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

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

Background. Current clinical treatment for spinal instability requires invasive spinal fusion with cages and screw instrumentation. We previously reported a novel injectable hydrogel (Bgel), which supports the delivery and differentiation of mesenchymal stem cells (MSCs) to bone forming cells and supports bone formation in vivo. Here, we investigated whether this system could be utilised to induce bone formation within intervertebral disc tissue as a potential injectable spinal fusion approach. Methodology. Bovine and Human Nucleus pulpous tissue explants were injected with Bgel with and without MSCs. Tissue samples were cultured under hypoxia (5%) in standard culture media for 4 weeks. Cell viability, histological assessment of matrix deposition, calcium formation, and cell phenotype analysis using immunohistochemistry for NP matrix and bone markers. Results. Following injection of B-gel into tissue explants following culture for 4 weeks, cells were visualized within the regions of the B-gel. Demonstrating that native cells were able to migrate into regions of B-gel. Increased collagen deposition was seen in tissue explants injected with Bgel, with increased collagen type I and X but decreased collagen type II staining in explants injected with Bgel. Tissue explants, in the absence of Bgel, showed limited calcium deposition, which was increased in B-gel injected explants. Furthermore, disc cells increased expression of bone markers (alkaline phosphatase & osteocalcin), but decreased NP matrix (Aggrecan and Collagen type II) following Bgel injection. Conclusion. This system could have potential to support spinal fusion via direct injection into the disc. Conflict of interest: C Le Maitre & C Sammon are inventors on the hydrogel discussed. Funding: This work was funded by GrowMed Tech Proof of Concept funding

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

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

Background. Intervertebral disc (IVD) degeneration is a major cause of Low back pain (LBP). We have reported an injectable hydrogel (NPgel), which following injection into bovine NP explants, integrates with NP tissue and promotes NP cell differentiation of delivered mesenchymal stem cells (MSCs) without growth factors. Here we investigated the injection of NPgel+MSCs into bovine NP explants under degenerate culture conditions to mimic the in vivo environment of the degenerate IVD. Methods. hMSCs were incorporated within liquid NPgel and injected into bovine NP explants alongside controls. Explants were cultured for 6 weeks under hypoxia (5%) with ± calcium 5.0mM CaCl. 2. or IL-1β individually or in combination to mimic the degenerate microenvironment. Cell viability was assessed by caspase 3 immunohistochemistry. Histological and immunohistochemical analysis was performed to investigate altered matrix synthesis and matrix degrading enzyme expression. Results. CFSe positive hMSCs were identified in all NPgel injected explants and cell viability was maintained. The NPgel integrated with NP tissue and hMSCs produced matrix components: aggrecan, collagen type II and chondroitin sulphate in standard and degenerate culture conditions. Increased cellular immunopositivty for aggrecan and collagen type II as well as decreased cellular immunopositivity for degrading enzyme expression was observed within NP tissue removed from the injection site. Conclusion. MSCs incorporated within NPgel could be used to regenerate the NP and restore the healthy NP phenotype of degenerate NP cells as a treatment strategy for LBP. We are currently investigating the survival and differentiation capacity of hMSCs delivered via the NPgel into degenerate human NP explants and thus ascertain the future clinical success of this therapy. Conflicts of Interest: None. Funding: BMRC, MERI Sheffield Hallam University

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

Aims

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

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

Aims

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.

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.

Background. Degeneration of the intervertebral disc (IVD) is a major cause of Low back pain. We have recently reported a novel, injectable liquid L-pNIPAM-co-DMAc hydrogel (NPgel), which promote differentiation of MSCs to nucleus pulposus (NP) cells without the need for additional growth factors. Here, we investigated the behaviour of hMSCs incorporated within the hydrogel injected into NP tissue. Methods. hMSCs were injected either alone or within NPgel, into bovine NP tissue explants and maintained at 5% O. 2. for up to 6wks. Media alone and acellular NPgel were also injected into NP explants to serve as controls. Cell viability was assessed by Caspase 3 immunohistochemistry and the phenotype of injected hMSC was assessed by histology and immunohistochemistry. Mechanical properties were also assessed via dynamic mechanical analysis (DMA). Results. No significant difference in the elastic modulus was observed between NPgel injected NP tissue and media injected controls. CFSe positive hMSCs were identified in all injected tissue samples and cell viability was maintained. Where hMSCs were delivered via NPgel, the hydrogel integrated with native NP tissue and cells producing NP matrix components: aggrecan; collagen type II and chondroitin sulphate. Conclusion. hMSC incorporated within L-pNIPAM-co-DMAc hydrogel and injected into NP explants, integrate with native NP tissue and promote differentiation towards the NP phenotype; thus potentially could be used to regenerate the NP as a treatment strategy for LBP. No conflict of interest. Funding: BMRC, MERI Sheffield Hallam University

Purpose of study. This study aims to establish the micro-structure of the vertebral endplate and its interface with the adjacent bone and disc in fresh, unstained tissue so that the structure can be related to normal and pathological function. Background. The endplate is key in both the mechanics, anchoring and nutrition of the disc. Understanding the detailed structure of the normal and pathological endplate is important for understanding how it achieves its functions. Advancements in imaging technology continually allow for greater understanding of biological structures. The development of two-photon fluorescence (TPF) combined with second harmonic generation (SHG), allows for the imaging of relatively thick, fresh samples without the need for staining. Methods. Bovine tail sections were sampled from the central region of the disc/vertebra interface. Samples were ground to provide a flat surface with a cross section including bone, endplate and disc. Samples were imaged using both TPF and SHG and images analysed using ImageJ. Results. The results detailed the interface between the bone, cartilage and disc. The SHG images show how the collagen fibre arrangement changes between the disc, endplate and subchondral bone. Due to its highly fluorescent nature, the interface between the calcified and non-calcified tissue was clear on TPF images. Conclusion. The application of TPF and SHG allowed us to image the endplate and its interfaces with the bone and disc in fine detail. Characterisation of these structure in healthy tissue is key to understanding how they function and are a foundation for understanding pathological changes. No conflicts of interest. Funding obtained from Orthopaedic Research UK (Reff: 524)

Aims

Vertebral body tethering (VBT) is a non-fusion technique to correct scoliosis. It allows correction of scoliosis through growth modulation (GM) by tethering the convex side to allow concave unrestricted growth similar to the hemiepiphysiodesis concept. The other modality is anterior scoliosis correction (ASC) where the tether is able to perform most of the correction immediately where limited growth is expected.

Aims

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.

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).

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

Cite this article: Bone Joint Res 2013;2:169–78.

Aims

In a multicentre, randomised study of adolescents undergoing posterior spinal fusion for idiopathic scoliosis, we investigated the effect of adding gelatine matrix with human thrombin to the standard surgical methods of controlling blood loss.

The widespread use of MRI has revolutionised the diagnostic process for spinal disorders. A typical protocol for spinal MRI includes T1 and T2 weighted sequences in both axial and sagittal planes. While such an imaging protocol is appropriate to detect pathological processes in the vast majority of patients, a number of additional sequences and advanced techniques are emerging. The purpose of the article is to discuss both established techniques that are gaining popularity in the field of spinal imaging and to introduce some of the more novel ‘advanced’ MRI sequences with examples to highlight their potential uses.

Cite this article: Bone Joint J 2015;97-B:1683–92.