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Zhi Pan, Brian D. Lawrence, Mark I. Rosenblatt; Patterned Silk Fibroin Films Regulate the Cytoskeletal Dynamics and Gene Expression of Corneal Epithelial Cells. Invest. Ophthalmol. Vis. Sci. 2011;52(14):1944.
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© ARVO (1962-2015); The Authors (2016-present)
Silk fibroin is a promising biomaterial for tissue engineering in ophthalmic related applications due to its transparency, controllable material properties, and biocompatibility. Highly controlled surface topographies can be produced on the surface of silk films and provide contact cues to guide corneal epithelial cell migration. In order to further explore the underlying mechanisms of how the surface topographies regulate cell behaviors, we studied related signaling pathways and whether these topographic cues also induce any changes in gene expression.
Silk solution was cast upon microfabricated PDMS molds with the same feature size and different geometrical topographical patterns (lines, rings, and spiral) to produce ~30 um thick films that retained the negative image of the micropatterns. The films were then annealed to produce an insoluble material. A cell line of human corneal-limbal epithelium (HCLE) was transfected with plasmids encoding RFP-actin, CFP-tubulin and GFP-vinculin and seeded on patterned silk films. Living cell imaging was then performed during cell adhesion and migration. The activation and distribution of several Rho GTPases (RhoA, Rac1 and Cdc42) that are closely related to the cytoskeleton formation and remodeling were studied by western blotting and immunofluorescent staining. Real-time PCR was used to compare the expression level of genes related to cell differentiation.
The alignment of actin fibers and focal adhesions along patterns was observed as early as 2 hours after incubation. The dynamics of actin cytoskeletal contraction was significantly slower on patterned surfaces. Although the distribution and activity of Rac1 and Cdcd42 were not significantly changed by silk surface patterning, RhoA was more concentrated along the patterns. On silk/patterned silk surfaces, the gene expression levels of epithelial cell differentiation markers such as keratin 3 and involucrin were significantly reduced.
The results of this study indicate that non-patterned and micro-patterned bioengineered substrates composed of silk fibroin can significantly influence cell behavior via modulation of signaling pathways regulating cytoskeletal dynamics. External signals from silk substrates not only regulated cell phenotype functions but also profoundly influence the gene expression. Furthermore, findings from this study strongly suggest that silk fibroin films may maintain cells in a less differentiated state which would be very promising for ocular surface tissue engineering.
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