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.

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.

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.

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.

Low back pain (LBP), caused by intervertebral disc (IVD) degeneration represents one of the most significant socioeconomic conditions facing Western economies. Novel regenerative therapies, however, have the potential to restore function and relieve pain. We have previously shown that stimulation of adipose-derived stem cells (ASCs) with growth differentiation factor-6 (GDF6) promotes differentiation to nucleus pulposus (NP) cells of the IVD, offering a potential treatment for LBP. The aims of this study were to i) elucidate GDF6 cell surface receptor profile and signalling pathways to better understand mechanism of action; and (ii) develop a microparticle (MP) delivery system for GDF6 stimulation of ASCs. GDF6 receptor expression by ASCs (N=6) was profiled through western blot, immunofluorescence (IF) and flow cytometry. Signal transduction through Smad1/5/9 and non-Smad pathways following GDF6 (100ng/ml) stimulation was assessed using western blotting and confirmed using pathway specific blockers and type II receptor sub-unit knockdown using CRISPR. Release kinetics of GDF6 from MPs was calculated (BCA assay, ELISAs) and ASC differentiation to NP cells was assessed. BMPR profiling revealed high BMPR2 expression on ASCs. GDF6 stimulation of ASCs resulted in significant increases in Smad1/5/9 and Erk phosphorylation, but not p38 signalling. Blocking GDF6 signalling confirmed differentiation to NP cells required Smad phosphorylation, but not Erk. GDF6 release from MPs was controlled over 14days in vitro and demonstrated comparable NP-like differentiation to exogenous GDF6 delivery. This study elucidates the signalling mechanisms responsible for GDF6-induced ASC differentiation to NP cells and also demonstrates an effective and controllable release vehicle for GDF6.

Clinical trials are underway to elucidate a successful MSC-based therapy for the repair and regeneration of intervertebral disc (IVD) tissue. Currently, there is a lack of knowledge surrounding the relationship between naïve MSCs and the inflammatory microenvironment of the degenerate disc. To inform a phase II clinical trial, this study tests the hypothesis that cytokines, IL-1ß and TNFα regulate the expression of neuropeptides and neurotrophic factors from MSCs, thus exacerbating pain in those patients that have the presence of sensory nerve fibres within the IVD. Patient-matched MSCs derived from bone marrow (BM) or adipose (AD) tissue were stimulated with IL-1β (10ng/ml) or TNFα (10ng/ml) for 48 hours in either 21% or 5% O2. qRT-PCR was performed to assess expression of trophic factors involved in the survival or nerves (NGF & BDNF), blood vessels (VEGF) as well as pain related peptides (SP & CGRP) and inflammatory factors. Conditioned culture medium was analysed using ELISAs to identify secretion of soluble factors. IL-1β did not regulate neurotrophic factor expression from BM-MSCs under normoxic or hypoxic conditions. However, TNFα increased NGF, BDNF, SP and CGRP under normoxic conditions. In ADMSCs, VEGF was increased following IL-1β and TNFα stimulation; with TNFα also increasing NGF and CGRP under normoxic conditions. When exposed to hypoxia, the trophic effect of TNFα on human BM-MSCs was reduced. Overall this data suggests a role for priming or pre-stimulation of naïve MSCs prior to implantation to prevent exacerbation of pain from sensory nerve fibres.

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.

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.

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.

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.

Background

Signalling by growth differentiation factor 6 (GDF6/BMP13) has been implicated in the development and maintenance of healthy NP cell phenotypes and GDF6 mutations are associated with defective vertebral segmentation in Klippel-Feil syndrome. GDF6 may thus represent a promising biologic for treatment of IVD degeneration. This study aimed to investigate the effect of GDF6 in human NP cells and critical signal transduction pathways involved.

Background

Intervertebral disc degeneration is implicated as a major cause of chronic lower back pain. Current therapies for lower back pain are aimed purely at relieving the symptoms rather than targeting the underlying aberrant cell biology. As such focus has shifted to development of cell based alternatives. Notochordal cells are progenitors to the adult nucleus pulposus that display therapeutic potential. However, notochordal cell phenotype and suitable culture conditions for research or therapeutic application are poorly described. This study aims to develop a suitable culture system to allow comprehensive study of the notochordal phenotype.

