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Title: Matrix disruption and altered cell phenotypes in degenerated intervertebral discs
Author: Lama, Polly
Awarding Body: University of Bristol
Current Institution: University of Bristol
Date of Award: 2013
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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.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available