Silk fibroin protein enhances both in vitro and in vivo corneal epithelial wound healing through increased cell migration, adhesion, and proliferation.

Abstract

Purpose : Corneal epithelial wound healing involves a set of complex biological processes that work to restore the epithelium after injury. Biomaterials derived from Bombyx mori silk fibroin have been used in therapeutic applications to promote wound healing and tissue regeneration. Our study evaluated the effect of silk fibroin, in its solution form, on corneal epithelial cell behavior and regeneration in vitro, and corneal epithelial wound healing in an animal model.

Methods : We used human corneal limbal epithelial cells (HCLE) to evaluate if silk fibroin solution induces a significant effect on epithelial cell adhesion, migration and proliferation, and the concomitant effect on epithelial cell recover after injury. To study cellular adhesion, we utilized a parallel plate flow chamber and analyzed focal adhesion protein by immunofluorescence staining. Migration of epithelial cells after a scratch injury was quantitatively analyzed using time lapse imaging and the spreading of epithelial cells in response to treatment with silk fibroin solution was quantified using phase contrast imaging. Cell proliferation was evaluated using an MTT colorimetric assay as well as flow-cytometric cell cycle analysis. The effect of silk fibroin solution in promoting corneal epithelial wound healing in vivo, was analyzed in a rabbit corneal abrasion model.

Results : Our in vitro results using HCLE cells demosntarted that silk fibroin solution enhanced cell matric focal adhesions by over 95% compasred to control. Similarly, we observed a 50% increase in epithelial cell migration and a 60% enhancement in cell proliferation when cells were treated with silk fibroin solution. Epithelial wound closure showed a 2 fold increase in healing rate by 10 hours with 0.4% wt./vol. silk fibroin and a 30% improvement in wound closure by 15 hours. HCLE cells that were mitotically inhibited showed a 30% increase in wound closure when treated with silk fibroin solution. In our in vivo model, silk fibroin solution accelerated the recovery of the injured corneal epithelium.

Conclusions : Our findings indicate that silk fibroin solution induces major rearrangements on the cell cytoskeleton that may contribute to enhanced cell adhesion, migration and proliferation, leading to faster re-epithelialization of corneal wounds.

This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.