WorldWideScience

Sample records for chondrocytes

  1. Regulative mechanisms of chondrocyte adhesion

    DEFF Research Database (Denmark)

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

    2006-01-01

    -matrix interaction, as well as collagen type II expression in the cartilage graft after two weeks of in vitro cultivation. Basic fibroblast growth factor (bFGF) treated chondrocytes showed increased adhesion to collagen types I and II, fibronectin, and fibrinogen. Attachment to these investigated proteins......%) 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...... significantly enhanced cell proliferation. Matrix design in cartilage engineering must meet the biological demands of amplified cells, because adhesion of chondrocytes depends on their differentiation status and is regulated by bFGF....

  2. Chondrocyte physiopathology and drug efficacy.

    Science.gov (United States)

    Serni, U; Mannoni, A

    1991-01-01

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

  3. 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.

  4. Posttraumatic Chondrocyte Apoptosis in the Murine Xiphoid

    Science.gov (United States)

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

    2013-01-01

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

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

    Science.gov (United States)

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

    2005-11-01

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

  6. 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

  7. Effect of freezing on rabbit cultured chondrocytes

    Directory of Open Access Journals (Sweden)

    R.R Filgueiras

    2011-02-01

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

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

    Science.gov (United States)

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

    2013-01-01

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

  9. Oxygen tension affects lubricin expression in chondrocytes.

    Science.gov (United States)

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

    2014-10-01

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

  10. Increased chondrocyte adhesion on nanotubular anodized titanium.

    Science.gov (United States)

    Burns, Kevin; Yao, Chang; Webster, Thomas J

    2009-03-01

    Previous studies have demonstrated increased osteoblast (bone-forming cells) functions (including adhesion, synthesis of intracellular collagen, alkaline phosphatase activity, and deposition of calcium-containing minerals) on titanium anodized to possess nanometer features compared with their unanodized counterparts. Such titanium materials were anodized to possess novel nanotubes also capable of drug delivery. Since titanium has not only experienced wide spread commercial use in orthopedic but also in cartilage applications, the objective of the present in vitro study was for the first time to investigate chondrocyte (cartilage synthesizing cells) functions on titanium anodized to possess nanotubes. For this purpose, titanium was anodized in dilute hydrofluoric acid at 20 V for 20 min. Results showed increased chondrocyte adhesion on anodized titanium with nanotube structures compared with unanodized titanium. Importantly, the present study also provided evidence why. Since material characterization studies revealed significantly greater nanometer roughness and similar chemistry as well as crystallinity between nanotubular anodized and unanodized titanium, the results of the present study highlight the importance of the nanometer roughness provided by anodized nanotubes on titanium for enhancing chondrocyte adhesion. In this manner, the results of the present in vitro study indicated that anodization might be a promising quick and inexpensive method to modify the surface of titanium-based implants to induce better chondrocyte adhesion for cartilage applications.

  11. 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.

  12. Chondrocyte hypertrophy in skeletal development, growth, and disease.

    Science.gov (United States)

    Sun, Margaret Man-Ger; Beier, Frank

    2014-03-01

    Most of our bones form through the process of endochondral ossification, which is tightly regulated by the activity of the cartilage growth plate. Chondrocyte maturation through the various stages of growth plate physiology ultimately results in hypertrophy. Chondrocyte hypertrophy is an essential contributor to longitudinal bone growth, but recent data suggest that these cells also play fundamental roles in signaling to other skeletal cells, thus coordinating endochondral ossification. On the other hand, ectopic hypertrophy of articular chondrocytes has been implicated in the pathogenesis of osteoarthritis. Thus, a better understanding of the processes that control chondrocyte hypertrophy in the growth plate as well as in articular cartilage is required for improved management of both skeletal growth disorders and osteoarthritis. This review summarizes recent findings on the regulation of hypertrophic chondrocyte differentiation, the cellular mechanisms involved in hypertrophy, and the role of chondrocyte hypertrophy in skeletal physiology and pathophysiology.

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

    Science.gov (United States)

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

    2016-11-15

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

  14. 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.

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

    Science.gov (United States)

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

    2016-01-01

    Chondrocytes are the exclusive cells residing in cartilage and maintain the functionality of cartilage tissue. Series of biocomponents such as different growth factors, cytokines, and transcriptional factors regulate the mesenchymal stem cells (MSCs) differentiation to chondrocytes. The number of chondrocytes and dedifferentiation are the key limitations in subsequent clinical application of the chondrocytes. Different culture methods are being developed to overcome such issues. Using tissue engineering and cell based approaches, chondrocytes offer prominent therapeutic option specifically in orthopedics for cartilage repair and to treat ailments such as tracheal defects, facial reconstruction, and urinary incontinence. Matrix-assisted autologous chondrocyte transplantation/implantation is an improved version of traditional autologous chondrocyte transplantation (ACT) method. An increasing number of studies show the clinical significance of this technique for the chondral lesions treatment. Literature survey was carried out to address clinical and functional findings by using various ACT procedures. The current study was conducted to study the pharmacological significance and biomedical application of chondrocytes. Furthermore, it is inferred from the present study that long term follow-up studies are required to evaluate the potential of these methods and specific positive outcomes. PMID:27631002

  16. Fibroblast-like synoviocyte-chondrocyte interaction in cartilage degradation

    NARCIS (Netherlands)

    Steenvoorden, M.M.C.; Bank, R.A.; Ronday, H.K.; Toes, R.E.M.; Huizinga, T.W.J.; Groot, J. de

    2007-01-01

    Objective: In vitro models for joint diseases often focus on a single cell type, such as chondrocytes in osteoarthritis (OA) or fibroblast-like synoviocytes (synoviocytes) in rheumatoid arthritis (RA). However, these joint diseases affect the whole joint and interaction between chondrocytes and syno

  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 human nasal chondrocytes on macroporous microcarriers enhances redifferentiation

    NARCIS (Netherlands)

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

    2003-01-01

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

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

    NARCIS (Netherlands)

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

    2003-01-01

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

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

    Science.gov (United States)

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

    2008-02-12

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

  1. 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

  2. Autologous chondrocyte implantation in children and adolescents

    DEFF Research Database (Denmark)

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

    2013-01-01

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

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

    Science.gov (United States)

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

    2016-08-01

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

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

    Institute of Scientific and Technical Information of China (English)

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

    2013-01-01

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

  5. 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...... Repair Society (ICRS) rating, the percentage of patients rated A (normal) and B (nearly normal) increased from 33.9% preoperatively to 92.5% at 24 months after ACT-CS. IKDC and Lysholm scores increased from 50.1 points (SD, 13.4) and 60.5 points (SD, 9.4), respectively, to 76.1 points (SD, 15.2) (P...... CS as a salvage procedure. The rate of failures in patients with isolated cartilage defects was 5.9%. CONCLUSIONS: ACT-CS...

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

    Institute of Scientific and Technical Information of China (English)

    何清义; 李起鸿; 杨柳; 许建中

    2003-01-01

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

  7. Identification of the calcitonin receptor in osteoarthritic chondrocytes

    Directory of Open Access Journals (Sweden)

    Christensen Tjorbjoern

    2011-10-01

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

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

    OpenAIRE

    2010-01-01

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

  9. Linoleate impairs collagen synthesis in primary cultures of avian chondrocytes.

    Science.gov (United States)

    Watkins, B A; Xu, H; Turek, J J

    1996-06-01

    The effects of supplemental fatty acids, vitamin E (VIT E), and iron-induced oxidative stress on collagen synthesis, cellular injury, and lipid peroxidation were evaluated in primary cultures of avian epiphyseal chondrocytes. The treatments included oleic and linoleic acids (O or 50 microM) complexed with BSA and dl-alpha-tocopheryl acetate (VIT E at 0 or 100 microM). After 14 days of preculture, the chondrocytes were enriched with fatty acids for 8 days then cultured with VIT E for 2 days. The chondrocytes were then treated with ferrous sulfate (O or 20 microM) for 24 hr to induce oxidative stress. Collagen synthesis was the lowest and the activity of lactate dehydrogenase (LDH) was the highest in chondrocyte cultures treated with 50 microM linoleic acid and 0 VIT E. In contrast, VIT E supplemented at 100 microM partially restored collagen synthesis in the chondrocytes enriched with linoleic acid and lowered LDH activity in the media. The iron oxidative inducer significantly increased the values of thiobarbituric acid-reactive substances (TBARS) in the culture medium. The data showed that linoleic acid impaired chondrocyte cell function and caused cellular injury but that VIT E reversed these effects. Results from a previous study demonstrated that VIT E stimulated bone formation in chicks fed unsaturated fat, and the present findings in cultures of epiphyseal chondrocytes suggest that VIT E is important for chondrocyte function in the presence of polyunsaturated fatty acids. VIT E appears to be beneficial for growth cartilage biology and in optimizing bone growth.

  10. 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.

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

    Science.gov (United States)

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

    2016-01-01

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

  12. Customized biomaterials to augment chondrocyte gene therapy.

    Science.gov (United States)

    Aguilar, Izath Nizeet; Trippel, Stephen; Shi, Shuiliang; Bonassar, Lawrence J

    2017-02-07

    A persistent challenge in enhancing gene therapy is the transient availability of the target gene product. This is particularly true in tissue engineering applications. The transient exposure of cells to the product could be insufficient to promote tissue regeneration. Here we report the development of a new material engineered to have a high affinity for a therapeutic gene product. We focus on insulin-like growth factor-I (IGF-I) for its highly anabolic effects on many tissues such as spinal cord, heart, brain and cartilage. One of the ways that tissues store IGF-I is through a group of insulin like growth factor binding proteins (IGFBPs), such as IGFBP-5. We grafted the IGF-I binding peptide sequence from IGFBP-5 onto alginate in order to retain the endogenous IGF-I produced by transfected chondrocytes. This novel material bound IGF-I and released the growth factor for at least 30days in culture. We found that this binding enhanced the biosynthesis of transfected cells up to 19-fold. These data demonstrate the coordinated engineering of cell behavior and material chemistry to greatly enhance extracellular matrix synthesis and tissue assembly, and can serve as a template for the enhanced performance of other therapeutic proteins.

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

    NARCIS (Netherlands)

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

    2004-01-01

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

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

    NARCIS (Netherlands)

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

    2004-01-01

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

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

    NARCIS (Netherlands)

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

    2014-01-01

    Cartilage injury represents one of the most significant clinical conditions. Implantation of expanded autologous chondrocytes from noninjured compartments of the joint is a typical strategy for repairing cartilage. However, two-dimensional culture causes dedifferentiation of chondrocytes, making the

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

    Science.gov (United States)

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

    2015-05-22

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

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

    NARCIS (Netherlands)

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

    1992-01-01

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

  18. File list: His.Bon.10.AllAg.Chondrocytes [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available His.Bon.10.AllAg.Chondrocytes mm9 Histone Bone Chondrocytes SRX1035110,SRX1035109,S...RX1035113,SRX1035112,SRX1035111,SRX963261 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/His.Bon.10.AllAg.Chondrocytes.bed ...

  19. File list: Oth.Bon.50.AllAg.Chondrocytes [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Bon.50.AllAg.Chondrocytes mm9 TFs and others Bone Chondrocytes SRX1035108,SRX10...35114 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Oth.Bon.50.AllAg.Chondrocytes.bed ...

  20. File list: ALL.Bon.10.AllAg.Chondrocytes [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available ALL.Bon.10.AllAg.Chondrocytes mm9 All antigens Bone Chondrocytes SRX1035110,SRX1035...3261,SRX963262 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/ALL.Bon.10.AllAg.Chondrocytes.bed ...

  1. File list: ALL.Bon.05.AllAg.Chondrocytes [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available ALL.Bon.05.AllAg.Chondrocytes mm9 All antigens Bone Chondrocytes SRX1035118,SRX1035...3261,SRX963262 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/ALL.Bon.05.AllAg.Chondrocytes.bed ...

  2. File list: His.Bon.20.AllAg.Chondrocytes [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available His.Bon.20.AllAg.Chondrocytes mm9 Histone Bone Chondrocytes SRX1035110,SRX1035109,S...RX1035113,SRX1035112,SRX1035111,SRX963261 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/His.Bon.20.AllAg.Chondrocytes.bed ...

  3. File list: Oth.Bon.05.AllAg.Chondrocytes [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Bon.05.AllAg.Chondrocytes mm9 TFs and others Bone Chondrocytes SRX1035114,SRX10...35108 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Oth.Bon.05.AllAg.Chondrocytes.bed ...

  4. File list: Unc.Bon.20.AllAg.Chondrocytes [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Unc.Bon.20.AllAg.Chondrocytes mm9 Unclassified Bone Chondrocytes SRX1035118,SRX1035...116,SRX1035117 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Unc.Bon.20.AllAg.Chondrocytes.bed ...

  5. File list: ALL.Bon.20.AllAg.Chondrocytes [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available ALL.Bon.20.AllAg.Chondrocytes mm9 All antigens Bone Chondrocytes SRX1035110,SRX1035...3261,SRX963262 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/ALL.Bon.20.AllAg.Chondrocytes.bed ...

  6. File list: Unc.Bon.10.AllAg.Chondrocytes [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Unc.Bon.10.AllAg.Chondrocytes mm9 Unclassified Bone Chondrocytes SRX1035118,SRX1035...117,SRX1035116 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Unc.Bon.10.AllAg.Chondrocytes.bed ...

  7. File list: ALL.Bon.50.AllAg.Chondrocytes [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available ALL.Bon.50.AllAg.Chondrocytes mm9 All antigens Bone Chondrocytes SRX1035109,SRX1035...3261,SRX963262 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/ALL.Bon.50.AllAg.Chondrocytes.bed ...

  8. File list: Unc.Bon.50.AllAg.Chondrocytes [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Unc.Bon.50.AllAg.Chondrocytes mm9 Unclassified Bone Chondrocytes SRX1035118,SRX1035...117,SRX1035116 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Unc.Bon.50.AllAg.Chondrocytes.bed ...

  9. File list: Unc.Bon.05.AllAg.Chondrocytes [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Unc.Bon.05.AllAg.Chondrocytes mm9 Unclassified Bone Chondrocytes SRX1035118,SRX1035...117,SRX1035116 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Unc.Bon.05.AllAg.Chondrocytes.bed ...

  10. File list: His.Bon.05.AllAg.Chondrocytes [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available His.Bon.05.AllAg.Chondrocytes mm9 Histone Bone Chondrocytes SRX1035110,SRX1035111,S...RX1035109,SRX1035113,SRX1035112,SRX963261 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/His.Bon.05.AllAg.Chondrocytes.bed ...

  11. File list: His.Bon.50.AllAg.Chondrocytes [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available His.Bon.50.AllAg.Chondrocytes mm9 Histone Bone Chondrocytes SRX1035109,SRX1035110,S...RX1035113,SRX1035112,SRX1035111,SRX963261 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/His.Bon.50.AllAg.Chondrocytes.bed ...

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

    Science.gov (United States)

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

    2008-04-01

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

  13. Astaxanthin reduces matrix metalloproteinase expression in human chondrocytes.

    Science.gov (United States)

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

    2014-03-01

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

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

    Science.gov (United States)

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

    2015-08-01

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

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

    Directory of Open Access Journals (Sweden)

    Lin Jian-Hua

    2011-11-01

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

  16. Growth characteristics and functional changes in rat chondrocytes cultured in porous tantalum in vitro

    Directory of Open Access Journals (Sweden)

    Ling ZHANG

    2014-08-01

    Full Text Available Objective To evaluate the growth characteristics and functional changes in rat chondrocytes cultured in porous tantalum in vitro. Methods The chondrocytes isolated from cartilage of 3-week old SD rats were cultured in vitro, then the 2nd passage cells were identified and implanted in porous tantalum scaffolds with a density of 1×106 cells/ml. The morphological characteristics of the chondrocytes cultured in porous tantalum were observed under inverted microscope, scanning electron microscope (SEM and transmission electron microscope (TEM, and the content of glycosaminoglycan (GAG in the chondrocytes was measured by chromatometry. Results The harvested cells were identified as chondrocytes by type Ⅱ collagen immunocytochemical staining, toluidine blue staining and safranin-O staining. Many chondrocytes adhering to the edge of porous tantalum were found by inverted microscope. Observation under SEM showed that chondrocytes spread well on the surface and distributed in the holes of porous tantalum, and they proliferated and secreted some extracellular matrixes. TEM observation showed that the ultrastructure of chondrocytes cultured in porous tantalum was similar to that of normal chondrocytes. Chromatometry determination showed that the chondrocytes in porous tantalum could secrete GAG continuously. Conclusion Porous tantalum is shown to have a satisfactory biocompatibility with chondrocytes in vitro, and may be used as a scaffold for cartilage tissue engineering. DOI: 10.11855/j.issn.0577-7402.2014.06.08

  17. Chondrogenic potential of articular chondrocytes depends on their original location

    NARCIS (Netherlands)

    Bekkers, Joris E J; Saris, Daniel B F; Tsuchida, Anika Iris; van Rijen, Mattie H P; Dhert, Wouter J A; Creemers, Laura B

    2014-01-01

    OBJECTIVE: This study aimed to investigate the regenerative capacity of chondrocytes derived from debrided defect cartilage and healthy cartilage from different regions in the joint to determine the best cell source for regenerative cartilage therapies. METHODS: Articular cartilage was obtained from

  18. MicroRNA-33 suppresses CCL2 expression in chondrocytes.

    Science.gov (United States)

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

    2016-06-01

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

  19. Confocal microscopy indentation system for studying in situ chondrocyte mechanics.

    Science.gov (United States)

    Han, Sang-Kuy; Colarusso, Pina; Herzog, Walter

    2009-10-01

    Chondrocytes synthesize extracellular matrix molecules, thus they are essential for the development, adaptation and maintenance of articular cartilage. Furthermore, it is well accepted that the biosynthetic activity of chondrocytes is influenced by the mechanical environment. Therefore, their response to mechanical stimuli has been studied extensively. Much of the knowledge in this area of research has been derived from testing of isolated cells, cartilage explants, and fixed cartilage specimens: systems that differ in important aspects from chondrocytes embedded in articular cartilage and observed during loading conditions. In this study, current model systems have been improved by working with the intact cartilage in real time. An indentation system was designed on a confocal microscope that allows for simultaneous loading and observation of chondrocytes in their native environment. Cell mechanics were then measured under precisely controlled loading conditions. The indentation system is based on a light transmissible cylindrical glass indentor of 0.17 mm thickness and 1.64 mm diameter that is aligned along the focal axis of the microscope and allows for real time observation of live cells in their native environment. The system can be used to study cell deformation and biological responses, such as calcium sparks, while applying prescribed loads on the cartilage surface. It can also provide novel information on the relationship between cell loading and cartilage adaptive/degenerative processes in the intact tissue.

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

    Science.gov (United States)

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

    1993-09-15

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

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

    Directory of Open Access Journals (Sweden)

    V Nicolin

    2009-08-01

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

  2. Doxycycline inhibits collagen synthesis by differentiated articular chondrocytes.

    NARCIS (Netherlands)

    TeKoppele, J.M.; Beekman, B.; Verzijl, N.; Koopman, J.L.; Groot, J. de; Bank, R.A.

    1998-01-01

    Doxycycline (DOX) profoundly inhibited collagen synthesis by differentiated articular chondrocytes. At 25 microM, the rate of collagen synthesis was suppressed by more than 50% without affecting cell proliferation (DNA levels) and general protein synthesis (35S-Met and 35S-Cys incorporation). Steady

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2012-09-07

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

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

    Directory of Open Access Journals (Sweden)

    Ebrahim Esfandiary

    2008-01-01

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

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

    Science.gov (United States)

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

    2017-02-01

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

  6. Metabolic Effects of Avocado/Soy Unsaponifiables on Articular Chondrocytes

    Directory of Open Access Journals (Sweden)

    Louis Lippiello

    2008-01-01

    Full Text Available Avocado/soy unsaponifiable (ASU components are reported to have a chondroprotective effect by virtue of anti-inflammatory and proanabolic effects on articular chondrocytes. The identity of the active component(s remains unknown. In general, sterols, the major component of unsaponifiable plant material have been demonstrated to be anti-inflammatory in vitro and in animal models. These studies were designed to clarify whether the sterol content of ASU preparations were the primary contributors to biological activity in articular chondrocytes. ASU samples were analyzed by high pressure liquid chromatography (HPLC and GC mass spectrometry. The sterol content was normalized between diverse samples prior to in vitro testing on bovine chondrocytes. Anabolic activity was monitored by uptake of 35-sulfate into proteoglycans and quantitation of labeled hydroxyproline and proline content after incubation with labeled proline. Anti-inflammatory activity was assayed by measuring reduction of interleukin-1 (IL-1-induced synthesis of PGE2 and metalloproteases and release of label from tissue prelabeled with S-35.All ASU samples exerted a similar time-dependent up-regulation of 35-sulfate uptake in bovine cells reaching a maximum of greater than 100% after 72 h at sterol doses of 1–10 μg/ml. Non-collagenous protein (NCP and collagen synthesis were similarly up-regulated. All ASU were equally effective in dose dependently inhibiting IL-1-induced MMP-3 activity (23–37%, labeled sulfate release (15–23% and PGE2 synthesis (45–58%. Up-regulation of glycosaminoglycan and collagen synthesis and reduction of IL-1 effects in cartilage are consistent with chondroprotective activity. The similarity of activity of ASU from diverse sources when tested at equal sterol levels suggests sterols are important for biologic effects in articular chondrocytes.

  7. Metabolic Effects of Avocado/Soy Unsaponifiables on Articular Chondrocytes

    Science.gov (United States)

    Nardo, Joseph V.; Harlan, Robert; Chiou, Tiffany

    2008-01-01

    Avocado/soy unsaponifiable (ASU) components are reported to have a chondroprotective effect by virtue of anti-inflammatory and proanabolic effects on articular chondrocytes. The identity of the active component(s) remains unknown. In general, sterols, the major component of unsaponifiable plant material have been demonstrated to be anti-inflammatory in vitro and in animal models. These studies were designed to clarify whether the sterol content of ASU preparations were the primary contributors to biological activity in articular chondrocytes. ASU samples were analyzed by high pressure liquid chromatography (HPLC) and GC mass spectrometry. The sterol content was normalized between diverse samples prior to in vitro testing on bovine chondrocytes. Anabolic activity was monitored by uptake of 35-sulfate into proteoglycans and quantitation of labeled hydroxyproline and proline content after incubation with labeled proline. Anti-inflammatory activity was assayed by measuring reduction of interleukin-1 (IL-1)-induced synthesis of PGE2 and metalloproteases and release of label from tissue prelabeled with S-35.All ASU samples exerted a similar time-dependent up-regulation of 35-sulfate uptake in bovine cells reaching a maximum of greater than 100% after 72 h at sterol doses of 1–10 μg/ml. Non-collagenous protein (NCP) and collagen synthesis were similarly up-regulated. All ASU were equally effective in dose dependently inhibiting IL-1-induced MMP-3 activity (23–37%), labeled sulfate release (15–23%) and PGE2 synthesis (45–58%). Up-regulation of glycosaminoglycan and collagen synthesis and reduction of IL-1 effects in cartilage are consistent with chondroprotective activity. The similarity of activity of ASU from diverse sources when tested at equal sterol levels suggests sterols are important for biologic effects in articular chondrocytes. PMID:18604259

  8. Epigenetic Regulation of Chondrocyte Catabolism and Anabolism in Osteoarthritis

    OpenAIRE

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

    2015-01-01

    Osteoarthritis (OA) is one of the most prevalent forms of joint disorder, associated with a tremendous socioeconomic burden worldwide. Various non-genetic and lifestyle-related factors such as aging and obesity have been recognized as major risk factors for OA, underscoring the potential role for epigenetic regulation in the pathogenesis of the disease. OA-associated epigenetic aberrations have been noted at the level of DNA methylation and histone modification in chondrocytes. These epigenet...

  9. 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.

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

    Institute of Scientific and Technical Information of China (English)

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

    2005-01-01

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

  11. Chondrogenic differentiation of mouse embryonic stem cells promoted by mature chondrocytes

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    In order to direct embryonic stem (ES) cells to differentiate into chondrocytes, a chondrogenic envi-ronment provided by mature chondrocytes was investigated. Flk-1 positive cells sorted from pre-differentiated mouse ES cells were mixed with adult porcine articular chondrocytes, seeded on biodegradable scaffolds, and then implanted subcutaneously into nude mice. The cell-scaffold com-plexes formed cartilage tissues after 4 weeks, which was demonstrated by histology and anti-type II collagen antibody staining. Positive staining of mouse Major Histocompatibility Complex class I molecules confirmed that part of the chondrocytes were derived from mouse ES cells. The current study established a new approach for directing ES cell differentiation.

  12. Chondrogenic differentiation of mouse embryonic stem cells promoted by mature chondrocytes

    Institute of Scientific and Technical Information of China (English)

    XIE Feng; ZHANG WenJie; CHEN FanFan; ZHOU GuangDong; CUI Lei; LIU Wei; CAO YiLin

    2008-01-01

    In order to direct embryonic stem (ES) cells to differentiate into chondrocytes, a chondrogenic envi-ronment provided by mature chondrocytes was investigated. FIk-1 positive cells sorted from pre-differentiated mouse ES cells were mixed with adult porcine articular chondrocytes, seeded on biodegradable scaffolds, and then implanted subcutaneously into nude mice. The cell-scaffold com-plexes formed cartilage tissues after 4 weeks, which was demonstrated by histology and anti-type Ⅱ collagen antibody staining. Positive staining of mouse Major Histocompatibility Complex class Ⅰ molecules confirmed that part of the chondrocytes were derived from mouse ES cells. The current study established a new approach for directing ES cell differentiation.

  13. Chondrocyte Morphology in Stiff and Soft Agarose Gels and the Influence of Fetal Calf Serum.

    Science.gov (United States)

    Karim, Asima; Hall, Andrew C

    2017-05-01

    Changes to chondrocyte volume/morphology may have deleterious effects on extracellular matrix (ECM) metabolism potentially leading to cartilage deterioration and osteoarthritis (OA). The factors controlling chondrocyte properties are poorly understood, however, pericellular matrix (PCM) weakening may be involved. We have studied the density, volume, morphology, and clustering of cultured bovine articular chondrocytes within stiff (2% w/v) and soft (0.2% w/v) three-dimensional agarose gels. Gels with encapsulated chondrocytes were cultured in Dulbecco's Modified Eagle's Medium (DMEM; fetal calf serum (FCS) 1-10%;380 mOsm) for up to 7 days. Chondrocytes were fluorescently labeled after 1, 3, and 7 days with 5-chloromethylfluorescein-diacetate (CMFDA) and propidium iodide (PI) or 1,5-bis{[2-(di-methylamino)ethyl]amino}-4,8-dihydroxyanthracene-9,10-dione (DRAQ5) to identify cytoplasmic space or DNA and imaged by confocal laser scanning microscopy (CLSM). Chondrocyte density, volume, morphology, and clustering were quantified using Volocity™ software. In stiff gels after 7 d with 10% FCS, chondrocyte density remained unaffected and morphology was relatively normal with occasional cytoplasmic processes. However, in soft gels by day 1, chondrocyte volume increased (P = 0.0058) and by day 7, density increased (P = 0.0080), along with the percentage of chondrocytes of abnormal morphology (P stiff gels. FCS exacerbated changes to density (P stiffness and/or increased FCS concentrations promoted chondrocyte proliferation and clustering, increased cell volume, and stimulated abnormal morphology, producing similar changes to those occurring in OA. The increased penetration of factors in FCS into soft gels may be important in the development of these abnormal chondrocyte properties. J. Cell. Physiol. 232: 1041-1052, 2017. © 2016 Wiley Periodicals, Inc.

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

    Science.gov (United States)

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

    2014-09-01

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

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

    Directory of Open Access Journals (Sweden)

    Tarik Onur, Alexis Dang

    2014-09-01

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

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

    Science.gov (United States)

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

    2004-05-01

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

  17. Preservation of the chondrocyte's pericellular matrix improves cell-induced cartilage formation

    NARCIS (Netherlands)

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

    2010-01-01

    The extracellular matrix surrounding chondrocytes within a chondron is likely to affect the metabolic activity of these cells. In this study we investigated this by analyzing protein synthesis by intact chondrons obtained from different types of cartilage and compared this with chondrocytes. Chondro

  18. Preservation of the Chondrocyte's Pericellular Matrix Improves Cell-Induced Cartilage Formation

    NARCIS (Netherlands)

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

    2010-01-01

    The extracellular matrix surrounding chondrocytes within a chondron is likely to affect the metabolic activity of these cells. In this study we investigated this by analyzing protein synthesis by intact chondrons obtained from different types of cartilage and compared this with chondrocytes. Chondro

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

    Directory of Open Access Journals (Sweden)

    N Kaneshiro

    2007-05-01

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

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

    Institute of Scientific and Technical Information of China (English)

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

    2009-01-01

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

  1. Effect of oxygen tension on adult articular chondrocytes in microcarrier bioreactor culture

    NARCIS (Netherlands)

    Malda, J.; Brink, van den P.; Meeuwse, P.; Grojec, M.; Martens, D.E.; Tramper, J.; Riesle, J.; Blitterswijk, van C.A.

    2004-01-01

    Tissue-engineering approaches for cartilage repair hold promise for the treatment of cartilage defects. Various methods to prevent or reduce dedifferentiation during chondrocyte expansion are currently under investigation. In the present study we evaluated the effect of oxygen on chondrocyte prolife

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

    NARCIS (Netherlands)

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

    2012-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Eliane Antonioli

    2015-01-01

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

  4. Direct measurement of TRPV4 and PIEZO1 activity reveals multiple mechanotransduction pathways in chondrocytes

    Science.gov (United States)

    Rocio Servin-Vences, M; Moroni, Mirko; Lewin, Gary R; Poole, Kate

    2017-01-01

    The joints of mammals are lined with cartilage, comprised of individual chondrocytes embedded in a specialized extracellular matrix. Chondrocytes experience a complex mechanical environment and respond to changing mechanical loads in order to maintain cartilage homeostasis. It has been proposed that mechanically gated ion channels are of functional importance in chondrocyte mechanotransduction; however, direct evidence of mechanical current activation in these cells has been lacking. We have used high-speed pressure clamp and elastomeric pillar arrays to apply distinct mechanical stimuli to primary murine chondrocytes, stretch of the membrane and deflection of cell-substrate contacts points, respectively. Both TRPV4 and PIEZO1 channels contribute to currents activated by stimuli applied at cell-substrate contacts but only PIEZO1 mediates stretch-activated currents. These data demonstrate that there are separate, but overlapping, mechanoelectrical transduction pathways in chondrocytes. DOI: http://dx.doi.org/10.7554/eLife.21074.001 PMID:28135189

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

    OpenAIRE

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

    1993-01-01

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

  6. 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.

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

    Science.gov (United States)

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

    2017-02-28

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

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

    Science.gov (United States)

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

    2008-06-01

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

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

    Science.gov (United States)

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

    2014-10-16

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

  10. Study of cryopreservation of articular chondrocytes using the Taguchi method.

    Science.gov (United States)

    Lyu, Shaw-Ruey; Wu, Wei Te; Hou, Chien Chih; Hsieh, Wen-Hsin

    2010-04-01

    This study evaluates the effect of control factors on cryopreservation of articular cartilage chondrocytes using the Taguchi method. Freeze-thaw experiments based on the L(8)(2(7)) two-level orthogonal array of the Taguchi method are conducted, and ANOVA (analysis of variables) is adopted to determine the statistically significant control factors that affect the viability of the cell. Results show that the type of cryoprotectant, freezing rate, thawing rate, and concentration of cryoprotectant (listed in the order of influence) are the statistically significant control factors that affect the post-thaw viability. The end temperature and durations of the first and second stages of freezing do not affect the post-thaw viability. Within the ranges of the control factors studied in this work, the optimal test condition is found to be a freezing rate of 0.61+/-0.03 degrees C/min, a thawing rate of 126.84+/-5.57 degrees C/min, Me(2)SO cryoprotectant, and a cryoprotectant concentration of 10% (v/v) for maximum cell viability. In addition, this study also explores the effect of cryopreservation on the expression of type II collagen using immunocytochemical staining and digital image processing. The results show that the ability of cryopreserved chondrocytes to express type II collagen is reduced within the first five days of monolayer culture.

  11. PTHrP regulates chondrocyte maturation in condylar cartilage.

    Science.gov (United States)

    Rabie, A B M; Tang, G H; Xiong, H; Hägg, U

    2003-08-01

    PTHrP is a key factor regulating the pace of endochondral ossification during skeletal development. Mandibular advancement solicits a cascade of molecular responses in condylar cartilage. However, the pace of cellular maturation and its effects on condylar growth are still unknown. The purpose of this study was to evaluate the pattern of expression of PTHrP and correlate it to cellular dynamics of chondrocytes in condylar cartilage during natural growth and mandibular advancement. We fitted 35-day-old Sprague-Dawley rats with functional appliances. Experimental animals with matched controls were labeled with bromodeoxyuridine 3 days before their death, so that mesenchymal cell differentiation could be traced. Mandibular advancement increased the number of differentiated chondroblasts and subsequently increased the cartilage volume. Higher levels of PTHrP expression in experimental animals coincided with the slowing of chondrocyte hypertrophy. Thus, mandibular advancement promoted mesenchymal cell differentiation and triggered PTHrP expression, which retarded their further maturation to allow for more growth.

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

    Science.gov (United States)

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

    2002-01-01

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

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

    Science.gov (United States)

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

    2016-09-01

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

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

    Science.gov (United States)

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

    2013-06-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2012-07-20

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

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

    Directory of Open Access Journals (Sweden)

    Fei Zhu

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

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

    Science.gov (United States)

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

    2016-12-01

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

  18. Rapid Chondrocyte Isolation for Tissue Engineering Applications: The Effect of Enzyme Concentration and Temporal Exposure on the Matrix Forming Capacity of Nasal Derived Chondrocytes

    Directory of Open Access Journals (Sweden)

    Srujana Vedicherla

    2017-01-01

    Full Text Available Laboratory based processing and expansion to yield adequate cell numbers had been the standard in Autologous Disc Chondrocyte Transplantation (ADCT, Allogeneic Juvenile Chondrocyte Implantation (NuQu®, and Matrix-Induced Autologous Chondrocyte Implantation (MACI. Optimizing cell isolation is a key challenge in terms of obtaining adequate cell numbers while maintaining a vibrant cell population capable of subsequent proliferation and matrix elaboration. However, typical cell yields from a cartilage digest are highly variable between donors and based on user competency. The overall objective of this study was to optimize chondrocyte isolation from cartilaginous nasal tissue through modulation of enzyme concentration exposure (750 and 3000 U/ml and incubation time (1 and 12 h, combined with physical agitation cycles, and to assess subsequent cell viability and matrix forming capacity. Overall, increasing enzyme exposure time was found to be more detrimental than collagenase concentration for subsequent viability, proliferation, and matrix forming capacity (sGAG and collagen of these cells resulting in nonuniform cartilaginous matrix deposition. Taken together, consolidating a 3000 U/ml collagenase digest of 1 h at a ratio of 10 ml/g of cartilage tissue with physical agitation cycles can improve efficiency of chondrocyte isolation, yielding robust, more uniform matrix formation.

  19. bFGF influences human articular chondrocyte differentiation

    DEFF Research Database (Denmark)

    Schmal, H; Zwingmann, J; Fehrenbach, M

    2007-01-01

    FGF concentrations in supernatants of primary human articular chondrocytes peaked immediately after isolation and then declined. In a dose-dependent manner, bFGF enhanced cell amplification and viability. BFGF induced a decrease in the apoptotic cell population, while the number of proliferating cells remained...... in monolayer. bFGF-dependent cell proliferation, production of collagen type II and aggrecan were monitored 10 days after isolation. Furthermore, effect of bFGF on cell cycle, cell morphology, and mRNA expression of integrins and chondrogenic markers determined by real time PCR were analyzed. RESULTS: b...... unchanged. Supplementation of cell culture with bFGF reduced collagen type II mRNA by 49%, but increased expression of the integrin alpha(2) by 70%. bFGF did not significantly regulate the integrins alpha(1), alpha(5), alpha(10), alpha(v) and type I collagen. bFGF reduced the amount of collagen type II...

  20. Effects of Cryoprotective Agents on the Bovine Articular Chondrocyte Viability

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    Cryopreservation is the process of choice for long term preservation of cells and tissues. In this study, the effects of cryoprotective agents, dimethyl sulfoxide(DMSO), glycerol and 1,2-propanediol on the bovine articular chondrocyte viability were examined experimentally. The CPA was added at the concentrations of 0. 6. 0.9, 1.2 and 1.5 mol/I and at 4℃ and 37℃ and removed at 37℃ in one-step. CPA stepwise addition and removal at 0. 6 and 1. 2 mol/L and at 37℃ was also tested as an alternative protocol. Cell volume excursion during DMSO addition and removal was estimated and correlated well with cell survival rates. Solution makeup affects cell survival rate and a stepwise protocol can improve the cell survival rates significantly.

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

    Directory of Open Access Journals (Sweden)

    F Wolf

    2008-12-01

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

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

    Indian Academy of Sciences (India)

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

    2015-03-01

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

  3. Autologous chondrocyte implantation for treatment of cartilage defects of the knee

    DEFF Research Database (Denmark)

    Jungmann, Pia M; Salzmann, Gian M; Schmal, Hagen;

    2012-01-01

    BACKGROUND: Autologous chondrocyte implantation (ACI) is a well-established treatment option for isolated cartilage defects of the knee joint, providing satisfying outcome. However, cases of treatment failure with the need for surgical reintervention are reported; typical patient's individual and...

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2014-05-05

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

  5. The cytoskeleton of chondrocytes of Sepia officinalis (Mollusca, Cephalopoda: an immunocytochemical study

    Directory of Open Access Journals (Sweden)

    F Leone

    2009-06-01

    Full Text Available Our previous electron microscope study showed that chondrocytes from cephalopod cartilage possess a highly developed cytoskeleton and numerous cytoplasmic processes that ramify extensively through the tissue. We have now carried out a light microscope immunocytochemical study of chondrocytes from the orbital cartilage of Sepia officinalis to obtain indications as to the nature of the cytoskeletal components. We found clear positivity to antibodies against mammalian tubulin, vimentin, GFAP, and actin, but not keratin. The simultaneous presence of several cytoskeletal components is consistent with the hypothesis that cephalopod chondrocytes have the characteristics of both chondrocytes and osteocytes of vertebrates, which endow the tissue as a whole with some of the properties of vertebrate bone. We confirm, therefore, the presence in molluscs of the ubiquitous cytoskeletal proteins of metazoan cells that have remained highly conserved throughout phylogenetic evolution.

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

    Science.gov (United States)

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

    2015-06-25

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

  7. 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.

