| Cell behavior is strongly influenced by the microenvironment. In vitro studies have conclusively shown that chemistry, geometry, and mechanics have all been shown to influence or direct cellular proliferation, differentiation, and matrix formation. However, few systems exist that allow researchers to study the interaction of these factors. In this work, electrohydrodynamic jet (e-jet) printing is introduced as a method to pattern adhesion proteins on hydrogel substrates for cell culture. First, a new technique to fabricate polyacrylamide substrates was developed and optimized. Hydrogels were formulated with acrylic acid and activated with N-hydroxysuccinimide. Protein density was shown to depend on the amount of acrylic acid, providing a novel way to control ligand density. Second, e-jet was used to pattern extracellular matrix (ECM) proteins on activated polyacrylamide. Protein conjugation was verified with immunohistochemistry, and functionality has demonstrated with cell adhesion. Cells seeded on e-jet- patterned substrates were cultured up to four days, growing to confluence within printed patterns without spreading onto non-patterned regions. The substrates were next used to study the formation of "nodules," the fundamental unit of bone formation in vitro. Nodule structure was evaluated after four days in culture, and patterned substrates were shown to be compatible with traction force microscopy (TFM). These represented preliminary results for a larger study to evaluate how microenvironmental stiffness and geometry influence cytoskeletal contractility and ultimately bone formation. Finally, a novel method was introduced to pattern both stiffness and chemistry at subcellular resolution. Polyacrylamide spots printed with e-jet were backfilled with a second polymer mixture to create substrates with circular microwells. Finite element modeling (FEM) was used to show that microwell topography was a result of backfill contraction during exposure. The FE model was then used to make predictions for further substrate design. The techniques presented in this thesis represent highly flexible, high resolution methods for crafting substrates to study microenvironmental regulation of cell behavior. |
