Annulus Fibrosus Cells Research Articles

616. Disc Gene Therapy: Development of a Novel Inducible System To Regulate Expression of the Therapeutic Transgene TIMP1

Introduction: Gene therapy using the anti-catabolic factor Tissue Inhibitor of Metalloproteinase-1 (hTIMP1) has shown promising efficacy in treating disc degeneration. However, a major limitation of the current gene therapy technique is the use of a constitutive promoter to drive transgene expression that results in uncontrolled and excessive expression of therapeutic gene product which could result in undesirable side effects. To overcome this important limitation, we successfully developed a new gene therapy recombinant adeno-associated viral (rAAV)vector, rAAV-NFkB-hTIMP1, in which the therapeutic recombinant human TIMP1 gene is placed under the control of a promoter containing the NF-kB element. The rAAV-NFkB-hTIMP1 construct is designed to produce the therapeutic gene product TIMP1 only when the cells experience stress which activates the NF-kB pathway. Hence we expect using this strategy that hTIMP1 will be selectively expressed in cells under inflammatory conditions typically found in active degenerating discs and not under normal conditions, in which the therapeutic gene was not needed. In this study we tested our hypothesis that rabbit annulus fibrosus cells transfected with aserotype 2 of rAAV vector carrying a hTIMP1 gene will not express high level of hTIMP1 unless stimulated with the IL-1b.Methods: Cultured rabbit annulus fibrosus (rAF) cells were divided into six groups; “Control” (cell only); “rAAV-CMV-hTIMP1(cells transfected with AAV-CMV-hTIMP1 plasmid DNA)” and “rAAV-NFkB-hTIMP1(cells transfected with AAV-NFkB-hTIMP1 plasmid DNA)” treated with and without IL-1b (n=4 for each group). Production of hTIMP1 was determined by RT-PCR. To determine if MMP activity was inhibited by the produced TIMP1, MMP activity assay was measured by following cleavage of a fluorogenic substrate.Results: RT-PCR analysis demonstrated that the level of hTIMP1 transcription from cells and culture media transfected with rAAV-NFkB-hTIMP1 construct was greater than in the IL-1b stimulated condition compared to unstimulated control. The result shows that the level of MMP activity was decreased compared to baseline levels or cells exposed to IL-1.Discussion: The RT-PCR result showed cells transfected with AAV-NFkB-hTIMP1 plasmid DNA had significant increase in the hTIMP-1 mRNA expression after stimulation with IL-1b. These results demonstrate the novelty of this strategy in which the NF-kB element containing promoter ensures that the transgene hTIMP1 is expressed highly only under conditions of inflammation typically found in injured and degenerating discs which would act to block MMP-mediated matrix proteolytic destruction. Low levels of hTIMP1 were also observed under unstimulated conditions and the bioenergetic consequences of the cell are unclear.

Lidocaine and Bupivacaine Toxicity on Human Intervertebral Disc Cells in Vitro

Introduction The treatment and diagnostics of discogenic low back pain often employ local anesthetic agents. Lidocaine and bupivacaine are most commonly used for controlling and relieving the pain in interventional spine procedures. It is known that these local anesthetics may be cytotoxic to the intervertebral disc cells following the intradiscal injections and that they may provoke or accelerate the intervertebral disc degeneration. However, the potential effects of these agents still remain unclear, as many studies so far have been accomplished on animal cells. The purpose of the study was to evaluate the effect of various local anesthetics on human intervertebral disc cells in vitro and to compare the cytotoxicity of lidocaine, bupivacaine, and its mixture to saline control solution. Materials and Methods Annulus fibrosus and nucleus pulposus cells were isolated from human lumbar intervertebral disc fragments following discectomies. They were cultured in the microtiter plates and exposed to various concentrations of lidocaine, bupivacaine, as well as their mixture. The saline solution was used for control. Three different dilutions (undiluted, 1:2, and 1:4) of anesthetic agents were tested. The cells were treated for 6, 24, and 48 hours. They were then examined with the crystal violet cell staining. The percentage of live cells after treatment was determined. Results Human intervertebral disc cells demonstrated a time- and dose-dependent response to lidocaine and bupivacaine. Nucleus pulposus cells were more susceptible than annulus fibrosus cells to the toxic effects of both anesthetic agents. Lidocaine was the most toxic in all three tested concentrations (undiluted, 1:2, and 1:4) with the cell survival of 0, 10, and 20%, respectively. Bupivacaine presented less cytotoxicity than lidocaine with the survival rates of 10, 60, and 80%, respectively. Lidocaine–bupivacaine mixture showed an intermediate toxicological effect. The cell death was mainly caused by necrosis rather than apoptosis. Conclusions When incubating the human intervertebral disc cells in vitro with a combination of anesthetic agents commonly used for discography, the cytotoxic effects were observed in a dose- and time-dependent manner. According to our study, lidocaine and its mixtures should be avoided due to their high toxicity to the intervertebral disc cells. Bupivacaine was the least toxic, especially when used in 1:4 dilutions and may be thus recommended for the intradiscal diagnostics. It is assumed that the genesis of disc degeneration might be contributed also by the toxic effects of the anesthetics used, culminating to progressive tissue damage after the diagnostic measures.

