The development of an in vitro model of nucleus pulposus degeneration.
Published: 2005
Language: eng
Number of pages: 229
Description: Intervertebral disc (IVD) degeneration is very common, yet the etiology and disease process remain poorly understood. This thesis was initiated with the hypothesis that it would be possible to generate nucleus pulposus tissue  in vitro, suitable for use as a model system to characterize the intracellular mechanisms regulating the early stages of intervertebral disc degeneration. Nucleus pulposus cells were seeded at high density on the surface of a calcium polyphosphate (CPP) biomaterial and cultured up to 6 weeks. The cells formed a continuous layer of tissue with proteoglycan content and compressive mechanical properties similar to the native nucleus pulposus. This  in vitro-formed tissue was then used to study early degenerative changes induced by TNFalpha treatment, a cytokine that has been localized to the nucleus pulposus of degenerate non-herniated intervertebral discs. TNFalpha induced multiple cellular responses including: decreased aggrecan, link protein, and type II collagen gene expression, decreased synthesis of aggrecan and collagen, increased expression of MMP-1, -3, -13, ADAMTS-4/-5, and the induction of aggrecanase-mediated aggrecan degradation and gelatinase activity. In this model system, the induction of MMP and ADAMTS gene expression was differentially regulated by activation of ERK, p38, and JNK MAPK and NF-kappaB pathways. MMP-1 and -3 were regulated by JNK and p38, MMP-13 was regulated by JNK, p38 and NF-kappaB, and MT1-MMP was regulated by ERK. These studies demonstrated the novel role of NF-kappaB in the regulation of ADAMTS-4 and -5 expression. Furthermore, the data suggests that both ADAMTS-4/-5 and MT1-MMP primarily regulate ''aggrecanase'' activity and the subsequent loss of tissue proteoglycans. Finally, these studies demonstrate that TNFalpha induced the ERK-dependent activation of MMP-2 gelatinase activity in nucleus pulposus cells. This coincided with increased production of MT1-MMP, and a transient ERK-dependent increase in Egr-1 production. We further demonstrated that MMP-2 activation is mediated by Egr-1 transcriptional regulation of the MT1-MMP promoter. As regulation of proteoglycan degradation is essential to maintain intervertebral disc homeostasis in vivo, understanding the intracellular mechanisms governing activation of the proteolytic enzymes involved may be essential to delineate and eventually regulate of the process of pathological intervertebral disc degeneration.
Notes: Source: Dissertation Abstracts International, Volume: 66-10, Section: B, page: 5187.

Thesis (Ph.D.)--University of Toronto, 2005.

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