Effect of Dental Polymer Degree of Conversion on Oral Biofilms
Alison Kraigsley, Sheng Lin-Gibson, Nancy J. Lin
National Institute of Standards and Technology, Gaithersburg, MD
Polymeric composites are the primary materials used for dental restorations (“fillings”), replacing traditional amalgam due to their esthetic qualities and lack of mercury. However, failure at the tooth-composite interface typically occurs within 3-10 yrs, often due to secondary caries (recurrent tooth decay). Streptococcus mutans (S. mutans) attaches to surfaces in communities of cells (“biofilms”) and is known to have a significant role in the development of caries. However, quantification of the highly aggregative S. mutans biofilms is inherently challenging and currently lacks standard methods for evaluating biofilm-material interactions, such as the effects of composite properties on biofilm growth. Polymeric degree of conversion (DC), a critical property of dental composites, varies clinically and affects many material properties, including surface chemistry and soluble leachables. The objective of this research was to develop a method to quantify S. mutans biofilm growth under any growth condition and to apply that method to determine the effect of DC on S. mutans biofilms and the individual contributions of surface methacrylates and leachables.
The methylthiazolyldiphenyl-tetrazolium bromide (MTT) colorimetric dye was first validated for the determination of S. mutans biofilm metabolic activity. Using mature (24 h) biofilms grown under pathogenic conditions, the biofilm suspension was then diluted prior to treatment with MTT or treated in bulk then diluted. Regardless of whether the number of cells or MTT signal was diluted, the results produced a linear response indicating that the MTT assay is a robust technique for quantification of aggregative S. mutans biofilms.
Polymer disks of 50:50 (by mass) bisphenol A glycerolate dimethacrylate (bisGMA) and triethylene glycol dimethacrylate (TEGDMA) were photopolymerized between untreated glass slides for 7 s to 60 s per side, resulting in a DC range of 50 % to 76 % as quantified using near infrared spectroscopy. S. mutans biofilms were grown on polymer disks for 4 h and 24 h in medium containing 1 % sucrose by mass. Crystal violet (CV) was used to measure overall biomass, and MTT was used to quantify metabolic activity. The contributions of pendant methacrylate groups on the material surface and leachables released from the disks were decoupled by evaluating biofilms on surface-functionalized coverslips and in the presence of leachables in the absence of disks respectively.
Results from biofilms cultured on polymer disks with varying DC values demonstrated that metabolic activity decreased as DC decreased. Biomass, however, did not change as a function of DC, indicating a true decrease in metabolic activity (not a reduction in overall biofilm biomass). Surface chemistry had no significant effect on biofilm metabolic activity or biomass, whereas leachables from low DC polymers had a negative effect on metabolic activity similar to that seen in biofilms cultured directly on low DC polymers. In conclusion, the MTT assay was validated for use with S. mutans biofilms and used to determine that low DC decreases S. mutans biofilm metabolic activity, likely due to an increased amount of leachables. Additional studies are needed to further identify individual leachable components responsible for this effect.
Work was supported by NIDCR/NIST Interagency Agreement Y1-DE-7005-01.