A Comparative Study Of The Antibacterial Activity And Cytotoxicity Of Two Popular Quaternary Ammonium Antibacterial Monomers And The Exploration Of Mechanisms Underlying Their Cytotoxicity

Since dental caries has been recognized as an infectious disease induced bycariogenic bacteria, attempts to develop restorative materials possessingantibacterial effects have been an important topic in dental materials science.The conventional strategy of directly adding organic or inorganic antibacterialagents to existing restorative materials to provide the carrier material withantibacterial activity has recently been abondoned by researchers due to itslimited period of effectiveness and negative influences on the originalmechanical properties of the carrier material.The “immobilized bactericide” is a group of polymerizable antibacterial agents, whose antibacterial components can be immobilized in the carrier material bycovalent bonding. This technology enables non-agent-releasing typeantibacterial restoratives that can exert stable contact antibacterial acitivity inthe long term, and thus is recognized as a novel and promising strategy todevelop bioactive dental materials with antibacterial activities. Prof. Imazatofrom Osaka University synthesized the first immobilized bactericide, namelymethacryloyloxydodecylpyridinium bromide (MDPB), that can be utilized inresin-based dental materials,. Based on MDPB, the world’s first antibacterialdental adhesive systerm has been successfully invented and commercialized. Inrecent years, our research group also focused on studies about immobilizedbactericides, and successfully synthesized several antibacterial monomers.Similar to MDPB, the antibacterial monomers developed by our group alsorelies on their quaternary ammonium groups to exert potent antibacterialactivites, and thus, together with MDPB, are grouped as quaternary ammoniumantibacterial monomers. Through intense comparative studies, we identifiedmethacryloxylethyl cetyl dimethyl ammonium bromide (DMAE-CB) as apromising candidate for developing new antibacterial resin-based restorativematerials. The application of DMAE-CB in dental adhesives and fissure sealantshas been found to be effective to provide these resin-based materials with stableantibacterial activity without compromising their original mechanical properties.However, the successful clinical application of bio-active resin-based dentalmaterials with antibacterial activity not only depends on their antibacterialactivity and mechanical properties, but also relies on their biological safety.Therefore, study of the cytotoxicity and mechanisms underlying the cytotoxicityof quaternary ammonium monomers, and exploration of possible protectivestrategies against cytotoxicity are of primary importance. However, to date, no related study has been reported yet.1. PURPOSEThe main purpose of the present study was to:①study the antibacterialactivity and cytotoxicity of the two quaternary ammonium antibacterialmonomers, DMAE-CB and MDPB;②explore the mechanism underlying thecytotoxicity of quaternary ammonium antibacterial monomers;③identify thepossible protective agents against the cytotoxicity of quaternary ammoniummonomers. Hopefully, this study will be helpful for finding effective strategiesto improve the biocompatibility of antibacterial monomer-based bioactive dentalmaterials.2. METHODS2.1. Minimal inhibitory concentration (MIC) and minimal bactericidalconcentration (MBC) test, rapid killing studies were performed to investigate theantibacterial activity of DMAE-CB and MDPB, and to explore the possibleinfluence of N-acetyl-L-cysteine (NAC) on the antibacterial activity ofquaternary ammonium monomers.2.2. Live/Dead fluorescent staining was combined with confocal microscopicobservation to investigate the rapid killing effects of DMAE-CB and MDPBagainst adhering bacteria.2.3. MTT assay was performed to study the influence of qauternary ammoniumantibacterial monomers on the proliferation and viability of cells. MTT assaywas also used to explore the possible protective effects of NAC and colloidalplatinum nanoparticles (CPtN) against antibacterial monomer-induced celldeath.2.4. Alkaline phosphatase (ALP) activity was monitored to indicate the influence of antibacterial monomers on the differentiation of osteoblast-likecells, and also explore the possible protective effects of NAC.2.5. ALP-Von Kossa staining was performed to assess the influence ofquaternary ammonium monomers on the mineralization activity ofosteoblast-like cells.2.6. Fluorescent staining technique was combined with flow cytometry toinvestigate the influences of quaternary ammonium antibacatierla monomers onthe proliferation, cell cycle progression, apoptosis and necrosis of cells.2.7. Western blot