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Collagen II (collagen + ii)
Selected AbstractsControl of human articular chondrocyte differentiation by reduced oxygen tensionJOURNAL OF CELLULAR PHYSIOLOGY, Issue 3 2004Christopher L. Murphy Cell number is often a limiting factor in studies of chondrocyte physiology, particularly for human investigations. Chondrocytes can be readily proliferated in monolayer culture, however, differentiated phenotype is soon lost. We therefore endeavored to restore normal phenotype to human chondrocytes after serial passage in monolayer culture by manipulating cell morphology and oxygen tension towards the in vivo state. Third passage cells were encapsulated in alginate and exposed to either 20% or more physiologic 5% oxygen tensions. To assess cell phenotype, gene expression was measured using TaqMan real-time PCR. Encapsulated, primary chondrocytes cultured in 20% oxygen were used as a positive reference. Passaged human chondrocytes were fibroblastic in appearance and had lost normal phenotype as evidenced by a decrease in expression of collagen II, aggrecan, and sox9 genes of 66, 6, and 14 fold, respectively; with concomitant high expression of type I collagen (22 fold increase). A partial regaining of the differentiated phenotype was observed by encapsulation in 20% oxygen; however, even after 4 weeks, collagen II gene expression was not fully restored. Collagen II and aggrecan expression were increased, on average, 3 fold, in 5% oxygen tension compared to 20% cultures. Furthermore, matrix glycosaminoglycan (GAG) levels were significantly increased in reduced oxygen. In fact, after 4 weeks in 5% oxygen, encapsulated third passage cells had collagen II expression fully regained and aggrecan and sox9 levels actually exceeding primary cell levels in 20% oxygen. Our results show that the phenotype of serially passaged human articular chondrocytes is more fully restored by combining encapsulation with culture in more physiological levels of oxygen. Sox9, an essential transcription factor for chondrocyte differentiation is strongly implicated in this process since its expression was upregulated almost 27 fold. These findings have implications for the optimal conditions for the in vitro culture of chondrocytes. © 2004 Wiley-Liss, Inc. [source] The fate of transplanted xenogeneic bone marrow-derived stem cells in rat intervertebral discsJOURNAL OF ORTHOPAEDIC RESEARCH, Issue 3 2009Aiqun Wei Abstract Intervertebral disc degeneration is a major cause and a risk factor for chronic low back pain. The potential of using stem cells to treat disc degeneration has been raised. The aims of our study were to assess whether xenogeneic bone-marrow derived stem cells could survive in a rat disc degeneration model and to determine which cell types, if any, survived and differentiated into disc-like cells. Human bone-marrow derived CD34+ (hematopoietic progenitor cells) and CD34, (nonhematopoietic progenitor cells, including mesenchymal stem cells) cells were isolated, fluorescent-labeled, and injected into rat coccygeal discs. The rats were sacrificed at day 1, 10, 21, and 42. Treated discs were examined by histological and immunostaining techniques and compared to control discs. The survival of transplanted cells was further confirmed with a human nuclear specific marker. Fluorescent labeled CD34, cells were detected until day 42 in the nucleus pulposus of the injected discs. After 3 weeks these cells had differentiated into cells expressing chondrocytic phenotype (Collagen II and Sox-9). In contrast, the fluorescent labeled CD34+ cells could not be detected after day 21. No fluorescence-positive cells were detected in the noninjected control discs. Further, no inflammatory cells infiltrated the nucleus pulposus, even though these animals had not received immunosuppressive treatment. Our data provide evidence that transplanted human BM CD34, cells survived and differentiated within the relative immune privileged nucleus pulposus of intervertebral disc degeneration. © 2008 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 27:374,379, 2009 [source] Pellet culture elicits superior chondrogenic redifferentiation than alginate-based systemsBIOTECHNOLOGY PROGRESS, Issue 4 2009Peter Bernstein Abstract Although pellet culture and encapsulation of chondrocytes into gel-like biomaterials have lead to major advances in cartilage tissue engineering, a quantitative comparative characterization of cellular differentiation behavior during those cultivation procedures has not yet been performed. Our study