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Shin Ae Park, Vijaykrishna Raghuanthan, Sara Thomasy, Christopher Murphy; Covalently immobilized epidermal growth factor accelerates proliferation of human corneal epithelial cells. Invest. Ophthalmol. Vis. Sci. 2013;54(15):3895.
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Corneal wound healing involves the coordination of a number of complex processes including cell migration, cell proliferation, re-stratification, as well as matrix deposition and tissue remodeling. Cell migration and proliferation are greatly influenced by the presence of growth factors and delivery of cytoactive factors in a corneal wound remains a difficult challenge. Here, we investigated an in vitro model for the covalent immobilization of epidermal growth factor (EGF) to enhance corneal epithelial cell proliferation.
Unmodified glass coverslips or those modified with thio-silane or propyl silane were used as stimulants of a corneal wound bed. Disulfide bonds were reduced to expose sulfhydryl (-SH) groups using 10μM tris(2-carboxyethyl)phosphine (TCEP). Human recombinant EGF, modified with a heterobifunctional crosslinker (sulfo-SMCC) was covalently linked to the -SH groups of the silane. The cytoactivity of the covalently limmobilized EGF was investigated by measuring proliferation of human immortalized corneal epithelial cells (hTCEpi) using the MTT assay over days. Cytotoxicity of the reducing agent, TCEP, was also determined by MTT assay over a differential dose range (1 nM to 1 mM).
Proliferation of hTCEpi cells was significantly greater (>2 fold) when EGF was covalently linked to the surface in comparison with the non treated control groups (p < 0.05). TCEP was determined to be non-toxic for doses <100 μM and cell viability was comparable with control (p > 0.05).
Our data strongly demonstrate that TCEP can potentially be used as a safe reducing agent for covalent immobilization of cytoactive factors. Importantly, we successfully demonstrate that covalent immobilization of EGF (30 ng) potently enhances proliferation of hTCEpi cells in vitro. Covalent integration of EGF into corneal wounds, is a unique strategy to promote re-epithelialization using significantly less cyctoactive factor and dose frequency than topical application.
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