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

    PubMed

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

    2016-08-01

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

  2. Deciphering chondrocyte behaviour in matrix-induced autologous chondrocyte implantation to undergo accurate cartilage repair with hyaline matrix.

    PubMed

    Demoor, M; Maneix, L; Ollitrault, D; Legendre, F; Duval, E; Claus, S; Mallein-Gerin, F; Moslemi, S; Boumediene, K; Galera, P

    2012-06-01

    Since the emergence in the 1990s of the autologous chondrocytes transplantation (ACT) in the treatment of cartilage defects, the technique, corresponding initially to implantation of chondrocytes, previously isolated and amplified in vitro, under a periosteal membrane, has greatly evolved. Indeed, the first generations of ACT showed their limits, with in particular the dedifferentiation of chondrocytes during the monolayer culture, inducing the synthesis of fibroblastic collagens, notably type I collagen to the detriment of type II collagen. Beyond the clinical aspect with its encouraging results, new biological substitutes must be tested to obtain a hyaline neocartilage. Therefore, the use of differentiated chondrocytes phenotypically stabilized is essential for the success of ACT at medium and long-term. That is why researchers try now to develop more reliable culture techniques, using among others, new types of biomaterials and molecules known for their chondrogenic activity, giving rise to the 4th generation of ACT. Other sources of cells, being able to follow chondrogenesis program, are also studied. The success of the cartilage regenerative medicine is based on the phenotypic status of the chondrocyte and on one of its essential component of the cartilage, type II collagen, the expression of which should be supported without induction of type I collagen. The knowledge accumulated by the scientific community and the experience of the clinicians will certainly allow to relief this technological challenge, which influence besides, the validation of such biological substitutes by the sanitary authorities. Copyright © 2012 Elsevier Masson SAS. All rights reserved.

  3. Macrophage-inducing FasL on chondrocytes forms immune privilege in cartilage tissue engineering, enhancing in vivo regeneration.

    PubMed

    Fujihara, Yuko; Takato, Tsuyoshi; Hoshi, Kazuto

    2014-05-01

    To obtain stable outcomes in regenerative medicine, controlling inflammatory reactions is a requirement. Previously, auricular chondrocytes in tissue-engineered cartilage have been shown to express factors related to immune privilege including Fas ligand (FasL) in mice. Since elucidation of mechanism on immune privilege formed in cartilage regeneration may contribute to suppression of excessive inflammation, in this study, we investigated the function of FasL and induction of immune privilege in tissue-engineered cartilage using a mouse subcutaneous model. When cocultured, auricular chondrocytes of FasL-dysfunctional mice, C57BL/6JSlc-gld/gld (gld), induced less cell death and apoptosis of macrophage-like cells, RAW264, compared with chondrocytes of C57BL/6 mice (wild), suggesting that FasL on chondrocytes could induce the apoptosis of macrophages. Meanwhile, the viability of chondrocytes was hardly affected by cocultured RAW264, although the expression of type II collagen was decreased, indicating that macrophages could hamper the maturation of chondrocytes. Tissue-engineered cartilage containing gld chondrocytes exhibited greater infiltration of macrophages, with less accumulation of proteoglycan than did wild constructs. Analysis of the coculture medium identified G-CSF as an inducer of FasL on chondrocytes, and G-CSF-treated tissue-engineered cartilage showed less infiltration of macrophages, with increased formation of cartilage after transplantation. The interactions between chondrocytes and macrophages may increase G-CSF secretion in macrophages and induce FasL on chondrocytes, which in turn induce the apoptosis of macrophages and suppress tissue reactions, promoting the maturation of tissue-engineered cartilage. These findings provide scientific insight into the mechanism of autologous chondrocyte transplantation, which could be applied as a novel strategy for cartilage tissue engineering.

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2016-01-01

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

  6. Bacterial lipopolysaccharides induce in vitro degradation of cartilage matrix through chondrocyte activation.

    PubMed Central

    Jasin, H E

    1983-01-01

    The present studies demonstrate that bacterial lipopolysaccharides (LPS) induce cartilage matrix degradation in live explants in organ culture. Quintuplicate bovine nasal fibrocartilage explants cultured for 8 d with three different purified LPS preparations derived from Escherichia coli and Salmonella typhosa at concentrations ranging from 1.0 to 25.0 micrograms/ml resulted in matrix proteoglycan depletion of 33.3 +/- 5.8 to 92.5 +/- 2.0% (medium control depletion 17.7 +/- 0.7 to 32.4 +/- 1.4%). Matrix degradation depended on the presence of live chondrocytes because frozen-thawed explants incubated with LPS failed to show any proteoglycan release. Moreover, the addition of Polymyxin B (25 micrograms/ml) to live explants incubated with LPS abolished matrix release, whereas Polymyxin B had no effect on the matrix-degrading activity provided by blood mononuclear cell factors. A highly purified Lipid A preparation induced matrix degradation at a concentration of 0.01 micrograms/ml. Cartilage matrix collagen and proteoglycan depletion also occurred with porcine articular cartilage explants (collagen release: 18.3 +/- 3.5%, medium control: 2.1 +/- 0.5%; proteoglycan release: 79.0 +/- 5.9%, medium control: 28.8 +/- 4.8%). Histochemical analysis of the cultured explants confirmed the results described above. Gel chromatography of the proteoglycans released in culture indicated that LPS induced significant degradation of the high molecular weight chondroitin sulfate-containing aggregates. These findings suggest that bacterial products may induce cartilage damage by direct stimulation of chondrocytes. This pathogenic mechanism may play a role in joint damage in septic arthritis and in arthropathies resulting from the presence of bacterial products derived from the gastrointestinal tract. Images PMID:6358260

  7. Intracellular calcium oscillations in articular chondrocytes induced by basic calcium phosphate crystals lead to cartilage degradation.

    PubMed

    Nguyen, C; Lieberherr, M; Bordat, C; Velard, F; Côme, D; Lioté, F; Ea, H-K

    2012-11-01

    Basic calcium phosphate (BCP) crystals, including octacalcium phosphate (OCP), carbonated-apatite (CA) and hydroxyapatite (HA) crystals are associated with destructive forms of osteoarthritis. Mechanisms of BCP-induced cartilage breakdown remain incompletely understood. We assessed the ability of BCP to induce changes in intracellular calcium (iCa(2+)) content and oscillations and the role of iCa(2+) in BCP-induced cartilage degradation. Bovine articular chondrocytes (BACs) and bovine cartilage explants (BCEs) were stimulated with BCP or monosodium urate (MSU) crystals. iCa(2+) levels were determined by spectrofluorimetry and oscillations by confocal microscopy. mRNA expression of matrix metalloproteinase 3 (MMP-3), a disintegrin and metalloprotease with thrombospondin-like motifs 4 (ADAMTS-4) and ADAMTS-5 was assessed by quantitative real-time PCR. Glycosaminoglycan (GAG) release was measured in the supernatants of BCE cultures. All three BCP crystals significantly increased iCa(2+) content. OCP also induced iCa(2+) oscillations. Rate of BACs displaying iCa(2+) oscillations increased over time, with a peak after 20 min of stimulation. OCP-induced iCa(2+) oscillations involved both extracellular Ca(2+) (eCa(2+)) influx and iCa(2+) stores. Indeed, OCP-induced iCa(2+) oscillations decreased rapidly in Ca(2+)-free medium. Both voltage- and non-voltage-dependent Ca(2+) channels were involved in eCa(2+) influx. BCP crystal-induced variation in iCa(2+) content was associated with BCP crystal-induced cartilage matrix degradation. However, iCa²(+) was not associated with OCP crystal-induced mRNA expression of MMP-3, ADAMTS-4 or ADAMTS-5. BCP crystals can induce variation in iCa(2+) content and oscillations in articular chondrocytes. Furthermore, BCP crystal-induced changes in iCa(2+) content play a pivotal role in BCP catabolic effects on articular cartilage. Copyright © 2012 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.

  8. Calcium regulates cyclic compression-induced early changes in chondrocytes during in vitro cartilage tissue formation.

    PubMed

    Raizman, Igal; De Croos, J N Amritha; Pilliar, Robert; Kandel, Rita A

    2010-10-01

    A single application of cyclic compression (1kPa, 1Hz, 30min) to bioengineered cartilage results in improved tissue formation through sequential catabolic and anabolic changes mediated via cell shape changes that are regulated by α5β1 integrin and membrane-type metalloprotease (MT1-MMP). To determine if calcium was involved in this process, the role of calcium in regulating cell shape changes, MT1-MMP expression and integrin activity in response to mechanical stimulation was examined. Stimulation-induced changes in cell shape and MT1-MMP expression were abolished by chelation of extracellular calcium, and this effect was reversed by re-introduction of calcium. Spreading was inhibited by blocking stretch-activated channels (with gadolinium), while retraction was prevented by blocking the L-Type voltage-gated channel (with nifedipine); both compounds inhibited MT1-MMP upregulation. Calcium A23187 ionophore restored cellular response further supporting a role for these channels. Calcium regulated the integrin-mediated signalling pathway, which was facilitated through Src kinase. Both calcium- and integrin-mediated pathways converged on ERK-MAPK in response to stimulation. While both integrins and calcium signalling mediate chondrocyte mechanotransduction, calcium appears to play the major regulatory role. Understanding the underlying molecular mechanisms involved in chondrocyte mechanotransduction may lead to the development of improved bioengineered cartilage.

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

    PubMed Central

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

    2002-01-01

    Interleukin-1 is considered a central mediator of cartilage loss in osteoarthritis in several species, however an equine recombinant form of this cytokine is not readily available for in vitro use in equine osteoarthritis research. Equine recombinant interleukin-1β was cloned and expressed and its effects on the expression and activity of selected chondrocytic proteins implicated in cartilage matrix degradation were characterized. Reverse transcriptase polymerase chain reaction methods were used to amplify the entire coding region of the equine IL-1β mRNA, which was cloned into an expression vector, expressed in E. coli, and purified using a Ni2+ chromatographic method. The effects of the recombinant peptide on chondrocyte gene expression were determined by Northern blotting using RNA from equine chondrocyte cultures hybridized to probes for matrix metalloproteinases (MMP 1, MMP 3, MMP 13), tissue inhibitor of matrix metalloproteinases 1 (TIMP 1) and cyclooxygenase 2 (COX 2). Effects on selected mediators of cartilage degradation (nitrite concentrations and MMP activity) were determined using conditioned medium from reIL-1β-treated equine cartilage explant cultures. A recombinant peptide of approximately 21 kd was obtained. Northern blotting analyses revealed a marked up-regulation of expression of all MMPs, TIMP 1, and COX 2 in mRNA from treated chondrocytes. Furthermore, cartilage explants exposed to reIL-1β had augmented collagenase/gelatinase and stromelysin activities as well as increased concentration of nitrite in conditioned media. The development of a biologically active, species-specific IL-1β provides a valuable tool in the study of osteoarthritis pathophysiology and its treatment in horses. PMID:11858644

  10. Modulation of IL-1-induced cartilage injury by NO synthase inhibitors: a comparative study with rat chondrocytes and cartilage entities

    PubMed Central

    Cipolletta, Christine; Jouzeau, Jean-Yves; Gegout-Pottie, Pascale; Presle, Nathalie; Bordji, Karim; Netter, Patrick; Terlain, Bernard

    1998-01-01

    Nitric oxide (NO) is produced in diseased joints and may be a key mediator of IL-1 effects on cartilage. Therefore, we compared the potency of new [aminoguanidine (AG), S-methylisothiourea (SMT), S-aminoethylisothiourea (AETU)] and classical [Nω-monomethyl-L-arginine (L-NMMA), Nω-nitro-L-arginine methyl ester (L-NAME)] NO synthase (NOS) inhibitors on the inhibitory effect of recombinant human interleukin-1β (rhIL-1β) on rat cartilage anabolism. Three different culture systems were used: (1) isolated chondrocytes encapsulated in alginate beads; (2) patellae and (3) femoral head caps. Chondrocyte beads and cartilage entities were incubated in vitro for 48 h in the presence of rhIL-1β with a daily change of incubation medium to obtain optimal responses on proteoglycan synthesis and NO production. Proteoglycan synthesis was assessed by incorporation of radiolabelled sodium sulphate [Na235SO4] and NO production by cumulated nitrite release during the period of study. Chondrocytes and patellae, as well as femoral head caps, responded concentration-dependently to IL-1β challenge (0 to 250 U ml−1 and 0 to 15 U ml−1 respectively) by a large increase in nitrite level and a marked suppression of proteoglycan synthesis. Above these concentrations of IL-1β (2500 U ml−1 and 30 U ml−1 respectively), proteoglycan synthesis plateaued whereas nitrite release still increased thus suggesting different concentration-response curves. When studying the effect of NOS inhibitors (1 to 1000 μM) on NO production by cartilage cells stimulated with IL-1β (25 U ml−1 or 5 U ml−1), we observed that: (i) their ability to reduce nitrite level decreased from chondrocytes to cartilage samples, except for L-NMMA and AETU; (ii) they could be roughly classified in the following rank order of potency: AETU>L-NMMA⩾SMT>AG⩾L-NAME and (iii) AETU was cytotoxic when used in the millimolar range. When studying the effect of NOS inhibitors on proteoglycan

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

    PubMed

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

    2013-12-01

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

  12. CCN1 Regulates Chondrocyte Maturation and Cartilage Development

    PubMed Central

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

    2016-01-01

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

  13. CCN1 Regulates Chondrocyte Maturation and Cartilage Development.

    PubMed

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

    2016-03-01

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

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

    PubMed

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

    2005-11-01

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

  15. [Cartilage biopsy for autologous chondrocyte implantation (ACI)].

    PubMed

    Pestka, J M; Salzmann, G M; Südkamp, N P; Niemeyer, P

    2013-06-01

    Autologous chondrocyte implantation (ACI) is an established two-step procedure for the treatment of full-thickness cartilage defects of the knee. Cartilage harvest from the affected knee joint represents the first step of this procedure and is essential for further in vitro expansion of autologous chondrocytes. Nevertheless, the cartilage biopsy process itself is underrepresented in the scientific literature and currently there is only a limited amount of data available addressing this process. Biopsy location as well as the technique itself and instruments used for cartilage collection are not well defined and only little standardisation can be found. The article describes the relevant aspects of the biopsy in the context of ACI with regard to the literature available. Follow-up studies to better define and standardise the cartilage biopsy process are thus required.

  16. Fate of Meckel's cartilage chondrocytes in ocular culture

    SciTech Connect

    Richman, J.M.; Diewert, V.M.

    1988-09-01

    Modulation of the chondrocyte phenotype was observed in an organ culture system using Meckel's cartilage. First branchial arch cartilage was dissected from fetal rats of 16- and 17-day gestation. Perichondrium was mechanically removed, cartilage was split at the rostral process, and each half was grafted into the anterior chamber of an adult rat eye. The observed pattern of development in nonirradiated specimens was the following: hypertrophy of the rostral process and endochondral-type ossification, fibrous atrophy in the midsection, and mineralization of the malleus and incus. A change in matrix composition of the implanted cartilage was demonstrated with immunofluorescence staining for cartilage-specific proteoglycan (CSPG). After 15 days of culture, CSPG was found in the auricular process but not in the midsection or rostral process. In order to mark the implanted cells and follow their fate, cartilage was labeled in vitro with (3H)thymidine (3H)TdR). Immediately after labeling 20% of the chondrocytes contained (3H)TdR. After culturing for 5 days, 20% of the chondrocytes were still labeled and 10% of the osteogenic cells also contained radioactive label. The labeling index decreased in both cell types with increased duration of culture. Multinucleated clast-type cells did not contain label. Additional cartilages not labeled with (3H)TdR were exposed to between 20000 and 6000 rad of gamma irradiation before ocular implantation. Irradiated cartilage did not hypertrophy or form bone but a fibrous region developed in the midsection. Cells of the host animal were not induced to form bone around the irradiated cartilage. Our studies suggest that fully differentiated chondrocytes of Meckel's cartilage have the capacity to become osteocytes, osteoblasts, and fibroblasts.

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

    PubMed

    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.

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

    PubMed

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

    2015-06-25

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

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

    PubMed

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

    2015-05-22

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

  20. Autophagy Modulates Articular Cartilage Vesicle Formation in Primary Articular Chondrocytes*

    PubMed Central

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

    2015-01-01

    Chondrocyte-derived extracellular organelles known as articular cartilage vesicles (ACVs) participate in non-classical protein secretion, intercellular communication, and pathologic calcification. Factors affecting ACV formation and release remain poorly characterized; although in some cell types, the generation of extracellular vesicles is associated with up-regulation of autophagy. We sought to determine the role of autophagy in ACV production by primary articular chondrocytes. Using an innovative dynamic model with a light scatter nanoparticle counting apparatus, we determined the effects of autophagy modulators on ACV number and content in conditioned medium from normal adult porcine and human osteoarthritic chondrocytes. Healthy articular chondrocytes release ACVs into conditioned medium and show significant levels of ongoing autophagy. Rapamycin, which promotes autophagy, increased ACV numbers in a dose- and time-dependent manner associated with increased levels of autophagy markers and autophagosome formation. These effects were suppressed by pharmacologic autophagy inhibitors and short interfering RNA for ATG5. Caspase-3 inhibition and a Rho/ROCK inhibitor prevented rapamycin-induced increases in ACV number. Osteoarthritic chondrocytes, which are deficient in autophagy, did not increase ACV number in response to rapamycin. SMER28, which induces autophagy via an mTOR-independent mechanism, also increased ACV number. ACVs induced under all conditions had similar ecto-enzyme specific activities and types of RNA, and all ACVs contained LC3, an autophagosome-resident protein. These findings identify autophagy as a critical participant in ACV formation, and augment our understanding of ACVs in cartilage disease and repair. PMID:25869133

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

    PubMed Central

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

    2016-01-01

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

  2. Expansion of human articular chondrocytes and formation of tissue-engineered cartilage: a step towards exploring a potential use of matrix-induced cell therapy.

    PubMed

    Munirah, S; Samsudin, O C; Aminuddin, B S; Ruszymah, B H I

    2010-10-01

    sox-9 were significantly higher in FD10GFs when compared to control. After implantation, 'chondrocytes-fibrin' constructs exhibited firm, white, smooth and glistening cartilage-like properties. FD10GFs constructs formed better quality cartilage-like tissue than FD constructs in term of overall cartilaginous tissue formation, cells organization and extracellular matrix distribution in the specimens. Cartilaginous tissue formation was confirmed by the presence of lacunae and cartilage-isolated cells embedded within basophilic ground substance. Presence of proteoglycan was confirmed by positive Safranin O staining. Collagen type II exhibited immunopositivity at the pericellular and inter-territorial matrix area. Chondrogenic properties of the construct were further confirmed by the expression of genes encoding collagen type II, aggrecan core protein and sox9. In conclusion, FD10GFs promotes the proliferation of chondrocytes and formation of good quality 'chondrocytes-fibrin' constructs which may have potential use of matrix-induced cell implantation. Copyright © 2010 Elsevier Ltd. All rights reserved.

  3. Induction of cartilage integration by a chondrocyte/collagen-scaffold implant.

    PubMed

    Pabbruwe, Moreica B; Esfandiari, Ehsanollah; Kafienah, Wael; Tarlton, John F; Hollander, Anthony P

    2009-09-01

    The integration of implanted cartilage is a major challenge for the success of tissue engineering protocols. We hypothesize that in order for effective cartilage integration to take place, matrix-free chondrocytes must be induced to migrate between the two tissue surfaces. A chondrocyte/collagen-scaffold implant system was developed as a method of delivering dividing cells at the interface between two cartilage surfaces. Chondrocytes were isolated from bovine nasal septum and seeded onto both surfaces of a collagen membrane to create the chondrocyte/collagen-scaffold implant. A model of two cartilage discs and the chondrocyte/collagen-scaffold sandwiched in between was used to effect integration in vitro. The resulting tissue was analysed histologically and biomechanically. The cartilage-implant-cartilage sandwich appeared macroscopically as one continuous piece of tissue at the end of 40 day cultures. Histological analysis showed tissue continuum across the cartilage-scaffold interface. The integration was dependent on both cells and scaffold. Fluorescent labeling of implanted chondrocytes demonstrated that these cells invade the surrounding mature tissue and drive a remodelling of the extracellular matrix. Using cell-free scaffolds we also demonstrated that some chondrocytes migrated from the natural cartilage into the collagen scaffold. Quantification of integration levels using a histomorphometric repair index showed that the chondrocyte/collagen-scaffold implant achieved the highest repair index compared to controls, reflected functionally through increased tensile strength. In conclusion, cartilage integration can be achieved using a chondrocyte/collagen-scaffold implant that permits controlled delivery of chondrocytes to both host and graft mature cartilage tissues. This approach has the potential to be used therapeutically for implantation of engineered tissue.

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

    PubMed Central

    2011-01-01

    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 < 0.05). Supporting these gene expression results, IL-1β-induced cartilage matrix breakdown, as evidenced by GAG release from cartilage explants, was also significantly blocked (p < 0.05). Moreover, in the presence of herbal-Leucine mixture (HLM) up-regulation of ACAN and COL2A1 expression in IL-1β-stimulated OA chondrocytes was also noted (p < 0.05). The inhibitory effects of HLM were mediated by inhibiting the activation of nuclear factor (NF)-kB in human OA chondrocytes in presence of IL-1β. Conclusion Our data

  5. From gristle to chondrocyte transplantation: treatment of cartilage injuries

    PubMed Central

    Lindahl, Anders

    2015-01-01

    This review addresses the progress in cartilage repair technology over the decades with an emphasis on cartilage regeneration with cell therapy. The most abundant cartilage is the hyaline cartilage that covers the surface of our joints and, due to avascularity, this tissue is unable to repair itself. The cartilage degeneration seen in osteoarthritis causes patient suffering and is a huge burden to society. The surgical approach to cartilage repair was non-existing until the 1950s when new surgical techniques emerged. The use of cultured cells for cell therapy started as experimental studies in the 1970s that developed over the years to a clinical application in 1994 with the introduction of the autologous chondrocyte transplantation technique (ACT). The technology is now spread worldwide and has been further refined by combining arthroscopic techniques with cells cultured on matrix (MACI technology). The non-regenerating hypothesis of cartilage has been revisited and we are now able to demonstrate cell divisions and presence of stem-cell niches in the joint. Furthermore, cartilage derived from human embryonic stem cells and induced pluripotent stem cells could be the base for new broader cell treatments for cartilage injuries and the future technology base for prevention and cure of osteoarthritis. PMID:26416680

  6. From gristle to chondrocyte transplantation: treatment of cartilage injuries.

    PubMed

    Lindahl, Anders

    2015-10-19

    This review addresses the progress in cartilage repair technology over the decades with an emphasis on cartilage regeneration with cell therapy. The most abundant cartilage is the hyaline cartilage that covers the surface of our joints and, due to avascularity, this tissue is unable to repair itself. The cartilage degeneration seen in osteoarthritis causes patient suffering and is a huge burden to society. The surgical approach to cartilage repair was non-existing until the 1950s when new surgical techniques emerged. The use of cultured cells for cell therapy started as experimental studies in the 1970s that developed over the years to a clinical application in 1994 with the introduction of the autologous chondrocyte transplantation technique (ACT). The technology is now spread worldwide and has been further refined by combining arthroscopic techniques with cells cultured on matrix (MACI technology). The non-regenerating hypothesis of cartilage has been revisited and we are now able to demonstrate cell divisions and presence of stem-cell niches in the joint. Furthermore, cartilage derived from human embryonic stem cells and induced pluripotent stem cells could be the base for new broader cell treatments for cartilage injuries and the future technology base for prevention and cure of osteoarthritis.

  7. Increasing the Dose of Autologous Chondrocytes Improves Articular Cartilage Repair

    PubMed Central

    Guillén-García, Pedro; Rodríguez-Iñigo, Elena; Guillén-Vicente, Isabel; Caballero-Santos, Rosa; Guillén-Vicente, Marta; Abelow, Stephen; Giménez-Gallego, Guillermo

    2014-01-01

    Background: We hypothesized that implanting cells in a chondral defect at a density more similar to that of the intact cartilage could induce them to synthesize matrix with the features more similar to that of the uninjured one. Methods: We compared the implantation of different doses of chondrocytes: 1 million (n = 5), 5 million (n = 5), or 5 million mesenchymal cells (n = 5) in the femoral condyle of 15 sheep. Tissue generated by microfracture at the trochlea, and normal cartilage from a nearby region, processed as the tissues resulting from the implantation, were used as references. Histological and molecular (expression of type I and II collagens and aggrecan) studies were performed. Results: The features of the cartilage generated by implantation of mesenchymal cells and elicited by microfractures were similar and typical of a poor repair of the articular cartilage (presence of fibrocartilage, high expression of type I collagen and a low mRNA levels of type II collagen and aggrecan). Nevertheless, in the samples obtained from tissues generated by implantation of chondrocytes, hyaline-like cartilage, cell organization, low expression rates of type I collagen and high levels of mRNA corresponding to type II collagen and aggrecan were observed. These histological features, show less variability and are more similar to those of the normal cartilage used as control in the case of 5 million cells implantation than when 1 million cells were used. Conclusions: The implantation of autologous chondrocytes in type I/III collagen membranes at high density could be a promising tool to repair articular cartilage. PMID:26069691

  8. Collagen VI enhances cartilage tissue generation by stimulating chondrocyte proliferation.

    PubMed

    Smeriglio, Piera; Dhulipala, Lakshmi; Lai, Janice H; Goodman, Stuart B; Dragoo, Jason L; Smith, Robert L; Maloney, William J; Yang, Fan; Bhutani, Nidhi

    2015-02-01

    Regeneration of human cartilage is inherently inefficient. Current cell-based approaches for cartilage repair, including autologous chondrocytes, are limited by the paucity of cells, associated donor site morbidity, and generation of functionally inferior fibrocartilage rather than articular cartilage. Upon investigating the role of collagen VI (Col VI), a major component of the chondrocyte pericellular matrix (PCM), we observe that soluble Col VI stimulates chondrocyte proliferation. Interestingly, both adult and osteoarthritis chondrocytes respond to soluble Col VI in a similar manner. The proliferative effect is, however, strictly due to the soluble Col VI as no proliferation is observed upon exposure of chondrocytes to immobilized Col VI. Upon short Col VI treatment in 2D monolayer culture, chondrocytes maintain high expression of characteristic chondrocyte markers like Col2a1, agc, and Sox9 whereas the expression of the fibrocartilage marker Collagen I (Col I) and of the hypertrophy marker Collagen X (Col X) is minimal. Additionally, Col VI-expanded chondrocytes show a similar potential to untreated chondrocytes in engineering cartilage in 3D biomimetic hydrogel constructs. Our study has, therefore, identified soluble Col VI as a biologic that can be useful for the expansion and utilization of scarce sources of chondrocytes, potentially for autologous chondrocyte implantation. Additionally, our results underscore the importance of further investigating the changes in chondrocyte PCM with age and disease and the subsequent effects on chondrocyte growth and function.

  9. C/EBPβ and RUNX2 cooperate to degrade cartilage with MMP-13 as the target and HIF-2α as the inducer in chondrocytes.

    PubMed

    Hirata, Makoto; Kugimiya, Fumitaka; Fukai, Atsushi; Saito, Taku; Yano, Fumiko; Ikeda, Toshiyuki; Mabuchi, Akihiko; Sapkota, Bishwa Raj; Akune, Toru; Nishida, Nao; Yoshimura, Noriko; Nakagawa, Takumi; Tokunaga, Katsushi; Nakamura, Kozo; Chung, Ung-il; Kawaguchi, Hiroshi

    2012-03-01

    To elucidate the molecular mechanism underlying the endochondral ossification process during the skeletal growth and osteoarthritis (OA) development, we examined the signal network around CCAAT/enhancer-binding protein-β (C/EBPβ, encoded by CEBPB), a potent regulator of this process. Computational predictions and a C/EBP motif-reporter assay identified RUNX2 as the most potent transcriptional partner of C/EBPβ in chondrocytes. C/EBPβ and RUNX2 were induced and co-localized in highly differentiated chondrocytes during the skeletal growth and OA development of mice and humans. The compound knockout of Cebpb and Runx2 in mice caused growth retardation and resistance to OA with decreases in cartilage degradation and matrix metalloproteinase-13 (Mmp-13) expression. C/EBPβ and RUNX2 cooperatively enhanced promoter activity of MMP13 through specific binding to a C/EBP-binding motif and an osteoblast-specific cis-acting element 2 motif as a protein complex. Human genetic studies failed to show the association of human CEBPB gene polymorphisms with knee OA, nor was there a genetic variation around the identified responsive region in the human MMP13 promoter. However, hypoxia-inducible factor-2α (HIF-2α), a functional and genetic regulator of knee OA through promoting endochondral ossification, was identified as a potent and functional inducer of C/EBPβ expression in chondrocytes by the CEBPB promoter assay. Hence, C/EBPβ and RUNX2, with MMP-13 as the target and HIF-2α as the inducer, control cartilage degradation. This molecular network in chondrocytes may represent a therapeutic target for OA.

  10. Induction of cartilage integration by a chondrocyte/collagen-scaffold implant

    PubMed Central

    Pabbruwe, Moreica B.; Esfandiari, Ehsanollah; Kafienah, Wael; Tarlton, John F.; Hollander, Anthony P.

    2009-01-01

    The integration of implanted cartilage is a major challenge for the success of tissue engineering protocols. We hypothesize that in order for effective cartilage integration to take place, matrix-free chondrocytes must be induced to migrate between the two tissue surfaces. A chondrocyte/collagen-scaffold implant system was developed as a method of delivering dividing cells at the interface between two cartilage surfaces. Chondrocytes were isolated from bovine nasal septum and seeded onto both surfaces of a collagen membrane to create the chondrocyte/collagen-scaffold implant. A model of two cartilage discs and the chondrocyte/collagen-scaffold sandwiched in between was used to effect integration in vitro. The resulting tissue was analysed histologically and biomechanically. The cartilage–implant–cartilage sandwich appeared macroscopically as one continuous piece of tissue at the end of 40 day cultures. Histological analysis showed tissue continuum across the cartilage–scaffold interface. The integration was dependent on both cells and scaffold. Fluorescent labeling of implanted chondrocytes demonstrated that these cells invade the surrounding mature tissue and drive a remodelling of the extracellular matrix. Using cell-free scaffolds we also demonstrated that some chondrocytes migrated from the natural cartilage into the collagen scaffold. Quantification of integration levels using a histomorphometric repair index showed that the chondrocyte/collagen-scaffold implant achieved the highest repair index compared to controls, reflected functionally through increased tensile strength. In conclusion, cartilage integration can be achieved using a chondrocyte/collagen-scaffold implant that permits controlled delivery of chondrocytes to both host and graft mature cartilage tissues. This approach has the potential to be used therapeutically for implantation of engineered tissue. PMID:19539365

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

    PubMed Central

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

    2013-01-01

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

  12. Elastic cartilage reconstruction by transplantation of cultured hyaline cartilage-derived chondrocytes.

    PubMed

    Mizuno, M; Takebe, T; Kobayashi, S; Kimura, S; Masutani, M; Lee, S; Jo, Y H; Lee, J I; Taniguchi, H

    2014-05-01

    Current surgical intervention of craniofacial defects caused by injuries or abnormalities uses reconstructive materials, such as autologous cartilage grafts. Transplantation of autologous tissues, however, places a significant invasiveness on patients, and many efforts have been made for establishing an alternative graft. Recently, we and others have shown the potential use of reconstructed elastic cartilage from ear-derived chondrocytes or progenitors with the unique elastic properties. Here, we examined the differentiation potential of canine joint cartilage-derived chondrocytes into elastic cartilage for expanding the cell sources, such as hyaline cartilage. Articular chondrocytes are isolated from canine joint, cultivated, and compared regarding characteristic differences with auricular chondrocytes, including proliferation rates, gene expression, extracellular matrix production, and cartilage reconstruction capability after transplantation. Canine articular chondrocytes proliferated less robustly than auricular chondrocytes, but there was no significant difference in the amount of sulfated glycosaminoglycan produced from redifferentiated chondrocytes. Furthermore, in vitro expanded and redifferentiated articular chondrocytes have been shown to reconstruct elastic cartilage on transplantation that has histologic characteristics distinct from hyaline cartilage. Taken together, cultured hyaline cartilage-derived chondrocytes are a possible cell source for elastic cartilage reconstruction. Crown Copyright © 2014. Published by Elsevier Inc. All rights reserved.

  13. Functional Properties of Chondrocytes and Articular Cartilage using Optical Imaging to Scanning Probe Microscopy.

    PubMed

    Xia, Yang; Darling, Eric M; Herzog, Walter

    2017-10-03

    Mature chondrocytes in adult articular cartilage vary in number, size, and shape, depending on their depth in the tissue, location in the joint, and source species. Chondrocytes are the primary structural, functional, and metabolic unit in articular cartilage, the loss of which will induce fatigue to the extracellular matrix (ECM), eventually leading to failure of the cartilage and impairment of the joint as a whole. This brief review focuses on the functional and biomechanical studies of chondrocytes and articular cartilage, using microscopic imaging from optical microscopies to scanning probe microscopy. Three topics are covered in this review, including the functional studies of chondrons by optical imaging (unpolarized and polarized light and infrared light, two-photon excitation microscopy), the probing of chondrocytes and cartilage directly using microscale measurement techniques, and different imaging approaches that can measure chondrocyte mechanics and chondrocyte biological signaling under in situ and in vivo environments. Technical advancement in chondrocyte research during recent years has enabled new ways to study the biomechanical and functional properties of these cells and cartilage. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

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

    PubMed Central

    Klinger, P.; Ferrazzi, F.; Hotfiel, T.; Swoboda, B.; Aigner, T.

    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. PMID:28191465

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

    PubMed

    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.

  16. The Identification of CD163 Expressing Phagocytic Chondrocytes in Joint Cartilage and Its Novel Scavenger Role in Cartilage Degradation

    PubMed Central

    Wu, Yaoping; Qiu, Zhong Ying; He, Jianjun; Cao, Yunxin; Hu, Jintao; Zhu, Han; Niu, Li-Na; Cao, Xu; Yang, Kun; Wang, Mei-Qing

    2013-01-01

    Background Cartilage degradation is a typical characteristic of arthritis. This study examined whether there was a subset of phagocytic chondrocytes that expressed the specific macrophage marker, CD163, and investigated their role in cartilage degradation. Methods Cartilage from the knee and temporomandibular joints of Sprague-Dawley rats was harvested. Cartilage degradation was experimentally-induced in rat temporomandibular joints, using published biomechanical dental methods. The expression levels of CD163 and inflammatory factors within cartilage, and the ability of CD163+ chondrocytes to conduct phagocytosis were investigated. Cartilage from the knees of patients with osteoarthritis and normal cartilage from knee amputations was also investigated. Results In the experimentally-induced degrading cartilage from temporomandibular joints, phagocytes were capable of engulfing neighboring apoptotic and necrotic cells, and the levels of CD163, TNF-α and MMPs were all increased (P<0.05). However, the levels of ACP-1, NO and ROS, which relate to cellular digestion capability were unchanged (P>0.05). CD163+ chondrocytes were found in the cartilage mid-zone of temporomandibular joints and knee from healthy, three-week old rats. Furthermore, an increased number of CD163+ chondrocytes with enhanced phagocytic activity were present in Col-II+ chondrocytes isolated from the degraded cartilage of temporomandibular joints in the eight-week experimental group compared with their age-matched controls. Increased number with enhanced phagocytic activity of CD163+ chondrocytes were also found in isolated Col-II+ chondrocytes stimulated with TNF-α (P<0.05). Mid-zone distribution of CD163+ cells accompanied with increased expression of CD163 and TNF-α were further confirmed in the isolated Col-II+ chondrocytes from the knee cartilage of human patients with osteoarthritis, in contrast to the controls (both P<0.05). Conclusions An increased number of CD163+ chondrocytes with

  17. The identification of CD163 expressing phagocytic chondrocytes in joint cartilage and its novel scavenger role in cartilage degradation.

    PubMed

    Jiao, Kai; Zhang, Jing; Zhang, Mian; Wei, Yuying; Wu, Yaoping; Qiu, Zhong Ying; He, Jianjun; Cao, Yunxin; Hu, Jintao; Zhu, Han; Niu, Li-Na; Cao, Xu; Yang, Kun; Wang, Mei-Qing

    2013-01-01

    Cartilage degradation is a typical characteristic of arthritis. This study examined whether there was a subset of phagocytic chondrocytes that expressed the specific macrophage marker, CD163, and investigated their role in cartilage degradation. Cartilage from the knee and temporomandibular joints of Sprague-Dawley rats was harvested. Cartilage degradation was experimentally-induced in rat temporomandibular joints, using published biomechanical dental methods. The expression levels of CD163 and inflammatory factors within cartilage, and the ability of CD163(+) chondrocytes to conduct phagocytosis were investigated. Cartilage from the knees of patients with osteoarthritis and normal cartilage from knee amputations was also investigated. In the experimentally-induced degrading cartilage from temporomandibular joints, phagocytes were capable of engulfing neighboring apoptotic and necrotic cells, and the levels of CD163, TNF-α and MMPs were all increased (P<0.05). However, the levels of ACP-1, NO and ROS, which relate to cellular digestion capability were unchanged (P>0.05). CD163(+) chondrocytes were found in the cartilage mid-zone of temporomandibular joints and knee from healthy, three-week old rats. Furthermore, an increased number of CD163(+) chondrocytes with enhanced phagocytic activity were present in Col-II(+) chondrocytes isolated from the degraded cartilage of temporomandibular joints in the eight-week experimental group compared with their age-matched controls. Increased number with enhanced phagocytic activity of CD163(+) chondrocytes were also found in isolated Col-II(+) chondrocytes stimulated with TNF-α (P<0.05). Mid-zone distribution of CD163(+) cells accompanied with increased expression of CD163 and TNF-α were further confirmed in the isolated Col-II(+) chondrocytes from the knee cartilage of human patients with osteoarthritis, in contrast to the controls (both P<0.05). An increased number of CD163(+) chondrocytes with enhanced phagocytic activity

  18. Effects of short-term glucocorticoid treatment on changes in cartilage matrix degradation and chondrocyte gene expression induced by mechanical injury and inflammatory cytokines

    PubMed Central

    2011-01-01

    Introduction Traumatic joint injury damages cartilage and causes adjacent joint tissues to release inflammatory cytokines, increasing the risk of developing osteoarthritis. The main objective of this study was to determine whether the combined catabolic effects of mechanical injury, tumor necrosis factor alpha (TNFα) and interleukin-6 (IL-6)/soluble IL-6 receptor (sIL-6R) on cartilage could be abolished by short-term treatment with glucocorticoids such as dexamethasone. Methods In an initial dexamethasone-dose-response study, bovine cartilage explants were treated with TNFα and increasing concentrations of dexamethasone. Bovine and human cartilage explants were then subjected to individual and combined treatments with TNFα, IL-6/sIL-6R and injury in the presence or absence of dexamethasone. Treatment effects were assessed by measuring glycosaminoglycans (GAG) release to the medium and synthesis of proteoglycans. Additional experiments tested whether pre-exposure of cartilage to dexamethasone could prevent GAG loss and inhibition of biosynthesis induced by cytokines, and whether post-treatment with dexamethasone could diminish the effects of pre-established cytokine insult. Messenger ribonucleic acid (mRNA) levels for genes involved in cartilage homeostasis (proteases, matrix molecules, cytokines, growth and transcription factors) were measured in explants subjected to combined treatments with injury, TNFα and dexamethasone. To investigate mechanisms associated with dexamethasone regulation of chondrocyte metabolic response, glucocorticoid receptor (GR) antagonist (RU486) and proprotein convertase inhibitor (RVKR-CMK) were used. Results Dexamethasone dose-dependently inhibited GAG loss and the reduction in biosynthesis caused by TNFα. The combination of mechanical injury, TNFα and IL-6/sIL-6R caused the most severe GAG loss; dexamethasone reduced this GAG loss to control levels in bovine and human cartilage. Additionally, dexamethasone pre-treatment or post

  19. Autologous chondrocyte implantation: superior biologic properties of hyaline cartilage repairs.

    PubMed

    Henderson, Ian; Lavigne, Patrick; Valenzuela, Herminio; Oakes, Barry

    2007-02-01

    Information regarding the quality of autologous chondrocyte implantation repair is needed to determine whether the current autologous chondrocyte implantation surgical technology and the subsequent biologic repair processes are capable of reliably forming durable hyaline or hyaline-like cartilage in vivo. We report and analyze the properties and qualities of autologous chondrocyte implantation repairs. We evaluated 66 autologous chondrocyte implantation repairs in 57 patients, 55 of whom had histology, indentometry, and International Cartilage Repair Society repair scoring at reoperation for mechanical symptoms or pain. International Knee Documentation Committee scores were used to address clinical outcome. Maximum stiffness, normalized stiffness, and International Cartilage Repair Society repair scoring were higher for hyaline articular cartilage repairs compared with fibrocartilage, with no difference in clinical outcome. Reoperations revealed 32 macroscopically abnormal repairs (Group B) and 23 knees with normal-looking repairs in which symptoms leading to arthroscopy were accounted for by other joint disorders (Group A). In Group A, 65% of repairs were either hyaline or hyaline-like cartilage compared with 28% in Group B. Autologous chondrocyte repairs composed of fibrocartilage showed more morphologic abnormalities and became symptomatic earlier than hyaline or hyaline-like cartilage repairs. The hyaline articular cartilage repairs had biomechanical properties comparable to surrounding cartilage and superior to those associated with fibrocartilage repairs.

  20. Platelets promote cartilage repair and chondrocyte proliferation via ADP in a rodent model of osteoarthritis.

    PubMed

    Zhou, Qi; Xu, Chunhua; Cheng, Xingyao; Liu, Yangyang; Yue, Ming; Hu, Mengjiao; Luo, Dongjiao; Niu, Yuxi; Ouyang, Hongwei; Ji, Jiansong; Hu, Hu

    2016-01-01

    Osteoarthritis (OA) is the most common age-related degenerative joint disease and platelet-rich plasma (PRP) has been shown to be beneficial in OA. Therefore, in this study, we aimed to investigate the effects of platelets on chondrocytes and the underlying mechanisms. Anabolic and catabolic activity and the proliferation rate of chondrocytes were evaluated after co-culture with platelets. Chondrocyte gene expression was measured by real-time PCR. Chondrocyte protein expression and phosphorylation were measured by western blot. Chondrocytes treated with or without platelets were transplanted into a rat model of OA induced by intra-articular injection of monosodium iodoacetate and the repair of articular cartilage was evaluated macroscopically and histologically. Platelets significantly promoted the proliferation of chondrocytes, while mildly influencing anabolic and catabolic activity. Chondrocytes co-cultured with platelets showed significantly increased production of bone morphogenetic protein 7 (BMP7). The autocrine/paracrine effect of BMP7 was responsible for the increased proliferation of chondrocytes, via the ERK/CDK1/cyclin B1 signaling pathway. Transplantation of platelet-treated chondrocytes showed better cartilage repair in the OA model. Platelet-derived ADP was identified as the major mediator to promote the production of BMP7 and the proliferation of chondrocytes, through the ADP receptor P2Y1. Finally, direct injection of α,β-methyleneadenosine-5'-diphosphate into OA joints also enhanced cartilage repair. This study has identified that platelet-derived ADP, but not ATP, is the key mediator for platelet-promoted chondrocyte proliferation and cartilage repair in osteoarthritis. This finding may provide a key explanation for the therapeutic effect of platelets in OA and help shaping a strategy to improve OA therapy.

  1. PGA-associated heterotopic chondrocyte cocultures: implications of nasoseptal and auricular chondrocytes in articular cartilage repair.

    PubMed

    El Sayed, K; Marzahn, U; John, T; Hoyer, M; Zreiqat, H; Witthuhn, A; Kohl, B; Haisch, A; Schulze-Tanzil, G

    2013-01-01

    The availability of autologous articular chondrocytes remains a limiting issue in matrix assisted autologous chondrocyte transplantation. Non-articular heterotopic chondrocytes could be an alternative autologous cell source. The aims of this study were to establish heterotopic chondrocyte cocultures to analyze cell-cell compatibilities and to characterize the chondrogenic potential of nasoseptal chondrocytes compared to articular chondrocytes. Primary porcine and human nasoseptal and articular chondrocytes were investigated for extracellular cartilage matrix (ECM) expression in a monolayer culture. 3D polyglycolic acid- (PGA) associated porcine heterotopic mono- and cocultures were assessed for cell vitality, types II, I, and total collagen-, and proteoglycan content. The type II collagen, lubricin, and Sox9 gene expressions were significantly higher in articular compared with nasoseptal monolayer chondrocytes, while type IX collagen expression was lower in articular chondrocytes. Only β1-integrin gene expression was significantly inferior in humans but not in porcine nasoseptal compared with articular chondrocytes, indicating species-dependent differences. Heterotopic chondrocytes in PGA cultures revealed high vitality with proteoglycan-rich hyaline-like ECM production. Similar amounts of type II collagen deposition and type II/I collagen ratios were found in heterotopic chondrocytes cultured on PGA compared to articular chondrocytes. Quantitative analyses revealed a time-dependent increase in total collagen and proteoglycan content, whereby the differences between heterotopic and articular chondrocyte cultures were not significant. Nasoseptal and auricular chondrocytes monocultured in PGA or cocultured with articular chondrocytes revealed a comparable high chondrogenic potential in a tissue engineering setting, which created the opportunity to test them in vivo for articular cartilage repair. Copyright © 2011 John Wiley & Sons, Ltd.

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed Central

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

    2012-01-01

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

  4. Nasal chondrocytes and fibrin sealant for cartilage tissue engineering.

    PubMed

    Vinatier, C; Gauthier, O; Masson, M; Malard, O; Moreau, A; Fellah, B H; Bilban, M; Spaethe, R; Daculsi, G; Guicheux, J

    2009-04-01

    Hybrid constructs associating a biodegradable matrix and autologous chondrocytes hold promise for the treatment of articular cartilage defects. In this context, our objective was to investigate the potential use of nasal chondrocytes associated with a fibrin sealant for the treatment of articular cartilage defects. The phenotype of primary nasal chondrocytes (NC) from human (HNC) and rabbit (RNC) origin were characterized by RT-PCR. The ability of constructs associating fibrin sealant and NC to form a cartilaginous tissue in vivo was investigated, firstly in a subcutaneous site in nude mice and secondly in an articular cartilage defect in rabbit. HNC express type II collagen and aggrecan, the two major hallmarks of a chondrocytic phenotype. Furthermore, when injected subcutaneously into nude mice within a fibrin sealant, these chondrocytes were able to form a cartilage-like tissue. Our data indicate that RNC also express type II collagen and aggrecan and maintained their phenotype in three-dimensional culture within a fibrin sealant. Moreover, treatment of rabbit articular cartilage defects with autologous RNC embedded in a fibrin sealant led to the formation of a hyalin-like repair tissue. The use of fibrin sealant containing hybrid autologous NC therefore appears as a promising approach for cell-based therapy of articular cartilage. Copyright 2008 Wiley Periodicals, Inc.

  5. Passaged human chondrocytes accumulate extracellular matrix when induced by bovine chondrocytes.

    PubMed

    Ahmed, Nazish; Taylor, Drew W; Wunder, Jay; Nagy, Andras; Gross, Allan E; Kandel, Rita A

    2010-03-01

    A source of sufficient number of cells is a major limiting factor for cartilage tissue engineering. To circumvent this problem, we developed a co-culture method to induce redifferentiation in bovine articular chondrocytes, which had undergone dedifferentiation following serial passage in monolayer culture. In this study we determine whether human osteoarthritic (OA) and non-diseased passaged dedifferentiated chondrocytes will respond similarly. Human passaged chondrocytes were co-cultured for 4 weeks with primary bovine chondrocytes and their redifferentiation status was determined. Afterwards the cells were cultured either independently or in co-culture with cryopreserved passaged cells for functional analysis. The co-culture of passaged cells with primary chondrocytes resulted in reversion of their phenotype towards articular chondrocytes, as shown by increased gene expression of type II collagen and COMP, decreased type I collagen expression and extracellular matrix formation in vitro. Furthermore, this redifferentiation was stable, as those cells not only formed hyaline-like cartilage tissue when grown on their own but also they could induce redifferentiation of passaged chondrocytes in co-culture. These data suggest that it may be possible to use autologous chondrocytes obtained from osteoarthritic cartilage to form tissue suitable to use for cartilage repair. Copyright (c) 2009 John Wiley & Sons, Ltd.

  6. The circadian clock in murine chondrocytes regulates genes controlling key aspects of cartilage homeostasis.

    PubMed

    Gossan, Nicole; Zeef, Leo; Hensman, James; Hughes, Alun; Bateman, John F; Rowley, Lynn; Little, Christopher B; Piggins, Hugh D; Rattray, Magnus; Boot-Handford, Raymond P; Meng, Qing-Jun

    2013-09-01

    To characterize the circadian clock in murine cartilage tissue and identify tissue-specific clock target genes, and to investigate whether the circadian clock changes during aging or during cartilage degeneration using an experimental mouse model of osteoarthritis (OA). Cartilage explants were obtained from aged and young adult mice after transduction with the circadian clock fusion protein reporter PER2::luc, and real-time bioluminescence recordings were used to characterize the properties of the clock. Time-series microarrays were performed on mouse cartilage tissue to identify genes expressed in a circadian manner. Rhythmic genes were confirmed by quantitative reverse transcription-polymerase chain reaction using mouse tissue, primary chondrocytes, and a human chondrocyte cell line. Experimental OA was induced in mice by destabilization of the medial meniscus (DMM), and articular cartilage samples were microdissected and subjected to microarray analysis. Mouse cartilage tissue and a human chondrocyte cell line were found to contain intrinsic molecular circadian clocks. The cartilage clock could be reset by temperature signals, while the circadian period was temperature compensated. PER2::luc bioluminescence demonstrated that circadian oscillations were significantly lower in amplitude in cartilage from aged mice. Time-series microarray analyses of the mouse tissue identified the first circadian transcriptome in cartilage, revealing that 615 genes (∼3.9% of the expressed genes) displayed a circadian pattern of expression. This included genes involved in cartilage homeostasis and survival, as well as genes with potential importance in the pathogenesis of OA. Several clock genes were disrupted in the early stages of cartilage degeneration in the DMM mouse model of OA. These results reveal an autonomous circadian clock in chondrocytes that can be implicated in key aspects of cartilage biology and pathology. Consequently, circadian disruption (e.g., during aging

  7. Characteristics of tissue-engineered cartilage from human auricular chondrocytes.

    PubMed

    Park, Stephen S; Jin, Hong Ryul; Chi, David H; Taylor, Ray S

    2004-05-01

    This study was done to define the mechanical and histological properties of tissue-engineered cartilage (TEC) derived from human chondrocytes and to compare these findings with those of native cartilage. Chondrocytes were obtained from 10 human auricular cartilages and seeded onto a biodegradable template of polyglycolic acid and poly L-lactic acid. Each template was shaped into a 1 cm x 2 cm rectangle. The templates were implanted in athymic mice for 8 weeks. Eight human auricular cartilages were used for comparison. Mechanical analysis with a tensile testing device provided values of ultimate tensile strength (UTS), stiffness, and resilience. Statistical analysis was performed with the Student's t-test. Histological assessment was done with hematoxylin-eosin staining along with other special stains. The TEC had UTS of 2.07 MPa, stiffness of 3.7 MPa, and resilience of 0.37 J/m3. The control specimens had UTS of 2.18 MPa, stiffness of 5.11 MPa, and resilience of 0.42 J/m3. No statistical difference was found between the experimental and control groups for each of the three parameters. Histological analysis showed mature cartilage with characteristic collagen, glycosaminoglycans, and elastin in the TEC. The neo-cartilage showed slightly smaller size and more irregular distribution of chondrocytes and unique fibrous capsule formation with peripheral infiltration of fibrous tissue. This study showed that the mechanical qualities of TEC from human chondrocytes are similar to those of native auricular cartilage. It suggests that the engineered cartilage from human chondrocytes may have sufficient strength and durability for clinical uses. The histological findings revealed some differences with neo-cartilage.

  8. In vitro cartilage regeneration from proliferated adult elastic chondrocytes.

    PubMed

    Terada, Shinichi; Fuchs, Julie R; Yoshimoto, Hiroshi; Fauza, Dario O; Vacanti, Joseph P

    2005-08-01

    The purpose of this study was to investigate cellular feasibility in the proliferation and differentiation status of adult chondrocytes for cartilage regeneration in comparison to fetal chondrocytes. Primary cells were isolated from adult (n = 6) and fetal (n = 6) sheep ear cartilages and expanded in 10% fetal bovine serum (FBS) containing Ham's F12 medium, in which adult and fetal cell proliferation rates were compared using a WST-1 assay kit. Approximately 4 million cells were seeded onto each 1 x 1 x 0.2-cm (200 microL) nonwoven fabric scaffold made from polyglycolic acid. Cell/polymer constructs were cultured in serum-free DMEM/F12 medium supplemented with 5 ng/mL TGF-beta2 and 5 ng/mL des(1-3)IGF-I (adult chondrocytes, group A) or in 10% FBS containing Ham's F12 medium (adult chondrocytes, group B, and fetal chondrocytes, group C) as controls in a rotating bioreactor for 6 weeks. The proliferation assay showed that fetal cells had a significantly better growth potential than did adult cells. Histology and extracellular matrix analyses revealed that groups A and C qualitatively displayed better matrix deposition than did group B. In conclusion, although adult sheep elastic chondrocytes had less growth potential than did fetal cells, the serum-free medium supplemented with growth factors significantly enhanced the production of cartilage matrix secreted from proliferated adult sheep elastic chondrocytes.

  9. Enhanced apoptotic and reduced protective response in chondrocytes following endoplasmic reticulum stress in osteoarthritic cartilage

    PubMed Central

    Takada, Koji; Hirose, Jun; Senba, Kei; Yamabe, Soichiro; Oike, Yuichi; Gotoh, Tomomi; Mizuta, Hiroshi

    2011-01-01

    Endoplasmic reticulum (ER) stress has been shown to participate in many disease pathologies. Although recent reports have demonstrated that ER stress in chondrocytes is present in human osteoarthritis (OA), its role in the pathology of cartilage degeneration, such as chondrocyte apoptosis, remains unclear. In the present study, we investigated the expression of phosphorylated PERK (pPERK), ubiquitin (Ub), GRP78, CHOP, phosphorylated JNK (pJNK) and cleaved caspase-3 (C-CASP3) and the mRNA splicing of XBP1 (XBP1 splicing) in human OA cartilage by immunohistochemistry and RT-PCR. Additionally, human chondrocytes were treated with several concentrations of tunicamycin, an ER stress inducer, to assess the impact of ER stress on the mRNA expression of CHOP, XBP1 splicing and apoptosis, as determined by real-time PCR, RT-PCR and ELISA analyses respectively. In human OA cartilage, the number of chondrocytes expressing pPERK, Ub, CHOP and pJNK positively correlated with cartilage degeneration and the number of C-CASP3-positive chondrocytes. XBP1 splicing and GRP78 expression in severe OA containing the greatest number of C-CASP3-positive chondrocytes were similar to the levels in mild OA, however, XBP1 splicing was higher in moderate OA than in mild and severe OA. Tunicamycin dose dependently increased CHOP expression and apoptosis of cultured chondrocytes. Although tunicamycin upregulated XBP1 splicing in cultured chondrocytes, its impact on XBP1 splicing was weakened at higher concentrations. In conclusion, the present results indicate that ER stress may contribute to chondrocyte apoptosis along with OA progression, which was closely associated with an enhanced apoptotic response and a reduced protective response by the cells. PMID:21294793

  10. Human Cartilage-Derived Progenitor Cells From Committed Chondrocytes for Efficient Cartilage Repair and Regeneration.

    PubMed

    Jiang, Yangzi; Cai, Youzhi; Zhang, Wei; Yin, Zi; Hu, Changchang; Tong, Tong; Lu, Ping; Zhang, Shufang; Neculai, Dante; Tuan, Rocky S; Ouyang, Hong Wei

    2016-06-01

    Articular cartilage is not a physiologically self-renewing tissue. Injury of cartilage often progresses from the articular surface to the subchondral bone, leading to pathogenesis of tissue degenerative diseases, such as osteoarthritis. Therapies to treat cartilage defects using autologous chondrocyte-based tissue engineering have been developed and used for more than 20 years; however, the challenge of chondrocyte expansion in vitro remains. A promising cell source, cartilage stem/progenitor cells (CSPCs), has attracted recent attention. Because their origin and identity are still unclear, the application potential of CSPCs is under active investigation. Here we have captured the emergence of a group of stem/progenitor cells derived from adult human chondrocytes, highlighted by dynamic changes in expression of the mature chondrocyte marker, COL2, and mesenchymal stromal/stem cell (MSC) marker, CD146. These cells are termed chondrocyte-derived progenitor cells (CDPCs). The stem cell-like potency and differentiation status of CDPCs were determined by physical and biochemical cues during culture. A low-density, low-glucose 2-dimensional culture condition (2DLL) was critical for the emergence and proliferation enhancement of CDPCs. CDPCs showed similar phenotype as bone marrow mesenchymal stromal/stem cells but exhibited greater chondrogenic potential. Moreover, the 2DLL-cultured CDPCs proved efficient in cartilage formation both in vitro and in vivo and in repairing large knee cartilage defects (6-13 cm(2)) in 15 patients. These findings suggest a phenotype conversion between chondrocytes and CDPCs and provide conditions that promote the conversion. These insights expand our understanding of cartilage biology and may enhance the success of chondrocyte-based therapies. Injury of cartilage, a non-self-repairing tissue, often progresses to pathogenesis of degenerative joint diseases, such as osteoarthritis. Although tissue-derived stem cells have been shown to

  11. Human Cartilage-Derived Progenitor Cells From Committed Chondrocytes for Efficient Cartilage Repair and Regeneration

    PubMed Central

    Jiang, Yangzi; Cai, Youzhi; Zhang, Wei; Yin, Zi; Hu, Changchang; Tong, Tong; Lu, Ping; Zhang, Shufang; Neculai, Dante

    2016-01-01

    Articular cartilage is not a physiologically self-renewing tissue. Injury of cartilage often progresses from the articular surface to the subchondral bone, leading to pathogenesis of tissue degenerative diseases, such as osteoarthritis. Therapies to treat cartilage defects using autologous chondrocyte-based tissue engineering have been developed and used for more than 20 years; however, the challenge of chondrocyte expansion in vitro remains. A promising cell source, cartilage stem/progenitor cells (CSPCs), has attracted recent attention. Because their origin and identity are still unclear, the application potential of CSPCs is under active investigation. Here we have captured the emergence of a group of stem/progenitor cells derived from adult human chondrocytes, highlighted by dynamic changes in expression of the mature chondrocyte marker, COL2, and mesenchymal stromal/stem cell (MSC) marker, CD146. These cells are termed chondrocyte-derived progenitor cells (CDPCs). The stem cell-like potency and differentiation status of CDPCs were determined by physical and biochemical cues during culture. A low-density, low-glucose 2-dimensional culture condition (2DLL) was critical for the emergence and proliferation enhancement of CDPCs. CDPCs showed similar phenotype as bone marrow mesenchymal stromal/stem cells but exhibited greater chondrogenic potential. Moreover, the 2DLL-cultured CDPCs proved efficient in cartilage formation both in vitro and in vivo and in repairing large knee cartilage defects (6–13 cm2) in 15 patients. These findings suggest a phenotype conversion between chondrocytes and CDPCs and provide conditions that promote the conversion. These insights expand our understanding of cartilage biology and may enhance the success of chondrocyte-based therapies. Significance Injury of cartilage, a non-self-repairing tissue, often progresses to pathogenesis of degenerative joint diseases, such as osteoarthritis. Although tissue-derived stem cells have been shown

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

    PubMed Central

    Dewan, Ashvin K.; Gibson, Matthew A.; Elisseeff, Jennifer H.; Trice, Michael E.

    2014-01-01

    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. PMID:25210707

  13. Processed xenogenic cartilage as innovative biomatrix for cartilage tissue engineering: effects on chondrocyte differentiation and function.

    PubMed

    Schwarz, Silke; Elsaesser, Alexander F; Koerber, Ludwig; Goldberg-Bockhorn, Eva; Seitz, Andreas M; Bermueller, Christian; Dürselen, Lutz; Ignatius, Anita; Breiter, Roman; Rotter, Nicole

    2015-12-01

    One key point in the development of new bioimplant matrices for the reconstruction and replacement of cartilage defects is to provide an adequate microenvironment to ensure chondrocyte migration and de novo synthesis of cartilage-specific extracellular matrix (ECM). A recently developed decellularization and sterilization process maintains the three-dimensional (3D) collagen structure of native septal cartilage while increasing matrix porosity, which is considered to be crucial for cartilage tissue engineering. Human primary nasal septal chondrocytes were amplified in monolayer culture and 3D-cultured on processed porcine nasal septal cartilage scaffolds. The influence of chondrogenic growth factors on neosynthesis of ECM proteins was examined at the protein and gene expression levels. Seeding experiments demonstrated that processed xenogenic cartilage matrices provide excellent environmental properties for human nasal septal chondrocytes with respect to cell adhesion, migration into the matrix and neosynthesis of cartilage-specific ECM proteins, such as collagen type II and aggrecan. Matrix biomechanical stability indicated that the constructs retrieve full stability and function during 3D culture for up to 42 days, proportional to collagen type II and GAG production. Thus, processed xenogenic cartilage offers a suitable environment for human nasal chondrocytes and has promising potential for cartilage tissue engineering in the head and neck region.

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

    PubMed

    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.

