sequenced. The sequences in the 5'-flanking region of the α- and β-MYHC-encoding genes diverge extensively from one another, suggesting that expression of the α- and β-MYHC genes is independently regulated

  8. The human protein disulfide isomerase gene family

    Directory of Open Access Journals (Sweden)

    Galligan James J

    2012-07-01

    Full Text Available Abstract Enzyme-mediated disulfide bond formation is a highly conserved process affecting over one-third of all eukaryotic proteins. The enzymes primarily responsible for facilitating thiol-disulfide exchange are members of an expanding family of proteins known as protein disulfide isomerases (PDIs. These proteins are part of a larger superfamily of proteins known as the thioredoxin protein family (TRX. As members of the PDI family of proteins, all proteins contain a TRX-like structural domain and are predominantly expressed in the endoplasmic reticulum. Subcellular localization and the presence of a TRX domain, however, comprise the short list of distinguishing features required for gene family classification. To date, the PDI gene family contains 21 members, varying in domain composition, molecular weight, tissue expression, and cellular processing. Given their vital role in protein-folding, loss of PDI activity has been associated with the pathogenesis of numerous disease states, most commonly related to the unfolded protein response (UPR. Over the past decade, UPR has become a very attractive therapeutic target for multiple pathologies including Alzheimer disease, Parkinson disease, alcoholic and non-alcoholic liver disease, and type-2 diabetes. Understanding the mechanisms of protein-folding, specifically thiol-disulfide exchange, may lead to development of a novel class of therapeutics that would help alleviate a wide range of diseases by targeting the UPR.

  9. Human Intellectual Disability Genes Form Conserved Functional Modules in Drosophila

    Science.gov (United States)

    Oortveld, Merel A. W.; Keerthikumar, Shivakumar; Oti, Martin; Nijhof, Bonnie; Fernandes, Ana Clara; Kochinke, Korinna; Castells-Nobau, Anna; van Engelen, Eva; Ellenkamp, Thijs; Eshuis, Lilian; Galy, Anne; van Bokhoven, Hans; Habermann, Bianca; Brunner, Han G.; Zweier, Christiane; Verstreken, Patrik; Huynen, Martijn A.; Schenck, Annette

    2013-01-01

    Intellectual Disability (ID) disorders, defined by an IQ below 70, are genetically and phenotypically highly heterogeneous. Identification of common molecular pathways underlying these disorders is crucial for understanding the molecular basis of cognition and for the development of therapeutic intervention strategies. To systematically establish their functional connectivity, we used transgenic RNAi to target 270 ID gene orthologs in the Drosophila eye. Assessment of neuronal function in behavioral and electrophysiological assays and multiparametric morphological analysis identified phenotypes associated with knockdown of 180 ID gene orthologs. Most of these genotype-phenotype associations were novel. For example, we uncovered 16 genes that are required for basal neurotransmission and have not previously been implicated in this process in any system or organism. ID gene orthologs with morphological eye phenotypes, in contrast to genes without phenotypes, are relatively highly expressed in the human nervous system and are enriched for neuronal functions, suggesting that eye phenotyping can distinguish different classes of ID genes. Indeed, grouping genes by Drosophila phenotype uncovered 26 connected functional modules. Novel links between ID genes successfully predicted that MYCN, PIGV and UPF3B regulate synapse development. Drosophila phenotype groups show, in addition to ID, significant phenotypic similarity also in humans, indicating that functional modules are conserved. The combined data indicate that ID disorders, despite their extreme genetic diversity, are caused by disruption of a limited number of highly connected functional modules. PMID:24204314

  10. Nucleotide sequence of a human tRNA gene heterocluster

    International Nuclear Information System (INIS)

    Chang, Y.N.; Pirtle, I.L.; Pirtle, R.M.

    1986-01-01

    Leucine tRNA from bovine liver was used as a hybridization probe to screen a human gene library harbored in Charon-4A of bacteriophage lambda. The human DNA inserts from plaque-pure clones were characterized by restriction endonuclease mapping and Southern hybridization techniques, using both [3'- 32 P]-labeled bovine liver leucine tRNA and total tRNA as hybridization probes. An 8-kb Hind III fragment of one of these γ-clones was subcloned into the Hind III site of pBR322. Subsequent fine restriction mapping and DNA sequence analysis of this plasmid DNA indicated the presence of four tRNA genes within the 8-kb DNA fragment. A leucine tRNA gene with an anticodon of AAG and a proline tRNA gene with an anticodon of AGG are in a 1.6-kb subfragment. A threonine tRNA gene with an anticodon of UGU and an as yet unidentified tRNA gene are located in a 1.1-kb subfragment. These two different subfragments are separated by 2.8 kb. The coding regions of the three sequenced genes contain characteristic internal split promoter sequences and do not have intervening sequences. The 3'-flanking region of these three genes have typical RNA polymerase III termination sites of at least four consecutive T residues

  11. Origins of De Novo Genes in Human and Chimpanzee.

    Science.gov (United States)

    Ruiz-Orera, Jorge; Hernandez-Rodriguez, Jessica; Chiva, Cristina; Sabidó, Eduard; Kondova, Ivanela; Bontrop, Ronald; Marqués-Bonet, Tomàs; Albà, M Mar

    2015-12-01

    The birth of new genes is an important motor of evolutionary innovation. Whereas many new genes arise by gene duplication, others originate at genomic regions that did not contain any genes or gene copies. Some of these newly expressed genes may acquire coding or non-coding functions and be preserved by natural selection. However, it is yet unclear which is the prevalence and underlying mechanisms of de novo gene emergence. In order to obtain a comprehensive view of this process, we have performed in-depth sequencing of the transcriptomes of four mammalian species--human, chimpanzee, macaque, and mouse--and subsequently compared the assembled transcripts and the corresponding syntenic genomic regions. This has resulted in the identification of over five thousand new multiexonic transcriptional events in human and/or chimpanzee that are not observed in the rest of species. Using comparative genomics, we show that the expression of these transcripts is associated with the gain of regulatory motifs upstream of the transcription start site (TSS) and of U1 snRNP sites downstream of the TSS. In general, these transcripts show little evidence of purifying selection, suggesting that many of them are not functional. However, we find signatures of selection in a subset of de novo genes which have evidence of protein translation. Taken together, the data support a model in which frequently-occurring new transcriptional events in the genome provide the raw material for the evolution of new proteins.

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

    International Nuclear Information System (INIS)

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

    2008-01-01

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

  13. Automated Identification of Core Regulatory Genes in Human Gene Regulatory Networks.

    Directory of Open Access Journals (Sweden)

    Vipin Narang

    Full Text Available Human gene regulatory networks (GRN can be difficult to interpret due to a tangle of edges interconnecting thousands of genes. We constructed a general human GRN from extensive transcription factor and microRNA target data obtained from public databases. In a subnetwork of this GRN that is active during estrogen stimulation of MCF-7 breast cancer cells, we benchmarked automated algorithms for identifying core regulatory genes (transcription factors and microRNAs. Among these algorithms, we identified K-core decomposition, pagerank and betweenness centrality algorithms as the most effective for discovering core regulatory genes in the network evaluated based on previously known roles of these genes in MCF-7 biology as well as in their ability to explain the up or down expression status of up to 70% of the remaining genes. Finally, we validated the use of K-core algorithm for organizing the GRN in an easier to interpret layered hierarchy where more influential regulatory genes percolate towards the inner layers. The integrated human gene and miRNA network and software used in this study are provided as supplementary materials (S1 Data accompanying this manuscript.

  14. Characterization of the human gene (TBXAS1) encoding thromboxane synthase.

    Science.gov (United States)

    Miyata, A; Yokoyama, C; Ihara, H; Bandoh, S; Takeda, O; Takahashi, E; Tanabe, T

    1994-09-01

    The gene encoding human thromboxane synthase (TBXAS1) was isolated from a human EMBL3 genomic library using human platelet thromboxane synthase cDNA as a probe. Nucleotide sequencing revealed that the human thromboxane synthase gene spans more than 75 kb and consists of 13 exons and 12 introns, of which the splice donor and acceptor sites conform to the GT/AG rule. The exon-intron boundaries of the thromboxane synthase gene were similar to those of the human cytochrome P450 nifedipine oxidase gene (CYP3A4) except for introns 9 and 10, although the primary sequences of these enzymes exhibited 35.8% identity each other. The 1.2-kb of the 5'-flanking region sequence contained potential binding sites for several transcription factors (AP-1, AP-2, GATA-1, CCAAT box, xenobiotic-response element, PEA-3, LF-A1, myb, basic transcription element and cAMP-response element). Primer-extension analysis indicated the multiple transcription-start sites, and the major start site was identified as an adenine residue located 142 bases upstream of the translation-initiation site. However, neither a typical TATA box nor a typical CAAT box is found within the 100-b upstream of the translation-initiation site. Southern-blot analysis revealed the presence of one copy of the thromboxane synthase gene per haploid genome. Furthermore, a fluorescence in situ hybridization study revealed that the human gene for thromboxane synthase is localized to band q33-q34 of the long arm of chromosome 7. A tissue-distribution study demonstrated that thromboxane synthase mRNA is widely expressed in human tissues and is particularly abundant in peripheral blood leukocyte, spleen, lung and liver. The low but significant levels of mRNA were observed in kidney, placenta and thymus.

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

    Science.gov (United States)

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

    2010-03-30

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

  16. Gene Transfer and Molecular Cloning of the Human NGF Receptor

    Science.gov (United States)

    Chao, Moses V.; Bothwell, Mark A.; Ross, Alonzo H.; Koprowski, Hilary; Lanahan, Anthony A.; Buck, C. Randall; Sehgal, Amita

    1986-04-01

    Nerve growth factor (NGF) and its receptor are important in the development of cells derived from the neural crest. Mouse L cell transformants have been generated that stably express the human NGF receptor gene transfer with total human DNA. Affinity cross-linking, metabolic labeling and immunoprecipitation, and equilibrium binding with 125I-labeled NGF revealed that this NGF receptor had the same size and binding characteristics as the receptor from human melanoma cells and rat PC12 cells. The sequences encoding the NGF receptor were molecularly cloned using the human Alu repetitive sequence as a probe. A cosmid clone that contained the human NGF receptor gene allowed efficient transfection and expression of the receptor.

  17. Sex-Dependent Gene Expression in Human Pluripotent Stem Cells

    Directory of Open Access Journals (Sweden)

    Daniel Ronen

    2014-08-01

    Full Text Available Males and females have a variety of sexually dimorphic traits, most of which result from hormonal differences. However, differences between male and female embryos initiate very early in development, before hormonal influence begins, suggesting the presence of genetically driven sexual dimorphisms. By comparing the gene expression profiles of male and X-inactivated female human pluripotent stem cells, we detected Y-chromosome-driven effects. We discovered that the sex-determining gene SRY is expressed in human male pluripotent stem cells and is induced by reprogramming. In addition, we detected more than 200 differentially expressed autosomal genes in male and female embryonic stem cells. Some of these genes are involved in steroid metabolism pathways and lead to sex-dependent differentiation in response to the estrogen precursor estrone. Thus, we propose that the presence of the Y chromosome and specifically SRY may drive sex-specific differences in the growth and differentiation of pluripotent stem cells.

  18. Chromosomal localization of the human vesicular amine transporter genes

    Energy Technology Data Exchange (ETDEWEB)

    Peter, D.; Finn, P.; Liu, Y.; Roghani, A.; Edwards, R.H.; Klisak, I.; Kojis, T.; Heinzmann, C.; Sparkes, R.S. (UCLA School of Medicine, Los Angeles, CA (United States))

    1993-12-01

    The physiologic and behavioral effects of pharmacologic agents that interfere with the transport of monoamine neurotransmitters into vesicles suggest that vesicular amine transport may contribute to human neuropsychiatric disease. To determine whether an alteration in the genes that encode vesicular amine transport contributes to the inherited component of these disorders, the authors have isolated a human cDNA for the brain transporter and localized the human vesciular amine transporter genes. The human brain synaptic vesicle amine transporter (SVAT) shows unexpected conservation with rat SVAT in the regions that diverge extensively between rat SVAT and the rat adrenal chromaffin granule amine transporter (CGAT). Using the cloned sequences with a panel of mouse-human hybrids and in situ hybridization for regional localization, the adrenal CGAT gene (or VAT1) maps to human chromosome 8p21.3 and the brain SVAT gene (or VAT2) maps to chromosome 10q25. Both of these sites occur very close to if not within previously described deletions that produce severe but viable phenotypes. 26 refs., 3 figs., 1 tab.

  19. Characterization of human septic sera induced gene expression modulation in human myocytes

    Science.gov (United States)

    Hussein, Shaimaa; Michael, Paul; Brabant, Danielle; Omri, Abdelwahab; Narain, Ravin; Passi, Kalpdrum; Ramana, Chilakamarti V.; Parrillo, Joseph E.; Kumar, Anand; Parissenti, Amadeo; Kumar, Aseem

    2009-01-01

    To gain a better understanding of the gene expression changes that occurs during sepsis, we have performed a cDNA microarray study utilizing a tissue culture model that mimics human sepsis. This study utilized an in vitro model of cultured human fetal cardiac myocytes treated with 10% sera from septic patients or 10% sera from healthy volunteers. A 1700 cDNA expression microarray was used to compare the transcription profile from human cardiac myocytes treated with septic sera vs normal sera. Septic sera treatment of myocytes resulted in the down-regulation of 178 genes and the up-regulation of 4 genes. Our data indicate that septic sera induced cell cycle, metabolic, transcription factor and apoptotic gene expression changes in human myocytes. Identification and characterization of gene expression changes that occur during sepsis may lead to the development of novel therapeutics and diagnostics. PMID:19684886

  20. Does Platelet-Rich Plasma Freeze-Thawing Influence Growth Factor Release and Their Effects on Chondrocytes and Synoviocytes?

    Directory of Open Access Journals (Sweden)

    Alice Roffi

    2014-01-01

    Full Text Available PRP cryopreservation remains a controversial point. Our purpose was to investigate the effect of freezing/thawing on PRP molecule release, and its effects on the metabolism of chondrocytes and synoviocytes. PRP was prepared from 10 volunteers, and a half volume underwent one freezing/thawing cycle. IL-1β, HGF, PDGF AB/BB, TGF-β1, and VEGF were assayed 1 hour and 7 days after activation. Culture media of chondrocytes and synoviocytes were supplemented with fresh or frozen PRP, and, at 7 days, proliferation, gene expression, and secreted proteins levels were evaluated. Results showed that in the freeze-thawed PRP the immediate and delayed molecule releases were similar or slightly lower than those in fresh PRP. TGF-β1 and PDGF AB/BB concentrations were significantly reduced after freezing both at 1 hour and at 7 days, whereas HGF concentration was significantly lower in frozen PRP at 7 days. In fresh PRP IL-1β and HGF concentrations underwent a significant further increase after 7 days. Similar gene expression was found in chondrocytes cultured with both PRPs, whereas in synoviocytes HGF gene expression was higher in frozen PRP. PRP cryopreservation is a safe procedure, which sufficiently preserves PRP quality and its ability to induce proliferation and the production of ECM components in chondrocytes and synoviocytes.

