| This dissertation encompasses an investigation of cell-produced extracellular matrix and its role in microvessel development. A fibrous, decellularized matrix from human lung fibroblasts was created. This three-dimensional matrix contained fibronectin, tenascin-C, collagens I, IV, and VI, decorin, and versican. Structural and physical properties of the matrix were examined with unique image processing tools. In comparison to the other proteins examined, fibronectin was described as relatively distinct in its localization and structure in this matrix. An approach to interrogate the micromechanics of natural extracellular matrix was developed utilizing atomic force microscopy. This data quantitatively described the fibroblast-derived matrix as mechanically compliant and heterogeneous. Since substrate biochemical and physical properties are known to affect cell function, this description of natural fibroblast-derived extracellular matrix is important for understanding the native in vivo microenvironment and providing insight into matrix features to be implemented in tissue engineering scaffolds. The fibroblast-derived matrix provided a novel platform to study endothelial morphogenesis. The three-dimensional endothelial cell-matrix adhesions contained mechanosensitive tyrosine phosphorylation events that were preferentially interacting with fibronectin, the unique component of the matrix. After 24 hours, the endothelial cells remodeled the matrix and formed microvessels with lumens, without the addition of growth factors or angiogenic agents. Key signaling events in microvessel development, such as exposure of the 70 kDa fibronectin domain and endothelial cell membrane type 1 matrix metalloproteinase expression, were identified and the basis for a new hypothesis for vessel formation was formed. In addition, the microvessel orientation was manipulated and directed by the alignment of the fibroblast-derived matrix. The matrix alignment was controlled with a micropatterned substrate. This data demonstrated the importance of extracellular matrix organization for instructing cell morphogenesis. In summary, the work presented in this dissertation established a strong foundation for using fibroblast-derived matrix as a tissue engineering scaffold to study cell morphogenesis. In addition, these findings provided significant contributions to the fields of tissue engineering and developmental biology by creating a scaffold to study the instructive signals within natural extracellular matrix and providing insights into signaling mechanisms involved in vascular development. |
