Aims. This study aimed, through bioinformatics analysis and in vitro experiment validation, to identify the key extracellular proteins of intervertebral disc degeneration (IDD). Methods. The gene expression profile of GSE23130 was downloaded from the Gene Expression Omnibus (GEO) database. Extracellular protein-differentially expressed genes (EP-DEGs) were screened by protein annotation databases, and we used Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) to analyze the functions and pathways of EP-DEGs. STRING and Cytoscape were used to construct protein-protein interaction (PPI) networks and identify hub EP-DEGs. NetworkAnalyst was used to analyze transcription factors (TFs) and microRNAs (miRNAs) that regulate hub EP-DEGs. A search of the Drug Signatures Database (DSigDB) for hub EP-DEGs revealed multiple drug molecules and drug-target interactions. Results. A total of 56 EP-DEGs were identified in the differential expression analysis. EP-DEGs were enriched in the extracellular structure organization, ageing, collagen-activated signalling pathway, PI3K-Akt signalling pathway, and AGE-RAGE signalling pathway. PPI network analysis showed that the top ten hub EP-DEGs are closely related to IDD. Correlation analysis also demonstrated a significant correlation between the ten hub EP-DEGs (p＜0.05), which were selected to construct TF–gene interaction and TF–miRNA coregulatory networks. In addition, ten candidate drugs were screened for the treatment of IDD. Conclusion. The findings clarify the roles of extracellular proteins in IDD and highlight their potential as promising novel therapeutic targets. Cite this article: Bone Joint Res 2023;12(9):522–535

Aims. Interleukin (IL)-1β is one of the major pathogenic regulators during the pathological development of intervertebral disc degeneration (IDD). However, effective treatment options for IDD are limited. Suramin is used to treat African sleeping sickness. This study aimed to investigate the pharmacological effects of suramin on mitigating IDD and to characterize the underlying mechanism. Methods. Porcine nucleus pulposus (NP) cells were treated with vehicle, 10 ng/ml IL-1β, 10 μM suramin, or 10 μM suramin plus IL-1β. The expression levels of catabolic and anabolic proteins, proinflammatory cytokines, mitogen-activated protein kinase (MAPK), and nuclear factor (NF)-κB-related signalling molecules were assessed by Western blotting, quantitative real-time polymerase chain reaction (qRT-PCR), and immunofluorescence analysis. Flow cytometry was applied to detect apoptotic cells. The ex vivo effects of suramin were examined using IDD organ culture and differentiation was analyzed by Safranin O-Fast green and Alcian blue staining. Results. Suramin inhibited IL-1β-induced apoptosis, downregulated matrix metalloproteinase (MMP)-3, MMP-13, a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)-4, and ADAMTS-5, and upregulated collagen 2A (Col2a1) and aggrecan in IL-1β-treated NP cells. IL-1β-induced inflammation, assessed by IL-1β, IL-8, and tumour necrosis factor α (TNF-α) upregulation, was alleviated by suramin treatment. Suramin suppressed IL-1β-mediated proteoglycan depletion and the induction of MMP-3, ADAMTS-4, and pro-inflammatory gene expression in ex vivo experiments. Conclusion. Suramin administration represents a novel and effectively therapeutic approach, which could potentially alleviate IDD by reducing extracellular matrix (ECM) deposition and inhibiting apoptosis and inflammatory responses in the NP cells. Cite this article: Bone Joint Res 2021;10(8):498–513

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

Intervertebral disc degeneration is a common cause of low-back pain, the musculoskeletal disorder with the largest impact world-wide. The complex disease is however not yet well understood, and no treatment is available. This is somewhat in contrast with osteoarthritis, a subject of more extensive research. Intervertebral disc degeneration may though be a type of osteoarthritis, as other vertebrates have a diarthrodial joint instead of an intervertebral disc. We describe the parallel in view of the anatomy, composition and degeneration of the intervertebral disc and articular joint. Not only different embryonic origin and anatomy suggest significant differences between the intervertebral disc and the synovial joint, but their biomechanical properties also partly differ, as articulation is one of the key properties of a synovial joint and does not occur in the intervertebral disc. However, both tissues provide flexibility and are able to endure compressive loads, and both cell behavior and extracellular matrix appear much the same, mainly existing of chondrocytes, proteoglycans and collagen type II, suggesting that the environment of the cell is more important to its behavior than embryonic origin. Moreover, great similarities are found in the inflammatory cytokines, which are mainly IL-1β and TNF-α, and matrix-degrading factors (i.e. MMPs and ADAMTSs) involved in the cascade of degeneration, resulting in overlapping clinical and radiological features such as loss of joint space, subchondral sclerosis, and the formation of osteophytes, causing pain and morning stiffness. Therefore, we state that disc degeneration can result in the osteoarthritic intervertebral disc. This point of view may enhance the synergy between both fields of research, and potentially provide new regenerative strategies for intervertebral disc degeneration

Objectives. Interleukin 18 (IL-18) is a regulatory cytokine that degrades the disc matrix. Bone morphogenetic protein-2 (BMP-2) stimulates synthesis of the disc extracellular matrix. However, the combined effects of BMP-2 and IL-18 on human intervertebral disc degeneration have not previously been reported. The aim of this study was to investigate the effects of the anabolic cytokine BMP-2 and the catabolic cytokine IL-18 on human nucleus pulposus (NP) and annulus fibrosus (AF) cells and, therefore, to identify potential therapeutic and clinical benefits of recombinant human (rh)BMP-2 in intervertebral disc degeneration. Methods. Levels of IL-18 were measured in the blood of patients with intervertebral disc degenerative disease and in control patients. Human NP and AF cells were cultured in a NP cell medium and treated with IL-18 or IL-18 plus BMP-2. mRNA levels of target genes were measured by real-time polymerase chain reaction, and protein levels of aggrecan, type II collagen, SOX6, and matrix metalloproteinase 13 (MMP13) were assessed by western blot analysis. Results. The serum level of patients (IL-18) increased significantly with the grade of IVD degeneration. There was a dramatic alteration in IL-18 level between the advanced degeneration (Grade III to V) group and the normal group (p = 0.008) Furthermore, IL-18 induced upregulation of the catabolic regulator MMP13 and downregulation of the anabolic regulators aggrecan, type II collagen, and SOX6 at 24 hours, contributing to degradation of disc matrix enzymes. However, BMP-2 antagonised the IL-18 induced upregulation of aggrecan, type II collagen, and SOX6, resulting in reversal of IL-18 mediated disc degeneration. Conclusions. BMP-2 is anti-catabolic in human NP and AF cells, and its effects are partially mediated through provocation of the catabolic effect of IL-18. These findings indicate that BMP-2 may be a unique therapeutic option for prevention and reversal of disc degeneration. Cite this article: S. Ye, B. Ju, H. Wang, K-B. Lee. Bone morphogenetic protein-2 provokes interleukin-18-induced human intervertebral disc degeneration. Bone Joint Res 2016;5:412–418. DOI: 10.1302/2046-3758.59.BJR-2016-0032.R1

Using deep learning and image processing technology, a standardized automatic quantitative analysis systerm of lumbar disc degeneration based on T2MRI is proposed to help doctors evaluate the prognosis of intervertebral disc (IVD) degeneration. A semantic segmentation network BianqueNet with self-attention mechanism skip connection module and deep feature extraction module is proposed to achieve high-precision segmentation of intervertebral disc related areas. A quantitative method is proposed to calculate the signal intensity difference (SI) in IVD, average disc height (DH), disc height index (DHI), and disc height-to-diameter ratio (DHR). According to the correlation analysis results of the degeneration characteristic parameters of IVDs, 1051 MRI images from four hospitals were collected to establish the quantitative ranges for these IVD parameters in larger population around China. The average dice coefficients of the proposed segmentation network for vertebral bodies and intervertebral discs are 97.04% and 94.76%, respectively. The designed parameters of intervertebral disc degeneration have a significant negative correlation with the Modified Pfirrmann Grade. This procedure is suitable for different MRI centers and different resolution of lumbar spine T2MRI (ICC=.874~.958). Among them, the standard of intervertebral disc signal intensity degeneration has excellent reliability according to the modified Pfirrmann Grade (macroF1=90.63%~92.02%). we developed a fully automated deep learning-based lumbar spine segmentation network, which demonstrated strong versatility and high reliability to assist residents on IVD degeneration grading by means of IVD degeneration quantitation

Purpose of study and background. Clinical researchers use Pfirrmann classification for grading intervertebral disc degeneration radiologically. Basic researchers have access to morphology and instead use the Thompson score. The aim of this study was to assess the inter-observer reliability of both classifications, along with their correlation. Methods and Results. We obtained T2-weighted MR images of 80 human lumbar intervertebral discs with various stages of degeneration to assess the Pfirrmann-score. Then the discs were dissected midsagittally to obtain the Thompson-score. The observers were typical users of both grading systems: a spine surgeon, radiology resident, orthopaedic resident, and a basic scientist, all experts on intervertebral disc degeneration. Cohen's kappa (CK) was used to determine inter-observer reliability, and intra-class correlation (ICC) as a measure for the variation between the outcomes. For the Thompson score, the average CK was 0.366 and ICC score 0.873. The average inter-observer reliability for the Pfirrmann score was 0.214 (CK) and 0.790 (ICC). Comparing the grading systems, the intra-observer agreement was 0.240 (CK) and 0.685 (ICC). Conclusion. With substantial variation between observers, the inter-observer agreements for the Pfirrmann and Thompson grading systems were moderate. This may explain the poor relationship between radiological and clinical observations in patients and raises questions about the validity of the Pfirrmann score. The mediocre intra-observer agreement between the Pfirrmann and Thompson score shows that there is no clear definition of intervertebral disc degeneration. The field is in need for a new, objective and quantitative classification system to better define and evaluate disc degeneration. There are no conflicts of interest. Funded in part by Annafonds Netherlands and Dutch Spine Society

Intervertebral disc degeneration (IDD) is a major cause of low back pain, which affects 80% of the adult population at least once in their life. The pathophysiological conditions underlying IDD are still poorly understood. Genetic makeup, aging, smoking, physical inactivity and mechanical overloading, especially due to obesity, are among the strongest risk factors involved. Moreover, IDD is often associated with chronic inflammation within disc tissues, which increases matrix breakdown, glycosaminoglycan (GAG) loss and cell death. This micro-inflammatory environment is typical of several metabolic disorders, including diabetes mellitus (DM). As the etiopathogenesis of IDD in diabetic subjects remains scarcely understood, we hypothesised that this may be driven by a DM-induced inflammation leading to a combination of reduced GAG levels, decreased proteoglycan synthesis and increased matrix breakdown within the disc. The objective of the study was to investigate the pathogenesis of IDD in a murine model of type 1 DM (T1DM), namely non-obese diabetic (NOD) mouse. Total disc glycosaminoglycan (GAG) content, proteoglycan synthesis, aggrecan fragmentation mediated by matrix metalloproteinases (MMPs) and a Disintegrin and Metalloproteinase with Thrombospondin motifs (ADAMTS), glucose transporter (mGLUT1) gene expression and apoptosis (TUNEL assay) were assessed in NOD mice and wild-type euglycemic control mice. Spinal structural and molecular changes were analysed by micro-computed tomography (mCT), histological staining (Safranin-O and fast green) and quantitative immunofluorescence (anti-ADAMTS-4 and 5 antibodies). Statistical analysis was conducted considering the average of 35 samples ± standard error for each measurement, with 95% confidence intervals calculated to determine statistical significance (p-value < 0.05). IVDs of NOD mice showed increased disc apoptosis (p < 0.05) and higher aggrecan fragmentation mediated by ADAMTS (p < 0.05). However, ADAMTS-4 and −5 did not appear to be involved in this process. The total GAG content normalized to DNA and PG synthesis showed no statistically significant alterations, as well as Safranin O staining. Although not significantly, NOD mice showed reduced glucose uptake. In addition, the vertebral structure of NOD mice at mCT seemed not to be altered. These data demonstrate that DM may contribute to IDD by increasing aggrecan degradation and promoting cell apoptosis, which may represent early indicators of the involvement of DM in the pathogenesis of IDD

Current therapies for intervertebral disc degeneration are aimed at treating the pathologic and disabling conditions arising from discopathy rather than directly treating the underlying problem of disc degeneration. Our group is exploring the potential of cell therapy to repopulate the disc and stop the progressive loss of proteoglycans. Stem cells appear to be excellent candidates for this purpose, based on their ability to differentiate along multiple connective tissue lineages. The purpose of this study is to investigate the interaction between stem cells and nucleus polposus cells to test the feasibility of stem cell therapy for the treatment of disc degeneration. Human nucleus polposus cells (NPCs) were isolated from patients undergoing disc surgery and were co-cultured for 2 weeks with muscle-derived stem cells (MdSCs) from 3-week-old mdx mice in monolayer culture system at different ratio with or without added TGF-β1. Each well contained an admixture of cells with NPC-to-SC ratios of 0:100, 25:75, 50:50, 75:25, and 100:0. Proteoglycan synthesis and DNA content were measured. Co-culturing of NPCs with MdSCs in the monolayer culture system resulted in vigorous increases in proteoglycans synthesis as compared with NPCs alone and MdSCs alone both with and without TGF-β1. The increases were on the 200% for an NPC-to-MDSC ratio of 75:25. Addition of TGF-β1 to the NPC and MDSC co-cultures resulted in further increases up to 400%. DNA content also increased with co-culture. The data from this study show that there is a synergistic effect between stem cells and nNPC resulting in upregulated proteoglycan synthesis in vitro. The observed benefits of co-culture might be due either to stem cell plasticity, the stem cells trans-differentiation towards chondrocyte-like cells, or the stimulation of NPC by agents synthesised by stem cells or other mechanisms. Elucidation of the precise mechanisms of action may permit development of strategies to optimise the synergistic effects in vivo. These results support the feasibility of developing a stem cell therapy approach to treat and prevent intervertebral disc degeneration

Intervertebral disc (IVD) degeneration is responsible for severe clinical symptoms including chronic back pain. Galectins are a family of carbohydrate-binding proteins, some of which can induce functional disease markers in IVD cells and other musculoskeletal diseases. Galectins −4 and −8 were shown to trigger disease-promoting activity in chondrocytes but their effects on IVD cells have not been investigated yet. This study elucidates the role of galectin-4 and −8 in IVD degeneration. Immunohistochemical evidence for the presence of galectin-4 and −8 in the IVD was comparatively provided in specimens of 36 patients with spondylochondrosis, spondylolisthesis, or spinal deformity. Confocal microscopy revealed co-localization of galectin-4 and −8 in chondrocyte clusters of degenerated cartilage. The immunohistochemical presence of galectin-4 correlated with histopathological and clinical degeneration scores of patients, whereas galectin-8 did not show significant correlations. The specimens were separated into annulus fibrosus (AF), nucleus pulposus (NP) and endplate, which was confirmed histologically. Separate cell cultures of AF and NP (n=20) were established and characterized using cell type-specific markers. Potential binding sites for galectins including sialylated N-glycans and LacdiNAc structures were determined in AF and NP cells using LC/ESI-MS-MS. To assess galectin functions, cell cultures were treated with recombinant galectin-4 or −8, in comparison to IL-1β, and analyzed using RT-qPCR and In-cell Western blot. In vitro, both galectins triggered the induction of functional disease markers (CXCL8 and MMP3) on mRNA level and activated the nuclear factor-kB pathway. NP cells were significantly more responsive to galectin-8 and Il-1β than AF cells. Phosphorylation of p-65 was time-dependently induced by both galectins in both cell types to a comparable extent. Taken together, this study provides evidence for a functional role of glycobiological processes in IVD degeneration and highlights galectin-4 and −8 as regulators of pro-inflammatory and degrative processes in AF and NP cells

