Denture Cleanser Effects on In Vitro Denture Plaque Biofilms

Objectives: Regular cleaning of dentures is essential to good oral hygiene. Dentures harbour complex biofilms, which are more difficult to kill than equivalent planktonic bacteria. There is a need for realistic models of denture plaque biofilms to test product effects. This study aimed to develop reproducible, complex biofilms on PMMA denture acrylic, and to evaluate denture cleanser effects by a range of methods.
Methods: PMMA discs were sterilised using 70% ethanol. Denture plaque from several individuals without disease, was frozen and three such samples diluted 1:5 in artificial saliva (AS) and inoculated onto sterile PMMA acrylic discs in 24 well plate. Plates were placed in an anaerobic chamber at 37°C with shaking for 96 h, with AS changed after 48 h. Discs were gently washed with 1 mL PBS to remove non-adherent cells. Polident tablet was added to 150 mL of 40°C hard water and this solution used to treat biofilms. At 3 and 5min, discs were removed into Dey-Engley neutralising broth for 15 min. Biofilm was measured by Alamar Blue reduction, conventional viable counting, qPCR and live-dead fluorescence microscopy (Syto9 and Propidium Iodide) using standard protocols.
Results: Treatment of undefined biofilms grown on PMMA discs with Polident significantly (P<0.0001) reduced Alamar Blue activity to 14 and 13% following a 3 min and 5 min exposure, respectively, though no there was no significant difference between 3 and 5 min treatments. Total viable aerobes and anaerobes were both reduced by more than 5 logs (P>0.0005) at both treatment times. qPCR showed significantly reduced numbers at 3 and 5mins. Auto-fluorescence prevented clear conclusions in respect of live-dead staining.
Conclusions: Denture cleansers were effective in killing and removing in vitro-grown complex denture plaque biofilms, although different methods yielded different numerical results. Further work is needed to overcome limitations of standard live-dead visualisation methods.