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

    Science.gov (United States)

    Gradišnik, Lidija; Gorenjak, Mario; Vogrin, Matjaž

    2017-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Jakob Naranda

    2017-03-01

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

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

    Directory of Open Access Journals (Sweden)

    Madhushika Ratnayake

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

  11. Canine chondrocytes seeded in type I and type II collagen implants investigated in vitro.

    Science.gov (United States)

    Nehrer, S; Breinan, H A; Ramappa, A; Shortkroff, S; Young, G; Minas, T; Sledge, C B; Yannas, I V; Spector, M

    1997-01-01

    Synthetic and natural absorbable polymers have been used as vehicles for implantation of cells into cartilage defects to promote regeneration of the articular joint surface. Implants should provide a pore structure that allows cell adhesion and growth, and not provoke inflammation or toxicity when implanted in vivo. The scaffold should be absorbable and the degradation should match the rate of tissue regeneration. To facilitate cartilage repair the chemical structure and pore architecture of the matrix should allow the seeded cells to maintain the chondrocytic phenotype, characterized by synthesis of cartilage-specific proteins. We investigated the behavior of canine chondrocytes in two spongelike matrices in vitro: a collagen-glycosaminoglycan (GAG) copolymer produced from bovine hide consisting of type I collagen and a porous scaffold made of type II collagen by extraction of porcine cartilage. Canine chondrocytes were seeded on both types of matrices and cultured for 3 h, 7 days, and 14 days. The histology of chondrocyte-seeded implants showed a significantly higher percentage of cells with spherical morphology, consistent with chondrocytic morphology, in the type II sponge at each time point. Pericellular matrix stained for proteoglycans and for type II collagen after 14 days. Biochemical analysis of the cell seeded sponges for GAG and DNA content showed increases with time. At day 14 there was a significantly higher amount of DNA and GAG in the type II matrix. This is the first study that directly compares the behavior of chondrocytes in type I and type II collagen matrices. The type II matrix may be of value as a vehicle for chondrocyte implantation on the basis of the higher percentage of chondrocytes retaining spherical morphology and greater biosynthetic activity that was reflected in the greater increase of GAG content.

  12. 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.

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

    Science.gov (United States)

    Xu, Jiaming; Zhang, Changqing

    2014-08-01

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

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

    Science.gov (United States)

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

    2016-09-01

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

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

    Directory of Open Access Journals (Sweden)

    Michiyo Nasu

    2016-01-01

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

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

    Science.gov (United States)

    Kasper, S; Friesen, H G

    1986-01-01

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

  17. GROWTH DIFFERENTIATION FACTOR-5 STIMULATES THE GROWTH AND ANABOLIC METABOLISM OF ARTICULAR CHONDROCYTES

    Institute of Scientific and Technical Information of China (English)

    2005-01-01

    Objective To observe the effect of growth differentiation factor-5 (GDF-5) on the growth and anabolic metabolism of articular chondrocytes. Methods The articular chondrocytes isolated from rats were treated with various concentrations of rmGDF-5, and the growth of chondrocytes measured by MTT assay, the cellular cartilage matrices formation detected sulfated glycosaminoglycan by Alcian blue staining and type Ⅱcollagen by RT-PCR. Results After 7 days culture, MTT assay showed that GDF-5 enhanced the growth of chondrocytes in a dose-dependent manner, RT-PCR showed that GDF-5 clearly induced the synthesis of type Ⅱ collagen because of the col2a1 mRNA band more and more strong in a dose-dependent. Chondrocytes were cultured with GDF-5 for 14 days, the intensity of Alcian blue staining was greatly enhanced, especially, at a high concentration of blue-stainable material in a concentration-dependent manner and in a does-dependent manner. Conclusion GDF-5 enhanced the growth of mature articular chondrocytes, and stimulated the cellular cartilage matrices formation in mono-layer culture.

  18. Finite difference time domain model of ultrasound propagation in agarose scaffold containing collagen or chondrocytes.

    Science.gov (United States)

    Inkinen, Satu I; Liukkonen, Jukka; Malo, Markus K H; Virén, Tuomas; Jurvelin, Jukka S; Töyräs, Juha

    2016-07-01

    Measurement of ultrasound backscattering is a promising diagnostic technique for arthroscopic evaluation of articular cartilage. However, contribution of collagen and chondrocytes on ultrasound backscattering and speed of sound in cartilage is not fully understood and is experimentally difficult to study. Agarose hydrogels have been used in tissue engineering applications of cartilage. Therefore, the aim of this study was to simulate the propagation of high frequency ultrasound (40 MHz) in agarose scaffolds with varying concentrations of chondrocytes (1 to 32 × 10(6) cells/ml) and collagen (1.56-200 mg/ml) using transversely isotropic two-dimensional finite difference time domain method (FDTD). Backscatter and speed of sound were evaluated from the simulated pulse-echo and through transmission measurements, respectively. Ultrasound backscatter increased with increasing collagen and chondrocyte concentrations. Furthermore, speed of sound increased with increasing collagen concentration. However, this was not observed with increasing chondrocyte concentrations. The present study suggests that the FDTD method may have some applicability in simulations of ultrasound scattering and propagation in constructs containing collagen and chondrocytes. Findings of this study indicate the significant role of collagen and chondrocytes as ultrasound scatterers and can aid in development of modeling approaches for understanding how cartilage architecture affects to the propagation of high frequency ultrasound.

  19. 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.

  20. Normal age-related viscoelastic properties of chondrons and chondrocytes isolated from rabbit knee

    Institute of Scientific and Technical Information of China (English)

    DUAN Wang-ping; SUN Zhen-wei; LI Qi; LI Chun-jiang; WANG Li; CHEN Wei-yi; Jennifer Tickner; ZHENG Ming-hao; WEI Xiao-chun

    2012-01-01

    Background The mechanical microenvironment of the chondrocytes plays an important role in cartilage homeostasis and in the health of the joint.The pericellular matrix,cellular membrane of the chondrocytes,and their cytoskeletal structures are key elements in the mechanical environment.The aims of this study are to measure the viscoelastic properties of isolated chondrons and chondrocytes from rabbit knee cartilage using micropipette aspiration and to determine the effect of aging on these properties.Methods Three age groups of rabbit knees were evaluated:(1) young (2 months,n=10);(2) adult (8 months,n=10);and (3) old (31 months,n=10).Chondrocytes were isolated from the right knee cartilage and chondrons were isolated from left knees using enzymatic methods.Micropipette aspiration combined with a standard linear viscoelastic solid model was used to quantify changes in the viscoelastic properties of chondrons and chondrocytes within 2 hours of isolation.The morphology and structure of isolated chondrons were evaluated by optical microscope using hematoxylin and eosin staining and collagen-6 immunofluorescence staining.Results In response to an applied constant 0.3-0.4 kPa of negative pressure,all chondrocytes exhibited standard linear viscoelastic solid properties.Model predictions of the creep data showed that the average equilibrium modulus (E∞),instantaneous modulus (E0).and apparent viscosity (μ) of old chondrocytes was significantly lower than the young and adult chondrocytes (P<0.001);however,no difference was found between young and adult chondrocytes (P>0.05).The adult and old chondrons generally possessed a thicker pericellular matrix (PCM) with more enclosed cells.The young and adult chondrons exhibited the same viscoelastic creep behavior under a greater applied pressure (1.0-1.1kPa) without the deformation seen in the old chondrons.The viscoelastic properties (E∞,E0,and u) of young and adult chondrons were significantly greater than that observed

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

    Science.gov (United States)

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

    2010-02-01

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

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

    Directory of Open Access Journals (Sweden)

    Matsuo M

    2001-12-01

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

  3. Resveratrol protects rabbit articular chondrocyte against sodium nitroprusside-induced apoptosis via scavenging ROS.

    Science.gov (United States)

    Liang, Qian; Wang, Xiao-ping; Chen, Tong-sheng

    2014-09-01

    This study aims to investigate the mechanism by which resveratrol (RV) prevents sodium nitroprusside (SNP)-induced chondrocyte apoptosis, which is a characteristic feature of osteoarthritis (OA). Rabbit articular chondrocytes were pre-incubated with 100 μM RV for 18 h before 1.5 mM SNP co-treatment for 6 h. Cell viability was evaluated by CCK-8. Annexin V/PI double staining and Hoechst 33258 staining were used to determine the fashion of SNP-induced chondrocytes death. Mitochondrial membrane potential (ΔΨm) was measured by using flow cytometry (FCM) with TMRM and Rhodamine 123 staining. Intracellular reactive oxygen species (ROS) and nitric oxide (NO) levels were confirmed by FCM analysis with DCFH-DA and DAF-FM DA staining. Cytoskeleton proteins of chondrocytes co-stained with Actin-Trakcer Green and Tubulin-Trakcer Red were validated by confocal microscopy. SNP induced time- and dose-dependent chondrocytes apoptosis with decline of ΔΨm, activation of caspases as well as cytoskeletal remodeling. SNP induced a significant induction of both ROS and NO. RV remarkably prevented SNP-induced ROS production and apoptosis as well as cytoskeletal remodeling, but did not prevent SNP-induced NO production. Pretreatment with NO scavengers did not significantly prevent SNP-induced apoptosis and cytoskeletal remodeling. SNP induces NO-independent ROS production which dominates rabbit articular chondrocyte apoptosis, and RV protects chondrocytes against SNP-induced apoptosis via scavenging ROS instead of NO.

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

    Science.gov (United States)

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

    2016-05-01

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

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

    Science.gov (United States)

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

    2010-01-01

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

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

    Science.gov (United States)

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

    2016-01-01

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

  7. 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.

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

    Science.gov (United States)

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

    2015-01-01

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

  9. 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.

  10. Mechanical vibrations increase the proliferation of articular chondrocytes in high-density culture.

    Science.gov (United States)

    Kaupp, J A; Waldman, S D

    2008-07-01

    Tissue engineering is a promising approach for articular cartilage repair; however, it still has proven a challenge to produce tissue from the limited number of cells that can be extracted from a single individual. Relatively few cell expansion methods exist without the problems of dedifferentiation and/or loss of potency. Previously, it has been shown that mechanical vibrations can enhance chondrocyte proliferation in monolayer culture. Thus, it was hypothesized that chondrocytes grown in high-density culture would respond in a similar fashion while maintaining phenotypic stability. Isolated bovine articular chondrocytes were seeded in high-density culture on Millicell filters and subjected to mechanical vibrations 48 h after seeding. Mechanical vibrations enhanced chondrocyte proliferation at frequencies above 350 Hz, with the peak response occurring at a 1g amplitude for a duration of 30 min. Under these conditions, the gene expression of cartilage-specific and dedifferentiation markers (collagen II, collagen I, and aggrecan) were unchanged by the imposed stimulus. To determine the effect of accumulated extracellular matrix (ECM) on this proliferative response, selected cultures were stimulated under the same conditions after varying lengths of preculture. The amount of accumulated ECM (collagen and proteoglycans) decreased this proliferative response, with the cultures becoming insensitive to the stimulus after 1 week of preculture. Thus, mechanical vibration can serve as an effective means preferentially to stimulate the proliferation of chondrocytes during culture, but its effects appear to be limited to the early stages where ECM accumulation is at a minimum.

  11. Combining Targeted Metabolomic Data with a Model of Glucose Metabolism: Toward Progress in Chondrocyte Mechanotransduction

    Science.gov (United States)

    Salinas, Daniel; Carlson, Ross P.; McCutchen, Carley N.

    2017-01-01

    Osteoarthritis is a debilitating disease likely involving altered metabolism of the chondrocytes in articular cartilage. Chondrocytes can respond metabolically to mechanical loads via cellular mechanotransduction, and metabolic changes are significant because they produce the precursors to the tissue matrix necessary for cartilage health. However, a comprehensive understanding of how energy metabolism changes with loading remains elusive. To improve our understanding of chondrocyte mechanotransduction, we developed a computational model to calculate the rate of reactions (i.e. flux) across multiple components of central energy metabolism based on experimental data. We calculated average reaction flux profiles of central metabolism for SW1353 human chondrocytes subjected to dynamic compression for 30 minutes. The profiles were obtained solving a bounded variable linear least squares problem, representing the stoichiometry of human central energy metabolism. Compression synchronized chondrocyte energy metabolism. These data are consistent with dynamic compression inducing early time changes in central energy metabolism geared towards more active protein synthesis. Furthermore, this analysis demonstrates the utility of combining targeted metabolomic data with a computational model to enable rapid analysis of cellular energy utilization. PMID:28056047

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

    Science.gov (United States)

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

    2015-12-16

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

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

    Science.gov (United States)

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

    2009-07-01

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

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

    Science.gov (United States)

    Nazempour, A; Van Wie, B J

    2016-05-01

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

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

    Science.gov (United States)

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

    2015-08-14

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

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

    Science.gov (United States)

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

    2011-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Javier Conde

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

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

    Directory of Open Access Journals (Sweden)

    Richard Barrett-Jolley

    2012-04-01

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

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

    NARCIS (Netherlands)

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

    2013-01-01

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

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

    NARCIS (Netherlands)

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

    2004-01-01

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

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

    NARCIS (Netherlands)

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

    2011-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Trung Dung Nguyen

    2014-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Li-ke Luo

    2015-01-01

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

  4. The application of POSS nanostructures in cartilage tissue engineering: the chondrocyte response to nanoscale geometry.

    Science.gov (United States)

    Oseni, Adelola O; Butler, Peter E; Seifalian, Alexander M

    2015-11-01

    Despite extensive research into cartilage tissue engineering (CTE), there is still no scaffold ideal for clinical applications. Various synthetic and natural polymers have been investigated in vitro and in vivo, but none have reached widespread clinical use. The authors investigate the potential of POSS-PCU, a synthetic nanocomposite polymer, for use in CTE. POSS-PCU is modified with silsesquioxane nanostructures that improve its biological and physical properties. The ability of POSS-PCU to support the growth of ovine nasoseptal chondrocytes was evaluated against a polymer widely used in CTE, polycaprolactone (PCL). Scaffolds with varied concentrations of the POSS molecule were also synthesized to investigate their effect on chondrocyte growth. Chondrocytes were seeded onto scaffold disks (PCU negative control; POSS-PCU 2%, 4%, 6%, 8%; PCL). Cytocompatibilty was evaluated using cell viability, total DNA, collagen and GAG assays. Chondrocytes cultured on POSS-PCU (2% POSS) scaffolds had significantly higher viability than PCL scaffolds (p  0.05). POSS-PCU (6% and 8% POSS) had improved viability and proliferation over an 18 day culture period compared with 2% and 4% POSS-PCU (p < 0.0001). Increasing the percentage of POSS in the scaffolds increased the size of the pores found in the scaffolds (p < 0.05). POSS-PCU has excellent potential for use in CTE. It supports the growth of chondrocytes in vitro and the POSS modification significantly enhances the growth and proliferation of nasoseptal chondrocytes compared with traditional scaffolds such as PCL.

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

    Science.gov (United States)

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

    2012-06-01

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

  6. Lidocaine induces ROCK-dependent membrane blebbing and subsequent cell death in rabbit articular chondrocytes.

    Science.gov (United States)

    Maeda, Tsutomu; Toyoda, Futoshi; Imai, Shinji; Tanigawa, Hitoshi; Kumagai, Kousuke; Matsuura, Hiroshi; Matsusue, Yoshitaka

    2016-05-01

    Local anesthetics are administered intraarticularly for pain control in orthopedic clinics and surgeries. Although previous studies have shown that local anesthetics can be toxic to chondrocytes, the underlying cellular mechanisms remain unclear. The present study investigates acute cellular responses associated with lidocaine-induced toxicity to articular chondrocytes. Rabbit articular chondrocytes were exposed to lidocaine and their morphological changes were monitored with live cell microscopy. The viability of chondrocytes was evaluated using a fluorescence based LIVE/DEAD assay. Acute treatment of chondrocytes with lidocaine (3-30 mM) induced spherical protrusions on the cell surface (so called "membrane blebbing") in a time- and concentration-dependent manner. The concentration-response relationship for the lidocaine effect was shifted leftward by elevating extracellular pH, as expected for the non-ionized lidocaine being involved in the bleb formation. ROCK (Rho-kinase) inhibitors Y-27632 and fasudil completely prevented the lidocaine-induced membrane blebbing, suggesting that ROCK activation is required for bleb formation. Caspase-3 levels were unchanged by 10 mM lidocaine (p = 0.325) and a caspase inhibitor z-VAD-fmk did not affect the lidocaine-induced blebbing (p = 0.964). GTP-RhoA levels were significantly increased (p ROCK inhibitors or a myosin-II inhibitor blebbistatin (p ROCK-dependent membrane blebbing and thereby produces a cytotoxic effect on chondrocytes. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:754-762, 2016.

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

    Science.gov (United States)

    Melrose, J.

    2016-01-01

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

  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. Growth factor priming differentially modulates components of the extracellular matrix proteome in chondrocytes and synovium-derived stem cells.

    Directory of Open Access Journals (Sweden)

    Elena Alegre-Aguarón

    Full Text Available To make progress in cartilage repair it is essential to optimize protocols for two-dimensional cell expansion. Chondrocytes and SDSCs are promising cell sources for cartilage repair. We previously observed that priming with a specific growth factor cocktail (1 ng/mL transforming growth factor-β1, 5 ng/mL basic fibroblast growth factor, and 10 ng/mL platelet-derived growth factor-BB in two-dimensional culture, led to significant improvement in mechanical and biochemical properties of synovium-derived stem cell (SDSC-seeded constructs. The current study assessed the effect of growth factor priming on the proteome of canine chondrocytes and SDSCs. In particular, growth factor priming modulated the proteins associated with the extracellular matrix in two-dimensional cultures of chondrocytes and SDSCs, inducing a partial dedifferentiation of chondrocytes (most proteins associated with cartilage were down-regulated in primed chondrocytes and a partial differentiation of SDSCs (some collagen-related proteins were up-regulated in primed SDSCs. However, when chondrocytes and SDSCs were grown in pellet culture, growth factor-primed cells maintained their chondrogenic potential with respect to glycosaminoglycan and collagen production. In conclusion, the strength of the label-free proteomics technique is that it allows for the determination of changes in components of the extracellular matrix proteome in chondrocytes and SDSCs in response to growth factor priming, which could help in future tissue engineering strategies.

  10. Evidence for lysosomal exocytosis and release of aggrecan-degrading hydrolases from hypertrophic chondrocytes, in vitro and in vivo

    Directory of Open Access Journals (Sweden)

    Edward R. Bastow

    2012-02-01

    The abundant proteoglycan, aggrecan, is resorbed from growth plate cartilage during endochondral bone ossification, yet mice with genetically-ablated aggrecan-degrading activity have no defects in bone formation. To account for this apparent anomaly, we propose that lysosomal hydrolases degrade extracellular, hyaluronan-bound aggrecan aggregates in growth plate cartilage, and that lysosomal hydrolases are released from hypertrophic chondrocytes into growth plate cartilage via Ca2+-dependent lysosomal exocytosis. In this study we confirm that hypertrophic chondrocytes release hydrolases via lysosomal exocytosis in vitro and we show in vivo evidence for lysosomal exocytosis in hypertrophic chondrocytes during skeletal development. We show that lysosome-associated membrane protein 1 (LAMP1 is detected at the cell surface following in vitro treatment of epiphyseal chondrocytes with the calcium ionophore, ionomycin. Furthermore, we show that in addition to the lysosomal exocytosis markers, cathepsin D and β-hexosaminidase, ionomycin induces release of aggrecan- and hyaluronan-degrading activity from cultured epiphyseal chondrocytes. We identify VAMP-8 and VAMP7 as v-SNARE proteins with potential roles in lysosomal exocytosis in hypertrophic chondrocytes, based on their colocalisation with LAMP1 at the cell surface in secondary ossification centers in mouse tibiae. We propose that resorbing growth plate cartilage involves release of destructive hydrolases from hypertrophic chondrocytes, via lysosomal exocytosis.

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

    Science.gov (United States)

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

    2010-10-19

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

  12. 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...

  13. MRTF-A signaling regulates the acquisition of the contractile phenotype in dedifferentiated chondrocytes.

    Science.gov (United States)

    Parreno, Justin; Raju, Sneha; Wu, Po-Han; Kandel, Rita A

    2016-10-14

    Chondrocyte culture as a monolayer for cell number expansion results in dedifferentiation whereby expanded cells acquire contractile features and increased actin polymerization status. This study determined whether the actin polymerization based signaling pathway, myocardin-related transcription factor-a (MRTF-A) is involved in regulating this contractile phenotype. Serial passaging of chondrocytes in monolayer culture to passage 2 resulted in increased gene and protein expression of the contractile molecules alpha-smooth muscle actin, transgelin and vinculin compared to non-passaged, primary cells. This resulted in a functional change as passaged 2, but not primary, chondrocytes were capable of contracting type I collagen gels in a stress-relaxed contraction assay. These changes were associated with increased actin polymerization and MRTF-A nuclear localization. The involvement of actin was demonstrated by latrunculin B depolymerization of actin which reversed these changes. Alternatively cytochalasin D which activates MRTF-A increased gene and protein expression of α-smooth muscle actin, transgelin and vinculin, whereas CCG1423 which deactivates MRTF-A decreased these molecules. The involvement of MRTF-A signaling was confirmed by gene silencing of MRTF or its co-factor serum response factor. Knockdown experiments revealed downregulation of α-smooth muscle actin and transgelin gene and protein expression, and inhibition of gel contraction. These findings demonstrate that passaged chondrocytes acquire a contractile phenotype and that this change is modulated by the actin-MRTF-A-serum response factor signaling pathway.

  14. 3D Culture of Chondrocytes in Gelatin Hydrogels with Different Stiffness

    Directory of Open Access Journals (Sweden)

    Xiaomeng Li

    2016-07-01

    Full Text Available Gelatin hydrogels can mimic the microenvironments of natural tissues and encapsulate cells homogeneously, which makes them attractive for cartilage tissue engineering. Both the mechanical and biochemical properties of hydrogels can affect the phenotype of chondrocytes. However, the influence of each property on chondrocyte phenotype is unclear due to the difficulty in separating the roles of these properties. In this study, we aimed to study the influence of hydrogel stiffness on chondrocyte phenotype while excluding the role of biochemical factors, such as adhesion site density in the hydrogels. By altering the degree of methacryloyl functionalization, gelatin hydrogels with different stiffnesses of 3.8, 17.1, and 29.9 kPa Young’s modulus were prepared from the same concentration of gelatin methacryloyl (GelMA macromers. Bovine articular chondrocytes were encapsulated in the hydrogels and cultured for 14 days. The influence of hydrogel stiffness on the cell behaviors including cell viability, cell morphology, and maintenance of chondrogenic phenotype was evaluated. GelMA hydrogels with high stiffness (29.9 kPa showed the best results on maintaining chondrogenic phenotype. These results will be useful for the design and preparation of scaffolds for cartilage tissue engineering.

  15. 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.

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

    DEFF Research Database (Denmark)

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

    2003-01-01

    -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...

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

    Directory of Open Access Journals (Sweden)

    Belinda Pingguan-Murphy

    2012-08-01

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

  18. Polyethylene-glycol-modified single-walled carbon nanotubes for intra-articular delivery to chondrocytes.

    Science.gov (United States)

    Sacchetti, Cristiano; Liu-Bryan, Ru; Magrini, Andrea; Rosato, Nicola; Bottini, Nunzio; Bottini, Massimo

    2014-12-23

    Osteoarthritis (OA) is a common and debilitating degenerative disease of articular joints for which no disease-modifying medical therapy is currently available. Inefficient delivery of pharmacologic agents into cartilage-resident chondrocytes after systemic administration has been a limitation to the development of anti-OA medications. Direct intra-articular injection enables delivery of high concentrations of agents in close proximity to chondrocytes; however, the efficacy of this approach is limited by the fast clearance of small molecules and biomacromolecules after injection into the synovial cavity. Coupling of pharmacologic agents with drug delivery systems able to enhance their residence time and cartilage penetration can enhance the effectiveness of intra-articularly injected anti-OA medications. Herein we describe an efficient intra-articular delivery nanosystem based on single-walled carbon nanotubes (SWCNTs) modified with polyethylene glycol (PEG) chains (PEG-SWCNTs). We show that PEG-SWCNTs are capable to persist in the joint cavity for a prolonged time, enter the cartilage matrix, and deliver gene inhibitors into chondrocytes of both healthy and OA mice. PEG-SWCNT nanoparticles did not elicit systemic or local side effects. Our data suggest that PEG-SWCNTs represent a biocompatible and effective nanocarrier for intra-articular delivery of agents to chondrocytes.

  19. A new test method for the standardized evaluation of changes in the ultrastructure of chondrocytes.

    Science.gov (United States)

    Annefeld, M

    1985-01-01

    By means of a new ultrastructural test system, which is based on standardized morphometry and statistical evaluation, we are able for the first time to quantify changes in the cell metabolism of chondrocytes in the articular cartilage. To compare anti-inflammatory substances of different structure, different absorption characteristics and pharmacokinetics as regards their effect on rat cartilage, we used an equieffective dosage of the different anti-inflammatory drugs. Dexamethasone as steroidal and indomethacin and phenylbutazone as classical non-steroidal anti-inflammatory drugs (NSAIDs) were administered in an ED50 dosage referred to provoked arthritis over 12 weeks, using the same mode of administration. The standardized results of untreated rats weighing 300 and 450 g were used as the controls. Dexamethasone brings about massive degenerative changes in the ultrastructure of the vital chondrocyte. Under indomethacin and phenylbutazone the metabolic activity of the chondrocyte is inhibited to a much lesser extent. The damage to the chondrocyte after treatment with dexamethasone, indomethacin and phenylbutazone cannot be regarded as minimal but in some cases is tolerable as regards the benefit/risk ratio in the treatment of rheumatoid diseases.

  20. Chondrogenic potential of articular chondrocytes depends on their original location in the knee

    NARCIS (Netherlands)

    Bekkers, J.E.J.; Saris, D.B.F.; Tsuchida, A.I.; Rijen, van M.H.P.; Dhert, W.J.A.; Creemers, L.

    2014-01-01

    Objective: This study aimed to investigate the regenerative capacity of chondrocytes derived from debrided defect cartilage and healthy cartilage from different regions in the joint in order to determine the best cell source for regenerative cartilage therapies. Methods: Articular cartilage was obta

  1. Profilin 1 is required for abscission during late cytokinesis of chondrocytes

    Science.gov (United States)

    Böttcher, Ralph T; Wiesner, Sebastian; Braun, Attila; Wimmer, Reiner; Berna, Alejandro; Elad, Nadav; Medalia, Ohad; Pfeifer, Alexander; Aszódi, Attila; Costell, Mercedes; Fässler, Reinhard

    2009-01-01

    Profilins are key factors for dynamic rearrangements of the actin cytoskeleton. However, the functions of profilins in differentiated mammalian cells are uncertain because profilin deficiency is early embryonic lethal for higher eukaryotes. To examine profilin function in chondrocytes, we disrupted the profilin 1 gene in cartilage (Col2pfn1). Homozygous Col2pfn1 mice develop progressive chondrodysplasia caused by disorganization of the growth plate and defective chondrocyte cytokinesis, indicated by the appearance of binucleated cells. Surprisingly, Col2pfn1 chondrocytes assemble and contract actomyosin rings normally during cell division; however, they display defects during late cytokinesis as they frequently fail to complete abscission due to their inability to develop strong traction forces. This reduced force generation results from an impaired formation of lamellipodia, focal adhesions and stress fibres, which in part could be linked to an impaired mDia1-mediated actin filament elongation. Neither an actin nor a poly-proline binding-deficient profilin 1 is able to rescue the defects. Taken together, our results demonstrate that profilin 1 is not required for actomyosin ring formation in dividing chondrocytes but necessary to generate sufficient force for abscission during late cytokinesis. PMID:19262563

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

    Science.gov (United States)

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

    2013-04-01

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

  3. Evaluation of a mPEG-polyester-based hydrogel as cell carrier for chondrocytes.

    Science.gov (United States)

    Peng, Sydney; Yang, Shu-Rui; Ko, Chao-Yin; Peng, Yu-Shiang; Chu, I-Ming

    2013-11-01

    Temperature-sensitive hydrogels are attractive alternatives to porous cell-seeded scaffolds and is minimally invasive through simple injection and in situ gelling. In this study, we compared the performance of two types of temperature-sensitive hydrogels on chondrocytes encapsulation for the use of tissue engineering of cartilage. The two hydrogels are composed of methoxy poly(ethylene glycol)- poly(lactic-co-valerolactone) (mPEG-PVLA), and methoxy poly(ethylene glycol)-poly(lactic- co-glycolide) (mPEG-PLGA). Osmolarity and pH were optimized through the manipulation of polymer concentration and dispersion medium. Chondrocytes proliferation in mPEG-PVLA hydrogels was observed as well as accumulation of GAGs and collagen. On the other hand, chondrocytes encapsulated in mPEG-PLGA hydrogels showed low viability and chondrogenesis. Also, mPEG-PVLA hydrogel, which is more hydrophobic, retained physical integrity after 14 days while mPEG-PLGA hydrogel underwent full degradation due to faster hydrolysis rate and more pronounced acidic self-catalyzed degradation. The mPEG-PVLA hydrogel can be furthered tuned by manipulation of molecular weights to obtain hydrogels with different swelling and degradation characteristics, which may be useful as producing a selection of hydrogels compatible with different cell types. Taken together, these results demonstrate that mPEG-PVLA hydrogels are promising to serve as three-dimensional cell carriers for chondrocytes and potentially applicable in cartilage tissue engineering.

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

    Science.gov (United States)

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

    2016-03-25

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

  5. Chondrocyte BMP2 signaling plays an essential role in bone fracture healing.

    Science.gov (United States)

    Mi, Meng; Jin, Hongting; Wang, Baoli; Yukata, Kiminori; Sheu, Tzong-Jen; Ke, Qiao Han; Tong, Peijian; Im, Hee-Jeong; Xiao, Guozhi; Chen, Di

    2013-01-10

    The specific role of endogenous Bmp2 gene in chondrocytes and in osteoblasts in fracture healing was investigated by generation and analysis of chondrocyte- and osteoblast-specific Bmp2 conditional knockout (cKO) mice. The unilateral open transverse tibial fractures were created in these Bmp2 cKO mice. Bone fracture callus samples were collected and analyzed by X-ray, micro-CT, histology analyses, biomechanical testing and gene expression assays. The results demonstrated that the lack of Bmp2 expression in chondrocytes leads to a prolonged cartilage callus formation and a delayed osteogenesis initiation and progression into mineralization phase with lower biomechanical properties. In contrast, when the Bmp2 gene was deleted in osteoblasts, the mice showed no significant difference in the fracture healing process compared to control mice. These findings suggest that endogenous BMP2 expression in chondrocytes may play an essential role in cartilage callus maturation at an early stage of fracture healing. Our studies may provide important information for clinical application of BMP2.

  6. Repairing cartilage defects using chondrocyte and osteoblast composites developed using a bioreactor

    Institute of Scientific and Technical Information of China (English)

    SUN Shui; REN Qiang; WANG Dong; ZHANG Lei; WU Shuai; SUN Xi-tao

    2011-01-01

    Background Articular cartilage injury is a common disease, and the incidence of articular wear, degeneration, trauma and sports injury is increasing, which often lead to disability and reduced quality of life. Unfortunately repair of articular cartilage defects do not always provide satisfactory outcomes.Methods Chondrocyte and osteoblast composites were co-cultured using a bioreactor. The cartilage defects were treated with cell-β-tricalcium phosphate (β-TCP) composites implanted into osteochondral defects in dogs, in vivo, using mosaicplasty, by placing chondrocyte-β-TCP scaffold composites on top of the defect and osteoblast-β-TCP scaffold composites below the defect.Results Electron microscopy revealed that the induced chondrocytes and osteoblast showed fine adhesive progression and proliferation in the β-TCP scaffold. The repaired tissues in the experimental group maintained their thickness to the full depth of the original defects, as compared with the negative control group (q=12.3370, P <0.01; q=31.5393, P <0.01).Conclusions Perfusion culture provided sustained nutrient supply and gas exchange into the center of the large scaffold. This perfusion bioreactor enables the chondrocytes and osteoblasts to survive and proliferate in a three-dimensional scaffold.

  7. The effect of compressive loading magnitude on in situ chondrocyte calcium signaling.

    Science.gov (United States)

    Madden, Ryan M J; Han, Sang-Kuy; Herzog, Walter

    2015-01-01

    Chondrocyte metabolism is stimulated by deformation and is associated with structural changes in the cartilage extracellular matrix (ECM), suggesting that these cells are involved in maintaining tissue health and integrity. Calcium signaling is an initial step in chondrocyte mechanotransduction that has been linked to many cellular processes. Previous studies using isolated chondrocytes proposed loading magnitude as an important factor regulating this response. However, calcium signaling in the intact cartilage differs compared to isolated cells. The purpose of this study was to investigate the effect of loading magnitude on chondrocyte calcium signaling in intact cartilage. We hypothesized that the percentage of cells exhibiting at least one calcium signal increases with increasing load. Fully intact rabbit femoral condyle and patellar bone/cartilage samples were incubated in calcium-sensitive dyes and imaged continuously under compressive loads of 10-40 % strain. Calcium signaling was primarily associated with the dynamic loading phase and greatly increased beyond a threshold deformation of about 10 % nominal tissue strain. There was a trend toward more cells exhibiting calcium signaling as loading magnitude increased (p = 0.133). These results provide novel information toward identifying mechanisms underlying calcium-dependent signaling pathways related to cartilage homeostasis and possibly the onset and progression of osteoarthritis.

  8. Influence of cell quality on clinical outcome after autologous chondrocyte implantation

    DEFF Research Database (Denmark)

    Niemeyer, Philipp; Pestka, Jan M; Salzmann, Gian M;

    2012-01-01

    BACKGROUND: Several factors influence clinical outcome after autologous chondrocyte implantation (ACI) for the treatment of cartilage defects of the knee joint. HYPOTHESIS/PURPOSE: The aim of the present study was to investigate the influence of cell quality on clinical outcome after ACI. The hyp...

  9. Clinical outcomes after cell-seeded autologous chondrocyte implantation of the knee

    DEFF Research Database (Denmark)

    Pestka, Jan M; Bode, Gerrit; Salzmann, Gian;

    2014-01-01

    BACKGROUND: Autologous chondrocyte implantation (ACI) has been associated with satisfying results. Still, it remains unclear when success or failure after ACI can be estimated. PURPOSE: To evaluate the clinical outcomes of cell-seeded collagen matrix-supported ACI (ACI-Cs) for the treatment of ca...

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

    Directory of Open Access Journals (Sweden)

    Ashvin K. Dewan

    2014-01-01

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

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

    NARCIS (Netherlands)

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

    2013-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Kwok Yeung Tsang

    2007-03-01

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

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

    Science.gov (United States)

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

    2001-06-01

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

  14. 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.

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

    Directory of Open Access Journals (Sweden)

    Akira Ito

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

  16. Interleukin-1 inhibits osmotically-induced calcium signaling and volume regulation in articular chondrocytes

    Science.gov (United States)

    Pritchard, Scott; Votta, Bartholomew J.; Kumar, Sanjay; Guilak, Farshid

    2011-01-01

    OBJECTIVE Articular chondrocytes respond to osmotic stress with transient changes in cell volume and the intracellular concentration of calcium ion ([Ca2+]i). The goal of this study was to examine the hypothesis that interleukin-1 (IL-1), a pro-inflammatory cytokine associated with osteoarthritis, influences osmotically-induced Ca2+ signaling. METHODS Fluorescence ratio imaging was used to measure [Ca2+]i and cell volume in response to hypo- or hyper-osmotic stress in isolated porcine chondrocytes, with or without pre-exposure to 10 ng/ml IL-1α. Inhibitors of IL-1 (IL-1 receptor antagonist, IL-Ra), Ca2+ mobilization (thapsigargin, an inhibitor of Ca-ATPases), and cytoskeletal remodeling (Toxin B, an inhibitor of the Rho family of small GTPases) were used to determine the mechanisms involved in increased [Ca2+]i, F-actin remodeling, volume adaptation and active volume recovery. RESULTS In response to osmotic stress, chondrocytes exhibited transient increases in [Ca2+]i, generally followed by decaying oscillations. Pre-exposure to IL-1 significantly inhibited regulatory volume decrease following hypo-osmotic swelling and reduced the change in cell volume and the time to peak [Ca2+]i in response to hyper-osmotic stress, but did not affect the peak magnitudes of [Ca2+]i in those cells that did response. Co-treatment with IL-1Ra, thapsigargin, or Toxin B restored these responses to control levels. The effects were associated with alterations in F-actin organization. CONCLUSIONS IL-1 alters the normal volumetric and Ca2+ signaling response of chondrocytes to osmotic stress through mechanisms involving F-actin remodeling via small Rho GTPases. These findings provide further insights into the mechanisms by which IL-1 may interfere with normal physiologic processes in the chondrocyte, such as the adaptation or regulatory responses to mechanical and osmotic loading. PMID:18495501

  17. Aquaporin 1 contributes to chondrocyte apoptosis in a rat model of osteoarthritis.

    Science.gov (United States)

    Gao, Hangfei; Gui, Jiancao; Wang, Liming; Xu, Yan; Jiang, Yiqiu; Xiong, Mingyue; Cui, Yongguang

    2016-12-01

    Aquaporins (AQPs) have been found to be associated with a number of diseases. However, the role of AQP‑1 in the pathogenesis of osteoarthritis remains unclear. We previously found that AQP‑1 expression was upregulated in osteoarthritic cartilage and strongly correlated with caspase‑3 expression and activity. The aim of this study was to further investigate the association of AQP‑1 expression with chondrocyte apoptosis in a rat model of osteoarthritis, using RNA interference to knock down AQP‑1. For this purspose, 72 male Sprague‑Dawley rats were randomly assigned to 3 groups as follows: the control group not treated surgically (n=24), the sham‑operated group (n=24), and the osteoarthritis group (n=24). Osteoarthritis was induced by amputating the anterior cruciate ligament and medial collateral ligament and partially excising the medial meniscus. Chondrocytes from the rats with osteoarthritis were isolated and cultured. shRNAs were used to knock down AQP‑1 expression in the cultured chondrocytes. The expression of AQP‑1 and caspase‑3 was determined by reverse transcription-quantitative polymerase chain reaction. Caspase‑3 activity was measured using a caspase‑3 colorimetric assay. The rats in our model of osteoarthritis exhibited severe cartilage damage. The knockdown of AQP‑1 decreased caspase‑3 expression and activity in the cultured chondrocytes. In addition, the expression of AQP‑1 positively correlated with caspase‑3 expression and activity. Thus, the findings of our study, suggest that AQP‑1 promotes caspase‑3 activation and thereby contributes to chondrocyte apoptosis and to the development of osteoarthritis.

  18. 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.

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

    Science.gov (United States)

    Heywood, Hannah K; Lee, David A

    2010-01-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2007-09-15

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

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

    Science.gov (United States)

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

    2017-04-01

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

  2. 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

  3. Effects of RNAi-mediated inhibition of aggrecanase-1 and aggrecanase-2 on rat costochondral chondrocytes in vitro

    Institute of Scientific and Technical Information of China (English)

    Zheng-hui WANG; Zhuang-qun YANG; Xi-jing HE; Li WANG; Li-xia LI; Jun-bo TU

    2008-01-01

    Aim:Failure of transplanted cartilage or allogenic chondrocytes is attributed mainly to immunological rejection and cartilage degradation.A major feature is the loss of aggrecan from the cartilage matrix,primarily due to the action of the specific proteinases aggrecanase-1 and aggrecanase-2.The aim of this in vitro study was to determine whether the specific inhibition of aggrecanase-1 and aggrecanase-2 by RNAi would mitigate aggrecan loss from cultured chondrocytes.Methods:Expression plasmid vectors of shRNA targeting aggrecanase-1 and aggrecanase-2 were constructed and transfected into cultured rattus costochondral chondrocytes.The transfected cells were induced with interleukin-1 β (IL-1β).Gene mRNA levels were analyzed by RT-PCR.Aggrecan and collagen Ⅱ content were measured by immunohistochemistry and Western blotting.Results:As the chondrocytes underwent dedifferentiation,agggrecanase-1 increased significantly.The specific inhibition of aggrecanase-1 and aggrecanase-2 by RNAi had no negative effect on the morphology and growth velocity of the chondrocytes.The mRNA of aggrecanase-1 and aggrecanase-2 decreased significantly.The α-2-macroglobulin expression level was increased by the shRNA specific for aggrecanase-1.Other genes of the chondrocytic extracellular matrix were not affected.RNAi significantly increased the aggrecan and collagen Ⅱ content of chondrocytes treated with IL-1β.Conclusion:The results suggest that inhibition of aggrecanase-1 and aggrecanase-2 by RNAi can mitigate aggrecan degradation,without interfering with chondrocytic gene phenotype recovery.RNAi technology can be a useful tool for studying degenerative processes in cartilage.