Intervertebral disc (IVD) degeneration is the most frequent cause of Low Back Pain (LBP) affecting nearly 80% of the population [1]. Current treatments fail to restore a functional IVD or to provide a long-term solution, so, there is an urgent need for novel therapeutic strategies. We have defined the IVD extracellular matrix (ECM) profile, showing that the pro-regenerative molecules Collagen type XII and XIV, are uniquely expressed during fetal stages [2]. Now we propose the first fetal injectable biomaterial to regenerate the IVD. Fetal decellularized IVD scaffolds were recellularized with adult IVD cells and further implanted in vivo to evaluate their anti-angiogenic potential. Young decellularized IVD scaffolds were used as controls. Finally, a large scale protocol to produce a stable, biocompatible and easily injectable fetal IVD-based hydrogel was developed. Fetal scaffolds were more effective at promoting Aggrecan and Collagen type II expression by IVD cells. In a Chorioallantoid membrane assay, only fetal matrices showed an anti-angiogenic potential. The same was observed in vivo when the angiogenesis was induced by human NP cells. In this context, human NP cells were more effective in GAG synthesis within a fetal microenvironment. Vaccum-assisted perfusion decellularized IVDs were obtained, with high DNA removal and sGAG retention. Hydrogel pre-solution passed through 21-30G needles. IVD cells seeded on the hydrogels initially decreased metabolic activity, but increased up to 70% at day 7, while LDH assay revealed cytotoxicity always below 30%. This study will open new avenues for the establishment of a disruptive treatment for IVD degeneration with a positive impact on the angiogenesis associated with LBP, and on the improvement of patients’ quality of life. Acknowledgements: Financial support was obtained from EUROSPINE, ON Foundation and FCT (Fundação para a Ciência e a Tecnologia)

It has been proposed that intervertebral disc degeneration might be caused by low-grade infection. The purpose of the present study was to assess the incidence of herpes viruses in intervertebral disc specimens from patients with lumbar disc herniation. A polymerase chain reaction based assay was applied to screen for the DNA of eight different herpes viruses in 16 patients and two controls. DNA of at least one herpes virus was detected in 13 specimens (81.25%). Herpes Simplex Virus type-1 (HSV-1) was the most frequently detected virus (56.25%), followed by Cytomegalovirus (CMV) (37.5%). In two patients, co-infection by both HSV-1 and CMV was detected. All samples, including the control specimens, were negative for Herpes Simplex Virus type-2, Varicella Zoster Virus, Epstein Barr Virus, Human Herpes Viruses 6, 7 and 8. The absence of an acute infection was confirmed both at the serological and mRNA level. To our knowledge this is the first unequivocal evidence of the presence of herpes virus DNA in intervertebral disc specimens of patients with lumbar disc herniation suggesting the potential role of herpes viruses as a contributing factor to the pathogenesis of degenerative disc disease

Intervertebral disc (IVD) degeneration is a pathological process often associated with chronic back pain and considered a leading cause of disability worldwide. 1. During degeneration, progressive structural and biochemical changes occur, leading to blood vessel and nerve ingrowth and promoting discogenic pain. 2. 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 approach. 3. 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 performance. 4. 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. For any figures and tables, please contact the authors directly

Biomechanical overloading initiates intervertebral disc degeneration. We hypothesized that this is due to mechanosensitivity of the cells, which break down the extracellular matrix. Previously, we found that overloading in a loaded disc culture system causes upregulation of remodeling- and inflammatory gene expressions. Fourier Transform Infrared Spectroscopy is a novel technique to identify, visualize and quantify ECM. In this research, we first identified novel spectroscopic markers for disc degeneration, and then applied these markers to investigate the first steps into disc degeneration by overloading. In dataset 1, 18 discs of 9 goats were injected with chondroitinase ABC (degenerated) or not (control), and obducted 3 months after injection. This was used to find new spectroscopic markers for degeneration. In dataset 2, 42 goat discs were loaded with a physiological loading regime (50–150N) or overloading (50–400N) in a loaded disc culture system. In 18 of these discs, the cell activity was diminished in advance by freeze-thaw cycles and culturing on saline alone (non-vital group)). 24 additional discs were cultured in culture medium immediately post-mortem (vital group). Thereby, we are able to control whether the effect of the overloading is due to cell activity. The discs were fixed in formaldehyde, and 4 μm mid-sagittal were mounted to steel reflectance slides. Infrared spectroscopic mosaic images (23 × 57 images) were collected in transflectance mode at a spectral region of 1025–1150 cm. −1. Data was pre-processed by second derivative transformation and MCR-MALS with two factors. The two factors were transferable between datasets, confirming the reliability. The first factor represents proteoglycans, as confirmed by Saffrin-O staining. In dataset 1, the degenerated group had less proteoglycan factor overall, especially in the nucleus (p<0.05). The second factor was found to have a lower entropy (p<0.01), showing a disorganization in the matrix. In dataset 2, no significant reduction in proteoglycan was found due to overloading in any group. However, the entropy was lower in the overloaded vital group (p<0.05), but not in the overloaded non-vital group (p>0.5). Therefore, we conclude that infrared spectroscopy is a promising tool to investigate early disc degeneration. Overloading can cause changes in the extracellular matrix, but only due to cell activity. Entropy is an early marker for early disc degeneration, implying that cutting of the extracellular matrix by cell activity is the first step into intervertebral disc degeneration

Introduction: Osteoporotic fractures affect certain bones more than others, suggesting that systemic bone loss is not the only underlying cause. We have shown that age-related intervertebral disc degeneration causes the anterior vertebral body (VB) to be stress-shielded in erect postures, and yet severely loaded when the spine is flexed (1). We hypothesise that this unequal loading causes exaggerated bone loss from the anterior vertebral body, making it vulnerable to fracture when the spine is heavily loaded in a forward stooping (flexed) posture. Materials and Methods: Regional volumetric bone mineral density (BMD) was measured in 35 thoracolumbar motion segments (aged 64–92 yrs) using dual-energy x-ray absorptiometry. The distribution of compressive stress was measured along the mid-sagittal diameter of each intervertebral disc using a miniature pressure transducer. Stresses were integrated over area to give the compressive force acting on the anterior and posterior halves of the VB (1). Motion segment compressive strength was measured in moderate flexion. Results: BMD of the anterior half of the VB was 26% (STD 13%) lower than that of the posterior half (p< 0.0001), was correlated with % load on the anterior VB in erect posture (r. 2. =0.48, p< 0.0001), and was a better predictor of motion segment compressive strength (in flexion) than was BMD of the whole vertebral body (r. 2. = 0.79 compared to r. 2. = 0.59). Conclusion: These results clearly support our hypothesis. It appears that intervertebral disc degeneration leads to exaggerated bone loss from the anterior VB, leaving it more vulnerable to fracture when the spine is flexed. Future work aims to confirm this important result on a larger number of specimens, and to compare the relative importance of disc degeneration and overall bone loss on vertebral compressive strength. Pollintine P et al (2001). SBPR Annual Meeting, Bristol. Backcare Research Award 2002

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

Background. The association between lumbar intervertebral disc degeneration (LDD) and low back pain (LBP) is modest. We have recently shown that genetic propensity to pain is an effect modifier of the LDD-LBP relationship when LDD is defined as a summary score of LDD (LSUM), suggesting the association may be driven by individuals with the greatest genetic predisposition to pain. This study examined the association between individual spine magnetic resonance imaging (MRI)-determined LDD features and LBP in subgroups defined by genetic predisposition to pain. Method. We developed a polygenic risk score (PRS) for “genetic propensity to pain” defined as the number of non-back pain locations (head, face, neck/shoulder, stomach/abdomen, hip, and knee) with duration ≥3 months in 377,538 UK Biobank participants of European ancestry. This PRS was used to stratify TwinsUK MRI samples (n=645) into four strata of genetic propensity to pain. We examined the association between LBP and MRI features of lumbar disc height, disc signal intensity, disc bulge, and osteophytes with adjustments for age, sex, PRS strata, interaction terms for each MRI feature x PRS strata, and twin status. Results. We found significant effect modification of the LDD-LBP relationship by genetic propensity to pain for the lumbar MRI features of disc height (p=0.03 for the interaction term with highest quartile of genetically-predicted propensity to pain) and disc signal intensity (p=0.001), but not for disc bulge and osteophytes. Conclusion. Genetic propensity to pain modifies the association between individual LDD features and LBP and should be considered in LBP clinical studies. Conflicts of interest. No conflicts of interest. Sources of funding. No funding obtained. Acknowledgement. UKBB data were obtained under the project #18219. This paper is submitted to the Spine journal and is under review

Purpose of study and background. Mechanical overloading initiates intervertebral disc degeneration, presumably because cells break down the extracellular matrix (ECM). We used Fourier Transform Infrared Spectroscopy (FTIR) imaging to identify, visualize and quantify the ECM and aimed to identify spectroscopic markers for early disc degeneration. Methods and Results. In seven goats, one disc was injected with chondroitinase ABC (mild degeneration) and after three months compared to control. Ex vivo, 50 caprine discs received physiological loading (50–150N) or overloading (50–400N) in a loaded disc culture system. To determine whether ECM degeneration is due to cell activity, half of the discs was subjected to freeze-thaw cycles. Spectroscopic images were collected at 1000–1300 cm. −1. and analyzed using multivariate curve resolution analysis. In vivo, less proteoglycan was found in the degenerated group (p<0.05), especially in the nucleus. Collagen content was increased in the nucleus and anterior annulus, and had higher entropy (p<0.01), indicating matrix disorganization. In the ex vivo experiment, the proteoglycan/collagen ratio was decreased (p<0.05) in the vital group and there was an increase in collagen entropy (p<0.05). A significant interaction between loading and vitality was found in the amount of collagen (p<0.05), but not in the entropy. Conclusion. Three weeks of mild overloading causes measurable changes in the extracellular matrix. Increased collagen entropy indicates that remodeling of collagen is a first step into disc degeneration. We could not confirm, however, that increase in entropy was due to cell activity. FTIR imaging allows more detailed investigation of early disc degeneration than traditional measures. There are no conflicts of interest. Partially funded by Dutch Arthritis Funds, personal grant KSE

Back pain is a leading cause of disability worldwide and it is primarily considered to be triggered by intervertebral disc (IVD) degeneration (IVDD). Current treatments may improve pain and mobility, but carry high costs and fail to address IVD repair or regeneration. As no effective therapeutic approach has been proposed to restore inflamed and degenerated IVDs, there is the urgent need to clarify the key pathomechanism of IVDD, the involvement of inflammation, particularly complement activation in matrix catabolism, and how to target them towards tissue repair/regeneration. Mesenchymal stem cell (MSC)-based therapies have become the focus of several regenerative IVD studies. Although patients in clinical trials reported less pain after cell therapy, the long-term success of cell engraftment is unclear due to the hostile IVD environment. The mechanism-of-action of MSCs is mostly dependent on the secreted soluble factors. Moreover, priming of MSC with interleukin (IL)-1β modulates the secretome content, improving its anti-inflammatory and regenerative effect on IVDD organ culture models. MSC-derived extracellular vesicles (EVs) have also been shown to modulate human IVD cells towards a healthy IVD phenotype in vitro. However, the mechanisms involved in the effect of secretome and EVs, particularly with regard to immunomodulation and matrix metabolism, are not fully understood. Our work investigates the effects of secretome and EVs secreted by IL-1β-primed MSCs to impair IVD matrix degradation and/or improve matrix formation in IVDD

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)

Mesenchymal stem cells (MSCs) are exciting candidates for cellular repopulation and repair in intervertebral disc degeneration (IDD). Our purpose is to investigate the interaction between MSCs and nucleus polposus cells (NPCs) and to determine viability of MSC in the intervertebral disc (IVD). Human NPCs and hMSCs were co-cultured in pellet system at different ratios. Proteoglycans were measured and normalized with DNA content. Histological analysis were also performed. Rabbit MSCs from bone marrow were trasduced with LacZ reporter gene and were injected into a rabbit IVD. Rabbits were sacrificed postoperatively at 3, 6, 12 and 24 weeks. Histological analysis was performed. Co-culturing of hNPCs with hMSCs resulted in increases proteoglycans as compared with hNPCs alone. Histological examination of the injected IVDs revealed presence of MSCs without apparent decrease in numbers or diminishment of protein production at 3, 6, 12 and 24 weeks. The data from this study show that there is a synergistic effect between MSCs and NPCs resulting in upregulated proteoglycan synthesis in-vitro. MSC remain viable and continue to express an ex-vivo transduced protein for up to 24 weeks. These results suggest that MSCs can survive in the harsh environment of the IVD and may favourably modify ECM production

Purpose of study and background. Low back pain affects 80% of the population at some point in their lives with 40% of cases attributed to intervertebral disc (IVD) degeneration. A number of potential regenerative approaches are under investigation worldwide, however their translation to clinic is currently hampered by an appropriate model for testing prior to clinical trials. Therefore, a more representative large animal model for IVD degeneration is needed to mimic human degeneration. Here we investigate a caprine IVD degeneration model in a loaded disc culture system which can mimic the native loading environment of the disc. Methods and Results. Goat discs were excised and cultured in a bioreactor under diurnal, simulated-physiological loading (SPL) conditions, following 3 days pre load, IVDs were degenerated enzymatically for 2hrs and subsequently loaded for 10 days under physiological loading. A PBS injected group was used as controls. Disc deformation was continuously monitored and changes in disc height recovery quantified using stretched-exponential fitting. Histological staining was performed on caprine discs to assess extracellular matrix production and immunohistochemistry performed to determine expression of catabolic protein expression. The injection of collagenase and cABC induced mechanical behavior akin to that seen in human degeneration. A decrease in collagens and glycosaminoglycans (GAGs) was seen in enzyme injected discs, which was accompanied by increased cellular expression for degradative enzymes and catabolic cytokines. Conclusion. This model provides a reproducible model of IVD degeneration which mimics human degeneration. This model allows the testing of biomaterials and other potential treatments of IVD degeneration on a scale more representative of the human disc. There are no conflicts of interest. Funded by MRC and Versus Arthritis

Background. 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. Methods and Results. Immunohistochemistry for the senescence marker: p16. INK4A. 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. Conclusions. 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. No conflicts of interest. Sources of funding: Funded by ARUK and MRC

Background. Endplate defect is an MRI trait, found to be associated with intervertebral disc degeneration. There is a lack of understanding regarding the mechanism underlying lumbar disc degeneration (LDD). This large-scale longitudinal population-based study aimed to determine the order of appearance of degenerative change in the vertebral body and intervertebral disc, the influence of endplate degeneration on LBP and whether there is a genetic influence on endplate damage. Methods. Individuals from the TwinsUK spine study having longitudinal T2-weighted lumbar MRI scans at baseline (n=996) and a decade later (n=438) were included. LDD, vertebral endplate defect expressed as a total endplate (TEP) score and Modic change (MC) were assessed using standard techniques. Mixed-effects models were used to determine the association between spine pathology features adjusted for covariates. Endplate defect heritability was estimated using variance component analysis. Results. Significant association between endplate defect, LDD, MRI features of LDD and MC was observed. Endplate defect was independently associated with severe disabling LBP episodes. An association between LDD at baseline and MC at follow-up was shown at upper lumbar levels. TEP score was heritable with estimated additive genetic component A = 55.3% (95% CI 43.0–65.4). Conclusion. Endplate defect, LDD and MC are all independent risk factors for episodes of severe and disabling LBP. Longitudinal analysis showed LDD is followed by MC. Endplate defect has significant heritability. However, whether endplate defect triggers LDD or these pathological changes occur concurrently could not be determined conclusively. Conflicts of interest: none. Sources of Funding: This work was funded by the EU FP7 project Pain_Omics

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

Intervertebral discs (IVDs) degeneration is one of the major causes of back pain. Upon degeneration, the IVDs tissue become inflamed, and this inflammatory microenvironment may cause discogenic pain. Cellular senescence is a state of stable cell cycle arrest in response to a variety of cellular stresses including oxidative stress and adverse load. The accumulation of senescent IVDs cells in the tissue suggest a crucial role in the initiation and development of painful IVD degeneration. Senescent cells secrete an array of cytokines, chemokines, growth factors, and proteases known as the senescence-associated secretory phenotype (SASP). The SASP promote matrix catabolism and inflammation in IVDs thereby accelerating the process of degeneration. In this study, we quantified the level of senescence in degenerate and non-degenerate IVDs and we evaluated the potential of two natural compounds to remove senescent cells and promote overall matrix production of the remaining cells. Human IVDs were obtained from organ donors. Pellet or monolayer cultures were prepared from freshly isolated cells and cultured in the presence or absence of two natural compounds: Curcumin and its metabolite vanillin. Monolayer cultures were analyzed after four days and pellets after 21 days for the effect of senolysis. A cytotoxicity study was performed using Alamar blue assay. Following treatment, RNA was extracted, and gene expression of senescence and inflammatory markers was evaluated by real-time q-PCR using the comparative ΔΔCt method. Also, protein expression of p16, Ki-67 and Caspase-3 were evaluated in fixed pellets or monolayer cultures and total number of cells was counted on consecutive sections using DAPI and Hematoxylin. Proteoglycan content was evaluated using SafraninO staining or DMMB assay to measure sulfated glycosaminoglycan (sGAG) and antibodies were used to stain for collagen type II expression. We observed 40% higher level of senescent cells in degenerate compare to the non-degenerate discs form unrelated individuals and a 10% increase when we compare degenerate compare to the non-degenerate discs of the same individual. Using the optimal effective and safe doses, curcumin and vanillin cleared 15% of the senescent cells in monolayer and up to 80% in pellet cultures. Also, they increased the number of proliferating and apoptotic cells in both monolayer and pellets cultures. The increase in apoptotic cell number and caspase-3/7 activity was specific to degenerate cells. Following treatment, mRNA expression levels of SASP factors were decreased by four to 32-fold compared to the untreated groups. Senescent cell clearance decreased, protein expression of MMP-3 and −13 by 15 and 50% and proinflammatory cytokines levels of IL-1, IL-6 and IL-8 by 42, 63 and 58 %. Overall matrix content was increased following treatment as validated by an increase in proteoglycan content in pellet cultures and surrounding culture media. This work identifies novel senolytic drugs for the treatment of IVD degeneration. Senolytic drugs could provide therapeutic interventions that ultimately, decrease pain and provide a better quality of life of patients living with IVDs degeneration and low back pain

Degenerative disc disease (DDD) is a common cause of lower back pain. Calcification of the intervertebral disc (IVD) has been correlated with DDD, and is especially prevalent in scoliotic discs. The appearance of calcium deposits has been shown to increase with age, and its occurrence has been associated with several other disorders such as hyperparathyroidism, chondrocalcinosis, and arthritis. Trauma, vertebral fusion and infection have also been shown to increase the incidence of IVD calcification. Our data indicate that Ca²⁺ and expression of the extracellular calcium-sensing receptor (CaSR) are significantly increased in mild to severely degenerative human IVDs. In this study, we evaluated the effects of Ca²⁺ and CaSR on the degeneration and calcification of IVDs.