Fibrin-genipin annulus fibrosus sealant as a delivery system for anti-TNFα drug

Background contextIntervertebral discs (IVDs) are attractive targets for local drug delivery because they are avascular structures with limited transport. Painful IVDs are in a chronic inflammatory state. Although anti-inflammatories show poor performance in clinical trials, their efficacy treating IVD cells suggests that sustained, local drug delivery directly to painful IVDs may be beneficial. PurposeThe purpose of this study was to determine if genipin cross-linked fibrin (FibGen) with collagen Type I hollow spheres (CHS) can serve as a drug-delivery carrier for infliximab, the anti-tumor necrosis factor α (TNFα) drug. Infliximab was chosen as a model drug because of the known role of TNFα in increasing downstream production of several pro-inflammatory cytokines and pain mediators. Genipin cross-linked fibrin was used as drug carrier because it is adhesive, injectable, and slowly degrading hydrogel with the potential to seal annulus fibrosus (AF) defects. CHS allow simple and nondamaging drug loading and could act as a drug reservoir to improve sustained delivery. Study design/settingThis is a study of biomaterials and human AF cell culture to determine drug release kinetics and efficacy. MethodsInfliximab was delivered at low and high concentrations using FibGen with and without CHS. Gels were analyzed for structure, drug release kinetics, and efficacy treating human AF cells after release. ResultsFibrin showed rapid infliximab drug release but degraded quickly. CHS alone showed a sustained release profile, but the small spheres may not remain in a degenerated IVD with fissures. Genipin cross-linked fibrin showed steady and low levels of infliximab release that was increased when loaded with higher drug concentrations. Infliximab was bound in CHS when delivered within FibGen and was only released after enzymatic degradation. The infliximab released over 20 days retained its bioactivity as confirmed by the sustained reduction of interleukin (IL)-1β, IL-6, IL-8, and TNFα concentrations produced by AF cells. ConclusionsDirect mixing of infliximab into FibGen was the simplest drug-loading protocol capable of sustained release. Results show feasibility of using drug-loaded FibGen for delivery of infliximab and, in the context with the literature, show potential to seal AF defects and partially restore IVD biomechanics. Future investigations are required to determine if drug-loaded FibGen can effectively deliver drugs, seal AF defects, and promote IVD repair or prevent further IVD degeneration in vivo.

Intervertebral Disc Tissue Engineering with Natural Extracellular Matrix-Derived Biphasic Composite Scaffolds

Tissue engineering has provided an alternative therapeutic possibility for degenerative disc diseases. However, we lack an ideal scaffold for IVD tissue engineering. The goal of this study is to fabricate a novel biomimetic biphasic scaffold for IVD tissue engineering and evaluate the feasibility of developing tissue-engineered IVD in vitro and in vivo. In present study we developed a novel integrated biphasic IVD scaffold using a simple freeze-drying and cross-linking technique of pig bone matrix gelatin (BMG) for the outer annulus fibrosus (AF) phase and pig acellular cartilage ECM (ACECM) for the inner nucleus pulposus (NP) phase. Histology and SEM results indicated no residual cells remaining in the scaffold that featured an interconnected porous microstructure (pore size of AF and NP phase 401.4±13.1 μm and 231.6±57.2 μm, respectively). PKH26-labeled AF and NP cells were seeded into the scaffold and cultured in vitro. SEM confirmed that seeded cells could anchor onto the scaffold. Live/dead staining showed that live cells (green fluorescence) were distributed in the scaffold, with no dead cells (red fluorescence) being found. The cell—scaffold constructs were implanted subcutaneously into nude mice and cultured for 6 weeks in vivo. IVD-like tissue formed in nude mice as confirmed by histology. Cells in hybrid constructs originated from PKH26-labeled cells, as confirmed by in vivo fluorescence imaging system. In conclusion, the study demonstrates the feasibility of developing a tissue-engineered IVD in vivo with a BMG- and ACECM-derived integrated AF-NP biphasic scaffold. As well, PKH26 fluorescent labeling with in vivo fluorescent imaging can be used to track cells and analyse cell—scaffold constructs in vivo.

