Purpose of study and background

Low back pain affects 80% of the population at some point in their lives with 40% of cases attributed to intervertebral disc (IVD) degeneration. A number of potential regenerative approaches are under investigation worldwide, however their translation to clinic is currently hampered by an appropriate model for testing prior to clinical trials. Therefore, a more representative large animal model for IVD degeneration is needed to mimic human degeneration. Here we investigate a caprine IVD degeneration model in a loaded disc culture system which can mimic the native loading environment of the disc.

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.

Introduction

The intervertebral disc (IVD) is a highly hydrated and hyperosmotic tissue, water and salt content fluctuate daily due to mechanical loading. Resident IVD cells must adapt to this ever-changing osmotic environment, to maintain normal behaviour. However, during IVD degeneration the disc becomes permanently dehydrated and cells can no longer perform their correct function. Here, we investigated how human nucleus pulposus (NP) cells respond to altered osmolality with regards to cell size and the rate of water permeability, along with the potential involvement of aquaporins (AQPs) and transient receptor potential vanilloid (TRPV) membrane channels.

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.

Background

Intervertebral disc (IVD) degeneration is a major cause of low back pain (LBP). Degenerate discs are associated with accelerated cellular senescence. Cell senescence is associated with a secretory phenotype characterised by increased production of catabolic enzymes and cytokines. However to date, the mechanism of cell senescence within disc degeneration is unclear. Senescence can be induced by increased replication or induced by stress such as reactive oxygen species or cytokines. This study investigated the association of cellular senescence with markers of DNA damage and presence of cytoplasmic DNA (which in cancer cells has been shown to be a key regulator of the secretory phenotype), to determine mechanisms of senescence in disc degeneration.

Introduction

Injectable hydrogels via minimally invasive surgery reduce the risk of infection, scar formation and the cost of treatment. Degradation of the intervertebral disc (IVD) currently has no preventative treatment. An injectable hydrogel material could restore disc height, reinforce local mechanical properties, and promote tissue regeneration. We present a hydrogel material Laponite^® associated poly(N-isopropylacrylamide)-co-poly(dimethylacrylamide) (NPGel). Understanding how the components of this hydrogel system influence material properties, is crucial for tailoring treatment strategies for the IVD and other tissues.

The IVD is a highly hydrated, hyperosmolar tissue that allows the correct biomechanical function of the spine. When degenerated, water and ions are lost from the disc, especially within the central nucleus pulposus (NP), producing a hypoosmotic environment in which the resident cells can no longer function correctly, exacerbating the degenerative cascade. One potential way that IVD cells may adapt to their environment is through the expression and regulation of aquaporin (AQP) channels that control the movement of water in and out of cells. During human IVD degeneration AQP1 and 5 expression is decreased, highlighting AQPs may be of importance for the correct function of NP cells. The regulation of AQPs in NP cells by healthy and degenerate conditions, and the potential underlying molecular mechanisms, were investigated in both human and rat IVD cells. The gene and protein expression of AQP1 and AQP5 was upregulated by hyperosmotic conditions (425mOsm/kg H₂O) in rat and human NP cells. Lentiviral knockdown of tonicity enhancer binding protein (TonEBP), a transcription factor responsible for maintaining the function of NP cells, resulted in the loss of AQP1 and 5 gene expression under hyperosmotic conditions. The maintenance of the IVD environment and adaptation of cells is vital for the function of the IVD. The regulation of AQPs by physiological conditions and TonEBP suggests a role for these water channels related to the adaptation of disc cells to their environment, which is dysregulated during degeneration.

Intervertebral disc (IVD) degeneration is a major cause of low back pain (LBP). Degenerate discs are associated with accelerated cellular senescence. Cell senescence is associated with a secretory phenotype characterised by increased production of catabolic enzymes and cytokines. However, to date, the mechanism of cell senescence within disc degeneration is unclear. Senescence can be induced by increased replication or induced by stress such as reactive oxygen species or cytokines. This study investigated the association of cellular senescence with markers of DNA damage and presence of cytoplasmic DNA (which in cancer cells has been shown to be a key regulator of the secretory phenotype), to determine mechanisms of senescence in disc degeneration. Immunohistochemistry for the senescence marker: p16INK4A was firstly utilised to screen human intervertebral discs for discs displaying at least 30% immunopostivity. These discs were then subsequently analysed for immunopostivity for DNA damage markers γH2AX and cGAS and the presence of cytoplasmic DNA. The number of immunopositive cells for p16 INK4A positively correlated with the expression of γH2AX and cGAS. Senescent cells were also associated with the presence of cytoplasmic DNA. These new findings elucidated a role of cGAS and γH2AX as a link from genotoxic stress to cytokine expression which is associated with senescent cells. The findings indicate that cellular senescence in vivo is associated with DNA damage and presence of cytoplasmic DNA. Whether this DNA damage is a result of replicative senescence or stress induced is currently being investigated in vitro.

