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Disc Cells (disc + cell)
Selected AbstractsCollagen gene expression and mechanical properties of intervertebral disc cell,alginate culturesJOURNAL OF ORTHOPAEDIC RESEARCH, Issue 1 2001Anthony E. Baer Cells of the intervertebral disc have a limited capacity for matrix repair that may contribute to the onset and progression of degenerative disc changes. In this study, the biosynthetic capacity of cells isolated from specific regions of the porcine intervertebral disc was evaluated in vitro. Using a competitive reverse transcription-polymerase chain reaction technique, gene expression levels for types I and II collagen were quantified in cells cultured for up to 21 d in a three-dimensional alginate culture system and compared to levels obtained for cells in vivo. The mechanical properties of cell-alginate constructs were measured in compression and shear after periods of culture up to 16 weeks. Cells from the anulus fibrosus expressed the most type I collagen mRNA in vivo and in vitro, while cells from the transition zone expressed the most type II collagen mRNA in vivo and in vitro. Mechanical testing results indicate that a mechanically functional matrix did not form at any time during the culture period; rather, decreases of up to 50% were observed in the compressive and shear moduli of the cell,alginate constructs compared to alginate with no cells. Together with results of prior studies, these results suggest that intervertebral disc cells maintain characteristics of their phenotype when cultured in alginate, but the molecules they synthesize are not able to form a mechanically functional matrix in vitro. © 2001 Orthopaedic Research Society. Published by Elsevier Science Ltd. All rights reserved. [source] Disc structure function and its potential for repairINTERNATIONAL JOURNAL OF RHEUMATIC DISEASES, Issue 1 2002J. Melrose The intervertebral disc (IVD) is the largest predominantly avascular, aneural, alymphatic structure of the human body. It provides articulation between adjoining vertebral bodies and also acts as a weight-bearing cushion dissipating axially applied spinal loads. The IVD is composed of an outer collagen-rich annulus fibrosus (AF) and a central proteoglycan (PG)-rich nucleus pulposus (NP). Superior and inferior cartilaginous endplates (CEPs), thin layers of hyaline-like cartilage, cover the ends of the vertebral bodies. The AF is composed of concentric layers (lamellae) which contain variable proportions of type I and II collagen, this tissue has high tensile strength. The NP in contrast is a gelatinous PG-rich tissue which provides weight-bearing properties to the composite disc structure. With the onset of age, cells in the NP progressively die as this tissue becomes depleted of PGs, less hydrated and more fibrous as the disc undergoes an age-dependent fibrocartilaginous transformation. Such age-dependent cellular and matrix changes can decrease the discs' biomechanical competence and trauma can further lead to failure of structural components of the disc. Annular defects are fairly common and include vertebral rim-lesions, concentric (circumferential) annular tears (separation of adjacent annular lamellae) and radial annular tears (clefts which initiate within the NP). While vascular in-growth around annular tears has been noted, evidence from human post-mortem studies indicate they have a limited ability to undergo repair. Several experimental approaches are currently under evaluation for their ability to promote the repair of such annular lesions. These include growth of AF fibrochondrocytes on a resorbable polycaprolactone (PCL) bio-membrane.1 Sheets of fibrochondrocytes lay down type-I collagen and actin stress fibres on PCL. These matrix components are important for the spatial assembly of the collagenous lamella during annular development and correct phenotypic expression of cells in biomatrices.1 An alternative approach employs preparation of tissue engineered IVDs where AF and NP cells are separately cultured in polyglycolic acid and sodium alginate biomatrices, either separately or within a manifold designed to reproduce the required IVD dimensions for its use as a prospective implant device.2 AF and NP cells have also been grown on tissue culture inserts after their recovery from alginate bead culture to form plugs of tissue engineered