  1. The human cumulus--oocyte complex gene-expression profile

    Science.gov (United States)

    Assou, Said; Anahory, Tal; Pantesco, Véronique; Le Carrour, Tanguy; Pellestor, Franck; Klein, Bernard; Reyftmann, Lionel; Dechaud, Hervé; De Vos, John; Hamamah, Samir

    2006-01-01

    BACKGROUND The understanding of the mechanisms regulating human oocyte maturation is still rudimentary. We have identified transcripts differentially expressed between immature and mature oocytes, and cumulus cells. METHODS Using oligonucleotides microarrays, genome wide gene expression was studied in pooled immature and mature oocytes or cumulus cells from patients who underwent IVF. RESULTS In addition to known genes such as DAZL, BMP15 or GDF9, oocytes upregulated 1514 genes. We show that PTTG3 and AURKC are respectively the securin and the Aurora kinase preferentially expressed during oocyte meiosis. Strikingly, oocytes overexpressed previously unreported growth factors such as TNFSF13/APRIL, FGF9, FGF14, and IL4, and transcription factors including OTX2, SOX15 and SOX30. Conversely, cumulus cells, in addition to known genes such as LHCGR or BMPR2, overexpressed cell-tocell signaling genes including TNFSF11/RANKL, numerous complement components, semaphorins (SEMA3A, SEMA6A, SEMA6D) and CD genes such as CD200. We also identified 52 genes progressively increasing during oocyte maturation, comprising CDC25A and SOCS7. CONCLUSION The identification of genes up and down regulated during oocyte maturation greatly improves our understanding of oocyte biology and will provide new markers that signal viable and competent oocytes. Furthermore, genes found expressed in cumulus cells are potential markers of granulosa cell tumors. PMID:16571642

  2. Nomenclature for alleles of the human carboxylesterase 1 gene

    DEFF Research Database (Denmark)

    Rasmussen, Henrik B.; Madsen, Majbritt B.; Bjerre, Ditte

    2017-01-01

    The carboxylesterase 1 gene (CES1) in humans encodes a hydrolase, which is implicated in the metabolism of several commonly used drugs 1. This gene is located on chromosome 16 with a highly homologous pseudogene, CES1P1, in its proximity. A duplicated segment of CES1 replaces most of CES1P1 in some...... appears to be low 8,13. The formation of hybrids consisting of a gene and a related pseudogene has been reported for other genes than CES1. This includes the hybrids of the gene encoding cytochrome P450 2D6 (CYP2D6) and pseudogene CYP2D7, that is, the so-called CYP2D7/D6 hybrids 14......,15. These are categorized as CYP2D6 variants and not as variants of pseudogene CYP2D716....

  3. Gene expression in the aging human brain: an overview.

    Science.gov (United States)

    Mohan, Adith; Mather, Karen A; Thalamuthu, Anbupalam; Baune, Bernhard T; Sachdev, Perminder S

    2016-03-01

    The review aims to provide a summary of recent developments in the study of gene expression in the aging human brain. Profiling differentially expressed genes or 'transcripts' in the human brain over the course of normal aging has provided valuable insights into the biological pathways that appear activated or suppressed in late life. Genes mediating neuroinflammation and immune system activation in particular, show significant age-related upregulation creating a state of vulnerability to neurodegenerative and neuropsychiatric disease in the aging brain. Cellular ionic dyshomeostasis and age-related decline in a host of molecular influences on synaptic efficacy may underlie neurocognitive decline in later life. Critically, these investigations have also shed light on the mobilization of protective genetic responses within the aging human brain that help determine health and disease trajectories in older age. There is growing interest in the study of pre and posttranscriptional regulators of gene expression, and the role of noncoding RNAs in particular, as mediators of the phenotypic diversity that characterizes human brain aging. Gene expression studies in healthy brain aging offer an opportunity to unravel the intricately regulated cellular underpinnings of neurocognitive aging as well as disease risk and resiliency in late life. In doing so, new avenues for early intervention in age-related neurodegenerative disease could be investigated with potentially significant implications for the development of disease-modifying therapies.

  4. Mechanosensitive promoter region in the human HB-GAM gene

    DEFF Research Database (Denmark)

    Liedert, Astrid; Kassem, Moustapha; Claes, Lutz

    2009-01-01

    Mechanical loading is essential for maintaining bone mass in the adult skeleton. However, the underlying process of the transfer of the physical stimulus into a biochemical response, which is termed mechanotransduction is poorly understood. Mechanotransduction results in the modulation of gene...... cells. Analysis of the human HB-GAM gene upstream regulatory region with luciferase reporter gene assays revealed that the upregulation of HB-GAM expression occurred at the transcriptional level and was mainly dependent on the HB-GAM promoter region most upstream containing three potential AP-1 binding...

  5. Inhibition of Notch1 promotes hedgehog signalling in a HES1-dependent manner in chondrocytes and exacerbates experimental osteoarthritis.

    Science.gov (United States)

    Lin, Neng-Yu; Distler, Alfiya; Beyer, Christian; Philipi-Schöbinger, Ariella; Breda, Silvia; Dees, Clara; Stock, Michael; Tomcik, Michal; Niemeier, Andreas; Dell'Accio, Francesco; Gelse, Kolja; Mattson, Mark P; Schett, Georg; Distler, Jörg Hw

    2016-11-01

    Notch ligands and receptors have recently been shown to be differentially expressed in osteoarthritis (OA). We aim to further elucidate the functional role of Notch signalling in OA using Notch1 antisense transgenic (Notch1 AS) mice. Notch and hedgehog signalling were analysed by real-time PCR and immunohistochemistry. Notch-1 AS mice were employed as a model of impaired Notch signalling in vivo. Experimental OA was induced by destabilisation of the medial meniscus (DMM). The extent of cartilage destruction and osteophyte formation was analysed by safranin-O staining with subsequent assessment of the Osteoarthritis Research Society International (OARSI) and Mankin scores and µCT scanning. Collagen X staining was used as a marker of chondrocyte hypertrophy. The role of hairy/enhancer of split 1 (Hes-1) was investigated with knockdown and overexpression experiments. Notch signalling was activated in human and murine OA with increased expression of Jagged1, Notch-1, accumulation of the Notch intracellular domain 1 and increased transcription of Hes-1. Notch1 AS mice showed exacerbated OA with increases in OARSI scores, osteophyte formation, increased subchondral bone plate density, collagen X and osteocalcin expression and elevated levels of Epas1 and ADAM-TS5 mRNA. Inhibition of the Notch pathway induced activation of hedgehog signalling with induction of Gli-1 and Gli-2 and increased transcription of hedgehog target genes. The regulatory effects of Notch signalling on Gli-expression were mimicked by Hes-1. Inhibition of Notch signalling activates hedgehog signalling, enhances chondrocyte hypertrophy and exacerbates experimental OA including osteophyte formation. These data suggest that the activation of the Notch pathway may limit aberrant hedgehog signalling in OA. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/.

  6. Relation between HLA genes, human skin volatiles and attractiveness of humans to malaria mosquitoes

    NARCIS (Netherlands)

    Verhulst, N.O.; Beijleveld, H.; Qiu, Y.T.; Maliepaard, C.A.; Verduyn, W.; Haasnoot, G.W.; Claas, F.H.J.; Mumm, R.; Bouwmeester, H.J.; Takken, W.; Loon, van J.J.A.; Smallegange, R.C.

    2013-01-01

    Chemical cues are considered to be the most important cues for mosquitoes to find their hosts and humans can be ranked for attractiveness to mosquitoes based on the chemical cues they emit. Human leukocyte antigen (HLA) genes are considered to be involved in the regulation of human body odor and may

  7. Chondrogenesis of human adipose derived stem cells for future microtia repair using co-culture technique.

    Science.gov (United States)

    Goh, Bee See; Che Omar, Siti Nurhadis; Ubaidah, Muhammad Azhan; Saim, Lokman; Sulaiman, Shamsul; Chua, Kien Hui

    2017-04-01

    In conclusion, these result showed HADSCs could differentiate into chondrocytes-like cells, dependent on signaling induced by TGF-β3 and chondrocytes. This is a promising result and showed that HADSCs is a potential source for future microtia repair. The technique of co-culture is a positive way forward to assist the microtia tissue. Reconstructive surgery for the repair of microtia still remains the greatest challenge among the surgeons. Its repair is associated with donor-site morbidity and the degree of infection is inevitable when using alloplastic prosthesis with uncertain long-term durability. Thus, human adipose derived stem cells (HADSCs) can be an alternative cell source for cartilage regeneration. This study aims to evaluate the chondrogenic potential of HADSCs cultured with transforming growth factor-beta (TGF-β) and interaction of auricular chondrocytes with HADSCs for new cartilage generation. Multi-lineages differentiation features of HADSCs were monitored by Alcian Blue, Alizarin Red, and Oil Red O staining for chondrogenic, adipogenic, and osteogenic differentiation capacity, respectively. Further, HADSCs alone were culture in medium added with TGF-β3; and human auricular chondrocytes were interacted indirectly in the culture with and without TGF-βs for up to 21 days, respectively. Cell morphology and chondrogenesis were monitored by inverted microscope. For cell viability, Alamar Blue assay was used to measure the cell viability and the changes in gene expression of auricular chondrocyte markers were determined by real-time polymerase chain reaction analysis. For the induction of chondrogenic differentiation, HADSCs showed a feature of aggregation and formed a dense matrix of proteoglycans. Staining results from Alizirin Red and Oil Red O indicated the HADSCs also successfully differentiated into adipogenic and osteogenic lineages after 21 days. According to a previous study, HADSCs were strongly positive for the mesenchymal markers CD90, CD73

  8. Roles of the Y chromosome genes in human cancers

    Directory of Open Access Journals (Sweden)

    Tatsuo Kido

    2015-06-01

    Full Text Available Male and female differ genetically by their respective sex chromosome composition, that is, XY as male and XX as female. Although both X and Y chromosomes evolved from the same ancestor pair of autosomes, the Y chromosome harbors male-specific genes, which play pivotal roles in male sex determination, germ cell differentiation, and masculinization of various tissues. Deletions or translocation of the sex-determining gene, SRY, from the Y chromosome causes disorders of sex development (previously termed as an intersex condition with dysgenic gonads. Failure of gonadal development results not only in infertility, but also in increased risks of germ cell tumor (GCT, such as gonadoblastoma and various types of testicular GCT. Recent studies demonstrate that either loss of Y chromosome or ectopic expression of Y chromosome genes is closely associated with various male-biased diseases, including selected somatic cancers. These observations suggest that the Y-linked genes are involved in male health and diseases in more frequently than expected. Although only a small number of protein-coding genes are present in the male-specific region of Y chromosome, the impacts of Y chromosome genes on human diseases are still largely unknown, due to lack of in vivo models and differences between the Y chromosomes of human and rodents. In this review, we highlight the involvement of selected Y chromosome genes in cancer development in men.

  9. DRUMS: a human disease related unique gene mutation search engine.

    Science.gov (United States)

    Li, Zuofeng; Liu, Xingnan; Wen, Jingran; Xu, Ye; Zhao, Xin; Li, Xuan; Liu, Lei; Zhang, Xiaoyan

    2011-10-01

    With the completion of the human genome project and the development of new methods for gene variant detection, the integration of mutation data and its phenotypic consequences has become more important than ever. Among all available resources, locus-specific databases (LSDBs) curate one or more specific genes' mutation data along with high-quality phenotypes. Although some genotype-phenotype data from LSDB have been integrated into central databases little effort has been made to integrate all these data by a search engine approach. In this work, we have developed disease related unique gene mutation search engine (DRUMS), a search engine for human disease related unique gene mutation as a convenient tool for biologists or physicians to retrieve gene variant and related phenotype information. Gene variant and phenotype information were stored in a gene-centred relational database. Moreover, the relationships between mutations and diseases were indexed by the uniform resource identifier from LSDB, or another central database. By querying DRUMS, users can access the most popular mutation databases under one interface. DRUMS could be treated as a domain specific search engine. By using web crawling, indexing, and searching technologies, it provides a competitively efficient interface for searching and retrieving mutation data and their relationships to diseases. The present system is freely accessible at http://www.scbit.org/glif/new/drums/index.html. © 2011 Wiley-Liss, Inc.

  10. Nucleotide sequence of the human N-myc gene

    International Nuclear Information System (INIS)

    Stanton, L.W.; Schwab, M.; Bishop, J.M.

    1986-01-01

    Human neuroblastomas frequently display amplification and augmented expression of a gene known as N-myc because of its similarity to the protooncogene c-myc. It has therefore been proposed that N-myc is itself a protooncogene, and subsequent tests have shown that N-myc and c-myc have similar biological activities in cell culture. The authors have now detailed the kinship between N-myc and c-myc by determining the nucleotide sequence of human N-myc and deducing the amino acid sequence of the protein encoded by the gene. The topography of N-myc is strikingly similar to that of c-myc: both genes contain three exons of similar lengths; the coding elements of both genes are located in the second and third exons; and both genes have unusually long 5' untranslated regions in their mRNAs, with features that raise the possibility that expression of the genes may be subject to similar controls of translation. The resemblance between the proteins encoded by N-myc and c-myc sustains previous suspicions that the genes encode related functions

  11. A scale invariant clustering of genes on human chromosome 7

    Directory of Open Access Journals (Sweden)

    Kendal Wayne S

    2004-01-01

    Full Text Available Abstract Background Vertebrate genes often appear to cluster within the background of nontranscribed genomic DNA. Here an analysis of the physical distribution of gene structures on human chromosome 7 was performed to confirm the presence of clustering, and to elucidate possible underlying statistical and biological mechanisms. Results Clustering of genes was confirmed by virtue of a variance of the number of genes per unit physical length that exceeded the respective mean. Further evidence for clustering came from a power function relationship between the variance and mean that possessed an exponent of 1.51. This power function implied that the spatial distribution of genes on chromosome 7 was scale invariant, and that the underlying statistical distribution had a Poisson-gamma (PG form. A PG distribution for the spatial scattering of genes was validated by stringent comparisons of both the predicted variance to mean power function and its cumulative distribution function to data derived from chromosome 7. Conclusion The PG distribution was consistent with at least two different biological models: In the microrearrangement model, the number of genes per unit length of chromosome represented the contribution of a random number of smaller chromosomal segments that had originated by random breakage and reconstruction of more primitive chromosomes. Each of these smaller segments would have necessarily contained (on average a gamma distributed number of genes. In the gene cluster model, genes would be scattered randomly to begin with. Over evolutionary timescales, tandem duplication, mutation, insertion, deletion and rearrangement could act at these gene sites through a stochastic birth death and immigration process to yield a PG distribution. On the basis of the gene position data alone it was not possible to identify the biological model which best explained the observed clustering. However, the underlying PG statistical model implicated neutral

  12. Genetic Variants Contribute to Gene Expression Variability in Humans

    Science.gov (United States)

    Hulse, Amanda M.; Cai, James J.

