C. L. Le Maitre

Do intervertebral discs degenerate before they herniate, or after?

The belief that an intervertebral disc must degenerate before it can herniate has clinical and medicolegal significance, but lacks scientific validity. We hypothesised that tissue changes in herniated discs differ from those in discs that degenerate without herniation. Tissues were obtained at surgery from 21 herniated discs and 11 non-herniated discs of similar degeneration as assessed by the Pfirrmann grade. Thin sections were graded histologically, and certain features were quantified using immunofluorescence combined with confocal microscopy and image analysis. Herniated and degenerated tissues were compared separately for each tissue type: nucleus, inner annulus and outer annulus. Herniated tissues showed significantly greater proteoglycan loss (outer annulus), neovascularisation (annulus), innervation (annulus), cellularity/inflammation (annulus) and expression of matrix-degrading enzymes (inner annulus) than degenerated discs. No significant differences were seen in the nucleus tissue from herniated and degenerated discs. Degenerative changes start in the nucleus, so it seems unlikely that advanced degeneration caused herniation in 21 of these 32 discs. On the contrary, specific changes in the annulus can be interpreted as the consequences of herniation, when disruption allows local swelling, proteoglycan loss, and the ingrowth of blood vessels, nerves and inflammatory cells. In conclusion, it should not be assumed that degenerative changes always precede disc herniation. Cite this article: Bone Joint J 2013;95-B:1127-33.

Cannabinoid WIN-55,212-2 mesylate inhibits interleukin-1β induced matrix metalloproteinase and tissue inhibitor of matrix metalloproteinase expression in human chondrocytes

OBJECTIVE Interleukin-1β (IL-1β) is involved in the up-regulation of matrix metalloproteinases (MMPs) leading to cartilage degradation. Cannabinoids are anti-inflammatory and reduce joint damage in animal models of arthritis. This study aimed to determine a mechanism whereby the synthetic cannabinoid WIN-55,212-2 mesylate (WIN-55) may inhibit cartilage degradation. METHODS Effects of WIN-55 were studied on IL-1β stimulated production of MMP-3 and -13 and their inhibitors TIMP-1 and -2 in human chondrocytes. Chondrocytes were obtained from articular cartilage of patients undergoing total knee replacement. Chondrocytes were grown in monolayer and 3D alginate bead cultures. Real-time polymerase chain reaction (PCR) was used to determine the gene expression of MMP-3, -13, TIMP-1 and -2 and Enzyme Linked Immunosorbent Assay (ELISA) to measure the amount of MMP-3 and MMP-13 protein released into media. Immunocytochemistry was used to investigate the expression of cannabinoid receptors in chondrocyte cultures. RESULTS Treatment with WIN-55 alone or in combination with IL-1β, decreased or abolished MMP-3, -13, TIMP-1 and -2 gene expression in human chondrocyte monolayer and alginate bead cultures in both a concentration and time dependent manner. WIN-55 treatment alone, and in combination with IL-1β, reduced MMP-3 and -13 protein production by chondrocytes cultured in alginate beads. Immunocytochemistry demonstrated the expression of cannabinoid receptors in chondrocyte cultures. CONCLUSION Cannabinoid WIN-55 can reduce both basal and IL-1β stimulated gene and protein expression of MMP-3 and -13. However WIN-55 also decreased basal levels of TIMP-1 and -2 mRNA. These actions of WIN-55 suggest a mechanism by which cannabinoids may act to prevent cartilage breakdown in arthritis.

Thermally triggered injectable hydrogel, which induces mesenchymal stem cell differentiation to nucleus pulposus cells: Potential for regeneration of the intervertebral disc

There is an urgent need for new therapeutic options for low back pain, which target degeneration of the intervertebral disc (IVD). Here, we investigated a pNIPAM hydrogel system, which is liquid at 39°C ex vivo, where following injection into the IVD, body temperature triggers gelation. The combined effects of hypoxia (5% O2) and the structural environment of the hydrogel delivery system on the differentiation of human mesenchymal stem cells (hMSCs), towards an NP cell phenotype was investigated. hMSCs were incorporated into the liquid hydrogel, the mixture solidified and cultured for up to 6weeks under 21% O2 or 5% O2 where viability was maintained. Immunohistochemistry revealed significant increases in NP matrix components: aggrecan; collagen type II and chondroitin sulphate after culture for 1week in 5% O2, accompanied by increased matrix staining for proteoglycans and collagen, observed histologically. NP markers HIF1α, PAX1 and FOXF1 were also significantly increased where hMSC were incorporated into hydrogels with accelerated expression observed when cultured in 5% O2. hMSCs cultured under hypoxic conditions, which mimic the native disc microenvironment, accelerate differentiation of hMSCs within the hydrogel system, towards the NP phenotype without the need for chondrogenic inducing medium or additional growth factors, thus simplifying the treatment strategy for the repair of IVD degeneration.

