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Yuncin Luo, Michael Sun, Victor H Guaiquil, Mark Rosenblatt; Growth of human corneal epithelial cell on silk films with different nanotopographies.. Invest. Ophthalmol. Vis. Sci. 2017;58(8):147.
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© ARVO (1962-2015); The Authors (2016-present)
Silk fibroin films (SF) can be used as cellular scaffold for tissue repair. In this study, we used photolithography to create different patterns size on silk films to study cell morphology, cytoskeletal dynamics and growth of corneal epithelial cells.
Silk films were obtained by casting silk solution on polydimethyl siloxane molds with different nanotopographies produced by photolithography techniques. Human corneal limbal epithelial (HCLE) cells, and primary cultures of human corneal epithelial cell (HCEC) and mouse corneal epithelial cells (MCEC) were seeded on different silk film topographies with or without extracellular matrix (ECM) coating. Cell adhesion and growth were evaluated in a time dependent manner using light microscopy. Cells were fixed and stained for focal adhesion staining of vinculin and actin. Cells were imaged using spinning disk confocal microscopy. Focal adhesion(FA) and spreading of these cytoskeletal proteins was assessed and quantified using ImageJ software (NIH).
Silk film nanotopography significantly affected the attachment, FA localization, and actin cytoskeleton alignment of HCLE cells. At 4 h after seeding, HCLE cellular spreading on parallel line pattern and flat surfaces was 30% -50% higher than control glass surface. Distinct vinculin localization was observed along the edges for all patterned silk film topographies after 4 hours and 3 days incubation. In addition, FA and actin filament alignment with topography was better when pitch size is smaller. Primary MCEC were able to adhere and align with pitch size of 1.6um, 2 um, 4 um and flat SF in 3-days culture while no cell attachment or spreading was observed on glass surface. Their adherence increased as the patterned size decreased. Coating the SF with ECM provided better attachment in all topographies and under this conditions primary MCEC could proliferate and remains on SF.
Our results indicate that SF nanotopographies provide better cell adhesion and growth than flat surfaces. Addition of ECM increased the cellular adhesion to silk micro- and nanotopography and provides better growth of corneal epithelial cells.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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