Low-intensity pulsed ultrasound stimulates matrix metabolism of human annulus fibrosus cells mediated by transforming growth factor β1 and extracellular signal-regulated kinase pathway

Purpose: There are limited strategies to restore the damaged annulus fibrosus (AF) of the intervertebral disc. Low-intensity pulsed ultrasound (LIPUS) has positive effects on the proliferation of several types of cells and the repair of damage tissue in vivo. However, scientific evidence of therapeutic effects of LIPUS on AF cells remains limited. The purpose of this study is to evaluate the feasibility of applying LIPUS to the repair of the AF.Materials and methods: We used an in vitro model of human AF cells subjected to LIPUS stimulation to examine its effects on cell proliferation and matrix metabolism. Cell viability, synthesis of collagen and glycosaminoglycan (GAG), expression of matrix metalloproteinases (MMPs) and transforming growth factor β1 and pathways involving mitogen-activated protein kinases (MAPKs) were investigated.Results: LIPUS significantly enhanced proliferation of AF cells after 5 days of treatment. LIPUS with an intensity of 0.5 W/cm2 increased the collagen and GAG synthesis and decreased the expressions of MMP-1 and -3 of human AF cells. Real-time polymerase chain reactions and western blotting analysis revealed that LIPUS could increase transforming growth factor β1 (TGF-β1) and activate extracellular signal-regulated kinase (ERK) pathway. In addition, TGF-β receptor kinase inhibitor could suppress the ultrasound-induced alterations in cell viability and matrix metabolism.Conclusions: The findings suggested that LIPUS could be useful as a physical stimulation of cell metabolism for the repair of the AF.

Gene expression modulation in TGF-β3-mediated rabbit bone marrow stem cells using electrospun scaffolds of various stiffness.

Tissue engineering has recently evolved into a promising approach for annulus fibrosus (AF) regeneration. However, selection of an ideal cell source, which can be readily differentiated into AF cells of various regions, remains challenging because of the heterogeneity of AF tissue. In this study, we set out to explore the feasibility of using transforming growth factor-β3-mediated bone marrow stem cells (tBMSCs) for AF tissue engineering. Since the differentiation of stem cells significantly relies on the stiffness of substrate, we fabricated nanofibrous scaffolds from a series of biodegradable poly(ether carbonate urethane)-urea (PECUU) materials whose elastic modulus approximated that of native AF tissue. We cultured tBMSCs on PECUU scaffolds and compared their gene expression profile to AF-derived stem cells (AFSCs), the newly identified AF tissue-specific stem cells. As predicted, the expression of collagen-I in both tBMSCs and AFSCs increased with scaffold stiffness, whereas the expression of collagen-II and aggrecan genes showed an opposite trend. Interestingly, the expression of collagen-I, collagen-II and aggrecan genes in tBMSCs on PECUU scaffolds were consistently higher than those in AFSCs regardless of scaffold stiffness. In addition, the cell traction forces (CTFs) of both tBMSCs and AFSCs gradually decreased with scaffold stiffness, which is similar to the CTF change of cells from inner to outer regions of native AF tissue. Together, findings from this study indicate that tBMSCs had strong tendency to differentiate into various types of AF cells and presented gene expression profiles similar to AFSCs, thereby establishing a rationale for the use of tBMSCs in AF tissue engineering.

Low level light therapy modulates inflammatory mediators secreted by human annulus fibrosus cells during intervertebral disc degeneration in vitro.

