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M. Gorbet, X. Duan, L. Jones, H. Sheardown; Activation of Human Corneal Epithelial Cells on Collagen Gels. Invest. Ophthalmol. Vis. Sci. 2007;48(13):1891. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
When testing biocompatibility of artificial keratoprostheses, most studies focus on in vitro tests for cell adhesion and proliferation. Hence, there is a very limited understanding of how corneal cells interact with biomaterials, which significantly hinders progress in the development of better keratoprostheses. This study is the first step towards characterizing in vitro corneal cell interactions with various crosslinked collagen gel surfaces. Activation of epithelial corneal cells was characterized by the expression of ß1 integrin, ICAM-1 and Fas.
Four different types of collagen crosslinking were tested: thermally crosslinked, glutaraldehyde crosslinked (GTA), EDC and dendrimer crosslinked. Control surfaces included tissue culture-treated polystyrene (TCPS) and Biocoat (Collagen I treated TCPS). Immortalized human corneal epithelial cells (HCECs) in keratinocyte serum-free medium were seeded on the surfaces for up to 48 hours. At 4, 24 and 48 hours, cells were removed from the surfaces by trypsinization and incubated with fluorescently labelled antibodies against CD29 (ß1 integrin), CD54 (ICAM-1) and CD95 (Fas). They were then fixed and analysed by flow cytometry within 5 days.
No difference in corneal cell activation was observed after 4 hours. At 24 hours, HCECs interacting with the crosslinked collagen gels had reduced levels of ß1 integrin expression when compared to Biocoat or TCPS. After 48 hours, further reduction in ß1 integrin levels were observed with GTA and EDC crosslinked collagen gels. After 24-hours, a small percentage of corneal cells on collagen gels also started to express ICAM-1. With the GTA crosslinking, the number of HCECs expressing ICAM-1 continued to increase at 48 hours. The levels of Fas on cells remained at background levels at all times with all the surfaces tested.
This study demonstrates that adherent HCECs express different levels of activation depending on the collagen crosslinking procedure. Despite good cellular adhesion and proliferation, the reduced levels of ß1 integrin on the dendrimer surfaces suggest that it may not be as biocompatible as expected. Further studies will include longer contact times, the introduction of laminin on the collagen surfaces and other markers of cellular activation.
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