两种常用季铵盐型抗菌单体抗菌性能、细胞毒性的比较研究及其细胞毒性机理初探
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摘要
基于对细菌在龋病这一感染性疾病中关键作用的深入理解，近年来许多学者都致力于研发新型具有抗菌性能的功能性齿科材料。以往通过直接添加可溶性有机抗菌剂或无机抗菌剂对齿科材料进行抗菌改性的方法因抗菌剂的突释效应及对材料本身机械性能的不利影响已逐渐被学者们抛弃。
“可聚合抗菌剂”是一种具有聚合功能的抗菌剂，其能够通过共价键将抗菌功能基团稳固交联结合于基质材料，从而赋予基质材料不依赖活性成分释放的、稳定的接触抑菌功能，是研发新型抗菌齿科修复材料的新策略。日本的Imazato教授在国际上最先合成可用于齿科树脂基材料的可聚合抗菌剂甲基丙烯酰氧十二烷基溴吡啶（methacryloyloxydodecylpyridiniumbromide，MDPB）单体，并在此基础上研发出目前国际上唯一商品化的抗菌粘接系统。近年来，本人所在课题组在国内率先开展了可聚合抗菌剂的研究，合成了与MDPB结构类似的多种抗菌单体。这些抗菌单体均依赖其分子结构中的季铵基团发挥抗菌作用，因此被统称为季铵盐型抗菌单体。本课题组通过前期性能检测优选出甲基丙烯酰氧十二烷基溴吡啶（methacryloxylethyl cetyl dimethyl ammonium bromide，DMAE-CB）进行齿科树脂基材料抗菌改性研究，并证实将DMAE-CB添加到粘接剂或窝沟封闭剂中可以在不显著影响材料本身理化性能的同时赋予其稳定的抑菌功能。但是，基于抗菌单体的功能性齿科材料在临床上的成功应用不仅依赖于其良好的抗菌性能和理化性能，还在很大程度上取决于其生物安全性能。因此研究季铵盐型抗菌单体的细胞毒性及其引起细胞毒性的机理、并探索可能的保护措施具有重要的意义。但是目前尚无这方面的研究。
1.研究目的本研究首先以MDPB和DMAE-CB为代表，比较不同结构的季铵盐单体抗菌性能、细胞毒性的差异，并在此基础上探索其细胞毒性的机制并寻找可能的降低其细胞毒性的措施，为日后改善抗菌齿科树脂基材料的生物安全性、拓展其在深龋及牙髓保护方面的应用提供理论及实验支持。
2.主要研究方法：
2.1.通过最小抑菌浓度（Minimal inhibitory concentration，MIC）测试、最小杀菌浓度（Minimal bactericidal concentraion，MBC）测试、快速杀菌实验评价并比较两种季铵盐型抗菌单体DMAE-CB与MDPB的抗菌效应，并研究抗氧化剂N-乙酰半胱氨酸（N-acetyl-L-cysteine，NAC）对上述单体抗菌性能的可能影响。
2.2.采用死菌/活菌荧光染色和激光共聚焦显微镜观察比较DMAE-CB和MDPB对黏附状态细菌的快速杀灭作用。
2.3.采用3-(4,5-二甲基噻唑基-2)-2,5二苯基四氮唑溴盐法（MTT法）评价DMAE-CB与MDPB对细胞增殖及存活情况的影响，并研究抗氧化剂NAC和纳米铂凝胶颗粒（Colloidal platinum nanoparticles，CPtN）对抗菌单体细胞毒性的可能影响。
2.4.通过检测成骨细胞碱性磷酸酶（Alkaline phosphatase，ALP）的活性，评价DMAE-CB与MDPB对成骨细胞分化的影响，并探索NAC对上述单体引起的分化抑制的可能影响。
2.5.通过ALP-冯库萨染色检测两种季铵盐型抗菌单体DMAE-CB与MDPB对成骨细胞矿化功能的影响。
2.6.采用荧光染色和流式细胞仪分析技术，评价季铵盐型抗菌单体对细胞增殖、细胞分裂周期、细胞凋亡和坏死的影响，探索细胞内氧化应激及线粒体依赖性凋亡通路是否与抗菌单体的细胞毒性作用有关。
2.7.通过western blot技术，探索铵盐型抗菌对细胞内Bcl-2、Bax及细胞色素C等线粒体依赖性凋亡通路的相关分子的表达及分布的影响，明确线粒体依赖性凋亡通路在抗菌单体引起的细胞死亡中的作用。