was performed to study the expression pattern of Bcl-2andBax and the distribution of Cytochrome C after exposure to quaternaryammonium antibacterial monomers.3. RESULTS3.1. DMAE-CB exhibited lower MIC/MBC (MIC:3.91-7.81μg/mL; MBC:7.81μg/mL) values than MDPB (MIC:7.81-31.3μg/mL; MBC:125-250μg/mL),indicating that the former has higher intrinsic antibacterial activity than thelatter.3.2. Both tested quaternary ammonium antibacterial monomers can killplanktonic and adhering bacteria in a very rapid manner. DMAE-CB showedpotent rapid killing effects at relatively lower concentrations than MDPB.3.3. Both tested antibacterial monomers exerted negative influences on theproliferation and viability of osteoblast-like cells. The negative influence ofDMAE-CB was greater than that of MDPB, but comparable to that of Bis-GMAwhich is widely used in various resin-based dental materials.3.4. Both tested antibacterial monomers showed inhibitory effects on thedifferentiation of osteoblast-like cells. The differentiation function of osteoblast-like cells is more sensitive to antibacterial monomers than itsproliferation and viability.3.5. Both tested antibacterial monomers inhibited calcium accumulation ofosteoblast-like cells.3.6. Antibacteiral monomers induced cell cycle arrest, and caused apoptosis andnecrosis in the tested mouse fibroblast cells.3.7. Treatment with antibacterial monomers induced an over-production ofreactive oxygen species (ROS) in mouse fibroblast cells.3.8. Antibacterial monomers caused the loss the membrane potential in somemouse fibroblast cells, reduced the expression of Bcl-2while increased theexpression of Bax, and resulted in the translocation of Cytochrome C frommitochondria to cytosol. These results indicate that mitochondria-dependentapoptotic pathway is involved in the cytotoxic effects of quaternary ammoniumantibacterial monomers.3.9. The antioxidant NAC exhibited partial protective effects against quaternaryammonium antibacterial monomer-induced reduction of cell proliferation andviability. The other tested antioxidant CPtN didn’t show any protective effects,but rather enhanced the cytotoxicity.3.10. NAC at the concentration of10mM did not protect against antibacterialmonomer-induced inhibition osteoblast-like cell differentiation.3.11. NAC at the concentration that can protect against antibacterialmonomer-induced cytotoxicity (10mM) did not exhibit any negative influenceson the rapid killing effects of quaternary ammonium antibacterial monomers.4. CONCLUSIONS4.1. Quaternary ammonium antibacterial monomers can kill planktonic and adhering bacteria in a rapid manner. Compared with MDPB, DMAE-CB hasrelatively stronger intrinsic antibacterial activity.4.2. Quaternary ammonium antibacterial monomers have negative influences onthe viability, differentiation and mineralization of osteobalst-like cells. Thecytotoxic effects of these monomers are in proportion to their antibacterialactivity: the monomer with stronger antibacterial monomer also exhibits highercytotoxicity. Although the cytotoxicity of DMAE-CB was higher than that ofMDPB, it is comparable to that of the widely used cross-linking monomerBis-GMA.4.3. The cytotoxicity of quaternary ammonium monomers is related to theover-production of ROS, which can thereafter induce cell cycle arrest, apoptosisand necrosis.4.4. Quaternary ammonium monomers can activate the mitochondria-dependentapoptosis pathway and therefore induce apoptotic cell death.4.5. The antioxidant NAC is protective against quaternary ammoniummonomer-related reduction of cell viability, but can not reduce antibacterialmonomer-induced inhibition of cell differentiation. The other tested antioxidantCPtN enhanced the cytotoxicity of antibacterial monomers.4.6. The antioxidant NAC at the concentration that can protect againstcytotoxicity presented no negative influences on the antibacterial activity ofquaternary ammonium monomers.In conclusion, quaternary ammonium antibacterial monomers have reliable andrapid bactericidal effects. But at the same time, these monomers also havenegative effects on the viability, differentiation and mineralization of cells. Thecytotoxicity of quaternary ammonium antibacterial monomers is related to theover-production of ROS and disturbance of redox balance, and the antioxidant NAC can partially reduce the cytotoxicity of quaternary ammonium monomerswithout compromising their antibacterial properties. Based in these results, wepropose that the combined use of antioxidants and quaternary ammoniummonomers may improve the biocompatibility of antibacterial resin-basedmaterials, and thus may be a promising strategy to optimize the pulp protectiveeffects of antibacterial adhesives in the treatment of deep caries, andcaries-induced pulp exposure.