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Chronic back pain is associated with specific pathological changes in intervertebral discs, including fissuring of the annulus fibrosus, defects in the vertebral body endplates, and disc herniation. The greatest risk factors for disc pathology are age, spinal level, and genetic inheritance, and major cell-mediated changes include cell clustering, increased expression of matrix-degrading enzymes, neo-vascularisation and innervation. We hypothesise that cell-mediated events arc influenced by structural disruption of the extracellular matrix. Thus, the first aim of this thesis is to investigate spatial associations between focal matrix disruption and altered cell phenotype, in degenerated and painful intervertebral discs. The second aim is to explore causal relationships, by examining the consequences of matrix swelling in excised samples of disc tissue. Lumbar intervertebral disc tissues were obtained from 40 patients undergoing surgery for disc herniation, painful disc degeneration, or adolescent scoliosis (the last serving as nondegenerate controls). Histology of 5 um frozen sections was used to score: matrix fissuring, loss of glycosaminosoglycans (GAG), cell clustering, expression of MMPs (matrix degrading enzymes), apoptosis, inflammatory cell invasion, and in-growth of blood vessels and nerves. Immunofluorescence and con focal microscopy were used with 30um thick frozen sections, to quantify capillaries, nerves and MMPs. Antibodies included CD-31, PGP 9.5, Substance P, caspase-3, MMPs 1,2,3, integrin a5Bl, and denatured collagen I, II, and III. Mean values of score and quantified variables were compared ,between patient groups, with separate analyses for each tissue type: nucleus, inner annulus and outer annulus. Spatial associations were assessed using Spearman rank correlation. Causality was explored by allowing 11 disc samples to swell under various conditions of restraint, using a culture system at 37°C fitted with a wide-field microscope. Time lapse cinematography was used to record cell movements, and GAG loss (measured using DMMB assay) was used as a surrogate measure of swelling. Swollen tissues were then examined as described above. Compared to controls and degenerated in-situ discs, herniated tissues showed significantly greater: fissuring, GAG loss, neovascularisation, innervation, cellularity/inflammation, clustering, expression of MMPs, and increased apoptosis. Most differences were greatest in outer annulus tissue and least in nucleus tissue. Nerves and capillaries were associated with matrix fissuring" and were-never more than 888 I-lm (capillaries) and 236 !lm (nerves) from the nearest fissure. Cell clusters were associated with GAG loss, and were most prevalent in the inner annulus of herniated discs. Tissue swelling lead to matrix fissuring, denaturation of collagen, loss of GAGs into the tissue culture medium, increased clustering, exposure of integrin (a5B1) binding, and elevated expression of MMP-1. Results suggest that disruption of disc collagen allows focal swelling and GAG loss, followed by cellular phenotypic changes which lead to blood vessels and nerve ingrowths, and pain.

Intervertebral disc degeneration (IVDD) remains one of the commonest causes of chronic disability in the working years. It involves both structural disruption and cell mediated alteration in the extracellular matrix (ECM) composition of the disc. ECM changes differentiation between disc degeneration and the normal physiological ageing and between disrupted and non-disrupted discs is also not fully understood. The hypothesis of this dissertation, is that IVDD is initiated by micro-damage to disc tissues, followed by cellular attempts to repair which are impeded by some degree of tissue hypoxia, leading to synthesis of an "inferior collagen" and to progressive disc degeneration. The collagen turnover of the extracellular matrix in aged and degenerated as well as in disrupted and non-disrupted discs were determined by measuring the levels of proteolytic enzymes such as the matrix metalloproteinases (MMPs) and collagen cross links. Other ECM components such as sulphated glycosaminoglycans (sGAG) and water contents were also measured in both disc groups. The biochemical and thermal changes were compared in hypoxic and normoxic monolayer cell culture medium of cultured human fibroblasts. Aged discs were found to have greater level of matrix turnover associated with synthesis of poorly hydroxylated neo-collagen as well as different ECM composition, than degenerated discs which found to have increased level of MMPs that might facilitate cellular invasion into their disrupted matrix. Disrupted discs were found to have imbalanced collagen degradation (over synthesis) which might lead to a weak non- functional disc. Focal tissue disruption acquiesce swelling of tissue as well as GAG depletion, thus, altering the GAG/water ratio locally. Painful discs with structural disruption were found to have up-regulated MMPs which is thought to contribute to further annular weakening (radial annular tear) leading to the nuclear material to be forced outward (disc herniation) induced by trauma and/or axial load shift.

An association between painful lumbar intervertebral discs (IVDs) and innervation deep with the nucleus pulposus (NP) of the degenerate IVD has been highlighted by a number of studies. However the mechanisms responsible for driving this innervation and hence the pathogenesis of symptomatic IVD degeneration is complex. The extracellular matrix (ECM) component aggrecan has been shown to inhibit neurite outgrowth and a role for cell mteractions via the e production of soluble mediators from degenerate disc cells, hypothesised to be neurotrophic factors, has been proposed. This study was undertaken to investigate NP cell/neural cell interactions in relation to degeneration of the IVD.

Developing computational models of the human spine has been a hot topic in biornechanical research for a couple of decades in order to have an understanding of the behaviour of the whole spine and the individual spinal parts under various loading conditions. The objectives of this thesis are to develop a biofidefic multi-body model of the whole human spine especially for dynamic analysis of impact situations, such as frontal impact in a car crash, and to generate finite element (FE) models of the specific spinal parts to investigate causes of injury of the spinal components. As a proposed approach, the predictions of the multi-body model under dynamic impact loading conditions, such as reaction forces at lumbar motion segments, were utilised not only to have a better understanding of the gross kinetics and kinematics of the human spine, but also to constitute the boundary conditions for the finite element models of the selected spinal components. This novel approach provides a versatile, cost effective and powerful tool to analyse the behaviour of the spine under various loading conditions which in turn helps to develop a better understanding of injury mechanisms.