Annulus Fibrosus Cells Research Articles

In vivo evaluation of 3-dimensional PLGA/Atelocollagen/Fibrin scaffolds for intervertebral disc (IVD) regeneration

Poly (lactic-co-glycolic acid) (PLGA) is a widely used synthetic biomaterial in tissue engineering. Incorporation of natural atelocollagen and fibrin may provide PLGA a better surface property for cellular adhesion and proliferation. This paper aims to evaluate PLGA/Atelocollagen/Fibrin hybrid scaffolds seeded with IVD’s annulus fibrosus (AF) cells after in vivo implantation. Porous 3D PLGA were fabricated via solvent-casting and salt-leaching technique. PLGA was crosslinked with atelocollagen (PA). Fibrin was pipetted onto PLGA (PF) and PA (PAF) scaffolds. Rabbits’ AF cells were seeded onto each scaffold groups i.e. PLGA only, PA, PF and PAF. The “cells-scaffolds” constructs were cultured for three-weeks and implanted subcutaneously in athymic nude mice for four-week. All constructs were evaluated for gross morphology, histology, immunohistochemistry (IHC) against collagen I and II with PLGA construct as control group. Sulphated glycosaminoglycan (sGAG) and DNA contents were compared to that of monolayer AF culture. All groups exhibited significant reduction in size but showed increment in weight. Histology analysis indicated high cellular density, supported by high DNA content in all groups. The IHC showed presence of collagen I and II. The PAF showed a significantly higher sGAG content than the other groups. Degradation of PLGA did not hinder tissue formation. Atelocollagen and fibrin may contribute to a higher cellular proliferation and extracellular matrix production in PLGA. Further evaluation using gene expression shall be performed to study the intrinsic properties and possible mechanisms involved. PLGA/Atelocollagen/Fibrin hybrid scaffolds may become potential candidate for tissue engineering application.

Regulation of inflammation, neurotrophins, glycosylation, sensory nerve innervation and pain in intervertebral disc degeneration using an implantable hyaluronic acid hydrogel

Event Abstract Back to Event Regulation of inflammation, neurotrophins, glycosylation, sensory nerve innervation and pain in intervertebral disc degeneration using an implantable hyaluronic acid hydrogel Isma Liza Mohd Isa1, 2*, Akshay Srivastava1, Michelle Kilcoyne1, 3, Sunny A Abbah1, Oliver Carroll1, David Tiernan1, Peter Owens2, 4, Lokesh Joshi1, 3, Peter Dockery1, 2, 4, David P Finn1, 5 and Abhay Pandit1 1 National University of Ireland, Centre for Research in Medical Devices (CÚRAM), Ireland 2 National University of Ireland, Anatomy, Ireland 3 National University of Ireland, Glycoscience Group, Ireland 4 National University of Ireland, Centre for Microscopy and Imaging, Ireland 5 National University of Ireland, Pharmacology and Therapeutics, Galway Neuroscience Centre and Centre for Pain Research, Ireland Introduction: Symptomatic intervertebral disc (IVD) degeneration is mediated by an inflammation that increases neurotrophin production to sensitize innervation in the disc resulting in pain. High molecular weight hyaluronic acid (HA) hydrogels demonstrate potential as bioactive materials for IVD regeneration as they exert an anti-inflammatory[1], analgesic effect[2] and mimic the disc microenvironment[3]. It is hypothesized that HA hydrogels attenuate the inflammatory receptor and neurotrophins in an in vitro inflammation milieu, and that modulate inflammation, glycan expression and inhibit hyper-innervation associated with pain in an in vivo IVD injury model. Experimental Methods: Sodium hyaluronate 0.75% (w/v) was mixed in 1 ml distilled water with 4-arm PEG-amine 75 mM, N-hydroxysuccinimide (NHS) 15% (w/v) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) 9% (w/v). The spherical-shaped hydrogels were then obtained by pipetting a channel volume of 5 μl onto a hydrophobically modified glass slide. The cytotoxicity of HA hydrogels was tested on nucleus pulposus (NP) cells and the stability under hydrolytic and enzymatic degradation was tested. The effect of HA hydrogels was determined in inflamed NP cells by investigating inflammatory receptor IL-1R1, MyD88, and expression of NGF and BDNF mRNA. Further, all protocols of in vivo studies were pre-approved by the institutional Animal Care Research Ethics Committee (ACREC). Thirty (30) Sprague Dawley rats were randomized into three groups: sham (n=10), injury (n=10) and injury with treatment implantable HA hydrogel (n=10). The injury model was created in the coccygeal IVDs (Co4-Co5 and/or Co5-Co6) using a 1.5 mm diameter biopsy puncture to pierce 2 mm tissue depth. Nocifensive behavior of the rats in response to thermal, cold and mechanical stimuli will be observed at baseline day -1 and post-operative days 1, 7, 14, 28 and 56. The rats were then sacrificed to harvest the IVD and spinal cord for analyses. Pro-inflammatory cytokine IL-1β and molecular pain marker of c-fos, NGF and BDNF were determined by qRT-PCR. Immunohistochemistry was used to identify reactivity to GAP43 and CGRP protein, an innervation and sensory neuropeptide marker. Lectin histochemistry and histology methods were adopted to analyse glycan expression and tissue restoration in IVD. Results and Discussion: Hydrogel showed resistance to enzymatic degradation after 28 days. NP cells maintained a rounded shape and no statistical difference in metabolic activity. IL-1R1, MyD88, NGF and BDNF were suppressed in the presence of HA hydrogels (Figure 1). HA hydrogel in the in vivo injury model revealed a significant decrease of sensory innervation in NP and annulus fibrosus (AF) tissue (Figure 2). Sialic acids was identified in the extracellular matrix of both NP and AF. High mannose was expressed in AF and NP cells. The presence of α-galactose in the ECM of AF and NP was determined by GS-I-B4 (Figure 3). Conclusion: No cytotoxicity of HA hydrogels was observed in NP cells. A potential therapeutic effect of HA hydrogel was supported by data demonstrating a suppression of inflammation and neurotrophins in the inflammation model, and inhibition of in vivo hyper-innervation associated pain in response to the injury so as to promote disc regeneration. Majlis Amanah Rakyat (MARA) Malaysia and AO Research Institute – Collaborative Research Programme for providing financial support to this project. This publication has emanated from research supported in part by a research grant from Science Foundation Ireland (SFI) and is co-funded under the European Regional Development Fund under Grant Number 13/RC/2073.

