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Intervertebral Disc (intervertebral + disc)
Kinds of Intervertebral Disc Terms modified by Intervertebral Disc Selected AbstractsExpression 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, sensory nerves and neurotrophins: who is who in discogenic pain?JOURNAL OF ANATOMY, Issue 1 2010José García-Cosamalón Abstract The normal intervertebral disc (IVD) is a poorly innervated organ supplied only by sensory (mainly nociceptive) and postganglionic sympathetic (vasomotor efferents) nerve fibers. Interestingly, upon degeneration, the IVD becomes densely innervated even in regions that in normal conditions lack innervation. This increased innervation has been associated with pain of IVD origin. The mechanisms responsible for nerve growth and hyperinnervation of pathological IVDs have not been fully elucidated. Among the molecules that are presumably involved in this process are some members of the family of neurotrophins (NTs), which are known to have both neurotrophic and neurotropic properties and regulate the density and distribution of nerve fibers in peripheral tissues. NTs and their receptors are expressed in healthy IVDs but much higher levels have been observed in pathological IVDs, thus suggesting a correlation between levels of expression of NTs and density of innervation in IVDs. In addition, NTs also play a role in inflammatory responses and pain transmission by increasing the expression of pain-related peptides and modulating synapses of nociceptive neurons at the spinal cord. This article reviews current knowledge about the innervation of IVDs, NTs and NT receptors, expression of NTs and their receptors in IVDs as well as in the sensory neurons innervating the IVDs, the proinflammatory role of NTs, NTs as nociception regulators, and the potential network of discogenic pain involving NTs. [source] Effect of varying osmotic conditions on the response of bovine nucleus pulposus cells to growth factors and the activation of the ERK and Akt pathwaysJOURNAL OF ORTHOPAEDIC RESEARCH, Issue 10 2010Eleni Mavrogonatou Abstract Intervertebral disc and especially nucleus pulposus is characterized by low cellularity. Additionally, extreme variations in osmolality are observed in this tissue, as a result of its specific physicochemical environment, daily activities, or degeneration. In this study, we investigated the role of osmotic fluctuations in the proliferative response of nucleus pulposus cells to exogenous growth factors. In particular, we examined the effect of platelet-derived growth factor (PDGF) and insulin-like growth factor-I (IGF-I) on the proliferation of bovine nucleus pulposus cells and on the activation of the MEK/ERK and PI-3-K/Akt pathways under varying osmotic conditions, in an effort to understand the mechanisms regulating cell proliferation in the intact and the degenerated intervertebral disc. Exposure of cells to high osmolality restrained novel DNA synthesis induced by PDGF or IGF-I in a dose-dependent manner and reduced ERK and Akt activation stimulated by serum or isolated growth factors. Our findings indicate that hyperosmolality imposes a strict control in intervertebral disc cells' proliferation, while hypo-osmotic conditions prevailing in degenerated discs may offer a more permissive environment for cellular proliferation. Published by Wiley Periodicals, Inc. J Orthop Res 28:1276,1282, 2010 [source] Apoptosis of human intervertebral discs after trauma compares to degenerated discs involving both receptor-mediated and mitochondrial-dependent pathwaysJOURNAL OF ORTHOPAEDIC RESEARCH, Issue 7 2008Sven K. Tschoeke Abstract Post-traumatic disc degeneration with consecutive loss of reduction and kyphosis remains a debatable issue within both the operative and nonoperative treatment regimen of thoracolumbar spine fractures. Intervertebral disc (IVD) cell apoptosis has been suggested to play a vital role in promoting the degeneration process. To evaluate and compare apoptosis-regulating signaling mechanisms, IVDs were obtained from patients with thoracolumbar spine fractures (n,=,21), patients suffering from symptomatic IVD degeneration (n,=,6), and from patients undergoing surgical resection of a primary vertebral tumor (n,=,3 used as control samples). All tissues were prospectively analyzed in regards to caspase-3/7, -8, and -9 activity, apoptosis-receptor expression levels, and gene expression of the mitochondria-bound apoptosis-regulating proteins Bax and Bcl-2. Morphologic changes characteristic for apoptotic cell death were confirmed by H&E staining. Statistical significance was designated at p,<,0.05 using the Student's t -test. Both traumatic and degenerative IVD demonstrated a significant increase of caspase-3/7 activity with evident apoptosis. Although caspase-3/7 activation was significantly greater in degenerated discs, both showed equally significant activation of the initiator caspases 8 and 9. Traumatic IVD alone demonstrated a significant increase of the Fas receptor (FasR), whereas the TNF receptor I (TNFR I) was equally up-regulated in both morbid IVD groups. Only traumatic IVD showed distinct changes in up-regulated TNF expression, in addition to significantly down-regulated antiapoptotic Bcl-2 protein. Our results suggest that post-traumatic disc changes may be promoted and amplified by both the intrinsic mitochondria-mediated and extrinsic receptor-mediated apoptosis signaling pathways, which could be, in part, one possible explanation for developing subsequent disc degeneration. © 2008 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 26:999,1006, 2008 [source] An in vivo model of degenerative disc diseaseJOURNAL