Human donor lumbar spines of Thompson grade 2, 3 and 4 through organ donations within 24 hs after death. IVD cells, NP and AF, were isolated from tissue by sequential digestion with Pronase followed by Collagenase. Cells were expanded for 7 days under standard cell culture conditions. Immunohistochemistry was performed on IVD tissue to validate the grade and expression of CaSR. Free calcium levels were also measured and compared between grades. Immunocytochemistry, Western blotting and RT-qPCR were performed on cultured NP and AF cells to demonstrate expression of CaSR, matrix proteins aggrecan and collagen, catabolic enzymes and calcification markers. IVD cells were cultured in increasing concentrations of Ca²⁺ [1.0-5.0 mM], CaSR allosteric agonist (cincalcet, 1 uM), and IL-1b [5 ng/mL] for 7 days. Ex vivo IVD organ cultures were prepared using PrimeGrowth Disc Isolation System (Wisent Bioproducts, Montreal, Quebec). IVDs were cultured in 1.0, 2.5 mM Ca²⁺ or with cinacalcet for 21 days to determine effects on disc degeneration, calcification and biomechanics. Complex modulus and structural stiffness of disc tissues was determined using the MACH-1 mechanical testing system (Biomomentum, Laval, Quebec).

Ca²⁺ dose-dependently decreased matrix protein synthesis of proteoglycan and Col II in NP and AF cells, similar to treatment with IL-1b. (n = 4). Contrarily to IL-1b, Ca²⁺ and cincalcet did not significantly increase the expression of catabolic enzymes save ADAMTS5. Similar effects were observed in whole organ cultures, as Ca²⁺ and cinacalcet decreased proteoglycan and collagen content. Although both Ca²⁺ and cinacalcet increased the expression of alkaline phosphatase (ALP), only in Ca²⁺-treated IVDs was there evidence of calcium deposits in NP and AF tissues as determined by von Kossa staining. Biomechanical studies on Ca²⁺ and cinacalcet-treated IVDs demonstrated decreases in complex modulus (p<0.01 and p<0.001, respectively; n=5), however, only Ca²⁺-treated IVDs was there significant increases stiffness in NP and AF tissues (p<0.001 and p<0.05, respectively; n=3).

Our results suggest that changes in the local concentrations of calcium and activation of CaSR affects matrix protein synthesis, calcification and IVD biomechanics. Ca²⁺ may be a contributing factor in IVD degeneration and calcification.

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.

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.

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.

Purpose: Intervertebral disc (IVD) degeneration is associated with proteolytic degradation of proteoglycan aggregates present within the extracellular matrix of the disc. Link-N peptide is the N-terminal peptide of link protein, which stabilizes the proteoglycan aggregates. It is generated in vivo by proteolytic degradation during tissue turnover. We have previously shown that this peptide can stimulate the synthesis of proteoglycans and collagens by IVD cells in vitro. However, to date, there have been no reports on the effect of Link-N on the IVD in vivo. The purpose of the present study was to determine the effect of intradiscally administration of Link-N peptide on disc cell survival and extracellular matrix synthesis using a rabbit annular needle puncture model of IVD degeneration. Method: Twelve New Zealand white rabbits (~3.5 kg; 5–6 months old) received an annular puncture with an 18-gauge needle on 2 non-contiguous discs (L2–L3 and L4–L5). The disc (L3–L4) between the punctured discs and that above (L5–L6) was left intact as internal controls. Two weeks after the initial puncture, the anterior surfaces of the previously punctured discs (L2–L3 and L4–L5) were injected with either saline (10 μl/disc) or Link-N (100 μg in 10μl saline/disc) into the center of the NP. Disc height was radiographically monitored biweekly. After 12 weeks post-injection, all the rabbits were euthanized and the IVDs from both experimental groups were removed from each lumbar spine for biochemical analysis. The nucleus pulposus (NP) was separated from the annulus fibrosus (AF), the specimens weighed (wet weight), the content of DNA measured using PicoGreen, and the total contents of sulfated glycosaminoglycans (GAG) measured by the 1,9-dimethylmethylene blue (DMMB) assay. Results: Following needle puncture that initiates disc degeneration, the disc height index (DHI) decreased by about 25%. By 6 weeks after Link-N injection, the mean percent DHI of injected discs in the Link-N group was higher than in the saline group. This difference in mean percent DHI was maintained during the rest of the follow-up. Puncturing the IVD also led to a decrease in proteoglycan content in both the NP and the AF in saline-treated discs. Treatment with Link-N stimulated proteoglycan synthesis (GAG) in both the NP and AF by about 20%. Link-N did not cause an increase in the DNA content of the discs. Conclusion: Results of the present study show that Link-N can stimulate proteoglycan production in vivo when administered to degenerate disc. This stimulation occurs in both the NP and AF of the disc and in the absence of any effect on cell division. The changes observed with Link-N on proteoglycan synthesis are similar to those reported after injection of osteogenic protein-1 (OP-1) Thus, Link-N appears to be equally effective at stimulating repair of the IVD in vivo. One major advantage of Link-N over OP-1 for therapeutic use is the large saving in cost, Link-N being about 400 times cheaper than OP-1

The purpose of this study was to investigate the possible relationship between matrix metalloproteinase-3 (MMP-3) promoter 5A/6A polymorphism and intervertebral disc (IVD) degeneration in the older generation. One of the important steps in IVD degeneration is disc matrix degradation by matrix degrading enzymes such as MMPs. MMP-3 is one of the potent proteoglycan degrading enzymes and has been suggested to play an important role in IVD degradation. A common 5A/6A polymorphism in the promoter region of the human MMP-3 gene has been identified. This polymorphism was reported to be involved in the regulation of MMP-3 gene expression (the 5A allele has 2-fold higher promoter activity than 6A). We now hypothesize that IVD degeneration is associated with MMP-3 promoter 5A/6A polymorphism. Forty-nine elderly Japanese volunteers (mean age 74.3 years, range 64–94 years) were studied. Each lumbar disc was graded according to the radiographic classification system of IVD degeneration described by Kellgren and Lawrence. The 5A/6A polymorphism was determined with both single strand conformation polymorphism (SSCP) and polymerase chain reaction with allele-specific primers (AS-PCR). Two subjects (4%) with 5A5A genotype, 16 (33%) with 5A6A, and 31 (63%) with 6A6A were observed. Genotype was totally independent of age and sex. There was a significantly larger number of IVDs graded 2 and higher in the 5A/5A+5A/6A than in the 6A/6A (p< 0.05). The degenerative scores of lumber discs were also distributed more highly in the 5A/5A+5A/6A than in the 6A/6A (p=0.0029). Many environmental factors have been reported to accelerate IVD degeneration. Recently, genetic factors have also been highlighted as possible risk factors. The 5A allele of the human MMP-3 promoter is a possible risk factor for acceleration of IVD degeneration in people aged over 64 years old. We conclude that MMP-3 plays a key role in the degeneration of IVD in the older generation

Introduction: Intervertebral disc (IVD) degeneration involves loss of disc matrix leading to instability and pain. Autologous cells are the ideal choice for bioengineering a new IVD, but removal of cells from the IVD is problematic. Our aim was to direct mesenchymal stromal cells (MSCs) down a chondrocytic lineage to mimic disc chondrocyte phenotype. Methods: MSCs were either maintained in monolayer, pelleted into micromass aggregates or transferred to alginate beads. Pellet cultures were used in immunohis-tochemistry for type II collagen and aggrecan and in situ hybridisation for SOX-9 mRNA. Monolayer and alginate cells were cultured in the presence or absence of chondrogenic medium for 4 and 11 days. Monolayer cultured MSCs were also transfected with a SOX-9 adenovirus and cultured in the presence or absence of TGF-_1. Realtime quantitative PCR was used to analyse expression of chondrocyte markers. Results: IHC showed increased expression of type II collagen and aggrecan in pellet cultures, while ISH showed that SOX-9 was not expressed by monolayer MSCs, but increased after pelleting. Realtime PCR using alginate-cultured MSCs showed down regulation of type I collagen mRNA expression and up-regulation of SOX-9 that was increased by chondrocgenic medium. SOX-9 transduced monolayer MSCs showed increased type II collagen, aggrecan, SOX-6 and SOX-9 mRNA over controls, while type I collagen levels showed no significant change. Stimulation of transfected MSCs with TGF-_1 showed similar increases in chondrocyte genes. Discussion & conclusions: Adult human MSCs were induced to differentiate along a chondrocytic phenotype, which was mediated by culture conditions. Alginate and pellet culture produce a cell that has more chondrogenic characteristics than monolayer cells. SOX-9 transduced monolayer MSCs appeared to produce a more chondrocytic phenotype which was modulated by TGF-_1. Results suggest SOX-9 transfected monolayer MSCs may be used as a source of chondrocytes for repair of degenerate IVD

Current medical treatments for IVD degeneration rely on conservative therapies or surgery. Surgical treatments (e.g. spinal fusion,) have shown satisfactory results in alleviating pain, but long-term clinical outcomes remain poor. Thus, there is an urgent need for alternative cell based regenerative therapies focussed on correcting the underlying pathogenesis of IVD degeneration. However, for these to be successful an appropriate cell source for implantation, together with a suitable growth factor to direct cell differentiation and formation of a functional matrix must be identified. Additionally, extensive in vitro studies are needed to establish and support further pre-clinical and potential commercial development. We have demonstrated that stimulation of both BM-MSCs and AD-MSCs with GDF6 results in improved differentiation to a nucleus pulposus (NP)-like phenotype and synthesis of proteoglycan rich matrix with micromechanical properties akin to the healthy IVD. Significantly, these studies have highlighted that AD-MSCs are the more appropriate cell source. Furthermore, our studies have shown hat GDF6 has anabolic effects on degenerate human NP cells, stimulating adoption of a more normal NP phenotype and increasing appropriate atrix synthesis. This suggests that delivery of GDF6 as part of an MSC-based therapy may be beneficial both in directing lineage-specific MSC differentiation, but also in restoring a more anabolic phenotype in native NP cells, thereby having a dual regenerative effect.

Introduction

Injectable hydrogels via minimally invasive surgery reduce the risk of infection, scar formation and the cost of treatment. Degradation of the intervertebral disc (IVD) currently has no preventative treatment. An injectable hydrogel material could restore disc height, reinforce local mechanical properties, and promote tissue regeneration. We present a hydrogel material Laponite^® associated poly(N-isopropylacrylamide)-co-poly(dimethylacrylamide) (NPGel). Understanding how the components of this hydrogel system influence material properties, is crucial for tailoring treatment strategies for the IVD and other tissues.

Background

Mesenchymal stem cells (MSCs) are undergoing evaluation as a potential new therapy for immune and inflammatory-mediated conditions such as IVD degeneration (IDD). Both adipose (ASCs) and bone-marrow (BMSCs) derived MSCs have been widely used in this regard. The optimal tissue source and expansion conditions required to exploit the regenerative capacity of these cells are not yet fully elucidated. In addition the phenotypic response of transplanted cells to the disease environment is not well understood. In this study, ASCs and BMSCs were exposed to a combination of hypoxic conditioning and selected inflammatory mediators, conditions that mimic the microenvironment of the degenerate IVD, in an effort to understand their therapeutic potency for in vivo administration.

Our study establishes a rabbit model of disc degeneration which requires neither a chemical nor physical injury to the disc. Disc degeneration similar to that seen in man was created at levels proximal (L4-L5) and caudal (L7-S1) to a simulated lumbar fusion and was studied for up to nine months after arthrodesis.

Loss of the normal parallel arrangement of collagen bundles within the annular lamellae was observed in intervertebral discs adjacent to the fusion at three months. By six months there was further disorganisation as well as loss of distinction between the lamellae themselves. By nine months the structure of the disc had been replaced by disorganised fibrous tissue, and annular tears were seen. There was an initial cellular proliferative response followed by loss of chondrocytes and notochordal cells in the nucleus pulposus. Degeneration was accompanied by a decrease in the monomer size of proteoglycans. Narrowing of the disc space, endplate sclerosis and the formation of osteophytes at adjacent disc spaces were observed radiologically.

Intervertebral discs (IVDs) are fibrocartilagenous ovoids located between the vertebral bodies of the spine that provide the sole source of flexibility in that structure. IVDs are clinically very important as degeneration has been shown to be strongly associated with lower back pain, sciatica, and disc herniation: potentially disabling conditions that affect a very large section of the UK population.

The aetiology of disc degeneration is poorly understood although upregulation of matrix metalloproteinase (MMP) activity is thought to be involved. Degradation products of the extra-cellular matrix are known to increase MMP production and activity in other tissues. This project concentrated on examining the effects of degredation products of elastin. Elastin fragments (κ-elastin peptides) have been shown to upregulate mRNA levels and increase expression of pro-MMP-1 in human skin fibroblasts, cells that are thought to be similar to those residing in the annulus fibrosus of intervertebral discs. This study examined their effect on disc cells and on skin fibroblasts.

Total MMP-2 and -7 activity produced by cells extracted from the annulus fibrosus of bovine intervertebral disc cells and cultured for 24 hours with 0–300μg/ml κ-elastin was determined using fluorimetric and zymographic analyses. κ-elastin was prepared from bovine ligamentum nuchae or bovine intervertebral discs.

Culture with κ-elastin prepared from bovine ligamentum nuchae caused skin and disc cell potential pro-MMP-2 activity to increase in a dose-dependent manner; the potential pro-MMP-2 activity of both cell types is more than doubled when cultured with 300μg/ml κ-elastin.

These findings suggest that in the bovine disc, matrix breakdown may cause a feedback loop with degraded elastin stimulating disc cells to increase production of pro-MMP-2, with the possibility of further degrading elastin and other proteins and contributing to IVD breakdown.

We studied 52 patients, each with a lumbosacral transitional vertebra. Using MRI we found that the lumbar discs immediately above the transitional vertebra were significantly more degenerative and those between the transitional vertebrae and the sacrum were significantly less degenerative compared with discs at other levels. We also performed an anatomical study using 70 cadavers. We found that the iliolumbar ligament at the level immediately above the transitional vertebra was thinner and weaker than it was in cadavers without a lumbosacral transitional vertebra.

