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.