Meier, Lee A.2009-06-122009-06-122009-04-08http://purl.umn.edu/51033Additional contributors: Justin Weinbaum; Richard Beck; Zeeshan Syedain; Cary Valley; Robert Tranquillo (faculty mentor).The ultimate goal of the field of tissue engineering is the ability to develop engineered tissues that exhibit physiologically relevant dimensions and mechanical properties such that utilization in a clinical setting is possible. While the most common approaches in this field have resulted in significant progress toward this goal's realization, in order to fully understand the cell developmental processes that guide tissue formation in vitro, as well as in vivo, a modified approach is necessary. The field of systems biology is emerging with the hope of solving this problem. Its purpose is to better understand complex cellular signaling interactions through mathematical modeling in order to develop statistical correlations within large sets of data obtained through interrogation of the biological system of interest. Developing statistical correlations between externally induced cellular signaling events and the resulting tissue phenotype can aid in elucidation of a predictive method for understanding how a cell population will respond to varying degrees of stimulation. The question posed is: if one knows the cell/tissue culture input stimulation(s), can the resulting tissue qualities be accurately predicted? A statistical method known as discriminant partial least squares regression is commonly employed for such analyses. This type of analysis relies on construction of a matrix (X) describing the signaling events induced within a population of cells in response to varying degrees of stimulation, as well as a second matrix (Y) describing the observed cellular response in terms of expressed phenotype. It is hypothesized that a solution to the expression Y=f(X) provides a well-defined description of the connected signaling events within the system of interest. Least squares regression methods have been empirically proven effective for such previously mentioned purposes. Neonatal human dermal fibroblasts (NHDF) and Fischer rat vascular smooth muscle cells (SMC) are of primary interest for this experiment. Two-dimensional cell monolayers were utilized initially and the experimental procedure will be later extrapolated to vascular, three-dimensional, fibrin-based tissue equivalents, in particular: tunica media-equivalents (ME). The Flexcell International FX-4000 cell culturing system was used to subject cultured monolayers to periods of cyclic distention/strain, as well as varying degrees of growth factor and supplement stimulation. Following distention and biochemical stimulation, cultures were lysed and the soluble fraction isolated. Subsequently, total protein content was determined and the samples were enriched for their phosphoproteins. Mass spectrometry was then employed to quantify the enzymatic activity within individual populations of cells. This analytical medium has elucidated significant phosphoprotein mass profile distinction between differentially stimulated cell populations. Statistical correlations between the input stimulation events and the resulting cell population's qualities such as the phosphoprotein mass profiles, total collagen and elastin content, as well as total cell number will be determined. From this, a cell-culture stimulation paradigm of steepest-ascent towards developmentally- and mechanically-optimized tissue-equivalents will be employed.en-USCollege of Biological SciencesDepartment of Biochemistry, Molecular Biology and BiophysicsInstitute of TechnologyDepartment of Biomedical EngineeringDepartment of Chemical Engineering and Materials SciencePresentation