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Sample records for chondrocytes

  1. Simvastatin inhibits CD44 fragmentation in chondrocytes.

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

    2016-08-15

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

  2. Articular chondrocyte metabolism and osteoarthritis

    Energy Technology Data Exchange (ETDEWEB)

    Leipold, H.R.

    1989-01-01

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

  3. Biochemical and proteomic characterization of alkaptonuric chondrocytes.

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

    2012-09-01

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

  4. Effect of freezing on rabbit cultured chondrocytes

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    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

  5. Optimization of transport media for human chondrocytes

    International Nuclear Information System (INIS)

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

  6. Oxygen tension affects lubricin expression in chondrocytes.

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

    2014-10-01

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

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

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

    2016-01-01

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

  8. Chondrocytic Atf4 regulates osteoblast differentiation and function via Ihh

    OpenAIRE

    Wang, Weiguang; Lian, Na; Ma., Yun; LI, LINGZHEN; Gallant, Richard C.; Elefteriou, Florent; Yang, Xiangli

    2012-01-01

    Atf4 is a leucine zipper-containing transcription factor that activates osteocalcin (Ocn) in osteoblasts and indian hedgehog (Ihh) in chondrocytes. The relative contribution of Atf4 in chondrocytes and osteoblasts to the regulation of skeletal development and bone formation is poorly understood. Investigations of the Atf4–/–;Col2a1-Atf4 mouse model, in which Atf4 is selectively overexpressed in chondrocytes in an Atf4-null background, demonstrate that chondrocyte-derived Atf4 regulates osteog...

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

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

    1999-01-01

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

  10. Chondrocyte behavior on nanostructured micropillar polypropylene and polystyrene surfaces

    International Nuclear Information System (INIS)

    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 α1(II), procollagen α1(X), and procollagen α2(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 α1(II) appeared to decrease in passaged chondrocytes. However, the expressions of procollagen α2(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 repair. • Cell culture

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

    International Nuclear Information System (INIS)

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

  12. Effect of hyaluronic acid on chondrocyte apoptosis

    OpenAIRE

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

    2015-01-01

    OBJECTIVE: To determine the percentage of apoptotic cells in a contusion model of osteoarthritis (OA) and to assess whether intra-articular injection of high doses of hyaluronic acid (HA) immediately after trauma reduces chondrocyte apoptosis. METHODS: Forty knees from adult rabbits were impacted thrice with a 1 kg block released through a 1 meter tall cylinder (29.4 Joules). Subsequently, 2 mL of HA was injected in one knee and 2 mL saline in the contra-lateral knee. Medication were administ...

  13. 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.

  14. Catabolic effects of muramyl dipeptide on rabbit chondrocytes

    International Nuclear Information System (INIS)

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

  15. 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.

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

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

    1992-04-01

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

  17. IFT88 influences chondrocyte actin organization and biomechanics

    OpenAIRE

    Z. Wang; Wann, A.K.T.; Thompson, C L; Hassen, A.; Wang, W; Knight, M.M.

    2016-01-01

    Summary Objectives Primary cilia are microtubule based organelles which control a variety of signalling pathways important in cartilage development, health and disease. This study examines the role of the intraflagellar transport (IFT) protein, IFT88, in regulating fundamental actin organisation and mechanics in articular chondrocytes. Methods The study used an established chondrocyte cell line with and without hypomorphic mutation of IFT88 (IFT88orpk). Confocal microscopy was used to quantif...

  18. Primary cilia attenuate hedgehog signalling in neoplastic chondrocytes

    OpenAIRE

    Ho, L.; Ali, S A; Al-Jazrawe, M; R. Kandel; Wunder, J S; Alman, B. A.

    2012-01-01

    Primary cilia can act as either a negative or positive regulator of the hedgehog (Hh) signaling pathway. Many cartilage tumors are characterized by abnormal activation of the Hh pathway. Here, we report that the presence of primary cilia occurs at a low frequency (12.4%) in neoplastic chondrocytes from malignant human chondrosarcomas, compared with chondrocytes from normal articular cartilage (67.7%). To determine the function of primary cilia in cartilaginous neoplasia, we studied benign car...

  19. The first experience of autologous chondrocytes transplantation after lumbar microdiscectomy

    OpenAIRE

    Pedachenko, Eugene; Khyzhnyak, Mykhaylo; Gorbatyuk, Kostyantyn; Pedachenko, Yuriy; Krasilenko, Elena; Shabliy, Volodymyr

    2014-01-01

    The purpose. To develop and provide into clinical practice the hi-tech method of autologous chondrocytes transplantation for treatment of patients with intervertebral discs herniation after lumbar microdiscectomy.Material and methods. The chondrocytes were isolated from tissues of intervertebral disc hernia, cultivated and preserved, and administrated as percutaneous puncture in the operated intervertebral disc (3 months after microdiscectomy).We plan to study the influence of transplanted au...

  20. Role of CCN2 in Amino Acid Metabolism of Chondrocytes.

    Science.gov (United States)

    Murase, Yurika; Hattori, Takako; Aoyama, Eriko; Nishida, Takashi; Maeda-Uematsu, Aya; Kawaki, Harumi; Lyons, Karen M; Sasaki, Akira; Takigawa, Masaharu; Kubota, Satoshi

    2016-04-01

    CCN2/connective tissue growth factor (CTGF) is a multi-functional molecule that promotes harmonized development and regeneration of cartilage through its matricellular interaction with a variety of extracellular biomolecules. Thus, deficiency in CCN2 supply profoundly affects a variety of cellular activities including basic metabolism. A previous study showed that the expression of a number of ribosomal protein genes was markedly enhanced in Ccn2-null chondrocytes. Therefore, in this study, we analyzed the impact of CCN2 on amino acid and protein metabolism in chondrocytes. Comparative metabolome analysis of the amino acids in Ccn2-null and wild-type mouse chondrocytes revealed stable decreases in the cellular levels of all of the essential amino acids. Unexpectedly, uptake of such amino acids was rather enhanced in Ccn2-null chondrocytes, and the addition of exogenous CCN2 to human chondrocytic cells resulted in decreased amino acid uptake. However, as expected, amino acid consumption by protein synthesis was also accelerated in Ccn2-null chondrocytes. Furthermore, we newly found that expression of two genes encoding two glycolytic enzymes, as well as the previously reported Eno1 gene, was repressed in those cells. Considering the impaired glycolysis and retained mitochondrial membrane potential in Ccn2-null chondrocytes, these findings suggest that Ccn2 deficiency induces amino acid shortage in chondrocytes by accelerated amino acid consumption through protein synthesis and acquisition of aerobic energy. Interestingly, CCN2 was found to capture such free amino acids in vitro. Under physiological conditions, CCN2 may be regulating the levels of free amino acids in the extracellular matrix of cartilage. J. Cell. Biochem. 117: 927-937, 2016. © 2015 Wiley Periodicals, Inc. PMID:26364758

  1. Antioxidant effect of bisphosphonates and simvastatin on chondrocyte lipid peroxidation

    International Nuclear Information System (INIS)

    The objective of this study was to evaluate the effect of bisphosphonates (BPs) and simvastatin on chondrocyte lipid peroxidation. For this purpose, a flow cytometrical method using C11-BODIPY581/591 was developed to detect hydroperoxide-induced lipid peroxidation in chondrocytes. Tertiary butylhydroperoxide (t-BHP) induced a time and concentration dependent increase in chondrocyte lipid peroxidation. Addition of a Fe2+/EDTA complex to t-BHP or hydrogen peroxide (H2O2) clearly enhanced lipid peroxidation. The lipophilic simvastatin demonstrated a small inhibition in the chondrocyte lipid peroxidation. None of three tested BPs (clodronate, pamidronate, and risedronate) had an effect on chondrocyte lipid peroxidation induced by t-BHP. However, when Fe2+/EDTA complex was added to t-BHP or H2O2, BPs inhibited the lipid peroxidation process varying from 25% to 58%. This study demonstrates that BPs have antioxidant properties as iron chelators, thereby inhibiting the chondrocyte lipid peroxidation. These findings add evidence to the therapeutic potential of bisphosphonates and statins in rheumatoid arthritis

  2. CCN1 Regulates Chondrocyte Maturation and Cartilage Development.

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

    2016-03-01

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

  3. The interplay between chondrocyte redifferentiation pellet size and oxygen concentration.

    Directory of Open Access Journals (Sweden)

    Betul Kul Babur

    Full Text Available Chondrocytes dedifferentiate during ex vivo expansion on 2-dimensional surfaces. Aggregation of the expanded cells into 3-dimensional pellets, in the presence of induction factors, facilitates their redifferentiation and restoration of the chondrogenic phenotype. Typically 1×10(5-5×10(5 chondrocytes are aggregated, resulting in "macro" pellets having diameters ranging from 1-2 mm. These macropellets are commonly used to study redifferentiation, and recently macropellets of autologous chondrocytes have been implanted directly into articular cartilage defects to facilitate their repair. However, diffusion of metabolites over the 1-2 mm pellet length-scales is inefficient, resulting in radial tissue heterogeneity. Herein we demonstrate that the aggregation of 2×10(5 human chondrocytes into micropellets of 166 cells each, rather than into larger single macropellets, enhances chondrogenic redifferentiation. In this study, we describe the development of a cost effective fabrication strategy to manufacture a microwell surface for the large-scale production of micropellets. The thousands of micropellets were manufactured using the microwell platform, which is an array of 360×360 µm microwells cast into polydimethylsiloxane (PDMS, that has been surface modified with an electrostatic multilayer of hyaluronic acid and chitosan to enhance micropellet formation. Such surface modification was essential to prevent chondrocyte spreading on the PDMS. Sulfated glycosaminoglycan (sGAG production and collagen II gene expression in chondrocyte micropellets increased significantly relative to macropellet controls, and redifferentiation was enhanced in both macro and micropellets with the provision of a hypoxic atmosphere (2% O2. Once micropellet formation had been optimized, we demonstrated that micropellets could be assembled into larger cartilage tissues. Our results indicate that micropellet amalgamation efficiency is inversely related to the time cultured as

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

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

    2015-12-01

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

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

    International Nuclear Information System (INIS)

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

  6. Derepression of miRNA-138 contributes to loss of the human articular chondrocyte phenotype

    OpenAIRE

    Seidl, C; Martinez-Sanchez, A; Murphy, CL

    2016-01-01

    Objective: To investigate the function of microRNA-138 (miR-138) in human articular chondrocytes (HACs). Methods: The expression of miR-138 in intact cartilage and cultured chondrocytes and the effects of miR-138 overexpression on chondrocyte marker genes were investigated. Targets of miR-138 relevant to chondrocytes were identified and verified by overexpression of synthetic miRNA mimics and inhibitors, luciferase assays, chromatin immunoprecipitation, and RNA immunoprecipitation o...

  7. Studies of the humoral factors produced by layered chondrocyte sheets.

    Science.gov (United States)

    Hamahashi, K; Sato, M; Yamato, M; Kokubo, M; Mitani, G; Ito, S; Nagai, T; Ebihara, G; Kutsuna, T; Okano, T; Mochida, J

    2015-01-01

    The authors aimed to repair and regenerate articular cartilage with layered chondrocyte sheets, produced using temperature-responsive culture dishes. The purpose of this study was to investigate the humoral factors produced by layered chondrocyte sheets. Articular chondrocytes and synovial cells were harvested during total knee arthroplasty. After co-culture, the samples were divided into three groups: a monolayer, 7 day culture sheet group (group M); a triple-layered, 7 day culture sheet group (group L); and a monolayer culture group with a cell count identical to that of group L (group C). The secretion of collagen type 1 (COL1), collagen type 2 (COL2), matrix metalloproteinase-13 (MMP13), transforming growth factor-β (TGFβ), melanoma inhibitory activity (MIA) and prostaglandin E2 (PGE2) were measured by enzyme-linked immunosorbent assay (ELISA). Layered chondrocyte sheets produced the most humoral factors. PGE2 expression declined over time in group C but was significantly higher in groups M and L. TGFβ expression was low in group C but was significantly higher in groups M and L (p<0.05). Our results suggest that the humoral factors produced by layered chondrocyte sheets may contribute to cartilaginous tissue repair and regeneration. PMID:23165985

  8. Focal Adhesion Assembly Induces Phenotypic Changes and Dedifferentiation in Chondrocytes.

    Science.gov (United States)

    Shin, Hyunjun; Lee, Mi Nam; Choung, Jin Seung; Kim, Sanghee; Choi, Byung Hyune; Noh, Minsoo; Shin, Jennifer H

    2016-08-01

    The expansion of autologous chondrocytes in vitro is used to generate sufficient populations for cell-based therapies. However, during monolayer culture, chondrocytes lose inherent characteristics and shift to fibroblast-like cells as passage number increase. Here, we investigated passage-dependent changes in cellular physiology, including cellular morphology, motility, and gene and protein expression, as well as the role of focal adhesion and cytoskeletal regulation in the dedifferentiation process. We found that the gene and protein expression levels of both the focal adhesion complex and small Rho GTPases are upregulated with increasing passage number and are closely linked to chondrocyte dedifferentiation. The inhibition of focal adhesion kinase (FAK) but not small Rho GTPases induced the loss of fibroblastic traits and the recovery of collagen type II, aggrecan, and SOX9 expression levels in dedifferentiated chondrocytes. Based on these findings, we propose a strategy to suppress chondrogenic dedifferentiation by inhibiting the identified FAK or Src pathways while maintaining the expansion capability of chondrocytes in a 2D environment. These results highlight a potential therapeutic target for the treatment of skeletal diseases and the generation of cartilage in tissue-engineering approaches. J. Cell. Physiol. 231: 1822-1831, 2016. © 2015 Wiley Periodicals, Inc. PMID:26661891

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2010-10-22

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

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

    International Nuclear Information System (INIS)

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

  11. An ECHO in biology II: Insights in chondrocyte cell fate

    NARCIS (Netherlands)

    Schivo, S.; Scholma, J.; Huang, X.; Zhong, L.; Pol, van de J.C.; Karperien, H.B.J.; Langerak, R.; Post, J.N.

    2016-01-01

    Purpose: An intricate network of regulatory processes determines the chondrocyte cell fate during development and maintains tissue homeostasis. In the event of a disease such as OA, the regulatory network is critically compromised. To cure the disease, we need to restore the regulatory processes to

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

    Science.gov (United States)

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

    2016-01-01

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

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

    International Nuclear Information System (INIS)

    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

  14. 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. PMID:27427985

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

    International Nuclear Information System (INIS)

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

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

    Science.gov (United States)

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

    2016-04-22

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

  17. Implication of interleukin 18 in production of matrix metalloproteinases in articular chondrocytes in arthritis: direct effect on chondrocytes may not be pivotal

    OpenAIRE

    Dai, S.; Shan, Z; Nishioka, K.; Yudoh, K

    2005-01-01

    Objective: To clarify the effect of interleukin (IL) 18 on cartilage degeneration by studying the profile of IL18 receptor (IL18R) on chondrocytes and the direct effect of IL18 on production of matrix metalloproteinases (MMPs), aggrecanases, and tissue inhibitors of metalloproteinases (TIMPs) in articular chondrocytes.

  18. Yap1 Regulates Multiple Steps of Chondrocyte Differentiation during Skeletal Development and Bone Repair.

    Science.gov (United States)

    Deng, Yujie; Wu, Ailing; Li, Pikshan; Li, Gang; Qin, Ling; Song, Hai; Mak, Kinglun Kingston

    2016-03-01

    Hippo signaling controls organ size and tissue regeneration in many organs, but its roles in chondrocyte differentiation and bone repair remain elusive. Here, we demonstrate that Yap1, an effector of Hippo pathway inhibits skeletal development, postnatal growth, and bone repair. We show that Yap1 regulates chondrocyte differentiation at multiple steps in which it promotes early chondrocyte proliferation but inhibits subsequent chondrocyte maturation both in vitro and in vivo. Mechanistically, we find that Yap1 requires Teads binding for direct regulation of Sox6 expression to promote chondrocyte proliferation. In contrast, Yap1 inhibits chondrocyte maturation by suppression of Col10a1 expression through interaction with Runx2. In addition, Yap1 also governs the initiation of fracture repair by inhibition of cartilaginous callus tissue formation. Taken together, our work provides insights into the mechanism by which Yap1 regulates endochondral ossification, which may help the development of therapeutic treatment for bone regeneration. PMID:26923596

  19. Yap1 Regulates Multiple Steps of Chondrocyte Differentiation during Skeletal Development and Bone Repair

    Directory of Open Access Journals (Sweden)

    Yujie Deng

    2016-03-01

    Full Text Available Hippo signaling controls organ size and tissue regeneration in many organs, but its roles in chondrocyte differentiation and bone repair remain elusive. Here, we demonstrate that Yap1, an effector of Hippo pathway inhibits skeletal development, postnatal growth, and bone repair. We show that Yap1 regulates chondrocyte differentiation at multiple steps in which it promotes early chondrocyte proliferation but inhibits subsequent chondrocyte maturation both in vitro and in vivo. Mechanistically, we find that Yap1 requires Teads binding for direct regulation of Sox6 expression to promote chondrocyte proliferation. In contrast, Yap1 inhibits chondrocyte maturation by suppression of Col10a1 expression through interaction with Runx2. In addition, Yap1 also governs the initiation of fracture repair by inhibition of cartilaginous callus tissue formation. Taken together, our work provides insights into the mechanism by which Yap1 regulates endochondral ossification, which may help the development of therapeutic treatment for bone regeneration.

  20. Chondrocytes-Specific Expression of Osteoprotegerin Modulates Osteoclast Formation in Metaphyseal Bone

    OpenAIRE

    Baoli Wang; Hongting Jin; Bing Shu; Ranim R. Mira; Di Chen

    2015-01-01

    Bone marrow stromal cells/osteoblasts were originally thought to be the major player in regulating osteoclast differentiation through expressing RANKL/OPG cytokines. Recent studies have established that chondrocytes also express RANKL/OPG and support osteoclast formation. Till now, the in vivo function of chondrocyte-produced OPG in osteoclast formation and postnatal bone growth has not been directly investigated. In this study, chondrocyte-specific Opg transgenic mice were generated by using...

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

    OpenAIRE

    Shwu Jen Chang; Shyh Ming Kuo; Yen Ting Lin; Shan-Wei Yang

    2014-01-01

    The aim of this study was to investigate the biological effects of sex hormones (17 β -estradiol and testosterone) on rabbit articular chondrocytes from different genders. We cultured primary rabbit articular chondrocytes from both genders with varying concentration of sex hormones. We evaluate cell proliferation and biochemical functions by MTT and GAG assay. The chondrocyte function and phenotypes were analyzed by mRNA level using RT-PCR. Immunocytochemical staining was also used to evaluat...

  2. 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.

  3. Studies on a Novel Bioreactor Design for Chondrocyte Culture

    OpenAIRE

    Patil, Harshad; Chandel, Ishan Saurav; Rastogi, Amit K.; Srivastava, Pradeep

    2013-01-01

    A bioreactor system plays an important role in tissue engineering and enables reproduction and controlled changes in the environmental factor. The bioreactor provides technical means to perform controlled processes in safe and reduced reproducible generation of time. Cartilage cells were grown in vitro by mimicking the in vivo condition. The basic unit of cartilage, that is, chondrocyte, requires sufficient shear, strain, and hydrodynamic pressure for regular growth as it is nonvascular tissu...

  4. 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.

  5. Hyaluronan receptor-directed assembly of chondrocyte pericellular matrix

    OpenAIRE

    1993-01-01

    Initial assembly of extracellular matrix occurs within a zone immediately adjacent to the chondrocyte cell surface termed the cell- associated or pericellular matrix. Assembly within the pericellular matrix compartment requires specific cell-matrix interactions to occur, that are mediated via membrane receptors. The focus of this study is to elucidate the mechanisms of assembly and retention of the cartilage pericellular matrix proteoglycan aggregates important for matrix organization. Assemb...

  6. Hydrogen peroxide induces apoptosis via a mitochondrial pathway in chondrocytes

    Science.gov (United States)

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

    2012-03-01

    The degenerative joint disease such as osteoarthritis (OA) is closely associated with the death of chondrocytes in apoptosis fashion. Hydrogen peroxide (H2O2), higher expression following acute damage in OA patients, has been shown to be up-regulated during apoptosis in a bulk of experimental models. This study was aimed to explore the mechanism of H2O2-induced rabbit chondrocytes apoptosis. Articular cartilage was biopsied from the joints of 6 weeks old New Zealand rabbits. Cell Counting Kit (CCK-8) assay was used to assess the inhibitory effect of H2O2 on cell viability. H2O2 treatment induced a remarkable reduction of cell viability. We used flow cytometry to assess the form of cell death with Annexin-V/PI double staining, and found that H2O2 treatment induced apoptosis in a dose-and time-dependent manner. Exposure of chondrocytes to 1.5 mM of H2O2 for 2 h induced a burst apoptosis that can be alleviated by N-acetyl cysteine (NAC) pretreatment, an anti-oxidant amino-acid derivative. Loss of mitochondria membrane potential (▵Ψm) was evaluated using confocal microscopy imaging and flow cytometry (FCM). H2O2 treatment induced a marked reduction of ▵Ψm, and the abrupt disappearance of ▵Ψm occurred within 5 minutes. These results indicate that H2O2 induces a rapid apoptosis via a mitochondrial pathway in rabbit chondrocytes.

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

    Science.gov (United States)

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

    2016-09-01

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

  8. 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.

  9. 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.

  10. Regulation of collagenase inhibitor production in chondrosarcoma chondrocytes

    International Nuclear Information System (INIS)

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

  11. 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.

  12. Looping Mediated Interaction between the Promoter and 3′ UTR Regulates Type II Collagen Expression in Chondrocytes

    OpenAIRE

    Jash, Arijita; Yun, Kangsun; Sahoo, Anupama; So, Jae-Seon; Im, Sin-Hyeog

    2012-01-01

    Type II collagen is the major component of articular cartilage and is mainly synthesized by chondrocytes. Repeated sub-culturing of primary chondrocytes leads to reduction of type II collagen gene (Col2a1) expression, which mimics the process of chondrocyte dedifferentiation. Although the functional importance of Col2a1 expression has been extensively investigated, mechanism of transcriptional regulation during chondrocyte dedifferentiation is still unclear. In this study, we have investigate...

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

    NARCIS (Netherlands)

    Steenvoorden, Marjan Maria Claziena

    2007-01-01

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

  14. 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.

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

    Directory of Open Access Journals (Sweden)

    Eliane Antonioli

    2015-01-01

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

  16. Modulation of Apoptosis and Differentiation by the Treatment of Sulfasalazine in Rabbit Articular Chondrocytes

    Science.gov (United States)

    Lee, Won Kil; Kang, Jin Seok

    2016-01-01

    This study was conducted to examine the cellular regulatory mechanisms of sulfasalazine (SSZ) in rabbit articular chondrocytes treated with sodium nitroprusside (SNP). Cell phenotype was determined, and the MTT assay, Western blot analysis and immunofluorescence staining of type II collagen was performed in control, SNP-treated and SNP plus SSZ (50~200 μg/mL) rabbit articular chondrocytes. Cellular proliferation was decreased significantly in the SNP-treated group compared with that in the control (p < 0.01). SSZ treatment clearly increased the SNP-reduced proliferation levels in a concentration-dependent manner (p < 0.01). SNP treatment induced significant dedifferentiation and inflammation compared with control chondrocytes (p < 0.01). Type II collagen expression levels increased in a concentration-dependent manner in response to SSZ treatment but were unaltered in SNP-treated chondrocytes (p < 0.05 and < 0.01, respectively). Cylooxygenase-2 (COX-2) expression increased in a concentration-dependent manner in response to SSZ treatment but was unaltered in SNP-treated chondrocytes (p < 0.05). Immunofluorescence staining showed that SSZ treatment increased type II collagen expression compared with that in SNP-treated chondrocytes. Furthermore, phosphorylated extracellular regulated kinase (pERK) expression levels were decreased significantly in the SNP-treated group compared with those in control chondrocytes (p < 0.01). Expression levels of pERK increased in a concentration-dependent manner by SSZ but were unaltered in SNP-treated chondrocytes. pp38 kinase expression levels increased in a concentration-dependent manner by SSZ but were unaltered in control chondrocytes (p < 0.01). In summary, SSZ significantly inhibited nitric oxide-induced cell death and dedifferentiation, and regulated extracellular regulated kinases 1 and 2 and p38 kinase in rabbit articular chondrocytes. PMID:27123162

  17. Loss of the mammalian DREAM complex deregulates chondrocyte proliferation.

    Science.gov (United States)

    Forristal, Chantal; Henley, Shauna A; MacDonald, James I; Bush, Jason R; Ort, Carley; Passos, Daniel T; Talluri, Srikanth; Ishak, Charles A; Thwaites, Michael J; Norley, Chris J; Litovchick, Larisa; DeCaprio, James A; DiMattia, Gabriel; Holdsworth, David W; Beier, Frank; Dick, Frederick A

    2014-06-01

    Mammalian DREAM is a conserved protein complex that functions in cellular quiescence. DREAM contains an E2F, a retinoblastoma (RB)-family protein, and the MuvB core (LIN9, LIN37, LIN52, LIN54, and RBBP4). In mammals, MuvB can alternatively bind to BMYB to form a complex that promotes mitotic gene expression. Because BMYB-MuvB is essential for proliferation, loss-of-function approaches to study MuvB have generated limited insight into DREAM function. Here, we report a gene-targeted mouse model that is uniquely deficient for DREAM complex assembly. We have targeted p107 (Rbl1) to prevent MuvB binding and combined it with deficiency for p130 (Rbl2). Our data demonstrate that cells from these mice preferentially assemble BMYB-MuvB complexes and fail to repress transcription. DREAM-deficient mice show defects in endochondral bone formation and die shortly after birth. Micro-computed tomography and histology demonstrate that in the absence of DREAM, chondrocytes fail to arrest proliferation. Since DREAM requires DYRK1A (dual-specificity tyrosine phosphorylation-regulated protein kinase 1A) phosphorylation of LIN52 for assembly, we utilized an embryonic bone culture system and pharmacologic inhibition of (DYRK) kinase to demonstrate a similar defect in endochondral bone growth. This reveals that assembly of mammalian DREAM is required to induce cell cycle exit in chondrocytes. PMID:24710275

  18. Loss of the Mammalian DREAM Complex Deregulates Chondrocyte Proliferation

    Science.gov (United States)

    Forristal, Chantal; Henley, Shauna A.; MacDonald, James I.; Bush, Jason R.; Ort, Carley; Passos, Daniel T.; Talluri, Srikanth; Ishak, Charles A.; Thwaites, Michael J.; Norley, Chris J.; Litovchick, Larisa; DeCaprio, James A.; DiMattia, Gabriel; Holdsworth, David W.; Beier, Frank

    2014-01-01

    Mammalian DREAM is a conserved protein complex that functions in cellular quiescence. DREAM contains an E2F, a retinoblastoma (RB)-family protein, and the MuvB core (LIN9, LIN37, LIN52, LIN54, and RBBP4). In mammals, MuvB can alternatively bind to BMYB to form a complex that promotes mitotic gene expression. Because BMYB-MuvB is essential for proliferation, loss-of-function approaches to study MuvB have generated limited insight into DREAM function. Here, we report a gene-targeted mouse model that is uniquely deficient for DREAM complex assembly. We have targeted p107 (Rbl1) to prevent MuvB binding and combined it with deficiency for p130 (Rbl2). Our data demonstrate that cells from these mice preferentially assemble BMYB-MuvB complexes and fail to repress transcription. DREAM-deficient mice show defects in endochondral bone formation and die shortly after birth. Micro-computed tomography and histology demonstrate that in the absence of DREAM, chondrocytes fail to arrest proliferation. Since DREAM requires DYRK1A (dual-specificity tyrosine phosphorylation-regulated protein kinase 1A) phosphorylation of LIN52 for assembly, we utilized an embryonic bone culture system and pharmacologic inhibition of (DYRK) kinase to demonstrate a similar defect in endochondral bone growth. This reveals that assembly of mammalian DREAM is required to induce cell cycle exit in chondrocytes. PMID:24710275

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

    Directory of Open Access Journals (Sweden)

    Smith Logan B

    2012-12-01

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

  20. 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. PMID:11771670

  1. 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

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

    International Nuclear Information System (INIS)

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

  3. A poroviscohyperelastic model for numerical analysis of mechanical behavior of single chondrocyte.

    Science.gov (United States)

    Nguyen, Trung Dung; Oloyede, Adekunle; Gu, Yuantong

    2016-01-01

    The aim of this paper is to use a poroviscohyperelastic (PVHE) model, which is developed based on the porohyperelastic (PHE) model to explore the mechanical deformation properties of single chondrocytes. Both creep and relaxation responses are investigated by using finite element analysis models of micropipette aspiration and atomic force microscopy experiments, respectively. The newly developed PVHE model is compared thoroughly with the standard neo-Hookean solid and PHE models. It has been found that the PVHE can accurately capture both creep and stress relaxation behaviors of chondrocytes better than other two models. Hence, the PVHE is a promising model to investigate mechanical properties of single chondrocytes. PMID:25588670

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

    International Nuclear Information System (INIS)

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

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

    International Nuclear Information System (INIS)

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

  6. 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.

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

    DEFF Research Database (Denmark)

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

    2001-01-01

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

  8. 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.

  9. 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.

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

    International Nuclear Information System (INIS)

    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

  11. Inhibition of β-Catenin Signaling in Chondrocytes Induces Delayed Fracture Healing in Mice

    OpenAIRE

    Huang, Yang; Zhang, Xiaoling; Du, Kewei; Yang, Fei; Shi, Yu; Huang, Jingang; TANG, TINGTING; Chen, Di; DAI, KERONG

    2011-01-01

    Appropriate and controlled chondrogenesis and endochondral ossification play fundamental roles in the fracture healing cascade, a regenerative process involved in highly coordinated biological events, including the Wnt/β-catenin signaling pathway. To examine the role and importance of this pathway in chondrocytes, we studied bone repair of closed tibias fractures in Col2a1-ICAT transgenic mice, in which the Wnt/β-catenin signaling pathway is specially inhibited in chondrocytes. Radiological, ...

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

    OpenAIRE

    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, indic...

  13. Expression of Two Novel Alternatively Spliced COL2A1 Isoforms During Chondrocyte Differentiation

    OpenAIRE

    McAlinden, Audrey; Johnstone, Brian; Kollar, John; Kazmi, Najam; Hering, Thomas M.

    2007-01-01

    Alternative splicing of the type II procollagen gene (COL2A1) is developmentally-regulated during chondrogenesis. Type IIA procollagen (+ exon 2) is synthesized by chondroprogenitor cells while type IIB procollagen (- exon 2) is synthesized by differentiated chondrocytes. Here, we report expression of two additional alternatively spliced COL2A1 isoforms during chondrocyte differentiation of bone marrow derived mesenchymal stem cells (MSCs). One isoform, named IIC, contains only the first 34 n...

  14. Modulation of Apoptosis and Differentiation by the Treatment of Sulfasalazine in Rabbit Articular Chondrocytes

    OpenAIRE

    Lee, Won Kil; Kang, Jin Seok

    2016-01-01

    This study was conducted to examine the cellular regulatory mechanisms of sulfasalazine (SSZ) in rabbit articular chondrocytes treated with sodium nitroprusside (SNP). Cell phenotype was determined, and the MTT assay, Western blot analysis and immunofluorescence staining of type II collagen was performed in control, SNP-treated and SNP plus SSZ (50~200 μg/mL) rabbit articular chondrocytes. Cellular proliferation was decreased significantly in the SNP-treated group compared with that in the co...

  15. Chondroprotective Effect of Kartogenin on CD44-Mediated Functions in Articular Cartilage and Chondrocytes

    OpenAIRE

    Ono, Yohei; Ishizuka, Shinya; Knudson, Cheryl B.; Knudson, Warren

    2014-01-01

    Objective: A recent report identified the small molecule kartogenin as a chondrogenic and chondroprotective agent. Since changes in hyaluronan metabolism occur during cartilage degeneration in osteoarthritis, we began studies to determine whether there was a connection between extracellular hyaluronan, CD44–hyaluronan interactions and the effects of kartogenin on articular chondrocytes. Methods: Chondrocytes cultured in monolayers, bioengineered neocartilages, or cartilage explants were treat...

  16. Nanocomposite Scaffold for Chondrocyte Growth and Cartilage Tissue Engineering: Effects of Carbon Nanotube Surface Functionalization

    OpenAIRE

    Chahine, Nadeen O.; Collette, Nicole M.; Thomas, Cynthia B.; Genetos, Damian C.; Loots, Gabriela G

    2014-01-01

    The goal of this study was to assess the long-term biocompatibility of single-wall carbon nanotubes (SWNTs) for tissue engineering of articular cartilage. We hypothesized that SWNT nanocomposite scaffolds in cartilage tissue engineering can provide an improved molecular-sized substrate for stimulation of chondrocyte growth, as well as structural reinforcement of the scaffold's mechanical properties. The effect of SWNT surface functionalization (-COOH or -PEG) on chondrocyte viability and bioc...

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

    OpenAIRE

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

    2002-01-01

    Recent studies on osteoarthritis and the cartilage aging in our laboratory demonstrate that chronologic age correlates with molecular changes in human chondrocytes that affect cell cycle control and replicative life span. These findings indicate that age-related changes in chondrocytes may explain the heightened risk for development of primary osteoarthritis (OA) with increasing age. Concomitant studies of human chondrosarcoma suggest that these aging mechanisms may also play a role in preven...

  18. 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.

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

    Directory of Open Access Journals (Sweden)

    Xia Liu

    2014-01-01

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

  20. Stimulation by concanavalin A of cartilage-matrix proteoglycan synthesis in chondrocyte cultures

    Energy Technology Data Exchange (ETDEWEB)

    Yan, W.Q.; Nakashima, K.; Iwamoto, M.; Kato, Y. (Osaka Univ. (Japan))

    1990-06-15

    The effect of concanavalin A on proteoglycan synthesis by rabbit costal and articular chondrocytes was examined. Chondrocytes were seeded at low density and grown to confluency in medium supplemented with 10% fetal bovine serum, and then the serum concentration was reduced to 0.3%. At the low serum concentration, chondrocytes adopted a fibroblastic morphology. Addition of concanavalin A to the culture medium induced a morphologic alteration of the fibroblastic cells to spherical chondrocytes and increased by 3- to 4-fold incorporation of (35S)sulfate and (3H)glucosamine into large chondroitin sulfate proteoglycan that was characteristically found in cartilage. The stimulation of incorporation of labeled precursors reflected real increases in proteoglycan synthesis, as chemical analyses showed a 4-fold increase in the accumulation of macromolecules containing hexuronic acid in concanavalin A-maintained cultures. Furthermore, the effect of concanavalin A on (35S)sulfate incorporation into proteoglycans was greater than that of various growth factors or hormones. However, concanavalin A had smaller effects on (35S)sulfate incorporation into small proteoglycans and (3H)glucosamine incorporation into hyaluronic acid and chondroitinase AC-resistant glycosaminoglycans. Since other lectins tested, such as wheat germ agglutinin, lentil lectin, and phytohemagglutinin, had little effect on (35S)sulfate incorporation into proteoglycans, the concanavalin A action on chondrocytes seems specific. Although concanavalin A decreased (3H)thymidine incorporation in chondrocytes, the stimulation of proteoglycan synthesis could be observed in chondrocytes exposed to the inhibitor of DNA synthesis, cytosine arabinoside. These results indicate that concanavalin A is a potent modulator of proteoglycan synthesis by chondrocytes.

  1. Chondrocytes-Specific Expression of Osteoprotegerin Modulates Osteoclast Formation in Metaphyseal Bone.

    Science.gov (United States)

    Wang, Baoli; Jin, Hongting; Shu, Bing; Mira, Ranim R; Chen, Di

    2015-01-01

    Bone marrow stromal cells/osteoblasts were originally thought to be the major player in regulating osteoclast differentiation through expressing RANKL/OPG cytokines. Recent studies have established that chondrocytes also express RANKL/OPG and support osteoclast formation. Till now, the in vivo function of chondrocyte-produced OPG in osteoclast formation and postnatal bone growth has not been directly investigated. In this study, chondrocyte-specific Opg transgenic mice were generated by using type II collagen promoter. The Col2-Opg transgenic mice showed delayed formation of secondary ossification center and localized increase of bone mass in proximal metaphysis of tibiae. TRAP staining showed that osteoclast numbers were reduced in both secondary ossification center and proximal metaphysis. This finding was further confirmed by in vitro chondrocyte/spleen cell co-culture assay. In contrast, the mineral apposition rates were not changed in Col2-Opg transgenic mice. TUNEL staining revealed more apoptotic hypertrophic chondrocytes in the growth plate of Col2-Opg mice. Flow cytometry analysis showed fewer RANK-expressing cells in the marrow of Col2a1-Opg mice, suggesting the role of OPG in blocking the differentiation of early mesenchymal progenitors into RANK-expressing pre-osteoclasts. Our results demonstrated that OPG expression in chondrocyte increases bone mass in the proximal metaphysis of tibiae through negative regulation of osteoclast formation. PMID:26329493

  2. 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 PCU scaffolds compared with PCL (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 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. PMID:23576328

  3. 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. PMID:27475127

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

    Science.gov (United States)

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

    2016-09-01

    The external ear is composed of elastic cartilage. Microtia is a congenital malformation of the external ear that involves a small reduction in size or a complete absence. The aim of tissue engineering is to regenerate tissues and organs clinically implantable based on the utilization of cells and biomaterials. Remnants from microtia represent a source of cells for auricular reconstruction using tissue engineering. To examine the macromolecular architecture of microtia cartilage and behavior of chondrocytes, in order to enrich the knowledge of this type of cartilage as a cell reservoir. Auricular cartilage remnants were obtained from pediatric patients with microtia undergoing reconstructive procedures. Extracellular matrix composition was characterized using immunofluorescence and histological staining methods. Chondrocytes were isolated and expanded in vitro using a mechanical-enzymatic protocol. Chondrocyte phenotype was analyzed using qualitative PCR. Microtia cartilage preserves structural organization similar to healthy elastic cartilage. Extracellular matrix is composed of typical cartilage proteins such as type II collagen, elastin and proteoglycans. Chondrocytes displayed morphological features similar to chondrocytes derived from healthy cartilage, expressing SOX9, COL2 and ELN, thus preserving chondral phenotype. Cell viability was 94.6 % during in vitro expansion. Elastic cartilage from microtia has similar characteristics, both architectural and biochemical to healthy cartilage. We confirmed the suitability of microtia remnant as a reservoir of chondrocytes with potential to be expanded in vitro, maintaining phenotypical features and viability. Microtia remnants are an accessible source of autologous cells for auricular reconstruction using tissue engineering strategies. PMID:27566509

  5. Mutant activated FGFR3 impairs endochondral bone growth by preventing SOX9 downregulation in differentiating chondrocytes.

    Science.gov (United States)

    Zhou, Zi-Qiang; Ota, Sara; Deng, Chuxia; Akiyama, Haruhiko; Hurlin, Peter J

    2015-03-15

    Fibroblast growth factor receptor 3 (FGFR3) plays a critical role in the control of endochondral ossification, and bone growth and mutations that cause hyperactivation of FGFR3 are responsible for a collection of developmental disorders that feature poor endochondral bone growth. FGFR3 is expressed in proliferating chondrocytes of the cartilaginous growth plate but also in chondrocytes that have exited the cell cycle and entered the prehypertrophic phase of chondrocyte differentiation. Achondroplasia disorders feature defects in chondrocyte proliferation and differentiation, and the defects in differentiation have generally been considered to be a secondary manifestation of altered proliferation. By initiating a mutant activated knockin allele of FGFR3 (FGFR3K650E) that causes Thanatophoric Dysplasia Type II (TDII) specifically in prehypertrophic chondrocytes, we show that mutant FGFR3 induces a differentiation block at this stage independent of any changes in proliferation. The differentiation block coincided with persistent expression of SOX9, the master regulator of chondrogenesis, and reducing SOX9 dosage allowed chondrocyte differentiation to proceed and significantly improved endochondral bone growth in TDII. These findings suggest that a proliferation-independent and SOX9-dependent differentiation block is a key driving mechanism responsible for poor endochondral bone growth in achondroplasia disorders caused by mutations in FGFR3. PMID:25432534

  6. ACTIVITY OF CANONICAL WNT SIGNAL SYSTEM IN HYALINE CARTILAGE ARTICULAR CHONDROCYTES IN PROCESS OF SYNOVIAL JOINT DEVELOPMENT

    Directory of Open Access Journals (Sweden)

    A.O. Molotkov

    2009-03-01

    Full Text Available Canonical and non-canonical Wnt systems are essential regulators of chondrogenesis and bone development. However, the roles of these systems in synovial joint development are not well studied. To determine if canonical Wnt system is active in developing articular chondrocytes we used immunohistochemistry for в-galactosidase and doublecortin (cell-type specific marker for articular chondrocytes to double label sections through joint regions of E14.5, E18.5, P10 and adult mice. Here the following results are presented. Canonical Wnt signal system does not work in developing articular chondrocytes at early embryonic stages (E14.5; it is active in the articular chondrocytes at late embryonic stages (E16.5-E18.5 and during postnatal development (P7-P10, but is turned off again in the adult articular chondrocytes. These results suggest that canonical Wnt signaling is being regulated during articular chondrocytes differentiation and joint formation.

  7. 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.

  8. 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

  9. Compression regulates gene expression of chondrocytes through HDAC4 nuclear relocation via PP2A-dependent HDAC4 dephosphorylation.

    Science.gov (United States)

    Chen, Chongwei; Wei, Xiaochun; Wang, Shaowei; Jiao, Qiang; Zhang, Yang; Du, Guoqing; Wang, Xiaohu; Wei, Fangyuan; Zhang, Jianzhong; Wei, Lei

    2016-07-01

    Biomechanics plays a critical role in the modulation of chondrocyte function. The mechanisms by which mechanical loading is transduced into intracellular signals that regulate chondrocyte gene expression remain largely unknown. Histone deacetylase 4 (HDAC4) is specifically expressed in chondrocytes. Mice lacking HDAC4 display chondrocyte hypertrophy, ectopic and premature ossification, and die early during the perinatal period. HDAC4 has a remarkable ability to translocate between the cell's cytoplasm and nucleus. It has been established that subcellular relocation of HDAC4 plays a critical role in chondrocyte differentiation and proliferation. However, it remains unclear whether subcellular relocation of HDAC4 in chondrocytes can be induced by mechanical loading. In this study, we first report that compressive loading induces HDAC4 relocation from the cytoplasm to the nucleus of chondrocytes via stimulation of Ser/Thr-phosphoprotein phosphatases 2A (PP2A) activity, which results in dephosphorylation of HDAC4. Dephosphorylated HDAC4 relocates to the nucleus to achieve transcriptional repression of Runx2 and regulates chondrocyte gene expression in response to compression. Our results elucidate the mechanism by which mechanical compression regulates chondrocyte gene expression through HDAC4 relocation from the cell's cytoplasm to the nucleus via PP2A-dependent HDAC4 dephosphorylation. PMID:27106144

  10. Fibroblast Growth Factor 18 Increases the Trophic Effects of Bone Marrow Mesenchymal Stem Cells on Chondrocytes Isolated from Late Stage Osteoarthritic Patients

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    Zhenyu Zhang

    2014-01-01

    Full Text Available Coculture of mesenchymal stem cells with chondrocytes increases production of cartilaginous matrix. Chondrocytes isolated from late stage osteoarthritic patients usually lost their phenotype of producing cartilaginous matrix. Fibroblast growth factor 18 is believed to redifferentiate OA chondrocyte into functionally active chondrocytes. The aim of this study is to investigate the supportive effects of MSCs on OA chondrocytes and test if FGF18 could enhance the responsiveness of OA chondrocytes to the support of MSCs in a coculture system. Both pellet and transwell co-cultures were used. GAG quantification, hydroxyproline assay, and qPCR were performed. An ectopic models of cartilage formation was also applied. Our data indicated that, in pellets coculture of MSCs and OA chondrocytes, matrix production was increased in the presence of FGF18, comparing to the monoculture of chondrocytes. Results from transwell coculture study showed that expression of matrix producing genes in OA chondrocytes increased when cocultured with MSCs with FGF18 in culture medium, while hypertrophic genes were not changed by coculture. Finally, coimplantation of MSCs with OA chondrocytes produces more matrix than chondrocytes only. In conclusion, FGF18 can restore the responsiveness of OA chondrocytes to the trophic effects of MSCs. Coimplantation of MSCs and OA chondrocytes treated with FGF18 may be a good alternative cell source for regenerating cartilage tissue that is degraded during OA pathological changes.

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

    Science.gov (United States)

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

    2015-03-01

    Culturing chondrocytes under oxygen tension closely resembling their in vivo environment has been shown to have positive effects on matrix synthesis. In redifferentiation of expanded chondrocytes, hypoxia increased collagen type II expression. However, the mechanism by which hypoxia enhances redifferentiation is still unknown. We employed novel bioreactor technology to investigate the role of TGF-β, a growth factor heavily implicated in matrix production, in chondrocytes under hypoxia. Dedifferentiated chondrocytes in alginate were cultured for 48h under hypoxic (1% pO2) or normoxic (20%) conditions, using specialized bioreactor technology. Hypoxia induced gene expression (GDF1-, PHD3, HAS2, VEGF, COX2), chondrocyte markers (SOX9, COL2, COL1, AGC1 and MMP13), as well as components of the TGF-β signaling pathway (TGF-β isoforms, receptors, and downstream effectors) were analyzed by qPCR after 48h. In addition, protein expression of COL2 and TGF-β2 were evaluated. To further elucidate the involvement of the TGF-β2, we used siRNA and ALK5 inhibition. Hypoxic culture showed robust upregulation of hypoxic markers as well as upregulation of SOX9 and COL2 expression. Of all TGF-β isoforms, only TGF-β2 was upregulated under hypoxia on both gene and protein level. In addition, both type I receptors (ALK1 and ALK5) were upregulated under hypoxia, but type II and III receptors were not. TGF-β2 downregulation via siRNA abrogated the hypoxia-induced COL2 expression, as did ALK5 inhibition, giving a strong indication that this pathway is involved in chondrocyte redifferentiation under low oxygen tension. Hypoxic culture is a common approach for cartilage tissue engineering, but its underlying mechanisms are still poorly understood. Here, we show that increased TGF-β2 signaling through ALK5 plays a role in hypoxia-induced redifferentiation of chondrocytes. PMID:25621374

  12. Mesenchymal stromal cell-derived extracellular matrix influences gene expression of chondrocytes

    International Nuclear Information System (INIS)

    Decellularized extracellular matrix (ECM) has recently gained a lot of interest as an instructive biomaterial for regenerative medicine applications. In this study, the ability of adult human mesenchymal stem cell (hMSC)-derived ECM to rescue the phenotype of osteoarthritic (OA) chondrocytes and to further stimulate the differentiation of healthy (HL) chondrocytes was evaluated. ECMs were prepared by decellularizing hMSCs cultured in basic medium (BM) and chondrogenic medium (CM). The obtained ECM was then combined with a polymeric solution of Poly (ε-caprolactone) (PCL) dissolved in 1, 1, 1, 3, 3, 3-hexafluoro-2-propanol (HFIP) and electrospun meshes were fabricated. Electrospun ECM scaffolds were characterized using scanning electron microscopy (SEM) and picrosirius red staining was used to confirm the presence of collagen. OA and HL chondrocytes were cultured on scaffolds containing hMSC ECM in BM or CM and compared to PCL electrospun scaffolds without ECM. Metabolic activity and chondrogenic gene expression were assessed by Alamar blue assay and quantitative PCR (qPCR) analysis, respectively. The ECM presence resulted in a significant difference in chondrocyte metabolic activity compared to PCL scaffolds alone. HL chondrocytes cultured for 21 days in chondrogenic medium on electrospun scaffolds containing hMSC ECM from BM showed a significant increase in collagen II and aggrecan expression compared to hMSC ECM from CM and PCL scaffolds without ECM incorporation. No significant influence of hMSC ECM presence on the chondrogenic signature of OA chondrocytes was found. The influence of decellularized hMSC ECM on HL chondrocytes suggests that hMSC-derived ECM scaffolds are promising candidates for cartilage tissue engineering applications. (paper)

  13. Effects of PTHrP on chondrocytes of sika deer antler.

    Science.gov (United States)

    Guo, Bin; Wang, Shou-Tang; Duan, Cui-Cui; Li, Dang-Dang; Tian, Xue-Chao; Wang, Qu-Yuan; Yue, Zhan-Peng

    2013-11-01

    Parathyroid-hormone-related peptide (PTHrP) is an important regulator of chondrocyte differentiation in growth plates but little is known about its role in deer antler cartilage. The aim of the present study was to use the deer antler as a model to determine the possible role of PTHrP in regulating chondrocyte differentiation of deer antler. PTHrP and its receptor PTH1R mRNA were highly expressed in the perichondrium and cartilage of sika deer antler, as shown by in situ hybridization. Chondrocytes of deer antler were identified by toluidine blue staining of glycosaminoglycan and immunocytochemical staining of type II collagen (Col II). Treatment with PTHrP (1-34) reduced the expression of prehypertrophic chondrocyte marker Col IX and hypertrophic chondrocyte marker Col X. In order to confirm the mechanism of action of PTHrP, we initially examined the expression of cyclin D1, Bcl-2 and runt-related transcription factor 2 (Runx2) in sika deer antler by in situ hybridization and found that cyclin D1, Runx2 and Bcl-2 mRNA were also expressed in antler chondrocytes. Exogenous PTHrP induced the expression of cyclin D1 and Bcl-2 mRNA by various signalling pathways, whereas it inhibited Runx2 expression through PKA, p38MAPK, MEK and PI3K signalling pathways. Thus, PTHrP might promote the proliferation of antler chondrocytes and prevent their differentiation; it might furthermore influence the growth and development of sika deer antler. PMID:23824099

  14. 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).

  15. 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

    2016-12-01

    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). PMID:27178054

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

    Science.gov (United States)

    Shakibaei, Mehdi; Mobasheri, Ali; Buhrmann, Constanze

    2011-05-01

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

  17. An ovine in vitro model for chondrocyte-based scaffold-assisted cartilage grafts

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    Endres Michaela

    2012-11-01

    Full Text Available Abstract Background Scaffold-assisted autologous chondrocyte implantation is an effective clinical procedure for cartilage repair. From the regulatory point of view, the ovine model is one of the suggested large animal models for pre-clinical studies. The aim of our study was to evaluate the in vitro re-differentiation capacity of expanded ovine chondrocytes in biomechanically characterized polyglycolic acid (PGA/fibrin biomaterials for scaffold-assisted cartilage repair. Methods Ovine chondrocytes harvested from adult articular cartilage were expanded in monolayer and re-assembled three-dimensionally in PGA-fibrin scaffolds. De- and re-differentiation of ovine chondrocytes in PGA-fibrin scaffolds was assessed by histological and immuno-histochemical staining as well as by real-time gene expression analysis of typical cartilage marker molecules and the matrix-remodelling enzymes matrix metalloproteinases (MMP -1, -2 and −13 as well as their inhibitors. PGA scaffolds characteristics including degradation and stiffness were analysed by electron microscopy and biomechanical testing. Results Histological, immuno-histochemical and gene expression analysis showed that dedifferentiated chondrocytes re-differentiate in PGA-fibrin scaffolds and form a cartilaginous matrix. Re-differentiation was accompanied by the induction of type II collagen and aggrecan, while MMP expression decreased in prolonged tissue culture. Electron microscopy and biomechanical tests revealed that the non-woven PGA scaffold shows a textile structure with high tensile strength of 3.6 N/mm2 and a stiffness of up to 0.44 N/mm2, when combined with gel-like fibrin. Conclusion These data suggest that PGA-fibrin is suited as a mechanically stable support structure for scaffold-assisted chondrocyte grafts, initiating chondrogenic re-differentiation of expanded chondrocytes.