therefore aimed at answering the following question: is the redifferentiation pathway of chondrocytes altered by slight changes in the type of alginate biomaterial (pure alginate, alginate-fibrin, alginate-chitosan) and how do the cells behave in comparison to biomaterial-free (pellet) three-dimensional culturing? Monolayer-expanded chondrocytes from healthy adult porcine knee joints were cultivated in alginate, alginate-chitosan, alginate-fibrin beads and as pellets up to 4 weeks. Quantitative PCR and Immunohistology were used to assess chondrogenic markers. Alginate-fibrin,encapsulated chondrocytes behaved almost like monolayer chondrocytes. Alginate- and alginate-chitosan encapsulation lead to a low chondrogenic marker gene expression. Although all 3D-cultured chondrocytes showed a considerable amount of Sox9 expression, only pellet cultivation lead to a sufficient Collagen II expression. This puts the usage of alginate-cultivated cartilage tissue engineering constructs under question. Fibrin addition is not beneficial for chondrogenic differentiation. Sox9 and Collagen II behave differently, depending upon the surrounding 3D-environment. © 2009 American Institute of Chemical Engineers AIChE J, 2009 [source] Alpha2beta1 integrin is the major collagen-binding integrin expressed on human Th17 cellsEUROPEAN JOURNAL OF IMMUNOLOGY, Issue 10 2010Marc Boisvert Abstract Growing evidence indicates that collagen-binding integrins are important costimulatory molecules of effector T cells. In this study, we demonstrate that the major collagen-binding integrin expressed by human Th17 cells is alpha2beta1 (,2,1) or VLA-2, also known as the receptor for collagen I on T cells. Our results show that human naïve CD4+ T cells cultured under Th17 polarization conditions preferentially upregulate ,2,1 integrin rather than ,1,1 integrin, which is the receptor for collagen IV on T cells. Double staining analysis for integrin receptors and intracellular IL-17 showed that ,2 integrin but not ,1 integrin is associated with Th17 cells. Cell adhesion experiments demonstrated that Th17 cells attach to collagen I and collagen II using ,2,1 integrin but did not attach to collagen IV. Functional studies revealed that collagens I and II but not collagen IV costimulate the production of IL-17A, IL-17F and IFN-, by human Th17 cells activated with anti-CD3. These results identify ,2,1 integrin as the major collagen receptor expressed on human Th17 cells and suggest that it can be an important costimulatory molecule of Th17 cell responses. [source] Engagement of the CD137 (4-1BB) costimulatory molecule inhibits and reverses the autoimmune process in collagen-induced arthritis and establishes lasting disease resistanceIMMUNOLOGY, Issue 1 2004Juergen L. Foell Summary Agonistic antibodies against CD137 act as costimulators in the activation of CD8 T cells. They enhance the immune response against syngeneic tumour grafts and suppress T cell-dependent humoral immune responses in vivo. The present study was undertaken to determine whether suppression of antibody production by anti-CD137 mAb affects the development of collagen-induced arthritis (CIA). Male DBA/1J mice were immunized with bovine collagen II (CII) and treated with an agonistic anti-CD137 mAb or an isotype-matched control mAb. Mice were assessed regularly for macro- and microscopic signs of arthritis and for the appearance of collagen-specific antibody production. Interferon (IFN)-, determination, FACS analysis of splenocytes and histopathological joint examinations were performed after the animals were killed. Administration of anti-CD137 mAb at the time of collagen immunization blocked the development of disease and inhibited the humoral immune response against CII. Agonistic anti-CD137 mAb exhibited therapeutic efficacy even after the immune response to CII had succeeded and the disease became apparent. Furthermore, it induced a protective memory in the animals, enabling resistance to subsequent challenges with the pathogenic antigen. Our results suggest a key role for CD137 in the pathogenesis of CIA. This model provides insights into immunoregulatory conditions that control the pathogenesis of autoimmune diseases. [source] Generation of a scaffold free cartilage-like implant from a small amount of starting materialJOURNAL OF CELLULAR AND MOLECULAR MEDICINE, Issue 2 2006M. J. Stoddart Abstract Introduction: An autologous cellular based treatment of a traumatic cartilage injury requires a procedure whereby a biopsy of healthy cartilage is removed from the patient and the cells isolated