Development of 3-Dimensional skin infection models for the evaluation of antimicrobial strategies

Wound infection is major health problem, however, there is an urgent need for new anti-microbial strategies to evade the development of antibiotic resistance and local toxicity to skin cells, which hinders wound healing. This is currently hampered by the lack of in vitro models of skin infection; this thesis describes the development and validation of a 3D infected skin model for testing new anti-microbial strategies. The effectiveness of two potential anti-microbial strategies for infected wounds, together with effects they exert on mammalian skin cells was determined. Biocides known to have potential anti-microbial activity via free radical formation were investigated alone or in combination with Low Frequency Ultrasound (LFU). To study the biocidal effects, nitrocellulose membranes were used initially to form simple biofilms. A skin model was developed in vitro using DED scaffolds, HaCaT cells and commercially available primary fibroblasts. The 3D skin models were burnt, and infected with S. aureus or P. aeruginosa. Bacterial penetration and migration were determined and effects of biocides investigated. Effects on mammalian cells were determined by measuring skin cell death zones, and immunohistochemistry used to determine phenotype. Biocides reduced bacterial count, with greater effects on planktonic bacteria than on biofilms. Infection caused toxicity to the skin cells in the 3D model, which was greater for P. aeruginosa infection than S. aureus. Manuka honey was the most effective agent against bacterial infection, while associated with the least toxicity to skin cells. LFU decreased planktonic bacterial loads. An additive effect was observed when LFU was combined with biocides, enabling lower concentrations of biocides to be used, if combined with US, with less toxic effects on mammalian cells. Together the data presented in this thesis validates a novel 3D skin model which can be utilised to test new anti-microbial strategies in a robust manner in vitro.