Microcontact printing approaches to pattern the attachment of endothelial cells on silicone surfaces with microgrooves

Fluid shear stress (FSS) has been employed to create two-dimensional (2D) monolayers of endothelial cells (ECs) that resemble the organization of natural vasculature. However, shape-dependent EC properties that are independent of FSS remain largely undefined. More recently, surface micropatterning has been investigated as an approach to control the morphology and orientation of ECs using synthetic substratum. Most research studies have reported the effects of microtopography on sub-confluent layers of cells, which has been the standard in research investigations to date. In this thesis, surface micropatterning is used to mimic the natural EC vasculature in confluent layers of cells.Cell alignment and elongation of bovine aortic endothelial cells (BAECs) were studied on poly(dimethylsiloxane) (PDMS) surfaces consisting microgrooves of parallel channels and ridges with depths of 100 nm, 500 nm, 1 μm, and 5 μm. The silicone surfaces were preadsorbed with 10 μg/ml fibronectin (FN), an ECM protein, to encourage cell attachment. More than 70% of the cells aligned in the 500 nm and 1 μm depth microgrooves as compared to the BAECs cultured on unpatterned substrates, which showed no preferential alignment. Further, the 1 μm depth resulted in maximum elongation (> 3.0) of BAECs using Factor E, which quantifies morphological differences between cells on these microgrooves as compared to their counterparts on smooth silicone surfaces.The effects of microtopography-induced alignment on the spatial localization of caveolae were investigated. Caveolae are microdomains of the plasma membrane that contain and regulate a variety of signaling molecules, and hence play an important role in cell function. Immunostaining protocols were employed to characterize spatial localization of the endothelial nitric oxide synthase (eNOS) and its primary regulatory protein, caveolin- 1 (Cav-1). Analysis showed that the expression levels of eNOS and Cav-1 were significantly higher on 500 nm and 1 μm depth patterned surfaces. Based on the Mander’s coefficient of colocalization, the 1 μm depth exhibited the highest percent colocalization (R=76%) of eNOS and Cav-1. These signaling molecules were observed to align within the channels of the 5 μm depth microgrooves, and similar alignment was observed for actin filaments. This indicates possible interactions between eNOS and Cav- 1 with actin filaments. While PDMS microgrooves strongly influenced cell orientation and morphology, microcontact printing of fibronectin on smooth PDMS determined that these microgroove-based changes in cells are a result of the 2D surface geometry and the three-dimensional (3D) spatial arrangement of cells. In summary, PDMS substrates with patterned microgrooves provide a method for evaluating the interplay between cell orientation and spatial localization of membrane proteins, which may be patterned using microcontact printing techniques.M.S., Biomedical Engineering -- Drexel University, 201