Nucleus pulposus phenotypic markers to determine stem cell differentiation: fact or fiction?

Progress in mesenchymal stem cell (MSC) based therapies for nucleus pulposus (NP) regeneration are hampered by a lack of understanding and consensus of the normal NP cell phenotype. Despite the recent consensus paper on NP markers, there is still a need to further validate proposed markers. This study aimed to determine whether an NP phenotypic profile could be identified within a large population of mature NP samples.qRT-PCR was conducted to assess mRNA expression of 13 genes within human non-degenerate articular chondrocytes (AC) (n=10) and NP cells extracted from patients across a spectrum of histological degeneration grades (n=71). qRT-PCR results were used to select NP marker candidates for protein expression analysis.Differential expression at mRNA between AC and non-degenerate NP cells was only observed for Paired Box Protein 1 (PAX1) and Forkhead box F1 (FOXF1). In contrast no other previously suggested markers displayed differential expression between non-degenerate NP and AC at mRNA level. PAX1 and FOXF1 protein expression was significantly higher in the NP compared to annulus fibrosus (AF), cartilaginous endplate (CEP) and AC. In contrast Laminin-5 (LAM-332), Keratin-19 (KRT-19) and Hypoxia Inducible Factor 1 alpha (HIF1α) showed no differential expression in NP cells compared with AC cells.A marker which exclusively differentiates NP cells from AF and AC cells remains to be identified, raising the question: is the NP a heterogeneous population of cells? Or does the natural biological variation during IVD development, degeneration state and even the life cycle of cells make finding one definitive marker impossible?

Osteogenic differentiation of bone marrow stromal cells is hindered by the presence of intervertebral disc cells.

BackgroundClinical observations indicate that the presence of nucleus pulposus (NP) tissue during spinal fusion hinders the rate of disc ossification. While the underlying mechanism remains unknown, this observation could be due to incomplete removal of NP cells (NPCs) that secrete factors preventing disc calcification, such as bone morphogenetic protein (BMP) antagonists including noggin and members of the DAN (differential screening selected gene aberrative in neuroblastoma) family.MethodsMonolayer human bone marrow-derived mesenchymal stem cells (MSCs) were cocultured withNPCs and annulus fibrosus cells (AFCs) embedded in alginate for 21 days. At the end of coculture, MSCs were stained for mineral deposition by alizarin red, and relative expression of bone-related genes [Runt-related transcription factor 2, (RUNX2), Osteopontin (OPN), and Alkaline phosphatase (ALP)] and ALP activity were analyzed. Relative expression of three BMP antagonists, chordin (CHRD), gremlin (GREM1), and noggin (NOG), was determined in primary human NPCs and AFCs. These cells were also stained for Gremlin and Noggin by immunocytochemistry.ResultsAlizarin red staining showed that MSC osteogenesis in monolayer cultures was inhibited by coculture with NPCs or AFCs. ALP activity and RT-PCR analyses confirmed these results and demonstrated inhibition of osteogenesis of MSC in the presence of disc cells. NOG was significantly up-regulated in MSCs after coculture. Relative gene expression of intervertebral disc (IVD) cells showed higher expression of GREM1 in NPCs than in AFCs.ConclusionsWe show that primary IVD cells inhibit osteogenesis of MSCs. BMP inhibitors NOG, GREM1 and CHRD were expressed in IVD cells. GREM1 appears to be differentially expressed in NPCs and AFCs. Our results have implications for the design and development of treatments for non-union in spinal fusion.

