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Sample records for bovine articular chondrocytes

  1. 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.

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

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    William Hester

    2012-01-01

    Full Text Available Objectives. The objective of this paper is to determine if hyaluronan affects bupivacaine’s anesthetic function. Methods. Whole cell patch clamp recordings were performed on bovine articular chondrocytes cultured in 60 mm dishes. The chondrocytes were treated with phosphate-buffered saline (control group, 7.5 mg/mL hyaluronan (Orthovisc, 0.25% bupivacaine, or a mixture of 7.5 mg/mL hyaluronan and 0.25% bupivacaine. Outward currents were elicited by step depolarization from −90 mV to 150 mV with 5 mV increments and holding for 200 ms. Results. The amplitude of outward currents elicited at 150 mV was 607.1±135.4 pA (mean ± standard error in the chondrocytes treated with phosphate buffered saline, 550.0±194.9 pA in the chondrocytes treated with hyaluronan, 18.4±8.3 pA in the chondrocytes treated with bupivacaine, and 12.8±2.6 pA in the chondrocytes treated with a mixture of hyaluronan and bupivacaine. Conclusion. Hyaluronan does not affect bupivacaine’s inhibitory action on the potassium channel activities in bovine articular chondrocytes. This finding suggests that intra-articular injection of a mixture of hyaluronan and bupivacaine may not affect the anesthetic effects of bupivacaine.

  3. The effect of collagen degradation on chondrocyte volume and morphology in bovine articular cartilage following a hypotonic challenge.

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    Turunen, S M; Lammi, M J; Saarakkala, S; Han, S-K; Herzog, W; Tanska, P; Korhonen, R K

    2013-06-01

    Collagen degradation is one of the early signs of osteoarthritis. It is not known how collagen degradation affects chondrocyte volume and morphology. Thus, the aim of this study was to investigate the effect of enzymatically induced collagen degradation on cell volume and shape changes in articular cartilage after a hypotonic challenge. Confocal laser scanning microscopy was used for imaging superficial zone chondrocytes in intact and degraded cartilage exposed to a hypotonic challenge. Fourier transform infrared microspectroscopy, polarized light microscopy, and mechanical testing were used to quantify differences in proteoglycan and collagen content, collagen orientation, and biomechanical properties, respectively, between the intact and degraded cartilage. Collagen content decreased and collagen orientation angle increased significantly (p < 0.05) in the superficial zone cartilage after collagenase treatment, and the instantaneous modulus of the samples was reduced significantly (p < 0.05). Normalized cell volume and height 20 min after the osmotic challenge (with respect to the original volume and height) were significantly (p < 0.001 and p < 0.01, respectively) larger in the intact compared to the degraded cartilage. These findings suggest that the mechanical environment of chondrocytes, specifically collagen content and orientation, affects cell volume and shape changes in the superficial zone articular cartilage when exposed to osmotic loading. This emphasizes the role of collagen in modulating cartilage mechanobiology in diseased tissue.

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

    National Research Council Canada - National Science Library

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

    2014-01-01

    ...-estradiol and testosterone) on rabbit articular chondrocytes from different genders. We cultured primary rabbit articular chondrocytes from both genders with varying concentration of sex hormones...

  6. Superficial cells are self-renewing chondrocyte progenitors, which form the articular cartilage in juvenile mice.

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    Li, Lei; Newton, Phillip T; Bouderlique, Thibault; Sejnohova, Marie; Zikmund, Tomas; Kozhemyakina, Elena; Xie, Meng; Krivanek, Jan; Kaiser, Jozef; Qian, Hong; Dyachuk, Vyacheslav; Lassar, Andrew B; Warman, Matthew L; Barenius, Björn; Adameyko, Igor; Chagin, Andrei S

    2017-03-01

    Articular cartilage has little regenerative capacity. Recently, genetic lineage tracing experiments have revealed chondrocyte progenitors at the articular surface. We further characterized these progenitors by using in vivo genetic approaches. Histone H2B-green fluorescent protein retention revealed that superficial cells divide more slowly than underlying articular chondrocytes. Clonal genetic tracing combined with immunohistochemistry revealed that superficial cells renew their number by symmetric division, express mesenchymal stem cell markers, and generate chondrocytes via both asymmetric and symmetric differentiation. Quantitative analysis of cellular kinetics, in combination with phosphotungstic acid-enhanced micro-computed tomography, showed that superficial cells generate chondrocytes and contribute to the growth and reshaping of articular cartilage. Furthermore, we found that cartilage renewal occurs as the progeny of superficial cells fully replace fetal chondrocytes during early postnatal life. Thus, superficial cells are self-renewing progenitors that are capable of maintaining their own population and fulfilling criteria of unipotent adult stem cells. Furthermore, the progeny of these cells reconstitute adult articular cartilage de novo, entirely substituting fetal chondrocytes.-Li, L., Newton, P. T., Bouderlique, T., Sejnohova, M., Zikmund, T., Kozhemyakina, E., Xie, M., Krivanek, J., Kaiser, J., Qian, H., Dyachuk, V., Lassar, A. B., Warman, M. L., Barenius, B., Adameyko, I., Chagin, A. S. Superficial cells are self-renewing chondrocyte progenitors, which form the articular cartilage in juvenile mice. © FASEB.

  7. The biological effects of sex hormones on rabbit articular chondrocytes from different genders.

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

    2014-01-01

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

  8. Xanthan gum protects rabbit articular chondrocytes against sodium nitroprusside-induced apoptosis in vitro.

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    Chen, Qixin; Mei, Xifan; Han, Guanying; Ling, Peixue; Guo, Bin; Guo, Yuewei; Shao, Huarong; Wang, Guan; Cui, Zan; Bai, Yuxin; Xu, Fang

    2015-10-20

    We have previously reported that intra-articular injection of xanthan gum (XG) could significantly ameliorate the degree of joint cartilage degradation and pain in experimental osteoarthritis (OA) model in vivo. In this present study, we evaluated the protective effect of XG against Sodium nitroprusside (SNP)-induced rabbit articular chondrocytes apoptosis in vitro. Rabbit articular chondrocytes were incubated with various concentrations of XG for 24h prior to 0.5mmol/L SNP co-treatment for 24h. The proliferation of chondrocytes was analyzed using MTT assay. The chondrocytes early apoptosis rates were evaluated using Annexin V-FITC/PI flow cytometry. The morphology of apoptosis chondrocytes were observed by scanning electron microscopy (SEM). The loss/disruption of mitochondrial membrane potential was detected using rhodamin 123 by confocal microscope. The concentration of prostaglandin E2 (PGE2) in cell culture supernatants was evaluated using ELISA assay. The results showed that XG could significantly reverse SNP-reduced cell proliferation and inhibited cell early apoptosis rate in a dose-dependent manner. XG alleviated loss/disruption of mitochondrial membrane potential and decreased the PGE2 level of chondrocytes cell culture supernatants in SNP-induced chondrocytes. These results of the present research strongly suggest that XG can protect rabbit articular chondrocytes against SNP-induced apoptosis in vitro. Copyright © 2015 Elsevier Ltd. All rights reserved.

  9. Interaction of chondrocytes, extracellular matrix and growth factors: relevance for articular cartilage tissue engineering.

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    Kraan, P.M. van der; Buma, P.; Kuppevelt, A.H.M.S.M. van; Berg, W.B. van den

    2002-01-01

    The abundant extracellular matrix of articular cartilage has to be maintained by a limited number of chondrocytes. Vice versa, the extracellular matrix has an important role in the regulation of chondrocyte function. OBJECTIVE: In this review we discuss the role of the extracellular matrix in the

  10. Comparison between Chondrogenic Markers of Differentiated Chondrocytes from Adipose Derived Stem Cells and Articular Chondrocytes In Vitro

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    Mohmmad Mardani

    2013-06-01

    Full Text Available   Objective(s: Osteoarthritis is one of the most common diseases in middle-aged population in the world. Cartilage tissue engineering (TE has been presented as an effort to introduce the best combination of cells, biomaterial scaffolds and stimulating growth factors to produce a cartilage tissue similar to the natural articular cartilage. In this study, the chondrogenic potential of adipose derived stem cells (ADSCs was compared with natural articular chondrocytes cultured in alginate scaffold.   Materials and Methods: Human ADSCs were obtained from subcutaneous adipose tissue and human articular chondrocytes from non-weight bearing areas of knee joints. Cells were seeded in 1.5% alginate and cultured in chondrogenic media for three weeks with and without TGFβ3. The genes expression of types II and X collagens was assessed by Real Time PCR and the amount of aggrecan (AGC and type I collagen measured by ELISA and the content of glycosaminoglycan evaluated by GAG assay. Results: Our findings showed that type II collagen, GAG and AGC were expressed, in differentiated ADSCs. Meanwhile, they produced a lesser amount of types II and X collagens but more AGC, GAG and type I collagen in comparison with natural chondrocytes (NCs. Conclusion: Further attempt should be carried out to optimize achieving type II collagen in DCs, as much as, natural articular chondrocytes and decline of the production of type I collagen in order to provide efficient hyaline cartilage after chondrogenic induction, prior to the usage of harvested tissues in clinical trials.

  11. The effect of dexamethasone and triiodothyronine on terminal differentiation of primary bovine chondrocytes and chondrogenically differentiated mesenchymal stem cells.

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    Thomas M Randau

    Full Text Available The newly evolved field of regenerative medicine is offering solutions in the treatment of bone or cartilage loss and deficiency. Mesenchymal stem cells, as well as articular chondrocytes, are potential cells for the generation of bone or cartilage. The natural mechanism of bone formation is that of endochondral ossification, regulated, among other factors, through the hormones dexamethasone and triiodothyronine. We investigated the effects of these hormones on articular chondrocytes and chondrogenically differentiated mesenchymal stem cells, hypothesizing that these hormones would induce terminal differentiation, with chondrocytes and differentiated stem cells being similar in their response. Using a 3D-alginate cell culture model, bovine chondrocytes and chondrogenically differentiated stem cells were cultured in presence of triiodothyronine or dexamethasone, and cell proliferation and extracellular matrix production were investigated. Collagen mRNA expression was measured by real-time PCR. Col X mRNA and alkaline phosphatase were monitored as markers of terminal differentiation, a prerequisite of endochondral ossification. The alginate culture system worked well, both for the culture of chondrocytes and for the chondrogenic differentiation of mesenchymal stem cells. Dexamethasone led to an increase in glycosaminoglycan production. Triiodothyronine increased the total collagen production only in chondrocytes, where it also induced signs of terminal differentiation, increasing both collagen X mRNA and alkaline phosphatase activity. Dexamethasone induced terminal differentiation in the differentiated stem cells. The immature articular chondrocytes used in this study seem to be able to undergo terminal differentiation, pointing to their possible role in the onset of degenerative osteoarthritis, as well as their potential for a cell source in bone tissue engineering. When chondrocyte-like cells, after their differentiation, can indeed be moved on

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

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

    2017-01-01

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

  13. Leptin protects rat articular chondrocytes from cytotoxicity induced by TNF-α in the presence of cyclohexamide.

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    Lee, S W; Rho, J H; Lee, S Y; Kim, J H; Cheong, J-H; Kim, H Y; Jeong, N Y; Chung, W T; Yoo, Y H

    2015-12-01

    Although leptin appears to be an important local and systemic factor influencing cartilage homeostasis, the role of leptin in chondrocyte death is largely unknown. Tumor necrosis factor α (TNF-α) is a pro-inflammatory cytokine that plays a central role in the pathogenesis of articular diseases. This study examines whether leptin modulates TNF-α-induced articular chondrocyte death. Primary rat articular chondrocytes were isolated from knee joint cartilage slices. To induce cell death, the chondrocytes were treated with TNF-α. To examine whether leptin modulates the extent of TNF-α-mediated chondrocyte death, the cells were pretreated with leptin for 3 h before TNF-α treatment followed by viability analysis. To examine the mechanism by which leptin modulates the extent of TNF-α-mediated chondrocyte death, we utilized mitochondrial membrane potential (MMP) measurements, flow cytometry, nuclear morphology observation, co-immunoprecipitation, western blot analysis and confocal microscopy. We demonstrated that leptin suppresses TNF-α induced chondrocyte death. We further found that apoptosis partially contributes to TNF-α induced chondrocyte death while necroptosis primarily contributes to TNF-α induced chondrocyte death. In addition, we observed that leptin exerts anti-TNF-α toxicity via c-jun N-terminal kinase (JNK) in rat articular chondrocytes. Based on our findings, we suggest that the leptin present in the articular joint fluid protects articular chondrocytes against cumulative mechanical load and detrimental stresses throughout a lifetime, delaying the onset of degenerative changes in chondrocytes. We can further hypothesize that leptin protects articular chondrocytes against destructive stimuli even in the joints of osteoarthritis (OA) patients. Copyright © 2015 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.

  14. Hyaline Cartilage Tissue Is Formed through the Co-culture of Passaged Human Chondrocytes and Primary Bovine Chondrocytes

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

    2012-01-01

    To circumvent the problem of a sufficient number of cells for cartilage engineering, the authors previously developed a two-stage culture system to redifferentiate monolayer culture-expanded dedifferentiated human articular chondrocytes by co-culture with primary bovine chondrocytes (bP0). The aim of this study was to analyze the composition of the cartilage tissue formed in stage 1 and compare it with bP0 grown alone to determine the optimal length of the co-culture stage of the system. Biochemical data show that extracellular matrix accumulation was evident after 2 weeks of co-culture, which was 1 week behind the bP0 control culture. By 3 to 4 weeks, the amounts of accumulated proteoglycans and collagens were comparable. Expression of chondrogenic genes, Sox 9, aggrecan, and collagen type II, was also at similar levels by week 3 of culture. Immunohistochemical staining of both co-culture and control tissues showed accumulation of type II collagen, aggrecan, biglycan, decorin, and chondroitin sulfate in appropriate zonal distributions. These data indicate that co-cultured cells form cartilaginous tissue that starts to resemble that formed by bP0 after 3 weeks, suggesting that the optimal time to terminate the co-culture stage, isolate the now redifferentiated cells, and start stage 2 is just after 3 weeks. PMID:22610463

  15. Leptin Receptor JAK2/STAT3 Signaling Modulates Expression of Frizzled Receptors in Articular Chondrocytes

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    Ohba, Seigo; Lanigan, Thomas M.; Roessler, Blake J.

    2010-01-01

    Objective Differentiated articular chondrocytes express a functional isoform of the leptin receptor (LRb); however, leptin-LRb signaling in these cells is poorly understood. We hypothesized that leptin-LRb signaling in articular chondrocytes functions to modulate canonical Wnt signaling events by altering the expression of Frizzled receptors. Methods Human chondrocyte cell lines and primary articular chondrocytes were grown in serum containing growth media for 24 hrs, followed by a media change to DMEM containing 1% Nutridoma-SP to obtain a serum-deficient environment for 24 hours before treatment. Treatments included recombinant human leptin (10–100 nM), recombinant human IL-6 (0.3-3 nM), or recombinant human erythropoietin (10 mU/ml). Cells were harvested 30 min to 48 hrs after treatment and whole cell lysates were analyzed using immunoblots or luciferase assays. Results Treatment of cells with leptin resulted in activation of JAK2 and subsequent phosphorylation of specific tyrosine residues on LRb, followed by dose- and time-dependent increases in the expression of Frizzled-1 (Fzd1) and Frizzled-7 (Fzd7). Leptin-mediated increases in the expression of Fzd1 and Fzd7 were blocked by pre-treatment with the protein synthesis inhibitor cycloheximide or the JAK2 inhibitor AG490. Experiments using a series of hybrid erythropoietin extracellular domain-leptin intracellular domain receptors (ELR) harboring mutations of specific tyrosine residues in the cytoplasmic tail showed that increases in the expression of Fzd1 and Fzd7 were dependent on LRb-mediated phosphorylation of STAT3, but not ERK1/2 or STAT5. Leptin pre-treatment of chondrocytes prior to Wnt-3a stimulation resulted in an increased magnitude of canonical Wnt signaling. Conclusion These experiments show that leptin-LRb signaling in articular chondrocytes modulates expression of canonical Wnt signaling receptors and suggests that direct cross-talk between these pathways is important in determining chondrocyte

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

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    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.

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

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    Leilei Zhong

    2015-08-01

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

  18. 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...

  19. 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

    -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......OBJECTIVE: Calcitonin has been suggested to have chondroprotective effects. One signaling pathway of calcitonin is via the second messenger cAMP. We undertook this study to investigate whether increased cAMP levels in chondrocytes would be chondroprotective. METHODS: Cartilage degradation...... was induced in bovine articular cartilage explants by 10 ng/ml oncostatin M (OSM) and 20 ng/ml tumor necrosis factor (TNF). In these cultures, cAMP levels were augmented by treatment with either forskolin (4, 16, or 64 microM) or 3-isobutyl-1-methyl xanthine (IBMX; 4, 16, or 64 microM). Cartilage degradation...

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

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    Jakob Naranda

    2017-03-01

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

  1. Surface modified PLGA/carbon nanofiber composite enhances articular chondrocyte functions

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    Park, Grace Eunseung

    Since articular cartilage has a limited self regeneration capability, alternative methods are needed for repairing cartilage defects. The purpose of the present in vitro study was to explore the effects of material surface properties and external stimulation on chondrocyte (cartilage-synthesizing cell) functions. Based on this information, a goal of this research was to propose a scaffold composite material for enhancing articular chondrocyte function. To improve functions of chondrocytes, material (namely, poly(lactic-co-glycolic acid); PLGA) surfaces were modified via chemical (NaOH) etching techniques. Chondrocytes were cultured on surface-modified 2-D PLGA films and 3-D PLGA tissue engineering scaffolds, which were created by a salt-leaching method. Carbon nanofibers were imprinted on PLGA matrices in an aligned pattern for controlled electrically-active surface features. Electrical stimulation was applied to expedite and enhance chondrocyte functions. Results demonstrated that both NaOH-treated 2-D and 3-D substrates enhanced chondrocyte functions (cell numbers as well as extracellular matrix production) compared to non-treated PLGA substrates. Furthermore, chondrocytes preferred to attach along the carbon nanofiber patterns imprinted on PLGA. Electrical stimulation also enhanced chondrocyte functions on carbon nanofiber/PLGA composites. Underlying material properties that may have enhanced chondrocyte functions include a more hydrophilic surface, surface energy differences due to the presence of carbon nanofibers, increased surface area, altered porosity, and a greater degree of nanometer roughness. Moreover, these altered surface properties positively influenced select protein adsorption that controlled subsequent chondrocyte adhesion. Collectively, this study provided a scaffold model for osteochondral defects that can be synthesized using the above techniques and a layer by layer approach to accommodate the property differences in each layer of natural

  2. Initiating human articular chondrocyte re-differentiation in a 3D system after 2D expansion.

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    Kudva, Abhijith K; Luyten, Frank P; Patterson, Jennifer

    2017-09-05

    Cartilage damage affects a large population via acute and chronic injury and disease. Since native cartilage does not self-renew, cartilage tissue engineering has gained traction as a potential treatment. However, a limiting factor is that the primary cell type in cartilage, the articular chondrocyte, tends to de-differentiate when grown on 2D surfaces for in vitro expansion. Thus, 3D systems are being developed and used to counter this loss of chondrogenic capabilities. We hypothesize that a 3D matrix that can be remodeled may be more supportive of the chondrogenic phenotype of encapsulated articular chondrocytes than a 2D surface and may allow for the re-differentiation of chondrocytes after 2D expansion. Hence, in this study, enzymatically degradable polyethylene glycol (PEG) hydrogels containing two different protease degradable peptide segments, with different degradation rates, were tested in combination with chondrogenic medium as a 3D in vitro culture system to better recapitulate the native environment of human articular chondrocytes (hACs). In addition, the effect of incorporation of the integrin binding ligand Arg-Gly-Asp (RGD) in the hydrogels was explored. Hydrogels crosslinked with a slower degrading crosslinker and not functionalized with RGD maintained hAC viability and led to increased GAG production and chondrogenic gene expression over time, suggesting that this system can initiate hAC re-differentiation after 2D expansion.

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

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    Richard Barrett-Jolley

    2012-04-01

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

  4. 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.

  5. 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 Lidocaine (30 mM) reduced the cell viability of isolated chondrocytes (p lidocaine induces 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. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.

  6. Characterization of articular chondrocytes isolated from 211 osteoarthritic patients.

    Science.gov (United States)

    Laganà, Matteo; Arrigoni, Chiara; Lopa, Silvia; Sansone, Valerio; Zagra, Luigi; Moretti, Matteo; Raimondi, Manuela Teresa

    2014-03-01

    We analyzed specific features of chondrocytes as cellular yield, cell doubling rates and the dependence between these parameters and patient-related data in a set of 211 osteoarthritic (OA) patients undergoing total joint replacement. For each patient the data available were joint type, age and gender. Knee samples chosen randomly among all biopsies were graded according to ICRS score. Patients' age ranged between 30 and 90 years with a mean age of 66 ± 9.7 years. Patients were divided into age classes and statistically significant differences in proliferation rate at passage 1 were found between chondrocytes derived from young and old donors, with the last ones characterized by a lower proliferation rate. A similar trend was observed for proliferation rate at passage 2. For all the samples, cellular yields ranged between 0.1 and 5.5 million cells/g of tissue. No significant correlation was observed between the level of cartilage degeneration (ICRS score) and cellular yield and proliferation rates. However, in samples with a high degree of cartilage degeneration (ICRS score 4) the cellular yield was lower compared to the other three groups (ICRS scores 1-3). In this study we performed a systematic characterization of basic parameters of chondrocytes originating from a wide group of OA patients. Considering the use of autologous chondrocytes in chondral treatments, the characterization of cell basic features may represent an important step to determine the quality of the cell source which is a major determinant in the outcome of cell-based therapies.

  7. Synthesis of collagen by bovine chondrocytes cultured in alginate; posttranslational modifications and cell-matrix interaction

    NARCIS (Netherlands)

    Beekman, B.; Verzijl, N.; Bank, R.A.; Von Der Mark, K.; TeKoppele, J.M.

    1997-01-01

    The extracellular matrix synthesized by articular chondrocytes cultured in alginate beads was investigated. Collagen levels increased sigmoidally with time and remained constant after 2 weeks of culture. The presence of cartilage-specific type II collagen was confirmed immunohistochemically.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1989-06-01

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

  9. bFGF influences human articular chondrocyte differentiation

    DEFF Research Database (Denmark)

    Schmal, H; Zwingmann, J; Fehrenbach, M

    2007-01-01

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

  10. Leptin in osteoarthritis: Focus on articular cartilage and chondrocytes.

    Science.gov (United States)

    Scotece, Morena; Mobasheri, Ali

    2015-11-01

    Osteoarthritis (OA) is a complex joint disorder with a number of underlying physical, biochemical, biomechanical and genetic causes. Obesity is considered to be one of the major risk factors for the development and progression of OA. It actively contributes to the inflammatory status and to cartilage degradation in the OA joints. Recent data suggests that metabolic factors produced by white adipose tissue, such as leptin, may provide a mechanistic link between obesity and OA, providing an explanation for the high prevalence of OA among obese and over-weight individuals. The unbalanced production of catabolic and anabolic mediators by chondrocytes, the only cell type present in cartilage, determines cartilage degradation, which is the central pathological feature of OA. Evidence is accumulating to support a key role for leptin in the pathogenesis and/or progression of OA. The goal of this focused review is to summarize the current knowledge on the role of leptin in OA with particular emphasis on the effects of this adipokine in cartilage and chondrocyte pathophysiology. Copyright © 2015 Elsevier Inc. All rights reserved.

  11. The Inorganic Pyrophosphate Transporter ANK Preserves the Differentiated Phenotype of Articular Chondrocyte*

    Science.gov (United States)

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

    2010-01-01

    The differentiated phenotype of chondrocyte is lost in pathological situations and after interleukin (IL)-1β challenge. Wnt proteins and the inorganic pyrophosphate (PPi) transporter Ank regulate the differentiation process in many cell types. We investigated the possible contribution of Ank and/or PPi to the maintenance of the differentiated chondrocyte phenotype with special care to Wnt signaling. Primary articular chondrocytes lost their phenotype upon IL-1β challenge, with cessation of type II collagen and Sox-9 expression. Ank expression and PPi transport were strongly reduced by IL-1β, whereas Wnt-5a was the only Wnt protein increased. Transient overexpression of Ank counteracted most of IL-1β effects on Type II collagen, Sox-9, and Wnt-5a expression. When resting chondrocytes were transfected with a siRNA against Ank, this reproduced the phenotype induced by IL-1β. In both cases, no markers for hypertrophic chondrocytes were detected. The conditioned supernatant from chondrocytes knocked-down for Ank contained Wnt-5a, which activated Tcf/Lef reporter plasmids and promoted translocation of β-catenin into the nucleus without activating the c-Jun N-terminal kinase (JNK) pathway. Supplementation with PPi compensated for most effects of Ank deficiency on Type II collagen, Sox-9, and Wnt-5 expression, both in IL-1β and Ank knock-down conditions. Phenotype changes induced by IL-1β were also supported by activation of the JNK pathway, but this latter was not sensitive to PPi supplementation. Altogether our data demonstrate that the transport of PPi by ANK contributed to the maintenance of the differentiated phenotype of chondrocyte by controlling the canonical Wnt pathway in a Wnt-5a-dependent manner. PMID:20133941

  12. Using Costal Chondrocytes to Engineer Articular Cartilage with Applications of Passive Axial Compression and Bioactive Stimuli.

    Science.gov (United States)

    Huwe, Le W; Sullan, Gurdeep K; Hu, Jerry C; Athanasiou, Kyriacos A

    2017-08-14

    Generating neocartilage with suitable mechanical integrity from a cell source that can circumvent chondrocyte scarcity is indispensable for articular cartilage regeneration strategies. Costal chondrocytes of the rib eliminate donor site morbidity in the articular joint, but it remains unclear how neocartilage formed from these cells responds to mechanical loading, especially if the intent is to use it in a load-bearing joint. In a series of three experiments, this study sought to determine efficacious parameters of passive axial compressive stimulation that would enable costal chondrocytes to synthesize mechanically robust cartilage. Experiment 1 determined a suitable time window for stimulation by its application during either the matrix synthesis phase, the maturation phase, or during both phases of the self-assembling process. The results showed that compressive stimulation at either time was effective in increasing instantaneous moduli by 92% and 87% in the synthesis and maturation phases, respectively. Compressive stimulation during both phases did not further improve properties beyond a one-time stimulation. The magnitude of passive axial compression was examined in Experiment 2 by applying 0, 3.3, 5.0, or 6.7 kPa stresses to the neocartilage. Unlike 6.7 kPa, both 3.3 and 5.0 kPa significantly increased neocartilage compressive properties by 42% and 48% over untreated controls, respectively. Experiment 3 examined how the passive axial compression regimen developed from the previous phases interacted with a bioactive regimen (transforming growth factor [TGF]-β1, chondroitinase ABC, and lysyl oxidase-like 2). Passive axial compression significantly improved the relaxation modulus compared with bioactive treatment alone. Furthermore, a combined treatment of compressive and bioactive stimulation improved the tensile properties of neocartilage 2.6-fold compared with untreated control. The ability to create robust articular cartilage from passaged costal

  13. The COX-2 Selective Blocker Etodolac Inhibits TNFα-Induced Apoptosis in Isolated Rabbit Articular Chondrocytes

    Directory of Open Access Journals (Sweden)

    Hiroshi Matsuura

    2013-09-01

    Full Text Available Chondrocyte apoptosis contributes to the disruption of cartilage integrity in osteoarthritis (OA. Recently, we reported that activation of volume-sensitive Cl− current (ICl,vol mediates cell shrinkage, triggering apoptosis in rabbit articular chondrocytes. A cyclooxygenase (COX blocker is frequently used for the treatment of OA. In the present study, we examined in vitro effects of selective blockers of COX on the TNFα-induced activation of ICl,vol in rabbit chondrocytes using the patch-clamp technique. Exposure of isolated chondrocytes to TNFα resulted in an obvious increase in membrane Cl− conductance. The TNFα-evoked Cl− current exhibited electrophysiological and pharmacological properties similar to those of ICl,vol. Pretreatment of cells with selective COX-2 blocker etodolac markedly inhibited ICl,vol activation by TNFα as well as subsequent apoptotic events such as apoptotic cell volume decrease (AVD and elevation of caspase-3/7 activity. In contrast, a COX-1 blocker had no effect on the decrease in cell volume or the increase in caspase-3/7 activity induced by TNFα. Thus, the COX-2-selective blocker had an inhibitory effect on TNFα-induced apoptotic events, which suggests that this drug would have efficacy for the treatment of OA.

  14. 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.

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

    Directory of Open Access Journals (Sweden)

    Pengzhen Wang

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

  16. 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.

  17. Study of the Mechanical Environment of Chondrocytes in Articular Cartilage Defects Repaired Area under Cyclic Compressive Loading

    Directory of Open Access Journals (Sweden)

    Hai-Ying Liu

    2017-01-01

    Full Text Available COMSOL finite element software was used to establish a solid-liquid coupling biphasic model of articular cartilage and a microscopic model of chondrocytes, using modeling to take into account the shape and number of chondrocytes in cartilage lacuna in each layer. The effects of cyclic loading at different frequencies on the micromechanical environment of chondrocytes in different regions of the cartilage were studied. The results showed that low frequency loading can cause stress concentration of superficial chondrocytes. Moreover, along with increased frequency, the maximum value of stress response curve of chondrocytes decreased, while the minimum value increased. When the frequency was greater than 0.2 Hz, the extreme value stress of response curve tended to be constant. Cyclic loading had a large influence on the distribution of liquid pressure in chondrocytes in the middle and deep layers. The concentration of fluid pressure changed alternately from intracellular to peripheral in the middle layer. Both the range of liquid pressure in the upper chondrocytes and the maximum value of liquid pressure in the lower chondrocytes in the same lacunae varied greatly in the deep layer. At the same loading frequency, the elastic modulus of artificial cartilage had little effect on the mechanical environment of chondrocytes.

  18. FUNCTIONS OF THE mTOR SIGNALING PATHWAY IN NORMAL ARTICULAR CARTILAGE CHONDROCYTES AND IN OSTEOARTHRITIS

    Directory of Open Access Journals (Sweden)

    E. V. Chetina

    2016-01-01

    Full Text Available Osteoarthritis  (OA is a chronic disease associated with pain, stiffness, limited mobility and joint inflammation, as well as articular cartilage destruction.  Recent studies have shown the importance  of chondrocyte  differentiation (hypertrophy as one of the mechanisms  of cartilage degradation in OA. This suggests that chondrocyte  metabolism undergoes the profound changes during cartilage resorption,  which are due to dysregulation of cell function. One of the major cellular metabolic regulators is the protein mTOR (mechanistic target of rapamycin that controls cell growth, proliferation, protein biosynthesis and integrates extracellular signals from growth factors and hormones with amino acid availability and intracellular energy status. The importance  of mTOR activity for articular cartilage destruction  in OAis confirmed by significant changes in the work of mTOR regulatory network that involves multiple intracellular (growth factors, adenosine triphosphate, oxygen availability, and autophagy and extracellular (glucose, amino acids, lipids, and hexosamine signals. Moreover, the altered expression of the mTOR gene in the blood of patients with OA is associated with either increased pain or synovitis, which indicates that there is a strong metabolic heterogeneity in patients with OA and a need for a differentiated therapeutic  approach. The above problems are discussed in this review.

  19. In vitro effects of meloxicam on metabolism in articular chondrocytes from dogs with naturally occurring osteoarthritis.

    Science.gov (United States)

    Budsberg, Steven C; Stoker, Aaron M; Johnston, Spencer A; Liska, William; Reno, Lisa R; Cook, James L

    2013-09-01

    To assess effects of in vitro meloxicam exposure on metabolism in articular chondrocytes from dogs with naturally occurring osteoarthritis. Femoral head cartilage from 16 dogs undergoing total hip replacement. Articular cartilage samples were obtained. Tissue sulfated glycosaminoglycan (SGAG), collagen, and DNA concentrations were measured. Collagen, SGAG, chondroitin sulfate 846, NO, prostaglandin E2 (PGE2), and matrix metalloproteinase (MMP)-2, MMP-3, MMP-9, and MMP-13 concentrations in culture medium were analyzed. Aggrecan, collagen II, MMP-2, MMP-3, MMP-9, MMP-13, ADAM metallopeptidase with thrombospondin type 1 motif (ADAMTS)-4, ADAMTS-5, tissue inhibitor of metalloproteinase (TIMP)-1, TIMP-2, TIMP-3, interleukin-1β, tumor necrosis factor-α, cyclooxygenase-1, cyclooxygenase-2, and inducible nitric oxide synthase gene expression were evaluated. Comparisons between tissues cultured without (control) and with meloxicam at concentrations of 0.3, 3.0, and 30.0 μg/mL for up to 30 days were performed by means of repeated-measures analysis. Meloxicam had no effect on chondrocyte SGAG, collagen, or DNA concentrations. Expression of ADAMTS-5 was significantly decreased in all groups on all days, compared with the day 0 value. On day 3, culture medium PGE2 concentrations were significantly lower in all meloxicam-treated groups, compared with values for controls, and values remained low. Culture medium MMP-3 concentrations were significantly lower on day 30 than on day 3 in all meloxicam-treated groups. Results suggested that in vitro meloxicam treatment of osteoarthritic canine cartilage for up to 30 days did not induce matrix degradation or stimulate MMP production. Meloxicam lowered PGE2 release from this tissue, and effects on tissue chondrocyte content and matrix composition were neutral.

  20. Advances in the Surgical Management of Articular Cartilage Defects: Autologous Chondrocyte Implantation Techniques in the Pipeline.

    Science.gov (United States)

    Stein, Spencer; Strauss, Eric; Bosco, Joseph

    2013-01-01

    The purpose of this review is to gain insight into the latest methods of articular cartilage implantation (ACI) and to detail where they are in the Food and Drug Administration approval and regulatory process. A PubMed search was performed using the phrase "Autologous Chondrocyte Implantation" alone and with the words second generation and third generation. Additionally, clinicaltrials.gov was searched for the names of the seven specific procedures and the parent company websites were referenced. Two-Stage Techniques: BioCart II uses a FGF2v1 culture and a fibrinogen, thrombin matrix, whereas Hyalograft-C uses a Hyaff 11 matrix. MACI uses a collagen I/III matrix. Cartipatch consists of an agarose-alginate hydrogel. Neocart uses a high-pressure bioreactor for culturing with a type I collagen matrix. ChondroCelect makes use of a gene expression analysis to predict chondrocyte proliferation and has demonstrated significant clinical improvement, but failed to show superiority to microfracture in a phase III trial. One Step Technique: CAIS is an ACI procedure where harvested cartilage is minced and implanted into a matrix for defect filling. As full thickness defects in articular cartilage continue to pose a challenge to treat, new methods of repair are being researched. Later generation ACI has been developed to address the prevalence of fibrocartilage with microfracture and the complications associated with the periosteal flap of first generation ACI such as periosteal hypertrophy. The procedures and products reviewed here represent advances in tissue engineering, scaffolds and autologous chondrocyte culturing that may hold promise in our quest to alter the natural history of symptomatic chondral disease.

  1. Production of serum amyloid A in equine articular chondrocytes and fibroblast-like synoviocytes treated with proinflammatory cytokines and its effects on the two cell types in culture

    DEFF Research Database (Denmark)

    Jacobsen, Stine; Ladefoged, Søren; Berg, Lise Charlotte

    2016-01-01

    OBJECTIVE: To investigate the role of the major equine acute phase protein serum amyloid A (SAA) in inflammation of equine intraarticular tissues. SAMPLE: Articular chondrocytes and fibroblast-like synoviocytes (FLSs) from 8 horses (4 horses/cell type). PROCEDURES: Chondrocytes and FLSs were...... in chondrocytes and FLSs. Isoforms of SAA in lysed chondrocytes and their culture medium corresponded to those previously detected in synovial fluid from horses with joint disease. When exposed to SAA, chondrocytes and FLSs had increased expression of IL-6, SAA, and MMP3, and chondrocytes had increased expression...

  2. The effect of alginate, hyaluronate and hyaluronate derivatives biomaterials on synthesis of non-articular chondrocyte extracellular matrix.

    Science.gov (United States)

    Gerard, C; Catuogno, C; Amargier-Huin, C; Grossin, L; Hubert, P; Gillet, P; Netter, P; Dellacherie, E; Payan, E

    2005-06-01

    Cartilage engineering consists of re-constructing functional cartilage by seeding chondrocytes in suitable biomaterials in vitro. The characteristics of neocartilage differ upon the type of biomaterial chosen. This study aims at determining the appropriate scaffold material for articular cartilage reconstruction using non articular chondrocytes harvested from rat sternum. For this purpose, the use of polysaccharide hydrogels such as alginate (AA) and hyaluronic acid (HA) was investigated. Several ratios of AA/HA were used as well as three derivatives obtained by chemical modification of HA (HA-C18, HA-C12(2.3), HA-C12(2.5)-TEG0.5). Sternal chondrocytes were successfully cultured in 3D alginate and alginate/HA scaffolds. HA retention in alginate beads was found to be higher in beads seeded with cells than in beads without cells. HA-C18 improved HA retention in beads but inhibited the chondrocyte synthesis process. Cell proliferation and metabolism were enhanced in all biomaterials when beads were mechanically agitated. Preliminary results have shown that the chondrocyte neo-synthesised matrix had acquired articular characteristics after 21 days culture.

  3. MiR-23a inhibited IL-17-mediated proinflammatory mediators expression via targeting IKKα in articular chondrocytes.

    Science.gov (United States)

    Hu, Junzheng; Zhai, Chenjun; Hu, Jiaojiao; Li, Zeng; Fei, Hao; Wang, Zhen; Fan, Weimin

    2017-02-01

    The inflammatory cytokine interleukin 17 (IL-17) is an important contributor of rheumatoid arthritis (RA) chronicity. Although several microRNAs (miRNAs) have been shown to regulate RA pathogenesis, the function of miRNAs in articular chondrocytes during rheumatoid arthritis pathogenesis is unclear. Here we showed that miR-23a was downregulated in articular cartilage tissues from rheumatoid arthritis patients. MiR-23a suppressed IL-17 inflammatory cytokine-induced NF-κB activation and several proinflammatory mediators expression, such as cytokine IL-6, chemokine MCP-1, and matrix metalloproteinase MMP-3 in articular chondrocytes. Furthermore, we found that the miR-23a expression was inversely correlated with IKKα expression in articular cartilage tissues from rheumatoid arthritis patients. We identified that IKKα was the direct target of miR-23a and miR-23a inhibited IL-17-mediated proinflammatory mediators expression via targeting the IKKα in primary articular chondrocytes. Together, our study provides the first evidence of a role for miR-23a regulated IL-17-mediated proinflammatory mediators expression in rheumatoid arthritis by directly targeting IKKα. Our findings provide novel evidence that may be useful for future studies exploring therapeutic approaches for rheumatoid arthritis by targeting miR-23a. Thus, miR-23a may be a common therapeutic target for rheumatoid arthritis. Copyright © 2016. Published by Elsevier B.V.

  4. Protective effects of the pericellular matrix of chondrocyte on articular cartilage against the development of osteoarthritis.

    Science.gov (United States)

    Liu, Chenlu; Wang, Beiyu; Xiao, Li; Li, Yefu; Xu, Lin; Zhao, Zhihe; Zhang, Lin

    2018-01-23

    Understanding the pathogenesis of osteoarthritis (OA) provides invaluable information in the search of therapeutic targets for the development of disease-modifying OA drugs. Emerging results from investigations demonstrate that the pericellular matrix of chondrocytes plays important roles in protecting articular cartilages from being degraded. Thus, maintaining the structural integrity of the pericellular matrix may be an effective approach to prevent the development of osteoarthritic joints. In this review article, we discuss the consequences of lacking one or more components of the pericellular matrix, and biological effects of the destruction of the pericellular matrix in the development of OA. We believe that more attention should be directed towards the pericellular matrix for the identification of novel biomarkers and therapeutic targets for the prevention and treatment of OA.

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

    Directory of Open Access Journals (Sweden)

    Eleonora Olivotto

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

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

    Science.gov (United States)

    Olivotto, Eleonora; Otero, Miguel; Astolfi, Annalisa; Platano, Daniela; Facchini, Annalisa; Pagani, Stefania; Flamigni, Flavio; Facchini, Andrea; Goldring, Mary B; Borzì, Rosa Maria; Marcu, Kenneth B

    2013-01-01

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

  7. Effects of meloxicam compared to acetylsalicylic acid in human articular chondrocytes.

    Science.gov (United States)

    Bassleer, C; Magotteaux, J; Geenen, V; Malaise, M

    1997-01-01

    Meloxicam is a new nonsteroidal anti-inflammatory drug (NSAID) derived from enolic acid, which has displayed potent anti-inflammatory properties in animal studies combined with low gastrointestinal toxicity. Other NSAIDs have been shown, in vitro, to have a variety of effects on cartilage repair processes in diseased articular cartilage. The aim of this study was to ascertain the effects of meloxicam on some of these processes using in vitro models. Acetylsalicylic acid, a NSAID whose characteristics have been previously elucidated in the models, was used as an active comparator. The effects of meloxicam were different from those of acetylsalicylic acid on chondrocyte clusters. At pharmacologically active concentrations, meloxicam was a potent inhibitor of prostaglandin-E2 production. However, all chondroformative processes were unaffected by meloxicam as indicated by a lack of effect on DNA synthesis and on type-II collagen and proteoglycan levels in chondrocyte culture medium and clusters, while acetylsalicylic acid decreased proteoglycan production and cell proliferation. Consequently, these in vitro findings suggest that meloxicam does not adversely affect the reparative processes active within the cartilage matrix of a diseased joint. This study represents a sound basis for future studies to establish the effects of meloxicam on osteoarthritis disease progression.

  8. 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

    -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 (Pconcentration-dependently inhibited by calcitonin, 65% protection.......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...

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

    Science.gov (United States)

    Fernandes, Amilton M; Herlofsen, Sarah R; Karlsen, Tommy A; Küchler, Axel M; Fløisand, Yngvar; Brinchmann, Jan E

    2013-01-01

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

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

    Science.gov (United States)

    Pettersson, Sofia; Wetterö, Jonas; Tengvall, Pentti; Kratz, Gunnar

    2011-12-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2011-12-15

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

  12. 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.

  13. 3D Dynamic Culture of Rabbit Articular Chondrocytes Encapsulated in Alginate Gel Beads Using Spinner Flasks for Cartilage Tissue Regeneration

    Directory of Open Access Journals (Sweden)

    Feiyue Xu

    2014-01-01

    Full Text Available Cell-based therapy using chondrocytes for cartilage repair suffers from chondrocyte dedifferentiation. In the present study, the effects of an integrated three-dimensional and dynamic culture on rabbit articular chondrocytes were investigated. Cells (passages 1 and 4 were encapsulated in alginate gel beads and cultured in spinner flasks in chondrogenic and chondrocyte growth media. Subcutaneous implantation of the cell-laden beads was performed to evaluate the ectopic chondrogenesis. It was found that cells remained viable after 35 days in the three-dimensional dynamic culture. Passage 1 cells demonstrated a proliferative growth in both media. Passage 4 cells showed a gradual reduction in DNA content in growth medium, which was attenuated in chondrogenic medium. Deposition of glycosaminoglycans (GAG was found in all cultures. While passage 1 cells generally produced higher amounts of GAG than passage 4 cells, GAG/DNA became similar on day 35 for both cells in growth media. Interestingly, GAG/DNA in growth medium was greater than that in chondrogenic medium for both cells. Based on GAG quantification and gene expression analysis, encapsulated passage 1 cells cultured in growth medium displayed the best ectopic chondrogenesis. Taken together, the three-dimensional and dynamic culture for chondrocytes holds great potential in cartilage regeneration.

  14. Identification of phenotypic discriminating markers for intervertebral disc cells and articular chondrocytes.

    Science.gov (United States)

    Clouet, Johann; Grimandi, Gaël; Pot-Vaucel, Marianne; Masson, Martial; Fellah, Hakim B; Guigand, Lydie; Cherel, Yan; Bord, Eric; Rannou, François; Weiss, Pierre; Guicheux, Jérôme; Vinatier, Claire

    2009-11-01

    The present study was conducted to improve our knowledge of intervertebral disc (IVD) cell biology by comparing the phenotype of nucleus pulposus (NP) and annulus fibrosus (AF) cells with that of articular chondrocytes (ACs). Rabbit cells from NP and AF were isolated and their phenotype was compared with that of AC by real-time PCR analysis of type I (COL1A1), II (COL2A1) and V (COL5A1) collagens, aggrecan transcript (AGC1), matrix Gla protein (MGP) and Htra serine peptidase 1 (Htra1). Transcript analysis indicated that despite certain similarities, IVD cells exhibit distinct COL2A1/COL1A1 and COL2A1/AGC1 ratios as compared with AC. The expression pattern of COL5A1, MGP and Htra1 makes it possible to define a phenotypic signature for NP and AF cells. Our study shows that NP and AF cells exhibit a clearly distinguishable phenotype from that of AC. Type V collagen, MGP and HtrA1 could greatly help to discriminate among NP, AF and AC cells.

  15. Wnt signaling is involved in human articular chondrocyte de-differentiation in vitro

    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. Certain signaling pathways, such as the wnt pathway, are involved in cartilage pathology. Osteoarthritic chondrocytes undergo morphological and biochemical changes that lead to chondrocyte de-differentiation. We investigated

  16. Rosmarinic acid induces rabbit articular chondrocyte differentiation by decreases matrix metalloproteinase-13 and inflammation by upregulating cyclooxygenase-2 expression.

    Science.gov (United States)

    Eo, Seong-Hui; Kim, Song Ja

    2017-09-18

    Matrix metalloproteinases (MMPs) are known to play an important role in the degradation of the extracellular matrix and the pathological progression of osteoarthritis (OA). The natural polyphenolic compound rosmarinic acid (Ros. A) has been shown to suppress the inhibitory activity of matrix metalloproteinases (MMPs). However, the effects of Ros. A on OA have not been investigated. In the current study, primary articular chondrocytes were cultured from rabbit articular cartilage and treated with Ros. A. Phenotypic characterization was performed by western blotting to assess specific markers, prostaglandin E2 (PGE2) assays, and alcian blue staining to measure sulfated-proteoglycan production. We report that in rabbit articular chondrocytes, Ros. A increased type II collagen, sulfated-proteoglycan, cyclooxygenase-2 (COX-2), and PGE2 production in a dose- and time-dependent manner. Furthermore, Ros. A suppressed the expression of MMP-13. In addition, treatment with Ros A activated extracellular signal-regulated kinase (ERK)-1/2 and p38 kinase signaling pathways. Inhibition of MMP-13 enhanced Ros. A-induced type II collagen expression and sulfated-proteoglycan synthesis but COX-2 and PGE2 production were unchanged. Ros. A-mediated up-regulation of ERK phosphorylation was abolished by the MEK inhibitor, PD98059, which prevented induction of the associated inflammatory response. Inhibition of p38 kinase with SB203580 enhanced the increase in type II collagen expression via Ros. A-mediated down-regulation of MMP-13. Results suggest that ERK-1/2 regulates Ros. A-induced inflammation and that p38 regulates differentiation by inhibiting MMP-13 in rabbit articular chondrocytes.

  17. Resveratrol and N-acetylcysteine influence redox balance in equine articular chondrocytes under acidic and very low oxygen conditions.

    Science.gov (United States)

    Collins, John A; Moots, Robert J; Clegg, Peter D; Milner, Peter I

    2015-09-01

    Mature articular cartilage is an avascular tissue characterized by a low oxygen environment. In joint disease, acidosis and further reductions in oxygen levels occur, compromising cartilage integrity.This study investigated how acidosis and very low oxygen levels affect components of the cellular redox system in equine articular chondrocytesand whether the antioxidants resveratrol and N-acetylcysteine could modulate this system. We used articular chondrocytes isolated from nondiseased equine joints and cultured them in a 3-D alginate bead system for 48h in pH 7.2 or 6.2 in the absence or presence of the proinflammatory cytokine, interleukin-1β (10ng/ml).In addition, chondrocytes were cultured with resveratrol (10µM) or N-acetylcysteine (NAC) (2mM).Cell viability, glycosaminoglycan (GAG) release, mitochondrial membrane potential (ΔΨm), reactive oxygen species (ROS), GSH:GSSG ratio, and SOD1 and SOD2 protein expression were measured. Very low levels of oxygen (pH 6.2), and exposure to IL-1β led to reductions in cell viability, increased GAG release, alterations in ΔΨm and ROS levels, and reduced GSH:GSSG ratio. In addition, SOD1 and SOD2 protein expressions were reduced. Both resveratrol and NAC partially restored ΔΨm and ROS levels and prevented GAG release and cell loss and normalized SOD1 and SOD2 protein expression. In particular NAC was highly effective at restoring the GSH:GSSG ratio.These results show that the antioxidants resveratrol and N-acetylcysteine can counteract the redox imbalance in articular chondrocytes induced by low oxygen and acidic conditions. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

  18. 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. (c) 2010 Wiley-Liss, Inc.

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

    Directory of Open Access Journals (Sweden)

    Serena Guidotti

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

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

    Science.gov (United States)

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

    2015-01-01

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

  1. Autologous Chondrocyte Implantation in Osteoarthritic Surroundings

    DEFF Research Database (Denmark)

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

    2018-01-01

    . PURPOSE: To investigate the effects of tumor necrosis factor α (TNFα) and its potential inhibition by adalimumab on cartilage regeneration in an in vitro model of ACI. STUDY DESIGN: Controlled laboratory study. METHODS: Bovine articular chondrocytes were cultivated and transferred at passage 3 to fibrin-polyurethane...

  2. Articular cartilage and growth plate defects are associated with chondrocyte cytoskeletal abnormalities in Tg737orpk mice lacking the primary cilia protein polaris.

    Science.gov (United States)

    McGlashan, S R; Haycraft, C J; Jensen, C G; Yoder, B K; Poole, C A

    2007-05-01

    Primary cilia are highly conserved organelles found on almost all eukaryotic cells. Tg737(orpk) (orpk) mice carry a hypomorphic mutation in the Tg737 gene resulting in the loss of polaris, a protein essential for ciliogenesis. Orpk mice have an array of skeletal patterning defects and show stunted growth after birth, suggesting defects in appositional and endochondral development. This study investigated the association between orpk tibial long bone growth and chondrocyte primary cilia expression using histomorphometric and immunohistochemical analysis. Wild-type chondrocytes throughout the developing epiphysis and growth plate expressed primary cilia, which showed a specific orientation away from the articular surface in the first 7-10 cell layers. In orpk mice, primary cilia were identified on very few cells and were significantly shorter. Orpk chondrocytes also showed significant increases in cytoplasmic tubulin, a likely result of failed ciliary assembly. The growth plates of orpk mice were significantly smaller in length and width, with marked changes in cellular organization in the presumptive articular cartilage, proliferative and hypertrophic zones. Cell density at the articular surface and in the hypertrophic zone was significantly altered, suggesting defects in both appositional and endochondral growth. In addition, orpk hypertrophic chondrocytes showed re-organization of the F-actin network into stress fibres and failed to fully undergo hypertrophy, while there was a marked reduction in type X collagen sequestration. These data suggest that failure to form a functional primary cilium affects chondrocyte differentiation and results in delayed chondrocyte hypertrophy within the orpk growth plate.

  3. 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

  4. 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

  5. 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

  6. Biochemical investigation of the effects of human platelet releasates on human articular chondrocytes.

    Science.gov (United States)

    Spreafico, Adriano; Chellini, Federico; Frediani, Bruno; Bernardini, Giulia; Niccolini, Silvia; Serchi, Tommaso; Collodel, Giulia; Paffetti, Alessandro; Fossombroni, Vittorio; Galeazzi, Mauro; Marcolongo, Roberto; Santucci, Annalisa

    2009-12-01

    The aim of the present study was to demonstrate the mitogenic and differentiating properties of platelet-rich plasma releasates (PRPr) on human chondrocytes in mono- and three-dimensional cultures. In order to assess if PRPr supplementation could maintain the chondrocyte phenotype or at least inhibit the cell de-differentiation even after several days in culture, we performed a proteomic study on several cell cultures independently grown, for different periods of time, in culture medium with FCS, human serum (HS), and releasates obtained from PRP and platelet-poor plasma (PPP). We found that PRP treatment actually induced in chondrocytes the expression of proteins (some of which novel) involved in differentiation.