Thermally Triggered Hydrogel Injection Into Bovine Intervertebral Disc Tissue Explants Induces Differentiation Of Mesenchymal Stem Cells And Restores Mechanical Function

We previously reported a synthetic Laponite® crosslinked pNIPAM-co-DMAc (L-pNIPAM-co-DMAc) hydrogel which promotes differentiation of mesenchymal stem cells (MSCs) to nucleus pulposus (NP) cells without additional growth factors. The clinical success of this hydrogel is dependent on: integration with surrounding tissue; the capacity to restore mechanical function; as well as supporting the viability and differentiation of delivered MSCs. Bovine NP tissue explants were injected with media (control), human MSCs (hMSCs) alone, acellular L-pNIPAM-co-DMAc hydrogel or hMSCs incorporated within the L-pNIPAM-co-DMAc hydrogel and maintained at 5% O2 for 6weeks. Viability of native NP cells and delivered MSCs was maintained. Furthermore hMSCs delivered via the L-pNIPAM-co-DMAc hydrogel differentiated and produced NP matrix components: aggrecan, collagen type II and chondroitin sulphate, with integration of the hydrogel with native NP tissue. In addition L-pNIPAM-co-DMAc hydrogel injected into collagenase digested bovine discs filled micro and macro fissures, were maintained within the disc during loading and restored IVD stiffness. The mechanical support of the L-pNIPAM-co-DMAc hydrogel, to restore disc height, could provide immediate symptomatic pain relief, whilst the delivery of MSCs over time regenerates the NP extracellular matrix; thus the L-pNIPAM-co-DMAc hydrogel could provide a combined cellular and mechanical repair approach. STATEMENT OF SIGNIFICANCE Low back pain (LBP) is associated with degeneration of the intervertebral disc (IVD). We have previously described development of a jelly delivery system (hydrogel). This has the potential to deliver adult stem cells to the centre of the IVD, known as the nucleus pulposus (NP). Here, we have demonstrated that adult stem cells can be safely injected into the NP using small bore needles, reducing damage to the disc. Following injection the hydrogel integrates with surrounding NP tissue, promotes differentiation of stem cells towards disc cells and restores IVD mechanical function. The hydrogel could be used to restore mechanical function to the IVD and deliver cells to promote regeneration of the disc as a minimally invasive treatment for LBP.

Polyphenols act synergistically with doxorubicin and etoposide in leukaemia cell lines

The study aimed to assess the effects of polyphenols when used in combination with doxorubicin and etoposide, and to determine whether polyphenols sensitised leukaemia cells, causing inhibition of cell proliferation, cell cycle arrest and induction of apoptosis. This study is based on findings in solid cancer tumours, which have shown that polyphenols can sensitize cells to chemotherapy, and induce apoptosis and/or cell-cycle arrest. This could enable a reduction of chemotherapy dose and off-target effects, whilst maintaining treatment efficacy. Quercetin, apigenin, emodin, rhein and cis-stilbene were investigated alone and in combination with etoposide and doxorubicin in two lymphoid and two myeloid leukaemia cells lines. Measurements were made of ATP levels (using CellTiter-Glo assay) as an indication of total cell number, cell cycle progression (using propidium iodide staining and flow cytometry) and apoptosis (NucView caspase 3 assay and Hoechst 33342/propidium iodide staining). Effects of combination treatments on caspases 3, 8 and 9 activity were determined using Glo luminescent assays, glutathione levels were measured using the GSH-Glo Glutathione Assay and DNA damage determined by anti-γH2AX staining. Doxorubicin and etoposide in combination with polyphenols synergistically reduced ATP levels, induced apoptosis and increased S and/or G2/M phase cell cycle arrest in lymphoid leukaemia cell lines. However, in the myeloid cell lines the effects of the combination treatments varied; doxorubicin had a synergistic or additive effect when combined with quercetin, apigenin, emodin, and cis-stilbene, but had an antagonistic effect when combined with rhein. Combination treatment caused a synergistic downregulation of glutathione levels and increased DNA damage, driving apoptosis via caspase 8 and 9 activation. However, in myeloid cells where antagonistic effects were observed, this was associated with increased glutathione levels and a reduction in DNA damage and apoptosis. This study has demonstrated that doxorubicin and etoposide activity were enhanced by polyphenols in lymphoid leukaemia cells, however, differential responses were seen in myeloid cells with antagonistic responses seen in some combination therapies.

