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. Methods. Predicted whole-blood and skeletal muscle gene expression and genome-wide association study (GWAS) data from a DCS database were integrated, and functional summary-based imputation (FUSION) software was used on the integrated data. A transcriptome-wide association study (TWAS) was conducted using FUSION software to assess the association between predicted gene expression and DCS risk. The TWAS-identified genes were verified via comparison with differentially expressed genes (DEGs) in DCS RNA expression profiles in the Gene Expression Omnibus (GEO) (Accession Number: GSE153761). The Functional Mapping and Annotation (FUMA) tool for genome-wide association studies and Meta tools were used for gene functional enrichment and annotation analysis. Results. The TWAS detected 420 DCS genes with p < 0.05 in skeletal muscle, such as ribosomal protein S15A (RPS15A) (PTWAS = 0.001), and 110 genes in whole blood, such as selectin L (SELL) (PTWAS = 0.001). Comparison with the DCS RNA expression profile identified 12 common genes, including Apelin Receptor (APLNR) (PTWAS = 0.001, PDEG = 0.025). In total, 148 DCS-enriched Gene Ontology (GO) terms were identified, such as mast cell degranulation (GO:0043303); 15 DCS-enriched Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways were identified, such as the sphingolipid signalling pathway (ko04071). Nine terms, such as degradation of the extracellular matrix (R-HSA-1474228), were common to the TWAS enrichment results and the RNA expression profile. Conclusion. Our results identify putative susceptibility genes; these findings provide new ideas for exploration of the genetic mechanism of DCS development and new targets for preclinical intervention and clinical treatment. Cite this article: Bone Joint Res 2023;12(1):80–90

Aims. The aim of this study was to assess the accuracy of pedicle screw placement, as well as intraoperative factors, radiation exposure, and complication rates in adult patients with degenerative disorders of the thoracic and lumbar spines who have undergone robotic-navigated spinal surgery using a contemporary system. Methods. The authors reviewed the prospectively collected data on 196 adult patients who had pedicle screws implanted with robot-navigated assistance (RNA) using the Mazor X Stealth system between June 2019 and March 2022. Pedicle screws were implanted by one experienced spinal surgeon after completion of a learning period. The accuracy of pedicle screw placement was determined using intraoperative 3D fluoroscopy. Results. A total of 1,123 pedicle screws were implanted: 1,001 screws (89%) were placed robotically, 63 (6%) were converted from robotic placement to a freehand technique, and 59 (5%) were planned to be implanted freehand. Of the robotically placed screws, 942 screws (94%) were determined to be Gertzbein and Robbins grade A with median deviation of 0.8 mm (interquartile range 0.4 to 1.6). Skive events were noted with 20 pedicle screws (1.8%). No adverse clinical sequelae were noted in the 90-day follow-up. The mean fluoroscopic exposure per screw was 4.9 seconds (SD 3.8). Conclusion. RNA is highly accurate and reliable, with a low rate of abandonment once mastered. No adverse clinical sequelae occurred after implanting a large series of pedicle screws using the latest generation of RNA. Understanding of patient-specific anatomical features and the real-time intraoperative identification of risk factors for suboptimal screw placement have the potential to improve accuracy further. Cite this article: Bone Joint J 2023;105-B(5):543–550

