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Sample records for bovine articular chondrocytes

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

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

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

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

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    Tarik Onur, Alexis Dang

    2014-09-01

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

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

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    Siyuan Li

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

  4. Articular chondrocyte metabolism and osteoarthritis

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    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.

  5. 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.

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

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

    2015-05-22

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

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

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    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.

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

    Science.gov (United States)

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

    2008-01-01

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

  9. Co-administration of hyaluronic acid with local anaesthetics shows lower cytotoxicity than local anaesthetic treatment alone in bovine articular chondrocytes

    OpenAIRE

    Onur, T. S.; Sitron, C. S.; Dang, A

    2013-01-01

    Objective To study the effect of hyaluronic acid (HA) on local anaesthetic chondrotoxicity in vitro. Methods Chondrocytes were harvested from bovine femoral condyle cartilage and isolated using collagenase-containing media. At 24 hours after seeding 15 000 cells per well onto a 96-well plate, chondrocytes were treated with media (DMEM/F12 + ITS), PBS, 1:1 lidocaine (2%):PBS, 1:1 bupivacaine (0.5%):PBS, 1:1 lidocaine (2%):HA, 1:1 bupivacaine (0. 5%):HA, or 1:1 HA:PBS for one hour. Following tr...

  10. ROCK inhibitor prevents the dedifferentiation of human articular chondrocytes

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

    2012-03-30

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

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

    OpenAIRE

    2010-01-01

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

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

    NARCIS (Netherlands)

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

    2014-01-01

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

  13. Doxycycline inhibits collagen synthesis by differentiated articular chondrocytes.

    NARCIS (Netherlands)

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

    1998-01-01

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

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

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    Kaupp, J A; Waldman, S D

    2008-07-01

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

  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. Depth-varying density and organization of chondrocytes in immature and mature bovine articular cartilage assessed by 3d imaging and analysis

    Science.gov (United States)

    Jadin, Kyle D.; Wong, Benjamin L.; Bae, Won C.; Li, Kelvin W.; Williamson, Amanda K.; Schumacher, Barbara L.; Price, Jeffrey H.; Sah, Robert L.

    2005-01-01

    Articular cartilage is a heterogeneous tissue, with cell density and organization varying with depth from the surface. The objectives of the present study were to establish a method for localizing individual cells in three-dimensional (3D) images of cartilage and quantifying depth-associated variation in cellularity and cell organization at different stages of growth. Accuracy of nucleus localization was high, with 99% sensitivity relative to manual localization. Cellularity (million cells per cm3) decreased from 290, 310, and 150 near the articular surface in fetal, calf, and adult samples, respectively, to 120, 110, and 50 at a depth of 1.0 mm. The distance/angle to the nearest neighboring cell was 7.9 microm/31 degrees , 7.1 microm/31 degrees , and 9.1 microm/31 degrees for cells at the articular surface of fetal, calf, and adult samples, respectively, and increased/decreased to 11.6 microm/31 degrees , 12.0 microm/30 degrees , and 19.2 microm/25 degrees at a depth of 0.7 mm. The methodologies described here may be useful for analyzing the 3D cellular organization of cartilage during growth, maturation, aging, degeneration, and regeneration.

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

    DEFF Research Database (Denmark)

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

    2006-01-01

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

  18. bFGF influences human articular chondrocyte differentiation

    DEFF Research Database (Denmark)

    Schmal, H; Zwingmann, J; Fehrenbach, M

    2007-01-01

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

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

    Science.gov (United States)

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

    2010-04-01

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

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

    Institute of Scientific and Technical Information of China (English)

    Xu Peng; 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.

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

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    F Wolf

    2008-12-01

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

  2. 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.

  3. Hyaluronic acid-coated bovine serum albumin nanoparticles loaded with brucine as selective nanovectors for intra-articular injection

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    Chen Z

    2013-10-01

    Full Text Available Zhipeng Chen,* Juan Chen,* Li Wu, Weidong Li, Jun Chen, Haibo Cheng, Jinhuo Pan, Baochang CaiDepartment of Pharmacy, Nanjing University of Chinese Medicine, Nanjing, People's Republic of China*These authors contributed equally to this workObjective: To evaluate the potential of hyaluronic acid (HA-coated bovine serum albumin nanoparticles (BSANPs as a novel chondrocyte-targeting drug-delivery nanomedicine.Methods: The HA-BSANPs were characterized by dynamic light scattering, transmission electron microscopy, differential scanning calorimetry, and X-ray diffraction. Fluorescence imaging was used to visualize the distribution of nanoparticles after intra-articular injection. The chondrocyte-targeting efficiency and cellular uptake mechanism of HA-BSANPs were investigated using endocytic inhibitors.Results: HA-BSANPs were successfully prepared with HA coating the surface and amorphous drug in the core. Compared with BSANPs, HA-BSANPs exhibited improved uptake by chondrocytes through a receptor-mediated active uptake mechanism. The endocytosis process of BSANPs and HA-BSANPs involved clathrin-mediated endocytosis, caveolae-mediated endocytosis, and macropinocytosis. No apparent thickening or hyperplasia of the synovium was observed in either BSANPs or HA-BSANPs. The HA-BSANPs could reside in the articular cavity of rats for more than 14 days, which was significantly longer than BSANPs.Conclusion: HA-BSANPs are a promising carrier for articular-related diseases due to elongated articular residence and improved chondrocytic accumulation.Keywords: chondrocyte, intra-articular injection, hyaluronic acid, BSA, nanoparticles

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

    Institute of Scientific and Technical Information of China (English)

    2005-01-01

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

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

    Science.gov (United States)

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

    2014-12-23

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

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

    Science.gov (United States)

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

    2015-08-14

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

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

    Directory of Open Access Journals (Sweden)

    N Kaneshiro

    2007-05-01

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

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

    DEFF Research Database (Denmark)

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

    2007-01-01

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

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

    NARCIS (Netherlands)

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

    2014-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Li-ke Luo

    2015-01-01

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

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

    Science.gov (United States)

    Gradišnik, Lidija; Gorenjak, Mario; Vogrin, Matjaž

    2017-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Jakob Naranda

    2017-03-01

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

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

    Science.gov (United States)

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

    2003-01-01

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

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

    Science.gov (United States)

    Nazempour, A; Van Wie, B J

    2016-05-01

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

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

    Directory of Open Access Journals (Sweden)

    Richard Barrett-Jolley

    2012-04-01

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

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

    Science.gov (United States)

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

    2014-09-01

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

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

    Science.gov (United States)

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

    2016-05-01

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

  18. 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.

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

    Science.gov (United States)

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

    2001-06-01

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

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

    Science.gov (United States)

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

    2016-05-01

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

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

    Science.gov (United States)

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

    2010-01-01

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

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

    Science.gov (United States)

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

    2015-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Saliya Gurusinghe

    2014-01-01

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

  4. 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

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

    Science.gov (United States)

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

    2010-01-01

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

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

    Science.gov (United States)

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

    2011-01-01

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

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

    Science.gov (United States)

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

    2014-09-01

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

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

    Science.gov (United States)

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

    2013-02-08

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

  10. Mechanical Compression of Articular Cartilage Induces Chondrocyte Proliferation and Inhibits Proteoglycan Synthesis by Activation of the Erk Pathway: Implications for Tissue Engineering and Regenerative Medicine

    Science.gov (United States)

    Ryan, James A.; Eisner, Eric A.; DuRaine, Grayson; You, Zongbing; Reddi, A. Hari

    2013-01-01

    Articular cartilage is recalcitrant to endogenous repair and regeneration and thus a focus of tissue engineering and regenerative medicine strategies. A pre-requisite for articular cartilage tissue engineering is an understanding of the signal transduction pathways involved in mechanical compression during trauma or disease. We sought to explore the role of the extracellular signal-regulated kinase 1/2 (ERK 1/2) pathway in chondrocyte proliferation and proteoglycan synthesis following acute mechanical compression. Bovine articular cartilage explants were cultured with and without the ERK 1/2 pathway inhibitor PD98059. Cartilage explants were statically loaded to 40% strain at a strain rate of 1−sec for 5 seconds. Control explants were cultured under similar conditions but were not loaded. There were four experimental groups: 1) no load without inhibitor 2) no load with the inhibitor PD98059, 3) loaded without the inhibitor, and 4) loaded with the inhibitor PD98059. Explants were cultured for varying durations, from 5 minutes to 5 days. Explants were then analyzed by biochemical and immunohistochemical methods. Mechanical compression induced phosphorylation of ERK 1/2, and this was attenuated with the ERK 1/2 pathway inhibitor PD98059 in a dose-dependent manner. Chondrocyte proliferation was increased by mechanical compression. This effect was blocked by the inhibitor of the ERK 1/2 pathway. Mechanical compression also led to a decrease in proteoglycan synthesis that was reversed with inhibitor PD98059. In conclusion, the ERK 1/2 pathway is involved in the proliferative and biosynthetic response of chondrocytes following acute static mechanical compression. PMID:19177463

  11. 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.

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

    Science.gov (United States)

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

    2004-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Chun Chung-Woo

    2010-05-01

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

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

    Science.gov (United States)

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

    2014-04-01

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

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

    Directory of Open Access Journals (Sweden)

    M. Lehmann

    2013-11-01

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

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

    OpenAIRE

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

    2004-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Wan-Ying Lin

    2014-01-01

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

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

    Science.gov (United States)

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

    2016-09-01

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

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

    Science.gov (United States)

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

    2007-12-01

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

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

    Science.gov (United States)

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

    2011-08-01

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

  1. 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.

  2. Collagen metabolism of human osteoarthritic articular cartilage as modulated by bovine collagen hydrolysates.

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    Saskia Schadow

    Full Text Available Destruction of articular cartilage is a characteristic feature of osteoarthritis (OA. Collagen hydrolysates are mixtures of collagen peptides and have gained huge public attention as nutriceuticals used for prophylaxis of OA. Here, we evaluated for the first time whether different bovine collagen hydrolysate preparations indeed modulate the metabolism of collagen and proteoglycans from human OA cartilage explants and determined the chemical composition of oligopeptides representing collagen fragments. Using biophysical techniques, like MALDI-TOF-MS, AFM, and NMR, the molecular weight distribution and aggregation behavior of collagen hydrolysates from bovine origin (CH-Alpha®, Peptan™ B 5000, Peptan™ B 2000 were determined. To investigate the metabolism of human femoral OA cartilage, explants were obtained during knee replacement surgery. Collagen synthesis of explants as modulated by 0-10 mg/ml collagen hydrolysates was determined using a novel dual radiolabeling procedure. Proteoglycans, NO, PGE(2, MMP-1, -3, -13, TIMP-1, collagen type II, and cell viability were determined in explant cultures. Groups of data were analyzed using ANOVA and the Friedman test (n = 5-12. The significance was set to p≤0.05. We found that collagen hydrolysates obtained from different sources varied with respect to the width of molecular weight distribution, average molecular weight, and aggregation behavior. None of the collagen hydrolysates tested stimulated the biosynthesis of collagen. Peptan™ B 5000 elevated NO and PGE(2 levels significantly but had no effect on collagen or proteoglycan loss. All collagen hydrolysates tested proved not to be cytotoxic. Together, our data demonstrate for the first time that various collagen hydrolysates differ with respect to their chemical composition of collagen fragments as well as by their pharmacological efficacy on human chondrocytes. Our study underscores the importance that each collagen hydrolysate

  3. Both superficial and deep zone articular chondrocyte subpopulations exhibit the Crabtree effect but have different basal oxygen consumption rates.

    Science.gov (United States)

    Heywood, Hannah K; Knight, Martin M; Lee, David A

    2010-06-01

    In the absence of in vivo measurements, the oxygen concentration within articular cartilage is calculated from the balance between cellular oxygen consumption and mass transfer. Current estimates of the oxygen tension within articular cartilage are based on oxygen consumption data from full-depth tissue samples. However, superficial and deep cell subpopulations of articular cartilage express intrinsic metabolic differences. We test the hypothesis that the subpopulations differ with respect to their intrinsic oxygen consumption rate. Chondrocytes from the full cartilage thickness demonstrate enhanced oxygen consumption when deprived of glucose, consistent with the Crabtree phenomena. Chondrocyte subpopulations differ in the prevailing availability of oxygen and glucose, which decrease with distance from the cartilage-synovial fluid interface. Thus, we tested the hypothesis that the oxygen consumption of each subpopulation is modulated by nutrient availability, by examining the expression of the Crabtree effect. The deep cells had a greater oxygen consumption than the superficial cells (V(max) of 6.6 compared to 3.2 fmol/cell/h), consistent with our observations of mitochondrial volume (mean values 52.0 vs. 36.4 microm(3)/cell). Both populations expressed the Crabtree phenomena, with oxygen consumption increasing approximately 2.5-fold in response to glycolytic inhibition by glucose deprivation or 2-deoxyglucose. Over 90% of this increase was oligomycin-sensitive and thus accounted for by oxidative phosphorylation. The data contributes towards our understanding of chondrocyte energy metabolism and provides information valuable for the accurate calculation of the oxygen concentration that the cells experience in vivo. The work has further application to the optimisation of bioreactor design and engineered tissues.

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

    Science.gov (United States)

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

    2017-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Hirotaka Mutsuzaki

    2017-01-01

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

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

    NARCIS (Netherlands)

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

    2004-01-01

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

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

    Science.gov (United States)

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

    2014-10-03

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

  8. Fibrin and poly(lactic-co-glycolic acid hybrid scaffold promotes early chondrogenesis of articular chondrocytes: an in vitro study

    Directory of Open Access Journals (Sweden)

    Idrus Ruszymah BH

    2008-04-01

    Full Text Available Abstract Background Synthetic- and naturally derived- biodegradable polymers have been widely used to construct scaffolds for cartilage tissue engineering. Poly(lactic-co-glycolic acid (PLGA are bioresorbable and biocompatible, rendering them as a promising tool for clinical application. To minimize cells lost during the seeding procedure, we used the natural polymer fibrin to immobilize cells and to provide homogenous cells distribution in PLGA scaffolds. We evaluated in vitro chondrogenesis of rabbit articular chondrocytes in PLGA scaffolds using fibrin as cell transplantation matrix. Methods PLGA scaffolds were soaked in chondrocytes-fibrin suspension (1 × 106cells/scaffold and polymerized by dropping thrombin-calcium chloride (CaCl2 solution. PLGA-seeded chondrocytes was used as control. All constructs were cultured for a maximum of 21 days. Cell proliferation activity was measured at 1, 3, 7, 14 and 21 days in vitro using 3-(4,5-dimethylthiazole-2-yl-2-, 5-diphenyltetrazolium-bromide (MTT assay. Morphological observation, histology, immunohistochemistry (IHC, gene expression and sulphated-glycosaminoglycan (sGAG analyses were performed at each time point of 1, 2 and 3 weeks to elucidate in vitro cartilage development and deposition of cartilage-specific extracellular matrix (ECM. Results Cell proliferation activity was gradually increased from day-1 until day-14 and declined by day-21. A significant cartilaginous tissue formation was detected as early as 2-week in fibrin/PLGA hybrid construct as confirmed by the presence of cartilage-isolated cells and lacunae embedded within basophilic ECM. Cartilage formation was remarkably evidenced after 3 weeks. Presence of cartilage-specific proteoglycan and glycosaminoglycan (GAG in fibrin/PLGA hybrid constructs were confirmed by positive Safranin O and Alcian Blue staining. Collagen type II exhibited intense immunopositivity at the pericellular matrix. Chondrogenic properties were further

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

    Science.gov (United States)

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

    2013-11-01

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

  10. 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.

  11. Stimulation of Superficial Zone Protein/Lubricin/PRG4 by Transforming Growth Factor-β in Superficial Zone Articular Chondrocytes and Modulation by Glycosaminoglycans.

    Science.gov (United States)

    Cuellar, Araceli; Reddi, A Hari

    2015-07-01

    Superficial zone protein (SZP), also known as lubricin and proteoglycan 4 (PRG4), plays an important role in the boundary lubrication of articular cartilage and is regulated by transforming growth factor (TGF)-β. Here, we evaluate the role of cell surface glycosaminoglycans (GAGs) during TGF-β1 stimulation of SZP/lubricin/PRG4 in superficial zone articular chondrocytes. We utilized primary monolayer superficial zone articular chondrocyte cultures and treated them with various concentrations of TGF-β1, in the presence or absence of heparan sulfate (HS), heparin, and chondroitin sulfate (CS). The cell surface GAGs were removed by pretreatment with either heparinase I or chondroitinase-ABC before TGF-β1 stimulation. Accumulation of SZP/lubricin/PRG4 in the culture medium in response to stimulation with TGF-β1 and various exogenous GAGs was demonstrated by immunoblotting and quantitated by enzyme-linked immunosorbent assay. We show that TGF-β1 and exogenous HS enhanced SZP accumulation of superficial zone chondrocytes in the presence of surface GAGs. At the dose of 1 ng/mL of TGF-β1, the presence of exogenous heparin inhibited SZP accumulation whereas the presence of exogenous CS stimulated SZP accumulation in the culture medium. Enzymatic depletion of GAGs on the surface of superficial zone chondrocytes enhanced the ability of TGF-β1 to stimulate SZP accumulation in the presence of both exogenous heparin and CS. Collectively, these results suggest that GAGs at the surface of superficial zone articular chondrocytes influence the response to TGF-β1 and exogenous GAGs to stimulate SZP accumulation. Cell surface GAGs modulate superficial zone chondrocytes' response to TGF-β1 and exogenous HS.

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

    Science.gov (United States)

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

    2004-01-01

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

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

    Science.gov (United States)

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

    2016-01-01

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

  14. The Frictional Coefficient of Bovine Knee Articular Cartilage

    Institute of Scientific and Technical Information of China (English)

    Qian Shan-hua; Ge Shi-rong; Wang Qing-liang

    2006-01-01

    The normal displacement of articular cartilage was measured under load and in sliding, and the coefficient of friction during sliding was measured using a UMT-2 Multi-Specimen Test System. The maximum normal displacement under load and the start-up frictional coefficient have similar tendency of variation with loading time. The sliding speed does not significantly influence the frictional coefficient of articular cartilage.

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

    Science.gov (United States)

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

    2013-03-01

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

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

    Science.gov (United States)

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

    2004-01-01

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

  18. Cyclic mechanical load causes global translational arrest in articular chondrocytes: a process which is partially dependent upon PKR phosphorylation

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    C Lomas

    2011-09-01

    Full Text Available he cellular mechanisms by which articular cartilage responds to load are poorly understood, but such responses may involve regulation at the level of protein translation rather than synthesis of mRNA. We investigated the role of translational control in cyclically (0.5 Hz, 0.1 Hz and 0.05 Hz and statically loaded porcine articular cartilage explants. Messenger RNA was extracted for real time polymerase chain reaction (RT-PCR and newly synthesised proteins were measured by their incorporation of radiolabelled 35S[methionine/cysteine] or 35SO4. Some medium from loaded and unloaded explants was immunoblotted for type II collagen, CTGF and TIMP3. The pathways that control protein translation were investigated by immunoblotting explant lysates for PKR, PERK (PKR like endoplasmic reticulum kinase, eIF2a (eukaryotic initiation factor 2a, eEFs (eukaryotic elongation factors, and AMP-dependent kinase. Explants were also loaded in the presence of inhibitors of PKR, the fibroblast growth factor (FGF receptor and PI3 kinase. Cyclic loading caused complete global translational arrest as evidenced by a total suppression of new protein synthesis whilst maintaining mRNA levels. Translational arrest did not occur following static loading and was partly dependent upon the load frequency. There was a rebound increase in protein synthesis when labelling was performed after load had been withdrawn. Phosphorylation of PKR occurred in explants following cyclic load and inhibition of PKR modestly reversed suppression of newly synthesised proteins suggesting that PKR, at least in part, was responsible for loading induced translational arrest. These results show that translational control provides a rapid and potentially important mechanism for controlling the synthetic responses of articular chondrocytes in response to different types of mechanical load.

  19. 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

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

    Science.gov (United States)

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

    2013-12-01

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

  1. Methods for Producing Scaffold-Free Engineered Cartilage Sheets from Auricular and Articular Chondrocyte Cell Sources and Attachment to Porous Tantalum

    OpenAIRE

    Whitney, G. Adam; Mera, Hisashi; Weidenbecher, Mark; Awadallah, Amad; Mansour, Joseph M.; Dennis, James E.

    2012-01-01

    Abstract Scaffold-free cartilage engineering techniques may provide a simple alternative to traditional methods employing scaffolds. We previously reported auricular chondrocyte-derived constructs for use in an engineered trachea model; however, the construct generation methods were not reported in detail. In this study, methods for cartilage construct generation from auricular and articular cell sources are described in detail, and the resulting constructs are compared for use in a joint res...

  2. 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.

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

    Science.gov (United States)

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

    2015-01-01

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

  4. Preliminary investigation of intrinsic UV fluorescence spectroscopic changes associated with proteolytic digestion of bovine articular cartilage

    Science.gov (United States)

    Lewis, William; Padilla-Martinez, Juan-Pablo; Ortega-Martinez, Antonio; Franco, Walfre

    2016-03-01

    Degradation and destruction of articular cartilage is the etiology of osteoarthritis (OA), an entity second only to cardiovascular disease as a cause of disability in the United States. Joint mechanics and cartilage biochemistry are believed to play a role in OA; an optical tool to detect structural and chemical changes in articular cartilage might offer benefit for its early detection and treatment. The objective of the present study was to identify the spectral changes in intrinsic ultraviolet (UV) fluorescence of cartilage that occur after proteolytic digestion of cartilage. Bovine articular cartilage samples were incubated in varying concentrations of collagenase ranging from 10ug/mL up to 5mg/mL for 18 hours at 37°C, a model of OA. Pre- and post-incubation measurements were taken of the UV excitation-emission spectrum of each cartilage sample. Mechanical tests were performed to determine the pre- and post-digestion force/displacement ratio associated with indentation of each sample. Spectral changes in intrinsic cartilage fluorescence and stiffness of the cartilage were associated with proteolytic digestion. In particular, changes in the relative intensity of fluorescence peaks associated with pentosidine crosslinks (330 nm excitation, 390 nm emission) and tryptophan (290 nm excitation, 340 nm emission) were found to correlate with different degrees of cartilage digestion and cartilage stiffness. In principle, it may be possible to use UV fluorescence spectral data for early detection of damage to articular cartilage, and as a surrogate measure for cartilage stiffness.

  5. Methods for producing scaffold-free engineered cartilage sheets from auricular and articular chondrocyte cell sources and attachment to porous tantalum.

    Science.gov (United States)

    Whitney, G Adam; Mera, Hisashi; Weidenbecher, Mark; Awadallah, Amad; Mansour, Joseph M; Dennis, James E

    2012-08-01

    Scaffold-free cartilage engineering techniques may provide a simple alternative to traditional methods employing scaffolds. We previously reported auricular chondrocyte-derived constructs for use in an engineered trachea model; however, the construct generation methods were not reported in detail. In this study, methods for cartilage construct generation from auricular and articular cell sources are described in detail, and the resulting constructs are compared for use in a joint resurfacing model. Attachment of cartilage sheets to porous tantalum is also investigated as a potential vehicle for future attachment to subchondral bone. Large scaffold-free cartilage constructs were produced from culture-expanded chondrocytes from skeletally mature rabbits, and redifferentiated in a chemically-defined culture medium. Auricular constructs contained more glycosaminoglycan (39.6±12.7 vs. 9.7±1.9 μg/mg wet weight, mean and standard deviation) and collagen (2.7±0.45 vs. 1.1±0.2 μg/mg wet weight, mean and standard deviation) than articular constructs. Aggregate modulus was also higher for auricular constructs vs. articular constructs (0.23±0.07 vs. 0.12±0.03 MPa, mean and standard deviation). Attachment of constructs to porous tantalum was achieved by neocartilage ingrowth into tantalum pores. These results demonstrate that large scaffold-free neocartilage constructs can be produced from mature culture-expanded chondrocytes in a chemically-defined medium, and that these constructs can be attached to porous tantalum.

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

    Directory of Open Access Journals (Sweden)

    Thomas J Kean

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

  7. Cyclic tensile stretch load and oxidized low density lipoprotein synergistically induce lectin-like oxidized ldl receptor-1 in cultured bovine chondrocytes, resulting in decreased cell viability and proteoglycan synthesis.

    Science.gov (United States)

    Akagi, Masao; Nishimura, Shunji; Yoshida, Kohji; Kakinuma, Takumi; Sawamura, Tatsuya; Munakata, Hiroshi; Hamanishi, Chiaki

    2006-08-01

    Mechanical stimulation is known to be an essential factor in the regulation of cartilage metabolism. We tested the hypothesis that expression of lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) can be modulated by cyclic tensile stretch load in chondrocytes. Cyclic loading of repeated stretch stress at 10 cycles per minute with 10 kPa of stress for 6 h induced expression of LOX-1 to 2.6 times control in cultured bovine articular chondrocytes, equivalent to the addition of 10 microg/mL oxidized low density lipoprotein (ox-LDL) (2.4 times control). Application of the cyclic load to the chondrocytes along with 10 microg/mL ox-LDL resulted in synergistically increased LOX-1 expression to 6.3 times control. Individual application of cyclic loading and 10 microg/mL ox-LDL significantly suppressed chondrocytes viability (84.6% +/- 3.4% and 80.9% +/- 3.2% of control at 24 h, respectively; n = 3; p LDL (n = 3)]. Cyclic loading and 10 microg/mL ox-LDL synergistically affected cell viability and proteoglycan synthesis, which were significantly suppressed to 45.6% +/- 4.9% and 48.7% +/- 6.7% of control at 24 h, respectively (n = 3; p LDL). In this study, we demonstrated synergistic effects of cyclic tensile stretch load and ox-LDL on cell viability and proteoglycan synthesis in chondrocytes, which may be mediated through enhanced expression of LOX-1 and which has important implications in the progression of cartilage degeneration in osteoarthritis.

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

    NARCIS (Netherlands)

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

    2004-01-01

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

  9. Viscoelastic properties of bovine articular cartilage attached to subchondral bone at high frequencies

    Directory of Open Access Journals (Sweden)

    Shepherd Duncan ET

    2009-06-01

    Full Text Available Abstract Background Articular cartilage is a viscoelastic material, but its exact behaviour under the full range of physiological loading frequencies is unknown. The objective of this study was to measure the viscoelastic properties of bovine articular cartilage at loading frequencies of up to 92 Hz. Methods Intact tibial plateau cartilage, attached to subchondral bone, was investigated by dynamic mechanical analysis (DMA. A sinusoidally varying compressive force of between 16 N and 36 N, at frequencies from 1 Hz to 92 Hz, was applied to the cartilage surface by a flat indenter. The storage modulus, loss modulus and phase angle (between the applied force and the deformation induced were determined. Results The storage modulus, E', increased with increasing frequency, but at higher frequencies it tended towards a constant value. Its dependence on frequency, f, could be represented by, E' = Aloge (f + B where A = 2.5 ± 0.6 MPa and B = 50.1 ± 12.5 MPa (mean ± standard error. The values of the loss modulus (4.8 ± 1.0 MPa mean ± standard deviation were much less than the values of storage modulus and showed no dependence on frequency. The phase angle was found to be non-zero for all frequencies tested (4.9 ± 0.6°. Conclusion Articular cartilage is viscoelastic throughout the full range of frequencies investigated. The behaviour has implications for mechanical damage to articular cartilage and the onset of osteoarthritis. Storage modulus increases with frequency, until the plateau region is reached, and has a higher value than loss modulus. Furthermore, loss modulus does not increase with loading frequency. This means that more energy is stored by the tissue than is dissipated and that this effect is greater at higher frequencies. The main mechanism for this excess energy to be dissipated is by the formation of cracks.

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

    Science.gov (United States)

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

    2015-01-01

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

  11. Oxygen tension affects lubricin expression in chondrocytes.

    Science.gov (United States)

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

    2014-10-01

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

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

    DEFF Research Database (Denmark)

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

    2011-01-01

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

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

    Science.gov (United States)

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

    2017-02-01

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

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

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    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.

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

    NARCIS (Netherlands)

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

    2014-01-01

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

  16. The Inhibitory Effect of Salmon Calcitonin on Tri-Iodothyronine Induction of Early Hypertrophy in Articular Cartilage

    OpenAIRE

    Pingping Chen-An; Kim Vietz Andreassen; Kim Henriksen; Yadong Li; Morten Asser Karsdal; Anne-Christine Bay-Jensen

    2012-01-01

    OBJECTIVE: Salmon calcitonin has chondroprotective effect both in vitro and in vivo, and is therefore being tested as a candidate drug for cartilage degenerative diseases. Recent studies have indicated that different chondrocyte phenotypes may express the calcitonin receptor (CTR) differentially. We tested for the presence of the CTR in chondrocytes from tri-iodothyronin (T3)-induced bovine articular cartilage explants. Moreover, investigated the effects of human and salmon calcitonin on the ...

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

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    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.

  18. 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.

  19. Ionizing Radiation Induces Cellular Senescence of Articular Chondrocytes via Negative Regulation of SIRT1 by p38 Kinase

    Energy Technology Data Exchange (ETDEWEB)

    Hong, Eun Hee; Hwang, Sang Gu [Korea Institute of Radiological and Medical Sciences, Seoul (Korea, Republic of)

    2009-05-15

    Senescent cells exhibit irreversible growth arrest, large flat morphology, and up-regulated senescence-associated {beta}-galactosidase activity at pH 6.0. Several conditions, including oncogenic stress, oxidative stress, and DNA damage are associated with cellular senescence. Massive acute DNA double-strand breaks occurring as a result of mechanical and chemical stress can be repaired, but some DNA damage persists, eventually triggering premature senescence. Since ionizing radiation directly induces DBS, it is possible that cellular senescence is activated under these conditions. The biological events in chondrocytes following irradiation are poorly understood, and limited information is available on the molecular signal transduction mechanisms of cellular senescence at present. In this study, we identify SIRT1 as a target molecule of p38 kinase and demonstrate that the interactions between p38 kinase and SIRT1 protein play an important role in the regulation of cellular senescence in response to IR.

  20. 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

  1. Site-specific ultrasound reflection properties and superficial collagen content of bovine knee articular cartilage

    Energy Technology Data Exchange (ETDEWEB)

    Laasanen, Mikko S [Department of Clinical Physiology and Nuclear Medicine, Kuopio University Hospital and University of Kuopio, POB 1777, FIN-70211 Kuopio (Finland); Department of Surgery, Division of Orthopaedics and Traumatology, Jyvaeskylae Central Hospital, Keskussairaalantie 19, FIN-40620 Jyvaeskylae (Finland); Saarakkala, Simo [Department of Nuclear Medicine, Etelae-Savo Hospital District, Mikkeli Central Hospital, Porrassalmenkatu 35-37, 50100 Mikkeli (Finland); Toeyraes, Juha [Department of Clinical Neurophysiology, Kuopio University Hospital and University of Kuopio, POB 1777, FIN-70211 Kuopio (Finland); Rieppo, Jarno [Department of Anatomy, University of Kuopio, POB 1627, FIN-70211 Kuopio (Finland); Jurvelin, Jukka S [Department of Clinical Physiology and Nuclear Medicine, Kuopio University Hospital and University of Kuopio, POB 1777, FIN-70211 Kuopio (Finland); Department of Applied Physics, University of Kuopio, POB 1627, FIN-70211 Kuopio (Finland)

    2005-07-21

    Previous quantitative 2D-ultrasound imaging studies have demonstrated that the ultrasound reflection measurement of articular cartilage surface sensitively detects degradation of the collagen network, whereas digestion of cartilage proteoglycans has no significant effect on the ultrasound reflection. In this study, the first aim was to characterize the ability of quantitative 2D-ultrasound imaging to detect site-specific differences in ultrasound reflection and backscattering properties of cartilage surface and cartilage-bone interface at visually healthy bovine knee (n = 30). As a second aim, we studied factors controlling ultrasound reflection properties of an intact cartilage surface. The ultrasound reflection coefficient was determined in time (R) and frequency domains (IRC) at medial femoral condyle, lateral patello-femoral groove, medial tibial plateau and patella using a 20 MHz ultrasound imaging instrument. Furthermore, cartilage surface roughness was quantified by calculating the ultrasound roughness index (URI). The superficial collagen content of the cartilage was determined using a FT-IRIS-technique. A significant site-dependent variation was shown in cartilage thickness, ultrasound reflection parameters, URI and superficial collagen content. As compared to R and IRC, URI was a more sensitive parameter in detecting differences between the measurement sites. Ultrasound reflection parameters were not significantly related to superficial collagen content, whereas the correlation between R and URI was high. Ultrasound reflection at the cartilage-bone interface showed insignificant site-dependent variation. The current results suggest that ultrasound reflection from the intact cartilage surface is mainly dependent on the cartilage surface roughness and the collagen content has a less significant role.

  2. Calcium input potentiates the transforming growth factor (TGF)-beta1-dependent signaling to promote the export of inorganic pyrophosphate by articular chondrocyte.

    Science.gov (United States)

    Cailotto, Frederic; Reboul, Pascal; Sebillaud, Sylvie; Netter, Patrick; Jouzeau, Jean-Yves; Bianchi, Arnaud

    2011-06-03

    Transforming growth factor (TGF)-β1 stimulates extracellular PP(i) (ePP(i)) generation and promotes chondrocalcinosis, which also occurs secondary to hyperparathyroidism-induced hypercalcemia. We previously demonstrated that ANK was up-regulated by TGF-β1 activation of ERK1/2 and Ca(2+)-dependent protein kinase C (PKCα). Thus, we investigated mechanisms by which calcium could affect ePP(i) metabolism, especially its main regulating proteins ANK and PC-1 (plasma cell membrane glycoprotein-1). We stimulated articular chondrocytes with TGF-β1 under extracellular (eCa(2+)) or cytosolic Ca(2+) (cCa(2+)) modulations. We studied ANK, PC-1 expression (quantitative RT-PCR, Western blotting), ePP(i) levels (radiometric assay), and cCa(2+) input (fluorescent probe). Voltage-operated Ca(2+)-channels (VOC) and signaling pathways involved were investigated with selective inhibitors. Finally, Ank promoter activity was evaluated (gene reporter). TGF-β1 elevated cCa(2+) and ePP(i) levels (by up-regulating Ank and PC-1 mRNA/proteins) in an eCa(2+) dose-dependent manner. TGF-β1 effects were suppressed by cCa(2+) chelation or L- and T-VOC blockade while being mostly reproduced by ionomycin. In the same experimental conditions, the activation of Ras, the phosphorylation of ERK1/2 and PKCα, and the stimulation of Ank promoter activity were affected similarly. Activation of SP1 (specific protein 1) and ELK-1 (Ets-like protein-1) transcription factors supported the regulatory role of Ca(2+). SP1 or ELK-1 overexpression or blockade experiments demonstrated a major contribution of ELK-1, which acted synergistically with SP1 to activate Ank promoter in response to TGF-β1. TGF-β1 promotes input of eCa(2+) through opening of L- and T-VOCs, to potentiate ERK1/2 and PKCα signaling cascades, resulting in an enhanced activation of Ank promoter and ePP(i) production in chondrocyte.

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

    Science.gov (United States)

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

    2016-08-17

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

  4. Nondestructive fluorescence-based quantification of threose-induced collagen cross-linking in bovine articular cartilage

    Science.gov (United States)

    Kinnunen, Jussi; Kokkonen, Harri T.; Kovanen, Vuokko; Hauta-Kasari, Markku; Vahimaa, Pasi; Lammi, Mikko J.; Töyräs, Juha; Jurvelin, Jukka S.