  15. Smad6/Smurf1 overexpression in cartilage delays chondrocyte hypertrophy and causes dwarfism with osteopenia

    PubMed Central

    Horiki, Mitsuru; Imamura, Takeshi; Okamoto, Mina; Hayashi, Makoto; Murai, Junko; Myoui, Akira; Ochi, Takahiro; Miyazono, Kohei; Yoshikawa, Hideki; Tsumaki, Noriyuki

    2004-01-01

    Biochemical experiments have shown that Smad6 and Smad ubiquitin regulatory factor 1 (Smurf1) block the signal transduction of bone morphogenetic proteins (BMPs). However, their in vivo functions are largely unknown. Here, we generated transgenic mice overexpressing Smad6 in chondrocytes. Smad6 transgenic mice showed postnatal dwarfism with osteopenia and inhibition of Smad1/5/8 phosphorylation in chondrocytes. Endochondral ossification during development in these mice was associated with almost normal chondrocyte proliferation, significantly delayed chondrocyte hypertrophy, and thin trabecular bone. The reduced population of hypertrophic chondrocytes after birth seemed to be related to impaired bone growth and formation. Organ culture of cartilage rudiments showed that chondrocyte hypertrophy induced by BMP2 was inhibited in cartilage prepared from Smad6 transgenic mice. We then generated transgenic mice overexpressing Smurf1 in chondrocytes. Abnormalities were undetectable in Smurf1 transgenic mice. Mating Smad6 and Smurf1 transgenic mice produced double-transgenic pups with more delayed endochondral ossification than Smad6 transgenic mice. These results provided evidence that Smurf1 supports Smad6 function in vivo. PMID:15123739

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

    PubMed

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

    2015-07-17

    In ankylosing spondylitis (AS), joint remodeling leading to joint ankylosis involves cartilage fusion. Here, we analyzed whether chondrocyte hypertrophy is involved in cartilage fusion and subsequent joint remodeling in AS. We assessed the expression of chondrocyte hypertrophy markers runt-related transcription factor 2 (Runx2), type X collagen (COL10), matrix metalloproteinase 13 (MMP13), osteocalcin and beta-catenin and the expression of positive bone morphogenic proteins (BMPs) and negative regulators (dickkopf-1 (DKK-1)), sclerostin, (wingless inhibitory factor 1 (wif-1)) of chondrocyte hypertrophy in the cartilage of facet joints from patients with AS or osteoarthritis (OA) and from autopsy controls (CO) by immunohistochemistry. Sex determining region Y (SRY)-box 9 (Sox9) and type II collagen (COL2) expression was assessed as indicators of chondrocyte integrity and function. The percentage of hypertrophic chondrocytes expressing Runx2, COL10, MMP13, osteocalcin or beta-catenin was significantly increased in OA but not in AS joints compared to CO joints. Frequencies of sclerostin-positive and DKK-1-positive chondrocytes were similar in AS and CO. In contrast, wif-1- but also BMP-2- and BMP-7-expressing and Sox9-expressing chondrocytes were drastically reduced in AS joints compared to CO as well as OA joints whereas the percentage of COL2-expressing chondrocytes was significantly higher in AS joints compared to CO joints. We found no evidence for chondrocyte hypertrophy within hyaline cartilage of AS joints even in the presence of reduced expression of the wnt inhibitor wif-1 suggesting that chondrocyte hypertrophy is not a predominant pathway involved in joint fusion and remodeling in AS. In contrast, the reduced expression of Sox9, BMP-2 and BMP-7 concomitantly with induced COL2 expression rather point to disturbed cartilage homeostasis promoting cartilage degeneration in AS.

  17. Cartilage tissue engineering: molecular control of chondrocyte differentiation for proper cartilage matrix reconstruction.

    PubMed

    Demoor, Magali; Ollitrault, David; Gomez-Leduc, Tangni; Bouyoucef, Mouloud; Hervieu, Magalie; Fabre, Hugo; Lafont, Jérôme; Denoix, Jean-Marie; Audigié, Fabrice; Mallein-Gerin, Frédéric; Legendre, Florence; Galera, Philippe

    2014-08-01

    Articular cartilage defects are a veritable therapeutic problem because therapeutic options are very scarce. Due to the poor self-regeneration capacity of cartilage, minor cartilage defects often lead to osteoarthritis. Several surgical strategies have been developed to repair damaged cartilage. Autologous chondrocyte implantation (ACI) gives encouraging results, but this cell-based therapy involves a step of chondrocyte expansion in a monolayer, which results in the loss in the differentiated phenotype. Thus, despite improvement in the quality of life for patients, reconstructed cartilage is in fact fibrocartilage. Successful ACI, according to the particular physiology of chondrocytes in vitro, requires active and phenotypically stabilized chondrocytes. This review describes the unique physiology of cartilage, with the factors involved in its formation, stabilization and degradation. Then, we focus on some of the most recent advances in cell therapy and tissue engineering that open up interesting perspectives for maintaining or obtaining the chondrogenic character of cells in order to treat cartilage lesions. Current research involves the use of chondrocytes or progenitor stem cells, associated with "smart" biomaterials and growth factors. Other influential factors, such as cell sources, oxygen pressure and mechanical strain are considered, as are recent developments in gene therapy to control the chondrocyte differentiation/dedifferentiation process. This review provides new information on the mechanisms regulating the state of differentiation of chondrocytes and the chondrogenesis of mesenchymal stem cells that will lead to the development of new restorative cell therapy approaches in humans. This article is part of a Special Issue entitled Matrix-mediated cell behaviour and properties. Copyright © 2014 Elsevier B.V. All rights reserved.

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

    PubMed

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

    2013-02-08

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

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

    PubMed Central

    Akkiraju, Hemanth; Nohe, Anja

    2016-01-01

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

  20. Sodium nitroprusside induces apoptosis of rabbit chondrocytes

    NASA Astrophysics Data System (ADS)

    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.

  1. Expression Pattern and Role of Chondrocyte Clusters in Osteoarthritic Human Knee Cartilage

    PubMed Central

    Hoshiyama, Yoshiaki; Otsuki, Shuhei; Oda, Shuhei; Kurokawa, Yoshitaka; Nakajima, Mikio; Jotoku, Tsuyoshi; Tamura, Ryuichi; Okamoto, Yoshinori; Lotz, Martin K.; Neo, Masashi

    2015-01-01

    The purpose of this study was to investigate the site-specific expression pattern and the role of chondrocyte clusters in human OA knee. Cartilage explants were obtained from 45 varus knees of medial and lateral femoral condyle undergoing total knee replacement surgery. Cartilage degeneration, number of chondrocytes, and the cell arrangement were evaluated by live/dead assay and immunohistochemical analyses with antibodies of STRO-1, FGF2, and Ki-67. Chondrocytes from medial and lateral femoral condyle were cultured to compare the potential of cell proliferation and production of cartilaginous nodules. Finally, cartilage tissue from medial femoral condyle, which included cartilage cleft with chondrocyte clusters, was observed the histological alternation. As the results, chondrocyte density adjacent to severe cartilage degeneration was highest, whereas chondrocytes in lateral femoral condyle displayed low density with single type of cells. Over 80% of these chondrocyte clusters were survived, expressing STRO-1, FGF2, and Ki-67. Furthermore, chondrocyte clusters proliferated faster and produced more cartilaginous nodules than single type of chondrocytes. Cartilage clefts involving numerous chondrocyte clusters were filled with extracellular matrix during organ culture. In conclusion, chondrocyte clusters adjacent to severe cartilage degeneration have shown completely specific characteristics with progenitor and proliferative potential. Regulating chondrocyte clusters may offer new approaches to cartilage repair and OA therapy in the future. PMID:25691232

  2. Loading of Articular Cartilage Compromises Chondrocyte Respiratory Function

    PubMed Central

    Coleman, Mitchell C.; Ramakrishnan, Prem S.; Brouillette, Marc J.; Martin, James A.

    2015-01-01

    Objective Determine whether repeatedly overloading healthy cartilage disrupts mitochondrial function in a manner similar to that associated with osteoarthritis pathogenesis. Methods We exposed normal articular cartilage on bovine osteochondral explants to 1 day or 7 consecutive days of cyclic axial compression (0.25 or 1.0 MPa, 0.5 Hz, 3 hours) and evaluated effects on chondrocyte viability, ATP concentration, reactive oxygen species (ROS) production, indicators of oxidative stress, respiration, and mitochondrial membrane potential. Results Neither 0.25 nor 1.0 MPa cyclic compression caused extensive chondrocyte death, macroscopic tissue damage, or overt changes in stress-strain behavior. After one day of loading, differences in respiratory activities between the 0.25 and 1.0 MPa groups were minimal; after 7 loading days, however, respiratory activity and ATP levels were suppressed in the 1.0 MPa group relative to the 0.25 MPa group, an effect prevented with pretreatment with 10 mM N-acetylcysteine. These changes were accompanied by increased proton leakage and decreases in mitochondrial membrane potential as well as by increased ROS formation indicated by dihydroethidium staining and glutathione oxidation. Conclusion Repeated overloading leads to chondrocyte oxidant-dependent mitochondrial dysfunction. This mitochondrial dysfunction may contribute to destabilization of cartilage during various stages of OA in distinct ways by disrupting chondrocyte anabolic responses to mechanical stimuli. PMID:26473613

  3. Effects of co-culturing BMSCs and auricular chondrocytes on the elastic modulus and hypertrophy of tissue engineered cartilage.

    PubMed

    Kang, Ning; Liu, Xia; Guan, Yue; Wang, Jian; Gong, Fuxing; Yang, Xun; Yan, Li; Wang, Qian; Fu, Xin; Cao, Yilin; Xiao, Ran

    2012-06-01

    Co-culture of BMSCs and chondrocytes is considered as a promising strategy to generate tissue engineered cartilage as chondrocytes induce the chondrogenesis of BMSCs and inhibit the hypertrophy of engineered cartilage. Because the tissue specific stem/progenitor cells have been isolated from mature tissues including auricular cartilage, we hypothesized that adding stem cells to auricular chondrocytes in co-culture would also enhance the quality of engineered cartilage. In the present study, using the histological assay, biomechanical evaluation, and quantitative analysis of gene expression, we compared different strategies of auricular chondrocytes, BMSCs induction, and co-culture at different ratios on PGA/PLA scaffolds to construct tissue engineered elastic cartilage in vitro and in vivo. The up-regulation of RUNX2 and down-regulation of SOX9 were found in BMSCs chondrogenic induction group, which might imply a regulatory mechanism for the hypertrophy and potential osteogenic differentiation. Engineered cartilage in co-culture 5:5 group showed the densest elastic fibers and the highest Young's modulus, which were consistent with the expression profile of cartilage matrix-related genes including DCN and LOXL2 genes. Moreover, the better proliferative and chondrogenic potentials of engineered cartilage in co-culture 5:5 group were demonstrated by the stronger expression of Ki67 and Dlk1.

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

    PubMed

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

    1993-09-15

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

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

    PubMed Central

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

    1993-01-01

    We identified a rat cDNA that encodes cartilage homeoprotein 1 (Cart-1). The deduced amino acid sequence of Cart-1 contains a paired-type homeodomain. Northern blot hybridization and RNase protection assay revealed that Cart-1 RNA was present at high levels in a well-differentiated rat chondrosarcoma tumor and in a cell line derived from this tumor. Cart-1 RNA was detected in primary mouse and rat chondrocytes but not in various fibroblasts including mouse 10T1/2 cells, NIH 3T3 cells, BALB 3T3 cells, and rat skin fibroblasts. It was also undetectable in mouse C2 myoblasts, S194 myeloma cells, and embryonic stem cells. Cart-1 RNA was present at a very low level in tested but was not detected in other soft tissues of 8-week-old rats. In situ hybridization of rat embryos between 14.5 and 16.5 days post coitum revealed relatively high levels of Cart-1 RNA in condensed prechondrocytic mesenchymal cells and in early chondrocytes of cartilage primordia. The levels of Cart-1 RNA were lower in mature chondrocytes. No hybridization was observed in brain, spinal cord, heart, spleen, gastrointestinal tract, liver, and muscle. We speculate that Cart-1 has a role in chondrocyte differentiation. Images Fig. 1 Fig. 2 Fig. 3 PMID:7690966

  6. Transforming Growth Factor Beta Signaling Is Essential for the Autonomous Formation of Cartilage-Like Tissue by Expanded Chondrocytes

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2015-01-01

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

  8. Antirheumatic drug response signatures in human chondrocytes: potential molecular targets to stimulate cartilage regeneration

    PubMed Central

    Andreas, Kristin; Häupl, Thomas; Lübke, Carsten; Ringe, Jochen; Morawietz, Lars; Wachtel, Anja; Sittinger, Michael; Kaps, Christian

    2009-01-01

    Introduction Rheumatoid arthritis (RA) leads to progressive destruction of articular cartilage. This study aimed to disclose major mechanisms of antirheumatic drug action on human chondrocytes and to reveal marker and pharmacological target genes that are involved in cartilage dysfunction and regeneration. Methods An interactive in vitro cultivation system composed of human chondrocyte alginate cultures and conditioned supernatant of SV40 T-antigen immortalised human synovial fibroblasts was used. Chondrocyte alginate cultures were stimulated with supernatant of RA synovial fibroblasts, of healthy donor synovial fibroblasts, and of RA synovial fibroblasts that have been antirheumatically treated with disease-modifying antirheumatic drugs (DMARDs) (azathioprine, gold sodium thiomalate, chloroquine phosphate, and methotrexate), nonsteroidal anti-inflammatory drugs (NSAIDs) (piroxicam and diclofenac), or steroidal anti-inflammatory drugs (SAIDs) (methylprednisolone and prednisolone). Chondrocyte gene expression profile was analysed using microarrays. Real-time reverse transcription-polymerase chain reaction and enzyme-linked immunosorbent assay were performed for validation of microarray data. Results Genome-wide expression analysis revealed 110 RA-related genes in human chondrocytes: expression of catabolic mediators (inflammation, cytokines/chemokines, and matrix degradation) was induced, and expression of anabolic mediators (matrix synthesis and proliferation/differentiation) was repressed. Potential marker genes to define and influence cartilage/chondrocyte integrity and regeneration were determined and include already established genes (COX-2, CXCR-4, IL-1RN, IL-6/8, MMP-10/12, and TLR-2) and novel genes (ADORA2A, BCL2-A1, CTGF, CXCR-7, CYR-61, HSD11B-1, IL-23A, MARCKS, MXRA-5, NDUFA4L2, NR4A3, SMS, STS, TNFAIP-2, and TXNIP). Antirheumatic treatment with SAIDs showed complete and strong reversion of RA-related gene expression in human chondrocytes, whereas

  9. Lactoferrin inhibits dexamethasone-induced chondrocyte impairment from osteoarthritic cartilage through up-regulation of extracellular signal-regulated kinase 1/2 and suppression of FASL, FAS, and Caspase 3

    SciTech Connect

    Tu, Yihui; Xue, Huaming; Francis, Wendy; Davies, Andrew P.; Pallister, Ian; Kanamarlapudi, Venkateswarlu; Xia, Zhidao

    2013-11-08

    Highlights: •Dex exerts dose-dependant inhibition of HACs viability and induction of apoptosis. •Dex-induced impairment of chondrocytes was attenuated by rhLF. •ERK and FASL/FAS signaling are involved in the effects of rhLF. •OA patients with glucocorticoid-induced cartilage damage may benefit from treatment with rhLF. -- Abstract: Dexamethasone (Dex) is commonly used for osteoarthritis (OA) with excellent anti-inflammatory and analgesic effect. However, Dex also has many side effects following repeated use over prolonged periods mainly through increasing apoptosis and inhibiting proliferation. Lactoferrin (LF) exerts significantly anabolic effect on many cells and little is known about its effect on OA chondrocytes. Therefore, the aim of this study is to investigate whether LF can inhibit Dex-induced OA chondrocytes apoptosis and explore its possible molecular mechanism involved in. MTT assay was used to determine the optimal concentration of Dex and recombinant human LF (rhLF) on chondrocytes at different time and dose points. Chondrocytes were then stimulated with Dex in the absence or presence of optimal concentration of rhLF. Cell proliferation and viability were evaluated using MTT and LIVE/DEAD assay, respectively. Cell apoptosis was evaluated by multi-parameter apoptosis assay kit using both confocal microscopy and flow cytometry, respectively. The expression of extracellular signal-regulated kinase (ERK), FAS, FASL, and Caspase-3 (CASP3) at the mRNA and protein levels were examined by real-time polymerase chain reaction (PCR) and immunocytochemistry, respectively. The optimal concentration of Dex (25 μg/ml) and rhLF (200 μg/ml) were chosen for the following experiments. rhLF significantly reversed the detrimental effect of Dex on chondrocytes proliferation, viability, and apoptosis. In addition, rhLF significantly prevented Dex-induced down-regulation of ERK and up-regulation of FAS, FASL, and CASP3. These findings demonstrated that rhLF acts as

  10. Heterotopic autologous chondrocyte transplantation--a realistic approach to support articular cartilage repair?

    PubMed

    El Sayed, Karym; Haisch, Andreas; John, Thilo; Marzahn, Ulrike; Lohan, Anke; Müller, Riccarda D; Kohl, Benjamin; Ertel, Wolfgang; Stoelzel, Katharina; Schulze-Tanzil, Gundula

    2010-12-01

    Injured articular cartilage is limited in its capacity to heal. Autologous chondrocyte transplantation (ACT) is a suitable technique for cartilage repair, but it requires articular cartilage biopsies for sufficient autologous chondrocyte expansion in vitro. Hence, ACT is restricted by donor-site morbidity and autologous articular chondrocytes availability. The use of nonarticular heterotopic chondrocytes such as auricular, nasoseptal, or costal chondrocytes for ACT might overcome these limitations: heterotopic sources show lesser donor-site morbidity and a comparable extracellular cartilage matrix synthesis profile to articular cartilage. However, heterotopic (h)ACT poses a challenge. Particular tissue characteristics of heterotopic cartilage, divergent culturing peculiarities of heterotopic chondrocytes, and the advantages and drawbacks related to these diverse cartilage sources were critically discussed. Finally, available in vitro and in vivo experimental (h)ACT approaches were summarized. The quality of the cartilage engineered using heterotopic chondrocytes remains partly controversy due to the divergent methodologies and culture conditions used. While some encouraging in vivo results using (h)ACT have been demonstrated, standardized culturing protocols are strongly required. However, whether heterotopic chondrocytes implanted into joint cartilage defects maintain their particular tissue properties or can be adapted via tissue engineering strategies to fulfill regular articular cartilage functions requires further studies.

  11. [Allograft of cultured chondrocytes into articular cartilage defects in rabbits--experimental study of the repair of articular cartilage injuries].

    PubMed

    Tsuge, H; Sasaki, T; Susuda, K; Abe, K

    1983-08-01

    Articular cartilage defects were created by dill holes, 2 mm wide and 3 mm deep, through the articular cartilage into the subchondral bone in the patellar groove of the femur in mature rabbits. The defects received graft of cultured chondrocytes and the matrix obtained from the primary culture of chondrocytes isolated from the articular cartilage or auricular cartilage in immature rabbits. The isolated cells were cultured for 10 to 14 days. For graft, the cultured chondrocytes together with the matrix were detached from the culture chamber using rubber policemen and centrifuged. The repair of the grafted defects or defects without graft (control) was histologically studied 2 to 12 weeks after operation. The defects without the graft were progressively filled with fibrous tissue containing spindle shaped cells, fibers perpendicular to the surface, and matrix showing weak metachromasia with toluidin blue at 8 weeks. The defects received articular cartilage cell graft were occupied by new cartilage tissue consisting colonylike crumps of chondrocytes 2 weeks after operation. The crumps showed strong metachromasia with toluidin blue and strong stainability for safranin-O. By 4-8 weeks, the defects were filled with homogeneous cartilage. At 12 weeks, arrangement of the chondrocytes of the superficial layer of the new cartilage became columnar as seen in the normal articular cartilage. The defects received elastic cartilage cell graft were filled by reformed cartilage with chondrocytes surrounded by elastic fibers 2-12 weeks after operation. The results indicate that allograft of cultured chondrocytes with matrix into the articular cartilage defects accerated the repair process of the defects by formation of the new cartilage derived from the grafted chondrocytes.

  12. Induced pluripotent stem cells in cartilage repair

    PubMed Central

    Lietman, Steven A

    2016-01-01

    Articular cartilage repair techniques are challenging. Human embryonic stem cells and induced pluripotent stem cells (iPSCs) theoretically provide an unlimited number of specialized cells which could be used in articular cartilage repair. However thus far chondrocytes from iPSCs have been created primarily by viral transfection and with the use of cocultured feeder cells. In addition chondrocytes derived from iPSCs have usually been formed in condensed cell bodies (resembling embryoid bodies) that then require dissolution with consequent substantial loss of cell viability and phenotype. All of these current techniques used to derive chondrocytes from iPSCs are problematic but solutions to these problems are on the horizon. These solutions will make iPSCs a viable alternative for articular cartilage repair in the near future. PMID:27004161

  13. [Preliminary study on tissue-engineered cartilage with human dermal fibroblasts co-cultured with porcine chondrocytes in vitro].

    PubMed

    Liu, Xia; Zhou, Guang-dong; Liu, Wei; Cao, Yi-lin

    2009-11-01

    To explore the feasibility of constructing tissue-engineered cartilage with human dermal fibroblasts (HDFs) in vitro. Porcine articular chondrocytes and HDFs were isolated and in vitro expanded respectively. Then they were mixed at the ratio of 1:1 (chondrocytes: fibroblasts) . The mixed cells were seeded onto polyglycolic acid (PGA) scaffold at the ultimate concentration of 5.0 x 10(7)/ml as co-culture group. Chondrocytes and HDFs at the same ultimate concentration were seeded respectively onto the scaffold as chondrocyte group ( positive control group) and fibroblast group ( negative control group). The specimens were collected after in vitro culture for 8 weeks. Gross observation, histology and immunohistochemistry were used to evaluate the results. In chondrocyte group, the cell-scaffold constructs could maintain the original size and shape during in vitro culture. The new formed cartilage-like tissue had typical histological structure and extracellular matrix staining similar to normal cartilage. In co-culture group the constructs shrunk slightly at 8 weeks, cartilage-like tissue formed and GAG could be detected for strong expression by Safranin O staining. Furthermore, using the specific identification, a few HDFs derived cells were found to form lacuna structure at the peripheral area of cartilage-like tissue. In fibroblast group, the constructs deformed and shrunk gradually without mature cartilage lacuna in histology. The 3D-co-culture system can effectively induce the differentiation of HDFs to chondrocytes. The tissue-engineered cartilage can be constructed in vitro with the 3D-co-culture system.

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

    SciTech Connect

    Yan, W.Q.; Nakashima, K.; Iwamoto, M.; Kato, Y. )

    1990-06-15

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

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

    PubMed

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

    2011-01-01

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

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

    PubMed Central

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

    2011-01-01

    Osteoarthritis (OA) is characterized by degenerative changes within joints that involved quantitative and/or qualitative alterations of cartilage and synovial fluid lubricin, a mucinous glycoprotein secreted by synovial fibroblasts and chondrocytes. Modern therapeutic methods, including tissue-engineering techniques, have been used to treat mechanical damage of the articular cartilage but to date there is no specific and effective treatment. This study aimed at investigating lubricin immunohistochemical expression in cartilage explant from normal and OA patients and in cartilage constructions formed by Poly (ethylene glycol) (PEG) based hydrogels (PEG-DA) encapsulated OA chondrocytes. The expression levels of lubricin were studied by immunohistochemistry: i) in tissue explanted from OA and normal human cartilage; ii) in chondrocytes encapsulated in hydrogel PEGDA from OA and normal human cartilage. Moreover, immunocytochemical and western blot analysis were performed in monolayer cells from OA and normal cartilage. The results showed an increased expression of lubricin in explanted tissue and in monolayer cells from normal cartilage, and a decreased expression of lubricin in OA cartilage. The chondrocytes from OA cartilage after 5 weeks of culture in hydrogels (PEGDA) showed an increased expression of lubricin compared with the control cartilage. The present study demonstrated that OA chondrocytes encapsulated in PEGDA, grown in the scaffold and were able to restore lubricin biosynthesis. Thus our results suggest the possibility of applying autologous cell transplantation in conjunction with scaffold materials for repairing cartilage lesions in patients with OA to reduce at least the progression of the disease. PMID:22073377

  17. Aging and Osteoarthritis: The Role of Chondrocyte Senescence and Aging Changes in the Cartilage Matrix

    PubMed Central

    Loeser, Richard F.

    2009-01-01

    Summary Objective Age-related changes in multiple components of the musculoskeletal system may contribute to the well established link between aging and osteoarthritis (OA). This review focused on potential mechanisms by which age-related changes in the articular cartilage could contribute to the development of OA. Methods The peer-reviewed literature published prior to February 2009 in the PubMed database was searched using pre-defined search criteria. Articles, selected for their relevance to aging and articular chondrocytes or cartilage, were summarized. Results Articular chondrocytes exhibit an age-related decline in proliferative and synthetic capacity while maintaining the ability to produce pro-inflammatory mediators and matrix degrading enzymes. These findings are characteristic of the senescent secretory phenotype and are most likely a consequence of extrinsic stress-induced senescence driven by oxidative stress rather than intrinsic replicative senescence. Extracellular matrix changes with aging also contribute to the propensity to develop OA and include the accumulation of proteins modified by non-enzymatic glycation. Conclusion The effects of aging on chondrocytes and their matrix result in a tissue that is less able to maintain homeostasis when stressed, resulting in breakdown and loss of the articular cartilage, a hallmark of osteoarthritis. A better understanding of the basic mechanisms underlying senescence and how the process may be modified could provide novel ways to slow the development of osteoarthritis. PMID:19303469

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

    PubMed

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

    2016-09-01

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

  19. Calcium flux and endogenous calcium content in isolated mammalian growth-plate chondrocytes, hyaline-cartilage chondrocytes, and hepatocytes.

    PubMed

    Iannotti, J P; Brighton, C T; Stambough, J L; Storey, B T

    1985-01-01

    The role of chondrocyte mitochondria in endochondral ossification has been the subject of intensive investigation and controversy. The purpose of this study was to quantitate the endogenous calcium content and the maximum capacity for calcium accumulation and release in isolated mammalian growth-plate chondrocytes and hyaline-cartilage chondrocytes. The results indicated that the mitochondria of the isolated growth-plate and hyaline-cartilage chondrocytes possess a greater endogenous calcium content, a greater capacity for calcium accumulation, and a larger labile Ca+2 pool than do the mitochondria of hepatocytes. Growth-plate and hyaline-cartilage mitochondria had an endogenous calcium content of 908 and 142 nanomoles of Ca+2 per milligram of mitochondrial protein. The growth-plate mitochondria had a maximum calcium capacity of 5249 nanomoles of Ca+2 per milligram of mitochondrial protein. In comparison, the mitochondria of hepatocytes had a much smaller endogenous-calcium content and a smaller maximum Ca+2 capacity: twenty-one and 3262 nanomoles of Ca+2 per milligram of mitochondrial protein, respectively. The mitochondrial labile-calcium pool in both growth-plate and hyaline-cartilage chondrocytes was twofold greater than that in the mitochondria of hepatocytes. Chondrocyte mitochondria released approximately 2400 nanomoles of Ca+2 per milligram of mitochondrial protein, whereas hepatocyte mitochondria released 1200 nanomoles of Ca+2 per milligram. These results suggest that the chondrocyte mitochondria are specialized for calcium transport and are important in the calcification of the extracellular matrix of the growth plate.

  20. Use of flow cytometry to assess chondrocyte viability after laser reshaping of cartilage

    NASA Astrophysics Data System (ADS)

    Rasouli, Alexandre; Kim, Charlton C.; Basu, Reshmi; Wong, Brian J.

    2000-05-01

    Lasers have been shown to cause permanent shape change in cartilage via photothermally induced mechanical stress relaxation. While the biophysical properties of cartilage during laser irradiation have been studied, tissue viability following laser irradiation has not been fully characterized. In this study, cell viability staining and flow cytometry were used to determine chondrocyte viability following photothermal stress relaxation. Porcine septal cartilage slabs (10 X 25 X 1.5 mm) were irradiated with light from a Nd:YAG laser ((lambda) equals 1.32 micrometer, 25 W/cm2) while surface temperature, stress relaxation, and diffuse reflectance were recorded. Each slab received one, two, or three laser exposures (respective exposure times of 6.7, 7.2, 10 s), determined from measurements of diffuse reflectance, which correlate with mechanical stress relaxation. Irradiated samples were then divided into two groups analyzed immediately and at five days following laser exposure (the latter group was maintained in culture). Chondrocytes were isolated following serial enzymatic digestion with hyaluronidase, protease, and collagenase II for a total of 17 hours. Chondrocytes were then stained using SYTOR/DEAD RedTM (Molecular Probes; Eugene, OR) wherein live cells stained green (530 nm) and dead cells stained red (630 nm) when excited at 488 nm. A flow cytometer (FACScan, Becton Dickinson, Franklin Lakes, NJ) was then used to detect differential cell fluorescence; size; granularity; and the number of live cells, dead cells, and post irradiation debris in each treatment population. Nearly 60% of chondrocytes from reshaped cartilage samples isolated shortly after irradiation, were viable as determined using flow cytometry while non- irradiated controls were 100 percent viable. Specimens irradiated two or three times with the laser demonstrated increasing amounts of cellular debris along with a reduction in chondrocyte viability: 31 percent following two laser exposures, and 16

  1. Repair of articular cartilage defects in rabbits through tissue-engineered cartilage constructed with chitosan hydrogel and chondrocytes.

    PubMed

    Zhao, Ming; Chen, Zhu; Liu, Kang; Wan, Yu-qing; Li, Xu-dong; Luo, Xu-wei; Bai, Yi-guang; Yang, Ze-long; Feng, Gang

    2015-11-01

    In our previous work, we prepared a type of chitosan hydrogel with excellent biocompatibility. In this study, tissue-engineered cartilage constructed with this chitosan hydrogel and costal chondrocytes was used to repair the articular cartilage defects. Chitosan hydrogels were prepared with a crosslinker formed by combining 1,6-diisocyanatohexane and polyethylene glycol. Chitosan hydrogel scaffold was seeded with rabbit chondrocytes that had been cultured for one week in vitro to form the preliminary tissue-engineered cartilage. This preliminary tissue-engineered cartilage was then transplanted into the defective rabbit articular cartilage. There were three treatment groups: the experimental group received preliminary tissue-engineered cartilage; the blank group received pure chitosan hydrogels; and, the control group had received no implantation. The knee joints were harvested at predetermined time. The repaired cartilage was analyzed through gross morphology, histologically and immunohistochemically. The repairs were scored according to the international cartilage repair society (ICRS) standard. The gross morphology results suggested that the defects were repaired completely in the experimental group after twelve weeks. The regenerated tissue connected closely with subchondral bone and the boundary with normal tissue was fuzzy. The cartilage lacuna in the regenerated tissue was similar to normal cartilage lacuna. The results of ICRS gross and histological grading showed that there were significant differences among the three groups (P<0.05). Chondrocytes implanted in the scaffold can adhere, proliferate, and secrete extracellular matrix. The novel tissue-engineered cartilage constructed in our research can completely repair the structure of damaged articular cartilage.

  2. Chondrocyte death in mechanically injured articular cartilage--the influence of extracellular calcium.

    PubMed

    Amin, Anish K; Huntley, James S; Bush, Peter G; Simpson, A Hamish R W; Hall, Andrew C

    2009-06-01

    Calcium is thought to be an important regulator of chondrocyte death associated with articular cartilage injury. Our objective was to determine the influence of extracellular calcium on chondrocyte death following mechanical injury. Using a surgically relevant model of sharp mechanical injury (with a scalpel) and confocal laser scanning microscopy (CLSM), in situ chondrocyte death was quantified within the full thickness of articular cartilage as a function of medium calcium concentration and time (2.5 h and 7 days). Exposure of articular cartilage to calcium-free media (approximately 0 mM) significantly reduced superficial zone chondrocyte death after mechanical injury compared with exposure to calcium-rich media (2-20 mM, ANOVA at 2.5 h, p = 0.002). In calcium-rich media, although the extent of chondrocyte death increased with increasing medium calcium concentration, cell death remained localized to the superficial zone of articular cartilage over 7 days (ANOVA, p < 0.05). However, in calcium-free media, there was an increase in chondrocyte death within deeper zones of articular cartilage over 7 days. The early (within hours) chondroprotective effect in calcium-free media suggests that the use of joint irrigation solutions without added calcium may decrease chondrocyte death from mechanical injury during articular surgery. The delayed (within days) increase in chondrocyte death in calcium-free media supports the use of calcium supplementation in media used during cartilage culture for tissue engineering or transplantation.

  3. Pediatric auricular chondrocytes gene expression analysis in monolayer culture and engineered elastic cartilage.

    PubMed

    Ruszymah, B H I; Lokman, B S; Asma, A; Munirah, S; Chua, Kienhui; Mazlyzam, A L; Isa, M R; Fuzina, N H; Aminuddin, B S

    2007-08-01

    This study was aimed at regenerating autologous elastic cartilage for future use in pediatric ear reconstruction surgery. Specific attentions were to characterize pediatric auricular chondrocyte growth in a combination culture medium and to assess the possibility of elastic cartilage regeneration using human fibrin. Laboratory experiment using human pediatric auricular chondrocytes. Pediatric auricular chondrocytes growth kinetics and quantitative gene expression profile in three different types of media were compared in primary culture and subsequent three passages. Large-scale culture-expanded chondrocytes from the combination medium were then mixed with human fibrin for the formation of elastic cartilage via tissue engineering technique. The equal mixture of Ham's F12 and Dulbecco's Modified Eagle Medium (FD) promoted the best chondrocyte growth at every passage compared to the individual media. Chondrocytes differentiation index; ratio of type II to type I collagen gene expression level, aggrecan and elastin expression gradually decreased while passaging but they were then restored in engineered tissues after implantation. The engineered cartilage was glistening white in color and firm in consistency. Histological evaluation, immunohistochemistry analysis and quantitative gene expression assessment demonstrated that the engineered cartilage resemble the features of native elastic cartilage. Pediatric auricular chondrocytes proliferate better in the combination medium (FD) and the utilization of human fibrin as a biomaterial hold promises for the regeneration of an autologous elastic cartilage for future application in ear reconstructive surgery.

  4. Cartilage engineering using chondrocyte cell sheets and its application in reconstruction of microtia.

    PubMed

    Zhou, Libin; Ding, Ruiying; Li, Baowei; Han, Haolun; Wang, Hongnan; Wang, Gang; Xu, Bingxin; Zhai, Suoqiang; Wu, Wei

    2015-01-01

    The imperfections of scaffold materials have hindered the clinical application of cartilage tissue engineering. The recently developed cell-sheet technique is adopted to engineer tissues without scaffold materials, thus is considered being potentially able to overcome the problems concerning the scaffold imperfections. This study constructed monolayer and bilayer chondrocyte cell sheets and harvested the sheets with cell scraper instead of temperature-responsive culture dishes. The properties of the cultured chondrocyte cell sheets and the feasibility of cartilage engineering using the chondrocyte cell sheets was further investigated via in vitro and in vivo study. Primary extracellular matrix (ECM) formation and type II collagen expression was detected in the cell sheets during in vitro culture. After implanted into nude mice for 8 weeks, mature cartilage discs were harvested. The morphology of newly formed cartilage was similar in the constructs originated from monolayer and bilayer chondrocyte cell sheet. The chondrocytes were located within evenly distributed ovoid lacunae. Robust ECM formation and intense expression of type II collagen was observed surrounding the evenly distributed chondrocytes in the neocartilages. Biochemical analysis showed that the DNA contents of the neocartilages were higher than native human costal cartilage; while the contents of the main component of ECM, glycosaminoglycan and hydroxyproline, were similar to native human costal cartilage. In conclusion, the chondrocyte cell sheet constructed using the simple and low-cost technique is basically the same with the cell sheet cultured and harvested in temperature-responsive culture dishes, and can be used for cartilage tissue engineering.

  5. Cartilage engineering using chondrocyte cell sheets and its application in reconstruction of microtia

    PubMed Central

    Zhou, Libin; Ding, Ruiying; Li, Baowei; Han, Haolun; Wang, Hongnan; Wang, Gang; Xu, Bingxin; Zhai, Suoqiang; Wu, Wei

    2015-01-01

    The imperfections of scaffold materials have hindered the clinical application of cartilage tissue engineering. The recently developed cell-sheet technique is adopted to engineer tissues without scaffold materials, thus is considered being potentially able to overcome the problems concerning the scaffold imperfections. This study constructed monolayer and bilayer chondrocyte cell sheets and harvested the sheets with cell scraper instead of temperature-responsive culture dishes. The properties of the cultured chondrocyte cell sheets and the feasibility of cartilage engineering using the chondrocyte cell sheets was further investigated via in vitro and in vivo study. Primary extracellular matrix (ECM) formation and type II collagen expression was detected in the cell sheets during in vitro culture. After implanted into nude mice for 8 weeks, mature cartilage discs were harvested. The morphology of newly formed cartilage was similar in the constructs originated from monolayer and bilayer chondrocyte cell sheet. The chondrocytes were located within evenly distributed ovoid lacunae. Robust ECM formation and intense expression of type II collagen was observed surrounding the evenly distributed chondrocytes in the neocartilages. Biochemical analysis showed that the DNA contents of the neocartilages were higher than native human costal cartilage; while the contents of the main component of ECM, glycosaminoglycan and hydroxyproline, were similar to native human costal cartilage. In conclusion, the chondrocyte cell sheet constructed using the simple and low-cost technique is basically the same with the cell sheet cultured and harvested in temperature-responsive culture dishes, and can be used for cartilage tissue engineering. PMID:25755694

  6. Effects of osmotic challenges on membrane potential in human articular chondrocytes from healthy and osteoarthritic cartilage.

    PubMed

    Sánchez, Julio C; López-Zapata, Diego F

    2010-01-01

    Changes in external osmolarity arise from variations in mechanical loads on joints and may affect the homeostasis of chondrocytes, which are the only cell type responsible for matrix turnover. Accordingly, variations in membrane potential may affect cartilage production. The present study assessed the effects of variations in external osmolarity on membrane potential and the possible mechanisms responsible for this response. Membrane potential was measured by the patch clamp whole-cell technique using human articular chondrocytes freshly isolated from healthy and osteoarthritic cartilage. The membrane potential was -39±4 mV in articular human chondrocytes from healthy cartilage and -26±4 mV in those from osteoarthritic cartilage. Increasing the osmolarity produced a reversible hyperpolarization mediated by K+ efflux through BKCa channels in both groups of chondrocytes, but the response in osteoarthritic cells was significantly reduced; no other K+ pathways were involved in this effect. Alternatively, decreasing the osmolarity elicited depolarization in healthy chondrocytes but did not produce any response in chondrocytes from osteoarthritic cartilage. The depolarization was dependent on Na+ influx through Gd3+-sensitive stretch-activated cation channels and was independent of external Ca2+. The differential responses observed in chondrocytes from osteoarthritic cartilage suggest that disregulation on the responses to external osmolarity may be involved in the process that leads to the alterations in the cartilage structure observed in osteoarthritis.

  7. Engineering cartilage tissue by pellet coculture of chondrocytes and mesenchymal stromal cells.

    PubMed

    Wu, Ling; Post, Janine N; Karperien, Marcel

    2015-01-01

    Coculture of chondrocytes and mesenchymal stromal cells (MSCs) in pellets has been shown to be beneficial in engineering cartilage tissue in vitro. In these cultures trophic effects of MSCs increase the proliferation and matrix deposition of chondrocytes. Thus, large cartilage constructs can be made with a relatively small number of chondrocytes. In this chapter, we describe the methods for making coculture pellets of MSCs and chondrocytes. We also provide detailed protocols for analyzing coculture pellets with cell tracking, proliferation assays, species specific polymerase chain reactions (PCR), short tandem repeats analysis, and histological examination.

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

    PubMed

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

    2013-12-01

    Cartilage damage is frequently observed in advanced destructive gout. The aim of our study was to investigate the effects of monosodium urate monohydrate (MSU) crystals on chondrocyte viability and function. The alamarBlue assay and flow cytometry were used to assess the viability of primary human chondrocytes and cartilage explants following culture with MSU crystals. The number of dead chondrocytes in cartilage explants cultured with MSU crystals was quantified. Real-time PCR was used to determine changes in the relative mRNA expression levels of chondrocytic genes. The histological appearance of cartilage in joints affected by gout was also examined. MSU crystals rapidly reduced primary human chondrocyte and cartilage explant viability in a dose-dependent manner (p < 0.01 for both). Cartilage explants cultured with MSU crystals had a greater percentage of dead chondrocytes at the articular surface compared to untreated cartilage (p = 0.004). Relative mRNA expression of type II collagen and the cartilage matrix proteins aggrecan and versican was decreased in chondrocytes following culture with MSU crystals (p < 0.05 for all). However, expression of the degradative enzymes ADAMTS4 and ADAMTS5 was increased (p < 0.05 for both). In joints affected by gout, normal cartilage architecture was lost, with empty chondrocyte lacunae observed. MSU crystals have profound inhibitory effects on chondrocyte viability and function. Interactions between MSU crystals and chondrocytes may contribute to cartilage damage in gout through reduction of chondrocyte viability and promotion of a catabolic state.

  9. Characterization of auricular chondrocytes and auricular/articular chondrocyte co-cultures in terms of an application in articular cartilage repair.

    PubMed

    Kuhne, Maren; John, Thilo; El-Sayed, Karym; Marzahn, Ulrike; Aue, Annekatrin; Kohl, Benjamin; Stoelzel, Katharina; Ertel, Wolfgang; Blottner, Dieter; Haisch, Andreas; Schulze-Tanzil, Gundula

    2010-05-01

    Cartilage injury remains a challenge in orthopedic surgery as articular cartilage only has a limited capacity for intrinsic healing. Autologous chondrocyte transplantation (ACT) is a suitable technique for cartilage repair, but requires articular cartilage biopsies for autologous chondrocyte expansion. The use of heterotopic chondrocytes derived from non-articular cartilage sources such as auricular chondrocytes may be a novel approach for ACT. The aim of the study is to evaluate whether co-cultured articular/auricular chondrocytes exhibit characteristics comparable to articular chondrocytes. Analysis of the proliferation rate, extracellular cartilage matrix (ECM) gene and protein expression (type II and I collagen, elastin, lubricin), beta1-integrins and the chondrogenic transcription factor sox9 in articular/auricular chondrocytes was performed using RTD-PCR, flow cytometry, immunofluorescence microscopy and Western blot analysis. Additionally, three-dimensional (3D) chondrocyte mono- and co-cultures were established. The proliferative activity and elastin gene expression were lower and that of type II collagen and lubricin was higher in articular compared with auricular chondrocytes. The species generally did not influence the chondrocyte characteristics, with the exception of type I collagen and sox9 expression, which was higher in porcine but not in human articular chondrocytes compared with both types of auricular chondrocytes. beta1-integrin gene expression did not differ significantly between the chondrocyte types. The type II collagen gene and protein expression was higher in articular chondrocyte monocultures and was slightly higher in co-cultures compared with monocultured auricular chondrocytes. Both chondrocyte types survived in co-culture. Despite their differing expression profiles, co-cultures revealed some adjustment in the ECM expression of both chondrocyte types.

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

    PubMed

    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.

  11. Formation of cartilage in vivo with immobilized autologous rabbit auricular cultured chondrocytes in collagen matrices.

    PubMed

    Sanz, Ernesto; Peñas, Lucas; Lequerica, Juan L

    2007-05-01

    The availability of generated cartilage de novo is one of the needs of reconstructive surgery. In this study, the authors constructed a matrix formed by autologous immobilized chondrocytes using collagen gel as a scaffold. Furthermore, the ability of these matrices to engraft and generate new cartilage was examined. Biopsy specimens of elastic cartilage were surgically obtained from the ears of eight New Zealand White rabbits. After collagenase II digestion of cartilage, chondrocytes were isolated and propagated in culture medium. Chondrocytes were immobilized into bovine collagen lattices and implanted, replacing pieces of removed native cartilage. Five weeks after implantation, the rabbits were killed and the ears were examined macroscopically and analyzed by means of histochemical methods. The results show the formation of new cartilage from implanted lattices with chondrocytes. Gross analysis of the ears shows similarities in appearance, consistency, texture, and histology between native and new cartilage. Fluorescence of the nucleus from bisbenzimide-labeled chondrocytes was detected in newly formed tissue, pointing out its in vitro culture origin. No signs of an inflammatory reaction attributable to implants were found in either the control or the chondrocyte lattices. The authors suggest that this approach is of value for future clinical use.

  12. Passaged Adult Chondrocytes Can Form Engineered Cartilage with Functional Mechanical Properties: A Canine Model

    PubMed Central

    Ng, Kenneth W.; Lima, Eric G.; Bian, Liming; O'Conor, Christopher J.; Jayabalan, Prakash S.; Stoker, Aaron M.; Kuroki, Keiichi; Cook, Cristi R.; Ateshian, Gerard A.; Cook, James L.

    2010-01-01

    It was hypothesized that previously optimized serum-free culture conditions for juvenile bovine chondrocytes could be adapted to generate engineered cartilage with physiologic mechanical properties in a preclinical, adult canine model. Primary or passaged (using growth factors) adult chondrocytes from three adult dogs were encapsulated in agarose, and cultured in serum-free media with transforming growth factor-β3. After 28 days in culture, engineered cartilage formed by primary chondrocytes exhibited only small increases in glycosaminoglycan content. However, all passaged chondrocytes on day 28 elaborated a cartilage matrix with compressive properties and glycosaminoglycan content in the range of native adult canine cartilage values. A preliminary biocompatibility study utilizing chondral and osteochondral constructs showed no gross or histological signs of rejection, with all implanted constructs showing excellent integration with surrounding cartilage and subchondral bone. This study demonstrates that adult canine chondrocytes can form a mechanically functional, biocompatible engineered cartilage tissue under optimized culture conditions. The encouraging findings of this work highlight the potential for tissue engineering strategies using adult chondrocytes in the clinical treatment of cartilage defects. PMID:19845465

  13. Xiphoid process-derived chondrocytes: a novel cell source for elastic cartilage regeneration.

    PubMed

    Nam, Seungwoo; Cho, Wheemoon; Cho, Hyunji; Lee, Jungsun; Lee, EunAh; Son, Youngsook

    2014-11-01

    Reconstruction of elastic cartilage requires a source of chondrocytes that display a reliable differentiation tendency. Predetermined tissue progenitor cells are ideal candidates for meeting this need; however, it is difficult to obtain donor elastic cartilage tissue because most elastic cartilage serves important functions or forms external structures, making these tissues indispensable. We found vestigial cartilage tissue in xiphoid processes and characterized it as hyaline cartilage in the proximal region and elastic cartilage in the distal region. Xiphoid process-derived chondrocytes (XCs) showed superb in vitro expansion ability based on colony-forming unit fibroblast assays, cell yield, and cumulative cell growth. On induction of differentiation into mesenchymal lineages, XCs showed a strong tendency toward chondrogenic differentiation. An examination of the tissue-specific regeneration capacity of XCs in a subcutaneous-transplantation model and autologous chondrocyte implantation model confirmed reliable regeneration of elastic cartilage regardless of the implantation environment. On the basis of these observations, we conclude that xiphoid process cartilage, the only elastic cartilage tissue source that can be obtained without destroying external shape or function, is a source of elastic chondrocytes that show superb in vitro expansion and reliable differentiation capacity. These findings indicate that XCs could be a valuable cell source for reconstruction of elastic cartilage. ©AlphaMed Press.

  14. Xiphoid Process-Derived Chondrocytes: A Novel Cell Source for Elastic Cartilage Regeneration

    PubMed Central

    Nam, Seungwoo; Cho, Wheemoon; Cho, Hyunji; Lee, Jungsun

    2014-01-01

    Reconstruction of elastic cartilage requires a source of chondrocytes that display a reliable differentiation tendency. Predetermined tissue progenitor cells are ideal candidates for meeting this need; however, it is difficult to obtain donor elastic cartilage tissue because most elastic cartilage serves important functions or forms external structures, making these tissues indispensable. We found vestigial cartilage tissue in xiphoid processes and characterized it as hyaline cartilage in the proximal region and elastic cartilage in the distal region. Xiphoid process-derived chondrocytes (XCs) showed superb in vitro expansion ability based on colony-forming unit fibroblast assays, cell yield, and cumulative cell growth. On induction of differentiation into mesenchymal lineages, XCs showed a strong tendency toward chondrogenic differentiation. An examination of the tissue-specific regeneration capacity of XCs in a subcutaneous-transplantation model and autologous chondrocyte implantation model confirmed reliable regeneration of elastic cartilage regardless of the implantation environment. On the basis of these observations, we conclude that xiphoid process cartilage, the only elastic cartilage tissue source that can be obtained without destroying external shape or function, is a source of elastic chondrocytes that show superb in vitro expansion and reliable differentiation capacity. These findings indicate that XCs could be a valuable cell source for reconstruction of elastic cartilage. PMID:25205841

  15. The use of de-differentiated chondrocytes delivered by a heparin-based hydrogel to regenerate cartilage in partial-thickness defects.

    PubMed

    Kim, Mihye; Kim, Se Eun; Kang, Seong Soo; Kim, Young Ha; Tae, Giyoong

    2011-11-01

    Partial-thickness cartilage defects, with no subchondral bone injury, do not repair spontaneously, thus there is no clinically effective treatment for these lesions. Although the autologous chondrocyte transplantation (ACT) is one of the promising approaches for cartilage repair, it requires in vitro cell expansion to get sufficient cells, but chondrocytes lose their chondrogenic phenotype during expansion by monolayer culture, leading to de-differentiation. In this study, a heparin-based hydrogel was evaluated and optimized to induce cartilage regeneration with de-differentiated chondrocytes. First, re-differentiation of de-differentiated chondrocytes encapsulated in heparin-based hydrogels was characterized in vitro with various polymer concentrations (from 3 to 20 wt.%). Even under a normal cell culture condition (no growth factors or chondrogenic components), efficient re-differentiation of cells was observed with the optimum at 10 wt.% hydrogel, showing the complete re-differentiation within a week. Efficient re-differentiation and cartilage formation of de-differentiated cell/hydrogel construct were also confirmed in vivo by subcutaneous implantation on the back of nude mice. Finally, excellent cartilage regeneration and good integration with surrounding, similar to natural cartilage, was also observed by delivering de-differentiated chondrocytes using the heparin-based hydrogel in partial-thickness defects of rabbit knees whereas no healing was observed for the control defects. These results demonstrate that the heparin-based hydrogel is very efficient for re-differentiation of expanded chondrocytes and cartilage regeneration without using any exogenous inducing factors, thus it could serve as an injectable cell-carrier and scaffold for cartilage repair. Excellent chondrogenic nature of the heparin-based hydrogel might be associated with the hydrogel characteristic that can secure endogenous growth factors secreted from chondrocytes, which then can promote

  16. The potential of human allogeneic juvenile chondrocytes for restoration of articular cartilage.

    PubMed

    Adkisson, H Davis; Martin, James A; Amendola, Richard L; Milliman, Curt; Mauch, Kelsey A; Katwal, Arbindra B; Seyedin, Mitchell; Amendola, Annuziato; Streeter, Philip R; Buckwalter, Joseph A

    2010-07-01

    Donor-site morbidity, limited numbers of cells, loss of phenotype during ex vivo expansion, and age-related decline in chondrogenic activity present critical obstacles to the use of autologous chondrocyte implantation for cartilage repair. Chondrocytes from juvenile cadaveric donors may represent an alternative to autologous cells. Hypothesis/ The authors hypothesized that juvenile chondrocyte would show stronger and more stable chondrogenic activity than adult cells in vitro and that juvenile cells pose little risk of immunologic incompatibility in adult hosts. Controlled laboratory study. Cartilage samples were from juvenile (<13 years old) and adult (>13 years old) donors. The chondrogenic activity of freshly isolated human articular chondrocytes and of expanded cells after monolayer culture was measured by proteoglycan assay, gene expression analysis, and histology. Lymphocyte proliferation assays were used to assess immunogenic activity. Proteoglycan content in neocartilage produced by juvenile chondrocytes was 100-fold higher than in neocartilage produced by adult cells. Collagen type II and type IX mRNA in fresh juvenile chondrocytes were 100- and 700-fold higher, respectively, than in adult chondrocytes. The distributions of collagens II and IX were similar in native juvenile cartilage and in neocartilage made by juvenile cells. Juvenile cells grew significantly faster in monolayer cultures than adult cells (P = .002) and proteoglycan levels produced in agarose culture was significantly higher in juvenile cells than in adult cells after multiple passages (P < .001). Juvenile chondrocytes did not stimulate lymphocyte proliferation. These results document a dramatic age-related decline in human chondrocyte chondrogenic potential and show that allogeneic juvenile chondrocytes do not stimulate an immunologic response in vivo. Juvenile human chondrocytes have greater potential to restore articular cartilage than adult cells, and may be transplanted without the

  17. THE POTENTIAL OF HUMAN ALLOGENEIC JUVENILE CHONDROCYTES FOR RESTORATION OF ARTICULAR CARTILAGE

    PubMed Central

    Adkisson, H. Davis; Martin, James A.; Amendola, Richard L.; Milliman, Curt; Mauch, Kelsey A.; Katwal, Arbindra B.; Seyedin, Mitchell; Amendola, Annuziato; Streeter, Philip R.; Buckwalter, Joseph A.

    2013-01-01

    Background Donor site morbidity, limited numbers of cells, loss of phenotype during ex vivo expansion, and age-related decline in chondrogenic activity present critical obstacles to the use of autologous chondrocyte implantation for cartilage repair. Chondrocytes from juvenile cadaveric donors may represent an alternative to autologous cells. Hypothesis/Purpose The authors hypothesized that juvenile chondrocyte would show stronger and more stable chondrogenic activity than adult cells in vitro and that juvenile cells pose little risk of immunologic incompatibility in adult hosts. Study Design Controlled laboratory study. Methods Cartilage samples were from juvenile (<13 years old) and adult (> 13 years old) donors. The chondrogenic activity of freshly isolated human articular chondrocytes and of expanded cells after monolayer culture was measured by proteoglycan assay, gene expression analysis, and histology. Lymphocyte proliferation assays were used to assess immunogenic activity. Results Proteoglycan content in neocartilage produced by juvenile chondrocytes was 100-fold higher than in neocartilage produced by adult cells. Collagen type II and type IX mRNAs in fresh juvenile chondrocytes were 100- and 700-fold higher, respectively, than in adult chondrocytes. The distributions of collagens II and IX were similar in native juvenile cartilage and in neocartilage made by juvenile cells. Juvenile cells grew significantly faster in monolayer cultures than adult cells (p = 0.002) and proteoglycan levels produced in agarose culture was significantly higher in juvenile cells than in adult cells after multiple passages (p < 0.001). Juvenile chondrocytes did not stimulate lymphocyte proliferation. Conclusions These results document a dramatic age related decline in human chondrocyte chondrogenic potential and show that allogeneic juvenile chondrocytes do not stimulate an immunologic response in vivo. Clinical Relevance Juvenile human chondrocytes have greater potential to

  18. Role of insulin-transferrin-selenium in auricular chondrocyte proliferation and engineered cartilage formation in vitro.

    PubMed

    Liu, Xia; Liu, Jinchun; Kang, Ning; Yan, Li; Wang, Qian; Fu, Xin; Zhang, Yuanyuan; Xiao, Ran; Cao, Yilin

    2014-01-21

    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.

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

    PubMed

    Karsdal, Morten Asser; Sumer, Eren Ufuk; Wulf, Helle; Madsen, Suzi H; Christiansen, Claus; Fosang, Amanda J; Sondergaard, Bodil-Cecilie

    2007-05-01

    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. 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 was assessed by 1) quantification of C-terminal crosslinking telopeptide of type II collagen fragments (CTX-II), 2) matrix metalloproteinase (MMP)-mediated aggrecan degradation by (342)FFGV- G2 assay, 3) aggrecanase-mediated degradation by (374)ARGS-G2 assay, 4) release of sulfated glycosaminoglycans (sGAG) into culture medium, 5) immunohistochemistry with a monoclonal antibody recognizing the CTX-II epitope, and 6) toluidine blue staining of proteoglycans. MMP expression and activity were assessed by gelatin zymography. OSM and TNF induced an 8,000% increase in CTX-II compared with control (P < 0.001). Both forskolin and IBMX dose-dependently inhibited release of CTX-II (P < 0.001). OSM and TNF induced a 6-fold increase in (342)FFGV-G2, which was abrogated by forskolin and IBMX (by >80%). OSM and TNF stimulated MMP expression as visualized by zymography, and MMP expression was dose-dependently inhibited by forskolin and IBMX. The highest concentration of IBMX lowered cytokine-induced release of sGAG by 72%. 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 strongly inhibited cartilage degradation. Specific cAMP modulators in chondrocytes may be potential treatments for cartilage degenerative diseases.

  20. Biocompatibility of polysebacic anhydride microparticles with chondrocytes in engineered cartilage

    PubMed Central

    Ponnurangam, Sathish; O'Connell, Grace D.; Hung, Clark T.; Somasundaran, Ponisseril

    2015-01-01

    One of main challenges in developing clinically relevant engineered cartilage is overcoming limited nutrient diffusion due to progressive elaboration of extracellular matrix at the periphery of the construct. Macro-channels have been used to decrease the nutrient path-length; however, the channels become occluded with matrix within weeks in culture, reducing nutrient diffusion. Alternatively, microparticles can be imbedded throughout the scaffold to provide localized nutrient delivery. In this study, we evaluated biocompatibility of polysebacic anhydride (PSA) polymers and the effectiveness of PSA-based microparticles for short-term delivery of nutrients in engineered cartilage. PSA-based microparticles were biocompatible with juvenile bovine chondrocytes for concentrations up to 2mg/mL; however, cytotoxicity was observed at 20mg/mL. Cytotoxicity at high concentrations is likely due to intracellular accumulation of PSA degradation products and resulting lipotoxicity. Cytotoxicity of PSA was partially reversed in the presence of bovine serum albumin. In conclusion, the findings from this study demonstrate concentration-dependent biocompatibility of PSA-based microparticles and potential application as a nutrient delivery vehicle that can be imbedded in scaffolds for tissue engineering. PMID:26398146

  1. Karyotyping of human chondrocytes in scaffold-assisted cartilage tissue engineering.

    PubMed

    Trimborn, Marc; Endres, Michaela; Bommer, Christiane; Janke, Una; Krüger, Jan-Philipp; Morawietz, Lars; Kreuz, Peter C; Kaps, Christian

    2012-04-01

    Scaffold-assisted autologous chondrocyte implantation (ACI) is an effective clinical procedure for cartilage repair. The aim of our study was to evaluate the chromosomal stability of human chondrocytes subjected to typical cell culture procedures needed for regenerative approaches in polymer-scaffold-assisted cartilage repair. Chondrocytes derived from post mortem donors and from donors scheduled for ACI were expanded, cryopreserved and re-arranged in polyglycolic acid (PGA)-fibrin scaffolds for tissue culture. Chondrocyte redifferentiation was analyzed by electron microscopy, histology and gene expression analysis. Karyotyping was performed using GTG banding and fluorescence in situ hybridization on a single cell basis. Chondrocytes showed de- and redifferentiation accompanied by the formation of extracellular matrix and induction of typical chondrocyte marker genes like type II collagen in PGA-fibrin scaffolds. Post mortem chondrocytes showed up to 1.7% structural and high numbers of numerical (up to 26.7%) chromosomal aberrations, while chondrocytes from living donors scheduled for ACI showed up to 1.8% structural and up to 1.3% numerical alterations. Cytogenetically, cell culture procedures and PGA-fibrin scaffolds did not significantly alter chromosomal integrity of the chondrocyte genome. Human chondrocytes derived from living donors subjected to regenerative medicine cell culture procedures like cell expansion, cryopreservation and culture in resorbable polymer-based scaffolds show normal chromosomal integrity and normal karyotypes. Copyright © 2011 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

  2. Biomechanical study of the edge outgrowth phenomenon of encapsulated chondrocytic isogenous groups in the surface layer of hydrogel scaffolds for cartilage tissue engineering.

    PubMed

    Ng, Soon Seng; Su, Kai; Li, Chuan; Chan-Park, Mary B; Wang, Dong-An; Chan, Vincent

    2012-01-01

    In cartilage tissue engineering, hydrogel is widely used as the scaffold for hosting and culturing chondrocyte suspension during neo-tissue formation. In order to develop cultured chondrocytes into a functional cartilage equivalent, the hydrogel must provide an ideal microenvironment for the rapidly proliferating chondrocytes. At the same time, the essential functions of chondrocytes, such as the secretion of type II collagen and glycosaminoglycans, must be maintained. In these studies, we quantitatively characterize the mechanobiology underlying a newly discovered "edge flourish" phenomenon of cultured chondrocytes within a three-dimensional agarose hydrogel, which may ultimately nurture scaffold-free cartilaginous tissue regeneration. First, real-time microscopy was used to track the spatiotemporal distributions of chondrocytes at different focal planes. The chondrocytes were observed to exhibit abundant neo-tissue outgrowth and significant cartilaginous phenotype at the edge of the hydrogel compared to those inside the hydrogel bulk. Secondly, the hydrogel surface stresses induced by the encapsulated chondrocytes were characterized quantitatively in real time using the finite-element method. Finally, the real-time three-dimensional matrix deformations of agarose hydrogel under the influence of chondrocytes were measured using a multiple-particle tracking assay. Our results indicate that the mechanism of the "edge flourish" phenomenon is induced by the oriented outgrowth of chondrocytic isogenous groups located at the edge of hydrogel. These isogenous groups exhibit directed outgrowth towards the surface of the hydrogel and eventually generate substantial surface tension on the interface of hydrogel and medium. Ultimately, the encapsulated chondrocytes closest to the hydrogel/medium interface will spontaneously sprout out of the hydrogel and form a layer of rich proliferative and chondrocytic extracellular matrix secreting chondrocytes at the surface of the

  3. Induction and characterization of metallothionein in chicken epiphyseal growth plate cartilage chondrocytes.

    PubMed

    Sauer, G R; Nie, D; Wu, L N; Wuthier, R E

    1998-01-01

    Following exposure to cadmium or zinc, chickens were sacrificed and the liver, kidney, and bone epiphyseal growth plates harvested. When cytosolic extracts of the growth plate cartilage were fractionated by gel filtration chromatography, a protein with high metal-binding capacity and low ultraviolet (UV) absorbance eluted in the same position as liver metallothionein (MT) and a MT standard. Cd or Zn treatment resulted in a 25-fold or 5-fold induction in growth plate MT, respectively. In liver the greatest level of MT induction was seen with short-term Cd exposures. In contrast, MT levels in the growth plate increased as the duration of Cd exposure increased. Induction of MT in growth plate chondrocyte cell cultures was observed for media Cd concentrations of > or = 0.1 microM and Zn concentrations of > or = 100 microM. Basal and inducible levels of MT declined through the culture period and were lowest in the terminally differentiated mineralized late stages of the culture. Alkaline phosphatase activity was also lowest in the late-stage cultures, while total cellular protein increased throughout the culture period. Treatment of chondrocytes with Zn prior to Cd exposure resulted in a protective induction of MT. Pre-treatment of chondrocytes with dexamethasone resulted in suppressed synthesis of MT upon Cd exposure and greater Cd toxicity. Both Cd and Zn resulted in significantly increased levels of MT mRNA in chondrocyte cell cultures. Dexamethasone treatment resulted in an approximate 2- to 3-fold increase in MT mRNA. This is contrary to the finding that MT protein levels were decreased by dexamethasone. The findings suggest that an increased rate of MT degradation in dexamethasone-treated and late-stage chondrocyte cultures may be associated with the terminally differentiated phenotype.

