Background. Chronic low back pain is strongly linked to degeneration of the intervertebral disc (IVD), which currently lacks any targeted treatments. This study explores NPgel, a biomaterial combined with notochordal cells (NC), developmental precursor cells, as a potential solution. NCs, known for anti-catabolic effects on IVD cells, present a promising avenue for regenerating damaged IVD tissue. Methods. Bovine IVDs underwent enzymatic degeneration before NPgel (+/- NC) injection. Degenerated bovine IVDs were cultured under biomechanical loading for 21 days. Histology and immunohistochemistry assessed NC survival, phenotype, and matrix production. Within an in vivo sheep pilot study, NPgel (+/- NC) was injected into degenerated IVDs, blood was taken, and immune cell activation was monitored via flow cytometry over three months post-injection. Results. Within the ex vivo model, IVDs injected with NPgel (+/- NC) exhibited increased matrix expression and deposition. Viable NCs were detected post-culture, indicating survival and matrix production. In the in vivo model, NPgel injection into sheep IVDs did not significantly increase activation of immune cells compared to controls, suggesting no systemic inflammatory effects. Conclusion. NPgel, combined with NCs, shows promise for IVD regeneration. Ex vivo findings indicate NPgel supports NC survival and matrix production. Moreover, in vivo results demonstrate the absence of systemic immunogenic responses post-NPgel injection. This suggests NPgel's potential as a carrier for NCs in IVD regeneration therapy. These findings underscore NPgel's candidacy for further investigation in addressing chronic low back pain associated with IVD degeneration. Subsequent research, including long-term efficacy and safety evaluations, is imperative for clinical translation. Conflicts of interest. There are no conflicts of interest. Sources of funding. iPSpine, grant # 825925, Horizon 2020

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

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)

Background. While the human embryonic, foetal and juvenile intervertebral disc (IVD) is composed of large vacuolated notochordal cells, these morphologically distinct cells are lost with skeletal maturity being replaced by smaller nucleus pulpous cells. Notochordal cells are thought to be fundamental in maintaining IVD homeostasis and, hence, their loss in humans may be a key initiator of degeneration, leading ultimately to back pain. Therefore, it is essential to understand the human notochordal cell phenotype to enable the development of novel biological/regenerative therapies. Methods. CD24+ notochordal cells and CD24- sclerotomal cells were sorted from enzymatically-digested human foetal spines (7.5–14 WPC, n=5) using FACS. Sorting accuracy was validated using qPCR for known notochordal markers and Affymetrix cDNA microarrays performed. Differential gene expression was confirmed (qPCR) and Interactive Pathway Analysis (IPA) performed. Results. CD24+ve notochordal cells (mean 10.4%) and CD24-ve sclerotomal cells (mean 60.9% CD24-) were successfully sorted. Higher expression of notochordal markers CD24 and brachyury was identified in CD24+ve cells. Hierarchical clustering and PCA mapping revealed distinct differences in the gene expression profile of CD24+ and CD24- cells. Top notochordal markers were CD24, STMN2. RTN1, PRPH and CXCL12. IPA identified IL-1 receptor antagonist (IL-1RN) and noggin as master regulators of notochordal cell phenotype. Conclusions. This study has, for the first time, defined human foetal notochordal cell phenotype and identified important pathways and upstream regulators. In particular, IL-1RN and noggin are of interest as master regulators of notochordal cell function, suggesting vital roles for these molecules in IVD development and homeostasis. Conflicts of interest. No conflicts of interest. Sources of funding. We would like to acknowledge UKRMP Acellular Hub, MRC, NIHR Musculoskeletal BRU and The Rosetrees Trust for funding this research

This review provides a concise outline of the advances made in the care of patients and to the quality of life after a traumatic spinal cord injury (SCI) over the last century. Despite these improvements reversal of the neurological injury is not yet possible. Instead, current treatment is limited to providing symptomatic relief, avoiding secondary insults and preventing additional sequelae. However, with an ever-advancing technology and deeper understanding of the damaged spinal cord, this appears increasingly conceivable. A brief synopsis of the most prominent challenges facing both clinicians and research scientists in developing functional treatments for a progressively complex injury are presented. Moreover, the multiple mechanisms by which damage propagates many months after the original injury requires a multifaceted approach to ameliorate the human spinal cord. We discuss potential methods to protect the spinal cord from damage, and to manipulate the inherent inhibition of the spinal cord to regeneration and repair. Although acute and chronic SCI share common final pathways resulting in cell death and neurological deficits, the underlying putative mechanisms of chronic SCI and the treatments are not covered in this review.