OF ORTHOPAEDIC RESEARCH, Issue 1 2003Jason P. Norcross Although the precise etiology of low back pain is disputed, degeneration of the intervertebral disc is believed to play an important role. Many animal models have been described which reproduce the changes found in degenerative disc disease, but none allow for efficient, large-scale testing of purported therapeutic agents. The purpose of this study was to develop a simple animal model resembling degenerative disc disease using the intervertebral discs found in the tails of rats. The proximal two intervertebral discs in the tails of 20 rats were injected with either chondroitinase ABC or control (phosphate buffered saline, PBS). The tails were harvested at 2 weeks, and measurements were made of intervertebral disc height (measured radiographically and histologically), biomechanics (stiffness, hysteresis, and residual deformation), and histologic appearance. Treatment with chondroitinase ABC resulted in a significant loss in intervertebral disc height (radiographic intervertebral disc height, p < 0.001; histologic intervertebral disc height, p < 0.001) and significant increases in all biomechanical parameters (stiffness, p < 0.001; hysteresis, p < 0.006; residual deformation, p < 0.004) compared to PBS controls. Intervertebral discs treated with chondroitinase ABC had significantly lower histologic grades for each grading category (nucleus pulposus (NP), annulus fibrosus, and proteoglycan staining) compared to controls. The results of injury with chondroitinase ABC were similar to the findings in degenerative disc disease: reduced intervertebral disc height, diminished proteoglycan content, loss of NP cells, and increased stiffness of the disc. Thus, the model appears to be a reasonable tool for the preliminary in vivo evaluation of proposed treatments for degenerative disc disease. © 2002 Orthopaedic Research Society. Published by Elsevier Science Ltd. All rights reserved. [source] A preliminary in vitro study into the use of IL-1Ra gene therapy for the inhibition of intervertebral disc degenerationINTERNATIONAL JOURNAL OF EXPERIMENTAL PATHOLOGY, Issue 1 2006Christine L. Le Maitre Summary Conventional therapies for low back pain (LBP) are purely symptomatic and do not target the cause of LBP, which in approximately 40% of cases is caused by degeneration of the intervertebral disc (DIVD). Targeting therapies to inhibit the process of degeneration would be a potentially valuable treatment for LBP. There is increasing evidence for a role for IL-1 in DIVD. A natural inhibitor of IL-1 exists, IL-1Ra, which would be an ideal molecular target for inhibiting IL-1-mediated effects involved in DIVD and LBP. In this study, the feasibility of ex vivo gene transfer of IL-1Ra to the IVD was investigated. Monolayer and alginate cultures of normal and degenerate human intervertebral disc (IVD) cells were infected with an adenoviral vector carrying the IL-1Ra gene (Ad-IL-1Ra) and protein production measured using an enzyme-linked immunosorbent assay. The ability of these infected cells to inhibit the effects of IL-1 was also investigated. In addition, normal and degenerate IVD cells infected with Ad-IL-1Ra were injected into degenerate disc tissue explants and IL-1Ra production in these discs was assessed. This demonstrated that both nucleus pulposus and annulus fibrosus cells infected with Ad-IL-1Ra produced elevated levels of IL-1Ra for prolonged time periods, and these infected cells were resistant to IL-1. When the infected cells were injected into disc explants, IL-1Ra protein expression was increased which was maintained for 2 weeks of investigation. This in vitro study has shown that the use of ex vivo gene transfer to degenerate disc tissue is a feasible therapy for the inhibition of IL-1-mediated events during disc degeneration. [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] A comparative analysis of the differential spatial and temporal distributions of the large (aggrecan, versican) and small (decorin, biglycan, fibromodulin) proteoglycans of the intervertebral discJOURNAL OF ANATOMY, Issue 1 2001JAMES MELROSE This study provides a comparative analysis of the temporal and spatial distribution of 5 intervertebral disc (IVD) proteoglycans (PGs) in sheep. The main PGs in the 2 and 10 y old sheep groups were polydisperse chondroitin sulphate and keratan sulphate substituted species. Their proportions did not differ markedly either with spinal level or disc zone. In contrast, the fetal discs contained 2 slow migrating (by composite agarose polyacrylamide gel electrophoresis, CAPAGE), relatively monodisperse chondroitin sulphate-rich aggrecan species which were also identified by monoclonal antibody 7-D-4 to an atypical chondroitin sulphate isomer presentation previously found in chick limb bud, and shark cartilage. The main small PG detectable in the fetal discs was biglycan, whereas decorin predominated in the 2 and 10 y old IVD samples; its levels were highest in the outer annulus fibrosus (AF). Versican was most abundant in the AF of the fetal sheep group; it was significantly less abundant in the 2 and 10 y old groups. Furthermore, versican was immunolocalised between adjacent layers of annular lamellae suggesting that it may have some role in the provision of the viscoelastic properties to this tissue. Versican was also diffusely distributed throughout the nucleus pulposus of fetal IVDs, and its levels were significantly lower in adult IVD specimens. This is the first study to identify versican in ovine IVD tissue sections and confirmed an earlier study which demonstrated that ovine IVD cells synthesised versican in culture (Melrose et al. 2000). The