The influence of biomaterial properties on the phenotypic behavior of intervertebral disc cells in vitro and in vivo

The intervertebral disc (IVD) is a fibro-cartilaginous tissue that functions to support and distribute loads and permit motion of the spine. The disc is a composite structure consisting of the lamellar annulus fibrosus comprised of organized collagen fibers arranged concentrically around the gelatinous, semi-fluid nucleus pulposus (NP). Intervertebral disc degeneration has become a significant health concern in the United States. A tissue-engineered intradiscal replacement of the NP represents a potential strategy for treatment of disc degeneration. However, engineering approaches to treat disc degeneration require an understanding of disc cell biology and knowledge of biomaterial properties that modulate IVD cell function. This thesis project explored the effects of two- and three-dimensional culture conditions on the phenotypic behavior of IVD cells in vitro and in vivo. It was determined through gene expression and immunohistochemical analyses that primary IVD cells from 5-8 population doublings expanded on two-dimensional, tissue culture polystyrene were not significantly different from freshly isolated cells, such that they could be used to study the phenotypic behavior of these cells in vitro. It was also shown that the three-dimensional culture environment, whether in a fibrous or hydrated gel format, has a greater effect on the cellular phenotype than the distinct origin of the cells within the tissue. Building on these basic science investigations, additional work focused on developing a biomaterial scaffold that recapitulated the native environment of the disc and allowed for functional tissue growth. A photo-crosslinkable alginate hydrogel was successfully synthesized and characterized for NP cell encapsulation. Nucleus pulposus cells encapsulated in photo-crosslinked alginate hydrogels not only exhibited characteristic type II collagen and proteoglycan gene expression and accumulation, but were able to assemble a mechanically functional matrix. Unconfined compression testing of cell-seeded photo-crosslinked hydrogels after 8 weeks in vivo revealed that constructs had reached an equilibrium Young's modulus of 4.31+/-1.39 kPa, which is comparable that of native NP tissue. Taken together, the outcomes of this project have provided a cell culture paradigm for investigating IVD tissue formation as well as a new biomaterial scaffold for functional IVD repair. Moving forward, we hope to continue evaluating photo-crosslinked alginate hydrogels to determine optimal culture parameters for development of a biological NP replacement.