Development of Collagen Thin Films for Intervertebral Dis Cell Culture & Examination of Cell Phenotypes and Interactions

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Morschauser, Michael Anthony
Hsieh, Adam H
Intervertebral disc degeneration and the associated low back pain lead to a decrease in the quality of life for many individuals, as well as a substantial burden on the health care system. Current therapies are often aimed at resolving the symptoms of degenerative disc disease, rather than addressing the underlying causes of disc degeneration. Researchers have strived to develop cell-based therapies that will not only manage the associated symptoms of disc degeneration but also reverse the degenerative process. To do so, a thorough understanding of the function of each cell type of the disc, both in a healthy and degenerative state, must be reached. For this project, <italic>in vitro</italic> cell culture substrates for each disc cell phenotype were developed using collagen thin film technology. Hybrid films were created with both type I and type II collagen fibers, the presence of which were confirmed using fluorescently-labeled antibodies. The hybrid films, along with type I and type II collagen thin films, were used to characterize the annulus fibrosus and nucleus pulposus cell phenotypes by examining morphology and gene expression. Annulus fibrosus cell morphology was observed to be independent of collagen type, but instead on the presence of fibers. Time dependence was also noted. No statistically significant differences were noted for substrate dependent gene expression of either cell type. Various methods for separating the multiple cell types of the nucleus pulposus were evaluated, and filtration was chosen as the most acceptable. By culturing the two cell types together or individually and examining the gene expression trends, it was observed that chondrocyte-like cells and notochordal cells influence each other in vitro. This has significance regarding disc degeneration, as notochordal cells disappear from the disc. This work was able to develop novel substrates for culture of intervertebral disc cells and utilize these substrates to further characterize the distinct phenotypes of the different cell types found in the disc. It also examined the influence of the disappearance of notochordal cells from the disc has on the remaining chondrocyte-like cells' gene expression. This information could aid in the development of future cell-based therapies for intervertebral disc degeneration.&#8195;