Use of Tissue-Engineered Skin to Study In Vitro Biofilm Development


Address correspondence and reprint requests to: Robert S. Kirsner, MD, PhD, Department of Dermatology and Cutaneous Surgery, University of Miami School of Medicine, PO Box 016250 (R250), Miami, Florida 33136, or e-mail:


BACKGROUND Biofilms are aggregations of microorganisms that have been identified as potential pathogens in the chronicity of nonhealing wounds.

OBJECTIVE To develop an in vitro wound model to study biofilms using Graftskin, a tissue-engineered skin equivalent.

MATERIALS AND METHODS Graftskin constructs were divided into sections, and wounds were created on each section. Bacterial suspensions with a concentration of 106 CFU/mL were prepared from cultures of pathogenic isolates of Pseudomonas aeruginosa and Staphylococcus aureus. A 25-μL aliquot of each suspension was deposited in the center of wounds created on the Graftskin. Sections were incubated at various time points, and a biopsy was then taken from the wounded and inoculated area. Sections were visualized with light (hematoxylin and eosin) and epifluorescent microscopy (calcofluor white and ethidium bromide).

RESULTS Biofilm was observed on the wound model. Biofilm formation was dependent on time of Graftskin exposure to the bacteria. Biofilm was visualized in the S. aureus group at an earlier time point than in the P. aeruginosa group.

CONCLUSIONS We demonstrated biofilm formation in vitro using a wound model. This model may provide a basis on which future studies may explore therapeutic modalities to prevent and eradicate pathogenic bacterial biofilm.