Growth Factor and Interleukin Concentrations in Amniotic Membrane-Conditioned Medium

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 1 (TGF1), fibroblast growth factor basic (FGFb), and interleukins, IL-1, IL-6, IL-8, in AMM following different preparation methods. Methods: Amniotic membranes of 10 placentas were prepared and thereafter stored at -80 degrees 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 mu l DMEM culture medium was added. EGF, NGF, VEGF, TGF1, FGFb, IL-1, 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, TGF1, and IL-1 ss 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.