Intervertebral disc (IVD) degeneration is a pathological process often associated with chronic back pain and considered a leading cause of disability worldwide¹. During degeneration, progressive structural and biochemical changes occur, leading to blood vessel and nerve ingrowth and promoting discogenic pain². In the last decades, several cytokines have been applied to IVD cells in vitro to investigate the degenerative cascade. Particularly, IL-10 and IL-4 have been predicted as important anabolic factors in the IVD according to a regulatory network model based in silico approach³. Thus, we aim to investigate the potential presence and anabolic effect of IL-10 and IL-4 in human NP cells (in vitro) and explants (ex vivo) under hypoxia (5% O2) after a catabolic induction.

Primary human NP cells were expanded, encapsulated in 1.2% alginate beads (4 × 106 cells/ml) and cultured for two weeks in 3D for phenotype recovery while human NP explants were cultured for five days. Afterwards, both alginate and explant cultures were i) cultured for two days and subsequently treated with 10 ng/ml IL-10 or IL-4 (single treatments) or ii) stimulated with 0.1 ng/ml IL-1β for two days and subsequently treated with 10 ng/ml IL-10 or IL-4 (combined treatments).

The presence of IL-4 receptor, IL-4 and IL-10 was confirmed in human intact NP tissue (Fig 1). Additionally, IL-4 single and combined treatments induced a significant increase of proinflammatory protein secretion in vitro (Fig. 2A-C) and ex vivo (Fig. 2D and E). In contrast, no significant differences were observed in the secretome between IL-10 single and combined treatments compared to control group.

Overall, IL-4 containing treatments promote human NP cell and explant catabolism in contrast to previously reported IL-4 anti-inflammatory performance⁴. Thus, a possible pleiotropic effect of IL-4 could occur depending on the IVD culture and environmental condition.

Acknowledgements: This project was supported by the Marie Skłodowska Curie International Training Network “disc4all” under the grant agreement #955735.

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Intervertebral disc (IVD) degeneration (IDD) involves imbalance between the anabolic and the catabolic processes that regulate the extracellular matrix of its tissues. These processes are complex, and improved integration of knowledge is needed. Accordingly, we present a nucleus pulposus cell (NPC) regulatory network model (RNM) that integrates critical biochemical interactions in IVD regulation and can replicate experimental results. The RNM was built from a curated corpus of 130 specialized journal articles. Proteins were represented as nodes that interact through activation and inhibition edges. Semi-quantitative steady states (SS) of node activations were calculated. Then, a full factorial sensitivity analysis (SA) identified which out of the RNM 15 cytokines, and 4 growth factors affected most the structural proteins and degrading enzymes. The RNM was further evaluated against metabolic events measured in non-healthy human NP explant cultures, after 2 days of 1ng/ml IL-1B catabolic induction. The RNM represented successfully an anabolic basal SS, as expected in normal IVD. IL-1B was able to increase catabolic markers and angiogenic factors and decrease matrix proteins. Such activity was confirmed by the explant culture measurements. The SA identified TGF-β and IL1RA as the two most powerful rescue mediators. Accordingly, TGFβ signaling-based IDD treatments have been proposed and IL-1RA gene therapy diminished the expression of proteases. It resulted challenging to simulate rescue strategies by IL-10, but interestingly, IL-1B could not induce IL-10 expression in the explant cultures. Our RNM was confronted to independent in vitro measurements and stands for a unique model, to integrate soluble protein signaling and explore IDD.

Acknowledgements: European Commission (Disc4All-ITN-ETN-955735)

Low back pain affects 80% of the population with half of cases attributed to intervertebral disc (IVD) degeneration. However, the majority of treatments focus on pain management, with none targeting the underlying pathophysiological causes. PCRX-201 presents a novel gene therapy approach that addresses this issue. PCRX-201 codes for interleukin-1 receptor antagonist (IL-1Ra), the natural inhibitor of the pro-inflammatory cytokine IL-1, which orchestrates the catabolic degeneration of the IVD. Our objective here is to determine the ability of PCRX-201 to infect human nucleus pulposus (NP) cells and tissue to increase the production of IL-1Ra and assess downstream effects on catabolic protein production.