Intervertebral disc degeneration (IVD) is one of the important causes of low back pain and is associated with inflammation induced by interaction between macrophages and the human annulus fibrosus (AF) cells. Low-level light therapy (LLLT) has been widely known to regulate inflammatory reaction. However, the effect of LLLT on macrophage-mediated inflammation in the AF cells has not been studied till date. The aim of this study is to mimic the inflammatory microenvironment and to investigate the anti-inflammatory effect of LLLT at a range of wavelengths (405, 532 and 650nm) on the AF treated with macrophage-like THP-1 cells conditioned medium (MCM) containing proinflammatory cytokines and chemokines (interleukin-1beta, tumor necrosis factor-alpha, interleukin-6 and 8). We observed that AF cells exposed to MCM secrete significantly higher concentrations of IL-6, IL-8, IL-1β and TNF-α. LLLT markedly inhibited secretion of IL-6 at 405nm in a time-dependent manner. Level of IL-8 was significantly decreased at all wavelengths in a time-dependent manner. We showed that MCM can induce the inflammatory microenvironment in AF cells and LLLT selectively suppressed IL-6 and 8 levels. The results indicate that LLLT is a potential method of IVD treatment and provide insights into further investigation of its anti-inflammation effect on IVD.

Custom-tailored tissue engineered polycaprolactone scaffolds for total disc replacement

Degeneration of the intervertebral disc (IVD) represents a significant musculoskeletal disease burden. Tissue engineering has proposed several strategies comprising the use of biodegradable materials to prepare scaffolds that can present mechanical properties similar to those of native IVD tissues. However, this might be insufficient, since the patient’s intervertebral space geometry must be replicated to allow for appropriate implant fixation and integration. Herein, we propose the use of reverse engineering and rapid prototyping techniques with the goal of preparing custom-tailored annulus fibrosus scaffolds; these techniques have previously been applied to rabbit models. The IVD reverse-engineered architecture was obtained by means of microcomputed tomography acquisition and three-dimensional modelling, resulting in a computer-aided design (CAD) that replicates the original rabbit IVD. Later, a fused deposition-modelling three-dimensional printer was used to produce the scaffolds with different geometries provided by the CAD, using polycaprolactone (PCL) with 100% infill density. The microstructure of the PCL scaffolds was investigated by scanning electron microscopy (SEM), which allowed us to observe an adequate fusion adhesion between the layers. The SEM images revealed that, up to the point of moderate resolution, the porosities manually designed in the CAD model were successfully replicated. The PCL scaffolds’ three-dimensional architecture was also assessed by means of microcomputed tomography analysis. Compressive stiffness was determined using a mechanical testing system. Results showed higher values than those of human IVDs (5.9–6.7 kN mm−1 versus 1.2 kN mm−1, respectively). In vitro studies were performed to investigate the possible cytotoxicity of the polycaprolactone scaffolds’ leachables. The results showed that the custom-tailored PCL scaffolds do not have any deleterious cytotoxic effect over annulus fibrosus cells or the mouse lung fibroblast’s cell line. This study proposed a simple, rapid, and low-cost strategy to fabricate custom-tailored annulus fibrosus scaffolds. In the future, this strategy might be used in association with nucleus pulposus regeneration strategies to facilitate the development of tissue-engineered total disc replacement implants specific to each patient, with a goal of full IVD regeneration.

Defining the phenotype of young healthy nucleus pulposus cells: recommendations of the Spine Research Interest Group at the 2014 annual ORS meeting.

Low back pain is a major physical and socioeconomic problem. Degeneration of the intervertebral disc and especially that of nucleus pulposus (NP) has been linked to low back pain. In spite of much research focusing on the NP, consensus among the research community is lacking in defining the NP cell phenotype. A consensus agreement will allow easier distinguishing of NP cells from annulus fibrosus (AF) cells and endplate chondrocytes, a better gauge of therapeutic success, and a better guidance of tissue-engineering-based regenerative strategies that attempt to replace lost NP tissue. Most importantly, a clear definition will further the understanding of physiology and function of NP cells, ultimately driving development of novel cell-based therapeutic modalities. The Spine Research Interest Group at the 2014 Annual ORS Meeting in New Orleans convened with the task of compiling a working definition of the NP cell phenotype with hope that a consensus statement will propel disc research forward into the future. Based on evaluation of recent studies describing characteristic NP markers and their physiologic relevance, we make the recommendation of the following healthy NP phenotypic markers: stabilized expression of HIF-1α, GLUT-1, aggrecan/collagen II ratio >20, Shh, Brachyury, KRT18/19, CA12, and CD24.