  7. Evaluation of nonbiomedical and biomedical grade alginates for the transplantation of genetically modified articular chondrocytes to cartilage defects in a large animal model in vivo.

    Science.gov (United States)

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

    2011-04-01

    Genetically modified chondrocytes embedded in alginate improve cartilage repair in experimental models, and alginates are clinically used for articular chondrocyte transplantation. In the present study, we tested the hypothesis that the alginate system allows for sustained transgene expression in cartilage defects in a preclinical large animal model in vivo. Primary cultures of ovine articular chondrocytes were transfected with the Photinus pyralis luc or the Escherichia coli lacZ genes in monolayer culture in vitro using eight different nonviral compounds. Optimally transfected chondrocytes were encapsulated in spheres composed of nonbiomedical or biomedical grade alginates for evaluation of luciferase expression, cell numbers and viabilities in vitro. Transfected chondrocytes encapsulated in spheres comprised of the different alginates were then implanted into osteochondral defects in the knee joints of sheep to examine the profiles of transgene expression in vivo. Ovine articular chondrocytes were efficiently transfected with FuGENE 6. Transgene expression was detectable after encapsulation in the alginates over 21 days in vitro. Transplantation of genetically modified chondrocytes to cartilage defects in vivo resulted in maximal transgene expression on day 1 after transfection, with a decrease by day 21, the longest time point evaluated. Remarkably, the reduction in luciferase activity was less pronounced when biomedical grade alginates were employed, compared to nonbiomedical grade alginates, suggesting that such alginates might be better suited to support elevated transgene expression after transplantation of genetically modified chondrocytes. This approach may be of value to study the effects of potential therapeutic genes upon cartilage repair in a clinically relevant setting. Copyright © 2011 John Wiley & Sons, Ltd.

  8. Engineering biomechanically functional neocartilage derived from expanded articular chondrocytes through the manipulation of cell-seeding density and dexamethasone concentration.

    Science.gov (United States)

    Huang, Brian J; Huey, Daniel J; Hu, Jerry C; Athanasiou, Kyriacos A

    2017-08-01

    Recent work has established methods to engineer self-assembled, scaffold-free neocartilage from an expanded articular chondrocyte (AC) cell source. In continuing such work, the objective of the present study was to investigate the effects of cell-seeding density and dexamethasone concentration on these neocartilage constructs. Neocartilage discs (5 mm diameter) were formed by self-assembling passaged leporine articular chondrocytes into non-adherent agarose moulds. The cell-seeding densities (2, 3, 4, 5 and 6 million cells/construct) and dexamethasone concentrations (10 and 100 nm) in the culture medium were varied in a full-factorial study. After 4 weeks, the neocartilage constructs were assessed for morphological, biochemical and biomechanical properties. The cell-seeding density profoundly affected neocartilage properties. The two dexamethasone concentrations explored did not induce overall significant differences. Constructs formed using lower cell-seeding densities possessed much higher biochemical and biomechanical properties than constructs seeded with higher cell densities. Notably, the 2 million cells/construct group formed hyaline-like neocartilage with a collagen wet weight (WW) content of ~7% and a Young's modulus of ~4 MPa, representing the high end of values achieved in self-assembled neocartilage. Excitingly, the mechanical properties of these constructs were on a par with that of native cartilage tissues tested under similar conditions. Through optimization of cell-seeding density, this study shows for the first time the use of expanded ACs to form homogeneous self-assembled neocartilage with exceptionally high tensile strength. With such functional properties, these engineered neocartilage constructs provide a promising alternative for treating articular lesions. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

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

    NARCIS (Netherlands)

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

    2015-01-01

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

  10. Characterization of a specific somatomedin-c receptor on isolated bovine growth plate chondrocytes.

    Science.gov (United States)

    Trippel, S B; Van Wyk, J J; Foster, M B; Svoboda, M E

    1983-06-01

    The interaction of somatomedin (Sm) with growth plate chondrocytes (GPCs) is believed to be the primary stimulus of skeletal growth. Using techniques designed to disrupt as little as possible the phenotypic characteristics of GPCs, we have been able to obtain 3-4 x 10(8) viable cells from the major physes of one newborn calf. The availability of these cells plus essentially pure Sm-C/insulin-like growth factor I, the most GH-dependent Sm, has now made possible detailed studies of the interaction of this radiolabeled peptide with the GPC receptor and of the subsequent processing of this hormone by these cells. The enzymatic methods required to free GPCs from their matrix led to loss of receptors, followed by rapid receptor regeneration by de novo synthesis in suspension cultures. Binding of [125I]iodo-Sm-C to GPCs was time dependent and saturable, with optima at 15 C and pH 7.8. At 37 C, binding peaked at 90 min and declined thereafter. Multiplication-stimulating activity, insulin, and nerve growth factor were less potent than unlabeled Sm-C in competition with [125I]iodo-Sm-C for its receptor. Human GH, epidermal growth factor, and fibroblast growth factor failed to show competition even at 10(-6) M. Analysis of the fate of [125I]iodo-Sm-C bound to GPCs at 37 C provided evidence that this hormone is internalized and extruded from the cell in a partially degraded form. Scatchard analysis of [125I]iodo-Sm-C binding to GPCs and to chondrocytes isolated from articular cartilage revealed similar Ka values, but reproducibly 2-6 times more receptors on growth plate than on articular chondrocytes.

  11. 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.

  12. Production of serum amyloid A in equine articular chondrocytes and fibroblast-like synoviocytes treated with proinflammatory cytokines and its effects on the two cell types in culture.

    Science.gov (United States)

    Jacobsen, Stine; Ladefoged, Søren; Berg, Lise C

    2016-01-01

    To investigate the role of the major equine acute phase protein serum amyloid A (SAA) in inflammation of equine intraarticular tissues. Articular chondrocytes and fibroblast-like synoviocytes (FLSs) from 8 horses (4 horses/cell type). Chondrocytes and FLSs were stimulated in vitro for various periods up to 48 hours with cytokines (recombinant interleukin [IL]-1β, IL-6, tumor necrosis factor-α, or a combination of all 3 [IIT]) or with recombinant SAA. Gene expression of SAA, IL-6, matrix metalloproteinases (MMP)-1 and -3, and cartilage-derived retinoic acid-sensitive protein were assessed by quantitative real-time PCR assay; SAA protein was evaluated by immunoturbidimetry and denaturing isoelectric focusing and western blotting. All cytokine stimulation protocols increased expression of SAA mRNA and resulted in detectable SAA protein production in chondrocytes and FLSs. Isoforms of SAA in lysed chondrocytes and their culture medium corresponded to those previously detected in synovial fluid from horses with joint disease. When exposed to SAA, chondrocytes and FLSs had increased expression of IL-6, SAA, and MMP3, and chondrocytes had increased expression of MMP-1. Chondrocytes had decreased expression of cartilage-derived retinoic acid-sensitive protein. Upregulation of SAA in chondrocytes and FLSs stimulated with proinflammatory cytokines and the proinflammatory effects of SAA suggested that SAA may be involved in key aspects of pathogenesis of the joint inflammation in horses.

  13. Mixed cell therapy of bone marrow-derived mesenchymal stem cells and articular cartilage chondrocytes ameliorates osteoarthritis development.

    Science.gov (United States)

    Prasadam, Indira; Akuien, Akoy; Friis, Thor E; Fang, Wei; Mao, Xinzhan; Crawford, Ross W; Xiao, Yin

    2018-01-01

    Of the many cell-based treatments that have been tested in an effort to regenerate osteoarthritic articular cartilage, none have ever produced cartilage that compare with native hyaline cartilage. Studies show that different cell types lead to inconsistent results and for cartilage regeneration to be considered successful, there must be an absence of fibrotic tissue. Here we report of a series of experiments in which bone marrow-derived stem cells (BMSCs) and articular cartilage chondrocytes (ACCs) were mixed in a 1:1 ratio and tested for their ability to enhance cartilage regeneration in three different conditions: (1) in an in vitro differentiation model; (2) in an ex vivo cartilage defect model implanted subcutaneously in mice; and (3) as an intra-articular injection in a meniscectomy-induced OA model in rats. The mixed cells were compared with monocultures of BMSCs and ACCs. In all three experimental models there was significantly enhanced cartilage regeneration and decreased fibrosis in the mixed BMSCs+ACCs group compared with the monocultures. Molecular analysis showed a reduction in vascularization and hypertrophy, coupled with higher chondrogenic gene expression resulting from the BMSCs+ACCs treatment. Together, our data suggest that mixed BMSCs+ACCs treatment is highly chondro-protective and is more effective in regenerating damaged cartilage in both the ex vivo cartilage defect and post-trauma OA disease models. The results from this approach could potentially be used for regeneration of cartilage in OA patients.

  14. Calcium signaling of in situ chondrocytes in articular cartilage under compressive loading: Roles of calcium sources and cell membrane ion channels.

    Science.gov (United States)

    Lv, Mengxi; Zhou, Yilu; Chen, Xingyu; Han, Lin; Wang, Liyun; Lu, X Lucas

    2017-10-05

    Mechanical loading on articular cartilage can induce many physical and chemical stimuli on chondrocytes residing in the extracellular matrix (ECM). Intracellular calcium ([Ca2+ ]i ) signaling is among the earliest responses of chondrocytes to physical stimuli, but the [Ca2+ ]i signaling of in situ chondrocytes in loaded cartilage is not fully understood due to the technical challenges in [Ca2+ ]i imaging of chondrocytes in a deforming ECM. This study developed a novel bi-directional microscopy loading device that enables the record of transient [Ca2+ ]i responses of in situ chondrocytes in loaded cartilage. It was found that compressive loading significantly promoted [Ca2+ ]i signaling in chondrocytes with faster [Ca2+ ]i oscillations in comparison to the non-loaded cartilage. Seven [Ca2+ ]i signaling pathways were further investigated by treating the cartilage with antagonists prior to and/or during the loading. Removal of extracellular Ca2+ ions completely abolished the [Ca2+ ]i responses of in situ chondrocytes, suggesting the indispensable role of extracellular Ca2+ sources in initiating the [Ca2+ ]i signaling in chondrocytes. Depletion of intracellular Ca2+ stores, inhibition of PLC-IP3 pathway, and block of purinergic receptors on plasma membrane led to significant reduction in the responsive rate of cells. Three types of ion channels that are regulated by different physical signals, TRPV4 (osmotic and mechanical stress), T-type VGCCs (electrical potential), and mechanical sensitive ion channels (mechanical loading) all demonstrated critical roles in controlling the [Ca2+ ]i responses of in situ chondrocyte in the loaded cartilage. This study provided new knowledge about the [Ca2+ ]i signaling and mechanobiology of chondrocytes in its natural residing environment. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.

  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. Copyright © 2012 Orthopaedic Research Society.

  16. Age-related decrease in proteoglycan synthesis of human articular chondrocytes: the role of nonenzymatic glycation

    NARCIS (Netherlands)

    Degroot, J.; Verzijl, N.; Bank, R. A.; Lafeber, F. P.; Bijlsma, J. W.; TeKoppele, J. M.

    1999-01-01

    To examine the effect of nonenzymatic glycation of cartilage extracellular matrix on the synthetic activity of chondrocytes. The proteoglycan-synthesis rate (35SO4(2-) incorporation) and levels of advanced nonenzymatic glycation (determined by high-performance liquid chromatography measurement of

  17. Age-related decrease in proteoglycan synthesis of human articular chondrocytes: The role of nonenzymatic glycation

    NARCIS (Netherlands)

    Groot, J. de; Verzijl, N.; Bank, R.A.; Lafeber, F.P.J.G.; Bijlsma, J.W.J.; TeKoppele, J.M.

    1999-01-01

    Objective. To examine the effect of nonenzymatic glycation of cartilage extracellular matrix on the synthetic activity of chondrocytes. Methods. The proteoglycan-synthesis rate (35SO42- incorporation) and levels of advanced nonenzymatic glycation (determined by high-performance liquid chromatography

  18. Effects of electromagnetic fields on proteoglycan metabolism of bovine articular cartilage explants

    NARCIS (Netherlands)

    De Mattei, M; Pasello, M; Pellati, A; Stabellini, G; Massari, L; Gemmati, D; Caruso, A

    2003-01-01

    Electromagnetic field (EMF) exposure has been proposed for the treatment of osteoarthritis. In this study, we investigated the effects of EMF (75 Hz, 2,3 mT) on proteoglycan (PG) metabolism of bovine articular cartilage explants cultured in vitro, both under basal conditions and in the presence of

  19. 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

    2017-01-26

    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 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 immobilization group was higher than in the sham group at four and eight weeks after surgery (p immobilization group than that in the sham group at four and eight weeks after surgery (p 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.

  20. Platelet-Rich Plasma Attenuates 30-kDa Fibronectin Fragment-Induced Chemokine and Matrix Metalloproteinase Expression by Meniscocytes and Articular Chondrocytes.

    Science.gov (United States)

    Wang, Chih-Chien; Lee, Chian-Her; Peng, Yi-Jen; Salter, Donald M; Lee, Herng-Sheng

    2015-10-01

    Proteolytic fragments of fibronectin have catabolic effects on cartilage and menisci. Platelet-rich plasma (PRP) is increasingly being used to treat a range of joint conditions, but it is unknown whether PRP influences fibronectin fragment (FN-f) procatabolic activity. The procatabolic activity of FN-f on meniscocytes and articular chondrocytes is attenuated by cotreatment with PRP. Controlled laboratory study. Human meniscocytes were treated with FN-f (30 kDa) with or without PRP coincubation, and gene expression was analyzed by complementary DNA microarray analysis. Validation of altered expression of known and novel chemokine and protease genes was undertaken by real-time polymerase chain reaction (RT-PCR) in articular chondrocytes and meniscocytes. Chemokine release was assayed by enzyme-linked immunosorbent assay, and intracellular pathway signaling was evaluated by Western immunoblotting. Microarray analysis and RT-PCR showed increased expression of matrix metalloproteinase (MMP)1, MMP2, MMP3, MMP9, MMP13, interleukin (IL)-6, IL-8 (CXCL8), CCL5, CCL20, and CXCL10 chemokines in meniscocytes after treatment with FN-f. Upregulation of these genes was significantly attenuated by PRP. Similar results were seen with articular chondrocytes, although no changes in MMP2 or MMP9 levels were identified. PRP-induced suppression of gene expression was associated with activation of Akt and p44/p42. PRP treatment attenuates the 30-kDa FN-f-induced expression of a range of proinflammatory chemokines and MMPs, including IL-8, IL-6, CCL20, CCL5, CXCL10, MMP1, MMP3, and MMP13, by both meniscocytes and articular chondrocytes. These observations provide support for the use and further trials of PRP in management of cartilage and meniscal injuries. © 2015 The Author(s).

  1. In vitro chondrogenic commitment of human Wharton's jelly stem cells by co-culture with human articular chondrocytes.

    Science.gov (United States)

    Pereira, R C; Costa-Pinto, A R; Frias, A M; Neves, N M; Azevedo, H S; Reis, R L

    2017-06-01

    Wharton's jelly stem cells (WJSCs) are a potential source of transplantable stem cells in cartilage-regenerative strategies, due to their highly proliferative and multilineage differentiation capacity. We hypothesized that a non-direct co-culture system with human articular chondrocytes (hACs) could enhance the potential chondrogenic phenotype of hWJSCs during the expansion phase compared to those expanded in monoculture conditions. Primary hWJSCs were cultured in the bottom of a multiwell plate separated by a porous transwell membrane insert seeded with hACs. No statistically significant differences in hWJSCs duplication number were observed under either of the culture conditions during the expansion phase. hWJSCs under co-culture conditions show upregulations of collagen type I and II, COMP, TGFβ1 and aggrecan, as well as of the main cartilage transcription factor, SOX9, when compared to those cultured in the absence of chondrocytes. Chondrogenic differentiation of hWJSCs, previously expanded in co-culture and monoculture conditions, was evaluated for each cellular passage using the micromass culture model. Cells expanded in co-culture showed higher accumulation of glycosaminoglycans (GAGs) compared to cells in monoculture, and immunohistochemistry for localization of collagen type I revealed a strong detection signal when hWJSCs were expanded under monoculture conditions. In contrast, type II collagen was detected when cells were expanded under co-culture conditions, where numerous round-shaped cell clusters were observed. Using a micromass differentiation model, hWJSCs, previously exposed to soluble factors secreted by hACs, were able to express higher levels of chondrogenic genes with deposition of cartilage extracellular matrix components, suggesting their use as an alternative cell source for treating degenerated cartilage. Copyright © 2015 John Wiley & Sons, Ltd. Copyright © 2015 John Wiley & Sons, Ltd.

  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. Hydrostatic pressure in articular cartilage tissue engineering: from chondrocytes to tissue regeneration.

    Science.gov (United States)

    Elder, Benjamin D; Athanasiou, Kyriacos A

    2009-03-01

    Cartilage has a poor intrinsic healing response, and neither the innate healing response nor current clinical treatments can restore its function. Therefore, articular cartilage tissue engineering is a promising approach for the regeneration of damaged tissue. Because cartilage is exposed to mechanical forces during joint loading, many tissue engineering strategies use exogenous stimuli to enhance the biochemical or biomechanical properties of the engineered tissue. Hydrostatic pressure (HP) is emerging as arguably one of the most important mechanical stimuli for cartilage, although no optimal treatment has been established across all culture systems. Therefore, this review evaluates prior studies on articular cartilage involving the use of HP, with a particular emphasis on the treatments that appear promising for use in future studies. Additionally, this review addresses HP bioreactor design, chondroprotective effects of HP, the use of HP for chondrogenic differentiation, the effects of high pressures, and HP mechanotransduction.

  4. Hydrostatic Pressure in Articular Cartilage Tissue Engineering: From Chondrocytes to Tissue Regeneration

    OpenAIRE

    Elder, Benjamin D.; Athanasiou, Kyriacos A.

    2009-01-01

    Cartilage has a poor intrinsic healing response, and neither the innate healing response nor current clinical treatments can restore its function. Therefore, articular cartilage tissue engineering is a promising approach for the regeneration of damaged tissue. Because cartilage is exposed to mechanical forces during joint loading, many tissue engineering strategies use exogenous stimuli to enhance the biochemical or biomechanical properties of the engineered tissue. Hydrostatic pressure (HP) ...

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

    Science.gov (United States)

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

    2014-08-01

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

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

    Science.gov (United States)

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

    2016-04-01

    Osteoarthritis is a nonrheumatologic joint disease characterized by progressive degeneration of the cartilage extracellular matrix. Berberine (BBR) is an isoquinoline alkaloid used in traditional Chinese medicine, the majority of which is extracted from Huang Lian (Coptis chinensis). Although numerous studies have revealed the anticancer activity of BBR, its effects on normal cells, such as chondrocytes, and the molecular mechanisms underlying its actions remain elusive. Therefore, we examined the effects of BBR on rabbit articular chondrocytes, and the underlying molecular mechanisms, focusing on actin cytoskeletal reorganization. BBR induced dedifferentiation by inhibiting activation of phosphoinositide-3(PI3)-kinase/Akt and p38 kinase. Furthermore, inhibition of p38 kinase and PI3-kinase/Akt with SB203580 and LY294002, respectively, accelerated the BBR-induced dedifferentiation. BBR also caused actin cytoskeletal architecture reorganization and, therefore, we investigated if these effects were involved in the dedifferentiation. Disruption of the actin cytoskeleton by cytochalasin D reversed the BBR-induced dedifferentiation by activating PI3-kinase/Akt and p38 kinase. In contrast, the induction of actin filament aggregation by jasplakinolide accelerated the BBR-induced dedifferentiation via PI3-kinase/Akt inhibition and p38 kinase activation. Taken together, these data suggest that BBR strongly induces dedifferentiation, and actin cytoskeletal reorganization is a crucial requirement for this effect. Furthermore, the dedifferentiation activity of BBR appears to be mediated via PI3-kinase/Akt and p38 kinase pathways in rabbit articular chondrocytes. © 2016 by the Society for Experimental Biology and Medicine.

  7. Circular RNA expression profile of articular chondrocytes in an IL-1β-induced mouse model of osteoarthritis.

    Science.gov (United States)

    Zhou, Zhibin; Du, Di; Chen, Aimin; Zhu, Lei

    2018-02-20

    Osteoarthritis (OA) is a widely prevalent degenerative joint disease characterized by articular cartilage degradation and joint inflammation. The pathogenesis of OA remains unclear, leading to a lack of effective treatment. Previous studies have reported that circular RNAs (circRNAs) are involved in the development of various diseases. However, the function of circRNAs and their roles in OA is largely unknown. Therefore, we aimed to investigate changes in circRNA expression and predict their functions in OA by using bioinformatics analysis. An OA model was established in mouse articular chondrocytes (MACs) treated by interleukin-1β (IL-1β), and then the circRNA profile was screened by Next Generation Sequencing. By comparing circRNA expression in IL-1β- treated MACs and normal controls, differentially expressed circRNAs were identified during OA pathogenesis, and differential expression levels of selected circRNAs were validated by qRT-PCR. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were employed to predict the functions of these circRNAs. Because circRNAs can act as "miRNA sponges", we also constructed a circRNA-miRNA network to predict their functions. A total of 255 circRNAs were found to be differentially expressed in IL-1β-treated MACs (p≤0.05; fold-change≥2) from the expression of the normal controls. Among them, 119 circRNAs were significantly up-regulated, and the other 136 were down-regulated. Seven circRNAs were randomly selected to verify the reliability of these profiles by quantitative qRT-PCR. After obtaining the parental genes of differentially expressed circRNA, the top 30 enrichment GO entries and KEGG pathways were annotated. Then, two significantly differentially expressed circRNAs (mmu-circRNA-30365 and mmu-circRNA-36866) were identified and selected for further analysis, meanwhile a circRNA-miRNA regulation network was created and the top five most likely functional-related target miRNAs of

  8. 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.

  9. Influence of species and anatomical location on chondrocyte expansion

    Directory of Open Access Journals (Sweden)

    Hurtig Mark B

    2005-05-01

    Full Text Available Abstract Background Bovine articular cartilage is often used to study chondrocytes in vitro. It is difficult to correlate in vitro studies using bovine chondrocytes with in vivo studies using other species such as rabbits and sheep. The aim of this investigation was to study the effect of species, anatomical location and exogenous growth factors on chondrocyte proliferation in vitro. Methods Equine (EQ, bovine (BO and ovine (OV articular chondrocytes from metacarpophalangeal (fetlock (F, shoulder (S and knee (K joints were cultured in tissue culture flasks. Growth factors (rh-FGFb: 10 ng/ml; rh-TGFβ: 5 ng/ml were added to the cultures at days 2 and 4. On day 6, cells were counted and flow cytometry analysis was performed to determine cell size and granularity. A three factor ANOVA with paired Tukey's correction was used for statistical analysis. Results After 6 days in culture, cell numbers had increased in control groups of EQ-F, OV-S, OV-F and BO-F chondrocytes. The addition of rh-FGFb led to the highest increase in cell numbers in the BO-F, followed by EQ-F and OV-S chondrocytes. The addition of rh-TGFβ increased cell numbers in EQ-S and EQ-F chondrocytes, but showed nearly no effect on EQ-K, OV-K, OV-S, OV-F and BO-F chondrocytes. There was an overall difference with the addition of growth factors between the different species and joints. Conclusion Different proliferation profiles of chondrocytes from the various joints were found. Therefore, we recommend performing in vitro studies using the species and site where subsequent in vivo studies are planned.

  10. Influence of species and anatomical location on chondrocyte expansion

    Science.gov (United States)

    Akens, Margarete K; Hurtig, Mark B

    2005-01-01

    Background Bovine articular cartilage is often used to study chondrocytes in vitro. It is difficult to correlate in vitro studies using bovine chondrocytes with in vivo studies using other species such as rabbits and sheep. The aim of this investigation was to study the effect of species, anatomical location and exogenous growth factors on chondrocyte proliferation in vitro. Methods Equine (EQ), bovine (BO) and ovine (OV) articular chondrocytes from metacarpophalangeal (fetlock (F)), shoulder (S) and knee (K) joints were cultured in tissue culture flasks. Growth factors (rh-FGFb: 10 ng/ml; rh-TGFβ: 5 ng/ml) were added to the cultures at days 2 and 4. On day 6, cells were counted and flow cytometry analysis was performed to determine cell size and granularity. A three factor ANOVA with paired Tukey's correction was used for statistical analysis. Results After 6 days in culture, cell numbers had increased in control groups of EQ-F, OV-S, OV-F and BO-F chondrocytes. The addition of rh-FGFb led to the highest increase in cell numbers in the BO-F, followed by EQ-F and OV-S chondrocytes. The addition of rh-TGFβ increased cell numbers in EQ-S and EQ-F chondrocytes, but showed nearly no effect on EQ-K, OV-K, OV-S, OV-F and BO-F chondrocytes. There was an overall difference with the addition of growth factors between the different species and joints. Conclusion Different proliferation profiles of chondrocytes from the various joints were found. Therefore, we recommend performing in vitro studies using the species and site where subsequent in vivo studies are planned. PMID:15904515

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

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    Wertheimer Michael R

    2011-01-01

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

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

    Science.gov (United States)

    2011-01-01

    Background Recent evidence indicates that osteoarthritis (OA) may be a systemic disease since mesenchymal stem cells (MSCs) from OA patients express type X collagen, a marker of late stage chondrocyte hypertrophy (associated with endochondral ossification). We recently showed that the expression of type X collagen was suppressed when MSCs from OA patients were cultured on nitrogen (N)-rich plasma polymer layers, which we call "PPE:N" (N-doped plasma-polymerized ethylene, containing up to 36 atomic percentage (at.% ) of N. Methods In the present study, we examined the expression of type X collagen in fetal bovine growth plate chondrocytes (containing hypertrophic chondrocytes) cultured on PPE:N. We also studied the effect of PPE:N on the expression of matrix molecules such as type II collagen and aggrecan, as well as on proteases (matrix metalloproteinase-13 (MMP-13) and molecules implicated in cell division (cyclin B2). Two other culture surfaces, "hydrophilic" polystyrene (PS, regular culture dishes) and nitrogen-containing cation polystyrene (Primaria®), were also investigated for comparison. Results Results showed that type X collagen mRNA levels were suppressed when cultured for 4 days on PPE:N, suggesting that type X collagen is regulated similarly in hypertrophic chondrocytes and in human MSCs from OA patients. However, the levels of type X collagen mRNA almost returned to control value after 20 days in culture on these surfaces. Culture on the various surfaces had no significant effects on type II collagen, aggrecan, MMP-13, and cyclin B2 mRNA levels. Conclusion Hypertrophy is diminished by culturing growth plate chondrocytes on nitrogen-rich surfaces, a mechanism that is beneficial for MSC chondrogenesis. Furthermore, one major advantage of such "intelligent surfaces" over recombinant growth factors for tissue engineering and cartilage repair is potentially large cost-saving. PMID:21244651

  13. Naringenin regulates production of matrix metalloproteinases in the knee-joint and primary cultured articular chondrocytes and alleviates pain in rat osteoarthritis model

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    C.C. Wang

    Full Text Available Inflammation of cartilage is a primary symptom for knee-joint osteoarthritis. Matrix metalloproteinases (MMPs are known to play an important role in the articular cartilage destruction related to osteoarthritis. Naringenin is a plant-derived flavonoid known for its anti-inflammatory properties. We studied the effect of naringenin on the transcriptional expression, secretion and enzymatic activity of MMP-3 in vivo in the murine monosodium iodoacetate (MIA osteoarthritis model. The assessment of pain behavior was also performed in the MIA rats. The destruction of knee-joint tissues was analyzed microscopically. Moreover, the effect of naringenin was also studied in vitro in IL-1β activated articular chondrocytes. The transcriptional expression of MMP-3, MMP-1, MMP-13, thrombospondin motifs (ADAMTS-4 and ADAMTS-5 was also studied in primary cultured chondrocytes of rats. Naringenin caused significant reduction in pain behavior and showed marked improvement in the tissue morphology of MIA rats. Moreover, a significant inhibition of MMP-3 expression in MIA rats was observed upon treatment with naringenin. In the in vitro tests, naringenin caused a significant reduction in the transcriptional expression, secretion and enzymatic activity of the studied degradative enzymes. The NF-κB pathway was also found to be inhibited upon treatment with naringenin in vitro. Overall, the study suggests that naringenin alleviated pain and regulated the production of matrix-metalloproteinases via regulation of NF-κB pathway. Thus, naringenin could be a potent therapeutic option for the treatment of osteoarthritis.

  14. Naringenin regulates production of matrix metalloproteinases in the knee-joint and primary cultured articular chondrocytes and alleviates pain in rat osteoarthritis model.

    Science.gov (United States)

    Wang, C C; Guo, L; Tian, F D; An, N; Luo, L; Hao, R H; Wang, B; Zhou, Z H

    2017-03-23

    Inflammation of cartilage is a primary symptom for knee-joint osteoarthritis. Matrix metalloproteinases (MMPs) are known to play an important role in the articular cartilage destruction related to osteoarthritis. Naringenin is a plant-derived flavonoid known for its anti-inflammatory properties. We studied the effect of naringenin on the transcriptional expression, secretion and enzymatic activity of MMP-3 in vivo in the murine monosodium iodoacetate (MIA) osteoarthritis model. The assessment of pain behavior was also performed in the MIA rats. The destruction of knee-joint tissues was analyzed microscopically. Moreover, the effect of naringenin was also studied in vitro in IL-1β activated articular chondrocytes. The transcriptional expression of MMP-3, MMP-1, MMP-13, thrombospondin motifs (ADAMTS-4) and ADAMTS-5 was also studied in primary cultured chondrocytes of rats. Naringenin caused significant reduction in pain behavior and showed marked improvement in the tissue morphology of MIA rats. Moreover, a significant inhibition of MMP-3 expression in MIA rats was observed upon treatment with naringenin. In the in vitro tests, naringenin caused a significant reduction in the transcriptional expression, secretion and enzymatic activity of the studied degradative enzymes. The NF-κB pathway was also found to be inhibited upon treatment with naringenin in vitro. Overall, the study suggests that naringenin alleviated pain and regulated the production of matrix-metalloproteinases via regulation of NF-κB pathway. Thus, naringenin could be a potent therapeutic option for the treatment of osteoarthritis.

  15. Up-regulation of type II collagen gene by 17β-estradiol in articular chondrocytes involves Sp1/3, Sox-9, and estrogen receptor α.

    Science.gov (United States)

    Maneix, Laure; Servent, Aurélie; Porée, Benoît; Ollitrault, David; Branly, Thomas; Bigot, Nicolas; Boujrad, Noureddine; Flouriot, Gilles; Demoor, Magali; Boumediene, Karim; Moslemi, Safa; Galéra, Philippe

    2014-11-01

    The existence of a link between estrogen deprivation and osteoarthritis (OA) in postmenopausal women suggests that 17β-estradiol (17β-E2) may be a modulator of cartilage homeostasis. Here, we demonstrate that 17β-E2 stimulates, via its receptor human estrogen receptor α 66 (hERα66), type II collagen expression in differentiated and dedifferentiated (reflecting the OA phenotype) articular chondrocytes. Transactivation of type II collagen gene (COL2A1) by ligand-independent transactivation domain (AF-1) of hERα66 was mediated by "GC" binding sites of the -266/-63-bp promoter, through physical interactions between ERα, Sp1/Sp3, Sox9, and p300, as demonstrated in chromatin immunoprecipitation (ChIP) and Re-Chromatin Immuno-Precipitation (Re-ChIP) assays in primary and dedifferentiated cells. 17β-E2 and hERα66 increased the DNA-binding activities of Sp1/Sp3 and Sox-9 to both COL2A1 promoter and enhancer regions. Besides, Sp1, Sp3, and Sox-9 small interfering RNAs (siRNAs) prevented hERα66-induced transactivation of COL2A1, suggesting that these factors and their respective cis-regions are required for hERα66-mediated COL2A1 up-regulation. Our results highlight the genomic pathway by which 17β-E2 and hERα66 modulate Sp1/Sp3 heteromer binding activity and simultaneously participate in the recruitment of the essential factors Sox-9 and p300 involved respectively in the chondrocyte-differentiated status and COL2A1 transcriptional activation. These novel findings could therefore be attractive for tissue engineering of cartilage in OA, by the fact that 17β-E2 could promote chondrocyte redifferentiation. 17β-E2 up-regulates type II collagen gene expression in articular chondrocytes. An ERα66/Sp1/Sp3/Sox-9/p300 protein complex mediates this stimulatory effect. This heteromeric complex interacts and binds to Col2a1 promoter and enhancer in vivo. Our findings highlight a new regulatory mechanism for 17β-E2 action in chondrocytes. 17β-E2 might be an attractive

  16. Altered osmotic swelling behavior of proteoglycan-depleted bovine articular cartilage using high frequency ultrasound

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Q; Zheng, Y P; Leung, G; Mak, A F T [Department of Health Technology and Informatics, Hong Kong Polytechnic University, Kowloon, Hong Kong (China); Lam, W L; Guo, X [Department of Rehabilitation Sciences, Hong Kong Polytechnic University, Hong Kong (China); Lu, H B; Qin, L [Musculoskeletal Research Laboratory, Department of Orthopaedics and Traumatology, Chinese University of Hong Kong, Hong Kong (China)], E-mail: ypzheng@ieee.org

    2008-05-21

    Swelling behavior is an electrochemical mechanical property of articular cartilage. It plays an important role in weight bearing and joint lubrication. In this study, the altered transient and inhomogeneous swelling behavior of the degenerated articular cartilage was observed and quantified in situ using ultrasound. Three groups of bovine patellar articular cartilage samples (n = 10 x 3) were obtained and digested by trypsin for 10, 20 and 30 min respectively to mimic different levels of degeneration. The osmotic-free shrinkage and swelling behavior induced by changing the concentration of the bathing saline solution from 0.15 M to 2 M and then back to 0.15 M were characterized using high-frequency ultrasound (central frequency = 35 MHz) before and after digestion. It was found that the degenerated cartilage specimens showed a weaker shrinkage-swelling behavior compared with the normal cartilage samples. However, no significant differences in the peak shrinkage or swelling strains were observed between different groups. The absolute values of the peak shrinkage strain significantly (p < 0.05) decreased by 45.4%, 42.1% and 50.6% respectively after the trypsin digestion for 10, 20 and 30 min, but such significance was not demonstrated for the peak swelling strains. Due to the potential alterations in the collagen-PG matrix during trypsin digestion, the correlation between the swelling strain and the shrinkage strain of the degenerated samples changed slightly in comparison with the normal samples. The proposed ultrasound method has been successfully used to measure the transient and inhomogeneous swelling behavior of the degenerated articular cartilage and has the potential for the characterization of osteoarthritis.

  17. 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.

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

    Directory of Open Access Journals (Sweden)

    Lourdes Recha-Sancho

    2016-06-01

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

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

    Science.gov (United States)

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

    2010-01-01

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

  20. 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

  1. Senescence of chondrocytes in aging articular cartilage: GADD45β mediates p21 expression in association with C/EBPβ in senescence-accelerated mice.

    Science.gov (United States)

    Shimada, Hirofumi; Sakakima, Harutoshi; Tsuchimochi, Kaneyuki; Matsuda, Fumiyo; Komiya, Setsuro; Goldring, Mary B; Ijiri, Kosei

    2011-04-15

    Growth arrest and DNA damage-inducible protein 45β (GADD45β) is expressed in normal and early osteoarthritic articular cartilage. We recently reported that GADD45β enhances CCAAT/enhancer binding protein β (C/EBPβ) activation in vitro. This study was undertaken in order to determine whether GADD45β is expressed with C/EBPβ in aging articular cartilage. We also investigated whether the synergistic expression of GADD45β and C/EBPβ may be involved in the mechanism of chondrocyte senescence. Senescence-accelerated mice (SAMP1) were used as a model of aging. GADD45β, C/EBPβ, and p21 were analyzed by immunohistochemistry. A luciferase reporter assay using ATDC5 cells was performed in order to examine p21 as a target gene of the GADD45β/C/EBPβ cascade. GADD45β exhibited increased expression in the aging articular cartilage of SAMP1 mice compared to that in control mice. The co-localization of GADD45β and C/EBPβ was confirmed by double immunostaining. The synergistic mechanisms of GADD45β and C/EBPβ on the gene regulation of p21, a molecule related to cellular senescence, were verified by a p21-luciferase reporter assay. Co-expression of C/EBPβ and p21 was confirmed. These observations suggest that the synergism between GADD45β and C/EBPβ may play an important role in cellular senescence in the aging articular cartilage. Crown Copyright © 2011. Published by Elsevier GmbH. All rights reserved.

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

    Science.gov (United States)

    Pestka, Jan M; Schmal, Hagen; Salzmann, Gian; Hecky, Jochen; Südkamp, Norbert P; Niemeyer, Philipp

    2011-06-01

    Autologous chondrocyte implantation (ACI) is a well-established therapeutic option for the treatment of cartilage defects of the knee joint. Since information concerning the cellular aspects of ACI is still limited, the aim of the present study was to investigate relevant differences between chondrocyte quality after in vitro cultivation and possible correlations with patient-specific factors. 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. 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 rates of cartilage-specific markers when compared to older patients' chondrocytes. The present study identifies relevant differences concerning chondrocyte quality after in vitro expansion in a highly preselected study population of 252 patients that from a surgical point of view were eligible for ACI. With the exception of patients aged 20 years or younger, no patient-specific parameters could be identified which might allow anticipation of cell quality in individual patients.

  3. Effect of retinoic acid on proteoglycan turnover in bovine articular cartilage cultures

    Energy Technology Data Exchange (ETDEWEB)

    Campbell, M.A.; Handley, C.J.

    1987-10-01

    This paper describes proteoglycan catabolism by adult bovine articular cartilage treated with retinoic acid as a means of stimulating the loss of this macromolecule from the extracellular matrix of cartilage. Addition of retinoic acid (10(-12)-10(-6) M) to adult bovine articular cartilage which had been labeled with (/sup 35/S)sulfate for 6 h after 5 days in culture, resulted in a dose-dependent increase in the rate of loss of /sup 35/S-labeled proteoglycans from the matrix of the tissue. Concomitant with this loss was a decrease in the proteoglycan content of the tissue. Incubation of cultures treated with 1 microM retinoic acid, at 4 degrees C, or with 0.5 mM cycloheximide, resulted in a significant decrease in the rate of retinoic acid-induced loss of proteoglycans and demonstrated cellular involvement in this process. Analysis of the /sup 35/S-labeled proteoglycans remaining in the matrix showed that the percentage of radioactivity associated with the small proteoglycan species extracted from the matrix of articular cartilage explants labeled with (/sup 35/S)sulfate after 5 days in culture was 15% and this increased to 22% in tissue maintained in medium alone. In tissue treated with 1 microM retinoic acid for 6 days, the percentage of radioactivity associated with the small proteoglycan was 58%. Approximately 93% of the /sup 35/S-labeled proteoglycans released into the medium of control and retinoic acid-treated cultures was recovered in high density fractions after CsCl gradient centrifugation and eluted on Sepharose CL-2B as a broad peak with a Kav of 0.30-0.37. Less than 17% of these proteoglycans was capable of aggregating with hyaluronate. These results indicate that in both control and retinoic acid-treated cultures the larger proteoglycan species is lost to the medium at a greater rate than the small proteoglycan species. The effect of retinoic acid on proteoglycan turnover was shown to be reversible.

  4. 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

    OBJECTIVE: Autologous chondrocyte implantation (ACI) is a well-established therapeutic option for the treatment of cartilage defects of the knee joint. Since information concerning the cellular aspects of ACI is still limited, the aim of the present study was to investigate relevant differences b...

  5. Achyranthes bidentate saponins protect rat articular chondrocytes against interleukin-1β-induced inflammation and apoptosis in vitro

    Directory of Open Access Journals (Sweden)

    Xian-Xiang Xu

    2017-02-01

    Full Text Available Achyranthes bidentate Blume (Niuxi is often employed for treatment of arthritis in Traditional Chinese Medicine and possesses anti-inflammatory properties. Phytochemical and pharmacological studies proved the oleanane-type saponins to be the main bioactive principles. In the present study, protective effects of A. bidentata saponins (ABS on inflammation and apoptosis in interleukine-1β (IL-1β-induced chondrocytes were investigated. Rat chondrocytes were pretreated with ABS at 3 μg/mL, 10 μg/mL, and 30 μg/mL, and subsequently stimulated with IL-1β (10 ng/mL. Methylthiazolyldiphenyl-tetrazolium bromide assay and annexin V/propidium iodide dual staining demonstrated that ABS could protect IL-1β-induced chondrocyte injury. ABS suppressed IL-1β-induced apoptosis by suppressing the activation of caspase-3, inhibiting levels of proapoptotic proteins Bax and Bad, decreasing p53 protein phosphorylation, and promoting the expression of antiapoptotic protein Bcl-xL and proliferating cell nuclear antigen. IL-1β-induced inflammation and matrix degradation were also alleviated by ABS through the downregulation of the expressions of matrix metalloproteinases 3 and 9 and cyclooxygenase-2. Moreover, ABS inhibited IL-1β-induced nuclear factor κB activation in rat chondrocytes. We demonstrated, for the first time, the protective effects of ABS on IL-1β-stimulated chondrocytes and their molecular mechanisms. Thus, it is suggested that ABS might be a potential drug in the treatment of osteoarthritis.

  6. Measuring clinically relevant endpoints in a serum-free, three-dimensional, primary cell culture system of human osteoarthritic articular chondrocytes.

    Science.gov (United States)

    Bundens, Grace; Buckley, Andrea; Milton, LaBraya; Behling, Kathryn; Chmielewski, Sarah; Cho, Ellen; Lozano-Torres, Xiomara; Selim, Abdulhafez; Lackman, Richard; George-Weinstein, Mindy; Miller, Lawrence; D'Angelo, Marina

    2017-08-15

    Osteoarthritis (OA) is characterized by degeneration of articular cartilage within the joint, inflammation and pain. The purpose of this study was to develop a primary, serum free cell culture system of human osteoarthritic articular chondrocytes (HOACs) with which to study manifestations of the disease process. Joint tissues were obtained from OA patients undergoing total knee arthroplasty (TKA). HOACs isolated from the femoral condyles and tibial plateau of the same side were combined, plated in three-dimensional, alginate beads and cultured for five days in serum, hormone and protein free medium. More living cells were obtained from the femoral condyles than the tibial plateau. The optimal plating density was 2.5 × 106 cells/ml of alginate. The amounts of DNA, RNA, proteoglycans and total collagen were similar in cultures prepared from the sides of least and greatest pathology. More type 1 than type 2 collagen was detected in the medium on days 2 and 5. A greater percentage of type 1 than type 2 collagen was degraded. The inflammatory cytokine interleukin-1 beta was present in the medium and alginate associated matrix. Although variation in the metabolic profiles between subjects was observed, HOACs from all patients continued to reflect the OA phenotype for five days in culture. This serum free, three-dimensional primary culture system of HOACs provides a platform with which to measure clinically relevant endpoints of OA and screen potential disease modifying OA therapeutics. Copyright © 2017 Elsevier Inc. All rights reserved.

  7. 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

  8. Influence of 1α, 25-dihydroxyvitamin D3 [1, 25(OH)2D3] on the expression of Sox 9 and the transient receptor potential vanilloid 5/6 ion channels in equine articular chondrocytes.

    Science.gov (United States)

    Hdud, Ismail M; Loughna, Paul T

    2014-01-01

    Sox 9 is a major marker of chondrocyte differentiation. When chondrocytes are cultured in vitro they progressively de-differentiate and this is associated with a decline in Sox 9 expression. The active form of vitamin D, 1, 25 (OH)2D3 has been shown to be protective of cartilage in both humans and animals. In this study equine articular chondrocytes were grown in culture and the effects of 1, 25 (OH)2D3 upon Sox 9 expression examined. The expression of the transient receptor potential vanilloid (TRPV) ion channels 5 and 6 in equine chondrocytes in vitro, we have previously shown, is inversely correlated with de-differentiation. The expression of these channels in response to 1, 25 (OH)2D3 administration was therefore also examined. The active form of vitamin D (1, 25 (OH)2D3) when administered to cultured equine chondrocytes at two different concentrations significantly increased the expression of Sox 9 at both. In contrast 1, 25 (OH)2D3 had no significant effect upon the expression of either TRPV 5 or 6 at either the protein or the mRNA level. The increased expression of Sox 9, in equine articular chondrocytes in vitro, in response to the active form of vitamin D suggests that this compound could be utilized to inhibit the progressive de-differentiation that is normally observed in these cells. It is also supportive of previous studies indicating that 1α, 25-dihydroxyvitamin D3 can have a protective effect upon cartilage in animals in vivo. The previously observed correlation between the degree of differentiation and the expression levels of TRPV 5/6 had suggested that these ion channels may have a direct involvement in, or be modulated by, the differentiation process in vitro. The data in the present study do not support this.

  9. S100B + A1 CELISA: A Novel Potency Assay and Screening Tool for Redifferentiation Stimuli of Human Articular Chondrocytes.

    Science.gov (United States)

    Diaz-Romero, Jose; Kürsener, Sibylle; Kohl, Sandro; Nesic, Dobrila

    2017-06-01

    During monolayer expansion, a necessary step in autologous chondrocyte implantation, human articular chondrocytes (HAC) dedifferentiate and lose their capacity to produce stable hyaline cartilage. Determining HAC potency and learning how to trigger their redifferentiation would improve cell-based cartilage regeneration therapies. We previously identified S100B and S100A1 proteins as markers of HAC redifferentiation potential. Here, we aimed to: (i) demonstrate a correlation between S100B + A1-positive HAC in monolayer culture and their neochondrogenesis capacity in pellet culture; (ii) develop an S100B + A1 cell-based ELISA, and (iii) prove that S100B + A1 induction in HAC increases their chondrogenic capacity. Expression patterns of S100A1 and S100B were investigated in HAC during dedifferentiation (monolayer) or redifferentiation (pellet or high-osmolarity/BMP4 treatment in monolayer) using qRT-PCR, immunocytochemistry, or immunohistochemistry. A cell-based ELISA (CELISA) was developed as a 96-well microplate multiplex assay to measure S100B + A1 (chondrogenesis), alkaline phosphatase (hypertrophy), and DNA amount (normalization), and applied to HAC, bone marrow-derived mesenchymal stem cells and the chondrocytic cell line ATDC5. The direct correlation between the percentage of S100B + A1-positive HAC in monolayer and their neochondrogenesis in pellets validates S100B + A1 as a marker of chondrogenic potency. The S100B + A1-CELISA accurately determines HAC differentiation status, allows identification of chondrogenic stimuli, and permits the simultaneous monitoring of the undesirable hypertrophic phenotype. This novel assay offers a high-throughput, comprehensive and versatile approach for measuring cell chondrogenic potency and for identifying redifferentiation factors/conditions. HAC improved neochondrogenesis in pellets-induced with high-osmolarity and BMP4 treatment in monolayer-suggests that cell instruction prior to implantation

  10. Laser radiation effect on chondrocytes and intercellular matrix of costal and articular cartilage impregnated with magnetite nanoparticles.

    Science.gov (United States)

    Soshnikova, Yulia M; Shekhter, Anatoly B; Baum, Olga I; Shcherbakov, Evgeniy M; Omelchenko, Alexander I; Lunin, Valeriy V; Sobol, Emil N

    2015-03-01

    Magnetic nanoparticles with the ability to absorb laser radiation are the perspective agents for the early diagnostics and laser therapy of degenerative cartilage. The effect of starch stabilized magnetite nanoparticles (SSNPs) on the cartilage structure components has never been studied before. The aim of the work is to establish the Erbium:glass laser effect on costal and articular cartilage impregnated with SSNPs. Porcine articular and costal cartilage disks (2.0 mm in diameter and 1.5-2 mm in thickness) were impregnated with SSNPs and irradiated using a 1.56 μm laser in therapeutic laser setting. The one sample group underwent the second irradiation after the SSNPs impregnation. The samples were analyzed by the means of histology, histochemistry and transmission electron microscopy (TEM) to reveal the alterations of cells, glycosaminoglycans and collagen network. The irradiated cartilage demonstrates the higher content of cell alterations than the intact one due to the heat and mechanical affection in the course of laser irradiation. However the alterations are localized at the areas near the irradiated surfaces and not dramatic. The impregnation of SSNPs does not cause any additional cell alterations. For both costal and articular cartilage the matrix alterations of irradiated samples are not critical: there is the slight decrease in acid proteoglycan content at the irradiated areas while the collagen network is not altered. Distribution and localization of impregnated SSNPs is described: agglomerates of 150-230 nm are observed located at the borders between matrix and cell lacunas of articular cartilage; SSNPs of 15-45 nm are found in the collagen network of costal cartilage. It was shown that SSNPs do not appreciably affect the structural components of both articular and costal cartilage and can be safely used for the laser diagnostics and therapy. The area of structural alterations is diffuse and local as the result of the mechanical and heat

  11. 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.

  12. Calcium Input Potentiates the Transforming Growth Factor (TGF)-β1-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

    2011-01-01

    Transforming growth factor (TGF)-β1 stimulates extracellular PPi (ePPi) 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 Ca2+-dependent protein kinase C (PKCα). Thus, we investigated mechanisms by which calcium could affect ePPi metabolism, especially its main regulating proteins ANK and PC-1 (plasma cell membrane glycoprotein-1). We stimulated articular chondrocytes with TGF-β1 under extracellular (eCa2+) or cytosolic Ca2+ (cCa2+) modulations. We studied ANK, PC-1 expression (quantitative RT-PCR, Western blotting), ePPi levels (radiometric assay), and cCa2+ input (fluorescent probe). Voltage-operated Ca2+-channels (VOC) and signaling pathways involved were investigated with selective inhibitors. Finally, Ank promoter activity was evaluated (gene reporter). TGF-β1 elevated cCa2+ and ePPi levels (by up-regulating Ank and PC-1 mRNA/proteins) in an eCa2+ dose-dependent manner. TGF-β1 effects were suppressed by cCa2+ 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 Ca2+. 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 eCa2+ through opening of L- and T-VOCs, to potentiate ERK1/2 and PKCα signaling cascades, resulting in an enhanced activation of Ank promoter and ePPi production in chondrocyte. PMID:21471198

  13. 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.

  14. Young adult chondrocytes proliferate rapidly and produce a cartilaginous tissue at the gel-media interface in agarose cultures.

    Science.gov (United States)

    Tran-Khanh, Nicolas; Chevrier, Anik; Lascau-Coman, Viorica; Hoemann, Caroline D; Buschmann, Michael D

    2010-06-01

    Primary chondrocytes cultured in agarose can escape the gel, accumulate at the interface between agarose and the culture medium, and form an outgrowing tissue. These outgrowths can appear as voluminous cartilage-like nodules that have never been previously investigated. In the present study, bovine articular chondrocytes from three age groups (fetal, young adult, aged) were seeded and cultured in agarose to test the hypothesis that hyaline-like cartilage outgrowths develop at the interface by appositional growth, in an age-dependant manner. Macroscopic appearance, cell content, cell division, cytoskeletal morphology, and extracellular matrix (ECM) composition were analyzed. Fetal chondrocytes produced a fibrous interfacial tissue while aged chondrocytes produced ECM-poor cell clusters. In contrast young adult chondrocytes produced large cartilaginous outgrowths, rich in proteoglycan and collagen II, where cells in the central region displayed a chondrocyte morphology. Cell proliferation was confined to the peripheral edge of these outgrowths, where elongated cell morphology, cell-cell contacts, and cell extensions toward the culture medium were seen. Thus these voluminous cartilaginous outgrowths formed in an appositional growth process and only for donor chondrocytes from young adult animals. This system offers an interesting ability to proliferate chondrocytes in a manner that results in a chondrocyte morphology and a cartilaginous ECM in central regions of the outgrowing tissue. It also provides an in vitro model system to study neocartilage appositional growth.

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

    Science.gov (United States)

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

    2015-07-01

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

  16. 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.

  17. 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 gels. FCS exacerbated changes to density (P gel strength. Reduced gel 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. © 2016 Wiley Periodicals, Inc.

  18. chondrocytes for transplantation?

    African Journals Online (AJOL)

    condyle (usually from the posterior cuts) were also enzymatically digested as for the cartilage from the ... articular cartilage defects, they may provide an alternative source of chondrocytes for transplantation in cases .... collagene and anti-mouse HRP conjugated secondary antibody was used for type II collagen. Results.