variable distribution of the PGs identified in this study provides further evidence of differences in phenotypic expression of IVD cell populations during growth and development and further demonstrates the complexity of the PGs in this heterogeneous but intricately organised connective tissue. [source] Synthesis and characterization of injectable bioadhesive hydrogels for nucleus pulposus replacement and repair of the damaged intervertebral discJOURNAL OF BIOMEDICAL MATERIALS RESEARCH, Issue 2 2010J. Vernengo Abstract Bioadhesive polymers are natural or synthetic materials that can be used for soft tissue repair. The aim of this investigation was to develop an injectable, bioadhesive hydrogel with the potential to serve as a synthetic replacement for the nucleus pulposus of the intervertebral disc or as an annulus closure material. Branched copolymers of poly(N -isopropylacrylamide) (PNIPAAm) and poly(ethylene glycol) (PEG) were blended with poly(ethylene imine) (PEI). This three component injectable system can form a precipitated gel at physiological temperature due to the phase transition of PNIPAAm. The injection of glutaraldehyde into the gel core will adhere the implant to the surrounding tissues. 1H NMR results indicated the successful physical incorporation of PEI into the PNIPAAm-PEG network by blending. In addition, the covalent crosslinking between the amine functionalities on the PEI and the aldehyde functionalities on the glutaraldehyde was verified using FTIR difference spectroscopy. Mechanical characterization of these blends showed a significant increase (p < 0.05) in compressive modulus following glutaraldehyde injection. The in vitro bioadhesive force studies with porcine skin showed a significant increase (p < 0.05) in the mean maximum force of detachment for PNIPAAm-PEG/PEI gels when glutaraldehyde was injected into the gel core. The results of this study indicate that the reactivity between amines and aldehyde functionalities can be exploited to impart bioadhesive properties to PNIPAAm-PEG/PEI copolymers. © 2010 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 2010 [source] The effect of nucleus implant parameters on the compressive mechanics of the lumbar intervertebral disc: A finite element studyJOURNAL OF BIOMEDICAL MATERIALS RESEARCH, Issue 2 2009Abhijeet Joshi Abstract A simplified finite element model of the human lumbar intervertebral disc was utilized for understanding nucleus pulposus implant mechanics. The model was used to assess the effect of nucleus implant parameter variations on the resulting compressive biomechanics of the lumbar anterior column unit. The effects of nucleus implant material (modulus and Poisson's ratio) and geometrical (height and diameter) parameters on the mechanical behavior of the disc were investigated. The model predicted that variations in implant modulus contribute less to the compressive disc mechanics compared to the implant geometrical parameters, for the ranges examined. It was concluded that some threshold exists for the nucleus implant modulus, below which little variations in load,displacement behavior were shown. Compressive biomechanics were highly affected by implant volume (under-filling the nucleus cavity, line-to-line fit, or over-filling the nucleus cavity) with a greater restoration of compressive mechanics observed with the over-filled implant design. This work indicated the effect of nucleus implant parameter variations on the compressive mechanics of the human lumbar intervertebral disc and importance of the "fit and fill" effect of the nuclear cavity in the restoration of the human intervertebral disc mechanics in compression. These findings may have clinical significance for nucleus implant design. © 2009 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 2009 [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] PI3K/AKT regulates aggrecan gene expression by modulating Sox9 expression and activity in nucleus pulposus cells of the intervertebral discJOURNAL OF CELLULAR PHYSIOLOGY, Issue 3 2009Chin-Chang Cheng The goal of the investigation was to test the hypothesis that the phosphoinositide-3 kinase (PI3K)/AKT signaling pathway regulates the expression of the major extracellular matrix component of the intervertebral disc, aggrecan, in nucleus pulposus cells. Primary rat nucleus pulposus cells were treated with PI3K inhibitor to measure changes in gene and protein expression. In addition, cells were transfected with various luciferase reporter plasmids to investigate mechanisms of regulation of aggrecan gene expression. We found that treatment of nucleus pulposus cells with a PI3K inhibitor, LY294002 resulted in decreased expression of aggrecan and a reduction in deposition of sulfated glycosaminoglycans. Moreover, pharmacological suppression or co-expression of dominant negative (DN)-PI3K or DN-AKT resulted in downregulation of aggrecan promoter activity. Expression of constitutively active (CA)-PI3K significantly induced aggrecan promoter activity. We observed that PI3K maintained Sox9 gene expression and activity: inhibition of PI3K/AKT resulted in decreased Sox9 expression, lowered promoter activity, and mediated a reduction in Sox9 transcriptional activity. PI3K effects were independent of phosphorylation status of C-terminus transactivation domain (TAD) of Sox9. Finally, we noted that in nucleus pulposus cells, PI3K signaling controlled transactivation of p300 (p300-TAD activity), an important transcriptional co-activator of Sox9. Results of these studies demonstrate for the first time that PI3K/AKT signaling controls aggrecan gene expression, in part by modulating Sox9 expression and activity in cells of the nucleus pulposus. J. Cell. Physiol. 