Growth Factor and Interleukin Concentrations in Amniotic Membrane-Conditioned Medium

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Abstract
Purpose: Application of amniotic membrane-conditioned medium (AMM) eye drops is a potential treatment alternative for therapy-resistant corneal epithelial defects. Our purpose was to determine the concentration of growth factors epidermal growth factor (EGF), nerve growth factor (NGF), vascular endothelial growth factor (VEGF), transforming growth factor β1 (TGFβ1), fibroblast growth factor basic (FGFb), and interleukins, IL-1β, IL-6, IL-8, in AMM following different preparation methods. Methods: Amniotic membranes of 10 placentas were prepared and thereafter stored at −80°C using the standard method of our LIONS Cornea Bank. Following defreezing, amniotic membrane pieces with a standard size were inserted in a 12-well plate either complete or cut in small pieces, and 2000 µl DMEM culture medium was added. EGF, NGF, VEGF, TGFβ1, FGFb, IL-1β, IL-6, and IL-8 concentrations in the culture medium were determined following 8, 48, and 96 hours, and 1, 2, 3, and 4 weeks of incubation using enzyme-linked immunosorbent assay (ELISA). Results: Concentrations of NGF, VEGF, TGFβ1, and IL-1ß were beyond the detection limit at all time points. EGF concentrations were between 0.14 and 0.80 ng/g tissue, FGFb between 0.48 and 2.89 ng/g tissue, IL-6 between 0.11 and 1.41 ng/g tissue, and IL-8 between 0.32 and 6.18 ng/g tissue. A significant difference between both preparation methods was shown for the IL-6 concentration after 8 and 48 hours (p < 0.001; p = 0.01) and in IL-8 concentration after 8 and 96 hours and after 3 weeks (p = 0.02; p = 0.002; p = 0.027). Conclusion: AMM containing EGF and FGFb, and IL-6 and IL-8 AMM is a potential nonsurgical treatment alternative of therapy-resistant corneal epithelial defects. However, the most effective preparation method and the optimal harvesting time point are yet to be determined.

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