  18. Distinct Effect of TCF4 on the NFκB Pathway in Human Primary Chondrocytes and the C20/A4 Chondrocyte Cell Line

    NARCIS (Netherlands)

    Landman, E.B.M.; Periyasamy, P.C.; Blitterswijk, van C.A.; Post, J.N.; Karperien, M.

    2014-01-01

    Objective: Previous studies indicated a difference in crosstalk between canonical WNT pathway and nuclear factor-κB (NFκB) signaling in human and animal chondrocytes. To assess whether the differences found were dependent on cell types used, we tested the effect of WNT modulation on NFκB signaling i

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

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    Sung Won Lee

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

  20. G-protein stimulatory subunit alpha and Gq/11α G-proteins are both required to maintain quiescent stem-like chondrocytes

    OpenAIRE

    Chagin, Andrei S; Vuppalapati, Karuna K; Kobayashi, Tatsuya; Guo, Jun; Hirai, Takao; Chen, Min; Offermanns, Stefan; Lee S Weinstein; Kronenberg, Henry M.

    2014-01-01

    Round chondrocytes in the resting zone of the growth plate provide precursors for columnar chondrocytes and have stem-like properties. Here we demonstrate that these stem-like chondrocytes undergo apoptosis in the absence of the receptor (PPR) for parathyroid hormone-related protein. We examine the possible roles of heterotrimeric G-proteins activated by the PPR. Inactivation of the G-protein stimulatory α-subunit (Gsα) leads to accelerated differentiation of columnar chondrocytes, as seen in...

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

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    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.

  2. 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. PMID:26987846

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

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    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.

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

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    Leilei Zhong

    2015-08-01

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

  5. The differential effect of scaffold composition and architecture on chondrocyte response to mechanical stimulation.

    Science.gov (United States)

    Appelman, Taly P; Mizrahi, Joseph; Elisseeff, Jennifer H; Seliktar, Dror

    2009-02-01

    This study aims to explore the differential effect of scaffold composition and architecture on chondrogenic response to dynamic strain stimulation using encapsulating PEG-based hydrogels and primary bovine chondrocytes. Proteins and proteoglycans were conjugated to functionalized poly(ethylene glycol) (PEG) and immobilized in PEG hydrogels to create bio-synthetic materials to be used as scaffolds. Four different compositions were tested, including: PEG-Proteoglycan (PP), PEG-Fibrinogen (PF), PEG-Albumin (PA), and PEG only. Primary articular chondrocytes were encapsulated in the hydrogel scaffolds and subjected to 15% dynamic compressive strain stimulation at 1-Hz frequency for 28 days. Stimulation of PP, PF, PA and PEG constructs resulted in a respective increase in the unconfined true compressive modulus by 32%, 45.4%, 33.6%, and 28.2%, compared to their static controls. The PF showed a significantly larger relative increase in the modulus in comparison to all other scaffolds tested. These results support the hypothesis that mechanical stimulation and material bioactivity have a significant effect on the reported chondrocyte response. Similar trends were observed with the swelling ratio of the constructs. These findings indicate that while stimulation causes metabolic changes in chondrocytes seeded in PEG hydrogels, the matrix bioactivity has a significant role in enhancing chondrocyte mechanotransduction in encapsulating scaffolds subjected to physical deformations. PMID:19000634

  6. Effect of transforming growth factor-β3 on mono and multilayer chondrocytes.

    Science.gov (United States)

    Sefat, Farshid; Youseffi, Mansour; Khaghani, Seyed Ali; Soon, Chin Fhung; Javid, Farideh

    2016-07-01

    Articular cartilage is an avascular and flexible connective tissue found in joints. It produces a cushioning effect at the joints and provides low friction to protect the ends of the bones from wear and tear/damage. It has poor repair capacity and any injury can result pain and loss of mobility. Transforming growth factor-beta (TGF-β), a cytokine superfamily, regulates cell function, including differentiation and proliferation. Although the function of the TGF-βs in various cell types has been investigated, their function in cartilage repair is as yet not fully understood. The effect of TGF-β3 in biological regulation of primary chondrocyte was investigated in this work. TGF-β3 provided fibroblastic morphology to chondrocytes and therefore overall reduction in cell proliferation was observed. The length of the cells supplemented with TGF-β3 were larger than the cells without TGF-β3 treatment. This was caused by the fibroblast like cells (dedifferentiated chondrocytes) which occupied larger areas compared to cells without TGF-β3 addition. The healing process of the model wound closure assay of chondrocyte multilayer was slowed down by TGF-β3, and this cytokine negatively affected the strength of chondrocyte adhesion to the cell culture surface. PMID:27108397

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

    Science.gov (United States)

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

    2016-08-01

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

  8. Del1 Knockout Mice Developed More Severe Osteoarthritis Associated with Increased Susceptibility of Chondrocytes to Apoptosis

    Science.gov (United States)

    Wang, Zhen; Tran, Misha C.; Bhatia, Namrata J.; Hsing, Alexander W.; Chen, Carol; LaRussa, Marie F.; Fattakhov, Ernst; Rashidi, Vania; Jang, Kyu Yun; Choo, Kevin J.; Nie, Xingju; Mathy, Jonathan A.; Longaker, Michael T.; Dauskardt, Reinhold H.; Helms, Jill A.; Yang, George P.

    2016-01-01

    Objective We identified significant expression of the matricellular protein, DEL1, in hypertrophic and mature cartilage during development. We hypothesized that this tissue-specific expression indicated a biological role for DEL1 in cartilage biology. Methods Del1 KO and WT mice had cartilage thickness evaluated by histomorphometry. Additional mice underwent medial meniscectomy to induce osteoarthritis, and were assayed at 1 week for apoptosis by TUNEL staining and at 8 weeks for histology and OA scoring. In vitro proliferation and apoptosis assays were performed on primary chondrocytes. Results Deletion of the Del1 gene led to decreased amounts of cartilage in the ears and knee joints in mice with otherwise normal skeletal morphology. Destabilization of the knee led to more severe OA compared to controls. In vitro, DEL1 blocked apoptosis in chondrocytes. Conclusion Osteoarthritis is among the most prevalent diseases worldwide and increasing in incidence as our population ages. Initiation begins with an injury resulting in the release of inflammatory mediators. Excessive production of inflammatory mediators results in apoptosis of chondrocytes. Because of the limited ability of chondrocytes to regenerate, articular cartilage deteriorates leading to the clinical symptoms including severe pain and decreased mobility. No treatments effectively block the progression of OA. We propose that direct modulation of chondrocyte apoptosis is a key variable in the etiology of OA, and therapies aimed at preventing this important step represent a new class of regenerative medicine targets. PMID:27505251

  9. Elevation of IGFBP2 contributes to mycotoxin T-2-induced chondrocyte injury and metabolism.

    Science.gov (United States)

    Wang, Xiaoqing; Zhang, Yan; Chang, Yanhai; Duan, Dapeng; Sun, Zhengming; Guo, Xiong

    2016-09-01

    Kashin-Beck disease (KBD) is an endemic degenerative osteoarthropathy. The mycotoxin of T-2 toxin is extensively accepted as a major etiological contributor to KBD. However, its function and mechanism in KBD remains unclearly elucidated. Here, T-2 toxin treatment induced chondrocyte injury in a time- and dose-dependent manner by repressing cell viability and promoting cell necrosis and apoptosis. Importantly, T-2 suppressed the transcription of type II collagen and aggrecan, as well as the release of sulphated glycosaminoglycan (sGAG). Furthermore, exposure to T-2 enhanced the transcription of matrix metalloproteinases (MMPs), including MMP-1, -2, -3 and -9. In contrast to control groups, higher expression of insulin-like growth factor binding protein 2 (IGFBP2) was observed in chondrocytes from KBD patients. Interestingly, T-2 toxin caused a dramatical elevation of IGFBP2 expression in chondrocytes. Mechanism analysis corroborated that cessation of IGFBP2 expression alleviated T-2-induced damage to chondrocytes. Simultaneously, transfection with IGFBP2 siRNA also attenuated matrix synthesis and catabolism-related gene expressions of MMPs. Together, this study validated that T-2 toxin exposure might promote the progression of KBD by inducing chondrocyte injury, suppressing matrix synthesis and accelerating cellular catabolism through IGFBP2. Therefore, this research will elucidate a new insight about how T-2 toxin participate in the pathogenesis of KBD. PMID:27416762

  10. Dependence of light attenuation and backscattering on collagen concentration and chondrocyte density in agarose scaffolds

    International Nuclear Information System (INIS)

    Optical coherence tomography (OCT) has been applied for high resolution imaging of articular cartilage. However, the contribution of individual structural elements of cartilage on OCT signal has not been thoroughly studied. We hypothesize that both collagen and chondrocytes, essential structural components of cartilage, act as important light scatterers and that variation in their concentrations can be detected by OCT through changes in backscattering and attenuation. To evaluate this hypothesis, we established a controlled model system using agarose scaffolds embedded with variable collagen concentrations and chondrocyte densities. Using OCT, we measured the backscattering coefficient (µb) and total attenuation coefficient (µt) in these scaffolds. Along our hypothesis, light backscattering and attenuation in agarose were dependent on collagen concentration and chondrocyte density. Significant correlations were found between µt and chondrocyte density (ρ = 0.853, p < 0.001) and between µt and collagen concentration (ρ = 0.694, p < 0.001). µb correlated significantly with chondrocyte density (ρ = 0.504, p < 0.001) but not with collagen concentration (ρ = 0.103, p = 0.422) of the scaffold. Thus, quantitation of light backscattering and, especially, attenuation could be valuable when evaluating the integrity of soft tissues, such as articular cartilage with OCT. (paper)

  11. Characterization of chondrocyte sheets prepared using a co-culture method with temperature-responsive culture inserts.

    Science.gov (United States)

    Kokubo, Mami; Sato, Masato; Yamato, Masayuki; Mitani, Genya; Kutsuna, Toshiharu; Ebihara, Goro; Okano, Teruo; Mochida, Joji

    2016-06-01

    Conventional culture methods using temperature-responsive culture dishes require 4-5 weeks to prepare layered chondrocyte sheets that can be used in articular cartilage repair and regeneration. This study investigated whether the use of synovial tissue obtained from the same joint as the chondrocyte nutritive supply source could more quickly facilitate the preparation of chondrocyte sheets. After culturing derived synoviocytes and chondrocytes together (i.e. combined culture or co-culture) on temperature-responsive inserts, chondrocyte growth was assessed and a molecular analysis of the chondrocyte sheets was performed. Transplantable tissue could be obtained more quickly using this method (average 10.5 days). Real-time polymerase chain reaction and immunostaining of the three-layer chondrocyte sheets confirmed the significant expression of genes critical to cartilage maintenance, including type II collagen (COL2), aggrecan-1 and tissue metallopeptidase inhibitor 1. However, the expression of COL1, matrix metalloproteinase 3 (MMP3), MMP13 and A-disintegrin and metalloproteinase with thrombospondin motifs 5 was suppressed. The adhesive factor fibronectin-1 (FN1) was observed in all sheet layers, whereas in sheets generated using conventional preparation methods positive FN1 immunostaining was observed only on the surface of the sheets. The results indicate that synoviocyte co-cultures provide an optimal environment for the preparation of chondrocyte sheets for tissue transplantation and are particularly beneficial for shortening the required culture period. Copyright © 2013 John Wiley & Sons, Ltd. PMID:23868865

  12. PKCa Agonists Enhance the Protective Effect of Hyaluronic Acid on Nitric Oxide-Induced Apoptosis of Articular Chondrocytes in Vitro

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    Jian-lin Zhou

    2013-12-01

    The results may be showed that PKCa regulate the expresion of caspase-3, which contribute to the apoptosis of chondrocytes induced by NO. PKC α agonists enhance the protective effect of hyaluronic acid on nitric oxide-induced articular chondrocytes apoptosis.

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

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    Trung Dung Nguyen

    2014-01-01

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

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

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    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.

  15. Location of 64K collagen producer chondrocytes in developing chicken embryo tibiae

    International Nuclear Information System (INIS)

    The synthesis of a new low-molecular-weight collagen by cultured chicken embryo chondrocytes has been recently demonstrated. In this paper the authors report results on the location of chondrocytes synthesizing this new collagen (64K collagen) in the developing chicken embryo. The 64K collagen is synthesized in very large amounts by cells concentrated at the diaphysis of 9-day-old and at the epiphysis of 17-day-old embryo tibiae. These regions are characterized by a remodeling of the cartilage matrix leading to the replacement of the cartilage with bone tissue; therefore, this collagen appears to be a marker of a specific developmental stage of chondrocytes. The origin of cells competent for the synthesis of the 64K collagen is also discussed

  16. Initiation of Chondrocyte Self-Assembly Requires an Intact Cytoskeletal Network.

    Science.gov (United States)

    Lee, Jennifer K; Hu, Jerry C Y; Yamada, Soichiro; Athanasiou, Kyriacos A

    2016-02-01

    Self-assembly and self-organization have recently emerged as robust scaffold-free tissue engineering methodologies that can be used to generate various tissues, including cartilage, vessel, and liver. Self-assembly, in particular, is a scaffold-free platform for tissue engineering that does not require the input of exogenous energy to the system. Although self-assembly can generate functional tissues, most notably neocartilage, the mechanisms of self-assembly remain unclear. To study the self-assembling process, we used articular chondrocytes as a model to identify parameters that can affect this process. Specifically, the roles of cell-cell and cell-matrix adhesion molecules, surface-bound collagen, and the actin cytoskeletal network were investigated. Using time-lapse imaging, we analyzed the early stages of chondrocyte self-assembly. Within hours, chondrocytes rapidly coalesced into cell clusters before compacting to form tight cellular structures. Chondrocyte self-assembly was found to depend primarily on integrin function and secondarily on cadherin function. In addition, actin or myosin II inhibitors prevented chondrocyte self-assembly, suggesting that cell adhesion alone is not sufficient, but rather the active contractile actin cytoskeleton is essential for proper chondrocyte self-assembly and the formation of neocartilage. Better understanding of the self-assembly mechanisms allows for the rational modulation of this process toward generating neocartilages with improved properties. These findings are germane to understanding self-assembly, an emerging platform for tissue engineering of a plethora of tissues, especially as these neotissues are poised for translation. PMID:26729374

  17. Hyperosmolarity regulates SOX9 mRNA posttranscriptionally in human articular chondrocytes

    OpenAIRE

    Tew, Simon R.; Peffers, Mandy J.; McKay, Tristan R; Lowe, Emma T.; Khan, Wasim S; Hardingham, Timothy E.; Clegg, Peter D

    2009-01-01

    The transcription factor SOX9 regulates cartilage extracellular matrix gene expression and is essential for chondrocyte differentiation. We previously showed that activation of p38 MAPK by cycloheximide in human chondrocytes leads to stabilization of SOX9 mRNA (Tew SR and Hardingham TE. J Biol Chem 281: 39471–39479, 2006). In this study we investigated whether regulation of p38 MAPK caused by changes in osmotic pressure could control SOX9 mRNA levels expression by a similar mechanism. Primary...

  18. Derivation of Chondrocyte and Osteoblast Reporter Mouse Embryonic Stem Cell Lines

    OpenAIRE

    Fu, Yu; Maye, Peter

    2015-01-01

    With the establishment of methods that provide evidence for the generation of chondrocyte and osteoblast cell types from ESCs, there is a need for reagents that will enable their further characterization. Here we report on the derivation of chondrocyte and osteoblast reporter ESCs from previously generated and characterized transgenic mouse lines, Collagen type 2 alpha 1(Col2a1)-ECFP, Bone Sialoprotein (BSP)-Topaz, and BSP-Topaz/Dentin Matrix Protein 1 (DMP1)-Cherry dual reporter mice. Col2a1...

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

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    Natalya Krivoruchko

    2014-12-01

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

  20. Wnt induction of chondrocyte hypertrophy through the Runx2 transcription factor.

    Science.gov (United States)

    Dong, Yu-Feng; Soung, Do Y; Schwarz, Edward M; O'Keefe, Regis J; Drissi, Hicham

    2006-07-01

    We investigated the molecular mechanisms underlying canonical Wnt-mediated regulation of chondrocyte hypertrophy using chick upper sternal chondrocytes. Replication competent avian sarcoma (RCAS) viral over-expression of Wnt8c and Wnt9a, upregulated type X collagen (col10a1) and Runx2 mRNA expression thereby inducing chondrocyte hypertrophy. Wnt8c and Wnt9a strongly inhibited mRNA levels of Sox9 and type II collagen (col2a1). Wnt8c further enhanced canonical bone morphogenetic proteins (BMP-2)-induced expression of Runx2 and col10a1 while Wnt8c and Wnt9a inhibited TGF-beta-induced expression of Sox9 and col2a1. Over-expression of beta-catenin mimics the effect of Wnt8c and Wnt9a by upregulating Runx2, col10a1, and alkaline phosphatase (AP) mRNA levels while it inhibits col2a1 transcription. Western blot analysis shows that Wnt8c and beta-catenin also induces Runx2 protein levels in chondrocytes. Thus, our results indicate that activation of the canonical beta-catenin Wnt signaling pathway induces chondrocyte hypertrophy and maturation. We further investigated the effects of beta-catenin-TCF/Lef on Runx2 promoter. Co-transfection of lymphoid enhancer factor (Lef1) and beta-catenin in chicken upper sternal chondrocytes together with deletion constructs of the Runx2 promoter shows that the proximal region spanning the first 128 base pairs of this promoter is responsible for the Wnt-mediated induction of Runx2. Mutation of the TCF/Lef binding site in the -128 fragment of the Runx2 promoter resulted in loss of its responsiveness to beta-catenin. Additionally, gel-shift assay analyses determined the DNA/protein interaction of the TCF/Lef binding sites on the Runx2 promoter. Finally, our site-directed mutagenesis data demonstrated that the Runx2 site on type X collagen promoter is required for canonical Wnt induction of col10a1. Altogether we demonstrate that Wnt/beta-catenin signaling is regulated by TGF-beta and BMP-2 in chick upper sternal chondrocytes, and mediates

  1. Chondroprotective effects and mechanisms of resveratrol in advanced glycation end products-stimulated chondrocytes

    OpenAIRE

    Liu, Feng-Cheng; Hung, Li-Feng; Wu, Wan-Lin; Chang, Deh-Ming; Huang, Chuan-Yueh; Lai, Jenn-Haung; Ho, Ling-Jun

    2010-01-01

    Introduction Accumulation of advanced glycation end products (AGEs) in joints contributes to the pathogenesis of cartilage damage in osteoarthritis (OA). We aim to explore the potential chondroprotective effects of resveratrol on AGEs-stimulated porcine chondrocytes and cartilage explants. Methods Chondrocytes were isolated from pig joints. Activation of the IκB kinase (IKK)-IκBα-nuclear factor-kappaB (NF-κB) and c-Jun N-terminal kinase (JNK)/extracellular signal-regulated kinase (ERK)-activa...

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

    International Nuclear Information System (INIS)

    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/TNFR1 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, TNFR1, 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 TNFR1 was suppressed with its siRNA. The protein levels of TNFα, TNFR1 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, TNFR1 and caspase-12 increased after the chondrocytes were exposed to ofloxacin. The downregulated mRNA expressions of TNFR1, Caspase-8 and TRADD, and the decreased apoptosis rates of the ofloxacin-treated chondrocytes occurred after TNFR1–siRNA interference. Conclusions and implications: Ofloxacin-induced chondrocyte apoptosis in a time- and concentration-dependent fashion. TNF/TNFR1 pathway and ERs are involved in ofloxacin-induced apoptosis of juvenile canine chondrocytes in the early stage. - Highlights: • Chondrocyte apoptosis is induced by ofloxacin in a time- and concentration-dependent manners. • TNF/TNFR1

  3. Wnt/β-catenin signaling of cartilage canal and osteochondral junction chondrocytes and full thickness cartilage in early equine osteochondrosis.

    Science.gov (United States)

    Kinsley, Marc A; Semevolos, Stacy A; Duesterdieck-Zellmer, Katja F

    2015-10-01

    The objective of this study was to elucidate gene and protein expression of Wnt signaling molecules in chondrocytes of foals having early osteochondrosis (OC) versus normal controls. The hypothesis was that increased expression of components of Wnt signaling pathway in osteochondral junction (OCJ) and cartilage canal (CC) chondrocytes would be found in early OC when compared to controls. Paraffin-embedded osteochondral samples (7 OC, 8 normal) and cDNA from whole cartilage (7 OC, 10 normal) and chondrocytes surrounding cartilage canals and osteochondral junctions captured with laser capture microdissection (4 OC, 6 normal) were obtained from femoropatellar joints of 17 immature horses. Equine-specific Wnt signaling molecule mRNA expression levels were evaluated by two-step real-time qPCR. Spatial tissue protein expression of β-catenin, Wnt-11, Wnt-4, and Dkk-1 was determined by immunohistochemistry. There was significantly decreased Wnt-11 and increased β-catenin, Wnt-5b, Dkk-1, Lrp6, Wif-1, Axin1, and SC-PEP gene expression in early OC cartilage canal chondrocytes compared to controls. There was also significantly increased β-catenin gene expression in early OC osteochondral junction chondrocytes compared to controls. Based on this study, abundant gene expression differences in OC chondrocytes surrounding cartilage canals suggest pathways associated with catabolism and inhibition of chondrocyte maturation are targeted in early OC pathogenesis. PMID:25676127

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

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    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.

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

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    R.R. Filgueiras

    2014-02-01

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

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

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    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.

  7. Different hyaluronic acid morphology modulates primary articular chondrocyte behavior in hyaluronic acid-coated polycaprolactone scaffolds

    OpenAIRE

    Lebourg, Myriam; Rodenas Rochina, Joaquín; Sousa, Tiago; Mano, J. F.; Gómez Ribelles, J. L.

    2013-01-01

    Scaffolds for cartilage tissue engineering should promote both adequate biomechanical environment and chondrogenic stimulation. Hyaluronic acid (HA) has been used in cartilage engineering for its chondrogenic and chondroprotective properties, nevertheless its mechanical properties are limited. Influence of HA microstructure in chondrocyte response has not been addressed yet. In this work, polycaprolactone (PCL) scaffolds were modified using HA following two coating str...

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

    International Nuclear Information System (INIS)

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

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

    Science.gov (United States)

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

    2013-01-01

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

  10. 3D Hydrogel Scaffolds for Articular Chondrocyte Culture and Cartilage Generation.

    Science.gov (United States)

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

    2015-01-01

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

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

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    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.

  12. Effects of electromagnetic field frequencies on chondrocytes in 3D cell-printed composite constructs.

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    Yi, Hee-Gyeong; Kang, Kyung Shin; Hong, Jung Min; Jang, Jinah; Park, Moon Nyeo; Jeong, Young Hun; Cho, Dong-Woo

    2016-07-01

    In cartilage tissue engineering, electromagnetic field (EMF) therapy has been reported to have a modest effect on promoting cartilage regeneration. However, these studies were conducted using different frequencies of EMF to stimulate chondrocytes. Thus, it is necessary to investigate the effect of EMF frequency on cartilage formation. In addition to the stimulation, a scaffold is required to satisfy the characteristics of cartilage such as its hydrated and dense extracellular matrix, and a mechanical resilience to applied loads. Therefore, we 3D-printed a composite construct composed of a polymeric framework and a chondrocyte-laden hydrogel. Here, we observed frequency-dependent positive and negative effects on chondrogenesis using a 3D cell-printed cartilage tissue. We found that a frequency of 45 Hz promoted gene expression and secretion of extracellular matrix molecules of chondrocytes. In contrast, a frequency of 7.5 Hz suppressed chondrogenic differentiation in vitro. Additionally, the EMF-treated composite constructs prior to implantation showed consistent results with those of in vitro, suggesting that in vitro pre-treatment with different EMF frequencies provides different capabilities for the enhancement of cartilage formation in vivo. This correlation between EMF frequency and 3D-printed chondrocytes suggests the necessity for optimization of EMF parameters when this physical stimulus is applied to engineered cartilage. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 1797-1804, 2016. PMID:26991030

  13. Evaluation of Differentially Expressed Genes by Shear Stress in Human Osteoarthritic Chondrocytes In Vitro

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    Mel S. Lee

    2009-02-01

    Full Text Available Background: The pathogenesis of osteoarthritis is related to abnormal mechanical stressesthat alter cartilage metabolism and chondrocyte survival. Among themechanical stresses, shear stress is held responsible for the development ofarthritis.Methods: Monolayer cultures of human osteoarthritic chondrocytes were subjected tofluid-induced shear stress in vitro. A cDNA microarray technology was usedto screen the differentially regulated genes and quantitative real-time polymerasechain reaction (Q-RT-PCR was used to confirm the results. The significanceof the expression ratio for each gene was determined on the lowestassociated false discovery rate calculated from the changes of gene expressionin relation to the standard deviation of repeated measurements for thatgene.Results: Exposure of human osteoarthritic chondrocytes to shear stress (0.82 Pa for 2hours differentially regulated 373 and 227 clones in two independentmicroarray analyses with at least a 1.7-fold change. By comparing the differentiallyregulated clones, 14 upregulated and 6 downregulated genes wereidentified. Many of the differentially expressed genes were related to cellproliferation/differentiation (TGF-β, acidic FGF, cell survival/apoptosis(CYP1B1, BCL2L3, TNFRSF11B, chemokine ligands, ADM, and matrixhomeostasis (DCN, SDC2, MGP, WISP2.Conclusion: The gene expression patterns following shear stress show a high similarity tothe gene expression in the reparative process of osteoarthritis chondrocytes.Using microarray analysis, this study suggests a close interaction betweenshear stress and the pathogenesis of osteoarthritis.

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

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

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    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.

  16. 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

  17. NF-κB regulates Lef1 gene expression in chondrocytes

    International Nuclear Information System (INIS)

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

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

    Science.gov (United States)

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

    2016-06-01

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

  19. 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. PMID:24039062

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

    Science.gov (United States)

    Akkiraju, Hemanth; Nohe, Anja

    2016-01-01

    Articular cartilage (AC) covers the diarthrodial joints and is responsible for the mechanical distribution of loads across the joints. The majority of its structure and function is controlled by chondrocytes that regulate Extracellular Matrix (ECM) turnover and maintain tissue homeostasis. Imbalance in their function leads to degenerative diseases like Osteoarthritis (OA). OA is characterized by cartilage degradation, osteophyte formation and stiffening of joints. Cartilage degeneration is a consequence of chondrocyte hypertrophy along with the expression of proteolytic enzymes. Matrix Metalloproteinases (MMPs) and A Disintegrin and Metalloproteinase with Thrombospondin Motifs (ADAMTS) are an example of these enzymes that degrade the ECM. Signaling cascades involved in limb patterning and cartilage repair play a role in OA progression. However, the regulation of these remains to be elucidated. Further the role of stem cells and mature chondrocytes in OA progression is unclear. The progress in cell based therapies that utilize Mesenchymal Stem Cell (MSC) infusion for cartilage repair may lead to new therapeutics in the long term. However, many questions are unanswered such as the efficacy of MSCs usage in therapy. This review focuses on the role of chondrocytes in cartilage formation and the progression of OA. Moreover, it summarizes possible alternative therapeutic approaches using MSC infusion for cartilage restoration. PMID:27347486

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

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    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.

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

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    Mallein-Gerin Frédéric

    2008-09-01

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

  3. Adaptation of chondrocytes to low oxygen tension: relationship between hypoxia and cellular metabolism.

    Science.gov (United States)

    Rajpurohit, R; Koch, C J; Tao, Z; Teixeira, C M; Shapiro, I M

    1996-08-01

    In endochondral bone, the growth cartilage is the site of rapid growth. Since the vascular supply to the cartilage is limited, it is widely assumed that cells of the cartilage are hypoxic and that limitations in the oxygen supply regulate the energetic state of the maturing cells. In this report, we evaluate the effects of oxygen tension on chondrocyte energy metabolism, thiol status, and expression of transcription elements, HIF and AP-1. Imposition of an hypoxic environment on cultured chondrocytes caused a proportional increase in glucose utilization and elevated levels of lactate synthesis. Although we observed a statistical increase in the activities of phosphofructokinase, pyruvate kinase, lactate dehydrogenase, and creatine kinase after exposure to lowered oxygen concentrations, the effect was small. The cultured cells exhibited a decreased utilization of glutamine, possibly due to down regulation of mitochondrial function and inhibition of oxidative deamination. With respect to total energy generation, we noted that these cells are quite capable of maintaining the energy charge of the cell at low oxygen tensions. Indeed, no changes in the absolute quantity of adenine nucleotides or the energy charge ratio was observed. Hypoxia caused a decrease in the glutathione content of cultured chondrocytes and a concomitant rise in cell and medium cysteine levels. It is likely that the fall in cell glutathione level is due to decreased synthesis of the tripeptide under reduced oxygen stress and the limited supply of glutamate. The observed rise in cellular and medium cysteine levels probably reflects an increase in the rate of degradation of glutathione and a decrease in synthesis of the peptide. To explore how cells transduce these metabolic effects, gel retardation assays were used to study chondrocyte HIF and AP-1 binding activities. Chondrocyte nuclear preparations bound an HIF-oligonucleotide; however, at low oxygen tensions, no increase in HIF binding was

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

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    Eleonora Olivotto

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

  5. Epiphyseal chondrocyte secondary ossification centers require thyroid hormone activation of Indian hedgehog and osterix signaling.

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    Xing, Weirong; Cheng, Shaohong; Wergedal, Jon; Mohan, Subburaman

    2014-10-01

    Thyroid hormones (THs) are known to regulate endochondral ossification during skeletal development via acting directly in chondrocytes and osteoblasts. In this study, we focused on TH effects on the secondary ossification center (SOC) because the time of appearance of SOCs in several species coincides with the time when peak levels of TH are attained. Accordingly, micro-computed tomography (µCT) evaluation of femurs and tibias at day 21 in TH-deficient and control mice revealed that endochondral ossification of SOCs is severely compromised owing to TH deficiency and that TH treatment for 10 days completely rescued this phenotype. Staining of cartilage and bone in the epiphysis revealed that whereas all of the cartilage is converted into bone in the prepubertal control mice, this conversion failed to occur in the TH-deficient mice. Immunohistochemistry studies revealed that TH treatment of thyroid stimulating hormone receptor mutant (Tshr(-/-) ) mice induced expression of Indian hedgehog (Ihh) and Osx in type 2 collagen (Col2)-expressing chondrocytes in the SOC at day 7, which subsequently differentiate into type 10 collagen (Col10)/osteocalcin-expressing chondro/osteoblasts at day 10. Consistent with these data, treatment of tibia cultures from 3-day-old mice with 10 ng/mL TH increased expression of Osx, Col10, alkaline phosphatase (ALP), and osteocalcin in the epiphysis by sixfold to 60-fold. Furthermore, knockdown of the TH-induced increase in Osx expression using lentiviral small hairpin RNA (shRNA) significantly blocked TH-induced ALP and osteocalcin expression in chondrocytes. Treatment of chondrogenic cells with an Ihh inhibitor abolished chondro/osteoblast differentiation and SOC formation. Our findings indicate that TH regulates the SOC initiation and progression via differentiating chondrocytes into bone matrix-producing osteoblasts by stimulating Ihh and Osx expression in chondrocytes. PMID:24753031

  6. Crosstalk between adipose-derived stem cells and chondrocytes: when growth factors matter.

    Science.gov (United States)

    Zhong, Juan; Guo, Bin; Xie, Jing; Deng, Shuwen; Fu, Na; Lin, Shiyu; Li, Guo; Lin, Yunfeng; Cai, Xiaoxiao

    2016-01-01

    Adipose-derived stem cells (ASCs) and mesenchymal stem cells are promising for tissue repair because of their multilineage differentiation capacity. Our previous data confirmed that the implantation of mixed ASCs and chondrocytes into cartilage defects induced desirable in vivo healing outcomes. However, the paracrine action of ASCs on chondrocytes needs to be further elucidated. In this study, we established a co-culture system to achieve cell-to-cell and cell-to-tissue crosstalk and explored the soluble growth factors in both ASCs and chondrocytes supplemented with 1% fetal bovine serum to mimic the physiological microenvironment. In ASCs, we screened for growth factors by semi-quantitative PCR and quantitative real-time PCR and found that the expression of bone morphogenetic protein 2 (BMP-2), vascular endothelial growth factor B (VEGFB), hypoxia inducible factor-1α (HIF-1α), fibroblast growth factor-2 (FGF-2), and transforming growth factor-β1 significantly increased after co-culture in comparison with mono-culture. In chondrocytes, VEGFA was significantly enhanced after co-culture. Unexpectedly, the expression of collagen II and aggrecan was significantly down-regulated in the co-culture group compared with the mono-culture group. Meanwhile, among all the growth factors screened, we found that the BMP family members BMP-2, BMP-4, and BMP-5 were down-regulated and that VEGFB, HIF-1α, FGF-2, and PDGF were significantly decreased after co-culture. These results suggest that crosstalk between ASCs and chondrocytes is a pathway through the regulated growth factors that might have potential in cartilage repair and regeneration and could be useful for tissue engineering. PMID:26848404

  7. 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.

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

    Science.gov (United States)

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

    2016-09-01

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

  9. 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.

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

    International Nuclear Information System (INIS)

    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

  11. Dual effects of 17ß-oestradiol on interleukin 1ß-induced proteoglycan degradation in chondrocytes

    OpenAIRE

    Richette, P; Dumontier, M.; Francois, M; Tsagris, L.; Korwin-Zmijowska, C; Rannou, F; Corvol, M

    2004-01-01

    Objective: To determine whether 17ß-oestradiol (E2) modulates interleukin (IL) 1ß-induced proteoglycan degradation in chondrocytes, and to analyse the part played by metalloproteinases (MMPs) in this process.

  12. Effective implantation of autologous chondrocytes in a patient suffering from a painful and invalidating rizoarthrosis: a case report.

    Science.gov (United States)

    Carelli, Francesco; Sgherzi, Stefano; Sillani, Alessandro; Magris, Cecilia

    2009-01-01

    A 45-year-old patient, caucasian, affected by severe, painful and invalidating rizoarthrosis has been treated by implanting autologous chondrocytes, normally used for degenerative joint diseases of the knee and ankle. PMID:19918494

  13. Effective implantation of autologous chondrocytes in a patient suffering from a painful and invalidating rizoarthrosis: a case report

    OpenAIRE

    Carelli, Francesco; Sgherzi, Stefano; Sillani, Alessandro; Magris, Cecilia

    2009-01-01

    A 45-year-old patient, caucasian, affected by severe, painful and invalidating rizoarthrosis has been treated by implanting autologous chondrocytes, normally used for degenerative joint diseases of the knee and ankle.

  14. Myeloid CD34+CD13+ Precursor Cells Transdifferentiate into Chondrocyte-Like Cells in Atherosclerotic Intimal Calcification

    OpenAIRE

    Doehring, Lars Christian; Heeger, Christian; Aherrahrou, Zouhair; Kaczmarek, Piotr Maciel; Erdmann, Jeanette; Schunkert, Heribert; Ehlers, Eva-Maria

    2010-01-01

    Chondrogenic differentiation is pivotal in the active regulation of artery calcification. We investigated the cellular origin of chondrocyte-like cells in atherosclerotic intimal calcification of C57BL/6 LDLr−/− mice using bone marrow transplantation to trace ROSA26-LacZ-labeled cells. Immunohistochemical costaining of collagen type II with LacZ and leukocyte defining surface antigens was performed and analyzed by high-resolution confocal microscopy. Chondrocyte-like cells were detected in me...

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

    Directory of Open Access Journals (Sweden)

    James Claudine G

    2006-09-01

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

  16. 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

  17. 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.

  18. Morphological evidence of the shedding of chondrocytes from the articular surface in neonatal rats: relationship to the interlacunar network.

    Science.gov (United States)

    Cole, M B; Narine, K R; Ellinger, J

    1983-08-01

    The superficial zone of the femoral head articular cartilage of 5- to 15-day old rats was examined by light and electron microscopy for evidence of shedding into the joint space. Chondrocytes deepest in the superficial zone were round, surrounded by a capsule, and connected to adjacent chondrocytes by the interlacunar network, whereas cells in the middle of the zone appeared similar but with less cytoplasm. At the circular surface, chondrocytes were small, with pyknotic nuclei and poorly defined organelles. These cells occasionally protruded from the articular surface but maintained at least partial connection with the network and their capsule. Depressions in the articular surface were lined with material similar to that of the network and were the only locations found where the network did not terminate at a cell surface. This static evidence suggested at least two hypotheses: 1) Degenerating chondrocytes moved up through the superficial zone to the articular surface and were shed into the joint space. This movement may be facilitated by the network as part of neonatal cartilage development. 2) During joint formation, the surface of the articular cartilage was eroded down to the chondrocytes, which were exposed to the joint fluid, causing cell degeneration, death, and shedding. Evidence of cell shedding was rarely seen after 2 weeks of age. Likewise, the interlacunar network disappeared from the superficial zone during this period. A physiological as well as structural relationship may exist between the chondrocytes and interlacunar network. PMID:6625202

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2011-12-15

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

  20. The use of fibrin matrix-mixed gel-type autologous chondrocyte implantation in the treatment for osteochondral lesions of the talus

    OpenAIRE

    Lee, Kyung Tai; Kim, Jin Su; Young, Ki Won; Lee, Young Koo; Park, Young Uk; Kim, Yong Hoon; Cho, Hun ki

    2012-01-01

    Purpose This study assessed the clinical results and second-look arthroscopy after fibrin matrix-mixed gel-type autologous chondrocyte implantation to treat osteochondral lesions of the talus. Methods Chondrocytes were harvested from the cuboid surface of the calcaneus in 38 patients and cultured, and gel-type autologous chondrocyte implantation was performed with or without medial malleolar osteotomy. Preoperative American orthopedic foot and ankle society ankle-hind foot scores, visual anal...

  1. Quantitative analysis of rough endoplasmic reticulum in chondrocytes of articular and tracheal cartilage of rabbits following the systemic administration of hydrocortisone.

    OpenAIRE

    T. Itani; Kanai, K.; Watanabe, J.; Ogawa, R; Kanamura, S

    1992-01-01

    The rough endoplasmic reticulum (RER) in chondrocytes was analysed stereologically in articular cartilage of knee joints and in tracheal cartilage of rabbits injected intramuscularly with 5 mg/kg hydrocortisone daily for 4 wk. In articular cartilage, RER area per unit cytoplasmic volume decreased in chondrocytes in all (superficial, middle and deep) zones, although the volume of glycogen deposits per unit cytoplasmic volume increased in the middle and deep zones. RER area per chondrocyte also...

  2. 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.

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

    OpenAIRE

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

    2004-01-01

    We generated transgenic mice that express a constitutively active mutant of MEK1 in chondrocytes. These mice showed a dwarf phenotype similar to achondroplasia, the most common human dwarfism, caused by activating mutations in FGFR3. These mice displayed incomplete hypertrophy of chondrocytes in the growth plates and a general delay in endochondral ossification, whereas chondrocyte proliferation was unaffected. Immunohistochemical analysis of the cranial base in transgenic embryos showed redu...

  4. Linked decreases in Liver Kinase B1 and AMP-activated protein kinase activity modulate matrix catabolic responses to biomechanical injury in chondrocytes

    OpenAIRE

    Petursson, Freyr; Husa, Matt; June, Ron; Lotz, Martin; Terkeltaub, Robert; Liu-Bryan, Ru

    2013-01-01

    Abstract Introduction AMP-activated protein kinase (AMPK) maintains cultured chondrocyte matrix homeostasis in response to inflammatory cytokines. AMPK activity is decreased in human knee osteoarthritis (OA) chondrocytes. Liver kinase B1 (LKB1) is one of the upstream activators of AMPK. Hence, we examined the relationship between LKB1 and AMPK activity in OA and aging cartilages, and in chondrocytes subjected to inflammatory cytokine treatment and biomechanical compression injury, and p...

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

    Science.gov (United States)

    Wang, Pengzhen; Zhang, Fengjie; He, Qiling; Wang, Jianqi; Shiu, Hoi Ting; Shu, Yinglan; Tsang, Wing Pui; Liang, Shuang; Zhao, Kai; Wan, Chao

    2016-01-01

    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 marker genes

  6. 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

  7. 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.

  8. 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.

  9. Effect of a novel synthesized sulfonamido-based gallate-SZNTC on chondrocytes metabolism in vitro.

    Science.gov (United States)

    Liu, Qin; Li, Mu-Yan; Lin, Xiao; Lin, Cui-Wu; Liu, Bu-Ming; Zheng, Li; Zhao, Jin-Min

    2014-09-25

    The ideal therapeutic agent for treatment of osteoarthritis (OA) should have not only potent anti-inflammatory effect but also favorable biological properties to restore cartilage function. Gallic acid (GA) and its derivatives are anti-inflammatory agents reported to have an effect on OA (Singh et al., 2003) [1]. However, GA has much weaker antioxidant effects and inferior bioactivity compared with its derivatives. We modified GA with the introduction of sulfonamide to synthesize a novel sulfonamido-based gallate named sodium salt of 3,4,5-trihydroxy-N-[4-(thiazol-2-ylsulfamoyl)-phenyl]-benzamide (SZNTC) and analyzed its chondro-protective and pharmacological effects. Comparison of SZNTC with GA and sulfathiazole sodium (ST-Na) was also performed. Results showed that SZNTC could effectively inhibit the Interleukin-1 (IL-1)-mediated induction of metalloproteinase-1 (MMP-1) and MMP-3 and could induce the expression of tissue inhibitor of metalloproteinase-1 (TIMP-1), which demonstrated ability to reduce the progression of OA. SZNTC can also exert chondro-protective effects by promoting cell proliferation and maintaining the phenotype of articular chondrocytes, as evidenced by improved cell growth, enhanced synthesis of cartilage specific markers such as aggrecan, collagen II and Sox9. Expression of the collagen I gene was effectively down-regulated, revealing the inhibition of chondrocytes dedifferentiation by SZNTC. Hypertrophy that may lead to chondrocyte ossification was also undetectable in SZNTC groups. The recommended dose of SZNTC ranges from 3.91μg/ml to 15.64μg/ml, among which the most profound response was observed with 7.82μg/ml. In contrast, its source products of GA and ST-Na have a weak effect in the bioactivity of chondrocytes, which indicated the significance of this modification. This study revealed SZNTC as a promising novel agent in the treatment of chondral and osteochondral lesions. PMID:25130855

  10. Assessment of Growth Factor Treatment on Fibrochondrocyte and Chondrocyte Co-Cultures for TMJ Fibrocartilage Engineering

    OpenAIRE

    Kalpakci, Kerem N.; Kim, Eric J.; Athanasiou, Kyriacos A.

    2010-01-01

    Treatments for patients suffering from severe temporomandibular joint (TMJ) dysfunction are limited, motivating the development of strategies for tissue regeneration. In this study, co-cultures of fibrochondrocytes (FC) and articular chondrocytes (AC) were seeded in agarose wells, and supplemented with growth factors, to engineer tissue with biomechanical properties and ECM composition similar to native TMJ fibrocartilage. In the first phase, growth factors were applied alone and in combinati...

  11. Tauroursodeoxycholic acid suppresses endoplasmic reticulum stress in the chondrocytes of patients with osteoarthritis.

    Science.gov (United States)

    Liu, Chao; Cao, Yongping; Yang, Xin; Shan, Pengcheng; Liu, Heng

    2015-10-01

    The main pathogenic events in osteoarthritis (OA) include loss and abnormal remodeling of cartilage extracellular matrix. The present study aimed to evaluate the protective effect of tauroursodeoxycholic acid on chondrocyte apoptosis induced by endoplasmic reticulum (ER) stress. Articular cartilage tissues were collected from 18 patients who underwent total knee arthroplasty and were analyzed histologically. Subsequently, chondrocyte apoptosis was assessed by TUNEL. Quantitative polymerase chain reaction and western blot analysis were employed to evaluate gene and protein expression, respectively, of ER stress markers, including glucose‑regulated protein 78 (GRP78), growth arrest and DNA‑damage‑inducible gene 153 (GADD153) and caspase‑12 along with type II collagen. Chondrocytes obtained from osteoarthritis patients at different stages were cultured in three conditions including: No treatment (CON group), tunicamycin treatment to induce ER stress (ERS group) and tauroursodeoxycholic acid treatment after 4 h of tunicamycin (TDA group); and cell proliferation, apoptosis, function and ER stress level were assessed. Degradation of cartilage resulted in histological damage with more apoptotic cartilage cells observed. Of note, GRP78, GADD153 and caspase‑12 mRNA and protein expression increased gradually from grade I to III cartilage tissue, while type II collagen expression decreased. Tunicamycin induced ER stress, as shown by a high expression of ER stress markers, reduced cell proliferation, increased apoptosis and decreased synthesis of type II collagen. Notably, tauroursodeoxycholic acid treatment resulted in the improvement of tunicamycin‑induced ER stress. These results indicated that ER stress is highly involved in the tunicamycin‑induced apoptosis in chondrocytes, which can be prevented by tauroursodeoxycholic acid. PMID:26238983

  12. Femtosecond laser microstructuring and bioactive nanocoating of titanium surfaces in relation to chondrocyte growth

    Science.gov (United States)

    Ilgner, Justus; Biedron, Slavomir; Fadeeva, Elena; Chichkov, Boris; Klee, Doris; Loos, Anneke; Sowa-Söhle, Eveline; Westhofen, Martin

    2010-02-01

    Introduction: Titanium implants can be regarded as the current gold standard for restoration of sound transmission in the middle ear following destruction of the ossicular chain by chronic inflammation. Many efforts have been made to improve prosthesis design, while less attention had been given to the role of the interface. We present a study on chemical nanocoating on microstructured titanium contact surface with bioactive protein. Materials and Methods: Titanium samples of 5mm diameter and 0,25mm thickness were structured by means of a Ti:Sapphire femtosecond laser operating at 970nm with parallel lines of 5μm depth, 5μm width and 10μm inter-groove distance. In addition, various nanolayers were applied to titanium samples by aminosilanization, to which Star-Polyethylene glycole (Star-PEG) molecules plus biomarkers (e.g. RGD peptide sequence) were linked. Results: Chondrocytes could be cultured on microstructured surfaces without reduced rate of vital / dead cells compared to native surfaces. Chondrocytes also showed contact guidance by growing along ridges particularly on 5μm lines. On nanocoated titanium samples, first results showed a strong effect of Star-PEG suppressing unspecific protein absorption, while RGD peptide sequence did not promote chondrocyte cell growth. Discussion: According to these results, the idea of promoting cell growth on titanium prosthesis contact surfaces compared to non-contact surfaces (e.g. prosthesis shaft) by nanocoating is practicable. However, relative selectivity induced by microstructures for growth of chondrocytes compared to fibrocytes is subject to further evaluation.

  13. Hyaluronan fragments activate nitric oxide synthase and the production of nitric oxide by articular chondrocytes

    OpenAIRE

    Iacob, Stanca; Knudson, Cheryl B.

    2005-01-01

    Chondrocyte CD44 receptors anchor hyaluronan to the cell surface, enabling the assembly and retention of proteoglycan aggregates in the pericellular matrix. Hyaluronan–CD44 interactions also provide signaling important for maintaining cartilage homeostasis. Disruption of chondrocyte–hyaluronan contact alters CD44 occupancy, initiating alternative signaling cascades. Treatment with hyaluronan oligosaccharides is one approach to uncouple CD44 receptors from its native ligand, hyaluronan. In bov...

  14. Osteogenic differentiation of hypertrophic chondrocytes involves asymmetric cell divisions and apoptosis

    OpenAIRE

    1995-01-01

    We have investigated the early cellular events that take place during the change in lineage commitment from hypertrophic chondrocytes to osteoblast-like cells. We have induced this osteogenic differentiation by cutting through the hypertrophic cartilage of embryonic chick femurs and culturing the explants. Immunocytochemical characterization, [3H]thymidine pulse-chase labeling, in situ nick translation or end labeling of DNA breaks were combined with ultrastructural studies to characterize th...

  15. Recombinant equine interleukin-1β induces putative mediators of articular cartilage degradation in equine chondrocytes

    OpenAIRE

    Tung, J. T.; Fenton, J. I.; Arnold, C; Alexander, L.; Yuzbasiyan-Gurkan, V.; Venta, P J; Peters, T. L.; Orth, M W; Richardson, D. W.; Caron, J P

    2002-01-01

    Interleukin-1 is considered a central mediator of cartilage loss in osteoarthritis in several species, however an equine recombinant form of this cytokine is not readily available for in vitro use in equine osteoarthritis research. Equine recombinant interleukin-1β was cloned and expressed and its effects on the expression and activity of selected chondrocytic proteins implicated in cartilage matrix degradation were characterized. Reverse transcriptase polymerase chain reaction methods were u...

  16. Vav1 Regulates Mesenchymal Stem Cell Differentiation Decision Between Adipocyte and Chondrocyte via Sirt1.

    Science.gov (United States)

    Qu, Peng; Wang, Lizhen; Min, Yongfen; McKennett, Lois; Keller, Jonathan R; Lin, P Charles

    2016-07-01

    Mesenchymal stem cells (MSCs) are multipotent stromal cells residing in the bone marrow. MSCs have the potential to differentiate to adipocytes, chondrocytes, and other types of cells. In this study, we investigated the molecular mechanism that controls MSC cell fate decisions for differentiation. We found that Vav1, a guanine nucleotide exchange factor for Rho GTPase, was highly expressed in MSCs. Interestingly, loss of Vav1 in MSCs led to spontaneous adipogenic but impaired chondrogenic differentiation, and accordingly Vav1 null mice displayed an increase in fat content and a decrease in cartilage. Conversely, ectopic expression of Vav1 in MSCs reversed this phenotype, and led to enhanced MSC differentiation into chondrocyte but retarded adipogenesis. Mechanistically, loss of Vav1 reduced the level of Sirt1, which was responsible for an increase of acetylated PPARγ. As acetylation activates PPARγ, it increased C/EBPα expression and promoted adipogenesis. On the other hand, loss of Vav1 resulted in an increase of acetylated Sox9, a target of Sirt1. As acetylation represses Sox9 activity, it led to a dramatic reduction of collagen 2α1, a key regulator in chondrocyte differentiation. Finally, we found that Vav1 regulates Sirt1 in MSCs through Creb. Together this study reveals a novel function of Vav1 in regulating MSC cell fate decisions for differentiation through Sirt1. Sirt1 deacetylates PPARγ and Sox9, two key mediators that control adipocyte and chondrocyte differentiation. The acetylation status of PPARγ and Sox9 has opposite effects on its activity, thereby controlling cell fate decision. Stem Cells 2016;34:1934-1946. PMID:26990002

  17. 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

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

    OpenAIRE

    Li-ke Luo; Qing-jun Wei; Lei Liu; Li Zheng; Jin-min Zhao

    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 n...