cartilage.3 A key component in this latter strategy was the stimulation of the high density disc cell cultures with osteogenic protein-1 (OP-1) 200 ng/mL.3 This resulted in the production of tissue engineered AF and NP plugs with compositions, histochemical characteristics and biomechanical properties approaching those of the native disc tissues.2,3 Such materials hold reat promise in future applications as disc or annular implants. The introduction of appropriate genes into disc cells by gene transduction methodology using adenoviral vectors or ,gene-gun' delivery systems also holds considerable promise for the promotion of disc repair processes.4 Such an approach with the OP-1 gene is particularly appealing.5 The anchoring of discal implants to vertebral bodies has also been evaluated by several approaches. A 3D fabric based polyethylene biocomposite holds much promise as one such anchorage device6 while biological glues used to seal fibrocartilaginous structures such as the AF and meniscus8 following surgical intervention, also hold promise in this area. Several very promising new experimental approaches and strategies are therefore currently under evaluation for the improvement of discal repair. The aforementioned IVD defects are a common cause of disc failure and sites of increased nerve in-growth in symptomatic IVDs in man and are thus often sources of sciatic-type pain. Annular defects such as those described above have formerly been considered incapable of undergoing spontaneous repair thus a clear need exists for interventions which might improve on their repair. Based on the rapid rate of progress and the examples outlined above one may optimistically suggest that a successful remedy to this troublesome clinical entity will be developed in the not so distant future. References 1JohnsonWEBet al. (2001) Directed cytoskeletal orientation and intervertebral disc cell growth: towards the development of annular repair techniques. Trans Orthop Res Soc26, 894. 2MizunoHet al. (2001) Tissue engineering of a composite intervertebral disc. Trans Orthop Res Soc26, 78. 3MatsumotoTet al. (2001) Formation of transplantable disc shaped tissues by nucleus pulposus and annulus fibrosus cells: biochemical and biomechanical properties. Trans Orthop Res Soc26, 897. 4NishidaKet al. (2000) Potential applications of gene therapy to the treatment of intervertebral disc disorders. Clin Orthop Rel Res379 (Suppl), S234,S241. 5MatsumotoTet al. (2001) Transfer of osteogenic protein-1 gene by gene gun system promotes matrix synthesis in bovine intervertebral disc and articular cartilage cells. Trans Orthop Res Soc26, 30. 6ShikinamiY , Kawarada (1998) Potential application of a triaxial three-dimensional fabric (3-DF) as an implant. Biomaterials19, 617,35. [source] Expression of Acid-Sensing Ion Channel 3 (ASIC3) in Nucleus Pulposus Cells of the Intervertebral Disc Is Regulated by p75NTR and ERK Signaling,JOURNAL OF BONE AND MINERAL RESEARCH, Issue 12 2007Yoshiyasu Uchiyama Abstract Although a recent study has shown that skeletal tissues express ASICs, their function is unknown. We show that intervertebral disc cells express ASIC3; moreover, expression is uniquely regulated and needed for survival in a low pH and hypoeromsotic medium. These findings suggest that ASIC3 may adapt disc cells to their hydrodynamically stressed microenvironment. Introduction: The nucleus pulposus is an avascular, hydrated tissue that permits the intervertebral disc to resist compressive loads to the spine. Because the tissue is hyperosmotic and avascular, the pH of the nucleus pulposus is low. To determine the mechanisms by which the disc cells accommodate to the low pH and hypertonicity, the expression and regulation of the acid sensing ion channel (ASIC)3 was examined. Materials and Methods: Expression of ASICs in cells of the intervertebral disc was analyzed. To study its regulation, we cloned the 2.8-kb rat ASIC3 promoter and performed luciferase reporter assays. The effect of pharmacological inhibition of ASICs on disc cell survival was studied by measuring MTT and caspase-3 activities. Results: ASIC3 was expressed in discal tissues and cultured disc cells in vitro. Because studies of neuronal cells have shown that ASIC3 expression and promoter activity is induced by nerve growth factor (NGF), we examined the effect of NGF on nucleus pulposus