Enhancement of chondrogenesis by directing cellular condensation through chondroinductive microenvironments, and, Designed solid freeform fabricated scaffolds

Articular cartilage is a complex organ that is unique in its isolation from the body due to the absence of vasculature, lymphatic vessels, and nerves. Due to this isolation, it has poor regenerative properties because the repair mechanisms of the body that are usually elicited after an injury do not occur. A repair response is only generated when the underlying subchondral bone is penetrated. The infiltrating blood brings mesenchymal stem cells and growth factors required for growth and repair. It is through a combination of this response and embryonic limb morphogenesis that we model the research described in this thesis. The objective is to create conducive microenvironments for chondrogenic differentiation by using biochemical and biomaterial scaffolds that promote cellular condensation. Cellular condensation is a pivotal point during embryonic development where tissue-specific genes are upregulated and cellular differentiation follows only if the appropriate conditions have been met. Therefore, it is hypothesized that the formation of high-density cellular condensations directed by biomolecules (hyaluronic acid) and designed scaffold architecture, in the presence of chondroinductive growth factors, will provide an environment that enhances chondrogenesis by bone marrow stromal cells (BMSC) and chondrocytes in solid freeform fabricated (SFF) scaffolds.;Hyaluronic acid (HyA) is a ubiquitous glycosaminosglycan that is present during mesenchymal condensation. It facilitates cellular migration, proliferation, and also aids in the formation of aggregates. The addition of HyA to a collagen hydrogel induced cellular condensation and also increased chondrogenic differentiation of BMSC. The presence of HyA increased the amount of cartilage formation from 3% to 10% for BMSC and 29% to 63% for chondrocytes. Both BMSC and chondrocytes cultured in the HyA hydrogels also had greater amounts of sulfated glycosaminoglycans (sGAG) in the matrix, indicating that the extracellular matrix surrounding the cells is more hyaline-like.;Two designed scaffold pore architectures were tested for their chondroconductive properties: (1) a cubic pore with square channels and (2) an ellipsoid pore that mimics the size, shape, volume, and low permeability of micromass cultures, which have been shown to induce chondrogenic differentiation. The amount of cartilage formation was increased from 3% to 13% for BMSC and from 61% to 79% for chondrocytes cultured within the ellipsoid pored scaffolds. The ellipsoid pore geometry was able to stimulate greater areas of cellular condensation, which initiated more chondrogenesis. A combination of the chondroinductive HyA hydrogel and chondroconductive scaffold pore architecture was best able to enhance chondrogenesis by BMSC and chondrocytes. Although the HyA had a greater effect on chondrogenesis than the scaffold pore geometry, these results imply that in conjunction with the traditional chondroinductive environments, physical scaffold properties, such as permeability, porosity, and pore architecture, are able to further improve chondrogenesis. SFF technology is a powerful tool that allows the ability to design specific scaffold properties that can be tailored for many tissue engineering applications. The determination of the scaffold properties best able to stimulate chondrogenesis or the morphogenesis of any organ will be a significant advancement in tissue engineering.