17β-estradiol protects against apoptosis induced by interleukin-1β in rat nucleus pulposus cells by down-regulating MMP-3 and MMP-13.

In our previous study, 17β-estradiol was proved to protect rat annulus fibrosus cells against apoptosis induced by interleukin-1β (IL-1β). However, whether 17β-estradiol has protective effect on rat nucleus pulposus cells remains unclear. The purpose of this study was to further explore the effects of 17β-estradiol on rat nucleus pulposus cells based on IL-1β-induced apoptosis. TUNEL assay and Annexin V/PI double staining were used to detect apoptosis and revealed that IL-1β induced notable apoptosis, which was reversed by 17β-estradiol. Meanwhile, cell viability and binding ability were decreased by IL-1β, but activated caspase-3 was increased. However, all of the detected effects of IL-1β were eliminated by 17β-estradiol. Furthermore, real-time quantitative RT-PCR was used to further find that IL-1β downregulated expression level of type II collagen, aggrecan, tissue inhibitor of matrix metalloproteinase (TIMP)-1, while upregulated matrix metalloproteinase (MMP)-3, MMP-13 and Bcl-2, which was further confirmed by western blot. Finally, 17β-estradiol was proved to abolish the above negative effects of IL-1β. In summary, this work presented that IL-1β maybe induced apoptosis of rat nucleus pulposus cells, which was resisted by 17β-estradiol by down-regulating MMP-3 and MMP-13 via a mitochondrial pathway. This research provides a novel insight into the anti-apoptotic effect of 17β-estradiol on IL-1β-induced cytotoxicity, and may potentially lead to a better understanding of the clinical effects of 17β-estradiol, especially in terms of intervertebral disc degeneration.

Co-culture of annulus fibrosus cells and bone marrow mesenchymal stem cells.

To investigate the effect on cell proliferation and extracellular matrix expression of annulus fibrosus (AF) cells when co-cultured with bone marrow mesenchymal stem cells (BMSCs). Primary isolated rabbit BMSCs and AF cells were cultured and harvested cells were placed into a 15-mL centrifugal tube co-culture system in a ratio of 2:1 (A group), 1:1 (B group), and 1:2 (C group). Cell proliferation was evaluated using cell counting kit-8, and mRNA of collagen II and mucopolysaccharide was quantified using real-time polymerase chain reaction (PCR) on Days 7, 14, and 21. The cell count, synthesized collagen II and mucopolysaccharide increased in a time-dependent manner, with a peak at Day 21. Cells in Group B proliferated faster and synthesized more collagen II and mucopolysaccharide than groups A and C, where the difference was significant. AF cells and BMSCs in the ratio of 1:1, cultured using the centrifugal tube three-dimensional co-culture system showed that BMSCs can promote AF cell proliferation and extracellular matrix synthesis.

Effect of the mixture of bone marrow mesenchymal stromal cells and annulus fibrosus cells in repairing the degenerative discs of rabbits.

The aims of this study were to investigate the effect of a mixture of bone marrow mesenchymal stem cells (BMSCs) and annulus fibrosus cells (AFCs) in repairing the degenerative discs of rabbits, and to provide an experimental basis for its clinical application. BMSCs from rabbits were cultured in vitro and mixed with AFCs. The animal model of degenerative intervertebral disc was built by aspirating the nucleus pulposus (L3-4, L4-5, L5-6, and L6-7) through a posterolateral approach. Mixtures of BMSCs and AFCs (group A), BMSCs alone (B), or saline (C) were injected into test discs, and changes evaluated by plain radiographs, magnetic resonance imaging, and histology. After two weeks, each group showed typical internal disc disruption; stenosis of the intervertebral space, weakening T2 disc signal, decreased disc height and expression of type II collagen and glycosaminoglycan, and fibrosis of the nucleus pulposus. After cell transplantation, the disc heights in groups A and B were regained; however, they continued to decrease in group C. The transplanted cells survived in the discs, proliferated after transplantation, and produced copious matrix. Macroscopic and histological evaluations confirmed structural and nuclear preservation in cell-transplanted discs. The secretions and expressions of Type Ⅱ collagen and glycosaminoglycan in group A were statistically significant higher than those in group B (Type Ⅱ collagen, group A 141.6 ± 5.87, group B 139.8 ± 8.65, P = 0.004; glycosaminoglycan, group A 3.008 ± 0.35, group B 2.94 ± 0.29, P = 0.003). Expression of type II collagen and glycosaminoglycan was significantly greater in group A than group B. Therefore, co-transplantation of BMSCs and AFCs can restore the extracellular matrix, making this approach superior to transplantation of BMSCs alone, which may be beneficial for the therapy of intervertebral disc degeneration.

