Mesenchymal stem-cell based therapies have been proposed as novel treatments for intervertebral disc degeneration, a prevalent and disabling condition associated with back pain. The development of these treatment strategies, however, has been hindered by the incomplete understanding of the human nucleus pulposus phenotype and by an inaccurate interpretation and translation of animal to human research. This review summarises recent work characterising the nucleus pulposus phenotype in different animal models and in humans and integrates their findings with the anatomical and physiological differences between these species. Understanding this phenotype is paramount to guarantee that implanted cells restore the native functions of the intervertebral disc. Cite this article: Bone Joint Res 2013;2:169–78

Aims. Inflammatory response plays a pivotal role in the pathophysiological process of intervertebral disc degeneration (IDD). A20 (also known as tumour necrosis factor alpha-induced protein 3 (TNFAIP3)) is a ubiquitin-editing enzyme that restricts nuclear factor-kappa B (NF-κB) signalling. A20 prevents the occurrence of multiple inflammatory diseases. However, the role of A20 in the initiation of IDD has not been elucidated. The aim of the study was to investigate the effect of A20 in senescence of TNF alpha (TNF-α)-induced nucleus pulposus cells (NPCs). Methods. Immunohistochemical staining was performed to observe the expression of A20 in normal and degenerated human intervertebral discs. The NPCs were dissected from the tail vertebrae of healthy male Sprague-Dawley rats and were cultured in the incubator. In the experiment, TNF-α was used to mimic the inflammatory environment of IDD. The cell viability and senescence were examined to investigate the effect of A20 on TNF-α-treated NPCs. The expression of messenger RNA (mRNA)-encoding proteins related to matrix macromolecules (collagen II, aggrecan) and senescence markers (p53, p16). Additionally, NF-κB/p65 activity of NPCs was detected within different test compounds. Results. The expression of A20 was upregulated in degenerate human intervertebral discs. The A20 levels of NPCs in TNF-α inflammatory microenvironments were dramatically higher than those of the control group. TNF-α significantly decreased cell proliferation potency but increased senescence-associated beta-galactosidase (SA-β-Gal) activity, the expression of senescence-associated proteins, the synthesis of extracellular matrix, and G1 cycle arrest. The senescence indicators and NF-κB/p65 expression of A20 downregulated group treated with TNF-α were significantly upregulated compared to TNF-α-treated normal NPCs. Conclusion. A20 has a self-protective effect on the senescence of NPCs induced by TNF-α. The downregulation of A20 in NPCs exacerbated the senescence of NPCs induced by TNF-α. Cite this article:Bone Joint Res. 2020;9(5):225–235

Aims. This study was performed to explore the effect of melatonin on pyroptosis in nucleus pulposus cells (NPCs) and the underlying mechanism of that effect. Methods. This experiment included three patients diagnosed with lumbar disc herniation who failed conservative treatment. Nucleus pulposus tissue was isolated from these patients when they underwent surgical intervention, and primary NPCs were isolated and cultured. Western blotting, reverse transcription polymerase chain reaction, fluorescence staining, and other methods were used to detect changes in related signalling pathways and the ability of cells to resist pyroptosis. Results. Western blot analysis confirmed the expression of cleaved CASP-1 and melatonin receptor (MT-1A-R) in NPCs. The cultured NPCs were identified by detecting the expression of CD24, collagen type II, and aggrecan. After treatment with hydrogen peroxide, the pyroptosis-related proteins NLR family pyrin domain containing 3 (NLRP3), cleaved CASP-1, N-terminal fragment of gasdermin D (GSDMD-N), interleukin (IL)-18, and IL-1β in NPCs were upregulated, and the number of propidium iodide (PI)-positive cells was also increased, which was able to be alleviated by pretreatment with melatonin. The protective effect of melatonin on pyroptosis was blunted by both the melatonin receptor antagonist luzindole and the nuclear factor erythroid 2–related factor 2 (Nrf2) inhibitor ML385. In addition, the expression of the transcription factor Nrf2 was up- or downregulated when the melatonin receptor was activated or blocked by melatonin or luzindole, respectively. Conclusion. Melatonin protects NPCs against reactive oxygen species-induced pyroptosis by upregulating the transcription factor Nrf2 via melatonin receptors. Cite this article: Bone Joint Res 2023;12(3):202–211

