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. Methods. In the current study, the luciferase assay confirmed a binding site of bromodomain-containing protein 4 (BRD4) and Wnt family member 5A (WNT5A). Then we detected expression of miR-381, BRD4, and WNT5A in dorsal root ganglia (DRG) cells treated with MSC-isolated EVs and measured neuron apoptosis in culture by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining. A rat model of SCI was established to detect the in vivo effect of miR-381 and MSC-EVs on SCI. Results. We confirmed an interaction between miR-381 and BRD4, and showed that miR-381 overexpression inhibited the expression of BRD4 in DRG cells as well as the apoptosis of DRG cells through WNT5A via activation of Ras homologous A (RhoA)/Rho-kinase activity. Moreover, treatment of MSC-EVs rescued neuron apoptosis and promoted the recovery of SCI through inhibition of the BRD4/WNT5A axis. Conclusion. Taken altogether, miR-381 derived from MSC-EVs can promote the recovery of SCI through BRD4/WNT5A axis, providing a new perspective on SCI treatment. Cite this article: Bone Joint Res 2021;10(5):328–339

Background. Degeneration of the intervertebral disc (IVD) is a leading cause of lower back pain, and a significant clinical problem. Inflammation mediated by IL-1β and TNF-α drives IVD degeneration through promoting a phenotypic switch in the resident nucleus pulposus (NP) cells towards a more catabolic state, resulting in extracellular matrix degradation. Bone marrow mesenchymal stem cells (MSCs) produce bioactive factors that modulate local tissue microenvironments and their anti-inflammatory potential has been shown in numerous disease models. Thus MSCs offer a potential therapy for IVD degeneration. In a clinical setting, adipose-derived stem cells (ASCs) might represent an alternative and perhaps more appealing cell source. However, their anti-inflammatory properties remain poorly understood. Methods. Here we assess the anti-inflammatory properties of donor-matched human ASCs and MSCs using qPCR and western blotting. Results. We demonstrate that stimulating ASCs or MSCs with IL-1β and/or TNF-α elicits a strong anti-inflammatory response with increased expression of IL-1 receptor antagonist (IL-1Ra), cyclooxygenase-2 (COX-2) and the tissue protective protein tumour-necrosis factor stimulated gene-6 (TSG-6). ASCs produced significantly higher levels of IL-1Ra and TSG-6 than their matched MSCs at both gene and protein levels, indicating that ASCs are potentially a more potent anti-inflammatory cell type. This anti-inflammatory response was also observed upon co-culture with degenerate NP cells without exogenous cytokine. Signalling analyses suggested this difference between cell types might be mediated through differences in the activation of inflammation-associated transcription factors. Conclusion. These data indicate that the anti-inflammatory properties of ASCs may be useful in developing future therapies for IVD degeneration. No conflicts of interest. Sources of funding: EPSRC-MRC Centre for Doctoral Training in Regenerative Medicine (EP/L014904/1)

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

Background 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. 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. Methods and Results. Donor-matched ASCs and MSCs were pre-conditioned with either IL-1β (10ng/ml) or TNFα (10ng/ml) for 48 hours under hypoxic conditions (5% O. 2. ). Conditioned media was collected and 45 different immunomodulatory proteins were analysed using human magnetic Luminex® assay. Secreted levels of several key cytokines and chemokines, both pro- and anti-inflammatory, were significantly upregulated in ASCs and BMSCs following the conditioning regime. Under all conditions tested, ASCs expressed significantly higher levels of IL-4, IL-6, IL-10, IL-12, TGF-α, and GCSF compared to BMSCs. Pre-conditioning with TNFα resulted in significantly higher levels of IL-10 while preconditioning with IL-1β resulted in higher levels of IL-6, IL-12 and GCSF. Conclusion. These data suggest that pre-conditioned ASCs may have enhanced therapeutic potential in modulating IVD repair through the increased release of trophic factors that play a role in immunomodulation. Conflicts of interest: None. Sources of funding: Financial support for this research was provided by EU Horizon 2020 RESPINE grant (Project ID# 732163)