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

    Directory of Open Access Journals (Sweden)

    Hee-Jeong Im

    2009-10-01

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

  20. 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.

  1. 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.

    Science.gov (United States)

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

    2012-01-01

    The aim of this study was to characterize the anti-inflammatory mode of action of botanical extracts from rosehip (Rosa canina), willow bark (Salix alba), and nettle leaf (Urtica dioica) in an in vitro model of primary canine articular chondrocytes. Methods. The biological effects of the botanical extracts were studied in chondrocytes treated with IL-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.

  2. 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

    Science.gov (United States)

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

    2012-01-01

    The aim of this study was to characterize the anti-inflammatory mode of action of botanical extracts from rosehip (Rosa canina), willow bark (Salix alba), and nettle leaf (Urtica dioica) in an in vitro model of primary canine articular chondrocytes. Methods. The biological effects of the botanical extracts were studied in chondrocytes treated with IL-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. PMID:22474508

  3. 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

  4. Chondrocyte Apoptosis in the Pathogenesis of Osteoarthritis

    Science.gov (United States)

    Hwang, Hyun Sook; Kim, Hyun Ah

    2015-01-01

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

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

    Science.gov (United States)

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

    2016-05-01

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

  6. Adipose-Derived Stem Cells Cocultured with Chondrocytes Promote the Proliferation of Chondrocytes

    Directory of Open Access Journals (Sweden)

    Jie Shi

    2017-01-01

    Full Text Available Articular cartilage injury and defect caused by trauma and chronic osteoarthritis vascularity are very common, while the repair of injured cartilage remains a great challenge due to its limited healing capacity. Stem cell-based tissue engineering provides a promising treatment option for injured articular cartilage because of the cells potential for multiple differentiations. However, its application has been largely limited by stem cell type, number, source, proliferation, and differentiation. We hypothesized that (1 adipose-derived stem cells are ideal seed cells for articular cartilage repair because of their accessibility and abundance and (2 the microenvironment of articular cartilage could induce adipose-derived stem cells (ADSCs to differentiate into chondrocytes. In order to test our hypotheses, we isolated stem cells from rabbit adipose tissues and cocultured these ADSCs with rabbit articular cartilage chondrocytes. We found that when ADSCs were cocultured with chondrocytes, the proliferation of articular cartilage chondrocytes was promoted, the apoptosis of chondrocytes was inhibited, and the osteogenic and chondrogenic differentiation of ADSCs was enhanced. The study on the mechanism of this coculture system indicated that the role of this coculture system is similar to the function of TGF-β1 in the promotion of chondrocytes.

  7. Bone morphogenetic proteins and articular cartilage: To serve and protect or a wolf in sheep clothing's?

    NARCIS (Netherlands)

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

    2010-01-01

    OBJECTIVE: Alterations in chondrocyte differentiation and matrix remodeling play a central role in osteoarthritis (OA). Chondrocyte differentiation and remodeling are amongst others regulated by the so-called Bone Morphogenetic Proteins (BMPs). Although BMPs are considered protective for articular

  8. The repair of full-thickness articular cartilage defects. Immune responses to reparative tissue formed by allogeneic growth plate chondrocyte implants

    Energy Technology Data Exchange (ETDEWEB)

    Kawabe, N.; Yoshinao, M. (Department of Orthopaedics, Shimane Medical School, Izumo (Japan))

    1991-07-01

    Growth plate cartilage cultivated in vitro was attached with a fibrin clot to a full-thickness articular cartilage defect on knee joints in allogeneic New Zealand rabbits. The healing of the defects was assessed by gross examination, light microscopy, and immunologic analysis for 24 weeks. Immunologic assessment of cell-mediated immunity, cytotoxicity of a humoral antibody by a 51 chromium release assay, and immunofluorescence studies were carried out. During the first two weeks following grafting, healing was excellent in 11 of the 17 defects. From three to 24 weeks, 11 of 42 defects examined had good results. Host lymphocytes had accumulated around the allograft at two to 12 weeks. Most of the implanted cartilage grown in vitro died and was replaced by fibrous tissue. The immunologic studies suggested that the implanted cartilage began to degenerate two to three weeks after implantation partially because of a humoral immune response but more importantly because of cell-mediated cytotoxicity.

  9. Articular cartilage generation applying PEG-LA-DM/PEGDM copolymer hydrogels

    National Research Council Canada - National Science Library

    Zhao, Xing; Papadopoulos, Anestis; Ibusuki, Shinichi; Bichara, David A; Saris, Daniel B; Malda, J; Anseth, Kristi S; Gill, Thomas J; Randolph, Mark A

    2016-01-01

    ...) production for neocartilage formation. In this study, we demonstrated the feasibility of neocartilage regeneration using swine articular chondrocytes photoencapsualted into poly (ethylene glycol) dimethacrylate (PEGDM...

  10. Can microcarrier-expanded chondrocytes synthesize cartilaginous tissue in vitro?

    Science.gov (United States)

    Surrao, Denver C; Khan, Aasma A; McGregor, Aaron J; Amsden, Brian G; Waldman, Stephen D

    2011-08-01

    Tissue engineering is a promising approach for articular cartilage repair; however, it is challenging to produce adequate amounts of tissue in vitro from the limited number of cells that can be extracted from an individual. Relatively few cell expansion methods exist without the problems of de-differentiation and/or loss of potency. Recently, however, several studies have noted the benefits of three-dimensional (3D) over monolayer expansion, but the ability of 3D expanded chondrocytes to synthesize cartilaginous tissue constructs has not been demonstrated. Thus, the purpose of this study was to compare the properties of engineered cartilage constructs from expanded cells (monolayer and 3D microcarriers) to those developed from primary chondrocytes. Isolated bovine chondrocytes were grown for 3 weeks in either monolayer (T-Flasks) or 3D microcarrier (Cytodex 3) expansion culture. Expanded and isolated primary cells were then seeded in high density culture on Millicell™ filters for 4 weeks to evaluate the ability to synthesize cartilaginous tissue. While microcarrier expansion was twice as effective as monolayer expansion (microcarrier: 110-fold increase, monolayer: 52-fold increase), the expanded cells (monolayer and 3D microcarrier) were not effectively able to synthesize cartilaginous tissue in vitro. Tissues developed from primary cells were substantially thicker and accumulated significantly more extracellular matrix (proteoglycan content: 156%-292% increase; collagen content: 70%-191% increase). These results were attributed to phenotypic changes experienced during the expansion phase. Monolayer expanded chondrocytes lost their native morphology within 1 week, whereas microcarrier-expanded cells were spreading by 3 weeks of expansion. While the use of 3D microcarriers can lead to large cellular yields, preservation of chondrogenic phenotype during expansion is required in order to synthesize cartilaginous tissue.

  11. Effect of partial H2O-D2O replacement on the anisotropy of transverse proton spin relaxation in bovine articular cartilage.

    Directory of Open Access Journals (Sweden)

    Sirisha Tadimalla

    Full Text Available Anisotropy of transverse proton spin relaxation in collagen-rich tissues like cartilage and tendon is a well-known phenomenon that manifests itself as the "magic-angle" effect in magnetic resonance images of these tissues. It is usually attributed to the non-zero averaging of intra-molecular dipolar interactions in water molecules bound to oriented collagen fibers. One way to manipulate the contributions of these interactions to spin relaxation is by partially replacing the water in the cartilage sample with deuterium oxide. It is known that dipolar interactions in deuterated solutions are weaker, resulting in a decrease in proton relaxation rates. In this work, we investigate the effects of deuteration on the longitudinal and the isotropic and anisotropic contributions to transverse relaxation of water protons in bovine articular cartilage. We demonstrate that the anisotropy of transverse proton spin relaxation in articular cartilage is independent of the degree of deuteration, bringing into question some of the assumptions currently held over the origins of relaxation anisotropy in oriented tissues.

  12. 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.

  13. Noninvasive assessment of articular cartilage surface damage using reflected polarized light microscopy

    Science.gov (United States)

    Huynh, Ruby N.; Nehmetallah, George; Raub, Christopher B.

    2017-06-01

    Articular surface damage occurs to cartilage during normal aging, osteoarthritis, and in trauma. A noninvasive assessment of cartilage microstructural alterations is useful for studies involving cartilage explants. This study evaluates polarized reflectance microscopy as a tool to assess surface damage to cartilage explants caused by mechanical scraping and enzymatic degradation. Adult bovine articular cartilage explants were scraped, incubated in collagenase, or underwent scrape and collagenase treatments. In an additional experiment, cartilage explants were subject to scrapes at graduated levels of severity. Polarized reflectance parameters were compared with India ink surface staining, features of histological sections, changes in explant wet weight and thickness, and chondrocyte viability. The polarized reflectance signal was sensitive to surface scrape damage and revealed individual scrape features consistent with India ink marks. Following surface treatments, the reflectance contrast parameter was elevated and correlated with image area fraction of India ink. After extensive scraping, polarized reflectance contrast and chondrocyte viability were lower than that from untreated explants. As part of this work, a mathematical model was developed and confirmed the trend in the reflectance signal due to changes in surface scattering and subsurface birefringence. These results demonstrate the effectiveness of polarized reflectance microscopy to sensitively assess surface microstructural alterations in articular cartilage explants.

  14. Gene Expression Profiling Reveals Similarities between the Spatial Architectures of Postnatal Articular and Growth Plate Cartilage: e103061

    National Research Council Canada - National Science Library

    Michael Chau; Julian C Lui; Ellie B M Landman; Stephan-Stanislaw Späth; Andrea Vortkamp; Jeffrey Baron; Ola Nilsson

    2014-01-01

      Articular and growth plate cartilage are discrete tissues but arise from a common cartilaginous condensation and have comparable spatial architectures consisting of distinct layers of chondrocytes...

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

    NARCIS (Netherlands)

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

    2001-01-01

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

  16. The chondrocyte: biology and clinical application.

    Science.gov (United States)

    Lin, Zhen; Willers, Craig; Xu, Jiake; Zheng, Ming-Hao

    2006-07-01

    Chondrocyte is a unique cell type in articular cartilage tissue and is essential for cartilage formation and functionality. It arises from mesenchymal stem cells (MSCs) and is regulated by a series of cytokine and transcription factor interactions, including the transforming growth factor-beta super family, fibroblast growth factors, and insulin-like growth factor-1. To understand the biomechanisms of the chondrocyte differentiation process, various cellular model systems have been employed, such as primary chondrocyte culture, clonal normal cell lines (HCS-2/8, Ch-1, ATDC5, CFK-2, and RCJ3.1C5.18), and transformed clonal cell lines (T/C-28a2, T/C-28a4, C-28/I2, tsT/AC62, and HPV-16 E6/E7). Additionally, cell culture methods, including conventional monolayer culture, three-dimensional scaffold culture, bioreactor culture, pellet culture, and organ culture, have been established to create stable environments for the expansion, phenotypic maintenance, and subsequent biological study of chondrocytes for clinical application. Knowledge gained through these study systems has allowed for the use of chondrocytes in orthopedics for the treatment of cartilage injury and epiphyseal growth plate defects using tissue-engineering approaches. Furthermore, the potential of chondrocyte implantation for facial reconstruction, the treatment of long segmental tracheal defects, and urinary incontinence and vesicoureteral reflux are being investigated. This review summarizes the present study of chondrocyte biology and the potential uses of this cell in orthopedics and other disciplines.

  17. [Toxicity of antiseptics on chondrocytes in vitro].

    Science.gov (United States)

    Schaumburger, J; Beckmann, J; Springorum, H-R; Handel, M; Anders, S; Kalteis, T; Grifka, J; Rath, B

    2010-01-01

    Local antiseptics are commonly used for perioperative skin and wound disinfection and as solutions for joint lavage. Therefore, we examined if an intra-articular use of these antiseptics is possible by using an IN VITRO chondrocyte model. Articular chondrocytes harvested from 7 patients were cultured. After reaching 80% confluency different concentrations (0%, 1%, 10%, 50%, 100%) of polyhexanide, hydrogen peroxide and povidone-iodine were added for 5 minutes. Afterwards, the solution was removed and the chondrocytes were cultured for 24 hours. Subsequently the vitality and proliferation rate (DNA synthesis) were analysed with the WST-1 and BrdU tests. 1% povidone-iodine and 1% hydrogen peroxide solutions significantly (p=0.001) decreased the chondrocyte vitality as compared to our control group. There was no significant difference (p=0.71) after the application of 1% polyhexanide in the vitality ratios. A significant decrease in vitality was also observed after the application of 10% polyhexanide solution (p=0.001). Application of 1% povidone-iodine solution, 1% hydrogen peroxide solution and 10% polyhexanide revealed a decrease in the metabolic cell activity of 80% compared to our control group, whereas the activity was 65% (p=0.026) compared to the control group after application of 1% polyhexanide solution. Our results demonstrate the chondrotoxic effect of the tested antiseptic solutions in clinical used concentrations within short time points. Polyhexanide in a low concentrated solution (1%) was the antiseptic with the lowest influence on the vitality and the DNA synthesis of chondrocytes. Thus, this antiseptic solution seemed to be the best choice for intra-articular application. But overall, our study showed general limitations for the intra-articular use of local antiseptics. Copyright (c) Georg Thieme Verlag KG Stuttgart-New York.

  18. bovine

    African Journals Online (AJOL)

    of various breeds under local conditions of management. (Hale, 1974b). AdditionaIly, this procedure has been used to assess the production of LH by the bovine anterior pituitary in vitro and to study the relationships between this production and the activity of the pineal- hypothalamic axis (Hayes, Knight & Symington, 1974;.

  19. MR imaging of autologous chondrocyte implantation of the knee

    Energy Technology Data Exchange (ETDEWEB)

    James, S.L.J.; Connell, D.A.; Saifuddin, A.; Skinner, J.A.; Briggs, T.W.R. [RNOH Stanmore, Department of Radiology, Stanmore, Middlesex (United Kingdom)

    2006-05-15

    Autologous chondrocyte implantation (ACI) is a surgical technique that is increasingly being used in the treatment of full-thickness defects of articular cartilage in the knee. It involves the arthroscopic harvesting and in vitro culture of chondrocytes that are subsequently implanted into a previously identified chondral defect. The aim is to produce a repair tissue that closely resembles hyaline articular cartilage that gradually becomes incorporated, restoring joint congruity. Over the long term, it is hoped that this will prevent the progression of full-thickness articular cartilage defects to osteoarthritis. This article reviews the indications and operative procedure performed in ACI. Magnetic resonance imaging (MRI) sequences that provide optimal visualization of articular cartilage in the post-operative period are discussed. Normal appearances of ACI on MRI are presented along with common complications that are encountered with this technique. (orig.)

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

    Science.gov (United States)

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

    2017-02-01

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

  1. Sodium nitroprusside induces apoptosis of rabbit chondrocytes

    Science.gov (United States)

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

    2013-02-01

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

  2. Importance of Donor Chondrocyte Viability for Osteochondral Allografts.

    Science.gov (United States)

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

    2016-05-01

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

  3. The effect of matrix stiffness on biomechanical properties of chondrocytes.

    Science.gov (United States)

    Zhang, Quanyou; Yu, Yang; Zhao, Hucheng

    2016-10-01

    The behavior of chondrocytes is regulated by multiple mechanical microenvironmental cues. During development and degenerative disease of articular cartilage, as an external signal, the extracellular matrix stiffness of chondrocytes changes significantly, but whether and how this biophysical cue affects biomechanical properties of chondrocytes remain elusive. In the present study, we designed supporting-biomaterials as  mimics of native pericellular matrix to study the effect of matrix stiffness on chondrocyte morphology and F-actin distribution. Furthermore, the active mechanical behavior of chondrocytes during sensing and responding to different matrix stiffness was quantitatively investigated using atom force microscope technique and theoretical model. Our results indicated that stiffer matrix tends to increase the cell spreading area, the percentage of irregular cell shape distribution and mechanical parameters including elastic modulus (Eelastic), instantaneous modulus (E0), relaxed modulus (ER) and apparent viscosity (μ) of chondrocytes. Knowledge of matrix stiffness-dependent biomechanical behaviors of chondrocytes has important implications for optimizing matrix material and advancing chondrocyte-based applications for functional tissue engineering. © The Author 2016. Published by Oxford University Press on behalf of the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  4. 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

  5. Dynamic Mechanical Compression of Chondrocytes for Tissue Engineering: A Critical Review

    Directory of Open Access Journals (Sweden)

    Devon E. Anderson

    2017-12-01

    Full Text Available Articular cartilage functions to transmit and translate loads. In a classical structure–function relationship, the tissue resides in a dynamic mechanical environment that drives the formation of a highly organized tissue architecture suited to its biomechanical role. The dynamic mechanical environment includes multiaxial compressive and shear strains as well as hydrostatic and osmotic pressures. As the mechanical environment is known to modulate cell fate and influence tissue development toward a defined architecture in situ, dynamic mechanical loading has been hypothesized to induce the structure–function relationship during attempts at in vitro regeneration of articular cartilage. Researchers have designed increasingly sophisticated bioreactors with dynamic mechanical regimes, but the response of chondrocytes to dynamic compression and shear loading remains poorly characterized due to wide variation in study design, system variables, and outcome measurements. We assessed the literature pertaining to the use of dynamic compressive bioreactors for in vitro generation of cartilaginous tissue from primary and expanded chondrocytes. We used specific search terms to identify relevant publications from the PubMed database and manually sorted the data. It was very challenging to find consensus between studies because of species, age, cell source, and culture differences, coupled with the many loading regimes and the types of analyses used. Early studies that evaluated the response of primary bovine chondrocytes within hydrogels, and that employed dynamic single-axis compression with physiologic loading parameters, reported consistently favorable responses at the tissue level, with upregulation of biochemical synthesis and biomechanical properties. However, they rarely assessed the cellular response with gene expression or mechanotransduction pathway analyses. Later studies that employed increasingly sophisticated biomaterial-based systems, cells

  6. Dynamic Mechanical Compression of Chondrocytes for Tissue Engineering: A Critical Review.

    Science.gov (United States)

    Anderson, Devon E; Johnstone, Brian

    2017-01-01

    Articular cartilage functions to transmit and translate loads. In a classical structure-function relationship, the tissue resides in a dynamic mechanical environment that drives the formation of a highly organized tissue architecture suited to its biomechanical role. The dynamic mechanical environment includes multiaxial compressive and shear strains as well as hydrostatic and osmotic pressures. As the mechanical environment is known to modulate cell fate and influence tissue development toward a defined architecture in situ, dynamic mechanical loading has been hypothesized to induce the structure-function relationship during attempts at in vitro regeneration of articular cartilage. Researchers have designed increasingly sophisticated bioreactors with dynamic mechanical regimes, but the response of chondrocytes to dynamic compression and shear loading remains poorly characterized due to wide variation in study design, system variables, and outcome measurements. We assessed the literature pertaining to the use of dynamic compressive bioreactors for in vitro generation of cartilaginous tissue from primary and expanded chondrocytes. We used specific search terms to identify relevant publications from the PubMed database and manually sorted the data. It was very challenging to find consensus between studies because of species, age, cell source, and culture differences, coupled with the many loading regimes and the types of analyses used. Early studies that evaluated the response of primary bovine chondrocytes within hydrogels, and that employed dynamic single-axis compression with physiologic loading parameters, reported consistently favorable responses at the tissue level, with upregulation of biochemical synthesis and biomechanical properties. However, they rarely assessed the cellular response with gene expression or mechanotransduction pathway analyses. Later studies that employed increasingly sophisticated biomaterial-based systems, cells derived from different

  7. Articular cartilage bioreactors and bioprocesses.

    Science.gov (United States)

    Darling, Eric M; Athanasiou, Kyriacos A

    2003-02-01

    This review summarizes the major approaches for developing articular cartilage, using bioreactors and mechanical stimuli. Cartilage cells live in an environment heavily influenced by mechanical forces. The development of cartilaginous tissue is dependent on the environment that surrounds it, both in vivo and in vitro. Chondrocytes must be cultured in a way that gives them the proper concentration of nutrients and oxygen while removing wastes. A mechanical force must also be applied during the culturing process to produce a phenotypically correct tissue. Four main types of forces are currently used in cartilage-culturing processes: hydrostatic pressure, direct compression, "high"-shear fluid environments, and "low"-shear fluid environments. All these forces have been integrated into culturing devices that serve as bioreactors for articular cartilage. The strengths and weaknesses of each device and stimulus are explored, as is the future of cartilage bioreactors.

  8. Biomechanical evaluation of suture holding properties of native and tissue engineered articular cartilage

    Science.gov (United States)

    DuRaine, GD; Arzi, B; Lee, JK; Lee, CA; Responte, DJ; Hu, JC; Athanasiou, KA

    2014-01-01

    Objective The purpose of this study was to determine suture-holding properties of tissue engineered neocartilage relative to native articular cartilage. To this end, suture pull-out strength was quantified for native articular cartilage and for neocartilages possessing various mechanical properties. Methods Suture holding properties were examined in vitro and in vivo. Neocartilage from bovine chondrocytes was engineered using two sets of exogenous stimuli resulting in neotissue of different biochemical compositions. Compressive and tensile properties and glycosaminoglycan, collagen, and pyridinoline cross-link contents were assayed (study 1). Suture pull-out strength was compared between neocartilage constructs, and bovine and leporine native cartilage. Uniaxial pull-out test until failure was performed after passing 6-0 Vicryl through each tissue (study 2). Subsequently, neocartilage was implanted into a rabbit model to examine short-term suture holding ability in vivo (study 3). Results Neocartilage glycosaminoglycan and collagen content per wet weight reached 4.55% ± 1.62% and 4.21 ± 0.77%, respectively. Tensile properties for neocartilage constructs reached 2.6 ± 0.77 MPa for Young’s modulus and 1.39 ± 0.63 MPa for ultimate tensile strength. Neocartilage reached ~33% of suture pull-out strength of native articular cartilage. Neocartilage cross-link content reached 50% of native values, and suture pull-out strength correlated positively with cross-link content (R2=0.74). Neocartilage sutured into rabbit osteochondral defects was successfully maintained for 3 weeks. Conclusion This study shows that pyridinoline cross-links in neocartilage may be vital in controlling suture pull-out strength. Neocartilage produced in vitro with one-third of native tissue pull-out strength appears sufficient for construct suturing and retention in vivo. PMID:24848644

  9. 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

  10. [Influence of BMP-7 on chondrocyte secretion and expression of Col-II,AGG and Sox9 mRNA in porous tantalum-chondrocyte composites in vitro].

    Science.gov (United States)

    Zhang, H; Li, L; Wang, Q; Gan, H Q; Wang, H; Bi, C; Li, Q J; Wang, Z Q

    2015-04-18

    To study the influence of bone morphogenetic protein-7 (BMP-7) on chondrocyte secretion and expression of type II collagen (Col-II), aggrecan (AGG) and SRY-related high mobility group-box gene 9 (Sox9) mRNA in porous tantalum-chondrocyte composites. The articular chondrocytes were isolated from 3-week-old New Zealand immature rabbits and identified. The 2nd generation of chondrocytes with 1×10(6)/mL inoculate concentration was seeded in porous tantalum and divided into 4 groups, and control group (tantalum/chondrocyte), 50 μg/L BMP-7 group (50 μg/L BMP-7/tantalum/chondrocyte), 100 μg/L BMP-7 group (100 μg/L BMP-7/tantalum/chondrocyte), and 200 μg/L BMP-7 group (200 μg/L BMP-7/tantalum/chondrocyte). The proliferation of chondrocytes was measured by CCK-8 assay. The chondrocyte growth and morphology were observed by scanning electron microscopy (SEM). The synthesis of glycosaminoglycan (GAG) in chondrocytes was tested by dimethyl methylene blue (DMMB) colorimetric quantification method. Col-II, AGG and Sox9 mRNA in chondrocytes were detected by real-time PCR. The chondrocytes were spindle-shaped in 24 hours of primary cell culture and most cells became polygonal shaped in 4 days. The chondrocytes were affirmed by alcian blue, safranin O and Col-II immunocytochemistry staining. The result of CCK-8 assay showed that the level of cell proliferation in 100 μg/L BMP-7 groups were higher than those in the other groups (Ptantalum scaffolds with BMP-7 had better functions, by which cytoplasmic processes developed and extended to the surface and inner of porous tantalum by SEM observation. DMMB quantitative determination of GAG showed that GAG amount of chondrocytes in 100 μg/L BMP-7 groups was significantly higher than those in the other groups (Ptantalum/chondrocytes composites enhanced in vitro chondrocyte proliferation and extracellular matrix greatly, and can promote chondrogenic gene expression.

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

    Directory of Open Access Journals (Sweden)

    David Garciadiego-Cázares

    Full Text Available 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 differentiation in the articular cartilage. Members of the Transforming growth factor beta (Tgf-β Superfamily, 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 dedifferentiation during OA progression, while increase of α1, α2, and α3 integrin expression was related with fibrous areas in articular cartilage during OA. Hypertrophic chondrocytes expressed αV 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 the α5 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

  12. 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

  13. Role of polyamines in hypertrophy and terminal differentiation of osteoarthritic chondrocytes.

    Science.gov (United States)

    Facchini, A; Borzì, R M; Olivotto, E; Platano, D; Pagani, S; Cetrullo, S; Flamigni, F

    2012-02-01

    Polyamines are naturally occurring, positively charged polycations which are able to control several cellular processes in different cell types, by interacting with negatively charged compounds and structures within the living cell. Functional genomics in rodents targeting key biosynthetic or catabolic enzymes have revealed a series of phenotypic changes, many of them related to human diseases. Several pieces of evidence from the literature point at a role of polyamines in promoting chondrocyte differentiation, a process which is physiological in growth plate maturation or fracture healing, but has pathological consequences in articular chondrocytes, programmed to keep a maturational arrested state. Inappropriate differentiation of articular chondrocytes results in osteoarthritis. Thus, we have studied the effects of exogenously added spermine or spermidine in chondrocyte maturation recapitulated in 3D cultures, to tease out the effects on gene and protein expression of key chondrogenesis regulatory transcription factors, markers and effectors, as well as their posttranscriptional regulation. The results indicate that both polyamines are able to increase the rate and the extent of chondrogenesis, with enhanced collagen 2 deposition and remodeling with downstream generation of collagen 2 bioactive peptides. These were able to promote nuclear localization of RUNX-2, the pivotal transcription factor in chondrocyte hypertrophy and osteoblast generation. Indeed, samples stimulated with polyamines showed an enhanced mineralization, along with increased caspase activity, indicating increased chondrocyte terminal differentiation. In conclusion these results indicate that the polyamine pathway can represent a potential target to control and correct chondrocyte inappropriate maturation in osteoarthritis.

  14. Osteoprotegerin deficiency leads to deformation of the articular cartilage in femoral head.

    Science.gov (United States)

    Liu, Yi; Ge, Jianping; Chen, Danying; Weng, Yuteng; Du, Haiming; Sun, Yao; Zhang, Qi

    2016-10-01

    Osteoarthritis (OA) was a degenerative joint disease characterized by articular cartilage degradation and extensive remodeling of the subchondral bone. Multiple lines of evidence indicated that Osteoprotegerin (OPG), a member of TNF receptor superfamily that was expressed in the chondrocytes of articular cartilage and adjacent locations in the physiological setting, was involved in maintaining integrity of articular cartilage. OPG could prevent subchondral bone from resorption, and also protect cartilage from degradation. In this study, we used Osteoprotegerin-knockout mice (Opg-KO mice) to find out the role of OPG in articular cartilage. We examined articular cartilage in the femoral head of Opg-KO mice began in early adulthood using modern molecular and imaging methods. We found cartilage changes starting from adulthood and progressively with age, reminiscent of pathological changes in OA. Deficiency of OPG caused thinned articular cartilage and extensive remodeling of the subchondral bone in femoral head in comparison with wild-type mice (WT mice). Also, the articular cartilage of femoral head expressed significantly less of Aggrecan, Col-II and Col-X, but more Col-I and Matrix Metalloproteinases-13 (Mmp-13) than WT mice both at gene and protein level. Moreover, increased chondrocyte apoptosis and decreased chondrocyte proliferation were observed in femoral head of Opg-KO mice compared to WT mice. These data suggested that OPG played an important role in maintaining the homeostasis of articular cartilage of femoral head.

  15. Positive-feedback regulation of subchondral H-type vessel formation by chondrocyte promotes osteoarthritis development in mice.

    Science.gov (United States)

    Lu, Jiansen; Zhang, Haiyan; Cai, Daozhang; Zeng, Chun; Lai, Pinglin; Shao, Yan; Fang, Hang; Li, Delong; Ouyang, Jiayao; Zhao, Chang; Xie, Denghui; Huang, Bin; Yang, Jian; Jiang, Yu; Bai, Xiaochun

    2018-01-12

    Vascular-invasion-mediated interactions between activated articular chondrocytes and subchondral bone are essential for osteoarthritis (OA) development. Here, we determined the role of nutrient sensing mechanistic target of rapamycin complex 1 (mTORC1) signaling in the crosstalk across the bone cartilage interface and its regulatory mechanisms. Then mice with chondrocyte-specific mTORC1 activation (Tsc1 CKO and Tsc1 CKOER ) or inhibition (Raptor CKOER ) and their littermate controls were subjected to OA induced by destabilization of the medial meniscus (DMM) or not. DMM or Tsc1 CKO mice were treated with bevacizumab, a vascular endothelial growth factor (VEGF)-A antibody that blocks angiogenesis. Articular cartilage degeneration was evaluated using the Osteoarthritis Research Society International score. Immunostaining and western blotting were conducted to detect H-type vessels and protein levels in mice. Primary chondrocytes from mutant mice and ADTC5 cells were treated with interleukin-1β to investigate the role of chondrocyte mTORC1 in VEGF-A secretion and in vitro vascular formation. Clearly, H-type vessels were increased in subchondral bone in DMM-induced OA and aged mice. Cartilage mTORC1 activation stimulated VEGF-A production in articular chondrocyte and H-type vessel formation in subchondral bone. Chondrocyte mTORC1 promoted OA partially through formation of VEGF-A-stimulated subchondral H-type vessels. In particular, vascular-derived nutrients activated chondrocyte mTORC1, and stimulated chondrocyte activation and production of VEGF, resulting in further angiogenesis in subchondral bone. Thus a positive-feedback regulation of H-type vessel formation in subchondral bone by articular chondrocyte nutrient-sensing mTORC1 signaling is essential for the pathogenesis and progression of OA. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

  16. Articular Cartilage Aging-Potential Regenerative Capacities of Cell Manipulation and Stem Cell Therapy

    Directory of Open Access Journals (Sweden)

    Magdalena Krajewska-Włodarczyk

    2018-02-01

    Full Text Available Changes in articular cartilage during the aging process are a stage of natural changes in the human body. Old age is the major risk factor for osteoarthritis but the disease does not have to be an inevitable consequence of aging. Chondrocytes are particularly prone to developing age-related changes. Changes in articular cartilage that take place in the course of aging include the acquisition of the senescence-associated secretory phenotype by chondrocytes, a decrease in the sensitivity of chondrocytes to growth factors, a destructive effect of chronic production of reactive oxygen species and the accumulation of the glycation end products. All of these factors affect the mechanical properties of articular cartilage. A better understanding of the underlying mechanisms in the process of articular cartilage aging may help to create new therapies aimed at slowing or inhibiting age-related modifications of articular cartilage. This paper presents the causes and consequences of cellular aging of chondrocytes and the biological therapeutic outlook for the regeneration of age-related changes of articular cartilage.

  17. The importance of bicarbonate and nonbicarbonate buffer systems in batch and continuous flow bioreactors for articular cartilage tissue engineering.

    Science.gov (United States)

    Khan, Aasma A; Surrao, Denver C

    2012-05-01

    In cartilage tissue engineering an optimized culture system, maintaining an appropriate extracellular environment (e.g., pH of media), can increase cell proliferation and extracellular matrix (ECM) accumulation. We have previously reported on a continuous-flow bioreactor that improves tissue growth by supplying the cells with a near infinite supply of medium. Previous studies have observed that acidic environments reduce ECM synthesis and chondrocyte proliferation. Hence, in this study we investigated the combined effects of a continuous culture system (bioreactor) together with additional buffering agents (e.g., sodium bicarbonate [NaHCO₃]) on cartilaginous tissue growth in vitro. Isolated bovine chondrocytes were grown in three-dimensional cultures, either in static conditions or in a continuous-flow bioreactor, in media with or without NaHCO₃. Tissue constructs cultivated in the bioreactor with NaHCO₃-supplemented media were characterized with significantly increased (pbioreactor with NaHCO₃-supplemented media were significantly thicker than all other constructs (pTissue outgrowth was negligible in all other culturing conditions. Thus this study demonstrates the advantage of employing a continuous flow bioreactor coupled with NaHCO₃ supplemented media for articular cartilage tissue engineering.

  18. Modelling and Simulating the Adhesion and Detachment of Chondrocytes in Shear Flow

    Science.gov (United States)

    Hao, Jian; Pan, Tsorng-Whay; Rosenstrauch, Doreen

    Chondrocytes are typically studied in the environment where they normally reside such as the joints in hips, intervertebral disks or the ear. For example, in [SKE+99], the effect of seeding duration on the strength of chondrocyte adhesion to articulate cartilage has been studied in shear flow chamber since such adhesion may play an important role in the repair of articular defects by maintaining cells in positions where their biosynthetic products can contribute to the repair process. However, in this investigation, we focus mainly on the use of auricular chondrocytes in cardiovascular implants. They are abundant, easily and efficiently harvested by a minimally invasive technique. Auricular chondrocytes have ability to produce collagen type-II and other important extracellular matrix constituents; this allows them to adhere strongly to the artificial surfaces. They can be genetically engineered to act like endothelial cells so that the biocompatibility of cardiovascular prothesis can be improved. Actually in [SBBR+02], genetically engineered auricular chondrocytes can be used to line blood-contacting luminal surfaces of left ventricular assist device (LVAD) and a chondrocyte-lined LVAD has been planted into the tissue-donor calf and the results in vivo have proved the feasibility of using autologous auricular chondrocytes to improve the biocompatibility of the blood-biomaterial interface in LVADs and cardiovascular prothesis. Therefore, cultured chondrocytes may offer a more efficient and less invasive means of covering artificial surface with a viable and adherent cell layer.

  19. Adipose mesenchymal stem cells protect chondrocytes from degeneration associated with osteoarthritis.

    Science.gov (United States)

    Maumus, Marie; Manferdini, Cristina; Toupet, Karine; Peyrafitte, Julie-Anne; Ferreira, Rosanna; Facchini, Andrea; Gabusi, Elena; Bourin, Philippe; Jorgensen, Christian; Lisignoli, Gina; Noël, Danièle

    2013-09-01

    Our work aimed at evaluating the role of adipose stem cells (ASC) on chondrocytes from osteoarthritic (OA) patients and identifying the mediators involved. We used primary chondrocytes, ASCs from different sources and bone marrow mesenchymal stromal cells (MSC) from OA donors. ASCs or MSCs were co-cultured with chondrocytes in a minimal medium and using cell culture inserts. Under these conditions, ASCs did not affect the proliferation of chondrocytes but significantly decreased camptothecin-induced apoptosis. Both MSCs and ASCs from different sources allowed chondrocytes in the cocultures maintaining a stable expression of markers specific for a mature phenotype, while expression of hypertrophic and fibrotic markers was decreased. A number of factors known to regulate the chondrocyte phenotype (IL-1β, IL-1RA, TNF-α) and matrix remodeling (TIMP-1 and -2, MMP-1 and -9, TSP-1) were not affected. However, a significant decrease of TGF-β1 secretion by chondrocytes and induction of HGF secretion by ASCs was observed. Addition of a neutralizing anti-HGF antibody reversed the anti-fibrotic effect of ASCs whereas hypertrophic markers were not modulated. In summary, ASCs are an interesting source of stem cells for efficiently reducing hypertrophy and dedifferentiation of chondrocytes, at least partly via the secretion of HGF. This supports the interest of using these cells in therapies for osteo-articular diseases. Copyright © 2013 Elsevier B.V. All rights reserved.

  20. Inflammatory milieu cultivated Sema3A signaling promotes chondrocyte apoptosis in knee osteoarthritis.

    Science.gov (United States)

    Sun, Jie; Wei, Xuelei; Wang, Zengliang; Liu, Yunjiao; Lu, Jie; Lu, Yandong; Cui, Meng; Zhang, Xi; Li, Fangguo

    2018-03-01

    Osteoarthritis (OA) is the leading degenerative joint disease and featured by articular cartilage destruction, where chondrocyte apoptosis plays a critical role. Semaphorin-3A (Sema3A) has been implicated in OA chondrocyte physiology. In this study we aimed to uncover how Sema3A signaling is regulated in chondrocytes and investigate its role in OA chondrocyte survival. Here, we report that Sema3A and its receptor neuropilin-1 (Nrp1) are synchronously upregulated in cartilage chondrocytes of knee OA patients. Their expressions in chondrocytes could be induced by the stimulation of proinflammatory cytokines IL-1β and TNF-α and subsequent transcriptional activation orchestrated by C/EBPβ. The resulting excessive Sema3A signaling promotes chondrocyte apoptosis through impairing PI3K/Akt prosurvival signaling. These findings indicate a regulatory mechanism and a proapoptotic function of aberrant Sema3A signaling in OA chondrocytes, and suggest that targeting Sema3A signaling might interfere OA pathogenesis. © 2017 Wiley Periodicals, Inc.

  1. Treatment of osteoarthritis using a helper-dependent adenoviral vector retargeted to chondrocytes

    Directory of Open Access Journals (Sweden)

    Merry ZC Ruan

    2016-01-01

    Full Text Available Osteoarthritis (OA is a joint disease characterized by degeneration of the articular cartilage, subchondral bone remodeling, and secondary inflammation. It is among the top three causes of chronic disability, and currently there are no treatment options to prevent disease progression. The localized nature of OA makes it an ideal candidate for gene and cell therapy. However, gene and cell therapy of OA is impeded by inefficient gene transduction of chondrocytes. In this study, we developed a broadly applicable system that retargets cell surface receptors by conjugating antibodies to the capsid of helper-dependent adenoviral vectors (HDVs. Specifically, we applied this system to retarget chondrocytes by conjugating an HDV to an α-10 integrin monoclonal antibody (a10mab. We show that a10mab-conjugated HDV (a10mabHDV-infected chondrocytes efficiently in vitro and in vivo while detargeting other cell types. The therapeutic index of an intra-articular injection of 10mabHDV-expressing proteoglycan 4 (PRG4 into a murine model of post-traumatic OA was 10-fold higher than with standard HDV. Moreover, we show that PRG4 overexpression from articular, superficial zone chondrocytes is effective for chondroprotection in postinjury OA and that α-10 integrin is an effective protein for chondrocyte targeting.

  2. Osteoarthritis-derived chondrocytes are a potential source of multipotent progenitor cells for cartilage tissue engineering.

    Science.gov (United States)

    Oda, Tomoyuki; Sakai, Tadahiro; Hiraiwa, Hideki; Hamada, Takashi; Ono, Yohei; Nakashima, Motoshige; Ishizuka, Shinya; Matsukawa, Tetsuya; Yamashita, Satoshi; Tsuchiya, Saho; Ishiguro, Naoki

    2016-10-21

    The natural healing capacity of damaged articular cartilage is poor, rendering joint surface injuries a prime target for regenerative medicine. While autologous chondrocyte or mesenchymal stem cell (MSC) implantation can be applied to repair cartilage defects in young patients, no appropriate long-lasting treatment alternative is available for elderly patients with osteoarthritis (OA). Multipotent progenitor cells are reported to present in adult human articular cartilage, with a preponderance in OA cartilage. These facts led us to hypothesize the possible use of osteoarthritis-derived chondrocytes as a cell source for cartilage tissue engineering. We therefore analyzed chondrocyte- and stem cell-related markers, cell growth rate, and multipotency in OA chondrocytes (OACs) and bone marrow-derived MSCs, along with normal articular chondrocytes (ACs) as a control. OACs demonstrated similar phenotype and proliferation rate to MSCs. Furthermore, OACs exhibited multilineage differentiation ability with a greater chondrogenic differentiation ability than MSCs, which was equivalent to ACs. We conclude that chondrogenic capacity is not significantly affected by OA, and OACs could be a potential source of multipotent progenitor cells for cartilage tissue engineering. Copyright © 2016 Elsevier Inc. All rights reserved.

  3. 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.

  4. Primary Culture of Canine Growth Plate Chondrocytes as a Model of Biomineralization

    OpenAIRE

    Ryuji, HOSOKAWA; Kenji, KIKUZAKI; Daisuke, CHIBA; Yasumasa, AKAGAWA; Department of Removable Prosthodontics, Hiroshima University School of Dentistry; Department of Removable Prosthodontics, Hiroshima University School of Dentistry; Department of Removable Prosthodontics, Hiroshima University School of Dentistry; Department of Removable Prosthodontics, Hiroshima University School of Dentistry

    1999-01-01

    This study investigated the mineralization process in primary cultures of dog growth plate chondrocytes as a model of biomineralization. Chondrocytes were isolated from the growth plates of ribs of 1-week-old dogs. The chondrocytes were maintained at extremely high density (5x10^4 cells/well) in collagen-coated 96-well dishes in a-MEM supplemented with 10% fetal bovine serum and 50 μg/ml ascorbic acid. Mineralization was initiated between days 20 and 24; however, the addition of fibroblast gr...

  5. 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. © 2013 Wiley Periodicals, Inc.

  6. Effects of indigo carmine on human chondrocytes in vitro.

    Science.gov (United States)

    Zippelius, Timo; Hoburg, Arnd; Preininger, Bernd; Vörös, Pauline; Perka, Carsten; Matziolis, Georg; Röhner, Eric

    2013-01-01

    Joint infections following or accompanying superficious soft tissue infections are severe complication in orthopedic surgery. The use of intra-articular blue staining is a helpful method to visualize a fistula and to differentiate between superficial and intra-articular infections. Regarding this clinical implication data about the effects of indigo carmine, a frequently used blue staining substance, on cartilage is missing. The hypothesis of this study was that indigo carmine damages human chondrocytes in a time and concentration dependent manner. Human chondrocytes were isolated from donors with osteoarthritis who were treated with TKA. Cells were cultivated and treated with different concentrations of indigo carmine for 5 and 10 minutes. Morphologic damage was examined by light microscopy. Toxicity was quantified by counting vital cell number and lactate dehydrogenase (LDH) expression. Analysis by light microscopy showed defected cell structure and loss of cell number after treatment with 100% indigo carmine for 10 minutes. Treatment with 10% and 1% indigo carmine showed no significant cell defects and loss of cells. Counting vital cell number showed loss of vital cells after treatment with 100% and 10% indigo carmine for 10 minutes. LDH expression was significantly increased after treatment with 100% indigo carmine.Toxic effects were shown after treatment with indigo carmine. Therefore, it should be used in 1:100 dilution. This is both, sufficient for visualizing a fistula in a possible clinical application and could be protective for chondrocytes.

  7. 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 (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. Copyright © 2015 Elsevier Ltd. All rights reserved.

  8. Transforming growth factor β-induced superficial zone protein accumulation in the surface zone of articular cartilage is dependent on the cytoskeleton.

    Science.gov (United States)

    McNary, Sean M; Athanasiou, Kyriacos A; Reddi, A Hari

    2014-03-01

    The phenotype of articular chondrocytes is dependent on the cytoskeleton, specifically the actin microfilament architecture. Articular chondrocytes in monolayer culture undergo dedifferentiation and assume a fibroblastic phenotype. This process can be reversed by altering the actin cytoskeleton by treatment with cytochalasin. Whereas dedifferentiation has been studied on chondrocytes isolated from the whole cartilage, the effects of cytoskeletal alteration on specific zones of cells such as superficial zone chondrocytes are not known. Chondrocytes from the superficial zone secrete superficial zone protein (SZP), a lubricating proteoglycan that reduces the coefficient of friction of articular cartilage. A better understanding of this phenomenon may be useful in elucidating chondrocyte dedifferentiation in monolayer and accumulation of the cartilage lubricant SZP, with an eye toward tissue engineering functional articular cartilage. In this investigation, the effects of cytoskeletal modulation on the ability of superficial zone chondrocytes to secrete SZP were examined. Primary superficial zone chondrocytes were cultured in monolayer and treated with a combination of cytoskeleton modifying reagents and transforming growth factor β (TGFβ) 1, a critical regulator of SZP production. Whereas cytochalasin D maintains the articular chondrocyte phenotype, the hallmark of the superficial zone chondrocyte, SZP, was inhibited in the presence of TGFβ1. A decrease in TGFβ1-induced SZP accumulation was also observed when the microtubule cytoskeleton was modified using paclitaxel. These effects of actin and microtubule alteration were confirmed through the application of jasplakinolide and colchicine, respectively. As Rho GTPases regulate actin organization and microtubule polymerization, we hypothesized that the cytoskeleton is critical for TGFβ-induced SZP accumulation. TGFβ-mediated SZP accumulation was inhibited by small molecule inhibitors ML141 (Cdc42), NSC23766 (Rac1

  9. Developmental Mechanisms in Articular Cartilage Degradation in Osteoarthritis

    Directory of Open Access Journals (Sweden)

    Elena V. Tchetina

    2011-01-01

    Full Text Available Osteoarthritis is the most common arthritic condition, which involves progressive degeneration of articular cartilage. The most recent accomplishments have significantly advanced our understanding on the mechanisms of the disease development and progression. The most intriguing is the growing evidence indicating that extracellular matrix destruction in osteoarthritic articular cartilage resembles that in the hypertrophic zone of fetal growth plate during endochondral ossification. This suggests common regulatory mechanisms of matrix degradation in OA and in the development and can provide new approaches for the treatment of the disease by targeting reparation of chondrocyte phenotype.

  10. 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...... that the chondrocyte, like several other cell types of mesenchymal origin, is surrounded by the functional equivalent of a basement membrane. This structure is presumably involved in maintaining chondrocyte phenotype and viability and may well allow a new understanding of cartilage development and provide clues...

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

    Directory of Open Access Journals (Sweden)

    Carole Bougault

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

  12. Chondrogenic capability of osteoarthritic chondrocytes from the trapeziometacarpal and hip joints.

    Science.gov (United States)

    Lovati, Arianna B; Colombini, Alessandra; Recordati, Camilla; Ceriani, Cristina; Zagra, Luigi; Berzero, Gianfranco; Moretti, Matteo

    2016-03-01

    Osteoarthritis is the most common degenerative disease of joints like the hip and the trapeziometacarpal joint (rhizarthrosis). In this in vitro study, we compared the chondrogenesis of chondrocytes derived from the trapezium and the femoral head cartilage of osteoarthritic patients to have a deeper insight on trapezium chondrocyte behavior as autologous cell source for the repair of cartilage lesions in rhizarthrosis. Chondrocytes collected from trapezium and femoral head articular cartilage were cultured in pellets and analyzed for chondrogenic differentiation, cell proliferation, glycosaminoglycan production, gene expression of chondrogenic and fibrous markers, histological and immunohistochemical analyses. Our results showed a higher cartilaginous matrix deposition and a lower fibrocartilaginous phenotype of the femoral chondrocytes with respect to the trapezium chondrocytes assessed by a higher absolute glycosaminoglycan and type II collagen production, thus demonstrating a superior chondrogenic potential of the femoral with respect to the trapezium chondrocytes. The differences in chondrogenic potential between trapezium and femoral head chondrocytes confirmed a lower regenerative capability in the trapezium than in the femoral head cartilage due to the different environment and loading acting on these joints that affects the metabolism of the resident cells. This could represent a limitation to apply the cell therapy for rhizoarthrosis.

  13. Encapsulation of chondrocytes in high-stiffness agarose microenvironments for in vitro modeling of osteoarthritis mechanotransduction.

    Science.gov (United States)

    Jutila, Aaron A; Zignego, Donald L; Schell, William J; June, Ronald K

    2015-05-01

    In articular cartilage, chondrocytes reside within a gel-like pericellular matrix (PCM). This matrix provides a mechanical link through which joint loads are transmitted to chondrocytes. The stiffness of the PCM decreases in the most common degenerative joint disease, osteoarthritis. To develop a system for modeling the stiffness of both the healthy and osteoarthritic PCM, we determined the concentration-stiffness relationships for agarose. We extended these results to encapsulate chondrocytes in agarose of physiological stiffness. Finally, we assessed the relevance of stiffness for chondrocyte mechanotransduction by examining the biological response to mechanical loading for cells encapsulated in low- and high-stiffness gels. We achieved agarose equilibrium stiffness values as large as 51.3 kPa. At 4.0% agarose, we found equilibrium moduli of 34.3 ± 1.65 kPa, and at 4.5% agarose, we found equilibrium moduli of 35.7 ± 0.95 kPa. Cyclical tests found complex moduli of ~100-300 kPa. Viability was >96% for all studies. We observed distinct metabolomic responses in >500 functional small molecules describing changes in cell physiology, between primary human chondrocytes encapsulated in 2.0 and 4.5% agarose indicating that the gel stiffness affects cellular mechanotransduction. These data demonstrate both the feasibility of modeling the chondrocyte pericellular matrix stiffness and the importance of the physiological pericellular stiffness for understanding chondrocyte mechanotransduction.

  14. The role of autologous chondrocyte implantation in the treatment of symptomatic chondromalacia patellae.

    Science.gov (United States)

    Macmull, Simon; Jaiswal, Parag K; Bentley, George; Skinner, John A; Carrington, Richard W J; Briggs, Tim W R

    2012-07-01

    Chondromalacia patella is a distinct clinical entity of abnormal softening of the articular cartilage of the patella, which results in chronic retropatellar pain. Its aetiology is still unclear but the process is thought to be a due to trauma to superficial chondrocytes resulting in a proteolytic enzymic breakdown of the matrix. Our aim was to assess the effectiveness of autologous chondrocyte implantation on patients with a proven symptomatic retropatellar lesion who had at least one failed conventional marrow-stimulating therapy. We performed chondrocyte implantation on 48 patients: 25 received autologous chondrocyte implantation with a type I/III membrane (ACI-C) method (Geistlich Biomaterials, Wolhusen, Switzerland), and 23 received the Matrix-assisted Chondrocyte Implantation (MACI) technique (Genzyme, Kastrup, Denmark). Over a mean follow-up period of 40.3 months, there was a statistically significant improvement in subjective pain scoring using the visual analogue scale (VAS) and objective functional scores using the Modified Cincinnati Rating System (MCS) in both groups. Chondromalacia patellae lesions responded well to chondrocyte implantation. Better results occurred with MACI than with ACI-C. Excellent and good results were achieved in 40% of ACI-C patients and 57% of MACI patients, but success of chondrocyte implantation was greater with medial/odd-facet lesions. Given that the MACI procedure is technically easier and less time consuming, we consider it to be useful for treating patients with symptomatic chondral defects secondary to chondromalacia patellae.

  15. Advances in the Surgical Management of Articular Cartilage Defects

    OpenAIRE

    Stein, Spencer; Strauss, Eric; Bosco, Joseph

    2013-01-01

    Objective: The purpose of this review is to gain insight into the latest methods of articular cartilage implantation (ACI) and to detail where they are in the Food and Drug Administration approval and regulatory process. Design: A PubMed search was performed using the phrase ?Autologous Chondrocyte Implantation? alone and with the words second generation and third generation. Additionally, clinicaltrials.gov was searched for the names of the seven specific procedures and the parent company we...

  16. Tensorial Electrokinetics in Articular Cartilage

    Science.gov (United States)

    Reynaud, Boris; Quinn, Thomas M.

    2006-01-01

    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 ∼−7.5 μL/As in the range of 0–20% compression to −6.0 μL/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. PMID:16798804

  17. Effect of bone marrow-derived stem cells on chondrocytes from patients with osteoarthritis.

    Science.gov (United States)

    Zhang, Qiangzhi; Chen, Yong; Wang, Qiang; Fang, Chaoyong; Sun, Yu; Yuan, Tao; Wang, Yuebei; Bao, Rongni; Zhao, Ningjian

    2016-02-01

    Increasing numbers of individuals are suffering from osteoarthritis every year, and the directed intra-articular injection of bone marrow stem cells has provided a promising treatment strategy for osteoarthritis. Although a number of studies have demonstrated that intra-articular injection of bone marrow stem cells produced desirable results, the mechanism underlying this effect has not been elucidated. In the current study, the effect of bone marrow stem cells on chondrocytes from patients with osteoarthritis was observed in a co-culture system. Human chondrocytes were obtained from patients with osteoarthritis who underwent surgical procedures and bone marrow stem cells were obtained from bone marrow aspirates, and then the chondrocytes were then cultured alone or cocultured with bone marrow stem cells in 0.4-µm Transwell inserts. The differentiation and biological activity of chondrocytes in the culture system were measured, and the inflammatory factors and OA-associated markers were also measured. The results indicated that coculture with human bone marrow stem cells increases cell proliferation of chondrocytes and inhibits inflammatory activity in osteoarthritis.