  4. Chondrocyte differentiation for auricular cartilage reconstruction using a chitosan based hydrogel.

    PubMed

    García-López, J; Garciadiego-Cázares, D; Melgarejo-Ramírez, Y; Sánchez-Sánchez, R; Solís-Arrieta, L; García-Carvajal, Z; Sánchez-Betancourt, J I; Ibarra, C; Luna-Bárcenas, G; Velasquillo, C

    2015-12-01

    Tissue engineering with the use of biodegradable and biocompatible scaffolds is an interesting option for ear repair. Chitosan-Polyvinyl alcohol-Epichlorohydrine hydrogel (CS-PVA-ECH) is biocompatible and displays appropriate mechanical properties to be used as a scaffold. The present work, studies the potential of CS-PVA-ECH scaffolds seeded with chondrocytes to develop elastic cartilage engineered-neotissues. Chondrocytes isolated from rabbit and swine elastic cartilage were independently cultured onto CS-PVA-ECH scaffolds for 20 days to form the appropriate constructs. Then, in vitro cell viability and morphology were evaluated by calcein AM and EthD-1 assays and Scanning Electron Microscopy (SEM) respectively, and the constructs were implanted in nu/nu mice for four months, in order to evaluate the neotissue formation. Histological analysis of the formed neotissues was performed by Safranin O, Toluidine blue (GAG's), Verhoeff-Van Gieson (elastic fibers), Masson's trichrome (collagen) and Von Kossa (Calcium salts) stains and SEM. Results indicate appropriate cell viability, seeded with rabbit or swine chondrocyte constructs; nevertheless, upon implantation the constructs developed neotissues with different characteristics depending on the animal species from which the seeded chondrocytes came from. Neotissues developed from swine chondrocytes were similar to auricular cartilage, while neotissues from rabbit chondrocytes were similar to hyaline cartilage and eventually they differentiate to bone. This result suggests that neotissue characteristics may be influenced by the animal species source of the chondrocytes isolated.

  5. 3D Hydrogel Scaffolds for Articular Chondrocyte Culture and Cartilage Generation

    PubMed Central

    Yang, Fan; Bhutani, Nidhi

    2015-01-01

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

  6. Prolactin inhibits the apoptosis of chondrocytes induced by serum starvation.

    PubMed

    Zermeño, C; Guzmán-Morales, J; Macotela, Y; Nava, G; López-Barrera, F; Kouri, J B; Lavalle, C; de la Escalera, G Martínez; Clapp, C

    2006-05-01

    The apoptosis of chondrocytes plays an important role in endochondral bone formation and in cartilage degradation during aging and disease. Prolactin (PRL) is produced in chondrocytes and is known to promote the survival of various cell types. Here we show that articular chondrocytes from rat postpubescent and adult cartilage express the long form of the PRL receptor as revealed by immunohistochemistry of cartilage sections and by RT-PCR and Western blot analyses of the isolated chondrocytes. Furthermore, we demonstrate that PRL inhibits the apoptosis of these same chondrocytes cultured in low-serum. Chondrocyte apoptosis was measured by hypodiploid DNA content determined by flow cytometry and by DNA fragmentation evaluated by the ELISA and the TUNEL methods. The anti-apoptotic effect of PRL was dose-dependent and was prevented by heat inactivation. These data demonstrate that PRL can act as a survival factor for chondrocytes and that it has potential preventive and therapeutic value in arthropathies characterized by cartilage degradation.

  7. Capability of Cartilage Extract to In Vitro Differentiation of Rat Mesenchymal Stem Cells (MSCs) to Chondrocyte Lineage.

    PubMed

    Talakoob, Setareh; Joghataei, Mohammad Taghi; Parivar, Kazem; Bananej, Maryam; Sanadgol, Nima

    2015-01-01

    The importance of mesenchymal stem cells (MSCs), as adult stem cells (ASCs) able to divide into a variety of different cells is of utmost importance for stem cell researches. In this study, the ability of cartilage extract to induce differentiation of rat derived omentum tissue MSCs (rOT-MSCs) into chondrocyte cells (CCs) was investigated. After isolation of rOT-MSCs, they were co-cultured with different concentrations of hyaline cartilage extract and chondrocyte differentiation was monitored. Expression of MSCs markers was analyzed via flow cytometry. Moreover, expression of octamer- binding transcription factor-4 (Oct-4), Wilm's tumor suppressor gene-1 (WT-1), aggrecan (AG), collagen type-II (CT-II) and collagen type-X (CT-X) was analyzed using RT-PCR on 16, 18 and 21 days. Furthermore, immunocytochemistry and western blot were performed for CT-II production. Finally, proteoglycans (PGs) were examined using toluidine blue and alcian blue staining. The phenotypic characterization revealed the positive expression of CD90, CD44 and negative expression of CD45 in rOT-MSCs. These cells also expressed mRNA of Oct-4 and WT-1 as markers of omentum tissue. Differentiated rOT-MSCs in the presence of 20 µg/ ml cartilage extract expressed AG, CT-II, CT-X, and PGs as specific markers of CCs. These observations suggest that cartilage extract is potentially able to induce differentiation of MSCs into chondrocyte lineage and may be considered as an available source for imposing tissue healing on the damaged cartilage. More investigations are needed to prove in vivo cartilage repair via cartilage extract or its effective factors.

  8. Capability of Cartilage Extract to In Vitro Differentiation of Rat Mesenchymal Stem Cells (MSCs) to Chondrocyte Lineage

    PubMed Central

    Talakoob, Setareh; Joghataei, Mohammad Taghi; Parivar, Kazem; Bananej, Maryam; Sanadgol, Nima

    2015-01-01

    The importance of mesenchymal stem cells (MSCs), as adult stem cells (ASCs) able to divide into a variety of different cells is of utmost importance for stem cell researches. In this study, the ability of cartilage extract to induce differentiation of rat derived omentum tissue MSCs (rOT-MSCs) into chondrocyte cells (CCs) was investigated. After isolation of rOT-MSCs, they were co-cultured with different concentrations of hyaline cartilage extract and chondrocyte differentiation was monitored. Expression of MSCs markers was analyzed via flow cytometry. Moreover, expression of octamer- binding transcription factor-4 (Oct-4), Wilm's tumor suppressor gene-1 (WT-1), aggrecan (AG), collagen type-II (CT-II) and collagen type-X (CT-X) was analyzed using RT-PCR on 16, 18 and 21 days. Furthermore, immunocytochemistry and western blot were performed for CT-II production. Finally, proteoglycans (PGs) were examined using toluidine blue and alcian blue staining. The phenotypic characterization revealed the positive expression of CD90, CD44 and negative expression of CD45 in rOT-MSCs. These cells also expressed mRNA of Oct-4 and WT-1 as markers of omentum tissue. Differentiated rOT-MSCs in the presence of 20 µg/ ml cartilage extract expressed AG, CT-II, CT-X, and PGs as specific markers of CCs. These observations suggest that cartilage extract is potentially able to induce differentiation of MSCs into chondrocyte lineage and may be considered as an available source for imposing tissue healing on the damaged cartilage. More investigations are needed to prove in vivo cartilage repair via cartilage extract or its effective factors. PMID:25815278

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

    PubMed

    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.

  10. Repair of experimentally produced defects in rabbit articular cartilage by autologous chondrocyte transplantation

    SciTech Connect

    Grande, D.A.; Pitman, M.I.; Peterson, L.; Menche, D.; Klein, M.

    1989-01-01

    Using the knee joints of New Zealand White rabbits, a baseline study was made to determine the intrinsic capability of cartilage for healing defects that do not fracture the subchondral plate. A second experiment examined the effect of autologous chondrocytes grown in vitro on the healing rate of these defects. To determine whether any of the reconstituted cartilage resulted from the chondrocyte graft, a third experiment was conducted involving grafts with chondrocytes that had been labeled prior to grafting with a nuclear tracer. Results were evaluated using both qualitative and quantitative light microscopy. Macroscopic results from grafted specimens displayed a marked decrease in synovitis and other degenerative changes. In defects that had received transplants, a significant amount of cartilage was reconstituted (82%) compared to ungrafted controls (18%). Autoradiography on reconstituted cartilage showed that there were labeled cells incorporated into the repair matrix.

  11. Enhancing Chondrogenic Phenotype for Cartilage Tissue Engineering: Monoculture and Coculture of Articular Chondrocytes and Mesenchymal Stem Cells

    PubMed Central

    Hubka, Kelsea M.; Dahlin, Rebecca L.; Meretoja, Ville V.; Kasper, F. Kurtis

    2014-01-01

    Articular cartilage exhibits an inherently low rate of regeneration. Consequently, damage to articular cartilage often requires surgical intervention. However, existing treatments generally result in the formation of fibrocartilage tissue, which is inferior to native articular cartilage. As a result, cartilage engineering strategies seek to repair or replace damaged cartilage with an engineered tissue that restores full functionality to the impaired joint. These strategies often involve the use of chondrocytes, yet in vitro expansion and culture can lead to undesirable changes in chondrocyte phenotype. This review focuses on the use of articular chondrocytes and mesenchymal stem cells (MSCs) in either monoculture or coculture for the enhancement of chondrogenesis. Coculture strategies increasingly outperform their monoculture counterparts with regard to chondrogenesis and present unique opportunities to attain chondrocyte phenotype stability in vitro. Methods to prevent chondrocyte dedifferentiation and promote chondrocyte redifferentiation as well as to promote the chondrogenic differentiation of MSCs while preventing MSC hypertrophy are discussed. PMID:24834484

  12. Enhancing chondrogenic phenotype for cartilage tissue engineering: monoculture and coculture of articular chondrocytes and mesenchymal stem cells.

    PubMed

    Hubka, Kelsea M; Dahlin, Rebecca L; Meretoja, Ville V; Kasper, F Kurtis; Mikos, Antonios G

    2014-12-01

    Articular cartilage exhibits an inherently low rate of regeneration. Consequently, damage to articular cartilage often requires surgical intervention. However, existing treatments generally result in the formation of fibrocartilage tissue, which is inferior to native articular cartilage. As a result, cartilage engineering strategies seek to repair or replace damaged cartilage with an engineered tissue that restores full functionality to the impaired joint. These strategies often involve the use of chondrocytes, yet in vitro expansion and culture can lead to undesirable changes in chondrocyte phenotype. This review focuses on the use of articular chondrocytes and mesenchymal stem cells (MSCs) in either monoculture or coculture for the enhancement of chondrogenesis. Coculture strategies increasingly outperform their monoculture counterparts with regard to chondrogenesis and present unique opportunities to attain chondrocyte phenotype stability in vitro. Methods to prevent chondrocyte dedifferentiation and promote chondrocyte redifferentiation as well as to promote the chondrogenic differentiation of MSCs while preventing MSC hypertrophy are discussed.

  13. Cartilage-specific deletion of Mig-6 results in osteoarthritis-like disorder with excessive articular chondrocyte proliferation.

    PubMed

    Staal, Ben; Williams, Bart O; Beier, Frank; Vande Woude, George F; Zhang, Yu-Wen

    2014-02-18

    A deficiency of mitogen-inducible gene-6 (Mig-6) in mice leads to the development of an early-onset, osteoarthritis (OA)-like disorder in multiple synovial joints, underlying its importance in maintaining joint homeostasis. Here we determined what joint tissues Mig-6 is expressed in and what role chondrocytes play in the Mig-6-deficient OA-like disorder. A Mig-6/lacZ reporter mouse strain expressing β-galactosidase under the control of the Mig-6 gene promoter was generated to determine Mig-6 expression in joint tissues. By β-galactosidase staining, we demonstrated that Mig-6 was uniquely expressed in the cells across the entire surface of the synovial joint cavity, including chondrocytes in the superficial zone of articular cartilage and in the meniscus, as well as synovial lining cells. By crossing Mig-6-floxed mice to Col2a1-Cre transgenic mice, to generate cartilage-specific deletion of Mig-6, we demonstrated that deficiency of Mig-6 in the chondrocytes results in a joint phenotype that only partially recapitulates the OA-like disorder of the Mig-6-deficient mice: Ubiquitous deletion of Mig-6 led to the OA-like disorder in multiple joints, whereas cartilage-specific deletion affected the knees but rarely other joints. Furthermore, chondrocytes with Mig-6 deficiency showed excessive proliferative activities along with enhanced EGF receptor signaling in the articular cartilage and in the abnormally formed osteophytes. Our findings provide insight into the crucial requirement for Mig-6 in maintaining joint homeostasis and in regulating chondrocyte activities in the synovial joints. Our data also suggest that other cell types are required for fully developing the Mig-6-deficient OA-like disorder.

  14. Cartilage-specific deletion of Mig-6 results in osteoarthritis-like disorder with excessive articular chondrocyte proliferation

    PubMed Central

    Staal, Ben; Williams, Bart O.; Beier, Frank; Vande Woude, George F.; Zhang, Yu-Wen

    2014-01-01

    A deficiency of mitogen-inducible gene-6 (Mig-6) in mice leads to the development of an early-onset, osteoarthritis (OA)-like disorder in multiple synovial joints, underlying its importance in maintaining joint homeostasis. Here we determined what joint tissues Mig-6 is expressed in and what role chondrocytes play in the Mig-6–deficient OA-like disorder. A Mig-6/lacZ reporter mouse strain expressing β-galactosidase under the control of the Mig-6 gene promoter was generated to determine Mig-6 expression in joint tissues. By β-galactosidase staining, we demonstrated that Mig-6 was uniquely expressed in the cells across the entire surface of the synovial joint cavity, including chondrocytes in the superficial zone of articular cartilage and in the meniscus, as well as synovial lining cells. By crossing Mig-6–floxed mice to Col2a1-Cre transgenic mice, to generate cartilage-specific deletion of Mig-6, we demonstrated that deficiency of Mig-6 in the chondrocytes results in a joint phenotype that only partially recapitulates the OA-like disorder of the Mig-6–deficient mice: Ubiquitous deletion of Mig-6 led to the OA-like disorder in multiple joints, whereas cartilage-specific deletion affected the knees but rarely other joints. Furthermore, chondrocytes with Mig-6 deficiency showed excessive proliferative activities along with enhanced EGF receptor signaling in the articular cartilage and in the abnormally formed osteophytes. Our findings provide insight into the crucial requirement for Mig-6 in maintaining joint homeostasis and in regulating chondrocyte activities in the synovial joints. Our data also suggest that other cell types are required for fully developing the Mig-6–deficient OA-like disorder. PMID:24550287

  15. Cotransplantation of autologous bone marrow stromal cells and chondrocytes as a novel therapy for reconstruction of condylar cartilage.

    PubMed

    Dai, Jiewen; Wang, Xudong; Shen, Guofang

    2011-07-01

    Condylar cartilage is absolutely necessary for the normal function of temporomandibular joint (TMJ). Unfortunately, condylar cartilage defect or missing is also one of the common clinical problems. Repair or reconstruction of cartilage is always a hot topic. Cell based cartilage regeneration is suggested as novel therapies in cartilage tissue engineering, and autologous chondrocytes were initially regarded as the ideal cell source. However, there are some disadvantages such as its limited augmentation capability for culture in vitro and may differentiate to other types of cells. On the other hand, bone marrow stromal cells (BMSCs) have gained special interest in tissue engineering. Because they can be obtained easily, cause relatively minor trauma and show the potential of long-run ex vivo expansion capacity. What most important is their capacity of multi-directional differentiation. They can differentiate into a variety of other types of cells when there are supplement exogenous factors or genes, but their clinical use is limited by safety concerns such as toxicity, insertional teratogenic, uncontrollable gene expression. Fortunately, the chondrocytes microenvironment has been demonstrated that could induce BMSCs to structure cartilage when culture in vitro or reimplanted in nude mice subcutaneously area. So in this article, we hypothesize that cotransplantation of autologous BMSCs and chondrocytes, which coculture with extracellular scaffolds, is a novel therapy for reconstruction of TMJ condylar cartilage. In our strategy, advantages of two types of cells are utilized and shortcomings are avoided, which strongly improve the feasibility and clinical safety, finally bring great hope to the patients with TMJ disease. Copyright © 2011 Elsevier Ltd. All rights reserved.

  16. Calcitonin attenuates cartilage degeneration and nociception in an experimental rat model of osteoarthritis: role of TGF-β in chondrocytes

    PubMed Central

    Wen, Zhi-Hong; Tang, Chi-Chieh; Chang, Yi-Chen; Huang, Shi-Ying; Lin, Yen-You; Hsieh, Shih-Peng; Lee, Hsin-Pai; Lin, Sung-Chun; Chen, Wu-Fu; Jean, Yen-Hsuan

    2016-01-01

    We investigated the role of the calcitonin (Miacalcin) in the progression of osteoarthritis (OA) and in nociceptive behavior in an experimental rat model of OA and osteoporosis. OA was induced by anterior cruciate ligament transection (ACLT) of the right knee and by bilateral ovariectomy (OVX) in Wistar rats. Nociceptive behaviors (secondary mechanical allodynia and weight-bearing distribution of the hind paws) were analyzed prior to surgery and every week, beginning at 12 weeks after surgery, up to 20 weeks. At 20 weeks, histopathological studies were performed on the cartilage of the knee joints. Immunohistochemical analysis was performed to examine the effect of calcitonin on transforming growth factor (TGF)-β1 expression in articular cartilage chondrocytes. Rats subjected to ACLT + OVX surgery showed obvious OA changes in the joints. Animals subjected to ACLT + OVX and treated with calcitonin showed significantly less cartilage degeneration and improved nociceptive tests compared with animals subjected to ACLT + OVX surgeries alone. Moreover, calcitonin increased TGF-β1 expression in chondrocytes in ACLT + OVX-affected cartilage. Subcutaneous injection of calcitonin (1) attenuated the development of OA, (2) concomitantly reduced nociception, and (3) modulated chondrocyte metabolism, possibly by increasing cellular TGF-β1 expression. PMID:27345362

  17. Cartilage-specific β-CATENIN signaling regulates chondrocyte maturation, generation of ossification centers, and perichondrial bone formation during skeletal development

    PubMed Central

    Dao, Debbie Y.; Jonason, Jennifer H.; Zhang, Yongchun; Hsu, Wei; Chen, Di; Hilton, Matthew J.; O’Keefe, Regis J.

    2012-01-01

    The WNT/β-CATENIN signaling pathway is a critical regulator of chondrocyte and osteoblast differentiation during multiple phases of cartilage and bone development. While the importance of β-CATENIN signaling during the process of endochondral bone development has been previously appreciated using a variety of genetic models that manipulate β-CATENIN in skeletal progenitors and osteoblasts, genetic evidence demonstrating a specific role for β-CATENIN in committed growth plate chondrocytes has been less robust. To identify the specific role of cartilage-derived β-CATENIN in regulating cartilage and bone development, we studied chondrocyte-specific gain- and loss-of-function genetic mouse models using the tamoxifen-inducible Col2CreERT2 transgene in combination with β-cateninfx(exon3)/wt or β-cateninfx/fx floxed alleles, respectively. From these genetic models and biochemical data, three significant and novel findings were uncovered. First, cartilage-specific β-CATENIN signaling promotes chondrocyte maturation, possibly involving a BMP2 mediated mechanism. Second, cartilage-specific β–CATENIN facilitates primary and secondary ossification center formation via the induction of chondrocyte hypertrophy, possibly through enhanced MMP expression at sites of cartilage degradation, and potentially by enhancing IHH signaling activity to recruit vascular tissues. Finally, cartilage-specific β-CATENIN signaling promotes perichondrial bone formation possibly via a mechanism in which BMP2 and IHH paracrine signals synergize to accelerate perichondrial osteoblastic differentiation. The work presented here supports the concept that the cartilage-derived β-CATENIN signal is a central mediator for major events during endochondral bone formation, including chondrocyte maturation, primary and secondary ossification center development, vascularization, and perichondrial bone formation. PMID:22508079

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

    PubMed

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

    2014-06-01

    Previously, we had addressed the issues of shape control/maintenance of in vitro engineered human-ear-shaped cartilage. Thus, lack of applicable cell source had become a major concern that blocks clinical translation of this technology. Autologous microtia chondrocytes (MCs) and bone marrow stromal cells (BMSCs) were both promising chondrogenic cells that did not involve obvious donor site morbidity. However, limited cell availability of MCs and ectopic ossification of chondrogenically induced BMSCs in subcutaneous environment greatly restricted their applications in external ear reconstruction. The current study demonstrated that MCs possessed strong proliferation ability but accompanied with rapid loss of chondrogenic ability during passage, indicating a poor feasibility to engineer the entire ear using expanded MCs. Fortunately, the co-transplantation results of MCs and BMSCs (25% MCs and 75% BMSCs) demonstrated a strong chondroinductive ability of MCs to promote stable ectopic chondrogenesis of BMSCs in subcutaneous environment. Moreover, cell labeling demonstrated that BMSCs could transform into chondrocyte-like cells under the chondrogenic niche provided by co-cultured MCs. Most importantly, a human-ear-shaped cartilaginous tissue with delicate structure and proper elasticity was successfully constructed by seeding the mixed cells (MCs and BMSCs) into the pre-shaped biodegradable ear-scaffold followed by 12 weeks of subcutaneous implantation in nude mouse. These results may provide a promising strategy to construct stable ectopic cartilage with MCs and stem cells (BMSCs) for autologous external ear reconstruction. Copyright © 2014 Elsevier Ltd. All rights reserved.

  19. Influence of biological scaffold regulation on the proliferation of chondrocytes and the repair of articular cartilage

    PubMed Central

    Wang, Si-Qun; Xia, Jun; Chen, Jie; Lu, Jian-Xi; Wei, Yi-Bing; Chen, Fei-Yan; Huang, Gang-Yong; Shi, Jing-Sheng; Yu, Yong-Lin

    2016-01-01

    Purpose: To investigate the effects of hard tissue engineering scaffold (the material is β-TCP) with different micro-structures on the proliferation of chondrocytes, and the influence of its composite erythrocytes on the repair of articular cartilage defects. Methods: Rabbit cartilage cells were on β-TCP bioceramic scaffold with different micro-structures in vitro, the proliferation growth trend of chondrocytes within the scaffold was calculated, and a optimal micro-structure suitable for cartilage cell growth was determined. Composite chondrocytes were implanted into rabbit models of articular cartilage defects, and the repair situation was observed. Results: the bioceramic scaffold with an inner diameter of 120 μm and an aperture of 500-630 μm was suitable for the growth of cartilage cells. Scaffold loaded with second generation of cartilage cell suspension got a top histological score of 20.76±2.13, which was closely similar to that of normal cartilage. Conclusion: When loaded with the second generation of cartilage cells, the β-TCP biological ceramic scaffold with a pore size of 500-630 μm, and an inner diameter of 120 μm, shows a best repairing effect on animal articular cartilage defects. PMID:27904662

  20. Production of three-dimensional tissue-engineered cartilage through mutual fusion of chondrocyte pellets.

    PubMed

    Hoshi, K; Fujihara, Y; Mori, Y; Asawa, Y; Kanazawa, S; Nishizawa, S; Misawa, M; Numano, T; Inoue, H; Sakamoto, T; Watanabe, M; Komura, M; Takato, T

    2016-09-01

    In this study, the mutual fusion of chondrocyte pellets was promoted in order to produce large-sized tissue-engineered cartilage with a three-dimensional (3D) shape. Five pellets of human auricular chondrocytes were first prepared, which were then incubated in an agarose mold. After 3 weeks of culture in matrix production-promoting medium under 5.78g/cm(2) compression, the tissue-engineered cartilage showed a sufficient mechanical strength. To confirm the usefulness of these methods, a transplantation experiment was performed using beagles. Tissue-engineered cartilage prepared with 50 pellets of beagle chondrocytes was transplanted subcutaneously into the cell-donor dog for 2 months. The tissue-engineered cartilage of the beagles maintained a rod-like shape, even after harvest. Histology showed fair cartilage regeneration. Furthermore, 20 pellets were made and placed on a beta-tricalcium phosphate prism, and this was then incubated within the agarose mold for 3 weeks. The construct was transplanted into a bone/cartilage defect in the cell-donor beagle. After 2 months, bone and cartilage regeneration was identified on micro-computed tomography and magnetic resonance imaging. This approach involving the fusion of small pellets into a large structure enabled the production of 3D tissue-engineered cartilage that was close to physiological cartilage tissue in property, without conventional polyper scaffolds.

  1. Update on the biology of the chondrocyte and new approaches to treating cartilage diseases.

    PubMed

    Goldring, Mary B

    2006-10-01

    Osteoarthritis (OA) is a joint disease that involves degeneration of articular cartilage, limited intraarticular inflammation manifested by synovitis and changes in the subchondral bone. The aetiology of OA is largely unknown, but since it may involve multiple factors, including mechanical, biochemical and genetic factors, it has been difficult to identify unique targets for therapy. Chondrocytes, which are the unique cellular component of adult articular cartilage, are capable of responding to structural changes in the surrounding cartilage matrix. Since the initial stages of OA involve increased cell proliferation and synthesis of matrix proteins, proteinases and cytokines in the cartilage, laboratory investigations have focused on the chondrocyte as a target for therapeutic intervention. The capacity of the adult articular chondrocyte to regenerate the normal cartilage matrix architecture is limited, however, and the damage becomes irreversible unless the destructive process is interrupted. Current pharmacological interventions that address chronic pain are insufficient and no proven disease-modifying therapy is available. Identification of methods for early diagnosis is of key importance, since therapeutic interventions aimed at blocking or reversing structural damage will be more effective when there is the possibility of preserving normal homeostasis. At later stages, cartilage tissue engineering with or without gene therapy with anabolic factors will also require therapy to inhibit inflammation and block damage to newly repaired cartilage. This review will focus on experimental approaches currently under study that may lead to elucidation of effective strategies for therapy in OA, with emphasis on mediators that affect the function of chondrocytes and interactions with surrounding tissues.

  2. Comparative potential of juvenile and adult human articular chondrocytes for cartilage tissue formation in three-dimensional biomimetic hydrogels.

    PubMed

    Smeriglio, Piera; Lai, Janice H; Dhulipala, Lakshmi; Behn, Anthony W; Goodman, Stuart B; Smith, Robert L; Maloney, William J; Yang, Fan; Bhutani, Nidhi

    2015-01-01

    Regeneration of human articular cartilage is inherently limited and extensive efforts have focused on engineering the cartilage tissue. Various cellular sources have been studied for cartilage tissue engineering including adult chondrocytes, and embryonic or adult stem cells. Juvenile chondrocytes (from donors below 13 years of age) have recently been reported to be a promising cell source for cartilage regeneration. Previous studies have compared the potential of adult and juvenile chondrocytes or adult and osteoarthritic (OA) chondrocytes. To comprehensively characterize the comparative potential of young, old, and diseased chondrocytes, here we examined cartilage formation by juvenile, adult, and OA chondrocytes in three-dimensional (3D) biomimetic hydrogels composed of poly(ethylene glycol) and chondroitin sulfate. All three human articular chondrocytes were encapsulated in the 3D biomimetic hydrogels and cultured for 3 or 6 weeks to allow maturation and extracellular matrix formation. Outcomes were analyzed using quantitative gene expression, immunofluorescence staining, biochemical assays, and mechanical testing. After 3 and 6 weeks, juvenile chondrocytes showed a greater upregulation of chondrogenic gene expression than adult chondrocytes, while OA chondrocytes showed a downregulation. Aggrecan and type II collagen deposition and glycosaminoglycan accumulation were high for juvenile and adult chondrocytes but not for OA chondrocytes. Similar trend was observed in the compressive moduli of the cartilage constructs generated by the three different chondrocytes. In conclusion, the juvenile, adult and OA chondrocytes showed differential responses in the 3D biomimetic hydrogels. The 3D culture model described here may also provide a useful tool to further study the molecular differences among chondrocytes from different stages, which can help elucidate the mechanisms for age-related decline in the intrinsic capacity for cartilage repair.

  3. Cryopreservation Effect on Proliferative and Chondrogenic Potential of Human Chondrocytes Isolated from Superficial and Deep Cartilage

    PubMed Central

    Muiños-López, Emma; Rendal-Vázquez, Mª Esther; Hermida-Gómez, Tamara; Fuentes-Boquete, Isaac; Díaz-Prado, Silvia; Blanco, Francisco J

    2012-01-01

    Objectives: To compare the proliferative and chondrogenic potential of fresh and frozen chondrocytes isolated from superficial and deep articular cartilage biopsies. Materials and Methodology: The study included 12 samples of fresh and frozen healthy human knee articular cartilage. Cell proliferation was tested at 3, 6 and 9 days. Studies of mRNA quantification, protein expression and immunofluorescence for proliferation and chondrogenic markers were performed. Results: Stimulation of fresh and frozen chondrocytes from both superficial and deep cartilage with fetal bovine serum produced an increase in the proliferative capacity compared to the non-stimulated control group. In the stimulated fresh cells group, the proliferative capacity of cells from the deep biopsy was greater than that from cells from the superficial biopsy (0.046 vs 0.028, respectively, p<0.05). There was also a significant difference between the proliferative capacity of superficial zone fresh (0.028) and frozen (0.051) chondrocytes (p<0.05). CCND1 mRNA and protein expression levels, and immunopositivity for Ki67 revealed a higher proliferative capacity for fresh articular chondrocytes from deep cartilage. Regarding the chondrogenic potential, stimulated fresh cells showed higher SOX9 and Col II expression in chondrocytes from deep than from superficial zone (p<0.05, T student test). Conclusions: The highest rate of cell proliferation and chondrogenic potential of fresh chondrocytes was found in cells obtained from deep cartilage biopsies, whereas there were no statistically significant differences in proliferative and chondrogenic capacity between biopsy origins with frozen chondrocytes. These results indicate that both origin and cryopreservation affect the proliferative and chondrogenic potential of chondrocytes. PMID:22523526

  4. Disruption of glucocorticoid signaling in chondrocytes delays metaphyseal fracture healing but does not affect normal cartilage and bone development

    PubMed Central

    Tu, Jinwen; Henneicke, Holger; Zhang, Yaqing; Stoner, Shihani; Cheng, Tegan L.; Schindeler, Aaron; Chen, Di; Tuckermann, Jan; Cooper, Mark S.; Seibel, Markus J.; Zhou, Hong

    2014-01-01

    States of glucocorticoid excess are associated with defects in chondrocyte function. Most prominently there is a reduction in linear growth but delayed healing of fractures that require endochondral ossification to also occur. In contrast, little is known about the role of endogenous glucocorticoids in chondrocyte function. As glucocorticoids exert their cellular actions through the glucocorticoid receptor (GR), we aimed to elucidate the role of endogenous glucocorticoids in chondrocyte function in vivo through characterization of tamoxifen-inducible chondrocyte-specific GR knockout (chGRKO) mice in which the GR was deleted at various post-natal ages. Knee joint architecture, cartilage structure, growth plates, intervertebral discs, long bone length and bone micro-architecture were similar in chGRKO and control mice at all ages. Analysis of fracture healing in chGRKO and control mice demonstrated that in metaphyseal fractures, chGRKO mice formed a larger cartilaginous callus at 1 and 2 week post-surgery, as well as a smaller amount of well-mineralized bony callus at the fracture site 4 week post-surgery, when compared to control mice. In contrast, chondrocyte-specific GR knockout did not affect diaphyseal fracture healing. We conclude that endogenous GC signaling in chondrocytes plays an important role during metaphyseal fracture healing but is not essential for normal long bone growth. PMID:25193158

  5. Comparison of articular cartilage repair by autologous chondrocytes with and without in vitro cultivation.

    PubMed

    Chiang, Hongsen; Liao, Chun-Jen; Wang, Yao-Hong; Huang, Hsin-Yi; Chen, Chun-Nan; Hsieh, Chang-Hsun; Huang, Yi-You; Jiang, Ching-Chuan

    2010-04-01

    autologous chondrocyte implantation usually requires in vitro cell expansion before implantation. We compared the efficacy of cartilage regeneration by in vitro-expanded chondrocytes at high density and freshly harvested chondrocytes at low density. surgically created osteochondral defects at weight-bearing surface of femoral condyles of domestic pigs were repaired by biphasic cylindrical porous plugs of DL-poly-lactide-co-glycolide and beta-tricalcium phosphate. Plugs were seeded with autologous chondrocytes in its chondral phase, and press-fit to defects. Seeded cells were (1) in vitro-expanded chondrocytes harvested from stifle joint 3 weeks before implantation and (2) freshly harvested chondrocytes from recipient knee. Seeding densities were 70 x 10(6) and 7 x 10(6) cells/mL, respectively. Cell-free plugs served as control and defects remained untreated as null control. Outcome was examined at 6 months with International Cartilage Repair Society Scale. the two experimental groups were repaired by hyaline cartilage with collagen type II and Safranin-O. Tissue in control group was primarily fibrocartilage. No regeneration was found in null control. Experimental groups had higher mean International Cartilage Repair Society scores than control in surface, matrix, and cell distribution, but were comparable with control in cell viability, subchondral bone, and mineralization. No significant difference existed between two experimental groups in any of the six categories. Uni-axial indentation test revealed similar creeping stress-relaxation property as native cartilage on experimental, but not control, specimen. cartilage could regenerate in both experimental models, in comparable quality. Culture of chondrocytes before implantation is not necessary.

  6. Inflammation-induced chondrocyte hypertrophy is driven by receptor for advanced glycation end products.

    PubMed

    Cecil, Denise L; Johnson, Kristen; Rediske, John; Lotz, Martin; Schmidt, Ann Marie; Terkeltaub, Robert

    2005-12-15

    The multiligand receptor for advanced glycation end products (RAGE) mediates certain chronic vascular and neurologic degenerative diseases accompanied by low-grade inflammation. RAGE ligands include S100/calgranulins, a class of low-molecular-mass, calcium-binding polypeptides, several of which are chondrocyte expressed. Here, we tested the hypothesis that S100A11 and RAGE signaling modulate osteoarthritis (OA) pathogenesis by regulating a shift in chondrocyte differentiation to hypertrophy. We analyzed human cartilages and cultured human articular chondrocytes, and used recombinant human S100A11, soluble RAGE, and previously characterized RAGE-specific blocking Abs. Normal human knee cartilages demonstrated constitutive RAGE and S100A11 expression, and RAGE and S100A11 expression were up-regulated in OA cartilages studied by immunohistochemistry. CXCL8 and TNF-alpha induced S100A11 expression and release in cultured chondrocytes. Moreover, S100A11 induced cell size increase and expression of type X collagen consistent with chondrocyte hypertrophy in vitro. CXCL8-induced, IL-8-induced, and TNF-alpha-induced but not retinoic acid-induced chondrocyte hypertrophy were suppressed by treatment with soluble RAGE or RAGE-specific blocking Abs. Last, via transfection of dominant-negative RAGE and dominant-negative MAPK kinase 3, we demonstrated that S100A11-induced chondrocyte type X collagen expression was dependent on RAGE-mediated p38 MAPK pathway activation. We conclude that up-regulated chondrocyte expression of the RAGE ligand S100A11 in OA cartilage, and RAGE signaling through the p38 MAPK pathway, promote inflammation-associated chondrocyte hypertrophy. RAGE signaling thereby has the potential to contribute to the progression of OA.

  7. Effect of metal ions on calcifying growth plate cartilage chondrocytes.

    PubMed

    Litchfield, T M; Ishikawa, Y; Wu, L N; Wuthier, R E; Sauer, G R

    1998-04-01

    The effects of the trace metals zinc (Zn), manganese (Mn), and cadmium (Cd) on the metabolism of growth plate chondrocytes was examined using a mineralizing culture system. Supplementation of serum-free primary cultures of growth plate chondrocytes with 10-100 mu m Zn resulted in an increase in cell protein and greatly increased alkaline phosphatase (AP) activity; however, above 25 mu m Zn mineralization of the cultures was reduced. The effects of Zn on cellular protein and AP activity were enhanced by the addition of the albumin to the culture media. Removal of Zn from basal culture media resulted in recoverable reductions in cellular protein and AP activities. Cadmium was acutely toxic to chondrocyte cell cultures at concentrations above 5 mu m. Even at very low concentrations (0.25 mu m) Cd caused significant reductions in DNA, cellular protein, and matrix protein synthesis. In contrast, Cd had negligible effects on AP activity or culture mineralization. Manganese treatment (50 mu m) resulted in reduced levels of proteoglycan, cell protein, DNA synthesis, and collagen synthesis, although AP specific activity did not change. At 10 mu m, Mn significantly reduced mineralization but had only minor influence on other culture parameters. Both Zn (200 mu m) and Cd (0.1 mu m), but not Mn, induced the synthesis of metallothionein. The physiological and biochemical effects of specific metal ions is largely dependent on their physicochemical properties, especially their ligand affinities. Knowledge of these properties allows predictions to be made regarding whether the organic or the mineral phase are most likely to be affected in a mineralized tissue.

  8. Protective Mechanism of Articular Cartilage to Severe Loading: Roles of Lubricants, Cartilage Surface Layer, Extracellular Matrix and Chondrocyte

    NASA Astrophysics Data System (ADS)

    Murakami, Teruo; Sawae, Yoshinori; Ihara, Maki

    The natural synovial joints have excellent tribological performance known as very low friction and very low wear for various daily activities in human life. These functions are likely to be supported by the adaptive multimode lubrication mechanism, in which the various lubrication modes such as elastohydrodynamic lubrication, weeping, boundary and gel film lubrication appear to operate to protect articular cartilage, depending on the severity of the rubbing conditions. In this paper, various protective roles of synovial fluid, cartilage surface layer, extracellular matrix and chondrocyte to severe loading are described. In the first part, the protective mechanism by adsorbed films and underlying gel films was described on the basis of the frictional behaviors of articular cartilage against articular cartilage or glass. It was discussed that the replenishment of gel film removed during severe rubbing is likely to be controlled by supply of proteoglycan from the extracellular matrix, where the chondrocyte plays the main role in the metabolism. In the second part, the time-dependent local deformation of biphasic articular cartilage under constant total compressive strain condition was evaluated in the finite element analyses. The importance of clarification of actual stress-strain in chondrocyte was indicated in relation to the tribological property of articular cartilage.

  9. Standardized cartilage biopsies from the intercondylar notch for autologous chondrocyte implantation (ACI).

    PubMed

    Niemeyer, Philipp; Pestka, Jan M; Kreuz, Peter C; Salzmann, Gian M; Köstler, Wolfgang; Südkamp, Norbert P; Steinwachs, Matthias

    2010-08-01

    Autologous chondrocyte implantation (ACI) is an established therapy for the treatment of cartilage defects across the knee joint. Even though different techniques for initial biopsy have been described, the exact location, depth, and volume of the biopsy are chosen individually by the treating surgeon. This study evaluated 252 consecutive cartilage biopsies taken from the intercondylar notch with a standardized hollow cylinder system for the isolation and in vitro cultivation of human chondrocytes assigned to ACI. All biopsies were assessed for weight of total cartilage obtained, cartilage biopsy weight per cylinder, biopsy cylinder quality, and initial cell count after digestive cellular isolation as well as cell vitality. Parameters were correlated with individual patient parameters. Mean patient age was 35.1 years (median 35.9; range 14.7-56.4). Adequate amounts of cartilage assigned to chondrocyte in vitro cultivation could be harvested in all cases. The mean overall biopsy weight averaged 75.5 mg (SD +/- 44.9) and could be identified as main factor for initial cell number (mean 1.05E+05; SD +/- 7.44E+04). No correlation was found between the initial cell count and patient age (correlation coefficient r = 0.005) or grade of joint degeneration (r = 0.040). Concerning cell viability, a total of 4.4% (SD + 3.0) of the chondrocytes harvested were apoptotic. Cartilage biopsies from the intercondylar notch using a standardized hollow cylinder system provides a reliable, safe, and successful method to obtain articular cartilage for further in vitro cultivation of articular chondrocytes to achieve autologous chondrocyte transplantation.

  10. Impact of storage solution formulation during refrigerated storage upon chondrocyte viability and cartilage matrix.

    PubMed

    Wright, Gregory J; Brockbank, Kelvin G M; Rahn, Eliza; Halwani, Dina O; Chen, Zhen; Yao, Hai

    2014-01-01

    Various preservation solutions have been evaluated for longer hypothermic cartilage storage for tissue transplantation; however, the results are mixed. This research was carried out to determine whether phosphate-buffered saline (PBS) or organ preservation solutions would preserve both the extracellular matrix and chondrocytes of articular cartilage better than culture medium during refrigerated storage in the time frame that cartilage is stored for clinical use. Porcine cartilage plugs were stored, without the underlying bone, in culture medium with and without fetal bovine serum (FBS), PBS, Belzer's and Unisol solutions for 1 month at 4°C. Metabolic activity was tested using a resazurin reduction method, and matrix permeability was evaluated by measuring electrical conductivity. Storage in culture medium with 10% FBS was shown to provide good cartilage metabolic function for 7 days, decreasing to about 36% after 1 month of storage. There was no significant difference between samples stored in culture medium with and without FBS after 1 month of storage (p = 0.5005). Refrigerated storage of cartilage in PBS and two different solutions (Belzer's and Unisol) designed for optimal refrigerated tissue and organ storage results in loss of chondrocyte function and retention of matrix permeability. In contrast, the opposite, namely significantly better retention of chondrocyte function and loss of matrix permeability, was observed with culture medium. Future research should be focused on combining retention of chondrocyte function and matrix permeability by storage solution formulation.

  11. A chondrocyte infiltrated collagen type I/III membrane (MACI® implant) improves cartilage healing in the equine patellofemoral joint model.

    PubMed

    Nixon, A J; Rickey, E; Butler, T J; Scimeca, M S; Moran, N; Matthews, G L

    2015-04-01

    Autologous chondrocyte implantation (ACI) has improved outcome in long-term studies of joint repair in man. However, ACI requires sutured periosteal flaps to secure the cells, which precludes minimally-invasive implantation, and introduces complications with arthrofibrosis and graft hypertrophy. This study evaluated ACI on a collagen type I/III scaffold (matrix-induced autologous chondrocyte implantation; MACI(®)) in critical sized defects in the equine model. Chondrocytes were isolated from horses, expanded and seeded onto a collagen I/III membrane (ACI-Maix™) and implanted into one of two 15-mm defects in the femoral trochlear ridge of six horses. Control defects remained empty as ungrafted debrided defects. The animals were examined daily, scored by second look arthroscopy at 12 weeks, and necropsy examination 6 months after implantation. Reaction to the implant was determined by lameness, and synovial fluid constituents and synovial membrane histology. Cartilage healing was assessed by arthroscopic scores, gross assessment, repair tissue histology and immunohistochemistry, cartilage glycosaminoglycan (GAG) and DNA assay, and mechanical testing. MACI(®) implanted defects had improved arthroscopic second-look, gross healing, and composite histologic scores, compared to spontaneously healing empty defects. Cartilage GAG and DNA content in the defects repaired by MACI implant were significantly improved compared to controls. Mechanical properties were improved but remained inferior to normal cartilage. There was minimal evidence of reaction to the implant in the synovial fluid, synovial membrane, subchondral bone, or cartilage. The MACI(®) implant appeared to improve cartilage healing in a critical sized defect in the equine model evaluated over 6 months. Copyright © 2015 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.

  12. LRP4 induces extracellular matrix productions and facilitates chondrocyte differentiation.

    PubMed

    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.

  13. Magnetic resonance imaging of chondrocytes labeled with superparamagnetic iron oxide nanoparticles in tissue-engineered cartilage.

    PubMed

    Ramaswamy, Sharan; Greco, Jane B; Uluer, Mehmet C; Zhang, Zijun; Zhang, Zhuoli; Fishbein, Kenneth W; Spencer, Richard G

    2009-12-01

    The distribution of cells within tissue-engineered constructs is difficult to study through nondestructive means, such as would be required after implantation. However, cell labeling with iron-containing particles may prove to be a useful approach to this problem, because regions containing such labeled cells have been shown to be readily detectable using magnetic resonance imaging (MRI). In this study, we used the Food and Drug Administration-approved superparamagnetic iron oxide (SPIO) contrast agent Feridex in combination with transfection agents to label chondrocytes and visualize them with MRI in two different tissue-engineered cartilage constructs. Correspondence between labeled cell spatial location as determined using MRI and histology was established. The SPIO-labeling process was found not to affect the phenotype or viability of the chondrocytes or the production of major cartilage matrix constituents. We believe that this method of visualizing and tracking chondrocytes may be useful in the further development of tissue engineered cartilage therapeutics.

  14. Magnetic Resonance Imaging of Chondrocytes Labeled with Superparamagnetic Iron Oxide Nanoparticles in Tissue-Engineered Cartilage

    PubMed Central

    Ramaswamy, Sharan; Greco, Jane B.; Uluer, Mehmet C.; Zhang, Zijun; Zhang, Zhuoli; Fishbein, Kenneth W.

    2009-01-01

    The distribution of cells within tissue-engineered constructs is difficult to study through nondestructive means, such as would be required after implantation. However, cell labeling with iron-containing particles may prove to be a useful approach to this problem, because regions containing such labeled cells have been shown to be readily detectable using magnetic resonance imaging (MRI). In this study, we used the Food and Drug Administration–approved superparamagnetic iron oxide (SPIO) contrast agent Feridex in combination with transfection agents to label chondrocytes and visualize them with MRI in two different tissue-engineered cartilage constructs. Correspondence between labeled cell spatial location as determined using MRI and histology was established. The SPIO-labeling process was found not to affect the phenotype or viability of the chondrocytes or the production of major cartilage matrix constituents. We believe that this method of visualizing and tracking chondrocytes may be useful in the further development of tissue engineered cartilage therapeutics. PMID:19788362

  15. A Biosynthetic Scaffold that Facilitates Chondrocyte-Mediated Degradation and Promotes Articular Cartilage Extracellular Matrix Deposition

    PubMed Central

    Sridhar., Balaji V.; Dailing, Eric A.; Brock, J. Logan; Stansbury, Jeffrey W.; Randolph, Mark A.; Anseth, Kristi S.

    2015-01-01

    Articular cartilage remains a significant clinical challenge to repair because of its limited self-healing capacity. Interest has grown in the delivery of autologous chondrocytes to cartilage defects, and combining cell-based therapies with scaffolds that capture aspects of native tissue and allow cell-mediated remodeling could improve outcomes. Currently, scaffold-based therapies with encapsulated chondrocytes permit matrix production; however, resorption of the scaffold often does not match the rate of matrix production by chondrocytes, which can limit functional tissue regeneration. Here, we designed a hybrid biosynthetic system consisting of poly (ethylene glycol) (PEG) endcapped with thiols and crosslinked by norbornene-functionalized gelatin via a thiol-ene photopolymerization. The protein crosslinker was selected to facilitate chondrocyte-mediated scaffold remodeling and matrix deposition. Gelatin was functionalized with norbornene to varying degrees (~4–17 norbornenes/gelatin), and the shear modulus of the resulting hydrogels was characterized (<0.1–0.5 kPa). Degradation of the crosslinked PEG-gelatin hydrogels by chondrocyte-secreted enzymes was confirmed by gel permeation chromatography. Finally, chondrocytes encapsulated in these biosynthetic scaffolds showed significantly increased glycosaminoglycan deposition over just 14 days of culture, while maintaining high levels of viability and producing a distributed matrix. These results indicate the potential of a hybrid PEG-gelatin hydrogel to permit chondrocyte-mediated remodeling and promote articular cartilage matrix production. Tunable scaffolds that can easily permit chondrocyte-mediated remodeling may be useful in designing treatment options for cartilage tissue engineering applications. PMID:26900597

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

    PubMed

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

    2013-06-01

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

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

    PubMed

    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.

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

    PubMed

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

    2016-08-01

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

  19. Tissue responses against tissue-engineered cartilage consisting of chondrocytes encapsulated within non-absorbable hydrogel.

    PubMed

    Kanazawa, Sanshiro; Fujihara, Yuko; Sakamoto, Tomoaki; Asawa, Yukiyo; Komura, Makoto; Nagata, Satoru; Takato, Tsuyoshi; Hoshi, Kazuto

    2013-01-01

    To disclose the influence of foreign body responses raised against a non-absorbable hydrogel consisting of tissue-engineered cartilage, we embedded human/canine chondrocytes within agarose and transplanted them into subcutaneous pockets in nude mice and donor beagles. One month after transplantation, cartilage formation was observed in the experiments using human chondrocytes in nude mice. No significant invasion of blood cells was noted in the areas where the cartilage was newly formed. Around the tissue-engineered cartilage, agarose fragments, a dense fibrous connective tissue and many macrophages were observed. On the other hand, no cartilage tissue was detected in the autologous transplantation of canine chondrocytes. Few surviving chondrocytes were observed in the agarose and no accumulation of blood cells was observed in the inner parts of the transplants. Localizations of IgG and complements were noted in areas of agarose, and also in the devitalized cells embedded within the agarose. Even if we had inhibited the proximity of the blood cells to the transplanted cells, the survival of the cells could not be secured. We suggest that these cytotoxic mechanisms seem to be associated not only with macrophages but also with soluble factors, including antibodies and complements.

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

    NASA Astrophysics Data System (ADS)

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

    2013-12-01

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

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

    PubMed Central

    2012-01-01

    Background Scaffold-assisted autologous chondrocyte implantation is an effective clinical procedure for cartilage repair. From the regulatory point of view, the ovine model is one of the suggested large animal models for pre-clinical studies. The aim of our study was to evaluate the in vitro re-differentiation capacity of expanded ovine chondrocytes in biomechanically characterized polyglycolic acid (PGA)/fibrin biomaterials for scaffold-assisted cartilage repair. Methods Ovine chondrocytes harvested from adult articular cartilage were expanded in monolayer and re-assembled three-dimensionally in PGA-fibrin scaffolds. De- and re-differentiation of ovine chondrocytes in PGA-fibrin scaffolds was assessed by histological and immuno-histochemical staining as well as by real-time gene expression analysis of typical cartilage marker molecules and the matrix-remodelling enzymes matrix metalloproteinases (MMP) -1, -2 and −13 as well as their inhibitors. PGA scaffolds characteristics including degradation and stiffness were analysed by electron microscopy and biomechanical testing. Results Histological, immuno-histochemical and gene expression analysis showed that dedifferentiated chondrocytes re-differentiate in PGA-fibrin scaffolds and form a cartilaginous matrix. Re-differentiation was accompanied by the induction of type II collagen and aggrecan, while MMP expression decreased in prolonged tissue culture. Electron microscopy and biomechanical tests revealed that the non-woven PGA scaffold shows a textile structure with high tensile strength of 3.6 N/mm2 and a stiffness of up to 0.44 N/mm2, when combined with gel-like fibrin. Conclusion These data suggest that PGA-fibrin is suited as a mechanically stable support structure for scaffold-assisted chondrocyte grafts, initiating chondrogenic re-differentiation of expanded chondrocytes. PMID:23137017

  2. Communication between paired chondrocytes in the superficial zone of articular cartilage

    PubMed Central

    Chi, Simon S; Rattner, Jerome B; Matyas, John R

    2004-01-01

    The regeneration and repair of cartilage damaged by injury or disease, a major goal of orthopaedic science, depends on understanding the structure and function of both the extracellular matrix and the chondrocytes. In this study, we explored the in situ organization and potential interactions between chondrocytes in the superficial zone of adult rabbit articular cartilage. Some chondrocytes in this zone were observed close together and appeared to be paired whereas others were solitary. The shared surfaces of a chondrocyte pair were separated by a narrow plate of extracellular matrix, into which extended small cytoplasmic projections from both cells. Furthermore, the spatial distribution of major cellular landmarks, such as the nucleus and centrosome as well as some intracellular proteins such as connexin-43, tended to be mirrored about this matrix plate. Fluorescence recovery after photobleaching revealed the fluorescent dye calcein–AM dye can pass between paired cells, and that the passage of this dye can be inhibited by the gap junction blocker octanol. These results illustrate that rapid cellular communication is possible between cells in the superficial layer of adult articular cartilage, which challenges the current thinking that these chondrocytes function in isolation. PMID:15575885

  3. Mesenchymal stem cells downregulate articular chondrocyte differentiation in noncontact coculture systems: implications in cartilage tissue regeneration.

    PubMed

    Xu, Lei; Wang, Qi; Xu, Feiyue; Ye, Zhaoyang; Zhou, Yan; Tan, Wen-Song

    2013-06-01

    While chondrogenesis of mesenchymal stem cells (MSCs) in vitro has been extensively studied, their participation in cartilage tissue repair remains unresolved. This study was designed to elucidate if MSCs affect the phenotype of articular chondrocytes (ACs). A combination of noncontact coculture modes was developed. Human or rabbit MSCs and rabbit ACs (rACs) were encapsulated in alginate hydrogel beads [three-dimensional (3D)] or cultured in a monolayer [two-dimensional (2D)] and subsequently cocultured in the Transwell(®) system. After coculture, cell morphology, growth, deposition of the cartilaginous extracellular matrix (ECM), and gene expression of rACs were investigated. It was found that upon coculture without a cell-cell contact, both 2D and 3D cultured MSCs dramatically induced the morphological transformation of 2D cultured rACs from round to a spindle-like shape, and however inhibited the generation of cellular aggregates of 3D cultured rACs. Most strikingly, a coculture resulted in a significantly less deposition of the cartilaginous ECM, including glycosaminoglycans and collagen type II by both 2D and 3D cultured rACs. Importantly, similar observations were achieved for rACs cultured in an MSC-conditioned medium, confirming the definite paracrine interactions between MSCs and rACs. Based on the analysis of gene expression, this phenotypic change of rACs was not identical as the dedifferentiation. To the best of our knowledge, this is a first study demonstrating that MSCs could downregulate chondrocytic differentiation of ACs and warrants considerations in cartilage tissue repair.

  4. Autologous nasal chondrocytes delivered by injectable hydrogel for in vivo articular cartilage regeneration.

    PubMed

    Chen, Wenliang; Li, Changhua; Peng, Maoxiu; Xie, Bingju; Zhang, Lei; Tang, Xiaojun

    2017-08-16

    Cell based tissue engineering serves as a promising strategy for articular cartilage repair, which remains a challenge both for researchers and clinicians. The aim of this research was to assess the potential of autologous nasal chondrocytes (NCs) combined with alginate hydrogel as injectable constructs for rabbit articular cartilage repair. Autologous nasal chondrocytes were isolated from rabbit nasal septum, expanded either on monolayer or in 3D alginate hydrogel. In vitro, DNA quantification revealed that NCs can proliferate stable in 3D alginate matrix, but slower than that cultured in monolayer. Further, a higher synthesis rate of glycosaminoglycans (GAGs) was detected by GAG measurement in 3D alginate culture. Gene expression analysis at different time point (day 1, 7, 14) showed that 3D culture of NCs in alginate up-regulated chondrogenic markers (Col2A1, ACAN SOX9), meanwhile down-regulated dedifferentiation related gene (Col1A1). In vivo, autologous nasal chondrocytes combined with alginate hydrogel were used for repairing rabbit knee osteochondral defect (Alg + NC group). Histological staining indicated that Alg + NC group obtained superior and more hyaline-like repaired tissue both at 3 and 6 months after surgery. Mechanical analysis showed that the repaired tissue in the Alg + NC group possessed similar mechanical properties to the native cartilage. In conclusion, nasal chondrocytes appeared to be a very promising seed cell source for cartilage tissue engineering, and alginate hydrogel can serve as suitable delivery system.

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

    PubMed

    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.

  6. Effects of Cartilage Impact with and without Fracture on Chondrocyte Viability and the Release of Inflammatory Markers

    PubMed Central

    Stolberg-Stolberg, Josef A.; Furman, Bridgette D.; Garrigues, N. William; Lee, Jaewoo; Pisetsky, David S.; Stearns, Nancy A; DeFrate, Louis E.; Guilak, Farshid; Olson, Steven A.

    2014-01-01

    Post-traumatic arthritis (PTA) frequently develops after intra-articular fracture of weight bearing joints. Loss of cartilage viability and post-injury inflammation have both been implicated as possible contributing factors to PTA progression. In order to further investigate chondrocyte response to impact and fracture, we have developed a blunt impact model applying 70%, 80% or 90% surface-to-surface compressive strain with or without induction of an articular fracture in a cartilage explant model. Following mechanical loading, chondrocyte viability and apoptosis were assessed. Culture media were evaluated for the release of double-stranded DNA (dsDNA) and immunostimulatory activity via nuclear factor kappa B (NF-κB) activity in Toll-like receptor-expressing Ramos-Blue reporter cells. High compressive strains, with or without articular fracture, resulted in significantly reduced chondrocyte viability. Blunt impact at 70% strain induced a loss in viability over time through a combination of apoptosis and necrosis, whereas blunt impact above 80% strain caused predominantly necrosis. In the fracture model, a high level of primarily necrotic chondrocyte death occurred along the fracture edges. At sites away from the fracture, viability was not significantly different than controls. Interestingly, both dsDNA release and NF-κB activity in Ramos-Blue cells increased with blunt impact, but was only significantly increased in the media from fractured cores. This study indicates that the mechanism of trauma determines the type of chondrocyte death as well as the potential for post-injury inflammation. PMID:23620164

  7. Auricular cartilage repair using cryogel scaffolds loaded with BMP-7-expressing primary chondrocytes.

    PubMed

    Odabas, S; Feichtinger, G A; Korkusuz, P; Inci, I; Bilgic, E; Yar, A S; Cavusoglu, T; Menevse, S; Vargel, I; Piskin, E

    2013-10-01

    The loss of cartilage tissue due to trauma, tumour surgery or congenital defects, such as microtia and anotia, is one of the major concerns in head and neck surgery. Recently tissue-engineering approaches, including gene delivery, have been proposed for the regeneration of cartilage tissue. In this study, primary chondrocytes were genetically modified with plasmid-encoding bone morphogenetic protein-7 (BMP-7) via the commercially available non-viral Turbofect vector, with the aim of bringing ex vivo transfected chondrocytes to resynthesize BMP-7 in vitro as they would in vivo. Genetically modified cells were implanted into gelatin-oxidized dextran scaffolds and cartilage tissue formation was investigated in 15 × 15 mm auricular cartilage defects in vivo in 48 New Zealand (NZ) white rabbits for 4 months. The results were evaluated via histology and early gene expression. Early gene expression results indicated a strong effect of exogenous BMP-7 on matrix synthesis and chondrocyte growth. In addition, histological analysis results exhibited significantly better cartilage healing with BMP-7-modified (transfected) cells than in the non-modified (non-transfected) group and as well as the control.

  8. Association between the chondrocyte phenotype and the expression of adipokines and their receptors: evidence for a role of leptin but not adiponectin in the expression of cartilage-specific markers.

    PubMed

    Francin, Pierre-Jean; Guillaume, Cécile; Humbert, Anne-Claude; Pottie, Pascale; Netter, Patrick; Mainard, Didier; Presle, Nathalie

    2011-11-01

    Although extensive evidence support the key role of adipokines in cartilage homeostasis, contradictory data have been found for their expression and their effects in chondrocytes. This study was then undertaken to determine whether a phenotypic modulation may affect the expression of adipokines and their receptors in human chondrocytes. The expression of leptin, adiponectin and their receptors, as well as cartilage-specific genes was examined in chondrocytes obtained from patients with osteoarthritis either directly after cells harvest or after culture in monolayer or in alginate beads. The results showed major changes in the gene expression pattern after culture in monolayer with a shift from the adipokines to their receptors. Interestingly, this downregulation of adipokines was associated with a loss of chondrocyte phenotype, and chondrocytes recovered a cartilage-like expression profile of leptin and adiponectin when cultured in a tridimensional chondrocyte phenotype-inducing system, but ceased expressing their receptors. Further experiments clearly showed that leptin but not adiponectin promoted the expression of cartilage-specific markers through mitogen-activated protein kinase, Janus kinase and phosphatidylinositol-3 kinase signaling pathways. In conclusion, our data indicate that any phenotypic modulation could affect chondrocyte responsiveness to leptin or adiponectin, and provide evidence for an important role for leptin in regulating the expression of cartilage-specific markers.

  9. β-Defensin-4 (HBD-4) is expressed in chondrocytes derived from normal and osteoarthritic cartilage encapsulated in PEGDA scaffold.

    PubMed

    Musumeci, Giuseppe; Carnazza, Maria Luisa; Loreto, Corrado; Leonardi, Rosalia; Loreto, Carla

    2012-12-01

    Defensins are antibiotic peptides involved in host defense mechanisms, wound healing and tissue repair. Furthermore, they seem to play an important role in protection mechanisms in articular joints. The aim of this study was to investigate β-defensin-4 expression in chondrocytes taken from articular cartilage of knees of patients with osteoarthritis (OA) compared to normal cartilage, in vivo in explanted tissue, and in vitro in chondrocytes encapsulated in construct PEGDA hydrogels. The present investigation was conducted to try and elucidate the possible use of β-defensin-4 as a relevant marker for the eventual use of successive scaffold allografts, and to provide new insights for hydrogel PEGDA scaffold efficacy in re-differentiation or repair of OA chondrocytes in vitro. Articular cartilage specimens from OA cartilage and normal cartilage were assessed by histology, histochemistry, immunohistochemistry and Western blot analysis. The results showed strong β-defensin-4 immunoexpression in explanted tissue from OA cartilage and weak β-defensin-4 expression in control cartilage. The chondrocytes from OA cartilage after 4 weeks of culture in PEGDA hydrogels showed the formation of new hyaline cartilage and a decreased expression of β-defensin-4 immunostaining comparable to that of control cartilage. Our results suggest the possibility of applying autologous cell transplantation in conjunction with scaffold materials for repair of cartilage lesions in patients with OA using β-defensin-4 as a relevant marker. Copyright © 2012 Elsevier GmbH. All rights reserved.

