Inhibition of the Biofilm Formation of Anaerobic Bacteria Involved in Secondary Caries by Dental Adhesive

Abstract

Secondary caries is a type of carious lesion found at the margins of or next to an existing restoration after the filling has been used for a period of time. It generally arises from the formation of defects or cracks in the filling material after restoration. This can create gaps between the material and the tooth tissue, which will allow bacteria in the biofilm to enter the interface. Dental adhesives are commonly used to provide retention for composite cement or filling materials. A good adhesive should be able to prevent leakage along the restoration margin as well as resist the mechanical load of chewing pressure. Recently, the inclusion of calcium in the adhesive monomer has been produced as Bio-Coat Ca, and its antimicrobial property against some oral bacteria has been studied. No information was found on anaerobes. The aim of this study was to evaluate the antimicrobial potential of dental adhesive on the biofilm formation of anaerobic bacteria involved in secondary caries. An adhesive containing CMET (calcium salt of 4-methacryloxyethyl trimellitic acid) and 10-methacryloyloxydecyl dihydrogen calcium phosphate (MDCP) (Bio-Coat Ca, Sun Medical, Moriyama, Shiga, Japan) was applied to the flat-bottom surface of the saliva-coated 96-well plate. Then it was polymerized with LED light at 460 nm and sterile with UV light. Porphyromonas gingivalis ATCC 33277, Prevotella intermedia ATCC 25611, and Fusobacterium nucleatum ATCC 25586 were prepared as a suspension of approximately 1 × 108 CFU/mL and added to the well. The plate was left for 120 min at 37°C in a shaking incubator (120 r/min) to allow bacterial adhesion. After removing non-adherent cells, Schaedler broth was added and further incubated for 48-72 h to grow the biofilm. The culture medium was changed every 24 h. A biofilm formed on a 96-well plate surface without the adhesive was set up as a control. The amount of vital biofilm was assessed by the WST Microbial Cell Counting Kit (Dojindo Molecular Technologies, USA). All tests were triplicated performed and repeated three times. As a statistical analysis, the Mann-Whitney U test was applied. The results showed that dental adhesive exhibited significant anti-biofilm formation of P. gingivalis and F. nucleatum at a percent inhibition of 56% and 46%, respectively. On the other hand, no significant effect was found on P. intermedia. This was similar to our previous report on bacteria associated with primary caries, which revealed that the anti-biofilm effect of Bio-Coat Ca adhesive on Streptococcus mutans was 65% while no significant suppressive action was observed Lactobacillus casei and Actinomyces viscosus. The inhibitory effect of the adhesive was proposed to be the acidic characteristic of the monomers. This newly developed adhesive could be a promising material for the prevention of secondary caries. However, this study was done on the single-species biofilm formation in vitro and conducted in a short time. Long-term clinical studies are needed to evaluate the effect on the patients.