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To prevent infections local delivery of antibiotics is a useful tool. Especially in bone fractures, vancomycin impregnated bone cements are often used allowing high concentrations of antibiotics at the infection side without high serum concentrations. However, besides potential pathogens, cells involved in tissue regeneration may also be affected by the drug. We investigated the effect of vancomycin on the viability and functionality on osteoblasts, endothelial cells, fibroblasts and skeletal muscle cells. Our results show that the viability of all cells analyzed was reduced by vancomycin and that the observed effects were time and concentration dependent. The most pronounced toxic effect was detected on day three when even the lowest concentration of 0.01 mg/ml led to a significant decrease in proliferation compared to control. Functionality assays of osteoblasts and skeletal muscle cells revealed a sensitive reaction of the cells to the drug, indicating that vancomycin is toxic to these cells during the process of differentiation. These data suggest that the vancomycin administration is critical for cell survival and function. Therefore, the concentration of administered antibiotics needs to be carefully evaluated to find a balance between defense against pathogens and functionality of host cells and tissues.