Introduction: Intervertebral disc (IVD) degeneration is a major underlying factor in the pathogenesis of chronic low back pain. The healthy IVD is both avascular and aneural, however during symptomatic degeneration there is ingrowth of nociceptive nerve fibres and blood vessels into proximal regions of the IVD. Semaphorin 3A (sema3A) is an axonal guidance molecule with the ability to repel nerves. This study aimed to identify whether class 3 semaphorins were expressed by cells of the IVD and addresses the hypothesis that they may play a role in repelling axons surrounding the healthy disc thus maintaining its aneural condition.

Methods: Forty human IVD samples were investigated using RT-PCR and immunohistochemistry to identify the expression of sema3A, 3F and their receptors; neuropilins (1 & 2) and plexins (A1-4). Serial sections were stained for PGP9.5 and CD31 to correlate semaphorin expression with nerve and blood vessel ingrowth respectively.

Results: Sema3A protein, localised primarily to the OAF, was expressed highly in the healthy disc. In degenerate samples sema3A expression decreased significantly in this region, although chondrocyte clusters within the degenerate NP exhibited strong immunopositivity. mRNA for sema3A receptors was also identified in healthy and degenerate tissues. CD31 and PGP9.5 were expressed most highly in degenerate tissues correlating with low expression of sema3A.

Conclusions: This study is the first to establish the expression of semaphorins and their receptors in the human IVD with a decrease seen in the degenerate symptomatic IVD. Sema3A may therefore, amongst other roles, act as a ‘barrier’ to neuronal ingrowth into the healthy disc.

Conflicts of Interest: None declared

Sources of Funding: Arthritis Research Campaign

Background and Aims: Low back pain has been attributed to degeneration of the intervertebral disc (IVD). Increased evidence of senescence biomarkers, including the protein caveolin-1, during IVD degeneration has been demonstrated and linked with disease development rather than ageing per se, suggesting that a particular type of senescence, stress-induced premature senescence (SIPS), occurs in disc degeneration. SIPS can be induced by cytokines such as interleukin-1 (IL-1 Since IL-1 is known to be an important mediator of the catabolic events in IVD degeneration we sought to investigate whether IL-1 induces expression of the senescence biomarker caveolin-1 in IVD cells and whether its induction is associated with markers of cell senescence.

Methods: Human nucleus pulposus (NP) cells cultured in monolayer were treated for 24 hours with 10ng/ml IL-1 Quantitative real-time RT-PCR was used to assess gene expression for caveolin-1 and cell cycle inhibitors p53, p21 and p16INK4a. Cells were stained for senescence-associated-galactosidase and flow cytometry performed to analyse cell cycle position.

Results: IL-1 treatment induced transcription of caveolin-1 at 8 hours after the start of treatment. This coincided with increased expression of the cell cycle inhibitors p21 and p16INK4a expression at 2 hours and p21 and p53 at 8 hours. Flow cytometry revealed that IL-1 treatment caused a shift away from the S phase of the cell cycle and treated cells exhibited senescence-associated-galactosidase staining.

Conclusion: Our findings indicate that IL-1 induces caveolin expression and features of cellular senescence in human NP cells suggesting a role for IL-1 and caveolin-1 in SIPS within the human IVD.

Conflicts of Interest: None

Source of Funding: Furlong Research Charitable Foundation

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.

Objective and Background: This study investigated the effects of IL-1 on human intervertebral disc cells (IVD). IL-1 has been implicated in the degradation of IVD, in particular the up-regulation of Matrix Metalloproteinases (MMPs) and the down regulation of proteoglycan synthesis. However very little is known of the effects of IL-1 on human IVD cells. Here, we have investigated the effects of both IL-1 α and IL-1 β on nucleus pulposus (NP) and Annulus fibrosus (AF) cells isolated from human degenerate IVD.