Organotypic Cultures of Intervertebral Disc Cells: Responses to Growth Factors and Signaling Pathways Involved.

Intervertebral disc (IVD) degeneration is strongly associated with low back pain, a major cause of disability worldwide. An in-depth understanding of IVD cell physiology is required for the design of novel regenerative therapies. Accordingly, aim of this work was the study of IVD cell responses to mitogenic growth factors in a three-dimensional (3D) organotypic milieu, comprising characteristic molecules of IVD's extracellular matrix. In particular, annulus fibrosus (AF) cells were cultured inside collagen type-I gels, while nucleus pulposus (NP) cells in chondroitin sulfate A (CSA) supplemented collagen gels, and the effects of Platelet-Derived Growth Factor (PDGF), basic Fibroblast Growth Factor (bFGF), and Insulin-Like Growth Factor-I (IGF-I) were assessed. All three growth factors stimulated DNA synthesis in both AF and NP 3D cell cultures, with potencies similar to those observed previously in monolayers. CSA supplementation inhibited basal DNA synthesis rates, without affecting the response to growth factors. ERK and Akt were found to be phosphorylated following growth factor stimulation. Blockade of these two signaling pathways using pharmacologic inhibitors significantly, though not completely, inhibited growth factor-induced DNA synthesis. The proposed culture systems may prove useful for further in vitro studies aiming at future interventions for IVD regeneration.

Effect of Shentongzhuyu Decoction on p38MAPK Signal Pathway in Annulus Fibrosus Cells of Model Rats with Lumbar Disc Degeneration

Effect of Shentongzhuyu Decoction on p38MAPK Signal Pathway in Annulus Fibrosus Cells of Model Rats with Lumbar Disc Degeneration

Functional impact of integrin α5β1 on the homeostasis of intervertebral discs: a study of mechanotransduction pathways using a novel dynamic loading organ culture system

Background contextIntervertebral disc (IVD) degeneration, a major cause of low back pain, is considered to be induced by daily mechanical loading. Mechanical stress is widely known to affect cell survival and extracellular matrix metabolism in many cell types. Although the involvement of integrin α5β1 transmembrane mechanoreceptor in IVD degeneration has been reported, the precise function of integrin α5β1 remains obscure. PurposeTo reflect IVD tissue response to mechanical stress using a dynamic loading organ culture system and elucidate the functional impact of integrin α5β1 on the pathomechanism of IVD degeneration. Study designAn ex vivo study using a dynamic loading organ culture system. MethodsNinety-six rat IVD explants were examined. Intervertebral discs were subjected to 1.3 MPa, 1.0 Hz dynamic compressive load in the presence or absence of an Arg-Gly-Asp (RGD) peptide with affinity to the fibronectin binding-site of integrin α5β1. Cell viability and histomorphology were assessed. The localization of integrin α5β1 in the IVD was assessed by immunohistochemistry. Gene expression levels of IVD cells were evaluated using real-time reverse transcription-polymerase chain reaction. ResultsIn the nucleus pulposus (NP), cell density and viability were reduced by dynamic compressive load. Histologic degenerative alterations, mainly seen in the NP, were the morphologic changes of NP cells. In both NP and annulus fibrosus (AF), immunohistochemistry revealed localization of integrin α5β1 and that the messenger-RNA expression of integrin α5β1 was increased by dynamic load. Dynamic load induced a catabolic effect, the stimulation of matrix metalloproteinase-3 and -13 gene expressions by NP and AF cells. The RGD peptide partially blocked the histologic alterations and the catabolic effect. ConclusionsThe dynamic loading organ culture system simulated cellular responses to mechanical loading of the IVD. Our results suggest that IVD cells recognize the mechanical stress through RGD integrins, particularly the α5β1 subtype that is highly expressed in NP and AF cells. Further experiments using this system will provide information about pathomechanisms of IVD degeneration through the mechanotransduction pathways.

Activation of intervertebral disc cells by co-culture with notochordal cells, conditioned medium and hypoxia.

