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Wound healing is compromised by critical colonization and infection with bacteria. Hence, antimicrobial agents are used clinically to decrease the bacterial load and promote wound healing. Polihexanide (PHMB) has been found to be effective against a broad spectrum of micro-organisms and is increasingly utilized in rinsing solutions or in combination with wound dressings because of its good biocompatibility. In the present study, a co-culture of human keratinocytes and Staphylococcus aureus was established to serve as an in vitro model for infected wounds. Incubation of keratinocytes with increasing concentrations of S. aureus led to a dose-dependent decline of cell viability and proliferation. Lactate dehydrogenase release and interleukin-8 liberation were found to be elevated under these conditions. Polihexanide dose-dependently was able to protect keratinocytes from bacterial damage and re-establish normal human cell proliferation in vitro. Furthermore, a dressing consisting of biocellulose derived from Acetobacter xylinum with the addition of polihexanide was adept to safeguard keratinocytes against S. aureus. In conclusion, the co-culture system presented embodies a valuable tool as a model system for infected cells in a non-healing wound. Furthermore, the results obtained support the favorable function of polihexanide in the treatment of infected chronic wounds.