Effect of Honeycomb-Patterned Surface Topography on the Adhesion and Signal Transduction of Porcine Aortic Endothelial Cells

Abstract

Surface topography has vital roles in cellular response. Here, to investigate the mechanism behind cellular response to surface topography, we prepared honeycomb (HC)-patterned films from poly(epsilon-caprolactone) (PCL) with micropatterned surface topography by casting a polymer solution of water-immiscible solvent under high humidity. We characterized the adsorption of fibronectin (Fn) on the film using atomic force microscopy (AFM) and confocal laser scanning microscopy (CLSM). The response of porcine aortic endothelial cells (PAECs) to adsorbed Fn molecules onto HC-patterned films was observed by immunofluorescence labeling of vinculin and the actin fiber of PAECs cultured for 1 and 72 h in serum-free medium. The expression of focal adhesion kinase autophosphorylated at the tyrosine residue (pFAK) at 1 h culture was determined using an immunoprecipitation method. Fn adsorbed selectively around the pore edges to form ring-shaped aggregates. The immunostaining results revealed that PAECs adhered to the HC-patterned films at focal contact points localized around pore peripheries. These points correspond to adsorption sites of Fn. The expression of pFAK after 1 h on the HC-patterned film was 3 times higher than that on a corresponding flat film, indicating that the signaling mediated by the binding between Fn and the integrin receptor was more highly activated on the HC-patterned film. These results suggest that the cellular response to HC-patterned films (e.g., adhesion pattern and phosphorylation of FAK) originates from the regularly aligned adsorption pattern of Fn determined by the pore structure of the film.

Reversible on-demand cell alignment using reconfigurable microtopography

“…Some weaknesses of the system are: (i) the use of a relatively thick (∼8 mm) bottom substrate to provide mechanical support reduces the quality of optical micrographs and excludes use of short working distance, high numerical aperture objectives with inverted microscopes, (ii) difficulty in precisely reproducing the oxidation conditions leads to some variability in the wave sizes obtained from batch to batch (although the ability to create and recreate very similar features on the same substrate is excellent); and (iii) the use of a material, PDMS, that may adsorb lipophilic chemicals and alter the effective concentration of reagents for cell biological studies [34] (though the relatively large volumes of liquid to surface area in this device, compared to microfluidics systems, should mitigate this effect). Even though this paper focused exclusively on alignment of C2C12 myoblasts, many other cell types respond to topographical features, including other muscle cells [35,36], osteoblasts [37], endothelial cells [38], HeLa cells, hepatocytes and fibroblasts [39,40].…”

Cell adhesion and response to synthetic nanopatterned environments by steering receptor clustering and spatial location

“…Protein-surface interactions are highly dependent on surface properties such as charge, hydrophobicity and hydrophilicity (Bergkvist et al, 2003). These features directly determine the conformation of the adsorbed protein, which is vital for the maintenance of its biological activity (Yamamoto et al, 2007). While structural changes in ECM adhesive proteins during adsorption onto a substrate affect the molecular binding of these proteins to cell receptors and, in turn, mediate cell response (Garcia et al, 1999;Keselowsky et al, 2004), the number of available sites for the interaction with cell receptors cannot be controlled because of unpredictability of orientation and accessibility of the binding sites (Elbert et al, 2001).…”

Controlled cellular orientation on PLGA microfibers with defined diameters

“…In the body, cells are exposed to a 3-D environments which signals the cells about whether it should proliferate, differentiate, migrate or remain quiescent. Therefore, conventional 2-D patterns or micropatterned 3-D structures (like grooves) have limitations in mimicking the in vivo environments for tissue culture (Yamamoto et al 2007) or drug screening. (Mai et al 2007) Many components of the natural extracellular matrix (ECM) have a fibrous structure (L' Heureux et al 2006); thus the ability to understand cellular behavior on fibers would provide new opportunities in tissue formation and regeneration by enabling closer control of cell shape, adhesion and differentiation.…”