Introduction

During development the central disc contains large, vacuolated notochordal (NC) cells which in humans are replaced by mature nucleus pulposus (NP) cells during aging, but are maintained in certain breeds of dogs. During degeneration the disc becomes less hydrated which affects its normal function. Aquaporins (AQP) are a family of 13 transmembrane channel proteins that allow passage of water and are responsible for maintaining water homeostasis. AQP1, 2, 3 and 5 have been identified in the intervertebral disc (IVD). Here, expression of AQPs in human and canine IVDs to determine expression in NC v/s NP cells and whether expression changes during degeneration.

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.

Background

We have reported an injectable L-pNIPAM-co-DMAc hydrogel with hydroxyaptite nanoparticles (HAPna) which promotes mesenchymal stem cell (MSC) differentiation to bone cells without the need for growth factors. This hydrogel could potentially be used as an osteogenic and osteoconductive bone filler of spinal cages to improve vertebral body fusion. Here we investigated the biocompatibility and efficacy of the hydrogel in vivo using a proof of concept femur defect model.

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.

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

Auxetic materials have a negative poisons ratio, and a number of native biological tissues are proposed to possess auxetic properties. One such tissue is annulus fibrosus (AF), the fibrous outer layers of the intervertebral disc (IVD). However, few studies to date have investigated the potential of these materials as tissue engineering scaffolds. Here we describe the potential of manually converted polyurethane (PU) foams as three dimensional cellular scaffolds for AF repair.

Introduction

Within the intervertebral disc (IVD), nucleus pulposus (NP) cells reside within a unique microenvironment. Factors such as hypoxia, osmolality, pH and the presence of cytokines all dictate the function of NP cells and as such the cells must adapt to their environment to survive. Previously we have identified the expression of aquaporins (AQP) within human IVD tissue. AQPs allow the movement of water across the cell membrane and are important in cellular homeostasis. Here we investigated how AQP gene expression was regulated by the microenvironment of the IVD.

Introduction

The intervertebral disc (IVD) is a highly hydrated tissue which is reduced during degeneration leading to loss of function. Aquaporins (AQP) are a family of 13 (AQP0-12) transmembrane channel proteins that selectively allow the passage of water and other small molecules in and out of cells and are responsible for maintaining water homeostasis. AQP1, 2, 3 and 5 have been identified in the IVD. Here gene and protein expression of all 13 AQPs was investigated in a large cohort of human IVDs to investigate expression during IVD degeneration.

Summary Statement

IL-1β stimulation of human OA chondrocytes induces NFκB, ERK1/2, c-JUN, IκB and P38 signalling pathways. Pre-treatment with cannabinoid WIN-55 for 48 hours inhibits certain pathways, providing mechanisms for cannabinoids inhibitory actions on IL-1β induced cartilage degradation.

Matrix metalloproteinases (MMPs) are involved in extracellular matrix (ECM) breakdown in osteoarthritis (OA) and their expression is regulated by nuclear factor kappa B (NFκB). In addition signalling pathways ERK1/2, c-JUN, IκB and P38 are activated in OA and are induced by inflammatory cytokine interleukin 1 (IL-1). Cannabinoids have been shown to reduce joint damage in animal models of arthritis. Synthetic cannabinoid WIN-55, 212-2 mesylate (WIN-55) significantly reduces IL-1β induced expression of MMP-3 and -13 in human OA chondrocytes, indicating a possible mechanism via which cannabinoids may act to prevent ECM breakdown. Here the effects of WIN-55 on IL-1β induced NFκB, ERK1/2, c-JUN, IκB and P38 phosphorylation in human OA chondrocytes has been investigated.

