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P. Denk, M. Grueb; Interaction of Human Corneal Epithelial and Stromal Cells With Edc–Crosslinked Type I Collagen Films . Invest. Ophthalmol. Vis. Sci. 2006;47(13):3940.
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In the present study we investigated wether three–dimensional EDC–crosslinked collagen films are populated by human corneal epithelial cells and human fibroblasts in vitro. Furthermore we checked for deposition of basement membrane proteins by regenerating epithelium onto the collagen films during organ culture.
Three dimensional collagen films were chemically crosslinked using 1–ethyl–3–(3–dimethyl aminopropyl) carbodiimide (EDC). After removal of an anterior corneal stromal lamella, collagen films were sutured into the stromal defect of a human donor corneoscleral segment and incubated for 2 weeks in organ culture medium. After the incubation, the specimen were processed for light microscopy and immunohistochemistry in order to check for expression of fibronectin, laminin and collagen IV. In a second experimental model, cultured human corneal fibroblasts were seeded onto the collagen films in Boyden chambers and cultured in the presence of 10% FCS and PDGF–BB (50ng/ml). After 10 days of incubation, the cell–matrix–complex was prepared for light microscopy to evaluate fibroblast migration into the collagen films.
In the organ culture model, EDC–crosslinked collagen films were found to be completely covered by donor epithelium after 2 weeks of incubation. Epithelial cells formed two to three cell layers and expression of both laminin and fibronectin was observed under the regenerating epithelial cells. Both in the organ culture model and in the explant culture model we failed to detect fibroblast migration into the matrix.
The present study shows, that donor corneal epithelium regenerates over EDC–crosslinked three–dimensional collagen films which were sutured onto a corneoscleral donor segment. Regenerating epithelial cells deposit laminin and fibronectin to reconstruct a new basal membrane on the collagen matrix. It is therefore concluded, that the incorporation of epithelial cells and basal membrane into artificial corneal tissue is probably not necessary for artificial corneal to serve as an alternative to corneal allografts in the future.
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