Ana I. Teixeira

Epithelial contact guidance on well-defined micro- and nanostructured substrates

The human corneal basement membrane has a rich felt-like surface topography with feature dimensions between 20 nm and 200 nm. On the basis of these findings, we designed lithographically defined substrates to investigate whether nanotopography is a relevant stimulus for human corneal epithelial cells. We found that cells elongated and aligned along patterns of grooves and ridges with feature dimensions as small as 70 nm, whereas on smooth substrates, cells were mostly round. The percentage of aligned cells was constant on substrate tomographies with lateral dimensions ranging from the nano- to the micronscale, and increased with groove depth. The presence of serum in the culture medium resulted in a larger percentage of cells aligning along the topographic patterns than when no serum was added to the basal medium. When present, actin microfilaments and focal adhesions were aligned along the substrate topographies. The width of the focal adhesions was determined by the width of the ridges in the underlying substrate. This work documents that biologic length-scale topographic features that model features encountered in the native basement membrane can profoundly affect epithelial cell behavior.

Biological length scale topography enhances cell-substratum adhesion of human corneal epithelial cells

The basement membrane possesses a rich 3-dimensional nanoscale topography that provides a physical stimulus, which may modulate cell-substratum adhesion. We have investigated the strength of cell-substratum adhesion on nanoscale topographic features of a similar scale to that of the native basement membrane. SV40 human corneal epithelial cells were challenged by well-defined fluid shear, and cell detachment was monitored. We created silicon substrata with uniform grooves and ridges having pitch dimensions of 400-4000 nm using X-ray lithography. F-actin labeling of cells that had been incubated for 24 hours revealed that the percentage of aligned and elongated cells on the patterned surfaces was the same regardless of pitch dimension. In contrast, at the highest fluid shear, a biphasic trend in cell adhesion was observed with cells being most adherent to the smaller features. The 400 nm pitch had the highest percentage of adherent cells at the end of the adhesion assay. The effect of substratum topography was lost for the largest features evaluated, the 4000 nm pitch. Qualitative and quantitative analyses of the cells during and after flow indicated that the aligned and elongated cells on the 400 nm pitch were more tightly adhered compared to aligned cells on the larger patterns. Selected experiments with primary cultured human corneal epithelial cells produced similar results to the SV40 human corneal epithelial cells. These findings have relevance to interpretation of cell-biomaterial interactions in tissue engineering and prosthetic design.

Cell behavior on lithographically defined nanostructured substrates

Lithographically defined substrates offer unique opportunities for the study of cell behaviors, by allowing the presentation of controlled cell stimuli. We have investigated the effects of substrate topography on the behavior of human corneal epithelial cells using substrates patterned with grooves and ridges of well-defined dimensions. On each substrate, we included feature sizes ranging from the micrometer to the nanometer scale. This work was motivated by the fact that the surface that underlies epithelial tissues in vivo, the basement membrane, has a rich topography with features of nanoscale dimensions. We found that cells responded to topographic features as small as 70 nm wide by aligning with the pattern direction. Additionally, substrate topography affected internal organization of the cell, inducing the alignment of cytoskeletal elements (actin filaments) and adhesive structures (focal adhesions). Therefore, synthetic topographies with feature dimensions of the same length scale as the features ...