  4. Ear-Shaped Stable Auricular Cartilage Engineered from Extensively Expanded Chondrocytes in an Immunocompetent Experimental Animal Model.

    Science.gov (United States)

    Pomerantseva, Irina; Bichara, David A; Tseng, Alan; Cronce, Michael J; Cervantes, Thomas M; Kimura, Anya M; Neville, Craig M; Roscioli, Nick; Vacanti, Joseph P; Randolph, Mark A; Sundback, Cathryn A

    2016-02-01

    Advancement of engineered ear in clinical practice is limited by several challenges. The complex, largely unsupported, three-dimensional auricular neocartilage structure is difficult to maintain. Neocartilage formation is challenging in an immunocompetent host due to active inflammatory and immunological responses. The large number of autologous chondrogenic cells required for engineering an adult human-sized ear presents an additional challenge because primary chondrocytes rapidly dedifferentiate during in vitro culture. The objective of this study was to engineer a stable, human ear-shaped cartilage in an immunocompetent animal model using expanded chondrocytes. The impact of basic fibroblast growth factor (bFGF) supplementation on achieving clinically relevant expansion of primary sheep chondrocytes by in vitro culture was determined. Chondrocytes expanded in standard medium were either combined with cryopreserved, primary passage 0 chondrocytes at the time of scaffold seeding or used alone as control. Disk and human ear-shaped scaffolds were made from porous collagen; ear scaffolds had an embedded, supporting titanium wire framework. Autologous chondrocyte-seeded scaffolds were implanted subcutaneously in sheep after 2 weeks of in vitro incubation. The quality of the resulting neocartilage and its stability and retention of the original ear size and shape were evaluated at 6, 12, and 20 weeks postimplantation. Neocartilage produced from chondrocytes that were expanded in the presence of bFGF was superior, and its quality improved with increased implantation time. In addition to characteristic morphological cartilage features, its glycosaminoglycan content was high and marked elastin fiber formation was present. The overall shape of engineered ears was preserved at 20 weeks postimplantation, and the dimensional changes did not exceed 10%. The wire frame within the engineered ear was able to withstand mechanical forces during wound healing and neocartilage

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

    Science.gov (United States)

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

    2012-01-01

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

  6. Voltage-gated K+ currents in mouse articular chondrocytes regulate membrane potential.

    Science.gov (United States)

    Clark, Robert B; Hatano, Noriyuki; Kondo, Colleen; Belke, Darrell D; Brown, Barry S; Kumar, Sanjay; Votta, Bartholomew J; Giles, Wayne R

    2010-01-01

    Membrane currents and resting potential of isolated primary mouse articular chondrocytes maintained in monolayer cell culture for 1-9 days were recorded using patch clamp methods. Quantitative RT-PCR showed that the most abundantly expressed transcript of voltage-gated K(+) channels was for K(V)1.6, and immunological methods confirmed the expression of K(V)1.6 α-subunit proteins. These chondrocytes expressed a large time- and potential-dependent, Ca(2+)-independent 'delayed rectifier' K(+) current. Steady-state activation was well-fit by a Boltzmann function with a threshold near -50 mV, and a half-activation potential of -34.5 mV. The current was 50% blocked by 1.48 mM tetraethylammonium, 0.66 mM 4-aminopyridine and 20.6 nM α-dendrotoxin. The current inactivated very slowly at membrane potentials in the range of the resting potential of the chondrocytes. Resting membrane potential of the chondrocytes at room temperature (19-21°C) and in 5 mM external K(+) was -46.4 ± 1.3 mV (mean ± s.e.m; n = 23), near the 'foot' of the activation curve of this K(+) current. Resting potential was depolarized by an average of 4.2 ± 0.8 mV by 25 mM TEA, which blocked about 95% of the K(+) current. At a membrane potential of -50 mV, the apparent time constant of inactivation (tau(in)) was 37.9 s, and the 'steady-state' current level was 19% of that at a holding potential of -90 mV; at -40 mV, tau(in) was 20.3 s, and 'steady-state' current was 5% of that at -90 mV. These results demonstrate that in these primary cultured, mouse articular chondrocytes steady-state activation of a voltage-gated K(+) current contributes to resting membrane potential. However, this current is also likely to have a significant physiological role in repolarizing the chondrocyte following depolarizing stimuli that might occur in conditions of membrane stretch. For example, activation of TRP('transient receptor potential') non-specific cation channels in these cells during cyclic loading and unloading

  7. Osthole Inhibits Proliferation and Induces Catabolism in Rat Chondrocytes and Cartilage Tissue

    Directory of Open Access Journals (Sweden)

    Guoqing Du

    2015-08-01

    Full Text Available Background/Aims: Cartilage destruction is thought to be the major mediator of osteoarthritis. Recent studies suggest that inhibition of subchrondral bone loss by anti-osteoporosis (OP drug can protect cartilige erosion. Osthole, as a promising agent for treating osteoporosis, may show potential in treating osteoarthritis. The purpose of this study was to investigate whether Osthole affects the proliferation and catabolism of rat chondrocytes, and the degeneration of cartilage explants. Methods: Rat chondrocytes were treated with Osthole (0 μM, 6.25 μM, 12.5 μM, and 25 μM with or without IL1-β (10ng/ml for 24 hours. The expression levels of type II collagen and MMP13 were detected by western Blot. Marker genes for chondrocytes (A-can and Sox9, matrix metalloproteinases (MMPs, aggrecanases (ADAMTS5 and genes implicated in extracellular matrix catabolism were evaluated by qPCR. Cell proliferation was assessed by measuring proliferating cell nuclear antigen (PCNA expression and fluorescence activated cell sorter. Wnt7b/β-catenin signaling was also investigated. Cartilage explants from two-week old SD rats were cultured with IL-1β, Osthole and Osthole plus IL-1β for four days and glycosaminoglycan (GAG synthesis was assessed with toluidine blue staining and Safranine O/Fast Green FCF staining, collagen type II expression was detected by immunofuorescence. Results: Osthole reduced expression of chondrocyte markers and increased expression of MMP13, ADAMTS5 and MMP9 in a dose-dependent manner. Catabolic gene expression levels were further improved by Osthole plus IL-1β. Osthole inhibited chondrocyte proliferation. GAG synthesis and type II collagen were decreased in both the IL-1β groups and the Osthole groups, and significantly reduced by Osthole plus IL-1β. Conclusions: Our data suggested that Osthole increases the catabolism of rat chondrocytes and cartilage explants, this effect might be mediated through inhibiting Wnt7b

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

    Directory of Open Access Journals (Sweden)

    Tang Rining

    2012-09-01

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

  9. [The synergistic effect of amygdalin and HSYA on the IL-1beta induced endplate chondrocytes of rat intervertebral discs].

    Science.gov (United States)

    Niu, Kai; Zhao, Yong-Jian; Zhang, Lei; Li, Chen-Guang; Wang, Yong-Jun; Zheng, Wei-Chao

    2014-08-01

    The effect of amygdalin joint hydroxysafflor yellow A (HSYA) on the endplate chondrocytes derived from intervertebral discs of rats induced by IL-1beta and the possible mechanism were studied and explored. Chondrocytes were obtained from endplate of one-month SD rat intervertebral discs and cultured primary endplate chondrocytes. After identification, they were divided into normal group, induced group, amygdalin group, HSYA group and combined group. CCK-8 kit was adopted to detect the proliferation of the endplate chondrocytes. FCM was measured to detect the apoptosis. Real-time PCR method was adopted to observe the mRNA expression of Aggrecan, Col 2 alpha1, Col 10 alpha1, MMP-13 and the inflammatory cytokines IL-1beta. The protein expression of Col II, Col X was tested through immunofluorescence. Compared with the normal group, the proliferation of the endplate chondrocytes decreased while the apoptosis increased (P amygdalin group, HSYA group, the combined group could inhibit the apoptosis and promote the proliferation (P amygdalin and HSYA. Amygdalin joint HSYA could inhibit the degeneration of the endplate chondrocytes derived from intervertebral discs of rats induced by IL-1beta and better than the single use of amygdalin or HSYA.

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

    Science.gov (United States)

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

    2016-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Christopher J. Little

    2014-09-01

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

  12. Prolyl Hydroxylase Domain-Containing Protein 2 (Phd2) Regulates Chondrocyte Differentiation and Secondary Ossification in Mice

    Science.gov (United States)

    Cheng, Shaohong; Aghajanian, Patrick; Pourteymoor, Sheila; Alarcon, Catrina; Mohan, Subburaman

    2016-01-01

    Endochondral ossification plays an important role in the formation of the primary ossification centers (POCs) and secondary ossification centers (SOCs) of mammalian long bones. However, the molecular mechanisms that regulate POC and SOC formation are different. We recently demonstrated that Prolyl Hydroxylase Domain-containing Protein 2 (Phd2) is a key mediator of vitamin C effects on bone. We investigated the role of Phd2 on endochondral ossification of the epiphyses by conditionally deleting the Phd2 gene in osteoblasts and chondrocytes. We found that the deletion of Phd2 in osteoblasts did not cause changes in bone parameters in the proximal tibial epiphyses in 5 week old mice. In contrast, deletion of Phd2 in chondrocytes resulted in increased bone mass and bone formation rate (normalized to tissue volume) in long bone epiphyses, indicating that Phd2 expressed in chondrocytes, but not osteoblasts, negatively regulates secondary ossification of epiphyses. Phd2 deletion in chondrocytes elevated mRNA expression of hypoxia-inducible factor (HIF) signaling molecules including Hif-1α, Hif-2α, Vegfa, Vegfb, and Epo, as well as markers for chondrocyte hypertrophy and mineralization such as Col10, osterix, alkaline phosphatase, and bone sialoprotein. These data suggest that Phd2 expressed in chondrocytes inhibits endochondral ossification at the epiphysis by suppressing HIF signaling pathways. PMID:27775044

  13. 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.

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

    Science.gov (United States)

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

    2013-12-01

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

  15. Flavonoid Compound Icariin Activates Hypoxia Inducible Factor-1α in Chondrocytes and Promotes Articular Cartilage Repair.

    Directory of Open Access Journals (Sweden)

    Pengzhen Wang

    Full Text Available Articular cartilage has poor capability for repair following trauma or degenerative pathology due to avascular property, low cell density and migratory ability. Discovery of novel therapeutic approaches for articular cartilage repair remains a significant clinical need. Hypoxia is a hallmark for cartilage development and pathology. Hypoxia inducible factor-1alpha (HIF-1α has been identified as a key mediator for chondrocytes to response to fluctuations of oxygen availability during cartilage development or repair. This suggests that HIF-1α may serve as a target for modulating chondrocyte functions. In this study, using phenotypic cellular screen assays, we identify that Icariin, an active flavonoid component from Herba Epimedii, activates HIF-1α expression in chondrocytes. We performed systemic in vitro and in vivo analysis to determine the roles of Icariin in regulation of chondrogenesis. Our results show that Icariin significantly increases hypoxia responsive element luciferase reporter activity, which is accompanied by increased accumulation and nuclear translocation of HIF-1α in murine chondrocytes. The phenotype is associated with inhibiting PHD activity through interaction between Icariin and iron ions. The upregulation of HIF-1α mRNA levels in chondrocytes persists during chondrogenic differentiation for 7 and 14 days. Icariin (10-6 M increases the proliferation of chondrocytes or chondroprogenitors examined by MTT, BrdU incorporation or colony formation assays. Icariin enhances chondrogenic marker expression in a micromass culture including Sox9, collagen type 2 (Col2α1 and aggrecan as determined by real-time PCR and promotes extracellular matrix (ECM synthesis indicated by Alcian blue staining. ELISA assays show dramatically increased production of aggrecan and hydroxyproline in Icariin-treated cultures at day 14 of chondrogenic differentiation as compared with the controls. Meanwhile, the expression of chondrocyte catabolic

  16. The ECM-Cell Interaction of Cartilage Extracellular Matrix on Chondrocytes

    Directory of Open Access Journals (Sweden)

    Yue Gao

    2014-01-01

    Full Text Available Cartilage extracellular matrix (ECM is composed primarily of the network type II collagen (COLII and an interlocking mesh of fibrous proteins and proteoglycans (PGs, hyaluronic acid (HA, and chondroitin sulfate (CS. Articular cartilage ECM plays a crucial role in regulating chondrocyte metabolism and functions, such as organized cytoskeleton through integrin-mediated signaling via cell-matrix interaction. Cell signaling through integrins regulates several chondrocyte functions, including differentiation, metabolism, matrix remodeling, responses to mechanical stimulation, and cell survival. The major signaling pathways that regulate chondrogenesis have been identified as wnt signal, nitric oxide (NO signal, protein kinase C (PKC, and retinoic acid (RA signal. Integrins are a large family of molecules that are central regulators in multicellular biology. They orchestrate cell-cell and cell-matrix adhesive interactions from embryonic development to mature tissue function. In this review, we emphasize the signaling molecule effect and the biomechanics effect of cartilage ECM on chondrogenesis.

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

    Institute of Scientific and Technical Information of China (English)

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

    2007-01-01

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

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

    Science.gov (United States)

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

    2013-01-01

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

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

    Science.gov (United States)

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

    2013-10-01

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

  20. 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

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

    Science.gov (United States)

    Hyzy, Sharon L.; Doroudi, Maryam; Williams, Joseph K.; Gall, Ken; Boyan, Barbara D.; Schwartz, Zvi

    2017-01-01

    Stem cell fate has been linked to the mechanical properties of their underlying substrate, affecting mechanoreceptors and ultimately leading to downstream biological response. Studies have used polymers to mimic the stiffness of extracellular matrix as well as of individual tissues and shown mesenchymal stem cells (MSCs) could be directed along specific lineages. In this study, we examined the role of stiffness in MSC differentiation to two closely related cell phenotypes: osteoblast and chondrocyte. We prepared four methyl acrylate/methyl methacrylate (MA/MMA) polymer surfaces with elastic moduli ranging from 0.1 MPa to 310 MPa by altering monomer concentration. MSCs were cultured in media without exogenous growth factors and their biological responses were compared to committed chondrocytes and osteoblasts. Both chondrogenic and osteogenic markers were elevated when MSCs were grown on substrates with stiffness <10 MPa. Like chondrocytes, MSCs on lower stiffness substrates showed elevated expression of ACAN, SOX9, and COL2 and proteoglycan content; COMP was elevated in MSCs but reduced in chondrocytes. Substrate stiffness altered levels of RUNX2 mRNA, alkaline phosphatase specific activity, osteocalcin, and osteoprotegerin in osteoblasts, decreasing levels on the least stiff substrate. Expression of integrin subunits α1, α2, α5, αv, β1, and β3 changed in a stiffness- and cell type-dependent manner. Silencing of integrin subunit beta 1 (ITGB1) in MSCs abolished both osteoblastic and chondrogenic differentiation in response to substrate stiffness. Our results suggest that substrate stiffness is an important mediator of osteoblastic and chondrogenic differentiation, and integrin β1 plays a pivotal role in this process. PMID:28095466

  2. A mutation in TRPV4 results in altered chondrocyte calcium signaling in severe metatropic dysplasia.

    Science.gov (United States)

    Hurd, Lauren; Kirwin, Susan M; Boggs, Mary; Mackenzie, William G; Bober, Michael B; Funanage, Vicky L; Duncan, Randall L

    2015-10-01

    Transient receptor potential cation channel, subfamily V, member 4 (TRPV4) is a polymodal modulated non-selective cation channel required for normal development and maintenance of bone and cartilage. Heterozygous mutations of this channel cause a variety of channelopathies, including metatropic dysplasia (MD). We analyzed the effect of a novel TRPV4 mutation c.2398G>A, p.Gly800Asp on intracellular calcium ([Ca(2+) ]i ) regulation in chondrocytes and compared this response to chondrocytes with a frequently observed mutation, c.2396C>T, p.Pro799Leu. We observed temperature-dependent [Ca(2+) ]i oscillations in both intact and MD chondrocytes however, MD mutations exhibited increased peak magnitudes of [Ca(2+) ]i during oscillations. We also found increased baseline [Ca(2+) ]i in MD primary cells, as well as increased [Ca(2+) ]i response to either hypotonic swelling or the TRVP4-specific agonist, GSK1016790A. Oscillations and stimulation responses were blocked with the TRPV4-specific antagonist, GSK205. Analysis of [Ca(2+) ]i response kinetics showed that MD chondrocytes had increased frequency of temperature-sensitive oscillations, and the magnitude and duration of [Ca(2+) ]i responses to given stimuli. Duration of the response of the p.Gly800Asp mutation to stimulation was greater than for the p.Pro799Leu mutation. These experiments show that this region of the channel is essential for proper [Ca(2+) ]i regulation. These studies of primary cells from patients show how both mutant and WT TRPV4 channels regulate cartilage and bone development. © 2015 Wiley Periodicals, Inc.

  3. 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.

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

    Science.gov (United States)

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

    2013-01-01

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

  5. 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.

  6. Effects of cytokines, growth factors and drugs on matrix metalloproteinases activities of osteoarthritic chondrocytes and synoviocytes

    Institute of Scientific and Technical Information of China (English)

    GUAN Jian-long; HAN Xing-hai; SHI Gui-ying; YUAN Guo-hua

    2001-01-01

    Objective: To evaluate the effects of some cytokines, TGF-β1 and drugs on matrix metalloproteinases (MMPs) activities in culture medium of arthritic chondrocytes and synoviocytes. Methods: The chondrocyte and synoviocyte monolayers isolated from the cartilages and synovial fluids in 10 knee OA patients were treated with IL-1β TGF-β1, TNF-α, diclofenac acid, dexamethasone or doxycycline individually and together for 72 h. Zymography was used to determine the activities of MMP-2 and -9. Results: The chondrocyte monolayers produced MMP-2 and -9, while the synoviocytes only produced MMP-2. The MMP-9 activity was markedly enhanced by IL-1β TNF-α and diclofenac. IL-1β was the most effective stimulus, and had synergistic effect with TNF-α or diclifenac. MMP-2 activity was not affected. Doxcycline, TGF-β1 and dexamethasone could depress the activities of MMP-9 and MMP-2, and antagonize the enhancing effect of IL-1β TNF-α or diclofenac. Conclusion: IL-1β and TNF-α may play important roles degrading OA cartilage, while TGF-β1 and doxycycline may be protective factors.

  7. 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.

  8. Enhanced redifferentiation of chondrocytes on microperiodic silk/gelatin scaffolds: toward tailor-made tissue engineering.

    Science.gov (United States)

    Das, Sanskrita; Pati, Falguni; Chameettachal, Shibu; Pahwa, Shikha; Ray, Alok R; Dhara, Santanu; Ghosh, Sourabh

    2013-02-11

    Direct-write assembly allows rapid fabrication of complex three-dimensional (3D) architectures, such as scaffolds simulating anatomical shapes, avoiding the need for expensive lithographic masks. However, proper selection of polymeric ink composition and tailor-made viscoelastic properties are critically important for smooth deposition of ink and shape retention. Deposition of only silk solution leads to frequent clogging due to shear-induced β-sheet crystallization, whereas optimized viscoelastic property of silk-gelatin blends facilitate the flow of these blends through microcapillary nozzles of varying diameter. This study demonstrates that induction of controlled changes in scaffold surface chemistry, by optimizing silk-gelatin ratio, can govern cell proliferation and maintenance of chondrocyte morphology. Microperiodic silk-gelatin scaffolds can influence postexpansion redifferentiation of goat chondrocytes by enhancing Sox-9 gene expression, aggregation, and driving cartilage matrix production, as evidenced by upregulation of collagen type II and aggrecan expression. The strategy for optimizing redifferentiation of chondrocytes can offer valuable consideration in scaffold-based cartilage repair strategies.

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

    Science.gov (United States)

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

    2017-03-25

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

  10. Andrographolide enhances proliferation and prevents dedifferentiation of rabbit articular chondrocytes: an in vitro study.

    Science.gov (United States)

    Luo, Li-Ke; Wei, Qing-Jun; Liu, Lei; Zheng, Li; Zhao, Jin-Min

    2015-01-01

    As the main active constituent of Andrographis paniculata that was applied in treatment of many diseases including inflammation in ancient China, andrographolide (ANDRO) was found to facilitate reduction of edema and analgesia in arthritis. This suggested that ANDRO may be promising anti-inflammatory agent to relieve destruction and degeneration of cartilage after inflammation. In this study, the effect of ANDRO on rabbit articular chondrocytes in vitro was investigated. Results showed that not more than 8 μM ANDRO did no harm to chondrocytes (P ANDRO groups comparing to the control (P ANDRO could promote expression of aggrecan, collagen II, and Sox9 genes while downregulating expression of collagen I gene (P 0.05). The viability assay, hematoxylin-eosin, safranin O, and immunohistochemical staining also showed better performances in ANDRO groups. As to the doses, 3 μM ANDRO showed the best performance. The results indicate that ANDRO can accelerate proliferation of rabbit articular chondrocytes in vitro and meanwhile maintain the phenotype, which may provide valuable references for further exploration on arthritis.

  11. Effect of Carnosine in Experimental Arthritis and on Primary Culture Chondrocytes

    Directory of Open Access Journals (Sweden)

    S. Ponist

    2016-01-01

    Full Text Available Carnosine’s (CARN anti-inflammatory potential in autoimmune diseases has been but scarcely investigated as yet. The aim of this study was to evaluate the therapeutic potential of CARN in rat adjuvant arthritis, in the model of carrageenan induced hind paw edema (CARA, and also in primary culture of chondrocytes under H2O2 injury. The experiments were done on healthy animals, arthritic animals, and arthritic animals with oral administration of CARN in a daily dose of 150 mg/kg b.w. during 28 days as well as animals with CARA treated by a single administration of CARN in the same dose. CARN beneficially affected hind paw volume and changes in body weight on day 14 and reduced hind paw swelling in CARA. Markers of oxidative stress in plasma and brain (malondialdehyde, 4-hydroxynonenal, protein carbonyls, and lag time of lipid peroxidation and also activity of gamma-glutamyltransferase were significantly corrected by CARN. CARN also reduced IL-1alpha in plasma. Suppression of intracellular oxidant levels was also observed in chondrocytes pretreated with CARN. Our results obtained on two animal models showed that CARN has systemic anti-inflammatory activity and protected rat brain and chondrocytes from oxidative stress. This finding suggests that CARN might be beneficial for treatment of arthritic diseases.

  12. MicroRNA-181b regulates articular chondrocytes differentiation and cartilage integrity.

    Science.gov (United States)

    Song, Jinsoo; Lee, Myeungsu; Kim, Dongkyun; Han, Jiyeon; Chun, Churl-Hong; Jin, Eun-Jung

    2013-02-08

    MicroRNAs are endogenous gene regulators that have been implicated in various developmental and pathological processes. However, the precise identities and functions of the miRNAs involved in cartilage development are not yet well understood. Here, we report that miR-181b regulates chondrocyte differentiation and maintains cartilage integrity, and is thus a potent therapeutic target. MiR-181b was significantly down-regulated during chondrogenic differentiation of TGF-β3-stimulated limb mesenchymal cells, but it was significantly up-regulated in osteoarthritic chondrocytes isolated from the cartilage of osteoarthritis patients. The use of a mimic or an inhibitor to alter miR-181b levels in chondroblasts and articular chondrocytes showed that attenuation of miR-181b reduced MMP-13 expression while inducing type II collagen expression. Furthermore, over-expression of anti-miR-181b significantly reduced the cartilage destruction caused by DMM surgery in mice. In sum, our data suggest that miR-181b is a negative regulator of cartilage development, and that inhibition of miR-181b could be an effective therapeutic strategy for cartilage-related disease.

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

    Institute of Scientific and Technical Information of China (English)

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

    2004-01-01

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

  14. Vanadate proliferative and anti-mineralogenic effects are mediated by MAPK and PI-3K/Ras/Erk pathways in a fish chondrocyte cell line.

    Science.gov (United States)

    Tiago, Daniel M; Cancela, M Leonor; Aureliano, Manuel; Laizé, Vincent

    2008-04-16

    We recently reported proliferative and anti-mineralogenic effects of vanadate on fish chondrocytes and here we investigate the signalling pathways associated with these effects. Our data show that vanadate stimulates chondrocyte proliferation through the MAPK pathway, using signalling mechanisms similar to those used by IGF-1, while it inhibits chondrocyte differentiation/mineralization through a putative PI-3K/Ras/Erk signalling, a pathway shared with insulin. Our data also suggest that vanadate impairs ECM mineralization not only by interfering with regulatory pathways but also by inhibiting enzymatic activity of ALP. Finally, this work provides additional evidence for the conservation, throughout evolution, of mechanisms regulating chondrocyte proliferation and differentiation.

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

    Science.gov (United States)

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

    1990-09-01

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

  16. TNF Accelerates Death of Mandibular Condyle Chondrocytes in Rats with Biomechanical Stimulation-Induced Temporomandibular Joint Disease

    Science.gov (United States)

    Zhang, Hongyun; Zhang, Jing; Jing, Lei; Liao, Lifan; Wang, Meiqing

    2015-01-01

    Objective To determine if temporomandibular joint chondrocyte apoptosis is induced in rats with dental biomechanical stimulation and what a role TNF takes. Methods Thirty-two rats were divided into 4 groups (n = 8/group) and exposed to incisor mal-occlusion induced by unilateral anterior crossbite biomechanical stimulation. Two groups were sampled at 2 or 4 weeks. The other two groups were treated with local injections of a TNF inhibitor or PBS into the temporomandibular joints area at 2 weeks and then sampled at 4 weeks. Twenty-four rats either served as unilateral anterior crossbite mock operation controls (n = 8/group) with sampling at 2 or 4 weeks or received a local injection of the TNF inhibitor at 2 weeks with sampling at 4 weeks. Chondrocytes were isolated from the temporomandibular joints of 6 additional rats and treated with TNF in vitro. Joint samples were assessed using Hematoxylin&eosin, Safranin O, TUNEL and immunohistochemistry staining, real-time PCR, fluorogenic activity assays and Western blot analyses. The isolated chondrocytes were also analyzed by flow cytometry. Results Unilateral anterior crossbite stimulation led to temporomandibular joint cartilage degradation, associated with an increase in TUNEL-positive chondrocytes number, caspase-9 expression levels, and the release of cytochrome c from mitochondria at 2 weeks without changes in TNF and caspase-8 levels until after 4 weeks. TNF stimulated apoptosis of the isolated chondrocytes and up-regulated caspase-8 expression, but did not change caspase-9 expression levels. Local injection of TNF inhibitor down-regulated caspase-8 expression and reduced TUNEL-positive cell number, but did not reverse cartilage thickness reduction, caspase-9 up-regulation or cytochrome c release. Conclusions Unilateral anterior crossbite stimulation induces mitochondrion-mediated apoptosis of articular chondrocytes. TNF accelerated the unilateral anterior crossbite induced chondrocytes apoptosis via death

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

    Science.gov (United States)

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

    2015-01-01

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

  18. TNF Accelerates Death of Mandibular Condyle Chondrocytes in Rats with Biomechanical Stimulation-Induced Temporomandibular Joint Disease.

    Directory of Open Access Journals (Sweden)

    Hongxu Yang

    Full Text Available To determine if temporomandibular joint chondrocyte apoptosis is induced in rats with dental biomechanical stimulation and what a role TNF takes.Thirty-two rats were divided into 4 groups (n = 8/group and exposed to incisor mal-occlusion induced by unilateral anterior crossbite biomechanical stimulation. Two groups were sampled at 2 or 4 weeks. The other two groups were treated with local injections of a TNF inhibitor or PBS into the temporomandibular joints area at 2 weeks and then sampled at 4 weeks. Twenty-four rats either served as unilateral anterior crossbite mock operation controls (n = 8/group with sampling at 2 or 4 weeks or received a local injection of the TNF inhibitor at 2 weeks with sampling at 4 weeks. Chondrocytes were isolated from the temporomandibular joints of 6 additional rats and treated with TNF in vitro. Joint samples were assessed using Hematoxylin&eosin, Safranin O, TUNEL and immunohistochemistry staining, real-time PCR, fluorogenic activity assays and Western blot analyses. The isolated chondrocytes were also analyzed by flow cytometry.Unilateral anterior crossbite stimulation led to temporomandibular joint cartilage degradation, associated with an increase in TUNEL-positive chondrocytes number, caspase-9 expression levels, and the release of cytochrome c from mitochondria at 2 weeks without changes in TNF and caspase-8 levels until after 4 weeks. TNF stimulated apoptosis of the isolated chondrocytes and up-regulated caspase-8 expression, but did not change caspase-9 expression levels. Local injection of TNF inhibitor down-regulated caspase-8 expression and reduced TUNEL-positive cell number, but did not reverse cartilage thickness reduction, caspase-9 up-regulation or cytochrome c release.Unilateral anterior crossbite stimulation induces mitochondrion-mediated apoptosis of articular chondrocytes. TNF accelerated the unilateral anterior crossbite induced chondrocytes apoptosis via death-receptor pathway. However, anti

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

    Institute of Scientific and Technical Information of China (English)

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

    2013-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Atsuo Kawakita

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

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

    Directory of Open Access Journals (Sweden)

    He X

    2015-03-01

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

  2. Determinants of microstructural load transfer in cartilage tissue from chondrocyte culture

    Science.gov (United States)

    Fedewa, Michelle Marie

    2000-10-01

    The goals of this research were to (i) develop a tissue model system for studying the microstructure of matrix produced by chondrocytes, (ii) characterize the biochemical and mechanical properties of the chondrocyte culture tissue, (iii) evaluate the response of the chondrocyte culture tissue to various stimulants (retinoic acid, interleukin-1beta, and xyloside), (iv) investigate the roles of proteoglycan and collagen in the tearing and tensile properties of a chondrocyte culture tissue, and (v) develop a finite element model of the chondrocyte culture tissue microstructure to study its tensile pre-failure properties. The roles of proteoglycan and collagen were explored by experimentation using a cultured cartilage tissue, and by development of a theoretical finite element model which related the cartilage tissue microstructure to its macroscopic properties. Tear and tensile testing was performed. Failure testing is valuable because it is known that cracks exist and propagate from the cartilage surface in osteoarthritic joints. It was found that collagen was important for providing the material stiffness of the cultured tissue, and that both collagen and proteoglycan were important for providing the tear toughness of the tissue. It was also found that as the collagen density or collagen material stiffness increased, the material stiffness of the cultured tissue increased, and as the proteoglycan or collagen densities increased, the tear toughness of the tissue increased. A three-dimensional finite element microstructural model of cartilage was developed, consisting of linear elastic collagen fibrils embedded in a linear viscoelastic proteoglycan solid matrix. Fluid flow in the cartilage matrix was not included in this model. Viscoelastic time dependent behavior was an appropriate model for the cartilage. The results of this model were comparable to the experimental results, as well as to past continuum models of cartilage. Collagen and proteoglycan material moduli

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

    Directory of Open Access Journals (Sweden)

    Wang ZH

    2014-05-01

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

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

    Directory of Open Access Journals (Sweden)

    Muhammad Farooq Rai

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

  5. Effect of the disruption of three cytoskeleton components on chondrocyte metabolism in rabbit knee cartilage

    Institute of Scientific and Technical Information of China (English)

    Duan Wangping; Wei Lei; Cao Xiaoming; Guo Heng; Wang Lei; Hao Yongzhuang; Wei Xiaochun

    2014-01-01

    Background Chondrocytes' phenotype and biosynthesis of matrix are dependent on having an intact cytoskeletal structure.Microfilaments,microtubules,and intermediate filaments are three important components of the cytoskeletal structure of chondrocytes.The aims of this study were to determine and compare the effects of the disruption of these three cytoskeletal elements on the apoptosis and matrix synthesis by rabbit knee chondrocytes in vitro.Methods Chondrocytes were isolated from full-thickness knee cartilage of two-month-old rabbits using enzymatic methods (n=24).The isolated cells were stabilized for three days and then exposed to low,medium,and high doses of chemical agents that disrupt the three principal cytoskeletal elements of interest:colchicine for microtubules,acrylamide for intermediate filaments,and cytochalasin D for actin microfilaments.A group of control cells were treated with carrier.Early apoptosis was assessed using the Annexin-FITC binding assay by flow cytometry on days 1 and 2 after exposure to the disrupting chemical agents.The components and distribution of the cytoskeleton within the cells were analyzed by laser scanning confocal microscopy (LSCM) with immunofluorescence staining on day 3.The mRNA levels of aggrecan (AGG) and type Ⅱ collagen (Col-2) and their levels in culture medium were analyzed using real-time PCR and enzymelinked immunosorbent serologic assay (ELISA) on days 3,6,and 9.Results In the initial drug-dose-response study,there was no significant difference in the vitality of cells treated with 0.1 μmol/L colchicine,2.5 mmol/L acrylamide,and 10 μg/L cytochalasin D for two days when compared with the control group of cells.The concentrations of colchicine and acrylamide treatment selected above significantly decreased the number of viable cells over the nine-day culture and disrupted significantly more cell nuclei.Real-time PCR and ELISA results showed that the mRNA levels and medium concentrations of AGG and Col-2 were

  6. Autologous chondrocyte implantation in the knee: systematic review and economic evaluation.

    Science.gov (United States)

    Mistry, Hema; Connock, Martin; Pink, Joshua; Shyangdan, Deepson; Clar, Christine; Royle, Pamela; Court, Rachel; Biant, Leela C; Metcalfe, Andrew; Waugh, Norman

    2017-01-01

    BACKGROUND The surfaces of the bones in the knee are covered with articular cartilage, a rubber-like substance that is very smooth, allowing frictionless movement in the joint and acting as a shock absorber. The cells that form the cartilage are called chondrocytes. Natural cartilage is called hyaline cartilage. Articular cartilage has very little capacity for self-repair, so damage may be permanent. Various methods have been used to try to repair cartilage. Autologous chondrocyte implantation (ACI) involves laboratory culture of cartilage-producing cells from the knee and then implanting them into the chondral defect. OBJECTIVE To assess the clinical effectiveness and cost-effectiveness of ACI in chondral defects in the knee, compared with microfracture (MF). DATA SOURCES A broad search was done in MEDLINE, EMBASE, The Cochrane Library, NHS Economic Evaluation Database and Web of Science, for studies published since the last Health Technology Assessment review. REVIEW METHODS Systematic review of recent reviews, trials, long-term observational studies and economic evaluations of the use of ACI and MF for repairing symptomatic articular cartilage defects of the knee. A new economic model was constructed. Submissions from two manufacturers and the ACTIVE (Autologous Chondrocyte Transplantation/Implantation Versus Existing Treatment) trial group were reviewed. Survival analysis was based on long-term observational studies. RESULTS Four randomised controlled trials (RCTs) published since the last appraisal provided evidence on the efficacy of ACI. The SUMMIT (Superiority of Matrix-induced autologous chondrocyte implant versus Microfracture for Treatment of symptomatic articular cartilage defects) trial compared matrix-applied chondrocyte implantation (MACI(®)) against MF. The TIG/ACT/01/2000 (TIG/ACT) trial compared ACI with characterised chondrocytes against MF. The ACTIVE trial compared several forms of ACI against standard treatments, mainly MF. In the SUMMIT

  7. Potential of Raloxifene in reversing osteoarthritis-like alterations in rat chondrocytes: An in vitro model study

    Indian Academy of Sciences (India)

    Aysegul Kavas; Seda Tuncay Cagatay; Sreeparna Banerjee; Dilek Keskin; Aysen Tezcaner

    2013-03-01

    The aim of this study was to investigate the effects of Raloxifene (Ral) on degeneration-related changes in osteoarthritis (OA)-like chondrocytes using two- and three-dimensional models. Five-azacytidine (Aza-C) was used to induce OA-like alterations in rat articular chondrocytes and the model was verified at molecular and macrolevels. Chondrocytes were treated with Ral (1, 5 and 10 M) for 10 days. Caspase-3 activity, gene expressions of aggrecan, collagen II, alkaline phosphatase (ALP), collagen X, matrix metalloproteinases (MMP-13, MMP-3 and MMP-2), and MMP-13, MMP-3 and MMP-2 protein expressions were studied in two-dimensional model. Matrix deposition and mechanical properties of agarose-chondrocyte discs were evaluated in three-dimensional model. One M Ral reduced expression of OA-related genes, decreased apoptosis, and MMP-13 and MMP-3 protein expressions. It also increased aggrecan and collagen II gene expressions relative to untreated OA-like chondrocytes. In three-dimensional model, 1 M Ral treatment resulted in increased collagen deposition and improved mechanical properties, although a significant increase for sGAG was not observed. In summation, 1 M Ral improved matrix-related activities, whereas dose increment reversed these effects except ALP gene expression and sGAG deposition. These results provide evidence that low-dose Ral has the potential to cease or reduce the matrix degeneration in OA.

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

    Directory of Open Access Journals (Sweden)

    G. Musumeci

    2011-09-01

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

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

    Science.gov (United States)

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

    2014-09-01

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

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

    Directory of Open Access Journals (Sweden)

    Rainer Beckmann

    2014-09-01

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

  11. 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.

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

    Science.gov (United States)

    Lu, Shijin; Xiao, Xungang; Cheng, Minghua

    2015-01-01

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

  13. 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.

  14. Evaluation of Magnetic Nanoparticle-Labeled Chondrocytes Cultivated on a Type II Collagen–Chitosan/Poly(Lactic-co-Glycolic) Acid Biphasic Scaffold

    OpenAIRE

    Juin-Yih Su; Shi-Hui Chen; Yu-Pin Chen; Wei-Chuan Chen

    2017-01-01

    Chondral or osteochondral defects are still controversial problems in orthopedics. Here, chondrocytes labeled with magnetic nanoparticles were cultivated on a biphasic, type II collagen–chitosan/poly(lactic-co-glycolic acid) scaffold in an attempt to develop cultures with trackable cells exhibiting growth, differentiation, and regeneration. Rabbit chondrocytes were labeled with magnetic nanoparticles and characterized by scanning electron microscopy (SEM), transmission electron (TEM) microsco...

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

    NARCIS (Netherlands)

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

    2015-01-01

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

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

    NARCIS (Netherlands)

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

    2011-01-01

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

  17. In vitro cell quality of articular chondrocytes assigned for autologous implantation in dependence of specific patient characteristics

    DEFF Research Database (Denmark)

    Pestka, Jan M; Schmal, Hagen; Salzmann, Gian;

    2011-01-01

    between chondrocyte quality after in vitro cultivation and possible correlations with patient-specific factors. DESIGN: Cell quality of 252 consecutive ACI patients was assessed after chondrocyte in vitro expansion by determination of the expression of cartilage relevant surface marker CD44 and cartilage......-specific differentiation markers (aggrecan and collagen type II). All cell quality parameters were correlated with patient-specific parameters, such as age, size and defect location, number of defects and grade of joint degeneration according to the Kellgren-Lawrence classification. RESULTS: Neither the expression of CD44......, aggrecan or collagen type II nor cell density or viability after proliferation seemed to correlate with the grade of joint degeneration, defect aetiology or patient gender. However, chondrocytes harvested from the knee joints of patients at less than 20 years of age showed significantly higher expression...