Instability of the vertebral segment above the transitional vertebra because of a weak iliolumbar ligament could lead to subsequent disc degeneration which may occur earlier than at other disc levels. Some stability between the transitional vertebra and the sacrum could be preserved by the formation of either an articulation or by bony union between the vertebra and the sacrum through its transverse process. This may protect the disc from further degeneration in the long term.

Background and aims:

T2D is postulated to be an important aetiological factor for lumbar disc degeneration (LDD), which itself has a well documented relationship with low back pain. Obesity increases risk of both T2D, low back pain and LDD. Connective tissue modification has been reported in hyperglycaemia, but the epidemiology of LDD in T2D has not been described to date.

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.

Degenerate disc disease is a major cause of low back pain, yet its aetiology is still poorly understood. The intervertebral disc is the largest avascular structure in the body. Cells of the nucleus pulposus, therefore, rely on diffusion of oxygen & nutrients down concentration gradients from peripheral vessels in the cartilage end-plates. Thus, there is a low oxygen tension and cellular respiration is largely anaerobic.

The purpose of this study was to examine the effects of inflammation, hypoxia and acidosis on degeneration and pro-inflammatory mediator production in virgin porcine nucleus pulposus cultures.

Intervertebral discs were harvested from normal 6-month old agricultural pigs slaughtered for other purposes. Nucleus pulposus was contained within the annulus until further dissection under sterile conditions in the laboratory was performed. Nucleus pulposus was harvested, diced and divided into 200mg samples. Samples were incubated under optimal conditions.

Discs were cultured in 5μg/ml E. coli lipopolysaccharide, in a hypoxic environment or at low pH. IL-6, IL-8 and LDH assays were performed by ELISA, in accordance with manufacturer’s instructions.

Time and dose-response curves were generated for each experiment (results not shown). Results at 72 hours incubation are tabulated below:

These results confirm that nucleus pulposus is a biochemically active tissue capable of producing pro-inflammatory mediators in response to environmental stresses. IL-6 and IL-8 are both involved in the inflammatory cascade, causing chemotaxis of neutrophils and macrophages to the area. IL-8 itself causes hyperalgesia. Acidotic and inflammatory conditions, but not hypoxia, stimulated cytokine release. This may indicate a protective reduction in cellular activity in reduced oxygen environments. Necrosis, as measured by LDH production, was negligible.

Anatomisches Institut der Georg-August-Universität Göttingen, Germany

Biomedical Research Centre, Dept. of Orthopaedic Surgery, Academic Hospital, Pretoria, South Africa

To date, no animal model for disc degeneration has gained much acceptance, mostly due to the fact that most animals are quadrupeds and thus lack basic biomechanical characteristics of human spines. An adequate model would be of invaluable interest for degeneration related research.

In a standardized series of animal experiments in 18 adult Minipigs and 20 adult Cercopithecus aethiops monkeys all animals obtained nucleotomy in one lumbar FSU from a ret-roperitoneal approach and were sacrificed at last 24 weeks afterwards. The Minipigs were x-rayed at time of sacrifice, the monkeys prior to operation and at termination of the experiment. Vice versa, the Minipigs obtained intradiscal pressure recordings at these occasions. The Minipig spines were formol fixed whereas the monkey spines were harvested after perfusion with PBS, fresh frozen, and obtained CT and MRI scans prior to thawing, fixation and comprehensive histological evaluation.

The lumbar FSU of Minipig and Cercopithecus mainly consists of the same elements as in man. There are certain differences concerning the porcine endplates which ossify as an epiphyseal-like formation with ossification starting in its center, different from the so-called “Randleiste”. Whereas the operative procedure in the Minipigs came in handy, in the Cercopithecus monkey it proved to be demanding, though feasible, due to relatively wide transverse processes and thick psoas muscle structures. The psoas could not be easily detached and needed to be split instead, thus directly exposing the segment nerves. The histological, standard radiological, CT, MRI, and mechanical observations were very similar to those which can be made during the natural aging process of the disc in man.

Both animal models are recommendable for further research: Cercopithecus FSUs are more difficult to expose. Logistic reasons may favour Minipigs in Europe. In case of fusion related experiments the use of primates yet seems inevitable.

We attempted to correlate the findings of MRI and discography in patients with low back pain, examining 108 lumbar intervertebral discs in 33 consecutive patients. MRI results were assessed from the intensity and shape of the signal obtained from the central part of the disc. Discography was classified according to the pattern of contrast material, the pressure accepted and the pain reproduced. All discs which were abnormal on MRI had altered patterns on discography, but 18 of the 60 discs with normal MRI had abnormal discograms. Of 39 asymptomatic discs, 33 had normal MRI signals and 24 had normal discograms. None of the 15 discs showing severe degeneration on MRI sustained high levels of intradiscal pressure, but only six of the 60 discs giving normal MRI had low pressure. With current techniques, discography is more accurate than MRI for the detection of annular pathology: a normal MRI does not exclude significant changes in the peripheral structure of the intervertebral disc which can produce low back pain.

Introduction: Disc degeneration causes structural and biochemical tissue changes resulting in altered stresses that may affect vertebral bone remodelling. We hypothesized that disc degeneration alters vertebral cortical strains and disc mechanics of the motion segment, with and without the presence of zygapophyseal joints.

Methods: Twenty human lumbar functional spinal units (FSUs) were strain gauged on the lateral and anterior vertebral cortices, below the inferior endplate. Each FSU was preloaded overnight (0.2 MPa) in a bath and subjected to dynamic compression (1 MPa), flexion/extension/lateral bending (500N + 5 Nm), and axial rotation (5 Nm), before and after removal of the zygapophyseal joints. After testing, discs were macroscopically assessed and graded (1–4) for degeneration. Stiffness, phase angle (energy absorption) and principal strains were calculated. ANOVAs with the dependent variable of principal strain/stiffness/phase angle versus disc grade were performed for each testing direction.

Results: Assessment of disc degenerative condition revealed six grade 2 discs, eight grade 3, and six grade 4. Age and degeneration were highly correlated (r=0.80, P< 0.0001). The effect of disc grade on stiffness was significant overall in most loading directions, before and after removal of zygapophyseal joints (P< 0.008), apart for axial rotation (P> 0.587). Post-hoc multiple comparisons for all loading directions apart for axial rotation revealed that the stiffness of grade 4 discs was significantly larger than grades 2 and 3 discs in most loading directions.

For phase angle (approximate magnitude 5°), no significant overall effects due to degeneration were found across any loading direction (P> 0.2). ANOVA analyses on maximum/minimum principal strains found no significant effect due to disc grade (P> 0.063). However, a small number of significant effects due to disc grade were found at particular strain gauge locations for the isolated disc in flexion, the intact FSU in extension, and the intact FSU/isolated disc in right lateral bending.

Discussion: This study represents the first of its kind to investigate the effects of disc degeneration on vertebral bone cortical strain and disc mechanical properties. Significant increases in stiffness were found with increasing degeneration in all test directions apart for axial rotation. Changes in disc stiffness were consistent with other studies and may be a result of the structural and biochemical changes within the disc that accompany the degenerative process.

The non-significant small phase angles suggest that the disc behaves more like an elastic solid than a poroelastic material, and that dehydration associated with degeneration does not adversely affect damping. Principal strains were not significantly affected by disc degeneration overall, suggesting that the cortical shell adjacent to the disc-endplate boundary maintains a relatively homeostatic condition, with more dramatic architectural changes probably occurring within the trabecular bone. Applications of this research include providing important validation data for analytical/finite element models of the intact FSU and isolated disc segment, and a better understanding of the magnitudes of cortical strains that need to be maintained in order to avoid damaging vertebral bone stress-shielding effects after treatments for disc degeneration.

Introduction: Osteoporotic fractures in elderly people are usually attributed to hormonal changes and inactivity. But why should the anterior vertebral body be affected so often?

Materials and Methods: Forty-one cadaveric thoraco-lumbar motion segments aged 62–94 yrs were loaded to simulate upright and flexed postures. A pressure transducer was used to measure “stress” inside the disc, and calculations showed how compressive loading was distributed between the neural arch, and the anterior and posterior halves of the vertebral body. Compressive strength was measured in flexed posture. Regional volumetric bone mineral density (BMD) and histomorpho-metric parameters were measured.

Results: Upright posture. Compressive load-bearing by the neural arch increased with grade of disc degeneration, averaging 52+25% in specimens with grade 3 or 4 discs. In these same specimens, the anterior half of the vertebral body resisted only 16+18% of the applied load. Relative unloading of the anterior vertebral body was associated with low BMD and with histomorphometric evidence of inferior bone quality. Flexed posture. Flexion always transferred loading to the anterior half of the vertebral body, so that it resisted 55+17% in specimens with grade 3/4 discs. Compressive strength measured in this posture was most closely proportional to BMD in the anterior vertebral body (r² = 0.75), and inversely proportional to neural arch load-bearing in the upright posture (r² = 0.39).

Conclusion: Disc degeneration causes the anterior vertebral body to be unloaded in habitual upright postures, reducing bone density and quality within it. This predisposes to wedge fracture when the spine is flexed.

Introduction

Disc degeneration is often scored using macroscopic and microscopic scoring systems. Although reproducible, these scores may not accurately reflect declining function in a degenerated disc. Accordingly, we compared macroscopic and microscopic degeneration scores with measurements of disc function.

We investigated the relationship between spinopelvic parameters and disc degeneration in young adult patients with spondylolytic spondylolisthesis. A total of 229 men with a mean age of 21 years (18 to 26) with spondylolytic spondylolisthesis were identified. All radiological measurements, including pelvic incidence, sacral slope, pelvic tilt, lumbar lordosis, sacral inclination, lumbosacral angle (LSA), and sacrofemoral distance, were calculated from standing lateral lumbosacral radiographs. The degree of intervertebral disc degeneration was classified using a modified Pfirrmann scale. We analysed the spinopelvic parameters according to disc level, degree of slip and disc degeneration.

There were significant positive correlations between the degree of slip and pelvic incidence (p = 0.009), sacral slope (p = 0.003) and lumbar lordosis (p = 0.010). The degree of slip and the LSA were correlated with disc degeneration (p < 0.001 and p = 0.003, respectively). There was also a significant difference between the degree of slip (p < 0.001) and LSA (p = 0.006) according to the segmental level of disc degeneration.

Cite this article: Bone Joint J 2013;95-B:1239–43.

Aims

The presence of facet tropism has been correlated with an elevated susceptibility to lumbar disc pathology. Our objective was to evaluate the impact of facet tropism on chronic lumbosacral discogenic pain through the analysis of clinical data and finite element modelling (FEM).

Cite this article: Bone Joint Res 2023;12(3):199–201.

Intervertebral disc degeneration can lead to physical disability and significant pain, while the present therapeutics still fail to biochemically and biomechanically restore the tissue. Stem cell-based therapy in treating intervertebral disc (IVD) degeneration is promising while transplanting cells alone might not be adequate for effective regeneration. Recently, gene modification and 3D-printing strategies represent promising strategies to enhanced therapeutic efficacy of MSC therapy. In this regard, we hypothesized that the combination of thermosensitive chitosan hydrogel and adipose derived stem cells (ADSCs) engineered with modRNA encoding Interleukin − 4 (IL-4) can inhibit inflammation and promote the regeneration of the degenerative IVD. Rat ADSCs were acquired from adipose tissue and transfected with modRNAs. First, the kinetics and efficacy of modRNA-mediated gene transfer in mouse ADSCs were analyzed in vitro. Next, we applied an indirect co-culture system to analyze the pro-anabolic potential of IL-4 modRNA engineered ADSCs (named as IL-4-ADSCs) on nucleus pulposus cells. ModRNA transfected mouse ADSCs with high efficiency and the IL-4 modRNA-transfected ADSCs facilitated burst-like production of bio-functional IL-4 protein. In vitro, IL-4-ADSCs induced increased anabolic markers expression of nucleus pulposus cells in inflammation environment compared to untreated ADSCs. These findings collectively supported the therapeutic potential of the combination of thermosensitive chitosan hydrogel and IL-4-ADSCs for intervertebral disc degeneration management. Histological and in vivo validation are now being conducted

In the last decades, the use of artificial intelligence (AI) has been increasingly investigated in intervertebral disc degeneration (IDD) and chronic low back pain (LBP) research. To date, several AI-based cutting-edge technologies, such as computer vision, computer-assisted diagnosis, decision support system and natural language processing have been utilized to optimize LBP prevention, diagnosis, and treatment. This talk will provide an outline on contemporary AI applications to IDD and LBP research, with a particular attention towards actual knowledge gaps and promising innovative tools

Monomeric C reactive protein (mCRP) presents important proinflammatory effects in endothelial cells, leukocytes, or chondrocytes. However, CRP in its pentameric form exhibits weak anti-inflammatory activity. It is used as a biomarker to follow severity and progression in infectious or inflammatory diseases, such as intervertebral disc degeneration (IVDD). This work assesses for the first time the mCRP effects in human intervertebral disc cells, trying to verify the pathophysiological relevance and mechanism of action of mCRP in the etiology and progression of IVD degeneration. We demonstrated that mCRP induces the expression of multiple proinflammatory and catabolic factors, like 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), in human annulus fibrosus (AF) and nucleus pulposus (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 signaling of mCRP. Our results indicate that the effect of mCRP is persistent and sustained, regardless of the proinflammatory environment, as it was similar in healthy and degenerative human primary AF cells. This is the first article that demonstrates the localization of mCRP in intravertebral disc cells of the AF and NP and that provides evidence for the functional activity of mCRP in healthy and degenerative human AF and NP disc cells

Tryfonidou leads the Horizon 2020 consortium (iPSpine; 2019–2023) bringing a transdisciplinary team of 21 partners together to address the challenges and bottlenecks of iPS-based advanced therapies towards their transition to the clinic. Here, chronic back pain due to intervertebral disc degeneration is employed as a show case. The project develops the iPS-technology and designed smart biomaterials to carry, protect and instruct the iPS cells within the degenerate disc environment. This work will be presented including ongoing activities focus on translating the developed methodology and tools towards clinically relevant animal models. The consortium optimized the protocol for the differentiated iPS-notochordal-like cells (iPS-NLCs) and shortlisted two biomaterials shortlisted based on their physicochemical, cytotoxicity, biomechanical and biocompatibility testing. Both were shown to be safe and have been tested with the progenitors of iPS-NLCs. An advanced platform (e.g., the dynamic loading bioreactor for disc tissue) was used to evaluate their performance: the biomaterials supported the iPS-NLC progenitors after injection into the degenerate disc and seem to also support their maturation towards NLCs. Furthermore, we confirmed the capacity of these cells to survive inside degenerated discs at 30 days upon injection in sheep, whereafter we continued with their evaluation at 3 months post-injection. We achieved full evaluation of the sheep spines, including biomechanical analysis using the portable spine biomechanics tester prior analysis at the macro- and microscopic, and biochemical level

Background. Intervertebral disc degeneration (DD) is a complex age-related condition that constitutes the main risk factor for disabling back pain. DD is assessed using different traits extracted from MR imaging (MRI), normally combined to give summary measures (e.g. Pfirmann score). The aetiology of DD is poorly understood and despite its high heritability (75%), the precise genetic predisposition is yet to be defined. Genome wide association study (GWAS) is used to discover genetic variants associated with a disease or phenotype. It tests variants across the whole genome. It requires large samples to provide adequate but unfortunately there is poor availability of spine imaging data due to the high cost of MRI. We have adopted new methods to examine different MRI traits independently and use the information of those traits to boost GWAS power using specialized statistical software for jointly analyse correlated traits. Methods/Results. We examined DD MRI features disc narrowing, disc bulge, disc signal intensity and osteophyte formation in the TwinsUK cohort who had undergone T2-weighted sagittal spine MRI. GWAS were performed on the four traits. MTAG software was used to boost single trait GWAS power using the information in the other trait GWAS. 9 different loci were identified. Conclusions. Preliminary results suggest genes GDF6, SP1/SP7 are associated with individual trait signal intensity. In addition, novel associated genes with potential for shedding new light on pathogenic mechanisms are identified. Additional cohorts will be included in the design as a replication to test reproducibility of the results. Conflicts of interest: No conflicts of interest. Sources of funding: Funded by Disc4All, EU Horizon 2020, MSCA-2020-ITN-ETN GA: 955735