and expanded by monolayer passage. This increases the cell number to required levels but also leads to a de-differentiation of the cells. We aim to produce a scaffold-free, de-novo implant from a biopsy of cartilage. Methods: Bovine chondrocytes were isolated from a small biopsy and expanded. The chondrocytic phenotype of the monolayer expanded cells was recovered during a period of culture in alginate and the effect of factors such as IGF1, TFG,1 and dexamethasone was investigated. Results: During the alginate culture period a pre-treatment with IGF1 and dexamethasone was shown to have little effect. IGF1 however increased the glycosaminoglycan/DNA (GAG/DNA) content on day 14 to 84.95±5ng/ng compared with 37.3±1.8ng/ng in the controls (P <0.001). 35S labeling demonstrated an increased GAG synthesis in the presence of IGF1 (P < 0.001). IGF1 also induced a increase of DNA content 1383±314ng/bead compared to 512±19ng/bead in the controls (P < 0.001). The cells were released from the alginate and cultured in a silicon mould for a further 14 days to obtain a three dimensional implant. Releasing the cells from the alginate and casting in a mould produced an implant of defined shape which contained no foreign material. After 31 days of culture the implants contained 152.4±13.14ng/ng GAG/DNA and 42.93±10.23ng/ng collagen II. Discussion: We believe alginate released chondrocytes provide a real alternative to artificial scaffolds. [source] Control of human articular chondrocyte differentiation by reduced oxygen tensionJOURNAL OF CELLULAR PHYSIOLOGY, Issue 3 2004Christopher L. Murphy Cell number is often a limiting factor in studies of chondrocyte physiology, particularly for human investigations. Chondrocytes can be readily proliferated in monolayer culture, however, differentiated phenotype is soon lost. We therefore endeavored to restore normal phenotype to human chondrocytes after serial passage in monolayer culture by manipulating cell morphology and oxygen tension towards the in vivo state. Third passage cells were encapsulated in alginate and exposed to either 20% or more physiologic 5% oxygen tensions. To assess cell phenotype, gene expression was measured using TaqMan real-time PCR. Encapsulated, primary chondrocytes cultured in 20% oxygen were used as a positive reference. Passaged human chondrocytes were fibroblastic in appearance and had lost normal phenotype as evidenced by a decrease in expression of collagen II, aggrecan, and sox9 genes of 66, 6, and 14 fold, respectively; with concomitant high expression of type I collagen (22 fold increase). A partial regaining of the differentiated phenotype was observed by encapsulation in 20% oxygen; however, even after 4 weeks, collagen II gene expression was not fully restored. Collagen II and aggrecan expression were increased, on average, 3 fold, in 5% oxygen tension compared to 20% cultures. Furthermore, matrix glycosaminoglycan (GAG) levels were significantly increased in reduced oxygen. In fact, after 4 weeks in 5% oxygen, encapsulated third passage cells had collagen II expression fully regained and aggrecan and sox9 levels actually exceeding primary cell levels in 20% oxygen. Our results show that the phenotype of serially passaged human articular chondrocytes is more fully restored by combining encapsulation with culture in more physiological levels of oxygen. Sox9, an essential transcription factor for chondrocyte differentiation is strongly implicated in this process since its expression was upregulated almost 27 fold. These findings have implications for the optimal conditions for the in vitro culture of chondrocytes. © 2004 Wiley-Liss, Inc. [source] Gene expression in skeletal tissues: application of laser capture microdissectionJOURNAL OF MICROSCOPY, Issue 1 2005D. Benoyahu Summary Tissue differentiation is based on the expression of transcription factors, receptors for cytokines, and nuclear receptors that regulate a specific phenotype. The purpose of this study was to select cells from various skeletal tissues in order to analyse differential gene expression of cells in the native environment in vivo. It is a difficult task to obtain cells from skeletal tissues, such as cartilage, periost, bone and muscle, that are structured together and do not exist as individual organs. We used laser capture microdissection which permits the selection and isolation of individual cells from tissue sections. The RNA isolated from these tissues was used for reverse transcriptase-polymerase chain reactions for molecular analysis. We analysed the expression of transcription