cells. Surprisingly, ASIC3 promoter activity did not increase after NGF treatment. The absence of induction was linked to nonexpression of tropomyosin-related kinase A (TrkA), a high-affinity NGF receptor, although a modest expression of p75NTR was seen. When treated with p75NTR antibody or transfected with dominant negative-p75NTR plasmid, there was significant suppression of ASIC3 basal promoter activity. To further explore the downstream mechanism of control of ASIC3 basal promoter activity, we blocked p75NTR and measured phospho extracellular matrix regulated kinase (pERK) levels. We found that DN-p75NTR suppressed NGF mediated transient ERK activation. Moreover, inhibition of ERK activity by dominant negative-mitogen activated protein kinase kinase (DN-MEK) resulted in a dose-dependent suppression of ASIC3 basal promoter activity, whereas overexpression of constitutively active MEK1 caused an increase in ASIC3 promoter activity. Finally, to gain insight in the functional importance of ASIC3, we suppressed ASIC activity in nucleus pulposus cells. Noteworthy, under both hyperosmotic and acidic conditions, ASIC3 served to promote cell survival and lower the activity of the pro-apoptosis protein, caspase-3. Conclusions: Results of this study indicate that NGF serves to maintain the basal expression of ASIC3 through p75NTR and ERK signaling in discal cells. We suggest that ASIC3 is needed for adaptation of the nucleus pulposus and annulus fibrosus cells to the acidic and hyperosmotic microenvironment of the intervertebral disc. [source] MEK/ERK Signaling Controls Osmoregulation of Nucleus Pulposus Cells of the Intervertebral Disc by Transactivation of TonEBP/OREBP,JOURNAL OF BONE AND MINERAL RESEARCH, Issue 7 2007Tsung-Ting Tsai Abstract Earlier studies have shown that intervertebral disc cells express TonEBP, a transcription factor that permits adaptation to osmotic stress and regulates aggrecan gene expression. However, the mechanism of hyperosmotic activation of TonEBP in disc cells is not known. Results of this study show that hypertonic activation of ERK signaling regulates transactivation activity of TonEBP, modulating its function. Introduction: In an earlier report, we showed that tonicity enhancer binding protein (TonEBP) positively regulates aggrecan gene expression in disc cells, thereby autoregulating its osmotic environment. Although these studies indicated that the cells of the nucleus pulposus were optimally adapted to a hyperosmotic state, the mechanism by which the cells transduce the osmotic stress was not delineated. The primary goal of this study was to test the hypothesis that, in a hyperosmotic medium, the extracellular signal-regulated kinase (ERK) signaling pathway regulated TonEBP activity. Materials and Methods: Nucleus pulposus cells were maintained in isotonic or hypertonic media, and MAPK activation and TonEBP expression were analyzed. To study the role of MAPK in regulation of TonEBP function, gel shift and luciferase reporter assays were performed. ERK expression in cells was modulated by using expression plasmids or siRNA, and transactivation domain (TAD)-TonEBP activity was studied. Results: We found that hypertonicity resulted in phosphorylation and activation of ERK1/2 proteins and concomitant activation of C terminus TAD activity of ELK-1, a downstream transcription factor. In hypertonic media, treatment with ERK and p38 inhibitors resulted in downregulation of TonE promoter activity of TauT and HSP-70 and decreased binding of TonEBP to TonE motif. Similarly, forced expression of DN-ERK and DN-p38 in nucleus pulposus cells suppressed TauT and HSP-70 reporter gene activity. Finally, we noted that ERK was needed for transactivation of TonEBP. Expression of DN-ERK significantly suppressed, whereas, WT-ERK and CA-MEK1 enhanced, TAD activity of TonEBP. Experiments performed with HeLa cells indicated that the ERK signaling pathway also served a major role in regulating the osmotic response in nondiscal cells. Conclusions: Together, these studies showed that adaptation of the nucleus pulposus cells to their hyperosmotic milieu is dependent on activation of the ERK and p38- MAPK pathways acting through TonEBP and its target genes. [source] Intervertebral disc cell response to dynamic compression is age and frequency dependent,JOURNAL OF ORTHOPAEDIC RESEARCH, Issue 6 2009Casey L. Korecki Abstract The