    2012-09-01

    Extensive collagen cross-linking affects the mechanical competence of articular cartilage: it can make the cartilage stiffer and more brittle. The concentrations of the best known cross-links, pyridinoline and pentosidine, can be accurately determined by destructive high-performance liquid chromatography (HPLC). We explore a nondestructive evaluation of cross-linking by using the intrinsic fluorescence of the intact cartilage. Articular cartilage samples from bovine knee joints were incubated in threose solution for 40 and 100 h to increase the collagen cross-linking. Control samples without threose were also prepared. Excitation-emission matrices at wavelengths of 220 to 950 nm were acquired from the samples, and the pentosidine and pyridinoline cross-links and the collagen concentrations were determined using HPLC. After the threose treatment, pentosidine and lysyl pyridinole (LP) concentrations increased. The intrinsic fluorescence, excited below 350 nm, decreased and was related to pentosidine [r=-0.90, 240/325 nm (excitation/emission)] or LP (r=-0.85, 235/285 nm) concentrations. Due to overlapping, the changes in emission could not be linked specifically to the recorded cross-links. However, the fluorescence signal enabled a nondestructive optical estimate of changes in the pentosidine and LP cross-linking of intact articular cartilage.

  5. Research progress of the effects of compression stimulation on the metabolism of articular chondrocytes and ;mechanical properties%压缩刺激影响关节软骨细胞代谢和力学特性的研究进展

    Institute of Scientific and Technical Information of China (English)

    苑伟; 段王平; 卫小春

    2013-01-01

    Articular cartilages endure a variety of dynamic stresses in a complex physiological environment. As the main force of normal human articular movement, the pressure can change the biological and mechanical properties of chondrocytes, including changes in the stress and strain of articular cartilage tissues, hydrostatic pressure, interstitial fluid flow, flow energy, osmotic pressure, changes of transferring between tissues and cell deformations and so on. Because of the specificity of the mechanical environment, the changes of the mechanical properties of chondrocytes will have an effect on the physiological functions of articular cartilages. It is difficult for articular cartilage injuries caused by many factors to repair themselves, which has brought great difficulties to the clinical treatment. There are many traditional methods of repairing cartilage defects clinically, while some shortcomings exist. Thanks to the development of tissue engineering technology, there is a new idea and method for the regeneration and repair of articular cartilage defects. Scaffold materials are mainly utilized to provide a space structure for the proliferation and matrix secretion of chondrocytes, and a kind of structure is formed similar to normal cartilage tissues to transplant and repair articular cartilage defects. Many factors get involved in the construction of tissue-engineered cartilages with good functions, such as seed cells, scaffold materials, culture conditions and so on, among which mechanical stimulation has a particularly important effect on the metabolism of articular cartilage matrixes and their mechanical properties. Therefore, an intensive study of the mechanical properties of chondrocytes will further clarify a series of processes needed to maintain normal physiological functions, such as the stress conditions and responses. There will be positive signiifcance for the understanding of the biological properties of chondrocytes, the effects of compression

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

    Science.gov (United States)

    Karim, Asima; Hall, Andrew C

    2017-05-01

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

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

    Directory of Open Access Journals (Sweden)

    Hee-Jeong Im

    2009-10-01

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

  8. 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.

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

    Science.gov (United States)

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

    2013-04-01

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

  10. Effects of chondroitin sulfate and sodium hyaluronate on chondrocytes and extracellular matrix of articular cartilage in dogs with degenerative joint disease Efeitos do sulfato de condroitina e do hialuronato de sódio nos condrócitos e na matriz extracelular na cartilagem articular de cães com doença articular degenerativa

    Directory of Open Access Journals (Sweden)

    G. Gonçalves

    2008-02-01

    Full Text Available Samples of articular cartilage of femur, tibia and patella of 15 dogs with experimentally induced degenerative joint disease (DJD were microscopically analyzed. Animals were distributed into three groups (n=5: the control group received no medication; the second group was treated with chondroitin sulfate and the third received sodium hyaluronate. Samples were processed and stained with HE and toluidine blue for morphological evaluation. The metabolic and proliferative activity of the chondrocytes was evaluated by the measurement of nucleolar organizer regions (NORs after impregnation by silver nitrate. Significant differences were not observed (P>0.05 in the morphology among the groups, however, the group treated with sodium hyaluronate had a higher score suggesting a trend to a greater severity of the lesions. Significant differences were not observed (P>0.05 in the measurement of NORs, cells and NORs/cells among the groups. Although differences were not significant, sodium hyaluronate group showed higher NOR and cell counts which suggested an increase of the proliferation rate of chondrocytes. In addition, a higher NOR/cell ratio in the group treated with chondroitin sulfate suggested that this drug may have stimulated the metabolic activity of the chondrocytes, minimizing the lesions resulting from DJD.Foram utilizadas amostras de cartilagem articular do fêmur, tíbia e patela de 15 cães com doença articular degenerativa (DAD, induzida experimentalmente. Foram constituídos três grupos de cinco animais: grupo 1 - controle, não medicado; grupo 2 - tratado com sulfato de condroitina e grupo 3 - tratado com hialuronato de sódio. As amostras foram processadas e coradas pelas técnicas de HE e de azul de toluidina para avaliação das alterações morfológicas, e impregnadas pelo nitrato de prata para análise da atividade metabólica e/ou proliferativa dos condrócitos, por meio da visualização e quantificação de regiões organizadoras

  11. Induction of increased cAMP levels in articular chondrocytes blocks matrix metalloproteinase-mediated cartilage degradation, but not aggrecanase-mediated cartilage degradation

    DEFF Research Database (Denmark)

    Karsdal, Morten Asser; Sumer, Eren Ufuk; Wulf, Helle

    2007-01-01

    was assessed by 1) quantification of C-terminal crosslinking telopeptide of type II collagen fragments (CTX-II), 2) matrix metalloproteinase (MMP)-mediated aggrecan degradation by (342)FFGV- G2 assay, 3) aggrecanase-mediated degradation by (374)ARGS-G2 assay, 4) release of sulfated glycosaminoglycans (s......-dependently inhibited by forskolin and IBMX. The highest concentration of IBMX lowered cytokine-induced release of sGAG by 72%. CONCLUSION: Levels of cAMP in chondrocytes play a key role in controlling catabolic activity. Increased cAMP levels in chondrocytes inhibited MMP expression and activity and consequently...

  12. Safranin O reduces loss of glycosaminoglycans from bovine articular cartilage during histological specimen preparation.

    Science.gov (United States)

    Király, K; Lammi, M; Arokoski, J; Lapveteläinen, T; Tammi, M; Helminen, H; Kiviranta, I

    1996-02-01

    The ability of Safranin O, added to fixation and decalcification solutions, to prevent the escape of glycosaminoglycans (GAGs) from small cartilage tissue blocks during histological processing of cartilage has been studied. GAGs in the fixatives and decalcifying solutions used and those remaining in the 1 mm3 cubes of cartilage were assayed biochemically. The quantity of GAGs remaining in the cartilage cubes were determined from Safranin O-stained sections using videomicroscopy or microspectrophotometry. A quantity (10.6%) of GAGs were lost during a conventional 4% buffered formaldehyde fixation (48 h) and a subsequent decalcification in 10% EDTA (12 days) at 4 degrees C. Roughly one-quarter of the total GAG loss occurred during the 48 h fixation, and three-quarters during the 12 days of decalcification. Inclusion of 4% formaldehyde in the decalcification fluid decreased the loss of GAGs to 6.2%. The presence of 0.5% Safranin O in the fixative reduced this loss to 3.4%. When 0.5% Safranin O was included in the fixative and 4% formaldehyde in the decalcification solution, Safranin O staining of the histological sections increased on average by 13.5%. After fixation in the presence of 0.5% Safranin O, there was no difference in the staining intensities when decalcification was carried out in the presence of either Safranin O or formaldehyde, or both. It took 24 h for Safranin O to penetrate into the deep zone of articular cartilage, warranting a fixation period of at least this long. In conclusion, the addition of Safranin O to the fixative and either Safranin O or formaldehyde in the following decalcification fluid, markedly reduces the loss of GAGs from small articular cartilage explants during histological processing. However, for immunohistochemical studies, Safranin O cannot be included in the processing solutions, because it may interfere.

  13. 99Tc-亚甲基二膦酸盐对胶原诱导性关节炎大鼠滑膜及软骨凋亡相关因子的影响%Effects of 99Tc-MDP on synoviocytes and articular chondrocytes apoptosis associated factors on CIA rats

    Institute of Scientific and Technical Information of China (English)

    胡绍先; 孔芳; 柯丹; 苏敏; 雷小妹; 涂巍; 沈桂芬; 何培根

    2009-01-01

    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 chon-drocytes. 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 in-jected 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 orMTX. (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 < 0.001]. In the synovioeytes of 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 <0.001), while the level of bax was slightly increased and the ratio of bcl-2/bax was significantly decreased. (3)The expression level of bcl-2 and bax in the articular chondro-cytes of CIA model group [(20.20 ± 2.78) %, (36.40 ± 1.67) %] was significantly higher than control group [(9.91±4.09)%, (6.71 ±3.50)%; q=10.51, 37.01, allP<0.001]. Compared with CIA model group, the expression level of bcl-2 in articular chondrocytes of 99Tc-MDP group [(26. 58 ± 2

  14. 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

  15. Evidence for a negative Pasteur effect in articular cartilage.

    Science.gov (United States)

    Lee, R B; Urban, J P

    1997-01-01

    Uptake of external glucose and production of lactate were measured in freshly-excised bovine articular cartilage under O2 concentrations ranging from 21% (air) to zero (N2-bubbled). Anoxia (O2 concentration Pasteur effect in bovine articular cartilage. Anoxia also suppressed glycolysis in articular cartilage from horse, pig and sheep. Inhibitors acting on the glycolytic pathway (2-deoxy-D-glucose, iodoacetamide or fluoride) strongly decreased aerobic lactate production and ATP concentration, consistent with the belief that articular cartilage obtains its principal supply of ATP from substrate-level phosphorylation in glycolysis. Azide or cyanide lowered the ATP concentration in aerobic cartilage to approximately the same extent as did anoxia but, because glycolysis (lactate production) was also inhibited by these treatments, the importance of any mitochondrial ATP production could not be assessed. A negative Pasteur effect would make chondrocytes particularly liable to suffer a shortage of energy under anoxic conditions. Incorporation of [35S]sulphate into proteoglycan was severely curtailed by treatments, such as anoxia, which decreased the intracellular concentration of ATP.

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

    Science.gov (United States)

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

    1997-01-01

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

  17. 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.

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

    Science.gov (United States)

    Sun, Margaret Man-Ger; Beier, Frank

    2014-03-01

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

  19. 3D Culture of Chondrocytes in Gelatin Hydrogels with Different Stiffness

    Directory of Open Access Journals (Sweden)

    Xiaomeng Li

    2016-07-01

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

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

    NARCIS (Netherlands)

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

    2003-01-01

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

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

    NARCIS (Netherlands)

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

    2003-01-01

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

  2. Identification of the calcitonin receptor in osteoarthritic chondrocytes

    Directory of Open Access Journals (Sweden)

    Christensen Tjorbjoern

    2011-10-01

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

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

    Directory of Open Access Journals (Sweden)

    Adel Tekari

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

  4. 组织工程化软骨细胞和骨髓间充质干细胞用于修复同种异体关节软骨缺损%Tissue engineered chondrocytes and bone marrow mesenchymal stem cells for the repair of articular cartilage defects

    Institute of Scientific and Technical Information of China (English)

    孙皓; 左健

    2012-01-01

    BACKGROUND: As the articular cartilage almost has no self-repair capacity, and in clinic, the repair on it mainly depends on the autologous or allogenic cartilage transplantation, perichondrium or periosteal transplantation and the chondrocytes transplantation. The limitation of autologous cartilage source and the chronic immune rejection of allograft cartilage may eventually lead to the poor prognosis. The cartilage repaired by perichondrium or periosteum transplantation is easy to degenerate which may lead to a poor repair result.OBJECTIVE: To review the research progress of tissue engineered chondrocytes , bone marrow mesenchymal stem cells and the co-culture of them on the repair of allogeneic cartilage defects.METHODS: A computer-based search on the PubMed database and CNKI database from January 1994 to January 2012 was performed for the articles on tissue engineered chondrocytes and bone marrow mesenchymal stem cells for the repair of allograft articular cartilage defects. The English key words were "cartilage defect, allograft, chondrocyte, mesenchymal stem cells, bone marrow mesenchymal stem cells" and the Chinese keywords were "cartilage defect, allograft, chondrocyte, bone marrow mesenchymal stem cells". The repetitive articles and the articles not in English or Chinese were eliminated, and finally, a total of 35 articles were included to review.RESULTS AND CONCLUSION: With the continuous improvement of in vitro cell culture methods, chondrocytes can be isolated from the tough cartilage, and a large number of high-purity chondrocytes and new chondrocytes can be obtained. Due to the low proliferative capacity of the chondrocytes, subculture may easily lead to aging and dedifferentiation; however, the content of bone marrow mesenchymal stem cells is low in adult bone marrow, with the increasing of the passages number, the chondrogenic potential is significantly decreased. When the bone marrow mesenchymal stem cells co-cultured with chondrocytes, they can

  5. 微管蛋白破坏对软骨细胞代谢功能的影响%Disassembly of tubulin cytoskeleton disrupts the homeostasis of articular cartilage chondrocytes

    Institute of Scientific and Technical Information of China (English)

    郭恒; 段王平; 李琦; 曹晓明; 王磊; 卫小春

    2011-01-01

    Objective To investigate the influence of tubulin disassembly on the in vitro metabolism of articular chondrocytes. Methods Eight New Zealand rabbits aged 2 months were sacrificed by air embolism. The full-thickness cartilages were harvested from both knees under sterile conditions. Then 0. 4% pronase and 0. 025% Ⅱ collagenase were used to digest for primary chondrocytes. The cells were cultured for 3 days after attachment and then divided into the control and experimental groups. The control group continued culturing with primary medium and the experimental group with colchicine, a tubulin destructive & 2, the early apoptosis of two cell groups was 0. 1 μmol/L. At Days 1 agent,at a final concentration of assayed with phosphatidylserine ( Annexin Ⅴ). At Day 6, the morphological changes of cells were observed by hematoxylin and eosin staining. At Days 3, 6 & 9, the expression levels of type Ⅱ collagen, proteoglycan and MMP (matrix metalloproteinase)-13 mRNA were measured by real time quantitative fluorescent reverse transcription-polymerase chain reaction (real time RT-PCR). Meanwhile at Days 3, 6 & 9, the cultured supernatant of each group was obtained to detect the contents of type Ⅱ collagen and proteoglycan by the methods of ELISA (enzyme-linked immunosorbent assay) and Alcian blue. Results At Day 2, the early cell apoptosis rate of the experimental group was higher than that of the control group ( P <0. 05 ). At Day 6 post-dosing, the experimental cell morphology was found to be irregular and polygonal. Hyperchromatic nucleus and division phase increased while matrix cell decreased. At Days 3, 6 & 9, the expressions of collagen and proteoglycan mRNA in the experimental group decreased versus those in the control group (P <0. 05). The level of MMP-13 mRNA showed no significant changes in the control group at Day 3. And it 0. 01). Meanwhile, type Ⅱ collagen and glycosaminoglycan were & 9 (P<significantly increased at Days 6 & 9 than those in the

  6. The inhibitory effect of salmon calcitonin on tri-iodothyronine induction of early hypertrophy in articular cartilage.

    Directory of Open Access Journals (Sweden)

    Pingping Chen-An

    Full Text Available OBJECTIVE: Salmon calcitonin has chondroprotective effect both in vitro and in vivo, and is therefore being tested as a candidate drug for cartilage degenerative diseases. Recent studies have indicated that different chondrocyte phenotypes may express the calcitonin receptor (CTR differentially. We tested for the presence of the CTR in chondrocytes from tri-iodothyronin (T3-induced bovine articular cartilage explants. Moreover, investigated the effects of human and salmon calcitonin on the explants. METHODS: Early chondrocyte hypertrophy was induced in bovine articular cartilage explants by stimulation over four days with 20 ng/mL T3. The degree of hypertrophy was investigated by molecular markers of hypertrophy (ALP, IHH, COLX and MMP13, by biochemical markers of cartilage turnover (C2M, P2NP and AGNxII and histology. The expression of the CTR was detected by qPCR and immunohistochemistry. T3-induced explants were treated with salmon or human calcitonin. Calcitonin down-stream signaling was measured by levels of cAMP, and by the molecular markers. RESULTS: Compared with untreated control explants, T3 induction increased expression of the hypertrophic markers (p<0.05, of cartilage turnover (p<0.05, and of CTR (p<0.01. Salmon, but not human, calcitonin induced cAMP release (p<0.001. Salmon calcitonin also inhibited expression of markers of hypertrophy and cartilage turnover (p<0.05. CONCLUSIONS: T3 induced early hypertrophy of chondrocytes, which showed an elevated expression of the CTR and was thus a target for salmon calcitonin. Molecular marker levels indicated salmon, but not human, calcitonin protected the cartilage from hypertrophy. These results confirm that salmon calcitonin is able to modulate the CTR and thus have chondroprotective effects.

  7. The Effects of Vitamin D3 on Proliferation and Apoptosis of Primary Cultured Chondrocytes from Human Articular Cartilage%活性维生素D3对体外原代培养人骨关节炎软骨细胞的影响

    Institute of Scientific and Technical Information of China (English)

    张良; 郭艾

    2012-01-01

    Objective To obtain articular chondrocyte from patients with osteoarthritis by primary culture,to explore the relationship between different concentrate [1, 25-(OH)2D3]and proliferation, apoptosis of chondrocytes. Methods Human articular chondrocytes were obtained by modified digestive method with enzyme in vitro and inden-tified by alkaline phosphatase (AKP) staining. OA Chondrocytes were cultured in medium with different concentrate [1,25-(OH)2D3]. MMT method was used to assay proliferation of chondrocytes. Optical density(OD) at 490nm was determined by ELISA. Flow cytometry was used to assay the apoptosis ratio at different time point. Results 10~5 u-mol/L[l ,25-(OH)2D3]can promote the proliferation of chondrocytes obviously,and the proper react time point is 48 hour(P<0. 01). At the same time 10-5 umol/L[1, 25-(OH)2D3]can inhibit the apoptosis ratio obviously at 48 hour time point. Conclusion To promote the proliferation and inhibit the apoptosis of OA chondrocytes in vitro,the better concentration of [1,25-(OH)2D3]is 1×10-5 umol/L. 48 hour after[1,25-(OH)2D3]added is the best time for the experiment objective.%目的 探讨不同浓度的1α,25二羟基维生素D3[1,25 -(OH)2D3]对体外培养的人骨关节炎患者关节软骨细胞的增殖及凋亡率的影响.方法 酶二步消化法体外分离培养人软骨细胞,以碱性磷酸酶染色法鉴定.加入不同剂量的[1,25 (OH)2D3],通过噻唑蓝比色试验检测细胞存活和增殖情况,以及用流式细胞仪检测软骨细胞在不同药物浓度、不同作用时间的凋亡率.结论 [1,25 (OH)2D3]作用于入骨关节炎软骨细胞的最佳作用浓度为1×10-5umol/L,最佳作用时间点为48h.高浓度的[1,25-(OH)2D3]能明显促进细胞坏死,极低浓度的[1,25 -(OH)2D3]对软骨细胞增殖、凋亡无明显影响.

  8. Gel structure has an impact on pericellular and extracellular matrix deposition, which subsequently alters metabolic activities in chondrocyte-laden PEG hydrogels.

    Science.gov (United States)

    Nicodemus, G D; Skaalure, S C; Bryant, S J

    2011-02-01

    While designing poly(ethylene glycol) hydrogels with high moduli suitable for in situ placement is attractive for cartilage regeneration, the impact of a tighter crosslinked structure on the organization and deposition of the matrix is not fully understood. The objectives of this study were to characterize the composition and spatial organization of new matrix as a function of gel crosslinking and study its impact on chondrocytes in terms of anabolic and catabolic gene expression and catabolic activity. Bovine articular chondrocytes were encapsulated in hydrogels with three crosslinking densities (compressive moduli 60, 320 and 590 kPa) and cultured for 25 days. Glycosaminoglycan production increased with culture time and was greatest in the gels with lowest crosslinking. Collagens II and VI, aggrecan, link protein and decorin were localized to pericellular regions in all gels, but their presence decreased with increasing gel crosslinking. Collagen II and aggrecan expression were initially up-regulated in gels with higher crosslinking, but increased similarly up to day 15. Matrix metalloproteinase (MMP)-1 and MMP-13 expression were elevated (∼25-fold) in gels with higher crosslinking throughout the study, while MMP-3 was unaffected by gel crosslinking. The presence of aggrecan and collagen degradation products confirmed MMP activity. These findings indicate that chondrocytes synthesized the major cartilage components within PEG hydrogels, however, gel structure had a significant impact on the composition and spatial organization of the new tissue and on how chondrocytes responded to their environment, particularly with respect to their catabolic expression.

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

    Science.gov (United States)

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

    2015-04-13

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

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

    Science.gov (United States)

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

    2013-01-01

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

  11. 低氧对脂肪干细胞和关节软骨细胞三维共培养成软骨能力的影响%Hypoxia effects on the chondrogenic differentiation of three-dimensional co-cultured adipose-derived stem cells and articular chondrocytes

    Institute of Scientific and Technical Information of China (English)

    戴兵; 徐海艇; 金海东; 陈辉; 蔡建武; 范时洋; 潘骏

    2014-01-01

    背景:多项体内外研究表明低氧和共培养均促进干细胞向软骨细胞方向分化。  目的:观察低氧对脂肪干细胞和关节软骨细胞三维共培养成软骨能力的影响。  方法:脂肪干细胞和关节软骨细胞二者按3∶1比例混合,以5×1010 L-1接种于聚乳酸-羟基乙酸共聚物/明胶支架上,分别在常氧(体积分数为20% O2)、低氧(体积分数为5% O2  结果与结论:低氧组苏木精-伊红染色显示大量细胞及细胞外基质生成,阿尔新蓝染色显示有大量糖胺多糖生成,免疫组化显示Ⅱ型胶原表达强阳性,且DNA、糖胺聚糖、羟脯氨酸等各项指标均高于常氧组。表明低氧促进脂肪干细胞和关节软骨细胞共培养成软骨分化。  行组织学分析,阿尔新蓝染色鉴定糖胺多糖的合成,免疫组化鉴定Ⅱ型胶原的表达,并测定各组支架-细胞复合物的DNA、糖胺聚糖、羟脯氨酸含量。%BACKGROUND:Many in vivo and in vitro experiments indicate that hypoxic co-cultures promote stem cells differentiate into chondrocytes. OBJECTIVE:To evaluate the influence of hypoxia on the chondrogenic differentiation of three-dimensional co-cultured adipose-derived stem cells and articular chondrocytes. METHODS:Adipose-derived stem cells and articular chondrocytes were mixed at the ratio of 3:1, then the mixed cells were seeded onto poly(lactic-co-glycolic acid)-gelatin scaffold at the ultimate concentration of 5.0×1010/L. The cells were cultured in normoxia (20%O 2 ) and hypoxic (5%O 2 ) conditions for 6 weeks. After culture, hematoxylin and eosin staining was performed for histological structure analysis, and alcian blue staining was used to evaluate glycosaminoglycan synthesis. Type II col agen expression was detected by immunohistochemistry staining. The content of DNA, glycosaminoglycan and hydroxyproline in the scaffold-cellcomplex was measured. RESULTS AND CONCLUSION:In the hypoxia group

  12. Human stem cells and articular cartilage regeneration.

    Science.gov (United States)

    Inui, Atsuyuki; Iwakura, Takashi; Reddi, A Hari

    2012-11-05

    The regeneration of articular cartilage damaged due to trauma and posttraumatic osteoarthritis is an unmet medical need. Current approaches to regeneration and tissue engineering of articular cartilage include the use of chondrocytes, stem cells, scaffolds and signals, including morphogens and growth factors. Stem cells, as a source of cells for articular cartilage regeneration, are a critical factor for articular cartilage regeneration. This is because articular cartilage tissue has a low cell turnover and does not heal spontaneously. Adult stem cells have been isolated from various tissues, such as bone marrow, adipose, synovial tissue, muscle and periosteum. Signals of the transforming growth factor beta superfamily play critical roles in chondrogenesis. However, adult stem cells derived from various tissues tend to differ in their chondrogenic potential. Pluripotent stem cells have unlimited proliferative capacity compared to adult stem cells. Chondrogenesis from embryonic stem (ES) cells has been studied for more than a decade. However, establishment of ES cells requires embryos and leads to ethical issues for clinical applications. Induced pluripotent stem (iPS) cells are generated by cellular reprogramming of adult cells by transcription factors. Although iPS cells have chondrogenic potential, optimization, generation and differentiation toward articular chondrocytes are currently under intense investigation.

  13. Human Stem Cells and Articular Cartilage Regeneration

    Directory of Open Access Journals (Sweden)

    A. Hari Reddi

    2012-11-01

    Full Text Available  The regeneration of articular cartilage damaged due to trauma and posttraumatic osteoarthritis is an unmet medical need. Current approaches to regeneration and tissue engineering of articular cartilage include the use of chondrocytes, stem cells, scaffolds and signals, including morphogens and growth factors. Stem cells, as a source of cells for articular cartilage regeneration, are a critical factor for articular cartilage regeneration. This is because articular cartilage tissue has a low cell turnover and does not heal spontaneously. Adult stem cells have been isolated from various tissues, such as bone marrow, adipose, synovial tissue, muscle and periosteum. Signals of the transforming growth factor beta superfamily play critical roles in chondrogenesis. However, adult stem cells derived from various tissues tend to differ in their chondrogenic potential. Pluripotent stem cells have unlimited proliferative capacity compared to adult stem cells. Chondrogenesis from embryonic stem (ES cells has been studied for more than a decade. However, establishment of ES cells requires embryos and leads to ethical issues for clinical applications. Induced pluripotent stem (iPS cells are generated by cellular reprogramming of adult cells by transcription factors. Although iPS cells have chondrogenic potential, optimization, generation and differentiation toward articular chondrocytes are currently under intense investigation.

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

    Science.gov (United States)

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

    2004-05-01

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

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

    Science.gov (United States)

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

    2010-02-01

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

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

    Institute of Scientific and Technical Information of China (English)

    刘丰; 彭昊; 周建林; 方洪松; 邓爽; 杨骁; 翁金清

    2015-01-01

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

  17. One intra-articular injection of hyaluronan prevents cell death and improves cell metabolism in a model of injured articular cartilage in the rabbit

    NARCIS (Netherlands)

    Jansen, Edwin J. P.; Ernans, Pieter J.; Douw, Conny M.; Guidemond, Nick A.; Van Rhijn, Lodewijk W.; Bulstra, Sjoerd K.; Kuijer, Roell

    2008-01-01

    The purpose of this study was to determine the effect of one intra-articular injection of hyaluronan on chondrocyte death and metabolism in injured cartilage. Twenty-three 6-month-old rabbits received partial-thickness articular cartilage defects created on each medial femoral condyle. In order to e

  18. 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.

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

    Institute of Scientific and Technical Information of China (English)

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

    2008-01-01

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

  20. Chondrocyte behavior on nanostructured micropillar polypropylene and polystyrene surfaces

    Energy Technology Data Exchange (ETDEWEB)

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

    2014-10-01

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

  1. [Structure of the articular cartilage in the middle aged].

    Science.gov (United States)

    Kop'eva, T N; Mul'diiarov, P Ia; Bel'skaia, O B; Pastel', V B

    1983-10-01

    In persons 17-83 years of age having no articular disorders 39 samples of the patellar articular cartilage, the articulated surface and the femoral head have been studied histochemically, histometrically and electron microscopically. Age involution of the articular cartilage is revealed after 40 years of age as a progressive decrease in chondrocytes density in the superficial and (to a less degree) in the intermediate zones. This is accompanied with a decreasing number of 3- and 4-cellular lacunae and with an increasing number of unicellular and hollow lacunae. In some chondrocytes certain distrophic and necrotic changes are revealed. In the articular matrix the zone with the minimal content of glycosaminoglycans becomes thicker and keratansulfate content in the territorial matrix of the cartilage deep zone grows large.

  2. 应用生物蛋白胶与胚胎软骨细胞混合移植修复兔膝关节实验性软骨缺损区%Repair of experimental defects of articular cartilage in rabbits with homografts of fibrin sealant and embryonic chondrocytes

    Institute of Scientific and Technical Information of China (English)

    陆敏安; 杨渊; 肖增明; 李世德

    2005-01-01

    BACKGROUND:Research about the repair of articular cartilage with heterograft chondrocytes is frequently reported, but the method may cause immune rejection. Since the embryonic cells possess lower antigenicity and stronger proliferation capability, it is hoped that they can be used as a novel carrier substitute in tissue engineering research.DESIGN: A randomized grouping observation and comparative experiment.SETTING: Histological Embryonic Laboratory in Guangxi Medical University.MATERIALS: A big white adult New Zealand rabbit pregnant for 4 weeks was adopted; and another 24 big white adult New Zealand rabbits were selected, with no limitationin whether they were female or male and with a body mass of 2 to 2.5 kg.METHODS: This experiment was carried out at the Histological Embryonic Laboratory in Guangxi Medical University between December 2000and June 2002. The models of defects in articular cartilage were made artificially in femur medial malleolus of the mature rabbits. In the experimental group, defects were repaired by the implantation of Fibrin Sealant and embryonic chondrocytes mixture, but for the control group, only Fibrin Sealant was implanted or nothing was done about the defect. The restoration of articular cartilage defect was then observed 4,8 and 12 weeks after the operation, and was scored according to modified Pineda's method. The standard consists of 5 items, I.e., cellular morphology, matrix staining, surfacing smoothness, cartilage thickness and host union. 0 refers to normal and the higher the score is, the more serious the pathological changes are.MAIN OUTCOME MEASURES: ①The general observation of rabbit knee joint; ② Histological observation of rabbit knee joints; ③ Histological semi-quantitative score of articular cartilage; ④ Appraisal of the curative effect of articular cartilage defects.RESULTS: Totally 24 rabbits were enrolled in this experiment and all entered the stage of result analysis. ① The general observation of rabbit

  3. 透明质酸/壳聚糖/pEGFP纳米粒介导体外基因转染关节软骨细胞与滑膜细胞的比较%Hyaluronic acid/chitosan/pEGFP nanoparticles mediated gene transfection of articular chondrocytes and synoviocytes in vitro

    Institute of Scientific and Technical Information of China (English)

    吕璐璐; 卢华定; 陆慧琼; 张富程; 赵慧清

    2012-01-01

    observed under scanning electron microscopy. The sizes and zeta - potentials of the nanoparticles were measured by a Marven - nano laser diffractometer. The binding of pDNA with chitosan and the release of pDNA from HA/CS/pDNA nanoparticles were e-valuated by agarose gel electrophoresis analysis. The gene transfection experiments in vitro were performed with rabbits chondro-cytes and synoviocytes. The gene transfection efficiency was measured by flow cytometry and fluorescence microscope. [ Re-Sults] HA/CS/pDNA nanoparticles were mainly spherical with an average size of ( 142. 5 ± 20. 3) nm, zeta - potential of (25.99 ± 8.48) mV and polydispersity index of (0.283 ± 0.089) . The agarose gel electrophoresis analysis confirmed that they can effeclively protect pDNA from degradation against Dnase L By adjusting the pH value to 7. 5 or even higher, or adding chi-tosanase, the release of pDNA from the nanoparticles could be promoted Gene transfection in vitro proved that HA/CS/pDNA nanoparticles were efficient in transfecting rabbits chondrocytes and synoviocytes, and the expressions of green fluorescent proteins were observed under fluorescent microscope. And their transfection efficiency against chondrocytes were much higher than that of synoviocytes, and also, were much higher than that of the naked pDNA or the CS/pDNA when transfected chondrocytes.[Conclusion] HA/CS/pDNA nanoparticles were effective novel non - viral gene transfer vectors, which could mediate gene transfering of articular chondrocytes and synoviocytes. Although their transfection efficiency was cell dependent, they provided potential targeting non - viral gene transfer vectors for joint disease.

  4. Repair of articular cartilage defects in rabbits through tissue-engineered cartilage constructed with chitosan hydrogel and chondrocytes%新型壳聚糖水凝胶结合软骨细胞修复兔关节软骨缺损的实验研究

    Institute of Scientific and Technical Information of China (English)

    Ming ZHAO; Zhu CHEN; Kang LIU; Yu-qing WAN; Xu-dong LI; Xu-wei LUO; Yi-guang BAI; Ze-long YANG; Gang FENG

    2015-01-01

    Objective: In our previous work, we prepared a type of chitosan hydrogel with excelent biocompatibility. In this study, tissue-engineered cartilage constructed with this chitosan hydrogel and costal chondrocytes was used to repair the articular cartilage defects. Methods: Chitosan hydrogels were prepared with a crosslinker formed by com-bining 1,6-disocyanatohexane and polyethylene glycol. Chitosan hydrogel scaffold was seeded with rabbit chondro-cytes that had been cultured for one weekin vitro to form the preliminary tissue-engineered cartilage. This preliminary tissue-engineered cartilage was then transplanted into the defective rabbit articular cartilage. There were three treatment groups: the experimental group received preliminary tissue-engineered cartilage; the blank group received pure chitosan hydrogels; and, the control group had received no implantation. The knee joints were harvested at predetermined time. The repaired cartilage was analyzed through gross morphology, histologicaly and immuno-histochemicaly. The repairs were scored according to the international cartilage repair society (ICRS) standard. Results: The gross morphology results suggested that the defects were repaired completely in the experimental group after twelve weeks. The regenerated tissue connected closely with subchondral bone and the boundary with normal tissue was fuzzy. The cartilage lacuna in the regenerated tissue was similar to normal cartilage lacuna. The results of ICRS gross and histological grading showed that there were significant differences among the three groups (P  创新点:利用自主研发的具有良好生物相容性和稳定性的壳聚糖水凝胶与软骨细胞,在体外初步构建组织工程软骨,并尝试利用其修复缺损的关节软骨,从而为关节软骨缺损的修复提供了一种新的治疗方法。  方法:取兔肋软骨体外培养扩增,获得P2代软骨细胞,将其种植到冻干的壳聚糖水凝胶上,体

  5. Tissue Engineering Based Therapy for Articular Cartilage Defects - A New Approach

    Directory of Open Access Journals (Sweden)

    Abraham S

    2007-01-01

    Full Text Available Background: Articular cartilage, the load-bearing tissue in diarthrodial joints, when damaged due to trauma could lead to osteoarthritis. At present Autologous Cartilage Implantation is an established method in which patients own chondrocytes are isolated and then implanted after in vitro expansion over the affected area with bovine or porcine collagen matrix. This procedure results in more of Collagen Type I during in vitro expansion, which eventually becomes fibrocartilage. Also it requires growth factors. We have in this study tried growing human Chondrocytes without growth factors using synthetic scaffolds to grow more Collagen Type II Materials and Methods: Human cartilage specimens were harvested through arthroscopy from the non-weight bearing area of the knee joint from 13 patients who underwent surgical procedures of the knee joint after getting their informed consent. The tissues were transported in saline taking 1 hour to laboratory and subjected to digestion with Collagenase type II for 16~18 Hrs. The chondrocyte cells obtained after dissociation were divided into two groups for culture. Gr. I were embedded in a Thermogelation polymer (TGP and Gr. II in basal culture media (DMEM + Ascorbic Acid without using any growth factors. The Group II cells were viable only for 4 weeks and then started degenerating. The TGP-Chondrocytes scaffolds were grown for 16 weeks and the specimens were harvested at 4, 8, 12 and 16-week intervals and their morphology and molecular characteristics were studied by H&E staining, S-100 protein analysis and RT-PCR.Results: Human chondrocytes could be cultured in both TGP (group I and Basal culture media (group II. The Gr. I cells were viable upto the 16th week while the Group II chondrocytes started degenerating after the 4 week. Both the groups were proven positive for S-100 protein, a Chondrocyte specific marker protein; Gr. II specimens after 4 weeks, and Gr. I specimens after 4, 8, 12 and 16 weeks. RT

  6. The properties of bioengineered chondrocyte sheets for cartilage regeneration

    Directory of Open Access Journals (Sweden)

    Ota Naoshi

    2009-03-01

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

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

    Science.gov (United States)

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

    2015-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Elena Vasil'evna Chetina

    2010-01-01

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

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

    NARCIS (Netherlands)

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

    2011-01-01

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

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

    Science.gov (United States)

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

    2008-04-01

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

  11. Induction of advanced glycation end products and alterations of the tensile properties of articular cartilage

    NARCIS (Netherlands)

    Chen, A.C.; Temple, M.M.; Ng, D.M.; Verzijl, N.; Groot, J. de; TeKoppele, J.M.; Sah, R.L.