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

    Science.gov (United States)

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

    2016-12-13

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

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

    Science.gov (United States)

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

    2003-01-01

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

  20. Evaluating Joint Morbidity after Chondral Harvest for Autologous Chondrocyte Implantation (ACI)

    Science.gov (United States)

    McCarthy, Helen S.; Richardson, James B.; Parker, Jane C. E.; Roberts, Sally

    2016-01-01

    Objective To establish if harvesting cartilage to source chondrocytes for autologous chondrocyte implantation (ACI) results in donor site morbidity. Design Twenty-three patients underwent ACI for chondral defects of either the ankle or the hip. This involved cartilage harvest from the knee (stage I), chondrocyte expansion in the laboratory and implantation surgery (stage II) into the affected joint. Prior to chondral harvest, no patient had sought treatment for their knee. Lysholm knee scores were completed prior to chondral harvest and annually post-ACI. Histological analyses of the donor site were performed at 12.3 ± 1.5 months for 3 additional patients who had previously had ACI of the knee. Results The median preoperative Lysholm score was 100, with no significant differences observed at either 13.7±1.7 months or 4.8±1.8 years postharvest (median Lysholm scores 91.7 and 87.5, respectively). Patients whose cartilage was harvested from the central or medial trochlea had a significantly higher median Lysholm score at latest follow-up (97.9 and 93.4, respectively), compared with those taken from the intercondylar notch (median Lysholm score 66.7). The mean International Cartilage Repair Society (ICRS) II histological score for the biopsies taken from the donor site of 3 additional knee ACI patients was 117 ± 10 (maximum score 140). Conclusions This study suggests that the chondral harvest site in ACI is not associated with significant joint morbidity, at least up to 5 years postharvest. However, one should carefully consider the location for chondral harvest as this has been shown to affect knee function in the longer term. PMID:26958313

  1. 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.

  2. Subculture of chondrocytes on a collagen type I-coated substrate with suppressed cellular dedifferentiation.

    Science.gov (United States)

    Kino-Oka, Masahiro; Yashiki, Shino; Ota, Yuka; Mushiaki, Yuko; Sugawara, Katsura; Yamamoto, Takeyuki; Takezawa, Toshiaki; Taya, Masahito

    2005-01-01

    To evaluate the degree of cellular dedifferentiation, subculture of chondrocytes was conducted on a surface coated with collagen type I at a density of 1.05 mg/cm(2). In the primary culture, most of the cells were round in shape on the collagen (CL) substrate, whereas fibroblastic and partially extended cells were dominant on the polystyrene plastic (PS) substrate. Stereoscopic observation revealed that the round-shaped cells on the CL substrate were hemispherical with nebulous and punctuated F-actin filaments, whereas the fibroblastic cells on the PS substrate were flattened with fully developed stress fibers. This suggested that cell polarization was suppressed during culture on the former substrate. Although serial passages of chondrocytes through subcultures on the CL and PS substrates caused a decrease in the number of round-shaped cells, the morphological change was appreciably suppressed on the CL substrate, as compared with that on the PS substrate. It was found that only round-shaped cells formed collagen type II, which supports the view that cellular dedifferentiation can be suppressed to some extent on the CL substrate. Three-dimensional cultures in collagen gel were performed with cells isolated freshly and passaged on the CL or PS substrate. Cell density at 21 days in the culture of cells passaged on the CL substrate was comparable to that in the culture of freshly isolated cells, in spite of a significant reduction in cell density observed in the culture of cells passaged on the PS substrate. In addition, histological analysis revealed that the expression of glycosaminoglycans and collagen type II was of significance in the collagen gel with cells passaged on the CL substrate, and likewise in the gel with freshly isolated cells. This indicated that the CL substrate could offer a monolayer culture system for expanding chondrocyte cells.

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

    Science.gov (United States)

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

    2015-05-01

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

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

    Institute of Scientific and Technical Information of China (English)

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

    2011-01-01

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

  5. 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.

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

    Science.gov (United States)

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

    2015-01-01

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

  7. 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

  8. 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.

  9. 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.

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

    Science.gov (United States)

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

    2011-11-01

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

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

    Science.gov (United States)

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

    2007-12-01

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

  12. MicroRNA-146a Induced by Hypoxia Promotes Chondrocyte Autophagy through Bcl-2

    Directory of Open Access Journals (Sweden)

    Fei Zhang

    2015-10-01

    Full Text Available Background/Aims: There have been many studies on the etiology of osteoarthritis (OA with regard to the function of inflammatory cytokines, the process of cartilage degradation, the function of miR-146a, hypoxia stimulation and autophagy in OA chondrocytes, but there have been no reports on the relationship between miR-146a and autophagy in cartilage, especially under hypoxia. This study aimed to confirm the relationship of miR-146a and autophagy in cartilage under hypoxia. Methods: Chondrocytes were treated by hypoxia gradients, and the main factors including HIF-1α, HIF-2α, miR-146a and Bcl-2 and autophagy markers ULK-1, ATG-5 were detected by quantitative PCR (Q-PCR and western blotting. The autophagy marker LC-3 was detected by immunofluorescence. The reciprocal effects between miR-146a and Bcl-2 were confirmed by several combinations of shRNAs and adenovirus-gene systems followed by Q-PCR and western blot detection. Results: Hypoxia maintained the chondrocytes phenotype and promoted autophagy and miR-146a expression via HIF-1α, but not HIF-2α, while miR-146a did not reversely affect HIF-1α. The autophagy induced by hypoxia through HIF-1α, miR-146a and Bcl-2. Simply, hypoxia induced HIF-1α, and HIF-1α increased miR-146a, but miR-146a suppressed Bcl-2, an autophagy inhibitor. While Bcl-2 affected neither HIF-1α nor miR-146a. The absence of both HIF-1α and miR-146a or Bcl-2 over-expression inhibited hypoxia-induced autophagy. Conclusion: HIF-1α, miR-146a and Bcl-2 play crucial roles during hypoxia-induced autophagy, Hypoxia, HIF-1α and miR-146a promote chondrocytes autophagy via depressing Bcl-2. We conclude that miR-146a may serve as a novel therapeutic target for protecting cartilage from degeneration in OA.

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

    Science.gov (United States)

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

    2017-02-16

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

  14. Transforming growth factor beta signaling is essential for the autonomous formation of cartilage-like tissue by expanded chondrocytes.

    Directory of Open Access Journals (Sweden)

    Adel Tekari

    Full Text Available Cartilage is a tissue with limited self-healing potential. Hence, cartilage defects require surgical attention to prevent or postpone the development of osteoarthritis. For cell-based cartilage repair strategies, in particular autologous chondrocyte implantation, articular chondrocytes are isolated from cartilage and expanded in vitro to increase the number of cells required for therapy. During expansion, the cells lose the competence to autonomously form a cartilage-like tissue, that is in the absence of exogenously added chondrogenic growth factors, such as TGF-βs. We hypothesized that signaling elicited by autocrine and/or paracrine TGF-β is essential for the formation of cartilage-like tissue and that alterations within the TGF-β signaling pathway during expansion interfere with this process. Primary bovine articular chondrocytes were harvested and expanded in monolayer culture up to passage six and the formation of cartilage tissue was investigated in high density pellet cultures grown for three weeks. Chondrocytes expanded for up to three passages maintained the potential for autonomous cartilage-like tissue formation. After three passages, however, exogenous TGF-β1 was required to induce the formation of cartilage-like tissue. When TGF-β signaling was blocked by inhibiting the TGF-β receptor 1 kinase, the autonomous formation of cartilage-like tissue was abrogated. At the initiation of pellet culture, chondrocytes from passage three and later showed levels of transcripts coding for TGF-β receptors 1 and 2 and TGF-β2 to be three-, five- and five-fold decreased, respectively, as compared to primary chondrocytes. In conclusion, the autonomous formation of cartilage-like tissue by expanded chondrocytes is dependent on signaling induced by autocrine and/or paracrine TGF-β. We propose that a decrease in the expression of the chondrogenic growth factor TGF-β2 and of the TGF-β receptors in expanded chondrocytes accounts for a decrease

  15. 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

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

    Science.gov (United States)

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

    2014-06-01

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

  17. Potential of an injectable chitosan/starch/beta-glycerol phosphate hydrogel for sustaining normal chondrocyte function.

    Science.gov (United States)

    Ngoenkam, Jatuporn; Faikrua, Atchariya; Yasothornsrikul, Sukkid; Viyoch, Jarupa

    2010-05-31

    An injectable hydrogel for chondrocyte delivery was developed by blending chitosan and starch derived from various sources with beta-glycerol phosphate (beta-GP) in the expectation that it would retain a liquid state at room temperature and gel at raised temperatures. Rheological investigation indicated that the system consisting of chitosan derived from crab shell and corn starch at 4:1 by weight ratio (1.53%, w/v of total polymers), and 6.0% (w/v) beta-GP (C/S/GP system) exhibited the sharpest sol-gel transition at 37+/-2 degrees C. The C/S/GP hydrogel was gradually degraded by 67% within 56 days in PBS containing 0.02 mg/ml lysozyme. The presence of starch in the system increased the water absorption of the hydrogel when compared to the system without starch. SEM observation revealed to the interior structure of the C/S/GP hydrogel having interconnected pore structure (average pore size 26.4 microm) whereas the pore size of the hydrogel without starch was 19.8 microm. The hydrogel also showed an ability to maintain chondrocyte phenotype as shown by cell morphology and expression of type II collagen mRNA and protein. In vivo study revealed that the gel was formed rapidly and localized at the injection site.

  18. Natural polysaccharides promote chondrocyte adhesion and proliferation on magnetic nanoparticle/PVA composite hydrogels.

    Science.gov (United States)

    Hou, Ruixia; Nie, Lei; Du, Gaolai; Xiong, Xiaopeng; Fu, Jun

    2015-08-01

    This paper aims to investigate the synergistic effects of natural polysaccharides and inorganic nanoparticles on cell adhesion and growth on intrinsically cell non-adhesive polyvinyl alcohol (PVA) hydrogels. Previously, we have demonstrated that Fe2O3 and hydroxyapatite (nHAP) nanoparticles are effective in increasing osteoblast growth on PVA hydrogels. Herein, we blended hyaluronic acid (HA) and chondroitin sulfate (CS), two important components of cartilage extracellular matrix (ECM), with Fe2O3/nHAP/PVA hydrogels. The presence of these natural polyelectrolytes dramatically increased the pore size and the equilibrium swelling ratio (ESR) while maintaining excellent compressive strength of hydrogels. Chondrocytes were seeded and cultured on composite PVA hydrogels containing Fe2O3, nHAP and Fe2O3/nHAP hybrids and Fe2O3/nHAP with HA or CS. Confocal laser scanning microscopy (CLSM) and cell counting kit-8 (CCK-8) assay consistently confirmed that the addition of HA or CS promotes chondrocyte adhesion and growth on PVA and composite hydrogels. Particularly, the combination of HA and CS exhibited further promotion to cell adhesion and proliferation compared with any single polysaccharide. The results demonstrated that the magnetic composite nanoparticles and polysaccharides provided synergistic promotion to cell adhesion and growth. Such polysaccharide-augmented composite hydrogels may have potentials in biomedical applications.

  19. Gel-type autologous chondrocyte (Chondron™ implantation for treatment of articular cartilage defects of the knee

    Directory of Open Access Journals (Sweden)

    Chun Chung-Woo

    2010-05-01

    Full Text Available Abstract Background Gel-type autologous chondrocyte (Chondron™ implantations have been used for several years without using periosteum or membrane. This study involves evaluations of the clinical results of Chondron™ at many clinical centers at various time points during the postoperative patient follow-up. Methods Data from 98 patients with articular cartilage injury of the knee joint and who underwent Chondron™ implantation at ten Korean hospitals between January 2005 and November 2008, were included and were divided into two groups based on the patient follow-up period, i.e. 13~24-month follow-up and greater than 25-month follow-up. The telephone Knee Society Score obtained during telephone interviews with patients, was used as the evaluation tool. Results On the tKSS-A (telephone Knee Society Score-A, the score improved from 43.52 ± 20.20 to 89.71 ± 13.69 (P Conclusion Gel-type autologous chondrocyte implantation for chondral knee defects appears to be a safe and effective method for both decreasing pain and improving knee function.

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

    Institute of Scientific and Technical Information of China (English)

    Madaí A GóMEZ-CAMARILLO; Juan B.KOURI

    2005-01-01

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

  1. Effects of electromagnetic fields on the metabolism of lubricin of rat chondrocytes.

    Science.gov (United States)

    Wang, Wei; Li, Wenkai; Song, Mingyu; Wei, Sheng; Liu, Chaoxu; Yang, Yong; Wu, Hua

    2016-01-01

    Electromagnetic fields (EMFs) can improve pain, stiffness and physical function in osteoarthritis (OA) patients and have been proposed for the treatment of OA. However, the precise mechanisms involved in this process are still not fully understood. In the present study, we investigated the effects of exposure for different durations with 75 Hz, 2.3 mT sinusoidal EMFs (SEMFs) on the metabolism of lubricin of rat chondrocytes cultured in vitro. Our results showed that SEMFs exposure promoted lubricin synthesis in a time-dependent manner, and the expression of transforming growth factor (TGF)-β1 was also enhanced after SEMFs treatment. The up-regulation effect of the expression of lubricin under SEMF was partly reduced by SB431542, an inhibitor of TGF-RI kinase. The Smad pathway was also investigated in our study. Smad2 synthesis was higher in EMF-exposed condition than in controls, whereas no effects were observed on inhibitory Smads (Smad6 and Smad7) production. Altogether, these data suggest that SEMF exposure can promote lubricin synthesis of rat chondrocytes in a time-dependent manner and that the TGF-β/Smads signaling pathway plays a partial role.

  2. Chitosan-Coated Collagen Membranes Promote Chondrocyte Adhesion, Growth, and Interleukin-6 Secretion

    Directory of Open Access Journals (Sweden)

    Nabila Mighri

    2015-11-01

    Full Text Available Designing scaffolds made from natural polymers may be highly attractive for tissue engineering strategies. We sought to produce and characterize chitosan-coated collagen membranes and to assess their efficacy in promoting chondrocyte adhesion, growth, and cytokine secretion. Porous collagen membranes were placed in chitosan solutions then crosslinked with glutaraldehyde vapor. Fourier transform infrared (FTIR analyses showed elevated absorption at 1655 cm-1 of the carbon–nitrogen (N=C bonds formed by the reaction between the (NH2 of the chitosan and the (C=O of the glutaraldehyde. A significant peak in the amide II region revealed a significant deacetylation of the chitosan. Scanning electron microscopy (SEM images of the chitosan-coated membranes exhibited surface variations, with pore size ranging from 20 to 50 µm. X-ray photoelectron spectroscopy (XPS revealed a decreased C–C groups and an increased C–N/C–O groups due to the reaction between the carbon from the collagen and the NH2 from the chitosan. Increased rigidity of these membranes was also observed when comparing the chitosan-coated and uncoated membranes at dried conditions. However, under wet conditions, the chitosan coated collagen membranes showed lower rigidity as compared to dried conditions. Of great interest, the glutaraldehyde-crosslinked chitosan-coated collagen membranes promoted chondrocyte adhesion, growth, and interleukin (IL-6 secretion. Overall results confirm the feasibility of using designed chitosan-coated collagen membranes in future applications, such as cartilage repair.

  3. Mimicking of Chondrocyte Microenvironment Using In Situ Forming Dendritic Polyglycerol Sulfate-Based Synthetic Polyanionic Hydrogels.

    Science.gov (United States)

    Dey, Pradip; Schneider, Tobias; Chiappisi, Leonardo; Gradzielski, Michael; Schulze-Tanzil, Gundula; Haag, Rainer

    2016-04-01

    A stable polymeric network that mimics the highly polyanionic extracellular cartilage matrix still remains a great challenge. The main aim of this study is to present the synthesis of dendritic polyglycerol sulfate (dPGS)-based in situ forming hydrogels using strain promoted azide-alkyne cycloaddition reactions. A real time rheological study has been used to characterize the hydrogel properties. The viability of encapsulated human chondrocytes in the different hydrogels are monitored using live-dead staining. Furthermore, type I and II collagen gene have been analyzed. Hydrogels with elastic moduli ranging from 1 to 5 kPa have been prepared by varying the dPGS amount. The chondrocyte viability in dPGS hydrogels is found to be higher than in pure PEG and alginate-based hydrogels after 21 d. The higher cell viability in the dPGS engineered hydrogels can be explained by the fact that dPGS can interact with different proteins responsible for cell growth and proliferation.

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

    Science.gov (United States)

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

    2012-02-01

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

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

    Science.gov (United States)

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

    2012-01-01

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

  6. Melanoma inhibitory activity, a biomarker related to chondrocyte anabolism, is reversibly suppressed by proinflammatory cytokines in rheumatoid arthritis

    NARCIS (Netherlands)

    Vandooren, B.; Cantaert, T.; van Lierop, M.J.; Bos, E.; de Rycke, L.; Veys, E.M.; de Keyser, F.; Bresnihan, B.; Luyten, F.P.; Verdonk, P.C.; Tak, P.P.; Boots, A.H.; Baeten, D.

    2009-01-01

    Objective: In mice, melanoma inhibitory activity (MIA) is a chondrocyte-specific molecule with similar regulation to collagen type II. As MIA is a small secreted protein, its value as cartilage biomarker in human inflammatory arthritis was assessed. Methods: MIA tissue distribution was studied by qu

  7. In vitro and in vivo characterization of nonbiomedical- and biomedical-grade alginates for articular chondrocyte transplantation.

    Science.gov (United States)

    Heiligenstein, Susanne; Cucchiarini, Magali; Laschke, Matthias W; Bohle, Rainer M; Kohn, Dieter; Menger, Michael D; Madry, Henning

    2011-08-01

    Alginate is a key hydrogel for cartilage tissue engineering. Here, we systematically evaluated four biomedical- and two nonbiomedical-grade alginates for their capacity to support the in vitro culture and in vivo transplantation of articular chondrocytes. Chondrocytes in all ultrapure alginates maintained high cell viability. Spheres composed of biomedical-grade, low-viscosity, high-mannuronic acid content alginate showed the lowest decrease in size over time. Biomedical-grade, low-viscosity, high-guluronic acid content alginate allowed for optimal cell proliferation. Biomedical-grade, medium-viscosity, high-mannuronic acid content alginate promoted the highest production of proteoglycans. When transplanted into osteochondral defects in the knee joint of sheep in vivo, empty spheres were progressively surrounded by a granulation tissue. In marked contrast with these observations, all alginate spheres carrying allogeneic chondrocytes were gradually invaded by a granulation tissue containing multinucleated giant cells, lymphocytes, and fibroblasts, regardless whether they were based on biomedical- or nonbiomedical-grade alginates. After 21 days in vivo, transplanted chondrocytes were either viable or underwent necrosis, and apoptosis played a minor role in their early fate. The individual characteristics of these alginates may be valuable to tailor specific experimental and clinical strategies for cartilage tissue engineering.

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

    Institute of Scientific and Technical Information of China (English)

    2005-01-01

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

  9. PP2A-mediated dephosphorylation of p107 plays a critical role in chondrocyte cell cycle arrest by FGF.

    Directory of Open Access Journals (Sweden)

    Victoria Kolupaeva

    Full Text Available FGF signaling inhibits chondrocyte proliferation, a cell type-specific response that is the basis for several genetic skeletal disorders caused by activating FGFR mutations. This phenomenon requires the function of the p107 and p130 members of the Rb protein family, and p107 dephosphorylation is one of the earliest distinguishing events in FGF-induced growth arrest. To determine whether p107 dephoshorylation played a critical role in the chondrocyte response to FGF, we sought to counteract this process by overexpressing in RCS chondrocytes the cyclin D1/cdk4 kinase complex. CyclinD/cdk4-expressing RCS cells became resistant to FGF-induced p107 dephosphorylation and growth arrest, and maintained significantly high levels of cyclin E/cdk2 activity and of phosphorylated p130 at later times of FGF treatment. We explored the involvement of a phosphatase in p107 dephosphorylation. Expression of the SV40 small T-Ag, which inhibits the activity of the PP2A phosphatase, or knockdown of the expression of the PP2A catalytic subunit by RNA interference prevented p107 dephosphorylation and FGF-induced growth arrest of RCS cells. Furthermore, an association between p107 and PP2A was induced by FGF treatment. Our data show that p107 dephosphorylation is a key event in FGF-induced cell cycle arrest and indicate that in chondrocytes FGF activates the PP2A phosphatase to promote p107 dephosphorylation.

  10. Endoplasmic reticulum stress inhibits collagen synthesis independent of collagen-modifying enzymes in different chondrocyte populations and dermal fibroblasts

    NARCIS (Netherlands)

    Vonk, Lucienne A.; Doulabi, Behrouz Zandieh; Huang, Chun-Ling; Helder, Marco N.; Everts, Vincent; Bank, Ruud A.

    2010-01-01

    Chondrocytes respond to glucose deprivation with a decreased collagen synthesis due to disruption of a proper functioning of the endoplasmic reticulum (ER): ER stress. Since the mechanisms involved in the decreased synthesis are unknown, we have investigated whether chaperones and collagen-modifying

  11. 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.

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

    Science.gov (United States)

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

    2017-02-01

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

  13. 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.

  14. The Study of the Frequency Effect of Dynamic Compressive Loading on Primary Articular Chondrocyte Functions Using a Microcell Culture System

    Directory of Open Access Journals (Sweden)

    Wan-Ying Lin

    2014-01-01

    Full Text Available Compressive stimulation can modulate articular chondrocyte functions. Nevertheless, the relevant studies are not comprehensive. This is primarily due to the lack of cell culture apparatuses capable of conducting the experiments in a high throughput, precise, and cost-effective manner. To address the issue, we demonstrated the use of a perfusion microcell culture system to investigate the stimulating frequency (0.5, 1.0, and 2.0 Hz effect of compressive loading (20% and 40% strain on the functions of articular chondrocytes. The system mainly integrates the functions of continuous culture medium perfusion and the generation of pneumatically-driven compressive stimulation in a high-throughput micro cell culture system. Results showed that the compressive stimulations explored did not have a significant impact on chondrocyte viability and proliferation. However, the metabolic activity of chondrocytes was significantly affected by the stimulating frequency at the higher compressive strain of 40% (2 Hz, 40% strain. Under the two compressive strains studied, the glycosaminoglycans (GAGs synthesis was upregulated when the stimulating frequency was set at 1 Hz and 2 Hz. However, the stimulating frequencies explored had no influence on the collagen production. The results of this study provide useful fundamental insights that will be helpful for cartilage tissue engineering and cartilage rehabilitation.

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

    Science.gov (United States)

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

    2013-01-01

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

  16. Simvastatin induces differentiation of rabbit articular chondrocytes via the ERK-1/2 and p38 kinase pathways.

    Science.gov (United States)

    Han, Yohan; Kim, Song Ja

    2016-08-15

    Statins are competitive inhibitors of hydroxy-methyl-glutaryl Coenzyme A (HMG-CoA) reductase, a key enzyme involved in the conversion of HMG-CoA to the cholesterol precursor mevalonate. Some statins, such as simvastatin (simvastatin), have been shown to have anti-cancer and anti-inflammatory effects, reducing cartilage degradation in osteoarthritic rabbits in vivo. However, the regulatory mechanisms undergirding simvastatin mediated chondrocyte differentiation have not been well elucidated. Thus, we investigated the action and mechanism of simvastatin on differentiation of rabbit articular chondrocytes through western blot analyses, RT-PCR, and immunohistochemical (IHC) and immunofluorescence (IF) staining. Simvastatin treatment was found to induce type II collagen expression and sulfated-proteoglycan synthesis in a dose- and time-dependent manner. Indeed, RT-PCR revealed increased expression of type II collagen on treatment with simvastatin. Both IHC and IF staining indicated differentiation of chondrocytes. Simvastatin treatment reduced activation of ERK-1/2 and stimulated activation of p38 kinase. Inhibition of ERK-1/2 with PD98059 enhanced simvastatin induced differentiation, whereas inhibition of p38 kinase with SB203580 inhibited simvastatin induced differentiation. Simvastatin treatment also inhibits loss of type II collagen in serial monolayer culture. Collectively, our results indicate that ERK-1/2 and p38 kinase regulate simvastatin-induced differentiation of chondrocytes in opposing manners. Thus, these findings suggest that simvastatin may be a potential therapeutic drug for osteoarthritis.

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

    Science.gov (United States)

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

    2016-04-01

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

  18. The major basement membrane components localize to the chondrocyte pericellular matrix--a cartilage basement membrane equivalent?

    DEFF Research Database (Denmark)

    Kvist, Alexander J.; Nyström, Alexander; Hultenby, Kjell;

    2007-01-01

    In this study, we demonstrate that articular cartilage chondrocytes are surrounded by the defining basement membrane proteins laminin, collagen type IV, nidogen and perlecan, and suggest that these form the functional equivalent of a basement membrane. We found by real-time PCR that mouse chondro...... to the progression of degenerative joint disorders....

  19. Optimization of dual effects of Mg-1Ca alloys on the behavior of chondrocytes and osteoblasts in vitro

    Institute of Scientific and Technical Information of China (English)

    Yana Dou; Ayeesha Mujeeb; Yufeng Zheng; Zigang Ge

    2014-01-01

    Mg ions can enhance the proliferation and redifferentiation of chondrocytes and the osteogenic differentiation of osteoblasts at specific concentrations, respectively. However, degradation of Mg alloys at varying degradation rates could lead to complex changes in the surrounding tissue environment, such as changes in the dynamic concentration of Mg ions and subsequent pH value. Considering the above mentioned factors, the comprehensive effects of Mg alloys on chondrocytes and osteoblasts behaviors have not yet been optimized. In this study, we evaluated the effects of Mg–1Ca microspheres on cell behavior with an aim to optimize conditions favorable for both cell types. Cells were cultured with Mg–1Ca microspheres prepared using the following concentrations:250μg/ml, 500μg/ml and 1000μg/ml. At specific time points, cytotoxicity, expression of specific genes and extracellular matrix deposition by cells (Alizarin Red Staining of osteoblasts and Alcian blue staining for chondrocytes) were evaluated. The experimental results revealed that Mg–1Ca microspheres prepared at a concentration of 250μg/ml were optimum for both cell types, where chondrocytes were found to be in hypertrophy state while osteoblasts in close proximity to the microspheres showed osteogenetic differentiation. Interestingly, a slight change in osteoblasts behavior was observed nearer to and at a relative distance away from Mg–1Ca microspheres, an important observation for administering the application of microspheres as potential scaffolds.

  20. Tracheal reconstruction using chondrocytes seeded on a poly(L-lactic-co-glycolic acid)-fibrin/hyaluronan.

    Science.gov (United States)

    Hong, Hyun Jun; Chang, Jae Won; Park, Ju-Kyeong; Choi, Jae Won; Kim, Yoo Suk; Shin, Yoo Seob; Kim, Chul-Ho; Choi, Eun Chang

    2014-11-01

    Reconstruction of trachea is still a clinical dilemma. Tissue engineering is a recent and promising concept to resolve this problem. This study evaluated the feasibility of allogeneic chondrocytes cultured with fibrin/hyaluronic acid (HA) hydrogel and degradable porous poly(L-lactic-co-glycolic acid) (PLGA) scaffold for partial tracheal reconstruction. Chondrocytes from rabbit articular cartilage were expanded and cultured with fibrin/HA hydrogel and injected into a 5 × 10 mm-sized, curved patch-shape PLGA scaffold. After 4 weeks in vitro culture, the scaffold was implanted on a tracheal defect in eight rabbits. Six and 10 weeks postoperatively, the implanted sites were evaluated by bronchoscope and radiologic and histologic analyses. Ciliary beat frequency (CBF) of regenerated epithelium was also evaluated. None of the eight rabbits showed any sign of respiratory distress. Bronchoscopic examination did not reveal stenosis of the reconstructed trachea and the defects were completely recovered with respiratory epithelium. Computed tomography scan showed good luminal contour of trachea. Histologic data showed that the implanted chondrocytes successfully formed neocartilage with minimal granulation tissue. CBF of regenerated epithelium was similar to that of normal epithelium. Partial tracheal defect was successfully reconstructed anatomically and functionally using allogeneic chondrocytes cultured with PLGA-fibrin/HA composite scaffold.

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

    NARCIS (Netherlands)

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

    2014-01-01

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

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

    Science.gov (United States)

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

    2015-01-01

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

  3. 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

  4. Niacinamide therapy for osteoarthritis--does it inhibit nitric oxide synthase induction by interleukin 1 in chondrocytes?

    Science.gov (United States)

    McCarty, M F; Russell, A L

    1999-10-01

    Fifty years ago, Kaufman reported that high-dose niacinamide was beneficial in osteoarthritis (OA) and rheumatoid arthritis. A recent double-blind study confirms the efficacy of niacinamide in OA. It may be feasible to interpret this finding in the context of evidence that synovium-generated interleukin-1 (IL-1), by inducing nitric oxide (NO) synthase and thereby inhibiting chondrocyte synthesis of aggrecan and type II collagen, is crucial to the pathogenesis of OA. Niacinamide and other inhibitors of ADP-ribosylation have been shown to suppress cytokine-mediated induction of NO synthase in a number of types of cells; it is therefore reasonable to speculate that niacinamide will have a comparable effect in IL-1-exposed chondrocytes, blunting the anti-anabolic impact of IL-1. The chondroprotective antibiotic doxycycline may have a similar mechanism of action. Other nutrients reported to be useful in OA may likewise intervene in the activity or synthesis of IL-1. Supplemental glucosamine can be expected to stimulate synovial synthesis of hyaluronic acid; hyaluronic acid suppresses the anti-catabolic effect of IL-1 in chondrocyte cell cultures, and has documented therapeutic efficacy when injected intra-articularly. S-adenosylmethionine (SAM), another proven therapy for OA, upregulates the proteoglycan synthesis of chondrocytes, perhaps because it functions physiologically as a signal of sulfur availability. IL-1 is likely to decrease SAM levels in chondrocytes; supplemental SAM may compensate for this deficit. Adequate selenium nutrition may down-regulate cytokine signaling, and ample intakes of fish oil can be expected to decrease synovial IL-1 production; these nutrients should receive further evaluation in OA. These considerations suggest that non-toxic nutritional regimens, by intervening at multiple points in the signal transduction pathways that promote the synthesis and mediate the activity of IL-1, may provide a substantially superior alternative to NSAIDs

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

    Directory of Open Access Journals (Sweden)

    Annalisa Facchini

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

  6. A cell shrinkage artefact in growth plate chondrocytes with common fixative solutions: importance of fixative osmolarity for maintaining morphology

    Directory of Open Access Journals (Sweden)

    MY Loqman

    2010-05-01

    Full Text Available The remarkable increase in chondrocyte volume is a major determinant in the longitudinal growth of mammalian bones. To permit a detailed morphological study of hypertrophic chondrocytes using standard histological techniques, the preservation of normal chondrocyte morphology is essential. We noticed that during fixation of growth plates with conventional fixative solutions, there was a marked morphological (shrinkage artifact, and we postulated that this arose from the hyper-osmotic nature of these solutions. To test this, we fixed proximal tibia growth plates of 7-day-old rat bones in either (a paraformaldehyde (PFA; 4%, (b glutaraldehyde (GA; 2% with PFA (2% with ruthenium hexamine trichloride (RHT; 0.7%, (c GA (2% with RHT (0.7%, or (d GA (1.3% with RHT (0.5% and osmolarity adjusted to a ‘physiological’ level of ~280mOsm. Using conventional histological methods, confocal microscopy, and image analysis on fluorescently-labelled fixed and living chondrocytes, we then quantified the extent of cell shrinkage and volume change. Our data showed that the high osmolarity of conventional fixatives caused a shrinkage artefact to chondrocytes. This was particularly evident when whole bones were fixed, but could be markedly reduced if bones were sagittally bisected prior to fixation. The shrinkage artefact could be avoided by adjusting the osmolarity of the fixatives to the osmotic pressure of normal extracellular fluids (~280mOsm. These results emphasize the importance of fixative osmolarity, in order to accurately preserve the normal volume/morphology of cells within tissues.

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

    Science.gov (United States)

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

    2014-04-01

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

  8. The effect of chemically defined medium on spontaneous calcium signaling of in situ chondrocytes during long-term culture.

    Science.gov (United States)

    Zhou, Yilu; Park, Miri; Cheung, Enoch; Wang, Liyun; Lu, X Lucas

    2015-04-13

    Chemically defined serum-free medium has been shown to better maintain the mechanical integrity of articular cartilage explants than serum-supplemented medium during long-term in vitro culture, but little is known about its effect on cellular mechanisms. We hypothesized that the chemically defined culture medium could regulate the spontaneous calcium signaling of in situ chondrocytes, which may modulate the cellular metabolic activities. Bovine cartilage explants were cultured in chemically defined serum-free or serum-supplemented medium for four weeks. The spontaneous intracellular calcium ([Ca(2+)]i) signaling of in situ chondrocytes was longitudinally measured together along with the biomechanical properties of the explants. The spontaneous [Ca(2+)]i oscillations in chondrocytes were enhanced at the initial exposure of serum-supplemented medium, but were significantly dampened afterwards. In contrast, cartilage explants in chemically defined medium preserved the level of calcium signaling, and showed more responsive cells with higher and more frequent [Ca(2+)]i peaks throughout the four week culture in comparison to those in serum medium. Regardless of the culture medium that the explants were exposed, a positive correlation was detected between the [Ca(2+)]i responsive rate and the stiffness of cartilage (Spearman's rank correlation coefficient=0.762). A stable pattern of [Ca(2+)]i peaks was revealed for each chondrocyte, i.e., the spatiotemporal features of [Ca(2+)]i peaks from a cell were highly consistent during the observation period (15 min). This study showed that the beneficial effect of chemically defined culture of cartilage explants is associated with the spontaneous [Ca(2+)]i signaling of chondrocytes in cartilage.

  9. MRI evaluation of a new scaffold-based allogenic chondrocyte implantation for cartilage repair

    Energy Technology Data Exchange (ETDEWEB)

    Dhollander, A.A.M., E-mail: Aad.Dhollander@Ugent.b [Department of Orthopaedic Surgery and Traumatology, Ghent University Hospital, De Pintelaan 185, 1P5, B9000 Gent (Belgium); Huysse, W.C.J., E-mail: Wouter.Huysse@Ugent.b [Department of Radiology, Ghent University Hospital, De Pintelaan 185, -1K12 IB, B9000 Gent (Belgium); Verdonk, P.C.M., E-mail: pverdonk@yahoo.co [Department of Orthopaedic Surgery and Traumatology, Ghent University Hospital, De Pintelaan 185, 1P5, B9000 Gent (Belgium); Verstraete, K.L., E-mail: Koenraad.Verstraete@Ugent.b [Department of Radiology, Ghent University Hospital, De Pintelaan 185, -1K12 IB, B9000 Gent (Belgium); Verdonk, R., E-mail: Rene.Verdonk@Ugent.b [Department of Orthopaedic Surgery and Traumatology, Ghent University Hospital, De Pintelaan 185, 1P5, B9000 Gent (Belgium); Verbruggen, G., E-mail: Gust.Verbruggen@Ugent.b [Laboratory of Connective Tissue Biology, Department of Rheumatology, Ghent University Hospital, De Pintelaan 185, Ghent (Belgium); Almqvist, K.F., E-mail: Fredrik.Almqvist@Ugent.b [Department of Orthopaedic Surgery and Traumatology, Ghent University Hospital, De Pintelaan 185, 1P5, B9000 Gent (Belgium)

    2010-07-15

    Aim: The present study was designed to evaluate the implantation of alginate beads containing human mature allogenic chondrocytes for the treatment of symptomatic cartilage defects of the knee. MRI was used for the morphological analysis of cartilage repair. The correlation between MRI findings and clinical outcome was also studied. Methods: A biodegradable, alginate-based biocompatible scaffold containing human mature allogenic chondrocytes was used for the treatment of symptomatic chondral and osteochondral lesions in the knee. Twenty-one patients were prospectively evaluated with use of the Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) and the Visual Analogue Scale (VAS) for pain preoperatively and at 3, 6, 9 and 12 months of follow-up. Of the 21 patients, 12 had consented to follow the postoperative MRI evaluation protocol. MRI data were analyzed based on the original MOCART (Magnetic Resonance Observation of Cartilage Repair Tissue) and modified MOCART scoring system. The correlation between the clinical outcome and MRI findings was evaluated. Results: A statistically significant clinical improvement became apparent after 6 months and patients continued to improve during the 12 months of follow-up. One of the two MRI scoring systems that were used, showed a statistically significant deterioration of the repair tissue at 1 year of follow-up. Twelve months after the operation complete filling or hypertrophy was found in 41.6%. Bone-marrow edema and effusion were seen in 41.7% and 25% of the study patients, respectively. We did not find a consistent correlation between the MRI criteria and the clinical results. Discussion: The present study confirmed the primary role of MRI in the evaluation of cartilage repair. Two MOCART-based scoring systems were used in a longitudinal fashion and allowed a practical and morphological evaluation of the repair tissue. However, the correlation between clinical outcome and MRI findings was poor. Further

  10. Age-Independent Cartilage Generation for Synovium-Based Autologous Chondrocyte Implantation.

    Science.gov (United States)

    Hunziker, Ernst B; Lippuner, Kurt; Keel, Marius J B; Shintani, Nahoko

    2015-07-01

    The articular cartilage layer of synovial joints is commonly lesioned by trauma or by a degenerative joint disease. Attempts to repair the damage frequently involve the performance of autologous chondrocyte implantation (ACI). Healthy cartilage must be first removed from the joint, and then, on a separate occasion, following the isolation of the chondrocytes and their expansion in vitro, implanted within the lesion. The disadvantages of this therapeutic approach include the destruction of healthy cartilage-which may predispose the joint to osteoarthritic degeneration-the necessarily restricted availability of healthy tissue, the limited proliferative capacity of the donor cells-which declines with age-and the need for two surgical interventions. We postulated that it should be possible to induce synovial stem cells, which are characterized by high, age-independent, proliferative and chondrogenic differentiation capacities, to lay down cartilage within the outer juxtasynovial space after the transcutaneous implantation of a carrier bearing BMP-2 in a slow-release system. The chondrocytes could be isolated on-site and immediately used for ACI. To test this hypothesis, Chinchilla rabbits were used as an experimental model. A collagenous patch bearing BMP-2 in a slow-delivery vehicle was sutured to the inner face of the synovial membrane. The neoformed tissue was excised 5, 8, 11 and 14 days postimplantation for histological and histomorphometric analyses. Neoformed tissue was observed within the outer juxtasynovial space already on the 5th postimplantation day. It contained connective and adipose tissues, and a central nugget of growing cartilage. Between days 5 and 14, the absolute volume of cartilage increased, attaining a value of 12 mm(3) at the latter juncture. Bone was deposited in measurable quantities from the 11th day onwards, but owing to resorption, the net volume did not exceed 1.5 mm(3) (14th day). The findings confirm our hypothesis. The quantity of

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

    Science.gov (United States)

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

    2014-07-07

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

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

    Science.gov (United States)

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

    2008-01-01

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

  13. Calcium pentosan polysulfate directly inhibits enzymatic activity of ADAMTS4 (aggrecanase-1) in osteoarthritic chondrocytes.