maintenance of the intervertebral disc extracellular matrix is regulated by mechanical loading, nutrition, and the accumulation of matrix proteins and cytokines that are affected by both aging and degeneration. Evidence suggests that cellular aging may lead to alterations in the quantity and quality of extracellular matrix produced. The aims of this study were to examine the role of loading and maturation (a subset of aging), and the interaction between these two factors in intervertebral disc cell gene expression and biosynthesis in a controlled 3D culture environment. Cells were isolated from young (4,6 months) and mature (18,24 months) bovine caudal annulus fibrosus and nucleus pulposus tissue. Isolated cells were seeded into alginate and dynamically compressed for 7 days at either 0.1, 1, or 3 Hz or maintained as a free-swelling control. After 7 days, DNA and sulfated glycosaminoglycan contents were analyzed along with real time, quantitative reverse transcription-polymerase chain reaction analysis for collagen types I and II, aggrecan, and matrix metalloproteinase-3 gene expression. Results suggest that maturation plays an important role in intervertebral disc homeostasis and influences the cell response to mechanical loading. While isolated intervertebral disc cells responded to mechanical compression in 3D culture, the effect of loading frequency was minimal. Altered cellular phenotype and biosynthesis rates appear to be an attribute of the cell maturation process, potentially independent of changes in cellular microenvironment associated with lost nutrition and disc degeneration. Mature cells may have a decreased capacity to create or retain extracellular matrix components in response to mechanical loading compared to young cells. © 2008 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 27: 800,806, 2009 [source] TNF-,,induced NF-,B signaling reverses age-related declines in VEGF induction and angiogenic activity in intervertebral disc tissuesJOURNAL OF ORTHOPAEDIC RESEARCH, Issue 2 2009Tetsuro Ohba Abstract We previously demonstrated that VEGF and its receptors were expressed in human herniated discs (HD). TNF-, induced VEGF, resulting in neovascularization of disc tissues in a model of HD. The goal of the current research was to investigate the precise role of TNF-,,induced VEGF and the mechanism of angiogenesis in disc tissues. We performed ELISAs, Western blots, and immunohistological examinations to assess the role of TNF-,,induced VEGF using organ disc cultures with wild type, TNF receptor 1-null (TNF-RInull), or TNF receptor 2-null (TNF-RIInull) mice. VEGF induction was inhibited when we used TNF-RInull -derived disc tissues. NF-,B pathway inhibitors also strongly suppressed VEGF induction. Thus, TNF-, induced VEGF expression in disc cells primarily through the NF-,B pathway. In addition, VEGF immunoreactivity was detected predominantly in annulus fibrosus cells and increased after TNF-, stimulation. TNF-, treatment also resulted in CD31 expression on endothelial cells and formation of an anastomosing network. In contrast, angiogenic activity was strongly inhibited in the presence of NF-,B inhibitors or anti-VEGF antibody. Our data show angiogenesis activity in disc tissues is regulated by VEGF and the NF-,B pathway, both of which are induced by TNF-,. The level of angiogenic activity in disc tissues was closely related to aging. Because neovascularization of HD is indispensable for HD resorption, the prognosis of HD and the rate of the resorption process in patients may vary as a function of the patient's age. © 2008 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 27:229,235, 2009 [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] Collagen gene expression and mechanical properties of intervertebral disc cell,alginate culturesJOURNAL OF ORTHOPAEDIC RESEARCH, Issue 1 2001Anthony E. Baer Cells of the intervertebral disc have a limited capacity for matrix repair that may contribute to the onset and progression of degenerative disc changes. In this study, the biosynthetic capacity of cells isolated from specific regions of the porcine intervertebral disc was evaluated in vitro. Using a competitive reverse transcription-polymerase chain reaction technique, gene expression levels for types I and II collagen were quantified in cells cultured for up to 21 d in a three-dimensional alginate culture