Characterisation of intervertebral discs using MID-IR spectroscopic imaging

Introduction Lower back pain affects millions of people worldwide, and has been linked to degenerative changes in the intervertebral disc (IVD) of the spine. In the “NPmimetic” project, a multidisciplinary team has come together to create a biomimetic nanopolymer based implant and develop a minimally invasive therapy to reconstruct and regenerate diseased nucleus pulposus (NP). The biodegradable nanofibers of the implant can also be designed to carry anti-inflammatory drugs, which can be released in situ promoting healing and preventing inflammation (http://npmimetic.com/). An IVD consists primarily of a proteoglycan-water gel embedded in a randomly arranged collagen network in the NP, and highly ordered collagen lamella in the annulus fibrosus (AF). Disc cells in the AF are elongated parallel to the collagen fibers and produce predominantly collagen I in the outer AF in response to deformation. NP cells are responsive to hydrostatic pressure and synthesize mostly proteoglycan and collagen II. In adults, the cell density in an IVD is very low and cell phenotypes can change in response to altered matrix and stress distribution.1 For successful regeneration, tissue integrity together with the right mechanical environment is essential for normal cell function. To develop suitable biomimetic implants a thorough characterization profile, that can be used as an aspirational target is important. In this study, we use Fourier transform infrared (FTIR) microscopic imaging to obtain chemical maps of control and in-vivo CABC-degenerated goat IVDs.2,3 Materials and Methods Goat IVDs were kindly provided from VU University Medical Centre (VUMC), Amsterdam. The IVDs were formalin fixed (10%, overnight), paraffin embedded, and 4 µm sections were mounted on custom- made reflective steel slides. FTIR microscopy in transflection mode was used to generate chemical distribution maps from unstained paraffin sections. One microscopic image covers only a very small area (350 × 350 µm) of an IVD sample (∼2.8 × 2.3 cm, transverse section). Bigger areas are measured by sequential sample movement and image acquisition covering a user-defined mosaic image area. In this example, an area of 80 × 64 images was measured resulting in a total of 10,720 IR spectra per IVD section. FTIR mosaic imaging generally generates many thousands of data points. A major challenge is handling and analyzing such large and chemically complex datasets to extract meaningful information. However, using iterative multivariate curve resolution (MCR) techniques on the reduced data matrix from principal component analysis analysis of second derivative spectra it is possible to deconvolute highly overlapping infrared peaks into single contributions of different molecular species. Results The chemical identity of the extracted component using an iterative MCR algorithm is determined by comparing the extracted spectral profiles with the spectral profiles of reference materials for proteoglycan and collagen. Spectral features matching typical proteoglycan and collagen spectral characteristics are observed. The distribution of the extracted MCR factors for collagen and proteoglycan are compared with consecutive IVD sections, which were stained using traditional histological methods; Masson trichrome staining for collagen and Alcian blue staining for proteoglycan. A good match can be observed between the distribution maps of collagen and proteoglycans derived from FTIR microscopic imaging measurements and the traditionally stained sections. Conclusion FTIR microscopic imaging in transflection mode has been successfully used to generate molecular images of the collagen and proteoglycan distribution of natural IVD material without the use of contrast-enhancing agents. The use of second derivative spectra together with the application of iterative MCR algorithms shows great potential to enhance the chemical specificity of FTIR mosaic imaging and opens up the possibility to distinguish between different proteclycans and proteins as well as their secondary structure, which usually are expressed only through minor peak shifts. Acknowledgments We thank the European Union (NPMIMETIC ref 246351) for 7th Framework Program funding. Disclosure of Interest None declared References Adams MA, Roughley PJ. What is intervertebral disc degeneration, and what causes it? Spine 2006;31(18):2151–2161 Hoogendoorn RJW, Wuisman PIJM, Smit TH, Everts VE, Helder MN. Experimental intervertebral disc degeneration induced by chondroitinase ABC in the goat. Spine 2007;32(17):1816–1825 Hoogendoorn RJW, Helder MN, Kroeze RJ, Bank RA, Smit TH, Wuisman PI. Reproducible long-term disc degeneration in a large animal model. Spine 2008;33(9):949–954