Cyclic tensile stress of human annulus fibrosus cells induces MAPK activation: involvement in proinflammatory gene expression

To study the role of mitogen-activated protein kinases (MAPKs) in human annulus fibrosus (AF) cells subjected to cyclic tensile stress (CTS). An invitro system for CTS studies was established using AF cultures on fibronectin-coated silicone dishes. MAPK phosphorylation was studied by western analysis, while gene expression was followed by qRT-PCR. DNA synthesis was assessed by both tritiated thymidine incorporation and flow cytometry, and collagen synthesis using tritiated proline incorporation and the protease-free collagenase method. All three MAPKs studied, i.e., ERK, SAPK/JNK, and p38 were found to be phosphorylated immediately after CTS application within physiological range. A second wave of phosphorylation appeared at later time points. MAPK activation was elevated at higher CTS magnitudes, but independent of the frequency. CTS did not stimulate DNA synthesis neither extracellular matrix turnover, but it stimulated the proinflammatory genes, COX-2, IL-6, and IL-8. This stimulation was more intense at the highest magnitude (8%) testedand at the median frequency (1Hz) and time interval (12h). Blocking of ERK, SAPK/JNK, and p38 MAPK inhibited the CTS-induced stimulation of COX-2 and IL-8, while IL-6 expression was mediated only by SAPK/JNK and p38 MAPK. We have described for the first time the activation of MAPKs in human AF cells in response to CTSand showed that it drives an inflammatory reaction. These observations shed light on the mechanisms of intervertebral disc (IVD) cell responses to mechanical stress, contributing to the understanding of disc pathophysiologyand possibly to the design of novel therapeutic interventions.

Excessive reactive oxygen species are therapeutic targets for intervertebral disc degeneration.

IntroductionOxidative stress has been reported to be involved in numerous human diseases, including musculoskeletal disorders such as osteoarthritis. However, the interaction between intervertebral disc (IVD) degeneration and oxidative stress is not well understood. The purpose of the present study was to elucidate the contribution of oxidative stress to IVD degeneration and the efficacy of antioxidant treatment for degenerative discs.MethodsThe expression level of an oxidative stress marker, nitrotyrosine, was assessed by immunohistochemistry and Western blotting. For evaluating intracellular reactive oxygen species (ROS) levels and oxidative stress in rat annulus fibrosus (AF) cells, flow cytometry and luciferase assay with an OKD48 construct were performed. The grade of IVD degeneration was assessed by magnetic resonance imaging and histological analysis.ResultsA high frequency of nitrotyrosine-positive cells was observed in rat and human degenerative discs. mRNA expression of catabolic factors such as tumor necrosis factor-alpha (TNF-alpha), matrix metalloprotease-3 (MMP-3), and cyclooxygenase-2 (COX-2) was significantly induced by treatment with H2O2 or buthionine sulfoximine, whereas that of aggrecan, an important chondrogenic proteoglycan, was reduced in a dose-dependent manner. Treatment with mitogen-activated protein kinase (MAPK) inhibitors blocked the inductive effect of excessive ROS on COX-2 mRNA expression. Western blotting confirmed the phosphorylation of MAPKs in H2O2 and BSO-treated AF cells. Conversely, we showed that TNF-α induced oxidative stress with increased intracellular ROS levels in AF cells. Treatment with the antioxidant N-acetyl cysteine (NAC) abrogated the catabolic effect of excessive ROS and TNF-alpha in vitro. Finally, we showed that oral administration of NAC prevented IVD degeneration in rat degenerative model.ConclusionsA positive feedback loop was formed between excessive ROS and TNF-alpha in AF cells. Thus, oxidative stress contributes to the progression of IVD degeneration and NAC can be a therapeutic option for IVD degeneration.Electronic supplementary materialThe online version of this article (doi:10.1186/s13075-015-0834-8) contains supplementary material, which is available to authorized users.

Gene Expression Profiling Identifies Interferon Signalling Molecules and IGFBP3 in Human Degenerative Annulus Fibrosus.