system and compared to levels obtained for cells in vivo. The mechanical properties of cell-alginate constructs were measured in compression and shear after periods of culture up to 16 weeks. Cells from the anulus fibrosus expressed the most type I collagen mRNA in vivo and in vitro, while cells from the transition zone expressed the most type II collagen mRNA in vivo and in vitro. Mechanical testing results indicate that a mechanically functional matrix did not form at any time during the culture period; rather, decreases of up to 50% were observed in the compressive and shear moduli of the cell,alginate constructs compared to alginate with no cells. Together with results of prior studies, these results suggest that intervertebral disc cells maintain characteristics of their phenotype when cultured in alginate, but the molecules they synthesize are not able to form a mechanically functional matrix in vitro. © 2001 Orthopaedic Research Society. Published by Elsevier Science Ltd. All rights reserved. [source] Notochordal intervertebral disc cells: Sensitivity to nutrient deprivationARTHRITIS & RHEUMATISM, Issue 4 2009Thorsten Guehring Objective The nucleus pulposus (NP) of the intervertebral disc develops from the notochord. Humans and other species in which notochordal cells (NCs) disappear to be replaced by chondrocyte-like mature NP cells (MNPCs) frequently develop disc degeneration, unlike other species that retain NCs. The reasons for NC disappearance are unknown. In humans, the change in cell phenotype (to MNPCs) coincides with changes that decrease nutrient supply to the avascular disc. We undertook this study to test the hypothesis that the consequent nutrient stress could be associated with NC disappearance. Methods We measured cell densities and metabolic rates in 3-dimensional cultures of porcine NCs and bovine MNPCs, and we determined survival rates under conditions of nutrient deprivation. We used scanning electron microscopy to examine end plate porosity of discs with NCs and those with MNPCs. Nutrient,metabolite profiles and cell viability were calculated as a function of cell density and disc size in a consumption/diffusion mathematical model. Results NCs were more active metabolically and more susceptible to nutrient deprivation than were MNPCs. Hypoxia increased rates of glycolysis in NCs but not in MNPCs. Higher end plate porosity in discs with NCs suggested greater nutrient supply in keeping with higher nutritional demands. Mathematical simulations and experiments using an analog disc diffusion chamber indicated that a fall in nutrient concentrations resulting from increased diffusion distance during growth and/or a fall in blood supply through end plate changes could instigate NC disappearance. Conclusion NCs demand more energy and are less resistant to nutritional stress than MNPCs, which may shed light on the fate of NCs in humans. This provides important information about prospective NC tissue engineering approaches. [source] Genetic link between p53 and genes required for formation of the zonula adherens junctionCANCER SCIENCE, Issue 5 2004Masamitsu Yamaguchi Ectopic expression of human p53 in Drosophila eye imaginal disc cells induces apoptosis and results in a rough eye phenotype in the adult flies. We have screened Drosophila stocks to identify mutations that enhance or suppress the p53-induced rough eye phenotype. One of the dominant enhancers of the p53-induced rough eye phenotype corresponds to a loss-of-function mutation of the crumbs gene, which is essential for the biogenesis of the zonula adherens junction and the establishment of apical polarity in epithelial cells. Enhancement of p53-induced apoptosis in the eye imaginal discs by a half-reduction of the crumbs gene dose was confirmed by a TUNEL method. Furthermore, mutations of genes for Shotgun (Drosophila E-cadherin) and Armadillo (Drosophila,-catenin), the two main components of the adherens junction, also strongly enhanced the p53-induced rough eye phenotype. These results suggest that human p53 senses subtle abnormality at the adherens junction or in signals derived from the junction, and consequently induces apoptosis to remove abnormal cells from tissue. Thus p53 likely plays a role as a guardian of the tissue not only by sensing the damaged DNA, but also by sensing signals from the adherens junction. [source] | ||||||||||