    2013-01-01

    Expression quantitative trait loci (eQTL) studies have established convincing relationships between genetic variants and gene expression. Most of these studies focused on the mean of gene expression level, but not the variance of gene expression level (i.e., gene expression variability). In the present study, we systematically explore genome-wide association between genetic variants and gene expression variability in humans. We adapt the double generalized linear model (dglm) to simultaneously fit the means and the variances of gene expression among the three possible genotypes of a biallelic SNP. The genomic loci showing significant association between the variances of gene expression and the genotypes are termed expression variability QTL (evQTL). Using a data set of gene expression in lymphoblastoid cell lines (LCLs) derived from 210 HapMap individuals, we identify cis-acting evQTL involving 218 distinct genes, among which 8 genes, ADCY1, CTNNA2, DAAM2, FERMT2, IL6, PLOD2, SNX7, and TNFRSF11B, are cross-validated using an extra expression data set of the same LCLs. We also identify ∼300 trans-acting evQTL between >13,000 common SNPs and 500 randomly selected representative genes. We employ two distinct scenarios, emphasizing single-SNP and multiple-SNP effects on expression variability, to explain the formation of evQTL. We argue that detecting evQTL may represent a novel method for effectively screening for genetic interactions, especially when the multiple-SNP influence on expression variability is implied. The implication of our results for revealing genetic mechanisms of gene expression variability is discussed. PMID:23150607

  13. Evolutionary Conservation in Genes Underlying Human Psychiatric Disorders

    Directory of Open Access Journals (Sweden)

    Lisa Michelle Ogawa

    2014-05-01

    Full Text Available Many psychiatric diseases observed in humans have tenuous or absent analogs in other species. Most notable among these are schizophrenia and autism. One hypothesis has posited that these diseases have arisen as a consequence of human brain evolution, for example, that the same processes that led to advances in cognition, language, and executive function also resulted in novel diseases in humans when dysfunctional. Here, the molecular evolution of genes associated with these and other psychiatric disorders are compared among species. Genes associated with psychiatric disorders are drawn from the literature and orthologous sequences are collected from eleven primate species (human, chimpanzee, bonobo, gorilla, orangutan, gibbon, macaque, baboon, marmoset, squirrel monkey, and galago and thirty one non-primate mammalian species. Evolutionary parameters, including dN/dS, are calculated for each gene and compared between disease classes and among species, focusing on humans and primates compared to other mammals and on large-brained taxa (cetaceans, rhinoceros, walrus, bear, and elephant compared to their small-brained sister species. Evidence of differential selection in primates supports the hypothesis that schizophrenia and autism are a cost of higher brain function. Through this work a better understanding of the molecular evolution of the human brain, the pathophysiology of disease, and the genetic basis of human psychiatric disease is gained.

  14. Primary cilia modulate Ihh signal transduction in response to hydrostatic loading of growth plate chondrocytes.

    Science.gov (United States)

    Shao, Yvonne Y; Wang, Lai; Welter, Jean F; Ballock, R Tracy

    2012-01-01

    Indian hedgehog (Ihh) is a key component of the regulatory apparatus governing chondrocyte proliferation and differentiation in the growth plate. Recent studies have demonstrated that the primary cilium is the site of Ihh signaling within the cell, and that primary cilia are essential for bone and cartilage formation. Primary cilia are also postulated to act as mechanosensory organelles that transduce mechanical forces acting on the cell into biological signals. In this study, we used a hydrostatic compression system to examine Ihh signal transduction under the influence of mechanical load. Our results demonstrate that hydrostatic compression increased both Ihh gene expression and Ihh-responsive Gli-luciferase activity. These increases were aborted by disrupting the primary cilia structure with chloral hydrate. These results suggest that growth plate chondrocytes respond to hydrostatic loading by increasing Ihh signaling, and that the primary cilium is required for this mechano-biological signal transduction to occur. Copyright © 2011 Elsevier Inc. All rights reserved.

  15. Primary Cilia Modulate IHH Signal Transduction in Response to Hydrostatic Loading of Growth Plate Chondrocytes

    Science.gov (United States)

    Shao, Y, Yvonne Y.; Wang, Lai; Welter, J, Jean F.; Ballock, R. Tracy

    2011-01-01

    Indian Hedgehog (Ihh) is a key component of the regulatory apparatus governing chondrocyte proliferation and differentiation in the growth plate. Recent studies have demonstrated that the primary cilium is the site of Ihh signaling within the cell, and that primary cilia are essential for bone and cartilage formation. Primary cilia are also postulated to act as mechanosensory organelles that transduce mechanical forces acting on the cell into biological signals. In this study, we used a hydrostatic compression system to examine Ihh signal transduction under the influence of mechanical load. Our results demonstrate that hydrostatic compression increased both Ihh gene expression and Ihh-responsive Gli-luciferase activity. These increases were aborted by disrupting the primary cilia structure with chloral hydrate. These results suggest that growth plate chondrocytes respond to hydrostatic loading by increasing Ihh signaling, and that the primary cilium is required for this mechano-biological signal transduction to occur. PMID:21930256

  16. 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.

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

    Directory of Open Access Journals (Sweden)

    Abdul-Rehman Phull

    2016-01-01

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

  18. Expansion of bovine chondrocytes on microcarriers enhances redifferentiation

    NARCIS (Netherlands)

    Malda, J.; van Blitterswijk, Clemens; Grojec, M.; Martens, D.E.; Tramper, J.; Riesle, J.U.

    2003-01-01

    Functional cartilage implants for orthopedic surgery or in vitro tissue evaluation can be created from expanded chondrocytes and biodegradable scaffolds. Expansion of chondrocytes in two-dimensional culture systems results in their dedifferentiation. The hallmark of this process is the switch of

  19. Biomarkers of Chondrocyte Apoptosis and Autophagy in Osteoarthritis

    Science.gov (United States)

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

    2015-01-01

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

  20. The human oxytocin gene promoter is regulated by estrogens.

    Science.gov (United States)

    Richard, S; Zingg, H H

    1990-04-15

    Gonadal steroids affect brain function primarily by altering the expression of specific genes, yet the specific mechanisms by which neuronal target genes undergo such regulation are unknown. Recent evidence suggests that the expression of the neuropeptide gene for oxytocin (OT) is modulated by estrogens. We therefore examined the possibility that this regulation occurred via a direct interaction of the estrogen-receptor complex with cis-acting elements flanking the OT gene. DNA-mediated gene transfer experiments were performed using Neuro-2a neuroblastoma cells and chimeric plasmids containing portions of the human OT gene 5'-glanking region linked to the chloramphenicol acetyltransferase gene. We identified a 19-base pair region located at -164 to -146 upstream of the transcription start site which is capable of conferring estrogen responsiveness to the homologous as well as to a heterologous promoter. The hormonal response is strictly dependent on the presence of intracellular estrogen receptors, since estrogen induced stimulation occurred only in Neuro-2a cells co-transfected with an expression vector for the human estrogen receptor. The identified region contains a novel imperfect palindrome (GGTGACCTTGACC) with sequence similarity to other estrogen response elements (EREs). To define cis-acting elements that function in synergism with the ERE, sequences 3' to the ERE were deleted, including the CCAAT box, two additional motifs corresponding to the right half of the ERE palindrome (TGACC), as well as a CTGCTAA heptamer similar to the "elegans box" found in Caenorhabditis elegans. Interestingly, optimal function of the identified ERE was fully independent of these elements and only required a short promoter region (-49 to +36). Our studies define a molecular mechanism by which estrogens can directly modulate OT gene expression. However, only a subset of OT neurons are capable of binding estrogens, therefore, direct action of estrogens on the OT gene may be

  1. Identifying human disease genes through cross-species gene mapping of evolutionary conserved processes.

    Directory of Open Access Journals (Sweden)

    Martin Poot

    2011-05-01

    Full Text Available Understanding complex networks that modulate development in humans is hampered by genetic and phenotypic heterogeneity within and between populations. Here we present a method that exploits natural variation in highly diverse mouse genetic reference panels in which genetic and environmental factors can be tightly controlled. The aim of our study is to test a cross-species genetic mapping strategy, which compares data of gene mapping in human patients with functional data obtained by QTL mapping in recombinant inbred mouse strains in order to prioritize human disease candidate genes.We exploit evolutionary conservation of developmental phenotypes to discover gene variants that influence brain development in humans. We studied corpus callosum volume in a recombinant inbred mouse panel (C57BL/6J×DBA/2J, BXD strains using high-field strength MRI technology. We aligned mouse mapping results for this neuro-anatomical phenotype with genetic data from patients with abnormal corpus callosum (ACC development.From the 61 syndromes which involve an ACC, 51 human candidate genes have been identified. Through interval mapping, we identified a single significant QTL on mouse chromosome 7 for corpus callosum volume with a QTL peak located between 25.5 and 26.7 Mb. Comparing the genes in this mouse QTL region with those associated with human syndromes (involving ACC and those covered by copy number variations (CNV yielded a single overlap, namely HNRPU in humans and Hnrpul1 in mice. Further analysis of corpus callosum volume in BXD strains revealed that the corpus callosum was significantly larger in BXD mice with a B genotype at the Hnrpul1 locus than in BXD mice with a D genotype at Hnrpul1 (F = 22.48, p<9.87*10(-5.This approach that exploits highly diverse mouse strains provides an efficient and effective translational bridge to study the etiology of human developmental disorders, such as autism and schizophrenia.

  2. Cognitive genomics: Linking genes to behavior in the human brain

    Directory of Open Access Journals (Sweden)

    Genevieve Konopka

    2017-02-01

    Full Text Available Correlations of genetic variation in DNA with functional brain activity have already provided a starting point for delving into human cognitive mechanisms. However, these analyses do not provide the specific genes driving the associations, which are complicated by intergenic localization as well as tissue-specific epigenetics and expression. The use of brain-derived expression datasets could build upon the foundation of these initial genetic insights and yield genes and molecular pathways for testing new hypotheses regarding the molecular bases of human brain development, cognition, and disease. Thus, coupling these human brain gene expression data with measurements of brain activity may provide genes with critical roles in brain function. However, these brain gene expression datasets have their own set of caveats, most notably a reliance on postmortem tissue. In this perspective, I summarize and examine the progress that has been made in this realm to date, and discuss the various frontiers remaining, such as the inclusion of cell-type-specific information, additional physiological measurements, and genomic data from patient cohorts.

  3. Law-medicine interfacing: patenting of human genes and mutations.

    Science.gov (United States)

    Fialho, Arsenio M; Chakrabarty, Ananda M

    2011-08-01

    Mutations, Single Nucleotide Polymorphisms (SNPs), deletions and genetic rearrangements in specific genes in the human genome account for not only our physical characteristics and behavior, but can lead to many in-born and acquired diseases. Such changes in the genome can also predispose people to cancers, as well as significantly affect the metabolism and efficacy of many drugs, resulting in some cases in acute toxicity to the drug. The testing of the presence of such genetic mutations and rearrangements is of great practical and commercial value, leading many of these genes and their mutations/deletions and genetic rearrangements to be patented. A recent decision by a judge in the Federal District Court in the Southern District of New York, has created major uncertainties, based on the revocation of BRCA1 and BRCA2 gene patents, in the eligibility of all human and presumably other gene patents. This article argues that while patents on BRCA1 and BRCA2 genes could be challenged based on a lack of utility, the patenting of the mutations and genetic rearrangements is of great importance to further development and commercialization of genetic tests that can save human lives and prevent suffering, and should be allowed.

  4. 5-Aza-2'-deoxycytidine acts as a modulator of chondrocyte hypertrophy and maturation in chick caudal region chondrocytes in culture.

    Science.gov (United States)

    Haq, Samina Hyder

    2016-06-01

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

  5. Gene expression and adaptive noncoding changes during human evolution.

    Science.gov (United States)

    Babbitt, Courtney C; Haygood, Ralph; Nielsen, William J; Wray, Gregory A

    2017-06-05

    Despite evidence for adaptive changes in both gene expression and non-protein-coding, putatively regulatory regions of the genome during human evolution, the relationship between gene expression and adaptive changes in cis-regulatory regions remains unclear. Here we present new measurements of gene expression in five tissues of humans and chimpanzees, and use them to assess this relationship. We then compare our results with previous studies of adaptive noncoding changes, analyzing correlations at the level of gene ontology groups, in order to gain statistical power to detect correlations. Consistent with previous studies, we find little correlation between gene expression and adaptive noncoding changes at the level of individual genes; however, we do find significant correlations at the level of biological function ontology groups. The types of function include processes regulated by specific transcription factors, responses to genetic or chemical perturbations, and differentiation of cell types within the immune system. Among functional categories co-enriched with both differential expression and noncoding adaptation, prominent themes include cancer, particularly epithelial cancers, and neural development and function.

  6. Death and resurrection of the human IRGM gene.

    Directory of Open Access Journals (Sweden)

    Cemalettin Bekpen

    2009-03-01

    Full Text Available Immunity-related GTPases (IRG play an important role in defense against intracellular pathogens. One member of this gene family in humans, IRGM, has been recently implicated as a risk factor for Crohn's disease. We analyzed the detailed structure of this gene family among primates and showed that most of the IRG gene cluster was deleted early in primate evolution, after the divergence of the anthropoids from prosimians ( about 50 million years ago. Comparative sequence analysis of New World and Old World monkey species shows that the single-copy IRGM gene became pseudogenized as a result of an Alu retrotransposition event in the anthropoid common ancestor that disrupted the open reading frame (ORF. We find that the ORF was reestablished as a part of a polymorphic stop codon in the common ancestor of humans and great apes. Expression analysis suggests that this change occurred in conjunction with the insertion of an endogenous retrovirus, which altered the transcription initiation, splicing, and expression profile of IRGM. These data argue that the gene became pseudogenized and was then resurrected through a series of complex structural events and suggest remarkable functional plasticity where alleles experience diverse evolutionary pressures over time. Such dynamism in structure and evolution may be critical for a gene family locked in an arms race with an ever-changing repertoire of intracellular parasites.