Aims. In this investigation, we administered oxidative stress to nucleus pulposus cells (NPCs), recognized DNA-damage-inducible transcript 4 (DDIT4) as a component in intervertebral disc degeneration (IVDD), and devised a hydrogel capable of conveying small interfering RNA (siRNA) to IVDD. Methods. An in vitro model for oxidative stress-induced injury in NPCs was developed to elucidate the mechanisms underlying the upregulation of DDIT4 expression, activation of the reactive oxygen species (ROS)-thioredoxin-interacting protein (TXNIP)-NLRP3 signalling pathway, and nucleus pulposus pyroptosis. Furthermore, the mechanism of action of small interfering DDIT4 (siDDIT4) on NPCs in vitro was validated. A triplex hydrogel named siDDIT4@G5-P-HA was created by adsorbing siDDIT4 onto fifth-generation polyamidoamine (PAMAM) dendrimer using van der Waals interactions, and then coating it with hyaluronic acid (HA). In addition, we established a rat puncture IVDD model to decipher the hydrogel’s mechanism in IVDD. Results. A correlation between DDIT4 expression levels and disc degeneration was shown with human nucleus pulposus and needle-punctured rat disc specimens. We confirmed that DDIT4 was responsible for activating the ROS-TXNIP-NLRP3 axis during oxidative stress-induced pyroptosis in rat nucleus pulposus in vitro. Mitochondria were damaged during oxidative stress, and DDIT4 contributed to mitochondrial damage and ROS production. In addition, siDDIT4@G5-P-HA hydrogels showed good delivery activity of siDDIT4 to NPCs. In vitro studies illustrated the potential of the siDDIT4@G5-P-HA hydrogel for alleviating IVDD in rats. Conclusion. DDIT4 is a key player in mediating pyroptosis and IVDD in NPCs through the ROS-TXNIP-NLRP3 axis. Additionally, siDDIT4@G5-P-HA hydrogel has been found to relieve IVDD in rats. Our research offers an innovative treatment option for IVDD. Cite this article: Bone Joint Res 2024;13(5):247–260

Aims. CRP is an acute-phase protein that is used as a biomarker to follow severity and progression in infectious and inflammatory diseases. Its pathophysiological mechanisms of action are still poorly defined. CRP in its pentameric form exhibits weak anti-inflammatory activity. The monomeric isoform (mCRP) exerts potent proinflammatory properties in chondrocytes, endothelial cells, and leucocytes. No data exist regarding mCRP effects in human intervertebral disc (IVD) cells. This work aimed to verify the pathophysiological relevance of mCRP in the aetiology and/or progression of IVD degeneration. Methods. We investigated the effects of mCRP and the signalling pathways that are involved in cultured human primary annulus fibrosus (AF) cells and in the human nucleus pulposus (NP) immortalized cell line HNPSV-1. We determined messenger RNA (mRNA) and protein levels of relevant factors involved in inflammatory responses, by quantitative real-time polymerase chain reaction (RT-qPCR) and western blot. We also studied the presence of mCRP in human AF and NP tissues by immunohistochemistry. Results. We demonstrated that mCRP increases nitric oxide synthase 2 (NOS2), cyclooxygenase 2 (COX2), matrix metalloproteinase 13 (MMP13), vascular cell adhesion molecule 1 (VCAM1), interleukin (IL)-6, IL-8, and Lipocalin 2 (LCN2) expression in human AF and NP cells. We also showed that nuclear factor-κβ (NF-κβ), extracellular signal-regulated kinase 1/2 (ERK1/2), and phosphoinositide 3-kinase (PI3K) are at play in the intracellular signalling of mCRP. Finally, we demonstrated the presence of mCRP in human AF and NP tissues. Conclusion. Our results indicate, for the first time, that mCRP can be localized in IVD tissues, where it triggers a proinflammatory and catabolic state in degenerative and healthy IVD cells, and that NF-κβ signalling may be implicated in the mediation of this mCRP-induced state. Cite this article: Bone Joint Res 2023;12(3):189–198