Glutathione is key to the synergistic enhancement of doxorubicin and etoposide by polyphenols in leukaemia cell lines

Recently published in Nature: Cell Death and Discovery, Mahbub et al.1 have demonstrated that polyphenols can synergistically enhance the action of the topoisomerase II inhibitors: doxorubicin and etoposide in leukaemia cells. A reduction of glutathione (GSH) was strongly associated with sensitising cells to the pro-apoptotic effects of polyphenols when used in combination with doxorubicin or etoposide. Importantly, when polyphenols and topoisomerase II inhibitors were combined, it was possible to induce a synergistic decrease in cell proliferation (measured as ATP levels), cell-cycle arrest and induction of apoptosis in leukaemia cell lines.

Use of hydrogel scaffolds to develop an in vitro 3D culture model of human intestinal epithelium

The human intestinal cell lines: Caco-2 and HT29-MTX cells have been used extensively in 2D and 3D cell cultures as simple models of the small intestinal epithelium in vitro. This study aimed to investigate the potential of three hydrogel scaffolds to support the 3D culture of Caco-2 and HT29-MTX cells and critically assess their use as scaffolds to stimulate villi formation to model a small intestinal epithelium in vitro. Here, alginate, l-pNIPAM, and l-pNIPAM-co-DMAc hydrogels were investigated. The cells were suspended within or layered on these hydrogels and maintained under static or dynamic culture conditions for up to 21days. Caco-2 cell viability was increased when layered on the synthetic hydrogel scaffolds, but reduced when suspended within the synthetic hydrogels. In contrast, HT29-MTX cells remained viable when suspended within or layered on all 3D cultures. Interestingly, cells cultured in and on the alginate hydrogel scaffolds formed multilayer spheroid structures, whilst the cells layered on synthetic hydrogels formed villus-like structures. Immunohistochemistry staining demonstrated positive expression of enterocyte differentiation markers and goblet cell marker. In conclusion, l-pNIPAM hydrogel scaffolds supported both cell lines and induced formation of villus-like structures when cells were layered on and cultured under dynamic conditions. The ability of the l-pNIPAM to recapitulate the 3D structure and differentiate main cell types of human intestinal villi may deliver a potential alternative in vitro model for studying intestinal disease and for drug testing. STATEMENT OF SIGNIFICANCE Forty percent of hospital referrals are linked to disorders of the digestive tract. Current studies have utilised animal models or simple cultures of isolated cells which do not behave in the same manner as human intestine. Thus new models are required which more closely mimic the behaviour of intestinal cells. Here, we tested a number of scaffolds and conditions to develop a cell culture model which closely represents the 3D environment seen within the human small intestine. We successfully created structures seen within the intestine which have not previously been possible with other culture models. These models could be used to investigate tissue engineering, drug discovery, and used asan alternative to in vivo animal models in drug toxicity studies.

Human Degenerate Discs Show Increased Activation of Intracellular Signalling Pathways of IL-1