  7. Structure of gene and pseudogenes of human apoferritin H

    Energy Technology Data Exchange (ETDEWEB)

    Costanzo, F; Colombo, M; Staempfli, S; Santoro, C; Marone, M; Frank, K; Delius, H; Cortese, R

    1986-01-24

    Ferritin is composed of two subunits, H and L. cDNA's coding for these proteins from human liver, lymphocytes and from the monocyte-like cell line U937 have been cloned and sequenced. Southern blot analysis on total human DNA reveals that there are many DNA segments hybridizing to the apoferritin H and L cDNA probes. In view of the tissue heterogeneity of ferritin molecules, it appeared possible that apoferritin molecules could be coded by a family of genes differentially expressed in various tissues. In this paper, the authors describe the cloning and sequencing of the gene coding for human apoferritin H. This gene has three introns; the exon sequence is identical to that of cDNAs isolated from human liver, lymphocytes, HeLa cells and endothelial cells. In addition they show that at least 15 intronless pseudogenes exist, with features suggesting that there were originated by reverse transcription and insertion. On the basis of these results they conclude that only one gene is responsible for the synthesis of the majority of apoferritin H mRNA in various tissues examined, and that probably all the other DNA segments hybridizing with apoferritin cDNA are pseudogenes.

  8. Gene expression profiles in adenosine-treated human mast cells ...

    African Journals Online (AJOL)

    Gene expression profiles in adenosine-treated human mast cells. ... SW Kang, JE Jeong, CH Kim, SH Choi, SH Chae, SA Jun, HJ Cha, JH Kim, YM Lee, YS ... beta 4, ring finger protein, high-mobility group, calmodulin 2, RAN binding protein, ...

  9. Ethical perception of human gene in transgenic banana | Amin ...

    African Journals Online (AJOL)

    Transgenic banana has been developed to prevent hepatitis B through vaccination. Its production seems to be an ideal alternative for cheaper vaccines. The objective of this paper is to assess the ethical perception of transgenic banana which involved the transfer of human albumin gene, and to compare their ethical ...

  10. Global patterns of diversity and selection in human tyrosinase gene.

    Science.gov (United States)

    Hudjashov, Georgi; Villems, Richard; Kivisild, Toomas

    2013-01-01

    Global variation in skin pigmentation is one of the most striking examples of environmental adaptation in humans. More than two hundred loci have been identified as candidate genes in model organisms and a few tens of these have been found to be significantly associated with human skin pigmentation in genome-wide association studies. However, the evolutionary history of different pigmentation genes is rather complex: some loci have been subjected to strong positive selection, while others evolved under the relaxation of functional constraints in low UV environment. Here we report the results of a global study of the human tyrosinase gene, which is one of the key enzymes in melanin production, to assess the role of its variation in the evolution of skin pigmentation differences among human populations. We observe a higher rate of non-synonymous polymorphisms in the European sample consistent with the relaxation of selective constraints. A similar pattern was previously observed in the MC1R gene and concurs with UV radiation-driven model of skin color evolution by which mutations leading to lower melanin levels and decreased photoprotection are subject to purifying selection at low latitudes while being tolerated or even favored at higher latitudes because they facilitate UV-dependent vitamin D production. Our coalescent date estimates suggest that the non-synonymous variants, which are frequent in Europe and North Africa, are recent and have emerged after the separation of East and West Eurasian populations.

  11. Designer Babies? Teacher Views on Gene Technology and Human Medicine.

    Science.gov (United States)

    Schibeci, Renato

    1999-01-01

    Summarizes the views of a sample of primary and high school teachers on the application of gene technology to human medicine. In general, high school teachers are more positive about these developments than primary teachers, and both groups of teachers are more positive than interested lay publics. Highlights ways in which this topic can be…

  12. A human gut microbial gene catalogue established by metagenomic sequencing

    DEFF Research Database (Denmark)

    dos Santos, Marcelo Bertalan Quintanilha; Sicheritz-Pontén, Thomas; Nielsen, Henrik Bjørn

    2010-01-01

    To understand the impact of gut microbes on human health and well-being it is crucial to assess their genetic potential. Here we describe the Illumina-based metagenomic sequencing, assembly and characterization of 3.3 million non-redundant microbial genes, derived from 576.7 gigabases of sequence...

  13. Gene therapy in nonhuman primate models of human autoimmune disease

    NARCIS (Netherlands)

    t'Hart, B. A.; Vervoordeldonk, M.; Heeney, J. L.; Tak, P. P.

    2003-01-01

    Before autoimmune diseases in humans can be treated with gene therapy, the safety and efficacy of the used vectors must be tested in valid experimental models. Monkeys, such as the rhesus macaque or the common marmoset, provide such models. This publication reviews the state of the art in monkey

  14. Mutational landscape of the human Y chromosome-linked genes ...

    Indian Academy of Sciences (India)

    Mutational landscape of the human Y chromosome-linked genes and loci in patients with hypogonadism. Deepali Pathak, Sandeep Kumar Yadav, Leena Rawal and Sher Ali. J. Genet. 94, 677–687. Table 1. Details showing age, sex, karyotype, clinical features and diagnosis results of the patients with H. Hormone profile.

  15. Contemporary Animal Models For Human Gene Therapy Applications.

    Science.gov (United States)

    Gopinath, Chitra; Nathar, Trupti Job; Ghosh, Arkasubhra; Hickstein, Dennis Durand; Nelson, Everette Jacob Remington

    2015-01-01

    Over the past three decades, gene therapy has been making considerable progress as an alternative strategy in the treatment of many diseases. Since 2009, several studies have been reported in humans on the successful treatment of various diseases. Animal models mimicking human disease conditions are very essential at the preclinical stage before embarking on a clinical trial. In gene therapy, for instance, they are useful in the assessment of variables related to the use of viral vectors such as safety, efficacy, dosage and localization of transgene expression. However, choosing a suitable disease-specific model is of paramount importance for successful clinical translation. This review focuses on the animal models that are most commonly used in gene therapy studies, such as murine, canine, non-human primates, rabbits, porcine, and a more recently developed humanized mice. Though small and large animals both have their own pros and cons as disease-specific models, the choice is made largely based on the type and length of study performed. While small animals with a shorter life span could be well-suited for degenerative/aging studies, large animals with longer life span could suit longitudinal studies and also help with dosage adjustments to maximize therapeutic benefit. Recently, humanized mice or mouse-human chimaeras have gained interest in the study of human tissues or cells, thereby providing a more reliable understanding of therapeutic interventions. Thus, animal models are of great importance with regard to testing new vector technologies in vivo for assessing safety and efficacy prior to a gene therapy clinical trial.

  16. 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.

  17. A human-specific de novo protein-coding gene associated with human brain functions.

    Directory of Open Access Journals (Sweden)

    Chuan-Yun Li

    2010-03-01

    Full Text Available To understand whether any human-specific new genes may be associated with human brain functions, we computationally screened the genetic vulnerable factors identified through Genome-Wide Association Studies and linkage analyses of nicotine addiction and found one human-specific de novo protein-coding gene, FLJ33706 (alternative gene symbol C20orf203. Cross-species analysis revealed interesting evolutionary paths of how this gene had originated from noncoding DNA sequences: insertion of repeat elements especially Alu contributed to the formation of the first coding exon and six standard splice junctions on the branch leading to humans and chimpanzees, and two subsequent substitutions in the human lineage escaped two stop codons and created an open reading frame of 194 amino acids. We experimentally verified FLJ33706's mRNA and protein expression in the brain. Real-Time PCR in multiple tissues demonstrated that FLJ33706 was most abundantly expressed in brain. Human polymorphism data suggested that FLJ33706 encodes a protein under purifying selection. A specifically designed antibody detected its protein expression across human cortex, cerebellum and midbrain. Immunohistochemistry study in normal human brain cortex revealed the localization of FLJ33706 protein in neurons. Elevated expressions of FLJ33706 were detected in Alzheimer's brain samples, suggesting the role of this novel gene in human-specific pathogenesis of Alzheimer's disease. FLJ33706 provided the strongest evidence so far that human-specific de novo genes can have protein-coding potential and differential protein expression, and be involved in human brain functions.

  18. Recent advances in human gene-longevity association studies

    DEFF Research Database (Denmark)

    De Benedictis, G; Tan, Q; Jeune, B

    2001-01-01

    This paper reviews the recent literature on genes and longevity. The influence of genes on human life span has been confirmed in studies of life span correlation between related individuals based on family and twin data. Results from major twin studies indicate that approximately 25......% of the variation in life span is genetically determined. Taking advantage of recent developments in molecular biology, researchers are now searching for candidate genes that might have an influence on life span. The data on unrelated individuals emerging from an ever-increasing number of centenarian studies makes...... this possible. This paper summarizes the rich literature dealing with the various aspects of the influence of genes on individual survival. Common phenomena affecting the development of disease and longevity are discussed. The major methodological difficulty one is confronted with when studying the epidemiology...

  19. Cartilage-selective genes identified in genome-scale analysis of non-cartilage and cartilage gene expression

    Directory of Open Access Journals (Sweden)

    Cohn Zachary A

    2007-06-01

    Full Text Available Abstract Background Cartilage plays a fundamental role in the development of the human skeleton. Early in embryogenesis, mesenchymal cells condense and differentiate into chondrocytes to shape the early skeleton. Subsequently, the cartilage anlagen differentiate to form the growth plates, which are responsible for linear bone growth, and the articular chondrocytes, which facilitate joint function. However, despite the multiplicity of roles of cartilage during human fetal life, surprisingly little is known about its transcriptome. To address this, a whole genome microarray expression profile was generated using RNA isolated from 18–22 week human distal femur fetal cartilage and compared with a database of control normal human tissues aggregated at UCLA, termed Celsius. Results 161 cartilage-selective genes were identified, defined as genes significantly expressed in cartilage with low expression and little variation across a panel of 34 non-cartilage tissues. Among these 161 genes were cartilage-specific genes such as cartilage collagen genes and 25 genes which have been associated with skeletal phenotypes in humans and/or mice. Many of the other cartilage-selective genes do not have established roles in cartilage or are novel, unannotated genes. Quantitative RT-PCR confirmed the unique pattern of gene expression observed by microarray analysis. Conclusion Defining the gene expression pattern for cartilage has identified new genes that may contribute to human skeletogenesis as well as provided further candidate genes for skeletal dysplasias. The data suggest that fetal cartilage is a complex and transcriptionally active tissue and demonstrate that the set of genes selectively expressed in the tissue has been greatly underestimated.

  20. Human gene therapy and imaging in neurological diseases

    International Nuclear Information System (INIS)

    Jacobs, Andreas H.; Winkler, Alexandra; Castro, Maria G.; Lowenstein, Pedro

    2005-01-01

    Molecular imaging aims to assess non-invasively disease-specific biological and molecular processes in animal models and humans in vivo. Apart from precise anatomical localisation and quantification, the most intriguing advantage of such imaging is the opportunity it provides to investigate the time course (dynamics) of disease-specific molecular events in the intact organism. Further, molecular imaging can be used to address basic scientific questions, e.g. transcriptional regulation, signal transduction or protein/protein interaction, and will be essential in developing treatment strategies based on gene therapy. Most importantly, molecular imaging is a key technology in translational research, helping to develop experimental protocols which may later be applied to human patients. Over the past 20 years, imaging based on positron emission tomography (PET) and magnetic resonance imaging (MRI) has been employed for the assessment and ''phenotyping'' of various neurological diseases, including cerebral ischaemia, neurodegeneration and brain gliomas. While in the past neuro-anatomical studies had to be performed post mortem, molecular imaging has ushered in the era of in vivo functional neuro-anatomy by allowing neuroscience to image structure, function, metabolism and molecular processes of the central nervous system in vivo in both health and disease. Recently, PET and MRI have been successfully utilised together in the non-invasive assessment of gene transfer and gene therapy in humans. To assess the efficiency of gene transfer, the same markers are being used in animals and humans, and have been applied for phenotyping human disease. Here, we review the imaging hallmarks of focal and disseminated neurological diseases, such as cerebral ischaemia, neurodegeneration and glioblastoma multiforme, as well as the attempts to translate gene therapy's experimental knowledge into clinical applications and the way in which this process is being promoted through the use of

  1. Low-intensity pulsed ultrasound stimulates cell proliferation, proteoglycan synthesis and expression of growth factor-related genes in human nucleus pulposus cell line

    Directory of Open Access Journals (Sweden)

    Y Kobayashi

    2009-06-01

    Full Text Available Low-intensity pulsed ultrasound (LIPUS stimulation has been shown to effect differentiation and activation of human chondrocytes. A study involving stimulation of rabbit disc cells with LIPUS revealed upregulation of cell proliferation and proteoglycan (PG synthesis. However, the effect of LIPUS on human nucleus pulposus cells has not been investigated. In the present study, therefore, we investigated whether LIPUS stimulation of a human nucleus pulposus cell line (HNPSV-1 exerted a positive effect on cellular activity. HNPSV-1 cells were encapsulated in 1.2% sodium alginate solution at 1x105 cells/ml and cultured at 10 beads/well in 6-well plates. The cells were stimulated for 20 min each day using a LIPUS generator, and the effects of LIPUS were evaluated by measuring DNA and PG synthesis. Furthermore, mRNA expression was analyzed by cDNA microarray using total RNA extracted from the cultured cells. Our study revealed no significant difference in cell proliferation between the control and the ultrasound treated groups. However, PG production was significantly upregulated in HNPSV cells stimulated at intensities of 15, 30, 60, and 120 mW/cm2 compared with the control. The results of cDNA array showed that LIPUS significantly stimulated the gene expression of growth factors and their receptors (BMP2, FGF7, TGFbetaR1 EGFRF1, VEGF. These findings suggest that LIPUS stimulation upregulates PG production in human nucleus pulposus cells by the enhancement of several matrix-related genes including growth factor-related genes. Safe and non-invasive stimulation using LIPUS may be a useful treatment for delaying the progression of disc degeneration.

  2. Polycythemia in transgenic mice expressing the human erythropoietin gene

    International Nuclear Information System (INIS)

    Semenza, G.L.; Traystman, M.D.; Gearhart, J.D.; Antonarakis, S.E.

    1989-01-01

    Erythropoietin is a glycoprotein hormone that regulates mammalian erythropoiesis. To study the expression of the human erythropoietin gene, EPO, 4 kilobases of DNA encompassing the gene with 0.4 kilobase of 5' flanking sequence and 0.7 kilobase of 3' flanking sequence was microinjected into fertilized mouse eggs. Transgenic mice were generated that are polycythemic, with increased erythrocytic indices in peripheral blood, increased numbers of erythroid precursors in hematopoietic tissue, and increased serum erythropoietin levels. Transgenic homozygotes show a greater degree of polycythemia than do heterozygotes as well as striking extramedullary erythropoiesis. Human erythropoietin RNA was found not only in fetal liver, adult liver, and kidney but also in all other transgenic tissues analyzed. Anemia induced increased human erythropoietin RNA levels in liver but not kidney. These transgenic mice represent a unique model of polycythemia due to increased erythropoietin levels

  3. Homology of yeast photoreactivating gene fragment with human genomic digests

    International Nuclear Information System (INIS)

    Meechan, P.J.; Milam, K.M.; Cleaver, J.E.

