Title: Tissue engineering of the intervertebral disc
Description: That defects in the nucleus pulposus show little spontaneous healing is a function of the low cell density and avascularity of the intervertebral disc. In the absence of large migratory cell source and a provisional fibrin matrix in the defect, there is little opportunity to repopulate the area with native cells capable of synthesizing new tissue. The primary goal of this thesis was to address these barriers to disc regeneration by developing a cell-seeded, biodegradable scaffold implant in vitro for use in future in vivo regeneration studies. A secondary objective was to identify whether or not a lubricating molecule demonstrated to impede integrative tissue repair in articular cartilage is present in the disc in order to inform future in vivo studies. Toward the first objective, four factors were investigated as potential regulators of chondrogenesis in the scaffolds, including (1) cell density, (2) scaffold material composition, (3) scaffold pore size and cross-linking, and (4) oxygen concentration in the growth environment. Biochemical, histological, and immunohistochemical techniques were used to evaluate the effects of these variables on GAG and type II collagen contents within the scaffolds. Experimental results showed that decreasing scaffold cross-link density and increasing cell density induced greater chondrogenesis as demonstrated by a more chondrocytic cell morphology and greater tissue formation, with increased GAG densities and greater qualitative type II collagen contents.

On the other hand, the presence of GAG in the collagen scaffold and hypoxic culture environments appeared to reduce the degree of chondrogenesis. Pore size in the range test had little effect. Toward the second objective, the presence and distribution of a specific glycoprotein lubricant, termed lubricin, was investigated using immunohistochemical techniques. Experimental results showed that lubricin was indeed present within the cells and tissues of normal caprine and human intervertebral disc sections. In the goat, lubricin staining was limited to a small fraction of cells and tissues in the annulus fibrosus only, whereas in the human samples lubricin staining was present in all disc tissues and its extent varied inversely with outward radial position. Moreover, lubricin was identified in pathologic human tissues coating exposed surfaces.