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

Low back pain (LBP) is the main cause of disability worldwide and is primarily triggered by intervertebral disc degeneration (IDD). Although several treatment options exist, no therapeutic tool has demonstrated to halt the progressive course of IDD. Therefore, several clinical trials are being conducted to investigate different strategies to regenerate the intervertebral disc, with numerous studies not reaching completion nor being published. The aim of this study was to analyze the publication status of clinical trials on novel regenerative treatments for IDD by funding source and identify critical obstacles preventing their conclusion. Prospective clinical trials investigating regenerative treatments for IDD and registered on . ClinicalTrials.gov. were included. Primary outcomes were publication status and investigational treatment funding. Fisher's exact test was utilized to test the association for categorical variables between groups. 25 clinical trials were identified. Among these, only 6 (24%) have been published. The most common source of funding was university (52%), followed by industry (36%) and private companies (12%). Investigational treatments included autologous (56%) or allogeneic (12%) products alone or in combination with a carrier or delivery system (32%). The latter were more likely utilized in industry or privately funded studies (Fig. 1, p=0.0112). No significant difference was found in terms of funding regarding the publication status of included trials (Table 1, p=0.9104). Most clinical trials investigating regenerative approaches for the treatment of IDD were never completed nor published. This is likely due to multiple factors, including difficult enrollment, high dropout rate, and publication bias. 3. More accurate design and technical support from stakeholders and clinical research organization (CROs) may likely increase the quality of future clinical trials in the field. For any figures or tables, please contact the authors directly

Despite promising results in attempting intervertebral disc regeneration, intradiscal cell transplantation is affected by several drawbacks, including poor viability in the harsh disc environment, low cost-effectiveness, and immunogenic/tumorigenic concerns. Recently, the development of cell-free approaches is gaining increasing interest in the field, with a particular regard towards extracellular vesicles (EVs). Nucleus pulposus cell (NPC) progenitors characterized by Tie2 expression have shown a higher chondrogenic differentiation potential compared to MSCs. The aim of this study was to investigate the putative regenerative effects of EVs isolated from Tie2-overexpressing NPC progenitors on degenerative NPCs. NPCs were isolated from young donors and underwent an optimized culture protocol to maximize Tie2 expression (NPCs. Tie2+. ) or a standard protocol (NPCs. STD. ). Following EV characterization, NPC isolated from patients affected by intervertebral disc degeneration (IDD) were treated with either NPCs. Tie2+. -EVs or NPCs. STD. -EVs. Cell proliferation and viability were assessed with the CCK-8 assay. Cell apoptosis and necrosis were evaluated with the Annexin V/PI assay. Cell senescence was investigated with b-galactosidase staining. EV uptake was assessed with PKH26 staining of EVs under confocal microscopy. Treatment with EVs isolated from young NPC donors significantly increased degenerative NPC viability, especially in samples treated with NPCs. Tie2+. -EVs. Likewise, NPCs. Tie2+. -EVs significantly reduced cell senescence and did not show to exert necrotic nor apoptotic effects on recipient cells. Furthermore, EV uptake was successfully observed in all treated cells. NPCs. Tie2+. -EVs demonstrated to significantly enhance degenerative NPC viability, senescence and apoptosis. The use of committed progenitors naturally residing the in the nucleus pulposus may optimize EV regenerative properties and constitute the basis for a new therapy for IDD

Objectives. This study aims to investigate whether bacteria are present in intervertebral discs (IVDs) and their influence. Causality between chronic infection of the IVD and its degenerative process gained great interest recently. Granville Smith et al. (2021) identified 36 articles from 34 research studies investigating bacteria in IVDs, from these 27 studies found, Cutibacterium acnes being the most abundant. However, whether bacteria identified were present in vivo or if they represent contamination remains unclear. Methods. Human IVD tissue was fixed in paraffin and Immunohistochemical stained for Gram-positive bacteria. NP cells in monolayer have been stimulated with LPS (0.1–50 µg/ml) and Peptidoglycan (0.1–50 µg/ml) for 24, 48 and 72 hrs to investigate their influence. The concentration of proinflammatory and catabolic cytokines in the media is being measured using ELISA. RNA extracted and RT-qPCR utilised for factors associated with disc degeneration matrix genes, matrix degrading enzymes, cytokines, neurotrophic factors and angiogenic factors. Results. Bacteria were detected within IVD tissue. Bacteria was internalized by the NP cells and influenced the nuclei morphology. Preliminary results of the exposure of NP cells to bacterial components demonstrate that ADAMTS4 as well as IL-8 were showed an increase in gene expression after LPS and peptidoglycan treatment compared to the non-treated control. Underlining these results, IL-8 protein was increased in treated groups, whereas peptidoglycan treated groups showed a dose dependence. Conclusion. This study demonstrates that Gram positive bacteria are present within the IVD. The exposure of NP cells to peptidoglycans indicates that bacterial components trigger a stress response. Conflicts of Interest: No conflict of interest. Sources of Funding: This project is part of the Disc4All Training network to advance integrated computational simulations in translational medicine, applies to intervertebral disc degeneration and funded by Horizon 2020 (H2020-MSCA-ITN-ETN-2020 GA: 955735)

Intervertebral disc degeneration (IDD), the main cause of low back pain, is closely related to the inflammatory microenvironment in the nucleus pulposus (NP). Tumor necrosis factor-α (TNF-α) plays an important role in inflammation-related metabolic disturbance of NP cells. Melatonin has been proven to regulate the metabolism of NP cells, but whether it can protect NP cells from TNF-α-induced damage is still unclear. Therefore, this study aims to investigate the role and specific mechanism of melatonin on regulating the metabolism of NP cells in the inflammatory microenvironment. Human primary NP cells were treated with or without vehicle, TNF-α and melatonin. And the metabolic markers were also detected by western blotting and RT-qPCR. The activity of NF-κB signaling and Hippo/YAP signaling were assessed by western blotting and immunofluorescence. Membrane receptors inhibitors, pathway inhibitors, lentiviral infection, plasmids transfection and immunoprecipitation were used to explore the specific mechanism of melatonin. In vivo, the rat IDD model were constructed and melatonin was injected intraperitoneally to evaluate its therapeutical effect on IDD. We demonstrated that melatonin could alleviate the development of IDD in a rat model and reverse TNF-α–impaired metabolism of NP cells in vitro. Further investigation revealed that the protective effects of melatonin on NP cells mainly rely on MTNR1B, which subsequently activates Gαi2 protein. The activation of Gαi2 could upregulate the yes-associated protein (YAP) level, resulting in anabolic enhancement of NP cells. In addition, melatonin-mediated YAP upregulation increased the expression of IκBα and suppressed the TNF-α–induced activation of the NF-κB pathway, thereby inhibiting the catabolism of NP cells. Our results revealed that melatonin can reverse TNF-α–impaired metabolism of NP cells via the MTNR1B/Gαi2/YAP axis and suggested that melatonin can be used as a potential therapeutic drug in the treatment of IDD

Introduction. Multiple studies have identified Cutibacterium acnes (C.acnes) and other microbes in intervertebral disc tissue using 16S DNA Sequencing and microbial cultures. However, it remains unclear whether these bacteria are native to the discs or result from perioperative contamination. Our study aimed to detect Gram-positive bacteria in non-herniated human disc samples and explore correlations with Toll-like receptors (TLR) 2, TLR4, NLRP3, and Gasdermin D. Methods. Immunohistochemical staining was conducted on 75 human IVD samples for Gram-positive bacteria, S. aureus, C.acnes, TLR2, TLR4, NLRP3, and Gasdermin D. Cell detection and classification were performed using QuPath. NP cells were treated with Lipopolysaccharide (LPS) and Peptidoglycan (PGN) in monolayer and alginate beads for up to 72 hours, followed by secretome analysis using Luminex. Statistical analysis included Kruskal-Wallis, Dunn's multiple comparison test, and Pearson correlation. Results. Immunohistochemical staining revealed Gram-positive bacteria exclusively within cells, with C. acnes positivity ranging from 5–99% and correlating with patient age (r=0.41, p= 0.007). TLR2 positivity ranged from 5–99% and TLR4 from 3–72%, showing a strong correlation (r= 0.62, p= 1.5e-006). Females with mid-degenerative grades exhibited significantly decreased TLR2 expression compared to those without degeneration signs. Treatment with LPS and PGN increased catabolic cyto- and chemokines associated with IVD degeneration. Conclusion. In conclusion, this study confirms Gram-positive bacteria presence in non-herniated human disc samples and highlights their role in triggering a catabolic response in disc cells. No conflicts of interest.  . Sources of funding. This project is part of the Disc4All Training network to advance integrated computational simulations in translational medicine, applies to intervertebral disc degeneration and funded by Horizon 2020 (H2020-MSCA-ITN-ETN-2020 GA: 955735)

Intervertebral disc degeneration (IDD) affects more than 80% of the population all over the world. Current strategies for the treatment of IDD are based on conservative or surgical procedures with the aim of relieving pain. Mesenchymal stem cell (MSC) transplantation has emerged as a promising therapy in recent decades, but studies showed that the particularly hostile microenvironment in the intervertebral disc (IVD) can compromise cells survival rate. The use of exosomes, extracellular vesicles released by various cell types, possess considerable economic advantages including low immunogenicity and toxicity. Exosomes allow intercellular communication by conveying functional proteins, RNA, miRNA and lipids between cells. The purpose of this study is to assess the therapeutic effects of exosomes derived from Wharton Jelly mesenchymal stromal cells (WJ-MSC) on human nucleuspulposus cells (hNPC) in an in vitro 3D culture model. Exosomes (exos) were isolated by tangential flow filtration of WJ-MSC conditioned media and characterized by: quantification with BCA test; morphological observation with TEM, surface marker expression by WB and size evaluation by NTA. Confocal microscopy has been used to identify exosomes marked with PKH26 and monitor fusion and/or incorporation in hNPC. hNPC were isolated from waste surgical material from patients undergoing discectomy (n = 5), expanded, encapsulated in alginate beads and treated with: culture medium (control group); WJ-MSC exos (WJ-exos) at different concentrations (10 μg/ml, 50 μg/ml and 100 μg/ml). They were then analysed for: cell proliferation (Trypan Blu); viability (Live/Dead Assay); quantification of nitrites (Griess) and glycosaminoglycans, GAG (DMBB). The hNPC in alginate beads treated for 7 days were included in paraffin and histologically analysed to determine the presence of extracellular matrix (ECM) components. Finally, the expression levels of catabolic and anabolic genes were evaluated through real-time polymerase chain reaction (qPCR). All concentrations of WJ-exos under exam were capable to induce a significant increase in cell proliferation after 10 and 14 days of treatment (p < 0.01 and p < 0.001, respectively). Live/Dead assay showed a decrease in cell death at 50 μg/ml of WJ-exos (p < 0.05). While cellular oxidative stress indicator, nitrite production, was reduced in a dose-dependent way and statistically significant only with 100 μg/ml of WJ-exos (p < 0.05). WJ-exos at 10 and 100 μg/ml induced a significant increase in GAG content (p < 0.05; p < 0.01, respectively) confirmed by Alcian Blu staining. Exos derived from WJ-MSC modulated gene expression levels by increasing expression of ACAN and SOX-9 genes and reducing significantly of IL-6, MMP-1, MMP-13 and ADAMTS-5 levels (p < 0.05; p < 0.01) compared to the control group. Our results supported the potential use of exosomes from WJ-MSC for the treatment of IDD. Exosomes improved hNPC growth, attenuated ECM degradation and reduced oxidative stress and inflammation. This study offers a new scenario in IVD regeneration, promoting the potential use of extracellular vesicles as an alternative strategy to cell therapy

Osteoarthritis (OA) is a painful and disabling chronic condition that constitutes a major challenge to health care worldwide. There is currently no cure for OA and the analgesic pharmaceuticals available do not offer adequate and sustained pain relief, often being associated with significant undesirable side effects. Another disease associated with degenerating joints is Intervertebral disc degeneration (IVDD) which is a leading cause of chronic back pain and loss of function. It is characterized by the loss of extracellular matrix, specifically proteoglycan and collagen, tissue dehydration, fissure development and loss of disc height, inflammation, endplate sclerosis, cell death and hyperinnervation of nociceptive nerve fibers. The adult human IVD seems incapable of intrinsic repair and there are currently no proven treatments to prevent, stop or even retard disc degeneration. Fusion is currently the most common surgical treatment of symptomatic disc disease. However, radiographic follow-up studies have revealed that many patients develop adjacent segment disc degeneration due to altered spine biomechanics. The development of safe and efficacious disease modifying OA drugs (DMOADs) that treat pain and inflammation in joints will improve our ability to control the disease. I addition, a biologic treatment of IVDD is desirable. This presentation will provide an overview of recent advances and future prospects of a multimodal biologic treatment of OA, and IVDD. We will focus on Link N, a naturally occurring peptide representing the N terminal region of link protein and the first 1–8 residues of Link N (short Link N, sLN) responsible for the biologic therapy in question

Introduction and Objective. Low back pain (LBP) is a disorder strongly associated with intervertebral disc degeneration (IDD) with an important impact on the quality of life of affected people. To date, LBP treatment is based on conservative methods with the aim to reduce back pain without restoring the degenerative environment of the disc. The main cause of IDD is the drastic reduction of the proteoglycan content within the nucleus pulposus (NP), eventually leading to the loss of disc water content, micro-architecture, biochemical and mechanical properties. A promising approach for disc regeneration is represented by the transplantation of mesenchymal stromal cells (MSCs). The exact mechanism remains unknown. Growing evidence suggests that MSCs can influence cells and modulate cells’ behaviour by secreting a set of bioactive factors. MSCs secretome is composed of several molecules such as soluble protein, lipids, nucleic acids and extracellular vesicles (EVs) involved in inflammation, immunomodulation, cell survival and intercellular communication. The aim of this study was to evaluate the in vitro effects of MSCs secretome on human NP cells (hNPCs) in a 3D culture model with and without inflammatory stimulus. Materials and Methods. MSCs secretome was collected from bone marrow-MSCs (BM-MSCs) and adipose tissue-MSCs (ASCs) after centrifugation and obtained by culturing cells without fetal bovine serum (FBS) for 48 hours. hNPCs were isolated from surgical specimens through digestion with type II collagenase, culture expanded in vitro, encapsulated in alginate beads (three-dimensional culture system) and treated with growth medium (controls), BM-MSCs or ASCs secretome with or without interleukin-1 beta (IL-1b). After 7 days, total RNA was extracted and reverse-transcribed. Gene expression levels of catabolic and anabolic genes were analyzed through real time-polymerase chain reaction (qPCR). Cell proliferation and glycosaminoglycan (GAG) production was assessed by flow cytometry and 1,9-dimethylmethylene blue (DMMB), respectively. hNPCs in alginate beads were stained with Live/Dead assay and detected using confocal immunofluorescence microscopy. Data were analyzed using Graphpad prism 8 and expressed as mean ± S.D. One-way ANOVA analysis was used to compare differences among the groups under exam. Results. Our results reported an increase of hNPCs proliferation after treatment with both MSCs-secretomes. In detail, cell proliferation levels increased at 7 days after ASC-secretome (p ≤ 0,05) and BM-secretome (p ≥ 0,05) treatment compared to control. Live/dead staining showed that cell death was reduced by BM-secretome (p ≤ 0,05); in combined treatment of BM-secretome with IL1b 10ng/mL (p ≤ 0,05) at 7 days compared to control. There is not a significant difference between treated and untreated hNPCs’ GAG synthesis. In addition, gene expression levels resulted to be modulated by MSCs-secretomes under study compared to controls. Conclusions. Although the cell-therapy may be considered an attractive and safe option, MSCs require long and expensive processes. In conclusion, our experimental conditions supported as BM-MSCs and ASCs secretomes could represent cell-free alternative approaches in IDD, overcoming translational limits of cell therapy to the clinical practice