    1984-01-01

    Enzymatic photoreactivation of UV-induced DNA lesions has been demonstrated for a variety of prokaryotic and eukaryotic organisms. Its presence in placental mammals, however, has not been clearly established. The authors attempted to resolve this question by assaying for the presence (or absence) of sequences in human DNA complimentary to a fragment of the photoreactivating gene from S. cerevisiae that has recently been cloned. In another study, DNA from human, chick E. coli and yeast cells was digested with either HindIII of BglII, electrophoresed on a 0.5% agarose gel, transferred (Southern blot) to a nylon membrane and probed for homology against a Sau3A restriction fragment from S. cerevisiae that compliments phr/sup -/ cells. Hybridization to human DNA digests was observed only under relatively non-stringent conditions indicating the gene is not conserved in placental mammals. These results are correlated with current literature data concerning photoreactivating enzymes

  4. Delivery of the Sox9 gene promotes chondrogenic differentiation of human umbilical cord blood-derived mesenchymal stem cells in an in vitro model

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Z.H. [Department of Otolaryngology - Head and Neck Surgery, The Second Hospital, Xi' an Jiaotong University, Xi' an (China); Li, X.L. [Department of Dermatology, The Second Hospital, Xi' an Jiaotong University, Xi' an (China); He, X.J. [Department of Orthopedics, The Second Hospital, Xi' an Jiaotong University, Xi' an (China); Wu, B.J.; Xu, M. [Department of Otolaryngology - Head and Neck Surgery, The Second Hospital, Xi' an Jiaotong University, Xi' an (China); Chang, H.M. [Department of Otolaryngology - Head and Neck Surgery, Affiliated Hospital of Xi' an Medical University, Xi' an (China); Zhang, X.H. [Department of Otolaryngology - Head and Neck Surgery, The Second Hospital, Xi' an Jiaotong University, Xi' an (China); Xing, Z. [Department of Clinical Dentistry, Faculty of Dentistry, Center for Clinical Dental Research, University of Bergen, Bergen (Norway); Jing, X.H.; Kong, D.M.; Kou, X.H.; Yang, Y.Y. [Department of Otolaryngology - Head and Neck Surgery, The Second Hospital, Xi' an Jiaotong University, Xi' an (China)

    2014-03-18

    SRY-related high-mobility-group box 9 (Sox9) gene is a cartilage-specific transcription factor that plays essential roles in chondrocyte differentiation and cartilage formation. The aim of this study was to investigate the feasibility of genetic delivery of Sox9 to enhance chondrogenic differentiation of human umbilical cord blood-derived mesenchymal stem cells (hUC-MSCs). After they were isolated from human umbilical cord blood within 24 h after delivery of neonates, hUC-MSCs were untreated or transfected with a human Sox9-expressing plasmid or an empty vector. The cells were assessed for morphology and chondrogenic differentiation. The isolated cells with a fibroblast-like morphology in monolayer culture were positive for the MSC markers CD44, CD105, CD73, and CD90, but negative for the differentiation markers CD34, CD45, CD19, CD14, or major histocompatibility complex class II. Sox9 overexpression induced accumulation of sulfated proteoglycans, without altering the cellular morphology. Immunocytochemistry demonstrated that genetic delivery of Sox9 markedly enhanced the expression of aggrecan and type II collagen in hUC-MSCs compared with empty vector-transfected counterparts. Reverse transcription-polymerase chain reaction analysis further confirmed the elevation of aggrecan and type II collagen at the mRNA level in Sox9-transfected cells. Taken together, short-term Sox9 overexpression facilitates chondrogenesis of hUC-MSCs and may thus have potential implications in cartilage tissue engineering.

  5. Delivery of the Sox9 gene promotes chondrogenic differentiation of human umbilical cord blood-derived mesenchymal stem cells in an in vitro model

    Directory of Open Access Journals (Sweden)

    Z.H. Wang

    2014-04-01

    Full Text Available SRY-related high-mobility-group box 9 (Sox9 gene is a cartilage-specific transcription factor that plays essential roles in chondrocyte differentiation and cartilage formation. The aim of this study was to investigate the feasibility of genetic delivery of Sox9 to enhance chondrogenic differentiation of human umbilical cord blood-derived mesenchymal stem cells (hUC-MSCs. After they were isolated from human umbilical cord blood within 24 h after delivery of neonates, hUC-MSCs were untreated or transfected with a human Sox9-expressing plasmid or an empty vector. The cells were assessed for morphology and chondrogenic differentiation. The isolated cells with a fibroblast-like morphology in monolayer culture were positive for the MSC markers CD44, CD105, CD73, and CD90, but negative for the differentiation markers CD34, CD45, CD19, CD14, or major histocompatibility complex class II. Sox9 overexpression induced accumulation of sulfated proteoglycans, without altering the cellular morphology. Immunocytochemistry demonstrated that genetic delivery of Sox9 markedly enhanced the expression of aggrecan and type II collagen in hUC-MSCs compared with empty vector-transfected counterparts. Reverse transcription-polymerase chain reaction analysis further confirmed the elevation of aggrecan and type II collagen at the mRNA level in Sox9-transfected cells. Taken together, short-term Sox9 overexpression facilitates chondrogenesis of hUC-MSCs and may thus have potential implications in cartilage tissue engineering.

  6. Delivery of the Sox9 gene promotes chondrogenic differentiation of human umbilical cord blood-derived mesenchymal stem cells in an in vitro model

    International Nuclear Information System (INIS)

    Wang, Z.H.; Li, X.L.; He, X.J.; Wu, B.J.; Xu, M.; Chang, H.M.; Zhang, X.H.; Xing, Z.; Jing, X.H.; Kong, D.M.; Kou, X.H.; Yang, Y.Y.

    2014-01-01

    SRY-related high-mobility-group box 9 (Sox9) gene is a cartilage-specific transcription factor that plays essential roles in chondrocyte differentiation and cartilage formation. The aim of this study was to investigate the feasibility of genetic delivery of Sox9 to enhance chondrogenic differentiation of human umbilical cord blood-derived mesenchymal stem cells (hUC-MSCs). After they were isolated from human umbilical cord blood within 24 h after delivery of neonates, hUC-MSCs were untreated or transfected with a human Sox9-expressing plasmid or an empty vector. The cells were assessed for morphology and chondrogenic differentiation. The isolated cells with a fibroblast-like morphology in monolayer culture were positive for the MSC markers CD44, CD105, CD73, and CD90, but negative for the differentiation markers CD34, CD45, CD19, CD14, or major histocompatibility complex class II. Sox9 overexpression induced accumulation of sulfated proteoglycans, without altering the cellular morphology. Immunocytochemistry demonstrated that genetic delivery of Sox9 markedly enhanced the expression of aggrecan and type II collagen in hUC-MSCs compared with empty vector-transfected counterparts. Reverse transcription-polymerase chain reaction analysis further confirmed the elevation of aggrecan and type II collagen at the mRNA level in Sox9-transfected cells. Taken together, short-term Sox9 overexpression facilitates chondrogenesis of hUC-MSCs and may thus have potential implications in cartilage tissue engineering

  7. A Gene Implicated in Activation of Retinoic Acid Receptor Targets Is a Novel Renal Agenesis Gene in Humans

    DEFF Research Database (Denmark)

    Brophy, Patrick D.; Rasmussen, Maria; Parida, Mrutyunjaya

    2017-01-01

    investigations have identified several gene variants that cause RA, including EYA1, LHX1, and WT1 However, whereas compound null mutations of genes encoding α and γ retinoic acid receptors (RARs) cause RA in mice, to date there have been no reports of variants in RAR genes causing RA in humans. In this study, we...... in humans....

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

    Directory of Open Access Journals (Sweden)

    Salvador Fernández-Arroyo

    2015-10-01

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

  9. Network Analysis of Human Genes Influencing Susceptibility to Mycobacterial Infections

    Science.gov (United States)

    Lipner, Ettie M.; Garcia, Benjamin J.; Strong, Michael

    2016-01-01

    Tuberculosis and nontuberculous mycobacterial infections constitute a high burden of pulmonary disease in humans, resulting in over 1.5 million deaths per year. Building on the premise that genetic factors influence the instance, progression, and defense of infectious disease, we undertook a systems biology approach to investigate relationships among genetic factors that may play a role in increased susceptibility or control of mycobacterial infections. We combined literature and database mining with network analysis and pathway enrichment analysis to examine genes, pathways, and networks, involved in the human response to Mycobacterium tuberculosis and nontuberculous mycobacterial infections. This approach allowed us to examine functional relationships among reported genes, and to identify novel genes and enriched pathways that may play a role in mycobacterial susceptibility or control. Our findings suggest that the primary pathways and genes influencing mycobacterial infection control involve an interplay between innate and adaptive immune proteins and pathways. Signaling pathways involved in autoimmune disease were significantly enriched as revealed in our networks. Mycobacterial disease susceptibility networks were also examined within the context of gene-chemical relationships, in order to identify putative drugs and nutrients with potential beneficial immunomodulatory or anti-mycobacterial effects. PMID:26751573

  10. Serial Analysis of Gene Expression: Applications in Human Studies

    Directory of Open Access Journals (Sweden)

    Tuteja Renu

    2004-01-01

    Full Text Available Serial analysis of gene expression (SAGE is a powerful tool, which provides quantitative and comprehensive expression profile of genes in a given cell population. It works by isolating short fragments of genetic information from the expressed genes that are present in the cell being studied. These short sequences, called SAGE tags, are linked together for efficient sequencing. The frequency of each SAGE tag in the cloned multimers directly reflects the transcript abundance. Therefore, SAGE results in an accurate picture of gene expression at both the qualitative and the quantitative levels. It does not require a hybridization probe for each transcript and allows new genes to be discovered. This technique has been applied widely in human studies and various SAGE tags/SAGE libraries have been generated from different cells/tissues such as dendritic cells, lung fibroblast cells, oocytes, thyroid tissue, B-cell lymphoma, cultured keratinocytes, muscles, brain tissues, sciatic nerve, cultured Schwann cells, cord blood-derived mast cells, retina, macula, retinal pigment epithelial cells, skin cells, and so forth. In this review we present the updated information on the applications of SAGE technology mainly to human studies.

  11. XBP1-Independent UPR Pathways Suppress C/EBP-β Mediated Chondrocyte Differentiation in ER-Stress Related Skeletal Disease.

    Directory of Open Access Journals (Sweden)

    Trevor L Cameron

    2015-09-01

    Full Text Available Schmid metaphyseal chondrodysplasia (MCDS involves dwarfism and growth plate cartilage hypertrophic zone expansion resulting from dominant mutations in the hypertrophic zone collagen, Col10a1. Mouse models phenocopying MCDS through the expression of an exogenous misfolding protein in the endoplasmic reticulum (ER in hypertrophic chondrocytes have demonstrated the central importance of ER stress in the pathology of MCDS. The resultant unfolded protein response (UPR in affected chondrocytes involved activation of canonical ER stress sensors, IRE1, ATF6, and PERK with the downstream effect of disrupted chondrocyte differentiation. Here, we investigated the role of the highly conserved IRE1/XBP1 pathway in the pathology of MCDS. Mice with a MCDS collagen X p.N617K knock-in mutation (ColXN617K were crossed with mice in which Xbp1 was inactivated specifically in cartilage (Xbp1CartΔEx2, generating the compound mutant, C/X. The severity of dwarfism and hypertrophic zone expansion in C/X did not differ significantly from ColXN617K, revealing surprising redundancy for the IRE1/XBP1 UPR pathway in the pathology of MCDS. Transcriptomic analyses of hypertrophic zone cartilage identified differentially expressed gene cohorts in MCDS that are pathologically relevant (XBP1-independent or pathologically redundant (XBP1-dependent. XBP1-independent gene expression changes included large-scale transcriptional attenuation of genes encoding secreted proteins and disrupted differentiation from proliferative to hypertrophic chondrocytes. Moreover, these changes were consistent with disruption of C/EBP-β, a master regulator of chondrocyte differentiation, by CHOP, a transcription factor downstream of PERK that inhibits C/EBP proteins, and down-regulation of C/EBP-β transcriptional co-factors, GADD45-β and RUNX2. Thus we propose that the pathology of MCDS is underpinned by XBP1 independent UPR-induced dysregulation of C/EBP-β-mediated chondrocyte differentiation

  12. Exploring the potential relevance of human-specific genes to complex disease

    Directory of Open Access Journals (Sweden)

    Cooper David N

    2011-01-01

    Full Text Available Abstract Although human disease genes generally tend to be evolutionarily more ancient than non-disease genes, complex disease genes appear to be represented more frequently than Mendelian disease genes among genes of more recent evolutionary origin. It is therefore proposed that the analysis of human-specific genes might provide new insights into the genetics of complex disease. Cross-comparison with the Human Gene Mutation Database (http://www.hgmd.org revealed a number of examples of disease-causing and disease-associated mutations in putatively human-specific genes. A sizeable proportion of these were missense polymorphisms associated with complex disease. Since both human-specific genes and genes associated with complex disease have often experienced particularly rapid rates of evolutionary change, either due to weaker purifying selection or positive selection, it is proposed that a significant number of human-specific genes may play a role in complex disease.

  13. A combined approach for the assessment of cell viability and cell functionality of human fibrochondrocytes for use in tissue engineering.

    Directory of Open Access Journals (Sweden)

    Ingrid Garzón

    Full Text Available Temporo-mandibular joint disc disorders are highly prevalent in adult populations. Autologous chondrocyte implantation is a well-established method for the treatment of several chondral defects. However, very few studies have been carried out using human fibrous chondrocytes from the temporo-mandibular joint (TMJ. One of the main drawbacks associated to chondrocyte cell culture is the possibility that chondrocyte cells kept in culture tend to de-differentiate and to lose cell viability under in in-vitro conditions. In this work, we have isolated human temporo-mandibular joint fibrochondrocytes (TMJF from human disc and we have used a highly-sensitive technique to determine cell viability, cell proliferation and gene expression of nine consecutive cell passages to determine the most appropriate cell passage for use in tissue engineering and future clinical use. Our results revealed that the most potentially viable and functional cell passages were P5-P6, in which an adequate equilibrium between cell viability and the capability to synthesize all major extracellular matrix components exists. The combined action of pro-apoptotic (TRAF5, PHLDA1 and anti-apoptotic genes (SON, HTT, FAIM2 may explain the differential cell viability levels that we found in this study. These results suggest that TMJF should be used at P5-P6 for cell therapy protocols.

  14. A combined approach for the assessment of cell viability and cell functionality of human fibrochondrocytes for use in tissue engineering.