    Science.gov (United States)

    Takizawa, Masayuki; Yatabe, Taku; Okada, Aiko; Chijiiwa, Miyuki; Mochizuki, Satsuki; Ghosh, Peter; Okada, Yasunori

    2008-08-20

    Aggrecanases that include ADAMTS1, 4, 5, 8, 9 and 15 are considered to play key roles in aggrecan degradation in osteoarthritic cartilage. Here we demonstrate that calcium pentosan polysulfate (CaPPS) directly inhibits the aggrecanase activity of ADAMTS4 without affecting the mRNA expression of the ADAMTS species in interleukin-1alpha-stimulated osteoarthritic chondrocytes. Synthetic peptides corresponding to specific regions of the thrombospondin type 1 repeat, cysteine-rich or spacer domain of ADAMTS4 inhibit the binding to immobilized CaPPS. These data suggest that CaPPS could function as chondroprotective agent for the treatment of osteoarthritis by inhibition of ADAMTS4 through interaction with the C-terminal ancillary domain.

  14. 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

    -extracellular matrix interactions in the growth plate. INTRODUCTION: The disintegrin and metalloprotease ADAM12 is expressed in both osteoblasts and osteoclasts, suggesting a regulatory role of ADAM12 in bone. However, thus far, no in vivo function of ADAM12 in the skeleton has been reported. MATERIALS AND METHODS......: 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...... in mice expressing higher levels of the transgene than in a lower-expressing line. Histological analysis revealed no alterations in the growth plate organization, but mean growth plate width was increased. Both the cellular incorporation of bromodeoxyuridine and the width of the collagen type X...

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

    Science.gov (United States)

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

    2014-01-22

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

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

    Directory of Open Access Journals (Sweden)

    Chao Pin-Zhir

    2011-11-01

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

  17. Experimental study of millimeter wave-induced differentiation of bone marrow mesenchymal stem cells into chondrocytes.

    Science.gov (United States)

    Wu, Guang-Wen; Liu, Xian-Xiang; Wu, Ming-Xia; Zhao, Jin-Yan; Chen, Wen-Lie; Lin, Ru-Hui; Lin, Jiu-Mao

    2009-04-01

    Low power millimeter wave irradiation is widely used in clinical medicine. We describe the effects of this treatment on cultured mesenchymal stem cells (MSCs) and attempted to identify the underlying mechanism. Cells cultured using the whole marrow attachment culture method proliferated dispersedly or in clones. Flow cytometric analyses showed that the MSCs were CD90 positive, but negative for CD45. The negative control group (A) did not express detectable levels of Cbfa1 or Sox9 mRNA at any time point, while cells in the millimeter wave-induced groups (B and C) increasingly expressed both genes after the fourth day post-induction. Statistical analysis showed that starting on the fourth day post-induction, there were very significant differences in the expression of Cbfa1 and Sox9 mRNA between groups A and B as well as A and C at any given time point, between treated groups B and C after identical periods of induction, and within each treated group at different induction times. Transition electron microscopy analysis showed that the rough endoplasmic reticulum of cells in the induced groups was richer and more developed than in cells of the negative control group, and that the shape of cells shifted from long-spindle to near ellipse. Toluidine blue staining revealed heterochromia in the cytoplasm and extracellular matrix of cells in the induced groups, whereas no obvious heterochromia was observed in negative control cells. Induced cells also exhibited positive immunohistochemical staining of collagen II, in contrast to the negative controls. These results show that millimeter wave treatment successfully induced MSCs to differentiate as chondrocytes and the extent of differentiation increased with treatment duration. Our findings suggest that millimeter wave irradiation can be employed as a novel non-drug inducing method for the differentiation of MSCs into chondrocytes.

  18. Matrix-induced autologous chondrocyte implantation addressing focal chondral defect in adolescent knee

    Institute of Scientific and Technical Information of China (English)

    DAI Xue-song; CAI You-zhi

    2012-01-01

    Background Matrix-induced autologous chondrocyte implantation(MACI)is the third generation tissue-engineering technique for the treatment of full-thickness articular cartilage defects.The aim of this study was to describe this new technique and the postoperative findings in adolescent knee with focal chondral defect.Methods The MACI consists of diagnostic arthroscopy and cartilage harvest,chondrocyte culture and seeding in tissue-engineering collagenous membrane,and implantation of the scaffold.Clinical outcome at minimum 1-year follow-up was assessed in seven patients(mean age(16.6±1.5)years;14-19 years)with full-thickness cartilage defects,with International Knee Documentation Committee(IKDC)score,the International Cartilage Repair Society(ICRS)score and the Knee Injury and Osteoarthritis Outcome Score(KOOS).Besides,MR imaging was performed with T1 and T2-weighted imaging and three-dimensional spoiled gradient-recalled(3D-SPGR)MR imaging.Results Clinical evaluation showed significant improvement and MRI analysis showed that the structure was homogeneous and the implant surface was regular and intact in six patients,but irregular in one.Of all the seven patients,the cartilage defect site was nearly totally covered by the implanted scaffold.Conclusions These results indicated that MACl technique is an option for cartilage defect in adolescent knee joint,especially large defect of over 2 cm2.Long-term assessment is necessary to determine the true value of this technique.

  19. Effects of the mycotoxin nivalenol on bovine articular chondrocyte metabolism in vitro.

    Directory of Open Access Journals (Sweden)

    Siyuan Li

    Full Text Available OBJECTIVE: Kashin-Beck Disease (KBD is an endemic, age-related degenerative osteoarthropathy and its cause is hypothesised to involve Fusarium mycotoxins. This study investigated the Fusarium mycotoxin Nivalenol (NIV on the metabolism of bovine articular chondrocytes in vitro. DESIGN: The effect 0.0-0.5 µg/ml NIV on transcript levels of types I and II collagen, aggrecan, matrix metalloproteinases (MMPs, a disintegrin and metalloproteinase with thrombospondin motif (ADAMTS and the tissue inhibitors of MMPs (TIMPs was investigated using quantitative PCR. Amounts of sulphated glycosaminoglycans, MMPs and TIMPs were assessed using the Dimethylmethylene Blue assay, gelatin zymography and reverse gelatin zymography respectively. Cytoskeletal organisation was analysed using confocal microscopy and cytoskeletal gene and protein levels were measured by quantitative PCR and Western blot analysis, respectively. RESULTS: NIV caused a dose-dependent increase in aggrecan transcription with a concomitant retention of sGAG in the cell lysate. Furthermore, NIV significantly increased MMPs-2, -3 & -9, ADAMTS-4 and -5, and TIMP-2 and -3 transcript levels but inhibited type I collagen, MMP 1 and TIMP 1 mRNA levels. NIV promoted extensive cytoskeletal network remodelling, particularly with vimentin where a dose-dependent peri-nuclear aggregation occurred. CONCLUSION: NIV exposure to chondrocytes decreased matrix deposition, whilst enhancing selective catabolic enzyme production, suggesting its potential for induction of cellular catabolism. This NIV-induced extracellular matrix remodelling may be due to extensive remodelling/disassembly of the cytoskeletal elements. Collectively, these findings support the hypothesis that trichothecene mycotoxins, and in particular NIV, have the potential to induce matrix catabolism and propagate the pathogenesis of KBD.

  20. Environmental regulation of type X collagen production by cultures of limb mesenchyme, mesectoderm, and sternal chondrocytes.

    Science.gov (United States)

    Solursh, M; Jensen, K L; Reiter, R S; Schmid, T M; Linsenmayer, T F

    1986-09-01

    We have examined whether the production of hypertrophic cartilage matrix reflecting a late stage in the development of chondrocytes which participate in endochondral bone formation, is the result of cell lineage, environmental influence, or both. We have compared the ability of cultured limb mesenchyme and mesectoderm to synthesize type X collagen, a marker highly selective for hypertrophic cartilage. High density cultures of limb mesenchyme from stage 23 and 24 chick embryos contain many cells that react positively for type II collagen by immunohistochemistry, but only a few of these initiate type X collagen synthesis. When limb mesenchyme cells are cultured in or on hydrated collagen gels or in agarose (conditions previously shown to promote chondrogenesis in low density cultures), almost all initiate synthesis of both collagen types. Similarly, collagen gel cultures of limb mesenchyme from stage 17 embryos synthesize type II collagen and with some additional delay type X collagen. However, cytochalasin D treatment of subconfluent cultures on plastic substrates, another treatment known to promote chondrogenesis, induces the production of type II collagen, but not type X collagen. These results demonstrate that the appearance of type X collagen in limb cartilage is environmentally regulated. Mesectodermal cells from the maxillary process of stages 24 and 28 chick embryos were cultured in or on hydrated collagen gels. Such cells initiate synthesis of type II collagen, and eventually type X collagen. Some cells contain only type II collagen and some contain both types II and X collagen. On the other hand, cultures of mandibular processes from stage 29 embryos contain chondrocytes with both collagen types and a larger overall number of chondrogenic foci than the maxillary process cultures. Since the maxillary process does not produce cartilage in situ and the mandibular process forms Meckel's cartilage which does not hypertrophy in situ, environmental influences

  1. Induction of heat shock protein 70 (Hsp70) by proteasome inhibitor MG 132 protects articular chondrocytes from cellular death in vitro and in vivo.

    Science.gov (United States)

    Grossin, Laurent; Etienne, Stéphanie; Gaborit, Nadège; Pinzano, Astrid; Cournil-Henrionnet, Christel; Gerard, Catherine; Payan, Elisabeth; Netter, Patrick; Terlain, Bernard; Gillet, Pierre

    2004-01-01

    The aim of this work was to determine whether Hsp70 overexpression via proteasome inhibitor MG132 was able to protect chondrocytes towards mono-iodoacetate (MIA) cytotoxicity both in vitro and in vivo. In vitro, overexpression of Hsp70 via MG132 was significantly able to protect chondrocytes from MIA toxicity (MTT/LDH analyses). Hsp70 essentially mediated this chondroprotective effect as demonstrated by antisense strategy. In vivo, chondrocytic overexpression of Hsp70, after a preventive intra-articular injection of MG132 in rat knee, was sufficient to decrease the severity of OA-induced MIA lesions, as demonstrated histologically and biochemically. In conclusion, intracellular overexpression of Hsp70, through proteasome inhibition, could be an interesting tool in protecting chondrocytes from cellular injuries, either necrotic or apoptotic in nature, and thus might be a novel chondroprotective modality in rat experimental OA.

  2. YKL-39 (chitinase 3-like protein 2), but not YKL-40 (chitinase 3-like protein 1), is up regulated in osteoarthritic chondrocytes

    OpenAIRE

    Knorr, T; Obermayr, F; Bartnik, E.; A. Zien; Aigner, T

    2003-01-01

    Methods: cDNA array and online quantitative polymerase chain reaction (PCR) were used to measure mRNA expression levels of YKL-39 and YKL-40 in chondrocytes in normal, early degenerative, and late stage osteoarthritic cartilage samples.

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

    OpenAIRE

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

    2004-01-01

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

  4. 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...

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

    NARCIS (Netherlands)

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

    2015-01-01

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

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

    NARCIS (Netherlands)

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

    2015-01-01

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

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

    Science.gov (United States)

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

    2015-01-01

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

  8. 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.

  9. In vitro effects of sodium hyaluronate on the proliferation and the apoptosis in chondrocytes from patients with Kashin-Beck disease and osteoarthritis

    Institute of Scientific and Technical Information of China (English)

    Zongqiang Gao; Xiong Guo; Chen Duan; Weijuan Ma; Peng Xu; Ruiyu Liu; Qisheng Gu; Junchang Chen

    2009-01-01

    Objective:To identify the in vitro effects of sodium hyaluronate(HA) on the proliferation and the apoptosis of chondrocytes from patients with Kashin-Beck disease(KBD) and osteoarthritis(OA). Methods:Samples of articular cartilages from KBD and OA patients, as well as healthy volunteers(6 subjects in each of the 3 groups) were dissected, digested with collagenase and the cells cultured in monolayers. Chondrocytes from each sample were assigned to an untreated group and two HA-treated groups: H0(no HA), H100(HA, 0.1 g/L) and H500(HA, 0.5 g/L). The first passage chondrocytes were used to observe proliferation using the MTT assay, and apoptosis by flow cytometry through Annexin V/PI staining. Results:HA promoted proliferation of chondrocytes in all the three groups, and in KBD and OA groups, for cells cultured for 4 and 6 days, H500 significantly promoted the cell proliferation. The apoptotic rates of both KBD and OA group chondrocytes were in the order H500 < HA100 < H0. Conclusion:Sodium hyaluronate administration has a dose-dependendent vitro effect to promote proliferation and inhibit apoptosis of chondrocytes from patients with KBD and OA.

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

    Science.gov (United States)

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

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

  11. The effects of interleukin-1b in modulating osteoclast-conditioned medium’s influence on gelatinases in chondrocytes through mitogen-activated protein kinases

    Institute of Scientific and Technical Information of China (English)

    Xiao-Xiao Cai

    2015-01-01

    Osteoarthritis is recognised to be an interactive pathological process involving the cartilage, subchondral bone and synovium. The signals from the synovium play an important role in cartilage metabolism, but little is known regarding the influence of the signalling from bone. Additionally, the collagenases and stromelysin-1 are involved in cartilage catabolism through mitogen-activated protein kinase (MAPK) signalling, but the role of the gelatinases has not been elucidated. Here, we studied the influence of osteoclastic signals on chondrocytes by characterising the expression of interleukin-1b (IL-1b)-induced gelatinases through MAPK signalling. We found that osteoclast-conditioned media attenuated the gelatinase activity in chondrocytes. However, IL-1b induced increased levels of gelatinase activity in the conditioned media group relative to the mono-cultured chondrocyte group. More specifically, IL-1b restored high levels of gelatinase activity in c-Jun N-terminal kinase inhibitor-pretreated chondrocytes in the conditioned media group and led to lower levels of gelatinase activity in extracellular signal-regulated kinase or p38 inhibitor-pretreated chondrocytes. Gene expression generally correlated with protein expression. Taken together, these results show for the first time that signals from osteoclasts can influence gelatinase activity in chondrocytes. Furthermore, these data show that IL-1b restores gelatinase activity through MAPK inhibitors;this information can help to increase the understanding of the gelatinase modulation in articular cartilage.

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

    DEFF Research Database (Denmark)

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

    2006-01-01

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

  13. Possible recruitment of osteoblastic precursor cells from hypertrophic chondrocytes during initial osteogenesis in cartilaginous limbs of young rats.

    Science.gov (United States)

    Franzen, A; Oldberg, A; Solursh, M

    1989-08-01

    The appearance of the bone phenotype during rat embryogenesis was studied by in situ hybridization using a cDNA clone to osteopontin. Radiolabeled sense and antisense RNA probes were prepared from the osteopontin cDNA by in vitro transcription. The probes were used to hybridize paraffin sections of the cartilaginous diaphysis from embryonic rats at day 17 of gestation. The hybridization pattern was analyzed by autoradiography. Hybridization with the antisense probe gave patterns of silver grain labeling, indicating the presence of osteopontin mRNA among the hypertrophic chondrocytes. No silver grains could be detected in the corresponding region following hybridization of consecutive sections with the sense probe, showing the specificity of the technique being used. Whether these results indicate that the osteopontin gene is transiently expressed by hypertrophic chondrocytes or that osteopontin is an early marker for osteoblastic precursor cells will have to be explored further.

  14. First-generation versus second-generation autologous chondrocyte implantation for treatment of cartilage defects of the knee

    DEFF Research Database (Denmark)

    Niemeyer, Philipp; Salzmann, Gian; Feucht, Matthias;

    2014-01-01

    membrane was utilized in second generation ACI. To date, however, no study has proven the superiority of this modification in terms of long-term clinical outcome. The purpose of this matched-pair analysis was therefore to compare the clinical long-term outcome of first and second generation ACI...... treated with first generation ACI. In both groups, four patients (17.4%) received surgical reintervention during follow-up. CONCLUSIONS: The use of a collagen membrane in combination with autologous chondrocytes (second generation ACI) leads to superior clinical long-term outcome compared to first......PURPOSE: Since the introduction of autologous chondrocyte implantation (ACI) for the treatment of cartilage defects, the initial technique has undergone several modifications. Whereas an autologous periosteum flap was used for defect coverage in first generation ACI, a standardized collagen...

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

    Science.gov (United States)

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

    2010-04-01

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

  16. Increased classical endoplasmic reticulum stress is sufficient to reduce chondrocyte proliferation rate in the growth plate and decrease bone growth.

    Directory of Open Access Journals (Sweden)

    Louise H W Kung

    Full Text Available Mutations in genes encoding cartilage oligomeric matrix protein and matrilin-3 cause a spectrum of chondrodysplasias called multiple epiphyseal dysplasia (MED and pseudoachondroplasia (PSACH. The majority of these diseases feature classical endoplasmic reticulum (ER stress and activation of the unfolded protein response (UPR as a result of misfolding of the mutant protein. However, the importance and the pathological contribution of ER stress in the disease pathogenesis are unknown. The aim of this study was to investigate the generic role of ER stress and the UPR in the pathogenesis of these diseases. A transgenic mouse line (ColIITgcog was generated using the collagen II promoter to drive expression of an ER stress-inducing protein (Tgcog in chondrocytes. The skeletal and histological phenotypes of these ColIITgcog mice were characterised. The expression and intracellular retention of Tgcog induced ER stress and activated the UPR as characterised by increased BiP expression, phosphorylation of eIF2α and spliced Xbp1. ColIITgcog mice exhibited decreased long bone growth and decreased chondrocyte proliferation rate. However, there was no disruption of chondrocyte morphology or growth plate architecture and perturbations in apoptosis were not apparent. Our data demonstrate that the targeted induction of ER stress in chondrocytes was sufficient to reduce the rate of bone growth, a key clinical feature associated with MED and PSACH, in the absence of any growth plate dysplasia. This study establishes that classical ER stress is a pathogenic factor that contributes to the disease mechanism of MED and PSACH. However, not all the pathological features of MED and PSACH were recapitulated, suggesting that a combination of intra- and extra-cellular factors are likely to be responsible for the disease pathology as a whole.

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

    Directory of Open Access Journals (Sweden)

    Emeric Limagne

    2016-05-01

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

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

    Science.gov (United States)

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

    2005-01-01

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

  19. Knee chondral lesions treated with autologous chondrocyte transplantation in a tridimensional matrix: clinical evaluation at 1-year follow-up

    OpenAIRE

    Vilchez, Félix; Lara, Jorge; Álvarez-Lozano, Eduardo; Cuervo, Carlos E.; Mendoza, Oscar F.; Acosta-Olivo, Carlos A.

    2009-01-01

    Background Despite the many studies on chondral injury repair, no outcomes have been evaluated with the Western Ontario and McMaster (WOMAC) Universities osteoarthritis index, the Knee Injury and Osteoarthritis Outcome Score (KOOS), and the Oxford Knee Score, all of which are specific for evaluating the presence of osteoarthritis. Materials and methods We evaluated the clinical progress of patients following autologous chondrocyte implantation (ACI) performed by our Bone and Tissue Bank using...

  20. Conditional Deletion of Fgfr3 in Chondrocytes leads to Osteoarthritis-like Defects in Temporomandibular Joint of Adult Mice.

    Science.gov (United States)

    Zhou, Siru; Xie, Yangli; Li, Wei; Huang, Junlan; Wang, Zuqiang; Tang, Junzhou; Xu, Wei; Sun, Xianding; Tan, Qiaoyan; Huang, Shuo; Luo, Fengtao; Xu, Meng; Wang, Jun; Wu, Tingting; Chen, Liang; Chen, Hangang; Su, Nan; Du, Xiaolan; Shen, Yue; Chen, Lin

    2016-04-04

    Osteoarthritis (OA) in the temporomandibular joint (TMJ) is a common degenerative disease in adult, which is characterized by progressive destruction of the articular cartilage. To investigate the role of FGFR3 in the homeostasis of TMJ cartilage during adult stage, we generated Fgfr3(f/f); Col2a1-CreER(T2) (Fgfr3 cKO) mice, in which Fgfr3 was deleted in chondrocytes at 2 months of age. OA-like defects were observed in Fgfr3 cKO TMJ cartilage. Immunohistochemical staining and quantitative real-time PCR analyses revealed a significant increase in expressions of COL10, MMP13 and AMAMTS5. In addition, there was a sharp increase in chondrocyte apoptosis at the Fgfr3 cKO articular surface, which was accompanied by a down-regulation of lubricin expression. Importantly, the expressions of RUNX2 and Indian hedgehog (IHH) were up-regulated in Fgfr3 cKO TMJ. Primary Fgfr3 cKO chondrocytes were treated with IHH signaling inhibitor, which significantly reduced expressions of Runx2, Col10, Mmp13 and Adamts5. Furthermore, the IHH signaling inhibitor partially alleviated OA-like defects in the TMJ of Fgfr3 cKO mice, including restoration of lubricin expression and improvement of the integrity of the articular surface. In conclusion, our study proposes that FGFR3/IHH signaling pathway plays a critical role in maintaining the homeostasis of TMJ articular cartilage during adult stage.

  1. Glucose concentration and medium volume influence cell viability and glycosaminoglycan synthesis in chondrocyte-seeded alginate constructs.

    Science.gov (United States)

    Heywood, Hannah K; Bader, Dan L; Lee, David A

    2006-12-01

    Increasing the thickness of tissue-engineered cartilage is associated with loss of chondrocyte viability and biosynthetic activity at the tissue center. Exceptionally high volumes of culture medium, however, can maintain cellularity and glycosaminoglycan synthesis throughout 4-mm-thick constructs. We hypothesized that glucose supplementation could replicate the augmentation of tissue formation achieved by medium volume. Chondrocyte-alginate constructs (40x10(6) cells/mL) were cultured for 14 days in 0.4-6.4 mL/10(-6) cells of either low- (5.1 mM) or high- (20.4 mM) glucose medium. Glucose was critical to chondrocyte viability, and glucose uptake increased significantly (P cells of low-glucose medium had a mass of 172 +/- 6.1 mg and glycosaminoglycan (GAG) content of 0.32 +/- 0.03 mg (mean +/- standard deviation). A 4-fold increase in medium volume increased the final construct mass by 44% and GAG content by 207%. An equivalent increase in glucose supply in the absence of volume change increased these parameters by just 10% and 73%, respectively. A similar trend was observed from 0.8 to 3.2 mL/10(-6) cells, when maximal values of construct GAG content and mass were obtained. Therefore, medium volume remains an important consideration for the optimal culture of tissue-engineered cartilage.

  2. Low intensity pulse ultrasound stimulate chondrocytes growth in a 3-D alginate scaffold through improved porosity and permeability.

    Science.gov (United States)

    Guo, Gepu; Lu, Lu; Ji, Hongfei; Ma, Yong; Dong, Rui; Tu, Juan; Guo, Xiasheng; Qiu, Yuanyuan; Wu, Junru; Zhang, Dong

    2015-04-01

    A 3-D scaffold culture system has been used to promote in producing functional chondrocytes for repairing damaged cartilage. In the present study, the low intensity pulse ultrasound (LIPUS) (P(-)=0, 0.055, 0.085 and 0.11 MPa) was applied to improve the porosity and permeability of a 3-D alginate scaffold which was beneficial for the nutrition supply and metabolism during cell growth in 3-D alginate scaffold. The porosity and permeability of the scaffold was quantitatively analyzed based on scanning electron microscopy examination and fluorescence image observation. The results suggest that, for the scaffold exposed to LIPUS, its porosity and permeability could be significantly enhanced by the increasing LIPUS amplitude, which might be induced by the microstreaming shear stress generated by ultrasound-driven microbubble oscillations. Furthermore, the assessments of cell proliferation and collagen II expression confirmed that chondrocytes growth could be effectively promoted in 3-D alginate scaffolds treated by LIPUS, because of the improved scaffold porosity and permeability might benefit cell growth space and nutrition supply. It should also be noticed that appropriate LIPUS driving parameters should be adapted to achieve optimized chondrocytes culture effect in 3-D alginate scaffold.

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

    Science.gov (United States)

    Qu, Yanlong; Zhou, Li; Wang, Chunlei

    2017-02-01

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

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

    Directory of Open Access Journals (Sweden)

    Shun-Fu Chang

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

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

    Science.gov (United States)

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

    2016-05-04

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

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

    Directory of Open Access Journals (Sweden)

    Silny Jiri

    2010-08-01

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

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

    Directory of Open Access Journals (Sweden)

    Sandy A van Gool

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

  8. Outcomes of Autologous Chondrocyte Implantation in the Knee following Failed Microfracture

    Science.gov (United States)

    Riff, Andrew Joseph; Yanke, Adam Blair; Tilton, Annemarie K.; Cole, Brian J.

    2016-01-01

    Objectives: Marrow stimulation techniques such as drilling or microfracture are first-line treatment options for symptomatic cartilage defects of the knee. For young patients who have failed microfracture, cartilage restoration techniques such as autologous chondrocyte implantation (ACI), OATS, and osteochondral allograft and are frequently employed. Nevertheless, there a few reports in the literature evaluating the results of ACI following failed microfracture and those available suggest inferior outcomes compared to primary ACI. This study was performed to evaluate the clinical outcomes of autologous chondrocyte implantation (ACI) following failed microfracture in the knee and compare these outcomes to those of primary ACI. Methods: Patients were identified who underwent autologous chondrocyte implantation for symptomatic chondral lesions of the knee refractory to previous microfracture. Postoperative data were collected using several subjective scoring systems (Noyes, Tegner, Lysholm, IKDC, KOOS, SF12). An age-matched cohort of 103 patients who underwent primary ACI of the knee was used as a control group. Statistics were performed in a paired manner using a Student’s t-test for ordinal data and chi-square test for categorical data. Results: Ninety-two patients met the inclusion criteria. The average patient age was 30.1 years (range, 14-49 years) at the time of ACI. The average duration from microfracture to ACI was 21.2 months (range, 1-88 months). ACI was performed in the tibiofemoral compartment in 42 patients, the patellofemoral compartments in 38 patients, and in both in 12 patients. The primary lesion treated with ACI involved the MFC in 38 patients, the trochlea in 25 patients, the patella in 19 patients, and the LFC in 10 patients. The lesions averaged 467mm3 in the trochlea, 445mm3 in the LFC, 265mm3 in the patella, and 295mm3 in the patella. Nineteen patients underwent concurrent ACI to multiple lesions. Thirty-one patients underwent concomitant

  9. Influence of Knee Immobilization on Chondrocyte Apoptosis and Histological Features of the Anterior Cruciate Ligament Insertion and Articular Cartilage in Rabbits

    Science.gov (United States)

    Mutsuzaki, Hirotaka; Nakajima, Hiromi; Wadano, Yasuyoshi; Furuhata, Syogo; Sakane, Masataka

    2017-01-01

    This study examined the influence of immobilization on chondrocyte apoptosis and histological features of the anterior cruciate ligament (ACL) insertion and knee articular cartilage in rabbits. Forty-eight male Japanese white rabbits were assigned to an immobilization (n = 24) or sham (n = 24) group. Rabbits in the immobilization group underwent complete unilateral surgical knee immobilization and rabbits in the sham group underwent a sham surgery. The average thickness of the glycosaminoglycan (GAG) stained red area by safranin O staining, the chondrocyte apoptosis rate and the chondrocyte proliferation rate in the cartilage layer in the ACL insertion and the articular cartilage of the medial tibial condyle were measured at one, two, four and eight weeks in six animals from each group. In the ACL insertion, the chondrocyte apoptosis rate was higher in the immobilization group than in the sham group at two and eight weeks after surgery (p < 0.05). The chondrocyte proliferation rate gradually decreased from two weeks to eight weeks in the immobilization group. The GAG layer was thinner in the immobilization group than in the sham group at two, four and eight weeks after surgery (p < 0.05). In the articular cartilage, the chondrocyte apoptosis rate in the immobilization group was higher than in the sham group at four and eight weeks after surgery (p < 0.05). The GAG layer was significantly thinner in the immobilization group than that in the sham group at four and eight weeks after surgery (p < 0.05). Knee immobilization significantly increased chondrocyte apoptosis at two and eight weeks after surgery in the ACL insertion and at four and eight weeks after surgery in the articular cartilage of the medial tibial condyle, and decreased GAG layer thickness from two to eight weeks after surgery in the ACL insertion and from four to eight weeks after surgery in the articular cartilage. PMID:28134763

  10. Hyperbaric oxygen protects mandibular condylar chondrocytes from interleukin-1β-induced apoptosis via the PI3K/AKT signaling pathway

    Science.gov (United States)

    Chen, Hang; Wu, Gaoyi; Sun, Qi; Dong, Yabing; Zhao, Huaqiang

    2016-01-01

    Objectives: Mandibular condylar chondrocyte apoptosis is mainly responsible for the development and progression of temporomandibular joint osteoarthritis (TMJ-OA). Interleukin-1β (IL-1β) generally serves an agent that induces chondrocyte apoptosis. Hyperbaric oxygen (HBO) treatment increases proteoglycan synthesis in vivo. We explore the protective effect of HBO on IL-1β-induced mandibular condylar chondrocyte apoptosis in rats and the potential molecular mechanisms. Methods: Chondrocytes were isolated from the TMJ of 3-4-week old Sprague-Dawley rats. The Cell Counting Kit-8 (CCK-8) assay was used to determine cell viability. The phosphorylated phosphoinositide-3 kinase (p-PI3K), phosphorylated AKT (p-Akt), type II collagen (COL2), and aggrecan (AGG) content was detected by immunofluorescence, immunocytochemistry and western blotting. The expression of Pi3k, Akt, Col2 and Agg mRNA was measured using real-time quantitative polymerase chain reaction (RT-qPCR). Results: HBO inhibited the cytotoxicity and apoptosis induced by IL-1β (10 ng/mL) in the mandibular condylar chondrocytes. HBO also decreased the IL-1β activity that decreased p-PI3K and p-AKT levels, and increased COL2 and AGG expression, with the net effect of suppressing extracellular matrix degradation. Conclusions: These data suggest that HBO may protect mandibular condylar chondrocytes against IL-1β-induced apoptosis via the PI3K/AKT signaling pathway, and that it may promote the expression of mandibular condylar chondrocyte extracellular matrix through the PI3K/AKT signaling pathway. PMID:27904712

  11. Influence of Knee Immobilization on Chondrocyte Apoptosis and Histological Features of the Anterior Cruciate Ligament Insertion and Articular Cartilage in Rabbits

    Directory of Open Access Journals (Sweden)

    Hirotaka Mutsuzaki

    2017-01-01

    Full Text Available This study examined the influence of immobilization on chondrocyte apoptosis and histological features of the anterior cruciate ligament (ACL insertion and knee articular cartilage in rabbits. Forty-eight male Japanese white rabbits were assigned to an immobilization (n = 24 or sham (n = 24 group. Rabbits in the immobilization group underwent complete unilateral surgical knee immobilization and rabbits in the sham group underwent a sham surgery. The average thickness of the glycosaminoglycan (GAG stained red area by safranin O staining, the chondrocyte apoptosis rate and the chondrocyte proliferation rate in the cartilage layer in the ACL insertion and the articular cartilage of the medial tibial condyle were measured at one, two, four and eight weeks in six animals from each group. In the ACL insertion, the chondrocyte apoptosis rate was higher in the immobilization group than in the sham group at two and eight weeks after surgery (p < 0.05. The chondrocyte proliferation rate gradually decreased from two weeks to eight weeks in the immobilization group. The GAG layer was thinner in the immobilization group than in the sham group at two, four and eight weeks after surgery (p < 0.05. In the articular cartilage, the chondrocyte apoptosis rate in the immobilization group was higher than in the sham group at four and eight weeks after surgery (p < 0.05. The GAG layer was significantly thinner in the immobilization group than that in the sham group at four and eight weeks after surgery (p < 0.05. Knee immobilization significantly increased chondrocyte apoptosis at two and eight weeks after surgery in the ACL insertion and at four and eight weeks after surgery in the articular cartilage of the medial tibial condyle, and decreased GAG layer thickness from two to eight weeks after surgery in the ACL insertion and from four to eight weeks after surgery in the articular cartilage.

  12. An additive manufacturing-based PCL-alginate-chondrocyte bioprinted scaffold for cartilage tissue engineering.

    Science.gov (United States)

    Kundu, Joydip; Shim, Jin-Hyung; Jang, Jinah; Kim, Sung-Won; Cho, Dong-Woo

    2015-11-01

    Regenerative medicine is targeted to improve, restore or replace damaged tissues or organs using a combination of cells, materials and growth factors. Both tissue engineering and developmental biology currently deal with the process of tissue self-assembly and extracellular matrix (ECM) deposition. In this investigation, additive manufacturing (AM) with a multihead deposition system (MHDS) was used to fabricate three-dimensional (3D) cell-printed scaffolds using layer-by-layer (LBL) deposition of polycaprolactone (PCL) and chondrocyte cell-encapsulated alginate hydrogel. Appropriate cell dispensing conditions and optimum alginate concentrations for maintaining cell viability were determined. In vitro cell-based biochemical assays were performed to determine glycosaminoglycans (GAGs), DNA and total collagen contents from different PCL-alginate gel constructs. PCL-alginate gels containing transforming growth factor-β (TGFβ) showed higher ECM formation. The 3D cell-printed scaffolds of PCL-alginate gel were implanted in the dorsal subcutaneous spaces of female nude mice. Histochemical [Alcian blue and haematoxylin and eosin (H&E) staining] and immunohistochemical (type II collagen) analyses of the retrieved implants after 4 weeks revealed enhanced cartilage tissue and type II collagen fibril formation in the PCL-alginate gel (+TGFβ) hybrid scaffold. In conclusion, we present an innovative cell-printed scaffold for cartilage regeneration fabricated by an advanced bioprinting technology.

  13. SHP2-Deficiency in Chondrocytes Deforms Orofacial Cartilage and Ciliogenesis in Mice.

    Science.gov (United States)

    Kamiya, Nobuhiro; Shen, Jingling; Noda, Kazuo; Kitami, Megumi; Feng, Gen-Sheng; Chen, Di; Komatsu, Yoshihiro

    2015-11-01

    Congenital orofacial abnormalities are clinically seen in human syndromes with SHP2 germline mutations such as LEOPARD and Noonan syndrome. Recent studies demonstrate that SHP2-deficiency leads to skeletal abnormalities including scoliosis and cartilaginous benign tumor metachondromatosis, suggesting that growth plate cartilage is a key tissue regulated by SHP2. The role and cellular mechanism of SHP2 in the orofacial cartilage, however, remains unknown. Here, we investigated the postnatal craniofacial development by inducible disruption of Shp2 in chondrocytes. Shp2 conditional knockout (cKO) mice displayed severe deformity of the mandibular condyle accompanied by disorganized, expanded cartilage in the trabecular bone region, enhanced type X collagen, and reduced Erk production. Interestingly, the length of primary cilia, an antenna like organelle sensing environmental signaling, was significantly shortened, and the number of primary cilia was reduced in the cKO mice. The expression levels of intraflagellar transports (IFTs), essential molecules in the assembly and function of primary cilia, were significantly decreased. Taken together, lack of Shp2 in orofacial cartilage led to severe defects of ciliogenesis through IFT reduction, resulting in mandibular condyle malformation and cartilaginous expansion. Our study provides new insights into the molecular pathogenesis of SHP2-deficiency in cartilage and helps to understand orofacial and skeletal manifestations seen in patients with SHP2 mutations.

  14. Detection, imaging, and kinetics of sub-micron organelles of chondrocytes by multiple beam interference microscopy

    Science.gov (United States)

    Joshi, Narahari V.; Medina, Honorio; Barboza, J. M.; Colantuoni, Gladys; Quintero, Maritza

    2004-07-01

    Chondrocytes, obtained from testosterone treated human articular cartilage, were examined by a recently developed Multiple Beam Interference Microscopy (MBIM) attached to a confocal set up, Video-enhanced differential interference microphotography and also by cinematography. In the MBIM, the intensity of the transmitted pattern is given by the Airy function which increases the contrast dramatically as the coefficient of the reflectance of the parallel plates increases. Moreover, in this configuration, the beam passes several times through a specific organelle and increases its optical path difference both because of the increase in the trajectory and refractive index (high density) of the organelle. The improved contrast enhances the resolving power of the system and makes visible several structural details of sub micron dimensions like nucleolus, retraction fibers, podia, etc. which are not possible to reveal with such a clarity by conventional techniques such as bright field, phase contrast or DIC. This technique permits to detect the oscillatory and rotational motions of unstained cilia for the first time. The frequency of oscillations was found to be 0.8 Hz.

  15. Laminins and Nidogens in the Pericellular Matrix of Chondrocytes: Their Role in Osteoarthritis and Chondrogenic Differentiation.

    Science.gov (United States)

    Schminke, Boris; Frese, Jenny; Bode, Christa; Goldring, Mary B; Miosge, Nicolai

    2016-02-01

    The aim of this study was to investigate the role of laminins and nidogen-2 in osteoarthritis (OA) and their potential to support chondrogenic differentiation. We applied immunohistochemistry, electron microscopy, siRNA, quantitative RT-PCR, Western blot, and proteome analysis for the investigation of cartilage tissue and isolated chondrocytes in three-dimensional culture obtained from patients with late-stage knee OA and nidogen-2 knockout mice. We demonstrate that subunits of laminins appear in OA cartilage and that nidogen-2-null mice exhibit typical osteoarthritic features. Chondrogenic progenitor cells (CPCs) produced high levels of laminin-α1, laminin-α5, and nidogen-2 in their pericellular matrix, and laminin-α1 enhanced collagen type II and reduced collagen type I expression by cultured CPCs. Nidogen-2 increased SOX9 gene expression. Knockdown of nidogen-2 reduced SOX9 expression, whereas it up-regulated RUNX2 expression. This study reveals that the influence of the pericellular matrix on CPCs is important for the expression of the major regulator transcription factors, SOX9 and RUNX2. Our novel findings that laminins and nidogen-2 drive CPCs toward chondrogenesis may help in the elucidation of new treatment strategies for cartilage tissue regeneration.