Degenerate human NP cells and tissue explants were infected with PCRX-201 at 0 or 3000 multiplicities of infection (MOI) and subsequently cultured for 5 days in monolayer (n=7), 21 days in alginate beads (n=6) and 14 days in tissue explants (n=5). Cell culture supernatant was collected throughout culture duration and downstream targets associated with pain and degeneration were assessed using ELISA.

IL-1Ra production was increased in NP cells and tissue infected with PCRX-201. The production of downstream catabolic proteins such as IL-1β, IL-6, MMP3, ADAMTS4 and VEGF was decreased in both 3D-cultured NP cells and tissue explants.

Here, we have demonstrated that a novel gene therapy, PCRX-201, is able to infect and increase the production of IL-1Ra in degenerate NP cells and tissue in vitro. The increase of IL-1Ra also resulted in a decrease in the production of a number of pro-inflammatory and catabolic proteins, suggesting PCRX-201 enables the inhibition of IL-1-driven IVD degeneration. At present, no treatments for IVD degeneration target the underlying pathology. The ability of FX201 to elicit anti-catabolic responses is promising and warrants further investigation in vitro and in vivo, to determine the efficacy of this exciting, novel gene therapy.

Previous research has shown catabolic cell signalling induced by TNF-α and IL-1β within intervertebral (IVD) cells. However, these studies have investigated this in 2D monolayer cultures, and under hyper-physiological doses. Thus, we aim to revisit the catabolic responses of bovine IVD cells in vitro in 3D culture under increasing doses of TNF-α or IL-1β stimulation at three different timepoints.

Primary bovine nucleus pulposus (NP) and annulus fibrosus (AF) cells were isolated and expanded for two weeks. Subsequently, NP and AF cells were encapsulated in 1.2% alginate beads (4 × 106 cells/ml) and cultured for two weeks for phenotype recovery. Re-differentiated cells were stimulated with 0.1, 1 and 10 ng/ml TNF-α or with 0.01, 0.1 and 10 ng/ml IL-1β for one week. Beads were collected on the stimulation day (Day 0) and on Day 1 and 7 after stimulation.

A dose-dependent upregulation of catabolic markers was observed in both cell types after one day of TNF-α or IL-1β stimulation. 10 ng/ml TNF-α stimulation induced a significant upregulation (p<0.05) of ADAMTS4, MMP3 and MMP13 in AF cells after one day of stimulation. Similarly, MMP3 upregulation showed a strong trend (p=0.0643) in NP cells. However, no effects on expression were seen after seven days. In addition, no significant difference between treatments in COL2, COL1 and ACAN expression was observed, and cell viability was not reduced at any time point, regardless of the treatment.

We demonstrate a dose-dependent upregulation of catabolic markers in NP and AF cells under TNF-α or IL-1β stimulation, with a significant upregulation of ADAMTS4, MMP3 and MMP13 genes in AF cells after one day of treatment. Notably, after seven days of treatment, the dose-dependent effects were no longer observed possibly due to an adaptation mechanism of IVD cells to counter the metabolic shift.

The effect of high-fat diet and testosterone replacement therapy upon bone remodelling was investigated in orchiectomised male APOE-/- mice.

Mice were split in to three groups: sham surgery + placebo treatment (control, n=9), orchiectomy plus placebo treatment (n=8) and orchiectomy plus testosterone treatment (n=10). Treatments were administered via intramuscular injection once a fortnight for 17 weeks before sacrifice at 25 weeks of age. Tibiae were scanned ex-vivo using µCT followed by post-analysis histology and immunohistochemistry.

Previously presented µCT data demonstrated orchiectomised, placebo treated mice exhibited significantly reduced trabecular bone volume, number, thickness and BMD compared to control mice despite no significant differences in body weight. Trabecular parameters were rescued back to control levels in orchiectomised mice treated with testosterone. No significant differences were observed in the cortical bone.

Assessment of TRAP stained FFPE sections revealed no significant differences in osteoclast or osteoblast number along the endocortical surface. IHC assessment of osteoprotegerin (OPG) expression in osteoblasts is to be quantified alongside markers of osteoclastogenesis including RANK and RANKL.

Results support morphological analysis of cortical bone where no change in cortical bone volume or density between groups is in line with no significant change in osteoblast or osteoclast number and percentage across all three groups.