Low back pain is a major cause of disability especially for people between 20 and 50 years of age. As a costly healthcare problem, it imposes a serious socio-economic burden. Current surgical therapies fail to replace the normal disc in facilitating spinal movements and absorbing load. The focus of regenerative medicine is on identifying biomarkers and signalling pathways to improve our understanding about cascades of disc degeneration and allow for the design of specific therapies. We hypothesized that comparing microarray profiles from degenerative and non-degenerative discs will lead to the identification of dysregulated signalling and pathophysiological targets. Microarray data sets were generated from human annulus fibrosus cells and analysed using IPA ingenuity pathway analysis. Gene expression values were validated by qRT-PCR, and respective proteins were identified by immunohistochemistry. Microarray analysis revealed 238 differentially expressed genes in the degenerative annulus fibrosus. Seventeen of the dysregulated molecular markers showed log2-fold changes greater than ±1.5. Various dysregulated cellular functions, including cell proliferation and inflammatory response, were identified. The most significant canonical pathway induced in degenerative annulus fibrosus was found to be the interferon pathway. This study indicates interferon-alpha signalling pathway activation with IFIT3 and IGFBP3 up-regulation, which may affect cellular function in human degenerative disc.

Beta1 integrin inhibits apoptosis induced by cyclic stretch in annulus fibrosus cells via ERK1/2 MAPK pathway.

Low back pain is associated with intervertebral disc degeneration (IVDD) due to cellular loss through apoptosis. Mechanical factors play an important role in maintaining the survival of the annulus fibrosus (AF) cells and the deposition of extracellular matrix. However, the mechanisms that excessive mechanical forces lead to AF cell apoptosis are not clear. The present study was to look for how AF cells sense mechanical changes. In vivo experiments, the involvement of mechanoreceptors in apoptosis was examined by RT-PCR and/or immunoblotting in the lumbar spine of rats subjected to unbalanced dynamic and static forces. In vitro experiments, we investigated apoptotic signaling pathways in untransfected and transfected AF cells with the lentivirus vector for rat β1 integrin overexpression after cyclic stretch. Apoptosis in AF cells was assessed using flow cytometry, Hoechst 33258 nuclear staining. Western blotting was used to analyze expression of β1 integrin and caspase-3 and ERK1/2 MAPK signaling molecules. In the rat IVDD model, unbalanced dynamic and static forces induced apoptosis of disc cells, which corresponded to decreased expression of β1 integrin. Cyclic stretch-induced apoptosis in rat AF cells correlated with the activation of caspase-3 and with decreased levels of β1 integrin and the phosphorylation levels of ERK1/2 activation level. However, the overexpression of β1 integrin in AF cells ameliorated cyclic stretch-induced apoptosis and decreased caspase-3 activation. Furthermore, ERK1/2-specific inhibitor promotes apoptosis in vector β1-infected AF cells. These results suggest that the disruption of β1 integrin signaling may underlie disc cell apoptosis induced by mechanical stress. Further work is necessary to fully elucidate the pathophysiological mechanisms that underlie IVDD caused by unbalanced dynamic and static forces.

Induction of proinflammatory cytokine production in intervertebral disc cells by macrophage-like THP-1 cells requires mitogen-activated protein kinase activity

To determine the role played by mitogen-activated protein kinase (MAPK) signaling in the interactions between macrophages and intervertebral disc (IVD) cells, it was hypothesized that MAPK inhibition would modulate the production of the proinflammatory cytokines associated with inflammatory reaction in IVD cells. Human annulus fibrosus (AF) and nucleus pulposus (NP) cells were cocultured with phorbol myristate acetate-stimulated macrophage-like THP-1 cells, with and without SB202190 (a p38-α and -β inhibitor), SP600125 (a c-Jun N-terminal kinase [JNK] inhibitor), and PD98059 (an extracellular signal-regulated kinase [ERK] 1/2 inhibitor). The cytokines in conditioned media from cocultured and macrophage-exposed (nemotic) cells were assayed using enzyme-linked immunosorbent assays (ELISAs). Interleukin (IL)-6 and IL-8 were secreted in greater quantities by the cocultured cells compared with naive IVD cells and macrophages (MΦ) cultured alone. The tumor necrosis factor (TNF)- α and IL-6 levels produced by the NP cells cocultured with MΦs (NP-MΦ) were significantly lower than those produced by AF cells cocultured with MΦs (AF-MΦ). SB202190 dose-dependently suppressed IL-6 secretion by AF-MΦ and NP-MΦ cocultures, and 10 μM SB202190 significantly decreased IL-6 and IL-8 production in nemotic AF and NP pellets. SP600125 at 10 μM significantly suppressed the production of TNF α IL-6. and IL-8 in AF-MΦ and NP-MΦ cocultures and significantly suppressed IL-1β production in the NP-MΦ coculture. Administration of 10 μM PD98059 significantly decreased IL-6 levels in the AF-MΦ coculture, and decreased the levels of TNF α and IL-8 in both the AF-MΦ and NP-MΦ cocultures. The present study shows that inhibitors of p38 MAPK effectively controlled IL-6 production during inflammatory reactions and that JNK and ERK1/2 inhibitors successfully suppressed the production of major proinflammatory cytokines during interactions between macrophages and IVD cells. Therefore, selective blockade of these signals may serve as a therapeutic approach to symptomatic IVD degeneration.

