Development of In-vitro Skin Models for Thermal Injury; to Investigate the Impact of Commensal Staphylococcus species on Wound Healing

Skin is the largest organ of the body protecting our internal organs from physical injury, microbial invasion and infection. Healthy skin homeostasis relies on the interaction between the skin’s physical properties, its immunity and microflora. When the skin is damaged it cannot maintain homeostasis or protect the body from microbial invasion and has a dynamic process to close the wound as quickly as possible. Thermal injury is a serious type of trauma, where significant burn wounds need specific care to reduce morbidity and mortality. Across New Zealand and Australia a total 2656 patients were hospitalised with burn injuries between 2012 and 2013, of these patients 24 died from burn wound related complications. The role of commensal bacteria on wound healing is not well understood. Commensal bacteria, in particular coagulase negative staphylococci (CoNS), on the skin and associated appendages colonise the wounds surface within the first 48 hours after wounding. They are generally dismissed when identified in infections, as known pathogens such as Staphylococcus aureus (S. aureus), Pseudomonas aeruginosa and Escherichia coli would also be identified. However CoNS can are known to cause nosocomial infections such as foreign body blood stream infections. It is clinically important to be able to distinguish colonizing and infecting bacteria in a wound so appropriate treatment can be carried out. This study establishes in vitro skin thermal injury models, 2 and 3 dimensional (2D and 3D) to investigate the effects on CoNS on wound healing. The attachment and protein expression profiles (e.g. matrix metalloproteinases, MMPs) of S. lugdunensis, S. haemolyticus, S. hominis and S. capitis on epithelial cells were compared to the most abundant skin commensal S. epidermidis and commonly isolated pathogen S. aureus. 3D skin rafts grown on Transwell inserts better represented the structure and the environment of human skin compared to the 2D monolayer. A burn technique was established using 3 mm diameter copper rod at 80°C that created reproducible burns in both models, which could effectively heal in a manner reminiscent of re-epithelialization. The 3D model demonstrated a repair process similar to in vivo skin, including an increase in MMP-2 and MMP-9 in response to injury. The addition of each CoNS, apart from S. epidermidis, to the wound significantly prevented wound closure (P< 0.05) in a low nutrient environment when bacterial numbers exceeded 105 CFU/mL, but not to the same extent in a high nutrient environment. Although the CoNS were less pathogenic in this model than S. aureus, S. lugdunensis did show similar attachment traits to S. aureus with heavy colonisation at the wounds edge. All the CoNS had increased biofilm forming potential compared to S. epidermidis. Interestingly all bacteria investigated reduced MMP-2 and MMP-9 expression by the epithelial cells across both models. This is the first report of decreased MMP activity in response to bacteria in wounds. The findings in this thesis suggest that CoNS have the potential to impair wound healing