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Sample records for axin2 regulates chondrocyte

  1. Coordinated action of Axin1 and Axin2 suppresses β-catenin to regulate muscle stem cell function.

    PubMed

    Figeac, Nicolas; Zammit, Peter S

    2015-08-01

    The resident stem cells of skeletal muscle are satellite cells, which are regulated by both canonical and non-canonical Wnt pathways. Canonical Wnt signalling promotes differentiation, and is controlled at many levels, including via Axin1 and Axin2-mediated β-catenin degradation. Axin1 and Axin2 are thought equivalent suppressors of canonical Wnt signalling, although Axin2 is also a Wnt target gene. We show that Axin1 expression was higher in proliferating satellite cells, while Axin2 was up-regulated during differentiation. siRNA-mediated Axin1 knockdown changed cell morphology, suppressed proliferation and promoted myogenic differentiation. Simultaneous knockdown of both Axin1 and β-catenin rescued proliferation and partially, premature differentiation. Surprisingly, retroviral-mediated overexpression of Axin2 was unable to compensate for knockdown of Axin1 in satellite cells, indicating that Axin1 and Axin2 are not fully redundant. Isolated satellite cells from Axin2-null mice also had no major phenotype. However, siRNA-mediated knockdown of Axin1 in Axin2-null cells strongly inhibited proliferation, while inducing differentiation, clear nuclear localisation of β-catenin, up-regulation of canonical Wnt target genes (Axin2, Lef1, Tcf4, Pitx2c and Lgr5) and activation of a TCF reporter construct. Again, concomitant knockdown of Axin1 and β-catenin in Axin2-null satellite cells rescued morphology and proliferation, but only partially prevented precocious differentiation. Thus, Axin1 and Axin2 do not have equivalent functions in satellite cells, but are both involved in repression of Wnt/β-catenin signalling to maintain proliferation and contribute to controlling timely myogenic differentiation. PMID:25866367

  2. Barx2 and Pax7 Regulate Axin2 Expression in Myoblasts by Interaction with β-Catenin and Chromatin Remodelling.

    PubMed

    Hulin, Julie-Ann; Nguyen, Thi Diem Tran; Cui, Shuang; Marri, Shashikanth; Yu, Ruth T; Downes, Michael; Evans, Ronald M; Makarenkova, Helen; Meech, Robyn

    2016-08-01

    Satellite cells are the resident stem cells of skeletal muscle; quiescent in adults until activated by injury to generate proliferating myoblasts. The canonical Wnt signalling pathway, mediated by T-cell factor/lymphoid enhancer factor (TCF/LEF) and β-catenin effector proteins, controls myoblast differentiation in vitro, and recent work suggests that timely termination of the Wnt/β-catenin signal is important for normal adult myogenesis. We recently identified the Barx2 and Pax7 homeobox proteins as novel components of the Wnt effector complex. Here, we examine molecular and epigenetic mechanisms by which Barx2 and Pax7 regulate the canonical Wnt target gene Axin2, which mediates critical feedback to terminate the transcriptional response to Wnt signals. Barx2 is recruited to the Axin2 gene via TCF/LEF binding sites, recruits β-catenin and the coactivator GRIP-1, and induces local H3K-acetylation. Barx2 also promotes nuclear localization of β-catenin. Conversely, Pax7 represses Axin2 promoter/intron activity and inhibits Barx2-mediated H3K-acetylation via the corepressor HDAC1. Wnt3a not only induces Barx2 mRNA, but also stabilises Barx2 protein in myoblasts; conversely, Wnt3a potently inhibits Pax7 protein expression. As Barx2 promotes myogenic differentiation and Pax7 suppresses it, this novel posttranscriptional regulation of Barx2 and Pax7 by Wnt3a may be involved in the specification of differentiation-competent and -incompetent myoblast populations. Finally, we propose a model for dual function of Barx2 downstream of Wnt signals: activation of myogenic target genes in association with canonical myogenic regulatory factors, and regulation of the negative feedback loop that limits the response of myoblasts to Wnt signals via direct interaction of Barx2 with the TCF/β-catenin complex. Stem Cells 2016;34:2169-2182. PMID:27144473

  3. SOX7 co-regulates Wnt/β-catenin signaling with Axin-2: both expressed at low levels in breast cancer.

    PubMed

    Liu, Huidi; Mastriani, Emilio; Yan, Zi-Qiao; Yin, Si-Yuan; Zeng, Zheng; Wang, Hong; Li, Qing-Hai; Liu, Hong-Yu; Wang, Xiaoyu; Bao, Hong-Xia; Zhou, Yu-Jie; Kou, Jun-Jie; Li, Dongsheng; Li, Ting; Liu, Jianrui; Liu, Yongfang; Yin, Lin; Qiu, Li; Gong, Liling; Liu, Shu-Lin

    2016-01-01

    SOX7 as a tumor suppressor belongs to the SOX F gene subfamily and is associated with a variety of human cancers, including breast cancer, but the mechanisms involved are largely unclear. In the current study, we investigated the interactions between SOX7 and AXIN2 in their co-regulation on the Wnt/β-catenin signal pathway, using clinical specimens and microarray gene expression data from the GEO database, for their roles in breast cancer. We compared the expression levels of SOX7 and other co-expressed genes in the Wnt/β-catenin pathway and found that the expression of SOX7, SOX17 and SOX18 was all reduced significantly in the breast cancer tissues compared to normal controls. AXIN2 had the highest co-relativity with SOX7 in the Wnt/β-catenin signaling pathway. Clinicopathological analysis demonstrated that the down-regulated SOX7 was significantly correlated with advanced stages and poorly differentiated breast cancers. Consistent with bioinformatics predictions, SOX7 was correlated positively with AXIN2 and negatively with β-catenin, suggesting that SOX7 and AXIN2 might play important roles as co-regulators through the Wnt-β-catenin pathway in the breast tissue to affect the carcinogenesis process. Our results also showed Smad7 as the target of SOX7 and AXIN2 in controlling breast cancer progression through the Wnt/β-catenin signaling pathway. PMID:27188720

  4. SOX7 co-regulates Wnt/β-catenin signaling with Axin-2: both expressed at low levels in breast cancer

    PubMed Central

    Liu, Huidi; Mastriani, Emilio; Yan, Zi-Qiao; Yin, Si-Yuan; Zeng, Zheng; Wang, Hong; Li, Qing-Hai; Liu, Hong-Yu; Wang, Xiaoyu; Bao, Hong-Xia; Zhou, Yu-Jie; Kou, Jun-Jie; Li, Dongsheng; Li, Ting; Liu, Jianrui; Liu, Yongfang; Yin, Lin; Qiu, Li; Gong, Liling; Liu, Shu-Lin

    2016-01-01

    SOX7 as a tumor suppressor belongs to the SOX F gene subfamily and is associated with a variety of human cancers, including breast cancer, but the mechanisms involved are largely unclear. In the current study, we investigated the interactions between SOX7 and AXIN2 in their co-regulation on the Wnt/β-catenin signal pathway, using clinical specimens and microarray gene expression data from the GEO database, for their roles in breast cancer. We compared the expression levels of SOX7 and other co-expressed genes in the Wnt/β-catenin pathway and found that the expression of SOX7, SOX17 and SOX18 was all reduced significantly in the breast cancer tissues compared to normal controls. AXIN2 had the highest co-relativity with SOX7 in the Wnt/β-catenin signaling pathway. Clinicopathological analysis demonstrated that the down-regulated SOX7 was significantly correlated with advanced stages and poorly differentiated breast cancers. Consistent with bioinformatics predictions, SOX7 was correlated positively with AXIN2 and negatively with β-catenin, suggesting that SOX7 and AXIN2 might play important roles as co-regulators through the Wnt-β-catenin pathway in the breast tissue to affect the carcinogenesis process. Our results also showed Smad7 as the target of SOX7 and AXIN2 in controlling breast cancer progression through the Wnt/β-catenin signaling pathway. PMID:27188720

  5. Expression Pattern of Axin2 During Chicken Development

    PubMed Central

    Eckei, Gesa; Böing, Marion; Brand-Saberi, Beate; Morosan-Puopolo, Gabriela

    2016-01-01

    Canonical Wnt-signalling is well understood and has been extensively described in many developmental processes. The regulation of this signalling pathway is of outstanding relevance for proper development of the vertebrate and invertebrate embryo. Axin2 provides a negative-feedback-loop in the canonical Wnt-pathway, being a target gene and a negative regulator. Here we provide a detailed analysis of the expression pattern in the development of the chicken embryo. By performing in-situ hybridization on chicken embryos from stage HH 04+ to HH 32 we detected a temporally and spatially restricted dynamic expression of Axin2. In particular, data about the expression of Axin2 mRNA in early embryogenesis, somites, neural tube, limbs, kidney and eyes was obtained. PMID:27680024

  6. Nuclear AXIN2 represses MYC gene expression

    SciTech Connect

    Rennoll, Sherri A.; Konsavage, Wesley M.; Yochum, Gregory S.

    2014-01-03

    Highlights: •AXIN2 localizes to cytoplasmic and nuclear compartments in colorectal cancer cells. •Nuclear AXIN2 represses the activity of Wnt-responsive luciferase reporters. •β-Catenin bridges AXIN2 to TCF transcription factors. •AXIN2 binds the MYC promoter and represses MYC gene expression. -- Abstract: The β-catenin transcriptional coactivator is the key mediator of the canonical Wnt signaling pathway. In the absence of Wnt, β-catenin associates with a cytosolic and multi-protein destruction complex where it is phosphorylated and targeted for proteasomal degradation. In the presence of Wnt, the destruction complex is inactivated and β-catenin translocates into the nucleus. In the nucleus, β-catenin binds T-cell factor (TCF) transcription factors to activate expression of c-MYC (MYC) and Axis inhibition protein 2 (AXIN2). AXIN2 is a member of the destruction complex and, thus, serves in a negative feedback loop to control Wnt/β-catenin signaling. AXIN2 is also present in the nucleus, but its function within this compartment is unknown. Here, we demonstrate that AXIN2 localizes to the nuclei of epithelial cells within normal and colonic tumor tissues as well as colorectal cancer cell lines. In the nucleus, AXIN2 represses expression of Wnt/β-catenin-responsive luciferase reporters and forms a complex with β-catenin and TCF. We demonstrate that AXIN2 co-occupies β-catenin/TCF complexes at the MYC promoter region. When constitutively localized to the nucleus, AXIN2 alters the chromatin structure at the MYC promoter and directly represses MYC gene expression. These findings suggest that nuclear AXIN2 functions as a rheostat to control MYC expression in response to Wnt/β-catenin signaling.

  7. A Novel AXIN2 Missense Mutation Is Associated with Non-Syndromic Oligodontia

    PubMed Central

    Zhan, Yuan; Feng, Hailan

    2015-01-01

    Oligodontia is defined as the congenital absence of six or more permanent teeth, excluding the third molars. Oligodontia may contribute to masticatory dysfunction, speech alteration, aesthetic problems and malocclusion. Numerous gene mutations have been association with oligodontia. In the present study, we identified a de novo AXIN2 missense mutation (c.314T>G) in a Chinese individual with non-syndromic oligodontia. This mutation results in the substitution of Val at residue 105 for Gly (p.Val105Gly); residue 105 is located in the highly conserved regulator of G protein signaling (RGS) domain of the AXIN2 protein. This is the first report indicating that a mutation in the RGS domain of AXIN2 is responsible for non-syndromic oligodontia. Our study supports the relationship between AXIN2 mutation and non-syndromic oligodontia and extends the mutation spectrum of the AXIN2 gene. PMID:26406231

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

    PubMed

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

    2013-08-01

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

  9. Growth factor transgenes interactively regulate articular chondrocytes.

    PubMed

    Shi, Shuiliang; Mercer, Scott; Eckert, George J; Trippel, Stephen B

    2013-04-01

    Adult articular chondrocytes lack an effective repair response to correct damage from injury or osteoarthritis. Polypeptide growth factors that stimulate articular chondrocyte proliferation and cartilage matrix synthesis may augment this response. Gene transfer is a promising approach to delivering such factors. Multiple growth factor genes regulate these cell functions, but multiple growth factor gene transfer remains unexplored. We tested the hypothesis that multiple growth factor gene transfer selectively modulates articular chondrocyte proliferation and matrix synthesis. We tested the hypothesis by delivering combinations of the transgenes encoding insulin-like growth factor I (IGF-I), fibroblast growth factor-2 (FGF-2), transforming growth factor beta1 (TGF-β1), bone morphogenetic protein-2 (BMP-2), and bone morphogenetic protien-7 (BMP-7) to articular chondrocytes and measured changes in the production of DNA, glycosaminoglycan, and collagen. The transgenes differentially regulated all these chondrocyte activities. In concert, the transgenes interacted to generate widely divergent responses from the cells. These interactions ranged from inhibitory to synergistic. The transgene pair encoding IGF-I and FGF-2 maximized cell proliferation. The three-transgene group encoding IGF-I, BMP-2, and BMP-7 maximized matrix production and also optimized the balance between cell proliferation and matrix production. These data demonstrate an approach to articular chondrocyte regulation that may be tailored to stimulate specific cell functions, and suggest that certain growth factor gene combinations have potential value for cell-based articular cartilage repair.

  10. Axin2 as regulatory and therapeutic target in newborn brain injury and remyelination

    PubMed Central

    Fancy, Stephen P.J.; Harrington, Emily P.; Yuen, Tracy J.; Silbereis, John C.; Zhao, Chao; Baranzini, Sergio E.; Bruce, Charlotte C.; Otero, Jose J.; Huang, Eric J.; Nusse, Roel; Franklin, Robin J.M.; Rowitch, David H.

    2011-01-01

    Permanent damage to white matter tracts, comprising axons and myelinating oligodendrocytes, is an important component of newborn brain injuries that cause cerebral palsy and cognitive disabilities as well as multiple sclerosis (MS) in adults. However, regulatory factors relevant in human developmental myelin disorders and in myelin regeneration are unclear. Here, we report expression of AXIN2 in immature oligodendrocyte progenitor cells (OLP) within white matter lesions of human newborns with neonatal hypoxic-ischemic and gliotic brain damage, as well as active MS lesions in adults. Axin2 is a target of Wnt transcriptional activation that feeds back negatively on the pathway, promoting β-catenin degradation. We show Axin2 function is essential for normal kinetics of remyelination. Small molecule inhibitor XAV939, which targets enzymatic activity of Tankyrase, acts to stabilize Axin2 levels in OLP from brain and spinal cord and accelerates their differentiation and myelination after hypoxic and demyelinating injury. Together, these findings indicate that Axin2 is an essential regulator of remyelination and that it might serve as a pharmacological checkpoint in this process. PMID:21706018

  11. Axin2 as regulatory and therapeutic target in newborn brain injury and remyelination.

    PubMed

    Fancy, Stephen P J; Harrington, Emily P; Yuen, Tracy J; Silbereis, John C; Zhao, Chao; Baranzini, Sergio E; Bruce, Charlotte C; Otero, Jose J; Huang, Eric J; Nusse, Roel; Franklin, Robin J M; Rowitch, David H

    2011-08-01

    Permanent damage to white matter tracts, comprising axons and myelinating oligodendrocytes, is an important component of brain injuries of the newborn that cause cerebral palsy and cognitive disabilities, as well as multiple sclerosis in adults. However, regulatory factors relevant in human developmental myelin disorders and in myelin regeneration are unclear. We found that AXIN2 was expressed in immature oligodendrocyte progenitor cells (OLPs) in white matter lesions of human newborns with neonatal hypoxic-ischemic and gliotic brain damage, as well as in active multiple sclerosis lesions in adults. Axin2 is a target of Wnt transcriptional activation that negatively feeds back on the pathway, promoting β-catenin degradation. We found that Axin2 function was essential for normal kinetics of remyelination. The small molecule inhibitor XAV939, which targets the enzymatic activity of tankyrase, acted to stabilize Axin2 levels in OLPs from brain and spinal cord and accelerated their differentiation and myelination after hypoxic and demyelinating injury. Together, these findings indicate that Axin2 is an essential regulator of remyelination and that it might serve as a pharmacological checkpoint in this process.

  12. Targeted repression of AXIN2 and MYC gene expression using designer TALEs

    SciTech Connect

    Rennoll, Sherri A.; Scott, Samantha A.; Yochum, Gregory S.

    2014-04-18

    Highlights: • We designed TALE–SID fusion proteins to target AXIN2 and MYC. • TALE–SIDs bound the chromosomal AXIN2 and MYC genes and repressed their expression. • TALE–SIDs repress β-catenin{sup S45F}-dependent AXIN2 and MYC transcription. - Abstract: Designer TALEs (dTALEs) are chimeric transcription factors that can be engineered to regulate gene expression in mammalian cells. Whether dTALEs can block gene transcription downstream of signal transduction cascades, however, has yet to be fully explored. Here we tested whether dTALEs can be used to target genes whose expression is controlled by Wnt/β-catenin signaling. TALE DNA binding domains were engineered to recognize sequences adjacent to Wnt responsive enhancer elements (WREs) that control expression of axis inhibition protein 2 (AXIN2) and c-MYC (MYC). These custom DNA binding domains were linked to the mSin3A interaction domain (SID) to generate TALE–SID chimeric repressors. The TALE–SIDs repressed luciferase reporter activity, bound their genomic target sites, and repressed AXIN2 and MYC expression in HEK293 cells. We generated a novel HEK293 cell line to determine whether the TALE–SIDs could function downstream of oncogenic Wnt/β-catenin signaling. Treating these cells with doxycycline and tamoxifen stimulates nuclear accumulation of a stabilized form of β-catenin found in a subset of colorectal cancers. The TALE–SIDs repressed AXIN2 and MYC expression in these cells, which suggests that dTALEs could offer an effective therapeutic strategy for the treatment of colorectal cancer.

  13. Axis Inhibition Protein 2 (AXIN2) Polymorphisms and Tooth Agenesis

    PubMed Central

    Callahan, N; Modesto, A; Meira, R; Seymen, F; Patir, A; Vieira, AR

    2009-01-01

    Tooth agenesis is a common congenital disorder that affects almost 20 percent of the world’s population. A number of different genes have been shown to be associated with cases of tooth agenesis including AXIN2, IRF6, FGFR1, MSX1, PAX9, and TGFA. Of particular interest is AXIN2, which was linked to two families segregating oligodontia and colorectal cancer. We studied two collections of families affected with tooth agenesis and tested them for association with AXIN2. Significant association between tooth agenesis and AXIN2 was found (p = 0.02) in cases with at least one missing incisor. Our work further supports a role of AXIN2 in human tooth agenesis and for the first time suggests AXIN2 is involved in sporadic forms of common incisor agenesis. Future studies should identify which specific tooth agenesis subphenotypes are consequence of AXIN2 genetic variations. A subset of these cases could have an increased susceptibility for colon cancer or other types of tumors and this knowledge would have significant clinical implications. PMID:18790474

  14. AXIN2 is Associated With Papillary Thyroid Carcinoma

    PubMed Central

    Liu, Xin; Li, Shuang; Lin, Xuejun; Yan, Kangkang; Zhao, Longyu; Yu, Qiong; Liu, Xiaodong

    2016-01-01

    Background: Findings of recent studies have demonstrated a rapid increase of the incidence of papillary thyroid carcinoma (PTC), which accounts for nearly 80% of thyroid cancers. Objectives: The aim of this study was to explore the association between AXIN2 gene polymorphism and papillary thyroid carcinoma (PTC). Patients and Methods: 106 blood samples (56 PTC patients and 50 healthy controls) were drawn from China-Japan Union Hospital in Jilin province, China, during October 2010 to March 2011. A case-control study was designed to examine the association between AXIN2 and PTC. Seven tag single nucleotide polymorphisms (tag SNPs) in AXIN2 were selected and genotyped. Frequencies of different genotypes and alleles were analyzed between the patients and the controls, using the R × C column contingency table χ2 test. The possible association of haplotypes constructed by the combined effects of two or more loci with PTC was analyzed through the UNPHASED 3.1.4 program. Results: Rs11655966, rs3923086 and rs7591 of AXIN2 showed significant associations with PTC (P < 0.05). The result of haplotypes analysis showed that rs11655966-rs3923086-rs4791169 had statistically significant differences between the two groups (P < 0.05). Conclusions: Together with the functions of the target genes, we further elucidated that AXIN2 is associated with papillary thyroid carcinoma in the Chinese Han population. PMID:27168945

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

    PubMed

    Murata, Koichi; Kitaori, Toshiyuki; Oishi, Shinya; Watanabe, Naoki; Yoshitomi, Hiroyuki; Tanida, Shimei; Ishikawa, Masahiro; Kasahara, Takashi; Shibuya, Hideyuki; Fujii, Nobutaka; Nagasawa, Takashi; Nakamura, Takashi; Ito, Hiromu

    2012-01-01

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

  16. 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

  17. Epigenetic regulation in chondrocyte phenotype maintenance for cell-based cartilage repair

    PubMed Central

    Duan, Li; Liang, Yujie; Ma, Bin; Zhu, Weimin; Wang, Daping

    2015-01-01

    Loss of hyaline chondrocyte phenotype during the monolayer culture in vitro is a major obstacle for cell-based articular cartilage repair. Increasing evidence implicates an important role of the epigenetic regulation in maintaining the chondrocyte phenotype. DNA methylation, histone modifications and microRNAs have all been shown to contribute to chondrocyte dedifferentiation and hypertrophy. Moreover, the interplay among epigenetic regulators forms a complicated epigenetic network in regulating chondrocyte dedifferentiation. This review provides a detailed overview of the epigenetic regulation in maintaining the chondrocyte phenotype for chondrocyte-based cartilage repair. PMID:26807163

  18. Mutations in AXIN2 cause familial tooth agenesis and predispose to colorectal cancer.

    PubMed

    Lammi, Laura; Arte, Sirpa; Somer, Mirja; Jarvinen, Heikki; Lahermo, Paivi; Thesleff, Irma; Pirinen, Sinikka; Nieminen, Pekka

    2004-05-01

    Wnt signaling regulates embryonic pattern formation and morphogenesis of most organs. Aberrations of regulation of Wnt signaling may lead to cancer. Here, we have used positional cloning to identify the causative mutation in a Finnish family in which severe permanent tooth agenesis (oligodontia) and colorectal neoplasia segregate with dominant inheritance. Eleven members of the family lacked at least eight permanent teeth, two of whom developed only three permanent teeth. Colorectal cancer or precancerous lesions of variable types were found in eight of the patients with oligodontia. We show that oligodontia and predisposition to cancer are caused by a nonsense mutation, Arg656Stop, in the Wnt-signaling regulator AXIN2. In addition, we identified a de novo frameshift mutation 1994-1995insG in AXIN2 in an unrelated young patient with severe tooth agenesis. Both mutations are expected to activate Wnt signaling. The results provide the first evidence of the importance of Wnt signaling for the development of dentition in humans and suggest that an intricate control of Wnt-signal activity is necessary for normal tooth development, since both inhibition and stimulation of Wnt signaling may lead to tooth agenesis. Our findings introduce a new gene for hereditary colorectal cancer and suggest that tooth agenesis may be an indicator of cancer susceptibility. PMID:15042511

  19. Mutations in AXIN2 cause familial tooth agenesis and predispose to colorectal cancer.

    PubMed

    Lammi, Laura; Arte, Sirpa; Somer, Mirja; Jarvinen, Heikki; Lahermo, Paivi; Thesleff, Irma; Pirinen, Sinikka; Nieminen, Pekka

    2004-05-01

    Wnt signaling regulates embryonic pattern formation and morphogenesis of most organs. Aberrations of regulation of Wnt signaling may lead to cancer. Here, we have used positional cloning to identify the causative mutation in a Finnish family in which severe permanent tooth agenesis (oligodontia) and colorectal neoplasia segregate with dominant inheritance. Eleven members of the family lacked at least eight permanent teeth, two of whom developed only three permanent teeth. Colorectal cancer or precancerous lesions of variable types were found in eight of the patients with oligodontia. We show that oligodontia and predisposition to cancer are caused by a nonsense mutation, Arg656Stop, in the Wnt-signaling regulator AXIN2. In addition, we identified a de novo frameshift mutation 1994-1995insG in AXIN2 in an unrelated young patient with severe tooth agenesis. Both mutations are expected to activate Wnt signaling. The results provide the first evidence of the importance of Wnt signaling for the development of dentition in humans and suggest that an intricate control of Wnt-signal activity is necessary for normal tooth development, since both inhibition and stimulation of Wnt signaling may lead to tooth agenesis. Our findings introduce a new gene for hereditary colorectal cancer and suggest that tooth agenesis may be an indicator of cancer susceptibility.

  20. Wnt/β-catenin signaling via Axin2 is required for myogenesis and, together with YAP/Taz and Tead1, active in IIa/IIx muscle fibers.

    PubMed

    Huraskin, Danyil; Eiber, Nane; Reichel, Martin; Zidek, Laura M; Kravic, Bojana; Bernkopf, Dominic; von Maltzahn, Julia; Behrens, Jürgen; Hashemolhosseini, Said

    2016-09-01

    Canonical Wnt/β-catenin signaling plays an important role in myogenic differentiation, but its physiological role in muscle fibers remains elusive. Here, we studied activation of Wnt/β-catenin signaling in adult muscle fibers and muscle stem cells in an Axin2 reporter mouse. Axin2 is a negative regulator and a target of Wnt/β-catenin signaling. In adult muscle fibers, Wnt/β-catenin signaling is only detectable in a subset of fast fibers that have a significantly smaller diameter than other fast fibers. In the same fibers, immunofluorescence staining for YAP/Taz and Tead1 was detected. Wnt/β-catenin signaling was absent in quiescent and activated satellite cells. Upon injury, Wnt/β-catenin signaling was detected in muscle fibers with centrally located nuclei. During differentiation of myoblasts expression of Axin2, but not of Axin1, increased together with Tead1 target gene expression. Furthermore, absence of Axin1 and Axin2 interfered with myoblast proliferation and myotube formation, respectively. Treatment with the canonical Wnt3a ligand also inhibited myotube formation. Wnt3a activated TOPflash and Tead1 reporter activity, whereas neither reporter was activated in the presence of Dkk1, an inhibitor of canonical Wnt signaling. We propose that Axin2-dependent Wnt/β-catenin signaling is involved in myotube formation and, together with YAP/Taz/Tead1, associated with reduced muscle fiber diameter of a subset of fast fibers. PMID:27578179

  1. Loss of Axin2 Causes Ocular Defects During Mouse Eye Development

    PubMed Central

    Alldredge, Ashley; Fuhrmann, Sabine

    2016-01-01

    Purpose The scaffold protein Axin2 is an antagonist and universal target of the Wnt/β-catenin pathway. Disruption of Axin2 may lead to developmental eye defects; however, this has not been examined. The purpose of this study was to investigate the role of Axin2 during ocular and extraocular development in mouse. Methods Animals heterozygous and homozygous for a Axin2lacZ knock-in allele were analyzed at different developmental stages for reporter expression, morphology as well as for the presence of ocular and extraocular markers using histologic and immunohistochemical techniques. Results During early eye development, the Axin2lacZ reporter was expressed in the periocular mesenchyme, RPE, and optic stalk. In the developing retina, Axin2lacZ reporter expression was initiated in ganglion cells at late embryonic stages and robustly expressed in subpopulations of amacrine and horizontal cells postnatally. Activation of the Axin2lacZ reporter overlapped with labeling of POU4F1, PAX6, and Calbindin. Germline deletion of Axin2 led to variable ocular phenotypes ranging from normal to severely defective eyes exhibiting microphthalmia, coloboma, lens defects, and expanded ciliary margin. These defects were correlated with abnormal tissue patterning in individual affected tissues, such as the optic fissure margins in the ventral optic cup and in the expanded ciliary margin. Conclusions Our results reveal a critical role for Axin2 during ocular development, likely by restricting the activity of the Wnt/β-catenin pathway. PMID:27701636

  2. RAGE, receptor of advanced glycation endoproducts, negatively regulates chondrocytes differentiation.

    PubMed

    Kosaka, Tatsuya; Fukui, Rino; Matsui, Mio; Kurosaka, Yuko; Nishimura, Haruka; Tanabe, Motoki; Takakura, Yuuki; Iwai, Keisuke; Waki, Takuya; Fujita, Takashi

    2014-01-01

    RAGE, receptor for advanced glycation endoproducts (AGE), has been characterized as an activator of osteoclastgenesis. However, whether RAGE directly regulates chondrocyte proliferation and differentiation is unclear. Here, we show that RAGE has an inhibitory role in chondrocyte differentiation. RAGE expression was observed in chondrocytes from the prehypertrophic to hypertrophic regions. In cultured cells, overexpression of RAGE or dominant-negative-RAGE (DN-RAGE) demonstrated that RAGE inhibited cartilaginous matrix production, while DN-RAGE promoted production. Additionally, RAGE regulated Ihh and Col10a1 negatively but upregulated PTHrP receptor. Ihh promoter analysis and real-time PCR analysis suggested that downregulation of Cdxs was the key for RAGE-induced inhibition of chondrocyte differentiation. Overexpression of the NF-κB inhibitor I-κB-SR inhibited RAGE-induced NF-κB activation, but did not influence inhibition of cartilaginous matrix production by RAGE. The inhibitory action of RAGE was restored by the Rho family GTPases inhibitor Toxin B. Furthermore, inhibitory action on Ihh, Col10a1 and Cdxs was reproduced by constitutively active forms, L63RhoA, L61Rac, and L61Cdc42, but not by I-κB-SR. Cdx1 induced Ihh and Col10a1 expressions and directly interacted with Ihh promoter. Retinoic acid (RA) partially rescued the inhibitory action of RAGE. These data combined suggests that RAGE negatively regulates chondrocyte differentiation at the prehypertrophic stage by modulating NF-κB-independent and Rho family GTPases-dependent mechanisms.

  3. RAGE, Receptor of Advanced Glycation Endoproducts, Negatively Regulates Chondrocytes Differentiation

    PubMed Central

    Kurosaka, Yuko; Nishimura, Haruka; Tanabe, Motoki; Takakura, Yuuki; Iwai, Keisuke; Waki, Takuya; Fujita, Takashi

    2014-01-01

    RAGE, receptor for advanced glycation endoproducts (AGE), has been characterized as an activator of osteoclastgenesis. However, whether RAGE directly regulates chondrocyte proliferation and differentiation is unclear. Here, we show that RAGE has an inhibitory role in chondrocyte differentiation. RAGE expression was observed in chondrocytes from the prehypertrophic to hypertrophic regions. In cultured cells, overexpression of RAGE or dominant-negative-RAGE (DN-RAGE) demonstrated that RAGE inhibited cartilaginous matrix production, while DN-RAGE promoted production. Additionally, RAGE regulated Ihh and Col10a1 negatively but upregulated PTHrP receptor. Ihh promoter analysis and real-time PCR analysis suggested that downregulation of Cdxs was the key for RAGE-induced inhibition of chondrocyte differentiation. Overexpression of the NF-κB inhibitor I-κB-SR inhibited RAGE-induced NF-κB activation, but did not influence inhibition of cartilaginous matrix production by RAGE. The inhibitory action of RAGE was restored by the Rho family GTPases inhibitor Toxin B. Furthermore, inhibitory action on Ihh, Col10a1 and Cdxs was reproduced by constitutively active forms, L63RhoA, L61Rac, and L61Cdc42, but not by I-κB-SR. Cdx1 induced Ihh and Col10a1 expressions and directly interacted with Ihh promoter. Retinoic acid (RA) partially rescued the inhibitory action of RAGE. These data combined suggests that RAGE negatively regulates chondrocyte differentiation at the prehypertrophic stage by modulating NF-κB-independent and Rho family GTPases-dependent mechanisms. PMID:25275461

  4. Association of AXIN2 with Non-syndromic Oral Clefts in Multiple Populations

    PubMed Central

    Letra, A.; Bjork, B.; Cooper, M.E.; Szabo-Rogers, H.; Deleyiannis, F.W.B.; Field, L.L.; Czeizel, A.E.; Ma, L.; Garlet, G.P.; Poletta, F.A.; Mereb, J.C.; Lopez-Camelo, J.S.; Castilla, E.E.; Orioli, I.M.; Wendell, S.; Blanton, S.H.; Liu, K.; Hecht, J.T.; Marazita, M.L.; Vieira, A.R.; Silva, R.M.

    2012-01-01

    We have previously shown the association of AXIN2 with oral clefts in a US population. Here, we expanded our study to explore the association of 11 AXIN2 markers in 682 cleft families from multiple populations. Alleles for each AXIN2 marker were tested for transmission distortion with clefts by means of the Family-based Association Test. We observed an association with SNP rs7224837 and all clefts in the combined populations (p = 0.001), and with SNP rs3923086 and cleft lip and palate in Asian populations (p = 0.004). We confirmed our association findings in an additional 528 cleft families from the United States (p < 0.009). We tested for gene-gene interaction between AXIN2 and additional cleft susceptibility loci. We assessed and detected Axin2 mRNA and protein expression during murine palatogenesis. In addition, we also observed co-localization of Axin2 with Irf6 proteins, particularly in the epithelium. Our results continue to support a role for AXIN2 in the etiology of human clefting. Additional studies should be performed to improve our understanding of the biological mechanisms linking AXIN2 to oral clefts. PMID:22370446

  5. Self-renewing diploid Axin2+ cells fuel homeostatic renewal of the liver

    PubMed Central

    Wang, Bruce; Zhao, Ludan; Fish, Matt; Logan, Catriona Y.; Nusse, Roel

    2015-01-01

    Summary The source of new hepatocytes in the uninjured liver has remained an open question. By lineage tracing using the Wnt-responsive gene Axin2, we identify a population of proliferating and self-renewing cells adjacent to the central vein in the liver lobule. These pericentral cells express the early liver progenitor marker Tbx3, are diploid, and thus differ from mature hepatocytes, which are mostly polyploid. The descendants of pericentral cells differentiate into Tbx3-negative, polyploid hepatocytes and can replace all hepatocytes along the liver lobule during homeostatic renewal. Adjacent central vein endothelial cells provide Wnt signals that maintain the pericentral cells, thereby constituting the niche. Thus, we identify a cell population in the liver that subserves homeostatic hepatocyte renewal, characterize its anatomical niche, and identify molecular signals that regulate its activity. PMID:26245375

  6. Self-renewing diploid Axin2(+) cells fuel homeostatic renewal of the liver.

    PubMed

    Wang, Bruce; Zhao, Ludan; Fish, Matt; Logan, Catriona Y; Nusse, Roel

    2015-08-13

    The source of new hepatocytes in the uninjured liver has remained an open question. By lineage tracing using the Wnt-responsive gene Axin2 in mice, we identify a population of proliferating and self-renewing cells adjacent to the central vein in the liver lobule. These pericentral cells express the early liver progenitor marker Tbx3, are diploid, and thereby differ from mature hepatocytes, which are mostly polyploid. The descendants of pericentral cells differentiate into Tbx3-negative, polyploid hepatocytes, and can replace all hepatocytes along the liver lobule during homeostatic renewal. Adjacent central vein endothelial cells provide Wnt signals that maintain the pericentral cells, thereby constituting the niche. Thus, we identify a cell population in the liver that subserves homeostatic hepatocyte renewal, characterize its anatomical niche, and identify molecular signals that regulate its activity.

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

    PubMed

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

    2016-03-01

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

  8. Notch signaling indirectly promotes chondrocyte hypertrophy via regulation of BMP signaling and cell cycle arrest

    PubMed Central

    Shang, Xifu; Wang, Jinwu; Luo, Zhengliang; Wang, Yongjun; Morandi, Massimo M.; Marymont, John V.; Hilton, Matthew J.; Dong, Yufeng

    2016-01-01

    Cell cycle regulation is critical for chondrocyte differentiation and hypertrophy. Recently we identified the Notch signaling pathway as an important regulator of chondrocyte proliferation and differentiation during mouse cartilage development. To investigate the underlying mechanisms, we assessed the role for Notch signaling regulation of the cell cycle during chondrocyte differentiation. Real-time RT-PCR data showed that over-expression of the Notch Intracellular Domain (NICD) significantly induced the expression of p57, a cell cycle inhibitor, in chondrocytes. Flow cytometric analyses further confirmed that over-expression of NICD in chondrocytes enhances the G0/G1 cell cycle transition and cell cycle arrest. In contrast, treatment of chondrocytes with the Notch inhibitor, DAPT, decreased both endogenous and BMP2-induced SMAD 1/5/8 phosphorylation and knockdown of SMAD 1/5/8 impaired NICD-induced chondrocyte differentiation and p57 expression. Co-immunoprecipitation using p-SMAD 1/5/8 and NICD antibodies further showed a strong interaction of these proteins during chondrocyte maturation. Finally, RT-PCR and Western blot results revealed a significant reduction in the expression of the SMAD-related phosphatase, PPM1A, following NICD over-expression. Taken together, our results demonstrate that Notch signaling induces cell cycle arrest and thereby initiates chondrocyte hypertrophy via BMP/SMAD-mediated up-regulation of p57. PMID:27146698

  9. Notch signaling controls chondrocyte hypertrophy via indirect regulation of Sox9

    PubMed Central

    Kohn, Anat; Rutkowski, Timothy P; Liu, Zhaoyang; Mirando, Anthony J; Zuscik, Michael J; O’Keefe, Regis J; Hilton, Matthew J

    2015-01-01

    RBPjk-dependent Notch signaling regulates both the onset of chondrocyte hypertrophy and the progression to terminal chondrocyte maturation during endochondral ossification. It has been suggested that Notch signaling can regulate Sox9 transcription, although how this occurs at the molecular level in chondrocytes and whether this transcriptional regulation mediates Notch control of chondrocyte hypertrophy and cartilage development is unknown or controversial. Here we have provided conclusive genetic evidence linking RBPjk-dependent Notch signaling to the regulation of Sox9 expression and chondrocyte hypertrophy by examining tissue-specific Rbpjk mutant (Prx1Cre;Rbpjkf/f), Rbpjk mutant/Sox9 haploinsufficient (Prx1Cre;Rbpjkf/f;Sox9f/+), and control embryos for alterations in SOX9 expression and chondrocyte hypertrophy during cartilage development. These studies demonstrate that Notch signaling regulates the onset of chondrocyte maturation in a SOX9-dependent manner, while Notch-mediated regulation of terminal chondrocyte maturation likely functions independently of SOX9. Furthermore, our in vitro molecular analyses of the Sox9 promoter and Notch-mediated regulation of Sox9 gene expression in chondrogenic cells identified the ability of Notch to induce Sox9 expression directly in the acute setting, but suppresses Sox9 transcription with prolonged Notch signaling that requires protein synthesis of secondary effectors. PMID:26558140

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

    PubMed

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

    2010-12-01

    Alternations in cartilage chondrocyte phenotype characteristic by the decreased type II collagen and aggrecan together with increased type X collagen synthesis serve as a beacon for osteoarthritis progression. However, little is known about the underlying molecular mechanisms. The current study seeks to discover molecules that involved in osteoarthritic chondrocytes phenotype regulation. Differential proteomics was generated with two-dimensional gel electrophoresis between normal articular cartilage (NAC) and advanced osteoarthritic cartilage (AOC). Those differentially expressed proteins were identified by mass spectrometry. The down-regulation of a neuronal silencer, the REST corepressor (CoREST) in AOC, was verified by Western blot. CoREST silencing was performed in primarily cultured NAC chondrocytes with specific siRNA to reveal the possible involvement of CoREST repression in chondrocyte phenotypic genes modulation. Ninteen differentially expressed proteins were screened and identified. Among these proteins, CoREST, HHL, and zinc finger protein 155 were estimated to be possible gene modulators. CoREST protein level was verified to be down-regulated by 69.5% (p < 0.001) in AOC. In response to CoREST knock-down by 64.8% (p < 0.001) in NAC chondrocytes, the gene expression level of the chondrocyte terminal differentiation marker gene, collagen X was found to be up-regulated by 40.0% (p = 0.017), whereas the chondrocyte differentiation phenotypic genes, collagen II and aggrecan were down-regulated by 71.4% (p < 0.001) and 57.6% (p < 0.001), respectively. The results indicate that the silencing of CoREST by siRNA transfection in NAC may reflect CoREST repression in AOC, which results in phenotypic genes modulation and suggests a homeostatic role of this transcription factor in articular chondrocyte.

  11. Regulation of chondrocyte differentiation by the actin cytoskeleton and adhesive interactions.

    PubMed

    Woods, Anita; Wang, Guoyan; Beier, Frank

    2007-10-01

    Chondrocyte differentiation is a multi-step process characterized by successive changes in cell morphology and gene expression. In addition to tight regulation by numerous soluble factors, these processes are controlled by adhesive events. During the early phase of the chondrocyte life cycle, cell-cell adhesion through molecules such as N-cadherin and neural cell adhesion molecule (N-CAM) is required for differentiation of mesenchymal precursor cells to chondrocytes. At later stages, for example in growth plate chondrocytes, adhesion signaling from extracellular matrix (ECM) proteins through integrins and other ECM receptors such as the discoidin domain receptor (DDR) 2 (a collagen receptor) and Annexin V is necessary for normal chondrocyte proliferation and hypertrophy. Cell-matrix interactions are also important for chondrogenesis, for example through the activity of CD44, a receptor for Hyaluronan and collagens. The roles of several signaling molecules involved in adhesive signaling, such as integrin-linked kinase (ILK) and Rho GTPases, during chondrocyte differentiation are beginning to be understood, and the actin cytoskeleton has been identified as a common target of these adhesive pathways. Complete elucidation of the pathways connecting adhesion receptors to downstream effectors and the mechanisms integrating adhesion signaling with growth factor- and hormone-induced pathways is required for a better understanding of physiological and pathological skeletal development.

  12. Regulation of collagenase inhibitor production in chondrosarcoma chondrocytes

    SciTech Connect

    Harper, J.; Harper, E.

    1987-05-01

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

  13. mTORC1 regulates PTHrP to coordinate chondrocyte growth, proliferation and differentiation

    PubMed Central

    Yan, Bo; Zhang, Zhongmin; Jin, Dadi; Cai, Chen; Jia, Chunhong; Liu, Wen; Wang, Ting; Li, Shengfa; Zhang, Haiyan; Huang, Bin; Lai, Pinglin; Wang, Hua; Liu, Anling; Zeng, Chun; Cai, Daozhang; Jiang, Yu; Bai, Xiaochun

    2016-01-01

    Precise coordination of cell growth, proliferation and differentiation is essential for the development of multicellular organisms. Here, we report that although the mechanistic target of rapamycin complex 1 (mTORC1) activity is required for chondrocyte growth and proliferation, its inactivation is essential for chondrocyte differentiation. Hyperactivation of mTORC1 via TSC1 gene deletion in chondrocytes causes uncoupling of the normal proliferation and differentiation programme within the growth plate, resulting in uncontrolled cell proliferation, and blockage of differentiation and chondrodysplasia in mice. Rapamycin promotes chondrocyte differentiation and restores these defects in mutant mice. Mechanistically, mTORC1 downstream kinase S6K1 interacts with and phosphorylates Gli2, and releases Gli2 from SuFu binding, resulting in nuclear translocation of Gli2 and transcription of parathyroid hormone-related peptide (PTHrP), a key regulator of bone development. Our findings demonstrate that dynamically controlled mTORC1 activity is crucial to coordinate chondrocyte proliferation and differentiation partially through regulating Gli2/PTHrP during endochondral bone development. PMID:27039827

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

    PubMed

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

    2016-07-01

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

  15. IFT80 is essential for chondrocyte differentiation by regulating hedgehog and Wnt signaling pathways

    PubMed Central

    Wang, Changdong; Yuan, Xue; Yang, Shuying

    2013-01-01

    Partial mutation of intraflagellar transport 80 (IFT80) in humans causes Jeune asphyxiating thoracic dystrophy (JATD) and short-rib polydactyly (SRP) syndrome type III. These diseases are autosomal recessive chondrodysplasias that share clinical similarities, including shortened long bones and constricted thoracic cage. However, the role and mechanism of IFT80 in the regulation of chondrocyte differentiation and function remain largely unknown. We hypothesize that IFT80 is required for the formation and function of cilia and plays a critical role in chondrogenic differentiation by regulating Hedgehog (Hh) and Wingless (Wnt) signaling pathways. To test this hypothesis, we first analyzed the IFT80 expression pattern and found that IFT80 was predominantly expressed in growth plate chondrocytes and during chondrogenic differentiation. Silencing IFT80 impaired cilia formation and chondrogenic differentiation in mouse bone marrow derived stromal cells (BMSCs), and decreased the expression of chondrocyte marker genes—collagen II and aggrecan. Additionally, silencing IFT80 down-regulated Hh signaling activity whereas up-regulated Wnt signaling activity. The overexpression of Gli2 in IFT80-silenced cells promoted chondrogenesis and recovered the chondrogenic deficiency from IFT80 silencing. Overall, our results demonstrate that IFT80 is essential for chondrocyte differentiation by regulating the Hh and Wnt signaling pathways. PMID:23333501

  16. Regulation of human mesenchymal stem cells differentiation into chondrocytes in extracellular matrix-based hydrogel scaffolds.

    PubMed

    Du, Mingchun; Liang, Hui; Mou, Chenchen; Li, Xiaoran; Sun, Jie; Zhuang, Yan; Xiao, Zhifeng; Chen, Bing; Dai, Jianwu

    2014-02-01

    To induce human mesenchymal stem cells (hMSCs) to differentiate into chondrocytes in three-dimensional (3D) microenvironments, we developed porous hydrogel scaffolds using the cartilage extracellular matrix (ECM) components of chondroitin sulfate (CS) and collagen (COL). The turbidity and viscosity experiments indicated hydrogel could form through pH-triggered co-precipitation when pH=2-3. Enzyme-linked immunosorbent assay (ELISA) confirmed the hydrogel scaffolds could controllably release growth factors as envisaged. Transforming growth factor-β (TGF-β) was released to stimulate hMSCs differentiation into chondrocytes; and then collagen binding domain-basic fibroblast growth factor (CBD-bFGF) was released to improve the differentiation and preserve the chondrocyte phenotype. In in vitro cell culture experiments, the differentiation processes were compared in different microenvironments: 2D culture in culture plate as control, 3D culture in the fabricated scaffolds without growth factors (CC), the samples with CBD-bFGF (CC-C), the samples with TGF-β (CC-T), the samples with CBD-bFGF/TGF-β (CC-CT). Real-time polymerase chain reaction (RT-PCR) revealed the hMSC marker genes of CD44 and CD105 decreased; at the same time the chondrocyte marker genes of collagen type II and aggrecan increased, especially in the CC-CT sample. Immunostaining results further confirmed the hMSC marker protein of CD 44 disappeared and the chondrocyte marker protein of collagen type II emerged over time in the CC-CT sample. These results imply the ECM-based hydrogel scaffolds with growth factors can supply suitable 3D cell niches for hMSCs differentiation into chondrocytes and the differentiation process can be regulated by the controllably released growth factors. PMID:24231133

  17. Protein kinase B and extracellular signal-regulated kinase contribute to the chondroprotective effect of morroniside on osteoarthritis chondrocytes

    PubMed Central

    Cheng, Liang; Zeng, Guoqing; Liu, Zejun; Zhang, Bing; Cui, Xu; Zhao, Honghai; Zheng, Xinpeng; Song, Gang; Kang, Jian; Xia, Chun

    2015-01-01

    Despite extensive studies on the multifaceted roles of morroniside, the main active constituent of iridoid glycoside from Corni Fructus, the effect of morroniside on osteoarthritis (OA) chondrocytes remains poorly understood. Here, we investigated the influence of morroniside on cultured human OA chondrocytes and a rat experimental model of OA. The results showed that morroniside enhanced the cell viability and the levels of proliferating cell nuclear antigen expression (PCNA), type II collagen and aggrecan in human OA chondrocytes, indicating that morroniside promoted chondrocyte survival and matrix synthesis. Furthermore, different doses of morroniside activated protein kinase B (AKT) and extracellular signal-regulated kinase (ERK) in human OA chondrocytes, and in turn, triggered AKT/S6 and ERK/P70S6K/S6 pathway, respectively. The PI3K/AKT inhibitor LY294002 or the MEK/ERK inhibitor U0126 attenuated the effect of morroniside on human OA chondrocytes, indicating that the activation of AKT and ERK contributed to the regulation of morroniside in human OA chondrocytes. In addition, the intra-articular injection of morroniside elevated the level of proteoglycans in cartilage matrix and the thickness of articular cartilage in a rat experimental model of OA, with the increase of AKT and ERK activation. As a consequence, morroniside has chondroprotective effect on OA chondrocytes, and may have the therapeutic potential for OA treatment. PMID:25754021

  18. TRPV4-mediated mechanotransduction regulates the metabolic response of chondrocytes to dynamic loading

    PubMed Central

    O’Conor, Christopher J.; Leddy, Holly A.; Benefield, Halei C.; Liedtke, Wolfgang B.; Guilak, Farshid

    2014-01-01

    Mechanical loading of joints plays a critical role in maintaining the health and function of articular cartilage. The mechanism(s) of chondrocyte mechanotransduction are not fully understood, but could provide important insights into new physical or pharmacologic therapies for joint diseases. Transient receptor potential vanilloid 4 (TRPV4), a Ca2+-permeable osmomechano-TRP channel, is highly expressed in articular chondrocytes, and loss of TRPV4 function is associated with joint arthropathy and osteoarthritis. The goal of this study was to examine the hypothesis that TRPV4 transduces dynamic compressive loading in articular chondrocytes. We first confirmed the presence of physically induced, TRPV4-dependent intracellular Ca2+ signaling in agarose-embedded chondrocytes, and then used this model system to study the role of TRPV4 in regulating the response of chondrocytes to dynamic compression. Inhibition of TRPV4 during dynamic loading prevented acute, mechanically mediated regulation of proanabolic and anticatabolic genes, and furthermore, blocked the loading-induced enhancement of matrix accumulation and mechanical properties. Furthermore, chemical activation of TRPV4 by the agonist GSK1016790A in the absence of mechanical loading similarly enhanced anabolic and suppressed catabolic gene expression, and potently increased matrix biosynthesis and construct mechanical properties. These findings support the hypothesis that TRPV4-mediated Ca2+ signaling plays a central role in the transduction of mechanical signals to support cartilage extracellular matrix maintenance and joint health. Moreover, these insights raise the possibility of therapeutically targeting TRPV4-mediated mechanotransduction for the treatment of diseases such as osteoarthritis, as well as to enhance matrix formation and functional properties of tissue-engineered cartilage as an alternative to bioreactor-based mechanical stimulation. PMID:24474754

  19. NF-{kappa}B regulates Lef1 gene expression in chondrocytes

    SciTech Connect

    Yun, Kangsun; Choi, Yoo Duk; Nam, Jong Hee; Park, Zeeyoung; Im, Sin-Hyeog . E-mail: imsh@gist.ac.kr

    2007-06-08

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

  20. Millimeter wave promotes the synthesis of extracellular matrix and the proliferation of chondrocyte by regulating the voltage-gated K+ channel.

    PubMed

    Li, Xihai; Liu, Chao; Liang, Wenna; Ye, Hongzhi; Chen, Wenlie; Lin, Ruhui; Li, Zuanfang; Liu, Xianxiang; Wu, Mingxia

    2014-07-01

    Previously, we reported that millimeter wave promoted the chondrocyte proliferation by pushing cell cycle progression. Activation of K(+) channels plays an essential role in the stimulating of extracellular matrix (ECM) synthesis and the cell proliferation in chondrocytes. While it is unclear if millimeter wave enhances ECM synthesis and proliferation of chondrocytes by regulating K(+) channel activity, we here investigated the effects of millimeter waves on ECM synthesis, chondrocyte proliferation and ion channels in the primary chondrocyte culture. We found that millimeter waves led to the increase of chondrocyte viability, the morphological changes of chondrocyte, and the F-actin distortion and remodeling. Ultrastructural analysis showed that treated chondrocytes contained an expansion of mitochondria and granular endoplasmic reticulum, and a high number of cytoplasmic vesicles in the cytoplasm compared to untreated cells, suggesting millimeter waves increased the energy metabolism and protein synthesis of chondrocytes. The analysis of differential ion channels' genes expression further showed an obvious increase of Kcne1, Kcnj3 and Kcnq2. To determine the role of voltage-gated K(+) channel in chondrocyte, we blocked the voltage-gated K(+) channel with 10 mM tetraethylammonium (TEA) and treated chondrocytes with millimeter waves. The results indicated that TEA significantly negated the promotion of millimeter waves for the ECM synthesis and chondrocyte proliferation. Our results support the hypothesis that millimeter waves promote the synthesis of ECM and the proliferation of chondrocyte by regulating the voltage-gated K(+) channel.

  1. MicroRNA-381 Regulates Chondrocyte Hypertrophy by Inhibiting Histone Deacetylase 4 Expression.

    PubMed

    Chen, Weishen; Sheng, Puyi; Huang, Zhiyu; Meng, Fangang; Kang, Yan; Huang, Guangxin; Zhang, Zhiqi; Liao, Weiming; Zhang, Ziji

    2016-01-01

    Chondrocyte hypertrophy, regulated by Runt-related transcription factor 2 (RUNX2) and matrix metalloproteinase 13 (MMP13), is a crucial step in cartilage degeneration and osteoarthritis (OA) pathogenesis. We previously demonstrated that microRNA-381 (miR-381) promotes MMP13 expression during chondrogenesis and contributes to cartilage degeneration; however, the mechanism underlying this process remained unclear. In this study, we observed divergent expression of miR-381 and histone deacetylase 4 (HDAC4), an enzyme that directly inhibits RUNX2 and MMP13 expression, during late-stage chondrogenesis of ATDC5 cells, as well as in prehypertrophic and hypertrophic chondrocytes during long bone development in E16.5 mouse embryos. We therefore investigated whether this miRNA regulates HDAC4 expression during chondrogenesis. Notably, overexpression of miR-381 inhibited HDAC4 expression but promoted RUNX2 expression. Moreover, transfection of SW1353 cells with an miR-381 mimic suppressed the activity of a reporter construct containing the 3'-untranslated region (3'-UTR) of HDAC4. Conversely, treatment with a miR-381 inhibitor yielded increased HDAC4 expression and decreased RUNX2 expression. Lastly, knockdown of HDAC4 expression resulted in increased RUNX2 and MMP13 expression in SW1353 cells. Collectively, our results indicate that miR-381 epigenetically regulates MMP13 and RUNX2 expression via targeting of HDAC4, thereby suggesting the possibilities of inhibiting miR-381 to control chondrocyte hypertrophy and cartilage degeneration. PMID:27563877

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

    PubMed Central

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

    2012-01-01

    Type II collagen is the major component of articular cartilage and is mainly synthesized by chondrocytes. Repeated sub-culturing of primary chondrocytes leads to reduction of type II collagen gene (Col2a1) expression, which mimics the process of chondrocyte dedifferentiation. Although the functional importance of Col2a1 expression has been extensively investigated, mechanism of transcriptional regulation during chondrocyte dedifferentiation is still unclear. In this study, we have investigated the crosstalk between cis-acting DNA element and transcription factor on Col2a1 expression in primary chondrocytes. Bioinformatic analysis revealed the potential regulatory regions in the Col2a1 genomic locus. Among them, promoter and 3′ untranslated region (UTR) showed highly accessible chromatin architecture with enriched recruitment of active chromatin markers in primary chondrocytes. 3′ UTR has a potent enhancer function which recruits Lef1 (Lymphoid enhancer binding factor 1) transcription factor, leading to juxtaposition of the 3′ UTR with the promoter through gene looping resulting in up-regulation of Col2a1 gene transcription. Knock-down of endogenous Lef1 level significantly reduced the gene looping and subsequently down-regulated Col2a1 expression. However, these regulatory loci become inaccessible due to condensed chromatin architecture as chondrocytes dedifferentiate which was accompanied by a reduction of gene looping and down-regulation of Col2a1 expression. Our results indicate that Lef1 mediated looping between promoter and 3′ UTR under the permissive chromatin architecture upregulates Col2a1 expression in primary chondrocytes. PMID:22815835

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

    PubMed Central

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

    2016-01-01

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

  4. MicroRNA-381 Regulates Chondrocyte Hypertrophy by Inhibiting Histone Deacetylase 4 Expression

    PubMed Central

    Chen, Weishen; Sheng, Puyi; Huang, Zhiyu; Meng, Fangang; Kang, Yan; Huang, Guangxin; Zhang, Zhiqi; Liao, Weiming; Zhang, Ziji

    2016-01-01

    Chondrocyte hypertrophy, regulated by Runt-related transcription factor 2 (RUNX2) and matrix metalloproteinase 13 (MMP13), is a crucial step in cartilage degeneration and osteoarthritis (OA) pathogenesis. We previously demonstrated that microRNA-381 (miR-381) promotes MMP13 expression during chondrogenesis and contributes to cartilage degeneration; however, the mechanism underlying this process remained unclear. In this study, we observed divergent expression of miR-381 and histone deacetylase 4 (HDAC4), an enzyme that directly inhibits RUNX2 and MMP13 expression, during late-stage chondrogenesis of ATDC5 cells, as well as in prehypertrophic and hypertrophic chondrocytes during long bone development in E16.5 mouse embryos. We therefore investigated whether this miRNA regulates HDAC4 expression during chondrogenesis. Notably, overexpression of miR-381 inhibited HDAC4 expression but promoted RUNX2 expression. Moreover, transfection of SW1353 cells with an miR-381 mimic suppressed the activity of a reporter construct containing the 3′-untranslated region (3′-UTR) of HDAC4. Conversely, treatment with a miR-381 inhibitor yielded increased HDAC4 expression and decreased RUNX2 expression. Lastly, knockdown of HDAC4 expression resulted in increased RUNX2 and MMP13 expression in SW1353 cells. Collectively, our results indicate that miR-381 epigenetically regulates MMP13 and RUNX2 expression via targeting of HDAC4, thereby suggesting the possibilities of inhibiting miR-381 to control chondrocyte hypertrophy and cartilage degeneration. PMID:27563877

  5. Smad2 and Smad3 Regulate Chondrocyte Proliferation and Differentiation in the Growth Plate

    PubMed Central

    Wang, Weiguang; Song, Buer; Anbarchian, Teni; Shirazyan, Anna

    2016-01-01

    TGFβs act through canonical and non-canonical pathways, and canonical signals are transduced via Smad2 and Smad3. However, the contribution of canonical vs. non-canonical pathways in cartilage is unknown because the role of Smad2 in chondrogenesis has not been investigated in vivo. Therefore, we analyzed mice in which Smad2 is deleted in cartilage (Smad2CKO), global Smad3-/- mutants, and crosses of these strains. Growth plates at birth from all mutant strains exhibited expanded columnar and hypertrophic zones, linked to increased proliferation in resting chondrocytes. Defects were more severe in Smad2CKO and Smad2CKO;Smad3-/- (Smad2/3) mutant mice than in Smad3-/- mice, demonstrating that Smad2 plays a role in chondrogenesis. Increased levels of Ihh RNA, a key regulator of chondrocyte proliferation and differentiation, were seen in prehypertrophic chondrocytes in the three mutant strains at birth. In accordance, TGFβ treatment decreased Ihh RNA levels in primary chondrocytes from control (Smad2fx/fx) mice, but inhibition was impaired in cells from mutants. Consistent with the skeletal phenotype, the impact on TGFβ-mediated inhibition of Ihh RNA expression was more severe in Smad2CKO than in Smad3-/- cells. Putative Smad2/3 binding elements (SBEs) were identified in the proximal Ihh promoter. Mutagenesis demonstrated a role for three of them. ChIP analysis suggested that Smad2 and Smad3 have different affinities for these SBEs, and that the repressors SnoN and Ski were differentially recruited by Smad2 and Smad3, respectively. Furthermore, nuclear localization of the repressor Hdac4 was decreased in growth plates of Smad2CKO and double mutant mice. TGFβ induced association of Hdac4 with Smad2, but not with Smad3, on the Ihh promoter. Overall, these studies revealed that Smad2 plays an essential role in the development of the growth plate, that both Smads 2 and 3 inhibit Ihh expression in the neonatal growth plate, and suggested they accomplish this by binding to

  6. Microfluidics-based optimization of neuroleukin-mediated regulation of articular chondrocyte proliferation

    PubMed Central

    TIAN, KANG; ZHONG, WEILIANG; ZHANG, YINGQIU; YIN, BAOSHENG; ZHANG, WEIGUO; LIU, HAN

    2016-01-01

    Due to the low proliferative and migratory capacities of chondrocytes, cartilage repair remains a challenging clinical problem. Current therapeutic strategies for cartilage repair result in unsatisfactory outcomes. Autologous chondrocyte implantation (ACI) is a cell based therapy that relies on the in vitro expansion of healthy chondrocytes from the patient, during which proliferation-promoting factors are frequently used. Neuroleukin (NLK) is a multifunctional protein that possesses growth factor functions, and its expression has been associated with cartilage development and bone regeneration, however its direct role in chondrocyte proliferation remains to be fully elucidated. In the current study, the role of NLK in chondrocyte proliferation in vitro in addition to its potential to act as an exogenous factor during ACI was investigated. Furthermore, the concentration of NLK for in vitro chondrocyte culture was optimized using a microfluidic device. An NLK concentration of 12.85 ng/ml was observed to provide optimal conditions for the promotion of chondrocyte proliferation. Additionally, NLK stimulation resulted in an increase in type II collagen synthesis by chondrocytes, which is a cartilaginous secretion marker and associated with the phenotype of chondrocytes. Together these data suggest that NLK is able to promote cell proliferation and type II collagen synthesis during in vitro chondrocyte propagation, and thus may serve as an exogenous factor for ACI. PMID:26573126

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

    PubMed

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

    2016-09-01

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

  8. Axin2 marks quiescent hair follicle bulge stem cells that are maintained by autocrine Wnt/β-catenin signaling.

    PubMed

    Lim, Xinhong; Tan, Si Hui; Yu, Ka Lou; Lim, Sophia Beng Hui; Nusse, Roeland

    2016-03-15

    How stem cells maintain their identity and potency as tissues change during growth is not well understood. In mammalian hair, it is unclear how hair follicle stem cells can enter an extended period of quiescence during the resting phase but retain stem cell potential and be subsequently activated for growth. Here, we use lineage tracing and gene expression mapping to show that the Wnt target gene Axin2 is constantly expressed throughout the hair cycle quiescent phase in outer bulge stem cells that produce their own Wnt signals. Ablating Wnt signaling in the bulge cells causes them to lose their stem cell potency to contribute to hair growth and undergo premature differentiation instead. Bulge cells express secreted Wnt inhibitors, including Dickkopf (Dkk) and secreted frizzled-related protein 1 (Sfrp1). However, the Dickkopf 3 (Dkk3) protein becomes localized to the Wnt-inactive inner bulge that contains differentiated cells. We find that Axin2 expression remains confined to the outer bulge, whereas Dkk3 continues to be localized to the inner bulge during the hair cycle growth phase. Our data suggest that autocrine Wnt signaling in the outer bulge maintains stem cell potency throughout hair cycle quiescence and growth, whereas paracrine Wnt inhibition of inner bulge cells reinforces differentiation.