  16. Strenuous Treadmill Running Induces a Chondrocyte Phenotype in Rat Achilles Tendons

    Science.gov (United States)

    Xu, Shao-Yong; Li, Shu-Fen; Ni, Guo-Xin

    2016-01-01

    Background Although tendinopathy is common, its underlying pathogenesis is poorly understood. This study aimed to investigate the possible pathogenesis of tendinopathy. Material/Methods In this study, a total of 24 rats were randomly and evenly divided into a control (CON) group and a strenuous treadmill running (STR) group. Animals in the STR group were subjected to a 12-week treadmill running protocol. Subsequently, all Achilles tendons were harvested to perform histological observation or biochemical analyses. Results Histologically, hypercellularity and round cells, as well as disorganized collagen fibrils, were presented in rat Achilles tendon sections from the STR group. Furthermore, our results showed that the expression of aggrecan, collagen type II (Col II), and Sex-Determining Region Y Box 9 (Sox 9) were markedly increased in the STR group compared with that in the CON group. Additionally, the mRNA expression of bone morphogenetic protein-2 (BMP-2) and biglycan was significantly up-regulated in the STR group in contrast to that in CON group. Conclusions These results suggest that a 12-week strenuous treadmill running regimen can induce chondrocyte phenotype in rat Achilles tendons through chondrogenic differentiation of tendon stem cells (TSCs) by BMP-2 signaling. PMID:27742920

  17. Chondrocyte Behavior on Micropatterns Fabricated Using Layer-by-Layer Lift-Off: Morphological Analysis

    Directory of Open Access Journals (Sweden)

    Jameel Shaik

    2013-01-01

    Full Text Available Cell patterning has emerged as an elegant tool in developing cellular arrays, bioreactors, biosensors, and lab-on-chip devices and for use in engineering neotissue for repair or regeneration. In this study, micropatterned surfaces were created using the layer-by-layer lift-off (LbL-LO method for analyzing canine chondrocytes response to patterned substrates. Five materials were chosen based on our previous studies. These included: poly(dimethyldiallylammonium chloride (PDDA, poly(ethyleneimine (PEI, poly(styrene sulfonate (PSS, collagen, and chondroitin sulfate (CS. The substrates were patterned with these five different materials, in five and ten bilayers, resulting in the following multilayer nanofilm architectures: (PSS/PDDA5, (PSS/PDDA10; (CS/PEI4/CS, (CS/PEI9/CS; (PSS/PEI5, (PSS/PEI10; (PSS/Collagen5, (PSS/Collagen10; (PSS/PEI4/PSS, (PSS/PEI9/PSS. Cell characterization studies were used to assess the viability, longevity, and cellular response to the configured patterned multilayer architectures. The cumulative cell characterization data suggests that cell viability, longevity, and functionality were enhanced on micropatterned PEI, PSS, collagen, and CS multilayer nanofilms suggesting their possible use in biomedical applications.

  18. The optimization of porous polymeric scaffolds for chondrocyte/atelocollagen based tissue-engineered cartilage.

    Science.gov (United States)

    Tanaka, Yoko; Yamaoka, Hisayo; Nishizawa, Satoru; Nagata, Satoru; Ogasawara, Toru; Asawa, Yukiyo; Fujihara, Yuko; Takato, Tsuyoshi; Hoshi, Kazuto

    2010-06-01

    To broaden the clinical application of cartilage regenerative medicine, we should develop an implant-type tissue-engineered cartilage with firmness and 3-D structure. For that, we attempted to use a porous biodegradable polymer scaffold in the combination with atelocollagen hydrogel, and optimized the structure and composition of porous scaffold. We administered chondrocytes/atelocollagen mixture into the scaffolds with various kinds of porosities (80-95%) and pore sizes (0.3-2.0 mm), consisting of PLLA or related polymers (PDLA, PLA/CL and PLGA), and transplanted the constructs in the subcutaneous areas of nude mice. The constructs using scaffolds of excessively large pore sizes (>1 mm) broke out on the skin and impaired the host tissue. The scaffold with the porosity of 95% and pore size of 0.3 mm could effectively retain the cells/gel mixture and indicated a fair cartilage regeneration. Regarding the composition, the tissue-engineered cartilage was superior in PLGA and PLLA to that in PLA/CA and PDLA. The latter two showed the dense accumulation of macrophages, which may deteriorate the cartilage regeneration. Although PLGA or PLLA has been currently recommended for the scaffold of cartilage, the polymer for which biodegradation was exactly synchronized to the cartilage regeneration would improve the quality of the tissue-engineered cartilage.

  19. 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.

  20. A bit of give and take: the relationship between the extracellular matrix and the developing chondrocyte.

    Science.gov (United States)

    Behonick, Danielle J; Werb, Zena

    2003-11-01

    The extracellular matrix (ECM), once thought to be a static structural component of tissues, is now known to play a complex and dynamic role in a variety of cellular functions in a number of diverse tissues. A significant body of literature attests to the ability of the ECM to communicate both spatial and temporal information to adherent cells, thereby directing cell behavior via interactions between the ECM and cell-surface receptors. Moreover, volumes of experimental data show that a great deal of communication travels in the opposite direction, from the cell to the ECM, allowing for regulation of the cues transmitted by the ECM. As such, the ECM, with respect to its components and their organization, is not a fixed reflection of the state the local microenvironment in which a cell finds itself at a particular time, but rather is able to respond to and effect changes in its local microenvironment. As an example of the developmental consequences of ECM interactions, this review gives an overview of the 'give and take' relationship between the ECM and the cells of the developing skeletal elements, in particular, the chondrocyte.

  1. Superficial Collagen Fibril Modulus and Pericellular Fixed Charge Density Modulate Chondrocyte Volumetric Behaviour in Early Osteoarthritis

    Directory of Open Access Journals (Sweden)

    Petri Tanska

    2013-01-01

    Full Text Available The aim of this study was to investigate if the experimentally detected altered chondrocyte volumetric behavior in early osteoarthritis can be explained by changes in the extracellular and pericellular matrix properties of cartilage. Based on our own experimental tests and the literature, the structural and mechanical parameters for normal and osteoarthritic cartilage were implemented into a multiscale fibril-reinforced poroelastic swelling model. Model simulations were compared with experimentally observed cell volume changes in mechanically loaded cartilage, obtained from anterior cruciate ligament transected rabbit knees. We found that the cell volume increased by 7% in the osteoarthritic cartilage model following mechanical loading of the tissue. In contrast, the cell volume decreased by 4% in normal cartilage model. These findings were consistent with the experimental results. Increased local transversal tissue strain due to the reduced collagen fibril stiffness accompanied with the reduced fixed charge density of the pericellular matrix could increase the cell volume up to 12%. These findings suggest that the increase in the cell volume in mechanically loaded osteoarthritic cartilage is primarily explained by the reduction in the pericellular fixed charge density, while the superficial collagen fibril stiffness is suggested to contribute secondarily to the cell volume behavior.

  2. Composite articular cartilage engineered on a chondrocyte-seeded aliphatic polyurethane sponge.

    Science.gov (United States)

    Liu, Yanchun; Webb, Ken; Kirker, Kelly R; Bernshaw, Nicole J; Tresco, Patrick A; Gray, Steven D; Prestwich, Glenn D

    2004-01-01

    To circumvent the reconstructive disadvantages inherent in resorbable polyglycolic acid (PGA)/polylactic acid (PLA) used in cartilage engineering, a nonresorbable, and nonreactive polyurethane sponge (Tecoflex sponge, TS) was studied as both a cell delivery device and as an internal support scaffolding. The in vitro viability and proliferation of porcine articular chondrocytes (PACs) in TS, and the in vivo generation of new articular cartilage and long-term resorption, were examined. The initial cell attachment rate was 40%, and cell density increased more than 5-fold after 12 days of culture in vitro. PAC-loaded TS blocks were implanted into nude mice, became opalescent, and resembled native cartilage at weeks 12 and 24 postimplantation. The mass and volume of newly formed cartilage were not significantly different at week 24 from samples harvested at week 6 or week 12. Safranin O-fast green staining revealed that the specimens from cell-loaded TS groups at week 12 and week 24 consisted of mature cartilage. Collagen typing revealed that type II collagen was present in all groups of tissue-engineered cartilage. In conclusion, the implantation of PAC-TS resulted in composite tissue-engineered articular cartilage with TS as an internal support. Long-term observation (24 weeks) of mass and volume showed no evidence of resorption.

  3. 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.

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

    Directory of Open Access Journals (Sweden)

    Wei-Ping Chen

    2015-05-01

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

  5. Mesenchymal Stem Cells Reshape and Provoke Proliferation of Articular Chondrocytes by Paracrine Secretion

    Science.gov (United States)

    Xu, Lei; Wu, Yuxi; Xiong, Zhimiao; Zhou, Yan; Ye, Zhaoyang; Tan, Wen-Song

    2016-09-01

    Coculture between mesenchymal stem cells (MSCs) and articular chondrocytes (ACs) represents a promising strategy for cartilage regeneration. This study aimed at elaborating how ACs were regulated by MSCs. Rabbit ACs (rACs) and rabbit MSCs (rMSCs) were seeded separately in a Transwell system to initiate non-contact coculture in growth medium without chondrogenic factors. Cell morphology, cell proliferation, production of extracellular matrix (ECM), and gene expression of rACs were characterized. Upon coculture, rACs underwent a morphological transition from a rounded or polygonal shape into a fibroblast-like one and proliferation was provoked simultaneously. Such effects were dependent on the amount of rMSCs. Along with these changes, ECM production and gene expression of rACs were also perturbed. Importantly, when a ROCK inhibitor (Y27632) was supplemented to coculture, the effects except that on cell proliferation were inhibited, suggesting the involvement of RhoA/ROCK signaling. By applying an inhibitor (BIBF1120) of VEGFR1/2/3, FGFR1/2/3 and PDGFRα/β in coculture, or supplementing FGF-1, VEGF-A and PDGFbb in monoculture, it was confirmed that the paracrine factors by rMSCs mediated the compounding effects on rACs. These findings shed light on MSCs-ACs interactions and might confer an insight view on cell-based cartilage regeneration.

  6. Evaluation of Magnetic Nanoparticle-Labeled Chondrocytes Cultivated on a Type II Collagen–Chitosan/Poly(Lactic-co-Glycolic Acid Biphasic Scaffold

    Directory of Open Access Journals (Sweden)

    Juin-Yih Su

    2017-01-01

    Full Text Available Chondral or osteochondral defects are still controversial problems in orthopedics. Here, chondrocytes labeled with magnetic nanoparticles were cultivated on a biphasic, type II collagen–chitosan/poly(lactic-co-glycolic acid scaffold in an attempt to develop cultures with trackable cells exhibiting growth, differentiation, and regeneration. Rabbit chondrocytes were labeled with magnetic nanoparticles and characterized by scanning electron microscopy (SEM, transmission electron (TEM microscopy, and gene and protein expression analyses. The experimental results showed that the magnetic nanoparticles did not affect the phenotype of chondrocytes after cell labeling, nor were protein and gene expression affected. The biphasic type II collagen–chitosan/poly(lactic-co-glycolic acid scaffold was characterized by SEM, and labeled chondrocytes showed a homogeneous distribution throughout the scaffold after cultivation onto the polymer. Cellular phenotype remained unaltered but with increased gene expression of type II collagen and aggrecan, as indicated by cell staining, indicating chondrogenesis. Decreased SRY-related high mobility group-box gene (Sox-9 levels of cultured chondrocytes indicated that differentiation was associated with osteogenesis. These results are encouraging for the development of techniques for trackable cartilage regeneration and osteochondral defect repair which may be applied in vivo and, eventually, in clinical trials.

  7. Advanced oxidation protein products induce chondrocyte apoptosis via receptor for advanced glycation end products-mediated, redox-dependent intrinsic apoptosis pathway.

    Science.gov (United States)

    Wu, Qian; Zhong, Zhao-Ming; Zhu, Si-Yuan; Liao, Cong-Rui; Pan, Ying; Zeng, Ji-Huan; Zheng, Shuai; Ding, Ruo-Ting; Lin, Qing-Song; Ye, Qing; Ye, Wen-Bin; Li, Wei; Chen, Jian-Ting

    2016-01-01

    Pro-inflammatory cytokine-induced chondrocyte apoptosis is a primary cause of cartilage destruction in the progression of rheumatoid arthritis (RA). Advanced oxidation protein products (AOPPs), a novel pro-inflammatory mediator, have been confirmed to accumulate in patients with RA. However, the effect of AOPPs accumulation on chondrocyte apoptosis and the associated cellular mechanisms remains unclear. The present study demonstrated that the plasma formation of AOPPs was enhanced in RA rats compared with normal. Then, chondrocyte were treated with AOPPs-modified rat serum albumin (AOPPs-RSA) in vitro. Exposure of chondrocyte to AOPPs activated nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and increased expression of NADPH oxidase subunits, which was mediated by receptor for advanced glycation end products (RAGE), but not scavenger receptor CD36. Moreover, AOPPs challenge triggered NADPH oxidase-dependent ROS generation which induced mitochondrial dysfunction and endoplasmic reticulum stress resulted in activation of caspase family that eventually lead to apoptosis. Lastly, blockade of RAGE, instead of CD36, largely attenuated these signals. Our study demonstrated first time that AOPPs induce chondrocyte apoptosis via RAGE-mediated and redox-dependent intrinsic apoptosis pathway in vitro. These data implicates that AOPPs may represent a novel pathogenic factor that contributes to RA progression. Targeting AOPPs-triggered cellular mechanisms might emerge as a promising therapeutic option for patients with RA.

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

    Science.gov (United States)

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

    2013-02-01

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

  9. SOX9 directly Regulates CTGF/CCN2 Transcription in Growth Plate Chondrocytes and in Nucleus Pulposus Cells of Intervertebral Disc.

    Science.gov (United States)

    Oh, Chun-do; Yasuda, Hideyo; Zhao, Weiwei; Henry, Stephen P; Zhang, Zhaoping; Xue, Ming; de Crombrugghe, Benoit; Chen, Di

    2016-07-20

    Several lines of evidence indicate that connective tissue growth factor (CTGF/CCN2) stimulates chondrocyte proliferation and maturation. Given the fact that SOX9 is essential for several steps of the chondrocyte differentiation pathway, we asked whether Ctgf (Ccn2) is the direct target gene of SOX9. We found that Ctgf mRNA was down-regulated in primary sternal chondrocytes from Sox9(flox/flox) mice infected with Ad-CMV-Cre. We performed ChIP-on-chip assay using anti-SOX9 antibody, covering the Ctgf gene from 15 kb upstream of its 5'-end to 10 kb downstream of its 3'-end to determine SOX9 interaction site. One high-affinity interaction site was identified in the Ctgf proximal promoter by ChIP-on-chip assay. An important SOX9 regulatory element was found to be located in -70/-64 region of the Ctgf promoter. We found the same site for SOX9 binding to the Ctgf promoter in nucleus pulposus (NP) cells. The loss of Sox9 in growth plate chondrocytes in knee joint and in NP cells in intervertebral disc led to the decrease in CTGF expression. We suggest that Ctgf is the direct target gene of SOX9 in chondrocytes and NP cells. Our study establishes a strong link between two regulatory molecules that have a major role in cartilaginous tissues.

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

    Science.gov (United States)

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

    2007-01-01

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

  11. Both superficial and deep zone articular chondrocyte subpopulations exhibit the Crabtree effect but have different basal oxygen consumption rates.

    Science.gov (United States)

    Heywood, Hannah K; Knight, Martin M; Lee, David A

    2010-06-01

    In the absence of in vivo measurements, the oxygen concentration within articular cartilage is calculated from the balance between cellular oxygen consumption and mass transfer. Current estimates of the oxygen tension within articular cartilage are based on oxygen consumption data from full-depth tissue samples. However, superficial and deep cell subpopulations of articular cartilage express intrinsic metabolic differences. We test the hypothesis that the subpopulations differ with respect to their intrinsic oxygen consumption rate. Chondrocytes from the full cartilage thickness demonstrate enhanced oxygen consumption when deprived of glucose, consistent with the Crabtree phenomena. Chondrocyte subpopulations differ in the prevailing availability of oxygen and glucose, which decrease with distance from the cartilage-synovial fluid interface. Thus, we tested the hypothesis that the oxygen consumption of each subpopulation is modulated by nutrient availability, by examining the expression of the Crabtree effect. The deep cells had a greater oxygen consumption than the superficial cells (V(max) of 6.6 compared to 3.2 fmol/cell/h), consistent with our observations of mitochondrial volume (mean values 52.0 vs. 36.4 microm(3)/cell). Both populations expressed the Crabtree phenomena, with oxygen consumption increasing approximately 2.5-fold in response to glycolytic inhibition by glucose deprivation or 2-deoxyglucose. Over 90% of this increase was oligomycin-sensitive and thus accounted for by oxidative phosphorylation. The data contributes towards our understanding of chondrocyte energy metabolism and provides information valuable for the accurate calculation of the oxygen concentration that the cells experience in vivo. The work has further application to the optimisation of bioreactor design and engineered tissues.

  12. Changes in collagens and chondrocytes in the temporomandibular joint cartilage in growing rats fed a liquid diet.

    Science.gov (United States)

    Uekita, Hiroki; Takahashi, Shigeru; Domon, Takanori; Yamaguchi, Taihiko

    2015-11-01

    The temporomandibular joint (TMJ) of growing rats fed a soft diet is reported to be smaller in size and to have thinner condyle and glenoid fossa cartilage than rats fed a solid diet. The aim of this study was to determine the effect of a soft diet on the collagens and chondrocytes in the growing TMJ cartilage. Forty-eight male Wistar rats were divided into a control group fed a solid diet and an experimental group fed a liquid diet for 1-8 weeks. After the experimental period, the TMJs were harvested and examined histologically, immunohistochemically for collagen types I, II, and X, and with transmission electron microscopy. The condylar cartilage in the experimental rats showed weak immunoreactions for three types of collagens compared with the controls. The ultrastructure had fewer fine collagen fibrils in the experimental rats compared with that of the controls. The glenoid fossa cartilage in the experimental rats showed narrower Alcian blue-positive areas than the control staining. The immunoreactions for three types of collagen in the experimental rats were also weaker than those of the controls. The chondrocytes in the experimental rats appeared dark, had extended thin cytoplasmic processes, and had formed gap junctions, as assessed by transmission electron microscopy. Fewer fine collagen fibrils, but thick bands of collagen fibrils were observed in the glenoid fossa of the experimental cartilage. The results of the present study showed that a liquid diet had deleterious effects on the quality and quantity of collagens and chondrocytes in the TMJ cartilage in growing rats.

  13. Comparative, osteochondral defect repair: Stem cells versus chondrocytes versus Bone Morphogenetic Protein-2, solely or in combination

    Directory of Open Access Journals (Sweden)

    R Reyes

    2013-07-01

    Full Text Available Full-thickness articular cartilage damage does not resolve spontaneously. Studies with growth factors, implantation of autologous chondrocytes and mesenchymal stem cells have led to variable, to some extent inconsistent, results. This work compares osteochondral knee-defect repair in rabbits upon implantation of a previously described alginate/(poly(lactic-co-glycolic acid (PLGA osteochondral scaffold in distinct conditions. Systems were either in vitro pre-cultured with a small number of allogeneic chondrocytes under fibroblast growth factor (FGF-2 stimulation or the same amount of allogeneic, marrow derived, mesenchymal stem cells (without any pre-differentiation, or loaded with microsphere-encapsulated bone morphogenetic protein (BMP-2 within the alginate layer, or holding combinations of one or the other cell type with BMP-2. The experimental limit was 12 weeks, because a foregoing study with this release system had shown a maintained tissue response for at least 24 weeks post-operation. After only 6 weeks, histological analyses revealed newly formed cartilage-like tissue, which resembled the adjacent, normal cartilage in cell as well as BMP-2 treated defects, but cell therapy gave higher histological scores. This advantage evened out until 12 weeks. Combinations of cells and BMP-2 did not result in any additive or synergistic effect. Equally efficient osteochondral defect repair was achieved with chondrocyte, stem cell, and BMP-2 treatment. Expression of collagen X and collagen I, signs of ongoing ossification, were histologically undetectable, and the presence of aggrecan protein indicated cartilage-like tissue. In conclusion, further work should demonstrate whether spatiotemporally controlled, on-site BMP-2 release alone could become a feasible therapeutic approach to repair large osteochondral defects.

  14. Characterization of healthy and osteoarthritic chondrocyte cell patterns on phase contrast CT images of the knee cartilage matrix

    Science.gov (United States)

    Nagarajan, Mahesh B.; Coan, Paola; Huber, Markus B.; Yang, Chien-Chun; Glaser, Christian; Reiser, Maximilian F.; Wismüller, Axel

    2012-03-01

    The current approach to evaluating cartilage degeneration at the knee joint requires visualization of the joint space on radiographic images where indirect cues such as joint space narrowing serve as markers for osteoarthritis. A recent novel approach to visualizing the knee cartilage matrix using phase contrast CT imaging (PCI-CT) was shown to allow direct examination of chondrocyte cell patterns and their subsequent correlation to osteoarthritis. This study aims to characterize chondrocyte cell patterns in the radial zone of the knee cartilage matrix in the presence and absence of osteoarthritic damage through both gray-level co-occurrence matrix (GLCM) derived texture features as well as Minkowski Functionals (MF). Thirteen GLCM and three MF texture features were extracted from 404 regions of interest (ROI) annotated on PCI images of healthy and osteoarthritic specimens of knee cartilage. These texture features were then used in a machine learning task to classify ROIs as healthy or osteoarthritic. A fuzzy k-nearest neighbor classifier was used and its performance was evaluated using the area under the ROC curve (AUC). The best classification performance was observed with the MF features 'perimeter' and 'Euler characteristic' and with GLCM correlation features (f3 and f13). With the experimental conditions used in this study, both Minkowski Functionals and GLCM achieved a high classification performance (AUC value of 0.97) in the task of distinguishing between health and osteoarthritic ROIs. These results show that such quantitative analysis of chondrocyte patterns in the knee cartilage matrix can distinguish between healthy and osteoarthritic tissue with high accuracy.

  15. 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

  16. Relationship between the chondrocyte maturation cycle and the endochondral ossification in the diaphyseal and epiphyseal ossification centers.

    Science.gov (United States)

    Pazzaglia, Ugo E; Congiu, Terenzio; Sibilia, Valeria; Pagani, Francesca; Benetti, Anna; Zarattini, Guido

    2016-09-01

    The chondrocyte maturation cycle and endochondral ossification were studied in human, fetal cartilage Anlagen and in postnatal meta-epiphyses. The relationship between the lacunar area, the inter-territorial fibril network variations, and calcium phosphorus nucleation in primary and secondary ossification centers were assessed using light microscopy and scanning electron microscopy (SEM) morphometry. The Anlage topographic, zonal classification was derived from the anatomical nomenclature of the completely developed long bone (diaphysis, metaphyses and epiphyses). A significant increase in the chondrocyte lacunar area was documented in the Anlage of epiphyseal zones 4 and 3 to zone 2 (metaphysis) and zone 1 (diaphysis), with the highest variation from zone 2 to zone 1. An inverse reduction in the intercellular matrix area and matrix interfibrillar empty space was also documented. These findings are consistent with the osmotic passage of free cartilage water from the interfibrillar space into the swelling chondrocytes, which increased the ion concentrations to a critical threshold for mineral precipitation in the matrix. The mineralized cartilage served as a scaffold for osteoblast apposition both in primary and secondary ossification centers and in the metaphyseal growth plate cartilage, though at different periods of bone Anlage development and with distinct patterns for each zone. All developmental processes shared a common initial pathway but progressed at different rates, modes and organization in diaphysis, metaphysis and epiphysis. In the ossification phase the developing vascular supply appeared to play a key role in determining the cortical or trabecular structure of the long bones. J. Morphol. 277:1187-1198, 2016. © 2016 Wiley Periodicals, Inc.

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

    Directory of Open Access Journals (Sweden)

    Kürşat Oğuz Yaykaşlı

    2014-02-01

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

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

    Directory of Open Access Journals (Sweden)

    Padma P Srinivasan

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

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

    Science.gov (United States)

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

    2004-01-01

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

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

    Science.gov (United States)

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

    2016-07-01

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

  1. Stimulation of Superficial Zone Protein/Lubricin/PRG4 by Transforming Growth Factor-β in Superficial Zone Articular Chondrocytes and Modulation by Glycosaminoglycans.

    Science.gov (United States)

    Cuellar, Araceli; Reddi, A Hari

    2015-07-01

    Superficial zone protein (SZP), also known as lubricin and proteoglycan 4 (PRG4), plays an important role in the boundary lubrication of articular cartilage and is regulated by transforming growth factor (TGF)-β. Here, we evaluate the role of cell surface glycosaminoglycans (GAGs) during TGF-β1 stimulation of SZP/lubricin/PRG4 in superficial zone articular chondrocytes. We utilized primary monolayer superficial zone articular chondrocyte cultures and treated them with various concentrations of TGF-β1, in the presence or absence of heparan sulfate (HS), heparin, and chondroitin sulfate (CS). The cell surface GAGs were removed by pretreatment with either heparinase I or chondroitinase-ABC before TGF-β1 stimulation. Accumulation of SZP/lubricin/PRG4 in the culture medium in response to stimulation with TGF-β1 and various exogenous GAGs was demonstrated by immunoblotting and quantitated by enzyme-linked immunosorbent assay. We show that TGF-β1 and exogenous HS enhanced SZP accumulation of superficial zone chondrocytes in the presence of surface GAGs. At the dose of 1 ng/mL of TGF-β1, the presence of exogenous heparin inhibited SZP accumulation whereas the presence of exogenous CS stimulated SZP accumulation in the culture medium. Enzymatic depletion of GAGs on the surface of superficial zone chondrocytes enhanced the ability of TGF-β1 to stimulate SZP accumulation in the presence of both exogenous heparin and CS. Collectively, these results suggest that GAGs at the surface of superficial zone articular chondrocytes influence the response to TGF-β1 and exogenous GAGs to stimulate SZP accumulation. Cell surface GAGs modulate superficial zone chondrocytes' response to TGF-β1 and exogenous HS.

  2. Nicotine-induced retardation of chondrogenesis through down-regulation of IGF-1 signaling pathway to inhibit matrix synthesis of growth plate chondrocytes in fetal rats

    Energy Technology Data Exchange (ETDEWEB)

    Deng, Yu; Cao, Hong; Cu, Fenglong [Department of Orthopedic Surgery, Zhongnan Hospital of Wuhan University, Wuhan 430071 (China); Xu, Dan [Department of Pharmacology, Basic Medical School of Wuhan University, Wuhan 430071 (China); Research Center of Food and Drug Evaluation, Wuhan University, Wuhan 430071 (China); Lei, Youying [Department of Pharmacology, Basic Medical School of Wuhan University, Wuhan 430071 (China); Tan, Yang [Department of Orthopedic Surgery, Zhongnan Hospital of Wuhan University, Wuhan 430071 (China); Magdalou, Jacques [UMR 7561 CNRS-Nancy Université, Faculté de Médicine, Vandoeuvre-lès-Nancy (France); Wang, Hui [Department of Pharmacology, Basic Medical School of Wuhan University, Wuhan 430071 (China); Research Center of Food and Drug Evaluation, Wuhan University, Wuhan 430071 (China); Chen, Liaobin, E-mail: lbchen@whu.edu.cn [Department of Orthopedic Surgery, Zhongnan Hospital of Wuhan University, Wuhan 430071 (China)

    2013-05-15

    Previous studies have confirmed that maternal tobacco smoking causes intrauterine growth retardation (IUGR) and skeletal growth retardation. Among a multitude of chemicals associated with cigarette smoking, nicotine is one of the leading candidates for causing low birth weights. However, the possible mechanism of delayed chondrogenesis by prenatal nicotine exposure remains unclear. We investigated the effects of nicotine on fetal growth plate chondrocytes in vivo and in vitro. Rats were given 2.0 mg/kg·d of nicotine subcutaneously from gestational days 11 to 20. Prenatal nicotine exposure increased the levels of fetal blood corticosterone and resulted in fetal skeletal growth retardation. Moreover, nicotine exposure induced the inhibition of matrix synthesis and down-regulation of insulin-like growth factor 1 (IGF-1) signaling in fetal growth plates. The effects of nicotine on growth plates were studied in vitro by exposing fetal growth plate chondrocytes to 0, 1, 10, or 100 μM of nicotine for 10 days. Nicotine inhibited matrix synthesis and down-regulated IGF-1 signaling in chondrocytes in a concentration-dependent manner. These results suggest that prenatal nicotine exposure induces delayed chondrogenesis and that the mechanism may involve the down-regulation of IGF-1 signaling and the inhibition of matrix synthesis by growth plate chondrocytes. The present study aids in the characterization of delayed chondrogenesis caused by prenatal nicotine exposure, which might suggest a candidate mechanism for intrauterine origins of osteoporosis and osteoarthritis. - Highlights: ► Prenatal nicotine-exposure could induce delayed chondrogenesis in fetal rats. ► Nicotine inhibits matrix synthesis of fetal growth plate chondrocytes. ► Nicotine inhibits IGF-1 signaling pathway in fetal growth plate chondrocytes.

  3. HIF-1 α had pivotal effects on downregulation of miR-210 decreasing viability and inducing apoptosis in hypoxic chondrocytes.

    Science.gov (United States)

    Chang, Zhiqiang; Huo, Lifeng; Wu, Yimin; Zhang, Pei

    2014-01-01

    Hypoxia-inducible factor 1-alpha (HIF-1 α ) and some microRNA (miRNAs) play pivotal roles in response to hypoxia-related physiologic and pathophysiologic responses. Up to date, the regulatory mechanisms of these molecules were largely unknown in chondrocytes. In this study, to study the mechanisms of degradation and homeostasis of chondrocytes, the effects of miRNAs and HIF-1 α on chondrocytes in physiologic environment were investigated. We found that the overexpression of miR-210 and HIF-1 α was present on hypoxia in C28/I2 human chondrocytes significantly by qRT-PCR and western plot. Further study displayed that miR-210 played positive role as a promoter in regulation and its regulated molecules (bcl-xl and PHD-2) in C28/I2 cells on hypoxia by silenced miR-210, silenced HIF-1 α , and adding miR-210. Moreover, downregulated miR-210 could significantly repress the viability and increase the apoptosis in C28/I2 cells on hypoxia, compared to those on normoxia. Furthermore, miR-210 could not modulate viability and apoptosis in C28/I2 cells with the HIF-1 α knockdown on hypoxia and normoxia. Taken together, this study demonstrated that the MiR-210 was involved in an HIF-1 α -dependent way in C28/I2 human chondrocytes for the first time. It also suggested that miR-210 downregulation decreased viability and induced apoptosis in hypoxic chondrocytes depending on HIF-1 α .

  4. HIF-1α had Pivotal Effects on Downregulation of miR-210 Decreasing Viability and Inducing Apoptosis in Hypoxic Chondrocytes

    Directory of Open Access Journals (Sweden)

    Zhiqiang Chang

    2014-01-01

    Full Text Available Hypoxia-inducible factor 1-alpha (HIF-1α and some microRNA (miRNAs play pivotal roles in response to hypoxia-related physiologic and pathophysiologic responses. Up to date, the regulatory mechanisms of these molecules were largely unknown in chondrocytes. In this study, to study the mechanisms of degradation and homeostasis of chondrocytes, the effects of miRNAs and HIF-1α on chondrocytes in physiologic environment were investigated. We found that the overexpression of miR-210 and HIF-1α was present on hypoxia in C28/I2 human chondrocytes significantly by qRT-PCR and western plot. Further study displayed that miR-210 played positive role as a promoter in regulation and its regulated molecules (bcl-xl and PHD-2 in C28/I2 cells on hypoxia by silenced miR-210, silenced HIF-1α, and adding miR-210. Moreover, downregulated miR-210 could significantly repress the viability and increase the apoptosis in C28/I2 cells on hypoxia, compared to those on normoxia. Furthermore, miR-210 could not modulate viability and apoptosis in C28/I2 cells with the HIF-1α knockdown on hypoxia and normoxia. Taken together, this study demonstrated that the MiR-210 was involved in an HIF-1α-dependent way in C28/I2 human chondrocytes for the first time. It also suggested that miR-210 downregulation decreased viability and induced apoptosis in hypoxic chondrocytes depending on HIF-1α.

  5. Hydrostatic Compress Force Enhances the Viability and Decreases the Apoptosis of Condylar Chondrocytes through Integrin-FAK-ERK/PI3K Pathway

    Science.gov (United States)

    Ma, Dandan; Kou, Xiaoxing; Jin, Jing; Xu, Taotao; Wu, Mengjie; Deng, Liquan; Fu, Lusi; Liu, Yi; Wu, Gang; Lu, Haiping

    2016-01-01

    Reduced mechanical stimuli in many pathological cases, such as hemimastication and limited masticatory movements, can significantly affect the metabolic activity of mandibular condylar chondrocytes and the growth of mandibles. However, the molecular mechanisms for these phenomena remain unclear. In this study, we hypothesized that integrin-focal adhesion kinase (FAK)-ERK (extracellular signal–regulated kinase)/PI3K (phosphatidylinositol-3-kinase) signaling pathway mediated the cellular response of condylar chondrocytes to mechanical loading. Primary condylar chondrocytes were exposed to hydrostatic compressive forces (HCFs) of different magnitudes (0, 50, 100, 150, 200, and 250 kPa) for 2 h. We measured the viability, morphology, and apoptosis of the chondrocytes with different treatments as well as the gene, protein expression, and phosphorylation of mechanosensitivity-related molecules, such as integrin α2, integrin α5, integrin β1, FAK, ERK, and PI3K. HCFs could significantly increase the viability and surface area of condylar chondrocytes and decrease their apoptosis in a dose-dependent manner. HCF of 250 kPa resulted in a 1.51 ± 0.02-fold increase of cell viability and reduced the ratio of apoptotic cells from 18.10% ± 0.56% to 7.30% ± 1.43%. HCFs could significantly enhance the mRNA and protein expression of integrin α2, integrin α5, and integrin β1 in a dose-dependent manner, but not ERK1, ERK2, or PI3K. Instead, HCF could significantly increase phosphorylation levels of FAK, ERK1/2, and PI3K in a dose-dependent manner. Cilengitide, the potent integrin inhibitor, could dose-dependently block such effects of HCFs. HCFs enhances the viability and decreases the apoptosis of condylar chondrocytes through the integrin-FAK-ERK/PI3K pathway. PMID:27827993

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

    Science.gov (United States)

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

    2014-06-01

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

  7. Col2CreERT2, A MOUSE MODEL FOR A CHONDROCYTE-SPECIFIC AND INDUCIBLE GENE DELETION

    OpenAIRE

    2014-01-01

    In 2007 and 2008, we published two articles reporting a tamoxifen (TM)-inducible, chondrocyte-specific gene-targeting mouse model in which the expression of CreERT2 is driven by the type II collagen promoter (Col2CreERT2). The fusion protein is specifically expressed and translocated into the nucleus upon TM administration, which in turn triggers gene recombination. Since then, this animal model has become a powerful tool to study the molecular mechanism of skeletal development and degenerati...

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2013-02-15

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

  9. Fibrin and poly(lactic-co-glycolic acid hybrid scaffold promotes early chondrogenesis of articular chondrocytes: an in vitro study

    Directory of Open Access Journals (Sweden)

    Idrus Ruszymah BH

    2008-04-01

    Full Text Available Abstract Background Synthetic- and naturally derived- biodegradable polymers have been widely used to construct scaffolds for cartilage tissue engineering. Poly(lactic-co-glycolic acid (PLGA are bioresorbable and biocompatible, rendering them as a promising tool for clinical application. To minimize cells lost during the seeding procedure, we used the natural polymer fibrin to immobilize cells and to provide homogenous cells distribution in PLGA scaffolds. We evaluated in vitro chondrogenesis of rabbit articular chondrocytes in PLGA scaffolds using fibrin as cell transplantation matrix. Methods PLGA scaffolds were soaked in chondrocytes-fibrin suspension (1 × 106cells/scaffold and polymerized by dropping thrombin-calcium chloride (CaCl2 solution. PLGA-seeded chondrocytes was used as control. All constructs were cultured for a maximum of 21 days. Cell proliferation activity was measured at 1, 3, 7, 14 and 21 days in vitro using 3-(4,5-dimethylthiazole-2-yl-2-, 5-diphenyltetrazolium-bromide (MTT assay. Morphological observation, histology, immunohistochemistry (IHC, gene expression and sulphated-glycosaminoglycan (sGAG analyses were performed at each time point of 1, 2 and 3 weeks to elucidate in vitro cartilage development and deposition of cartilage-specific extracellular matrix (ECM. Results Cell proliferation activity was gradually increased from day-1 until day-14 and declined by day-21. A significant cartilaginous tissue formation was detected as early as 2-week in fibrin/PLGA hybrid construct as confirmed by the presence of cartilage-isolated cells and lacunae embedded within basophilic ECM. Cartilage formation was remarkably evidenced after 3 weeks. Presence of cartilage-specific proteoglycan and glycosaminoglycan (GAG in fibrin/PLGA hybrid constructs were confirmed by positive Safranin O and Alcian Blue staining. Collagen type II exhibited intense immunopositivity at the pericellular matrix. Chondrogenic properties were further

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

    Directory of Open Access Journals (Sweden)

    Hanaoka Teruyasu

    2011-08-01

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

  11. 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.

  12. Co-administration of hyaluronic acid with local anaesthetics shows lower cytotoxicity than local anaesthetic treatment alone in bovine articular chondrocytes

    OpenAIRE

    Onur, T. S.; Sitron, C. S.; Dang, A

    2013-01-01

    Objective To study the effect of hyaluronic acid (HA) on local anaesthetic chondrotoxicity in vitro. Methods Chondrocytes were harvested from bovine femoral condyle cartilage and isolated using collagenase-containing media. At 24 hours after seeding 15 000 cells per well onto a 96-well plate, chondrocytes were treated with media (DMEM/F12 + ITS), PBS, 1:1 lidocaine (2%):PBS, 1:1 bupivacaine (0.5%):PBS, 1:1 lidocaine (2%):HA, 1:1 bupivacaine (0. 5%):HA, or 1:1 HA:PBS for one hour. Following tr...

  13. [The effect of steroidal and nonsteroidal anti-inflammatory agents on the ultrastructure of chondrocytes in the rat. Electron microscope and morphometric study].

    Science.gov (United States)

    Annefeld, M; Raiss, R; Cleres, C

    1984-01-01

    In order to obtain indications regarding the different influences of antiinflammatory drugs on the articular cartilage, the effects of a steroidal drug (dexamethasone) and of nonsteroidal antiinflammatory substances (indometacin and phenylbutazone) on the ultrastructure of the chondrocyte were examined in comparison with tiaprofenic acid (Surgam). Using an equiefficient dosage of the active principles, referred to the ED50 in the model of adjuvant arthritis in the rat, the synthetic apparatus of the chondrocytes was stimulated by tiaprofenic acid while it was diminished by indometacin and phenylbutazone and particularly by dexamethasone.

  14. Induction of increased cAMP levels in articular chondrocytes blocks matrix metalloproteinase-mediated cartilage degradation, but not aggrecanase-mediated cartilage degradation

    DEFF Research Database (Denmark)

    Karsdal, Morten Asser; Sumer, Eren Ufuk; Wulf, Helle

    2007-01-01

    was assessed by 1) quantification of C-terminal crosslinking telopeptide of type II collagen fragments (CTX-II), 2) matrix metalloproteinase (MMP)-mediated aggrecan degradation by (342)FFGV- G2 assay, 3) aggrecanase-mediated degradation by (374)ARGS-G2 assay, 4) release of sulfated glycosaminoglycans (s......-dependently inhibited by forskolin and IBMX. The highest concentration of IBMX lowered cytokine-induced release of sGAG by 72%. CONCLUSION: Levels of cAMP in chondrocytes play a key role in controlling catabolic activity. Increased cAMP levels in chondrocytes inhibited MMP expression and activity and consequently...