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

    Science.gov (United States)

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

    2016-01-01

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

  20. In vitro evidence for effects of magnesium supplementation on quinolone-treated horse and dog chondrocytes.

    Science.gov (United States)

    Egerbacher, M; Wolfesberger, B; Gabler, C

    2001-03-01

    Quinolones and magnesium deficiency cause similar lesions in joint cartilage of young animals. Chondrocytes cultivated in the presence of quinolones and in Mg-free medium show severe alterations in cytoskeleton and decreased ability to adhere to the culture dish. We investigated whether Mg2+ supplementation can prevent quinolone-mediated effects on chondrocytes in vitro. Chondrocytes cultivated in Dulbecco's modified Eagle's medium/HAM's F-12 medium were treated with ciprofloxacin (80 and 160 microg/ml) and enrofloxacin (100 and 150 microg/ml). Mg2+ was added at a concentration of 0.0612 mg/ml (MgCl) and 0.0488 mg/ml (MgSO4) or a triple dose. In addition, cells were cultivated in Mg-free medium and accordingly treated with Mg2+ supplementation. After 5 days in culture, the number of adherent cells per milliliter was determined. The number of chondrocytes in quinolone-treated groups decreased to 12-36% that of the control group within the culture period. With Mg2+ supplementation, the number of attached cells increased to 40-70% that of control cells. The threefold dose of Mg2+ led to better results than did the single dose. Cell proliferation tested by immunohistochemical staining with Ki67 (clone MIB5) decreased from 70% in control groups to 55%, 48%, and 30% in enrofloxacin-treated groups in a concentration dependent manner (50, 100, and 150 microg/ml). Addition of Mg2+ did not increase the rate of cell proliferation. These results suggest that a great part of quinolone-induced damage is due to magnesium complex formation, as Mg2+ supplementation is able to reduce the effects in vitro. However, quinolone effects on cell proliferation seem to be an independent process that is not influenced by magnesium supplementation. PMID:11280370

  1. Encapsulation and survival of a chondrocyte cell line within xanthan gum derivative

    OpenAIRE

    Mendes, Ana C.; Baran, Erkan T.; Pereira, Rui C.; Azevedo, Helena S.; Reis, Rui L.

    2011-01-01

    A chemical derivative of xanthan gum polysaccharide is investigated as a new artificial matrix for the encapsulation of chondrocytic cells. Toward this goal, a novel micro-droplet generator is developed to produce microcapsules. Microcapsules with an average diameter of 500 mm, smooth surface, and homogeneous size distribution are obtained. ATDC5 cells encapsulated in carboxymethyl xanthan (CMX) microcapsules remain viable and are observed to proliferate for prolonged ...

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

    Science.gov (United States)

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

    2016-05-01

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

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

    Directory of Open Access Journals (Sweden)

    Salvador Fernández-Arroyo

    2015-10-01

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

  4. 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.

  5. Comparison of proteomic datasets from hypertrophic chondrocytes in response to ER stress.

    Science.gov (United States)

    Kudelko, Mateusz; Sharma, Rakesh; Cheah, Kathryn S E; Chan, Danny

    2016-06-01

    Cartilage proteomics is challenging due to the dominance of poorly soluble matrix components and limited available tissue. Using a "spatial" strategy coupled to MS/MS analysis we have specifically labeled and extracted hypertrophic chondrocytes within the growth plate providing thus a comprehensive proteomic map of normal hypertrophic chondrocytes. Furthermore our established 13del mouse model in which the activation of ER stress did not lead to apoptosis of the hypertrophic cells allowed us to address the natural consequences of ER stress in vivo. Thus our data provide also an overview of proteomic changes occurring in cells under ER stress. Associated with the published study [1] this dataset article provided the detailed information of experimental designing, methods, features as well as the raw data of mass spectrometry (MS) identification. Furthermore the data presented here allow the reader to assert the extent of proteomic changes occurring under ER stress in hypertrophic chondrocytes as well as address the data technical reproducibility in both wild type and stress condition. The mass spectrometry proteomics data can be fully accessed from the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD002125. PMID:27014728

  6. Cartilage-derived extracellular matrix extract promotes chondrocytic phenotype in three-dimensional tissue culture.

    Science.gov (United States)

    Youngstrom, Daniel W; Cakstina, Inese; Jakobsons, Eriks

    2016-05-01

    Cell transplantation is a promising regenerative therapy for cartilage degeneration. However, obtaining sufficient numbers of cells for this purpose is a challenge, due a lack of autologous donor tissue and the difficulty of culturing chondrocytes in vitro. Tissue engineering strategies that induce or maintain chondrocytic phenotype may solve these problems by (1) broadening the range of available donor tissue, and (2) facilitating the expansion of these cells while controlling phenotypic drift. In this study, bone marrow-derived mesenchymal stem cells (MSCs) and cartilage-derived cells (CDCs) were cultured on composite hydrogels containing agarose and homogenized cartilage extracellular matrix (ECM). MSCs cultured on agarose-ECM scaffolds did not show significant signs of chondrogenic differentiation in the absence of additional cues. However, CDCs cultured on agarose-ECM scaffolds proliferated more rapidly than their ECM-free counterparts and MSCs, while retaining chondrocytic morphology. These results were corroborated via expression of cartilage marker genes: in autologous constructs, SOX 9 expression was upregulated by 12.6 ± 5.3-fold, and COL II was upregulated by 2.0 ± 0.3-fold. Agarose-ECM composite hydrogels are therefore useful for expanding partially differentiated CDCs for applications in regenerative medicine. PMID:25707441

  7. Bioluminescence imaging of chondrocytes in rabbits by intraarticular injection of D-luciferin

    International Nuclear Information System (INIS)

    Luciferase is one of the most commonly used reporter enzymes in the field of in vivo optical imaging. D-luciferin, the substrate for firefly luciferase has very high cost that allows this kind of experiment limited to small animals such as mice and rats. In this current study, we validated local injection of D-luciferin in the articular capsule for bioluminescence imaging in rabbits. Chondrocytes were cultured and infected by replication-defective adenoviral vector encoding firefly luciferase (Fluc). Chondrocytes expressing Fluc were injected or implanted in the left knee joint. The rabbits underwent optical imaging studies after local injection of D-luciferin at 1, 5, 7, 9 days after cellular administration. We sought whether optimal imaging signals was could be by a cooled CCD camera after local injection of D-luciferin. Imaging signal was not observed from the left knee joint after intraperitoneal injection of D-luciferin (15 mg/kg), whereas it was observed after intraarticular injection. Photon intensity from the left knee joint of rabbits was compared between cell injected and implanted groups after intraarticular injection of D-luciferin. During the period of imaging studies, photon intensity of the cell implanted group was 5-10 times higher than that of the cell injected group. We successfully imaged chondrocytes expressing Fluc after intraarticular injection of D-luciferin. This technique may be further applied to develop new drugs for knee joint disease

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

    Science.gov (United States)

    Ponist, S; Drafi, F; Kuncirova, V; Mihalova, D; Rackova, L; Danisovic, L; Ondrejickova, O; Tumova, I; Trunova, O; Fedorova, T; Bauerova, K

    2016-01-01

    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. PMID:26885252

  9. 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.

  10. 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.

  11. 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

  12. 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

  13. 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

  14. 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

  15. Overexpression of Galnt3 in Chondrocytes Resulted in Dwarfism Due to the Increase of Mucin-type O-Glycans and Reduction of Glycosaminoglycans*

    Science.gov (United States)

    Yoshida, Carolina Andrea; Kawane, Tetsuya; Moriishi, Takeshi; Purushothaman, Anurag; Miyazaki, Toshihiro; Komori, Hisato; Mori, Masako; Qin, Xin; Hashimoto, Ayako; Sugahara, Kazuyuki; Yamana, Kei; Takada, Kenji; Komori, Toshihisa

    2014-01-01

    Galnt3, UDP-N-acetyl-α-d-galactosamine:polypeptide N-acetylgalactosaminyltransferase 3, transfers N-acetyl-d-galactosamine to serine and threonine residues, initiating mucin type O-glycosylation of proteins. We searched the target genes of Runx2, which is an essential transcription factor for chondrocyte maturation, in chondrocytes and found that Galnt3 expression was up-regulated by Runx2 and severely reduced in Runx2−/− cartilaginous skeletons. To investigate the function of Galnt3 in chondrocytes, we generated Galnt3−/− mice and chondrocyte-specific Galnt3 transgenic mice under the control of the Col2a1 promoter-enhancer. Galnt3−/− mice showed a delay in endochondral ossification and shortened limbs at embryonic day 16.5, suggesting that Galnt3 is involved in chondrocyte maturation. Galnt3 transgenic mice presented dwarfism, the chondrocyte maturation was retarded, the cell cycle in chondrocytes was accelerated, premature chondrocyte apoptosis occurred, and the growth plates were disorganized. The binding of Vicia villosa agglutinin, which recognizes the Tn antigen (GalNAc-O-Ser/Thr), was drastically increased in chondrocytes, and aggrecan (Acan) was highly enriched with Tn antigen. However, safranin O staining, which recognizes glycosaminoglycans (GAGs), and Acan were severely reduced. Chondroitin sulfate was reduced in amount, but the elongation of chondroitin sulfate chains had not been severely disturbed in the isolated GAGs. These findings indicate that overexpression of Galnt3 in chondrocytes caused dwarfism due to the increase of mucin-type O-glycans and the reduction of GAGs, probably through competition with xylosyltransferases, which initiate GAG chains by attaching O-linked xylose to serine residues, suggesting a negative effect of Galnt family proteins on Acan deposition in addition to the positive effect of Galnt3 on chondrocyte maturation. PMID:25107907

  16. Overexpression of Galnt3 in chondrocytes resulted in dwarfism due to the increase of mucin-type O-glycans and reduction of glycosaminoglycans.

    Science.gov (United States)

    Yoshida, Carolina Andrea; Kawane, Tetsuya; Moriishi, Takeshi; Purushothaman, Anurag; Miyazaki, Toshihiro; Komori, Hisato; Mori, Masako; Qin, Xin; Hashimoto, Ayako; Sugahara, Kazuyuki; Yamana, Kei; Takada, Kenji; Komori, Toshihisa

    2014-09-19

    Galnt3, UDP-N-acetyl-α-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase 3, transfers N-acetyl-D-galactosamine to serine and threonine residues, initiating mucin type O-glycosylation of proteins. We searched the target genes of Runx2, which is an essential transcription factor for chondrocyte maturation, in chondrocytes and found that Galnt3 expression was up-regulated by Runx2 and severely reduced in Runx2(-/-) cartilaginous skeletons. To investigate the function of Galnt3 in chondrocytes, we generated Galnt3(-/-) mice and chondrocyte-specific Galnt3 transgenic mice under the control of the Col2a1 promoter-enhancer. Galnt3(-/-) mice showed a delay in endochondral ossification and shortened limbs at embryonic day 16.5, suggesting that Galnt3 is involved in chondrocyte maturation. Galnt3 transgenic mice presented dwarfism, the chondrocyte maturation was retarded, the cell cycle in chondrocytes was accelerated, premature chondrocyte apoptosis occurred, and the growth plates were disorganized. The binding of Vicia villosa agglutinin, which recognizes the Tn antigen (GalNAc-O-Ser/Thr), was drastically increased in chondrocytes, and aggrecan (Acan) was highly enriched with Tn antigen. However, safranin O staining, which recognizes glycosaminoglycans (GAGs), and Acan were severely reduced. Chondroitin sulfate was reduced in amount, but the elongation of chondroitin sulfate chains had not been severely disturbed in the isolated GAGs. These findings indicate that overexpression of Galnt3 in chondrocytes caused dwarfism due to the increase of mucin-type O-glycans and the reduction of GAGs, probably through competition with xylosyltransferases, which initiate GAG chains by attaching O-linked xylose to serine residues, suggesting a negative effect of Galnt family proteins on Acan deposition in addition to the positive effect of Galnt3 on chondrocyte maturation. PMID:25107907

  17. 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.

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

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    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.

  19. 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.

  20. Edited by SONG Shuang-mingEffect of basic fibroblast growth factor and hyaluronic acid on proliferation of rabbit chondrocytes in vitro

    Institute of Scientific and Technical Information of China (English)

    沈雁; 李斯明; 唐毅; 钟灿灿; 梁佩红; 陈鸿辉

    2004-01-01

    Objective: To investigate the effect of basic fibroblast growth factor (bFGF) and hyaluronic acid (HA) on the proliferation of rabbit chondrocytes in vitro.Methods: Chondrocytes from the knee joints of New Zealand white rabbits were cultured. bFGF or HA or both were added into the culture medium respectively, and the proliferation of the chondrocytes was measured with MTT 3-(4, 5-dimethylthiazol-2-yl) 2, 5-diphenyl-tetra-zolium bromide. (MTT, Sigma, M2128).Results: Basic fibroblast growth factor (10 ng/ml) with low concentration of fetal bovine serum in the culture medium promoted the proliferation of chondrocytes significantly, and this effect reached its maximum when concentration of bFGF reached 50 ng/ml. HA itself had no effect on the proliferation of chondrocytes. However, when bFGF was used in combination with HA, especially when the concentration of bFGF was 50-500 ng/ml and that of HA was 10-50 ng/ml, the effect on the proliferation of chondrocytes was much more than when bFGF or HA was used alone. Conclusions: bFGF can promote the proliferation of chondrocytes. HA, which has no effect on the proliferation of the cells, can maintain a normal growth of chondrocytes. When bFGF is used in combination with HA, more proliferation is obtained.

  1. 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.

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

    Science.gov (United States)

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

    2006-01-01

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

  3. Xenotransplantation of pig chondrocytes: therapeutic potential and barriers for cartilage repair.

    Science.gov (United States)

    Sommaggio, R; Uribe-Herranz, M; Marquina, M; Costa, C

    2016-01-01

    Transplantation may be the best option for the repair of many cartilage lesions including early osteoarthritis. Currently, autologous and allogeneic chondrocytes are grafted into cartilage defects to treat selected patients with moderate clinical success. However, their limited use justifies exploring novel therapies for cartilage repair. Xenotransplantation could become a solution by offering high cell availability, quality and genetic engineering capabilities. The rejection process of xenogeneic cartilage is thus being elucidated in order to develop counteractive strategies. Initial studies determined that pig cartilage xenografts are rejected by a slow process comprising humoral and cellular responses in which the galactose α1,3-galactose antigen participates. Since then, our group has identified key mechanisms of the human response to pig chondrocytes (PCs). In particular, human antibody and complement contribute to PC rejection by inducing a pro-inflammatory milieu. Furthermore, PCs express and up-regulate molecules which are functionally relevant for a variety of cellular immune responses (SLA-I, the potent co-stimulatory molecule CD86, and adhesion molecules VCAM-1 and ICAM-1). These participate by triggering a T cell response, as well as supporting a prominent role of the innate immune responses led by natural killer (NK) cells and monocytes/macrophages. Human NK cells lyse PCs by using selected NK activating receptors, whereas human monocytes are activated by PCs to secrete cytokines and chemokines. All this knowledge sets the bases for the development of genetic engineering approaches designed to avert rejection of xenogeneic chondrocytes and leads the way to developing new clinical applications for cartilage repair. PMID:27377665

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

    International Nuclear Information System (INIS)

    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® 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: ► Human-like collagen was first used to prepare cartilage tissue engineering scaffold. ► Genipin, a natural biological cross-linking agent, was introduced to treat scaffold. ► We chose market product as a control.

  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. 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.

  7. Three-year clinical outcome after chondrocyte transplantation using a hyaluronan matrix for cartilage repair

    Energy Technology Data Exchange (ETDEWEB)

    Nehrer, S. [Department of Orthopedics, Medical University of Vienna, Waehringer Guertel 18-20, A-1090 Vienna (Austria)]. E-mail: stefan.nehrer@meduniwien.ac.at; Domayer, S. [Department of Orthopedics, Medical University of Vienna, Waehringer Guertel 18-20, A-1090 Vienna (Austria); Dorotka, R. [Department of Orthopedics, Medical University of Vienna, Waehringer Guertel 18-20, A-1090 Vienna (Austria); Schatz, K. [Department of Orthopedics, Medical University of Vienna, Waehringer Guertel 18-20, A-1090 Vienna (Austria); Bindreiter, U. [Department of Orthopedics, Medical University of Vienna, Waehringer Guertel 18-20, A-1090 Vienna (Austria); Kotz, R. [Department of Orthopedics, Medical University of Vienna, Waehringer Guertel 18-20, A-1090 Vienna (Austria)

    2006-01-15

    Repair of articular cartilage represents a significant clinical problem and although various new techniques - including the use of autologous chondrocytes - have been developed within the last century the clinical efficacy of these procedures is still discussed controversially. Although autologous chondrocyte transplantation (ACT) has been widely used with success, it has several inherent limitations, including its invasive nature and problems related to the use of the periosteal flap. To overcome these problems autologous chondrocytes transplantation combined with the use of biodegradable scaffolds has received wide attention. Among these, a hyaluronan-based scaffold has been found useful for inducing hyaline cartilage regeneration. In the present study, we have investigated the mid-term efficacy and safety of Hyalograft[reg] C grafts in a group of 36 patients undergoing surgery for chronic cartilage lesions of the knee. Clinical Outcome was assessed prospectively before and at 12, 24, and 36 months after surgery. No major adverse events have been reported during the 3-year follow-up. Significant improvements of the evaluated scores were observed (P < 0.02) at 1 year and a continued increase of clinical performance was evident at 2 and 3 years follow-up. Patients under 30 years of age with single lesions showed statistically significant improvements at all follow-up visits compared to those over 30 with multiple defects (P < 0.01). Hyalograft[reg] C compares favorably with classic ACT and is particularly indicated in younger patients with single lesions. The graft can be implanted through a miniarthrotomy and needs no additional fixation with sutures except optional fibrin gluing at the defect borders. These results suggest that Hyalograft[reg] C is a valid alternative to ACT.

  8. 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

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

    International Nuclear Information System (INIS)

    Quinolones (QNs)-induced arthropathy is an important toxic effect in immature animals leading to restriction of their therapeutic use in pediatrics. However, the exact mechanism still remains unclear. Recently, we have demonstrated that ofloxacin, a typical QN, induces apoptosis of alginate microencapsulated juvenile rabbit joint chondrocytes by disturbing the β1 integrin functions and inactivating the ERK/MAPK signaling pathway. In this study, we extend our initial observations to further elucidate the mechanism(s) of ofloxacin-induced apoptosis by utilizing specific caspase inhibitors. Pretreatment with both caspase-9-specific inhibitor zLEHD-fmk and caspase-8 inhibitor zIETD-fmk attenuated ofloxacin-induced apoptosis and activation of caspase-3 of chondrocyte in a concentration-dependent manner, as determined by fluorescent dye staining, enzyme activity assay and immunoblotting. Furthermore, the activation of caspase-9, -8 and -3 stimulated by ofloxacin was significantly inhibited in the presence of zIETD-fmk while pretreatment with zLEHD-fmk only blocked the activation of caspase-9 and -3. Ofloxacin also stimulated a concentration-dependent translocation of cytochrome c from mitochondria into the cytosol and a decrease of mitochondrial transmembrane potential, which was completely inhibited by zIETD-fmk. In addition, ofloxacin was found to increase the level of Bax, tBid, p53 in a concentration- and time-dependent manner. Taken together, The current results indicate that the caspase-8-dependent mitochondrial pathway is primarily involved in the ofloxacin-induced apoptosis of microencapsulated juvenile rabbit joint chondrocytes

  10. 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.

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

    International Nuclear Information System (INIS)

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

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

    Directory of Open Access Journals (Sweden)

    Dusanic Daliborka

    2012-01-01

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

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

    Czech Academy of Sciences Publication Activity Database

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

    2010-01-01

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

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

    OpenAIRE

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

    2011-01-01

    Objectives. To compare the ability of different cyclodextrin polysulphate (CDPS) derivatives to affect human articular cartilage cell metabolism in vitro. Methods. OA chondrocytes were cultured in alginate and exposed to 5 µg/ml of 2,3,6-tri-O-methyl-β-cyclodextrin (ME-CD), 2,3-di-O-methyl-6-sulphate-β-cyclodextrin (ME-CD-6-S), 2,6-di-O-methyl-3-sulphate-β-cyclodextrin (ME-CD-3-S), (2-carboxyethyl)-β-CDPS (CE-CDPS), (2-hydroxypropyl)-β-CDPS (HP-CDPS), 6-monoamino-6-monodeoxy-β-CDPS (MA-CDPS) ...

  15. Nitric oxide compounds have different effects profiles on human articular chondrocyte metabolism

    OpenAIRE

    de Andrés, María C.; Maneiro, Emilia; Martín, Miguel A.; Arenas, Joaquín; Francisco J Blanco

    2013-01-01

    Introduction The pathogenesis of osteoarthritis (OA) is characterized by the production of high amounts of nitric oxide (NO), as a consequence of up-regulation of chondrocyte-inducible nitric oxide synthase (iNOS) induced by inflammatory cytokines. NO donors represent a powerful tool for studying the role of NO in the cartilage in vitro. There is no consensus about NO effects on articular cartilage in part because the differences between the NO donors available. The aim of this work is to com...

  16. Nanocomposite scaffold for chondrocyte growth and cartilage tissue engineering: effects of carbon nanotube surface functionalization.

    Science.gov (United States)

    Chahine, Nadeen O; Collette, Nicole M; Thomas, Cynthia B; Genetos, Damian C; Loots, Gabriela G

    2014-09-01

    The goal of this study was to assess the long-term biocompatibility of single-wall carbon nanotubes (SWNTs) for tissue engineering of articular cartilage. We hypothesized that SWNT nanocomposite scaffolds in cartilage tissue engineering can provide an improved molecular-sized substrate for stimulation of chondrocyte growth, as well as structural reinforcement of the scaffold's mechanical properties. The effect of SWNT surface functionalization (-COOH or -PEG) on chondrocyte viability and biochemical matrix deposition was examined in two-dimensional cultures, in three-dimensional (3D) pellet cultures, and in a 3D nanocomposite scaffold consisting of hydrogels+SWNTs. Outcome measures included cell viability, histological and SEM evaluation, GAG biochemical content, compressive and tensile biomechanical properties, and gene expression quantification, including extracellular matrix (ECM) markers aggrecan (Agc), collagen-1 (Col1a1), collagen-2 (Col2a1), collagen-10 (Col10a1), surface adhesion proteins fibronectin (Fn), CD44 antigen (CD44), and tumor marker (Tp53). Our findings indicate that chondrocytes tolerate functionalized SWNTs well, with minimal toxicity of cells in 3D culture systems (pellet and nanocomposite constructs). Both SWNT-PEG and SWNT-COOH groups increased the GAG content in nanocomposites relative to control. The compressive biomechanical properties of cell-laden SWNT-COOH nanocomposites were significantly elevated relative to control. Increases in the tensile modulus and ultimate stress were observed, indicative of a tensile reinforcement of the nanocomposite scaffolds. Surface coating of SWNTs with -COOH also resulted in increased Col2a1 and Fn gene expression throughout the culture in nanocomposite constructs, indicative of increased chondrocyte metabolic activity. In contrast, surface coating of SWNTs with a neutral -PEG moiety had no significant effect on Col2a1 or Fn gene expression, suggesting that the charged nature of the -COOH surface

  17. Chlorogenic acid suppresses interleukin-1β-induced inflammatory mediators in human chondrocytes

    OpenAIRE

    Chen, Wei-Ping; Wu, Li-Dong

    2014-01-01

    We investigated the anti-inflammatory properties of chlorogenic acid (CGA) in interleukin-1β-induced chondrocytes. The nitric oxide (NO) and prostaglandin E2 (PGE2) were detected by Griess and Enzyme-linked immunosorbent assay (ELISA) respectively. Quantitative real-time PCR and western blot were performed to measure the expression of inducible NO synthase (iNOS) and cyclooxygenase (COX)-2. Our results indicate that CGA inhibited the production of NO and PGE2 as well as the expression of iNOS...

  18. ESE-1 Is a Potent Repressor of Type II Collagen Gene (COL2A1) Transcription In Human Chondrocytes

    OpenAIRE

    Peng, Haibing; TAN, LUJIAN; Osaki, Makoto; Zhan, Yumei; Ijiri, Kosei; Tsuchimochi, Kaneyuki; Otero, Miguel; Wang, Hong; CHOY, BOB K.; GRALL, FRANCK T.; Gu, Xuesong; Libermann, Towia A; Oettgen, Peter; Goldring, Mary B.

    2008-01-01

    The epithelium-specific ETS (ESE)-1 transcription factor is induced in chondrocytes by interleukin-1β (IL-1β). We reported previously that early activation of EGR-1 by IL-1β results in suppression of the proximal COL2A1 promoter activity by displacement of Sp1 from GC boxes. Here we report that ESE-1 is a potent transcriptional suppressor of COL2A1 promoter activity in chondrocytes and accounts for the sustained, NF-κB-dependent inhibition by IL-1β. Of the ETS factors tested, this response wa...

  19. Generation of a Transgenic Mouse Model With Chondrocyte-Specific and Tamoxifen-Inducible Expression of Cre Recombinase

    OpenAIRE

    Chen, Mo; Lichtler, Alexander C.; Sheu, Tzong-Jen; Xie, Chao; Zhang, Xinping; O’Keefe, Regis J.; Chen, Di

    2007-01-01

    Postnatal cartilage development and growth are regulated by key growth factors and signaling molecules. To fully understand the function of these regulators, an inducible and chondrocyte-specific gene deletion system needs to be established to circumvent the perinatal lethality. In this report, we have generated a transgenic mouse model (Col2a1-CreERT2) in which expression of the Cre recombinase is driven by the chondrocyte-specific col2a1 promoter in a tamoxifen-inducible manner. To determin...

  20. Hyaluronan Does Not Affect Bupivacaine’s Inhibitory Action on Voltage-Gated Potassium Channel Activities in Bovine Articular Chondrocytes

    OpenAIRE

    William Hester; Jinnan Yang; Guo-Yong Wang; Sen Liu; Michael J O'Brien; Savoie, Felix H.; Zongbing You

    2012-01-01

    Objectives. The objective of this paper is to determine if hyaluronan affects bupivacaine's anesthetic function. Methods. Whole cell patch clamp recordings were performed on bovine articular chondrocytes cultured in 60 mm dishes. The chondrocytes were treated with phosphate-buffered saline (control group), 7.5 mg/mL hyaluronan (Orthovisc), 0.25% bupivacaine, or a mixture of 7.5 mg/mL hyaluronan and 0.25% bupivacaine. Outward currents were elicited by step depolarization from −90 mV to 150 mV ...

  1. 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

  2. The canonical Wnt signaling pathway promotes chondrocyte differentiation in a Sox9-dependent manner

    International Nuclear Information System (INIS)

    To better understand the role of the canonical Wnt signaling pathway in cartilage development, we adenovirally expressed a constitutively active (Canada) or a dominant negative (dn) form of lymphoid enhancer factor-1 (LEF-1), the main nuclear effector of the pathway, in undifferentiated mesenchymal cells, chondrogenic cells, and primary chondrocytes, and examined the expression of markers for chondrogenic differentiation and hypertrophy. caLEF-1 and LiCl, an activator of the canonical pathway, promoted both chondrogenic differentiation and hypertrophy, whereas dnLEF-1 and the gene silencing of β-catenin suppressed LiCl-promoted effects. To investigate whether these effects were dependent on Sox9, a master regulator of cartilage development, we stimulated Sox9-deficient ES cells with the pathway. caLEF-1 and LiCl promoted both chondrogenic differentiation and hypertrophy in wild-type, but not in Sox9-deficient, cells. The response of Sox9-deficient cells was restored by the adenoviral expression of Sox9. Thus, the canonical Wnt signaling pathway promotes chondrocyte differentiation in a Sox9-dependent manner

  3. 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.

  4. Comparison of three types of chondrocytes in collagen scaffolds for cartilage tissue engineering

    Energy Technology Data Exchange (ETDEWEB)

    Zhang Lu [Department of Plastic and Reconstructive Surgery, Shanghai Tissue Engineering Center, Shanghai 9th People' s Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai (China); Spector, Myron, E-mail: luzhangmd@gmail.co [Tissue Engineering, VA Boston Healthcare System, Boston, MA (United States)

    2009-08-15

    The objective of this study was to compare the chondrogenesis in type I and II collagen scaffolds seeded with chondrocytes from three types of cartilage, after four weeks of culture: auricular (AU), articular (AR) and meniscal (ME). Related aims were to investigate the expression of a contractile muscle actin isoform, alpha-smooth muscle actin (SMA), in the cells in the scaffold and to determine the presence of a lubricating glycoprotein, lubricin, in the constructs. Adult goat AU, AR and ME chondrocytes were seeded into two types of collagen scaffolds: type II collagen and type I/III collagen. After four weeks of culture, the constructs were prepared for histochemical and immunohistochemical analysis of the distribution of glycosaminoglycan (GAG), types I and II collagen, elastin, SM and lubricin. AU constructs contained substantially more tissue than the AR and ME samples. The AU constructs exhibited neocartilage, but no elastin. There were no notable differences between the type I and II collagen scaffolds. Novel findings were the expression of SMA by the AU cells in the scaffolds and the presence of lubricin in the AR and AU constructs. AU cells have the capability to produce cartilage in collagen scaffolds under conditions in which there is little histogenesis by AR and ME cells.

  5. 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.

  6. ISOLATION AND INDUCTION OF RABBIT BONE MARROW MESENCHYMAL STEM CELLS TO EXPRESS CHONDROCYTIC PHENOTYPE

    Institute of Scientific and Technical Information of China (English)

    尹战海; 刘淼; 王金堂; 曹峻岭; 张璟; 郑钧

    2002-01-01

    Objective To isolate rabbit bone marrow mesenchymal stem cells (MSCs), and observe the inducing effect of growth factors on MSCs to express chondrocytic phenotype. Methods MSCs were seperated from bone marrow of New Zealand rabbit. TGF-β1, IGF-I, Vitamin C and dexamethasone were added into culture medium to induce proliferation and differention of MSCs. Procollagen α1(Ⅱ) mRNA in cells was detected by RT-PCR to observe the chondrogenous effect of inducing factors. ALP in culture medium was detected by automatic biochemical analyser, and lipid droplet in cells was stained by Sudan Ⅲ to clarify whether these factors given had osteogenic and adipogenic potential. Results Expression of articular cartilage specific procollagen α1 (Ⅱ)mRNA was promoted by inducing factors-TGF-β1, IGF-I, Vitamine C and dexamethasone; elevated level of ALP in culture medium and lipid droplet in cells were also detected. Whereas ALP level was decreased and lipid stain were negative in groups without dexamethasone. Conclusion ① Expression of chondrocytic phenotype by MSCs could be induced by the synergistic action of TGF-β1, IGF-I and Vitamine C. ② Dexmathasone had osteogenic and adipogenic potential, it should not be chosen to induce chondrogenic differention of MSCs.

  7. Prostaglandin E2 regulates the expression of connective tissue growth factor (CTGF/CCN2 in human osteoarthritic chondrocytes via the EP4 receptor

    Directory of Open Access Journals (Sweden)

    Nakamura Hiroshi

    2010-01-01

    Full Text Available Abstract Background The regulatory mechanisms of the expression of connective tissue growth factor/CCN family member 2 (CTGF/CCN2 in human articular chondrocytes have not been clarified. We investigated the effect of prostaglandin E2 (PGE2 on CTGF/CCN2 expression in chondrocytes. Findings Articular cartilage samples were obtained from patients with osteoarthritis (OA and chondrocytes were isolated and cultured in vitro. Chondrocytes were stimulated with PGE2, PGE receptor (EP-specific agonists, or interleukin (IL-1. CTGF expression was analyzed using quantitative polymerase chain reaction, Western blot, and enzyme-linked immunosorbent assay. The inhibitory effects of EP receptor antagonists (for EP2 and EP4 against PGE2 stimulation were also investigated. Stimulation of chondrocytes with PGE2 or IL-1 significantly suppressed CTGF expression. The suppressive effect of PGE2 was reproduced by EP2/EP4 receptor agonists but not by EP1/EP3 receptor agonists, and was partially blocked by an EP4 receptor antagonist, suggesting that the EP4 receptor has a dominant role. Conclusions PGE2 may be involved in the regulation of CTGF/CCN2 expression in human articular chondrocytes via the EP4 receptor. Elucidation of EP4-mediated signaling in chondrocytes may contribute to a better understanding of the effects of PGE2 in arthritis.

  8. Articular chondrocytes and mesenchymal stem cells seeded on biodegradable scaffolds for the repair of cartilage in a rat osteochondral defect model.

    Science.gov (United States)

    Dahlin, Rebecca L; Kinard, Lucas A; Lam, Johnny; Needham, Clark J; Lu, Steven; Kasper, F Kurtis; Mikos, Antonios G

    2014-08-01

    This work investigated the ability of co-cultures of articular chondrocytes and mesenchymal stem cells (MSCs) to repair articular cartilage in osteochondral defects. Bovine articular chondrocytes and rat MSCs were seeded in isolation or in co-culture onto electrospun poly(ɛ-caprolactone) (PCL) scaffolds and implanted into an osteochondral defect in the trochlear groove of 12-week old Lewis rats. Additionally, a blank PCL scaffold and untreated defect were investigated. After 12 weeks, the extent of cartilage repair was analyzed through histological analysis, and the extent of bone healing was assessed by quantifying the total volume of mineralized bone in the defect through microcomputed tomography. Histological analysis revealed that the articular chondrocytes and co-cultures led to repair tissue that consisted of more hyaline-like cartilage tissue that was thicker and possessed more intense Safranin O staining. The MSC, blank PCL scaffold, and empty treatment groups generally led to the formation of fibrocartilage repair tissue. Microcomputed tomography revealed that while there was an equivalent amount of mineralized bone formation in the MSC, blank PCL, and empty treatment groups, the defects treated with chondrocytes or co-cultures had negligible mineralized bone formation. Overall, even with a reduced number of chondrocytes, co-cultures led to an equal level of cartilage repair compared to the chondrocyte samples, thus demonstrating the potential for the use of co-cultures of articular chondrocytes and MSCs for the in vivo repair of cartilage defects. PMID:24927682

  9. Novel Elements of the Chondrocyte Stress Response Identified Using an in Vitro Model of Mouse Cartilage Degradation.

    Science.gov (United States)

    Wilson, Richard; Golub, Suzanne B; Rowley, Lynn; Angelucci, Constanza; Karpievitch, Yuliya V; Bateman, John F; Fosang, Amanda J

    2016-03-01

    The destruction of articular cartilage in osteoarthritis involves chondrocyte dysfunction and imbalanced extracellular matrix (ECM) homeostasis. Pro-inflammatory cytokines such as interleukin-1α (IL-1α) contribute to osteoarthritis pathophysiology, but the effects of IL-1α on chondrocytes within their tissue microenvironment have not been fully evaluated. To redress this we used label-free quantitative proteomics to analyze the chondrocyte response to IL-1α within a native cartilage ECM. Mouse femoral heads were cultured with and without IL-1α, and both the tissue proteome and proteins released into the media were analyzed. New elements of the chondrocyte response to IL-1α related to cellular stress included markers for protein misfolding (Armet, Creld2, and Hyou1), enzymes involved in glutathione biosynthesis and regeneration (Gstp1, Gsto1, and Gsr), and oxidative stress proteins (Prdx2, Txn, Atox1, Hmox1, and Vnn1). Other proteins previously not associated with the IL-1α response in cartilage included ECM components (Smoc2, Kera, and Crispld1) and cysteine proteases (cathepsin Z and legumain), while chondroadherin and cartilage-derived C-type lectin (Clec3a) were identified as novel products of IL-1α-induced cartilage degradation. This first proteome-level view of the cartilage IL-1α response identified candidate biomarkers of cartilage destruction and novel targets for therapeutic intervention in osteoarthritis. PMID:26794603

  10. Chondrocytes expressing intracellular collagen type II enter the cell cycle and co-express collagen type I in monolayer culture.

    Science.gov (United States)

    Tekari, Adel; Luginbuehl, Reto; Hofstetter, Willy; Egli, Rainer J

    2014-11-01

    For autologous chondrocyte transplantation, articular chondrocytes are harvested from cartilage tissue and expanded in vitro in monolayer culture. We aimed to characterize with a cellular resolution the synthesis of collagen type II (COL2) and collagen type I (COL1) during expansion in order to further understand why these cells lose the potential to form cartilage tissue when re-introduced into a microenvironment that supports chondrogenesis. During expansion for six passages, levels of transcripts encoding COL2 decreased to COL2/COL1-double positive phenotype during expansion, and the COL2 positive cells were able to enter the cell cycle. While the fraction of COL2 positive cells decreased from 70% to 95%. In parallel to the decrease of the fraction of COL2 positive cells, the cells' potential to form cartilage-like tissue in pellet cultures steadily decreased. Intracellular staining for COL2 enables for characterization of chondrocyte lineage cells in more detail with a cellular resolution, and it may allow predicting the effectiveness of expanded chondrocytes to form cartilage-like tissue. PMID:25043137

  11. 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.

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

    NARCIS (Netherlands)

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

    2001-01-01

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

  13. Effect of Ginkgo biloba extract on matrix metalloproteinase-3 expression in a rat model of chondrocyte injury.

    Science.gov (United States)

    Zeng, J Z; Ma, L F; Meng, H; Yu, H M; Zhang, Y K; Guo, A

    2015-01-01

    A rat model with cartilage chondrocyte injury was established using interleukin-1β (IL-1β) to investigate the effect of Ginkgo biloba extract (EGb) on matrix metalloproteinase-3 (MMP-3) expression. Rat chondrocytes were extracted and randomly divided into six groups: control group, IL-1β (model) group, IL-1β + dexamethasone group, and IL-1β + EGb group (both high and low dose groups). Reverse transcriptase polymerase chain reaction and enzyme-linked immunosorbent assay were used to detect MMP-3 expression. Compared to the MMP-3 mRNA level in the control group, MMP-3 mRNA level significantly increased in the model group (P < 0.05). The application of dexamethasone or EGb significantly decreased the MMP-3 mRNA level (P < 0.05). MMP-3 mRNA and protein levels decreased in the EGb-treated group, especially in the high-dose group, compared to those in the dexamethasone group (P < 0.05). EGb may reduce MMP-3 production during IL-1β-induced chondrocyte damage and protect chondrocytes to some extent, with better efficacy at high doses. PMID:26782475

  14. 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. PMID:26499345

  15. 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.

  16. DEC2 is a negative regulator for the proliferation and differentiation of chondrocyte lineage-committed mesenchymal stem cells.

    Science.gov (United States)

    Sasamoto, Tomoko; Fujimoto, Katsumi; Kanawa, Masami; Kimura, Junko; Takeuchi, Junpei; Harada, Naoko; Goto, Noriko; Kawamoto, Takeshi; Noshiro, Mitsuhide; Suardita, Ketut; Tanne, Kazuo; Kato, Yukio

    2016-09-01

    Differentiated embryo chondrocyte 2 (DEC2) is a basic helix-loop-helix-Orange transcription factor that regulates cell differentiation in various mammalian tissues. DEC2 has been shown to suppress the differentiation of mesenchymal stem cells (MSCs) into myocytes and adipocytes. In the present study, we examined the role of DEC2 in the chondrogenic differentiation of human MSCs. The overexpression of DEC2 exerted minimal effects on the proliferation of MSCs in monolayer cultures with the growth medium under undifferentiating conditions, whereas it suppressed increases in DNA content, glycosaminoglycan content, and the expression of several chondrocyte-related genes, including aggrecan and type X collagen alpha 1, in MSC pellets in centrifuge tubes under chondrogenic conditions. In the pellets exposed to chondrogenesis induction medium, DEC2 overexpression downregulated the mRNA expression of fibroblast growth factor 18, which is involved in the proliferation and differentiation of chondrocytes, and upregulated the expression of p16INK4, which is a cell cycle inhibitor. These findings suggest that DEC2 is a negative regulator of the proliferation and differentiation of chondrocyte lineage-committed mesenchymal cells. PMID:27430159

  17. 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. PMID:27475840

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

    Science.gov (United States)

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

    2010-06-01

    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 enzymes are affected by glucose deprivation. Chondrocytes obtained from nucleus pulposus, annulus fibrosus, articular cartilage, and meniscus and dermal fibroblasts were cultured under control conditions or exposed to the ER stress-inducing treatments of tunicamycin addition or glucose withdrawal. Both treatments resulted in an up-regulation of the gene expression of the ER stress markers in all cell types, but dermal fibroblasts showed a delayed response to glucose deprivation. Collagen gene expression was down-regulated, and less collagen protein was present in the cells under both ER stress-inducing conditions. The expression levels of the prolyl 4-hydroxylases were either not affected (P4ha3) or increased (P4ha1 and P4ha2), the levels of the lysyl hydroxylases decreased, and the N-propeptidase Adamts2 decreased. Both treatments induced apoptosis. Chondrocytes respond more quickly to glucose deprivation, but it appears that chondrocytes can cope better with tunicamycin-induced ER stress than fibroblasts. Although collagen synthesis was inhibited by the treatments, some collagen-modifying enzymes and chaperones were up-regulated, suggesting that there is no causal relation between them. PMID:20555395

  19. 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

  20. 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

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

    Directory of Open Access Journals (Sweden)

    Wertheimer Michael R

    2011-01-01

    Full Text Available Abstract Background Recent evidence indicates that osteoarthritis (OA may be a systemic disease since mesenchymal stem cells (MSCs from OA patients express type X collagen, a marker of late stage chondrocyte hypertrophy (associated with endochondral ossification. We recently showed that the expression of type X collagen was suppressed when MSCs from OA patients were cultured on nitrogen (N-rich plasma polymer layers, which we call "PPE:N" (N-doped plasma-polymerized ethylene, containing up to 36 atomic percentage (at.% of N. Methods In the present study, we examined the expression of type X collagen in fetal bovine growth plate chondrocytes (containing hypertrophic chondrocytes cultured on PPE:N. We also studied the effect of PPE:N on the expression of matrix molecules such as type II collagen and aggrecan, as well as on proteases (matrix metalloproteinase-13 (MMP-13 and molecules implicated in cell division (cyclin B2. Two other culture surfaces, "hydrophilic" polystyrene (PS, regular culture dishes and nitrogen-containing cation polystyrene (Primaria®, were also investigated for comparison. Results Results showed that type X collagen mRNA levels were suppressed when cultured for 4 days on PPE:N, suggesting that type X collagen is regulated similarly in hypertrophic chondrocytes and in human MSCs from OA patients. However, the levels of type X collagen mRNA almost returned to control value after 20 days in culture on these surfaces. Culture on the various surfaces had no significant effects on type II collagen, aggrecan, MMP-13, and cyclin B2 mRNA levels. Conclusion Hypertrophy is diminished by culturing growth plate chondrocytes on nitrogen-rich surfaces, a mechanism that is beneficial for MSC chondrogenesis. Furthermore, one major advantage of such "intelligent surfaces" over recombinant growth factors for tissue engineering and cartilage repair is potentially large cost-saving.

  2. 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.

  3. The influence of biological motifs and dynamic mechanical stimulation in hydrogel scaffold systems on the phenotype of chondrocytes.

    Science.gov (United States)

    Appelman, Taly P; Mizrahi, Joseph; Elisseeff, Jennifer H; Seliktar, Dror

    2011-02-01

    Primary bovine chondrocytes and PEG-based hydrogels were used to investigate the effects of scaffold composition and architecture on the cellular response to large dynamic compressive strain stimulation. Proteins and proteoglycans were conjugated to functionalized poly(ethylene glycol) (PEG) and immobilized in PEG hydrogels to create bio-synthetic scaffolds. Second passage articular chondrocytes were encapsulated into four different scaffold compositions: PEG-Proteoglycan (PP), PEG-Fibrinogen (PF), PEG-Albumin (PA), and PEG only and subjected to 15% dynamic compressive strain at 1-Hz frequency. Cellular response was evaluated in terms of cell number, glycosaminoglycans (GAGs), collagen type II and collagen type I accumulation in the constructs following 24h and 28 days of stimulated and static culture. Stimulation of the constructs resulted in an increase in the cell number in all scaffolds, with no statistical difference measured among them. Dynamic stimulation of PP, PF, PA and PEG constructs resulted in a respective increase in the GAGs by 33%, 53.4%, 240.5%, and 284.5%, compared to their static controls. The permissive PEG and PA scaffolds showed a significantly larger relative increase in the GAGs in comparison to the other scaffolds tested. Collagen type II content in the PF, PA and PEG constructs increased by 78%, 1266% and 896% respectively, compared to their static controls. Permissive constructs showed a significantly larger relative increase and final absolute values of GAGs and type II collagen, compared to the PF constructs. Immunostaining for collagen type I, an indicator for chondrocyte de-differentiation, indicated that stimulation inhibited its production. Correlation maps between scaffold properties highlighted the major differences between permissive and instructive scaffolds. These results support the hypothesis that both compressive strain and scaffold bioactivity have an important effect on the chondrocyte metabolic response to mechanical

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

    Science.gov (United States)

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

    2016-07-19

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

  5. Measurement of the chondrocyte membrane permeability to Me2SO, glycerol and 1,2-propanediol.

    Science.gov (United States)

    Xu, Xia; Cui, Zhanfeng; Urban, Jill P G

    2003-09-01

    The addition of cryopreservative agents (CPAs) to chondrocytes and natural and engineered cartilage is critical to protect the cells and tissues from freezing damage during cryopreservation, but this may cause cell damage, e.g. by osmotic shock. The damage could be minimized by the control of the cell volume excursion with the knowledge of cell membrane permeability. In this study, the cell volume responses of chondrocytes to three commonly used CPAs were evaluated using a perfusion microscope stage. The osmotic response of chondrocytes was measured to the perfusion with 1.4 M dimethyl sulfoxide (Me2SO), 1,2-propanediol and glycerol at 21 degrees C. Cell volumes and their transients were determined with image analysis. The cell membrane permeability parameters, including the hydraulic conductivity (Lp), the CPA permeability (omega) and the reflection coefficients (sigma) in the Kedem-Katchalsky (K-K) model, and the Lp and omega in the two-parameter model were determined. The correlated K-K parameters at 21 degrees C were Lp=0.166 +/- 0.035, 0.149 +/- 0.061, 0.212 +/- 0.041 microm/min atm, omega=(7.630 +/- 0.174) x 10(-2), (1.428 +/- 0.627) x 10(-2), (2.744 +/- 0.775) x 10(-2) microm/s and sigma=0.91 +/- 0.09, 0.82 +/- 0.11, 0.88 +/- 0.10 for Me(2)SO, glycerol and 1,2-propanediol, respectively. For the two-parameter model, the parameter values were Lp=0.163 +/- 0.040, 0.128 +/- 0.031, 0.169 +/- 0.025 microm/min atm, omega=(7.881 +/- 0.178) x 10(-2), (1.529 +/- 0.525) x 10(-2), (3.716 +/- 0.493) x 10(-2) microm/s for Me2SO, glycerol and 1,2-propanediol, respectively. No significant difference in the predictions of cell volume excursion during CPA addition was observed when using either the K-K model or the two-parameter model and it was hence advised to adopt the simple two-parameter model in the evaluation. The measured parameters can be used to optimise the CPA addition and removal protocols to maximize the cell survival during cryopreservation. PMID:12835070

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

    International Nuclear Information System (INIS)

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

  7. Delayed hypertrophic differentiation of epiphyseal chondrocytes contributes to failed secondary ossification in mucopolysaccharidosis VII dogs.

    Science.gov (United States)

    Peck, Sun H; O'Donnell, Philip J M; Kang, Jennifer L; Malhotra, Neil R; Dodge, George R; Pacifici, Maurizio; Shore, Eileen M; Haskins, Mark E; Smith, Lachlan J

    2015-11-01

    Mucopolysaccharidosis (MPS) VII is a lysosomal storage disorder characterized by deficient β-glucuronidase activity, which leads to the accumulation of incompletely degraded glycosaminoglycans (GAGs). MPS VII patients present with severe skeletal abnormalities, which are particularly prevalent in the spine. Incomplete cartilage-to-bone conversion in MPS VII vertebrae during postnatal development is associated with progressive spinal deformity and spinal cord compression. The objectives of this study were to determine the earliest postnatal developmental stage at which vertebral bone disease manifests in MPS VII and to identify the underlying cellular basis of impaired cartilage-to-bone conversion, using the naturally-occurring canine model. Control and MPS VII dogs were euthanized at 9 and 14 days-of-age, and vertebral secondary ossification centers analyzed using micro-computed tomography, histology, qPCR, and protein immunoblotting. Imaging studies and mRNA analysis of bone formation markers established that secondary ossification commences between 9 and 14 days in control animals, but not in MPS VII animals. mRNA analysis of differentiation markers revealed that MPS VII epiphyseal chondrocytes are unable to successfully transition from proliferation to hypertrophy during this critical developmental window. Immunoblotting demonstrated abnormal persistence of Sox9 protein in MPS VII cells between 9 and 14 days-of-age, and biochemical assays revealed abnormally high intra and extracellular GAG content in MPS VII epiphyseal cartilage at as early as 9 days-of-age. In contrast, assessment of vertebral growth plates and primary ossification centers revealed no significant abnormalities at either age. The results of this study establish that failed vertebral bone formation in MPS VII can be traced to the failure of epiphyseal chondrocytes to undergo hypertrophic differentiation at the appropriate developmental stage, and suggest that aberrant processing of Sox9 protein

  8. Three-dimensional cell culture of chondrocytes on modified di-phenylalanine scaffolds.

    Science.gov (United States)

    Jayawarna, V; Smith, A; Gough, J E; Ulijn, R V

    2007-06-01

    The design of self-assembled peptide-based structures for three-dimensional cell culture and tissue repair has been a key objective in biomaterials science for decades. In search of the simplest possible peptide system that can self-assemble, we discovered that combinations of di-peptides that are modified with aromatic stacking ligands could form nanometre-sized fibres when exposed to physiological conditions. For example, we demonstrated that a number of Fmoc (fluoren-9-ylmethyloxycarbonyl) modified di- and tri-peptides form highly ordered hydrogels via hydrogen-bonding and pi-pi interactions from the fluorenyl rings. These highly hydrated gels allowed for cell proliferation of chondrocytes in three dimensions [Jayawarna, Ali, Jowitt, Miller, Saiani, Gough and Ulijn (2006) Adv. Mater. 18, 611-614]. We demonstrated that fibrous architecture and physical properties of the resulting materials were dictated by the nature of the amino acid building blocks. Here, we report the self-assembly process of three di-phenylalanine analogues, Fmoc-Phe-Phe-OH, Nap (naphthalene)-Phe-Phe-OH and Cbz (benzyloxycarbonyl)-Phe-Phe-OH, to compare and contrast the self-assembly properties and cell culture conditions attributable to their protecting group difference. Fibre morphology analysis of the three structures using cryo-SEM (scanning electron microscopy) and TEM (transmission electron microscopy) suggested fibrous structures with dramatically varying fibril dimensions, depending on the aromatic ligand used. CD and FTIR (Fourier-transform IR) data confirmed beta-sheet arrangements in all three samples in the gel state. The ability of these three new hydrogels to support cell proliferation of chondrocytes was confirmed for all three materials. PMID:17511646

  9. 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. PMID:19288021

  10. 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.