3.主要研究结果
3.1. DMAE-CB的MIC（3.91-7.81μg/mL）和MBC（7.81μg/mL）值低于MDPB（MIC：7.81-31.3μg/mL；MBC：125-250μg/mL），提示DMAE-CB抗菌性能强于MDPB。
3.2.季铵盐型抗菌单体对游离及黏附状态下的变形链球菌均具有快速杀灭作用，且DMAE-CB的作用强于MDPB。
3.3.季铵盐型抗菌单体对成骨细胞的增殖和存活具有抑制作用，DMAE-CB的抑制作用强于MDPB，但与常用牙科单体Bis-GMA的抑制作用相当。
3.4.季铵盐型抗菌单体对成骨细胞的分化具有抑制作用，且较低浓度的上述抗菌单体即可引起细胞分化的显著抑制。
3.5.季铵盐型抗菌单体对成骨细胞的矿化功能具有抑制作用，可显著减少钙沉淀的形成。
3.6.季铵盐型抗菌单体可引起小鼠成纤维细胞细胞周期的紊乱，并使其通过凋亡和坏死两种方式发生死亡。
3.7.季铵盐型抗菌单体可引起小鼠成纤维细胞内活性氧（Reactive oxygenspecies，ROS）水平呈时间及浓度依赖性上升。
3.8.季铵盐型抗菌单体可引起小鼠成纤维细胞内线粒体膜电位的丧失、下调抑凋亡分子Bcl-2的表达，上调促凋亡分子Bax的表达，并促进细胞色素C从线粒体向细胞质转位，提示抗菌单体激活了线粒体依赖性凋亡通路。
3.9.抗氧化剂NAC对季铵盐型抗菌单体的细胞毒性具有一定的缓解作用，但抗氧化剂CPtN却增强了其毒性。
3.10.抗氧化剂NAC对季铵盐型抗菌单体引起的成骨细胞分化的抑制没有显著影响。
3.11.抗氧化剂NAC在可降低细胞毒性的浓度（10mM）下对DMAE-CB和MDPB的快速杀菌活性没有显著不利影响。
4.主要结论
4.1.季铵盐型抗菌单体对游离及黏附状态下的细菌均有快速杀菌作用，且DMAE-CB的抗菌性能强于MDPB。
4.2.季铵盐型抗菌单体对成骨细胞的增殖、分化和矿化有不利影响，且DMAE-CB的毒性稍大于MDPB，但DMAE-CB对细胞增殖的影响与常用交联单体Bis-GMA相当。
4.3.季铵盐型抗菌单体的细胞毒性与ROS过量产生有一定关系，并可以导致细胞周期的紊乱、凋亡及坏死的发生。
4.4.季铵盐型抗菌单体可以激活线粒体介导的凋亡通路，并导致部分细胞通过凋亡模式发生死亡。
4.5.抗氧化剂NAC对季铵盐型抗菌单体引起的细胞死亡具有一定的缓解作用，却对其引起的细胞分化抑制作用无显著影响。另一种抗氧化剂CPtN会增强季铵盐型抗菌单体的细胞毒性。
4.6.抗氧化剂NAC在可降低细胞毒性的浓度（10mM）下对季铵盐型抗菌单体的快速杀菌作用没有不利影响。
综上所述，季铵盐型抗菌单体具有稳定、可靠的快速杀菌作用，同时，其对细胞的增殖、分化、矿化等功能有一定的不利影响。季铵盐型抗菌单体的细胞毒性与细胞内氧化还原平衡的破坏有一定关联。使用抗氧化剂NAC可在不影响抗菌单体抗菌性能的基础上缓解其细胞毒性。基于上述结果我们推测，使用抗氧化剂可以提高含季铵盐抗菌单体的抗菌树脂基材料的生物安全性，进而进一步改善抗菌树脂基材料在深龋及龋源型露髓的保髓治疗方面的效果。
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. PURPOSE
The 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. METHODS
2.1. 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. RESULTS
3.1. 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. CONCLUSIONS
4.1. 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.