Discogenic low back pain is a common cause of disability, but its pathogenesis is poorly understood. We collected 19 specimens of lumbar intervertebral discs from 17 patients with discogenic low back pain during posterior lumbar interbody fusion, 12 from physiologically ageing discs and ten from normal control discs. We investigated the histological features and assessed the immunoreactive activity of neurofilament (NF200) and neuropeptides such as substance P (SP) and vasoactive-intestinal peptide (VIP) in the nerve fibres. The distinct histological characteristic of the painful disc was the formation of a zone of vascularised granulation tissue from the nucleus pulposus to the outer part of the annulus fibrosus along the edges of the fissures. SP-, NF- and VIP-immunoreactive nerve fibres in the painful discs were more extensive than in the control discs. Growth of nerves deep into the annulus fibrosus and nucleus pulposus was observed mainly along the zone of granulation tissue in the painful discs. This suggests that the zone of granulation tissue with extensive innervation along the tears in the posterior part of the painful disc may be responsible for causing the pain of discography and of discogenic low back pain

Introduction. Given the predominant functional role which aggrecan has in the intervertebral disc, particularly within the nucleus pulposus, it is necessary to evaluate the quality of aggrecan produced by cells within tissue engineered disc constructs. The aim here was to characterise the nanostructure of aggrecan synthesised by nucleus pulposus cells treated with growth differentiation factor [GDF]-6) seeded in hydrogels in comparison to aggrecan isolated from healthy disc. Methods. Aggrecan was isolated from bovine nucleus pulposus (NP) tissue (n=3 [<18 months old]) and primary bovine NP cells cultured with (+GDF6) or without GDF6 (−GDF6) for 28 days (n=2) in type I collagen hydrogels. Isolated aggrecan monomers were visualised by atomic force microscopy and categorised as either intact (globular domains visible at both the N and C termini) or non-intact. Core protein contour length (L. CP. ) was calculated for intact molecules. The proportion of non-intact/fragmented to intact aggrecan and the molecular area of all monomers was determined. Results. Very few aggrecan molecules were intact (1.3% in NP compared to 4.3% +GDF6 and 0% -GDF6). There was no significant difference in the mean L. CP. between NP (389 ± 37 nm) compared to +GDF6 (379.2 ± 26 nm) or the molecular area between NP (3560 ± 2179 nm. 2. ) and –GDF6 (3586 ± 2071 nm. 2. ). However, the molecular area in both cases was significantly lower than +GDF6 (4774 ± 3715 nm. 2. ) p≤0.0001. Discussion & conclusions. Aggrecan structure can be altered by culture conditions. GDF6 treatment promoted the synthesis of more intact monomers, with greater over all molecular area. Conflicts of interest: None. Funding: Impact Research Scholarship and the Presidents Doctoral Scholarship, provided by the University of Manchester

Background. An improved understanding of intervertebral disc (IVD) structure and function is required for treatment development. Loading induces micro-fractures at the interface between the nucleus pulposus (NP) and the annulus fibrosus (AF), which is hypothesized to induce a cascade of cellular changes leading to degeneration. However, there is limited understanding of the structural relationship between the NP and AF at this interface and particularly response to load. Here, X-ray scattering is utilised to provide hierarchical morphometric information of collagen structure across the IVD, especially the interface region under load. Methodology. IVDs were imaged using the I22 SAXS/WAXS beamline at Diamond Light Source. Peaks associated with the D-banded structure of collagen fibrils were fitted to quantify their azimuthal distribution, as well the magnitude and direction of internal strains under static and applied strain (0–20%). Results. IVD tissue regions exhibited structural “AF-like” and “NP-like” fingerprints. Demonstrating high internal strains on collagen fibres particularly within the NP region of the disc. AF and NP regions showed distinct collagen orientation and internal strains with an apparent lack of bracing structure seen at the interface between the differential mechanical tissues. X-ray scattering under tensile strain provided structural information at high resolution, with clear differences observed between normal and degenerate discs under load. Conclusion. X ray scattering has been utilised to develop an improved understanding of collagen structure across the intervertebral disc which can be utilised to gain an increased understanding of load induced propagation of micro fissures and disc degeneration. Conflict of Interest: No conflict of interest. Funding: BioPro Network, UCL for funding this study through support from the MRC (MR/R025673/1)