  9. Axin2 marks quiescent hair follicle bulge stem cells that are maintained by autocrine Wnt/β-catenin signaling.

    PubMed

    Lim, Xinhong; Tan, Si Hui; Yu, Ka Lou; Lim, Sophia Beng Hui; Nusse, Roeland

    2016-03-15

    How stem cells maintain their identity and potency as tissues change during growth is not well understood. In mammalian hair, it is unclear how hair follicle stem cells can enter an extended period of quiescence during the resting phase but retain stem cell potential and be subsequently activated for growth. Here, we use lineage tracing and gene expression mapping to show that the Wnt target gene Axin2 is constantly expressed throughout the hair cycle quiescent phase in outer bulge stem cells that produce their own Wnt signals. Ablating Wnt signaling in the bulge cells causes them to lose their stem cell potency to contribute to hair growth and undergo premature differentiation instead. Bulge cells express secreted Wnt inhibitors, including Dickkopf (Dkk) and secreted frizzled-related protein 1 (Sfrp1). However, the Dickkopf 3 (Dkk3) protein becomes localized to the Wnt-inactive inner bulge that contains differentiated cells. We find that Axin2 expression remains confined to the outer bulge, whereas Dkk3 continues to be localized to the inner bulge during the hair cycle growth phase. Our data suggest that autocrine Wnt signaling in the outer bulge maintains stem cell potency throughout hair cycle quiescence and growth, whereas paracrine Wnt inhibition of inner bulge cells reinforces differentiation. PMID:26903625

  10. Axin2 marks quiescent hair follicle bulge stem cells that are maintained by autocrine Wnt/β-catenin signaling

    PubMed Central

    Lim, Xinhong; Tan, Si Hui; Yu, Ka Lou; Lim, Sophia Beng Hui; Nusse, Roeland

    2016-01-01

    How stem cells maintain their identity and potency as tissues change during growth is not well understood. In mammalian hair, it is unclear how hair follicle stem cells can enter an extended period of quiescence during the resting phase but retain stem cell potential and be subsequently activated for growth. Here, we use lineage tracing and gene expression mapping to show that the Wnt target gene Axin2 is constantly expressed throughout the hair cycle quiescent phase in outer bulge stem cells that produce their own Wnt signals. Ablating Wnt signaling in the bulge cells causes them to lose their stem cell potency to contribute to hair growth and undergo premature differentiation instead. Bulge cells express secreted Wnt inhibitors, including Dickkopf (Dkk) and secreted frizzled-related protein 1 (Sfrp1). However, the Dickkopf 3 (Dkk3) protein becomes localized to the Wnt-inactive inner bulge that contains differentiated cells. We find that Axin2 expression remains confined to the outer bulge, whereas Dkk3 continues to be localized to the inner bulge during the hair cycle growth phase. Our data suggest that autocrine Wnt signaling in the outer bulge maintains stem cell potency throughout hair cycle quiescence and growth, whereas paracrine Wnt inhibition of inner bulge cells reinforces differentiation. PMID:26903625

  11. Cathepsin B expression and down-regulation by gene silencing and antisense DNA in human chondrocytes.

    PubMed Central

    Zwicky, Roman; Müntener, Kathrin; Goldring, Mary B; Baici, Antonio

    2002-01-01

    Cathepsin B, a marker of the dedifferentiated chondrocyte phenotype, contributes to cartilage destruction in osteoarthritis and pathological proteolysis in rheumatoid arthritis and cancer. In search of possible means for neutralizing the action of this enzyme, we compared its expression, biosynthesis and distribution in articular chondrocytes and two lines of immortalized human chondrocytes. Native articular chondrocytes in primary culture and the polyclonal T/C-28a2 chondrocyte cell line were similar with respect to the number of endosomes and lysosomes, the distribution of three alternatively spliced cathepsin B mRNA forms, and the cathepsin B activity. In contrast, the clonal C-28/I2 cell line contained four times higher levels of intracellular cathepsin B activity, slightly higher numbers of endosomes and lysosomes, and uniform distribution of all three cathepsin B transcripts and thus resembled subcultured chondrocytes at an early stage of dedifferentiation. Transfection of T/C-28a2 chondrocytes with double-stranded cathepsin B mRNA resulted in inhibition of cathepsin B biosynthesis by up to 70% due to RNA interference, and single-stranded antisense DNAs of various sizes decreased cathepsin B biosynthesis by up to 78%. An antisense oligonucleotide designed to hybridize to the end of cathepsin B's exons 1 and the beginning of exon 3 was successful in specifically inhibiting the mRNA splice variant lacking exon 2. These results indicate that cathepsin B expression and activity may be targeted for gene silencing by RNA interference and antisense DNA in chondrocytes. Furthermore, the differential expression and distribution of cathepsin B and presence of the necessary molecular apparatus for gene silencing in the immortalized human chondrocyte cell lines indicate that they may serve as a useful model for studying the function of relevant enzymes in cartilage pathologies. PMID:12086583

  12. Suppression of Nkx3.2 by phosphatidylinositol-3-kinase signaling regulates cartilage development by modulating chondrocyte hypertrophy

    PubMed Central

    Kim, Jeong-Ah; Im, Suhjean; Cantley, Lewis C.; Kim, Dae-Won

    2016-01-01

    Phosphatidylinositol-3-kinase (PI3K) is a key regulator of diverse biological processes including cell proliferation, migration, survival, and differentiation. While a role of PI3K in chondrocyte differentiation has been suggested, its precise mechanisms of action are poorly understood. Here we show that PI3K signaling can down-regulate Nkx3.2 at both mRNA and protein levels in various chondrocyte cultures in vitro. In addition, we have intriguingly found that p85β, not p85α, is specifically employed as a regulatory subunit for PI3K-mediated Nkx3.2 suppression. Furthermore, we found that regulation of Nkx3.2 by PI3K requires Rac1–PAK1, but not Akt, signaling downstream of PI3K. Finally, using embryonic limb bud cultures, ex vivo long bone cultures, and p85β knockout mice, we demonstrated that PI3K-mediated suppression of Nkx3.2 in chondrocytes plays a role in the control of cartilage hypertrophy during skeletal development in vertebrates. PMID:26363466

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

    SciTech Connect

    Mayo, Jaime L.; Holden, Devin N.; Barrow, Jeffery R.; Bridgewater, Laura C.

    2009-08-01

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

  14. Millimeter Wave Treatment Inhibits Apoptosis of Chondrocytes via Regulation Dynamic Equilibrium of Intracellular Free Ca2+

    PubMed Central

    Ye, Jinxia; Wu, Guangwen; Li, Xihai; Li, Zuanfang; Zheng, Chunsong; Liu, Xianxiang; Ye, Hongzhi

    2015-01-01

    The molecular mechanisms of TNF-α-induced apoptosis of chondrocyte and the role of Ca2+ mediating the effects of MW on TNF-α-induced apoptosis of chondrocytes remained unclear. In this study, we investigated the molecular mechanism underlying inhibiting TNF-α-induced chondrocytes apoptosis of MW. MTT assay, DAPI, and flow cytometry demonstrated that MW significantly increased cell activity and inhibited chromatin condensation accompanying the loss of plasma membrane asymmetry and the collapse of mitochondrial membrane potential. Our results also indicated that MW reduced the elevation of [Ca2+]i in chondrocytes by LSCM. Moreover, MW suppressed the protein levels of calpain, Bax, cytochrome c, and caspase-3, while the expressions of Bcl-2, collagen II, and aggrecan were increased. Our evidences indicated that MW treatment inhibited the apoptosis of chondrocytes through depression of [Ca2+]i. It also inhibited calpain activation, which mediated Bax cleavage and cytochrome c release and initiated the apoptotic execution phase. In addition, MW treatment increased the expression of collagen II and aggrecan of chondrocytes. PMID:25705239

  15. Ionic osmolytes and intracellular calcium regulate tissue production in chondrocytes cultured in a 3D charged hydrogel.

    PubMed

    Farnsworth, Nikki L; Mead, Benjamin E; Antunez, Lorena R; Palmer, Amy E; Bryant, Stephanie J

    2014-11-01

    The goal of this study was to investigate the role of fixed negative charges in regulating cartilage-like tissue production by chondrocytes under static and dynamic three-dimensional culture, and to determine whether intracellular calcium ([Ca(2+)]i) is involved in mediating this response. Initial experiments using the 3D neutral hydrogel were conducted in static isotonic culture with ionic and non-ionic osmolytes added to the culture medium. Tissue production by bovine chondrocytes with non-ionic osmolytes was 1.9-fold greater than with ionic osmolytes, suggesting that the ionic nature of the osmolyte is an important regulator of tissue production. To investigate fixed negative charges, a 3D culture system containing encapsulated chondrocytes was employed based on a synthetic and neutral hydrogel platform within which negatively charged chondroitin sulfate was incorporated in a controlled manner. Incorporation of negative charges did not affect the mechanical properties of the hydrogel; however, intracellular ion concentration was elevated from the culture medium (330 mOsm) and estimated to be similar to that in ~400 mOsm culture medium. With dynamic loading, GAG synthesis decreased by 26% in neutral hydrogels cultured in 400mOsm medium, and increased by 26% in charged gels cultured in 330 mOsm. Treatment of chondrocyte-seeded hydrogels with the Ca(2+) chelator BAPTA-AM decreased GAG synthesis by 32-46% and was similar among all conditions, suggesting multiple roles for Ca(2+) mediated tissue production including with ionic osmolytes. In conclusion, findings from this study suggest that a dynamic ionic environment regulates tissue synthesis and points to [Ca(2+)]i signaling as a potential mediator. PMID:25128592

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

    PubMed Central

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

    2015-01-01

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

  17. 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

  18. Hypoxia inducible factor 1 alpha down-regulates type i collagen through Sp3 transcription factor in human chondrocytes.

    PubMed

    Duval, Elise; Bouyoucef, Mouloud; Leclercq, Sylvain; Baugé, Catherine; Boumédiene, Karim

    2016-09-01

    Cartilage engineering is one challenging issue in regenerative medicine. Low oxygen tension or hypoxia inducible factor-1 (HIF-1α) gene therapy are promising strategies in the field of cartilage repair. Previously, we showed that hypoxia and its mediator HIF-1 regulate matrix genes expression (collagens and aggrecan). Here, we investigated the molecular mechanism involved in the regulation of type I collagen (COL1A1) by HIF-1 in human articular chondrocytes. We show that HIF-1α reduces COL1A1 transcription, through a distal promoter (-2300 to -1816 bp upstream transcription initiation site), containing two GC boxes that bind Sp transcription factors (Sp1/Sp3). Sp1 acts as a positive regulator but is not induced by HIF-1. COL1A1 inhibition caused by HIF-1 implies only Sp3, which accumulates and competes Sp1 binding on COL1A1 promoter. Additionally, Sp3 ectopic expression inhibits COL1A1, while Sp3 knockdown counteracts the downregulation of COL1A1 induced by HIF-1. In conclusion, we established a new regulatory model of COL1A1 regulation by HIF-1, and bring out its relationship with Sp3 transcription factor. In a fundamental level, these findings give insights in the mechanisms controlling COL1A1 gene expression. This may be helpful to improve strategies to impair type I collagen expression during chondrocyte differentiation for cartilage engineering. © 2016 IUBMB Life, 68(9):756-763, 2016. PMID:27521280

  19. Two-pore domain K⁺ channels regulate membrane potential of isolated human articular chondrocytes.

    PubMed

    Clark, Robert B; Kondo, Colleen; Belke, Darrell D; Giles, Wayne R

    2011-11-01

    Potassium channels that regulate resting membrane potential (RMP) of human articular chondrocytes (HACs) of the tibial joint maintained in short-term (0-3 days) non-confluent cell culture were studied using patch-clamp techniques. Quantitative PCR showed that transcripts of genes for two-pore domain K(+) channels (KCNK1, KCNK5 and KCNK6), and 'BK' Ca(2+)-activated K(+) channels (KCNMA1) were abundantly expressed. Immunocytological methods detected α-subunits for BK and K(2p)5.1 (TASK-2) K(+) channels. Electrophysiological recordings identified three distinct K(+) currents in isolated HACs: (i) a voltage- and time-dependent 'delayed rectifier', blocked by 100 nM α-dendrotoxin, (ii) a large 'noisy' voltage-dependent current that was blocked by low concentrations of tetraethylammonium (TEA; 50% blocking dose = 0.15 mM) and iberiotoxin (52% block, 100 nM) and (iii) a voltage-independent 'background' K(+) current that was blocked by acidic pH (5.5-6), was increased by alkaline pH (8.5), and was not blocked by TEA, but was blocked by the local anaesthetic bupivacaine (0.25 mM). The RMP of isolated HACs was very slightly affected by 5 mM TEA, which was sufficient to block both voltage-dependent K(+) currents, suggesting that these currents probably contributed little to maintaining RMP under 'resting' conditions (i.e. low internal [Ca(2+)]). Increases in external K(+) concentration depolarized HACs by 30 mV in response to a 10-fold increase in [K(+)], indicating a significant but not exclusive role for K(+) current in determining RMP. Increases in external [K(+)] in voltage-clamped HACs revealed a voltage-independent K(+) current whose inward current magnitude increased with external [K(+)]. Block of this current by bupivacaine (0.25-1 mM) in 5 and 25 mM external [K(+)] resulted in a large (8-25 mV) depolarization of RMP. The biophysical and pharmacological properties of the background K(+) current, together with expression of mRNA and α-subunit protein for TASK-2

  20. MicroRNA-127-5p regulates osteopontin expression and osteopontin-mediated proliferation of human chondrocytes

    PubMed Central

    Tu, Min; Li, Yusheng; Zeng, Chao; Deng, Zhenhan; Gao, Shuguang; Xiao, Wenfeng; Luo, Wei; Jiang, Wei; Li, Liangjun; Lei, Guanghua

    2016-01-01

    The aim of this study was to determine the specific microRNA (miRNA) that regulates expression of osteopontin (OPN) in osteoarthritis (OA). The potential regulatory miRNAs for OPN messenger RNA (mRNA) were predicted by miRNA prediction programs. Among eight potential regulatory miRNAs, miR-220b, miR-513a-3p and miR-548n increased, while miR-181a, miR-181b, miR-181c, miR-181d and miR-127-5p decreased in OA patients. miRNA-127-5p mimics suppressed OPN production as well as the activity of a reporter construct containing the 3′-UTR of human OPN mRNA. In addition, mutation of miR-127-5p binding site in the 3′-UTR of OPN mRNA abolished miR-127-5p-mediated repression of reporter activity. Conversely, treatment with miR-127-5p inhibitor increased reporter activity and OPN production. Interestingly, miR-127-5p inhibited proliferation of chondrocytes through OPN. In conclusion, miRNA-127-5p is an important regulator of OPN in human chondrocytes and may contribute to the development of OA. PMID:27126955

  1. Interleukin-1β induced Stress Granules Sequester COX-2 mRNA and Regulates its Stability and Translation in Human OA Chondrocytes

    PubMed Central

    Ansari, Mohammad Y.; Haqqi, Tariq M.

    2016-01-01

    Enhanced and immediate expression of cyclooxygenase-2 (COX-2) mRNA is observed in IL-1β-stimulated OA chondrocytes but the synthesis of protein found significantly delayed. Here we investigated the role of stress granules (SGs), ribonucleoprotein complexes that regulate mRNA translation, in the delayed translation of COX-2 mRNAs in IL-1β-stimulated OA chondrocytes. Stimulation of human chondrocytes with IL-1β activated the stress response genes and the phosphorylation of eIF2α that triggered the assembly of SGs. Using combined immunofluorescence staining of SGs markers and COX-2 protein, RNA fluorescence in situ hybridization and RNA immunoprecipitation, the COX-2 mRNAs were found sequestered in SGs in IL-1β-stimulated OA chondrocytes. No increase in COX-2 protein expression was observed during the persistence of SGs but enhanced expression of COX-2 protein was noted upon clearance of the SGs. Inhibition of SGs clearance blocked COX-2 mRNA translation whereas blocking the assembly of SGs by TIA-1 depletion resulted in rapid and increased production of COX-2 and PGE2. Our findings show for the first time assembly of SGs and sequestration of COX-2 mRNAs in human OA chondrocytes under pathological conditions. Post-transcriptional regulation of COX-2 mRNAs translation by SGs indicates a role in IL-1β-mediated catabolic response that could be therapeutically targeted in OA. PMID:27271770

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

    SciTech Connect

    Deng, Yu; Cao, Hong; Cu, Fenglong; Xu, Dan; Lei, Youying; Tan, Yang; Magdalou, Jacques; Wang, Hui; Chen, Liaobin

    2013-05-15

    Previous studies have confirmed that maternal tobacco smoking causes intrauterine growth retardation (IUGR) and skeletal growth retardation. Among a multitude of chemicals associated with cigarette smoking, nicotine is one of the leading candidates for causing low birth weights. However, the possible mechanism of delayed chondrogenesis by prenatal nicotine exposure remains unclear. We investigated the effects of nicotine on fetal growth plate chondrocytes in vivo and in vitro. Rats were given 2.0 mg/kg·d of nicotine subcutaneously from gestational days 11 to 20. Prenatal nicotine exposure increased the levels of fetal blood corticosterone and resulted in fetal skeletal growth retardation. Moreover, nicotine exposure induced the inhibition of matrix synthesis and down-regulation of insulin-like growth factor 1 (IGF-1) signaling in fetal growth plates. The effects of nicotine on growth plates were studied in vitro by exposing fetal growth plate chondrocytes to 0, 1, 10, or 100 μM of nicotine for 10 days. Nicotine inhibited matrix synthesis and down-regulated IGF-1 signaling in chondrocytes in a concentration-dependent manner. These results suggest that prenatal nicotine exposure induces delayed chondrogenesis and that the mechanism may involve the down-regulation of IGF-1 signaling and the inhibition of matrix synthesis by growth plate chondrocytes. The present study aids in the characterization of delayed chondrogenesis caused by prenatal nicotine exposure, which might suggest a candidate mechanism for intrauterine origins of osteoporosis and osteoarthritis. - Highlights: ► Prenatal nicotine-exposure could induce delayed chondrogenesis in fetal rats. ► Nicotine inhibits matrix synthesis of fetal growth plate chondrocytes. ► Nicotine inhibits IGF-1 signaling pathway in fetal growth plate chondrocytes.

  3. Reciprocal inhibition between miR-26a and NF-κB regulates obesity-related chronic inflammation in chondrocytes

    PubMed Central

    Xie, Qingyun; Wei, Meng; Kang, Xia; Liu, Da; Quan, Yi; Pan, Xianming; Liu, Xiling; Liao, Dongfa; Liu, Jinbiao; Zhang, Bo

    2015-01-01

    Obesity is causally linked to osteoarthritis (OA), with the mechanism being not fully elucidated. miRNAs (miRs) are pivotal regulators of various diseases in multiple tissues, including inflammation in the chondrocytes. In the present study, we for the first time identified the expression of miR-26a in mouse chondrocytes. Decreased level of miR-26a was correlated to increased chronic inflammation in the chondrocytes and circulation in obese mouse model. Mechanistically, we demonstrated that miR-26a attenuated saturated free fatty acid-induced activation of NF-κB (p65) and production of proinflammatory cytokines in chondrocytes. Meanwhile, NF-κB (p65) also suppressed miR-26a production by directly binding to a predicted NF-κB binding element in the promoter region of miR-26a. Finally, we observed a negative correlation between NF-κB and miR-26a in human patients with osteoarthritis. Thus, we identified a reciprocal inhibition between miR-26a and NF-κB downstream of non-esterified fatty acid (NEFA) signalling in obesity-related chondrocytes. Our findings provide a potential mechanism linking obesity to cartilage inflammation. PMID:26182366

  4. Apigenin Regulates Interleukin-1β-Induced Production of Matrix Metalloproteinase Both in the Knee Joint of Rat and in Primary Cultured Articular Chondrocytes

    PubMed Central

    Park, Jin Sung; Kim, Dong Kyu; Shin, Hyun-Dae; Lee, Hyun Jae; Jo, Ho Seung; Jeong, Jin Hoon; Choi, Young Lac; Lee, Choong Jae; Hwang, Sun-Chul

    2016-01-01

    We examined whether apigenin affects the gene expression, secretion and activity of matrix metalloproteinase-3 (MMP-3) in primary cultured rabbit articular chondrocytes, as well as in vivo production of MMP-3 in the knee joint of rat to evaluate the potential chondroprotective effects of apigenin. Rabbit articular chondrocytes were cultured in a monolayer, and reverse transcription - polymerase chain reaction (RT-PCR) was used to measure interleukin-1β (IL-1β)-induced expression of MMP-3, MMP-1, MMP-13, a disintegrin and metalloproteinase with thrombospondin motifs-4 (ADAMTS-4), and ADAMTS-5. In rabbit articular chondrocytes, the effects of apigenin on IL-1β-induced secretion and proteolytic activity of MMP-3 were investigated using western blot analysis and casein zymography, respectively. The effect of apigenin on MMP-3 protein production was also examined in vivo. In rabbit articular chondrocytes, apigenin inhibited the gene expression of MMP-3, MMP-1, MMP-13, ADAMTS-4, and ADAMTS-5. Furthermore, apigenin inhibited the secretion and proteolytic activity of MMP-3 in vitro, and inhibited production of MMP-3 protein in vivo. These results suggest that apigenin can regulate the gene expression, secretion, and activity of MMP-3, by directly acting on articular chondrocytes. PMID:26902085

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

    PubMed Central

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

    2016-01-01

    Several lines of evidence indicate that connective tissue growth factor (CTGF/CCN2) stimulates chondrocyte proliferation and maturation. Given the fact that SOX9 is essential for several steps of the chondrocyte differentiation pathway, we asked whether Ctgf (Ccn2) is the direct target gene of SOX9. We found that Ctgf mRNA was down-regulated in primary sternal chondrocytes from Sox9flox/flox mice infected with Ad-CMV-Cre. We performed ChIP-on-chip assay using anti-SOX9 antibody, covering the Ctgf gene from 15 kb upstream of its 5′-end to 10 kb downstream of its 3′-end to determine SOX9 interaction site. One high-affinity interaction site was identified in the Ctgf proximal promoter by ChIP-on-chip assay. An important SOX9 regulatory element was found to be located in −70/−64 region of the Ctgf promoter. We found the same site for SOX9 binding to the Ctgf promoter in nucleus pulposus (NP) cells. The loss of Sox9 in growth plate chondrocytes in knee joint and in NP cells in intervertebral disc led to the decrease in CTGF expression. We suggest that Ctgf is the direct target gene of SOX9 in chondrocytes and NP cells. Our study establishes a strong link between two regulatory molecules that have a major role in cartilaginous tissues. PMID:27436052

  6. 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

  7. Heme oxygenase-1 regulates matrix metalloproteinase MMP-1 secretion and chondrocyte cell death via Nox4 NADPH oxidase activity in chondrocytes.

    PubMed

    Rousset, Francis; Nguyen, Minh Vu Chuong; Grange, Laurent; Morel, Françoise; Lardy, Bernard

    2013-01-01

    Interleukin-1β (IL-1β) activates the production of reactive oxygen species (ROS) and secretion of MMPs as well as chondrocyte apoptosis. Those events lead to matrix breakdown and are key features of osteoarthritis (OA). We confirmed that in human C-20/A4 chondrocytes the NADPH oxidase Nox4 is the main source of ROS upon IL-1β stimulation. Since heme molecules are essential for the NADPH oxidase maturation and activity, we therefore investigated the consequences of the modulation of Heme oxygenase-1 (HO-1), the limiting enzyme in heme catabolism, on the IL-1β signaling pathway and more specifically on Nox4 activity. Induction of HO-1 expression decreased dramatically Nox4 activity in C-20/A4 and HEK293 T-REx™ Nox4 cell lines. Unexpectedly, this decrease was not accompanied by any change in the expression, the subcellular localization or the maturation of Nox4. In fact, the inhibition of the heme synthesis by succinylacetone rather than heme catabolism by HO-1, led to a confinement of the Nox4/p22(phox) heterodimer in the endoplasmic reticulum with an absence of redox differential spectrum highlighting an incomplete maturation. Therefore, the downregulation of Nox4 activity by HO-1 induction appeared to be mediated by carbon monoxide (CO) generated from the heme degradation process. Interestingly, either HO-1 or CO caused a significant decrease in the expression of MMP-1 and DNA fragmentation of chondrocytes stimulated by IL-1β. These results all together suggest that a modulation of Nox4 activity via heme oxygenase-1 may represent a promising therapeutic tool in osteoarthritis. PMID:23840483

  8. Heme Oxygenase-1 Regulates Matrix Metalloproteinase MMP-1 Secretion and Chondrocyte Cell Death via Nox4 NADPH Oxidase Activity in Chondrocytes

    PubMed Central

    Rousset, Francis; Nguyen, Minh Vu Chuong; Grange, Laurent; Morel, Françoise; Lardy, Bernard

    2013-01-01

    Interleukin-1β (IL-1β) activates the production of reactive oxygen species (ROS) and secretion of MMPs as well as chondrocyte apoptosis. Those events lead to matrix breakdown and are key features of osteoarthritis (OA). We confirmed that in human C-20/A4 chondrocytes the NADPH oxidase Nox4 is the main source of ROS upon IL-1β stimulation. Since heme molecules are essential for the NADPH oxidase maturation and activity, we therefore investigated the consequences of the modulation of Heme oxygenase-1 (HO-1), the limiting enzyme in heme catabolism, on the IL-1β signaling pathway and more specifically on Nox4 activity. Induction of HO-1 expression decreased dramatically Nox4 activity in C-20/A4 and HEK293 T-REx™ Nox4 cell lines. Unexpectedly, this decrease was not accompanied by any change in the expression, the subcellular localization or the maturation of Nox4. In fact, the inhibition of the heme synthesis by succinylacetone rather than heme catabolism by HO-1, led to a confinement of the Nox4/p22phox heterodimer in the endoplasmic reticulum with an absence of redox differential spectrum highlighting an incomplete maturation. Therefore, the downregulation of Nox4 activity by HO-1 induction appeared to be mediated by carbon monoxide (CO) generated from the heme degradation process. Interestingly, either HO-1 or CO caused a significant decrease in the expression of MMP-1 and DNA fragmentation of chondrocytes stimulated by IL-1β. These results all together suggest that a modulation of Nox4 activity via heme oxygenase-1 may represent a promising therapeutic tool in osteoarthritis. PMID:23840483

  9. Master regulator for chondrogenesis, Sox9, regulates transcriptional activation of the endoplasmic reticulum stress transducer BBF2H7/CREB3L2 in chondrocytes.

    PubMed

    Hino, Kenta; Saito, Atsushi; Kido, Miori; Kanemoto, Soshi; Asada, Rie; Takai, Tomoko; Cui, Min; Cui, Xiang; Imaizumi, Kazunori

    2014-05-16

    The endoplasmic reticulum (ER) stress transducer, box B-binding factor 2 human homolog on chromosome 7 (BBF2H7), is a basic leucine zipper (bZIP) transmembrane transcription factor. This molecule is activated in response to ER stress during chondrogenesis. The activated BBF2H7 accelerates cartilage matrix protein secretion through the up-regulation of Sec23a, which is responsible for protein transport from the ER to the Golgi apparatus and is a target of BBF2H7. In the present study, we elucidated the mechanisms of the transcriptional activation of Bbf2h7 in chondrocytes. The transcription of Bbf2h7 is regulated by Sex determining region Y-related high-mobility group box 9 (Sox9), a critical factor for chondrocyte differentiation that facilitates the expression of one of the major cartilage matrix proteins Type II collagen (Col2), through binding to the Sox DNA-binding motif in the Bbf2h7 promoter. BBF2H7 is activated as a transcription factor in response to physiological ER stress caused by abundant synthesis of cartilage matrix proteins, and consequently regulates the secretion of cartilage matrix proteins. Taken together, our findings demonstrate novel regulatory mechanisms of Sox9 for controlling the secretion of cartilage matrix proteins through the activation of BBF2H7-Sec23a signaling during chondrogenesis.

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

    PubMed Central

    Terauchi, Koh; Kobayashi, Hajime; Yatabe, Kanaka; Yui, Naoko; Fujiya, Hiroto; Niki, Hisateru; Musha, Haruki; Yudoh, Kazuo

    2016-01-01

    Aging is one of the major pathologic factors associated with osteoarthritis (OA). Recently, numerous reports have demonstrated the impact of sirtuin-1 (Sirt1), which is the NAD-dependent deacetylase, on human aging. It has been demonstrated that Sirt1 induces osteogenic and chondrogenic differentiation of mesenchymal stem cells. However, the role of Sirt1 in the OA chondrocytes still remains unknown. We postulated that Sirt1 regulates a hypertrophic chondrocyte lineage and degeneration of articular cartilage through the activation of osteogenic transcriptional activator Runx2 and matrix metalloproteinase (MMP)-13 in OA chondrocytes. To verify whether sirtuin-1 (Sirt1) regulates chondrocyte activity in OA, we studied expressions of Sirt1, Runx2 and production of MMP-13, and their associations in human OA chondrocytes. The expression of Sirt1 was ubiquitously observed in osteoarthritic chondrocytes; in contrast, Runx2 expressed in the osteophyte region in patients with OA and OA model mice. OA relating catabolic factor IL-1βincreased the expression of Runx2 in OA chondrocytes. OA chondrocytes, which were pretreated with Sirt1 inhibitor, inhibited the IL-1β-induced expression of Runx2 compared to the control. Since the Runx2 is a promotor of MMP-13 expression, Sirt1 inactivation may inhibit the Runx2 expression and the resultant down-regulation of MMP-13 production in chondrocytes. Our findings suggest thatSirt1 may regulate the expression of Runx2, which is the osteogenic transcription factor, and the production of MMP-13 from chondrocytes in OA. Since Sirt1 activity is known to be affected by several stresses, including inflammation and oxidative stress, as well as aging, SIRT may be involved in the development of OA. PMID:27367673

  11. VEGF-independent cell-autonomous functions of HIF-1α regulating oxygen consumption in fetal cartilage are critical for chondrocyte survival.

    PubMed

    Maes, Christa; Araldi, Elisa; Haigh, Katharina; Khatri, Richa; Van Looveren, Riet; Giaccia, Amato J; Haigh, Jody J; Carmeliet, Geert; Schipani, Ernestina

    2012-03-01

    Fetal growth plate cartilage is nonvascularized, and chondrocytes largely develop in hypoxic conditions. We previously found that mice lacking the hypoxia-inducible transcription factor HIF-1α in cartilage show massive death of centrally located, hypoxic chondrocytes. A similar phenotype was observed in mice with genetic ablation of either all or specifically the diffusible isoforms of vascular endothelial growth factor (VEGF), a prime angiogenic target of HIF-1α. Here, we assessed whether VEGF is a critical downstream component of the HIF-1α-dependent survival pathway in chondrocytes. We used a genetic approach to conditionally overexpress VEGF164 in chondrocytes lacking HIF-1α, evaluating potential rescuing effects. The effectiveness of the strategy was validated by showing that transgenic expression of VEGF164 in Col2-Cre;VEGF(f/f) mice stimulated angiogenesis in the perichondrium, fully corrected the excessive hypoxia of VEGF-deficient chondrocytes, and completely prevented chondrocyte death. Yet, similarly crossed double-mutant embryos lacking HIF-1α and overexpressing VEGF164 in the growth plate cartilage still displayed a central cell death phenotype, albeit slightly delayed and less severe compared with mice exclusively lacking HIF-1α. Transgenic VEGF164 induced massive angiogenesis in the perichondrium, yet this only partially relieved the aberrant hypoxia present in HIF-1α-deficient cartilage and thereby likely inflicted only a partial rescue effect. In fact, excessive hypoxia and failure to upregulate phosphoglycerate-kinase 1 (PGK1), a key enzyme of anaerobic glycolytic metabolism, were among the earliest manifestations of HIF-1α deficiency in cartilaginous bone templates, and reduced PGK1 expression was irrespective of transgenic VEGF164. These findings suggest that HIF-1α activates VEGF-independent cell-autonomous mechanisms to sustain oxygen levels in the challenged avascular cartilage by reducing oxygen consumption. Hence, regulation of the

  12. Regulation by glucocorticoids of cell differentiation and insulin-like growth factor binding protein production in cultured fetal rat nasal chondrocytes.

    PubMed

    Nadra, Reem; Menuelle, Pierrette; Chevallier, Sylviana; Berdal, Ariane

    2003-04-01

    Glucocorticoids (GCs) modulate insulin-like growth factor action in cartilage through mechanisms that are complex and insufficiently defined, especially in the context of cranio-facial growth. Because the family of IGF-binding proteins (IGFBP-1 to -6) is important in the regulation of IGF availability and bioactivity, we examined the effect of GCs on chondrocyte differentiation in correlation with IGFBP production in cultured fetal rat chondrocytes isolated from nasal septum cartilage of fetal rat. Dexamethasone (DEX) effects were tested before and at the onset of extracellular matrix maturation. DEX induced a dose-dependent increase in the size of cartilage nodule formed, (45)Ca incorporation into extracellular matrix, alkaline phosphatase activity, and sulfatation of glycosaminoglycans, maximal effects being obtained with a 10-mM DEX concentration. The IGFBPs produced by cultured chondrocytes were characterized in culture medium which had been conditioned for 24 h under serum-free conditions by these cells. Western ligand blotting with a mixture of [(125)I]IGF-I and -II revealed bands of 20, 24, 29, a 31-32 kDa doublet and a 39-41 kDa triplet which were differently regulated by DEX. Immunoblotting showed that following DEX exposure, IGFBP-3 and -6 were up-regulated whereas IGFBP-2, -5, and the 24 kDa band were down-regulated. The effect of DEX on both differentiation and IGFBP production showed a same dependence, and developed when extracellular matrix maturation had been just induced. The results obtained in this chondrocyte culture system show that production of IGFBPs is modulated by DEX at physiological concentrations thus regulating IGF availability and action, a control which could promote the primordial role of the rat nasal septum in craniofacial growth.

  13. Collagen VI regulates pericellular matrix properties, chondrocyte swelling, and mechanotransduction in articular cartilage

    PubMed Central

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

    2015-01-01

    Objective 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 examined 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, transient receptor potential vanilloid 4 (TRPV4), osmotically-induced chondrocyte swelling, and pericellular matrix (PCM) mechanical properties. Methods 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. Results 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. Conclusion In contrast to our original hypothesis, type VI collagen was not required for TRPV4-mediated Ca2+ 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 suggest that alterations in PCM properties, as may occur with aging or osteoarthritis, can influence mechanotransduction via TRPV4 or other ion channels. PMID:25604429

  14. MicroRNA-602 and microRNA-608 regulate sonic hedgehog expression via target sites in the coding region in human chondrocytes

    PubMed Central

    Akhtar, Nahid; Makki, Mohammad Shahidul; Haqqi, Tariq M.

    2015-01-01

    Objective Hedgehog(Hh) signaling has recently been associated with cartilage degradation in osteoarthritis(OA). As interleukin-1β(IL-1β) is a critical mediator of OA pathogenesis, here we determined whether IL-1β induces the expression of sonic hedgehog(SHH) and its regulation by microRNAs in human chondrocytes. Methods SHH protein expression in human OA-cartilage and in an animal model of OA was determined by immunohistochemistry and immunofluorescence respectively. Gene and protein expression in IL-1β or SHH-stimulated chondrocytes was determined by TaqMan assays and immunoblotting respectively. Effect of overexpression of miR-602 and miR-608 or their anatgomirs on SHH expression was evaluated by transient transfections of human chondrocytes and HEK-293 cells. Role of signaling pathways was evaluated using small molecule inhibitors. Binding of miRNAs with the putative “seed sequence” in the SHH mRNA was validated with a SHH luciferase reporter assay. Results Expression of SHH, PTCH-1, GLI-1, HHIP, MMP-13, and COL10A1 was high in damaged OAcartilage. Expression of SHH was inversely correlated with the expression of miR-608 in damaged cartilage and in IL-1β-stimulated chondrocytes. Transfection with miR-608 or miR-602 mimics inhibited the reporter activity and mutation of the miRNAs “seed sequences” abolished the repression of reporter activity. Overexpression of miR-602 or miR-608 inhibited the expression of SHH mRNA and protein and this was abrogated by antagomirs. Stimulation with SHH-protein up-regulated the MMP-13 expression and inhibition of Hh signaling blocked MMP-13 expression in OA chondrocytes. Conclusions miR-602 and miR-608 are important regulators of SHH expression in chondrocytes and their suppression by IL-1β may contribute to the enhanced expression of SHH and MMP-13 in OA. PMID:25385442

  15. Prostaglandin (PG)D2 and 15-deoxy-Δ12,14-PGJ2, but not PGE2, Mediate Shear-Induced Chondrocyte Apoptosis via Protein Kinase A-dependent Regulation of Polo-like Kinases

    PubMed Central

    Zhu, Fei; Wang, Pu; Kontrogianni-Konstantopoulos, Aikaterini; Konstantopoulos, Konstantinos

    2010-01-01

    Excessive mechanical loading of cartilage producing hydrostatic stress, tensile strain and fluid flow leads to chondrocyte apoptosis and osteoarthritis. High fluid flow induces cyclooxygenase-2 (COX-2) expression in sheared chondrocytes, which suppresses their antioxidant capacity and contributes to apoptosis. The pivotal role of COX-2 in shear-induced chondrocyte apoptosis and the conflicting literature data on the roles of prostaglandin (PG)E2, PGD2 and its metabolite 15-deoxy-Δ12,14-PGJ2 (15d-PGJ2) in chondrocyte apoptosis prompted us to investigate which COX-2-derived PG is involved in this process. We show that exogenously added PGD2 and 15d-PGJ2, but not PGE2, diminish the viability of human T/C-28a2 chondrocytes under static conditions. In agreement with these observations, knockdown of L-PGD synthase (L-PGDS) abolishes shear-induced chondrocyte apoptosis. Using cDNA microarrays in conjunction with clustering algorithms, we propose a novel signaling pathway by which high fluid shear mediates COX-2/L-PGDS-dependent chondrocyte apoptosis, which is validated by molecular interventions. We demonstrate that L-PGDS controls the downregulation of Protein Kinase A (PKA), which in turn regulates Polo-like kinase1 (Plk1) and Plk3. Plks target p53, which controls the transcription of p53 effectors (TP53INPs, FAS and Bax) involved in chondrocyte apoptosis. Reconstructing the signaling network regulating chondrocyte apoptosis may provide insights to optimize conditions for culturing artificial cartilage in bioreactors and for developing therapeutic strategies for arthritic disorders. PMID:20150912

  16. Lysophosphatidic Acid Enhanced the Angiogenic Capability of Human Chondrocytes by Regulating Gi/NF-kB-Dependent Angiogenic Factor Expression

    PubMed Central

    Chen, Kai-Hua; Hong, Chang-Zern; Chang, Pey-Jium; Hsu, Hung-Chih

    2014-01-01

    Lysophosphatidic acid (LPA) has been found to mediate myeloid differentiation, stimulate osteogenesis, alter cell proliferation and migration, and inhibit apoptosis in chondrocytes. The effect of LPA on the angiogenic capability of chondrocytes is not clear. This study aimed to investigate its effect on the angiogenic capability of human chondrocytes and the underlying mechanism of these effects. Human chondrocyte cell line, CHON-001, commercialized human chondrocytes (HC) derived from normal human articular cartilage, and human vascular endothelial cells (HUVECs) were used as cell models in this study. The angiogenic capability of chondrocytes was determined by capillary tube formation, monolayer permeability, cell migration, and cell proliferation. An angiogenesis protein array kit was used to evaluate the secretion of angiogenic factors in conditioned medium. Angiogenin, insulin-like growth factor-binding protein 1 (IGFBP-1), interleukin (IL)-8, monocyte chemoattractant protein-1 (MCP-1), matrix metalloproteinase (MMP)-9, and vascular endothelial growth factor (VEGF) mRNA and protein expressions were evaluated by Q-RT-PCR and EIA, respectively. LPA receptor (LPAR) expression was determined by RT-PCR. Signaling pathways were clarified using inhibitors, Western blot analysis, and reporter assays. The LPA treatment promoted the angiogenic capability of CHON-001 cells and HC, resulting in enhanced HUVEC capillary tube formation, monolayer permeability, migration, and cell growth. Angiogenin, IGFBP-1, IL-8, MCP-1, MMP-9, and VEGF mRNA and protein expressions were significantly enhanced in LPA-treated chondrocytes. LPA2, 3, 4 and 6 were expressed in CHON-001 and HC cells. Pretreatment with the Gi/o type G protein inhibitor, pertussis toxin (PTX), and the NF-kB inhibitor, PDTC, significantly inhibited LPA-induced angiogenin, IGFBP-1, IL-8, MCP-1, MMP-9, and VEGF expressions in chondrocytes. The PTX pretreatment also inhibited LPA-mediated NF-kB activation, suggesting

  17. 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

  18. Effects of UCP4 on the Proliferation and Apoptosis of Chondrocytes: Its Possible Involvement and Regulation in Osteoarthritis.

    PubMed

    Huang, Zhongming; Li, Junhua; Du, Shaohua; Chen, Guangnan; Qi, Yiying; Huang, Ligang; Xiao, Luwei; Tong, Peijian

    2016-01-01

    Reactive oxygen species (ROS)-induced chondrocytes apoptosis plays a key role in osteoarthritis (OA) pathogenesis. Uncoupling protein 4 (UCP4) can protect cells against oxidative stress via reducing ROS production and cell apoptosis. Here, silencing of UCP4 in primary chondrocytes significantly inhibited cell survival, but induced ROS production and cell apoptosis. UCP4 mRNA of cartilage tissues was decreased in osteoarthritis patients, which was negatively correlated with synovial fluid (SF) leptin concentration. Moreover, leptin treatment (5, 10 and 20 ng/ml) of primary cultured chondrocytes significantly decreased mRNA and protein levels of UCP4, but increased ROS production and cell apoptosis in a dose-dependent manner. The effects of leptin treatment (20 ng/ml) on chondrocytes was partially reversed by ectopic expression of UCP4. More importantly, intraarticularly injection of UCP4 adenovirus remarkably alleviate OA progression and cell apoptosis in a rat OA model induced by anterior cruciate ligament transection (ACLT). In conclusion, UCP4, whose expression was suppressed by leptin, may be involved in the ROS production and apoptosis of chondrocytes, thus contributing to the OA pathogenesis. PMID:26934480

  19. Effects of UCP4 on the Proliferation and Apoptosis of Chondrocytes: Its Possible Involvement and Regulation in Osteoarthritis

    PubMed Central

    Huang, Zhongming; Li, Junhua; Du, Shaohua; Chen, Guangnan; Qi, Yiying; Huang, Ligang; Xiao, Luwei; Tong, Peijian

    2016-01-01

    Reactive oxygen species (ROS)-induced chondrocytes apoptosis plays a key role in osteoarthritis (OA) pathogenesis. Uncoupling protein 4 (UCP4) can protect cells against oxidative stress via reducing ROS production and cell apoptosis. Here, silencing of UCP4 in primary chondrocytes significantly inhibited cell survival, but induced ROS production and cell apoptosis. UCP4 mRNA of cartilage tissues was decreased in osteoarthritis patients, which was negatively correlated with synovial fluid (SF) leptin concentration. Moreover, leptin treatment (5, 10 and 20 ng/ml) of primary cultured chondrocytes significantly decreased mRNA and protein levels of UCP4, but increased ROS production and cell apoptosis in a dose-dependent manner. The effects of leptin treatment (20 ng/ml) on chondrocytes was partially reversed by ectopic expression of UCP4. More importantly, intraarticularly injection of UCP4 adenovirus remarkably alleviate OA progression and cell apoptosis in a rat OA model induced by anterior cruciate ligament transection (ACLT). In conclusion, UCP4, whose expression was suppressed by leptin, may be involved in the ROS production and apoptosis of chondrocytes, thus contributing to the OA pathogenesis. PMID:26934480

  20. 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

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

    SciTech Connect

    Oh, Jung-Hoon; Park, Seung-Yoon; Crombrugghe, Benoit de; Kim, Jung-Eun

    2012-02-24

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

  2. Regulation of type-II collagen gene expression during human chondrocyte de-differentiation and recovery of chondrocyte-specific phenotype in culture involves Sry-type high-mobility-group box (SOX) transcription factors.

    PubMed Central

    Stokes, D G; Liu, G; Dharmavaram, R; Hawkins, D; Piera-Velazquez, S; Jimenez, S A

    2001-01-01

    During ex vivo growth as monolayer cultures, chondrocytes proliferate and undergo a process of de-differentiation. This process involves a change in morphology and a change from expression of chondrocyte-specific genes to that of genes that are normally expressed in fibroblasts. Transfer of the monolayer chondrocyte culture to three-dimensional culture systems induces the cells to re-acquire a chondrocyte-specific phenotype and produce a cartilaginous-like tissue in vitro. We investigated mechanisms involved in the control of the de-differentiation and re-differentiation process in vitro. De-differentiated chondrocytes re-acquired their chondrocyte-specific phenotype when cultured on poly-(2-hydroxyethyl methacrylate) (polyHEMA) as assayed by morphology, reverse transcriptase PCR of chondrocyte-specific mRNA, Western-blot analysis and chondrocyte-specific promoter activity. Essentially, full recovery of the chondrocyte-specific phenotype was observed when cells that had been cultured for 4 weeks on plastic were transferred to culture on polyHEMA. However, after subsequent passages on plastic, the phenotype recovery was incomplete or did not occur. The activity of a gene reporter construct containing the promoter and enhancer from the human type-II collagen gene (COL2A1) was modulated by the culture conditions, so that its transcriptional activity was repressed in monolayer cultures and rescued to some extent when the cells were switched to polyHEMA cultures. The binding of Sry-type high-mobility-group box (SOX) transcription factors to the enhancer region was modulated by the culture conditions, as were the mRNA levels for SOX9. A transfected human type-II collagen reporter construct was activated in de-differentiated cells by ectopic expression of SOX transcription factors. These results underscore the overt change in phenotype that occurs when chondrocytes are cultured as monolayers on tissue-culture plastic substrata. PMID:11716775

  3. Functional analysis of diastrophic dysplasia sulfate transporter. Its involvement in growth regulation of chondrocytes mediated by sulfated proteoglycans.

    PubMed

    Satoh, H; Susaki, M; Shukunami, C; Iyama, K; Negoro, T; Hiraki, Y

    1998-05-15

    Mutations in the diastrophic dysplasia sulfate transporter (DTDST) gene constitute a family of recessively inherited osteochondrodysplasias including achondrogenesis type 1B, atelosteogenesis type II, and diastrophic dysplasia. However, the functional properties of the gene product have yet to be elucidated. We cloned rat DTDST cDNA from rat UMR-106 osteoblastic cells. Northern blot analysis suggested that cartilage and intestine were the major expression sites for DTDST mRNA. Analysis of the genomic sequence revealed that the rat DTDST gene was composed of at least five exons. Two distinct transcripts were expressed in chondrocytes due to alternative utilization of the third exon, corresponding to an internal portion of the 5'-untranslated region of the cDNA. Injection of rat and human DTDST cRNA into Xenopus laevis oocytes induced Na+-independent sulfate transport. Transport activity of the expressed DTDST was markedly inhibited by extracellular chloride and bicarbonate. In contrast, canalicular Na+-independent sulfate transporter Sat-1 required the presence of extracellular chloride in the cRNA-injected oocytes. The activity profile of sulfate transport in growth plate chondrocytes was studied in the extracellular presence of various anions and found substantially identical to DTDST expressed in oocytes. Thus, sulfate transport of chondrocytes is dominantly dependent on the DTDST system. Finally, we demonstrate that undersulfation of proteoglycans by the chlorate treatment of chondrocytes significantly impaired growth response of the cells to fibroblast growth factor, suggesting a role for DTDST in endochondral bone formation. PMID:9575183

  4. Allogeneic Articular Chondrocyte Transplantation Down Regulates IL-8 Gene Expression in the Degenerating Rabbit Intervertebral Disc in Vivo

    PubMed Central

    Zhang, Yejia; Chee, Ana; Shi, Peng; Wang, Rui; Moss, Isaac; Chen, Er-Yun; He, Tong-Chuan; An, Howard S.

    2014-01-01

    Objective To investigate if repopulating the degenerating intervertebral disc (IVD) with articular chondrocytes (ACs) will decrease inflammation and restore disc structure. In this study, we aimed to determine if well-differentiated AC alone or transduced with adenovirus overexpressing BMP-7 gene may survive and inhibit inflammation or repair disc structure in the degenerating rabbit IVD. Design This was a biological study in a rabbit IVD-injury model in vivo. Dual cell tracking methods (IR dye-labeling and adenovirus transduction) were used to demonstrate the viability of allogeneic AC injected into degenerating rabbit IVDs. Interleukin (IL)-8 gene expression was determined via real-time PCR. Infiltrating inflammatory cells (macrophages, T-cells or neutrophils) were examined with immunohistochemistry. The IVDs were also examined by routine histology. Results ACs labeled with infrared (IR) dye were detected in the degenerating IVDs at both 2 and 8 weeks after injection. At the 2-week time point, IL-8 gene expression was comparable in IVDs injected with chondrocytes and in intact discs as control (P=0.647), while its expression in IVDs injected with saline increased 50fold (p=0.028). Transgene expression of red fluorescent protein, β-galactosidase, and BMP-7 diminished at 8 weeks post injection. IVDs injected with chondrocytes overexpressing hBMP-7 did not show lower IL-8 gene expression or improved histology. Macrophages were consistently detected by immunohistochemistry in the cartilage formation around the needle insertion sites in both the saline and chondrocyte groups, while neither T cells nor neutrophils were detected. Conclusions Allogeneic rabbit AC survived in the degenerating rabbit IVDs for at least 8 weeks. Cell treatment resulted in reduced IVD inflammation, but did not significantly improve IVD structure. PMID:25133623

  5. 17 beta-estradiol-BSA conjugates and 17 beta-estradiol regulate growth plate chondrocytes by common membrane associated mechanisms involving PKC dependent and independent signal transduction.

    PubMed

    Sylvia, V L; Walton, J; Lopez, D; Dean, D D; Boyan, B D; Schwartz, Z

    2001-01-01

    Nuclear receptors for 17 beta-estradiol (E(2)) are present in growth plate chondrocytes from both male and female rats and regulation of chondrocytes through these receptors has been studied for many years; however, recent studies indicate that an alternative pathway involving a membrane receptor may also be involved in the cell response. E(2) was found to directly affect the fluidity of chondrocyte membranes derived from female, but not male, rats. In addition, E(2) activates protein kinase C (PKC) in a nongenomic manner in female cells, and chelerythrine, a specific inhibitor of PKC, inhibits E(2)-dependent alkaline phosphatase activity and proteoglycan sulfation in these cells, indicating PKC is involved in the signal transduction mechanism. The aims of the present study were: (1) to examine the effect of a cell membrane-impermeable 17 beta-estradiol-bovine serum albumin conjugate (E(2)-BSA) on chondrocyte proliferation, differentiation, and matrix synthesis; (2) to determine the pathway that mediates the membrane effect of E(2)-BSA on PKC; and (3) to compare the action of E(2)-BSA to that of E(2). Confluent, fourth passage resting zone (RC) and growth zone (GC) chondrocytes from female rat costochondral cartilage were treated with 10(-9) to 10(-7) M E(2) or E(2)-BSA and changes in alkaline phosphatase specific activity, proteoglycan sulfation, and [(3)H]-thymidine incorporation measured. To examine the pathway of PKC activation, chondrocyte cultures were treated with E(2)-BSA in the presence or absence of GDP beta S (inhibitor of G-proteins), GTP gamma S (activator of G-proteins), U73122 or D609 (inhibitors of phospholipase C [PLC]), wortmannin (inhibitor of phospholipase D [PLD]) or LY294002 (inhibitor of phosphatidylinositol 3-kinase). E(2)-BSA mimicked the effects of E(2) on alkaline phosphatase specific activity and proteoglycan sulfation, causing dose-dependent increases in both RC and GC cell cultures. Both forms of estradiol inhibited [(3)H

  6. Shikonin inhibits inflammation and chondrocyte apoptosis by regulation of the PI3K/Akt signaling pathway in a rat model of osteoarthritis

    PubMed Central

    Fu, Daijie; Shang, Xifu; Ni, Zhe; Shi, Guoguang

    2016-01-01

    Shikonin has previously been shown to have antitumor, anti-inflammatory, antiviral and extensive pharmacological effects. The aim of the present study was to explore whether the protective effect of shikonin is mediated via the inhibition of inflammation and chondrocyte apoptosis, and to elucidate the potential molecular mechanisms in a rat model of osteoarthritis. A model of osteoarthritis was established in healthy male Sprague-Dawley rats and 10 mg/kg/day shikonin was administered intraperitoneally for 4 days. It was found that shikonin treatment significantly inhibited inflammatory reactions in the rats with osteoarthritis. Osteoarthritis was found to significantly increase interleukin (IL)-1β, tumor necrosis factor (TNF)-α and inducible nitric oxide synthase (iNOS) levels compared with those in the sham group. However, shikonin treatment significantly inhibited the increases in IL-1β, TNF-α and iNOS levels in the rats with osteoarthritis. Furthermore, caspase-3 activity and cyclooxygenase (COX)-2 protein expression were significantly increased and phosphorylated Akt protein expression was greatly suppressed in rats with osteoarthritis when compared with the sham group. Shikonin administration attenuated the changes in caspase-3 activity and COX-2 expression and Akt phosphorylation in rats with osteoarthritis. These results indicate that shikonin inhibits inflammation and chondrocyte apoptosis by regulating the phosphoinositide 3-kinase/Akt signaling pathway in a rat model of osteoarthritis. PMID:27703516

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

    PubMed

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

    2013-01-01

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

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

    PubMed

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

    2016-01-01

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

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

    PubMed

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

    2016-01-01

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

  10. Evc is a positive mediator of Ihh-regulated bone growth that localises at the base of chondrocyte cilia.

    PubMed

    Ruiz-Perez, Victor L; Blair, Helen J; Rodriguez-Andres, M Elena; Blanco, Maria Jose; Wilson, Amy; Liu, Yu-Ning; Miles, Colin; Peters, Heiko; Goodship, Judith A

    2007-08-01

    EVC is a novel protein mutated in the human chondroectodermal dysplasia Ellis-van Creveld syndrome (EvC; OMIM: 225500). We have inactivated Evc in the mouse and show that Evc(-/-) mice develop an EvC-like syndrome, including short ribs, short limbs and dental abnormalities. lacZ driven by the Evc promoter revealed that Evc is expressed in the developing bones and the orofacial region. Antibodies developed against Evc locate the protein at the base of the primary cilium. The growth plate of Evc(-/-) mice shows delayed bone collar formation and advanced maturation of chondrocytes. Indian hedgehog (Ihh) is expressed normally in the growth plates of Evc(-/-) mice, but expression of the Ihh downstream genes Ptch1 and Gli1 was markedly decreased. Recent studies have shown that Smo localises to primary cilia and that Gli3 processing is defective in intraflagellar transport mutants. In vitro studies using Evc(-/-) cells demonstrate that the defect lies downstream of Smo. Chondrocyte cilia are present in Evc(-/-) mice and Gli3 processing appears normal by western blot analysis. We conclude that Evc is an intracellular component of the hedgehog signal transduction pathway that is required for normal transcriptional activation of Ihh target genes.