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

    Directory of Open Access Journals (Sweden)

    Thomas J Kean

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

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

    Science.gov (United States)

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

    2013-03-01

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

  17. Biotin-conjugated anti-CD44 antibody-avidin binding system for the improvement of chondrocyte adhesion to scaffolds.

    Science.gov (United States)

    Lin, Hong; Zhou, Jian; Shen, Longxiang; Ruan, Yuhui; Dong, Jian; Guo, Changan; Chen, Zhengrong

    2014-04-01

    The clinical need for improved treatment options for patients with cartilage injuries has motivated tissue-engineering studies aimed at the in vitro generation of cell-based implants with functional properties. The success of tissue-engineered repair of cartilage may depend on the rapid and efficient adhesion of transplanted cells to the scaffold. In the present study, chondrocyte-scaffold constructs were engineered by planting porcine chondrocytes into nonporous chitosan membranes and 3D porous chitosan scaffolds that were treated with or without biotin-conjugated anti-CD44 antibody-avidin binding system and avidin-biotin binding system. The spreading area, cell exfoliation rates, cell proliferation rates, histological analysis, DNA and glycosaminoglycan (GAG) content, and mRNA expression were investigated to evaluate the efficiency of biotin-conjugated anti-CD44 antibody-avidin binding system for the improvement of cell adhesion to scaffolds in the cartilage tissue. The results showed that the biotin-conjugated anti-CD44 antibody-avidin binding system improved cell adhesion to scaffolds effectively. These studies suggest that this binding system has the potential to provide improved tissue-engineered cartilage for clinical applications.

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

    Science.gov (United States)

    Chen, Hongwei; Tian, Yun

    2017-01-16

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

  19. Syndecan-3: a cell-surface heparan sulfate proteoglycan important for chondrocyte proliferation and function during limb skeletogenesis.

    Science.gov (United States)

    Pacifici, Maurizio; Shimo, Tsuyoshi; Gentili, Chiara; Kirsch, Thorsten; Freeman, Theresa A; Enomoto-Iwamoto, Motomi; Iwamoto, Masahiro; Koyama, Eiki

    2005-01-01

    Syndecans are single-pass integral membrane components that serve as co-receptors for growth factors and cytokines and can elicit signal transduction via their cytoplasmic tails. We review here previous studies from our groups on syndecan-3 biology and function in the growth plates of developing long bones in chick and mouse embryos. Gain- and loss-of-function data indicate that syndecan-3 has important roles in restricting mitotic activity to the proliferative zone of growth plate and may do so in close cooperation and interaction with the signaling molecule Indian hedgehog (IHH). Biochemical and protein-modeling data suggest a dimeric/oligomeric syndecan-3 configuration on the chondrocyte's cell surface. Analyses of embryos misexpressing syndecan-3 or lacking IHH provide further clues on syndecan-3/IHH interdependence and interrelationships. The data and the conclusions reached provide insights into mechanisms fine-tuning chondrocyte proliferation, maturation, and function in the developing and growing skeleton and into how abnormalities in these fundamental mechanisms may subtend human congenital pathologies, including osteochondromas in hereditary multiple exostoses syndrome.

  20. 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.

  1. The Study on Biocompatibility of Porous nHA/PLGA Composite Scaffolds for Tissue Engineering with Rabbit Chondrocytes In Vitro

    Directory of Open Access Journals (Sweden)

    Lei Chen

    2013-01-01

    Full Text Available Objective. To examine the biocompatibility of a novel nanohydroxyapatite/poly[lactic-co-glycolic acid] (nHA/PLGA composite and evaluate its feasibility as a scaffold for cartilage tissue engineering. Methods. Chondrocytes of fetal rabbit were cultured with nHA/PLGA scaffold in vitro and the cell viability was assessed by MTT assay first. Cells adhering to nHA/PLGA scaffold were then observed by inverted microscope and scanning electron microscope (SEM. The cell cycle profile was analyzed by flow cytometry. Results. The viability of the chondrocytes on the scaffold was not affected by nHA/PLGA comparing with the control group as it was shown by MTT assay. Cells on the surface and in the pores of the scaffold increased in a time-dependent manner. Results obtained from flow cytometry showed that there was no significant difference in cell cycle profiles between the coculture group and control (P>0.05. Conclusion. The porous nHA/PLGA composite scaffold is a biocompatible and good kind of scaffold for cartilage tissue engineering.

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

    Science.gov (United States)

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

    2014-10-03

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

  3. Biological Knee Reconstruction With Concomitant Autologous Chondrocyte Implantation and Meniscal Allograft Transplantation

    Science.gov (United States)

    Ogura, Takahiro; Bryant, Tim; Minas, Tom

    2016-01-01

    Background: Treating articular cartilage defects and meniscal deficiency is challenging. Although some short- to mid-term follow-up studies report good clinical outcomes after concurrent autologous chondrocyte implantation (ACI) and meniscal allograft transplantation (MAT), longer follow-up is needed. Purpose: To evaluate mid- to long-term outcomes after combined ACI with MAT. Study Design: Case series; Level of evidence, 4. Methods: We performed a retrospective review of prospectively gathered data from patients who had undergone ACI with MAT between 1999 and 2013. A single surgeon treated 18 patients for symptomatic full-thickness chondral defects with meniscal deficiency. One patient was lost to follow-up. Thus, 17 patients (18 knees; mean age, 31.7 years) were evaluated over a mean 7.9-year follow-up (range, 2-16 years). A mean 1.8 lesions per knee were treated over a total surface area of 7.6 cm2 (range, 2.3-21 cm2) per knee. Seventeen lateral and 1 medial MATs were performed. Survival was analyzed using the Kaplan-Meier method. The modified Cincinnati Knee Rating Scale, Western Ontario and McMaster Universities Osteoarthritis Index, visual analog scale, and Short Form–36 were used to evaluate clinical outcomes. Patients also self-reported knee function and satisfaction. Standard radiographs were scored for Kellgren-Lawrence (K-L) grade. Results: Both 5- and 10-year survival rates were 75%. Outcomes for 6 knees were considered failures. Of the 6 failures, 4 knees were converted to arthroplasty and the other 2 knees underwent biological revision surgery. Of the 12 successfully operated knees, all clinical measures significantly improved postoperatively. Ten patients representing 11 of the 12 knees rated outcomes for their knees as good or excellent, and 1 rated their outcome as fair. Eight patients representing 9 of the 12 knees were satisfied with the procedure. There was no significant osteoarthritis progression based on K-L grading from preoperatively to a

  4. Intermediate- to Long-Term Results of Combined Anterior Cruciate Ligament Reconstruction and Autologous Chondrocyte Implantation

    Science.gov (United States)

    Pike, Andrew N.; Bryant, Tim; Ogura, Takahiro; Minas, Tom

    2017-01-01

    Background: Cartilage injury associated with anterior cruciate ligament (ACL) ruptures is common; however, relatively few reports exist on concurrent cartilage repair with ACL reconstruction. Autologous chondrocyte implantation (ACI) has been utilized successfully for treatment of moderate to large chondral defects. Hypothesis: ACL insufficiency with relatively large chondral defects may be effectively managed with concurrent ACL reconstruction and ACI. Study Design: Case series; Level of evidence, 4. Methods: Patients undergoing concurrent ACL primary or revision reconstruction with ACI of single or multiple cartilage defects were prospectively evaluated for a minimum 2 years. Pre- and postoperative outcome measures included the modified Cincinnati Rating Scale (MCRS), Western Ontario and McMaster Universities Osteoarthritis Index, visual analog pain scales, and postsurgery satisfaction surveys. ACI graft failure or persistent pain without functional improvement were considered treatment failures. Results: Twenty-six patients were included, with 13 primary and 13 revision ACL reconstructions performed. Mean defect total surface area was 8.4 cm2, with a mean follow-up of 95 months (range, 24-240 months). MCRS improved from 3.62 ± 1.42 to 5.54 ± 2.32, Western Ontario and McMaster Universities Osteoarthritis Index from 45.31 ± 17.27 to 26.54 ± 17.71, and visual analog pain scale from 6.19 ± 1.27 to 3.65 ± 1.77 (all Ps <.001). Eight patients were clinical failures, 69% of patients were improved at final follow-up, and 92% stated they would likely undergo the procedure again. No outcome correlation was found with regard to age, body mass index, sex, defect size/number, follow-up time, or primary versus revision ACL reconstruction. In subanalysis, revision ACL reconstructions had worse preoperative MCRS scores and greater defect surface areas. However, revision MCRS score improvements were greater, resulting in similar final functional scores when compared with

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

    Science.gov (United States)

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

    2011-11-01

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

  6. Low-Frequency High-Magnitude Mechanical Strain of Articular Chondrocytes Activates p38 MAPK and Induces Phenotypic Changes Associated with Osteoarthritis and Pain

    Directory of Open Access Journals (Sweden)

    Derek H. Rosenzweig

    2014-08-01

    Full Text Available Osteoarthritis (OA is a debilitating joint disorder resulting from an incompletely understood combination of mechanical, biological, and biochemical processes. OA is often accompanied by inflammation and pain, whereby cytokines associated with chronic OA can up-regulate expression of neurotrophic factors such as nerve growth factor (NGF. Several studies suggest a role for cytokines and NGF in OA pain, however the effects of changing mechanical properties in OA tissue on chondrocyte metabolism remain unclear. Here, we used high-extension silicone rubber membranes to examine if high mechanical strain (HMS of primary articular chondrocytes increases inflammatory gene expression and promotes neurotrophic factor release. HMS cultured chondrocytes displayed up-regulated NGF, TNFα and ADAMTS4 gene expression while decreasing TLR2 expression, as compared to static controls. HMS culture increased p38 MAPK activity compared to static controls. Conditioned medium from HMS dynamic cultures, but not static cultures, induced significant neurite sprouting in PC12 cells. The increased neurite sprouting was accompanied by consistent increases in PC12 cell death. Low-frequency high-magnitude mechanical strain of primary articular chondrocytes in vitro drives factor secretion associated with degenerative joint disease and joint pain. This study provides evidence for a direct link between cellular strain, secretory factors, neo-innervation, and pain in OA pathology.

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

    Science.gov (United States)

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

    2016-04-01

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

  8. Gel structure has an impact on pericellular and extracellular matrix deposition, which subsequently alters metabolic activities in chondrocyte-laden PEG hydrogels.

    Science.gov (United States)

    Nicodemus, G D; Skaalure, S C; Bryant, S J

    2011-02-01

    While designing poly(ethylene glycol) hydrogels with high moduli suitable for in situ placement is attractive for cartilage regeneration, the impact of a tighter crosslinked structure on the organization and deposition of the matrix is not fully understood. The objectives of this study were to characterize the composition and spatial organization of new matrix as a function of gel crosslinking and study its impact on chondrocytes in terms of anabolic and catabolic gene expression and catabolic activity. Bovine articular chondrocytes were encapsulated in hydrogels with three crosslinking densities (compressive moduli 60, 320 and 590 kPa) and cultured for 25 days. Glycosaminoglycan production increased with culture time and was greatest in the gels with lowest crosslinking. Collagens II and VI, aggrecan, link protein and decorin were localized to pericellular regions in all gels, but their presence decreased with increasing gel crosslinking. Collagen II and aggrecan expression were initially up-regulated in gels with higher crosslinking, but increased similarly up to day 15. Matrix metalloproteinase (MMP)-1 and MMP-13 expression were elevated (∼25-fold) in gels with higher crosslinking throughout the study, while MMP-3 was unaffected by gel crosslinking. The presence of aggrecan and collagen degradation products confirmed MMP activity. These findings indicate that chondrocytes synthesized the major cartilage components within PEG hydrogels, however, gel structure had a significant impact on the composition and spatial organization of the new tissue and on how chondrocytes responded to their environment, particularly with respect to their catabolic expression.

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

    DEFF Research Database (Denmark)

    Harkness, Linda; Taipaleenmaki, Hanna; Mahmood, Amer

    2009-01-01

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

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

    NARCIS (Netherlands)

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

    2012-01-01

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

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

    Science.gov (United States)

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

    2015-02-01

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

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

    Science.gov (United States)

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

    2016-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Bo Qiu

    2016-01-01

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

  14. Sp1 upregulates the proximal promoter activity of the mouse collagen α1(XI) gene (Col11a1) in chondrocytes.

    Science.gov (United States)

    Watanabe, Keijirou; Hida, Mariko; Sasaki, Takako; Yano, Hiroyuki; Kawano, Kenji; Yoshioka, Hidekatsu; Matsuo, Noritaka

    2016-02-01

    Type XI collagen is a cartilage-specific extracellular matrix, and is important for collagen fibril formation and skeletal morphogenesis. We have previously reported that NF-Y regulated the proximal promoter activity of the mouse collagen α1(XI) gene (Col11a1) in chondrocytes (Hida et. al. In Vitro Cell. Dev. Biol. Anim. 2014). However, the mechanism of the Col11a1 gene regulation in chondrocytes has not been fully elucidated. In this study, we further characterized the proximal promoter activity of the mouse Col11a1 gene in chondrocytes. Cell transfection experiments with deletion and mutation constructs indicated that the downstream region of the NF-Y binding site (-116 to +1) is also necessary to regulate the proximal promoter activity of the mouse Col11a1 gene. This minimal promoter region has no TATA box and GC-rich sequence; we therefore examined whether the GC-rich sequence (-96 to -67) is necessary for the transcription regulation of the Col11a1 gene. Luciferase assays using a series of mutation constructs exhibited that the GC-rich sequence is a critical element of Col11a1 promoter activity in chondrocytes. Moreover, in silico analysis of this region suggested that one of the most effective candidates was transcription factor Sp1. Consistent with the prediction, overexpression of Sp1 significantly increased the promoter activity. Furthermore, knockdown of Sp1 expression by siRNA transfection suppressed the proximal promoter activity and the expression of endogenous transcript of the mouse Col11a1 gene. Taken together, these results indicate that the transcription factor Sp1 upregulates the proximal promoter activity of the mouse Col11a1 gene in chondrocytes.

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

    Science.gov (United States)

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

    2004-01-01

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

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

    Science.gov (United States)

    Qiu, Bo; Gong, Ming; He, Qi-Ting

    2016-01-01

    This paper investigates the protective effect of interleukin-1 receptor antagonist (IL-1Ra) released from hyaluronic acid chitosan (HA-CS) microspheres in a controlled manner on IL-1β-induced inflammation and apoptosis in chondrocytes. The IL-1Ra release kinetics was characterized by an initial burst release, which was reduced to a linear release over eight days. Chondrocytes were stimulated with 10 ng/ml IL-1β and subsequently incubated with HA-CS-IL-1Ra microspheres. The cell viability was decreased by IL-1β, which was attenuated by HA-CS-IL-1Ra microspheres as indicated by an MTT assay. ELISA showed that HA-CS-IL-1Ra microspheres inhibited IL-1β-induced inflammation by attenuating increases in NO2− and prostaglandin E2 levels as well as increase in glycosaminoglycan release. A terminal deoxyribonucleotide transferase deoxyuridine triphosphate nick-end labeling assay revealed that the IL-1β-induced chondrocyte apoptosis was decreased by HA-CS-IL-1Ra microspheres. Moreover, HA-CS-IL-1Ra microspheres blocked IL-1β-induced chondrocyte apoptosis by increasing B-cell lymphoma 2 (Bcl-2) and decreasing Bcl-2-associated X protein and caspase-3 expressions at mRNA and protein levels, as indicated by reverse-transcription quantitative polymerase chain reaction and western blot analysis, respectively. The results of the present study indicated that HA-CS-IL-1Ra microspheres as a controlled release system of IL-1Ra possess potential anti-inflammatory and antiapoptotic properties in rat chondrocytes due to their ability to regulate inflammatory factors and apoptosis associated genes. PMID:27872853

  17. Resveratrol protects bone marrow mesenchymal stem cell derived chondrocytes cultured on chitosan-gelatin scaffolds from the inhibitory effect of interleukin-1β

    Institute of Scientific and Technical Information of China (English)

    Ming LEI; Shi-qing LIU; Yu-lan LIU

    2008-01-01

    Aim: To investigate the effects of resveratrol on interleukin-lbeta (IL-1β) induced catabolism in bone marrow mesenchymal stem cell (MSC) derived chon-drocytes cultured on chitosan-gelatin scaffolds (CGS). Methods: The chondro-genesis of alginate-encapsulated MSCs was evaluated by toluidine blue staining, RT-PCR, and immunostaing. MSC-derived chondrocyte morphology cultured on CGS was evaluated by a scanning electron microscope (SEM) and a laser confocal microscope (LCM). When these cells on CGS were pre-stimulated with IL-1β or co-treated with IL-1β and resveratrol in the absence and presence of the specific β1-integrin blocking antibody, collagen type Ⅱ, aggrecan, matrix metalloproteinase-13 (MMP-13) expression, and the translocation of nuclear factor kappaB (NF-κB) were analyzed by Western blot analysis. Results: Transforming growth factor beta 3 (TGF-β3) combined with insulin-like growth factor Ⅰ (IGF-Ⅰ) induced the cartilage-specific collagen type Ⅱ, aggrecan expression and extracellular matrix (ECM) accumulation at the end of a 3-week culture. CGS supported those differentiated chondrocytes' attachment, proliferation, migration, and ECM formation. When those cells cultured on CGS were stimulated with IL-1β alone, collagen type Ⅱ and aggrecan expression was inhibited. However, MMP-13 expression increased. Resveratrol reversed the catabolic effects by reducing the translocation of NF-κB. A specific β1-integrin blocking antibody abrogated the effects of resveratrol on IL-1β stimulated MSC-derived chondrocytes. Conclusion: These results indicated that resveratrol acta as a NF-κB inhibitor to protect MSC-derived chondrocytes on the CGS from the IL-1β catabolism and these effects are mediated by β1-integrin.

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

    Directory of Open Access Journals (Sweden)

    Anna Santoro

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

  19. Col2CreERT2, a mouse model for a chondrocyte-specific and inducible gene deletion

    Directory of Open Access Journals (Sweden)

    M Chen

    2014-10-01

    Full Text Available In 2007 and 2008, we published two articles reporting a tamoxifen (TM-inducible, chondrocyte-specific gene-targeting mouse model in which the expression of CreERT2 is driven by the type II collagen promoter (Col2CreERT2. The fusion protein is specifically expressed and translocated into the nucleus upon TM administration, which in turn triggers gene recombination. Since then, this animal model has become a powerful tool to study the molecular mechanism of skeletal development and degenerative cartilage diseases, including knee joint osteoarthritis (OA, temporomandibular joint (TMJ OA, and intervertebral disc (IVD degeneration. In this review article, we summarise the application of Col2CreERT2 mice and discuss the potential usage of this animal model in a broad spectrum of cartilage development and molecular pathology studies.

  20. Ionizing Radiation Induces Cellular Senescence of Articular Chondrocytes via Negative Regulation of SIRT1 by p38 Kinase

    Energy Technology Data Exchange (ETDEWEB)

    Hong, Eun Hee; Hwang, Sang Gu [Korea Institute of Radiological and Medical Sciences, Seoul (Korea, Republic of)

    2009-05-15

    Senescent cells exhibit irreversible growth arrest, large flat morphology, and up-regulated senescence-associated {beta}-galactosidase activity at pH 6.0. Several conditions, including oncogenic stress, oxidative stress, and DNA damage are associated with cellular senescence. Massive acute DNA double-strand breaks occurring as a result of mechanical and chemical stress can be repaired, but some DNA damage persists, eventually triggering premature senescence. Since ionizing radiation directly induces DBS, it is possible that cellular senescence is activated under these conditions. The biological events in chondrocytes following irradiation are poorly understood, and limited information is available on the molecular signal transduction mechanisms of cellular senescence at present. In this study, we identify SIRT1 as a target molecule of p38 kinase and demonstrate that the interactions between p38 kinase and SIRT1 protein play an important role in the regulation of cellular senescence in response to IR.

  1. Long-term T2 and Qualitative MRI Morphology After First-Generation Knee Autologous Chondrocyte Implantation

    DEFF Research Database (Denmark)

    Salzmann, Gian M; Erdle, Benjamin; Porichis, Stella;

    2014-01-01

    significantly correlated with the mKOSS (P quantitative imaging data and clinical function. Qualitative imaging data are much better correlated to functional outcomes.......BACKGROUND: There are several reports on long-term clinical outcomes after autologous chondrocyte implantation (ACI) for knee cartilage defect treatment. Few published articles have evaluated defect quality using quantitative magnetic resonance (MR) imaging techniques. PURPOSE: To evaluate clinical...... outcomes and the quality of repair tissue (RT) after first-generation periosteum-covered ACI (ACI-P) using qualitative MR outcomes and T2-weighted relaxation times. STUDY DESIGN: Case series; Level of evidence, 4. METHODS: All patients (n = 86) who underwent knee joint ACI-P (from 1997 through 2001...

  2. Evaluation of chondrocyte growth in the highly porous scaffolds made by fused deposition manufacturing (FDM) filled with type II collagen.

    Science.gov (United States)

    Yen, Hung-Jen; Tseng, Ching-Shiow; Hsu, Shan-Hui; Tsai, Ching-Lin

    2009-06-01

    Highly porous poly(D,L-lactide-co-glycolide) (PLGA) scaffolds for cartilage tissue engineering were fabricated in this study using the fused deposition manufacturing (FDM) process and were further modified by type II collagen. The average molecular weight of PLGA decreased to about 60% of the original value after the melt-extrusion process. Type II collagen exhibited sponge-like structure and filled the macroporous FDM scaffolds. An increase of the fiber spacing resulted in an increase of the porosity. The storage modulus of FDM scaffolds with a large fiber spacing was comparable to that of the native porcine articular cartilage. Although the FDM hybrid scaffolds were swollen in various extents after 28 days of in vitro culture, the seeded chondrocytes were well distributed in the interior of the scaffolds with a large fiber spacing and neocartilage was formed around the scaffolds. The study also suggested that a low processing temperature may be required to produce PLGA precision scaffolds using FDM.

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

    Science.gov (United States)

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

    2007-07-01

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

  4. Sclareol exerts anti-osteoarthritic activities in interleukin-1β-induced rabbit chondrocytes and a rabbit osteoarthritis model.

    Science.gov (United States)

    Zhong, Ying; Huang, Yi; Santoso, Marcel B; Wu, Li-Dong

    2015-01-01

    Sclareol is a natural product initially isolated form Salvia sclarea which possesses immune-regulation and anti-inflammatory activities. However, the anti-osteoarthritic properties of sclareol have not been investigated. The present study is aimed at evaluating the potential effects of sclareol in interleukin-1β (IL-1β)-induced rabbit chondrocytes as well as an experimental rabbit knee osteoarthritis model induced by anterior cruciate ligament transection (ACLT). Cultured rabbit chondrocytes were pretreated with 1, 5 and 10 μg/mL sclareol for 1 h and followed by stimulation of IL-1β (10 ng/mL) for 24 h. Gene expression of matrix metalloproteinase-1 (MMP-1), MMP-3, MMP-13, tissue inhibitors of metalloproteinase-1 (TIMP-1), inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX)-2 was determined by quantitative real-time polymerase chain reaction (qRT-PCR). MMP-3, TIMP-1, iNOS and COX-2 proteins were measured by Western blotting. Enzyme-linked immunosorbent assay (ELISA) was applied for nitric oxide (NO) and prostaglandin E2 (PGE2) assessment. For the in vivo study, rabbits received six weekly 0.3 mL sclareol (10 μg/mL) intra-articular injections in the knees four weeks after ACLT surgery. Cartilage was harvested for measurement of MMP-1, MMP-3, MMP-13, TIMP-1, iNOS and COX-2 by qRT-PCR, while femoral condyles were used for histological evaluation. The in vitro results we obtained showed that sclareol inhibited the MMPs, iNOS and COX-2 expression on mRNA and protein levels, while increased the TIMP-1 expression. And over-production of NO and PGE2 was also suppressed. For the in vivo study, both qRT-PCR results and histological evaluation confirmed that sclareol ameliorated cartilage degradation. Hence, we speculated that sclareol may be an ideal approach for treating osteoarthritis.

  5. Study on the shape, phenotype and intercellular communication of chondrocyte%软骨细胞形态、表型与胞间通讯的研究

    Institute of Scientific and Technical Information of China (English)

    张文涛; 卢世璧

    2004-01-01

    BACKGROUND: The chondrocytes cultured in vitro will grow in a thin layer and lose their pheootype, assuming fibroblast. There is no report about the relationship between the phenotype and intercellular communication of chondrocytes.OBJECTIVE: To study the proliferation, matrix synthesis, relationship between fluorescence stain intake and gap junction of various form chondrocytes cultured in vitro.DESIGN: An experimental study based on diagnosis was conducted.SETTING and PARTICIPANTS: The experiment was conducted in the Institute of Orthopaedics, and Institute of Basic Sciences, General Hospital of PLA. The subjects were rabbit joint cartilages and human femoral head cartilages.INTERVENTIONS: Rabbit chondrocytes cultured in vitro were stained with hematoxylin-eosin(HE) and light green-safranin O respectively. The chondrocyte' s size was measured with microscope and the intracellular fluorescence intensity was measured with confocal laser microscope. The fluorescence stain in chondrocyte was quenched with laser.MAIN OUTCOME MEASURES: Appearance, size and intracellular fluorescence intensity of chondrocytes.RESULTS: The chondrocytes became larger and lost their spherical form.Then their proliferation was accelerated. The average projection area was 1755.1 μm2 with the difference of 50 times. The matrix synthesis and gap junction disappeared. The intake of CFDA-AM increased in spherical chondrocytes(average 2057/30 cells) compared with the decreased intake in flat chondrocytes(average 80/30 cells) . There was intercellular communication in spherical rabbit chondrocytes in vitro and human chondrocytes in lacuna of cartilage germinal layer.CONCLUSION: There are certain relationships among the density, form,phenotype and gap junction in chondrocytes. The phenotype may be determined by chondrocytes form.%背景:在体外培养时软骨细胞会像成纤维细胞样成片生长并失去表型,软骨细胞胞间通讯与细胞表型的关系尚无报告.目的:了解体外

  6. Antioxidant effects of betulin on porcine chondrocyte behavior in gelatin/C6S/C4S/HA modified tricopolymer scaffold

    Energy Technology Data Exchange (ETDEWEB)

    Lin, Wen-Yang; Lin, Feng-Huei [Institute of Biomedical Engineering, National Taiwan University, Taipei, Taiwan (China); Sadhasivam, S., E-mail: rahulsbio@yahoo.co.in [Institute of Biomedical Engineering, National Taiwan University, Taipei, Taiwan (China); Savitha, S. [Institute of Biomedical Engineering, National Taiwan University, Taipei, Taiwan (China)

    2010-05-10

    The antioxidant effects of betulin on porcine chondrocytes cultured in gelatin/C6S/C4S/HA modified tricopolymer scaffold for a period of 4 weeks was investigated. The porous structure of the scaffold and cell attachment was observed by scanning electron microscopy (SEM). Biochemical measures of necrosis, cell proliferation, sulfated glycosaminoglycans (sGAG) content and extracellular matrix related gene expressions were quantitatively evaluated. The cell proliferation data showed good cellular viability in tricopolymer scaffold and increased optical density for total DNA demonstrated that the cells continued to proliferate inside the scaffold. The sGAG production indicated chondrogenic differentiation. Chondrocytes treated with betulin expressed transcripts encoding type II collagen, aggrecan, and decorin. To conclude, the substantiated results supported cell proliferation, production of extracellular matrix proteins and down-regulation of matrix metalloproteases and cytokine, in betulin treated scaffolds.

  7. Chondrocyte deformations as a function of tibiofemoral joint loading predicted by a generalized high-throughput pipeline of multi-scale simulations.

    Directory of Open Access Journals (Sweden)

    Scott C Sibole

    Full Text Available Cells of the musculoskeletal system are known to respond to mechanical loading and chondrocytes within the cartilage are not an exception. However, understanding how joint level loads relate to cell level deformations, e.g. in the cartilage, is not a straightforward task. In this study, a multi-scale analysis pipeline was implemented to post-process the results of a macro-scale finite element (FE tibiofemoral joint model to provide joint mechanics based displacement boundary conditions to micro-scale cellular FE models of the cartilage, for the purpose of characterizing chondrocyte deformations in relation to tibiofemoral joint loading. It was possible to identify the load distribution within the knee among its tissue structures and ultimately within the cartilage among its extracellular matrix, pericellular environment and resident chondrocytes. Various cellular deformation metrics (aspect ratio change, volumetric strain, cellular effective strain and maximum shear strain were calculated. To illustrate further utility of this multi-scale modeling pipeline, two micro-scale cartilage constructs were considered: an idealized single cell at the centroid of a 100×100×100 μm block commonly used in past research studies, and an anatomically based (11 cell model of the same volume representation of the middle zone of tibiofemoral cartilage. In both cases, chondrocytes experienced amplified deformations compared to those at the macro-scale, predicted by simulating one body weight compressive loading on the tibiofemoral joint. In the 11 cell case, all cells experienced less deformation than the single cell case, and also exhibited a larger variance in deformation compared to other cells residing in the same block. The coupling method proved to be highly scalable due to micro-scale model independence that allowed for exploitation of distributed memory computing architecture. The method's generalized nature also allows for substitution of any macro

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

    DEFF Research Database (Denmark)

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

    2016-01-01

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

  9. Methods for Producing Scaffold-Free Engineered Cartilage Sheets from Auricular and Articular Chondrocyte Cell Sources and Attachment to Porous Tantalum

    OpenAIRE

    Whitney, G. Adam; Mera, Hisashi; Weidenbecher, Mark; Awadallah, Amad; Mansour, Joseph M.; Dennis, James E.

    2012-01-01

    Abstract Scaffold-free cartilage engineering techniques may provide a simple alternative to traditional methods employing scaffolds. We previously reported auricular chondrocyte-derived constructs for use in an engineered trachea model; however, the construct generation methods were not reported in detail. In this study, methods for cartilage construct generation from auricular and articular cell sources are described in detail, and the resulting constructs are compared for use in a joint res...

  10. Chondrocyte outgrowth into a gelatin scaffold in a single impact load model of damage/repair – effect of BMP-2

    Directory of Open Access Journals (Sweden)

    Vincent Thea

    2007-12-01

    Full Text Available Abstract Background Articular cartilage has little capacity for repair in vivo, however, a small number of studies have shown that, in vitro, a damage/repair response can be induced. Recent work by our group has shown that cartilage can respond to single impact load and culture by producing repair cells on the articular surface. The purpose of this study was to identify whether chondrocyte outgrowth into a 3D scaffold could be observed following single impact load and culture. The effect of bone morphogenic-2 (BMP-2 on this process was investigated. Methods Cartilage explants were single impact loaded, placed within a scaffold and cultured for up to 20 days +/- BMP-2. Cell numbers in the scaffold, on and extruding from the articular surface were quantified and the immunohistochemistry used to identify the cellular phenotype. Results Following single impact load and culture, chondrocytes were observed in a 3D gelatin scaffold under all culture conditions. Chondrocytes were also observed on the articular surface of the cartilage and extruding out of the parent cartilage and on to the cartilage surface. BMP-2 was demonstrated to quantitatively inhibit these events. Conclusion These studies demonstrate that articular chondrocytes can be stimulated to migrate out of parent cartilage following single impact load and culture. The addition of BMP-2 to the culture medium quantitatively reduced the repair response. It may be that the inhibitory effect of BMP-2 in this experimental model provides a clue to the apparent inability of articular cartilage to heal itself following damage in vivo.

  11. Effect of Transforming Growth Factor Beta (TGF Beta) and Vitamin D3 Metabolites on Protein Kinase C Mediated Signal Transduction in Rat Costochondral Chondrocyte Cultures.

    Science.gov (United States)

    1995-05-01

    that catalyze the transfer of phosphate groups from one compound to another ( Lehninger , 1982). Protein kinase C (PKC) has been intensively studied...metabolism ( Lehninger , 1982). In vitamin D deficient chicks the growth zone of the cartilage fails to mineralize. When given vitamin D (Atkin et al., 1985...chondrocyte cultures. Calcif. Tissue Int., 47:230-236. Lehninger , A.L. (1982) In: Principles of Biochemistry. New York: Worth Publishers, pp.377- 380

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

    Directory of Open Access Journals (Sweden)

    Naila Rasheed

    2016-01-01

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

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

    Science.gov (United States)

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

    2014-02-01

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

  14. Chondrogenic Potency Analyses of Donor-Matched Chondrocytes and Mesenchymal Stem Cells Derived from Bone Marrow, Infrapatellar Fat Pad, and Subcutaneous Fat

    Directory of Open Access Journals (Sweden)

    John Garcia

    2016-01-01

    Full Text Available Autologous chondrocyte implantation (ACI is a cell-based therapy that has been used clinically for over 20 years to treat cartilage injuries more efficiently in order to negate or delay the need for joint replacement surgery. In this time, very little has changed in the ACI procedure, but now many centres are considering or using alternative cell sources for cartilage repair, in particular mesenchymal stem cells (MSCs. In this study, we have tested the chondrogenic potential of donor-matched MSCs derived from bone marrow (BM, infrapatellar fat pad (FP, and subcutaneous fat (SCF, compared to chondrocytes. We have confirmed that there is a chondrogenic potency hierarchy ranging across these cell types, with the most potent being chondrocytes, followed by FP-MSCs, BM-MSCs, and lastly SCF-MSCs. We have also examined gene expression and surface marker profiles in a predictive model to identify cells with enhanced chondrogenic potential. In doing so, we have shown that Sox-9, Alk-1, and Coll X expressions, as well as immunopositivity for CD49c and CD39, have predictive value for all of the cell types tested in indicating chondrogenic potency. The findings from this study have significant clinical implications for the refinement and development of novel cell-based cartilage repair strategies.

  15. Chondrogenic Potency Analyses of Donor-Matched Chondrocytes and Mesenchymal Stem Cells Derived from Bone Marrow, Infrapatellar Fat Pad, and Subcutaneous Fat

    Science.gov (United States)

    Garcia, John; McCarthy, Helen S.; Roberts, Sally; Richardson, James B.

    2016-01-01

    Autologous chondrocyte implantation (ACI) is a cell-based therapy that has been used clinically for over 20 years to treat cartilage injuries more efficiently in order to negate or delay the need for joint replacement surgery. In this time, very little has changed in the ACI procedure, but now many centres are considering or using alternative cell sources for cartilage repair, in particular mesenchymal stem cells (MSCs). In this study, we have tested the chondrogenic potential of donor-matched MSCs derived from bone marrow (BM), infrapatellar fat pad (FP), and subcutaneous fat (SCF), compared to chondrocytes. We have confirmed that there is a chondrogenic potency hierarchy ranging across these cell types, with the most potent being chondrocytes, followed by FP-MSCs, BM-MSCs, and lastly SCF-MSCs. We have also examined gene expression and surface marker profiles in a predictive model to identify cells with enhanced chondrogenic potential. In doing so, we have shown that Sox-9, Alk-1, and Coll X expressions, as well as immunopositivity for CD49c and CD39, have predictive value for all of the cell types tested in indicating chondrogenic potency. The findings from this study have significant clinical implications for the refinement and development of novel cell-based cartilage repair strategies. PMID:27781068

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

    Science.gov (United States)

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

    2016-01-01

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

  17. Evc works in chondrocytes and osteoblasts to regulate multiple aspects of growth plate development in the appendicular skeleton and cranial base.

    Science.gov (United States)

    Pacheco, María; Valencia, María; Caparrós-Martín, José A; Mulero, Francisca; Goodship, Judith A; Ruiz-Perez, Victor L

    2012-01-01

    Ellis-van Creveld syndrome protein homolog (Evc) was previously shown to mediate expression of Indian hedgehog (Ihh) downstream targets in chondrocytes. Consequently disruption of the Ihh/Pthrp axis was demonstrated in Evc(-/-) mice, but the full extent of Evc involvement in endochondral development was not totally characterized. Herein we have examined further the Evc(-/-) growth plate in a homogeneous genetic background and show that Evc promotes chondrocyte proliferation, chondrocyte hypertrophy and the differentiation of osteoblasts in the perichondrium, hence implicating Evc in both Pthrp-dependent and Pthrp-independent Ihh functions. We also demonstrate that Evc, which localizes to osteoblast primary cilia, mediates Hedgehog (Hh) signaling in the osteoblast lineage. In spite of this, bone collar development is mildly affected in Evc(-/-) mutants. The onset of perichondrial osteoblastogenesis is delayed at the initial stages of endochondral ossification in Evc(-/-) mice, and in later stages, the leading edge of expression of osteoblast markers and Wnt/β-catenin signaling components is located closer to the primary spongiosa in the Evc(-/-) perichondrium owing to impaired osteoblast differentiation. Additionally we have used Ptch1-LacZ reporter mice to learn about the different types of Hh-responsive cells that are present in the perichondrium of normal and Evc(-/-) mice. Evc mediates Hh target gene expression in inner perichondrial cells, but it is dispensable in the external layers of the perichondrium. Finally, we report cranial base defects in Evc(-/-) mice and reveal that Evc is essential for intrasphenoidal synchondrosis development.

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

    Science.gov (United States)

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

    2009-09-01

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

  19. 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.

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

    Directory of Open Access Journals (Sweden)

    Haqqi Tariq M

    2011-08-01

    Full Text Available Abstract Background Conventional treatments for the articular diseases are often effective for symptom relief, but can also cause significant side effects and do not slow the progression of the disease. Several natural substances have been shown to be effective at relieving the symptoms of osteoarthritis (OA, and preliminary evidence suggests that some of these compounds may exert a favorable influence on the course of the disease. The objective of this study was to investigate the anti-inflammatory/chondroprotective potential of a Herbal and amino acid mixture containing extract of the Uncaria tomentosa, Boswellia spp., Lepidium meyenii and L-Leucine on the IL-1β-induced production of nitric oxide (NO, glycosaminoglycan (GAG, matrix metalloproteinases (MMPs, aggrecan (ACAN and type II collagen (COL2A1 in human OA chondrocytes and OA cartilage explants. Methods Primary OA chondrocytes or OA cartilage explants were pretreated with Herbal-Leucine mixture (HLM, 1-10 μg/ml and then stimulated with IL-1β (5 ng/ml. Effect of HLM on IL-1β-induced gene expression of iNOS, MMP-9, MMP-13, ACAN and COL2A1 was verified by real time-PCR. Estimation of NO and GAG release in culture supernatant was done using commercially available kits. Results HLM tested in these in vitro studies was found to be an effective anti-inflammatory agent, as evidenced by strong inhibition of iNOS, MMP-9 and MMP-13 expression and NO production in IL-1β-stimulated OA chondrocytes (p Leucine mixture (HLM up-regulation of ACAN and COL2A1 expression in IL-1β-stimulated OA chondrocytes was also noted (p Conclusion Our data suggests that HLM could be chondroprotective and anti-inflammatory agent in arthritis, switching chondrocyte gene expression from catabolic direction towards anabolic and regenerative, and consequently this approach may be potentially useful as a new adjunct therapeutic/preventive agent for OA or injury recovery.