  18. Coumestrol Counteracts Interleukin-1β-Induced Catabolic Effects by Suppressing Inflammation in Primary Rat Chondrocytes.

    Science.gov (United States)

    You, Jae-Seek; Cho, In-A; Kang, Kyeong-Rok; Oh, Ji-Su; Yu, Sang-Joun; Lee, Gyeong-Je; Seo, Yo-Seob; Kim, Su-Gwan; Kim, Chun Sung; Kim, Do Kyung; Im, Hee-Jeong; Kim, Jae-Sung

    2017-02-01

    In the present study, we investigated the anti-catabolic effects of coumestrol, a phytoestrogen derived from herbal plants, against interleukin-1β-induced cartilage degeneration in primary rat chondrocytes and articular cartilage. Coumestrol did not affect the viability of human normal oral keratinocytes and primary rat chondrocytes treated for 24 h and 21 days, respectively. Although coumestrol did not significantly increase the proteoglycan contents in long-term culture, it abolished the interleukin-1β-induced loss of proteoglycans in primary rat chondrocytes and knee articular cartilage. Furthermore, coumestrol suppressed the expression of matrix-degrading enzymes such as matrix metalloproteinase-13, -3, and -1 in primary rat chondrocytes stimulated with interleukin-1β. Moreover, the expression of catabolic factors such as nitric oxide synthase, cyclooxygenase-2, prostaglandin E 2 , and inflammatory cytokines in interleukin-1β-stimulated primary rat chondrocytes was suppressed by coumestrol. In summary, these results indicate that coumestrol counteracts the catabolic effects induced by interleukin-1β through the suppression of inflammation. Therefore, based on its biological activity and safety profile, coumestrol could be used as a potential anti-catabolic biomaterial for osteoarthritis.

  19. REST corepressor (CoREST) repression induces phenotypic gene regulation in advanced osteoarthritic chondrocytes.

    Science.gov (United States)

    Xiao, Jun; Li, Tao; Wu, Zhihong; Shi, Zhanjun; Chen, Jianting; Lam, Stephen K L; Zhao, Zandong; Yang, Lanbo; Qiu, Guixing

    2010-12-01

    Alternations in cartilage chondrocyte phenotype characteristic by the decreased type II collagen and aggrecan together with increased type X collagen synthesis serve as a beacon for osteoarthritis progression. However, little is known about the underlying molecular mechanisms. The current study seeks to discover molecules that involved in osteoarthritic chondrocytes phenotype regulation. Differential proteomics was generated with two-dimensional gel electrophoresis between normal articular cartilage (NAC) and advanced osteoarthritic cartilage (AOC). Those differentially expressed proteins were identified by mass spectrometry. The down-regulation of a neuronal silencer, the REST corepressor (CoREST) in AOC, was verified by Western blot. CoREST silencing was performed in primarily cultured NAC chondrocytes with specific siRNA to reveal the possible involvement of CoREST repression in chondrocyte phenotypic genes modulation. Ninteen differentially expressed proteins were screened and identified. Among these proteins, CoREST, HHL, and zinc finger protein 155 were estimated to be possible gene modulators. CoREST protein level was verified to be down-regulated by 69.5% (p 71.4% (p < 0.001) and 57.6% (p < 0.001), respectively. The results indicate that the silencing of CoREST by siRNA transfection in NAC may reflect CoREST repression in AOC, which results in phenotypic genes modulation and suggests a homeostatic role of this transcription factor in articular chondrocyte. © 2010 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.

  20. Clinical potential and challenges of using genetically modified cells for articular cartilage repair

    Science.gov (United States)

    Madry, Henning; Cucchiarini, Magali

    2011-01-01

    Articular cartilage defects do not regenerate. Transplantation of autologous articular chondrocytes, which is clinically being performed since several decades, laid the foundation for the transplantation of genetically modified cells, which may serve the dual role of providing a cell population capable of chondrogenesis and an additional stimulus for targeted articular cartilage repair. Experimental data generated so far have shown that genetically modified articular chondrocytes and mesenchymal stem cells (MSC) allow for sustained transgene expression when transplanted into articular cartilage defects in vivo. Overexpression of therapeutic factors enhances the structural features of the cartilaginous repair tissue. Combined overexpression of genes with complementary mechanisms of action is also feasible, holding promises for further enhancement of articular cartilage repair. Significant benefits have been also observed in preclinical animal models that are, in principle, more appropriate to the clinical situation. Finally, there is convincing proof of concept based on a phase I clinical gene therapy study in which transduced fibroblasts were injected into the metacarpophalangeal joints of patients without adverse events. To realize the full clinical potential of this approach, issues that need to be addressed include its safety, the choice of the ideal gene vector system allowing for a long-term transgene expression, the identification of the optimal therapeutic gene(s), the transplantation without or with supportive biomaterials, and the establishment of the optimal dose of modified cells. As safe techniques for generating genetically engineered articular chondrocytes and MSCs are available, they may eventually represent new avenues for improved cell-based therapies for articular cartilage repair. This, in turn, may provide an important step toward the unanswered question of articular cartilage regeneration. PMID:21674822

  1. Cartilage tissue engineering and bioreactor systems for the cultivation and stimulation of chondrocytes.

    Science.gov (United States)

    Schulz, Ronny Maik; Bader, Augustinus

    2007-04-01

    Damage to and degeneration of articular cartilage is a major health issue in industrialized nations. Articular cartilage has a particularly limited capacity for auto regeneration. At present, there is no established therapy for a sufficiently reliable and durable replacement of damaged articular cartilage. In this, as well as in other areas of regenerative medicine, tissue engineering methods are considered to be a promising therapeutic component. Nevertheless, there remain obstacles to the establishment of tissue-engineered cartilage as a part of the routine therapy for cartilage defects. One necessary aspect of potential tissue engineering-based therapies for cartilage damage that requires both elucidation and progress toward practical solutions is the reliable, cost effective cultivation of suitable tissue. Bioreactors and associated methods and equipment are the tools with which it is hoped that such a supply of tissue-engineered cartilage can be provided. The fact that in vivo adaptive physical stimulation influences chondrocyte function by affecting mechanotransduction leads to the development of specifically designed bioreactor devices that transmit forces like shear, hydrostatic pressure, compression, and combinations thereof to articular and artificial cartilage in vitro. This review summarizes the basic knowledge of chondrocyte biology and cartilage dynamics together with the exploration of the various biophysical principles of cause and effect that have been integrated into bioreactor systems for the cultivation and stimulation of chondrocytes.

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

    Science.gov (United States)

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

    2017-01-01

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

  3. 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

  4. 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.

  5. Improved Chondrotoxic Profile of Liposomal Bupivacaine Compared With Standard Bupivacaine After Intra-articular Infiltration in a Porcine Model.

    Science.gov (United States)

    Shaw, K Aaron; Moreland, Colleen; Jacobs, Jeremy; Hire, Justin M; Topolski, Richard; Hoyt, Nathan; Parada, Stephen A; Cameron, Craig D

    2017-10-01

    Increasingly, liposomal bupivacaine is being used with multimodal pain management strategies. In vitro investigations have shown decreased chondrotoxicity profiles for liposomal bupivacaine; however, there is no evidence regarding its in vivo effects. Hypothesis/Purpose: This study sought to investigate the in vivo chondrotoxicity of liposomal bupivacaine, hypothesizing that there would be increased chondrocyte viability after exposure to liposomal bupivacaine when compared with standard bupivacaine. Controlled laboratory study. Eight juvenile, female Yorkshire cross piglets underwent a lateral stifle joint injection with either 1.3% liposomal bupivacaine or 0.5% bupivacaine. Injections were performed on one joint per animal with no injection to the contralateral knee, which served as the control. Chondrocyte viability was assessed 1 week after injection with a live-dead staining protocol and histologic examination. Significant chondrocyte death was seen with the live-dead staining in the bupivacaine group (33% nonviable cells) in comparison with liposomal bupivacaine (6.2%) and control (5.8%) groups ( P model. Although bupivacaine demonstrated decreased chondrocyte viability on a cellular level, histologically there were no changes. This study highlights the dichotomy between fluorescent staining and histologic appearance of articular chondrocytes in short-term analyses of viability. This study supports the peri-articular application of liposomal bupivacaine in the setting of preserved articular cartilage. A single injection of standard bupivacaine did not produce histologic changes in the articular cartilage.

  6. Oxidative stress-induced apoptosis and matrix loss of chondrocytes is inhibited by eicosapentaenoic acid.

    Science.gov (United States)

    Sakata, Shuhei; Hayashi, Shinya; Fujishiro, Takaaki; Kawakita, Kohei; Kanzaki, Noriyuki; Hashimoto, Shingo; Iwasa, Kenjiro; Chinzei, Nobuaki; Kihara, Shinsuke; Haneda, Masahiko; Ueha, Takeshi; Nishiyama, Takayuki; Kuroda, Ryosuke; Kurosaka, Masahiro

    2015-03-01

    Eicosapentaenoic acid (EPA) is an antioxidant and n-3 polyunsaturated fatty acid that reduces the production of inflammatory cytokines. We evaluated the role of EPA in chondrocyte apoptosis and degeneration. Normal human chondrocytes were treated with EPA and sodium nitroprusside (SNP). Expression of metalloproteinases (MMPs) was detected by real-time polymerase chain reaction (PCR) and that of apoptosis-related proteins was detected by western blotting. Chondrocyte apoptosis was detected by flow cytometry. C57BL/6J mice were used for the detection of MMP expression by immunohistochemistry and for investigation of chondrocyte apoptosis. EPA inhibited SNP-induced chondrocyte apoptosis, caspase 3 and poly(ADP-ribose) polymerase cleavage, phosphorylation of p38 MAPK and p53, and expression of MMP3 and MMP13. Intra-articular injection of EPA prevented the progression of osteoarthritis (OA) by inhibiting MMP13 expression and chondrocyte apoptosis. EPA treatment can control oxidative stress-induced OA progression, and thus may be a new approach for OA therapy. © 2014 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.

  7. Comprehensive characterization of chondrocyte cultures in plasma and whole blood biomatrices for cartilage tissue engineering.

    Science.gov (United States)

    Schulz, Ronny M; Haberhauer, Marcus; Zernia, Göran; Pösel, Claudia; Thümmler, Christian; Somerson, Jeremy S; Huster, Daniel

    2014-07-01

    Many synthetic polymers and biomaterials have been used as matrices for 3D chondrocyte seeding and transplantation in the field of cartilage tissue engineering. To develop a fully autologous carrier for chondrocyte cultivation, we examined the feasibility of allogeneic plasma and whole blood-based matrices and compared them to agarose constructs. Primary articular chondrocytes isolated from 12-month-old pigs were embedded into agarose, plasma and whole blood matrices and cultivated under static-free swelling conditions for up to four weeks. To evaluate the quality of the synthesized extracellular matrix (ECM), constructs were subjected to weekly examinations using histological staining, spectrophotometry, immunohistochemistry and biochemical analysis. In addition, gene expression of cartilage-specific markers such as aggrecan, Sox9 and collagen types I, II and X was determined by RT-PCR. Chondrocyte morphology was assessed via scanning electron microscopy and viability staining, including proliferation and apoptosis assays. Finally, (13)  C NMR spectroscopy provided further evidence of synthesis of ECM components. It was shown that chondrocyte cultivation in allogeneic plasma and whole-blood matrices promoted sufficient chondrocyte viability and differentiation behaviour, resulting in neo-formation of a hyaline-like cartilage matrix. Copyright © 2012 John Wiley & Sons, Ltd.

  8. Mesenchymal Stem Cells for Treating Articular Cartilage Defects and Osteoarthritis.

    Science.gov (United States)

    Wang, Yu; Yuan, Mei; Guo, Quan-yi; Lu, Shi-bi; Peng, Jiang

    2015-01-01

    Articular cartilage damage and osteoarthritis are the most common joint diseases. Joints are prone to damage caused by sports injuries or aging, and such damage regularly progresses to more serious joint disorders, including osteoarthritis, which is a degenerative disease characterized by the thinning and eventual wearing out of articular cartilage, ultimately leading to joint destruction. Osteoarthritis affects millions of people worldwide. Current approaches to repair of articular cartilage damage include mosaicplasty, microfracture, and injection of autologous chondrocytes. These treatments relieve pain and improve joint function, but the long-term results are unsatisfactory. The long-term success of cartilage repair depends on development of regenerative methodologies that restore articular cartilage to a near-native state. Two promising approaches are (i) implantation of engineered constructs of mesenchymal stem cell (MSC)-seeded scaffolds, and (ii) delivery of an appropriate population of MSCs by direct intra-articular injection. MSCs may be used as trophic producers of bioactive factors initiating regenerative activities in a defective joint. Current challenges in MSC therapy are the need to overcome current limitations in cartilage cell purity and to in vitro engineer tissue structures exhibiting the required biomechanical properties. This review outlines the current status of MSCs used in cartilage tissue engineering and in cell therapy seeking to repair articular cartilage defects and related problems. MSC-based technologies show promise when used to repair cartilage defects in joints.

  9. Autologous Chondrocyte Implantation in Osteoarthritic Surroundings: TNFα and Its Inhibition by Adalimumab in a Knee-Specific Bioreactor.

    Science.gov (United States)

    Ossendorff, Robert; Grad, Sibylle; Stoddart, Martin J; Alini, Mauro; Schmal, Hagen; Südkamp, Norbert; Salzmann, Gian M

    2017-11-01

    Autologous chondrocyte implantation (ACI) fails in up to 20% of cases. Advanced intra-articular degeneration paired with an inflammatory environment may be closely related to implantation failure. Certain cytokines have been identified to play a major role during early osteoarthritis. To investigate the effects of tumor necrosis factor α (TNFα) and its potential inhibition by adalimumab on cartilage regeneration in an in vitro model of ACI. Controlled laboratory study. Bovine articular chondrocytes were cultivated and transferred at passage 3 to fibrin-polyurethane scaffolds. Constructs were loaded by compression (10%-20% scaffold height) and shear (±25°) in a fully characterized multiaxial load (L) bioreactor to simulate clinical ACI or were subjected to free swelling (FS) conditions for a duration of 2 weeks. TNFα (20 ng/mL), adalimumab (10 µg/mL), or both were added to the medium. To assess the outcome, DNA, GAG (glycosaminoglycan), and total collagen were quantified, and gene expression of anabolic (collagen 2, aggrecan, cartilage oligomeric protein, proteoglycan 4), catabolic (matrix metalloproteinases [MMP] 3 and 13), dedifferentiation (collagen 1), and hypertrophy (collagen 10) markers and proinflammatory cytokines (TNFα, IL-1β) was analyzed. Histological evaluation was performed with safranin O/fast green, toluidine blue, and immunohistochemistry of collagen 1 and 2. Apoptosis was analyzed by immunolabeling of anti-active caspase 3. For statistical evaluation, nonparametric tests were chosen with a significance level of P < .05. A general downregulation of anabolic and upregulation of catabolic markers was detected in the TNFα groups. Collagen 2 was suppressed by TNFα (FS, P = .029; L, P = .006), while MMP 3 was significantly upregulated (FS, P = .035; L, P = .001). Dynamic loading induced a chondrogenic response, which could not fully antagonize the effect of the cytokine. Adalimumab antagonized all effects of TNFα. The histological and

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

    Science.gov (United States)

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

    2012-06-05

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

  11. Directed differentiation of induced pluripotent stem cells into chondrogenic lineages for articular cartilage treatment

    Directory of Open Access Journals (Sweden)

    Michał Lach

    2014-09-01

    Full Text Available In recent years, increases in the number of articular cartilage injuries caused by environmental factors or pathological conditions have led to a notable rise in the incidence of premature osteoarthritis. Osteoarthritis, considered a disease of civilization, is the leading cause of disability. At present, standard methods for treating damaged articular cartilage, including autologous chondrocyte implantation or microfracture, are short-term solutions with important side effects. Emerging treatments include the use of induced pluripotent stem cells, a technique that could provide a new tool for treatment of joint damage. However, research in this area is still early, and no optimal protocol for transforming induced pluripotent stem cells into chondrocytes has yet been established. Developments in our understanding of cartilage developmental biology, together with the use of modern technologies in the field of tissue engineering, provide an opportunity to create a complete functional model of articular cartilage.

  12. 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.

  13. Hyperosmolarity normalises serum-induced changes to chondrocyte properties in a model of cartilage injury.

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    Karim, A; Hall, A C

    2016-03-29

    Partial-thickness cartilage injuries do not heal effectively, potentially leading to degeneration as occurs in post-traumatic osteoarthritis (PTOA). The role of chondrocytes could be crucial in determining the nature of the repair; however, their response to this injury is poorly understood. We have utilised an in vitro bovine osteochondral partial-thickness scalpel injury model and determined chondrocyte properties at and distant from the injury in the presence/absence of (a) serum-free DMEM (340 mOsm), (b) synovial fluid DMEM (SF-DMEM), (c) foetal calf serum DMEM (FCS-DMEM), (d) hyperosmolar serum-free DMEM (600 mOsm), or (e) hyperosmolar FCS-DMEM for up to two weeks. Chondrocytes were fluorescently-labelled with 5-chloromethylfluorescein-diacetate (CMFDA)/propidium iodide (PI) for live/dead cells and imaged using confocal microscopy. Quantitative data were obtained on chondrocyte properties (cell volume, clusters, morphology) at and distant from the injury. In serum-free DMEM, chondrocyte morphology at the injury remained unaffected throughout culture. However, with SF-DMEM or FCS-DMEM the chondrocytes displayed an increase in volume (p serum-free DMEM. Cluster formation and shape changes during FCS-DMEM culture were more pronounced than with SF-DMEM. SF-DMEM or FCS-DMEM stimulated these changes to chondrocytes at the injury with only small effects on distant cells. Hyperosmolarity inhibited the morphological and volume changes to chondrocytes induced by FCS-DMEM (p serum-free DMEM. Raised osmolarity may therefore have benefit in preserving the morphological phenotype of chondrocytes at the site of injury, and thus promote more effective integrative repair in partial-thickness cartilage injury.

  14. Disruption of Phosphoinositide-Specific Phospholipases Cγ1 Contributes to Extracellular Matrix Synthesis of Human Osteoarthritis Chondrocytes

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    Guoqing Zeng

    2014-07-01

    Full Text Available Osteoarthritis (OA is a degenerative joint disease characterized by articular cartilage degradation including extracellular matrix (ECM degradation and cell loss. It is known that phosphoinositide-specific phospholipase γ1 (PLCγ1 can trigger several signaling pathways to regulate cell metabolism. However, whether this kinase is expressive and active in human OA chondrocytes and its role in the pathological progression of OA have not been investigated. The current study was designed to investigate the PLCγ1 expression in human OA cartilage, and whether PLCγ1 was involved in the ECM synthesis had been further explored using cultured human OA chondrocytes. Our results indicated that PLCγ1 was highly expressed in human OA chondrocytes. In our further study using the cultured human OA chondrocytes, the results demonstrated that the disruption of PLCγ1 by its inhibitor, U73122, and siRNA contributed to the ECM synthesis of human OA chondrocytes through regulating the expression of ECM-related signaling molecules, including MMP-13, Col II, TIMP1, Sox-9, and AGG. Furthermore, PLCγ1/IP3/Ca(2+/CaMK II signaling axis regulated the ECM synthesis of human chondrocytes through triggering mTOR/P70S6K/S6 pathway. In summary, our results suggested that PLC-γ1 activities played an important role in the ECM synthesis of human OA chondrocytes, and may serve as a therapeutic target for treating OA.

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

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    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.

  16. Adenovirus-mediated osteoprotegerin ameliorates cartilage destruction by inhibiting proteoglycan loss and chondrocyte apoptosis in rats with collagen-induced arthritis.

    Science.gov (United States)

    Feng, Zhi-yun; He, Zhen-nian; Zhang, Bin; Li, Yi-qiao; Guo, Jian; Xu, Yuan-lin; Han, Ming-yuan; Chen, Zhong

    2015-10-01

    Our aim is to elucidate the effects of osteoproteogerin (OPG) on cartilage destruction in rats as a model of collagen-induced arthritis (CIA). To establish the CIA model, Sprague Dawley rats were injected with bovine type II collagen solution subcutaneously via the tails. Adenovirus-mediated OPG (Ad-OPG) was then injected intra-articularly either at the beginning of CIA (early OPG treatment) or one week after CIA establishment (late OPG treatment); vehicle or Ad-green fluorescent protein were injected as controls. The rats were killed 4 weeks after treatment. Ankle-joint sections were obtained for histology. Serum samples were collected for enzyme-linked immunosorbent assay. Safranin O staining showed that proteoglycan loss was inhibited in the early and late Ad-OPG groups. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling staining revealed that both early and late Ad-OPG treatments significantly prevented chondrocyte apoptosis in CIA rats. Furthermore, disintegrin and metalloproteinase with thrombospondin motif-5 expression decreased remarkably in the early and late OPG treatment groups. However, the cartilage destruction score, cartilage oligomeric matrix protein level and caspase-3 expression were only decreased in the early Ad-OPG treatment group. Additionally, ankle-joint swelling and the interleukin-1β expression level in CIA rats were not notably altered by Ad-OPG treatment. Taken together, our results suggest that early Ad-OPG treatment has potent protective effects against cartilage destruction during rheumatoid arthritis progression, mainly by reducing proteoglycan loss and chondrocyte apoptosis.

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

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

    2014-01-01

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

  18. Simultaneous Magnetic Resonance Imaging and Consolidation Measurement of Articular Cartilage

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    Robert Mark Wellard

    2014-05-01

    Full Text Available Magnetic resonance imaging (MRI offers the opportunity to study biological tissues and processes in a non-disruptive manner. The technique shows promise for the study of the load-bearing performance (consolidation of articular cartilage and changes in articular cartilage accompanying osteoarthritis. Consolidation of articular cartilage involves the recording of two transient characteristics: the change over time of strain and the hydrostatic excess pore pressure (HEPP. MRI study of cartilage consolidation under mechanical load is limited by difficulties in measuring the HEPP in the presence of the strong magnetic fields associated with the MRI technique. Here we describe the use of MRI to image and characterize bovine articular cartilage deforming under load in an MRI compatible consolidometer while monitoring pressure with a Fabry-Perot interferometer-based fiber-optic pressure transducer.

  19. 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...

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

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    Salinas, Daniel; Minor, Cody A; Carlson, Ross P; McCutchen, Carley N; Mumey, Brendan M; June, Ronald K

    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.

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

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    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.

  2. 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.

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

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    Sébastien Flajollet

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

  4. Regulative mechanisms of chondrocyte adhesion

    DEFF Research Database (Denmark)

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

    2006-01-01

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

  5. INJURED ARTICULAR CARTILAGE REPAIR

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    Ariana Barlič

    2008-02-01

    Surveys show that the most frequently used surgical methods are mosaicplasty and bonemarrow stimulation with microfracturing. The efficacy of the autologous chondrocyte implantationmethod should be superior to microfracturing on a long run. Especially when(regeneration of the hyaline cartilage instead of fibrous tissue (fibrocartilage is concerned.However, it has not been scientifically proved yet

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

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    Natalya Krivoruchko

    2014-12-01

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

  7. Advances in autologous chondrocyte implantation and related techniques for cartilage repair.

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    Foldager, Casper Bindzus

    2013-04-01

    Articular cartilage is a specialized tissue exhibiting low intrinsic capabilities of regeneration or healing after injury. Autologous chondrocyte implantation (ACI) and scaffold-supported ACI are often used for treatment of larger chondral defects (> 2 cm2). These utilize open surgery re-implantation of ex vivo cultured autologous chondrocytes harvested as a biopsy arthroscopically in a prior surgery. This two-step procedure is an advanced and expensive treatment that despite high expectations have failed to regenerate articular cartilage in a consistent and predictable fashion, and as many as 25% the operated of patients have dissatisfactory outcomes. The objective of the present thesis was to address and investigate methods for optimizing the steps involved in the ACI and scaffold-supported ACI treatment including chondrocyte culture environment, chondrocyte labeling and tracking, improved biomaterials, and cell seeding densities. We hypothesized that these areas were eligible for targeted optimization, which has been addressed in the five papers constituting the work performed in the present thesis. The first two studies address the in vitro cell expansion of chondrocytes before re-implantation. After validation of hypoxia-suitable housekeeping genes for quantitative gene expression analysis using previously validated algorithms (study 1) the effect of combined hypoxic- and 3D culture on human chondrocytes gene expression was investigated (study 2). An in vitro experiment was performed to determine the effect on gene expression of an intracellular superparamagnetic labeling agent for 1.5T MRI-tracking of alginate-embedded human chondrocytes (study 3). We further performed a literature study, reviewing the cell seeding densities of the implanted chondrocytes used in clinically available cell transplantation-based treatments for cartilage repair (study 4). Finally, we tested the addition of dermatan sulfate to a clinically approved methoxy-polyethen-glycol (MPEG

  8. 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.

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

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    P. Klinger

    2017-01-01

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

  10. Dynamic mechanical loading enhances functional properties of tissue-engineered cartilage using mature canine chondrocytes.

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    Bian, Liming; Fong, Jason V; Lima, Eric G; Stoker, Aaron M; Ateshian, Gerard A; Cook, James L; Hung, Clark T

    2010-05-01

    The concept of cartilage functional tissue engineering (FTE) has promoted the use of physiologic loading bioreactor systems to cultivate engineered tissues with load-bearing properties. Prior studies have demonstrated that culturing agarose constructs seeded with primary bovine chondrocytes from immature joints, and subjected to dynamic deformation, produced equilibrium compressive properties and proteoglycan content matching the native tissue. In the process of translating these results to an adult canine animal model, it was found that protocols previously successful with immature bovine primary chondrocytes did not produce the same successful outcome when using adult canine primary chondrocytes. The objective of this study was to assess the efficacy of a modified FTE protocol using adult canine chondrocytes seeded in agarose hydrogel and subjected to dynamic loading. Two modes of dynamic loading were applied to constructs using custom bioreactors: unconfined axial compressive deformational loading (DL; 1 Hz, 10% deformation) or sliding contact loading (Slide; 0.5 Hz, 10% deformation). Loading for 3 h daily was initiated on day 0, 14, or 28 (DL0, DL14, DL28, and Slide14). Constructs with applied loading (both DL and Slide) exhibited significant increases in Young's modulus compared with free-swelling control as early as day 28 in culture (p engineered constructs compare favorably with (and exceed in some cases) those of native canine knee (patella groove and condyle) cartilage. Our findings successfully demonstrate an FTE strategy incorporating clinically relevant, adult chondrocytes and gel scaffold for engineering cartilage replacement tissue. These results, using continuous growth factor supplementation, are in contrast to our previously reported studies with immature chondrocytes where the sequential application of dynamic loading after transient transforming growth factor-beta3 application was found to be a superior culture protocol. Sliding, which simulates

  11. Vitamin D and Its Effects on Articular Cartilage and Osteoarthritis.

    Science.gov (United States)

    Garfinkel, Rachel J; Dilisio, Matthew F; Agrawal, Devendra K

    2017-06-01

    Osteoarthritis (OA) currently affects 10% of the American population. There has been a recent push to determine exactly what causes OA and how it can be treated most effectively. Serum vitamin D levels have been associated with OA and may have an effect on articular cartilage remodeling. To critically review the published research on the effect of vitamin D on articular cartilage and the development of OA as well as on the mechanism behind cartilage regeneration and degeneration. Review. A systematic search of PubMed and the Web of Science was performed for relevant studies published in the English language through April 30, 2016, using the terms vitamin D , articular cartilage , and osteoarthritis . On a molecular level, 1α,25(OH) 2 D 3 , the activated form of vitamin D, plays a role in articular cartilage degeneration. Vitamin D binds to vitamin D receptors, triggering a signaling cascade that leads to chondrocyte hypertrophy. In clinical trials, vitamin D deficiency poses a risk factor for OA, and those with decreased cartilage thickness are more likely to be vitamin D-insufficient. The role of vitamin D supplementation in the treatment or prevention of OA remains uncertain. More research is needed to reconcile these conflicting findings.

  12. 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

  13. Articular Cartilage Regeneration: An Update of Possible Treatment Approaches

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    Ray Marks

    2017-08-01

    Full Text Available BACKGROUND: Osteoarthritis, a widespread chronically disabling disorder primarily affecting articular cartilage is said to be irreversible. Researchers have however, been examining processes and methods of promoting articular cartilage repair for some time. QUESTIONS: Can a case be made for the possibility of restoring osteoarthritic cartilage? How advanced is this undertaking? What barriers exist in translating basic studies in the clinical realm? What physical modalities are deemed efficacious in promoting cartilage structure? METHODS: All relevant publications detailing articular cartilage repair themes in the leading databases were examined. Specific emphasis was placed on a broad array of efforts and observations concerning articular cartilage and its repair. Articles of historic significance and more current strategies designed to foster cartilage repair were focused on, and reported in narrative form. Ideas extracted from the voluminous literature were those that answered one or more of the key questions driving this research. RESULTS: Numerous attempts have been made over time to foster cartilage repair, using a variety of approaches such as creating artificial cartilage, and transplanting stem cells into damaged cartilage to promote repair. Most current strategies are forged in laboratories and do not always account for the complex disease process, and the importance mechanical and inflammatory determinants play in the disease. However, manipulating biophysical, and biomechanical stimuli favorably is likely to hold promise for attenuating destruction of/or for fostering cartilage viability and repair, even in the presence of adverse osteoarthritic cartilage tissue changes. CONCLUSION: More work is needed to examine the key upstream determinants leading to articular cartilage destruction, and to enhancing the viability of the tissue. Employing carefully construed therapeutic strategies known to impact articular cartilage homeostasis

  14. Extracorporeal shock waves down-regulate the expression of interleukin-10 and tumor necrosis factor-alpha in osteoarthritic chondrocytes

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    Lapadula Giovanni

    2008-01-01

    Full Text Available Abstract Background The purpose of this study was to investigate the effects of extra corporeal shock waves (ESW therapy on the metabolism of healthy and osteoarthritic human chondrocytes, and particularly on the expression of IL-10, TNF-alpha and beta1 integrin. Methods Human adult articular cartilage was obtained from 9 patients (6 male and 3 females, with primary knee osteoarthritis (OA, undergoing total joint replacement and from 3 young healthy donors (HD (2 males, 1 female with joint traumatic fracture. After isolation, chondrocytes underwent ESW treatment (electromagnetic generator system, MINILITH SL1, STORZ MEDICAL at different parameters of impulses, energy levels and energy flux density. After that, chondrocytes were cultured in 24-well plate in DMEM supplemented with 10% FCS for 48 hours and then beta1 integrin surface expression and intracellular IL-10 and TNF-alpha levels were evaluated by flow-cytometry. Results At baseline, osteoarthritic chondrocytes expressed significantly lower levels of beta1 integrin and higher levels and IL-10 and TNF-alpha levels. Following ESW application, while beta1 integrin expression remain unchanged, a significant decrease of IL-10 and TNF-alpha intracellular levels was observed both in osteoarthritic and healthy chondrocytes. IL-10 levels decreased at any impulses and energy levels, while a significant reduction of TNF-alpha was mainly found at middle energies. Conclusion Our study confirmed that osteoarthritic chondrocytes express low beta1 integrin and high TNF-alpha and IL-10 levels. Nonetheless, ESW treatment application down-regulate the intracellular levels of TNF-alpha and IL-10 by chondrocytes, suggesting that ESW might restore TNF-alpha and IL-10 production by osteoarthritic chondrocytes at normal levels. However, further in vivo and in vitro studies are necessary to establish if ESW can represent a viable option in the treatment of OA.

  15. MMP-13 loss associated with impaired ECM remodelling disrupts chondrocyte differentiation by concerted effects on multiple regulatory factors

    Science.gov (United States)

    Borzi, Rosa Maria; Olivotto, Eleonora; Pagani, Stefania; Vitellozzi, Roberta; Neri, Simona; Battistelli, Michela; Falcieri, Elisabetta; Facchini, Annalisa; Flamigni, Flavio; Penzo, Marianna; Platano, Daniela; Santi, Spartaco; Facchini, Andrea; Marcu, Kenneth B

    2010-01-01

    Purpose To link MMP-13 activity and ECM remodeling to alterations in regulatory factors leading to a disruption in chondrocyte homeostasis. Methods Matrix-metalloproteinase-13 (MMP-13) expression was ablated in primary human chondrocytes by stable retrotransduction of short-hairpin RNAs. The effects of MMP-13 KD on key regulators of chondrocyte differentiation (Sox9, Runx2 and β-catenin), and angiogenesis (VEGF) were scored at the protein (immunohistochemistry or western blot) and RNA (real time PCR) levels in high density monolayer and micromass cultures under mineralizing conditions. Effects on cellular viability in conjunction with chondrocyte progression towards a hypertrophic-like state were assessed in micromass cultures. Alterations in Sox9 subcellular distribution were assessed by confocal microscopy in micromass cultures and also in OA cartilage. Results Differentiation of control chondrocyte micromasses progressed up to a terminal phase, with calcium deposition in conjunction with reduced cell viability and scant ECM. MMP-13 knock-down (KD) impaired ECM remodeling and suppressed differentiation in conjunction with reduced levels of Runx2, β-catenin and VEGF. MMP-13 levels in vitro and ECM remodeling in vitro and in vivo were linked to changes in Sox9 sub-cellular localization. Sox9 was largely excluded from the nuclei of chondrocytes with MMP-13 remodeled or degraded ECM, and exhibited an intranuclear staining pattern in chondrocytes with impaired MMP-13 activity in vitro or with more intact ECM in vivo. Conclusions MMP-13 loss leads to a break-down in primary human articular chondrocyte differentiation by altering the expression of multiple regulatory factors. PMID:20506238

  16. Matrix metalloproteinase 13 loss associated with impaired extracellular matrix remodeling disrupts chondrocyte differentiation by concerted effects on multiple regulatory factors.

    Science.gov (United States)

    Borzí, Rosa Maria; Olivotto, Eleonora; Pagani, Stefania; Vitellozzi, Roberta; Neri, Simona; Battistelli, Michela; Falcieri, Elisabetta; Facchini, Annalisa; Flamigni, Flavio; Penzo, Marianna; Platano, Daniela; Santi, Spartaco; Facchini, Andrea; Marcu, Kenneth B

    2010-08-01

    To link matrix metalloproteinase 13 (MMP-13) activity and extracellular matrix (ECM) remodeling to alterations in regulatory factors leading to a disruption in chondrocyte homeostasis. MMP-13 expression was ablated in primary human chondrocytes by stable retrotransduction of short hairpin RNA. The effects of MMP-13 knockdown on key regulators of chondrocyte differentiation (SOX9, runt-related transcription factor 2 [RUNX-2], and beta-catenin) and angiogenesis (vascular endothelial growth factor [VEGF]) were scored at the protein level (by immunohistochemical or Western blot analysis) and RNA level (by real-time polymerase chain reaction) in high-density monolayer and micromass cultures under mineralizing conditions. Effects on cellular viability in conjunction with chondrocyte progression toward a hypertrophic-like state were assessed in micromass cultures. Alterations in SOX9 subcellular distribution were assessed using confocal microscopy in micromass cultures and also in osteoarthritic cartilage. Differentiation of control chondrocyte micromasses progressed up to a terminal phase, with calcium deposition in conjunction with reduced cell viability and scant ECM. MMP-13 knockdown impaired ECM remodeling and suppressed differentiation in conjunction with reduced levels of RUNX-2, beta-catenin, and VEGF. MMP-13 levels in vitro and ECM remodeling in vitro and in vivo were linked to changes in SOX9 subcellular localization. SOX9 was largely excluded from the nuclei of chondrocytes with MMP-13-remodeled or -degraded ECM, and exhibited an intranuclear staining pattern in chondrocytes with impaired MMP-13 activity in vitro or with more intact ECM in vivo. MMP-13 loss leads to a breakdown in primary human articular chondrocyte differentiation by altering the expression of multiple regulatory factors.

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

    Science.gov (United States)

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

    2017-09-01

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

  18. Cell Viability in Arthroscopic Versus Open Autologous Chondrocyte Implantation.

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    Biant, Leela C; Simons, Michiel; Gillespie, Trudi; McNicholas, Michael J

    2017-01-01

    Autologous chondrocyte implantation (ACI) is an effective method of repair of articular cartilage defects. It is a 2-stage operation, with the second stage most commonly performed via mini-arthrotomy. Arthroscopic ACI is gaining popularity, as it is less invasive and may accelerate early rehabilitation. However, handling and manipulation of the implant have been shown to cause chondrocyte cell death. To assess the number and viability of cells delivered via an open versus arthroscopic approach in ACI surgery. Controlled laboratory study. Sixteen ACI surgeries were performed on young cadaveric knees by 2 experienced surgeons: 8 via mini-arthrotomy and 8 arthroscopically. Live and dead cells were stained and counted on implants after surgery. The cell number and viability were assessed using confocal laser scanning microscopy. Surgery was timed from knife to skin until the end of cycling the knee 10 times after implantation of the cell-membrane construct. On receipt of cell membranes after transportation from the laboratory, ≥92% of the cells were viable. There were significantly more remaining cells (8.47E+07 arthroscopic vs 1.41E+08 open; P arthroscopic vs 37.34% open; P arthroscopic technique. Open surgery was of a significantly shorter duration (6 vs 32 minutes; P arthroscopic technique. The viability of cells delivered for ACI via an arthroscopic approach was 16 times less than via an open approach. The mini-arthrotomy approach is recommended until long-term clinical comparative data are available.

  19. Viability of human chondrocytes in an ex vivo model in relation to temperature and cartilage depth.

    Science.gov (United States)

    Drobnic, M; Mars, T; Alibegović, A; Bole, V; Balazic, J; Grubic, Z; Brecelj, J

    2005-01-01

    Chondrocytes in human articular cartilage remain viable post-mortem. It has however not been established yet how the storage temperature affects their survival, which is essential information when post-mortem cartilage is used for toxicologic studies. Our aim was to construct a simple model of explanted knee cartilage and to test the influences of time and temperature on the viability of chondrocytes in the ex vivo conditions. Osteochondral cylinders were procured from the cadaveric femoral condyles. The cylinders were embedded in water-tight rubber tubes, which formed separate chondral and osteal compartments. Tubes were filled with normal saline, without additives, to keep chondrocytes under close-to-normal conditions. The samples were divided into two groups stored at 4 degrees C and 35 degrees C, respectively. Three samples of each of these two groups were analysed at the time of removal, and then three and nine days later. Images of Live-Dead staining were scanned by a confocal laser microscope. Count of viable chondrocytes in four regions, from surface to bone, was obtained using image analysis software. The regression model revealed that the number of viable chondrocytes decreased every day by 19% and that an increase in temperature by 1 degree C decreased their viability by 5.8%. The temperature effect fell by 0.2 percentage points for every 100 microm from the surface to the bone. Herein we demonstrate that chondrocytes remain viable in the ex vivo model of human knee cartilage long enough to be able to serve as a model for toxicologic studies. Their viability is, however, significantly influenced by time and temperature.

  20. 1,25-dihydroxyvitamin D3 Activates MMP13 Gene Expression in Chondrocytes through p38 MARK Pathway

    OpenAIRE

    Chen, Dafu; Li, Yang; Dai, Xuejun; Zhou, Xinhua; Tian, Wei; Zhou, Yixin; Zou, Xuenong; Zhang, Chi

    2013-01-01

    Osteoarthritis (OA) is the most prevalent degenerative joint disease. The highly regulated balance of matrix synthesis and degradation is disrupted in OA, leading to progressive breakdown of articular cartilage. The molecular events and pathways involved in chondrocyte disfunction of cartilage in OA are not fully understood. It is known that 1,25-dihydroxyvitamin D? (1,25-(OH)2D3) is synthesized by macrophages derived from synovial fluid of patients with inflammatory arthritis. Vitmain D rece...

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

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    Mallein-Gerin Frédéric

    2008-09-01

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

  2. 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.

  3. [Preliminary study of primarily cultured C57 articular cartilage transfected with plasmid IDO-EGFP by lipofectamine].

    Science.gov (United States)

    Duan, Xiao-Hong; He, Xian-Hui; Cui, Peng-Cheng; Wang, Xiao-Yan; Wu, Ming-Ming; Shi, Jian-Bo; Xu, Geng; Jiang, Xun

    2007-12-01

    To determine the transfection efficiency and transient expression of pIDO-EGFP gene in primarily cultured C57 articular cartilage of mice, and to establish a transfection method of the primarily cultured articular cartilage in mice. Plasmid IDO-EGFP was amplified in Escherichia coli. The primarily cultured mouse chondrocytes which were initially obtained from articular cartilage were cultured in vitro and transfected with pIDO-EGFP by lipofectamine2000 reagent under optimized condition. Transfection process and transient expression were evaluated by fluorescent microscopy and laser scanning confocal microscopy (LSCM), and transfection efficiency was determined by flow cytometry. There was obvious expression of EGFP at 24 h after transfection. The transfection efficiency of pIDO-EGFP into primarily cultured mouse chondrocytes reached 36.43% at 48 hours and the transfection did not affect the process of cell adherence. IDO gene has been successfully transfected into primarily cultured chondrocytes by means of lipofectamine2000 reagent and the chondrocytes can survive in vitro. Satisfactory efficiency of transient transfection can be reached under optimized condition, which will provide a basis for gene introduction and modification of tissue engineered cartilage.

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

    Science.gov (United States)

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

    2017-02-01

    The objective of this study was to test the hypothesis that platelet-rich plasma (PRP) pretreatment on a poly-lactic-co-glycolic acid (PLGA) mesh scaffold enhances the healing capacity of the meniscus with human chondrocyte-seeded scaffolds in vivo, even when the seeded number of cells was reduced from 10 million to one million. A flexible PLGA mesh scaffold was pretreated with PRP using a centrifugal technique. One million human articular chondrocytes were seeded onto the scaffold by dynamic oscillation. After 7 days, scaffolds were placed between human meniscal discs and were implanted subcutaneously in nude mice for 6 weeks (n = 16/group). Fluorescence microscopy demonstrated uniform attachment of the chondrocytes throughout the scaffolds 24 h following seeding. Cell attachment analysis revealed a significantly increased number of chondrocytes on PRP-pretreated than non-treated scaffolds (p < 0.05). Field emission scanning electron microscopy revealed chondrocytes attached to the PRP-pretreated scaffolds interconnecting their cellular processes with the fibrin 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 < 0.05). These findings suggest that human articular chondrocytes on PRP-pretreated PLGA 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. Copyright © 2014 John Wiley & Sons, Ltd.

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

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    Jun Tian

    2016-02-01

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

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

    Science.gov (United States)

    Tian, Jun; Li, Yan

    2016-01-01

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

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

    Science.gov (United States)

    Tian, Jun; Li, Yan

    2016-01-01

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

  8. 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.

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

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    R.R. Filgueiras

    2014-02-01

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

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

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    Hemanth Akkiraju

    2015-12-01

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

  11. Regulation of chondrocyte functions by transient receptor potential cation channel V6 in osteoarthritis.

    Science.gov (United States)

    Song, Tengfei; Ma, Jun; Guo, Lei; Yang, Peng; Zhou, Xuhui; Ye, Tianwen

    2017-11-01

    Transient receptor potential vanilloid (TRPV) channels function to maintain the dynamic balance of calcium signaling and calcium metabolism in bones. The goal of this study was to determine the potential role of TRPV6 in regulation of chondrocytes. The level of TRPV6 expression was analyzed by western blot in articular cartilage derived from the knee joints of osteoarthritis (OA) rat models and OA patients. Bone structure and osteoarthritic changes in the knee joints of TRPV6 knockout mice were examined using micro-computed and histological analysis at the age of 6 and 12 months old. Furthermore, to investigate the effects of TRPV6 on chondrocyte extracellular matrix secretion, the release of matrix degrading enzymes, cell proliferation, and apoptosis, we decreased and increased TRPV6 expression in chondrocytes with lentiviral constructs encoding shRNA targeting TRPV6 and encoding TRPV6, respectively. The results showed that the level of TRPV6 expression in an OA rat model was markedly down-regulated. TRPV6 knockout mice showed severe osteoarthritis changes, including cartilage fibrillation, eburnation, and loss of proteoglycans. In addition, deficiency of TRPV6 clearly affected chondrocyte function, such as extracellular matrix secretion, the release of matrix degrading enzymes, cell proliferation, and apoptosis. Taken together, our results implicated that TRPV6 channel, as a chondro-protective factor, was involved in the pathogenesis of OA. © 2017 Wiley Periodicals, Inc.

  12. [Peri-articular ossifications].

    Science.gov (United States)

    Guastavino, V; Dijs, H; Herheyen, G; Driessens, M

    1990-01-01

    Restriction of motion of the joints due to peri-articular ossifications constitute a major problem in the rehabilitation of patients with transverse spinal cord lesion. The therapeutic possibilities as well as the preventive measurements are described and illustrated with a few case reports.

  13. 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

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

    Science.gov (United States)

    Liu, Shuyun; Huang, Jingxiang; Guo, Weimin; Chen, Jifeng; Zhang, Li; Zhao, Bin; Peng, Jiang; Wang, Aiyuan; Wang, Yu; Xu, Wenjing; Lu, Shibi; Yuan, Mei; Guo, Quanyi

    2014-01-01

    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. PMID:24959581

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

    Directory of Open Access Journals (Sweden)

    Yue Gao

    2014-01-01

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

  16. The effects of cross-linking of collagen-glycosaminoglycan scaffolds on compressive stiffness, chondrocyte-mediated contraction, proliferation and biosynthesis.

    Science.gov (United States)

    Lee, C R; Grodzinsky, A J; Spector, M

    2001-12-01

    The healing of articular cartilage defects may be improved by the use of implantable three-dimensional matrices. The present study investigated the effects of four cross-linking methods on the compressive stiffness of collagen-glycosaminoglycan (CG) matrices and the interaction between adult canine articular chondrocytes and the matrix: dehydrothermal treatment (DHT), ultraviolet irradiation (UV), glutaraldehyde treatment (GTA), and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDAC). The degree and kinetics of chondrocyte-mediated contraction, chondrocyte proliferation, and protein and glycosaminoglycan synthesis were evaluated over a four-week period in vitro. Cell-mediated contraction of the matrices varied with cross-linking: the most compliant DHT and UV matrices contracted the most (60% reduction in matrix diameter) and stiffest EDAC matrices contracted the least (30% reduction in matrix diameter). All cross-linking protocols permitted cell proliferation and matrix synthesis as measured by DNA content and radiolabeled sulfate and proline incorporation, respectively. During the first week in culture, a lower level of proliferation was seen in the GTA matrices but over the four-week culture period, the GTA and EDAC matrices provided for the greatest cell proliferation. On day 2, there was a significantly lower rate of 3H-proline incorporation in the GTA matrices (pcollagen in DHT and UV matrices at the early time points. These findings serve as a foundation for future studies of tissue engineering of articular cartilage and the association of chondrocyte contraction and the processes of mitosis and biosynthesis.

  17. Transcriptional activation in chondrocytes submitted to hydrostatic pressure.

    Science.gov (United States)

    Sironen, R; Elo, M; Kaarniranta, K; Helminen, H J; Lammi, M J

    2000-01-01

    At present, only a little is known about the transcriptional regulation in chondrocytes submitted to various physicomechanical factors known to exist in articular cartilage. Recently, we have investigated the effects of hydrostatic pressure on transcriptional control in chondrocytes using human chondrosarcoma and immortalized chondrocyte cell lines for the experiments. Hydrostatic pressure was applied on the cells in a special computer-controlled, water-filled pressure chamber, where cyclic and static pressures up to 32 MPa can be created. Differential display RT-PCR and probing of cDNA arrays are the methods we have used to study differential gene expression due to hydrostatic pressure. By differential display RT-PCR experiments, we have observed several differentially expressed cDNA bands under continuous 30 MPa hydrostatic pressure, while 30 MPa cyclic pressure at 1 Hz produced much fewer changes. In the first phase of our studies, we have focused on the effects of 30 MPa hydrostatic pressure because it causes a unique hsp70-mediated stress response in immortalized chondrocytes. Differential display RT-PCR screening provided us with several clones that derive from low-abundance mRNAs, such as death-associated protein 3 (DAP3), a nucleotide-binding protein which increases due to interferon-gamma induced cell death; PTZ-17 (or p311), a seizure-related protein; H-NUC, a nuclear DNA binding protein; and one new gene of unknown function. In Northern blots, an induction was confirmed for the new gene, DAP3 and PTZ-17 were down-regulated in some but not in all parallel experiments; however, basal level of H-NUC mRNA was too low to be detected in Northern blots. We then chose to widen our screening to a number of known genes arrayed as cDNA blots. Under 30 MPa continuous hydrostatic pressure, four different time points were chosen (0, 3, 6 and 24 h) for the experiments. The screening of 588 cDNAs showed 15 up-regulated and 6 down-regulated genes. Consistently with our

  18. Irrigating solutions used in arthroscopy and their effect on articular cartilage. An in vivo study

    Energy Technology Data Exchange (ETDEWEB)

    Arciero, R.A.; Little, J.S.; Liebenberg, S.P.; Parr, T.J.

    1986-11-01

    The effect of arthroscopic irrigating solutions on articular cartilage was determined by the use of an animal model. Rabbit knee joints were irrigated continuously for two hours with either normal saline, Ringer's lactate, or sterile water. Subsequently, the rate of incorporation of /sup 35/SO/sub 4/ by articular cartilage was used to measure the effect of the irrigants on chondrocyte metabolism. In addition, the irrigated groups were compared to an unirrigated control group. There was no significant difference in /sup 35/SO/sub 4/ incorporation between the groups. This suggested that none of the irrigating solutions used in this study adversely affected articular cartilage function. On the basis of these findings, it appears that normal saline, Ringer's lactate, and sterile water can be safely used as irrigating solutions during most arthroscopic procedures.

  19. 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)

    Mayer, Nathalie; Lopa, Silvia; 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.

  20. Chondrocyte migration affects tissue-engineered cartilage integration by activating the signal transduction pathways involving Src, PLCγ1, and ERK1/2.

    Science.gov (United States)

    Lu, Yiming; Xu, Yang; Yin, Zhaowei; Yang, Xiaofei; Jiang, Yiqiu; Gui, Jianchao

    2013-11-01

    To determine the signal transduction pathways involved in chondrocyte migration and their effects on cartilage integration in autologous chondrocyte implantation. Articular chondrocytes were divided into three inhibitor groups pretreated with different inhibitors to Src, phospholipase Cγ1 (PLCγ1), and extracellular signal-regulated kinase (ERK)1/2 signaling pathways and one control group pretreated with vehicle. The effect of these pathways on chondrocyte migration was first explored by Boyden chamber assay, and then by an in vitro cell/ring integration model. Chondrocyte migration was visualized and quantified by cell tracking, and the activity of Src, PLCγ1, and ERK1/2 was determined by Western blotting. The effect of these pathways on cartilage integration was evaluated histologically, biochemically, and biomechanically. Boyden chamber assay revealed that the number of migrated cells was significantly increased in the control group without inhibitors. In an in vitro integration model, the implanted chondrocytes were observed to migrate through the interface and infiltrate into the native cartilage. Additionally, chondrocyte migration could be improved in the absence of inhibitors After 4 weeks of culture, the control group demonstrated a significantly higher cellularity, larger amount of chemical content deposition, stronger extracellular matrix staining in the integration zone, and higher integrative strength as compared to the inhibitor groups. Western blotting demonstrated that the Src-PLCγ1-ERK1/2 signaling pathway was promoted in the integration process. This study is the first to show that the Src-PLCγ1-ERK1/2 signaling transduction pathway is involved in cartilage tissue integration by affecting chondrocyte migration. Our results raise the importance of the chondrocyte migration enhancement therapy or the development of new agents specifically targeting the pathways to ensure long-term functionality of the restored joint surface.

  1. Dual effect of platelet lysate on human articular cartilage: a maintenance of chondrogenic potential and a transient proinflammatory activity followed by an inflammation resolution.