Future work will include further IHC assessment of bone remodelling and adiposity, as well as utilisation of mechanical testing to establish the effects of observed morphological differences in bone upon mechanical properties. Additionally, the effects of hormone treatments upon murine-derived bone cells will be investigated to provide mechanistic insights.

An increasing elderly population means joint replacement surgery numbers are projected to increase, with associated complications such as periprosthetic joint infections (PJI) also rising. PJI are particularly challenging due to antimicrobial resistant biofilm development on implant surfaces and surrounding tissues, with treatment typically involving invasive surgeries and systemic antibiotic delivery. Consequently, functionalisation of implant surfaces to prevent biofilm formation is a major research focus. This study characterises clinically relevant antimicrobials including gentamicin, clindamycin, daptomycin, vancomycin and caspofungin within a silica-based, biodegradable sol-gel coating for prosthetic devices.

Antimicrobial activity of the coatings against clinically relevant microorganisms was assessed via disc diffusion assays, broth microdilution culture methods and the MBEC assay used to determine anti-biofilm activity. Human and bovine cells were cultured in presence of antimicrobial sol-gel to determine cytotoxicity using Alamar blue and antibiotic release was measured by LC-MS. Biodegradability in physiological conditions was assayed by FT-IR, ICP-MS and measuring mass change. Effect of degradation products on osteogenesis were studied by culturing mesenchymal stem cells in the presence of media in which sol-gel samples had been immersed.

Antimicrobial-loaded coatings showed strong activity against a wide range of clinically relevant bacterial and fungal pathogens with no loss of activity from antibiotic alone. The sol-gel coating demonstrated controlled release of antimicrobials and initial sol-gel coatings showed no loss of viability on MSCs with gentamicin containing coatings. Current work is underway investigating cytotoxicity of sol-gel compositions against MG-63 cells and primary osteoblasts.

This research forms part of an extended study into a promising antimicrobial delivery strategy to prevent PJI. The implant coating has potential to advance PJI infection prevention, reducing future burden upon healthcare costs and patient wellbeing.

This study aimed to characterise the microarchitecture of bone in different species of animal leading to the development of a physiologically relevant 3D printed cellular model of trabecular (Tb) and cortical bone (CB). Using high resolution micro-computed tomography (μ-CT) bone samples from multiple species were scanned and analysed before creating in silico models for 3D printing. Biologically relevant printing materials with physical characteristics similar to that of in vivo bone will be selected and tested for printability.

Porcine and murine bone samples were scanned using μ-CT, with a resolution of 4.60 μM for murine and 11 μM for porcine and reconstructed to determine the architectural properties of both Tb and CB independently. A region of interest, 1 mm in height, will be used to generate an in-silico 3D model with dimensions (10 mm³) and suitable resolution before being translated into printable G code using CAD assisted software.

A 1 mm section of each bone was analysed, to determine the differences in the microarchitecture with the intent of setting a benchmark for the developmental 3D in vitro model to be comparable against. In contrast, porcine caudal vertebrae (PCV) have an increased volume due to the size of the bone sample. Interestingly, BV/TR for Tb is similar between species in all samples except murine femur. Murine tibia and PCV have a similar Tb. number and thickness, however different SMI shape and separation.

μ-CT scanning and analysis permits tessellation of the 3D output which will lead to the generation of an in silico printable model. Biomaterials are currently under optimisation to allow printability and shape integrity to reflect the morphological and physiological properties of bone.

A key cause of low back pain is the degeneration of the intervertebral disc (IVD). Causality between infection of the IVD and its degenerative process gained great interest over the last decade. Granville Smith et al. (2021) identified 36 articles from 34 research studies investigating bacteria in human IVDs. Bacteria was identified in 27 studies, whereas 9 attributed bacterial presence to contamination. Cutibacterium acnes was the most abundant, followed by coagulase-negative staphylococcus. However, whether bacteria identified were present in vivo or represent perioperative contamination remains unclear. This study investigated whether bacteria are present in IVDs and what potential effects they may have on native disc cells.

