Tissue engineering offers the possibility of creating functional replacements for diseased human organs. A key factor in producing useful tissues is the availability of materials to promote and control cellular activity. Ideally suited would be materials that transmit biological signals (in a controlled manner) that induce cells to attach, spread, grow, differentiate, and eventually organize to form a tissue. Fibronectin is a matrix protein known to induce cell adhesion and growth through its cell binding site. For this reason, materials coated with fibronectin have found use as tissue engineering substrata. A problem is that current placement procedures often result in a distribution of protein orientations on the surface, leaving a large fraction of binding sites inaccessible to cells. Our hypothesis is that if fibronectin could be bound to a surface with a preferential orientation that directs the cell binding site away from the surface, cell attachment and growth would be enhanced and precise control over tissue growth would become possible. In this proposed work, affinity, electric field, and antibody linking techniques will be developed for engineering orientationally controlled fibronectin coatings. Initial work will investigate control of fibronectin orientation using affinity techniques. An affinity ligand will be attached to a material's surface and the bio-specific interaction between the ligand and fibronectin will be exploited to orient the protein so that its active segment faces upwards (towards contacting cells). Ligands investigated here will include monoclonal antibodies and metal chelates. An integrated optical biosensor will be used to provide continuous measurements of the density and thickness of the interfacial protein. Since extensive surface modification may be disfavored in some applications for cost or biocompatibility reasons, new techniques for controlling protein orientation will also be investigated. The application of a perpendicular electric field during chemical adsorption will be used to align the protein and induce its adsorption. Antibody- linking techniques will be used to induce the protein to chemically adsorb with the active site facing away from the surface. The ability of the engineered fibronectin layers in promote and control cellular activity will be measured by observing the attachment, spreading, and growth of endothelial cells using an optical microscope. To improve our understanding of affinity, electric field, and linked-antibody adsorption, and to assist in the rational design of fibronectin coated materials, an entropy sampling Monte Carlo computer simulation of a coarse grained model fibronectin will be performed.
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