056 In Vitro Evaluation of Biofilm Development on Tissue Engineered Skin

Abstract

Bacteria reside in various forms, including planktonic, or free‐floating, or as biofilms, which are a tightly adherent, potentially more treatment‐resistant form. Biofilms, composed in part as polysaccharide matrices, can act as protection for bacteria by shielding them from antimicrobials and the innate immune system. It is estimated that bacterial biofilm are responsible for 65–80% of all chronic infections and may be responsible in part for non‐healing of chronic wounds.An advance in the treatment of non‐healing wounds has lead to the development of human skin equivalents (HSE), which can successfully treat many non‐healing wounds. Better understanding the interaction between bacteria and HSE might improve patient outcomes. Studies involving planktonic E. coli and HSE have demonstrated the growth of bacterial colonies only on the dermal surface. Growth was not seen on the epidermal layer of the HSE, possibly due in part to the expression of innate antimicrobial peptides called human β defensin‐2 (hBD‐2), which are expressed by HSE keratinocytes. The objective of this study was to determine if biofilm formation could occur on a bioengineered HSE.We used a commercially available HSE. Three (3) mm full‐thickness incisions were made in a triangular section of HSE. Each section was inoculated on the epidermal aspect with 1.0 × 105CFU per gram of Pseudomonas aeruginosa at 35 degrees Celsius for specified time points. Sections of HSE were sampled in areas of injury at various time points. Biopsy sections were processed for histologic analysis with H&E and epifluorescent microscopy to visualize Pseudomonas aeruginosa. We found that biofilm formation occurred at multiple time points on HSE. Colonies of adherent bacteria were visualized within the injured area of the epidermal layer of HSE by H & E staining. Eplifluoresecnt microscopy using calcofluor white staining revealed the characteristic exopolysaccharide (EPS) matrix of biofilm.Visualization of the expression of human β defensin‐2 (hBD‐2) with HSE after biofilm formation is underway. This knowledge will help to understand the role of bacterial biofilms within HSE and their effect on subsequent healing.