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. Methods. We conducted a transcriptome-wide association study (TWAS) of spinal canal stenosis by integrating genome-wide association study summary statistics (including 661 cases and 178,065 controls) derived from Biobank Japan, and pre-computed gene expression weights of skeletal muscle and whole blood implemented in FUSION software. To verify the TWAS results, the candidate genes were furthered compared with messenger RNA (mRNA) expression profiles of LSS to screen for common genes. Finally, Metascape software was used to perform enrichment analysis of the candidate genes and common genes. Results. TWAS identified 295 genes with permutation p-values < 0.05 for skeletal muscle and 79 genes associated for the whole blood, such as RCHY1 (PTWAS = 0.001). Those genes were enriched in 112 gene ontology (GO) terms and five Kyoto Encyclopedia of Genes and Genomes pathways, such as ‘chemical carcinogenesis - reactive oxygen species’ (LogP value = −2.139). Further comparing the TWAS significant genes with the differentially expressed genes identified by mRNA expression profiles of LSS found 18 overlapped genes, such as interleukin 15 receptor subunit alpha (IL15RA) (PTWAS = 0.040, PmRNA = 0.010). Moreover, 71 common GO terms were detected for the enrichment results of TWAS and mRNA expression profiles, such as negative regulation of cell differentiation (LogP value = −2.811). Conclusion. This study revealed the genetic mechanism behind the pathological changes in LSS, and may provide novel insights for the early diagnosis and intervention of LSS. Cite this article: Bone Joint Res 2023;12(6):387–396

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

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

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

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)

We previously reported that osteoblasts at the curve apex in adolescent idiopathic scoliosis (AIS) exhibit a differential phenotype, compared to non-curve osteoblasts(1). However, the Hueter-Volkmann principle on vertebral body growth in spinal deformities (2) suggests this could be secondary to altered biomechanics. This study examined whether non-curve osteoblasts subjected to mechanical strain resemble the transcriptomic phenotype of curve apex osteoblasts. Facet spinal tissue was collected perioperatively from three sites, (i) the concave and (ii) convex side at the curve apex and (iii) from outside the curve (non-curve) from six AIS female patients (age 13–18 years; NRES 19/WM/0083). Non-curve osteoblasts were subjected to strain using a 4-point bending device. Osteoblast phenotype was determined by RNA sequencing and bioinformatic pathway analysis. RNAseq revealed that curve apex osteoblasts exhibited a differential transcriptome, with 1014 and 1301 differentially expressed genes (DEGs; p<0.05, fold-change >1.5) between convex/non-curve and concave/non-curve sites respectively. Non-curve osteoblasts subjected to strain showed increased protein expression of the mechanoresponsive biomarkers COX2 and C-Fos. Comparing unstimulated vs strain-induced non-curve osteoblasts, 423 DEGs were identified (p<0.05, fold-change >1.5). Of these DEGs, only 5% and 6% were common to the DEGs found at either side of the curve apex, compared to non-curve cells. Bioinformatic analysis of these strain-induced DEGs revealed a different array of canonical signalling pathways and cellular processes, to those significantly affected in cells at the curve apex. Mechanical strain of AIS osteoblasts in vitro did not induce the differential transcriptomic phenotype of AIS osteoblasts at the curve apex

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

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This study was performed to explore the effect of melatonin on pyroptosis in nucleus pulposus cells (NPCs) and the underlying mechanism of that effect.

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

Purpose and Background. The intervertebral disc is constantly subjected to forces generated by movement. But degeneration can disrupt normal biomechanics, generating uneven and complex loading patterns. Evidence suggests that these forces are converted into voltages through different mechanisms, such as streaming potentials. This implicates voltage-gated ion channels in the biological remodelling response of the disc to loading. These signalling pathways have not been studied, and this incomplete understanding of disc mechanotransduction may hinder regenerative therapies. The purpose of this study is to identify and determine the role of voltage-gated ion channels in the intervertebral disc and to investigate any changes in degeneration. Methods and Results. Primary bovine and human disc cells were cultured in monolayer or alginate beads for experiments. Cells were treated with altered osmolarity alone or in combination with IL-1β. Ion flux was measured through calcium influx and will be further investigated using the xCelligence RTCA CardioECR. Immunohistochemistry was performed on human and bovine discs to evaluate expression levels of ion channels. RNA was extracted from bovine NP cells and will be analysed through PCR/Microarray for gene expression. Conclusions. Preliminary results show that the Ca. v. 2.2 channel is expressed across the human disc, and is altered by degree of degeneration. Treatment with IL-1β may partly hinder the increase in calcium signalling of disc cells in response to lower osmolarity conditions. The presence of voltage-gated ion channels in the disc has been demonstrated for the first time. The role of these channels will be investigated through measuring ion flux with channel inhibitors across different culture treatments. No conflicts of interest exist. This research was supported by funding from the Society for Back Pain Research through the Travel Award 2019 and from the Irish Research Council under the Government of Ireland Postgraduate Scholarship Programme (GOIPG/2018/2416)

