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Orthopaedic Proceedings
Vol. 85-B, Issue SUPP_III | Pages 240 - 240
1 Mar 2003
Le Maitre C Williamson B Ross R Freemont A Hoyland J
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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.


Orthopaedic Proceedings
Vol. 85-B, Issue SUPP_III | Pages 240 - 240
1 Mar 2003
Le Maitre CL Baird P Williamson B Ross R Freemont A Hoyland J
Full Access

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.


Orthopaedic Proceedings
Vol. 85-B, Issue SUPP_III | Pages 240 - 240
1 Mar 2003
Watkins A Hoyland J Le Maitre C Baird P Ross R Williamson B Freemont A
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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.


Orthopaedic Proceedings
Vol. 85-B, Issue SUPP_III | Pages 239 - 240
1 Mar 2003
Le Maitre C Williamson B Ross R Freemont A Hoyland J
Full Access

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.