Proliferation and Growth Behavior of Annulus Fibrosus Cells on Hesperidin Loaded Poly(lactide-co-glycolic acid) Scaffold

항산화와 항염증으로 잘 알려진 헤스페리딘은 디스크 재생을 위해 효과적인 지지체로 제작하기 위해 사용하였다. 지지체는 poly(lactide-co-glycolic acid) (PLGA)에 헤스페리딘(0, 3, 5, 10%)을 첨가하여 염 침출법으로 제작하였고, 섬유륜 세포(AF)를 파종한 후 세포의 변화를 연구하였다. SEM, WST 그리고 RT-PCR 분석을 통해 세포의 부착과 증식, 세포의 표현형 분석을 실시하였다. 또한 면역조직화학염색을 통하여 AF세포의 생체 내 거동을 확인하였다. 그 결과, 5% 헤스페리딘이 함유된 PLGA 지지체가 가장 좋은 세포의 형태와 생체 적합성을 보여, 디스크 재생을 위한 지지체로서의 활용 가능성을 확인하였다.

Regulation of 3D Axonal Outgrowth from Cultured DRG by Annulus Fibrosus Cells

Regulation of 3D Axonal Outgrowth from Cultured DRG by Annulus Fibrosus Cells

Protective effects of paeoniflorin against FasL-induced apoptosis of intervertebral disc annulus fibrosus cells via Fas-FasL signalling pathway.

In the present study, we demonstrate that the degeneration of intervertebral discs is caused by ageing and apoptosis of matrix cells. Apoptosis is as essential as the function of proteoglycan synthesis in assessing the possible degeneration of intervertebral discs; paeoniflorin (PF) induces cytoprotective effects on various types of cells. In this study, the function of PF in inhibiting Fas ligand (FasL)-induced apoptosis in annulus fibrosus cells was assessed, and the correlation between apoptosis and the Fas-FasL pathway was determined. Annulus fibrosus cells were derived from the intervertebral discs of 1-month-old Sprague Dawley rats; the cells were characterised by toluidine blue staining and subjected to apoptosis with FasL. PF was diluted to various concentrations and added to annulus fibrosus cells at various times. The impact of PF and FasL on cell apoptosis of annulus fibrosus cells was determined by flow cytometry. Western blot analysis was performed to determine the protein expression levels of Fas and caspase-3. The percentages of apoptotic annulus fibrosus cells as well as the expression levels of caspase-3 and Fas were significantly reduced following treatment with 208, 20.8 or 2.08 µM PF. PF inhibits the activation of the Fas-FasL signal pathway and decreases FasL-induced apoptosis of annulus fibrosus cells.

Substance P Receptor Antagonist Suppresses Inflammatory Cytokine Expression in Human Disc Cells.

Laboratory study. To evaluate whether blockade of the Substance P (SP) NK1R attenuates its proinflammatory effect on human intervertebral disc cells (IVD), and to evaluate the signaling pathways associated with SP. SP and its receptors are expressed in human IVD cells, and cause upregulation of inflammatory mediators; however, the effects of blocking these receptors have not been studied in human IVD cells. Human annulus fibrosus (AF) and nucleus pulposus (NP) cells were expanded in monolayer, and then suspended in alginate beads. The alginate beads were treated with culture medium first containing a high affinity NK1R antagonist (L-760735) at different concentrations, and then with medium containing both NK1R antagonist and SP at 2 concentrations. Ribonucleic acid was isolated and transcribed into cDNA. Quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) was performed to evaluate expression of interleukin (IL)-1β, IL-6, and IL-8. Western blot analysis was performed to examine levels of the phosphorylated p38 mitogen-activated protein kinase (MAPK), extracellular signal regulated kinase 1/2 (ERK1/2) and nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB p65). The cells were pretreated with specific inhibitors of p38 (SB203580), ERK1/2 (PD98059), and p65 (SM7368) and then stimulated with SP. We detected expression of NK1R, neurokinin receptor 2 (NK2R), and neurokinin receptor 3 (NK3R) in AF and NP cells. Treatment of disc cells with the NK1R antagonist was able to suppress expression of IL-1β, IL-6, and IL-8 in a dose-dependent manner. SP stimulation increased phosphorylation of p38-MAPK and ERK1/2, but not of NFκB p65. This indicates that p38-MAPK and ERK1/2 control SP-induced cytokine expression independently from NF-kB p65. Inhibition of p38 and ERK1/2 activation reduced SP-induced IL-6 production in human disc cells. NK1R is responsible for the proinflammatory effect of SP on IVD cells and this effect can be blocked by preventing binding of SP to NK1R. This study shows for the first time that SP mediates signaling in disc cells through NK1R and that SP activates the proinflammatory p38-MAPK and ERK1/2 pathways. 4.