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

    Directory of Open Access Journals (Sweden)

    Chao Pin-Zhir

    2011-11-01

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

  12. Cartilage in facet joints of patients with ankylosing spondylitis (AS) shows signs of cartilage degeneration rather than chondrocyte hypertrophy: implications for joint remodeling in AS

    OpenAIRE

    Bleil, Janine; Sieper, Joachim; Maier, Rene; Schlichting, Uwe; Hempfing, Axel; Syrbe, Uta; Appel, Heiner

    2015-01-01

    Introduction In ankylosing spondylitis (AS), joint remodeling leading to joint ankylosis involves cartilage fusion. Here, we analyzed whether chondrocyte hypertrophy is involved in cartilage fusion and subsequent joint remodeling in AS. Methods We assessed the expression of chondrocyte hypertrophy markers runt-related transcription factor 2 (Runx2), type X collagen (COL10), matrix metalloproteinase 13 (MMP13), osteocalcin and beta-catenin and the expression of positive bone morphogenic protei...

  13. 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.

  14. Saponin-rich fraction from Clematis chinensis Osbeck roots protects rabbit chondrocytes against nitric oxide-induced apoptosis via preventing mitochondria impairment and caspase-3 activation

    OpenAIRE

    Wu, Wenjun; Gao, Xinghua; Xu, Xianxiang; Luo, Yubin; Liu, Mei; Xia, Yufeng; Dai, Yue

    2012-01-01

    Our previous study reported that the saponin-rich fraction from Clematis chinensis Osbeck roots (SFC) could effectively alleviate experimental osteoarthritis induced by monosodium iodoacetate in rats through protecting articular cartilage and inhibiting local inflammation. The present study was performed to investigate the preventive effects of SFC on articular chondrocyte, and explore the underlying mechanisms. Primary rabbit chondrocytes were cultured and exposed to sodium nitroprusside (SN...

  15. Cell-Engineered Human Elastic Chondrocytes Regenerate Natural Scaffold In Vitro and Neocartilage with Neoperichondrium in the Human Body Post-Transplantation

    OpenAIRE

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

    2012-01-01

    We have developed a unique method that allows us to culture large volumes of chondrocyte expansion from a small piece of human elastic cartilage. The characteristic features of our culturing method are that fibroblast growth factor-2 (FGF2), which promotes proliferation of elastic chondrocytes, is added to a culture medium, and that cell-engineering techniques are adopted in the multilayered culture system that we have developed.1–4 We have subsequently discovered that once multilayered chond...

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

    International Nuclear Information System (INIS)

    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

  17. Effects of non-steroidal anti-inflammatory drugs on cell proliferation and death in cultured epiphyseal-articular chondrocytes of fetal rats

    International Nuclear Information System (INIS)

    Previous reports indicated that non-steroidal anti-inflammatory drugs (NSAIDs) suppress bone repair. Our previous study further found that ketorolac delayed the endochondral bone formation, and the critical effective timing was at the early stage of repair. Furthermore, we found that NSAIDs suppressed proliferation and induced cell death of cultured osteoblasts. In this study, we hypothesized that chondrocytic proliferation and death, which plays an important role at the early stage of endochondral bone formation, might be affected by NSAIDs. Non-selective NSAIDs, indomethacin, ketorolac, diclofenac and piroxicam; cyclooxygenase-2 (COX-2) selective NSAIDs, celecoxib and DFU (an analog of rofecoxib); prostaglandins (PGs), PGE1, PGE2 and PGF2α; and each NSAID plus each PG were tested. The effects of NSAIDs on proliferation, cell cycle kinetics, cytotoxicity and cell death of epiphyseal-articular chondrocytes of fetal rats were examined. The results showed that all the tested NSAIDs, except DFU, inhibited thymidine incorporation of chondrocytes at a concentration range (10-8 to 10-4 M) covering the theoretic therapeutic concentrations. Cell cycle was arrested by NSAIDs at the G /G1 phase. Upon a 24 h treatment, LDH leakage and cell death (both apoptosis and necrosis) were significantly induced by the four non-selective NSAIDs in chondrocyte cultures. However, COX-2 inhibitors revealed non-significant effects on cytotoxicity of chondrocytes except higher concentration of celecoxib (10-4 M). Replenishments of PGE1, PGE2 or PGF2α could not reverse the effects of NSAIDs on chondrocytic proliferation and cytotoxicity. In this study, we found that therapeutic concentrations of non-selective NSAIDs caused proliferation suppression and cell death of chondrocytes, suggesting these adverse effects may be one of the reasons that NSAIDs delay the endochondral ossification during bone repair found in previous studies. Furthermore, these effects of NSAIDs may act via PG

  18. Saponin-rich fraction from Clematis chinensis Osbeck roots protects rabbit chondrocytes against nitric oxide-induced apoptosis via preventing mitochondria impairment and caspase-3 activation.

    Science.gov (United States)

    Wu, Wenjun; Gao, Xinghua; Xu, Xianxiang; Luo, Yubin; Liu, Mei; Xia, Yufeng; Dai, Yue

    2013-03-01

    Our previous study reported that the saponin-rich fraction from Clematis chinensis Osbeck roots (SFC) could effectively alleviate experimental osteoarthritis induced by monosodium iodoacetate in rats through protecting articular cartilage and inhibiting local inflammation. The present study was performed to investigate the preventive effects of SFC on articular chondrocyte, and explore the underlying mechanisms. Primary rabbit chondrocytes were cultured and exposed to sodium nitroprusside (SNP), a NO donor. After treatment with different concentrations of SFC (30, 100, 300, 1,000 μg/ml) for 24 h, nucleic morphology, apoptotic rate, mitochondrial function and caspase-3 activity of chondrocytes were examined. The results showed that SNP induced remarkable apoptosis of rabbit chondrocytes evidenced by Hoechst 33258 staining and flow cytometry analysis, and SFC prevented the apoptosis in a concentration-dependent manner. Further studies indicated that SFC could prevent the depolarization of mitochondrial membrane potential (∆ψm) in SNP-treated chondrocytes and suppress the activation of caspase-3. It can be concluded that the protection of SFC on articular chondrocytes is associated with the anti-apoptosis effects via inhibiting the mitochondrion impairment and caspase-3 activation. PMID:22821055

  19. 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.

  20. TNF/TNFR signal transduction pathway-mediated anti-apoptosis and anti-inflammatory effects of sodium ferulate on IL-1β-induced rat osteoarthritis chondrocytes in vitro

    OpenAIRE

    Qin, Jun.; Shang, Liang; Ping, An-song; Li, Jing; Li, Xiao-Jun; Yu, Hong; Magdalou, Jacques; Chen, Liao-bin; Wang, Hui

    2012-01-01

    Introduction Sodium ferulate (SF) is a natural component of traditional Chinese herbs. Our previous study shows that SF has a protective effect on osteoarthritis (OA). The objective of this study was to investigate the effect of SF on the TNF/TNF receptor (TNFR) signal transduction pathway of rat OA chondrocytes. Methods Primary rat articular chondrocytes were co-treated with IL-1β and SF. Chondrocyte apoptosis was assessed by fluorescein isothiocyanate-annexin V/propidium iodide assay. The P...

  1. Chondrocyte-Specific Inhibition of β-Catenin Signaling Leads to Dysplasia of the Caudal Vertebrae in Mice

    OpenAIRE

    Shu, Bing; Li, Tian-Fang; Li, Xiao-Feng; Tang, De-Zhi; Zhang, Yejia; Shi, Qi; Wang, Yong-Jun; Chen, Di

    2013-01-01

    Study Design. To inhibit β-catenin specifically signaling in chondrocytes Col2-ICAT transgenic mice were generated. Anomalies in caudal vertebrae were detected during embryonic and postnatal stages of Col2-ICAT transgenic mice. Objective. To determine the role of canonical β-catenin signaling in caudal vertebral development. Summary of Background Data. β-catenin signaling plays a critical role in skeletal development. Col2-ICAT transgenic mice were generated to selectively block β-catenin sig...

  2. Evaluation of the effect of antiarthritic drugs on the secretion of proteoglycans by lapine chondrocytes using a novel assay procedure.

    OpenAIRE

    Collier, S; P. Ghosh

    1989-01-01

    A new method is described for separating free 35SO4-- from 35SO4 labelled proteoglycans synthesised by rabbit articular chondrocytes cultured in the presence of excess 35SO4--. The procedure uses the low solubility product of barium sulphate to remove, by precipitation, free 35SO4-- from culture medium. Optimum recovery of 35SO4 labelled proteoglycans was achieved after papain digestion to release 35SO4-glycosaminoglycans, and addition of chondroitin sulphate before the precipitation step. Us...

  3. FGF23 Suppresses Chondrocyte Proliferation in the Presence of Soluble α-Klotho both in Vitro and in Vivo*

    Science.gov (United States)

    Kawai, Masanobu; Kinoshita, Saori; Kimoto, Akihito; Hasegawa, Yasuhiro; Miyagawa, Kazuaki; Yamazaki, Miwa; Ohata, Yasuhisa; Ozono, Keiichi; Michigami, Toshimi

    2013-01-01

    Fibroblast growth factor-23 (FGF23) is well established to play crucial roles in the regulation of phosphate homeostasis. X-linked hypophosphatemic rickets (XLH) is characterized by impaired mineralization and growth retardation associated with elevated circulating FGF23 levels. Administration of phosphate and calcitriol is effective in improving growth retardation, but is not sufficient to fully reverse impaired growth, suggesting the existence of a disease-specific mechanism in the development of growth retardation in addition to dysregulated phosphate metabolism. However, the precise mechanisms of growth retardation in XLH remain elusive. Here, we postulated that FGF23 suppressed chondrocyte proliferation in the presence of soluble α-Klotho (sKL). In vitro and ex vivo studies revealed that FGF23 formed a protein complex with sKL through KL1 internal repeat and suppressed the linear growth of metatarsals in the presence of sKL, which was antagonized by co-incubation with neutralizing antibodies against FGF23 or by knocking-down FGFR3 expression. Additionally, FGF23 binding to FGFR3 was enhanced in the presence of sKL. Histologically, the length of the proliferating zone was diminished and was associated with decreased chondrocyte proliferation. FGF23/sKL suppressed Indian hedgehog (Ihh) expression and administration of Ihh protein partially rescued the suppressive effect of FGF23/sKL on metatarsal growth. Intraperitoneal administration of sKL in Hyp mice, a murine model for XLH, caused a decrease in the length of the proliferating zone associated with decreased chondrocyte proliferation without altering circulating phosphate levels. These findings suggest that suppression of chondrocyte proliferation by FGF23 could have a causative role in the development of growth retardation in XLH. PMID:23235154

  4. Genetic inactivation of ERK1 and ERK2 in chondrocytes promotes bone growth and enlarges the spinal canal

    OpenAIRE

    Sebastian, Arjun; Matsushita, Takehiko; Kawanami, Aya; Mackem, Susan; Landreth, Gary; Murakami, Shunichi

    2010-01-01

    Activating mutations in FGFR3 cause the most common forms of human dwarfism: achondroplasia and thanatophoric dysplasia. In mouse models of achondroplasia, recent studies have implicated the ERK MAPK pathway, a pathway activated by FGFR3, in creating reduced bone growth. Our recent studies have indicated that increased Fgfr3 and ERK MAPK signaling in chondrocytes also causes premature synchondrosis closure in the cranial base and vertebrae, accounting for the sometimes fatal stenosis of the f...

  5. Reactive oxygen species induce expression of vascular endothelial growth factor in chondrocytes and human articular cartilage explants

    OpenAIRE

    Fay, Jakob; Varoga, Deike; Wruck, Christoph J.; Kurz, Bodo; Goldring, Mary B.; Pufe, Thomas

    2006-01-01

    Vascular endothelial growth factor (VEGF) promotes cartilage-degrading pathways, and there is evidence for the involvement of reactive oxygen species (ROS) in cartilage degeneration. However, a relationship between ROS and VEGF has not been reported. Here, we investigate whether the expression of VEGF is modulated by ROS. Aspirates of synovial fluid from patients with osteoarthritis (OA) were examined for intra-articular VEGF using ELISA. Immortalized C28/I2 chondrocytes and human knee cartil...

  6. Phenotypic Characterization of Mycoplasma synoviae Induced Changes in the Metabolic and Sensitivity Profile of In Vitro Infected Chicken Chondrocytes

    OpenAIRE

    Daliborka Dušanić; Dušan Benčina; Mojca Narat; Irena Oven

    2014-01-01

    In infectious synovitis caused by Mycoplasma synoviae chicken chondrocytes (CCH) may come into direct contact with these bacteria that are also capable of invading CCH in vitro. In this study, phenotype microarrays were used to evaluate the influence of Mycoplasma synoviae on the global metabolic activity of CCH. Therefore, CCH were cultured in the presence of 504 individual compounds, spotted in wells of 11 phenotype microarrays for eukaryotic cells, and exposed to Mycoplasma synoviae membra...

  7. Wnt/β-Catenin and Retinoic Acid Receptor Signaling Pathways Interact to Regulate Chondrocyte Function and Matrix Turnover*

    OpenAIRE

    Yasuhara, Rika; Yuasa, Takahito; Williams, Julie A.; Byers, Stephen W.; Shah, Salim; Pacifici, Maurizio; Iwamoto, Masahiro; Enomoto-Iwamoto, Motomi

    2009-01-01

    Activation of the Wnt/β-catenin and retinoid signaling pathways is known to tilt cartilage matrix homeostasis toward catabolism. Here, we investigated possible interactions between these pathways. We found that all-trans-retinoic acid (RA) treatment of mouse epiphyseal chondrocytes in culture did increase Wnt/β-catenin signaling in the absence or presence of exogenous Wnt3a, as revealed by lymphoid enhancer factor/T-cell factor/β-catenin reporter activity and β-catenin nuclear accumulation. T...

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

    Science.gov (United States)

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

    2016-01-01

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

  9. Polysaccharide from Angelica sinensis protects chondrocytes from H2O2-induced apoptosis through its antioxidant effects in vitro.

    Science.gov (United States)

    Zhuang, Chao; Xu, Nan-Wei; Gao, Gong-Ming; Ni, Su; Miao, Kai-Song; Li, Chen-Kai; Wang, Li-Ming; Xie, Hong-Guang

    2016-06-01

    This study aimed to explore the protective effects of Angelica sinensis polysaccharide (ASP) on rat chondrocyte injury induced by hydrogen peroxide (H2O2). Rat chondrocytes were cultured and treated with different concentrations of ASP alone or in combination with H2O2, and they were measured with cell viability, apoptosis, release of inflammatory cytokines, such as interleukin-1beta (IL-1β) and tumor necrosis factor-alpha (TNF-α), activity of superoxide dismutase (SOD), and catalase (CAT), and levels of malondialdehyde (MDA) production, respectively. In addition, quantitative real-time reverse transcription polymerase chain reaction was used to estimate the relative expression levels of osteoarthritis (OA)-associated genes, such as collagen type II (Col2a1), aggrecan, SOX9, matrix metalloproteinase (MMP)-1, -3, and -9, as well as tissue inhibitor of matrix metalloproteinase (TIMP)-1, respectively. Results indicated that ASP protected chondrocytes from H2O2-induced oxidative stress and subsequent cell injury through its antioxidant, antiapoptotic and anti-inflammatory effects in vitro. Our study suggests that ASP could become a therapeutic supplementation for the treatment of OA. PMID:26893055

  10. 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.

  11. Monotropein exerts protective effects against IL-1β-induced apoptosis and catabolic responses on osteoarthritis chondrocytes.

    Science.gov (United States)

    Wang, Feng; Wu, Longhuo; Li, Linfu; Chen, Siyi

    2014-12-01

    Osteoarthritis, characterized by a loss of articular cartilage accompanied with inflammation, is the most common age-associated degenerative disease. Monotropein, an iridoids glycoside isolated from the roots of Morinda officinalis How, has been demonstrated to exhibit anti-inflammatory activity. In the present study, monotropein was firstly to exhibit cartilage protective activity by down regulating the pro-inflammatory cytokines in the knee synovial fluid in vivo. The anti-apoptotic and anti-catabolic effects of monotropein on rat OA chondrocytes treated by IL-1β were investigated in vitro. In cultured chondrocytes, monotropein attenuated apoptosis in a dose-dependent manner in response to IL-1β stimulation. Moreover, treatment with monotropein, the expressions of MMP-3 and MMP-13 were significantly decreased, the expression of COL2A1 was increased. Taken together, these findings suggested that monotropein exerted anti-apoptosis and anti-catabolic activity in chondrocytes, which might support its possible therapeutic role in OA. PMID:25466264

  12. 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.

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

    Science.gov (United States)

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

    2015-12-01

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

  14. Skeletal Characterization of Smurf2-Deficient Mice and In Vitro Analysis of Smurf2-Deficient Chondrocytes.

    Science.gov (United States)

    Huang, Henry; Veien, Eric S; Zhang, Hong; Ayers, David C; Song, Jie

    2016-01-01

    Overexpression of Smad ubiquitin regulatory factor 2 (Smurf2) in chondrocytes was reported to cause spontaneous osteoarthritis (OA) in mice. However, it is unclear whether Smurf2 is involved in bone and cartilage homeostasis and if it is required for OA pathogenesis. Here we characterized age-related changes in the bone and articular cartilage of Smurf2-deficient (MT) mice by microCT and histology, and examined whether reduced Smurf2 expression affected the severity of OA upon surgical destabilization of the medial meniscus (DMM). Using immature articular chondrocytes (iMAC) from MT and wild-type (WT) mice, we also examined how Smurf2 deficiency affects chondrogenic and catabolic gene expressions and Smurf2 and Smurf1 proteins upon TGF-β3 or IL-1β treatment in culture. We found no differences in cortical, subchondral and trabecular bone between WT and MT in young (4 months) and old mice (16-24 months). The articular cartilage and age-related alterations between WT and MT were also similar. However, 2 months following DMM, young MT showed milder OA compared to WT (~70% vs ~30% normal or exhibiting only mild OA cartilage phenotype). The majority of the older WT and MT mice developed moderate/severe OA 2 months after DMM, but a higher subset of aged MT cartilage (27% vs. 9% WT) remained largely normal. Chondrogenic gene expression (Sox9, Col2, Acan) trended higher in MT iMACs than WT with/without TGF-β3 treatment. IL-1β treatment suppressed chondrgenic gene expression, but Sox9 expression in MT remained significantly higher than WT. Smurf2 protein in WT iMACs increased upon TGF-β3 treatment and decreased upon IL-1β treatment in a dose-dependent manner. Smurf1 protein elevated more in MT than WT upon TGF-β3 treatment, suggesting a potential, but very mild compensatory effect. Overall, our data support a role of Smurf2 in regulating OA development but suggest that inhibiting Smurf2 alone may not be sufficient to prevent or consistently mitigate post-traumatic OA

  15. Skeletal Characterization of Smurf2-Deficient Mice and In Vitro Analysis of Smurf2-Deficient Chondrocytes.

    Directory of Open Access Journals (Sweden)

    Henry Huang

    Full Text Available Overexpression of Smad ubiquitin regulatory factor 2 (Smurf2 in chondrocytes was reported to cause spontaneous osteoarthritis (OA in mice. However, it is unclear whether Smurf2 is involved in bone and cartilage homeostasis and if it is required for OA pathogenesis. Here we characterized age-related changes in the bone and articular cartilage of Smurf2-deficient (MT mice by microCT and histology, and examined whether reduced Smurf2 expression affected the severity of OA upon surgical destabilization of the medial meniscus (DMM. Using immature articular chondrocytes (iMAC from MT and wild-type (WT mice, we also examined how Smurf2 deficiency affects chondrogenic and catabolic gene expressions and Smurf2 and Smurf1 proteins upon TGF-β3 or IL-1β treatment in culture. We found no differences in cortical, subchondral and trabecular bone between WT and MT in young (4 months and old mice (16-24 months. The articular cartilage and age-related alterations between WT and MT were also similar. However, 2 months following DMM, young MT showed milder OA compared to WT (~70% vs ~30% normal or exhibiting only mild OA cartilage phenotype. The majority of the older WT and MT mice developed moderate/severe OA 2 months after DMM, but a higher subset of aged MT cartilage (27% vs. 9% WT remained largely normal. Chondrogenic gene expression (Sox9, Col2, Acan trended higher in MT iMACs than WT with/without TGF-β3 treatment. IL-1β treatment suppressed chondrgenic gene expression, but Sox9 expression in MT remained significantly higher than WT. Smurf2 protein in WT iMACs increased upon TGF-β3 treatment and decreased upon IL-1β treatment in a dose-dependent manner. Smurf1 protein elevated more in MT than WT upon TGF-β3 treatment, suggesting a potential, but very mild compensatory effect. Overall, our data support a role of Smurf2 in regulating OA development but suggest that inhibiting Smurf2 alone may not be sufficient to prevent or consistently mitigate post

  16. Age-related differential gene and protein expression in postnatal cartilage canal and osteochondral junction chondrocytes.

    Science.gov (United States)

    Duesterdieck-Zellmer, Katja; Semevolos, Stacy; Kinsley, Marc; Riddick, Tara

    2015-01-01

    Wnt/β-catenin, Indian hedgehog (Ihh)/Parathyroid-related peptide (PTHrP) and retinoid signaling pathways regulate cartilage differentiation, growth, and function during development and play a key role in endochondral ossification. The objective of this study was to elucidate the gene and protein expression of signaling molecules of these regulatory pathways in chondrocytes surrounding cartilage canals and the osteochondral junction during neonatal and pre-adolescent development. This study revealed cell-specific and age-related differences in gene and protein expression of signaling molecules of these regulatory pathways. A trend for higher gene expression of PTHrP along the cartilage canals and Ihh along the osteochondral junction suggests the presence of paracrine feedback in articular-epiphyseal cartilage. Differential expression of canonical (β-catenin, Wnt-4, Lrp4, Lrp6) and noncanonical Wnt signaling (Wnt-5b, Wnt-11) and their inhibitors (Dkk1, Axin1, sFRP3, sFRP5, Wif-1) surrounding the cartilage canals and osteochondral junction provides evidence of the complex interactions occurring during endochondral ossification. PMID:25479004

  17. The mechanism of inhibition of endothelin-1-induced stimulation of DNA synthesis in rat articular chondrocytes.

    Science.gov (United States)

    Khatib, A M; Ribault, D; Quintero, M; Barbara, A; Fiet, J; Mitrovic, D R

    1997-09-19

    Endothelin-1 (ET-1) is a potent mitogen for rat articular chondrocytes (AC) in short term culture (24 h). Prolonged incubation (72 h) of AC with ET-1 resulted in inhibition of [3H]thymidine incorporation. This inhibition seemed to be mediated by prostaglandins (PGs) released in response to ET-1, since indomethacin (INDO) enhanced ET-1-induced [3H]thymidine incorporation. In agreement with this hypothesis, exogenous prostaglandins (PGE2, PGF2alpha and TxB2) blocked all basal, ET-1-induced and ET-1 induced-INDO-enhanced [3H]thymidine incorporation and ET-1 stimulated PGE2 release in a time and concentration-dependent manner. INDO also blocked cGMP production and 6-anilino-5,8-quinolinedione, a relatively specific inhibitor of cGMP formation, enhanced the stimulation and suppressed the inhibition of ET-1-induced DNA synthesis. In addition, 8-bromo-cGMP, an analogue of cGMP, blocked at all time periods studied, both basal and ET-1-induced incorporations of [3H]thymidine. Thus, PGs produced in response to ET-1 counteract the ET-1-induced stimulation of [3H]thymidine incorporation into rat AC by increasing cGMP production. PMID:9324043

  18. Culture of bovine articular chondrocytes in funnel-like collagen-PLGA hybrid sponges

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    Lu Hongxu; Ko, Young-Gwang; Kawazoe, Naoki; Chen Guoping, E-mail: Guoping.Chen@nims.go.jp [Tissue Regeneration Materials Unit, International Center for Materials Nanoarchitectonics (MANA), National Institute for Materials Science, 1-1 Namiki, Tsukuba, Ibaraki 305-0044 (Japan)

    2011-08-15

    Three-dimensional porous scaffolds play an important role in tissue engineering and regenerative medicine. Structurally, these porous scaffolds should have an open and interconnected porous architecture to facilitate a homogeneous cell distribution. Moreover, the scaffolds should be mechanically strong to support new tissue formation. We developed a novel type of funnel-like collagen sponge using embossing ice particulates as a template. The funnel-like collagen sponges could promote the homogeneous cell distribution, ECM production and chondrogenesis. However, the funnel-like collagen sponges deformed during cell culture due to their weak mechanical strength. To solve this problem, we reinforced the funnel-like collagen sponges with a knitted poly(D,L-lactic-co-glycolic acid) (PLGA) mesh by hybridizing these two types of materials. The hybrid scaffolds were used to culture bovine articular chondrocytes. The cell adhesion, distribution, proliferation and chondrogenesis were investigated. The funnel-like structure promoted the even cell distribution and homogeneous ECM production. The PLGA knitted mesh protected the scaffold from deformation during cell culture. Histological and immunohistochemical staining and cartilaginous gene expression analyses revealed the cartilage-like properties of the cell/scaffold constructs after in vivo implantation. The hybrid scaffold, composed of a funnel-like collagen sponge and PLGA mesh, would be a useful tool for cartilage tissue engineering.

  19. 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. PMID:25919282

  20. Selective enrichment of microRNAs in extracellular matrix vesicles produced by growth plate chondrocytes.

    Science.gov (United States)

    Lin, Zhao; Rodriguez, Nicholas E; Zhao, Junjun; Ramey, Allison N; Hyzy, Sharon L; Boyan, Barbara D; Schwartz, Zvi

    2016-07-01

    Matrix vesicles (MVs) are membrane organelles found in the extracellular matrix of calcifying cells, which contain matrix processing enzymes and regulate the extracellular environment via action of these enzymes. It is unknown whether MVs are also exosomic mediators of cell-cell communication via transfer of RNA material, and specifically, microRNA (miRNA). We investigated the presence of RNA in MVs isolated from cultures of costochondral growth zone chondrocytes. Our results showed that the average yield of MV RNA was 1.93±0.78ng RNA/10(4) cells, which was approximately 0.1% of the parent cell's total RNA. MV RNA was well-protected from RNase by the lipid membrane and was highly enriched in small RNA molecules compared to cells. Moreover, coding and non-coding small RNAs in MVs were in proportions that differed from parent cells. Enrichment of specific miRNAs was consistently observed in all three miRNA detection platforms that we used, suggesting that miRNAs are selectively packaged into MVs. MV-enriched miRNAs were related to different signaling pathways associated with bone formation. This study suggests a significant role for MVs as "matrisomes" in cell-cell communication in cartilage and bone development via transfer of specific miRNAs. PMID:27080510

  1. Anti-apoptotic Activity of Ginsenoside Rb1 in Hydrogen Peroxide-treated Chondrocytes: Stabilization of Mitochondria and the Inhibition of Caspase-3.

    Science.gov (United States)

    Na, Ji-Young; Kim, Sokho; Song, Kibbeum; Lim, Kyu-Hee; Shin, Gee-Wook; Kim, Jong-Hoon; Kim, Bumseok; Kwon, Young-Bae; Kwon, Jungkee

    2012-07-01

    Chondrocyte apoptosis has been recognized as an important factor in the pathogenesis of osteoarthritis (OA). Hydrogen peroxide (H2O2), which produces reactive oxygen species, reportedly induces apoptosis in chondrocytes. The ginsenoside Rb1 (GRb1) is the principal component in ginseng and has been shown to have a variety of biological activities, such as anti-arthritis, anti-inflammation, and anti-tumor activities. In this study, we evaluated the effects of G-Rb1 on the mitochondrial permeability transition (MPT) and caspase-3 activity of chondrocyte apoptosis induced by H2O2. Cultured rat articular chondrocytes were exposed to H2O2 with or without G-Rb1 and assessed for viability, MPT, Bcl-xL/Bax expression, caspase-3 activity, and apoptosis. The co-treatment with G-Rb1 showed an inhibition of MPT, caspase-3 activity, and cell death. Additionally, the levels of the apoptotic protein Bax were significantly lower and the levels of the anti-apoptotic protein Bcl-xL were higher compared with H2O2 treatment alone. The results of this study demonstrate that G-Rb1 protects chondrocytes against H2O2-induced apoptosis, at least in part via the inhibition of MPT and caspase-3 activity. These results demonstrate that G-Rb1 is a potentially useful drug for the treatment of OA patients. PMID:23717124

  2. STUDY ON THE EFFECT OF T-2 TOXIN AND SELENIUM ON CD44 EXPRESSION IN THE CULTURED HUMAN FETAL CHONDROCYTES IN VITRO

    Institute of Scientific and Technical Information of China (English)

    谢龙; 曹峻岭; 岳燕; 朱建宏; 张增铁; 张富军; 李思远

    2003-01-01

    Objective To investigate the effect on the structure of reestablished cartilage in vitro and CD44 expression on chondrocytes and compare the inducing effect on the reestablished cartilage in vitro between cortical bone matrix gelatin and cancellous bone matrix gelatin. Methods To plant human fetal chondrocytes on the BMG, the damage of the cultured chondrocytes was observed by the optical microscope (HE staining). The immunohistochemistry of CD44 was quantitative analysis by the image collection and analysis system. Results With the increasing concentration of T-2 toxin, the damage of chondroytes was more and more evident and CD44 expression was lowered. After adding selenium, the damage was relieved and CD44 expression increased. The density of chondrocytes on the cortical bone matrix gelatin was much higher than that on the cancellous bone matrix gelatin. Conclusion T-2 toxin can lower the CD44 expression on the chondrocytes and adding selenium can relieve the damage caused by T-2toxin and increased CD44 expression. The inducing effect on reestablished cartilage in vitro of cortical bone matrix gelatin was much higher than that of cancellous bone matrix gelatin.

  3. The DNA Repair Enzyme Apurinic/Apyrimidinic Endonuclease (Apex Nuclease 2 Has the Potential to Protect against Down-Regulation of Chondrocyte Activity in Osteoarthritis

    Directory of Open Access Journals (Sweden)

    Naoko Yui

    2014-08-01

    Full Text Available Apurinic/apyrimidinic endonuclease 2 (Apex 2 plays a critical role in DNA repair caused by oxidative damage in a variety of human somatic cells. We speculated that chondrocyte Apex 2 may protect against the catabolic process of articular cartilage in osteoarthritis (OA. Higher levels of Apex 2 expression were histologically observed in severely compared with mildly degenerated OA cartilage from STR/OrtCrlj mice, an experimental model which spontaneously develops OA. The immunopositivity of Apex 2 was significantly correlated with the degree of cartilage degeneration. Moreover, the OA-related catabolic factor interleukin-1β induced the expression of Apex 2 in chondrocytes, while Apex 2 silencing using small interfering RNA reduced chondrocyte activity in vitro. The expression of Apex 2 in chondrocytes therefore appears to be associated with the degeneration of articular cartilage and could be induced by an OA-related catabolic factor to protect against the catabolic process of articular cartilage. Our findings suggest that Apex 2 may have the potential to prevent the catabolic stress-mediated down-regulation of chondrocyte activity in OA.

  4. Comparative repair capacity of knee osteochondral defects using regenerated silk fiber scaffolds and fibrin glue with/without autologous chondrocytes during 36 weeks in rabbit model.

    Science.gov (United States)

    Kazemnejad, Somaieh; Khanmohammadi, Manijeh; Mobini, Sahba; Taghizadeh-Jahed, Masoud; Khanjani, Sayeh; Arasteh, Shaghayegh; Golshahi, Hannaneh; Torkaman, Giti; Ravanbod, Roya; Heidari-Vala, Hamed; Moshiri, Ali; Tahmasebi, Mohammad-Naghi; Akhondi, Mohammad-Mehdi

    2016-06-01

    The reconstruction capability of osteochondral (OCD) defects using silk-based scaffolds has been demonstrated in a few studies. However, improvement in the mechanical properties of natural scaffolds is still challengeable. Here, we investigate the in vivo repair capacity of OCD defects using a novel Bombyx mori silk-based composite scaffold with great mechanical properties and porosity during 36 weeks. After evaluation of the in vivo biocompatibility and degradation rate of these scaffolds, we examined the effectiveness of these fabricated scaffolds accompanied with/without autologous chondrocytes in the repair of OCD lesions of rabbit knees after 12 and 36 weeks. Moreover, the efficiency of these scaffolds was compared with fibrin glue (FG) as a natural carrier of chondrocytes using parallel clinical, histopathological and mechanical examinations. The data on subcutaneous implantation in mice showed that the designed scaffolds have a suitable in vivo degradation rate and regenerative capacity. The repair ability of chondrocyte-seeded scaffolds was typically higher than the scaffolds alone. After 36 weeks of implantation, most parts of the defects reconstructed by chondrocytes-seeded silk scaffolds (SFC) were hyaline-like cartilage. However, spontaneous healing and filling with a scaffold alone did not eventuate in typical repair. We could not find significant differences between quantitative histopathological and mechanical data of SFC and FGC. The fabricated constructs consisting of regenerated silk fiber scaffolds and chondrocytes are safe and suitable for in vivo repair of OCD defects and promising for future clinical trial studies. PMID:26822846

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

    International Nuclear Information System (INIS)

    As part of a study to identify novel transcriptional regulators of chondrogenesis-related gene expression, we have cloned and characterized cDNA for zinc-finger protein 470 (ZNF470), the human ortholog of which encodes a 717 amino acid residue protein containing 17 Cys2His2 zinc-finger domains, as well as KRAB-A and KRAB-B motifs. The cDNA library used to isolate the initial ZNF470 clone was prepared from human bone marrow-derived mesenchymal progenitor cells at an intermediate stage of chondrogenic differentiation. We have determined the intron-exon structure of the human ZNF470 gene, which has been mapped to a zinc-finger cluster in a known imprinted region of human chromosome 19q13.4. ZNF470 is expressed at high levels in human testis and is expressed at low or undetectible levels in other adult tissues. Human ZNF470 expressed in mammalian cells as an EGFP fusion protein localizes predominantly to the nucleus, consistent with a role in transcriptional regulation. ZNF470, analyzed by quantitative real time PCR, was transiently expressed before the maximal expression of COL2A1 during chondrogenic differentiation in vitro. We have also characterized the bovine ortholog of human ZNF470, which encodes a 508 amino acid residue protein having 10 zinc-finger domains. A bovine ZNF470 cDNA clone was used to examine expression of ZNF470 in bovine articular chondrocytes treated with retinoic acid to stimulate dedifferentiation. Bovine ZNF470 expression was undetectable in freshly isolated bovine articular chondrocytes, but was dramatically upregulated in dedifferentiated retinoic acid-treated chondrocytes. These results, in two model systems, suggest a possible role for ZNF470 in the regulation of chondrogenesis-specific gene expression

  6. Ginkgo biloba extract individually inhibits JNK activation and induces c-Jun degradation in human chondrocytes: potential therapeutics for osteoarthritis.

    Directory of Open Access Journals (Sweden)

    Ling-Jun Ho

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

  7. Interleukin-1β induces fibroblast growth factor 2 expression and subsequently promotes endothelial progenitor cell angiogenesis in chondrocytes.

    Science.gov (United States)

    Chien, Szu-Yu; Huang, Chun-Yin; Tsai, Chun-Hao; Wang, Shih-Wei; Lin, Yu-Min; Tang, Chih-Hsin

    2016-05-01

    Arthritis is a process of chronic inflammation that results in joint damage. IL (interleukin)-1β is an inflammatory cytokine that acts as a key mediator of cartilage degradation, and is abundantly expressed in arthritis. Neovascularization is one of the pathological characteristics of arthritis. However, the role of IL-1β in the angiogenesis of chondrocytes remains unknown. In the present study, we demonstrate that stimulating chondrocytes (ATDC5) with IL-1β increased the expression of FGF (fibroblast growth factor)-2, a potent angiogenic inducer, and then promoted EPC (endothelial progenitor cell) tube formation and migration. In addition, FGF-2-neutralizing antibody abolished ATDC5-conditional medium-mediated angiogenesis in vitro, as well as its angiogenic effects in the CAM (chick chorioallantoic membrane) assay and Matrigel plug nude mice model in vivo. IHC (immunohistochemistry) staining from a CIA (collagen-induced arthritis) mouse model also demonstrates that arthritis increased the expression of IL-1β and FGF-2, as well as EPC homing in articular cartilage. Moreover, IL-1β-induced FGF-2 expression via IL-1RI (type-1 IL-1 receptor), ROS (reactive oxygen species) generation, AMPK (AMP-activated protein kinase), p38 and NF-κB (nuclear factor κB) pathway has been demonstrated. On the basis of these findings, we conclude that IL-1β promotes FGF-2 expression in chondrocytes through the ROS/AMPK/p38/NF-κB signalling pathway and subsequently increases EPC angiogenesis. Therefore IL-1β serves as a link between inflammation and angiogenesis during arthritis. PMID:26811540

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

    Science.gov (United States)

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

    2012-01-01

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

  9. A suppressive effect of prostaglandin E2 on the expression of SERPINE1/plasminogen activator inhibitor-1 in human articular chondrocytes: An in vitro pilot study

    Directory of Open Access Journals (Sweden)

    Kayo Masuko

    2009-04-01

    Full Text Available Kayo Masuko1, Minako Murata2, Naoya Suematsu1, Kazuki Okamoto1, Kazuo Yudoh2, Hiroyuki Shimizu3, Moroe Beppu3, Hiroshi Nakamura4, Tomohiro Kato11Department of Biochemistry; 2Department of Frontier Medicine, Institute of Medical Science; 3Department of Orthopedic Surgery, St. Marianna University School of Medicine, Kawasaki-shi, Kanagawa, Japan; 4Department of Joint Disease and Rheumatism, Nippon Medical School, Bunkyo-ku, Tokyo, JapanAbstract: Prostaglandin E2 (PGE2 is expressed in articular joints with inflammatory arthropathy and may exert catabolic effects leading to cartilage degradation. As we observed in a preliminary experiment that PGE2 suppressed the expression of SERPINE1/plasminogen activator inhibitor (PAI-1 mRNA in chondrocytes, we focused on the effect of PGE2 on PAI-1 in a panel of cultured chondrocytes obtained from osteoarthritic patients. Specifically, articular cartilage specimens were obtained from patients with osteoarthritis who underwent joint surgery. Isolated chondrocytes were cultured in vitro as a monolayer and stimulated with PGE2. Stimulated cells and culture supernatants were analyzed using Western blotting and enzyme-linked immunosorbent assay. The results confirmed that the in vitro PGE2 stimulation suppressed the expression of PAI-1 in the tested chondrocyte samples. The inhibitory effect was partly abrogated by an antagonist of EP4 receptor of PGE2, but not by an EP2 antagonist. Although PGE2 induced activations of mitogen-activated protein kinases (MAPK, blocking of the MAPK did not abrogate the suppressive effect of PGE2, implying a distinct signaling pathway. In summary, prostaglandin is suggested to modulate the plasminogen system in chondrocytes. Further elucidation of the interaction might open a new avenue to understand the degradative process of cartilage.Keywords: chondrocyte, prostaglandin, PGE2, PAI-1

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

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    Padma P Srinivasan

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

  11. Nicotine-induced retardation of chondrogenesis through down-regulation of IGF-1 signaling pathway to inhibit matrix synthesis of growth plate chondrocytes in fetal rats

    International Nuclear Information System (INIS)

    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

  12. 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.

  13. 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.

  14. The transcription factor Lc-Maf participates in Col27a1 regulation during chondrocyte maturation

    International Nuclear Information System (INIS)

    The transcription factor Lc-Maf, which is a splice variant of c-Maf, is expressed in cartilage undergoing endochondral ossification and participates in the regulation of type II collagen through a cartilage-specific Col2a1 enhancer element. Type XXVII and type XI collagens are also expressed in cartilage during endochondral ossification, and so enhancer/reporter assays were used to determine whether Lc-Maf could regulate cartilage-specific enhancers from the Col27a1 and Col11a2 genes. The Col27a1 enhancer was upregulated over 4-fold by Lc-Maf, while the Col11a2 enhancer was downregulated slightly. To confirm the results of these reporter assays, rat chondrosarcoma (RCS) cells were transiently transfected with an Lc-Maf expression plasmid, and quantitative RT-PCR was performed to measure the expression of endogenous Col27a1 and Col11a2 genes. Endogenous Col27a1 was upregulated 6-fold by Lc-Maf overexpression, while endogenous Col11a2 was unchanged. Finally, in situ hybridization and immunohistochemistry were performed in the radius and ulna of embryonic day 17 mouse forelimbs undergoing endochondral ossification. Results demonstrated that Lc-Maf and Col27a1 mRNAs are coexpressed in proliferating and prehypertrophic regions, as would be predicted if Lc-Maf regulates Col27a1 expression. Type XXVII collagen protein was also most abundant in prehypertrophic and proliferating chondrocytes. Others have shown that mice that are null for Lc-Maf and c-Maf have expanded hypertrophic regions with reduced ossification and delayed vascularization. Separate studies have indicated that Col27a1 may serve as a scaffold for ossification and vascularization. The work presented here suggests that Lc-Maf may affect the process of endochondral ossification by participating in the regulation of Col27a1 expression.

  15. Dynamic mechanical properties of the tissue-engineered matrix associated with individual chondrocytes.

    Science.gov (United States)

    Lee, Bobae; Han, Lin; Frank, Eliot H; Chubinskaya, Susan; Ortiz, Christine; Grodzinsky, Alan J

    2010-02-10

    The success of cell-based tissue engineering approaches in restoring biological function will be facilitated by a comprehensive fundamental knowledge of the temporal evolution of the structure and properties of the newly synthesized matrix. Here, we quantify the dynamic oscillatory mechanical behavior of the engineered matrix associated with individual chondrocytes cultured in vitro for up to 28 days in alginate scaffolds. The magnitude of the complex modulus (|E*|) and phase shift (delta) were measured in culture medium using Atomic Force Microscopy (AFM)-based nanoindentation in response to an imposed oscillatory deformation (amplitude approximately 5nm) as a function of frequency (f=1-316Hz), probe tip geometry (2.5microm radius sphere and 50nm radius square pyramid), and in the absence and presence of growth factors (GF, insulin growth factor-1, IGF-1, and osteogenic protein-1, OP-1). |E*| for all conditions increased nonlinearly with frequency dependence approximately f(1/2) and ranged between approximately 1 and 25kPa. This result, along with theoretical calculations of the characteristic poroelastic relaxation frequency, f(p), (approximately 50-90Hz) suggested that this time-dependent behavior was governed primarily by fluid flow-dependent poroelasticity, rather than flow-independent viscoelastic processes associated with the solid matrix. |E*(f)| increased, (f) decreased, and the hydraulic permeability, k, decreased with time in culture and with growth factor treatment. This trend of a more elastic-like response was thought to be associated with increased macromolecular biosynthesis, density, and a more mature matrix structure/organization. PMID:19889416

  16. Studies on the hyaluronate binding properties of newly synthesized proteoglycans purified from articular chondrocyte cultures

    Energy Technology Data Exchange (ETDEWEB)

    Sandy, J.D.; Plaas, A.H.

    1989-06-01

    Primary cultures of rabbit articular chondrocytes have been maintained for 10 days and labeled with (35S)sulfate, (3H)leucine, and (35S)cysteine in pulse-chase protocols to study the structure and hyaluronate binding properties of newly synthesized proteoglycan monomers. Radiolabeled monomers were purified from medium and cell-layer fractions by dissociative CsCl gradient centrifugation with bovine carrier monomer, and analyzed for hyaluronate binding affinity on Sepharose CL-2B in 0.5 M Na acetate, 0.1% Triton X-100, pH 6.8. Detergent was necessary to prevent self-association of newly synthesized monomers during chromatography. Monomers secreted during a 30-min pulse labeling with (35S)sulfate had a low affinity relative to carrier. Those molecules released into the medium during the first 12 h of chase remained in the low affinity form whereas those retained by the cell layer rapidly acquired high affinity. In cultures where more than 90% of the preformed cell-layer proteoglycan was removed by hyaluronidase digestion before radiolabeling the newly synthesized low affinity monomers also rapidly acquired high affinity if retained in the cell layer. Cultures labeled with amino acid precursors were used to establish the purity of monomer preparations and to isolate core proteins for study. Leucine- or cysteine-labeled core proteins derived from either low or high affinity monomer preparations migrated as a single major species on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with electrophoretic mobility very similar to that of core protein derived from extracted proteoglycan monomer. Purified low affinity monomers were converted to the high affinity form by treatment at pH 8.6; however, this change was prevented by guanidinium-HCl at concentrations above 0.8 M.

  17. Meclozine facilitates proliferation and differentiation of chondrocytes by attenuating abnormally activated FGFR3 signaling in achondroplasia.

    Directory of Open Access Journals (Sweden)

    Masaki Matsushita

    Full Text Available Achondroplasia (ACH is one of the most common skeletal dysplasias with short stature caused by gain-of-function mutations in FGFR3 encoding the fibroblast growth factor receptor 3. We used the drug repositioning strategy to identify an FDA-approved drug that suppresses abnormally activated FGFR3 signaling in ACH. We found that meclozine, an anti-histamine drug that has long been used for motion sickness, facilitates chondrocyte proliferation and mitigates loss of extracellular matrix in FGF2-treated rat chondrosarcoma (RCS cells. Meclozine also ameliorated abnormally suppressed proliferation of human chondrosarcoma (HCS-2/8 cells that were infected with lentivirus expressing constitutively active mutants of FGFR3-K650E causing thanatophoric dysplasia, FGFR3-K650M causing SADDAN, and FGFR3-G380R causing ACH. Similarly, meclozine alleviated abnormally suppressed differentiation of ATDC5 chondrogenic cells expressing FGFR3-K650E and -G380R in micromass culture. We also confirmed that meclozine alleviates FGF2-mediated longitudinal growth inhibition of embryonic tibia in bone explant culture. Interestingly, meclozine enhanced growth of embryonic tibia in explant culture even in the absence of FGF2 treatment. Analyses of intracellular FGFR3 signaling disclosed that meclozine downregulates phosphorylation of ERK but not of MEK in FGF2-treated RCS cells. Similarly, meclozine enhanced proliferation of RCS cells expressing constitutively active mutants of MEK and RAF but not of ERK, which suggests that meclozine downregulates the FGFR3 signaling by possibly attenuating ERK phosphorylation. We used the C-natriuretic peptide (CNP as a potent inhibitor of the FGFR3 signaling throughout our experiments, and found that meclozine was as efficient as CNP in attenuating the abnormal FGFR3 signaling. We propose that meclozine is a potential therapeutic agent for treating ACH and other FGFR3-related skeletal dysplasias.

  18. Effects of mesenchymal stem cells on interleukin-1β-treated chondrocytes and cartilage in a rat osteoarthritic model.

    Science.gov (United States)

    Tang, Jilei; Cui, Weiding; Song, Fanglong; Zhai, Chenjun; Hu, Hansheng; Zuo, Qiang; Fan, Weimin

    2015-08-01

    In the present study, the effects and mechanisms of mesenchymal stem cells (MSCs) on interleukin (IL)-1β-stimulated rat chondrocytes, as well as cartilage from a rat model of osteoarthritis (OA) induced by anterior cruciate ligament transection and medial meniscectomy were investigated. Confluent rat chondrocytes were treated with IL-1β (10 ng/ml), then cultured indirectly with or without MSCs at a ratio of 2:1. Total RNA and protein were collected at various time-points, and western blot and reverse transcription-quantitative polymerase chain reaction analyses were used to investigate the expression of type II collagen (Col2), aggrecan, matrix metalloproteinase-13 (MMP-13) and cyclooxygenase-2 (COX-2). The activation of extracellular signal-regulated kinases 1/2 (ERK1/2), c-Jun N-terminal kinase (JNK), p38 mitogen-activated protein kinase (MAPK), nuclear factor-κB (NF-κB) p65 and inhibitory-κ-B-α (IκBα) were also assessed by western blotting. In addition, the in vivo effects of MSCs in a rat OA model were assessed by histology and western blot analysis. The results indicated that in vitro, IL-1β markedly upregulated the expression of MMP-13, COX-2, phosphorylated ERK1/2, JNK, p38 MAPK and NF-κB p65, and inhibited the expression of Col2, aggrecan and IκBα. Conversely, MSCs enhanced the expression of Col2, aggrecan and IκBα, and inhibited the expression of MMP-13 and NF-κB p65 in IL-1β-stimulated rat chondrocytes. In vivo histological and western blot analyses revealed analogous results to the in vitro findings. The results of the present study demonstrated that MSCs suppressed the inflammatory response and extracellular matrix degradation in IL-1β‑induced rat chondrocytes, as well as cartilage in a osteoarthritic rat model, in part via the NF-κB signaling pathway. PMID:25892273

  19. The dimerization domain of SOX9 is required for transcription activation of a chondrocyte-specific chromatin DNA template

    OpenAIRE

    Coustry, Françoise; Oh, Chun-do; Hattori, Takako; Maity, Sankar N.; de Crombrugghe, Benoit; Yasuda, Hideyo

    2010-01-01

    Mutations in SOX9, a gene essential for chondrocyte differentiation cause the human disease campomelic dysplasia (CD). To understand how SOX9 activates transcription, we characterized the DNA binding and cell-free transcription ability of wild-type SOX9 and a dimerization domain SOX9 mutant. Whereas formation of monomeric mutant SOX9–DNA complex increased linearly with increasing SOX9 concentrations, formation of a wild-type SOX9–DNA dimeric complex increased more slowly suggesting a more sig...

  20. Col2CreERT2, A MOUSE MODEL FOR A CHONDROCYTE-SPECIFIC AND INDUCIBLE GENE DELETION

    OpenAIRE

    M. Chen; S. Li; Xie, W.; Wang, B; Chen, D.

    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...

  1. 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

  2. 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

  3. Comparison of the cellular composition of two different chondrocyte-seeded biomaterials and the results of their transplantation in humans.

    Science.gov (United States)

    Horák, M; Handl, M; Podškubka, A; Kaňa, R; Adler, J; Povýšil, C

    2014-01-01

    Our study compares the histological and immunohistochemical cellular composition of two different chondrocyte-seeded biomaterials and the results of their transplantation. Our study cohort included 21 patients, comprising 19 men and two women with a mean age of 32 years, who were affected by single chondral lesions of the femoral condyles. These patients were enrolled in our study and treated with arthroscopic implantation of the tissue Hyalograft C and/or Brno culture. Brno culture bioengineered with a fibrin-based scaffold contains round cells showing features of differentiated chondrocytes expressing S-100 protein and α-smooth muscle actin. In contrast, in the case of Hyalograft C, the scaffold was made up of a fibrillar network composed of biomaterial fibres of the esters of hyaluronic acid and cells resembling fibroblasts and myofibroblasts and expressing only α-smooth muscle actin. The average size of the defects was 2.5 cm2. Patients were evaluated using the standardized guidelines of the International Knee Documentation Committee. During the comparison of bioptic samples obtained from both patient cohorts, we did not observe any important differences in the histological makeup of the newly formed cartilage. The histological analysis of these two groups of homogeneous patients shows that this bioengineered approach, under proper indications, may offer favourable and stable clinical results over time, in spite of the different matrix and cellular composition of the two transplants used. PMID:24594051

  4. Engineered allogeneic chondrocyte pellet for reconstruction of fibrocartilage zone at bone-tendon junction--a preliminary histological observation.