221: 668,676, 2009. © 2009 Wiley-Liss, Inc. [source] Effects of high-intensity focused ultrasound on the intervertebral disc: A potential therapy for disc herniationsJOURNAL OF CLINICAL ULTRASOUND, Issue 7 2006Carina Forslund PhD Abstract Purpose. To determine the potential application of high-intensity focused ultrasound for the minimally invasive treatment of herniated intervertebral discs by developing a probe that produces sufficiently high temperature locally to shrink collagen fibers (65,75°C). Materials and Methods. A 5-mm ultrasound probe was produced with a geometric focal length of 15 mm. The probe produced 2.5 W of acoustic power and was operated at a frequency of 4.1 MHz. Measurements of temperature increase were performed in discs from bovine tails. In vivo experiments were performed to assess histologic changes in the disc as well as in nerve root and muscle. Results. Sufficient temperature increase to produce collagen shrinkage was observed close to the focus of the ultrasound. Temperature measurements in vertebral end plates showed a temperature increase of only 4°C after 60-second exposure of the disc. In vivo experiments revealed histologic changes in the disc consistent with collagen shrinkage, with no adverse effects seen in surrounding tissues. Conclusions. The experiments demonstrated the feasibility of high-intensity focused ultrasound in the treatment of contained herniated discs. This technique has several advantages over other thermal treatment modalities. © 2006 Wiley Periodicals, Inc. J Clin Ultrasound 34:330,338, 2006 [source] Effect of cell number on mesenchymal stem cell transplantation in a canine disc degeneration modelJOURNAL OF ORTHOPAEDIC RESEARCH, Issue 10 2010Kenji Serigano Abstract Transplantation of mesenchymal stem cells (MSCs) inhibits the progression of disc degeneration in animal models. We know of no study to determine the optimal number of cells to transplant into the degenerated intervertebral disc (IVD). To determine the optimal donor cell number for maximum benefit, we conducted an in vivo study using a canine disc degeneration model. Autologous MSCs were transplanted into degenerative discs at 105, 106, or 107,cells per disc. The MSC-transplanted discs were evaluated for 12 weeks using plain radiography, magnetic resonance imaging, and gross and microscopic evaluation. Preservation of the disc height, annular structure was seen in MSC-transplantation groups compared to the operated control group with no MSC transplantation. Result of the number of remaining transplanted MSCs, the survival rate of NP cells, and apoptosis of NP cells in transplanted discs showed both structural microenvironment and abundant extracellular matrix maintained in 106 MSCs transplanted disc, while less viable cells were detected in 105 MSCs transplanted and more apoptotic cells in 107 MSCs transplanted discs. The results of this study demonstrate that the number of cells transplanted affects the regenerative capability of MSC transplants in experimentally induced degenerating canine discs. It is suggested that maintenance of extracellular matrix by its production from transplanted cells and/or resident cells is important for checking the progression of structural disruption that leads to disc degeneration. Published by Wiley Periodicals, Inc. J Orthop Res 28:1267,1275, 2010 [source] Effect of varying osmotic conditions on the response of bovine nucleus pulposus cells to growth factors and the activation of the ERK and Akt pathwaysJOURNAL OF ORTHOPAEDIC RESEARCH, Issue 10 2010Eleni Mavrogonatou Abstract Intervertebral disc and especially nucleus pulposus is characterized by low cellularity. Additionally, extreme variations in osmolality are observed in this tissue, as a result of its specific physicochemical environment, daily activities, or degeneration. In this study, we investigated the role of osmotic fluctuations in the proliferative response of nucleus pulposus cells to exogenous growth factors. In particular, we examined the effect of platelet-derived growth factor (PDGF) and insulin-like growth factor-I (IGF-I) on the proliferation of bovine nucleus pulposus cells and on the activation of the MEK/ERK and PI-3-K/Akt pathways under varying osmotic conditions, in an effort to understand the mechanisms regulating cell proliferation in the intact and the degenerated intervertebral disc. Exposure of cells to high osmolality restrained novel DNA synthesis induced by PDGF or IGF-I in a dose-dependent manner and reduced ERK and Akt activation stimulated by serum or isolated growth factors. Our findings indicate that hyperosmolality imposes a strict control in intervertebral disc cells' proliferation, while hypo-osmotic conditions prevailing in degenerated discs may offer a more permissive environment for cellular proliferation. Published by Wiley Periodicals, Inc. J Orthop Res 28:1276,1282, 2010 [source] Effects of cyclic dynamic tensile strain on previously compressed inner annulus fibrosus and nucleus pulposus cells of human intervertebral disc,an in vitro studyJOURNAL OF ORTHOPAEDIC RESEARCH, Issue 4 2010Hwan Tak Hee Abstract Our objective was to investigate whether dynamic tensile strain on previously compressed human intervertebral disc (IVD) cells can restore the biosynthetic effects of collagen and glycosaminoglycan. Inner annulus fibrosus (AF) and nucleus pulposus (NP) tissues of adolescent idiopathic scoliosis cases undergoing thoracoscopic discectomy and fusion were cultured on compressive plates. Compressive stress was applied using 0.4 MPa at 1 Hz, for 2 h twice a day for 7 days, to the inner AF and NP tissues, followed by equibiaxial