    Science.gov (United States)

    Garzón, Ingrid; Carriel, Victor; Marín-Fernández, Ana Belén; Oliveira, Ana Celeste; Garrido-Gómez, Juan; Campos, Antonio; Sánchez-Quevedo, María Del Carmen; Alaminos, Miguel

    2012-01-01

    Temporo-mandibular joint disc disorders are highly prevalent in adult populations. Autologous chondrocyte implantation is a well-established method for the treatment of several chondral defects. However, very few studies have been carried out using human fibrous chondrocytes from the temporo-mandibular joint (TMJ). One of the main drawbacks associated to chondrocyte cell culture is the possibility that chondrocyte cells kept in culture tend to de-differentiate and to lose cell viability under in in-vitro conditions. In this work, we have isolated human temporo-mandibular joint fibrochondrocytes (TMJF) from human disc and we have used a highly-sensitive technique to determine cell viability, cell proliferation and gene expression of nine consecutive cell passages to determine the most appropriate cell passage for use in tissue engineering and future clinical use. Our results revealed that the most potentially viable and functional cell passages were P5-P6, in which an adequate equilibrium between cell viability and the capability to synthesize all major extracellular matrix components exists. The combined action of pro-apoptotic (TRAF5, PHLDA1) and anti-apoptotic genes (SON, HTT, FAIM2) may explain the differential cell viability levels that we found in this study. These results suggest that TMJF should be used at P5-P6 for cell therapy protocols.

  15. Clone and expression of human transferrin receptor gene: a marker gene for magnetic resonance imaging

    International Nuclear Information System (INIS)

    Li Li; Liu Lizhi; Lv Yanchun; Liu Xuewen; Cui Chunyan; Wu Peihong; Liu Qicai; Ou Shanxing

    2007-01-01

    Objective: To clone human transferrin receptor (hTfR) gene and construct expression vector producing recombination protein. Methods: Human transferrin receptor gene cDNA was amplified by RT-PCR from human embryonic liver and lung tissue. Recombinant pcDNA3-hTfR and pEGFP-Cl-hTfR plasmids were constructed and confirmed by DNA sequencing. These plasmids were stably transfected into the HEK293 cells. The protein expression in vitro was confirmed by Western Blot. The efficiency of expression and the location of hTfR were also investigated by fluorescence microscopy and confocal fluorescence microscopy. Results: The full length cDNA of hTfR gene (2332 bp) was cloned and sequenced. The hTfR (190 000) was overexpressed in transfected HEK293 cells by Western blot analysis. Fluorescence micrographs displayed that the hTfR was expressed at high level and located predominantly in the cell surface. Conclusions: Human transferrin receptor (hTfR) gene has been successfully cloned and obtained high-level expression in HEK293 cells, and the recombination protein of hTfR distributed predominantly in the cell membrane. (authors)

  16. Importance of Donor Chondrocyte Viability for Osteochondral Allografts.

    Science.gov (United States)

    Cook, James L; Stannard, James P; Stoker, Aaron M; Bozynski, Chantelle C; Kuroki, Keiichi; Cook, Cristi R; Pfeiffer, Ferris M

    2016-05-01

    Osteochondral allograft (OCA) transplantation provides a biological treatment option for functional restoration of large articular cartilage defects in multiple joints. While successful outcomes after OCA transplantation have been linked to viable donor chondrocytes, the importance of donor cell viability has not been comprehensively validated. To use a canine model to determine the importance of donor chondrocyte viability at the time of implantation with respect to functional success of femoral condylar OCAs based on radiographic, gross, cell viability, histologic, biochemical, and biomechanical outcome measures. Controlled laboratory study. After approval was obtained from the institutional animal care and use committee, adult female dogs (N = 16) were implanted with 8-mm cylindrical OCAs from male dogs in the lateral and medial femoral condyles of 1 knee. OCAs were preserved for 28 or 60 days after procurement, and chondrocyte viability was quantified before implantation. Two different storage media, temperatures, and time points were used to obtain a spectrum of percentage chondrocyte viability at the time of implantation. A successful outcome was defined as an OCA that was associated with graft integration, maintenance of hyaline cartilage, lack of associated cartilage disorder, and lack of fibrillation, fissuring, or fibrous tissue infiltration of the allograft based on subjective radiographic, gross, and histologic assessments at 6 months after implantation. Chondrocyte viability ranged from 23% to 99% at the time of implantation. All successful grafts had >70% chondrocyte viability at the time of implantation, and no graft with chondrocyte viability <70% was associated with a successful outcome. Live-dead stained sections and histologic findings with respect to cell morphological features suggested that successful grafts were consistently composed of viable chondrocytes in lacunae, while grafts that were not successful were composed of nonviable

  17. Characterization of human septic sera induced gene expression modulation in human myocytes

    OpenAIRE

    Hussein, Shaimaa; Michael, Paul; Brabant, Danielle; Omri, Abdelwahab; Narain, Ravin; Passi, Kalpdrum; Ramana, Chilakamarti V.; Parrillo, Joseph E.; Kumar, Anand; Parissenti, Amadeo; Kumar, Aseem

    2009-01-01

    To gain a better understanding of the gene expression changes that occurs during sepsis, we have performed a cDNA microarray study utilizing a tissue culture model that mimics human sepsis. This study utilized an in vitro model of cultured human fetal cardiac myocytes treated with 10% sera from septic patients or 10% sera from healthy volunteers. A 1700 cDNA expression microarray was used to compare the transcription profile from human cardiac myocytes treated with septic sera vs normal sera....

  18. Developmental gene expression profiles of the human pathogen Schistosoma japonicum

    Directory of Open Access Journals (Sweden)

    McManus Donald P

    2009-03-01

    Full Text Available Abstract Background The schistosome blood flukes are complex trematodes and cause a chronic parasitic disease of significant public health importance worldwide, schistosomiasis. Their life cycle is characterised by distinct parasitic and free-living phases involving mammalian and snail hosts and freshwater. Microarray analysis was used to profile developmental gene expression in the Asian species, Schistosoma japonicum. Total RNAs were isolated from the three distinct environmental phases of the lifecycle – aquatic/snail (eggs, miracidia, sporocysts, cercariae, juvenile (lung schistosomula and paired but pre-egg laying adults and adult (paired, mature males and egg-producing females, both examined separately. Advanced analyses including ANOVA, principal component analysis, and hierarchal clustering provided a global synopsis of gene expression relationships among the different developmental stages of the schistosome parasite. Results Gene expression profiles were linked to the major environmental settings through which the developmental stages of the fluke have to adapt during the course of its life cycle. Gene ontologies of the differentially expressed genes revealed a wide range of functions and processes. In addition, stage-specific, differentially expressed genes were identified that were involved in numerous biological pathways and functions including calcium signalling, sphingolipid metabolism and parasite defence. Conclusion The findings provide a comprehensive database of gene expression in an important human pathogen, including transcriptional changes in genes involved in evasion of the host immune response, nutrient acquisition, energy production, calcium signalling, sphingolipid metabolism, egg production and tegumental function during development. This resource should help facilitate the identification and prioritization of new anti-schistosome drug and vaccine targets for the control of schistosomiasis.

  19. IGF-1R signaling in chondrocytes modulates growth plate development by interacting with the PTHrP/Ihh pathway.

    Science.gov (United States)

    Wang, Yongmei; Cheng, Zhiqiang; Elalieh, Hashem Z; Nakamura, Eiichiro; Nguyen, Minh-Thanh; Mackem, Susan; Clemens, Thomas L; Bikle, Daniel D; Chang, Wenhan

    2011-07-01

    Systemic derangements and perinatal death of generalized insulin-like growth factor 1 (IGF-1) and IGF-1 receptor (IGF-1R) knockout mice preclude definitive assessment of IGF-1R actions in growth-plate (GP) chondrocytes. We generated cartilage-specific Igf1r knockout ((Cart) Igf1r(-/-)) mice to investigate local control of chondrocyte differentiation in the GP by this receptor. These mice died shortly after birth and showed disorganized chondrocyte columns, delayed ossification and vascular invasion, decreased cell proliferation, increased apoptosis, and increased expression of parathyroid hormone-related protein (Pthrp) RNA and protein in their GPs. The increased Pthrp expression in the knockout GPs likely was due to an increase in gene transcription, as determined by the increased activity of a LacZ reporter that was inserted downstream of the endogenous PTHrP promoter and bred into the knockout mice. To circumvent the early death of (Cart) Igf1r(-/-) mice and investigate the role of IGF-1R during postnatal growth, we made tamoxifen (Tam)-inducible, cartilage-specific Igf1r knockout ((TamCart) Igf1r(-/-)) mice. At 2 weeks of age and 7 to 8 days after Tam injection, the (TamCart) Igf1r(-/-) mice showed growth retardation with a disorganized GP, reduced chondrocyte proliferation, decreased type 2 collagen and Indian Hedgehog (Ihh) expression, but increased expression of PTHrP. Consistent with in vivo observations, in vitro knockout of the Igf1r gene by adenoviral expression of Cre recombinase suppressed cell proliferation, promoted apoptosis, and increased Pthrp expression. Our data indicate that the IGF-1R in chondrocytes controls cell growth, survival, and differentiation in embryonic and postnatal GPs in part by suppression of Pthrp expression. Copyright © 2011 American Society for Bone and Mineral Research.

  20. Human amyloid beta protein gene locus: HaeIII RFLP

    Energy Technology Data Exchange (ETDEWEB)

    Taylor, J E; Gonzalez-DeWhitt, P A; Fuller, F; Cordell, B; Frossard, P M [California Biotechnology Inc., Mountain View (USA); Tinklenberg, J R; Davies, H D; Eng, L F; Yesavage, J A [Stanford Univ. School of Medicine, Palo Alto, CA (USA)

    1988-07-25

    A 2.2 kb EcoRI-EcoRI fragment from the 5{prime} end of the human amyloid beta protein cDNA was isolated from a human fibroblast cDNA library and subcloned into pGEM3. HaeIII (GGCC) detects 6 invariant bands at 0.5 kb, 1.0 kb, 1.1 kb, 1.3 kb, 1.4 kb and 1.6 kb and a two-allele polymorphism with bands at either 1.9 kb or 2.1 kb. Its frequency was studied in 50 North Americans. Human amyloid beta protein gene mapped to the long arm of chromosome 21 (21q11.2-21q21) by Southern blot analysis of human-rodent somatic cell hybrids. Co-dominant segregation was observed in two families (15 individuals).

  1. Cloning and chromosomal localization of the three human syntrophin genes

    Energy Technology Data Exchange (ETDEWEB)

    Feener, C.A.; Anderson, M.D.S.; Selig, S. [Children`s Hospital, Boston, MA (United States)] [and others

    1994-09-01

    Dystrophin, the protein product the Duchenne muscular dystrophy locus, is normally found to be associated with a complex of proteins. Among these dystrophin-associated proteins are the syntrophins, a group of 59 kDa membrane-associated proteins. When the syntrophins are purified based upon their association with dystrophin, they have been shown previously to form two distinct groups, the acidic ({alpha}) and basic ({beta}) forms. Based on peptide and rodent cDNA sequences, three separate syntrophin genes have been cloned and characterized from human tissues. The predicted amino acid sequences from these cDNA reveal that these proteins are related but are distinct with respect to charge, as predicted from their biochemistry. The family consists of one acidic ({alpha}-syntrophin, analogous to mouse syntrophin-1) and two basic ({beta}{sub 1}-syntrophin; and {beta}{sub 2}-syntrophin, analogous to mouse syntrophin-2) genes. Each of the three genes are widely expressed in a variety of human tissues, but the relative abundance of the three are unique with respect to each other. {alpha}-syntrophin is expressed primarily in skeletal muscle and heart as a single transcript. {beta}{sub 1}-syntrophin is expressed widely in up to five distinct transcript sizes, and is most abundant in brain. The human chromosomal locations of the three syntrophins are currently being mapped. {beta}{sub 1}-syntrophin maps to chromosome 8q23-24 and {beta}{sub 2}-syntrophin to chromosome 16. The {alpha}-syntrophin gene will be mapped accordingly. Although all three genes are candidates for neuromuscular diseases, the predominant expression of {alpha}-syntrophin in skeletal muscle and heart makes it a strong candidate to be involved in a neuromuscular disease.

  2. RNA-Guided Activation of Pluripotency Genes in Human Fibroblasts

    DEFF Research Database (Denmark)

    Xiong, Kai; Zhou, Yan; Blichfeld, Kristian Aabo

    2017-01-01

    -associated protein 9 (dCas9)-VP64 (CRISPRa) alone, or a combination of dCas9-VP64 and MS2-P65-HSF1 [synergistic activation mediator (SAM) system] mediated activation of five pluripotency genes: KLF4 (K), LIN28 (L), MYC (M), OCT4 (O), and SOX2 (S) in human cells (HEK293T, HeLa, HepG2, and primary fibroblasts...... could be obtained from these SAM fibroblasts. In conclusion, our study showed that CRISPR/Cas9-based ATFs are potent to activate and maintain transcription of endogenous human pluripotent genes. However, future improvements of the system are still required to improve activation efficiency and cellular...

  3. BcII RFLP for the human vimentin gene

    Energy Technology Data Exchange (ETDEWEB)

    Marcus, E M; Smith, B A; Telenius, H; Ponder, B A.J.; Mathew, C G.P. [Haddow Laboratories, Surrey (England); Landsvater, R M; Buys, C H.C.M. [State Univ. of Groningen (Netherlands); Ferrari, S [Temple University Medical School, Philadelphia, PA (USA)

    1988-09-26

    A 1.1 kb cDNA clone (hp4F1) encoding the human vimentin gene was identified in a human library by screening with 4F1, a hamster vimentin cDNA. BcII (TGATCA) recognizes a two allele polymorphism: bands A1 at 8.1 kb, and A2 at 3.6 kb. The allele frequency was determined in 47 unrelated Caucasian individuals. The RFLP was mapped to chromosome 10pter-10q23 using somatic cell hybrids and to 10p13 by in situ hybridization. Co-dominant segregation was observed in 2 informative families.

  4. Potential Effects of Horizontal Gene Exchange in the Human Gut.

    Science.gov (United States)

    Lerner, Aaron; Matthias, Torsten; Aminov, Rustam

    2017-01-01

    Many essential functions of the human body are dependent on the symbiotic microbiota, which is present at especially high numbers and diversity in the gut. This intricate host-microbe relationship is a result of the long-term coevolution between the two. While the inheritance of mutational changes in the host evolution is almost exclusively vertical, the main mechanism of bacterial evolution is horizontal gene exchange. The gut conditions, with stable temperature, continuous food supply, constant physicochemical conditions, extremely high concentration of microbial cells and phages, and plenty of opportunities for conjugation on the surfaces of food particles and host tissues, represent one of the most favorable ecological niches for horizontal gene exchange. Thus, the gut microbial system genetically is very dynamic and capable of rapid response, at the genetic level, to selection, for example, by antibiotics. There are many other factors to which the microbiota may dynamically respond including lifestyle, therapy, diet, refined food, food additives, consumption of pre- and probiotics, and many others. The impact of the changing selective pressures on gut microbiota, however, is poorly understood. Presumably, the gut microbiome responds to these changes by genetic restructuring of gut populations, driven mainly via horizontal gene exchange. Thus, our main goal is to reveal the role played by horizontal gene exchange in the changing landscape of the gastrointestinal microbiome and potential effect of these changes on human health in general and autoimmune diseases in particular.