    Science.gov (United States)

    Wong, Margaret W N; Qin, Lin; Tai, Jenny K O; Lee, Simon K M; Leung, K S; Chan, K M

    2004-08-15

    This study examined histologically the potential of using allogeneic cultured chondrocyte pellet (CCP) in enhancing bone-tendon junction (BTJ) healing using a rabbit partial patellectomy model. Chondrocytes isolated from the cartilaginous ribs of 6-week-old New Zealand white rabbits were cultured for 14 days to form CCP. Partial patellectomy was performed on 30 18-week-old rabbits. After removal of the distal third patella, the BTJ gap was repaired surgically with or without CCP interposition. Four samples of patella-patellar tendon complexes (PPTC) for each group were harvested each at 8, 12, and 16 weeks; and two additional PPTC for each group were harvested at 2, 4, and 6 weeks for early observation of fibrocartilage zone regeneration, histologically. Results showed that CCP interposition demonstrated earlier structural integration at the BTJ after 8, 12, and 16 weeks of healing, and formation of a fibrocartilage zone like structure, compared with control specimens. In addition, no immune rejection was observed in CCP experimental group. The results suggested that CCP had a stimulatory effect on BTJ healing. This bioengineering approach might have potential clinical application in treatment of difficult BTJ healing. However, systemic histomorphometric, immunological tests, and biomechanical evaluations are needed before any clinical trials. PMID:15264320

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

    Science.gov (United States)

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

    2010-11-01

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

  6. Influence of cytochalasin D-induced changes in cell shape on proteoglycan synthesis by cultured articular chondrocytes

    International Nuclear Information System (INIS)

    There is growing evidence that cell shape regulates both proliferation and differentiated gene expression in a variety of cell types. The authors have explored the relationship between the morphology of articular chondrocytes in culture and the amount and type of proteoglycan they synthesize, using cytochalasin D to induce reversible cell rounding. When chondrocytes were prevented from spreading or when spread cells were induced to round up, 35SO4 incorporation into proteoglycan was stimulated. Incorporation into the cell layer was stimulated more than into the medium. When the cells were allowed to respread by removing cytochalasin D, proteoglycan synthesis returned to control levels. Cytochalasin D-induced stimulation of 35SO4 incorporation reflected an increase in core protein synthesis rather than lengthening of glycosaminoglycan chains, because [3H]serine incorporation into core protein was also stimulated. Cytochalasm D-treatment of cells in suspension caused no further stimulation of 35SO4 incorporation, suggesting that the observed effects were due to cell rounding rather than exposure to cytochalasin D per se

  7. Autologous chondrocyte implantation (ACI for the treatment of large and complex cartilage lesions of the knee

    Directory of Open Access Journals (Sweden)

    Ossendorf Christian

    2011-05-01

    Full Text Available Abstract Background Complex cartilage lesions of the knee including large cartilage defects, kissing lesions, and osteoarthritis (OA represent a common problem in orthopaedic surgery and a challenging task for the orthopaedic surgeon. As there is only limited data, we performed a prospective clinical study to investigate the benefit of autologous chondrocyte implantation (ACI for this demanding patient population. Methods Fifty-one patients displaying at least one of the criteria were included in the present retrospective study: (1. defect size larger than 10 cm2; (2. multiple lesions; (3. kissing lesions, cartilage lesions Outerbridge grade III-IV, and/or (4. mild/moderate osteoarthritis (OA. For outcome measurements, the International Cartilage Society's International Knee Documentation Committee's (IKDC questionnaire, as well as the Cincinnati, Tegner, Lysholm and Noyes scores were used. Radiographic evaluation for OA was done using the Kellgren score. Results and Discussion Patient's age was 36 years (13-61, defects size 7.25 (3-17.5 cm2, previous surgical procedures 1.94 (0-8, and follow-up 30 (12-63 months. Instruments for outcome measurement indicated significant improvement in activity, working ability, and sports. Mean ICRS grade improved from 3.8 preoperatively to grade 3 postoperatively, Tegner grade 1.4 enhanced to grade 3.39. The Cincinnati score enhanced from 25.65 to 66.33, the Lysholm score from 33.26 to 64.68, the Larson score from 43.59 to 79.31, and Noyes score from 12.5 to 46.67, representing an improvement from Cincinnati grade 3.65 to grade 2.1. Lysholm grade 4 improved to grade 3.33, and Larson grade 3.96 to 2.78 (Table 1, (p Table 1 Mean scores and grades at surgery (Tx and at follow-up Tx Follow-up Score Grade Score Grade ICRS 4 3 Tegner 1 3 Noyes 13 47 Cincinnati 26 4 66 2 Lysholm 33 4 65 3 Larson 44 4 79 3 Conclusion Our results suggest that ACI provides mid-term results in patients with complex cartilage lesions of

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

    Czech Academy of Sciences Publication Activity Database

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

    2010-01-01

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

  9. 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.

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

    Science.gov (United States)

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

    2012-01-01

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

  11. A post-translational modification cascade employing HDAC9-PIASy-RNF4 axis regulates chondrocyte hypertrophy by modulating Nkx3.2 protein stability.

    Science.gov (United States)

    Choi, Hye-Jeong; Kwon, Seongran; Kim, Dae-Won

    2016-09-01

    While Nkx3.2/Bapx1 promotes chondrogenic differentiation and plays a role in maintaining chondrocyte viability and suppressing chondrocyte hypertrophy, the regulatory mechanisms of Nkx3.2 remain poorly understood. Here we show that p300- and HDAC9-induced Nkx3.2 acetylation and de-acetylation, respectively, play critical roles in controlling Nkx3.2 protein stability. In addition, we also found that HDAC9-dependent de-acetylation of Nkx3.2 triggers PIASy-mediated sumoylation and subsequent RNF4-mediated SUMO-targeted ubiquitination. Furthermore, we demonstrate that Nkx3.2 regulation by HDAC9 can be linked to the management of chondrocyte survival and hypertrophic maturation during cartilage development. Finally, our results together reveal a novel mechanism of protein stability control involving complex interplay between acetylation, de-acetylation, sumoylation, and ubiquitination, and suggest that this post-translational modification of Nkx3.2 employing HDAC9-PIASy-RNF4 axis plays a crucial role in controlling chondrocyte viability and hypertrophic maturation during skeletal development in vertebrates. PMID:27312341

  12. 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

  13. 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. PMID:26846886

  14. 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.

  15. Effects of 99Tc-MDP on synoviocytes and articular chondrocytes apoptosis associated factors on CIA rats

    International Nuclear Information System (INIS)

    Objective: Collagen induced arthritis (CIA) rats is an animal model of human rheumatoid arthritis (RA). It is widely used in research of the pathogenesis and the therapeutic targets of RA. This paper was to investigate the therapeutic action of 99Tc-methylene diphosphonic acid (MDP) on CIA rats and its effects on the expression of apoptosis associated factor bcl-2 and bax in synoviocytes and articular chondrocytes. Methods: CIA rat models were carried out by subcutaneous injection with bovine collagen II and incomplete Freud's adjuvant. Rats were divided into four groups: control group, CIA model group (the CIA rats were infused with physiological saline via tail vein daily), 99Tc-MDP group (the C1A rats were injected with 99Tc-Mi)P 0.04 μg 99Tc/kg via tail vein daily) and methotrexate (MTX) group (the CIA rats were injected with MTX 1 mg/kg via tail vein weekly). The signs of arthritis were evaluated by arthritis index (AI) scores. Immunohistochemistry was performed to detect the expression of bcl-2 and bax in synoviocytes and articular chondrocytes. SPSS 13.0 was used for data analysis. Results: (1) The signs of arthritis, AI scores and pathological changes of arthrosynovitis in CIA rats were significantly improved by 99Tc-MDP or MTX. (2) The expression of bcl-2 and box in the synoviocytes of CIA model group [(39.30 ± 0.53) %, (27.37 ± 2.45)%] was significantly increased compared with control group [(7.56 ± 1. 18)% , (6.14 ± 1.71) % ; q = 46.27, 24.57, all P 99Tc-M DP group and MTX group, the level of bcl-2 was remarkably decreased [(30.24 ± 2.09) %, (27.25 ± 3.33) %] compared with CIA model group (q = 13.20, 17.56, all P 99Tc-MDP group [(26. 58 ± 2. 52) %] and MTX group [(27.06 ± 1.92) %] was remarksbly increased [(24.26 ± 2.75) %, (23.53 ± 0.74) % ; q = 6.53, 7.01, all P 99Tc-MDP could improve the signs of arthritis, meanwhile regulate the expression of bcl-2 and bax in synoviocytes and articular ehondrocytes, suggesting that one of the therapeutic

  16. 99mTc-labeled chondroitin sulfate-uptake by chondrocytes and cartilage. Potential agent for osteoarthritis imaging?

    International Nuclear Information System (INIS)

    Aim: Chondroitin sulfate (CS) is an endogenous component of cartilage proteoglycan which could monitor osteoarthritic cartilage degradation after radiolabeling. This substance is used in the treatment of human osteoarthritis as a slow acting symptomatic drug (CONDROSULF; Sanova Pharma, Vienna; Ibsa, Switzerland). Material and Methods: Radiolabeling of CS was performed using 99mTcO4-/stannous chloride in 0.50 M sodium acetate buffer at pH 5.0. The quality control of the tracer was performed using ITLC-SG chromatography and 0.2 M saline in 10% ethanol as solvent to detect colloid content. Aluminium oxide IB-F TLC-sheets and ethanol as solvent were used to estimate free pertechnetate. For uptake studies cultured human chondrocytes and age-matched cartilage were used. Uptake of the tracer in chondrocytes was studied in monolayer and in suspension cultures at 370C. Uptake was monitored for a total of 120-180 minutes, samples being drawn every 10 minutes. Because the commercially available drug Condrosulf contains calcium stearate as additive to improve the resorption of the drug, we investigated also the uptake with and without additive. Results: The tracer was stable over 6h period after labeling (95% of the radiochemical purity). In plasma the stability was lower amounting to 75%. Viability of chondrocytes after incubation with either CS-preparation was found by trypan blue exclusion to be above 95 %. Uptake of the tracer performed in monolayer ± additives was low and amounted to 0.5%±0.05%, n=6. The cells were saturated already after an incubation interval of 10 minutes. In suspension cultures a maximal uptake of 1.0%±0.1%, n=6 and 5.9%±0.65%, n=6 was found, without and with additives, respectively, the saturation was achieved after 100 min. Thus, not only the resorption of the drug is enhanced by Ca-stearate, but also uptake increases in presence of this additive. Using human rib cartilage the uptake of the tracer was much higher amounting to 4.9%±2.3%, n=6

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

    Directory of Open Access Journals (Sweden)

    Collodel G

    2013-03-01

    Full Text Available Giulia Collodel,1 Antonella Fioravanti,2 Nicola Antonio Pascarelli,2 Antonello Lamboglia,2 Vania Fontani,3 Margherita Maioli,3–5 Sara Santaniello,4,5 Gianfranco Pigliaru,4,5 Alessandro Castagna,3 Elena Moretti,1 Francesca Iacoponi,1 Salvatore Rinaldi,3 Carlo Ventura3,5,6 1Department of Biomedical Sciences (Applied Biology, 2Department of Clinical Medicine and Immunological Sciences (Rheumatology Unit, University of Siena, Siena, Italy; 3Department of Regenerative Medicine, Rinaldi Fontani Institute, Florence, Italy; 4Department of Biomedical Sciences, University of Sassari, Sassari, Italy; 5Laboratory of Molecular Biology and Stem Cell Engineering, National Institute of Biostructures and Biosystems, Bologna, Italy; 6Cardiovascular Department, S Orsola-Malpighi Hospital, University of Bologna, Bologna, Italy Purpose: Osteoarthritis (OA is a degenerative disease characterized by a progressive loss of articular cartilage extracellular matrix and is due to functional impairments occurring in chondrocytes. In previous works, we highlighted that Regenerative Tissue Optimization (TO-RGN treatment with radioelectric asymmetric conveyer (REAC technology influenced the gene expression profiles controlling stem cell differentiation and the pluripotency of human skin-derived fibroblasts in vitro. Since interleukin-1 beta signaling has been implicated in the induction and progression of this disease (through metalloproteinase-3 synthesis and nitric oxide production, we investigated whether REAC TO-RGN might influence the biochemical and morphological changes induced by interleukin-1 beta in normal and OA chondrocytes. Methods: The induction of metalloproteinase-3 and proteoglycan synthesis was evaluated by a solid-phase enzyme-amplified sensitivity immunoassay, and nitric oxide production was evaluated with the Griess method. Ultrastructural features were observed by transmission electron microscopy. Results: REAC TO-RGN treatment decreased nitric oxide

  18. CartiGenea®-AC: A Mesenchymal stem cells enriched Autologous Chondrocytes for the Treatment of patients with cartilaginous defects on a New Drug-Cell Combinatory Effect Prediction Algorithm on the Cell Based on Chondro defects Gene Expression and Dose-Response Curve.

    OpenAIRE

    Grigoriadis Ioannis

    2015-01-01

    CartiGenea®-AC: Chondrocytes, the predominant cell type within AC, synthesize matrix components. Because AC lacks a major vascular supply, lymphatic drainage, and nervous system innervation, chondrocytes function under avascular, anaerobic conditions, obtaining nutrients by diffusion from synovial fluid. Within AC, metabolic and morphologic profiles of deep-zone chondrocytes are distinct from those populating the superficial tangential zone. The factors responsible for this variation are ...

  19. Antioxidant effects of betulin on porcine chondrocyte behavior in gelatin/C6S/C4S/HA modified tricopolymer scaffold

    International Nuclear Information System (INIS)

    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.

  20. The polyamine analogue N1,N11-diethylnorspermine can induce chondrocyte apoptosis independently of its ability to alter metabolism and levels of natural polyamines.

    Science.gov (United States)

    Stanic', Ivana; Facchini, Annalisa; Borzì, Rosa Maria; Stefanelli, Claudio; Flamigni, Flavio

    2009-04-01

    We have been investigating the effects of natural polyamines and polyamine analogues on the survival and apoptosis of chondrocytes, which are cells critical for cartilage integrity. Treatment of human C-28/I2 chondrocytes with N(1),N(11)-diethylnorspermine (DENSPM), a polyamine analogue with clinical relevance as an experimental anticancer agent, rapidly induced spermidine/spermine N(1)-acetyltransferase (SSAT) and spermine oxidase (SMO), key enzymes of polyamine catabolism and down-regulated ornithine decarboxylase, the first enzyme of polyamine biosynthesis, thus depleting all main polyamines within 24 h. The treatment with DENSPM did not provoke cell death and caspase activation when given alone for 24 h, but caused a caspase-3 and -9 dependent apoptosis in chondrocytes further exposed to cycloheximide (CHX). In other cellular models, enhanced polyamine catabolism or polyamine depletion has been implicated as mechanisms involved in DENSPM-related apoptosis. However, the simultaneous addition of DENSPM and CHX rapidly increased caspase activity in C-28/I2 cells in the absence of SSAT and SMO induction or significant reduction of polyamine levels. Moreover, caspase activation induced by DENSPM plus CHX was not prevented by a N(1)-acetylpolyamine oxidase (PAO)/SMO inhibitor, and depletion of all polyamines obtained by specific inhibitors of polyamine biosynthesis did not reproduce DENSPM effects in the presence of CHX. DENSPM/CHX-induced apoptosis was associated with changes in the amount or activation of signalling kinases, Akt and MAPKs, and increased uptake of DENSPM. In conclusion, the results suggest that DENSPM can favour apoptosis in chondrocytes independently of its effects on polyamine metabolism and levels. PMID:19097065

  1. 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...

  2. 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.

  3. Andrographolide Exerts Chondroprotective Activity in Equine Cartilage Explant and Suppresses Interleukin-1β-Induced MMP-2 Expression in Equine Chondrocyte Culture

    OpenAIRE

    Tangyuenyong, Siriwan; Viriyakhasem, Nawarat; Peansukmanee, Siriporn; Kongtawelert, Prachya; Ongchai, Siriwan

    2014-01-01

    Cartilage erosion in degenerative joint diseases leads to lameness in affected horses. It has been reported that andrographolide from Andrographis paniculata inhibited cartilage matrix-degrading enzymes. This study aimed to explore whether this compound protects equine cartilage degradation in the explant culture model and to determine its effect on matrix metalloproteinase-2 (MMP-2) expression, a matrix-degrading enzyme, in equine chondrocyte culture. Equine articular cartilage explant cultu...

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

    Science.gov (United States)

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

    2015-12-01

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

  5. Berberine induces dedifferentiation by actin cytoskeleton reorganization via phosphoinositide 3-kinase/Akt and p38 kinase pathways in rabbit articular chondrocytes.

    Science.gov (United States)

    Yu, Seon-Mi; Cho, Hongsik; Kim, Gwang-Hoon; Chung, Ki-Wha; Seo, Sung-Yum; Kim, Song-Ja

    2016-04-01

    Osteoarthritis is a nonrheumatologic joint disease characterized by progressive degeneration of the cartilage extracellular matrix. Berberine (BBR) is an isoquinoline alkaloid used in traditional Chinese medicine, the majority of which is extracted from Huang Lian (Coptis chinensis). Although numerous studies have revealed the anticancer activity of BBR, its effects on normal cells, such as chondrocytes, and the molecular mechanisms underlying its actions remain elusive. Therefore, we examined the effects of BBR on rabbit articular chondrocytes, and the underlying molecular mechanisms, focusing on actin cytoskeletal reorganization. BBR induced dedifferentiation by inhibiting activation of phosphoinositide-3(PI3)-kinase/Akt and p38 kinase. Furthermore, inhibition of p38 kinase and PI3-kinase/Akt with SB203580 and LY294002, respectively, accelerated the BBR-induced dedifferentiation. BBR also caused actin cytoskeletal architecture reorganization and, therefore, we investigated if these effects were involved in the dedifferentiation. Disruption of the actin cytoskeleton by cytochalasin D reversed the BBR-induced dedifferentiation by activating PI3-kinase/Akt and p38 kinase. In contrast, the induction of actin filament aggregation by jasplakinolide accelerated the BBR-induced dedifferentiation via PI3-kinase/Akt inhibition and p38 kinase activation. Taken together, these data suggest that BBR strongly induces dedifferentiation, and actin cytoskeletal reorganization is a crucial requirement for this effect. Furthermore, the dedifferentiation activity of BBR appears to be mediated via PI3-kinase/Akt and p38 kinase pathways in rabbit articular chondrocytes. PMID:26851252

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

    Science.gov (United States)

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

    2015-07-01

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

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

    International Nuclear Information System (INIS)

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

  8. 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.

  9. 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.

  10. 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

  11. Three-Dimensional Matrix-Induced Autologous Chondrocytes Implantation for Osteochondral Lesions of the Talus: Midterm Results

    Directory of Open Access Journals (Sweden)

    B. Magnan

    2012-01-01

    Full Text Available Introduction. We evaluate the midterm results of thirty patients who underwent autologous chondrocytes implantation for talus osteochondral lesions treatment. Materials and Methods. From 2002 to 2009, 30 ankles with a mean lesion size of 2,36 cm2 were treated. We evaluated patients using American Orthopaedic Foot and Ankle Surgery and Coughlin score, Van Dijk scale, recovering time, and Musculoskeletal Outcomes Data Evaluation and Management System. Results. The mean AOFAS score varied from 36.9 to 83.9 at follow-up. Average of Van Dijk scale was 141.1. Coughlin score was excellent/good in 24 patients. MOCART score varied from 6.3 to 3.8. Discussion. This matrix is easy to handle conformable to the lesion and apply by arthroscopy. No correlation between MRI imaging and clinical results is found. Conclusions. Our results, compared with those reported in literature with other surgical procedures, show no superiority evidence for our technique compared to the others regarding the size of the lesions.

  12. 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.

  13. Decreased synthesis of extracellular matrix components by chondrocytes cultured in scorbutic guinea pig serum plus vitamin C

    Energy Technology Data Exchange (ETDEWEB)

    Oyamada, I.; Bird, T.A.; Peterkofsky, B.

    1986-05-01

    The authors have previously shown that cartilage collagen (col) and proteoglycan (PG) syntheses decreased coordinately in guinea pig (GP) receiving a vitamin C deficient (C-def) diet for more than 2 weeks. The defects were related to the fasting state induced during the 3rd and 4th weeks of scurvy, rather than to the role of ascorbate in proline (pro) hydroxylation. These results and the generalized effect on col synthesis suggested the involvement of humoral factors. Sera from normal (NGPS) and scorbutic (SGPS) GP supported growth of BALB 3T3 cells under conditions where EGF plus insulin (or IGF I) were required, for up to 2 weeks after initiation of the C-def diet. Thereafter activity was lost from SGPS. The ability of NGPS and 4-week-SGPS, both with added ascorbate, to maintain normal rates of col and PG syntheses in cultured chick embryo chondrocytes was measured by incorporation of (/sup 14/C)pro into collagenase digestible protein and (/sup 35/S)sulfate into PG. Although pro hydroxylation and col secretion were normal in cells cultured with SGPS for 2 days, col and PG synthetic rates were decreased to 40-50% of rates in cells cultured in NGPS, with no change in the types of col and PG synthesized. These results provide evidence that depletion of growth factors during the fasting stage of scurvy may cause decreased synthesis of extracellular matrix components.

  14. Assay for inorganic pyrophosphate in chondrocyte culture using anion-exchange high-performance liquid chromatography and radioactive orthophosphate labeling

    International Nuclear Information System (INIS)

    A method is described for determination of inorganic pyrophosphate (PPi) in cell culture medium and in rabbit articular chondrocytes grown in the presence of radioactive orthophosphate (32Pi). Intra- and extracellular 32PPi formed was measured using high-performance liquid chromatographic (HPLC) separation of the PPi from orthophosphate (Pi) and other phosphate-containing compounds. The chromatographic separation on a weak anion-exchange column is based on the extent to which various phosphate compounds form complexes with Mg2+ at low pH and the rate at which such formation occurs. These complexes are eluted more readily than the uncomplexed compounds. Best results were obtained using a simultaneous gradient of Mg2+ ions and ionic strength. In this case separation of small amounts of PPi from a large excess of Pi was possible without prior removal of Pi or extraction of the PPi fraction. The assay is also useful for measurement of inorganic pyrophosphatase activity. The sensitivity of the assay depends on the specific activity of the added 32Pi and on the culture conditions, but is comparable with the most sensitive of the enzymatic assays. Sample preparation, particularly deproteinization, proved to be of importance. The losses of PPi which occur during procedures of this sort due to hydrolysis and coprecipitation were quantitated

  15. 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.

  16. Mitogenic and metabolic actions of epidermal growth factor on rat articular chondrocytes: modulation by fetal calf serum, transforming growth factor-beta, and tyrphostin.

    Science.gov (United States)

    Ribault, D; Khatib, A M; Panasyuk, A; Barbara, A; Bouizar, Z; Mitrovic, R D

    1997-01-15

    The effects of human recombinant epidermal growth factor (EGF) on rat articular chondrocytes from humeral and femoral head cartilage of 21-day-old Wistar rats were analyzed. The cells were cultured under standard conditions as monolayers. Cell proliferation was studied by [3H]thymidine incorporation and determination of DNA content, proteoglycan synthesis by [35S]sulfate incorporation, and collagen synthesis by [3H]proline incorporation. The presence of specific receptors was confirmed by [125I]-EGF binding and that of EGF and EGF-receptor (EGF-R) mRNA by reverse transcription and the polymerase chain reaction. EGF (0.5-2.5 ng/ml) stimulated [3H]thymidine incorporation and increased DNA content of cultures. The effect was strongest when serum concentration was low ( or =7.5%) serum concentrations. The EGF-induced effect on deoxynucleic acid synthesis was inhibited by transforming growth factor-beta and tyrphostin, a tyrosine kinase inhibitor that blocks the phosphorylation of tyrosine residues on EGF-R. Cultured rat articular chondrocytes possess a single class of high-affinity binding sites (Kd 0.18 nM). There were about 4.5 x 10(9) binding sites per microgram of DNA or about 37,800 binding sites per cell with 8.3 pg DNA per cell. Cultured cells contained EGF mRNA and EGF-R mRNA. Incubation of cells with EGF for 24 h decreased the EGF mRNA transcripts and increased the EGF-R mRNA levels. These findings suggest that EGF probably takes part in the regulation of chondrocyte activity under normal and presumably pathological conditions. PMID:9016808

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

    Directory of Open Access Journals (Sweden)

    Bobrowski Paul

    2006-04-01

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

  18. 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.)

  19. Diffusion-weighted imaging for the follow-up of patients after matrix-associated autologous chondrocyte transplantation

    International Nuclear Information System (INIS)

    Objective: To evaluate the use of diffusion-weighted imaging (DWI) for the assessment of cartilage maturation in patients after matrix-associated autologous chondrocyte transplantation (MACT). Materials and methods: Fifteen patients after MACT were examined by 3.0-T magnetic-resonance-tomography; the examination was up to 13 month after surgery in group 1, and later than 13 month after surgery in group 2. Both groups had a follow-up one-year later. DWI was acquired using a steady-state gradient-echo sequence. Mean values of the diffusion quotients of regions of interest within cartilage repair tissue and of reference regions were assessed. Each region-of-interest was subdivided into a deep, and a superficial area. Results: Mean diffusion quotients of cartilage repair tissues were 1.44 (baseline), and 1.44 (follow-up). Mean diffusion quotients of reference tissues were 1.29 (baseline) and 1.28 (follow-up). At the follow-up diffusion quotients of cartilage repair tissue were significantly higher than those of reference cartilage. In group 1 the diffusion quotients were significantly lower at the follow-up (1.45 versus 1.65); in group 2 no statistically significant differences between follow-up (1.39) and baseline (1.41) were found. Reference cartilages and cartilage repair tissues of group 2 showed a decrease of diffusion quotients from the deep to the superficial area being stable at the follow-up. In group 1 initially a significant increase (1.49 versus 1.78) of the diffusion quotients from deep to superficial area of the cartilage repair tissue was found changing into a decrease (1.65 versus 1.52) at the follow-up. Conclusions: DWI detected changes of diffusion within cartilage repair tissue that may reflect cartilage maturation. Changes in diffusity occurred up to two years after surgery and were stable later. Zonal variations within cartilage could be measured.

  20. Pharmacological effects of a C-phycocyanin-based multicomponent nutraceutical in an in-vitro canine chondrocyte model of osteoarthritis.

    Science.gov (United States)

    Martinez, Stephanie E; Chen, Yufei; Ho, Emmanuel A; Martinez, Steven A; Davies, Neal M

    2015-07-01

    Multicomponent nutraceuticals are becoming increasingly popular treatments or adjunctive therapies for osteoarthritis in veterinary medicine despite lack of evidence of efficacy for many products. The objective of this study was to evaluate the anti-inflammatory and antioxidant activities of a commercially available C-phycocyanin-based nutraceutical and select constituent ingredients in an in-vitro model of canine osteoarthritis. Normal canine articular chondrocytes were used in an in-vitro model of osteoarthritis. Inflammatory conditions were induced using interleukin-1β. The nutraceutical preparation as a whole, its individual constituents, as well as carprofen were evaluated at concentrations of 0 to 250 μg/mL for reduction of the following inflammatory mediators and indicators of catabolism of the extracellular matrix: prostaglandin E2 (PGE2), tumor necrosis factor-α (TFN-α), interleukin-6 (IL-6), metalloproteinase-3 (MMP-3), nitric oxide, and sulfated glycosaminoglycans (sGAGs). Validated, commercially available assay kits were used for quantitation of inflammatory mediators. The antioxidant capacities, as well as cyclooxygenase-1 (COX-1), cyclooxygenase-2 (COX-2), and lipoxygenase (LOX) inhibitory activities of the whole nutraceutical preparation and select constituents, were also assessed using validated commercially available assay kits. The antioxidant capacity of the nutraceutical and constituents was concentration-dependent. The nutraceutical and constituents appear to display anti-inflammatory activity primarily through the inhibition of COX-2. The nutraceutical displayed similar strength to carprofen in reducing TNF-α, IL-6, MMP-3, nitric oxide, and sGAGs at select concentration ranges. The C-phycocyanin (CPC)-based nutraceutical and constituents may be able to mediate 3 primary pathogenic mechanisms of osteoarthritis: inflammation, chondral degeneration, and oxidative stress in vitro. The nutraceutical may be clinically useful in veterinary

  1. MR appearance of autologous chondrocyte implantation in the knee: correlation with the knee features and clinical outcome

    International Nuclear Information System (INIS)

    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.)

  2. 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. PMID:24777663

  3. 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

    OpenAIRE

    Srinivasan, Padma P.; Parajuli, Ashutosh; Price, Christopher; Wang, Liyun; Duncan, Randall L.; Kirn-Safran, Catherine B.

    2015-01-01

    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-VS...

  4. 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

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

    OpenAIRE

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

    2012-01-01

    The aim of this study was to characterize the anti-inflammatory mode of action of botanical extracts from rosehip (Rosa canina), willow bark (Salix alba), and nettle leaf (Urtica dioica) in an in vitro model of primary canine articular chondrocytes. Methods. The biological effects of the botanical extracts were studied in chondrocytes treated with IL-1 beta for up to 72h. Expression of collagen type II, cartilage-specific proteoglycan (CSPG), beta 1-integrin, SOX-9, COX-2, and MMP-9 and MMP-1...

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

    OpenAIRE

    Mehdi Shakibaei; David Allaway; Simone Nebrich; Ali Mobasheri

    2012-01-01

    The aim of this study was to characterize the anti-inflammatory mode of action of botanical extracts from rosehip (Rosa canina), willow bark (Salix alba), and nettle leaf (Urtica dioica) in an in vitro model of primary canine articular chondrocytes. Methods. The biological effects of the botanical extracts were studied in chondrocytes treated with IL-1β for up to 72 h. Expression of collagen type II, cartilage-specific proteoglycan (CSPG), β1-integrin, SOX-9, COX-2, and MMP-9 and MMP-13 was e...

  7. 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. PMID:24711445

  8. Biocompatibility and biomechanical characteristics of loofah based scaffolds combined with hydroxyapatite, cellulose, poly-l-lactic acid with chondrocyte-like cells.

    Science.gov (United States)

    Cecen, Berivan; Kozaci, Leyla Didem; Yuksel, Mithat; Ustun, Ozcan; Ergur, Bekir Ugur; Havitcioglu, Hasan

    2016-12-01

    The current study reports the biocompatibility and biomechanical characteristics of loofah-based scaffolds combined with hydroxyapatite (HA), cellulose, poly-l-lactic acid (PLLA) with chondrocytes-like cells. Scanning electron microscope (SEM) micrographs of the scaffolds showed that the addition of PLLA usually resulted in an increase in cell's attachment on scaffolds. Mechanical and elemental analyzes were assessed using tensile test and Energy Dispersive X-ray spectrometry (EDS), respectively. In summary, we showed that the loofah+PLLA+HA scaffolds perform significantly better than other loofah-based scaffolds employed in terms of increasing a diversity of mechanical properties including tensile strength and Young's modulus. Based on the analysis of the differential scanning calorimetry (DSC) thermograms and EDS spectrums that give an idea about the calcium phosphate (CaP) ratios, the improvement in the mechanical properties could principally be recognized to the strong interaction formed between loofah, PLLA and HA. The viability of chondrocytes on loofah-based scaffolds was analyzed by XTT tests. However, none of the scaffolds have proved to be toxic in metabolic activity. The histological evaluation obtained by hematoxylin and eosin (H&E), Masson trichrome, toluidine blue and immunohistochemistry methods showed that cells in all scaffolds produced extracellular matrix that defined proteoglycan and type I-II collagens. The results of this study suggest that the loofah-based scaffold with desirable properties can be considered as an ideal candidate for cartilage tissue engineering applications. PMID:27612733

  9. The effect of intrauterine exposure to tetracycline on the population of hypertrophic chondrocytes in the growth plate of long bones in mice

    International Nuclear Information System (INIS)

    The hypertrophic chondrocytes is one factor that determines the size of growth plates. Tetracycline, a broad spectrum antibiotic is known to result in limb shortening following prenatal exposure in mice. The effects of 12mg/kg body weight of tetracycline on the size and number of hypertrophic chondrocytes of the growth plate of mice was investigated in the study. Oral tetracycline at 12mg/kg body weight was administered to pregnant mice between 11th and 15th day of gestation. The foetuses were harvested on the 20th day of gestation. The humeri of the foetuses were fixed in formal saline and histological slides were made using Toluidine blue as the stain. Measurements were made with the use of a micrometer. No gross anomaly or significant growth retardation was revealed. However, a significant reduction of humeral length was observed (T-cal=7.359, P>0.05) with associated significant cell depletion, particularly in the distal growth plate. There is however, no significant difference in the cell population in the proximal growth plate and the cellular density of both proximal and distal plates. At the aforementioned dose, tetracycline does not appear to cause any significant growth retardation or foetal anomaly. However, it causes a cell-depleting effect particularly on the distal growth plate. This shows that the cell depleting effect of tetracycline is anatomical site dependent. (author)

  10. Co-electrospun gelatin-poly(L-lactic acid) scaffolds: Modulation of mechanical properties and chondrocyte response as a function of composition

    International Nuclear Information System (INIS)

    Bio-synthetic scaffolds of interspersed poly(L-lactic acid) (PLLA) and gelatin (GEL) fibers are fabricated by co-electrospinning. Tailored PLLA/GEL compositions are obtained and GEL crosslinking with genipin provides for the maintenance of good fiber morphology. Scaffold tensile mechanical properties are intermediate between those of pure PLLA and GEL and vary as a function of PLLA content. Primary human chondrocytes grown on the scaffolds exhibit good proliferation and increased values of the differentiation parameters, especially for intermediate PLLA/GEL compositions. Mineralization tests enable the deposition of a uniform layer of poorly crystalline apatite onto the scaffolds, suggesting potential applications involving cartilage as well as cartilage–bone interface tissue engineering. - Highlights: • Bio-synthetic scaffolds of PLLA and gelatin are produced by co-electrospinning. • Scaffolds with tailored PLLA–gelatin composition are fabricated. • PLLA/gelatin ratio controls scaffold mechanical properties and mineralization. • Chondrocyte proliferation and differentiation are modulated. • Scaffolds are suitable for cartilage–bone interface tissue engineering

  11. Co-electrospun gelatin-poly(L-lactic acid) scaffolds: Modulation of mechanical properties and chondrocyte response as a function of composition

    Energy Technology Data Exchange (ETDEWEB)

    Torricelli, Paola [Preclinical and Surgical Studies Laboratory, Codivilla Putti Research Institute, Rizzoli Orthopaedic Institute, via di Barbiano, 1/10, 40136 Bologna (Italy); Laboratory of Biocompatibility, Innovative Technologies and Advanced Therapies—Department Rizzoli Research, Innovation, Technology, via di Barbiano, 1/10, 40136 Bologna (Italy); Gioffrè, Michela; Fiorani, Andrea; Panzavolta, Silvia [Department of Chemistry “G. Ciamician” and National Consortium of Materials Science and Technology (INSTM, Bologna RU), University of Bologna (Italy); Gualandi, Chiara [Department of Chemistry “G. Ciamician” and National Consortium of Materials Science and Technology (INSTM, Bologna RU), University of Bologna (Italy); Advanced Mechanics and Materials—Interdepartmental Center for Industrial Research (AMM ICIR), University of Bologna (Italy); Fini, Milena [Preclinical and Surgical Studies Laboratory, Codivilla Putti Research Institute, Rizzoli Orthopaedic Institute, via di Barbiano, 1/10, 40136 Bologna (Italy); Laboratory of Biocompatibility, Innovative Technologies and Advanced Therapies—Department Rizzoli Research, Innovation, Technology, via di Barbiano, 1/10, 40136 Bologna (Italy); Focarete, Maria Letizia, E-mail: marialetizia.focarete@unibo.it [Department of Chemistry “G. Ciamician” and National Consortium of Materials Science and Technology (INSTM, Bologna RU), University of Bologna (Italy); Health Sciences and Technologies—Interdepartmental Center for Industrial Research (HST-ICIR) (Italy); Bigi, Adriana [Department of Chemistry “G. Ciamician” and National Consortium of Materials Science and Technology (INSTM, Bologna RU), University of Bologna (Italy)

    2014-03-01

    Bio-synthetic scaffolds of interspersed poly(L-lactic acid) (PLLA) and gelatin (GEL) fibers are fabricated by co-electrospinning. Tailored PLLA/GEL compositions are obtained and GEL crosslinking with genipin provides for the maintenance of good fiber morphology. Scaffold tensile mechanical properties are intermediate between those of pure PLLA and GEL and vary as a function of PLLA content. Primary human chondrocytes grown on the scaffolds exhibit good proliferation and increased values of the differentiation parameters, especially for intermediate PLLA/GEL compositions. Mineralization tests enable the deposition of a uniform layer of poorly crystalline apatite onto the scaffolds, suggesting potential applications involving cartilage as well as cartilage–bone interface tissue engineering. - Highlights: • Bio-synthetic scaffolds of PLLA and gelatin are produced by co-electrospinning. • Scaffolds with tailored PLLA–gelatin composition are fabricated. • PLLA/gelatin ratio controls scaffold mechanical properties and mineralization. • Chondrocyte proliferation and differentiation are modulated. • Scaffolds are suitable for cartilage–bone interface tissue engineering.

  12. Protein phosphorylation on tyrosine restores expression and glycosylation of cyclooxygenase-2 by 2-deoxy-D-glucose-caused endoplasmic reticulum stress in rabbit articular chondrocyte

    Directory of Open Access Journals (Sweden)

    Seon-Mi Yu

    2012-05-01

    Full Text Available 2-deoxy-D-glucose(2DG-caused endoplasmic reticulum (ERstress inhibits protein phosphorylation at tyrosine residues.However, the accurate regulatory mechanisms, which determinethe inflammatory response of chondrocytes to ER stress via proteintyrosine phosphorylation, have not been systematicallyevaluated. Thus, in this study, we examined whether proteinphosphorylation at tyrosine residues can modulate the expressionand glycosylation of COX-2, which is reduced by 2DG-inducedER stress. We observed that protein tyrosine phosphatase (PTP inhibitors,sodium orthovanadate (SOV, and phenylarsine oxide(PAO significantly decreased expression of ER stress inducibleproteins, glucose-regulated protein 94 (GRP94, and CCAAT/ enhancer-binding-protein- related gene (GADD153, which was inducedby 2DG. In addition, we demonstrated that SOV and PAOnoticeably restored the expression and glycosylation of COX-2 aftertreatment with 2DG. These results suggest that protein phosphorylationof tyrosine residues plays an important role in theregulation of expression and glycosylation during 2DG-inducedER stress in rabbit articular chondrocytes. [BMB reports 2012;45(5: 317-322

  13. Differential regulation of IGF-binding proteins in rabbit costal chondrocytes by IGF-I and dexamethasone.

    Science.gov (United States)

    Koedam, J A; Hoogerbrugge, C M; Van Buul-Offers, S C

    2000-06-01

    Cartilage is a primary target tissue for the IGFs. The mitogenic activity of these peptides is regulated by a family of high-affinity IGF-binding proteins (IGFBP-1 to -6). We characterized the IGFBPs produced by cultured chondrocytes derived from rib cartilage of prepubertal rabbits. Culture medium, which had been conditioned by these cells for 48 h showed bands of 22 kDa, 24 kDa and a 31/32 kDa doublet by Western ligand blotting with [(125)I]IGF-II. When the cells were grown in the presence of increasing amounts of IGF-I or IGF-II, the 31/32 kDa doublet increased in intensity (reaching a plateau of about 11-fold stimulation between 2 and 10 nM IGF-I). The 22 kDa and 24 kDa bands increased only slightly while a 26 kDa band became faintly visible. By Western immunoblotting the 31/32 kDa doublet was identified as IGFBP-5. An IGF-I analog with reduced affinity for IGFBPs, Long-R3 IGF-I, also induced IGFBP-5, while insulin was less effective (2.2-fold stimulation at 10 nM). IGF-I protected IGFBP-5 against proteolytic degradation by conditioned medium. IGF-I also enhanced the level of IGFBP-5 mRNA. LY294002, a specific inhibitor of the intracellular signaling molecule phosphatidylinositol 3-kinase, inhibited stimulation of IGFBP-5 by IGF-I. Dexamethasone suppressed IGFBP-5 (by 70% at 20 nM) but, at the same time, a 39/41 kDa doublet (presumably IGFBP-3) was induced. IGFBP-5 has been shown in several cell types to enhance the mitogenic activity of IGF-I. IGFBP-3 generally acts as a growth inhibitor. Therefore, the differential effects of dexamethasone on these regulatory proteins could account, at least in part, for the growth-arresting effect of this glucocorticoid. PMID:10828839

  14. Embryo-chondrocyte expressed gene 1, downregulating hypoxia-inducible factor 1α, is another marker of lung tumor hypoxia

    Institute of Scientific and Technical Information of China (English)

    Li ZHANG; Qing-quan LI

    2007-01-01

    Aim: To explore the mechanism of differentiated embryo-chondrocyte expressed gene 1 (DEC 1) in the lung adenocarcinoma A549 cell line and its relationship with hypoxia-inducible factor la (HIF-1α). Methods: DEC1 and HIF-1α protein levels were detected in lung adenocarcinoma tissues by immunohistochemistry. A549cells were cultured at hypoxia. MTT assay was used to determine the effect of cell viability. The apoptotic analysis was determined by flow cytometry. RT-PCR and immunocytochemistry were carried out to observe the DEC1 and HIF-1α mRNA and protein expression. HIF-1α mRNA and protein levels were detected in the stably transfected pcDNA-DEC1+/-A549 cells by RT-PCR and Western blotting.DEC1 mRNA and protein levels were detected in the stably transfected pcDNA-DEC1+/- A549 cells with or without HIF-1α antisense oligonucleotide treatment by RT-PCR and Western blotting. Results: DEC1 was specifically located in 40(48.8%) lung adenocarcinoma tissues. The results of the MTT test showed the growth of the stably transfected pcDNA-DEC+ A549 cells was higher than those of A549 cells and the stably transfected pcDNA-DEC- A549 cells. Hypoxia also induced the apoptosis of A549 cells and the stably transfected pcDNA-DEC+/-A549 cells. Hypoxia increased the mRNA and protein levels of DEC1 and HIF-1α.Both HIF- 1α mRNA and protein levels decreased in the stably transfected pcDNA-DEC+ cells, but HIF-1α antisense oligonucleotide had no effect on DEC1 in the stably transfected pcDNA-DEC+/-A549 cells. Conclusion: DEC1 is a marker of tumor hypoxia. DEC1 downregulates the HIF-1α at both mRNA and protein levels at hypoxia in lung adenocarcinoma A549 cells.

  15. Novel methods for the quantification of changes in actin organization in chondrocytes using fluorescent imaging and linear profiling.

    Science.gov (United States)

    Qusous, Ala; Parker, Eleanor; Geewan, Corinne; Kapasi, Arva; Getting, Stephen J; Hucklebridge, Frank; Keshavarz, Tajalli; Kerrigan, Mark J P

    2012-07-01

    We present three novel reproducible methodologies for the quantification of changes in actin organization from microscope images. Striation and integrative analysis were devised for the investigation of trans-cellular filaments and F-actin localization, respectively, in response to physiological or mechanical actin-modulatory conditions. Additionally, the Parker-Qusous (PQ) formula was developed as a measure of total quantity of F-actin, independent of cell volume changes, whereby fluorescence intensity was divided by the cube root of cell volume, squared. Values obtained were quantified in Mauricean Units (Mu; pixel/μm(3)). Upon isolation, there was a 49% decrease in total F-actin fluorescence from 1.91 ± 0.16 pixel/μm(3) (Mu) to 0.95 ± 0.55 Mu, whereas upon culture, an apparent increase in total fluorescence was deemed insignificant due to an increase in average cell volume, with a rise, however, in striation units (StU) from 1 ± 1 to 5 ± 1 StU/cell, and a decrease in percentage cortical fluorescence to 30.45% ± 1.52% (P = 7.8 × 10(-5)). Freshly isolated chondrocytes exhibited a decrease in total F-actin fluorescence to 0.61 ± 0.05 Mu and 0.32 ± 0.02 Mu, 10 min posthypertonic and hypotonic challenges, respectively. Regulatory volume decrease was inhibited in the presence of REV5901 with maintenance of actin levels at 1.15 Mu. Following mechanical impact in situ, there was a reduction in total F-actin fluorescence to 0.95 ± 0.08 Mu and 0.74 ± 0.06 Mu under isotonic and hypotonic conditions, respectively, but not under hypertonic conditions. We report simple methodologies for quantification of changes in actin organization, which will further our understanding of the role of actin in various cellular stress responses. These techniques can be applied to better quantify changes in localization of various proteins using fluorescent labeling. PMID:22514026

  16. 威灵仙提取物干预膝骨关节炎软骨细胞的生长活力★%Effect of radix clematidis extract on viability of knee osteoarthritis chondrocytes

    Institute of Scientific and Technical Information of China (English)

    徐扬; 桂鉴超; 高峰; 徐燕; 王黎明; 陆一鸣; 尹昭伟

    2013-01-01

      背景:近年来研究显示关节软骨细胞过度凋亡是骨性关节炎开始和进展的关键因素,利用药物抑制软骨细胞凋亡来控制骨性关节炎的进展是现在的研究热点。而威灵仙在国内临床治疗骨关节炎时取得了较确切的疗效,但是其具体的作用机制是否通过抑制软骨细胞凋亡来实现尚未见相关报道。目的:观察中药威灵仙提取物对膝骨关节炎软骨细胞生长活力的影响。方法:取骨关节炎晚期行膝关节置换的关节软骨,剪碎后,采用酶消化法消化细胞,将处于对数生长期第3代细胞随机分组,实验组分别加入0.01,0.05,0.1,0.5,1.0 g/L威灵仙提取物培养基,对照组仅加入普通培养基进行培养。Live/Dead染色法测定人软骨细胞活力指数,TUNEL染色法测定人软骨细胞凋亡指数。结果与结论:0.05,0.1 g/L威灵仙提取物能明显提高软骨细胞活力,而0.5,1.0 g/L威灵仙提取物反而降低了软骨细胞活力,其活力指数与对照组比较差异均有显著性意义(P <0.05);0.01,0.05,0.1 g/L威灵仙提取物能有效抑制软骨细胞凋亡,1.0 g/L威灵仙提取物反而促进了软骨细胞凋亡,其凋亡指数与对照组比较差异均有显著性意义(P <0.05)。结果可见威灵仙提取物在合适的质量浓度时能有效提高人软骨细胞活力,抑制人软骨细胞凋亡,尤以0.1 g/L时效果最佳。%BACKGROUND:In recent years, studies have shown that excessive apoptosis of articular chondrocytes is the key factor for the start and progress of osteoarthritis. To study the progress in the use of drugs that inhibit apoptosis of chondrocytes to control osteoarthritis has become a hotspot. Clematis has obtained exact effect in the domestic clinical treatment of osteoarthritis, but the specific mechanism underlying inhibition of chondrocytes apoptosis has not been reported. OBJECTIVE:To observe the effect of radix

  17. Skeletal Mineralization Deficits and Impaired Biogenesis and Function of Chondrocyte-Derived Matrix Vesicles in Phospho1(-/-) and Phospho1/Pi t1 Double-Knockout Mice.

    Science.gov (United States)

    Yadav, Manisha C; Bottini, Massimo; Cory, Esther; Bhattacharya, Kunal; Kuss, Pia; Narisawa, Sonoko; Sah, Robert L; Beck, Laurent; Fadeel, Bengt; Farquharson, Colin; Millán, José Luis

    2016-06-01

    We have previously shown that ablation of either the Phospho1 or Alpl gene, encoding PHOSPHO1 and tissue-nonspecific alkaline phosphatase (TNAP) respectively, lead to hyperosteoidosis, but that their chondrocyte-derived and osteoblast-derived matrix vesicles (MVs) are able to initiate mineralization. In contrast, the double ablation of Phospho1 and Alpl completely abolish initiation and progression of skeletal mineralization. We argued that MVs initiate mineralization by a dual mechanism: PHOSPHO1-mediated intravesicular generation of inorganic phosphate (Pi ) and phosphate transporter-mediated influx of Pi . To test this hypothesis, we generated mice with col2a1-driven Cre-mediated ablation of Slc20a1, hereafter referred to as Pi t1, alone or in combination with a Phospho1 gene deletion. Pi t1(col2/col2) mice did not show any major phenotypic abnormalities, whereas severe skeletal deformities were observed in the [Phospho1(-/-) ; Pi t1(col2/col2) ] double knockout mice that were more pronounced than those observed in the Phospho1(-/-) mice. Histological analysis of [Phospho1(-/-) ; Pi t1(col2/col2) ] bones showed growth plate abnormalities with a shorter hypertrophic chondrocyte zone and extensive hyperosteoidosis. The [Phospho1(-/-) ; Pi t1(col2/col2) ] skeleton displayed significant decreases in BV/TV%, trabecular number, and bone mineral density, as well as decreased stiffness, decreased strength, and increased postyield deflection compared to Phospho1(-/-) mice. Using atomic force microscopy we found that ∼80% of [Phospho1(-/-) ; Pi t1(col2/col2) ] MVs were devoid of mineral in comparison to ∼50% for the Phospho1(-/-) MVs and ∼25% for the WT and Pi t1(col2/col2) MVs. We also found a significant decrease in the number of MVs produced by both Phospho1(-/-) and [Phospho1(-/-) ; Pi t1(col2/col2) ] chondrocytes. These data support the involvement of phosphate transporter 1, hereafter referred to as Pi T-1, in the initiation of skeletal mineralization and

  18. ECHO: the executable chondrocyte

    NARCIS (Netherlands)

    Scholma, J.; Schivo, S.; Kerkhofs, J.; Langerak, R.; Karperien, M.; Pol, van de J.; Geris, L.; Post, J.N.

    2014-01-01

    Introduction: We recently presented ANIMO (Analysis of Networks with Interactive Modeling), a software tool for modeling dynamic molecular networks for use by biologists [1, 2]. We used ANIMO to generate a computational model of articular cartilage. Materials and methods: Based on a largescale lite

  19. Modelling and simulation of the chondrocyte cell growth, glucose consumption and lactate production within a porous tissue scaffold inside a perfusion bioreactor

    Directory of Open Access Journals (Sweden)

    Md. Shakhawath Hossain

    2015-03-01

    Full Text Available Mathematical and numerical modelling of the tissue culture process in a perfusion bioreactor is able to provide insight into the fluid flow, nutrients and wastes transport, dynamics of the pH value, and the cell growth rate. Knowing the complicated interdependence of these processes is essential for optimizing the culture process for cell growth. This paper presents a resolved scale numerical simulation, which allows one not only to characterize the supply of glucose inside a porous tissue scaffold in a perfusion bioreactor, but also to assess the overall culture condition and predict the cell growth rate. The simulation uses a simplified scaffold that consists of a repeatable unit composed of multiple strands. The simulation results explore some problematic regions inside the simplified scaffold where the concentration of glucose becomes lower than the critical value for the chondrocyte cell viability and the cell growth rate becomes significantly reduced.

  20. Integration of Stem Cell to Chondrocyte-Derived Cartilage Matrix in Healthy and Osteoarthritic States in the Presence of Hydroxyapatite Nanoparticles.

