Spatially controlled engineering of myocardial tissue

Thesis (Ph. D.)--University of Washington, 2001Developmental biology and tissue regeneration processes utilize specific spatial adhesive cues to direct the assembly of cells into complex, organized tissues. Microfabrication methods provide analogous technologies to accurately control the displacement of proteins and cells in vitro to mimic their natural spatial organization. Thus, we have investigated how spatially controlled engineering of protein interfaces can be used to direct cellular response and the organized assembly of tissues. Adhesive extracellular matrix proteins were micropatterned directly by microcontact printing onto a number of different substrates and shown to spatially control the attachment of various anchorage dependent mammalian cell types. Specifically, micropatterned lanes of laminin were used to guide the adhesion and organization of cardiomyocytes on polymeric surfaces such that they exhibited a more mature and fully differentiated phenotype. Morphological features of native myocardium, such as the dimensions of individual cardiomyocytes, myofibril assembly and organization, intercalated disk localization and synchronous contractile behavior, were reproduced by micropatterned cultures of cardiomyocytes on polystyrene substrates. The true myocardium, however, is an organized, heterogeneous tissue composed of various cell types, thus a spatially defined co-culture system was developed, utilizing an engineered streptavidin mutant, to facilitate secondary cell adhesion between patterned rows of cardiomyocytes. Comparable cardiomyocyte patterns were also achieved on thin films of resorbable polymers (i.e. PLGA and biodegradable polyurethane), in order to engineer organized sheets of cardiomyocytes. Dense, highly aligned layers of cardiomyocytes could contract thin elastomeric polyurethane films and were successfully grafted onto the surface of the heart in nude mice. This method represents a novel therapeutic approach to potentially repair infarcted myocardium by transplanting spatially organized layers of cardiac tissue. In addition to tissue engineering applications, patterned cardiomyocyte cultures are also applicable to in vitro cell biology and physiological studies or diagnostic applications, such as pharmacological screening, that require more accurate reproduction of native myocardial architecture