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

Abstract

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.

Fig. 1

Characterization of nanostructured surfaces. Scanning electron microscopy image of a cross-sectional view of the silicon structures created by X-ray lithography. p, pitch; g, groove size; r, ridge size; e, etch depth. Scale bar: 400 nm.

Fig. 2

(A) Schematic of flow cell and image capture and analysis system. Fluid is circulated by the peristaltic pump, through the damping vessel and into the parallel plate flow chamber to yield uniform shear on the substratum. The damping vessel serves to reduce pulsatile flow. (B) Profile of the calculated volumetric flow and associated wall shear stress that cells were exposed to. The circulating fluid for this profile was DPBS solution.

Fig. 3

SEM images of adherent SV40-HCECs on the surfaces. (A) Planar surface. (B) Patterned surfaces: (i) 400 nm pitch, (ii) 4000 nm pitch. (C) Tip of an elongated cell: (i) 400 nm pitch, (ii) 4000 nm pitch.

Fig. 4

Images of SV40-HCECs on the patterned and planar surfaces prior to and after exposure to uniform fluid shear. Before Shear: (A) 400 nm pitch, (C) 4000 nm pitch (E) planar surface. Insets in A, C and E show typical cell morphology on these surfaces. AfterFlow (maximum wall shear stress of 80 Pa): (B) 400 nm pitch, (D) 4000 nm pitch, (F) planar surface. Scale bar: 100 μm.

Fig. 5.

Percentage of adherent cells increases with decreasing pitch size. (A) SV40-HCEC adhesion using DPBS solution as circulating medium and a stepped-up fluid shear stress profile. Percentage of cell attachment on the seven topographical areas at all the wall shear stresses sampled. Mean±s.e.m. n=4. (ii) Percentage of cell attachment on the seven topographical areas at 40 and 80 Pa wall shear stress. Mean±s.e.m. n=4. Total cell count for each topographical area was approximately 160. Asterisks indicates significantly different means (P≤0.05). (B) SV40-HCEC adhesion using SHEM media solution as the circulating medium and a constant fluid shear stress of 10 Pa. Representative populations of cells before (i) and after (ii) fluid shear. (C) Comparison between primary and SV40 human corneal epithelial cell attachment on the 400 nm pitch after fluid shear as shown in Fig. 2B.

Fig. 5.

Percentage of adherent cells increases with decreasing pitch size. (A) SV40-HCEC adhesion using DPBS solution as circulating medium and a stepped-up fluid shear stress profile. Percentage of cell attachment on the seven topographical areas at all the wall shear stresses sampled. Mean±s.e.m. n=4. (ii) Percentage of cell attachment on the seven topographical areas at 40 and 80 Pa wall shear stress. Mean±s.e.m. n=4. Total cell count for each topographical area was approximately 160. Asterisks indicates significantly different means (P≤0.05). (B) SV40-HCEC adhesion using SHEM media solution as the circulating medium and a constant fluid shear stress of 10 Pa. Representative populations of cells before (i) and after (ii) fluid shear. (C) Comparison between primary and SV40 human corneal epithelial cell attachment on the 400 nm pitch after fluid shear as shown in Fig. 2B.

Fig. 6.

Time-lapse images of cells on the 400 nm pitch, 4000 nm pitch and planar surface as they experience fluid shear stress: (A,F,K) No Flow (static conditions). (B-O) Wall shear stress of 20 Pa (B,G,L), 40 Pa (C,H,M), 60 Pa (D,I,N), 80 Pa (E,J,O). Arrowheads indicate position of detached cells. Scale bar: 50 μm.

Fig. 7.

Populations of oriented and elongated cells before and after flow and the number of cells still adherent at maximum wall shear stress. The numbers are based on 100 adherent cells before flow. The total number of cells analyzed was approximately 200 for each type of topography sampled. Mean±s.e.m. n=4.

Fig. 8

The effect of surface discontinuities on cell adhesion on the different feature sizes. Both the average number of discontinuities that a cell interfaced and the number of adherent cells present after exposure to a wall shear stress of 10 Pa for 15 minutes are presented as a functions of surface topography. The number of surface discontinuities that a cell spanned was determined from SEM images of cells on the different topographical areas. For SEM analysis the total number of cells examined was 25. Mean±s.e.m. 4≤n≤7.