Tissue engineering is a promising solution to the problematic healing of cartilage defects. The purpose of this thesis was to establish a foundation for the development of a collagen-glycosaminoglycan (CG) scaffold for articular cartilage tissue engineering by exploring the behavior of passaged chondrocytes in the CG scaffolds under the influence of a variety of environmental factors.

Using in vitro studies, the first two parts of the thesis evaluated the effects of the physical environment on the behavior of adult, passaged chondrocytes in the CG scaffold. Scaffold cross-linking procedures increased cross-link density and scaffold stiffness and increased resistance to cell-mediated degradation and contraction as follows:​ dehydrothermal treatment (DHT) < ultraviolet irradiation (UV) < gluteraldehyde (GTA) < carbodiimide (EDAC). EDAC scaffolds also provided for the highest levels of cell proliferation and protein and GAG synthesis throughout a 4-week culture. Static mechanical compression (0-50% strain) applied to cell-seeded EDAC cross-linked scaffolds decreased rates of protein and GAG synthesis while dynamic compression (3% sine amplitude, 0.1 Hz) increased rates of biosynthesis over a 24-hour period. These results were similar to those of prior studies of loading of intact cartilage explants. Unlike the explant studies, however, dynamic compression failed to increase the accumulation of matrix molecules within the construct compared to unloaded ("free-swelling") controls because of a large increase in the release of newly synthesized macromolecules into the media.

To evaluate the in vivo performance of the chondrocyte-seeded EDAC crosslinked CG scaffold, repair tissue formed 15 weeks after implantation of a 4-week in vitro cultured construct was evaluated. The majority of the repair tissue was hyaline and fibrocartilaginous. However, it displayed decreased levels of type II collagen and GAG staining compared to normal articular cartilage, and had a compressive stiffness that was 20-fold lower than normal.

Finally, in anticipation of future work utilizing gene therapy to improve cartilage repair, the CG scaffolds were modified for direct delivery of genetic material to cells in situ. Scaffold cross-linking and plasmid pH altered the ability of the CG scaffolds to carry plasmid DNA to local cells. EDAC cross-linked scaffolds and scaffolds prepared with plasmid at a neutral pH bound the lowest amount of DNA but, over two to eight week in vitro culture periods, these scaffolds led to higher levels of gene expression compared to non- or DHT cross-linked scaffolds and plasmid preparations at an acidic pH (pH 2.5).

Although current knowledge is not sufficient to successfully repair articular cartilage wounds, the understanding of the responsiveness of passaged chondrocytes in CG scaffolds gained in this thesis can be used to further the development of this system. In brief, the construct that is recommended for future investigation as an implant for articular cartilage repair is a chondrocyte-seeded, EDAC cross-linked CG scaffold cultured in vitro under dynamic compression prior to implantation.