Methods: Human IVD tissue was obtained from disc replacement surgery and separated into NP and AF tissue, cells were cultured within an alginate bead system for 5 weeks before treatment with IL-1 α and IL-1 β for 48 hours. Following treatment, RNA was extracted and Real time RT-PCR was performed to investigate gene expression of IL-1 gene family, matrix proteins and degrading enzymes MMPs and ADAMTS.

Results: Interleukin 1 α showed a more potent response than IL-1 β and in addition NP cells were more sensitive than AF cells. In summary, IL-1 showed a positive feedback loop causing an up-regulation of α and β genes. IL-1 Ra was also up-regulated but to a lesser extent than IL-1 α and IL-1 β. A negative feedback loop was seen with inhibition of the IL-1 receptor gene upon treatment with IL-1. MMPs and ADAMTS showed up-regulation upon treatment with IL-1. In addition IL-1 down regulated the matrix protein’s collagen type II and Aggrecan.

Conclusions: This study demonstrates that IL-1 causes up-regulation in discal cells of the major degrading enzymes involved in discal degeneration, and a down regulation of the major matrix components within the IVD. Suggesting that IL-1 plays a major in process of discal degeneration.

Objective and Background: Interleukin 1 has been implicated in the progression of degenerative disc disease, however little data is available on the expression and production of IL-1 within degenerate discal cells. A few studies, have investigated herniated disc tissue but the results from these studies have been inconsistent. This study investigated the gene expression of IL-1 α, β, Ra and the receptor type I in discs removed at surgery from 7 prolapsed, 3 Scoliosis and 15 Degenerative discs (DD). In addition immunohistochemistry (IHC) was used to localise IL-1 α and IL-1 β within normal, and degenerate discs.

Methods: Human IVD tissue was obtained from disc replacement surgery and separated into nucleus pulposus (NP) and annulus fibrosus (AF) tissue, cell isolation using collagenase treatment was carried out, and RNA extraction on the cells performed immediately. Real time RT-PCR was then used to investigate gene expression of IL-1 gene family. IHC for IL-1 α and IL-1 β was also performed on paraffin embedded normal and degenerate disc samples.

Results: Expression of the IL-1 family genes was present at low levels within prolapsed disc samples. In contrast levels within scoliosis patents were the highest of the 3 disease states, however in both prolapsed discs and those from scoliosis patients a balance of IL-1 α/β to IL-1 Ra existed. Within samples from DD this balance was lost, with levels of IL-1 α and IL-1 β greatly exceeding levels of IL-1 Ra. In addition levels of IL-1 α and β showed an increase with age and were highest in those samples from the AF than the NP. IHC demonstrated both IL-1 α and IL-1 β protein within the NP and AF cells of the degenerate discs.

Conclusion: This study has demonstrated the mRNA expression of all members of the IL-1 family within IVD and in addition the chondrocytes within the disc produced IL-1 α and IL-1 β protein. The imbalance of IL-1 α/β to IL-1 Ra within those samples from degenerate discs but not prolapsed or scoliotic discs suggests a role for IL-1 within discal degeneration.

Aims: The intervertebral disc (IVD) consists of three structurally distinct areas; a nucleus pulposus (NP), annulus fibrosus (AF) and two cartilage endplates that together form a functional unit that allow flexibility of the spinal column and load transfer from adjoining vertebrae. The NP and AF contain cells that are phenotypically similar to chondrocytes found in articular cartilage. They also produce the 2 major matrix components aggrecan and collagen-type I and II. One feature of IVD degeneration is breakdown of the cartilage matrix. Using soluble growth factors could stimulate new matrix formation and help regenerate degraded discal cartilage. The aim of this study was to demonstrate the presence of four growth factor receptors within the IVD.

Methods and Results: Using immunohistochemsitry, we targeted expression of four growth factor receptors, (BMPRII, FGFR3, IGFR-1 and TGFβII), in biopsies taken from normal and degenerate IVD. Receptor expression was scored across regions of the disc using a peer-reviewed system that assessed the proportion of cells expressing a particular antigen and the average level of expression for those cells. For FGFR3, IGFR-1 and TGFβII, cells of the outer part of NP and inner AF expressed significantly higher receptor levels. The expression BMPRII deviated from that pattern and was present at higher levels in the inner and outer NP than in the AF. Although there were significant differences between FGFR3 expression in normal and degenerate biopsies, that was not the case for the other receptors. Growth factor receptor expression was also detectable on the ingrowing neurons and blood vessels that characterise part of the disease aetiology.