cyclic tensile strain to deform the released cells onto the plate's flexible bottom. With 10% elongation at a rate of 1 Hz, for 2 h twice a day for 7 days, a significant increase in the level of collagen and glycosaminoglycan of the previously compressed inner AF, as well as the level of glycosaminoglycan of the previously compressed NP cells were found. The DNA content and number of endoplasmic reticulum under transmission electron micrograph of the previously compressed inner AF and NP cell were also significantly increased. The results suggested that equibiaxial cyclic tensile strain at a rate of 1 Hz with 10% tensile strain was capable of increasing collagen and glycosaminoglycan synthesis of previously compressed inner AF cells, and glycosaminoglycan synthesis of previously compressed NP cells. © 2009 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 28:503,509, 2010 [source] The presence and distribution of lubricin in the caprine intervertebral discJOURNAL OF ORTHOPAEDIC RESEARCH, Issue 10 2008Kristy M. Shine Abstract Lubricin is a large, multifunctional glycoprotein that is known to play a role as a boundary lubricant in diarthrodial joint articulation. The hypothesis of this study was that lubricin is present in the intervertebral disc in a distribution consistent with serving to facilitate interlamellar tribology. The objectives were to: (1) determine the distribution of lubricin in the normal caprine disc; and (2) investigate the synthesis of lubricin by caprine annulus fibrosus (AF) and nucleus pulposus (NP) cells in vitro, using immunohistochemical methods. Caprine lumbar intervertebral discs from five levels and four animals were studied. Positive staining revealed the presence of the lubricin in the outer AF of nearly all samples. No staining was present in the inner AF or the NP. Within the outer AF, lubricin was prominent in the layers separating lamellae and in the extracellular matrix of the lamellae. Some of the AF cells within the lubricin-positive regions demonstrated intracellular lubricin staining, suggesting that these cells may be synthesizing the lubricin protein observed. Immunohistochemistry performed on monolayer cultures of primary AF and NP cells demonstrated intracellular lubricin staining in both cell types. Thus, lubricin is selectively present in the outer caprine intervertebral disc AF, and its distribution suggests that it may play a role in interlamellar tribology. Cells from both the annulus and nucleus were found capable of synthesizing lubricin in vitro, suggesting that these cells may be a potential source of the glycoprotein under some conditions. © 2008 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 26:1398,1406, 2008 [source] Experimental and model determination of human intervertebral disc osmoviscoelasticityJOURNAL OF ORTHOPAEDIC RESEARCH, Issue 8 2008Y. Schroeder Abstract Finite element (FE) models have become an important tool to study load distribution in the healthy and degenerated disc. However, model predictions require accurate constitutive laws and material properties. As the mechanical properties of the intervertebral disc are regulated by its biochemical composition and fiber-reinforced structure, the relationship between the constitutive behavior of the tissue and its composition requires careful consideration. While numerous studies have investigated the annulus fibrosus compressive and tensile properties, specific conditions required to determine model parameters for the osmoviscoelastic model are unavailable. Therefore, the objectives of this study were (1) to complement the existing material testing in the literature with confined compression and tensile tests on human annulus fibrosus and (2) to use these data, together with existing nucleus pulposus compression data to tune a composition-based, osmoviscoelastic material constitutive law. The osmoviscoelastic material constitutive law and the experimental data were used to describe the fiber and nonfiber properties of the human disc. The compressive material properties of normal disc tissue were Gm,=,1.23 MPa, M,=,1.57, and ,,=,1.964,×,10,16 m4/Ns; the tensile fiber material parameters were E0,=,77.0 MPa; E,,=,500 MPa, and ,,=,1.8,×,103 MPa,s. The goodness of fit ranged from 0.88 to 0.96 for the four experimental conditions evaluated. The constitutive law emphasized the interdependency of the strong swelling ability of the tissue and the viscoelastic nature of the collagen fibers. This is especially important for numerical models to further study the load sharing behavior with regard to disc degeneration and regeneration. © 2008 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 26:1141,1146, 2008 [source] Functional integrin subunits regulating cell,matrix interactions in the intervertebral discJOURNAL OF ORTHOPAEDIC RESEARCH, Issue 6 2007Christopher L. Gilchrist Abstract Cellular interactions with the extracellular matrix are key factors regulating cell survival, differentiation, and response to environmental stimuli in cartilagenous tissues. Much is known about the extracellular matrix proteins in the intervertebral disc (IVD) and their variations with region, age, or degenerative state of the tissue. In contrast, little is known of the integrin cell surface receptors that directly bind to and interact with these matrix proteins in the IVD. In almost all tissues, these integrin-mediated cell,matrix interactions are important for transducing environmental cues arising from mechanical stimuli, matrix degradation fragments, and cytokines into intracellular signals. In this study, cells from the nucleus pulposus and anulus fibrosus regions of porcine IVDs were analyzed via flow cytometry