    Science.gov (United States)

    Pereira, Rui Cruz; Scaranari, Monica; Benelli, Roberto; Strada, Paolo; Reis, Rui L; Cancedda, Ranieri; Gentili, Chiara

    2013-06-01

    Platelet-rich plasma (PRP), a cocktail of platelet growth factors and bioactive proteins, has been proposed as a therapeutic agent to restore damaged articular cartilage. We report the biological effect of the platelet lysate (PL), a PRP derivative, on primary human articular chondrocytes cultured under both physiological and inflammatory conditions. When added to the culture medium, PL induced a strong mitogenic response in the chondrocytes. The in vitro expanded cell population maintained a chondrogenic redifferentiation potential as revealed by micromass culture in vitro and ectopic cartilage formation in vivo. Further, in chondrocytes cultured in the presence of the proinflammatory cytokine interleukin-1α (IL-1α), the PL induced a drastic enhancement of the synthesis of the cytokines IL-6 and IL-8 and of neutrophil-gelatinase associated lipocalin, a lipocalin expressed during chondrocyte differentiation and inflammation. These events were mediated by the p38 MAP kinase and NF-κB pathways. We observed that inflammatory stimuli activated phospo-MAP kinase-activated protein kinase 2, a direct target of p38. The proinflammatory effect of the PL was a transient phenomenon; after an initial upregulation, we observed significant reduction of the NF-κB activity together with the repression of the inflammatory enzyme cyclooxygenase-2. Moreover, the medium of chondrocytes cultured in the simultaneous presence of PL and IL-1α, showed a significant enhancement of the chemoattractant activity versus untreated chondrocytes. Our findings support the concept that the platelet products have a direct beneficial effect on articular chondrocytes and could drive in sequence a transient activation and the resolution of the inflammatory process, thus providing a rational for their use as therapeutic agents in cartilage inflammation and damage.

  2. Spatial regulation of bone morphogenetic proteins (BMPs) in postnatal articular and growth plate cartilage

    Science.gov (United States)

    Garrison, Presley; Yue, Shanna; Hanson, Jeffrey; Baron, Jeffrey; Lui, Julian C.

    2017-01-01

    Articular and growth plate cartilage both arise from condensations of mesenchymal cells, but ultimately develop important histological and functional differences. Each is composed of three layers—the superficial, mid and deep zones of articular cartilage and the resting, proliferative and hypertrophic zones of growth plate cartilage. The bone morphogenetic protein (BMP) system plays an important role in cartilage development. A gradient in expression of BMP-related genes has been observed across growth plate cartilage, likely playing a role in zonal differentiation. To investigate the presence of a similar expression gradient in articular cartilage, we used laser capture microdissection (LCM) to separate murine growth plate and articular cartilage from the proximal tibia into their six constituent zones, and used a solution hybridization assay with color-coded probes (nCounter) to quantify mRNAs for 30 different BMP-related genes in each zone. In situ hybridization and immunohistochemistry were then used to confirm spatial expression patterns. Expression gradients for Bmp2 and 6 were observed across growth plate cartilage with highest expression in hypertrophic zone. However, intracellular BMP signaling, assessed by phospho-Smad1/5/8 immunohistochemical staining, appeared to be higher in the proliferative zone and prehypertrophic area than in hypertrophic zone, possibly due to high expression of Smad7, an inhibitory Smad, in the hypertrophic zone. We also found BMP expression gradients across the articular cartilage with BMP agonists primarily expressed in the superficial zone and BMP functional antagonists primarily expressed in the deep zone. Phospho-Smad1/5/8 immunohistochemical staining showed a similar gradient. In combination with previous evidence that BMPs regulate chondrocyte proliferation and differentiation, the current findings suggest that BMP signaling gradients exist across both growth plate and articular cartilage and that these gradients may

  3. Doxycycline inhibits collagen synthesis by bovine chondrocytes cultured in alginate

    NARCIS (Netherlands)

    Beekman, B.; Verzijl, N.; Roos, J.A.D.M.de; Koopman, J.L.; Tekoppele, J.M.

    1997-01-01

    Doxycycline is known for its ability to inhibit matrix metalloproteinases (MMPs), a family of enzymes that play a role in cartilage breakdown in arthritides. Its prophylactic effect in reducing joint degradation in osteoarthritis is mainly attributed to this property. In this study, we show that

  4. Repair of cartilage defects in osteoarthritis rats with induced pluripotent stem cell derived chondrocytes.

    Science.gov (United States)

    Zhu, Yanxia; Wu, Xiaomin; Liang, Yuhong; Gu, Hongsheng; Song, Kedong; Zou, Xuenong; Zhou, Guangqian

    2016-11-09

    The incapacity of articular cartilage (AC) for self-repair after damage ultimately leads to the development of osteoarthritis. Stem cell-based therapy has been proposed for the treatment of osteoarthritis (OA) and induced pluripotent stem cells (iPSCs) are becoming a promising stem cell source. Three steps were developed to differentiate human iPSCs into chondrocytes which were transplanted into rat OA models induced by monosodium iodoacetate (MIA). After 6 days embryonic body (EB) formation and 2 weeks differentiation, the gene and protein expression of Col2A1, GAG and Sox9 has significantly increased compare to undifferentiated hiPSCs. After 15 weeks transplantation, no immune responses were observed, micro-CT showed gradual engraftment and the improvement of subchondrol plate integrity, and histological examinations demonstrated articular cartilage matrix production. hiPSC could be an efficient and clinically translatable approach for cartilage tissue regeneration in OA cartilages.

  5. Intra-articular injection of the selective cyclooxygenase-2 inhibitor meloxicam (Mobic) reduces experimental osteoarthritis and nociception in rats.

    Science.gov (United States)

    Wen, Z-H; Tang, C-C; Chang, Y-C; Huang, S-Y; Chen, C-H; Wu, S-C; Hsieh, S-P; Hsieh, C-S; Wang, K-Y; Lin, S-Y; Lee, H-L; Lee, C-H; Kuo, H-C; Chen, W-F; Jean, Y-H

    2013-12-01

    To study the effect of intra-articular injection of meloxicam (Mobic) on the development of osteoarthritis (OA) in rats and examine concomitant changes in nociceptive behavior and the expression of mitogen-activated protein kinases (MAPKs) in articular cartilage chondrocytes. OA was induced in Wistar rats by right anterior cruciate ligament transection (ACLT); the left knee was not treated. The OA + meloxicam (1.0 mg) group was injected intra-articularly in the ACLT knee with 1.0 mg of meloxicam once a week for 5 consecutive weeks starting 5 weeks after ACLT. The OA + meloxicam (0.25 mg) group was treated similarly with 0.25 mg meloxicam. The sham group underwent arthrotomy only and received vehicle of 0.1 mL sterile 0.9% saline injections, whereas the naive rats in meloxicam-only groups were treated similarly with 1.0- and 0.25-mg meloxicam. Nociception was measured as secondary mechanical allodynia and hind paw weight-bearing distribution at before (pre-) and 5, 10, 15, and 20 weeks post-ACLT. Histopathology of the cartilage and synovia was examined 20 weeks after ACLT. Immunohistochemical analysis was performed to examine the effect of meloxicam on MAPKs (p38, c-Jun N-terminal kinase (JNK), and extracellular signal-regulated kinase (ERK)) expression in the articular cartilage chondrocytes. OA rats receiving intra-articular meloxicam treatment showed significantly less cartilage degeneration and synovitis than saline-treated controls. Nociception were improved in the OA + meloxicam groups compared with the OA group. Moreover, meloxicam attenuated p38 and JNK but enhanced ERK expression in OA-affected cartilage. Intra-articular injection of meloxicam (1) attenuates the development of OA, (2) concomitantly reduces nociception, and (3) modulates chondrocyte metabolism, possibly through inhibition of cellular p38 and JNK, but enhances ERK expression. Crown Copyright © 2013. Published by Elsevier Ltd. All rights reserved.

  6. 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.

  7. 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.

  8. Chondrocyte and mesenchymal stem cell-based therapies for cartilage repair in osteoarthritis and related orthopaedic conditions.

    Science.gov (United States)

    Mobasheri, Ali; Kalamegam, Gauthaman; Musumeci, Giuseppe; Batt, Mark E

    2014-07-01

    Osteoarthritis (OA) represents a final and common pathway for all major traumatic insults to synovial joints. OA is the most common form of degenerative joint disease and a major cause of pain and disability. Despite the global increase in the incidence of OA, there are no effective pharmacotherapies capable of restoring the original structure and function of damaged articular cartilage. Consequently cell-based and biological therapies for osteoarthritis (OA) and related orthopaedic disorders have become thriving areas of research and development. Autologous chondrocyte implantation (ACI) has been used for treatment of osteoarticular lesions for over two decades. Although chondrocyte-based therapy has the capacity to slow down the progression of OA and delay partial or total joint replacement surgery, currently used procedures are associated with the risk of serious adverse events. Complications of ACI include hypertrophy, disturbed fusion, delamination, and graft failure. Therefore there is significant interest in improving the success rate of ACI by improving surgical techniques and preserving the phenotype of the primary chondrocytes used in the procedure. Future tissue-engineering approaches for cartilage repair will also benefit from advances in chondrocyte-based repair strategies. This review article focuses on the structure and function of articular cartilage and the pathogenesis of OA in the context of the rising global burden of musculoskeletal disease. We explore the challenges associated with cartilage repair and regeneration using cell-based therapies that use chondrocytes and mesenchymal stem cells (MSCs). This paper also explores common misconceptions associated with cell-based therapy and highlights a few areas for future investigation. Copyright © 2014 The Authors. Published by Elsevier Ireland Ltd.. All rights reserved.

  9. Transient Receptor Potential Vanilloid 5 Mediates Ca2+ Influx and Inhibits Chondrocyte Autophagy in a Rat Osteoarthritis Model

    Directory of Open Access Journals (Sweden)

    Yingliang Wei

    2017-05-01

    Full Text Available Background: Autophagy, a self-protective mechanism of chondrocytes, has become a promising target to impede the progress of osteoarthritis (OA. Autophagy is regulated by cytosolic Ca2+ activity and may thus be modified by the Ca2+ permeable transient receptor potential channel vanilloid 5 (TRPV5. Therefore, we investigated the potential role of TRPV5 in mediating Ca2+ influx and in inhibiting chondrocyte autophagy in a rat OA model. Methods: The rat OA model was assessed by macroscopic and histological analyses. light chain 3B (LC3B immunolocalization was detected by immunohistochemistry. TRPV5, LC3B and calmodulin in OA articular cartilage were assessed by real time polymerase chain reaction (RT-PCR and western blotting. TRPV5 small interfering RNA (TRPV5 siRNA were transfected into rat primary chondrocyte then the calmodulin and LC3B was detected by immunofluorescence. The functionality of the TRPV5 was assessed by Ca2+ influx. Western blot was used to measure autophagy-related proteins. Results: We constructed a monosodium iodoacetate (MIA -induced rat OA model and found that ruthenium red (TRPV5 inhibitor slowed the progression of joint destruction. We found that the TRPV5 and calmodulin were up-regulated but LC3B was down-regulated in articular cartilage following prolonged progression of OA. Furthermore, the up-regulated TRPV5 channel caused an increase in the Ca2+ influx in chondrocytes. The up-regulation of TRPV5 stimulated Ca2+ influx, which inhibited autophagy by increasing the production of calmodulin, phosphorylation of calmodulin dependent protein kinases II (p-CAMK II, phosphorylation of Beclin1 (p-Beclin1, and protein of B-cell lymphoma-2 (Bcl-2, and attenuating ratio of LC3-II/ LC3-. Conclusion: Up-regulated TRPV5 as an initiating factor inhibited chondrocyte autophagy via the mediation of Ca2+ influx.

  10. Assessment of strategies to increase chondrocyte viability in cryopreserved human osteochondral allografts: evaluation of the glycosylated hydroquinone, arbutin.

    Science.gov (United States)

    Rosa, S C; Gonçalves, J; Judas, F; Lopes, C; Mendes, A F

    2009-12-01

    Allogeneic cartilage is used to repair damaged areas of articular cartilage, requiring the presence of living chondrocytes. So far, no preservation method can effectively meet that purpose. Identification of more effective cryoprotective agents (CPAs) can contribute to this goal. The aim of this study was to determine whether the glycosylated hydroquinone, arbutin, alone or in combination with low concentrations of other CPAs, has cryoprotective properties towards human articular cartilage. Human tibial plateaus were procured from multi-organ donors, with the approval of the Ethics Committee of the University Hospital of Coimbra. The tibial plateaus were treated with or without arbutin (50 or 100mM), alone or in combination with various concentrations of dimethyl sulfoxide (DMSO) and glycerol, for 0.5-1.5h/37 degrees C, then frozen at -20 degrees C and 24h later transferred to a biofreezer at -80 degrees C. Two to 3 months later, thawing was achieved by immersion in cell culture medium at 37 degrees C/1h. Chondrocyte viability was assessed before and after freeze-thawing using a colorimetric assay based on the cell's metabolic activity and fluorescent dyes to evaluate cell membrane integrity. Before freezing, chondrocyte metabolic activity was identical in all the conditions tested. After freeze-thawing, the highest activity, corresponding to 34.2+/-2.1% of that in the Fresh Control, was achieved in tibial plateaus incubated in 50mM arbutin for 1h whereas in those left untreated it was 11.1+/-4.7. Addition of DMSO and glycerol to arbutin did not increase chondrocyte viability any further. Fluorescence microscopy confirmed these results and showed that living chondrocytes were mainly restricted to the superficial cartilage layers. Arbutin seems to be an effective cryoprotective agent for osteochondral allografts with potential benefits over DMSO and glycerol.

  11. 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.

  12. Rapamycin Maintains the Chondrocytic Phenotype and Interferes with Inflammatory Cytokine Induced Processes

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    Andrea De Luna-Preitschopf

    2017-07-01

    Full Text Available Osteoarthritis (OA is hallmarked by a progressive degradation of articular cartilage. Besides risk factors including trauma, obesity or genetic predisposition, inflammation has a major impact on the development of this chronic disease. During the course of inflammation, cytokines such as tumor necrosis factor-alpha(TNF-α and interleukin (IL-1β are secreted by activated chondrocytes as well as synovial cells and stimulate the production of other inflammatory cytokines and matrix degrading enzymes. The mTORC1 inhibitor rapamycin is a clinical approved immunosuppressant and several studies also verified its chondroprotective effects in OA. However, the effect of blocking the mechanistic target of rapamycin complex (mTORC1 on the inflammatory status within OA is not well studied. Therefore, we aimed to investigate if inhibition of mTORC1 by rapamycin can preserve and sustain chondrocytes in an inflammatory environment. Patient-derived chondrocytes were cultured in media supplemented with or without the mTORC1 inhibitor rapamycin. To establish an inflammatory environment, either TNF-α or IL-1β was added to the media (=OA-model. The chondroprotective and anti-inflammatory effects of rapamycin were evaluated using sulfated glycosaminoglycan (sGAG release assay, Caspase 3/7 activity assay, lactate dehydrogenase (LDH assay and quantitative real time polymerase chain reaction (PCR. Blocking mTORC1 by rapamycin reduced the release and therefore degradation of sGAGs, which are components of the extracellular matrix secreted by chondrocytes. Furthermore, blocking mTORC1 in OA chondrocytes resulted in an enhanced expression of the main chondrogenic markers. Rapamycin was able to protect chondrocytes from cell death in an OA-model shown by reduced Caspase 3/7 activity and diminished LDH release. Furthermore, inhibition of mTORC1 preserved the chondrogenic phenotype of OA chondrocytes, but also reduced inflammatory processes within the OA-model. This study

  13. Ongoing studies of cell-based therapies for articular cartilage defects in Japan

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    Ogura T

    2014-12-01

    Full Text Available Takahiro Ogura,1 Akihiro Tsuchiya,2 Shuichi Mizuno1 1Department of Orthopedic Surgery, Brigham and Women’s Hospital and Harvard Medical School, Boston, MA, USA; 2Funabashi Orthopaedic Hospital Sports Medicine Center, Funabashi, Chiba, Japan Abstract: Recently, cell-based therapies have generated great interest in the repair of articular cartilage defects and degeneration. Surgical treatments for these indications have multiple options, including marrow stimulation, osteochondral autograft transplant, and autologous chondrocyte implantation. The autologous chondrocyte implantation technique has been improved using a cell scaffold and other devices. Meanwhile, advanced cell-based therapies, including cultured stem cell treatment, have been studied in clinical trials. Most studies have been designed and authorized by institutional review boards and/or the regulatory agencies of the investigators’ countries. For cellular products in regenerative medicine, regulations of many countries are amenable to expedited approval. This paper aims to provide an update on ongoing and prospective cell-based therapies, focusing on articular cartilage injury at designated institutions authorized by the Japanese Pharmaceutical and Medical Device Agency. Keywords: autologous chondrocyte implantation, mesenchymal stem cell, knee joint

  14. Structural Dynamics of Chondrocytes during Culturing.

    Science.gov (United States)

    Omelyanenko, N P; Rodionov, S A

    2017-12-01

    We performed comparative analysis of the morphology of chondrocytes in normal cartilage, after their isolation from the tissue, and at different stages of culturing; structural dynamics of cells during culturing was also studied. Significant morphological differences in chondrocytes at the specified stages of their preparation to in vivo use were revealed. Pronounced structural changes (blebbing and cytoplasm swelling) were found in chondrocytes before their implantation, which can affect the formation of cartilage regenerate. The study was performed using light microscopy methods including time-lapse recording of the cell cultures with differential interference Nomarski contrasting combined with transmission electron microscopy.

  15. Subtractive gene expression profiling of articular cartilage and mesenchymal stem cells: serpins as cartilage-relevant differentiation markers.

    Science.gov (United States)

    Boeuf, S; Steck, E; Pelttari, K; Hennig, T; Buneb, A; Benz, K; Witte, D; Sültmann, H; Poustka, A; Richter, W

    2008-01-01

    Mesenchymal stem cells (MSCs) are a population of cells broadly discussed to support cartilage repair. The differentiation of MSCs into articular chondrocytes is, however, still poorly understood on the molecular level. The aim of this study was to perform an almost genome-wide screen for genes differentially expressed between cartilage and MSCs and to extract new markers useful to define chondrocyte differentiation stages. Gene expression profiles of MSCs (n=8) and articular cartilage from OA patients (n=7) were compared on a 30,000 cDNA-fragment array and differentially expressed genes were extracted by subtraction. Expression of selected genes was assessed during in vitro chondrogenic differentiation of MSCs and during dedifferentiation of expanded chondrocytes using quantitative and semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR). Protein secretion was measured by enzyme-linked immunosorbent assay. Eighty-seven genes were differentially expressed between MSCs and cartilage with a more than three-fold difference. Sixty-seven of them were higher expressed in cartilage and among them 15 genes were previously not detected in cartilage. Differential expression was confirmed for 69% of 26 reanalysed genes by RT-PCR. The profiles of three unknown transcripts and six protease-related molecules were characterised during differentiation. SERPINA1 and SERPINA3 mRNA expression correlated with chondrogenic differentiation of MSCs and dedifferentiation of chondrocytes, and SERPINA1 protein levels in culture supernatants could be correlated alike. cDNA-array analysis identified SERPINA1 and A3 as new differentiation-relevant genes for cartilage. Since SERPINA1 secretion correlated with both chondrogenesis of MSCs and dedifferentiation during chondrocyte expansion, it represents an attractive marker for refinement of chondrocyte differentiation.

  16. In Vivo Articular Cartilage Regeneration Using Human Dental Pulp Stem Cells Cultured in an Alginate Scaffold: A Preliminary Study

    Directory of Open Access Journals (Sweden)

    Manuel Mata

    2017-01-01

    Full Text Available Osteoarthritis is an inflammatory disease in which all joint-related elements, articular cartilage in particular, are affected. The poor regeneration capacity of this tissue together with the lack of pharmacological treatment has led to the development of regenerative medicine methodologies including microfracture and autologous chondrocyte implantation (ACI. The effectiveness of ACI has been shown in vitro and in vivo, but the use of other cell types, including bone marrow and adipose-derived mesenchymal stem cells, is necessary because of the poor proliferation rate of isolated articular chondrocytes. In this investigation, we assessed the chondrogenic ability of human dental pulp stem cells (hDPSCs to regenerate cartilage in vitro and in vivo. hDPSCs and primary isolated rabbit chondrocytes were cultured in chondrogenic culture medium and found to express collagen II and aggrecan. Both cell types were cultured in 3% alginate hydrogels and implanted in a rabbit model of cartilage damage. Three months after surgery, significant cartilage regeneration was observed, particularly in the animals implanted with hDPSCs. Although the results presented here are preliminary, they suggest that hDPSCs may be useful for regeneration of articular cartilage.

  17. Response of human chondrocytes and mesenchymal stromal cells to a decellularized human dermis

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    Giavaresi Gianluca

    2013-01-01

    Full Text Available Abstract Background Although progress has been made in the treatment of articular cartilage lesions, they are still a major challenge because current techniques do not provide satisfactory long-term outcomes. Tissue engineering and the use of functional biomaterials might be an alternative regenerative strategy and fulfill clinical needs. Decellularized extracellular matrices have generated interest as functional biologic scaffolds, but there are few studies on cartilage regeneration. The aim of this study was to evaluate in vitro the biological influence of a newly developed decellularized human dermal extracellular matrix on two human primary cultures. Methods Normal human articular chondrocytes (NHAC-kn and human mesenchymal stromal cells (hMSC from healthy donors were seeded in polystyrene wells as controls (CTR, and on decellularized human dermis batches (HDM_derm for 7 and 14 days. Cellular proliferation and differentiation, and anabolic and catabolic synthetic activity were quantified at each experimental time. Histology and scanning electron microscopy were used to evaluate morphology and ultrastructure. Results Both cell cultures had a similar proliferation rate that increased significantly (p p p p p p p p Conclusions The results obtained showed that in in vitro conditions HDM_derm behaves as a suitable scaffold for the growth of both well-differentiated chondrocytes and undifferentiated mesenchymal cells, thus ensuring a biocompatible and bioactive substrate. Further studies are mandatory to test the use of HDM_derm with tissue engineering to assess its therapeutic and functional effectiveness in cartilage regeneration.

  18. Human Developmental Chondrogenesis as a Basis for Engineering Chondrocytes from Pluripotent Stem Cells

    Science.gov (United States)

    Wu, Ling; Bluguermann, Carolina; Kyupelyan, Levon; Latour, Brooke; Gonzalez, Stephanie; Shah, Saumya; Galic, Zoran; Ge, Sundi; Zhu, Yuhua; Petrigliano, Frank A.; Nsair, Ali; Miriuka, Santiago G.; Li, Xinmin; Lyons, Karen M.; Crooks, Gay M.; McAllister, David R.; Van Handel, Ben; Adams, John S.; Evseenko, Denis

    2013-01-01

    Summary Joint injury and osteoarthritis affect millions of people worldwide, but attempts to generate articular cartilage using adult stem/progenitor cells have been unsuccessful. We hypothesized that recapitulation of the human developmental chondrogenic program using pluripotent stem cells (PSCs) may represent a superior approach for cartilage restoration. Using laser-capture microdissection followed by microarray analysis, we first defined a surface phenotype (CD166low/negCD146low/negCD73+CD44lowBMPR1B+) distinguishing the earliest cartilage committed cells (prechondrocytes) at 5–6 weeks of development. Functional studies confirmed these cells are chondrocyte progenitors. From 12 weeks, only the superficial layers of articular cartilage were enriched in cells with this progenitor phenotype. Isolation of cells with a similar immunophenotype from differentiating human PSCs revealed a population of CD166low/negBMPR1B+ putative cartilage-committed progenitors. Taken as a whole, these data define a developmental approach for the generation of highly purified functional human chondrocytes from PSCs that could enable substantial progress in cartilage tissue engineering. PMID:24371811

  19. Investigation of polarization-sensitive optical coherence tomography towards the study of microstructure of articular cartilage

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    Kasaragod, Deepa; Lu, Zenghai; Le Maitre, Christine; Wilkinson, J. Mark; Matcher, Stephen

    2013-03-01

    This paper highlights the extended Jones matrix calculus based multi-angle study carried out to understand the depth dependent structural orientation of the collagen fibers in articular cartilage using polarization-sensitive optical coherence tomography (PS-OCT). A 3D lamellar model for the collagen fiber orientation, with a quadratic profile for the arching of the collagen fibers in transitional zone which points towards an ordered arrangement of fibers in that zone is the basis of the organization architecture of collagen fibers in articular cartilage. Experimental data for both ex-vivo bovine fetlock and human patellar cartilage samples are compared with theoretical predictions, with a good quantitative agreement for bovine and a reasonable qualitative agreement for human articular cartilage samples being obtained

  20. Glucose adsorption to chitosan membranes increases proliferation of human chondrocyte via mammalian target of rapamycin complex 1 and sterol regulatory element-binding protein-1 signaling.

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    Chang, Shun-Fu; Huang, Kuo-Chin; Cheng, Chin-Chang; Su, Yu-Ping; Lee, Ko-Chao; Chen, Cheng-Nan; Chang, Hsin-I

    2017-10-01

    Osteoarthritis (OA) is currently still an irreversible degenerative disease of the articular cartilage. Recent, dextrose (d-glucose) intraarticular injection prolotherapy for OA patients has been reported to benefit the chondrogenic stimulation of damaged cartilage. However, the detailed mechanism of glucose's effect on cartilage repair remains unclear. Chitosan, a naturally derived polysaccharide, has recently been investigated as a surgical or dental dressing to control breeding. Therefore, in this study, glucose was adsorbed to chitosan membranes (CTS-Glc), and the study aimed to investigate whether CTS-Glc complex membranes could regulate the proliferation of human OA chondrocytes and to explore the underlying mechanism. Human OA and SW1353 chondrocytes were used in this study. The experiments involving the transfection of cells used SW1353 chondrocytes. A specific inhibitor and siRNAs were used to investigate the mechanism underlying the CTS-Glc-regulated proliferation of human chondrocytes. We found that CTS-Glc significantly increased the proliferation of both human OA and SW1353 chondrocytes comparable to glucose- or chitosan-only stimulation. The role of mammalian target of rapamycin complex 1 (mTORC1) signaling, including mTOR, raptor, and S6k proteins, has been demonstrated in the regulation of CTS-Glc-increased human chondrocyte proliferation. mTORC1 signaling increased the expression levels of maturated SREBP-1 and FASN and then induced the expressions of cell cycle regulators, that is, cyclin D, cyclin-dependent kinase-4 and -6 in human chondrocytes. This study elucidates the detailed mechanism behind the effect of CTS-Glc complex membranes in promoting chondrocyte proliferation and proposes a possible clinical application of the CTS-Glc complex in the dextrose intraarticular injection of OA prolotherapy in the future to attenuate the pain and discomfort of OA patients. © 2017 Wiley Periodicals, Inc.

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

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    Zejun Liu

    2015-08-01

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

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

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    Pedersen Christian

    2010-04-01

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

  3. Tumor Proteins D52 and D54 Have Opposite Effects on the Terminal Differentiation of Chondrocytes

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    Chihiro Ito

    2017-01-01

    Full Text Available The tumor protein D (TPD family consists of four members, TPD52, TPD53, TPD54, and TPD55. The physiological roles of these genes in normal tissues, including epidermal and mesenchymal tissues, have rarely been reported. Herein, we examined the expression of TPD52 and TPD54 genes in cartilage in vivo and in vitro and investigated their involvement in the proliferation and differentiation of chondrocytes in vitro. TPD52 and TPD54 were uniformly expressed in articular cartilage and trabecular bone and were scarcely expressed in the epiphyseal growth plate. In MC3T3E-1 cells, the expressions of TPD52 and TPD54 were increased in a differentiation-dependent manner. In contrast, their expressions were decreased in ATDC5 cells. In ATDC5 cells, overexpression of TPD52 decreased alkaline phosphatase (ALPase activity, while knock-down of TPD52 showed little effect. In contrast, overexpression of TPD54 enhanced ALPase activity, Ca2+ deposition, and the expressions of type X collagen and ALPase genes, while knock-down of TPD54 reduced them. The results revealed that TPD52 inhibits and that TPD54 promotes the terminal differentiation of a chondrocyte cell line. As such, we report for the first time the important roles of TPD52 and TPD54, which work oppositely, in the terminal differentiation of chondrocytes during endochondral ossification.

  4. Rosmarinic acid down-regulates NO and PGE2 expression via MAPK pathway in rat chondrocytes.

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    Chen, We-Ping; Jin, Guo-Jun; Xiong, Yan; Hu, Peng-Fei; Bao, Jia-Peng; Wu, Li-Dong

    2017-09-25

    Rosmarinic acid (RosA) is a water-soluble polyphenol, which can be isolated from many herbs such as orthosiphon diffuses and rosmarinus officinalis. Previous studies have shown that RosA possesses various biological properties. In this study, we investigate the anti-osteoarthritic effects of RosA in rat articular chondrocytes. Chondrocytes were pre-treated with RosA, followed by the stimulation of IL-1β. Real-time PCR and Western blot were performed to detect the expression of matrix metalloproteinase (MMP)-1, MMP-3 and MMP-13. Nitric oxide and PGE2 production were measured by Griess reagent and enzyme-linked immunosorbent assay (ELISA). The expression of mitogen-activated protein kinase (MAPK) and nuclear factor-κB (NF-κB) was also investigated by Western blot analysis. We found that RosA down-regulated the MMPs expression as well as nitric oxide and PGE2 production in IL-1β-induced chondrocytes. In addition, RosA inhibited p38 and JNK phosphorylation as well as p65 translocation. The results suggest that RosA may be considered a possible agent in the treatment of OA. © 2017 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

  5. A kinetic modeling of chondrocyte culture for manufacture of tissue-engineered cartilage.

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    Kino-Oka, Masahiro; Maeda, Yoshikatsu; Yamamoto, Takeyuki; Sugawara, Katsura; Taya, Masahito

    2005-03-01

    For repairing articular cartilage defects, innovative techniques based on tissue engineering have been developed and are now entering into the practical stage of clinical application by means of grafting in vitro cultured products. A variety of natural and artificial materials available for scaffolds, which permit chondrocyte cells to aggregate, have been designed for their ability to promote cell growth and differentiation. From the viewpoint of the manufacturing process for tissue-engineered cartilage, the diverse nature of raw materials (seeding cells) and end products (cultured cartilage) oblige us to design a tailor-made process with less reproducibility, which is an obstacle to establishing a production doctrine based on bioengineering knowledge concerning growth kinetics and modeling as well as designs of bioreactors and culture operations for certification of high product quality. In this article, we review the recent advances in the manufacturing of tissue-engineered cartilage. After outlining the manufacturing processes for tissue-engineered cartilage in the first section, the second and third sections, respectively, describe the three-dimensional culture of chondrocytes with Aterocollagen gel and kinetic model consideration as a tool for evaluating this culture process. In the final section, culture strategy is discussed in terms of the combined processes of monolayer growth (ex vivo chondrocyte cell expansion) and three-dimensional growth (construction of cultured cartilage in the gel).

  6. Extracellular and intracellular mechanisms of mechanotransduction in three-dimensionally embedded rat chondrocytes.

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    Shioji, Suguru; Imai, Shinji; Ando, Kosei; Kumagai, Kousuke; Matsusue, Yoshitaka

    2014-01-01

    Articular cartilage homeostasis involves modulation of chondrocyte matrix synthesis in response to mechanical stress (MS). We studied extracellular and intracellular mechanotransduction pathways mediating this response. We first confirmed rapid up-regulation of the putative chondro-protective cytokine, interleukin (IL)-4, as an immediate response to MS. We then studied the role of IL-4 by investigating responses to exogenous IL-4 or a specific IL-4 inhibitor, combined with MS. Next we investigated the intracellular second messengers. Since chondrocyte phenotype alters according to the extracellular environment, we characterized the response to mechanotransduction in 3-dimensionally embedded chondrocytes. Expression of aggrecan and type II collagen was significantly up-regulated by exogenous IL-4 whereas MS-induced matrix synthesis was inhibited by an IL-4 blocker. Further, MS-induced matrix synthesis was completely blocked by a p38 MAPK inhibitor, while it was only partially blocked by inhibitors of other putative second messengers. IL-4 mediates an extracellular pathway of mechanotransduction, perhaps via an autocrine/paracrine loop, while p38 mediates an intracellular pathway prevalent only in a 3-dimensional environment.

  7. [Low-intensity pulsed ultrasound promotes extracellular matrix synthesis of human osteoarthritis chondrocytes].

    Science.gov (United States)

    DU, Dengkui; Chen, Shirong; Yi, Gang; Wang, Pan; Tang, Ying; Zheng, Liwen; Chen, Junnan

    2016-11-01

    Objective To investigate the effect of low-intensity pulsed ultrasound (LIPUS) on the extracellular matrix synthesis of human osteoarthritis (OA) chondrocytes and explore the underlying mechanism. Methods Human osteoarthritis chondrocytes were collected from abandoned articular cartilage. Then the cells were cultured and identified by toluidine blue staining and immunocytochemical staining of type 2 collagen. The passage 2 cells were randomly divided into 3 groups: control OA group, 30 mW/cm(2) LIPUS-treated OA group, 30 mW/cm(2) LIPUS combined with 5 μmol/L LY294002-treated OA group. LIPUS treatment was performed for 20 minutes per day, totally 7 days. The mRNA levels of Col2, aggrecan and matrix metalloprotease 13 (MMP-13) were determined by quantitative real-time PCR. The protein levels of Col2, aggrecan, Akt, p-Akt and MMP-13 were evaluated by Western blotting. Results Compared with the control OA group, the expressions of Col2 and aggrecan at both mRNA and protein levels significantly increased, and MMP-13 significantly reduced in the LIPUS-treated OA group. The p-Akt protein level was significantly elevated after LIPUS stimulation, but there was no significant difference in the Akt protein levels between the two groups. Moreover, LY294002, an inhibitor of PI3K/Akt, significantly suppressed the biological effect activated by LIPUS. Conclusion LIPUS enhances the synthesis and inhibits the degradation of the extracellular matrix in human osteoarthritis chondrocytes.

  8. Fibromodulin is expressed by both chondrocytes and osteoblasts during fetal bone development.

    Science.gov (United States)

    Gori, F; Schipani, E; Demay, M B

    Fibromodulin, a keratan-sulfate proteoglycan, was first isolated in articular cartilage and tendons. We have identified fibromodulin as a gene regulated during BMP-2-induced differentiation of a mouse prechondroblastic cell line. Because expression of fibromodulin during endochondral bone formation has not been studied, we examined whether selected cells of the chondrocytic and osteoblastic lineage expressed fibromodulin. Fibromodulin mRNA was detected in conditionally immortalized murine bone marrow stromal cells, osteoblasts, and growth plate chondrocytes, as well as in primary murine calvarial osteoblasts. We, therefore, investigated the temporo-spatial expression of fibromodulin in vivo during endochondral bone formation by in situ hybridization. Fibromodulin was first detected at 15.5 days post coitus (dpc) in the perichondrium and proliferating chondrocytes. Fibromodulin mRNA was also detected at 15.5 dpc in the bone collar and periosteum. At later time points fibromodulin was expressed in the primary spongiosa and the endosteum. To determine whether fibromodulin was expressed during intramembranous bone formation as well, in situ hybridization was performed on calvariae. Fibromodulin mRNA was present in calvarial osteoblasts from 15.5 dpc. These results demonstrate that fibromodulin is developmentally expressed in cartilage and bone cells during endochondral and intramembranous ossification. These findings suggest that this extracellular matrix protein plays a role in both endochondral and intramembranous bone formation. Copyright 2001 Wiley-Liss, Inc.

  9. Epigenetic regulation of leptin affects MMP‐13 expression in osteoarthritic chondrocytes: possible molecular target for osteoarthritis therapeutic intervention

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    Iliopoulos, D; Malizos, K N; Tsezou, A

    2007-01-01

    Objective To investigate whether epigenetic mechanisms can regulate leptin's expression and affect its downstream targets as metalloproteinases 3,9,13 in osteoarthritic chondrocytes. Methods DNA methylation in leptin promoter was measured by DNA bisulfite sequencing, and mRNA expression levels were measured by real‐time quantitative PCR in osteoarthritic as well as in normal cartilage. Osteoarthritic articular cartilage samples were obtained from two distinct locations of the knee (n = 15); from the main defective area of maximum load (advanced osteoarthritis (OA)) and from adjacent macroscopically intact regions (minimal OA). Using small interference RNA, we tested if leptin downregulation would affect matrix metalloproteinase (MMP)‐13 activity. We also evaluated the effect of the demethylating agent, 5′‐Aza‐2‐deoxycytidine (AZA) and of the histone deacetylase inhibitor trichostatin A (TSA) on leptin expression in chondrocyte cultures. Furthermore, we performed chromatin immunoprecipitation in leptin's promoter area. Results We found a correlation between leptin expression and DNA methylation and also that leptin controls MMP‐13 activity in chondrocytes. Leptin's downregulation with small interference RNA inhibited MMP‐13 expression dramatically. After 5‐AZA application in normal chondrocytes, leptin's methylation was decreased, while its expression was upregulated, and MMP‐13 was activated. Furthermore, TSA application in normal chondrocyte cultures increased leptin's expression. Also, chromatin immunoprecipitation in leptin's promoter after TSA treatment revealed that histone H3 lysines 9 and 14 were acetylated. Conclusion We found that epigenetic mechanisms regulate leptin's expression in chondrocytes affecting its downstream target MMP‐13. Small interference RNA against leptin deactivated directly MMP‐13, which was upregulated after leptin's epigenetic reactivation, raising the issue of leptin's therapeutic potential for

  10. Epigenetic regulation of leptin affects MMP-13 expression in osteoarthritic chondrocytes: possible molecular target for osteoarthritis therapeutic intervention.

    Science.gov (United States)

    Iliopoulos, D; Malizos, K N; Tsezou, A

    2007-12-01

    To investigate whether epigenetic mechanisms can regulate leptin's expression and affect its downstream targets as metalloproteinases 3,9,13 in osteoarthritic chondrocytes. DNA methylation in leptin promoter was measured by DNA bisulfite sequencing, and mRNA expression levels were measured by real-time quantitative PCR in osteoarthritic as well as in normal cartilage. Osteoarthritic articular cartilage samples were obtained from two distinct locations of the knee (n = 15); from the main defective area of maximum load (advanced osteoarthritis (OA)) and from adjacent macroscopically intact regions (minimal OA). Using small interference RNA, we tested if leptin downregulation would affect matrix metalloproteinase (MMP)-13 activity. We also evaluated the effect of the demethylating agent, 5'-Aza-2-deoxycytidine (AZA) and of the histone deacetylase inhibitor trichostatin A (TSA) on leptin expression in chondrocyte cultures. Furthermore, we performed chromatin immunoprecipitation in leptin's promoter area. We found a correlation between leptin expression and DNA methylation and also that leptin controls MMP-13 activity in chondrocytes. Leptin's downregulation with small interference RNA inhibited MMP-13 expression dramatically. After 5-AZA application in normal chondrocytes, leptin's methylation was decreased, while its expression was upregulated, and MMP-13 was activated. Furthermore, TSA application in normal chondrocyte cultures increased leptin's expression. Also, chromatin immunoprecipitation in leptin's promoter after TSA treatment revealed that histone H3 lysines 9 and 14 were acetylated. We found that epigenetic mechanisms regulate leptin's expression in chondrocytes affecting its downstream target MMP-13. Small interference RNA against leptin deactivated directly MMP-13, which was upregulated after leptin's epigenetic reactivation, raising the issue of leptin's therapeutic potential for osteoarthritis.

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

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    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.

  12. Low oxygen conditions promote synergistic increases in chondrogenesis during co-culture of human osteoarthritic stem cells and chondrocytes.

    Science.gov (United States)

    Critchley, Susan E; Eswaramoorthy, Rajalakshmanan; Kelly, Daniel J

    2017-11-26

    There has been increased interest in co-cultures of stem cells and chondrocytes for cartilage tissue engineering as there are the limitations associated with using either cell type alone. Drawbacks associated with the use of chondrocytes include the limited numbers of cells available for isolation from damaged or diseased joints, their dedifferentiation during in vitro expansion and a diminished capacity to synthesise cartilage-specific extracellular matrix components with age and disease. This has motivated the use of adult stem cells with either freshly isolated or culture expanded chondrocytes for cartilage repair applications, however the ideal combination of cells and environmental conditions for promoting robust chondrogenesis remains unclear. In this study, we compared the effect of combining a small number of freshly isolated or culture expanded human chondrocytes with infrapatellar fat pad derived stem cells (FPSCs) from osteoarthritic donors on chondrogenesis in altered oxygen (5% or 20%) and growth factor supplementation (TGF-β3 only or TGF-β3 and BMP-7) conditions. Both co-cultures, but particularly those including freshly isolated chondrocytes, were found to promote cell proliferation and enhanced matrix accumulation compared to the use of FPSCs alone, resulting in the development of a tissue that was compositionally more similar to that of the native articular cartilage. Local oxygen levels were found to impact chondrogenesis in co-cultures, with more robust increases in proteoglycan and collagen deposition observed at 5% O2 . Additionally, collagen type I synthesis was supressed in co-cultures maintained at low oxygen conditions. This study demonstrates that a co-culture of freshly isolated human chondrocytes and FPSCs promotes robust chondrogenesis and thus is a promising cell combination for cartilage tissue engineering. This article is protected by copyright. All rights reserved.

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

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    Koh Terauchi

    2016-06-01

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

  14. Intra-Articular Injections of Polyphenols Protect Articular Cartilage from Inflammation-Induced Degradation: Suggesting a Potential Role in Cartilage Therapeutics

    Science.gov (United States)

    Natarajan, Venkatachalam; Madhan, Balaraman; Tiku, Moti L.

    2015-01-01

    Arthritic diseases, such as osteoarthritis and rheumatoid arthritis, inflict an enormous health care burden on society. Osteoarthritis, a degenerative joint disease with high prevalence among older people, and rheumatoid arthritis, an autoimmune inflammatory disease, both lead to irreversible structural and functional damage to articular cartilage. The aim of this study was to investigate the effect of polyphenols such as catechin, quercetin, epigallocatechin gallate, and tannic acid, on crosslinking type II collagen and the roles of these agents in managing in vivo articular cartilage degradation. The thermal, enzymatic, and physical stability of bovine articular cartilage explants following polyphenolic treatment were assessed for efficiency. Epigallocatechin gallate and tannic acid-treated explants showed >12 °C increase over native cartilage in thermal stability, thereby confirming cartilage crosslinking. Polyphenol-treated cartilage also showed a significant reduction in the percentage of collagen degradation and the release of glycosaminoglycans against collagenase digestion, indicating the increase physical integrity and resistance of polyphenol crosslinked cartilage to enzymatic digestion. To examine the in vivo cartilage protective effects, polyphenols were injected intra-articularly before (prophylactic) and after (therapeutic) the induction of collagen-induced arthritis in rats. The hind paw volume and histomorphological scoring was done for cartilage damage. The intra-articular injection of epigallocatechin gallate and tannic acid did not significantly influence the time of onset or the intensity of joint inflammation. However, histomorphological scoring of the articular cartilage showed a significant reduction in cartilage degradation in prophylactic- and therapeutic-groups, indicating that intra-articular injections of polyphenols bind to articular cartilage and making it resistant to degradation despite ongoing inflammation. These studies establish

  15. Effect of freezing on rabbit cultured chondrocytes

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    R.R Filgueiras

    2011-02-01

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

  16. Characterization of the chondrocyte secretome in photoclickable poly(ethylene glycol) hydrogels.

    Science.gov (United States)

    Schneider, Margaret C; Barnes, Christopher A; Bryant, Stephanie J

    2017-09-01

    Poly(ethylene glycol) (PEG) hydrogels are highly tunable platforms that are promising cell delivery vehicles for chondrocytes and cartilage tissue engineering. In addition to characterizing the type of extracellular matrix (ECM) that forms, understanding the types of proteins that are secreted by encapsulated cells may be important. Thus, the objectives for this study were to characterize the secretome of chondrocytes encapsulated in PEG hydrogels and determine whether the secretome varies as a function of hydrogel stiffness and culture condition. Bovine chondrocytes were encapsulated in photoclickable PEG hydrogels with a compressive modulus of 8 and 46 kPa and cultured under free swelling or dynamic compressive loading conditions. Cartilage ECM deposition was assessed by biochemical assays and immunohistochemistry. The conditioned medium was analyzed by liquid chromatography-tandem mass spectrometry. Chondrocytes maintained their phenotype within the hydrogels and deposited cartilage-specific ECM that increased over time and included aggrecan and collagens II and VI. Analysis of the secretome revealed a total of 64 proteins, which were largely similar among all experimental conditions. The identified proteins have diverse functions such as biological regulation, response to stress, and collagen fibril organization. Notably, many of the proteins important to the assembly of a collagen-rich cartilage ECM were identified and included collagen types II(α1), VI (α1, α2, and α3), IX (α1), XI (α1 and α2), and biglycan. In addition, many of the other identified proteins have been reported to be present within cell-secreted exosomes. In summary, chondrocytes encapsulated within photoclickable PEG hydrogels secrete many types of proteins that diffuse out of the hydrogel and which have diverse functions, but which are largely preserved across different hydrogel culture environments. Biotechnol. Bioeng. 2017;114: 2096-2108. © 2017 Wiley Periodicals, Inc. © 2017

  17. Development of a cartilage composite utilizing porous tantalum, fibrin, and rabbit chondrocytes for treatment of cartilage defect.

    Science.gov (United States)

    Jamil, Kamal; Chua, Kien-Hui; Joudi, Samad; Ng, Sook-Luan; Yahaya, Nor Hamdan

    2015-02-07

    Functional tissue engineering has emerged as a potential means for treatment of cartilage defect. Development of a stable cartilage composite is considered to be a good option. The aim of the study was to observe whether the incorporation of cultured chondrocytes on porous tantalum utilizing fibrin as a cell carrier would promote cartilage tissue formation. Rabbit articular chondrocytes were cultured and seeded onto tantalum with fibrin as temporary matrix in a composite, which was divided into three groups. The first group was kept in vitro while a total of 12 constructs were implanted into the dorsum of mice for the second and third groups. The implanted tissues were harvested after 4 weeks (second group) and after 8 weeks (third group). Specific characteristic of cartilage growth were studied by histological and biochemical assessment, immunohistochemistry, and quantitative PCR analysis. Histological and biochemical evaluation of the formed cartilage using hematoxylin and eosin and Alcian blue staining showed lacunae chondrocytes embedded in the proteoglycan rich matrix. Dimethylmethylene blue assay demonstrated high glycosaminoglycans content in the removed tissue following 8 weeks of implantation. Immunohistochemistry results showed the composites after implantation expressed high collagen type II. Quantitative PCR results confirmed a significant increase in cartilage associated genes expression (collagen type II, AggC, Sox 9) after implantation. Tantalum scaffold with fibrin as cell carrier promotes chondrocyte proliferation and cartilaginous tissue formation. Producing hyaline cartilage within a stable construct of tantalum and fibrin has a potential for treatment of cartilage defect.

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

    DEFF Research Database (Denmark)

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

    2010-01-01

    PURPOSE: We report the 2-year clinical results and identify prognostic factors in patients treated with autologous chondrocyte transplantation by use of a collagen membrane to seed the chondrocytes (ACT-CS). METHODS: This is a prospective study of 59 patients who were treated with ACT-CS and foll...

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

    DEFF Research Database (Denmark)

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

    2017-01-01

    Purpose: The use of passaged chondrocytes is the current standard for autologous chondrocyte implantation (ACI). De-differentiation due to amplification and donor site morbidity are known drawbacks highlighting the need for alternative cell sources. Methods: Via clinically validated flow cytometr...

  20. Hyaluronan suppresses lidocaine-induced apoptosis of human chondrocytes in vitro by inhibiting the p53-dependent mitochondrial apoptotic pathway

    Science.gov (United States)

    Lee, Yoon-Jin; Kim, Soo A; Lee, Sang-Han

    2016-01-01

    Aim: Intra-articular injection of local anesthetics (LAs) is a common procedure for therapeutic purposes. However, LAs have been found toxic to articular cartilage, and hyaluronan may attenuate this toxicity. In this study we investigated whether hyaluronan attenuated lidocaine-induced chondrotoxicity, and if so, to elucidate the underlying mechanisms. Methods: Human chondrocyte cell line SW1353 and newly isolated murine chondrocytes were incubated in culture medium containing hyaluronan and/or lidocaine for 72 h. Cell viability was evaluated using MTT assay. Cell apoptosis was detected with DAPI staining, caspase 3/7 activity assay and flow cytometry. Cell cycle distributions, ROS levels and mitochondrial membrane potential (ΔΨm) were determined using flow cytometry. The expression of p53 and p53-regulated gene products was measured with Western blotting. Results: Lidocaine (0.005%−0.03%) dose-dependently decreased the viability of SW1353 cells. This local anesthetic (0.015%, 0.025%) induced apoptosis, G2/M phase arrest and loss of ΔΨm, and markedly increased ROS production in SW1353 cells. Hyaluronan (50−800 μg/mL) alone did not affect the cell viability, but co-treatment with hyaluronan (200 μg/mL) significantly attenuated lidocaine-induced apoptosis and other abnormalities in SW1353 cells. Furthermore, co-treatment with lidocaine and hyaluronan significantly decreased the levels of p53 and its transcription targets Bax and p21 in SW1353 cells, although treatment with lidocaine alone did not significantly change these proteins. Similar results were obtained in ex vivo cultured murine chondrocytes. Conclusion: Hyaluronan suppresses lidocaine-induced apoptosis of human chondrocytes in vitro through inhibiting the p53-dependent mitochondrial apoptotic pathway. PMID:27041463

  1. 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

  2. The distribution of YKL-40 in osteoarthritic and normal human articular cartilage

    DEFF Research Database (Denmark)

    Volck, B; Ostergaard, K; Johansen, J S

    1999-01-01

    YKL-40, also called human cartilage glycoprotein-39, is a major secretory protein of human chondrocytes in cell culture. YKL-40 mRNA is expressed by cartilage from patients with rheumatoid arthritis, but is not detectable in normal human cartilage. The aim was to investigate the distribution of YKL......-40 in osteoarthritic (n=9) and macroscopically normal (n=5) human articular cartilage, collected from 12 pre-selected areas of the femoral head, to discover a potential role for YKL-40 in cartilage remodelling in osteoarthritis. Immunohistochemical analysis showed that YKL-40 staining was found...

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

    Science.gov (United States)

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

    2015-01-01

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

  4. 1,25-Dihydroxyvitamin D3 activates MMP13 gene expression in chondrocytes through p38 MARK pathway.

    Science.gov (United States)

    Chen, Dafu; Li, Yang; Dai, Xuejun; Zhou, Xinhua; Tian, Wei; Zhou, Yixin; Zou, Xuenong; Zhang, Chi

    2013-01-01

    Osteoarthritis (OA) is the most prevalent degenerative joint disease. The highly regulated balance of matrix synthesis and degradation is disrupted in OA, leading to progressive breakdown of articular cartilage. The molecular events and pathways involved in chondrocyte disfunction of cartilage in OA are not fully understood. It is known that 1,25-dihydroxyvitamin D₃(1,25-(OH)2D3) is synthesized by macrophages derived from synovial fluid of patients with inflammatory arthritis. Vitmain D receptor is expressed in chondrocytes within osteoarthritic cartilage, suggesting a contributory role of 1,25-(OH)2D3 in the aberrant behavior of chondrocytes in OA. However, the physiological function of 1,25-(OH)2D3 on chondrocytes in OA remains obscure. Effect of 1,25-(OH)2D3 on gene expression in chondrocytes was investigated in this study. We found that 1,25-(OH)2D3 activated MMP13 expression in a dose-dependent and time-dependent manner, a major enzyme that targets cartilage for degradation. Interestingly, a specific mitogen-activated protein kinase p38 inhibitor SB203580, but not JNK kinase inhibitor SP600125, abrogated 1,25-(OH)2D3 activation of MMP13 expression. 1,25-(OH)2D3-induced increase in MMP13 protein level was in parallel with the phosphorylation of p38 in chondrocytes. To further address the effect of 1,25-(OH)2D3 on MMP13 expression, transfection assays were used to show that 1,25-(OH)2D3 activated the MMP13 promoter reporter expression. MMP13 is known to target type II collagen and aggrecan for degradation, two major components of cartilage matrix. We observed that the treatment of 1,25-(OH)2D3 in chondrocytes results in downregulation of both type II collagen and aggrecan while MMP13 was upregulated. Taken together, we provide the first evidence to demonstrate that 1,25-(OH)2D3 activates MMP13 expression through p38 pathway in chondrocytes. Since MMP13 plays a major role in cartilage degradation in OA, we speculate that the ability of 1,25-(OH)2D3 to

  5. 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.