factors (cFOS, cbfa1, MyoD), receptors for cytokines, nuclear receptors, alkaline phosphatase and the structural proteins osteocalcin and collagen II. The results obtained demonstrate differential patterns of gene expression according to the tissue arrangement in their native in vivo environment, with reliable interpretation of the functions of the analysed genes in the context of intact skeletal tissue physiology. [source] p38 MAPK inhibition modulates rabbit nucleus pulposus cell response to IL-1,JOURNAL OF ORTHOPAEDIC RESEARCH, Issue 7 2008Rebecca K. Studer Abstract Analysis of disc gene expression implicated IL-1 in the development of intervertebral disc degeneration (IDD) in a rabbit stab model. The purpose of these studies is to determine the role of p38 Mitogen Activated Protein Kinase (p38 MAPK) signaling in nucleus pulposus cell response to IL-1, and to compare rabbit nucleus pulposus (rNP) cell responses to IL-1 activation with those in a stab model of disc degeneration. NP cells maintained in alginate bead culture were exposed to IL-1, with or without p38 MAPK inhibition. RNA was isolated for reverse transcription polymerase chain reaction (RT-PCR) analysis of gene expression, conditioned media analyzed for accumulation of nitric oxide (NO) and prostaglandin E-2 (PGE-2), and proteoglycan synthesis measured after 10 days. IL-1 upregulation of mRNA for cycloxygenase-2 (COX-2), matrix metalloproteinase-3 (MMP-3), IL-1, and IL-6, was blunted by p38 inhibition while downregulation of matrix proteins (collagen I, collagen II, aggrecan) and insulin-like-growth-factor I (IFG-1) was also reversed. mRNA for tissue inhibitor of matrixmetalloproteinase-1 (TIMP-1) was modestly increased by IL-1, while those for Transforming Growth Factor-, (TGF-,) SOX-9, and versican remained unchanged. Blocking p38 MAPK reduced IL-1 induced NO and PGE-2 accumulation and partially restored proteoglycan synthesis. p38 MAPK inhibition in control cells increased mRNA for matrix proteins (aggrecan, collagen II, versican, collagen I) and anabolic factors (IGF-1, TGF, and SOX-9) from 50% to 120%, decreased basal PGE-2 accumulation, but had no effect on message for TIMP-1, MMP-3, or COX-2. Inhibition of p38 MAPK in cytokine-activated disc cells blunts gene expression and production of factors associated with inflammation, pain, and disc matrix catabolism while reversing IL-1 downregulation of matrix protein gene expression and proteoglycan synthesis. The results support the hypothesis that IL-1 could be responsible for many of the mRNA changes seen in rabbit NP in the stab model of disc degeneration, and uphold the concept that development of molecular techniques to block p38 MAPK could provide a therapeutic approach to slow the course of intervertebral disc degeneration. © 2008 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 26:991,998, 2008 [source] Type II collagen modulates the composition of extracellular matrix synthesized by articular chondrocytesJOURNAL OF ORTHOPAEDIC RESEARCH, Issue 2 2003Wen-Ning Qi Abstract The articular cartilage extracellular matrix (ECM) interfaces with chondrocytes and influences many biological processes important to cartilage homeostasis and repair. The alginate bead culture system can be viewed as a model of cartilage repair in which the chondrocyte attempts to recreate the pericellular matrix while maintaining a differentiated phenotype. The purpose of this study was to evaluate the alteration in epitopes of proteoglycan and tenascin synthesized by chondrocytes in the presence of exogenous extracellular type II collagen. We evaluated the effects on four biomarkers associated with the creation of the denovo matrix using ELISA and immunohistochemistry: keratan sulfate epitope (5D4), 3B3(,) neoepitope of chondroitin-6- sulfate, 3B3(+) chondroitinase-generatedepitope of chondroitin-6-sulfate, and tenascin-C expression. TGF-,1 stimulated the production of 3B3(+), 5D4, and tenascin-C in a dose-dependent manner and decreased 3B3(,) levels. Following the addition of exogenous type II collagen, 3B3(,) increased and tenascin-C decreased but did not change the direction of TGF-,1 effects. In contrast, 5D4 expression decreased in the presence of collagen II as TGF-,1 increased to 10 ng/ml. Interestingly, the amount of 3B3(+) epitope was not affected by the incorporation of type II collagen. Immunohistochemistry found there was no significant difference in distribution of these biomarkers in the presence and absence of extracellular type II collagen incorporation. These results elucidate the subtle