“可聚合抗菌剂”是一种具有聚合功能的抗菌剂，其能够通过共价键将抗菌功能基团稳固交联结合于基质材料，从而赋予基质材料不依赖活性成分释放的、稳定的接触抑菌功能，是研发新型抗菌齿科修复材料的新策略。日本的Imazato教授在国际上最先合成可用于齿科树脂基材料的可聚合抗菌剂甲基丙烯酰氧十二烷基溴吡啶（methacryloyloxydodecylpyridiniumbromide，MDPB）单体，并在此基础上研发出目前国际上唯一商品化的抗菌粘接系统。近年来，本人所在课题组在国内率先开展了可聚合抗菌剂的研究，合成了与MDPB结构类似的多种抗菌单体。这些抗菌单体均依赖其分子结构中的季铵基团发挥抗菌作用，因此被统称为季铵盐型抗菌单体。本课题组通过前期性能检测优选出甲基丙烯酰氧十二烷基溴吡啶（methacryloxylethyl cetyl dimethyl ammonium bromide，DMAE-CB）进行齿科树脂基材料抗菌改性研究，并证实将DMAE-CB添加到粘接剂或窝沟封闭剂中可以在不显著影响材料本身理化性能的同时赋予其稳定的抑菌功能。但是，基于抗菌单体的功能性齿科材料在临床上的成功应用不仅依赖于其良好的抗菌性能和理化性能，还在很大程度上取决于其生物安全性能。因此研究季铵盐型抗菌单体的细胞毒性及其引起细胞毒性的机理、并探索可能的保护措施具有重要的意义。但是目前尚无这方面的研究。
1.研究目的本研究首先以MDPB和DMAE-CB为代表，比较不同结构的季铵盐单体抗菌性能、细胞毒性的差异，并在此基础上探索其细胞毒性的机制并寻找可能的降低其细胞毒性的措施，为日后改善抗菌齿科树脂基材料的生物安全性、拓展其在深龋及牙髓保护方面的应用提供理论及实验支持。
2.主要研究方法：
2.1.通过最小抑菌浓度（Minimal inhibitory concentration，MIC）测试、最小杀菌浓度（Minimal bactericidal concentraion，MBC）测试、快速杀菌实验评价并比较两种季铵盐型抗菌单体DMAE-CB与MDPB的抗菌效应，并研究抗氧化剂N-乙酰半胱氨酸（N-acetyl-L-cysteine，NAC）对上述单体抗菌性能的可能影响。
2.2.采用死菌/活菌荧光染色和激光共聚焦显微镜观察比较DMAE-CB和MDPB对黏附状态细菌的快速杀灭作用。
2.3.采用3-(4,5-二甲基噻唑基-2)-2,5二苯基四氮唑溴盐法（MTT法）评价DMAE-CB与MDPB对细胞增殖及存活情况的影响，并研究抗氧化剂NAC和纳米铂凝胶颗粒（Colloidal platinum nanoparticles，CPtN）对抗菌单体细胞毒性的可能影响。
2.4.通过检测成骨细胞碱性磷酸酶（Alkaline phosphatase，ALP）的活性，评价DMAE-CB与MDPB对成骨细胞分化的影响，并探索NAC对上述单体引起的分化抑制的可能影响。
2.5.通过ALP-冯库萨染色检测两种季铵盐型抗菌单体DMAE-CB与MDPB对成骨细胞矿化功能的影响。
2.6.采用荧光染色和流式细胞仪分析技术，评价季铵盐型抗菌单体对细胞增殖、细胞分裂周期、细胞凋亡和坏死的影响，探索细胞内氧化应激及线粒体依赖性凋亡通路是否与抗菌单体的细胞毒性作用有关。
2.7.通过western blot技术，探索铵盐型抗菌对细胞内Bcl-2、Bax及细胞色素C等线粒体依赖性凋亡通路的相关分子的表达及分布的影响，明确线粒体依赖性凋亡通路在抗菌单体引起的细胞死亡中的作用。
3.主要研究结果
3.1. DMAE-CB的MIC（3.91-7.81μg/mL）和MBC（7.81μg/mL）值低于MDPB（MIC：7.81-31.3μg/mL；MBC：125-250μg/mL），提示DMAE-CB抗菌性能强于MDPB。