    2002-01-01

    Objective. To determine whether increasing advanced glycation end products (AGEs) in bovine articular cartilage to levels present in aged human cartilage modulates the tensile biomechanical properties of the tissue. Methods. Adult bovine articular cartilage samples were incubated in a buffer solutio

  12. MicroRNA-33 suppresses CCL2 expression in chondrocytes.

    Science.gov (United States)

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

    2016-06-01

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

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

    Science.gov (United States)

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

    2009-10-01

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

  14. 纳米硒、亚硒酸钠对大骨节病关节软骨细胞凋亡和活性氧的影响%Effects of selenium nanoparticles and sodium selenite on apoptosis and reactive oxygen species in chondrocytes from articular cartilage in patients with Kashin-Beck disease

    Institute of Scientific and Technical Information of China (English)

    姚筱; 马玮娟; 常彦海; 郭团茂; 吴翠艳; 郭雄

    2015-01-01

    Objective To compare the effects of selenium nanoparticles (Nano-Se) and sodium selenium (Na2SeO3) on apoptosis and reactive oxygen species (ROS) of articular chondrocytes from patients with Kashin-Beck Disease (KBD) in vitro,and provide a scientific basis for preventing KBD.Methods The subjects with KBD were diagnosed on National Clinical Diagnostic Criteria of KBD (WS/T207-2010),articular cartilage from 8 patients undertaken joint replacement operation were collected.In vitro,chondrocytes were treated with concentration of 0,25,50,100,200,300,400 and 500 μg/L of Nano-Se and Na2SeO3 for 5 d,respectively.Cell growth was detected by MTT assay,and the highest concentration and time corresponding to the highest survival rate of Nano-Se and Na2SeO3 were used in the following experiment.KBD chondrocytes were treated with Nano-Se and Na2SeO3,and divided into control group,Na2SeO3 group,Nano-Se group according to the randomized design.Each group had 8 cases.The cell apoptosis and ROS were detected by flow cytometry.Results The optimal intervention concentration of Nano-Se and Na2SeO3 was 100 and 400 μg/L,respectively.The optimal intervention time of NanoSe and Na2SeO3 both was 3 days.There was a significant decrease in the total and terminal apoptosis,ROS level of chondrocytes in Nano-Se group [(4.67 ± 0.89)%,(1.51 ± 0.48)%,(56.04 ± 4.81)%] and Na2SeO3 group [(7.07 ±0.25)%,(4.37 ± 0.37)%,(87.13 ± 6.60)%] compared with those of control group [(9.95 ± 0.38)%,(6.93 ± 0.42)%,(125.17 ± 16.60)%,all P < 0.01].The difference of early apoptotic rate among control group,Na2SeO3 group,NanoSe group [(3.02 ± 0.41)%,(2.7 ± 0.46)%,(3.16 ± 0.56)%] was not statistically significant (F =2.11,P =0.35).Conclusion Appropriate concentration of Nano-Se can significantly decrease oxidative stress of KBD chondrocytes and inhibit apoptosis compared to Na2SeO3.%目的 比较纳米硒(Nano-Se)、亚硒酸钠(Na2SeO3)对体外培养的大骨节病关节软骨

  15. 牛关节软骨中Ⅱ型胶原和硫酸软骨素的综合提取与表征%Extraction and Characterization of Type H Collagen and Chondroitin Sulfate From Bovine Articular Cartilage

    Institute of Scientific and Technical Information of China (English)

    毕彩霞; 王瑞瑞; 金京国; 李德富

    2013-01-01

    In the present study,fresh bovine articular cartilage is used as raw material to extract type Ⅱ collagen by acid and pepsin method after degreasing and decalcification.The residue is used to extract chondroitin sulfate by dilute alkali and trypsin method.Then the FT-IR,AFM and thermal gravimetric analysis are used to detect the structure and characteristic of type Ⅱ collagen and chondroitin sulfate.The results show that this extraction process does not damage the original native conformation structure of type Ⅱ collagen and chondroitin sulfate,and chondroitin sulfate has a high purity.%以新鲜牛关节软骨为原料,脱脂脱钙后采用乙酸与胃蛋白酶相结合的方法提取Ⅱ型胶原,然后采用稀碱与胰蛋白酶相结合的方法提取剩余残渣中的硫酸软骨素,并采用FT-IR、AFM和热重分析对所得Ⅱ型胶原和硫酸软骨素进行检测.结果表明,该方法提取得到的Ⅱ型胶原和硫酸软骨素均很好的保持了其天然结构,而且所得硫酸软骨素具有较高的纯度.

  16. Computational aspects in mechanical modeling of the articular cartilage tissue.

    Science.gov (United States)

    Mohammadi, Hadi; Mequanint, Kibret; Herzog, Walter

    2013-04-01

    This review focuses on the modeling of articular cartilage (at the tissue level), chondrocyte mechanobiology (at the cell level) and a combination of both in a multiscale computation scheme. The primary objective is to evaluate the advantages and disadvantages of conventional models implemented to study the mechanics of the articular cartilage tissue and chondrocytes. From monophasic material models as the simplest form to more complicated multiscale theories, these approaches have been frequently used to model articular cartilage and have contributed significantly to modeling joint mechanics, addressing and resolving numerous issues regarding cartilage mechanics and function. It should be noted that attentiveness is important when using different modeling approaches, as the choice of the model limits the applications available. In this review, we discuss the conventional models applicable to some of the mechanical aspects of articular cartilage such as lubrication, swelling pressure and chondrocyte mechanics and address some of the issues associated with the current modeling approaches. We then suggest future pathways for a more realistic modeling strategy as applied for the simulation of the mechanics of the cartilage tissue using multiscale and parallelized finite element method.

  17. 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.

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

    Science.gov (United States)

    Xu, Jiaming; Zhang, Changqing

    2014-08-01

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

  19. Improving Joint Function Using Photochemical Hydrogels for Articular Surface Repair

    Science.gov (United States)

    2014-10-01

    type I collagen (11.4 mg/mL) was obtained from BD Biosciences, Bedford, MA. Ham F12, 10% fetal bovine serum. 1% antibiotic /antimycotic liquid and 1...mixed in an Eppendorf tube with chondrocyte media [Ham F12, 10% fetal bovine serum (Gibco), 1% antibiotic /antimycotic liquid, 1% MEM nones- sential...solution in Ham F-12 medium (Life Sciences, Grand Island, NY) containing 1% Anti-Anti ( antibiotic , antimycotic, Life Sciences) overnight at 37C

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

    Science.gov (United States)

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

    2014-02-01

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

  1. Tensorial electrokinetics in articular cartilage.

    Science.gov (United States)

    Reynaud, Boris; Quinn, Thomas M

    2006-09-15

    Electrokinetic phenomena contribute to biomechanical functions of articular cartilage and underlie promising methods for early detection of osteoarthritic lesions. Although some transport properties, such as hydraulic permeability, are known to become anisotropic with compression, the direction-dependence of cartilage electrokinetic properties remains unknown. Electroosmosis experiments were therefore performed on adult bovine articular cartilage samples, whereby fluid flows were driven by electric currents in directions parallel and perpendicular to the articular surface of statically compressed explants. Magnitudes of electrokinetic coefficients decreased slightly with compression (from approximately -7.5 microL/As in the range of 0-20% compression to -6.0 microL/As in the 35-50% range) consistent with predictions of microstructure-based models of cartilage material properties. However, no significant dependence on direction of the electrokinetic coupling coefficient was detected, even for conditions where the hydraulic permeability tensor is known to be anisotropic. This contrast may also be interpreted using microstructure-based models, and provides insights into structure-function relationships in cartilage extracellular matrix and physical mediators of cell responses to tissue compression. Findings support the use of relatively simple isotropic modeling approaches for electrokinetic phenomena in cartilage and related materials, and indicate that measurement of electrokinetic properties may provide particularly robust means for clinical evaluation of cartilage matrix integrity.

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

    Indian Academy of Sciences (India)

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

    2015-03-01

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

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

    Science.gov (United States)

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

    2015-06-25

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

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

    Science.gov (United States)

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

    2017-02-28

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

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

    Science.gov (United States)

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

    2016-09-01

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

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

    Institute of Scientific and Technical Information of China (English)

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

    2011-01-01

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

  7. A study of crystalline biomaterials for articular cartilage bioengineering

    Energy Technology Data Exchange (ETDEWEB)

    Gross-Aviv, Talia [Department of Biotechnology Engineering, Ben-Gurion University of the Negev, Beer Sheva, 84105 (Israel)], E-mail: taliag@bgu.ac.il; DiCarlo, Bryan B. [Department of Bioengineering, Rice University, Houston, TX 77003 (United States)], E-mail: bdicarlo@rice.edu; French, Margaret M. [Department of Bioengineering, Rice University, Houston, TX 77003 (United States)], E-mail: mmfrench@rice.edu; Athanasiou, Kyriacos A. [Department of Bioengineering, Rice University, Houston, TX 77003 (United States)], E-mail: athanasiou@rice.edu; Vago, Razi [Department of Biotechnology Engineering, Ben-Gurion University of the Negev, Beer Sheva, 84105 (Israel)], E-mail: rvago@bgu.ac.il

    2008-12-01

    This study examines the suitability of marine origin coral species, Porites lutea (POR) and the hydrozoan Millepora dichotoma (MIL), for use as novel three dimensional growth matrices in the field of articular cartilage tissue engineering. Therefore, mesenchymal stem cells (MSCs) and chondrocytes were grown on the skeletal material obtained from each of these two organisms to investigate their potential use as three dimensional scaffolding for cartilage tissue growth. Chondrogenic induction of MSCs was achieved by addition of transforming growth factor-{beta}1 (TGF-{beta}1) and insulin growth factor-I (IGF-I). Cell adherence, proliferation, differentiation and tissue development were investigated through six weeks of culture. Cartilage tissue growth and chondrocytic phenotype maintenance of each cell type were examined by cell morphology, histochemical analyses, expression of collagen type II and quantitative measures of glycosaminoglycan (GAG) content. The MSCs and the chondrocytes were shown good adherence to the scaffolds and maintenance of the chondrocytic phenotype in the initial stages of culture. However after two weeks of culture on MIL and three weeks on POR these cultures began to exhibit signs of further differentiation and phenotypic loss. The shown results indicated that POR was a better substrate for chondrocytes phenotype maintenance than MIL. We believe that surface modification of POR combined with mechanical stimuli will provide a suitable environment for chondrogenic phenotype maintenance. Further investigation of POR and other novel coralline biomatrices is indicated and warranted in the field of cartilage tissue engineering applications.

  8. 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

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

    DEFF Research Database (Denmark)

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

    2007-01-01

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

  10. Transcriptional profiling differences for articular cartilage and repair tissue in equine joint surface lesions

    Directory of Open Access Journals (Sweden)

    Stromberg Arnold J

    2009-09-01

    Full Text Available Abstract Background Full-thickness articular cartilage lesions that reach to the subchondral bone yet are restricted to the chondral compartment usually fill with a fibrocartilage-like repair tissue which is structurally and biomechanically compromised relative to normal articular cartilage. The objective of this study was to evaluate transcriptional differences between chondrocytes of normal articular cartilage and repair tissue cells four months post-microfracture. Methods Bilateral one-cm2 full-thickness defects were made in the articular surface of both distal femurs of four adult horses followed by subchondral microfracture. Four months postoperatively, repair tissue from the lesion site and grossly normal articular cartilage from within the same femorotibial joint were collected. Total RNA was isolated from the tissue samples, linearly amplified, and applied to a 9,413-probe set equine-specific cDNA microarray. Eight paired comparisons matched by limb and horse were made with a dye-swap experimental design with validation by histological analyses and quantitative real-time polymerase chain reaction (RT-qPCR. Results Statistical analyses revealed 3,327 (35.3% differentially expressed probe sets. Expression of biomarkers typically associated with normal articular cartilage and fibrocartilage repair tissue corroborate earlier studies. Other changes in gene expression previously unassociated with cartilage repair were also revealed and validated by RT-qPCR. Conclusion The magnitude of divergence in transcriptional profiles between normal chondrocytes and the cells that populate repair tissue reveal substantial functional differences between these two cell populations. At the four-month postoperative time point, the relative deficiency within repair tissue of gene transcripts which typically define articular cartilage indicate that while cells occupying the lesion might be of mesenchymal origin, they have not recapitulated differentiation to

  11. Induction of advanced glycation end products and alterations of the tensile properties of articular cartilage

    OpenAIRE

    Chen, A C; Temple, M.M.; Ng, D.M.; Verzijl, N; de Groot, J.; TeKoppele, J.M.; Sah, R.L.

    2002-01-01

    Objective. To determine whether increasing advanced glycation end products (AGEs) in bovine articular cartilage to levels present in aged human cartilage modulates the tensile biomechanical properties of the tissue. Methods. Adult bovine articular cartilage samples were incubated in a buffer solution with ribose to induce the formation of AGEs or in a control solution. Portions of cartilage samples were assayed for biochemical indices of AGEs and tested to assess their tensile biomechanical p...

  12. Repairing articular cartilage defects with tissue-engineering cartilage in rabbits

    Institute of Scientific and Technical Information of China (English)

    SONG Hong-xing; LI Fo-bao; SHEN Hui-liang; LIAO Wei-ming; LIU Miao; WANG Min; CAO Jun-ling

    2006-01-01

    Objective: To investigate the effect of cancellous bone matrix gelatin (BMG) engineered with allogeneic chondrocytes in repairing articular cartilage defects in rabbits.Methods: Chondrocytes were seeded onto three-dimensional cancellous BMG and cultured in vitro for 12 days to prepare BMG-chondrocyte complexes. Under anesthesia with 2.5% pentobarbital sodium (1 ml/kg body weight), articular cartilage defects were made on the right knee joints of 38 healthy New Zealand white rabbits (regardless of sex, aged 4-5 months and weighing 2.5-3 kg) and the defects were then treated with 2.5 % trypsin.Then BMG-chondrocyte complex (Group A, n=18 ),BMG ( Group B, n=10), and nothing ( Group C, n=10)were implanted into the cartilage defects, respectively. The repairing effects were assessed by macroscopic, histologic,transmission electron microscopic (TEM) observation,immunohistochemical examination and in situ hybridization detection, respectively, at 2, 4, 8, 12 and 24 weeks after operation.Results: Cancellous BMG was degraded within 8 weeks after operation. In Group A, lymphocyte infiltration was observed around the graft. At 24 weeks after operation, the cartilage defects were repaired by cartilage tissues and the articular cartilage and subchondral bone were soundly healed. Proteoglycan and type Ⅱ collagen were detected in the matrix of the repaired tissues by Safranin-O staining and immunohistochemical staining,respectively. In situ hybridization proved gene expression of type Ⅱ collagen in the cytoplasm of chondrocytes in the repaired tissues. TEM observation showed that chondrocytes and cartilage matrix in repaired tissues were almost same as those in the normal articular cartilage. In Group B, the defects were repaired by cartilage-fibrous tissues. In Group C, the defects were repaired only by fibrous tissues.Conclusions : Cancellous BMG can be regarded as the natural cell scaffolds for cartilage tissue engineering.Articular cartilage defects can be repaired by

  13. 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.

  14. The Functions of BMP3 in Rabbit Articular Cartilage Repair

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    Zhe Zhang

    2015-10-01

    Full Text Available Bone morphogenetic proteins (BMPs play important roles in skeletal development and repair. Previously, we found fibroblast growth factor 2 (FGF2 induced up-regulation of BMP2, 3, 4 in the process of rabbit articular cartilage repair, which resulted in satisfactory repair effects. As BMP2/4 show a clearly positive effect for cartilage repair, we investigated the functions of BMP3 in rabbit articular cartilage repair. In this paper, we find that BMP3 inhibits the repair of partial-thickness defect of articular cartilage in rabbit by inducing the degradation of extracellular matrix, interfering with the survival of chondrocytes surrounding the defect, and directly inhibiting the expression of BMP2 and BMP4. Meanwhile BMP3 suppress the repair of full-thickness cartilage defect by destroying the subchondral bone through modulating the proliferation and differentiation of bone marrow stem cells (BMSCs, and directly increasing the expression of BMP4. Although BMP3 has different functions in the repair of partial and full-thickness defects of articular cartilage in rabbit, the regulation of BMP expression is involved in both of them. Together with our previous findings, we suggest the regulation of the BMP signaling pathway by BMP3 is essential in articular cartilage repair.

  15. Doublecortin May Play a Role in Defining Chondrocyte Phenotype

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    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.

  16. Suppression of adverse angiogenesis in an albumin-based hydrogel for articular cartilage and intervertebral disc regeneration

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    B Scholz

    2010-07-01

    Full Text Available An injectable polyethylene glycol-crosslinked albumin gel (AG supplemented with hyaluronic acid as a matrix for autologous chondrocyte implantation was evaluated with regard to its impact on angiogenesis. Healthy articular cartilage and intervertebral discs (IVD are devoid of blood vessels, whereas pathological blood vessel formation augments degeneration of both theses tissues. In contrast to human endothelial cells, primary human articular chondrocytes encapsulated in the AG retained their viability. Endothelial cells did not adhere to the gel surface to a significant extent nor did they proliferate in vitro. The AG did not release any diffusible toxic components. Contrary to MatrigelTM employed as positive control, the AG prevented endothelial chemoinvasion in Transwell filter assays even in the presence of a chemotactic gradient of vascular endothelial growth factor. In ovo, the AG exhibited a barrier function for blood vessels of the chick chorioallantoic membrane. Subcutaneous implantation of human IVD chondrocytes enclosed in the albumin gel into immunodeficient mice revealed a complete lack of angiogenesis inside the gel after two weeks. At the same time, the IVD chondrocytes within the gel remained vital and displayed a characteristic gene expression pattern as judged from aggrecan, collagen type I and type II mRNA levels. In summary, aiming at articular cartilage and IVD regeneration the albumin gel promises to be a beneficial implant matrix for chondrocytes simultaneously exhibiting non-permissive properties for adverse endothelial cells.

  17. The relationship between ultra-short telomeres, aging of articular cartilage and the development of human hip osteoarthritis

    DEFF Research Database (Denmark)

    Harbo, M; Delaisse, J M; Kjaersgaard-Andersen, P;

    2013-01-01

    Ultra-short telomeres caused by stress-induced telomere shortening are suggested to induce chondrocyte senescence in human osteoarthritic knees. Here we have further investigated the role of ultra-short telomeres in the development of osteoarthritis (OA) and in aging of articular cartilage in human...

  18. Progression of articular cartilage degeneration after application of muscle stretch.

    Science.gov (United States)

    Dias, Carolina Náglio Kalil; Renner, Adriana Frias; dos Santos, Anderson Amaro; Vasilceac, Fernando Augusto; Mattiello, Stela Márcia

    2012-01-01

    The aim of study was to evaluate the progression of the ankle articular cartilage alterations after a post-immobilization muscle stretching. Twenty-nine Wistar rats were separated into five groups: C--control, S--stretched, SR--stretch recovery, IS--immobilized and stretched, and ISR--immobilized stretched recovery. The immobilization was maintained for 4 weeks and the left ankle was then stretched manually through a full dorsal flexion for 10 times for 60 s with a 30 s interval between each 60 s period, 7 days/week for 3 weeks. The recovery period was of 7 weeks. At the end of the experiment, the left ankles were removed, processed in paraffin, and stained in hematoxylin-eosin and safranin O. Two blinded observers evaluated the articular cartilage using the Mankin grading system (cellularity, chondrocyte cloning, and proteoglycan content) through light microscopy, and performed the morphometry (cellularity, total thickness, non-calcified thickness, and calcified thickness measures). Both the Mankin grading system and the morphometric analysis showed that the ISR group presented the most increased cellularity among the groups. The IS and SR groups showed the highest proteoglycan loss, and the ISR group showed the same content of proteoglycan observed in the C group. No significant differences were found in the chondrocyte cloning, the total cartilage thickness, the non-calcified cartilage thickness, and the calcified cartilage thickness among the groups. The results suggest that the cartilage can recover the proteoglycan loss caused by immobilization and stretching, probably because of the increased chondrocyte density. Therefore, the ankle articular cartilage responded as to repair the metabolic deficits.

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

    Science.gov (United States)

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

    2013-01-01

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

  20. Effects of Articular Cartilage Constituents on Phosphotungstic Acid Enhanced Micro-Computed Tomography

    Science.gov (United States)

    Karhula, Sakari S.; Finnilä, Mikko A.; Lammi, Mikko J.; Ylärinne, Janne H.; Kauppinen, Sami; Rieppo, Lassi; Pritzker, Kenneth P. H.; Nieminen, Heikki J.; Saarakkala, Simo

    2017-01-01

    Contrast-enhanced micro-computed tomography (CEμCT) with phosphotungstic acid (PTA) has shown potential for detecting collagen distribution of articular cartilage. However, the selectivity of the PTA staining to articular cartilage constituents remains to be elucidated. The aim of this study was to investigate the dependence of PTA for the collagen content in bovine articular cartilage. Adjacent bovine articular cartilage samples were treated with chondroitinase ABC and collagenase to degrade the proteoglycan and the collagen constituents in articular cartilage, respectively. Enzymatically degraded samples were compared to the untreated samples using CEμCT and reference methods, such as Fourier-transform infrared imaging. Decrease in the X-ray attenuation of PTA in articular cartilage and collagen content was observed in cartilage depth of 0–13% and deeper in tissue after collagen degradation. Increase in the X-ray attenuation of PTA was observed in the cartilage depth of 13–39% after proteoglycan degradation. The X-ray attenuation of PTA-labelled articular cartilage in CEμCT is associated mainly with collagen content but the proteoglycans have a minor effect on the X-ray attenuation of the PTA-labelled articular cartilage. In conclusion, the PTA labeling provides a feasible CEμCT method for 3D characterization of articular cartilage. PMID:28135331

  1. 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.

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

    Science.gov (United States)

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

    2016-04-04

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

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

    Science.gov (United States)

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

    2017-01-01

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

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

    Science.gov (United States)

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

    2015-12-16

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

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

    Science.gov (United States)

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

    2011-01-01

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

  6. Condrocalcinose articular familiar Familial articular chondrocalcinosis

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    Mittermayer Barreto Santiago

    2004-08-01

    Full Text Available Condrocalcinose articular familiar é uma condição clínica caracterizada pela deposição de cristais de pirofosfato de cálcio no líquido sinovial e cartilagens articulares levando à artrite. Descrevemos três membros de uma família com condrocalcinose cujo quadro clínico era caracterizado por artrite intermitente em dois e artrite crônica lembrando artrite reumatóide em um. A avaliação radiológica mostrou calcificações em cartilagens de diversas articulações, particularmente de joelhos. A utilização de colchicina foi suficiente para prevenir as crises de artrite em dois pacientes e o paciente com a forma crônica necessitou uso contínuo de antiinflamatórios não-hormonais. Embora aparentemente rara no Brasil, não afastamos a possibilidade desse dado estar subestimado e sugerimos que seja realizada uma avaliação radiológica articular dos familiares de todo paciente com diagnóstico de condrocalcinose esporádica.Familial articular chondrocalcinosis is a disorder characterized by deposition of calcium pyrophosphate dihydrate crystal in synovial fluid and articular cartilage that can cause joint pain and arthritis. We have identified three members of the same family with chondrocalcinosis. The clinical features of the disease were intermittent episodes of arthritis in two patients and polyarthritis resembling rheumatoid arthritis in one member. The radiological evaluation showed calcification in several joints, particularly in cartilages of the knees. Therapy with colchicine was enough to prevent arthritic crisis in two patients and continous NSAID use was necessary to control symptoms in the last one. Familial chondrocalcinosis seems to be rare in Brazil, but we do not exclude the possibility that this figure is underestimated and suggest that in cases of sporadic chondrocalcinosis other members of the family should be fully investigated.

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

    Science.gov (United States)

    Kasper, S; Friesen, H G

    1986-01-01

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

  8. Chondrogenic Differentiation of Human Adipose-Derived Stem Cells: A New Path in Articular Cartilage Defect Management?

    Directory of Open Access Journals (Sweden)

    Jan-Philipp Stromps

    2014-01-01

    Full Text Available According to data published by the Centers for Disease Control and Prevention, over 6 million people undergo a variety of medical procedures for the repair of articular cartilage defects in the U.S. each year. Trauma, tumor, and age-related degeneration can cause major defects in articular cartilage, which has a poor intrinsic capacity for healing. Therefore, there is substantial interest in the development of novel cartilage tissue engineering strategies to restore articular cartilage defects to a normal or prediseased state. Special attention has been paid to the expansion of chondrocytes, which produce and maintain the cartilaginous matrix in healthy cartilage. This review summarizes the current efforts to generate chondrocytes from adipose-derived stem cells (ASCs and provides an outlook on promising future strategies.

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

    Directory of Open Access Journals (Sweden)

    Sébastien Flajollet

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

  10. Hypotonic challenge modulates cell volumes differently in the superficial zone of intact articular cartilage and cartilage explant.

    Science.gov (United States)

    Turunen, Siru M; Lammi, Mikko J; Saarakkala, Simo; Koistinen, Arto; Korhonen, Rami K

    2012-05-01

    The objective of this study was to evaluate the effect of sample preparation on the biomechanical behaviour of chondrocytes. We compared the volumetric and dimensional changes of chondrocytes in the superficial zone (SZ) of intact articular cartilage and cartilage explant before and after a hypotonic challenge. Calcein-AM labelled SZ chondrocytes were imaged with confocal laser scanning microscopy through intact cartilage surfaces and through cut surfaces of cartilage explants. In order to clarify the effect of tissue composition on cell volume changes, Fourier Transform Infrared microspectroscopy was used for estimating the proteoglycan and collagen contents of the samples. In the isotonic medium (300 mOsm), there was a significant difference (p integrity of the mechanical environment of chondrocytes.

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

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    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.

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

    Directory of Open Access Journals (Sweden)

    Natalya Krivoruchko

    2014-12-01

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

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

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    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.

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

    Science.gov (United States)

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

    2014-06-01

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

  15. 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

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    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

  16. Effect of low-level helium-neon laser therapy on histological and ultrastructural features of immobilized rabbit articular cartilage.

    Science.gov (United States)

    Bayat, Mohammad; Ansari, Enayatallah; Gholami, Narges; Bayat, Aghdas

    2007-05-25

    The present study investigates whether low-level helium-neon laser therapy can increase histological parameters of immobilized articular cartilage in rabbits or not. Twenty five rabbits were divided into three groups: the experiment group, which received low-level helium-neon laser therapy with 13J/cm(2) three times a week after immobilization of their right knees; the control group which did not receive laser therapy after immobilization of their knees; and the normal group which received neither immobilization nor laser therapy. Histological and electron microscopic examinations were performed at 4 and 7 weeks after immobilization. Depth of the chondrocyte filopodia in four-week immobilized experiment group, and depth of articular cartilage in seven-week immobilized experiment group were significantly higher than those of relevant control groups (exact Fisher test, p=0.001; student's t-test, p=0.031, respectively). The surfaces of articular cartilages of the experiment group were relatively smooth, while those of the control group were unsmooth. It is therefore concluded that low-level helium-neon laser therapy had significantly increased the depth of the chondrocyte filopodia in four-week immobilized femoral articular cartilage and the depth of articular cartilage in seven-week immobilized knee in comparison with control immobilized articular cartilage.

  17. [Intra-articular injections].

    Science.gov (United States)

    Chapelle, Ch

    2015-09-01

    It is not unusual for a specialist or general practitioner to be presented with a pathology which necessitates the use of an intra-articular injection of corticosteroids, hyaluronic acid or a local anaesthetic. It would seem to be interesting to update and to precise the techniques and methods of intraarticular injections which have appeared in recent international publications, when we know that 30 % of the injections given into the knee and so called "dry" are incorrect and, therefore, inefficient. The indication of an articular injection depends, firstly, on the diagnosis which should be done with great care; after which should be an objective analysis complete with secondary effects linked to both the injection and the product used. The conditions of asepsis, the choice of needles and quantities of the injection and even the ways of the injections should be reviewed in detail. The last studies clearly question the secondary effects of the cartilage degradations of the cortisone given as an intra-articular injection and shows its efficiency on the pain and inflammatory phenomonen in osteoarthritis. Studies on hyaluronic acid are often contradictory going from a modest result to an important pain relief but it is necessary to be aware that the objective criteria are difficult to interpret. The use of local anaesthetics in intra-articular is limited by the few indications in view of the major risk of aggravating the pre-existing lesions by the disappearing signs of pain.

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

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    Xingfu Li

    2016-01-01

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

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

    Science.gov (United States)

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

    2017-03-25

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

  20. Chondrocyte outgrowth into a gelatin scaffold in a single impact load model of damage/repair – effect of BMP-2

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    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.

  1. Deficiency of Thrombospondin-4 in Mice Does Not Affect Skeletal Growth or Bone Mass Acquisition, but Causes a Transient Reduction of Articular Cartilage Thickness.

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    Anke Jeschke

    Full Text Available Although articular cartilage degeneration represents a major public health problem, the underlying molecular mechanisms are still poorly characterized. We have previously utilized genome-wide expression analysis to identify specific markers of porcine articular cartilage, one of them being Thrombospondin-4 (Thbs4. In the present study we analyzed Thbs4 expression in mice, thereby confirming its predominant expression in articular cartilage, but also identifying expression in other tissues, including bone. To study the role of Thbs4 in skeletal development and integrity we took advantage of a Thbs4-deficient mouse model that was analyzed by undecalcified bone histology. We found that Thbs4-deficient mice do not display phenotypic differences towards wildtype littermates in terms of skeletal growth or bone mass acquisition. Since Thbs4 has previously been found over-expressed in bones of Phex-deficient Hyp mice, we additionally generated Thbs4-deficient Hyp mice, but failed to detect phenotypic differences towards Hyp littermates. With respect to articular cartilage we found that Thbs4-deficient mice display transient thinning of articular cartilage, suggesting a protective role of Thbs4 for joint integrity. Gene expression analysis using porcine primary cells revealed that Thbs4 is not expressed by synovial fibroblasts and that it represents the only member of the Thbs gene family with specific expression in articular, but not in growth plate chondrocytes. In an attempt to identify specific molecular effects of Thbs4 we treated porcine articular chondrocytes with human THBS4 in the absence or presence of conditioned medium from porcine synovial fibroblasts. Here we did not observe a significant influence of THBS4 on proliferation, metabolic activity, apoptosis or gene expression, suggesting that it does not act as a signaling molecule. Taken together, our data demonstrate that Thbs4 is highly expressed in articular chondrocytes, where its

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

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    Akira Ito

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

  3. Acute and chronic response of articular cartilage to Ho:YAG laser irradiation

    Science.gov (United States)

    Trauner, Kenneth B.; Nishioka, Norman S.; Flotte, Thomas J.; Patel, Dinesh K.

    1992-06-01

    A Ho:YAG laser system operating at a wavelength of 2.1 microns has recently been introduced for use in arthroscopic surgery. The acceptability of this new tool will be determined not only by its ability to resect tissue, but also by its long term effects on articular surfaces. In order to investigate these issues further, we performed two studies to evaluate the acute and chronic effects of the laser on cartilaginous tissue. We evaluated the acute, in vitro effects of 2.1 micron laser irradiation on articular and fibrocartilage. This included the measurement of ablation efficiency, ablation threshold and thermal damage in both meniscus and articular cartilage. To document the chronic effects on articular cartilage in vivo, we next performed a ten week healing study. Eight sheep weighing 30 - 40 kg underwent bilateral arthrotomy procedures. Multiple full thickness and partial thickness defects were created. Animals were sacrificed at 0, 2, 4, and 10 weeks. The healing study demonstrated: (1) no healing of full or partial thickness defects at 10 weeks with hyaline cartilage; (2) fibrocartilaginous granulation tissue filling full thickness defects at two and four weeks, but no longer evident at ten weeks; (3) chondrocyte necrosis extending to greater than 900 microns distal to ablation craters at four weeks with no evidence of repair at later dates; and (4) chondrocyte hyperplasia at the borders of the damage zone at two weeks but no longer evident at later sacrifice dates.

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

    Science.gov (United States)

    Annefeld, M

    1985-01-01

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

  5. 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.

  6. Dynamic compressive properties of bovine knee layered tissue

    Science.gov (United States)

    Nishida, Masahiro; Hino, Yuki; Todo, Mitsugu

    2015-09-01

    In Japan, the most common articular disease is knee osteoarthritis. Among many treatment methodologies, tissue engineering and regenerative medicine have recently received a lot of attention. In this field, cells and scaffolds are important, both ex vivo and in vivo. From the viewpoint of effective treatment, in addition to histological features, the compatibility of mechanical properties is also important. In this study, the dynamic and static compressive properties of bovine articular cartilage-cancellous bone layered tissue were measured using a universal testing machine and a split Hopkinson pressure bar method. The compressive behaviors of bovine articular cartilage-cancellous bone layered tissue were examined. The effects of strain rate on the maximum stress and the slope of stress-strain curves of the bovine articular cartilage-cancellous bone layered tissue were discussed.

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

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    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. Regulative mechanisms of chondrocyte adhesion

    DEFF Research Database (Denmark)

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

    2006-01-01

    -matrix interaction, as well as collagen type II expression in the cartilage graft after two weeks of in vitro cultivation. Basic fibroblast growth factor (bFGF) treated chondrocytes showed increased adhesion to collagen types I and II, fibronectin, and fibrinogen. Attachment to these investigated proteins......%) increased. A cartilage construct was developed based on a clinically established collagen type I scaffold. In this matrix, more than 80% of the cells could be immobilized by mechanisms of adhesion, filtration, and cell entrapment. Confocal laser microscopy revealed focal adhesion sites as points of cell...... significantly enhanced cell proliferation. Matrix design in cartilage engineering must meet the biological demands of amplified cells, because adhesion of chondrocytes depends on their differentiation status and is regulated by bFGF....

  9. Chondrocyte physiopathology and drug efficacy.

    Science.gov (United States)

    Serni, U; Mannoni, A

    1991-01-01

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

  10. Cyclooxygenases (COX-1 and COX-2) for tissue engineering of articular cartilage--from a developmental model to first results of tissue and scaffold expression.

    Science.gov (United States)

    Brochhausen, Christoph; Zehbe, Rolf; Gross, Ulrich; Libera, Jeanette; Schubert, Helmut; Nüsing, Rolf M; Klaus, Günter; Kirkpatrick, C James

    2008-01-01

    Tissue engineering of articular cartilage remains an ongoing challenge. Since tissue regeneration recapitulates ontogenetic processes the growth plate can be regarded as an innovative model to target suitable signalling molecules and growth factors for the tissue engineering of cartilage. In the present study we analysed the expression of cyclooxygenases (COX) in a short-term chondrocyte culture in gelatin-based scaffolds and in articular cartilage of rats and compared it with that in the growth plate. Our results demonstrate the strong cellular expression of COX-1 but only a focal weak expression of COX-2 in the seeded scaffolds. Articular cartilage of rats expresses homogeneously COX-1 and COX-2 with the exception of the apical cell layer. Our findings indicate a functional role of COX in the metabolism of articular chondrocytes. The expression of COX in articular cartilage and in the seeded scaffolds opens interesting perspectives to improve the proliferation and differentiation of chondrocytes in scaffold materials by addition of specific receptor ligands of the COX system.

  11. Effects of intra-articular injection of mesenchymal stem cells associated with platelet-rich plasma in a rabbit model of osteoarthritis.