Conclusion: In conclusion, all of the receptors were found in the IVD, predominantly within the NP, suggesting that, addition of the ligands for these receptors may elicit a physiological response from disc chondrocytes.

Objectives and Background: This study investigated a simple, novel, in vitro culture system which enables the in situ investigation of human intervertebral disc (IVD) cell function in healthy and diseased IVD in explant culture. Studies investigating the function of cells in IVD tissue are scarce. Whilst there is a paucity of realistic animal models of human IVD disease and in vivo study of human tissue remains impracticable, the only possible approaches remain in-situ molecular biology applied to tissue sections of biopsied tissue, which suffers from lacking a dynamic dimension. Or in vitro studies, of which cell culture lacks physiological relevance and explant cultures are subject to loss of tissue integrity and altered cellular behaviour. We have investigated a system that preserves the structure of the tissue and cellular phenotype within an explant culture system.

Methods: Human IVD tissue was obtained from disc replacement surgery and separated into nucleus pulposus (NP) and annulus fibrosus (AF) tissue, which was then cultured in either a Perspex ring or unconstrained in tissue culture medium for up to 3 weeks. The effectiveness of this system to maintain tissue integrity and cell function was tested using microscopy and either tinctoral histochemistry or immunohistochemistry.

Results: Unconstrained in medium, IVD tissue expanded and structural integrity was disturbed. The number of cells expressing type I collagen increased and aggrecan decreased by comparison with directly harvested tissue. In contrast the tissue in the Perspex rings maintained its structure and at the end of 3 weeks the cellular parameters were the same as in the newly harvested tissue.

Conclusions: This is the first reported system to preserve cell function of discal explants for long periods in tissue culture. This system will be a useful tool for a wide range of investigations of IVD biology that have not hitherto been possible.

Purpose and Background: We have previously reported our investigations of nerve ingrowth into intervertebral discs (IVD) from patients with mechanical low back pain. We have shown that in discs that are painful on discography (pain level discs) nerves actively grow into the deep annulus fibrosus and nucleus pulposus. Nerve ingrowth accompanies blood vessel ingrowth and advances into the nucleus pulposus from the end plate. The morphology and neurochemistry of these nerves indicate them to be nociceptive.

The growth of non-myelinated pain fibres in other settings is regulated by the cytokine Nerve Growth Factor (NGF). In this study, we have investigated the production and distribution of NGF, or more particularly its active isoform – NGF-β, and its receptors, in diseased intervertebral discs in order to establish whether this cytokine might be responsible for the observed nerve ingrowth in this situation.

Methods: Tissue sections of 21 pain level, 15 non-pain level diseased and 12 normal intervertebral discs, taken at the time of spinal surgery, and from cadavers, were probed by radioactive in situ hybridisation (ISH) for expression of NGF-β, and by immunohistochemistry (IHC) for its high and low affinity receptors (trk-A and p75 respectively). In addition, either serial sections were stained with cell specific markers (CD31 – endothelial cell, PGP9.5 – neurones, GAP43 – actively growing nerves) or sections were doubled stained (two antibodies or both ISH and IHC).

Results: We have demonstrated that NGF-β is synthesised by the endothelial cells of blood vessels growing into the IVD from the end plate. The high affinity receptor is expressed by those small nerve fibres that accompany the vessels and in their offshoots in pain level discs that are growing from perivascular nerves into the disc. In addition to their expressing the nerve specific molecule PGP9.5, the trk-A positive cells also express the nerve growth associated protein GAP43.

Conclusion: The data indicate that nerve ingrowth into IVD is regulated by NGF-β. We have localised this production to the endothelial cells of ingrowing blood vessels. NGF-β is a potential therapeutic target for the management of back pain.