BackgroundNotochordal cells (NC) remain in the focus of research for regenerative therapy for the degenerated intervertebral disc (IVD) due to their progenitor status. Recent findings suggested their regenerative action on more mature disc cells, presumably by the secretion of specific factors, which has been described as notochordal cell conditioned medium (NCCM). The aim of this study was to determine NC culture conditions (2D/3D, fetal calf serum, oxygen level) that lead to significant IVD cell activation in an indirect co-culture system under normoxia and hypoxia (2% oxygen).MethodsPorcine NC was kept in 2D monolayer and in 3D alginate bead culture to identify a suitable culture system for these cells. To test stimulating effects of NC, co-cultures of NC and bovine derived coccygeal IVD cells were conducted in a 1:1 ratio with no direct cell contact between NC and bovine nucleus pulposus cell (NPC) or annulus fibrosus cells (AFC) in 3D alginate beads under normoxia and hypoxia (2%) for 7 and 14 days. As a positive control, NPC and AFC were stimulated with NC-derived conditioned medium (NCCM). Cell activity, glycosaminoglycan (GAG) content, DNA content and relative gene expression was measured. Mass spectrometry analysis of the NCCM was conducted.ResultsWe provide evidence by flow cytometry that monolayer culture is not favorable for NC culture with respect to maintaining NC phenotype. In 3D alginate culture, NC activated NPC either in indirect co-culture or by addition of NCCM as indicated by the gene expression ratio of aggrecan/collagen type 2. This effect was strongest with 10% fetal calf serum and under hypoxia. Conversely, AFC seemed unresponsive to co-culture with pNC or to the NCCM. Further, the results showed that hypoxia led to decelerated metabolic activity, but did not lead to a significant change in the GAG/DNA ratio. Mass spectrometry identified connective tissue growth factor (CTGF, syn. CCN2) in the NCCM.ConclusionsOur results confirm the requirement to culture NC in 3D to best maintain their phenotype, preferentially in hypoxia and with the supplementation of FCS in the culture media. Despite these advancements, the ideal culture condition remains to be identified.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2474-15-422) contains supplementary material, which is available to authorized users.

Inner and outer annulus fibrosus cells exhibit differentiated phenotypes and yield changes in extracellular matrix protein composition in vitro on a polycarbonate urethane scaffold.

Since there is currently no optimal treatment for chronic neck or back pain that restores full spine functionality and disc height, recent research has focused on developing a regenerative medicine approach. This requires a better understanding of the phenotype nature of the different cell types present in disc tissues. In particular, there is very little known about the differentiated states of cells that co-exist within the annulus fibrosus (AF), despite the critical importance of this tissue in maintaining disc functionality. Maintenance of the differentiated states of these AF cells is imperative to the production of appropriate extracellular matrix (ECM) macromolecules and to the engineering of functional AF tissues. The objective of this study was to determine whether inner annulus fibrosus (IAF) cells when grown on polycarbonate urethane (PU) scaffolds in vitro will produce ECM molecules characteristic of IAF cells and different from outer annulus fibrosus (OAF) cells. OAF and IAF cells isolated from bovine coccygeal intervertebral disc were grown on nanofibrous PU for approximately 14 days. The effect of culture time on ECM gene expression, DNA content, and the synthesis and retention of proteoglycans and collagens were evaluated for both OAF and IAF cells. The ECM accumulated was also characterized by immunostaining and Western blot. The tensile strengths of the tissue/scaffolds were evaluated at 14 days. Both OAF and IAF cells both attached to PU and had similar DNA contents over time. IAF cells maintained relatively higher levels of COL2A1, ACAN and VCAN gene expression, and relatively lower levels of COL1A1 gene expression when compared with OAF cells, by 10 days of culture. IAF cells synthesized and retained similar amounts of total collagen and proteoglycans when compared with OAF cells. While both OAF and IAF cells accumulated type I collagen, only IAF cells accumulated type II collagen. Both cell types accumulated similar amounts of aggrecan but IAF cells accumulated higher amounts of versican as determined by immunostaining. In conclusion, nanofibrous PU scaffolds enabled the maintenance of most of the characteristic features of the IAF cell phenotype, and were different from those of OAF cells.