Purpose of study and background. We have previously reported the development of injectable hydrogels for potential disc regeneration (NPgel) or bone formation which could be utilized in spinal fusion (Bgel). As there are multiple sources of mesenchymal stem cells (MSCs), this study investigated the incorporation of patient matched hMSCs derived from adipose tissue (AD) and bone marrow (BM) to determine their ability to differentiate within both hydrogel systems under different culture conditions. Methods and Results. Human fat pad and bone marrow derived MSCs were isolated from femoral heads of patients undergoing hip replacement surgery for osteoarthritis with informed consent. MSCs were encapsulated into either NPgel or Bgel and cultured for up to 6 weeks in 5% (NPgel) or 21% (Bgel) O. 2. Histology and immunohistochemistry was utilized to determine phenotype. Both fat and bone marrow derived MSCs, were able to differentiate into both cell lineages. NPgel culture conditions increased expression of matrix components such as collagen II and aggrecan and NP phenotypic markers FOXF1 and PAX1, whereas Bgel induced expression of collagen I and osteopontin, indicative of osteogenic differentiation. Conclusion. NPgel and Bgel were able to differentiate patient derived MSCs from different sources into both NP and osteogenic lineages, which may give rise to novel treatment strategies for IVD degeneration and spinal fusion, enabling choice for cell source according to patients' circumstances and needs. C Le Maitre and C Sammon hold a patent for the hydrogel described. Funded by MRC and Versus Arthritis

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

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. Methods. hMSCs were encapsulated in NPgel and cultured for 4 weeks under hypoxia (5%) with ± calcium (2.5mM and 5.0mM CaCl. 2. ), IL-1β and TNFα either individually or in combination to mimic the degenerate microenvironment. Cell viability was assessed by Alamar blue assay. Histological and immunohistochemical analysis investigated altered matrix and matrix degrading enzyme expression. Results. Viability of hMSCs was maintained under all culture conditions. Matrix deposition of glycosaminoglycans were observed under all conditions, MMP13 expression was upregulated by calcium but not by pro-inflammatory cytokines IL-1β and TNFα. Conclusions. We are developing an in vitro modelling system which can be used to test novel therapies within a degenerate microenvironment. Interestingly, our preliminary findings suggest calcium is a major contributor to regulating MMP13 in this model system. Investigating the degenerate niche will identify targets for inhibition to provide the correct niche to promote regeneration of the IVD. No conflict of interest. Funding: BMRC, MERI Sheffield Hallam University, for joint funding the Daphne Jackson Trust fellowship

Background. Stem cell therapy has been suggested as a potential regenerative strategy to treat IVD degeneration and GDF6 has been shown to differentiate adipose-derived stem cells (ASCs) into an NP-like phenotype. However, for clinical translation, a delivery system is required to ensure controlled and sustained GDF6 release. This study aimed to investigate the encapsulation of GDF6 inside novel microparticles (MPs) to control delivery and assess the effect of the released GDF6 on NP-like differentiation of human ASCs. Methods. GDF6 release from PLGA-PEG-PLGA MPs over 14 days was determined using BCA and ELISA. The effect of MP loading density on collagen gel formation was assessed through SEM and histological staining. ASCs were cultured in collagen hydrogels for 14 days with GDF6 delivered exogenously or via microspheres. ASC differentiation was assessed by qPCR for NP markers, glycosaminoglycan production (DMMB) and immunohistochemistry. Results. GDF6 release from MPs was controlled over 14 days equivalently to exogenous addition. SEM and histology confirmed that MPs were distributed throughout gels and that gel formation was not disrupted. In 3D cultures, GDF6 release from microspheres elicited equivalent ASC differentiation and NP-like matrix formation compared to exogenous delivery in media, indicating activity was not affected by MP encapsulation. Conclusions. This study demonstrates the effective encapsulation and controlled delivery of GDF6, which was able to maintain its activity and induce ASC differentiation into an NP-like phenotype and production of an NP-like ECM. Delivery of GDF6 microspheres in combination with ASCs is a promising strategy for IVD regeneration and treatment of back pain. 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