    Science.gov (United States)

    Hermeto, L C; DeRossi, R; Oliveira, R J; Pesarini, J R; Antoniolli-Silva, A C M B; Jardim, P H A; Santana, A E; Deffune, E; Rinaldi, J C; Justulin, L A

    2016-09-02

    The current study aims to evaluate the macroscopic and histological effects of autologous mesenchymal stem cells (MSC) and platelet-rich plasma on knee articular cartilage regeneration in an experimental model of osteoarthritis. Twenty-four rabbits were randomly divided into four groups: control group, platelet-rich plasma group, autologous MSC undifferentiated group, and autologous MSC differentiated into chondrocyte group. Collagenase solution was used to induce osteoarthritis, and treatments were applied to each group at 6 weeks following osteoarthritis induction. After 60 days of therapy, the animals were euthanized and the articular surfaces were subjected to macroscopic and histological evaluations. The adipogenic, chondrogenic, and osteogenic differentiation potentials of MSCs were evaluated. Macroscopic and histological examinations revealed improved tissue repair in the MSC-treated groups. However, no difference was found between MSC-differentiated and undifferentiated chondrocytes. We found that MSCs derived from adipose tissue and platelet-rich plasma were associated with beneficial effects in articular cartilage regeneration during experimental osteoarthritis.

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

    Science.gov (United States)

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

    2016-01-01

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

  13. 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.

  14. 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.

  15. [The effect of steroidal and nonsteroidal anti-inflammatory agents on the ultrastructure of chondrocytes in the rat. Electron microscope and morphometric study].

    Science.gov (United States)

    Annefeld, M; Raiss, R; Cleres, C

    1984-01-01

    In order to obtain indications regarding the different influences of antiinflammatory drugs on the articular cartilage, the effects of a steroidal drug (dexamethasone) and of nonsteroidal antiinflammatory substances (indometacin and phenylbutazone) on the ultrastructure of the chondrocyte were examined in comparison with tiaprofenic acid (Surgam). Using an equiefficient dosage of the active principles, referred to the ED50 in the model of adjuvant arthritis in the rat, the synthetic apparatus of the chondrocytes was stimulated by tiaprofenic acid while it was diminished by indometacin and phenylbutazone and particularly by dexamethasone.

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

    Science.gov (United States)

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

    2015-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Hongxu Yang

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

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

    Science.gov (United States)

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

    2017-01-01

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

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

    Science.gov (United States)

    Heywood, Hannah K; Lee, David A

    2010-01-01

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

  20. Condensed cellular seeded collagen gel as an improved biomaterial for tissue engineering of articular cartilage.

    Science.gov (United States)

    Mueller-Rath, Ralf; Gavénis, Karsten; Andereya, Stefan; Mumme, Torsten; Albrand, Monique; Stoffel, Marcus; Weichert, Dieter; Schneider, Ulrich

    2010-01-01

    Three-dimensional autologous chondrocyte implantation based on collagen gel as matrix scaffold has become a clinically applied treatment for focal defects of articular cartilage. However, the low biomechanical properties of collagen gel makes intraoperative handling difficult and creates the risk of early damages to the vulnerable implant. The aim of the study was to create a stabilized form of collagen gel and to evaluate its biomechanical and biochemical properties.Collagen type-I gel was seeded with human articular chondrocytes. 20 samples were subject to condensation which was achieved mechanically by compression and filtration. Control samples were left uncondensed. From both types of gels 10 samples were used for initial biomechanical evaluation by means of unconfined compression and 10 samples were cultivated under standard conditions in vitro. Following cultivation the samples were evaluated by conventional histology and immunohistochemistry. The proliferation rate was calculated and matrix gene expression was quantified by real-time PCR.The biomechanical tests revealed a higher force carrying capacity of the condensed specimens. Strain rate dependency and relaxation was seen in both types of collagen gel representing viscoelastic material properties. Cells embedded within the condensed collagen gel were able to produce extracellular matrix proteins and showed proliferation.Condensed collagen gel represents a mechanically improved type of biomaterial which is suitable for three-dimensional autologous chondrocyte implantation.

  1. 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.

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

    Institute of Scientific and Technical Information of China (English)

    Xiao-Xiao Cai

    2015-01-01

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

  3. The biochemical content of articular cartilage: an original MRI approach.

    Science.gov (United States)

    Loeuille, Damien; Olivier, Pierre; Watrin, Astrid; Grossin, Laurent; Gonord, Patrick; Guillot, Geneviève; Etienne, Stéphanie; Blum, Alain; Netter, Patrick; Gillet, Pierre

    2002-01-01

    The MR aspect of articular cartilage, that reflects the interactions between protons and macromolecular constituents, is affected by the intrinsic tissue structure (water content, the content of matrix constituents, collagen network organization), imager characteristics, and acquisition parameters. On the T1-weighted sequences, the bovine articular cartilage appears as an homogeneous tissue in high signal intensity, whatever the age of animals considered, whereas on the T2-weighted sequences, the articular bovine cartilage presents variations of its imaging pattern (laminar appearance) well correlated to the variations of its histological and biochemical structure. The T2 relaxation time measurement (T2 mapping), which reflects quantitatively the signal intensity variations observed on T2 weighted sequences, is a way to evaluate more precisely the modifications of cartilage structure during the aging and maturation processes (rat's study). This technique so far confined to experimental micro-imagers is now developed on clinical imagers. Consequently, it may permit to depict the early stages of osteoarthritic disease (OA) or to evaluate the chondroprotective effect of drugs.

  4. Regeneration of Articular Cartilage in Lizard Knee from Resident Stem/Progenitor Cells.

    Science.gov (United States)

    Alibardi, Lorenzo

    2015-09-01

    The epiphysis of femur and tibia in the lizard Podarcis muralis can extensively regenerate after injury. The process involves the articular cartilage and metaphyseal (growth) plate after damage. The secondary ossification center present between the articular cartilage and the growth plate is replaced by cartilaginous epiphyses after about one month of regeneration at high temperature. The present study analyzes the origin of the chondrogenic cells from putative stem cells located in the growing centers of the epiphyses. The study is carried out using immunocytochemistry for the detection of 5BrdU-labeled long retaining cells and for the localization of telomerase, an enzyme that indicates stemness. The observations show that putative stem cells retaining 5BrdU and positive for telomerase are present in the superficial articular cartilage and metaphyseal growth plate located in the epiphyses. This observation suggests that these areas represent stem cell niches lasting for most of the lifetime of lizards. In healthy long bones of adult lizards, the addition of new chondrocytes from the stem cells population in the articular cartilage and the metaphyseal growth plate likely allows for slow, continuous longitudinal growth. When the knee is injured in the adult lizard, new populations of chondrocytes actively producing chondroitin sulfate proteoglycan are derived from these stem cells to allow for the formation of completely new cartilaginous epiphyses, possibly anticipating the re-formation of secondary centers in later stages. The study suggests that in this lizard species, the regenerative ability of the epiphyses is a pre-adaptation to the regeneration of the articular cartilage.

  5. Regeneration of Articular Cartilage in Lizard Knee from Resident Stem/Progenitor Cells

    Directory of Open Access Journals (Sweden)

    Lorenzo Alibardi

    2015-09-01

    Full Text Available The epiphysis of femur and tibia in the lizard Podarcis muralis can extensively regenerate after injury. The process involves the articular cartilage and metaphyseal (growth plate after damage. The secondary ossification center present between the articular cartilage and the growth plate is replaced by cartilaginous epiphyses after about one month of regeneration at high temperature. The present study analyzes the origin of the chondrogenic cells from putative stem cells located in the growing centers of the epiphyses. The study is carried out using immunocytochemistry for the detection of 5BrdU-labeled long retaining cells and for the localization of telomerase, an enzyme that indicates stemness. The observations show that putative stem cells retaining 5BrdU and positive for telomerase are present in the superficial articular cartilage and metaphyseal growth plate located in the epiphyses. This observation suggests that these areas represent stem cell niches lasting for most of the lifetime of lizards. In healthy long bones of adult lizards, the addition of new chondrocytes from the stem cells population in the articular cartilage and the metaphyseal growth plate likely allows for slow, continuous longitudinal growth. When the knee is injured in the adult lizard, new populations of chondrocytes actively producing chondroitin sulfate proteoglycan are derived from these stem cells to allow for the formation of completely new cartilaginous epiphyses, possibly anticipating the re-formation of secondary centers in later stages. The study suggests that in this lizard species, the regenerative ability of the epiphyses is a pre-adaptation to the regeneration of the articular cartilage.

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

    Directory of Open Access Journals (Sweden)

    Belinda Pingguan-Murphy

    2012-08-01

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

  7. The structure and function of the pericellular matrix of articular cartilage.

    Science.gov (United States)

    Wilusz, Rebecca E; Sanchez-Adams, Johannah; Guilak, Farshid

    2014-10-01

    Chondrocytes in articular cartilage are surrounded by a narrow pericellular matrix (PCM) that is both biochemically and biomechanically distinct from the extracellular matrix (ECM) of the tissue. While the PCM was first observed nearly a century ago, its role is still under investigation. In support of early hypotheses regarding its function, increasing evidence indicates that the PCM serves as a transducer of biochemical and biomechanical signals to the chondrocyte. Work over the past two decades has established that the PCM in adult tissue is defined biochemically by several molecular components, including type VI collagen and perlecan. On the other hand, the biomechanical properties of this structure have only recently been measured. Techniques such as micropipette aspiration, in situ imaging, computational modeling, and atomic force microscopy have determined that the PCM exhibits distinct mechanical properties as compared to the ECM, and that these properties are influenced by specific PCM components as well as disease state. Importantly, the unique relationships among the mechanical properties of the chondrocyte, PCM, and ECM in different zones of cartilage suggest that this region significantly influences the stress-strain environment of the chondrocyte. In this review, we discuss recent advances in the measurement of PCM mechanical properties and structure that further increase our understanding of PCM function. Taken together, these studies suggest that the PCM plays a critical role in controlling the mechanical environment and mechanobiology of cells in cartilage and other cartilaginous tissues, such as the meniscus or intervertebral disc.

  8. Posttraumatic Chondrocyte Apoptosis in the Murine Xiphoid

    Science.gov (United States)

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

    2013-01-01

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

  9. [Effects of SL-1010 (sodium hyaluronate with high molecular weight) on experimental osteoarthritis induced by intra-articularly applied papain in guinea pigs].

    Science.gov (United States)

    Tanaka, H; Kitoh, Y; Katsuramaki, T; Tanaka, M; Kitabayashi, N; Fujimori, S; Umemoto, J; Namba, K

    1992-07-01

    Effects of SL-1010 on the experimental osteoarthritis (OA) produced by intra-articular injection of papain, proteolytic enzyme, in the knee joint of the guinea pigs were histologically and biochemically investigated. In addition, experimental conditions to produce OA in guinea pig knee joint were also examined, since papain-induced OA has been mainly studied in rabbits. Six weeks after intra-articular injection of papain (1%, 0.1 ml), there were inflammatory reactions of the synovial membrane, degenerative changes in chondrocytes and the matrix of the articular cartilage, a decrease in the Safranin-O staining intensity and lowering of sulfated glycosaminoglycan. Electronmicroscopic observations revealed that the amorphous layer had disappeared and large bundles of unit collagen fibers and larger collagen fibers had appeared in the cartilage matrix. In the OA model, SL-1010 reduced the inflammatory reactions of the synovial membrane, inhibited development of degenerative changes in chondrocytes and the matrix of the articular cartilage and recovered the Safranin-O staining intensity. The sulfated glycosaminoglycan contents in the cartilage was significantly increased in the SL-1010-treated group, compared with the control group. The electromicroscopically observed charges in the papain-injected knee joint of the control group were rarely detected in the SL-1010-treated group. These results suggest that SL-1010 inhibits degenerative changes in the chondrocytes and the matrix probably by reducing synovial inflammation and protection of the cartilage in the OA model of guinea pigs.

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

    Science.gov (United States)

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

    2005-11-01

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

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

    Directory of Open Access Journals (Sweden)

    Pedersen Christian

    2010-04-01

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

  12. 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.

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

    Science.gov (United States)

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

    2011-11-01

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

  14. 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.

  15. Effect of low-power helium-neon laser irradiation on 13-week immobilized articular cartilage of rabbits.

    Science.gov (United States)

    Bayat, Mohammad; Ansari, Anayatallah; Hekmat, Hossien

    2004-09-01

    Influence of low-power (632.8 nm, Helium-Neon, 13 J/cm2, three times a week) laser on 13-week immobilized articular cartilage was examined with rabbits knee model. Number of chondrocytes and depth of articular cartilage of experimental group were significantly higher than those of sham irradiated group. Surface morphology of sham-irradiated group had rough prominences, fibrillation and lacunae but surface morphology of experimental group had more similarities to control group than to sham irradiated group. There were marked differences between ultrastructure features of control group and experimental group in comparison with sham irradiated group. Low-power Helium-Neon laser irradiation on 13-week immobilized knee joints of rabbits neutrilized adverse effects of immobilization on articular cartilage.

  16. POSSIBILITIES OF CURRENT CELLULAR TECHNOLOGIES FOR ARTICULAR CARTILAGE REPAIR (ANALYTICAL REVIEW

    Directory of Open Access Journals (Sweden)

    M. S. Bozhokin

    2016-01-01

    Full Text Available Despite a wide variety of surgical procedures utilized in clinical practice for treatment of articular cartilage lesions, the search for other options of articular reconstruction remains a relevant and open issue at the current stage of medicine and biotechnologies development. The recent years demonstrated a strong belief in cellular methods of hyaline cartilage repair such as implantation of autologous chondrocytes (ACI or cultures of mesenchymal stem cells (MSC including techniques for genetic modification of cells.The purpose of presented review is to summarize the published scientific data on up to date results of perspective cellular technologies for articular cartilage repair that are being developed. Autologous chondrocyte transplantation originally performed by Swedish researchers in 1987 is considered the first clinically applied technique for restoration of hyaline cartilage using cellular technologies. However, the transplanted cell culture featured low proliferative capacity and inability to form a regenerate resistant to high physical activity. Another generation of methods originated at the turn of the century utilized mesenchymal stem cells instead of autologous chondrocytes. Preparation of MSCs is a less invasive procedure compared to chondrocytes harvesting and the culture is featured by a higher proliferative ability. Researchers use various biodegradable carriers (matrices to secure cell fixation. Despite good clinical mid-term outcomes the transplanted tissue-engineering structures deteriorate with time due to cellular de-differentiation. Next generation of techniques being currently under pre-clinical studies is featured by the preliminary chondrogenic modification of transplanted cell culture. Usage of various growth factors, modified cell product and gene-activated matrices allow to gain a stable regulatory and key proteins synthesis and achieve a focused influence on regenerate's chondrogenic proliferation and in result

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

    Science.gov (United States)

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

    2017-02-01

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

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

    Science.gov (United States)

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

    2014-11-01

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

  19. Effect of freezing on rabbit cultured chondrocytes

    Directory of Open Access Journals (Sweden)

    R.R Filgueiras

    2011-02-01

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

  20. CCN family member 2/connective tissue growth factor (CCN2/CTGF has anti-aging effects that protect articular cartilage from age-related degenerative changes.

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    Shinsuke Itoh

    Full Text Available To examine the role of connective tissue growth factor CCN2/CTGF (CCN2 in the maintenance of the articular cartilaginous phenotype, we analyzed knee joints from aging transgenic mice (TG overexpressing CCN2 driven by the Col2a1 promoter. Knee joints from 3-, 14-, 40-, and 60-day-old and 5-, 12-, 18-, 21-, and 24-month-old littermates were analyzed. Ccn2-LacZ transgene expression in articular cartilage was followed by X-gal staining until 5 months of age. Overexpression of CCN2 protein was confirmed through all ages in TG articular cartilage and in growth plates. Radiographic analysis of knee joints showed a narrowing joint space and other features of osteoarthritis in 50% of WT, but not in any of the TG mice. Transgenic articular cartilage showed enhanced toluidine blue and safranin-O staining as well as chondrocyte proliferation but reduced staining for type X and I collagen and MMP-13 as compared with those parameters for WT cartilage. Staining for aggrecan neoepitope, a marker of aggrecan degradation in WT articular cartilage, increased at 5 and 12 months, but disappeared at 24 months due to loss of cartilage; whereas it was reduced in TG articular cartilage after 12 months. Expression of cartilage genes and MMPs under cyclic tension stress (CTS was measured by using primary cultures of chondrocytes obtained from wild-type (WT rib cartilage and TG or WT epiphyseal cartilage. CTS applied to primary cultures of mock-transfected rib chondrocytes from WT cartilage and WT epiphyseal cartilage induced expression of Col1a1, ColXa1, Mmp-13, and Mmp-9 mRNAs; however, their levels were not affected in CCN2-overexpressing chondrocytes and TG epiphyseal cartilage. In conclusion, cartilage-specific overexpression of CCN2 during the developmental and growth periods reduced age-related changes in articular cartilage. Thus CCN2 may play a role as an anti-aging factor by stabilizing articular cartilage.

  1. Morphological Study: Ultrastructural Aspects of Articular Cartilage and Subchondral Bone in Patients Affected by Post-Traumatic Shoulder Instability.

    Science.gov (United States)

    Baudi, Paolo; Catani, Fabio; Rebuzzi, Manuela; Ferretti, Marzia; Smargiassi, Alberto; Campochiaro, Gabriele; Serafini, Fabio; Palumbo, Carla

    2016-12-16

    Post-traumatic shoulder instability is a frequent condition in active population, representing one of most disabling pathologies, due to altered balance involving joints. No data are so far available on early ultrastructural osteo-chondral damages, associated with the onset of invalidating pathologies, like osteoarthritis-OA. Biopsies of glenoid articular cartilage and sub-chondral bone were taken from 10 adult patients underwent arthroscopic stabilization. Observations were performed under Transmission Electron Microscopy-TEM in tangential, arcuate and radial layers of the articular cartilage and in the sub-chondral bone. In tangential and arcuate layers chondrocytes display normal and very well preserved ultrastructure, probably due to the synovial liquid supply; otherwise, throughout the radial layer (un-calcified and calcified) chondrocytes show various degrees of degeneration; occasionally, in the radial layer evidences of apoptosis/autophagy were also observed. Concerning sub-chondral bone, osteocytes next to the calcified cartilage also show signs of degeneration, while osteocytes farther from the osteo-chondral border display normal ultrastructure, probably due to the bone vascular supply. The ultrastructural features of the osteo-chondral complex are not age-dependent. This study represents the first complete ultrastructural investigation of the articular osteo-chondral complex in shoulder instability, evaluating the state of preservation/viability of both chondrocytes and osteocytes throughout the successive layers of articular cartilage and sub-chondral bone. Preliminary observations here collected represent the morphological basis for further deepening of pathogenesis related to shoulder instability, enhancing the relationship between cell shape and microenvironment; in particular, they could be useful in understanding if the early surgical treatment in shoulder instability could avoid the onset of OA. Anat Rec, 300:12-15, 2017. © 2016 Wiley

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

    Science.gov (United States)

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

    2013-02-01

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

  3. THE FUNCTIONAL EFFECTIVENESS OF A CELL-ENGINEERED CONSTRUCT FOR THE REGENERATION OF ARTICULAR CARTILAGE

    Directory of Open Access Journals (Sweden)

    V. I. Sevastianov

    2015-01-01

    Full Text Available The aim of this study is an analysis of the functional effectiveness of a biomedical cell product consisting of a biopolymer microheterogeneous collagen-containing hydrogel (BMCH, human adipose-derived mesenchymal stromal cells (hADMSCs, and chondrogenic induction medium in the regeneration of articular cartilage. Materials and methods. The test model of the adjuvant arthritis was used (female Soviet Chinchilla rabbits with the further development into osteoarthrosis (OA combined with the clinical, biochemical, radiological, and histochemical trials. Results. On Day 92 of the OA model it has been found that the intra-articular introduction of a BMCH with hADMSCs into the left knee joint (n = 3 30 days after the OA modeling, as opposed to the right joint (negative control, n = 3, stimulates the regenerative processes of the cartilaginous tissue structure characterized by the formation of chondrocyte «columns», the emergence of isogenic groups in the intracellular matrix and the regeneration of its structure. Upon the intra-articular introduction of a BMCH (n = 3 such effects are markedly less pronounced. Conclusions. A significant regenerative potential of a cell-engineered construct of human articular tissue (CEC ATh has been proven. It is possible to presume that biostimulating properties of CEC ATh are due to the activating effect of a biomedical cell product on the stem cell migration processes from the surrounding tissue into the injured area with their subsequent differentiation. 

  4. Ultrasonic quantitation of superficial degradation of articular cartilage.

    Science.gov (United States)

    Saarakkala, Simo; Töyräs, Juha; Hirvonen, Jani; Laasanen, Mikko S; Lappalainen, Reijo; Jurvelin, Jukka S

    2004-06-01

    Ultrasound (US) has been suggested as a means for the quantitative detection of early osteoarthrotic changes in articular cartilage. In this study, the ability of quantitative US 2-D imaging (20 MHz) to reveal superficial changes in bovine articular cartilage after mechanical or enzymatic degradation was investigated in vitro. Mechanical degradation was induced by grinding samples against an emery paper with the grain size of 250 microm, 106 microm, 45 microm or 23 microm. For enzymatic degradation, samples were digested with collagenase, trypsin or chondroitinase ABC. Variations of the US reflection coefficient induced by the degradation were investigated. Furthermore, two novel parameters, the US roughness index (URI) and the spatial variation of the US reflection coefficient (SVR), were established to quantitate the integrity of the cartilage surface. Statistically significant decreases (p < 0.05) in US reflection coefficient were observed after mechanical degradations or enzymatic digestion with collagenase. Increases (p < 0.05) in URI were also revealed after these treatments. We conclude that quantitative US imaging may be used to detect collagen disruption and increased roughness in the articular surface. These structural damages are typical of early osteoarthrosis.

  5. Transport phenomena in articular cartilage cryopreservation as predicted by the modified triphasic model and the effect of natural inhomogeneities.

    Science.gov (United States)

    Abazari, Alireza; Thompson, Richard B; Elliott, Janet A W; McGann, Locksley E

    2012-03-21

    Knowledge of the spatial and temporal distribution of cryoprotective agent (CPA) is necessary for the cryopreservation of articular cartilage. Cartilage dehydration and shrinkage, as well as the change in extracellular osmolality, may have a significant impact on chondrocyte survival during and after CPA loading, freezing, and thawing, and during CPA unloading. In the literature, Fick's law of diffusion is commonly used to predict the spatial distribution and overall concentration of the CPA in the cartilage matrix, and the shrinkage and stress-strain in the cartilage matrix during CPA loading are neglected. In this study, we used a previously described biomechanical model to predict the spatial and temporal distributions of CPA during loading. We measured the intrinsic inhomogeneities in initial water and fixed charge densities in the cartilage using magnetic resonance imaging and introduced them into the model as initial conditions. We then compared the prediction results with the results obtained using uniform initial conditions. The simulation results in this study demonstrate the presence of a significant mechanical strain in the matrix of the cartilage, within all layers, during CPA loading. The osmotic response of the chondrocytes to the cartilage dehydration during CPA loading was also simulated. The results reveal that a transient shrinking occurs to different levels, and the chondrocytes experience a significant decrease in volume, particularly in the middle and deep zones of articular cartilage, during CPA loading.

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

    Science.gov (United States)

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

    2014-07-07

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

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

    Science.gov (United States)

    McCarty, M F; Russell, A L

    1999-10-01

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

  8. Poly(dopamine) coating of scaffolds for articular cartilage tissue engineering.

    Science.gov (United States)

    Tsai, Wei-Bor; Chen, Wen-Tung; Chien, Hsiu-Wen; Kuo, Wei-Hsuan; Wang, Meng-Jiy

    2011-12-01

    A surface modification technique based on poly(dopamine) deposition developed from oxidative polymerization of dopamine is known to promote cell adhesion to several cell-resistant substrates. In this study this technique was applied to articular cartilage tissue engineering. The adhesion and proliferation of rabbit chondrocytes were evaluated on poly(dopamine)-coated polymer films, such as polycaprolactone, poly(L-lactide), poly(lactic-co-glycolic acid) and polyurethane, biodegradable polymers that are commonly used in tissue engineering. Cell adhesion was significantly increased by merely 15 s of dopamine incubation, and 4 min incubation was enough to reach maximal cell adhesion, a 1.35-2.69-fold increase compared with that on the untreated substrates. Cells also grew much faster on the poly(dopamine)-coated substrates than on untreated substrates. The increase in cell affinity for poly(dopamine)-coated substrates was demonstrated via enhancement of the immobilization of serum adhesive proteins such as fibronectin. When the poly(dopamine)-coating technique was applied to three-dimensional (3-D) polyurethane scaffolds, the proliferation of chondrocytes and the secretion of glycosaminoglycans were increased compared with untreated scaffolds. Our results show that the deposition of a poly(dopamine) layer on 3-D porous scaffolds is a simple and promising strategy for articular cartilage tissue engineering, and may be applied to other types of tissue engineering.

  9. Mesenchymal stem cells as a potent cell source for articular cartilage regeneration

    Institute of Scientific and Technical Information of China (English)

    Mohamadreza; Baghaban; Eslaminejad; Elham; Malakooty; Poor

    2014-01-01

    Since articular cartilage possesses only a weak capac-ity for repair, its regeneration potential is considered one of the most important challenges for orthopedic surgeons. The treatment options, such as marrow stimulation techniques, fail to induce a repair tissue with the same functional and mechanical properties of native hyaline cartilage. Osteochondral transplantation is considered an effective treatment option but is as-sociated with some disadvantages, including donor-site morbidity, tissue supply limitation, unsuitable mechani-cal properties and thickness of the obtained tissue. Although autologous chondrocyte implantation results in reasonable repair, it requires a two-step surgical pro-cedure. Moreover, chondrocytes expanded in culture gradually undergo dedifferentiation, so lose morpho-logical features and specialized functions. In the search for alternative cells, scientists have found mesenchymal stem cells(MSCs) to be an appropriate cellular mate-rial for articular cartilage repair. These cells were origi-nally isolated from bone marrow samples and further investigations have revealed the presence of the cells in many other tissues. Furthermore, chondrogenic dif-ferentiation is an inherent property of MSCs noticedat the time of the cell discovery. MSCs are known to exhibit homing potential to the damaged site at which they differentiate into the tissue cells or secrete a wide spectrum of bioactive factors with regenerative proper-ties. Moreover, these cells possess a considerable im-munomodulatory potential that make them the general donor for therapeutic applications. All of these topics will be discussed in this review.

  10. 24R,25-Dihydroxyvitamin D3 Protects against Articular Cartilage Damage following Anterior Cruciate Ligament Transection in Male Rats

    Science.gov (United States)

    Boyan, Barbara D.; Hyzy, Sharon L.; Pan, Qingfen; Scott, Kayla M.; Coutts, Richard D.; Healey, Robert; Schwartz, Zvi

    2016-01-01

    Osteoarthritis (OA) in humans is associated with low circulating 25-hydroxyvitamin D3 [25(OH)D3]. In vitamin D replete rats, radiolabeled 24R,25-dihydroxyvitamin D3 [24R,25(OH)2D3] accumulates in articular cartilage following injection of [3H]-25(OH)D3. Previously, we showed that 24R,25(OH)2D3 blocks chondrocyte apoptosis via phospholipase D and p53, suggesting a role for 24R,25(OH)2D3 in maintaining cartilage health. We examined the ability of 24R,25(OH)2D3 to prevent degenerative changes in articular cartilage in an OA-like environment and the potential mechanisms involved. In vitro, rat articular chondrocytes were treated with IL-1β with and without 24R,25(OH)2D3 or 1α,25(OH)2D3. 24R,25(OH)2D3 but not 1α,25(OH)2D3 blocked the effects of IL-1β in a dose-dependent manner, and its effect was partially mediated through the TGF-β1 signaling pathway. In vivo, unilateral anterior cruciate ligament transections were performed in immunocompetent rats followed by intra-articular injections of 24R,25(OH)2D3 or vehicle (t = 0, 7, 14, 21 days). Tissues were harvested on day 28. Joints treated with vehicle had changes typical of OA whereas joints treated with 24R,25(OH)2D3 had less articular cartilage damage and levels of inflammatory mediators. These results indicate that 24R,25(OH)2D3 protects against OA, and suggest that it may be a therapeutic approach for preventing trauma-induced osteoarthritis. PMID:27575371

  11. Postnatal development of articular cartilage

    NARCIS (Netherlands)

    Turnhout, van M.C.

    2010-01-01

    Articular cartilage (AC) is the thin layer of tissue that covers the ends of the bones in the synovial joints in mammals. Functional adult AC has depth-dependent mechanical properties that are not yet present at birth. These depth-dependent mechanical properties in adult life are the result of a dep

  12. Structural Variations in Articular Cartilage Matrix Are Associated with Early-Onset Osteoarthritis in the Spondyloepiphyseal Dysplasia Congenita (Sedc Mouse

    Directory of Open Access Journals (Sweden)

    Robert E. Seegmiller

    2013-08-01

    Full Text Available Heterozgyous spondyloepiphyseal dysplasia congenita (sedc/+ mice expressing a missense mutation in col2a1 exhibit a normal skeletal morphology but early-onset osteoarthritis (OA. We have recently examined knee articular cartilage obtained from homozygous (sedc/sedc mice, which express a Stickler-like phenotype including dwarfism. We examined sedc/sedc mice at various levels to better understand the mechanistic process resulting in OA. Mutant sedc/sedc, and control (+/+ cartilages were compared at two, six and nine months of age. Tissues were fixed, decalcified, processed to paraffin sections, and stained with hematoxylin/eosin and safranin O/fast green. Samples were analyzed under the light microscope and the modified Mankin and OARSI scoring system was used to quantify the OA-like changes. Knees were stained with 1C10 antibody to detect the presence and distribution of type II collagen. Electron microscopy was used to study chondrocyte morphology and collagen fibril diameter. Compared with controls, mutant articular cartilage displayed decreased fibril diameter concomitant with increases in size of the pericellular space, Mankin and OARSI scores, cartilage thickness, chondrocyte clustering, proteoglycan staining and horizontal fissuring. In conclusion, homozygous sedc mice are subject to early-onset knee OA. We conclude that collagen in the mutant’s articular cartilage (both heterozygote and homozygote fails to provide the normal meshwork required for matrix integrity and overall cartilage stability.

  13. Snorc is a novel cartilage specific small membrane proteoglycan expressed in differentiating and articular chondrocytes

    DEFF Research Database (Denmark)

    Heinonen, J; Taipaleenmäki, H; Roering, P;

    2011-01-01

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

  14. 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

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

    Science.gov (United States)

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

    2012-02-01

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

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

    Science.gov (United States)

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

    2012-01-01

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

  17. Increased chondrocyte adhesion on nanotubular anodized titanium.

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

    2009-03-01

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

  18. Classification of primary articular chondrocalcinosis.

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    Zitnan, D; Sitaj, S

    1979-01-01

    Based on long-term observations the authors submit a categorization of primary (hereditary and solitary) articular chondrocalcinosis into three different sub-populations. Attention is drawn to the fact that the extent of the qualitative disorder of the articular cartilage, obviously conditioned genetically, is linked with the age factor and determines the quantitative differences of pyrophosphate arthropathy in primary chondrocalcinosis. In young age, as a rule in the third decade, severe polyarticular condrocalcinosis (first sub-population) develops which causes relatively soon invalidity, in middle age (5th and 6th decade) milder condrocalcinosis develops (second sub-population) which combines with extraarticular, tendinous and tissue calcifacations, and finally in advanced age oligoarticular chondrocalcinosis develops (third sub-population) which is usually associated with ankylosing hyperostosis of the spine. Articular chondrocalcinosis (CCA) which we described by this term as a special metabolic arthropathy which occurs in families and solitary and which we defined as a special nosological unit (35, 36,) has become generally known and firmly established in rheumatology. As ensues from numerous publications, primary (idiopathic) CCA which comprises the hereditary and solitary (sporadic) form is characterized by pyrophosphate arthropathy which develops on articular cartilages not damaged by another process (13, 25, 26, 37); on the other hand as secondary CCA we consider pyrophosphate arthropathies which are associated with metabolic, endocrine or other diseases (9, 30). The common sign of both basic forms of CCA is the presence of microcrystals of calcium pyrophosphate dihydrate (CaPD) in articular cartilages, synovial fluid, or other articular structures (capsules, tendons, ligaments), characterized originally by McCarty et al. (11, 18) and later by other authors (2, 23, 27, 32). In addition to semantic (terminological) problems there were also questions of the

  19. Effects of Chondroitinase ABC-Mediated Proteoglycan Digestion on Decellularization and Recellularization of Articular Cartilage.

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    Catherine A Bautista

    Full Text Available Articular cartilage has a limited capacity to heal itself and thus focal defects often result in the development of osteoarthritis. Current cartilage tissue engineering strategies seek to regenerate injured tissue by creating scaffolds that aim to mimic the unique structure and composition of native articular cartilage. Decellularization is a novel strategy that aims to preserve the bioactive factors and 3D biophysical environment of the native extracellular matrix while removing potentially immunogenic factors. The purpose of this study was to develop a procedure that can enable decellularization and recellularization of intact articular cartilage matrix. Full-thickness porcine articular cartilage plugs were decellularized with a series of freeze-thaw cycles and 0.1% (w/v sodium dodecyl sulfate detergent cycles. Chondroitinase ABC (ChABC was applied before the detergent cycles to digest glycosaminoglycans in order to enhance donor chondrocyte removal and seeded cell migration. Porcine synovium-derived mesenchymal stem cells were seeded onto the decellularized cartilage scaffolds and cultured for up to 28 days. The optimized decellularization protocol removed 94% of native DNA per sample wet weight, while collagen content and alignment were preserved. Glycosaminoglycan depletion prior to the detergent cycles increased removal of nuclear material. Seeded cells infiltrated up to 100 μm into the cartilage deep zone after 28 days in culture. ChABC treatment enhances decellularization of the relatively dense, impermeable articular cartilage by reducing glycosaminoglycan content. ChABC treatment did not appear to affect cell migration during recellularization under static, in vitro culture, highlighting the need for more dynamic seeding methods.

  20. TGF-ß1 enhances the BMP-2-induced chondrogenesis of bovine synovial explants and arrests downstream differentiation at an early stage of hypertrophy.

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    Nahoko Shintani

    Full Text Available BACKGROUND: Synovial explants furnish an in-situ population of mesenchymal stem cells for the repair of articular cartilage. Although bone morphogenetic protein 2 (BMP-2 induces the chondrogenesis of bovine synovial explants, the cartilage formed is neither homogeneously distributed nor of an exclusively hyaline type. Furthermore, the downstream differentiation of chondrocytes proceeds to the stage of terminal hypertrophy, which is inextricably coupled with undesired matrix mineralization. With a view to optimizing BMP-2-induced chondrogenesis, the modulating influences of fibroblast growth factor 2 (FGF-2 and transforming growth factor beta 1 (TGF-ß1 were investigated. METHODOLOGY/PRINCIPAL FINDINGS: Explants of bovine calf metacarpal synovium were exposed to BMP-2 (200 ng/ml for 4 (or 6 weeks. FGF-2 (10 ng/ml or TGF-ß1 (10 ng/ml was introduced at the onset of incubation and was present either during the first week of culturing alone or throughout its entire course. FGF-2 enhanced the BMP-2-induced increase in metachromatic staining for glycosaminoglycans (GAGs only when it was present during the first week of culturing alone. TGF-ß1 enhanced not only the BMP-2-induced increase in metachromasia (to a greater degree than FGF-2, but also the biochemically-assayed accumulation of GAGs, when it was present throughout the entire culturing period; in addition, it arrested the downstream differentiation of cells at an early stage of hypertrophy. These findings were corroborated by an analysis of the gene- and protein-expression levels of key cartilaginous markers and by an estimation of individual cell volume. CONCLUSIONS/SIGNIFICANCE: TGF-ß1 enhances the BMP-2-induced chondrogenesis of bovine synovial explants, improves the hyaline-like properties of the neocartilage, and arrests the downstream differentiation of cells at an early stage of hypertrophy. With the prospect of engineering a mature, truly articular type of cartilage in the context of

  1. The effects of ascorbic acid on cartilage metabolism in guinea pig articular cartilage explants.

    Science.gov (United States)

    Clark, Amy G; Rohrbaugh, Amy L; Otterness, Ivan; Kraus, Virginia B

    2002-03-01

    Ascorbic acid has been associated with the slowing of osteoarthritis progression in guinea pig and man. The goal of this study was to evaluate transcriptional and translational regulation of cartilage matrix components by ascorbic acid. Guinea pig articular cartilage explants were grown in the presence of L-ascorbic acid (L-Asc), D-isoascorbic acid (D-Asc), sodium L-ascorbate (Na L-Asc), sodium D-isoascorbate (Na D-Asc), or ascorbyl-2-phosphate (A2P) to isolate and analyze the acidic and nutrient effects of ascorbic acid. Transcription of type II collagen, prolyl 4-hydroxylase (alpha subunit), and aggrecan increased in response to the antiscorbutic forms of ascorbic acid (L-Asc, Na L-Asc, and A2P) and was stereospecific to the L-forms. Collagen and aggrecan synthesis also increased in response to the antiscorbutic forms but only in the absence of acidity. All ascorbic acid forms tended to increase oxidative damage over control. This was especially true for the non-nutrient D-forms and the high dose L-Asc. Finally, we investigated the ability of chondrocytes to express the newly described sodium-dependent vitamin C transporters (SVCTs). We identified transcripts for SVCT2 but not SVCT1 in guinea pig cartilage explants. This represents the first characterization of SVCTs in chondrocytes. This study confirms that ascorbic acid stimulates collagen synthesis and in addition modestly stimulates aggrecan synthesis. These effects are exerted at both transcriptional and post-transcriptional levels. The stereospecificity of these effects is consistent with chondrocyte expression of SVCT2, shown previously to transport L-Asc more efficiently than D-Asc. Therefore, this transporter may be the primary mechanism by which the L-forms of ascorbic acid enter the chondrocyte to control matrix gene activity.