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

    NASA Astrophysics Data System (ADS)

    Fedewa, Michelle Marie

    2000-10-01

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

  11. Reconstruction of Hyaline Cartilage Deep Layer Properties in 3-Dimensional Cultures of Human Articular Chondrocytes.

    PubMed

    Nanduri, Vibudha; Tattikota, Surendra Mohan; T, Avinash Raj; Sriramagiri, Vijaya Rama Rao; Kantipudi, Suma; Pande, Gopal

    2014-06-01

    Articular cartilage (AC) injuries and malformations are commonly noticed because of trauma or age-related degeneration. Many methods have been adopted for replacing or repairing the damaged tissue. Currently available AC repair methods, in several cases, fail to yield good-quality long-lasting results, perhaps because the reconstructed tissue lacks the cellular and matrix properties seen in hyaline cartilage (HC). To reconstruct HC tissue from 2-dimensional (2D) and 3-dimensional (3D) cultures of AC-derived human chondrocytes that would specifically exhibit the cellular and biochemical properties of the deep layer of HC. Descriptive laboratory study. Two-dimensional cultures of human AC-derived chondrocytes were established in classical medium (CM) and newly defined medium (NDM) and maintained for a period of 6 weeks. These cells were suspended in 2 mm-thick collagen I gels, placed in 24-well culture inserts, and further cultured up to 30 days. Properties of chondrocytes, grown in 2D cultures and the reconstructed 3D cartilage tissue, were studied by optical and scanning electron microscopic techniques, immunohistochemistry, and cartilage-specific gene expression profiling by reverse transcription polymerase chain reaction and were compared with those of the deep layer of native human AC. Two-dimensional chondrocyte cultures grown in NDM, in comparison with those grown in CM, showed more chondrocyte-specific gene activity and matrix properties. The NDM-grown chondrocytes in 3D cultures also showed better reproduction of deep layer properties of HC, as confirmed by microscopic and gene expression analysis. The method used in this study can yield cartilage tissue up to approximately 1.6 cm in diameter and 2 mm in thickness that satisfies the very low cell density and matrix composition properties present in the deep layer of normal HC. This study presents a novel and reproducible method for long-term culture of AC-derived chondrocytes and reconstruction of cartilage

  12. The C-terminal domain of connexin43 modulates cartilage structure via chondrocyte phenotypic changes

    PubMed Central

    Gago-Fuentes, Raquel; Bechberger, John F.; Varela-Eirin, Marta; Varela-Vazquez, Adrian; Acea, Benigno; Fonseca, Eduardo

    2016-01-01

    Chondrocytes in cartilage and bone cells population express connexin43 (Cx43) and gap junction intercellular communication (GJIC) is essential to synchronize cells for coordinated electrical, mechanical, metabolic and chemical communication in both tissues. Reduced Cx43 connectivity decreases chondrocyte differentiation and defective Cx43 causes skeletal defects. The carboxy terminal domain (CTD) of Cx43 is located in the cytoplasmic side and is key for protein functions. Here we demonstrated that chondrocytes from the CTD-deficient mice, K258stop/Cx43KO and K258stop/K258stop, have reduced GJIC, increased rates of proliferation and reduced expression of collagen type II and proteoglycans. We observed that CTD-truncated mice were significantly smaller in size. Together these results demonstrated that the deletion of the CTD negatively impacts cartilage structure and normal chondrocyte phenotype. These findings suggest that the proteolytic cleavage of the CTD under pathological conditions, such as under the activation of metalloproteinases during tissue injury or inflammation, may account for the deleterious effects of Cx43 in cartilage and bone disorders such as osteoarthritis. PMID:27682878

  13. Sprifermin (rhFGF18) enables proliferation of chondrocytes producing a hyaline cartilage matrix.

    PubMed

    Gigout, A; Guehring, H; Froemel, D; Meurer, A; Ladel, C; Reker, D; Bay-Jensen, A C; Karsdal, M A; Lindemann, S

    2017-08-18

    Fibroblast growth factor (FGF) 18 has been shown to increase cartilage volume when injected intra-articularly in animal models of osteoarthritis (OA) and in patients with knee OA (during clinical development of the recombinant human FGF18, sprifermin). However, the exact nature of this effect is still unknown. In this study, we aimed to investigate the effects of sprifermin at the cellular level. A combination of different chondrocyte culture systems was used and the effects of sprifermin on proliferation, the phenotype and matrix production were evaluated. The involvement of MAPKs in sprifermin signalling was also studied. In monolayer, we observed that sprifermin promoted a round cell morphology and stimulated both cellular proliferation and Sox9 expression while strongly decreasing type I collagen expression. In 3D culture, sprifermin increased the number of matrix-producing chondrocytes, improved the type II:I collagen ratio and enabled human OA chondrocytes to produce a hyaline extracellular matrix (ECM). Furthermore, we found that sprifermin displayed a 'hit and run' mode of action, with intermittent exposure required for the compound to fully exert its anabolic effect. Finally, sprifermin appeared to signal through activation of ERK. Our results indicate that intermittent exposure to sprifermin leads to expansion of hyaline cartilage-producing chondrocytes. These in vitro findings are consistent with the increased cartilage volume observed in the knees of OA patients after intra-articular injection with sprifermin in clinical studies. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

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

    PubMed

    Nazempour, A; Van Wie, B J

    2016-05-01

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

  15. Type VI Collagen Regulates Pericellular Matrix Properties, Chondrocyte Swelling, and Mechanotransduction in Mouse Articular Cartilage.

    PubMed

    Zelenski, Nicole A; Leddy, Holly A; Sanchez-Adams, Johannah; Zhang, Jinzi; Bonaldo, Paolo; Liedtke, Wolfgang; Guilak, Farshid

    2015-05-01

    Mechanical factors play a critical role in the physiology and pathology of articular cartilage, although the mechanisms of mechanical signal transduction are not fully understood. We undertook this study to test the hypothesis that type VI collagen is necessary for mechanotransduction in articular cartilage by determining the effects of type VI collagen knockout on the activation of the mechano-osmosensitive, calcium-permeable channel TRPV4 (transient receptor potential vanilloid channel 4) as well as on osmotically induced chondrocyte swelling and pericellular matrix (PCM) mechanical properties. Confocal laser scanning microscopy was used to image TRPV4-mediated calcium signaling and osmotically induced cell swelling in intact femora from 2- and 9-month-old wild-type (WT) and type VI collagen-deficient (Col6a1(-/-)) mice. Immunofluorescence-guided atomic force microscopy was used to map PCM mechanical properties based on the presence of perlecan. Hypo-osmotic stress-induced TRPV4-mediated calcium signaling was increased in Col6a1(-/-) mice relative to WT controls at 2 months. Col6a1(-/-) mice exhibited significantly increased osmotically induced cell swelling and decreased PCM moduli relative to WT controls at both ages. In contrast to our original hypothesis, type VI collagen was not required for TRPV4-mediated Ca(2+) signaling; however, knockout of type VI collagen altered the mechanical properties of the PCM, which in turn increased the extent of cell swelling and osmotically induced TRPV4 signaling in an age-dependent manner. These findings emphasize the role of the PCM as a transducer of mechanical and physicochemical signals, and they suggest that alterations in PCM properties, as may occur with aging or osteoarthritis, can influence mechanotransduction via TRPV4 or other ion channels. © 2015, American College of Rheumatology.

  16. Evaluating Osteoarthritic Chondrocytes through a Novel 3-Dimensional In Vitro System for Cartilage Tissue Engineering and Regeneration.

    PubMed

    Li, Hanwei; Davison, Noel; Moroni, Lorenzo; Feng, Felicia; Crist, Joshua; Salter, Erin; Bingham, Clifton O; Elisseeff, Jennifer

    2012-04-01

    To characterize and evaluate osteoarthritic (OA) chondrocytes, in comparison to normal chondrocytes, through a novel 3-dimensional (3-D) culture system, poly(ethylene-glycol) diacrylate (PEGDA). The cytokine interleukin 1β (IL-1β) was also used to simulate an in vitro OA model. Normal and OA chondrocytes were cultured in monolayer and analyzed for changes in cartilage-specific gene expressions due to passage number. Then, cells were encapsulated in PEGDA to evaluate phenotype and matrix production capabilities through the in vitro culture system. Characterization was conducted with polymerase chain reaction (PCR), biochemical analyses, and histological staining. 3-D encapsulated chondrocytes (human and bovine) were also treated with IL-1β to characterize how the cytokine affects gene transcription and extracellular matrix (ECM) content. In 2-dimensional monolayer, anabolic genes were down-regulated significantly in both normal and OA chondrocytes. In 3-D culture, OA chondrocytes demonstrated significantly higher expressions of catabolic genes when compared to normal cells. Differentiation medium resulted in significantly more matrix production than growth medium from OA chondrocytes, indicated through histological staining. In addition, normal chondrocytes responded more significantly to exogenous administration of IL-1β than OA chondrocytes. Temporary initial stimulation of IL-1β to OA chondrocytes resulted in comparable gene expressions to untreated cells after 3 weeks of in vitro culture. Our findings demonstrate the use of OA chondrocytes in tissue engineering and their significance for potential future cartilage regeneration research through their matrix production capabilities and the use of a hydrogel culture system.

  17. Mechanical Impact Induces Cartilage Degradation via Mitogen Activated Protein Kinases

    PubMed Central

    Ding, Lei; Heying, Emily; Nicholson, Nathan; Stroud, Nicolas J.; Homandberg, Gene A.; Guo, Danping; Buckwalter, Joseph A.; Martin, James A.

    2010-01-01

    Objective To determine the activation of MAP kinases in and around cartilage subjected to mechanical damage and to determine the effects of their inhibitors on impaction induced chondrocyte death and cartilage degeneration. Design The phosphorylation of MAP kinases was examined with confocal microscopy and immunoblotting. The effects of MAP kinase inhibitors on impaction-induced chondrocyte death and proteoglycan loss were determined with fluorescent microscopy and DMMB assay. The expression of catabolic genes at mRNA levels was examined with quantitative real time PCR. Results Early p38 activation was detected at 20 min and 1 hr post-impaction. At 24 hr, enhanced phosphorylation of p38 and ERK1/2 was visualized in chondrocytes from in and around impact sites. The phosphorylation of p38 was increased by 3.0-fold in impact sites and 3.3-fold in adjacent cartilage. The phosphorylation of ERK-1 was increased by 5.8-fold in impact zone and 5.4-fold in adjacent cartilage; the phosphorylation of ERK-2 increased by 4.0-fold in impacted zone and 3.6-fold in adjacent cartilage. Furthermore, the blocking of p38 pathway did not inhibit impaction-induced ERK activation. The inhibition of p38 or ERK pathway significantly reduced injury-related chondrocyte death and proteoglycan losses. Quantative Real-time PCR analysis revealed that blunt impaction significantly up-regulated MMP-13, TNF-α, and ADAMTS-5 expression. Conclusion These findings implicate p38 and ERK MAPKs in the post injury spread of cartilage degeneration and suggest that the risk of PTOA following joint trauma could be decreased by blocking their activities, which might be involved in up-regulating expressions of MMP-13, ADAMTS-5, and TNF-α. PMID:20813194

  18. Conditional Deletion of the Phd2 Gene in Articular Chondrocytes Accelerates Differentiation and Reduces Articular Cartilage Thickness

    PubMed Central

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

    2017-01-01

    Based on our findings that PHD2 is a negative regulator of chondrocyte differentiation and that hypoxia signaling is implicated in the pathogenesis of osteoarthritis, we investigated the consequence of disruption of the Phd2 gene in chondrocytes on the articular cartilage phenotype in mice. Immunohistochemistry detected high expression of PHD2 in the superficial zone (SZ), while PHD3 and HIF-1α (target of PHD2) are mainly expressed in the middle-deep zone (MDZ). Conditional deletion of the Phd2 gene (cKO) in chondrocytes accelerated the transition of progenitors to hypertrophic (differentiating) chondrocytes as revealed by reduced SZ thickness, and increased MDZ thickness, as well as increased chondrocyte hypertrophy. Immunohistochemistry further revealed decreased levels of progenitor markers but increased levels of hypertrophy markers in the articular cartilage of the cKO mice. Treatment of primary articular chondrocytes, in vitro, with IOX2, a specific inhibitor of PHD2, promoted articular chondrocyte differentiation. Knockdown of Hif-1α expression in primary articular chondrocytes using lentiviral vectors containing Hif-1α shRNA resulted in reduced expression levels of Vegf, Glut1, Pgk1, and Col10 compared to control shRNA. We conclude that Phd2 is a key regulator of articular cartilage development that acts by inhibiting the differentiation of articular cartilage progenitors via modulating HIF-1α signaling. PMID:28349987

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

    NASA Astrophysics Data System (ADS)

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

    2012-03-01

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

  20. An injectable cellulose-based hydrogel for the transfer of autologous nasal chondrocytes in articular cartilage defects.

    PubMed

    Vinatier, C; Gauthier, O; Fatimi, A; Merceron, C; Masson, M; Moreau, A; Moreau, F; Fellah, B; Weiss, P; Guicheux, J

    2009-03-01

    Articular cartilage has a low capacity for spontaneous repair. To promote the repair of this tissue, the transfer of autologous chondrocytes using a three-dimensional matrix appears promising. In this context, the aim of the present work was to investigate the potential use of autologous rabbit nasal chondrocytes (RNC) associated with an injectable self-setting cellulose-based hydrogel (Si-HPMC). Firstly, the influence of Si-HPMC on chondrocytic phenotype was investigated by real-time PCR for specific chondrocyte markers (type II collagen and aggrecan) and type I collagen. Thereafter, autologous RNC were amplified in vitro for 4 weeks before transplantation with Si-HPMC into a rabbit articular cartilage defect followed by analysis 6 weeks later. Implants were histologically characterized for the presence of sulfated GAG and type II collagen. Transcripts analysis indicated that dedifferentiated RNC recovered expression of the main chondrocytic markers after in vitro three-dimensional culture within Si-HPMC. Histological analysis of autologous RNC transplanted in an articular cartilage defect revealed the formation of repair tissue with a histological organization similar to that of healthy articular cartilage. In addition, immunohistological analysis of type II collagen suggested that the repair tissue was a hyaline-like cartilage. Si-HPMC hydrogel associated with nasal chondrocytes therefore appears a promising injectable tissue engineering device for the repair of articular cartilage.

  1. Hyaline cartilage tissue is formed through the co-culture of passaged human chondrocytes and primary bovine chondrocytes.

    PubMed

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

    2012-08-01

    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.

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

    PubMed

    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 E2, 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.

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

    PubMed

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

    2010-06-01

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

  4. [Chondrocyte glycosyltransferases: new pharmacological targets for degenerative diseases of articular cartilage?].

    PubMed

    Magdalou, Jacques; Ouzzine, Mohamed; Netter, Patrick; Fournel-Gigleux, Sylvie

    2006-10-01

    Arthritis, osteoarthritis and other degenerative diseases characterized by cartilage deterioration are the most prevalent chronic human health disorders. Despite their major socioeconomic impact there is still no satisfactory treatment. Their frequency is increasing with the lengthening of life expectancy, creating a major public health challenge for coming years. It is important to diagnose such diseases at an early stage and to develop new effective therapies. We are attempting to develop new therapeutic approaches in this context, keeping in mind that cartilage is one of the few human tissues which is unable to regenerate. We intend to identify and characterize key proteins involved in the biosynthesis of cartilage matrix components. One innovative strategy consists of gene transfer, triggering overexpression of native or recombinant factors that can stimulate chondrocyte anabolic activity in order to promote cartilage repair The loss of matrix components, and especially glycosaminoglycans (GAG), is the earliest event in cartilage degeneration. We therefore looked at glycosyltransferases, and especially galactose beta1,3-glucuronosyltransferase-I (GlcAT-1), which catalyses one of the first steps in GAG biosynthesis. We found that any variation in GlcAT-I activity in chondrocytes or cartilage explants (overexpression, or repression with antisense RNA) affected the GAG content of cartilage. Interestingly, overexpression of this enzyme completely counteracted the GAG depletion produced by the proinflammatory cytokine interleukin 1-beta. The neosynthesized GAG was qualitatively identical to that present in the original cartilage matrix. These results are encouraging for therapeutic approaches based on gene transfer We also investigated the structure-function relationship of human recombinant GlcAT-I upon expression in the methyltrophic yeast Pichia pastoris. This allowed us to determine the molecular basis of the recognition of the donor and acceptor substrates of

  5. Regenerative Potential of Tissue-Engineered Nasal Chondrocytes in Goat Articular Cartilage Defects.

    PubMed

    Mumme, Marcus; Steinitz, Amir; Nuss, Katja M; Klein, Karina; Feliciano, Sandra; Kronen, Peter; Jakob, Marcel; von Rechenberg, Brigitte; Martin, Ivan; Barbero, Andrea; Pelttari, Karoliina

    2016-11-01

    Nasal chondrocytes (NC) were previously demonstrated to remain viable and to participate in the repair of articular cartilage defects in goats. Here, we investigated critical features of tissue-engineered grafts generated by NC in this large animal model, namely cell retention at the implantation site, architecture and integration with adjacent tissues, and effects on subchondral bone changes. In this study, isolated autologous goat NC (gNC) and goat articular chondrocytes (gAC, as control) were expanded, green fluorescent protein-labelled and seeded on a type I/III collagen membrane. After chondrogenic differentiation, tissue-engineered grafts were implanted into chondral defects (6 mm in diameter) in the stifle joint for 3 or 6 months. At the time of explantation, surrounding tissues showed no or very low (only in the infrapatellar fat pad <0.32%) migration of the grafted cells. In repair tissue, gNC formed typical structures of articular cartilage, such as flattened cells at the surface and column-like clusters in the middle layers. Semi-quantitative histological evaluation revealed efficient integration of the grafted tissues with the adjacent native cartilage and underlying subchondral bone. A significantly increased subchondral bone area, as a sign for the onset of osteoarthritis, was observed following treatment of cartilage defects with gAC-, but not with gNC-grafts. Our results reinforce the use of NC-based engineered tissue for articular cartilage repair and preliminarily indicate their potential for the treatment of early osteoarthritic defects.

  6. Bushen Zhuangjin decoction inhibits TM-induced chondrocyte apoptosis mediated by endoplasmic reticulum stress.

    PubMed

    Lin, Pingdong; Weng, Xiaping; Liu, Fayuan; Ma, Yuhuan; Chen, Houhuang; Shao, Xiang; Zheng, Wenwei; Liu, Xianxiang; Ye, Hongzhi; Li, Xihai

    2015-12-01

    Chondrocyte apoptosis triggered by endoplasmic reticulum (ER) stress plays a vital role in the pathogenesis of osteoarthritis (OA). Bushen Zhuangjin decoction (BZD) has been widely used in the treatment of OA. However, the cellular and molecular mechanisms responsible for the inhibitory effects of BZD on chondrocyte apoptosis remain to be elucidated. In the present study, we investigated the effects of BZD on ER stress-induced chondrocyte apoptosis using a chondrocyte in vitro model of OA. Chondrocytes obtained from the articular cartilage of the knee joints of Sprague Dawley (SD) rats were detected by immunohistochemical staining for type Ⅱ collagen. The ER stress-mediated apoptosis of tunicamycin (TM)‑stimulated chondrocytes was detected using 4-phenylbutyric acid (4‑PBA). We found that 4‑PBA inhibited TM-induced chondrocyte apoptosis, which confirmed the successful induction of chondrocyte apoptosis. BZD enhanced the viability of the TM-stimulated chondrocytes in a dose- and time-dependent manner, as shown by MTT assay. The apoptotic rate and the loss of mitochondrial membrane potential (ΔΨm) of the TM-stimulated chondrocytes treated with BZD was markedly decreased compared with those of chondrocytes not treated with BZD, as shown by 4',6-diamidino-2-phenylindole (DAPI) staining, Annexin V-FITC binding assay and JC-1 assay. To further elucidate the mechanisms responsible for the inhibitory effects of BZD on TM‑induced chondrocyte apoptosis mediated by ER stress, the mRNA and protein expression levels of binding immunoglobulin protein (Bip), X‑box binding protein 1 (Xbp1), activating transcription factor 4 (Atf4), C/EBP‑homologous protein (Chop), caspase‑9, caspase-3, B-cell lymphoma 2 (Bcl-2) and Bcl-2-associated X protein (Bax) were measured by reverse transcription-polymerase chain reaction (RT-PCR) and western blot analysis. In the TM-stimulated chondrocytes treated with BZD, the mRNA and protein expression levels of Bip, Atf4, Chop, caspase

  7. Bushen Zhuangjin decoction inhibits TM-induced chondrocyte apoptosis mediated by endoplasmic reticulum stress

    PubMed Central

    LIN, PINGDONG; WENG, XIAPING; LIU, FAYUAN; MA, YUHUAN; CHEN, HOUHUANG; SHAO, XIANG; ZHENG, WENWEI; LIU, XIANXIANG; YE, HONGZHI; LI, XIHAI

    2015-01-01

    Chondrocyte apoptosis triggered by endoplasmic reticulum (ER) stress plays a vital role in the pathogenesis of osteoarthritis (OA). Bushen Zhuangjin decoction (BZD) has been widely used in the treatment of OA. However, the cellular and molecular mechanisms responsible for the inhibitory effects of BZD on chondrocyte apoptosis remain to be elucidated. In the present study, we investigated the effects of BZD on ER stress-induced chondrocyte apoptosis using a chondrocyte in vitro model of OA. Chondrocytes obtained from the articular cartilage of the knee joints of Sprague Dawley (SD) rats were detected by immunohistochemical staining for type II collagen. The ER stress-mediated apoptosis of tunicamycin (TM)-stimulated chondrocytes was detected using 4-phenylbutyric acid (4-PBA). We found that 4-PBA inhibited TM-induced chondrocyte apoptosis, which confirmed the successful induction of chondrocyte apoptosis. BZD enhanced the viability of the TM-stimulated chondrocytes in a dose- and time-dependent manner, as shown by MTT assay. The apoptotic rate and the loss of mitochondrial membrane potential (ΔΨm) of the TM-stimulated chondrocytes treated with BZD was markedly decreased compared with those of chondrocytes not treated with BZD, as shown by 4′,6-diamidino-2-phenylindole (DAPI) staining, Annexin V-FITC binding assay and JC-1 assay. To further elucidate the mechanisms responsible for the inhibitory effects of BZD on TM-induced chondrocyte apoptosis mediated by ER stress, the mRNA and protein expression levels of binding immunoglobulin protein (Bip), X-box binding protein 1 (Xbp1), activating transcription factor 4 (Atf4), C/EBP-homologous protein (Chop), caspase-9, caspase-3, B-cell lymphoma 2 (Bcl-2) and Bcl-2-associated X protein (Bax) were measured by reverse transcription-polymerase chain reaction (RT-PCR) and western blot analysis. In the TM-stimulated chondrocytes treated with BZD, the mRNA and protein expression levels of Bip, Atf4, Chop, caspase-9, caspase-3

  8. In vitro glycation of articular cartilage alters the biomechanical response of chondrocytes in a depth-dependent manner.

    PubMed

    Fick, J M; Huttu, M R J; Lammi, M J; Korhonen, R K

    2014-10-01

    To determine if increasing cartilage cross-links through in vitro glycation of cartilage explants can alter the biomechanical response of chondrocytes to compressive deformation. Bovine osteochondral explants were either incubated with cell culture solution supplemented with (n = 7) or without (n = 7) ribose for 42 h in order to induce glycation. Deformation-induced changes in cell volume, dimensions and local tissue strains were determined through confocal laser scanning microscopy (CLSM) and the use of a custom built micro-compression device. Osteochondral explants were also utilized to demonstrate changes in depth-wise tissue properties, biomechanical tissue properties and cross-links such as pentosidine (Pent), hydroxylysyl pyridinoline (HP) and lysyl pyridinoline (LP). The ribose treated osteochondral samples experienced reduced cell volume deformation in the upper tissue zone by ∼ 8% (P = 0.005), as compared the control samples, through restricting cell expansion. In the deeper tissue zone, cell volume deformation was increased by ∼ 12% (P < 0.001) via the transmission of mechanical signals further into the tissue depth. Biomechanical testing of the ribose treated osteochondral samples demonstrated an increase in the equilibrium and dynamic strain dependent moduli (P < 0.001 and P = 0.008, respectively). The biochemical analysis revealed an increase in Pent cross-links (P < 0.001). Depth-wise tissue property analyses revealed increased levels of carbohydrate content, greater levels of fixed charge density and an increased carbohydrate to protein ratio from 6 to 16%, 55-100% and 72-79% of the normalized tissue thickness (from the surface), respectively, in the ribose-treated group (P < 0.05). In vitro glycation alters the biomechanical response of chondrocytes in cartilage differently in upper and deeper zones, offering possible insights into how aging could alter cell deformation behavior in cartilage. Copyright © 2014 Osteoarthritis Research Society

  9. Combined 3D and hypoxic culture improves cartilage-specific gene expression in human chondrocytes.

    PubMed

    Foldager, Casper B; Nielsen, Anna B; Munir, Samir; Ulrich-Vinther, Michael; Søballe, Kjeld; Bünger, Cody; Lind, Martin

    2011-04-01

    In vitro expansion of autologous chondrocytes is an essential part of many clinically used cartilage repair treatments. Native chondrocytes reside in a 3-dimensional (3D) network and are exposed to low levels of oxygen. We compared monolayer culture to combined 3D and hypoxic culture using quantitative gene expression analysis. Cartilage biopsies were collected from the intercondylar groove in the distal femur from 12 patients with healthy cartilage. Cells were used for either monolayer or scaffold culture. The scaffolds were clinically available MPEG-PLGA scaffolds (ASEED). After harvesting of cells for baseline investigation, the remainder was divided into 3 groups for incubation in conditions of normoxia (21% oxygen), hypoxia (5% oxygen), or severe hypoxia (1% oxygen). RNA extractions were performed 1, 2, and 6 days after the baseline time point, respectively. Quantitative RT-PCR was performed using assays for RNA encoding collagen types 1 and 2, aggrecan, sox9, ankyrin repeat domain-37, and glyceraldehyde-3-phosphate dehydrogenase relative to 2 hypoxia-stable housekeeping genes. Sox9, aggrecan, and collagen type 2 RNA expression increased with reduced oxygen. On day 6, the expression of collagen type 2 and aggrecan RNA was higher in 3D culture than in monolayer culture. Our findings suggest that there was a combined positive effect of 3D culture and hypoxia on cartilage-specific gene expression. The positive effects of 3D culture alone were not detected until day 6, suggesting that seeding of chondrocytes onto a scaffold for matrix-assisted chondrocyte implantation should be performed earlier than 2 days before implantation.

  10. Human chondrocyte cultures as models of cartilage-specific gene regulation.

    PubMed

    Otero, Miguel; Favero, Marta; Dragomir, Cecilia; Hachem, Karim El; Hashimoto, Ko; Plumb, Darren A; Goldring, Mary B

    2012-01-01

    The human adult articular chondrocyte is a unique cell type that has reached a fully differentiated state as an end point of development. Within the cartilage matrix, chondrocytes are normally quiescent and maintain the matrix constituents in a low-turnover state of equilibrium. Isolated chondrocytes in culture have provided useful models to study cellular responses to alterations in the environment such as those occurring in different forms of arthritis. However, expansion of primary chondrocytes in monolayer culture results in the loss of phenotype, particularly if high cell density is not maintained. This chapter describes strategies for maintaining or restoring differentiated phenotype by culture in suspension, gels, or scaffolds. Techniques for assessing phenotype involving primarily the analysis of synthesis of cartilage-specific matrix proteins as well as the corresponding mRNAs are also described. Approaches for studying gene regulation, including transfection of promoter-driven reporter genes with expression vectors for transcriptional and signaling regulators, chromatin immunoprecipitation, and DNA methylation are also described.

  11. Intermittent compressive strain may reduce aggrecanase expression in cartilage: a study of chondrocytes in agarose gel.

    PubMed

    Mio, Kensuke; Saito, Seiji; Tomatsu, Taisuke; Toyama, Yoshiaki

    2005-04-01

    We tested the hypothesis that intermittent compressive strain reduces the catabolic actions of interleukin-1 beta on chondrocyte metabolism at the gene expression level. We investigated the effects of intermittent compressive strain on mRNA expression in bovine chondrocytes cultured in agarose gel supplemented with or without interleukin-1 beta. Fifteen percent compressive strain amplitude was applied to agarose-chondrocyte constructs at a frequency of 1 Hz. In the absence of interleukin-1 beta, the strain caused an increase in the mRNA levels of anabolic factors like aggrecan and Type II collagen, compared with the levels of anabolic factors in unstrained constructs. These results suggest that the strain may stimulate matrix production in normal cartilage. Interleukin-1 beta is a powerful catabolic agent; in unstrained agarose-chondrocyte constructs, interleukin-1 beta caused a decrease in the mRNA levels of anabolic factors. However, interleukin-1 beta also caused an increase in the mRNA of catabolic factors like aggrecanase-1, aggrecanase-2, and matrix metalloproteinase-3. In the presence of interleukin-1 beta, the strain reduced the mRNA levels of aggrecanase-1 and aggrecanase-2. These results suggest that intermittent compressive strain may protect cartilage by suppressing the expression of aggrecanase-1 and aggrecanase-2, which are thought to be the major matrix-degrading enzymes responsible for cleaving aggrecan.

  12. Scaffold-free cartilage tissue engineering with a small population of human nasoseptal chondrocytes.

    PubMed

    Chiu, Loraine L Y; To, William T H; Lee, John M; Waldman, Stephen D

    2017-03-01

    Cartilage tissue engineering is a promising approach to provide suitable materials for nasal reconstruction; however, it typically requires large numbers of cells. We have previously shown that a small number of chondrocytes cultivated within a continuous flow bioreactor can elicit substantial tissue growth, but translation to human chondrocytes is not trivial. Here, we aimed to demonstrate the application of the bioreactor to generate large-sized tissues from a small population of primary human nasoseptal chondrocytes. Experimental study. Chondrocytes were cultured in the bioreactor using different medium compositions, with varying amounts of serum and with or without growth factors. Resulting engineered tissues were analyzed for physical properties, biochemical composition, tissue microstructure, and protein localization. Bioreactor-cultivated constructs grown with serum and growth factors (basic fibroblast growth factor and transforming growth factor beta 2) had greater thickness, as well as DNA and glycosaminoglycan (GAG) contents, compared to low serum and no growth factor controls. These constructs also showed the most intense proteoglycan and collagen II staining. The combination of bioreactor conditions, serum, and growth factors allowed the generation of large, thick scaffold-free human cartilaginous tissues that resembled the native nasoseptal cartilage. There also may be implications for patient selection in future clinical applications of these engineered tissues because their GAG content decreased with donor age. NA. Laryngoscope, 127:E91-E99, 2017. © 2016 The American Laryngological, Rhinological and Otological Society, Inc.

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

    PubMed

    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

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

    PubMed

    He, Xiaomin; Feng, Bei; Huang, Chuanpei; Wang, Hao; Ge, Yang; Hu, Renjie; Yin, Meng; Xu, Zhiwei; Wang, Wei; Fu, Wei; Zheng, Jinghao

    2015-01-01

    Electrospinning has recently received considerable attention, showing notable potential as a novel method of scaffold fabrication for cartilage engineering. The aim of this study was to use a coculture strategy of chondrocytes combined with electrospun gelatin/polycaprolactone (GT/PCL) membranes, instead of pure chondrocytes, to evaluate the formation of cartilaginous tissue. We prepared the GT/PCL membranes, seeded bone marrow stromal cell (BMSC)/chondrocyte cocultures (75% BMSCs and 25% chondrocytes) in a sandwich model in vitro, and then implanted the constructs subcutaneously into nude mice for 12 weeks. Gross observation, histological and immunohistological evaluation, glycosaminoglycan analyses, Young's modulus measurement, and immunofluorescence staining were performed postimplantation. We found that the coculture group formed mature cartilage-like tissue, with no statistically significant difference from the chondrocyte group, and labeled BMSCs could differentiate into chondrocyte-like cells under the chondrogenic niche of chondrocytes. This entire strategy indicates that GT/PCL membranes are also a suitable scaffold for stem cell-based cartilage engineering and may provide a potentially clinically feasible approach for cartilage repairs.

  15. Parathyroid hormone 1-34 reduces dexamethasone-induced terminal differentiation in human articular chondrocytes.

    PubMed

    Chang, Ling-Hua; Wu, Shun-Cheng; Chen, Chung-Hwan; Wang, Gwo-Jaw; Chang, Je-Ken; Ho, Mei-Ling

    2016-08-10

    Intra-articular injection of dexamethasone (Dex) is occasionally used to relieve pain and inflammation in osteoarthritis (OA) patients. Dex induces terminal differentiation of chondrogenic mesenchymal stem cells in vitro and causes impaired longitudinal skeletal growth in vivo. Parathyroid hormone 1-34 (PTH 1-34) has been shown to reverse terminal differentiation of osteoarthritic articular chondrocytes. We hypothesized that Dex induces terminal differentiation of articular chondrocytes and that this effect can be mitigated by PTH 1-34 treatment. We tested the effect of Dex on terminal differentiation in human articular chondrocytes and further tested if PTH 1-34 reverses the effects. We found that Dex treatment downregulated chondrogenic-induced expressions of SOX-9, collagen type IIa1 (Col2a1), and aggrecan and reduced synthesis of cartilaginous matrix (Col2a1 and sulfated glycosaminoglycan) synthesis. Dex treatment upregulated chondrocyte hypertrophic markers of collagen type X and alkaline phosphatase at mRNA and protein levels, and it increased the cell size of articular chondrocytes and induced cell death. These results indicated that Dex induces terminal differentiation of articular chondrocytes. To test whether PTH 1-34 treatment reverses Dex-induced terminal differentiation of articular chondrocytes, PTH 1-34 was co-administered with Dex. Results showed that PTH 1-34 treatment reversed both changes of chondrogenic and hypertrophic markers in chondrocytes induced by Dex. PTH 1-34 also decreased Dex-induced cell death. PTH 1-34 treatment reduces Dex-induced terminal differentiation and apoptosis of articular chondrocytes, and PTH 1-34 treatment may protect articular cartilage from further damage when received Dex administration.

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

    PubMed Central

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

    2016-01-01

    The mechanical environment is crucial for intervertebral disc degeneration (IDD). However, the mechanisms underlying the regulation of cartilage endplate (CEP) calcification by altered matrix stiffness remain unclear. In this study, we found that matrix stiffness of CEP was positively correlated with the degree of IDD, and stiff matrix, which mimicked the severe degeneration of CEP, promoted inorganic phosphate-induced calcification in CEP chondrocytes. Co-expression analysis of the miRNA and mRNA profiles showed that increasing stiffness resulted in up-regulation of miR-20a and down-regulation of decreased ankylosis protein homolog (ANKH) during inorganic phosphate-induced calcification in CEP chondrocytes. Through a dual luciferase reporter assay, we confirmed that miR-20a directly targets 3′-untranslated regions of ANKH. The inhibition of miR-20a attenuated the calcium deposition and calcification-related gene expression, whereas the overexpression of miR-20a enhanced calcification in CEP chondrocytes on stiff matrix. The rescue of ANKH expression restored the decreased pyrophosphate efflux and inhibited calcification. In clinical samples, the levels of ANKH expression were inversely associated with the degeneration degree of CEP. Thus, our findings demonstrate that the miR-20a/ANKH axis mediates the stiff matrix- promoted CEP calcification, suggesting that miR-20a and ANKH are potential targets in restraining the progression of IDD. PMID:27142968

  17. Transplantation of autologous chondrocytes ex-vivo expanded using Thermoreversible Gelation Polymer in a rabbit model of articular cartilage defect.

    PubMed

    Arumugam, Sivaraman; Bhupesh Karthik, Balasubramanyan; Chinnuswami, Rajeswar; Mori, Yuichi; Yoshioka, Hiroshi; Senthilkumar, Rajappa; Mathaiyan, Rajmohan; Ramalingam, Karthick; Senthilkumar, Preethy; Abraham, Samuel J K

    2017-06-01

    Graft failure due to de-differentiation of the chondrocytes during in vitro culture and after transplantation is a major hurdle in Autologous Chondrocyte Implantation (ACI). We, herein, report the transplantation of autologous chondrocytes ex vivo expanded using a Thermo-reversible Gelation Polymer (TGP) in a rabbit model. A full thickness chondral defect was created in one of the knee joints in each of the six rabbits of the study and autologous chondrocytes in vitro expanded using TGP scaffold were transplanted after 10 weeks. H & E staining of the biopsy after 6 months revealed maintenance of articular cartilage phenotype.

  18. Alterations in the Young's modulus and volumetric properties of chondrocytes isolated from normal and osteoarthritic human cartilage.

    PubMed

    Jones, W R; Ting-Beall, H P; Lee, G M; Kelley, S S; Hochmuth, R M; Guilak, F

    1999-02-01

    The mechanical environment of the chondrocyte is an important factor that influences the maintenance of the articular cartilage extracellular matrix. Previous studies have utilized theoretical models of chondrocytes within articular cartilage to predict the stress-strain and fluid flow environments around the cell, but little is currently known regarding the cellular properties which are required for implementation of these models. The objectives of this study were to characterize the mechanical behavior of primary human chondrocytes and to determine the Young's modulus of chondrocytes from non-osteoarthritic ('normal') and osteoarthritic cartilage. A second goal was to quantify changes in the volume of isolated chondrocytes in response to mechanical deformation. The micropipette aspiration technique was used to measure the deformation of a single chondrocyte into a glass micropipette in response to a prescribed pressure. The results of this study indicate that the human chondrocyte behaves as a viscoelastic solid. No differences were found between the Young's moduli of normal (0.65+/-0.63 kPa, n = 44) and osteoarthritic chondrocytes (0.67+/-0.86 kPa, n = 69, p = 0.93). A significant difference in cell volume was observed immediately and 600 s after complete aspiration of the cell into the pipette (p < 0.001), and the magnitude of this volume change between normal (11+/-11%, n = 40) and osteoarthritic (20+/-11%, n = 41) chondroctyes was significantly different at both time points (p < 0.002). This finding suggests that chondrocytes from osteoarthritic cartilage may have altered volume regulation capabilities in response to mechanical deformation. The mechanical and volumetric properties determined in this study will be of use in analytical and finite element models of chondrocyte-matrix interactions in order to better predict the mechanical environment of the cell in vivo.

  19. Similar Properties of Chondrocytes from Osteoarthritis Joints and Mesenchymal Stem Cells from Healthy Donors for Tissue Engineering of Articular Cartilage

    PubMed Central

    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. PMID:23671648

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

    PubMed

    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.

  1. Human chondrocyte migration behaviour to guide the development of engineered cartilage.

    PubMed

    O'Connell, Grace D; Tan, Andrea R; Cui, Victoria; Bulinski, J Chloe; Cook, James L; Attur, Mukundan; Abramson, Steven B; Ateshian, Gerard A; Hung, Clark T

    2017-03-01

    Tissue-engineering techniques have been successful in developing cartilage-like tissues in vitro using cells from animal sources. The successful translation of these strategies to the clinic will likely require cell expansion to achieve sufficient cell numbers. Using a two-dimensional (2D) cell migration assay to first identify the passage at which chondrocytes exhibited their greatest chondrogenic potential, the objective of this study was to determine a more optimal culture medium for developing three-dimensional (3D) cartilage-like tissues using human cells. We evaluated combinations of commonly used growth factors that have been shown to promote chondrogenic growth and development. Human articular chondrocytes (AC) from osteoarthritic (OA) joints were cultured in 3D environments, either in pellets or encapsulated in agarose. The effect of growth factor supplementation was dependent on the environment, such that matrix deposition differed between the two culture systems. ACs in pellet culture were more responsive to bone morphogenetic protein (BMP2) alone or combinations containing BMP2 (i.e. BMP2 with PDGF or FGF). However, engineered cartilage development within agarose was better for constructs cultured with TGFβ3. These results with agarose and pellet culture studies set the stage for the development of conditions appropriate for culturing 3D functional engineered cartilage for eventual use in human therapies. Copyright © 2015 John Wiley & Sons, Ltd.

  2. Effects of CCN3 on rat cartilage endplate chondrocytes cultured under serum deprivation in vitro

    PubMed Central

    DING, LEI; WU, JINGPING; LI, DEFANG; WANG, HOULEI; ZHU, BIN; LU, WEI; XU, GUOXIONG

    2016-01-01

    The presence of apoptotic cells and loss of extracellular matrix (ECM) are common characteristics of degenerated cartilage endplates (CEPs). In addition, therapeutic efficacy is hampered by an incomplete understanding regarding the mechanisms underlying CEP homeostasis and degeneration. The CCN proteins have recently emerged as important regulators of cell-ECM interactions, and have been identified as key mediators of nucleus pulposus ECM composition and tissue homeostasis. However, whether CCN3 is associated with CEP homeostasis has yet to be elucidated. The present study aimed to investigate the effects of CCN3 on the apoptosis and ECM synthesis of CEP cells cultured under serum deprivation. Rat CEP cells were confirmed to be of the chondrocytic phenotype by toluidine blue staining. The mRNA expression levels of CCN3 were markedly increased, and a dose-dependent increase of apoptotic rate was detected under serum deprivation conditions following treatment with recombinant CCN3, whereas CCN3 did not exert a proapoptotic effect on cells cultured under normal conditions. Furthermore, CCN3-treated cells exhibited a decrease in the expression levels of aggrecan and collagen II in both groups. These results suggested that CCN3 may act as a regulator, rather than an initiator, of serum deprivation-induced cellular apoptosis, and that CCN3 has a catabolic effect on the mediation of ECM synthesis under both normal and serum deprivation conditions. Therefore, CCN3 may represent a novel therapeutic target for the prevention of CEP degeneration. PMID:26795879

  3. Regulation of human chondrocyte function through direct inhibition of cartilage master regulator SOX9 by microRNA-145 (miRNA-145).

    PubMed

    Martinez-Sanchez, Aida; Dudek, Katarzyna A; Murphy, Chris L

    2012-01-06

    Articular cartilage enables weight bearing and near friction-free movement in the joints. Critical to its function is the production of a specialized, mechanocompetent extracellular matrix controlled by master regulator transcription factor SOX9. Mutations in SOX9 cause campomelic dysplasia, a haploinsufficiency disorder resulting in severe skeletal defects and dwarfism. Although much is understood about how SOX9 regulates cartilage matrix synthesis and hence joint function, how this master regulator is itself regulated remains largely unknown. Here we identify a specific microRNA, miR-145, as a direct regulator of SOX9 in normal healthy human articular chondrocytes. We show that miR-145 directly represses SOX9 expression in human cells through a unique binding site in its 3'-UTR not conserved in mice. Modulation of miR-145 induced profound changes in the human chondrocyte phenotype. Specifically, increased miR-145 levels cause greatly reduced expression of critical cartilage extracellular matrix genes (COL2A1 and aggrecan) and tissue-specific microRNAs (miR-675 and miR-140) and increased levels of the hypertrophic markers RUNX2 and MMP13, characteristic of changes occurring in osteoarthritis. We propose miR-145 as an important regulator of human chondrocyte function and a new target for cartilage repair.

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

    PubMed

    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

  5. Chondrocyte Apoptosis Is Not Essential for Cartilage Calcification: Evidence From an In Vitro Avian Model

    PubMed Central

    Pourmand, Eric P.; Binderman, Itzhak; Doty, Stephen B.; Kudryashov, Valery; Boskey, Adele L.

    2006-01-01

    The calcification of cartilage is an essential step in the process of normal bone growth through endochondral ossification. Chondrocyte apoptosis is generally observed prior to the transition of calcified cartilage to bone. There are, however, contradictory reports in the literature as to whether chondrocyte apoptosis is a precursor to cartilage calcification, a co-event, or occurs after calcification. The purpose of this study was to test the hypothesis that chondrocyte apoptosis is not a requirement for initial calcification using a cell culture system that mimics endochondral ossification. Mesenchymal stem cells harvested from Stages 21-23 chick limb buds were plated as micro-mass cultures in the presence of 4 mM inorganic phosphate (mineralizing conditions). The cultures were treated with either an apoptosis inhibitor or stimulator and compared to un-treated controls before the start of calcification on day 7. Inhibition of apoptosis with the caspase inhibitor Z-Val-Ala-Asp (O-Me)-fluoromethylketone (Z-VAD-fmk) caused no decreases in calcification as indicated by radioactive calcium uptake or Fourier transform infrared (FT-IR) analysis of mineral properties. When apoptosis was inhibited, the cultures showed more robust histological features (including more intense staining for proteoglycans, and more intact cells within the nodules as well as along the periphery of the cells as compared to untreated controls), more proliferation as noted by bromo-deoxyuridine (BrdU) labeling, decreases in terminal deoxynucleotidyl transferase (Tdt)-mediated dUTP nick-end labeling (TUNEL) staining, and fewer apoptotic bodies in electron microscopy. Stimulation of apoptosis with 40-120 nM staurosporine prior to the onset of calcification resulted in inhibition of calcium accretion, with the extent of total calcium uptake significantly decreased, the amount of matrix deposition impaired, and the formation of abnormal mineral crystals. These results indicate that chondrocyte

  6. Chondrocyte apoptosis is not essential for cartilage calcification: evidence from an in vitro avian model.

    PubMed

    Pourmand, Eric P; Binderman, Itzhak; Doty, Stephen B; Kudryashov, Valery; Boskey, Adele L

    2007-01-01

    The calcification of cartilage is an essential step in the process of normal bone growth through endochondral ossification. Chondrocyte apoptosis is generally observed prior to the transition of calcified cartilage to bone. There are, however, contradictory reports in the literature as to whether chondrocyte apoptosis is a precursor to cartilage calcification, a co-event, or occurs after calcification. The purpose of this study was to test the hypothesis that chondrocyte apoptosis is not a requirement for initial calcification using a cell culture system that mimics endochondral ossification. Mesenchymal stem cells harvested from Stages 21-23 chick limb buds were plated as micro-mass cultures in the presence of 4 mM inorganic phosphate (mineralizing conditions). The cultures were treated with either an apoptosis inhibitor or stimulator and compared to un-treated controls before the start of calcification on day 7. Inhibition of apoptosis with the caspase inhibitor Z-Val-Ala-Asp (O-Me)-fluoromethylketone (Z-VAD-fmk) caused no decreases in calcification as indicated by radioactive calcium uptake or Fourier transform infrared (FT-IR) analysis of mineral properties. When apoptosis was inhibited, the cultures showed more robust histological features (including more intense staining for proteoglycans, and more intact cells within the nodules as well as along the periphery of the cells as compared to untreated controls), more proliferation as noted by bromo-deoxyuridine (BrdU) labeling, decreases in terminal deoxynucleotidyl transferase (Tdt)-mediated dUTP nick-end labeling (TUNEL) staining, and fewer apoptotic bodies in electron microscopy. Stimulation of apoptosis with 40-120 nM staurosporine prior to the onset of calcification resulted in inhibition of calcium accretion, with the extent of total calcium uptake significantly decreased, the amount of matrix deposition impaired, and the formation of abnormal mineral crystals. These results indicate that chondrocyte

  7. Alpha B-Crystallin Protects Rat Articular Chondrocytes against Casein Kinase II Inhibition-Induced Apoptosis.

    PubMed

    Lee, Sung Won; Rho, Jee Hyun; Lee, Sang Yeob; Yoo, Seung Hee; Kim, Hye Young; Chung, Won Tae; Yoo, Young Hyun

    2016-01-01

    Although alpha (α)B-crystallin is expressed in articular chondrocytes, little is known about its role in these cells. Protein kinase casein kinase 2 (CK2) inhibition induces articular chondrocyte death. The present study examines whether αB-crystallin exerts anti-apoptotic activity in articular chondrocytes. Primary rat articular chondrocytes were isolated from knee joint slices. Cells were treated with CK2 inhibitors with or without αB-crystallin siRNA. To examine whether the silencing of αB-crystallin sensitizes rat articular chondrocytes to CK2 inhibition-induced apoptosis, we assessed apoptosis by performing viability assays, mitochondrial membrane potential measurements, flow cytometry, nuclear morphology observations, and western blot analysis. To investigate the mechanism by which αB-crystallin modulates the extent of CK2 inhibition-mediated chondrocyte death, we utilized confocal microscopy to observe the subcellular location of αB-crystallin and its phosphorylated forms and performed a co-immunoprecipitation assay to observe the interaction between αB-crystallin and CK2. Immunochemistry was employed to examine αB-crystallin expression in cartilage obtained from rats with experimentally induced osteoarthritis (OA). Our results demonstrated that silencing of αB-crystallin sensitized rat articular chondrocytes to CK2 inhibitor-induced apoptosis. Furthermore, CK2 inhibition modulated the expression and subcellular localization of αB-crystallin and its phosphorylated forms and dissociated αB-crystallin from CK2. The population of rat articular chondrocytes expressing αB-crystallin and its phosphorylated forms was reduced in an experimentally induced rat model of OA. In summary, αB-crystallin protects rat articular chondrocytes against CK2 inhibition-induced apoptosis. αB-crystallin may represent a suitable target for pharmacological interventions to prevent OA.

  8. Alpha B-Crystallin Protects Rat Articular Chondrocytes against Casein Kinase II Inhibition-Induced Apoptosis

    PubMed Central

    Rho, Jee Hyun; Lee, Sang Yeob; Yoo, Seung Hee; Kim, Hye Young; Chung, Won Tae; Yoo, Young Hyun

    2016-01-01

    Although alpha (α)B-crystallin is expressed in articular chondrocytes, little is known about its role in these cells. Protein kinase casein kinase 2 (CK2) inhibition induces articular chondrocyte death. The present study examines whether αB-crystallin exerts anti-apoptotic activity in articular chondrocytes. Primary rat articular chondrocytes were isolated from knee joint slices. Cells were treated with CK2 inhibitors with or without αB-crystallin siRNA. To examine whether the silencing of αB-crystallin sensitizes rat articular chondrocytes to CK2 inhibition-induced apoptosis, we assessed apoptosis by performing viability assays, mitochondrial membrane potential measurements, flow cytometry, nuclear morphology observations, and western blot analysis. To investigate the mechanism by which αB-crystallin modulates the extent of CK2 inhibition-mediated chondrocyte death, we utilized confocal microscopy to observe the subcellular location of αB-crystallin and its phosphorylated forms and performed a co-immunoprecipitation assay to observe the interaction between αB-crystallin and CK2. Immunochemistry was employed to examine αB-crystallin expression in cartilage obtained from rats with experimentally induced osteoarthritis (OA). Our results demonstrated that silencing of αB-crystallin sensitized rat articular chondrocytes to CK2 inhibitor-induced apoptosis. Furthermore, CK2 inhibition modulated the expression and subcellular localization of αB-crystallin and its phosphorylated forms and dissociated αB-crystallin from CK2. The population of rat articular chondrocytes expressing αB-crystallin and its phosphorylated forms was reduced in an experimentally induced rat model of OA. In summary, αB-crystallin protects rat articular chondrocytes against CK2 inhibition-induced apoptosis. αB-crystallin may represent a suitable target for pharmacological interventions to prevent OA. PMID:27851782

  9. Dietary lipids modify the fatty acid composition of cartilage, isolated chondrocytes and matrix vesicles.

    PubMed

    Xu, H; Watkins, B A; Adkisson, H D

    1994-09-01

    The effects of dietary lipids on the fatty acid composition of hyaline cartilage, epiphyseal chondrocytes (EC) and matrix vesicles (MV) were evaluated in chicks. A basal semipurified diet was fed to chicks containing one of the following lipid sources at 70 g/kg: soybean oil, butter+corn oil, margarine+corn oil or menhaden oil+corn oil (MEC). Articular and epiphyseal growth cartilage were isolated from the proximal tibiotarsus; EC and MV were subsequently released by trypsin (EC 3.4.21.4) and collagenase (EC 3.4.24.3) digestion followed by ultracentrifugation. The fatty acid composition of polar lipids in chick epiphyseal cartilage at three and six weeks, as well as articular cartilage, EC and MV at eight weeks of age revealed the presence of high levels of saturated and monounsaturated fatty acids (up to 85.5%) but low levels of n-6 polyunsaturated fatty acids (PUFA) (2.6-10.2%). Mead acid (20:3n-9, > 3%) was also present in cartilage, EC and MV lipids, and was unaffected by the dietary lipid treatments. Total n-3 PUFA concentrations were the highest in cartilage, EC and MV of chicks consuming MEC. Feeding MEC lowered the levels of 20:4n-6 in cartilage, but increased 20:5n-3 levels. The data are consistent with those reported previously which showed that cartilage tissues are low in n-6 PUFA and that they contain 20:3n-9. We furthermore demonstrated that the PUFA composition of cartilage can be modified by dietary lipids.

  10. Proliferation of rabbit chondrocyte and inhibition of IL-1β-induced apoptosis through MEK/ERK signaling by statins.

    PubMed

    Zhou, Bin; Chen, Deheng; Xu, Huazi; Zhang, Xiaolei

    2017-02-01

    Chondrocyte plays a critical role in endochondral ossification and cartilage repair by maintaining the cartilaginous matrix. Statins have been widely used to lower the cholesterol level in patients with cardiovascular disorders. Previous research has demonstrated potential role of statins in chondrocyte proliferation. This study addresses the proliferation-regulatory effect of lovastatin in rabbit chondrocytes as well as the underlying signaling mechanisms, thereby exploring its potential application in chondrocyte-related disorders, such as cartilage damage and osteoarthritis. Rabbit chondrocytes were treated with lovastatin at multiple concentrations, and the proliferation rate was measured by CCK-8 test. The results showed significant increase in chondrocyte proliferation under lovastatin treatment. Using real-time quantitative PCR, it was observed that the expression levels of COL2A1, SOX-9, Caspase-3, and MMP-3 genes were significantly changed by lovastatin treatment. Western blotting analysis showed that the abundance of COL2A1, SOX-9, MEK1/2, p-MEK1/2, ERK1/2, p-ERK1/2, Caspase-3, and MMP-3 proteins was also significantly influenced by lovastatin treatment. Interleukine-1 beta (IL-1β) is involved in the progression of osteoarthritis (OA) by inducing articular cartilage and chondrocyte aging and senescence. In this study, we observed that lovastatin treatment inhibited IL-1β-induced chondrocyte apoptosis, while the combined treatment of lovastatin and U0126 evidently offset the apoptosis-inhibiting effect of lovastatin in chondrocyte proliferation. The expressional level and protein abundance of COL2A1, SOX-9, MEK1/2, p-MEK1/2, ERK1/2, p-ERK1/2, caspase-3, and MMP-3 genes showed significant alterations under the combined treatment. Together, our results suggested that lovastatin significantly promoted proliferation and inhibited the IL-1β-induced apoptosis in rabbit chondrocytes, which was mediated by the MEK/ERK signaling.

  11. [Autologous chondrocyte transplantation for treatment of cartilage defects of the knee joint. Clinical results].

    PubMed

    Erggelet, C; Browne, J E; Fu, F; Mandelbaum, B R; Micheli, L J; Mosely, J B

    2000-01-01

    Cartilage defects in the knee joint are common and have a bad tendency for healing due to the limited regeneration of hyaline cartilage. Surgeons have an ample choice of various operative treatment measures. Especially for the treatment of larger lesions first results of autologous chondrocyte transplantation (ACT) were published in 1994 [3]. Autologous chondrocytes are isolated from an arthoscopically harvested cartilage biopsy, cultured in vitro and implanted in the defect under a periostal flap in a second procedure. In an international multicenter study 1,051 patients treated with ACT between 6/95 and 12/98 were documented with follow-up examinations after 12 months (588 patients), 24 months (220 patients) and 36 months (40 patients). The majority of the defects (61.2%) were localized on the medial femoral condyle, measuring 4.6 cm2 and mostly described as grade III/IV lesions. The clinical evaluation was performed using a modified Cincinnati knee rating system independently for clinician and patient. Evaluations showed an increase from 3.35 to 6.25 after 24 months and from 3.10 to 6.77 in a scale from 1 (bad) to 10 (excellent). ACT favours defects of the femur with an improvement rate of 85%. Adverse events possibly related to ACT were described in 4.8% of the patients. Diagnostic second-look arthroscopies are included in the reoperation rate of 5.1%. The presented data indicate autologous chondrocyte transplantation as an effective and safe option for the treatment of large full thickness cartilage defects in the knee joint.

  12. Early induction of a prechondrogenic population allows efficient generation of stable chondrocytes from human induced pluripotent stem cells

    PubMed Central

    Lee, Jieun; Taylor, Sarah E. B.; Smeriglio, Piera; Lai, Janice; Maloney, William J.; Yang, Fan; Bhutani, Nidhi

    2015-01-01

    Regeneration of human cartilage is inherently inefficient; an abundant autologous source, such as human induced pluripotent stem cells (hiPSCs), is therefore attractive for engineering cartilage. We report a growth factor-based protocol for differentiating hiPSCs into articular-like chondrocytes (hiChondrocytes) within 2 weeks, with an overall efficiency >90%. The hiChondrocytes are stable and comparable to adult articular chondrocytes in global gene expression, extracellular matrix production, and ability to generate cartilage tissue in vitro and in immune-deficient mice. Molecular characterization identified an early SRY (sex-determining region Y) box (Sox)9low cluster of differentiation (CD)44lowCD140low prechondrogenic population during hiPSC differentiation. In addition, 2 distinct Sox9-regulated gene networks were identified in the Sox9low and Sox9high populations providing novel molecular insights into chondrogenic fate commitment and differentiation. Our findings present a favorable method for generating hiPSC-derived articular-like chondrocytes. The hiChondrocytes are an attractive cell source for cartilage engineering because of their abundance, autologous nature, and potential to generate articular-like cartilage rather than fibrocartilage. In addition, hiChondrocytes can be excellent tools for modeling human musculoskeletal diseases in a dish and for rapid drug screening.—Lee, J., Taylor, S. E. B., Smeriglio, P., Lai, J., Maloney, W. J., Yang, F., Bhutani, N. Early induction of a prechondrogenic population allows efficient generation of stable chondrocytes from human induced pluripotent stem cells. PMID:25911615

  13. Ectopic bone formation during tissue-engineered cartilage repair using autologous chondrocytes and novel plasma-derived albumin scaffolds.

    PubMed

    Robla Costales, David; Junquera, Luis; García Pérez, Eva; Gómez Llames, Sara; Álvarez-Viejo, María; Meana-Infiesta, Álvaro

    2016-10-01

    The aims of this study were twofold: first, to evaluate the production of cartilaginous tissue in vitro and in vivo using a novel plasma-derived scaffold, and second, to test the repair of experimental defects made on ears of New Zealand rabbits (NZr) using this approach. Scaffolds were seeded with chondrocytes and cultured in vitro for 3 months to check in vitro cartilage production. To evaluate in vivo cartilage production, a chondrocyte-seeded scaffold was transplanted subcutaneously to a nude mouse. To check in vivo repair, experimental defects made in the ears of five New Zealand rabbits (NZr) were filled with chondrocyte-seeded scaffolds. In vitro culture produced mature chondrocytes with no extracellular matrix (ECM). Histological examination of redifferentiated in vitro cultures showed differentiated chondrocytes adhered to scaffold pores. Subcutaneous transplantation of these constructs to a nude mouse produced cartilage, confirmed by histological study. Experimental cartilage repair in five NZr showed cartilaginous tissue repairing the defects, mixed with calcified areas of bone formation. It is possible to produce cartilaginous tissue in vivo and to repair experimental auricular defects by means of chondrocyte cultures and the novel plasma-derived scaffold. Further studies are needed to determine the significance of bone formation in the samples. Copyright © 2016 European Association for Cranio-Maxillo-Facial Surgery. Published by Elsevier Ltd. All rights reserved.

  14. Cartilage repair with chondrocytes in fibrin hydrogel and MPEG polylactide scaffold: an in vivo study in goats.

    PubMed

    Lind, Martin; Larsen, Allan; Clausen, Christian; Osther, Kurt; Everland, Hanne

    2008-07-01

    Polylactic acid polymers have been used extensively as biomaterials and have shown promising properties for cartilage tissue engineering. Numerous scaffold materials exist and the optimal scaffold needs to be identified. We have tried to assess the possibilities for cartilage repair by the use of two different scaffold techniques; autologous chondrocytes in a fibrin hydrogel and a novel MPEG-PLGA scaffold, where autologous chondrocytes are immobilized within the MPEG-PLGA scaffold by a fibrin hydrogel. Twenty adult goats were used for the study. A 6 mm circular full-thickness cartilage defect was created in both medial femoral condyles. The defects were randomized to the following four treatment groups. (1) Empty defect (control). (2) Subchondral drilling (control). (3) Fibrin hydrogel with autologous chondrocytes. (4) Fibrin hydrogel/chondrocyte solution in a MPEG-PLGA porous scaffold. Animals were followed for 4 month. Eight defects in each treatment group completed the study. ICRS macroscopic scoring (0-12). Indentation test was performed to assess stiffness of repair tissue. Histological analyses was performed using O'Driscoll and Pineda cartilage scores as well as percentage tissue filling of the defects. The MPEG-PLGA/chondrocytes scaffold was the superior treatment modality based on the macroscopic surface score, histological scores and defect filling. The mechanical test demonstrated no difference between treatment groups. The MPEG-PLGA/chondrocyte composite demonstrated significantly better cartilage repair response than empty defects, osteochondral drilling and fibrin hydrogel with chondrocytes. The novel MPEG-PLGA scaffold in combination with chondrocytes need further studies with respect to longer follow-up times.

  15. Synthesis of cartilage matrix by mammalian chondrocytes in vitro. III. Effects of ascorbate

    PubMed Central

    1984-01-01

    Chondrocytes isolated from bovine articular cartilage were plated at high density and grown in the presence or absence of ascorbate. Collagen and proteoglycans, the major matrix macromolecules synthesized by these cells, were isolated at times during the course of the culture period and characterized. In both control and ascorbate-treated cultures, type II collagen and cartilage proteoglycans accumulated in the cell-associated matrix. Control cells secreted proteoglycans and type II collagen into the medium, whereas with time in culture, ascorbate-treated cells secreted an increasing proportion of types I and III collagens into the medium. The ascorbate-treated cells did not incorporate type I collagen into the cell-associated matrix, but continued to accumulate type II collagen in this compartment. Upon removal of ascorbate, the cells ceased to synthesize type I collagen. Morphological examination of ascorbate-treated and control chondrocyte culture revealed that both collagen and proteoglycans were deposited into the extracellular matrix. The ascorbate-treated cells accumulated a more extensive matrix that was rich in collagen fibrils and ruthenium red-positive proteoglycans. This study demonstrated that although ascorbate facilitates the formation of an extracellular matrix in chondrocyte cultures, it can also cause a reversible alteration in the phenotypic expression of those cells in vitro. PMID:6501411

  16. Immunological response to tissue-engineered cartilage derived from auricular chondrocytes and a PLLA scaffold in transgenic mice.