to quantify integrin expression levels upon isolation and after monolayer culture. Assays of cell attachment to collagens, fibronectin, and laminin were performed after functional blocking of select integrin subunits to evaluate the role of specific integrins in cell attachment. In situ distribution and co-localization of integrins and laminin were also characterized. Results identify integrin receptors critical for IVD cell interactions with collagens (,1,1) and fibronectin (,5,1). Additionally, dramatic differences in cell,laminin interactions were observed between cells of the nucleus and anulus regions, including differences in ,6 integrin expression, cell adhesion to laminin, and in situ pericellular environments. These findings suggest laminin,cell interactions may be important and unique to the nucleus pulposus region of the IVD. The results of this study provide new information on functional cell,matrix interactions in tissues of the IVD. © 2006 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 25: 829,840, 2007 [source] Nucleus pulposus glycosaminoglycan content is correlated with axial mechanics in rat lumbar motion segmentsJOURNAL OF ORTHOPAEDIC RESEARCH, Issue 9 2006John I. Boxberger Abstract The unique biochemical composition and structure of the intervertebral disc allow it to support load, permit motion, and dissipate energy. With degeneration, both the biochemical composition and mechanical behavior of the disc are drastically altered, yet quantitative relationships between the biochemical changes and overall motion segment mechanics are lacking. The objective of this study was to determine the contribution of nucleus pulposus glycosaminoglycan content, which decreases with degeneration, to mechanical function of a rat lumbar spine motion segment in axial loading. Motion segments were treated with varying doses of Chondroitinase-ABC (to degrade glycosaminoglycans) and loaded in axial cyclic compression-tension, followed by compressive creep. Nucleus glycosaminoglycan content was significantly correlated (p,<,0.05) with neutral zone mechanical behavior, which occurs in low load transition between tension and compression (stiffness: r,=,0.59; displacement: r,=,,0.59), and with creep behavior (viscous parameter ,1: r,=,0.34; short time constant ,1: r,=,0.46). These results indicate that moderate decreases in nucleus glycosaminoglycan content consistent with early human degeneration affect overall mechanical function of the disc. These decreases may expose the disc to altered internal stress and strain patterns, thus contributing through mechanical or biological mechanisms to the degenerative cascade. © 2006 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res [source] An in vivo model of degenerative disc diseaseJOURNAL OF ORTHOPAEDIC RESEARCH, Issue 1 2003Jason P. Norcross Although the precise etiology of low back pain is disputed, degeneration of the intervertebral disc is believed to play an important role. Many animal models have been described which reproduce the changes found in degenerative disc disease, but none allow for efficient, large-scale testing of purported therapeutic agents. The purpose of this study was to develop a simple animal model resembling degenerative disc disease using the intervertebral discs found in the tails of rats. The proximal two intervertebral discs in the tails of 20 rats were injected with either chondroitinase ABC or control (phosphate buffered saline, PBS). The tails were harvested at 2 weeks, and measurements were made of intervertebral disc height (measured radiographically and histologically), biomechanics (stiffness, hysteresis, and residual deformation), and histologic appearance. Treatment with chondroitinase ABC resulted in a significant loss in intervertebral disc height (radiographic intervertebral disc height, p < 0.001; histologic intervertebral disc height, p < 0.001) and significant increases in all biomechanical parameters (stiffness, p < 0.001; hysteresis, p < 0.006; residual deformation, p < 0.004) compared to PBS controls. Intervertebral discs treated with chondroitinase ABC had significantly lower histologic grades for each grading category (nucleus pulposus (NP), annulus fibrosus, and proteoglycan staining) compared to controls. The results of injury with chondroitinase ABC were similar to the findings in degenerative disc disease: reduced intervertebral disc height, diminished proteoglycan content, loss of NP cells, and increased stiffness of the disc. Thus, the model appears to be a reasonable tool for the preliminary in vivo evaluation of proposed treatments for degenerative disc disease. © 2002 Orthopaedic Research Society. Published by Elsevier Science Ltd. All rights reserved. [source] The characterization of versican and its message in human articular cartilage and intervertebral discJOURNAL OF ORTHOPAEDIC RESEARCH, Issue 2 2002Robert Sztrolovics Splicing variation of the versican message and size heterogeneity of the versican core protein were analyzed in human articular cartilage and intervertebral disc. Splicing variation of the message was studied by PCR analysis to detect the presence or absence of exons 7 and 8, which encode large chondroitin sulfate attachment regions. At all ages in normal cartilage from the third trimester fetus to the mature adult, the presence of the versican isoform possessing exon 8 but not exon 7 (V1) could be readily detected. The message isoforms possessing neither exon 7 nor 8 (V3) or both exons 7 and 8 (V0) were only detectable in the fetus, and the isoform possessing only exon 7 (V2) was never detected. In osteoarthritic cartilage and in adult intervertebral disc the versican message