Photodynamic inactivation of fibroblasts and inhibition of Staphylococcus epidermidis adhesion and biofilm formation by toluidine blue O

Abstract

Treating skin and soft tissue infections of severe limb traumas can be challenging. Crucial concerns focus on inhibiting biofilm formation by antibiotic-resistant bacteria, and preventing scar formation by fibroblastic hyperproliferation. The local use of toluidine blue O (TBO)-mediated photodynamic therapy (PDT) may be a promising strategy for treating such lesions. The present study used Staphylococcus epidermidis (strain ATCC 35984) to assess the effects of TBO-PDT on bacterial adherence and biofilm formation, using confocal laser scanning microscopy (CLSM), tissue culture plating (TCP) and scanning electron microscopy (SEM). Primary human fibroblast cells were used to evaluate the cytotoxicity of TBO-PDT using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and CLSM. Six different treatment groups were investigated: Medium only [tryptone soy broth (TSB) or Dulbecco's modified Eagle's medium (DMEM)]; red light control (light dose, 30 J/cm2); TBO group (50 mM TBO); TBO-PDT1 (TBO irradiated with 10 J/cm2); TBO-PDT2 (TBO irradiated with 20 J/cm2); and TBO-PDT3 (TBO irradiated with 30 J/cm2). The results of the S. epidermidis adhesion assay indicated that the TSB, light and TBO groups exhibited significant bacterial adherence, compared with the TBO-PDT groups. Analysis of biofilm formation revealed significant light dose-dependent differences between the TBO-PDT groups and the TSB, light, and TBO groups. Furthermore, SEM indicated fewer colony masses in the TBO-PDT groups compared with the control groups. The MTT assay for fibroblastic cell toxicity demonstrated ~1.1, 4.6, 14.5, 29.7 and 43.4% reduction in optical density for the light, TBO, TBO-PDT1, TBO-PDT2 and TBO-PDT3 groups, respectively, compared with the DMEM control group. There was no difference in toxicity between the light and control groups, however, there were significant differences among the TBO-PDT groups. Finally, alterations in fibroblast morphology and cell spreading were revealed by CLSM, following TBO-PDT treatment. TBO-PDT inhibited bacterial adhesion and biofilm formation, and exhibited significant cytotoxic effects on human fibroblasts. These results indicate that the local use of TBO-PDT in limb lesions may be a useful treatment method for inhibiting bacterial biofilm formation and fibroblastic hyperproliferation, which may prevent infectious hypertrophic scar formation.