Extended PGRM Method to Evaluate Toothpaste Effects in vivo

Objectives: this exploratory study extends the PGRM to examine effects on Volatile Sulfur Compound (VSC) production, and protease activity, following treatment with Aquafresh Fresh & Minty (AFM) or Aquafresh Ultimate, (AU, containing a novel zinc chloride/o-cymen-5-ol antimicrobial system) toothpaste slurries.   

Methods: 6 subjects were recruited/consented in accordance with an ethically approved protocol.  Subjects avoided oral hygiene for 10 hours prior to participation.  Plaque samples were taken prior to treatment from one quadrant of the mouth using a sterile cotton wool swab.  Subjects then rinsed with one treatment (toothpaste slurries 1.6g toothpaste/4.8ml water) for 1 timed minute.  Further plaque samples were taken from alternate quadrants at 15, 30 and 60 minutes post treatment.  All samples were standardised to OD_600nm of 0.2.  Plaque regrowth was measured by turbidity and glycolysis by pH after 4h incubation.  Further aliquots of plaque samples were dispensed into vials and supplemented with cysteine/methionine, and VSCs measured at 2hrs by headspace GC.  Further aliquots in microtitre plates were supplemented with BAPNA, and protease activity assayed by ΔOD_405nm. 

Results: Plaque glycolysis was reduced to a greater extent by AU treatment (1.02 pH unit compared to 1.32 pH unit for AFM at 30 minutes post treatment).  Regrowth was reduced by 6.5-fold at 1hr for AU, compared to 4.6-fold for AFM.  Protease activity was reduced to a greater extent by AU than by AFM (62% of baseline for AU vs. 25% of baseline for AFM at 30 mins).  VSC production was reduced by both toothpaste treatments, but experimental procedures still need to be optimised for this assay.

Conclusions: the extended PGRM model shows promise in evaluating the effects of actives on relevant, disease-associated microbial activities in vivo after oral care product treatments.