Objectives. 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. Methodology. 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. Results. Histological analysis revealed that NCs survive in the biomaterials after four weeks and maintained cell clustering in NPgel, Albugel and dNCM/NPgel. NPgel and Albugel maintained NC cell markers and extracellular matrix. NC containing constructs excreted more GAGs over the four weeks than the acellular controls. Conclusion. 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. Conflicts of interest: No conflicts of interest. Sources of funding: This project has received funding from the European Union's Horizon 2020 research and innovation programme under grant agreement No 825925

Percutaneous nucleotomy is a relatively new technique for treating lumbar disc herniation. There is no agreement as to the volume of disc material to be removed. A long-term study of clinical and radiological data from patients treated by percutaneous nucleotomy was designed to identify the factors associated with favourable and unfavourable outcomes. We studied 42 patients for at least ten years; the mean follow-up was 10.9 years. They were divided into two subgroups to assess the value of preserving the nucleus pulposus in the central area of the disc. The overall success rate for both subgroups was 50%. A decrease in disc height on plain radiography and a decrease in signal intensity on MRI were observed more infrequently in patients in whom the nucleus pulposus in the central area of the disc had been preserved, than in those in whom it had been extensively removed. These adverse radiological findings correlated closely with increased low back pain during the first one to two years after operation and a poorer overall outcome. We conclude that percutaneous nucleotomy is most likely to be successful when the central area of the disc is preserved

Although interlaminar endoscopic lumbar discectomy (IELD) is considered to be less invasive than microscopic lumbar discectomy (MLD) in treatment of lumbar herniated nucleus pulposus, the radiologic change of multifidus muscles by each surgery has rarely been reported. The aim of the present study was to compare the quantitative and qualitative changes of multifidus muscles between two surgical approaches and to analyze the correlation between various parameters of multifidus muscles and long term surgical outcome. 21 patients who received MLD and 18 patients who received IELD in a single tertiary hospital were enrolled and their preoperative, postoperative (≤15 days), and follow-up (≥6 months) MRIs were analyzed. The cross-sectional area (CSA) and fatty degeneration rate (FD) were quantitatively estimated at the level of surgery. The correlations among CSA, FD, body mass index, follow-up visual analogue scale(VAS) and Oswestry Disability Index(ODI) were assessed. Mean intervals of postoperative MRI and follow-up MRI from surgery were 3.0±3.7 days and 14.5±10.7 months, respectively. During the follow-up period, VAS was improved from 7.1±1.3 to 2.1±1.8 in MLD and from 8.2±1.4 to 2.2±1.8 in IELD. In cases of MLD, comparing with preoperative MRI, ipsilateral CSA was significantly increased in postoperative MRI (795.6mm. 2. vs. 906.5mm. 2. , p<0.01), but it was not significantly different in follow-up MRI (795.6mm. 2. vs. 814.4mm. 2. , p=1.00). However, in case of IELD, the ipsilateral CSAs in preoperative, postoperative, and follow-up periods were 892.0 mm. 2. , 909.3 mm. 2. , and 900.3 mm. 2. , respectively. These changes were not significant over time (p=0.691). The ipsilateral FDs were not significantly changed between preoperative and follow-up periods in both MLD (21.4% vs. 20.9%, p=0.81) and IELD groups (23.5% vs. 21.8%, p=0.19). The increment of ipsilateral CSA had significant correlations with follow-up ODI (r=−0.368, p=0.02). Comparing with IELD, MLD induced more surgical trauma on multifidus muscle in postoperative period, but the muscular damage was recovered in follow-up period. IELD can minimize surgical trauma on multifidus muscle showing similar pain relief as MLD. Favorable surgical outcome in follow-up period may be related to increment of multifidus muscle volume. Figure 1. (A-C) The multifidus muscles in preoperative, postoperative, and follow-up periods, respectively, in patient with MLD. Comparing with preoperative period, the CSA of right multifidus muscle (ipsilateral side) was increased in postoperative period, but recovered in follow-up period. (D-F) The multifidus muscle in preoperative, postoperative, and follow-up periods, respectively, in patient with IELD. The CSA of left multifidus muscles (ipsilateral side) was not significantly changed over time. Comparing preoperative MRIs with follow-up MRIs, the FDs of multifidus muscles were not significantly changed regardless of surgical technique. Figure 2. The CSA was measured by marking region of interest (ROI) and FD was measured by calculating the rate of pixels beyond the threshold in ROI. All measurements were performed using ImageJ software (version 1.52a, National Institutes of Health, Bethesda, Maryland, USA). For any figures or tables, please contact the authors directly