    Science.gov (United States)

    Dua, Rupak; Comella, Kristin; Butler, Ryan; Castellanos, Glenda; Brazille, Bryn; Claude, Andrew; Agarwal, Arvind; Liao, Jun; Ramaswamy, Sharan

    2016-01-01

    We investigated the effectiveness of integrating tissue engineered cartilage derived from human bone marrow derived stem cells (HBMSCs) to healthy as well as osteoarthritic cartilage mimics using hydroxyapatite (HA) nanoparticles immersed within a hydrogel substrate. Healthy and diseased engineered cartilage from human chondrocytes (cultured in agar gels) were integrated with human bone marrow stem cell (HBMSC)-derived cartilaginous engineered matrix with and without HA, and evaluated after 28 days of growth. HBMSCs were seeded within photopolymerizable poly (ethylene glycol) diacrylate (PEGDA) hydrogels. In addition, we also conducted a preliminary in vivo evaluation of cartilage repair in rabbit knee chondral defects treated with subchondral bone microfracture and cell-free PEGDA with and without HA. Under in vitro conditions, the interfacial shear strength between tissue engineered cartilage derived from HBMSCs and osteoarthritic chondrocytes was significantly higher (p < 0.05) when HA nanoparticles were incorporated within the HBMSC culture system. Histological evidence confirmed a distinct spatial transition zone, rich in calcium phosphate deposits. Assessment of explanted rabbit knees by histology demonstrated that cellularity within the repair tissues that had filled the defects were of significantly higher number (p < 0.05) when HA was used. HA nanoparticles play an important role in treating chondral defects when osteoarthritis is a co-morbidity. We speculate that the calcified layer formation at the interface in the osteoarthritic environment in the presence of HA is likely to have attributed to higher interfacial strength found in vitro. From an in vivo standpoint, the presence of HA promoted cellularity in the tissues that subsequently filled the chondral defects. This higher presence of cells can be considered important in the context of accelerating long-term cartilage remodeling. We conclude that HA nanoparticles play an important role in engineered

  1. Integration of Stem Cell to Chondrocyte-Derived Cartilage Matrix in Healthy and Osteoarthritic States in the Presence of Hydroxyapatite Nanoparticles.

    Directory of Open Access Journals (Sweden)

    Rupak Dua

    Full Text Available We investigated the effectiveness of integrating tissue engineered cartilage derived from human bone marrow derived stem cells (HBMSCs to healthy as well as osteoarthritic cartilage mimics using hydroxyapatite (HA nanoparticles immersed within a hydrogel substrate. Healthy and diseased engineered cartilage from human chondrocytes (cultured in agar gels were integrated with human bone marrow stem cell (HBMSC-derived cartilaginous engineered matrix with and without HA, and evaluated after 28 days of growth. HBMSCs were seeded within photopolymerizable poly (ethylene glycol diacrylate (PEGDA hydrogels. In addition, we also conducted a preliminary in vivo evaluation of cartilage repair in rabbit knee chondral defects treated with subchondral bone microfracture and cell-free PEGDA with and without HA. Under in vitro conditions, the interfacial shear strength between tissue engineered cartilage derived from HBMSCs and osteoarthritic chondrocytes was significantly higher (p < 0.05 when HA nanoparticles were incorporated within the HBMSC culture system. Histological evidence confirmed a distinct spatial transition zone, rich in calcium phosphate deposits. Assessment of explanted rabbit knees by histology demonstrated that cellularity within the repair tissues that had filled the defects were of significantly higher number (p < 0.05 when HA was used. HA nanoparticles play an important role in treating chondral defects when osteoarthritis is a co-morbidity. We speculate that the calcified layer formation at the interface in the osteoarthritic environment in the presence of HA is likely to have attributed to higher interfacial strength found in vitro. From an in vivo standpoint, the presence of HA promoted cellularity in the tissues that subsequently filled the chondral defects. This higher presence of cells can be considered important in the context of accelerating long-term cartilage remodeling. We conclude that HA nanoparticles play an important role in

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

    Directory of Open Access Journals (Sweden)

    R Willumeit

    2007-03-01

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

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

    Directory of Open Access Journals (Sweden)

    S Giovannini

    2010-10-01

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

  4. Dedifferentiated Human Articular Chondrocytes Redifferentiate to a Cartilage-Like Tissue Phenotype in a Poly(ε-Caprolactone/Self-Assembling Peptide Composite Scaffold

    Directory of Open Access Journals (Sweden)

    Lourdes Recha-Sancho

    2016-06-01

    Full Text Available Adult articular cartilage has a limited capacity for growth and regeneration and, with injury, new cellular or biomaterial-based therapeutic platforms are required to promote repair. Tissue engineering aims to produce cartilage-like tissues that recreate the complex mechanical and biological properties found in vivo. In this study, a unique composite scaffold was developed by infiltrating a three-dimensional (3D woven microfiber poly (ε-caprolactone (PCL scaffold with the RAD16-I self-assembling nanofibers to obtain multi-scale functional and biomimetic tissue-engineered constructs. The scaffold was seeded with expanded dedifferentiated human articular chondrocytes and cultured for four weeks in control and chondrogenic growth conditions. The composite constructs were compared to control constructs obtained by culturing cells with 3D woven PCL scaffolds or RAD16-I independently. High viability and homogeneous cell distribution were observed in all three scaffolds used during the term of the culture. Moreover, gene and protein expression profiles revealed that chondrogenic markers were favored in the presence of RAD16-I peptide (PCL/RAD composite or alone under chondrogenic induction conditions. Further, constructs displayed positive staining for toluidine blue, indicating the presence of synthesized proteoglycans. Finally, mechanical testing showed that constructs containing the PCL scaffold maintained the initial shape and viscoelastic behavior throughout the culture period, while constructs with RAD16-I scaffold alone contracted during culture time into a stiffer and compacted structure. Altogether, these results suggest that this new composite scaffold provides important mechanical requirements for a cartilage replacement, while providing a biomimetic microenvironment to re-establish the chondrogenic phenotype of human expanded articular chondrocytes.

  5. 不同浓度硝普钠诱导兔软骨细胞凋亡模型的比较研究%Rabbit Chondrocytes Apoptosis Models Induced by Different Concentrations of SNP

    Institute of Scientific and Technical Information of China (English)

    陈启鑫; 韩冠英; 郭斌; 郭跃伟

    2015-01-01

    目的:比较不同浓度硝普钠( SNP)诱导兔软骨细胞凋亡模型,为进一步探讨药物对软骨细胞凋亡的调控作用提供理论支持。方法2014年9—12月选取4周龄健康雄性新西兰大耳白兔8只,体质量200~250 g,体外分离培养并鉴定兔软骨细胞,采用不同浓度SNP (0、0.0625、0.125、0.25、0.5、1、2 mmol/L)诱导兔软骨细胞,分别于12、24、48 h采用四甲基偶氮唑盐比色试验法( MTT法)和末端脱氧核苷酸转移的dUTP缺口末端标记法( TUNEL法)评价并比较不同浓度SNP诱导软骨细胞凋亡模型,记录兔软骨细胞增殖情况( OD值)和兔软骨细胞凋亡率。结果不同浓度SNP诱导的兔软骨细胞在不同时间点的 OD值比较,差异均有统计学意义( P<0.05)。 SNP浓度为0.0625、0.125 mmol/L培养12、24、48 h时兔软骨细胞OD值高于SNP浓度为0 mmol/L ( P<0.05); SNP浓度为0.25、0.5、1、2 mmol/L培养12、24、48 h时兔软骨细胞OD值均低于SNP浓度为0 mmol/L; SNP浓度为0.25、0.5、1、2 mmol/L培养12、24、48 h时兔软骨细胞OD值在不同浓度间两两比较,差异均有统计学意义( P<0.05)。不同浓度SNP诱导的兔软骨细胞在不同时间点的凋亡率比较,差异均有统计学意义( P<0.05)。 SNP浓度为0.25、0.5、1、2 mmol/L培养12、24、48 h时兔软骨细胞凋亡率均高于SNP浓度为0 mmol/L ( P<0.05);且不同浓度间两两比较,差异有统计学意义(P<0.05)。结论 SNP浓度为0.25~2 mmol/L且诱导12、24、48 h时可制备不同严重程度的软骨细胞凋亡模型,为进一步探讨药物对软骨细胞凋亡的调控作用提供了理论支持。%Objective To compare the chondrocytes apoptosis models induced by different concentrations of sodium nitroprusside ( SNP) , in order to provide theoretical support for the further investigation of the regulating effect of drugs on

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2011-02-18

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

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

    International Nuclear Information System (INIS)

    Research highlights: → Glucosamine and a NF-kB inhibitor reduce inflammation in OA. → Cytokine induced demethylation of CpG site in IL1β promoter prevented by glucosamine. → Glucosamine and NF-kB inhibitor have epigenetic effects on human chondrocytes. -- Abstract: Objective: Idiopathic osteoarthritis is the most common form of osteoarthritis (OA) world-wide and remains the leading cause of disability and the associated socio-economic burden in an increasing aging population. Traditionally, OA has been viewed as a degenerative joint disease characterized by progressive destruction of the articular cartilage and changes in the subchondral bone culminating in joint failure. However, the etiology of OA is multifactorial involving genetic, mechanical and environmental factors. Treatment modalities include analgesia, joint injection with steroids or hyaluronic acid, oral supplements including glucosamine and chondroitin sulfate, as well as physiotherapy. Thus, there is significant interest in the discovery of disease modifying agents. One such agent, glucosamine (GlcN) is commonly prescribed even though the therapeutic efficacy and mechanism of action remain controversial. Inflammatory cytokines, including IL-1β, and proteinases such as MMP-13 have been implicated in the pathogenesis and progression of OA together with an associated CpG demethylation in their promoters. We have investigated the potential of GlcN to modulate NF-kB activity and cytokine-induced abnormal gene expression in articular chondrocytes and, critically, whether this is associated with an epigenetic process. Method: Human chondrocytes were isolated from the articular cartilage of femoral heads, obtained with ethical permission, following fractured neck of femur surgery. Chondrocytes were cultured for 5 weeks in six separate groups; (i) control culture, (ii) cultured with a mixture of 2.5 ng/ml IL-1β and 2.5 ng/ml oncostatin M (OSM), (iii) cultured with 2 mM N-acetyl GlcN (Sigma

  8. Growth and behavior of chondrocytes on nano engineered surfaces and construction of micropatterned co-culture platforms using layer-by-layer platforms using layer-by-layer assembly lift-off method

    Science.gov (United States)

    Shaik, Jameel

    Several approaches such as self-assembled monolayers and layer-by-layer assembled multilayer films are being used as tools to study the interactions of cells with biomaterials in vitro. In this study, the layer-by-layer assembly approach was used to create monolayer, bilayer, trilayer, five, ten and twenty-bilayer beds of eleven different biomaterials. The various biomaterials used were poly(styrene-sulfonate), fibronectin, poly-L-lysine, poly-D-lysine, laminin, bovine serum albumin, chondroitin sulfate, poly(ethyleneimine), polyethylene glycol amine, collagen and poly(dimethyldiallyl-ammonium chloride) with unmodified tissue-culture polystyrene as standard control. Three different cell lines---primary bovine articular chondrocytes, and two secondary cell lines, human chondrosarcoma cells and canine chondrocytes were used in these studies. Chondrocyte morphology and attachment, viability, proliferation, and functionality were determined using bright field microscopy, the Live/Dead viability assay, MTT assay, and immunocytochemistry, respectively. Atomic force microscopy of the nanofilms indicated an increase in surface roughness with increasing number of layers. The most important observations from the studies on primary bovine articular chondrocytes were that these cells exhibited increasing viability and cell metabolic activity with increasing number of bilayers. The increase in viability was more pronounced than the increase in cell metabolic activity. Also, bovine chondrocytes on bilayers of poly(dimethyldiallyl-ammonium chloride, poly-L-lysine, poly(styrene-sulfonate), and bovine serum albumin were substantially bigger in size and well-attached when compared to the cells grown on monolayer and trilayers. Lactate dehydrogenase assay performed on chondrosarcoma cells grown on 5- and 10-bilayer multilayer beds indicated that the 10-bilayer beds had reduced cytotoxicity compared to the 5-bilayer beds. MTT assay performed on canine chondrocytes grown on 5-, 10

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

    Institute of Scientific and Technical Information of China (English)

    欧阳晴晴; 赵进军; 杨敏

    2014-01-01

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

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

    Science.gov (United States)

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

    2015-06-01

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

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

    Directory of Open Access Journals (Sweden)

    Angelica Rossi Sartori-Cintra

    2012-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Mehdi Shakibaei

    2012-01-01

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

  13. Cissus quadrangularis inhibits IL-1β induced inflammatory responses on chondrocytes and alleviates bone deterioration in osteotomized rats via p38 MAPK signaling

    Directory of Open Access Journals (Sweden)

    Kanwar JR

    2015-06-01

    Full Text Available Jagat R Kanwar,1 Rasika M Samarasinghe,1 Kuldeep Kumar,2 Ramesh Arya,2 Sanjeev Sharma,2 Shu-Feng Zhou,3 Sreenivasan Sasidharan,4 Rupinder K Kanwar11Nanomedicine-Laboratory of Immunology and Molecular Biomedical Research (NLIMBR, School of Medicine (SoM, Molecular and Medical Research (MMR Strategic Research Centre, Faculty of Health, Geelong Technology Precinct (GTP, Deakin University, Waurn Ponds, VIC, Australia; 2Ayurvedic College, Paprola, Kangra, Himachal Pradesh, India; 3Department of Pharmaceutical Sciences, College of Pharmacy, University of South Florida, Tampa, FL, USA; 4Institute for Research in Molecular Medicine (INFORMM, Universiti Sains Malaysia, Penang, MalaysiaIntroduction: Inflammatory mediators are key players in the pathogenesis of osteoarthritis (OA and bone destruction. Conventional drugs suppress symptomatic activity and have no therapeutic influence on disease. Cissus quadrangularis and Withania somnifera are widely used for the treatment of bone fractures and wounds; however, the cellular and molecular mechanisms regulated by these herbals are still unclear.Methods: We established an in vitro OA culture model by exposing human chondrocytes to proinflammatory cytokine and interleukin (IL-1β for 36 hours prior to treatment with the herbals: C. quadrangularis, W. somnifera, and the combination of the two herbals. Cell viability, toxicity, and gene expression of OA modifying agents were examined. In addition, expression of survivin, which is crucial for cell growth, was analyzed. In vivo work on osteotomized rats studied the bone and cartilage regenerative effects of C. quadrangularis, W. somnifera, and the combination therapy.Results: Exposure of chondrocytes to IL-1β induced significant toxicity and cell death. However, herbal treatment alleviated IL-1β induced cell toxicity and upregulated cell growth and proliferation. C. quadrangularis inhibited gene expression of cytokines and matrix metalloproteinases, known to

  14. Debridement of cartilage lesions before autologous chondrocyte implantation by open or transarthroscopic techniques: a comparative study using post-mortem materials.

    Science.gov (United States)

    Drobnic, M; Radosavljevic, D; Cör, A; Brittberg, M; Strazar, K

    2010-04-01

    We compared the quality of debridement of chondral lesions performed by four arthroscopic (SH, shaver; CU, curette; SHCU, shaver and curette; BP, bipolar electrodes) and one open technique (OPEN, scalpel and curette) which are used prior to autologous chondrocyte implantation (ACI). The ex vivo simulation of all five techniques was carried out on six juvenile equine stifle joints. The OPEN, SH and SHCU techniques were tested on knees harvested from six adult human cadavers. The most vertical walls with the least adjacent damage to cartilage were obtained with the OPEN technique. The CU and SHCU methods gave inferior, but still acceptable results whereas the SH technique alone resulted in a crater-like defect and the BP method undermined the cartilage wall. The subchondral bone was severely violated in all the equine samples which might have been peculiar to this model. The predominant depth of the debridement in the adult human samples was at the level of the calcified cartilage. Some minor penetrations of the subchondral end-plate were induced regardless of the instrumentation used. Our study suggests that not all routine arthroscopic instruments are suitable for the preparation of a defect for ACI. We have shown that the preferred debridement technique is either open or arthroscopically-assisted manual curettage. The use of juvenile equine stifles was not appropriate for the study of the cartilage-subchondral bone interface. PMID:20357342

  15. A novel injectable thermoresponsive and cytocompatible gel of poly(N-isopropylacrylamide) with layered double hydroxides facilitates siRNA delivery into chondrocytes in 3D culture.

    Science.gov (United States)

    Yang, Hsiao-yin; van Ee, Renz J; Timmer, Klaas; Craenmehr, Eric G M; Huang, Julie H; Öner, F Cumhur; Dhert, Wouter J A; Kragten, Angela H M; Willems, Nicole; Grinwis, Guy C M; Tryfonidou, Marianna A; Papen-Botterhuis, Nicole E; Creemers, Laura B

    2015-09-01

    Hybrid hydrogels composed of poly(N-isopropylacrylamide) (pNIPAAM) and layered double hydroxides (LDHs) are presented in this study as novel injectable and thermoresponsive materials for siRNA delivery, which could specifically target several negative regulators of tissue homeostasis in cartilaginous tissues. Effectiveness of siRNA transfection using pNIPAAM formulated with either MgAl-LDH or MgFe-LDH platelets was investigated using osteoarthritic chondrocytes. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an endogenous model gene to evaluate the extent of silencing. No significant adverse effects of pNIPAAM/LDH hydrogels on cell viability were noticed. Cellular uptake of fluorescently labeled siRNA was greatly enhanced (>75%) in pNIPAAM/LDH hydrogel constructs compared to alginate, hyaluronan and fibrin gels, and was absent in pNIPAAM hydrogel without LDH platelets. When using siRNA against GAPDH, 82-98% reduction of gene expression was found in both types of pNIPAAM/LDH hydrogel constructs after 6 days of culturing. In the pNIPAAM/MgAl-LDH hybrid hydrogel, 80-95% of GAPDH enzyme activity was reduced in parallel with gene. Our findings show that the combination of a cytocompatible hydrogel and therapeutic RNA oligonucleotides is feasible. Thus it might hold promise in treating degeneration of cartilaginous tissues by providing supporting scaffolds for cells and interference with locally produced degenerative factors. PMID:26022968

  16. 碱性成纤维细胞生长因子转染兔关节软骨细胞的研究%Transfection of basic fibroblast growth factor in rabbits' articular chondrocytes

    Institute of Scientific and Technical Information of China (English)

    曾庆彩; 鲍丽娟; 龙源深; 杨炎馨; 董文其; 李明

    2004-01-01

    背景:软骨细胞体外培养困难和表型难以维持是软骨组织工程研究的一大难题.目的:探讨碱性成纤维细胞生长因子(basic Fibroblast Growth Factor,bFGF)基因转染兔关节软骨细胞后对培养的关节软骨细胞形态、分裂增殖及代谢等方面的影响.设计:完全随机对照实验研究.地点和方法:实验在解放军第一军医大学热带军队卫生学系完成,对象为兔软骨细胞(3周龄新西兰新生兔购于第一军医大学实验动物中心).干预:将bFGF基因克隆于真核表达载体pHβ0AP-1中,构建重组真核表达载体pHβ-bFGF,转染兔关节软骨细胞.G418筛选阳性克隆,检测阳性细胞bFGF基因的表达水平.测定培养软骨细胞的DNA含量、糖醛酸含量、软骨细胞增殖情况及进行细胞周期分析.主要观察指标:DNA含量、糖醛酸含量、软骨细胞增殖情况及细胞周期分析.结果:bFGF基因转染软骨细胞表型未见显著变化;bFGF基因转染组、载体对照组、空白对照组DNA含量分别为(77.37±6 21),(40.39±4.33),(33.77±4.25)μg/瓶(P<0.01),糖醛酸含量分别为(308.8±10.2),(77.9±8.7),(80.2±10.5)μg/瓶(P<0.01),软骨细胞G1期分别为59.3±2.1,69.5±4.0,73.1±3.9(P<0.05).结论:bFGF转染关节软骨细胞后,可显著促进细胞分裂增殖并缩短细胞周期,为软骨组织工程研究提供新的技术路线及理论基础.%BACKGROUND: The difficulties in the culture in vitro of chondrocytes and in the maintenance of phenotype are big problems in the research of cartilage engineering.OBJECTIVE: To discuss the impact of basic fibroblast growth factor(bFGF)gene on the morphology, division, proliferation and metabolism of chondrocytes in culture after the transfection in articular chondrocytes of rabbits.DESIGN: A complete randomized controlled study was conducted.SETTING and PARTICIPANTS: The study was completed in the Department of Military Tropical Medicine and Hygiene, First Military Medical

  17. EFFECTS OF AGE ON VISCOELASTICITY AND DEFORMATION RECOVERY OF ARTICULAR CHONDROCYTES%软骨细胞黏弹性及恢复变形与年龄相关性研究

    Institute of Scientific and Technical Information of China (English)

    张全有; 陈维毅; 卫小春; 李春江; 刘琳琳

    2009-01-01

    The aim of this study was to characterize the age-related changes of viscoelastic properties and deformation recovery of rabbit chondrocytes in natural development of articular cartilage. The micropipette aspiration combined with a standard linear viscoelastic solid model was used to quantify all parameters of chondrocytes from different age groups. Results indicate that the viscoelastic properties of chondrocytes in old group exhibited a significantly lower instantaneous modulus (E_0), equilibrium modulus (E_∞), and apparent viscosity (μ) compared with those of young group (p 0.1). The process of creep and deformation recovery of chondrocytes has changed significantly during natural development. The time t_E that old chondrocytes need to reach equilibrium is significantly less than young and adult ones (p 0.05). At the same time, maximal creep displacement L_M in old group dramatically higher than young and adult group (p 0.05). Comparisons of the deformation recovery ratio of different age groups before 8 seconds have shown that the ratio value of young group is significantly higher than those of adult and old ones (p 0.05). Additionally, there were no significant correlation between the viscoelastic parameters and the ratio of the cell to micropipette diameter. These results may be helpful for chondrocyte-based cartilage tissue engineering.%本文旨在表征年龄对软骨细胞在自然生长过程中的黏弹性和恢复变形能力的影响.结果表明:年龄对软骨细胞黏弹性及其恢复变形能力产生显著影响,老年组软骨细胞各项黏弹性参数值均明显高于幼年和中年组软骨细胞(p0.05);老年组软骨细胞蠕变达到平衡态所需时间t_E显著小于幼年和中年组(p0.05).老年组软骨细胞最大蠕变位移L_M显著大于幼年和中年组(p0.05).老年组软骨细胞恢复变形时间t_R显著大于幼年和中年组(p0.05).恢复变形前8s的分析发现,幼年组软骨细胞恢复变形率K_y显

  18. THEEMPIRICAL STUDY TO THE PATHOLOGICAL CHANGES OF ACETABULAR CHONDROCYTE IN THE DEVELOPMENTAL DISLOCATION OF THE HIP%发育性髋脱位髋臼软骨细胞病理学改变的实验研究

    Institute of Scientific and Technical Information of China (English)

    韦宜山; 刘万林; 丁良甲; 王炳海; 白锐; 李岱鹤; 赵振群

    2012-01-01

    Objective: To investigate the pathological changes of acetabular chondrocyte in the developmental dislocation of the hip( DDH). Methods: 20 rabbits of 4-week-old which female and male were not restricted had been made for the models. The back limb that the hip was flexured and the knee was extened then fixed with a plaster cast was made for DDH model group and the right side without fixationas the control group. Pelvis anteroposterior X-rays had been made on the models before the fixation and after8 - weeks fixation. The femoral head dislocation or not by shenton ' s line was discontinuity or by the femoral head was at the extabottom or extraupper quadrant of the Perkin squarse. Observing the changes of general shape of bilateral acetabular and the changes of chondrocyte, then observing the apoptosis of acetabular chondrocyte in 12 successful models. Results: Success rate of DDH models were60% ( 12/20). Hip X-ray of experimental side shown that the femoral head was dislocation toward the extabottom or extraupper quadrant of the Perkin squarse, the acetabular angle of the experimental was significantly increased than the control side(P<0. 05). The experimental side was found that the acetabulum became narrowing and fiied with soft tissue and the color of cartilage changed into dark,the chondrocytes were sparse and in a mess. Transmission electron microscopy results shown that the chromatin of acetabular chondrocytes were margination and condensation, the nuclear shape was irregular, the cytoplasmic vacuoles were present. Apoptosis rate of acetabular chondrocytes in experimental side was higher than the control side(P<0. 05). Conclusion; Excessive apoptosis of acetabular chondroctes may take part in the regulation of acetabular cartilage dysplasia in DDH.%目的:探讨发育性髋脱位(DDH)髋关节结构内髋臼软骨细胞的病理学变化.方法:选取出生4W的新西兰大耳白兔20只,雌雄兼用,采用兔后肢屈髋伸膝位管型石膏固定制作DDH

  19. 三氧化二砷对体外培养大鼠软骨细胞增殖和凋亡的影响%Effects of arsenic trioxide on rat primary chondrocytes in vitro

    Institute of Scientific and Technical Information of China (English)

    王娇; 崔洋; 刘伟东; 孟红梅

    2013-01-01

    Objective To study the cell viability,proliferation and apoptosis of cultured rat primary chondrocytes exposed to different doses of arsenic trioxide (As2O3) in vitro.Methods The third generation of primary cultured chondrocytes were treated with As2O3 at 0.5,1.0,2.0,4.0 and 8.0 μmol/L for 24,48 and 72 h.The cell vitality was detected with Cell Counting Kits-8 and the proliferation and apoptosis of chondrocytes were detected by flow cytometry.Results Compared to the control group,the vitality of chondrocytes exposed to each concentrations of As2O3 was inhibited (except the group at 0.5 μmnol/L for 24 h).Both of the distribution of cell cycle and the rate of apoptosis of chondrocytes analyzed by flow cytometry showed that As2O3 induced G2 cell-cycle arrest and increased apoptotic rate.Conclusion As2O3 could inhibit the proliferation and promote the apoptosis of rat primary chondrocytes in vitro.%目的 探讨三氧化二砷(As2O3)对体外培养大鼠软骨细胞活力、增殖和凋亡的影响.方法 采用细胞培养的方法,原代培养1~3天Wistar大鼠的关节软骨细胞,取第3代细胞进行实验,按染砷剂量不同分为0(对照)、0.5、1.0、2.0、4.0、8.0 μmol/L组.采用细胞增殖与毒性检测方法(CCK-8法),在染砷24、48、72 h测定细胞活力变化;流式细胞仪检测砷对软骨细胞周期及凋亡的影响.结果 与对照组相比,各浓度染砷大鼠软骨细胞(除0.5 μmol/L 24 h无统计学意义)活力均被抑制(P<0.01);流式细胞仪分析结果显示,As2O3将大鼠软骨细胞周期阻滞于G2期(P<0.05),使细胞凋亡率明显增加(P<0.05).结论 As2O3可以抑制体外培养大鼠软骨细胞的增殖,促进细胞凋亡.

  20. Synergistic Chondroprotective Effect of α-Tocopherol, Ascorbic Acid, and Selenium as well as Glucosamine and Chondroitin on Oxidant Induced Cell Death and Inhibition of Matrix Metalloproteinase-3—Studies in Cultured Chondrocytes

    Directory of Open Access Journals (Sweden)

    Anne-Christi Graeser

    2009-12-01

    Full Text Available Overproduction of reactive oxygen species and impaired antioxidant defence accompanied by chronic inflammatory processes may impair joint health. Pro-inflammatory cytokines such as interleukin-1β (IL-1β and tumor necrosis factor alpha (TNF-α stimulate the expression of metalloproteinases which degrade the extracellular matrix. Little is known regarding the potential synergistic effects of natural compounds such as α-tocopherol (α-toc, ascorbic acid (AA and selenium (Se on oxidant induced cell death. Furthermore studies regarding the metalloproteinase-3 inhibitory activity of glucosamine sulfate (GS and chondroitin sulfate (CS are scarce. Therefore we have studied the effect of α-toc (0.1–2.5 µmol/L, AA (10–50 µmol/L and Se (1–50 nmol/L on t-butyl hydroperoxide (t-BHP, 100–500 µmol/L-induced cell death in SW1353 chondrocytes. Furthermore we have determined the effect of GS and CS alone (100–500 µmol/L each and in combination on MMP3 mRNA levels and MMP3 secretion in IL-1β stimulated chondrocytes. A combination of α-toc, AA, and Se was more potent in counteracting t-BHP-induced cytotoxicity as compared to the single compounds. Similarly a combination of CS and GS was more effective in inhibiting MMP3 gene expression and secretion than the single components. The inhibition of MMP3 secretion due to GS plus CS was accompanied by a decrease in TNF-α production. Combining natural compounds such as α-toc, AA, and Se as well as GS and CS seems to be a promising strategy to combat oxidative stress and cytokine induced matrix degradation in chondrocytes.

  1. 透明质酸对人骨关节炎软骨细胞保护机制的探讨%Protective effects and mechanism of hyaluronic acid on human osteoarthritis chondrocytes

    Institute of Scientific and Technical Information of China (English)

    李化光; 刘华; 杨新明; 张林西

    2012-01-01

    Objective To study the effects and mechanism of hyaluronic acid on human osteoarthritis chondrocytes. Methods Mitochondrial activity was measured by methylthiazolyl tetrazolium test. Mitochondrial membrane protein (MMP) , apoptosis and [ Ca2+ ] I were measured by flow cytometry. Results Hyaluronic acid significantly increased MMP and mitochondrial activity, and decreased [Ca2+]I and apoptosis rate in human osteoarthritis chondrocytes. Conclusions Hyaluronic acid might inhibit the declines of MMP and mitochondrial activity, and has an inhibitory effect on osteoarthritis chondrocytes apoptosis. The mechanism might be related to inhibiting intracellular calcium overload.%目的:探讨透明质酸对人骨关节炎软骨细胞的作用机制.方法:应用细胞培养、四唑盐比色实验检测细胞线粒体活性,流式细胞术检测线粒体膜电位、细胞凋亡百分率和细胞内游离钙离子浓度.结果:透明质酸能明显提高人骨关节炎软骨细胞线粒体活性,抬高线粒体膜电位,降低细胞凋亡率和胞内游离钙浓度.结论:透明质酸可抑制人骨关节炎软骨细胞线粒体膜电位的降低,从而具有稳定线粒体膜电位的作用,抑制细胞凋亡的发生,这种作用可能与其能抑制软骨细胞内钙超载有关.

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

    Science.gov (United States)

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

    2001-07-01

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

  3. Melittin has a chondroprotective effect by inhibiting MMP-1 and MMP-8 expressions via blocking NF-κB and AP-1 signaling pathway in chondrocytes.

    Science.gov (United States)

    Jeong, Yun-Jeong; Shin, Jae-Moon; Bae, Young-Seuk; Cho, Hyun-Ji; Park, Kwan-Kyu; Choe, Jung-Yoon; Han, Sang-Mi; Moon, Sung-Kwon; Kim, Wun-Jae; Choi, Yung Hyun; Kim, Cheorl-Ho; Chang, Hyeun-Wook; Chang, Young-Chae

    2015-04-01

    Bee venom is a natural ingredient produced by the honey bee (Apis mellifera), and has been widely used in China, Korea and Japan as a traditional medicine for various diseases such as arthritis, rheumatism, and skin diseases However, the regulation of the underlying molecular mechanisms of the anti-arthritis by bee venom and its major peptides is largely unknown. In this study, we investigated the potential molecular mechanisms underlying the anti-arthritis effect of bee venom and its major peptides, melittin and apamin, in tumor necrosis factor-α (TNF-α) responsive C57BL/6 mice chondrocyte cells. The bee venom and melittin significantly and selectively suppressed the TNF-α-mediated decrease of type II collagen expression, whereas the apamin had no effects on the type II collagen expression. We, furthermore, found that the bee venom and melittin inhibited the protein expression of matrix metalloproteinase (MMP)-1 and MMP-8, which suggests that the chondroprotective effect of bee venom may be caused by melittin. The inhibitory effects of melittin on the TNF-α-induced MMP-1 and MMP-8 protein expression were regulated by the inhibition of NF-kB and AP-1. In addition, melittin suppressed the TNF-α-induced phosphorylation of Akt, JNK and ERK1/2, but did not affect the phosphorylation of p38 kinase. These results suggest that melittin suppresses TNF-α-stimulated decrease of type II collagen expression by the inhibiting MMP-1 and MMP-8 through regulation of the NF-kB and AP-1 pathway and provision of a novel role for melittin in anti-arthritis action. PMID:25708656

  4. T2 mapping and dGEMRIC after autologous chondrocyte implantation with a fibrin-based scaffold in the knee: Preliminary results

    Energy Technology Data Exchange (ETDEWEB)

    Domayer, S.E. [Department of Orthopedics, Medical University of Vienna, Waehringer Guertel 18-20, A 1090 Vienna (Austria); MR Centre of Excellence, Department of Radiodiagnostics, Medical University of Vienna, Lazarettgasse 14, A-1090 Vienna (Austria)], E-mail: stephan.domayer@meduniwien.ac.at; Welsch, G.H. [MR Centre of Excellence, Department of Radiodiagnostics, Medical University of Vienna, Lazarettgasse 14, A-1090 Vienna (Austria); Nehrer, S. [Centre of Regenerative Medicine, Danube University of Krems, Dr.-Karl-Dorrek-Strasse, 30 A-3500 Krems (Austria)], E-mail: stefan.nehrer@donau-uni.ac.at; Chiari, C.; Dorotka, R. [Department of Orthopedics, Medical University of Vienna, Waehringer Guertel 18-20, A 1090 Vienna (Austria); Szomolanyi, P. [MR Centre of Excellence, Department of Radiodiagnostics, Medical University of Vienna, Lazarettgasse 14, A-1090 Vienna (Austria); Institute of Measurement Science, Slovak Academy of Sciences, Dubravska cesta 9, 841 04 Bratislava (Slovakia); Mamisch, T.C. [Department of Orthopedics, Inselspital, University of Bern, 3010 Bern (Switzerland); Yayon, A. [ProChon Biotech Ltd., Weizmann Science Park, Nes Ziona (Israel); Trattnig, S. [MR Centre of Excellence, Department of Radiodiagnostics, Medical University of Vienna, Lazarettgasse 14, A-1090 Vienna (Austria)], E-mail: siegfried.trattnig@meduniwien.ac.at

    2010-03-15

    Objective: To assess repair tissue (RT) after the implantation of BioCart{sup TM}II, an autologous chondrocyte implantation (ACI) technique with a fibrin-hyaluronan polymer as scaffold. T2 mapping and delayed Gadolinium Enhanced Magnetic Resonance Imaging of Cartilage (dGEMRIC) were used to gain first data on the biochemical properties of BioCart{sup TM}II RT in vivo. Methods: T2 mapping and dGEMRIC were performed at 3 T in five patients (six knee joints) who had undergone ACI 15-27 months before. T2 maps were obtained using a pixel wise, mono-exponential non-negative least squares fit analysis. For quantitative T1 mapping a dual flip angle 3D GRE sequence was used and T1 maps were calculated pre- and post-contrast using IDL software. Subsequent region of interest analysis was carried out in comparison with morphologic MRI. Results: A spatial variation of T2 values in both hyaline, normal cartilage (NC) and RT was found. Mean RT T2 values and mean NC T2 values did not differ significantly. Relative T2 values were calculated from global RT and NC T2 and showed a small range (0.84-1.07). The relative delta relaxation rates (r{delta}R1) obtained from the T1 maps had a wider range (0.77-4.91). Conclusion: T2 mapping and dGEMRIC provided complementary information on the biochemical properties of the repair tissue. BioCart{sup TM}II apparently can provide RT similar to hyaline articular cartilage and may become a less-invasive alternative to ACI with a periosteal flap.

  5. Age-related decrease in the responsiveness of rat articular chondrocytes to EGF is associated with diminished number and affinity for the ligand of cell surface binding sites.

    Science.gov (United States)

    Ribault, D; Habib, M; Abdel-Majid, K; Barbara, A; Mitrovic, D

    1998-01-12

    The effect of age on the responsiveness of articular chondrocytes (AC) to epidermal growth factor (EGF) was examined. Cells were isolated by digesting cartilage fragments from the humeral and femoral heads of 21-day old, 8- and 14-month old rats with collagenase. The cells were cultured under standard conditions, as monolayers. DNA synthesis was measured by [3H]thymidine incorporation and cell proliferation by the DNA content of subconfluent cultures. [125I]EGF binding and the amounts of EGF and EGF-receptor mRNAs were determined using confluent cells. DNA synthesis was decreased with age of animals. EGF stimulated DNA synthesis in cultures in 1- and 8-month old rats at low serum concentrations (< 5%), and in cultures in 14-month old animals at high serum concentrations. It also increased 5-day DNA content of cultures compared to serum-treated controls but this effect was weak in cultures in 14-month old rats. The number of high affinity binding sites for [125I]EGF decreased from 37,800 in the 1-month old to 1950 in the 14-month old rat AC. The apparent dissociation constant (Kd) also decreased with age: 0.18 nmol/l in the 1-month old; 0.12 nmol/l in the 8-month old; and 0.07 nmol/l in the 14-month old cells. AC in older rats contained more EGF mRNA and less EGF-receptor mRNA. Incubation of the cells with EGF resulted in down regulation of the EGF- and upregulation of EGF-receptor mRNA expressions. These findings show the age-related quantitative and qualitative alterations in EGF and EGF-receptor which may account, at least in part, for the diminished responsiveness of senescent AC to EGF. PMID:9509392

  6. 人颈椎椎体终板软骨细胞退变模型的建立及其意义%Establishment and significance of an in vitro model of degeneration of human cervical endplate chondrocytes

    Institute of Scientific and Technical Information of China (English)

    徐宏光; 彭红心; 程加峰; 吕坤

    2011-01-01

    目的 建立人颈椎椎体终板软骨细胞退变模型,观察人正常颈椎椎体和退变颈椎椎体终板软骨细胞的形态及表征.方法 选择2010年7月至2011年7月49例颈椎骨折、脱位(19例)及颈椎病(30例)患者术中取出的颈椎终板软骨,用酶消化法分别分离培养人正常颈椎椎体终板软骨细胞(对照组)和退变颈椎椎体终板软骨细胞(颈椎病组);用倒置显微镜和HE染色法观察细胞形态学变化;四甲基偶氮唑蓝(MTT)法绘制细胞生长曲线;甲苯蓝染色及反转录-PCR(RT-PCR)法对终板软骨细胞进行鉴定;RT-PCR法检测终板软骨细胞特征性基因蛋白多糖、Ⅱ型胶原及Ⅰ型胶原的表达.结果 人颈椎椎体终板软骨细胞表达特征性蛋白多糖、Ⅱ型胶原及Ⅰ型胶原,其生长情况及细胞表型类似于关节软骨细胞.对照组原代终板软骨细胞以多角形为主,增殖速度较快;而颈椎病组原代终板软骨细胞以梭形为主,细胞增殖速度较慢.颈椎病组原代终板软骨细胞表达的蛋白多糖基因(0.695 ±0.052)和Ⅱ型胶原基因(0.726 ±0.035)均低于对照组(0.950±0.032、0.907±0.078,t=7.263、3.681,P=0.002、0.021),Ⅰ型胶原基因则高于对照组(0.795±0.028比0.552±0.070,t=-5.560,P=0.005).结论 成功建立了人颈椎椎体终板软骨细胞退变模型,为椎间盘退变机制研究提供了较好的细胞学基础,解决了以前一直以动物细胞模型为研究对象的局限性.%Objective To establish an in vitro model of degeneration of human cervical endplate chondrocytes and observe the morphology and phenotypes of endplate chondrocytes in normal and degenerative cervical vertebral endplates.Methods Cartilage endplates of 49 patients were divided into control group ( n =19 ) with cervical vertebral fracture or dislocation and experiment group ( n =30) with cervical spondylotic myelopathy.Endplate chondrocytes were isolated by enzyme digestion and cultured in vitro

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

    Directory of Open Access Journals (Sweden)

    Paul-Emile Poleni

    2010-01-01

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

  8. Transplante autólogo de condrócitos: relato de três casos Autologous chondrocyte implantation: series of 3 cases

    Directory of Open Access Journals (Sweden)

    Riccardo Gomes Gobbi

    2010-01-01

    Full Text Available A cartilagem hialina recobre as superfícies articulares e tem um papel importante na redução da fricção e da carga mecânica das articulações sinoviais, como o joelho. Este tecido não é suprido de vasos, nervos ou circulação linfática, o que pode ser uma das razões pela qual a cartilagem articular tem uma péssima capacidade de cicatrização. As lesões condrais, quando atingem o osso subcondral (lesão osteocondral, não cicatrizam e podem progredir para artrose com o passar do tempo. Em pacientes jovens, o tratamento dos defeitos condrais do joelho ainda é um desafio, principalmente as lesões maiores de 4cm. Uma das opções de tratamento nesses pacientes é o transplante autólogo de condrócitos, que por não violar o osso subcondral e por reparar o defeito com tecido semelhante à cartilagem hialina, teria a vantagem teórica de ser mais biológico e mecanicamente superior, quando comparado a outras técnicas. Descreveremos nesse artigo a experiência do Instituto de Ortopedia e Traumatologia do Hospital das Clínicas da Universidade de São Paulo (IOT-HCFMUSP com o transplante autólogo de condrócitos (ACI, através do relato de três casos.Hyaline cartilage in the surface of synovial joints plays an important role in lowering stress and attrition in joints such as the knee. This tissue has no blood vessels, nerves, nor lymphatic drainage, which in part explains why articular cartilage has such poor capacity for healing. Chondral lesions reaching the subchondral bone (osteochondral lesions do not heal and may progress to osteoarthritis as time passes. In young patients, treatment of such defects is challenging, especially in lesions larger than 4 cm. One option in young adults is the autologous chondrocyte implantation, capable of filling the defect with tissue similar to hyaline cartilage without violating the subchondral bone. Theoretically, it has biological and mechanical advantages over other surgical options. In this

  9. Initial results of in vivo high-resolution morphological and biochemical cartilage imaging of patients after matrix-associated autologous chondrocyte transplantation (MACT) of the ankle

    International Nuclear Information System (INIS)

    The aim of this study was to use morphological as well as biochemical (T2 and T2* relaxation times and diffusion-weighted imaging (DWI)) magnetic resonance imaging (MRI) for the evaluation of healthy cartilage and cartilage repair tissue after matrix-associated autologous chondrocyte transplantation (MACT) of the ankle joint. Ten healthy volunteers (mean age, 32.4 years) and 12 patients who underwent MACT of the ankle joint (mean age, 32.8 years) were included. In order to evaluate possible maturation effects, patients were separated into short-term (6-13 months) and long-term (20-54 months) follow-up cohorts. MRI was performed on a 3.0-T magnetic resonance (MR) scanner using a new dedicated eight-channel foot-and-ankle coil. Using high-resolution morphological MRI, the magnetic resonance observation of cartilage repair tissue (MOCART) score was assessed. For biochemical MRI, T2 mapping, T2* mapping, and DWI were obtained. Region-of-interest analysis was performed within native cartilage of the volunteers and control cartilage as well as cartilage repair tissue in the patients subsequent to MACT. The overall MOCART score in patients after MACT was 73.8. T2 relaxation times (∝50 ms), T2* relaxation times (∝16 ms), and the diffusion constant for DWI (∝1.3) were comparable for the healthy volunteers and the control cartilage in the patients after MACT. The cartilage repair tissue showed no significant difference in T2 and T2* relaxation times (p≥0.05) compared to the control cartilage; however, a significantly higher diffusivity (∝1.5; p<0.05) was noted in the cartilage repair tissue. The obtained results suggest that besides morphological MRI and biochemical MR techniques, such as T2 and T2* mapping, DWI may also deliver additional information about the ultrastructure of cartilage and cartilage repair tissue in the ankle joint using high-field MRI, a dedicated multichannel coil, and sophisticated sequences. (orig.)

  10. 发育性髋脱位髋臼软骨细胞凋亡的实验研究%The empirical study to acetabular chondrocyte apoptosis in the developmental dislocation of the hip.

    Institute of Scientific and Technical Information of China (English)

    韦宜山; 刘万林; 丁良甲; 王炳海; 白锐; 李岱鹤; 赵振群

    2012-01-01

    Objective To investigate the correlation of the apoptosis of acetabular chondrocyte and expression of Bcl-2 in the developmental dislocation of the hip ( DDH). Method 20 rabbits of 4-week-old which female and male were not restricted had been made for the models. The back limb that the hip was flex-ured and the knee was extened then fixed with a plaster cast was made for DDH model group and the right side without fixation as the control group. Pelvis anteroposterior X-rays had been made on the models before the fixation and after 8-weeks fixation. The femoral head dislocation or not by shenton' s line was discontinuity or by the femoral head was at the extabottom or extraupper quadrant of the Perkin squarse. 12 successful models were sacrificed at once. Observing the changes of general shape of bilateral acetabular and the changes of chondrocyte , then observing the apoptosis and expression of Bel - 2 of acetabular chondrocyte. Results Success rate of DDH models were 60% (12/20). Hip X-ray of experimental side shown that the superior margin of acetabu-lum was blunting, the femoral head was dislocation toward the extabottom or extraupper quadrant of the Perkin squarse, the acetabular angle of the experimental was significantly increased than the control side ( P < 0. 05 ) . The experimental side was found that the acetabulum became narrowing and fiied with soft tissue and the color of cartilage changed into dark, the chondrocytes were sparse and in a mess . Transmission electron microscopy results shown that the chromatin of acetabular chondrocytes were margination and condensation , the nuclear shape was irregular, the cytoplasmic vacuoles were present. Apoptosis rate of acetabular chondrocytes in experimental side was higher than the control side ( P < 0. 05 ) . The expression of Bcl-2 of acetabular chondrocytes in experimental side was lower than the control side ( P < 0. 05 ) , apoptosis and Bcl-2 expression of acetabular chondroctes were positive correlation in

  11. Pulsed electromagnetic fields increased the anti-inflammatory effect of A₂A and A₃ adenosine receptors in human T/C-28a2 chondrocytes and hFOB 1.19 osteoblasts.

    Directory of Open Access Journals (Sweden)

    Fabrizio Vincenzi

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

  12. Fabrication of Larynx-Shape Porous PHBHH for Cartilage Tissue Engineering and the Cytocompatibility with Chondrocytes%组织工程PHBHH多孔材料喉支架的制备与细胞相容性研究

    Institute of Scientific and Technical Information of China (English)

    孙安科; 胡平; 李万同; 孟庆延; 陈伟; 唐维维

    2011-01-01

    Objective To explore the possibility of using [poly(3 - hydroxybutyrate - co - 3- hydroxy-hexanoate) ,PHBHH] as biomaterials for cartilage tissue engineering of larynx and observe the cytocompatibility between PHBHH and chondrocytes. Methods Larynx-shape porous PHBHH models was fabricated by means of solvent casting,compression molding and paniculate leaching techniques; and the porosity of PHBHH was measured by liquid (alcohol) replacement. The larynx-shape PHBHH models were evaluated by morphological observation. PHBHH porousness, and the attachment, growth condition and distribution between PHBHH and chondrocytes were evaluated by morphological observation and scanning electron microscopy. Results Larynx-shape porous PHBHH models with edges and corners of laryngeal cartilage appeared to be hollow half-trumpet shape and its porosity was 92%±2%. The porous PHBHH looked like netwok. The chondrocytes equallyl attached to the surface of porous PHBHH, filled within porousness, and secrected a large quantity of extracellular matrix, Conclusion Porous PHBHH may be used as biomaterials to fabricate larynx-shape tissue engineering models with suitable mechan-ical support and porosity, and had good cytocompatibility with chondrocytes in vitro.%目的 探索组织工程多孔生物材料喉支架形态塑形物的制备技术,体外观察其细胞相容性,为进一步探寻软骨组织工程适宜生物材料提供实验依据.方法 以新型生物材料聚羟基丁酸酯与聚羟基己酸酯共聚物[ poly(3-hydroxybutyrate- co-3- hydroxyhexanoate),PHBHH,M=60万]为原料,氯化钠盐粒为致孔剂,采用溶剂浇铸、模压成形和颗粒滤沥技术制备多孔PHBHH喉支架形态塑形物,以乙醇置换法测定其孔隙率,负载软骨细胞后体外共同培养,通过大体形态、扫描电镜观察材料模压成形效果、材料孔隙连续性及其与软骨细胞粘附、分布和生长情况.结果 模压成形的组织工程PHBHH材料塑形物类似喉

  13. Induction of type Ⅱ collagen phenotype in transformed human articular chondrocytes%诱导转化人关节软骨细胞Ⅱ型胶原表型的有效方式

    Institute of Scientific and Technical Information of China (English)

    何清义; 李起鸿; 许建中

    2004-01-01

    BACKGROUND: Soft agar suspended culture is the main method for inducing the dedifferentiated chondrocytes to reexpress type Ⅱ collagen.OBJECTIVE: To induce collagen phenotype in dedifferentiated transformed human articular chondrocytes with centrifuge tube aggregate culture.DESIGN: A non-random uncontrolled study was conducted.SETTING and PARTICIPANTS: The experiment was completed in the Department of Orthopaedics, Southwest Hospital, Third Military Medical University. Subjects were transformed human articular chondrocytes, products of Hyclone Company, USA.INTERVENTIONS: The 30th, 40th 50th generation of dedifferentiated transformed chondrocytes (DTCs) were digested and cultivated in centrifuge tube. The expression of type Ⅰ, Ⅱ, Ⅲ collagen and the production of extracellular matrix from these cells were compared under the circumstances of monolayer culture and centrifuge tube aggregate culture with ordinary medium and BAI induced medium(bone morphogenetic protein 2 +ascorbate +insulin).MAIN OUTCOME MEASURES: Immunohistochemical staining of type Ⅰ, Ⅱ, Ⅲ collagen; mRNA expression of type Ⅱ collagen.RESULTS: In monolayer culture with ordinary medium, only type Ⅰ collagen was expressed in the DTCs, while in aggregate culture with BAI induced medium, massive type Ⅱ collagen and extracellular matrix were expressed in the DTCs.CONCLUSION: Centrifuge tube aggregate culture and BAI induced medium are effective manners in inducing DTCs to express type Ⅱ collagen.%背景:诱导去分化软骨细胞重新表达Ⅱ型胶原主要基于软琼脂悬浮培养法.目的:用离心管聚集体培养(aggregate culture)诱导去分化转化人关节细胞的Ⅱ型胶原.设计:非随机非对照实验研究.地点和对象:实验在第三军医大学西南医院骨科完成,对象为转化人关节软骨细胞,美国Hyclone公司产品.干预:将体外长期培养的第30,40,50代去分化转化软骨细胞消化后进行离心管培养,比较细胞在单层培

  14. 转腺病毒-人骨形态发生蛋白7软骨细胞分泌的透明质酸和Ⅱ型胶原%Secretion of type Ⅱ collagen and hyaluronic acid in chondrocytes transfected by adenovirus-bone morphogenetic protein 7

    Institute of Scientific and Technical Information of China (English)

    张洁; 刘巍; 朱新辉; 周怡

    2011-01-01

    BACKGROUND: Chondrocytes are limited in tissue engineering due to their poor self-dividing capacity and defifferentiation.OBJECTIVE: To investigate the expression of bone morphogenetic protein 7 (BMP-7) in chondrocytes transfected by adenovirus BMP-7 and the effects of transfection on chondrocyte secretion of type Ⅱ collagen and hyaluronic acid.METHODS: Adenoviral vector containing BMP-7 was prepared and then transfected into rabbit passage chondrocytes. BMP-7 mRNA and protein expressions in chondrocytes were detected. Changes in type Ⅱ collagen and hyaluronic acid in chondrocytes were determined.RESULTS AND CONCLUSION: At 48 and 72 hours after transfection, RT -PCR and western blot analysis showed that BMP-7 mRNA and protein expressions in the chondrocytes were increased; RT-PCR and ELISA showed the type Ⅱ collagen and hyaluronic acid in the chondrocytes were also obviously increased. These findings suggest that BMP-7 can be successfully transfected into chondrocytes by adenovirus and promote the secretion of type Ⅱ collagen and hyaluronic acid.%背景:软骨细胞自身分裂能力不强和去分化现象限制了其在组织工程中的应用.目的:观察腺病毒-骨形态发生蛋白7转染兔软骨细胞后骨形态发生蛋白7的表达及其对软骨细胞分泌Ⅱ型胶原和透明质酸功能的影响.方法:包装骨形态发生蛋白7腺病毒载体,将其转染至兔第2代软骨细胞.检测骨形态发生蛋白7 mRNA及蛋白的表达;检测软骨细胞中Ⅱ型胶原和透明质酸的变化.结果与结论:转染腺病毒-骨形态发生蛋白7后48,72 h,RT-PCR和Western blot方法显示软骨细胞表达的骨形态发生蛋白7 mRNA和蛋白均明显增加,RT-PCR和ELISA显示软骨细胞分泌的Ⅱ型胶原和透明质酸也显著增加.说明应用腺病毒可成功将骨形态发生蛋白7转染至兔软骨细胞,并能促进软骨细胞分泌Ⅱ型胶原和透明质酸.