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

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    Shun-Fu Chang

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

  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.

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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.

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

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

    2016-07-01

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

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

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    Koichi Murata

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

  6. Intra-articular chondroma of the knee

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    Talwalkar, S.C.; Kambhampati, S.B.S.; Lang Stevenson, A.I. [Oldchurch Hospital, Romford, Essex (United Kingdom); Whitehouse, R. [Manchester University, Department of Radiology, Manchester (United Kingdom); Freemont, A. [University of Manchester, Department of Osteoarticular Pathology, Manchester (United Kingdom)

    2005-06-01

    Chondromas are tumours that develop in relation to the periosteum and, although they are common around the knee, most reports deal with soft tissue chondromas in para-articular locations or intracortical tumours in extra-articular regions. We report a rare case of an intra-articular chondroma in a 16-year-old boy of Asian origin developing in the region of the medial femoral condyle of the femur and extending into the femoral sulcus and the patellofemoral joint. (orig.)

  7. Tribology approach to the engineering and study of articular cartilage.

    Science.gov (United States)

    Wimmer, Markus A; Grad, Sibylle; Kaup, Thomas; Hänni, Markus; Schneider, Erich; Gogolewski, Sylwester; Alini, Mauro

    2004-01-01

    This study has been based on the assumption that articular motion is an important aspect of mechanotransduction in synovial joints. For this reason a new bioreactor concept, able to reproduce joint kinematics more closely, has been designed. The prototype consists of a rotating scaffold and/or cartilage pin, which is pressed onto an orthogonally rotating ball. By oscillating pin and ball in phase difference, elliptical displacement trajectories are generated that are similar to the motion paths occurring in vivo. Simultaneously, dynamic compression may be applied with a linear actuator, while two-step-motors generate the rotation of pin and ball. The whole apparatus is placed in an incubator. The control station is located outside. Preliminary investigations at the gene expression level demonstrated promising results. Compared with free-swelling control and/or simply compression-loaded samples, chondrocyte-seeded scaffolds as well as nasal cartilage explants exposed to interface motion both showed elevated levels of cartilage oligomeric matrix protein mRNA. The final design of the bioreactor will include four individual stations in line, which will facilitate the investigation of motion-initiated effects at the contacting surfaces in more detail.

  8. Articular cartilage repair and the evolving role of regenerative medicine

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    Pieter K Bos

    2010-10-01

    Full Text Available Pieter K Bos1, Marloes L van Melle1, Gerjo JVM van Osch1,21Department of Orthopaedic Surgery, Erasmus MC, Rotterdam, the Netherlands; 2Department of Otorhinolaryngology, Erasmus MC, Rotterdam, the NetherlandsAbstract: Among the growing applications of regenerative medicine, clinical articular cartilage repair has now been used for 2 decades and forms a successful example of translational medicine. Cartilage is characterized by a limited intrinsic repair capacity following injury. Articular cartilage defects cause symptoms, are not spontaneously repaired, and are generally believed to result in early osteoarthritis. Marrow stimulation techniques, osteochondral transplantation, and cell-based therapies, such as autologous chondrocyte implantation (ACI and use of mesenchymal stem cells (MSCs, are used for tissue regeneration, symptom relief, and prevention of further joint degeneration. The exact incidence of cartilage defects and the natural outcome of joints with these lesions are unclear. Currently available cartilage repair techniques are designed for defect treatment in otherwise healthy joints and limbs, mostly in young adults. The natural history studies presented in this review estimated that the prevalence of cartilage lesions in this patient group ranges from 5% to 11%. The background and results from currently available randomized clinical trials of the three mostly used cartilage repair techniques are outlined in this review. Osteochondral transplantation, marrow stimulation, and ACI show improvement of symptoms with an advantage for cell-based techniques, but only a suggestion that risk for joint degeneration can be reduced. MSCs, characterized by their good proliferative capacity and the potential to differentiate into different mesenchymal lineages, form an attractive alternative cell source for cartilage regeneration. Moreover, MSCs provide a regenerative microenvironment by the secretion of bioactive factors. This trophic activity

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

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    Emeric Limagne

    2016-05-01

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

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

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    Naila Rasheed

    2016-01-01

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

  11. Biochemical composition of the superficial layer of articular cartilage.

    Science.gov (United States)

    Crockett, R; Grubelnik, A; Roos, S; Dora, C; Born, W; Troxler, H

    2007-09-15

    To gain more information on the mechanism of lubrication in articular joints, the superficial layer of bovine articular cartilage was mechanically removed in a sheet of ice that formed on freezing the cartilage. Freeze-dried samples contained low concentrations of chondroitin sulphate and protein. Analysis of the protein by SDS PAGE showed that the composition of the sample was comparable to that of synovial fluid (SF). Attenuated total reflection infrared (ATR-IR) spectroscopy of the dried residue indicated that the sample contained mostly hyaluronan. Moreover, ATR-IR spectroscopy of the upper layer of the superficial layer, adsorbed onto silicon, showed the presence of phospholipids. A gel could be formed by mixing hyaluronan and phosphatidylcholine in water with mechanical properties similar to those of the superficial layer on cartilage. Much like the superficial layer of natural cartilage, the surface of this gel became hydrophobic on drying out. Thus, it is proposed that the superficial layer forms from hyaluronan and phospholipids, which associate by hydrophobic interactions between the alkyl chains of the phospholipids and the hydrophobic faces of the disaccharide units in hyaluronan. This layer is permeable to material from the SF and the cartilage, as shown by the presence of SF proteins and chondroitin sulphate. As the cartilage dries out after removal from the joint, the phospholipids migrate towards the surface of the superficial layer to reduce the surface tension. It is also proposed that the highly efficient lubrication in articular joints can, at least in part, be attributed to the ability of the superficial layer to adsorb and hold water on the cartilage surface, thus creating a highly viscous boundary protection.

  12. Phenotypes of articular disc cells in the rat temporomandibular joint as demonstrated by immunohistochemistry for nestin and GFAP.

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    Miyako, Hitoshi; Suzuki, Akiko; Nozawa-Inoue, Kayoko; Magara, Jin; Kawano, Yoshiro; Ono, Kazuhiro; Maeda, Takeyasu

    2011-10-01

    The articular disc is a dense collagenous tissue containing disc cells that are phenotypically described as chondrocyte-like cells or fibrochondrocytes. Despite the possible existence of these phenotypes in systemic joints, little is known about the detailed classification of the articular disc cells in the temporomandibular joint. In this immunocytochemical study we examined the localization and distribution patterns of nestin and glial fibrillary acidic protein (GFAP) in the articular disc of the rat temporomandibular joint at postnatal day 1, and weeks 1, 2, 4 and 8, based on the status of tooth eruption and occlusion. Nestin and GFAP are intermediate filament proteins whose expression patterns are closely related to cell differentiation and cell migration. Both types of immunopositive cell greatly increased postnatally to a stable level after postnatal week 4, but they showed different distribution patterns and cell morphologies. Nestin-reactive disc cells, which were characterized by a meagre cytoplasm and thin cytoplasmic processes, were scattered in the articular disc, whereas GFAP-positive cells, characterized by broader processes, existed exclusively in the deeper area. In mature discs, the major proportion of articular disc cells exhibited GFAP immunoreactivity. Furthermore, a double-immunostaining demonstrated that the nestin-negative cells, consisting of GFAP-positive and -negative cells, exhibited immunoreactions for heat shock protein 25. These findings indicate that the articular disc cells comprise at least three types in the rat temporomandibular joint and suggest that their expressions closely relate to mechanical loading forces within the joint, including occlusal force, as observed through postnatal development.

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

    Science.gov (United States)

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

    2016-11-15

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

  14. The use of fibrin and poly(lactic-co-glycolic acid hybrid scaffold for articular cartilage tissue engineering: an in vivo analysis

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    S Munirah

    2008-02-01

    Full Text Available Our preliminary results indicated that fibrin and poly(lactic-co-glycolic acid (PLGA hybrid scaffold promoted early chondrogenesis of articular cartilage constructs in vitro. The aim of this study was to evaluate in vivo cartilaginous tissue formation by chondrocyte-seeded fibrin/PLGA hybrid scaffolds. PLGA scaffolds were soaked carefully, in chondrocyte-fibrin suspension, and polymerized by dropping thrombin-calcium chloride (CaCl2 solution. PLGA-seeded chondrocytes were used as a control. Resulting constructs were implanted subcutaneously, at the dorsum of nude mice, for 4 weeks. Macroscopic observation, histological evaluation, gene expression and sulphated-glycosaminoglycan (sGAG analyses were performed at each time point of 1, 2 and 4 weeks post-implantation. Cartilaginous tissue formation in fibrin/PLGA hybrid construct was confirmed by the presence of lacunae and cartilage-isolated cells embedded within basophilic ground substance. Presence of proteoglycan and glycosaminoglycan (GAG in fibrin/PLGA hybrid constructs was confirmed by positive Safranin O and Alcian Blue staining. Collagen type II exhibited intense immunopositivity at the pericellular matrices. Chondrogenic properties were further demonstrated by the expression of gene encoded cartilage-specific markers, collagen type II and aggrecan core protein. The sGAG production in fibrin/PLGA hybrid constructs was higher than in the PLGA group. In conclusion, fibrin/PLGA hybrid scaffold promotes cartilaginous tissue formation in vivo and may serve as a potential cell delivery vehicle and a structural basis for articular cartilage tissue-engineering.

  15. The study on the mechanical characteristics of articular cartilage in simulated microgravity

    Science.gov (United States)

    Niu, Hai-Jun; Wang, Qing; Wang, Yue-Xiang; Li, Ang; Sun, Lian-Wen; Yan, Yan; Fan, Fan; Li, De-Yu; Fan, Yu-Bo

    2012-10-01

    The microgravity environment of a long-term space flight may induce acute changes in an astronaut's musculo-skeletal systems. This study explores the effects of simulated microgravity on the mechanical characteristics of articular cartilage. Six rats underwent tail suspension for 14 days and six additional rats were kept under normal earth gravity as controls. Swelling strains were measured using high-frequency ultrasound in all cartilage samples subject to osmotic loading. Site-specific swelling strain data were used in a triphasic theoretical model of cartilage swelling to determine the uniaxial modulus of the cartilage solid matrix. No severe surface irregularities were found in the cartilage samples obtained from the control or tail-suspended groups. For the tail-suspended group, the thickness of the cartilage at a specified site, as determined by ultrasound echo, showed a minor decrease. The uniaxial modulus of articular cartilage at the specified site decreased significantly, from (6.31 ± 3.37)MPa to (5.05 ± 2.98)MPa ( p < 0.05). The histology-stained image of a cartilage sample also showed a reduced number of chondrocytes and decreased degree of matrix staining. These results demonstrated that the 14 d simulated microgravity induced significant effects on the mechanical characteristics of articular cartilage. This study is the first attempt to explore the effects of simulated microgravity on the mechanical characteristics of articular cartilage using an osmotic loading method and a triphasic model. The conclusions may provide reference information for manned space flights and a better understanding of the effects of microgravity on the skeletal system.

  16. The study on the mechanical characteristics of articular cartilage in simulated microgravity

    Institute of Scientific and Technical Information of China (English)

    Hai-Jun Niu; Qing Wang; Yue-Xiang Wang; Ang Li; Lian-Wen Sun; Yan Yan; Fan Fan; De-Yu Li; Yu-Bo Fan

    2012-01-01

    The microgravity environment of a long-term space flight may induce acute changes in an astronaut's musculo-skeletal systems.This study explores the effects of simulated microgravity on the mechanical characteristics of articular cartilage.Six rats underwent tail suspension for 14 days and six additional rats were kept under normal earth gravity as controls.Swelling strains were measured using high-frequency ultrasound in all cartilage samples subject to osmotic loading.Site-specific swelling strain data were used in a triphasic theoretical model of cartilage swelling to determine the uniaxial modulus of the cartilage solid matrix.No severe surface irregularities were found in the cartilage samples obtained from the control or tail-suspended groups.For the tail-suspended group,the thickness of the cartilage at a specified site,as determined by ultrasound echo,showed a minor decrease.The uniaxial modulus of articular cartilage at the specified site decreased significantly,from (6.31 ± 3.37) MPa to (5.05 ± 2.98) MPa (p < 0.05).The histology-stained image of a cartilage sample also showed a reduced number of chondrocytes and decreased degree of matrix staining.These results demonstrated that the 14 d simulated microgravity induced significant effects on the mechanical characteristics of articular cartilage.This study is the first attempt to explore the effects of simulated microgravity on the mechanical characteristics of articular cartilage using an osmotic loading method and a triphasic model.The conclusions may provide reference information for manned space flights and a better understanding of the effects of microgravity on the skeletal system.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2012-02-24

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

  18. Protocols for the in vitro design of animal articular cartilage based on tissue engineering methods

    Directory of Open Access Journals (Sweden)

    Diego Correa

    2012-10-01

    Full Text Available The articular cartilage is the structure that covers the joint ends. It has some specific tasks crucial to the correct joint physiology. It may experience a large amount of injuries that could generate considerable disabilities. Unfortunately its selfrepair capacity is too limited; therefore, many treatments have been developed with partial success, given the suboptimal biomechanical behavior of the resultant tissue. Given that, Tissue Engineering offers an alternative, based on the design of a new tissue with biological and biomechanical features which resembles the native tissue. In this work, the authors describe the methodologies followed to accomplish that goal, studying the chondrocytes harvesting, the cellular cultures, the scaffold seeding processes, the mechanical stimulation and the structural and biomechanical evaluation. Finally, exposed some of the preliminary results, as a experimental validation of the methods proposed are.

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

    Science.gov (United States)

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

    2016-01-01

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

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

    NARCIS (Netherlands)

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

    2007-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Abdul-Rehman Phull

    2016-01-01

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

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

    Science.gov (United States)

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

    2008-02-12

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

  3. 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.

  4. Connective tissue growth factor and articular cartilage repair%结缔组织生长因子与关节软骨的修复

    Institute of Scientific and Technical Information of China (English)

    张世松; 张志峰; 黄健

    2015-01-01

    BACKGROUND:Connective tissue growth factor has the potential to stimulate the differentiation of mesenchymal cels into chondrocytes, can promote the proliferation and differentiation of chondrocytes, and also can promote the expression of type II colagen and proteoglycan in the articular cartilage. So, the connective tissue growth factor plays an important role in the articular cartilage repair along with other growth factors. OBJECTIVE: To focus on the structure of connective tissue growth factor, its function in articular cartilage repair and the interactions with other factors. METHODS: A search across the databases of PubMed (January 1980 to July 2014) was performed, with the key words of “connective tissue growth factor, connective tissue growth factor and articular cartilage, articular cartilage damage, articular cartilage repairment” in English and “articular cartilage injury” in Chinese. Studies with the obsolete, repetitive or unrelated content were excluded. A total of 32 papers were included in thi study. RESULTS AND CONCLUSION: Connective tissue growth factor has the potential to stimulate the differentiation of mesenchymal cels into chondrocytes, can promote the proliferation, differentiation and maturity of chondrocytes, can maintain the extracelular matrix synthesis and balance, and also can promote the expression of type II colagen and proteoglycan in the articular cartilage. Along with other growth factors, connective tissue growth factor exerts an important role in the articular cartilage repair. Connective tissue growth factor is the key factor of chondrocyte growth, proliferation and differentiation, which is throughout the process of cartilage repair. Studies have shown that articular chondrocytes from patients with osteoarthritis show an increase in the number positively correlated with the expressions of fibroblast growth factor 1 and connective tissue growth factor. Connective tissue growth factor interacts with articular cartilage

  5. Autologous chondrocyte implantation in children and adolescents

    DEFF Research Database (Denmark)

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

    2013-01-01

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

  6. Estabilidade articular: abordagem biomecânica

    OpenAIRE

    Alex Sandra Oliveira de Cerqueira Soares

    2015-01-01

    A instabilidade articular é responsável pelo desenvolvimento de lesões degenerativas incapacitantes que comprometem o desempenho funcional. Compreender os processos desenvolvidos para estabilização dinâmica articular é um desafio para pesquisadores das mais diversas áreas. O presente estudo propõe o uso da abordagem Biomecânica para reconhecer os mecanismos relacionadas ao processo de estabilização dinâmica articular, por meio de três diferentes condições experimentais. No experimento 1 foi a...

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

    Science.gov (United States)

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

    2015-02-01

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

  8. The effects of physical activity on apoptosis and lubricin expression in articular cartilage in rats with glucocorticoid-induced osteoporosis.

    Science.gov (United States)

    Musumeci, Giuseppe; Loreto, Carla; Leonardi, Rosalia; Castorina, Sergio; Giunta, Salvatore; Carnazza, Maria Luisa; Trovato, Francesca Maria; Pichler, Karin; Weinberg, Annelie Martina

    2013-05-01

    Glucocorticoids are considered the most powerful anti-inflammatory and immunomodulating drugs. However, a number of side-effects are well documented in different diseases, including articular cartilage, where increases or decreases in the synthesis of hormone-dependent extracellular matrix components are seen. The objective of this study has been to test the effects of procedures or drugs affecting bone metabolism on articular cartilage in rats with prednisolone-induced osteoporosis and to evaluate the outcomes of physical activity with treadmill and vibration platform training on articular cartilage. The animals were divided into 5 groups, and bone and cartilage evaluations were performed using whole-body scans and histomorphometric analysis. Lubricin and caspase-3 expression were evaluated by immunohistochemistry, Western blot analysis and biochemical analysis. These results confirm the beneficial effect of physical activity on the articular cartilage. The effects of drug therapy with glucocorticoids decrease the expression of lubricin and increase the expression of caspase-3 in the rats, while after physical activity the values return to normal compared to the control group. Our findings suggest that it might be possible that mechanical stimulation in the articular cartilage could induce the expression of lubricin, which is capable of inhibiting caspase-3 activity, preventing chondrocyte death. We can assume that the physiologic balance between lubricin and caspase-3 could maintain the integrity of cartilage. Therefore, in certain diseases such as osteoporosis, mechanical stimulation could be a possible therapeutic treatment. With our results we can propose the hypothesis that physical activity could also be used as a therapeutic treatment for cartilage disease such as osteoarthritis.

  9. Contrast Agent-Enhanced Computed Tomography of Articular Cartilage: Association with Tissue Composition and Properties

    Energy Technology Data Exchange (ETDEWEB)

    Silvast, T.S.; Jurvelin, J.S.; Aula, A.S.; Lammi, M.J.; Toeyraes, J. (Dept. of Clinical Neurophysiology, Kuopio Univ. Hospital, Kuopio (Finland))

    2009-01-15

    Background: Contrast agent-enhanced computed tomography may enable the noninvasive quantification of glycosaminoglycan (GAG) content of articular cartilage. It has been reported that penetration of the negatively charged contrast agent ioxaglate (Hexabrix) increases significantly after enzymatic degradation of GAGs. However, it is not known whether spontaneous degradation of articular cartilage can be quantitatively detected with this technique. Purpose: To investigate the diagnostic potential of contrast agent-enhanced cartilage tomography (CECT) in quantification of GAG concentration in normal and spontaneously degenerated articular cartilage by means of clinical peripheral quantitative computed tomography (pQCT). Material and Methods: In this in vitro study, normal and spontaneously degenerated adult bovine cartilage (n=32) was used. Bovine patellar cartilage samples were immersed in 21 mM contrast agent (Hexabrix) solution for 24 hours at room temperature. After immersion, the samples were scanned with a clinical pQCT instrument. From pQCT images, the contrast agent concentration in superficial as well as in full-thickness cartilage was calculated. Histological and functional integrity of the samples was quantified with histochemical and mechanical reference measurements extracted from our earlier study. Results: Full diffusion of contrast agent into the deep cartilage was found to take over 8 hours. As compared to normal cartilage, a significant increase (11%, P<0.05) in contrast agent concentration was seen in the superficial layer of spontaneously degenerated samples. Significant negative correlations were revealed between the contrast agent concentration and the superficial or full-thickness GAG content of tissue (|R|>0.5, P<0.01). Further, pQCT could be used to measure the thickness of patellar cartilage. Conclusion: The present results suggest that CECT can be used to diagnose proteoglycan depletion in spontaneously degenerated articular cartilage with a

  10. Evaluation of chondrocyte growth in the highly porous scaffolds made by fused deposition manufacturing (FDM) filled with type II collagen.

    Science.gov (United States)

    Yen, Hung-Jen; Tseng, Ching-Shiow; Hsu, Shan-Hui; Tsai, Ching-Lin

    2009-06-01

    Highly porous poly(D,L-lactide-co-glycolide) (PLGA) scaffolds for cartilage tissue engineering were fabricated in this study using the fused deposition manufacturing (FDM) process and were further modified by type II collagen. The average molecular weight of PLGA decreased to about 60% of the original value after the melt-extrusion process. Type II collagen exhibited sponge-like structure and filled the macroporous FDM scaffolds. An increase of the fiber spacing resulted in an increase of the porosity. The storage modulus of FDM scaffolds with a large fiber spacing was comparable to that of the native porcine articular cartilage. Although the FDM hybrid scaffolds were swollen in various extents after 28 days of in vitro culture, the seeded chondrocytes were well distributed in the interior of the scaffolds with a large fiber spacing and neocartilage was formed around the scaffolds. The study also suggested that a low processing temperature may be required to produce PLGA precision scaffolds using FDM.

  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. 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.

  13. Imaging of intra-articular osteoid osteoma

    Energy Technology Data Exchange (ETDEWEB)

    Allen, S.D.; Saifuddin, A. E-mail: asaifuddin@aol.com

    2003-11-01

    Intra-articular osteoid osteoma accounts for approximately 13% of all osteoid osteomas and presents as a monoarthropathy. Radiographs commonly do not identify the nidus, and in this event, MRI is likely to be the next imaging investigation. MRI may show a variety of appearances depending upon the age of the lesion. This article illustrates the imaging features of intra-articular osteoid osteoma, with emphasis on MRI. CT remains the investigation of choice for identifying the nidus.

  14. Canine articular cartilage regeneration using mesenchymal stem cells seeded on platelet rich fibrin

    Science.gov (United States)

    Shams Asenjan, K.; Dehdilani, N.; Parsa, H.

    2017-01-01

    Objectives Mesenchymal stem cells have the ability to differentiate into various cell types, and thus have emerged as promising alternatives to chondrocytes in cell-based cartilage repair methods. The aim of this experimental study was to investigate the effect of bone marrow derived mesenchymal stem cells combined with platelet rich fibrin on osteochondral defect repair and articular cartilage regeneration in a canine model. Methods Osteochondral defects were created on the medial femoral condyles of 12 adult male mixed breed dogs. They were either treated with stem cells seeded on platelet rich fibrin or left empty. Macroscopic and histological evaluation of the repair tissue was conducted after four, 16 and 24 weeks using the International Cartilage Repair Society macroscopic and the O’Driscoll histological grading systems. Results were reported as mean and standard deviation (sd) and compared at different time points between the two groups using the Mann-Whitney U test, with a value regeneration. It is postulated that platelet rich fibrin creates a suitable environment for proliferation and differentiation of stem cells by releasing endogenous growth factors resulting in creation of a hyaline-like reparative tissue. Cite this article: D. Kazemi, K. Shams Asenjan, N. Dehdilani, H. Parsa. Canine articular cartilage regeneration using mesenchymal stem cells seeded on platelet rich fibrin: Macroscopic and histological assessments. Bone Joint Res 2017;6:98–107. DOI: 10.1302/2046-3758.62.BJR-2016-0188.R1. PMID:28235767

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

    DEFF Research Database (Denmark)

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

    2010-01-01

    PURPOSE: We report the 2-year clinical results and identify prognostic factors in patients treated with autologous chondrocyte transplantation by use of a collagen membrane to seed the chondrocytes (ACT-CS). METHODS: This is a prospective study of 59 patients who were treated with ACT-CS...... Repair Society (ICRS) rating, the percentage of patients rated A (normal) and B (nearly normal) increased from 33.9% preoperatively to 92.5% at 24 months after ACT-CS. IKDC and Lysholm scores increased from 50.1 points (SD, 13.4) and 60.5 points (SD, 9.4), respectively, to 76.1 points (SD, 15.2) (P...... CS as a salvage procedure. The rate of failures in patients with isolated cartilage defects was 5.9%. CONCLUSIONS: ACT-CS...

  16. Comparative, osteochondral defect repair: Stem cells versus chondrocytes versus Bone Morphogenetic Protein-2, solely or in combination

    Directory of Open Access Journals (Sweden)

    R Reyes

    2013-07-01

    Full Text Available Full-thickness articular cartilage damage does not resolve spontaneously. Studies with growth factors, implantation of autologous chondrocytes and mesenchymal stem cells have led to variable, to some extent inconsistent, results. This work compares osteochondral knee-defect repair in rabbits upon implantation of a previously described alginate/(poly(lactic-co-glycolic acid (PLGA osteochondral scaffold in distinct conditions. Systems were either in vitro pre-cultured with a small number of allogeneic chondrocytes under fibroblast growth factor (FGF-2 stimulation or the same amount of allogeneic, marrow derived, mesenchymal stem cells (without any pre-differentiation, or loaded with microsphere-encapsulated bone morphogenetic protein (BMP-2 within the alginate layer, or holding combinations of one or the other cell type with BMP-2. The experimental limit was 12 weeks, because a foregoing study with this release system had shown a maintained tissue response for at least 24 weeks post-operation. After only 6 weeks, histological analyses revealed newly formed cartilage-like tissue, which resembled the adjacent, normal cartilage in cell as well as BMP-2 treated defects, but cell therapy gave higher histological scores. This advantage evened out until 12 weeks. Combinations of cells and BMP-2 did not result in any additive or synergistic effect. Equally efficient osteochondral defect repair was achieved with chondrocyte, stem cell, and BMP-2 treatment. Expression of collagen X and collagen I, signs of ongoing ossification, were histologically undetectable, and the presence of aggrecan protein indicated cartilage-like tissue. In conclusion, further work should demonstrate whether spatiotemporally controlled, on-site BMP-2 release alone could become a feasible therapeutic approach to repair large osteochondral defects.

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

    Science.gov (United States)

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

    2015-07-01

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

  18. Focal adhesion kinase and mitogen-activated protein kinases are involved in chondrocyte activation by the 29-kDa amino-terminal fibronectin fragment.

    Science.gov (United States)

    Gemba, Takefumi; Valbracht, Jean; Alsalameh, Saifeddin; Lotz, Martin

    2002-01-11

    The 29-kDa amino-terminal fibronectin fragment (FN-f) has a potent chondrolytic effect and is thought to be involved in cartilage degradation in arthritis. However, little is known about signal transduction pathways that are activated by FN-f. Here we demonstrated that FN-f induced nitric oxide (NO) production from human articular chondrocytes. Expression of inducible nitric-oxide synthase (iNOS) mRNA and NO production were observed at 6 and 48 h after FN-f treatment, respectively. Interleukin-1beta (IL-1beta) mRNA up-regulation was stimulated by FN-f in human chondrocytes. To address the possibility that FN-f-induced NO release is mediated by IL-1beta production, the effect of IL-1 receptor antagonist (IL-1ra) was determined. IL-1ra partially inhibited FN-f-induced NO release although it almost completely inhibited IL-1beta-induced NO release. Tyrosine phosphorylation of focal adhesion kinase was induced transiently by FN-f treatment. Blocking antibodies to alpha(5) or beta(1) integrin and Arg-Gly-Asp-containing peptides did not inhibit FN-f-induced NO production. PP2, a Src family kinase inhibitor, or cytochalasin D, which selectively disrupts the network of actin filaments, inhibited both FAK phosphorylation and NO production induced by FN-f, but the phosphatidylinositol 3-kinase inhibitor wortmannin had no effect. Analysis of mitogen-activated protein kinases (MAPK) showed activation of extracellular signal-regulated kinase (ERK), c-Jun NH(2)-terminal kinase, and p38 MAPK. High concentrations of SB203580, which inhibit both JNK and p38 MAPK, and PD98059 a selective inhibitor of MEK1/2 that blocks ERK activation, inhibited FN-f induced NO production. These data suggest that focal adhesion kinase and MAPK mediate FN-f induced activation of human articular chondrocytes.

  19. Transplantatation of scaffold-free spheroids composed of synovium-derived cells and chondrocytes for the treatment of cartilage defects of the knee

    Directory of Open Access Journals (Sweden)

    J-I Lee

    2011-11-01

    Full Text Available Autologous chondrocyte implantation (ACI is the treatment of choice for osteoarthritis. However, to regenerate articular cartilage using this method, the procedure paradoxically demands that the cell source of the articular chondrocytes (ACs for ex vivo expansion be from the patient’s own healthy cartilage, which can result in donor site morbidity. Accordingly, it is essential to develop a substitute for AC. In the present study, we investigated whether synovium-derived cells (SYs could be used as a partial replacement for ACs in ACI. ACs and SYs from the knees of rabbits were isolated and cultured, and the growth rates of the cells were compared. To manufacture the cellular transplants, we developed a high-density suspension-shaking culture method (HDSS, which circulates the cells in culture media, promoting self-assembly of scaffold-free cellular aggregates. ACs and SYs were mixed in various ratios using HDSS. Injectable cellular transplants were harvested and transplanted into full-thickness osteochondral defects. Simultaneously, histological evaluations were conducted with toluidine blue and safranin O, and immunohistochemistry of collagen type I and II was conducted. Gene expression to evaluate chondrocyte-specific differentiation was also performed. We successfully prepared a large quantity of spheroids (spheroidal cell aggregates in a short time using mixed ACs and SYs, for all cellular composition ratios. Our data showed that the minimal therapeutic unit for the transplants contributed to in situ regeneration of cartilage. In summary, SYs can be used as a replacement for ACs in clinical cases of ACI in patients with broad areas of osteoarthritic lesions.

  20. Working conditions of bipolar radiofrequency on human articular cartilage repair following thermal injury during arthroscopy

    Institute of Scientific and Technical Information of China (English)

    Huang Yuelong; Zhang Yujun; Ding Xiaoquan; Liu Songyang; Sun Tiezheng

    2014-01-01

    Background The thermal injury during bipolar radiofrequercy results in chondrocyte death that limits cartilage repair.The purpose was to determine the effects of various factors of bipolar radiofrequency on human articular cartilage after thermal injury,offering suitable working conditions for bipolar radiofrequency during arthroscopy.Methods Osteochondral explants from 28 patients undergoing total knee arthroplasty (TKA) in Department of Orthopaedic,Peking University Reople's Hospital from October 2013 to May 2014,were harvested and treated using bipolar radiofrequency in a light contact mode under the following conditions:various power setting of levels 2,4 and 6; different durations of 2 seconds,5 seconds and 10 seconds; irrigation with fluids of different temperatures of 4℃,22℃,and 37℃; two different bipolar radiofrequency probes ArthroCare TriStar 50 and Paragon T2.The percentage of cell death and depth of cell death were quantified with laser confocal microscopy.The content of proteoglycan elution at different temperatures was determined by spectrophotometer at 530 nm.Results Chondrocyte mortality during the treatment time of 2 seconds and power setting of level 2 was significantly lower than that with long duration or in higher level groups (time:P=0.001; power:P=0.001).The percentage of cell death after thermal injury was gradually reduced by increasing the temperature of the irrigation solutions (P=0.003),the depth of dead chondrocytes in the 37℃ solution group was significantly less than those in the 4℃ and 22℃ groups (P=0.001).The proteoglycan elution was also gradually reduced by increasing the temperature (P=0.004).Compared with the ArthroCare TriStar 50 group,the percentage of cell death in the Paragon T2 group was significantly decreased (P=0.046).Conclusions Thermal chondroplasty with bipolar radiofrequency resulted in defined margins of chondrocyte death under controlled conditions.The least cartilage damage during thermal chondroplasty

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

    Science.gov (United States)

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

    1996-06-01

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

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

    Science.gov (United States)

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

    2016-01-01

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

  3. Imaging of the cervical articular pillar

    Energy Technology Data Exchange (ETDEWEB)

    Yeomans, E. [Orange Base Hospital, Orange, NSW (Australia)

    1998-12-01

    The cervical articular pillar, due to the complex anatomical structure of the cervical spine, is not well demonstrated in routine plain radiographic views. Dedicated views have been devised to demonstrate the pillar, yet their performance has abated considerably since the inception of Computed Tomography (CT) in the 1970`s. It is the consideration that CT does not image the articular pillar with a 10 per cent accuracy that poses the question: Is there still a need for plain radiography of the cervical articular pillar? This paper studies the anatomy, plain radiography, and incidence of injury to the cervical articular pillar. It discusses (with reference to current and historic literature) the efficacy of current imaging protocols in depicting this injury. It deals with plain radiography, CT, complex tomography, and Magnetic Resonance Imaging (MRI) of the cervical spine to conclude there may still be a position in current imaging protocols for plain radiography of the cervical articular pillar. Copyright (1998) Australian Institute of Radiography 43 refs., 5 figs.

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

    Science.gov (United States)

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

    2004-07-01

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

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

    Directory of Open Access Journals (Sweden)

    Wei-Ping Chen

    2015-05-01

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

  6. Development of artificial articular cartilage

    Indian Academy of Sciences (India)

    Biswajit Bera

    2009-10-01

    The present study describes the development of artificial articular cartilage on the basis of mimicking structural gel properties and mechanical gel properties of natural articular cartilage. It is synthesized from PVA/Si nanocomposite containing 20% Tetra ethoxy silane (TEOS) by sol–gel method. Mechanical strength of Poly(vinyl alcohol), PVA is improved up to 35 MPa. Manufacturing method is adopted considering colloidal stability of nano silica particle in PVA sol at specific pH = 1. An adhesive is also prepared from PVA/Si nanocomposite containing 40% TEOS for firm attachment of artificial articular cartilage on underlying bone with high bond strength.