Study purpose and background. Novel regenerative therapies have the potential to restore function and relieve pain in patients with low back pain (LBP) caused by intervertebral disc (IVD) degeneration. We have previously shown that stimulation of adipose-derived stem cells (ASCs) with growth differentiation factor-6 (GDF6) promotes differentiation into nucleus pulposus (NP) cells of the IVD, which have potential for IVD regeneration. We have also shown that GDF6 stimulation activates the Smad1/5/8 and ERK1/2 signalling cascades. The aim of this study was to progress our understanding of the immediate/early response mechanisms in ASCs (N=3) which may direct GDF6-induced differentiation. Methods and results. RNAseq was used to perform transcriptome-wide analysis across a 12-hour time course, post-stimulation. Gene ontology analysis revealed greater transcription factor and biological processes activity at 2hrs than at the 6hr and 12hr time points, where molecular and cellular activities appeared to stabilise. Interestingly, a number of lineage determining genes were identified as differentially expressed and work is ongoing to investigate whether the early response genes are maintained throughout differentiation, or whether they are responsible for early NP lineage commitment. Conclusion. This study is the first transcriptome-wide analysis on GDF6-mediated stimulation of ASCs, elucidating important early response mechanisms involved in directing appropriate differentiation. Identification of additional key markers and signalling pathways of differentiation will allow improved selection of ASCs for IVD regeneration. ‘No conflicts of interest’. Funding sources: NIHR Manchester Biomedical Research Centre and The RoseTrees Trust

Introduction. The intervertebral disc (IVD) is a highly hydrated and hyperosmotic tissue, water and salt content fluctuate daily due to mechanical loading. Resident IVD cells must adapt to this ever-changing osmotic environment, to maintain normal behaviour. However, during IVD degeneration the disc becomes permanently dehydrated and cells can no longer perform their correct function. Here, we investigated how human nucleus pulposus (NP) cells respond to altered osmolality with regards to cell size and the rate of water permeability, along with the potential involvement of aquaporins (AQPs) and transient receptor potential vanilloid (TRPV) membrane channels. Methods. Water permeability of NP cells exposed to altered osmolality (225–525mOsm/kg) in the presence or absence of AQP and TRPV channel inhibitors was investigated with the cell-permeable calcein-AM fluorescent dye, and cell size determined using microscopy and flow cytometry. Results. Human NP cells modulate their size and water permeability in response to altered osmolality. Inhibiting channel proteins, specifically AQP4, modified NP cell responses to altered osmolality. Conclusion. IVD cells must regulate their size in order to survive and function within an osmotically challenging environment. Here, we demonstrated that NP cells alter their size and permeability in response to altered osmolality which enables them to adapt to their environment. Furthermore these processes were shown to be dependent at least in part by AQP4 expression, which we have previously shown to be decreased during disc degeneration. This potentially highlights novel ways to restore NP cell and overall IVD function by modulating AQPs in the disc. No conflicts of interest. Funded by BMRC, Sheffield Hallam University

Background. Degeneration of the intervertebral disc (IVD) is a leading cause of lower back pain, and a significant clinical problem. Inflammation mediated by IL-1β and TNF-α drives IVD degeneration through promoting a phenotypic switch in the resident nucleus pulposus (NP) cells towards a more catabolic state, resulting in extracellular matrix degradation. Bone marrow mesenchymal stem cells (MSCs) produce bioactive factors that modulate local tissue microenvironments and their anti-inflammatory potential has been shown in numerous disease models. Thus MSCs offer a potential therapy for IVD degeneration. In a clinical setting, adipose-derived stem cells (ASCs) might represent an alternative and perhaps more appealing cell source. However, their anti-inflammatory properties remain poorly understood. Methods. Here we assess the anti-inflammatory properties of donor-matched human ASCs and MSCs using qPCR and western blotting. Results. We demonstrate that stimulating ASCs or MSCs with IL-1β and/or TNF-α elicits a strong anti-inflammatory response with increased expression of IL-1 receptor antagonist (IL-1Ra), cyclooxygenase-2 (COX-2) and the tissue protective protein tumour-necrosis factor stimulated gene-6 (TSG-6). ASCs produced significantly higher levels of IL-1Ra and TSG-6 than their matched MSCs at both gene and protein levels, indicating that ASCs are potentially a more potent anti-inflammatory cell type. This anti-inflammatory response was also observed upon co-culture with degenerate NP cells without exogenous cytokine. Signalling analyses suggested this difference between cell types might be mediated through differences in the activation of inflammation-associated transcription factors. Conclusion. These data indicate that the anti-inflammatory properties of ASCs may be useful in developing future therapies for IVD degeneration. No conflicts of interest. Sources of funding: EPSRC-MRC Centre for Doctoral Training in Regenerative Medicine (EP/L014904/1)