Introduction Intervertebral disc degeneration is implicated in 40% of low back pain cases. Interleukin 1 (IL-1) is known to be important in the pathogenesis of intervertebral disc (IVD) degeneration, here we investigated the intracellular signaling pathways activated by IL-1 and determined the activation status of these pathways in native IVD tissues highlighting potential pathways for new therapies. Materials and Methods Human nucleus pulposus cells (NP) removed during discetomy for nerve root pain were stimulated with IL-1 for 30 minutes. The activation of ERK1/2, p38, c-jun, and IκB were determined using cell-based enzyme-linked immunosorbent assays, in addition pNFκB localisation in stimulated cells was determined using immunohistochemistry. Localisation of phosphorylated c-jun, p38, ERK1/2, and NFκB together with IL-1 was investigated within paraffin embedded sections of human IVD to investigate the presence of active pathways in vivo. Pretreatment with inhibitors of p38, c-jun, and NFκB for 30 minutes before stimulation with IL-1 (10 ng/mL) for 48 hours was investigated to determine the ability of individual signaling inhibitors to block the catabolic responses induced by IL-1. Results IL-1-induced activation of p38, MAPK, ERK ½, JNK/c-jun, and NFκB signaling pathways in human NP cells. IVD tissue samples displayed immunopositive staining for phosphorylated c-jun, ERK1/2, NFκB, and p38, immunopositivity was significantly increased within degenerate discs, particularly in those discs which also expressed high levels of IL-1. Inhibition of individual signaling pathways demonstrated differential regulation of the pleathora of IL-1-induced catabolic events, with different signaling molecules shown to regulate matrix metalloproteases, cytokines, and factors involved in innervation and angiogenesis of the disc. Conclusion Here, we have shown that the signaling pathways activated by IL-1 in vitro display increased activation in vivo during disc degeneration. Inhibitors of these pathways demonstrated multiple signaling pathways were involved in the pleathora of IL-1 actions. Thus, inhibition of signaling pathways could be a novel mechanism to inhibit 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. Disclosure of Interest None declared

Recombinant IL-1Ra Stimulation of Native NP Cells Inhibits a Multitude of Pathological Features

Introduction Interleukin-1 (IL-1) has been implicated in the pathogenesis of disc degeneration, with increased levels seen during degeneration without a concordant increase in its natural inhibitor: IL-1 receptor antagonist (IL-1Ra). IL-1 has been shown to stimulate a pleathora of catabolic actions including matrix degrading enzymes, cytokines, chemokines, and factors which promote nerve and blood vessel ingrowth. Here, we investigated the effect of the natural inhibitor of IL-1, to determine its ability to inhibit catabolic events regulated by natively produced IL-1 in degenerate human nucleus pulposus (NP) cells. Materials and Methods Human NP cells were isolated via collagenase digestion from four human intervertebral disc (IVD) obtained from degenerate disc samples from patients undergoing discetomy. Following expansion in monolayer culture, cells were transferred to alginate bead cultures at passage two and maintained for 2 weeks before stimulation with IL-1Ra to enable redifferentiation to take place. Cells were treated with IL-1Ra at 1, 10, 100 pg/mL and 1, 10, 100 ng/mL for 48 hours. Following stimulation, cells were released from alginate beads and RNA isolated using Trizol reagent. cDNA synthesized and real-time PCR used to determine the effects on a wide range of gene targets including matrix metalloproteases (MMPs), cytokines, chemokines, and factors involved in nerve ingrowth (NGF), pain pathways (substance P), and angiogenesis (vascular endothelial growth factor, VEGF). Results Baseline levels of all factors investigated varied between patients as did the overall responsiveness to IL-1Ra stimulation. IL-1Ra stimulation resulted in decreased gene expression for several MMPs including MMPs 3 and 13; cytokines including IL-1 and IL-6; chemokines including IL-8, CCL2, and CXCL3. NGF, substance P, and VEGF were also all decreased by IL-1Ra stimulation. Effective doses were target and patient-specific and while the highest dose of IL-1Ra often resulted in decreased expression of catabolic factors in all patients, lowest doses (1.10 pg/mL IL-1Ra) resulted in stimulation of some catabolic responses in some patients, for example, IL-6 and IL-8 were increased in two out of four patient samples investigated at low doses of IL-1Ra. Conclusion IL-1 has been implicated as a key factor in the pathogenesis of disc degeneration, however till date it is not fully understood whether inhibition of this cytokine in native disc cells can inhibit the plethora of pathogenic factors linked to IL-1. This study has demonstrated that treatment of NP cells with IL-1Ra results in decreased expression of MMPs, cytokines, chemokines, nerve, and blood vessel growth factors and neurotrophic factors, suggesting IL-1Ra could be a useful agent in the prevention of further degeneration to create the correct tissue niche to support tissue regeneration. However, patient responses were seen to be variable, with some patients displaying increased expression of some catabolic factors at low doses of IL-1Ra, thus selection of patients and carefully controlled drug release would be essential to enable such a therapy to be deployed successfully. Disclosure of Interest None declared