Primary human chondrocytes were obtained from articular cartilage removed from patients with symptomatic OA during total knee replacement (Ethic approval:SMB002). Cartilage tissue was graded macroscopically 0–4 using the Outerbridge Classification method. Chondrocytes isolated from grade 2 cartilage and cultured in monolayer were pre-treated with 10 μM WIN-55 for 1 hour prior to stimulation with 10 ng/ml IL-1β for 30 minutes for investigation of NFκB, c-JUN, IκB and P38 phosphorylation. In addition chondrocytes were pre-treated with 10 μM WIN-55 for 30 minutes, 1, 3, 6, 24 and 48 hours prior to 10 ng/ml IL-1β stimulation for 30 minutes to investigate ERK1/2 phosphorylation.

Dimethyl sulfoxide (DMSO) was used as a vehicle control at 0.1%. Immunocytochemistry was used to investigate the phosphorylation and translocation of NFκB. ERK1/2, c-JUN, IκB, and P38 activation was investigated using cell based ELISA. Immunocytochemical analysis showed chondrocytes stimulated with IL-1β induced NFκB phosphorylation and translocation to the nucleus.

Chondrocytes treated with IL-1β with WIN-55 for 1 hour pre-treatment showed no inhibition of the IL-1β induced NFκB phosphorylation and translocation to the nucleus. WIN-55 treatment alone for 1 hour stimulated NFκB phosphorylation in the cytoplasm but not the nucleus. ELISA showed that phosphorylation of ERK1/2, c-JUN, IκB, and P38 was significantly induced by IL-1β following 30 minutes stimulation (p<0.05). Pre-treatment with WIN-55 for 1 hour had no significant effect on this IL-1β induced phosphorylation. However WIN-55 pre-treatment for 48 hours prior to IL-1β stimulation for 30 minutes, resulted in a significant decrease in ERK1/2 phosphorylation compared to IL-1β stimulation alone (p<0.05).

WIN-55 treatment alone for 1 hour significantly induced c-JUN phosphorylation (p<0.05), but had no effect on IκB and P38 phosphorylation compared to DMSO control. IL-1β stimulation of ERK1/2 phosphorylation was not significantly affected by WIN-55 pre-treatment of 30 minutes, 1, 3, 6 and 24 hours. WIN-55 treatment alone for 48 hours significantly reduced ERK1/2 phosphorylation compared to DMSO control (p<0.05). WIN-55 treatment alone for 30 min, 1, 3, 6 and 24 hours had no significant effect on ERK1/2 phosphorylation compared to DMSO control. The results show that following 48 hours pre-treatment WIN-55 inhibits IL-1β induced ERK1/2 phosphorylation in human OA chondrocytes. Thus inhibitory effects of cannabinoids on IL-1β induced cartilage degradation may be mediated via modulation of ERK1/2 signalling.

Summary

Cytokines produced within the degenerate disc induce expression of neurotrophic factors and pain related peptides which could be important in nerve ingrowth and pain sensitisation leading to low back pain.

The intervertebral disc (IVD) is considered the largest aneural and avascular structure within the human body, yet during degeneration vascularisation of the IVD is seen to be accompanied by nociceptive nerves. Low back pain is a highly debilitating condition affecting around 80% of the population, 40% of which are attributed to IVD degeneration. Discogenic pain was largely thought to be a result of irritation and compression of the nerve root, yet recent data suggests that pain may be attributed to the sensitisation of sensory nerves by the synthesis of pain related peptides, calcitonin gene related peptide (CGRP) and substance P. It is known that cytokines and chemokines produced by nucleus pulposus cells elicit various effects including the production of matrix degrading enzymes, and decreased matrix molecules. Here, we investigate the hypothesis that cytokines regulate both neurotrophic factor and pain related peptide synthesis within nucleus pulposus and nerve cells which may elicit algesic effects.

Real-Time PCR was performed to investigate gene expression of the neurotrophic factors NGF, BDNF, NT3 and their receptors Trk A, B and C along with Substance P and CGRP on directly extracted RNA from human NP cells and NP cells cultured in alginate for 2 weeks prior to treatment for 48hours with IL-1, IL-6 or TNFα at 0–100ng/mL. Similarly SH-SY5Y neuroblastoma cells were differentiated in retinoic acid for 7 days prior to stimulation with IL-1, IL-6 or TNFα at 0ng/mL and 10ng/mL for 48hours. Immunohistochemistry was used to localise neurotrophic factor receptors Trk A, B and C in both degenerate discs and neuronal cells.

