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.

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.

Background: Low back pain (LBP) is a major cause of disability. However, current treatments are often empirical and few are directed at the underlying disorder, altered discal cell metabolism, which precipitates the problem. The use of gene therapy to manipulate discal metabolism to treat LBP is an interesting possibility. The Intervertebral disc (IVD) is a therapeutic target in LBP, and one approach to gene therapy would be to isolate IVD chondrocytes (IVDC) and transfer genes ex vivo into these cells. Subsequent reinjection of these genetically altered cells into the lumbar IVD, would permit the expression of the transgene in vivo, generating the therapeutic protein within the IVD.

Methods: To test the viability of this approach, we isolated human IVDC from patients undergoing surgery, grew them ex vivo and transfected them with the marker gene LacZ, using an adenovirus vector and the CMV promoter. Expression of the gene was then measured using X-gal staining for the gene product _-galactosidase. Post infection, some cells were treated with forskolin for 24 hours to assess whether expression of the transgene could be manipulated.

Results: IVDC infected with adenovirus/CMV-LacZ showed maximal LacZ expression 2 days post infection, with almost 50% of cells displaying X-gal positivity. Cells maintained a low level of expression for the remaining 12 days of the study. Control cultures showed no LacZ expression. Cells treated with forskolin after infection with adenovirus/CMV-LacZ exhibited 4 times the level of _-galactosidase activity seen in unstimulated cultures.

Conclusion: This study shows that human IVDC can be transfected with a foreign gene using the adenovirus vector. The gene transduction of a therapeutic gene into IVDC could provide a long lasting effect. In addition, the use of inducible promoters could allow for the autoregulation of gene expression.