Interfacial biomolecular engineering: Controlling material-protein-cell interactions through micropatterning.

摘要

Emerging biomolecular patterning techniques provide us with powerful tools to interrogate complex interfacial biological phenomena through the controlled placement of relevant molecules at microscale and nanoscale resolutions. Characterization methods such as optical imaging and scanning force microscopies (SFM) additionally leverage our ability to understand the molecular signals present in model surfaces and our ability to interpret biological and molecular interactions at submicrometer scales. In this work, chemical and protein micropatterning methods have been applied to investigate the phenomena of protein spatial-temporal adsorption kinetics on heterogeneous surfaces, in vitro protein deposition bioactivity and resulting effects on cell adhesion and phenotypic expression, and neuronal integration of and adaptation to graded permissive and inhibitory signals.; Protein binding kinetics and spatial distribution were imaged and quantified on micropatterned heterogeneous organosilane films employing a fluorescence microscopy (FM) and image analysis technique. A novel dual protein patterning method was developed for cell choice assays and characterized through SFM, FM, and protein integrin epitope presentation. Protein submicroscale organization and epitope availability were then related to neuronal cell attachment and neurite outgrowth behavior in the presence of permissive, nonpermissive, and inhibitory signals. Based on observations from these studies, a microfabrication process was devised to print multiple distinct densities of the inhibitory proteoglycan aggrecan in the form of micro-island arrays on a single homogeneous growth promoting laminin field. Time lapse video microscopy, integrin receptor labeling and functional block, FM, and total internal reflection fluorescence microscopy, were used to investigate neuronal responses to graded and changing inhibitory substratum bound signals during growth cone signal integration, pathfinding, and adaptation.; It was found that protein interactions with submicroscale surface heterogeneities provides for an accurate model of interfacial protein adsorption, that nonspecific adsorption proceeds at faster rates and results in lower surface densities than do molecular specific driven binding events, that the method of laminin deposition in the presence of aggrecan and final integrin presentation are powerful predictors of neurite outgrowth, and that neuronal adaptation to aggrecan is dose and time dependent. Through these experiments, the utility of micropatterning is demonstrated for several unique biological phenomena.