  5. Potential Effects of Horizontal Gene Exchange in the Human Gut

    Directory of Open Access Journals (Sweden)

    Aaron Lerner

    2017-11-01

    Full Text Available Many essential functions of the human body are dependent on the symbiotic microbiota, which is present at especially high numbers and diversity in the gut. This intricate host–microbe relationship is a result of the long-term coevolution between the two. While the inheritance of mutational changes in the host evolution is almost exclusively vertical, the main mechanism of bacterial evolution is horizontal gene exchange. The gut conditions, with stable temperature, continuous food supply, constant physicochemical conditions, extremely high concentration of microbial cells and phages, and plenty of opportunities for conjugation on the surfaces of food particles and host tissues, represent one of the most favorable ecological niches for horizontal gene exchange. Thus, the gut microbial system genetically is very dynamic and capable of rapid response, at the genetic level, to selection, for example, by antibiotics. There are many other factors to which the microbiota may dynamically respond including lifestyle, therapy, diet, refined food, food additives, consumption of pre- and probiotics, and many others. The impact of the changing selective pressures on gut microbiota, however, is poorly understood. Presumably, the gut microbiome responds to these changes by genetic restructuring of gut populations, driven mainly via horizontal gene exchange. Thus, our main goal is to reveal the role played by horizontal gene exchange in the changing landscape of the gastrointestinal microbiome and potential effect of these changes on human health in general and autoimmune diseases in particular.

  6. Gene expression of manganese superoxide dismutase in human glioma cells

    Directory of Open Access Journals (Sweden)

    Novi S. Hardiany

    2010-02-01

    Full Text Available Aim This study analyze the MnSOD gene expression as endogenous antioxidant in human glioma cells compared with leucocyte cells as control.Methods MnSOD gene expression of 20 glioma patients was analyzed by measuring the relative expression of mRNA and enzyme activity of MnSOD in brain and leucocyte cells. The relative expression of mRNA MnSOD was determined by using quantitative Real Time RT-PCR and the enzyme activity of MnSOD using biochemical kit assay (xantine oxidase inhibition. Statistic analysis for mRNA and enzyme activity of MnSOD was performed using Kruskal Wallis test.Results mRNA of MnSOD in glioma cells of 70% sample was 0.015–0.627 lower, 10% was 1.002-1.059 and 20% was 1.409-6.915 higher than in leucocyte cells. Also the specific activity of MnSOD enzyme in glioma cells of 80% sample showed 0,064-0,506 lower and 20% sample was 1.249-2.718 higher than in leucocyte cells.Conclusion MnSOD gene expression in human glioma cells are significantly lower than its expression in leucocytes cells. (Med J Indones 2010; 19:21-5Keywords : MnSOD, glioma, gene expression

  7. Iterative design of peptide-based hydrogels and the effect of network electrostatics on primary chondrocyte behavior.

    Science.gov (United States)

    Sinthuvanich, Chomdao; Haines-Butterick, Lisa A; Nagy, Katelyn J; Schneider, Joel P

    2012-10-01

    Iterative peptide design was used to generate two peptide-based hydrogels to study the effect of network electrostatics on primary chondrocyte behavior. MAX8 and HLT2 peptides have formal charge states of +7 and +5 per monomer, respectively. These peptides undergo triggered folding and self-assembly to afford hydrogel networks having similar rheological behavior and local network morphologies, yet different electrostatic character. Each gel can be used to directly encapsulate and syringe-deliver cells. The influence of network electrostatics on cell viability after encapsulation and delivery, extracellular matrix deposition, gene expression, and the bulk mechanical properties of the gel-cell constructs as a function of culture time was assessed. The less electropositive HLT2 gel provides a microenvironment more conducive to chondrocyte encapsulation, delivery, and phenotype maintenance. Cell viability was higher for this gel and although a moderate number of cells dedifferentiated to a fibroblast-like phenotype, many retained their chondrocytic behavior. As a result, gel-cell constructs prepared with HLT2, cultured under static in vitro conditions, contained more GAG and type II collagen resulting in mechanically superior constructs. Chondrocytes delivered in the more electropositive MAX8 gel experienced a greater degree of cell death during encapsulation and delivery and the remaining viable cells were less prone to maintain their phenotype. As a result, MAX8 gel-cell constructs had fewer cells, of which a limited number were capable of laying down cartilage-specific ECM. Published by Elsevier Ltd.

  8. Muscle gene expression patterns in human rotator cuff pathology.

    Science.gov (United States)

    Choo, Alexander; McCarthy, Meagan; Pichika, Rajeswari; Sato, Eugene J; Lieber, Richard L; Schenk, Simon; Lane, John G; Ward, Samuel R

    2014-09-17

    Rotator cuff pathology is a common source of shoulder pain with variable etiology and pathoanatomical characteristics. Pathological processes of fatty infiltration, muscle atrophy, and fibrosis have all been invoked as causes for poor outcomes after rotator cuff tear repair. The aims of this study were to measure the expression of key genes associated with adipogenesis, myogenesis, and fibrosis in human rotator cuff muscle after injury and to compare the expression among groups of patients with varied severities of rotator cuff pathology. Biopsies of the supraspinatus muscle were obtained arthroscopically from twenty-seven patients in the following operative groups: bursitis (n = 10), tendinopathy (n = 7), full-thickness rotator cuff tear (n = 8), and massive rotator cuff tear (n = 2). Quantitative polymerase chain reaction (qPCR) was performed to characterize gene expression pathways involved in myogenesis, adipogenesis, and fibrosis. Patients with a massive tear demonstrated downregulation of the fibrogenic, adipogenic, and myogenic genes, indicating that the muscle was not in a state of active change and may have difficulty responding to stimuli. Patients with a full-thickness tear showed upregulation of fibrotic and adipogenic genes; at the tissue level, these correspond to the pathologies most detrimental to outcomes of surgical repair. Patients with bursitis or tendinopathy still expressed myogenic genes, indicating that the muscle may be attempting to accommodate the mechanical deficiencies induced by the tendon tear. Gene expression in human rotator cuff muscles varied according to tendon injury severity. Patients with bursitis and tendinopathy appeared to be expressing pro-myogenic genes, whereas patients with a full-thickness tear were expressing genes associated with fatty atrophy and fibrosis. In contrast, patients with a massive tear appeared to have downregulation of all gene programs except inhibition of myogenesis. These data highlight the

  9. Robust, synergistic regulation of human gene expression using TALE activators.

    Science.gov (United States)

    Maeder, Morgan L; Linder, Samantha J; Reyon, Deepak; Angstman, James F; Fu, Yanfang; Sander, Jeffry D; Joung, J Keith

    2013-03-01

    Artificial activators designed using transcription activator-like effector (TALE) technology have broad utility, but previous studies suggest that these monomeric proteins often exhibit low activities. Here we demonstrate that TALE activators can robustly function individually or in synergistic combinations to increase expression of endogenous human genes over wide dynamic ranges. These findings will encourage applications of TALE activators for research and therapy, and guide design of monomeric TALE-based fusion proteins.

  10. Genome-wide associations of gene expression variation in humans.

    Directory of Open Access Journals (Sweden)

    Barbara E Stranger

    2005-12-01

    Full Text Available The exploration of quantitative variation in human populations has become one of the major priorities for medical genetics. The successful identification of variants that contribute to complex traits is highly dependent on reliable assays and genetic maps. We have performed a genome-wide quantitative trait analysis of 630 genes in 60 unrelated Utah residents with ancestry from Northern and Western Europe using the publicly available phase I data of the International HapMap project. The genes are located in regions of the human genome with elevated functional annotation and disease interest including the ENCODE regions spanning 1% of the genome, Chromosome 21 and Chromosome 20q12-13.2. We apply three different methods of multiple test correction, including Bonferroni, false discovery rate, and permutations. For the 374 expressed genes, we find many regions with statistically significant association of single nucleotide polymorphisms (SNPs with expression variation in lymphoblastoid cell lines after correcting for multiple tests. Based on our analyses, the signal proximal (cis- to the genes of interest is more abundant and more stable than distal and trans across statistical methodologies. Our results suggest that regulatory polymorphism is widespread in the human genome and show that the 5-kb (phase I HapMap has sufficient density to enable linkage disequilibrium mapping in humans. Such studies will significantly enhance our ability to annotate the non-coding part of the genome and interpret functional variation. In addition, we demonstrate that the HapMap cell lines themselves may serve as a useful resource for quantitative measurements at the cellular level.

  11. Genome-Wide Associations of Gene Expression Variation in Humans.

    Directory of Open Access Journals (Sweden)

    2005-12-01

    Full Text Available The exploration of quantitative variation in human populations has become one of the major priorities for medical genetics. The successful identification of variants that contribute to complex traits is highly dependent on reliable assays and genetic maps. We have performed a genome-wide quantitative trait analysis of 630 genes in 60 unrelated Utah residents with ancestry from Northern and Western Europe using the publicly available phase I data of the International HapMap project. The genes are located in regions of the human genome with elevated functional annotation and disease interest including the ENCODE regions spanning 1% of the genome, Chromosome 21 and Chromosome 20q12-13.2. We apply three different methods of multiple test correction, including Bonferroni, false discovery rate, and permutations. For the 374 expressed genes, we find many regions with statistically significant association of single nucleotide polymorphisms (SNPs with expression variation in lymphoblastoid cell lines after correcting for multiple tests. Based on our analyses, the signal proximal (cis- to the genes of interest is more abundant and more stable than distal and trans across statistical methodologies. Our results suggest that regulatory polymorphism is widespread in the human genome and show that the 5-kb (phase I HapMap has sufficient density to enable linkage disequilibrium mapping in humans. Such studies will significantly enhance our ability to annotate the non-coding part of the genome and interpret functional variation. In addition, we demonstrate that the HapMap cell lines themselves may serve as a useful resource for quantitative measurements at the cellular level.

  12. Gene expression, nucleotide composition and codon usage bias of genes associated with human Y chromosome.

    Science.gov (United States)

    Choudhury, Monisha Nath; Uddin, Arif; Chakraborty, Supriyo

    2017-06-01

    Analysis of codon usage pattern is important to understand the genetic and evolutionary characteristics of genomes. We have used bioinformatic approaches to analyze the codon usage bias (CUB) of the genes located in human Y chromosome. Codon bias index (CBI) indicated that the overall extent of codon usage bias was low. The relative synonymous codon usage (RSCU) analysis suggested that approximately half of the codons out of 59 synonymous codons were most frequently used, and possessed a T or G at the third codon position. The codon usage pattern was different in different genes as revealed from correspondence analysis (COA). A significant correlation between effective number of codons (ENC) and various GC contents suggests that both mutation pressure and natural selection affect the codon usage pattern of genes located in human Y chromosome. In addition, Y-linked genes have significant difference in GC contents at the second and third codon positions, expression level, and codon usage pattern of some codons like the SPANX genes in X chromosome.

  13. Spina Bifida: Pathogenesis, Mechanisms, and Genes in Mice and Humans

    Directory of Open Access Journals (Sweden)

    Siti W. Mohd-Zin

    2017-01-01

    Full Text Available Spina bifida is among the phenotypes of the larger condition known as neural tube defects (NTDs. It is the most common central nervous system malformation compatible with life and the second leading cause of birth defects after congenital heart defects. In this review paper, we define spina bifida and discuss the phenotypes seen in humans as described by both surgeons and embryologists in order to compare and ultimately contrast it to the leading animal model, the mouse. Our understanding of spina bifida is currently limited to the observations we make in mouse models, which reflect complete or targeted knockouts of genes, which perturb the whole gene(s without taking into account the issue of haploinsufficiency, which is most prominent in the human spina bifida condition. We thus conclude that the need to study spina bifida in all its forms, both aperta and occulta, is more indicative of the spina bifida in surviving humans and that the measure of deterioration arising from caudal neural tube defects, more commonly known as spina bifida, must be determined by the level of the lesion both in mouse and in man.

  14. Promoter Methylation Analysis of IDH Genes in Human Gliomas

    International Nuclear Information System (INIS)

    Flanagan, Simon; Lee, Maggie; Li, Cheryl C. Y.; Suter, Catherine M.; Buckland, Michael E.

    2012-01-01

    Mutations in isocitrate dehydrogenase (IDH)-1 or -2 are found in the majority of WHO grade II and III astrocytomas and oligodendrogliomas, and secondary glioblastomas. Almost all described mutations are heterozygous missense mutations affecting a conserved arginine residue in the substrate binding site of IDH1 (R132) or IDH2 (R172). But the exact mechanism of IDH mutations in neoplasia is not understood. It has been proposed that IDH mutations impart a “toxic gain-of-function” to the mutant protein, however a dominant-negative effect of mutant IDH has also been described, implying that IDH may function as a tumor suppressor gene. As most, if not all, tumor suppressor genes are inactivated by epigenetic silencing, in a wide variety of tumors, we asked if IDH1 or IDH2 carry the epigenetic signature of a tumor suppressor by assessing cytosine methylation at their promoters. Methylation was quantified in 68 human brain tumors, including both IDH-mutant and IDH wildtype, by bisulfite pyrosequencing. In all tumors examined, CpG methylation levels were less than 8%. Our data demonstrate that inactivation of IDH function through promoter hypermethylation is not common in human gliomas and other brain tumors. These findings do not support a tumor suppressor role for IDH genes in human gliomas.

  15. STAT3 Target Genes Relevant to Human Cancers

    International Nuclear Information System (INIS)

    Carpenter, Richard L.; Lo, Hui-Wen

    2014-01-01

    Since its discovery, the STAT3 transcription factor has been extensively studied for its function as a transcriptional regulator and its role as a mediator of development, normal physiology, and pathology of many diseases, including cancers. These efforts have uncovered an array of genes that can be positively and negatively regulated by STAT3, alone and in cooperation with other transcription factors. Through regulating gene expression, STAT3 has been demonstrated to play a pivotal role in many cellular processes including oncogenesis, tumor growth and progression, and stemness. Interestingly, recent studies suggest that STAT3 may behave as a tumor suppressor by activating expression of genes known to inhibit tumorigenesis. Additional evidence suggested that STAT3 may elicit opposing effects depending on cellular context and tumor types. These mixed results signify the need for a deeper understanding of STAT3, including its upstream regulators, parallel transcription co-regulators, and downstream target genes. To help facilitate fulfilling this unmet need, this review will be primarily focused on STAT3 downstream target genes that have been validated to associate with tumorigenesis and/or malignant biology of human cancers

  16. Update of the human and mouse Fanconi anemia genes.

    Science.gov (United States)

    Dong, Hongbin; Nebert, Daniel W; Bruford, Elspeth A; Thompson, David C; Joenje, Hans; Vasiliou, Vasilis

    2015-11-24

    Fanconi anemia (FA) is a recessively inherited disease manifesting developmental abnormalities, bone marrow failure, and increased risk of malignancies. Whereas FA has been studied for nearly 90 years, only in the last 20 years have increasing numbers of genes been implicated in the pathogenesis associated with this genetic disease. To date, 19 genes have been identified that encode Fanconi anemia complementation group proteins, all of which are named or aliased, using the root symbol "FANC." Fanconi anemia subtype (FANC) proteins function in a common DNA repair pathway called "the FA pathway," which is essential for maintaining genomic integrity. The various FANC mutant proteins contribute to distinct steps associated with FA pathogenesis. Herein, we provide a review update of the 19 human FANC and their mouse orthologs, an evolutionary perspective on the FANC genes, and the functional significance of the FA DNA repair pathway in association with clinical disorders. This is an example of a set of genes--known to exist in vertebrates, invertebrates, plants, and yeast--that are grouped together on the basis of shared biochemical and physiological functions, rather than evolutionary phylogeny, and have been named on this basis by the HUGO Gene Nomenclature Committee (HGNC).