Introduction. Intervertebral disc degeneration (IVDD) associated with low back pain is a major contributor to global disability. Current treatments are poorly efficient in the long-term resulting in medical complications. Therefore, minimally invasive injectable therapies are required to repopulate damaged tissues and aid regeneration. Among injectable biomaterials, self-assembling peptide hydrogels (SAPHs) represent potential candidates as 3D cell carriers. Moreover, the advent of graphene-related materials has opened the route for the fabrication of graphene-containing hydrogel nanocomposites to direct cellular fate. Here, we incorporated graphene oxide (GO) within a SAPH to develop a biocompatible and injectable hydrogel to be used as cell carrier to treat IVDD. Methods and results. Hydrogel morphology and mechanical properties have been investigated showing high mechanical properties (G'=12kPa) comparable with human native nucleus pulposus (NP) tissue (G'=10kPa), along with ease of handling and injectability in dry and body fluid conditions. Hydrogel nanocomposites resulted biocompatible for the encapsulation of bovine NP cells, showing higher viability (>80%) and metabolic activity in 3D cell culture over 7 days, compared to GO-free hydrogels. Moreover, GO has demonstrated to bind TGF-β3 biomolecules with high efficiency, suggesting the use of GO as local reservoir of growth factors within the injected hydrogel to promote extracellular matrix deposition and tissue repair. Conclusions. Our results show that incorporation of GO within the SAPH improves cell viability and metabolic activity. Furthermore, its tissue-mimicking mechanical properties and chemical tunability make it a promising candidate as injectable carrier of NP cells for the treatment of IVDD. Part of this work has been published (DOI: 10.1016/j.actbio.2019.05.004). Conflicts of interests: No conflicts of interest. Sources of funding: The authors thank the EPSRC & MRC CDT in Regenerative Medicine for its financial support (EP/L014904/1)

Aims

This study aimed, through bioinformatics analysis and in vitro experiment validation, to identify the key extracellular proteins of intervertebral disc degeneration (IDD).

Introduction. Primary cilia are singular structures containing a microtubule-based axoneme which are believed to not only be mechanosensitive but also to co-ordinate many cell functions via signalling pathways including Hedgehog and Wnt. Primary cilia have previously been described on cells of mouse intervertebral discs (IVDs), but not in bovine or human IVDs. Our aim was to examine primary cilia in these species. Methods. Nucleus pulposus cells were obtained from cows with no overt disc degeneration and patients following spine surgery (for herniations and/or degenerative disc disease) and cultured until confluent before maintaining with or without serum for 24h. Primary cilia were visualised with antibodies to the axoneme (acetylated α-tubulin and Arl13b) and/or the basal body (pericentrin) using fluorescent secondary antibodies and ≥200 cells per sample were counted. Results. Primary cilia were detected in the majority of disc cells (81.2±4.1% and 54.8±28.7% with and without serum depletion, respectively, in bovine and 78.9±0.3% and 89.8±7.4% in human cells). Some cilia demonstrated abnormalities, such as bulbous tips or breaks in the axonome. Conclusion. This is the first report of primary cilia being present on human and bovine IVD cells. There remain many other aspects to be investigated, for example, their length has been shown to alter in osteoarthritic chondrocytes. If this, or the incidence of abnormalities, differs in cells from normal and abnormal discs, it could suggest new pathways of disc degeneration, as these organelles are key to so many cell functions. Conflicts of interest: None. Supported by the Orthopaedic Institute Ltd