Background. Currently, there is a focus on the development of cell based therapies to treat intervertebral disc (IVD) degeneration, particularly for regenerating/repairing the central region, the nucleus pulposus (NP). Recently, we demonstrated that GDF6 promotes NP-like differentiation in mesenchymal stem cells (MSCs). However, bone marrow- (BM-MSCs) and adipose- (Ad-MSCs) showed differential responses to GDF6, with Ad-MSCs adopting a more NP-like phenotype. Here, we investigated GDF6 signalling in BM-MSCs and Ad-MSCs, with the aim to improve future IVD stem cell therapies. Methods. GDF6 receptor expression in patient-matched BM-MSCs and Ad-MSCs (N=6) was profiled through western blot and immunocytochemistry (ICC). GDF6 signal transduction was investigated through stimulation with 100 ng ml. −1. GDF6 for defined time periods. Subsequently smad1/5/9 phosphorylation and alternative non-smad pathway activation (phospho-p38; phospho-Erk1/2) was analysed (western blot, ELISA). Their role in inducing NP-like gene expression in Ad-MSCs was examined through pathway specific inhibitors. Results. Western blot and ICC established that BMPR profiles differed between MSC populations; specifically, BMPR2 (a GDF6 receptor) expression, was significantly higher in Ad-MSCs (p<0.05). ELISA and western blot analysis showed that smad1/5/9 phosphorylation was significantly higher in Ad-MSCs following GDF6 stimulation (p<0.05). GDF6 stimulation also phosphorylated p38 and Erk1/2 pathways. Blocking of both smad and non-smad pathways resulted in variation of GDF6 induced NP-like gene expression. Conclusions. The upregulation of BMPR2 in Ad-MSCs and corresponding differences in smad1/5/9 and non-smad pathway phosphorylation in response to GDF6 indicates an enhanced discogenic potential in Ad-MSCs, suggesting they may be more suitable for GDF6 mediated cellular IVD regeneration. 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

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

Purpose of study and background

IVD degeneration is a major cause of Low back pain. We have previously reported an injectable hydrogel (NPgel), which induces differentiation of human MSCs to disc cells and integrates with NP tissue following injection in vitro. However, the translation of this potential treatment strategy into clinic is dependent on survival and differentiation of MSCs into disc cells within the degenerate IVD. Here, we investigated the viability and differentiation of hMSCs incorporated into NPgel cultured under conditions mimicking the healthy and degenerate microenvironment of the disc.

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.

Background

For bone grafting procedures, the use of autologous bone is considered the gold standard, as it is has a better healing capacity compared to other alternatives as allograft and synthetic bone substitutes. However, as there are several drawbacks related to autografting (infection, nerve- or vascular damage, chronic pain problems, abdominal herniation), there has been a targeted effort to improve the healing capacities of synthetic bone substitutes.

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

Background. We have previously reported an injectable hydrogel (NPgel), which could deliver patients own stem cells, via small bore needles, decreasing damage to the annulus fibrosus. NPgel drives differentiation to NP cells and can inhibit the degenerate niche. However, clinical success of NPgel is dependent on the capacity to inject NPgel into naturally degenerate human discs, restore mechanical function to the IVD, prevent extrusion during loading and induce regeneration. This study assessed injectability of NPgel into human IVD, influence on mechanical properties, regeneration ability in an ex vivo culture system and retention under failure testing. Methodology. Cadaveric human discs were used to calculate disc height and to determine Youngs Modulus during simulated walking pre and post injection of NPgel, extrusion testing performed. Whole human IVDs were injected with NPgel +/− human BMPCs and maintained in culture under physiological loading regime for 4 weeks. Pre and post culture MRI imaging and in line biomechanical characteristics determined. Histology and immunochemistry performed for anabolic and catabolic factors. Results. NPgel injection significantly increased disc height and Youngs modulus with no extrusion observed during failure testing. T1ρ intensity was increased during culture in those injected with NPgel +/− cells compared to non-injected discs, and biomechanical restoration. Histological analysis has demonstrated excellent tissue attachment to the injected gel, and cellular migration into acellular gel systems. With increased matrix production and decreased catabolic factor expression. Conclusion. These results provide essential proof of concept data supporting the use of NPgel as an injectable therapy for disc regeneration. Conflict of interest: C Le Maitre & C Sammon are inventors on the hydrogel discussed. Funding: This work was funded by MRC and Versus Arthritis

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.

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.

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.

Aims

The aim of this study was to report the long-term prognosis of patients with multiple Langerhans cell histiocytosis (LCH) involving the spine, and to analyze the risk factors for progression-free survival (PFS).