pattern was the same as that observed in the normal adult with only the isoform possessing exon 8 being detected. Core protein heterogeneity was studied by immunoblotting following enzymic removal of the glycosaminoglycan chains from the proteoglycan, using an antibody recognizing the globular G1 region of versican. All articular cartilage extracts from the fetus to the mature adult contained multiple core protein sizes of greater than 200 kDa. The adult cartilage extracts tended to have an increased proportion of the smaller sized core proteins and osteoarthritic cartilage possessed similar core protein sizes to the normal adult. In contrast, intervertebral disc at all post-natal ages showed a greater range of size heterogeneity with a prominent component of about 50 kDa. The abundance of this component increased if the samples were treated with keratanase prior to analysis, suggesting that the G1 region of versican in disc can be substituted with keratan sulfate. The increased presence of versican in the disc relative to articular cartilage may suggest a more pronounced functional role for this proteoglycan, particularly in the nucleus pulposus. © 2002 Orthopaedic Research Society. Published by Elsevier Science Ltd. All rights reserved. [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] Treatment of a prolapsed lumbar intervertebral disc in a ferretJOURNAL OF SMALL ANIMAL PRACTICE, Issue 10 2004D. Lu A seven-month-old, male ferret had acute paraplegia and radiographs showed signs of disc prolapse between the second and third lumbar vertebrae (L2/3). Hemilaminectomy was performed to decompress the spinal cord. Histological examination revealed that the extradural material was consistent with annulus fibrosus and the L2/3 articular facets were enlarged as a result of bone remodelling. The ferret became ambulatory one month postoperatively. Five months postoperatively, the ferret had normal posture with mild proprioceptive deficits in the pelvic limbs, and fusion of the L2 and L3 vertebral bodies. [source] Intervertebral Disc Biacuplasty for the Treatment of Lumbar Discogenic Pain: Results of a Six-Month Follow-UpPAIN MEDICINE, Issue 1 2008Leonardo Kapural MD ABSTRACT Objective., Intradiscal biacuplasty (IDB) is a novel bipolar cooled radiofrequency system for the treatment of degenerative disk disease. We present the results of a pilot trial with 6-month follow-up. Design, Setting, Patients, and Interventions., Fifteen patients, 22,55 years old, underwent one- or two-level IDB treatment of their painful lumbar discs. All had chronic low back pain >6 months, back pain exceeding leg pain, concordant pain on provocative discography, disc height >50% of control, and evidence of single- or two-level degenerative disc disease without evidence of additional changes on magnetic resonance imaging. IDB was performed under fluoroscopy using two radiofrequency probes positioned bilaterally in the intervertebral disc. Thirteen patients completed follow-up questionnaires at 1, 3, and 6 months. Pain disability was evaluated with Oswestry and Short Form (SF)-36 questionnaires. Results., Median visual analog scale pain scores were reduced from 7 (95% confidence interval [CI] 6, 8) to 4 (2, 5) cm at 1 month, and remained at 3 (2, 5) cm at 6 months. The Oswestry improved from 23.3 (SD 7.0) to 16.5 (6.8) points at 1 month and remained similar after 6 months. The SF-36 Physical Functioning scores improved from 51 (18) to 70 (16) points after 6 months, while the SF-36 Bodily Pain score improved from 38 (15) to 54 (23) points. Daily opioid use did not change significantly from baseline: from 40 (95% CI 40, 120) before IDB to 5 (0, 40) mg of morphine sulfate equivalent 6 months after IDB. No procedure-related complications were detected. Conclusions., Patients showed improvements in several pain assessment measures after undergoing IDB for discogenic pain. A randomized controlled study is warranted and needed to address the efficacy of the procedure. [source] Disc Related and Non-Disc Related Disorders of the Thoracic SpinePAIN PRACTICE, Issue 2 2001Phillip S. Sizer Jr. MEd Abstract: Different anatomical structures and pathophysiological functions can be responsible for lumbar pain, each producing a distinctive clinical profile. Pain can arise from the intervertebral disc, either acutely as a primary disc related disorder, or as result of the degradation associated with chronic internal disc disruption. In either case, greatest pain provocation will be associated with movements and functions in the sagittal plane. Lumbar pain can also arise from afflictions within the zygapophyseal joint mechanism, as a result of synovitis or chondropathy. Either of these conditions will produce the greatest pain provocation during three-dimensional movements, due to maximal stress to either the synovium or joint cartilage. Finally, patients can experience different symptoms associated with irritation to the dural sleeve, dorsal root ganglion, or chemically irritated lumbar nerve root. Differential diagnosis of these conditions requires a thorough examination and provides information that can assist the clinician in selecting appropriate management strategies. [source] Enhancement of intervertebral disc cell senescence by WNT/,-catenin signaling,induced matrix metalloproteinase expressionARTHRITIS & RHEUMATISM, Issue 10 2010Akihiko Hiyama Objective To determine whether intervertebral disc (IVD) cells express ,-catenin and to assess the role of the WNT/,-catenin signaling pathway in cellular senescence and aggrecan synthesis. Methods The expression of ,-catenin messenger RNA (mRNA) and protein in rat IVD cells was assessed by using several