Introduction: Age-related hormonal changes and inactivity lead to systemic bone loss and osteoporotic fractures. However, it is not clear why the vertebral body should be affected so often, or why its anterior region should characteristically sustain a “wedge” deformity. We hypothesise that intervertebral disc degeneration in elderly spines leads to altered spinal load-sharing in such a manner that the anterior region of the vertebral body becomes vulnerable to injury. Methods: Forty thoraco-lumbar “motion segments”, consisting of two vertebrae and the intervening disc and ligaments, were obtained from cadaver spines aged 62–94 yrs. Volumetric bone mineral density (BMD) was measured for various regions of each vertebra using a Lunar Piximus DXA scanner. The distribution of the applied compressive force (1.5 kN) between the anterior and posterior halves of the vertebral body was calculated by pulling a needle-mounted pressure transducer along the sagittal midline of the adjacent disc. Pressure measurements were integrated over area to give force. Anterior and posterior disc forces were subtracted from the applied 1.5 kN to indicate loading of the neural arch. Measurements were repeated with the specimens positioned to simulate various postures in life. The strength of each motion segment was determined by compressing it to failure while positioned in a forward stooped posture. Disc and vertebral morphology were assessed from radiographs, and from digital photographs of tissue sections. Results: Load-bearing by the neural arch in erect posture increased in the presence of intervertebral disc degeneration, and was inversely proportional to the average height of the disc (P< 0.01). High neural arch load-bearing was associated with relatively low BMD in the anterior vertebral body (P< 0.01), and with low compressive strength (P< 0.0001). BMD in the anterior region of the vertebral body was the best univariate predictor of compressive strength (R. 2. = 0.78). Stepwise multiple linear regression showed that 86% of the variance in compressive strength could be explained by the following: anterior vertebral body BMD, vertebral body X-sectional area, and neural arch load-bearing (% of applied load). Forcing age, gender and spinal level into the model did little to improve the prediction. Discussion and Conclusions: Results strongly support our hypothesis. Evidently, intervertebral disc degeneration and narrowing cause the neural arch to “stress shield” the anterior vertebral body whenever the spine is held erect. This leads to reduced BMD in the anterior vertebral body, weakening the spine when it is loaded in a stooped posture. The small age-dependence of results can be attributed to the relatively narrow age range of specimens tested. Vertebral fracture risk can best be assessed from BMD measured in the anterior half of the vertebral body

The use of stem cells transplanted into the intervertebral disc (IVD) is a promising regenerative approach to treat intervertebral disc degeneration (IDD). The aim of this study was to assess the effect of a hydrogel composed of hyaluronic acid (HA) and platelet-rich plasma (PRP) loaded with human mesenchymal stem cells (hMSCs), on IVD extracellular matrix synthesis and nucleus pulposus (NP) marker expression in a whole IVD culture model. HA was blended with batroxobin (BTX), a gelling agent activated in presence of PRP to construct a hydrogel. Bovine IVDs (n=25) were nucleotomised and filled with 1×10. 6. or 2×10. 6. hMSCs suspended in ∼150 mL of the PRP/HA/BTX hydrogel. IVDs harvested at day 0 and nucleotomised IVDs with no hMSCs and/or hydrogel were used as controls. hMSCs alone or encapsulated in the hydrogel were also cultured in well plates to examine the effect of the IVD microenvironment on hMSCs. After 1 week, tissue structure, scaffold integration and gene expression of anabolic (collagen type I, collagen type II and aggrecan), catabolic (matrix metalloproteinase 3 – MMP-3 –, MMP-13 and a disintegrin and metalloproteinase with thrombospondin motifs 4) and NP cell (cytokeratin 19, carbonic anhydrase 12, cluster of differentiation 24) markers were assessed. Histological analysis showed a good integration of the scaffold within the NP area with cell repopulation. At the gene expression level, the hMSC-loaded hydrogels demonstrated to increase disc cell anabolic and catabolic marker expression and promoted hMSC differentiation towards a NP cell phenotype. This study demonstrated that the HA/PRP/BTX may represent a valid carrier for hMSCs being capable of stimulating cell activity and NP marker expression as well as achieving a good integration with the surrounding tissues

Intervertebral disc degeneration (IDD) affects more than 80% of the population and is often linked to a reduction of the proteoglycan content within the nucleus pulposus (NP). The nutritional decline and accumulation of degraded matrix products promote the inflammatory process favoring the onset of disease. Several regenerative approaches based on cell therapy have been explored. Recently, paracrine factors and extracellular vesicles (EVs) such as exosomes have been described to play a fundamental role in the cross-talk between mesenchymal stem cells (MSCs) and NP in the microenvironment. EVs vehicule different molecules: proteins, nucleic acids and lipids involved in intercellular communication regulating the homeostasis of recipient cells. Therefore, MSCs-derived exosomes are an interesting emerging tool for cell-free therapies in IDD. The aim of this study was to evaluate the in vitro effects of MSCs derived exosomes on human NP cells (hNPCs). Exosomes were isolated through a multistep ultracentrifugation of bone marrow-MSCs (BM-MSCs) conditioned media (CM), obtained by culturing BM-MSCs without fetal bovine serum (FBS) for 48 hours. Exosomal morphology was characterized by transmission electron microscope (TEM). The exosomes were quantified by bicinchoninic acid assay (BCA) and cryopreserved at –80 °C. hNPCs derived from surgical speciments digested with type II collagenase. After culture expansion in vitro, hNPCs in alginate beads (three-dimensional culture system) were treated with growth medium (controls), exosomes, CM, interleukin-1 beta (IL-1b), IL-1b plus exosomes, IL-1b plus CM. After 24 hours, total RNA was extracted and reverse-transcribed. Gene expression levels of catabolic and anabolic genes were analyzed through real time-polymerase chain reaction (qPCR). TEM analysis confirmed the cup-shaped vescicles in our preparations. Gene expression levels resulted to be modulated by both exosomes and CM compared to controls. In addition, both treatments were capable to alter the inflammatory stimuli of IL-1b. Interestingly, exosomes were able to change anabolic and catabolic gene expression levels differently from CM. In our experimental conditions, both exosomes and CM from BM-MSCs could be an interesting alternative strategy in intervertebral disc regeneration, overcoming the costs and translational limits of cell therapy to the clinical practice

In our study, the aims were to describe the changes in the appearance of the lumbar spine on MRI in elite fast bowlers during a follow-up period of one year, and to determine whether these could be used to predict the presence of a stress fracture of the posterior elements. We recruited 28 elite fast bowlers with a mean age of 19 years (16 to 24) who were training and playing competitively at the start of the study. They underwent baseline MRI (season 1) and further scanning (season 2) after one year to assess the appearance of the lumbar intervertebral discs and posterior bony elements. The incidence of low back pain and the amount of playing and training time lost were also recorded. In total, 15 of the 28 participants (53.6%) showed signs of acute bone stress on either the season 1 or season 2 MR scans and there was a strong correlation between these findings and the later development of a stress fracture (p < 0.001). The prevalence of intervertebral disc degeneration was relatively low. There was no relationship between disc degeneration on the season 1 MR scans and subsequent stress fracture. Regular lumbar MR scans of asymptomatic elite fast bowlers may be of value in detecting early changes of bone stress and may allow prompt intervention aimed at preventing a stress fracture and avoiding prolonged absence from cricket

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)

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

Backgrounds and aim. Low back pain resulting from Intervertebral 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. Thus, we aimed to investigate and compare their physiological behaviour in in vitro niche that mimics the healthy and degenerated intervertebral disc environment. Methodology. Porcine NC cells were encapsulated in 3D alginate beads to maintain their phenotype then cultured in media to mimic the healthy and degenerate disc environment, together with control NC media for 1 week. Following which viability using PI and Calcein AM, RNA extraction and RT-PCR for NC cell markers, anabolic and catabolic genes analysed. Proteomic analysis was also performed using Digiwest technology. Results. A small increase in 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 NCs 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. Discussion. 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 initial work has been then translated to investigate the potential use of iPSCs differentiated into notochordal like cells as potential regenerative cell sources. Conflicts of interest: No conflicts of interest. Sources of funding: This project has received funding from the European Union Horizon 2020 research and innovation programme under grant agreement No 825925

Intervertebral disc (IVD) degeneration occurs with aging, leading to low back pain (LBP), which is one of the leading conditions of disability worldwide. With the lack of effective treatment, decellularized extracellular matrix (dECM) – based biomaterials have been proposed for IVD regeneration. However, the impact of donor ages on tissue repair had never been explored before in the disc field. Therefore, we aimed to address this question. For that, a decellularization protocol for bovine nucleus pulposus (NP) of different aged donors (fetus, young and old) was optimized by testing several detergents (SDS and Triton). The process efficiency was evaluated in terms of DNA and cell removal, as well as ECM preservation. Afterwards, dECMs were repopulated with bovine NP cells and cultured ex vivo. At day 7, cell behavior, ECM de novo synthesis and remodeling were evaluated [1]. Moreover, dECMs’ inflammatory response was assessed after in vivo CAM assay. Finally, inflammatory and angiogenic cytokines were analyzed in the conditioned media-derived from dECMs by using a cytokine array. As results, an optimal decellularization protocol (SDS 0.1%, 1h), efficient at removing cells and DNA from bovine NPs, while preserving ECM cues of native tissues, was developed. After repopulation, aggrecan increased in younger NPs, while collagen 2 decreased which may be indicative of matrix remodeling [1]. After in vivo CAM assay, fetal dECMs showed the highest inflammatory response. Finally, no statistically significant changes of cytokines were detected in the matrices, despite for a trend of higher IFN-α, IFN-γ and LIF in fetal dECMs, IL-1β in young dECMs and Decorin in old dECMs. Overall, this work uncovered the importance of tissue donor ages for tissue regenerative purpose, opening new avenues for the development of appropriate therapeutic strategies for IVD degeneration. Acknowledgments: FCT, EUROSPINE, ON Foundation

Intervertebral disc (IVD) degeneration is inadequately understood due to the lack of in vitro systems that fully mimic the mechanical and biological complexity of this organ. We have recently made an advancement by developing a bioreactor able to simulate physiological, multiaxial IVD loading and maintain the biological environment in ex vivo IVD models [1]. To validate this new bioreactor system, we simulated natural spine movement by loading 12 bovine IVDs under a combination of static compression (0.1 MPa), cyclic flexion/extension (±3˚, ±6˚ or 0-6˚) and cyclic torsion (±2˚, ±4˚ or 0-4˚) for more than 10’000 (0.2 Hz) or 100’000 (1 Hz) cycles over 14 days. A higher number of cycles increased the release of glycosaminoglycans and nitric oxide, as an inflammation marker, whereas fewer cycles maintained these two factors at physiological levels. All applied protocols upregulated the expression of MMP13 in the outermost annulus fibrosus (AF), indicating a collagen degradation response. This was supported by fissures observed in the AF after a longer loading duration. Increasing loading cycles induced high cell death in the nucleus pulposus and inner AF, while with fewer cycles, high cell viability was maintained in all IVD regions, irrespective of the magnitude of rotation. Less frequent multiaxial loading maintains IVD homeostasis while more frequent loading initiates an IVD degenerative profile. Specifically, the morphological and molecular changes were localized in the AF, which can be associated with combined flexion/extension and torsion. More loading cycles induced region-specific cell death and a higher release of extracellular matrix molecules from the innermost IVD regions, likely associated with longer exposure to static compression. Altogether, we demonstrated the advantages of the multiaxial bioreactor to study region-specific response in the IVD, which will allow a more profound investigation of IVD degeneration under different combinations of motions

Objective. Modic changes (MC) is a form of intervertebral disc degeneration visible as subchondral and vertebral bone marrow changes on spine magnetic resonance (MR). Their etiology is not understood, but microbial infection may be involved for some subtypes. This study set out to test for an association between MC and gut microbiome in a population sample. Methods. Presence of MC was evaluated in lumbar MR images and gut microbiome assessed using 16S sequencing in TwinsUK dataset (N=309). Cases were identified by the presence of MC of any type, while controls were those without MC. Amplicon sequence variants (ASVs) have been obtained for 16S sequences followed by relative abundance calculation and centred log-ratio transformation. Linear mixed-effects models were applied to test for association between the ASVs at different taxon levels and MC adjusting for technical covariates and demographics. Results. Nominally significant (p<0.05) associations with MC were obtained for 6 ASVs annotated to species level (min p = 0.0016 for Sanguibacteroides justesenii), 8 ASVs annotated to genus level (min p = 0.0091 for Syntrophomonas), and 2 ASVs annotated to family level (min p = 0.0099 for Syntrophomonadaceae). None of the associations were significant after correction for multiple testing. Also, no statistically significant difference in microbial diversity was found between MC cases and controls. Conclusions. The results of this pilot study provide limited evidence of association between MC and gut microbiome. Further studies including MC stratified by subtype are warranted as well as studies based on advanced metagenome sequencing rather than 16S approach. No conflicts of interest. The study was supported by Versus Arthritis grant # 21227

Objective. Modic changes (MC), a form of intervertebral disc degeneration visible as subchondral and vertebral bone marrow changes on spine magnetic resonance (MR), are known to be associated with low back pain. This study aimed to identify genes contributing to the development of MC using genome-wide association study. Methods. Presence of MC was evaluated in lumbar MR images in the Northern Finland Birth Cohort 1966 (NFBC1966, N=1182) and TwinsUK (N=647). Genome-wide association analyses were carried out in the cohorts separately using a linear regression model fitted to test for additive effects of SNPs and adjusting for age, sex, BMI, and either family relatedness via a kinship matrix (TwinsUK) or population stratification using principal components (NFBC1966). Meta-analysis of the two studies was carried out using the inverse-variance weighting approach. Results. A locus associated with MC reaching genome-wide significance (p<5e-8) was found on chromosome 9 with the lead SNP rs1934268 in intron 6 of the PTPRD gene. The SNP is located in the region of binding for a number of transcription factors which are involved in the development of the musculoskeletal system and spine cord. Conclusions. The first GWAS of MC has identified a likely functional intronic locus in PTPRD on chromosome 9 implicating musculoskeletal development. This work sheds light on the genesis of MC and paves the way for further studies on the shared genetic factors underlying the various features of spine degeneration. No conflicts of interest. Sources of Funding: The study was supported by EU FP7 project PainOMICs (grant agreement #602736), University of Oulu (grant #24000692), Oulu University Hospital (grant #24301140), and the European Regional Development Fund (grant # 539/2010 A31592). MBF, MK, and SS contributed equally to this study

Low back pain is thought to relate to intervertebral disc (IVD) degeneration. Although the mechanisms have not been clearly identified, exhaustion of nucleus pulposus cells and their producing matrix is regarded as one cause. The matrix of the IVD is continuously replenished and remodeled by tissue-specialized cells and are crucial in supporting the IVD function. However, due to aging, trauma, and genetic and lifestyle factors, the cells can lose their potency and viability, thereby limiting their collective matrix production capacity. We have discovered the link between loss of angiopoietin-1 receptor (Tie2)-positive human NP progenitor cells (NPPC) and IVD degeneration. Tie2+ cells were characterized as undifferentiated cells with multipotency and possessing high self-renewal abilities. Thus we and others have proposed Tie2+ NPPC as a potent cell source for regenerative cell therapies against IVD degeneration. However, their utilization is hindered by low Tie2-expressing cell yields from NP tissue, in particular from commonly available older and degenerated tissue sources. Moreover, NPPC show a rapid Tie2 decrease due to cell differentiation as part of standard culture processes. As such, a need exists to optimize or develop new culture methods that enable the maintenance of Tie2-expressing NPPC. Trials to overcome these difficulties will be shared

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

Aims

Degenerative cervical spondylosis (DCS) is a common musculoskeletal disease that encompasses a wide range of progressive degenerative changes and affects all components of the cervical spine. DCS imposes very large social and economic burdens. However, its genetic basis remains elusive.