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

    Science.gov (United States)

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

    2016-01-01

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

  8. Diffusion and near-equilibrium distribution of MRI and CT contrast agents in articular cartilage

    Science.gov (United States)

    Silvast, Tuomo S.; Kokkonen, Harri T.; Jurvelin, Jukka S.; Quinn, Thomas M.; Nieminen, Miika T.; Töyräs, Juha

    2009-11-01

    Charged contrast agents have been used both in vitro and in vivo for estimation of the fixed charge density (FCD) in articular cartilage. In the present study, the effects of molecular size and charge on the diffusion and equilibrium distribution of several magnetic resonance imaging (MRI) and computed tomography (CT) contrast agents were investigated. Full thickness cartilage disks (Ø = 4.0 mm, n = 64) were prepared from fresh bovine patellae. Contrast agent (gadopentetate: Magnevist®, gadodiamide: Omniscan™, ioxaglate: Hexabrix™ or sodium iodide: NaI) diffusion was allowed either through the articular surface or through the deep cartilage. CT imaging of the samples was conducted before contrast agent administration and after 1, 5, 9, 16, 25 and 29 h (and with three samples after 2, 3, 4 and 5 days) diffusion using a clinical peripheral quantitative computed tomography (pQCT) instrument. With all contrast agents, the diffusion through the deep cartilage was slower when compared to the diffusion through the articular surface. With ioxaglate, gadopentetate and gadodiamide it took over 29 h for diffusion to reach the near-equilibrium state. The slow diffusion of the contrast agents raise concerns regarding the validity of techniques for FCD estimation, as these contrast agents may not reach the equilibrium state that is assumed. However, since cartilage composition, i.e. deep versus superficial, had a significant effect on diffusion, imaging of the nonequilibrium diffusion process might enable more accurate assessment of cartilage integrity.

  9. 77 FR 29914 - Bovine Spongiform Encephalopathy; Importation of Bovines and Bovine Products

    Science.gov (United States)

    2012-05-21

    ... RIN 0579-AC68 Bovine Spongiform Encephalopathy; Importation of Bovines and Bovine Products AGENCY... live bovines and products derived from bovines with regard to bovine spongiform encephalopathy. This... with regard to bovine spongiform encephalopathy. Comments on the proposed rule were required to......

  10. Intra-articular angiolipoma of the knee: a case report

    Directory of Open Access Journals (Sweden)

    Nishimori Makoto

    2010-04-01

    Full Text Available Abstract We report a case of intra-articular angiolipoma of the knee. This case report describes our experience in excising an intra-articular angiolipoma of the knee joint. Complete resection under arthroscopy was performed in a 30-year-old man. Two years after the surgery, no evidence of recurrence was seen. Intra-articular angiolipomas should be considered in the differential diagnosis of intra-articular masses in adolescents with recurrent hemarthrosis without trauma.

  11. Preparation of Articular Cartilage Specimens for Scanning Electron Microscopy.

    Science.gov (United States)

    Stupina, T A

    2016-08-01

    We developed and adapted a technology for preparation of articular cartilage specimens for scanning electron microscopy. The method includes prefixation processing, fixation, washing, and dehydration of articular cartilage specimens with subsequent treatment in camphene and air-drying. The technological result consists in prevention of deformation of the articular cartilage structures. The method is simpler and cheaper than the known technologies.

  12. Customized biomaterials to augment chondrocyte gene therapy.

    Science.gov (United States)

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

    2017-02-07

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

  13. Real-time electro-mechano-acoustic imaging for monitoring interactions between trypsin and different inhibitors in articular cartilage.

    Science.gov (United States)

    Zheng, Yong-Ping; Wang, Qing; Butt, Yoki Kwok Chu

    2011-03-01

    The purpose of this study was to observe the real-time interactions between trypsin and various inhibitors in articular cartilage in vitro using a novel electro-mechano-acoustic imaging method. Monitored in real-time, articular cartilage specimens from bovine patellae were first treated with trypsin to reach half proteoglycan depletion (Phase I), then the trypsin solution was replaced with (i) physiological saline buffer (PS), (ii) fetal bovine serum (FBS), (iii) protease inhibitor cocktail (PI) and (iv) 10% formalin (F), respectively, to observe their effects on residual digestion (Phase II). Ultrasound radio frequency signals from the articular cartilage were used to form a M-mode image, where the interface between trypsin digested and intact cartilage tissues could be observed with an additional echo generated. The inhibition time, the digestion depth and digestion fraction were measured for each specimen. The results showed that the dilution of trypsin using saline solution was not sufficient to stop the enzyme action instantly. Although groups FBS and PI had a similar inhibition time of approximately 1.5 h, their digestion depth was obviously different (0.25±0.03 and 0.06±0.06 mm, respectively). In contrast, formalin only took <30 min to stop the trypsin digestion with almost no further digestion. The results demonstrated that the current system was capable of monitoring the trypsin digestion and inhibition process in real time. Also, different chemicals affected the residual trypsin digestion to different degrees.

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

    NARCIS (Netherlands)

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

    2004-01-01

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

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

    NARCIS (Netherlands)

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

    2004-01-01

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

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

    NARCIS (Netherlands)

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

    2014-01-01

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

  17. Advanced Strategies for Articular Cartilage Defect Repair

    Directory of Open Access Journals (Sweden)

    Fergal J. O'Brien

    2013-02-01

    Full Text Available Articular cartilage is a unique tissue owing to its ability to withstand repetitive compressive stress throughout an individual’s lifetime. However, its major limitation is the inability to heal even the most minor injuries. There still remains an inherent lack of strategies that stimulate hyaline-like articular cartilage growth with appropriate functional properties. Recent scientific advances in tissue engineering have made significant steps towards development of constructs for articular cartilage repair. In particular, research has shown the potential of biomaterial physico-chemical properties significantly influencing the proliferation, differentiation and matrix deposition by progenitor cells. Accordingly, this highlights the potential of using such properties to direct the lineage towards which such cells follow. Moreover, the use of soluble growth factors to enhance the bioactivity and regenerative capacity of biomaterials has recently been adopted by researchers in the field of tissue engineering. In addition, gene therapy is a growing area that has found noteworthy use in tissue engineering partly due to the potential to overcome some drawbacks associated with current growth factor delivery systems. In this context, such advanced strategies in biomaterial science, cell-based and growth factor-based therapies that have been employed in the restoration and repair of damaged articular cartilage will be the focus of this review article.

  18. Intra-articular delivery of sinomenium encapsulated by chitosan microspheres and photo-crosslinked GelMA hydrogel ameliorates osteoarthritis by effectively regulating autophagy.

    Science.gov (United States)

    Chen, Pengfei; Xia, Chen; Mei, Sheng; Wang, Jiying; Shan, Zhi; Lin, Xianfeng; Fan, Shunwu

    2016-03-01

    Reduced expression of autophagy regulators has been observed in pathological cartilage in humans and mice. The present study aimed to investigate the synergistic therapeutic effect of promotion of chondrocyte autophagy via exposure to sinomenium (SIN) encapsulated by chitosan microspheres (CM-SIN) and photo-crosslinked gelatin methacrylate (GelMA) hydrogel, with the goal of evaluating CM-SIN as a treatment for patients with osteoarthritis. First, we fabricated and characterized GelMA hydrogels and chitosan microspheres. Next, we measured the effect of SIN on cartilage matrix degradation induced by IL1-β in chondrocytes and an ex vivo model. SIN ameliorated the pathological changes induced by IL1-β at least partially through activation of autophagy. Moreover, we surgically induced osteoarthritis in mice, which were injected intra-articularly with CM-SIN and GelMA. Cartilage matrix degradation and chondrocyte autophagy were evaluated 4 and 8 weeks after surgery. Treatment with the combination of CM-SIN and GelMA retarded the progression of surgically induced OA. SIN ameliorated cartilage matrix degradation at least partially by inducing autophagy in vivo. Our results demonstrate that injection of the combination of GelMA hydrogel and CM-SIN could be a promising strategy for treating patients with osteoarthritis.

  19. Body weight independently affects articular cartilage catabolism.

    Science.gov (United States)

    Denning, W Matt; Winward, Jason G; Pardo, Michael Becker; Hopkins, J Ty; Seeley, Matthew K

    2015-06-01

    Although obesity is associated with osteoarthritis, it is unclear whether body weight (BW) independently affects articular cartilage catabolism (i.e., independent from physiological factors that also accompany obesity). The primary purpose of this study was to evaluate the independent effect of BW on articular cartilage catabolism associated with walking. A secondary purpose was to determine how decreased BW influenced cardiovascular response due to walking. Twelve able-bodied subjects walked for 30 minutes on a lower-body positive pressure treadmill during three sessions: control (unadjusted BW), +40%BW, and -40%BW. Serum cartilage oligomeric matrix protein (COMP) was measured immediately before (baseline) and after, and 15 and 30 minutes after the walk. Heart rate (HR) and rate of perceived exertion (RPE) were measured every three minutes during the walk. Relative to baseline, average serum COMP concentration was 13% and 5% greater immediately after and 15 minutes after the walk. Immediately after the walk, serum COMP concentration was 14% greater for the +40%BW session than for the -40%BW session. HR and RPE were greater for the +40%BW session than for the other two sessions, but did not differ between the control and -40%BW sessions. BW independently influences acute articular cartilage catabolism and cardiovascular response due to walking: as BW increases, so does acute articular cartilage catabolism and cardiovascular response. These results indicate that lower-body positive pressure walking may benefit certain individuals by reducing acute articular cartilage catabolism, due to walking, while maintaining cardiovascular response. Key pointsWalking for 30 minutes with adjustments in body weight (normal body weight, +40% and -40% body weight) significantly influences articular cartilage catabolism, measured via serum COMP concentration.Compared to baseline levels, walking with +40% body weight and normal body weight both elicited significant increases in

  20. Age-related changes in the articular cartilage of the stifle joint in non-working and working German Shepherd dogs.

    Science.gov (United States)

    Francuski, J V; Radovanović, A; Andrić, N; Krstić, V; Bogdanović, D; Hadzić, V; Todorović, V; Lazarević Macanović, M; Sourice Petit, S; Beck-Cormier, S; Guicheux, J; Gauthier, O; Kovacević Filipović, M

    2014-11-01

    The aims of this study were to define age-related histological changes in the articular cartilage of the stifle joint in non-chondrodystrophic dogs and to determine whether physical activity has a positive impact on preservation of cartilage structure during ageing. Twenty-eight German shepherd dogs were included in the study. These dogs had no evidence of joint inflammation as defined by clinical assessment, radiology and synovial fluid analysis (specifically absence of synovial fluid serum amyloid A). The dogs were grouped as young working (n ¼ 4), young non-working (n ¼ 5), aged working (n ¼ 13) and aged non-working (n ¼ 6) animals. Gross changes in the stifle joints were recorded and biopsy samples of femoral and tibial articular cartilage were evaluated for thickness; chondrocyte number, density, surface area and morphology; isogenous group morphology; tidemark integrity; subchondral bone structure; presence of proteoglycans/ glycosaminoglycans; and expression of type I, II and X collagens. The major age-related changes, not related to type of physical activity, included elevated chondrocyte density and thinning of tibial cartilage and increased chondrocyte surface area in the superficial and intermediate zone of the femoral cartilage. There was also expression of type X collagen in the femoral and tibial calcified and non-calcified cartilage; however, type X collagen was not detected in the superficial zone of old working dogs. Therefore, ageing, with or without physical activity, leads to slight cartilage degeneration, while physical activity modulates the synthesis of type X collagen in the superficial cartilage zone, partially preserving the structure of hyaline cartilage.

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

    NARCIS (Netherlands)

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

    1992-01-01

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

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

    Lifescience Database Archive (English)

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

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

    Lifescience Database Archive (English)

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  16. 15-Deoxy-delta(12,14)-prostaglandin J(2) inhibits IL-1beta-induced IKK enzymatic activity and IkappaBalpha degradation in rat chondrocytes through a PPARgamma-independent pathway.

    Science.gov (United States)

    Boyault, Sandrine; Bianchi, Arnaud; Moulin, David; Morin, Sylvie; Francois, Mathias; Netter, Patrick; Terlain, Bernard; Bordji, Karim

    2004-08-13

    Peroxisome proliferator-activated receptor gamma (PPARgamma) ligands have been shown to inhibit the effects of proinflammatory cytokines such as interleukin-1beta (IL-1beta). This cytokine plays a key role in articular pathophysiologies by inducing the production of inflammatory mediators such as nitric oxide (NO) and prostaglandin E(2) (PGE(2)). We previously demonstrated that 15d-PGJ(2) was more potent than troglitazone to counteract IL-1beta effects on chondrocytes. Here, we studied the action of 15d-PGJ(2) on intracellular targets in nuclear factor-kappaB (NF-kappaB) signalling pathway in IL-1beta treated rat chondrocytes. We found that 15d-PGJ(2) decreased inhibitor kappaBalpha (IkappaBalpha) degradation but not its phosphorylation by specifically inhibiting IkappaB kinase beta (IKKbeta), but not IKKalpha, enzymatic activity. We further evaluated the involvement of PPARgamma in the anti-inflammatory action of its ligands. In chondrocytes overexpressing functional PPARgamma protein, 15d-PGJ(2) pre-treatment inhibited inducible NO synthase and COX-2 mRNA expression, nitrite and PGE(2) production, p65 translocation and NF-kappaB activation. Troglitazone or rosiglitazone pre-treatment had no effect. 15d-PGJ(2) exhibited the same effect in chondrocytes overexpressing mutated PPARgamma protein. These results suggest that 15d-PGJ(2) exerts its anti-inflammatory effect in rat chondrocytes by a PPARgamma-independent mechanism, which can be conferred to a partial inhibition of IkappaBalpha degradation.

  17. Astaxanthin reduces matrix metalloproteinase expression in human chondrocytes.

    Science.gov (United States)

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

    2014-03-01

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

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

    Science.gov (United States)

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

    2015-08-01

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

  19. Effect of Stratified Culture Compared to Confluent Culture in Monolayer on Proliferation and Differentiation of Human Articular Chondrocytes

    NARCIS (Netherlands)

    Hendriks, Jeanine; Riesle, Jens; Blitterswijk, van Clemens A.

    2006-01-01

    With conventional tissue culture of cells, it is generally assumed that when the available 2D substrate is fully occupied, growth ceases or is greatly reduced.However, in naturewound repairmostly involves proliferation of cells that are attracted to the defect site in a 3D environment.Hence, prolife

  20. Use of micro-computed tomography to evaluate the effects of exercise on preventing the degeneration of articular cartilage in tail-suspended rats

    Science.gov (United States)

    Luan, Hui-Qin; Sun, Lian-Wen; Huang, Yun-Fei; Wu, Xin-tong; Niu, Haijun; Liu, Hong; Fan, Yu-Bo

    2015-07-01

    Space flight has been shown to induce bone loss and muscle atrophy, which could initiate the degeneration of articular cartilage. Countermeasures to prevent bone loss and muscle atrophy have been explored, but few spaceflight or ground-based studies have focused on the effects on cartilage degeneration. In this study, we investigated the effects of exercise on articular cartilage deterioration in tail-suspended rats. Thirty-two female Sprague-Dawley rats were randomly divided into four groups (n = 8 in each): tail suspension (TS), tail suspension plus passive motion (TSP), tail suspension plus active exercise (TSA), and control (CON) groups. In the TS, TSP, and TSA groups, the rat hindlimbs were unloaded for 21 days by tail suspension. Next, the cartilage thickness and volume, and the attenuation coefficient of the distal femur were evaluated by micro-computed tomography (μCT). Histological analysis was used to assess the surface integrity of the cartilage, cartilage thickness, and chondrocytes. The results showed that: (1) the cartilage thickness on the distal femur was significantly lower in the TS and TSP groups compared with the CON and TSA groups; (2) the cartilage volume in the TS group was significantly lower compared with the CON, TSA, and TSP groups; and (3) histomorphology showed that the chondrocytes formed clusters where the degree of matrix staining was lower in the TS and TSP groups. There were no significant differences between any of these parameters in the CON and TSA groups. The cartilage thickness measurements obtained by μCT and histomorphology correlated well. In general, tail suspension could induce articular cartilage degeneration, but active exercise was effective in preventing this degeneration in tail-suspended rats.

  1. 骨髓基质细胞诱导分化修复髁突软骨面缺失的实验研究%An experimental study of bone marrow stromal cells differentiating to chondrocytes for repairing temporomandibular joint condylar cartilage defects

    Institute of Scientific and Technical Information of China (English)

    黄跃; 沈国芳; 王旭东; 杨辛; 张秀丽; 蒋欣泉

    2012-01-01

    PURPOSE: To repair the temporomandibular joint condylar cartilaginous defects by the differentiated bone marrow stroma cells combined with chondrocytes. METHODS: Fifteen goats were randomly divided into 2 groups.The experimental group included 9 goats, autologous BMSCs combined with chondrocytes were mixed with pluronic F-127 and implanted into goat temporomandibular joint condylar articular cartilage defects. The control group included 6 goats, pluronic F-127 gel was implanted into their temporomandibular joint condylar articular cartilage defects.The goats were killed 4,8,12 weeks postoperatively.The reconstructive articular cartilage was evaluated by HE staining and immunostaining of type Ⅱ collagen. RESULTS: The experimental group could reconstruct the articular cartilage after 4 weeks. 12 weeks later the articular cartilage did not degenerate. The control group could not reconstruct the articular cartilage. CONCLUSIONS: BMSCs can be differentiated into chondrocytes and play an important role in repairing articular cartilage defects. Supported by Phosphor Program of Science and Technology Commission of Shanghai Municipality (04QMX1424) and Key Project in Social Development Sponsored by Fujian Provincial Department of Science and Technology (2009Y0016).%目的:采用骨髓基质细胞(BMSCs)体内修复髁突软骨全层缺失.方法:15只山羊,9只作为实验组,将BMSCs和少量软骨细胞(7∶3比例混合)按5×107/mL与生物可降解材料复合后,植入山羊髁突软骨全层缺失处;对照组6只山羊,髁突软骨全层缺失区植入支架材料,分别于术后4、8、12周每个时间段取材3只实验动物,2只对照组动物;2组分别用HE染色、Ⅱ型胶原分泌的免疫组化法进行评价.结果:实验组术后4周,山羊髁突软骨缺失区能形成成熟的软骨组织,12周时软骨未退变.对照组不能形成成熟的软骨组织.结论:骨髓基质细胞在自体软骨细胞基质的诱导下,可以修复山羊颞下

  2. 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.

  3. The minor collagens in articular cartilage

    DEFF Research Database (Denmark)

    Luo, Yunyun

    2017-01-01

    Articular cartilage is a connective tissue consisting of a specialized extracellular matrix (ECM) that dominates the bulk of its wet and dry weight. Type II collagen and aggrecan are the main ECM proteins in cartilage. However, little attention has been paid to less abundant molecular components......, especially minor collagens, including type IV, VI, IX, X, XI, XII, XIII, and XIV, etc. Although accounting for only a small fraction of the mature matrix, these minor collagens not only play essential structural roles in the mechanical properties, organization, and shape of articular cartilage, but also...... fulfil specific biological functions. Genetic studies of these minor collagens have revealed that they are associated with multiple connective tissue diseases, especially degenerative joint disease. The progressive destruction of cartilage involves the degradation of matrix constituents including...

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

    Directory of Open Access Journals (Sweden)

    Lin Jian-Hua

    2011-11-01

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

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

    Directory of Open Access Journals (Sweden)

    Ling ZHANG

    2014-08-01

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

  6. Intact Pericellular Matrix of Articular Cartilage Is Required for Unactivated Discoidin Domain Receptor 2 in the Mouse Model

    Science.gov (United States)

    Xu, Lin; Polur, Ilona; Servais, Jacqueline M.; Hsieh, Sirena; Lee, Peter L.; Goldring, Mary B.; Li, Yefu

    2011-01-01

    Increased expression of the discoidin domain receptor 2 (DDR2) results from its interaction with collagen type II. This induces expression of matrix metalloproteinase (MMP)-13, leading to osteoarthritis (OA). To investigate the impact of the pericellular matrix of chondrocytes on DDR2, we generated a mouse model with inducible overexpression of DDR2 in cartilage. Conditional overexpression of DDR2 in mature mouse articular cartilage was controlled via the cartilage oligomeric matrix protein promoter using the Tet-Off-inducible system. Doxycycline was withdrawn at 1 month of age, and knee joints were examined at 2, 3, and 4 months of age. Microsurgery was performed on 3-month-old transgenic mice overexpressing DDR2 to destabilize the medial meniscus, and serial paraffin sections were examined at 2, 4, 8, and 12 weeks after surgery. DDR2 expression increased in the knee joints of transgenic mice. However, the increased DDR2 did not induce MMP-13 expression. No OA-like changes were observed in the transgenic mice at the age of 4 months. When transgenic mice were subjected to destabilizing of the medial meniscus, we observed accelerated progression to OA, which was associated with DDR2 activation. Therefore, conditionally overexpressing DDR2 in the mature articular cartilage of mouse knee joints requires activation to induce OA, and altered biomechanical stress can accelerate the onset of cartilage loss and progression to OA in transgenic mice. PMID:21855682

  7. Intact pericellular matrix of articular cartilage is required for unactivated discoidin domain receptor 2 in the mouse model.

    Science.gov (United States)

    Xu, Lin; Polur, Ilona; Servais, Jacqueline M; Hsieh, Sirena; Lee, Peter L; Goldring, Mary B; Li, Yefu

    2011-09-01

    Increased expression of the discoidin domain receptor 2 (DDR2) results from its interaction with collagen type II. This induces expression of matrix metalloproteinase (MMP)-13, leading to osteoarthritis (OA). To investigate the impact of the pericellular matrix of chondrocytes on DDR2, we generated a mouse model with inducible overexpression of DDR2 in cartilage. Conditional overexpression of DDR2 in mature mouse articular cartilage was controlled via the cartilage oligomeric matrix protein promoter using the Tet-Off-inducible system. Doxycycline was withdrawn at 1 month of age, and knee joints were examined at 2, 3, and 4 months of age. Microsurgery was performed on 3-month-old transgenic mice overexpressing DDR2 to destabilize the medial meniscus, and serial paraffin sections were examined at 2, 4, 8, and 12 weeks after surgery. DDR2 expression increased in the knee joints of transgenic mice. However, the increased DDR2 did not induce MMP-13 expression. No OA-like changes were observed in the transgenic mice at the age of 4 months. When transgenic mice were subjected to destabilizing of the medial meniscus, we observed accelerated progression to OA, which was associated with DDR2 activation. Therefore, conditionally overexpressing DDR2 in the mature articular cartilage of mouse knee joints requires activation to induce OA, and altered biomechanical stress can accelerate the onset of cartilage loss and progression to OA in transgenic mice.

  8. Pure waterjet drilling of articular bone

    OpenAIRE

    Biskup, Christian; Dunnen, Steven den; Kraaij, Gert; Kerkhoffs, Gino M. M. J.; Tuijthof, Gabrielle J. M.

    2015-01-01

    The clinical application of waterjet technology for machining tough human tissues, such as articular bone, has advantages, as it produces clean sharp cuts without tissue heating. Additionally, water supply is possible via flexible tubing, which enables minimally invasive surgical access. This pilot study investigates whether drilling bony tissue with pure waterjets is feasible. Water pressures between 20 and 120 MPa with an orifice of 0.6 mm were used to create waterjets to drill blind boring...

  9. Oxygen, nitric oxide and articular cartilage

    Directory of Open Access Journals (Sweden)

    B Fermor

    2007-04-01

    Full Text Available Molecular oxygen is required for the production of nitric oxide (NO, a pro-inflammatory mediator that is associated with osteoarthritis and rheumatoid arthritis. To date there has been little consideration of the role of oxygen tension in the regulation of nitric oxide production associated with arthritis. Oxygen tension may be particularly relevant to articular cartilage since it is avascular and therefore exists at a reduced oxygen tension. The superficial zone exists at approximately 6% O2, while the deep zone exists at less than 1% O2. Furthermore, oxygen tension can alter matrix synthesis, and the material properties of articular cartilage in vitro.The increase in nitric oxide associated with arthritis can be caused by pro-inflammatory cytokines and mechanical stress. Oxygen tension significantly alters endogenous NO production in articular cartilage, as well as the stimulation of NO in response to both mechanical loading and pro-inflammatory cytokines. Mechanical loading and pro-inflammatory cytokines also increase the production of prostaglandin E2 (PGE2. There is a complex interaction between NO and PGE2, and oxygen tension can alter this interaction. These findings suggest that the relatively low levels of oxygen within the joint may have significant influences on the metabolic activity, and inflammatory response of cartilage as compared to ambient levels. A better understanding of the role of oxygen in the production of inflammatory mediators in response to mechanical loading, or pro-inflammatory cytokines, may aid in the development of strategies for therapeutic intervention in arthritis.

  10. Sclareol exerts anti-osteoarthritic activities in interleukin-1β-induced rabbit chondrocytes and a rabbit osteoarthritis model.

    Science.gov (United States)

    Zhong, Ying; Huang, Yi; Santoso, Marcel B; Wu, Li-Dong

    2015-01-01

    Sclareol is a natural product initially isolated form Salvia sclarea which possesses immune-regulation and anti-inflammatory activities. However, the anti-osteoarthritic properties of sclareol have not been investigated. The present study is aimed at evaluating the potential effects of sclareol in interleukin-1β (IL-1β)-induced rabbit chondrocytes as well as an experimental rabbit knee osteoarthritis model induced by anterior cruciate ligament transection (ACLT). Cultured rabbit chondrocytes were pretreated with 1, 5 and 10 μg/mL sclareol for 1 h and followed by stimulation of IL-1β (10 ng/mL) for 24 h. Gene expression of matrix metalloproteinase-1 (MMP-1), MMP-3, MMP-13, tissue inhibitors of metalloproteinase-1 (TIMP-1), inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX)-2 was determined by quantitative real-time polymerase chain reaction (qRT-PCR). MMP-3, TIMP-1, iNOS and COX-2 proteins were measured by Western blotting. Enzyme-linked immunosorbent assay (ELISA) was applied for nitric oxide (NO) and prostaglandin E2 (PGE2) assessment. For the in vivo study, rabbits received six weekly 0.3 mL sclareol (10 μg/mL) intra-articular injections in the knees four weeks after ACLT surgery. Cartilage was harvested for measurement of MMP-1, MMP-3, MMP-13, TIMP-1, iNOS and COX-2 by qRT-PCR, while femoral condyles were used for histological evaluation. The in vitro results we obtained showed that sclareol inhibited the MMPs, iNOS and COX-2 expression on mRNA and protein levels, while increased the TIMP-1 expression. And over-production of NO and PGE2 was also suppressed. For the in vivo study, both qRT-PCR results and histological evaluation confirmed that sclareol ameliorated cartilage degradation. Hence, we speculated that sclareol may be an ideal approach for treating osteoarthritis.

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

    Institute of Scientific and Technical Information of China (English)

    赵忠; 刘德春; 宋阳春

    2013-01-01

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

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

    Science.gov (United States)

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

    1993-09-15

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

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

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    V Nicolin

    2009-08-01

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

  14. Microstructure of Temporomandibular Joint Cartilage after Intra-Articular Betamethasone Sodium Phosphate/Betamethasone Dipropionate Injection during the Early Stage of Experimental Osteoarthrosis

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    Irina N. Kostina

    2014-06-01

    Full Text Available Objective: to study the morphological changes in cartilage after a single intra-articular betamethasone sodium phosphate (BSP/betamethasone dipropionate (BDP injection during the early stage of experimental osteoarthrosis (OA of the temporomandibular joint (TMJ. Material and Methods: The experiment was performed on 18 male rabbits aged 6 months .The first group consisted of 9 healthy rabbits. The second group included 9 rabbits with mechanically induced TMJ OA. For 5 days, 3 hours daily, a load (with a force of 200N on the TMJ was imposed. In the left TMJ of the second group of rabbits, betamethasone was injected intra-articularly in different doses: 0.1 ml (n=3, 0.3 ml (n=3, and 0.5 ml (n=3. The right TMJ was used for comparison. A combined anesthesia was applied each experimental day. Rabbits of both groups were sacrificed on days 7, 14, and 30 with introductory combined anesthesia and intravenous injection of Zoletil 100® 20 mg/kg to stop their breathing. Results: Betamethasone caused destruction of the chondrocytes, fragmentation of collagen fibers, deficit of proteoglycans (PGs and glycosaminoglycans (GAGs, thinning of the cartilage, and contributed to the progression of TMJ OA. Conclusion: The optimal dose of BSP/BDP for intra-articular injection in the early stages of TMJ OA must be within the range of 0.1-0.3 ml|0.7-1.5 mg.

  15. Autophagy effects on chondrocyte survival and cartilage damage%自噬与软骨细胞生存及软骨损伤

    Institute of Scientific and Technical Information of China (English)

    张谢卓; 徐超; 伊力哈木·托合提

    2015-01-01

    AND CONCLUSION: Intra-articular chondrocytes can response to the changes in the microenvironment so as to adjust the extracelular matrix metabolism and maintain the biological function of articular cartilage. Hypoxic environment in which chondrocytes eixt is an important factor to causes autophagy. Autophagy is a normal balance or protection mechanism of chondrocytes. Studies on the correlation of autophagy with cartilage damage have made considerable progress in recent years, but stil in its infancy. Atg discovery at the molecular level deepens the understanding of autophagy, but the induction of cartilage autophagy pathway, signal transduction, and their effects on the survival of chondrocytes are not clear yet, which need further studies.

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

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    Chua Kien-Hui

    2013-01-01

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

  17. Melanocortin 1 receptor-signaling deficiency results in an articular cartilage phenotype and accelerates pathogenesis of surgically induced murine osteoarthritis.

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    Julia Lorenz

    Full Text Available Proopiomelanocortin-derived peptides exert pleiotropic effects via binding to melanocortin receptors (MCR. MCR-subtypes have been detected in cartilage and bone and mediate an increasing number of effects in diathrodial joints. This study aims to determine the role of MC1-receptors (MC1 in joint physiology and pathogenesis of osteoarthritis (OA using MC1-signaling deficient mice (Mc1re/e. OA was surgically induced in Mc1re/e and wild-type (WT mice by transection of the medial meniscotibial ligament. Histomorphometry of Safranin O stained articular cartilage was performed with non-operated controls (11 weeks and 6 months and 4/8 weeks past surgery. µCT-analysis for assessing epiphyseal bone architecture was performed as a longitudinal study at 4/8 weeks after OA-induction. Collagen II, ICAM-1 and MC1 expression was analysed by immunohistochemistry. Mc1re/e mice display less Safranin O and collagen II stained articular cartilage area compared to WT prior to OA-induction without signs of spontaneous cartilage surface erosion. This MC1-signaling deficiency related cartilage phenotype persisted in 6 month animals. At 4/8 weeks after OA-induction cartilage erosions were increased in Mc1re/e knees paralleled by weaker collagen II staining. Prior to OA-induction, Mc1re/e mice do not differ from WT with respect to bone parameters. During OA, Mc1re/e mice developed more osteophytes and had higher epiphyseal bone density and mass. Trabecular thickness was increased while concomitantly trabecular separation was decreased in Mc1re/e mice. Numbers of ICAM-positive chondrocytes were equal in non-operated 11 weeks Mc1re/e and WT whereas number of positive chondrocytes decreased during OA-progression. Unchallenged Mc1re/e mice display smaller articular cartilage covered area without OA-related surface erosions indicating that MC1-signaling is critical for proper cartilage matrix integrity and formation. When challenged with OA, Mc1re/e mice develop a more

  18. In vitro and in vivo co-culture of chondrocytes and bone marrow stem cells in photocrosslinked PCL-PEG-PCL hydrogels enhances cartilage formation.

    Science.gov (United States)

    Ko, Chao-Yin; Ku, Kuan-Lin; Yang, Shu-Rui; Lin, Tsai-Yu; Peng, Sydney; Peng, Yu-Shiang; Cheng, Ming-Huei; Chu, I-Ming

    2016-10-01

    Chondrocytes (CH) and bone marrow stem cells (BMSCs) are sources that can be used in cartilage tissue engineering. Co-culture of CHs and BMSCs is a promising strategy for promoting chondrogenic differentiation. In this study, articular CHs and BMSCs were encapsulated in PCL-PEG-PCL photocrosslinked hydrogels for 4 weeks. Various ratios of CH:BMSC co-cultures were investigated to identify the optimal ratio for cartilage formation. The results thus obtained revealed that co-culturing CHs and BMSCs in hydrogels provides an appropriate in vitro microenvironment for chondrogenic differentiation and cartilage matrix production. Co-culture with a 1:4 CH:BMSC ratio significantly increased the synthesis of GAGs and collagen. In vivo cartilage regeneration was evaluated using a co-culture system in rabbit models. The co-culture system exhibited a hyaline chondrocyte phenotype with excellent regeneration, resembling the morphology of native cartilage. This finding suggests that the co-culture of these two cell types promotes cartilage regeneration and that the system, including the hydrogel scaffold, has potential in cartilage tissue engineering. Copyright © 2013 John Wiley & Sons, Ltd.

  19. Cartilage storage at 4 °C with regular culture medium replacement benefits chondrocyte viability of osteochondral grafts in vitro.

    Science.gov (United States)

    Qi, Jianhong; Hu, Zunjie; Song, Hongqiang; Chen, Bin; Xie, Di; Zhou, Lu; Zhang, Yanming

    2016-09-01

    Maintenance of articular cartilage allografts in culture media is a common method of tissue storage; however, the technical parameters of graft storage remain controversial. In this study, we examined the optimal temperature and culture medium exchange rate for the storage of osteochondral allografts in vitro. Cylindrical osteochondral grafts (n = 120), harvested from the talar joint surface of ten Boer goats, were randomly classified into four groups and stored under the following conditions: Group A1 was maintained at 4 °C in culture medium that was refreshed every 2 days; Group A2 was maintained at 4 °C in the same culture medium, without refreshing; Group B1, was maintained at 37 °C in culture medium that was refreshed every 2 days; Group B2, was maintained at 37 °C in the same culture medium, without refreshing. Chondrocyte viability in the grafts was determined by ethidium bromide/fluorescein diacetate staining on days 7, 21, and 35. Proteoglycan content was measured by Safranin-O staining. Group A1 exhibited the highest chondrocyte survival rates of 90.88 %, 88.31 % and 78.69 % on days 7, 21, and 35, respectively. Safranin O staining revealed no significant differences between groups on days 21 and 35. These results suggest that storage of osteochondral grafts at 4 °C with regular culture medium replacement should be highly suitable for clinical application.

  20. Inhibitory effects of pentosan polysulfate sodium on MAP-kinase pathway and NF-κB nuclear translocation in canine chondrocytes in vitro.

    Science.gov (United States)

    Sunaga, Takafumi; Oh, Namgil; Hosoya, Kenji; Takagi, Satoshi; Okumura, Masahiro

    2012-06-01

    Pentosan polysulfate sodium (PPS) has a heparin-like structure and is purificated from the plant of European beech wood. PPS has been used for the treatment of interstitial cystitis for human patients. Recent years, it was newly recognised that PPS reduce pain and inflammation of OA. The molecular biological mechanism of PPS to express its clinical effects is not fully understood. The purpose of the present study is to investigate a mechanism of action of PPS on inflammatory reaction of chondrocytes in vitro. It was evaluated that effects of PPS on interleukin (IL)-1β-induced phosphorylation of mitogen-actiated protein kinases (MAPKs), such as p38, extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK), nuclear translocation of nuclear factor-kappa B (NF-κB), and matrix metalloproteinase (MMP)-3 production in cultured articular chondrocytes. As a result, in the presence of PPS existence, IL-1β-induced phosphorylation of p38 and ERK were certainly inhibited, while JNK phosphorylation was not affected. Nuclear translocation of NF-κB and MMP-3 production were suppressed by PPS pretreatment prior to IL-1β stimulation. In conclusion, it is strongly suggested that PPS treatment prevents inflammatory intracellular responses induced by IL-1 β through inhibition of phosphorylation of certain MAPKs, p38 and ERK and then nuclear translocation of NF-κB in cultured chondrocytes. These PPS properties may contribute to suppressive consequence of catabolic MMP-3 synthesis. These data might translate the clinical efficacy as PPS treatment could inhibit the cartilage catabolism and related clinical symptoms of OA in dogs.

