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T. Ng, K. Bujold, R. Redmond, J. D. Zieske; Development of an Artificial Corneal Bandage Using Primary Human Corneal Fibroblasts. Invest. Ophthalmol. Vis. Sci. 2008;49(13):2943. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
To make an artificial corneal bandage from corneal fibroblast secreted matrix in order to protect an injured cornea.
Primary human corneal fibroblasts were isolated from human donors, cultured, propagated in vitro and characterized. The cells were seeded in transwell inserts and then allowed to grow for 4 weeks. During this time the cells stratified and produced an extra-cellular matrix to form a 3-D construct. A 2-mm superficial keratectomy was made in the central cornea of BALB/c mice to expose the stroma. Rose Bengal (0.05%) was applied to the exposed corneal stromal surface, and the 3-D construct or corneal bandage was laid down on the stroma and irradiated with a green laser (532nm) for 180 seconds. Prior to transplantation, the artificial corneal bandage was labeled with DTAF. The attachment of the corneal bandage was examined by the presence of the DTAF on days 1, 7 and 14. The mice were sacrificed and the eyes were processed and examined by immunofluorescence for the presence of infiltrating immune cells with anti-CD45.
The isolated corneal cells expressed keratan sulfate proteoglycan (KSPG), Ki67 and CD90; however, they did not express keratin AE1/3 nor HLA DR. After forming the 3-D construct, the KSPG positive cells laid down extra-cellular matrix components, including collagen VI, fibronectin and thrombospondin-1. The Rose Bengal and green laser irradiation made a tight bond between the bandage and the corneal stroma. The DTAF labeled collagen stayed on the corneal stromal surface up to day 14 and was covered by epithelium by day 3. CD45 positive immune cells were found on day 1 in the limbus and central cornea in eyes +/- the artificial bandage. However, by day 3, the number of immune cells decreased in the corneas with the bandage; whereas, the corneas without bandage still maintained numerous CD45 positive cells.
The artificial corneal bandage provides a good barrier for protecting the injured corneal stroma.
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