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Kai B. Kang, Brigette Cole, Brian D. Lawrence, Aihong Liu, Mark I. Rosenblatt; Morphology and Alignment of Primary Human Corneal Epithelial Cells (HCEC) and a Human Corneal Limbal Epithelial (HCLE) Cell Line on Nano- and Micropatterned Silk Films. Invest. Ophthalmol. Vis. Sci. 2012;53(14):1816.
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© ARVO (1962-2015); The Authors (2016-present)
The corneal epithelial basement membrane has topographical features that may provide biophysical cues to direct the adherence, migration and proliferation of epithelial cells. Silk fibroin is a promising biomaterial for tissue engineering in ophthalmic related applications due to its transparency, biocompatibility and stability. In this study, patterns were created on silk films to explore how varying topographic pitch alters epithelial cell morphology,and cytoskeletal dynamics.
Silicon wafers with parallel ridge widths and spacing of 2,000 nm, 1,000 nm and 800 nm were produced. PDMS molds were produced from these surfaces and used to pattern silk film dried upon their surfaces. HCECs harvested from donor corneas and HCLE cell line were cultured onto silk films. After 72 hours of incubation, phase contrast images were taken to study cell alignment, density and shape on each substrate. Cell cytoskeletal structures were studied by immunofluorescent staining.
HCEC and HCLE cell alignment to the surface pattern was the greatest on 1,000 nm features (64% and 89% respectively, n=50), while cells aligned either parallel on 2,000 nm or perpendicular on 1,000 nm and 800 nm feature width dimensions. On the 1,000 nm features, elongated HCEC and HCLE cells exhibited the highest shape factors (ratio of major axis over minor axis) of 3.4 and 4.5 respectively, versus 1.7 and 1.9 on flat silk surfaces (p<0.001). For both HCEC and HCLE cells, cell density after 72 hours of culture was less on the 1,000 nm films in comparison to all other silk substrates (p<0.001).For both HCEC and HCLE cells, fluorescent microscopy of f-actin staining showed cell cytoskeleton alignment either in parallel (2,000 nm) or perpendicular (1,000 nm and 800 nm) to the long feature axis. Z-stack projection of vinculin staining indicated increased focal adhesion formation localized on the cellular basal surface as feature pitch decreased.
The results of the study indicate that patterned substrates composed of silk fibroin can influence cell morphology and shape via modulation of cytoskeletal dynamics. Findings from this study suggest that silk film feature pitch size may play a role in modulating cell characteristics and also offer a promising customizable material for use in ocular surface repair.
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