Development and Characterization of a Collagen-Based Three-Dimensional In Vitro Model to Mimic Biofilm Formation in a Wound Bed.

Abstract

Current studies using in vitro biofilm culturing systems have been instrumental at elucidating wound biofilm formation but fail to account for the diverse environment that bacteria are exposed to within the host. In the current study, we recapitulated this wound biofilm microenvironment by creating a hydrogel composed of collagen, thrombin, fibrinogen, meat broth, and FBS and subsequently infected the scaffolds with UAMS-1. We characterized the material properties of the hydrogel (noninfected) and found no significant differences in the storage modulus when fibrin was added to the collagen hydrogel. When infected with UAMS-1, temporal growth and polysaccharide formation were observed through plating, SEM, and histological staining, indicative of biofilm formation. PCR analysis revealed heightened expression of adhesion-associated genes with no increase in expression of metabolic genes, indicating significant increase in the formation of a robust biofilm over time. Vancomycin was ineffective in eradicating the already-developed biofilm, whereas the total CFUs in rifampin-treated models decreased significantly compared to those in the untreated group. Although it was not significant, an increase in SCVs was observed in the rifampin-treated group, suggesting that rifampin may create a harsher environment against the Staphylococcus aureus, allowing the increase in more resistant bacteria. The persistence of an infection in our rifampin-treated 3D in vitro wound model indicates an increased similarity to the host environment compared to that of a static biofilm model.