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

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

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

  2. T-2毒素致胎儿软骨细胞凋亡的观察%Effect of T-2 toxin on apoptosis of fetus chondrocytes

    Institute of Scientific and Technical Information of China (English)

    杨天府; 贾志强; 沈彬

    2001-01-01

    目的 探讨T-2毒素与软骨细 胞凋亡的关系。方法 水囊引产胎儿的软骨细胞体外单层培养。T-2 毒素浓度分为0,5,10,20和40 μg/L 5组,第1代软骨细胞接种后培养4 d,加入不同浓 度的T-2毒素,作用16 h。应用TUNEL法和流式细胞技术定性和定量检测软骨细胞凋 亡。同时观察T-2毒素对软骨细胞增殖的影响。结果 各组细胞在加入T-2毒素后,胞体明显皱缩,T-2 毒素浓度越大,皱缩越严重。流式细胞仪检测与TdT中介脱氧尿苷三磷酸末端标记(TU NEL)法检测均发现,当T-2毒素在0~10 μg/L之间时,T-2毒素浓度越大,凋亡细胞越多。结论 T-2毒素对软骨细胞的增殖有明显的抑制作用,并与T-2毒素浓度呈剂量—效应关系。T-2毒素可以引起体外培养的软骨细胞凋亡,且与T-2 毒素浓度有一定关系。%Objective To investigate the effect of T-2 toxin on apoptosis of chondrocytes.Methods Chondrocytes which were obtained from aborted fetal were cultured in vitro.Four days later,these chondrocytes were exposed to T-2 toxin in different concetrations for 16 hours.According to the concentratio ns,five experimental groups were divided:0,5,10,20,40 μg/L.Then TUNEL staining and Flowcytometry were used to detect the apoptosis of chondrocytes qualitativel y and quantitatively,the effect of T-2 toxin on proliferation of chondrocytes were also observed.Results After being exposed to T-2 toxin,the body of chondrocytes shrinked obviously and there was a dose-dependent relationship bet ween the toxin concentration and the degree of shrink.The concentration of T-2 toxin changed from 0 μg/L to 10 ng/ml,the number of apoptosis increased.Conclusions  T-2 toxin can inhibit the proliferation of chondroyte significantly in a dose-depenent manner. T-2 toxin can induce the apoptosis of chondrocyte and the numbers of apoptosis is proportionate to the concentration of T-2 toxin in particular

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

    Directory of Open Access Journals (Sweden)

    Jean-Edouard Ombetta

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

  4. Implants composed of carbon fiber mesh and bone-marrow-derived, chondrocyte-enriched cultures for joint surface reconstruction.

    Science.gov (United States)

    Robinson, D; Efrat, M; Mendes, D G; Halperin, N; Nevo, Z

    1993-01-01

    The current study integrates two distinct approaches in joint resurfacing into a combined type of implant, composed of carbon fiber mesh impregnated and coated with a hyaluronic-acid-based delivery substance containing cultured cells. Rabbit autogeneic chondrocyte-enriched cultures obtained from mesenchymal stem cells (chondroprogenitor cells) derived from adult rabbit bone marrow were grown in vitro under conditions favoring chondrogenesis. The improvement in quality of repair when a combined implant containing both cells and a carbon scaffold was used, in comparison to the utilization of carbon fiber mesh alone, was clearly demonstrated using clinical, histological, biochemical, and biomechanical examinations. Evaluations of the joints were performed at 6 weeks and 6 months after implantation. The repair tissue in the cell-implanted joints consisted of a typical hyaline cartilage, which was more cellular and thicker than the repair tissue in the hyaluronic-acid-impregnated carbon-fiber-implanted control joints. The hyaline cartilage in the experimental group formed a superficial layer above the carbon fibers, flush with the joint surface. In the controls, in which carbon fiber and the delivery substance alone were implanted, a histologically and biochemically fibrous tissue that was inferior biomechanically to the new cartilage was formed by the cells containing implants.

  5. Second-generation autologous chondrocyte transplantation: MRI findings and clinical correlations at a minimum 5-year follow-up

    Energy Technology Data Exchange (ETDEWEB)

    Kon, E. [Biomechanics Laboratory, III Clinic, Rizzoli Orthopaedic Institute, Via Di Barbiano 1/10, 40136 Bologna (Italy); Di Martino, A., E-mail: a.dimartino@biomec.ior.it [Biomechanics Laboratory, III Clinic, Rizzoli Orthopaedic Institute, Via Di Barbiano 1/10, 40136 Bologna (Italy); Filardo, G. [Biomechanics Laboratory, III Clinic, Rizzoli Orthopaedic Institute, Via Di Barbiano 1/10, 40136 Bologna (Italy); Tetta, C.; Busacca, M. [Radiology, Rizzoli Orthopaedic Institute, Bologna (Italy); Iacono, F. [Biomechanics Laboratory, III Clinic, Rizzoli Orthopaedic Institute, Via Di Barbiano 1/10, 40136 Bologna (Italy); Delcogliano, M. [Orthopaedic Departement San Carlo di Nancy Hospital, Rome (Italy); Albisinni, U. [Radiology, Rizzoli Orthopaedic Institute, Bologna (Italy); Marcacci, M. [Biomechanics Laboratory, III Clinic, Rizzoli Orthopaedic Institute, Via Di Barbiano 1/10, 40136 Bologna (Italy)

    2011-09-15

    Objective: To evaluate the clinical outcome of hyaluronan-based arthroscopic autologous chondrocyte transplantation at a minimum of 5 years of follow-up and to correlate it with the MRI evaluation parameters. Methods: Fifty consecutive patients were included in the study and evaluated clinically using the Cartilage Standard Evaluation Form as proposed by ICRS and the Tegner score. Forty lesions underwent MRI evaluation at a minimum 5-year follow-up. For the description and evaluation of the graft, we employed the MOCART-scoring system. Results: A statistically significant improvement in all clinical scores was observed at 2 and over 5 years. The total MOCART score and the signal intensity (3D-GE-FS) of the repair tissue were statistically correlated to the IKDC subjective evaluation. Larger size of the treated cartilage lesions had a negative influence on the degree of defect repair and filling, the integration to the border zone and the subchondral lamina integrity, whereas more intensive sport activity had a positive influence on the signal intensity of the repair tissue, the repair tissue surface, and the clinical outcome. Conclusion: Our findings confirm the durability of the clinical results obtained with Hyalograft C and the usefulness of MRI as a non-invasive method for the evaluation of the repaired tissue and the outcome after second-generation autologous transplantation over time.

  6. 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.

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

    Science.gov (United States)

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

    2011-12-01

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

  8. Autophagy effects on chondrocyte survival and cartilage damage%自噬与软骨细胞生存及软骨损伤

    Institute of Scientific and Technical Information of China (English)

    张谢卓; 徐超; 伊力哈木·托合提

    2015-01-01

    背景:自噬是细胞通过溶酶体途径处理内源性底物的过程,它普遍存在于机体细胞中,又被看作是细胞Ⅱ型程序性死亡,自噬可能是正常软骨细胞的一种保护或平衡机制.目的:就自噬与软骨及其损伤相关性的最新研究进展进行讨论,旨在对自噬在软骨及其损伤修复中的作用有一个更好的理解.方法:应用计算机检索中国知网、万方数据库及PubMed数据库最近20年有关自噬与软骨损伤方面的文献,中文检索词为"自噬、软骨、软骨细胞",英文检索词为"autophagy、cartilage、chondrocytes、beclin1、LC3".结果与结论:软骨细胞能感受关节内微环境变化而做出应答,以调整细胞基质代谢,维持关节软骨生物学功能,而软骨细胞所处低氧环境是引起细胞自噬的重要因素.自噬是正常软骨细胞的一种平衡或者保护机制,虽然自噬与软骨及其损伤相关性在近些年的研究中取得了长足的进步,但不得不承认其仍处于初级阶段,在分子水平上一些 Atg 的发现加深了对自噬的认识,但在软骨中如何诱导自噬途径,自噬信号是如何传导的,对软骨细胞生存会产生怎样的影响等方面的了解还不够丰富,有待广大学者进一步研究.%BACKGROUND:Autophagy is the celular process of lysosomal pathway processing by endogenous substrates, which exists in the body cels and has been considered as type II programmed cel death. Autophagy may be a protective or balancing mechanism of normal chondrocytes. OBJECTIVE:To discuss the latest research progress in autophagy and cartilage damage aiming to better understanding the role of autophagy in cartilage damage and repair. METHODS:A computer-based search of CNKI, Wanfang database and PubMed database was performed for articles relevant to autophagy and cartilage damage published in recent 20 years with the key words of "autophagy, cartilage, chondrocytes, beclin1, LC3" in Chinese and English. RESULTS

  9. 17 beta-estradiol-BSA conjugates and 17 beta-estradiol regulate growth plate chondrocytes by common membrane associated mechanisms involving PKC dependent and independent signal transduction.

    Science.gov (United States)

    Sylvia, V L; Walton, J; Lopez, D; Dean, D D; Boyan, B D; Schwartz, Z

    2001-01-01

    Nuclear receptors for 17 beta-estradiol (E(2)) are present in growth plate chondrocytes from both male and female rats and regulation of chondrocytes through these receptors has been studied for many years; however, recent studies indicate that an alternative pathway involving a membrane receptor may also be involved in the cell response. E(2) was found to directly affect the fluidity of chondrocyte membranes derived from female, but not male, rats. In addition, E(2) activates protein kinase C (PKC) in a nongenomic manner in female cells, and chelerythrine, a specific inhibitor of PKC, inhibits E(2)-dependent alkaline phosphatase activity and proteoglycan sulfation in these cells, indicating PKC is involved in the signal transduction mechanism. The aims of the present study were: (1) to examine the effect of a cell membrane-impermeable 17 beta-estradiol-bovine serum albumin conjugate (E(2)-BSA) on chondrocyte proliferation, differentiation, and matrix synthesis; (2) to determine the pathway that mediates the membrane effect of E(2)-BSA on PKC; and (3) to compare the action of E(2)-BSA to that of E(2). Confluent, fourth passage resting zone (RC) and growth zone (GC) chondrocytes from female rat costochondral cartilage were treated with 10(-9) to 10(-7) M E(2) or E(2)-BSA and changes in alkaline phosphatase specific activity, proteoglycan sulfation, and [(3)H]-thymidine incorporation measured. To examine the pathway of PKC activation, chondrocyte cultures were treated with E(2)-BSA in the presence or absence of GDP beta S (inhibitor of G-proteins), GTP gamma S (activator of G-proteins), U73122 or D609 (inhibitors of phospholipase C [PLC]), wortmannin (inhibitor of phospholipase D [PLD]) or LY294002 (inhibitor of phosphatidylinositol 3-kinase). E(2)-BSA mimicked the effects of E(2) on alkaline phosphatase specific activity and proteoglycan sulfation, causing dose-dependent increases in both RC and GC cell cultures. Both forms of estradiol inhibited [(3)H

  10. Inhibitory effects of pentosan polysulfate sodium on MAP-kinase pathway and NF-κB nuclear translocation in canine chondrocytes in vitro.

    Science.gov (United States)

    Sunaga, Takafumi; Oh, Namgil; Hosoya, Kenji; Takagi, Satoshi; Okumura, Masahiro

    2012-06-01

    Pentosan polysulfate sodium (PPS) has a heparin-like structure and is purificated from the plant of European beech wood. PPS has been used for the treatment of interstitial cystitis for human patients. Recent years, it was newly recognised that PPS reduce pain and inflammation of OA. The molecular biological mechanism of PPS to express its clinical effects is not fully understood. The purpose of the present study is to investigate a mechanism of action of PPS on inflammatory reaction of chondrocytes in vitro. It was evaluated that effects of PPS on interleukin (IL)-1β-induced phosphorylation of mitogen-actiated protein kinases (MAPKs), such as p38, extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK), nuclear translocation of nuclear factor-kappa B (NF-κB), and matrix metalloproteinase (MMP)-3 production in cultured articular chondrocytes. As a result, in the presence of PPS existence, IL-1β-induced phosphorylation of p38 and ERK were certainly inhibited, while JNK phosphorylation was not affected. Nuclear translocation of NF-κB and MMP-3 production were suppressed by PPS pretreatment prior to IL-1β stimulation. In conclusion, it is strongly suggested that PPS treatment prevents inflammatory intracellular responses induced by IL-1 β through inhibition of phosphorylation of certain MAPKs, p38 and ERK and then nuclear translocation of NF-κB in cultured chondrocytes. These PPS properties may contribute to suppressive consequence of catabolic MMP-3 synthesis. These data might translate the clinical efficacy as PPS treatment could inhibit the cartilage catabolism and related clinical symptoms of OA in dogs.

  11. Focal adhesion kinase and mitogen-activated protein kinases are involved in chondrocyte activation by the 29-kDa amino-terminal fibronectin fragment.

    Science.gov (United States)

    Gemba, Takefumi; Valbracht, Jean; Alsalameh, Saifeddin; Lotz, Martin

    2002-01-11

    The 29-kDa amino-terminal fibronectin fragment (FN-f) has a potent chondrolytic effect and is thought to be involved in cartilage degradation in arthritis. However, little is known about signal transduction pathways that are activated by FN-f. Here we demonstrated that FN-f induced nitric oxide (NO) production from human articular chondrocytes. Expression of inducible nitric-oxide synthase (iNOS) mRNA and NO production were observed at 6 and 48 h after FN-f treatment, respectively. Interleukin-1beta (IL-1beta) mRNA up-regulation was stimulated by FN-f in human chondrocytes. To address the possibility that FN-f-induced NO release is mediated by IL-1beta production, the effect of IL-1 receptor antagonist (IL-1ra) was determined. IL-1ra partially inhibited FN-f-induced NO release although it almost completely inhibited IL-1beta-induced NO release. Tyrosine phosphorylation of focal adhesion kinase was induced transiently by FN-f treatment. Blocking antibodies to alpha(5) or beta(1) integrin and Arg-Gly-Asp-containing peptides did not inhibit FN-f-induced NO production. PP2, a Src family kinase inhibitor, or cytochalasin D, which selectively disrupts the network of actin filaments, inhibited both FAK phosphorylation and NO production induced by FN-f, but the phosphatidylinositol 3-kinase inhibitor wortmannin had no effect. Analysis of mitogen-activated protein kinases (MAPK) showed activation of extracellular signal-regulated kinase (ERK), c-Jun NH(2)-terminal kinase, and p38 MAPK. High concentrations of SB203580, which inhibit both JNK and p38 MAPK, and PD98059 a selective inhibitor of MEK1/2 that blocks ERK activation, inhibited FN-f induced NO production. These data suggest that focal adhesion kinase and MAPK mediate FN-f induced activation of human articular chondrocytes.

  12. Transplantatation of scaffold-free spheroids composed of synovium-derived cells and chondrocytes for the treatment of cartilage defects of the knee

    Directory of Open Access Journals (Sweden)

    J-I Lee

    2011-11-01

    Full Text Available Autologous chondrocyte implantation (ACI is the treatment of choice for osteoarthritis. However, to regenerate articular cartilage using this method, the procedure paradoxically demands that the cell source of the articular chondrocytes (ACs for ex vivo expansion be from the patient’s own healthy cartilage, which can result in donor site morbidity. Accordingly, it is essential to develop a substitute for AC. In the present study, we investigated whether synovium-derived cells (SYs could be used as a partial replacement for ACs in ACI. ACs and SYs from the knees of rabbits were isolated and cultured, and the growth rates of the cells were compared. To manufacture the cellular transplants, we developed a high-density suspension-shaking culture method (HDSS, which circulates the cells in culture media, promoting self-assembly of scaffold-free cellular aggregates. ACs and SYs were mixed in various ratios using HDSS. Injectable cellular transplants were harvested and transplanted into full-thickness osteochondral defects. Simultaneously, histological evaluations were conducted with toluidine blue and safranin O, and immunohistochemistry of collagen type I and II was conducted. Gene expression to evaluate chondrocyte-specific differentiation was also performed. We successfully prepared a large quantity of spheroids (spheroidal cell aggregates in a short time using mixed ACs and SYs, for all cellular composition ratios. Our data showed that the minimal therapeutic unit for the transplants contributed to in situ regeneration of cartilage. In summary, SYs can be used as a replacement for ACs in clinical cases of ACI in patients with broad areas of osteoarthritic lesions.

  13. Mechanical Compression of Articular Cartilage Induces Chondrocyte Proliferation and Inhibits Proteoglycan Synthesis by Activation of the Erk Pathway: Implications for Tissue Engineering and Regenerative Medicine

    Science.gov (United States)

    Ryan, James A.; Eisner, Eric A.; DuRaine, Grayson; You, Zongbing; Reddi, A. Hari

    2013-01-01

    Articular cartilage is recalcitrant to endogenous repair and regeneration and thus a focus of tissue engineering and regenerative medicine strategies. A pre-requisite for articular cartilage tissue engineering is an understanding of the signal transduction pathways involved in mechanical compression during trauma or disease. We sought to explore the role of the extracellular signal-regulated kinase 1/2 (ERK 1/2) pathway in chondrocyte proliferation and proteoglycan synthesis following acute mechanical compression. Bovine articular cartilage explants were cultured with and without the ERK 1/2 pathway inhibitor PD98059. Cartilage explants were statically loaded to 40% strain at a strain rate of 1−sec for 5 seconds. Control explants were cultured under similar conditions but were not loaded. There were four experimental groups: 1) no load without inhibitor 2) no load with the inhibitor PD98059, 3) loaded without the inhibitor, and 4) loaded with the inhibitor PD98059. Explants were cultured for varying durations, from 5 minutes to 5 days. Explants were then analyzed by biochemical and immunohistochemical methods. Mechanical compression induced phosphorylation of ERK 1/2, and this was attenuated with the ERK 1/2 pathway inhibitor PD98059 in a dose-dependent manner. Chondrocyte proliferation was increased by mechanical compression. This effect was blocked by the inhibitor of the ERK 1/2 pathway. Mechanical compression also led to a decrease in proteoglycan synthesis that was reversed with inhibitor PD98059. In conclusion, the ERK 1/2 pathway is involved in the proliferative and biosynthetic response of chondrocytes following acute static mechanical compression. PMID:19177463

  14. Methods for producing scaffold-free engineered cartilage sheets from auricular and articular chondrocyte cell sources and attachment to porous tantalum.

    Science.gov (United States)

    Whitney, G Adam; Mera, Hisashi; Weidenbecher, Mark; Awadallah, Amad; Mansour, Joseph M; Dennis, James E

    2012-08-01

    Scaffold-free cartilage engineering techniques may provide a simple alternative to traditional methods employing scaffolds. We previously reported auricular chondrocyte-derived constructs for use in an engineered trachea model; however, the construct generation methods were not reported in detail. In this study, methods for cartilage construct generation from auricular and articular cell sources are described in detail, and the resulting constructs are compared for use in a joint resurfacing model. Attachment of cartilage sheets to porous tantalum is also investigated as a potential vehicle for future attachment to subchondral bone. Large scaffold-free cartilage constructs were produced from culture-expanded chondrocytes from skeletally mature rabbits, and redifferentiated in a chemically-defined culture medium. Auricular constructs contained more glycosaminoglycan (39.6±12.7 vs. 9.7±1.9 μg/mg wet weight, mean and standard deviation) and collagen (2.7±0.45 vs. 1.1±0.2 μg/mg wet weight, mean and standard deviation) than articular constructs. Aggregate modulus was also higher for auricular constructs vs. articular constructs (0.23±0.07 vs. 0.12±0.03 MPa, mean and standard deviation). Attachment of constructs to porous tantalum was achieved by neocartilage ingrowth into tantalum pores. These results demonstrate that large scaffold-free neocartilage constructs can be produced from mature culture-expanded chondrocytes in a chemically-defined medium, and that these constructs can be attached to porous tantalum.

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

    Science.gov (United States)

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

    2017-02-27

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

  16. MR appearance of autologous chondrocyte implantation in the knee: correlation with the knee features and clinical outcome

    Energy Technology Data Exchange (ETDEWEB)

    Takahashi, Tomoki [Department of Radiology and Institute of Orthopaedics, Oswestry, Shropshire (United Kingdom); Kumamoto University, Department of Orthopaedic and Neuro-Musculoskeletal Surgery, Kumamoto (Japan); Tins, Bernhard; McCall, Iain W.; Ashton, Karen [Department of Radiology and Institute of Orthopaedics, Oswestry, Shropshire (United Kingdom); Robert Jones and Agnes Hunt Orthopaedic Hospital NHS Trust, Department of Diagnostic Imaging, Oswestry, Shropshire (United Kingdom); Richardson, James B. [Department of Radiology and Institute of Orthopaedics, Oswestry, Shropshire (United Kingdom); RJAH Orthopaedic Hospital, Institute of Orthopaedics, Oswestry, Shropshire (United Kingdom); Takagi, Katsumasa [Department of Radiology and Institute of Orthopaedics, Oswestry, Shropshire (United Kingdom); Kumamoto Aging Research Institute, Kumamoto (Japan)

    2006-01-01

    To relate the magnetic resonance imaging (MRI) appearance of autologous chondrocyte implantation (ACI) in the knee in the 1st postoperative year with other knee features on MRI and with clinical outcome. Forty-nine examinations were performed in 49 patients at 1 year after ACI in the knee. Forty-one preoperative magnetic resonance (MR) examinations were also available. The grafts were assessed for smoothness, thickness in comparison with that of adjacent cartilage, signal intensity, integration to underlying bone and adjacent cartilage, and congruity of subchondral bone. Presence of overgrowth and bone marrow appearance beneath the graft were also assessed. Presence of osteophyte formation, further cartilage defects, appearance of the cruciate ligaments and the menisci were also recorded. An overall graft score was constructed, using the graft appearances. This was correlated with the knee features and the Lysholm score, a clinical self-assessment score. The data were analysed by a Kruskal-Wallis H test followed by a Mann-Whitney U test with Bonferroni correction as post-hoc test. Of 49 grafts, 32 (65%) demonstrated complete defect filling 1 year postoperatively. General overgrowth was seen in eight grafts (16%), and partial overgrowth in 13 grafts (26%). Bone marrow change underneath the graft was seen; oedema was seen in 23 grafts (47%), cysts in six grafts (12%) and sclerosis in two grafts (4%). Mean graft score was 8.7 (of maximal 12) (95% CI 8.0-9.5). Knees without osteophyte formation or additional other cartilage defects (other than the graft site) had a significantly higher graft score than knees with multiple osteophytes (P=0.0057) or multiple further cartilage defects (P=0.014). At 1 year follow-up improvement in the clinical scores was not significantly different for any subgroup. (orig.)

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

    Directory of Open Access Journals (Sweden)

    D Vonwil

    2010-11-01

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

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

    Directory of Open Access Journals (Sweden)

    Petar D. Petrov

    2015-11-01

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

  19. Sulfation of p-nitrophenyl-N-acetyl-beta-D-galactosaminide with a microsomal fraction from cultured chondrocytes

    Energy Technology Data Exchange (ETDEWEB)

    Habuchi, O.; Conrad, H.E.

    1985-10-25

    Chick embryo chondrocyte microsomes containing intact Golgi vesicles took up 3'-phosphoadenosine-5'-phospho(TVS)sulfate ((TVS)PAPS) in a time- and temperature-dependent, substrate-saturable manner. When (TVS)PAPS and p-nitrophenyl-N-acetyl-beta-D-galactosaminide (pNP-GalNAc) were added to the incubation in the absence of detergent, the microsomes catalyzed the transfer of sulfate from (TVS)PAPS to pNP-GalNAc to form pNP-GalNAc-6-TVSO4. The apparent Km values for PAPS in the uptake and the pNP-GalNAc sulfation reactions were 2 X 10(-7) and 2 X 10(-6) M, respectively. The sulfation of pNP-GalNAc by the microsomal preparation was inhibited by detergent. The microsomal fraction also catalyzed the transfer of sulfate from (TVS)PAPS to oligosaccharides prepared from chondroitin. However, in contrast to the sulfation of pNP-GalNAc, the rate of sulfation of these oligosaccharides was low in the absence of detergent and was markedly stimulated when detergent was added. Sulfation of pNP-GalNAc by the freeze-thawed microsomes was inhibited when the octasaccharide prepared from chondroitin was present in the reaction mixture. As the PAPS that had been internalized in the microsomal vesicles was consumed in the sulfation of pNP-GalNAc, more (TVS)PAPS was taken up and the sulfated pNP-GalNAc was released from the vesicles. These observations suggest that pNP-GalNAc may serve as a model membrane-permeable substrate for study of the 6-sulfo-transferase reaction involved in sulfation of chondroitin sulfate in intact Golgi vesicles.

  20. Cyclic mechanical load causes global translational arrest in articular chondrocytes: a process which is partially dependent upon PKR phosphorylation

    Directory of Open Access Journals (Sweden)

    C Lomas

    2011-09-01

    Full Text Available he cellular mechanisms by which articular cartilage responds to load are poorly understood, but such responses may involve regulation at the level of protein translation rather than synthesis of mRNA. We investigated the role of translational control in cyclically (0.5 Hz, 0.1 Hz and 0.05 Hz and statically loaded porcine articular cartilage explants. Messenger RNA was extracted for real time polymerase chain reaction (RT-PCR and newly synthesised proteins were measured by their incorporation of radiolabelled 35S[methionine/cysteine] or 35SO4. Some medium from loaded and unloaded explants was immunoblotted for type II collagen, CTGF and TIMP3. The pathways that control protein translation were investigated by immunoblotting explant lysates for PKR, PERK (PKR like endoplasmic reticulum kinase, eIF2a (eukaryotic initiation factor 2a, eEFs (eukaryotic elongation factors, and AMP-dependent kinase. Explants were also loaded in the presence of inhibitors of PKR, the fibroblast growth factor (FGF receptor and PI3 kinase. Cyclic loading caused complete global translational arrest as evidenced by a total suppression of new protein synthesis whilst maintaining mRNA levels. Translational arrest did not occur following static loading and was partly dependent upon the load frequency. There was a rebound increase in protein synthesis when labelling was performed after load had been withdrawn. Phosphorylation of PKR occurred in explants following cyclic load and inhibition of PKR modestly reversed suppression of newly synthesised proteins suggesting that PKR, at least in part, was responsible for loading induced translational arrest. These results show that translational control provides a rapid and potentially important mechanism for controlling the synthetic responses of articular chondrocytes in response to different types of mechanical load.

  1. In vitro chondrocyte toxicity following long-term, high-dose exposure to Gd-DTPA and a novel cartilage-targeted MR contrast agent

    Energy Technology Data Exchange (ETDEWEB)

    Midura, Sharon; Midura, Ronald J. [Cleveland Clinic, Biomedical Engineering, Lerner Research Institute, Cleveland, OH (United States); Schneider, Erika [Cleveland Clinic, Imaging Institute, A21, Cleveland, OH (United States); NitroSci Pharmaceuticals, New Berlin, WI (United States); Rosen, Gerald M. [University of Maryland School of Pharmacy, Department of Pharmaceutical Sciences, Baltimore, MD (United States); NitroSci Pharmaceuticals, New Berlin, WI (United States); Winalski, Carl S. [Cleveland Clinic, Biomedical Engineering, Lerner Research Institute, Cleveland, OH (United States); Cleveland Clinic, Imaging Institute, A21, Cleveland, OH (United States)

    2017-01-15

    To determine the concentrations exhibiting toxicity of a cartilage-targeted magnetic resonance imaging contrast agent compared with gadopentetate dimeglumine (Gd-DT-PA) in chondrocyte cultures. A long-term Swarm rat chondrosarcoma chondrocyte-like cell line was exposed for 48 h to 1.0-20 mM concentrations of diaminobutyl-linked nitroxide (DAB4-DLN) citrate, 1.0-20 mM Gd-DTPA, 1.0 μM staurosporine (positive control), or left untreated. Cell appearance, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays of metabolic activity, quantitative PicoGreen assays of DNA content, and calcein-AM viability assays were compared. At 1.0-7.5 mM, minimal decrease in cell proliferation was found for both agents. At all doses of both agents, cell culture appearances were similar after 24 h of treatment. At the higher doses, differences in cell culture appearance were found after 48 h of treatment, with dose-dependent declines in chondrocyte populations for both agents. Concentration-dependent declines in DNA content and calcein fluorescence were found after 48 h of treatment, but beginning at a lower dose of DAB4-DLN citrate than Gd-DTPA. Dose-dependent decreases in MTT staining (cell metabolism) were apparent for both agents, but larger effects were evident at a lower dose for DAB-DLN citrate. Poor MTT staining of cells exposed for 48 h to 20 mM DAB4-DLN citrate probably indicates dead or dying cells. The minimal effect of the long-term exposure of model chondrocyte cell cultures to DAB4-DLN citrate and Gd-DTPA concentrations up to 7.5 mM (3x typical arthrographic administration) is supporting evidence that these doses are acceptable for MR arthrography. The findings are reassuring given that the experimental exposure to the contrast agents at sustained concentrations was much longer than when used clinically. (orig.)

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

    Directory of Open Access Journals (Sweden)

    Huh Jeong-Eun

    2012-12-01

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

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

    Science.gov (United States)

    2012-01-01

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

  4. The paracrine feedback loop between vitamin D₃ (1,25(OH)₂D₃) and PTHrP in prehypertrophic chondrocytes.

    Science.gov (United States)

    Bach, Frances C; Rutten, Kirsten; Hendriks, Kristyanne; Riemers, Frank M; Cornelissen, Peter; de Bruin, Alain; Arkesteijn, Ger J; Wubbolts, Richard; Horton, William A; Penning, Louis C; Tryfonidou, Marianna A

    2014-12-01

    The endocrine feedback loop between vitamin D3(1,25(OH)2D3) and parathyroid hormone (PTH) plays a central role in skeletal development. PTH-related protein (PTHrP) shares homology and its receptor (PTHR1) with PTH. The aim of this study was to investigate whether there is a functional paracrine feedback loop between 1,25(OH)2D3 and PTHrP in the growth plate, in parallel with the endocrine feedback loop between 1,25(OH)2D3 and PTH. This was investigated in ATDC5 cells treated with 10(-8) M 1,25(OH)2D3 or PTHrP, Col2-pd2EGFP transgenic mice, and primary Col2-pd2EGFP growth plate chondrocytes isolated by FACS, using RT-qPCR, Western blot, PTHrP ELISA, chromatin immunoprecipitation (ChIP) assay, silencing of the 1,25(OH)2D3 receptor (VDR), immunofluorescent staining, immunohistochemistry, and histomorphometric analysis of the growth plate. The ChIP assay confirmed functional binding of the VDR to the PTHrP promoter, but not to the PTHR1 promoter. Treatment with 1,25(OH)2D3 decreased PTHrP protein production, an effect which was prevented by silencing of the VDR. Treatment with PTHrP significantly induced VDR production, but did not affect 1α- and 24-hydroxylase expression. Hypertrophic differentiation was inhibited by PTHrP and 1,25(OH)2D3 treatment. Taken together, these findings indicate that there is a functional paracrine feedback loop between 1,25(OH)2D3 and PTHrP in the growth plate. 1,25(OH)2D3 decreases PTHrP production, while PTHrP increases chondrocyte sensitivity to 1,25(OH)2D3 by increasing VDR production. In light of the role of 1,25(OH)2D3 and PTHrP in modulating chondrocyte differentiation, 1,25(OH)2D3 in addition to PTHrP could potentially be used to prevent undesirable hypertrophic chondrocyte differentiation during cartilage repair or regeneration.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2013-11-08

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

  6. Glucosamine modulates IL-1-induced activation of rat chondrocytes at a receptor level, and by inhibiting the NF-kappa B pathway.

    Science.gov (United States)

    Gouze, J N; Bianchi, A; Bécuwe, P; Dauça, M; Netter, P; Magdalou, J; Terlain, B; Bordji, K

    2002-01-16

    We recently reported that glucosamine reversed the decrease in proteoglycan synthesis and in UDP-glucuronosyltransferase I mRNA expression induced by interleukin-1 beta (IL-1 beta) [Arthritis Rheum. 44 (2001) 351-360]. In the present work, we show that glucosamine does not exert the same effects when chondrocytes were stimulated with reactive oxygen species (ROS). In order to better understand its mechanism of action, we determined if glucosamine could prevent the binding of IL-1 beta to its cellular receptors or could interfere with its signaling pathway at a post-receptor level. Addition of glucosamine to rat chondrocytes treated with IL-1 beta or with ROS decreased the activation of the nuclear factor kappa B, but not the activator protein-1. After treatment with IL-1 beta, glucosamine increased the expression of mRNA encoding the type II IL-1 beta receptor. These results emphasize the potential role of two regulating proteins of the IL-1 beta signaling pathway that could account for the beneficial effect of glucosamine in osteoarthritis.

  7. In vitro and in vivo co-culture of chondrocytes and bone marrow stem cells in photocrosslinked PCL-PEG-PCL hydrogels enhances cartilage formation.

    Science.gov (United States)

    Ko, Chao-Yin; Ku, Kuan-Lin; Yang, Shu-Rui; Lin, Tsai-Yu; Peng, Sydney; Peng, Yu-Shiang; Cheng, Ming-Huei; Chu, I-Ming

    2016-10-01

    Chondrocytes (CH) and bone marrow stem cells (BMSCs) are sources that can be used in cartilage tissue engineering. Co-culture of CHs and BMSCs is a promising strategy for promoting chondrogenic differentiation. In this study, articular CHs and BMSCs were encapsulated in PCL-PEG-PCL photocrosslinked hydrogels for 4 weeks. Various ratios of CH:BMSC co-cultures were investigated to identify the optimal ratio for cartilage formation. The results thus obtained revealed that co-culturing CHs and BMSCs in hydrogels provides an appropriate in vitro microenvironment for chondrogenic differentiation and cartilage matrix production. Co-culture with a 1:4 CH:BMSC ratio significantly increased the synthesis of GAGs and collagen. In vivo cartilage regeneration was evaluated using a co-culture system in rabbit models. The co-culture system exhibited a hyaline chondrocyte phenotype with excellent regeneration, resembling the morphology of native cartilage. This finding suggests that the co-culture of these two cell types promotes cartilage regeneration and that the system, including the hydrogel scaffold, has potential in cartilage tissue engineering. Copyright © 2013 John Wiley & Sons, Ltd.

  8. Cartilage storage at 4 °C with regular culture medium replacement benefits chondrocyte viability of osteochondral grafts in vitro.

    Science.gov (United States)

    Qi, Jianhong; Hu, Zunjie; Song, Hongqiang; Chen, Bin; Xie, Di; Zhou, Lu; Zhang, Yanming

    2016-09-01

    Maintenance of articular cartilage allografts in culture media is a common method of tissue storage; however, the technical parameters of graft storage remain controversial. In this study, we examined the optimal temperature and culture medium exchange rate for the storage of osteochondral allografts in vitro. Cylindrical osteochondral grafts (n = 120), harvested from the talar joint surface of ten Boer goats, were randomly classified into four groups and stored under the following conditions: Group A1 was maintained at 4 °C in culture medium that was refreshed every 2 days; Group A2 was maintained at 4 °C in the same culture medium, without refreshing; Group B1, was maintained at 37 °C in culture medium that was refreshed every 2 days; Group B2, was maintained at 37 °C in the same culture medium, without refreshing. Chondrocyte viability in the grafts was determined by ethidium bromide/fluorescein diacetate staining on days 7, 21, and 35. Proteoglycan content was measured by Safranin-O staining. Group A1 exhibited the highest chondrocyte survival rates of 90.88 %, 88.31 % and 78.69 % on days 7, 21, and 35, respectively. Safranin O staining revealed no significant differences between groups on days 21 and 35. These results suggest that storage of osteochondral grafts at 4 °C with regular culture medium replacement should be highly suitable for clinical application.

  9. Early in situ changes in chondrocyte biomechanical responses due to a partial meniscectomy in the lateral compartment of the mature rabbit knee joint.

    Science.gov (United States)

    Fick, J M; P Ronkainen, A; Madden, R; Sawatsky, A; Tiitu, V; Herzog, W; Korhonen, R K

    2016-12-08

    We determined the biomechanical responses of chondrocytes to indentation at specific locations within the superficial zone of cartilage (i.e. patellar, femoral groove, femoral condylar and tibial plateau sites) taken from female New Zealand white rabbits three days after a partial meniscectomy in the lateral compartment of a knee joint. Confocal laser scanning microscopy combined with a custom indentation system was utilized to image chondrocyte responses at sites taken from ten contralateral and experimental knee joints. Cell volume, height, width and depth changes, global, local axial and transverse strains and Young׳s moduli were determined. Histological assessment was performed and proteoglycan content from the superficial zone of each site was determined. Relative to contralateral group cells, patellar, femoral groove and lateral femoral condyle cells in the experimental group underwent greater volume decreases (p volume decreases (p 0.05), femoral groove, medial femoral condyle and medial tibial plateau experimental sites (p < 0.05). The findings suggest: (i) cell biomechanical responses to cartilage loading in the rabbit knee joint can become altered as early as 3 days after a partial meniscectomy, (ii) are site-specific, and (iii) occur before alterations in tissue mechanics or changes detectable with histology.

  10. Master regulator for chondrogenesis, Sox9, regulates transcriptional activation of the endoplasmic reticulum stress transducer BBF2H7/CREB3L2 in chondrocytes.

    Science.gov (United States)

    Hino, Kenta; Saito, Atsushi; Kido, Miori; Kanemoto, Soshi; Asada, Rie; Takai, Tomoko; Cui, Min; Cui, Xiang; Imaizumi, Kazunori

    2014-05-16

    The endoplasmic reticulum (ER) stress transducer, box B-binding factor 2 human homolog on chromosome 7 (BBF2H7), is a basic leucine zipper (bZIP) transmembrane transcription factor. This molecule is activated in response to ER stress during chondrogenesis. The activated BBF2H7 accelerates cartilage matrix protein secretion through the up-regulation of Sec23a, which is responsible for protein transport from the ER to the Golgi apparatus and is a target of BBF2H7. In the present study, we elucidated the mechanisms of the transcriptional activation of Bbf2h7 in chondrocytes. The transcription of Bbf2h7 is regulated by Sex determining region Y-related high-mobility group box 9 (Sox9), a critical factor for chondrocyte differentiation that facilitates the expression of one of the major cartilage matrix proteins Type II collagen (Col2), through binding to the Sox DNA-binding motif in the Bbf2h7 promoter. BBF2H7 is activated as a transcription factor in response to physiological ER stress caused by abundant synthesis of cartilage matrix proteins, and consequently regulates the secretion of cartilage matrix proteins. Taken together, our findings demonstrate novel regulatory mechanisms of Sox9 for controlling the secretion of cartilage matrix proteins through the activation of BBF2H7-Sec23a signaling during chondrogenesis.

  11. Controllable promotion of chondrocyte adhesion and growth on PVA hydrogels by controlled release of TGF-β1 from porous PLGA microspheres.

    Science.gov (United States)

    Nie, Lei; Zhang, Guohua; Hou, Ruixia; Xu, Haiping; Li, Yaping; Fu, Jun

    2015-01-01

    Poly(vinyl alcohol) (PVA) hydrogels have been candidate materials for cartilage tissue engineering. However, the cell non-adhesive nature of PVA hydrogels has been a limit. In this paper, the cell adhesion and growth on PVA hydrogels were promoted by compositing with transform growth factor-β1 (TGF-β1) loaded porous poly(D,L-lactide-co-glycolide) (PLGA) microspheres. The porous microspheres were fabricated by a modified double emulsion method with bovine serum albumin (BSA) as porogen. The average pore size of microspheres was manipulated by changing the BSA/PLGA ratio. Such controllable porous structures effectively influenced the encapsulation efficiency (Eencaps) and release profile of TGF-β1. By compositing PVA hydrogels with such TGF-β1-loaded PLGA microspheres, chondrocyte adhesion and proliferation were significantly promoted in a controllable manner, as confirmed by fluorescent imaging and quantitative CCK-8 assay. That is, the chondrocyte proliferation was favored by using PLGA microspheres with high Eencaps of TGF-β1 or by increasing the PLGA microsphere content in the hydrogels. These results demonstrated a facile method to improve the cell adhesion and growth on the intrinsically cell non-adhesive PVA hydrogels, which may find applications in cartilage substitution.

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

    Science.gov (United States)

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

    2016-01-01

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