Immunohistochemical staining for Gram positive bacteria was performed on human IVD tissue to identify presence and characterise bacterial species. Nucleus pulposus (NP) cells in monolayer and 3D alginate were stimulated with LPS and Peptidoglycan (0.1-50 µg/ml) for 48hrs. Following stimulation qPCR for factors associated with disc degeneration including matrix genes, matrix degrading enzymes, cytokines, neurotrophic factors and angiogenic factors and conditioned media collected for ELISA and luminex analysis

Gram positive bacteria was detected within human IVD tissue. Internalisation of bacteria by NP cells influenced the cell and nuclei morphology. Preliminary results of exposure of NP cells to bacterial components indicate that LPS as well as Peptidoglycan increase IL-8 and ADAMTS-4 gene expression following 48 hours of stimulation with a dose response seen for IL-8 induction by peptidoglycan compared to the control group. Underlining these results, IL-8 protein release was increased for treated groups compared to non-treated control. Further analysis is underway investigating other output measures and additional biological repeats.

This study has demonstrated bacteria are present within IVD cells within IVD tissue removed from degenerate IVD and is determining the potential influence of these on disc degeneration.

Low back pain resulting from Interertebral disc (IVD) degeneration is a serious worldwide problem, with poor treatment options available. Notochordal (NC) cells, are a promising therapeutic cell source with anti-catabolic and regenerative effect. However, their behaviour in the harsh degenerate environment is unknown.

Porcine NC cells (pNCs), and Human NP cells from degenerate IVDs were cultured in alginate beads to maintain phenotype. Cells were cultured alone or in combination, or co-stimulated with notochordal cell condition media (NCCM), in media to mimic the healthy and degenerate disc environment, together with controls for up to 1 week. Following culture viability, qPCR and proteomic analysis using Digiwest was performed.

A small increase in pNC cell death was observed in degenerated media compared to standard and healthy media, with a further decrease seen when cultured with IL-1β. Whilst no significant differences were seen in phenotypic marker expression in pNCs cultured in any media at gene level (ACAN, KRT8, KRT18, FOXA2, COL1A1 and Brachyury). Preliminary Digiwest analysis showed increased protein production for Cytokeratin 18, src and phosphorylated PKC but a decrease in fibronectin in degenerated media compared to standard media. Human NP cells cultured with NCCM, showed a decrease in IL-8 production compared to human NP cells alone when cultured in healthy media. However, gene expression analysis (ACAN, VEGF, MMP3 and IL-1β) demonstrated no significant difference between NP only and NP+NCCM groups.

Studying the behaviour of the NCs in in vitro conditions that mimic the in vivo healthy or degenerate niche will help us to better understand their potential for therapeutic approaches. The potential use of NC cell sources for regenerative therapies can then be translated to investigate the potential use of iPSCs differentiated into NC cells as a regenerative cell source.

Low back pain is strongly associated with degeneration of the intervertebral disc (IVD). During degeneration, altered matrix synthesis and increased matrix degradation, together with accompanied cell loss is seen particularly in the nucleus pulposus (NP). It has been proposed that notochordal (NC) cells, embryonic precursors for the cells within the NP, could be utilized for mediating IVD regeneration. However, injectable biomaterials are likely to be required to support their phenotype and viability within the degenerate IVD. Therefore, viability and phenotype of NC cells were analysed and compared within biomaterial carriers subjected to physiological oxygen conditions over a four-week period were investigated.

Porcine NC cells were incorporated into three injectable hydrogels: NPgel (a L-pNIPAM-co-DMAc hydrogel), NPgel with decellularized NC-matrix powder (dNCM) and Albugel (an albumin/ hyaluronan hydrogel). The NCs and biomaterials constructs were cultured for up to four weeks under 5% oxygen (n=3 biological repeats). Histological, immunohistochemical and glycosaminoglycans (GAG) analysis were performed to investigate NC viability, phenotype and extracellular matrix synthesis and deposition.

Histological analysis revealed that NCs survive in the biomaterials after four weeks and maintained cell clustering in NPgel, Albugel and dNCM/NPgel with maintenance of morphology and low caspase 3 staining. NPgel and Albugel maintained NC cell markers (brachyury and cytokeratin 8/18/19) and extracellular matrix (collagen type II and aggrecan). Whilst Brachyury and Cytokeratin were decreased in dNCM/NPgel biomaterials, Aggrecan and Collagen type II was seen in acellular and NC containing dNCM/NPgel materials. NC containing constructs excreted more GAGs over the four weeks than the acellular controls.

NC cells maintain their phenotype and characteristic features in vitro when encapsulated into biomaterials. NC cells and biomaterial construct could potentially become a therapy to treat and regenerate the IVD.