3.2.季铵盐型抗菌单体对游离及黏附状态下的变形链球菌均具有快速杀灭作用，且DMAE-CB的作用强于MDPB。
3.3.季铵盐型抗菌单体对成骨细胞的增殖和存活具有抑制作用，DMAE-CB的抑制作用强于MDPB，但与常用牙科单体Bis-GMA的抑制作用相当。
3.4.季铵盐型抗菌单体对成骨细胞的分化具有抑制作用，且较低浓度的上述抗菌单体即可引起细胞分化的显著抑制。
3.5.季铵盐型抗菌单体对成骨细胞的矿化功能具有抑制作用，可显著减少钙沉淀的形成。
3.6.季铵盐型抗菌单体可引起小鼠成纤维细胞细胞周期的紊乱，并使其通过凋亡和坏死两种方式发生死亡。
3.7.季铵盐型抗菌单体可引起小鼠成纤维细胞内活性氧（Reactive oxygenspecies，ROS）水平呈时间及浓度依赖性上升。
3.8.季铵盐型抗菌单体可引起小鼠成纤维细胞内线粒体膜电位的丧失、下调抑凋亡分子Bcl-2的表达，上调促凋亡分子Bax的表达，并促进细胞色素C从线粒体向细胞质转位，提示抗菌单体激活了线粒体依赖性凋亡通路。
3.9.抗氧化剂NAC对季铵盐型抗菌单体的细胞毒性具有一定的缓解作用，但抗氧化剂CPtN却增强了其毒性。
3.10.抗氧化剂NAC对季铵盐型抗菌单体引起的成骨细胞分化的抑制没有显著影响。
3.11.抗氧化剂NAC在可降低细胞毒性的浓度（10mM）下对DMAE-CB和MDPB的快速杀菌活性没有显著不利影响。
4.主要结论
4.1.季铵盐型抗菌单体对游离及黏附状态下的细菌均有快速杀菌作用，且DMAE-CB的抗菌性能强于MDPB。
4.2.季铵盐型抗菌单体对成骨细胞的增殖、分化和矿化有不利影响，且DMAE-CB的毒性稍大于MDPB，但DMAE-CB对细胞增殖的影响与常用交联单体Bis-GMA相当。
4.3.季铵盐型抗菌单体的细胞毒性与ROS过量产生有一定关系，并可以导致细胞周期的紊乱、凋亡及坏死的发生。
4.4.季铵盐型抗菌单体可以激活线粒体介导的凋亡通路，并导致部分细胞通过凋亡模式发生死亡。
4.5.抗氧化剂NAC对季铵盐型抗菌单体引起的细胞死亡具有一定的缓解作用，却对其引起的细胞分化抑制作用无显著影响。另一种抗氧化剂CPtN会增强季铵盐型抗菌单体的细胞毒性。
4.6.抗氧化剂NAC在可降低细胞毒性的浓度（10mM）下对季铵盐型抗菌单体的快速杀菌作用没有不利影响。
综上所述，季铵盐型抗菌单体具有稳定、可靠的快速杀菌作用，同时，其对细胞的增殖、分化、矿化等功能有一定的不利影响。季铵盐型抗菌单体的细胞毒性与细胞内氧化还原平衡的破坏有一定关联。使用抗氧化剂NAC可在不影响抗菌单体抗菌性能的基础上缓解其细胞毒性。基于上述结果我们推测，使用抗氧化剂可以提高含季铵盐抗菌单体的抗菌树脂基材料的生物安全性，进而进一步改善抗菌树脂基材料在深龋及龋源型露髓的保髓治疗方面的效果。
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. PURPOSE
The 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. METHODS
2.1. 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. RESULTS
3.1. 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. CONCLUSIONS
4.1. 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.
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