Method: Planktonic cells of S. mutans (ATCC 25175) were grown overnight in 5 ml of filtered brain heart infusion (BHI) in 3% sucrose at 37°C under 5% CO2. For biofilm generation, 100 μl of S. mutans culture was seeded in 6-well polystyrene plates and cultured with 6 ml of BHI for 48 h. Biofilm was then treated for 24 h with BHI supplemented with 6 ml of a BAG extract derived from 1 g of BAG in 20 ml DI-H2O agitated for 24 h. The BAG powder (85mol% SiO2, 11mol% CaO, 4%P2O5) was sol-gel processed in our lab. Biofilms were harvested and wet weights from control, i.e no BAG, vs. BAG-treated wells were within 1%. The RNA was extracted using sonication, enzymatic digestion, and mechanical disruption and transcribed to cDNA (Qiagen Sciences, MD, USA). Gene expression of the virulence genes, gtfB and gtfC, was quantified by PCR and compared to housekeeping gene 16S. Primers were designed against sequences in S. mutans UA159 and then confirmed to amplify genes in 25175.
Result: Densitometry scanning of resulting gels (NIH ImageJ) confirmed each group expressed equal amounts of the housekeeping gene. There was less PCR product of both virulence genes, gtfB/C, from biofilm treated with BAG than untreated controls. Expression of gtfB and gtfC genes decreased 34% and 16%, respectively, in the presence of BAG.
Conclusion: Treatment with dissolution products from BAG may be an effective method of reducing biofilm growth. Future studies will focus on biofilm derived from 25175 grown on dental composite restorations.