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First published online June 28, 2004
doi: 10.1242/10.1242/jcs.01146

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

Nancy W. Karuri¹, Sara Liliensiek², Ana I. Teixeira¹, George Abrams², Sean Campbell², Paul F. Nealey^1,*, and Christopher J. Murphy^2,*,

¹ Department of Chemical Engineering, School of Veterinary Medicine, University of Wisconsin, Madison, WI 53706, USA
² Department of Surgical Sciences, School of Veterinary Medicine, University of Wisconsin, Madison, WI 53706, USA

View larger version (122K): [in a new window]   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.

View larger version (19K): [in a new window]   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.

View larger version (141K): [in a new window]   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.

View larger version (96K): [in a new window]   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.

View larger version (28K): [in a new window]   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 (P0.05). (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.

View larger version (95K): [in a new window]   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.

View larger version (18K): [in a new window]   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.

View larger version (25K): [in a new window]   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 n7.

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