  11. 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

  12. MicroRNA-9 regulates the development of knee osteoarthritis through the NF-kappaB1 pathway in chondrocytes

    PubMed Central

    Gu, Ronghe; Liu, Ning; Luo, Simin; Huang, Weiguo; Zha, Zhengang; Yang, Jie

    2016-01-01

    Abstract It has been suggested that microRNA-9 (miR-9) is associated with the development of knee osteoarthritis (OA). This study was aimed to investigate the association between the mechanism of miR-9 targeting nuclear factor kappa-B1 (NF-κB1) and the proliferation and apoptosis of knee OA chondrocytes. Cartilage samples were collected from 25 patients with knee OA and 10 traumatic amputees, and another 15 OA rat models, together with 15 rats without knee OA lesions were also established. MiR-9 expressions in both knee OA cartilage and normal cartilage samples were detected using quantitative real-time PCR. The expressions of related genes (NF-κB1, IL-6, and MMP-13) in the two groups were also detected. Dual luciferase reporter gene assay was employed to examine the effect of miR-9 on the luciferase activity of NF-κB1 3′UTR. Knee OA chondrocytes were transfected with miR-9 mimics, miR-9 inhibitor, and NF-κB1 siRNA, respectively, and changes in cellular proliferation and apoptosis were detected via MTT assay and flow cytometric analysis, respectively. Western blotting assay was used to detect the expressions of NF-κB1, interleukin-6 (IL-6), and matrix metalloproteinase-13 (MMP-13). According to results from human OA samples and rat OA models, miR-9 was significantly downregulated in knee OA cartilage tissues compared with normal cartilage tissues (P < 0.01). The expressions of NF-κB1, IL-6, and MMP-13 in knee OA cartilage tissues were significantly higher than those in normal cartilage tissues (P < 0.01). Dual luciferase reporter gene assay showed that miR-9 could bind to the 3′UTR of NF-κB1 and significantly inhibit the luciferase activity by 37% (P < 0.01). Upregulation of miR-9 or downregulation of NF-κB1 could promote cell proliferation and suppress cell apoptosis. Conclusively, downregulated miR-9 can facilitate proliferation and antiapoptosis of knee OA chondrocytes by directly binding to NF-kB1, implying that stimulating miR-9

  13. MicroRNA-9 regulates the development of knee osteoarthritis through the NF-kappaB1 pathway in chondrocytes.

    PubMed

    Gu, Ronghe; Liu, Ning; Luo, Simin; Huang, Weiguo; Zha, Zhengang; Yang, Jie

    2016-09-01

    It has been suggested that microRNA-9 (miR-9) is associated with the development of knee osteoarthritis (OA). This study was aimed to investigate the association between the mechanism of miR-9 targeting nuclear factor kappa-B1 (NF-κB1) and the proliferation and apoptosis of knee OA chondrocytes.Cartilage samples were collected from 25 patients with knee OA and 10 traumatic amputees, and another 15 OA rat models, together with 15 rats without knee OA lesions were also established. MiR-9 expressions in both knee OA cartilage and normal cartilage samples were detected using quantitative real-time PCR. The expressions of related genes (NF-κB1, IL-6, and MMP-13) in the two groups were also detected. Dual luciferase reporter gene assay was employed to examine the effect of miR-9 on the luciferase activity of NF-κB1 3'UTR. Knee OA chondrocytes were transfected with miR-9 mimics, miR-9 inhibitor, and NF-κB1 siRNA, respectively, and changes in cellular proliferation and apoptosis were detected via MTT assay and flow cytometric analysis, respectively. Western blotting assay was used to detect the expressions of NF-κB1, interleukin-6 (IL-6), and matrix metalloproteinase-13 (MMP-13).According to results from human OA samples and rat OA models, miR-9 was significantly downregulated in knee OA cartilage tissues compared with normal cartilage tissues (P < 0.01). The expressions of NF-κB1, IL-6, and MMP-13 in knee OA cartilage tissues were significantly higher than those in normal cartilage tissues (P < 0.01). Dual luciferase reporter gene assay showed that miR-9 could bind to the 3'UTR of NF-κB1 and significantly inhibit the luciferase activity by 37% (P < 0.01). Upregulation of miR-9 or downregulation of NF-κB1 could promote cell proliferation and suppress cell apoptosis.Conclusively, downregulated miR-9 can facilitate proliferation and antiapoptosis of knee OA chondrocytes by directly binding to NF-kB1, implying that stimulating miR-9 expressions might

  14. PTHrP and Indian hedgehog control differentiation of growth plate chondrocytes at multiple steps.

    PubMed

    Kobayashi, Tatsuya; Chung, Ung-Il; Schipani, Ernestina; Starbuck, Michael; Karsenty, Gerard; Katagiri, Takenobu; Goad, Dale L; Lanske, Beate; Kronenberg, Henry M

    2002-06-01

    In developing murine growth plates, chondrocytes near the articular surface (periarticular chondrocytes) proliferate, differentiate into flat column-forming proliferating cells (columnar chondrocytes), stop dividing and finally differentiate into hypertrophic cells. Indian hedgehog (Ihh), which is predominantly expressed in prehypertrophic cells, stimulates expression of parathyroid hormone (PTH)-related peptide (PTHrP) which negatively regulates terminal chondrocyte differentiation through the PTH/PTHrP receptor (PPR). However, the roles of PTHrP and Ihh in regulating earlier steps in chondrocyte differentiation are unclear. We present novel mouse models with PPR abnormalities that help clarify these roles. In mice with chondrocyte-specific PPR ablation and mice with reduced PPR expression, chondrocyte differentiation was accelerated not only at the terminal step but also at an earlier step: periarticular to columnar differentiation. In these models, upregulation of Ihh action in the periarticular region was also observed. In the third model in which the PPR was disrupted in about 30% of columnar chondrocytes, Ihh action in the periarticular chondrocytes was upregulated because of ectopically differentiated hypertrophic chondrocytes that had lost PPR. Acceleration of periarticular to columnar differentiation was also noted in this mouse, while most of periarticular chondrocytes retained PPR signaling. These data suggest that Ihh positively controls differentiation of periarticular chondrocytes independently of PTHrP. Thus, chondrocyte differentiation is controlled at multiple steps by PTHrP and Ihh through the mutual regulation of their activities. PMID:12050144

  15. Regulation of xylosyltransferase I gene expression by interleukin 1β in human primary chondrocyte cells: mechanism and impact on proteoglycan synthesis.

    PubMed

    Khair, Mostafa; Bourhim, Mustapha; Barré, Lydia; Li, Dong; Netter, Patrick; Magdalou, Jacques; Fournel-Gigleux, Sylvie; Ouzzine, Mohamed

    2013-01-18

    Xylosyltransferase I (XT-I) is an essential enzyme of proteoglycan (PG) biosynthesis pathway catalyzing the initial and rate-limiting step in glycosaminoglycan chain assembly. It plays a critical role in the regulation of PG synthesis in cartilage; however, little is known about underlying mechanism. Here, we provide evidence that, in human primary chondrocytes, IL-1β regulates XT-I gene expression into an early phase of induction and a late phase of down-regulation. Based on promoter deletions, the region up to -850 bp was defined as a major element of XT-I gene displaying both constitutive and IL-1β-regulated promoter activity. Point mutation and signaling analyses revealed that IL-1β-induced promoter activity is achieved through AP-1 response elements and mediated by SAP/JNK and p38 signaling pathways. Transactivation and chromatin immunoprecipitation assays indicated that AP-1 is a potent transactivator of XT-I promoter and that IL-1β-induced activity is mediated through increased recruitment of AP-1 to the promoter. Finally, we show that Sp3 is a repressor of XT-I promoter and bring evidence that the repressive effect of IL-1β during the late phase is mediated through Sp3 recruitment to the promoter. This suggests that modulation of Sp3 in cartilage could prevent IL-1β inhibition of PG synthesis and limit tissue degradation.

  16. Considerations on the use of ear chondrocytes as donor chondrocytes for cartilage tissue engineering.

    PubMed

    Van Osch, Gerjo J V M; Mandl, Erik W; Jahr, Holger; Koevoet, Wendy; Nolst-Trenité, Gilbert; Verhaar, Jan A N

    2004-01-01

    Articular cartilage is often used for research on cartilage tissue engineering. However, ear cartilage is easier to harvest, with less donor-site morbidity. The aim of this study was to evaluate whether adult human ear chondrocytes were capable of producing cartilage after expansion in monolayer culture. Cell yield per gram of cartilage was twice as high for ear than for articular cartilage. Moreover, ear chondrocytes proliferated faster. Cell proliferation could be further stimulated by the use of serum-free medium with Fibroblast Growth Factor 2 (FGF2) in stead of medium with 10% serum. To evaluate chondrogenic capacity, multiplied chondrocytes were suspended in alginate and implanted subcutaneously in athymic mice. After 8 weeks the constructs demonstrated a proteoglycan-rich matrix that contained collagen type II. Constructs of ear chondrocytes showed a faint staining for elastin. Quantitative RT-PCR revealed that expression of collagen type II was 2-fold upregulated whereas expression of collagen type I was 2-fold down regulated in ear chondrocytes expanded in serum-free medium with FGF2 compared to serum-containing medium. Expression of alkaline phosphatase and collagen type X were low indicating the absence of terminal differentiation. We conclude that ear chondrocytes can be used as donor chondrocytes for cartilage tissue engineering. Furthermore, it may proof to be a promising alternative cell source to engineer cartilage for articular repair.

  17. Runx2 inhibits chondrocyte proliferation and hypertrophy through its expression in the perichondrium

    PubMed Central

    Hinoi, Eiichi; Bialek, Peter; Chen, You-Tzung; Rached, Marie-Therese; Groner, Yoram; Behringer, Richard R.; Ornitz, David M.; Karsenty, Gerard

    2006-01-01

    The perichondrium, a structure made of undifferentiated mesenchymal cells surrounding growth plate cartilage, regulates chondrocyte maturation through poorly understood mechanisms. Analyses of loss- and gain-of-function models show that Twist-1, whose expression in cartilage is restricted to perichondrium, favors chondrocyte maturation in a Runx2-dependent manner. Runx2, in turn, enhances perichondrial expression of Fgf18, a regulator of chondrocyte maturation. Accordingly, compound heterozygous embryos for Runx2 and Fgf18 deletion display the same chondrocyte maturation phenotype as Fgf18-null embryos. This study identifies a transcriptional basis for the inhibition of chondrocyte maturation by perichondrium and reveals that Runx2 fulfills antagonistic functions during chondrogenesis. PMID:17050674

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

    PubMed

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

    2015-06-01

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

  19. 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.

  20. Human immunodeficiency virus type 1 enhancer-binding protein 3 is essential for the expression of asparagine-linked glycosylation 2 in the regulation of osteoblast and chondrocyte differentiation.

    PubMed

    Imamura, Katsuyuki; Maeda, Shingo; Kawamura, Ichiro; Matsuyama, Kanehiro; Shinohara, Naohiro; Yahiro, Yuhei; Nagano, Satoshi; Setoguchi, Takao; Yokouchi, Masahiro; Ishidou, Yasuhiro; Komiya, Setsuro

    2014-04-01

    Human immunodeficiency virus type 1 enhancer-binding protein 3 (Hivep3) suppresses osteoblast differentiation by inducing proteasomal degradation of the osteogenesis master regulator Runx2. In this study, we tested the possibility of cooperation of Hivep1, Hivep2, and Hivep3 in osteoblast and/or chondrocyte differentiation. Microarray analyses with ST-2 bone stroma cells demonstrated that expression of any known osteochondrogenesis-related genes was not commonly affected by the three Hivep siRNAs. Only Hivep3 siRNA promoted osteoblast differentiation in ST-2 cells, whereas all three siRNAs cooperatively suppressed differentiation in ATDC5 chondrocytes. We further used microarray analysis to identify genes commonly down-regulated in both MC3T3-E1 osteoblasts and ST-2 cells upon knockdown of Hivep3 and identified asparagine-linked glycosylation 2 (Alg2), which encodes a mannosyltransferase residing on the endoplasmic reticulum. The Hivep3 siRNA-mediated promotion of osteoblast differentiation was negated by forced Alg2 expression. Alg2 suppressed osteoblast differentiation and bone formation in cultured calvarial bone. Alg2 was immunoprecipitated with Runx2, whereas the combined transfection of Runx2 and Alg2 interfered with Runx2 nuclear localization, which resulted in suppression of Runx2 activity. Chondrocyte differentiation was promoted by Hivep3 overexpression, in concert with increased expression of Creb3l2, whose gene product is the endoplasmic reticulum stress transducer crucial for chondrogenesis. Alg2 silencing suppressed Creb3l2 expression and chondrogenesis of ATDC5 cells, whereas infection of Alg2-expressing virus promoted chondrocyte maturation in cultured cartilage rudiments. Thus, Alg2, as a downstream mediator of Hivep3, suppresses osteogenesis, whereas it promotes chondrogenesis. To our knowledge, this study is the first to link a mannosyltransferase gene to osteochondrogenesis.

  1. 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.

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

    PubMed

    Phull, Abdul-Rehman; Eo, Seong-Hui; Abbas, Qamar; Ahmed, Madiha; Kim, Song Ja

    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. 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

  4. Effect of retinoic acid on protein synthesis by foetal bovine chondrocytes in high-density culture: down-regulation of the glucose-regulated protein, GRP-78, and type II collagen.

    PubMed Central

    Freyria, A M; Ronzière, M C; Boutillon, M M; Herbage, D

    1995-01-01

    The effect of 0.1-10 microM retinoic acid (RA) on foetal bovine chondrocytes was investigated in high-density cultures (0.6 x 10(6) cells/cm2). After 5 days of culture in ascorbate-free medium, control chondrocytes presented a typical rounded shape and synthesized type II, IX, XI and III collagens. After RA treatment on days 2-5 of culture, the cells exhibited a fibroblast-like shape and decreased synthesis of total protein (48%) and pepsinresistant proteins (60%) as determined by [35S]methionine labelling. Addition of RA was not followed by the expression of type I collagen, but induced quantitative changes in the synthesis of cartilage-specific collagens (II, IX and XI) as measured by direct autoradiography of the corresponding bands after SDS/PAGE. The main change was in type II collagen synthesis, with a 80% decrease in the cell-layer fraction and a 89% decrease in culture-medium fraction; inhibition of type IX and XI collagen synthesis was limited to 25 and 31% respectively. Modifications to intracellular proteins induced by RA were determined by using two-dimensional electrophoresis associated with a computerized imaging system. Synthesis of one of the more abundant proteins (pI 4.8; 78 kDa) was decreased by 75% after RA treatment. This protein was characterized by micro-sequencing as the glucose-regulated protein 78 (GRP 78). It was reported previously to bind denatured collagen and mutated type I procollagen molecule and to function as a molecular chaperone for collagen molecules. It remains to demonstrate whether the parallel down-regulation of GRP 78 and type II collagen observed here corresponds to a co-ordinate regulation of these two proteins. Images Figure 1 Figure 2 Figure 3 PMID:7832751

  5. Chondrocyte channel transcriptomics

    PubMed Central

    Lewis, Rebecca; May, Hannah; Mobasheri, Ali; Barrett-Jolley, Richard

    2013-01-01

    To date, a range of ion channels have been identified in chondrocytes using a number of different techniques, predominantly electrophysiological and/or biomolecular; each of these has its advantages and disadvantages. Here we aim to compare and contrast the data available from biophysical and microarray experiments. This letter analyses recent transcriptomics datasets from chondrocytes, accessible from the European Bioinformatics Institute (EBI). We discuss whether such bioinformatic analysis of microarray datasets can potentially accelerate identification and discovery of ion channels in chondrocytes. The ion channels which appear most frequently across these microarray datasets are discussed, along with their possible functions. We discuss whether functional or protein data exist which support the microarray data. A microarray experiment comparing gene expression in osteoarthritis and healthy cartilage is also discussed and we verify the differential expression of 2 of these genes, namely the genes encoding large calcium-activated potassium (BK) and aquaporin channels. PMID:23995703

  6. Differentiated embryo chondrocyte 1 (DEC1) is a novel negative regulator of hepatic fibroblast growth factor 21 (FGF21) in aging mice.

    PubMed

    Fujita, Yu; Makishima, Makoto; Bhawal, Ujjal K

    2016-01-15

    Human differentiated embryo chondrocyte expressed gene 1 (DEC1) is frequently used as a marker of senescence in vivo. Fibroblast growth factor 21 (FGF21), a novel endocrine-like member of the FGF superfamily, is highly expressed in the liver, and FGF21-transgenic mice have extended lifespans. Thus, we hypothesized that FGF21 may play a role in the DEC1-mediated aging process. In this study, DEC1 knockout (KO) mice were used to characterize the mechanism by which FGF21 protects mice from aging. Aging is strongly diminished in DEC1 KO mice, which is reflected by decreased lipid levels and oxidative stress, leading to an amelioration of liver function and structure. The expression of FGF21 decreased with aging in wild-type (WT) mice, whereas ATF4, Phospho-ERK and Phospho-p38 expression was maintained and was accompanied by a compensatory rise of FGF21 mRNA and protein expression in DEC1 KO mice. Over-expression of DEC1 markedly abolished the hepatic expression of FGF21, and siRNA-mediated inhibition of endogenous DEC1 increased the expression of FGF21. DEC1 further diminished the expression of ATF4 in HepG2 cells over-expressing DEC1. The induction of FGF21 and ATF4 at the mRNA and protein levels during the course of aging supports the view that DEC1 KO mice are able to restore the age-related imbalance of metabolism. Collectively, the data obtained in this study suggest that DEC1 is a novel negative regulator of hepatic FGF21 expression.

  7. Perinatal exposure to vitamin A differentially regulates chondrocyte growth and the expression of aggrecan and matrix metalloprotein genes in the femur of neonatal rats.

    PubMed

    Zhang, Yao; Wray, Amanda E; Ross, A Catharine

    2012-04-01

    Vitamin A (VA) and its active form, retinoic acid (RA), are regulators of skeletal development. In the present study, we investigated if maternal VA intake during pregnancy and lactation, as well as direct oral supplementation of neonates with VA + RA (VARA) in early life, alters neonatal bone formation and chondrocyte gene expression. Offspring of dams fed 3 levels of VA (marginal, adequate, and supplemented) for 10 wk were studied at birth (P0) and postnatal day 7 (P7). One-half of the newborns received an oral supplement of VARA on P1, P4, and P7. Tissues were collected on P0 and 6 h after the last dose on P7. Pup plasma and liver retinol concentrations were increased by both maternal VA intake and VARA (P < 0.01). Although maternal VA did not affect bone mineralization as assessed by von Kossa staining, newborn femur length was increased with maternal VA (P < 0.05). VARA supplementation of neonates increased the length of the hypertrophic zone only in VA-marginal pups, close to that in neonates from VA-adequate dams, suggesting VARA caused a catching up of growth that was limited by low maternal VA intake. Maternal diet did not alter type X nor type II collagen mRNA. However, VARA-treated pups from VA-supplemented dams had reduced mRNA for aggrecan, a major component of cartilage matrix, and increased mRNA for matrix metalloproteinase (MMP)13, which catalyzes the degradation of aggrecan and collagens. These results suggest that moderately high maternal VA intake combined with neonatal VARA supplementation can reduce the ratio of aggrecan:MMP, which may unfavorably alter early bone development.

  8. Effect of thiram on avian growth plate chondrocytes in culture.

    PubMed

    Rasaputra, Komal Singh; Liyanage, Rohana; Lay, Jackson O; Slavik, Michael F; Rath, Narayan C

    2013-02-01

    Thiram is a dithiocarbamate pesticide that causes tibial dyschondroplasia (TD), a growth plate defect, in poultry. Deaths of transitional zone chondrocytes appear to interrupt endochondral bone development leading to the broadening of growth plate. The mechanism of action of thiram on chondrocytes is not well understood. Since proteins play major roles in different aspects of cell's metabolism, growth, and survival, the objective of this study was to find whether thiram produces proteomic changes that could impair the development of chondrocytes. The chondrocytes, isolated from proximal tibial growth plates, were cultured with or without a sub-lethal concentration of thiram for 48 hr, and the cell proteins were extracted, and subjected to 2-D gel electrophoresis. The gel images were compared and statistically evaluated using Melanie software to identify differentially expressed protein spots. Of a total of 72 identifiable spots 3 were down-regulated and 2 up-regulated in thiram treated chondrocytes. In-gel trypsin digestion of the protein spots followed by their characterization by matrix-assisted laser desorption ionization-time-of- flight (MALDI-TOF) mass spectrometry identified 25 spots comprising of 23 proteins. Two of 3 down-regulated proteins were identified as a heat shock protein 70 (HSP 70) and a GALE (UDP-galactose-4 epimerase) protein isoform I. The up-regulated proteins were Serpin H1, a protein involved in collagen metabolism and a redox sensor NmrA-like (NMRAL) family domain protein-1. Both GALE and NMRAL proteins are implicated in energy metabolism and redox regulation whereas the HSP 70 protects cells against stress, and implicated in chondrocyte hypertrophy, an important event in endochondral bone formation. The failure of chondrocyte protective mechanisms such as associated with protection against cellular stress and energy metabolism appear to be the likely cause for chondrocyte death induced by thiram.

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

    SciTech Connect

    Ikeda, Kazuhiro; Tsukui, Tohru; Imazawa, Yukiko; Horie-Inoue, Kuniko; Inoue, Satoshi

    2012-09-07

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

  10. ATF3 deficiency in chondrocytes alleviates osteoarthritis development.

    PubMed

    Iezaki, Takashi; Ozaki, Kakeru; Fukasawa, Kazuya; Inoue, Makoto; Kitajima, Shigetaka; Muneta, Takeshi; Takeda, Shu; Fujita, Hiroyuki; Onishi, Yuki; Horie, Tetsuhiro; Yoneda, Yukio; Takarada, Takeshi; Hinoi, Eiichi

    2016-08-01

    Activating transcription factor 3 (Atf3) has been implicated in the pathogenesis of various diseases, including cancer and inflammation, as well as in the regulation of cell proliferation and differentiation. However, the involvement of Atf3 in developmental skeletogenesis and joint disease has not been well studied to date. Here, we show that Atf3 is a critical mediator of osteoarthritis (OA) development through its expression in chondrocytes. ATF3 expression was markedly up-regulated in the OA cartilage of both mice and humans. Conditional deletion of Atf3 in chondrocytes did not result in skeletal abnormalities or affect the chondrogenesis, but alleviated the development of OA generated by surgically inducing knee joint instability in mice. Inflammatory cytokines significantly up-regulated Atf3 expression through the nuclear factor-kB (NF-kB) pathway, while cytokine-induced interleukin-6 (Il6) expression was repressed, in ATF3-deleted murine and human chondrocytes. Mechanistically, Atf3 deficiency decreased cytokine-induced Il6 transcription in chondrocytes through repressing NF-kB signalling by the attenuation of the phosphorylation status of IkB and p65. These findings suggest that Atf3 is implicated in the pathogenesis of OA through modulation of inflammatory cytokine expression in chondrocytes, and the feed-forward loop of inflammatory cytokines/NF-kB/Atf3 in chondrocytes may be a novel therapeutic target for the treatment for OA. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

  11. A pathway to bone: signaling molecules and transcription factors involved in chondrocyte development and maturation

    PubMed Central

    Kozhemyakina, Elena; Lassar, Andrew B.; Zelzer, Elazar

    2015-01-01

    Decades of work have identified the signaling pathways that regulate the differentiation of chondrocytes during bone formation, from their initial induction from mesenchymal progenitor cells to their terminal maturation into hypertrophic chondrocytes. Here, we review how multiple signaling molecules, mechanical signals and morphological cell features are integrated to activate a set of key transcription factors that determine and regulate the genetic program that induces chondrogenesis and chondrocyte differentiation. Moreover, we describe recent findings regarding the roles of several signaling pathways in modulating the proliferation and maturation of chondrocytes in the growth plate, which is the ‘engine’ of bone elongation. PMID:25715393

  12. Evc works in chondrocytes and osteoblasts to regulate multiple aspects of growth plate development in the appendicular skeleton and cranial base.

    PubMed

    Pacheco, María; Valencia, María; Caparrós-Martín, José A; Mulero, Francisca; Goodship, Judith A; Ruiz-Perez, Victor L

    2012-01-01

    Ellis-van Creveld syndrome protein homolog (Evc) was previously shown to mediate expression of Indian hedgehog (Ihh) downstream targets in chondrocytes. Consequently disruption of the Ihh/Pthrp axis was demonstrated in Evc(-/-) mice, but the full extent of Evc involvement in endochondral development was not totally characterized. Herein we have examined further the Evc(-/-) growth plate in a homogeneous genetic background and show that Evc promotes chondrocyte proliferation, chondrocyte hypertrophy and the differentiation of osteoblasts in the perichondrium, hence implicating Evc in both Pthrp-dependent and Pthrp-independent Ihh functions. We also demonstrate that Evc, which localizes to osteoblast primary cilia, mediates Hedgehog (Hh) signaling in the osteoblast lineage. In spite of this, bone collar development is mildly affected in Evc(-/-) mutants. The onset of perichondrial osteoblastogenesis is delayed at the initial stages of endochondral ossification in Evc(-/-) mice, and in later stages, the leading edge of expression of osteoblast markers and Wnt/β-catenin signaling components is located closer to the primary spongiosa in the Evc(-/-) perichondrium owing to impaired osteoblast differentiation. Additionally we have used Ptch1-LacZ reporter mice to learn about the different types of Hh-responsive cells that are present in the perichondrium of normal and Evc(-/-) mice. Evc mediates Hh target gene expression in inner perichondrial cells, but it is dispensable in the external layers of the perichondrium. Finally, we report cranial base defects in Evc(-/-) mice and reveal that Evc is essential for intrasphenoidal synchondrosis development.

  13. Disruption of endogenous perlecan function improves differentiation of rat articular chondrocytes in vitro.

    PubMed

    Nakamura, Ryosuke; Nakamura, Fumio; Fukunaga, Shigeharu

    2015-04-01

    Heparan sulfate (HS) and heparan sulfate proteoglycans (HSPG) are necessary for normal cartilage development and chondrocyte differentiation. However, recent studies demonstrated that HSPG accelerate dedifferentiation and catabolism in chondrocytes from degenerative cartilage. In this study, we investigated the inhibitory effect of HSPG on chondrocyte differentiation in vitro. Rat articular chondrocytes were cultured at low (0.3 × 10(4) cells/cm(2) ) and high (1.5 × 10(5) cells/cm(2) ) density in the presence or absence of heparitinase I, an HS degrading enzyme. Cells cultured at low density dedifferentiated and exhibited an elongated morphology, and treatment with heparitinase I precluded cell elongation. Conversely, populations of chondrocytes cultured at high density exhibited either a dedifferentiated or differentiated phenotype. Glycosaminoglycan accumulation increased in heparitinase I-treated cells. To determine the function of perlecan, an important HSPG for cartilage development, in chondrocyte differentiation, rat chondrocyte cultures were exposed to an anti-perlecan antiserum to inhibit perlecan function. Western blotting analysis indicated that preventing perlecan activity increased type II collagen synthesis. Our results suggest that HSPG are negative regulators of chondrocyte differentiation in vitro and that perlecan contributes to chondrocyte dedifferentiation in vitro.

  14. Inorganic pyrophosphate generation by transforming growth factor-beta-1 is mainly dependent on ANK induction by Ras/Raf-1/extracellular signal-regulated kinase pathways in chondrocytes.

    PubMed

    Cailotto, Frederic; Bianchi, Arnaud; Sebillaud, Sylvie; Venkatesan, Narayanan; Moulin, David; Jouzeau, Jean-Yves; Netter, Patrick

    2007-01-01

    ANK is a multipass transmembrane protein transporter thought to play a role in the export of intracellular inorganic pyrophosphate and so to contribute to the pathophysiology of chondrocalcinosis. As transforming growth factor-beta-1 (TGF-beta1) was shown to favor calcium pyrophosphate dihydrate deposition, we investigated the contribution of ANK to the production of extracellular inorganic pyrophosphate (ePPi) by chondrocytes and the signaling pathways involved in the regulation of Ank expression by TGF-beta1. Chondrocytes were exposed to 10 ng/mL of TGF-beta1, and Ank expression was measured by quantitative polymerase chain reaction and Western blot. ePPi was quantified in cell supernatants. RNA silencing was used to define the respective roles of Ank and PC-1 in TGF-beta1-induced ePPi generation. Finally, selective kinase inhibitors and dominant-negative/overexpression plasmid strategies were used to explore the contribution of several signaling pathways to Ank induction by TGF-beta1. TGF-beta1 strongly increased Ank expression at the mRNA and protein levels, as well as ePPi production. Using small interfering RNA technology, we showed that Ank contributed approximately 60% and PC-1 nearly 20% to TGF-beta1-induced ePPi generation. Induction of Ank by TGF-beta1 required activation of the extracellular signal-regulated kinase (ERK) pathway but not of p38-mitogen-activated protein kinase or of protein kinase A. In line with the general protein kinase C (PKC) inhibitor calphostin C, Gö6976 (a Ca2+-dependent PKC inhibitor) diminished TGF-beta1-induced Ank expression by 60%, whereas a 10% inhibition was observed with rottlerin (a PKCdelta inhibitor). These data suggest a regulatory role for calcium in TGF-beta1-induced Ank expression. Finally, we demonstrated that the stimulatory effect of TGF-beta1 on Ank expression was inhibited by the suppression of the Ras/Raf-1 pathway, while being enhanced by their constitutive activation. Transient overexpression of Smad 7, an

  15. Effects of hexosamines and omega-3/omega-6 fatty acids on pH regulation by interleukin 1-treated isolated bovine articular chondrocytes.

    PubMed

    Tattersall, Amanda L; Wilkins, Robert J

    2008-06-01

    Previous work has shown that interleukin 1 (IL-1) increases the activity of acid extruders in articular chondrocytes, while the H+-adenosine triphosphatase (ATPase) inhibitor bafilomycin can prevent aggrecanase-mediated cartilage degradation. The H+ transport induced by IL-1 may therefore be required for proteinase activity. In the present study, the effects of hexosamines and fish oils on H+-ATPase activity have been characterised for isolated bovine articular chondrocytes. Cells isolated in the presence of IL-1 were acidified, and the fraction of acid extrusion mediated by Na+-H+ exchange and an H+-ATPase were determined using specific inhibitors. Exposure to IL-1 significantly enhanced both components of acid extrusion. Co-incubation with glucosamine or mannosamine attenuated the H+-ATPase fraction of efflux. The addition of glucosamine at 9 h after exposure to IL-1--when H+-ATPase activation is already apparent--was also able to abolish H+-ATPase activity, implying that hexosamines do not exert effects at the level of protein synthesis. Co-incubation with the glucose transport inhibitor phloretin elicited similar effects to the hexosamines, suggesting that modulation of adenosine triphosphate levels may underlie their effects on H+-ATPase function. The omega-3 fish oil linolenic acid but not the omega-6 fish oil linoleic acid reduced H+-ATPase activity to levels seen in IL-1-untreated cells, although total efflux remained elevated, as a result of an enhanced H+ leak. These observations support a model whereby IL-1 stimulates an H+-ATPase-dependent system, possibly involved in aggrecanase activation, which appears to be one of the target mechanisms interrupted by dietary supplements reported to have symptom-modifying effects on osteoarthritis.

  16. Prolonged Application of High Fluid Shear to Chondrocytes Recapitulates Gene Expression Profiles Associated with Osteoarthritis

    PubMed Central

    Zhu, Fei; Wang, Pu; Lee, Norman H.; Goldring, Mary B.; Konstantopoulos, Konstantinos

    2010-01-01

    Background Excessive mechanical loading of articular cartilage producing hydrostatic stress, tensile strain and fluid flow leads to irreversible cartilage erosion and osteoarthritic (OA) disease. Since application of high fluid shear to chondrocytes recapitulates some of the earmarks of OA, we aimed to screen the gene expression profiles of shear-activated chondrocytes and assess potential similarities with OA chondrocytes. Methodology/Principal Findings Using a cDNA microarray technology, we screened the differentially-regulated genes in human T/C-28a2 chondrocytes subjected to high fluid shear (20 dyn/cm2) for 48 h and 72 h relative to static controls. Confirmation of the expression patterns of select genes was obtained by qRT-PCR. Using significance analysis of microarrays with a 5% false discovery rate, 71 and 60 non-redundant transcripts were identified to be ≥2-fold up-regulated and ≤0.6-fold down-regulated, respectively, in sheared chondrocytes. Published data sets indicate that 42 of these genes, which are related to extracellular matrix/degradation, cell proliferation/differentiation, inflammation and cell survival/death, are differentially-regulated in OA chondrocytes. In view of the pivotal role of cyclooxygenase-2 (COX-2) in the pathogenesis and/or progression of OA in vivo and regulation of shear-induced inflammation and apoptosis in vitro, we identified a collection of genes that are either up- or down-regulated by shear-induced COX-2. COX-2 and L-prostaglandin D synthase (L-PGDS) induce reactive oxygen species production, and negatively regulate genes of the histone and cell cycle families, which may play a critical role in chondrocyte death. Conclusions/Significance Prolonged application of high fluid shear stress to chondrocytes recapitulates gene expression profiles associated with osteoarthritis. Our data suggest a potential link between exposure of chondrocytes/cartilage to abnormal mechanical loading and the pathogenesis/progression of OA

  17. 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. PMID:26661891

  18. 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. PMID:25869133

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

    SciTech Connect

    Liu, Qi; Wan, Qilong; Yang, Rongtao; Zhou, Haihua; Li, Zubing

    2012-07-20

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

  1. FOXO transcription factors support oxidative stress resistance in human chondrocytes

    PubMed Central

    Akasaki, Yukio; Alvarez-Garcia, Oscar; Saito, Masahiko; Caramés, Beatriz; Iwamoto, Yukihide; Lotz, Martin K.

    2014-01-01

    Objectives A major signaling pathway that regulates cellular aging is the Insulin/IGF-1/Pl3k/Akt/forkhead-box class O (FOXO) transcription factor axis. Previously, we observed that FOXO factors are dysregulated in aged and OA cartilage. The objective of this study was to investigate the impact of downregulated FOXOs on chondrocytes. Methods Small interference RNAs (siRNAs) for FOXO1 and FOXO3 were transfected into human articular chondrocytes. Cell viability following treatment with the oxidant tert-Butyl hydroperoxide (t-BHP) was measured by MTT assay. Caspase-3/7 activation and apoptotic cell were examined. Gene and protein expression of antioxidant proteins and autophagy related proteins and changes in inflammatory mediators following treatment with IL-1β were analyzed. Cells transfected with FOXO plasmids were also analyzed. Results Cell viability was significantly reduced by siFOXO under treatment with t-BHP. Apoptosis accompanied by caspase activation was significantly induced in FOXO-siRNA transfected chondrocytes. Knock-down of FOXO1 and FOXO1+3 resulted in significant reductions of GPX-1, catalase, LC3, Beclin1, and SIRT1 proteins following treatment with t-BHP. In contrast, constitutive active form of FOXO 3 increased cell viability while inducing GPX1, Beclin1, and LC3 in response to t-BHP. Expression and production of ADAMTS-4 and Chemerin were significantly increased in FOXO-siRNA transfected chondrocytes. Conclusions Reduced expression of FOXO transcription factors in chondrocytes increased susceptibility to cell death induced by oxidative stress. This was associated with reduced antioxidant proteins and autophagy related proteins. Our data provide evidence for a key role of FOXO transcription factors as regulators of chondrocyte oxidative stress resistance and tissue homeostasis. PMID:25186470

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

    PubMed

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

    2013-09-01

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

  4. MicroRNA-33 suppresses CCL2 expression in chondrocytes.

    PubMed

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

    2016-06-01

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

  5. Leptin Receptor Metabolism Disorder in Primary Chondrocytes from Adolescent Idiopathic Scoliosis Girls

    PubMed Central

    Wang, Yun-Jia; Yu, Hong-Gui; Zhou, Zhen-Hai; Guo, Qiang; Wang, Long-Jie; Zhang, Hong-Qi

    2016-01-01

    To investigate the underlying mechanisms of low metabolic activity of primary chondrocytes obtained from girls with adolescent idiopathic scoliosis (AIS); AIS is a spine-deforming disease that often occurs in girls. AIS is associated with a lower bone mass than that of healthy individuals and osteopenia. Leptin was shown to play an important role in bone growth. It can also regulate the function of chondrocytes. Changes in leptin and Ob-R levels in AIS patients have been reported in several studies. The underlying mechanisms between the dysfunction of peripheral leptin signaling and abnormal chondrocytes remain unclear; The following parameters were evaluated in AIS patients and the control groups: total serum leptin levels; Ob-R expression in the plasma membrane of primary chondrocytes; JAK2 and STAT3 phosphorylation status. Then, we inhibited the lysosome and proteasome and knocked down clathrin heavy chain (CHC) expression in primary chondrocytes isolated from girls with AIS and evaluated Ob-R expression. We investigated the effects of leptin combined with a lysosome inhibitor or CHC knockdown in primary chondrocytes obtained from AIS patients; Compared with the controls, AIS patients showed similar total serum leptin levels, reduced JAK2 and STAT3 phosphorylation, and decreased cartilage matrix synthesis in the facet joint. Lower metabolic activity and lower membrane expression of Ob-R were observed in primary chondrocytes from the AIS group than in the controls. Lysosome inhibition increased the total Ob-R content but had no effect on the membrane expression of Ob-R or leptin’s effects on AIS primary chondrocytes. CHC knockdown upregulated the membrane Ob-R levels and enhanced leptin’s effects on AIS primary chondrocytes; The underlying mechanism of chondrocytes that are hyposensitive to leptin in some girls with AIS is low plasma membrane Ob-R expression that results from an imbalance between the rate of receptor endocytosis and the insertion of newly

  6. Leptin Receptor Metabolism Disorder in Primary Chondrocytes from Adolescent Idiopathic Scoliosis Girls.

    PubMed

    Wang, Yun-Jia; Yu, Hong-Gui; Zhou, Zhen-Hai; Guo, Qiang; Wang, Long-Jie; Zhang, Hong-Qi

    2016-01-01

    To investigate the underlying mechanisms of low metabolic activity of primary chondrocytes obtained from girls with adolescent idiopathic scoliosis (AIS); AIS is a spine-deforming disease that often occurs in girls. AIS is associated with a lower bone mass than that of healthy individuals and osteopenia. Leptin was shown to play an important role in bone growth. It can also regulate the function of chondrocytes. Changes in leptin and Ob-R levels in AIS patients have been reported in several studies. The underlying mechanisms between the dysfunction of peripheral leptin signaling and abnormal chondrocytes remain unclear; The following parameters were evaluated in AIS patients and the control groups: total serum leptin levels; Ob-R expression in the plasma membrane of primary chondrocytes; JAK2 and STAT3 phosphorylation status. Then, we inhibited the lysosome and proteasome and knocked down clathrin heavy chain (CHC) expression in primary chondrocytes isolated from girls with AIS and evaluated Ob-R expression. We investigated the effects of leptin combined with a lysosome inhibitor or CHC knockdown in primary chondrocytes obtained from AIS patients; Compared with the controls, AIS patients showed similar total serum leptin levels, reduced JAK2 and STAT3 phosphorylation, and decreased cartilage matrix synthesis in the facet joint. Lower metabolic activity and lower membrane expression of Ob-R were observed in primary chondrocytes from the AIS group than in the controls. Lysosome inhibition increased the total Ob-R content but had no effect on the membrane expression of Ob-R or leptin's effects on AIS primary chondrocytes. CHC knockdown upregulated the membrane Ob-R levels and enhanced leptin's effects on AIS primary chondrocytes; The underlying mechanism of chondrocytes that are hyposensitive to leptin in some girls with AIS is low plasma membrane Ob-R expression that results from an imbalance between the rate of receptor endocytosis and the insertion of newly

  7. Biological and Chemical Removal of Primary Cilia Affects Mechanical Activation of Chondrogenesis Markers in Chondroprogenitors and Hypertrophic Chondrocytes

    PubMed Central

    Deren, Matthew E.; Yang, Xu; Guan, Yingjie; Chen, Qian

    2016-01-01

    Chondroprogenitors and hypertrophic chondrocytes, which are the first and last stages of the chondrocyte differentiation process, respectively, are sensitive to mechanical signals. We hypothesize that the mechanical sensitivity of these cells depends on the cell surface primary cilia. To test this hypothesis, we removed the primary cilia by biological means with transfection with intraflagellar transport protein 88 (IFT88) siRNA or by chemical means with chloral hydrate treatment. Transfection of IFT88 siRNA significantly reduced the percentage of ciliated cells in both chondroprogenitor ATDC5 cells as well as primary hypertrophic chondrocytes. Cyclic loading (1 Hz, 10% matrix deformation) of ATDC5 cells in three-dimensional (3D) culture stimulates the mRNA levels of chondrogenesis marker Type II collagen (Col II), hypertrophic chondrocyte marker Type X collagen (Col X), and a molecular regulator of chondrogenesis and chondrocyte hypertrophy bone morphogenetic protein 2 (BMP-2). The reduction of ciliated chondroprogenitors abolishes mechanical stimulation of Col II, Col X, and BMP-2. In contrast, cyclic loading stimulates Col X mRNA levels in hypertrophic chondrocytes, but not those of Col II and BMP-2. Both biological and chemical reduction of ciliated hypertrophic chondrocytes reduced but failed to abolish mechanical stimulation of Col X mRNA levels. Thus, primary cilia play a major role in mechanical stimulation of chondrogenesis and chondrocyte hypertrophy in chondroprogenitor cells and at least a partial role in hypertrophic chondrocytes. PMID:26861287

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

    SciTech Connect

    Saha, Sushmita; Kirkham, Jennifer; Wood, David; Curran, Stephen; Yang, Xuebin

    2010-10-22

    a difference in the temporal expression of chondrogenic markers which were up regulated in chondrogenic medium compared to levels in basal medium. Of the three cell types studied, adult chondrocytes offer a more promising cell source for cartilage tissue engineering. This comparative study revealed differences between the microenvironment of all three cell types and provides useful information to inform cell-based therapies for cartilage regeneration.

  9. Lucigenin-dependent chemiluminescence in articular chondrocytes.

    PubMed

    Rathakrishnan, C; Tiku, M L

    1993-08-01

    We were recently able to measure intracellular levels of hydrogen peroxide within normal articular chondrocytes using the trapped indicator 2',7'-dichlorofluorescein diacetate. Further studies have shown that stimulated chondrocytes produce luminol-dependent chemiluminescence, suggesting that these cells produce hydrogen peroxide and singlet oxygen. In the present study, we have investigated the lucigenin-dependent chemiluminescence response in normal articular chondrocytes. Chondrocytes either in suspension or adhered to cover slips showed lucigenin-dependent chemiluminescence. There was a dose-dependent increase in chemiluminescence response when chondrocytes were incubated with soluble stimuli like phorbol-myristate-acetate, concanavalin A, and f-met-leu-phe. Catalase and the metabolic inhibitor, sodium azide, which inhibits the enzyme myeloperoxidase, had no inhibitory effect on lucigenin-dependent chemiluminescence production. Only the antioxidant, superoxide dismutase, prevented lucigenin-dependent chemiluminescence, indicating that this assay measures the production of superoxide anions by chondrocytes. We confirmed that chondrocytes release superoxide radicals using the biochemical assay of ferricytochrome c reduction. Since cartilage tissue is semi-transparent, we were able to measure chemiluminescence response in live cartilage tissue, showing that chondrocytes which are embedded within the matrix can also generate superoxide anion radicals. Reactive oxygen intermediates have been shown to play a significant role in the degradation of matrix in arthritis. Our previous and present studies suggest that oxygen radicals produced by chondrocytes may be an important mechanism by which chondrocytes induce cartilage matrix degradation.

  10. Crucial Role of Elovl6 in Chondrocyte Growth and Differentiation during Growth Plate Development in Mice

    PubMed Central

    Kikuchi, Manami; Matsuzaka, Takashi; Ishii, Kiyoaki; Nakagawa, Yoshimi; Takayanagi, Misa; Yamada, Nobuhiro; Shimano, Hitoshi

    2016-01-01

    ELOVL family member 6, elongation of very long chain fatty acids (Elovl6) is a microsomal enzyme, which regulates the elongation of C12-16 saturated and monounsaturated fatty acids. Elovl6 has been shown to be associated with various pathophysiologies including insulin resistance, atherosclerosis, and non-alcoholic steatohepatitis. To investigate a potential role of Elovl6 during bone development, we here examined a skeletal phenotype of Elovl6 knockout (Elovl6-/-) mice. The Elovl6-/- skeleton was smaller than that of controls, but exhibited no obvious patterning defects. Histological analysis revealed a reduced length of proliferating and an elongated length of hypertrophic chondrocyte layer, and decreased trabecular bone in Elovl6-/- mice compared with controls. These results were presumably due to a modest decrease in chondrocyte proliferation and accelerated differentiation of cells of the chondrocyte lineage. Consistent with the increased length of the hypertrophic chondrocyte layer in Elovl6-/- mice, Collagen10α1 was identified as one of the most affected genes by ablation of Elovl6 in chondrocytes. Furthermore, this elevated expression of Collagen10α1 of Elovl6-null chondrocytes was likely associated with increased levels of Foxa2/a3 and Mef2c mRNA expression. Relative increases in protein levels of nuclear Foxa2 and cytoplasmic histone deacethylase 4/5/7 were also observed in Elovl6 knockdown cells of the chondrocyte lineage. Collectively, our data suggest that Elovl6 plays a critical role for proper development of embryonic growth plate. PMID:27467521

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

    PubMed

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

    2016-09-01

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

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

    PubMed

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

    2016-09-01

    Due to its evascular, aneural, and alymphatic conditions, articular cartilage shows extremely poor regenerative ability. Thus, directing chondrocyte toward a desired location and function by utilizing the mechanical cues of biomaterials is a promising approach for effective tissue regeneration. However, chondrocytes cultured on Petri dish will lose their typical phenotype which may lead to compromised results. Therefore, we fabricated polydimethylsiloxane (PDMS) materials with various stiffness as culture substrates. Cell morphology and focal adhesion of chondrocytes displayed significant changes. The cytoskeletal tension of the adherent cells observed by average myosin IIA fluorescent intensity increased as stiffness of the underlying substrates decreased, consistent with the alteration of chondrocyte phenotype in our study. Immunofluorescent images and q-PCR results revealed that chondrocyte cultured on soft substrates showed better chondrocyte functionalization by more type II collagen and aggrecan expression, related to the lowest mRNA level of Rac-1, RhoA, ROCK-1, and ROCK-2. Taken together, this work not only points out that matrix elasticity can regulate chondrocyte functionalization via RhoA/ROCK pathway, but also provides new prospect for biomechanical control of cell behavior in cell-based cartilage regeneration. PMID:27534990

  13. [Molecular mechanisms of cartilage formation and chondrocyte maturation].

    PubMed

    Tamamura, Yoshihiro; Iwamoto, Masahiro

    2004-07-01

    Cartilage plays multiple roles in vertebrate animals. In an embryonic stage and early postnatal life, cartilage is important not only as a structural support of early embryo but also as a template of endochondral bone. In a later postnatal life, cartilage provides smooth joint movement and tissue elasticity. A number of critical signaling molecules that regulate cartilage formation and chondrocytes maturation in endochondral bone formation have been identified to date. The interplay of those important molecules is also actively studied. However, several fundamental questions still remain unsolved. What signal initiates mesenchymal cell condensation? Does condensation enough to make cells competent for BMP-induced chondrogenesis? Is there chondrocyte stem cell in cartilage? Likewise, it is not known which factor triggers chondrocytes maturation. In this review article, we summarized the action of several key factors including BMP, hedgehog, PTHrP, and Wnt in condensation, chondrogenenic differentiation and maturation of chondrocytes. Towards further understanding of above fundamental questions, this review article also tried to propose future direction of cartilage biology research. PMID:15577071

  14. Identification and characterization of the novel Col10a1 regulatory mechanism during chondrocyte hypertrophic differentiation

    PubMed Central

    Gu, J; Lu, Y; Li, F; Qiao, L; Wang, Q; Li, N; Borgia, J A; Deng, Y; Lei, G; Zheng, Q

    2014-01-01

    The majority of human skeleton develops through the endochondral pathway, in which cartilage-forming chondrocytes proliferate and enlarge into hypertrophic chondrocytes that eventually undergo apoptosis and are replaced by bone. Although at a terminal differentiation stage, hypertrophic chondrocytes have been implicated as the principal engine of bone growth. Abnormal chondrocyte hypertrophy has been seen in many skeletal dysplasia and osteoarthritis. Meanwhile, as a specific marker of hypertrophic chondrocytes, the type X collagen gene (COL10A1) is also critical for endochondral bone formation, as mutation and altered COL10A1 expression are often accompanied by abnormal chondrocyte hypertrophy in many skeletal diseases. However, how the type X collagen gene is regulated during chondrocyte hypertrophy has not been fully elucidated. We have recently demonstrated that Runx2 interaction with a 150-bp mouse Col10a1 cis-enhancer is required but not sufficient for its hypertrophic chondrocyte-specific reporter expression in transgenic mice, suggesting requirement of additional Col10a1 regulators. In this study, we report in silico sequence analysis of this 150-bp enhancer and identification of its multiple binding factors, including AP1, MEF2, NFAT, Runx1 and TBX5. Using this enhancer as bait, we performed yeast one-hybrid assay and identified multiple candidate Col10a1-interacting genes, including cyclooxygenase 1 (Cox-1) and Cox-2. We have also performed mass spectrometry analysis and detected EF1-alpha, Fus, GdF7 and Runx3 as components of the specific complex formed by the cis-enhancer and nuclear extracts from hypertrophic MCT (mouse chondrocytes immortalized with large T antigen) cells that express Col10a1 abundantly. Notably, some of the candidate genes are differentially expressed in hypertrophic MCT cells and have been associated with chondrocyte hypertrophy and Runx2, an indispensible Col10a1 regulator. Intriguingly, we detected high-level Cox-2 expression in

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

    PubMed Central

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

    2012-01-01

    Objective: 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. Methods: 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. Results: 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. Conclusions: 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. PMID:26069626

  16. Dynamic Compression of Chondrocyte-Agarose Constructs Reveals New Candidate Mechanosensitive Genes

    PubMed Central

    Bougault, Carole; Aubert-Foucher, Elisabeth; Paumier, Anne; Perrier-Groult, Emeline; Huot, Ludovic; Hot, David; Duterque-Coquillaud, Martine; Mallein-Gerin, Frédéric

    2012-01-01

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

  17. Simvastatin inhibits CD44 fragmentation in chondrocytes.

    PubMed

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

    2016-08-15

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

  18. Articular chondrocyte metabolism and osteoarthritis

    SciTech Connect

    Leipold, H.R.

    1989-01-01

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

  19. CCN2 as a Novel Molecule Supporting Energy Metabolism of Chondrocytes

    PubMed Central

    Maeda-Uematsu, Aya; Kubota, Satoshi; Kawaki, Harumi; Kawata, Kazumi; Miyake, Yoshiaki; Hattori, Takako; Nishida, Takashi; Moritani, Norifumi; Lyons, Karen M.; Iida, Seiji; Takigawa, Masaharu

    2014-01-01

    CCN2/connective tissue growth factor (CTGF) is a unique molecule that promotes both chondrocytic differentiation and proliferation through its matricellular interaction with a number of extracellular biomolecules. This apparently contradictory functional property of CCN2 suggests its certain role in basic cellular activities such as energy metabolism, which is required for both proliferation and differentiation. Comparative metabolomic analysis of costal chondrocytes isolated from wild-type and Ccn2-null mice revealed overall impaired metabolism in the latter. Among the numerous metabolites analyzed, stable reduction in the intracellular level of ATP, GTP, CTP, or UTP was observed, indicating a profound role of CCN2 in energy metabolism. Particularly, the cellular level of ATP was decreased by more than 50% in the Ccn2-null chondrocytes. The addition of recombinant CCN2 (rCCN2) to cultured Ccn2-null chondrocytes partly redeemed the cellular ATP level attenuated by Ccn2 deletion. Next, in order to investigate the mechanistic background that mediates the reduction in ATP level in these Ccn2-null chondrocytes, we performed transcriptome analysis. As a result, several metabolism-associated genes were found to have been up-regulated or down-regulated in the mutant mice. Up-regulation of a number of ribosomal protein genes was observed upon Ccn2 deletion, whereas a fewgenes required for aerobic and anaerobic ATP production were down-regulated in the Ccn2-null chondrocytes. Among such genes, reduction in the expression of the enolase 1 gene was of particular note. These findings uncover a novel functional role of CCN2 as a metabolic supporter in the growth-plate chondrocytes, which is required for skeletogenesis in mammals. PMID:24288211

  20. Cytotoxic T lymphocytes recognize and lyse chondrocytes under inflammatory, but not non-inflammatory conditions.

    PubMed

    Cohen, E Suzanne; Bodmer, Helen C

    2003-05-01

    The human major histocompatibility complex (MHC) class I allele HLA-B27 is strongly associated with seronegative spondyloarthropathies including ankylosing spondylitis and reactive arthritis. Although of unknown aetiology, one hypothesis suggests that a cytotoxic T cell (CTL) response against a self-antigen at sites of inflammation, such as entheses or joints may be involved. The chondrocyte is one of the major specialized cell types found both in articular cartilage and cartilaginous entheses and therefore is a possible source of such an antigen. CTL recognition of these cells is a potential mechanism for inflammation and cartilage damage, both through direct lysis of chondrocytes and the secretion of pro-inflammatory cytokines such as tumour necrosis factor and interferon-gamma (IFN-gamma). We test the feasibility of this hypothesis by examining the ability of chondrocytes to present antigen to CTL in vitro. Chondrocytes isolated from the ribcages of mice did not constitutively express detectable levels of MHC class I by fluorescence-activated cell sorting analysis. In addition, they were resistant to lysis by alloreactive and influenza A virus nucleoprotein (NP)-specific CTL. However, treatment of chondrocytes with IFN-gamma up-regulated MHC class I expression and rendered the cells susceptible to lysis by CTL. Similarly, IFN-gamma-treated chondrocytes infected with influenza A virus were recognized by NP-specific CTL, though with variable efficiency. Thus, we suggest that under certain circumstances CTL-mediated lysis of chondrocytes is potentially a potent mechanism for cartilage damage in vivo, but that low levels of MHC class I on healthy chondrocytes protects from immune recognition in health. PMID:12709012

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

    PubMed Central

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

    2015-01-01

    Hypertrophic differentiation of chondrocytes is a main barrier in application of mesenchymal stem cells (MSCs) for cartilage repair. In addition, hypertrophy occurs occasionally in osteoarthritis (OA). Here we provide a comprehensive review on recent literature describing signal pathways in the hypertrophy of MSCs-derived in vitro differentiated chondrocytes and chondrocytes, with an emphasis on the crosstalk between these pathways. Insight into the exact regulation of hypertrophy by the signaling network is necessary for the efficient application of MSCs for articular cartilage repair and for developing novel strategies for curing OA. We focus on articles describing the role of the main signaling pathways in regulating chondrocyte hypertrophy-like changes. Most studies report hypertrophic differentiation in chondrogenesis of MSCs, in both human OA and experimental OA. Chondrocyte hypertrophy is not under the strict control of a single pathway but appears to be regulated by an intricately regulated network of multiple signaling pathways, such as WNT, Bone morphogenetic protein (BMP)/Transforming growth factor-β (TGFβ), Parathyroid hormone-related peptide (PTHrP), Indian hedgehog (IHH), Fibroblast growth factor (FGF), Insulin like growth factor (IGF) and Hypoxia-inducible factor (HIF). This comprehensive review describes how this intricate signaling network influences tissue-engineering applications of MSCs in articular cartilage (AC) repair, and improves understanding of the disease stages and cellular responses within an OA articular joint. PMID:26287176

  2. Morphological, genetic and phenotypic comparison between human articular chondrocytes and cultured chondrocytes.

    PubMed

    Mata-Miranda, Mónica Maribel; Martinez-Martinez, Claudia María; Noriega-Gonzalez, Jesús Emmanuel; Paredes-Gonzalez, Luis Enrique; Vázquez-Zapién, Gustavo Jesús

    2016-08-01

    Articular cartilage is an avascular and aneural tissue with limited capacity for regeneration. On large articular lesions, it is recommended to use regenerative medicine strategies, like autologous chondrocyte implantation. There is a concern about morphological changes that chondrocytes suffer once they have been isolated and cultured. Due to the fact that there is little evidence that compares articular cartilage chondrocytes with cultured chondrocytes, in this research we proposed to obtain chondrocytes from human articular cartilage, compare them with themselves once they have been cultured and characterize them through genetic, phenotypic and morphological analysis. Knee articular cartilage samples of 10 mm were obtained, and each sample was divided into two fragments; a portion was used to determine gene expression, and from the other portion, chondrocytes were obtained by enzymatic disaggregation, in order to be cultured and expanded in vitro. Subsequently, morphological, genetic and phenotypic characteristics were compared between in situ (articular cartilage) and cultured chondrocytes. Obtained cultured chondrocytes were rounded in shape, possessing a large nucleus with condensed chromatin and a clear cytoplasm; histological appearance was quite similar to typical chondrocyte. The expression levels of COL2A1 and COL10A1 genes were higher in cultured chondrocytes than in situ chondrocytes; moreover, the expression of COL1A1 was almost undetectable on cultured chondrocytes; likewise, COL2 and SOX9 proteins were detected by immunofluorescence. We concluded that chondrocytes derived from adult human cartilage cultured for 21 days do not tend to dedifferentiate, maintaining their capacity to produce matrix and also retaining their synthesis capacity and morphology.

  3. Growth Factor Priming Differentially Modulates Components of the Extracellular Matrix Proteome in Chondrocytes and Synovium-Derived Stem Cells

    PubMed Central

    Xiong, Jennifer C.; Colligan, Ryan M.; Bulinski, J. Chloë; Cook, James L.; Ateshian, Gerard A.; Brown, Lewis M.; Hung, Clark T.

    2014-01-01

    To make progress in cartilage repair it is essential to optimize protocols for two-dimensional cell expansion. Chondrocytes and SDSCs are promising cell sources for cartilage repair. We previously observed that priming with a specific growth factor cocktail (1 ng/mL transforming growth factor-β1, 5 ng/mL basic fibroblast growth factor, and 10 ng/mL platelet-derived growth factor-BB) in two-dimensional culture, led to significant improvement in mechanical and biochemical properties of synovium-derived stem cell (SDSC)-seeded constructs. The current study assessed the effect of growth factor priming on the proteome of canine chondrocytes and SDSCs. In particular, growth factor priming modulated the proteins associated with the extracellular matrix in two-dimensional cultures of chondrocytes and SDSCs, inducing a partial dedifferentiation of chondrocytes (most proteins associated with cartilage were down-regulated in primed chondrocytes) and a partial differentiation of SDSCs (some collagen-related proteins were up-regulated in primed SDSCs). However, when chondrocytes and SDSCs were grown in pellet culture, growth factor-primed cells maintained their chondrogenic potential with respect to glycosaminoglycan and collagen production. In conclusion, the strength of the label-free proteomics technique is that it allows for the determination of changes in components of the extracellular matrix proteome in chondrocytes and SDSCs in response to growth factor priming, which could help in future tissue engineering strategies. PMID:24516581

  4. Dielectric Characterization of Costal Cartilage Chondrocytes

    PubMed Central

    Stacey, Michael W.; Sabuncu, Ahmet Can; Beskok, Ali

    2013-01-01

    Background Chondrocytes respond to biomechanical and bioelectrochemical stimuli by secreting appropriate extracellular matrix proteins that enables the tissue to withstand the large forces it experiences. Although biomechanical aspects of cartilage are well described, little is known of the bioelectrochemical responses. The focus of this study is to identify bioelectrical characteristics of human costal cartilage cells using dielectric spectroscopy. Methods Dielectric spectroscopy allows non-invasive probing of biological cells. An in house computer program is developed to extract dielectric properties of human costal cartilage cells from raw cell suspension impedance data measured by a microfluidic device. The dielectric properties of chondrocytes are compared with other cell types in order to comparatively assess the electrical nature of chondrocytes. Results The results suggest that electrical cell membrane characteristics of chondrocyte cells are close to cardiomyoblast cells, cells known to possess an array of active ion channels. The blocking effect of the non-specific ion channel blocker gadolinium is tested on chondrocytes with a significant reduction in both membrane capacitance and conductance. Conclusions We have utilized a microfluidic chamber to mimic biomechanical events through changes in bioelectrochemistry and described the dielectric properties of chondrocytes to be closer to cells derived from electrically excitably tissues General significance and interest The studydescribes dielectric characterization of human costal chondrocyte cells using physical tools, where results and methodology can be used to identify potential anomalies in bioelectrochemical responses that may lead to cartilage disorders. PMID:24016606

  5. Hydrostatic pressure influences HIF-2 alpha expression in chondrocytes.

    PubMed

    Inoue, Hiroaki; Arai, Yuji; Kishida, Tsunao; Terauchi, Ryu; Honjo, Kuniaki; Nakagawa, Shuji; Tsuchida, Shinji; Matsuki, Tomohiro; Ueshima, Keiichirou; Fujiwara, Hiroyoshi; Mazda, Osam; Kubo, Toshikazu

    2015-01-01

    Hypoxia-inducible factor (HIF)-2α is considered to play a major role in the progression of osteoarthritis. Recently, it was reported that pressure amplitude influences HIF-2α expression in murine endothelial cells. We examined whether hydrostatic pressure is involved in expression of HIF-2α in articular chondrocytes. Chondrocytes were cultured and stimulated by inflammation or hydrostatic pressure of 0, 5, 10, or 50 MPa. After stimulation, heat shock protein (HSP) 70, HIF-2α, nuclear factor kappa B (NF-κB), matrix metalloproteinase (MMP)-13, MMP-3, and vascular endothelial growth factor (VEGF) gene expression were evaluated. The levels of all gene expression were increased by inflammatory stress. When chondrocytes were exposed to a hydrostatic pressure of 5 MPa, HIF-2α, MMP-13, and MMP-3 gene expression increased significantly although those of HSP70 and NF-κB were not significantly different from the control group. In contrast, HIF-2α gene expression did not increase under a hydrostatic pressure of 50 MPa although HSP70 and NF-κB expression increased significantly compared to control. We considered that hydrostatic pressure of 5 MPa could regulate HIF-2α independent of NF-κB, because the level of HIF-2α gene expression increased significantly without upregulation of NF-κB expression at 5 MPa. Hydrostatic pressure may influence cartilage degeneration, inducing MMP-13 and MMP-3 expression through HIF-2α. PMID:25569085

  6. DEXAMETHASONE PROMOTES CPPD CRYSTAL FORMATION BY ARTICULAR CHONDROCYTES

    PubMed Central

    Fahey, Mark; Mitton, Elizabeth; Muth, Emily; Rosenthal, Ann K.

