Antibacterial Effects Of A Dental Adhesive Incorporating A Quaternary Ammonium Monomer

Dental caries is a microbial disease that continues to pose a worldwide health problem, and usually requires fissure sealant for prevention and dental restoration for treatment. In clinical practice, resin-based materials enjoy high reputation and increased popularity, mostly due to the excellent aesthetic performance, improved mechanical properties and elevated bonding efficiency. However, resin-based materials are reported to accumulate more dental biofilm than other restorative materials both in vitro, and in vivo; gap formation can be observed between the adhesive resin and the primed dentin, or between the adhesive resin and the hybrid layer, according to the investigations on the resin/dentin interface of several commercial bonding systems. The clinical performance of resin-based restorations may therefore be threatened by the occurrence of secondary caries, which are usually generated by the penetration and subsequent propagation of cariogenic bacteria along microgaps between tooth tissue and the restoration, and have long been considered as the most common reason for the replacement of restorations. Therefore, attempts of functionalizing adhesive system with antibacterial activity have been made to ensure the biological sealing of the restoration even when microleakage occurs.A number of reports described the addition of antibacterial components, such as antibiotics, inorganic agents and fluorides, in the constituents of dental adhesive system. However, since antibacterial agents are only simply dispersed in matrix phase, it is impossible to get a strict control of the release kinetics. Moreover, the bonding properties of the carrier material may also be compromised by the constant releasing of antibacterial agents. To overcome the disadvantages caused by agent leaching-out, polymerizable cationic monomers, which can be covalently bound within the polymer matrix, are introduced to render dental adhesive system with antibacterial activity. The integration of polymerizable antibacterial monomer to the resin matrix of adhesive system has proved to be effective for providing non-volatile, chemically stable and long-lasting antibacterial activity, which is considered to be beneficial for reducing the risk of secondary caries and improving the longevity of restorations. Accordingly, Methacryloxylethyl cetyl dimethyl ammonium chloride (DMAE-CB), a polymerizable cationic monomer containing a quaternary ammonium for antibacterial effect and a methacryloyl group for integration with resin matrix, was synthesized in our previous research and has been demonstrated to be effective against oral pathogenic bacteria, therefore being selected as a tentative monomer for functionalizing dental adhesive systems with antibacterial property.Moreover, in the assessment of non-leaching antibacterial activity, planktonic bacteria are usually employed as tested subjects with agar plate diffusion test, bactericide test on the contact surface, bacteria attachment test, etc. In clinical application, however, dental materials will be challenged with biofilm, a complex ecosystem, in which bacteria are organized in a three-dimensional structure and enclosed within an extracellular matrix. Compared with their planktonic counterparts, bacteria harbored in biofilm display higher resistance to antibacterial agents, which is attributed in part to the spatial structure, and in part to the alteration of a number of genes expressed in response to the proximity of a specific surface. Thus, investigating antibacterial activity with biofilm, which can mimic oral environment closely, would provide inference concerning the potential clinic performance of antibacterial activity.1 The main objectives:In this study, DMAE-CB was incorporated into a commercial dental adhesive, to evaluate the antibacterial activity of this DMAE-CB-incorporated adhesive after being cured against planktonic Streptococcus mutans (S. mutans), S. mutans biofilm and microcosm dental biofilm, and to investigate the possible antibacterial mechanism of immobilized cationic monomers.2 The main methods:2.1 The effect of the cured DMAE-CB-incorporated adhesive on the growth of planktonic S. mutans was determined by film contact test.2.2 The influence of aging treatment or saliva treatment on the antibacterial efficiency of the modified adhesive was evaluated.2.3 The bacterial growth in the eluent of the modified adhesive was investigated with spectrophotometry and growth kinetic analysis.2.4 The effects of the cured adhesives on the membrane integrity of S. mutans were investigated using confocal laser scanning microscopy (CLSM) in conjunction with fluorescent indicators.2.5 The effects of the cured DMAE-CB-incorporated adhesive on the accumulation of in vitro S. mutans biofilm and microcosm dental biofilm were investigated with spectrophotometry. 