    PubMed

    Fujihara, Yuko; Takato, Tsuyoshi; Hoshi, Kazuto

    2010-02-01

    The immune response against biomaterials in tissue-engineered constructs could potentially worsen the outcome of tissue regeneration, but immunological reactions between host and donor in tissue-engineered constructs remain to be clarified. In the present study, we syngenically transplanted tissue-engineered cartilage constructs consisting of C57BL/6 mice auricular chondrocytes and poly-l-lactic acid scaffolds (MW:200,000) into EGFP transgenic mice of C57BL/6 background, and evaluated the response by the localization of donor-derived and host-derived cells, the latter of which were distinguished by the presence of EGFP. While donor-derived cells constituted the areas of regenerated cartilage, host-derived cells were increased in number for the initial two weeks, and then decreased and excluded to non-cartilage areas thereafter. Furthermore, EGFP positivity was mostly co-localized with that of F4/80, suggesting most of the host-derived cells in the tissue-engineered constructs could be macrophages. Immunohistochemical staining of the tissue-engineered cartilage constructs revealed expression of factors related to immune privilege in chondrocytes, such as macrophage migration inhibitory factor (MIF), fas ligand (FasL) and others. Co-culture of chondrocytes and macrophages in vitro increased the expression of MIF and FasL in the chondrocytes, suggesting that chondrocytes in tissue-engineered cartilage constructs could regulate the actions of host-derived macrophages by expressing factors related to immune privilege.

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

    PubMed

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

    2015-11-01

    Despite extensive research into cartilage tissue engineering (CTE), there is still no scaffold ideal for clinical applications. Various synthetic and natural polymers have been investigated in vitro and in vivo, but none have reached widespread clinical use. The authors investigate the potential of POSS-PCU, a synthetic nanocomposite polymer, for use in CTE. POSS-PCU is modified with silsesquioxane nanostructures that improve its biological and physical properties. The ability of POSS-PCU to support the growth of ovine nasoseptal chondrocytes was evaluated against a polymer widely used in CTE, polycaprolactone (PCL). Scaffolds with varied concentrations of the POSS molecule were also synthesized to investigate their effect on chondrocyte growth. Chondrocytes were seeded onto scaffold disks (PCU negative control; POSS-PCU 2%, 4%, 6%, 8%; PCL). Cytocompatibilty was evaluated using cell viability, total DNA, collagen and GAG assays. Chondrocytes cultured on POSS-PCU (2% POSS) scaffolds had significantly higher viability than PCL scaffolds (p < 0.001). Total DNA, collagen and sGAG protein were also greater on POSS-PCU scaffolds compared with PCL (p > 0.05). POSS-PCU (6% and 8% POSS) had improved viability and proliferation over an 18 day culture period compared with 2% and 4% POSS-PCU (p < 0.0001). Increasing the percentage of POSS in the scaffolds increased the size of the pores found in the scaffolds (p < 0.05). POSS-PCU has excellent potential for use in CTE. It supports the growth of chondrocytes in vitro and the POSS modification significantly enhances the growth and proliferation of nasoseptal chondrocytes compared with traditional scaffolds such as PCL.

  18. OA cartilage derived chondrocytes encapsulated in poly(ethylene glycol) diacrylate (PEGDA) for the evaluation of cartilage restoration and apoptosis in an in vitro model.

    PubMed

    Musumeci, G; Loreto, C; Carnazza, M L; Strehin, I; Elisseeff, J

    2011-10-01

    Osteoarthritis (OA) is characterized by cartilage attrition, subchondral bone remodeling, osteophyte formation and synovial inflammation. Perturbed homeostasis caused by inflammation, oxidative stress, mitochondrial dysfunction and proapoptotic/antiapoptotic dysregulation is known to impair chondrocyte survival in joint microenvironments and contribute to OA pathogenesis. However, the molecular mechanisms underlying the programmed cell death (apoptosis) of chondral cells are not yet well defined. The present study was conducted to evaluate apoptosis of chondrocytes from knee articular cartilage of patients with OA. The aim of this study was to investigate and compare the apoptosis through the expression of caspase-3 in tissue explants, in cells cultured in monolayer, and in cells encapsulated in a hydrogel (PEGDA) scaffold. Chondrocytes were also studied following cell isolation and encapsulation in poly(ethylene glycol) diacrylate (PEGDA) hydrogels. Specifically, articular cartilage specimens were assessed by histology (Hematoxlyn and Eosin) and histochemistry (Safranin-O and Alcian Blue). The effector of apoptosis caspase-3 was studied through immunohistochemistry, immunocytochemistry and immunofluorescence. DNA strand breaks were evaluated in freshly isolated chondrocytes from human OA cartilage using the TUNEL assay, and changes in nuclear morphology of apoptotic cells were detected by staining with Hoechst 33258. The results showed an increased expression of caspase-3 in tissue explants, in pre-confluent cells and after four passages in culture, and a decreased expression of caspase-3 comparable to control cartilage in cells encapsulated in hydrogels (PEGDA) after 5 weeks in culture. The freshly isolated chondrocytes were TUNEL positive. The chondrocytes after 5 weeks of culture in hydrogels (PEGDA) showed the formation of new hyaline cartilage with increased cell growth, cellular aggregations and extracellular matrix (ECM) production. This is of particular

  19. The optimal conditions of chondrocyte isolation and its seeding in the preparation for cartilage tissue engineering.

    PubMed

    Yonenaga, Kazumichi; Nishizawa, Satoru; Fujihara, Yuko; Asawa, Yukiyo; Sanshiro, Kanazawa; Nagata, Satoru; Takato, Tsuyoshi; Hoshi, Kazuto

    2010-12-01

    To optimize the chondrocyte numbers obtained after collagenase digestion for cartilage tissue engineering, we examined the enzyme concentration and incubation time for collagenase digestion. The appropriate cell density in the chondrocyte primary culture was also verified. The collagenase digestion conditions that maximized the viable cell numbers were 24 h in 0.15% and 0.3% collagenase, 6 h in 0.6%, and 4 h in 1.2%, leading to ∼5 × 10⁵ cells from 0.05 g. When seeded at 10,000 cells/cm², all of these cells became almost confluent within 1 week. Cells digested in 0.3% for 24 h or 0.6% for 6 h and seeded at 3000 cells/cm² may also be acceptable, and similarly reached confluence within 1 week. Results regarding expression of the p53, tumor necrosis factor-α, and interleukin-1β genes, as well as apoptosis enzyme-linked immunosorbent assay results, show that excessive collagenase exposure may decrease chondrocyte viability or activity. We recommend a 24-h incubation in 0.3% collagenase or 6 h in 0.6% collagenase, and a cell-seeding density of 3000-10,000 cells/cm². These conditions maximize the harvest of isolated chondrocytes from a small amount of biopsied tissue and significantly aid in obtaining a large quantity of cultured cells in a short period.

  20. [Quantitative analysis of chondrocyte distribution patterns in hyaline cartilage tissue--a comparison of different models].

    PubMed

    Ranke, T P; Ranke, I; Schwandtke, A; Rother, P

    1989-01-01

    The purpose of our investigations were aging changes in hyaline cartilage of the trachea and of the larynx. 42 samples of carina tracheae and 29 samples of arytenoid cartilage were used in both organs from the newborn age up to the age of 91 a. We have examined patterns of cell distribution with the help of the computer programme "Dichte" ("density") using the following methods of stochastic geometry: product density, L function, and others. Product density allows for a reliable destination between a regular pattern of cells (hard-core distribution), a random pattern (soft-core distribution), and a typical clustering of chondrocytes. 2 examples of arytenoid cartilage have been selected to demonstrate the possibilities of interpretation of product density supported by the L function. Soft-core and in few cases hard-core distribution have been found both with the carina tracheae and the cartilago arytaenoidea in the 1st decade of life. Beginning with the 2nd decade of life, the typical clustering of chondrocytes have been confirmed with both organs.

  1. Cell Seeding Densities in Autologous Chondrocyte Implantation Techniques for Cartilage Repair.

    PubMed

    Foldager, Casper Bindzus; Gomoll, Andreas H; Lind, Martin; Spector, Myron

    2012-04-01

    Cartilage repair techniques have been among the most intensively investigated treatments in orthopedics for the past decade, and several different treatment modalities are currently available. Despite the extensive research effort within this field, the generation of hyaline cartilage remains a considerable challenge. There are many parameters attendant to each of the cartilage repair techniques that can affect the amount and types of reparative tissue generated in the cartilage defect, and some of the most fundamental of these parameters have yet to be fully investigated. For procedures in which in vitro-cultured autologous chondrocytes are implanted under a periosteal or synthetic membrane cover, or seeded onto a porous membrane or scaffold, little is known about how the number of cells affects the clinical outcome. Few published clinical studies address the cell seeding density that was employed. The principal objective of this review is to provide an overview of the cell seeding densities used in cell-based treatments currently available in the clinic for cartilage repair. Select preclinical studies that have informed the use of specific cell seeding densities in the clinic are also discussed.

  2. OXIDANT CONDITIONING PROTECTS CARTILAGE FROM MECHANICALLY-INDUCED DAMAGE

    PubMed Central

    Ramakrishnan, Prem; Hecht, Benjamin; Pedersen, Doug; Lavery, Matthew; Maynard, Jerry; Buckwalter, Joseph; Martin, James

    2013-01-01

    Articular cartilage degeneration in osteoarthritis has been linked to abnormal mechanical stresses that are known to cause chondrocyte apoptosis and metabolic derangement in in vitro models. Evidence implicating oxidative damage as the immediate cause of these harmful effects suggests that the anti-oxidant defenses of chondrocytes might influence their tolerance for mechanical injury. Based on evidence that anti-oxidant defenses in many cell types are stimulated by moderate oxidant exposure, we hypothesized that oxidant pre-conditioning would reduce acute chondrocyte death and proteoglycan depletion in cartilage explants after exposure to abnormal mechanical stresses. Porcine cartilage explants were treated every 48 hours with tert-butyl hydrogen peroxide (tBHP) at non-lethal concentrations (25, 100, 250, 500 µM) for a varying number of times (1, 2 or 4) prior to a bout of unconfined axial compression (5 MPa, 1 Hz, 1800 cycles). When compared with untreated controls, tBHP had significant positive effects on post-compression viability, lactate production, and proteoglycan losses. Overall, the most effective regime was 100 µM tBHP applied 4 times. RNA analysis revealed significant effects of 100 µM tBHP on gene expression. Catalase, hypoxia-inducible factor-1alpha (HIF-1α), and glyceraldehyde 6-phosphate dehydrogenase (GAPDH) were significantly increased relative to untreated controls in explants treated 4 times with 100 µM tBHP, a regime that also resulted in a significant decrease in matrix metalloproteinase-3 (MMP-3) expression. These findings demonstrate that repeated exposure of cartilage to sub-lethal concentrations of peroxide can moderate the acute effects of mechanical stress, a conclusion supported by evidence of peroxide-induced changes in gene expression that could render chondrocytes more resistant to oxidative damage. PMID:20058262

  3. Cartilage biomarkers in hemodialysis patients and the effect of beta2-microglobulin on articular chondrocytes.

    PubMed

    Sunk, I-G; Demetriou, D; Szendroedi, J; Amoyo, L; Raffetseder, A; Hörl, W H; Sunder-Plassmann, G; Smolen, J S; Bobacz, K

    2008-11-01

    Dialysis-related amyloidosis (DRA) is a severe complication of maintenance hemodialysis (HD). Given the predominant deposition of beta(2)-microglobulin (beta2m) fibrils on articular cartilage in early DRA, we investigated the significance of beta2m and its relationship to distinct cartilage biomarkers in early DRA diagnosis in HD patients. Furthermore, we assessed the effects of beta2m on articular chondrocytes in vitro. Serum samples from 133 patients were collected before and after HD. Type II collagen cleavage product (C2C), procollagen II c-propeptide (CPII), aggrecan chondroitin sulfate 846 epitope (CS-486) and cartilage oligomeric matrix protein (COMP) levels were determined by enzyme-linked immunosorbent assay. Primary bovine articular chondrocytes were cultured as monolayers and incubated with beta2m at 1.5mg/l and 20mg/l. Cartilage glucosaminoglycan synthesis was measured by [(35)S]sulfate incorporation. mRNA expression of interleukin (IL)-1beta, matrix metalloproteinases (MMPs)-3 and -9 was measured by reverse-transcriptase polymerase chain reaction (RT-PCR). Incubation with beta2m at 20mg/l significantly decreased matrix biosynthesis. PCR analysis revealed an increase of IL-1beta, as well as MMPs-3 and -9 on the mRNA level. C2C/CPII, CS-486 and COMP levels were increased only in a subset of patients without a significant correlation with beta2m concentrations. A subgroup analysis elucidated an increase in type II collagen degradation during the first years of HD, as shown by the elevation of C2C/CPII ratio. beta2m exerted anti-anabolic effects on articular chondrocytes in vitro and might be involved in cartilage degradation in HD patients. beta2m serum levels, however, did not reflect cartilage degradation in DRA. The assessment of C2C/CPII, CS-486 or COMP concentrations apparently has minor relevance in DRA diagnosis in HD patients. However, the increased type II collagen breakdown within 5 years after HD onset possibly mirrors the early stages of DRA

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

    PubMed

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

    2016-10-22

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

  5. Viability of chondrocytes seeded onto a collagen I/III membrane for matrix-induced autologous chondrocyte implantation.

    PubMed

    Hindle, Paul; Hall, Andrew C; Biant, Leela C

    2014-11-01

    Cell viability is crucial for effective cell-based cartilage repair. The aim of this study was to determine the effect of handling the membrane during matrix-induced autologous chondrocyte implantation surgery on the viability of implanted chondrocytes. Images were acquired under five conditions: (i) Pre-operative; (ii) Handled during surgery; (iii) Cut edge; (iv) Thumb pressure applied; (v) Heavily grasped with forceps. Live and dead cell stains were used. Images were obtained for cell counting and morphology. Mean cell density was 6.60 × 10(5) cells/cm(2) (5.74-7.11 × 10(5) ) in specimens that did not have significant trauma decreasing significantly in specimens that had been grasped with forceps (p < 0.001) or cut (p = 0.004). Cell viability on delivery grade membrane was 75.1%(72.4-77.8%). This dropped to 67.4%(64.1-69.7%) after handling (p = 0.002), 56.3%(51.5-61.6%) after being thumbed (p < 0.001) and 28.8%(24.7-31.2%) after crushing with forceps (p < 0.001). When cut with scissors there was a band of cell death approximately 275 µm in width where cell viability decreased to 13.7%(10.2-18.2%, p < 0.001). Higher magnification revealed cells without the typical rounded appearance of chondrocytes. We found that confocal laser-scanning microscope (CLSM) can be used to quantify and image the fine morphology of cells on a matrix-induced autologous chondrocyte implantation (MACI) membrane. Careful handling of the membrane is essential to minimise chondrocyte death during surgery. © 2014 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.

  6. Toxicity of antiseptics against chondrocytes: what is best for the cartilage in septic joint surgery?

    PubMed

    Röhner, Eric; Kolar, Paula; Seeger, Joern B; Arnholdt, Joerg; Thiele, Kathi; Perka, Carsten; Matziolis, Georg

    2011-11-01

    In septic joint surgery, the most frequently used antiseptics are polyhexanide, hydrogen peroxide and taurolidine. The aim of this study was to examine the effects of these antiseptics on viability of human chondrocytes. Our hypothesis was that antiseptics and supplemental irrigation with sodium chloride lavage are less toxic on human chondrocytes than treatment with antiseptics only. Primary human chondrocytes were isolated and cultured from six donated human knee joints. Polyhexanide, hydrogen peroxide or taurolidine were added to the cultures. Toxicity analysis was performed by visualisation of cell structure using light microscopy and LDH activity. The determination of vital cells and total cell numbers of chondrocytes treated with antiseptics partly followed by irrigation with sodium chloride solution was performed by using Casy Cell-Counter. Light microscopic data revealed a defect in cell structure after addition of antiseptics. We showed a significant increase of LDH enzyme activity after the treatment with polyhexanide or taurolidine. After treatment with antiseptics followed by sodium chloride solution a significant increase of vital and total cell numbers resulted in comparison with the chondrocytes that were only treated with antiseptics. The data show that treatment with polyhexanid, hydrogen peroxide or taurolidine induces cell death of human chondroctes in vitro. The application of sodium chloride solution after the treatment with polyhexanide and hydrogen peroxide possibly has a protective effect on chondrocyte viability.

  7. Fetal Mesenchymal Stromal Cells Differentiating towards Chondrocytes Acquire a Gene Expression Profile Resembling Human Growth Plate Cartilage

    PubMed Central

    Leijten, Jeroen C. H.; Decker, Eva; Sticht, Carsten; van Houwelingen, Johannes C.; Goeman, Jelle J.; Kleijburg, Carin; Scherjon, Sicco A.; Gretz, Norbert; Wit, Jan Maarten; Rappold, Gudrun; Post, Janine N.; Karperien, Marcel

    2012-01-01

    We used human fetal bone marrow-derived mesenchymal stromal cells (hfMSCs) differentiating towards chondrocytes as an alternative model for the human growth plate (GP). Our aims were to study gene expression patterns associated with chondrogenic differentiation to assess whether chondrocytes derived from hfMSCs are a suitable model for studying the development and maturation of the GP. hfMSCs efficiently formed hyaline cartilage in a pellet culture in the presence of TGFβ3 and BMP6. Microarray and principal component analysis were applied to study gene expression profiles during chondrogenic differentiation. A set of 232 genes was found to correlate with in vitro cartilage formation. Several identified genes are known to be involved in cartilage formation and validate the robustness of the differentiating hfMSC model. KEGG pathway analysis using the 232 genes revealed 9 significant signaling pathways correlated with cartilage formation. To determine the progression of growth plate cartilage formation, we compared the gene expression profile of differentiating hfMSCs with previously established expression profiles of epiphyseal GP cartilage. As differentiation towards chondrocytes proceeds, hfMSCs gradually obtain a gene expression profile resembling epiphyseal GP cartilage. We visualized the differences in gene expression profiles as protein interaction clusters and identified many protein clusters that are activated during the early chondrogenic differentiation of hfMSCs showing the potential of this system to study GP development. PMID:23144774

  8. Local IL-13 gene transfer prior to immune-complex arthritis inhibits chondrocyte death and matrix-metalloproteinase-mediated cartilage matrix degradation despite enhanced joint inflammation

    PubMed Central

    Nabbe, Karin CAM; van Lent, Peter LEM; Holthuysen, Astrid EM; Sloëtjes, Annet W; Koch, Alisa E; Radstake, Timothy RDJ; van den Berg, Wim B

    2005-01-01

    During immune-complex-mediated arthritis (ICA), severe cartilage destruction is mediated by Fcγ receptors (FcγRs) (mainly FcγRI), cytokines (e.g. IL-1), and enzymes (matrix metalloproteinases (MMPs)). IL-13, a T helper 2 (Th2) cytokine abundantly found in synovial fluid of patients with rheumatoid arthritis, has been shown to reduce joint inflammation and bone destruction during experimental arthritis. However, the effect on severe cartilage destruction has not been studied in detail. We have now investigated the role of IL-13 in chondrocyte death and MMP-mediated cartilage damage during ICA. IL-13 was locally overexpressed in knee joints after injection of an adenovirus encoding IL-13 (AxCAhIL-13), 1 day before the onset of arthritis; injection of AxCANI (an empty adenoviral construct) was used as a control. IL-13 significantly increased the amount of inflammatory cells in the synovial lining and the joint cavity, by 30% to 60% at day 3 after the onset of ICA. Despite the enhanced inflammatory response, chondrocyte death was diminished by two-thirds at days 3 and 7. The mRNA level of FcγRI, a receptor shown to be crucial in the induction of chondrocyte death, was significantly down-regulated in synovium. Furthermore, MMP-mediated cartilage damage, measured as neoepitope (VDIPEN) expression using immunolocalization, was halved. In contrast, mRNA levels of MMP-3, -9, -12, and -13 were significantly higher and IL-1 protein, which induces production of latent MMPs, was increased fivefold by IL-13. This study demonstrates that IL-13 overexpression during ICA diminished both chondrocyte death and MMP-mediated VDIPEN expression, even though joint inflammation was enhanced. PMID:15743487

  9. Autologous chondrocyte implantation for cartilage injury treatment in Chiang Mai University Hospital: a case report.

    PubMed

    Wongtriratanachai, Prasit; Pruksakorn, Dumnoensun; Pothacharoen, Peraphan; Nimkingratana, Puwapong; Pattamapaspong, Nuttaya; Phornphutkul, Chanakarn; Setsitthakun, Sasiwariya; Fongsatitkul, Ladda; Phrompaet, Sureeporn

    2013-11-01

    Autologous chondrocyte implantation (ACI) has become one of the standard procedures for articular cartilage defect treatment. This technique provides a promising result. However the procedural process requires an approach of several steps from multidisciplinary teams. Although the success of this procedure has been reported from Srinakharinvirot University since 2007, the application of ACI is still limited in Thailand due to the complexity of processes and stringent quality control. This report is to present the first case of the cartilage defect treatment using the first generation-ACI under Chiang Mai University's (CMU) own facility and Ethics Committee. This paper also reviews the process of biotechnology procedures, patient selection, surgical, and rehabilitation techniques. The success of the first case is an important milestone for the further development of the CMU Human Translational Research Laboratory in near future.

  10. Different ratios of bone marrow mesenchymal stem cells and chondrocytes used in tissue-engineered cartilage and its application for human ear-shaped substitutes in vitro.

    PubMed

    Kang, Ning; Liu, Xia; Yan, Li; Wang, Qian; Cao, Yilin; Xiao, Ran

    2013-01-01

    The application of chondrocyte-based cartilage tissue engineering is limited because of the lack of autologous cartilage sources and chondrocyte dedifferentiation after in vitro expansion. Coculture of bone marrow mesenchymal stem cells (BMSCs) and chondrocytes has been a promising strategy for cartilage engineering as chondrocytes can provide a chondrogenic environment for BMSCs. However, there are no systematic comparison studies for engineered cartilage constructed using different mixing ratios of BMSCs and chondrocytes, and the most effective mixing ratio with the lowest number of chondrocytes is unknown. Here, we set a gradient of mixing ratios of BMSCs to chondrocytes for an in vitro coculture system and compared the shape retention and quality of the engineered cartilage using macroscopic and histological assays, glycosaminoglycan content assessment and immunohistochemical staining of type II collagen, biomechanical evaluation and hypertrophy-related gene expression analysis. The results showed that at least 30% chondrocytes were required to generate cartilage tissue with satisfactory shape and quality. Therefore, we preliminarily assessed the feasibility of engineering a human ear-shaped substitute using a coculture system with a 7:3 ratio of BMSCs to chondrocytes. After 8 weeks of in vitro culture, the precise architecture of the human ear-shaped construct was well maintained with the typical cartilaginous composition confirmed by histological assays.

  11. Rebamipide attenuates pain severity and cartilage degeneration in a rat model of osteoarthritis by downregulating oxidative damage and catabolic activity in chondrocytes.

    PubMed

    Moon, S-J; Woo, Y-J; Jeong, J-H; Park, M-K; Oh, H-J; Park, J-S; Kim, E-K; Cho, M-L; Park, S-H; Kim, H-Y; Min, J-K

    2012-11-01

    The objectives were to investigate the in vivo effects of treatment with rebamipide on pain severity and cartilage degeneration in an experimental model of rat osteoarthritis (OA) and to explore its mode of action. OA was induced in rats by intra-articular injection of monosodium iodoacetate (MIA). Oral administration of rebamipide was initiated on the day of MIA injection, 3 or 7 days after. Limb nociception was assessed by measuring the paw withdrawal latency and threshold. We analyzed the samples macroscopically and histomorphologically, and used immunohistochemistry to investigate the expression of matrix metalloproteinase-13 (MMP-13), interleukin-1β (IL-1β), hypoxia-inducible factor-2α (HIF-2α), inducible nitric oxide synthase (iNOS), and nitrotyrosine in knee joints. Real-time quantitative reverse transcription-polymerase chain reaction was used to quantify the mRNA for catabolic and anticatabolic factors in human OA chondrocytes. Rebamipide showed an antinociceptive property and attenuated cartilage degeneration. Rebamipide reduced the expression of MMP-13, IL-1β, HIF-2α, iNOS, and nitrotyrosine in OA cartilage in a dose-dependent manner. Nitrotyrosine expression in the subchondral bone region was decreased in the rebamipide-treated joints. mRNA expression of MMP-1, -3, and -13, and ADAMTS5 was attenuated in IL-1β-stimulated human OA chondrocytes. By contrast, rebamipide induced the mRNA expression of tissue inhibitor of metalloproteinase-1 and -3. The results show the inhibitory effects of rebamipide on pain production and cartilage degeneration in experimentally induced OA. The suppression of oxidative damage and the restoration of extracellular matrix homeostasis of articular chondrocyte suggest that rebamipide is a potential therapeutic strategy for OA. Crown Copyright © 2012. Published by Elsevier Ltd. All rights reserved.

  12. Shape of chondrocytes within articular cartilage affects the solid but not the fluid microenvironment under unconfined compression.

    PubMed

    Guo, Hongqiang; Torzilli, Peter A

    2016-01-01

    Metabolic activity of the chondrocytes in articular cartilage is strongly related to their zone-specific shape and the composition and mechanical properties of their surrounding extracellular matrix (ECM). However the mechanisms by which cell shape influences the response of the ECM microenvironment to mechanical loading is yet to be elucidated. This relationship was studied using a biphasic multiscale finite element model of different shaped chondrocytes in the superficial and deep zones of the ECM during unconfined stress relaxation. For chondrocytes in the superficial zone, increasing the cell's initial aspect ratio (length/height) increased the deformation and solid stresses of the chondrocyte and pericellular matrix (PCM) during the loading phase; for chondrocytes in the deep zone the effect of the cell shape on the solid microenvironment was time and variable dependent. However, for superficial and deep zone chondrocytes the cell shape did not affect the fluid pressure and fluid shear stress. These results suggest that mechanotransduction of chondrocytes in articular cartilage may be regulated through the solid phase rather than the fluid phase, and that high stresses and deformations in the solid microenvironment in the superficial zone may be essential for the zone-specific biosynthetic activity of the chondrocyte. The biphasic multiscale computational analysis suggests that maintaining the cell shape is critical for regulating the microenvironment and metabolic activity of the chondrocyte in tissue engineering constructs. We investigated the effect of chondrocyte shape on the cellular microenvironment using a biphasic multiscale finite element analysis. Our study showed that cell shapes affects the solid but not the fluid microenvironment of the chondrocyte, and that maintaining the cell shape is critical for regulating the microenvironment and metabolic activity of the chondrocyte in native cartilage and tissue engineering constructs. As far as we know

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

    PubMed

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

    2016-12-01

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

  14. Chondrocyte Apoptosis in the Pathogenesis of Osteoarthritis

    PubMed Central

    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

  15. Sclerostin Immunoreactivity Increases in Cortical Bone Osteocytes and Decreases in Articular Cartilage Chondrocytes in Aging Mice.

    PubMed

    Thompson, Michelle L; Jimenez-Andrade, Juan Miguel; Mantyh, Patrick W

    2016-03-01

    Sclerostin is a 24-kDa secreted glycoprotein that has been identified as a negative modulator of new bone formation and may play a major role in age-related decline in skeletal function. Although serum levels of sclerostin markedly increase with age, relatively little is known about whether cells in the skeleton change their expression of sclerostin with aging. Using immunohistochemistry and confocal microscopy, we explored sclerostin immunoreactivity (sclerostin-IR) in the femurs of 4-, 9-, and 24-month-old adult C3H/HeJ male mice. In the femur, the only two cell types that expressed detectable levels of sclerostin-IR were bone osteocytes and articular cartilage chondrocytes. At three different sites along the diaphysis of the femur, only a subset of osteocytes expressed sclerostin-IR and the percentage of osteocytes that expressed sclerostin-IR increased from approximately 36% to 48% in 4- vs. 24-month-old mice. In marked contrast, in the same femurs, there were ~40% fewer hypertrophic chondrocytes of articular cartilage that expressed sclerostin-IR when comparing 24- vs. 4-month-old mice. Understanding the mechanism(s) that drive these divergent changes in sclerostin-IR may provide insight into understanding and treating the age-related decline of the skeleton. © 2016 The Histochemical Society.

  16. Sclerostin Immunoreactivity Increases in Cortical Bone Osteocytes and Decreases in Articular Cartilage Chondrocytes in Aging Mice

    PubMed Central

    Thompson, Michelle L.; Jimenez-Andrade, Juan Miguel; Mantyh, Patrick W.

    2015-01-01

    Sclerostin is a 24-kDa secreted glycoprotein that has been identified as a negative modulator of new bone formation and may play a major role in age-related decline in skeletal function. Although serum levels of sclerostin markedly increase with age, relatively little is known about whether cells in the skeleton change their expression of sclerostin with aging. Using immunohistochemistry and confocal microscopy, we explored sclerostin immunoreactivity (sclerostin-IR) in the femurs of 4-, 9-, and 24-month-old adult C3H/HeJ male mice. In the femur, the only two cell types that expressed detectable levels of sclerostin-IR were bone osteocytes and articular cartilage chondrocytes. At three different sites along the diaphysis of the femur, only a subset of osteocytes expressed sclerostin-IR and the percentage of osteocytes that expressed sclerostin-IR increased from approximately 36% to 48% in 4- vs. 24-month-old mice. In marked contrast, in the same femurs, there were ~40% fewer hypertrophic chondrocytes of articular cartilage that expressed sclerostin-IR when comparing 24- vs. 4-month-old mice. Understanding the mechanism(s) that drive these divergent changes in sclerostin-IR may provide insight into understanding and treating the age-related decline of the skeleton. PMID:26701970

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

    PubMed

    Zhang, Lu; Spector, Myron

    2009-08-01

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

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2016-02-01

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

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

    PubMed

    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

  1. Self-assembling peptide hydrogel fosters chondrocyte extracellular matrix production and cell division: Implications for cartilage tissue repair

    NASA Astrophysics Data System (ADS)

    Kisiday, J.; Jin, M.; Kurz, B.; Hung, H.; Semino, C.; Zhang, S.; Grodzinsky, A. J.

    2002-07-01

    Emerging medical technologies for effective and lasting repair of articular cartilage include delivery of cells or cell-seeded scaffolds to a defect site to initiate de novo tissue regeneration. Biocompatible scaffolds assist in providing a template for cell distribution and extracellular matrix (ECM) accumulation in a three-dimensional geometry. A major challenge in choosing an appropriate scaffold for cartilage repair is the identification of a material that can simultaneously stimulate high rates of cell division and high rates of cell synthesis of phenotypically specific ECM macromolecules until repair evolves into steady-state tissue maintenance. We have devised a self-assembling peptide hydrogel scaffold for cartilage repair and developed a method to encapsulate chondrocytes within the peptide hydrogel. During 4 weeks of culture in vitro, chondrocytes seeded within the peptide hydrogel retained their morphology and developed a cartilage-like ECM rich in proteoglycans and type II collagen, indicative of a stable chondrocyte phenotype. Time-dependent accumulation of this ECM was paralleled by increases in material stiffness, indicative of deposition of mechanically functional neo-tissue. Taken together, these results demonstrate the potential of a self-assembling peptide hydrogel as a scaffold for the synthesis and accumulation of a true cartilage-like ECM within a three-dimensional cell culture for cartilage tissue repair.

  2. Surface zone articular chondrocytes modulate the bulk and surface mechanical properties of the tissue-engineered cartilage.

    PubMed

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

    2014-12-01

    The central hypothesis of functional tissue engineering is that an engineered construct can serve as a viable replacement tissue in vivo by replicating the structure and function of native tissue. In the case of articular cartilage, this requires the reproduction of the bulk mechanical and surface lubrication properties of native hyaline cartilage. Cartilage tissue engineering has primarily focused on achieving the bulk mechanical properties of native cartilage such as the compressive aggregate modulus and tensile strength. A scaffold-free self-assembling process has been developed that produces engineered cartilage with compressive properties approaching native tissue levels. Thus, the next step in this process is to begin addressing the friction coefficient and wear properties of these engineered constructs. The superficial zone protein (SZP), also known as lubricin or PRG4, is a boundary mode lubricant that is synthesized by surface zone (SZ) articular chondrocytes. Under conditions of high loading and low sliding speeds, SZP reduces friction and wear at the articular surface. The objective of this investigation was to determine whether increasing the proportion of SZ chondrocytes in cartilage constructs, in the absence of external stimuli such as growth factors and mechanical loading, would enhance the secretion of SZP and improve their frictional properties. In this study, cartilage constructs were engineered through a self-assembling process with varying ratios of SZ and middle zone (MZ) chondrocytes (SZ:MZ): 0:100, 25:75, 50:50, 75:25, and 100:0. Constructs containing different ratios of SZ and MZ chondrocytes did not significantly differ in the glycosaminoglycan composition or compressive aggregate modulus. In contrast, tensile properties and collagen content were enhanced in nearly all constructs containing greater amounts of SZ chondrocytes. Increasing the proportion of SZ chondrocytes had the hypothesized effect of improving the synthesis and secretion

  3. The cytotoxicity of bupivacaine, ropivacaine, and mepivacaine on human chondrocytes and cartilage.

    PubMed

    Breu, Anita; Rosenmeier, Katharina; Kujat, Richard; Angele, Peter; Zink, Wolfgang

    2013-08-01

    Intraarticular injections of local anesthetics are frequently used as part of multimodal pain regimens. However, recent data suggest that local anesthetics affect chondrocyte viability. In this study, we assessed the chondrotoxic effects of mepivacaine, ropivacaine, and bupivacaine. We hypothesized that specific cytotoxic potencies directly correlate with analgesic potencies, and that cytotoxic effects in intact cartilage are different than in osteoarthritic tissue. Human articular chondrocytes were exposed to equal and equipotent concentrations of bupivacaine, ropivacaine, and mepivacaine for 1 hour. Cell viability, apoptosis, and necrosis were determined at predefined time points using flow cytometry, live-dead staining, and caspase detection. Intact and osteoarthritic human cartilage explants were treated with equipotent concentrations of named drugs to determine cell viability applying fluorescence microscopy. Chondrotoxic effects increased from ropivacaine to mepivacaine to bupivacaine in a time-dependent and concentration-dependent manner. Compared with control, bupivacaine 0.5% decreased chondrocyte viability to 78% ± 9% (P = 0.0183) 1 hour and 16% ± 10% (P < 0.0001) 24 hours later, as determined by live-dead staining in monolayer cultures. Viability rates were reduced to 80% ± 7% (P = 0.0475) 1 hour and 80% ± 10% (P = 0.0095) 24 hours after treatment with ropivacaine 0.75%. After exposure to mepivacaine 2%, viable cells were scored 36% ± 6% (P < 0.0001) after 1 hour and 30% ± 11% (P < 0.0001) after 24 hours. Ropivacaine treatment was less chondrotoxic than bupivacaine (P = 0.0006) and mepivacaine exposure (P = 0.0059). Exposure to concentrations up to 0.25% of bupivacaine, 0.5% of ropivacaine, and 0.5% of mepivacaine did not reveal significant chondrotoxicity in flow cytometry. However, chondrotoxicity did not correlate with potency of local anesthetics. Immediate cell death was mainly due to necrosis followed by apoptosis. Cellular death rates were

  4. In Vitro Expression of the Extracellular Matrix Components Aggrecan, Collagen Types I and II by Articular Cartilage-Derived Chondrocytes.

    PubMed

    Schneevoigt, J; Fabian, C; Leovsky, C; Seeger, J; Bahramsoltani, M

    2017-02-01

    The extracellular matrix (ECM) of hyaline cartilage is perfectly suited to transmit articular pressure load to the subchondral bone. Pressure is transferred by a high amount of aggrecan-based proteoglycans and collagen type II fibres in particular. After any injury, the hyaline cartilage is replaced by fibrocartilage, which is low in proteoglycans and contains collagen type I predominantly. Until now, long-term results of therapeutic procedures including cell-based therapies like autologous chondrocyte transplantation (ACT) lead to a replacement tissue meeting the composition of fibrocartilage. Therefore, it is of particular interest to discover how and to what extent isolation and in vitro cultivation of chondrocytes affect the cells and their expression of ECM components. Hyaline cartilage-derived chondrocytes were cultivated in vitro and observed microscopically over a time period of 35 days. The expression of collagen type I, collagen type II and aggrecan was analysed using RT-qPCR and Western blot at several days of cultivation. Chondrocytes presented a longitudinal shape for the entire cultivation period. While expression of collagen type I prevailed within the first days, only prolonged cultivation led to an increase in collagen type II and aggrecan expression. The results indicate that chondrocyte isolation and in vitro cultivation lead to a dedifferentiation at least to the stage of chondroprogenitor cells.

  5. Implantation of rAAV5-IGF-I Transduced Autologous Chondrocytes Improves Cartilage Repair in Full-thickness Defects in the Equine Model

    PubMed Central

    Ortved, Kyla F; Begum, Laila; Mohammed, Hussni O; Nixon, Alan J

    2015-01-01

    Cartilage injury often precipitates osteoarthritis which has driven research to bolster repair in cartilage impact damage. Autologous chondrocytes transduced with rAAV5-IGF-I were evaluated in chondral defects in a well-established large animal model. Cartilage was harvested from the talus of 24 horses; chondrocytes were isolated and stored frozen. Twenty million cells were cultured and transduced with 105 AAV vg/cell prior to implantation. Chondrocytes from eight horses were transduced with rAAV5-IGF-I, chondrocytes from eight horses with rAAV5-GFP, and chondrocytes from eight horses were not transduced. A 15 mm full-thickness chondral defect was created arthroscopically in the lateral trochlear ridge of the femur in both femoropatellar joints. Treated defects were filled with naive or gene-enhanced chondrocytes, in fibrin vehicle. Control defects in the opposite limb received fibrin alone. rAAV5-IGF-I transduced chondrocytes resulted in significantly better healing at 8 week arthroscopy and 8 month necropsy examination when compared to controls. At 8 months, defects implanted with cells expressing IGF-I had better histological scores compared to control defects and defects repaired with naive chondrocytes. This included increased chondrocyte predominance and collagen type II, both features of hyaline-like repair tissue. The equine model closely approximates human cartilage healing, indicating AAV-mediated genetic modification of chondrocytes may be clinically beneficial to humans. PMID:25311491

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

    PubMed

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

    2007-05-01

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

  7. Hyperosmolaric contrast agents in cartilage tomography may expose cartilage to overload-induced cell death.

    PubMed

    Turunen, M J; Töyräs, J; Lammi, M J; Jurvelin, J S; Korhonen, R K

    2012-02-02

    In clinical arthrographic examination, strong hypertonic contrast agents are injected directly into the joint space. This may reduce the stiffness of articular cartilage, which is further hypothesized to lead to overload-induced cell death. We investigated the cell death in articular cartilage while the tissue was compressed in situ in physiological saline solution and in full strength hypertonic X-ray contrast agent Hexabrix(TM). Samples were prepared from bovine patellae and stored in Dulbecco's Modified Eagle's Medium overnight. Further, impact tests with or without creep were conducted for the samples with contact stresses and creep times changing from 1 MPa to 10 MPa and from 0 min to 15 min, respectively. Finally, depth-dependent cell viability was assessed with a confocal microscope. In order to characterize changes in the biomechanical properties of cartilage as a result of the use of Hexabrix™, stress-relaxation tests were conducted for the samples immersed in Hexabrix™ and phosphate buffered saline (PBS). Both dynamic and equilibrium modulus of the samples immersed in Hexabrix™ were significantly (p<0.05) lower than those of the samples immersed in PBS. Cartilage samples immersed in physiological saline solution showed load-induced cell death primarily in the superficial and middle zones. However, under high 8-10 MPa contact stresses, the samples immersed in full strength Hexabrix™ showed significantly (p<0.05) higher number of dead cells than the samples compressed in physiological saline, especially in the deep zone of cartilage. In conclusion, excessive loading stresses followed by tissue creep might increase the risk for chondrocyte death in articular cartilage when immersed in hypertonic X-ray contrast agent, especially in the deep zone of cartilage.

  8. Excess maternal and postnatal thyroxine alters chondrocyte numbers and the composition of the extracellular matrix of growth cartilage in rats.

    PubMed

    Ribeiro, Lorena Gabriela Rocha; Silva, Juneo Freitas; Ocarino, Natália de Melo; de Melo, Eliane Gonçalves; Serakides, Rogéria

    2017-02-13

    Purpose/Aim: The aim of this study was to evaluate the effects of excess maternal and postnatal thyroxine on chondrocytes and the extracellular matrix (ECM) of growth cartilage. We used 16 adult female Wistar rats divided into two groups: thyroxine treatment and control. From weaning to 40 days of age, offspring of the treated group (n = 8) received L-thyroxine. Plasma free T4 was measured. Histomorphometric analysis was performed on thyroids and femurs of all offspring. Alcian blue histochemical staining and real-time reverse transcription polymerase chain reaction measurements of gene expression levels of Sox9, Runx2, Aggrecan, Col I, Col II, Alkaline phosphatase, Mmp2, Mmp9, and Bmp2 were performed. Data were analyzed for statistical significance by student's t-test. Excess maternal and postnatal thyroxine reduced the intensity of Alcian blue staining, altered the number of chondrocytes in proliferative and hypertrophic zones in growth cartilage, and reduced the gene expression of Sox9, Mmp2, Mmp9, Col II, and Bmp2 in the growth cartilage of all offspring. Additionally, excess thyroxine altered the gene expression of Runx2, Aggrecan and Col I, and this effect was dependent on age. Excess thyroxine in neonates suppresses chondrocyte proliferation, stimulates chondrocyte hypertrophy and changes the ECM composition by reducing the amount of proteoglycans and glycosaminoglycans (GAGs). Prolonged exposure to excess thyroxine suppresses chondrocyte activity in general, with a severe reduction in the proteoglycan content of cartilage and the expression of gene transcripts essential for endochondral growth and characteristics of the chondrocyte phenotype.

  9. Cartilage Defect Treatments: With or without Cells? Mesenchymal Stem Cells or Chondrocytes? Traditional or Matrix-Assisted? A Systematic Review and Meta-Analyses

    PubMed Central

    Deng, Zhantao; Jin, Jiewen; Zhao, Jianning; Xu, Haidong

    2016-01-01

    Articular cartilage defects have been addressed by using multiple strategies. In the last two decades, promising new strategies by using assorted scaffolds and cell sources to induce tissue regeneration have emerged, such as autologous chondrocyte implantation (ACI) and mesenchymal stem cell implantation (MSCI). However, it is still controversial in the clinical strategies when to choose these treatments. Thus, we conducted a systematic review and meta-analyses to compare the efficacy and safety of different cartilage treatments. In our study, 17 studies were selected to compare different treatments for cartilage defects. The results of meta-analyses indicated that cell-based cartilage treatments showed significant better efficacy than cell-free treatments did (OR: 4.27, 95% CI: 2.19–8.34; WMD: 10.11, 95% CI: 2.69–16.53). Another result indicated that MACT had significant better efficacy than traditional ACI did (OR: 0.49, 95% CI: 0.30–0.82). Besides, the incidence of graft hypertrophy of MACT was slightly lower than that of traditional ACI (OR: 2.43, 95% CI: 1.00–5.94). Current data showed that the cell-based treatments and MACT are better options for cartilage treatments, but more well-designed comparative studies are still needed to enhance our understanding of different treatments for cartilage defects. PMID:26839570

  10. Temperature changes and chondrocyte death during drilling in a bovine cartilage model and chondroprotection by modified irrigation solutions.

    PubMed

    Farhan-Alanie, Muhamed M H; Hall, Andrew C

    2014-11-01

    Drilling into cartilage/bone is often required for orthopaedic surgery. While drilling into bone has been studied, the response of cartilage has received little attention. We have measured cartilage and drill bit temperatures during drilling and quantified the zone of chondrocyte death (ZCD) around the hole in the presence/absence of irrigation solutions. Drilling was performed using a 1.5-mm orthopaedic drill bit applied to bovine metatarsophalangeal joints and temperatures recorded by infrared camera. Osteochondral explants were then incubated with 5-chloromethylfluorescein diacetate (CMFDA) and propidium iodide (PI) to label living/dead chondrocytes respectively. The width of the ZCD was quantified by confocal laser scanning microscopy (CLSM) and image analysis. Without irrigation, the ZCD following drilling for two seconds was 135 ± 15 μm and this increased (>fourfold, P < 0.001) with five seconds of drilling. Irrigation reduced the ZCD following drilling for both two and five seconds (P < 0.05, P < 0.001 respectively) to the same level (approx. 60 μm). Without irrigation, drill bit and cartilage temperature increased rapidly to >265 and 119 °C respectively, whereas the camera saturated at >282 °C during drilling for five seconds. With irrigation, the drill bit temperature was significantly reduced during drilling for two and five seconds (approx. 90 °C) with negligible change in cartilage temperature. Drilling while irrigating with hyperosmotic saline (600 mOsm) reduced (P < 0.01) the ZCD compared to saline, whereas chondrocyte death was increased (P < 0.01) by Ca(2+) saline (5 mM). Reducing temperature during drilling by irrigation markedly suppressed, but did not abolish chondrocyte death. Optimising the irrigation solution by raising osmolarity and reducing Ca(2+) content significantly reduced chondrocyte death during drilling and may be clinically beneficial.

  11. Characterization of human mesenchymal stem cell-engineered cartilage: analysis of its ultrastructure, cell density and chondrocyte phenotype compared to native adult and fetal cartilage.

    PubMed

    Hillel, Alexander T; Taube, Janis M; Cornish, Toby C; Sharma, Blanka; Halushka, Marc; McCarthy, Edward F; Hutchins, Grover M; Elisseeff, Jennifer H

    2010-01-01

    The production of engineered cartilage from mesenchymal stem cells is a rapidly developing field. Potential applications include the treatment of degenerative joint disease as well as the treatment of traumatic and surgical bone injury. Prior to clinical application, however, further characterization of the morphology, ultrastructure, biocompatibility, and performance of the engineered tissue is warranted. To achieve this, human mesenchymal stem cells (hMSCs) were grown in vitro in pellet culture for 3 weeks in chondrogenic medium conditions. The resultant engineered cartilage was compared to native adult and fetal tissue. Routine histology, special stains, and ultrastructural and quantitative histomorphometric analyses were performed. The engineered tissue demonstrated a similar chondrocyte phenotype, collagen fibril appearance, and matrix distribution when compared to native cartilage. By histomorphometric analysis, the cell density of the engineered cartilage was between that of native fetal and adult cartilage. The cell-to-matrix ratio and cellular area fraction of engineered cartilage samples was significantly greater than in adult samples, but indistinguishable from fetal cartilage samples, supporting the hypothesis that hMSC-engineered cartilage regeneration may mimic fetal cartilage development.

  12. Long-term repetitive mechanical loading of the knee joint by in vivo muscle stimulation accelerates cartilage degeneration and increases chondrocyte death in a rabbit model.

    PubMed

    Horisberger, Monika; Fortuna, Rafael; Valderrabano, Victor; Herzog, Walter

    2013-06-01

    Excessive chronic loading is thought to be one factor responsible for the onset of osteoarthritis. For example, studies using treadmill running have shown an increased risk for osteoarthritis, thereby suggesting that muscle-induced joint loading may play a role in osteoarthritis onset and progression. However, in these studies, muscle-induced loading was not carefully quantified. Here, we present a model of controlled muscular loading which allows for the accurate quantification of joint loading. The aim of this study was to evaluate the effects of long-term, cyclic, isometric and dynamic, muscle-induced joint loading of physiologic magnitude but excessive intensity on cartilage integrity and cell viability in the rabbit knee. 24 rabbits were divided into an (i) eccentric, (ii) concentric, or (iii) isometric knee extensor contraction group (50 min of cyclic, submaximal stimulation 3 times/week for four weeks=19,500 cycles) controlled by the stimulation of a femoral nerve cuff electrode on the right hind limb. The contralateral knee was used as a non-loaded control. The knee articular cartilages were analysed by confocal microscopy for chondrocyte death, and histologically for Mankin Score, cartilage thickness and cell density. All loaded knees had significantly increased cell death rates and Mankin Scores compared to the non-loaded joints. Cartilage thicknesses did not systematically differ between loaded and control joints. Chondrocyte death and Mankin Scores were significantly increased in the loaded joints, thereby linking muscular exercise of physiologic magnitude but excessive intensity to cartilage degeneration and cell death in the rabbit knee. Copyright © 2013 Elsevier Ltd. All rights reserved.

  13. Effects of resveratrol on collagen type II protein in the superficial and middle zone chondrocytes of porcine articular cartilage.

    PubMed

    Maepa, Makwese; Razwinani, Mapula; Motaung, Shirley

    2016-02-03

    Resveratrol (RSV) was first isolated in 1940 from the roots of white hellebore (Veratrum grandiflorum (Maxim. ex Miq) O. Loes) and in 1963 from the roots of Japanese knotweed (Polygonum cuspidatum Siebold & Zucc.). These species have been used traditionally to treat arthritis, gout or inflammation. RSV (3,5,4-trihydroxystilbene) is a polyphenolic phytoalexin compound found in various plants, such as grape vines, berries, peanuts, seeds and roots; the highest concentration is in the skin of red grapes. This component of red wine has potent anti-inflammatory properties and may reduce the side effects of non-steroidal anti-inflammatory drugs that are currently used for pain amelioration in osteoarthritis (OA). In early degeneration of articular cartilage, which may lead to OA there is a loss of the tensile properties, indicative of damage to the fibrillar network. Damage to this fibrillar meshwork, made up of primarily collagen type II (90-95%), may be a critical event in the pathology of many arthritides, due in part to the very slow rate of collagen turnover within the cartilage. Collagen type II is the pre-dominant protein of the cartilage middle zone matrix mainly responsible for tensile strength of articular cartilage. The aim of the study was to investigate the effects of RSV on the expression of collagen type II from the superficial and middle zone chondrocytes of porcine articular cartilage. Porcine articular chondrocytes were isolated from the superficial and middle zone of articular cartilage, cultured as monolayers in serum-free chemically defined medium for four days. Effects of RSV on porcine articular chondrocytes were studied by assessing expression of collagen type II mRNA by RT-PCR and protein levels of collagen type II by ELISA; as well as localisation of collagen type II on cartilage tissue sections using immunohistochemistry. RSV significantly stimulated the expression of collagen type II at the mRNA and protein levels in the superficial and middle

  14. Cell compaction influences the regenerative potential of passaged bovine articular chondrocytes in an ex vivo cartilage defect model.

    PubMed

    Schmutzer, Michael; Aszodi, Attila

    2017-04-01

    The loss and degradation of articular cartilage tissue matrix play central roles in the process of osteoarthritis (OA). New models for evaluating cartilage repair/regeneration are thus of great value for transferring various culture systems into clinically relevant situations. The repair process can be better monitored in ex vivo systems than in in vitro cell cultures. I have therefore established an ex vivo defect model prepared from bovine femoral condyles for evaluating cartilage repair by the implantation of cells cultured in various ways, e.g., monolayer-cultured cells or suspension or pellet cultures of articular bovine chondrocytes representing different cell compactions with variable densities of chondrocytes. I report that the integrin subunit α10 was significantly upregulated in suspension-cultured bovine chondrocytes at passage P2 compared with monolayer-cultured cells at P1 (p = 0.0083) and P2 (p < 0.05). Suspension-cultured cells did not promote cartilage repair when compared with implanted monolayer-cultured chondrocytes and pellets: 24.0 ± 0.66% for suspension cells, 46.4 ± 2.9% for monolayer cells, and 127.64 ± 0.90% for pellets (p < 0.0001) of the original defect volume (percentage of defect). Additional cultivation with chondrogenesis-promoting growth factors TGF-β1 and BMP-2 revealed an enhancing effect on cartilage repair in all settings. The advantage and innovation of this system over in vitro differentiation (e.g., micromass, pellet) assays is the possibility of examining and evaluating cartilage regeneration in an environment in which implanted cells are embedded within native surrounding tissue at the defect site. Such ex vivo explants might serve as a better model system to mimic clinical situations.

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

    Yang, Hongxu; Zhang, Mian; Wang, Xin; Zhang, Hongyun; Zhang, Jing; Jing, Lei; Liao, Lifan; Wang, Meiqing

    2015-01-01

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

  17. Chondrocytes are released as viable cells during cartilage resorption associated with the formation of intrachondral canals in the rat tibial epiphysis.

    PubMed

    Alvarez, Jesús; Costales, Lorena; López-Muñiz, Alfonso; López, José M

    2005-06-01

    The development of cartilage canals is the first event of the ossification of the epiphyses in mammals. Canal formation differs from vascular invasion during primary ossification, since the former involves resorption of resting cartilage and is uncoupled from bone deposition. To learn more about the fate of resorbed chondrocytes during this process, we have carried out structural, cell proliferation, and in situ hybridization studies during the first stages of ossification of the rat tibial proximal epiphysis. Results concerning the formation of the cartilage canals implied the release of resting chondrocytes from the cartilage matrix to the canal cavity. Released chondrocytes had a well-preserved structure, expressed type-II collagen, and maintained the capacity to divide. All these data suggested that chondrocytes released into the canals remained viable for a specific time. Analysis of the proliferative activity at different regions of the cartilage canals showed that the percentage of proliferative chondrocytes at areas of active cartilage resorption was significantly higher than that in zones of low resorption. These results are consistent with the hypothesis that resting chondrocytes surrounding canals have a role in supplying cells for the development of the secondary ossification center. Since released chondrocytes are at an early stage of differentiation greatly preceding their entry into the apoptotic pathway and are exposed to a specific matrix, cellular, and humoral microenvironment, they might differentiate to other cell types and contribute to the ossification of the epiphysis.

  18. Matrigel scaffold combined with Ad-hBMP7-transfected chondrocytes improves the repair of rabbit cartilage defect

    PubMed Central

    Xia, Xiaopeng; Li, Jing; Xia, Bo; Yang, Huilin; Zhang, Dongmei; Zhou, Bin; Zhang, Jie; Zhou, Man; Liu, Fan

    2017-01-01

    The aim of this study was to explore an effective method for the repair of cartilage defects using chitosan/glycerophosphate (C/GP) gel- and Matrigel-engineered human bone morphogenetic protein 7 (hBMP7)-expressing chondrocytes. Rabbit chondrocytes were obtained, cultured in vitro and transfected with an adenovirus containing hBMP7 and green fluorescent protein (Ad-hBMP7-GFP). The expression of hBMP7 in the transfected cells was tested by reverse transcription-polymerase chain reaction (RT-PCR) and western blotting. The phenotype of the transfected cells was evaluated by detecting the yields of collagen II and hyaluronic acid using RT-PCR and enzyme-linked immunosorbent assay (ELISA). The growth of chondrocytes in the C/GP gel and Matrigel was accessed by measuring the cell growth rate, hematoxylin and eosin (H&E) staining and observation under a scanning microscope. Twelve adult male New Zealand white rabbits were randomly divided into three groups. Two cartilage defects were created in the rabbits' knees by aseptic surgery. Group A (n=4) did not receive any treatment, group B (n=4) were treated with C/GP gel and Matrigel-engineered Ad-mock-GFP-transfected chondrocytes, and group C (n=4) were treated with C/GP gel and Matrigel-engineered Ad-hBMP7-GFP-transfected chondrocytes. Rabbits were sacrificed at 4 weeks after transplantation, and the repair effect was measured by the Wakitani scoring method. On the basis of the RT-PCR and western blot results, hBMP7 was efficiently overexpressed in the Ad-hBMP7-GFP-transfected chondrocytes. The ELISA results showed that the yields of collagen II and hyaluronic acid in Ad-hBMP7-GFP-transfected chondrocytes were significantly higher than those in Ad-mock-GFP-transfected chondrocytes. Chondrocytes have a better morphology and arrangement in a Matrigel scaffold than in C/GP, as assessed by H&E staining and scanning microscopy. According to the Wakitani score, Matrigel combined with Ad-hBMP7-GFP-transfected chondrocytes

  19. Second-generation arthroscopic autologous chondrocyte implantation for the treatment of degenerative cartilage lesions.

    PubMed

    Filardo, Giuseppe; Kon, Elizaveta; Di Martino, Alessandro; Patella, Silvio; Altadonna, Giulio; Balboni, Federica; Bragonzoni, Laura; Visani, Andrea; Marcacci, Maurilio

    2012-09-01

    Degenerative cartilage lesions present a negative joint environment, which may have a negative effect on the process of cartilage regeneration. The aim of this study is to analyze the clinical outcome obtained with the treatment for isolated degenerative knee cartilage lesions by second-generation arthroscopic autologous chondrocyte implantation (ACI). Fifty-eight consecutive patients affected by focal degenerative chondral lesions of the femoral condyles and trochlea were treated by second-generation arthroscopic ACI. The mean age at surgery was 34.7 ± 9.1 years and the average defect size was 2.3 ± 0.9 cm(2). The patients were prospectively evaluated with IKDC, EQ-VAS, and Tegner scores preoperatively, at 2 and 6 years. A statistically significant improvement was observed in all scores from the basal evaluation to the final follow-up. The IKDC subjective score improved from 39.3 ± 13.6 to 68.8 ± 22.7 and 68.5 ± 23.9 at the 2- and 6-year follow-ups, respectively, with a significant improvement (P < 0.0005) and stable results over time; the same trend was confirmed by the EQ-VAS and Tegner scores. The worst results were found in patients with a low physical activity level, women, and those having undergone previous surgery, whereas the symptom duration before surgery did not influence the final outcome. The failure rate was 18.5%. Despite a significant improvement, the results were lower with respect to the outcome reported in different study populations, and the number of failures was markedly higher, too. Tissue-engineered cartilage implantation is a promising approach for the treatment of degenerative chondral lesions, but graft properties, besides mechanical and biochemical joint environment, have to be improved. Case series, Level IV.

  20. PTHrP Overexpression Partially Inhibits a Mechanical Strain-Induced Arthritic Phenotype in Chondrocytes

    PubMed Central

    Wang, Dean; Taboas, Juan M.; Tuan, Rocky S.

    2010-01-01

    Objective Cell-based tissue engineering strategies are currently in clinical use and continue to be developed at a rapid pace for the repair of cartilage defects. Regardless of the repair methodology, chondrocytes within newly regenerated cartilage remain susceptible to the abnormal inflammatory and mechanical environments that underlie osteoarthritic disease, likely compromising the implant’s integration, function, and longevity. The present study investigates the use of parathyroid hormone-related peptide (PTHrP) overexpression for chondroprotection. Design Bovine articular chondrocytes were transfected with human PTHrP (hPTHrP) constructs (1-141 or 1-173) and subjected to injurious cyclic tensile strain (CTS; 0.5 Hz and 16% elongation) for 48 hours. mRNA expression of matrix remodeling, inflammatory signaling, hypertrophic, and apoptotic genes were examined with real-time reverse transcription polymerase chain reaction. Nitric oxide (NO) and prostaglandin E2 (PGE2) production were measured using the Griess assay and enzyme immunoassay, respectively. Results CTS induced an arthritic phenotype in articular chondrocytes as indicated by increased gene expression of collagenases and aggrecanases and increased production of NO and PGE2. Additionally, CTS increased collagen type X (Col10a1) mRNA expression, whereas overexpression of either hPTHrP isoform inhibited CTS-induced Col10a1 gene expression. However, hPTHrP 1-141 augmented CTS-induced NO and PGE2 production, and neither hPTHrP isoform had any significant effect on apoptotic genes. Conclusions Our results suggest that chondrocytes overexpressing PTHrP resist mechanical strain-induced hypertrophic-like changes. Therapeutic PTHrP gene transfer may be considered for chondroprotection applications in newly regenerated cartilage. PMID:21087676

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

    PubMed

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

    2013-01-01

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

  2. Autologous chondrocyte implantation for cartilage repair: monitoring its success by magnetic resonance imaging and histology

    PubMed Central

    Roberts, Sally; McCall, Iain W; Darby, Alan J; Menage, Janis; Evans, Helena; Harrison, Paul E; Richardson, James B

    2003-01-01

    Autologous chondrocyte implantation is being used increasingly for the treatment of cartilage defects. In spite of this, there has been a paucity of objective, standardised assessment of the outcome and quality of repair tissue formed. We have investigated patients treated with autologous chondrocyte implantation (ACI), some in conjunction with mosaicplasty, and developed objective, semiquantitative scoring schemes to monitor the repair tissue using MRI and histology. Results indicate repair tissue to be on average 2.5 mm thick. It was of varying morphology ranging from predominantly hyaline in 22% of biopsy specimens, mixed in 48%, through to predominantly fibrocartilage, in 30%, apparently improving with increasing time postgraft. Repair tissue was well integrated with the host tissue in all aspects viewed. MRI scans provide a useful assessment of properties of the whole graft area and adjacent tissue and is a noninvasive technique for long-term follow-up. It correlated with histology (P = 0.02) in patients treated with ACI alone. PMID:12716454

  3. Concise Review: Mesenchymal Stem Cells for Functional Cartilage Tissue Engineering: Taking Cues from Chondrocyte-Based Constructs.

    PubMed

    Tan, Andrea R; Hung, Clark T

    2017-04-01

    Osteoarthritis, the most prevalent form of joint disease, afflicts 9% of the U.S. population over the age of 30 and costs the economy nearly $100 billion annually in healthcare and socioeconomic costs. It is characterized by joint pain and dysfunction, though the pathophysiology remains largely unknown. Due to its avascular nature and limited cellularity, articular cartilage exhibits a poor intrinsic healing response following injury. As such, significant research efforts are aimed at producing engineered cartilage as a cell-based approach for articular cartilage repair. However, the knee joint is mechanically demanding, and during injury, also a milieu of harsh inflammatory agents. The unforgiving mechano-chemical environment requires tissue replacements that are capable of bearing such burdens. The use of mesenchymal stem cells (MSCs) for cartilage tissue engineering has emerged as a promising cell source due to their ease of isolation, capacity to readily expand in culture, and ability to undergo lineage-specific differentiation into chondrocytes. However, to date, very few studies utilizing MSCs have successfully recapitulated the structural and functional properties of native cartilage, exposing the difficult process of uniformly differentiating stem cells into desired cell fates and maintaining the phenotype during in vitro culture and after in vivo implantation. To address these shortcomings, here, we present a concise review on modulating stem cell behavior, tissue development and function using well-developed techniques from chondrocyte-based cartilage tissue engineering. Stem Cells Translational Medicine 2017;6:1295-1303.