    2008-01-01

    Objective Calcium pyrophosphate dihydrate crystals (CPPD) are commonly found in osteoarthritic joints and correlate with a poor prognosis. Intra-articular corticosteroids, such as dexamethasone (Dxm), are commonly used therapies for osteoarthritis with or without CPPD deposition. Dxm has variable effects in mineralization models. We investigated the effects of Dxm on CPPD crystal formation in a well established tissue culture model. Methods Porcine articular chondrocytes were incubated with ATP to generate CPPD crystals. Chondrocytes incubated with or without ATP were exposed to 1–100 nM Dxm in the presence of 45Ca. Mineralization was measured by 45Ca uptake in the cell layer. We also investigated the effect of Dxm on mineralization-regulating enzymes such as alkaline phosphatase, NTPPPH and transglutaminase. Results Dxm significantly increased ATP-induced mineralization by articular chondrocytes. While alkaline phosphatase and NTPPPH activities were unchanged by Dxm, transglutaminase activity increased in a clear dose responsive manner. Levels of factor XIIIA mRNA and protein were increased by Dxm, while type II Tgase protein was unchanged. Transglutaminase inhibitors suppressed Dxm-induced increases in CPPD crystal formation. Conclusion These findings suggest a potential for Dxm to contribute to pathologic mineralization in cartilage and reinforce a central role for the transglutaminase enzymes in CPPD crystal formation. PMID:19132782

  7. Hydrostatic Pressure Influences HIF-2 Alpha Expression in Chondrocytes

    PubMed Central

    Inoue, Hiroaki; Arai, Yuji; Kishida, Tsunao; Terauchi, Ryu; Honjo, Kuniaki; Nakagawa, Shuji; Tsuchida, Shinji; Matsuki, Tomohiro; Ueshima, Keiichirou; Fujiwara, Hiroyoshi; Mazda, Osam; Kubo, Toshikazu

    2015-01-01

    Hypoxia-inducible factor (HIF)-2α is considered to play a major role in the progression of osteoarthritis. Recently, it was reported that pressure amplitude influences HIF-2α expression in murine endothelial cells. We examined whether hydrostatic pressure is involved in expression of HIF-2α in articular chondrocytes. Chondrocytes were cultured and stimulated by inflammation or hydrostatic pressure of 0, 5, 10, or 50 MPa. After stimulation, heat shock protein (HSP) 70, HIF-2α, nuclear factor kappa B (NF-κB), matrix metalloproteinase (MMP)-13, MMP-3, and vascular endothelial growth factor (VEGF) gene expression were evaluated. The levels of all gene expression were increased by inflammatory stress. When chondrocytes were exposed to a hydrostatic pressure of 5 MPa, HIF-2α, MMP-13, and MMP-3 gene expression increased significantly although those of HSP70 and NF-κB were not significantly different from the control group. In contrast, HIF-2α gene expression did not increase under a hydrostatic pressure of 50 MPa although HSP70 and NF-κB expression increased significantly compared to control. We considered that hydrostatic pressure of 5 MPa could regulate HIF-2α independent of NF-κB, because the level of HIF-2α gene expression increased significantly without upregulation of NF-κB expression at 5 MPa. Hydrostatic pressure may influence cartilage degeneration, inducing MMP-13 and MMP-3 expression through HIF-2α. PMID:25569085

  8. Wnts produced by Osterix-expressing osteolineage cells regulate their proliferation and differentiation

    PubMed Central

    Tan, Si Hui; Senarath-Yapa, Kshemendra; Chung, Michael T.; Longaker, Michael T.; Wu, Joy Y.; Nusse, Roeland

    2014-01-01

    Wnt signaling is a critical regulator of bone development, but the identity and role of the Wnt-producing cells are still unclear. We addressed these questions through in situ hybridization, lineage tracing, and genetic experiments. First, we surveyed the expression of all 19 Wnt genes and Wnt target gene Axin2 in the neonatal mouse bone by in situ hybridization, and demonstrated—to our knowledge for the first time—that Osterix-expressing cells coexpress Wnt and Axin2. To track the behavior and cell fate of Axin2-expressing osteolineage cells, we performed lineage tracing and showed that they sustain bone formation over the long term. Finally, to examine the role of Wnts produced by Osterix-expressing cells, we inhibited Wnt secretion in vivo, and observed inappropriate differentiation, impaired proliferation, and diminished Wnt signaling response. Therefore, Osterix-expressing cells produce their own Wnts that in turn induce Wnt signaling response, thereby regulating their proliferation and differentiation. PMID:25422448

  9. 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.

  10. ROCK inhibitor prevents the dedifferentiation of human articular chondrocytes

    SciTech Connect

    Matsumoto, Emi; Furumatsu, Takayuki; Kanazawa, Tomoko; Tamura, Masanori; Ozaki, Toshifumi

    2012-03-30

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

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

    PubMed

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

    2016-07-01

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

  12. Native Chondrocyte Viability during Cartilage Lesion Progression

    PubMed Central

    Ganguly, Kumkum; McRury, Ian D.; Goodwin, Peter M.; Morgan, Roy E.; Augé, Wayne K.

    2010-01-01

    Objective: Early surgical intervention for articular cartilage disease is desirable before full-thickness lesions develop. As early intervention treatments are designed, native chondrocyte viability at the treatment site before intervention becomes an important parameter to consider. The purpose of this study is to evaluate native chondrocyte viability in a series of specimens demonstrating the progression of articular cartilage lesions to determine if the chondrocyte viability profile changes during the evolution of articular cartilage disease to the level of surface fibrillation. Design: Osteochondral specimens demonstrating various degrees of articular cartilage damage were obtained from patients undergoing knee total joint replacement. Three groups were created within a patient harvest based on visual and tactile cues commonly encountered during surgical intervention: group 1, visually and tactilely intact surfaces; group 2, visually intact, tactilely soft surfaces; and group 3, surface fibrillation. Confocal laser microscopy was performed following live/dead cell viability staining. Results: Groups 1 to 3 demonstrated viable chondrocytes in all specimens, even within the fibrillated portions of articular cartilage, with little to no evidence of dead chondrocytes. Chondrocyte viability profile in articular cartilage does not appear to change as disease lesion progresses from normal to surface fibrillation. Conclusions: Fibrillated partial-thickness articular cartilage lesions are a good therapeutic target for early intervention. These lesions retain a high profile of viable chondrocytes and are readily diagnosed by visual and tactile cues during surgery. Early intervention should be based on matrix failure rather than on more aggressive procedures that further corrupt the matrix and contribute to chondrocyte necrosis of contiguous untargeted cartilage. PMID:26069561

  13. Perspective. Osteoclastogenesis and growth plate chondrocyte differentiation: emergence of convergence.

    PubMed

    Odgren, Paul R; Philbrick, William M; Gartland, Alison

    2003-01-01

    A "bone" is really a dynamic and highly interactive complex of many cell and tissue types. In particular, for the majority of skeletal elements to develop and grow, the process of endochondral ossification requires a constantly moving interface between cartilage, invading blood vessels, and bone. A great deal has been learned in recent years about the regulation of chondrocyte proliferation and differentiation by hormones, growth factors, and physiologic stimuli during skeletal development and growth. Likewise, the discovery that colony stimulating factor-1 (CSF-1, or M-CSF) and receptor activator of NF-kappaB ligand (RANKL, a tumor necrosis factor superfamily member also called TRANCE, ODF, OPGL, and TNFSF11) are pivotal in communicating from osteoblasts to osteoclasts has led to deeper insights into bone growth, turnover, and maintenance. Little is known, however, about how these two quite different systems communicate to solve the problem of providing integrated, continuous mechanical support during the dynamic invasion of cartilage by bone that characterizes endochondral bone growth. Evidence has accumulated in recent years that provides insight into the communication between growing bone and cartilage in the form of a subset of osteopetrotic mutations, which share a lack of osteoclasts and an accompanying chondrodysplasia of the growth plate. These mutations thus implicate some of the same gene products in regulating chondrocyte differentiation and bone resorption. We also consider expression studies of some known growth plate regulators, such as parathyroid hormone-related protein (PTHrP) and Indian hedgehog (Ihh), in light of this and propose a model in which the osteoclastogenic factors act also on chondrocytes, but downstream of PTRrP and Ihh in regulating proliferation and differentiation, and after early morphogenic patterns are established.

  14. IFT88 influences chondrocyte actin organization and biomechanics

    PubMed Central

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

    2016-01-01

    Summary Objectives Primary cilia are microtubule based organelles which control a variety of signalling pathways important in cartilage development, health and disease. This study examines the role of the intraflagellar transport (IFT) protein, IFT88, in regulating fundamental actin organisation and mechanics in articular chondrocytes. Methods The study used an established chondrocyte cell line with and without hypomorphic mutation of IFT88 (IFT88orpk). Confocal microscopy was used to quantify F-actin and myosin IIB organisation. Viscoelastic cell and actin cortex mechanics were determined using micropipette aspiration with actin dynamics visualised in live cells transfected with LifeACT-GFP. Results IFT88orpk cells exhibited a significant increase in acto-myosin stress fibre organisation relative to wild-type (WT) cells in monolayer and an altered response to cytochalasin D. Rounded IFT88orpk cells cultured in suspension exhibited reduced cortical actin expression with reduced cellular equilibrium modulus. Micropipette aspiration resulted in reduced membrane bleb formation in IFT88orpk cells. Following membrane blebbing, IFT88orpk cells exhibited slower reformation of the actin cortex. IFT88orpk cells showed increased actin deformability and reduced cortical tension confirming that IFT regulates actin cortex mechanics. The reduced cortical tension is also consistent with the reduced bleb formation. Conclusions This study demonstrates for the first time that the ciliary protein IFT88 regulates fundamental actin organisation and the stiffness of the actin cortex leading to alterations in cell deformation, mechanical properties and blebbing in an IFT88 chondrocyte cell line. This adds to the growing understanding of the role of primary cilia and IFT in regulating cartilage biology. PMID:26493329

  15. Biochemical and Proteomic Characterization of Alkaptonuric Chondrocytes

    PubMed Central

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

    2012-01-01

    Alkaptonuria (AKU) is a rare genetic disease associated with the accumulation of homogentisic acid (HGA) and its oxidized/polymerized products which leads to the deposition of melanin-like pigments (ochronosis) in connective tissues. Although numerous case reports have described ochronosis in joints, little is known on the molecular mechanisms leading to such a phenomenon. For this reason, we characterized biochemically chondrocytes isolated from the ochronotic cartilage of AKU patients. Based on the macroscopic appearance of the ochronotic cartilage, two sub-populations were identified: cells coming from the black portion of the cartilage were referred to as “black” AKU chondrocytes, while those coming from the white portion were referred to as “white” AKU chondrocytes. Notably, both AKU chondrocytic types were characterized by increased apoptosis, NO release, and levels of pro-inflammatory cytokines. Transmission electron microscopy also revealed that intracellular ochronotic pigment deposition was common to both “white” and “black” AKU cells. We then undertook a proteomic and redox-proteomic analysis of AKU chondrocytes which revealed profound alterations in the levels of proteins involved in cell defence, protein folding, and cell organization. An increased post-translational oxidation of proteins, which also involved high molecular weight protein aggregates, was found to be particularly relevant in “black” AKU chondrocytes. J. Cell. Physiol. 227: 3333–3343, 2012. © 2011 Wiley Periodicals, Inc. PMID:22213341

  16. Transamidation by Transglutaminase 2 Transforms S100A11 Calgranulin into a Procatabolic Cytokine for Chondrocytes1

    PubMed Central

    Cecil, Denise L.; Terkeltaub, Robert

    2008-01-01

    In osteoarthritis (OA), low-grade joint inflammation promotes altered chondrocyte differentiation and cartilage catabolism. S100/calgranulins share conserved calcium-binding EF-hand domains, associate noncovalently as homodimers and heterodimers, and are secreted and bind receptor for advanced glycation end products (RAGE). Chondrocyte RAGE expression and S100A11 release are stimulated by IL-1β in vitro and increase in OA cartilage in situ. Exogenous S100A11 stimulates chondrocyte hypertrophic differentiation. Moreover, S100A11 is covalently cross-linked by transamidation catalyzed by transglutaminase 2 (TG2), itself an inflammation-regulated and redox stress-inducible mediator of chondrocyte hypertrophic differentiation. In this study, we researched mouse femoral head articular cartilage explants and knee chondrocytes, and a soluble recombinant double point mutant (K3R/Q102N) of S100A11 TG2 transamidation substrate sites. Both TG2 and RAGE knockout cartilage explants retained IL-1β responsiveness. The K3R/Q102N mutant of S100A11 retained the capacity to bind to RAGE and chondrocytes but lost the capacity to signal via the p38 MAPK pathway or induce chondrocyte hypertrophy and glycosaminoglycans release. S100A11 failed to induce hypertrophy, glycosaminoglycan release, and appearance of the aggrecanase neoepitope NITEGE in both RAGE and TG2 knockout cartilages. We conclude that transamidation by TG2 transforms S100A11 into a covalently bonded homodimer that acquires the capacity to signal through the p38 MAPK pathway, accelerate chondrocyte hypertrophy and matrix catabolism, and thereby couple inflammation with chondrocyte activation to potentially promote OA progression. PMID:18523305

  17. 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.

  18. Constitutive E2F1 Overexpression Delays Endochondral Bone Formation by Inhibiting Chondrocyte Differentiation

    PubMed Central

    Scheijen, Blanca; Bronk, Marieke; van der Meer, Tiffany; Bernards, René

    2003-01-01

    Longitudinal bone growth results from endochondral ossification, a process that requires proliferation and differentiation of chondrocytes. It has been shown that proper endochondral bone formation is critically dependent on the retinoblastoma family members p107 and p130. However, the precise functional roles played by individual E2F proteins remain poorly understood. Using both constitutive and conditional E2F1 transgenic mice, we show that ubiquitous transgene-driven expression of E2F1 during embryonic development results in a dwarf phenotype and significantly reduced postnatal viability. Overexpression of E2F1 disturbs chondrocyte maturation, resulting in delayed endochondral ossification, which is characterized by reduced hypertrophic zones and disorganized growth plates. Employing the chondrogenic cell line ATDC5, we investigated the effects of enforced E2F expression on the different phases of chondrocyte maturation that are normally required for endochondral ossification. Ectopic E2F1 expression strongly inhibits early- and late-phase differentiation of ATDC5 cells, accompanied by diminished cartilage nodule formation as well as decreased type II collagen, type X collagen, and aggrecan gene expression. In contrast, overexpression of E2F2 or E2F3a results in only a marginal delay of chondrocyte maturation, and increased E2F4 levels have no effect. These data are consistent with the notion that E2F1 is a regulator of chondrocyte differentiation. PMID:12724423

  19. A role of glypican4 and wnt5b in chondrocyte stacking underlying craniofacial cartilage morphogenesis

    PubMed Central

    Sisson, Barbara E.; Dale, Rodney M.; Mui, Stephanie R.; Topczewska, Jolanta M.

    2015-01-01

    The Wnt/Planar Cell Polarity (PCP) pathway controls cell morphology and behavior during animal development. Several zebrafish mutants were identified as having perturbed Wnt/PCP signaling. Many of these mutants have defects in craniofacial formation. To better understand the role that Wnt/PCP plays in craniofacial development we set out to identify which of the mutants, known to be associated with the Wnt/PCP pathway, perturb head cartilage formation by disrupting chondrocyte morphology. Here we demonstrate that while vang-like 2 (vangl2), wnt11 and scribbled (scrib) mutants have severe craniofacial morphogenesis defects they do not display the chondrocyte stacking and intercalation problems seen in glypican 4 (gpc4) and wnt5b mutants. The function of Gpc4 or Wnt5b appears to be important for chondrocyte organization, as the neural crest in both mutants is specified, undergoes migration, and differentiates into the same number of cells to compose the craniofacial cartilage elements. We demonstrate that Gpc4 activity is required cell autonomously in the chondrocytes and that the phenotype of single heterozygous mutants is slightly enhanced in embryos double heterozygous for wnt5b and gpc4. This data suggests a novel mechanism for Wnt5b and Gpc4 regulation of chondrocyte behavior that is independent of the core Wnt/PCP molecules and differs from their collaborative action of controlling cell movements during gastrulation. PMID:26459057

  20. The role of BKCa channels on hyperpolarization mediated by hyperosmolarity in human articular chondrocytes.

    PubMed

    Sánchez, Julio C; López-Zapata, Diego F

    2011-03-01

    Chondrocytes, the only cell in cartilage, are subjected to hyperosmotic challenges continuously since extracellular osmolarity in articular cartilage increases in response to mechanical loads during joint movement. Hyperosmolarity can affect membrane transport, and it is possible that load modulates matrix synthesis through alterations in intracellular composition. In the present study, the effects of hyperosmotic challenges were evaluated using the whole-cell patch clamp technique, whole cell mode on freshly isolated human and bovine articular chondrocytes. In human chondrocytes, hypertonicity induced the activation of outward Ca(2+)-sensitive K(+) currents, which were inhibited by iberiotoxin and TEA-Cl. The current induced by hypertonic switching (osmolarity from 300 to 400 mOsm/l) caused cell hyperpolarization (from -39 mV to -70 mV) with a reversal potential of -96 ± 7 mV. These results suggest a role for Ca(2+)-activated K(+) channels in human articular chondrocytes, leading to hyperpolarization as a consequence of K(+) efflux through these channels. These channels could have a role in the articular chondrocyte's response to a hyperosmotic challenge and matrix metabolism regulation by load.

  1. Comparing effects of perfusion and hydrostatic pressure on gene profiles of human chondrocyte.

    PubMed

    Zhu, Ge; Mayer-Wagner, Susanne; Schröder, Christian; Woiczinski, Matthias; Blum, Helmut; Lavagi, Ilaria; Krebs, Stefan; Redeker, Julia I; Hölzer, Andreas; Jansson, Volkmar; Betz, Oliver; Müller, Peter E

    2015-09-20

    Hydrostatic pressure and perfusion have been shown to regulate the chondrogenic potential of articular chondrocytes. In order to compare the effects of hydrostatic pressure plus perfusion (HPP) and perfusion (P) we investigated the complete gene expression profiles of human chondrocytes under HPP and P. A simplified bioreactor was constructed to apply loading (0.1 MPa for 2 h) and perfusion (2 ml) through the same piping by pressurizing the medium directly. High-density monolayer cultures of human chondrocytes were exposed to HPP or P for 4 days. Controls (C) were maintained in static cultures. Gene expression was evaluated by sequencing (RNAseq) and quantitative real-time PCR analysis. Both treatments changed gene expression levels of human chondrocytes significantly. Specifically, HPP and P increased COL2A1 expression and decreased COL1A1 and MMP-13 expression. Despite of these similarities, RNAseq revealed a list of cartilage genes including ACAN, ITGA10 and TNC, which were differentially expressed by HPP and P. Of these candidates, adhesion related molecules were found to be upregulated in HPP. Both HPP and P treatment had beneficial effects on chondrocyte differentiation and decreased catabolic enzyme expression. The study provides new insight into how hydrostatic pressure and perfusion enhance cartilage differentiation and inhibit catabolic effects.

  2. Pterosin B prevents chondrocyte hypertrophy and osteoarthritis in mice by inhibiting Sik3

    PubMed Central

    Yahara, Yasuhito; Takemori, Hiroshi; Okada, Minoru; Kosai, Azuma; Yamashita, Akihiro; Kobayashi, Tomohito; Fujita, Kaori; Itoh, Yumi; Nakamura, Masahiro; Fuchino, Hiroyuki; Kawahara, Nobuo; Fukui, Naoshi; Watanabe, Akira; Kimura, Tomoatsu; Tsumaki, Noriyuki

    2016-01-01

    Osteoarthritis is a common debilitating joint disorder. Risk factors for osteoarthritis include age, which is associated with thinning of articular cartilage. Here we generate chondrocyte-specific salt-inducible kinase 3 (Sik3) conditional knockout mice that are resistant to osteoarthritis with thickened articular cartilage owing to a larger chondrocyte population. We also identify an edible Pteridium aquilinum compound, pterosin B, as a Sik3 pathway inhibitor. We show that either Sik3 deletion or intraarticular injection of mice with pterosin B inhibits chondrocyte hypertrophy and protects cartilage from osteoarthritis. Collectively, our results suggest Sik3 regulates the homeostasis of articular cartilage and is a target for the treatment of osteoarthritis, with pterosin B as a candidate therapeutic. PMID:27009967

  3. 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

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

    PubMed

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

    2016-08-01

    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

  5. Mesenchymal stem cells display different gene expression profiles compared to hyaline and elastic chondrocytes

    PubMed Central

    Zhai, Li-Jie; Zhao, Ke-Qing; Wang, Zhi-Qiang; Feng, Ya; Xing, Shuang-Chun

    2011-01-01

    Cartilage has a poor intrinsic repair capacity, requiring surgical intervention to effect biological repair. Tissue engineering technologies or regenerative medicine strategies are currently being employed to address cartilage repair. Mesenchymal stem cells (MSCs) are considered to be an excellent cell source for this application. However, the different gene expression profiles between the MSCs and differentiated cartilage remain unclear. In this report, we first examined the gene expression profiles between the MSCs, hyaline and elastic chondrocytes, and then identify candidate genes, which may be important in the process of MSC differentiation into hyaline and elastic cartilage. Several hundred differentially expressed genes were screened initially by microarray, including 417 simultaneously up-regulated genes in both hyaline and elastic chondrocytes, with 313 down-regulated genes. Several genes were identified that were up-regulated in hyaline chondrocytes while down-regulated in elastic chondrocytes. Both RT-PCR and western blot analysis were consistent with those results obtained by microarray analysis. Chondromodulinl (Chm1) was found to be highly expressed in MSCs differentiating to hyaline and elastic cartilage. Both collagen type II, alpha 1 (Col2a1) and cartilage homeo protein 1 (Cart1) were also highly upregulated and may be important early differentiation of MSCs to hyaline cartilage. PMID:21394289

  6. 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.

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

    PubMed

    Lu, Shijin; Xiao, Xungang; Cheng, Minghua

    2015-01-01

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

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

    PubMed

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

    2006-07-01

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

  9. 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

  10. Chondrocytes Utilize a Cholesterol-Dependent Lipid Translocator To Externalize Phosphatidylserine†

    PubMed Central

    Damek-Poprawa, Monika; Golub, Ellis; Otis, Linda; Harrison, Gerald; Phillips, Christine; Boesze-Battaglia, Kathleen

    2016-01-01

    During endochondral ossification, growth plate chondrocytes release plasma membrane (PM) derived matrix vesicles (MV), which are the site of initial hydroxyapatite crystal formation. MV constituents which facilitate the mineralization process include the integral membrane ectoenzymes alkaline phosphatase (ALPase) and nucleotide pyrophosphatase phosphodiesterase (NPP1/PC-1), along with a phosphatidylserine- (PS-) rich membrane surface that binds annexins and calcium, resulting in enhanced calcium entry into MV. In this study, we determined that chick growth plate MV were highly enriched in membrane raft microdomains containing high levels of cholesterol, glycophosphatidylinositol- (GPI-) anchored ALPase, and phosphatidylserine (PS) localized to the external leaflet of the bilayer. To determine how such membrane microdomains arise during chondrocyte maturation, we explored the role of PM cholesterol-dependent lipid assemblies in regulating the activities of lipid translocators involved in the externalization of PS. We first isolated and determined the composition of detergent-resistant membranes (DRMs) from chondrocyte PM. DRMs isolated from chondrocyte PM were enhanced in ganglioside 1 (GM1) and cholesterol as well as GPI-anchored ALPase. Furthermore, these membrane domains were enriched in PS (localized to the external leaflet of the bilayer) and had significantly higher ALPase activity than non-cholesterol-enriched domains. To understand the role of cholesterol-dependent lipid assemblies in the externalization of PS, we measured the activities of two lipid transporters involved in PS externalization, aminophospholipid translocase (APLT) and phospholipid scramblase (PLSCR1), during maturation of a murine chondrocytic cell line, N1511. In this report, we provide the first evidence that maturing chondrocytes express PLSCR1 and have scramblase activity. We propose that redistribution of PS is dependent on an increase in phospholipid scramblase activity and a decrease

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

    PubMed

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

    2015-12-16

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

  12. Follistatin in chondrocytes: the link between TRPV4 channelopathies and skeletal malformations

    PubMed Central

    Leddy, Holly A.; McNulty, Amy L.; Lee, Suk Hee; Rothfusz, Nicole E.; Gloss, Bernd; Kirby, Margaret L.; Hutson, Mary R.; Cohn, Daniel H.; Guilak, Farshid; Liedtke, Wolfgang

    2014-01-01

    Point mutations in the calcium-permeable TRPV4 ion channel have been identified as the cause of autosomal-dominant human motor neuropathies, arthropathies, and skeletal malformations of varying severity. The objective of this study was to determine the mechanism by which TRPV4 channelopathy mutations cause skeletal dysplasia. The human TRPV4V620I channelopathy mutation was transfected into primary porcine chondrocytes and caused significant (2.6-fold) up-regulation of follistatin (FST) expression levels. Pore altering mutations that prevent calcium influx through the channel prevented significant FST up-regulation (1.1-fold). We generated a mouse model of theTRPV4V620I mutation, and found significant skeletal deformities (e.g., shortening of tibiae and digits, similar to the human disease brachyolmia) and increases in Fst/TRPV4 mRNA levels (2.8-fold). FST was significantly up-regulated in primary chondrocytes transfected with 3 different dysplasia-causing TRPV4 mutations (2- to 2.3-fold), but was not affected by an arthropathy mutation (1.1-fold). Furthermore, FST-loaded microbeads decreased bone ossification in developing chick femora (6%) and tibiae (11%). FST gene and protein levels were also increased 4-fold in human chondrocytes from an individual natively expressing the TRPV4T89I mutation. Taken together, these data strongly support that up-regulation of FST in chondrocytes by skeletal dysplasia-inducing TRPV4 mutations contributes to disease pathogenesis.—Leddy, H. A., McNulty, A. L., Lee, S. H., Rothfusz, N. E., Gloss, B., Kirby, M. L., Hutson, M. R., Cohn, D. H., Guilak, F., Liedtke, W. Follistatin in chondrocytes: the link between TRPV4 channelopathies and skeletal malformations. PMID:24577120

  13. 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

  14. The ciliary Evc/Evc2 complex interacts with Smo and controls Hedgehog pathway activity in chondrocytes by regulating Sufu/Gli3 dissociation and Gli3 trafficking in primary cilia.

    PubMed

    Caparrós-Martín, Jose A; Valencia, María; Reytor, Edel; Pacheco, María; Fernandez, Margarita; Perez-Aytes, Antonio; Gean, Esther; Lapunzina, Pablo; Peters, Heiko; Goodship, Judith A; Ruiz-Perez, Victor L

    2013-01-01

    Hedgehog (Hh) signaling is involved in patterning and morphogenesis of most organs in the developing mammalian embryo. Despite many advances in understanding core components of the pathway, little is known about how the activity of the Hh pathway is adjusted in organ- and tissue-specific developmental processes. Mutations in EVC or EVC2 disrupt Hh signaling in tooth and bone development. Using mouse models, we show here that Evc and Evc2 are mutually required for localizing to primary cilia and also for maintaining their normal protein levels. Consistent with Evc and Evc2 functioning as a complex, the skeletal phenotypes in either single or double homozygous mutant mice are virtually indistinguishable. Smo translocation to the cilium was normal in Evc2-deficient chondrocytes following Hh activation with the Smo-agonist SAG. However, Gli3 recruitment to cilia tips was reduced and Sufu/Gli3 dissociation was impaired. Interestingly, we found Smo to co-precipitate with Evc/Evc2, indicating that in some cells Hh signaling requires direct interaction of Smo with the Evc/Evc2 complex. Expression of a dominantly acting Evc2 mutation previously identified in Weyer's acrodental dysostosis (Evc2Δ43) caused mislocalization of Evc/Evc2Δ43 within the cilium and also reproduced the Gli3-related molecular defects observed in Evc2(-/-) chondrocytes. Moreover, Evc silencing in Sufu(-/-) cells attenuated the output of the Hh pathway, suggesting that Evc/Evc2 also promote Hh signaling in the absence of Sufu. Together our data reveal that the Hh pathway involves Evc/Evc2-dependent modulations that are necessary for normal endochondral bone formation.

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

    PubMed Central

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

    2016-01-01

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

  16. 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

  17. Thyroid-specific gene expression in chondrocytes.

    PubMed

    Endo, Toyoshi; Kobayashi, Tetsuro

    2011-12-16

    Previously, we demonstrated that Runx2 (Cbfa1/AML3), a chondrocyte-specific transcription factor, is expressed in thyroid glands of mice, where it stimulates expression of the thyroglobulin (Tg) gene. Here, we reverse transcribed thyroid transcription factor-1 (TTF-1), Pax-8, Tg, thyroid peroxidase (TPO) and Na(+)/I(-) symporter (NIS) cDNAs from mouse trachea and bronchus RNA samples, but were unable to recover these cDNAs from mouse liver RNA samples. Tg mRNA levels in trachea and bronchus were about 5.1% and 2.1% of those in thyroid glands. ATDC-5 cells, cultured chondrocytes, expressed about 30-fold more Tg mRNA than undifferentiated cells. Gel shift and Tg gene reporter assay revealed that TTF-1 stimulated Tg gene expression in these cells. These results indicate that chondrocytes turn on some aspects of the thyroid gene expression program and that TTF-1 plays important roles in Tg gene expression in chondrocyte. PMID:21945616

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

    PubMed

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

    2016-01-01

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

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

    PubMed Central

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

    2016-01-01

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

  20. Loss of the Mammalian DREAM Complex Deregulates Chondrocyte Proliferation

    PubMed Central

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

    2014-01-01

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

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

    PubMed

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

    2014-06-01

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

  2. Effect of JJYMD-C, a novel synthetic derivative of gallic acid, on proliferation and phenotype maintenance in rabbit articular chondrocytes in vitro

    PubMed Central

    Xu, G.J.; Lu, Z.H.; Lin, X.; Lin, C.W.; Zheng, L.; Zhao, J.M.

    2014-01-01

    Tissue engineering encapsulated cells such as chondrocytes in the carrier matrix have been widely used to repair cartilage defects. However, chondrocyte phenotype is easily lost when chondrocytes are expanded in vitro by a process defined as “dedifferentiation”. To ensure successful therapy, an effective pro-chondrogenic agent is necessary to overcome the obstacle of limited cell numbers in the restoration process, and dedifferentiation is a prerequisite. Gallic acid (GA) has been used in the treatment of arthritis, but its biocompatibility is inferior to that of other compounds. In this study, we modified GA by incorporating sulfamonomethoxine sodium and synthesized a sulfonamido-based gallate, JJYMD-C, and evaluated its effect on chondrocyte metabolism. Our results showed that JJYMD-C could effectively increase the levels of the collagen II, Sox9, and aggrecan genes, promote chondrocyte growth, and enhance secretion and synthesis of cartilage extracellular matrix. On the other hand, expression of the collagen I gene was effectively down-regulated, demonstrating inhibition of chondrocyte dedifferentiation by JJYMD-C. Hypertrophy, as a characteristic of chondrocyte ossification, was undetectable in the JJYMD-C groups. We used JJYMD-C at doses of 0.125, 0.25, and 0.5 µg/mL, and the strongest response was observed with 0.25 µg/mL. This study provides a basis for further studies on a novel agent in the treatment of articular cartilage defects. PMID:25003544

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

    PubMed Central

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

    2015-01-01

    Thyroid hormones (TH) are known to regulate endochondral ossification during skeletal development via acting directly in chondrocytes and osteoblasts. In this study, we focused on TH effects on the secondary ossification center (SOC), since the time of appearance of SOCs in several species coincides with the time when peak levels of TH are attained. Accordingly, μCT evaluation of femurs and tibias at day 21 in TH-deficient and control mice revealed that endochondral ossification of SOCs is severely compromised due to TH deficiency and that TH treatment for 10 days completely rescued this phenotype. Staining of cartilage and bone in the epiphysis revealed that while all of the cartilage is converted into bone in the prepubertal control mice, this conversion failed to occur in the TH-deficient mice. Immunohistochemistry studies revealed that TH treatment of Tshr−/− mice induced expression of Ihh and Osx in Col2 expressing chondrocytes in the SOC at day 7 which subsequently differentiate into Col10/osteocalcin expressing chondro-osteoblasts at day 10. Consistent with these data, treatment of tibia cultures from 3-day old mice with10 ng/ml TH increased expression of Osx, Col10, ALP and osteocalcin in the epiphysis by 6–60 fold. Furthermore, knockdown of the TH-induced increase in Osx expression using lentiviral shRNA significantly blocked TH-induced ALP and osteocalcin expression in chondrocytes. Treatment of chondrogenic cells with an Ihh inhibitor abolished chondro-osteoblast differentiation and SOC formation. Our findings indicate that TH regulates the SOC initiation and progression via differentiating chondrocytes into bone matrix producing osteoblasts by stimulating Ihh and Osx expression in chondrocytes. PMID:24753031

  4. Physiological levels of hydrocortisone maintain an optimal chondrocyte extracellular matrix metabolism

    PubMed Central

    Wang, J; Elewaut, D; Hoffman, I; Veys, E; Verbruggen, G

    2004-01-01

    Objective: To investigate the effects of physiological doses of hydrocortisone on synthesis and turnover of cell associated matrix (CAM) by human chondrocytes obtained from normal articular cartilage. Methods: Human articular cartilage cells were obtained from visually intact cartilage of the femoral condyles of five donors and maintained in culture for one week to reach equilibrium in accumulated CAM compounds. 0, 0.05, 0.20, and 1.0 µg/ml hydrocortisone was added to the nutrient media during the entire culture period. Cells were liberated and levels of CAM aggrecan, type II collagen, and fibronectin, of intracellular IGF-1, IL1α and ß, and of their respective plasma membrane bound receptors IGFR1, IL1RI, and the decoy receptor IL1RII, were assayed by flow cytometry. Results: In comparison with controls, hydrocortisone treated chondrocytes, at all concentrations, expressed significantly higher plasma membrane bound IGFR1. Intracellular IGF-1 levels remained unchanged. Together with these changes, reflecting an increased ability to synthesise extracellular matrix (ECM) macromolecules, hydrocortisone treated cells expressed significantly higher amounts of the plasma membrane bound decoy IL1RII. Concurrently, intracellular IL1α and ß levels and membrane bound IL1RI were down regulated. Levels of CAM aggrecan, type II collagen, and fibronectin were significantly up regulated in the chondrocytes treated with hydrocortisone. Conclusion: 0.05 µg/ml hydrocortisone treated chondrocytes had decreased catabolic signalling pathways and showed an enhanced ability to synthesise ECM macromolecules. Because IL1 activity was decreased and the expression of IL1RII decoy receptor enhanced, more of the ECM macromolecules produced remained accumulated in the CAM of the chondrocytes. The effects were obtained at doses comparable with physiological plasma levels of hydrocortisone in humans. PMID:14672893

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

    PubMed

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

    2015-01-01

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

  6. Smpd3 Expression in both Chondrocytes and Osteoblasts Is Required for Normal Endochondral Bone Development.

    PubMed

    Li, Jingjing; Manickam, Garthiga; Ray, Seemun; Oh, Chun-do; Yasuda, Hideyo; Moffatt, Pierre; Murshed, Monzur

    2016-09-01

    Sphingomyelin phosphodiesterase 3 (SMPD3), a lipid-metabolizing enzyme present in bone and cartilage, has been identified to be a key regulator of skeletal development. A homozygous loss-of-function mutation called fragilitas ossium (fro) in the Smpd3 gene causes poor bone and cartilage mineralization resulting in severe congenital skeletal deformities. Here we show that Smpd3 expression in ATDC5 chondrogenic cells is downregulated by parathyroid hormone-related peptide through transcription factor SOX9. Furthermore, we show that transgenic expression of Smpd3 in the chondrocytes of fro/fro mice corrects the cartilage but not the bone abnormalities. Additionally, we report the generation of Smpd3(flox/flox) mice for the tissue-specific inactivation of Smpd3 using the Cre-loxP system. We found that the skeletal phenotype in Smpd3(flox/flox); Osx-Cre mice, in which the Smpd3 gene is ablated in both late-stage chondrocytes and osteoblasts, closely mimics the skeletal phenotype in fro/fro mice. On the other hand, Smpd3(flox/flox); Col2a1-Cre mice, in which the Smpd3 gene is knocked out in chondrocytes only, recapitulate the fro/fro mouse cartilage phenotype. This work demonstrates that Smpd3 expression in both chondrocytes and osteoblasts is required for normal endochondral bone development. PMID:27325675

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

    PubMed

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

    2016-03-25

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

  8. Chondrocyte spheroids on microfabricated PEG hydrogel surface and their noninvasive functional monitoring

    NASA Astrophysics Data System (ADS)

    Otsuka, Hidenori; Nagamura, Masako; Kaneko, Akie; Kutsuzawa, Koichi; Sakata, Toshiya; Miyahara, Yuji

    2012-12-01

    A two-dimensional microarray of 10 000 (100 × 100) chondrocyte spheroids was constructed with a 100 μm spacing on a micropatterned gold electrode that was coated with poly(ethylene glycol) (PEG) hydrogels. The PEGylated surface as a cytophobic region was regulated by controlling the gel structure through photolithography. In this way, a PEG hydrogel was modulated enough to inhibit outgrowth of chondrocytes from a cell adhering region in the horizontal direction, which is critical for inducing formation of three-dimensional chondrocyte aggregations (spheroids) within 24 h. We further report noninvasive monitoring of the cellular functional change at the cell membrane using a chondrocyte-based field effect transistor. This measurement is based on detection of extracellular potential change induced as a result of the interaction between extracellular matrix protein secreted from spheroid and substrate at the cell membrane. The interface potential change at the cell membrane/gate interface can be monitored during the differentiation of spheroids without any labeling materials. Our measurements of the time evolution of the interface potential provide important information for understanding the uptake kinetics for cellular differentiation.

  9. MicroRNA-411 inhibited matrix metalloproteinase 13 expression in human chondrocytes

    PubMed Central

    Wang, Guodong; Zhang, Yuanmin; Zhao, Xiaowei; Meng, Chunyang; Ma, Longfei; Kong, Ying

    2015-01-01

    Osteoarthritis (OA) is the most common joint degenerative disease affecting the joint structure, leading to loss of joint function and tissue destruction. Recent studies have demonstrated that miRNAs are involved in many pathological conditions, including OA. The study was to investigate the role of miR-411 in the pathogenesis of OA. The expression of miR-411 was downregulated in OA cartilage compared with in normal cartilage. Conversely, the expression of MMP-13 was upregulated in OA cartilage compared with in normal cartilage. IL-1β treatment repressed miR-411 expression in chondrocytes. Moreover, we identified MMP-13 as a direct target gene of miR-411 in chondrocytes and overexpression of miR-411 inhibited the MMP-13 expression. Furthermore, overexpression of miR-411 increased the expression of type II collagen and type IV collagen expression in chondrocytes. MiR-411 is a crucial regulator of MMP-13 in chondrocytes and may response to the development of OA. PMID:26692943

  10. Lysyl oxidase-like-2 (LOXL2) is a major isoform in chondrocytes and is critically required for differentiation.

    PubMed

    Iftikhar, Mussadiq; Hurtado, Paola; Bais, Manish V; Wigner, Nate; Stephens, Danielle N; Gerstenfeld, Louis C; Trackman, Philip C

    2011-01-14

    The lysyl oxidase family is made up of five members: lysyl oxidase (LOX) and lysyl oxidase-like 1-4 (LOXL1-LOXL4). All members share conserved C-terminal catalytic domains that provide for lysyl oxidase or lysyl oxidase-like enzyme activity; and more divergent propeptide regions. LOX family enzyme activities catalyze the final enzymatic conversion required for the formation of normal biosynthetic collagen and elastin cross-links. The importance of lysyl oxidase enzyme activity to normal bone development has long been appreciated, but regulation and roles for specific LOX isoforms in bone formation in vivo is largely unexplored. Fracture healing recapitulates aspects of endochondral bone development. The present study first investigated the expression of all LOX isoforms in fracture healing. A remarkable coincidence of LOXL2 expression with the chondrogenic phase of fracture healing was found, prompting more detailed analyses of LOXL2 expression in normal growth plates, and LOXL2 expression and function in developing ATDC5 chondrogenic cells. Data show that LOXL2 is expressed by pre-hypertrophic and hypertrophic chondrocytes in vivo, and that LOXL2 expression is regulated in vitro as a function of chondrocyte differentiation. Moreover, LOXL2 knockdown studies in vitro show that LOXL2 expression is required for ATDC5 chondrocyte cell line differentiation through regulation of SNAIL and SOX9, important transcription factors that control chondrocyte differentiation. Taken together, data provide evidence that LOXL2, like LOX, is a multifunctional protein. LOXL2 promotes chondrocyte differentiation by mechanisms that are likely to include roles as both a regulator and an effector of chondrocyte differentiation.

  11. Interleukin 1 beta suppresses transforming growth factor-induced inorganic pyrophosphate (PPi) production and expression of the PPi-generating enzyme PC-1 in human chondrocytes.

    PubMed Central

    Lotz, M; Rosen, F; McCabe, G; Quach, J; Blanco, F; Dudler, J; Solan, J; Goding, J; Seegmiller, J E; Terkeltaub, R

    1995-01-01

    Articular cartilage chondrocytes have the unique ability to elaborate large amounts of extracellular pyrophosphate (PPi), and transforming growth factor beta (TGF beta) appears singular among cartilage regulatory factors in stimulating PPi production. TGF beta caused a time and dose-dependent increase in intracellular and extracellular PPi in human articular chondrocyte cultures. TGF beta and interleukin 1 beta (IL-1 beta) antagonistically regulate certain chondrocyte functions. IL-1 beta profoundly inhibited basal and TGF beta-induced PPi elaboration. To address mechanisms involved with the regulation of PPi synthesis by IL-1 beta and TGF beta, we analyzed the activity of the PPi-generating enzyme NTP pyrophosphohydrolase (NTPPPH) and the PPi-hydrolyzing enzyme alkaline phosphatase. Human chondrocyte NTPPPH activity was largely attributable to plasma cell membrane glycoprotein 1, PC-1. Furthermore, TGF beta induced comparable increases in the activity of extracellular PPi, intracellular PPi, and cellular NTPPPH and in the levels of PC-1 protein and mRNA in chondrocytes as well as a decrease in alkaline phosphatase. All of these TGF beta-induced responses were completely blocked by IL-1 beta. Thus, IL-1 beta may be an important regulator of mineralization in chondrocytes by inhibiting TGF beta-induced PPi production and PC-1 expression. Images Fig. 4 Fig. 5 PMID:7479785

  12. Bovine achondrogenesis: evidence for defective chondrocyte differentiation.

    PubMed

    Horton, W A; Jayo, M J; Leipold, H W; Machado, M A; Campbell, D; Ahmed, S

    1987-01-01

    A survey study of growth cartilage abnormalities in bovine bone dysplasias revealed that a disorder in Holstein cattle called bulldog calf closely resembles human achondrogenesis Type II. Substantial amounts of Type I collagen and other non Type II collagens were detected in the bulldog cartilage which was comprised primarily of extensive vascular canals and cells having the characteristics of hypertrophic and degenerative chondrocytes normally found in the growth plate. It is proposed that chondrocytes throughout the bulldog growth cartilage prematurely differentiate into hypertrophic cells that degenerate and predispose the cartilage to vascular invasion and the formation of cartilage canals. The presence of these canals probably accounts for most of the observed collagen abnormalities. PMID:3606909

  13. Changes in the Chondrocyte and Extracellular Matrix Proteome during Post-natal Mouse Cartilage Development*

    PubMed Central

    Wilson, Richard; Norris, Emma L.; Brachvogel, Bent; Angelucci, Constanza; Zivkovic, Snezana; Gordon, Lavinia; Bernardo, Bianca C.; Stermann, Jacek; Sekiguchi, Kiyotoshi; Gorman, Jeffrey J.; Bateman, John F.

    2012-01-01

    cartilage development. Although the multifunctional chaperone BiP was not differentially expressed, enzymes and chaperones required specifically for collagen biosynthesis, such as the prolyl 3-hydroxylase 1, cartilage-associated protein, and peptidyl prolyl cis-trans isomerase B complex, were down-regulated during maturation. Conversely, the lumenal proteins calumenin, reticulocalbin-1, and reticulocalbin-2 were significantly increased, signifying a shift toward calcium binding functions. This first proteomic analysis of cartilage development in vivo reveals the breadth of protein expression changes during chondrocyte maturation and ECM remodeling in the mouse femoral head. PMID:21989018

  14. The ClC-7 Chloride Channel Is Downregulated by Hypoosmotic Stress in Human Chondrocytes.

    PubMed

    Kurita, Takashi; Yamamura, Hisao; Suzuki, Yoshiaki; Giles, Wayne R; Imaizumi, Yuji

    2015-07-01

    Articular chondrocytes in osteoarthritis (OA) patients are exposed to hypoosmotic stress because the osmolality of this synovial fluid is significantly decreased. Hypoosmotic stress can cause an efflux of Cl(-) and an associated decrease of cell volume. We have previously reported that a Cl(-) conductance contributes to the regulation of resting membrane potential and thus can alter intracellular Ca(2+) concentration ([Ca(2+)]i) in human chondrocytes. The molecular identity and pathologic function of these Cl(-) channels, however, remained to be determined. Here, we show that the ClC-7 Cl(-) channel is strongly expressed in a human chondrocyte cell line (OUMS-27) and that it is responsible for Cl(-) currents that are activated by extracellular acidification (pH 5.0). These acid-sensitive currents are inhibited by 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS; IC50 = 13 μM) and are markedly reduced by small-interfering RNA-induced knockdown of ClC-7. DIDS hyperpolarized these chondrocytes, and this was followed by an increase in [Ca(2+)]i. ClC-7 knockdown caused a similar hyperpolarization of the membrane potential. Short-term culture (48 hours) in hypoosmotic medium (270 mOsm) reduced the expression of ClC-7 and decreased the acid-sensitive currents. Interestingly, these hypoosmotic culture conditions, or ClC-7 knockdown, resulted in enhanced cell death. Taken together, our results show that the significant hyperpolarization due to ClC-7 impairment in chondrocytes can significantly increase [Ca(2+)]i and cell death. Thus, downregulation of ClC-7 channels during the hypoosmotic stress that accompanies OA progression is one important concept of the complex etiology of OA. These findings suggest novel targets for therapeutic intervention(s) and drug development for OA.

  15. miR-139 modulates MCPIP1/IL-6 expression and induces apoptosis in human OA chondrocytes

    PubMed Central

    Makki, Mohammad Shahidul; Haqqi, Tariq M

    2015-01-01

    IL-6 is an inflammatory cytokine and its overexpression plays an important role in osteoarthritis (OA) pathogenesis. Expression of IL-6 is regulated post-transcriptionally by MCPIP1. The 3′ untranslated region (UTR) of MCPIP1 mRNA harbors a miR-139 ‘seed sequence', therefore we examined the post-transcriptional regulation of MCPIP1 by miR-139 and its impact on IL-6 expression in OA chondrocytes. Expression of miR-139 was found to be high in the damaged portion of the OA cartilage compared with unaffected cartilage from the same patient and was also induced by IL-1β in OA chondrocytes. Inhibition of miR-139 decreased the expression of IL-6 mRNA by 38% and of secreted IL-6 protein by 40%. However, overexpression of miR-139 increased the expression of IL-6 mRNA by 36% and of secreted IL-6 protein by 56%. These data correlated with altered expression profile of MCPIP1 in transfected chondrocytes. Studies with a luciferase reporter construct confirmed the interactions of miR-139 with the ‘seed sequence' located in the 3′ UTR of MCPIP mRNA. Furthermore, miR-139 overexpression increased the catabolic gene expression but expression of anabolic markers remained unchanged. Overexpression of miR-139 also induced apoptosis in OA chondrocytes. Importantly, we also discovered that IL-6 is a potent inducer of miR-139 expression in OA chondrocytes. These findings indicate that miR-139 functions as a post-transcriptional regulator of MCPIP1 expression and enhances IL-6 expression, which further upregulates miR-139 expression in OA chondrocytes. These results support our hypothesis that miR-139-mediated downregulation of MCPIP1 promotes IL-6 expression in OA. Therefore, targeting miR-139 could be therapeutically beneficial in the management of OA. PMID:26450708

  16. 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.

  17. Clinical Outcome 3 Years After Autologous Chondrocyte Implantation Does Not Correlate With the Expression of a Predefined Gene Marker Set in Chondrocytes Prior to Implantation but Is Associated With Critical Signaling Pathways

    PubMed Central

    Stenberg, Johan; de Windt, Tommy S.; Synnergren, Jane; Hynsjö, Lars; van der Lee, Josefine; Saris, Daniel B.F.; Brittberg, Mats; Peterson, Lars; Lindahl, Anders

    2014-01-01

    Background: There is a need for tools to predict the chondrogenic potency of autologous cells for cartilage repair. Purpose: To evaluate previously proposed chondrogenic biomarkers and to identify new biomarkers in the chondrocyte transcriptome capable of predicting clinical success or failure after autologous chondrocyte implantation. Study Design: Controlled laboratory study and case-control study; Level of evidence, 3. Methods: Five patients with clinical improvement after autologous chondrocyte implantation and 5 patients with graft failures 3 years after implantation were included. Surplus chondrocytes from the transplantation were frozen for each patient. Each chondrocyte sample was subsequently thawed at the same time point and cultured for 1 cell doubling, prior to RNA purification and global microarray analysis. The expression profiles of a set of predefined marker genes (ie, collagen type II α1 [COL2A1], bone morphogenic protein 2 [BMP2], fibroblast growth factor receptor 3 [FGFR3], aggrecan [ACAN], CD44, and activin receptor–like kinase receptor 1 [ACVRL1]) were also evaluated. Results: No significant difference in expression of the predefined marker set was observed between the success and failure groups. Thirty-nine genes were found to be induced, and 38 genes were found to be repressed between the 2 groups prior to autologous chondrocyte implantation, which have implications for cell-regulating pathways (eg, apoptosis, interleukin signaling, and β-catenin regulation). Conclusion: No expressional differences that predict clinical outcome could be found in the present study, which may have implications for quality control assessments of autologous chondrocyte implantation. The subtle difference in gene expression regulation found between the 2 groups may strengthen the basis for further research, aiming at reliable biomarkers and quality control for tissue engineering in cartilage repair. Clinical Relevance: The present study shows the possible

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

    PubMed

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

    2015-10-01

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

  19. 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

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

    SciTech Connect

    Heywood, H.K. Lee, D.A.

    2008-08-22

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

  1. Joint aging and chondrocyte cell death

    PubMed Central

    Grogan, Shawn P; D’Lima, Darryl D

    2010-01-01

    Articular cartilage extracellular matrix and cell function change with age and are considered to be the most important factors in the development and progression of osteoarthritis. The multifaceted nature of joint disease indicates that the contribution of cell death can be an important factor at early and late stages of osteoarthritis. Therefore, the pharmacologic inhibition of cell death is likely to be clinically valuable at any stage of the disease. In this article, we will discuss the close association between diverse changes in cartilage aging, how altered conditions influence chondrocyte death, and the implications of preventing cell loss to retard osteoarthritis progression and preserve tissue homeostasis. PMID:20671988

  2. Chondrocyte behavior on nanostructured micropillar polypropylene and polystyrene surfaces.

    PubMed

    Prittinen, Juha; Jiang, Yu; Ylärinne, Janne H; Pakkanen, Tapani A; Lammi, Mikko J; Qu, Chengjuan

    2014-10-01

    This study was aimed to investigate whether patterned polypropylene (PP) or polystyrene (PS) could enhance the chondrocytes' extracellular matrix (ECM) production and phenotype maintenance. Bovine primary chondrocytes were cultured on smooth PP and PS, as well as on nanostructured micropillar PP (patterned PP) and PS (patterned PS) for 2 weeks. Subsequently, the samples were collected for fluorescein diacetate-based cell viability tests, for immunocytochemical assays of types I and II collagen, actin and vinculin, for scanning electronic microscopic analysis of cell morphology and distribution, and for gene expression assays of Sox9, aggrecan, procollagen α1(II), procollagen α1(X), and procollagen α2(I) using quantitative RT-PCR assays. After two weeks of culture, the bovine primary chondrocytes had attached on both patterned PP and PS, while practically no adhesion was observed on smooth PP. However, the best adhesion of the cells was on smooth PS. The cells, which attached on patterned PP and PS surfaces synthesized types I and II collagen. The chondrocytes' morphology was extended, and an abundant ECM network formed around the attached chondrocytes on both patterned PP and PS. Upon passaging, no significant differences on the chondrocyte-specific gene expression were observed, although the highest expression level of aggrecan was observed on the patterned PS in passage 1 chondrocytes, and the expression level of procollagen α1(II) appeared to decrease in passaged chondrocytes. However, the expressions of procollagen α2(I) were increased in all passaged cell cultures. In conclusion, the bovine primary chondrocytes could be grown on patterned PS and PP surfaces, and they produced extracellular matrix network around the adhered cells. However, neither the patterned PS nor PP could prevent the dedifferentiation of chondrocytes. PMID:25175232

  3. The Effect of Ultrasound Stimulation on the Cytoskeletal Organization of Chondrocytes Seeded In 3D Matrices

    PubMed Central

    Noriega, Sandra; Hasanova, Gulnara; Subramanian, Anuradha

    2013-01-01

    The impact of low intensity diffuse ultrasound (LIDUS) stimulation on the cytoskeletal organization of chondrocytes seeded in 3D scaffolds was evaluated. Chondrocytes seeded on 3D chitosan matrices were exposed to LIDUS at 5.0 MHz (~15kPa, 51-secs, 4-applications/day) in order to study the organization of actin, tubulin and vimentin. The results showed that actin presented a cytosolic punctuated distribution, tubulin presented a quasi parallel organization of microtubules whereas vimentin distribution was unaffected. Chondrocytes seeded on 3D scaffolds responded to US stimulation by the disruption of actin stress fibers and were sensitive to the presence of ROCK inhibitor (Y27632). The gene expression of ROCK-I, a key element in the formation of stress fibers and mDia1, was significantly up-regulated under the application of US. We conclude that the results of both the cytoskeletal analyses and gene expression support the argument that the presence of punctuated actin upon US stimulation was accompanied by the up-regulation of the RhoA/ROCK pathway. PMID:22987069

  4. 5-Aza-2'-deoxycytidine acts as a modulator of chondrocyte hypertrophy and maturation in chick caudal region chondrocytes in culture

    PubMed Central

    2016-01-01

    This study was carried out to explore the effect of DNA hypomethylation on chondrocytes phenotype, in particular the effect on chondrocyte hypertrophy, maturation, and apoptosis. Chondrocytes derived from caudal region of day 17 embryonic chick sterna were pretreated with hypomethylating drug 5-aza-2'-deoxycytidine for 48 hours and then maintained in the normal culture medium for up to 14 days. Histological studies showed distinct morphological changes occurred in the pretreated cultures when compared to the control cultures. The pretreated chondrocytes after 7 days in culture became bigger in size and acquired more flattened fibroblastic phenotype as well as a loss of cartilage specific extracellular matrix. Scanning electron microscopy at day 7 showed chondrocytes to have increased in cell volume and at day 14 in culture the extracellular matrix of the pretreated cultures showed regular fibrillar structure heavily embedded with matrix vesicles, which is the characteristic feature of chondrocyte hypertrophy. Transmission electron microscopic studies indicated the terminal fate of the hypertrophic cells in culture. The pretreated chondrocytes grown for 14 days in culture showed two types of cells: dark cells which had condense chromatin in dark patches and dark cytoplasm. The other light chondrocytes appeared to be heavily loaded with endoplasmic reticulum indicative of very active protein and secretory activity; their cytoplasm had large vacuoles and disintegrating cytoplasm. The biosynthetic profile showed that the pretreated cultures were actively synthesizing and secreting type X collagen and alkaline phosphatase as a major biosynthetic product. PMID:27382512

  5. The transcription factor Sox9 has essential roles in successive steps of the chondrocyte differentiation pathway and is required for expression of Sox5 and Sox6

    PubMed Central

    Akiyama, Haruhiko; Chaboissier, Marie-Christine; Martin, James F.; Schedl, Andreas; de Crombrugghe, Benoit

    2002-01-01

    To examine whether the transcription factor Sox9 has an essential role during the sequential steps of chondrocyte differentiation, we have used the Cre/loxP recombination system to generate mouse embryos in which either Sox9 is missing from undifferentiated mesenchymal cells of limb buds or the Sox9 gene is inactivated after chondrogenic mesenchymal condensations. Inactivation of Sox9 in limb buds before mesenchymal condensations resulted in a complete absence of both cartilage and bone, but markers for the different axes of limb development showed a normal pattern of expression. Apoptotic domains within the developing limbs were expanded, suggesting that Sox9 suppresses apoptosis. Expression of Sox5 and Sox6, two other Sox genes involved in chondrogenesis, was no longer detected. Moreover, expression of Runx2, a transcription factor needed for osteoblast differentiation, was also abolished. Embryos, in which Sox9 was deleted after mesenchymal condensations, exhibited a severe generalized chondrodysplasia, similar to that in Sox5; Sox6 double-null mutant mice. Most cells were arrested as condensed mesenchymal cells and did not undergo overt differentiation into chondrocytes. Furthermore, chondrocyte proliferation was severely inhibited and joint formation was defective. Although Indian hedgehog, Patched1, parathyroid hormone-related peptide (Pthrp), and Pth/Pthrp receptor were expressed, their expression was down-regulated. Our experiments further suggested that Sox9 is also needed to prevent conversion of proliferating chondrocytes into hypertrophic chondrocytes. We conclude that Sox9 is required during sequential steps of the chondrocyte differentiation pathway. PMID:12414734

  6. A novel role for integrin-linked kinase in periodic mechanical stress-mediated ERK1/2 mitogenic signaling in rat chondrocytes.

    PubMed

    Song, Huanghe; Liang, Wenwei; Xu, Shun; Li, Zeng; Chen, Zhefeng; Cui, Weiding; Zhou, Jinchun; Wang, Qing; Liu, Feng; Fan, Weimin

    2016-07-01

    In recent years, a variety of studies have been performed to investigate the cellular responses of periodic mechanical stress on chondrocytes. Integrin β1-mediated ERK1/2 activation was proven to be indispensable in periodic mechanical stress-induced chondrocyte proliferation and matrix synthesis. However, other signal proteins responsible for the mitogenesis of chondrocytes under periodic mechanical stress remain incompletely understood. In the current investigation, we probed the roles of integrin-linked kinase (ILK) signaling in periodic mechanical stress-induced chondrocyte proliferation and matrix synthesis. We found that upon periodic mechanical stress induction, ILK activity increased significantly. Depletion of ILK with targeted shRNA strongly inhibited periodic mechanical stress-induced chondrocyte proliferation and matrix synthesis. In addition, pretreatment with a blocking antibody against integrin β1 resulted in a remarkable decrease in ILK activity in cells exposed to periodic mechanical stress. Furthermore, inhibition of ILK with its target shRNA significantly suppressed ERK1/2 activation in relation to periodic mechanical stress. Based on the above results, we identified ILK as a crucial regulator involved in the integrin β1-ERK1/2 signal cascade responsible for periodic mechanical stress-induced chondrocyte proliferation and matrix synthesis.

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

    SciTech Connect

    Ryu, Jae-Sung; Jung, Yeon-Hwa; Cho, Mi-Young; Yeo, Jee Eun; Choi, Yun-Jin; Kim, Yong Il; Koh, Yong-Gon

    2014-05-16

    Highlights: • Co-culture of hSDMSCs with SNP-stimulated chondrocytes improves anti-inflammation. • Co-culture system produces IGF-1. • Co-culture system suppresses inflammatory genes expression. • Co-culture system improves cell proliferation. • Exogenous IGF-1 inhibits inflammatory activity in SNP-stimulated chondrocytes. - Abstract: Rheumatoid arthritis (RA) and osteoarthritis (OA) are primarily chronic inflammatory diseases. Mesenchymal stem cells (MSCs) have the ability to differentiate into cells of the mesodermal lineage, and to regulate immunomodulatory activity. Specifically, MSCs have been shown to secrete insulin-like growth factor 1 (IGF-1). The purpose of the present study was to examine the inhibitory effects on inflammatory activity from a co-culture of human synovium-derived mesenchymal stem cells (hSDMSCs) and sodium nitroprusside (SNP)-stimulated chondrocytes. First, chondrocytes were treated with SNP to generate an in vitro model of RA or OA. Next, the co-culture of hSDMSCs with SNP-stimulated chondrocytes reduced inflammatory cytokine secretion, inhibited expression of inflammation activity-related genes, generated IGF-1 secretion, and increased the chondrocyte proliferation rate. To evaluate the effect of IGF-1 on inhibition of inflammation, chondrocytes pre-treated with IGF-1 were treated with SNP, and then the production of inflammatory cytokines was analyzed. Treatment with IGF-1 was shown to significantly reduce inflammatory cytokine secretion in SNP-stimulated chondrocytes. Our results suggest that hSDMSCs offer a new strategy to promote cell-based cartilage regeneration in RA or OA.

