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Chau Vo, Hyun Jong Lee, Gabriella Fernandes-Cunha, David Myung; Engineering an electrospun nanofiber to direct corneal epithelial cell proliferation and morphology. Invest. Ophthalmol. Vis. Sci. 2019;60(9):906.
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© ARVO (1962-2015); The Authors (2016-present)
Despite its protective role to the eye, the cornea is highly vulnerable to injuries. This project aims to fabricate a scaffold to facilitate corneal epithelial wound healing by providing mechanical support and stimulating cell migration in the damaged cornea. We investigated three different fiber alignments—aligned, randomly aligned, and radially aligned—and sought to optimize their properties for corneal epithelial cell (CEC) proliferation and morphology.
At 15kV, randomly aligned fibers (RnF) and aligned fibers (AF) scaffolds were fabricated via electrospinning (RnF at 100 rpm, AF at 800rpm); radially aligned fibers (RdF) were made with ring-and-central-point electrodes. Fibers were crosslinked in glutaraldehyde vapor in 24 hours and visualized under scanning electron microscopy (SEM). The fibers were then cut into circles with diameter 10 mm and sterilized under UV light before being transferred to a 24-well plate. The scaffolds (n=10 for each type) were coated with collagen (300 mL) for 30 minutes. Immortalized CECs (2x10^4) were seeded to each scaffold and 500 mL of KSFM medium containing growth factor (EGF) and bovine pituitary extract (BPE). The media was changed every other day. After 2 and 5 days, the scaffolds (n=3) were fixed with PFA and stained with F-actin dye and DAPI. The fixed cells were then visualized under an inverted microscope to observe cell morphology. The remaining 2 samples were used to assess proliferation using MTT assay.
SEM showed desired fiber alignments were achieved. The average fiber diameter was 1 mm. MTT assay showed no statistically significant difference in CEC proliferation across the three fiber alignments. However, immunostaining and visualization under an inverted microscope showed significant differences in cell morphologies. Cells on wet plate had round morphologies. Cells on AF and RdF had elongated morphologies because the local fiber alignments were similar for both scaffolds. Cells on RnF orientated their lamellipodia in different directions based on fiber orientation.
We showed that fiber alignment does not affect cell proliferation per MTT assay. However, it does affect cell elongation along the nanofibers. Future studies include investigating whether RdF fabricated via electrospinning can provide a more suitable migratory environment than AF or RnF to direct peripheral cells toward a central wound site.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.
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