The Effects of Platelet-Rich Plasma on Halting the Progression in Porcine Intervertebral Disc Degeneration.

Disc degeneration and the subsequent herniation and/or rupture of the intervertebral disc (IVD) are due to a failure of the extracellular matrix of the annulus to contain the contents of the nucleus. This results from inadequate maintenance of the matrix components as well as the proteolytic activity of matrix metalloproteinases (MMPs) that degrade matrix molecules. Arresting progression of disc degeneration in the annulus holds greater clinical potential at this point than prevention of its onset in the nucleus. Therefore, in this study, we have therapeutic aims that would decrease levels of the cytokines and growth factors that indirectly lead to disc degeneration via stimulating MMP and increase levels of several beneficial growth factors, such as transforming growth factor-β, with the addition of platelet-rich plasma (PRP) that would stimulate cell growth and matrix synthesis. For this study, we attempted to address these imbalances of metabolism by using tumor necrosis factor-α treated annulus fibrosus cells isolated from porcine IVD tissue and incubating the cells in a growth factor rich environment with PRP. These results indicate that the PRP in vitro increased the production of the major matrix components (type II collagen and aggrecan) and decreased the inhibitory collagenase MMP-1. This application will address a therapeutic approach for intervening early in the degenerative process.

The effect of the fibre orientation of electrospun scaffolds on the matrix production of rabbit annulus fibrosus-derived stem cells.

Annulus fibrosus (AF) tissue engineering has recently received increasing attention as a treatment for intervertebral disc (IVD) degeneration; however, such engineering remains challenging because of the remarkable complexity of AF tissue. In order to engineer a functional AF replacement, the fabrication of cell-scaffold constructs that mimic the cellular, biochemical and structural features of native AF tissue is critical. In this study, we fabricated aligned fibrous polyurethane scaffolds using an electrospinning technique and used them for culturing AF-derived stem/progenitor cells (AFSCs). Random fibrous scaffolds, also prepared via electrospinning, were used as a control. We compared the morphology, proliferation, gene expression and matrix production of AFSCs on aligned scaffolds and random scaffolds. There was no apparent difference in the attachment or proliferation of cells cultured on aligned scaffolds and random scaffolds. However, compared to cells on random scaffolds, the AFSCs on aligned scaffolds were more elongated and better aligned, and they exhibited higher gene expression and matrix production of collagen-I and aggrecan. The gene expression and protein production of collagen-II did not appear to differ between the two groups. Together, these findings indicate that aligned fibrous scaffolds may provide a favourable microenvironment for the differentiation of AFSCs into cells similar to outer AF cells, which predominantly produce collagen-I matrix.

Expression of matrix factors in the process of neovascularization of intervertebral disc

<sec><title>OBJECTIVE:</title><p> To investigate the effects of proteins products of endothelial cells (ECs) on the annulus fibrosus (AF) cell metabolism in an in vitro culture.</p></sec><sec><title>METHODS:</title><p>Human AF cells were expanded in monolayer cultures and treated with proteins from the medium of cell line HMEC-1 (Human Microvascular Endothelial Cells) (125µg/ml). After 72h of treatment RNA was isolated from AF cells for analysis of gene expression and the culture medium was collected for protein expression analysis.</p></sec><sec><title>RESULTS:</title><p> The qRT-PCR analysis demonstrated increased gene expression of matrix metalloproteinases (MMPs) in AF cells treated with protein products of endothelial cells compared with cells from control group of AF cells: MMP-1 243.10 times (p<0.05), MMP-2 1.37 time (p<0.05), MMP-3 39.83 times (p<0.05) and MMP-13 5.70 times (p<0.05). In contrast, tissue inhibitors of metalloproteinases (TIMPs) were suppressed; TIMP-2 (0.55 time) (p<0.05) and TIMP-3 (0.60 time) (p<0.05) in the exposed groups. The expression of aggrecan gene (0.83 time) (p<0.05), an important extracellular matrix component, was also reduced. MMP-1 and MMP-3 detection was performed, confirming the results of PCR by Western Blot technique.</p></sec><sec><title>CONCLUSIONS:</title><p> In this study, we observed that the proteins produced by ECs induced the MMPs expression and suppressed the TIMPs as well as the aggrecan in primary cells of the human intervertebral disc, targeting the development of potential treatments for intervertebral disc degeneration and associated discogenic pain.</p></sec>

Ropivacaine- and bupivacaine-induced death of rabbit annulus fibrosus cells in vitro: involvement of the mitochondrial apoptotic pathway

