Connective tissue disease and injury have a significant effect on quality of life and in severe cases can prove lethal. Both disease and injury exhibit alterations to normal tissue structure, but the effects of these alterations are not fully understood. Native tissues contain both structural proteins that provide tissue support and cells that actively maintain tissue function; the driving hypothesis of this research is that altered tissue structure associated with injury and disease causes functional changes in cell-matrix interactions and cell phenotype. This research addresses significant problems for studying the functional effects of altered tissue structure: most experimental models to study cell-matrix interactions and cell behavior have been two-dimensional, composed of non-biological materials, or defined by a uniform structure. To address these problems, collagen- based surrogate materials with controllable structural features at multiple length scales will be developed, and these materials will be used as an experimental model to study cellular behavior across a range of structural alterations. In Aim 1, I will expand the capabilities of surrogate material manufacturing methods to allow controllable modification of surrogate structure at multiple scales relevant to the structural organization of collagen in native tissues. Material testing and quantitative 3D microscopy will be used to measure the effects of these structural alterations on surrogate material behavior. In Aim 2, the tissue surrogate materials will be used to study cellular response of primary fibroblasts (cells native to connective tissues) and adipose derived mesenchymal stem cells (cells that can differentiate into the cell populations of native tissues) to structural alterations in a three-dimensional, heterogeneous environment representative of physiological tissues. The structural alterations will represent the structural changes exhibited by diseased and injured tissues. Cellular contribution to surrogate mechanical behavior will be measured by material testing and quantitative 3D microscopy. Mechanisms of cell-matrix interactions will be probed by immunostaining of cell adhesion molecules. Finally, the genetic phenotype of cells will be measured by changes in gene expression of tendon-, bone-, muscle- and vasculature-specific genes. Together, these analyses will characterize changes in functional cell behavior related to altered surrogate material structure. This research will provide new insight to the effect of altered tissue structure in disease and injury, will identify parameters for the design of tissue engineered scaffolds, and provide new materials for use within tissue engineering.