NGF expression was present in normal and degenerate disc samples, however only degenerate discs expressed the high affinity receptor TrkA. Similarly Trk B was present in 22% of normal samples increasing to 100% expression within degenerate disc samples. All cytokines increased expression of NGF in NP cells (P≤0.05). TNFα also increased BDNF significantly, whereas no significant affects were seen in NT3 expression in NP cells. Trk B expression was significantly increased by IL-1 and TNFα treatment of NP cells. Conversely Trk C was down regulated by IL-6. Substance P was significantly increased by IL-1 and TNFα treatments whilst IL-6 and TNFα increased CGRP expression in NP cells. In SH-SY5Y cells, IL-1 significantly increased BDNF whilst IL-6 and TNFα failed to induce significant differences in neurotrophic factors. All cytokines increased Trk expression in the nerve cell line; however this failed to reach significance. Immunohistochemistry confirmed the presence of Trk receptors within the neuronal cell line.

Here we have demonstrated that a number of cytokines known to be up regulated during disc degeneration and disc prolapse, induce expression of various neurotrophic factors, their receptors and pain related peptides within human NP cells, as well as SH-SY5Y cells. This data suggests that the presence and production of cytokines within the degenerate disc may be responsible for nerve ingrowth and sensitisation of nerves which may result in discogenic pain.

Summary

Anabolic and catabolic signalling processes within IVDs display overlapping pathways, however some pathways were identified as selective to catabolic signalling and inhibition of one of these pathways inhibited some of the catabolic factors induced by IL-1 although NFkB inhibition also affected anabolic expression.

Degeneration of intervertebral discs (IVDs) is implicated in 40% of low back pain cases. In the normal disc the balance between anabolic and catabolic processes are carefully balanced. During degeneration this balance is lost in favour of catabolic processes which lead to degradation of the IVD, infiltration of blood vessels and nerves and release of cytokines which sensitise nerves to pain. Interleukin 1 (IL-1) is known to be important in the pathogenesis of IVD degeneration, here we investigated the intracellular signalling pathways activated by IL-1 and those activated by an anabolic factor (CDMP-1) to investigate differential pathways.

Human nucleus pulposus cells (NP) removed during discetomy for nerve root pain were stimulated with IL-1 or CDMP-1 for 30 minutes. Site-specific phosphorylation of 46 signalling molecules were identified using R&D proteome array. The activation of ERK1/2, p38, c-jun, and IkB were confirmed using cell based ELISAs, in addition pNFκB localisation in stimulated cells was determined using immunohistochemisty. Pre-treatment with inhibitors to p38, and NFkB for 30 minutes, followed by stimulation with IL-1 (10ng/mL) or CDMP-1 (10ng/mL) for 24 hours was investigated to determine effects on anabolic and catabolic factors. In addition localisation of phosphorylated c-jun, p38 and NFkB were investigated within paraffin embedded sections of human IVD to investigate the presence of active pathways in vivo.

Twenty intracellular signalling pathways were activated following CDMP-1 treatment and 8 signalling pathways activated by IL-1. Of note key classical IL-1 signalling pathways p38 MAPK, ERK 1/2 and JNK were activated by IL-1, however of these ERK 1/2 particularly was also activated by CDMP-1, whilst p38 and c-jun were only activated by IL-1. IL-1 induced activation of NFkB signalling to a greater extent than CDMP-1, these results were confirmed by the ‘in cell ELISAs’. IVD tissue samples displayed immunopositive staining for phosphorylated c-jun, NFkB and p38. Inhibition of p38 signalling inhibited IL-1 induced MMP 13 expression, but had little effect on the induction of IL-8. However inhibitors of NFkB signalling pathway failed to inhibit the induction of MMP 13 but abrogated the induced IL-6 and IL-8 expression. IL-1 induced a complete aberration of aggrecan expression by NP cells in alginate culture, this effect was partly inhibited by p38 MAPK inhibitor but was completely restored by inhibiting NFkB signalling. However the aggrecan expressed in CDMP-1 treated cells was decreased by inhibiting NFkB but not p38.

Here, we have shown that anabolic and catabolic signalling processes within IVDs show a number of overlapping pathways, however a number of differential pathways were identified and inhibition of p38 MAPK and NFkB pathways inhibited a number of catabolic processes investigated which were induced by IL-1. Thus inhibition of signalling pathways could be a novel mechanism of inhibiting catabolic processes which could hold promise to inhibit degeneration at early stages of disease but also create the correct tissue niche to promote regeneration of the disc.

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.