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Brian D. Lawrence, Zhi Pan, Aihong Liu, Mark I. Rosenblatt; Guided Vinculin Formation Using Patterned Silk Film Substrates. Invest. Ophthalmol. Vis. Sci. 2012;53(14):2206.
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© ARVO (1962-2015); The Authors (2016-present)
The transparent nature, robust mechanical properties, and non-inflammatory properties make silk films a promising material choice for use in ocular surface repair. Freestanding silk films possessing flat, parallel line and concentric ring topographies were generated and utilized for in vitro assessment of human corneal limbal-epithelial (HCLE) cell responses. Vinculin and actin localization and expression were assessed to determine the effects of silk film surface features on cytoskeleton development.
Micro patterns were produced upon silicon wafers using photolithography techniques, which the negative image was reproduced on cast polydimethyl siloxane (PDMS) molds. Silk solution was then cast upon the various patterned PDMS molds, allowed to dry, water-annealed, and then seeded with the HCLE cell line. Films were fixed at 4 hours and 3 days post-seeding, and then stained for vinculin and actin structures using antibody and phalloidin fluorescent probes respectively. Samples were then imaged using fluorescent microscopy and images were further processed using deconvolution Hyugens Professional software package (SVI, Netherlands). Vinculin structure size, number, and localization were assessed and quantified using ImageJ software (NIH). Protein expression was assessed using western blot analysis and infrared responsive anitbodies (LI-COR Biosciences).
Silk film topography significantly affected initial HCLE attachment, focal adhesion (FA) localization, and actin cytoskeleton alignment. Parallel line patterned surfaces displayed a 50% increase in initial cell attachment corresponding to an over 2-fold increase in FA localization when compared to the ringed surface features and flat silk substrates. In addition, distinct localization of FA formation was observed along the edges for all patterned silk film topographies. However, vinculin and actin expression remained consistent upon the various topographies.
Silk film feature topography aids in directing corneal epithelial cell responses and cytoskeleton development in vitro. Vinculin localization formed almost entirely at the edge surface of the various feature patterns. In addition, feature geometry appeared to play a role in guiding vinculin morphology and overall actin alignment. Protein expression remained consistent between the various patterns indicating that silk film surface features induce a localizing effect rather than altering internal protein expression. These results indicate that silk film surface design can be used to induce varying effects upon cultured corneal epithelial cell lines.
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