biochemical differences in ECM synthesized by chondrocytes in the presence of type II collagen and further characterize the role played by ECM in the TGF-,1 regulation of the articular cartilage physiology. © 2002 Orthopaedic Research Society. Published by Elsevier Science Ltd. All rights reserved. [source] Characterization and multilineage differentiation of embryonic stem cells derived from a buffalo parthenogenetic embryoMOLECULAR REPRODUCTION & DEVELOPMENT, Issue 10 2007Hathaitip Sritanaudomchai Abstract Embryonic stem (ES) cells derived from mammalian embryos have the ability to form any terminally differentiated cell of the body. We herein describe production of parthenogenetic buffalo (Bubalus Bubalis) blastocysts and subsequent isolation of an ES cell line. Established parthenogenetic ES (PGES) cells exhibited diploid karyotype and high telomerase activity. PGES cells showed remarkable long-term proliferative capacity providing the possibility for unlimited expansion in culture. Furthermore, these cells expressed key ES cell-specific markers defined for primate species including stage-specific embryonic antigen-4 (SSEA-4), tumor rejection antigen-1-81 (TRA-1-81), and octamer-binding transcription factor 4 (Oct-4). In vitro, in the absence of a feeder layer, cells readily formed embryoid bodies (EBs). When cultured for an extended period of time, EBs spontaneously differentiated into derivatives of three embryonic germ layers as detected by PCR for ectodermal (nestin, oligodendrocytes, and tubulin), mesodermal (scleraxis, ,- skeletal actin, collagen II, and osteocalcin) and endodermal markers (insulin and ,- fetoprotein). Differentiation of PGES cells toward chondrocyte lineage was directed by supplementing serum-containing media with ascorbic acid, ,-glycerophosphate, and dexamethasone. Moreover, when PGES cells were injected into nude mice, teratomas with derivatives representing all three embryonic germ layers were produced. Our results suggest that the cell line isolated from a parthenogenetic blastocyst holds properties of ES cells, and can be used as an in vitro model to study the effects of imprinting on cell differentiation and as an a invaluable material for extensive molecular studies on imprinted genes. Mol. Reprod. Dev. 74: 1295,1302, 2007. © 2007 Wiley-Liss, Inc. [source] Discoidin domain receptor 2 mediates the collagen II-dependent release of interleukin-6 in primary human chondrocytes,THE JOURNAL OF PATHOLOGY, Issue 2 2009Andreas R Klatt Abstract We deciphered constituent parts of a signal transduction cascade that is initiated by collagen II and results in the release of various pro-inflammatory cytokines, including interleukin-6 (IL-6), in primary human chondrocytes. This cascade represents a feed-forward mechanism whereby cartilage matrix degradation is exacerbated by the mutually inducing effect of released collagen II fragments and pro-inflammatory cytokines. We previously proposed discoidin domain receptor 2 as a central mediator in this event. Since this cascade plays a prominent role in the pathogenesis of osteoarthritis, our study further investigates the hypothesis that discoidin domain receptor 2 is a candidate receptor for collagen II, and that transcription factor NF,B, lipid kinase PI3K, and the MAP kinases are constituent parts of this very signal transduction cascade. To accomplish this, we selectively knocked down the molecules of interest in primary human chondrocytes, induced the specified cascade by incubating primary human chondrocytes with collagen II, and observed the outcome, specifically the changes in interleukin-6 release. Knockdown was performed by siRNA-mediated gene silencing in the case of discoidin domain receptor 2 (DDR2) or by using specific inhibitors for the remainder of the molecules. Results indicated that discoidin domain receptor 2 mediates the collagen II-dependent release of interleukin-6 in primary human chondrocytes and that MAP kinases p38, JNK and ERK, as well as transcription factor NF,B, are integral components of intracellular collagen II signalling. Given the detrimental role of these molecules in osteoarthritis, our findings provide new targets for more specific therapeutics, which may have fewer side effects than those currently applied. Copyright © 2009 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. [source] Induction of chondrogenesis from human embryonic stem cells without embryoid body formation by bone morphogenetic protein 7 and transforming growth factor ,1ARTHRITIS & RHEUMATISM, Issue 12 2009Toshiyuki Nakagawa Objective Human