Untersuchungen zur Zytoprotektion bei der Anwendung von Wundantiseptika mittels Lumineszenzmessung und Durchflusszytometrie

Antiseptic agents are often used in wound care. Tissue compatibility is in addition to an antimicrobial effect an important qualification for the choice of an optimal active component. There are several in vitro cytotoxicity tests for many antiseptic agents. For the registration of a pharmaceutical product you have to make animal experiments to test in vivo cytotoxicity. Between in vivo and in vitro tests there are substantial differences. Normally antiseptic agents are in vitro much better compatible for tissue. In this dissertation a cell culture model was used to give some explanation for this differences. The established and common antiseptic agents octenidine dihydrochloride 0,1%, chlorhexidine digluconate 1,0% and polyhexanide-solution concentrated 0,1% and 0,2% were tested. All this concentrations are standard concentrations. To simulate a wound immortalised ceratinocytes (HaCaT-cells) were used. Albumin 5%, human serum and fetal calf serum (FCS) were also used, because of the expectated cytoprotective effect. With the methods of an ATP-assay by luminescence measurement und fluorescence activated cell sorting (FACS) the cytotoxicity of the substances in presence of a cytoprotective addition was tested. It was also tested if there is still an antimicrobial effect. Boths methods of cytotoxicity measurement showed in presence of a cytoprotective addition differences between the different antiseptic agents. With ATP-measurement there was a bigger contingent of surviving cells when using octenidine dihydrochloride 0,1% than with chlorhexidine digluconate 1,0%. This happened at several measuring times and with several cytoprotective additions. Polyhexanide 0,1% and 0,2% were better compatible for the cells than the other substances using human serum as cytoprotective addition. Comparing the cytoprotective additions FCS had the best cytoprotective effect followed by human serum and albumin 5%. With FACS-measurement polyhexanide 0,1% and 0,2% and octenidine dihydrochloride 0,1% showed very good cell compatibility using all cytoprotective additions. With the application of chlorhexidine digluconate 1,0% there was only a small cytoprotctive effect. The comparison of the cytoprotective additions showed the same cytoprotective order as the ATP-method. Because of the different number of the used cells (1.000 at ATP-measurement vs. 500.000 at FACS) it is only possible to compare the results qualitativly. A sufficient antimicrobial effect was showed for all tested antiseptic agents with all cytoprotective agents. The cytoprotective effect correlated with the protein concentration of the addition. Fetal calf serum (FCS) with the chief ingredient BSA (bovine serum albumin) showed the best cytoprotective effect by human serum and albumin 5%. It was possible to give an explanation for the differences between the antiseptic agents by using images of albumin 5% with antiseptic agents in an electron microscope. With polyhexanide and octenidine dihydrochloride big aggregates of protein and antiseptic agent could be seen. Polyhexanide showed complexes of this protein/antiseptic agent aggregates. Chlorhexidine digluconate showed only very small aggregates. It is possible to hypothesise that the cytoprotective effect of albumin 5% under application of antiseptic agents depends on size, number and grade of aggregation and complexation of protein/antiseptic agent aggregates.