  1. Lubrication mode analysis of articular cartilage using Stribeck surfaces.

    Science.gov (United States)

    Gleghorn, Jason P; Bonassar, Lawrence J

    2008-01-01

    Lubrication of articular cartilage occurs in distinct modes with various structural and biomolecular mechanisms contributing to the low-friction properties of natural joints. In order to elucidate relative contributions of these factors in normal and diseased tissues, determination and control of lubrication mode must occur. The objectives of these studies were (1) to develop an in vitro cartilage on glass test system to measure friction coefficient, mu; (2) to implement and extend a framework for the determination of cartilage lubrication modes; and (3) to determine the effects of synovial fluid on mu and lubrication mode transitions. Patellofemoral groove cartilage was linearly oscillated against glass under varying magnitudes of compressive strain utilizing phosphate buffered saline (PBS) and equine and bovine synovial fluid as lubricants. The time-dependent frictional properties were measured to determine the lubricant type and strain magnitude dependence for the initial friction coefficient (mu(0)=mu(t-->0)) and equilibrium friction coefficient (mu(eq)=mu(t-->infinity)). Parameters including tissue-glass co-planarity, normal strain, and surface speed were altered to determine the effect of the parameters on lubrication mode via a 'Stribeck surface'. Using this testing apparatus, cartilage exhibited biphasic lubrication with significant influence of strain magnitude on mu(0) and minimal influence on mu(eq), consistent with hydrostatic pressurization as reported by others. Lubrication analysis using 'Stribeck surfaces' demonstrated clear regions of boundary and mixed modes, but hydrodynamic or full film lubrication was not observed even at the highest speed (50mm/s) and lowest strain (5%).

  2. Multi-parametric MRI characterization of enzymatically degraded articular cartilage.

    Science.gov (United States)

    Nissi, Mikko J; Salo, Elli-Noora; Tiitu, Virpi; Liimatainen, Timo; Michaeli, Shalom; Mangia, Silvia; Ellermann, Jutta; Nieminen, Miika T

    2016-07-01

    Several laboratory and rotating frame quantitative MRI parameters were evaluated and compared for detection of changes in articular cartilage following selective enzymatic digestion. Bovine osteochondral specimens were subjected to 44 h incubation in control medium or in collagenase or chondroitinase ABC to induce superficial collagen or proteoglycan (glycosaminoglycan) alterations. The samples were scanned at 9.4 T for T1 , T1 Gd (dGEMRIC), T2 , adiabatic T1 ρ , adiabatic T2 ρ , continuous-wave T1 ρ , TRAFF2 , and T1 sat relaxation times and for magnetization transfer ratio (MTR). For reference, glycosaminoglycan content, collagen fibril orientation and biomechanical properties were determined. Changes primarily in the superficial cartilage were noted after enzymatic degradation. Most of the studied parameters were sensitive to the destruction of collagen network, whereas glycosaminoglycan depletion was detected only by native T1 and T1 Gd relaxation time constants throughout the tissue and by MTR superficially. T1 , adiabatic T1 ρ , adiabatic T2 ρ , continuous-wave T1 ρ , and T1 sat correlated significantly with the biomechanical properties while T1 Gd correlated with glycosaminoglycan staining. The findings indicated that most of the studied MRI parameters were sensitive to both glycosaminoglycan content and collagen network integrity, with changes due to enzymatic treatment detected primarily in the superficial tissue. Strong correlation of T1 , adiabatic T1ρ , adiabatic T2 ρ , continuous-wave T1 ρ , and T1 sat with the altered biomechanical properties, reflects that these parameters were sensitive to critical functional properties of cartilage. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:1111-1120, 2016.

  3. An intra-articular salmon calcitonin-based nanocomplex reduces experimental inflammatory arthritis.

    Science.gov (United States)

    Ryan, Sinéad M; McMorrow, Jason; Umerska, Anita; Patel, Hetal B; Kornerup, Kristin N; Tajber, Lidia; Murphy, Evelyn P; Perretti, Mauro; Corrigan, Owen I; Brayden, David J

    2013-04-28

    Prolonged inappropriate inflammatory responses contribute to the pathogenesis of rheumatoid arthritis (RA) and to aspects of osteoarthritis (OA). The orphan nuclear receptor, NR4A2, is a key regulator and potential biomarker for inflammation and represents a potentially valuable therapeutic target. Both salmon calcitonin (sCT) and hyaluronic acid (HA) attenuated activated mRNA expression of NR4A1, NR4A2, NR4A3, and matrix metalloproteinases (MMPs) 1, 3 and 13 in three human cell lines: SW1353 chondrocytes, U937 and THP-1 monocytes. Ad-mixtures of sCT and HA further down-regulated expression of NR4A2 compared to either agent alone at specific concentrations, hence the rationale for their formulation in nanocomplexes (NPs) using chitosan. The sCT released from NP stimulated cAMP production in human T47D breast cancer cells expressing sCT receptors. When NP were injected by the intra-articular (I.A.) route to the mouse knee during on-going inflammatory arthritis of the K/BxN serum transfer model, joint inflammation was reduced together with NR4A2 expression, and local bone architecture was preserved. These data highlight remarkable anti-inflammatory effects of sCT and HA at the level of reducing NR4A2 mRNA expression in vitro. Combining them in NP elicits anti-arthritic effects in vivo following I.A. delivery.

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

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    Kürşat Oğuz Yaykaşlı

    2014-02-01

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

  5. Intra-Articular Osteoid Osteoma Mimicking Juvenile Arthritis

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    Sidi Yaya Traore

    2014-01-01

    Full Text Available In case of intra-articular osteoid osteoma, misdiagnosis as juvenile arthritis may occur, delaying adequate treatment. We report cases of intra-articular osteoid osteomas in children that were misdiagnosed and initially inappropriately treated with intra-articular corticoid injection. Diagnosis of osteoid osteoma was finally given by CT-scan and appropriate treatment by radiofrequency ablation or surgical ablation was performed. Clinicians and radiologists should be aware of the potentially confusing clinical and imaging findings associated with intra-articular osteoid osteoma.

  6. Metric analysis of loading magnitudes at articular and non-articular weight-bearing surfaces in human calcaneus.

    Science.gov (United States)

    Mahato, Niladri Kumar; Murthy, S Sathiya Narayana

    2013-03-01

    The calcaneus is axially loaded at its articular interface with the talus. A large bulk of this load is transmitted to the ground across the non-articular tubercles at the plantar surface of the bone. A small part of the incumbent load sustained by the calcaneus is directed towards the forefoot at the calcaneo-cuboid junction. This study investigates the proportion of load distributed across the articular and non-articular surfaces of the calcaneus. The present study demonstrates strong and significant correlation between some of the load bearing variables and suggests the need for further investigations to understand the effect of angular aspects of axial loading on the calcaneus. Accounting for the relative distribution of weight across the articular and non-articular areas may enable us to appreciate the internal trabecular structure of the calcaneus in light of its clinical importance.

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

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

    2012-09-07

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

  8. Intra-articular methotrexate associated to lipid nanoemulsions: anti-inflammatory effect upon antigen-induced arthritis

    Directory of Open Access Journals (Sweden)

    Mello SB

    2013-02-01

    Full Text Available Suzana BV Mello,1 Elaine R Tavares,2 Adriana Bulgarelli,2 Eloisa Bonfá,1 Raul C Maranhão2,31Rheumatology Division, Faculdade de Medicina da Universidade de São Paulo, São Paulo, Brazil; 2Lipid Metabolism Laboratory, the Heart Institute (INCOR of the Medical School Hospital, São Paulo, Brazil; 3Faculty of Pharmaceutical Sciences, University of São Paulo, São Paulo, BrazilObjective: Commercial methotrexate formulations (MTX have poor anti-inflammatory action for intra-articular treatment of rheumatoid arthritis. Our aim was to investigate whether an association between methotrexate and lipidic nanoemulsions (LDE could improve MTX intra-articular action.Methods: For its association to LDE, MTX was previously esterified with dodecyl bromide. LDE-MTX was prepared by high pressure homogenization. Antigen-induced arthritis (AIA was achieved in rabbits sensitized with methylated bovine serum albumin, and the rabbits were subsequently intra-articularly injected with the antigen. Twenty-four hours after AIA induction, groups of four to nine rabbits were intra-articularly injected with increasing doses (0.0625–0.5 µmol/kg of LDE-MTX, and were compared to treatment with 0.5 µmol/kg commercial MTX, LDE alone, and saline (controls. Synovial fluid was collected 48 hours after AIA induction for analysis of protein leakage and cell content. Synovial membranes were collected for histopathology. Uptake of LDE labeled with 3H-cholesteryl ether by the synovial tissue was also determined.Results: Uptake of radioactive LDE by arthritic joints was 2.5-fold greater than by normal joints. Treatment with intra-articular LDE-MTX elicited a clear dose response pattern by reducing the synovial leukocyte infiltrate (P = 0.004 and protein leakage (P = 0.032 when compared with arthritic non-treated joints. In contrast, the intra-articular injection of commercial MTX and LDE did not reduce leukocyte infiltrate or protein leakage. Toxicity to treatment was not observed

  9. Quantitative ultrasound imaging detects degenerative changes in articular cartilage surface and subchondral bone

    Science.gov (United States)

    Saarakkala, Simo; Laasanen, Mikko S.; Jurvelin, Jukka S.; Töyräs, Juha

    2006-10-01

    Previous studies have suggested that quantitative ultrasound imaging could sensitively diagnose degeneration of the articular surface and changes in the subchondral bone during the development of osteoarthrosis (OA). We have recently introduced a new parameter, ultrasound roughness index (URI), for the quantification of cartilage surface roughness, and successfully tested it with normal and experimentally degraded articular surfaces. In this in vitro study, the applicability of URI was tested in bovine cartilage samples with spontaneously developed tissue degeneration. Simultaneously, we studied the sensitivity of quantitative ultrasound imaging to detect degenerative changes in the cartilage-bone interface. For reference, histological degenerative grade of the cartilage samples was determined. Mechanical reference measurements were also conducted. Cartilage surface roughness (URI) was significantly (p < 0.05) higher in histologically degenerated samples with inferior mechanical properties. Ultrasound reflection at the cartilage-bone interface was also significantly (p < 0.05) increased in degenerated samples. Furthermore, it was quantitatively confirmed that ultrasound attenuation in the overlying cartilage significantly affects the measured ultrasound reflection values from the cartilage-bone interface. To conclude, the combined ultrasound measurement of the cartilage surface roughness and ultrasound reflection at the cartilage-bone interface complement each other, and may together enable more sensitive and quantitative diagnosis of early OA or follow up after surgical cartilage repair.

  10. Quantitative ultrasound imaging detects degenerative changes in articular cartilage surface and subchondral bone

    Energy Technology Data Exchange (ETDEWEB)

    Saarakkala, Simo [Department of Nuclear Medicine, Etelae-Savo Hospital District, Mikkeli Central Hospital, Porrassalmenkatu 35-37, 50100 Mikkeli (Finland); Laasanen, Mikko S [Information Technology R and D Unit, Engineering Kuopio, Savonia Polytechnic, POB 1188, FIN-70211 Kuopio (Finland); Jurvelin, Jukka S [Department of Physics, University of Kuopio, POB 1627, FIN-70211 Kuopio (Finland); Toeyraes, Juha [Department of Clinical Neurophysiology, Kuopio University Hospital and University of Kuopio, POB 1777, FIN-70211 Kuopio (Finland)

    2006-10-21

    Previous studies have suggested that quantitative ultrasound imaging could sensitively diagnose degeneration of the articular surface and changes in the subchondral bone during the development of osteoarthrosis (OA). We have recently introduced a new parameter, ultrasound roughness index (URI), for the quantification of cartilage surface roughness, and successfully tested it with normal and experimentally degraded articular surfaces. In this in vitro study, the applicability of URI was tested in bovine cartilage samples with spontaneously developed tissue degeneration. Simultaneously, we studied the sensitivity of quantitative ultrasound imaging to detect degenerative changes in the cartilage-bone interface. For reference, histological degenerative grade of the cartilage samples was determined. Mechanical reference measurements were also conducted. Cartilage surface roughness (URI) was significantly (p < 0.05) higher in histologically degenerated samples with inferior mechanical properties. Ultrasound reflection at the cartilage-bone interface was also significantly (p < 0.05) increased in degenerated samples. Furthermore, it was quantitatively confirmed that ultrasound attenuation in the overlying cartilage significantly affects the measured ultrasound reflection values from the cartilage-bone interface. To conclude, the combined ultrasound measurement of the cartilage surface roughness and ultrasound reflection at the cartilage-bone interface complement each other, and may together enable more sensitive and quantitative diagnosis of early OA or follow up after surgical cartilage repair.

  11. Wear and damage of articular cartilage with friction against orthopedic implant materials.

    Science.gov (United States)

    Oungoulian, Sevan R; Durney, Krista M; Jones, Brian K; Ahmad, Christopher S; Hung, Clark T; Ateshian, Gerard A

    2015-07-16

    The objective of this study was to measure the wear response of immature bovine articular cartilage tested against glass or alloys used in hemiarthroplasties. Two cobalt chromium alloys and a stainless steel alloy were selected for these investigations. The surface roughness of one of the cobalt chromium alloys was also varied within the range considered acceptable by regulatory agencies. Cartilage disks were tested in a configuration that promoted loss of interstitial fluid pressurization to accelerate conditions believed to occur in hemiarthroplasties. Results showed that considerably more damage occurred in cartilage samples tested against stainless steel (10 nm roughness) and low carbon cobalt chromium alloy (27 nm roughness) compared to glass (10 nm) and smoother low or high carbon cobalt chromium (10 nm). The two materials producing the greatest damage also exhibited higher equilibrium friction coefficients. Cartilage damage occurred primarily in the form of delamination at the interface between the superficial tangential zone and the transitional middle zone, with much less evidence of abrasive wear at the articular surface. These results suggest that cartilage damage from frictional loading occurs as a result of subsurface fatigue failure leading to the delamination. Surface chemistry and surface roughness of implant materials can have a significant influence on tissue damage, even when using materials and roughness values that satisfy regulatory requirements.

  12. Quantification of the optical surface reflection and surface roughness of articular cartilage using optical coherence tomography

    Energy Technology Data Exchange (ETDEWEB)

    Saarakkala, Simo; Wang Shuzhe; Huang Yanping; Zheng Yongping [Department of Health Technology and Informatics, Hong Kong Polytechnic University, Hong Kong (China)], E-mail: simo.saarakkala@uku.fi, E-mail: ypzheng@ieee.org

    2009-11-21

    Optical coherence tomography (OCT) is a promising new technique for characterizing the structural changes of articular cartilage in osteoarthritis (OA). The calculation of quantitative parameters from the OCT signal is an important step to develop OCT as an effective diagnostic technique. In this study, two novel parameters for the quantification of optical surface reflection and surface roughness from OCT measurements are introduced: optical surface reflection coefficient (ORC), describing the amount of a ratio of the optical reflection from cartilage surface with respect to that from a reference material, and OCT roughness index (ORI) indicating the smoothness of the cartilage surface. The sensitivity of ORC and ORI to detect changes in bovine articular cartilage samples after enzymatic degradations of collagen and proteoglycans using collagenase and trypsin enzymes, respectively, was tested in vitro. A significant decrease (p < 0.001) in ORC as well as a significant increase (p < 0.001) in ORI was observed after collagenase digestion. After trypsin digestion, no significant changes in ORC or ORI were observed. To conclude, the new parameters introduced were demonstrated to be feasible and sensitive to detect typical OA-like degenerative changes in the collagen network. From the clinical point of view, the quantification of OCT measurements is of great interest since OCT probes have been already miniaturized and applied in patient studies during arthroscopy or open knee surgery in vivo. Further studies are still necessary to demonstrate the clinical capability of the introduced parameters for naturally occurring early OA changes in the cartilage.

  13. Quantification of the optical surface reflection and surface roughness of articular cartilage using optical coherence tomography

    Science.gov (United States)

    Saarakkala, Simo; Wang, Shu-Zhe; Huang, Yan-Ping; Zheng, Yong-Ping

    2009-11-01

    Optical coherence tomography (OCT) is a promising new technique for characterizing the structural changes of articular cartilage in osteoarthritis (OA). The calculation of quantitative parameters from the OCT signal is an important step to develop OCT as an effective diagnostic technique. In this study, two novel parameters for the quantification of optical surface reflection and surface roughness from OCT measurements are introduced: optical surface reflection coefficient (ORC), describing the amount of a ratio of the optical reflection from cartilage surface with respect to that from a reference material, and OCT roughness index (ORI) indicating the smoothness of the cartilage surface. The sensitivity of ORC and ORI to detect changes in bovine articular cartilage samples after enzymatic degradations of collagen and proteoglycans using collagenase and trypsin enzymes, respectively, was tested in vitro. A significant decrease (p < 0.001) in ORC as well as a significant increase (p < 0.001) in ORI was observed after collagenase digestion. After trypsin digestion, no significant changes in ORC or ORI were observed. To conclude, the new parameters introduced were demonstrated to be feasible and sensitive to detect typical OA-like degenerative changes in the collagen network. From the clinical point of view, the quantification of OCT measurements is of great interest since OCT probes have been already miniaturized and applied in patient studies during arthroscopy or open knee surgery in vivo. Further studies are still necessary to demonstrate the clinical capability of the introduced parameters for naturally occurring early OA changes in the cartilage.

  14. 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.

  15. [Extra-articular manifestations of seronegative spondylarthritis].

    Science.gov (United States)

    Cammelli, Daniele

    2006-05-01

    Seronegative spondylarthritis are frequently characterised by extra-articular manifestations. They are frequently in recurrent uveitis. Between the cutaneous manifestations should be mentioned erythema nodosum, typical of inflammatory bowel diseases, and keratoderma blenorrhagicum, in the Reiter's syndrome. Cardiac complications in ankylosing spondylitis (AS) include aortic valvular regurgitation and arrhythmia and, more rarely, mitral valvulopathy, cardiomyopathy and pericarditis. Pulmonary involvement in AS includes ventilatory restrictive syndrome and fibro-bullous disease of the apex. Vertebral osteoporosis is a very important extra-articular manifestation because of the possibility of spontaneous fractures of the vertebrae. Central neurological manifestations include medullary compression from cervical sub-luxation while the most important peripheral involvements are lumbar stenosis and the cauda equina syndrome. Type AA amyloidosis is a rare late complication of the AS, possible cause of death especially in patients with aggressive disease. Kidney complications can be observed as consequences of prolonged anti-inflammatory therapy, but the most frequent renal complications are amyloidosis and mesangial IgA segmental and focal glomerulonephritis.

  16. PRP and Articular Cartilage: A Clinical Update

    Directory of Open Access Journals (Sweden)

    Antonio Marmotti

    2015-01-01

    Full Text Available The convincing background of the recent studies, investigating the different potentials of platelet-rich plasma, offers the clinician an appealing alternative for the treatment of cartilage lesions and osteoarthritis. Recent evidences in literature have shown that PRP may be helpful both as an adjuvant for surgical treatment of cartilage defects and as a therapeutic tool by intra-articular injection in patients affected by osteoarthritis. In this review, the authors introduce the trophic and anti-inflammatory properties of PRP and the different products of the available platelet concentrates. Then, in a complex scenario made of a great number of clinical variables, they resume the current literature on the PRP applications in cartilage surgery as well as the use of intra-articular PRP injections for the conservative treatment of cartilage degenerative lesions and osteoarthritis in humans, available as both case series and comparative studies. The result of this review confirms the fascinating biological role of PRP, although many aspects yet remain to be clarified and the use of PRP in a clinical setting has to be considered still exploratory.

  17. PRP and Articular Cartilage: A Clinical Update

    Science.gov (United States)

    Rossi, Roberto; Castoldi, Filippo; Michielon, Gianni

    2015-01-01

    The convincing background of the recent studies, investigating the different potentials of platelet-rich plasma, offers the clinician an appealing alternative for the treatment of cartilage lesions and osteoarthritis. Recent evidences in literature have shown that PRP may be helpful both as an adjuvant for surgical treatment of cartilage defects and as a therapeutic tool by intra-articular injection in patients affected by osteoarthritis. In this review, the authors introduce the trophic and anti-inflammatory properties of PRP and the different products of the available platelet concentrates. Then, in a complex scenario made of a great number of clinical variables, they resume the current literature on the PRP applications in cartilage surgery as well as the use of intra-articular PRP injections for the conservative treatment of cartilage degenerative lesions and osteoarthritis in humans, available as both case series and comparative studies. The result of this review confirms the fascinating biological role of PRP, although many aspects yet remain to be clarified and the use of PRP in a clinical setting has to be considered still exploratory. PMID:26075244

  18. Toward patient-specific articular contact mechanics.

    Science.gov (United States)

    Ateshian, Gerard A; Henak, Corinne R; Weiss, Jeffrey A

    2015-03-18

    The mechanics of contacting cartilage layers is fundamentally important to understanding the development, homeostasis and pathology of diarthrodial joints. Because of the highly nonlinear nature of both the materials and the contact problem itself, numerical methods such as the finite element method are typically incorporated to obtain solutions. Over the course of five decades, we have moved from an initial qualitative understanding of articular cartilage material behavior to the ability to perform complex, three-dimensional contact analysis, including multiphasic material representations. This history includes the development of analytical and computational contact analysis methods that now provide the ability to perform highly nonlinear analyses. Numerical implementations of contact analysis based on the finite element method are rapidly advancing and will soon enable patient-specific analysis of joint contact mechanics using models based on medical image data. In addition to contact stress on the articular surfaces, these techniques can predict variations in strain and strain through the cartilage layers, providing the basis to predict damage and failure. This opens up exciting areas for future research and application to patient-specific diagnosis and treatment planning applied to a variety of pathologies that affect joint function and cartilage homeostasis.

  19. 77 FR 20319 - Bovine Spongiform Encephalopathy; Importation of Bovines and Bovine Products

    Science.gov (United States)

    2012-04-04

    ...; ] DEPARTMENT OF AGRICULTURE Animal and Plant Health Inspection Service 9 CFR Part 93 RIN 0579-AC68 Bovine Spongiform Encephalopathy; Importation of Bovines and Bovine Products Correction In proposed rule...

  20. 78 FR 73993 - Bovine Spongiform Encephalopathy; Importation of Bovines and Bovine Products

    Science.gov (United States)

    2013-12-10

    ... Health Inspection Service 9 CFR Parts 92, 93, 94, 95, 96, and 98 RIN 0579-AC68 Bovine Spongiform Encephalopathy; Importation of Bovines and Bovine Products Corrections In rule document 2013-28228 appearing...

  1. Epigenetic Regulation of Chondrocyte Catabolism and Anabolism in Osteoarthritis

    OpenAIRE

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

    2015-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Jean-Edouard Ombetta

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

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

    Science.gov (United States)

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

    2007-05-04

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

  4. Unlocking the bovine genome

    Directory of Open Access Journals (Sweden)

    Worley Kim C

    2009-04-01

    Full Text Available Abstract The draft genome sequence of cattle (Bos taurus has now been analyzed by the Bovine Genome Sequencing and Analysis Consortium and the Bovine HapMap Consortium, which together represent an extensive collaboration involving more than 300 scientists from 25 different countries.

  5. Efeitos dos glicosaminoglicanos e sulfato de condroitina A sobre a cartilagem articular normal e com doença articular degenerativa em cães Glycosaminoglycans and chondroitin sulphate "A" effects on normal and osteoarthritic articular cartilage in dogs

    Directory of Open Access Journals (Sweden)

    N.T. Vieira

    2010-10-01

    Full Text Available Avaliaram-se os efeitos dos precursores dos glicosaminoglicanos (GAG e do sulfato de condroitina A (SC sobre a histomorfometria da cartilagem articular normal ou de cartilagem de cães com doença articular degenerativa (DAD experimental. Os grupos experimentais constituíram-se de animais com articulação direita normal, que não foi submetida a procedimento cirúrgico, e com articulação esquerda osteoartrótica e que foi submetida à intervenção cirúrgica. Os grupos foram subdivididos em animais com articulação não tratada e tratada, portanto: normais (N (n=5, NGAG (n=5 e NSC (n=4; e osteoartróticos (O (n=5, OGAG (n=5 e OSC (n=4. Secções de cartilagens do fêmur, da tíbia e da patela foram utilizadas neste estudo. Nos normais (N, NGAG e NSC, não se encontraram lesões que caracterizassem a DAD, embora tenha havido diminuição na celularidade nos de NGAG e NSC, em relação a N. Foram observadas alterações em graus variáveis entre os grupos osteoartróticos. Houve redução acentuada dos condrócitos no grupo O em comparação aos normais enquanto os grupos osteoartróticos tratados apresentaram celularidade semelhante aos normais tratados. Estes resultados foram confirmados pela análise do índice de proporção (IP, que se mostrou elevado em O, indicando menor síntese de proteoglicanos. Não houve diferença significativa entre os IPs dos grupos osteoartróticos tratados (OGAG, OSC apesar do comportamento distinto do OSC ao assemelhar-se aos grupos N e NSC. Estes resultados sugeriram que o SC agiu na cartilagem osteoartrótica de maneira mais eficaz, reduzindo a perda de proteoglicanos e estimulando a viabilidade celular e a atividade metabólica.The effects of precursors of glycosaminoglycans (GAG and chondroitin sulphate A (CS on the histomorphometry of normal articular cartilage and with experimental degenerative joint disease (DJD in dogs were evaluated. The groups were constituted as follows: normal joints were not

  6. Camel and bovine chymosin

    DEFF Research Database (Denmark)

    Langholm Jensen, Jesper; Mølgaard, Anne; Navarro Poulsen, Jens Christian;

    2013-01-01

    Bovine and camel chymosin are aspartic peptidases that are used industrially in cheese production. They cleave the Phe105-Met106 bond of the milk protein κ-casein, releasing its predominantly negatively charged C-terminus, which leads to the separation of the milk into curds and whey. Despite...... having 85% sequence identity, camel chymosin shows a 70% higher milk-clotting activity than bovine chymosin towards bovine milk. The activities, structures, thermal stabilities and glycosylation patterns of bovine and camel chymosin obtained by fermentation in Aspergillus niger have been examined...... interactions arising from variation in the surface charges and the greater malleability both in domain movements and substrate binding contribute to the better milk-clotting activity of camel chymosin towards bovine milk....

  7. Experimental study on tissue engineering resurfacing of articular cartilage defect%组织工程修复关节软骨缺损的实验研究

    Institute of Scientific and Technical Information of China (English)

    胡怀建; 雍宜民; 沈惠良

    2001-01-01

    目的用组织工程的方法,将新生关节软骨细胞先在高分子聚乳酸材料上进行体外培养,然后移植于关节软骨缺损处,以达到修复关节软骨缺损的目的。方法取新生新西兰兔关节软骨细胞培养于高分子聚乳酸材料支架上。将16只兔的32个膝关节股骨髁处人工造成一4mm×6mm大小缺损,并随机分为二组。Ⅰ组10只兔,20个关节缺损,用软骨细胞-高分子聚乳酸移植修复。Ⅱ组6只兔,左膝用软骨细胞-高分子聚乳酸移植体修复,表面覆盖单层软骨细胞;右膝仅用高分子聚乳酸材料修复。术后分别于4、8、12、24周取标本进行大体、光镜、组织化学的方法进行结果评估。结果用软骨细胞-高分子聚乳酸移植体能修复关节软骨缺损,且为透明软骨。新生软骨组织胶原定型为Ⅱ型胶原。结论用软骨细胞-高分子聚乳酸移植体移植,关节软骨缺损能获得透明软骨修复。%Objective Tissue engineering by chondrocyte transplantation with biodegradable polylactic acid scaffolds and new cartilage formation after in vitro culture is developed as a new attempt to repair articular cartilage defects.Methods Chondrocytes from newborn New Zealand rabbits were cultured on polylactic acid implants in vitro.4mm×6mm defects were created artificially at 32 knee joints of 16 rabbits and were randomized into 2 groups.20 joint defects of 10 rabbits in Group 1 were repaired by chondrocyte-seeded polylactic acid implants.In group 2,the left knee joint defects were repaired by chondrocyte-seeded polylactic acid implants while the right knee defects were repaired by polylactic acid implants only.Specimens were collected and assessed grossly,by light microscopy and histochemically at the 4,8,12 and 24th week after operation.Results Joint defects were repaired by chondrocyte-seeded polylactic acid implants and the new cartilages were hyalinic.The collagen component in the new

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

    Science.gov (United States)

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

    2014-09-01

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

  9. Effects of intra articular tramadol on articular cartilage and synovium of rats

    Directory of Open Access Journals (Sweden)

    Musa Kola

    2015-12-01

    Full Text Available Objective: To investigate the effects of intra articular tramadol injection on articular cartilage and synovium in rat knee joint. Methods: After Animal Ethical Committee approval, a total of 20 Sprague-Dawley rats were used and divided into 4 groups. Each group was composed of 5 rats. 0.2 ml of tramadol HCl was injected into the right knee joints and left knee joints of all the rats were considered as control. Control side joints received saline injection. Rats were sacrificed with ketamin on 1st, 7th, 14th and 21st days and knee joints were removed. Obtained tissue samples were decalcified and were dyed with Hematoxylin-eosin and Masson’s trichrome stain and examined by light microscopy for the presence of inflammation in periarticular area and synovia. Results: Congestion of synovial veins and perivascular cell infiltration were observed in tramadol group on days 1 and 7 (P < 0.05. The inflammation process was replaced by fibrosis on day 14 and fibrosis was significantly decreased on day 21. Conclusion: Intra articular tramadol should be used cautiously, keeping in mind that it may cause synovial inflammation in early phases and fibrosis in late phases. J Clin Exp Invest 2015; 6 (4: 337-342

  10. Articular cartilage repair with recombinant human type II collagen/polylactide scaffold in a preliminary porcine study.

    Science.gov (United States)

    Muhonen, Virpi; Salonius, Eve; Haaparanta, Anne-Marie; Järvinen, Elina; Paatela, Teemu; Meller, Anna; Hannula, Markus; Björkman, Mimmi; Pyhältö, Tuomo; Ellä, Ville; Vasara, Anna; Töyräs, Juha; Kellomäki, Minna; Kiviranta, Ilkka

    2016-05-01

    The purpose of this study was to investigate the potential of a novel recombinant human type II collagen/polylactide scaffold (rhCo-PLA) in the repair of full-thickness cartilage lesions with autologous chondrocyte implantation technique (ACI). The forming repair tissue was compared to spontaneous healing (spontaneous) and repair with a commercial porcine type I/III collagen membrane (pCo). Domestic pigs (4-month-old, n = 20) were randomized into three study groups and a circular full-thickness chondral lesion with a diameter of 8 mm was created in the right medial femoral condyle. After 3 weeks, the chondral lesions were repaired with either rhCo-PLA or pCo together with autologous chondrocytes, or the lesion was only debrided and left untreated for spontaneous repair. The repair tissue was evaluated 4 months after the second operation. Hyaline cartilage formed most frequently in the rhCo-PLA treatment group. Biomechanically, there was a trend that both treatment groups resulted in better repair tissue than spontaneous healing. Adverse subchondral bone reactions developed less frequently in the spontaneous group (40%) and the rhCo-PLA treated group (50%) than in the pCo control group (100%). However, no statistically significant differences were found between the groups. The novel rhCo-PLA biomaterial showed promising results in this proof-of-concept study, but further studies will be needed in order to determine its effectiveness in articular cartilage repair. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:745-753, 2016.

  11. Bovine Herpesvirus 4 infections and bovine mastitis

    NARCIS (Netherlands)

    Wellenberg, Gerardus Johannus

    2002-01-01

    Mastitis is an often occurring disease in dairy cattle with an enormous economic impact for milk producers worldwide. Despite intensive research, which is historically based on the detection of bacterial udder pathogens, still around 20-35% of clinical cases of bovine mastitis have an unknown aetiol

  12. [Familial articular chondrocalcinosis: study of an Alsatian family].

    Science.gov (United States)

    Netter, P; Loeuille, D; Jouzeau, J Y; Gillet, P; Peterschmitt, J; Pourel, J; Gaucher, A

    2001-01-01

    Familial articular chondrocalcinosis is a chronic articular disease characterized by acute intermittent attacks of arthritis, presence of calcium pyrophosphate dihydrate crystal in synovial fluid, cartilage and periarticular soft tissue and by x rays calcium deposition in articular cartilage. A family originating from Alsace, with an autosomal dominant transmission has been studied. As in English and Argentinean families, a linkage to the short arm of chromosome 5p has been found. These results suggest that a defective gene at this location may be related to the chondrocalcinosis in these families.

  13. Investigation of the performance of articular cartilage and synthetic biomaterials in multi-directional sliding motion as in orthopedic implants

    Science.gov (United States)

    Schwartz, Christian John

    The performance of several synthetic biomaterials and bovine articular cartilage were investigated in terms of their suitability for use as articulating surfaces in artificial joints. The Dual-Axis Wear Simulator (DAWS), a wear testing machine that simulates conditions in a synovial joint, was designed and fabricated to enable investigators to measure the wear of such materials in multi-directional sliding while immersed in a bovine serum lubricant solution. This machine was used initially to determine the wear mechanisms and wear amounts of ultra-high molecular weight polyethylene (UHMWPE), polytetrafluoroethylene (PTFE), polyoxymethylene (POM), and the compliant elastomer Pellethane(TM) 2363-80A. It was found that the compliant material produced lower wear. Dynamic mechanical analysis was used to determine that bovine articular cartilage had a very significant amount of viscoelasticity to support static loads and damp impact loads. Furthermore, the use of a compliant counterface led to lower wear in the cartilage as compared to a rigid counterface. Pt-Zr quasicrystals were used as fillers in UHMWPE, and the wear, stiffness, and impact toughness of the filled polymer were shown to be comparable or better than those of UHMWPE that had been irradiation crosslinked. Crosslinked UHMWPE was investigated for its susceptibility to oxidative degradation and increased wear. It was found that thermal stabilization of the polymer could be eliminated if a mild amount crosslinking was used. Furthermore, there was no degradation in wear resistance of mildly crosslinked and non-stabilized UHMWPE even after accelerated aging. Based on the results of this work and lessons learned about compliance and wear resistance, blends were produced by using surface-activated UHMWPE particles as fillers in elastomeric PUR. The blends showed better wear resistance than UHMWPE, as well as increased stiffness and damping over PUR. The results of this work indicated that there is great potential

  14. 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

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

    NARCIS (Netherlands)

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

    2010-01-01

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

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

    NARCIS (Netherlands)

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

    2010-01-01

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

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

    NARCIS (Netherlands)

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

    2012-01-01

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

  18. 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.

  19. 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

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

    Science.gov (United States)

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

    2017-01-01

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

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

    OpenAIRE

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

    1993-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Chongwei Chen

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

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

    Science.gov (United States)

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

    2008-06-01

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

  4. Controllable promotion of chondrocyte adhesion and growth on PVA hydrogels by controlled release of TGF-β1 from porous PLGA microspheres.

    Science.gov (United States)

    Nie, Lei; Zhang, Guohua; Hou, Ruixia; Xu, Haiping; Li, Yaping; Fu, Jun

    2015-01-01

    Poly(vinyl alcohol) (PVA) hydrogels have been candidate materials for cartilage tissue engineering. However, the cell non-adhesive nature of PVA hydrogels has been a limit. In this paper, the cell adhesion and growth on PVA hydrogels were promoted by compositing with transform growth factor-β1 (TGF-β1) loaded porous poly(D,L-lactide-co-glycolide) (PLGA) microspheres. The porous microspheres were fabricated by a modified double emulsion method with bovine serum albumin (BSA) as porogen. The average pore size of microspheres was manipulated by changing the BSA/PLGA ratio. Such controllable porous structures effectively influenced the encapsulation efficiency (Eencaps) and release profile of TGF-β1. By compositing PVA hydrogels with such TGF-β1-loaded PLGA microspheres, chondrocyte adhesion and proliferation were significantly promoted in a controllable manner, as confirmed by fluorescent imaging and quantitative CCK-8 assay. That is, the chondrocyte proliferation was favored by using PLGA microspheres with high Eencaps of TGF-β1 or by increasing the PLGA microsphere content in the hydrogels. These results demonstrated a facile method to improve the cell adhesion and growth on the intrinsically cell non-adhesive PVA hydrogels, which may find applications in cartilage substitution.

  5. Fibrin glue mixed with gelatin/hyaluronic acid/chondroitin-6-sulfate tri-copolymer for articular cartilage tissue engineering: the results of real-time polymerase chain reaction.