embryonic stem cells (ESCs) provide an unlimited supply of pluripotent cells for articular cartilage tissue engineering and regenerative medicine applications. Articular cartilage is an avascular tissue with precise polarity and organization comprising 3 distinct functional zones: surface, middle, and deep. To date, attempts at differentiating human ESCs into articular chondrocytes have been unsuccessful. The majority of studies have focused on chondrogenic (but not specifically articular cartilage) differentiation. Furthermore, previous investigations of induction of chondrogenesis by human ESCs required embryoid body formation; however, embryoid body formation often results in heterogeneous differentiation. The present study was undertaken to determine the in vitro chondrogenic potential of bone morphogenetic protein 7 (BMP-7) and transforming growth factor ,1 (TGF,1),induced human ESC differentiation toward the articular cartilage phenotype. Methods Dissociated single human ESCs were cultured and passaged on a gelatin-coated flask. The human ESCs were cultured as an aggregate in a pellet culture system for 14 days in basal chondrogenic medium (CM), CM with TGF,1, CM with BMP-7, or CM with both TGF,1 and BMP-7. Results The size and wet weight of the cartilage pellets and glycosaminoglycan levels increased, with the smallest, intermediate, and greatest increases, respectively, observed with CM plus TGF,1 treatment, CM plus BMP-7 treatment, and CM plus TGF,1 and BMP-7 treatment (compared with CM treatment alone). The largest size and highest weight of the pellet was in the group in which TGF,1 and BMP-7 were added to the medium. However, expression of the genes for cartilage-specific aggrecan and type II collagen II, as assessed by determination of messenger RNA levels, was highest in the BMP-7,treated group. Superficial zone protein (SZP)/lubricin, a marker of the superficial zone articular chondrocyte, was not detectable under identical culture conditions. Conclusion These results demonstrate an efficient and reproducible model system of human ESC-induced chondrogenesis, using a novel direct plating method in which intervening embryoid body formation does not occur. Further work is needed for optimization of conditions to obtain the articular cartilage phenotype that includes the superficial zone marker as demonstrated by SZP/lubricin synthesis. [source] Lentivirus-mediated knockdown of aggrecanase-1 and -2 promotes chondrocyte-engineered cartilage formation in vitroBIOTECHNOLOGY & BIOENGINEERING, Issue 4 2010Zheng-Hui Wang Abstract Chondrocyte-based tissue engineering has emerged as a promising approach for repair of injured cartilage tissues that have a poor self-healing capacity. However, this technique faces a major limitation: dedifferentiation of chondrocytes occurs following several passages in culture. Aggrecan, a major component of cartilage extracellular matrix, plays an essential role in chondrocyte differentiation. The aim of this study is to determine whether inhibition of chondrocyte aggrecanases, key degradative enzymes for aggrecan in cartilage, could benefit chondrocyte differentiation and the preservation of chondrocyte phenotype within a long-term period. Lentivirus-mediated RNA interference (RNAi) was employed to target both aggrecanase-1 and -2 in primary rat chondrocytes, and the transduced cells were seeded into chitosan,gelatin three-dimensional scaffolds. Histological, morphological, and biochemical analyses were performed at 1,8 weeks post-implantation to study chondrocyte survival, differentiation, and function. We found that lentivirus-mediated RNAi notably decreased the abundance of aggrecanase transcripts in chondrocytes but did not affect cell viability. Most importantly, compared to the control constructs seeded with untransduced chondrocytes, the aggrecanase inhibition increased chondrocyte proliferation and reinforced the production of glycosaminoglycans and total collagen, indicative of chondrocyte differentiation. The mRNA expression of chondrocyte marker genes (collagen II and aggrecan) was enhanced by aggrecanase silencing relative to the control. Together our data demonstrate that inhibition of endogenous aggrecanases facilitates chondrocyte differentiation and chondrocyte-engineered cartilage formation in vitro. The combination of lentiviral delivery system and genetic manipulation techniques provides a useful tool for modulation of chondrocyte phenotype in cartilage engineering. Biotechnol. Bioeng. 2010;107:730,736. © 2010 Wiley Periodicals, Inc. [source] | |||||||||||||