  6. Cell Death in Chondrocytes, Osteoblasts, and Osteocytes

    Directory of Open Access Journals (Sweden)

    Toshihisa Komori

    2016-12-01

    Full Text Available Cell death in skeletal component cells, including chondrocytes, osteoblasts, and osteocytes, plays roles in skeletal development, maintenance, and repair as well as in the pathogenesis of osteoarthritis and osteoporosis. Chondrocyte proliferation, differentiation, and apoptosis are important steps for endochondral ossification. Although the inactivation of P53 and RB is involved in the pathogenesis of osteosarcomas, the deletion of p53 and inactivation of Rb are insufficient to enhance chondrocyte proliferation, indicating the presence of multiple inhibitory mechanisms against sarcomagenesis in chondrocytes. The inflammatory processes induced by mechanical injury and chondrocyte death through the release of danger-associated molecular patterns (DAMPs are involved in the pathogenesis of posttraumatic osteoarthritis. The overexpression of BCLXL increases bone volume with a normal structure and maintains bone during aging by inhibiting osteoblast apoptosis. p53 inhibits osteoblast proliferation and enhances osteoblast apoptosis, thereby reducing bone formation, but also exerts positive effects on osteoblast differentiation through the Akt–FoxOs pathway. Apoptotic osteocytes release ATP, which induces the receptor activator of nuclear factor κ-B ligand (Rankl expression and osteoclastogenesis, from pannexin 1 channels. Osteocyte death ultimately results in necrosis; DAMPs are released to the bone surface and promote the production of proinflammatory cytokines, which induce Rankl expression, and osteoclastogenesis is further enhanced.

  7. Galectin-1 and galectin-3 expression in equine mesenchymal stromal cells (MSCs, synovial fibroblasts and chondrocytes, and the effect of inflammation on MSC motility

    Directory of Open Access Journals (Sweden)

    Heidi L. Reesink

    2017-11-01

    Full Text Available Abstract Background Mesenchymal stromal cells (MSCs can be used intra-articularly to quell inflammation and promote cartilage healing; however, mechanisms by which MSCs mitigate joint disease remain poorly understood. Galectins, a family of β-galactoside binding proteins, regulate inflammation, adhesion and cell migration in diverse cell types. Galectin-1 and galectin-3 are proposed to be important intra-articular modulators of inflammation in both osteoarthritis and rheumatoid arthritis. Here, we asked whether equine bone marrow-derived MSCs (BMSCs express higher levels of galectin-1 and -3 relative to synovial fibroblasts and chondrocytes and if an inflammatory environment affects BMSC galectin expression and motility. Methods Equine galectin-1 and -3 gene expression was quantified using qRT-PCR in cultured BMSCs, synoviocytes and articular chondrocytes, in addition to synovial membrane and articular cartilage tissues. Galectin gene expression, protein expression, and protein secretion were measured in equine BMSCs following exposure to inflammatory cytokines (IL-1β 5 and 10 ng/mL, TNF-α 25 and 50 ng/mL, or LPS 0.1, 1, 10 and 50 μg/mL. BMSC focal adhesion formation was assessed using confocal microscopy, and BMSC motility was quantified in the presence of inflammatory cytokines (IL-1β or TNF-α and the pan-galectin inhibitor β-lactose (100 and 200 mM. Results Equine BMSCs expressed 3-fold higher galectin-1 mRNA levels as compared to cultured synovial fibroblasts (p = 0.0005 and 30-fold higher galectin-1 (p < 0.0001 relative to cultured chondrocytes. BMSC galectin-1 mRNA expression was significantly increased as compared to carpal synovial membrane and articular cartilage tissues (p < 0.0001. IL-1β and TNF-α treatments decreased BMSC galectin gene expression and impaired BMSC motility in dose-dependent fashion but did not alter galectin protein expression. β-lactose abrogated BMSC focal adhesion formation and inhibited

  8. Pharmacoproteomic study of three different chondroitin sulfate compounds on intracellular and extracellular human chondrocyte proteomes.

    Science.gov (United States)

    Calamia, Valentina; Fernández-Puente, Patricia; Mateos, Jesús; Lourido, Lucía; Rocha, Beatriz; Montell, Eulália; Vergés, Josep; Ruiz-Romero, Cristina; Blanco, Francisco J

    2012-06-01

    Chondroitin sulfate (CS) is a symptomatic slow acting drug for osteoarthritis (OA) widely used for the treatment of this highly prevalent disease, characterized by articular cartilage degradation. However, little is known about its mechanism of action, and recent large scale clinical trials have reported variable results on OA symptoms. Herein, we aimed to study the modulations in the intracellular proteome and the secretome of human articular cartilage cells (chondrocytes) treated with three different CS compounds, with different origin or purity, by two complementary proteomic approaches. Osteoarthritic cells were treated with 200 μg/ml of each brand of CS. Quantitative proteomics experiments were carried out by the DIGE and stable isotope labeling with amino acids in cell culture (SILAC) techniques, followed by LC-MALDI-MS/MS analysis. The DIGE study, carried out on chondrocyte whole cell extracts, led to the detection of 46 spots that were differential between conditions in our study: 27 were modulated by CS1, 4 were modulated by CS2, and 15 were modulated by CS3. The SILAC experiment, carried out on the subset of chondrocyte-secreted proteins, allowed us to identify 104 different proteins. Most of them were extracellular matrix components, and 21 were modulated by CS1, 13 were modulated by CS2, and 9 were modulated by CS3. Each of the studied compounds induces a characteristic protein profile in OA chondrocytes. CS1 displayed the widest effect but increased the mitochondrial superoxide dismutase, the cartilage oligomeric matrix protein, and some catabolic or inflammatory factors like interstitial collagenase, stromelysin-1, and pentraxin-related protein. CS2 and CS3, on the other hand, increased a number of structural proteins, growth factors, and extracellular matrix proteins. Our study shows how, from the three CS compounds tested, CS1 induces the activation of inflammatory and catabolic pathways, whereas CS2 and CS3 induce an anti-inflammatory and anabolic

  9. Cdc42 is essential for both articular cartilage degeneration and subchondral bone deterioration in experimental osteoarthritis.

    Science.gov (United States)

    Hu, Xinhua; Ji, Xing; Yang, Mengting; Fan, Shihao; Wang, Jirong; Lu, Meiping; Shi, Wei; Mei, Liu; Xu, Chengyun; Fan, Xueying; Hussain, Musaddique; Du, Jingyu; Wu, Junsong; Wu, Ximei

    2018-01-03

    Cdc42, a member of Rho family small GTPases, is critical for cartilage development. We investigated the roles of Cdc42 in osteoarthritis and explored the potential mechanism underlying Cdc42-mediated articular cartilage degeneration and subchondral bone deterioration. Cdc42 is highly expressed in both articular cartilage and subchondral bone in a mouse osteoarthritis model with surgical destabilisation of the medial meniscus (DMM) in the knee joints. Specifically, genetic disruption of Cdc42, knockdown of Cdc42 expression, or inhibition of Cdc42 activity robustly attenuates the DMM-induced destruction, hypertrophy, high expression of matrix metallopeptidase-13 and collagen X, and activation of Stat3 in articular cartilages. Notably, genetic disruption of Cdc42, knockdown of Cdc42 expression or inhibition of Cdc42 activity significantly restored the increased numbers of mesenchymal stem cells, osteoprogenitors, osteoblasts, osteoclasts, and neovascularised vessels, the increased bone mass, and the activated Erk1/2, Smad1/5 and Smad2 in subchondral bone of DMM-operated mice. Mechanistically, Cdc42 mediates interleukin-1β-induced interleukin-6 production and subsequent Jak/Stat3 activation to regulate chondrocytic inflammation, and also lies upstream of Erk/Smads to regulate subchondral bone remodelling during transform growth factor-β1 signalling. Cdc42 is apparently required for both articular cartilage degeneration and subchondral bone deterioration of osteoarthritis, thus, interventions targeting Cdc42 have potential in osteoarthritic therapy. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

  10. Effects of initial cell seeding in self assembly of articular cartilage.

    Science.gov (United States)

    Revell, Christopher M; Reynolds, Catherine E; Athanasiou, Kyriacos A

    2008-09-01

    Current forays into tissue engineering of articular cartilage in vitro using the self-assembling method have produced constructs possessing significant extracellular matrix and resulting mechanical properties. However, large numbers of native articular chondrocytes are necessary to produce functional engineered cartilage; all previous work with the self-assembling process has used 5.5 x 10(6) cells/construct. In this study, the effects of initial cell seeding (0.25-11 x 10(6) cells/construct) on tissue quality were investigated. Results showed that tissue engineered articular cartilage was formed, when using at least 2 million cells/construct, possessing dimensional, compositional, and compressive properties approaching those of native tissue. It was noted that higher seeding contributed to thicker constructs with larger diameters and had a significant effect on resulting biochemical and biomechanical properties. It was further observed that aggregate modulus increased with increased seeding. By combining gross morphological, histological, biochemical, and biomechanical results, an optimal initial seeding for the self-assembling process of 3.75 x 10(6) cells/construct was identified. This finding enhances the translatability of this tissue engineering process by reducing the number of cells needed for tissue engineering of articular cartilage by 32% while maintaining essential tissue properties.

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

    Science.gov (United States)

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

    2013-08-09

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

  12. [Bionic design of articular cartilage].

    Science.gov (United States)

    Qin, Jun; Zhang, Wenguang; Wu, Gang; Wang, Chengtao

    2008-02-01

    Natural articular cartilage is well known as a special connective tissue with multiple effects and functions, which are important and irreplaceable, in human synovial joints. Biomedical, histological and pathological characteristics of articular cartilage, as well as biomaterial, biomechanical and bio-tribological properties thereof, are summarized from a novel aspect of bionics. Bionic design of articualr cartilage at macro-level and micro-level is carried out from three aspects, i.e., structure, material, and function; and a bionic design model of articular cartilage is set up. As a result, this basic research would be helpful to providing theoretical and practical basis for innovational design and manufacturing of new-style artificial joint with "soft-cushion bearing", and of bionic artificial cartilage.

  13. Conditional deletion of Tgfbr2 in hypertrophic chondrocytes delays terminal chondrocyte differentiation.

    Science.gov (United States)

    Sueyoshi, Tatsuya; Yamamoto, Koji; Akiyama, Haruhiko

    2012-07-01

    Transforming growth factor β (Tgfb) signaling plays an important role in endochondral ossification. Previous studies of mice in which the Tgfb type II receptor gene (Tgfbr2) was deleted in the limb bud mesenchymal cells or differentiated chondrocytes showed defects in the development of the long bones or the axial skeleton, respectively. Here, we generated mouse embryos in which the Tgfbr2 gene was ablated in hypertrophic chondrocytes. These mice exhibited delays in both the hypertrophic conversion of proliferating chondrocytes and the subsequent terminal chondrocyte differentiation. The expression domains of Col10a1, Matrix metalloproteinase 13, and Osteopontin were small, and the expression of Vascular endothelial growth factor and Platelet endothelial cell adhesion molecule was downregulated. The calcification of the bone collar in the mutant mice was markedly delayed and the periosteum was thin, possibly because of the downregulation of Indian hedgehog expression. We conclude that Tgfb signaling in hypertrophic chondrocytes positively regulates terminal chondrocyte differentiation, angiogenesis in calcified cartilage, and osteogenesis in the bone collar, at least partly through Indian hedgehog signaling in vivo. Copyright © 2012 International Society of Matrix Biology. Published by Elsevier B.V. All rights reserved.

  14. Growth Plate Chondrocytes' Morphology and Intrauterine ...

    African Journals Online (AJOL)

    Background: Longitudinal bone growth is dependent on the state of the chondrocytes and the extracellular matrix of the growth plate. Hypervitaminosis A is known to result in limb shortening in several models, this anomaly has been related to the early closure of the epiphyseal plate. The specific contribution of the different ...

  15. 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...

  16. Caprine articular, meniscus and intervertebral disc cartilage : An integral analysis of collagen network and chondrocytes

    NARCIS (Netherlands)

    Vonk, Lucienne A.; Kroeze, Robert Jan; Doulabi, Behrouz Zandieh; Hoogendoorn, Roel J.; Huang, ChunLing; Helder, Marco N.; Everts, Vincent; Bank, Ruud A.

    Cartilage is a tissue with only limited reparative capacities. A small part of its volume is composed of cells, the remaining part being the hydrated extracellular matrix (ECM) with collagens and proteoglycans as its main constituents. The functioning of cartilage depends heavily on its ECM.

  17. Caprine articular, meniscus and intervertebral disc cartilage: an integral analysis of collagen network and chondrocytes

    NARCIS (Netherlands)

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

    2010-01-01

    Cartilage is a tissue with only limited reparative capacities. A small part of its volume is composed of cells, the remaining part being the hydrated extracellular matrix (ECM) with collagens and proteoglycans as its main constituents. The functioning of cartilage depends heavily on its ECM.

  18. 24R,25-Dihydroxyvitamin D3 Protects against Articular Cartilage Damage following Anterior Cruciate Ligament Transection in Male Rats.

    Directory of Open Access Journals (Sweden)

    Barbara D Boyan

    Full Text Available Osteoarthritis (OA in humans is associated with low circulating 25-hydroxyvitamin D3 [25(OHD3]. In vitamin D replete rats, radiolabeled 24R,25-dihydroxyvitamin D3 [24R,25(OH2D3] accumulates in articular cartilage following injection of [3H]-25(OHD3. Previously, we showed that 24R,25(OH2D3 blocks chondrocyte apoptosis via phospholipase D and p53, suggesting a role for 24R,25(OH2D3 in maintaining cartilage health. We examined the ability of 24R,25(OH2D3 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(OH2D3 or 1α,25(OH2D3. 24R,25(OH2D3 but not 1α,25(OH2D3 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(OH2D3 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(OH2D3 had less articular cartilage damage and levels of inflammatory mediators. These results indicate that 24R,25(OH2D3 protects against OA, and suggest that it may be a therapeutic approach for preventing trauma-induced osteoarthritis.

  19. Clinical Trial and In Vitro Study for the Role of Cartilage and Synovia in Acute Articular Infection.

    Science.gov (United States)

    Langenmair, Elia R; Kubosch, Eva J; Salzmann, Gian M; Beck, Samuel; Schmal, Hagen

    2015-01-01

    Osteoarthritis is a long-term complication of acute articular infections. However, the roles of cartilage and synovia in this process are not yet fully understood. Patients with acute joint infections were enrolled in a prospective clinical trial and the cytokine composition of effusions compared in patients with arthroplasty (n = 8) or with intact joints (n = 67). Cytokines and cell function were also analyzed using a human in vitro model of joint infection. Synovial IL-1β levels were significantly higher in patients with arthroplasty (p = 0.004). Higher IL-1β concentrations were also found in the in vitro model without chondrocytes (p < 0.05). The anti-inflammatory cytokines IL-4 and IL-10 were consistently expressed in vivo and in vitro, showing no association with the presence of cartilage or chondrocytes. In contrast, FasL levels increased steadily in vitro, reaching higher levels without chondrocytes (p < 0.05). Likewise, the viability of synovial fibroblasts (SFB) during infection was higher in the presence of chondrocytes. The cartilage-metabolism markers aggrecan and bFGF were at higher concentrations in intact joints, but also synthesized by SFB. Our data suggest an anti-inflammatory effect of cartilage associated with the SFBs' increased resistance to infections, which displayed the ability to effectively synthesize cartilage metabolites.The trial is registered with DRKS 00003536, MISSinG.

  20. Clinical Trial and In Vitro Study for the Role of Cartilage and Synovia in Acute Articular Infection

    Directory of Open Access Journals (Sweden)

    Elia R. Langenmair

    2015-01-01

    Full Text Available Objective. Osteoarthritis is a long-term complication of acute articular infections. However, the roles of cartilage and synovia in this process are not yet fully understood. Methods. Patients with acute joint infections were enrolled in a prospective clinical trial and the cytokine composition of effusions compared in patients with arthroplasty (n = 8 or with intact joints (n = 67. Cytokines and cell function were also analyzed using a human in vitro model of joint infection. Results. Synovial IL-1β levels were significantly higher in patients with arthroplasty (p = 0.004. Higher IL-1β concentrations were also found in the in vitro model without chondrocytes (p < 0.05. The anti-inflammatory cytokines IL-4 and IL-10 were consistently expressed in vivo and in vitro, showing no association with the presence of cartilage or chondrocytes. In contrast, FasL levels increased steadily in vitro, reaching higher levels without chondrocytes (p < 0.05. Likewise, the viability of synovial fibroblasts (SFB during infection was higher in the presence of chondrocytes. The cartilage-metabolism markers aggrecan and bFGF were at higher concentrations in intact joints, but also synthesized by SFB. Conclusions. Our data suggest an anti-inflammatory effect of cartilage associated with the SFBs’ increased resistance to infections, which displayed the ability to effectively synthesize cartilage metabolites.The trial is registered with DRKS 00003536, MISSinG.

  1. Effects of exercise on chondrocyte viability and subchondral bone sclerosis in the distal third metacarpal and metatarsal bones of young horses.

    Science.gov (United States)

    Dykgraaf, Susanne; Firth, Elwyn C; Rogers, Christopher W; Kawcak, Christopher E

    2008-10-01

    The objective was to determine the effects of early exercise on the articular cartilage and subchondral bone at specific sites of the distal third metacarpal and metatarsal bones of 12 young Thoroughbred horses allowed free choice exercise at pasture. Six of the horses had additional controlled exercise 5 days per week from mean age of 21+/-20 days of age (range: 3-83 days) until 17.1 months of age. Confocal laser scanning microscopy was used to quantify viable and non-viable chondrocytes. Proteoglycan scoring and modified Mankin scoring was performed and subchondral bone mineral density measured by computed tomography. The number of viable chondrocytes was significantly greater in the conditioned group, which also had a higher Safranin O/Fast Green (SOFG) score than did the group which could exercise only at pasture. There was no difference in mean bone mineral density between groups, nor was there relationship between subchondral bone mineral density and chondrocyte viability. The apparent beneficial effects of early conditioning exercise may support the use of exercise to optimise development of articular cartilage in young individuals.

  2. Spatial and temporal changes of subchondral bone proceed to articular cartilage degeneration in rats subjected to knee immobilization.

    Science.gov (United States)

    Xu, Lei; Li, Zhe; Lei, Lei; Zhou, Yue-Zhu; Deng, Song-Yun; He, Yong-Bin; Ni, Guo-Xin

    2016-03-01

    This study was aimed to investigate the spatial and temporal changes of subchondral bone and its overlying articular cartilage in rats following knee immobilization. A total of 36 male Wistar rats (11-13 months old) were assigned randomly and evenly into 3 groups. For each group, knee joints in 6 rats were immobilized unilaterally for 1, 4, or 8 weeks, respectively, while the remaining rats were allowed free activity and served as external control groups. For each animal, femurs at both sides were dissected after sacrificed. The distal part of femur was examined by micro-CT. Subsequently, femoral condyles were collected for further histological observation and analysis. For articular cartilage, significant changes were observed only at 4 and 8 weeks of immobilization. The thickness of articular cartilage and chondrocytes numbers decreased with time. However, significant changes in subchondral bone were defined by micro-CT following immobilization in a time-dependent manner. Immobilization led to a thinner and more porous subchondral bone plate, as well as a reduction in trabecular thickness and separation with a more rod-like architecture. Changes in subchondral bone occurred earlier than in articular cartilage. More importantly, immobilization-induced changes in subchondral bone may contribute, at least partially, to changes in its overlying articular cartilage. © 2016 Wiley Periodicals, Inc.

  3. Genipin-cross-linked collagen/chitosan biomimetic scaffolds for articular cartilage tissue engineering applications.

    Science.gov (United States)

    Yan, Le-Ping; Wang, Ying-Jun; Ren, Li; Wu, Gang; Caridade, Sofia G; Fan, Jia-Bing; Wang, Ling-Yun; Ji, Pei-Hong; Oliveira, Joaquim M; Oliveira, João T; Mano, João F; Reis, Rui L

    2010-11-01

    In this study, genipin-cross-linked collagen/chitosan biodegradable porous scaffolds were prepared for articular cartilage regeneration. The influence of chitosan amount and genipin concentration on the scaffolds physicochemical properties was evaluated. The morphologies of the scaffolds were characterized by scanning electron microscope (SEM) and cross-linking degree was investigated by ninhydrin assay. Additionally, the mechanical properties of the scaffolds were assessed under dynamic compression. To study the swelling ratio and the biostability of the collagen/chitosan scaffold, in vitro tests were also carried out by immersion of the scaffolds in PBS solution or digestion in collagenase, respectively. The results showed that the morphologies of the scaffolds underwent a fiber-like to a sheet-like structural transition by increasing chitosan amount. Genipin cross-linking remarkably changed the morphologies and pore sizes of the scaffolds when chitosan amount was less than 25%. Either by increasing the chitosan ratio or performing cross-linking treatment, the swelling ratio of the scaffolds can be tailored. The ninhydrin assay demonstrated that the addition of chitosan could obviously increase the cross-linking efficiency. The degradation studies indicated that genipin cross-linking can effectively enhance the biostability of the scaffolds. The biocompatibility of the scaffolds was evaluated by culturing rabbit chondrocytes in vitro. This study demonstrated that a good viability of the chondrocytes seeded on the scaffold was achieved. The SEM analysis has revealed that the chondrocytes adhered well to the surface of the scaffolds and contacted each other. These results suggest that the genipin-cross-linked collagen/chitosan matrix may be a promising formulation for articular cartilage scaffolding.

  4. Adipose stem cells differentiated chondrocytes regenerate damaged cartilage in rat model of osteoarthritis.

    Science.gov (United States)

    Latief, Noreen; Raza, Fahad Ali; Bhatti, Fazal-Ur-Rehman; Tarar, Moazzam Nazir; Khan, Shaheen N; Riazuddin, Sheikh

    2016-05-01

    Transplantation of mesenchymal stem cells (MSCs) or autologous chondrocytes has been shown to repair damages to articular cartilage due to osteoarthritis (OA). However, survival of transplanted cells is considerably reduced in the osteoarthritic environment and it affects successful outcome of the transplantation of the cells. Differentiated chrondroytes derived from adipose stem cells have been proposed as an alternative source and our study investigated this possibility in rats. We investigated the regenerative potential of ADSCs and DCs in osteoarthritic environment in the repair of cartilage in rats. We found that ADSCs maintained fibroblast morphology in vitro and also expressed CD90 and CD29. Furthermore, ADSCs differentiated into chondrocytes, accompanied by increased level of proteoglycans and expression of chondrocytes specific genes, such as, Acan, and Col2a1. Histological examination of transplanted knee joints showed regeneration of cartilage tissue compared to control OA knee joints. Increase in gene expression for Acan, Col2a1 with concomitant decrease in the expression of Col1a1 suggested formation of hyaline like cartilage. A significant increase in differentiation index was observed in DCs and ADSCs transplanted knee joints (P = 0.0110 vs. P = 0.0429) when compared to that in OA control knee joints. Furthermore, transplanted DCs showed increased proliferation along with reduction in apoptosis as compared to untreated control. In conclusion, DCs showed better survival and regeneration potential as compared with ADSCs in rat model of OA and thus may serve a better option for regeneration of osteoarthritic cartilage. © 2016 International Federation for Cell Biology.

  5. Genetic engineering strategies to prevent the effects of antibody and complement on xenogeneic chondrocytes.

    Science.gov (United States)

    Sommaggio, R; Bello-Gil, D; Pérez-Cruz, M; Brokaw, J L; Máñez, R; Costa, C

    2015-11-18

    Advances in animal transgenesis may allow using xenogeneic chondrocytes in tissue-engineering applications for clinical cartilage repair. Porcine cartilage is rejected by humoral and cellular mechanisms that could be overcome by identifying key molecules triggering rejection and developing effective genetic-engineering strategies. Accordingly, high expression of α1,2-fucosyltransferase (HT) in xenogeneic cartilage protects from galactose α1,3-galactose (Gal)-mediated antibody responses. Now, we studied whether expression of a complement inhibitor provides further protection. First, porcine articular chondrocytes (PAC) were isolated from non-transgenic, single and double transgenic pigs expressing HT and moderate levels of human CD59 (hCD59) and their response to human serum was assessed. High recombinant expression of human complement regulatory molecules hCD59 and hDAF was also attained by retroviral transduction of PAC for further analyses. Complement activation on PAC after exposure to 20 % human serum for 24 hours mainly triggered the release of pro-inflammatory cytokines IL-6 and IL-8. Transgenic expression of HT and hCD59 did not suffice to fully counteract this effect. Nevertheless, the combination of blocking anti-Gal antibodies (or C5a) and high hCD59 levels conferred very high protection. On the contrary, high hDAF expression attained the most dramatic reduction in IL-6/IL-8 secretion by a single strategy, but the additional inhibition of anti-Gal antibodies or C5a did not provide further improvement. Notably, we demonstrate that both hCD59 and hDAF inhibit anaphylatoxin release in this setting. In conclusion, our study identifies genetic-engineering approaches to prevent humoral rejection of xenogeneic chondrocytes for use in cartilage repair.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2006-01-15

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

  7. 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

  8. The Importance of the mTOR Regulatory Network in Chondrocyte Biology and Osteoarthritis

    Directory of Open Access Journals (Sweden)

    Elena V. Tchetina

    2014-07-01

    Full Text Available Osteoarthritis (OA is a chronic disorder associated mainly with pain, limited range of motion, stiffness, joint inflammation, and articular cartilage (AC destruction. Recent studies demonstrated the involvement of chondrocyte differentiation (hypertrophy as one of the mechanisms of cartilage degradation in OA. This indicates the involvement of profound alterations in chondrocyte metabolism in the course of cartilage resorption orchestrated by principal changes in the regulation of cellular function. Mammalian target of rapamycin (mTOR controls critical cellular processes such as growth, proliferation, and protein synthesis, and integrates extracellular signals from growth factors and hormones with amino acid availability and intracellular energy status. The importance of mTOR activity during AC destruction in OA is supported by considerable alterations in the mTOR regulatory network, involving multiple intracellular (availability of growth factors, adenosine triphosphate [ATP], and oxygen as well as autophagy and extracellular (glucose, amino acid, lipid, and hexosamine signals. Moreover, variable mTOR gene expression in the peripheral blood of OA patients is associated with increases in pain or synovitis, and indicates a profound metabolic dissimilarity among patients that might require differential approaches to treatment. These issues are discussed in the present review article.

  9. Polysulfated glycosaminoglycan accelerates net synthesis of collagen and glycosaminoglycans by arthritic equine cartilage tissues and chondrocytes.

    Science.gov (United States)

    Glade, M J

    1990-05-01

    Low molecular weight polysulfated glycosaminoglycan (PSGAG) stimulated net collagen and glycosaminoglycan synthesis by normal and arthritic equine fetlock cartilage tissues in organ culture. Arthritic tissues were more sensitive to PSGAG stimulation. The rates of cartilage-specific type-II collagen and chondroitin sulfate-rich glycosaminoglycan synthesis by confluent chondrocyte cell cultures obtained from normal and arthritic equine cartilage tissues were increased by 25 and 50 mg of PSGAG/ml. Cells from arthritic cartilage were also more sensitive to the presence of PSGAG. In addition, concentrations of PSGAG (25 and 50 mg/ml) approximate to those in synovial fluid after intra-articular injection of 250 mg of PSGAG inhibited the rate of collagen and glycosaminoglycan degradation in cell culture. These findings suggest that PSGAG may have a role in the healing of mild cartilage degeneration by encouraging the production of replacement hyaline matrix materials, while delaying their subsequent degradation. In contrast, growth of cell cultures was inhibited by PSGAG, suggesting that these compounds may fail to stimulate chondrocyte replication, a prerequisite for tissue regeneration. Nonetheless, these observations provide direct evidence of a truly chondroprotective role for low molecular weight PSGAG in the treatment of equine degenerative joint disease.

  10. Human platelet-rich plasma improves the nesting and differentiation of human chondrocytes cultured in stabilized porous chitosan scaffolds.

    Science.gov (United States)

    Sancho-Tello, Maria; Martorell, Sara; Mata Roig, Manuel; Milián, Lara; Gámiz-González, M A; Gómez Ribelles, Jose Luis; Carda, Carmen

    2017-01-01

    The clinical management of large-size cartilage lesions is difficult due to the limited regenerative ability of the cartilage. Different biomaterials have been used to develop tissue engineering substitutes for cartilage repair, including chitosan alone or in combination with growth factors to improve its chondrogenic properties. The main objective of this investigation was to evaluate the benefits of combining activated platelet-rich plasma with a stabilized porous chitosan scaffold for cartilage regeneration. To achieve this purpose, stabilized porous chitosan scaffolds were prepared using freeze gelation and combined with activated platelet-rich plasma. Human primary articular chondrocytes were isolated and cultured in stabilized porous chitosan scaffolds with and without combination to activated platelet-rich plasma. Scanning electron microscopy was used for the morphological characterization of the resulting scaffolds. Cell counts were performed in hematoxylin and eosin-stained sections, and type I and II collagen expression was evaluated using immunohistochemistry. Significant increase in cell number in activated platelet-rich plasma/stabilized porous chitosan was found compared with stabilized porous chitosan scaffolds. Chondrocytes grown on stabilized porous chitosan expressed high levels of type I collagen but type II was not detectable, whereas cells grown on activated platelet rich plasma/stabilized porous chitosan scaffolds expressed high levels of type II collagen and type I was almost undetectable. In summary, activated platelet-rich plasma increases nesting and induces the differentiation of chondrocytes cultured on stabilized porous chitosan scaffolds.

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

    Science.gov (United States)

    Chang, Shun-Fu; Hsieh, Rong-Ze; Huang, Kuo-Chin; Chang, Cheng Allen; Chiu, Fang-Yao; Kuo, Hsing-Chun; Chen, Cheng-Nan; Su, Yu-Ping

    2015-01-01

    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.

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

    Directory of Open Access Journals (Sweden)

    Shun-Fu Chang

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

  13. H2O2 INDUCES APOPTOSIS OF RABBIT CHONDROCYTES VIA BOTH THE EXTRINSIC AND THE CASPASE-INDEPENDENT INTRINSIC PATHWAYS

    Directory of Open Access Journals (Sweden)

    CAIPING ZHUANG

    2013-07-01

    Full Text Available Osteoarthritis (OA, one of the most common joint diseases with unknown etiology, is characterized by the progressive destruction of articular cartilage and the apoptosis of chondrocytes. The purpose of this study is to elucidate the molecular mechanisms of H2O2-mediated rabbit chondrocytes apoptosis. CCK-8 assay showed that H2O2 treatment induced a remarkable reduction of cell viability, which was further verified by the remarkable phosphatidylserine externalization after H2O2 treatment for 1 h, the typical characteristics of apoptosis. H2O2 treatment induced a significant dysfunction of mitochondrial membrane potential (ΔΨm, but did not induce casapse-9 activation, indicating that H2O2 treatment induced caspase-independent intrinsic apoptosis that was further verified by the fact that silencing of AIF but not inhibiting caspase-9 potently prevented H2O2-induced apoptosis. H2O2 treatment induced a significant increase of caspase-8 and -3 activation, and inhibition of caspase-8 or -3 significantly prevented H2O2-induced apoptosis, suggesting that the extrinsic pathway played an important role. Collectively, our findings demonstrate that H2O2 induces apoptosis via both the casapse-8-mediated extrinsic and the caspase-independent intrinsic apoptosis pathways in rabbit chondrocytes.

  14. Cartilage tissue formation from dedifferentiated chondrocytes by codelivery of BMP-2 and SOX-9 genes encoding bicistronic vector.

    Science.gov (United States)

    Cha, Byung-Hyun; Kim, Jae-Hwan; Kang, Sun-Woong; Do, Hyun-Jin; Jang, Ju-Woong; Choi, Yon Rak; Park, Hansoo; Kim, Byung-Soo; Lee, Soo-Hong

    2013-01-01

    Articular cartilage, when damaged by degenerative disease or trauma, has limited ability for self-repair. Recently, many trials have demonstrated that gene therapy combined with tissue engineering techniques would be a promising approach for cartilage regeneration. Bone morphogenetic protein 2 (BMP-2) is an important signal for upregulation of osteogenesis and chondrogenesis of stem cells. Sex-determining region Y box gene 9 (SOX-9) has also been reported as one of the key transcription factors for chondrogenesis. We hypothesized that codelivery of BMP-2 and SOX-9 genes would result in improved efficiency of recovery of normal chondrogenic properties in dedifferentiated chondrocytes. To this aim, we constructed a bicistronic vector encoding the BMP-2 and SOX-9 genes linked to the "self-cleaving" 2A peptide sequence. After gene delivery to dedifferentiated chondrocytes using a microporator transfection system, we confirmed over 65% delivery efficiency of the BMP-2 and SOX-9 genes. According to RT-PCR analysis and Alcian blue staining, simultaneous delivery of BMP-2/SOX-9 resulted in significantly increased expression of chondrogenesis-related markers (type II collagen and aggrecan) and GAG matrix formation compared with individual delivery of the BMP-2 or SOX-9 gene. Six weeks after in vivo transplantation, BMP-2/SOX-9 genes also showed a significant increase in cartilage formation compared with the BMP-2 or SOX-9 gene. These results demonstrate that codelivery of two chondrogenic lineage-determining genes can enhance normal chondrogenic properties of dedifferentiated chondrocytes followed by improved cartilage formation.

  15. A hexadecylamide derivative of hyaluronan (HYMOVIS®) has superior beneficial effects on human osteoarthritic chondrocytes and synoviocytes than unmodified hyaluronan

    Science.gov (United States)

    2013-01-01

    Background Intra-articular hyaluronan (HA) injection provides symptomatic benefit in the treatment of osteoarthritis (OA). Previously we found superior beneficial effects in a large animal OA model of a hexadecylamide derivative compared with unmodified HA of the same initial molecular weight. The current study sought to define possible molecular mechanisms whereby this enhanced relief of symptoms was occurring. Methods Chondrocytes and synovial fibroblasts were isolated from tissues of patients undergoing arthroplasty for knee OA. Monolayer cultures of cells were treated with 0, 0.5, 1.0 or 1.5 mg/mL of unmodified HA (500–730 kDa) or a hexadecylamide derivative of HA of the same initial molecular weight (HYADD4®-G; HYMOVIS®) simultaneously or 1 hour before incubation with interleukin (IL)-1beta (2 ng/mL). Cultures were terminated 15 or 30 minutes later (chondrocytes and synovial fibroblasts, respectively) for quantitation of phosphorylated-(p)-JNK, p-NFkappaB, p-p38, or at 24 hours for quantitation of gene expression (MMP1 &13, ADAMTS4 &5, TIMP1 &3, CD44, COL1A1 &2A1, ACAN, PTGS2, IL6, TNF) and matrix metalloproteinase (MMP)-13 activity. Results The hexadecylamide derivative of HA had significantly better amelioration of IL-1beta-induced gene expression of key matrix degrading enzymes (MMP1, MMP13, ADAMTS5), and inflammatory mediators (IL6, PTGS2) by human OA chondrocytes and synovial fibroblasts. Pre-incubation of cells with the derivatized HA for 1 hour prior to IL-1beta exposure significantly augmented the inhibition of MMP1, MMP13, ADAMTS4 and IL6 expression by chondrocytes. The reduction in MMP13 mRNA by the amide derivative of HA was mirrored in reduced MMP-13 protein and enzyme activity in IL-1beta-stimulated chondrocytes. This was associated in part with a greater inhibition of phosphorylation of the cell signalling molecules JNK, p38 and NF-kappaB. Conclusions The present studies have demonstrated several potential key mechanisms whereby the

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

    Science.gov (United States)

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

    2015-09-01

    We determined if the epidermal growth factor receptor ligand HB-EGF is produced in cartilage and if it regulates chondrocyte anabolic or catabolic activity. HB-EGF expression was measured by quantitative PCR using RNA isolated from mouse knee joint tissues and from normal and osteoarthritis (OA) human chondrocytes. Immunohistochemistry was performed on normal and OA human cartilage and meniscus sections. Cultured chondrocytes were treated with fibronectin fragments (FN-f) as a catabolic stimulus and osteogenic protein 1 (OP-1) as an anabolic stimulus. Effects of HB-EGF on cell signaling were analyzed by immunoblotting of selected signaling proteins. MMP-13 was measured in conditioned media, proteoglycan synthesis was measured by sulfate incorporation, and matrix gene expression by quantitative PCR. HB-EGF expression was increased in 12-month old mice at 8 weeks after surgery to induce OA and increased amounts of HB-EGF were noted in human articular cartilage from OA knees. FN-f stimulated chondrocyte HB-EGF expression and HB-EGF stimulated chondrocyte MMP-13 production. However, HB-EGF was not required for FN-f stimulation of MMP-13 production. HB-EGF activated the ERK and p38 MAP kinases and stimulated phosphorylation of Smad1 at an inhibitory serine site which was associated with inhibition of OP-1 mediated proteoglycan synthesis and reduced aggrecan (ACAN) but not COL2A1 expression. HB-EGF is a new factor identified in OA cartilage that promotes chondrocyte catabolic activity while inhibiting anabolic activity suggesting it could contribute to the catabolic-anabolic imbalance seen in OA cartilage. Copyright © 2015 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.

  17. Local intra-articular injection of resveratrol delays cartilage degeneration in C57BL/6 mice by inducing autophagy via AMPK/mTOR pathway.

    Science.gov (United States)

    Qin, Na; Wei, Liwei; Li, Wuyin; Yang, Wei; Cai, Litao; Qian, Zhuang; Wu, Shufang

    2017-07-01

    Autophagy is an essential cellular homeostasis mechanism that was found to be compromised in aging and osteoarthritis (OA) cartilage. Previous studies showed that resveratrol can effectively regulate autophagy in other cells. The purpose of this study was to determine whether the chondroprotective effect of resveratrol was related to chondrocyte autophagy and to elucidate underlying mechanisms. OA model was induced by destabilization of the medial meniscus (DMM) in 10-week-old male mice. OA mice were treated with resveratrol with/without 3-MA for 8 weeks beginning 4 weeks after surgery. The local intra-articular injection of resveratrol delayed articular cartilage degradation in DMM-induced OA by OARSI scoring systems and Safranin O-fast green. Resveratrol treatment increased Unc-51-like kinase1, Beclin1, microtubule-associated protein light chain 3, hypoxia inducible factor-1α, phosphorylated AMPK, collagen-2A1, Aggrecan expressions, but decreased hypoxia inducible factor-2α, phosphorylated mTOR, matrix metalloproteinases13 and a disintegrin and metalloproteinase with thrombospondin motifs 5 expressions. The effects of resveratrol were obviously blunted by 3-MA except HIF and AMPK. These findings indicate that resveratrol intra-articular injection delayed articular cartilage degeneration and promoted chondrocyte autophagy in an experimental model of surgical DMM-induced OA, in part via balancing HIF-1α and HIF-2α expressions and thereby regulating AMPK/mTOR signaling pathway. Copyright © 2017 The Authors. Production and hosting by Elsevier B.V. All rights reserved.

  18. Local intra-articular injection of resveratrol delays cartilage degeneration in C57BL/6 mice by inducing autophagy via AMPK/mTOR pathway

    Directory of Open Access Journals (Sweden)

    Na Qin

    2017-07-01

    Full Text Available Autophagy is an essential cellular homeostasis mechanism that was found to be compromised in aging and osteoarthritis (OA cartilage. Previous studies showed that resveratrol can effectively regulate autophagy in other cells. The purpose of this study was to determine whether the chondroprotective effect of resveratrol was related to chondrocyte autophagy and to elucidate underlying mechanisms. OA model was induced by destabilization of the medial meniscus (DMM in 10-week-old male mice. OA mice were treated with resveratrol with/without 3-MA for 8 weeks beginning 4 weeks after surgery. The local intra-articular injection of resveratrol delayed articular cartilage degradation in DMM-induced OA by OARSI scoring systems and Safranin O-fast green. Resveratrol treatment increased Unc-51-like kinase1, Beclin1, microtubule-associated protein light chain 3, hypoxia inducible factor-1α, phosphorylated AMPK, collagen-2A1, Aggrecan expressions, but decreased hypoxia inducible factor-2α, phosphorylated mTOR, matrix metalloproteinases13 and a disintegrin and metalloproteinase with thrombospondin motifs 5 expressions. The effects of resveratrol were obviously blunted by 3-MA except HIF and AMPK. These findings indicate that resveratrol intra-articular injection delayed articular cartilage degeneration and promoted chondrocyte autophagy in an experimental model of surgical DMM-induced OA, in part via balancing HIF-1α and HIF-2α expressions and thereby regulating AMPK/mTOR signaling pathway.

  19. Intra-articular dexamethasone to inhibit the development of post-traumatic osteoarthritis.

    Science.gov (United States)

    Grodzinsky, Alan J; Wang, Yang; Kakar, Sanjeev; Vrahas, Mark S; Evans, Christopher H

    2017-03-01

    Injury to the joint provokes a number of local pathophysiological changes, including synthesis of inflammatory cytokines, death of chondrocytes, breakdown of the extra-cellular matrix of cartilage, and reduced synthesis of matrix macromolecules. These processes combine to engender the subsequent development of post-traumatic osteoarthritis (PTOA). To prevent this from happening, it is necessary to inhibit these disparate responses to injury; given their heterogeneity, this is challenging. However, dexamethasone has the necessary pleiotropic properties required of a drug for this purpose. Using in vitro models, we have shown that low doses of dexamethasone sustain the synthesis of cartilage proteoglycans while inhibiting their breakdown after injurious compression in the presence or absence of inflammatory cytokines. Under these conditions, dexamethasone is non-toxic and maintains the viability of chondrocytes exposed chronically to such cytokines as interleukin (IL) -1, IL-6, and tumor necrosis factor-α. Moreover, the anti-inflammatory properties of dexamethasone have been appreciated for decades. In view of this information, we have initiated a pilot clinical study to determine whether a single, intra-articular injection of dexamethasone into the wrist shows promise in preventing PTOA after intra-articular fracture of the distal radius. Suppressing the various etiopathophysiological responses to injury in the joint is an attractive strategy for lowering the clinical burden of PTOA. The intra-articular administration of dexamethasone soon after injury offers a simple and inexpensive means of accomplishing this. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:406-411, 2017. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.

  20. Berberine prevents nitric oxide-induced rat chondrocyte apoptosis and cartilage degeneration in a rat osteoarthritis model via AMPK and p38 MAPK signaling.

    Science.gov (United States)

    Zhou, Yan; Liu, Shi-Qing; Yu, Ling; He, Bin; Wu, Shi-Hao; Zhao, Qi; Xia, Shao-Qiang; Mei, Hong-Jun

    2015-09-01

    Chondrocyte apoptosis is an important mechanism involved in osteoarthritis (OA). Berberine (BBR), a plant alkaloid derived from Chinese medicine, is characterized by multiple pharmacological effects, such as anti-inflammatory and anti-apoptotic activities. This study aimed to evaluate the chondroprotective effect and underlying mechanisms of BBR on sodium nitroprusside (SNP)-stimulated chondrocyte apoptosis and surgically-induced rat OA model. The in vitro results revealed that BBR suppressed SNP-stimulated chondrocyte apoptosis as well as cytoskeletal remodeling, down-regulated expressions of inducible nitric oxide synthase (iNOS) and caspase-3, and up-regulated Bcl-2/Bax ratio and Type II collagen (Col II) at protein levels, which were accompanied by increased adenosine monophosphate-activated protein kinase (AMPK) phosphorylation and decreased phosphorylation of p38 mitogen-activated protein kinase (MAPK). Furthermore, the anti-apoptotic effect of BBR was blocked by AMPK inhibitor Compound C (CC) and adenosine-9-β-D-arabino-furanoside (Ara A), and enhanced by p38 MAPK inhibitor SB203580. In vivo experiment suggested that BBR ameliorated cartilage degeneration and exhibited an anti-apoptotic effect on articular cartilage in a rat OA model, as demonstrated by histological analyses, TUNEL assay and immunohistochemical analyses of caspase-3, Bcl-2 and Bax expressions. These findings suggest that BBR suppresses SNP-stimulated chondrocyte apoptosis and ameliorates cartilage degeneration via activating AMPK signaling and suppressing p38 MAPK activity.

  1. Modeling the transport of cryoprotective agents in articular cartilage for cryopreservation

    Science.gov (United States)

    Torqabeh, Alireza Abazari

    Loading vitrifiable concentrations of cryoprotective agents is an important step for cryopreservation of biological tissues by vitrification for research and transplantation purposes. This may be done by immersing the tissue in a cryoprotective agent (CPA) solution, and increasing the concentration, continuously or in multiple steps, and simultaneously decreasing the temperature to decrease the toxicity effects of the cryoprotective agent on the tissue cellular system. During cryoprotective agent loading, osmotic water movement from the tissue to the surrounding solution, and the resultant tissue shrinkage and stress-strain in the tissue matrix as well as on the cellular system can significantly alter the outcome of the cryopreservation protocol. In this thesis, a biomechanical model for articular cartilage is developed to account for the transport of the cryoprotective agent, the nonideal-nondilute properties of the vitrifiable solutions, the osmotic water movement and the resultant tissue shrinkage and stress-strain in the tissue matrix, and the osmotic volume change of the chondrocytes, during cryoprotective agent loading in the cartilage matrix. Four essential transport parameters needed for the model were specified, the values of which were obtained uniquely by fitting the model to experimental data from porcine articular cartilage. Then, it was shown that using real nonuniform initial distributions of water and fixed charges in cartilage, measured separately in this thesis using MRI, in the model can significantly affect the model predictions. The model predictions for dimethyl sulfoxide diffusion in porcine articular cartilage were verified by comparing to spatially and temporally resolved measurements of dimethyl sulfoxide concentration in porcine articular cartilage using a spectral MRI technique, developed for this purpose and novel to the field of cryobiology. It was demonstrated in this thesis that the developed mathematical model provides a novel tool

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

    Directory of Open Access Journals (Sweden)

    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.

  3. Study of the collagen structure in the superficial zone and physiological state of articular cartilage using a 3D confocal imaging technique

    Directory of Open Access Journals (Sweden)

    Zheng Ming H

    2008-07-01

    the collagen network in the superficial zone during early physiological alteration of articular cartilage. The fibre confocal imaging technology used in this study has allowed developing confocal arthroscopy for in vivo studying the chondrocytes in different depth of articular cartilage. Therefore, the current study has potential to develop an in vivo 3D histology for diagnosis of early osteoarthritis.

  4. Biodistribution and Immunogenicity of Allogeneic Mesenchymal Stem Cells in a Rat Model of Intraarticular Chondrocyte Xenotransplantation

    Directory of Open Access Journals (Sweden)

    Maribel Marquina

    2017-11-01

    Full Text Available Xenogeneic chondrocytes and allogeneic mesenchymal stem cells (MSC are considered a potential source of cells for articular cartilage repair. We here assessed the immune response triggered by xenogeneic chondrocytes when injected intraarticularly, as well as the immunoregulatory effect of allogeneic bone marrow-derived MSC after systemic administration. To this end, a discordant xenotransplantation model was established by injecting three million porcine articular chondrocytes (PAC into the femorotibial joint of Lewis rats and monitoring the immune response. First, the fate of MSC injected using various routes was monitored in an in vivo imaging system. The biodistribution revealed a dependency on the injection route with MSC injected intravenously (i.v. succumbing early after 24 h and MSC injected intraperitoneally (i.p. lasting locally for at least 5 days. Importantly, no migration of MSC to the joint was detected in rats previously injected with PAC. MSC were then administered either i.v. 1 week before PAC injection or i.p. 3 weeks after to assess their immunomodulatory function on humoral and adaptive immune parameters. Anti-PAC IgM and IgG responses were detected in all PAC-injected rats with a peak at week 2 postinjection and reactivity remaining above baseline levels by week 18. IgG2a and IgG2b were the predominant and long-lasting IgG subtypes. By contrast, no anti-MSC antibody response was detected in the cohort injected with MSC only, but infusion of MSC before PAC injection temporarily augmented the anti-PAC antibody response. Consistent with a cellular immune response to PAC in PAC-injected rats, cytokine/chemokine profiling in serum by antibody array revealed a distinct pattern relative to controls characterized by elevation of multiple markers at week 2, as well as increases in proliferation in draining lymph nodes. Notably, systemic administration of allogeneic MSC under the described conditions did not diminish the immune

  5. hWJECM-Derived Oriented Scaffolds with Autologous Chondrocytes for Rabbit Cartilage Defect Repairing.

    Science.gov (United States)

    Zhao, Peng; Liu, Shuyun; Bai, Yuhe; Lu, Shibi; Peng, Jiang; Zhang, Li; Huang, Jingxiang; Zhao, Bin; Xu, Wenjing; Guo, Quanyi

    2018-02-02

    Previously, we synthesized an articular cartilage extracellular matrix (ECM)-derived oriented scaffold for cartilage tissue engineering, which was biomimetic in terms of structure and biochemical composition. However, the limit resource of the cartilage-derived ECM is a hindrance for its application. In this study, we developed a new material for cartilage tissue engineering-human umbilical cord Wharton's jelly-derived ECM (hWJECM). The hWJECM has an abundant resource and similar biochemistry with cartilage ECM, and the use of it is not associated with ethical controversy. We adopted the method previously used in cartilage ECM-derived oriented scaffold preparation to generate the oriented hWJECM-derived scaffold, and the scaffold properties were tested in vitro and in vivo. The three-dimensional scaffold has a porous and well-oriented structure, with a mean pore diameter of ∼104 μm. Scanning electron microscopy and cell viability staining results demonstrated that the oriented scaffold has good biocompatibility and cell alignment. In addition, we used functional autologous chondrocytes to seed the hWJECM-derived oriented scaffold and tested the efficacy of the cell-scaffold constructs to repair the full-thickness articular cartilage defect in a rabbit model. Defects of 4 mm diameter were generated in the patellar grooves of the femurs of both knees and were implanted with chondrocyte-scaffold constructs (group A) or scaffolds alone (group B); rabbits with untreated defects were used as a control (group C). Six months after surgery, all defects in group A were filled completely with repaired tissue, and most of which were hyaline cartilage. In contrast, the defects in group B were filled partially with repaired tissue, and approximately half of these repaired tissues were hyaline cartilage. The defects in group C were only filled with fibrotic tissue. Histological grading score of group A was lower than those of groups B and C. Quantification of

  6. Baicalin prevents the apoptosis of endplate chondrocytes by inhibiting the oxidative stress induced by H2O2.

    Science.gov (United States)

    Pan, Yutao; Chen, Di; Lu, Qingyou; Liu, Lifeng; Li, Xia; Li, Zengchun

    2017-09-01

    Osteoarthritis (OA) is a degenerative disease of articular cartilage. The pathogenesis of OA remains to be fully elucidated, and several studies have found that oxidative stress is important in its pathogenesis. Baicalin is well known and has already been investigated for its role of inhibiting the oxidative stress pathway. Thus, the present study aimed to investigate the role of baicalin on the inhibition of oxidative stress in endplate chondrocytes induced by hydrogen peroxide (H2O2). Following treatment of endplate chondrocytes with different doses of H2O2 with or without baicalin for different incubation durations, a CCK‑8 assay and Annexin V/PI staining were used to measure the cell proliferation and apoptotic rates to identify the optimal experimental conditions. Subsequently, for examining the effects and underlying mechanism of baicalin on oxidative stress, the protein expression levels of cleaved‑poly (ADP‑ribose) polymerase (PARP), B‑cell lymphoma‑2‑associated X protein (Bax) and pro‑caspase‑3 were analyzed using western blot analysis, intracellular anti‑oxidant activities, including those of malondialdehyde (MDA), superoxide dismutase (SOD) and nitric oxide (NO), were quantified, and the levels of endothelial nitric oxide synthase (eNOS) were examined using reverse transcription‑polymerase chain reaction analysis. The results revealed that the oxidative stress of endplate chondrocytes induced by 0.5 mM H2O2 for 4 h were the most appropriate conditions for experiments, and pretreatment with 100 µmol/l baicalin for 1 h effectively reversed the effect of H2O2 on the endplate chondrocytes. In addition, Annexin V/PI staining demonstrated that the cell death induced by H2O2 was apoptotic, and baicalin reversed the apoptosis induced by oxidative stress. H2O2 activated PARP cleavage, and the expression of Bax and pro‑caspase‑3; however, baicalin inhibited the expression of these apoptotic signaling indicators. Baicalin also reduced

  7. Intra-articular enzyme replacement therapy with rhIDUA is safe, well-tolerated, and reduces articular GAG storage in the canine model of mucopolysaccharidosis type I.

