To mimic the uniformly elongated endothelium in natural linear vessels, bovine aortic endothelial cells (BAECs) are cultured on micro- to nanogrooved, model poly(dimethylsiloxane) (PDMS) substrates preadsorbed with about 300 ng/cm(2) of fibronectin. BAEC alignment, elongation, and projected area were investigated for channel depths of 200 nm, 500 nm, 1 microm, and 5 microm, as well as smooth surfaces. Except for the 5 microm case, the ridge and channel widths were held nearly constant about 3.5 microm. With increasing channel depth, the percentage of aligned BAECs increased by factors of 2, 2, 1.8, and 1.7 for 1, 4, 24, and 48 h. Maximum alignment, about 90%, was observed for 1 microm deep channels at 1 h. The alignment of BAECs on grooved PDMS was maintained at least until cells reached near confluence. F-actin and vinculin at focal adhesions also aligned with channel direction. Analysis of confocal microscopy images showed that focal adhesions localized at corners and along the sidewalls of 1-microm deep channels. In contrast, focal adhesions could not form on the bottom of the 5-microm deep channels. Cell proliferation was similar on grooved and smooth substrates. In summary, PDMS substrates engraved with micro- and nanochannels provide a powerful method for investigating the interplay between topography and cell/cytoskeletal alignment.

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The aim of this study was to investigate how topographic cues derived from a substrate containing three-dimensional microtopography interact with fluid shear stress in directing endothelial cell migration. Subconfluent bovine aortic endothelial cells were seeded on fibronectin-coated poly(dimethylsiloxane) substrates patterned with a combinatorial array of parallel and orthogonal microgrooves ranging from 2 to 5 m in width at a constant depth of 1 m. During a 4-h time-lapse observation in the absence of flow, the majority of the prealigned cells migrated parallel to the grooves with the distribution of their focal adhesions (FAs) depending on the groove width. No change in this migratory pattern was observed after the cells were exposed to moderate shear stress (13.5 dyn/cm 2 ), irrespective of groove direction with respect to flow. After 4-h exposure to high shear stress (58 dyn/cm 2 ) parallel to the grooves, the cells continued to migrate in the direction of both grooves and flow. By contrast, when microgrooves were oriented perpendicular to flow, most cells migrated orthogonal to the grooves and downstream with flow. Despite the change in the migration direction of the cells under high shear stress, most FAs and actin microfilaments maintained their original alignment parallel to the grooves, suggesting that topographic cues were more effective than those derived from shear stress in guiding the orientation of cytoskeletal and adhesion proteins during the initial exposure to flow. endothelial cell alignment; shear stress; focal adhesion ENDOTHELIAL CELL MIGRATION plays a critical role in vascular remodeling processes such as angiogenesis, vasculogenesis, and wound healing (24,25,27,30,35). The fluid shear stress experienced by vascular endothelial cells in the in vivo hemodynamic milieu provides an important mechanical cue that can direct cell migration and induce the activation of biochemical processes (8,15,35). Integrins (16, 38), focal adhesion (FA) proteins (24, 26, 27, 37), cytoskeletal components (6, 16, 18, 19, 23, 27, 28, 34, 37), regulatory proteins (13, 35, 41), and intracellular ion concentrations (12, 28) are amongst the molecular components that regulate the morphological changes and the physiological responses of endothelial cell migration upon exposure to shear stress. Therefore, the modulation of endothelial cell migration in such vascular remodeling processes requires an understanding of how the cells interact and respond to the shear stress environment.In physical terms, cell migration proceeds in three coordinated steps: 1) membrane extension and formation of FAs at the leading edge, 2) forward movement of the cell body through contraction of the actin cytoskeleton by myosin-based motors, and 3) detachment of the trailing edge of the cell, which completes the cycle of the migration process (25,30,35). When cultured cells are exposed to a steady, laminar flow, lamellipodial protrusions develop within minutes from the cell periphery in the direction of flow (27, 29), followed by the recrui...

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Endothelialization of synthetic surfaces has been challenging with limited success thus far. We investigated the hypothesis that covalent attachment of cholesterol to polyurethane via the urethane nitrogen groups would create a high-affinity surface for attachment and adhesion of endothelial cells. Cholesterol was covalently bound to the polyether polyurethane, Tecothane, by first derivatizing the polyurethane nitrogen groups with bromoalkyl side chains, followed by reacting mercapto-cholesterol to the bromoalkyl sites. Cholesterol-modified polyurethane demonstrated a qualitatively smoother surface per atomic force microscopy than nonmodified and increased surface energy (contact angle measurements) compared with unmodified polyurethane. Cell attachment assays showed a significantly greater number of attached bovine arterial endothelial cells (p = 0.0003) after 45 min of seeding on cholesterol-modified polyurethane versus unmodified polyurethane. Bovine arterial endothelial cells cultivated on cholesterol-modified Tecothane showed significantly greater levels of cell retention compared with unmodified Tecothane when exposed to arterial level shear stress for 2 h (25 dynes/cm2) with 90.0 +/- 6.23% cells remaining adherent compared with unmodified polyurethane, 41.4 +/- 11.7%, p = 0.0070. Furthermore, ovine endothelial precursors, obtained as blood outgrowth endothelial cells, were seeded on cholesterol-modified polyurethane and exposed to 25 dynes/cm2 shear conditions for 2 h, with the retention of 90.30 +/- 3.25% of seeded cells versus unmodified polyurethane, which retained only 4.56 +/- 0.85% (p < 0.001). It is concluded that covalently linking cholesterol to polyurethane results in improved material properties that permit increased endothelial cell retention compared with unmodified polyurethane.