An Innovative Oral Ex Vivo Biofilm Model for Antimicrobial Investigations

The methodical work describes all the necessary steps for establishing a stable oral ex vivo biofilm using saliva and crevicular plaque samples from periodontal healthy donors. First, cover slips were preconditioned with saliva supernatants and subsequently inoculated with crevicular plaque suspensions. Ex vivo biofilm formation was characterized by confocal laser scanning microscopy (cLSM) after 1, 4, 24, 48 and 72 h of anaerobic cultivation. Exemplarily, the inhibitory characteristics of blackcurrant fruit extracts [all-fruit juice (AFJ); alcoholic fraction from berry skins (AFBS)] were observed on 1, 4 and 24 h-aged ex vivo biofilms. Chlorhexidine (CHX, 0.2%) served as positive control. After direct contact (3 min), biofilms were dispersed, plated onto agar and anaerobically cultivated for 24 h. Early ex vivo biofilms (1 h-biofilm) showed scattered microbial colonies. After 4 h of cultivation, a multilayered biofilm was formed. Biofilm mass gradually increased, displaying a complex polymicrobial structure after 24 h. At 72 h, the biofilms had a dense three-dimensional appearance. Treatment with AFJ and CHX was more efficient in inhibiting biofilm growth compared to AFBS. Early biofilms (1 h, 4 h) were more susceptible to AFJ and CHX compared to 24 h-biofilms. The introduced model can be recommended for testing the efficiency of plaque-controlling agents.