Electroacupuncture inhibits annulus fibrosis cell apoptosis in vivo via TNF-α-TNFR1-caspase-8 and integrin β1/Akt signaling pathways

ObjectiveTo examine whether electroacupuncture (EA) treatment inhibited cell apoptosis of intervertebral annulus fibrosis (AF) via tumor necrosis factor-α (NF-α-tumor necrosis factor receptor 1 (TNFR1)-caspase-8 and integrin β1/Akt signaling pathways in a rat model of cervical intervertebral disc degeneration caused by unbalanced dynamic and static forces. MethodsThirty-two Sprague-Dawley rats were included in this study, of which 24 rats underwent surgery to induce cervical intervertebral disc degeneration, while eight rats received EA treatment at Dazhui (GV 14). Immunohistochemical staining was used to detect TNF-α, TNFR1, and caspase-8. Apoptosis of AF cells was examined with terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) staining. The mRNA and protein expression levels of integrin β1 and Akt were evaluated with real-time polymerase chain reaction and western blot analysis, respectively. ResultsTreatment with EA decreased TUNEL-positive AF cells and lowered TNF-α, TNFR1 and caspase-8 positive cells compared with control groups. EA treatment also increased integrin β1 and Akt mRNA and protein levels compared with controls. ConclusionTreatment with EA inhibits AF cell apoptosis through suppression of the TNF-α-TNFR1-caspase-8 signal pathway and increases the expression of integrin β1 and Akt. EA may be a good alternative therapy for treating cervical spondylosis.

Nerve growth factor increases MMP9 activity in annulus fibrosus cells by upregulating lipocalin 2 expression.

Nerve growth factor (NGF) expression and activity is important in chronic lower back pain but may also act as a pro-catabolic factor in the pathogenesis of intervertebral disc (IVD) degeneration. Lipocalin 2 (Lcn2) expression in IVD was upregulated by NGF stimulation in our previous study. The current study was undertaken to identify potential mechanisms of the latter effect including potential interactions between Lcn2 and matrix metalloproteinase 9 (MMP9). Rat annulus fibrosus (AF) cells were stimulated by NGF and subjected to microarray analysis, subsequent real-time PCR, western immunoblotting, and immunofluorescence. Cells were treated with NGF in the absence or presence of the NGF inhibitor Ro 08-2750. Zymography and functional MMP9 assays were used to determine MMP9 activity, whilst the dimethyl-methylene blue assay was used to quantify the release of glycosaminoglycans (GAGs) reflecting catabolic effects following NGF treatment. Immunoprecipitation with immunoblotting was used to identify interactions between MMP9 and Lcn2. Increased expression of Lcn2 gene and protein following NGF stimulation was confirmed by microarray analysis, real-time PCR, western blot and immunofluorescence. Zymography showed that NGF enhanced 125-kDa gelatinase activity, identified as a Lcn2/MMP9 complex by immunoprecipitation and immunoblotting. Functional assays showed increased MMP9 activity and GAG release in the presence of NGF. The effects of NGF were neutralized by the presence of Ro 08-2750. NGF upregulates Lcn2 expression and increases MMP9 activity in AF cells; processes which are likely to potentiate degeneration of AF tissue in vivo. Anti-NGF treatment may have benefit for management of pain relief and slowing down progression of AF tissue degeneration.

ATP promotes extracellular matrix biosynthesis of intervertebral disc cells.

We have recently found a high accumulation of extracellular adenosine triphosphate (ATP) in the center of healthy porcine intervertebral discs (IVD). Since ATP is a powerful extracellular signaling molecule, extracellular ATP accumulation might regulate biological activities in the IVD. The objective of this study was therefore to investigate the effects of extracellular ATP on the extracellular matrix (ECM) biosynthesis of porcine IVD cells isolated from two distinct anatomical regions: the annulus fibrosus (AF) and nucleus pulposus (NP). ATP treatment significantly promotes ECM deposition and corresponding gene expression (aggrecan and type II collagen) by both cell types in three-dimensional agarose culture. A significant increase in ECM accumulation has been found in AF cells at a lower ATP treatment level (20 μM) compared with NP cells (100 μM), indicating that AF cells are more sensitive to extracellular ATP than NP cells. NP cells also exhibit higher ECM accumulation and intracellular ATP than AF cells under control and treatment conditions, suggesting that NP cells are intrinsically more metabolically active. Moreover, ATP treatment also augments the intracellular ATP level in NP and AF cells. Our findings suggest that extracellular ATP not only promotes ECM biosynthesis via a molecular pathway, but also increases energy supply to fuel that process.