Current biomaterials are designed to be passive in nature to prevent the initiation of adverse immune responses upon contact with biological substances. While this approach of inertness is still a crucial design component for some applications, the possibility of engineering desired cell responses in the local environment of the material exists and is of particular interest in implantable devices and tissue engineered constructs. Fundamental knowledge of the relationships between cell adhesion and gross cell behavior will provide key design criteria for the creation of advanced biomaterials that induced locally controlled cellular responses. This work investigates the possibility of engineering cell behavior by limiting adhesion site maturation. Chemically-defined nanoislands of fibronectin were created using a combination of nanosphere lithography and an orthogonal surface functionalization strategy. Investigation of the adhesive and cytoskeletal components of cells cultured on these surfaces demonstrates that chemically-defined nanopatterns provide an upper size limit to adhesion site growth which in turn influences the degree of cytoskeletal formation. The imposed restriction on adhesion site growth results in the formation of a relatively higher number of more evenly distributed, small adhesions throughout the cell body. The adhesive behavior can be tuned by changing the nanopattern properties with respect to their size, spacing, and density. Furthermore, it is demonstrated that the observed differences in cell adhesion as imposed by the nanopatterned surfaces induces changes in gross cell behavior with respect to spreading, proliferation, and motility. The results presented here parallel observations documented in cells cultured on elastic surfaces and indicate that intracellular signaling cascades initiated and governed by cellular adhesion sites are sensitive to adhesion size/maturation and possibly the amount of force generated locally at these adhesion sites. The conclusions drawn from these studies give insight into the possibility of implementing nanostructured biomaterials for cell engineering purposes and provide design criteria for the next generation of tissue engineered constructs. / text

published_or_final_version / Physics / Master / Master of Philosophy

Cells rely on time-dependent binding and activation by the ECM to initiate downstream signal transduction. It is unknown whether adhesion to a ligand is required throughout various cell processes, or only during a specified time period ("temporal threshold”). Current approaches to ligand presentation often comprise of static, constant densities of ligands. In contrast, natural cell adhesive interactions with ECMs exhibit spatiotemporal patterns of binding and activation. Therefore, a key to future research in controlling cell-material interactions will be the development of materials that can respond to external stimuli. The objective of this project is to engineer biomaterials that present a UV-labile caged-Arginine-Glycine-Aspartic Acid (RGD) ligand and evaluate the effects on cell activities. RGD is the minimal adhesive sequence of fibronectin. By dynamically modulating adhesive ligand presentation, the effects of temporal control on cell processes can be elucidated. In this caged-peptide, a photo-labile group adjacent to the aspartic acid residue of RGD effectively “masks” a cyclo(RGDfk) peptide. Upon UV irradiation (360 nm), the caging group is released thereby restoring the adhesive activity of the peptide. By having unparalleled spatiotemporal control of RGD ligand presentation, we demonstrated two novel tools for discovery: 1) in vivo ligand presentation to probe downstream tissue behavior and cell infiltration to biomaterial implants, and 2) in vitro ligand presentation in situ using confocal-based live cell microscopy to investigate real-time vinculin recruitment and cell traction force generation. These studies represented the first demonstration of triggerable adhesive ligand presentation in vivo and demonstrated the utility of caged-compounds for probing specific receptor-ligand responses on highly defined PEG-based hydrogels. Triggerable in vitro ligand presentation, combined with traction force microscopy, demonstrated a new research tool for investigating focal adhesion formation and downstream force generation. Taken in whole, these results provide previously unknown insights into the importance of spatiotemporal control of adhesive ligands and created novel new research platforms for future discovery.

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