    Science.gov (United States)

    Chou, Cheng-Hung; Cheng, Winston T K; Kuo, Tzong-Fu; Sun, Jui-Sheng; Lin, Feng-Huei; Tsai, Jui-Che

    2007-09-01

    Autologous fibrin glue has been demonstrated as a potential scaffold with very good biocompatibility for neocartilage formation. However, fibrin glue has been reported not to provide enough mechanical strength, but with many growth factors to interfere the tissue growth. Gelatin/hyaluronic acid/chondroitin-6-sulfate (GHC6S) tri-copolymer sponge has been prepared as scaffold for cartilage tissue engineering and showed very good results, but problems of cell seeding and cell distribution troubled the researchers. In this study, GHC6S particles would be added into the fibrin glue to provide better mechanical strength, better cell distribution, and easier cell seeding, which would be expected to improve cartilage regeneration in vitro. Porcine cryo-precipitated fibrinogen and thrombin prepared from prothrombin activated by 10% CaCl(2) solution were used in two groups. One is the fibrin glue group in which porcine chondrocytes were mixed with thrombin-fibrinogen solution, which was then converted into fibrin glue. The other is GHC6S-fibrin glue in which GHC6S particles were added into the thrombin-fibrinogen solution with porcine chondrocytes. After culturing for 1-2 weeks, the chondrocytes cultured in GHC6S-fibrin glue showed a round shape with distinct lacuna structure and showed positive in S-100 protein immunohistochemical stain. The related gene expressions of tissue inhibitor of metalloproteinases-1, matrix metalloproteinase-2, MT1-MMP, aggrecan, decorin, type I, II, X collagen, interleukin-1 beta, transforming growth factor-beta 1 (TGF-beta1), and Fas-associating death domain were checked by real-time PCR. The results indicated that the chondrocytes cultured in GHC6S-fibrin glue would effectively promote extracellular matrix (ECM) secretion and inhibit ECM degradation. The evidence could support that GHC6S-fibrin glue would be a promising scaffold for articular cartilage tissue engineering.

  6. The Immunosuppressant FTY720 (Fingolimod enhances Glycosaminoglycan depletion in articular cartilage

    Directory of Open Access Journals (Sweden)

    Stradner Martin H

    2011-12-01

    Full Text Available Abstract Background FTY720 (Fingolimod is a novel immunosuppressive drug investigated in clinical trials for organ transplantation and multiple sclerosis. It acts as a functional sphingosine-1-phosphate (S1P receptor antagonist, thereby inhibiting the egress of lymphocytes from secondary lymphoid organs. As S1P is able to prevent IL-1beta induced cartilage degradation, we examined the direct impact of FTY720 on cytokine induced cartilage destruction. Methods Bovine chondrocytes were treated with the bioactive phosphorylated form of FTY720 (FTY720-P in combination with IL-1beta or TNF-alpha. Expression of MMP-1,-3.-13, iNOS and ADAMTS-4,-5 and COX-2 was evaluated using quantitative real-time PCR and western blot. Glycosaminoglycan depletion from cartilage explants was determined using a 1,9-dimethylene blue assay and safranin O staining. Results FTY720-P significantly reduced IL-1beta and TNF-alpha induced expression of iNOS. In contrast FTY720-P increased MMP-3 and ADAMTS-5 mRNA expression. Furthermore depletion of glycosaminoglycan from cartilage explants by IL-1beta and TNF-alpha was significantly enhanced by FTY720-P in an MMP-3 dependent manner. Conclusions Our results suggest that FTY720 may enhance cartilage degradation in pro-inflammatory environment.

  7. Pharmacological influence of antirheumatic drugs on proteoglycans from interleukin-1 treated articular cartilage.

    Science.gov (United States)

    Steinmeyer, J; Daufeldt, S

    1997-06-01

    The purpose of this study was to examine whether drugs used in the treatment of arthritic disorders possess any inhibitory potential on the proteoglycanolytic activities of matrix metalloproteinases (MMPs), and to determine whether drugs which inhibit these enzymes also modulate the biosynthesis and release of proteoglycans (PGs) from interleukin-1-(IL-1) treated articular cartilage explants. The cartilage-bone marrow extract and the glycosaminoglycan-peptide complex (DAK-16) dose-dependently inhibited MMP proteoglycanases in vitro when tested at concentrations ranging from 0.5 to 55 mg/mL, displaying an IC50 value of 31.78 mg/mL and 10.64 mg/mL (1.9 x 10[-4] M) respectively. (R,S)-N-[2-[2-(hydroxyamino)-2-oxoethyl]-4-methyl-1-oxopentyl++ +]-L-leucyl-L-phenylalaninamide (U-24522) proved to be a potent inhibitor of MMP proteoglycanases (IC50 value 1.8 x 10[-9] M). None of the other tested drugs, such as possible chondroprotective drugs, nonsteroidal anti-inflammatory drugs (NSAIDs), disease modifying antirheumatic drugs (DMARDs), glucocorticoids and angiotensin-converting enzyme inhibitors tested at a concentration of 10(-4) M displayed any significant inhibition. Only U-24522, tested at a concentration ranging from 10(-4) to 10(-6) M, significantly inhibited the IL-1-induced augmentation of PG loss from cartilage explants into the nutrient media, whereas DAK-16 and the cartilage-bone marrow extract were ineffective. DAK-16 and the cartilage-bone marrow extract did not modulate the IL-1-mediated reduced biosynthesis and aggregability of PGs by the cartilage explants. The addition of 10(-5) M U-24522, however, partially maintained the aggregability of PGs ex vivo. In our experiments, both possible chondroprotective drugs as well as U-24522 demonstrated no cytotoxic effects on chondrocytes.

  8. Mesenchymal stem cells in regenerative medicine: Focus on articular cartilage and intervertebral disc regeneration.

    Science.gov (United States)

    Richardson, Stephen M; Kalamegam, Gauthaman; Pushparaj, Peter N; Matta, Csaba; Memic, Adnan; Khademhosseini, Ali; Mobasheri, Reza; Poletti, Fabian L; Hoyland, Judith A; Mobasheri, Ali

    2016-04-15

    Musculoskeletal disorders represent a major cause of disability and morbidity globally and result in enormous costs for health and social care systems. Development of cell-based therapies is rapidly proliferating in a number of disease areas, including musculoskeletal disorders. Novel biological therapies that can effectively treat joint and spine degeneration are high priorities in regenerative medicine. Mesenchymal stem cells (MSCs) isolated from bone marrow (BM-MSCs), adipose tissue (AD-MSCs) and umbilical cord (UC-MSCs) show considerable promise for use in cartilage and intervertebral disc (IVD) repair. This review article focuses on stem cell-based therapeutics for cartilage and IVD repair in the context of the rising global burden of musculoskeletal disorders. We discuss the biology MSCs and chondroprogenitor cells and specifically focus on umbilical cord/Wharton's jelly derived MSCs and examine their potential for regenerative applications. We also summarize key components of the molecular machinery and signaling pathways responsible for the control of chondrogenesis and explore biomimetic scaffolds and biomaterials for articular cartilage and IVD regeneration. This review explores the exciting opportunities afforded by MSCs and discusses the challenges associated with cartilage and IVD repair and regeneration. There are still many technical challenges associated with isolating, expanding, differentiating, and pre-conditioning MSCs for subsequent implantation into degenerate joints and the spine. However, the prospect of combining biomaterials and cell-based therapies that incorporate chondrocytes, chondroprogenitors and MSCs leads to the optimistic view that interdisciplinary approaches will lead to significant breakthroughs in regenerating musculoskeletal tissues, such as the joint and the spine in the near future.

  9. Resposta das estruturas articulares do joelho de ratos pós-imobilização Response of the knee’s joint structures in rats after immobilization

    Directory of Open Access Journals (Sweden)

    Deisy Carla Cararo

    2009-03-01

    euthanized and the articulations were histologically analyzed. The results were tested through a paired t test with a 5% significance level. Results: In both groups of immobilization (G7 and G14, a reduction on the cartilage (G7: p=0,0006; G14: p=0,006 and subchondral (G7: p=0,0003; G14: p=0,002 bone thickness were observed, as well as an increase of the epiphyseal cartilage thickness (G7: p=0,001;G14: p=0,05 and the number of chondrocytes in the articular cartilage (G7=0,004; G14=0,034. In the 14-day group of immobilization it was also observed an increase of isogenic group population. Conclusion: With the results of this study, we can conclude that both after 7 and 14 days of immobilization, structural alterations occurs in the knee articulation, for instance thickness reduction in the articular cartilage of the subchondral bone and an increase of the epiphyseal cartilage thickness, affecting negatively the articular integrity.

  10. Biological Knee Reconstruction With Concomitant Autologous Chondrocyte Implantation and Meniscal Allograft Transplantation

    Science.gov (United States)

    Ogura, Takahiro; Bryant, Tim; Minas, Tom

    2016-01-01

    Background: Treating articular cartilage defects and meniscal deficiency is challenging. Although some short- to mid-term follow-up studies report good clinical outcomes after concurrent autologous chondrocyte implantation (ACI) and meniscal allograft transplantation (MAT), longer follow-up is needed. Purpose: To evaluate mid- to long-term outcomes after combined ACI with MAT. Study Design: Case series; Level of evidence, 4. Methods: We performed a retrospective review of prospectively gathered data from patients who had undergone ACI with MAT between 1999 and 2013. A single surgeon treated 18 patients for symptomatic full-thickness chondral defects with meniscal deficiency. One patient was lost to follow-up. Thus, 17 patients (18 knees; mean age, 31.7 years) were evaluated over a mean 7.9-year follow-up (range, 2-16 years). A mean 1.8 lesions per knee were treated over a total surface area of 7.6 cm2 (range, 2.3-21 cm2) per knee. Seventeen lateral and 1 medial MATs were performed. Survival was analyzed using the Kaplan-Meier method. The modified Cincinnati Knee Rating Scale, Western Ontario and McMaster Universities Osteoarthritis Index, visual analog scale, and Short Form–36 were used to evaluate clinical outcomes. Patients also self-reported knee function and satisfaction. Standard radiographs were scored for Kellgren-Lawrence (K-L) grade. Results: Both 5- and 10-year survival rates were 75%. Outcomes for 6 knees were considered failures. Of the 6 failures, 4 knees were converted to arthroplasty and the other 2 knees underwent biological revision surgery. Of the 12 successfully operated knees, all clinical measures significantly improved postoperatively. Ten patients representing 11 of the 12 knees rated outcomes for their knees as good or excellent, and 1 rated their outcome as fair. Eight patients representing 9 of the 12 knees were satisfied with the procedure. There was no significant osteoarthritis progression based on K-L grading from preoperatively to a

  11. The effect of extracellular pH on matrix turnover by cells of the bovine nucleus pulposus

    OpenAIRE

    Razaq, Sajjad; Wilkins, Robert J.; Urban, Jill P. G.

    2003-01-01

    It has long been known that very acidic conditions can be found in degenerate discs. The effect of these acid conditions on matrix turnover are, however, unknown. This study aimed to examine the effect of acidity on production of matrix components and on agents which break down the matrix in order to gain insight into the effect of pathological values of pH on matrix turnover. Cells were isolated from the nucleus of bovine discs and from bovine articular cartilage, embedded in alginate beads ...

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

    Science.gov (United States)

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

    2014-10-16

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

  13. Cultura de condrócitos em arcabouço tridimensional: hidrogel de alginato Chondrocyte cultures in tridimensional scaffold: alginate hydrogel

    Directory of Open Access Journals (Sweden)

    Renata Aparecida de Camargo Bittencourt

    2009-01-01

    Full Text Available OBJETIVOS: O presente estudo teve como objetivo cultivar condrócitos retirados da articulação do joelho de coelhos encapsulados em hidrogel de alginato (HA e caracterizar a produção de matriz extracelular (ECM. MÉTODOS: A cartilagem articular foi removida do joelho de coelhos, com três a seis meses, fragmentada em pedaços de 1mm e submetida à digestão enzimática. Uma concentração de 1x106 céls/mL foram ressuspensas em uma solução de alginato de sódio a 1,5% (w/v, em seguida fez-se o processo de gelatinização em CaCl2 (102 mM, permitindo a formação do HA e cultivo em meio DMEM-F12 durante quatro semanas. A distribuição das células e a ECM foram acessadas através das secções histológicas coradas com e azul de toluidina hematoxilina e eosina (HE. RESULTADOS: Houve um aumento no número e na viabilidade dos condrócitos durante as quatro semanas de cultura. Através das análises histológicas dos HAs corados com azul de toluidina e HE foi possível observar a distribuição definida dos condrócitos no hidrogel, assemelhando-se a grupos isógenos e formação de matriz territorial. CONCLUSÃO: Este estudo demonstrou a eficiência do HA como arcabouço para ser usado na cultura de condrócitos, constituindo uma alternativa no reparo de lesões na cartilagem articular.OBJECTIVES: The aim of this study was to culture chondrocytes from knee joint cartilage of rabbits encapsulated in alginate hydrogel (HA and to characterize the production of extracelular matrix (ECM. METHODS: Joint cartilage was obtained from rabbits' knees, three to six months old, fragmented into 1-mm pieces and submitted to enzymatic digestion. A concentration of 1x106 cells/mL were re-suspended into a 1.5% (w/v sodium alginate solution, followed by gel formation process with CaCl2 (102 mM, allowing HA to build for culturing it into a DMEM-F12 medium for four weeks. The distribution of cells and ECM were assessed from histological slices stained toluidine

  14. Endoplasmic Reticulum Stress Occurs in Osteoarthritis Chondrocytes%骨关节炎软骨细胞发生内质网应激

    Institute of Scientific and Technical Information of China (English)

    谢靖; 杨冠

    2013-01-01

    Objective: To investigate whether endoplasmic reticulum stress is present in osteoarthritis chondro-cytes. Methods: The tissue sections of human osteoarthritis cartilage samples from arthroplastic knee joints and normal ones were examined for the expression of endoplasmic reticulum stress marker immunoglobulin heavy chain binding protein(BiP) by immunohistochemistry. Besides, mice subjected to transection of the medial meniscus in the knee joint were employed as the experimental osteoarthritis models. The slides of the mouse knee joints were stained by Safrainin-O, scored by the Mankin grading system and detected for the BiP expression 1, 3 and 6 weeks after surgical operation, respectively. Results: In all human osteoarthritis samples, BiP expression in chondro-cytes was significantly increased. In mouse model of experimental osteoarthritis, all the joints after transection of the medial meniscus exhibited articular cartilage damage and developed severe osteoarthritis phenotype as revealed by Safranin-O staining. The Mankin score of experimental joints was increasingly higher than that of control joints. In addition, the number of chondrocytes positive for BiP expression increased in the articular cartilage of ex-perimental joints. Conclusion: In human osteoarthritis samples and mouse model of experimental osteoarthritis, evi-dent endoplasmic reticulum stress occurs in osteoarthritis chondrocytes.%目的:研究骨关节炎软骨细胞是否发生内质网应激现象。方法:对关节置换术后的人类骨关节炎软骨标本和正常关节软骨标本切片进行内质网应激标志分子免疫球蛋白重链结合蛋白(BiP)的免疫组织化学检测;对小鼠膝关节进行半月板切断术诱发实验性骨关节炎,在术后1、3和6周取材,对组织切片进行番红花“O”染色、Mankin评分及BiP的免疫组织化学检测。结果:所有人类骨关节炎标本中软骨细胞BiP的表达明显升高。番红花“O”染色

  15. Arthoscopy La artroscopia y las lesiones articulares

    Directory of Open Access Journals (Sweden)

    Raúl J. Naranjo

    1991-01-01

    Full Text Available

    A general view is presented on arthroscopy; its history, indications and limitations are described and analyzed; its advantages in comparison with open surgery are emphasized.

    La artroscopia es un procedimiento que permite, mediante un instrumento óptico, evaluar el Interior de las cavidades articulares. Comenzó a desarrollarse como un procedimiento diagnóstico y pronto sus grandes ventajas frente a las exploraciones abiertas aceleraron el desarrollo de las técnicas y del instrumental. La minimización del trauma a los tejidos y la menor morbilidad posoperatoria permiten que la deambulación y la recuperación funcional sean precoces lo cual, sumado a un mejor resultado estético, ha colocado a la artroscopia como procedimiento de elección para el diagnóstico y el tratamiento de múltiples estados patológicos intraarticulares.

  16. PTHrP regulates chondrocyte maturation in condylar cartilage.

    Science.gov (United States)

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

    2003-08-01

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

  17. 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.

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

    Science.gov (United States)

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

    2003-06-01

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

  19. The influence of collagen network integrity on the accumulation of gadolinium-based MR contrast agents in articular cartilage

    Energy Technology Data Exchange (ETDEWEB)

    Wiener, Edzard; Schmidt, C.; Diederichs, G. [Charite - Universitaetsmedizin Berlin (Germany). Inst. fuer Radiologie; Settles, M. [Klinikum rechts der Isar, Muenchen (Germany). Inst. fuer Roentgendiagnostik; Weirich, G. [Klinikum Rechts der Isar, Muenchen (Germany). Inst. fuer Pathologie und Pathologische Anatomie

    2011-03-15

    Delayed gadolinium-enhanced MR imaging of cartilage is used to quantify the proteoglycan loss in early osteoarthritis. It is assumed that T 1 after Gd-DTPA administration in the near equilibrium state reflects selective proteoglycan loss from cartilage. To investigate the influence of the collagen network integrity on contrast accumulation, the relaxation rates {delta}R1 and {delta}R2 were compared after Gd-DTPA administration in a well established model of osteoarthritis. Collagen or proteoglycan depletion was induced by the proteolytic enzymes papain and collagenase in healthy bovine patellar cartilage. Using a dedicated MRI sequence, T{sub 1} and T{sub 2} maps were simultaneously acquired before and 11 h after Gd-DTPA administration. Depth-dependent profiles of {delta}R1 and {delta}R2 were calculated in healthy, proteoglycan and collagen-depleted articular cartilage and the mean values of different cartilage layers were compared using the Mann-Whitney-U test. In superficial layers (1 mm) there was no significant difference (p > 0.05) in either {delta}R1 or {delta}R2 between proteoglycan-depleted (16.6 {+-} 1.2 s{sup -1}, 15.9 {+-} 1.0 s{sup -1}) and collagen-depleted articular cartilage (15.3 {+-} 0.9 s{sup -1}, 15.5 {+-} 0.9 s{sup -1}). In deep layers (3 mm) both parameters were significantly higher (p = 0.005, 0.03) in proteoglycan-depleted articular cartilage (12.3 {+-} 1.1 s{sup -1}, 9.8 {+-} 0.8 s{sup -1}) than in collagen-depleted articular cartilage (9.1 {+-} 1.1 s{sup -1}, 8.7 {+-} 0.7 s{sup -1}). Both proteoglycan loss and alterations in the collagen network influence the accumulation of Gd-DTPA in articular cartilage with significant differences between superficial and deep cartilage layers. (orig.)

  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.

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

    Science.gov (United States)

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

    2013-06-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2012-07-20

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

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

    Science.gov (United States)

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

    2016-12-01

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

  4. Kinematic biomechanical assessment of human articular cartilage transplants in the knee using 3-T MRI: an in vivo reproducibility study

    Energy Technology Data Exchange (ETDEWEB)

    Juras, Vladimir; Szomolanyi, Pavol [Medical University of Vienna, Department of Radiodiagnostics, MR Centre of Excellence, Vienna (Austria); Slovak Academy of Sciences, Department of Imaging Methods, Institute of Measurement Science, Bratislava (Slovakia); Ludwig Boltzmann Institute for Clinical and Experimental Traumatology, Austrian Cluster for Tissue Regeneration, Vienna (Austria); Welsch, Goetz H.; Pinker, Katja; Trattnig, Siegfried [Medical University of Vienna, Department of Radiodiagnostics, MR Centre of Excellence, Vienna (Austria); Ludwig Boltzmann Institute for Clinical and Experimental Traumatology, Austrian Cluster for Tissue Regeneration, Vienna (Austria); Millington, Steven [Royal National Orthopaedic Hospital, London, Stanmore (United Kingdom); Mamisch, Tallal C. [Inselspital, Orthopedic Surgery Department, Berne (Switzerland)

    2009-05-15

    The aims of this study were to examine the clinical feasibility and reproducibility of kinematic MR imaging with respect to changes in T{sub 2} in the femoral condyle articular cartilage. We used a flexible knee coil, which allows acquisition of data in different positions from 40 flexion to full extension during MR examinations. The reproducibility of T{sub 2} measurements was evaluated for inter-rater and inter-individual variability and determined as a coefficient of variation (CV) for each volunteer and rater. Three different volunteers were measured twice and regions of interest (ROIs) were selected by three raters at different time points. To prove the clinical feasibility of this method, 20 subjects (10 patients and 10 age- and sex-matched volunteers) were enrolled in the study. Inter-rater variability ranged from 2 to 9 and from 2 to 10% in the deep and superficial zones, respectively. Mean inter-individual variability was 7% for both zones. Different T{sub 2} values were observed in the superficial cartilage zone of patients compared with volunteers. Since repair tissue showed a different behavior in the contact zone compared with healthy cartilage, a possible marker for improved evaluation of repair tissue quality after matrix-associated autologous chondrocyte transplantation (MACT) may be available and may allow biomechanical assessment of cartilage transplants. (orig.)

  5. Sexing Bovine Embryos Using PCR Amplification of Bovine SRY Sequence

    Institute of Scientific and Technical Information of China (English)

    曾溢滔; 张美兰; 陈美珏; 周霞娣; 黄英; 任兆瑞; 黄淑帧; 胡明信; 吴学清; 高建明; 张斌; 徐慧如

    1994-01-01

    This study analyses the bovine SRY DNA sequence by direct sequencing procedure, followed by the designation of the PCR primers specific for bovine SRY. Using PCR amplification of bovine SRY gene, the embryo sex was determined. The results of the embryo sex identification were confirmed after the embryo transfer and pregnancies.

  6. Vulnerability of the Superficial Zone of Immature Articular Cartilage to Compressive Injury

    Energy Technology Data Exchange (ETDEWEB)

    Rolauffs, R.; Muehleman, C; Li, J; Kurz, B; Kuettner, K; Frank, E; Grodzinsky, A

    2010-01-01

    The zonal composition and functioning of adult articular cartilage causes depth-dependent responses to compressive injury. In immature cartilage, shear and compressive moduli as well as collagen and sulfated glycosaminoglycan (sGAG) content also vary with depth. However, there is little understanding of the depth-dependent damage caused by injury. Since injury to immature knee joints most often causes articular cartilage lesions, this study was undertaken to characterize the zonal dependence of biomechanical, biochemical, and matrix-associated changes caused by compressive injury. Disks from the superficial and deeper zones of bovine calves were biomechanically characterized. Injury to the disks was achieved by applying a final strain of 50% compression at 100%/second, followed by biomechanical recharacterization. Tissue compaction upon injury as well as sGAG density, sGAG loss, and biosynthesis were measured. Collagen fiber orientation and matrix damage were assessed using histology, diffraction-enhanced x-ray imaging, and texture analysis. Injured superficial zone disks showed surface disruption, tissue compaction by 20.3 {+-} 4.3% (mean {+-} SEM), and immediate biomechanical impairment that was revealed by a mean {+-} SEM decrease in dynamic stiffness to 7.1 {+-} 3.3% of the value before injury and equilibrium moduli that were below the level of detection. Tissue areas that appeared intact on histology showed clear textural alterations. Injured deeper zone disks showed collagen crimping but remained undamaged and biomechanically intact. Superficial zone disks did not lose sGAG immediately after injury, but lost 17.8 {+-} 1.4% of sGAG after 48 hours; deeper zone disks lost only 2.8 {+-} 0.3% of sGAG content. Biomechanical impairment was associated primarily with structural damage. The soft superficial zone of immature cartilage is vulnerable to compressive injury, causing superficial matrix disruption, extensive compaction, and textural alteration, which results

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

    Institute of Scientific and Technical Information of China (English)

    关鹏; 赵伟; 张全有; 谢静; 尹利军; 赵虎成; 许建文

    2015-01-01

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

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

    Directory of Open Access Journals (Sweden)

    D Vonwil

    2010-11-01

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

  9. Enzootic bovine leukosis and Bovine leukemia virus

    OpenAIRE

    Amauri Alcindo Alfieri; Alice Fernandes Alfieri; Luis Álvaro Leuzzi Junior

    2004-01-01

    All over de World the Enzootic Bovine Leukosis is a important viral infection in cattle herds. This revision points out topics relative to the etiological agent, clinical signals, diagnosis methods, control and prophylaxis of the infection.A Leucose Enzoótica Bovina é uma infecção viral amplamente disseminada em rebanhos bovinos de todo o mundo. Esta revisão tem por objetivo apresentar tópicos relacionados ao agente etiológico, à doença clínica e aos métodos de diagnóstico, controle e profila...

  10. Neurophysiological basis for neurogenic-mediated articular cartilage anabolism alteration.

    Science.gov (United States)

    Gouze-Decaris, E; Philippe, L; Minn, A; Haouzi, P; Gillet, P; Netter, P; Terlain, B

    2001-01-01

    This study was designed to investigate the pathways involved in neurogenic-mediated articular cartilage damage triggered by a nonsystemic distant subcutaneous or intra-articular inflammation. The cartilage damage was assessed 24 h after subcutaneous or intra-articular complete Freund's adjuvant (CFA) injection measuring patellar proteoglycan (PG) synthesis (ex vivo [Na(2)(35)SO(4)] incorporation) in 96 Wistar rats. Unilateral subcutaneous or intra-articular injection of CFA induced significant decrease (25-29%) in PG synthesis in both patellae. Chronic administration of capsaicin (50 mg. kg(-1). day(-1) during 4 days), which blunted the normal response of C fiber stimulation, prevented the bilateral significant decrease in cartilage synthesis. Similarly, intrathecal injection of MK-801 (10 nmol/day during 5 days), which blocked the glutamatergic synaptic transmission at the dorsal horn of signal originating in primary afferent C fibers, eliminated the CFA-induced PG synthesis decrease in both patellae. Chemical sympathectomy, induced by guanethidine (12.5 mg. kg(-1). day(-1) during 6 wk), also prevented PG synthesis alteration. Finally, compression of the spinal cord at the T3-T5 level had a similar protective effect on the reduction of [Na(2)(35)SO(4)] incorporation. It is concluded that the signal that triggers articular cartilage synthesis damage induced by a distant local inflammation 1) is transmitted through the afferent C fibers, 2) makes glutamatergic synaptic connections with the preganglionic neurons of the sympathetic system, and 3) involves spinal and supraspinal pathways.

  11. 17 beta-estradiol-BSA conjugates and 17 beta-estradiol regulate growth plate chondrocytes by common membrane associated mechanisms involving PKC dependent and independent signal transduction.

    Science.gov (United States)

    Sylvia, V L; Walton, J; Lopez, D; Dean, D D; Boyan, B D; Schwartz, Z

    2001-01-01

    Nuclear receptors for 17 beta-estradiol (E(2)) are present in growth plate chondrocytes from both male and female rats and regulation of chondrocytes through these receptors has been studied for many years; however, recent studies indicate that an alternative pathway involving a membrane receptor may also be involved in the cell response. E(2) was found to directly affect the fluidity of chondrocyte membranes derived from female, but not male, rats. In addition, E(2) activates protein kinase C (PKC) in a nongenomic manner in female cells, and chelerythrine, a specific inhibitor of PKC, inhibits E(2)-dependent alkaline phosphatase activity and proteoglycan sulfation in these cells, indicating PKC is involved in the signal transduction mechanism. The aims of the present study were: (1) to examine the effect of a cell membrane-impermeable 17 beta-estradiol-bovine serum albumin conjugate (E(2)-BSA) on chondrocyte proliferation, differentiation, and matrix synthesis; (2) to determine the pathway that mediates the membrane effect of E(2)-BSA on PKC; and (3) to compare the action of E(2)-BSA to that of E(2). Confluent, fourth passage resting zone (RC) and growth zone (GC) chondrocytes from female rat costochondral cartilage were treated with 10(-9) to 10(-7) M E(2) or E(2)-BSA and changes in alkaline phosphatase specific activity, proteoglycan sulfation, and [(3)H]-thymidine incorporation measured. To examine the pathway of PKC activation, chondrocyte cultures were treated with E(2)-BSA in the presence or absence of GDP beta S (inhibitor of G-proteins), GTP gamma S (activator of G-proteins), U73122 or D609 (inhibitors of phospholipase C [PLC]), wortmannin (inhibitor of phospholipase D [PLD]) or LY294002 (inhibitor of phosphatidylinositol 3-kinase). E(2)-BSA mimicked the effects of E(2) on alkaline phosphatase specific activity and proteoglycan sulfation, causing dose-dependent increases in both RC and GC cell cultures. Both forms of estradiol inhibited [(3)H

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

    Directory of Open Access Journals (Sweden)

    Srujana Vedicherla

    2017-01-01

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

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

    DEFF Research Database (Denmark)

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

    2012-01-01

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

  14. 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.

  15. 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.

  16. REGENERATION OF ARTICULAR CARTILAGE UNDER THE IMPLANTATION OF BONE MATRIX

    Directory of Open Access Journals (Sweden)

    Yuri M. Iryanov, Nikolay A. Kiryanov, Olga V. Dyuriagina , Tatiana Yu. Karaseva, Evgenii A. Karasev

    2015-07-01

    Full Text Available Background: The damage or loss of articular cartilage is costly medical problem. The purpose of this work – morphological analysis of reparative chondrogenesis when implanted in the area of the knee joint cartilage of granulated mineralized bone matrix. Material and Methods: The characteristic features of the knee cartilage regeneration studied experimentally in pubertal Wistar rats after modeling a marginal perforated defect and implantation of granulated mineralized bone matrix obtained according to original technology without heat and demineralizing processing into the injury zone. Results: This biomaterial established to have pronounced chondro- and osteoinductive properties, and to provide prolonged activation of reparative process, accelerated organotypical remodeling and restoration of the articular cartilage injured. Conclusion: The data obtained demonstrate the efficacy of МВМ in clinical practice for the treatment of diseases and injuries of the articular cartilage.

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

    Directory of Open Access Journals (Sweden)

    Madhushika Ratnayake

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

  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. Bovine milk glycome.

    Science.gov (United States)

    Tao, N; DePeters, E J; Freeman, S; German, J B; Grimm, R; Lebrilla, C B

    2008-10-01

    Bovine milk oligosaccharides have several potentially important biological activities including the prevention of pathogen binding to the intestinal epithelial and as nutrients for beneficial bacteria. It has been suggested that milk oligosaccharides are an important source of complex carbohydrates as supplements for the food and the pharmaceutical industries. However, only a small number of structures of bovine milk oligosaccharides (bMO) are known. There have been no systematic studies on bMO. High-performance mass spectrometry and separation methods are used to evaluate bMO, and nearly 40 oligosaccharides are present in bovine milk. Bovine milk oligosaccharides are composed of shorter oligomeric chains than are those in human milk. They are significantly more anionic with nearly 70%, measured abundances, being sialylated. Additionally, bMO are built not only on the lactose core (as are nearly all human milk oligosaccharides), but also on lactose amines. Sialic acid residues include both N-acetyl and N-glycolylneuraminic acid, although the former is significantly more abundant.

  20. Bovine milk exosome proteome

    Science.gov (United States)

    Exosomes are 40-100 nm membrane vesicles of endocytic origin and are found in blood, urine, amniotic fluid, bronchoalveolar lavage (BAL) fluid, as well as human and bovine milk. Exosomes are extracellular organelles important in intracellular communication/signaling, immune function, and biomarkers ...

  1. Intervet Symposium: bovine neosporosis

    NARCIS (Netherlands)

    Schetters, T.; Dubey, J.P.; Adrianarivo, A.; Frankena, K.; Romero, J.J.; Pérez, E.; Heuer, C.; Nicholson, C.; Russell, D.; Weston, J.

    2004-01-01

    This article summarises the most relevant data of presentations delivered at the 19th International Conference of the World Association for the Advancement of Veterinary Parasitology (WAAVP) held in New Orleans, LA, USA, from 10 to 14 August 2003) in a symposium session on bovine neosporosis. The sy

  2. Bovine Spongiform Encephalopathy

    Science.gov (United States)

    Bovine spongiform encephalopathy (BSE) is caused by a novel contagion, known to as a prion. Prions are proteins capable of converting a normal cellular protein into a prion, thereby propagating an infection. BSE is the first known prion zoonotic. As such it has attracted broad scientific and, to a r...

  3. Experimental articular cartilage repair in the Göttingen minipig

    DEFF Research Database (Denmark)

    Christensen, Bjørn Borsøe; Foldager, Casper Bindzus; Olesen, Morten Lykke;

    2015-01-01

    BACKGROUND: A gold standard treatment for articular cartilage injuries is yet to be found, and a cost-effective and predictable large animal model is needed to bridge the gap between in vitro studies and clinical studies. Ideally, the animal model should allow for testing of clinically relevant...... treatments and the biological response should be reproducible and comparable to humans. This allows for a reliable translation of results to clinical studies.This study aimed at verifying the Göttingen minipig as a pre-clinical model for articular cartilage repair by testing existing clinical cartilage...

  4. Intra-Articular Osteotomy for Distal Humerus Malunion

    Directory of Open Access Journals (Sweden)

    René K. Marti

    2009-01-01

    Full Text Available Intra-articular osteotomy is considered in the rare case of malunion after a fracture of the distal humerus to restore humeral alignment and gain a functional arc of elbow motion. Traumatic and iatrogenic disruption of the limited blood flow to the distal end of the humerus resulting in avascular necrosis of capitellum or trochlea is a major pitfall of the this technically challenging procedure. Two cases are presented which illustrate the potential problems of intra-articular osteotomy for malunion of the distal humerus.

  5. El concepto de contacto articular alternativo de la rodilla

    OpenAIRE

    1994-01-01

    La observación radioscópica de la interlínea articular de rodillas de cadáveres sin anomalías anatómicas durante movimientos de abducción y adducción, realizada en estudios previos del autor, permitió apreciar la aparición de pérdida de contacto entre las superficies articulares mediales o laterales respectivamente. El presente artículo tuvo como objetivo rememorar dichos estudios y documentar las apreciaciones visuales mediante el análisis de neumoartrografías seriada...

  6. Articular Cartilage Repair Through Muscle Cell-Based Tissue Engineering

    Science.gov (United States)

    2010-03-01

    fferentiation of s tem c ells is also an i mportant i ssue t o c onsider e specially f or t he persistence of the regenerate cartilage. Based on these...tap water for 10 minutes and counterstained with nuclear fast red. Differentiation of MDSCs into chondrocytes. Pellets in OCT blocks were sectioned and...into Alcian blue solution for 30 minutes. The slides were rinsed with running tap water for 10 minutes and counterstained with nuclear fast red

  7. 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.

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

    Directory of Open Access Journals (Sweden)

    Michiyo Nasu

    2016-01-01

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

  9. Characterization of enzymatically induced degradation of articular cartilage using high frequency ultrasound

    Science.gov (United States)

    Töyräs, J.; Rieppo, J.; Nieminen, M. T.; Helminen, H. J.; Jurvelin, J. S.

    1999-11-01

    Ultrasound may provide a quantitative technique for the characterization of cartilage changes typical of early osteoarthrosis. In this study, specific changes in bovine articular cartilage were induced using collagenase and chondroitinase ABC, enzymes that selectively degrade collagen fibril network and digest proteoglycans, respectively. Changes in cartilage structure and properties were quantified using high frequency ultrasound, microscopic analyses and mechanical indentation tests. The ultrasound reflection coefficient of the physiological saline-cartilage interface (R1) decreased significantly (-96.4%, p<0.01) in the collagenase digested cartilage compared to controls. Also a significantly lower ultrasound velocity (-6.2%, p<0.01) was revealed after collagenase digestion. After chondroitinase ABC digestion, a new acoustic interface at the depth of the enzyme penetration front was detected. Cartilage thickness, as determined with ultrasound, showed a high, linear correlation (R = 0.943, n = 60, average difference 0.073 mm (4.0%)) with the thickness measured by the needle-probe method. Both enzymes induced a significant decrease in the Young's modulus of cartilage (p<0.01). Our results indicate that high frequency ultrasound provides a sensitive technique for the analysis of cartilage structure and properties. Possibly ultrasound may be utilized in vivo as