The IVD is a highly hydrated, hyperosmolar tissue that allows the correct biomechanical function of the spine. When degenerated, water and ions are lost from the disc, especially within the central nucleus pulposus (NP), producing a hypoosmotic environment in which the resident cells can no longer function correctly, exacerbating the degenerative cascade. One potential way that IVD cells may adapt to their environment is through the expression and regulation of aquaporin (AQP) channels that control the movement of water in and out of cells. During human IVD degeneration AQP1 and 5 expression is decreased, highlighting AQPs may be of importance for the correct function of NP cells. The regulation of AQPs in NP cells by healthy and degenerate conditions, and the potential underlying molecular mechanisms, were investigated in both human and rat IVD cells. The gene and protein expression of AQP1 and AQP5 was upregulated by hyperosmotic conditions (425mOsm/kg H₂O) in rat and human NP cells. Lentiviral knockdown of tonicity enhancer binding protein (TonEBP), a transcription factor responsible for maintaining the function of NP cells, resulted in the loss of AQP1 and 5 gene expression under hyperosmotic conditions. The maintenance of the IVD environment and adaptation of cells is vital for the function of the IVD. The regulation of AQPs by physiological conditions and TonEBP suggests a role for these water channels related to the adaptation of disc cells to their environment, which is dysregulated during degeneration.

Intervertebral disc (IVD) degeneration is a major cause of low back pain (LBP). Degenerate discs are associated with accelerated cellular senescence. Cell senescence is associated with a secretory phenotype characterised by increased production of catabolic enzymes and cytokines. However, to date, the mechanism of cell senescence within disc degeneration is unclear. Senescence can be induced by increased replication or induced by stress such as reactive oxygen species or cytokines. This study investigated the association of cellular senescence with markers of DNA damage and presence of cytoplasmic DNA (which in cancer cells has been shown to be a key regulator of the secretory phenotype), to determine mechanisms of senescence in disc degeneration. Immunohistochemistry for the senescence marker: p16INK4A was firstly utilised to screen human intervertebral discs for discs displaying at least 30% immunopostivity. These discs were then subsequently analysed for immunopostivity for DNA damage markers γH2AX and cGAS and the presence of cytoplasmic DNA. The number of immunopositive cells for p16 INK4A positively correlated with the expression of γH2AX and cGAS. Senescent cells were also associated with the presence of cytoplasmic DNA. These new findings elucidated a role of cGAS and γH2AX as a link from genotoxic stress to cytokine expression which is associated with senescent cells. The findings indicate that cellular senescence in vivo is associated with DNA damage and presence of cytoplasmic DNA. Whether this DNA damage is a result of replicative senescence or stress induced is currently being investigated in vitro.

Summary Statement

IL-1β stimulation of human OA chondrocytes induces NFκB, ERK1/2, c-JUN, IκB and P38 signalling pathways. Pre-treatment with cannabinoid WIN-55 for 48 hours inhibits certain pathways, providing mechanisms for cannabinoids inhibitory actions on IL-1β induced cartilage degradation.

Matrix metalloproteinases (MMPs) are involved in extracellular matrix (ECM) breakdown in osteoarthritis (OA) and their expression is regulated by nuclear factor kappa B (NFκB). In addition signalling pathways ERK1/2, c-JUN, IκB and P38 are activated in OA and are induced by inflammatory cytokine interleukin 1 (IL-1). Cannabinoids have been shown to reduce joint damage in animal models of arthritis. Synthetic cannabinoid WIN-55, 212-2 mesylate (WIN-55) significantly reduces IL-1β induced expression of MMP-3 and -13 in human OA chondrocytes, indicating a possible mechanism via which cannabinoids may act to prevent ECM breakdown. Here the effects of WIN-55 on IL-1β induced NFκB, ERK1/2, c-JUN, IκB and P38 phosphorylation in human OA chondrocytes has been investigated.

Primary human chondrocytes were obtained from articular cartilage removed from patients with symptomatic OA during total knee replacement (Ethic approval:SMB002). Cartilage tissue was graded macroscopically 0–4 using the Outerbridge Classification method. Chondrocytes isolated from grade 2 cartilage and cultured in monolayer were pre-treated with 10 μM WIN-55 for 1 hour prior to stimulation with 10 ng/ml IL-1β for 30 minutes for investigation of NFκB, c-JUN, IκB and P38 phosphorylation. In addition chondrocytes were pre-treated with 10 μM WIN-55 for 30 minutes, 1, 3, 6, 24 and 48 hours prior to 10 ng/ml IL-1β stimulation for 30 minutes to investigate ERK1/2 phosphorylation.