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

    PubMed Central

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

    2016-01-01

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

  5. Lithium chloride prevents interleukin-1β induced cartilage degradation and loss of mechanical properties.

    PubMed

    Thompson, Clare L; Yasmin, Habiba; Varone, Anna; Wiles, Anna; Poole, C Antony; Knight, Martin M

    2015-10-01

    Osteoarthritis is a chronic degenerative disease that affects the articular cartilage. Recent studies have demonstrated that lithium chloride exhibits significant efficacy as a chondroprotective agent, blocking cartilage degradation in response to inflammatory cytokines. However, conflicting literature suggests lithium may affect the physicochemical properties of articular cartilage and thus long-term exposure may negatively affect the mechanical functionality of this tissue. This study aims to investigate the effect of lithium chloride on the biomechanical properties of healthy and interleukin-1β treated cartilage in vitro and examines the consequences of long-term exposure to lithium on cartilage health in vivo. Bovine cartilage explants were treated with lithium chloride for 12 days. Chondrocyte viability, matrix catabolism and the biomechanical properties of bovine cartilage explants were not significantly altered following treatment. Consistent with these findings, long term-exposure (9 months) to dietary lithium did not induce osteoarthritis in rats, as determined by histological staining. Moreover, lithium chloride did not induce the expression of catabolic enzymes in human articular chondrocytes. In an inflammatory model of cartilage destruction, lithium chloride blocked interleukin-1β signaling in the form of nitric oxide and prostaglandin E2 release and prevented matrix catabolism such that the loss of mechanical integrity observed with interleukin-1β alone was inhibited. This study provides further support for lithium chloride as a novel compound for the treatment of osteoarthritis.

  6. Standardized butanol fraction of WIN-34B suppresses cartilage destruction via inhibited production of matrix metalloproteinase and inflammatory mediator in osteoarthritis human cartilage explants culture and chondrocytes

    PubMed Central

    2012-01-01

    Background WIN-34B is a novel Oriental medicine, which represents the n-butanol fraction prepared from dried flowers of Lonicera japonica Thunb and dried roots of Anemarrhena asphodeloides BUNGE. The component herb of WIN-34B is used for arthritis treatment in East Asian countries. The aim of this study was to determine the cartilage-protective effects and mechanisms of WIN-34B and its major phenolic compounds, chlorogenic acid and mangiferin, in osteoarthritis (OA) human cartilage explants culture and chondrocytes. Methods The investigation focused on whether WIN-34B and its standard compounds protected cartilage in interleukin (IL)-1β-stimulated cartilage explants culture and chondrocytes derived from OA patients. Also, the mechanisms of WIN-34B on matrix metalloproteinases (MMPs), tissue inhibitor of matrix metalloproteinases (TIMPs), inflammatory mediators, and mitogen-activated protein kinases (MAPKs) pathways were assessed. Results WIN-34B was not cytotoxic to cultured cartilage explants or chondrocytes. WIN-34B dose-dependently inhibited the release of glycosaminoglycan and type II collagen, increased the mRNA expression of aggrecan and type II collagen, and recovered the intensity of proteoglycan and collagen by histological analysis in IL-1β-stimulated human cartilage explants culture. The cartilage protective effect of WIN-34B was similar to or better than that of chlorogenic acid and mangiferin. Compared to chlorogenic acid and mangiferin, WIN-34B displayed equal or greater decreases in the levels of MMP-1, MMP-3, MMP-13, ADAMTS-4, and ADAMTS-5, and markedly up-regulated TIMP-1 and TIMP-3. WIN-34B inhibited inflammatory mediators involved in cartilage destruction, such as prostaglandin E2, nitric oxide, tumor necrosis factor-alpha, and IL-1β. The phosphorylation of extracellular signal-regulated kinase, c-Jun N-terminal kinase (JNK), and p38 was significantly reduced by WIN-34B treatment, while phosphorylation of JNK was only inhibited by chlorogenic

  7. Berberine ameliorates cartilage degeneration in interleukin-1β-stimulated rat chondrocytes and in a rat model of osteoarthritis via Akt signalling

    PubMed Central

    Zhao, Honghai; Zhang, Tongen; Xia, Chun; Shi, Lei; Wang, Shaojie; Zheng, Xinpeng; Hu, Tianhui; Zhang, Bing

    2014-01-01

    Berberine, a plant alkaloid used in Chinese medicine, has broad cell-protective functions in a variety of cell lines. Chondrocyte apoptosis contributes to the pathogenesis of cartilage degeneration in osteoarthritis (OA). However, little is known about the effect and underlying mechanism of berberine on OA chondrocytes. Here, we assessed the effects of berberine on cartilage degeneration in interleukin-1β (IL-1β)-stimulated rat chondrocytes and in a rat model of OA. The results of an MTT assay and western blotting analysis showed that berberine attenuated the inhibitory effect of IL-1β on the cell viability and proliferating cell nuclear antigen expression in rat chondrocytes. Furthermore, berberine activated Akt, which triggered p70S6K/S6 pathway and up-regulated the levels of aggrecan and Col II expression in IL-1β-stimulated rat chondrocytes. In addition, berberine increased the level of proteoglycans in cartilage matrix and the thickness of articular cartilage, with the elevated levels of Col II, p-Akt and p-S6 expression in a rat OA model, as demonstrated by histopathological and immunohistochemistry techniques. The data thus strongly suggest that berberine may ameliorate cartilage degeneration from OA by promoting cell survival and matrix production of chondrocytes, which was partly attributed to the activation of Akt in IL-1β-stimulated articular chondrocytes and in a rat OA model. The resultant chondroprotective effects indicate that berberine merits consideration as a therapeutic agent in OA. PMID:24286347

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

    PubMed

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

    2015-11-01

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

  9. Changes in collagens and chondrocytes in the temporomandibular joint cartilage in growing rats fed a liquid diet.

    PubMed

    Uekita, Hiroki; Takahashi, Shigeru; Domon, Takanori; Yamaguchi, Taihiko

    2015-11-01

    The temporomandibular joint (TMJ) of growing rats fed a soft diet is reported to be smaller in size and to have thinner condyle and glenoid fossa cartilage than rats fed a solid diet. The aim of this study was to determine the effect of a soft diet on the collagens and chondrocytes in the growing TMJ cartilage. Forty-eight male Wistar rats were divided into a control group fed a solid diet and an experimental group fed a liquid diet for 1-8 weeks. After the experimental period, the TMJs were harvested and examined histologically, immunohistochemically for collagen types I, II, and X, and with transmission electron microscopy. The condylar cartilage in the experimental rats showed weak immunoreactions for three types of collagens compared with the controls. The ultrastructure had fewer fine collagen fibrils in the experimental rats compared with that of the controls. The glenoid fossa cartilage in the experimental rats showed narrower Alcian blue-positive areas than the control staining. The immunoreactions for three types of collagen in the experimental rats were also weaker than those of the controls. The chondrocytes in the experimental rats appeared dark, had extended thin cytoplasmic processes, and had formed gap junctions, as assessed by transmission electron microscopy. Fewer fine collagen fibrils, but thick bands of collagen fibrils were observed in the glenoid fossa of the experimental cartilage. The results of the present study showed that a liquid diet had deleterious effects on the quality and quantity of collagens and chondrocytes in the TMJ cartilage in growing rats.

  10. Repair of bone defects in vivo using tissue engineered hypertrophic cartilage grafts produced from nasal chondrocytes.

    PubMed

    Bardsley, Katie; Kwarciak, Agnieska; Freeman, Christine; Brook, Ian; Hatton, Paul; Crawford, Aileen

    2017-01-01

    The regeneration of large bone defects remains clinically challenging. The aim of our study was to use a rat model to use nasal chondrocytes to engineer a hypertrophic cartilage tissue which could be remodelled into bone in vivo by endochondral ossification. Primary adult rat nasal chondrocytes were isolated from the nasal septum, the cell numbers expanded in monolayer culture and the cells cultured in vitro on polyglycolic acid scaffolds in chondrogenic medium for culture periods of 5-10 weeks. Hypertrophic differentiation was assessed by determining the temporal expression of key marker genes and proteins involved in hypertrophic cartilage formation. The temporal changes in the genes measured reflected the temporal changes observed in the growth plate. Collagen II gene expression increased 6 fold by day 7 and was then significantly downregulated from day 14 onwards. Conversely, collagen X gene expression was detectable by day 14 and increased 100-fold by day 35. The temporal increase in collagen X expression was mirrored by increases in alkaline phosphatase gene expression which also was detectable by day 14 with a 30-fold increase in gene expression by day 35. Histological and immunohistochemical analysis of the engineered constructs showed increased chondrocyte cell volume (31-45 μm), deposition of collagen X in the extracellular matrix and expression of alkaline phosphatase activity. However, no cartilage mineralisation was observed in in vitro culture of up to 10 weeks. On subcutaneous implantation of the hypertrophic engineered constructs, the grafts became vascularised, cartilage mineralisation occurred and loss of the proteoglycan in the matrix was observed. Implantation of the hypertrophic engineered constructs into a rat cranial defect resulted in angiogenesis, mineralisation and remodelling of the cartilage tissue into bone. Micro-CT analysis indicated that defects which received the engineered hypertrophic constructs showed 38.48% in bone volume

  11. High seeding density of human chondrocytes in agarose produces tissue-engineered cartilage approaching native mechanical and biochemical properties.

    PubMed

    Cigan, Alexander D; Roach, Brendan L; Nims, Robert J; Tan, Andrea R; Albro, Michael B; Stoker, Aaron M; Cook, James L; Vunjak-Novakovic, Gordana; Hung, Clark T; Ateshian, Gerard A

    2016-06-14

    Animal cells have served as highly controllable model systems for furthering cartilage tissue engineering practices in pursuit of treating osteoarthritis. Although successful strategies for animal cells must ultimately be adapted to human cells to be clinically relevant, human chondrocytes are rarely employed in such studies. In this study, we evaluated the applicability of culture techniques established for juvenile bovine and adult canine chondrocytes to human chondrocytes obtained from fresh or expired osteochondral allografts. Human chondrocytes were expanded and encapsulated in 2% agarose scaffolds measuring ∅3-4mm×2.3mm, with cell seeding densities ranging from 15 to 90×10(6)cells/mL. Subsets of constructs were subjected to transient or sustained TGF-β treatment, or provided channels to enhance nutrient transport. Human cartilaginous constructs physically resembled native human cartilage, and reached compressive Young's moduli of up to ~250kPa (corresponding to the low end of ranges reported for native knee cartilage), dynamic moduli of ~950kPa (0.01Hz), and contained 5.7% wet weight (%/ww) of glycosaminoglycans (≥ native levels) and 1.5%/ww collagen. We found that the initial seeding density had pronounced effects on tissue outcomes, with high cell seeding densities significantly increasing nearly all measured properties. Transient TGF-β treatment was ineffective for adult human cells, and tissue construct properties plateaued or declined beyond 28 days of culture. Finally, nutrient channels improved construct mechanical properties, presumably due to enhanced rates of mass transport. These results demonstrate that our previously established culture system can be successfully translated to human chondrocytes.

  12. Honokiol, a low molecular weight natural product, prevents inflammatory response and cartilage matrix degradation in human osteoarthritis chondrocytes.

    PubMed

    Chen, Ying Ju; Tsai, Keh Sung; Chan, Ding Cheng; Lan, Kuo Cheng; Chen, Cheng Feng; Yang, Rong Sen; Liu, Shing Hwa

    2014-04-01

    Proinflammatory cytokine interleukin-1β (IL-1β) stimulates several mediators of cartilage degradation and plays an important role in the pathogenesis of osteoarthritis (OA). Honokiol, a low molecular weight natural product isolated from the Magnolia officinalis, has been shown to possess anti-inflammatory effect. Here, we used an in vitro model of cartilage inflammation to investigate the therapeutic potential of honokiol in OA. Human OA chondrocytes were cultured and pretreated with honokiol (2.5-10 µM) with or without IL-1β (10 ng/ml). Nitric oxide (NO) production was quantified by Griess reagent. Prostaglandin (PG)E2 , metalloproteinase-13 (MMP-13), and interleukin-6 (IL-6) productions were quantified by enzyme-linked immunosorbent assay. The expressions of collagen II, cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS), and nuclear factor κB (NF-κB)-related signaling molecules were determined by Western blotting. Our data showed that IL-1β markedly stimulated the expressions of iNOS and COX-2 and the productions of NO, PGE2 , and IL-6, which could be significantly reversed by honokiol. Honokiol could also suppress the IL-1β-triggered activation of IKK/IκBα/NF-κB signaling pathway. Moreover, honokiol significantly inhibited the IL-1β-induced MMP-13 production and collagen II reduction. Taken together, the present study suggests that honokiol may have a chondroprotective effect and may be a potential therapeutic choice in the treatment of OA patients. © 2013 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.

  13. In vitro effects of platelet-rich gel supernatants on histology and chondrocyte apoptosis scores, hyaluronan release and gene expression of equine cartilage explants challenged with lipopolysaccharide.

    PubMed

    Carmona, Jorge U; Ríos, Diana L; López, Catalina; Álvarez, María E; Pérez, Jorge E; Bohórquez, Mabel E

    2016-07-01

    Platelet-rich plasma (PRP) preparations are a common treatment in equine osteoarthritis (OA). However, there are controversies regarding the ideal concentration of platelets and leukocytes in these biological substances necessary to induce an adequate anti-inflammatory and anabolic response in articular cartilage. The aims were to study the influence of leukocyte- and platelet-rich gel (L-PRG) and pure platelet-rich gel (P-PRG) supernatants on the histological changes of cartilage, the degree of chondrocyte apoptosis, the production of hyaluronan (HA) and the gene expression of nuclear factor kappa beta (NFkβ), matrix metalloproteinase 13 (MMP-13), a disintegrin and metalloproteinase with thrombospondin motifs 4 (ADAMTS-4), collagen type I alpha 1 (COL1A1), collagen type II alpha 1 (COL2A1) and cartilage oligomeric matrix protein (COMP) in normal cartilage explants (CEs) challenged with lipopolysaccharide (LPS). Overall, 25 % L-PRG supernatant (followed in order of importance by, 50 % P-PRG, 25 % P-PRG and 50 % L-PRG) represented the substance with the most important anti-inflammatory and anabolic effect. 25 % P-PRG supernatant presented important anabolic effects, but it induced a more severe chondrocyte apoptosis than the other evaluated substances. 25 % L-PRG supernatant presented the best therapeutic profile. Our results demonstrate that the biological variability of PRP preparations makes their application rather challenging. Additional in vivo research is necessary to know the effect of PRP preparations at different concentrations.

  14. Paracrine effects of human adipose-derived mesenchymal stem cells in inflammatory stress-induced senescence features of osteoarthritic chondrocytes

    PubMed Central

    Platas, Julia; Guillén, Maria Isabel; del Caz, Maria Dolores Pérez; Gomar, Francisco; Castejón, Miguel Angel; Mirabet, Vicente; Alcaraz, Maria José

    2016-01-01

    Aging and exposure to stress would determine the chondrocyte phenotype in osteoarthritis (OA). In particular, chronic inflammation may contribute to stress-induced senescence of chondrocytes and cartilage degeneration during OA progression. Recent studies have shown that adipose-derived mesenchymal stem cells exert paracrine effects protecting against degenerative changes in chondrocytes. We have investigated whether the conditioned medium (CM) from adipose-derived mesenchymal stem cells may regulate senescence features induced by inflammatory stress in OA chondrocytes. Our results indicate that CM down-regulated senescence markers induced by interleukin-1β including senescence-associated β-galactosidase activity, accumulation of γH2AX foci and morphological changes with enhanced formation of actin stress fibers. Treatment of chondrocytes with CM also decreased the production of oxidative stress, the activation of mitogen-activated protein kinases, and the expression of caveolin-1 and p21. The effects of CM were related to the reduction in p53 acetylation which would be dependent on the enhancement of Sirtuin 1 expression. Therefore, CM may exert protective effects in degenerative joint conditions by countering the premature senescence of OA chondrocytes induced by inflammatory stress. PMID:27490266

  15. 4-Hydroxynonenal induces apoptosis in human osteoarthritic chondrocytes: the protective role of glutathione-S-transferase

    PubMed Central

    Vaillancourt, France; Fahmi, Hassan; Shi, Qin; Lavigne, Patrick; Ranger, Pierre; Fernandes, Julio C; Benderdour, Mohamed

    2008-01-01

    Introduction 4-Hydroxynonenal (HNE) is one of the most abundant and reactive aldehydes of lipid peroxidation products and exerts various effects on intracellular and extracellular signalling cascades. We have previously shown that HNE at low concentrations could be considered as an important mediator of catabolic and inflammatory processes in osteoarthritis (OA). In the present study, we focused on characterizing the signalling cascade induced by high HNE concentration involved in cell death in human OA chondrocytes. Methods Markers of apoptosis were quantified with commercial kits. Protein levels were evaluated by Western blotting. Glutathione (GSH) and ATP levels were measured with commercial kits. Glucose uptake was assessed by 2-deoxy-D-[3H]-glucose. The role of GSH-S-transferase A4-4 (GSTA4-4) in controlling HNE-induced chondrocyte apoptosis was investigated by chondrocyte transfection with small interfering RNA (siRNA) or with the expression vector of GSTA4-4. Results Our data showed that HNE at concentrations of up to 10 μM did not alter cell viability but was cytotoxic at concentrations of greater than or equal to 20 μM. HNE-induced chondrocyte death exhibited several classical hallmarks of apoptosis, including caspase activation, cytochrome c and apoptosis-induced factor release from mitochondria, poly (ADP-ribose) polymerase cleavage, Bcl-2 downregulation, Bax upregulation, and DNA fragmentation. Our study of signalling pathways revealed that HNE suppressed pro-survival Akt kinase activity but, in contrast, induced Fas/CD95 and p53 expression in chondrocytes. All of these effects were inhibited by an antioxidant, N-acetyl-cysteine. Analysis of cellular energy and redox status showed that HNE induced ATP, NADPH, and GSH depletion and inhibited glucose uptake and citric acid cycle activity. GSTA4-4 ablation by the siRNA method augmented HNE cytotoxicity, but, conversely, its overexpression efficiently protected chondrocytes from HNE-induced cell death

  16. Interleukin-1 Acts via the JNK-2 Signaling Pathway to Induce Aggrecan Degradation by Human Chondrocytes.

    PubMed

    Ismail, Heba M; Yamamoto, Kazuhiro; Vincent, Tonia L; Nagase, Hideaki; Troeberg, Linda; Saklatvala, Jeremy

    2015-07-01

    Aggrecan enables articular cartilage to bear load and resist compression. Aggrecan loss occurs early in osteoarthritis and rheumatoid arthritis and can be induced by inflammatory cytokines such as interleukin-1 (IL-1). IL-1 induces cleavage of specific aggrecans characteristic of the ADAMTS proteinases. The aim of this study was to identify the intracellular signaling pathways by which IL-1 causes aggrecan degradation by human chondrocytes and to investigate how aggrecanase activity is controlled by chondrocytes. We developed a cell-based assay combining small interfering RNA (siRNA)-induced knockdown with aggrecan degradation assays. Human articular chondrocytes were overlaid with bovine aggrecan after transfection with siRNAs against molecules of the IL-1 signaling pathway. After IL-1 stimulation, released aggrecan fragments were detected with AGEG and ARGS neoepitope antibodies. Aggrecanase activity and tissue inhibitor of metalloproteinases 3 levels were measured by enzyme-linked immunosorbent assay. Low-density lipoprotein receptor-related protein 1 (LRP-1) shedding was analyzed by Western blotting. ADAMTS-5 is a major aggrecanase in human chondrocytes, regulating aggrecan degradation in response to IL-1. The tumor necrosis factor receptor-associated 6 (TRAF-6)/transforming growth factor β-activated kinase 1 (TAK-1)/MKK-4 signaling axis is essential for IL-1-induced aggrecan degradation, while NF-κB is not. Of the 3 MAPKs (ERK, p38, and JNK), only JNK-2 showed a significant role in aggrecan degradation. Chondrocytes constitutively secreted aggrecanase, which was continuously endocytosed by LRP-1, keeping the extracellular level of aggrecanase low. IL-1 induced aggrecanase activity in the medium in a JNK-2-dependent manner, possibly by reducing aggrecanase endocytosis, because IL-1 caused JNK-2-dependent shedding of LRP-1. The signaling axis TRAF-6/TAK-1/MKK-4/JNK-2 mediates IL-1-induced aggrecanolysis. The level of aggrecanase is controlled by its

  17. Geranylgeranylacetone suppresses hydrogen peroxide-induced apoptosis of osteoarthritic chondrocytes.

    PubMed

    Yoda, Masaki; Sakai, Tadahiro; Mitsuyama, Hirohito; Hiraiwa, Hideki; Ishiguro, Naoki

    2011-11-01

    Osteoarthritis (OA) is a common disease, afflicting many sufferers with both pain and functional disorders. Various therapies have been attempted for OA, but no fully effective treatment has been established yet. Apoptosis of chondrocytes caused by reactive oxygen species (ROS) has been considered important in the pathogenesis of OA. The progression of OA may be prevented by suppressing apoptosis of chondrocytes. Geranylgeranylacetone (GGA) has been used as an anti-ulcer drug in Japan for more than 20 years. Several recent studies have shown that GGA can induce heat shock protein (HSP) and exert cytoprotective actions on a large variety of cells and tissues. In this study, we investigated the effects of GGA on the apoptosis of OA chondrocytes induced by hydrogen peroxide (H(2)O(2)). Human isolated OA chondrocytes were cultured in the absence or presence of GGA. Cell viability, caspase 3/7 and 9 activities, HSP70 mRNA and protein expressions were examined, and morphological analyses were conducted after exposure of cells to H(2)O(2) to induce apoptosis. Geranylgeranylacetone dose-dependently reversed the H(2)O(2)-induced decrease in cell viability. It was recognized that GGA rendered OA chondrocytes resistant to H(2)O(2)-induced apoptosis from Hoechst 33342 staining and TUNEL staining. Caspases 3 and 9 were activated by addition of H(2)O(2), and GGA suppressed this H(2)O(2)-induced activation of both caspases. H(2)O(2)-induced induction of HSP70 was enhanced in OA chondrocytes by pretreatment with GGA. The results showed that GGA can suppress apoptosis of chondrocytes and enhance production of HSP70. This study is the first, to our knowledge, to demonstrate that GGA protects OA chondrocytes from H(2)O(2)-induced apoptosis, at least in part by enhancing HSP70 production. These results indicate that GGA is a potentially useful drug for the treatment of OA.

  18. Characterization of healthy and osteoarthritic chondrocyte cell patterns on phase contrast CT images of the knee cartilage matrix

    NASA Astrophysics Data System (ADS)

    Nagarajan, Mahesh B.; Coan, Paola; Huber, Markus B.; Yang, Chien-Chun; Glaser, Christian; Reiser, Maximilian F.; Wismüller, Axel

    2012-03-01

    The current approach to evaluating cartilage degeneration at the knee joint requires visualization of the joint space on radiographic images where indirect cues such as joint space narrowing serve as markers for osteoarthritis. A recent novel approach to visualizing the knee cartilage matrix using phase contrast CT imaging (PCI-CT) was shown to allow direct examination of chondrocyte cell patterns and their subsequent correlation to osteoarthritis. This study aims to characterize chondrocyte cell patterns in the radial zone of the knee cartilage matrix in the presence and absence of osteoarthritic damage through both gray-level co-occurrence matrix (GLCM) derived texture features as well as Minkowski Functionals (MF). Thirteen GLCM and three MF texture features were extracted from 404 regions of interest (ROI) annotated on PCI images of healthy and osteoarthritic specimens of knee cartilage. These texture features were then used in a machine learning task to classify ROIs as healthy or osteoarthritic. A fuzzy k-nearest neighbor classifier was used and its performance was evaluated using the area under the ROC curve (AUC). The best classification performance was observed with the MF features 'perimeter' and 'Euler characteristic' and with GLCM correlation features (f3 and f13). With the experimental conditions used in this study, both Minkowski Functionals and GLCM achieved a high classification performance (AUC value of 0.97) in the task of distinguishing between health and osteoarthritic ROIs. These results show that such quantitative analysis of chondrocyte patterns in the knee cartilage matrix can distinguish between healthy and osteoarthritic tissue with high accuracy.

  19. Loading-induced changes in synovial fluid affect cartilage metabolism.

    PubMed

    Van den Hoogen, B M; van de Lest, C H; van Weeren, P R; Lafeber, F P; Lopes-Cardozo, M; van Golde, L M; Barneveld, A

    1998-06-01

    The purpose of this study was to determine whether changes in the synovial fluid (SF) induced by in vivo loading can induce an alteration in the metabolic activity of chondrocytes in vitro. Therefore, SF was collected from ponies after a period of box rest and after they had exercise for a week. Normal, unloaded articular cartilage explants were cultured in 20% solutions of these SFs for 4 days and chondrocyte activity was determined by glycosaminoglycan (GAG) turnover. In explants cultured in post-exercise SF, GAG synthesis was enhanced and GAG release was diminished when compared to cultures in pre-exercise SF. SF analysis showed that levels of insulin-like growth factors (IGF-I and IGF-II) tended to be higher in post-exercise SF, while no differences were found in metalloproteinase activity, hyaluronic acid and protein concentrations. This study showed that anabolic effects of joint loading on cartilage are, at least partially, mediated by alterations in the SF.

  20. Inhibition of phosphate-induced apoptosis in resting zone chondrocytes by thrombin peptide 508.

    PubMed

    Zhong, Ming; Carney, Darrell H; Ryaby, James T; Schwartz, Zvi; Boyan, Barbara D

    2009-01-01

    Growth plate chondrocytes are susceptible to apoptosis. Terminally differentiated chondrocytes are deleted via apoptosis, which primes the growth plate to vascular invasion and subsequent bone formation. Whether less differentiated resting zone chondrocytes are subject to the same mechanism that governs the apoptotic pathway of more differentiated growth zone chondrocytes is not known. In our current study, we demonstrated that inorganic phosphate, a key inducer of growth plate chondrocyte apoptosis, also causes apoptosis in resting zone chondrocytes, via a pathway similar to the one in growth zone chondrocytes. Our results demonstrated that the conditions that cause growth plate chondrocyte apoptosis lie in the external environment, instead of the differences in differentiation state.

  1. Biostable scaffolds of polyacrylate polymers implanted in the articular cartilage induce hyaline-like cartilage regeneration in rabbits.

    PubMed

    Sancho-Tello, María; Forriol, Francisco; Martín de Llano, José J; Antolinos-Turpin, Carmen; Gómez-Tejedor, José A; Gómez Ribelles, José L; Carda, Carmen

    2017-07-05

    To study the influence of scaffold properties on the organization of in vivo cartilage regeneration. Our hypothesis was that stress transmission to the cells seeded inside the pores of the scaffold or surrounding it, which is highly dependent on the scaffold properties, determines the differentiation of both mesenchymal cells and dedifferentiated autologous chondrocytes. 4 series of porous scaffolds made of different polyacrylate polymers, previously seeded with cultured rabbit chondrocytes or without cells, were implanted in cartilage defects in rabbits. Subchondral bone was injured during the surgery to allow blood to reach the implantation site and fill the scaffold pores. At 3 months after implantation, excellent tissue regeneration was obtained, with a well-organized layer of hyaline-like cartilage at the condylar surface in most cases of the hydrophobic or slightly hydrophilic series. The most hydrophilic material induced the poorest regeneration. However, no statistically significant difference was observed between preseeded and non-preseeded scaffolds. All of the materials used were biocompatible, biostable polymers, so, in contrast to some other studies, our results were not perturbed by possible effects attributable to material degradation products or to the loss of scaffold mechanical properties over time due to degradation. Cartilage regeneration depends mainly on the properties of the scaffold, such as stiffness and hydrophilicity, whereas little difference was observed between preseeded and non-preseeded scaffolds.

  2. Mechanical properties and structure-function relationships in articular cartilage repaired using IGF-I gene-enhanced chondrocytes.

    PubMed

    Griffin, Darvin J; Ortved, Kyla F; Nixon, Alan J; Bonassar, Lawrence J

    2016-01-01

    Several studies have demonstrated the benefits of IGF-I gene therapy in enhancing the histologic and biochemical content of cartilage repaired by chondrocyte transplantation. However, there is little to no data on the mechanical performance of IGF-I augmented cartilage grafts. This study evaluated the compressive properties of full-thickness chondral defects in the equine femur repaired with and without IGF-I gene therapy. Animals were randomly assigned to one of three study cohorts based on chondrocyte treatment provided in each defect: (i) IGF-I gene delivered by recombinant adeno-associated virus (rAAV)-5; (ii) AAV-5 delivering GFP as a reporter; (iii) naïve cells without virus. In each case, the opposite limb was implanted with a fibrin carrier without cells. Samples were prepared for confined compression testing to measure the aggregate modulus and hydraulic permeability. All treatment groups, regardless of cell content or transduction, had mechanical properties inferior to native cartilage. Overexpression of IGF-I increased modulus and lowered permeability relative to other treatments. Investigation of structure-property relationships revealed that Ha and k were linearly correlated with GAG content but logarithmically correlated with collagen content. This provides evidence that IGF-I gene therapy can improve healing of articular cartilage and can greatly increase the mechanical properties of repaired grafts.

  3. 3D Bioprinting Human Chondrocytes with Nanocellulose-Alginate Bioink for Cartilage Tissue Engineering Applications.

    PubMed

    Markstedt, Kajsa; Mantas, Athanasios; Tournier, Ivan; Martínez Ávila, Héctor; Hägg, Daniel; Gatenholm, Paul

    2015-05-11

    The introduction of 3D bioprinting is expected to revolutionize the field of tissue engineering and regenerative medicine. The 3D bioprinter is able to dispense materials while moving in X, Y, and Z directions, which enables the engineering of complex structures from the bottom up. In this study, a bioink that combines the outstanding shear thinning properties of nanofibrillated cellulose (NFC) with the fast cross-linking ability of alginate was formulated for the 3D bioprinting of living soft tissue with cells. Printability was evaluated with concern to printer parameters and shape fidelity. The shear thinning behavior of the tested bioinks enabled printing of both 2D gridlike structures as well as 3D constructs. Furthermore, anatomically shaped cartilage structures, such as a human ear and sheep meniscus, were 3D printed using MRI and CT images as blueprints. Human chondrocytes bioprinted in the noncytotoxic, nanocellulose-based bioink exhibited a cell viability of 73% and 86% after 1 and 7 days of 3D culture, respectively. On the basis of these results, we can conclude that the nanocellulose-based bioink is a suitable hydrogel for 3D bioprinting with living cells. This study demonstrates the potential use of nanocellulose for 3D bioprinting of living tissues and organs.

  4. Loading-induced changes in synovial fluid affect cartilage metabolism.

    PubMed

    van de Lest, C H; van den Hoogen, B M; van Weeren, P R

    2000-01-01

    The object of this study was to determine whether changes in the synovial fluid (SF) induced by in vivo loading can alter the metabolic activity of chondrocytes in vitro, and, if so, whether insulin-like growth factor-I (IGF-I) is responsible for this effect. Therefore, SF was collected from ponies after a period of box rest and after they had been exercised for a week. Normal, unloaded articular cartilage explants were cultured in 20% solutions of these SFs for 4 days and chondrocyte bioactivity was determined by glycosaminoglycan (GAG) turnover (i.e., the incorporation of 35SO4 into GAG and the release of GAG into the medium). Furthermore, the extent to which the bioactivity is IGF-I-dependent was determined in a cartilage explant culture in 20% SF, in the presence and absence of anti-IGF-I antibodies. In explants cultured in post-exercise SF, GAG synthesis was enhanced and GAG release was diminished when compared to cultures in pre-exercise SF. SF analysis showed that IGF-I and IGFBP-3 levels were increased in post-exercise SF. There was a positive correlation between IGF-I levels and proteoglycan synthesis, but no correlation between IGF-I levels and proteoglycan release. Addition of anti-IGF-I antibodies significantly inhibited stimulation of proteoglycan synthesis in explants cultured in SF with 40%. However, there was no difference in inhibition of proteoglycan synthesis between pre- and post-exercise SF which indicated that the relative contribution of IGF-I in the stimulating effect of SF did not change. Proteoglycan release was not influenced by the presence of anti-IGF-I antibodies. It is concluded that chondrocyte metabolic activity is at least partially regulated by changes in the SF induced by in vivo loading. Exercise altered the SF in a way that it had a favourable effect on cartilage PG content by enhancing the PG synthesis and reducing the PG breakdown. IGF-I is an important contributor to the overall stimulating effect of SF on cartilage

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

    PubMed

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

    2016-10-01

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

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

    PubMed Central

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

    2016-01-01

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

  7. Transplantation of autologous chondrocytes seeded on a fibrin/hyaluronan composite gel into tracheal cartilage defects in rabbits: preliminary results.

    PubMed

    Hong, Hyun Jun; Lee, Jin Seok; Choi, Jae Won; Min, Byoung-Hyun; Lee, Han-Bin; Kim, Chul-Ho

    2012-11-01

    Reconstruction of tracheal defects is one of the most difficult procedures in head and neck surgery. To date, various reconstructing techniques have been used with no consensus on the best approach. This study investigated the feasibility of using a fibrin/hyaluronic acid (HA) composite gel with autologous chondrocytes for tracheal reconstruction. Chondrocytes from autologous rabbit auricular cartilages were expanded and seeded into a culture dish at high density to form stable tracheal cartilages mechanically using a fibrin/HA composite gel. A 1-cm long by 0.5-cm wide defect was created by a scalpel on the cervical tracheae of six rabbits. Tissue-engineered cartilages using fibrin/HA composite were trimmed and fixed to the defect boundaries with tissuecol. Postoperatively, the site was evaluated endoscopically, histologically, radiologically, and functionally. None of the six rabbits showed signs of respiratory distress. Postoperatively, in all cases, rigid telescopic examination showed that the implanted scaffolds were completely covered with regenerated mucosa without granulation or stenosis. Histologically, the grafts showed no signs of inflammatory reaction and were covered with ciliated epithelium. Even when grafts were broken and migrated from their original insertion site, the implanted cartilages were well preserved. However, the grafts did show signs of mechanical failure at the implantation site. The beat frequency of ciliated epithelium on implants was very similar to that of normal respiratory mucosa. In conclusion, implants with autologous chondrocytes cultured with fibrin/HA showed good tracheal luminal contour, functional epithelial regeneration, and preservation of neocartilage without inflammation but lacked adequate mechanical stability. © 2012, Copyright the Authors. Artificial Organs © 2012, International Center for Artificial Organs and Transplantation and Wiley Periodicals, Inc.

  8. Low Shear Stress Attenuates COX-2 Expression Induced by Resistin in Human Osteoarthritic Chondrocytes.

    PubMed

    Su, Yu-Ping; Chen, Cheng-Nan; Chang, Hsin-I; Huang, Kuo-Chin; Cheng, Chin-Chang; Chiu, Fang-Yao; Lee, Ko-Chao; Lo, Chun-Min; Chang, Shun-Fu

    2017-06-01

    Low shear stress has been proposed to play a reparative role in modulating cartilage homeostasis. Recently, epidemiological studies have found a positive correlation between the resistin level in serum and synovial fluid and osteoarthritis (OA) severity in patients. However, the effect of moderate shear stress on the catabolic stimulation of resistin in OA chondrocytes remains unclear. Hence, this study was to investigate whether low shear stress could regulate resistin-induced catabolic cyclooxygenase (COX)-2 expression in human OA chondrocytes and the underlying mechanism. Human OA chondrocytes and SW1353 chondrosarcoma cells were used in this study. Two modes of low shear stress (2 dyn/cm(2) ), pre-shear and post-shear, were applied to the chondrocytes. A specific activator and siRNAs were used to investigate the mechanism of low shear stress-regulated COX-2 expression of resistin induction. We found that human OA chondrocytes exposed to different modes of low shear stress elicit an opposite effect on resistin-induced COX-2 expression: pre-shear for a short duration attenuates the resistin effect by inhibiting the transcription factor nuclear factor (NF)-κB-p65 subunit and the cAMP response element binding protein; however, post-shear over a longer duration enhances the resistin effect by activating only the NF-κB-p65 subunit. Moreover, our results demonstrated that the regulation of both shear modes in resistin-stimulated COX-2 expression occurs through increasing AMP-activated protein kinase activation and then sirtuin 1 expression. This study elucidates the detailed mechanism of low shear stress regulating the resistin-induced catabolic COX-2 expression and indicates a possible reparative role of moderate shear force in resistin-stimulated OA development. J. Cell. Physiol. 232: 1448-1457, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  9. Enhanced hyaline cartilage matrix synthesis in collagen sponge scaffolds by using siRNA to stabilize chondrocytes phenotype cultured with bone morphogenetic protein-2 under hypoxia.

    PubMed

    Legendre, Florence; Ollitrault, David; Hervieu, Magalie; Baugé, Catherine; Maneix, Laure; Goux, Didier; Chajra, Hanane; Mallein-Gerin, Frédéric; Boumediene, Karim; Galera, Philippe; Demoor, Magali

    2013-07-01

    Cartilage healing by tissue engineering is an alternative strategy to reconstitute functional tissue after trauma or age-related degeneration. However, chondrocytes, the major player in cartilage homeostasis, do not self-regenerate efficiently and lose their phenotype during osteoarthritis. This process is called dedifferentiation and also occurs during the first expansion step of autologous chondrocyte implantation (ACI). To ensure successful ACI therapy, chondrocytes must be differentiated and capable of synthesizing hyaline cartilage matrix molecules. We therefore developed a safe procedure for redifferentiating human chondrocytes by combining appropriate physicochemical factors: hypoxic conditions, collagen scaffolds, chondrogenic factors (bone morphogenetic protein-2 [BMP-2], and insulin-like growth factor I [IGF-I]) and RNA interference targeting the COL1A1 gene. Redifferentiation of dedifferentiated chondrocytes was evaluated using gene/protein analyses to identify the chondrocyte phenotypic profile. In our conditions, under BMP-2 treatment, redifferentiated and metabolically active chondrocytes synthesized a hyaline-like cartilage matrix characterized by type IIB collagen and aggrecan molecules without any sign of hypertrophy or osteogenesis. In contrast, IGF-I increased both specific and noncharacteristic markers (collagens I and X) of chondrocytes. The specific increase in COL2A1 gene expression observed in the BMP-2 treatment was shown to involve the specific enhancer region of COL2A1 that binds the trans-activators Sox9/L-Sox5/Sox6 and Sp1, which are associated with a decrease in the trans-inhibitors of COL2A1, c-Krox, and p65 subunit of NF-kappaB. Our procedure in which BMP-2 treatment under hypoxia is associated with a COL1A1 siRNA, significantly increased the differentiation index of chondrocytes, and should offer the opportunity to develop new ACI-based therapies in humans.

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

    PubMed

    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 < 0.05). The chondrocyte proliferation rate gradually decreased from two weeks to eight weeks in the immobilization group. The GAG layer was thinner in the immobilization group than in the sham group at two, four and eight weeks after surgery (p < 0.05). In the articular cartilage, the chondrocyte apoptosis rate in the immobilization group was higher than in the sham group at four and eight weeks after surgery (p < 0.05). The GAG layer was significantly thinner in the immobilization group than that in the sham group at four and eight weeks after surgery (p < 0.05). Knee immobilization significantly increased chondrocyte apoptosis at two and eight weeks after surgery in the ACL insertion and at four and eight weeks after surgery in the articular cartilage of the medial tibial condyle, and decreased GAG layer thickness from two to eight weeks after surgery in the ACL insertion and from four to eight weeks after surgery in the articular cartilage.

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

    PubMed Central

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

    2017-01-01

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

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

    PubMed Central

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

    2016-01-01

    The lipid mediator lysophosphatidic acid (LPA) signals via six distinct G protein-coupled receptors to mediate both unique and overlapping biological effects, including cell migration, proliferation and survival. LPA is produced extracellularly by autotaxin (ATX), a secreted lysophospholipase D, from lysophosphatidylcholine. ATX-LPA receptor signaling is essential for normal development and implicated in various (patho)physiological processes, but underlying mechanisms remain incompletely understood. Through gene targeting approaches in zebrafish and mice, we show here that loss of ATX-LPA1 signaling leads to disorganization of chondrocytes, causing severe defects in cartilage formation. Mechanistically, ATX-LPA1 signaling acts by promoting S-phase entry and cell proliferation of chondrocytes both in vitro and in vivo, at least in part through β1-integrin translocation leading to fibronectin assembly and further extracellular matrix deposition; this in turn promotes chondrocyte-matrix adhesion and cell proliferation. Thus, the ATX-LPA1 axis is a key regulator of cartilage formation. PMID:27005960

  13. Differential Response of Chondrocytes and Chondrogenic-Induced Mesenchymal Stem Cells to C1-OH Tributanoylated N-Acetylhexosamines

    PubMed Central

    Coburn, Jeannine M.; Bernstein, Nicholas; Bhattacharya, Rahul; Aich, Udayanath; Yarema, Kevin J.; Elisseeff, Jennifer H.

    2013-01-01

    Articular cartilage has a limited ability to self-repair because of its avascular nature and the low mitotic activity of the residing chondrocytes. There remains a significant need to develop therapeutic strategies to increase the regenerative capacity of cells that could repair cartilage. Multiple cell types, including chondrocytes and mesenchymal stem cells, have roles in articular cartilage regeneration. In this study, we evaluated a platform technology of multiple functionalized hexosamines, namely 3,4,6-O-tributanoylated-N-acetylgalactosamine (3,4,6-O-Bu3GalNAc), 3,4,6-O-tributanoylated-N-acetylmannosamine (3,4,6-O-Bu3ManNAc) and 3,4,6-O-Bu3GlcNAc, with the potential ability to reduce NFκB activity. Exposure of IL-1β-stimulated chondrocytes to the hexosamine analogs resulted in increased expression of ECM molecules and a corresponding improvement in cartilage-specific ECM accumulation. The greatest ECM accumulation was observed with 3,4,6-O-Bu3GalNAc. In contrast, mesenchymal stem cells (MSCs) exposed to 3,4,6-O-Bu3GalNAc exhibited a dose dependent decrease in chondrogenic differentation as indicated by decreased ECM accumulation. These studies established the disease modification potential of a hexosamine analog platform on IL-1β-stimulated chondrocytes. We determined that the modified hexosamine with the greatest potential for disease modification is 3,4,6-O-Bu3GalNAc. This effect was distinctly different with 3,4,6-O-Bu3GalNAc exposure to chondrogenic-induced MSCs, where a decrease in ECM accumulation and differentiation was observed. Furthermore, these studies suggest that NFκB pathway plays a complex role cartilage repair. PMID:23516573

  14. α5β1 integrin induces the expression of noncartilaginous procollagen gene expression in articular chondrocytes cultured in monolayers

    PubMed Central

    2013-01-01

    Introduction Articular chondrocytes undergo an obvious phenotypic change when cultured in monolayers. During this change, or dedifferentiation, the expression of type I and type III procollagen is induced where normal chondrocytes express little type I and type III procollagen. In this study, we attempted to determine the mechanism(s) for the induction of such procollagen expression in dedifferentiating chondrocytes. Methods All experiments were performed using primary-cultured human articular chondrocytes under approval of institutional review boards. Integrin(s) responsible for the induction of type I and type III procollagen expression were specified by RNAi experiments. The signal pathway(s) involved in the induction were determined by specific inhibitors and RNAi experiments. Adenovirus-mediated experiments were performed to identify a small GTPase regulating the activity of integrins in dedifferentiating chondrocytes. The effect of inhibition of integrins on dedifferentiation was investigated by experiments using echistatin, a potent disintegrin. The effect of echistatin was investigated first with monolayer-cultured chondrocytes, and then with pellet-cultured chondrocytes. Results In dedifferentiating chondrocytes, α5β1 integrin was found to be involved in the induction of type I and type III procollagen expression. The induction was known to be mediated by v-akt murine thymoma viral oncogene homolog (AKT) signaling. Among the three AKT isoforms, AKT1 seemed to be most involved in the signaling. Elated RAS viral (r-ras) oncogene homolog (RRAS) was considered to regulate the progression of dedifferentiation by modulating the affinity and avidity of α5β1 integrin to ligands. Echistatin inhibited dedifferentiation of monolayer-cultured chondrocytes. Furthermore, the matrix formed by pellet-cultured chondrocytes more closely resembled that of normal cartilage compared with the controls. Conclusions The result of this study has shown, for the first time, that

  15. Mechanobiological implications of articular cartilage crystals.

    PubMed

    Carlson, Alyssa K; McCutchen, Carley N; June, Ronald K

    2017-03-01

    Calcium crystals exist in both pathological and normal articular cartilage. The prevalence of these crystals dramatically increases with age, and crystals are typically found in osteoarthritic cartilage and synovial fluid. Relatively few studies have examined the effects of crystals on cartilage biomechanics or chondrocyte mechanotransduction. The purpose of this review is to describe how crystals could influence cartilage biomechanics and mechanotransduction in osteoarthritis. Crystals are found in both loaded and unloaded regions of articular cartilage. Exogenous crystals, in combination with joint motion, result in substantial joint inflammation. Articular cartilage vesicles promote crystal formation, and these vesicles are found near the periphery of chondrocytes. Crystallographic studies report monoclinic symmetry for synthetic crystals, suggesting that crystals will have a large stiffness compared with the cartilage extracellular matrix, the pericellular matrix, or the chondrocyte. This stiffness imbalance may cause crystal-induced dysregulation of chondrocyte mechanotransduction promoting both aging and osteoarthritis chondrocyte phenotypes. Because of their high stiffness compared with cartilage matrix, crystals likely alter chondrocyte mechanotransduction, and high concentrations of crystals within cartilage may alter macroscale biomechanics. Future studies should focus on understanding the mechanical properties of joint crystals and developing methods to understand how crystals affect chondrocyte mechanotransduction.

  16. TWIST1 induces MMP3 expression through up-regulating DNA hydroxymethylation and promotes catabolic responses in human chondrocytes

    PubMed Central

    Hasei, Joe; Teramura, Takeshi; Takehara, Toshiyuki; Onodera, Yuta; Horii, Takuro; Olmer, Merissa; Hatada, Izuho; Fukuda, Kanji; Ozaki, Toshifumi; Lotz, Martin K.; Asahara, Hiroshi

    2017-01-01

    The objective was to investigate the levels of TWIST1 in normal and OA cartilage and examine its role in regulating gene expression in chondrocytes. Human cartilage tissues and chondrocytes were obtained at autopsy from normal knee joints and from OA-affected joints at the time of total knee arthroplasty. TWIST1 expression was increased in human OA knee cartilage compared to normal knee cartilage. TWIST1 induced matrix metalloproteinase 3 (MMP3) expression without direct binding to MMP3 promoter and increased the 5-hydroxymethylcytosine (5hmC) level at the MMP3 promoter. The effect of TWIST1 on expression of TET family (TET1, 2 and 3) was measured in stable TWIST1 transfected TC28 cells, and TET1 expression was up-regulated. TWIST1 dependent upregulation of Mmp3 expression was suppressed in Tet triple KO fibroblast derived from mouse ES cells. Increased TWIST1 expression is a feature of OA-affected cartilage. We identified a novel mechanism of catabolic reaction where TWIST1 up-regulates MMP3 expression by enriching 5hmC levels at the MMP3 promoter via TET1 induction. These findings implicate TWIST1 as an important factor regulating OA related gene expression. Clarifying epigenetic mechanisms of 5hmC induced by TWIST1 is a critical molecule to understanding OA pathogenesis. PMID:28220902

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

    PubMed

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

    2015-02-01

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

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

    PubMed Central

    Limagne, Emeric; Lançon, Allan; Delmas, Dominique; Cherkaoui-Malki, Mustapha; Latruffe, Norbert

    2016-01-01

    State of the art. Osteoarthritis (OA) is a chronic articular disease characterized by cartilage degradation and osteophyte formation. OA physiopathology is multifactorial and involves mechanical and hereditary factors. So far, there is neither preventive medicine to delay cartilage breakdown nor curative treatment. Objectives. To investigate pro-inflammatory paracrine interactions between human primary chondrocytes and macrophages following interleukin-1-β (IL-1β) treatment; to evaluate the molecular mechanism responsible for the inhibitory effect of resveratrol. Results. The activation of NF-κB in chondrocytes by IL-1β induced IL-6 secretion. The latter will then activate STAT3 protein in macrophages. Moreover, STAT3 was able to positively regulate IL-6 secretion, as confirmed by the doubling level of IL-6 in the coculture compared to macrophage monoculture. These experiments confirm the usefulness of the coculture model in the inflammatory arthritis-linked process as a closer biological situation to the synovial joint than separated chondrocytes and macrophages. Il also demonstrated the presence of an inflammatory amplification loop induced by IL-1β. Resveratrol showed a strong inhibitory effect on the pro-inflammatory marker secretion. The decrease of IL-6 secretion is dependent on the NFκB inhibition in the chondrocytes. Such reduction of the IL-6 level can limit STAT3 activation in the macrophages, leading to the interruption of the inflammatory amplification loop. Conclusion. These results increase our understanding of the anti-inflammatory actions of resveratrol and open new potential approaches to prevent and treat osteoarthritis. PMID:27187448

  19. Laser-induced regeneration of cartilage

    NASA Astrophysics Data System (ADS)

    Sobol, Emil; Shekhter, Anatoly; Guller, Anna; Baum, Olga; Baskov, Andrey

    2011-08-01

    Laser radiation provides a means to control the fields of temperature and thermo mechanical stress, mass transfer, and modification of fine structure of the cartilage matrix. The aim of this outlook paper is to review physical and biological aspects of laser-induced regeneration of cartilage and to discuss the possibilities and prospects of its clinical applications. The problems and the pathways of tissue regeneration, the types and features of cartilage will be introduced first. Then we will review various actual and prospective approaches for cartilage repair; consider possible mechanisms of laser-induced regeneration. Finally, we present the results in laser regeneration of joints and spine disks cartilages and discuss some future applications of lasers in regenerative medicine.

  20. In vitro development of personalized cartilage microtissues uncovers an individualized differentiation capacity of human chondrocytes.

    PubMed

    Martin, Frank; Lehmann, Mario; Sack, Ulrich; Anderer, Ursula

    2017-01-01

    Personalized features in the treatment of knee injuries and articular replacement therapies play an important role in modern life with increasing demand. Therefore, cell-based therapeutic approaches for the regeneration of traumatic defects of cartilage tissue were developed. However, great variations in the quality of repair tissue or therapeutic outcome were observed. The aim of the study was to capture and visualize individual differentiation capacities of chondrocytes derived from different donors with regard to a possible personal regeneration capacity using a cell-based therapy. The redifferentiation potential of monolayer cultured cells was analyzed in a scaffold-free three-dimensional tissue model. Furthermore, stimulating options using cartilage maturation factors such as L-ascorbic acid and transforming growth factor beta 2 (TGF-β2) on this process were of special interest. Cells and tissues were analyzed via histological and immunohistochemical methods. Gene expression was measured by quantitative real-time polymerase chain reaction. In monolayer culture, cells from all donors showed an almost identical differentiation profile. In contrast, the differentiation state of cartilage-like three-dimensional microtissues revealed clear differences with respect to individual donors. Analyses at the protein and mRNA levels showed high variations regarding cartilage-typical matrix components (e.g. proteoglycans, collagen type II) and intracellular proteins (e.g. S100). Interestingly, only donor chondrocytes with a basic tendency to re-differentiate in a three-dimensional environment were able to increase this tissue-specific maturation when exposed to L-ascorbic acid and/or TGF-β2. Our approach revealed clear-cut possibilities for classification of individual donors into responders or non-responders. On the basis of these results an in vitro platform could be designed to discriminate responders from non-responders. This in vitro three-dimensional test system

  1. [The dose dependent effect of glycosaminoglycan peptide complex on corticosteroid-induced disordered metabolism in cartilage tissue of rats].

    PubMed

    Annefeld, M

    1989-01-01

    Systemic corticosteroid treatment induces morphological and functional changes in the articular cartilage similar to those in human osteoarthritis. In animal experiments the dexamethasone-induced inhibition of chondrocyte metabolism can be reduced in a dose-related manner by concomitant treatment with glycosaminoglycan-peptide complexes (GP-C)***). The metabolic changes in cartilage tissues of the joint and Processus Xiphoideus measured quantitatively by 35S-sulphate incorporation are comparable. The results indicate that GP-C could also have a dose-related effect on human osteoarthritic cartilage.

  2. Immunohistochemical study of collagen types I and II and procollagen IIA in human cartilage repair tissue following autologous chondrocyte implantation.

    PubMed

    Roberts, S; Menage, J; Sandell, L J; Evans, E H; Richardson, J B

    2009-10-01

    This study has assessed the relative proportions of type I and II collagens and IIA procollagen in full depth biopsies of repair tissue in a large sample of patients treated with autologous chondrocyte implantation (ACI). Sixty five full depth biopsies were obtained from knees of 58 patients 8-60 months after treatment by ACI alone (n=55) or in combination with mosaicplasty (n=10). In addition articular cartilage was examined from eight individuals (aged 10-50) as controls. Morphology and semi-quantitative immunohistochemistry for collagen types I and II and procollagen IIA in the repair tissue were studied. Repair cartilage thickness was 2.89+/-1.5 mm and there was good basal integration between the repair cartilage, calcified cartilage and subchondral bone. Sixty five percent of the biopsies were predominantly fibrocartilage (mostly type I collagen and IIA procollagen), 15% were hyaline cartilage (mostly type II collagen), 17% were of mixed morphology and 3% were fibrous tissue (mostly type I collagen). Type II collagen and IIA procollagen were usually found in the lower regions near the bone and most type II collagen was present 30-60 months after treatment. The presence of type IIA procollagen in the repair tissue supports our hypothesis that this is indicative of a developing cartilage, with the ratio of type II collagen:procollagen IIA increasing from <2% in the first two years post-treatment to 30% three to five years after treatment. This suggests that cartilage repair tissue produced following ACI treatment, is likely to take some years to mature.

  3. Cultured chondrocyte and porcine cartilage-derived substance (PCS) construct as a possible dorsal augmentation material in rhinoplasty: A preliminary animal study.

    PubMed

    Kim, Yoo Suk; Park, Do-Yang; Cho, Yong Hyun; Chang, Jae Won; Choi, Jae Won; Park, Joo Kyung; Min, Byung Hyun; Shin, Yoo Seob; Kim, Chul Ho

    2015-05-01

    As there is no single ideal material for dorsal augmentation in rhinoplasty, there has been a continuing need for the development of improved materials. Therefore, we aimed to evaluate the outcome of using a novel tissue-engineered construct composed of autologous chondrocytes cultured with a porcine cartilage-derived substance (PCS) scaffold as an augmentation material in rhinoplasty. A scaffold derived from decellularized and powdered porcine articular cartilage was prepared. The rabbit articular cartilage was used as the source of homologous chondrocytes, which were expanded and cultured with the PCS scaffold for 7 weeks. The chondrocyte-PCS constructs were then surgically implanted on the nasal dorsum of six rabbits. Four and eight weeks after implantation, the gross morphology, radiologic images, and histologic features of the site of implant were analyzed. The rabbits showed no signs of postoperative inflammation and infection. The degree of dorsal augmentation was maintained during the 8-week postoperative observation period. Postoperative histologic examinations showed chondrocyte proliferation without an inflammatory response. However, neo-cartilage formation from the constructs was not confirmed. The biocompatibility and structural features of tissue-engineered chondrocyte-PCS constructs indicate their potential as candidate dorsal augmentation material for use in rhinoplasty.

  4. Licofelone (ML-3000), a dual inhibitor of 5-lipoxygenase and cyclooxygenase, reduces the level of cartilage chondrocyte death in vivo in experimental dog osteoarthritis: inhibition of pro-apoptotic factors.

    PubMed

    Boileau, Christelle; Martel-Pelletier, Johanne; Jouzeau, Jean-Yves; Netter, Patrick; Moldovan, Florina; Laufer, Stefan; Tries, Susanne; Pelletier, Jean-Pierre

    2002-07-01

    To evaluate in vivo therapeutic efficacy of licofelone, a novel competitive dual inhibitor of 5-lipoxygenase (5-LOX) and cyclooxygenase (COX) in chondrocyte death in the canine ligament transection model of osteoarthritis (OA), and to explore its effect on factors involved in the apoptotic phenomenon, i.e., caspase-3, COX-2, and inducible nitric oxide synthase (iNOS). Cartilage specimens were obtained from 3 experimental groups of dogs: Group 1, dogs subjected to sectioning of the anterior cruciate ligament of the right knee and given placebo treatment; Groups 2 and 3, operated dogs that received oral treatment with licofelone (2.5 or 5.0 mg/kg/day, respectively) for 8 weeks starting immediately after surgery. All dogs were killed 8 weeks postsurgery. The cartilage level of chondrocyte death was detected by TUNEL reaction. Cartilage distribution of caspase-3, COX-2, and iNOS was documented by immunohistochemistry using specific antibodies, and other levels were quantified by morphometric analysis. In cartilage specimens from placebo treated dogs a large number of chondrocytes in the superficial layers stained positive for TUNEL reaction. Treatment with therapeutic concentrations of licofelone (2.5 and 5.0 mg/kg/day) markedly reduced the level of chondrocyte apoptosis to the same extent in both therapeutic groups (p < 0.0001, p < 0.002, respectively). In these groups, the levels of caspase-3, COX-2, and iNOS in cartilage from both condyles and plateaus were also significantly decreased (p < 0.0001, p < 0.0001, p < 0.0002, respectively) compared to the control (placebo) group. Licofelone is an effective treatment in vivo, capable of reducing the level of OA chondrocyte death. This effect is likely mediated by a decrease in the level of caspase-3 activity, which may be related to the reduced production of 2 major factors involved in chondrocyte apoptosis, NO and prostaglandin E2. These findings may explain some of the mechanisms by which licofelone reduces the

  5. Circular RNA Related to the Chondrocyte ECM Regulates MMP13 Expression by Functioning as a MiR-136 ‘Sponge’ in Human Cartilage Degradation

    PubMed Central

    Liu, Qiang; Zhang, Xin; Hu, Xiaoqing; Dai, Linghui; Fu, Xin; Zhang, Jiying; Ao, Yingfang

    2016-01-01

    Circular RNAs (circRNAs) are involved in the development of various diseases, but there is little knowledge of circRNAs in osteoarthritis (OA). The aim of study was to identify circRNA expression in articular cartilage and to explore the function of chondrocyte extracellular matrix (ECM)-related circRNAs (circRNA-CER) in cartilage. To identify circRNAs that are specifically expressed in cartilage, we compared the expression of circRNAs in OA cartilage with that in normal cartilage. Bioinformatics was employed to predict the interaction of circRNAs and mRNAs in cartilage. Loss-of-function and rescue experiments for circRNA-CER were performed in vitro. A total of 71 circRNAs were differentially expressed in OA and normal cartilage. CircRNA-CER expression increased with interleukin-1 and tumor necrosis factor levels in chondrocytes. Silencing of circRNA-CER using small interfering RNA suppressed MMP13 expression and increased ECM formation. CircRNA-CER could compete for miR-136 with MMP13. Our results demonstrated that circRNA-CER regulated MMP13 expression by functioning as a competing endogenous RNA (ceRNA) and participated in the process of chondrocyte ECM degradation. We propose that circRNA-CER could be used as a potential target in OA therapy. PMID:26931159

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

    PubMed Central

    2017-01-01

    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. PMID:28133485

  7. Loss of Vhl in cartilage accelerated the progression of age-associated and surgically induced murine osteoarthritis

    PubMed Central

    Weng, Tujun; Xie, Yangli; Yi, Lingxian; Huang, Junlan; Luo, Fengtao; Du, Xiaolan; Chen, Liang; Liu, Chongyang; Chen, Di; Chen, Lin

    2014-01-01

    Objective To investigate the role of Vhl in maintaining the integrity of articular cartilage and in the development of experimental osteoarthritis (OA). Method Histology of articular cartilage and subchondral bone in both Vhl cKO and WT mice were analyzed by histopathology and micro-CT. Articular cartilage destruction and proteoglycan loss were scored in aged (12-month-old) mice as well as in mice with surgically induced OA. Apoptosis of cartilage in age-related and surgically induced OA was detected with TUNNEL assay. Expressions of VHL, Fas, LC-3, HIF-1α, HIF-2α, p-mTOR and MMP-13 in the knee joints were analyzed by immunostaining. Results No gross differences in cartilage were observed between Vhl cKO and WT mice at age 4 months. However, Vhl cKO mice displayed accelerated age-associated spontaneous OA and surgically induced OA. Cartilage destruction and proteoglycan loss were observed in the absence of Vhl. In addition, inactivation of Vhl resulted in up-regulation of HIF-2α and increased chondrocyte apoptosis and decreased expression of autophagy during OA development. Immunohistochemical staining also showed that Vhl deficiency led to increased expression of Fas, p-mTOR and MMP-13, and those genes were associated with cell apoptosis, autophagy and cartilage matrix breakdown, respectively. Conclusion Loss of Vhl in adult articular cartilage is associated with earlier dysregulation of cartilage homeostasis, characterized by an increased chondrocyte apoptosis, compromised chondrocyte autophagy, and an accelerated age-related and surgery-induced OA development. These results highlight the novel role of Vhl in maintaining joint homeostasis and OA development. PMID:24999110

  8. RNA Interference and BMP-2 Stimulation Allows Equine Chondrocytes Redifferentiation in 3D-Hypoxia Cell Culture Model: Application for Matrix-Induced Autologous Chondrocyte Implantation.

