Abstract
Degeneration of the intervertebral disc (IVD) has been associated with the presentation of back pain, a common but serious disorder with high economic impact from lost work time and medical care costs. Considerable research has been undertaken to elucidate the underlying mechanisms and to resolve a strategy for treatment. Despite the growth in knowledge, however, reliable solutions are still elusive. While a surgical approach to fuse vertebrae and relieve pain has had success, it is preferable to realize a less invasive and traumatic procedure to restore the disc. Towards treatment of the ailment, research in the field of tissue engineering has been moving forward rapidly and promises a biological approach to treat disc degeneration.For the construction of an implantable biological device that reverses degeneration, a method is required for obtaining large numbers of IVD cells in optimal condition. Monolayer culture offers the potential to expand cells for engineering an effective implant; however, it is well known that the cellular phenotype changes from the native state during the culture period. The focus of this dissertation study is to develop and demonstrate the utility of a novel method of evaluating cultured IVD cells with flow cytometry. This method discriminates between cell populations in the preparations and examines the response of porcine IVD cells to culture conditions by measuring phenotypic attributes of the cells, namely extracellular matrix (ECM) component production and surface marker expression. The central hypotheses of the dissertation are: (1) the newly developed flow cytometric method can detect the presence of intracellular collagen molecules in IVD cells; (2) the detection system can quantify the amount of intracellular collagen on a relative basis; and (3) changes are quantifiable in the levels of extracellular matrix molecules and additional surface molecules as a result of culture conditions.Optimization of the flow cytometric procedure was undertaken to maximize the measured signals and improve the discrimination between the cultured cells as well as evaluating antibodies for the ability to quantitate ECM component distributions within the cell populations. Collagen type I, collagen type II and aggrecan ECM components, along with key surface markers, CD24 and CD44, were analyzed for differences in response to culture. Size distribution of fresh and cultured cells is also presented.This is the first study to show intracellular staining of collagen in IVD cells using flow cytometry and to discern multiple cell populations within a single sample. The method is sensitive enough to show changes in responses of cells to various culture conditions, and variations in extracellular matrix production and surface marker levels were demonstrated in statistically significant models. A unique feature of the flow cytometric screening tool is the ability of separating cells of different phenotypes by size and antigen expression. The methods presented may prove useful in future research of tissue engineered cells that rely on specific phenotypic patterns and show potential for large-scale screening and quality control of implantable cellular materials.