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

    PubMed

    Liu, Zejun; Cai, Heguo; Zheng, Xinpeng; Zhang, Bing; Xia, Chun

    2015-01-01

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

  9. Inhibitory effects of SRT1720 on the apoptosis of rabbit chondrocytes by activating SIRT1 via p53/bax and NF-κB/PGC-1α pathways.

    PubMed

    Liu, Bi; Lei, Ming; Hu, Tao; Yu, Fei; Xiao, De-Ming; Kang, Hao

    2016-06-01

    SRT1720, a new discovered drug, was reported to activate silent information regulator 1 (SIRT1) and inhibit the chondrocyte apoptosis. However, the underlying mechanism remains elusive. In the present study, the chondrocytes were extracted from the cartilage tissues of New Zealand white rabbits, cultured in the presence of sodium nitroprusside (SNP) (2.5 mmol/L) and divided into five groups: 1, 5, 10, and 20 μmol/L SRT1720 groups and blank control group (0 μmol/L SRT1720). MTT assay was used to detect the chondrocyte viability and proliferation, and DAPI staining and flow cytometry to measure the chondrocyte apoptosis. The expression levels of SIRT1, p53, NF-κB/p65, Bax, and peroxisome proliferator-activated receptor gamma coactivator 1-α (PGC-1α) were detected by Western blotting and the expression levels of SIRT1, type II collagen, and aggrecan mRNA by RT-PCR. The results showed that in the SRT1720-treated groups, the nuclei of chondrocytes were morphologically intact and had uniform chromatin. In the blank control group, nuclear rupture into debris was observed in chondrocytes. With the SRT1720 concentration increasing, the chondrocyte viability increased, the apoptosis rate decreased, the protein expression levels of SIRT1 and PGC-1α and the mRNA expression levels of type II collagen and aggrecan increased ({ptP}<0.05), and the expression levels of p53, NF-κB and bax decreased (P<0.05). It was suggested that SRT1720 inhibits chondrocyte apoptosis by activating the expression of SIRT1 via p53/bax and NF-κB/PGC-1α pathways. PMID:27376802

  10. The Role of PPARγ in Advanced Glycation End Products-Induced Inflammatory Response in Human Chondrocytes

    PubMed Central

    Li, Yu-qing; Chen, Cheng; Cai, Wei; Zeng, Yue-lin

    2015-01-01

    Objective Advances made in the past ten years highlight the notion that peroxisome proliferator-activated receptors gamma (PPARγ) has protective properties in the pathophysiology of osteoarthritis (OA). The aim of this study was to define the roles of PPARγ in AGEs-induced inflammatory response in human chondrocytes. Methods Primary human chondrocytes were stimulated with AGEs in the presence or absence of neutralizing antibody against RAGE (anti-RAGE), MAPK specific inhibitors and PPARγ agonist pioglitazone. The expression of IL-1, MMP-13, TNF-α, PPARγ, nuclear NF-κB p65 and cytosol IκBα was determined by western blotting and real-time PCR. Results AGEs could enhance the expression of IL-1, TNF-α, and MMP-13, but the level of PPARγ was decreased in a time- and dose-dependent manner, which was inhibited by anti-RAGE, SB203580 (P38 MAPK specific inhibitor) and SP600125 (a selective inhibitor of JNK). PPARγ agonist pioglitazone could inhibit the effects of AGEs-induced inflammatory response and PPARγ down-regulation. In human chondrocytes, AGEs could induce cytosol IκBα degradation and increase the level of nuclear NF-κB p65, which was inhibited by PPARγ agonist pioglitazone. Conclusions In primary human chondrocytes, AGEs could down-regulate PPARγ expression and increase the inflammatory mediators, which could be reversed by PPARγ agonist pioglitazone. Activation of RAGE by AGEs triggers a cascade of downstream signaling, including MAPK JNK/ p38, PPARγ and NF-κB. Taken together, PPARγ could be a potential target for pharmacologic intervention in the treatment of OA. PMID:26024533

  11. TIP39/parathyroid hormone type 2 receptor signaling is a potent inhibitor of chondrocyte proliferation and differentiation

    PubMed Central

    Panda, Dibiyendu; Goltzman, David; Jüppner, Harald

    2009-01-01

    Tuberoinfundibular peptide of 39 residues (TIP39) is a member of the parathyroid hormone (PTH) family of peptide hormones that exerts its function by interacting with the PTH type 2 receptor (PTH2R). Presently, no known function has been attributed to this signaling pathway in the developing skeleton. We observed that TIP39 and PTH2R were present in the newborn mouse growth plate, with the receptor localizing in the resting zone whereas ligand expression was restricted exclusively in prehypertrophic and hypertrophic chondrocytes. By 8 wk of life, PTH2R, and to a lesser degree TIP39, immunoreactivity was present in articular chondrocytes. We therefore sought to investigate the role of TIP39/PTH2R signaling in chondrocytes by generating stably transfected CFK2 chondrocytic cells overexpressing PTH2R (CFK2R). TIP39 treatment of CFK2R clones in culture inhibited their proliferation by restricting cells at the G0/G1 phase of the cell cycle, coupled with decreased expression and activity of cyclin-dependent kinases Cdk2 and Cdk4, while p21, an inhibitor of Cdks, was upregulated. In addition, TIP39 treatment decreased expression of differentiation markers in these cells associated with marked alterations in extracellular matrix and metalloproteinase expression. Transcription of Sox9, the master regulator of cartilage differentiation, was reduced in TIP39-treated CFK2R clones. Moreover, Sox9 promoter activity, as measured by luciferase reporter assay, was markedly diminished after TIP39 treatment. In summary, our results show that TIP39/PTH2R signaling inhibits proliferation and alters differentiation of chondrocytes by modulating SOX9 expression, thereby substantiating the functional significance of this signaling pathway in chondrocyte biology. PMID:19706789

  12. TIP39/parathyroid hormone type 2 receptor signaling is a potent inhibitor of chondrocyte proliferation and differentiation.

    PubMed

    Panda, Dibiyendu; Goltzman, David; Jüppner, Harald; Karaplis, Andrew C

    2009-11-01

    Tuberoinfundibular peptide of 39 residues (TIP39) is a member of the parathyroid hormone (PTH) family of peptide hormones that exerts its function by interacting with the PTH type 2 receptor (PTH2R). Presently, no known function has been attributed to this signaling pathway in the developing skeleton. We observed that TIP39 and PTH2R were present in the newborn mouse growth plate, with the receptor localizing in the resting zone whereas ligand expression was restricted exclusively in prehypertrophic and hypertrophic chondrocytes. By 8 wk of life, PTH2R, and to a lesser degree TIP39, immunoreactivity was present in articular chondrocytes. We therefore sought to investigate the role of TIP39/PTH2R signaling in chondrocytes by generating stably transfected CFK2 chondrocytic cells overexpressing PTH2R (CFK2R). TIP39 treatment of CFK2R clones in culture inhibited their proliferation by restricting cells at the G(0)/G(1) phase of the cell cycle, coupled with decreased expression and activity of cyclin-dependent kinases Cdk2 and Cdk4, while p21, an inhibitor of Cdks, was upregulated. In addition, TIP39 treatment decreased expression of differentiation markers in these cells associated with marked alterations in extracellular matrix and metalloproteinase expression. Transcription of Sox9, the master regulator of cartilage differentiation, was reduced in TIP39-treated CFK2R clones. Moreover, Sox9 promoter activity, as measured by luciferase reporter assay, was markedly diminished after TIP39 treatment. In summary, our results show that TIP39/PTH2R signaling inhibits proliferation and alters differentiation of chondrocytes by modulating SOX9 expression, thereby substantiating the functional significance of this signaling pathway in chondrocyte biology.

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

    PubMed Central

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

    2009-01-01

    Summary Postnatal cartilage development and growth are regulated by key growth factors and signaling molecules. To fully understand the function of these regulators, an inducible and chondrocyte-specific gene deletion system needs to be established to circumvent the perinatal lethality. In this report, we have generated a transgenic mouse model (Col2a1-CreERT2) in which expression of the Cre recombinase is driven by the chondrocyte-specific col2a1 promoter in a tamoxifen-inducible manner. To determine the specificity and efficiency of the Cre recombination, we have bred Col2a1-CreERT2 mice with Rosa26R reporter mice. The X-Gal staining showed that the Cre recombination is specifically achieved in cartilage tissues with tamoxifen-induction. In vitro experiments of chondrocyte cell culture also demonstrate the 4-hydroxy tamoxifen-induced Cre recombination. These results demonstrate that Col2a1-CreERT2 transgenic mice can be used as a valuable tool for an inducible and chondrocyte-specific gene deletion approach. PMID:17211877

  14. 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.

  15. Fibroblast growth factor is an inhibitor of chondrocyte terminal differentiation

    SciTech Connect

    Kato, Y.; Iwamoto, M. )

    1990-04-05

    The effects of basic fibroblast growth factor (bFGF) on terminal differentiation of chondrocytes and cartilage-matrix calcification were investigated. Rabbit growth-plate chondrocytes maintained as a pelleted mass in a centrifuge tube produced an abundant proteoglycan matrix during the matrix-maturation stage, yielding a cartilage-like tissue. Thereafter, they terminally differentiated to hypertrophic chondrocytes which produced high levels of alkaline phosphatase. These cells induced extensive calcification of the matrix in the absence of additional phosphate. Addition of bFGF to the chondrocyte cultures abolished the increases in alkaline phosphatase activity, {sup 45}Ca deposition, and the calcium content. These effects were dose-dependent, reversible, and observed in the presence of cytosine arabinoside, an inhibitor of DNA synthesis. The inhibitory effects could be observed only when chondrocytes were exposed to bFGF in a transition period between the matrix-maturation and hypertrophic stages. As chondrocytes differentiated to hypertrophic cells, bFGF became less effective in inhibiting the expression of the mineralization-related phenotypes. The present study also shows that although the rate of ({sup 35}S)sulfate incorporation into large, chondroitin sulfate proteoglycan in the cell-matrix fraction is very high during the matrix-maturation stage, it abruptly decreases by 90% after terminal differentiation. Furthermore, the terminal differentiation-associated decrease in proteoglycan synthesis was delayed by bFGF. These results provide evidence that bFGF inhibits terminal differentiation of chondrocytes and calcification.

  16. In vivo cartilage formation from growth factor modulated articular chondrocytes.

    PubMed

    Bradham, D M; Horton, W E

    1998-07-01

    Recent procedures for autologous repair of cartilage defects may be difficult in elderly patients because of the loss of stem cells and chondrocytes that occurs with age and the slow in vitro proliferation of chondrocytes from aged cartilage. In this study secondary chondroprogenitor cells were obtained by modulating the phenotype of articular chondrocytes with growth factors and stimulating the proliferation of these cells in culture. Chondrocytes isolated from the articular cartilage of mature New Zealand White rabbits were exposed to a combination of transforming growth factor beta and basic fibroblast growth factor treatment. These cells ceased the production of Collagen II (a marker for the chondrocyte phenotype) and underwent a 136-fold increase in cell number. Next, the cells were placed in high density culture and reexpressed the chondrocyte phenotype in vitro and formed hyaline cartilage in an in vivo assay. Primary chondrocytes obtained from articular cartilage of elderly humans could be manipulated in a similar fashion in vitro. These human secondary chondroprogenitor cells formed only cartilage tissue when assayed in vivo and in tissue bioreactors. This approach may be essential for autologous repair of degenerated articular cartilage in elderly patients with osteoarthritis.

  17. Expression of Angiotensin II Receptor-1 in Human Articular Chondrocytes

    PubMed Central

    Kawakami, Yuki; Matsuo, Kosuke; Murata, Minako; Yudoh, Kazuo; Nakamura, Hiroshi; Shimizu, Hiroyuki; Beppu, Moroe; Inaba, Yutaka; Saito, Tomoyuki; Kato, Tomohiro; Masuko, Kayo

    2012-01-01

    Background. Besides its involvement in the cardiovascular system, the renin-angiotensin-aldosterone (RAS) system has also been suggested to play an important role in inflammation. To explore the role of this system in cartilage damage in arthritis, we investigated the expression of angiotensin II receptors in chondrocytes. Methods. Articular cartilage was obtained from patients with osteoarthritis, rheumatoid arthritis, and traumatic fractures who were undergoing arthroplasty. Chondrocytes were isolated and cultured in vitro with or without interleukin (IL-1). The expression of angiotensin II receptor types 1 (AT1R) and 2 (AT2R) mRNA by the chondrocytes was analyzed using reverse transcription-polymerase chain reaction (RT-PCR). AT1R expression in cartilage tissue was analyzed using immunohistochemistry. The effect of IL-1 on AT1R/AT2R expression in the chondrocytes was analyzed by quantitative PCR and flow cytometry. Results. Chondrocytes from all patient types expressed AT1R/AT2R mRNA, though considerable variation was found between samples. Immunohistochemical analysis confirmed AT1R expression at the protein level. Stimulation with IL-1 enhanced the expression of AT1R/AT2R mRNA in OA and RA chondrocytes. Conclusions. Human articular chondrocytes, at least partially, express angiotensin II receptors, and IL-1 stimulation induced AT1R/AT2R mRNA expression significantly. PMID:23346400

  18. 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

  19. 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

  20. A novel synthesized sulfonamido-based gallic acid--LDQN-C: effects on chondrocytes growth and phenotype maintenance.

    PubMed

    Lu, Zhenhui; Wei, Shixiu; Wu, Huayu; Lin, Xiao; Lin, Cuiwu; Liu, Buming; Zheng, Li; Zhao, Jinmin

    2014-12-01

    Chondrocyte based therapy is promising to treat symptomatic chondral and osteochondral lesions. Growth factors to accelerate the proliferation and retain the phenotype of chondrocytes in vitro are imperative. However, the high cost and rapid degradation of growth factors limited their further application. Therefore, it is significant to find substitutes that can preserve chondrocytes phenotype and ensure sufficient cells for cytotherapy. Antioxidant and anti-inflammatory agents or their derivatives that have effect on arthritis may be an alternative. In this study, we synthesized sulfonamido-based gallate - LDQN-C and investigated its effect on rat articular chondrocytes through examination of the cell proliferation, morphology, viability, glycosaminoglycans (GAGs) synthesis and cartilage specific gene expression. Results showed that LDQN-C could enhance secretion and synthesis of cartilage extracellular matrix (ECM) by up-regulating expression levels of aggrecan, collagen II and Sox9 genes compared to the GA treated group and control group. Expression of collagen type II was effectively up-regulated while collagen I was down-regulated, which demonstrated that the inhibition of chondrocytes dedifferentiation by LDQN-C. Range of 1.36×10(-9)M to 1.36×10(-7)M is recommended dose of LDQN-C, among which the most profound response was observed with 1.36×10(-8)M. GA at concentration of 0.125μg/mL was compared. This study might provide a basis for the development of a novel agent for the treatment of articular cartilage defect.

  1. Effect of Fiber Diameter on the Spreading, Proliferation and Differentiation of Chondrocytes on Electrospun Chitosan Matrices

    PubMed Central

    Noriega, Sandra E.; Hasanova, Gulnara I.; Schneider, Min Jeong; Larsen, Gustavo F.; Subramanian, Anuradha

    2012-01-01

    Tissue-engineered neocartilage with appropriate biomechanical properties holds promise not only for graft applications but also as a model system for controlled studies of chondrogenesis. Our objective in the present research study is to better understand the impact of fiber diameter on the cellular activity of chondrocytes cultured on nanofibrous matrices. By using the electrospinning process, fibrous scaffolds with fiber diameters ranging from 300 nm to 1 μm were prepared and the physicomechanical properties of the scaffolds were characterized. Bovine articular chondrocytes were then seeded and maintained on the scaffolds for 7 and 14 days in culture. An upregulation in the gene expression of collagen II was noted with decreasing fiber diameters. For cells that were cultured on scaffolds with a mean fiber diameter of 300 nm, a 2-fold higher ratio of collagen II/collagen I was noted when compared to cells cultured on sponge-like scaffolds prepared by freeze drying and lyophilization. Integrin (α5, αv, β1) gene expression was also observed to be influenced by matrix morphology. Our combined results suggest that matrix geometry can regulate and promote the retention of the chondrocyte genotype. PMID:21540560

  2. Expression of voltage dependent potassium currents in freshly dissociated rat articular chondrocytes.

    PubMed

    Ponce, Arturo

    2006-01-01

    The electrophysiological properties of voltage dependent potassium channels from freshly dissociated rat articular chondrocytes were studied. The resting membrane potential (-42.7+/-2.0 mV) was significantly depolarized by increasing concentrations of external potassium. No change was observed when external chloride concentration was varied. Addition of TEA, 4AP, alpha-Dendrotoxin and charybdotoxin depolarized resting membrane potential. Whole cell patch clamp studies revealed the presence of outwardly rectifying currents whose kinetic and pharmacological properties suggest the expression of voltage dependent potassium channels. Two kinds of currents were observed under the same experimental conditions. The first one, most frequently observed (80%), starts activating near -50 mV, with V(1/2)=-18 mV, G(max)=0.30 pS/pF. The second kind was observed in only 10% of cases; It activates near -40 mV, with(1/2)=+28.35 mV, G(max)=0.28 pS/pF pA/pF and does not inactivates. Inactivating currents were significantly inhibited by TEA (IC(50)=1.45 mM), 4AP (IC(50)=0.64 mM), CTX (IC(50) = 10 nM), alpha-Dendrotoxin (IC(50) < 100 nM) and Margatoxin (IC(50)=28.5 nM). These results show that rat chondrocytes express voltage dependent potassium currents and suggest a role of voltage-dependent potassium channels in regulating membrane potential of rat chondrocytes.

  3. Runx1 Activities in Superficial Zone Chondrocytes, Osteoarthritic Chondrocyte Clones and Response to Mechanical Loading

    PubMed Central

    LeBlanc, Kimberly T.; Walcott, Marie E.; Gaur, Tripti; O’Connell, Shannon L.; Basil, Kirti; Tadiri, Christina P.; Mason-Savas, April; Silva, Jason A.; van Wijnen, Andre J.; Stein, Janet L.; Stein, Gary S; Ayers, David C.; Lian, Jane B.; Fanning, Paul J.

    2015-01-01

    Objective Runx1, the hematopoietic lineage determining transcription factor, is present in perichondrium and chondrocytes. Here we addressed Runx1 functions, by examining expression in cartilage during mouse and human osteoarthritis (OA) progression and in response to mechanical loading. Methods Spared and diseased compartments in knees of OA patients and in mice with surgical destabilization of the medial meniscus were examined for changes in expression of Runx1 mRNA (Q-PCR) and protein (immunoblot, immunohistochemistry). Runx1 levels were quantified in response to static mechanical compression of bovine articular cartilage. Runx1 function was assessed by cell proliferation (Ki67, PCNA) and cell type phenotypic markers. Results Runx1 is enriched in superficial zone (SZ) chondrocytes of normal bovine, mouse, and human tissues. Increasing loading conditions in bovine cartilage revealed a positive correlation with a significant elevation of Runx1. Runx1 becomes highly expressed at the periphery of mouse OA lesions and in human OA chondrocyte ‘clones’ where Runx1 co-localizes with Vcam1, the mesenchymal stem cell (MSC) marker and lubricin (Prg4), a cartilage chondroprotective protein. These OA induced cells represent a proliferative cell population, Runx1 depletion in MPCs decreases cell growth, supporting Runx1 contribution to cell expansion. Conclusion The highest Runx1 levels in SZC of normal cartilage suggest a function that supports the unique phenotype of articular chondrocytes, reflected by upregulation under conditions of compression. We propose Runx1 co-expression with Vcam1 and lubricin in murine cell clusters and human ‘clones’ of OA cartilage, participate in a cooperative mechanism for a compensatory anabolic function. PMID:25078095

  4. The life cycle of chondrocytes in the developing skeleton

    PubMed Central

    Shum, Lillian; Nuckolls, Glen

    2002-01-01

    Cartilage serves multiple functions in the developing embryo and in postnatal life. Genetic mutations affecting cartilage development are relatively common and lead to skeletal malformations, dysfunction or increased susceptibility to disease or injury. Characterization of these mutations and investigation of the molecular pathways in which these genes function have contributed to an understanding of the mechanisms regulating skeletal patterning, chondrogenesis, endochondral ossification and joint formation. Extracellular growth and differentiation factors including bone morphogenetic proteins, fibroblast growth factors, parathyroid hormone-related peptide, extracellular matrix components, and members of the hedgehog and Wnt families provide important signals for the regulation of cell proliferation, differentiation and apoptosis. Transduction of these signals within the developing mesenchymal cells and chondrocytes results in changes in gene expression mediated by transcription factors including Smads, Msx2, Sox9, signal transducer and activator of transcription (STAT), and core-binding factor alpha 1. Further investigation of the interactions of these signaling pathways will contribute to an understanding of cartilage growth and development, and will allow for the development of strategies for the early detection, prevention and treatment of diseases and disorders affecting the skeleton. PMID:11879545

  5. Chondrogenesis, chondrocyte differentiation, and articular cartilage metabolism in health and osteoarthritis.

    PubMed

    Goldring, Mary B

    2012-08-01

    Chondrogenesis occurs as a result of mesenchymal cell condensation and chondroprogenitor cell differentiation. Following chondrogenesis, the chondrocytes remain as resting cells to form the articular cartilage or undergo proliferation, terminal differentiation to chondrocyte hypertrophy, and apoptosis in a process termed endochondral ossification, whereby the hypertrophic cartilage is replaced by bone. Human adult articular cartilage is a complex tissue of matrix proteins that varies from superficial to deep layers and from loaded to unloaded zones. A major challenge to efforts to repair cartilage by stem cell-based and other tissue-engineering strategies is the inability of the resident chondrocytes to lay down a new matrix with the same properties as it had when it was formed during development. Thus, understanding and comparing the mechanisms of cartilage remodeling during development, osteoarthritis (OA), and aging may lead to more effective strategies for preventing cartilage damage and promoting repair. The pivotal proteinase that marks OA progression is matrix metalloproteinase 13 (MMP-13), the major type II collagen-degrading collagenase, which is regulated by both stress and inflammatory signals. We and other investigators have found that there are common mediators of these processes in human OA cartilage. We also observe temporal and spatial expression of these mediators in early through late stages of OA in mouse models and are analyzing the consequences of knockout or transgenic overexpression of critical genes. Since the chondrocytes in adult human cartilage are normally quiescent and maintain the matrix in a low turnover state, understanding how they undergo phenotypic modulation and promote matrix destruction and abnormal repair in OA may to lead to identification of critical targets for therapy to block cartilage damage and promote effective cartilage repair. PMID:22859926

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

    PubMed

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

    2015-10-01

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

  7. Deletion of IFT80 Impairs Epiphyseal and Articular Cartilage Formation Due to Disruption of Chondrocyte Differentiation

    PubMed Central

    Yuan, Xue; Yang, Shuying

    2015-01-01

    Intraflagellar transport proteins (IFT) play important roles in cilia formation and organ development. Partial loss of IFT80 function leads Jeune asphyxiating thoracic dystrophy (JATD) or short-rib polydactyly (SRP) syndrome type III, displaying narrow thoracic cavity and multiple cartilage anomalies. However, it is unknown how IFT80 regulates cartilage formation. To define the role and mechanism of IFT80 in chondrocyte function and cartilage formation, we generated a Col2α1; IFT80f/f mouse model by crossing IFT80f/f mice with inducible Col2α1-CreER mice, and deleted IFT80 in chondrocyte lineage by injection of tamoxifen into the mice in embryonic or postnatal stage. Loss of IFT80 in the embryonic stage resulted in short limbs at birth. Histological studies showed that IFT80-deficient mice have shortened cartilage with marked changes in cellular morphology and organization in the resting, proliferative, pre-hypertrophic, and hypertrophic zones. Moreover, deletion of IFT80 in the postnatal stage led to mouse stunted growth with shortened growth plate but thickened articular cartilage. Defects of ciliogenesis were found in the cartilage of IFT80-deficient mice and primary IFT80-deficient chondrocytes. Further study showed that chondrogenic differentiation was significantly inhibited in IFT80-deficient mice due to reduced hedgehog (Hh) signaling and increased Wnt signaling activities. These findings demonstrate that loss of IFT80 blocks chondrocyte differentiation by disruption of ciliogenesis and alteration of Hh and Wnt signaling transduction, which in turn alters epiphyseal and articular cartilage formation. PMID:26098911

  8. Deletion of IFT80 Impairs Epiphyseal and Articular Cartilage Formation Due to Disruption of Chondrocyte Differentiation.

    PubMed

    Yuan, Xue; Yang, Shuying

    2015-01-01

    Intraflagellar transport proteins (IFT) play important roles in cilia formation and organ development. Partial loss of IFT80 function leads Jeune asphyxiating thoracic dystrophy (JATD) or short-rib polydactyly (SRP) syndrome type III, displaying narrow thoracic cavity and multiple cartilage anomalies. However, it is unknown how IFT80 regulates cartilage formation. To define the role and mechanism of IFT80 in chondrocyte function and cartilage formation, we generated a Col2α1; IFT80f/f mouse model by crossing IFT80f/f mice with inducible Col2α1-CreER mice, and deleted IFT80 in chondrocyte lineage by injection of tamoxifen into the mice in embryonic or postnatal stage. Loss of IFT80 in the embryonic stage resulted in short limbs at birth. Histological studies showed that IFT80-deficient mice have shortened cartilage with marked changes in cellular morphology and organization in the resting, proliferative, pre-hypertrophic, and hypertrophic zones. Moreover, deletion of IFT80 in the postnatal stage led to mouse stunted growth with shortened growth plate but thickened articular cartilage. Defects of ciliogenesis were found in the cartilage of IFT80-deficient mice and primary IFT80-deficient chondrocytes. Further study showed that chondrogenic differentiation was significantly inhibited in IFT80-deficient mice due to reduced hedgehog (Hh) signaling and increased Wnt signaling activities. These findings demonstrate that loss of IFT80 blocks chondrocyte differentiation by disruption of ciliogenesis and alteration of Hh and Wnt signaling transduction, which in turn alters epiphyseal and articular cartilage formation. PMID:26098911

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

    PubMed

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

    2013-10-01

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

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

    PubMed

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

    2014-09-25

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

  11. A Qualitative Model of the Differentiation Network in Chondrocyte Maturation: A Holistic View of Chondrocyte Hypertrophy.

    PubMed

    Kerkhofs, Johan; Leijten, Jeroen; Bolander, Johanna; Luyten, Frank P; Post, Janine N; Geris, Liesbet

    2016-01-01

    Differentiation of chondrocytes towards hypertrophy is a natural process whose control is essential in endochondral bone formation. It is additionally thought to play a role in several pathophysiological processes, with osteoarthritis being a prominent example. We perform a dynamic analysis of a qualitative mathematical model of the regulatory network that directs this phenotypic switch to investigate the influence of the individual factors holistically. To estimate the stability of a SOX9 positive state (associated with resting/proliferation chondrocytes) versus a RUNX2 positive one (associated with hypertrophy) we employ two measures. The robustness of the state in canalisation (size of the attractor basin) is assessed by a Monte Carlo analysis and the sensitivity to perturbations is assessed by a perturbational analysis of the attractor. Through qualitative predictions, these measures allow for an in silico screening of the effect of the modelled factors on chondrocyte maintenance and hypertrophy. We show how discrepancies between experimental data and the model's results can be resolved by evaluating the dynamic plausibility of alternative network topologies. The findings are further supported by a literature study of proposed therapeutic targets in the case of osteoarthritis. PMID:27579819

  12. A Qualitative Model of the Differentiation Network in Chondrocyte Maturation: A Holistic View of Chondrocyte Hypertrophy

    PubMed Central

    Kerkhofs, Johan; Leijten, Jeroen; Bolander, Johanna; Luyten, Frank P.; Post, Janine N.; Geris, Liesbet

    2016-01-01

    Differentiation of chondrocytes towards hypertrophy is a natural process whose control is essential in endochondral bone formation. It is additionally thought to play a role in several pathophysiological processes, with osteoarthritis being a prominent example. We perform a dynamic analysis of a qualitative mathematical model of the regulatory network that directs this phenotypic switch to investigate the influence of the individual factors holistically. To estimate the stability of a SOX9 positive state (associated with resting/proliferation chondrocytes) versus a RUNX2 positive one (associated with hypertrophy) we employ two measures. The robustness of the state in canalisation (size of the attractor basin) is assessed by a Monte Carlo analysis and the sensitivity to perturbations is assessed by a perturbational analysis of the attractor. Through qualitative predictions, these measures allow for an in silico screening of the effect of the modelled factors on chondrocyte maintenance and hypertrophy. We show how discrepancies between experimental data and the model’s results can be resolved by evaluating the dynamic plausibility of alternative network topologies. The findings are further supported by a literature study of proposed therapeutic targets in the case of osteoarthritis. PMID:27579819

  13. 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.

  14. Effects of weak, low-frequency pulsed electromagnetic fields (BEMER type) on gene expression of human mesenchymal stem cells and chondrocytes: an in vitro study.

    PubMed

    Walther, Markus; Mayer, Florian; Kafka, Wolf; Schütze, Norbert

    2007-01-01

    In vitro effects of electromagnetic fields appear to be related to the type of electromagnetic field applied. Previously, we showed that human osteoblasts display effects of BEMER type electromagnetic field (BTEMF) on gene regulation. Here, we analyze effects of BTEMF on gene expression in human mesenchymal stem cells and chondrocytes. Primary mesenchymal stem cells from bone marrow and the chondrocyte cell line C28I2 were stimulated 5 times at 12-h intervals for 8 min each with BTEMF. RNA from treated and control cells was analyzed for gene expression using the affymetrix chip HG-U133A. A limited number of regulated gene products from both cell types mainly affect cell metabolism and cell matrix structure. There was no increased expression of cancer-related genes. RT-PCR analysis of selected transcripts partly confirmed array data. Results indicate that BTEMF in human mesenchymal stem cells and chondrocytes provide the first indications to understanding therapeutic effects achieved with BTEMF stimulation.

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

    PubMed

    Han, Yohan; Kim, Song Ja

    2016-08-15

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

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

    PubMed

    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

  17. 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

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

    PubMed

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

    1996-06-01

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

  19. Telomerase Activity in Articular Chondrocytes Is Lost after Puberty

    PubMed Central

    Wilson, Brooke; Novakofski, Kira D.; Donocoff, Rachel Sacher; Liang, Yan-Xiang Amber

    2014-01-01

    Objective: Telomere length and telomerase activity are important indicators of cellular senescence and replicative ability. Loss of telomerase is associated with ageing and the development of osteoarthritis. Implantation of telomerase-positive cells, chondrocytes, or stem cells expressing a normal chondrocyte phenotype is desired for cartilage repair procedures. The objective of this study was to identify at what age chondrocytes and at what passage bone marrow–derived mesenchymal stem cells (MSCs) become senescent based on telomerase activity. The effect of osteogenic protein–1 (OP-1) or interleukin-1α (IL-1α) treatment on telomerase activity in chondrocytes was also measured to determine the response to anabolic or catabolic stimuli. Methods: Articular cartilage was collected from horses (n = 12) aged 1 month to 18 years. Chondrocytes from prepubescent horses (<15 months) were treated with OP-1 or IL-1α. Bone marrow aspirate from adult horses was collected and cultured for up to 10 days to isolate MSCs. Telomerase activity was measured using the TeloTAGGG Telomerase PCR ELISA kit. Results: Chondrocytes from prepubescent horses were positive for telomerase activity. Treatment with IL-1α resulted in a decrease in chondrocyte telomerase activity; however, treatment with OP-1 did not change telomerase activity. One MSC culture sample was positive for telomerase activity on day 2; all samples were negative for telomerase activity on day 10. Conclusions: These results suggest that chondrocytes from prepubescent donors are potentially more suitable for cartilage repair procedures and that telomerase activity is diminished by anabolic and catabolic cytokine stimulation. If MSCs are utilized in cartilage repair, minimal passaging should be performed prior to implantation. PMID:26069700

  20. Membrane channel gene expression in human costal and articular chondrocytes.

    PubMed

    Asmar, A; Barrett-Jolley, R; Werner, A; Kelly, R; Stacey, M

    2016-04-01

    Chondrocytes are the uniquely resident cells found in all types of cartilage and key to their function is the ability to respond to mechanical loads with changes of metabolic activity. This mechanotransduction property is, in part, mediated through the activity of a range of expressed transmembrane channels; ion channels, gap junction proteins, and porins. Appropriate expression of ion channels has been shown essential for production of extracellular matrix and differential expression of transmembrane channels is correlated to musculoskeletal diseases such as osteoarthritis and Albers-Schönberg. In this study we analyzed the consistency of gene expression between channelomes of chondrocytes from human articular and costal (teenage and fetal origin) cartilages. Notably, we found 14 ion channel genes commonly expressed between articular and both types of costal cartilage chondrocytes. There were several other ion channel genes expressed only in articular (6 genes) or costal chondrocytes (5 genes). Significant differences in expression of BEST1 and KCNJ2 (Kir2.1) were observed between fetal and teenage costal cartilage. Interestingly, the large Ca(2+) activated potassium channel (BKα, or KCNMA1) was very highly expressed in all chondrocytes examined. Expression of the gap junction genes for Panx1, GJA1 (Cx43) and GJC1 (Cx45) was also observed in chondrocytes from all cartilage samples. Together, this data highlights similarities between chondrocyte membrane channel gene expressions in cells derived from different anatomical sites, and may imply that common electrophysiological signaling pathways underlie cellular control. The high expression of a range of mechanically and metabolically sensitive membrane channels suggest that chondrocyte mechanotransduction may be more complex than previously thought. PMID:27116676

  1. Membrane channel gene expression in human costal and articular chondrocytes

    PubMed Central

    Asmar, A.; Barrett-Jolley, R.; Werner, A.; Kelly, R.; Stacey, M.

    2016-01-01

    ABSTRACT Chondrocytes are the uniquely resident cells found in all types of cartilage and key to their function is the ability to respond to mechanical loads with changes of metabolic activity. This mechanotransduction property is, in part, mediated through the activity of a range of expressed transmembrane channels; ion channels, gap junction proteins, and porins. Appropriate expression of ion channels has been shown essential for production of extracellular matrix and differential expression of transmembrane channels is correlated to musculoskeletal diseases such as osteoarthritis and Albers-Schönberg. In this study we analyzed the consistency of gene expression between channelomes of chondrocytes from human articular and costal (teenage and fetal origin) cartilages. Notably, we found 14 ion channel genes commonly expressed between articular and both types of costal cartilage chondrocytes. There were several other ion channel genes expressed only in articular (6 genes) or costal chondrocytes (5 genes). Significant differences in expression of BEST1 and KCNJ2 (Kir2.1) were observed between fetal and teenage costal cartilage. Interestingly, the large Ca2+ activated potassium channel (BKα, or KCNMA1) was very highly expressed in all chondrocytes examined. Expression of the gap junction genes for Panx1, GJA1 (Cx43) and GJC1 (Cx45) was also observed in chondrocytes from all cartilage samples. Together, this data highlights similarities between chondrocyte membrane channel gene expressions in cells derived from different anatomical sites, and may imply that common electrophysiological signaling pathways underlie cellular control. The high expression of a range of mechanically and metabolically sensitive membrane channels suggest that chondrocyte mechanotransduction may be more complex than previously thought. PMID:27116676

  2. Cytokine networking of chondrocyte dedifferentiation in vitro and its implications for cell-based cartilage therapy

    PubMed Central

    Duan, Li; Ma, Bin; Liang, Yujie; Chen, Jielin; Zhu, Weimin; Li, Mingtao; Wang, Daping

    2015-01-01

    Autologous chondrocyte implantation (ACI) is a golden treatment for large defects of the knee joint without osteoarthritis or other complications. Despite notable progresses, generation of a stable chondrocyte phenotype using progenitor cells remains a main obstacle for chondrocyte-based cartilage treatment. Monolayer chondrocyte expansion in vitro is accompanied by chondrocyte dedifferentiation, which produces a non-specific mechanically inferior extracellular matrix (ECM) unsuitable for ACI. In-depth understanding of the molecular events during chondrocyte dedifferentiation is required to maintain the capacity of in vitro expanded chondrocytes to produce hyaline cartilage-specific ECM. This review discusses key cytokines and signaling pathways involved in chondrocyte dedifferentiation from the standpoint of catabolism and anabolism. Some potential therapeutic strategies are also presented to counteract chondrocyte dedifferentiation for cell-based cartilage therapy. PMID:25901191

  3. The properties of bioengineered chondrocyte sheets for cartilage regeneration

    PubMed Central

    Mitani, Genya; Sato, Masato; Lee, Jeong IK; Kaneshiro, Nagatoshi; Ishihara, Miya; Ota, Naoshi; Kokubo, Mami; Sakai, Hideaki; Kikuchi, Tetsutaro; Mochida, Joji

    2009-01-01

    Background Although the clinical results of autologous chondrocyte implantation for articular cartilage defects have recently improved as a result of advanced techniques based on tissue engineering procedures, problems with cell handling and scaffold imperfections remain to be solved. A new cell-sheet technique has been developed, and is potentially able to overcome these obstacles. Chondrocyte sheets applicable to cartilage regeneration can be prepared with this cell-sheet technique using temperature-responsive culture dishes. However, for clinical application, it is necessary to evaluate the characteristics of the cells in these sheets and to identify their similarities to naive cartilage. Results The expression of SOX 9, collagen type 2, 27, integrin α10, and fibronectin genes in triple-layered chondrocyte sheets was significantly increased in comparison to those in conventional monolayer culture and in a single chondrocyte sheet, implying a nature similar to ordinary cartilage. In addition, immunohistochemistry demonstrated that collagen type II, fibronectin, and integrin α10 were present in the triple-layered chondrocyte sheets. Conclusion The results of this study indicate that these chondrocyte sheets with a consistent cartilaginous phenotype and adhesive properties may lead to a new strategy for cartilage regeneration. PMID:19267909

  4. 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

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

    SciTech Connect

    Dombrecht, E.J.; De Tollenaere, C.B.; Aerts, K.; Cos, P.; Schuerwegh, A.J.; Bridts, C.H.; Van Offel, J.F.; Ebo, D.G.; Stevens, W.J. . E-mail: immuno@ua.ac.be; De Clerck, L.S.

    2006-09-22

    The objective of this study was to evaluate the effect of bisphosphonates (BPs) and simvastatin on chondrocyte lipid peroxidation. For this purpose, a flow cytometrical method using C11-BODIPY{sup 581/591} was developed to detect hydroperoxide-induced lipid peroxidation in chondrocytes. Tertiary butylhydroperoxide (t-BHP) induced a time and concentration dependent increase in chondrocyte lipid peroxidation. Addition of a Fe{sup 2+}/EDTA complex to t-BHP or hydrogen peroxide (H{sub 2}O{sub 2}) clearly enhanced lipid peroxidation. The lipophilic simvastatin demonstrated a small inhibition in the chondrocyte lipid peroxidation. None of three tested BPs (clodronate, pamidronate, and risedronate) had an effect on chondrocyte lipid peroxidation induced by t-BHP. However, when Fe{sup 2+}/EDTA complex was added to t-BHP or H{sub 2}O{sub 2}, BPs inhibited the lipid peroxidation process varying from 25% to 58%. This study demonstrates that BPs have antioxidant properties as iron chelators, thereby inhibiting the chondrocyte lipid peroxidation. These findings add evidence to the therapeutic potential of bisphosphonates and statins in rheumatoid arthritis.

  6. Mustn1 is expressed during chondrogenesis and is necessary for chondrocyte proliferation and differentiation in vitro

    PubMed Central

    Gersch, Robert P.; Hadjiargyrou, Michael

    2009-01-01

    Mustn1 encodes a small nuclear protein expressed specifically in the musculoskeletal system that was originally identified as a strongly up-regulated gene during bone regeneration, especially in fracture callus proliferating chondrocytes. Further experiments were undertaken to investigate its expression and role during chondrogenesis. Initially, whole mount mouse in situ hybridization was carried out and revealed Mustn1 expression in areas of active chondrogenesis that included limb buds, branchial arches and tail bud. To elucidate its function, experiments were carried out to perturb Mustn1 by overexpression and silencing in the pre-chondrocytic RCJ3.1C5.18 (RCJ) cell line. In these cells, Mustn1 is normally differentially regulated, with a spike in expression 2 days after induction of differentiation. Further, Mustn1 was successfully overexpressed in multiple RCJ cell lines by ~2–6 fold, and reduced to ~32–52% in silenced cell lines as compared to parental Mustn1 levels. Overexpressing, silenced, control, and parental RCJ cell lines were assayed for proliferation and differentiation. No statistically significant changes were observed in either proliferation or proteoglycan production when Mustn1 overexpressing lines were compared to parental and control. By contrast, both proliferation rate and differentiation were significantly reduced in Mustn1 silenced cell lines. Specifically, RNAi silenced cell lines showed reductions in populations of ~55–75%, and also ~34–40% less matrix (proteoglycan) production as compared to parental and random control lines. Further, this reduction in matrix production was accompanied by significant downregulation of chondrogenic marker genes, such as Sox9, Collagen type II (Col II), and Collagen type X (Col X). Lastly, reintroduction of Mustn1 into a silenced cell line rescued this phenotype, returning proliferation rate, matrix production, and chondrogenic marker gene expression back to parental levels. Taken together these

  7. 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

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

    PubMed

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

    2015-04-13

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

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

    PubMed

    McCarty, M F; Russell, A L

    1999-10-01

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

  10. The Effect of Chemically Defined Medium on Spontaneous Calcium Signaling of In Situ Chondrocytes during Long-term Culture

    PubMed Central

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

    2015-01-01

    Chemically defined serum-free medium has been shown to maintain the mechanical integrity of articular cartilage explants better than serum-supplemented medium during long-term in vitro culture, but little is known about its effect on cellular mechanisms. We hypothesized that the chemically defined culture medium can regulate the spontaneous calcium signaling of in situ chondrocytes, which may modulate the cellular metabolic activities. Bovine cartilage explants were cultured in chemically defined serum-free or serum-supplemented medium for four weeks. The spontaneous intracellular calcium ([Ca2+]i) signaling of in situ chondrocytes was longitudinally measured together along with the biomechanical properties of the explants. The spontaneous [Ca2+]i oscillations in chondrocytes were enhanced at the initial exposure of serum-supplemented medium, but were significantly dampened afterwards. In contrast, cartilage explants in chemically defined medium preserved the level of calcium signaling, and showed more responsive cells with higher and more frequent [Ca2+]i peaks after one to four week culture in comparison to those in serum medium. Regardless of the culture medium that the explants were exposed, a positive correlation was detected between the [Ca2+]i responsive rate and the stiffness of cartilage (Spearman's rank correlation coefficient = 0.762). A stable pattern of [Ca2+]i peaks was revealed for each chondrocyte, i.e., the spatiotemporal features of [Ca2+]i peaks from a cell were highly consistent during the observation period (15 minutes). This study showed that the beneficial effect of chemically defined culture of cartilage explants is associated with the spontaneous [Ca2+]i signaling of chondrocytes in cartilage. PMID:25700610

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

    PubMed Central

    2011-01-01

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

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

    PubMed Central

    YAN, SHIJU; WANG, MENG; ZHAO, JIAN; ZHANG, HONGTAO; ZHOU, CHENGPEI; JIN, LEI; ZHANG, YINGLONG; QIU, XIUCHUN; MA, BAOAN; FAN, QINGYU

    2016-01-01

    Osteoarthritis (OA) is the most prevalent degenerative joint disease with multifactorial etiology caused by risk factors such as ageing, obesity and trauma. Previously, it was reported that the inhibition of microRNA-34a (miR-34a) may reduce rat chondrocyte apoptosis induced by IL-1β, whereas the molecular mechanism and the role of miR-34a in human chondrocyte as well as in OA progression remains to be determined. In the current study, using MTT, luciferase reporter assays and western blot analysis we identified that miR-34a was upregulated while silent information regulator 1 (SIRT1) was inhibited in chondrocytes from 12 OA patients compared with healthy chondrocytes from 10 trauma amputees. Overexpression of miR-34a promoted apoptosis and inhibited cell proliferation in human chondrocytes. Transfection with miR-34a mimic inhibited SIRT1 expression, which attenuated the deacetylation of p53, leading to the upregulation of Bax and downregulation of Bcl-2. Furthermore, results from the western blot analysis and luciferase reporter assay demonstrated that SIRT1 was directly regulated by miR-34a in human chondrocytes. A rat model of OA was induced through anterior cruciate ligament transection and medial meniscus resection (ACLT+MMx). The results showed that the intra-articular injection of lentiviral vector encoding anti-miR-34a sequence effectively ameliorated the progression of OA. The results suggest that miR-34a has a crucial role in the pathogenesis of OA through direct regulation of the SIRT1/p53 signaling pathway and serves as a potential therapeutic target of OA. PMID:27247228

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

    PubMed

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

    2016-07-01

    Osteoarthritis (OA) is the most prevalent degenerative joint disease with multifactorial etiology caused by risk factors such as ageing, obesity and trauma. Previously, it was reported that the inhibition of microRNA-34a (miR-34a) may reduce rat chondrocyte apoptosis induced by IL-1β, whereas the molecular mechanism and the role of miR-34a in human chondrocyte as well as in OA progression remains to be determined. In the current study, using MTT, luciferase reporter assays and western blot analysis we identified that miR-34a was upregulated while silent information regulator 1 (SIRT1) was inhibited in chondrocytes from 12 OA patients compared with healthy chondrocytes from 10 trauma amputees. Overexpression of miR-34a promoted apoptosis and inhibited cell proliferation in human chondrocytes. Transfection with miR-34a mimic inhibited SIRT1 expression, which attenuated the deacetylation of p53, leading to the upregulation of Bax and downregulation of Bcl-2. Furthermore, results from the western blot analysis and luciferase reporter assay demonstrated that SIRT1 was directly regulated by miR-34a in human chondrocytes. A rat model of OA was induced through anterior cruciate ligament transection and medial meniscus resection (ACLT+MMx). The results showed that the intra‑articular injection of lentiviral vector encoding anti-miR‑34a sequence effectively ameliorated the progression of OA. The results suggest that miR-34a has a crucial role in the pathogenesis of OA through direct regulation of the SIRT1/p53 signaling pathway and serves as a potential therapeutic target of OA. PMID:27247228

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

    PubMed

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

    2016-07-01

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

  15. The Interplay between Chondrocyte Redifferentiation Pellet Size and Oxygen Concentration

    PubMed Central

    Babur, Betul Kul; Ghanavi, Parisa; Levett, Peter; Lott, William B.; Klein, Travis; Cooper-White, Justin J.; Crawford, Ross; Doran, Michael R.

    2013-01-01

    Chondrocytes dedifferentiate during ex vivo expansion on 2-dimensional surfaces. Aggregation of the expanded cells into 3-dimensional pellets, in the presence of induction factors, facilitates their redifferentiation and restoration of the chondrogenic phenotype. Typically 1×105–5×105 chondrocytes are aggregated, resulting in “macro” pellets having diameters ranging from 1–2 mm. These macropellets are commonly used to study redifferentiation, and recently macropellets of autologous chondrocytes have been implanted directly into articular cartilage defects to facilitate their repair. However, diffusion of metabolites over the 1–2 mm pellet length-scales is inefficient, resulting in radial tissue heterogeneity. Herein we demonstrate that the aggregation of 2×105 human chondrocytes into micropellets of 166 cells each, rather than into larger single macropellets, enhances chondrogenic redifferentiation. In this study, we describe the development of a cost effective fabrication strategy to manufacture a microwell surface for the large-scale production of micropellets. The thousands of micropellets were manufactured using the microwell platform, which is an array of 360×360 µm microwells cast into polydimethylsiloxane (PDMS), that has been surface modified with an electrostatic multilayer of hyaluronic acid and chitosan to enhance micropellet formation. Such surface modification was essential to prevent chondrocyte spreading on the PDMS. Sulfated glycosaminoglycan (sGAG) production and collagen II gene expression in chondrocyte micropellets increased significantly relative to macropellet controls, and redifferentiation was enhanced in both macro and micropellets with the provision of a hypoxic atmosphere (2% O2). Once micropellet formation had been optimized, we demonstrated that micropellets could be assembled into larger cartilage tissues. Our results indicate that micropellet amalgamation efficiency is inversely related to the time cultured as discreet

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

    PubMed Central

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

    2008-01-01

    Alternative splicing of the type II procollagen gene (COL2A1) is developmentally-regulated during chondrogenesis. Type IIA procollagen (+ exon 2) is synthesized by chondroprogenitor cells while type IIB procollagen (- exon 2) is synthesized by differentiated chondrocytes. Here, we report expression of two additional alternatively spliced COL2A1 isoforms during chondrocyte differentiation of bone marrow derived mesenchymal stem cells (MSCs). One isoform, named IIC, contains only the first 34 nucleotides of exon 2 by use of an alternative 5’ splice site, resulting in a premature termination codon and possible nonsense-mediated decay of IIC mRNA. Low levels of the IIC isoform were detected by RT-PCR and Southern analysis of COL2A1 cDNA amplified from differentiating rabbit and human MSCs. A second novel transcript, named IID, arises by use of another 5’ alternative splice site in intron 2. The IID isoform contains exon 2 plus 3 nucleotides, resulting in the insertion of an additional amino acid. The IID isoform was co-expressed with the IIA isoform during chondrogenesis, and was approximately one-third as abundant. Deletion of the IIC alternative 5’ splice site from a COL2A1 mini-gene construct resulted in a significant increase in the IIA:IIB ratio. A mutant mini-gene that inhibited production of the IID isoform, however, had differential effects on the production of the IIA and IIB isoforms: this was apparently related to the differentiation status of the cell type used. These data suggest that COL2A1 mRNA abundance and other aspects of chondrocyte differentiation may be regulated by the use of these previously undetermined alternative splice sites. PMID:18023161

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

    PubMed

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

    2015-05-01

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

  18. 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

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

    PubMed

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

    2016-05-04

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

  20. Lead alters parathyroid hormone-related peptide and transforming growth factor-beta1 effects and AP-1 and NF-kappaB signaling in chondrocytes.

    PubMed

    Zuscik, Michael J; Pateder, Dhruv B; Puzas, J Edward; Schwarz, Edward M; Rosier, Randy N; O'Keefe, Regis J

    2002-07-01

    The skeletal system is an important target for lead toxicity. One of the impacts of lead in the skeleton, the inhibition of axial bone development, is likely due to its effect on the normal progression of chondrocyte maturation that is central to the process of endochondral ossification. Since little is known about the effect of lead on chondrocyte function/maturation, its impact on (1) growth factor-induced proliferation, (2) expression of maturation-specific markers type X collagen and BMP-6, and (3) the activity of AP-1 and NF-kappaB was examined in chick growth plate and sternal chondrocyte models. Exposure to lead alone (1-30 microM) resulted in a dose-dependent inhibition of thymidine incorporation in growth plate chondrocytes. Lead also blunted the stimulation of thymidine incorporation by parathyroid hormone-related peptide (PTHrP) and transforming growth factor-beta1 (TGF-beta1), two critical regulators of chondrocyte maturation. Lead (1 and 10 microM), TGF-beta1 (3 ng/ml) and PTHrP (10(-7) M) all significantly inhibited the expression of type X collagen, a marker of chondrocyte terminal differentiation. However, when in combination, lead completely reversed the inhibition of type X collagen by PTHrP and TGF-beta1. The effect of lead on BMP-6. an inducer of terminal differentiation. was also examined. Independently, lead and TGF-beta1 were without effect on BMP-6 expression, but PTHrP significantly suppressed it. Comparatively, lead did not alter PTHrP-mediated suppression of BMP-6, but in combination with TGF-beta1. BMP-6 expression was increased 3-fold. To determine if lead effects on signaling might play a role in facilitating these events, the impact of lead on NF-kappaB and AP-1 signaling was assessed using luciferase reporter constructs in sternal chondrocytes. Lead had no effect on the AP-1 reporter, but it dose-dependently inhibited the NF-kappaB reporter. PTHrP, which signals through AP-1, did not activate the NF-kappaB reporter and did not affect

  1. Aging-related differences in chondrocyte viscoelastic properties.

    PubMed

    Steklov, Nikolai; Srivastava, Ajay; Sung, K L P; Chen, Peter C; Lotz, Martin K; D'Lima, Darryl D

    2009-06-01

    The biomechanical properties of articular cartilage change profoundly with aging. These changes have been linked with increased potential for cartilage degeneration and osteoarthritis. However, less is known about the change in biomechanical properties of chondrocytes with increasing age. Cell stiffness can affect mechanotransduction pathways and may alter cell function. We measured aging-related changes in the biomechanical properties of chondrocytes. Human chondrocytes were isolated from knee articular cartilage within 48 hours after death or from osteochondral specimens obtained from knee arthroplasty. Cells were divided into two age groups: between 18 and 35 years (18 - 35); and greater than 55 years (55+) of age. The 55+ group was further subdivided based on visual grade of osteoarthritis: normal (N) or osteoarthritic (OA). The viscoelastic properties of the cell were measured using the previously described micropipette cell aspiration technique. The equilibrium modulus, instantaneous modulus, and apparent viscosity were significantly higher in the 55+ year age group than in the 18 - 35 age group. On the other hand, no differences were found in the equilibrium modulus, instantaneous modulus, or apparent viscosity between the N and OA groups. The increase in cell stiffness can be attributed to altered mechanical properties of the cell membrane, the cytoplasm, or the cytoskeleton. Increased stiffness has been reported in osteoarthritic chondrocytes, which in turn has been attributed to the actin cytoskeleton. A similar mechanism may be responsible for our finding of increased stiffness in aging chondrocytes. With advancing age, changes in the biomechanical properties of the cell could alter molecular and biochemical responses.

  2. ECM stiffness primes the TGFβ pathway to promote chondrocyte differentiation

    PubMed Central

    Allen, Jessica L.; Cooke, Margaret E.; Alliston, Tamara

    2012-01-01

    Cells encounter physical cues such as extracellular matrix (ECM) stiffness in a microenvironment replete with biochemical cues. However, the mechanisms by which cells integrate physical and biochemical cues to guide cellular decision making are not well defined. Here we investigate mechanisms by which chondrocytes generate an integrated response to ECM stiffness and transforming growth factor β (TGFβ), a potent agonist of chondrocyte differentiation. Primary murine chondrocytes and ATDC5 cells grown on 0.5-MPa substrates deposit more proteoglycan and express more Sox9, Col2α1, and aggrecan mRNA relative to cells exposed to substrates of any other stiffness. The chondroinductive effect of this discrete stiffness, which falls within the range reported for articular cartilage, requires the stiffness-sensitive induction of TGFβ1. Smad3 phosphorylation, nuclear localization, and transcriptional activity are specifically increased in cells grown on 0.5-MPa substrates. ECM stiffness also primes cells for a synergistic response, such that the combination of ECM stiffness and exogenous TGFβ induces chondrocyte gene expression more robustly than either cue alone through a p38 mitogen-activated protein kinase–dependent mechanism. In this way, the ECM stiffness primes the TGFβ pathway to efficiently promote chondrocyte differentiation. This work reveals novel mechanisms by which cells integrate physical and biochemical cues to exert a coordinated response to their unique cellular microenvironment. PMID:22833566

  3. Identification of Chondrocyte-Binding Peptides by Phage Display

    PubMed Central

    Cheung, Crystal S.F.; Lui, Julian C.; Baron, Jeffrey

    2016-01-01

    As an initial step toward targeting cartilage tissue for potential therapeutic applications, we sought cartilage-binding peptides using phage display, a powerful technology for selection of peptides that bind to molecules of interest. A library of phage displaying random 12-amino acid peptides was iteratively incubated with cultured chondrocytes to select phage that bind cartilage. The resulting phage clones demonstrated increased affinity to chondrocytes by ELISA, when compared to a wild-type, insertless phage. Furthermore, the selected phage showed little preferential binding to other cell types, including primary skin fibroblast, myocyte and hepatocyte cultures, suggesting a tissue-specific interaction. Immunohistochemical staining revealed that the selected phage bound chondrocytes themselves and the surrounding extracellular matrix. FITC-tagged peptides were synthesized based on the sequence of cartilage-binding phage clones. These peptides, but not a random peptide, bound cultured chondrocytes, and extracelluar matrix. In conclusion, using phage display, we identified peptide sequences that specifically target chondrocytes. We anticipate that such peptides may be coupled to therapeutic molecules to provide targeted treatment for cartilage disorders. PMID:23440926

  4. Surface topography regulates wnt signaling through control of primary cilia structure in mesenchymal stem cells

    NASA Astrophysics Data System (ADS)

    McMurray, R. J.; Wann, A. K. T.; Thompson, C. L.; Connelly, J. T.; Knight, M. M.

    2013-12-01

    The primary cilium regulates cellular signalling including influencing wnt sensitivity by sequestering β-catenin within the ciliary compartment. Topographic regulation of intracellular actin-myosin tension can control stem cell fate of which wnt is an important mediator. We hypothesized that topography influences mesenchymal stem cell (MSC) wnt signaling through the regulation of primary cilia structure and function. MSCs cultured on grooves expressed elongated primary cilia, through reduced actin organization. siRNA inhibition of anterograde intraflagellar transport (IFT88) reduced cilia length and increased active nuclear β-catenin. Conversely, increased primary cilia assembly in MSCs cultured on the grooves was associated with decreased levels of nuclear active β-catenin, axin-2 induction and proliferation, in response to wnt3a. This negative regulation, on grooved topography, was reversed by siRNA to IFT88. This indicates that subtle regulation of IFT and associated cilia structure, tunes the wnt response controlling stem cell differentiation.