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

    Science.gov (United States)

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

    2010-03-30

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

  16. Improved Osteoblast and Chondrocyte Adhesion and Viability by Surface-Modified Ti6Al4V Alloy with Anodized TiO2 Nanotubes Using a Super-Oxidative Solution

    Directory of Open Access Journals (Sweden)

    Ernesto Beltrán-Partida

    2015-03-01

    Full Text Available Titanium (Ti and its alloys are amongst the most commonly-used biomaterials in orthopedic and dental applications. The Ti-aluminum-vanadium alloy (Ti6Al4V is widely used as a biomaterial for these applications by virtue of its favorable properties, such as high tensile strength, good biocompatibility and excellent corrosion resistance. TiO2 nanotube (NTs layers formed by anodization on Ti6Al4V alloy have been shown to improve osteoblast adhesion and function when compared to non-anodized material. In his study, NTs were grown on a Ti6Al4V alloy by anodic oxidation for 5 min using a super-oxidative aqueous solution, and their in vitro biocompatibility was investigated in pig periosteal osteoblasts and cartilage chondrocytes. Scanning electron microscopy (SEM, energy dispersion X-ray analysis (EDX and atomic force microscopy (AFM were used to characterize the materials. Cell morphology was analyzed by SEM and AFM. Cell viability was examined by fluorescence microscopy. Cell adhesion was evaluated by nuclei staining and cell number quantification by fluorescence microscopy. The average diameter of the NTs was 80 nm. The results demonstrate improved cell adhesion and viability at Day 1 and Day 3 of cell growth on the nanostructured material as compared to the non-anodized alloy. In conclusion, this study evidences the suitability of NTs grown on Ti6Al4V alloy using a super-oxidative water and a short anodization process to enhance the adhesion and viability of osteoblasts and chondrocytes. The results warrant further investigation for its use as medical implant materials.

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

    Science.gov (United States)

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

    2016-02-01

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

  18. Expression of human insulin-like growth factor Ⅰ mediated by retrovirus in chondrocytes%逆转录病毒载体介导人胰岛素样生长因子-Ⅰ在软骨细胞中的表达

    Institute of Scientific and Technical Information of China (English)

    宋之明; 张绍昆; 孙洋; 任琦

    2011-01-01

    Objective To transfect the rabbit chondrocytes using a recombinant retroviral vector containing hIGF-I cDNA, and to investigate the expression of exogenous hIGF-I gene in the chondrocytes and its effects on cell biological behavior. Methods The retroviral vector pLNC-IGF-GFP containing targeted gene was transfected into rabbit articular chondrocytes. The positive chondrocytes clones, which were G418-resistant, were selected by G418. The expression of hIGF-I mRNA and protein in chondrocytes were determined by RT-PCR and immunohistochemical staining, respectively. And the expression of GFP gene was observed under fluorescence microscope. Proliferation and phenotype of transfected chondrocytes were tested by MTF, flow cytometry, immunocytochemistry, dimethyl methylene blue method and ELISA. Results The stable expression levels of hIGF-I in transfected chondrocytes were confirmed by RT-PCR and immunocytochemical analysis, and GFP expression was established under fluorescence microscope to be observed green fluorescence. The proliferation of chondrocytes and the secretion of collagen Ⅱ and proteoglycan were higher than those of the same time point in non-transfected chondrocytes within 6 w after transfection ( P <0. 01 ). Conclusions 2hIGF-I gene can be effectively transfered into the rabbit chondrocytes mediated by retroviral vector and is expressed stably. Meanwhile, the transfected cells proliferate actively. It may provide a basis for further study of gene therapy for cartilage defects.%目的 探讨逆转录病毒载体介导人胰岛索样生长因子-Ⅰ(human insulin-like growth factor-I,hIGF-I)体外转染兔软骨细胞后软骨细胞hIGF-I的表达情况及其生物学行为变化.方法 将构建的含有目的 基因的逆转录病毒载体pLNc-IGF-GFP体外转染兔关节软骨细胞,经G418筛选阳性克隆,采用RT-PCR及免疫化学染色检测转染软骨细胞中hIGF-I的表达情况,并在荧光显微镜下观察标记基

  19. Efeito do Plasma Rico em Plaquetas na apoptose pós-traumática de condrócitos Effect of Platelet-Rich Plasma on impact-induced chondrocyte apoptosis

    Directory of Open Access Journals (Sweden)

    Márcia Uchôa de Rezende

    2011-04-01

    Full Text Available OBJETIVO: Avaliar se a injeção intra-articular de Plasma rico em plaquetas (PRP pode reduzir a apoptose pós-traumática de condrócitos. MÉTODOS: Foi desenvolvido um estudo experimental duplo-cego com quatro joelhos de coelhos adultos. Após a anestesia, os animais foram submetidos à contusão padronizada dos joelhos. Depois foi injetado 1ml de PRP humano nos dois joelhos esquerdos e 1ml de solução fisiológica (SF nos dois joelhos direitos. Os dois coelhos foram mantidos no mesmo ambiente sob controle de temperatura, de atividades diárias e de alimentação. A eutanásia dos animais ocorreu dez dias após a intervenção e foram realizadas biópsias da cartilagem de cada joelho. As peças foram preparadas para análise em microscopia eletrônica (ME. RESULTADOS: Quatro preparados para ME foram obtidos, cada um correspondendo a um joelho. Os joelhos-PRP apresentaram as taxas de apoptose de 47,62% (50/105 e de 48,36% (59/122, respectivamente. Nos joelhos-SF as taxas de apoptose foram, respectivamente, 56,67% (17/30 e 70,40% (88/125. A diferença do índice de apoptose nos joelhos-PRP (48,02% e nos joelhos-SF (67,74% foi significante (pOBJECTIVE: To evaluate if the injection of intra-articular platelet-rich plasma (PRP can reduce impact-induced chondrocyte apoptosis. METHODS: A double-blind experimental study was developed in four knees of two adult rabbits. Each knee was injured after anesthesia. Subsequently, 1ml PRP was injected in the right knees and 1ml of normal saline (NS in the left knees. The animals were euthanized ten days after the intervention. All cartilage was removed from the 4 knees and prepared for analysis in electron microscopy (EM. RESULTS: Four EM samples were obtained. The PRP-injected knees showed apoptosis rates of 47,62% (50/105 and 48,36% (59/122, respectively. NS-injected knees showed 56.67% (17/30 and 70.40% (88/125 of apoptosis. PRP-injected knees had statistically significant less apoptosis (48.02% than NS

  20. 软骨细胞发生和分化基因与大鼠肝再生的相关性%Relevance of Rat Liver Regeneration with the Genes of Chondrocyte Genesis and Differentiation

    Institute of Scientific and Technical Information of China (English)

    赵利峰; 李鹏鸽; 徐存拴

    2007-01-01

    肝脏由多种细胞构成,肝再生与细胞分化密切相关,细胞分化受基因转录水平调控.为在基因转录水平了解软骨细胞的发生和分化相关基因在大鼠肝再生中作用,通过搜集网站资料和查阅相关论文等方法获得参与软骨细胞发生和分化的基因,用Rat Genome 230 2.0芯片检测它们在大鼠肝再生(liver regeneration,LR)中表达情况,用比较真、假手术中基因表达差异确定肝再生相关基因.初步证实上述基因中23个基因与肝再生相关.肝再生启动(PH后0.5~4 h)、G0/G1过渡(PH后4~6 h)、细胞增殖(PH后6~66 h)、细胞分化和组织结构功能重建(PH后72~168 h)等四个阶段起始表达的基因数为15、4、8和0;基因的总表达次数为15、10、22和17.表明相关基因主要在肝再生启动阶段起始表达,在不同阶段发挥作用.它们共表达上调100次、下调77次,分为17种表达方式,表明肝再生中软骨细胞发生和分化相关基因活动多样和复杂.根据本文研究结果推测,上述基因不仅调节软骨细胞发生和分化,而且参与肝再生的生理生化活动.%Liver contains many cell types. Liver regeneration is closely related to cell differentiation, which is regulated by some genes. To investigate the actions of the genes participating in chondrocyte genesis and differentiation on rat liver regeneration at transcriptional level, the aforementioned genes were screened out via collecting database data and retrieving the related thesis. The Rat Genome 230 2.0 array was applied to the measurement of expression changes of them in rat regenerating liver. The liver regeneration-associated genes were determined by comparing differences in gene expression between partial hepatectomy (PH) and sham operation (SO). It was 23 among the above genes that were related to liver regeneration. In initiation phase of liver regeneration (0.5~4 h after PH), G0/G1 (4~6 h after PH), cell proliferation (6~66 h

  1. 透明质酸对人骨关节炎软骨细胞骨桥蛋白和CD44表达的影响%Effects of hyaluronic acid on osteopontin mRNA and CD44 mRNA expression in human osteoarthritic chondrocytes

    Institute of Scientific and Technical Information of China (English)

    周斌; 张方杰; 罗伟; 高曙光; 曾超; 熊依林; 李宇晟; 雷光华

    2014-01-01

    背景:软骨进行性破坏为晚期骨关节炎的特征性病变,骨关节炎相关因子透明质酸、骨桥蛋白、CD44在骨关节炎软骨中表达增加。  目的:通过透明质酸干预体外培养的人膝骨关节炎软骨细胞,探讨透明质酸对人膝骨关节炎软骨细胞 CD44与骨桥蛋白表达的影响。  方法:将软骨标本进行体外培养获取纯化的软骨细胞,分为3组:空白对照组、透明质酸干预组(100 mg/L)和透明质酸酶干预组(200 mg/L)。培养48 h后,采用Real-time Q PCR检测软骨细胞骨桥蛋白mRNA,CD44 mRNA表达水平。用SPSS 17.0统计软件包分析骨桥蛋白mRNA和CD44 mRNA经透明质酸干预前后表达的差异。  结果与结论:透明质酸组的骨桥蛋白 mRNA表达水平较空白组高,透明质酸酶组的骨桥蛋白 mRNA表达水平较空白组低;透明质酸组及透明质酸酶组的CD44 mRNA表达水平均较空白组低。结果提示透明质酸可以上调骨关节炎软骨细胞骨桥蛋白的表达;透明质酸在骨关节炎软骨细胞内对 CD44表达的影响具有双相性,其影响结果可能与透明质酸的相对分子质量有关。%BACKGROUND:Progressive fracture of the cartilage is considered the characteristic lesion in later osteoarthritis, the expression of osteoarthritis-related factors such as hyaluronic acid, osteopontin and CD44 in osteoarthritic cartilage is increased. OBJECTIVE:To investigate the effect of hyaluronic acid on the expression of osteopontin mRNA and CD44 mRNA of chondrocytes in the in vitro cultured chondrocytes of patients with knee osteoarthritis. METHODThe cartilage samples obtained from osteoarthritic patients were cultured and purified into acquire chondrocytes in vitro, and the cells were divided into three groupblank control group, hyaluronic acid (100 mg/mL) group and hyaluronidase (200 mg/mL) group. After 48 hours of cellculture, real-time quantitative polymerase chain reaction

  2. 胶原/羟基磷灰石复合支架负载软骨细胞构建组织工程软骨%Construction of tissue engineering cartilage with collagen/hydroxyapatite composite scaffolds loaded chondrocytes in vitro

    Institute of Scientific and Technical Information of China (English)

    卢华定; 蔡道章; 吴刚; 曾春

    2006-01-01

    BACKGROUND: A new composite scaffold for cartilage tissue engineer ing has been employed to culture chondrocytes and overcome many limits related to traditional scaffolds, such as poor biocompatibility, inferior mechanical property, inappropriate biodegradability, and simplex structure which can not match layered structure of articular cartilage, etc. The new composite scaffolds provided a new approach for the research of cartilage tissue engineering.OBJECTIVE: To evaluate the feasibility and value of layered cylindrical collagen/hydroxyapatite (HA) composite for cartilage tissue engineering by observing how it absorbs chondrocytes and affects its cellular characteristics.DESIGN: Completely randomized design and controlled experimental study.SETTING: Department of Orthopaedics, Third Hospital Affiliated to Sun Yat-sen University, and College of Material Science, South China University of Technology.MATERIALS: The experiment was conducted at the central experimental laboratory of the Third Hospital Affiliated to Sun Yat-sen University from June to November 2004. One two-week-old male healthy New Zealand rabbit,which was bred in 20 ℃ and 40% humidity, was used in this experiment.METHODS: ①Right amount of deionized water was added into HA, collagen I solution was added to disperse HA, then carbodiimide was added in the mixture at a proportion for getting the collagen/HA composite at different ratios. Pour to the certain mould in successive layers. The upper layer was pure collagen and the bottom was pure HA. The prepared layered cylindrical collagen/HA composite was put into the ultra low temperature freezer, lyophilized, and sterilized by ethylene oxide for the following procedures. ② Chondrocytes of juvenal rabbit were isolated and multiplied in vitro, then chondrocytes were seeded onto porous collagen/HA composite scaffold and cultured. The effects of composite scaffold on chondrocytes'morphological changes, proliferation, and function were evaluated through

  3. Chondrocyte growth dynamics and spatial pattern formation

    OpenAIRE

    Palumberi, Viviana

    2010-01-01

    In this thesis we study the generation of patterns in cell culture refering to the important work of Elsdale where fibroblast cultures were analyzed to investigate how densely packed cells organize. A mathematical model was introduced in Edelstein-Keshet and Ermentrout (1990) to prove that the pattern formation can be caused by the mere interactions of individual cells, although it is a population phenomena. Until then the formation of structures was only attributed to other m...

  4. Implante de condrócitos homólogos em defeitos osteocondrais de cães: padronização da técnica e avaliação histopatológica Homologous articular chondrocytes implantation in osteochondral defects of dogs: technique and histopathological evaluation standardization

    Directory of Open Access Journals (Sweden)

    L.S. Iamaguti

    2013-02-01

    Full Text Available Padronizou-se a metodologia para cultura de condrócitos em cães e avaliou-se seu implante em lesões osteocondrais, utilizando-se a membrana biossintética de celulose (MBC como revestimento. Dez cães, adultos e clinicamente sadios, foram submetidos à artrotomia das articulações fêmoro-tíbio-patelares. Defeitos de 4mm de diâmetro e profundidade foram induzidos no sulco troclear de ambos os membros. MBC foi aplicada na base e na superfície das lesões. Os defeitos do membro direito foram preenchidos com condrócitos homólogos cultivados formando o grupo-tratado (GT; os do membro esquerdo, sem implante celular, foram designados grupo-controle (GC. A evolução pós-operatória foi analisada com especial interesse nos processos de reparação da lesão, por meio de histomorfometria e imuno-histoquímica para colágeno tipo II e sulfato de condroitina. A cultura de condrócitos homólogos apresentou alta densidade e taxa de viabilidade. Observou-se integridade do tecido neoformado com a cartilagem adjacente na avaliação histológica, em ambos os grupos. Na imuno-histoquímica, verificou-se predomínio de colágeno tipo II no GT. Morfometricamente, não houve diferença significativa entre o tecido fibroso e o fibrocartilaginoso entre os grupos. A cultura de condrócitos homólogos de cães foi exequível. O tecido neoformado apresentou qualidade discretamente superior associado ao implante homólogo de condrócitos, contudo não promoveu reparação por cartilagem hialina.The aim of the study is to standardize the methodology to achieve canine chondrocytes culture, and evaluate its implant on osteochondral defects made in the femoral trochlear sulcus of dogs, using the cellulose biosynthetic membrane (CBM as coating. Ten healthy adult dogs without locomotor disorders were used. All animals were submitted to arthrotomy of stifle joints and defects of four millimeters in diameter x four millimeters deep were done in the femoral trochlear

  5. Disturbance of nivalenol on expression of adhesion molecule CD44 on surface of cultured chondrocytes%雪腐镰刀菌烯醇能干扰培养软骨细胞表面黏附分子CD44的表达

    Institute of Scientific and Technical Information of China (English)

    孙健; 曹峻岭; 杨旭东; 孟列素

    2006-01-01

    BACKGROUND: Deficit of nivalenol (NIV) and selenium (Se) is related with kashin-beck disease (KBD) to certain extent. Hyaluronic acid (HA) metabolism affects directly the polymerization of proteoglycans (PG) and normal structure and function of cartilage. The integration with HA receptor on surface of cartilage tissue is the key link in HA metabolism. Being the main receptor of HA on chondrocytic membrane, CD44 expression impacts directly HA metabolism, further affects cartilage matrix metabolism, which is extremely important to maintaining the structure and function of cartilage matrix.OBJECTIVE: To probe into the injury and protection of related etiology of KBD to target tissue cells and the mechanism on degenerative necrosis of chondrocytes.DESIGN: Randomized controlled observation was designed.SETTING: Department of Genetics and Molecular-Biology of Medical College of Xi' an Jiaotong University.MATERIALS: The experiment was performed in Key Laboratory Room on Ministry of Education associated with Environment and Disease of Xian Jiaotong University from October 2002 to July 2004. One New Zealand pedigree young rabbit aged 30 days was employed and its humerus, femurs and tibia were cut out in surgery.METHODS: With cell culture, the model of bone tissue was reconstructed in vitro, in which, NIV of various concentrations, KBD suspicious infectious agent and Se, the protective factor were added. HA receptor CD44 on chondrocytic membrane and soluble CD44 in cell culture solution were determined.MAIN OUTCOME MEASURES: ① Microscopic observation of adhesion molecule CD44 on chondrocytic surface. ② Soluble CD44 in chongrocytic culture solution.RESULTS: ① Microscopic observation of adhesion molecule CD44 on chondrocytic surface: CD expression in chondrocytic membrane was decreased with increasing of NIV concentration and it was in tendency of increasing with Se added. ② Soluble CD44 in chongrocytic culture solution:The concentration of soluble CD44 in cell culture

  6. Repairing Osteochondral Defect of Rabbits with Nano-HA/CS Scaffold Composite with Chondrocyte by Boot-shaped Structure%纳米HA/CS支架穿“靴”复合软骨样细胞修复兔关节软骨和软骨下骨缺损

    Institute of Scientific and Technical Information of China (English)

    李劼若; 屠美; 郑力恒; 谭文成; 查振刚; 郇松玮; 刘宁; 张国威; 吴昊; 林宏生; 姚平; 张嘉晴

    2012-01-01

    [Objective] To observe the condition of constructing the tissue engineered chondral composite constructing by co culturing the chondrocyte which were induced from bone marrow stem cells (BMSCs) and nano-hydroxyapatile/chitosan scaffold (nano-HA/CS), under a boot-shaped structure after compositing. The effect of the composite on repairing the osteochondral defect of rabbits' knee were observed. [Methods]The technique of combining the in situ hybridization and freeze-drying was taken to fabricate nano-HA/CS scaffold. The cylindrical boot-shaped structure were made of polytetrafluoroethylene. The chondrocyte were induced from BMSCs by cultivating in the chondrogenic differentiation medium. Then diey were seeded into the bottom of the nano-HA/CS scaffold whose top was coverd by the boot-shaped structure. The composite was cultivated in the chondrogenic differentiation medium for 2 weeks. The structure of nano-HA/CS scaffold and composite was observed by scanning electron microscope. The osteochondral defects in the epicondyle of femur, which was 4 mm in diameter and 8 mm in depth, were constructed in 30 New Zealand white rabbits. The rabbits were divided into three groups, experimental group (composite induced with boot-shaped structure grafted into the defect), control group (composite induced without boot-shaped structure), and blank group (with no implant). Finally the curative effect of specimens after 4 and 12 weeks were evaluated respectively. [Result] BMSCs can be induced to chondrocyte after cultivated in the chondrogenie differentiation medium. The nano-HA/CS scaffold with 92% interstice ratio and 125 (un diameter had a good adhesion with chondrocyte. In general, cartilage-like tissue were found in experimental group and control group, while fibrous tissue were found in blank group where the defect was still obvious. The HE staining showed that the cartilage defect was repaired by chondrocyte partly and the bone defect was repaired by osteoblasts partly in the

  7. Repairing allogenic thyroid cartilage defects using poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) seeded with chondrocytes%聚羟基烷酸酯聚合物负载软骨细胞修复同种异体喉软骨缺损

    Institute of Scientific and Technical Information of China (English)

    孙安科; 李万同; 刘松波; 张贺; 孙伟; 陈伟; 史春海; 唐维维

    2013-01-01

    ,直接应用初级组织工程软骨组织可节省时间、成本、工作量及操作环节,避免二次皮下手术的痛苦,是比较实用的方法之一。%BACKGROUND:A great development has been achieved in essential research on tissue engineered cartilage. However, its real application in otolaryngology has been rarely reported. It is faced with the topic to explore the simple and convenient method of repairing laryngeal cartilage by tissue engineering technique. OBJECTIVE:To compare the effect of porous spongy poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) seeded with chondrocytes or using senior tissue engineered cartilage in repairing al ogenic thyroid cartilage defects.METHODS:Chondrocytes at passage 3 were harvested from infant rabbits within 3 days. Porous spongy poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) seeded with chondrocytes composites were made by tissue engineering technique. The chondrocyte-poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) composites were co-cultured in vitro to form junior tissue engineered cartilage. And then respectively used for repairing the thyroid cartilage defects and directly transplanted with junior tissue engineered cartilage (experimental group A, n=5), or firstly the junior tissue engineered cartilage to be implanted subcutaneously for a period of time to further maturity for relative senior tissue engineered cartilage and secondly to be transplanted (experimental group B, n=5) into adult New Zealand white rabbits. Simple poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) sponge scaffold (control group A, n=4) and chondrocyte suspensions(control group B, n=4) were used as reparative materials in defect areas as control groups. Final y, the reparative effect was respectively studied grossly and histological y at 4 weeks (experimental group B) and 8 weeks (experimental group A, control group A and control group B) after transplantation. RESULTS AND CONCLUSION:The cartilage defects were wel repaired in the

  8. AGEs对兔软骨细胞TNF-α和MMP-13表达的影响及其机制研究%The effects of advanced glycation end products on expression of tumor necrosis factor-αand matrix metalloproteinase-13 in rabbit chondrocytes and its mechanism

    Institute of Scientific and Technical Information of China (English)

    陈铖; 马翅; 张莹; 肖钧; 蔡巍; 谭海涛

    2013-01-01

    目的:探讨晚期糖基化终末产物(AGEs)对兔软骨细胞肿瘤坏死因子-α(TNF-α)和基质金属蛋白酶-13(MMP-13)的影响及可能机制。方法:不同浓度的AGEs与兔软骨细胞共孵育48h后采用RT-PCR方法检测TNF-α和MMP-13的mRNA表达量,试剂盒方法检测过氧化氢酶(CAT)、超氧化物歧化酶(SOD)活性及丙二醛(MDA)水平,荧光探针法检测细胞内活性氧(ROS)水平。AGEs与兔软骨细胞共孵育的同时,分别加入AGEs受体的抗体(Anti-RAGE)及核因子-κB(NF-κB)的特异性阻断剂PDTC处理,同法检测TNF-α和MMP-13的mRNA表达量。结果:与AGEs共孵育48h后兔软骨细胞TNF-α及MMP-13表达明显增多,CAT、SOD活性降低,MDA、ROS含量增多,均呈浓度依赖性;分别加入Anti-RAGE 及PDTC 处理后软骨细胞TNF-α及MMP-13表达明显低于AGEs单独处理组(P0.05)。结论:AGEs能诱导软骨细胞TNF-α和MMP-13表达增多,其机制与激活RAGE,诱导ROS生成增多,激活NF-κB信号通路有关。%Objective To detect the effects of advanced glycation end products (AGEs) on expression of tu-mor necrosis factor-α(TNF-α) and matrix metalloproteinase-13(MMP-13) in rabbit chondrocytes and its mecha-nism. Methods The chondrocytes were incubated with different concentrations of AGEs for 48h, the expression of TNF-αand MMP-13 mRNA were detected by reverse transcription polymerase chain reaction(RT-PCR),the lev-el of catalase (CAT), Malondialdehyde (MDA) and the activity of superoxide dismutase (SOD) were detected by kits, and the level of ROS is measured by the method of Fluorescent probe. The effect of anti-RAGE(Receptor for AGEs) and PDTC(the inhibitor of NF-KB) on the expression of TNF-αand MMP-13 induced by AGEs were also measured by RT-PCR. Results After chondrocytes were incubated with AGEs, dose-dependly increased the ex-pression of TNF-αand MMP-13 (P0.05). Conclusion AGEs can induce the TNF-αand MMP-13 expression. It's mechanism may

  9. 体外诱导骨髓间充质干细胞定向分化为软骨细胞的实验研究%In Vitro Induction of Directional Differentiation of Bone Marrow Mesenchymal Stem Cells towards Chondrocytes

    Institute of Scientific and Technical Information of China (English)

    谭荣邦; 史宏灿

    2013-01-01

    目的 体外分离培养、扩增骨髓间充质干细胞(mesenchymal stem cells,MSCs),诱导其定向分化为软骨细胞,为MSCs应用于临床、构建组织工程气管软骨提供实验依据. 方法 采用全骨髓培养+贴壁纯化法分离培养MSCs,流式细胞仪鉴定MSCs,以转化生长因子βl(transforming growth factor p1,TGF-β1)、胰岛素样生长因子l(insulin-likegrowthfactor1,IGF-1)作为主要的诱导因子诱导MSCs定向分化为软骨细胞.21d后用Ⅱ型胶原免疫细胞化学染色法鉴定诱导出的细胞,在光学显微镜和电子显微镜下观察MSCs诱导前、后的细微和超微结构改变.结果 诱导21 d后Ⅱ型胶原免疫细胞化学染色呈阳性.光学显微镜和电子显微镜检查显示:MSCs呈梭形,诱导后细胞增大,呈多边形、星形、三角形.透射电子显微镜显示:诱导后细胞器丰富,胞核增大,核仁增多. 结论 MSCs诱导分化为软骨细胞的超微结构表现为细胞器丰富、胞核增大、核仁增多,提示细胞分化、代谢功能旺盛.%Objective To isolate,culture and expand bone marrow mesenchymal stem cells (MSCs) in vitro,induce MSCs to differentiate directionally towards chondrocytes,and provide experimental basis for clinical application of MSCs and construction of tissue engineering tracheal cartilage.Methods Cultured MSCs were isolated from bone marrow of Sprague-Dawley rats,purified using adherence separation,and identified by flow cytometry analysis.Transforming growth factor β1 (TGF-β1)and insulin-like growth factor 1 (IGF-1)were used as main induction factors to induce MSCs to differentiate directionally towards chondrocytes.The expression of collagen type Ⅱ was evaluated by immunocytochemical staining 21 days after induction.Light microscope and electron microscope were used to observe tiny and ultrastructural changes of the cells before and after induction.Results The expression of collagen type Ⅱ was positive by immunocytochemical staining 21

  10. Effects of 5-Aza-CdR on interleukin-1β, transforming growth factor-β and nitric oxide synthase expression in human chondrocyte%5-氮杂-2'-脱氧胞苷对软骨细胞中炎性介质和一氧化氮合酶表达的影响

    Institute of Scientific and Technical Information of China (English)

    赵丽珂; 王钱; 张春媚; 黄慈波

    2015-01-01

    Objective To investigate the effects of 5-Aza-CdR (methylation transferase inhibitor) on gene expression levels of interleukin-1β (IL-1β),transforming growth factor-β (TGF-β) and nitric oxide synthase (NOS) in chondrocytes.Methods Chondrocytes from patients with osteoarthritis (OA) (n=22),rheumatoid arthritis (RA) (n=3) or trauma without rheumatic diseases (n=10) were collected and cultured.The mRNA expression levels of IL-1β,TGF-β and NOS were detected by real-time polymerase chain reaction (RT-PCR).Chondrocytes in trauma group were treated with 5-Aza-CdR(10μmol/L) for 72 h,and the mRNA and protein expression levels of IL-1β,TGF-β and NOS were detected by RT-PCR and ELISA,respectively.Results The mRNA expression levels of IL-1β,TGF-β and NOS were increased in OA and RA group as compared with trauma group (P<0.05),while they had no differences between OA and RA groups.After treated with 5-Aza-CdR,the mRNA and protein expression levels of IL-1β,TGF-β and NOS in chondrocytes rised in trauma group as compared with pretreatment (all P<0.05).Conclusions The mRNA expression levels of IL-1β,TGF-β and NOS in chondroytes are higher in OA and RA patients.5-Aza-CdR could increase the mRNA and protein expression levels of IL-1β,TGF-β and NOS by inducing relative gene methylation,which suggests demethylation might play a role in OA pathogenesis by influncing the inflammatory signal pathway or cell apoptosis.%目的 探讨甲基化抑制剂5-氮杂-2'-脱氧胞苷(5-Aza-CdR)对软骨细胞白介素1β(IL-1β)、转化生长因子β(TGF-β)和一氧化氮合酶(NOS)基因表达的影响. 方法 收集骨关节炎患者(OA组,22例)、类风湿关节炎患者(RA组,3例)及外伤患者(对照组,10例)软骨标本并进行细胞培养,检测软骨细胞中IL-1β、TGF-β和NOS mRNA表达水平;用甲基化抑制剂5-Aza-CdR(浓度为10 μmol/L)培养对照组正常软骨细胞72 h,检测软骨细胞IL-1β、TGF-β和NOS mRNA及蛋白表达水平. 结果

  11. Hydrophilicity and adsorptivity of a novel scaffold twice embedded by lecithin and poly-L-lysine to human nasal septum chondrocytes%卵磷脂、多聚赖氨酸二次包埋新型支架材料的亲水性及对人鼻中隔软骨细胞的吸附力

    Institute of Scientific and Technical Information of China (English)

    程友; 王秋萍; 江满杰; 吴昆旻; 薛飞; 陈伟; 季俊峰; 李泽卿

    2007-01-01

    吸附力(人鼻中隔软骨细胞悬液在支架上是否飘动).②细胞生长繁殖及基质产生情况.结果:①单纯支架加入人鼻中隔软骨细胞悬液后悬液先呈球型附在支架表面,然后缓慢地扩散到支架空隙内.培养期间相差显微镜观察见细胞成团地附在支架纤维表面,晃动培养皿可见细胞团漂浮不定[黏附率为(21±3.7)%],随着培养时间延长无基质产生.②卵磷脂+多聚赖氨酸包埋的支架加入人鼻中隔软骨细胞悬液后悬液迅速均匀地扩散到支架空隙内[黏附率为(89±5.6)%,高于单纯支架组],表明支架有较强的亲水性;相差显微镜下见软骨细胞以单个或群落状均匀分布于支架纤维之间及吸附在支架纤维表面,晃动培养皿细胞团无飘动,散落在培养皿底部的细胞较少,表明支架对细胞有较强的吸附力;培养1周,支架纤维间有呈蜘蛛网状基质产生,随着培养时间延长,基质产生旺盛.结论:以卵磷脂和多聚赖氨酸共同包埋的聚丙交酯-乙交酯共聚物/甲壳胺无纺布的新型生物支架,亲水性良好,对人鼻中隔软骨细胞的吸附力明显增强,并可促进软骨细胞分泌基质.%BACKGROUND:Chitosan is a kind of natural biomaterial and is characterized by great biocompatibility, progressive degeneration and absorption and excellent mechanical property; however, whether it may become an ideal cytoskeleton in the engineering of cartilage tissue or not should be researched further.OBJECTIVE: To observe the hydrophilicity and adsorptivity to human nasal septum chondrocytes and the effect of its function of a novel scaffold made by [poly (dl-lactide-co-glycolide)] (PLGA)/chitosan nonwoven cloth embedded with lecithin (LEC) and poly-L-lysine (PLYS).DESIGN: Blank control study.SETTING: Department of Otolaryngology-Head and Neck Surgery, Nanjing General Hospital of Nanjing Military Area Command of Chinese PLA.MATERIALS: The experiment was carried out in the Department

  12. 低强度脉冲超声介导威灵仙干预兔膝关节软骨细胞增殖及Ⅱ型胶原和转化生长因子β1的表达%Effect of low-intensity pulsed ultrasound mediated Clematis chinensis Osbeck on the proliferation and expression of type II collagen and transforming growth factor-beta1 of rabbit knee articular chondrocytes

    Institute of Scientific and Technical Information of China (English)

    马勇; 郭杨; 屠娟; 成吉华; 郭各朴; 黄桂成

    2014-01-01

    BACKGROUND:Studies have shown that Clematis chinensis Osbeck can promote articular chondrocyte proliferation, maintain and promote the synthesis of cartilage proteoglycan and type II col agen in chondrocytes. Low-intensity pulsed ultrasound can effectively improve the carticular chondrocyte proliferation, increase the cellmembrane permeability, and promote chemicals or genes delivery, so as to improve the biological effects of chemicals. OBJECTIVE:To observe the effect of low-intensity pulsed ultrasound mediated Clematis chinensis Osbeck on the proliferation of cartilage cells and the expression of type II col agen and transforming growth factor (TGF)-β1 of rabbit knee articular chondrocytes cultured in vitro, and explore the effect and mechanism of Clematis chinensis Osbeck mediated by low-intensity pulsed ultrasound on cartilage damage. METHODThe rabbit knee articular chondrocytes were isolated and cultured in vitro. cells of the second generation in logarithmic growth phase were randomly divided into four groupcontrol group, low-intensity pulsed ultrasound (LIPUS) group, Clematis chinensis Osbeck (CCO) group, and CCO+LIPUS group. After cells were cultured under different conditions for 3 days, cellproliferation was measured with cellCount Kit-8 assay. The secretion of type II col agen was detected by cytochemical staining method, and the expression of TGF-β1 was assayed by western blot analysis. RESULTS AND CONCLUSION:The number of cells in the other three groups were significantly higher than that in control group at 3 days after culture (P  目的:观察低强度脉冲超声介导威灵仙对体外培养的兔膝关节软骨细胞增殖、Ⅱ型胶原及转化生长因子β1表达的影响,探讨低强度脉冲超声介导威灵仙在软骨损伤修复中的作用及机制。  方法:体外分离培养兔膝关节软骨细胞,取处于对数生长期的2代细胞,随机分为4组:对照组,低强度脉冲超声组,威灵仙组,威

  13. Early efficacy study of matrix-induced autologous chondrocyte implantation repairing knee joint cartilage injury%基质诱导自体软骨细胞移植修复膝关节软骨损伤的早期疗效

    Institute of Scientific and Technical Information of China (English)

    王庆; 黄华扬; 张涛; 郑小飞; 李凭跃; 沈洪园; 陈加荣

    2016-01-01

    目的:探讨基质诱导自体软骨细胞移植修复膝关节软骨损伤的可行性及早期疗效。方法回顾性分析2012年4月至2013年3月13例单侧膝关节局灶性软骨缺损患者资料,男11例,女2例;年龄19~37岁,平均27.5岁;膝关节软骨缺损面积2.3~7.5 cm2,平均4.2 cm2;国际软骨损伤修复协会(ICRS )分级为Ⅲ级3例,Ⅳ级10例,均出现膝关节疼痛症状[视觉模拟评分(visual analogue scale, VAS)>3分]。13例患者均使用基质诱导软骨细胞移植技术进行软骨细胞移植。术后进行规范化功能康复锻炼。结果术后随访1年,1例患者因术后6.5个月下楼梯时扭伤膝关节致半月板损伤行关节镜下半月板修补术而剔除该患者术后12个月的评分,以避免结果偏倚。膝关节活动度,术后3个月(123.1°±8.0°)较术前(135.4°±5.7°)减少,膝关节损伤和骨关节炎评分(knee injury and use osteoarthritis outcome score, KOOS)的5个子集均较术前降低,Lysholm评分[(65.7±9.4)分]较术前[(71.2±12.3)分]无明显变化,国际膝关节评分委员会评分(International Knee Documentation Committee, IKDC)[(26.1±3.9)分]较术前[(43.5±6.5)分]减少;术后6、12个月的膝关节活动度(136.1°±6.1°、135.1°±3.6°)、Lysholm评分[(80.6±9.6)分、(86.6±9.2)分]、IKDC评分[(53.3±5.8)分、(62.8±7.2)分]、KOOS评分均较术前明显提高。术后12个月软骨修复组织磁共振评分[(73.3±17.9)分]较术前[(51.5±12.6)分]明显提高。结论基质诱导自体软骨细胞移植技术可有效修复膝关节软骨损伤,改善膝关节功能,具有良好的近期疗效。%Objective To study the feasibility and early efficacy of matrix⁃induced autologous chondrocyte implantation repairing knee joint cartilage injury. Methods The Matrix⁃induced autologous chondrocyte implantation was used to repair knee joint

  14. Notochordal cells maintain the proliferation and phenotype of chondrocyte-like cells in the disc nucleus pulposus%脊索细胞维持椎间盘髓核软骨样细胞增殖与表型的研究进展

    Institute of Scientific and Technical Information of China (English)

    杨哲; 李树文

    2016-01-01

    BACKGROUND:The immature disc nucleus pulposus is composed of notochordal cels, but there is no notochordal cel in the mature human intervertebral disc, in which the notochordal cels are replaced by chondrocyte-like cels. It is very important to comprehend the disappearance of the notochordal cels; however, it is stil unknown at present. OBJECTIVE: To elaborate the feasibility of notochordal cels to maintain the proliferation and phenotype of chondrocyte-like cels and to induce the cartilage-like differentiation of bone marrow mesenchymal stem cels. METHODS: The first author used the computer to retrieve PubMed and Wanfang databases using the key words of “notochord cels; nucleus pulposus cels; identify” in English and Chinese, respectively. Totaly 9 896 relevant articles published from January 1999 to August 2015 were retrieved. Repetitive studies were excluded, and finaly 36 articles were in accordance with the inclusion criteria. RESULTS AND CONCLUSION:Now, the main functions of notochordal cels are to promote synthesis of extracelular matrix in the nucleus pulposus, induce directional differentiation of mesenchymal cels into nucleus pulposus cels or act as “seed cels” to form the nucleus pulposus cels. The presence and disappearance of notochordal cels is related to intervertebral disc degeneration. Cel apoptosis is involved in static compressionviadeath receptor signals, and then leads to intervertebral disc degeneration. fas ligand can mediate the reduction of notochordal cels, and hypoxia-inducible factor can induce spinal cord injury thereby triggering cel death and complete disappearance of nucleus pulposus. The measurement and verification of immune makers of notochordal cels, CK-8, CK-18 and galectin-3, can benefit to the identification and isolation of notochordal cels, and thereby help the studies on cel growth and differentiation, function and its mechanism of apoptosis.%背景:未成熟的椎间盘髓核是由脊索细胞所组成,但在

  15. 成纤维细胞生长因子和胰岛素促小鼠软骨细胞增殖分化的效应%Effect of basic fibroblast growth factor and insulin on the proliferation and differentiation of mouse chondrocytes

    Institute of Scientific and Technical Information of China (English)

    史剑波; 江逊; 狄静芳; 许庚; 崔蕴霞

    2005-01-01

    背景:根据软骨为单一类型细胞组成的无血管组织,对氧和营养的供应要求较低,同种免疫性较低,体内功能较简单,易于在体外构建成可供移植的人工移植细胞系等特点,从建立用于组织工程的通用人类同种软骨细胞系着手,为人工组织和人工器官走向标准化和批量生产奠定基础.目的:探讨碱性成纤维细胞生长因子(basic fibroblast growth factor,bFGF)和胰岛素在原代培养软骨细胞的增殖过程中的效应,为组织工程中细胞因子的应用与作用进行评估.设计:以细胞为研究对象,分组对照重复观察测量.单位:一所大学的组织移植与免疫实验室.材料:实验于2002-11/2003-05在暨南大学组织移植与免疫教育部重点实验室完成,研究对象为随机获取的培养的软骨细胞.方法:在低血清培养基培养条件下进行小鼠原代软骨细胞培养,采用WST1比色法和荧光染色法观察不同浓度bFGF和胰岛素对软骨细胞增殖的影响.主要观察指标:主要结局:①bFGF对小鼠原代软骨细胞增殖影响.②胰岛素对小鼠原代软骨细胞增殖的影响.次要结局:软骨细胞形态学观察.结果:原代软骨细胞在4 g/L胎牛血清培养基培养条件下,不同浓度的bFGF和胰岛素对软骨细胞的增殖有剂量相关性.形态学观察表明,bFGF和胰岛素不仅促进细胞增殖,而且可使细胞分泌基质增多.结论:胰岛素和bFGF均能促进软骨细胞的增殖.%BACKGROUND: Based on the characteristics of cartilage tissue, such as consisting of single type of cells, the cartilage cells or chondrocyte, absence of blood vessel, rather low consumption level of oxygen and nutrition, low level of allo-immunocompetence and simple function in vivo, it seems to be easy for cartilage cell lines to be established for tissue and cell transplantation. We want to set up a cell line with the purpose of current use in tissue engineering in vitro. It will provide the basis

  16. Chondrocyte Cultivationin Hyaluronan-Tyramine Cross-Linked Hydrogel

    Czech Academy of Sciences Publication Activity Database

    Kučera, L.; Weinfurterová, R.; Dvořáková, J.; Kučera, J.; Pravda, M.; Foglarová, M.; Svik, K.; Klein, P.; Velebný, V.; Kubala, Lukáš

    2015-01-01

    Roč. 64, č. 13 (2015), s. 661-674. ISSN 0091-4037 Institutional support: RVO:68081707 Keywords : Biocompatibility * hyaluronate * scaffold Subject RIV: BO - Biophysics Impact factor: 3.568, year: 2014

  17. Loss of the Mammalian DREAM Complex Deregulates Chondrocyte Proliferation

    OpenAIRE

    Forristal, Chantal; Henley, Shauna A; MacDonald, James I.; Bush, Jason R.; Ort, Carley; Daniel T. Passos; Talluri, Srikanth; Ishak, Charles A.; Thwaites, Michael J.; Norley, Chris J.; Litovchick, Larisa; DeCaprio, James A.; DiMattia, Gabriel; Holdsworth, David W.; Beier, Frank

    2014-01-01

    Mammalian DREAM is a conserved protein complex that functions in cellular quiescence. DREAM contains an E2F, a retinoblastoma (RB)-family protein, and the MuvB core (LIN9, LIN37, LIN52, LIN54, and RBBP4). In mammals, MuvB can alternatively bind to BMYB to form a complex that promotes mitotic gene expression. Because BMYB-MuvB is essential for proliferation, loss-of-function approaches to study MuvB have generated limited insight into DREAM function. Here, we report a gene-targeted mouse model...

  18. On the evolutionary relationship between chondrocytes and osteoblasts

    OpenAIRE

    Gómez-Picos, Patsy; Eames, B. Frank

    2015-01-01

    Vertebrates are the only animals that produce bone, but the molecular genetic basis for this evolutionary novelty remains obscure. Here, we synthesize information from traditional evolutionary and modern molecular genetic studies in order to generate a working hypothesis on the evolution of the gene regulatory network (GRN) underlying bone formation. Since transcription factors are often core components of GRNs (i.e., kernels), we focus our analyses on Sox9 and Runx2. Our argument centers on ...

  19. On the evolutionary relationship between chondrocytes and osteoblasts

    OpenAIRE

    Patsy eGomez-Picos; B Frank eEames

    2015-01-01

    Vertebrates are the only animals that produce bone, but the molecular genetic basis for this evolutionary novelty remains obscure. Here, we synthesize information from traditional evolutionary and modern molecular genetic studies in order to generate a working hypothesis on the evolution of the gene regulatory network (GRN) underlying bone formation. To make this argument, we focus on three skeletal tissues that comprise the majority of the vertebrate skeleton: immature cartilage, mature cart...

  20. Effects of three kinds of nonsteroidal anti-inflammatory drugs on expressions of IL-1β,TNF-α and IL-1α in osteoarthritic chondrocytes of Wistar rats%3种非甾体抗炎药对大鼠骨关节炎关节软骨细胞IL-1β、TNF-α、IL-1α表达的影响

    Institute of Scientific and Technical Information of China (English)

    欧云生; 安洪; 谭超; 刘显宏

    2013-01-01

    目的:探讨长期应用塞来昔布、布洛芬、吲哚美辛对大鼠骨关节炎(osteoarthritis,OA)关节软骨细胞白细胞介素-1β(interleukin-1β,IL-1β)、肿瘤坏死因子-α(tumor necrosis factor,TNF-α)、细胞白细胞介素-1 α(interleukin-1α,IL-1α)表达的影响.方法:采用左侧跟腱切除术建立Wistar大鼠同侧膝关节OA模型,分4组,每日给赛来昔布24 mg/kg(celecoxib,CE)组、布洛芬72 mg/kg(ibuprofen,IBP)组、吲哚美辛9 mg/kg(indomethacin,IN)组及生理盐水(normal saline,NS)组.用免疫组织化学法观察3、6、9个月后关节软骨细胞IL-1β、TNF-α、IL-1α表达的变化.结果:①连续用药3月,CE对软骨细胞IL-1β、TNF-α表达无明显影响,抑制IL-1α的表达;IBP促进软骨细胞IL-1β、TNF-α的表达,对IL-1α的表达无明显影响;IN轻度促进软骨细胞IL-1β表达,增强TNF-α的表达,对IL-1α的表达无明显影响.②连续用药6月,CE对软骨细胞IL-1β表达无明显影响,轻度抑制TNF-α、IL-1α表达;IBP轻度促进软骨细胞IL-1β的表达,促进TNF-α表达,轻度抑制IL-1α的表达;IN轻度抑制软骨细胞IL-1β,抑制TNF-α表达,轻度抑制IL-1α的表达.③连续用药9月,CE明显抑制软骨细胞IL-1β的表达,抑制TNF-α、IL-1α的表达;IBP抑制软骨细胞IL-1β、TNF-α的表达,轻度抑制IL-1α的表达.结论:CE抑制软骨细胞IL-1β、TNF-α、IL-1α的表达,可减轻OA关节软骨的损害,IBP、IN在不同时期对软骨细胞IL-1β、TNF-α、IL-1α表达的影响有所不同,其总体抑制效应不明显.%Objective:To explore the effects of celecoxib, ibuprofen and indomethcin on expressions of interleukin-1β(IL-1β) ,tumor necrosis factor-α (TNF-α) and interleukin-lα (IL-lα) in osteoarthritic chondrocytes of Wistar rats. Methods:One hundred and thirty Wistar rats were randomly divided into 4 groups; celecoxib (CE) group, ibuprofen (IBP) group, indomethacin group(IN) and normal saline(NS) group. Osteoarthritis

  1. Glycoconjugate expression of chondrocytes and perichondrium during hyaline cartilage development in the rat.

    OpenAIRE

    Zschäbitz, A; Krahn, V; Gabius, H J; Weiser, H; Khaw, A; Biesalski, H. K.; Stofft, E

    1995-01-01

    Alterations in the expression of glycoconjugate structures during cartilage development in the chondrocranium, nasal skeleton, Meckel's cartilage, limb buds, vertebral bodies and ribs were investigated comparatively in 13 to 21-d-old rat embryos. The binding patterns of 24 biotinylated lectins were analysed in serial sections and compared with results obtained using histochemical methods. Proteoglycan distribution, assessed by conventional staining procedures, was not associated with lectin b...

  2. Exosomes from IL-1β stimulated synovial fibroblasts induce osteoarthritic changes in articular chondrocytes

    OpenAIRE

    Kato, Tomohiro; Miyaki, Shigeru; Ishitobi, Hiroyuki; Nakamura, Yoshihiro; Nakasa, Tomoyuki; Lotz, Martin K.; Ochi, Mitsuo

    2014-01-01

    Introduction Osteoarthritis (OA) is a whole joint disease, and characterized by progressive degradation of articular cartilage, synovial hyperplasia, bone remodeling and angiogenesis in various joint tissues. Exosomes are a type of microvesicles (MVs) that may play a role in tissue-tissue and cell-cell communication in homeostasis and diseases. We hypothesized that exosomes function in a novel regulatory network that contributes to OA pathogenesis and examined the function of exosomes in comm...

  3. A chemically-defined protocol for generating chondrocytes from human embryonic stem cells

    OpenAIRE

    sprotocols

    2015-01-01

    Progress in generating robust differentiation protocols for efficient and scalable production of defined cell lineages from human embryonic stem cells (hESc) has been slow. Amongst the obstacles to be addressed are those inherent to standard hESc culture and differentiation practices including the use of feeder cells, serum and animal-derived matrices. These components are biologically complex, undefined and highly variable between batches, inhibiting the development of consistently reproduci...

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

    OpenAIRE

    Wang ZH; Li XL; He XJ; Zhang XH; Yang ZQ; Xu M; Wu BJ; Tu JB; Luo HN; Yan J

    2014-01-01

    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 Jiaot...

  5. Immobilization of thrombocytes on PCL nanofibres enhances chondrocyte proliferation in vitro

    Czech Academy of Sciences Publication Activity Database

    Jakubová, Radka; Míčková, Andrea; Buzgo, M.; Rampichová, Michala; Prosecká, Eva; Tvrdík, D.; Amler, Evžen

    2011-01-01

    Roč. 44, č. 2 (2011), s. 183-191. ISSN 0960-7722 R&D Projects: GA ČR GAP304/10/1307; GA AV ČR IAA500390702; GA MŠk 7E09088 Grant ostatní: GA MŠk(CZ) 2B06130; GA MŠk(CZ) 1M0510; GA ČR(CZ) GA202/09/1151; GA MŠk(CZ) GA UK119009; GA MŠk(CZ) GAUK96610; GA MŠk(CZ) GAUK119209; GA MŠk(CZ) GAUK97110; GA MŠk(CZ) GAUK80009 Institutional research plan: CEZ:AV0Z50390512; CEZ:AV0Z50390703 Keywords : polycaprolactone * mesenchymal stem cells * matrix Subject RIV: BO - Biophysics Impact factor: 2.521, year: 2011

  6. Accumulation of advanced glycation end products decreases collagen turnover by bovine chondrocytes

    NARCIS (Netherlands)

    Groot, J. de; Verzijl, N.; Budde, M.; Bijlsma, J.W.J.; Lafeber, F.P.J.G.; TeKoppele, J.M.

