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The Epitheliotrophic Capacity of Serum or Plasma Eyedrops Depends on the Preparation
1Herminghaus P., 2Hartwig D., 1Harloff S., 1Dibbelt L., 3Wedel T., 1Geerling G.,
1Medizinische Universität zu Lübeck, Klinik für Augenheilkunde (Lübeck)
2Medizinische Universität zu Lübeck, Institut für Immunologie und Transfusionsmedizin (Lübeck)
3Medizinische Universität zu Lübeck, Institut für Anatomie (Lübeck)
Purpose: Eyedrops made from autologous serum are a new therapeutic modality for the treatment of ocular surface disorders, such as persistend epithelial defects and dry eyes. Growth factors, fibronectin and vitamins are supposed to be the factors responsible for the epitheliotrophic character of serum. Thrombocytes are a major source of growth factors in blood. The clotting process as well as the G-force used to prepare the eyedrops may influence the concentration of these factors and thus epitheliotrophic capacity of autologous blood products.
Method: Full blood was collected from 20 healthy subjectsas and serum and plasma centrifuged at 500 G and 3000 G each. The concentration of EGF, PDGF, TGF-b and fibronectin were quantifiied by means of routine ELISA-techniques. Vitamin A was measured with HPLC. SV-40 immortalised human corneal keratinocytes were cultured in 96-well plates at 37°C, 5% CO2 with a fully defined culture medium. At 30% confluence the culture medium was withdrawn and the cells incubated for 24 hours with one of the blood products prepared. Proliferation was quantified by means of a luminescence-based ATP-assay in dose- and time-response experiment. Migration of the cell was examined with a colony-dispersion-assay and differentiation was visualised by means of scanning electron microscopy.
Results: Levels of TGF-b and PDGF were significantly higher in blood products prepared with a high G-force. Serum contained significantly more EGF, PDGF and vitamin A as plasma. Proliferation and migration of human corneal epithelial cells were best supported with serum prepared at a high G-force. Differentiation revealed not significant differences. The epitheliotrophic capacity of plasma was allways inferior than the equivalent serum product.
Conclusions: The epitheliotrophic capacity of blood product depends upon thrombocytic growth factors. The clotting process and the subsequent centrifugation determine the epitheliotrophic characteristics of plasma and serum. Plasma is less appropriate to treat ocular surface disorders than serum. The preparation of serum eyedrops has to be fully defined prior to controlled clinical studies.