  17. Sarcoptes scabiei mites modulate gene expression in human skin equivalents.

    Directory of Open Access Journals (Sweden)

    Marjorie S Morgan

    Full Text Available The ectoparasitic mite, Sarcoptes scabiei that burrows in the epidermis of mammalian skin has a long co-evolution with its hosts. Phenotypic studies show that the mites have the ability to modulate cytokine secretion and expression of cell adhesion molecules in cells of the skin and other cells of the innate and adaptive immune systems that may assist the mites to survive in the skin. The purpose of this study was to identify genes in keratinocytes and fibroblasts in human skin equivalents (HSEs that changed expression in response to the burrowing of live scabies mites. Overall, of the more than 25,800 genes measured, 189 genes were up-regulated >2-fold in response to scabies mite burrowing while 152 genes were down-regulated to the same degree. HSEs differentially expressed large numbers of genes that were related to host protective responses including those involved in immune response, defense response, cytokine activity, taxis, response to other organisms, and cell adhesion. Genes for the expression of interleukin-1α (IL-1α precursor, IL-1β, granulocyte/macrophage-colony stimulating factor (GM-CSF precursor, and G-CSF precursor were up-regulated 2.8- to 7.4-fold, paralleling cytokine secretion profiles. A large number of genes involved in epithelium development and keratinization were also differentially expressed in response to live scabies mites. Thus, these skin cells are directly responding as expected in an inflammatory response to products of the mites and the disruption of the skin's protective barrier caused by burrowing. This suggests that in vivo the interplay among these skin cells and other cell types, including Langerhans cells, dendritic cells, lymphocytes and endothelial cells, is responsible for depressing the host's protective response allowing these mites to survive in the skin.

  18. Sarcoptes scabiei Mites Modulate Gene Expression in Human Skin Equivalents

    Science.gov (United States)

    Morgan, Marjorie S.; Arlian, Larry G.; Markey, Michael P.

    2013-01-01

    The ectoparasitic mite, Sarcoptes scabiei that burrows in the epidermis of mammalian skin has a long co-evolution with its hosts. Phenotypic studies show that the mites have the ability to modulate cytokine secretion and expression of cell adhesion molecules in cells of the skin and other cells of the innate and adaptive immune systems that may assist the mites to survive in the skin. The purpose of this study was to identify genes in keratinocytes and fibroblasts in human skin equivalents (HSEs) that changed expression in response to the burrowing of live scabies mites. Overall, of the more than 25,800 genes measured, 189 genes were up-regulated >2-fold in response to scabies mite burrowing while 152 genes were down-regulated to the same degree. HSEs differentially expressed large numbers of genes that were related to host protective responses including those involved in immune response, defense response, cytokine activity, taxis, response to other organisms, and cell adhesion. Genes for the expression of interleukin-1α (IL-1α) precursor, IL-1β, granulocyte/macrophage-colony stimulating factor (GM-CSF) precursor, and G-CSF precursor were up-regulated 2.8- to 7.4-fold, paralleling cytokine secretion profiles. A large number of genes involved in epithelium development and keratinization were also differentially expressed in response to live scabies mites. Thus, these skin cells are directly responding as expected in an inflammatory response to products of the mites and the disruption of the skin’s protective barrier caused by burrowing. This suggests that in vivo the interplay among these skin cells and other cell types, including Langerhans cells, dendritic cells, lymphocytes and endothelial cells, is responsible for depressing the host’s protective response allowing these mites to survive in the skin. PMID:23940705

  19. [Construction of porous hydroxyapatite (HA) block loaded with cultured chondrocytes].

    Science.gov (United States)

    Yan, M; Dang, G

    1999-07-01

    To construct a kind of bone healing enhancing implant with cultured chondrocytes bound to hydroxyapatite (HA). Chondrocytes were obtained from the costicartilage of rat and were cultured on the porous HA blocks, 3 mm x 3 mm x 4 mm size, for three and seven days. Scanning electron micrograph was taken to show whether the cells grew outside and inside the pore of HA block. The cells cultured on tiny glass sheet for 2 days were used to prove where the cells come from by in situ hybridization technique with alpha1 (II) cDNA probe. Scanning electron micrographs showed that the pores of the HA surface and inside of the blocks are filled with cultured cells, especially the longer cultured block. The cells were chondrocytes confirmed by in situ hybridization. The porous HA can be used as cell cultured substrate and chondrocyte can adhere and proliferate inside the porous HA block.

  20. Latent Transforming Growth Factor-beta1 Functionalised Electrospun Scaffolds Promote Human Cartilage Differentiation: Towards an Engineered Cartilage Construct

    Directory of Open Access Journals (Sweden)

    Erh-Hsuin Lim

    2013-11-01

    Full Text Available BackgroundTo overcome the potential drawbacks of a short half-life and dose-related adverse effects of using active transforming growth factor-beta 1 for cartilage engineering, a cell-mediated latent growth factor activation strategy was developed incorporating latent transforming growth factor-β1 (LTGF into an electrospun poly(L-lactide scaffold.MethodsThe electrospun scaffold was surface modified with NH3 plasma and biofunctionalised with LTGF to produce both random and orientated biofunctionalised electrospun scaffolds. Scaffold surface chemical analysis and growth factor bioavailability assays were performed. In vitro biocompatibility and human nasal chondrocyte gene expression with these biofunctionalised electrospun scaffold templates were assessed. In vivo chondrogenic activity and chondrocyte gene expression were evaluated in athymic rats.ResultsChemical analysis demonstrated that LTGF anchored to the scaffolds was available for enzymatic, chemical and cell activation. The biofunctionalised scaffolds were non-toxic. Gene expression suggested chondrocyte re-differentiation after 14 days in culture. By 6 weeks, the implanted biofunctionalised scaffolds had induced highly passaged chondrocytes to re-express Col2A1 and produce type II collagen.ConclusionsWe have demonstrated a proof of concept for cell-mediated activation of anchored growth factors using a novel biofunctionalised scaffold in cartilage engineering. This presents a platform for development of protein delivery systems and for tissue engineering.

  1. Rabbit articular cartilage defects treated by allogenic chondrocyte transplantation

    OpenAIRE

    Boopalan, P. R. J. V. C.; Sathishkumar, Solomon; Kumar, Senthil; Chittaranjan, Samuel

    2006-01-01

    Articular cartilage defects have a poor capacity for repair. Most of the current treatment options result in the formation of fibro-cartilage, which is functionally inferior to normal hyaline articular cartilage. We studied the effectiveness of allogenic chondrocyte transplantation for focal articular cartilage defects in rabbits. Chondrocytes were cultured in vitro from cartilage harvested from the knee joints of a New Zealand White rabbit. A 3 mm defect was created in the articular cartilag...

  2. Inhibition of Chondrocyte Hypertrophy of Osteoarthritis by Disruptor Peptide

    Science.gov (United States)

    2017-07-01

    with PTHR and inhibits the pathogenic beta-catenin- mediated PTHR signaling switch. In Aim 2, we will define the role of disruptor peptide in...confirmed that disruptor peptide conjugated to penetratin can enter cells. Importantly, disruptor peptide can reverse the beta-catenin- mediated PTH... mediated PTHR signaling switch in chondrocytes. Mouse primary chondrocytes express both β-catenin and PTHR. Our data showed that Pen-dis- pep

  3. Co-culture of chondrocytes and bone marrow mesenchymal stem cells in vitro enhances the expression of cartilaginous extracellular matrix components

    Directory of Open Access Journals (Sweden)

    Chang Qing

    2011-04-01

    Full Text Available Chondrocytes and bone marrow mesenchymal stem cells (BMSCs are frequently used as seed cells in cartilage tissue engineering. In the present study, we determined if the co-culture of rabbit articular chondrocytes and BMSCs in vitro promotes the expression of cartilaginous extracellular matrix and, if so, what is the optimal ratio of the two cell types. Cultures of rabbit articular chondrocytes and BMSCs were expanded in vitro and then cultured individually or at a chondrocyte:BMSC ratio of 4:1, 2:1, 1:1, 1:2, 1:4 for 21 days and cultured in DMEM/F12. BMSCs were cultured in chondrogenic induction medium. Quantitative real-time RT-PCR and Western blot were used to evaluate gene expression. In the co-cultures, type II collagen and aggrecan expression increased on days 14 and 21. At the mRNA level, the expression of type II collagen and aggrecan on day 21 was much higher in the 4:1, 2:1, and 1:1 groups than in either the articular chondrocyte group or the induced BMSC group, and the best ratio of co-culture groups seems to be 2:1. Also on day 21, the expression of type II collagen and aggrecan proteins in the 2:1 group was much higher than in all other groups. The results demonstrate that the co-culture of rabbit chondrocytes and rabbit BMSCs at defined ratios can promote the expression of cartilaginous extracellular matrix. The optimal cell ratio appears to be 2:1 (chondrocytes:BMSCs. This approach has potential applications in cartilage tissue engineering since it provides a protocol for maintaining and promoting seed-cell differentiation and function.

  4. Co-culture of chondrocytes and bone marrow mesenchymal stem cells in vitro enhances the expression of cartilaginous extracellular matrix components.

    Science.gov (United States)

    Qing, Chang; Wei-ding, Cui; Wei-min, Fan

    2011-04-01

    Chondrocytes and bone marrow mesenchymal stem cells (BMSCs) are frequently used as seed cells in cartilage tissue engineering. In the present study, we determined if the co-culture of rabbit articular chondrocytes and BMSCs in vitro promotes the expression of cartilaginous extracellular matrix and, if so, what is the optimal ratio of the two cell types. Cultures of rabbit articular chondrocytes and BMSCs were expanded in vitro and then cultured individually or at a chondrocyte:BMSC ratio of 4:1, 2:1, 1:1, 1:2, 1:4 for 21 days and cultured in DMEM/F12. BMSCs were cultured in chondrogenic induction medium. Quantitative real-time RT-PCR and Western blot were used to evaluate gene expression. In the co-cultures, type II collagen and aggrecan expression increased on days 14 and 21. At the mRNA level, the expression of type II collagen and aggrecan on day 21 was much higher in the 4:1, 2:1, and 1:1 groups than in either the articular chondrocyte group or the induced BMSC group, and the best ratio of co-culture groups seems to be 2:1. Also on day 21, the expression of type II collagen and aggrecan proteins in the 2:1 group was much higher than in all other groups. The results demonstrate that the co-culture of rabbit chondrocytes and rabbit BMSCs at defined ratios can promote the expression of cartilaginous extracellular matrix. The optimal cell ratio appears to be 2:1 (chondrocytes:BMSCs). This approach has potential applications in cartilage tissue engineering since it provides a protocol for maintaining and promoting seed-cell differentiation and function.

  5. The signs of differentiation of chondrocytes in the formation of early cartilage lesions in the elderly

    Directory of Open Access Journals (Sweden)

    Elena Vasil'evna Chetina

    2011-01-01

    Results. The activity of collagen type II cleavage was shown to be increased in the area of age-related OA-like cartilage lesions. This was accompanied by the high expression of collagenases of metalloproteinases (MMP 1, 14 (MT1-MMP, aggrecanases - desintegrin and MMP with thrombospondin type 1 motif (ADAMTS 5, the cytokines of interleukins (IL 1α/β and tumor necrosis factor-α (TNF-α, as well as the genes associated with chondrocyte hypertrophy of type X collagen (C0L10A1, MMP 13 and 9, Indian hedgehog (Ihh and cas-pase 3 in the immediate vicinity of a lesion area. At the same time, there was a high expression of growth factors associated with the proliferation phase of chondrocytes, namely: parathyroid hormone-related peptide (PTHrP, fibroblast growth factor-2 (FGF-2, transforming growth factor β1/2 (TGF-β1/2, as well as macromolecules of matrix of type II collagen (C0L2A1 and aggrecan in both the areas adjacent to the lesions and at a considerable distance from their center. However, these areas showed no higher collagen cleavage activity. Nether higher collagen cleavage, nor excess expression of the genes examined were observed in the absolutely intact cartilage areas. Conclusion. Our studies have indicated that the area of very early age-related OA-like focal cartilage lesions exhibits enhanced type II collagen cleavage that is attended by the expression of the genes associated with chondrocyte differentiation in the embryonic growth plate.

  6. Gene expression profiling in the inductive human hematopoietic microenvironment

    International Nuclear Information System (INIS)

    Zhao Yongjun; Chen, Edwin; Li Liheng; Gong Baiwei; Xie Wei; Nanji, Shaherose; Dube, Ian D.; Hough, Margaret R.

    2004-01-01

    Human hematopoietic stem cells (HSCs) and their progenitors can be maintained in vitro in long-term bone marrow cultures (LTBMCs) in which constituent HSCs can persist within the adherent layers for up to 2 months. Media replenishment of LTBMCs has been shown to induce transition of HSCs from a quiescent state to an active cycling state. We hypothesize that the media replenishment of the LTBMCs leads to the activation of important regulatory genes uniquely involved in HSC proliferation and differentiation. To profile the gene expression changes associated with HSC activation, we performed suppression subtractive hybridization (SSH) on day 14 human LTBMCs following 1-h media replenishment and on unmanipulated controls. The generated SSH library contained 191 differentially up-regulated expressed sequence tags (ESTs), the majority corresponding to known genes related to various intracellular processes, including signal transduction pathways, protein synthesis, and cell cycle regulation. Nineteen ESTs represented previously undescribed sequences encoding proteins of unknown function. Differential up-regulation of representative genes, including IL-8, IL-1, putative cytokine 21/HC21, MAD3, and a novel EST was confirmed by semi-quantitative RT-PCR. Levels of fibronectin, G-CSF, and stem cell factor also increased in the conditioned media of LTBMCs as assessed by ELISA, indicating increased synthesis and secretion of these factors. Analysis of our library provides insights into some of the immediate early gene changes underlying the mechanisms by which the stromal elements within the LTBMCs contribute to the induction of HSC activation and provides the opportunity to identify as yet unrecognized factors regulating HSC activation in the LTBMC mil