Introduction. During development the central disc contains large, vacuolated notochordal (NC) cells which in humans are replaced by mature nucleus pulposus (NP) cells during aging, but are maintained in certain breeds of dogs. During degeneration the disc becomes less hydrated which affects its normal function. Aquaporins (AQP) are a family of 13 transmembrane channel proteins that allow passage of water and are responsible for maintaining water homeostasis. AQP1, 2, 3 and 5 have been identified in the intervertebral disc (IVD). Here, expression of AQPs in human and canine IVDs to determine expression in NC v/s NP cells and whether expression changes during degeneration. Methods. Gene expression of all 13 AQPs, were investigated in 102 human NP samples using RT-qPCR. AQPs which were expressed at gene level were further investigated by Immunohistochemistry in human and canine IVD samples. Results. At gene level, AQP0, 1, 2, 3, 4, 5, 6, 7 and 9 were expressed in both non-degenerate and degenerate tissue. For the first time, protein expression of AQP0, 4, 6, 7 and 9 was identified in human IVD tissue, AQP 1, 4 and 5 protein expression decreased during degeneration whilst AQP 7 was increased. AQPs were also expressed in canine IVD tissue, particularly within NC cell populations. Conclusion. Hydration of the IVD is vital for its correct biomechanical function and water loss is associated with degeneration. The presence of many AQP isoforms within NC and NP cells may suggest multiple roles related to the development, survival and adaptation of native cells, and physiology of the healthy IVD. No conflicts of interest. Funded by BMRC, Sheffield Hallam University

Background. Intervertebral disc degeneration is implicated as a major cause of chronic lower back pain. Current therapies for lower back pain are aimed purely at relieving the symptoms rather than targeting the underlying aberrant cell biology. As such focus has shifted to development of cell based alternatives. Notochordal cells are progenitors to the adult nucleus pulposus that display therapeutic potential. However, notochordal cell phenotype and suitable culture conditions for research or therapeutic application are poorly described. This study aims to develop a suitable culture system to allow comprehensive study of the notochordal phenotype. Methods & Results. Porcine notochordal cells were isolated from 6 week post natal discs using dissection and enzymatic digestion and cultured in vitro under different conditions: (1)DMEM vs αMEM (2)laminin-521, fibronectin, gelatin and untreated tissue culture plastic (3)2% 02 vs normoxia (4)αMEM (300 mOsm/L) vs αMEM (400 mOsm/L). Notochordal cells were cultured in alginate beads as a control. Adherence, cell viability, morphology and expression of known notochordal markers (CD24, KRT8, KRT18, KRT19 and T) were assessed throughout the culture period. Use of αMEM media and laminin-521 coated surfaces displayed the greatest cell adherence, viability and retention of notochordal cell morphology and gene expression, which was further enhanced through culture in hypoxia and hyperosmolar media mimicking the intervertebral disc niche. Conclusions. Assessment of the therapeutic potential of notochordal cells is potentially valuable to development of a cell based therapy for chronic lower back pain. Our model has provided a system in which notochordal cells can be studied extensively. Conflicts of Interest: None. Funding obtained from the Henry Smith Charity, London

Introduction. Current strategies to treat back pain address the symptoms but not the underlying cause. Here we are investigating a novel hydrogel material (NPgel) which can promote MSC differentiation to Nucleus pulposus cells. Current in vitro studies have only explored conditions that mimic the native disc microenvironment. Here, we aim to determine the stem cells regenerative capacity under conditions that mimic the degenerate environment seen during disc degeneration. Methods. hMSCs were encapsulated in NPgel and cultured for 4 weeks under hypoxia (5%) with ± calcium (2.5mM and 5.0mM CaCl. 2. ), IL-1β and TNFα either individually or in combination to mimic the degenerate microenvironment. Cell viability was assessed by Alamar blue assay. Histological and immunohistochemical analysis investigated altered matrix and matrix degrading enzyme expression. Results. Viability of hMSCs was maintained under all culture conditions. Matrix deposition of glycosaminoglycans were observed under all conditions, MMP13 expression was upregulated by calcium but not by pro-inflammatory cytokines IL-1β and TNFα. Conclusions. We are developing an in vitro modelling system which can be used to test novel therapies within a degenerate microenvironment. Interestingly, our preliminary findings suggest calcium is a major contributor to regulating MMP13 in this model system. Investigating the degenerate niche will identify targets for inhibition to provide the correct niche to promote regeneration of the IVD. No conflict of interest. Funding: BMRC, MERI Sheffield Hallam University, for joint funding the Daphne Jackson Trust fellowship