The purposes of this study were to assess whether local anesthetics (LAs), such as ropivacaine and bupivacaine, could induce apoptosis of rabbit annulus fibrosus (AF) cells in vitro and further to explore the possible underlying mechanism. Rabbit AF cells at second passage were treated with saline solution and various concentrations of LAs. Apoptosis of AF cells were examined by cell counting kit-8 (CCK-8), Annexin V assays, Hoechst 33342 staining, and Caspase-3, -9 activity assays. The expression of apoptosis-related markers was detected by real-time PCR (RT-PCR) and Western Blot. The JC-1 staining was used to evaluate the change of mitochondrial membrane potential (MMP). Moreover, the levels of reactive oxygen species (ROS) were determined with fluorescent probe DCFH-DA. The results of flow cytometry indicated that LAs could induce apoptosis of rabbit AF cells in a dose-dependent manner. Apoptosis was confirmed by cell morphology, condensed nuclei and activation of Caspase-3 and -9. In addition, the molecular data showed that LAs could significantly up-regulate the expression of Bax, accompanied by a significant down-regulation of Bcl-2 expression. Furthermore, we also observed that LAs resulted in alteration of MMP and accumulation of intracellular ROS in AF cells. Blockade of ROS production by N-acetyl-l-cysteine (NAC) inhibited LAs-induced apoptosis. These findings suggest that LAs in clinically relevant concentrations could induce apoptosis of rabbit AF cells in vitro, and the mitochondrial pathway was, at least in part, involved in the LAs-mediated apoptosis. Further investigations focusing on the potential cytotoxicity of LAs on IVD cells are needed.

High Glucose-Induced Oxidative Stress Accelerates Premature Stress-Induced Senescence of Young Intervertebral Disc Cells

Introduction Diabetes mellitus (DM) is a major health problem worldwide. Approximately 10% of all DM cases are type 1 (Juvenile onset) and the prevalence of type 1 DM among the under 20s continuously rise. DM is known to be an important etiologic factor for premature disc degeneration. Previous studies have reported higher incidence of degenerative disc diseases in patients with DM, relatively young age, than in the patients without DM. A glucose-mediated increase of oxidative stress is a major causative factor for the development of diseases associated with DM through senescence. However, little is known about relationship among DM, mitochondrial damage, oxidative stress, senescence of young intervertebral disc cells, and disc degeneration. The aim of this study was to investigate the effect of high glucose on mitochondrial damage, oxidative stress, senescence of nucleus pulposus (NP) cells, and disc degeneration. Material and Methods Young nucleus pulposus (NP) and annulus fibrosus (AF) cells were isolated from 4-week-old rats, cultured and placed in either 10% FBS (normal control) or 10% FBS plus two high glucose concentrations (0.1 and 0.2 M) (experimental conditions) for 1 and 3 days. We identified and quantified the mitochondrial damage (mitochondrial transmembrane potential) and reactive oxygen species (ROS) for 1 and 3 days. We also identified and quantified the occurrence of senescence and telomerase activity. Finally, we determined the expressions of proteins related to replicative senescence (p53–p21) and stress-induced senescence (p16–pRB) pathways. Results Two high glucoses enhanced the disruption of mitochondrial transmembrane potential (mitochondrial damage) in young NP and AF cells for 1 and 3 days compared with normal control. Two high glucoses increased the occurrence of senescence of young NP and AF cells in a dose- and time-dependent manner. Telomerase activity declined in a dose- and time-dependent manner. Both high glucoses increased the expression of p16 and pRB proteins in young NP and AF cells for 1 and 3 days. However, compared to normal control, the expressions of p53 and p21 proteins were decreased in young NP and AF cells treated with both high glucoses for 1 and 3 days. Conclusion The current study demonstrated that high glucose-induced oxidative stress accelerates premature stress-induced senescence of young NP and AF cells in a dose- and time-dependent manner rather than replicative stress. This may result in dysfunction of young NP and AF cells, leading to accelerated premature disc degeneration. Therefore, the prevention of excessive generation of oxidative stress by strict blood glucose control is important to prevent or to delay premature disc degeneration in young patients with DM.

Mechanical Injury to Human Intervertebral Discs and Isolated Cells Initiates Events Implicated in Degeneration and Pain