    PubMed

    Rakic, Rodolphe; Bourdon, Bastien; Hervieu, Magalie; Branly, Thomas; Legendre, Florence; Saulnier, Nathalie; Audigié, Fabrice; Maddens, Stéphane; Demoor, Magali; Galera, Philippe

    2017-08-24

    As in humans, osteoarthritis (OA) causes considerable economic loss to the equine industry. New hopes for cartilage repair have emerged with the matrix-associated autologous chondrocyte implantation (MACI). Nevertheless, its limitation is due to the dedifferentiation occurring during the chondrocyte amplification phase, leading to the loss of its capacity to produce a hyaline extracellular matrix (ECM). To enhance the MACI therapy efficiency, we have developed a strategy for chondrocyte redifferentiation, and demonstrated its feasibility in the equine model. Thus, to mimic the cartilage microenvironment, the equine dedifferentiated chondrocytes were cultured in type I/III collagen sponges for 7 days under hypoxia in the presence of BMP-2. In addition, chondrocytes were transfected by siRNA targeting Col1a1 and Htra1 mRNAs, which are overexpressed during dedifferentiation and OA. To investigate the quality of the neo-synthesized ECM, specific and atypical cartilage markers were evaluated by RT-qPCR and Western blot. Our results show that the combination of 3D hypoxia cell culture, BMP-2 (Bone morphogenetic protein-2), and RNA interference, increases the chondrocytes functional indexes (Col2a1/Col1a1, Acan/Col1a1), leading to an effective chondrocyte redifferentiation. These data represent a proof of concept for this process of application, in vitro, in the equine model, and will lead to the improvement of the MACI efficiency for cartilage tissue engineering therapy in preclinical/clinical trials, both in equine and human medicine.

  9. Is autologous chondrocyte implantation (ACI) an adequate treatment option for repair of cartilage defects in paediatric patients?

    PubMed

    Kaszkin-Bettag, Marietta

    2013-08-01

    Cartilage lesions in the knee of juvenile patients require an effective repair to regain life-long functional activity of the joint. Autologous chondrocyte implantation (ACI) is discussed to be advantageous over other methods for cartilage repair regarding long-term outcome. ACI has successfully been applied in juvenile patients, although currently recommended for patients ≥18 years of age. Only few controlled clinical trials present evidence of efficacy and safety of ACI in adolescent patients. ACI products have to undergo the process of a marketing authorisation application, including the submission of a paediatric investigation plan (PIP). Data from prospective clinical studies or retrospective collection of long-term data in paediatric patients should be submitted for risk-benefit evaluation by the Paediatric Committee (PDCO).

  10. Measuring microscale strain fields in articular cartilage during rapid impact reveals thresholds for chondrocyte death and a protective role for the superficial layer

    PubMed Central

    Bartell, Lena R.; Fortier, Lisa A.; Bonassar, Lawrence J.; Cohen, Itai

    2015-01-01

    Articular cartilage is a heterogeneous soft tissue that dissipates and distributes loads in mammalian joints. Though robust, cartilage is susceptible to damage from loading at high rates or magnitudes. Such injurious loads have been implicated in degenerative changes, including chronic osteoarthritis (OA), which remains a leading cause of disability in developed nations. Despite decades of research, mechanisms of OA initiation after trauma remain poorly understood. Indeed, although bulk cartilage mechanics are measurable during impact, current techniques cannot access microscale mechanics at those rapid time scales. We aimed to address this knowledge gap by imaging the microscale mechanics and corresponding acute biological changes of cartilage in response to rapid loading. In this study, we utilized fast-camera and confocal microscopy to achieve roughly 85 μm spatial resolution of the cartilage deformation during a rapid (~3 ms), localized impact and the chondrocyte death following impact. Our results showed that, at these high rates, strain and chondrocyte death were highly correlated (p<0.001) with a threshold of 8% microscale strain norm before any cell death occurred. Additionally, chondrocyte death had developed by two hours after impact, suggesting a time frame for clinical therapeutics. Moreover, when the superficial layer was removed, strain – and subsequently chondrocyte death – penetrated deeper into the samples (p<0.001), suggesting a protective role for the superficial layer of articular cartilage. Combined, these results provide insight regarding the detailed biomechanics that drive early chondrocyte damage after trauma and emphasize the importance of understanding cartilage and its mechanics on the microscale. PMID:26150096

  11. BMP-2, Hypoxia, and COL1A1/HtrA1 siRNAs Favor Neo-Cartilage Hyaline Matrix Formation in Chondrocytes

    PubMed Central

    Ollitrault, David; Legendre, Florence; Drougard, Carole; Briand, Mélanie; Benateau, Hervé; Goux, Didier; Chajra, Hanane; Poulain, Laurent; Hartmann, Daniel; Vivien, Denis; Shridhar, Vijayalakshmi; Baldi, Alfonso; Mallein-Gerin, Frédéric; Boumediene, Karim; Demoor, Magali

    2015-01-01

    Osteoarthritis (OA) is an irreversible pathology that causes a decrease in articular cartilage thickness, leading finally to the complete degradation of the affected joint. The low spontaneous repair capacity of cartilage prevents any restoration of the joint surface, making OA a major public health issue. Here, we developed an innovative combination of treatment conditions to improve the human chondrocyte phenotype before autologous chondrocyte implantation. First, we seeded human dedifferentiated chondrocytes into a collagen sponge as a scaffold, cultured them in hypoxia in the presence of a bone morphogenetic protein (BMP), BMP-2, and transfected them with small interfering RNAs targeting two markers overexpressed in OA dedifferentiated chondrocytes, that is, type I collagen and/or HtrA1 serine protease. This strategy significantly decreased mRNA and protein expression of type I collagen and HtrA1, and led to an improvement in the chondrocyte phenotype index of differentiation. The effectiveness of our in vitro culture process was also demonstrated in the nude mouse model in vivo after subcutaneous implantation. We, thus, provide here a new protocol able to favor human hyaline chondrocyte phenotype in primarily dedifferentiated cells, both in vitro and in vivo. Our study also offers an innovative strategy for chondrocyte redifferentiation and opens new opportunities for developing therapeutic targets. PMID:24957638

  12. BMP-2, hypoxia, and COL1A1/HtrA1 siRNAs favor neo-cartilage hyaline matrix formation in chondrocytes.

    PubMed

    Ollitrault, David; Legendre, Florence; Drougard, Carole; Briand, Mélanie; Benateau, Hervé; Goux, Didier; Chajra, Hanane; Poulain, Laurent; Hartmann, Daniel; Vivien, Denis; Shridhar, Vijayalakshmi; Baldi, Alfonso; Mallein-Gerin, Frédéric; Boumediene, Karim; Demoor, Magali; Galera, Philippe

    2015-02-01

    Osteoarthritis (OA) is an irreversible pathology that causes a decrease in articular cartilage thickness, leading finally to the complete degradation of the affected joint. The low spontaneous repair capacity of cartilage prevents any restoration of the joint surface, making OA a major public health issue. Here, we developed an innovative combination of treatment conditions to improve the human chondrocyte phenotype before autologous chondrocyte implantation. First, we seeded human dedifferentiated chondrocytes into a collagen sponge as a scaffold, cultured them in hypoxia in the presence of a bone morphogenetic protein (BMP), BMP-2, and transfected them with small interfering RNAs targeting two markers overexpressed in OA dedifferentiated chondrocytes, that is, type I collagen and/or HtrA1 serine protease. This strategy significantly decreased mRNA and protein expression of type I collagen and HtrA1, and led to an improvement in the chondrocyte phenotype index of differentiation. The effectiveness of our in vitro culture process was also demonstrated in the nude mouse model in vivo after subcutaneous implantation. We, thus, provide here a new protocol able to favor human hyaline chondrocyte phenotype in primarily dedifferentiated cells, both in vitro and in vivo. Our study also offers an innovative strategy for chondrocyte redifferentiation and opens new opportunities for developing therapeutic targets.

  13. Hyperosmotic stress-induced apoptotic signaling pathways in chondrocytes.

    PubMed

    Racz, Boglarka; Reglodi, Dora; Fodor, Barnabas; Gasz, Balazs; Lubics, Andrea; Gallyas, Ferenc; Roth, Erzsebet; Borsiczky, Balazs

    2007-06-01

    Articular chondrocytes have a well-developed osmoregulatory system that enables cells to survive in a constantly changing osmotic environment. However, osmotic loading exceeding that occurring under physiological conditions severely compromises chondrocyte function and leads to degenerative changes. The aim of the present study was to investigate the form of cell death and changes in apoptotic signaling pathways under hyperosmotic stress using a primary chondrocyte culture. Cell viability and apoptosis assays performed with annexin V and propidium iodide staining showed that a highly hyperosmotic medium (600 mOsm) severely reduced chondrocyte viability and led mainly to apoptotic cell death, while elevating osmotic pressure within the physiological range caused no changes compared to isosmotic conditions. Western blot analysis revealed that a 600 mOsm hyperosmotic environment induced the activation of proapoptotic members of the mitogen-activated protein kinase family such as c-Jun N-terminal kinase (JNK) and p38, and led to an increased level of extracellular signal regulated kinase (ERK1/2). Hyperosmotic stress also induced the activation of caspase-3. In summary, our results show that hyperosmotic stress leads to mainly apoptotic cell death via the involvement of proapoptotic signaling pathways in a primary chondrocyte culture.

  14. Glycophenotyping of osteoarthritic cartilage and chondrocytes by RT-qPCR, mass spectrometry, histochemistry with plant/human lectins and lectin localization with a glycoprotein

    PubMed Central

    2013-01-01

    Introduction This study aimed to characterize the glycophenotype of osteoarthritic cartilage and human chondrocytes. Methods Articular knee cartilage was obtained from nine osteoarthritis (OA) patients. mRNA levels for 27 glycosyltransferases were analyzed in OA chondrocytes using RT-qPCR. Additionally, N- and O-glycans were quantified using mass-spectrometry. Histologically, two cartilage areas with Mankin scores (MS) either ≤4 or ≥9 were selected from each patient representing areas of mild and severe OA, respectively. Tissue sections were stained with (1) a selected panel of plant lectins for probing into the OA glycophenotype, (2) the human lectins galectins-1 and -3, and (3) the glycoprotein asialofetuin (ASF) for visualizing β-galactoside-specific endogenous lectins. Results We found that OA chondrocytes expressed oligomannosidic structures as well as non-, mono- and disialylated complex-type N-glycans, and core 2 O-glycans. Reflecting B4GALNT3 mRNA presence in OA chondrocytes, LacdiNAc-terminated structures were detected. Staining profiles for plant and human lectins were dependent on the grade of cartilage degeneration, and ASF-positive cells were observed in significantly higher rates in areas of severe degeneration. Conclusions In summary, distinct aspects of the glycome in OA cartilage are altered with progressing degeneration. In particular, the alterations measured by galectin-3 and the pan-galectin sensor ASF encourage detailed studies of galectin functionality in OA. PMID:24289744

  15. Glycophenotyping of osteoarthritic cartilage and chondrocytes by RT-qPCR, mass spectrometry, histochemistry with plant/human lectins and lectin localization with a glycoprotein.

    PubMed

    Toegel, Stefan; Bieder, Daniela; André, Sabine; Altmann, Friedrich; Walzer, Sonja M; Kaltner, Herbert; Hofstaetter, Jochen G; Windhager, Reinhard; Gabius, Hans-Joachim

    2013-10-04

    This study aimed to characterize the glycophenotype of osteoarthritic cartilage and human chondrocytes. Articular knee cartilage was obtained from nine osteoarthritis (OA) patients. mRNA levels for 27 glycosyltransferases were analyzed in OA chondrocytes using RT-qPCR. Additionally, N- and O-glycans were quantified using mass-spectrometry. Histologically, two cartilage areas with Mankin scores (MS) either ≤ 4 or ≥ 9 were selected from each patient representing areas of mild and severe OA, respectively. Tissue sections were stained with (1) a selected panel of plant lectins for probing into the OA glycophenotype, (2) the human lectins galectins-1 and -3, and (3) the glycoprotein asialofetuin (ASF) for visualizing β-galactoside-specific endogenous lectins. We found that OA chondrocytes expressed oligomannosidic structures as well as non-, mono- and disialylated complex-type N-glycans, and core 2 O-glycans. Reflecting B4GALNT3 mRNA presence in OA chondrocytes, LacdiNAc-terminated structures were detected. Staining profiles for plant and human lectins were dependent on the grade of cartilage degeneration, and ASF-positive cells were observed in significantly higher rates in areas of severe degeneration. In summary, distinct aspects of the glycome in OA cartilage are altered with progressing degeneration. In particular, the alterations measured by galectin-3 and the pan-galectin sensor ASF encourage detailed studies of galectin functionality in OA.

  16. Repair of articular cartilage defect by autologous transplantation of basic fibroblast growth factor gene-transduced chondrocytes with adeno-associated virus vector.

    PubMed

    Yokoo, Naoki; Saito, Tomoyuki; Uesugi, Masaaki; Kobayashi, Naomi; Xin, Ke-Qin; Okuda, Kenji; Mizukami, Hiroaki; Ozawa, Keiya; Koshino, Tomihisa

    2005-01-01

    To examine the effects of basic fibroblast growth factor (bFGF) gene-transduced chondrocytes on the repair of articular cartilage defects. LacZ gene or bFGF gene was transduced into primary isolated rabbit chondrocytes with the use of a recombinant adeno-associated virus (AAV) vector. These gene-transduced chondrocytes were embedded in collagen gel and transplanted into a full-thickness defect in the articular cartilage of the patellar groove of a rabbit. The efficiency of gene transduction was assessed according to the percentage of LacZ-positive cells among the total number of living cells. The concentration of bFGF in the culture supernatant was measured by enzyme-linked immunosorbent assay to confirm the production by bFGF gene-transduced chondrocytes. At 4, 8, and 12 weeks after transplantation, cartilage repair was evaluated histologically and graded semiquantitatively using a histologic scoring system ranging from 0 (complete regeneration) to 14 (no regeneration) points. LacZ gene expression by chondrocytes was maintained until 8 weeks in >85% of the in vitro population. LacZ-positive cells were found at the transplant sites for at least 4 weeks after surgery. The mean concentration of bFGF was significantly increased in bFGF gene-transduced cells compared with control cells (P < 0.01). Semiquantitative histologic scoring indicated that the total score was significantly lower in the bFGF-transduced group than in the control group throughout the observation period. These results demonstrated that gene transfer to chondrocytes by an ex vivo method was established with the AAV vector, and transplantation of bFGF gene-transduced chondrocytes had a clear beneficial effect on the repair of rabbit articular cartilage defects.

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

    PubMed

    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.

  18. Prospective evaluation of serum biomarker levels and cartilage repair by autologous chondrocyte transplantation and subchondral drilling in a canine model

    PubMed Central

    Nganvongpanit, Korakot; Pothacharoen, Peraphan; Chaochird, Patama; Klunklin, Kasisin; Warrit, Kanawee; Settakorn, Jongkolnee; Pattamapaspong, Nuttaya; Luevitoonvechkij, Sirichai; Arpornchayanon, Olarn; Kongtawelert, Prachya; Pruksakorn, Dumnoensun

    2009-01-01

    Introduction The purpose of this study was to evaluate serum chondroitin sulfate (CS) and hyaluronic acid (HA) levels and the capability of cartilage repair of full-thickness cartilage defects after treatment with two different fundamental surgical techniques: autologous chondrocyte transplantation (AC) and subchondral drilling (SD). Methods A 4-mm-diameter full-thickness cartilage defect was created in each of 10 skeletally mature male outbred dogs. The dogs were randomly separated into two groups. Groups A and B were treated with AC and SD, respectively. An evaluation was made at the 24th week of the experiment. Serum was analyzed prospectively – preoperatively and at 6-week intervals – for CS and HA levels by enzyme-linked immunosorbent assay (ELISA) and ELISA-based assays, respectively. Results The cartilage repair assessment score (median ± standard deviation) of group A (9.5 ± 2.5) was significantly higher than that of group B (2.5 ± 1.3) (P < 0.05). Group A also demonstrated a better quality of hyaline-like cartilage repair. Prospective analysis of serum WF6 and HA levels between the two groups did not show any significant difference. Serum WF6 levels at the 24th week of the experiment had a negative correlation (r = -0.69, P < 0.05) with the cartilage repair assessment score, whereas serum HA levels tended to correlate positively (r = 0.46, 0.1

    cartilage marker levels. AC treatment demonstrated a smoother surface, less fissure, better border integration, and a more reliable outcome of repairing cartilage. Moreover, a decreasing level of serum WF6, which correlated with good quality of the repairing tissue at the end of the follow-up period, was found predominantly in the AC group. Serum WF6 therefore should be further explored as a sensitive marker for the noninvasive therapeutic evaluation of cartilage repair procedures. PMID

  19. [iPS cells for the generation of cartilage and for regenerative medicine and disease modeling of cartilage diseases].

    PubMed

    Tsumaki, Noriyuki

    2016-04-01

    The development of induced pluripotent stem cells(iPSCs)has enabled the acquisition of patient-specific chondrocytes by converting somatic cells, such as dermal fibroblasts or blood cells, from patients to iPSCs and then differentiating them toward chondrocytes. We can further generate cartilage tissue from iPSC-derived chondrocytes. Studies on iPSC-derived chondrocytes/cartilage for the regeneration of articular cartilage injury are ongoing. These studies will in the future use autologous iPSCs and allogenic iPSCs from an iPSC stock prepared from donor cells. Drug discovery research for related diseases such as skeletal dysplasia is also being conducted.

  20. Effects of serum and compressive loading on the cartilage matrix synthesis and spatiotemporal deposition around chondrocytes in 3D culture.

    PubMed

    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.

  1. Remobilization causes site-specific cyst formation in immobilization-induced knee cartilage degeneration in an immobilized rat model.

    PubMed

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

    2016-06-01

    An understanding of the articular cartilage degenerative process is necessary for the prevention and treatment of joint disease. The present study aimed to examine how long-term immobilization-induced cartilage degeneration is aggravated by remobilization. Sixty 8-week-old male Wistar rats were used in this study. The unilateral knee joint was immobilized using an external fixator for 8 weeks. The rats were killed at 0 and 3 days, and at 1, 2, 4 and 8 weeks after removing the fixator. After the rats were killed, the maximum knee extension angles were measured. Histological sections at the medial mid-condylar region (non-contact, transitional and contact regions of the femur and tibia) were prepared and scored. The cartilage thickness and number of chondrocytes were measured, and CD44 and Col2-3/4c expression levels were assessed immunohistochemically. The histological assessment revealed progressive aggravation of cartilage degeneration in the transitional region, with a decreased number of chondrocytes and CD44-positive chondrocytes as well as poor scoring over time, particularly in the tibia. Cyst formation was confirmed in the transitional region of the tibia at 8 weeks post-remobilization. The cartilage thickness in the transitional region was thicker than that in the contact region, particularly in the tibia. Col2-3/4c expression was observed in the non-contact and transitional regions, and the knee extension angle was recovered. In conclusion, immobilization-induced cartilage degeneration was aggravated by remobilization over time in the transitional region, followed by observations of a decreased number of chondrocytes and morphological disparity between different cartilage regions. © 2016 The Authors. Journal of Anatomy published by John Wiley & Sons Ltd on behalf of Anatomical Society.

  2. Analysis of clones from a human cartilage cDNA library provides insight into chondrocyte gene expression and identifies novel candidate genes for the osteochondrodysplasias.

    PubMed

    Krakow, Deborah; Sebald, Eiman T; Pogue, Robert; Rimoin, Lauren P; King, Lily; Cohn, Daniel H

    2003-05-01

    To begin to define the gene expression pattern in fetal cartilage and to identify uncharacterized candidate genes for the osteochondrodysplasias, we analyzed clones from a fetal cartilage cDNA library. Sequence analysis of 420 cDNA clones identified 210 clones derived from established genes but, for many of them, expression in cartilage had not been previously reported. Among the established genes were 14 genes known to produce skeletal abnormalities in either humans or mice when mutated. Thirty-two uncharacterized genes and their respective chromosomal positions were also identified. To further understand the expression profile of these genes in fetal cartilage, we constructed a cDNA microarray utilizing the clones. The microarray was used to determine which genes had higher expression in cartilage as compared with dedifferentiated, cultured chondrocytes. Many of the established genes, as well as five of the uncharacterized genes, had increased expression in cartilage, suggesting an important role for these genes in the differentiated state of chondrocytes. These data provide new candidate genes for the osteochondrodysplasias and demonstrate the usefulness of cartilage cDNA microarrays in expanding our understanding of the complexity of fetal cartilage gene expression.

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

    PubMed

    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.

  4. Autophagy Protects Advanced Glycation End Product-Induced Apoptosis and Expression of MMP-3 and MMP-13 in Rat Chondrocytes

    PubMed Central

    Wu, Tianlong

    2017-01-01

    Aging is one of the most prominent risk factors for the pathological progression of osteoarthritis (OA). One feature of age-related changes in OA is advanced glycation end products (AGEs) accumulation in articular cartilage. Autophagy plays a cellular housekeeping role by removing dysfunctional cellular organelles and proteins. However, the relationship between autophagy and AGE-associated OA is unknown. The aim of this study is to determine whether autophagy participates in the pathology of AGE-treated chondrocytes and to investigate the exact role of autophagy in AGE-induced cell apoptosis and expression of matrix metalloproteinase- (MMP-) 3 and MMP-13. AGEs induced notable apoptosis that was detected by Annexin V/PI double-staining, and the upregulation of MMP-3 and MMP-13 was confirmed by Western blotting. Autophagy-related proteins were also determined by Western blotting, and chondrocytes were transfected with mCherry-GFP-LC3B-adenovirus to monitor autophagic flux. As a result, autophagy significantly increased in chondrocytes and peaked at 6 h. Furthermore, rapamycin (RA) attenuated AGE-induced apoptosis and expression of MMP-3 and MMP-13 by autophagy activation. In contrast, pretreatment with autophagy inhibitor 3-methyladenine (3-MA) enhanced the abovementioned effects of AGEs. We therefore demonstrated that autophagy is linked with AGE-related pathology in rat chondrocytes and plays a protective role in AGE-induced apoptosis and expression of MMP-3 and MMP-13. PMID:28265573

  5. Cotransfected human chondrocytes: over-expression of IGF-I and SOX9 enhances the synthesis of cartilage matrix components collagen-II and glycosaminoglycans.

    PubMed

    Simental-Mendía, M; Lara-Arias, J; Álvarez-Lozano, E; Said-Fernández, S; Soto-Domínguez, A; Padilla-Rivas, G R; Martínez-Rodríguez, H G

    2015-12-01

    Damage to cartilage causes a loss of type II collagen (Col-II) and glycosaminoglycans (GAG). To restore the original cartilage architecture, cell factors that stimulate Col-II and GAG production are needed. Insulin-like growth factor I (IGF-I) and transcription factor SOX9are essential for the synthesis of cartilage matrix, chondrocyte proliferation, and phenotype maintenance. We evaluated the combined effect of IGF-I and SOX9 transgene expression on Col-II and GAG production by cultured human articular chondrocytes. Transient transfection and cotransfection were performed using two mammalian expression plasmids (pCMV-SPORT6), one for each transgene. At day 9 post-transfection, the chondrocytes that were over-expressing IGF-I/SOX9 showed 2-fold increased mRNA expression of the Col-II gene, as well as a 57% increase in Col-II protein, whereas type I collagen expression (Col-I) was decreased by 59.3% compared with controls. The production of GAG by these cells increased significantly compared with the controls at day 9 (3.3- vs 1.8-times, an increase of almost 83%). Thus, IGF-I/SOX9 cotransfected chondrocytes may be useful for cell-based articular cartilage therapies.

  6. Treatment of focal degenerative cartilage defects with polymer-based autologous chondrocyte grafts: four-year clinical results

    PubMed Central

    Kreuz, Peter C; Müller, Sebastian; Ossendorf, Christian; Kaps, Christian; Erggelet, Christoph

    2009-01-01

    Introduction Second-generation autologous chondrocyte implantation with scaffolds stabilizing the grafts is a clinically effective procedure for cartilage repair. In this ongoing prospective observational case report study, we evaluated the effectiveness of BioSeed®-C, a cell-based cartilage graft based on autologous chondrocytes embedded in fibrin and a stable resorbable polymer scaffold, for the treatment of clinical symptomatic focal degenerative defects of the knee. Methods Clinical outcome after 4-year clinical follow-up was assessed in 19 patients with preoperatively radiologically confirmed osteoarthritis and a Kellgren-Lawrence score of 2 or more. Clinical scoring was performed before implantation of the graft and 6, 12, and 48 months after implantation using the Lysholm score, the Knee injury and Osteoarthritis Outcome Score (KOOS), the International Knee Documentation Committee (IKDC) score, and the International Cartilage Repair Society (ICRS) score. Cartilage regeneration and articular resurfacing were assessed by magnetic resonance imaging (MRI) 4 years after implantation of the autologous cartilage graft. Results Significant improvement (P < 0.05) of the Lysholm and ICRS scores was observed as early as 6 months after implantation of BioSeed®-C and remained stable during follow-up. The IKDC score showed significant improvement compared with the preoperative situation at 12 and 48 months (P < 0.05). The KOOS showed significant improvement in the subclasses pain, activities of daily living, and knee-related quality of life 6 months as well as 1 and 4 years after implantation of BioSeed®-C in osteoarthritic defects (P < 0.05). MRI analysis showed moderate to complete defect filling with a normal to incidentally hyperintense signal in 16 out of 19 patients treated with BioSeed®-C. Two patients without improvement in the clinical and MRI scores received a total knee endoprosthesis after 4 years. Conclusions The results show that the good clinical

  7. Molecular differentiation in epiphyseal and physeal cartilage. Prominent role for gremlin in maintaining hypertrophic chondrocytes in epiphyseal cartilage.

    PubMed

    Shapiro, Frederic; Flynn, Evelyn; Calicchio, Monica L

    2009-12-18

    We have studied hypertrophic and immediately adjacent pre-hypertrophic chondrocytes at the same stage of histologic development in 7 day old post-natal Balb/C mouse physes and epiphyses. Laser capture microdissection (LCM) and GeneChip microarray analysis compared the molecular composition of the two hypertrophic chondrocyte regions. Molecules upregulated in dramatically higher levels in the epiphysis were gremlin (58-fold), epidermal growth factor-containing fibulin-like extracellular matrix protein 1 (25-fold), and frizzled related protein (6.4-fold and 5.7-fold). Molecules upregulated in higher levels in the physis were proline arginine-rich end leucine-rich repeat protein (PRELP) (15.6-fold), pyrophosphatase (inorganic) 1 (10-fold) and hedgehog-interacting protein (7.3-fold). Immunocytochemistry for gremlin confirmed specific localization patterns. This study indicates a critical site-specific role for hypertrophic chondrocytes with different synthesis patterns in separate regions even though they appear structurally the same and are at the same stage of development.

  8. Laser-structured bacterial nanocellulose hydrogels support ingrowth and differentiation of chondrocytes and show potential as cartilage implants.

    PubMed

    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

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

    PubMed

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

    2016-10-01

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

  10. Hypoxia in cartilage: HIF-1α is essential for chondrocyte growth arrest and survival

    PubMed Central

    Schipani, Ernestina; Ryan, Heather E.; Didrickson, Susanna; Kobayashi, Tatsuya; Knight, Melissa; Johnson, Randall S.

    2001-01-01

    Breakdown or absence of vascular oxygen delivery is a hallmark of many common human diseases, including cancer, myocardial infarction, and stroke. The chief mediator of hypoxic response in mammalian tissues is the transcription factor hypoxia-inducible factor 1 (HIF-1), and its oxygen-sensitive component HIF-1α. A key question surrounding HIF-1α and the hypoxic response is the role of this transcription factor in cells removed from a functional vascular bed; in this regard there is evidence indicating that it can act as either a survival factor or induce growth arrest and apoptosis. To study more closely how HIF-1α functions in hypoxia in vivo, we used tissue-specific targeting to delete HIF-1α in an avascular tissue: the cartilaginous growth plate of developing bone. We show here the first evidence that the developmental growth plate in mammals is hypoxic, and that this hypoxia occurs in its interior rather than at its periphery. As a result of this developmental hypoxia, cells that lack HIF-1α in the interior of the growth plate die. This is coupled to decreased expression of the CDK inhibitor p57, and increased levels of BrdU incorporation in HIF-1α null growth plates, indicating defects in HIF-1α-regulated growth arrest occurs in these animals. Furthermore, we find that VEGF expression in the growth plate is regulated through both HIF-1α-dependent and -independent mechanisms. In particular, we provide evidence that VEGF expression is up-regulated in a HIF-1α-independent manner in chondrocytes surrounding areas of cell death, and this in turn induces ectopic angiogenesis. Altogether, our findings have important implications for the role of hypoxic response and HIF-1α in development, and in cell survival in tissues challenged by interruption of vascular flow; they also illustrate the complexities of HIF-1α response in vivo, and they provide new insights into mechanisms of growth plate development. PMID:11691837

  11. Inhibition of microRNA-449a prevents IL-1β-induced cartilage destruction via SIRT1.

    PubMed

    Park, K W; Lee, K-M; Yoon, D S; Park, K H; Choi, W J; Lee, J W; Kim, S-H

    2016-12-01

    SIRT1 has anti-inflammatory as well as protective effects in chondrocytes. The object of this study was to investigate whether microRNA-449a regulates expression of SIRT1, which inhibits expression of catabolic genes in IL-1β-induced cartilage destruction. MicroRNA-449a expression was determined in OA chondrocytes and IL-1β-induced chondrocytes by real-time PCR. MicroRNA-449a binding sites on the 3'-UTR of SIRT1 mRNA and binding site conservation were examined using microRNA target prediction tools. SIRT1-overexpressing or knockdown chondrocytes were transfected with microRNA-449a or anti-microRNA-449a mimic and stimulated by IL-1β. Expression of catabolic and anabolic genes was examined by real-time PCR and western blotting. Finally, positive effects of anti-microRNA-449a on expression of these genes were confirmed by western analysis of OA chondrocytes. Expression of microRNA-449a was increased in OA chondrocytes and IL-1β-induced chondrocytes. MMP-13 expression was enhanced, whereas type II collagen and SIRT1 expression were decreased in IL-1β-induced chondrocytes. SIRT1 overexpression resulted in decreased expression of catabolic genes such as MMPs and ADAMTSs in response to IL-1β, but these effects were moderated by microRNA-449a. Suppression of microRNA-449a by anti-microRNA-449a inhibited expression of catabolic genes despite IL-1β stimulation, but these effects were abolished in SIRT1 knockdown chondrocytes. Furthermore, expression of catabolic genes was decreased and expression of type II collagen as well as SIRT1 was restored by anti-microRNA-449a in OA chondrocytes as well as in IL-1β-induced chondrocytes. Silencing of microRNA-449a had a protective effect, inhibiting catabolic gene expression and restoring anabolic gene expression, by targeting SIRT1 in IL-1β-induced cartilage destruction. Copyright © 2016 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.

  12. Esculetin inhibits cartilage resorption induced by interleukin 1α in combination with oncostatin M

    PubMed Central

    Elliott, S; Rowan, A; Carrere, S; Koshy, P; Catterall, J; Cawston, T

    2001-01-01

    OBJECTIVE—To determine if a new inhibitor, esculetin (EST), can block resorption of cartilage.
METHODS—Interleukin 1α (IL1α, 0.04-5 ng/ml) and oncostatin M (OSM, 0.4-50 ng/ml) were used to stimulate the release of proteoglycan and collagen from bovine nasal cartilage and human articular cartilage in explant culture. Proteoglycan and collagen loss were assessed by dimethylmethylene blue and hydroxyproline assays, respectively. Collagenase levels were measured by assay of bioactivity and by enzyme linked immunosorbent assay (ELISA). The effects of EST on the expression of matrix metalloproteinases (MMPs) and tissue inhibitor of metalloproteinase-1 (TIMP-1) in the transformed human chondrocyte cell line T/C28a4 were assessed by northern blot analysis. TIMP-1 protein levels were assayed by ELISA. The effect of EST on the MMP-1 promoter was assessed using a promoter-luciferase construct in transient transfection studies.
RESULTS—EST inhibited proteoglycan and collagen resorption in a dose dependent manner with significant decreases seen at 66 µM and 100 µM EST, respectively. Collagenolytic activity was significantly decreased in bovine nasal cartilage cultures. In human articular cartilage, EST also inhibited IL1α + OSM stimulated resorption and decreased MMP-1 levels. TIMP-1 levels were not altered compared with controls. In T/C28a4 chondrocytes the IL1α + OSM induced expression of MMP-1, MMP-3, and MMP-13 mRNA was reduced to control levels by 250 µM EST. TIMP-1 mRNA levels were unaffected by EST treatment. All cytokine stimulation of an MMP-1 luciferase-promoter construct was lost in the presence of the inhibitor.
CONCLUSION—EST inhibits degradation of bovine nasal cartilage and human articular cartilage stimulated to resorb with IL1α + OSM.

 PMID:11156550

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

    PubMed

    Zhang, Zhongwen; Zhong, Xin; Ji, Huiru; Tang, Zibin; Bai, Jianpeng; Yao, Minmin; Hou, Jianlei; Zheng, Minghao; Wood, David J; Sun, Jiazhi; Zhou, Shu-Feng; Liu, Aibing

    2014-01-01

    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 cm(2). 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-to-excellent filling of the chondral defect. Most (95%) of the MACI grafts were isointense and 5% were slightly hyperintense. Histologic evaluation at 15 and 24 months showed predominantly hyaline cartilage in newly generated tissue. There were no postoperative complications in any patients and no adverse events related to the MACI operation. This 2-year study has confirmed that MACI is safe and effective with the advantages of a simple technique and significant clinical improvements. Further functional and mechanistic studies with longer follow-up are needed to validate the efficacy and safety of MACI in patients with articular cartilage injuries.

  14. Periodic Mechanical Stress INDUCES Chondrocyte Proliferation and Matrix Synthesis via CaMKII-Mediated Pyk2 Signaling.

    PubMed

    Liang, Wenwei; Li, Zeng; Wang, Zhen; Zhou, Jinchun; Song, Huanghe; Xu, Shun; Cui, Weiding; Wang, Qing; Chen, Zhefeng; Liu, Feng; Fan, Weimin

    2017-01-01

    Periodic mechanical stress can promote chondrocyte proliferation and matrix synthesis to improve the quality of tissue-engineered cartilage. Although the integrin β1-ERK1/2 signal cascade has been implicated in periodic mechanical stress-induced mitogenic effects in chondrocytes, the precise mechanisms have not been fully established. The current study was designed to probe the roles of CaMKII and Pyk2 signaling in periodic mechanical stress-mediated chondrocyte proliferation and matrix synthesis. Chondrocytes were subjected to periodic mechanical stress, proliferation was assessed by direct cell counting and CCK-8 assay; gene expressions were analyzed using quantitative real-time PCR, protein abundance by Western blotting. Mechanical stress, markedly enhanced the phosphorylation levels of Pyk2 at Tyr402 and CaMKII at Thr286. Both suppression of Pyk2 with Pyk2 inhibitor PF431396 or Pyk2 shRNA and suppression of CaMKII with CaMKII inhibitor KN-93 or CaMKII shRNA blocked periodic mechanical stress-induced chondrocyte proliferation and matrix synthesis. Additionally, either pretreatment with KN-93 or shRNA targeted to CaMKII prevented the activation of ERK1/2 and Pyk2 under conditions of periodic mechanical stress. Interestingly, in relation to periodic mechanical stress, in the context of Pyk2 inhibition with PF431396 or its targeted shRNA, only the phosphorylation levels of ERK1/2 were abrogated, while CaMKII signal activation was not affected. Moreover, the phosphorylation levels of CaMKII- Thr286 and Pyk2- Tyr402 were abolished after pretreatment with blocking antibody against integrinβ1 exposed to periodic mechanical stress. Our results collectively indicate that periodic mechanical stress promotes chondrocyte proliferation and matrix synthesis through the integrinβ1-CaMKII-Pyk2-ERK1/2 signaling cascade. © 2017 The Author(s). Published by S. Karger AG, Basel.

  15. Pathways of load-induced cartilage damage causing cartilage degeneration in the knee after meniscectomy.

    PubMed

    Wilson, W; van Rietbergen, B; van Donkelaar, C C; Huiskes, R

    2003-06-01

    Results of both clinical and animal studies show that meniscectomy often leads to osteoarthritic degenerative changes in articular cartilage. It is generally assumed that this process of cartilage degeneration is due to changes in mechanical loading after meniscectomy. It is, however, not known why and where this cartilage degeneration starts. Load induced cartilage damage is characterized as either type (1)--damage without disruption of the underlying bone or calcified cartilage layer--or type (2), subchondral fracture with or without damage to the overlying cartilage. We asked the question whether cartilage degeneration after meniscectomy is likely to be initiated by type (1) and/or type (2) cartilage damage. To investigate that we applied an axisymmetric biphasic finite element analysis model of the knee joint. In this model the articular cartilage layers of the tibial and the femoral condyles, the meniscus and the bone underlying the articular cartilage of the tibia plateau were included. The model was validated with data from clinical studies, in which the effects of meniscectomy on contact areas and pressures were measured. It was found that both the maximal values and the distributions of the shear stress in the articular cartilage changed after meniscectomy, and that these changes could lead to both type (1) and type (2) cartilage damage. Hence it likely that the cartilage degeneration seen after meniscectomy is initiated by both type (1) and type (2) cartilage damage.

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

    PubMed

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

    2016-05-15

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

  17. Osteoarthritic cartilage explants affect extracellular matrix production and composition in cocultured bone marrow-derived mesenchymal stem cells and articular chondrocytes

    PubMed Central

    2014-01-01

    Introduction In the present study, we established a novel in vitro coculture model to evaluate the influence of osteoarthritis (OA) cartilage explants on the composition of newly produced matrix and chondrogenic differentiation of human bone marrow-derived mesenchymal stem cells (BMSCs) and the phenotype of OA chondrocytes. In addition, we included a “tri-culture” model, whereby a mixture of BMSCs and chondrocytes was cultured on the surface of OA cartilage explants. Methods Gene expression analysis, protein and glycosaminoglycan (GAG) assays, dot-blot, immunofluorescence, and biomechanical tests were used to characterize the properties of newly generated extracellular matrix (ECM) from chondrocytes and chondrogenically differentiated BMSCs and a mix thereof. We compared articular cartilage explant cocultures with BMSCs, chondrocytes, and mixed cultures (chondrocytes and BMSCs 1:1) embedded in fibrin gels with fibrin gel-embedded cells cultured without cartilage explants (monocultures). Results In general, co- and tri-cultured cell regimens exhibited reduced mRNA and protein levels of collagens I, II, III, and X in comparison with monocultures, whereas no changes in GAG synthesis were observed. All co- and tri-culture regimens tended to exhibit lower Young’s and equilibrium modulus compared with monocultures. In contrast, aggregate modulus and hydraulic permeability seemed to be higher in co- and tri-cultures. Supernatants of cocultures contained significant higher levels of interleukin-1 beta (IL-1β), IL-6, and IL-8. Stimulation of monocultures with IL-1β and IL-6 reduced collagen gene expression in BMSCs and mixed cultures in general but was often upregulated in chondrocytes at late culture time points. IL-8 stimulation affected BMSCs only. Conclusions Our results suggest an inhibitory effect of OA cartilage on the production of collagens. This indicates a distinct modulatory influence that affects the collagen composition of the de novo-produced ECM from

  18. AMPK deficiency in chondrocytes accelerated the progression of instability-induced and ageing-associated osteoarthritis in adult mice.

    PubMed

    Zhou, Sheng; Lu, Wanli; Chen, Liang; Ge, Qiting; Chen, Dongyang; Xu, Zhihong; Shi, Dongquan; Dai, Jin; Li, Jianxin; Ju, Huangxian; Cao, Yi; Qin, Jinzhong; Chen, Shuai; Teng, Huajian; Jiang, Qing

    2017-02-22

    Osteoarthritis (OA) is a progressive degenerative disease of the joints that is associated with both joint injury and ageing. Here, we investigated the role of the energy sensor AMP-activated protein kinase (AMPK) in maintaining a healthy state of articular cartilage and in OA development. Using cartilage-specific, tamoxifen-inducible AMPKα1 conditional knockout (AMPKα1 cKO), AMPKα2 conditional knockout (AMPKα2 cKO) and AMPKα1α2 conditional double knockout (AMPKα cDKO) mice, we found that compared with wild-type (WT) littermates, mutant mice displayed accelerated severity of surgically induced OA, especially AMPKα cDKO mice. Furthermore, male but not female AMPKα cDKO mice exhibited severely spontaneous ageing-associated OA lesions at 12 months of age. The chondrocytes isolated from AMPKα cDKO mice resulted in an enhanced interleukin-1β (IL-1β)-stimulated catabolic response. In addition, upregulated expression of matrix metalloproteinase-3 (MMP-3), MMP-13 and phospho-nuclear factor-κB (phospho-NF-κB) p65 and increased levels of apoptotic markers were detected in the cartilage of AMPKα cDKO mice compared with their WT littermates in vivo. Thus, our findings suggest that AMPK activity in chondrocytes is important in maintaining joint homeostasis and OA development.

  19. AMPK deficiency in chondrocytes accelerated the progression of instability-induced and ageing-associated osteoarthritis in adult mice

    PubMed Central

    Zhou, Sheng; Lu, Wanli; Chen, Liang; Ge, Qiting; Chen, Dongyang; Xu, Zhihong; Shi, Dongquan; Dai, Jin; Li, Jianxin; Ju, Huangxian; Cao, Yi; Qin, Jinzhong; Chen, Shuai; Teng, Huajian; Jiang, Qing

    2017-01-01

    Osteoarthritis (OA) is a progressive degenerative disease of the joints that is associated with both joint injury and ageing. Here, we investigated the role of the energy sensor AMP-activated protein kinase (AMPK) in maintaining a healthy state of articular cartilage and in OA development. Using cartilage-specific, tamoxifen-inducible AMPKα1 conditional knockout (AMPKα1 cKO), AMPKα2 conditional knockout (AMPKα2 cKO) and AMPKα1α2 conditional double knockout (AMPKα cDKO) mice, we found that compared with wild-type (WT) littermates, mutant mice displayed accelerated severity of surgically induced OA, especially AMPKα cDKO mice. Furthermore, male but not female AMPKα cDKO mice exhibited severely spontaneous ageing-associated OA lesions at 12 months of age. The chondrocytes isolated from AMPKα cDKO mice resulted in an enhanced interleukin-1β (IL-1β)-stimulated catabolic response. In addition, upregulated expression of matrix metalloproteinase-3 (MMP-3), MMP-13 and phospho-nuclear factor-κB (phospho-NF-κB) p65 and increased levels of apoptotic markers were detected in the cartilage of AMPKα cDKO mice compared with their WT littermates in vivo. Thus, our findings suggest that AMPK activity in chondrocytes is important in maintaining joint homeostasis and OA development. PMID:28225087

  20. The Novel Small Leucine-rich Protein Chondroadherin-like (CHADL) Is Expressed in Cartilage and Modulates Chondrocyte Differentiation*

    PubMed Central

    Tillgren, Viveka; Ho, James C. S.; Önnerfjord, Patrik; Kalamajski, Sebastian

    2015-01-01

    The constitution and biophysical properties of extracellular matrices can dramatically influence cellular phenotype during development, homeostasis, or pathogenesis. These effects can be signaled through a differentially regulated assembly of collagen fibrils, orchestrated by a family of collagen-associated small leucine-rich proteins (SLRPs). In this report, we describe the tissue-specific expression and function of a previously uncharacterized SLRP, chondroadherin-like (CHADL). We developed antibodies against CHADL and, by immunohistochemistry, detected CHADL expression mainly in skeletal tissues, particularly in fetal cartilage and in the pericellular space of adult chondrocytes. In situ hybridizations and immunoblots on tissue lysates confirmed this tissue-specific expression pattern. Recombinant CHADL bound collagen in cell culture and inhibited in vitro collagen fibrillogenesis. After Chadl shRNA knockdown, chondrogenic ATDC5 cells increased their differentiation, indicated by increased transcript levels of Sox9, Ihh, Col2a1, and Col10a1. The knockdown increased collagen II and aggrecan deposition in the cell layers. Microarray analysis of the knockdown samples suggested collagen receptor-related changes, although other upstream effects could not be excluded. Together, our data indicate that the novel SLRP CHADL is expressed in cartilaginous tissues, influences collagen fibrillogenesis, and modulates chondrocyte differentiation. CHADL appears to have a negative regulatory role, possibly ensuring the formation of a stable extracellular matrix. PMID:25451920

  1. Glucosamine Hydrochloride but Not Chondroitin Sulfate Prevents Cartilage Degradation and Inflammation Induced by Interleukin-1α in Bovine Cartilage Explants

    PubMed Central

    Bascoul-Colombo, Cécile; Garaiova, Iveta; Plummer, Sue F.; Harwood, John L.; Caterson, Bruce; Hughes, Clare E

    2016-01-01

    Objective Glucosamine hydrochloride (GH) and chondroitin sulfate (CS) are commonly used for the treatment of osteoarthritis (OA). The aim of this study was to assess their effects, alone and in combination, on preventing aggrecan degradation and inflammation in an in vitro model of OA. Design To test the effects of GH and/or CS as a preventative treatment, cartilage explants were pretreated with the compound(s) using concentrations that showed no detrimental effect on chondrocyte viability. Interleukin-1α (IL-1α) was added to induce cartilage degradation, supernatant and explants were analyzed for proteoglycan degradation products, aggrecanase mRNA expression and activity, and for the release of inflammatory markers. Results Following treatment with IL-1α, 2 mg/mL dose of GH pretreatment was associated with a reduction of glycosaminoglycan release, reduced generation of the pathological interglobular domain aggrecan catabolites, decreased mRNA levels of ADAMTS-4 and -5 and reduced activity of ADAMTS-4. In contrast, CS alone did not have a significant effect on IL-1α-induced cartilage degradation and the addition of 0.4 mg/mL CS to 2 mg/mL GH did not further inhibit IL-1α-induced activity. Pretreatment with 2 mg/mL GH also reduced the release of inflammatory markers, prostaglandin E2 and nitric oxide induced by IL-1α while CS did not have a significant effect. Conclusions The results suggest that GH prevents cartilage degradation mediated by aggrecanases ADAMTS-4 and -5, and may also reduce inflammation. This could be part of the mechanisms by which GH is effective in maintaining joint integrity and function, and preventing or delaying early symptoms of OA. PMID:26958319

  2. Sex-specific response of rat costochondral cartilage growth plate chondrocytes to 17β-estradiol involves differential regulation of plasma membrane associated estrogen receptors.

    PubMed

    Elbaradie, Khairat B Y; Wang, Yun; Boyan, Barbara D; Schwartz, Zvi

    2013-05-01

    Both male and female rat growth plate chondrocytes express estrogen receptors (ERs); however 17β-estradiol (E2) induces membrane responses leading to activation of phospholipase A2 (PLA2), phospholipase C (PLC), prostaglandin E2 (PGE2) production, protein kinase C (PKC), and ultimately mitogen protein kinase (MAPK) only in female cells. This study investigated if these sex-specific responses are due to differences in the actual ERs or in downstream signaling. Western blots and flow cytometry of costochondral cartilage resting zone chondrocytes (RCs) showed 2-3 times more ERα in plasma membranes (PMs) from female cells than male cells. Tunicamycin blocked E2-dependent ER-translocation to the PM, indicating palmitoylation was required. Co-immunoprecipitation showed E2 induced complex formation between ER isoforms only in female RCs. To examine if the lack of response in PKC and PGE2 in males is due to differences in signaling, we examined involvement of ERs and the role of PLC and PLA2. Selective ERα (propylpyrazole triol, PPT) and ERβ (diarylproprionitrile, DPN) agonists activated PKC in female RCs only. The PLC inhibitor, U73122 blocked E2's effect on PKC and the cytosolic PLA2 inhibitor, AACOCF3 inhibited the effect on PGE2 in female RCs, confirming involvement of PLC and PLA2 in the mechanism. The PLC activator, m-3M3FβS activated PKC and PLAA peptide increased PGE2 levels in male and female RCs, showing that the signaling pathways are present. These data indicate that differences in membrane ER amount, localization, translocation and interaction are responsible for the sexual dimorphic response to E2.

  3. Interleukin 17 induces cartilage collagen breakdown: novel synergistic effects in combination with proinflammatory cytokines

    PubMed Central

    Koshy, P; Henderson, N; Logan, C; Life, P; Cawston, T; Rowan, A

    2002-01-01

    Objective: To investigate whether interleukin 17 (IL17), derived specifically from T cells, can promote type II collagen release from cartilage. The ability of IL17 to synergise with other proinflammatory mediators to induce collagen release from cartilage, and what effect anti-inflammatory agents had on this process, was also assessed. Methods: IL17 alone, or in combination with IL1, IL6, oncostatin M (OSM), or tumour necrosis factor α (TNFα), was added to bovine nasal cartilage explant cultures. Proteoglycan and collagen release were determined. Collagenolytic activity was determined by bioassay. Chondroprotective effects of IL4, IL13, transforming growth factor ß1 (TGFß1) and insulin-like growth factor-1 (IGF1) were assessed by inclusion in the explant cultures. Results: IL17 alone stimulated a dose dependent release of proteoglycan and type II collagen from bovine nasal cartilage explants. Suboptimal doses of IL17 synergised potently with TNFα, IL1, OSM, and IL6 to promote collagen degradation. This collagen release was completely inhibited by tissue inhibitor of metalloproteinase-1 and BB-94 (a synthetic metalloproteinase inhibitor), and was significantly reduced by IL4, IL13, TGFß1, and IGF1. In IL17 treated chondrocytes, mRNA expression for matrix metalloproteinase (MMP)-1, MMP-3, and MMP-13 was detected. Moreover, a synergistic induction of these MMPs was seen when IL17 was combined with other proinflammatory cytokines. Conclusions: IL17 can, alone and synergistically in combination with other proinflammatory cytokines, promote chondrocyte mediated MMP dependent type II collagen release from cartilage. Because levels of all these proinflammatory cytokines are raised in rheumatoid synovial fluids, this study suggests that IL17 may act as a potent upstream mediator of cartilage collagen breakdown in inflammatory joint diseases. PMID:12117676

  4. Tamoxifen induces permanent growth arrest through selective induction of apoptosis in growth plate chondrocytes in cultured rat metatarsal bones.

    PubMed

    Chagin, Andrei S; Karimian, Elham; Zaman, Farasat; Takigawa, Masaharu; Chrysis, Dionisios; Sävendahl, Lars

    2007-05-01

    Estrogen affects skeletal growth and promotes growth plate fusion in humans. High doses of estrogen have been used to limit growth in girls with predicted extreme tall stature; a treatment which has been associated with severe side effects. Selective estrogen receptor modulators (SERMs) could potentially be used as an alternative treatment. We chose to study the effects of Tamoxifen (Tam), a first generation SERM that has been used in the treatment of pubertal gynecomastia or McCune-Albright syndrome. Cultured fetal rat metatarsal bones were used to study the effects of Tam on longitudinal bone growth. In sectioned bones, chondrocyte apoptosis and proliferation were analyzed by TUNEL assay and BrdU incorporation, respectively. We also used a human chondrocytic cell line, HSC-2/8, to study the effects of Tam on apoptosis (FACS analysis and Cell Death detection ELISA) and caspase activation (caspase substrate cleavage and Western immunoblotting). Tam caused a dose-dependent growth retardation of cultured metatarsal bones. No catch-up growth was observed after Tam was removed from the culture medium. Detailed analysis of sectioned growth plate cartilage revealed increased apoptosis of chondrocytes within the resting and hypertrophic zones. HCS-2/8 cells also underwent apoptosis upon Tam treatment. Tam-induced apoptosis was caspase-dependent and completely abrogated by either caspase-8 or -9 inhibitors. A substrate assay revealed that caspase-8 is first activated followed by caspase-9 and -3. Finally, FasL secretion was stimulated by Tam and blocking of either FasL or Fas decreased Tam-induced apoptosis in chondrocytes. We here describe a novel mechanism of tamoxifen-induced apoptosis in chondrocytes, involving the activation of caspases and the FasL/Fas pathway, which diminishes the potential for bone growth.

  5. [Autologous chondrocyte implantation (ACI) for cartilage defects of the knee: a guideline by the working group "Tissue Regeneration" of the German Society of Orthopaedic Surgery and Traumatology (DGOU)].

    PubMed

    Niemeyer, P; Andereya, S; Angele, P; Ateschrang, A; Aurich, M; Baumann, M; Behrens, P; Bosch, U; Erggelet, C; Fickert, S; Fritz, J; Gebhard, H; Gelse, K; Günther, D; Hoburg, A; Kasten, P; Kolombe, T; Madry, H; Marlovits, S; Meenen, N M; Müller, P E; Nöth, U; Petersen, J P; Pietschmann, M; Richter, W; Rolauffs, B; Rhunau, K; Schewe, B; Steinert, A; Steinwachs, M R; Welsch, G H; Zinser, W; Albrecht, D

    2013-02-01

    Autologous chondrocyte transplantation/implantation (ACT/ACI) is an established and recognised procedure for the treatment of localised full-thickness cartilage defects of the knee. The present review of the working group "Clinical Tissue Regeneration" of the German Society of Orthopaedics and Traumatology (DGOU) describes the biology and function of healthy articular cartilage, the present state of knowledge concerning potential consequences of primary cartilage lesions and the suitable indication for ACI. Based on current evidence, an indication for ACI is given for symptomatic cartilage defects starting from defect sizes of more than 3-4 cm2; in the case of young and active sports patients at 2.5 cm2. Advanced degenerative joint disease is the single most important contraindication. The review gives a concise overview on important scientific background, the results of clinical studies and discusses advantages and disadvantages of ACI.

  6. Non-enzymatic glycation of chondrocyte-seeded collagen gels for cartilage tissue engineering.

    PubMed

    Roy, Rani; Boskey, Adele L; Bonassar, Lawrence J

    2008-11-01

    Collagen glycated with ribose (250 mM) in solution (pre-glycation) and as a gel (post-glycation) was seeded with chondrocytes and the effects of glycation on chondrocyte matrix assembly in culture were determined. Pre-glycation enhanced GAG accumulation significantly over controls at both 2 and 4 weeks (p < 0.05), although at both time points there were no statistical differences in cell number between pre-glycated and control gels. The increased proteoglycan accumulation was shown to be in part due to significantly increased GAG retention by the pre-glycated constructs (p < 0.05). Total collagen content in these pre-glycated gels was also significantly higher than unglycated gels at 4 weeks (p < 0.05). With post-glycation of collagen gels, chondrocyte number and GAG accumulation were all significantly lower than controls (p < 0.05). Post-glycation also inhibited GAG retention by the constructs (p < 0.05). Given these results, pre-glycation may be an improved processing method for collagen gels for tissue engineering techniques.

  7. Suture compression induced midpalatal suture chondrocyte apoptosis with increased caspase-3, caspase-9, Bad, Bak, Bax and Bid expression.

    PubMed

    Lan, Tingting; Zhao, Hanchi; Xiang, Bilu; Wang, Jun; Liu, Yang

    2017-07-22

    Previous studies found bone resorption and chondrocytes loss in mouse models of mid-palatal suture when given continuous compressive force, although chondrocytes response remained unknown. Herein, we design this study to determine how continuous compression force induces chondrocytes apoptosis. Thirty C57BL/6 male mice (aged 6 weeks) were randomly assigned into controls (not ligated to a spring), blank controls (ligated with no compression) and the compression group (ligated with 20-g compression). After 4 d, palatal tissues were sampled and stained by TB and safranin-O. Tunel staining measured the percentage of apoptotic chondrocytes, and immunohistochemistry was performed to label apoptosis-associated proteins (e.g., Bcl-2, Bcl-xl, Bax, Bak, Bid, Bad, caspase-3, caspase-8 and caspase-9). Intergroup comparison was made by the rank sum test, and P < 0.05 was defined as statistical significance. After 7d of induction, TB and safranin-O staining revealed that the cartilage area in the compression group was significantly decreased, while the control group remained largely unaltered. Tunel staining showed that apoptotic cell numbers in the mid-palatal suture were significantly higher than the control group. Immunohistochemistry showed that mice in the compression group had significantly increased expression of caspase-3, caspase-9, Bad, Bak, Bax and Bid; However, caspase-8 remained unaltered. No expression of Bcl-2 and Bcl-xl was detected. Continuous compression force induces chondrocytes apoptosis in the mid-palatal suture. This process might be associated with the mitochondrial pathway. Copyright © 2017 Elsevier Inc. All rights reserved.

  8. Hsp90β inhibition modulates nitric oxide production and nitric oxide-induced apoptosis in human chondrocytes

    PubMed Central

    2011-01-01

    Background Hsp90β is a member of the Hsp90 family of protein chaperones. This family plays essential roles in the folding, maturation and activity of many proteins that are involved in signal transduction and transcriptional regulation. The role of this protein in chondrocytes is not well understood, although its increase in osteoarthritic cells has been reported. The present study aimed to explore the role of Hsp90β in key aspects of OA pathogenesis. Methods Human OA chondrocytes were isolated from cartilage obtained from patients undergoing joint replacement surgery, and primary cultured. Cells were stimulated with proinflammatory cytokines (IL-1β or TNF-α) and nitric oxide donors (NOC-12 or SNP). For Hsp90β inhibition, two different chemical inhibitors (Geldanamycin and Novobiocin) were employed, or siRNA transfection procedures were carried out. Gene expression was determined by real-time PCR, apoptosis was quantified by flow cytometry and ELISA, and nitric oxide (NO) production was evaluated by the Griess method. Indirect immunofluorescence assays were performed to evaluate the presence of Hsp90β in stimulated cells. Results Hsp90β was found to be increased by proinflammatory cytokines. Inhibition of Hsp90β by the chemicals Geldanamycin (GA) and Novobiocin (NB) caused a dose-dependent decrease of the NO production induced by IL-1β in chondrocytes, up to basal levels. Immunofluorescence analyses demonstrate that the NO donors NOC-12 and SNP also increased Hsp90β. Chemical inhibition or specific gene silencing of this chaperone reduced the DNA condensation and fragmentation, typical of death by apoptosis, that is induced by NO donors in chondrocytes. Conclusions The present results show how Hsp90β modulates NO production and NO-mediated cellular death in human OA chondrocytes. PMID:22004293

  9. Oxygen tension affects lubricin expression in chondrocytes.

    PubMed

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

    2014-10-01

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

  10. Basic fibroblast growth factor as a selective inducer of matrix Gla protein gene expression in proliferative chondrocytes.

    PubMed Central

    Stheneur, Chantal; Dumontier, Marie-France; Guedes, Claudie; Fulchignoni-Lataud, Marie-Claude; Tahiri, Khadija; Karsenty, Gerard; Corvol, Marie Thérèse

    2003-01-01

    Matrix Gla protein (MGP) is a member of the vitamin K-dependent gamma carboxylase protein family expressed in cartilage. Insulin-like growth factor I (IGF1) stimulates chondrocyte differentiation, whereas basic fibroblast growth factor (FGF2) acts in an opposite manner. We explored the differential expression and regulation by IGF1 and FGF2 of the MGP gene during chondrocyte differentiation. We used a primary culture system of rabbit epiphyseal chondrocytes to show that MGP mRNA is mainly expressed during serum-induced proliferation. Much lower MGP mRNA content is observed in post-mitotic chondrocytes, which newly express alpha 1X procollagen mRNA, a marker of late-differentiated cells. From studies of a series of growth factors, it was shown that IGF1 decreased chondrocyte MGP transcripts, whereas FGF2 had the opposite effect. FGF2 stimulated chondrocyte MGP production in a dose- and time-dependent manner at the mRNA and protein levels. FGF2 acted in a dose- and time-dependent manner, reaching a maximum at 10 ng/ml at 20 h. The protein synthesis inhibitor cycloheximide did not modify FGF2 action, in agreement with a direct effect. Actinomycin D abolished FGF2-induced stimulation, strongly suggesting that FGF2 modulated MGP gene transcription. We transiently transfected chondrocytes with a construct containing the mouse MGP promoter from -5000 to -168 base pairs, relative to the transcription start site of the gene linked to the luciferase gene (MGP-Luc). In transfected cells, FGF2 stimulated luciferase activity up to sevenfold while IGF1 had no effect. Hence, FGF2 induces transcription of the MGP gene via the 5'-flanking region of the gene. Using a series of deleted MGP-Luc constructs, we identified a sequence of 748 base pairs which was sufficient for transcriptional activation by FGF2. These results led us to postulate that the inhibitory chondrogenic action of FGF2 involves a mechanism whereby MGP gene transcription and protein are induced. PMID:12230429

  11. Autologous collagen induced chondrogenesis (ACIC: Shetty-Kim technique) - A matrix based acellular single stage arthroscopic cartilage repair technique.

    PubMed

    Shetty, Asode Ananthram; Kim, Seok Jung; Shetty, Vishvas; Jang, Jae Deog; Huh, Sung Woo; Lee, Dong Hwan

    2016-01-01

    The defects of articular cartilage in the knee joint are a common degenerative disease and currently there are several established techniques to treat this problem, each with their own advantages and shortcomings. Autologous chondrocyte implantation is the current gold standard but the technique is expensive, time-consuming and most versions require two stage procedures and an arthrotomy. Autologous collagen induced chondrogenesis (ACIC) is a single-stage arthroscopic procedure and we developed. This method uses microfracture technique with atelocollagen mixed with fibrin gel to treat articular cartilage defects. We introduce this ACIC techniques and its scientific background.

  12. Biomarkers of Chondrocyte Apoptosis and Autophagy in Osteoarthritis

    PubMed Central

    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

  13. A Review of the Combination of Experimental Measurements and Fibril-Reinforced Modeling for Investigation of Articular Cartilage and Chondrocyte Response to Loading

    PubMed Central

    Wilson, Wouter; Isaksson, Hanna; Jurvelin, Jukka S.; Herzog, Walter; Korhonen, Rami K.