Introduction and Objective. Low back pain (LBP) is a major cause of long-term disability in adults worldwide and it is frequently attributed to intervertebral disc (IVD) degeneration. So far, no consensus has been reached regarding appropriate treatment and LBP management outcomes remain disappointing. Spine unloading or traction protocols are common non-surgical approaches to treat LBP. These treatments are widely used and result in pain relief, decreased disability or reduced need for surgery. However, the underlying mechanisms -namely, the IVD unloading mechanobiology- have not yet been studied. The aim of this first study was to assess the feasibility of IVD unloading in a large animal organ culture set-up and evaluate its impact on mechanobiology. Materials and Methods. Bovine tail discs (diameter 16.1 mm ± 1.2 mm), including the endplates, were isolated and prepared for culture. Beside the day0 sample that was processed directly, three other discs were cultured for 3 days and processed on day4. One disc was loaded in the bioreactor according to a previously established physiological (compressive) loading protocol (2h/day, 0.2Hz). The two other discs were embedded in biocompatible resin, leaving the cartilage endplate free to permit nutrient diffusion, and fitted in the traction holder; one of these discs was kept in free swelling conditions, whereas the second was submitted to cyclic traction loading (2h/day, 0.2Hz) corresponding to 30% of the animal body weight corrected for organ culture. Results. The cell viability assessed on lactate dehydrogenase and ethidium homodimer stained histological slides was not different between the three cultured discs. This means that the disc viability was not affected neither by the embedding, nor by the traction itself. Compared to the physiologically loaded disc, the gene expression of COL1, COL2 and ACAN was higher in the nucleus pulposus and inner annulus fibrosus of the traction treated disc. In the outer annulus fibrosus of this disc TAGLN and MKX were higher expressed upon traction than in the physiologically loaded disc. Conclusions. Based on these preliminary data, we can conclude that large animal organ culture allows effective unloading of the disc, while preserving cell viability and modulating cellular gene expression responses. This sets the ground for future experiments and opens the door to an evidence-based improvement of clinical spine traction protocols and LBP management overall

Introduction and Objective. Intervertebral disc (IVD) degeneration accompanying with low back pain is a serious worldwide problem. Even though, surgical treatments are available for pain relief, there is an urgent need to establish enduring cell-based remedies. Notochordal (NC) cells as the ancestor of nucleus pulposus (NP) cells in human IVD are a promising therapeutic target. It has been reported that the loss of NC cells after childhood could promote the onset of disc degeneration. Thus, we firstly, aimed to optimise the culture of NC cells in vitro without using the FCS in alginate (3D) culture systems, secondly, investigate their behaviour in healthy and degenerate niche and lastly, co-culture these cells with degenerated NP cells to assess their regeneration potentials. Materials and Methods. Porcine NC cells were extracted using pronase treatment followed by overnight digestion in 0.01% collagenase II. After extraction, cells were culture in 1.2% alginate beads (gold standard 3D culture) in either low glucose DMEM or αMEM medium. Cells were harvested after 24 hours, 1 week and 2 weeks for gene expression analysis and formalin fixed paraffin embedding. Quantitative Real-Time PCR and Immuno-staining were performed for analysis of NC markers (KRT18, FOXA2 and T) and COL I as a negative marker. Next, NC cells were cultured in healthy and degenerate medium to assess their viability and behaviour. Results. A mixed phenotype of NC and NP cells was observed in alginate bead cultures. NC phenotype was observed within all culture conditions with production of GAGs and maintenance of vacuolated phenotype. Gene expression analysis showed no significant difference between the culture of NC cells in low glucose DMEM and αMEM medium. Interestingly, NC cell viability was maintained in both healthy and degenerate media, despite observing more dead cells in degenerate conditions. Current investigations are comparing the behaviour of NC cells in healthy and degenerate niche. Conclusions. Investigating the preservation of NC phenotype in alginate culture and studying their behaviour between healthy and degenerate conditions would lead us to better understand their characteristics in different niches and how we can further use them in therapeutic purposes for disc degeneration

Introduction and Objective. Intervertebral disc (IVD) degeneration is one of the major contributors to low back pain, the leading cause of disability worldwide. This multifactorial pathological process involves the degradation of the extracellular matrix, inflammation, and cell loss due to apoptosis and senescence. While the deterioration of the extracellular matrix and cell loss lead to structural collapse of the IVD, increased levels of inflammation result in innervation and the development of pain. Amongst the known regulators of inflammation, toll-like receptors (TLRs) and more specifically TLR-2 have been shown to be specifically relevant in IVD degeneration. As strong post-transcriptional regulators, microRNAs (miRNAs) and their dysregulation has been connected to multiple pathologies, including degenerative diseases such as osteoarthritis and IVD degeneration. However, the role of miRNAs in TLR signalling in the IVD is still poorly understood and was hence investigated in this study. Materials and Methods. Human Nucleus pulposus (hNP) and Annulus fibrosus (hAF) cells (n=5) were treated with the TLR-2/6 specific agonist PAM2CSK4 (100 ng/mL for 6 hours) in order to activate the TLR2 signalling pathway. After the activation both miRNA and mRNA were isolated, followed by next-generation sequencing and qPCR analysis of proinflammatory cytokines respectively. Furthermore, cell supernatants were used to analyze the secretion of proinflammatory cytokines with enzyme-linked immunosorbent assay. TLR-2 knockdown (siRNA) cells were used as a control. Statistical analysis was conducted by performing Kolmogorov-Smirnov test and a two-tailed Student's t-test using GraphPad Prism version 9.0.2 for Windows (GraphPad Software, La Jolla California USA). Results. TLR-2 activation resulted in the induction of an inflammatory cell response, with a significant increase in gene expression of interleukin (IL)-6 (525 ± 180 fold change, p < 0.05) and IL-8 (7513 ± 1907 fold change, p < 0.05) and protein secretion of IL-6 (30.5 ± 8.1 pg/mL) and IL-8 (28.9 ± 5.4 pg/mL). TLR-2 activation was furthermore associated with changes in the miRNA profile of hNP and hAF cells. Specifically, we identified 10 differentially expressed miRNAs in response to TLR-2 activation, amongst which were miR-335–3p (1.45 log2 FC, p < 0.05), miR-125b-1–3p (0.55 log2 FC, p < 0.05), and miR-181a-3p (−1.05 log2 FC, p < 0.05). Conclusions. The identified miRNAs are known to be associated with osteoarthritis (miR-335-3p), inflammation and IVD degeneration (mir-125-1-3p and miR-181a-3p), but the link to TLR signalling has not been previously reported. Experiments to validate the identified miRNAs and elucidate their functional role are undergoing. The identification of these miRNAs provides an opportunity to further investigate miRNAs in the context of TLR activation and inflammation and to enhance our understanding of underlying molecular mechanisms behind disc degeneration, inflammation, and TLR dysregulation

Aims

Lumbar spinal stenosis (LSS) is a common skeletal system disease that has been partly attributed to genetic variation. However, the correlation between genetic variation and pathological changes in LSS is insufficient, and it is difficult to provide a reference for the early diagnosis and treatment of the disease.

Abstract. Objectives. Back pain will be experienced by 70–85% of all people at some point in their lives and is linked with intervertebral disc (IVD) degeneration. The aim of this study was to 1) compare 3D internal strains in degenerate and non-degenerate human IVD under axial compression and 2) to investigate whether there is a correlation between strain patterns and failure locations. Methods. 9.4T MR images were obtained of ten human lumbar IVD. Five were classed as degenerate (Pfirrmann = 3.6 ± 0.3) and five were classed as non-degenerate (Pfirrmann = 2.0 ± 0.2). MR Images were acquired before applying load (unloaded), after 1 kN of axial compression, and after compression to failure using a T2-weighted RARE sequence (resolution = 90 µm). Digital Volume Correlation was then used to quantify 3D strains within the IVDs, and failure locations were determined from analysis of the failure MRIs. Results. Average of axial strains were higher (p<0.05) in the degenerate samples compared to the non-degenerate (−3.4 vs-5.2%, respectively), particularly in the posterior and lateral annulus (−6.2 vs −3.6%, and −5.6 vs −3.5%, respectively). Maximum 3D compressive strains were higher (p<0.05) in the posterior annulus and nucleus regions of the degenerate discs compared to non-degenerate (−9.8 vs −6.2%, and −7.7 vs −5.5%, respectively). In all samples peak tensile and shear strains were observed close to the endplates. All samples failed through the endplates with fractures in the nucleus region in all non-degenerate samples, and fractures in the lateral annulus regions in all degenerate samples. Conclusion. Degeneration caused significant changes to strain distributions within IVDs, particularly at the lateral and posterior AF regions. A shift from endplate failure in the nucleus to the annulus region was observed which was also seen in peak axial internal strains demonstrating a possible correlation between internal IVD strains, and endplate failure locations. Declaration of Interest. (b) declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported:I declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research project

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.

Introduction. Decreasing endplate porosity has been proposed as a risk factor for intervertebral disc degeneration, because it interferes with disc metabolite transport. However, endplate porosity has recently been shown to increase with age and disc degeneration. We hypothesise that this increase reflects adaptive remodelling in response to altered loading from adjacent discs. Methods. Nineteen cadaver motion segments (61–98 yrs) were compressed to 1kN while a pressure-transducer was pulled across the mid-sagittal diameter of the disc. Stress profiles indicated nucleus (intradiscal) pressure (IDP) and maximum stress in the anterior and posterior annulus. Subsequently, micro-CT was used to evaluate endplate porosity along the antero-posterior diameter of the adjacent endplates. Data were analysed using ANOVA and linear regression. Results. Endplate porosity averaged 67% centrally (where IDP averaged 0.85MPa) and decreased steadily to 48% and 53% in the anterior and posterior periphery, where maximum stresses were 1.37MPa and 1.33MPa, respectively. In each region, porosity was inversely related to IDP (or maximum stress) with R. 2. = 0.49, 0.32 and 0.31 respectively (P<0.001 in each case). Porosity was 3% higher in the inferior (weaker) endplate of the disc compared to the superior endplate (P=0.07). Conclusion. In old spines, strong correlations between endplate porosity and stress in each anatomical region indicate mechanically-adaptive remodelling. Regional differences in endplate porosity (across the antero-posterior diameter) probably reflect the varying nutritional demands of nucleus and annulus, as well as adaptations to loading from an adjacent decompressed disc. In younger age-groups, high loading could possibly reduce endplate porosity, promoting disc degeneration

The action of fast bowling in the game of cricket is known to cause injuries to the lumbar spine. We studied a group of 16- to 18-year-old fast bowlers, selected for special training in Western Australia. All 24 had MR scans of the spine, 22 had radiographs and CT scans; in 20 the bowling technique was analysed biomechanically. There was a high incidence of back pain and this was always associated with a radiological abnormality. Pars interarticularis defects were diagnosed in 54% and intervertebral disc degeneration in 63%. Bowling actions which involved counter-rotation were associated with a higher incidence of both injuries

The pathophysiology of intervertebral disc degeneration has been extensively studied. Various factors have been suggested as influencing its aetiology, including mechanical factors, such as compressive loading, shear stress and vibration, as well as ageing, genetic, systemic and toxic factors, which can lead to degeneration of the disc through biochemical reactions. How are these factors linked? What is their individual importance? There is no clear evidence indicating whether ageing in the presence of repetitive injury or repetitive injury in the absence of ageing plays a greater role in the degenerative process. Mechanical factors can trigger biochemical reactions which, in turn, may promote the normal biological changes of ageing, which can also be accelerated by genetic factors. Degradation of the molecular structure of the disc during ageing renders it more susceptible to superimposed mechanical injuries. This review supports the theory that degeneration of the disc has a complex multifactorial aetiology. Which factors initiate the events in the degenerative cascade is a question that remains unanswered, but most evidence points to an age-related process influenced primarily by mechanical and genetic factors

1. At the present stage of our experience, when 150 patients have been analysed over a period of five years, the conclusion has been reached that anterior interbody fusion in the lower lumbar spine is a procedure which should be added to our surgical armamentarium for use in selected cases. 2. Patients suffering from chronic intervertebral disc degeneration whose main symptoms are recurrent incapacitating backache derive the most benefit from this procedure. 3. When used as a salvage operation in patients who have had previous unsuccessful laminectomy or posterior fusion, good results can be expected. 4. In patients with spondylolisthesis anterior interbody fusion should be confined to cases in which the vertebral bodies have not slipped forward more than one-third

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

Background. Intervertebral disc (IVD) degeneration is a major cause of low back pain (LBP). We have developed an injectable hydrogel (NPgel), which following injection into bovine IVD explants, integrates with IVD tissue and promotes disc cell differentiation of delivered mesenchymal stem cells (MSCs) without growth factors. Here, we investigated the injection of NPgel+MSCs into IVD explants under degenerate culture conditions. Methods and Results. The NPgel integrated with bovine and human degenerate Nucleus Pulposus (NP) tissue and hMSCs produced matrix components: aggrecan, collagen type II and chondroitin sulphate in standard and degenerate culture conditions. Significantly increased cellular immunopositivty for aggrecan was observed within native NP cells surrounding the site where NPgel+MSCs were injected (P≤0.05). In NP explants a significant decrease in catabolic factors were observed where NPgel+MSCs was injected in comparison to controls. Conclusions. In agreement with our previous findings NPgel was sufficient alone to induce NP cell differentiation of MSCs following injection into NP tissue explants. Here, we have shown that viability is maintained even in degenerate conditions. Injection of NPgel with MSCs increased aggrecan expression and reduced MMP3 and IL-1R1 expression by native NP cells. The NPgel with incorporated MSCs has the potential to regenerate the NP and provide mechanical support, whilst reducing the catabolic phenotype of degenerate NP cells, as a treatment strategy for IVD degeneration. No conflicts of interest. Sources of funding: Funded by ARUK and MRC

The safety of nucleus implants remains an open issue in the treatment of intervertebral disc degeneration. Post-operative migration and subsequent extrusion represent a high risk of potential unsatisfactory outcome. The effectiveness of additionally sewing a biointegrative nucleus implant into an annulus defect was investigated therefore in this experiment. Laminectomy preserving the facet joints was performed on seven human functional spinal units (FSU’s). A reproducible annulus defect of 6×6 mm was incised, followed by a standard nucleotomy procedure and subsequent introduction of the implants. These woven patches consist of biointegrative, absorbable polyglycolic acid (PGA), lyophilized with hyaluronic acid. The annulus sealing technique requires placing a PGA-patch adjacent to the inner annulus, fixed by sutures (Polysorb 3-0, Syneture) at its four corners. Unsealed annulus defects served as a control group. FSU’s were loaded with a bending torque of 5 to 7.5 Nm. Continual revolution of the specimen around its vertical axis resulted in a combination of lateral, dorsal and flexural bending. During application of loads, implant herniation level was determined every 1 000 cycles according to predefined criteria. Tests were stopped after reaching 20 000 cycles. Five of totally six sewed specimens withstood 20 000 load cycles, whereas only one of five not sewed specimens terminated successfully. Based on the Mann-Whitney test, significant increase in stability can be detected for the sewed procedure. Sewing a biointegrative annulus implant into an annulus defect improves nucleus implant containment. It remains to be shown whether this annulus sealing technique is also effective in highly degenerated annulus tissue. Furthermore, a minimally invasive implantation device is crucial for application in a clinical setting

Introduction: Modic changes are vertebral endplate changes visible in magnetic resonance imaging (MRI), which associate with degenerative intervertebral disc disease. Twin studies suggest that intervertebral disc degeneration and low back pain may be primarily explained by genetic factors. There are, however, no studies on genetic factors in Modic changes. Materials and methods: Eleven variations in eight genes (COL9A2, COL9A3, COL11A2, IL1A, IL1B, IL6, MMP-3 and VDR) were genotyped in an occupational cohort of 159 male train engineers and 69 male paper mill workers. All the study subjects were MRI scanned and evaluated for Modic changes. Results: Out of 228 subjects studied, 128 (56%) were found to have Modic change at one or more disc levels. 15% of them had exclusively Modic type I while 32% had exclusively Modic II changes. 10% of the subjects had both type I and type II changes. When single nucleotide polymorphisms (SNPs) were analyzed independently, none of them significantly associated with Modic changes. However, when the gene-gene interactions were evaluated IL1A and MMP-3 polymorphisms together associated with type II Modic changes (OR 3.2, 95% CI 1.2–8.5; p = 0.038). Furthermore, IL-1 gene cluster together with MMP-3 polymorphism associated significantly with type II Modic changes (OR = 8.14, 95% CI 1.72–38.44; p = 0.008). Discussion: This is the first study evaluating the role of genetic factors in relation to Modic changes. Genetic variations in IL-1 cluster and MMP-3 gene were found together to associate significantly with type II Modic changes