  5. Morphologic stages of the terminal hypertrophic chondrocyte of growth plate cartilage.

    PubMed

    Farnum, C E; Wilsman, N J

    1987-11-01

    Recent biochemical and morphologic evidence supports the concept that hypertrophic chondrocytes of growth plate cartilage are fully viable cells that play a major functional role in controlling endochondral ossification. However, events associated with chondrocyte death remain unknown. In this study we assess the viability of terminal hypertrophic chondrocytes in situ in an organ culture system viewed simultaneously with rectified Nomarski interference contrast optics and with vital staining under fluorescence optics. Second, we use two methods of optimal chemical fixation at the ultrastructural level to define morphologically distinct stages of the terminal hypertrophic chondrocyte, which we interpret as the stages preceding chondrocyte death. An analysis of serial sections at the light microscope level showed that terminal chondrocytes were found, with different probabilities, in three morphologically distinguishable stages. Seventy-five percent of all profiles were fully hydrated cells with an intact plasma membrane making direct contact with the pericellular matrix, a morphology identical to that of living terminal chondrocytes viewed in Nomarski optics. Approximately 1% of terminal chondrocytes, while still in a fully hydrated state, consistently made a direct asymmetrical contact of the plasma membrane with the last transverse septum. In 24% of the profiles, terminal chondrocytes were found as condensed cells that retained their attachment to the last transverse septum. The stages were not characteristic of chondrocytes positioned more proximally in the growth plate. We hypothesize that a condensed morphology eventually characterizes each hypertrophic chondrocyte, and we relate these observations to current hypotheses concerning the mechanism of death of hypertrophic chondrocytes. PMID:3425941

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

    PubMed

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

    2008-04-01

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

  7. Pericellular coat of chick embryo chondrocytes: structural role of hyaluronate

    PubMed Central

    1984-01-01

    Chondrocytes produce large pericellular coats in vitro that can be visualized by the exclusion of particles, e.g., fixed erythrocytes, and that are removed by treatment with Streptomyces hyaluronidase, which is specific for hyaluronate. In this study, we examined the kinetics of formation of these coats and the relationship of hyaluronate and proteoglycan to coat structure. Chondrocytes were isolated from chick tibia cartilage by collagenase-trypsin digestion and were characterized by their morphology and by their synthesis of both type II collagen and high molecular weight proteoglycans. The degree of spreading of the chondrocytes and the size of the coats were quantitated at various times subsequent to seeding by tracing phase-contrast photomicrographs of the cultures. After seeding, the chondrocytes attached themselves to the tissue culture dish and exhibited coats within 4 h. The coats reached a maximum size after 3-4 d and subsequently decreased over the next 2-3 d. Subcultured chondrocytes produced a large coat only if passaged before 4 d. Both primary and first passage cells, with or without coats, produced type II collagen but not type I collagen as determined by enzyme-linked immunosorbent assay. Treatment with Streptomyces hyaluronidase (1.0 mU/ml, 15 min), which completely removed the coat, released 58% of the chondroitin sulfate but only 9% of the proteins associated with the cell surface. The proteins released by hyaluronidase were not digestible by bacterial collagenase. Monensin and cycloheximide (0.01-10 microM, 48 h) caused a dose-dependent decrease in coat size that was linearly correlated to synthesis of cell surface hyaluronate (r = 0.98) but not chondroitin sulfate (r = 0.2). We conclude that the coat surrounding chondrocytes is dependent on hyaluronate for its structure and that hyaluronate retains a large proportion of the proteoglycan in the coat. PMID:6501414

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

    PubMed Central

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

    2016-01-01

    Osteoarthritis (OA) in the temporomandibular joint (TMJ) is a common degenerative disease in adult, which is characterized by progressive destruction of the articular cartilage. To investigate the role of FGFR3 in the homeostasis of TMJ cartilage during adult stage, we generated Fgfr3f/f; Col2a1-CreERT2 (Fgfr3 cKO) mice, in which Fgfr3 was deleted in chondrocytes at 2 months of age. OA-like defects were observed in Fgfr3 cKO TMJ cartilage. Immunohistochemical staining and quantitative real-time PCR analyses revealed a significant increase in expressions of COL10, MMP13 and AMAMTS5. In addition, there was a sharp increase in chondrocyte apoptosis at the Fgfr3 cKO articular surface, which was accompanied by a down-regulation of lubricin expression. Importantly, the expressions of RUNX2 and Indian hedgehog (IHH) were up-regulated in Fgfr3 cKO TMJ. Primary Fgfr3 cKO chondrocytes were treated with IHH signaling inhibitor, which significantly reduced expressions of Runx2, Col10, Mmp13 and Adamts5. Furthermore, the IHH signaling inhibitor partially alleviated OA-like defects in the TMJ of Fgfr3 cKO mice, including restoration of lubricin expression and improvement of the integrity of the articular surface. In conclusion, our study proposes that FGFR3/IHH signaling pathway plays a critical role in maintaining the homeostasis of TMJ articular cartilage during adult stage. PMID:27041063

  9. Effect of nitric oxide on mitochondrial respiratory activity of human articular chondrocytes

    PubMed Central

    Maneiro, E; Lopez-Armada, M; de Andres, M C; Carames, B; Martin, M; Bonilla, A; del Hoyo, P; Galdo, F; Arenas, J; Blanco, F

    2005-01-01

    Objective: To investigate the effect of nitric oxide (NO) on mitochondrial activity and its relation with the apoptosis of human articular chondrocytes. Materials and methods: Mitochondrial function was evaluated by analysing respiratory chain enzyme complexes, citrate synthase (CS) activities, and mitochondrial membrane potential (Δψm). The activities of the mitochondrial respiratory chain (MRC) complexes (complex I: NADH CoQ1 reductase, complex II: succinate dehydrogenase, complex III: ubiquinol cytochrome c reductase, complex IV: cytochrome c oxidase) and CS were measured in human articular chondrocytes isolated from normal cartilage. The Δψm was measured by 5,5',6,6'-tetracholoro-1,1',3,3'-tetraethylbenzimidazole carbocyanide iodide (JC-1) using flow cytometry. Apoptosis was analysed by flow cytometry. The mRNA expression of caspases was analysed by ribonuclease protection analysis and the detection of protein synthesis by western blotting. Sodium nitroprusside (SNP) was used as an NO compound donor. Results: SNP at concentrations higher than 0.5 mmol/l for 24 hours induced cellular changes characteristic of apoptosis. SNP elicited mRNA expression of caspase-3 and caspase-7 and down regulated bcl-2 synthesis in a dose and time dependent manner. Furthermore, 0.5 mM SNP induced depolarisation of the mitochondrial membrane at 5, 12, and 24 hours. Analysis of the MRC showed that at 5 hours, 0.5 mM SNP reduced the activity of complex IV by 33%. The individual inhibition of mitochondrial complex IV with azide modified the Δψm and induced apoptosis. Conclusions: This study suggests that the effect of NO on chondrocyte survival is mediated by its effect on complex IV of the MRC. PMID:15708893

  10. Chondrocytes Transdifferentiate into Osteoblasts in Endochondral Bone during Development, Postnatal Growth and Fracture Healing in Mice

    PubMed Central

    Zhou, Xin; von der Mark, Klaus; Henry, Stephen; Norton, William; Adams, Henry; de Crombrugghe, Benoit

    2014-01-01

    One of the crucial steps in endochondral bone formation is the replacement of a cartilage matrix produced by chondrocytes with bone trabeculae made by osteoblasts. However, the precise sources of osteoblasts responsible for trabecular bone formation have not been fully defined. To investigate whether cells derived from hypertrophic chondrocytes contribute to the osteoblast pool in trabecular bones, we genetically labeled either hypertrophic chondrocytes by Col10a1-Cre or chondrocytes by tamoxifen-induced Agc1-CreERT2 using EGFP, LacZ or Tomato expression. Both Cre drivers were specifically active in chondrocytic cells and not in perichondrium, in periosteum or in any of the osteoblast lineage cells. These in vivo experiments allowed us to follow the fate of cells labeled in Col10a1-Cre or Agc1-CreERT2 -expressing chondrocytes. After the labeling of chondrocytes, both during prenatal development and after birth, abundant labeled non-chondrocytic cells were present in the primary spongiosa. These cells were distributed throughout trabeculae surfaces and later were present in the endosteum, and embedded within the bone matrix. Co-expression studies using osteoblast markers indicated that a proportion of the non-chondrocytic cells derived from chondrocytes labeled by Col10a1-Cre or by Agc1-CreERT2 were functional osteoblasts. Hence, our results show that both chondrocytes prior to initial ossification and growth plate chondrocytes before or after birth have the capacity to undergo transdifferentiation to become osteoblasts. The osteoblasts derived from Col10a1-expressing hypertrophic chondrocytes represent about sixty percent of all mature osteoblasts in endochondral bones of one month old mice. A similar process of chondrocyte to osteoblast transdifferentiation was involved during bone fracture healing in adult mice. Thus, in addition to cells in the periosteum chondrocytes represent a major source of osteoblasts contributing to endochondral bone formation in vivo

  11. Chondrocytes transdifferentiate into osteoblasts in endochondral bone during development, postnatal growth and fracture healing in mice.

    PubMed

    Zhou, Xin; von der Mark, Klaus; Henry, Stephen; Norton, William; Adams, Henry; de Crombrugghe, Benoit

    2014-12-01

    One of the crucial steps in endochondral bone formation is the replacement of a cartilage matrix produced by chondrocytes with bone trabeculae made by osteoblasts. However, the precise sources of osteoblasts responsible for trabecular bone formation have not been fully defined. To investigate whether cells derived from hypertrophic chondrocytes contribute to the osteoblast pool in trabecular bones, we genetically labeled either hypertrophic chondrocytes by Col10a1-Cre or chondrocytes by tamoxifen-induced Agc1-CreERT2 using EGFP, LacZ or Tomato expression. Both Cre drivers were specifically active in chondrocytic cells and not in perichondrium, in periosteum or in any of the osteoblast lineage cells. These in vivo experiments allowed us to follow the fate of cells labeled in Col10a1-Cre or Agc1-CreERT2 -expressing chondrocytes. After the labeling of chondrocytes, both during prenatal development and after birth, abundant labeled non-chondrocytic cells were present in the primary spongiosa. These cells were distributed throughout trabeculae surfaces and later were present in the endosteum, and embedded within the bone matrix. Co-expression studies using osteoblast markers indicated that a proportion of the non-chondrocytic cells derived from chondrocytes labeled by Col10a1-Cre or by Agc1-CreERT2 were functional osteoblasts. Hence, our results show that both chondrocytes prior to initial ossification and growth plate chondrocytes before or after birth have the capacity to undergo transdifferentiation to become osteoblasts. The osteoblasts derived from Col10a1-expressing hypertrophic chondrocytes represent about sixty percent of all mature osteoblasts in endochondral bones of one month old mice. A similar process of chondrocyte to osteoblast transdifferentiation was involved during bone fracture healing in adult mice. Thus, in addition to cells in the periosteum chondrocytes represent a major source of osteoblasts contributing to endochondral bone formation in vivo

  12. Static and dynamic compressive strains influence nitric oxide production and chondrocyte bioactivity when encapsulated in PEG hydrogels of different crosslinking densities

    PubMed Central

    Villanueva, I; Hauschulz, DS; Mejic, D; Bryant, SJ

    2012-01-01

    Summary Objective Mechanical loading is an important regulator of chondrocytes; however, many of the mechanisms involved in chondrocyte mechanotransduction still remain unclear. Here, poly(ethylene glycol) (PEG) hydrogels are proposed as a model system to elucidate chondrocyte response due to cell deformation, which is controlled by gel crosslinking (ρx). Methods Bovine articular chondrocytes (50×106cells/ml) were encapsulated in gels with three ρx’s and subjected to static (15% strain) or dynamic (0.3Hz or 1Hz, 15% amplitude strain) loading for 48 hours. Cell deformation was examined by confocal microscopy. Cell response was assessed by total nitric oxide production (NO), proteoglycan (PG) synthesis (35SO42−-incorporation) and cell proliferation ([3H]-thymidine incorporation) (CP). Oxygen consumption was assessed using an oxygen biosensor. Results An increase in ρx led to lower water contents, higher compressive moduli, and higher cell deformations. Chondrocyte response was dependent on both loading regime and ρx. For example, under a static strain, NO was not affected, while CP and PG synthesis were inhibited in low ρx and stimulated in high ρx. Dynamic loading resulted in either no effect or an inhibitory effect on NO, CP, and PG synthesis. Overall, our results showed correlations between NO and CP and/or PG synthesis under static and dynamic (0.3 Hz) loading. This finding was attributed to the hypoxic environment that resulted from the high cell-seeding density. Conclusion This study demonstrates gel ρx and loading condition influence NO, CP, and PG synthesis. Under a hypoxic environment and certain loading conditions, NO appears to have a positive effect on chondrocyte bioactivity. PMID:18203631

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

    PubMed

    Cuellar, Araceli; Reddi, A Hari

    2015-07-01

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

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

    PubMed Central

    Cuellar, Araceli

    2015-01-01

    Superficial zone protein (SZP), also known as lubricin and proteoglycan 4 (PRG4), plays an important role in the boundary lubrication of articular cartilage and is regulated by transforming growth factor (TGF)-β. Here, we evaluate the role of cell surface glycosaminoglycans (GAGs) during TGF-β1 stimulation of SZP/lubricin/PRG4 in superficial zone articular chondrocytes. We utilized primary monolayer superficial zone articular chondrocyte cultures and treated them with various concentrations of TGF-β1, in the presence or absence of heparan sulfate (HS), heparin, and chondroitin sulfate (CS). The cell surface GAGs were removed by pretreatment with either heparinase I or chondroitinase-ABC before TGF-β1 stimulation. Accumulation of SZP/lubricin/PRG4 in the culture medium in response to stimulation with TGF-β1 and various exogenous GAGs was demonstrated by immunoblotting and quantitated by enzyme-linked immunosorbent assay. We show that TGF-β1 and exogenous HS enhanced SZP accumulation of superficial zone chondrocytes in the presence of surface GAGs. At the dose of 1 ng/mL of TGF-β1, the presence of exogenous heparin inhibited SZP accumulation whereas the presence of exogenous CS stimulated SZP accumulation in the culture medium. Enzymatic depletion of GAGs on the surface of superficial zone chondrocytes enhanced the ability of TGF-β1 to stimulate SZP accumulation in the presence of both exogenous heparin and CS. Collectively, these results suggest that GAGs at the surface of superficial zone articular chondrocytes influence the response to TGF-β1 and exogenous GAGs to stimulate SZP accumulation. Cell surface GAGs modulate superficial zone chondrocytes' response to TGF-β1 and exogenous HS. PMID:25398329

  15. In vitro chondrocyte behavior on porous biodegradable poly(e-caprolactone)/polyglycolic acid scaffolds for articular chondrocyte adhesion and proliferation.

    PubMed

    Jonnalagadda, John B; Rivero, Iris V; Dertien, Janet S

    2015-01-01

    In this study, poly(e-caprolactone)/polyglycolic acid (PCL/PGA) scaffolds for repairing articular cartilage were fabricated via solid-state cryomilling along with compression molding and porogen leaching. Four distinct scaffolds were fabricated using this approach by four independent cryomilling times. These scaffolds were assessed for their suitability to promote articular cartilage regeneration with in vitro chondrocyte cell culture studies. The scaffolds were characterized for pore size, porosity, swelling ratio, compressive, and thermal properties. Cryomilling time proved to significantly affect the physical, mechanical, and morphological properties of the scaffolds. In vitro bovine chondrocyte culture was performed dynamically for 1, 7, 14, 28, and 35 days. Chondrocyte viability and adhesion were tested using MTT assay and scanning electron microscopy micrographs. Glycosaminoglycan (GAG) and DNA assays were performed to investigate the extracellular matrix (ECM) formation and cell proliferation, respectively. PCL/PGA scaffolds demonstrated high porosity for all scaffold types. Morphological analysis and poly(ethylene oxide) continuity demonstrated the existence of a co-continuous network of interconnected pores with pore sizes appropriate for tissue engineering and chondrocyte ingrowth. While mean pore size decreased, water uptake and compressive properties increased with increasing cryomilling times. Compressive modulus of 12, 30, and 60 min scaffolds matched the compressive modulus of human articular cartilage. Viable cells increased besides increase in cell proliferation and ECM formation with progress in culture period. Chondrocytes exhibited spherical morphology on all scaffold types. The pore size of the scaffold affected chondrocyte adhesion, proliferation, and GAG secretion. The results indicated that the 12 min scaffolds delivered promising results for applications in articular cartilage repair.

  16. Clinical Outcomes of Characterized Chondrocyte Implantation

    PubMed Central

    Huylebroek, José; Van Der Bauwhede, Jan; Saris, Daniël; Veeckman, Geert; Bobic, Vladimir; Victor, Jan; Almqvist, Karl Fredrik; Verdonk, Peter; Fortems, Yves; Van Lommel, Nel; Haazen, Ludo

    2012-01-01

    Objective: To assess the clinical outcome of patients treated with autologous chondrocyte implantation using ChondroCelect in daily practice. Methods: The study is a cross-sectional analysis of an open-label, noninterventional cohort. The setting was a compassionate use program, involving 43 orthopaedic centers in 7 European countries. The participants were patients treated with ChondroCelect between October 13, 2004 and July 2, 2008. The measurements used were Clinical Global Impression–Improvement and –Efficacy and solicited adverse event reports. Results: Safety data were collected from 334 patients (90.3%), and effectiveness data were from 282 (76.2%) of the 370 patients treated. Mean age at baseline was 33.6 years (range, 12-57 years), 57% were male, and mean body mass index was 25 kg/m2. Mean follow-up was 2.2 years (range, 0.4-4.1 years). A femoral condyle lesion was reported in 66% (288/379) and a patellar lesion in 19% (84/379). Mean lesion size was 3.5 cm2; a collagen membrane was used in 92.4% (328/355). A therapeutic effect was reported in 89% (234/264) of patients overall and in 87% (40/46) of patellar lesion patients. Rates of much or very much improved patients were similar in patients with short- (<18 months: 71% [115/163]) and long-term follow-up (>18 months: 68% [70/103]) (P = 0.68) and were independent of lesion size (>4 cm2: 75.5% [37/49]; ≤4 cm2: 67.7% [111/164]) (P = 0.38). Adverse events were similar to those reported in the randomized trial with the same product, with more arthrofibrosis, more reduced joint mobility, and more crepitations reported in patellar lesions. Overall, less cartilage hypertrophy was noted, probably due to the use of a biological membrane cover. Conclusions: Implantation of ChondroCelect appeared to result in a positive benefit/risk ratio when used in an unselected heterogenous population, irrespective of the follow-up period, lesion size, and type of lesion treated. PMID:26069630

  17. Lithium chloride modulates chondrocyte primary cilia and inhibits Hedgehog signaling.

    PubMed

    Thompson, Clare L; Wiles, Anna; Poole, C Anthony; Knight, Martin M

    2016-02-01

    Lithium chloride (LiCl) exhibits significant therapeutic potential as a treatment for osteoarthritis. Hedgehog signaling is activated in osteoarthritis, where it promotes chondrocyte hypertrophy and cartilage matrix catabolism. Hedgehog signaling requires the primary cilium such that maintenance of this compartment is essential for pathway activity. Here we report that LiCl (50 mM) inhibits Hedgehog signaling in bovine articular chondrocytes such that the induction of GLI1 and PTCH1 expression is reduced ​ by 71 and 55%, respectively. Pathway inhibition is associated with a 97% increase in primary cilia length from 2.09 ± 0.7 μm in untreated cells to 4.06 ± 0.9 μm in LiCl-treated cells. We show that cilia elongation disrupts trafficking within the axoneme with a 38% reduction in Arl13b ciliary localization at the distal region of the cilium, consistent with the role of Arl13b in modulating Hedgehog signaling. In addition, we demonstrate similar increases in cilia length in human chondrocytes in vitro and after administration of dietary lithium to Wistar rats in vivo. Our data provide new insights into the effects of LiCl on chondrocyte primary cilia and Hedgehog signaling and shows for the first time that pharmaceutical targeting of the primary cilium may have therapeutic benefits in the treatment of osteoarthritis. PMID:26499268

  18. Collagen and chondrocyte concentrations control ultrasound scattering in agarose scaffolds.

    PubMed

    Inkinen, S; Liukkonen, J; Ylärinne, J H; Puhakka, P H; Lammi, M J; Virén, T; Jurvelin, J S; Töyräs, J

    2014-09-01

    Ultrasound imaging has been proposed for diagnostics of osteoarthritis and cartilage injuries in vivo. However, the specific contribution of chondrocytes and collagen to ultrasound scattering in articular cartilage has not been systematically studied. We investigated the role of these tissue structures by measuring ultrasound scattering in agarose scaffolds with varying collagen and chondrocyte concentrations. Ultrasound catheters with center frequencies of 9 MHz (7.1-11.0 MHz, -6 dB) and 40 MHz (30.1-45.3 MHz, -6 dB) were applied using an intravascular ultrasound device. Ultrasound backscattering quantified in a region of interest starting right below sample surface differed significantly (p < 0.05) with the concentrations of collagen and chondrocytes. An ultrasound frequency of 40 MHz, as compared with 9 MHz, was more sensitive to variations in collagen and chondrocyte concentrations. The present findings may improve diagnostic interpretation of arthroscopic ultrasound imaging and provide information necessary for development of models describing ultrasound propagation within cartilage. PMID:24972499

  19. Induction and prevention of chondrocyte hypertrophy in culture

    PubMed Central

    1989-01-01

    Primary chondrocytes from whole chick embryo sterna can be maintained in suspension culture stabilized with agarose for extended periods of time. In the absence of FBS, the cells remain viable only when seeded at high densities. They do not proliferate at a high rate but they deposit extracellular matrix with fibrils resembling those of authentic embryonic cartilage in their appearance and collagen composition. The cells exhibit many morphological and biochemical characteristics of resting chondrocytes and they do not produce collagen X, a marker for hypertrophic cartilage undergoing endochondral ossification. At low density, cells survive in culture without FBS when the media are conditioned by chondrocytes grown at high density. Thus, resting cartilage cells in agarose cultures can produce factors required for their own viability. Addition of FBS to the culture media leads to profound changes in the phenotype of chondrocytes seeded at low density. Cells form colonies at a high rate and assume properties of hypertrophic cells, including the synthesis of collagen X. They extensively deposit extracellular matrix resembling more closely that of adult rather than embryonic cartilage. PMID:2808534

  20. Effect of thiram on avian growth plate chondrocytes in culture

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Thiram (tetramethyl thiuram disulfide) is a general use pesticide. It causes tibial dyschondroplasia, a cartilage defect in poultry leading to growth plate deformation and lameness. The mechanism of its action on chondrocytes is not understood. Since proteins play significant role in development an...

  1. Inhibition of matrix metalloproteinase-3 and -13 synthesis induced by IL-1beta in chondrocytes from mice lacking microsomal prostaglandin E synthase-1.

    PubMed

    Gosset, Marjolaine; Pigenet, Audrey; Salvat, Colette; Berenbaum, Francis; Jacques, Claire

    2010-11-15

    Joint destruction in arthritis is in part due to the induction of matrix metalloproteinase (MMP) expression and their inhibitors, especially MMP-13 and -3, which directly degrade the cartilage matrix. Although IL-1β is considered as the main catabolic factor involved in MMP-13 and -3 expression, the role of PGE(2) remains controversial. The goal of this study was to determine the role of PGE(2) on MMP synthesis in articular chondrocytes using mice lacking microsomal PGE synthase-1 (mPGES-1), which catalyses the rate-limiting step of PGE(2) synthesis. MMP-3 and MMP-13 mRNA and protein expressions were assessed by real-time RT-PCR, immunoblotting, and ELISA in primary cultures of articular chondrocytes from mice with genetic deletion of mPGES-1. IL-1β-induced PGE(2) synthesis was dramatically reduced in mPGES-1(-/-) and mPGES-1(+/-) compared with mPGES-1(+/+) chondrocytes. A total of 10 ng/ml IL-1β increased MMP-3 and MMP-13 mRNA, protein expression, and release in mPGES-1(+/+) chondrocytes in a time-dependent manner. IL-1β-induced MMP-3 and MMP-13 mRNA expression, protein expression, and release decreased in mPGES-1(-/-) and mPGES-1(+/-) chondrocytes compared with mPGES-1(+/+) chondrocytes from 8 up to 24 h. Otherwise, MMP inhibition was partially reversed by addition of 10 ng/ml PGE(2) in mPGES-1(-/-) chondrocytes. Finally, in mPGES-1(-/-) chondrocytes treated by forskolin, MMP-3 protein expression was significantly decreased compared with wild-type, suggesting that PGE(2) regulates MMP-3 expression via a signaling pathway dependent on cAMP. These results demonstrate that PGE(2) plays a key role in the induction of MMP-3 and MMP-13 in an inflammatory context. Therefore, mPGES-1 could be considered as a critical target to counteract cartilage degradation in arthritis.

  2. Influence of cell printing on biological characters of chondrocytes

    PubMed Central

    Qu, Miao; Gao, Xiaoyan; Hou, Yikang; Shen, Congcong; Xu, Yourong; Zhu, Ming; Wang, Hengjian; Xu, Haisong; Chai, Gang; Zhang, Yan

    2015-01-01

    Objective: To establish a two-dimensional biological printing technique of chondrocytes and compare the difference of related biological characters between printed chondrocytes and unprinted cells so as to control the cell transfer process and keep cell viability after printing. Methods: Primary chondrocytes were obtained from human mature and fetal cartilage tissues and then were regularly sub-cultured to harvest cells at passage 2 (P2), which were adjusted to the single cell suspension at a density of 1×106/mL. The experiment was divided into 2 groups: experimental group P2 chondrocytes were transferred by rapid prototype biological printer (driving voltage value 50 V, interval in x-axis 300 μm, interval in y-axis 1500 μm). Afterwards Live/Dead viability Kit and flow cytometry were respectively adopted to detect cell viability; CCK-8 Kit was adopted to detect cell proliferation viability; immunocytochemistry, immunofluorescence and RT-PCR was employed to identify related markers of chondrocytes; control group steps were the same as the printing group except that cell suspension received no printing. Results: Fluorescence microscopy and flow cytometry analyses showed that there was no significant difference between experimental group and control group in terms of cell viability. After 7-day in vitro culture, control group exhibited higher O.D values than experimental group from 2nd day to 7th day but there was no distinct difference between these two groups (P>0.05). Inverted microscope observation demonstrated that the morphology of these two groups had no significant difference either. Similarly, Immunocytochemistry, immunofluorescence and RT-PCR assays also showed that there was no significant difference in the protein and gene expression of type II collagen and aggrecan between these two groups (P>0.05). Conclusion Cell printing has no distinctly negative effect on cell vitality, proliferation and phenotype of chondrocytes. Biological printing technique may

  3. DIFFERENTIAL DIELECTRIC RESPONSES OF CHONDROCYTE AND JURKAT CELLS IN ELECTROMANIPULATION BUFFERS

    PubMed Central

    Sabuncu, Ahmet C.; Asmar, Anthony J.; Stacey, Michael W.; Beskok, Ali

    2015-01-01

    Electromanipulation of cells as a label free cell manipulation and characterization tool has gained particular interest recently. However, the applicability of electromanipulation, particularly dielectrophoresis (DEP), to biological cells is limited to cells suspended in buffers containing lower amounts of salts relative to the physiological buffers. One might question the use of low conductivity buffers (LCB) for DEP separation, as cells are stressed in buffers lacking physiological levels of salt. In LCB, cells leak ions and undergo volume regulation. Therefore, cells exhibit time-dependent DEP response in LCB. In this work, cellular changes in LCB are assessed by dielectric spectroscopy, cell viability assay, and gene expression of chondrocytes and Jurkats. Results indicate leakage of ions from cells, increases in cytoplasmic conductivity, membrane capacitance and conductance. Separability factor, which defines optimum conditions for DEP cell separation, for the two cell types is calculated using the cellular dielectric data. Optimum DEP separation conditions change as cellular dielectric properties evolve in LCB. Genetic analyses indicate no changes in expression of ionic channel proteins for chondrocytes suspended in LCB. Retaining cellular viability might be important during dielectrophoretic separation, especially when cells are to be biologically tested at a downstream microfluidic component. PMID:25958778

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

    PubMed

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

    2016-01-01

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

  5. Differential dielectric responses of chondrocyte and Jurkat cells in electromanipulation buffers.

    PubMed

    Sabuncu, Ahmet C; Asmar, Anthony J; Stacey, Michael W; Beskok, Ali

    2015-07-01

    Electromanipulation of cells as a label-free cell manipulation and characterization tool has gained particular interest recently. However, the applicability of electromanipulation, particularly dielectrophoresis (DEP), to biological cells is limited to cells suspended in buffers containing lower amounts of salts relative to the physiological buffers. One might question the use of low conductivity buffers (LCBs) for DEP separation, as cells are stressed in buffers lacking physiological levels of salt. In LCB, cells leak ions and undergo volume regulation. Therefore, cells exhibit time-dependent DEP response in LCB. In this work, cellular changes in LCB are assessed by dielectric spectroscopy, cell viability assay, and gene expression of chondrocytes and Jurkats. Results indicate leakage of ions from cells, increases in cytoplasmic conductivity, membrane capacitance, and conductance. Separability factor, which defines optimum conditions for DEP cell separation, for the two cell types is calculated using the cellular dielectric data. Optimum DEP separation conditions change as cellular dielectric properties evolve in LCB. Genetic analyses indicate no changes in expression of ionic channel proteins for chondrocytes suspended in LCB. Retaining cellular viability might be important during dielectrophoretic separation, especially when cells are to be biologically tested at a downstream microfluidic component.

  6. Beta-arrestin 1 is involved in the catabolic response stimulated by hyaluronan degradation in mouse chondrocytes.

    PubMed

    Campo, Giuseppe M; Avenoso, Angela; D'Ascola, Angela; Scuruchi, Michele; Calatroni, Alberto; Campo, Salvatore

    2015-08-01

    Beta-arrestin-1 (β-arrestin-1) is an adaptor protein that functions in the termination of G-protein activation and seems to be involved in the mediation of the inflammatory response. Interleukin-1β (IL-1β) elicits the expression of inflammatory mediators through a mechanism involving hyaluronan (HA) degradation, thereby contributing to toll-like receptor 4 (TLR-4) and CD44 activation. Stimulation of both receptors induces nuclear factor kappaB (NF-kB) activation that, through transforming-growth-factor-activated-kinase-1 (TAK-1), in turn stimulates the inflammatory mediators of transcription. As β-arrestin-1 seems to play an inflammatory role in arthritis, we have investigated the involvement of β-arrestin-1 in a model of IL-1β-induced inflammatory response in mouse chondrocytes. IL-1β treatment significantly increases chondrocytes TLR-4, CD44, β-arrestin-1, TAK-1, and serine/threonine kinase (AKT) mRNA expression and related protein levels. NF-kB is also markedly activated with consequent tumor-necrosis-factor-alpha, interleukin-6, and inducible-nitric-oxide-synthase up-regulation. Treatment of IL-1β-stimulated chondrocytes with β-arrestin-1 and/or AKT and/or TAK-1-specific inhibitors significantly reduces all parameters, although the inhibitory effect exerted by TAK-1-mediated pathways is more effective than that of β-arrestin-1. β-Arrestin-1-induced NF-kB activation is mediated by the AKT pathway as shown by IL-1β-stimulated chondrocytes treated with AKT inhibitor. Finally, a specific HA-blocking peptide (Pep-1) has confirmed the inflammatory role of degraded HA as a mediator of the IL-1β-induced activation of β-arrestin-1. PMID:25673209

  7. 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

  8. Beta-arrestin 1 is involved in the catabolic response stimulated by hyaluronan degradation in mouse chondrocytes.

    PubMed

    Campo, Giuseppe M; Avenoso, Angela; D'Ascola, Angela; Scuruchi, Michele; Calatroni, Alberto; Campo, Salvatore

    2015-08-01

    Beta-arrestin-1 (β-arrestin-1) is an adaptor protein that functions in the termination of G-protein activation and seems to be involved in the mediation of the inflammatory response. Interleukin-1β (IL-1β) elicits the expression of inflammatory mediators through a mechanism involving hyaluronan (HA) degradation, thereby contributing to toll-like receptor 4 (TLR-4) and CD44 activation. Stimulation of both receptors induces nuclear factor kappaB (NF-kB) activation that, through transforming-growth-factor-activated-kinase-1 (TAK-1), in turn stimulates the inflammatory mediators of transcription. As β-arrestin-1 seems to play an inflammatory role in arthritis, we have investigated the involvement of β-arrestin-1 in a model of IL-1β-induced inflammatory response in mouse chondrocytes. IL-1β treatment significantly increases chondrocytes TLR-4, CD44, β-arrestin-1, TAK-1, and serine/threonine kinase (AKT) mRNA expression and related protein levels. NF-kB is also markedly activated with consequent tumor-necrosis-factor-alpha, interleukin-6, and inducible-nitric-oxide-synthase up-regulation. Treatment of IL-1β-stimulated chondrocytes with β-arrestin-1 and/or AKT and/or TAK-1-specific inhibitors significantly reduces all parameters, although the inhibitory effect exerted by TAK-1-mediated pathways is more effective than that of β-arrestin-1. β-Arrestin-1-induced NF-kB activation is mediated by the AKT pathway as shown by IL-1β-stimulated chondrocytes treated with AKT inhibitor. Finally, a specific HA-blocking peptide (Pep-1) has confirmed the inflammatory role of degraded HA as a mediator of the IL-1β-induced activation of β-arrestin-1.

  9. 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. PMID:27427985

  10. A Biphasic Multiscale Study of the Mechanical Microenvironment of Chondrocytes within Articular Cartilage under Unconfined Compression

    PubMed Central

    Guo, Hongqiang; Maher, Suzanne A.; Torzilli, Peter A.

    2014-01-01

    Computational analyses have been used to study the biomechanical microenvironment of the chondrocyte that cannot be assessed by in vitro experimental studies; yet all computational studies thus far have focused on the effect of zonal location (superficial, middle, and deep) on the mechanical microenvironment of chondrocytes. The aim of this paper was to study the effect of both zonal and radial locations on the biomechanical microenvironment of chondrocytes in inhomogeneous cartilage under unconfined stress relaxation. A biphasic multiscale approach was employed and nine chondrocytes in different locations were studied. Hyperelastic biphasic theory and depth-dependent aggregate modulus and permeability of articular cartilage were included in the models. It was found that both zonal and radial locations affected the biomechanical stresses and strains of the chondrocytes. Chondrocytes in the mid-radial location had increased volume during the early stage of the loading process. Maximum principal shear stress at the interface between the chondrocyte and the extracellular matrix (ECM) increased with depth, yet that at the ECM-pericellular matrix (PCM) interface had an inverse trend. Fluid pressure decreased with depth, while the fluid pressure difference between the top and bottom boundaries of the microscale model increased with depth. Regardless of location, fluid was exchanged between the chondrocyte, PCM, and ECM. These findings suggested that even under simple compressive loading conditions, the biomechanical microenvironment of the chondrocytes, PCM and ECM were spatially dependent. The current study provides new insight on chondrocyte biomechanics. PMID:24882738

  11. SERPINE2 Inhibits IL-1α-Induced MMP-13 Expression in Human Chondrocytes: Involvement of ERK/NF-κB/AP-1 Pathways

    PubMed Central

    Scotece, Morena; Abella, Vanessa; Lois, Ana; Lopez, Veronica; Pino, Jesus; Gomez, Rodolfo; Gomez-Reino, Juan J.; Gualillo, Oreste

    2015-01-01

    Objectives Osteoarthritis (OA) is a chronic joint disease, characterized by a progressive loss of articular cartilage. During OA, proinflammatory cytokines, such as interleukin IL-1, induce the expression of matrix metalloproteinases (MMPs) in chondrocytes, contributing thus to the extracellular matrix (ECM) degradation. Members of Serpine family, including plasminogen activator inhibitors have been reported to participate in ECM regulation. The aim of this study was to assess the expression of serpin peptidase inhibitor clade E member 2 (SERPINE2), under basal conditions and in response to increasing doses of IL-1α, in human cultured chondrocytes. We also examined the effects of SERPINE2 on IL-1α-induced MMP-13 expression. For completeness, the signaling pathway involved in this process was also explored. Methods SERPINE2 mRNA and protein expression were evaluated by RT-qPCR and western blot analysis in human T/C-28a2 cell line and human primary chondrocytes. These cells were treated with human recombinant SERPINE2, alone or in combination with IL-1α. ERK 1/2, NFκB and AP-1 activation were assessed by western blot analysis. Results Human cultured chondrocytes express SERPINE2 in basal condition. This expression increased in response to IL-1α stimulation. In addition, recombinant SERPINE2 induced a clear inhibition of MMP-13 expression in IL-1α-stimulated chondrocytes. This inhibitory effect is likely regulated through a pathway involving ERK 1/2, NF-κB and AP-1. Conclusions Taken together, these data demonstrate that SERPINE2 might prevent cartilage catabolism by inhibiting the expression of MMP-13, one of the most relevant collagenases, involved in cartilage breakdown in OA. PMID:26305372

  12. Effect of oxygen tension on tissue-engineered human nasal septal chondrocytes

    PubMed Central

    Twu, Chih-Wen; Reuther, Marsha S.; Briggs, Kristen K.; Sah, Robert L.; Masuda, Koichi

    2014-01-01

    Tissue-engineered nasal septal cartilage may provide a source of autologous tissue for repair of craniofacial defects. Although advances have been made in manipulating the chondrocyte culture environment for production of neocartilage, consensus on the best oxygen tension for in vitro growth of tissue-engineered cartilage has not been reached. The objective of this study was to determine whether in vitro oxygen tension influences chondrocyte expansion and redifferentiation. Proliferation of chondrocytes from 12 patients expanded in monolayer under hypoxic (5% or 10%) or normoxic (21%) oxygen tension was compared over 14 days of culture. The highest performing oxygen level was used for further expansion of the monolayer cultures. At confluency, chondrocytes were redifferentiated by encapsulation in alginate beads and cultured for 14 days under hypoxic (5 or 10%) or normoxic (21%) oxygen tension. Biochemical and histological properties were evaluated. Chondrocyte proliferation in monolayer and redifferentiation in alginate beads were supported by all oxygen tensions tested. Chondrocytes in monolayer culture had increased proliferation at normoxic oxygen tension (p = 0.06), as well as greater accumulation of glycosaminoglycan (GAG) during chondrocyte redifferentiation (p < 0.05). Chondrocytes released from beads cultured under all three oxygen levels showed robust accumulation of GAG and type II collagen with a lower degree of type I collagen immunoreactivity. Finally, formation of chondrocyte clusters was associated with decreasing oxygen tension (p < 0.05). Expansion of human septal chondrocytes in monolayer culture was greatest at normoxic oxygen tension. Both normoxic and hypoxic culture of human septal chondrocytes embedded in alginate beads supported robust extracellular matrix deposition. However, GAG accumulation was significantly enhanced under normoxic culture conditions. Chondrocyte cluster formation was associated with hypoxic oxygen tension. PMID:25565047

  13. Structural differences in epiphyseal and physeal hypertrophic chondrocytes

    PubMed Central

    Shapiro, Frederic; Flynn, Evelyn

    2015-01-01

    We have observed that epiphyseal and physeal hypertrophic chondrocytes in BALB/c mice show considerable differences of light microscopic and ultrastructural appearance, even when the cells are at the same stage of differentiation. In addition, cell structure maintenance improved with tissue preparation controlled for osmolarity and for membrane stabilization using 0.5% ruthenium hexammine trichloride (RHT) for both light microscopy (LM) and electron microscopy (EM) or 0.5% lanthanum nitrate for LM. Physeal hypertrophic chondrocytes showed a gradual increase in size closer to the metaphysis and a change in shape as cells elongated along the long axis. The nucleus remained central, with uniformly dispersed chromatin, and the rough endoplasmic reticulum (RER) was randomly dispersed throughout cytoplasm with little to no presence against the cell membrane. Even the lowermost cells showed thin elongated or dilated cisternae of RER and intact cell membranes. Epiphyseal chondrocytes remained circular to oval with no elongation. Nucleus and RER were positioned as a complete transcellular central nucleocytoplasmic column or as an incomplete bud with RER of the column/bud always continuous with RER peripherally against the intact cell membrane. RER was densely packed with parallel cisternae with adjacent cytoplasm empty of organelles but often filled with circular deposits of moderately electron-dense material consistent with fat. Optimal technique for LM involved fixation using glutaraldehyde (GA) 1.3%, paraformaldehyde (PFA) 1% and RHT 0.5% (mOsm 606) embedded in JB-4 plastic and stained with 0.5% toluidine blue. Optimal technique for EM used fixation with GA 1.3%, PFA 1%, RHT 0.5% and cacodylate buffer 0.03 M (mOsm 511) and post-fixation including 1% osmium tetroxide. These observations lead to the possibility that the same basic cell, the hypertrophic chondrocyte, has differing functional mechanisms at different regions of the developing bone. PMID:25987982

  14. Incomplete defect filling after third generation autologous chondrocyte implantation

    PubMed Central

    Pietschmann, Matthias F.; Ficklscherer, Andreas; Gülecyüz, Mehmet F.; Hammerschmid, Florian; Müller, Peter E.

    2016-01-01

    Introduction Third generation autologous chondrocyte implantation (ACI) is a suitable method for the treatment of cartilage defects in the knee joint. However, knowledge about the development of graft thickness and the clinical relevance of incomplete defect filling in the postoperative course is low. This prospective study analyses the graft integration into the surrounding cartilage, with special consideration of the graft thickness. Material and methods A total of 71 consecutive patients with 79 cartilage defects were treated with third generation autologous chondrocyte implantation (NOVOCART 3D) in the knee. Follow-up magnetic resonance imaging (MRI) was performed at 0.25, 0.5, 1 and 2 years. Graft thickness was measured compared to the surrounding healthy cartilage. The International Knee Documentation Committee (IKDC) scoring system and the visual analogue scale (VAS) were used for clinical evaluation. Cartilage defect filling was classified as the percentage of the surrounding cartilage. Results The average graft thickness showed a significant increase between 3 and 6 months after autologous chondrocyte implantation. Incomplete defect filling occurred in 44 (55.7%) cases. Of these, 33 cases showed incomplete defect filling grade I (> 75%), 10 cases were grade II (> 50%) and one case grade III (> 25%). Incomplete defect filling grade IV (< 25%) was not observed. Incomplete defect filling occurred significantly more often in women (p = 0.021), without worse clinical results. Conclusions Graft thickness after third generation autologous chondrocyte implantation shows increasing graft thickness over the period of 2 years postoperatively. A high rate of incomplete defect filling in the surrounding cartilage was observed, without worse clinical results. PMID:27478460

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

    PubMed

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

    2016-09-01

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

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

    PubMed

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

    2011-08-01

    Tissue engineering is a promising approach for articular cartilage repair; however, it is challenging to produce adequate amounts of tissue in vitro from the limited number of cells that can be extracted from an individual. Relatively few cell expansion methods exist without the problems of de-differentiation and/or loss of potency. Recently, however, several studies have noted the benefits of three-dimensional (3D) over monolayer expansion, but the ability of 3D expanded chondrocytes to synthesize cartilaginous tissue constructs has not been demonstrated. Thus, the purpose of this study was to compare the properties of engineered cartilage constructs from expanded cells (monolayer and 3D microcarriers) to those developed from primary chondrocytes. Isolated bovine chondrocytes were grown for 3 weeks in either monolayer (T-Flasks) or 3D microcarrier (Cytodex 3) expansion culture. Expanded and isolated primary cells were then seeded in high density culture on Millicell™ filters for 4 weeks to evaluate the ability to synthesize cartilaginous tissue. While microcarrier expansion was twice as effective as monolayer expansion (microcarrier: 110-fold increase, monolayer: 52-fold increase), the expanded cells (monolayer and 3D microcarrier) were not effectively able to synthesize cartilaginous tissue in vitro. Tissues developed from primary cells were substantially thicker and accumulated significantly more extracellular matrix (proteoglycan content: 156%-292% increase; collagen content: 70%-191% increase). These results were attributed to phenotypic changes experienced during the expansion phase. Monolayer expanded chondrocytes lost their native morphology within 1 week, whereas microcarrier-expanded cells were spreading by 3 weeks of expansion. While the use of 3D microcarriers can lead to large cellular yields, preservation of chondrogenic phenotype during expansion is required in order to synthesize cartilaginous tissue. PMID:21449621

  17. Thermally reversible colloidal gels for three-dimensional chondrocyte culture

    PubMed Central

    Lapworth, James W.; Hatton, Paul V.; Goodchild, Rebecca L.; Rimmer, Stephen

    2012-01-01

    Healthy cells are required in large numbers to form a tissue-engineered construct and primary cells must therefore be increased in number in a process termed ‘expansion’. There are significant problems with existing procedures, including cell injury and an associated loss of phenotype, but three-dimensional culture has been reported to offer a solution. Reversible gels, which allow for the recovery of cells after expansion would therefore have great value in the expansion of chondrocytes for tissue engineering applications, but they have received relatively little attention to date. In this study, we examined the synthesis and use of thermoresponsive polymers that form reversible three-dimensional gels for chondrocyte cell culture. A series of polymers comprising N-isopropylacrylamide (NIPAM) and styrene was synthesized before studying their thermoresponsive solution behaviour and gelation. A poly(NIPAM-co-styrene-graft-N-vinylpyrrolidone) variant was also synthesized in order to provide increased water content. Both random- and graft-copolymers formed particulate gels above the lower critical solution temperature and, on cooling, re-dissolved to allow enzyme-free cell recovery. Chondrocytes remained viable in all of these materials for 24 days, increased in number and produced collagen type II and glycosaminoglycans. PMID:21775322

  18. Cellular response of chondrocytes to magnesium alloys for orthopedic applications

    PubMed Central

    LIAO, YI; XU, QINGLI; ZHANG, JIAN; NIU, JIALING; YUAN, GUANGYIN; JIANG, YAO; HE, YAOHUA; WANG, XINLING

    2015-01-01

    In the present study, the effects of Mg-Nd-Zn-Zr (JDBM), brushite (CaHPO4·2H2O)-coated JDBM (C-JDBM), AZ31, WE43, pure magnesium (Mg) and Ti alloy (TC4) on rabbit chondrocytes were investigated in vitro. Adhesion experiments revealed the satisfactory morphology of chondrocytes on the surface of all samples. An indirect cytotoxicity test using MTT assay revealed that C-JDBM and TC4 exhibited results similar to those of the negative control, better than those obtained with JDBM, AZ31, WE43 and pure Mg (p<0.05). There were no statistically significant differences observed between the JDBM, AZ31, WE43 and pure Mg group (p>0.05). The results of indirect cell cytotoxicity and proliferation assays, as well as those of apoptosis assay, glycosaminoglycan (GAG) quantification, assessment of collagen II (Col II) levels and RT-qPCR revealed a similar a trend as was observed with MTT assay. These findings suggested that the JDBM alloy was highly biocompatible with chondrocytes in vitro, yielding results similar to those of AZ31, WE43 and pure Mg. Furthermore, CaHPO4·2H2O coating significantly improved the biocompatibility of this alloy. PMID:25975216

  19. Doublecortin May Play a Role in Defining Chondrocyte Phenotype

    PubMed Central

    Ge, Dongxia; Zhang, Qing-Song; Zabaleta, Jovanny; Zhang, Qiuyang; Liu, Sen; Reiser, Brendan; Bunnell, Bruce A.; Braun, Stephen E.; O’Brien, Michael J.; Savoie, Felix H.; You, Zongbing

    2014-01-01

    Embryonic development of articular cartilage has not been well understood and the role of doublecortin (DCX) in determination of chondrocyte phenotype is unknown. Here, we use a DCX promoter-driven eGFP reporter mouse model to study the dynamic gene expression profiles in mouse embryonic handplates at E12.5 to E13.5 when the condensed mesenchymal cells differentiate into either endochondral chondrocytes or joint interzone cells. Illumina microarray analysis identified a variety of genes that were expressed differentially in the different regions of mouse handplate. The unique expression patterns of many genes were revealed. Cytl1 and 3110032G18RIK were highly expressed in the proximal region of E12.5 handplate and the carpal region of E13.5 handplate, whereas Olfr538, Kctd15, and Cited1 were highly expressed in the distal region of E12.5 and the metacarpal region of E13.5 handplates. There was an increasing gradient of Hrc expression in the proximal to distal direction in E13.5 handplate. Furthermore, when human DCX protein was expressed in human adipose stem cells, collagen II was decreased while aggrecan, matrilin 2, and GDF5 were increased during the 14-day pellet culture. These findings suggest that DCX may play a role in defining chondrocyte phenotype. PMID:24758934

  20. Human Articular Chondrocytes Express Multiple Gap Junction Proteins

    PubMed Central

    Mayan, Maria D.; Carpintero-Fernandez, Paula; Gago-Fuentes, Raquel; Martinez-de-Ilarduya, Oskar; Wang, Hong-Zhang; Valiunas, Virginijus; Brink, Peter; Blanco, Francisco J.

    2014-01-01

    Osteoarthritis (OA) is the most common joint disease and involves progressive degeneration of articular cartilage. The aim of this study was to investigate if chondrocytes from human articular cartilage express gap junction proteins called connexins (Cxs). We show that human chondrocytes in tissue express Cx43, Cx45, Cx32, and Cx46. We also find that primary chondrocytes from adults retain the capacity to form functional voltage-dependent gap junctions. Immunohistochemistry experiments in cartilage from OA patients revealed significantly elevated levels of Cx43 and Cx45 in the superficial zone and down through the next approximately 1000 μm of tissue. These zones corresponded with regions damaged in OA that also had high levels of proliferative cell nuclear antigen. An increased number of Cxs may help explain the increased proliferation of cells in clusters that finally lead to tissue homeostasis loss. Conversely, high levels of Cxs in OA cartilage reflect the increased number of adjacent cells in clusters that are able to interact directly by gap junctions as compared with hemichannels on single cells in normal cartilage. Our data provide strong evidence that OA patients have a loss of the usual ordered distribution of Cxs in the damaged zones and that the reductions in Cx43 levels are accompanied by the loss of correct Cx localization in the nondamaged areas. PMID:23416160

  1. 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

  2. Optimal 3-D culture of primary articular chondrocytes for use in the Rotating Wall Vessel Bioreactor

    PubMed Central

    Mellor, Liliana F.; Baker, Travis L.; Brown, Raquel J.; Catlin, Lindsey W.; Oxford, Julia Thom

    2014-01-01

    INTRODUCTION Reliable culturing methods for primary articular chondrocytes are essential to study the effects of loading and unloading on joint tissue at the cellular level. Due to the limited proliferation capacity of primary chondrocytes and their tendency to dedifferentiate in conventional culture conditions, long-term culturing conditions of primary chondrocytes can be challenging. The goal of this study was to develop a suspension culturing technique that not only would retain the cellular morphology but also maintain gene expression characteristics of primary articular chondrocytes. METHODS Three-dimensional culturing methods were compared and optimized for primary articular chondrocytes in the rotating wall vessel bioreactor, which changes the mechanical culture conditions to provide a form of suspension culture optimized for low shear and turbulence. We performed gene expression analysis and morphological characterization of cells cultured in alginate beads, Cytopore-2 microcarriers, primary monolayer culture, and passaged monolayer cultures using reverse transcription-PCR and laser scanning confocal microscopy. RESULTS Primary chondrocytes grown on Cytopore-2 microcarriers maintained the phenotypical morphology and gene expression pattern observed in primary bovine articular chondrocytes, and retained these characteristics for up to 9 days. DISCUSSION Our results provide a novel and alternative culturing technique for primary chondrocytes suitable for studies that require suspension such as those using the rotating wall vessel bioreactor. In addition, we provide an alternative culturing technique for primary chondrocytes that can impact future mechanistic studies of osteoarthritis progression, treatments for cartilage damage and repair, and cartilage tissue engineering. PMID:25199120

  3. 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

  4. Arginine methyltransferase CARM1/PRMT4 regulates endochondral ossification

    PubMed Central

    Ito, Tatsuo; Yadav, Neelu; Lee, Jaeho; Furumatsu, Takayuki; Yamashita, Satoshi; Yoshida, Kenji; Taniguchi, Noboru; Hashimoto, Megumi; Tsuchiya, Megumi; Ozaki, Toshifumi; Lotz, Martin; Bedford, Mark T; Asahara, Hiroshi

    2009-01-01

    Background Chondrogenesis and subsequent endochondral ossification are processes tightly regulated by the transcription factor Sox9 (SRY-related high mobility group-Box gene 9), but molecular mechanisms underlying this activity remain unclear. Here we report that coactivator-associated arginine methyltransferase 1 (CARM1) regulates chondrocyte proliferation via arginine methylation of Sox9. Results CARM1-null mice display delayed endochondral ossification and decreased chondrocyte proliferation. Conversely, cartilage development of CARM1 transgenic mice was accelerated. CARM1 specifically methylates Sox9 at its HMG domain in vivo and in vitro. Arg-methylation of Sox9 by CARM1 disrupts interaction of Sox9 with beta-catenin, regulating Cyclin D1 expression and cell cycle progression of chondrocytes. Conclusion These results establish a role for CARM1 as an important regulator of chondrocyte proliferation during embryogenesis. PMID:19725955

  5. Octacalcium phosphate crystals directly stimulate expression of inducible nitric oxide synthase through p38 and JNK mitogen-activated protein kinases in articular chondrocytes

    PubMed Central

    Ea, Hang-Korng; Uzan, Benjamin; Rey, Christian; Lioté, Frédéric

    2005-01-01

    Basic calcium phosphate (BCP) crystals, including hydroxyapatite, octacalcium phosphate (OCP) and carbonate-apatite, have been associated with severe osteoarthritis and several degenerative arthropathies. Most studies have considered the chondrocyte to be a bystander in the pathogenesis of calcium crystal deposition disease, assuming that synovial cell cytokines were the only triggers of chondrocyte activation. In the present study we identified direct activation of articular chondrocytes by OCP crystals, which are the BCP crystals with the greatest potential for inducing inflammation. OCP crystals induced nitric oxide (NO) production and inducible nitric oxide synthase (NOS) mRNA expression by isolated articular chondrocytes and cartilage fragments, in a dose-dependent manner and with variations over time. OCP crystals also induced IL-1β mRNA expression. Using pharmacological and cytokine inhibitors, we observed that OCP crystals induced NO production and inducible NOS mRNA activation were regulated at both the transcriptional and the translational levels; were independent from IL-1β gene activation; and involved p38 and c-Jun amino-terminal kinase (JNK) mitogen-activated protein kinase (MAPK) pathways, as further confirmed by OCP crystal-induced p38 and JNK MAPK phosphorylation. Taken together, our data suggest that the transcriptional inducible NOS response to OCP crystals involved both the p38 and the JNK MAPK pathways, probably under the control of activator protein-1. NO, a major mediator of cartilage degradation, can be directly produced by BCP crystals in chondrocytes. Together with synovial activation, this direct mechanism may be important in the pathogenesis of destructive arthropathies triggered by microcrystals. PMID:16207333

  6. The intracellular Ca(2+)-pump inhibitors thapsigargin and cyclopiazonic acid induce stress proteins in mammalian chondrocytes.

    PubMed

    Cheng, T C; Benton, H P

    1994-07-15

    Primary cultures of mammalian articular chondrocytes respond to treatment with the intracellular Ca(2+)-pump inhibitors thapsigargin (TG) and cyclopiazonic acid by specific changes in protein synthesis consistent with a stress response. Two-dimensional gel electrophoresis of newly synthesized proteins confirmed that the response was consistent with the induction of glucose-regulated proteins. The effects of low-dose TG (10 nM), measured by changes in [35S]methionine labelling of newly synthesized proteins, can first be observed by 10 h and are maximal by 24 h. The pattern of changes induced by TG is shared with cyclopiazonic acid, but effects of both perturbants differ significantly from changes induced by heat shock. Upon removal of TG, normal protein synthesis is restored by 48 h. Immunoblots showed increased concentrations of the stress proteins HSP90, HSP72/73 and HSP60 in chondrocytes treated with TG, but induction of newly synthesized heat-shock proteins by TG was not apparent on [35S]methionine-labelled gels. The alterations in protein synthesis induced by Ca(2+)-pump inhibitors were unaffected by BAPTA-AM loading, which clamped cytosolic Ca2+ at resting levels. We conclude that inhibition of intracellular Ca(2+)-pump activity can elicit a stress response, which has important implications for the interpretation of chronic use of Ca(2+)-pump inhibitors. In particular, the activation of the cellular shock response should be considered in interpreting the regulation of protein synthesis and cell survival by Ca(2+)-pump inhibitors such as TG. PMID:8043004

  7. Matrix molecule influence on chondrocyte phenotype and proteoglycan 4 expression by alginate-embedded zonal chondrocytes and mesenchymal stem cells.

    PubMed

    Coates, Emily E; Riggin, Corinne N; Fisher, John P

    2012-12-01

    Articular cartilage resists load and provides frictionless movement at joint surfaces. The tissue is organized into the superficial, middle, deep, and calcified zones throughout its depth, each which serve distinct functions. Proteoglycan 4 (PRG4), found in the superficial zone, is a critical component of the joint's lubricating mechanisms. Maintenance of both the chondrocyte and zonal chondrocyte phenotype remain challenges for in vitro culture and tissue engineering. Here we investigate the expression of PRG4 mRNA and protein by primary bovine superficial zone chondrocytes, middle/deep zone chondrocytes, and mesenchymal stem cells encapsulated in alginate hydrogels with hyaluronic acid (HA) and chondroitin sulfate (CS) additives. Chondrogenic phenotype and differentiation markers are evaluated by mRNA expression, histochemical, and immunohistochemical staining. Results show middle/deep cells express no measurable PRG4 mRNA by day 7. In contrast, superficial zone cells express elevated PRG4 mRNA throughout culture time. This expression can be significantly enhanced up to 15-fold by addition of both HA and CS to scaffolds. Conversely, PRG4 mRNA expression is downregulated (up to 5-fold) by CS and HA in differentiating MSCs, possibly due to build up of entrapped protein. HA and CS demonstrate favorable effects on chondrogenesis by upregulating transcription factor Sox9 mRNA (up to 4.6-fold) and downregulating type I collagen mRNA (up to 18-fold). Results highlight the important relationship between matrix components and expression of critical lubricating proteins in an engineered cartilage scaffold. PMID:22674584

  8. Mitochondrial respiratory chain dysfunction modulates metalloproteases -1, -3 and -13 in human normal chondrocytes in culture

    PubMed Central

    2013-01-01

    Background Mitochondrion has an important role in the osteoarthritis (OA) pathology. We have previously demonstrated that the alteration of the mitochondrial respiratory chain (MRC) contributes to the inflammatory response of the chondrocyte. However its implication in the process of cartilage destruction is not well understood yet. In this study we have investigated the relationship between the MRC dysfunction and the regulation of metalloproteases (MMPs) in human normal chondrocytes in culture. Methods Human normal chondrocytes were isolated from human knees obtained form autopsies of donors without previous history of rheumatic disease. Rotenone, 3-Nitropropionic acid (NPA), Antimycin A (AA), Sodium azide and Oligomycin were used to inhibit the activity of the mitochondrial complexes I, II, III, IV and V respectively. The mRNA expression of MMPs -1, -3 and -13 was studied by real time PCR. The intracellular presence of MMP proteins was evaluated by western blot. The liberation of these proteins to the extracellular media was evaluated by ELISA. The presence of proteoglycans in tissue was performed with tolouidin blue and safranin/fast green. Immunohistochemistry was used for evaluating MMPs on tissue. Results Firstly, cells were treated with the inhibitors of the MRC for 24 hours and mRNA expression was evaluated. An up regulation of MMP-1 and -3 mRNA levels was observed after the treatment with Oligomycin 5 and 100 μg/ml (inhibitor of the complex V) for 24 hours. MMP-13 mRNA expression was reduced after the incubation with AA 20 and 60 μg/ml (inhibitor of complex III) and Oligomycin. Results were validated at protein level observing an increase in the intracellular levels of MMP-1 and -3 after Oligomycin 25 μg/ml stimulation [(15.20±8.46 and 4.59±1.83 vs. basal=1, respectively (n=4; *P<0.05)]. However, AA and Oligomycin reduced the intracellular levels of the MMP-13 protein (0.70±0.16 and 0.3±0.24, respectively vs. basal=1). In order to know whether the

  9. Transcription Factor Erg Variants and Functional Diversification of Chondrocytes during Limb Long Bone Development

    PubMed Central

    Iwamoto, Masahiro; Higuchi, Yoshinobu; Koyama, Eiki; Enomoto-Iwamoto, Motomi; Kurisu, Kojiro; Yeh, Helena; Abrams, William R.; Rosenbloom, Joel; Pacifici, Maurizio

    2000-01-01

    During limb development, chondrocytes located at the epiphyseal tip of long bone models give rise to articular tissue, whereas the more numerous chondrocytes in the shaft undergo maturation, hypertrophy, and mineralization and are replaced by bone cells. It is not understood how chondrocytes follow these alternative pathways to distinct fates and functions. In this study we describe the cloning of C-1-1, a novel variant of the ets transcription factor ch-ERG. C-1-1 lacks a short 27–amino acid segment located ∼80 amino acids upstream of the ets DNA binding domain. We found that in chick embryo long bone anlagen, C-1-1 expression characterizes developing articular chondrocytes, whereas ch-ERG expression is particularly prominent in prehypertrophic chondrocytes in the growth plate. To analyze the function of C-1-1 and ch-ERG, viral vectors were used to constitutively express each factor in developing chick leg buds and cultured chondrocytes. We found that virally driven expression of C-1-1 maintained chondrocytes in a stable and immature phenotype, blocked their maturation into hypertrophic cells, and prevented the replacement of cartilage with bone. It also induced synthesis of tenascin-C, an extracellular matrix protein that is a unique product of developing articular chondrocytes. In contrast, virally driven expression of ch-ERG significantly stimulated chondrocyte maturation in culture, as indicated by increases in alkaline phosphatase activity and deposition of a mineralized matrix; however, it had modest effects in vivo. The data show that C-1-1 and ch-ERG have diverse biological properties and distinct expression patterns during skeletogenesis, and are part of molecular mechanisms by which limb chondrocytes follow alternative developmental pathways. C-1-1 is the first transcription factor identified to date that appears to be instrumental in the genesis and function of epiphyseal articular chondrocytes. PMID:10893254

  10. 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.

  11. Endochondral Growth Defect and Deployment of Transient Chondrocyte Behaviors Underlie Osteoarthritis Onset in a Natural Murine Model

    PubMed Central

    Staines, K. A.; Madi, K.; Mirczuk, S. M.; Parker, S.; Burleigh, A.; Poulet, B.; Hopkinson, M.; Bodey, A. J.; Fowkes, R. C.; Farquharson, C.; Lee, P. D.