Introduction. Adolescent idiopathic scoliosis (AIS) is the most common form of spinal deformity. It occurs mainly in girls and progresses during pre-pubertal and pubertal growth, which is a crucial period for bone mass acquisition. The cause and molecular mechanisms of AIS are not clear; at present the consensus is that AIS has a multifactor cause, with many genetic factors. During the past 5 years, considerable effort has been devoted to identify a gene or genes that cause a predisposition to AIS. Many loci for this disorder have been mapped to different chromosome regions, but no genes have been clearly identified as being responsible for AIS, and, most importantly, the resulting protein defects remain to be shown. We aimed to identify the gene(s) that could be involved in AIS and to validate their involvement by both genetic and functional analyses. Methods. A large multiplex AIS French family was chosen for this study on the basis of clinical and radiological data. Whole genome genotyping of the 20 members of this family led to the mapping of a dominant disease-causing gene to two critical genomic intervals (Edery and colleagues, Eur J Hum Genet, accepted [2011]), but the causative mutation remains to be identified. In parallel, gene expression profiling was investigated by microarray analysis in RNA samples isolated from osteoblasts derived from healthy individuals and those with AIS. RNA samples were extracted from osteoblasts, purified, fluorescently labelled, and then hybridised to gene expression microarrays with the Illumina expression BeadChips technology containing more than 46 000 probes for the human genome (HumanHT-12). Data analysis in R version 2.10.1 (Bioconductor packages oligo and limma) was done, and genes that had at least 1·5-fold change in expression were considered differentially regulated relative to controls. AIS candidate genes within the critical intervals were selected on the basis of their mRNA expression in AIS individuals and by their known functions. The coding regions of these candidate genes were then sequenced to identify potential mutations. The biological activity of mutant proteins is under evaluation by in-vivo functional studies in zebrafish. Results. In the AIS family, a maximum LOD score of 3·01 was reached on two specific chromosomal regions. The interval lengths of these regions were 7cM and 12cM. These two regions contain several genes that might be responsible for AIS. Microarray analysis showed many genes that are differentially regulated in AIS osteoblasts compared with control osteoblasts. We recorded that 2·6% of the 24000 genes examined were upregulated in AIS osteoblasts, whereas 2·16% of them were downregulated. We observed a roughly 3-fold increase or decrease in the transcripts of many genes in AIS osteoblasts. Some of the differentially regulated genes are located within the two chromosomal candidate regions. The sequencing of the candidate genes' coding sequences was done on the family members. Sequence analysis showed two rare SNPs located on the coding regions of a gene that we called CH5G1. These two SNPs are located on the C-terminal region of the CH5G1 protein and affect its structure and probably its cellular activity and biological process leading to the disease. The C-terminal region of this protein interacts with the mRNA of a gene whose defects cause scoliosis as a secondary phenotype. The pathogenic nature of these SNPs is being investigated in the zebrafish model. The results suggest that CH5G1 gene's defects could be associated with AIS in this family. Conclusions. Identification of susceptibility genes for AIS will facilitate the understanding of underlying biochemical pathways (functional studies) and ultimately the development of specific therapies (pharmacological studies). This is likely to have important implications, since the cause of AIS is unknown. Acknowledgments. This study is supported by the Fondation Yves Cotrel, Institut de France

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Non-coding microRNA (miRNA) in extracellular vesicles (EVs) derived from mesenchymal stem cells (MSCs) may promote neuronal repair after spinal cord injury (SCI). In this paper we report on the effects of MSC-EV-microRNA-381 (miR-381) in a rodent model of SCI.