2.6 The influence of aging treatment on the anti-biofilm efficiency of the modified adhesive was evaluated.2.7 The initial formation and the mature process of S. mutans biofilm and microcosm dental biofilm on the modified adhesive were observed with scanning electron microscopy.2.8 The effects of the cured adhesives on the viability profiles of S. mutans biofilm and microcosm dental biofilm were investigated using CLSM in conjunction with fluorescent indicators.2.9 The relative level of gtf gene expression by S. mutans in the biofilm or planktonic bacteria in the incubation media was quantified using real-time reverse-transcription polymerase chain-reaction (real-time RT-PCR).3 The main results:3.1 The cured DMAE-CB-incorporated dental adhesive exhibited inhibitory effect on the growth of S. mutans (P < 0.05).3.2 After aging treatment or saliva treatment, the DMAE-CB-incorporated adhesive could still inhibit the growth of S. mutans significantly (P < 0.05).3.3 The eluent of DMAE-CB-incorporated adhesive didn't show detectable antibacterial activity (P > 0.05).3.4 After fluorescence-labeling, bacteria with integral membranes were dyed as green and those with compromised membranes were red. The specimen of negative control was densely populated with bacteria, while the DMAE-CB-incorporated adhesive and positive control retained fewer bacteria.3.5 The fluorescence analysis of CLSM images demonstrated that cured DMAE-CB-incorporated adhesive and positive control could hamper the adherence of S. mutans, and exert detrimental effect on bacterial membrane integrity (P < 0.05).3.6 The cured DMAE-CB-incorporated dental adhesive exhibited inhibitory effect on the formation of S. mutans biofilm and microcosm dental biofilm (P < 0.05).3.7 The anti-biofilm activity of the modified dental adhesive was not attenuated after 1-month aging process.3.8 After 4-h incubation, initial S. mutans biofilm was generated on the DMAE-CB-incorporated adhesive with sporadically scattered bacteria and extracelluar matrix; the S. mutans biofilms of 24-h incubation were incompact on the DMAE-CB-incorporated adhesive and positive control. A mount of bacteria debris could be detected in microcosm dental biofilm of 4-h incubation on modified adhesive; mature microcosm dental biofilm could be found after 24-h incubation on modified adhesive.3.9 The viability of S. mutans biofilm and microcosm dental biofilm was reduced on the surface of DMAE-CB-incorporated adhesive.3.10 S. mutans harbored in the biofilm on DMAE-CB-incorporated adhesive showed a pronounced suppression in gtfB and gtfC expression (P < 0.05) and an unaltered gtfD expression (P > 0.05).3.11 The gtf genes expression of the S. mutans cells, planktonically growing in the incubation media, remained unchanged among the three groups (P > 0.05).4 The main conclusions:4.1 The cured DMAE-CB-incorporated dental adhesive could inhibit the growth and adherence of S. mutans.4.2 The inhibitory effect of DMAE-CB-incorporated adhesive on growth of S. mutans could not be attenuated by aging process, indicating a long-lasting antibacterial activity.4.3 The inhibitory effect of DMAE-CB-incorporated adhesive on growth of S. mutans could not be attenuated by saliva treatment, indicating a promising usage of the modified adhesive in clinical practice.4.4 The DMAE-CB-incorporated adhesive could exert contact antibacterial activity without DMAE-CB monomer being leached out.4.5 The DMAE-CB-incorporated adhesive could exert detrimental effect on bacterial membrane integrity, providing possible explanation for the antibacterial activity of immobilized cationic monomers.4.6 The cured DMAE-CB-incorporated dental adhesive could exhibit inhibitory effect on the formation and accumulation of S. mutans biofilm and microcosm dental biofilm.4.7 The anti-biofilm activity of the modified dental adhesive could not be attenuated after 1-month aging process, substantiating a long-term antibacterial activity.4.8 The viability of S. mutans biofilm and microcosm dental biofilm could be suppressed on the cured DMAE-CB-incorporated dental adhesive.4.9 The DMAE-CB-incorporated adhesive could hamper the S. mutans biofilm formation via selectively modulating the expression of gtf genes in S. mutans.4.10 The selective modulation of gtf genes expression was confined within the biofilm, therefore excluding the possibility of the leaching-out of DMAE-CB monomers and confirming the contact antibacterial activity of immobilized cationic monomer.Conclusively, the incorporation of DMAE-CB can render the dental adhesive with contact antibacterial activity after polymerization via influencing the growth, adherence, and membrane integrity of S. mutans. Moreover, the DMAE-CB-incorporated adhesive can exhibit anti-biofilm activity via inhibiting the biofilm formation, reducing the viability of biofilm, and modulating the genetic expression of virulence factors in S. mutans.