    Science.gov (United States)

    Wang, Raymond Y; Aminian, Afshin; McEntee, Michael F; Kan, Shih-Hsin; Simonaro, Calogera M; Lamanna, William C; Lawrence, Roger; Ellinwood, N Matthew; Guerra, Catalina; Le, Steven Q; Dickson, Patricia I; Esko, Jeffrey D

    2014-08-01

    Treatment with intravenous enzyme replacement therapy and hematopoietic stem cell transplantation for mucopolysaccharidosis (MPS) type I does not address joint disease, resulting in persistent orthopedic complications and impaired quality of life. A proof-of-concept study was conducted to determine the safety, tolerability, and efficacy of intra-articular recombinant human iduronidase (IA-rhIDUA) enzyme replacement therapy in the canine MPS I model. Four MPS I dogs underwent monthly rhIDUA injections (0.58 mg/joint) into the right elbow and knee for 6 months. Contralateral elbows and knees concurrently received normal saline. No intravenous rhIDUA therapy was administered. Monthly blood counts, chemistries, anti-rhIDUA antibody titers, and synovial fluid cell counts were measured. Lysosomal storage of synoviocytes and chondrocytes, synovial macrophages and plasma cells were scored at baseline and 1 month following the final injection. All injections were well-tolerated without adverse reactions. One animal required prednisone for spinal cord compression. There were no clinically significant abnormalities in blood counts or chemistries. Circulating anti-rhIDUA antibody titers gradually increased in all dogs except the prednisone-treated dog; plasma cells, which were absent in all baseline synovial specimens, were predominantly found in synovium of rhIDUA-treated joints at study-end. Lysosomal storage in synoviocytes and chondrocytes following 6 months of IA-rhIDUA demonstrated significant reduction compared to tissues at baseline, and saline-treated tissues at study-end. Mean joint synovial GAG levels in IA-rhIDUA joints were 8.62 ± 5.86 μg/mg dry weight and 21.6 ± 10.4 μg/mg dry weight in control joints (60% reduction). Cartilage heparan sulfate was also reduced in the IA-rhIDUA joints (113 ± 39.5 ng/g wet weight) compared to saline-treated joints (142 ± 56.4 ng/g wet weight). Synovial macrophage infiltration, which was present in all joints at baseline, was

  8. Effects of oral administration of phenylbutazone to horses on in vitro articular cartilage metabolism.

    Science.gov (United States)

    Beluche, L A; Bertone, A L; Anderson, D E; Rohde, C

    2001-12-01

    To evaluate the effects of orally administered phenylbutazone on proteoglycan synthesis and chondrocyte inhibition by IL-1beta in articular cartilage explants of horses. 11 healthy 1- to 2-year-old horses. Horses were randomly assigned to the control (n = 5) or treated group (4.4 mg of phenylbutazone/kg of body weight, p.o., q 12 h; n = 6). Articular cartilage specimens were collected before treatment was initiated (day 0), after 14 days of treatment, and 2 weeks after cessation of treatment (day 30). Proteoglycan synthesis and stromelysin concentration in cartilage extracts were assessed after 72 hours of culture in medium alone or with recombinant human interleukin-1beta (IL-1beta; 0.1 ng/ml). On day 0, proteoglycan synthesis was significantly less in cartilage explants cultured in IL-1beta, compared with medium alone. Mean proteoglycan synthesis in explants collected on days 14 and 30 was significantly less in treated horses, compared with controls. However, incubation of explants from treated horses with IL-1beta did not result in a further decrease in proteoglycan synthesis. Significant differences in stromelysin concentration were not detected between or within groups. Oral administration of phenylbutazone for 14 days significantly decreased proteoglycan synthesis in articular culture explants from healthy horses to a degree similar to that induced by in vitro exposure to IL-1beta. Phenylbutazone should be used judiciously in athletic horses with osteoarthritis, because chronic administration may suppress proteoglycan synthesis and potentiate cartilage damage.

  9. Influence on chondrogenesis of human osteoarthritic chondrocytes in co-culture with donor-matched mesenchymal stem cells from infrapatellar fat pad and subcutaneous adipose tissue.

    Science.gov (United States)

    Lopa, S; Colombini, A; Sansone, V; Preis, F W Baruffaldi; Moretti, M

    2013-01-01

    Co-culture of mesenchymal stem cells (MSCs) and articular chondrocytes (ACs) has been proposed for autologous cartilage cell-based therapies, to overcome the issues associated to limited availability of articular chondrocytes (ACs). To evaluate the potentiality of a co-culture approach in aged osteoarthritic patients, MSCs from infrapatellar fat pad (IFP-MSCs) and knee subcutaneous adipose tissue (ASCs) were co-cultured with donor-matched osteoarthritic, expanded and cryopreserved, ACs in a 75%/25% ratio. Co-cultures were prepared also from nasal chondrocytes (NCs) to evaluate their possible use as an alternative to ACs. Pellets were differentiated for 14 days, using mono-cultures of each cell type as reference. Chondrogenic genes SOX9, COL2A1, ACAN were less expressed in co-cultures compared to ACs and NCs. Total GAGs content in co-cultures did not differ significantly from values predicted as the sum of each cell type contribution corrected for the co-culture ratio, as confirmed by histology. No significant differences were observed for GAGs/DNA in mono-cultures, demonstrating a reduced chondrogenic potential of ACs and NCs. In conclusion, a small percentage of expanded and cryopreserved ACs and NCs did not lead to IFP-MSCs and ASCs chondro-induction. Our results suggest that chondrogenic potential and origin of chondrocytes may play a relevant role in the outcome of co-cultures, indicating a need for further investigations to demonstrate their clinical relevance in the treatment of aged osteoarthritic patients.

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

    Science.gov (United States)

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

    2014-09-01

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

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

    Science.gov (United States)

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

    2011-07-01

    To compare the ability of different cyclodextrin polysulphate (CDPS) derivatives to affect human articular cartilage cell metabolism in vitro. OA chondrocytes were cultured in alginate and exposed to 5 µg/ml of 2,3,6-tri-O-methyl-β-cyclodextrin (ME-CD), 2,3-di-O-methyl-6-sulphate-β-cyclodextrin (ME-CD-6-S), 2,6-di-O-methyl-3-sulphate-β-cyclodextrin (ME-CD-3-S), (2-carboxyethyl)-β-CDPS (CE-CDPS), (2-hydroxypropyl)-β-CDPS (HP-CDPS), 6-monoamino-6-monodeoxy-β-CDPS (MA-CDPS) or β-CDPS for 5 days. Effects on IL-1-driven chondrocyte extracellular matrix (ECM) metabolism were assayed by analysis of the accumulation of aggrecan in the interterritorial matrix, IL-6 secretion and qPCR. MA-CDPS, HP-CDPS, CE-CDPS and CDPS were analysed for their in vitro effect on coagulation and their ability to activate platelets in an in vitro assay to detect possible cross-reactivity with heparin-induced thrombocytopenia (HIT) antibodies. The monosulphated cyclodextrins ME-CD-6-S and -3-S failed to affect aggrecan synthesis and IL-6 secretion by the OA chondrocytes. Polysulphated cyclodextrins MA-CDPS, HP-CDPS, CE-CDPS and CDPS at 5 µg/ml concentrations, on the other hand, significantly induced aggrecan production and repressed IL-6 release by the chondrocytes in culture. aPTT and PT for all derivatives were lengthened for polysaccharide concentrations >50 µg/ml. Five micrograms per millilitre of β-CDPS concentrations that significantly modulated ECM ground substance production in vitro did not affect aPTT or PT. Furthermore, CE-CDPS, in contrast to MA-CDPS, HP-CDPS and CDPS, did not significantly activate platelets, suggesting a minimal potential to induce HIT thromboembolic accidents in vivo. CE-CDPS is a new, structurally adjusted, sulphated β-cyclodextrin derivative with preserved chondroprotective capacity and a promising safety profile.

  12. 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.

  13. Osteophytes - an alternative source of chondrocytes for ...

    African Journals Online (AJOL)

    Materials and Methods: The scrapings from the cartilage mantle of osteophytes harvested during routine total knee replacement for osteoarthritis were enzymatically digested and grown in monolayer culture. Articular cartilage scrapings obtained from visually normal area of the femoral condyle (usually from the posterior ...

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

    Directory of Open Access Journals (Sweden)

    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

  15. 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.

  16. Aberrant hypertrophy in Smad3-deficient chondrocytes is rescued by restoring TAK1-ATF-2 signaling: a potential clinical implication for osteoarthritis

    Science.gov (United States)

    Li, Tian-Fang; Gao, Lin; Sheu, Tzong-Jen; Sampson, Erik R.; Flick, Lisa M.; Konttinen, Yrjo T.; Chen, Di; Schwarz, Edward M.; Zuscik, Michael J.; Jonason, Jennifer H.; O’Keefe, Regis J.

    2010-01-01

    Objective To investigate the biological significance of Smad3 in the progression of osteoarthritis (OA), the crosstalk between Smad3 and ATF-2 in the TGF-β signaling pathway, and the effects of ATF-2 overexpression and p38 activation in chondrocyte differentiation. Methods Joint disease in Smad3 knockout (Smad3−/−) mice was examined by micro-CT and histology. Numerous in vitro methods including immunostaining, real-time PCR, Western blotting, an ATF-2 DNA-binding assay and a p38 kinase activity assay were used to study the various signaling responses and protein interactions underlying the altered chondrocyte phenotype in Smad3−/− mice. Results Smad3−/− mice gradually developed an end-stage OA phenotype. TGF-β-induced TAK1-ATF-2 signaling was disrupted in Smad3−/− chondrocytes at the level of p38 MAP kinase activation resulting in reduced ATF-2 phosphorylation and transcriptional activity. Re-introduction of Smad3 into the Smad3−/− cells restored the normal p38 response to TGF-β. Phospho-p38 formed a complex with Smad3 by binding to the Smad3 MH1-linker domains. Additionally, Smad3 inhibited the dephosphorylation of p38 by MAP kinase phosphatase-1 (MKP-1). Both ATF-2 overexpression and p38 activation repressed type X collagen expression in wild type and Smad3−/− chondrocytes. p38 was detected in articular cartilage and perichondrium; articular and sternal chondrocytes expressed p38 isoforms α, β and γ, but not δ. Conclusions Smad3 is involved in both the onset and progression of OA. Loss of Smad3 abrogates TAK1-ATF-2 signaling, most likely by disrupting the Smad3-phospho-p38 complex and, thereby, promoting p38 dephosphorylation and inactivation by MKP-1. p38 and ATF-2 activation inhibit chondrocyte hypertrophy. Modulation of p38 isoform activity may provide a new therapeutic approach for OA. PMID:20506210

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

    Directory of Open Access Journals (Sweden)

    Abdul-Rehman Phull

    2016-01-01

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

  18. Experimentally induced cartilage degeneration treated by pulsed electromagnetic field stimulation; an in vitro study on bovine cartilage.

    Science.gov (United States)

    Veronesi, Francesca; Fini, Milena; Giavaresi, Gianluca; Ongaro, Alessia; De Mattei, Monica; Pellati, Agnese; Setti, Stefania; Tschon, Matilde

    2015-10-20

    Osteoarthritis (OA) is the final result of progressive alterations to articular cartilage structure, composition and cellularity, followed by an increase in the concentration of pro-inflammatory cytokines in joint synovial fluid. Even though the effect of pulsed electromagnetic field (PEMF) stimulation in counteracting OA progression and inflammation is of increasing interest, because of its anabolic and anti-inflammatory properties, the present study aimed to improve the knowledge on cartilage extracellular matrix (ECM) and chondrocyte changes related to the exposure of PEMF, from a histological and histomorphometric point of view. An in vitro OA model was realized, culturing bovine cartilage explants with a high dose of interleukin 1β (IL1β, 50 ng/ml) at different experimental times (24 h, and 7 and 21 days). The effects of PEMFs (75 Hz, 1.5 mT) were evaluated in cartilage explants treated with IL1β or not (control), in terms of cartilage structure, cellularity and proteoglycans, glycosaminoglycans, collagen II and transforming growth factor β1 synthesis by using histology, histomorphometry and immunohistochemistry. Making a comparison with control cartilage, IL1β-treated explants showed a decrease in cartilage matrix, structure and cellularity parameters. PEMFs were able to counteract the progression of OA acting on both cartilage cellularity and ECM in cartilage previously treated with IL1β. Normal distribution (Kolmogroc-Smirnov test) and homoscedasticity (Levene test) of data were verified, then, the non-parametric Kruskal Wallis test followed by Mann-Whiteny U test for pairwise comparisons were performed. The p-value was adjusted according to the Dunn-Sidak correction. These results, obtained by culturing and treating cartilage explants from two different joints, confirmed that PEMF stimulation can be used as adjuvant therapy to preserve cartilage from detrimental effects of high inflammatory cytokine levels during OA.

  19. Influence of Proteoglycan on Time-Dependent Mechanical Behaviors of Articular Cartilage under Constant Total Compressive Deformation

    Science.gov (United States)

    Murakami, Teruo; Sakai, Nobuo; Sawae, Yoshinori; Tanaka, Koji; Ihara, Maki

    Articular cartilage has biphasic property based on high water content. It is generally believed that the proteoglycan supports the compressive load, but the detailed loading mechanism has not yet been clarified. In this study, first we observed the changes in compressive stress and strain of articular cartilage under constant total compressive deflection. We evaluated the changes in modulus of elasticity, which was estimated from the stress-strain relation in equilibrium state. To examine the role of proteoglycan in compressed articular cartilage, we compared the time-dependent viscoelastic behaviors in both the intact cartilage and the cartilage treated with chondoroitinase ABC under constant total compressive deformation. We could confirm that the peak stress after compression and the modulus of elasticity at equilibrium were reduced after the digestion of proteoglycan. Next, we observed the changes in local strain in both articular cartilage specimens with and without chondroitinase treatment by monitoring the position of stained chondrocyte in the confocal laser scanning microscope. These visualized images indicated that the local strain changed time-dependently and depth-dependently. The digested cartilage showed the quicker change in movement and larger thinning in surface layer than the intact cartilage. These results indicate that the proteoglycan contributes to the compressive load-carrying capacity and controls the permeability.

  20. 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. Copyright 2007 Wiley Periodicals, Inc.

  1. Optical clearing of articular cartilage: a comparison of clearing agents

    Science.gov (United States)

    Bykov, Alexander; Hautala, Tapio; Kinnunen, Matti; Popov, Alexey; Karhula, Sakari; Saarakkala, Simo; Nieminen, Miika T.; Tuchin, Valery

    2015-07-01

    Optical clearing technique was applied to the problem of OCT imaging of articular cartilage and subchondral bone. We show that optical clearing significantly enhances visualization of articular cartilage and cartilage-bone interface. The effect of different clearing agents was analyzed. For the clearing, iohexol solution and propylene glycol (PG) were used. Clearing was performed in vitro at room temperature by immersion method. Cylindrical osteochondral samples (d=4.8mm) were drilled from bovine lateral femur and stored in phosphate-buffered saline at -20°C until clearing. Monitoring of clearing process was performed using high-speed spectral-domain OCT system providing axial resolution of 5.8μm at 930nm. Total duration of experiment was 90-100min to ensure saturation of clearing. We have shown that iohexol solution and PG are capable to optically clear articular cartilage enabling reliable characterization of cartilagebone interface with OCT. Being a low osmolarity agent, iohexol provides minimal changes to the thickness of cartilage sample. Clearing saturation time for the cartilage sample with the thickness of 0.9 mm measured with OCT is of 50 min. However, less than 15 min is enough to reliably detect the rear cartilage boundary. Alternatively, PG significantly (60%) reduces the cartilage thickness enabling better visualization of subchondral bone. It was observed that PG has higher clearing rate. The clearing saturation time is of 30 min, however less than 5 min is enough to detect cartilage-bone interface. We conclude that iohexol solution is superior for OCT imaging of cartilage and cartilage-bone interface, while PG suits better for subhondral bone visualization.

  2. Biomarkers of Chondrocyte Apoptosis and Autophagy in Osteoarthritis

    Science.gov (United States)

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

    2015-01-01

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

  3. The effect of ketorolac tromethamine, methylprednisolone, and platelet-rich plasma on human chondrocyte and tenocyte viability.

    Science.gov (United States)

    Beitzel, Knut; McCarthy, Mary Beth; Cote, Mark P; Apostolakos, John; Russell, Ryan P; Bradley, James; ElAttrache, Neal S; Romeo, Antony A; Arciero, Robert A; Mazzocca, Augustus D

    2013-07-01

    The purpose of this study was to evaluate the effect on cell viability of the isolated and combined use of allogeneic platelet-rich plasma (PRP) and ketorolac tromethamine on human chondrocytes and tenocytes in a highly controlled in vitro environment. PRP was produced from 8 subjects. Human chondrocytes (Lonza, Hopkinton, MA) and tenocytes isolated from samples of the long head of the biceps tendons were treated in culture with PRP, ketorolac tromethamine, and methylprednisolone, both alone and in combination. Control samples were treated in media containing 2% or 10% fetal bovine serum (FBS). Cells were exposed for 1 hour. Luminescence assays were obtained to examine cell viability after 24 hours and long-term effects on cell viability after 120 hours. Radioactive thymidine assay was used to measure proliferation after 120 hours. For chondrocytes, cell viability (120 hours) increased significantly with the treatment of PRP alone (43,949 ± 28,104 cells; P investigation into alternative treatment options such as combinations of PRP and ketorolac tromethamine. In vitro evaluation of their effect on cell viability might build a basis for further translational research and clinical application. Copyright © 2013 Arthroscopy Association of North America. Published by Elsevier Inc. All rights reserved.

  4. Effects of Medium and Temperature on Cellular Responses in the Superficial Zone of Hypo-Osmotically Challenged Articular Cartilage

    Directory of Open Access Journals (Sweden)

    Mikko Lammi

    2012-08-01

    Full Text Available Osmotic loading of articular cartilage has been used to study cell-tissue interactions and mechanisms in chondrocyte volume regulation in situ. Since cell volume changes are likely to affect cell’s mechanotransduction, it is important to understand how environmental factors, such as composition of the immersion medium and temperature affect cell volume changes in situ in osmotically challenged articular cartilage. In this study, chondrocytes were imaged in situ with a confocal laser scanning microscope (CLSM through cartilage surface before and 3 min and 120 min after a hypo-osmotic challenge. Samples were measured either in phosphate buffered saline (PBS, without glucose and Ca2+ or in Dulbecco’s modified Eagle’s medium (DMEM, with glucose and Ca2+, and at 21 °C or at 37 °C. In all groups, cell volumes increased shortly after the hypotonic challenge and then recovered back to the original volumes. At both observation time points, cell volume changes as a result of the osmotic challenge were similar in PBS and DMEM in both temperatures. Our results indicate that the initial chondrocyte swelling and volume recovery as a result of the hypo-osmotic challenge of cartilage are not dependent on commonly used immersion media or temperature.

  5. 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... products to revise the conditions for the importation of live bovines and products derived from bovines...

  6. 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.

  7. Inhibition of glycosaminoglycan incorporation influences collagen network formation during cartilage matrix production

    NARCIS (Netherlands)

    Bastiaansen-Jenniskens, Y.M.; Koevoet, W.; Jansen, K.M.B.; Verhaar, J.A.N.; Groot, J. de; Vanosch, G.J.V.M.

    2009-01-01

    To understand cartilage degenerative diseases and improve repair procedures, we investigate the influence of glycosaminoglycans (GAGs) on cartilage matrix biochemistry and functionality. Bovine articular chondrocytes were cultured in alginate beads with(out) para-nitrophenyl-beta-d-xyloside (PNPX)

  8. [Technological aspects of regenerative medicine and tissue engineering of articular cartilage].

    Science.gov (United States)

    Pörtner, R; Meenen, N M

    2010-12-01

    The main problem in the treatment of orthopaedic joint-surface defects will be solved by tissue engineering of cartilage implants. Entire biological osteochondral implants can be grown from autologous cells of the patient. The nutrition of articular cartilage is by diffusion only. Therefore the chondrocyte as the unique cell type is perfectly dedicated to the tissue culture approach. Engineering techniques of bioreactors are prerequisite for these biological and medical solutions. With our tissue engineering project for the generation of osteochondral constructs we demonstrate possibilities and characteristics of bioreactors for the modification of cell culture techniques and mechanical conditioning of cartilage tissue for fully operable implants. © Georg Thieme Verlag KG Stuttgart · New York.

  9. Matrix-induced autologous chondrocyte implantation for the treatment of chondral defects of the knees in Chinese patients

    Directory of Open Access Journals (Sweden)

    Zhang ZW

    2014-12-01

    Full Text Available Zhongwen Zhang,1 Xin Zhong,2 Huiru Ji,1 Zibin Tang,1 Jianpeng Bai,1 Minmin Yao,1 Jianlei Hou,1 Minghao Zheng,3 David J Wood,3 Jiazhi Sun,4 Shu-Feng Zhou,4,5 Aibing Liu6 1Department of Orthopedics, General Hospital of Chinese People’s Armed Police Forces (CAPF, Beijing; 2Department of MRI Center, General Hospital of CAPF, Beijing, People’s Republic of China; 3Center for Orthopedic Research, School of Surgery and Pathology, University of Western Australia, Perth, Western Australia, Australia; 4Department of Pharmaceutical Sciences, College of Pharmacy, University of South Florida, Tampa, FL, USA; 5Guizhou Provincial Key Laboratory for Regenerative Medicine, Stem Cell and Tissue Engineering Research Center and Sino–US Joint Laboratory for Medical Sciences, Guiyang Medical University, Guiyang, Guizhou; 6Medical Research Center, General Hospital of Chinese People’s Armed Police Forces (CAPF, Beijing, People’s Republic of China Abstract: Articular cartilage injury is the most common type of damage seen in clinical orthopedic practice. The matrix-induced autologous chondrocyte implant (MACI was developed to repair articular cartilage with an advance on the autologous chondrocyte implant procedure. This study aimed to evaluate whether MACI is a safe and efficacious cartilage repair treatment for patients with knee cartilage lesions. The primary outcomes were the Knee Injury and Osteoarthritis Outcome Score (KOOS domains and magnetic resonance imaging (MRI results, compared between baseline and postoperative months 3, 6, 12, and 24. A total of 15 patients (20 knees, with an average age of 33.9 years, had a mean defect size of 4.01 cm2. By 6-month follow-up, KOOS results demonstrated significant improvements in symptoms and knee-related quality of life. MRI showed significant improvements in four individual graft scoring parameters at 24 months postoperatively. At 24 months, 90% of MACI grafts had filled completely and 10% had good

  10. Effects of harvest and selected cartilage repair procedures on the physical and biochemical properties of articular cartilage in the canine knee.

    Science.gov (United States)

    Lee, C R; Grodzinsky, A J; Hsu, H P; Martin, S D; Spector, M

    2000-09-01

    This study utilizes a canine model to quantify changes in articular cartilage 15-18 weeks after a knee joint is subjected to surgical treatment of isolated chondral defects. Clinical and experimental treatment of articular cartilage defects may include implantation of matrix materials or cells, or both. Three cartilage repair methods were evaluated: microfracture, microfracture and implantation of a type-II collagen matrix, and implantation of an autologous chondrocyte-seeded collagen matrix. The properties of articular cartilage in other knee joints subjected to harvest of articular cartilage from the trochlear ridge (to obtain cells for the cell-seeded procedure) were also evaluated. Physical properties (thickness, equilibrium compressive modulus, dynamic compressive stiffness, and streaming potential) and biochemical composition (hydration, glycosaminoglycan content, and DNA content) of the cartilage from sites distant to the surgical treatment were compared with values measured for site-matched controls in untreated knee joints. No significant differences were seen in joints subjected to any of the three cartilage repair procedures. However, a number of changes were induced by the harvest operation. The largest changes (displaying up to 3-fold increases) were seen in dynamic stiffness and streaming potential of patellar groove cartilage from joints subjected to the harvest procedure. Whether the changes reported will lead to osteoarthritic degeneration is unknown, but this study provides evidence that the harvest procedure associated with autologous cell transplantation for treatment of chondral defects may result in changes in the articular cartilage in the joint.

  11. Antiviral effects of bovine interferons on bovine respiratory tract viruses.

    OpenAIRE

    Fulton, R W; Downing, M M; Cummins, J M

    1984-01-01

    The antiviral effects of bovine interferons on the replication of bovine respiratory tract viruses were studied. Bovine turbinate monolayer cultures were treated with bovine interferons and challenged with several bovine herpesvirus 1 strains, bovine viral diarrhea virus, parainfluenza type 3 virus, goat respiratory syncytial virus, bovine respiratory syncytial virus, bovine adenovirus type 7, or vesicular stomatitis virus. Treatment with bovine interferons reduced viral yield for each of the...

  12. Mesenchymal cell-based repair of large, full-thickness defects of articular cartilage.

    Science.gov (United States)

    Wakitani, S; Goto, T; Pineda, S J; Young, R G; Mansour, J M; Caplan, A I; Goldberg, V M

    1994-04-01

    Osteochondral progenitor cells were used to repair large, full-thickness defects of the articular cartilage that had been created in the knees of rabbits. Adherent cells from bone marrow, or cells from the periosteum that had been liberated from connective tissue by collagenase digestion, were grown in culture, dispersed in a type-I collagen gel, and transplanted into a large (three-by-six-millimeter), full-thickness (three-millimeter) defect in the weight-bearing surface of the medial femoral condyle. The contralateral knee served as a control: either the defect in that knee was left empty or a cell-free collagen gel was implanted. The periosteal and the bone-marrow-derived cells showed similar patterns of differentiation into articular cartilage and subchondral bone. Specimens of reparative tissue were analyzed with use of a semiquantitative histological grading system and by mechanical testing with employment of a porous indenter to measure the compliance of the tissue at intervals until twenty-four weeks after the operation. There was no apparent difference between the results obtained with the cells from the bone marrow and those from the periosteum. As early as two weeks after transplantation, the autologous osteochondral progenitor cells had uniformly differentiated into chondrocytes throughout the defects. This repair cartilage was subsequently replaced with bone in a proximal-to-distal direction, until, at twenty-four weeks after transplantation, the subchondral bone was completely repaired, without loss of overlying articular cartilage. The mechanical testing data were a useful index of the quality of the long-term repair. Twenty-four weeks after transplantation, the reparative tissue of both the bone-marrow and the periosteal cells was stiffer and less compliant than the tissue derived from the empty defects but less stiff and more compliant than normal cartilage. The current modalities for the repair of defects of the articular cartilage have many

  13. Infrared microspectroscopic determination of collagen cross-links in articular cartilage

    Science.gov (United States)

    Rieppo, Lassi; Kokkonen, Harri T.; Kulmala, Katariina A. M.; Kovanen, Vuokko; Lammi, Mikko J.; Töyräs, Juha; Saarakkala, Simo

    2017-03-01

    Collagen forms an organized network in articular cartilage to give tensile stiffness to the tissue. Due to its long half-life, collagen is susceptible to cross-links caused by advanced glycation end-products. The current standard method for determination of cross-link concentrations in tissues is the destructive high-performance liquid chromatography (HPLC). The aim of this study was to analyze the cross-link concentrations nondestructively from standard unstained histological articular cartilage sections by using Fourier transform infrared (FTIR) microspectroscopy. Half of the bovine articular cartilage samples (n=27) were treated with threose to increase the collagen cross-linking while the other half (n=27) served as a control group. Partial least squares (PLS) regression with variable selection algorithms was used to predict the cross-link concentrations from the measured average FTIR spectra of the samples, and HPLC was used as the reference method for cross-link concentrations. The correlation coefficients between the PLS regression models and the biochemical reference values were r=0.84 (p<0.001), r=0.87 (p<0.001) and r=0.92 (p<0.001) for hydroxylysyl pyridinoline (HP), lysyl pyridinoline (LP), and pentosidine (Pent) cross-links, respectively. The study demonstrated that FTIR microspectroscopy is a feasible method for investigating cross-link concentrations in articular cartilage.

  14. Direct Quantification of Solute Diffusivity in Agarose and Articular Cartilage Using Correlation Spectroscopy.

    Science.gov (United States)

    Shoga, Janty S; Graham, Brian T; Wang, Liyun; Price, Christopher

    2017-10-01

    Articular cartilage is an avascular tissue; diffusive transport is critical for its homeostasis. While numerous techniques have been used to quantify diffusivity within porous, hydrated tissues and tissue engineered constructs, these techniques have suffered from issues regarding invasiveness and spatial resolution. In the present study, we implemented and compared two separate correlation spectroscopy techniques, fluorescence correlation spectroscopy (FCS) and raster image correlation spectroscopy (RICS), for the direct, and minimally-invasive quantification of fluorescent solute diffusion in agarose and articular cartilage. Specifically, we quantified the diffusional properties of fluorescein and Alexa Fluor 488-conjugated dextrans (3k and 10k) in aqueous solutions, agarose gels of varying concentration (i.e. 1, 3, 5%), and in different zones of juvenile bovine articular cartilage explants (i.e. superficial, middle, and deep). In agarose, properties of solute diffusion obtained via FCS and RICS were inversely related to molecule size, gel concentration, and applied strain. In cartilage, the diffusional properties of solutes were similarly dependent upon solute size, cartilage zone, and compressive strain; findings that agree with work utilizing other quantification techniques. In conclusion, this study established the utility of FCS and RICS as simple and minimally invasive techniques for quantifying microscale solute diffusivity within agarose constructs and articular cartilage explants.

  15. Intensity-Dependent Effect of Treadmill Running on Knee Articular Cartilage in a Rat Model

    Directory of Open Access Journals (Sweden)

    Guo-Xin Ni

    2013-01-01

    Full Text Available Objective. To understand the changes of femoral cartilage in response to treadmill running with different intensities in the hope of differentiating “moderate” and “strenuous” running in a rat model. Method. A total of 24 male Wistar rats were randomly assigned into groups of sedentary (SED, low-intensity running (LIR, medium-intensity running (MIR, and high-intensity running (HIR. Rats in LIR, MIR, and HIR groups underwent 8 weeks’ treadmill running programs. After sacrificed, femoral condyles were collected to take histomorphometric analysis and immunohistochemistry for collagen II. Results. Gross and histological observation showed osteoarthritic changes in group HIR. In comparison to SED group, there was significant increase in cartilage thickness, number of chondrocytes, and GAG content in groups LIR and MIR. Conversely, decrease in cartilage thickness, chondrocyte number, and GAG content was found in rats of HIR group, without significant difference though. In addition, in comparison to SED group, HIR group exhibited disorganization of collagen fibril and significantly lower content of collagen type II. Conclusion. An intensity-dependent effect was suggested on the articular cartilage. Our results also demonstrated that running with low-to-medium intensity applied in the present study should be regarded as “moderate” running, whereas high-intensity running as “strenuous” running.

  16. Multiscale Mechanics of Articular Cartilage: Potentials and Challenges of Coupling Musculoskeletal, Joint, and Microscale Computational Models

    Science.gov (United States)

    Halloran, J. P.; Sibole, S.; van Donkelaar, C. C.; van Turnhout, M. C.; Oomens, C. W. J.; Weiss, J. A.; Guilak, F.; Erdemir, A.

    2012-01-01

    Articular cartilage experiences significant mechanical loads during daily activities. Healthy cartilage provides the capacity for load bearing and regulates the mechanobiological processes for tissue development, maintenance, and repair. Experimental studies at multiple scales have provided a fundamental understanding of macroscopic mechanical function, evaluation of the micromechanical environment of chondrocytes, and the foundations for mechanobiological response. In addition, computational models of cartilage have offered a concise description of experimental data at many spatial levels under healthy and diseased conditions, and have served to generate hypotheses for the mechanical and biological function. Further, modeling and simulation provides a platform for predictive risk assessment, management of dysfunction, as well as a means to relate multiple spatial scales. Simulation-based investigation of cartilage comes with many challenges including both the computational burden and often insufficient availability of data for model development and validation. This review outlines recent modeling and simulation approaches to understand cartilage function from a mechanical systems perspective, and illustrates pathways to associate mechanics with biological function. Computational representations at single scales are provided from the body down to the microstructure, along with attempts to explore multiscale mechanisms of load sharing that dictate the mechanical environment of the cartilage and chondrocytes. PMID:22648577

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

    Directory of Open Access Journals (Sweden)

    Wendan Cheng

    2017-03-01

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

  18. 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

  19. Biochanin-A antagonizes the interleukin-1β-induced catabolic inflammation through the modulation of NFκB cellular signaling in primary rat chondrocytes

    Energy Technology Data Exchange (ETDEWEB)

    Oh, Ji-Su [Department of Oral and Maxillofacial Surgery, Chosun University, Gwangju, 61452 (Korea, Republic of); Cho, In-A; Kang, Kyeong-Rok [Department of Dental Bioengineering, Chosun University, Gwangju, 61452 (Korea, Republic of); You, Jae-Seek [Department of Oral and Maxillofacial Surgery, Chosun University, Gwangju, 61452 (Korea, Republic of); Yu, Sang-Joun [Department of Periodontology, Chosun University, Gwangju, 61452 (Korea, Republic of); Lee, Gyeong-Je [Department of Prosthodontics, Chosun University, Gwangju, 61452 (Korea, Republic of); Seo, Yo-Seob [Department of Oral and Maxillofacial Radiology, Chosun University, Gwangju, 61452 (Korea, Republic of); Kim, Chun Sung; Kim, Do Kyung [Pre-Dentistry, School of Dentistry, Chosun University, Gwangju, 61452 (Korea, Republic of); Kim, Su-Gwan [Department of Oral and Maxillofacial Surgery, Chosun University, Gwangju, 61452 (Korea, Republic of); Seo, Young-Woo [Korea Basic Science Institute, Gwangju Center, Chonnam National University, Gwangju, 61186 (Korea, Republic of); Im, Hee-Jeong [Department of Biochemistry, Rush University Medical Center, Chicago, IL, 60612 (United States); Kim, Jae-Sung, E-mail: js_kim@chosun.ac.kr [Pre-Dentistry, School of Dentistry, Chosun University, Gwangju, 61452 (Korea, Republic of)

    2016-09-02

    Biochanin-A, a phytoestrogen derived from herbal plants, protected from the IL-1β-induced loss of proteoglycans through the suppression of matrix degrading enzymes such as matrix metalloproteinase (MMP)-13, MMP-3, MMP-1, and ADAMTS-5 in primary rat chondrocytes and the knee articular cartilage. It also suppressed the expression of IL-1β-induced catabolic factors such as nitric oxide synthase 2, cyclooxygenase-2, prostaglandin E{sub 2}, and inflammatory cytokines. Furthermore, biochanin-A suppressed the IL-1β-induced phosphorylation of NFκB, and inhibited its nuclear translocation in primary rat chondrocytes. These results indicate that biochanin-A antagonizes the IL-1β-induced catabolic effects through its anti-inflammatory activity that involves the modulation of NFκB signaling. - Highlights: • Biochanin-A is a phytoestrogen derived from medicinal plants. • It suppressed the IL-1β-induced matrix degrading enzymes and catabolic factors. • It inhibited IL-1β-induced proteoglycan loss in chondrocytes and cartilage tissues. • Its anti-catabolic effects were mediated by modulation of NFκB signaling. • It may be used as a potential anti-catabolic biomaterial for osteoarthritis.

  20. 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...

  1. L-Monomethyl-arginine decreases apoptosis of chondrocytes by ...

    African Journals Online (AJOL)

    Yomi

    2012-04-03

    ) linked an increased. *Corresponding author. E-mail: zongbao_wang@126.com. Tel: +86-021-38804518. number of degraded chondrocytes to disease severity. A subsequent study of a rabbit knee OA model by. Hashimoto ...

  2. Vascular endothelial growth factor activities on osteoarthritic chondrocytes.

    Science.gov (United States)

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

    2005-01-01

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

  3. Chondroprotective effect of zinc oxide nanoparticles in conjunction with hypoxia on bovine cartilage-matrix synthesis.

    Science.gov (United States)

    Mirza, Eraj Humayun; Pan-Pan, Chong; Wan Ibrahim, Wan Mohd Azhar Bin; Djordjevic, Ivan; Pingguan-Murphy, Belinda

    2015-11-01

    Articular cartilage is a tissue specifically adapted to a specific niche with a low oxygen tension (hypoxia), and the presence of such conditions is a key factor in regulating growth and survival of chondrocytes. Zinc deficiency has been linked to cartilage-related disease, and presence of Zinc is known to provide antibacterial benefits, which makes its inclusion attractive in an in vitro system to reduce infection. Inclusion of 1% zinc oxide nanoparticles (ZnONP) in poly octanediol citrate (POC) polymer cultured in hypoxia has not been well determined. In this study we investigated the effects of ZnONP on chondrocyte proliferation and matrix synthesis cultured under normoxia (21% O2 ) and hypoxia (5% O2 ). We report an upregulation of chondrocyte proliferation and sulfated glycosaminoglycan (S-GAG) in hypoxic culture. Results demonstrate a synergistic effect of oxygen concentration and 1% ZnONP in up-regulation of anabolic gene expression (Type II collagen and aggrecan), and a down regulation of catabolic (MMP-13) gene expression. Furthermore, production of transcription factor hypoxia-inducible factor 1A (HIF-1A) in response to hypoxic condition to regulate chondrocyte survival under hypoxia is not affected by the presence of 1% ZnONP. Presence of 1% ZnONP appears to act to preserve homeostasis of cartilage in its hypoxic environment. © 2015 Wiley Periodicals, Inc.

  4. 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

  5. Inhibition of Chondrocyte Hypertrophy of Osteoarthritis by Disruptor Peptide

    Science.gov (United States)

    2017-07-01

    ultimate human subject testing. 2. KEYWORDS: Osteoarthritis Parathyroid hormone-related protein PTH receptor -catenin Cell signaling...To further verify our results, we will repeat the experiments with mouse bone marrow-derived mesenchymal stem cell differentiation to chondrocytes...AWARD NUMBER: W81XWH-16-1-0325 TITLE: Inhibition of Chondrocyte Hypertrophy of Osteoarthritis by Disruptor Peptide PRINCIPAL INVESTIGATOR: Bin

  6. Supporting Biomaterials for Articular Cartilage Repair

    Science.gov (United States)

    Duarte Campos, Daniela Filipa; Drescher, Wolf; Rath, Björn; Tingart, Markus

    2012-01-01

    Orthopedic surgeons and researchers worldwide are continuously faced with the challenge of regenerating articular cartilage defects. However, until now, it has not been possible to completely mimic the biological and biochemical properties of articular cartilage using current research and development approaches. In this review, biomaterials previously used for articular cartilage repair research are addressed. Furthermore, a brief discussion of the state of the art of current cell printing procedures mimicking native cartilage is offered in light of their use as future alternatives for cartilage tissue engineering. Inkjet cell printing, controlled deposition cell printing tools, and laser cell printing are cutting-edge techniques in this context. The development of mimetic hydrogels with specific biological properties relevant to articular cartilage native tissue will support the development of improved, functional, and novel engineered tissue for clinical application. PMID:26069634

  7. SOX trio decrease in the articular cartilage with the advancement of osteoarthritis.

    Science.gov (United States)

    Lee, Jai-Sun; Im, Gun-Il

    2011-01-01

    SOX trio (SOX-5, SOX-6, and SOX-9) maintain the chondrocytic phenotypes and are vital for chondrogenesis in embryonic development. The purpose of this study is to investigate the change in the expression of SOX trio with the advancement of osteoarthritis (OA) in human articular cartilage (AC). Human OA samples from eight patients were obtained from the distal femoral condyles during total knee arthroplasty. Minimally OA cartilage taken from areas with no obvious surface defects on lateral condyles was compared with advanced OA cartilage obtained from areas within 1 cm of overt lesion located on medial condyle surface. SOX-5, SOX-6, and SOX-9 gene expressions significantly decreased by 41% (p = 0.047), 46% (p = 0.047), and 56% (p = 0.029) in advanced OA area compared with the minimally OA area. There was a significant decrease in aggrecan and type II collagen (COL2A1) gene expressions by 73% (p = 0.029) and 65% (p = 0.029), respectively, in advanced OA area compared with the minimally OA area. From Western blotting and immunohistochemistry, SOX-5, SOX-6, SOX-9, type II collagen, and aggrecan protein expressions also significantly decreased in advanced OA cartilage compared with minimally OA cartilage. DNA methylation study of SOX-9 promoter regions revealed no difference in the epigenetic status between the two areas. It is concluded that SOX trio gene and protein decreased with advancement of OA in human articular cartilage.

  8. Development of a Spring-Loaded Impact Device to Deliver Injurious Mechanical Impacts to the Articular Cartilage Surface

    Science.gov (United States)

    Alexander, Peter G.; Song, Yingjie; Taboas, Juan M.; Chen, Faye H.; Melvin, Gary M.; Manner, Paul A.

    2013-01-01

    Objective: Traumatic impacts on the articular joint surface in vitro are known to lead to degeneration of the cartilage. The main objective of this study was to develop a spring-loaded impact device that can be used to deliver traumatic impacts of consistent magnitude and rate and to find whether impacts cause catabolic activities in articular cartilage consistent with other previously reported impact models and correlated with the development of osteoarthritic lesions. In developing the spring-loaded impactor, the operating hypothesis is that a single supraphysiologic impact to articular cartilage in vitro can affect cartilage integrity, cell viability, sulfated glycosaminoglycan and inflammatory mediator release in a dose-dependent manner. Design: Impacts of increasing force are delivered to adult bovine articular cartilage explants in confined compression. Impact parameters are correlated with tissue damage, cell viability, matrix and inflammatory mediator release, and gene expression 24 hours postimpact. Results: Nitric oxide release is first detected after 7.7 MPa impacts, whereas cell death, glycosaminoglycan release, and prostaglandin E2 release are first detected at 17 MPa. Catabolic markers increase linearly to maximal levels after ≥36 MPa impacts. Conclusions: A single supraphysiologic impact negatively affects cartilage integrity, cell viability, and GAG release in a dose-dependent manner. Our findings showed that 7 to 17 MPa impacts can induce cell death and catabolism without compromising the articular surface, whereas a 17 MPa impact is sufficient to induce increases in most common catabolic markers of osteoarthritic degeneration. PMID:26069650

  9. Characterization of cutaneous and articular sensory neurons.

    Science.gov (United States)

    da Silva Serra, Ines; Husson, Zoé; Bartlett, Jonathan D; Smith, Ewan St John

    2016-01-01

    A wide range of stimuli can activate sensory neurons and neurons innervating specific tissues often have distinct properties. Here, we used retrograde tracing to identify sensory neurons innervating the hind paw skin (cutaneous) and ankle/knee joints (articular), and combined immunohistochemistry and electrophysiology analysis to determine the neurochemical phenotype of cutaneous and articular neurons, as well as their electrical and chemical excitability. Immunohistochemistry analysis using RetroBeads as a retrograde tracer confirmed previous data that cutaneous and articular neurons are a mixture of myelinated and unmyelinated neurons, and the majority of both populations are peptidergic. In whole-cell patch-clamp recordings from cultured dorsal root ganglion neurons, voltage-gated inward currents and action potential parameters were largely similar between articular and cutaneous neurons, although cutaneous neuron action potentials had a longer half-peak duration (HPD). An assessment of chemical sensitivity showed that all neurons responded to a pH 5.0 solution, but that acid-sensing ion channel (ASIC) currents, determined by inhibition with the nonselective acid-sensing ion channel antagonist benzamil, were of a greater magnitude in cutaneous compared to articular neurons. Forty to fifty percent of cutaneous and articular neurons responded to capsaicin, cinnamaldehyde, and menthol, indicating similar expression levels of transient receptor potential vanilloid 1 (TRPV1), transient receptor potential ankyrin 1 (TRPA1), and transient receptor potential melastatin 8 (TRPM8), respectively. By contrast, significantly more articular neurons responded to ATP than cutaneous neurons. This work makes a detailed characterization of cutaneous and articular sensory neurons and highlights the importance of making recordings from identified neuronal populations: sensory neurons innervating different tissues have subtly different properties, possibly reflecting different

  10. 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.

  11. Vitamin D prevents articular cartilage erosion by regulating collagen II turnover through TGF-β1 in ovariectomized rats.

    Science.gov (United States)

    Li, S; Niu, G; Wu, Y; Du, G; Huang, C; Yin, X; Liu, Z; Song, C; Leng, H

    2016-02-01

    To explore the effect of vitamin D on turnover of articular cartilage with ovariectomy (OVX) induced OA, and to investigate transforming growth factor-β1 (TGF-β1) as a possible underlying mechanism mediated by 1α,25(OH)2D3. Sixty-six rats were randomly allocated into seven groups: sham plus control diet (SHAM+CTL), OVX+CTL diet, sham plus vitamin D-deficient (VDD) diet, OVX+VDD diet, and three groups of ovariectomized rats treated with different doses of 1α,25(OH)2D3. The cartilage erosion and the levels of serum 17β-estradiol, 1α,25(OH)2D3 and C-telopeptide of type II collagen (CTX-II) were measured. TGF-β1, type II Collagen (CII), matrix metalloproteinases (MMP)-9,-13 in articular cartilage were assessed by immunohistochemistry. TGF-β1 and CTX-II expression were measured in articular cartilage chondrocytes treated with/without tumor necrosis factor (TNF-α), 1α,25(OH)2D3, and TGF-β receptor inhibitor (SB505124) in vitro. Cartilage erosion due to OVX was significantly reduced in a dose-dependent manner by 1α,25(OH)2D3 supplementation, and exacerbated by VDD. The expressions of TGF-β1 and CII in articular cartilage were suppressed by OVX and VDD, and rescued by 1α,25(OH)2D3 supplementation. The expression of MMP-9,-13 in articular cartilage increased with OVX and VDD, and decreased with 1α,25(OH)2D3 supplementation. In vitro experiments showed that 1α,25(OH)2D3 increased the TGF-β1 expression of TNF-α stimulated chondrocytes in a dose-dependent manner. 1α,25(OH)2D3 significantly counteracted the increased CTX-II release due to TNF-α stimulation, and this effect was significantly suppressed by SB505124. VDD aggravated cartilage erosion, and 1α,25(OH)2D3 supplementation showed protective effects in OVX-induced OA partly through the TGF-β1 pathway. Copyright © 2015 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.

  12. MicroRNA-142-3p Inhibits Chondrocyte Apoptosis and Inflammation in Osteoarthritis by Targeting HMGB1.

    Science.gov (United States)

    Wang, Xiuqin; Guo, Yanqing; Wang, Chunyan; Yu, Hong; Yu, Xiuxiang; Yu, Hongbo

    2016-10-01

    Osteoarthritis (OA) is a degenerative joint disease characterized by articular cartilage degradation and joint inflammation in which microRNAs are significantly involved. Previous studies have reported that miR-142-3p is a novel mediator of inflammatory signaling pathways, but whether miR-142-3p regulates OA remains unknown. In this study, we aimed to investigate the potential role of miR-142-3p in OA and the underlying molecular mechanism. We showed that miR-142-3p was significantly reduced in the articular cartilage tissues from experimental OA mice. The expression of miR-142-3p was also decreased in chondrocytes treated with lipopolysaccharide (LPS) in vitro. Moreover, the overexpression of miR-142-3p significantly inhibited cell apoptosis, nuclear factor (NF)-kB, and the production of proinflammatory cytokines, including interleukin (IL)-1, IL-6, and tumor necrosis factor (TNF)-α induced by LPS. Interestingly, bioinformatics analysis demonstrated that high mobility group box 1 (HMGB1), an important inflammatory mediator of OA, was predicted as a target of miR-142-3p, which was validated by dual-luciferase reporter assay. The high expression of HMGB1 in chondrocytes induced by LPS was significantly inhibited by miR-142-3p overexpression. Furthermore, the restoration of HMGB1 markedly abrogated the effect of miR-142-3p. In OA mice, the overexpression of miR-142-3p by lentivirus-mediated gene transfer significantly inhibited HMGB1 expression, NF-kB signaling, and proinflammatory cytokines. Moreover, the overexpression of miR-142-3p significantly alleviated OA progression in OA mice in vivo. Taken together, our study suggests that miR-142-3p inhibits chondrocyte apoptosis and inflammation in OA by inhibiting the HMGB1-mediated NF-kB signaling pathway. The overexpression of miR-142-3p impedes the OA progression in mice in vivo indicating that miR-142-3p is a potential molecular target for OA treatment.

  13. Articular manifestations in patients with Lyme disease.

    Science.gov (United States)

    Vázquez-López, María Esther; Díez-Morrondo, Carolina; Sánchez-Andrade, Amalia; Pego-Reigosa, Robustiano; Díaz, Pablo; Castro-Gago, Manuel

    To determine the percentage of Lyme patients with articular manifestations in NW Spain and to know their evolution and response to treatment. A retrospective study (2006-2013) was performed using medical histories of confirmed cases of Lyme disease showing articular manifestations. Clinical and laboratory characteristics, together with the treatment and evolution of the patients, were analysed. Seventeen out of 108 LD confirmed patients (15.7%) showed articular manifestations. Regarding those 17 patients, 64.7%, 29.4% and 5.9% presented arthritis, arthralgia and bursitis, respectively. The knee was the most affected joint. Articular manifestations were often associated to neurological, dermatological and cardiac pathologies. Otherwise, most patients were in Stage III. The 11.8% of the cases progressed to a recurrent chronic arthritis despite the administration of an appropriate treatment. Lyme disease patients showing articular manifestations should be included in the diagnosis of articular affections in areas of high risk of hard tick bite, in order to establish a suitable and early treatment and to avoid sequels. Copyright © 2015 Elsevier España, S.L.U. and Sociedad Española de Reumatología y Colegio Mexicano de Reumatología. All rights reserved.

  14. Influence of Intra-Articular Administration of Trichostatin A on Autologous Osteochondral Transplantation in a Rabbit Model

    Directory of Open Access Journals (Sweden)

    Huacheng Hou

    2015-01-01

    Full Text Available Autologous osteochondral transplantation (AOT is a method for articular cartilage repair. However, several disadvantages of this method have been reported, such as transplanted cartilage degeneration and the lack of a connection between the grafted and adjacent cartilage tissues. To evaluate the effect of intra-articular administration of trichostatin A (TSA on AOT, we conducted a case control study in a rabbit model. International Cartilage Repair Society (ICRS macroscopic scores, the modified O’Driscoll histology scores, and real-time PCR were utilized to evaluate the results. At 4 weeks, both macroscopic and histological assessments showed that there was no significant difference between the TSA and control groups. However, the mean macroscopic and histological scores for the TSA-treated group were significantly higher than the scores for the control group at 12 weeks. TSA was shown to directly reduce collagen type II (COL2, aggrecan, matrix metalloproteinase (MMP, and a disintegrin and metalloproteinase domain with thrombospondin motifs 5 (ADAMTS-5 expression and to simultaneously repress the upregulation of MMP-3, MMP-9, and MMP-13 levels induced by interleukin 1β (IL-1β in chondrocytes. In conclusion, TSA protects AOT grafts from degeneration, which may provide a benefit in the repair of articular cartilage injury.

  15. Therapeutic Effects of the Superoxide Dismutase Mimetic Compound Me2DO2A on Experimental Articular Pain in Rats

    Directory of Open Access Journals (Sweden)

    Lorenzo Di Cesare Mannelli

    2013-01-01

    Full Text Available Superoxide anion ( is overproduced in joint inflammation, rheumatoid arthritis, and osteoarthritis. Increased production leads to tissue damage, articular degeneration, and pain. In these conditions, the physiological defense against , superoxide dismutases (SOD are decreased. The complex MnL4 is a potent SOD mimetic, and in this study it was tested in inflammatory and osteoarticular rat pain models. In vivo protocols were approved by the animal Ethical Committee of the University of Florence. Pain was measured by paw pressure and hind limb weight bearing alterations tests. MnL4 (15 mg kg−1 acutely administered, significantly reduced pain induced by carrageenan, complete Freund’s adjuvant (CFA, and sodium monoiodoacetate (MIA. In CFA and MIA protocols, it ameliorated the alteration of postural equilibrium. When administered by osmotic pump in the MIA osteoarthritis, MnL4 reduced pain, articular derangement, plasma TNF alpha levels, and protein carbonylation. The scaffold ring was ineffective. MnL4 (10−7 M prevented the lipid peroxidation of isolated human chondrocytes when was produced by RAW 264.7. MnL4 behaves as a potent pain reliever in acute inflammatory and chronic articular pain, being its efficacy related to antioxidant property. Therefore MnL4 appears as a novel protective compound potentially suitable for the treatment of joint diseases.

