Browsing by Author "Secor, Patrick Robert"
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Item Impact of Staphylococcus aureus biofilm conditioned medium on inflammation and epithelialization in human keratinocytes(Montana State University - Bozeman, College of Letters & Science, 2011) Secor, Patrick Robert; Chairperson, Graduate Committee: Thomas Hughes.Chronic wounds are characterized by prolonged inflammation and failure to epithelialize and do not respond well to conventional treatment. Bacterial biofilms are a major impediment to wound healing. The effective treatment of chronic wounds requires a better understanding of the relationship between bacterial biofilms and human skin. Human keratinocytes are the most abundant cell type in the epidermis and play essential roles in the inflammatory and epithelialization phases of wound repair. The aim of this dissertation was to determine the effect of biofilm-conditioned medium (BCM) and planktonic-conditioned medium (PCM) on inflammatory and epithelialization processes in keratinocytes. The phylogeny of chronic wounds was characterized to select a model pathogen. Staphylococcus aureus was found to be prevalent in chronic wounds. A novel in vitro model was developed to facilitate host-pathogen investigations between S. aureus biofilms and human keratinocytes. S. aureus BCM contained fermentation products and metabolites that regulate virulence. After four hours of exposure to BCM, pro-inflammatory genes were upregulated in keratinocytes relative to PCM. ELISA analysis of cytokine production in BCM-treated keratinocytes confirmed that after four hours of exposure, cytokine levels were higher relative to PCM-treated keratinocytes. However after 24 hours of exposure, BCM stalled the production of cytokines, suppressed activation of the mitogen activated protein kinases JNK and p38, and induced the release of intracellular calcium in keratinocytes. Processes relating to epithelialization such as the disruption of cytoskeletal components and induction of apoptosis were induced by BCM in keratinocytes. BCM induced a distinct inflammatory response and inhibited processes related to epithelialization. Collectively, the results provide insight into the formation and persistence of chronic wounds. The use of biofilm-based models of disease such as the in vitro model described herein will aid in the development of new biofilm based treatment strategies, not only for chronic wound infections, but all biofilm-based disease.