real-time reverse transcription,polymerase chain reaction, Western blot, immunohistochemical, and immunofluorescence analyses. The effect of WNT/,-catenin on nucleus pulposus (NP) cells was examined by transfection experiments, an MTT assay, senescence-associated ,-galactosidase staining, a cell cycle analysis, and a transforming growth factor (TGF,)/bone morphogenetic protein (BMP) pathway,focused microarray analysis. Results We found that ,-catenin mRNA and protein were expressed in discs in vivo and that rat NP cells exhibited increased ,-catenin mRNA and protein upon stimulation with lithium chloride, a known activator of WNT signaling. LiCl treatment inhibited the proliferation of NP cells in a time- and dose-dependent manner. In addition, there was an increased level of cellular senescence in LiCl-treated cells. Long-term treatment with LiCl induced cell cycle arrest and promoted subsequent apoptosis in NP cells. Activation of WNT/,-catenin signaling also regulated the expression of aggrecan. We also demonstrated that WNT/,-catenin signaling induced the expression of matrix metalloproteinases (MMPs) and TGF, in NP cells. Conclusion The activation of WNT/,-catenin signaling promotes cellular senescence and may modulate MMP and TGF, signaling in NP cells. We hypothesize that the activation of WNT/,-catenin signaling may lead to an increased breakdown of the matrix, thereby promoting IVD degeneration. [source] Hypoxia-inducible factor regulation of ANK expression in nucleus pulposus cells: Possible implications in controlling dystrophic mineralization in the intervertebral discARTHRITIS & RHEUMATISM, Issue 9 2010Renata Skubutyte Objective Since nucleus pulposus cells reside under conditions of hypoxia, we determined if the expression of ANK, a pyrophosphate transporter, is regulated by the hypoxia-inducible factor (HIF) proteins. Methods Quantitative reverse transcription,polymerase chain reaction and Western blot analyses were used to measure ANK expression in nucleus pulposus cells from rats and humans. Transfections were performed to determine the effect of HIF-1/2 on ANK promoter activity. Results ANK was expressed in embryonic and mature rat discs. Oxygen-dependent changes in ANK expression in nucleus pulposus cells were minimal. However, silencing of HIF-1, and HIF-2, resulted in increased ANK expression and up-regulation of promoter activity. HIF-mediated suppression of ANK was validated by measuring promoter activity in HIF-1,,null embryonic fibroblasts. Under conditions of hypoxia, there was induction of promoter activity in the null cells as compared with the wild-type cells. Overexpression of HIF-1, and HIF-2, in nucleus pulposus cells resulted in a significant suppression of ANK promoter activity. Since the ANK promoter contains 2 hypoxia-responsive elements (HREs), we performed site-directed mutagenesis and measured promoter activity. We found that HIF-1 can bind to either of the HREs and can suppress promoter activity; in contrast, HIF-2 was required to bind to both HREs in order to suppress activity. Finally, analysis of human nucleus pulposus tissue showed that while ANK was expressed in normal tissue, there was increased expression of ANK along with alkaline phosphatase in the degenerated state. Conclusion Both HIF-1 and HIF-2 serve as negative regulators of ANK expression in the disc. We propose that baseline expression of ANK in the disc serves to prevent mineral formation under physiologic conditions. [source] Proinflammatory cytokine expression profile in degenerated and herniated human intervertebral disc tissuesARTHRITIS & RHEUMATISM, Issue 7 2010Mohammed F. Shamji Objective Prior reports document macrophage and lymphocyte infiltration with proinflammatory cytokine expression in pathologic intervertebral disc (IVD) tissues. Nevertheless, the role of the Th17 lymphocyte lineage in mediating disc disease remains uninvestigated. We undertook this study to evaluate the immunophenotype of pathologic IVD specimens, including interleukin-17 (IL-17) expression, from surgically obtained IVD tissue and from nondegenerated autopsy control tissue. Methods Surgical IVD tissues were procured from patients with degenerative disc disease (n = 25) or herniated IVDs (n = 12); nondegenerated autopsy control tissue was also obtained (n = 8) from the anulus fibrosus and nucleus pulposus regions. Immunohistochemistry was performed for cell surface antigens (CD68 for macrophages, CD4 for lymphocytes) and various cytokines, with differences in cellularity and target immunoreactivity scores analyzed between surgical tissue groups and between autopsy control tissue regions. Results Immunoreactivity for IL-4, IL-6, IL-12, and interferon-, (IFN,) was modest in surgical IVD tissue, although expression was higher in herniated IVD samples and virtually nonexistent in control samples. The Th17 lymphocyte product IL-17 was present in >70% of surgical tissue fields, and among control samples was detected rarely in anulus fibrosus regions and modestly in nucleus pulposus regions. Macrophages were prevalent in surgical tissues, particularly herniated IVD samples, and lymphocytes were expectedly scarce. Control tissue revealed lesser infiltration by macrophages and a near absence of lymphocytes. Conclusion Greater IFN, positivity, macrophage presence, and cellularity in herniated IVDs suggests a pattern of Th1 lymphocyte activation in this pathology. Remarkable pathologic IVD tissue expression of IL-17 is a novel finding that contrasts markedly with low levels of IL-17 in autopsy control tissue. These findings suggest involvement of Th17 lymphocytes in the pathomechanism of disc degeneration. [source] | ||||||||||||||||