Fundamental Research On A Pit And Fissure Sealant Incorporating A Quaternary Ammonium Monomer

Pit and fissure sealing is considered to be one of the most effective method of protecting teeth from dental caries. The composite resin and glass ionomer are main materials of sealant. Because the retention of the glass ionomer sealant as compared with the composite resin is poor, the composite resin is the most commonly used material in this procedure, due to its reasonable flowability, good wettability and improved physical properties. However, polymerization shrinkage of composite resin-based materials might facilitate the formation of gaps between the filling and cavity wall, providing space for bacterial invasion and proliferation, thereby leading to secondary caries and the failure of restorations. Additionally, incipient caries may inadvertently be sealed in with dental sealants and the fate of those bacteria within carious lesions is of significance. Therefore, if sealants could provide additional antibacterial protection, especially against cariogenic bacteria such as Streptococcus mutans (S. mutans), additional protection could be afforded to prevent subsequent deterioration at the sealant-tooth interface and caries initiation.Fluoride is well-known for its anticarious activity by inhibiting the biosynthetic metabolism of bacteria. Fluoride released from dental restorative materials affects caries formation by reducing demineralization, enhancing remineralization, interfering with plaque formation and inhibiting microbial metabolism. The addition of fluoride to pit and fissure sealants has been widely applied in dental materials. However, the dynamic release of fluoride is unstable, exhibiting a higher initial release and subsequent leaching-out at very low levels, thereby making the permanence and validity of fluoride release suspicious. Moreover, there is some debate about whether adding fluoride to a sealant actually prevents caries more effectively than the sealant alone. Aside from fluoride, some other antibacterial components, such as antibiotics and inorganic agents, have also been added into the dental materials. However, the release kinetics of those antibacterial agents is difficult to control strictly. Moreover, the constant release of antibacterial agents may damage the physical and chemical properties of the carrier material.In order to avoid the defects caused by solubility of the agent, the dental material with antibacterial activity could be produced by binding the polymerizable monomers into polymer matrix. Several reports have described the incorporation of a quaternary ammonium monomer, methacryloyloxydodecyl pyridinium bromide (MDPB), in composite resin or an adhesive system as a strategy for obtaining antibacterial properties. Accordingly, methacryloxylethyl cetyl dimethyl ammonium chloride (DMAE-CB), a polymerizable cationic monomer containing a quaternary ammonium for antibacterial effect, was synthesized by our research group, and the DMAE-CB-incorporated adhesive was demonstrated to exert antibacterial activity and an anti-biofilm effect against S. mutans.In the present study, DMAE-CB was incorporated into a commercial resin-based sealant material to obtain an experimental DMAE-CB-incorporated sealant for evaluation of antibacterial activity and various other properties. The hypotheses were that the mobilized DMAE-CB in the resin-based sealant would exhibit a stable and long-lasting antibacterial effect against S. mutans and that the addition of a small amount (1% w/w) of DMAE-CB would not affect the properties of the parent material.1 The main objectives:DMAE-CB was incorporated at 1% (w/w) into a clinically used sealant (Helioseal). Helioseal without DMAE-CB served as a negative control. Helioseal F, containing a fluoride-releasing resin, was used as a positive control. This aim of this study was to evaluate their antibacterial activity and properties including surface contact angle, degree of conversion, hardness and microleakage characteristics.2 The main methods:2.1 The inhibitory effect of the cured sealant on bacterial growth was evaluated by the film contact method, and the influence of aging treatment or saliva treatment of the modified sealant was also evaluated by the same method.2.2 The bacterial growth in the eluent of the modified sealant was investigated with spectrophotometry and growth kinetic analysis.2.3 The effects of the cured DMAE-CB-incorporated sealant on the S. mutans biofilm accumulation of in vitro investigated with spectrophotometry,and the microcosm of S. mutans biofilm were observed with scanning electron microscope spectrophotometry.2.4 The effects of the cured sealants on the membrane integrity of S. mutans were investigated using confocal laser scanning microscopy (CLSM) in conjunction with fluorescent indicators.2.5 The contact angles of the sealants on the enamel surface were measured with a camera based goniometer.2.6 The degree of conversion of the sealants was measured using Fourier transform infrared reflectance spectroscopy.2.7 Vickers microhardness of the cured sealants was measured using a Micro Hardness Tester.2.8 30 extracted sound human molar teeth were randomly divided into three groups. After application of the three groups of materials, the specimens were subjected to thermocycling and then immersed in 