Dimethyl sulfoxide (DMSO) was used as a vehicle control at 0.1%. Immunocytochemistry was used to investigate the phosphorylation and translocation of NFκB. ERK1/2, c-JUN, IκB, and P38 activation was investigated using cell based ELISA. Immunocytochemical analysis showed chondrocytes stimulated with IL-1β induced NFκB phosphorylation and translocation to the nucleus.

Chondrocytes treated with IL-1β with WIN-55 for 1 hour pre-treatment showed no inhibition of the IL-1β induced NFκB phosphorylation and translocation to the nucleus. WIN-55 treatment alone for 1 hour stimulated NFκB phosphorylation in the cytoplasm but not the nucleus. ELISA showed that phosphorylation of ERK1/2, c-JUN, IκB, and P38 was significantly induced by IL-1β following 30 minutes stimulation (p<0.05). Pre-treatment with WIN-55 for 1 hour had no significant effect on this IL-1β induced phosphorylation. However WIN-55 pre-treatment for 48 hours prior to IL-1β stimulation for 30 minutes, resulted in a significant decrease in ERK1/2 phosphorylation compared to IL-1β stimulation alone (p<0.05).

WIN-55 treatment alone for 1 hour significantly induced c-JUN phosphorylation (p<0.05), but had no effect on IκB and P38 phosphorylation compared to DMSO control. IL-1β stimulation of ERK1/2 phosphorylation was not significantly affected by WIN-55 pre-treatment of 30 minutes, 1, 3, 6 and 24 hours. WIN-55 treatment alone for 48 hours significantly reduced ERK1/2 phosphorylation compared to DMSO control (p<0.05). WIN-55 treatment alone for 30 min, 1, 3, 6 and 24 hours had no significant effect on ERK1/2 phosphorylation compared to DMSO control. The results show that following 48 hours pre-treatment WIN-55 inhibits IL-1β induced ERK1/2 phosphorylation in human OA chondrocytes. Thus inhibitory effects of cannabinoids on IL-1β induced cartilage degradation may be mediated via modulation of ERK1/2 signalling.

Summary

Cytokines produced within the degenerate disc induce expression of neurotrophic factors and pain related peptides which could be important in nerve ingrowth and pain sensitisation leading to low back pain.

The intervertebral disc (IVD) is considered the largest aneural and avascular structure within the human body, yet during degeneration vascularisation of the IVD is seen to be accompanied by nociceptive nerves. Low back pain is a highly debilitating condition affecting around 80% of the population, 40% of which are attributed to IVD degeneration. Discogenic pain was largely thought to be a result of irritation and compression of the nerve root, yet recent data suggests that pain may be attributed to the sensitisation of sensory nerves by the synthesis of pain related peptides, calcitonin gene related peptide (CGRP) and substance P. It is known that cytokines and chemokines produced by nucleus pulposus cells elicit various effects including the production of matrix degrading enzymes, and decreased matrix molecules. Here, we investigate the hypothesis that cytokines regulate both neurotrophic factor and pain related peptide synthesis within nucleus pulposus and nerve cells which may elicit algesic effects.

Real-Time PCR was performed to investigate gene expression of the neurotrophic factors NGF, BDNF, NT3 and their receptors Trk A, B and C along with Substance P and CGRP on directly extracted RNA from human NP cells and NP cells cultured in alginate for 2 weeks prior to treatment for 48hours with IL-1, IL-6 or TNFα at 0–100ng/mL. Similarly SH-SY5Y neuroblastoma cells were differentiated in retinoic acid for 7 days prior to stimulation with IL-1, IL-6 or TNFα at 0ng/mL and 10ng/mL for 48hours. Immunohistochemistry was used to localise neurotrophic factor receptors Trk A, B and C in both degenerate discs and neuronal cells.