    2013-01-01

    The function of articular cartilage depends on its structure and composition, sensitively impaired in disease (e.g. osteoarthritis, OA). Responses of chondrocytes to tissue loading are modulated by the structure. Altered cell responses as an effect of OA may regulate cartilage mechanotransduction and cell biosynthesis. To be able to evaluate cell responses and factors affecting the onset and progression of OA, local tissue and cell stresses and strains in cartilage need to be characterized. This is extremely challenging with the presently available experimental techniques and therefore computational modeling is required. Modern models of articular cartilage are inhomogeneous and anisotropic, and they include many aspects of the real tissue structure and composition. In this paper, we provide an overview of the computational applications that have been developed for modeling the mechanics of articular cartilage at the tissue and cellular level. We concentrate on the use of fibril-reinforced models of cartilage. Furthermore, we introduce practical considerations for modeling applications, including also experimental tests that can be combined with the modeling approach. At the end, we discuss the prospects for patient-specific models when aiming to use finite element modeling analysis and evaluation of articular cartilage function, cellular responses, failure points, OA progression, and rehabilitation. PMID:23653665

  14. NADPH-oxidase-driven oxygen radical production determines chondrocyte death and partly regulates metalloproteinase-mediated cartilage matrix degradation during interferon-γ-stimulated immune complex arthritis

    PubMed Central

    van Lent, Peter LEM; Nabbe, Karin CAM; Blom, Arjen B; Sloetjes, Annet; Holthuysen, Astrid EM; Kolls, Jay; Van De Loo, Fons AJ; Holland, Steven M; Van Den Berg, Wim B

    2005-01-01

    In previous studies we have found that FcγRI determines chondrocyte death and matrix metalloproteinase (MMP)-mediated cartilage destruction during IFN-γ-regulated immune complex arthritis (ICA). Binding of immune complexes (ICs) to FcγRI leads to the prominent production of oxygen radicals. In the present study we investigated the contribution of NADPH-oxidase-driven oxygen radicals to cartilage destruction by using p47phox-/- mice lacking a functional NADPH oxidase complex. Induction of a passive ICA in the knee joints of p47phox-/- mice resulted in a significant elevation of joint inflammation at day 3 when compared with wild-type (WT) controls as studied by histology. However, when IFN-γ was overexpressed by injection of adenoviral IFN-γ in the knee joint before ICA induction, a similar influx of inflammatory cells was found at days 3 and 7, comprising mainly macrophages in both mouse strains. Proteoglycan depletion from the cartilage layers of the knee joints in both groups was similar at days 3 and 7. Aggrecan breakdown in cartilage caused by MMPs was further studied by immunolocalisation of MMP-mediated neoepitopes (VDIPEN). VDIPEN expression in the cartilage layers of arthritic knee joints was markedly lower (between 30 and 60%) in IFN-γ-stimulated arthritic p47phox-/- mice at day 7 than in WT controls, despite significant upregulation of mRNA levels of various MMPs such as MMP-3, MMP-9, MMP-12 and MMP-13 in synovia and MMP-13 in cartilage layers as measured with quantitative RT-PCR. The latter observation suggests that oxygen radicals are involved in the activation of latent MMPs. Chondrocyte death, determined as the percentage of empty lacunae in articular cartilage, ranged between 20 and 60% at day 3 and between 30 and 80% at day 7 in WT mice, and was completely blocked in p47phox-/- mice at both time points. FcγRI mRNA expression was significantly lower, and FcγRII and FcγRIII were higher, in p47phox-/- mice than in controls. NADPH

  15. Wnt7a Inhibits IL-1β Induced Catabolic Gene Expression and Prevents Articular Cartilage Damage in Experimental Osteoarthritis

    PubMed Central

    Gibson, Averi L.; Hui Mingalone, Carrie K.; Foote, Andrea T.; Uchimura, Tomoya; Zhang, Ming; Zeng, Li

    2017-01-01

    Wnt7a is a protein that plays a critical role in skeletal development. However, its effect on cartilage homeostasis under pathological conditions is not known. In this study, we found a unique inverse correlation between Wnt7a gene expression and that of MMP and IL-1β in individual human OA cartilage specimens. Upon ectopic expression in primary human articular chondrocytes, Wnt7a inhibited IL-1β-induced MMP and iNOS gene expression. Western blot analysis indicated that Wnt7a induced both canonical Wnt signaling and NFAT and Akt non-canonical signaling. Interestingly, inhibiting the canonical and Akt pathway did not affect Wnt7a activity. However, inhibiting the NFAT pathway impaired Wnt7a’s ability to inhibit MMP expression, suggesting that Wnt7a requires NFAT signaling to exert this function. In vivo, intraarticular injection of lentiviral Wnt7a strongly attenuated articular cartilage damage induced by destabilization of the medial meniscus (DMM) OA-inducing surgery in mice. Consistently, Wnt7a also inhibited the progressive increase of joint MMP activity in DMM animals. These results indicate that Wnt7a signaling inhibits inflammatory stimuli-induced catabolic gene expression in human articular chondrocytes and is sufficient to attenuate MMP activities and promote joint cartilage integrity in mouse experimental OA, demonstrating a novel effect of Wnt7a on regulating OA pathogenesis. PMID:28165497

  16. Human adult chondrocytes express hepatocyte growth factor (HGF) isoforms but not HgF: potential implication of osteoblasts on the presence of HGF in cartilage.

    PubMed

    Guévremont, Melanie; Martel-Pelletier, Johanne; Massicotte, Frédéric; Tardif, Ginette; Pelletier, Jean-Pierre; Ranger, Pierre; Lajeunesse, Daniel; Reboul, Pascal

    2003-06-01

    HGF is increased in human OA cartilage, possibly from Ob's. RT-PCR shows HGF isoforms are differently regulated between chondrocytes and Ob. A paracrine cross-talk between subchondral bone and cartilage may occur during OA. Recently, hepatocyte growth factor (HGF) has been identified by immunohistochemistry in cartilage and more particularly in the deep zone of human osteoarthritic (OA) cartilage. By investigating HGF expression in cartilage, we found that chondrocytes did not express HGF; however, they expressed the two truncated isoforms, namely HGF/NK1 and HGF/NK2. Because the only other cells localized near the deep zone are osteoblasts from the subchondral bone plate, we hypothesized that they were expressing HGF. Indeed, we found that HGF was synthesized by osteoblasts from the subchondral bone plate. Moreover, OA osteoblasts produced five times more HGF than normal osteoblasts and almost no HGF/NK1, unlike normal osteoblasts. Because prostaglandin E2 (PGE2) and pro-inflammatory cytokines such as interleukin (IL)-1 and IL-6 are involved in OA progression, we investigated whether these factors impact HGF produced by normal osteoblasts. PGE2 was the only factor tested that was able to stimulate HGF synthesis. However, the addition of NS398, a selective inhibitor of cyclo-oxygenase-2 (COX-2) had no effect on HGF produced by OA osteoblasts. HGF/NK2 had a moderate stimulating effect on HGF production by normal osteoblasts, whereas osteocalcin was not modulated by either HGF or HGF/NK2. When investigating signaling routes that might be implicated in OA osteoblast-produced HGF, we found that protein kinase A was at least partially involved. In summary, this study raises the hypothesis that the HGF found in articular cartilage is produced by osteoblasts, diffuses into the cartilage, and may be implicated in the OA process.

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2016-01-01

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

  19. Human osteoarthritic chondrons outnumber patient- and joint-matched chondrocytes in hydrogel culture - future application in autologous cell-based OA cartilage repair?

    PubMed

    Rothdiener, Miriam; Uynuk-Ool, Tatiana; Südkamp, Norbert; Aurich, Matthias; Grodzinsky, Alan J; Kurz, Bodo; Rolauffs, Bernd

    2017-07-17

    Autologous chondrocyte implantation (ACI) is used in 34-60% for osteoarthritic (OA) cartilage defects, although ACI is neither recommended nor designed for OA. Envisioning a hydrogel-based ACI for OA that uses chondrons instead of classically used chondrocytes, we hypothesized that human OA-chondrons may outperform OA-chondrocytes. We compared patient- and joint surface-matched human OA-chondrons vs. OA-chondrocytes cultured for the first time in a hydrogel, using a self-assembling peptide system. We determined yield, viability, cell numbers, mRNA expression, GAPDH mRNA enzyme activity, collagen II synthesis (CPII) and degradation (C2C), and sGAG. Ex vivo, mRNA expression was comparable. Over time, significant differences in survival led to 3.4-fold higher OA-chondron numbers in hydrogels after two weeks (p=0.002). Significantly more enzymatically active GAPDH protein indicated higher metabolic activity. The number of cultures that expressed mRNA for collagen types I, VI, COMP, aggrecan, VEGF, TGF-β1, and FGF-2 (but not collagen types II, X) was different, resulting in a 3.5-fold higher number of expression-positive OA-chondron cultures (p<0.05). Measuring CPII and C2C per hydrogel, OA-chondron hydrogels synthesized more than they degraded collagen type II, the opposite was true for OA-chondrocytes. Per cell, OA-chondrons but not OA-chondrocytes displayed more synthesis than degradation. Thus, OA-chondrons displayed superior biosynthesis and mRNA expression of tissue engineering and phenotype-relevant genes. Moreover, human OA-chondrons displayed a significant survival advantage in hydrogel culture, whose presence, drastic extent, and time scale was novel and is clinically significant. Collectively, these data highlight the high potential of human OA-chondrons for OA-ACI, as they would outnumber and, thus, surpass OA-chondrocytes. This article is protected by copyright. All rights reserved.

  20. Limited Immunogenicity of Human Induced Pluripotent Stem Cell-Derived Cartilages

    PubMed Central

    Kimura, Takeshi; Yamashita, Akihiro; Ozono, Keiichi

    2016-01-01

    Articular cartilage damage does not spontaneously heal and could ultimately result in a loss of joint function. Damaged cartilage can be repaired with cell/tissue sources that are transplanted, however, autologous chondrocytes are limited in number as a cell source. Induced pluripotent stem cells (iPSCs) are a relatively new and abundant cell source and can be made from the patient, but at a considerable cost. Because cartilage is immunoprivileged tissue, allogeneic cartilages have been transplanted effectively without matching for human leukocyte antigen (HLA), but are difficult to acquire due to scarcity of donors. In this study, we examined the immunogenicity of human iPSC-derived cartilages (hiPS-Carts) in vitro to evaluate whether allogeneic hiPS-Carts can be a new cell/tissue source. The cells in hiPS-Carts expressed limited amounts of major histocompatibility complex (MHC) class I (HLA-ABC) and MHC class II (HLA-DRDQDP). Treatment with interferon γ (IFNγ) induced the expression of MHC class I, but not MHC class II in hiPS-Carts. A mixed lymphocyte reaction assay showed that hiPS-Carts stimulated the proliferation of neither T cells nor the activation of NK cells. Furthermore, hiPS-Carts suppressed the proliferation of T cells stimulated with interleukin 2 and phytohemagglutinin (PHA). Together with previously reported findings, these results suggest that hiPS-Carts are no more antigenic than human cartilage. Additionally, in combination with the fact that iPSCs are unlimitedly expandable and thus can supply unlimited amounts of iPS-Carts from even one iPSC line, they suggest that allogeneic hiPS-Carts are a candidate source for transplantation to treat articular cartilage damage. PMID:27762664

  1. Unilateral anterior crossbite induces aberrant mineral deposition in degenerative temporomandibular cartilage in rats.

    PubMed

    Zhang, M; Wang, H; Zhang, J; Zhang, H; Yang, H; Wan, X; Jing, L; Lu, L; Liu, X; Yu, S; Chang, W; Wang, M

    2016-05-01

    To investigate whether mechanical stress induces mineral deposits that contribute to matrix degradation at the onset of osteoarthritis (OA) in temporomandibular joint (TMJ) cartilage. Female Spraguee-Dawley rats were subjected to an unilateral anterior crossbite (UAC) procedure. Histology, electron microscopy, and energy dispersive spectrometer (EDS) were used to examine cartilage matrix structures and composition of mineral deposit in the affected TMJ cartilage. Protein and/or RNA expression of phenotypic markers and mineralization modulators and matrix degradation was analyzed by immunohistochemistry and/or real-time PCR. Synthetic basic calcium phosphate (BCP) and calcium pyrophosphate dehydrate (CPPD) crystals were used to stimulate ATDC5 cells for their impact on cell differentiation and gene expression. Fragmented and disorganized collagen fibers, expanded fibrous spaces, and enhancement of matrix vesicle production and mineral deposition were observed in matrices surrounding hypertrophic chondrocytes in cartilage as early as 2-weeks post-UAC and exacerbated with time. The mineral deposits in TMJ cartilage at 12- and 20-weeks post-UAC had Ca/P ratios of 1.42 and 1.44, which are similar to the ratios for BCP. The expression of mineralization inhibitors, NPP1, ANK, CD73, and Matrix gla protein (MGP) was decreased from 2 to 8 weeks post-UAC, so were the chondrogenic markers, Col-2, Col-X and aggrecan. In contrast, the expression of tissue-nonspecific alkaline phosphatase (TNAP) and MMP13 was increased 4-weeks post-UAC. Treating ADTC5 cells with BCP crystals increased MMPs and ADAMTS5 expression, but reduced matrix production in a time-dependent manner. UAC induces deposition of BCP-like minerals in osteoarthritic cartilage, which can stimulate matrix degradation by promoting the expression of cartilage-degrading enzymes to facilitate OA progression. Copyright © 2015 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.

  2. Cartilage graft engineering by co-culturing primary human articular chondrocytes with human bone marrow stromal cells.

    PubMed

    Sabatino, Maria Antonietta; Santoro, Rosaria; Gueven, Sinan; Jaquiery, Claude; Wendt, David James; Martin, Ivan; Moretti, Matteo; Barbero, Andrea

    2015-12-01

    Co-culture of mesenchymal stromal cells (MSCs) with articular chondrocytes (ACs) has been reported to improve the efficiency of utilization of a small number of ACs for the engineering of implantable cartilaginous tissues. However, the use of cells of animal origin and the generation of small-scale micromass tissues limit the clinical relevance of previous studies. Here we investigated the in vitro and in vivo chondrogenic capacities of scaffold-based constructs generated by combining primary human ACs with human bone marrow MSCs (BM-MSCs). The two cell types were cultured in collagen sponges (2 × 6 mm disks) at the BM-MSCs:ACs ratios: 100:0, 95:5, 75:25 and 0:100 for 3 weeks. Scaffolds freshly seeded or further precultured in vitro for 2 weeks were also implanted subcutaneously in nude mice and harvested after 8 or 6 weeks, respectively. Static co-culture of ACs (25%) with BM-MSCs (75%) in scaffolds resulted in up to 1.4-fold higher glycosaminoglycan (GAG) content than what would be expected based on the relative percentages of the different cell types. In vivo GAG induction was drastically enhanced by the in vitro preculture and maximal at the ratio 95:5 (3.8-fold higher). Immunostaining analyses revealed enhanced accumulation of type II collagen and reduced accumulation of type X collagen with increasing ACs percentage. Constructs generated in the perfusion bioreactor system were homogeneously cellularized. In summary, human cartilage grafts were successfully generated, culturing BM-MSCs with a relatively low fraction of non-expanded ACs in porous scaffolds. The proposed co-culture strategy is directly relevant towards a single-stage surgical procedure for cartilage repair. Copyright © 2012 John Wiley & Sons, Ltd.

  3. Synthesis rates and binding kinetics of matrix products in engineered cartilage constructs using chondrocyte-seeded agarose gels.

    PubMed

    Nims, Robert J; Cigan, Alexander D; Albro, Michael B; Hung, Clark T; Ateshian, Gerard A

    2014-06-27

    Large-sized cartilage constructs suffer from inhomogeneous extracellular matrix deposition due to insufficient nutrient availability. Computational models of nutrient consumption and tissue growth can be utilized as an efficient alternative to experimental trials to optimize the culture of large constructs; models require system-specific growth and consumption parameters. To inform models of the [bovine chondrocyte]-[agarose gel] system, total synthesis rate (matrix accumulation rate+matrix release rate) and matrix retention fractions of glycosaminoglycans (GAG), collagen, and cartilage oligomeric matrix protein (COMP) were measured either in the presence (continuous or transient) or absence of TGF-β3 supplementation. TGF-β3's influences on pyridinoline content and mechanical properties were also measured. Reversible binding kinetic parameters were characterized using computational models. Based on our recent nutrient supplementation work, we measured glucose consumption and critical glucose concentration for tissue growth to computationally simulate the culture of a human patella-sized tissue construct, reproducing the experiment of Hung et al. (2003). Transient TGF-β3 produced the highest GAG synthesis rate, highest GAG retention ratio, and the highest binding affinity; collagen synthesis was elevated in TGF-β3 supplementation groups over control, with the highest binding affinity observed in the transient supplementation group; both COMP synthesis and retention were lower than those for GAG and collagen. These results informed the modeling of GAG deposition within a large patella construct; this computational example was similar to the previous experimental results without further adjustments to modeling parameters. These results suggest that these nutrient consumption and matrix synthesis models are an attractive alternative for optimizing the culture of large-sized constructs.

  4. Cartilage ultrastructure after high pressure freezing, freeze substitution, and low temperature embedding. I. Chondrocyte ultrastructure--implications for the theories of mineralization and vascular invasion

    PubMed Central

    1984-01-01

    Electron microscopic examination of epiphyseal cartilage tissue processed by high pressure freezing, freeze substitution, and low temperature embedding revealed a substantial improvement in the preservation quality of intracellular organelles by comparison with the results obtained under conventional chemical fixation conditions. Furthermore, all cells throughout the epiphyseal plate, including the terminal chondrocyte adjacent to the region of vascular invasion, were found to be structurally integral. A zone of degenerating cells consistently observed in cartilage tissue processed under conventional chemical fixation conditions was not apparent. Hence, it would appear that cell destruction in this region occurs during chemical processing and is not a feature of cartilage tissue in the native state. Since these cells are situated in a region where tissue calcification is taking place, the implication is that the onset and progression of cartilage calcification are, at least partially, controlled by the chondrocytes themselves. The observation that the terminal cell adjacent to the zone of vascular invasion is viable has important implications in relation to the theory of vascular invasion. This may now require reconceptualization to accommodate the possibility that active cell destruction may be a precondition for vascular invasion. PMID:6707090

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

    PubMed Central

    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

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

    PubMed Central

    Chien, Szu-Yu; Huang, Chun-Yin; Tsai, Chun-Hao; Wang, Shih-Wei

    2016-01-01

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

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

    SciTech Connect

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

    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.

  8. Interleukin 1 suppresses expression of cartilage-specific types II and IX collagens and increases types I and III collagens in human chondrocytes.

    PubMed Central

    Goldring, M B; Birkhead, J; Sandell, L J; Kimura, T; Krane, S M

    1988-01-01

    In inflammatory diseases such as rheumatoid arthritis, functions of chondrocytes including synthesis of matrix proteins and proteinases are altered through interactions with cells of the infiltrating pannus. One of the major secreted products of mononuclear inflammatory cells is IL-1. In this study we found that recombinant human IL-1 beta suppressed synthesis of cartilage-specific type II collagen by cultured human costal chondrocytes associated with decreased steady state levels of alpha 1 (II) and alpha 1(IX) procollagen mRNAs. In contrast, IL-1 increased synthesis of types I and III collagens and levels of alpha 1(I), alpha 2(I), and alpha 1(III) procollagen mRNAs, as we described previously using human articular chondrocytes and synovial fibroblasts. This stimulatory effect of IL-1 was observed only when IL-1-stimulated PGE2 synthesis was blocked by the cyclooxygenase inhibitor indomethacin. The suppression of type II collagen mRNA levels by IL-1 alone was not due to IL-1-stimulated PGE2, since addition of indomethacin did not reverse, but actually potentiated, this inhibition. Continuous exposure of freshly isolated chondrocytes from day 2 of culture to approximately half-maximal concentrations of IL-1 (2.5 pM) completely suppressed levels of type II collagen mRNA and increased levels of types I and III collagen mRNAs, thereby reversing the ratio of alpha 1(II)/alpha 1(I) procollagen mRNAs from greater than 6.0 to less than 1.0 by day 7. IL-1, therefore, can modify, at a pretranslational level, the relative amounts of the different types of collagen synthesized in cartilage and thereby could be responsible for the inappropriate repair of cartilage matrix in inflammatory conditions. Images PMID:3264290

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

    PubMed

    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

  10. Autologous chondrocyte implantation for the treatment of cartilage lesions of the knee: a systematic review of randomized studies.

    PubMed

    Vasiliadis, Haris S; Wasiak, Jason; Salanti, Georgia

    2010-12-01

    Autologous chondrocyte implantation (ACI) techniques are becoming more popular for the treatment of full thickness cartilage lesions of the knee joint. However, there is no systematic information for the efficacy of the new generation ACI techniques compared to other treatment options. A systematic review of the existing evidence from randomized clinical trials of ACI treatment would contribute to understanding the advantages and limitations of this method and would inform the planning of future studies. Using pre-defined criteria, we searched a number of electronic databases to identify all the existing randomized control trials of any type of ACI treatment. Risk of bias was assessed and an analysis of the reported outcomes was performed. Information on the clinical efficacy and safety of ACI compared to other interventions was collected and presented. Nine trials were identified with 626 patients. Patients ranged from 15 to 52 years, and the size of treated lesions was between 1 and 22 cm(2). ACI was associated with improvement in clinical outcomes compared to baseline. However, the body of evidence did not suggest any superiority of ACI over other treatments. Complication rates were comparable between interventions except from an increased rate of graft hypertrophies after ACI with periosteum. ACI is an effective treatment for full thickness chondral defects of the knee, providing an improvement of clinical outcomes. However, there is insufficient data to say whether ACI is superior to other treatment strategies. More high quality studies and harmonization in the reported outcomes are needed before specific suggestions for practice can be made.

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

    PubMed

    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.

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

    PubMed

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

    2012-08-01

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

  13. Ursodeoxycholic Acid ameliorates pain severity and cartilage degeneration in monosodium iodoacetate-induced osteoarthritis in rats.

    PubMed

    Moon, Su-Jin; Jeong, Jeong-Hee; Jhun, Joo Yeon; Yang, Eun Ji; Min, Jun-Ki; Choi, Jong Young; Cho, Mi-La

    2014-02-01

    Osteoarthritis (OA) is a degenerative joint disease characterized by a progressive loss of cartilage. And, increased oxidative stress plays a relevant role in the pathogenesis of OA. Ursodeoxycholic acid (UDCA) is a used drug for liver diseases known for its free radical-scavenging property. The objectives of this study were to investigate the in vivo effects of UDCA on pain severity and cartilage degeneration using an experimental OA model and to explore its mode of actions. OA was induced in rats by intra-articular injection of monosodium iodoacetate (MIA) to the knee. Oral administration UDCA was initiated on the day of MIA injection. Limb nociception was assessed by measuring the paw withdrawal latency and threshold. Samples were analyzed macroscopically and histologically. Immunohistochemistry was used to investigate the expression of interleukin-1β (IL-1β), IL-6, nitrotyrosine and inducible nitric oxide synthase (iNOS) in knee joints. UDCA showed an antinociceptive property and attenuated cartilage degeneration. OA rats given oral UDCA significantly exhibited a decreased number of osteoclasts in subchondral bone legion compared with the vehicle-treated OA group. UDCA reduced the expression of IL-1β, IL-6, nitrotyrosine and iNOS in articular cartilage. UDCA treatment significantly attenuated the mRNA expression of matrix metalloproteinase-3 (MMP-3), -13, and ADAMTS5 in IL-1β-stimulated human OA chondrocytes. These results show the inhibitory effects of UDCA on pain production and cartilage degeneration in experimentally induced OA. The chondroprotective properties of UDCA were achieved by suppressing oxidative damage and inhibiting catabolic factors that are implicated in the pathogenesis of cartilage damage in OA.

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

    PubMed

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

    2014-09-01

    Calcification of end plate chondrocytes is a major cause of intervertebral disc (IVD) degeneration. However, the underlying molecular mechanism of end plate chondrocyte calcification is still unclear. The aim of this study was to clarify whether autophagy in end plate chondrocytes could protect the calcification of end plate chondrocytes. Previous studies showed that intermittent cyclic mechanical tension (ICMT) contributes to the calcification of end plate chondrocytes in vitro. While autophagy serves as a cell survival mechanism, the relationship of autophagy and induced end plate chondrocyte calcification by mechanical tension in vitro is unknown. Thus, we investigated autophagy, the expression of the autophagy genes, Beclin-1 and LC3, and rat end plate chondrocyte calcification by ICMT. The viability of end plate chondrocytes was examined using the LIVE/DEAD viability/cytotoxicity kit. The reverse transcription-polymerase chain reaction and western blotting were used to detect the expression of Beclin-1; LC3; type I, II and X collagen; aggrecan; and Sox-9 genes. Immunofluorescent and fluorescent microscopy showed decreased autophagy in the 10- and 20-day groups loaded with ICMT. Additionally, Alizarin red and alkaline phosphatase staining detected the palpable calcification of end plate chondrocytes after ICMT treatment. We found that increased autophagy induced by short-term ICMT treatment was accompanied by an insignificant calcification of end plate chondrocytes. To the contrary, the suppressive autophagy inhibited by long-term ICMT was accompanied by a more significant calcification. The process of calcification induced by ICMT was partially resisted by increased autophagy activity induced by rapamycin, implicating that autophagy may prevent end plate chondrocyte calcification. Copyright © 2014 Elsevier Inc. All rights reserved.

  15. Bone and cartilage repair by transplantation of induced pluripotent stem cells in murine joint defect model.

    PubMed

    Uto, Sakura; Nishizawa, Satoru; Takasawa, Yutaka; Asawa, Yukiyo; Fujihara, Yuko; Takato, Tsuyoshi; Hoshi, Kazuto

    2013-01-01

    The establishment of cartilage regenerative medicine has been an important issue in the clinical field, because cartilage has the poor ability of self-repair. Currently, tissue engineering using autologous chondrocytes has risen, but we should investigate more appropriate cell sources that can be obtained without any quantitative limitation. In this study, we focused on induced pluripotent stem (iPS) cells, in which the ethical hurdle does not seem higher than that of embryonic stem cells. Mouse iPS cells were transplanted into the mouse joint defect model of the knee. Strains of the transplants and hosts were arranged to be either closest (homology 75% in genetic background) or identical (100%). For transplantation, we embedded the iPS cells within the collagen hydrogel in order to obtain the effective administration of the cells into defects, which induced the differentiation of the iPS cells. At 8 weeks of transplantation, although the iPS cells with a 75% homology to the host in the genetic background tended to form teratoma, those of 100% showed a joint regeneration. GFP immunohistochemistry proved that the transplanted iPS cells were responsible for the bone and cartilage repair. Taking these results together, the iPS cells are regarded as a promising cell source for the cartilage tissue engineering.

  16. The high affinity ALK1-ligand BMP9 induces a hypertrophy-like state in chondrocytes that is antagonized by TGFβ1.

    PubMed

    van Caam, A; Blaney Davidson, E; Garcia de Vinuesa, A; van Geffen, E; van den Berg, W; Goumans, M-J; ten Dijke, P; van der Kraan, P

    2015-06-01

    In osteoarthritic cartilage, expression of the receptor ALK1 correlates with markers of deleterious chondrocyte hypertrophy. Recently, bone morphogenetic protein 9 (BMP9) was identified as a high affinity ligand for ALK1. Therefore, we studied if BMP9 signaling results in expression of hypertrophy markers in chondrocytes. Furthermore, because transforming growth factorß1 (TGFβ1) is a well known anti-hypertrophic factor, the interaction between BMP9 and TGFβ1 signaling was also studied. Primary chondrocytes were isolated from bovine cartilage and stimulated with BMP9 and/or TGFβ1 to measure intracellular signaling via pSmads with the use of Western blot. Expression of Smad-responsive genes or hypertrophy-marker genes was measured using qPCR. To confirm observations on TGFβ/Smad3 responsive genes, a Smad3-dependent CAGA12-luc transcriptional reporter assay was performed in the chondrocyte G6 cell line. In primary chondrocytes, BMP9 potently induced phosphorylation of Smad1/5 and Smad2 to a lesser extent. BMP9-induced Smad1/5 phosphorylation was rapidly (2 h) reflected in gene expression, whereas Smad2 phosphorylation was not. Remarkably, BMP9 and TGFβ1 dose-dependently synergized on Smad2 phosphorylation, and showed an additive effect on expression of Smad3-dependent genes like bSerpine1 after 24 h. The activation of the TGFβ/Smad3 signaling cascade was confirmed using the CAGA12-luc transcriptional reporter. BMP9 selectively induced bAlpl and bColX expression, which are considered early markers of cellular hypertrophy, but this was potently antagonized by addition of a low dose of TGFβ1. This study shows that in vitro in chondrocytes, BMP9 potently induces pSmad1/5 and a chondrocyte hypertrophy-like state, which is potently blocked by TGFβ1. This observation underlines the importance of TGFβ1 in maintenance of chondrocyte phenotype. Copyright © 2015 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.

  17. Hydromechanical stimulator for chondrocyte-seeded constructs in articular cartilage tissue engineering applications.

    PubMed

    Pourmohammadali, Homeyra; Chandrashekar, Naveen; Medley, John B

    2013-03-01

    Mechanical stimulation is a key technique used for controlling the mechanical properties of tissue engineered articular cartilage constructs proposed for defect repair. The present study introduces a new technical method and device for 'hydromechanical' stimulation of tissue engineered articular cartilage constructs. The stimulation consists of simultaneous cyclic compression, frictional shear from a sliding indenter contact and direct pressurized fluid perfusion. Each of these modes of mechanical loading has been shown by other research groups to effectively stimulate tissue engineered constructs. A device for applying these conditions was designed, developed and tested. Two sets (high and low perfusion flow rates) of three experiments were performed, each with two samples subjected to hydromechanical stimulation conditions (compression and friction forces along with perfusion). Two other samples from each set were subjected to just compression and dynamic frictional shear forces, and two more were used as controls (not stimulated). The average amount of glycosaminoglycan retained in the constructs after 3 weeks ranked from low to high as follows: controls, hydromechanical conditions with the low-flow rate, hydromechanical conditions with the high-flow rate and just compression plus dynamic frictional shear. Statistically significant differences were not detected. However, future studies would focus on glycosaminoglycan production in the superficial zone, measuring the glycosaminoglycan released to the nutrient media, and address altering the hydromechanical stimulation parameters using the results of the present study as guidance, in attempts to achieve statistically significant increases in glycosaminoglycan production compared with the controls.

  18. Salvianolic acid B inhibits IL-1β-induced inflammatory cytokine production in human osteoarthritis chondrocytes and has a protective effect in a mouse osteoarthritis model.

    PubMed

    Lou, Yiting; Wang, Chenggui; Zheng, Wenhao; Tang, Qian; Chen, Yu; Zhang, Xiaolei; Guo, Xiaoshan; Wang, Jianshun

    2017-05-01

    Osteoarthritis (OA) is a chronic progressive disease that has complicated mechanisms that involve inflammation and cartilage degradation. In this study, we investigated the anti-inflammatory action of Salvianolic acid B (Sal B) in both human OA chondrocytes and a mouse OA model that was induced by destabilization of the medial meniscus. In vitro, chondrocytes were pretreated with Sal B (0, 25, 50, 100μM) for 2h, then incubated with IL-1β (10ng/mL) for 24h. NO production was determined by Griess method and PGE2 was assessed by ELISA. The expression of INOS, COX-2, MMP-13, ADAMTS-5 and NF-κB-related signaling molecules were tested by Western blotting. Immunofluorescence staining was used to detect P65 nuclear translocation. In vivo, the mouse OA model received intraperitoneal-injection of either Sal B (25mg/kg) or saline every other day. Hematoxylin and Eosin, as well as Safranin-O-Fast green staining, were utilized to evaluate the severity of cartilage lesions up to 8weeks following the surgery. Sal B inhibited the over-production of NO and PGE2, while the elevated expression of INOS, COX-2, MMP-13 and ADAMTS-5 were reversed by Sal B in IL-1β-induced chondrocytes. In addition, IL-1β significantly induced phosphorylation of NF-κB signaling, and this phosphorylation response was blocked by Sal B. Immunofluorescence staining demonstrated that Sal B could suppress IL-1β-induced p65 nuclear translocation. In vivo, the cartilage in Sal B-treated mice exhibited less cartilage degradation and lower OARSI scores. Taken together, Sal B possesses great potential value as a therapeutic agent for OA treatment.

  19. [Repair of articular cartilage defects by autologous bone mesenchymal stem cells and allogeneic costal chondrocytes in the knee of Wuzhishan miniature pigs].

    PubMed

    Yang, Cheng; Ni, Jiangdong; Zhang, Shou; Fan, Zhongcheng

    2017-08-28

    To investigate the feasibility of construction of tissue engineered cartilage by co-culture of bone marrow mesenchymal stem cells (BMSCs) and costal chondrocytes (CCs), and to provide theoretical basis and experimental basis for clinical repair of articular cartilage defects by Wuzhishan miniature pig knee cartilage defects with co-cultured cells.
 Methods: Density gradient centrifugation method was used to isolate BMSCs from Wuzhishan miniature pig. The double enzyme digestion method was used to isolate CCs. The passage 3 generation of BMSCs and passage 2 generation of CCs were randomly divided into 3 groups: a co-culture group of BMSCs:CCs for 1:2 (Group A), a simple CCs (Group B), and a simple BMSCs (Group C). The cell growth curve was drawn, and the content of glycosaminoglycan (GAG) of external separation in chondrocytes was determined. The 12 Wuzhishan miniature pigs were randomly divided into a co-culture cells/collagen membrane experimental group, a collagen membrane control group and the blank group. In the co-culture cells/collagen membrane experimental group, the co-cultured cells/collagen membrane were implanted into the cartilage defects of the mandibular condyle; in the collagen membrane control group, only collagen membrane was implanted; while in the blank group, nothing was implanted. Six animals were sacrificed at 8 and 16 weeks after surgery respectively (2 animals in each group). General observation, cartilage histological score and histopathological examination were carried out.
 Results: The BMSCs and co-culture cells grew well. The biological activity of CCs was good. After 16 weeks of operation, the repair tissues in the co-cultured cells/collagen membrane experimental group showed hyaline cartilage features: smooth, flat, and integrated well with the surrounding cartilage and subchondral bone. The collagen membrane in the collagen membrane control group was fibrously repaired. Repair tissue gross score in the co-culture cells

  20. Cartilage Turnover Reflected by Metabolic Processing of Type II Collagen: A Novel Marker of Anabolic Function in Chondrocytes

    PubMed Central

    Gudmann, Natasja Stæhr; Wang, Jianxia; Hoielt, Sabine; Chen, Pingping; Siebuhr, Anne Sofie; He, Yi; Christiansen, Thorbjørn G.; Karsdal, Morten Asser; Bay-Jensen, Anne Christine

    2014-01-01

    The aim of this study was to enable measurement of cartilage formation by a novel biomarker of type II collagen formation. The competitive enzyme-linked immunosorbent assay (ELISA) Pro-C2 was developed and characterized for assessment of the beta splice variant of type II procollagen (PIIBNP). This is expected to originate primarily from remodeling of hyaline cartilage. A mouse monoclonal antibody (Mab) was raised in mouse, targeting specifically PIIBNP (QDVRQPG) and used in development of the assay. The specificity, sensitivity, 4-parameter fit and stability of the assay were tested. Levels of PIIBNP were quantified in human serum (0.6–2.2 nM), human amniotic fluid (163–188 nM) and sera from different animal species, e.g., fetal bovine serum (851–901 nM) with general good linearity (100% (SD 7.6) recovery) and good intra- and inter-assay variation (CV% < 10). Dose (0.1 to 100 ng/mL) and time (7, 14 and 21 days) dependent release of PIIBNP were evaluated in the conditioned medium from bovine cartilage explants (BEX) and human cartilage explants (HEX) upon stimulation with insulin-like growth factor (IGF-1), transforming growth factor (TGF)-β1 and fibroblastic growth factor-2 (FGF-2). TGF-β1 and IGF-1 in concentrations of 10–100 ng/mL significantly (p < 0.05) induced release of PIIBNP in BEX compared to conditions without treatment (WO). In HEX, IGF-1 100 ng/mL was able to induce a significant increase of PIIBNP after one week compared to WO. FGF-2 did not induce a PIIBNP release in our models. To our knowledge this is the first assay, which is able to specifically evaluate PIIBNP excretion. The Pro-C2 assay seems to provide a promising and novel marker of type II collagen formation. PMID:25329619

  1. Cartilage turnover reflected by metabolic processing of type II collagen: a novel marker of anabolic function in chondrocytes.

    PubMed

    Gudmann, Natasja Stæhr; Wang, Jianxia; Hoielt, Sabine; Chen, Pingping; Siebuhr, Anne Sofie; He, Yi; Christiansen, Thorbjørn G; Karsdal, Morten Asser; Bay-Jensen, Anne Christine

    2014-10-17

    The aim of this study was to enable measurement of cartilage formation by a novel biomarker of type II collagen formation. The competitive enzyme-linked immunosorbent assay (ELISA) Pro-C2 was developed and characterized for assessment of the beta splice variant of type II procollagen (PIIBNP). This is expected to originate primarily from remodeling of hyaline cartilage. A mouse monoclonal antibody (Mab) was raised in mouse, targeting specifically PIIBNP (QDVRQPG) and used in development of the assay. The specificity, sensitivity, 4-parameter fit and stability of the assay were tested. Levels of PIIBNP were quantified in human serum (0.6-2.2 nM), human amniotic fluid (163-188 nM) and sera from different animal species, e.g., fetal bovine serum (851-901 nM) with general good linearity (100% (SD 7.6) recovery) and good intra- and inter-assay variation (CV% < 10). Dose (0.1 to 100 ng/mL) and time (7, 14 and 21 days) dependent release of PIIBNP were evaluated in the conditioned medium from bovine cartilage explants (BEX) and human cartilage explants (HEX) upon stimulation with insulin-like growth factor (IGF-1), transforming growth factor (TGF)-β1 and fibroblastic growth factor-2 (FGF-2). TGF-β1 and IGF-1 in concentrations of 10-100 ng/mL significantly (p < 0.05) induced release of PIIBNP in BEX compared to conditions without treatment (WO). In HEX, IGF-1 100 ng/mL was able to induce a significant increase of PIIBNP after one week compared to WO. FGF-2 did not induce a PIIBNP release in our models. To our knowledge this is the first assay, which is able to specifically evaluate PIIBNP excretion. The Pro-C2 assay seems to provide a promising and novel marker of type II collagen formation.

  2. Regulation of Articular Chondrocyte Proliferation and Differentiation by Indian Hedgehog and Parathyroid Hormone-related Protein

    PubMed Central

    Chen, Xuesong; Macica, Carolyn; Nasiri, Ali; Broadus, Arthur E.

    2008-01-01

    Objective The chondrocytes of the epiphyseal growth zone are regulated by the Indian hedgehog (Ihh)-parathyroid hormone-related protein (PTHrP) axis. In weight-bearing joints, this growth zone comes to be subdivided by the secondary ossification center into distinct articular and growth cartilage structures. Here, we explored the cells of origin, localization, regulation of expression, and putative functions of Ihh and PTHrP in articular cartilage in the mouse. Methods We assessed Ihh and PTHrP expression in an allelic PTHrP-lacZ knockin mouse and several versions of PTHrP-null mice. Selected joints were unloaded surgically to examine load-induction of PTHrP and Ihh. Results The embryonic growth zone appears to serve as the source of PTHrP-expressing proliferative chondrocytes that populate both the forming articular cartilage and growth plate structures. In articular cartilage, these cells take the form of articular chondrocytes in the mid-zone. In PTHrP-knockout mice, mineralizing chondrocytes encroach upon developing articular cartilage but appear to be prevented from mineralizing the joint space by Ihh-driven surface chondrocyte proliferation. In growing and adult mice, PTHrP expression in articular chondrocytes is load-induced, and unloading is associated with rapid changes in PTHrP expression and articular chondrocyte differentiation. Conclusion We conclude that the PTHrP-Ihh axis participates in the maintenance of articular cartilage. Dysregulation of this system might contribute to the pathogenesis of arthritis. PMID:19035497

  3. Reconstruction of Alar Nasal Cartilage Defects Using a Tissue Engineering Technique Based on a Combined Use of Autologous Chondrocyte Micrografts and Platelet-rich Plasma: Preliminary Clinical and Instrumental Evaluation

    PubMed Central

    Scioli, Maria G.; Bielli, Alessandra; Orlandi, Augusto; Cervelli, Valerio

    2016-01-01

    Background: Developing cartilage constructs with injectability, appropriate matrix composition, and persistent cartilaginous phenotype remains an enduring challenge in cartilage repair. The combined use of autologous chondrocyte micrografts and platelet-rich plasma (PRP) is an alternative that opens a new era in this field. Methods: At the Department of Plastic and Reconstructive Surgery, University of Rome Tor Vergata, Italy, 11 patients underwent nasal alar reconstruction with chondrocyte micrografts gently poured onto PRP in solid form. A computed tomographic scan control was performed after 12 months. Pearson’s Chi-square test was used to investigate difference in cartilage density between native and newly formed cartilages. Results: The constructs of chondrocyte micrografts–PRP that were subcutaneously injected resulted in a persistent cartilage tissue with appropriate morphology, adequate central nutritional perfusion without central necrosis or ossification, and further augmented nasal dorsum without obvious contraction and deformation. Conclusion: This report demonstrated that chondrocyte micrografts derived from nasal septum poured onto PRP in solid form are useful for cartilage regeneration in patients with external nasal valve collapse. PMID:27826462

  4. Oleic/Palmitate Induces Apoptosis in Human Articular Chondrocytes via Upregulation of NOX4 Expression and ROS Production.

    PubMed

    Fu, Dapeng; Lu, Jianmin; Yang, Sheng

    2016-07-01

    The association between obesity and reactive oxygen species (ROS) production in osteoarthritis (OA) patients has already been identified. However, the specific mechanism underlying ROS production and OA progression has never been elucidated. Osteoarthritic cartilage was obtained from patients undergoing total hip arthroplasty, and chondrocytes were isolated from these tissues. The cells were treated with varying concentrations (10,100,500 μM, and 5 mM) of oleic/palmitate (O/P) mixture at different times, that is at 6, 24, and 48 h. Cell viability was determined using MTT assay. ROS production was detected using immunofluorescence and flow cytometry. The protein levels of NOX4 and cleaved-caspase3 were detected using Western blot assay. O/P significantly decreased cell viability at 10, 100, 500 μM, and 5 mM in a dose-dependent manner. Furthermore, the cell viability was reduced by 500 μM O/P mixture at 6, 24, and 48 h in a time-dependent manner. Pretreatment with 500 μM O/P significantly enhanced ROS production and cell apoptosis in chondrocytes. Furthermore, treatment with O/P mixture significantly enhanced the expression of NOX4 and caspase3 activation in a dose- and time- dependent manner. More importantly, inhibition of NOX4 could partially eliminate O/P-induced chondrocytes apoptosis by reducing ROS production. To conclude, O/P treatment enhances ROS production and cell apoptosis mainly by upregulating the protein levels of NOX4 and caspase3 activation in chondrocytes, indicating a potential therapeutic target of OA in obesity patients.

  5. Expression and physiological role of CCN4/Wnt-induced secreted protein 1 mRNA splicing variants in chondrocytes.

    PubMed

    Yanagita, Takeshi; Kubota, Satoshi; Kawaki, Harumi; Kawata, Kazumi; Kondo, Seiji; Takano-Yamamoto, Teruko; Tanaka, Shinji; Takigawa, Masaharu

    2007-04-01

    CCN4/Wnt-induced secreted protein 1 (WISP1) is one of the CCN (CTGF/Cyr61/Nov) family proteins. CCN members have typical structures composed of four conserved cysteine-rich modules and their variants lacking certain modules, generated by alternative splicing or gene mutations, have been described in various pathological conditions. Several previous reports described a CCN4/WISP1 variant (WISP1v) lacking the second module in a few malignancies, but no information concerning the production of WISP1 variants in normal tissue is currently available. The expression of CCN4/WISP1 mRNA and its variants were analyzed in a human chondrosarcoma-derived chondrocytic cell line, HCS-2/8, and primary rabbit growth cartilage (RGC) chondrocytes. First, we found WISP1v and a novel variant of WISP1 (WISP1vx) to be expressed in HCS-2/8, as well as full-length WISP1 mRNA. This new variant was lacking the coding regions for the second and third modules and a small part of the first module. To monitor the expression of CCN4/WISP1 mRNA along chondrocyte differentiation, RGC cells were cultured and sampled until they were mineralized. As a result, we identified a WISP1v ortholog in normal RGC cells. Interestingly, the WISP1v mRNA level increased dramatically along with terminal differentiation. Furthermore, overexpression of WISP1v provoked expression of an alkaline phosphatase gene that is a marker of terminal differentiation in HCS-2/8 cells. These findings indicate that WISP1v thus plays a critical role in chondrocyte differentiation toward endochondral ossification, whereas HCS-2/8-specific WISP1vx may be associated with the transformed phenotypes of chondrosarcomas.

  6. [Chondrogenesis of passaged chondrocytes induced by different dynamic loads in bioreactor].

    PubMed

    Wang, Ning; Chen, Jiying; Zhang, Guoqiang; Chai, Wei

    2013-07-01

    To investigate the effect of dynamic compression and rotation motion on chondrogenesis of the 3rd passage cell-loaded three-dimensional scaffold in a joint-specific bioreactor in vitro so as to provide theoretical basis of the autologous chondrocyte transplantation in clinical practice. Primary chondrocytes were isolated and cultured from the knee cartilage of 3-4 months old calves. The 3rd passage cells were seeded onto fibrin-polyurethane scaffolds (8 mm x 4 mm). Experiment included 5 groups: unloaded culture for 2 weeks (group A), direct load for 2 weeks (group B), unloaded culture for 4 weeks (group C), direct load for 4 weeks (group D), and unload for 2 weeks followed by load for 2 weeks (group E). The cell-scaffold was incubated in incubator (unload) or in a joint-specific bioreactor (load culture). At different time points, the samples were collected for DNA and glycosaminoglycan (GAG) quantification detect; mRNA expressions of chondrogenic marker genes such as collagen type I, collagen type II, Aggrecan, cartilage oligomeric matrix protein (COMP), and superficial zone protein (SZP) were detected by real-time quantitative PCR; and histology observations were done by toluidine blue staining and immunohistochemistry staining. No significant difference was found in DNA content, GAG content, and the ratio of GAG to DNA among 5 groups (P > 0.05). After load, there was a large number of GAG in the medium, and the GAG significantly increased with time (P < 0.05). The mRNA expression of collagen type I showed no significant difference among 5 groups (P > 0.05). The mRNA expression of collagen type II in group B was significantly increased when compared with group A (P < 0.01), and groups D and E were significantly higher than group C (P < 0.01); the mRNA expression of Aggrecan in groups D and E were significantly increased when compared with group C (P < 0.01), and group E was significantly higher than group D (P < 0.01); the mRNA expression of COMP in group B was

  7. CCAAT/enhancer binding protein β (C/EBPβ) regulates the transcription of growth arrest and DNA damage-inducible protein 45 β (GADD45β) in articular chondrocytes.

    PubMed

    Shimada, Hirofumi; Otero, Miguel; Tsuchimochi, Kaneyuki; Yamasaki, Satoshi; Sakakima, Harutoshi; Matsuda, Fumiyo; Sakasegawa, Megumi; Setoguchi, Takao; Xu, Lin; Goldring, Mary B; Tanimoto, Akihide; Komiya, Setsuro; Ijiri, Kosei

    2016-04-01

    Osteoarthritis (OA) is a whole joint disease characterized by cartilage degradation, which causes pain and disability in older adults. Our previous work showed that growth arrest and DNA damage-inducible protein 45 β (GADD45β) is upregulated in chondrocyte clusters in OA cartilage, especially in the early stage of this disease. CCAAT/enhancer binding protein β (C/EBPβ) is expressed in the hypertrophic growth plate chondrocytes and functions in synergy with GADD45β. Here, the presence and localization of these proteins was assessed by immunohistochemistry using articular cartilage from OA patients, revealing colocalization of C/EBPβ and GADD45β in OA chondrocytes. GADD45β promoter analysis was performed to determine whether C/EBPβ directly regulates GADD45β transcription. Furthermore, we analyzed the effect of C/EBPβ on Gadd45β gene regulation in articular chondrocytes in vivo and in vitro. Immunohistochemical analysis of C/ebpβ-haploinsufficient mice (C/ebpβ(+/-)) cartilage showed that C/ebpβ haploinsufficiency led to reduced Gadd45β gene expression in these cells. In vitro, we evaluated the effects of conditional C/EBPβ overexpression driven by the cartilage oligomeric matrix protein (Comp) promoter in mComp-tTA;pTRE-Tight-BI-DsRed-mC/ebpβ transgenic mice. C/EBPβ overexpression significantly stimulated Gadd45β gene expression in articular chondrocytes. Taken together, our data demonstrate that C/EBPβ plays a central role in controlling Gadd45β gene expression in these cells.

  8. Polymer Formulations for Cartilage Repair

    SciTech Connect

    Gutowska, Anna; Jasionowski, Marek; Morris, J. E.; Chrisler, William B.; An, Yuehuei H.; Mironov, V.

    2001-05-15

    Regeneration of destroyed articular cartilage can be induced by transplantation of cartilage cells into a defect. The best results are obtained with the use of autologus cells. However, obtaining large amounts of autologus cartilage cells causes a problem of creating a large cartilage defect in a donor site. Techniques are currently being developed to harvest a small number of cells and propagate them in vitro. It is a challenging task, however, due to the fact that ordinarily, in a cell culture on flat surfaces, chondrocytes do not maintain their in vivo phenotype and irreversibly diminish or cease the synthesis of aggregating proteoglycans. Therefore, the research is continuing to develop culture conditions for chondrocytes with the preserved phenotype.

  9. Enhanced nutrient transport improves the depth-dependent properties of tri-layered engineered cartilage constructs with zonal co-culture of chondrocytes and MSCs.

    PubMed

    Kim, Minwook; Farrell, Megan J; Steinberg, David R; Burdick, Jason A; Mauck, Robert L

    2017-08-01

    Biomimetic design in cartilage tissue engineering is a challenge given the complexity of the native tissue. While numerous studies have generated constructs with near-native bulk properties, recapitulating the depth-dependent features of native tissue remains a challenge. Furthermore, limitations in nutrient transport and matrix accumulation in engineered constructs hinders maturation within the central core of large constructs. To overcome these limitations, we fabricated tri-layered constructs that recapitulate the depth-dependent cellular organization and functional properties of native tissue using zonally derived chondrocytes co-cultured with MSCs. We also introduced porous hollow fibers (HFs) and HFs/cotton threads to enhance nutrient transport. Our results showed that tri-layered constructs with depth-dependent organization and properties could be fabricated. The addition of HFs or HFs/threads improved matrix accumulation in the central core region. With HF/threads, the local modulus in the deep region of tri-layered constructs nearly matched that of native tissue, though the properties in the central regions remained lower. These constructs reproduced the zonal organization and depth-dependent properties of native tissue, and demonstrate that a layer-by-layer fabrication scheme holds promise for the biomimetic repair of focal cartilage defects. Articular cartilage is a highly organized tissue driven by zonal heterogeneity of cells, extracellular matrix proteins and fibril orientations, resulting in depth-dependent mechanical properties. Therefore, the recapitulation of the functional properties of native cartilage in a tissue engineered construct requires such a biomimetic design of the morphological organization, and this has remained a challenge in cartilage tissue engineering. This study demonstrates that a layer-by-layer fabrication scheme, including co-cultures of zone-specific articular CHs and MSCs, can reproduce the depth-dependent characteristics

  10. Biochemical and biomechanical alterations in equine articular cartilage following an experimentally-induced synovitis.

    PubMed

    Palmer, J L; Bertone, A L; Malemud, C J; Mansour, J

    1996-06-01

    The effects of inflammation on the biochemical and biomechanical properties of articular cartilage at two sites (dorsal and palmar) from the radial facet of the equine third carpal bone were examined in response to a synovitis induced with Escherichia coli lipopolysaccharide (LPS). Four groups were studied. In group 1 synovitis was induced at time zero and evaluated at week 6. Group 2 was the sham-treated control for group 1. In group 3 synovitis was induced at time zero and evaluated at week 2. Group 4 was the sham-treated control for group 3. There was a significant increase (P < 0.05) in newly synthesized proteoglycan PG from both sites in group 3 as compared to the sham-treated groups and group 1. No significant difference in the endogenous PG concentration between groups or sites was detected. Sepharose CL-2B revealed two peaks of newly synthesized PG in all groups; an early peak (Kav 0.11-0.13) and a late peak (Kav 0.48-0.64). Newly synthesized PG profiles from sham-treated groups and group 3 were similar, but the group 3 PG profile exhibited a more pronounced early peak. Conversely, the PG profile from group 1 demonstrated a more prominent late peak. Electrophoresis and Western blot analysis of the pooled late PG peak fractions from the sham-treated and group 1 showed a single toluidine blue stained band from both sites which reacted with monoclonal antibody (MAb) 1C6. By contrast, the late peak from the palmar site in group 3 showed an additional faster moving component on composite gels which did not react with MAb 1C6. There was a significant decrease in Poisson's ratio and a significant increase in cartilage thickness in groups 1 and 3 which had received synovitis. The increase in cartilage thickness of groups 1 and 3 was also significantly affected by site (dorsal > palmar). There was no significant difference in aggregate modulus or permeability constant among groups. Primary joint inflammation induced by LPS alters the biochemical and biomechanical

  11. Chop (Ddit3) Is Essential for D469del-COMP Retention and Cell Death in Chondrocytes in an Inducible Transgenic Mouse Model of Pseudoachondroplasia

    PubMed Central

    Posey, Karen L.; Coustry, Francoise; Veerisetty, Alka C.; Liu, Peiman; Alcorn, Joseph L.; Hecht, Jacqueline T.

    2012-01-01

    Cartilage oligomeric matrix protein (COMP), a secreted glycoprotein synthesized by chondrocytes, regulates proliferation and type II collagen assembly. Mutations in the COMP gene cause pseudoachondroplasia and multiple epiphyseal dysplasia. Previously, we have shown that expression of D469del-COMP in transgenic mice causes intracellular retention of D469del-COMP, thereby recapitulating pseudoachondroplasia chondrocyte pathology. This inducible transgenic D469del-COMP mouse is the only in vivo model to replicate the critical cellular and clinical features of pseudoachondroplasia. Here, we report developmental studies of D469del-COMP-induced chondrocyte pathology from the prenatal period to adolescence. D469del-COMP retention was limited prenatally and did not negatively affect the growth plate until 3 weeks after birth. Results of immunostaining, transcriptome analysis, and qRT-PCR suggest a molecular model in which D469del-COMP triggers apoptosis during the first postnatal week. By 3 weeks (when most chondrocytes are retaining D469del-COMP), inflammation, oxidative stress, and DNA damage contribute to chondrocyte cell death by necroptosis. Importantly, by crossing the D469del-COMP mouse onto a Chop null background (Ddit3 null), thereby eliminating Chop, the unfolded protein response was disrupted, thus alleviating both D469del-COMP intracellular retention and premature chondrocyte cell death. Chop therefore plays a significant role in processes that mediate D469del-COMP retention. Taken together, these results suggest that there may be an optimal window before the induction of significant D469del-COMP retention during which endoplasmic reticulum stress could be targeted. PMID:22154935

  12. Disturbed Cartilage and Joint Homeostasis Resulting From a Loss of Mitogen-Inducible Gene 6 in a Mouse Model of Joint Dysfunction

    PubMed Central

    Pest, Michael A.; Russell, Bailey A.; Zhang, Yu-Wen; Jeong, Jae-Wook; Beier, Frank

    2017-01-01

    Objective Mitogen-inducible gene 6 (MIG-6) regulates epidermal growth factor receptor (EGFR) signaling in synovial joint tissues. Whole-body knockout of the Mig6 gene in mice has been shown to induce osteoarthritis and joint degeneration. To evaluate the role of chondrocytes in this process, Mig6 was conditionally deleted from Col2a1-expressing cell types in the cartilage of mice. Methods Bone and cartilage in the synovial joints of cartilage-specific Mig6-deleted (knockout [KO]) mice and control littermates were compared. Histologic staining and immunohistochemical analyses were used to evaluate joint pathology as well as the expression of key extracellular matrix and regulatory proteins. Calcified tissue in synovial joints was assessed by micro–computed tomography (micro-CT) and whole-skeleton staining. Results Formation of long bones was found to be normal in KO animals. Cartilage thickness and proteoglycan staining of articular cartilage in the knee joints of 12-week-old KO mice were increased as compared to controls, with higher cellularity throughout the tissue. Radiopaque chondro-osseous nodules appeared in the knees of KO animals by 12 weeks of age and progressed to calcified bone–like tissue by 36 weeks of age. Nodules were also observed in the spine of 36-week-old animals. Erosion of bone at ligament entheses was evident by 12 weeks of age, by both histologic and micro-CT assessment. Conclusion MIG-6 expression in chondrocytes is important for the maintenance of cartilage and joint homeostasis. Dysregulation of EGFR signaling in chondrocytes results in anabolic activity in cartilage, but erosion of ligament entheses and the formation of ectopic chondro-osseous nodules severely disturb joint physiology. PMID:24966136

  13. The mechanical microenvironment of high concentration agarose for applying deformation to primary chondrocytes.

    PubMed

    Zignego, Donald L; Jutila, Aaron A; Gelbke, Martin K; Gannon, Daniel M; June, Ronald K

    2014-06-27

    Cartilage and chondrocytes experience loading that causes alterations in chondrocyte biological activity. In vivo chondrocytes are surrounded by a pericellular matrix with a stiffness of ~25-200kPa. Understanding the mechanical loading environment of the chondrocyte is of substantial interest for understanding chondrocyte mechanotransduction. The first objective of this study was to analyze the spatial variability of applied mechanical deformations in physiologically stiff agarose on cellular and sub-cellular length scales. Fluorescent microspheres were embedded in physiologically stiff agarose hydrogels. Microsphere positions were measured via confocal microscopy and used to calculate displacement and strain fields as a function of spatial position. The second objective was to assess the feasibility of encapsulating primary human chondrocytes in physiologically stiff agarose. The third objective was to determine if primary human chondrocytes could deform in high-stiffness agarose gels. Primary human chondrocyte viability was assessed using live-dead imaging following 24 and 72h in tissue culture. Chondrocyte shape was measured before and after application of 10% compression. These data indicate that (1) displacement and strain precision are ~1% and 6.5% respectively, (2) high-stiffness agarose gels can maintain primary human chondrocyte viability of >95%, and (3) compression of chondrocytes in 4.5% agarose can induce shape changes indicative of cellular compression. Overall, these results demonstrate the feasibility of using high-concentration agarose for applying in vitro compression to chondrocytes as a model for understanding how chondrocytes respond to in vivo loading.

  14. Autologous chondrocyte implantation (ACI) for cartilage defects of the knee: A guideline by the working group "Clinical Tissue Regeneration" of the German Society of Orthopaedics and Trauma (DGOU).

    PubMed

    Niemeyer, P; Albrecht, D; Andereya, S; Angele, P; Ateschrang, A; Aurich, M; Baumann, M; Bosch, U; Erggelet, C; Fickert, S; Gebhard, H; Gelse, K; Günther, D; Hoburg, A; Kasten, P; Kolombe, T; Madry, H; Marlovits, S; Meenen, N M; Müller, P E; Nöth, U; Petersen, J P; Pietschmann, M; Richter, W; Rolauffs, B; Rhunau, K; Schewe, B; Steinert, A; Steinwachs, M R; Welsch, G H; Zinser, W; Fritz, J

    2016-06-01

    Autologous chondrocyte implantation (ACI) is an established and well-accepted procedure for the treatment of localised full-thickness cartilage defects of the knee. The present review of the working group "Clinical Tissue Regeneration" of the German Society of Orthopaedics and Trauma (DGOU) describes the biology and function of healthy articular cartilage, the present state of knowledge concerning therapeutic consequences of primary cartilage lesions and the suitable indication for ACI. Based on best available scientific evidence, an indication for ACI is given for symptomatic cartilage defects starting from defect sizes of more than three to four square centimetres; in the case of young and active sports patients at 2.5cm(2), while advanced degenerative joint disease needs to be considered as the most important contraindication. The present review gives a concise overview on important scientific background and the results of clinical studies and discusses the advantages and disadvantages of ACI. Non-systematic Review. Copyright © 2016 Elsevier B.V. All rights reserved.

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

    PubMed Central

    Sharma, H.; Pigott, R.

    1992-01-01

    The intercellular adhesion molecule-1 (ICAM-1) was found by immunostaining chondrocytes in cartilage from three patients with rheumatoid arthritis. Expression of ICAM-1 was restricted to chondrocytes in areas of erodedcartilage adjacent to the invading synovial tissue. Toluidine blue staining of these areas demonstrated severe depletion of the cartilage extracellular matrix. In areas of undamaged cartilage there was no ICAM-1 expression. Since ICAM-1 is not constitutively expressed on normal human articular cartilage, but could be induced in vitro by exogenous IL-1α, TNFα and IFNγ or by co-culturing cartilage with inflammatory rheumatoid synovium, we conclude that the induction of ICAM-1 on rheumatoid chondrocytes results from the synergistic action of a variety of cytokines produced by the inflammatory cells of the invading pannus. PMID:18475445

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

    PubMed Central

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

    2015-01-01

    Sclareol is a natural product initially isolated form Salvia sclarea which possesses immune-regulation and anti-inflammatory activities. However, the anti-osteoarthritic properties of sclareol have not been investigated. The present study is aimed at evaluating the potential effects of sclareol in interleukin-1β (IL-1β)-induced rabbit chondrocytes as well as an experimental rabbit knee osteoarthritis model induced by anterior cruciate ligament transection (ACLT). Cultured rabbit chondrocytes were pretreated with 1, 5 and 10 μg/mL sclareol for 1 h and followed by stimulation of IL-1β (10 ng/mL) for 24 h. Gene expression of matrix metalloproteinase-1 (MMP-1), MMP-3, MMP-13, tissue inhibitors of metalloproteinase-1 (TIMP-1), inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX)-2 was determined by quantitative real-time polymerase chain reaction (qRT-PCR). MMP-3, TIMP-1, iNOS and COX-2 proteins were measured by Western blotting. Enzyme-linked immunosorbent assay (ELISA) was applied for nitric oxide (NO) and prostaglandin E2 (PGE2) assessment. For the in vivo study, rabbits received six weekly 0.3 mL sclareol (10 μg/mL) intra-articular injections in the knees four weeks after ACLT surgery. Cartilage was harvested for measurement of MMP-1, MMP-3, MMP-13, TIMP-1, iNOS and COX-2 by qRT-PCR, while femoral condyles were used for histological evaluation. The in vitro results we obtained showed that sclareol inhibited the MMPs, iNOS and COX-2 expression on mRNA and protein levels, while increased the TIMP-1 expression. And over-production of NO and PGE2 was also suppressed. For the in vivo study, both qRT-PCR results and histological evaluation confirmed that sclareol ameliorated cartilage degradation. Hence, we speculated that sclareol may be an ideal approach for treating osteoarthritis. PMID:26045743

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

    PubMed

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

    2015-01-01

    Sclareol is a natural product initially isolated form Salvia sclarea which possesses immune-regulation and anti-inflammatory activities. However, the anti-osteoarthritic properties of sclareol have not been investigated. The present study is