Aims: As inflammation plays a key role in the etiology of intervertebral disc degeneration, we suggest a possible contribution of pro-inflammatory gene polymorphisms in the pathogenesis of adolescent idiopathic scoliosis (AIS). The nucleus pulposus of scoliotic discs responds to exogenous stimuli by secreting interleukin-6 (IL-6) and other inflammatory cytokines. The association between matrix metalloproteinases (MMPs) and disc degeneration has been reported by several investigators. A human MMP-3 promoter 5A/6A gene polymorphism regulates MMP-3 genes expression, while the G/C polymorphism of the promoter region of IL-6 gene influences levels and functional activity of the IL-6 protein. Methods: We conducted a case-control study to investigate whether the 5A/6A polymorphism of the MMP-3 gene and the G/C polymorphism of the promoter region of IL-6 gene were associated with susceptibility to AIS. Results: The frequency of the 5A/5A genotype of MMP-3 gene polymorphism in patients with scoliosis was almost 3 times higher than in controls (30.2 % vs 11.2 %, p 0.001) and the frequency of the G/G genotype of IL-6 gene polymorphism in patients with scoliosis was almost 2 times higher than in controls (52.8 % vs 26.2 %, p < 0.001). 5A/5A genotype of MMP-3 gene polymorphism and G/G genotype of IL-6 gene polymorphism are independently associated with an higher risk of scoliosis (odds ratio respectively 3.34 and 10.54). Conclusions: This is the first study that has evaluated the possibility that gene variants of IL-6 and MMPs might be associated with scoliosis and suggests that MMP-3 and IL-6 promoter polymorphisms constitute important factors for the genetic predisposition to scoliosis

Background and Purpose. Intervertebral disc (IVD) degeneration is a prominent cause of low back pain. IVD cells expressing angiopoietin-1 receptor Tie2 represent a progenitor cell population which decreases with progression of IVD degeneration. Homing of mesenchymal stem cells (MSCs) is a physiological mechanism aiming to enhance the regenerative capacity of the IVD. The purpose of this study was to assess the effect of MSC homing on the Tie2 positive IVD progenitor cell population, the IVD cell viability, and the proliferative phenotype of the IVD cells. Methods and Results. Human MSCs were isolated from bone marrow aspirates and labelled with fluorescent dye. Whole IVDs with endplates were harvested from bovine tails; MSCs were placed on the endplates. Human traumatic, degenerative and healthy IVD tissues were obtained from patients and organ donors. MSCs were added onto tissue samples. After 5 days, IVD cells were isolated. Percentages of Tie2 positive, DAPI positive (dead) and Ki-67 positive (proliferative) IVD cells were determined. MSC homing or co-culture significantly increased the proportion of Tie2 positive progenitor IVD cells in bovine and 7/10 human IVDs, decreased the fraction of dead IVD cells in bovine and 7/10 human IVDs, and induced a proliferative phenotype in bovine and 5/6 human IVDs. Conclusion. Stimulation of bovine and human IVDs by MSC homing resulted in an enhanced population of Tie2 positive IVD progenitor cells, induced a proliferative response and reduced IVD cell death. Hence, the interaction with recruited MSCs may contribute to an improved survival of IVD cells, helping to reverse or slow down an ongoing degenerative process. Conflicts of interest: The authors declare no conflicts of interest. Sources of funding: AO Foundation and AOSpine International

Low back pain is more common in women than men, yet most studies of intervertebral disc (IVD) degeneration do not address sex differences. In humans, there are sex differences in spinal anatomy and degenerative changes in biomechanics, and animal models of chronic pain have demonstrated sex differences in pain transduction. However, there are few studies investigating sex differences in annular puncture IVD degeneration models. IVD puncture is known to result in progressive biomechanical alterations, but whether these IVD changes correlate with pain is unknown. This study used a rat IVD injury model to determine if sex differences exist in mechanical allodynia, biomechanics, and the relationship between them, six weeks after IVD injury. Procedures were IACUC approved. 24 male & 24 female four-month-old Sprague-Dawley rats underwent a sham or annular puncture injury surgery (n=12 male, 12 female). In injury groups, three lumbar IVDs were each punctured three times with a needle, and injected with tumor necrosis factor-alpha. Mechanical allodynia was tested biweekly using von Frey filaments. Six weeks after IVD injury, rats were euthanized and motion segments were dissected for non-destructive axial tension-compression and torsional rotation biomechanical testing. Two-way ANOVA with Bonferroni corrections identified statistically significant differences (p < 0 .05) and correlations used Pearson's coefficient. Annular puncture injury induced a significant increase in mechanical allodynia compared to sham in male but not female rats up to six weeks after injury. There was a significant sex effect on both torque range and torsional stiffness, with males exhibiting greater stiffness and torque range than females. Tensile stiffness, compressive stiffness, and axial range of motion showed no sex difference. Males and females showed similar patterns of correlation between variables when sham and injury groups were analyzed together, but correlations were stronger in males. Most correlations were clustered within testing approach: axial biomechanics negatively correlated, torsional biomechanics positively correlated, and von Frey thresholds positively correlated. Surprisingly, mechanical allodynia did not correlate with any biomechanics after injury, and the axial and torsional biomechanics showed little correlation. This study demonstrates that males and females respond to IVD injury differently. Given the absence of correlation between pain and biomechanics, pain cannot be attributed completely to biomechanical changes. This may explain why spinal fusion surgery, an intervention limited to the spine, has produced inconsistent results and is controversial for patients with low back pain. Thus, in addressing low back pain, we must consider both spinal tissues and the nervous system. Further, the limited correlation between axial and torsional biomechanics indicates that IVD injury may have distinct effects on nucleus pulposus and annulus fibrosus. Biomechanics did not differ between sham and injury at week six, suggesting healing after injury. It remains possible that acute biomechanical changes may initiate chronic pain pathogenesis. We conclude that the observed sex differences demonstrate the need for inclusion of both males and females in IVD injury and pain studies, and suggest that males and females may require different treatments for conditions that appear similar

Background: The epidemiology of Schmorl nodes is based on post-mortem investigations. The proposed pathogenesis of the focal nodes is a bulging of the disc into the vertebral body depending of the degree of osteoporosis. Secondar peripheral osteophytes are formed at the annular insertion. Study design: A cross sectional epidemiological study of 4151 participants of the Copenhagen Osteoarthritis Study in 1993 with a 13 years follow with the Roland-Morris (R-MQ) back pain questionnaire. In 1993 standardized, lateral radiographs of the lumbar spine were recorded and the bone mineral density (BMD) was estimated by digital x-ray radiogrammetry of standardised hand x-rays. Methods: Statistical correlations were made between Schmorl nodes and low back pain in 1993, the R-MQ score, BMD and the presence of osteophytes, disc degeneration and endplate sclerosis. Results: There were 2610 women and 1538 men. At follow up 1190 women and 674 responded. In 196 cases one or more Schmorl nodes in the lumbar spine were found (women 3.7 %, men 6.5 %). A decreasing prevalence of Schmorl nodes by ages was found in both genders (p< 0.000). At the time of the radiographic examination participants with Schmorl nodes clamed of low back pain (p=0.003). The presence of nodes was without relation to osteophytosis, intervertebral disc degeneration or sclerosis of endplates (p> 0.14) in 1993. Neither was the R-MQ score at follow-up related to Schmorls nodes(p> 0.26). The presence of nodes was associated with higher BMD (mean 0.50 (SD 0.079) versus 0.53 (SD 0.081)(p=0.000), however the difference disappeared taking into account age at examination. Conclusion: This large scaled epidemiological study cannot confirm the hitherto hold opinion of the implication of the Schmorls nodes. The nodes are not associated with radiological degeneration and osteoporosis neither are they a predictor of lower back pain later in life

Intervertebral disc (IVD) degeneration plays a major role in low back pain which is the leading cause of disability. Current treatments in severe cases require surgical intervention often leading to adjacent segment degeneration. Injectable hydrogels have received much attention in recent years as scaffolds for seeding cells to replenish disc cellularity and restore disc properties and function. However, they generally present poor mechanical properties. In this study, we investigated several novel thermosensitive chitosan hydrogels for their ability to mimic the mechanical properties of the nucleus pulposus (NP) while being able to sustain the viability of NP cells, and retain proteoglycans. CH hydrogels were prepared by mixing the acidic chitosan solution (2% w/v) with various combinations of three gelling agents: sodium hydrogen carbonate (SHC) and/or beta-glycerophosphate (BGP) and/or phosphate buffer (PB) (either BGP0.4M, SHC0.075M-BGP0.1M, SHC0.075M-PB0.02M or SHC0.075M-PB0.04M). The gelation speed was assessed by following rheological properties within 1h at 37°C (strain 5% and 1Hz). The mechanical properties were characterized and compared with that of human NP tissues. Elastic properties of the hydrogels were studied by evaluating the secant modulus in unconfined compression. Equilibrium modulus was also measured, using an incremental stress-relaxation test 24h after gelation in unconfined compression (5% strain at 5%/s followed by 5min relaxation, five steps). Cells from bovine IVD were encapsulated in CH-based gels and maintained in culture for 14 days. Cytocompatibility was assessed by measuring cell viability, metabolism and DNA content. Glycosaminoglycan (GAG) synthesis (retained in the gel and released) was determined using DMMB assay. Finally injectability was tested using human cadaveric discs. Unconfined compression confirmed drastically enhanced mechanical properties compared to conventional CH-BGP hydrogels (secant Young modulus of 105 kPa for SHC0.075PB0.02 versus 3–6 kPa for BGP0.04). More importantly, SHC0.075PB0.02 and SHC0.075BGP0.1 hydrogels exhibited mechanical properties very similar to NP tissue. For instance, equilibrium modulus was 5.2±0.6 KPa for SHC0.075PB0.02 and 8±0.8 KPa for SHC0.075BGP0.1 compared to 6.1±1.7 KPa for human NP tissue. Rheological properties and gelation time (G′=G″ after less than 15 s at 37°C, and rapid increase of G') of these hydrogels also appear to be adapted to this application. Cell survival was greater than 80% in SHC0.075BGP0.1 and SHC0.075PB0.02 hydrogels. Cells encapsulated in the new formulations also showed significantly higher metabolic activity and DNA content after 14 days of incubation compared to cells encapsulated in BGP0.4 hydrogel. Cells encapsulated in SHC0.075BGP0.1 and SHC0.075PB0.02 produced significantly higher amounts of glycosaminoglycans (GAG) compared to cells encapsulated in SHC0.075PB0.04 and BGP0.4 hydrogels. The total amount of GAG was higher in SHC0.075BGP0.1 hydrogel compared to SHC0.075PB0.02. Interestingly, both the SHC0.075BGP0.1 and SHC0.075PB0.02 hydrogels retained similar amounts of GAG. Injectability through a 25G syringe, filling of nuclear clefts and good retention in human degenerated discs was demonstrated for SHC0.075PB0.02 hydrogel. SHC0.075BGP0.1 appears to be a particularly promising injectable scaffold for IVD repair by providing suitable structural environment for cell survival, ECM production and mechanical properties very similar to that of NP tissue

Aims: Recent advances in our understanding of intervertebral disc biology have led to develop novel treatments for intervertebral disc degeneration (IDD). With the ability to provide sustained delivery of a potentially therapeutic agent, gene therapy has shown much promise in regard to the treatment of IDD. The aims of this study are (part 1) to test efficacy in delaying course of IDD by intrediscal injection of adenoviral vectors carrying human BMP-2 and (part 2) to describe the application of an inducible system in order to modulate transgene expression. Methods: (Part 1) IDD was induced in 13 NZW rabbits by anterolateral stab. Three weeks post-stab, saline with or without virus was injected directly into stabbed lumbar discs. Group 1 (n=8) received Ad/hBMP-2 while group 2 (n=5) received saline only. Rabbits were followed longitudinally with MRIs and X-rays preoperatively for up to 12 weeks post-stab. ELISAs were done to confirm BMP-2 production. (Part 2) Human nucleus pulposus cells (NPC) were transduced with an adenoviral vector that expresses GFP under the control of a tetracycline (Ad/GFP. tet. ). Cells were cultutred with and without tetracycline. Transgene expression was assessed by detecting GFP signal with both the FACS and the fluorescent microscope. Results: (Part 1) By 12 weeks, the saline-injected discs had lost 49% of their MRI Index, in contrast to only a 25% decrease for the Ad/hBMP-2 treated discs. X-rays demonstrated no obvious bony intervertebral fusion in either group. ELISAs confirmed vigorous hBMP-2 production 3 weeks after therapeutic gene transfer. (Part 2) NPC expressed GFP after transduction. GFP positivity was not observed two days after administration of tetracycline. The cells expressed GFP again three days after removal of tetracycline. Discussion: The results of this study demonstrate the efficacy of vector-mediated BMP-2 gene transfer to alter the course of IDD in a reproducible animal model, as well as the potential to control transgene expression, improving safety

1. In this series of posterior onlay grafting with fresh autogenous bone and without internal fixation, in the treatment of non-infective structural lesions in the lumbo-sacral area, 71 per cent of the patients were relieved of their symptoms, but bony fusion was obtained in only 60 per cent. 2. It is probable that with this technique twelve weeks' immobilisation in a plaster bed is required. 3. Some failures are ascribed to the use of an insufficient quantity of bone or to poor apposition of the graft to its bed. 4. It is evident that the more vertebrae one attempts to fuse the more difficult it is to succeed. When the diseased area is successfully fused but an unnecessarily long graft has been used, a pseudarthrosis above the level of the pre-operative pathology may be the cause of persisting backache. For these reasons we believe that one should not attempt to graft more vertebrae than is necessary to stabilise the local lesion. 5. The complication rate, particularly from deep vein thrombosis, was high. This major complication could perhaps be overcome by using banked bone. 6. The indications for the operation are assessed as follows. It should be done for low back pain only when there is a definite diagnosis and a limited extent of structural pathology; one can then expect excellent results when successful fusion is achieved and also an appreciably high proportion of satisfied patients even when bony fusion has not been obtained, presumably because there is a fibrous union strong enough to stabilise the affected spine. It is inadvisable to undertake lumbar-lumbo-sacral fusion for intervertebral disc degeneration when there are more than three adjacent vertebrae involved, and in any case operation should be confined to the indicated extent

Summary Statement. Conventional imaging techniques lack the ability to objectively assess early stages of intervertebral disc degeneration, characterised by glycosaminoglycan loss. This study shows that MRI T2∗ mapping correlates positively with GAG content and that it provides continuous measurements for disc degeneration. Introduction. Early degenerative changes arise in the nucleus pulposus (NP) and are characterised by a loss of glycosaminoglycans (GAG). Early disc degeneration (DD) could possibly be treated with upcoming regenerative therapies (e.g. with stem cells and/or growth factors). In order to evaluate degeneration and treatments, a sensitive diagnostic tool is needed. While conventional magnetic resonance imaging (MRI) and x-ray techniques can detect late stages of DD, these techniques lack the ability to detect early degenerative changes. Recently, T2∗ mapping has been proposed as a new technique to evaluate early IVD degeneration, yet the correlation with GAG content and histological features has not been previously investigated. The objective of this study was to determine the value of T2∗ mapping in diagnosing DD by correlating this technique with the biochemical composition of IVDs. Materials & Methods. Six caprine lumbar spines obtained from an in vivo study and two healthy goat spines from the local abattoir, encompassing a total of 48 IVDs, were examined using sagittal standard T2-weighted and T2∗ mapping MRI protocols at 1.5 Tesla. Regions of interest (ROIs) were drawn on the T2∗ maps, covering the IVD. Based on T2 weighted MRI, discs were morphologically classified using the Pfirrmann score. Histological and macroscopic features were evaluated based on grading scales adapted for goat DD. Finally, GAG content was determined using colorimetric analysis (DMMB assay). Correlations between variables were analysed using Pearson correlation (r) coefficients (parametric data) or Spearman's rho (ρ) coefficients (non-parametric data). Results. The mean GAG content in the NP was 450 μg/mg dry weight (range 20–730 μg/mg dry weight) and the mean histological grade was 2.2 (range 0–6), corresponding with relatively mild disc degeneration. A linear positive correlation was observed between T2∗ and NP GAG content (r = 0.65, p < 0.001). T2∗ in the NP decreased linearly with increasing degeneration as assessed with macroscopic (ρ = 0.33, p < 0.05) and histological (ρ = −0.45, p < 0.05) grading, as well as with the Pfirrmann scoring system (ρ = −0.67, p < 0.001). Discussion. T2∗ mapping is a relatively new MRI technique which allows for measurements on a continuous scale, is acquired in less time than T2 mapping and minimises observer bias compared to grading systems. Although limited by a small sample size (n=48), this study showed a relatively good, linear correlation between T2∗ and GAG content in the NP, suggesting that T2∗ mapping may be an efficient and reliable tool for the objective assessment of proteoglycan content in early DD. Furthermore, with minor software modifications, it can be implemented on a standard 1.5 Tesla clinical MRI scanner. Future research should aim at optimizing the efficiency and user-friendliness of the T2∗ mapping protocol as well as yielding an even stronger correlation between T2∗ mapping and glycosaminoglycan content in human IVD tissue