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G Geerling, P Herminghaus, G Mueller, D Hartwig; Epitheliotrophic Capacity of Blood Products for Corneal Epithelium - Influence of Centrifugation Force and Clotting . Invest. Ophthalmol. Vis. Sci. 2002;43(13):53.
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Purpose: Eyedrops prepared from autologous serum are used to treat ocular surface disorders such as epithelial defects and dry eyes. It is supposed that growth factors, fibronectin and vitamins in serum support the viability of epithelial cells. Thrombocytes are a major source of growth factors in blood. The clotting process as well as the level of G-force used to prepare the blood product may therefore influence the level of its epitheliotrophic factors and growth supporting capacity. Methods: Following local ethics committee approval 400 ml of blood were taken from 10 healthy donors. From each sample serum and plasma centrifuged at 500 G and 3000 G were prepared. In these blood products the concentrations of EGF, PDGF, TGF-b, and Fibronectin were determined by means of enzyme linked immunosorbent assays and vitamin A was quantified with high pressure liquid chromatography. SV-40 immortalised human corneal keratinocytes were grown on a 96 well plate at 37°C, 5% CO2 using a fully defined culture medium. At 30% confluence the culture medium was exchanged for a blood product or a negative (PBS) or positive (culture medium) control. Intracellular ATP as a measure for growth was quantified with a luminescence based luciferin-luciferase based assay in dose and time-response experiments. Results: Mean and standard error for the proteins analysed are given in the table below (* = Significant difference between high and low speed sample. # = Significant difference between serum and plasma sample). Serum rather than plasma and low rather than high G-force were superior in maintaining levels of cellular ATP in culture. At lower concentrations of plasma and serum no significant difference was observed. Conclusion: The growth supporting potential of blood products depends upon the release of thrombocytic growth factors. The clotting process as well as the subsequent centrifugation influence the epitheliotrophic character of blood products and have to be standardised for future clinical trials. View OriginalDownload SlideView OriginalDownload Slide
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