NGF expression was present in normal and degenerate disc samples, however only degenerate discs expressed the high affinity receptor TrkA. Similarly Trk B was present in 22% of normal samples increasing to 100% expression within degenerate disc samples. All cytokines increased expression of NGF in NP cells (P≤0.05). TNFα also increased BDNF significantly, whereas no significant affects were seen in NT3 expression in NP cells. Trk B expression was significantly increased by IL-1 and TNFα treatment of NP cells. Conversely Trk C was down regulated by IL-6. Substance P was significantly increased by IL-1 and TNFα treatments whilst IL-6 and TNFα increased CGRP expression in NP cells. In SH-SY5Y cells, IL-1 significantly increased BDNF whilst IL-6 and TNFα failed to induce significant differences in neurotrophic factors. All cytokines increased Trk expression in the nerve cell line; however this failed to reach significance. Immunohistochemistry confirmed the presence of Trk receptors within the neuronal cell line.

Here we have demonstrated that a number of cytokines known to be up regulated during disc degeneration and disc prolapse, induce expression of various neurotrophic factors, their receptors and pain related peptides within human NP cells, as well as SH-SY5Y cells. This data suggests that the presence and production of cytokines within the degenerate disc may be responsible for nerve ingrowth and sensitisation of nerves which may result in discogenic pain.

Summary

Anabolic and catabolic signalling processes within IVDs display overlapping pathways, however some pathways were identified as selective to catabolic signalling and inhibition of one of these pathways inhibited some of the catabolic factors induced by IL-1 although NFkB inhibition also affected anabolic expression.

Degeneration of intervertebral discs (IVDs) is implicated in 40% of low back pain cases. In the normal disc the balance between anabolic and catabolic processes are carefully balanced. During degeneration this balance is lost in favour of catabolic processes which lead to degradation of the IVD, infiltration of blood vessels and nerves and release of cytokines which sensitise nerves to pain. Interleukin 1 (IL-1) is known to be important in the pathogenesis of IVD degeneration, here we investigated the intracellular signalling pathways activated by IL-1 and those activated by an anabolic factor (CDMP-1) to investigate differential pathways.

Human nucleus pulposus cells (NP) removed during discetomy for nerve root pain were stimulated with IL-1 or CDMP-1 for 30 minutes. Site-specific phosphorylation of 46 signalling molecules were identified using R&D proteome array. The activation of ERK1/2, p38, c-jun, and IkB were confirmed using cell based ELISAs, in addition pNFκB localisation in stimulated cells was determined using immunohistochemisty. Pre-treatment with inhibitors to p38, and NFkB for 30 minutes, followed by stimulation with IL-1 (10ng/mL) or CDMP-1 (10ng/mL) for 24 hours was investigated to determine effects on anabolic and catabolic factors. In addition localisation of phosphorylated c-jun, p38 and NFkB were investigated within paraffin embedded sections of human IVD to investigate the presence of active pathways in vivo.

Twenty intracellular signalling pathways were activated following CDMP-1 treatment and 8 signalling pathways activated by IL-1. Of note key classical IL-1 signalling pathways p38 MAPK, ERK 1/2 and JNK were activated by IL-1, however of these ERK 1/2 particularly was also activated by CDMP-1, whilst p38 and c-jun were only activated by IL-1. IL-1 induced activation of NFkB signalling to a greater extent than CDMP-1, these results were confirmed by the ‘in cell ELISAs’. IVD tissue samples displayed immunopositive staining for phosphorylated c-jun, NFkB and p38. Inhibition of p38 signalling inhibited IL-1 induced MMP 13 expression, but had little effect on the induction of IL-8. However inhibitors of NFkB signalling pathway failed to inhibit the induction of MMP 13 but abrogated the induced IL-6 and IL-8 expression. IL-1 induced a complete aberration of aggrecan expression by NP cells in alginate culture, this effect was partly inhibited by p38 MAPK inhibitor but was completely restored by inhibiting NFkB signalling. However the aggrecan expressed in CDMP-1 treated cells was decreased by inhibiting NFkB but not p38.

Here, we have shown that anabolic and catabolic signalling processes within IVDs show a number of overlapping pathways, however a number of differential pathways were identified and inhibition of p38 MAPK and NFkB pathways inhibited a number of catabolic processes investigated which were induced by IL-1. Thus inhibition of signalling pathways could be a novel mechanism of inhibiting catabolic processes which could hold promise to inhibit degeneration at early stages of disease but also create the correct tissue niche to promote regeneration of the disc.