1% methylene-blue dye. Following sectioning, specimens were examined under a sterecmicroscope and microleakage scores were assigned.2.9 Fluoride release from Helioseal F and Helioseal F(1% DMAE-CB) was evaluated at time intervals of 4, 8, 12, and 24 hours and 2, 3, 7, 14, 28, 56, and 112 days. Seven disks of each material were made and stored for equilibration in double distilled water at 37°C for the time of each measurement. The solution was analyzed for fluoride with a fluoride combination electrode.3 The main results:3.1 The bacterial growth was inhibited significantly by the DMAE-CB -incorporated sealant (P < 0.05); no significant difference was found between the negative control and the positive control (P > 0.05).3.2 The bacterial growth curves of S. mutans in the eluents of the three sealant groups were similar to that of the blank control in fresh BHI. There was no statistical difference in the intercept values among the four groups (P > 0.05). The slope values of the three sealant groups were significantly lower than that of the blank control, but the difference was not statistically significant (P > 0.05).3.3 The cured DMAE-CB-incorporated dental sealant could inhibit the formation of S. mutans biofilm on the material surface. After 4-h incubation, on the surface of DMAE-CB incorporated sealant, S. mutans were sporadically scattered with blurred contours; extracelluar matrix was scanty and deformed. After 24-h incubation the biofilm of DMAE-CB-incorporated group was incompact with long chains of S. mutans attached on extracellular scaffold.3.4 CLSM images showed that the live bacteria were dyed as green and those dead bacteria were red. The DMAE-CB-incorporated sealant retained fewer red and green patches.3.5 The cured DMAE-CB-incorporated sealant could inhibit the adherence of S. mutans by destroying bacterial membrane integrity (P < 0.05).3.6 The contact angles of the positive control were higher than those of the DMAE-CB-incorporated group and the negative control (P < 0.05), and the difference between the latter two groups was not significant (P > 0.05). Moreover, the contact angle with etching was lower than that without etching (P < 0.05).3.7 The degree of conversion of the positive control was higher than that of the DMAE-CB-incorporated group and the negative control (P < 0.05). There was no significant difference between the DMAE-CB-incorporated group and the negative control (P > 0.05).3.8 The microhardness of the positive control was higher than that of the DMAE-CB-incorporated group and the negative control (P < 0.05). There was no significant difference between the DMAE-CB-incorporated group and the negative control (P > 0.05).3.9 The incorporation of the antibacterial monomer DMAE-CB showed no more microleakage compared to the commercial sealant in which no antibacterial composite was used (P > 0.05). The microleakage score for the positive control was higher than that of the other two groups (P < 0.05).3.10 Fluoride was released from the evaluated sealants with a similar pattern. Both the materials released measurable amounts of fluoride throughout the test period, with considerable amounts of fluoride released in the first 24 h and especially during the first 4 h.4 The main conclusions:4.1 The DMAE-CB-incorporated sealant could inhibit the bacterial growth and the antibacterial effect was also showed after 2-month aging process or saliva treatment.4.2 The eluent of DMAE-CB-incorporated sealant did not have any greater influence on bacterial growth than its parent material and bioactive components didn't release into the medium.4.3 DMAE-CB-incorporated sealant was demonstrated to have inhibitory effects on in vitro biofilm accumulation of S. mutans. 4.4 The DMAE-CB-incorporated sealant could destroy bacterial membrane integrity and even killing bacteria.4.5 The incorporation of a small amount of DMAE-CB to the sealant Helioseal had no significant influence on the surface contact angle. The etching of the enamel surface is necessary to ensure the effectiveness of the sealant.4.6 The incorporation of DMAE-CB had no adverse influence on the curing behaviour of the Bis-GMA/TEGDMA-based sealant. The degree of conversion increases with increasing filler content of composite resin material.4.7 The incorporation of the antibacterial monomer DMAE-CB had no influence on the microhardness and the curing level of the cured sealants.4.8 The incorporation of the antibacterial monomer DMAE-CB had no influence on the ability to prevent microleakage. The unfilled fissures showed less microleakage than filled fissures.4.9 The incorporation of DMAE-CB had no adverse influence on the fluoride release from Helioseal F which containing a fluoride-releasing resin. The antibacterial activity of long-term and effectiveness by mean of the incorporation of antibacterial monomer is feasible way.Overall, the DMAE-CB-incorporated sealant after polymerization showed a contact antibacterial effect against S. mutans, which could inhibit the bacterial growth, biofilm accumulation and destroy bacterial membrane integrity. The antibacterial activity could be maintained after aging process or saliva treatment. Additionally, the incorporation of small amounts of the monomer DMAE-CB showed no influence on the contact angle, degree of conversion, hardness, microleakage characteristic of the parental material.