Introduction Painful degenerating intact human intervertebral discs (IVDs) produce elevated levels of Toll-like receptors (TLRs), cytokines, and neuropeptides but events regulating their expression are poorly understood. Mechanical overload has been proposed as one potential initiating mechanism. The aim of this study was to investigate the cellular responses in IVDs and isolated disc cells exposed to mechanical overload or strain. Methods Lumbar spine segments were harvested from consenting donors via the Transplant Quebec organ donation program in Montreal, Quebec, Canada. A total of nine discs from six donors were used in this study. Intact IVDs were prepared and cells isolated using a standard technique. Intact discs with cartilaginous endplates were loaded with a single ramp compression peaking at either approximately 0.2 MPa (noninjurious) or about 1.5 MPa (injurious) stress applied at a rate of 30% strain per second. Injurious compression protocol consistently yields endplate fracture. Discs were immediately put in low serum containing media after loading. For isolated cell experiments, 2 × 105 nucleus pulposus (NP) and annulus fibrosus (AF) cells were seeded on flexible or static silicone dishes. The flexible dishes were exposed to a 20% magnitude cyclic mechanical strain at 0.0001 Hz was applied to the cultures for 8 h/d over 2 days. Conditioned media was collected and RNA was isolated. Quantitative RT-PCR was performed to assess TLR, NGF, and TNF gene expression. Conditioned media from intact discs and isolated cells was applied to PC12 cells and neurite outgrowth was assessed. Commercially available cytokine arrays were used to investigate protein levels of cytokines released from intact discs and isolated cells. Results Elevated levels of proteoglycan, inflammatory cytokines, and pain mediators were released from intact discs exposed to injurious loading. The highest levels were found 3 days postinjury and the conditioned media caused significantly induced neurite outgrowth in PC12 cells compared with that of the control group. About 50% of the cells in the IVDs died as a result of the inflicted trauma. Isolated NP and AF cells were exposed to a high-magnitude strain at low frequency to preserve cell attachments and maintain cell viability. The protocol was applied to evaluate if the adverse mechanical stimulation of the cells or the induced cell death caused by the injurious loading of the intact IVDs was responsible for the increased expression of inflammatory cytokines and pain mediators. Our data showed that high-magnitude low-frequency strain is sufficient to induce expression inflammatory cytokines and pain mediators in the absence of cell death. We also found a small but significant upregulation of TLR 2 and 4 in both NP and AF cells. Conclusion Cell death and cytokine production combined with loss of glycosaminoglycan indicate that mechanical injury to intact IVDs and isolated cells initiates cellular processes leading to disc degeneration. Once the proteoglycan content in the matrix has been degraded, elevated levels of NGF could lead to neuronal ingrowth directly causing pain. Increased TLR expression induced by high strain may prime cells toward a degenerate phenotype and could be a novel therapeutic target for degenerative disc disease.

Link N as a Therapeutic Agent to Treat Pain Associated with Intervertebral Disc Degeneration

Introduction The intervertebral discs (IVDs) are the largest avascular tissues in the body characterized by an abundant extracellular matrix and low cell density, with innervation limited at the most peripheral annular layers. During IVD degeneration, the neurotrophins, nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF), can stimulate nerve growth and hyperinnervation of the inner annulus fibrosus (AF) and nucleus pulposus (NP), which further could lead to back pain. Back pain can potentially be suppressed by BMP4 supplementation to decrease the density of innervation. Link N is a naturally occurring peptide that can stimulate BMP4 in human IVDs. The aim of the present study was to determine the effect of Link N on NGF, BDNF, and substance P (SP) in human AF cells stimulated with cytokines or punctured bovine discs and determine the possible mechanisms related to its effect. Material and Methods Human lumbar spines were obtained through Héma-Québec harvesting organ donation within 24 hour after death. Annulus fibrosus (AF) cells were isolated from AF region of human IVDs with Thompson grade 2 of degeneration, cultured in monolayers and stimulated with TNFα (100 ng/mL) and IL1β (10 ng/mL) in the presence or absence of Link N (1 µg/mL) for 48 hours. Total RNA was isolated and gene expression of neurotrophins, substance P, and their receptors was measured using RT PCR. AF cells isolated from human IVDs with Thompson grade 4 were cultured for 48 hours with or without Link N (1 µg/mL) and the NGF release in the media was measured by Western blotting. Coccygeal IVDs from the tails of adult bovine steers (20–25 months) were isolated with the cartilaginous endplates. After 24 hours preconditioning in complete DMEM, the IVDs were treated (control, capsaicin [1.5 µg/mL), punctured by 16 G needle, punctured by 16 G needle and incubated with Link N [10 µg/mL]) and the disc culture media was collected at different time points for analysis. SP in the media was concentrated by solid phase extraction and was assayed by Elisa. Results Proinflammatory cytokines IL1β and TNFα have been shown to trigger the expression of NGF and SP, as well as promoting the degradation of the extracellular matrix in human IVD cells. Our results demonstrate that gene expression of NGF, BDNF, TrkA, and TrkB significantly decreased in human disc cells stimulated with either IL-1β or TNFα supplemented with Link N when compared with the control. Additionally, Link N was also able to inhibit NGF release in the media when AF grade 4 was stimulated with IL-1β. Link N supplementation also led to a significant decrease in SP after 24 hour in injured discs, when compared with controls. Conclusion Link N can suppress the stimulation of NGF, BDNF, and their receptors TrkA and TrkB, respectively, as well as the neurotransmitter SP in degenerate discs. This suggests that Link N has the potential to inhibit pain induced by neuronal innervation caused by disc degeneration. Thus, administration of Link N has the potential to not only repair the discs in early stages of the disease but also suppress pain.