  16. Glycogen Synthase Kinase-3β Inhibition Links Mitochondrial Dysfunction, Extracellular Matrix Remodelling and Terminal Differentiation in Chondrocytes.

    Science.gov (United States)

    Guidotti, S; Minguzzi, M; Platano, D; Santi, S; Trisolino, G; Filardo, G; Mariani, E; Borzì, R M

    2017-09-21

    Following inflammatory stimuli, GSK3 inhibition functions as a hub with pleiotropic effects leading to cartilage degradation. However, little is known about the effects triggered by its direct inhibition as well as the effects on mitochondrial pathology, that contributes to osteoarthritis pathogenesis. To this aim we assessed the molecular mechanisms triggered by GSK3β inactivating stimuli on 3-D (micromass) cultures of human articular chondrocytes. Stimuli were delivered either at micromass seeding (long term) or after maturation (short term) to explore "late" effects on terminal differentiation or "early" mitochondrial effects, respectively. GSK3β inhibition significantly enhanced mitochondrial oxidative stress and damage and endochondral ossification based on increased nuclear translocation of Runx-2 and β-catenin, calcium deposition, cell death and enhanced remodelling of the extracellular matrix as demonstrated by the increased collagenolytic activity of supernatants, despite unmodified (MMP-1) or even reduced (MMP-13) collagenase gene/protein expression. Molecular dissection of the underlying mechanisms showed that GSK3β inhibition achieved with pharmacological/silencing strategies impacted on the control of collagenolytic activity, via both decreased inhibition (reduced TIMP-3) and increased activation (increased MMP-10 and MMP-14). To conclude, the inhibition of GSK3β enhances terminal differentiation via concerted effects on ECM and therefore its activity represents a tool to keep articular cartilage homeostasis.

  17. Intra-articular transplantation of atsttrin-transduced mesenchymal stem cells ameliorate osteoarthritis development.

    Science.gov (United States)

    Xia, Qingqing; Zhu, Shouan; Wu, Yan; Wang, Jiaqiu; Cai, Youzhi; Chen, Pengfei; Li, Jie; Heng, Boon Chin; Ouyang, Hong Wei; Lu, Ping

    2015-05-01

    Osteoarthritis (OA) remains an intractable clinical challenge. Few drugs are available for reversing this degenerative disease, although some promising candidates have performed well in preclinical studies. Tumor necrosis factor α (TNFα) has been identified as a crucial effector modulating OA pathogenesis. This study aimed to investigate the therapeutic effects of Atsttrin, a novel TNFα blocker, on OA treatment. We developed genetically modified mesenchymal stem cells (MSCs) that expressed recombinant Atsttrin (named as MSC-Atsttrin). Expression levels of ADAMTS-5, MMP13, and iNOS of human chondrocytes were analyzed when cocultured with MSC-GFP/Atsttrin. OA animal models were induced by anterior cruciate ligament transection, and MSC-GFP/Atsttrin were injected into the articular cavity 1 week postsurgery. The results showed that MSC-Atsttrin significantly suppressed TNFα-driven up-regulation of matrix proteases and inflammatory factors. Intra-articular injection of MSC-Atsttrin prevented the progression of degenerative changes in the surgically induced OA mouse model. Additionally, levels of detrimental matrix hydrolases were significantly diminished. Compared with nontreated OA samples at 8 weeks postsurgery, the percentages of MMP13- and ADAMTS-5-positive cells were significantly reduced from 91.33% ± 9.87% to 24.33% ± 5.7% (p < .001) and from 91.33% ± 7.1% to 16.67% ± 3.1% (p < .001), respectively. Our results thus indicated that suppression of TNFα activity is an effective strategy for OA treatment and that intra-articular injection of MSCs-Atsttrin could be a promising therapeutic modality. ©AlphaMed Press.

  18. MicroRNAs and Autophagy: Fine Players in the Control of Chondrocyte Homeostatic Activities in Osteoarthritis

    Directory of Open Access Journals (Sweden)

    Stefania D’Adamo

    2017-01-01

    Full Text Available Osteoarthritis (OA is a debilitating degenerative disease of the articular cartilage with a multifactorial etiology. Aging, the main risk factor for OA development, is associated with a systemic oxidative and inflammatory phenotype. Autophagy is a central housekeeping system that plays an antiaging role by supporting the clearance of senescence-associated alterations of macromolecules and organelles. Autophagy deficiency has been related to OA pathogenesis because of the accumulation of cellular defects in chondrocytes. Microribonucleic acids (microRNAs or miRs are a well-established class of posttranscriptional modulators belonging to the family of noncoding RNAs that have been identified as key players in the regulation of cellular processes, such as autophagy, by targeting their own cognate mRNAs. Here, we present a state-of-the-art literature review on the role of miRs and autophagy in the scenario of OA pathogenesis. In addition, a comprehensive survey has been performed on the functional connections of the miR network and the autophagy pathway in OA by using “microRNA,” “autophagy,” and “osteoarthritis” as key words. Discussion of available evidence sheds light on some aspects that need further investigation in order to reach a more comprehensive view of the potential of this topic in OA.

  19. 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.

  20. PREVALENCE OF BOVINE (1)

    African Journals Online (AJOL)

    BACKGROUND: Tuberculosis is caused by a number of Mycobacterium species, of which Mycobacterium bovis, causing 'bovine tuberculosis' is ... KEY WORDS: Mycobacterium bovis, Zoonosis, Holeta, Ethiopia causing 'bovine tuberculosis ..... isolation of infected animals in which communal grazing and watering practiced.

  1. Repair of articular cartilage defects by tissue-engineered cartilage constructed with adipose-derived stem cells and acellular cartilaginous matrix in rabbits.

    Science.gov (United States)

    Wang, Z J; An, R Z; Zhao, J Y; Zhang, Q; Yang, J; Wang, J B; Wen, G Y; Yuan, X H; Qi, X W; Li, S J; Ye, X C

    2014-06-18

    After injury, inflammation, or degeneration, articular cartilage has limited self-repair ability. We aimed to explore the feasibility of repair of articular cartilage defects with tissue-engineered cartilage constructed by acellular cartilage matrices (ACMs) seeded with adipose-derived stem cells (ADSCs). The ADSCs were isolated from 3-month-old New Zealand albino rabbit by using collagenase and cultured and amplified in vitro. Fresh cartilage isolated from adult New Zealand albino rabbit were freeze-dried for 12 h and treated with Triton X-100, DNase, and RNase to obtain ACMs. ADSCs were seeded in the acellular cartilaginous matrix at 2x10(7)/mL, and cultured in chondrogenic differentiation medium for 2 weeks to construct tissue-engineered cartilage. Twenty-four New Zealand white rabbits were randomly divided into A, B, and C groups. Engineered cartilage was transplanted into cartilage defect position of rabbits in group A, group B obtained ACMs, and group C did not receive any transplants. The rabbits were sacrificed in week 12. The restored tissue was evaluated using macroscopy, histology, immunohistochemistry, and transmission electron microscopy (TEM). In the tissue-engineered cartilage group (group A), articular cartilage defects of the rabbits were filled with chondrocyte-like tissue with smooth surface. Immunohistochemistry showed type II-collagen expression and Alcian blue staining was positive. TEM showed chondrocytes in the recesses, with plenty of secretary matrix particles. In the scaffold group (group B), the defect was filled with fibrous tissue. No repaired tissue was found in the blank group (group C). Tissue-engineered cartilage using ACM seeded with ADSCs can help repair articular cartilage defects in rabbits.

  2. Contrast agent enhanced pQCT of articular cartilage

    Energy Technology Data Exchange (ETDEWEB)

    Kallioniemi, A S [Department of Physics, University of Kuopio, POB 1627, 70211 Kuopio (Finland); Jurvelin, J S [Department of Physics, University of Kuopio, POB 1627, 70211 Kuopio (Finland); Nieminen, M T [Department of Diagnostic Radiology, POB 50, 90029 OYS, Oulu University Hospital, Oulu (Finland); Lammi, M J [Department of Anatomy, Institute of Biomedicine, University of Kuopio, POB 1627, 70211 Kuopio (Finland); Toeyraes, J [Department of Physics, University of Kuopio, POB 1627, 70211 Kuopio (Finland)

    2007-02-21

    The delayed gadolinium enhanced MRI of cartilage (dGEMRIC) technique is the only non-invasive means to estimate proteoglycan (PG) content in articular cartilage. In dGEMRIC, the anionic paramagnetic contrast agent gadopentetate distributes in inverse relation to negatively charged PGs, leading to a linear relation between T{sub 1,Gd} and spatial PG content in tissue. In the present study, for the first time, contrast agent enhanced peripheral quantitative computed tomography (pQCT) was applied, analogously to dGEMRIC, for the quantitative detection of spatial PG content in cartilage. The suitability of two anionic radiographic contrast agents, gadopentetate and ioxaglate, to detect enzymatically induced PG depletion in articular cartilage was investigated. First, the interrelationships of x-ray absorption, as measured with pQCT, and the contrast agent solution concentration were investigated. Optimal contrast agent concentrations for the following experiments were selected. Second, diffusion rates for both contrast agents were investigated in intact (n = 3) and trypsin-degraded (n 3) bovine patellar cartilage. The contrast agent concentration of the cartilaginous layer was measured prior to and 2-27 h after immersion. Optimal immersion time for the further experiments was selected. Third, the suitability of gadopentetate and ioxaglate enhanced pQCT to detect the enzymatically induced specific PG depletion was investigated by determining the contrast agent concentrations and uronic acid and water contents in digested and intact osteochondral samples (n = 16). After trypsin-induced PG loss (-70%, p < 0.05) the penetration of gadopentetate and ioxaglate increased (p < 0.05) by 34% and 48%, respectively. Gadopentetate and ioxaglate concentrations both showed strong correlation (r = -0.95, r -0.94, p < 0.01, respectively) with the uronic acid content. To conclude, contrast agent enhanced pQCT provides a technique to quantify PG content in normal and experimentally

  3. Protective effect of Capparis spinosa on chondrocytes.

    Science.gov (United States)

    Panico, A M; Cardile, V; Garufi, F; Puglia, C; Bonina, F; Ronsisvalle, G

    2005-09-30

    The aim of the present study was to evaluate the in vitro chondroprotective effects of the lyophilised methanolic extract from flowering buds of Capparis Spinosa L (LECS). This plant, common to the Mediterranean basin, has been used by the traditional medicine for its diuretic and antihypertensive effects and also in certain pathological conditions related to uncontrolled lipid peroxidation. The extract contains many constituents, in particular some flavonoids (kaempferol and quercetin derivatives) and hydrocinammic acids with several known biological effects such as the anti-inflammatory and the antioxidant ones. In this study, we assayed the effect of LECS on human chondrocytes cultures stimulated by proinflammatory cytokine interleukin-1beta (IL-1beta) and we determined the production of key molecules released during chronic inflammatory events (nitric oxide, glycosaminoglycans, prostaglandins and reactive oxygen species). We observed that LECS was able to counteract the harmful effects induced by IL-1beta. This protection appeared to be greater than that elicited by indomethacin, which is usually employed in joint diseases. Since LECS possess a chondroprotective effect, it might be used in the management of cartilage damage during the inflammatory processes.

  4. Autologous chondrocyte implantation in children and adolescents.

    Science.gov (United States)

    Schmal, H; Pestka, J M; Salzmann, G; Strohm, P C; Südkamp, N P; Niemeyer, P

    2013-03-01

    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. Two hundred and sixty-seven patients undergoing ACI in the knee between 2006 and 2010 were included in this analysis. Cell characteristics and expression data of cartilage-specific surface markers as CD44, aggrecan and collagen type II were statistically analysed for age association. Epidemiological data of the defined groups were compared. Course of treatment was evaluated using MRI. A correlation analysis showed statistically significant associations between age and aggrecan or collagen type II expression in all patients patella was significantly more affected. Cartilage lesions were mainly caused by osteochondritis dissecans (OCD) and trauma. The Knee Osteoarthritis Scoring System in MRI reached 4.8 ± 2.3 points before, declining to 3.3 ± 2.3 points 6 and 12 months after the operation. 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. Case series, Level IV.

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

    NARCIS (Netherlands)

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

    2015-01-01

    Culturing chondrocytes under oxygen tension closely resembling their in vivo environment has been shown to have positive effects on matrix synthesis. In redifferentiation of expanded chondrocytes, hypoxia increased collagen type II expression. However, the mechanism by which hypoxia enhances

  6. Effects of extracellular matrices derived from different cell sources on chondrocyte functions.

    Science.gov (United States)

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

    2011-01-01

    Cell-derived extracellular matrices (ECMs) are a key factor in regulating cell functions in tissue engineering and regenerative medicine. The fact that cells are surrounded by their specific ECM in vivo elicits the need to elucidate the effects of ECM derived from different cell sources on cell functions. Here, three types of ECM were prepared by decellularizing cultured chondrocytes, fibroblasts, and mesenchymal stem cells (MSC) and used for chondrocyte culture to compare their effects on chondrocyte adhesion, proliferation, and differentiation. Chondrocyte adhesion to the chondrocyte-derived ECM was greater than those to the fibroblast- and MSC-derived ECM. Chondrocyte proliferation on the chondrocyte-derived ECM was lower than those on the fibroblast- and MSC-derived ECM. The ECM showed no evident effect on chondrocyte differentiation. The effects of ECM on cell functions depended on the cell source used to prepare the ECM. Copyright © 2011 American Institute of Chemical Engineers (AIChE).

  7. Pronounced biomaterial dependency in cartilage regeneration using nonexpanded compared with expanded chondrocytes

    NARCIS (Netherlands)

    Tsuchida, A.I.; Bekkers, J.E.J.; Beekhuizen, M.; Vonk, L.A.; Dhert, W.J.A.; Saris, Daniël B.F.; Creemers, L.B.

    2013-01-01

    We aimed to investigate freshly isolated compared with culture-expanded chondrocytes with respect to early regenerative response, cytokine production and cartilage formation in response to four commonly used biomaterials. Materials & methods: Chondrocytes were both directly and after expansion to

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

    Directory of Open Access Journals (Sweden)

    Jianmei Li

    2016-01-01

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

  9. LRP4 induces extracellular matrix productions and facilitates chondrocyte differentiation.

    Science.gov (United States)

    Asai, Nobuyuki; Ohkawara, Bisei; Ito, Mikako; Masuda, Akio; Ishiguro, Naoki; Ohno, Kinji

    2014-08-22

    Endochondral ossification is an essential step for skeletal development, which requires chondrocyte differentiation in growth cartilage. The low-density lipoprotein receptor-related protein 4 (LRP4), a member of LDLR family, is an inhibitor for Wnt signaling, but its roles in chondrocyte differentiation remain to be investigated. Here we found by laser capture microdissection that LRP4 expression was induced during chondrocyte differentiation in growth plate. In order to address the roles, we overexpressed recombinant human LRP4 or knocked down endogenous LRP4 by lentivirus in mouse ATDC5 chondrocyte cells. We found that LRP4 induced gene expressions of extracellular matrix proteins of type II collagen (Col2a1), aggrecan (Acan), and type X collagen (Col10a1), as well as production of total proteoglycans in ATDC5 cells, whereas LRP4 knockdown had opposite effects. Interestingly, LRP4-knockdown reduced mRNA expression of Sox9, a master regulator for chondrogenesis, as well as Dkk1, an extracellular Wnt inhibitor. Analysis of Wnt signaling revealed that LRP4 blocked the Wnt/β-catenin signaling activity in ATDC5 cells. Finally, the reduction of these extracellular matrix productions by LRP4-knockdown was rescued by a β-catenin/TCF inhibitor, suggesting that LRP4 is an important regulator for extracellular matrix productions and chondrocyte differentiation by suppressing Wnt/β-catenin signaling. Copyright © 2014 Elsevier Inc. All rights reserved.

  10. Roles of Chondrocytes in Endochondral Bone Formation and Fracture Repair

    Science.gov (United States)

    Hinton, R.J.; Jing, Y.; Jing, J.; Feng, J.Q.

    2016-01-01

    The formation of the mandibular condylar cartilage (MCC) and its subchondral bone is an important but understudied topic in dental research. The current concept regarding endochondral bone formation postulates that most hypertrophic chondrocytes undergo programmed cell death prior to bone formation. Under this paradigm, the MCC and its underlying bone are thought to result from 2 closely linked but separate processes: chondrogenesis and osteogenesis. However, recent investigations using cell lineage tracing techniques have demonstrated that many, perhaps the majority, of bone cells are derived via direct transformation from chondrocytes. In this review, the authors will briefly discuss the history of this idea and describe recent studies that clearly demonstrate that the direct transformation of chondrocytes into bone cells is common in both long bone and mandibular condyle development and during bone fracture repair. The authors will also provide new evidence of a distinct difference in ossification orientation in the condylar ramus (1 ossification center) versus long bone ossification formation (2 ossification centers). Based on our recent findings and those of other laboratories, we propose a new model that contrasts the mode of bone formation in much of the mandibular ramus (chondrocyte-derived) with intramembranous bone formation of the mandibular body (non-chondrocyte-derived). PMID:27664203

  11. 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.

  12. Effects of serum and compressive loading on the cartilage matrix synthesis and spatiotemporal deposition around chondrocytes in 3D culture.

    Science.gov (United States)

    Wu, Peihui; DeLassus, Elizabeth; Patra, Debabrata; Liao, Weiming; Sandell, Linda J

    2013-05-01

    The aim of this study was to investigate the effects of serum and compressive dynamic loading on the cartilaginous matrix spatiotemporal distribution around chondrocytes in vitro. Murine chondrocytes suspended in agarose were cultured in serum-free media or in varying concentrations of serum with or without compressive dynamic loading. Gene expression was assayed by quantitative polymerase chain reaction. Immunohistochemistry was performed for type II collagen and type VI collagen, aggrecan, or cartilage oligomeric matrix protein (COMP) to study the effect of serum and dynamic loading on the spatiotemporal distribution of cartilage matrix components. Chondrocytes in serum-free culture exhibited negligible differences in type II collagen, aggrecan, and COMP mRNA expression levels over 15 days of cultivation. However, higher serum concentrations decreased matrix gene expression. Expression of the matrix metalloproteinases (MMP)-3 and MMP-13 mRNA increased over time in serum-free or reduced serum levels, but was significantly suppressed in 10% fetal bovine serum (FBS). Compressive loading significantly stimulated MMP-3 expression on days 7 and 15. Immunohistochemical analysis demonstrated that maximum pericellular matrix deposition was achieved in 10% FBS culture in the absence of compressive loading. The pericellular distribution of type II and VI collagens, aggrecan, and COMP proteins tended to be more co-localized in the pericellular region from day 9 to day 21; compressive loading helped promote this co-localization of matrix proteins. The results of this study suggest that the quantity, quality, and spatial distribution of cartilaginous matrix can be altered by serum concentrations and compressive loading.

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

    Lifescience Database Archive (English)

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

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

    Lifescience Database Archive (English)

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

  15. Osmotic Challenge Drives Rapid and Reversible Chromatin Condensation in Chondrocytes

    Science.gov (United States)

    Irianto, Jerome; Swift, Joe; Martins, Rui P.; McPhail, Graham D.; Knight, Martin M.; Discher, Dennis E.; Lee, David A.

    2013-01-01

    Changes in extracellular osmolality have been shown to alter gene expression patterns and metabolic activity of various cell types, including chondrocytes. However, mechanisms by which physiological or pathological changes in osmolality impact chondrocyte function remain unclear. Here we use quantitative image analysis, electron microscopy, and a DNase I assay to show that hyperosmotic conditions (>400 mOsm/kg) induce chromatin condensation, while hypoosmotic conditions (100 mOsm/kg) cause decondensation. Large density changes (p condensation and decondensation during a daily loading cycle. The effect of changes in osmolality on nuclear morphology (p condensation (p condensation and osmolality was accurately modeled by a polymer gel model which, along with the rapid nature of the chromatin condensation (<20 s), reveals the basic physicochemical nature of the process. Alterations in chromatin structure are expected to influence gene expression and thereby regulate chondrocyte activity in response to osmotic changes. PMID:23442954

  16. 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.

  17. miR-139 is up-regulated in osteoarthritis and inhibits chondrocyte proliferation and migration possibly via suppressing EIF4G2 and IGF1R

    Energy Technology Data Exchange (ETDEWEB)

    Hu, Weihua; Zhang, Weikai; Li, Feng; Guo, Fengjing; Chen, Anmin, E-mail: chenanmin6072@126.com

    2016-05-27

    Osteoarthritis (OA) is one of the most progressive articular cartilage erosions. microRNAs (miRNAs) play pivotal roles in OA modulation, but the role of miR-139 in OA remains elusive. This study aims to reveal the effects and possible mechanism of miR-139 in OA and chondrocytes. The levels of miR-139 and its possible targets eukaryotic translation initiation factor 4 gamma 2 (EIF4G2) and insulin-like growth factor 1 receptor (IGF1R) were detected by qRT-PCR in the articular cartilages of 20 OA patients and 20 non-OA patients. Human chondrocyte CHON-001 cells were transfected with miR-139 mimic or inhibitor, as well as the siRNAs of EIF4G2 and IGF1R. Cell viability by MTT assay, proliferation by colony formation assay and migration by Transwell assay were performed. Results showed that miR-139 was up-regulated, while EIF4G2 and IGF1R mRNAs down-regulated in OA cartilages (P < 0.001), and negative correlations existed between the level of miR-139 and EIF4G2 or IGF1R. Overexpression of miR-139 in CHON-001 cells suppressed both mRNA and protein levels of EIF4G2 and IGF1R, and inhibited cell viability, colony formation number and cell migration, while miR-139 inhibitor induced the opposite effects. Knockdown of EIF4G2 or IGF1R in CHON-001 cells reversed the effects of miR-139 inhibitor on cell viability, colony formation and cell migration. These results indicate that miR-139 is capable of inhibiting chondrocyte proliferation and migration, thus being a possible therapeutic target for OA. The mechanism of miR-139 in chondrocytes may be related to its regulation on EIF4G2 and IGF1R.

  18. Spanish Experience in Autologous Chondrocyte Implantation

    Science.gov (United States)

    Pérez-Cachafeiro, Santiago; Ruano-Raviña, Alberto; Couceiro-Follente, José; Benedí-Alcaine, Jose Antonio; Nebot-Sanchis, Ignacio; Casquete-Román, Ciriaco; Bello-Prats, Santiago; Couceiro-Sánchez, Gonzalo; Blanco, Francisco J.

    2010-01-01

    Introduction: The Spanish Ministry of Health commissioned the Galician Agency for Health Technology Assessment to monitor and follow-up Autologous Chondrocyte Implantation (ACI) used to treat chondral lesions of the knee in Spain. The objective of this monitoring was to assess efficacy and safety of the technique. Design: One-hundred and eleven consecutive patients with knee chondral lesions were included in a multi-center study between January 2001 and January 2005. ACI was used in these patients as a second-line treatment option (or a first-line treatment option if the cause was Osteocondritis dissecans). The Cincinnati score and the Short Form 36 (SF-36) questionnaire were used to assess the patients’ self-reported satisfaction with the outcomes of ACI. A descriptive analysis was performed and non-parametric tests were used to establish correlations and compare results among subgroups. A multivariate analysis was also performed to measure the effect of different variables on changes in the condition of the knee. Results: Eighty men (72%) and 31 women (21%) with an age range from 16 to 49 years, underwent ACI surgery. Among these subjects, the most common previous first-line treatment was debridement (64 individuals, 74.4%). The mean size of the lesion treated with ACI was 3.82 cm2, and the most frequent location of the lesion was the inner femoral condyle (53.6%). The patient satisfaction was high or very high in 36 subjects (66.7%). Overall knee joint assessment improved from 4.32 points to 6.78. All SF-36 questionnaire categories improved, notably those related to physical condition. Conclusions: The results of this study indicate that ACI is safe; however, further studies are mandated to assess the efficacy of ACI compared to alternative treatment options. PMID:20148094

  19. Ankylosing spondylitis presenting as juxta-articular masses in females

    Energy Technology Data Exchange (ETDEWEB)

    Lindsley, H.B.; De Smet, A.A.; Neff, J.R.

    1987-02-01

    Juxta-articular inflammatory masses, sternomanubrial or sternoclavicular, were noted in two women who were subsequently found to have ankylosing spondylitis. The differential diagnosis of juxta-articular masses should include systemic rheumatic disorders as well as tumor or infection.

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

    Directory of Open Access Journals (Sweden)

    Xin Zhou

    2014-12-01

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

  1. Articular cartilage destruction in experimental inflammatory arthritis: Insulin-like growth factor-1 regulation of proteoglycan metabolism in chondrocytes

    NARCIS (Netherlands)

    Verschure, P. J.; van Noorden, C. J.; van Marle, J.; van den Berg, W. B.

    1996-01-01

    Rheumatoid arthritis, a disease of unknown aetiology, is characterized by joint inflammation and, in its later stages, cartilage destruction. Inflammatory mediators may exert not only suppression of matrix synthesis but also cartilage degradation, which eventually leads to severe cartilage

  2. 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

  3. The infrapatellar fat pad induces inflammatory and degradative effects in articular cells but not through leptin or adiponectin.

    Science.gov (United States)

    Gross, Jean-Baptiste; Guillaume, Cecile; Gegout-Pottie, Pascale; Reboul, Pascal; Jouzeau, Jean-Yves; Mainard, Didier; Presle, Nathalie

    2017-01-01

    Based on a novel approach suggesting a role of adipose tissue in osteoarthritis (OA), we aimed to determine whether the infrapatellar fat pad (IFP) may affect joint cell functions through adipokines. The conditioned media of IFP and subcutaneous adipose tissue from OA patients were used to determine the production of leptin and adiponectin, and to stimulate chondrocytes and fibroblast-like synoviocytes (FLS). Blocking experiments were carried out to evaluate the contribution of adipokines to IFP effects. The gene expression of inflammatory and degradative proteins, growth factors and components of the extracellular matrix, and the production of inflammatory mediators and metalloproteases were determined to evaluate cell response to fat-conditioned media. IFP releases elevated amounts of leptin and adiponectin independently of the body mass index and the gender. The conditioned media from IFP strongly induce the expression of inflammatory genes in both articular cells and the expression of degradative genes in chondrocytes, but remain ineffective in regulating the expression of aggrecan, type 2 collagen or growth factors. Blocking leptin or adiponectin does not change the cell response to IFP. A great variability between patients is found when compared the inflammatory activity of paired samples of IFP and subcutaneous adipose tissue. IFP may trigger both cartilage destruction and inflammation of the synovium, but not through leptin or adiponectin. The data suggest also that IFP may have specific inflammatory phenotypic features independent from the general phenotype found in obesity.

  4. The intra-articular injection of RANKL-binding peptides inhibits cartilage degeneration in a murine model of osteoarthritis

    Directory of Open Access Journals (Sweden)

    Md. Zahirul Haque Bhuyan

    2017-06-01

    Full Text Available We recently found that the receptor activator of NF-κB ligand (RANKL-binding peptide, OP3-4 stimulated the differentiation of both chondrocytes and osteoblasts. OP3-4 is also shown to inhibit cartilage degeneration. To clarify whether the peptide can inhibit cartilage degeneration without stimulating bone formation, we first performed a proliferation assay using C3H10T1/2 (the murine mesenchymal stem cell line, which is the common origin of both chondrocytes and osteoblasts. The RANKL-binding peptides, OP3-4 and W9, promoted cellular proliferation at 24 and 48 h, respectively. Next, we injected both peptides into the intra-articular space of the knee joints of mice with monosodium-iodoacetate (MIA-induced osteoarthritis to clarify the effects of the peptides on cartilage tissue. Twenty-five nine-week-old male C57BL/6J mice received injections of vehicle, or the same molar amount of W9, OP3-4, or a control peptide (which could not stimulate osteoblast differentiation on days 7, 14, and 21 after the injection of MIA. The mice were sacrificed on day 28. The histomorphometric analyses revealed that both peptides inhibited the degeneration of cartilage without enhancing bone formation activity. Our data suggest that the stimulation of mesenchymal cell proliferation by the RANKL-binding peptides might lead to the inhibition of cartilage degeneration.

  5. 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

  6. 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.

  7. Laser-structured bacterial nanocellulose hydrogels support ingrowth and differentiation of chondrocytes and show potential as cartilage implants.

    Science.gov (United States)

    Ahrem, Hannes; Pretzel, David; Endres, Michaela; Conrad, Daniel; Courseau, Julien; Müller, Hartmut; Jaeger, Raimund; Kaps, Christian; Klemm, Dieter O; Kinne, Raimund W

    2014-03-01

    The small size and heterogeneity of the pores in bacterial nanocellulose (BNC) hydrogels limit the ingrowth of cells and their use as tissue-engineered implant materials. The use of placeholders during BNC biosynthesis or post-processing steps such as (touch-free) laser perforation can overcome this limitation. Since three-dimensionally arranged channels may be required for homogeneous and functional seeding, three-dimensional (3-D) laser perforation of never-dried BNC hydrogels was performed. Never-dried BNC hydrogels were produced in different shapes by: (i) the cultivation of Gluconacetobacter xylinus (DSM 14666; synonym Komagataeibacter xylinus) in nutrient medium; (ii) the removal of bacterial residues/media components (0.1M NaOH; 30 min; 100 °C) and repeated washing (deionized water; pH 5.8); (iii) the unidirectional or 3-D laser perforation and cutting (pulsed CO2 Rofin SC × 10 laser; 220 μm channel diameter); and (iv) the final autoclaving (2M NaOH; 121 °C; 20 min) and washing (pyrogen-free water). In comparison to unmodified BNC, unidirectionally perforated--and particularly 3-D-perforated - BNC allowed ingrowth into and movement of vital bovine/human chondrocytes throughout the BNC nanofiber network. Laser perforation caused limited structural modifications (i.e. fiber or globular aggregates), but no chemical modifications, as indicated by Fourier transform infrared spectroscopy, X-ray photoelectron scattering and viability tests. Pre-cultured human chondrocytes seeding the surface/channels of laser-perforated BNC expressed cartilage-specific matrix products, indicating chondrocyte differentiation. 3-D-perforated BNC showed compressive strength comparable to that of unmodified samples. Unidirectionally or 3-D-perforated BNC shows high biocompatibility and provides short diffusion distances for nutrients and extracellular matrix components. Also, the resulting channels support migration into the BNC, matrix production and phenotypic stabilization of

  8. Insights on Molecular Mechanisms of Chondrocytes Death in Osteoarthritis

    Directory of Open Access Journals (Sweden)

    Edith Charlier

    2016-12-01

    Full Text Available Osteoarthritis (OA is a joint pathology characterized by progressive cartilage degradation. Medical care is mainly based on alleviating pain symptoms. Compelling studies report the presence of empty lacunae and hypocellularity in cartilage with aging and OA progression, suggesting that chondrocyte cell death occurs and participates to OA development. However, the relative contribution of apoptosis per se in OA pathogenesis appears complex to evaluate. Indeed, depending on technical approaches, OA stages, cartilage layers, animal models, as well as in vivo or in vitro experiments, the percentage of apoptosis and cell death types can vary. Apoptosis, chondroptosis, necrosis, and autophagic cell death are described in this review. The question of cell death causality in OA progression is also addressed, as well as the molecular pathways leading to cell death in response to the following inducers: Fas, Interleukin-1β (IL-1β, Tumor Necrosis factor-α (TNF-α, leptin, nitric oxide (NO donors, and mechanical stresses. Furthermore, the protective role of autophagy in chondrocytes is highlighted, as well as its decline during OA progression, enhancing chondrocyte cell death; the transition being mainly controlled by HIF-1α/HIF-2α imbalance. Finally, we have considered whether interfering in chondrocyte apoptosis or promoting autophagy could constitute therapeutic strategies to impede OA progression.

  9. Insights on Molecular Mechanisms of Chondrocytes Death in Osteoarthritis.

    Science.gov (United States)

    Charlier, Edith; Relic, Biserka; Deroyer, Céline; Malaise, Olivier; Neuville, Sophie; Collée, Julie; Malaise, Michel G; De Seny, Dominique

    2016-12-20

    Osteoarthritis (OA) is a joint pathology characterized by progressive cartilage degradation. Medical care is mainly based on alleviating pain symptoms. Compelling studies report the presence of empty lacunae and hypocellularity in cartilage with aging and OA progression, suggesting that chondrocyte cell death occurs and participates to OA development. However, the relative contribution of apoptosis per se in OA pathogenesis appears complex to evaluate. Indeed, depending on technical approaches, OA stages, cartilage layers, animal models, as well as in vivo or in vitro experiments, the percentage of apoptosis and cell death types can vary. Apoptosis, chondroptosis, necrosis, and autophagic cell death are described in this review. The question of cell death causality in OA progression is also addressed, as well as the molecular pathways leading to cell death in response to the following inducers: Fas, Interleukin-1β (IL-1β), Tumor Necrosis factor-α (TNF-α), leptin, nitric oxide (NO) donors, and mechanical stresses. Furthermore, the protective role of autophagy in chondrocytes is highlighted, as well as its decline during OA progression, enhancing chondrocyte cell death; the transition being mainly controlled by HIF-1α/HIF-2α imbalance. Finally, we have considered whether interfering in chondrocyte apoptosis or promoting autophagy could constitute therapeutic strategies to impede OA progression.

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

    Science.gov (United States)

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

    2011-03-01

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

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

    Directory of Open Access Journals (Sweden)

    V Nicolin

    2009-08-01

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

  12. Mechanical confinement regulates cartilage matrix formation by chondrocytes

    Science.gov (United States)

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

    2017-12-01

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

  13. Effects of Indigo Carmine on Human Chondrocytes In Vitro

    OpenAIRE

    Zippelius, Timo; Hoburg, Arnd; Preininger, Bernd; V?r?s, Pauline; Perka, Carsten; Matziolis, Georg; R?hner, Eric

    2013-01-01

    Joint infections following or accompanying superficious soft tissue infections are severe complication in orthopedic surgery. The use of intra-articular blue staining is a helpful method to visualize a fistula and to differentiate between superficial and intra-articular infections. Regarding this clinical implication data about the effects of indigo carmine, a frequently used blue staining substance, on cartilage is missing. The hypothesis of this study was that indigo carmine damages human c...

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

    Science.gov (United States)

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

    2015-01-01

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

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

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    Tatsuya Kosaka

    Full Text Available RAGE, receptor for advanced glycation endoproducts (AGE, has been characterized as an activator of osteoclastgenesis. However, whether RAGE directly regulates chondrocyte proliferation and differentiation is unclear. Here, we show that RAGE has an inhibitory role in chondrocyte differentiation. RAGE expression was observed in chondrocytes from the prehypertrophic to hypertrophic regions. In cultured cells, overexpression of RAGE or dominant-negative-RAGE (DN-RAGE demonstrated that RAGE inhibited cartilaginous matrix production, while DN-RAGE promoted production. Additionally, RAGE regulated Ihh and Col10a1 negatively but upregulated PTHrP receptor. Ihh promoter analysis and real-time PCR analysis suggested that downregulation of Cdxs was the key for RAGE-induced inhibition of chondrocyte differentiation. Overexpression of the NF-κB inhibitor I-κB-SR inhibited RAGE-induced NF-κB activation, but did not influence inhibition of cartilaginous matrix production by RAGE. The inhibitory action of RAGE was restored by the Rho family GTPases inhibitor Toxin B. Furthermore, inhibitory action on Ihh, Col10a1 and Cdxs was reproduced by constitutively active forms, L63RhoA, L61Rac, and L61Cdc42, but not by I-κB-SR. Cdx1 induced Ihh and Col10a1 expressions and directly interacted with Ihh promoter. Retinoic acid (RA partially rescued the inhibitory action of RAGE. These data combined suggests that RAGE negatively regulates chondrocyte differentiation at the prehypertrophic stage by modulating NF-κB-independent and Rho family GTPases-dependent mechanisms.

  16. RAGE, Receptor of Advanced Glycation Endoproducts, Negatively Regulates Chondrocytes Differentiation

    Science.gov (United States)

    Kurosaka, Yuko; Nishimura, Haruka; Tanabe, Motoki; Takakura, Yuuki; Iwai, Keisuke; Waki, Takuya; Fujita, Takashi

    2014-01-01

    RAGE, receptor for advanced glycation endoproducts (AGE), has been characterized as an activator of osteoclastgenesis. However, whether RAGE directly regulates chondrocyte proliferation and differentiation is unclear. Here, we show that RAGE has an inhibitory role in chondrocyte differentiation. RAGE expression was observed in chondrocytes from the prehypertrophic to hypertrophic regions. In cultured cells, overexpression of RAGE or dominant-negative-RAGE (DN-RAGE) demonstrated that RAGE inhibited cartilaginous matrix production, while DN-RAGE promoted production. Additionally, RAGE regulated Ihh and Col10a1 negatively but upregulated PTHrP receptor. Ihh promoter analysis and real-time PCR analysis suggested that downregulation of Cdxs was the key for RAGE-induced inhibition of chondrocyte differentiation. Overexpression of the NF-κB inhibitor I-κB-SR inhibited RAGE-induced NF-κB activation, but did not influence inhibition of cartilaginous matrix production by RAGE. The inhibitory action of RAGE was restored by the Rho family GTPases inhibitor Toxin B. Furthermore, inhibitory action on Ihh, Col10a1 and Cdxs was reproduced by constitutively active forms, L63RhoA, L61Rac, and L61Cdc42, but not by I-κB-SR. Cdx1 induced Ihh and Col10a1 expressions and directly interacted with Ihh promoter. Retinoic acid (RA) partially rescued the inhibitory action of RAGE. These data combined suggests that RAGE negatively regulates chondrocyte differentiation at the prehypertrophic stage by modulating NF-κB-independent and Rho family GTPases-dependent mechanisms. PMID:25275461

  17. Sulfato de condroitina e hialuronato de sódio no tratamento da doença articular degenerativa em cães: estudo histológico da cartilagem articular e membrana sinovial Chondroitin sulfate and sodium hyaluronate in the treatment of the degenerative joint disease in dogs: histological features of articular cartilage and synovium

    Directory of Open Access Journals (Sweden)

    E.G. Melo

    2008-02-01

    processo degenerativo da cartilagem articular. Não foi constatada ação favorável das drogas na membrana sinovial.Fifteen mongrel dogs, both genders, weighting from 18 to 25kg were used and Degenerative Joint Disease (DJD was induced through cranial cruciate ligament (CCrL artroscopical section. After three weeks, CCrL was reconstructed by Schawalder's (1989 technique. Then, dogs were distributed in three groups and the following protocols were used: group I, control, no other treatment but the CCrL reconstruction; group II received chondroitin sulfate 24mg per animal every five days, intramuscularly, in a total of six injections; and group III received sodium hyaluronate 20mg per animal every five days, intravenously, in a total of three injections. Clinical observation was done until 90 days after treatments. By that time, the articular cartilage and synovium were collected and their morphology was evaluated. In group I, the degenerative alterations of the DJD were the most intense. Thus, decrease of chondrocytes number, pannus, fibrillations, grooves, erosion, and irregular articular surface were observed on the cartilage. In group II, raise of chondrocytes number was observed, with increase of synthesis activity of matrix and decrease of lesions on the articular surface. There was an increase of chondrocytes in group III, but the cells were morphologically unviable. All the groups showed proliferation of the synovial membrane, with limpho-plasma cells infiltrated in subintim and perivascular. In groups I and III, the proliferation of synovium was abundant, with formation of pannus, flattened synoviocytes or synovium absent with granulation tissue. Those results suggest that the chondroitin sulfate stimulated the articular cartilage; decreasing or delaying the alterations of DJD, as well as, the sodium hyaluronate did not interfere on degenerative process in articular cartilage. No favorable action of these drugs in the synovial membrane was verified.

  18. 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 on...

  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 document...

  20. Chondrocyte death associated with human femoral osteochondral harvest as performed for mosaicplasty.

    Science.gov (United States)

    Huntley, J S; Bush, P G; McBirnie, J M; Simpson, A H; Hall, A C

    2005-02-01

    Autologous osteochondral transfer is an option for the treatment of articular defects. However, there are concerns about graft integration and the nature of the tissue forming the cartilage-cartilage bridge. Chondrocyte viability at graft and recipient edges is thought to be an important determinant of the quality of repair. The purpose of the present study was to evaluate early cell viability at the edges of osteochondral grafts from ex vivo human femoral condyles. Fresh human tissue was obtained from eleven knees at the time of total knee arthroplasty for the treatment of osteoarthritis. Osteochondral cylinders were harvested with use of a 4.5-mm-diameter mosaicplasty osteotome from regions of the anterolateral aspect of the femoral condyle that were macroscopically nondegenerate and histologically nonfibrillated. Plugs were assessed for marginal cell viability by means of confocal laser scanning microscopy. The diameter of the cartilaginous portion of the osteochondral plugs was a mean (and standard error of the mean) of 4.84 +/- 0.12 mm (as determined on the basis of three plugs). This value was approximately 300 microm greater than the measured internal diameter of the osteotome. There was a substantial margin of superficial zone cell death (mean thickness, 382 +/- 68.2 microm), with >99% cell viability seen more centrally (as determined on the basis of five plugs). Demiplugs were created by splitting the mosaicplasty explants with a fresh number-11 scalpel blade. The margin of superficial zone cell death at the curved edge was significantly greater than that at the site of the scalpel cut (390.3 +/- 18.8 microm compared with 34.8 +/- 3.2 microm; p = 0.0286). Similar findings were observed when the cartilage alone was breached and the bone was left intact, with the margin of superficial zone cell death being significantly greater than that obtained in association with the straight scalpel incision (268 +/- 38.9 microm compared with 41.3 +/- 13.4 microm; p = 0

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

    Directory of Open Access Journals (Sweden)

    Chua Kien-Hui

    2013-01-01

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

  2. Development and validation of a novel bioreactor system for load- and perfusion-controlled tissue engineering of chondrocyte-constructs.

    Science.gov (United States)

    Schulz, Ronny M; Wüstneck, Nico; van Donkelaar, Corrinus C; Shelton, Julia C; Bader, Augustinus

    2008-11-01

    Osteoarthritis is a severe socio-economical disease, for which a suitable treatment modality does not exist. Tissue engineering of cartilage transplants is the most promising method to treat focal cartilage defects. However, current culturing procedures do not yet meet the requirements for clinical implementation. This article presents a novel bioreactor device for the functional tissue engineering of articular cartilage which enables cyclic mechanical loading combined with medium perfusion over long periods of time, under controlled cultivation and stimulation conditions whilst ensuring system sterility. The closed bioreactor consists of a small, perfused, autoclavable, twin chamber culture device with a contactless actuator for mechanical loading. Uni-axial loading is guided by externally applied magnetic fields with real-time feedback-control from a platform load cell and an inductive proximity sensor. This precise measurement allows the development of the mechanical properties of the cultured tissue to be monitored in real-time. This is an essential step towards clinical implementation, as it allows accounting for differences in the culture procedure induced by patient-variability. This article describes, based on standard agarose hydrogels of 3 mm height and 10 mm diameter, the technical concept, implementation, scalability, reproducibility, precision, and the calibration procedures of the whole bioreactor instrument. Particular attention is given to the contactless loading system by which chondrocyte scaffolds can be compressed at defined loading frequencies and magnitudes, whilst maintaining an aseptic cultivation procedure. In a "proof of principle" experiment, chondrocyte seeded agarose gels were cultured for 21 days in the bioreactor system. Intermittent medium perfusion at a steady flow rate (0.5 mL/min) was applied. Sterility and cell viability (ds-DNA quantification and fluorometric live/dead staining) were preserved in the system. Flow induced shear

  3. 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.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2012-09-07

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

  5. Diagnosis of bovine neosporosis.

    Science.gov (United States)

    Dubey, J P; Schares, G

    2006-08-31

    The protozoan parasite Neospora caninum is a major cause of abortion in cattle. The diagnosis of neosporosis-associated mortality and abortion in cattle is difficult. In the present paper we review histologic, serologic, immunohistochemical, and molecular methods for dignosis of bovine neosporosis. Although not a routine method of diagnosis, methods to isolate viable N. caninum from bovine tissues are also reviewed.

  6. [The study of apoptosis factors released from laser injured cartilage inducing apoptosis of chondrocyte].

    Science.gov (United States)

    Zheng, Min; Grogan, Sp; Schaffner, T; Mainil-Varlet, P

    2007-06-05

    To explore the role of pro-apoptotic signals following tissue injury and how these may promote a progression of further cell death. Laser treated porcine articular cartilage disks were maintained in culture media. The collected media at various time periods (3, 6, 9, 12, 24 and 48 h), was called treated conditioned media (TCM). Non-laser treated cartilage disks were used to create control conditioned media (CCM). Each disk was subsequently maintained for 28 days and used in confocal microscopic assessment to document the progression of the damaged area. Isolated porcine chondrocytes were cultured in monolayer, and were exposed to TCM, CCM or normal culture medium (NM). As a positive inducer of apoptosis, the monolayer cells were exposed to UV radiation for 10 min and cultured in NM. Following 24 h exposure, the cells were harvested and stained with the appropriate combination of fluorescent dyes and processed via flow cytometry. All cultured cells exposed to TCM displayed a caspase-3 positive subpopulation, a loss of CMXRos, and with a reduced or lost NO signal. CCM exposure signals were comparable to the NM treatments with all having retained CMXRos, NO and without evidence of caspase-3 activity. UV treatment also induced a reduction in NO, but both CMXRos and caspase-3 positive, representing an earlier stage of apoptosis and suggesting that the mode of cell death via UV and TCM exposure are via different processes. The investigation of a dose (100%, 50%, 25% and 12.5%) and time (0.5, 1, 3, 9, 12 h) response to TCM exhibited that all treatments observed an increase in caspase-3 positive cells and a reduction in NO and CMXRos. The usefulness of FCM can be used in the study of cell viability and apoptosis. Such a system may be useful in the study of mechanisms of disease such as osteoarthritis, thus may be of practical use for the pharmaceutical industry for screening associated drugs.

  7. Osteoarthritis in horses - Part 2: a review of the intra-articular use of corticosteroids as a method of treatment

    Directory of Open Access Journals (Sweden)

    Maria Verônica de Souza

    Full Text Available ABSTRACT: The problem considered in this review is related to the frequent use of corticosteroids (COs (i.e. steroids in the practice of equine medicine and surgery as a therapeutic strategy in the treatment of degenerative joint disease, commonly known as osteoarthritis (OA. This disease is one of the most common among the equine musculoskeletal diseases, and is clinically characterized by pain, lameness, joint effusion at the initial stage, which usually reduces with the progression of the disease, and reduced physical performance. Although steroids are considered excellent drugs in the control of clinical signs resulting from osteoarthritis, they also influence cellular activity through activation of various signaling mechanisms. However, they can cause adverse effects when administered intra-articularly, since they are immunosuppressive drugs of many cell types. They are also incriminated as suppressors of the chondrocyte matrix synthetic activities, which may contribute to 'arthropathy by corticosteroids', which can also be associated with cumulative injury resulting from improper use (dose and frequency of application of already damaged joints. The objective of this article is to review information about the advantages and disadvantages of intra-articular COs for treatment of the disease in horses. In addition, some important information of other species is also presented.

  8. Intra-articular morphine in horses

    DEFF Research Database (Denmark)

    Lindegaard, Casper

    for pharmacological analysis were obtained repeatedly. Pain was evaluated by degree of lameness as well as using a visual analogue scale of pain intensity (VAS) and a composite measure pain scale (CMPS), developed for this purpose. Intra-articular injection of LPS elicited a marked synovitis resulting in lameness...... and pain. Intra-articularly administered morphine showed a significant analgesic effect as measured by reduced lameness scores, less administered rescue analgesia and lower pain scores. A significant anti-inflammatory effect was demonstrated by reduced joint swelling, reduced SF serum amyloid A (SAA...... that morphine pharmacokinetics in horses were similar to those reported in man. The developed CMPS showed good inter-observer agreement and it is suggested that this pain-scale can be employed for evaluation of orthopaedic pain in equine patients. A good analgesic and anti-inflammatory effect of IA morphine...

  9. 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...

  10. Applied biomechanics in articular injuries: perspectives in the basic investigation of articular injuries and clinical application.

    Science.gov (United States)

    Olson, Steven A; Brown, Thomas D; Athanasiou, Kyriacos A; Natoli, Roman M; Dirschl, Douglas R

    2011-01-01

    Joint injury is an important cause of arthritis. Although the treatment of injury, in general, has been widely studied, the contribution of injury to the development of posttraumatic arthritis is still a relatively understudied area. One of the most perplexing aspects of investigating articular injuries is the complex nature of the injury itself and the multiple facets of the injury mechanism that can potentially lead to the development of arthritis. A symposium by the Orthopaedic Research Society and the American Academy of Orthopaedic Surgeons was designed to examine the spectrum of basic science to clinical investigation in the role of biomechanics in the study of joint injury and subsequent posttraumatic arthritis. Four perspectives in the clinical aspects of managing articular injuries were investigated, including the clinical applications of basic science findings, the challenges and advancements in measuring and modeling articular fractures, the relationship of articular cartilage mechanical injuries and osteoarthritis, and the controlled creation of an intra-articular fracture to permit observations of the natural history of posttraumatic arthritis.

  11. Articular fetlock injuries in exercising horses.

    Science.gov (United States)

    Santschi, Elizabeth M

    2008-04-01

    Articular injuries to the fetlock joint can be categorized as injuries to the soft tissues (synovium, ligaments, cartilage) or bone (third metacarpus/metatarsus, first phalanx, proximal sesamoids). This article focuses on the traumatic injuries to the cartilage and bone from anatomic, functional, and pathophysiological perspectives. An understanding of fetlock motion and loading will assist clinicians in the diagnosis, treatment, and, most importantly, prevention of fetlock injury in working horses.

  12. Osteoarthritic human chondrocytes proliferate in 3D co-culture with mesenchymal stem cells in suspension bioreactors.

    Science.gov (United States)

    Khurshid, Madiha; Mulet-Sierra, Aillette; Adesida, Adetola; Sen, Arindom

    2017-07-28

    Osteoarthritis (OA) is a painful disease, characterized by progressive surface erosion of articular cartilage. The use of human articular chondrocytes (hACs) sourced from OA patients has been proposed as a potential therapy for cartilage repair, but this approach is limited by the lack of scalable methods to produce clinically relevant quantities of cartilage-generating cells. Previous studies in static culture have shown that hACs co-cultured with human mesenchymal stem cells (hMSCs) as 3D pellets can upregulate proliferation and generate neocartilage with enhanced functional matrix formation relative to that produced from either cell type alone. However, because static culture flasks are not readily amenable to scale up, scalable suspension bioreactors were investigated to determine if they could support the co-culture of hMSCs and OA hACs under serum-free conditions to facilitate clinical translation of this approach. When hACs and hMSCs (1:3 ratio) were inoculated at 20,000 cells/ml into 125-ml suspension bioreactors and fed weekly, they spontaneously formed 3D aggregates and proliferated, resulting in a 4.75-fold increase over 16 days. Whereas the apparent growth rate was lower than that achieved during co-culture as a 2D monolayer in static culture flasks, bioreactor co-culture as 3D aggregates resulted in a significantly lower collagen I to II mRNA expression ratio and more than double the glycosaminoglycan/DNA content (5.8 vs. 2.5 μg/μg). The proliferation of hMSCs and hACs as 3D aggregates in serum-free suspension culture demonstrates that scalable bioreactors represent an accessible platform capable of supporting the generation of clinical quantities of cells for use in cell-based cartilage repair. Copyright © 2017 John Wiley & Sons, Ltd.

  13. High resistance of the mechanical properties of the chondrocyte pericellular matrix to proteoglycan digestion by chondroitinase, aggrecanase, or hyaluronidase.