    2001-01-01

    The integrity of the collagen network is essential for articular cartilage to fulfill its function in load support and distribution. Damage to the collagen network is one of the first characteristics of osteoarthritis. Since extensive collagen damage is considered irreversible, it is crucial that ch

  7. Tritiated thymidine uptake in chondrocytes of chickens afflicted with tibial dyschondroplasia

    International Nuclear Information System (INIS)

    3H-Thymidine was localized in sections of growth-plate cartilage and associated tibial dyschondroplastic lesion by autoradiography. One hour after 3H-thymidine was injected, radioactivity was found in the proliferating zone; after 48 hr it was also in the hypertrophic zone, and by 96 hr it was present in cells that were 4 to 5 mm into the lesion. This indicates that the lesion develops from the growth plate itself. The life span of the cells in the growth plate appears to be about 48 hr

  8. Effects of diadenosine tetraphosphate on FGF9-induced chloride flux changes in achondroplastic chondrocytes

    OpenAIRE

    Huete, Fernando; Guzman-Aranguez, Ana; Ortín, Javier; Hoyle, Charles H. V.; Pintor, Jesús

    2011-01-01

    Achondroplasia, the most common type of dwarfism, is characterized by a mutation in the fibroblast growth factor receptor 3 (FGFR3). Achondroplasia is an orphan pathology with no pharmacological treatment so far. However, the possibility of using the dinucleotide diadenosine tetraphosphate (Ap4A) with therapeutic purposes in achondroplasia has been previously suggested. The pathogenesis involves the constitutive activation of FGFR3, resulting in altered biochemical and physiological processes...

  9. Interleukin-1 induced nitric oxide inhibits sulphation of glycosaminoglycan chains in human articular chondrocytes.

    Science.gov (United States)

    Hickery, M S; Bayliss, M T

    1998-10-23

    Incubation of human articular cartilage explants with interleukin-1alpha (IL-1alpha) inhibited the rate of [35S]sulphate incorporation into glycosaminoglycan (GAG) chains concomitant with an increase in nitric oxide (NO) production. Measurement of the [35S]sulphate showed that IL-1alpha inhibited the synthesis of both keratan sulphate and chondroitin sulphate (CS) chains to a similar extent. This effect was reversed by the NO synthase inhibitor Nomega-iminoethyl-l-ornithine (l-NIO). Analysis of alkali borohydride cleaved GAG chains showed that IL-1alpha had no effect on their size. Similarly when GAG chains were coupled to xyloside the size of the GAG chains attached to the exogenous acceptor decreased but IL-1alpha had no further effect on hydrodynamic size. IL-1alpha did, however, inhibit [35S]sulphate incorporation into xyloside-linked CS chains. In both experiments l-NIO reversed the inhibitory effect on sulphation. Disaccharide analysis of the [35S]GAG chains showed that IL-1alpha preferentially inhibited sulphation of the 6-sulphated isomer and that l-NIO reversed this effect. Thus, IL-1alpha-induced NO mediates the inhibition of sulphate incorporation and alters the sulphation pattern of newly synthesised GAG chains. PMID:9795242

  10. Chondrocytic potential of allogenic mesenchymal stem cells transplanted without immunosuppression to regenerate physeal defect in rabbits

    Czech Academy of Sciences Publication Activity Database

    Gál, P.; Nečas, A.; Plánka, L.; Kecová, H.; Křen, L.; Krupa, P.; Hlučilová, Jana; Usvald, Dušan

    2007-01-01

    Roč. 76, - (2007), s. 265-275. ISSN 0001-7213 R&D Projects: GA MŠk 2B06130 Grant ostatní: MZd(CZ) NR8483 Institutional research plan: CEZ:AV0Z50450515 Keywords : growth plate injury * bone bridge * limb deformity Subject RIV: GJ - Animal Vermins ; Diseases, Veterinary Medicine Impact factor: 0.687, year: 2007

  11. Nanotechnology and mesenchymal stem cells with chondrocytes in prevention of partial growth plate arrest in pigs

    Czech Academy of Sciences Publication Activity Database

    Plánka, L.; Srnec, R.; Rauser, P.; Starý, D.; Filová, Eva; Jančář, J.; Juhásová, Jana; Křen, J.; Nečas, A.; Gál, P.

    2012-01-01

    Roč. 156, č. 2 (2012), s. 128-134. ISSN 1213-8118 R&D Projects: GA MZd(CZ) NS9896 Institutional research plan: CEZ:AV0Z50390512; CEZ:AV0Z50450515 Institutional support: RVO:68378041 ; RVO:67985904 Keywords : mesenchymal stem cells * growth plate defect * bone bridge Subject RIV: FI - Traumatology, Orthopedics Impact factor: 0.990, year: 2012

  12. Snorc is a novel cartilage specific small membrane proteoglycan expressed in differentiating and articular chondrocytes

    DEFF Research Database (Denmark)

    Heinonen, J; Taipaleenmäki, H; Roering, P; Takatalo, M; Harkness, L; Sandholm, J; Uusitalo-Järvinen, H; Kassem, M; Kiviranta, I; Laitala-Leinonen, T; Säämänen, A-M

    2011-01-01

    expressed in Cos7 cells, and the cell lysate was studied for putative glycosaminoglycan attachment by digestion with chondroitinase ABC and Western blotting. RESULTS: The predicted molecule is a small, 121 amino acids long type I single-pass transmembrane chondroitin sulfate proteoglycan, that contains ER...... signal peptide, lumenal/extracellular domain with several threonines/serines prone to O-N-acetylgalactosamine modification, and a cytoplasmic tail with a Ying-yang site prone to phosphorylation or O-N-acetylglucosamine modification. It is highly conserved in mammals with orthologs in all vertebrate...... models demonstrated similar expression profiles with Sox9, Acan and Col2a1 and up-regulation by BMP-2. Based on its cartilage specific expression, the molecule was named Snorc, (Small NOvel Rich in Cartilage). CONCLUSION: A novel cartilage specific molecule was identified which marks the differentiating...

  13. Vitamin D receptors in the rheumatoid lesion: expression by chondrocytes, macrophages, and synoviocytes

    OpenAIRE

    Tetlow, L; Smith, S; Mawer, E; Woolley, D

    1999-01-01

    OBJECTIVES—The active form of vitamin D3, 1α,25 dihydroxyvitamin D3 (1,25D3), through its interaction with vitamin D receptors (VDR), is reported to effect a variety of anabolic and catabolic events, especially in bone and cartilage tissues. As cartilage degradation and tissue remodelling are characteristic features of the rheumatoid lesion, the distribution and expression of VDR at sites of cartilage erosion was examined.
METHODS—Immunolocalisation techniques using a rat monoclonal antibody ...

  14. MicroRNA-23a-3p promotes the development of osteoarthritis by directly targeting SMAD3 in chondrocytes.

    Science.gov (United States)

    Kang, Liang; Yang, Cao; Song, Yu; Liu, Wei; Wang, Kun; Li, Shuai; Zhang, Yukun

    2016-09-01

    Osteoarthritis (OA) is a common chronic degenerative joint disease. Progressive destruction of the integrity of articular cartilage is an important pathological feature, but treatment options that reverse this damage have not been developed. According to recent studies, microRNAs have important regulatory roles in the initiation and progression of OA. In the current study, the biological effects of miR-23a-3p and its expression in OA tissues were examined. We found that miR-23a-3p expression was obviously higher and SMAD3 expression was significantly lower in OA cartilage than in normal tissues. The hypomethylation status of CpG islands in the promoter region of miR-23a-3p was confirmed by methylation-specific polymerase chain reaction in OA cartilage tissues. Furthermore, a bioinformatics analysis and luciferase reporter assay identified SMAD3 as a target gene of miR-23a-3p and SMAD3 expression at both the protein and mRNA levels was inhibited by miR-23a-3p. A functional analysis demonstrated that miR-23a-3p overexpression suppresses type II collagen and aggrecan expression, while miR-23a-3p inhibition had the opposite effects. Small interfering RNA-mediated knockdown of SMAD3 reversed the effects of the miR-23a-3p inhibitor on the expression of type II collagen and aggrecan. Our results suggested that miR-23a-3p contributes to OA progression by directly targeting SMAD3, providing a potential therapeutic target for OA treatment. PMID:27318087

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

    OpenAIRE

    He X; Feng B; Huang C; Wang H; Ge Y; Hu R; Yin M; Xu Z.; Wang W; Fu W.; Zheng J

    2015-01-01

    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: Electrospinnin...

  16. Fibroblast growth factor inhibits interferon gamma-STAT1 and interleukin 6-STAT3 signaling in chondrocytes

    Czech Academy of Sciences Publication Activity Database

    Krejčí, Pavel; Procházková, Jiřina; Bryja, Vítězslav; Jelínková, P.; Pejchalová, K.; Kozubík, Alois; Thompson, L.M.; Wilcox, W.R.

    2009-01-01

    Roč. 21, č. 1 (2009), s. 151-160. ISSN 0898-6568 Institutional research plan: CEZ:AV0Z50040507; CEZ:AV0Z50040702 Keywords : fibroblast growth factor * cartilage * STAT1 Subject RIV: BO - Biophysics Impact factor: 4.094, year: 2009

  17. Targeted overexpression of parathyroid hormone-related peptide in chondrocytes causes chondrodysplasia and delayed endochondral bone formation.

    OpenAIRE

    Weir, E C; Philbrick, W M; Amling, M.; Neff, L A; Baron, R; Broadus, A E

    1996-01-01

    Parathyroid hormone-related peptide (PTHrP) was initially identified as a product of malignant tumors that mediates paraneoplastic hypercalcemia. It is now known that the parathyroid hormone (PTH) and PTHrP genes are evolutionarily related and that the products of these two genes share a common receptor, the PTH/PTHrP receptor. PTHrP and the PTH/PTHrP receptor are widely expressed in both adult and fetal tissues, and recent gene-targeting and disruption experiments have implicated PTHrP as a ...

  18. Lentiviral-mediated RNAi targeting caspase-3 inhibits apoptosis induced by serum deprivation in rat endplate chondrocytes in vitro

    International Nuclear Information System (INIS)

    Current studies find that degenerated cartilage endplates (CEP) of vertebrae, with fewer diffusion areas, decrease nutrient supply and accelerate intervertebral disc degeneration. Many more apoptotic cells have been identified in degenerated than in normal endplates, and may be responsible for the degenerated grade. Previous findings suggest that inhibition of apoptosis is one possible approach to improve disc regeneration. It is postulated that inhibition of CEP cell apoptosis may be responsible for the regeneration of endplates. Caspase-3, involved in the execution phase of apoptosis, is a candidate for regulating the apoptotic process. In the present study, CEP cells were incubated in 1% fetal bovine serum. Activated caspases were detected to identify the apoptotic pathway, and apoptosis was quantified by flow cytometry. Lentiviral caspase-3 short hairpin RNA (shRNA) was employed to study its protective effects against serum deprivation. Silencing of caspase-3 expression was quantified by reverse transcription-polymerase chain reaction and Western blots, and inhibition of apoptosis was quantified by flow cytometry. Serum deprivation increased apoptosis of rat CEP cells through activation of a caspase cascade. Lentiviral caspase-3 shRNA was successfully transduced into CEP cells, and specifically silenced endogenous caspase-3 expression. Surviving cells were protected by the downregulation of caspase-3 expression and activation. Thus, lentiviral caspase-3 shRNA-mediated RNAi successfully silenced endogenous caspase-3 expression, preventing inappropriate or premature apoptosis

  19. The paradox of FGFR3 signaling in skeletal dysplasia: Why chondrocytes growth arrest while other cells over proliferate

    Czech Academy of Sciences Publication Activity Database

    Krejčí, Pavel

    2014-01-01

    Roč. 759, JAN2014 (2014), s. 40-48. ISSN 1383-5742 Institutional support: RVO:68081707 Keywords : ONCOGENE-INDUCED SENESCENCE * FACIO-CUTANEOUS SYNDROME * ENDOCHONDRAL BONE-FORMATION Subject RIV: BO - Biophysics Impact factor: 6.213, year: 2014

  20. Lentiviral-mediated RNAi targeting caspase-3 inhibits apoptosis induced by serum deprivation in rat endplate chondrocytes in vitro

    Directory of Open Access Journals (Sweden)

    L. Ding

    2014-06-01

    Full Text Available Current studies find that degenerated cartilage endplates (CEP of vertebrae, with fewer diffusion areas, decrease nutrient supply and accelerate intervertebral disc degeneration. Many more apoptotic cells have been identified in degenerated than in normal endplates, and may be responsible for the degenerated grade. Previous findings suggest that inhibition of apoptosis is one possible approach to improve disc regeneration. It is postulated that inhibition of CEP cell apoptosis may be responsible for the regeneration of endplates. Caspase-3, involved in the execution phase of apoptosis, is a candidate for regulating the apoptotic process. In the present study, CEP cells were incubated in 1% fetal bovine serum. Activated caspases were detected to identify the apoptotic pathway, and apoptosis was quantified by flow cytometry. Lentiviral caspase-3 short hairpin RNA (shRNA was employed to study its protective effects against serum deprivation. Silencing of caspase-3 expression was quantified by reverse transcription-polymerase chain reaction and Western blots, and inhibition of apoptosis was quantified by flow cytometry. Serum deprivation increased apoptosis of rat CEP cells through activation of a caspase cascade. Lentiviral caspase-3 shRNA was successfully transduced into CEP cells, and specifically silenced endogenous caspase-3 expression. Surviving cells were protected by the downregulation of caspase-3 expression and activation. Thus, lentiviral caspase-3 shRNA-mediated RNAi successfully silenced endogenous caspase-3 expression, preventing inappropriate or premature apoptosis.

  1. Glucose transport and metabolism in chondrocytes: a key to understanding chondrogenesis, skeletal development and cartilage degradation in osteoarthritis

    OpenAIRE

    A. Mobasheri; Vannucci, S.J.; Bondy, C A; Carter, S D; Innes, J.F.; Arteaga, M F; E. Trujillo; Ferraz, I; Shakibaei, M.; Martín Vasallo, P.

    2002-01-01

    Despite the recognition that degenerative cartilage disorders like osteoarthritis (OA) and osteochondritis dissecans (OCD) may have nutritional abnormalities at the root of their pathogenesis, balanced dietary supplementation programs have played a secondary role in their management. This review emphasizes the importance and role of nutritional factors such as glucose and glucose-derived sugars (i.e. glucosamine sulfate and vitamin C) in the development, ma...

  2. Lentiviral-mediated RNAi targeting caspase-3 inhibits apoptosis induced by serum deprivation in rat endplate chondrocytes in vitro

    Energy Technology Data Exchange (ETDEWEB)

    Ding, L.; Wu, J.P. [Fudan University, Jinshan Hospital, Department of Orthopaedics, Shanghai, China, Department of Orthopaedics, Jinshan Hospital, Fudan University, Shanghai (China); Xu, G. [Fudan University, Jinshan Hospital, Center Laboratory, Shanghai, China, Center Laboratory, Jinshan Hospital, Fudan University, Shanghai (China); Zhu, B.; Zeng, Q.M.; Li, D.F.; Lu, W. [Fudan University, Jinshan Hospital, Department of Orthopaedics, Shanghai, China, Department of Orthopaedics, Jinshan Hospital, Fudan University, Shanghai (China)

    2014-05-09

    Current studies find that degenerated cartilage endplates (CEP) of vertebrae, with fewer diffusion areas, decrease nutrient supply and accelerate intervertebral disc degeneration. Many more apoptotic cells have been identified in degenerated than in normal endplates, and may be responsible for the degenerated grade. Previous findings suggest that inhibition of apoptosis is one possible approach to improve disc regeneration. It is postulated that inhibition of CEP cell apoptosis may be responsible for the regeneration of endplates. Caspase-3, involved in the execution phase of apoptosis, is a candidate for regulating the apoptotic process. In the present study, CEP cells were incubated in 1% fetal bovine serum. Activated caspases were detected to identify the apoptotic pathway, and apoptosis was quantified by flow cytometry. Lentiviral caspase-3 short hairpin RNA (shRNA) was employed to study its protective effects against serum deprivation. Silencing of caspase-3 expression was quantified by reverse transcription-polymerase chain reaction and Western blots, and inhibition of apoptosis was quantified by flow cytometry. Serum deprivation increased apoptosis of rat CEP cells through activation of a caspase cascade. Lentiviral caspase-3 shRNA was successfully transduced into CEP cells, and specifically silenced endogenous caspase-3 expression. Surviving cells were protected by the downregulation of caspase-3 expression and activation. Thus, lentiviral caspase-3 shRNA-mediated RNAi successfully silenced endogenous caspase-3 expression, preventing inappropriate or premature apoptosis.

  3. The Effects of Extracellular Matrix on Tissue Engineering Construction of Cartilage in Vitro

    Institute of Scientific and Technical Information of China (English)

    YU Li; LI Fa-tao; TANG Ming-qiao; YAN Wei-qun

    2006-01-01

    The effects of various cartilage extracellular matrix on the construction of rabbit growth plate cartilage tissue in vitro were studied. The results show that collagen, proteoglycan and hyaluronic acid can promote the growth of cultured chondrocytes but the effects of various cartilage extracellular matrix(ECM)on chondrocyte differentiation are different. Collagen can promote the hypertrophy of chondrocytes while proteoglycan and hyaluronic acid inhibit the transition of mature chondrocytes into hypertrophied chondrocytes.

  4. Smurf2 Induces Degradation of GSK-3β and Upregulates β-Catenin in Chondrocytes: A Potential Mechanism for Smurf2-Induced Degeneration of Articular Cartilage

    OpenAIRE

    Wu, Qiuqian; Jason H Huang; Sampson, Erik R.; Kim, Kyung-Ok; Zuscik, Michael J.; O’Keefe, Regis J.; Chen, Di; Rosier, Randy N.

    2009-01-01

    We have previously demonstrated that Smurf2 is highly expressed in human osteoarthritis (OA) tissue, and overexpression of Smurf2 under the control of the type II collagen promoter (Col2a1) induces an OA-like phenotype in aged Col2a1-Smurf2 transgenic mice, suggesting that Smurf2 is located upstream of a signal cascade which initiates OA development. However, the factors downstream of Smurf2 in this signal cascade and how Smurf2-induced OA is initiated are largely unknown. In this study, we f...

  5. Fuyuan Decoction Enhances SOX9 and COL2A1 Expression and Smad2/3 Phosphorylation in IL-1β-Activated Chondrocytes

    OpenAIRE

    Yudi Zhang; Rongheng Li; Yu Zhong; Sihan Zhang; Lingyun Zhou; Shike Shang

    2015-01-01

    Fuyuan Decoction (FYD), a herbal formula in China, has been widely used for osteoarthritis (OA) treatment. Herein, we determined the effects of FYD on the expression of transcription factor SOX9 and its target gene collagen type II, alpha 1 (COL2A1) as well as the activation of Smad2/3 in interleukin- (IL-) 1β-stimulated SW1353 chondrosarcoma cells. Serum-derived FYD (FYD-CS) was prepared to treat SW1353 cells with or without SB431542, a TGF-β1 receptor inhibitor. Cell cycle progression was t...

  6. Assessment of cartilage repair after chondrocyte transplantation with a fibrin-hyaluronan matrix – Correlation of morphological MRI, biochemical T2 mapping and clinical outcome

    International Nuclear Information System (INIS)

    Objective: To evaluate change over time of clinical scores, morphological MRI of cartilage appearance and quantitative T2 values after implantation with BioCart™II, a second generation matrix-assisted implantation system. Methods: Thirty-one patients were recruited 6–49 months post surgery for cartilage defect in the femoral condyle. Subjects underwent MRI (morphological and T2-mapping sequences) and completed the International Knee Documentation Committee (IKDC) questionnaire. MRI scans were scored using the MR Observation of Cartilage Repair Tissue (MOCART) system and cartilage T2-mapping values were registered. Analysis included correlation of IKDC scores, MOCART and T2 evaluation with each other, with implant age and with previous surgical intervention history. Results: IKDC score significantly correlated with MOCART score (r = −0.39, p = 0.031), inversely correlated with previous interventions (r = −0.39, p = 0.034) and was significantly higher in patients with longer follow-up time (p = 0.0028). MOCART score was slight, but not significantly higher in patients with longer term implants (p = 0.199). T2 values were significantly lower in patients with longer duration implants (p < 0.001). This trend was repeated in patients with previous interventions, although to a lesser extent. Conclusions: Significant improvement with time from BioCart™II implantation can be expected by IKDC scoring and MRI T2-mapping values. Patients with previous knee operations can also benefit from this procedure.

  7. A Comparison of the influence of material on in vitro cartilage tissue engineering with PCL, PGS, and POC 3D scaffold architecture seeded with chondrocytes

    OpenAIRE

    Jeong, Claire G.; Hollister, Scott J.

    2010-01-01

    The goal of this study was to determine material effects on cartilage regeneration for scaffolds with the same controlled architecture. The 3D polycaprolactone (PCL), poly (glycerol sebacate) (PGS), and poly (1,8 octanediol-co-citrate) (POC) scaffolds of the same design were physically characterized and tissue regeneration in terms of cell phenotype, cellular proliferation and differentiation, and matrix production were compared to find which material would be most optimal for cartilage regen...

  8. Dedifferentiated Human Articular Chondrocytes Redifferentiate to a Cartilage-Like Tissue Phenotype in a Poly(ε-Caprolactone)/Self-Assembling Peptide Composite Scaffold

    OpenAIRE

    Lourdes Recha-Sancho; Moutos, Franklin T.; Jordi Abellà; Farshid Guilak; Semino, Carlos E.

    2016-01-01

    Adult articular cartilage has a limited capacity for growth and regeneration and, with injury, new cellular or biomaterial-based therapeutic platforms are required to promote repair. Tissue engineering aims to produce cartilage-like tissues that recreate the complex mechanical and biological properties found in vivo. In this study, a unique composite scaffold was developed by infiltrating a three-dimensional (3D) woven microfiber poly (ε-caprolactone) (PCL) scaffold with the RAD16-I self-asse...

  9. Second-harmonic generation microscopy used to evaluate the effect of the dimethyl sulfoxide in the cryopreservation process in collagen fibers of differentiated chondrocytes

    Science.gov (United States)

    Andreoli-Risso, M. F.; Duarte, A. S. S.; Ribeiro, T. B.; Bordeaux-Rego, P.; Luzo, A.; Baratti, M. O.; Adur, J.; de Thomaz, A. A.; Pelegati, V. B.; Carvalho, H. F.; Cesar, C. L.; Kharmadayan, P.; Costa, F. F.; Olalla-Saad, S. T.

    2012-03-01

    Cartilaginous lesions are a significant public health problem and the use of adult stem cells represents a promising therapy for this condition. Cryopreservation confers many advantages for practitioners engaged in cell-based therapies. However, conventional slow freezing has always been associated with damage and mortality due to intracellular ice formation, cryoprotectant toxicity, and dehydration. The aim of this work is to observe the effect of the usual Dimethyl Sulfoxide (DMSO) cryopreservation process on the architecture of the collagen fiber network of chondrogenic cells from mesenchymal stem cells by Second Harmonic Generation (SHG) microscopy. To perform this study we used Mesenchymal Stem Cells (MSC) derived from adipose tissue which presents the capacity to differentiate into other lineages such as osteogenic, adipogenic and chondrogenic lineages. Mesenchymal stem cells obtained after liposuction were isolated digested by collagenase type I and characterization was carried out by differentiation of mesodermic lineages, and flow cytometry using specific markers. The isolated MSCs were cryopreserved by the DMSO technique and the chondrogenic differentiation was carried out using the micromass technique. We then compared the cryopreserved vs non-cryopreserved collagen fibers which are naturally formed during the differentiation process. We observed that noncryopreserved MSCs presented a directional trend in the collagen fibers formed which was absent in the cryopreserved MSCs. We confirmed this trend quantitatively by the aspect ratio obtained by Fast Fourier Transform which was 0.76 for cryopreserved and 0.52 for non-cryopreserved MSCs, a statistical significant difference.

  10. TCDD诱导软骨细胞细胞凋亡的研究%Studies On The Toxicity of TCDD on Apoptosis in Chondrocyte

    Institute of Scientific and Technical Information of China (English)

    赵玉红; 吴丽霞; 赵玉岩; 郭磊

    2007-01-01

    目的:探讨不同浓度的TCDD对体外培养软骨细胞程序性死亡过程的影响.方法:胰酶消化法体外培养兔的软骨细胞;软骨细胞株暴露于二(噁)英(TCDD)24 h(浓度为1×10-10~1×10-8mol/L);光镜和透射电镜观察软骨细胞株细胞形态变化;流式细胞仪检测软骨细胞株细胞凋亡率.结果:1×10-10~1×10-8mol/L的TCDD诱导软骨细胞出现核固缩、碎裂和凋亡小体,并随TCDD浓度的增加而更明显;TCDD诱导的软骨细胞凋亡呈剂量依赖效应,1×10-10~1×10-8mol/L浓度的TCDD作用24 h后,软骨细胞株凋亡率分别为11.75%±2.41%,19.5%±2.75%,27.5%±3.45%,对照组为5.21%±2.46%,差异有显著性(P<0.05).结论:TCDD可以诱导体外培养的软骨细胞发生细胞凋亡.

  11. A novel injectable thermoresponsive and cytocompatible gel of poly(N-isopropylacrylamide) with layered double hydroxides facilitates siRNA delivery into chondrocytes in 3D culture

    NARCIS (Netherlands)

    Yang, H.Y.; Ee, R.J. van; Timmer, K.; Craenmehr, E.G.M.; Huang, J.H.; Öner, C.; Dhert, W.J.A.; Kragten, A.H.M.; Willems, N.; Grinwis, G.C.M.; Tryfonidou, M.A.; Papen-Botterhuis, N.E.; Creemers, L.B.

    2015-01-01

    Hybrid hydrogels composed of poly(N-isopropylacrylamide) (pNIPAAM) and layered double hydroxides (LDHs) are presented in this study as novel injectable and thermoresponsive materials for siRNA delivery, which could specifically target several negative regulators of tissue homeostasis in cartilaginou

  12. Fisiopatología celular de la osteoartritis: el condrocito articular como protagonista = Osteoarthritis cellular pathophysiology: The articular chondrocyte as a central player

    Directory of Open Access Journals (Sweden)

    Sánchez Naranjo, Julio César

    2011-06-01

    Full Text Available La osteoartritis es una de las enfermedades más prevalentes y que más discapacidad produce en todo el mundo, lo que ocasiona costos altos para el paciente y la sociedad. En años recientes se ha venido obteniendo información importante sobre el funcionamiento normal del condrocito, la única célula presente en el cartílago articular y responsable de la síntesis de matriz extracelular. El condrocito responde a las condiciones fluctuantes del medio, generadas por los cambios de presión, modificando su composición iónica y alterando el transporte de solutos y agua en su membrana. Esta capacidad de respuesta es clave para el mantenimiento de la matriz extracelular y, por ende, de un cartílago funcional. Diversos factores relacionados con enfermedades crónicas metabólicas inician una cascada de eventos que termina con una respuesta inadecuada del condrocito ante la carga mecánica, lo cual lleva a un predominio del catabolismo de la matriz y a un cartílago defectuoso que es la base del desarrollo de la osteoartritis. En este proceso están implicadas diversas citocinas y hormonas que afectan la homeostasis del cartílago y que pueden constituirse en blancos terapéuticos prometedores.

  13. Steady-state diffusion imaging for MR in-vivo evaluation of reparative cartilage after matrix-associated autologous chondrocyte transplantation at 3 tesla-Preliminary results

    International Nuclear Information System (INIS)

    Objectives: To demonstrate the feasibility of time-reversed fast imaging with steady-state precession (FISP) called PSIF for diffusion-weighted imaging of cartilage and cartilage transplants in a clinical study. Material and Methods: In a cross-sectional study 15 patients underwent MRI using a 3D partially balanced steady-state gradient echo pulse sequence with and without diffusion weighting at two different time points after matrix-associated autologous cartilage transplantation (MACT). Mean diffusion quotients (signal intensity without diffusion-weighting divided by signal intensity with diffusion weighting) within the cartilage transplants were compared to diffusion quotients found in normal cartilage. Results: The global diffusion quotient found in repair cartilage was significantly higher than diffusion values in normal cartilage (p < 0.05). There was a decrease between the earlier and the later time point after surgery. Conclusions: In-vivo diffusion-weighted imaging based on the PSIF technique is possible. Our preliminary results show follow-up of cartilage transplant maturation in patients may provide additional information to morphological assessment

  14. Fisiopatología celular de la osteoartritis: el condrocito articular como protagonista = Osteoarthritis cellular pathophysiology: The articular chondrocyte as a central player

    OpenAIRE

    Sánchez Naranjo, Julio César; López Zapata, Diego Fernando

    2011-01-01

    La osteoartritis es una de las enfermedades más prevalentes y que más discapacidad produce en todo el mundo, lo que ocasiona costos altos para el paciente y la sociedad. En años recientes se ha venido obteniendo información importante sobre el funcionamiento normal del condrocito, la única célula presente en el cartílago articular y responsable de la síntesis de matriz extracelular. El condrocito responde a las condiciones fluctuantes del medio, generadas por los cambios de presión, modifican...

  15. Hyaluronan Oligosaccharides Induce Matrix Metalloproteinase 13 via Transcriptional Activation of NFκB and p38 MAP Kinase in Articular Chondrocytes*

    OpenAIRE

    Ohno, Shigeru; Im, Hee-Jeong; Knudson, Cheryl B.; Knudson, Warren

    2006-01-01

    Hyaluronan exerts a variety of biological effects on cells including changes in cell migration, proliferation, and matrix metabolism. However, the signaling pathways associated with the action of hyaluronan on cells have not been clearly defined. In some cells, signaling is induced by the loss of cell-hyaluronan interactions. The goal of this study was to use hyaluronan oligosaccharides as a molecular tool to explore the effects of changes in cell-hyaluronan interactions and determine the und...

  16. Fibroblast growth factor and canonical WNT/beta-catenin signaling cooperate in suppression of chondrocyte differentiation in experimental models of FGFR signaling in cartilage

    Czech Academy of Sciences Publication Activity Database

    Buchtová, Marcela; Oralová, Veronika; Aklian, A.; Mašek, J.; Veselá, I.; Ouyang, Z.; Obadalová, T.; Konečná, Ž.; Spoustová, T.; Pospíšilová, T.; Matula, P.; Vařecha, M.; Balek, L.; Gudernová, I.; Jelínková, I.; Ďuran, I.; Červenková, I.; Murakami, S.; Kozubík, Alois; Dvořák, P.; Bryja, Vítězslav; Krejčí, P.

    2015-01-01

    Roč. 1852, č. 5 (2015), s. 839-850. ISSN 0925-4439 R&D Projects: GA ČR GCP302/12/J059; GA ČR GBP302/12/G157; GA ČR(CZ) GA14-31540S Institutional support: RVO:67985904 ; RVO:68081707 Keywords : fibroblast growth factor receptor * FGFR3 * WNT Subject RIV: EA - Cell Biology Impact factor: 4.882, year: 2014

  17. 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

    cells when cultured as micromass pellets in a xeno-free system containing TGFbeta1. In conclusion, we identified dlk1/FA1 as a novel marker of chondroprogenitor cells that undergo embryonic lineage progression from proliferation to the prehypertrophic stage. Tracking dlk1/FA1 expression as a mesoderm...

  18. Interleukin-1ß-induced expression of the urokinase-type plasminogen activator receptor and its co-localization with MMPs in human articular chondrocytes

    OpenAIRE

    Schwab, W.; Schulze-Tanzil, G.; Mobasheri, A; Dressler, J.; Kotzsch, M.; Shakibaei, M.

    2004-01-01

    The urokinase-type plasminogen activator receptor (uPAR) plays a critical role in cartilage degradation during osteoarthritis as it regulates pericellular proteolysis mediated by serine proteinases. Another important family of proteinases responsible for ECM destruction in arthritis are the matrix metalloproteinases (MMPs). MMPs are regulated by IL- 1ß, a cytokine that plays a pivotal role in pathogenesis of osteoarthritis. This study was undertaken to address ...

  19. Vliv povrchového náboje polymerních hydrogelů na růst chondrocytů in-vitro

    Czech Academy of Sciences Publication Activity Database

    Lesný, P.; Přádný, Martin; Michálek, Jiří; Janoušek, P.; Kabelka, Z.

    2005-01-01

    Roč. 14, č. 2 (2005), s. 5-9. ISSN 1210-0447 R&D Projects: GA ČR GA304/02/0759 Institutional research plan: CEZ:AV0Z40500505 Keywords : macroporous hydrogels * 2-hydroxyethylmethacrylate, * tissue engineering Subject RIV: CD - Macromolecular Chemistry

  20. Partial rescue of postnatal growth plate abnormalities in Ihh mutants by expression of a constitutively active PTH/PTHrP receptor

    OpenAIRE

    Maeda, Yukiko; Schipani, Ernestina; Densmore, Michael J.; Lanske, Beate

    2009-01-01

    Indian hedgehog (Ihh) is essential for chondrocyte proliferation/differentiation and osteoblast differentiation during prenatal endochondral bone formation. Ihh expression in postnatal chondrocytes has a non-redundant role in maintaining a growth plate and sustaining trabecular bone after birth. Loss of Ihh in postnatal chondrocytes results in fusion of the growth plate and a decrease in trabecular bone. In order to normalize this abnormal chondrocyte phenotype and to investigate whether a pu...

  1. A Zinc Finger Transcription Factor, αA-Crystallin Binding Protein 1, Is a Negative Regulator of the Chondrocyte-Specific Enhancer of the α1(II) Collagen Gene

    OpenAIRE

    Tanaka, Kazuhiro; Matsumoto, Yoshihiro; Nakatani, Fumihiko; Iwamoto, Yukihide; Yamada, Yoshihiko

    2000-01-01

    Transcription of the type II collagen gene (Col2a1) is regulated by multiple cis-acting sites. The enhancer element, which is located in the first intron, is necessary for high-level and cartilage-specific expression of Col2a1. A mouse limb bud cDNA expression library was screened by the Saccharomyces cerevisiae one-hybrid screening method to identify protein factors bound to the enhancer. A zinc finger protein, αA-crystallin binding protein 1 (CRYBP1), which had been reported to bind to the ...

  2. 牛蒡子苷元对关节软骨细胞增殖及Ⅱ型胶原表达的影响%Effects of arctigenin on expression of articular chondrocytes proliferation and Ⅱ Collagen

    Institute of Scientific and Technical Information of China (English)

    陈世宣; 冯伟; 周一心; 曹彦俊; 卢远坚; 顾小华

    2015-01-01

    目的 观察牛蒡子苷元(arctigenin,ARC-G)对关节软骨细胞增殖及Ⅱ型胶原表达的影响.方法 采用酶消化法体外分离关节软骨细胞,取第1代软骨细胞分别接种于6孔培养板和96孔培养板中,实验分为空白组、牛蒡子苷元低浓度组(低浓度组)和牛蒡子苷元高浓度组(高浓度组).各组分别给予相应的干预措施,48 h后,以SABC免疫组织化学染色法观察软骨细胞Ⅱ型胶原的表达情况,以MTT法检测细胞的增殖情况,以酶联免疫吸附法(ELISA)检测细胞上清Ⅱ型胶原的含量.结果 ①与空白组比较,低浓度组和高浓度组在软骨细胞数量和阳性染色强度上均有所增加,且高浓度组优于低浓度组;②与空白组比较,低浓度组和高浓度组软骨细胞OD值及Ⅱ型胶原含量均明显提高(P<0.05),且高浓度组优于低浓度组(P<0.05).结论 牛蒡子苷元可明显促进体外培养关节软骨细胞的增殖和Ⅱ型胶原的表达,较好地维持软骨细胞表型,从而可有效用于骨关节炎的治疗.

  3. Combined role of type IX collagen and cartilage oligomeric matrix protein in cartilage matrix assembly: Cartilage oligomeric matrix protein counteracts type IX collagen-induced limitation of cartilage collagen fibril growth in mouse chondrocyte cultures

    NARCIS (Netherlands)

    Blumbach, K.; Bastiaansen-Jenniskens, Y.M.; Groot, J. de; Paulsson, M.; Osch, G.J.V.M. van; Zaucke, F.

    2009-01-01

    Objective. Defects in the assembly and composition of cartilage extracellular matrix are likely to result in impaired matrix integrity and increased susceptibility to cartilage degeneration. The aim of this study was to determine the functional interaction of the collagen fibril-associated proteins

  4. [Research on pericellular matrix properties for chondrcytes].

    Science.gov (United States)

    Han, Jun-liang; Duan, Wang-ping; Shi, Guang-hua; Yuan, Wei; Wei, Xiao-chun

    2015-06-01

    Pericellular matrix (PCM) is a narrow tissue region surrounding chondrocytes, which "chondron" with its enclosed cells. A number of studies suggested that PCM is rich in proteoglycans, collagen and fibronectin, and plays an important role in regulating microenvironment of chondrocytes. Direct measures of PCM properties through micropipette aspiration technique showed that PCM was different from mechanical property of chondrocytes and nature extracellular matrix. However, the function of PCM is not clear, and need further study. PMID:26255489

  5. Expandable Scaffold Improves Integration of Tissue-Engineered Cartilage: An In Vivo Study in a Rabbit Model.

    Science.gov (United States)

    Wang, Chen-Chie; Yang, Kai-Chiang; Lin, Keng-Hui; Liu, Yen-Liang; Yang, Ya-Ting; Kuo, Tzong-Fu; Chen, Ing-Ho

    2016-06-01

    One of the major limitations of tissue-engineered cartilage is poor integration of chondrocytes and scaffold structures with recipient tissue. To overcome this limitation, an expandable scaffold with a honeycomb-like structure has been developed using microfluidic technology. In this study, we evaluated the performance of this expandable gelatin scaffold seeded with rabbit chondrocytes in vivo. The chondrocyte/scaffold constructs were implanted into regions of surgically introduced cylindrical osteochondral defects in rabbit femoral condyles. At 2, 4, and 6 months postsurgery, the implanted constructs were evaluated by gross and histological examinations. As expected, the osteochondral defects, which were untreated or transplanted with blank scaffolds, showed no signs of repair, whereas the defects transplanted with chondrocyte/scaffold constructs showed significant cartilage regeneration. Furthermore, the expandable scaffolds seeded with chondrocytes had more regenerated cartilage tissue and better integration with the recipient tissue than autologous chondrocyte implantation. Biomechanical tests revealed that the chondrocyte/scaffold group had the highest compressive strength among all groups at all three time points and endured a similar compressive force to normal cartilage after 6 months of implantation. Histological examinations revealed that the chondrocytes were distributed uniformly within the scaffolds, maintained a normal phenotype, and secreted functional components of the extracellular matrix. Histomorphometric assessment showed a remarkable total interface of up to 87% integration of the expandable scaffolds with the host tissue at 6 months postoperation. In conclusion, the expandable scaffolds improved chondrocyte/scaffold construct integration with the host tissue and were beneficial for cartilage repair. PMID:27193498

  6. Preparing PCL/PLGA Hybrid Nanofiber Scaffold Capable of Controlled Releasing of Insulin for Cartilage Tissue Engineering Application

    Directory of Open Access Journals (Sweden)

    A Basiri

    2014-08-01

    Conclusion: Biological activity of insulin has been maintained during 22 days of controlled release from hybrid nanofiber PCL/PLGA scaffold. Chondrocytes were distributed evenly throughout the scaffold and revealed a rounded morphology. Therefore, this scaffold provides a suitable carrier for chondrocyte growth as well as formation of tissue engineered cartilage.

  7. EST Table: FS816912 [KAIKOcDNA[Archive

    Lifescience Database Archive (English)

    Full Text Available FS816912 E_FL_fmgV_49O17_F_0 10/09/28 56 %/182 aa ref|XP_001812240.1| PREDICTED: similar to clas ... (differentially expressed in chondrocytes) (mdec) (sharp ) [Tribolium castaneum] 10/09/09 50 %/173 aa FBpp01 ... (differentially expressed in chondrocytes) (mdec) (sharp ) [Tribolium castaneum] FS845099 fmgV ...

  8. EST Table: FS845099 [KAIKOcDNA[Archive

    Lifescience Database Archive (English)

    Full Text Available FS845099 E_FL_fner_22L02_F_0 11/12/09 GO hit GO:0005634(nucleus)|GO:0030528(transcription regula ... (differentially expressed in chondrocytes) (mdec) (sharp ) [Tribolium castaneum] 10/09/10 low homology 10/08 ... (differentially expressed in chondrocytes) (mdec) (sharp ) [Tribolium castaneum] FS845099 fner ...

  9. Colonies in engineered articular cartilage express superior differentiation.

    Science.gov (United States)

    Selvaratnam, L; Abd Rahim, S; Kamarul, T; Chan, K Y; Sureshan, S; Penafort, R; Ng, C L L

    2005-07-01

    In view of poor regeneration potential of the articular cartilage, in-vitro engineering of cartilage tissue offers a promising option for progressive joint disease. This study aims to develop a biologically engineered articular cartilage for autologous transplantation. The initial work involved determination of chondrocyte yield and viability, and morphological analysis. Cartilage was harvested from the knee, hip and shoulder joints of adult New Zealand white rabbits and chondrocytes were isolated by enzymatic digestion of the extra-cellular matrix before serial cultivation in DMEM/Ham's F12 media as monolayer cultures. No differences were noted in cell yield. Although chondrocytes viability was optimal (>93%) following harvest from native cartilage, their viability tended to be lowered on passaging. Chondrocytes aggregated in isogenous colonies comprising ovoid cells with intimate intracellular contacts and readily exhibited Safranin-O positive matrix; features typically associated with articular cartilage in-vivo. However, chondrocytes also existed concurrently in scattered bipolar/multipolar forms lacking Safranin-O expression. Therefore, early data demonstrated successful serial culture of adult chondrocytes with differentiated morphology seen in established chondrocyte colonies synthesizing matrix proteoglycans. PMID:16381284

  10. Three-dimensional chitin-based scaffolds from Verongida sponges (Demospongiae: Porifera). Part II: Biomimetic potential and applications.

    Science.gov (United States)

    Ehrlich, H; Steck, E; Ilan, M; Maldonado, M; Muricy, G; Bavestrello, G; Kljajic, Z; Carballo, J L; Schiaparelli, S; Ereskovsky, A; Schupp, P; Born, R; Worch, H; Bazhenov, V V; Kurek, D; Varlamov, V; Vyalikh, D; Kummer, K; Sivkov, V V; Molodtsov, S L; Meissner, H; Richter, G; Hunoldt, S; Kammer, M; Paasch, S; Krasokhin, V; Patzke, G; Brunner, E; Richter, W

    2010-08-01

    In order to evaluate the biomedical potential of three-dimensional chitinous scaffolds of poriferan origin, chondrocyte culturing experiments were performed. It was shown for the first time that freshly isolated chondrocytes attached well to the chitin scaffold and synthesized an extracellular matrix similar to that found in other cartilage tissue engineering constructs. Chitin scaffolds also supported deposition of a proteoglycan-rich extracellular matrix of chondrocytes seeded bioconstructs in an in vivo environment. We suggest that chitin sponge scaffolds, apart from the demonstrated biomedical applications, are highly optimized structures for use as filtering systems, templates for biomineralization as well as metallization in order to produce catalysts. PMID:20478334

  11. Effects of glucosamine on chondrocyte proteoglycan metabolism induced by interleukin-1 beta in human osteoarthritis cultured in vitro%氨基葡萄糖对白细胞介素1β诱导体外培养人骨关节炎软骨细胞蛋白聚糖代谢的影响★

    Institute of Scientific and Technical Information of China (English)

    杨建辉; 吕建国; 聂会勇; 申晓东

    2012-01-01

      背景:蛋白聚糖和胶原纤维的降解是骨关节炎发病的主要生理和病理学基础,氨基葡萄糖不仅可以减轻骨关节炎疼痛症状,同时可以延缓骨关节炎的病理改变.目的:了解氨基葡萄糖对白细胞介素1β诱导骨关节炎软骨细胞蛋白聚糖代谢的影响.方法:取骨关节炎患者的软骨细胞,分阶段酶消化法进行体外原代培养.在培养液中加入白细胞介素1β诱导剂,设立不含药兔血清对照组、白细胞介素1β对照组和加入不同浓度兔氨基葡萄糖含药血清的实验组.结果与结论:各浓度氨基葡萄糖含药血清组体外培养软骨细胞释放入培养液中糖胺聚糖百分比均值明显低于对照组(P <0.01),随氨基葡萄糖浓度的增加,糖胺聚糖百分比逐渐降低;蛋白聚糖合成标记物3B3含量的均值较对照组明显增高(P <0.01),与氨基葡萄糖浓度呈正相关;而蛋白聚糖降解标记物5D4含量的均值则正相反;氨基葡萄糖可以增加骨关节炎患者软骨细胞蛋白聚糖mRNA表达,减低基质金属蛋白酶1,3 mRNA表达.表明氨基葡萄糖可以抑制白细胞介素1β对骨关节炎患者软骨细胞蛋白聚糖代谢的促进作用,达到保护软骨,防止骨关节炎的目的.%10.3969/j.issn.2095-4344.2012.33.009

  12. 维药买朱尼含药血清对IL-1β作用下大鼠软骨细胞MMP-13、Type-Ⅱ Collagen表达的影响%Effect of Weiyao-maizhuni containing serum against rat chondrocyte MMP-13,type-Ⅱ collagen expression under the action of IL-1 β

    Institute of Scientific and Technical Information of China (English)

    刘振峰; 艾力江阿斯拉; 方锐; 洪汉刚; 孟庆才

    2012-01-01

    目的:观察维药买朱尼含药血清对IL-1β作用下体外培养的大鼠关节软骨细胞Type-Ⅱ Collagen、MMP-1、MMP-13表达的影响,并进一步探讨维药买朱尼防治OA的作用机制.方法:从1周龄SD大鼠关节软骨中分离培养原代软骨细胞,经鉴定后选用第二代细胞随机分为空白对照组、模型组、维药买朱尼组,培养第3d时模型组、维药买朱尼组采用细胞因子IL-1β(10ng/ml)继续培养,分别在培养后第24h、36h、48h采用Real Time PCR方法检测并分析各组软骨细胞中MMP-13、Type-Ⅱ Collagen的表达情况.结果:在第24h时各组MMP-13表达增加,Type-Ⅱ Collagen有一定降低,但无统计学差异;而在第36h和48h时空白对照组与模型组MMP-13、Type-Ⅱ Collagen表达差异均有统计学意义(P<0.05),维药买朱尼组较模型组MMP-13与Type-ⅡCollagen的表达在48h、72h时间点有显著性差异(P<0.05).结论:维药买朱尼可抑制IL-1β对Type-Ⅱ Collagen的降解和破坏作用,并能抑制IL-1β对MMP-13的诱导和激活作用.

  13. 氯胺酮对兔膝骨性关节炎软骨细胞Bcl-2及Bax表达的影响%Influence of Ketamine (KET,Ketamine) of intra-articular injection of rabbit chondrocytes and Bax expression

    Institute of Scientific and Technical Information of China (English)

    王雯; 王林; 陆巍

    2015-01-01

    Objective To explore the different concentrations of after the Bcl‐2 ,and the influence of Bax expres‐sion .Method The rest of rabbits with left knee immobilized in extension position to establish osteoarthritis models , then ,divided the models into model group ,ketamine60 group ,ketamine100 group and ketamine200 group in ran‐dom .They were received 0 .4 ml physiologic saline ,KET of 60μmol/L ,KET of 100μmol/L ,KET of 200μmol/L intra‐articular respectively ,two times a week for 4 weeks ,and sacrificed one week after last injection .To return af‐ter knee joint cartilage specimens corresponding processing ,using immunohistochemical method to observe the Bcl‐2 in the articular cartilage and the expression of bax .Result Compared with model group ,the injection of ketamine and promote cartilage cells apoptosis gene Bcl‐2 and Bax genes are changed (P<0 .05) .ketamine200 group of antiapop‐totic effect is the most significant (P<0 .05) .Conclusion Ketamine can suppress the cartilage cell apoptosis induced by osteoarthritis .%目的:观察不同浓度氯胺酮(Ketamine ,KET )家兔关节腔内注射后软骨细胞Bcl‐2及Bax表达的影响。方法通过管型石膏伸直位制动法造模成功后,随机分为模型组、氯胺酮60组、氯胺酮100组、氯胺酮200组,其中模型组、氯胺酮60组、氯胺酮100组、氯胺酮200组分别给予0.4 mL的生理盐水、KET(60μmol/L)、KET(100μmol/L)、KET(200μmol/L)关节腔内注射,每周2次,共进行4周,最后一次注射后1周处死动物。留取膝关节软骨标本作相应处理后,利用免疫组化法观察关节软骨中Bcl‐2及Bax的表达情况。结果与模型组比较,注射氯胺酮的软骨细胞促凋亡基因Bcl‐2及Bax均改变(P<0.05),其中氯胺酮200组的抗凋亡效果最为显著(P<0.05)。结论氯胺酮可以抑制骨关节炎诱导的软骨细胞凋亡。

  14. Optimizing a novel method for low intensity ultrasound in chondrogenesis induction

    Directory of Open Access Journals (Sweden)

    Hajar Shafaei

    2013-01-01

    Conclusion: Using LIUS resulted in early chondrogenesis in comparison with terminally differentiated chondrocytes by TGFβ. Therefore, LIUS might provide an applicable, safe, efficient, and cheap tool for chondrogenic differentiation of ASCs in cartilage tissue engineering.

  15. Activin receptor-like kinase receptors ALK5 and ALK1 are both required for TGFβ-induced chondrogenic differentiation of human bone marrow-derived mesenchymal stem cells

    NARCIS (Netherlands)

    L.M.G. De Kroon (Laurie M.G.); R. Narcisi (Roberto); E.N. Blaney Davidson (Esmeralda); M.A. Cleary (Mairéad); H.M. van Beuningen (Henk); W.J.L.M. Koevoet (Wendy J.L.M.); G.J.V.M. van Osch (Gerjo); P.M. van der Kraan (Peter)

    2015-01-01

    textabstractIntroduction Bone marrow-derived mesenchymal stem cells (BMSCs) are promising for cartilage regeneration because BMSCs can differentiate into cartilage tissue-producing chondrocytes. Transforming Growth Factor beta; (TGFbeta;) is crucial for inducing chondrogenic differentiation of BMSCs

  16. Activin Receptor-Like Kinase Receptors ALK5 and ALK1 Are Both Required for TGFbeta-Induced Chondrogenic Differentiation of Human Bone Marrow-Derived Mesenchymal Stem Cells

    NARCIS (Netherlands)

    Kroon, L.M.G. de; Narcisi, R.; Davidson, E.N.; Cleary, M.A.; Beuningen, H.M. van; Koevoet, W.J.; Osch, G.J. van; Kraan, P.M. van der

    2015-01-01

    INTRODUCTION: Bone marrow-derived mesenchymal stem cells (BMSCs) are promising for cartilage regeneration because BMSCs can differentiate into cartilage tissue-producing chondrocytes. Transforming Growth Factor beta (TGFbeta) is crucial for inducing chondrogenic differentiation of BMSCs and is known

  17. A HYBRID SCAFFOLD OF POLY(LACTIDE-CO-GLYCOLIDE) SPONGE FILLE