    2016-01-01

    Objective To explore whether aberrant transient chondrocyte behaviors occur in the joints of STR/Ort mice (which spontaneously develop osteoarthritis [OA]) and whether they are attributable to an endochondral growth defect. Methods Knee joints from STR/Ort mice with advanced OA and age‐matched CBA (control) mice were examined by Affymetrix microarray profiling, multiplex polymerase chain reaction (PCR) analysis, and immunohistochemical labeling of endochondral markers, including sclerostin and MEPE. The endochondral phenotype of STR/Ort mice was analyzed by histologic examination, micro–computed tomography, and ex vivo organ culture. A novel protocol for quantifying bony bridges across the murine epiphysis (growth plate fusion) using synchrotron x‐ray computed microtomography was developed and applied. Results Meta‐analysis of transcription profiles showed significant elevation in functions linked with endochondral ossification in STR/Ort mice (compared to CBA mice; P < 0.05). Consistent with this, immunolabeling revealed increased matrix metalloproteinase 13 (MMP‐13) and type X collagen expression in STR/Ort mouse joints, and multiplex quantitative reverse transcriptase–PCR showed differential expression of known mineralization regulators, suggesting an inherent chondrocyte defect. Support for the notion of an endochondral defect included accelerated growth, increased zone of growth plate proliferative chondrocytes (P < 0.05), and widespread type X collagen/MMP‐13 labeling beyond the expected hypertrophic zone distribution. OA development involved concomitant focal suppression of sclerostin/MEPE in STR/Ort mice. Our novel synchrotron radiation microtomography method showed increased numbers (P < 0.001) and mean areal growth plate bridge densities (P < 0.01) in young and aged STR/Ort mice compared to age‐matched CBA mice. Conclusion Taken together, our data support the notion of an inherent endochondral defect that is linked to growth dynamics and

  12. The dependence of autologous chondrocyte transplantation on varying cellular passage, yield and culture duration.

    PubMed

    Salzmann, Gian M; Sauerschnig, Martin; Berninger, Markus T; Kaltenhauser, Theresa; Schönfelder, Martin; Vogt, Stephan; Wexel, Gabriele; Tischer, Thomas; Sudkamp, Norbert; Niemeyer, Philipp; Imhoff, Andreas B; Schöttle, Philip B

    2011-09-01

    Matrix-assisted chondrocyte transplantation (m-ACI) still lacks any standardization in its execution in terms of cell passage (P), cell yield (C) and in vitro membrane-holding time (T). It was the goal of this study to analyze the effect of shifting cell culture parameters (P, C, T) on the in vitro as well as in vivo effort of a regulated animal m-ACI. Autologous rabbit knee articular chondrocytes were seeded within bilayer collagen I/III 3-D matrices in variation of P, C and T. Each time, 2 PCT-identical by 2 PCT-identical cell-matrix-constructs (CMC)/animal were created. Simultaneously 2 (PCT-distinct) were re-implanted (CMC-e) autologous into artificial trochlear pristine chondral defects in vivo to remain for 12 weeks while the remaining 2 were harvested (CMC-i) for immediate in vitro analysis at the time of transplantation of their identical twins. mRNA of both, CMC-e regenerates and CMC-i membranes, was analyzed for Collagen-1,-2,-10, COMP, Aggrecan, Sox9 expression by use of a mixed linear model, multiple regression analysis. Generally, CMC-i values were higher than CMC-e values for differentiation targets; the opposite was true for dedifferentiation targets. Regarding individual gene expression, in vivo regenerate cell-matrix properties were significantly dependent on initial cell-matrix in vitro values as a sign of linearity. The parameter membrane-holding time (T) had strongest effects on the resulting mRNA expression with slightly less impact of the parameter passage (P), whereas cell yield (C) had clearly less effects. Noting differences between in vitro and in vivo data, in general, optimal expression patterns concerning chondrogenic differentiation were achieved by few passages, medium cellular yield, short membrane-holding time. Clinical m-ACI may benefit from optimal orchestration of the cell culture parameters passage, yield and time. PMID:21592563

  13. 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.

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

    PubMed

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

    2004-05-01

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

  15. Redifferentiation of dedifferentiated chondrocytes in a novel three-dimensional microcavitary hydrogel.

    PubMed

    Zeng, Lei; Chen, Xiaofeng; Zhang, Qing; Yu, Feng; Li, Yuli; Yao, Yongchang

    2015-05-01

    Although chondrocytes exist in native cartilage all over the body, it is still a challenge to use them as therapeutic cells for cartilage tissue engineering (TE) because of their easy dedifferentiation in in vitro culture. An improved culture system to maintain the characteristics of chondrocytes or recover their chondrocytic phenotype should be developed. In this study, we have set up an innovative microcavitary alginate hydrogel in an easy way. We compared this culture system with the conventional hydrogel and found that the microcavitary hydrogel exhibited outstanding superiorities in helping the dedifferentiated chondrocytes recover the capability for synthesizing cartilaginous extracellular matrix. In addition, we explored the correlation between chondrocyte redifferentiation in microcavitary hydrogels and changes in p38 and Erk1/2 activity. Our findings indicated that this microcavitary hydrogel would be a promising culture system to provide sufficient competent cells for cartilage regeneration and TE.

  16. Linking cell shape, elasticity and fate: in vitro re-differentiation of chondrocytes

    NASA Astrophysics Data System (ADS)

    Yuan, Xiaofei; Chim, Yahua; Yin, Huabing

    2014-02-01

    Autologous chondrocyte transplantation (ACT) has become a promising method for repairing large articular defects. However, dedifferentiation of chondrocytes during cell expansion remains a major limitation for ACT procedures. In this study, we explore the potential of confining cell shape for re-differentiation of dedifferentiated bovine chondrocytes. A novel culture system, combining 2D micropatterning with 3D matrix formation, was developed to control and maintain individual chondrocyte's shape. Both collagen II synthesis and the mechanical properties of cells were monitored during re-differentiation. We show that a spherical morphology without cell spreading plays a limited role in induction of re-differentiation. Instead, isolated, dedifferentiated chondrocytes partially regain chondrogenic properties if they have an appropriate cell shape and limited spreading.

  17. 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

  18. Cyclic Equibiaxial Tensile Strain Alters Gene Expression of Chondrocytes via Histone Deacetylase 4 Shuttling

    PubMed Central

    Chen, Chongwei; Wei, Xiaochun; Lv, Zhi; Sun, Xiaojuan; Wang, Shaowei; Zhang, Yang; Jiao, Qiang; Wang, Xiaohu; Li, Yongping; Wei, Lei

    2016-01-01

    Objectives This paper aims to investigate whether equibiaxial tensile strain alters chondrocyte gene expression via controlling subcellular localization of histone deacetylase 4 (HDAC4). Materials and Methods Murine chondrocytes transfected with GFP-HDAC4 were subjected to 3 h cyclic equibiaxial tensile strain (CTS, 6% strain at 0.25 Hz) by a Flexcell® FX-5000™ Tension System. Fluorescence microscope and western blot were used to observe subcellular location of HDAC4. The gene expression was analyzed by real-time RT-PCR. The concentration of Glycosaminoglycans in culture medium was quantified by bimethylmethylene blue dye; Collagen II protein was evaluated by western blot. Cells phenotype was identified by immunohistochemistry. Cell viability was evaluated by live-dead cell detect kit. Okadaic acid, an inhibitor of HDAC4 nuclear relocation, was used to further validate whether HDAC4 nuclear relocation plays a role in gene expression in response to tension stimulation. Results 87.5% of HDAC4 was located in the cytoplasm in chondrocytes under no loading condition, but it was relocated to the nucleus after CTS. RT-PCR analysis showed that levels of mRNA for aggrecan, collagen II, LK1 and SOX9 were all increased in chondrocytes subjected to CTS as compared to no loading control chondrocytes; in contrast, the levels of type X collagen, MMP-13, IHH and Runx2 gene expression were decreased in the chondrocytes subjected to CTS as compared to control chondrocytes. Meanwhile, CTS contributed to elevation of glycosaminoglycans and collagen II protein, but did not change collagen I production. When Okadaic acid blocked HDAC4 relocation from the cytoplasm to nucleus, the changes of the chondrocytes induced by CTS were abrogated. There was no chondrocyte dead detected in this study in response to CTS. Conclusions CTS is able to induce HDAC4 relocation from cytoplasm to nucleus. Thus, CTS alters chondrocytes gene expression in association with the relocation of HDAC4 induced

  19. E74-like Factor 3 (ELF3) Impacts on Matrix Metalloproteinase 13 (MMP13) Transcriptional Control in Articular Chondrocytes under Proinflammatory Stress*

    PubMed Central

    Otero, Miguel; Plumb, Darren A.; Tsuchimochi, Kaneyuki; Dragomir, Cecilia L.; Hashimoto, Ko; Peng, Haibing; Olivotto, Eleonora; Bevilacqua, Michael; Tan, Lujian; Yang, Zhiyong; Zhan, Yumei; Oettgen, Peter; Li, Yefu; Marcu, Kenneth B.; Goldring, Mary B.

    2012-01-01

    Matrix metalloproteinase (MMP)-13 has a pivotal, rate-limiting function in cartilage remodeling and degradation due to its specificity for cleaving type II collagen. The proximal MMP13 promoter contains evolutionarily conserved E26 transformation-specific sequence binding sites that are closely flanked by AP-1 and Runx2 binding motifs, and interplay among these and other factors has been implicated in regulation by stress and inflammatory signals. Here we report that ELF3 directly controls MMP13 promoter activity by targeting an E26 transformation-specific sequence binding site at position −78 bp and by cooperating with AP-1. In addition, ELF3 binding to the proximal MMP13 promoter is enhanced by IL-1β stimulation in chondrocytes, and the IL-1β-induced MMP13 expression is inhibited in primary human chondrocytes by siRNA-ELF3 knockdown and in chondrocytes from Elf3−/− mice. Further, we found that MEK/ERK signaling enhances ELF3-driven MMP13 transactivation and is required for IL-1β-induced ELF3 binding to the MMP13 promoter, as assessed by chromatin immunoprecipitation. Finally, we show that enhanced levels of ELF3 co-localize with MMP13 protein and activity in human osteoarthritic cartilage. These studies define a novel role for ELF3 as a procatabolic factor that may contribute to cartilage remodeling and degradation by regulating MMP13 gene transcription. PMID:22158614

  20. Effects of weak, low-frequency pulsed electromagnetic fields (BEMER type) on gene expression of human mesenchymal stem cells and chondrocytes: an in vitro study.

    PubMed

    Walther, Markus; Mayer, Florian; Kafka, Wolf; Schütze, Norbert

    2007-01-01

    In vitro effects of electromagnetic fields appear to be related to the type of electromagnetic field applied. Previously, we showed that human osteoblasts display effects of BEMER type electromagnetic field (BTEMF) on gene regulation. Here, we analyze effects of BTEMF on gene expression in human mesenchymal stem cells and chondrocytes. Primary mesenchymal stem cells from bone marrow and the chondrocyte cell line C28I2 were stimulated 5 times at 12-h intervals for 8 min each with BTEMF. RNA from treated and control cells was analyzed for gene expression using the affymetrix chip HG-U133A. A limited number of regulated gene products from both cell types mainly affect cell metabolism and cell matrix structure. There was no increased expression of cancer-related genes. RT-PCR analysis of selected transcripts partly confirmed array data. Results indicate that BTEMF in human mesenchymal stem cells and chondrocytes provide the first indications to understanding therapeutic effects achieved with BTEMF stimulation. PMID:17886005

  1. Beta-arrestin-2 negatively modulates inflammation response in mouse chondrocytes induced by 4-mer hyaluronan oligosaccharide.

    PubMed

    Campo, Giuseppe M; Avenoso, Angela; D'Ascola, Angela; Scuruchi, Michele; Calatroni, Alberto; Campo, Salvatore

    2015-01-01

    Beta-arrestin-2 is an adaptor protein that terminates G protein activation and seems to be involved in the modulation of the inflammatory response. Small hyaluronan (HA) fragments, such as 4-mer HA oligosaccharides, are known to interact with the toll-like receptor-4 (TLR-4) with consequent activation of the nuclear factor kappaB (NF-kB) that in turn stimulates the inflammation response. NF-kB activation is mediated by different pathways, in particular by the transforming growth factor-activated kinase-1 (TAK-1). Conversely, increased levels of protein kinase A (PKA), induced by cyclic adenosine monophosphate (cAMP), seem to inhibit NF-kB activation. We studied the involvement and role of beta-arrestin-2 in mouse chondrocytes stimulated with 4-mer HA fragments. The exposure of chondrocytes to 4-mer HA produced a significant up-regulation in TLR-4, cAMP, beta-arrestin-2, TAK-1, protein 38 mitogen-activated protein kinase (p38MAPK), and PKA, both in terms of mRNA expression and of the related protein levels. NF-kB was significantly activated, thereby producing the transcription of pro-inflammatory mediators, including tumor necrosis factor alpha, interleukin-6, and interleukin-17. The treatment of 4-mer HA-stimulated chondrocytes with antibodies against beta-arrestin-2 and/or a specific PKA inhibitor, significantly increased the inflammatory response, while the treatment with a specific p38MAPK inhibitor significantly reduced the inflammatory response. Interestingly, the anti-inflammatory action exerted by beta-arrestin-2 appeared to be mediated in part through the direct inhibition of p38MAPK, preventing NF-kB activation, and in part through cAMP and PKA activation primed by G protein signaling, which exerted an inhibitory effect on NF-kB. Taken together, these results could be useful for future anti-inflammatory strategies. PMID:25318610

  2. Beta-arrestin-2 negatively modulates inflammation response in mouse chondrocytes induced by 4-mer hyaluronan oligosaccharide.

    PubMed

    Campo, Giuseppe M; Avenoso, Angela; D'Ascola, Angela; Scuruchi, Michele; Calatroni, Alberto; Campo, Salvatore

    2015-01-01

    Beta-arrestin-2 is an adaptor protein that terminates G protein activation and seems to be involved in the modulation of the inflammatory response. Small hyaluronan (HA) fragments, such as 4-mer HA oligosaccharides, are known to interact with the toll-like receptor-4 (TLR-4) with consequent activation of the nuclear factor kappaB (NF-kB) that in turn stimulates the inflammation response. NF-kB activation is mediated by different pathways, in particular by the transforming growth factor-activated kinase-1 (TAK-1). Conversely, increased levels of protein kinase A (PKA), induced by cyclic adenosine monophosphate (cAMP), seem to inhibit NF-kB activation. We studied the involvement and role of beta-arrestin-2 in mouse chondrocytes stimulated with 4-mer HA fragments. The exposure of chondrocytes to 4-mer HA produced a significant up-regulation in TLR-4, cAMP, beta-arrestin-2, TAK-1, protein 38 mitogen-activated protein kinase (p38MAPK), and PKA, both in terms of mRNA expression and of the related protein levels. NF-kB was significantly activated, thereby producing the transcription of pro-inflammatory mediators, including tumor necrosis factor alpha, interleukin-6, and interleukin-17. The treatment of 4-mer HA-stimulated chondrocytes with antibodies against beta-arrestin-2 and/or a specific PKA inhibitor, significantly increased the inflammatory response, while the treatment with a specific p38MAPK inhibitor significantly reduced the inflammatory response. Interestingly, the anti-inflammatory action exerted by beta-arrestin-2 appeared to be mediated in part through the direct inhibition of p38MAPK, preventing NF-kB activation, and in part through cAMP and PKA activation primed by G protein signaling, which exerted an inhibitory effect on NF-kB. Taken together, these results could be useful for future anti-inflammatory strategies.

  3. Chondrocyte-intrinsic Smad3 represses Runx2-inducible MMP-13 expression to maintain articular cartilage and prevent osteoarthritis

    PubMed Central

    Chen, Carol G.; Thuillier, Daniel; Chin, Emily N.; Alliston, Tamara

    2012-01-01

    Objective To identify mechanisms by which Smad3 maintains articular cartilage and prevents osteoarthritis. Methods A combination of in vivo and in vitro approaches was used to test the hypothesis that Smad3 represses Runx2-inducible gene expression to prevent articular cartilage degeneration. Col2-Cre;Smad3fl/fl mice allowed study of the chondrocyte-intrinsic role of Smad3, independently of its role in the perichondrium or other tissues. Primary Smad3fl/fl articular chondrocytes and ATDC5 chondroprogenitors were employed to evaluate Smad3 and Runx2 regulation of matrix metalloproteinase-13 (MMP-13) mRNA and protein expression. Results Chondrocyte-specific reduction of Smad3 causes progressive articular cartilage degeneration due to imbalanced cartilage matrix synthesis and degradation. In addition to reduced collagen II mRNA expression, Col2-Cre;Smad3fl/fl articular cartilage is severely deficient in collagen II and aggrecan protein, due to excessive MMP-13-mediated proteolysis of these key cartilage matrix constituents. Normally, TGF-β signals through Smad3 to confer a rapid and dynamic repression of Runx2-inducible MMP-13 expression. However, in the absence of Smad3, TGF-β signals through p38 and Runx2 to induce MMP-13 expression. Conclusion This work elucidates a mechanism by which Smad3 mutations in humans and mice cause cartilage degeneration and osteoarthritis. Specifically, Smad3 maintains the balance between cartilage matrix synthesis and degradation by inducing collagen II expression and repressing Runx2-inducible MMP-13 expression. Selective activation of TGF-β signaling through Smad3, rather than p38, may help to restore the balance between matrix synthesis and proteolysis that is lost in osteoarthritis. PMID:22674505

  4. 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. PMID:25054343

  5. Hypoxia promotes redifferentiation and suppresses markers of hypertrophy and degeneration in both healthy and osteoarthritic chondrocytes

    PubMed Central

    2013-01-01

    Introduction Hypoxia is considered to be a positive influence on the healthy chondrocyte phenotype and cartilage matrix formation. However, hypoxia-inducible factors (HIFs) have been implicated in the pathogenesis of osteoarthritis (OA). Thus, we assessed whether healthy and OA chondrocytes have distinct responses to oxygen, particularly with regard to hypertrophy and degradation during redifferentiation. Methods Monolayer-expanded healthy and OA chondrocytes were redifferentiated for 14 days in pellet cultures under standard (20% oxygen) or hypoxic (2% oxygen) conditions. Cartilage matrix gene expression, matrix quality and quantity, degradative enzyme expression and HIF expression were measured. Results In hypoxia, both healthy and OA chondrocytes had higher human collagen type II, α1 gene (COL2A1), and aggrecan (ACAN) expression and sulfated glycosaminoglycan (sGAG) accumulation, concomitant with lower human collagen type X, α1 gene (COL10A1), and human collagen type I, α1 gene (COL1A1), expression and collagen I extracellular accumulation. OA chondrocytes had significantly lower sGAGs/DNA than healthy chondrocytes, but only in high oxygen conditions. Hypoxia also caused significantly greater sGAG retention and hyaluronic acid synthase 2 (HAS2) expression by OA chondrocytes. Both healthy and OA chondrocytes had significantly lower expression of matrix metalloproteinases (MMPs) MMP1, MMP2, MMP3 and MMP13 in hypoxia and less active MMP2 enzyme, consistent with lower MMP14 expression. However, aggrecanase (ADAMTS4 and ADAMTS5) expression was significantly lowered by hypoxia only in healthy cells, and COL10A1 and MMP13 remained significantly higher in OA chondrocytes than in healthy chondrocytes in hypoxic conditions. HIF-1α and HIF-2α had similar expression profiles in healthy and OA cells, increasing to maximal levels early in hypoxia and decreasing over time. Conclusions Hypoxic culture of human chondrocytes has long been acknowledged to result in increased

  6. MR imaging of osteochondral grafts and autologous chondrocyte implantation

    PubMed Central

    Millington, S. A.; Szomolanyi, P.; Marlovits, S.

    2006-01-01

    Surgical articular cartilage repair therapies for cartilage defects such as osteochondral autograft transfer, autologous chondrocyte implantation (ACI) or matrix associated autologous chondrocyte transplantation (MACT) are becoming more common. MRI has become the method of choice for non-invasive follow-up of patients after cartilage repair surgery. It should be performed with cartilage sensitive sequences, including fat-suppressed proton density-weighted T2 fast spin-echo (PD/T2-FSE) and three-dimensional gradient-echo (3D GRE) sequences, which provide good signal-to-noise and contrast-to-noise ratios. A thorough magnetic resonance (MR)-based assessment of cartilage repair tissue includes evaluations of defect filling, the surface and structure of repair tissue, the signal intensity of repair tissue and the subchondral bone status. Furthermore, in osteochondral autografts surface congruity, osseous incorporation and the donor site should be assessed. High spatial resolution is mandatory and can be achieved either by using a surface coil with a 1.5-T scanner or with a knee coil at 3 T; it is particularly important for assessing graft morphology and integration. Moreover, MR imaging facilitates assessment of complications including periosteal hypertrophy, delamination, adhesions, surface incongruence and reactive changes such as effusions and synovitis. Ongoing developments include isotropic 3D sequences, for improved morphological analysis, and in vivo biochemical imaging such as dGEMRIC, T2 mapping and diffusion-weighted imaging, which make functional analysis of cartilage possible. PMID:16802126

  7. AUTOLOGOUS CHONDROCYTE TRANSPLANTATION-SERIES OF 3 CASES

    PubMed Central

    Gobbi, Riccardo Gomes; Demange, Marco Kawamura; Barreto, Ronald Bispo; Pécora, José Ricardo; Rezende, Múrcia Uchõa de; Filho, Tarcisio E.P Barros; Lombello, Christiane Bertachini

    2015-01-01

    Hyaline cartilage covers joint surfaces and plays an important role in reducing friction and mechanical loading on synovial joints such as the knee. This tissue is not supplied with blood vessels, nerves or lymphatic circulation, which may be one of the reasons why joint cartilage has such poor capacity for healing. Chondral lesions that reach the subchondral bone (osteochondral lesions) do not heal and may progress to arthrosis with the passage of time. In young patients, treatment of chondral defects of the knee is still a challenge, especially in lesions larger than 4 cm. One option for treating these patients is autologous chondrocyte transplantation/implantation. Because this treatment does not violate the subchondral bone and repairs the defect with tissue similar to hyaline cartilage, it has the theoretical advantage of being more biological, and mechanically superior, compared with other techniques. In this paper, we describe our experience with autologous chondrocyte transplantation/implantation at the Institute of Orthopedics and Traumatology, Hospital das Clínicas, University of Sâo Paulo, through a report on three cases. PMID:27022579

  8. Dynamic loading stimulates chondrocyte biosynthesis when encapsulated in charged hydrogels prepared from poly(ethylene glycol) and chondroitin sulfate.

    PubMed

    Villanueva, Idalis; Gladem, Sara K; Kessler, Jeff; Bryant, Stephanie J

    2010-01-01

    potentials and/or dynamic changes in osmolarity may be important regulators of chondrocytes while cell deformation and fluid flow appear to have less of an effect. PMID:19720146

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

    PubMed Central

    TANG, JILEI; CUI, WEIDING; SONG, FANGLONG; ZHAI, CHENJUN; HU, HANSHENG; ZUO, QIANG; FAN, WEIMIN

    2015-01-01

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

  10. 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.

  11. 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. PMID:25755694

  12. In vitro effect of a synthesized sulfonamido-based gallate on articular chondrocyte metabolism.

    PubMed

    Lin, Xiao; Zheng, Li; Liu, Qin; Liu, Buming; Jiang, Bingli; Peng, Xiaoyu; Lin, Cuiwu

    2014-06-01

    Autologous chondrocyte implantation (ACI) is a promising strategy for cartilage repair and reconstitution. However, limited cell numbers and the dedifferentiation of chondrocytes present major difficulties to the success of ACI therapy. Therefore, it is important to find effective pro-chondrogenic agents that restore these defects to ensure a successful therapy. In this study, we synthesized a sulfonamido-based gallate, namely N-[4-(4,6-dimethyl-pyrimidin-2-ylsulfamoyl)-phenyl]-3,4,5-trihydroxy-benzamide (EJTC), and investigated its effects on rabbit articular chondrocytes through an examination of its specific effects on cell proliferation, morphology, viability, GAG synthesis, and cartilage-specific gene expression. The results show that EJTC can effectively promote chondrocyte growth and enhance the secretion and synthesis of cartilage ECM by upregulating the expression levels of the aggrecan, collagen II, and Sox9 genes. The expression of the collagen I gene was effectively downregulated, which indicates that EJTC inhibits chondrocytes dedifferentiation. Chondrocyte hypertrophy, which may lead to chondrocyte ossification, was also undetectable in the EJTC-treated groups. The recommended dose of EJTC ranges from 3.125 μg/mL to 7.8125 μg/mL, and the most profound response was observed with 7.8125 μg/mL. This study may provide a basis for the development of a novel agent for the treatment of articular cartilage defects.

  13. Characterization of Chondrocyte Scaffold Carriers for Cell-based Gene Therapy in Articular Cartilage Repair

    PubMed Central

    Shui, Wei; Yin, Liangjun; Luo, Jeffrey; Li, Ruidong; Zhang, Wenwen; Zhang, Jiye; Huang, Wei; Hu, Ning; Liang, Xi; Deng, Zhong-Liang; Hu, Zhenming; Shi, Lewis; Luu, Hue H.; Haydon, Rex C.; He, Tong-Chuan; Ho, Sherwin

    2014-01-01

    Articular cartilage lesions in the knee are common injuries. Chondrocyte transplant represents a promising therapeutic modality for articular cartilage injuries. Here, we characterize the viability and transgene expression of articular chondrocytes cultured in 3-D scaffolds provided by four types of carriers. Articular chondrocytes are isolated from rabbit knees and cultured in four types of scaffolds: type I collagen sponge, fibrin glue, hyaluronan, and Open-cell PolyLactic Acid (OPLA). The cultured cells are transduced with adenovirus expressing green fluorescence protein (AdGFP) and luciferase (AdGL3-Luc). The viability and gene expression in the chondrocytes are determined with fluorescence microscopy and luciferase assay. Cartilage matrix production is assessed by Alcian blue staining. Rabbit articular chondrocytes are effectively infected by AdGFP and exhibited sustained GFP expression. All tested scaffolds support the survival and gene expression of the infected chondrocytes. However, the highest transgene expression is observed in the OPLA carrier. At four weeks, Alcian blue-positive matrix materials are readily detected in OPLA cultures. Thus, our results indicate that, while all tested carriers can support the survival of chondrocytes, OPLA supports the highest transgene expression and is the most conductive scaffold for matrix production, suggesting that OPLA may be a suitable scaffold for cell-based gene therapy of articular cartilage repairs. PMID:23629940

  14. IL-36α: a novel cytokine involved in the catabolic and inflammatory response in chondrocytes

    PubMed Central

    Conde, Javier; Scotece, Morena; Abella, Vanessa; Lois, Ana; López, Verónica; García-Caballero, Tomás; Pino, Jesús; Gómez-Reino, Juan Jesús; Gómez, Rodolfo; Lago, Francisca; Gualillo, Oreste

    2015-01-01

    Recent studies confer to IL-36α pro-inflammatory properties. However, little is known about the expression and function of IL-36α in cartilage. This study sought to analyze the expression of IL-36α in healthy and OA cartilage. Next, we determined the effects of recombinant IL-36α on catabolism and inflammation in chondrocytes. For completeness, part of the signaling pathway elicited by IL-36α was also explored. IL-36α expression was evaluated by immunohistochemistry and RT-qPCR. Expression of MMP-13, NOS2 and COX-2 was also determined in OA articular chondrocytes treated with recombinant IL-36α. IκB-α and P-p38 was explored by western blot. We observed a low constitutive expression of IL-36α in healthy human chondrocytes. However, OA chondrocytes likely expressed more IL-36α than healthy chondrocytes. In addition, immune cells infiltrated into the joint and PBMCs express higher levels of IL-36α in comparison to chondrocytes. OA chondrocytes, treated with IL-36α, showed significant increase in the expression of MMP-13, NOS2 and COX-2. Finally, IL-36α stimulated cells showed NFκB and p38 MAPK activated pathways. IL-36α acts as a pro-inflammatory cytokine at cartilage level, by increasing the expression of markers of inflammation and cartilage catabolism. Like other members of IL-1 family, IL-36α acts through the activation of NFκB and p38 MAPK pathway. PMID:26560022

  15. 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.

  16. Core Binding Factor β Plays a Critical Role During Chondrocyte Differentiation.

    PubMed

    Park, Na-Rae; Lim, Kyung-Eun; Han, Min-Su; Che, Xiangguo; Park, Clara Yongjoo; Kim, Jung-Eun; Taniuchi, Ichiro; Bae, Suk-Chul; Choi, Je-Yong

    2016-01-01

    Core binding factor β (Cbfβ) is a partner protein of Runx family transcription factors with minimally characterized function in cartilage. Here we address the role of Cbfβ in cartilage by generating chondrocyte-specific Cbfβ-deficient mice (Cbfb(Δch/Δch) ) from Cbfb-floxed mice crossed with mice expressing Cre from the Col2a1 promoter. Cbfb(Δch/Δch) mice died soon after birth and exhibited delayed endochondral bone formation, shorter appendicular skeleton length with increased proliferative chondrocytes, and nearly absent hypertrophic chondrocyte zones. Immunohistochemical and quantitative real-time PCR analyses showed that the number and size of proliferative chondrocytes increased and the expression of chondrocyte maturation markers at the growth plates, including Runx2, osterix, and osteopontin, significantly diminished in Cbfb(Δch/Δch) mice compared to wild type mice. With regard to signaling pathways, both PTHrP-Ihh and BMP signaling were compromised in Cbfb(Δch/Δch) mice. Mechanistically, Cbfβ deficiency in chondrocytes caused a decrease of protein levels of Runx transcription factors by accelerating polyubiquitination-mediated proteosomal degradation in vitro. Indeed, Runx2 and Runx3, but not Runx1, decreased in Cbfb(Δch/Δch) mice. Collectively, these findings indicate that Cbfβ plays a critical role for chondrocyte differentiation through stabilizing Runx2 and Runx3 proteins in cartilage. PMID:26058470

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

    PubMed

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

    2002-01-01

    Hypertrophic chondrocytes have important roles in promoting invasion of cartilage by blood vessels and its replacement with bone. However, it is unclear whether blood vessels exert reciprocal positive influences on chondrocyte maturation and function. Therefore, we implanted beads containing the antiangiogenic molecule squalamine around humeral anlagen in chick embryo wing buds and monitored the effects over time. Fluorescence microscopy showed that the drug diffused from the beads and accumulated in humeral perichondrial tissues, indicating that these tissues were the predominant targets of drug action. Diaphyseal chondrocyte maturation was indeed delayed in squalamine-treated humeri, as indicated by reduced cell hypertrophy and expression of type X collagen, transferrin, and Indian hedgehog (Ihh). Although reduced in amount, Ihh maintained a striking distribution in treated and control humeri, being associated with diaphyseal chondrocytes as well as inner perichondrial layer. These decreases were accompanied by lack of cartilage invasion and tartrate-resistant acid phosphatase-positive (TRAP+) cells and a significant longitudinal growth retardation. Recovery occurred at later developmental times, when in fact expression in treated humeri of markers such as matrix metalloproteinase 9 (MMP-9) and connective tissue growth factor (CTGF) appeared to exceed that in controls. Treating primary cultures of hypertrophic chondrocytes and osteoblasts with squalamine revealed no obvious changes in cell phenotype. These data provide evidence that perichondrial tissues and blood vessels in particular influence chondrocyte maturation in a positive manner and may cooperate with hypertrophic chondrocytes in dictating the normal pace and location of the transition from cartilage to bone. PMID:11771670

  18. 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.

  19. The potential of pulsed low intensity ultrasound to stimulate chondrocytes matrix synthesis in agarose and monolayer cultures.

    PubMed

    Vaughan, Natalie M; Grainger, James; Bader, Dan L; Knight, Martin M

    2010-12-01

    Pulsed low intensity ultrasound (PLIUS) has been used successfully for bone fracture repair and has therefore been suggested for cartilage regeneration. However, previous in vitro studies with chondrocytes show conflicting results as to the effect of PLIUS on the elaboration of extracellular matrix. This study tests the hypothesis that PLIUS, applied for 20 min/day, stimulates the synthesis of sulphated glycosaminoglycan (sGAG) by adult bovine articular chondrocytes cultured in either monolayer or agarose constructs. For both culture models, PLIUS at either 30 or 100 mW/cm(2) intensity had no net effect on the total sGAG content. Although PLIUS at 100 mW/cm(2) did induce a 20% increase in sGAG content at day 2 of culture in agarose, this response was lost by day 5. Intensities of 200 and 300 mW/cm(2) resulted in cell death probably due to heating from the ultrasound transducers. The lack of a sustained up-regulation of sGAG synthesis may reflect the suggestion that PLIUS only induces a stimulatory effect in the presence of a tissue injury response. These results suggest that PLIUS has a limited potential to provide an effective method of stimulating matrix production as part of a tissue engineering strategy for cartilage repair. PMID:20938751

  20. Withaferin A-Caused Production of Intracellular Reactive Oxygen Species Modulates Apoptosis via PI3K/Akt and JNKinase in Rabbit Articular Chondrocytes

    PubMed Central

    2014-01-01

    Withaferin A (WFA) is known as a constituent of Ayurvedic medicinal plant, Withania somnifera, and has been used for thousands of years. Although WFA has been used for the treatment of osteoarthritis (OA) and has a wide range of biochemical and pharmacologic activities, there are no findings suggesting its properties on chondrocytes or cartilage. The aim of the present study is to investigate the effects of WFA on apoptosis with focus on generation of intracellular reactive oxygen species (ROS). Here we showed that WFA significantly increased the generation of intracellular ROS in a dose-dependent manner. We also determined that WFA markedly leads to apoptosis as evidenced by accumulation of p53 by Western blot analysis. N-Acetyl-L-Cystein (NAC), an antioxidant, prevented WFA-caused expression of p53 and inhibited apoptosis of chondrocytes. We also found that WFA causes the activation of PI3K/Akt and JNKinase. Inhibition of PI3K/Akt and JNKinase with LY294002 (LY)/triciribine (TB) or SP600125 (SP) in WFA-treated cells reduced accumulation of p53 and inhibited fragmented DNA. Our findings suggested that apoptosis caused by WFA-induced intracellular ROS generation is regulated through PI3K/Akt and JNKinase in rabbit articular chondrocytes. Graphical Abstract PMID:25120312

  1. Sox9 Transcriptionally Represses Spp1 to Prevent Matrix Mineralization in Maturing Heart Valves and Chondrocytes

    PubMed Central

    Ediriweera, Hasini N.; Lincoln, Joy

    2011-01-01

    Sox9 is an SRY-related transcription factor required for expression of cartilaginous genes in the developing skeletal system and heart valve structures. In contrast to positively regulating cartilaginous matrix, Sox9 also negatively regulates matrix mineralization associated with bone formation. While the transcriptional activation of Sox9 target genes during chondrogenesis has been characterized, the mechanisms by which Sox9 represses osteogenic processes are not so clear. Using ChIP-on-chip and luciferase assays we show that Sox9 binds and represses transactivation of the osteogenic glycoprotein Spp1. In addition, Sox9 knockdown in post natal mouse heart valve explants and rib chondrocyte cultures promotes Spp1 expression and matrix mineralization, while attenuating expression of cartilage genes Type II Collagen and Cartilage Link Protein. Further, we show that Spp1 is required for matrix mineralization induced by Sox9 knockdown. These studies provide insights into the molecular mechanisms by which Sox9 prevents pathologic matrix mineralization in tissues that must remain cartilaginous. PMID:22046352

  2. The 45 kDa collagen-binding fragment of fibronectin induces matrix metalloproteinase-13 synthesis by chondrocytes and aggrecan degradation by aggrecanases.

    PubMed Central

    Stanton, Heather; Ung, Linh; Fosang, Amanda J

    2002-01-01

    Fragments of fibronectin occur naturally in vivo and are increased in the synovial fluid of arthritis patients. We have studied the 45 kDa fragment (Fn-f 45), representing the N-terminal collagen-binding domain of fibronectin, for its ability to modulate the expression of metalloproteinases by porcine articular chondrocytes in vitro. We report that stimulation of cultured chondrocytes, or cartilage explants, with Fn-f 45 increased the levels of matrix metalloproteinase-13 (MMP-13; collagenase-3) released into the conditioned medium in a dose-dependent manner. Increased levels of MMP-13 were due to stimulation of MMP-13 synthesis, rather than release of MMP-13 from accumulated matrix stores. Fn-f 45 also stimulated the synthesis of MMP-3 (stromelysin-1) from cultured chondrocytes and cartilage cultures. The Fn-f 45-induced increase in MMP-3 and MMP-13 synthesis occurred via an interleukin 1-independent mechanism, since the receptor antagonist of interleukin-1 was unable to block the increased synthesis. The gelatinases, MMP-2 and MMP-9, were not modulated by Fn-f 45 in these culture systems. Fn-f 45 also stimulated the release of aggrecan from cartilage explants into conditioned medium. Neoepitope antibodies specific for aggrecan fragments generated by MMPs or aggrecanases showed that the Fn-f 45-induced aggrecan loss was mediated by aggrecanases, and not by MMPs. Extracts of cultured cartilage contained elevated levels of the aggrecanase-derived ITEGE(373)-G1 domain, whereas levels of the matrix metalloproteinase-derived DIPEN(341)-G1 domain were unchanged. These studies show that Fn-f 45 can induce a catabolic phenotype in articular chondrocytes by up-regulating the expression of metalloproteinases specific for the degradation of collagen and aggrecan. PMID:11988091

  3. Differential effects of insulin-like growth factor I and growth hormone on developmental stages of rat growth plate chondrocytes in vivo.

    PubMed Central

    Hunziker, E B; Wagner, J; Zapf, J

    1994-01-01

    Skeletal growth depends upon enchondral ossification in growth plate cartilage, within which chondrocytes undergo well defined stages of maturation. We infused IGF-I or growth hormone (GH), two key regulators of skeletal growth, into hypophysectomized rats and compared their effects on growth plate chondrocyte differentiation using qualitative and quantitative autoradiography, stereology, and incident light fluorescence microscopy. Stem cell cycle time was shortened from 50 to 15 and 8 d after treatment with IGF-I and GH, respectively. Proliferating cell cycle time decreased from 11 to 4.5 and 3 d, and duration of the hypertrophic phase decreased from 6 to 4 and 2.8 d. Average matrix volume per cell at each differentiation stage was similar for normal, hormone-treated, and untreated hypophysectomized groups. Mean cell volume and cell height were significantly reduced by hypophysectomy at the proliferative and hypertrophic stages, but were restored to physiological values by IGF-I and GH. In contrast, cell productivity, i.e., increases in cell volume, height, and matrix production per unit of time, did not reach normal values with either IGF-I or GH, and this parameter was inversely proportional to cell cycle time or phase duration. IGF-I and GH are thus capable of stimulating growth plate chondrocytes at all stages of differentiation, albeit to variable degrees with respect to individual cell activities. Although it is generally accepted that GH acts at both the stem and proliferating phases of chondrocyte differentiation, our data represent the first evidence in vivo that IGF-I is also capable of stimulating stem cells. Images PMID:8132746

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

    SciTech Connect

    Sakurai, Atsuo; Okahashi, Nobuo; Maruyama, Fumito; Ooshima, Takashi; Hamada, Shigeyuki; Nakagawa, Ichiro

    2008-08-29

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

  5. Potassium channels of pig articular chondrocytes are blocked by propofol.

    PubMed

    Mozrzymas, J W; Visintin, M; Vittur, F; Ruzzier, F

    1994-07-15

    The effect of propofol on the voltage-activated potassium channels in pig articular chondrocytes was investigated. Propofol was found to reversibly block the potassium channels in a dose-dependent manner. The blocking effect was voltage-independent and the Hill coefficient was 1.85 +/- 0.18. No changes either in the slope conductance or in the single channel kinetics were observed. The half-blocking concentration (Ec50) was 6.0 +/- 0.49 microM which is much lower than the concentrations used to observe the scavenging effect of the drug in an artificial synovial fluid. Interestingly, Ec50 found in our experiments is also smaller than the blood concentration of propofol used in anaesthesia. These results show that propofol may strongly affect the potassium channels in some non-excitable cells.

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

    SciTech Connect

    Nguyen, Trung Dung; Gu, YuanTong

    2014-05-05

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

  7. 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

    2015-01-01

    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. PMID:26108578

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

    PubMed Central

    Fu, Yu; Maye, Peter

    2015-01-01

    With the establishment of methods that provide evidence for the generation of chondrocyte and osteoblast cell types from ESCs, there is a need for reagents that will enable their further characterization. Here we report on the derivation of chondrocyte and osteoblast reporter ESCs from previously generated and characterized transgenic mouse lines, Collagen type 2 alpha 1(Col2a1)-ECFP, Bone Sialoprotein (BSP)-Topaz, and BSP-Topaz/Dentin Matrix Protein 1 (DMP1)-Cherry dual reporter mice. Col2a1-ECFP is highly expressed in chondrocytes, while BSP-Topaz and DMP1-Cherry are highly expressed in osteoblasts and osteocytes, respectively. These new skeletal reporter mouse ESC lines will serve as valuable reagents to investigate the functionality of ESC derived chondrocyte and osteoblast cell types. PMID:25809957

  9. Effect of Heterotheca inuloides essential oil on rat cytoskeleton articular chondrocytes.

    PubMed

    Flores-San Martin, Denise; Perea-Flores, María de Jesús; Morales-López, Javier; Centeno-Alvarez, Mónica María; Pérez-Ishiwara, Guillermo; Pérez-Hernández, Nury; Pérez-Hernández, Elizabeth

    2013-01-01

    Osteoarthritis is characterised by progressive loss of articular cartilage through the increase of catabolic metalloproteinases, and chondrocyte cytoskeleton disruption has also been reported. In this regard, we studied the effect of Heterotheca inuloides essential oil (HIEO) on the distribution and immunolocalisation of actin, vimentin and tubulin of chondrocytes from cultured rat articular cartilage explants in the presence of the cytoskeleton disassembly agent acrylamide. After 48 h, chondrocytes treated with acrylamide showed changes in actin immunolocalisation and shrinkage, loss of tubulin compartmentalisation and vimentin collapse and redistribution. However, the immunostaining pattern of these three proteins in acrylamide- and HIEO-treated chondrocytes simultaneously retained their typical characteristics. These results suggest that HIEO promotes protein cytoskeleton reorganisation without providing a preventive effect of acrylamide-associated disassembly. However, it is also possible that HIEO prevents vimentin disorganisation by chemical interaction with acrylamide.

  10. Derivation of chondrocyte and osteoblast reporter mouse embryonic stem cell lines.

    PubMed

    Fu, Yu; Maye, Peter

    2015-01-01

    With the establishment of methods that provide evidence for the generation of chondrocyte and osteoblast cell types from ESCs, there is a need for reagents that will enable their further characterization. Here we report on the derivation of chondrocyte and osteoblast reporter ESCs from previously generated and characterized transgenic mouse lines, Collagen type 2 alpha 1(Col2a1)-ECFP, Bone Sialoprotein (BSP)-Topaz, and BSP-Topaz/Dentin Matrix Protein 1 (DMP1)-Cherry dual reporter mice. Col2a1-ECFP is highly expressed in chondrocytes, while BSP-Topaz and DMP1-Cherry are highly expressed in osteoblasts and osteocytes, respectively. These new skeletal reporter mouse ESC lines will serve as valuable reagents to investigate the functionality of ESC derived chondrocyte and osteoblast cell types. PMID:25809957

  11. Mechanical loading of in situ chondrocytes in lapine retropatellar cartilage after anterior cruciate ligament transection.

    PubMed

    Han, Sang-Kuy; Seerattan, Ruth; Herzog, Walter

    2010-06-01

    The aims of this study were (i) to quantify chondrocyte mechanics in fully intact articular cartilage attached to its native bone and (ii) to compare the chondrocyte mechanics for cells in healthy and early osteoarthritis (OA) tissue. We hypothesized that cells in the healthy tissue would deform less for given articular surface pressures than cells in the early OA tissue because of a loss of matrix integrity in early OA and the associated loss of structural integrity that is thought to protect chondrocytes. Chondrocyte dynamics were quantified by measuring the deformation response of the cells to controlled loading of fully intact cartilage using a custom-designed confocal indentation system. Early OA was achieved nine weeks following transection of the anterior cruciate ligament (ACL) in rabbit knees. Experiments were performed on the retropatellar cartilage of early OA rabbit knees (four joints and 48 cells), the corresponding intact contralateral control knees (four joints and 48 cells) and knees from normal control rabbits (four joints and 48 cells). Nine weeks following ACL transection, articular cartilage of the experimental joints showed substantial increases in thickness, and progression towards OA as assessed using histological grading. Local matrix strains in the superficial zone were greater for the experimental (38 +/- 4%) compared with the contralateral (27 +/- 5%) and normal (28 +/- 4%) joints (p = 0.04). Chondrocyte deformations in the axial and depth directions were similar during indentation loading for all experimental groups. However, cell width increased more for the experimental cartilage chondrocytes (12 +/- 1%) than the contralateral (6 +/- 1%) and normal control chondrocytes (6 +/- 1%; p < 0.001). On average, chondrocyte volume increased with indentation loading in the early OA cartilage (8 +/- 3%, p = 0.001), while it decreased for the two control groups (-8 +/- 2%, p = 0.002 for contralateral and -8 +/- 1%, p = 0.004 for normal controls

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

    PubMed

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

    2016-05-01

    Local anesthetics are administered intraarticularly for pain control in orthopedic clinics and surgeries. Although previous studies have shown that local anesthetics can be toxic to chondrocytes, the underlying cellular mechanisms remain unclear. The present study investigates acute cellular responses associated with lidocaine-induced toxicity to articular chondrocytes. Rabbit articular chondrocytes were exposed to lidocaine and their morphological changes were monitored with live cell microscopy. The viability of chondrocytes was evaluated using a fluorescence based LIVE/DEAD assay. Acute treatment of chondrocytes with lidocaine (3-30 mM) induced spherical protrusions on the cell surface (so called "membrane blebbing") in a time- and concentration-dependent manner. The concentration-response relationship for the lidocaine effect was shifted leftward by elevating extracellular pH, as expected for the non-ionized lidocaine being involved in the bleb formation. ROCK (Rho-kinase) inhibitors Y-27632 and fasudil completely prevented the lidocaine-induced membrane blebbing, suggesting that ROCK activation is required for bleb formation. Caspase-3 levels were unchanged by 10 mM lidocaine (p = 0.325) and a caspase inhibitor z-VAD-fmk did not affect the lidocaine-induced blebbing (p = 0.964). GTP-RhoA levels were significantly increased (p < 0.001), but Rho inhibitor-1 failed to suppress the membrane blebbing (p = 0.875). Lidocaine (30 mM) reduced the cell viability of isolated chondrocytes (p < 0.001) and in situ chondrocytes (p < 0.001). The chondrotoxicity was attenuated by pretreatment of cells with ROCK inhibitors or a myosin-II inhibitor blebbistatin (p < 0.001). These findings suggest that lidocaine induces ROCK-dependent membrane blebbing and thereby produces a cytotoxic effect on chondrocytes. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:754-762, 2016.

  13. 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.

  14. 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

  15. Altered Signaling in the G1 Phase Deregulates Chondrocyte Growth in a Mouse Model With Proteoglycan Undersulfation

    PubMed Central

    Leonardis, Fabio De; Monti, Luca; Gualeni, Benedetta; Tenni, Ruggero; Forlino, Antonella; Rossi, Antonio

    2014-01-01

    In several skeletal dysplasias defects in extracellular matrix molecules affect not only the structural and mechanical properties of cartilage, but also the complex network of signaling pathways involved in cell proliferation and differentiation. Sulfated proteoglycans, besides playing an important structural role in cartilage, are crucial in modulating the transport, diffusion, and interactions of growth factors with their specific targets, taking part in the regulation of signaling pathways involved in skeletal development and growth. In this work, we investigated by real time PCR and Western blots of the microdissected growth plate and by immunohistochemistry the molecular basis of reduced chondrocyte proliferation in the growth plate of the dtd mouse, a chondrodysplastic model with defective chondroitin sulfate proteoglycan sulfation of articular and growth plate cartilage. We detected activation of the Wnt pathway, leading to an increase in the non-phosphorylated form of nuclear β-catenin and subsequent up-regulation of cyclin D1 expression in the G1 phase of the cell cycle. β-Catenin was further stabilized by up-regulation of Smad3 expression through TGF-β pathway synergistic activation. We demonstrate that notwithstanding cyclin D1 expression increase, cell cycle progression is compromised in the G1 phase due to reduced phosphorylation of the pocket protein p130 leading to inhibition of transcription factors of the E2F family which are crucial for cell cycle progression and DNA replication. These data, together with altered Indian hedgehox signaling detected previously, explain at the molecular level the reduced chondrocyte proliferation rate of the dtd growth plate leading to reduced skeletal growth. J. Cell. Biochem. 115: 1779–1786, 2014. PMID:24820054

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

    PubMed Central

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

    2014-01-01

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

  17. Chitosan Enriched Three-Dimensional Matrix Reduces Inflammatory and Catabolic Mediators Production by Human Chondrocytes

    PubMed Central

    Oprenyeszk, Frederic; Sanchez, Christelle; Dubuc, Jean-Emile; Maquet, Véronique; Henrist, Catherine; Compère, Philippe; Henrotin, Yves

    2015-01-01

    This in vitro study investigated the metabolism of human osteoarthritic (OA) chondrocytes encapsulated in a spherical matrix enriched of chitosan. Human OA chondrocytes were encapsulated and cultured for 28 days either in chitosan-alginate beads or in alginate beads. The beads were formed by slowly passing dropwise either the chitosan 0.6%–alginate 1.2% or the alginate 1.2% solution through a syringe into a 102 mM CaCl2 solution. Beads were analyzed histologically after 28 days. Interleukin (IL)-6 and -8, prostaglandin (PG) E2, matrix metalloproteinases (MMPs), hyaluronan and aggrecan were quantified directly in the culture supernatant by specific ELISA and nitric oxide (NO) by using a colorimetric method based on the Griess reaction. Hematoxylin and eosin staining showed that chitosan was homogeneously distributed through the matrix and was in direct contact with chondrocytes. The production of IL-6, IL-8 and MMP-3 by chondrocytes significantly decreased in chitosan-alginate beads compared to alginate beads. PGE2 and NO decreased also significantly but only during the first three days of culture. Hyaluronan and aggrecan production tended to increase in chitosan-alginate beads after 28 days of culture. Chitosan-alginate beads reduced the production of inflammatory and catabolic mediators by OA chondrocytes and tended to stimulate the synthesis of cartilage matrix components. These particular effects indicate that chitosan-alginate beads are an interesting scaffold for chondrocytes encapsulation before transplantation to repair cartilage defects. PMID:26020773

  18. Calcium signaling in response to fluid flow by chondrocytes in 3D alginate culture.

    PubMed

    Degala, Satish; Williams, Rebecca; Zipfel, Warren; Bonassar, Lawrence J

    2012-05-01

    Quantifying the effects of mechanical loading on the metabolic response of chondrocytes is difficult due to complicated structure of cartilage ECM and the coupled nature of the mechanical stimuli presented to the cells. In this study we describe the effects of fluid flow, particularly hydrostatic pressure and wall shear stress, on the Ca(2+) signaling response of bovine articular chondrocytes in 3D culture. Using well-established alginate hydrogel system to maintain spherical chondrocyte morphology, we altered solid volume fraction to change scaffold mechanics. Fluid velocities in the bulk of the scaffolds were directly measured via an optical technique and scaffold permeability and aggregate modulus was characterized to quantify the mechanical stimuli presented to cells. Ca(2+) signaling response to direct perfusion of chondrocyte-seeded scaffolds increased monotonically with flow rate and was found more directly dependent on fluid velocity rather than shear stress or hydrostatic pressure. Chondrocytes in alginate scaffolds responded to fluid flow at velocities and shear stresses 2-3 orders of magnitude lower than seen in previous monolayer studies. Our data suggest that flow-induced Ca(2+) signaling response of chondrocytes in alginate culture may be due to mechanical signaling pathways, which is influenced by the 3D nature of cell shape.

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

    PubMed

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

    2016-07-01

    Measurement of ultrasound backscattering is a promising diagnostic technique for arthroscopic evaluation of articular cartilage. However, contribution of collagen and chondrocytes on ultrasound backscattering and speed of sound in cartilage is not fully understood and is experimentally difficult to study. Agarose hydrogels have been used in tissue engineering applications of cartilage. Therefore, the aim of this study was to simulate the propagation of high frequency ultrasound (40 MHz) in agarose scaffolds with varying concentrations of chondrocytes (1 to 32 × 10(6) cells/ml) and collagen (1.56-200 mg/ml) using transversely isotropic two-dimensional finite difference time domain method (FDTD). Backscatter and speed of sound were evaluated from the simulated pulse-echo and through transmission measurements, respectively. Ultrasound backscatter increased with increasing collagen and chondrocyte concentrations. Furthermore, speed of sound increased with increasing collagen concentration. However, this was not observed with increasing chondrocyte concentrations. The present study suggests that the FDTD method may have some applicability in simulations of ultrasound scattering and propagation in constructs containing collagen and chondrocytes. Findings of this study indicate the significant role of collagen and chondrocytes as ultrasound scatterers and can aid in development of modeling approaches for understanding how cartilage architecture affects to the propagation of high frequency ultrasound. PMID:27475127

  20. Growth requirements of low-density rabbit costal chondrocyte cultures maintained in serum-free medium.

    PubMed

    Kato, Y; Gospodarowicz, D

    1984-09-01

    The factors required for the active proliferation of low-density rabbit costal chondrocytes exposed to 9:1 (v/v) mixture of Dulbecco's modified Eagle's medium and Ham's F12 medium have been defined. Low-density primary cultures of rabbit costal chondrocytes proliferated actively when the medium was supplemented with high-density lipoprotein (300 micrograms/ml), transferrin (60 micrograms/ml), fibroblast growth factor (FGF) (1 ng/ml), hydrocortisone (10(-6) M), and epidermal growth factor (EGF) (30 ng/ml). Insulin, although it slightly decreased the final cell density, was required for reexpression of the cartilage phenotype at confluence. Optimal proliferation of low-density chondrocyte cultures was only observed when dishes were coated with an extracellular matrix (ECM) produced by cultured corneal endothelial cells, but not on plastic. Furthermore, serum-free chondrocyte cultures seeded at low density and maintained on ECM-coated dishes gave rise to a homogeneous cartilage-like tissue composed of spherical cells. These chondrocytes therefore seem to provide a good experimental system for analyzing factors involved in supporting proliferation of chondrocytes and their phenotypic expression.

  1. 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. PMID:27566509

  2. 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

  3. Effects of introducing cultured human chondrocytes into a human articular cartilage explant model.

    PubMed

    Secretan, Charles; Bagnall, Keith M; Jomha, Nadr M

    2010-02-01

    Articular cartilage (AC) heals poorly and effective host-tissue integration after reconstruction is a concern. We have investigated the ability of implanted chondrocytes to attach at the site of injury and to be incorporated into the decellularized host matrix adjacent to a defect in an in vitro human explant model. Human osteochondral dowels received a standardized injury, were seeded with passage 3 chondrocytes labelled with PKH 26 and compared with two control groups. All dowels were cultured in vitro, harvested at 0, 7, 14 and 28 days and assessed for chondrocyte adherence and migration into the region of decellularized tissue adjacent to the defects. Additional evaluation included cell viability, general morphology and collagen II production. Seeded chondrocytes adhered to the standardized defect and areas of lamina splendens disruption but did not migrate into the adjacent acellular region. A difference was noted in viable-cell density between the experimental group and one control group. A thin lattice-like network of matrix surrounded the seeded chondrocytes and collagen II was present. The results indicate that cultured human chondrocytes do indeed adhere to regions of AC matrix injury but do not migrate into the host tissue, despite the presence of viable cells. This human explant model is thus an effective tool for studying the interaction of implanted cells and host tissue. PMID:20012649

  4. The Chondrogenic Potential of Mesenchymal Cells and Chondrocytes from Osteoarthritic Subjects

    PubMed Central

    Agar, Gabriel; Blumenstein, Sara; Bar-Ziv, Yaron; Kardosh, Rami; Schrift-Tzadok, Michal; Gal-Levy, Ronit; Fischler, Tali; Goldschmid, Revital; Yayon, Avner

    2011-01-01

    Objective: The multipotential nature of stem or progenitor cells apparently makes them the ideal choice for any cell therapy, but this as yet remains to be proven. This study (30 subjects) was designed to compare the potential to repair articular cartilage of chondrocytes taken from different regions in osteoarthritic cartilage with that of mesenchymal stem cells prepared from bone marrow of the same subject. Design: Cartilage biopsies, bone marrow, and blood samples were taken from each of 30 individuals with chronic osteoarthritis (aged 62-85 years) undergoing total knee replacement. The chondrogenic potential of chondrocytes isolated from cartilage biopsies taken from different regions of osteoarthritic cartilage was compared with that of mesenchymal cells by quantitative analysis of several chondrocyte specific markers and an ex vivo cartilage differentiation assay. Results: Cartilage-derived articular chondrocytes are superior to bone marrow–derived cells when compared for their ex vivo chondrogenic potential. Interestingly, there was marked and significant difference in the expression of chondrocytic markers between chondrocytes derived from adjacent, visually distinct regions of the diseased cartilage. When cultured in the presence of a fibroblast growth factor 2 variant, all cell samples from both tissues showed a high degree of chondrogenic potential. Conclusions: Although bone marrow–derived mesenchymal cells, when supplemented with the appropriate chondrogenic factors, are a suitable source for autologous cartilage implantation, adult chondroprogenitor cells, particularly those from moderately affected regions of the osteoarthritic joints, demonstrate superior chondrogenic potential. PMID:26069568

  5. Opiates do not violate the viability and proliferative activity of human articular chondrocytes.

    PubMed

    Chechik, Ofir; Arbel, Ron; Salai, Moshe; Gigi, Roy; Beilin, Mark; Flaishon, Ron; Sever, Ronen; Khashan, Morsi; Ben-Tov, Tomer; Gal-Levy, Ronit; Yayon, Avner; Blumenstein, Sara

    2014-09-01

    Articular cartilage injuries present a challenge for the clinician. Autologous chondrocyte implantation embedded in scaffolds are used to treat cartilage defects with favorable outcomes. Autologous serum is often used as a medium for chondrocyte cell culture during the proliferation phase of the process of such products. A previous report showed that opiate analgesics (fentanyl, alfentanil and diamorphine) in the sera have a significant inhibitory effect on chondrocyte proliferation. In order to determine if opiates in serum inhibit chondrocyte proliferation, twenty two patients who underwent knee arthroscopy and were anesthetized with either fentanyl or remifentanil were studied. Blood was drawn before and during opiate administration and up to 2 h after its discontinuation. The sera were used as medium for in vitro proliferation of both cryopreserved and freshly isolated chondrocytes, and the number and viability of cells were measured. There was no difference in the yield or cell viability between the serum samples of patients anesthetized with fentanyl when either fresh or cryopreserved human articular chondrocytes (hACs) were used. Some non-significant reduction in the yield of cells was observed in the serum samples of patients anesthetized with remifentanil when fresh hAC were used. We conclude that Fentanyl in human autologous serum does not inhibit in vitro hAC proliferation. Remifentanil may show minimal inhibitory effect on in vitro fresh hAC proliferation.

  6. Passaged adult chondrocytes can form engineered cartilage with functional mechanical properties: a canine model.

    PubMed

    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; Hung, Clark T

    2010-03-01

    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-beta3. 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.

  7. Effects of manganese deficiency on chondrocyte development in tibia growth plate of Arbor Acres chicks.

    PubMed

    Wang, Jian; Wang, Zhen Yong; Wang, Zhao Jun; Liu, Ran; Liu, Shao Qiong; Wang, Lin

    2015-01-01

    The aim of this study was to investigate the effects of manganese (Mn) deficiency on chondrocyte development in tibia growth plate. Ninety 1-day-old Arbor Acres chicks were randomly divided into three groups and fed on control diet (60 mg Mn/kg diet) and manganese deficient diets (40 mg Mn/kg diet, manganese deficiency group I; 8.7 mg Mn/kg diet, manganese deficiency group II), respectively. The width of the proliferative zone of growth plate was measured by the microscope graticule. Chondrocyte apoptosis was estimated by TUNEL staining. Gene expression of p21 and Bcl-2, and expression of related proteins were analyzed by quantitative real time reverse transcription polymerase chain reaction and immunohistochemistry, respectively. Compared with the control group, manganese deficiency significantly decreased the proliferative zone width and Bcl-2 mRNA expression level, while significantly increased the apoptotic rates and the expression level of p21 gene in chondrocytes. The results indicate that manganese deficiency had a negative effect on chondrocyte development, which was mediated by the inhibition of chondrocyte proliferation and promotion of chondrocyte apoptosis.

  8. Altered responses of chondrocytes to nanophase PLGA/nanophase titania composites.

    PubMed

    Savaiano, Jennifer K; Webster, Thomas J

    2004-01-01

    Chondrocyte (cartilage-synthesizing cells) cell density and synthesis of select intracellular proteins by chondrocytes were investigated on novel nanophase poly-lactic/glycolic acid (PLGA) and titania composites in the present in vitro study. Nanophase PLGA films were created by chemically treating conventional (or micron-structured) PLGA films with 10N NaOH for 1h. Titania particle dimensions in ceramic compacts were controlled by utilizing either conventional (i.e., micron) or nanometer grain size titania. Composites of either conventional or nanophase PLGA with either conventional or nanophase titania at 70/30wt% were also created. Compared to surfaces with a conventional or micron topography, results provided the first evidence of stagnant confluent cell densities on nanostructured surfaces at time points between 1 and 7 days. Moreover, compared to surfaces with a conventional topography, increased chondrocyte intracellular synthesis of alkaline phosphatase and chondrocyte expressed protein-68 (proteins that have been correlated with the functions of chondrocytes) were observed on nanophase PLGA/nanophase titania composites. The present study, thus, provided the first evidence of different chondrocyte responses to nanostructured PLGA/nanophase titania composites; in light of other reports demonstrating increased functions of bone cells on the same materials, such data indicates that further investigation of these materials at the bone-cartilage interface should be conducted.

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

    PubMed

    Kasper, S; Friesen, H G

    1986-01-01

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

  10. Protocatechuic acid benefits proliferation and phenotypic maintenance of rabbit articular chondrocytes: An in vitro study

    PubMed Central

    LUO, LIKE; WEI, QINGJUN; LIU, LEI; LIN, XIAO; LIN, CUIWU; ZHENG, LI; ZHAO, JINMIN

    2015-01-01

    Numerous antioxidants exhibit antiarthritic effects due to their inhibitory effect on inflammatory factors. Certain antioxidants, such as protocatechuic acid (PCA) and its analogs, have been reported to be effective in the treatment of arthritis. However, the effect of PCA on chondro-protection may be alleviated due to the induction of apoptosis, as has been demonstrated in stomatocytes. To clearly determine the effect of PCA on the biological and cellular metabolism of rabbit articular chondrocytes in vitro, examinations of cytotoxicity, proliferation and morphology were performed, in addition to analyses of glycosaminoglycan (GAG) synthesis and the expression of cartilage-specific genes. The results revealed that PCA effectively promoted chondrocyte growth, the synthesis of the extracellular matrix and the mRNA expression of aggrecan, collagen II and Sox9, while downregulating the expression of the collagen I gene, a marker of chondrocyte dedifferentiation. In addition, hypertrophy, which may result in chondrocyte ossification, was not detected in the groups. Among the doses (range, 0.05–0.3 mmol/l) of PCA that promoted the proliferation of chondrocytes, a concentration of 0.125 mmol/l produced the optimum performance. The results indicated that PCA, particularly at a dose of 0.125 mmol/l, accelerated the proliferation of rabbit articular chondrocytes in vitro and maintained their phenotype. This study may provide a basis for further research concerning the treatment of cartilage defects. PMID:26136906

  11. Non-woven PGA/PVA fibrous mesh as an appropriate scaffold for chondrocyte proliferation.

    PubMed

    Rampichová, M; Koštáková, E; Filová, E; Prosecká, E; Plencner, M; Ocheretná, L; Lytvynets, A; Lukáš, D; Amler, E

    2010-01-01

    Non-woven textile mesh from polyglycolic acid (PGA) was found as a proper material for chondrocyte adhesion but worse for their proliferation. Neither hyaluronic acid nor chitosan nor polyvinyl alcohol (PVA) increased chondrocyte adhesion. However, chondrocyte proliferation suffered from acidic byproducts of PGA degradation. However, the addition of PVA and/or chitosan into a wet-laid non-woven textile mesh from PGA improved chondrocyte proliferation seeded in vitro on the PGA-based composite scaffold namely due to a diminished acidification of their microenvironment. This PVA/PGA composite mesh used in combination with a proper hydrogel minimized the negative effect of PGA degradation without dropping positive parameters of the PGA wet-laid non-woven textile mesh. In fact, presence of PVA and/or chitosan in the PGA-based wet-laid non-woven textile mesh even advanced the PGA-based wet-laid non-woven textile mesh for chondrocyte seeding and artificial cartilage production due to a positive effect of PVA in such a scaffold on chondrocyte proliferation.

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

    NASA Astrophysics Data System (ADS)

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

    2016-09-01

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

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

    PubMed

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

    2016-01-01

    Coculture between mesenchymal stem cells (MSCs) and articular chondrocytes (ACs) represents a promising strategy for cartilage regeneration. This study aimed at elaborating how ACs were regulated by MSCs. Rabbit ACs (rACs) and rabbit MSCs (rMSCs) were seeded separately in a Transwell system to initiate non-contact coculture in growth medium without chondrogenic factors. Cell morphology, cell proliferation, production of extracellular matrix (ECM), and gene expression of rACs were characterized. Upon coculture, rACs underwent a morphological transition from a rounded or polygonal shape into a fibroblast-like one and proliferation was provoked simultaneously. Such effects were dependent on the amount of rMSCs. Along with these changes, ECM production and gene expression of rACs were also perturbed. Importantly, when a ROCK inhibitor (Y27632) was supplemented to coculture, the effects except that on cell proliferation were inhibited, suggesting the involvement of RhoA/ROCK signaling. By applying an inhibitor (BIBF1120) of VEGFR1/2/3, FGFR1/2/3 and PDGFRα/β in coculture, or supplementing FGF-1, VEGF-A and PDGFbb in monoculture, it was confirmed that the paracrine factors by rMSCs mediated the compounding effects on rACs. These findings shed light on MSCs-ACs interactions and might confer an insight view on cell-based cartilage regeneration. PMID:27596239

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

    PubMed Central

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

    2016-01-01

    Coculture between mesenchymal stem cells (MSCs) and articular chondrocytes (ACs) represents a promising strategy for cartilage regeneration. This study aimed at elaborating how ACs were regulated by MSCs. Rabbit ACs (rACs) and rabbit MSCs (rMSCs) were seeded separately in a Transwell system to initiate non-contact coculture in growth medium without chondrogenic factors. Cell morphology, cell proliferation, production of extracellular matrix (ECM), and gene expression of rACs were characterized. Upon coculture, rACs underwent a morphological transition from a rounded or polygonal shape into a fibroblast-like one and proliferation was provoked simultaneously. Such effects were dependent on the amount of rMSCs. Along with these changes, ECM production and gene expression of rACs were also perturbed. Importantly, when a ROCK inhibitor (Y27632) was supplemented to coculture, the effects except that on cell proliferation were inhibited, suggesting the involvement of RhoA/ROCK signaling. By applying an inhibitor (BIBF1120) of VEGFR1/2/3, FGFR1/2/3 and PDGFRα/β in coculture, or supplementing FGF-1, VEGF-A and PDGFbb in monoculture, it was confirmed that the paracrine factors by rMSCs mediated the compounding effects on rACs. These findings shed light on MSCs-ACs interactions and might confer an insight view on cell-based cartilage regeneration. PMID:27596239

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2015-11-01

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

  17. Determination of the Poisson's ratio of the cell: recovery properties of chondrocytes after release from complete micropipette aspiration.

    PubMed

    Trickey, Wendy R; Baaijens, Frank P T; Laursen, Tod A; Alexopoulos, Leonidas G; Guilak, Farshid

    2006-01-01

    Chondrocytes in articular cartilage are regularly subjected to compression and recovery due to dynamic loading of the joint. Previous studies have investigated the elastic and viscoelastic properties of chondrocytes using micropipette aspiration techniques, but in order to calculate cell properties, these studies have generally assumed that cells are incompressible with a Poisson's ratio of 0.5. The goal of this study was to measure the Poisson's ratio and recovery properties of the chondrocyte by combining theoretical modeling with experimental measures of complete cellular aspiration and release from a micropipette. Chondrocytes isolated from non-osteoarthritic and osteoarthritic cartilage were fully aspirated into a micropipette and allowed to reach mechanical equilibrium. Cells were then extruded from the micropipette and cell volume and morphology were measured throughout the experiment. This experimental procedure was simulated with finite element analysis, modeling the chondrocyte as either a compressible two-mode viscoelastic solid, or as a biphasic viscoelastic material. By fitting the experimental data to the theoretically predicted cell response, the Poisson's ratio and the viscoelastic recovery properties of the cell were determined. The Poisson's ratio of chondrocytes was found to be 0.38 for non-osteoarthritic cartilage and 0.36 for osteoarthritic chondrocytes (no significant difference). Osteoarthritic chondrocytes showed an increased recovery time following full aspiration. In contrast to previous assumptions, these findings suggest that chondrocytes are compressible, consistent with previous studies showing cell volume changes with compression of the extracellular matrix.

  18. The effect of radio-frequency glow discharge treatment of polystyrene on the behavior of porcine chondrocytes in vitro.

    PubMed

    Tsai, Wei-Bor; Wei, Ta-Chin; Lin, Mei-Chiao; Wang, Jie-Ying; Chen, Chun-Hong

    2005-01-01

    The aim of this study was to determine the effects of physicochemical surface properties of tissue-culture substrata on chondrocyte behavior. Polystyrene was modified by radio-frequency glow discharge (RFGD) plasma treatment with various monomers. The changes in surface properties of the modified polystyrene were verified by ESCA and water contact angle measurements. Porcine chondrocytes were seeded on these surfaces and cultured for 5 days. After 5 days of culture, the number of chondrocytes was highest on the N2 plasma-treated surface, followed by the CH2/N2 plasma-treated surface, untreated polystyrene and CF4 plasma-treated surface. The number of chondrocytes decreased with increasing water contact angle. The surface chemical properties influenced the morphology and gene expression of cultured chondrocytes. The cells cultured on the CF4 plasma-treated surface retained a round morphology characteristic of chondrocytes after day 1, while most of the cells grown on the N2 plasma-treated surface or the untreated polystyrene showed a flattened morphology. Using RT-PCR, expression of type-I collagen could not be detected in the chondrocytes cultured on the CF4 plasma-treated surface and the CH2/N2 plasma-treated surface. In contrast, the chondrocytes grown on the N2 plasma-treated surface or the untreated polystyrene surface expressed type-I collagen mRNA. This study shows that modification by RFGD treatment could modulate chondrocyte culture and gene expression. PMID:16028591

  19. Protective Effect of Resveratrol against IL-1β-Induced Inflammatory Response on Human Osteoarthritic Chondrocytes Partly via the TLR4/MyD88/NF-κB Signaling Pathway: An “in Vitro Study”

    PubMed Central

    Liu, Li; Gu, Hailun; Liu, Huimin; Jiao, Yongliang; Li, Keyu; Zhao, Yue; An, Li; Yang, Jun

    2014-01-01

    Resveratrol is a natural polyphenolic compound that prevents inflammation in chondrocytes and animal models of osteoarthritis (OA) via yet to be defined mechanisms. The purpose of this study was to determine whether the protective effect of resveratrol on IL-1β-induced human articular chondrocytes was associated with the TLR4/MyD88/NF-κB signaling pathway by incubating human articular chondrocytes (harvested from osteoarthritis patients) with IL-1β before treatment with resveratrol. Cell viability was evaluated using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay and TNFα levels in culture supernatants were measured by ELISA(Enzymelinked immunosorbent assay). The levels of TLR4 and its downstream signaling targets (MyD88 and TRAF6) and IL-1β were assessed by measuring the levels of mRNA and protein expression by real-time RT-PCR and western blot analysis, respectively, in addition to assessing NF-κB activation. In addition, TLR4 siRNA was used to block TLR4 expression in chondrocytes further demonstrating that resveratrol prevented IL-1β-mediated inflammation by TLR4 inhibition. We found that resveratrol prevented IL-1β-induced reduction in cell viability. Stimulation of chondrocytes with IL-1β caused a significant up-regulation of TLR4 and its downstream targets MyD88 and TRAF6 resulting in NF-κB activation associated with the synthesis of IL-1β and TNFα. These IL-1β-induced inflammatory responses were all effectively reversed by resveratrol. Furthermore, activation of NF-κB in chondrocytes treated with TLR4 siRNA was significantly attenuated, but not abolished, and exposure to resveratrol further reduced NF-κB translocation. These data suggested that resveratrol prevented IL-1β-induced inflammation in human articular chondrocytes at least in part by inhibiting the TLR4/MyD88/NF-κB signaling pathway suggesting that resveratrol has the potential to be used as a nutritional supplement to counteract OA symptoms. PMID

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

    PubMed

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

    2006-08-01

    Mechanical stimulation is known to be an essential factor in the regulation of cartilage metabolism. We tested the hypothesis that expression of lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) can be modulated by cyclic tensile stretch load in chondrocytes. Cyclic loading of repeated stretch stress at 10 cycles per minute with 10 kPa of stress for 6 h induced expression of LOX-1 to 2.6 times control in cultured bovine articular chondrocytes, equivalent to the addition of 10 microg/mL oxidized low density lipoprotein (ox-LDL) (2.4 times control). Application of the cyclic load to the chondrocytes along with 10 microg/mL ox-LDL resulted in synergistically increased LOX-1 expression to 6.3 times control. Individual application of cyclic loading and 10 microg/mL ox-LDL significantly suppressed chondrocytes viability (84.6% +/- 3.4% and 80.9% +/- 3.2% of control at 24 h, respectively; n = 3; p < 0.05) and proteoglycan synthesis [81.0% +/- 7.1% and 85.7% +/- 5.2% of control at 24 h, respectively; p < 0.05 when compared with 94.6% +/- 4.6% for native-LDL (n = 3)]. Cyclic loading and 10 microg/mL ox-LDL synergistically affected cell viability and proteoglycan synthesis, which were significantly suppressed to 45.6% +/- 4.9% and 48.7% +/- 6.7% of control at 24 h, respectively (n = 3; p < 0.01 when compared with individual application of cyclic loading or 10 microg/mL ox-LDL). In this study, we demonstrated synergistic effects of cyclic tensile stretch load and ox-LDL on cell viability and proteoglycan synthesis in chondrocytes, which may be mediated through enhanced expression of LOX-1 and which has important implications in the progression of cartilage degeneration in osteoarthritis.

  1. Dual pathways to endochondral osteoblasts: a novel chondrocyte-derived osteoprogenitor cell identified in hypertrophic cartilage

    PubMed Central

    Park, Jung; Gebhardt, Matthias; Golovchenko, Svitlana; Perez-Branguli, Francesc; Hattori, Takako; Hartmann, Christine; Zhou, Xin; deCrombrugghe, Benoit; Stock, Michael; Schneider, Holm; von der Mark, Klaus

    2015-01-01

    According to the general understanding, the chondrocyte lineage terminates with the elimination of late hypertrophic cells by apoptosis in the growth plate. However, recent cell tracking studies have shown that murine hypertrophic chondrocytes can survive beyond “terminal” differentiation and give rise to a progeny of osteoblasts participating in endochondral bone formation. The question how chondrocytes convert into osteoblasts, however, remained open. Following the cell fate of hypertrophic chondrocytes by genetic lineage tracing using BACCol10;Cre induced YFP-reporter gene expression we show that a progeny of Col10Cre-reporter labelled osteoprogenitor cells and osteoblasts appears in the primary spongiosa and participates – depending on the developmental stage – substantially in trabecular, endosteal, and cortical bone formation. YFP+ trabecular and endosteal cells isolated by FACS expressed Col1a1, osteocalcin and runx2, thus confirming their osteogenic phenotype. In searching for transitory cells between hypertrophic chondrocytes and trabecular osteoblasts we identified by confocal microscopy a novel, small YFP+Osx+ cell type with mitotic activity in the lower hypertrophic zone at the chondro-osseous junction. When isolated from growth plates by fractional enzymatic digestion, these cells termed CDOP (chondrocyte-derived osteoprogenitor) cells expressed bone typical genes and differentiated into osteoblasts in vitro. We propose the Col10Cre-labeled CDOP cells mark the initiation point of a second pathway giving rise to endochondral osteoblasts, alternative to perichondrium derived osteoprogenitor cells. These findings add to current concepts of chondrocyte-osteocyte lineages and give new insight into the complex cartilage-bone transition process in the growth plate. PMID:25882555

  2. Investigation of the Effects of Extracellular Osmotic Pressure on Morphology and Mechanical Properties of Individual Chondrocyte.

    PubMed

    Nguyen, Trung Dung; Oloyede, Adekunle; Singh, Sanjleena; Gu, YuanTong

    2016-06-01

    It has been demonstrated that most cells of the body respond to osmotic pressure in a systematic manner. The disruption of the collagen network in the early stages of osteoarthritis causes an increase in water content of cartilage which leads to a reduction of pericellular osmolality in chondrocytes distributed within the extracellular environment. It is therefore arguable that an insight into the mechanical properties of chondrocytes under varying osmotic pressure would provide a better understanding of chondrocyte mechanotransduction and potentially contribute to knowledge on cartilage degeneration. In this present study, the chondrocyte cells were exposed to solutions with different osmolality. Changes in their dimensions and mechanical properties were measured over time. Atomic force microscopy (AFM) was used to apply load at various strain-rates and the force-time curves were logged. The thin-layer elastic model was used to extract the elastic stiffness of chondrocytes at different strain-rates and at different solution osmolality. In addition, the porohyperelastic (PHE) model was used to investigate the strain-rate-dependent responses under the loading and osmotic pressure conditions. The results revealed that the hypo-osmotic external environment increased chondrocyte dimensions and reduced Young's modulus of the cells at all strain-rates tested. In contrast, the hyper-osmotic external environment reduced dimensions and increased Young's modulus. Moreover, using the PHE model coupled with inverse FEA simulation, we established that the hydraulic permeability of chondrocytes increased with decreasing extracellular osmolality which is consistent with previous work in the literature. This could be due to a higher intracellular fluid volume fraction with lower osmolality.

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

    NASA Astrophysics Data System (ADS)

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

    2016-05-01

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

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

    PubMed

    Huang, Hao; Quan, Ying-Yao; Wang, Xiao-Ping; Chen, Tong-Sheng

    2016-12-01

    Gold nanoparticles (AuNPs) have been widely used in biomedical science including antiarthritic agents, drug loading, and photothermal therapy. In this report, we studied the effects of AuNPs with diameters of 3, 13, and 45 nm, respectively, on rabbit articular chondrocytes. AuNPs were capped with citrate and their diameter and zeta potential were measured by dynamic light scattering (DLS). Cell viability was evaluated by Cell Counting Kit-8 (CCK-8) assay after the rabbit articular chondrocytes were pre-incubated with 3, 13, and 45 nm AuNPs, respectively, for 24 h. Flow cytometry (FCM) analysis with annexin V/propidium iodide (PI) double staining and fluorescence imaging with Hoechst 33258 staining were used to determine the fashion of AuNPs-induced chondrocyte death. Further, 13 nm AuNPs (2 nM) significantly induced chondrocyte death accompanying apoptotic characteristics including mitochondrial damage, externalization of phosphatidylserine and nuclear concentration. However, 3 nm AuNPs (2 nM) and 45 nm (0.02 nM) AuNPs did not induce cytotoxicity in chondrocytes. Although 13 nm AuNPs (2 nM) increased the intracellular reactive oxygen species (ROS) level, pretreatment with Nacetyl cysteine (NAC), a ROS scavenger, did not prevent the cytotoxicity induced by 13 nm AuNPs, indicating that 13 nm AuNPs (2 nM) induced ROS-independent apoptosis in chondrocytes. These results demonstrate the size-dependent cytotoxicity of AuNPs in chondrocytes, which must be seriously considered when using AuNPs for treatment of osteoarthritis (OA). PMID:27178054

  5. Investigation of the Effects of Extracellular Osmotic Pressure on Morphology and Mechanical Properties of Individual Chondrocyte.

    PubMed

    Nguyen, Trung Dung; Oloyede, Adekunle; Singh, Sanjleena; Gu, YuanTong

    2016-06-01

    It has been demonstrated that most cells of the body respond to osmotic pressure in a systematic manner. The disruption of the collagen network in the early stages of osteoarthritis causes an increase in water content of cartilage which leads to a reduction of pericellular osmolality in chondrocytes distributed within the extracellular environment. It is therefore arguable that an insight into the mechanical properties of chondrocytes under varying osmotic pressure would provide a better understanding of chondrocyte mechanotransduction and potentially contribute to knowledge on cartilage degeneration. In this present study, the chondrocyte cells were exposed to solutions with different osmolality. Changes in their dimensions and mechanical properties were measured over time. Atomic force microscopy (AFM) was used to apply load at various strain-rates and the force-time curves were logged. The thin-layer elastic model was used to extract the elastic stiffness of chondrocytes at different strain-rates and at different solution osmolality. In addition, the porohyperelastic (PHE) model was used to investigate the strain-rate-dependent responses under the loading and osmotic pressure conditions. The results revealed that the hypo-osmotic external environment increased chondrocyte dimensions and reduced Young's modulus of the cells at all strain-rates tested. In contrast, the hyper-osmotic external environment reduced dimensions and increased Young's modulus. Moreover, using the PHE model coupled with inverse FEA simulation, we established that the hydraulic permeability of chondrocytes increased with decreasing extracellular osmolality which is consistent with previous work in the literature. This could be due to a higher intracellular fluid volume fraction with lower osmolality. PMID:26831866

  6. Matrix assisted autologous chondrocyte transplantation for cartilage treatment

    PubMed Central

    Kon, E.; Filardo, G.; Di Matteo, B.; Perdisa, F.; Marcacci, M.

    2013-01-01

    Objectives Matrix-assisted autologous chondrocyte transplantation (MACT) has been developed and applied in the clinical practice in the last decade to overcome most of the disadvantages of the first generation procedures. The purpose of this systematic review is to document and analyse the available literature on the results of MACT in the treatment of chondral and osteochondral lesions of the knee. Methods All studies published in English addressing MACT procedures were identified, including those that fulfilled the following criteria: 1) level I-IV evidence, 2) measures of functional or clinical outcome, 3) outcome related to cartilage lesions of the knee cartilage. Results The literature analysis showed a progressively increasing number of articles per year. A total of 51 articles were selected: three randomised studies, ten comparative studies, 33 case series and five case reports. Several scaffolds have been developed and studied, with good results reported at short to medium follow-up. Conclusions MACT procedures are a therapeutic option for the treatment of chondral lesions that can offer a positive outcome over time for specific patient categories, but high-level studies are lacking. Systematic long-term evaluation of these techniques and randomised controlled trials are necessary to confirm the potential of this treatment approach, especially when comparing against less ambitious traditional treatments. PMID:23610698

  7. Self-assembled octapeptide scaffolds for in vitro chondrocyte culture.

    PubMed

    Mujeeb, Ayeesha; Miller, Aline F; Saiani, Alberto; Gough, Julie E

    2013-01-01

    Nature has evolved a variety of creative approaches to many aspects of materials synthesis and microstructural control. Molecular self-assembly is a simple and efficient way to fabricate complex nanostructures such as hydrogels. We have recently investigated the gelation properties of a series of ionic-complementary peptides based on the alternation of non-polar hydrophobic and polar hydrophilic residues. In this work we focus on one specific octapeptide, FEFEFKFK (F, phenylalanine; E, glutamic acid; K, lysine). This peptide was shown to self-assemble in solution and form β-sheet-rich nanofibres which, above a critical gelation concentration, entangle to form a self-supporting hydrogel. The fibre morphology of the hydrogel was analysed using transmission electron microscopy and cryo-scanning electron microscopy illustrating a dense fibrillar network of nanometer size fibres. Oscillatory rheology results show that the hydrogel possesses visco-elastic properties. Bovine chondrocytes were used to assess the biocompatibility of the scaffolds over 21 days under two-dimensional (2-D) and three-dimensional (3-D) cell culture conditions, particularly looking at cell morphology, proliferation and matrix deposition. 2-D culture resulted in cell viability and collagen type I deposition. In 3-D culture the mechanically stable gel was shown to support the viability of cells, the retention of cell morphology and collagen type II deposition. Subsequently the scaffold may serve as a template for cartilage tissue engineering.

  8. Growth factor effects on costal chondrocytes for tissue engineering fibrocartilage.

    PubMed

    Johns, D E; Athanasiou, K A

    2008-09-01

    Tissue-engineered fibrocartilage could become a feasible option for replacing tissues such as the knee meniscus or temporomandibular joint disc. This study employed five growth factors (insulin-like growth factor-I, transforming growth factor-beta1, epidermal growth factor, platelet-derived growth factor-BB, and basic fibroblast growth factor) in a scaffoldless approach with costal chondrocytes, attempting to improve biochemical and mechanical properties of engineered constructs. Samples were quantitatively assessed for total collagen, glycosaminoglycans, collagen type I, collagen type II, cells, compressive properties, and tensile properties at two time points. Most treated constructs had lower biomechanical and biochemical properties than the controls with no growth factors, suggesting a detrimental effect, but the treatment with insulin-like growth factor-I tended to improve the constructs. Additionally, the 6-week time point was consistently better than that at 3 weeks, with total collagen, glycosaminoglycans, and aggregate modulus doubling during this time. Further optimization of the time in culture and exogenous stimuli will be important in making a more functional replacement tissue. PMID:18597118

  9. Chondrocyte distribution in the articular cartilage of human femoral condyles.

    PubMed Central

    Gilmore, R S; Palfrey, A J

    1988-01-01

    The distribution of chondrocytes throughout the total thickness of articular cartilage from the femoral condyles of infants, children and adults has been studied using serial sections cut parallel as well as perpendicular to the articular surface. The thickness of the articular cartilage was estimated in fixed sections. In one of the adult specimens, the thickness of the articular cartilage was estimated firstly by direct measurement of the cut surfaces of a series of blocks cut from both condyles and then from the number of parallel sections of the cartilage prepared from those blocks. Cell density was highest in the superficial zone of all specimens examined, declining to lower values in the deep zone of the cartilage. Within this pattern the infant specimens had the highest values for cell density and the adults the lowest, with values for children in an intermediate range. There was no significant variation in cell density across the condyles of the selected adult specimen. The absolute values for cartilage thickness depended on the method used, but in general total thickness was found to approximately double from late gestation to maturity. In the selected adult specimen, the cartilage was thickest just anterior and posterior to the main weight-bearing area of the condyles. PMID:3198480

  10. 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

  11. Bone-forming capacity of adult human nasal chondrocytes

    PubMed Central

    Pippenger, Benjamin E; Ventura, Manuela; Pelttari, Karoliina; Feliciano, Sandra; Jaquiery, Claude; Scherberich, Arnaud; Walboomers, X Frank; Barbero, Andrea; Martin, Ivan

    2015-01-01

    Nasal chondrocytes (NC) derive from the same multipotent embryological segment that gives rise to the majority of the maxillofacial bone and have been reported to differentiate into osteoblast-like cells in vitro. In this study, we assessed the capacity of adult human NC, appropriately primed towards hypertrophic or osteoblastic differentiation, to form bone tissue in vivo. Hypertrophic induction of NC-based micromass pellets formed mineralized cartilaginous tissues rich in type X collagen, but upon implantation into subcutaneous pockets of nude mice remained avascular and reverted to stable hyaline-cartilage. In the same ectopic environment, NC embedded into ceramic scaffolds and primed with osteogenic medium only sporadically formed intramembranous bone tissue. A clonal study could not demonstrate that the low bone formation efficiency was related to a possibly small proportion of cells competent to become fully functional osteoblasts. We next tested whether the cues present in an orthotopic environment could induce a more efficient direct osteoblastic transformation of NC. Using a nude rat calvarial defect model, we demonstrated that (i) NC directly participated in frank bone formation and (ii) the efficiency of survival and bone formation by NC was significantly higher than that of reference osteogenic cells, namely bone marrow-derived mesenchymal stromal cells. This study provides a proof-of-principle that NC have the plasticity to convert into bone cells and thereby represent an easily available cell source to be further investigated for craniofacial bone regeneration. PMID:25689393

  12. Association of Reduced Type IX Collagen Gene Expression in Human Osteoarthritic Chondrocytes With Epigenetic Silencing by DNA Hypermethylation

    PubMed Central

    Imagawa, Kei; de Andrés, María C; Hashimoto, Ko; Itoi, Eiji; Otero, Miguel; Roach, Helmtrud I; Goldring, Mary B; Oreffo, Richard O C

    2014-01-01

    Objective To investigate whether the changes in collagen gene expression in osteoarthritic (OA) human chondrocytes are associated with changes in the DNA methylation status in the COL2A1 enhancer and COL9A1 promoter. Methods Expression levels were determined using quantitative reverse transcription–polymerase chain reaction, and the percentage of DNA methylation was quantified by pyrosequencing. The effect of CpG methylation on COL9A1 promoter activity was determined using a CpG-free vector; cotransfections with expression vectors encoding SOX9, hypoxia-inducible factor 1α (HIF-1α), and HIF-2α were carried out to analyze COL9A1 promoter activities in response to changes in the methylation status. Chromatin immunoprecipitation assays were carried out to validate SOX9 binding to the COL9A1 promoter and the influence of DNA methylation. Results Although COL2A1 messenger RNA (mRNA) levels in OA chondrocytes were 19-fold higher than those in the controls, all of the CpG sites in the COL2A1 enhancer were totally demethylated in both samples. The levels of COL9A1 mRNA in OA chondrocytes were 6,000-fold lower than those in controls; 6 CpG sites of the COL9A1 promoter were significantly hypermethylated in OA patients as compared with controls. Treatment with 5-azadeoxycitidine enhanced COL9A1 gene expression and prevented culture-induced hypermethylation. In vitro methylation decreased COL9A1 promoter activity. Mutations in the 5 CpG sites proximal to the transcription start site decreased COL9A1 promoter activity. Cotransfection with SOX9 enhanced COL9A1 promoter activity; CpG methylation attenuated SOX9 binding to the COL9A1 promoter. Conclusion This first demonstration that hypermethylation is associated with down-regulation of COL9A1 expression in OA cartilage highlights the pivotal role of epigenetics in OA, involving not only hypomethylation, but also hypermethylation, with important therapeutic implications for OA treatment. PMID:25048791

  13. 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

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

    PubMed

    Ruan, Merry Zc; Cerullo, Vincenzo; Cela, Racel; Clarke, Chris; Lundgren-Akerlund, Evy; Barry, Michael A; Lee, Brendan Hl

    2016-01-01

    Osteoarthritis (OA) is a joint disease characterized by degeneration of the articular cartilage, subchondral bone remodeling, and secondary inflammation. It is among the top three causes of chronic disability, and currently there are no treatment options to prevent disease progression. The localized nature of OA makes it an ideal candidate for gene and cell therapy. However, gene and cell therapy of OA is impeded by inefficient gene transduction of chondrocytes. In this study, we developed a broadly applicable system that retargets cell surface receptors by conjugating antibodies to the capsid of helper-dependent adenoviral vectors (HDVs). Specifically, we applied this system to retarget chondrocytes by conjugating an HDV to an α-10 integrin monoclonal antibody (a10mab). We show that a10mab-conjugated HDV (a10mabHDV)-infected chondrocytes efficiently in vitro and in vivo while detargeting other cell types. The therapeutic index of an intra-articular injection of 10mabHDV-expressing proteoglycan 4 (PRG4) into a murine model of post-traumatic OA was 10-fold higher than with standard HDV. Moreover, we show that PRG4 overexpression from articular, superficial zone chondrocytes is effective for chondroprotection in postinjury OA and that α-10 integrin is an effective protein for chondrocyte targeting. PMID:27626040

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

    PubMed Central

    Ruan, Merry ZC; Cerullo, Vincenzo; Cela, Racel; Clarke, Chris; Lundgren-Akerlund, Evy; Barry, Michael A; Lee, Brendan HL

    2016-01-01

    Osteoarthritis (OA) is a joint disease characterized by degeneration of the articular cartilage, subchondral bone remodeling, and secondary inflammation. It is among the top three causes of chronic disability, and currently there are no treatment options to prevent disease progression. The localized nature of OA makes it an ideal candidate for gene and cell therapy. However, gene and cell therapy of OA is impeded by inefficient gene transduction of chondrocytes. In this study, we developed a broadly applicable system that retargets cell surface receptors by conjugating antibodies to the capsid of helper-dependent adenoviral vectors (HDVs). Specifically, we applied this system to retarget chondrocytes by conjugating an HDV to an α-10 integrin monoclonal antibody (a10mab). We show that a10mab-conjugated HDV (a10mabHDV)-infected chondrocytes efficiently in vitro and in vivo while detargeting other cell types. The therapeutic index of an intra-articular injection of 10mabHDV-expressing proteoglycan 4 (PRG4) into a murine model of post-traumatic OA was 10-fold higher than with standard HDV. Moreover, we show that PRG4 overexpression from articular, superficial zone chondrocytes is effective for chondroprotection in postinjury OA and that α-10 integrin is an effective protein for chondrocyte targeting. PMID:27626040

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

    PubMed

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

    2016-02-01

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

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

    NASA Astrophysics Data System (ADS)

    Puhakka, P. H.; Ylärinne, J. H.; Lammi, M. J.; Saarakkala, S.; Tiitu, V.; Kröger, H.; Virén, T.; Jurvelin, J. S.; Töyräs, J.

    2014-11-01

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

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

    PubMed

    Puhakka, P H; Ylärinne, J H; Lammi, M J; Saarakkala, S; Tiitu, V; Kröger, H; Virén, T; Jurvelin, J S; Töyräs, J

    2014-11-01

    Optical coherence tomography (OCT) has been applied for high resolution imaging of articular cartilage. However, the contribution of individual structural elements of cartilage on OCT signal has not been thoroughly studied. We hypothesize that both collagen and chondrocytes, essential structural components of cartilage, act as important light scatterers and that variation in their concentrations can be detected by OCT through changes in backscattering and attenuation. To evaluate this hypothesis, we established a controlled model system using agarose scaffolds embedded with variable collagen concentrations and chondrocyte densities. Using OCT, we measured the backscattering coefficient (µb) and total attenuation coefficient (µt) in these scaffolds. Along our hypothesis, light backscattering and attenuation in agarose were dependent on collagen concentration and chondrocyte density. Significant correlations were found between µt and chondrocyte density (ρ = 0.853, p < 0.001) and between µt and collagen concentration (ρ = 0.694, p < 0.001). µb correlated significantly with chondrocyte density (ρ = 0.504, p < 0.001) but not with collagen concentration (ρ = 0.103, p = 0.422) of the scaffold. Thus, quantitation of light backscattering and, especially, attenuation could be valuable when evaluating the integrity of soft tissues, such as articular cartilage with OCT. PMID:25310088

  19. Periodic rewetting enhances the viability of chondrocytes in human articular cartilage exposed to air.

    PubMed

    Pun, S Y; Teng, M S; Kim, H T

    2006-11-01

    Desiccation of articular cartilage during surgery is often unavoidable and may result in the death of chondrocytes, with subsequent joint degeneration. This study was undertaken to determine the extent of chondrocyte death caused by exposure to air and to ascertain whether regular rewetting of cartilage could decrease cell death. Macroscopically normal human cartilage was exposed to air for 0, 30, 60 or 120 minutes. Selected samples were wetted in lactated Ringer's solution for ten seconds every ten or 20 minutes. The viability of chondrocytes was measured after three days by Live/Dead staining. Chondrocyte death correlated with the length of exposure to air and the depth of the cartilage. Drying for 120 minutes caused extensive cell death mainly in the superficial 500 microm of cartilage. Rewetting every ten or 20 minutes significantly decreased cell death. The superficial zone is most susceptible to desiccation. Loss of superficial chondrocytes likely decreases the production of essential lubricating glycoproteins and contributes to subsequent degeneration. Frequent wetting of cartilage during arthrotomy is therefore essential.

  20. Biological Effects of the Herbal Plant-Derived Phytoestrogen Bavachin in Primary Rat Chondrocytes.

    PubMed

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

    2015-01-01

    The aim of this study was to examine the anabolic and anticatabolic functions of bavachin in primary rat chondrocytes. With bavachin treatment, chondrocytes survived for 21 d without cell proliferation, and the proteoglycan content and extracellular matrix increased. Short-term monolayer culture of chondrocytes showed that gene induction of both aggrecan and collagen type II, major extracellular matrix components, was significantly upregulated by bavachin. The expression and activities of cartilage-degrading enzymes such as matrix metalloproteinases and a disintegrin and metalloproteinase with thrombospondin motifs were inhibited significantly by bavachin, while tissue inhibitors of metalloprotease were significantly upregulated. Bavachin inhibits the expression of inducible nitric oxide synthase, a representative catabolic factor, and downregulated the expression of nitric oxide, cyclooxygenase-2, and prostaglandin E2 in a dose-dependent manner in chondrocytes. Our results suggest that the bavachin has anabolic and potent anticatabolic biological effects on chondrocytes, which may have considerable promise in treating articular cartilage degeneration in the future. PMID:26235583

  1. Metabolic characteristics of in vitro cultured human chondrocytes in relation to the histopathologic grade of osteoarthritis.

    PubMed

    Bulstra, S K; Buurman, W A; Walenkamp, G H; Van der Linden, A J

    1989-05-01

    Isolated human chondrocytes derived from healthy and osteoarthritic (OA) cartilage were cultured in high density in a newly designed microculture system. The severity of OA was graded according to a modified histopathologic score originally described by Mankin et al. Chondrocytes from adult patients with OA showed 35S-sulphate and 3H-thymidine incorporation in vitro, which increased with severity of the disease through Mankin 11-12. Incorporation rapidly declined after Mankin 11-12. Both matrix synthesis and cell proliferation were strongly reduced in the severe grades of OA. Histologic examination of the newly formed cartilage was only possible if the chondrocytes were derived from less severe grades of OA. Microscopy showed healthy chondrocytes surrounded by newly synthesized matrix, which stained well with specific dyes, indicating the ability of the cells to synthesize normal matrix components. The phenotype of human articular chondrocytes, derived from different grades of OA, was maintained in a high-density culture system. The data suggest dysregulation of the cell metabolism in OA cartilage. The increased cell metabolism was directly related to the histopathologic grade of OA. PMID:2706860

  2. 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

  3. 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.

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

    PubMed Central

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

    2016-01-01

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

  5. 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 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.

  6. Lead Induces an Osteoarthritis-like Phenotype in Articular Chondrocytes through Disruption of TGF-β Signaling

    PubMed Central

    Holz, Jonathan D.; Beier, Eric; Sheu, Tzong-Jen; Ubayawardena, Resika; Wang, Meina; Sampson, Erik R.; Rosier, Randy N.; Zuscik, Michael; Puzas, J. Edward

    2013-01-01

    Lead remains a significant environmental toxin, and we believe we may have identified a novel target of lead toxicity in articular chondrocytes. These cells are responsible for the maintenance of joint matrix, and do so under the regulation of TGF-β signaling. As lead is concentrated in articular cartilage, we hypothesize that it can disrupt normal chondrocyte phenotype through suppression of TGF-β signaling. These experiments examine the effects of lead exposure in vivo and in vitro at biologically-relevant levels, from 1nM–10µM on viability, collagen levels, matrix degrading enzyme activity, TGF-β signaling, and articular surface morphology. Our results indicate that viability was unchanged at levels ≤100µM Pb, but low and high level lead in vivo exposure resulted in fibrillation and degeneration of the articular surface. Lead treatment also decreased levels of type II collagen and increased type X collagen, in vivo and in vitro. Additionally, MMP13 activity increased in a dose-dependent manner. Active caspase3 and 8 were dose-dependently elevated, and treatment with 10µM Pb resulted in increases of 30% and 500%, respectively. Increasing lead treatment resulted in a corresponding reduction in TGF-β reporter activity, with a 95% reduction at 10µM. Levels of phosphoSmad2 and 3 were suppressed in vitro and in vivo and lead dose-dependently increased Smurf2. These changes closely parallel those seen in osteoarthritis. Over time this phenotypic shift could compromise maintenance of the joint matrix. PMID:22517267

  7. Synthesis and extracellular deposition of fibronectin in chondrocyte cultures. Response to the removal of extracellular cartilage matrix

    PubMed Central

    1978-01-01

    Fibronectin, the major cell surface glycoprotein of fibroblasts, is absent from differentiated cartilage matrix and chondrocytes in situ. However, dissociation of embryonic chick sternal cartilage with collagenase and trypsin, followed by inoculation in vitro reinitiates fibronectin synthesis by chondrocytes. Immunofluorescence microscopy with antibodies prepared against plasma fibronectin (cold insoluble globulin [CIG]) reveals fibronectin associated with the chondrocyte surface. Synthesis and secretion of fibronectin into the medium are shown by anabolic labeling with [35S]methionine or [3H]glycine, and identification of the secreted proteins by immunoprecipitation and sodium dodecyl sulfate (SDS)-disc gel electrophoresis. When chondrocytes are plated onto tissue culture dishes, the pattern of surface-associated fibronectin changes from a patchy into a strandlike appearance. Where epithelioid clones of polygonal chondrocytes develop, only short strands of fibronectin appear preferentially at cellular interfaces. This pattern is observed as long as cells continue to produce type II collagen that fails to precipitate as extracellular collagen fibers for some time in culture. Using the immunofluorescence double-labeling technique, we demonstrate that fibroblasts as well as chondrocytes which synthesize type I collagen and deposit this collagen as extracellular fibers show a different pattern of extracellular fibronectin that codistributes in large parts with collagen fibers. Where chondrocytes begin to accumulate extracellular cartilage matrix, fibronectin strands disappear. From these observations, we conclude (a) that chondrocytes synthesize fibronectin only in the absence of extracellular cartilage matrix, and (b) that fibronectin forms only short intercellular "stitches" in the absence of extracellular collagen fibers in vitro. PMID:363726

  8. Coordination of chondrocyte differentiation and joint formation by alpha5beta1 integrin in the developing appendicular skeleton.

    PubMed

    Garciadiego-Cázares, David; Rosales, Carlos; Katoh, Masaru; Chimal-Monroy, Jesús

    2004-10-01

    The control point by which chondrocytes take the decision between the cartilage differentiation program or the joint formation program is unknown. Here, we have investigated the effect of alpha5beta1 integrin inhibitors and bone morphogenetic protein (BMP) on joint formation. Blocking of alpha5beta1 integrin by specific antibodies or RGD peptide (arginine-glycine-aspartic acid) induced inhibition of pre-hypertrophic chondrocyte differentiation and ectopic joint formation between proliferating chondrocytes and hypertrophic chondrocytes. Ectopic joint expressed Wnt14, Gdf5, chordin, autotaxin, type I collagen and CD44, while expression of Indian hedgehog and type II collagen was downregulated in cartilage. Expression of these interzone markers confirmed that the new structure is a new joint being formed. In the presence of BMP7, inhibition of alpha5beta1 integrin function still induced the formation of the ectopic joint between proliferating chondrocytes and hypertrophic chondrocytes. By contrast, misexpression of alpha5beta1 integrin resulted in fusion of joints and formation of pre-hypertrophic chondrocytes. These facts indicate that the decision of which cell fate to make pre-joint or pre-hypertrophic is made on the basis of the presence or absence of alpha5beta1 integrin on chondrocytes.

  9. 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.

  10. Evaluation of thermoreversible polymers containing fibroblast growth factor 9 (FGF-9) for chondrocyte culture

    SciTech Connect

    Au, Angela; Ha, Jinny; Polotsky, Anna; Krzyminski, Karol J.; Gutowska, Anna; Hungerford, Davis S.; Frondoza, Carmelita G.

    2004-05-01

    We have evaluated a biomaterial to serve as a scaffold for the propagation and amplification of chondrocytes that promotes the original cellular phenotype of these cells. The goal of the present study was to investigate the use of thermally reversible polymer gels poly(NiPAAm-co-AAc), as a biocompatible supporting scaffold for the propagation of chondrocytic cells. The polymer gels at temperatures above its lower critical solution temperature (LCST) while liquefying at temperatures below its LCST of 34.5 C. Hence, the polymer, in its gelled form, has the ability to hold cells in situ, forming a matrix similar to the natural cellular environment or the extracellular matrix that comprises cartilage. We tested the hypothesis that the polymer gel promotes cell viability and function. Human osteoblast-like cells, nasal chondrocytes, and articular chondrocytes (1x105/150 ?l) were re-suspended in enriched DMEM media and were plated onto control (without gel) and gel containing 24-well plates. The plates were re-incubated at 37 C, 5% CO2 for the time-point of interest. Additional media was added to the plates and exchanged as needed. Following cell culture, cells were retrieved, enumerated, and cell viability was determined. Other aliquots of the cells were stained for morphological analysis while expression of chondrocyte markers including collagen type II and aggrecan were determined using Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR). The polymer gel was not cytotoxic as the cell number retrieved from three-dimensional culture gel was found to be one to two times higher than that retrieved from monolayer culture. Chondrocytes propagated in the thermo-reversible polymers expressed enhanced or maintained expression of collagen type II and aggrecan. Collagen type I expression was decreased or unaltered. The N-isopropylacrylamide and acrylic acid copolymer gel has potential use as a cell culture substrate and as a cell delivery vehicle.

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

    PubMed Central

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

    2014-01-01

    Objective: A recent report identified the small molecule kartogenin as a chondrogenic and chondroprotective agent. Since changes in hyaluronan metabolism occur during cartilage degeneration in osteoarthritis, we began studies to determine whether there was a connection between extracellular hyaluronan, CD44–hyaluronan interactions and the effects of kartogenin on articular chondrocytes. Methods: Chondrocytes cultured in monolayers, bioengineered neocartilages, or cartilage explants were treated with kartogenin with or without stimulation by IL-1β. Accumulation of matrix was visualized by a particle exclusion assay or by safranin O staining and release of sulfated glycosaminoglycans was determined. Production of aggrecanases and aggrecan G1-ITEGE neoepitope, fragmentation of CD44 and the SMAD1/5/8 signaling pathway were evaluated by western blotting. Results: Kartogenin treatment enhanced chondrocyte pericellular matrix assembly and retention in the presence of IL-1β. The chondroprotective effects of kartogenin on IL-1β-induced release of sulfated glycosaminoglycans from articular cartilage explants, reduction in safranin O staining of neocartilage discs as well as a reduction in aggrecan G1-ITEGE neoepitope in chondrocyte and explant cartilage cultures were observed. Kartogenin partially blocked the IL-1β-induced increased expression of ADAMTS-5. Additionally, kartogenin-treated articular chondrocytes exhibited a decrease in CD44 proteolytic fragmentation. However, kartogenin treatment did not enhance proteoglycan in control, non-IL-1β-treated cultures. Similarly, kartogenin enhanced the SMAD1 phosphorylation but only following pretreatment with IL-1β. Conclusion: These studies provide novel information on the chondroprotective function of kartogenin in adult articular cartilage. The effects of kartogenin are significant after activation of chondrocytic chondrolysis, which may occur following disruption of homeostasis maintained by hyaluronan–CD44

  12. Cartilage Abnormalities Associated with Defects of Chondrocytic Primary Cilia in Bardet-Biedl Syndrome Mutant Mice

    PubMed Central

    Kaushik, Anjan P.; Martin, James A.; Zhang, Qihong; Sheffield, Val C.; Morcuende, Jose A.

    2013-01-01

    SUMMARY Primary cilia are found on nearly every mammalian cell, including osteocytes, fibroblasts, and chondrocytes. However, the functions of primary cilia have not been extensively studied in these cells, particularly chondrocytes. Interestingly, defects in the primary cilium result in skeletal defects such as polydactyly in Bardet-Biedl Syndrome (BBS), a ciliary disorder that also results in obesity, retinopathy, and cognitive impairments (1–4). Wild-type mice and mutant mice of the ciliary proteins Bbs1, Bbs2, and Bbs6 were evaluated with respect to histological and biochemical differences in chondrocytes from articular cartilage and xiphoid processes. Using immunofluorescence microscopy, chondrocytic cilia were visualized from the load-bearing joints and non-load-bearing xiphoid processes. Significant differences in ciliary morphology were not identified between mutant and wild-type mice. However, after expanding chondrocytes in cell culture and implanting them in solid agarose matrix, it was seen that the fraction of ciliated cells in cultures from mutant mice was significantly lower than in the wild-type cultures (p<.05). In addition, in Safranin-O-stained whole joint sections, Bbs mutant mice had significantly lower articular joint thickness (p<.05) and lower proteoglycan content saturation (p<.05) than wild-type mice. Moreover, there were statistically significant differences of cell distribution between Bbs mutant and wild-type mice (p<.05), indicating that mutant articular cartilage had changes consistent with early signs of osteoarthritis. These data indicate that Bbs genes and their functions in the chondrocytic primary cilium are important for normal articular cartilage maintenance. PMID:19195025

  13. ROCK inhibition enhances aggrecan deposition and suppresses matrix metalloproteinase-3 production in human articular chondrocytes.

    PubMed

    Furumatsu, Takayuki; Matsumoto-Ogawa, Emi; Tanaka, Takaaki; Lu, Zhichao; Ozaki, Toshifumi

    2014-04-01

    Homeostasis of articular cartilage is maintained by a balance between catabolism and anabolism. Matrix metalloproteinase-3 (MMP-3) catabolism of cartilaginous extracellular matrix (ECM), including aggrecan (AGN), is an important factor in osteoarthritis progression. We previously reported that inhibition of Rho-associated coiled-coil forming kinase (ROCK), an effector of Rho family GTPases, activates the chondrogenic transcription factor SRY-type high-mobility-group box (SOX) 9 and prevents dedifferentiation of monolayer-cultured chondrocytes. We hypothesized that ROCK inhibition prevents chondrocyte dedifferentiation by altering the transcriptional balance between MMP-3 and AGN. Normal human articular chondrocytes were cultured in the presence or absence of ROCK inhibitor (ROCKi, Y-27632). Expression of MMP-3 and AGN during monolayer cultivation was assessed by quantitative real-time PCR and western blot analysis. Chondrogenic redifferentiation potential of ROCKi-treated chondrocytes was evaluated by immunohistological analysis of pellet cultures. ROCKi treatment suppressed MMP-3 expression in monolayer- and pellet-cultured chondrocytes but increased AGN expression. Chromatin immunoprecipitation revealed that the association between transcription factors E26 transformation specific (ETS)-1 and SOX9 and their target genes MMP-3 and AGN, respectively, was affected by ROCKi treatment. ROCKi decreased the association between ETS-1 and its binding sites on the MMP-3 promoter, whereas ROCKi promoted the interaction between SOX9 and the AGN promoter. Our results suggest that ROCK inhibition may have an important role in modulating the balance between degradation and synthesis of cartilaginous ECM, a finding that may facilitate development of techniques to prepare differentiated chondrocytes for cartilage regeneration therapy.

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

    PubMed

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

    2016-02-01

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

  15. Expression and cellular localization of human hyaluronidase-2 in articular chondrocytes and cultured cell lines

    PubMed Central

    Chow, G.; Knudson, C. B.; Knudson, W.

    2011-01-01

    Summary Objective There is debate whether hyaluronan (HA) can be enzymatically degraded within the extracellular matrix of cartilage and other tissues or whether its catabolism occurs strictly within the lysosomal compartment of chondrocytes and other cell types. Previous studies have suggested that one of the lysosomal hyaluronidases (hyaluronidase-2) can be expressed as a functionally-active glycosyl phosphatidylinositol-linked protein at the surface of mammalian cells. If this form of hyaluronidase expression occurs in chondrocytes, this could represent a possible mechanism for extracellular HA cleavage. Thus, which hyaluronidases are expressed and where was the objective of this study. Methods mRNA for hyaluronidases was quantified by reverse transcription-polymerase chain reaction (RT-PCR) and enzymatic activity by HA zymograms. Recombinant forms of hyaluronidase-2 were generated and expressed in model cell lines. A peptide-specific polyclonal antiserum was prepared to localize endogenous human hyaluronidase-2 in human articular chondrocytes. Results Hyaluronidase-2 is the principal mRNA transcript expressed by primary human articular chondrocytes as well as various model cell lines. Recombinant hyaluronidase-2, containing N-terminal or C-terminal epitope tags, was strictly localized intracellularly and not released by treatment with a phosphatidylinositol-specific phospholipase. Endogenous hyaluronidase-2 expressed by human chondrocytes as well as HeLa cells could only be detected following detergent permeabilization of the plasma membranes. Conclusions These data suggest that on chondrocytes and other cell types examined, hyaluronidase-2 is not present or functional at the external plasma membrane. Thus, local turnover of HA is dependent on receptor-mediated endocytosis and delivery to low pH intracellular organelles for its complete degradation. PMID:16600643

  16. Dose-injury relationships for cryoprotective agent injury to human chondrocytes.

    PubMed

    Fahmy, M D; Almansoori, K A; Laouar, L; Prasad, V; McGann, L E; Elliott, J A W; Jomha, N M

    2014-02-01

    Vitrification of articular cartilage (AC) could enhance tissue availability but requires high concentrations of cyroprotective agents (CPAs). This study investigated relative injuries caused by commonly used CPAs. We hypothesized that the in situ chondrocyte dose-injury relationships of five commonly used CPAs are nonlinear and that relative injuries could be determined by comparing cell death after exposure at increasing concentrations. Human AC samples were used from four patients undergoing total knee arthroplasty surgery. Seventy μm slices were exposed in a stepwise protocol to increasing concentrations of 5 CPAs (max = 8 M); dimethyl sulfoxide (Me(2)SO), glycerol (Gly), propylene glycol (PG), ethylene glycol (EG), and formamide (FM). Chondrocyte viability was determined by membrane integrity stains. Statistical analysis included t-tests and nonlinear least squares estimation methods. The dose-injury to chondrocytes relationships for all CPAs were found to be nonlinear (sigmoidal best fit). For the particular loading protocol in this study, the data identified the following CPA concentrations at which chondrocyte recoveries statistically deviated significantly from the control recovery; 1 M for Gly, 4 M for FM and PG, 6 M for Me(2)SO, and 7 M for EG. Comparison of individual means demonstrated that Gly exposure resulted in the lowest recovery, followed by PG, and then Me(2)SO, FM and EG in no specific order. The information from this study provides an order of damage to human chondrocytes in situ of commonly used CPAs for vitrification of AC and identifies threshold CPA concentrations for a stepwise loading protocol at which chondrocyte recovery is significantly decreased. In general, Gly and PG were the most damaging while DMSO and EG were among the least damaging. PMID:24269869

  17. Adult equine bone marrow stromal cells produce a cartilage-like ECM mechanically superior to animal-matched adult chondrocytes.

    PubMed

    Kopesky, P W; Lee, H-Y; Vanderploeg, E J; Kisiday, J D; Frisbie, D D; Plaas, A H K; Ortiz, C; Grodzinsky, A J

    2010-06-01

    Our objective was to evaluate the age-dependent mechanical phenotype of bone marrow stromal cell- (BMSC-) and chondrocyte-produced cartilage-like neo-tissue and to elucidate the matrix-associated mechanisms which generate this phenotype. Cells from both immature (2-4 month-old foals) and skeletally-mature (2-5 year-old adults) mixed-breed horses were isolated from animal-matched bone marrow and cartilage tissue, encapsulated in self-assembling-peptide hydrogels, and cultured with and without TGF-beta1 supplementation. BMSCs and chondrocytes from both donor ages were encapsulated with high viability. BMSCs from both ages produced neo-tissue with higher mechanical stiffness than that produced by either young or adult chondrocytes. Young, but not adult, chondrocytes proliferated in response to TGF-beta1 while BMSCs from both age groups proliferated with TGF-beta1. Young chondrocytes stimulated by TGF-beta1 accumulated ECM with 10-fold higher sulfated-glycosaminoglycan content than adult chondrocytes and 2-3-fold higher than BMSCs of either age. The opposite trend was observed for hydroxyproline content, with BMSCs accumulating 2-3-fold more than chondrocytes, independent of age. Size-exclusion chromatography of extracted proteoglycans showed that an aggrecan-like peak was the predominant sulfated proteoglycan for all cell types. Direct measurement of aggrecan core protein length and chondroitin sulfate chain length by single molecule atomic force microscopy imaging revealed that, independent of age, BMSCs produced longer core protein and longer chondroitin sulfate chains, and fewer short core protein molecules than chondrocytes, suggesting that the BMSC-produced aggrecan has a phenotype more characteristic of young tissue than chondrocyte-produced aggrecan. Aggrecan ultrastructure, ECM composition, and cellular proliferation combine to suggest a mechanism by which BMSCs produce a superior cartilage-like neo-tissue than either young or adult chondrocytes. PMID:20153827

  18. Microcontact printing of BMP-2 and its effect on human chondrocytes behavior

    NASA Astrophysics Data System (ADS)

    Pan, Chang-Jiang; Nie, Yu-Dong

    2010-01-01

    The present study is to investigate human chondrocytes behavior on microcontact printed bone morphogenetic protein-2 (BMP-2) lines on polystyrene (PS) surface. It was found that the cells aligned with BMP lines and expressed type II and VI collagen. The chondrocytes in vitro cultured on BMP lines were elongated, which resulted in altered cell morphology. Taking all these results into consideration, BMP-2 lines enhance cell adhesion, restrict spreading, and increase type II and VI collagen expression. The results represented in this study may be an approach to the problem of engineering reparative cartilage in vitro.

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

    PubMed Central

    Melrose, J.

    2016-01-01

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

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

    PubMed

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

    2012-06-01

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

  1. Alteration of viscoelastic properties is associated with a change in cytoskeleton components of ageing chondrocytes from rabbit knee articular cartilage.

    PubMed

    Duan, Wangping; Wei, Lei; Zhang, Juntao; Hao, Yongzhuang; Li, Chunjiang; Li, Hao; Li, Qi; Zhang, Quanyou; Chen, Weiyi; Wei, Xiaochun

    2011-12-01

    The cytoskeleton network is believed to play an important role in the biomechanical properties of the chondrocyte. Ours and other laboratories have demonstrated that chondrocytes exhibit a viscoelastic solid creep behavior in vitro and that viscoelastic properties decrease in osteoarthritic chondrocytes. In this study, we aimed to understand whether the alteration of viscoelastic properties is associated with changes in cytoskeleton components of ageing chondrocytes from rabbit knee articular cartilage. Three age groups were used for this study: young (2-months-old, N=23), adult (8-months-old, N=23), and old (31-months-old, N=23) rabbit groups. Cartilage structure and proteoglycan and type II collagen content were determined by H&E and Toluidine Blue staining, and type II collagen antibody. The detailed structure of the chondrocytes in all groups was visualized using transmission electron microscopy (TEM). Chondrocytes were isolated from full-thickness knee cartilage of rabbits from all groups and their viscoelastic properties were quantified within 2 hours of isolation using a micropipette aspiration technique combined with a standard linear viscoelastic solid model. The components and network of the cytoskeleton within the cells were analyzed by laser scanning confocal microscopy (LSCM) with immunofluorescence staining as well as real time PCR and western blotting. With ageing, articular cartilage contained less chondrocytes and less proteoglycans and type II collagen. TEM observations showed that the cell membranes were not clearly defined, organelles were fewer and the nuclei were deformed or shrunk in the old cells compared with the young and adult cells. In suspension, chondrocytes from all three age groups showed significant viscoelastic creep behavior, but the deformation rate and amplitude of old chondrocytes were increased under the same negative pressure when compared to young and adult chondrocytes. Viscoelastic properties of the old cells, including

  2. Sequential differentiation of mesenchymal stem cells in an agarose scaffold promotes a physis-like zonal alignment of chondrocytes.

    PubMed

    Schmitt, Jacqueline Frida; See, Kwee Hua; Hua, See Kwee; Yang, Zheng; Zheng, Yang; Hui, James Hoi Po; Po, James Hui Hoi; Lee, Eng Hin; Hin, Lee Eng

    2012-11-01

    Chondrocytes of the epiphyseal growth plate (physis) differentiate and mature in defined linear zones. The current study examines the differentiation of human bone marrow derived mesenchymal stem cells (hBMSCs) into zonal physeal cartilage. hBMSCs were embedded in an agarose scaffold with only the surface of the scaffold in direct contact with the culture medium. The cells were differentiated using a two-step system involving the sequential addition of TGFβ followed by BMP2. The resultant samples displayed a heterogenic population of physis-like collagen type 2 positive cells including proliferating chondrocytes and mature chondrocytes showing hypertrophy, expression of early bone markers and matrix mineralization. Histological analysis revealed a physis-like linear zonal alignment of chondrocytes in varying stages of differentiation. The less mature chondrocytes were seen at the base of the construct while hypertrophic chondrocytes and matrix mineralization was observed closer to the surface of the construct. The described differentiation protocol using hBMSCs in an agarose scaffold can be used to study the factors and conditions that influence the differentiation, proliferation, maturation, and zonal alignment of physeal chondrocytes. PMID:22517299

  3. Effects of allicin on the proliferation and cell cycle of chondrocytes

    PubMed Central

    Li, Tao; Shi, Hong-Yan; Hua, Yong-Xin; Gao, Chen; Xia, Qing; Yang, Guang; Li, Bin

    2015-01-01

    The present study demonstrates the effect of allicin on the proliferation and the cell cycle distribution of the chondrocytes. MTT assay and flow cytometry were used for the evaluation of the effect of allicin on cell proliferative and the cell cycle distribution, respectively of the chondrocytes. The reverse transcription polymerase chain reaction (RT-PCR) and western blot analysis were respectively used for the analysis of mRNA and protein expression levels of cyclin D1, CDK4 and CDK6. The results revea