季铵盐型抗菌单体改性牙本质粘接剂的抗菌性能研究
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摘要
复合树脂及粘接系统广泛应用于修复牙体组织缺损,改善牙齿颜色和外观。但因缺乏有效的抗菌性能,复合树脂材料表面容易聚积菌斑;树脂材料的聚合收缩和粘接不良可形成界面微渗漏,成为致龋菌的侵入通道。因此,继发龋常影响树脂修复体远期临床效果,是修复失败的重要原因。
目前,开发具有抗菌功能的复合树脂和粘接系统受到了国内外学者的广泛关注,希望借此减少继发龋的发生。有研究将抗菌剂添加至树脂基质,依赖抗菌剂的释放获得抗菌功能。但抗菌成分的添加和释放会影响材料的力学性能和美学效果,且获得的抗菌性能难以稳定持久。为克服上述抗菌改性方式的不足,研究者尝试开发可聚合抗菌单体用于树脂材料抗菌改性,将抗菌功能基团接枝共聚于树脂基质,发挥稳定的接触性抗菌作用,且无损材料的机械性能,具有良好的应用前景。据此,本课题组合成了多种可聚合季铵盐抗菌功能单体,其中甲基丙烯酰氧乙基一正十六烷基一二甲基氯化铵(methacryloxylethyl cetyl dimethyl ammonium chloride,DMAE-CB),对口腔常见细菌具有较强的杀菌作用,因此作为备选单体用于树脂材料抗菌改性。
评价修复材料抗菌效果的研究多局限于应用琼脂平板弥散法对抑菌环的观察,材料浸提液对细菌悬液的作用,以及材料对浮游状态细菌生长的影响等。上述实验均可用于检测材料的溶出性抗菌性能,但无法准确评价含有季铵盐抗菌单体的高分子牙科材料的接触性抗菌性能。此外,实验中采用浮游状态细菌进行抗菌研究,同口腔环境中的菌斑生物膜相差甚远。细菌在形成菌斑生物膜后,基因表达和生物学行为发生改变,耐药性显著提高。因此,体外培养生物膜,研究抗菌材料表面对生物膜形成和生态特征的影响,对于准确评估材料的临床应用前景尤为重要。
1主要研究目的:
将季铵盐抗菌单体DMAE-CB添加到商品化牙本质粘接剂Single Bond 2中,获得改性粘接剂作为实验组,并以Single Bond 2作为阴性对照,含有抗菌单体MDPB的粘接系统Clearfil Protect Bond作为阳性对照。评价DMAE-CB改性牙本质粘接剂固化后的抗菌效果,及其对菌斑生物膜形成和活性的影响,初步探索固定于高分子网络的季铵盐基团抑制细菌生长和生物膜形成的机理。
2主要研究方法:
2.1采用接触抑菌实验评价改性粘接剂的固化表面对变形链球菌生长的影响。
2.2研究老化处理和唾液处理对改性粘接剂固化表面抗菌性能的影响。
2.3分析变形链球菌在粘接剂浸提液中的生长动力学和倍增时间,检测材料浸提液的抗菌活性。
2.4利用荧光指示剂和激光共聚焦扫描显微镜评价改性粘接剂固化表面对变形链球菌胞膜完整性的影响,初步探索固定于高分子网络的季铵盐基团的抗菌机理。
2.5改性粘接剂固化表面进行变链生物膜和全菌生物膜的体外培养,通过光密度值评价改性材料对变链生物膜和全菌生物膜形成的影响。
2.6采用光密度值分析,评价改性粘接剂的固化表面经老化处理后对体外变链生物膜和全菌生物膜形成的影响。
2.7扫描电镜观察改性粘接剂表面变链生物膜和全菌生物膜初步形成和成熟过程,验证改性材料对生物膜形成的影响。
2.8应用荧光指示剂和激光共聚焦扫描显微镜研究改性材料表面变链生物膜和全菌生物膜的三维活性结构。
2.9改性材料表面培养变链生物膜,收集生物膜变链和孵育液中浮游变链,进行RNA抽提和实时定量RT-PCR分析,检测改性材料表面生物膜变链和孵育液中浮游变链的gtf基因表达水平,初步探索固定于高分子网络的季铵盐基团抑制生物膜形成的机理。
3主要研究结果:
3.1固化后的DMAE-CB改性粘接剂可抑制表面变形链球菌的生长,生长抑制率约为99%(P < 0.05)。
3.2老化处理或唾液处理后,改性材料表面仍表现出抗菌活性:细菌生长抑制率分别约为99% (P < 0.05)和90% (P < 0.05)。
3.3改性粘接剂的浸提液无抗菌活性:实验组和阴性对照组浸提液中变形链球菌指数生长期拟合直线的斜率及细菌的倍增时间均无显著差异(P > 0.05)。
3.4荧光染色标记后,细胞膜完整的细菌染为绿色,胞膜受损的细菌染为红色。阴性对照粘接剂固化表面细菌密集;改性粘接剂和阳性对照粘接剂表面细菌附着较少。
3.5荧光强度分析显示各组的总荧光强度FIt和红绿荧光强度比值FIr/FIg的差异显著(P < 0.05);阴性对照组的FIt值最高而FIr/FIg最低(P < 0.05),提示改性粘接剂组和阳性对照可以损伤变形链球菌胞膜的完整性,减少细菌粘附。
3.6同阴性对照相比,改性粘接剂和阳性对照组的固化表面可抑制表面变链生物膜和全菌生物膜的形成(P < 0.05)。
3.7老化处理后改性材料和阳性对照的表面,变链生物膜和全菌生物膜形成量较老化前均无显著提高(P > 0.05),仍表现出抑制表面生物膜形成的作用(P < 0.05)。
3.8扫描电镜观察发现:改性粘接剂表面变链生物膜孵育4 h,可见变链和细胞外基质散在分布;孵育24 h,形成松散的变链生物膜。改性粘接剂表面的全菌生物膜孵育4 h,可见细菌残骸;孵育24 h后,形成全菌生物膜。
3.9改性粘接剂表面的变链生物膜和全菌生物膜的厚度和总体活性均较阴性对照组下降。
3.10粘接剂表面生物膜变链的gtfBCD基因表达,三组之间有所差异:阳性对照组和改性粘接剂组的生物膜变链gtfB表达分别下降为阴性对照的23%和8.6% (P < 0.05),gtfC的表达分别下降为阴性对照的35.9 %和14.1 % (P < 0.05);而阳性对照组和改性粘接剂组gtfD的表达同阴性对照之间没有显著差异(P > 0.05)。
3.11试件孵育液中的浮游态细菌的gtfB、gtfC和gtfD的RNA拷贝数,三组之间均无显著差异(P > 0.05)。
4主要结论:
4.1 DMAE-CB改性粘接剂固化后可以抑制表面细菌生长和粘附。
4.2老化处理后DMAE-CB改性材料仍表现出抗菌活性,提示季铵盐单体改性牙本质粘接剂可以长期发挥抗菌作用。
4.3唾液预处理后DMAE-CB改性材料仍表现出抗菌活性,提示季铵盐单体改性粘接剂可在口腔环境中发挥抗菌作用,具有临床应用前景。
4.4排除了抗菌单体从改性粘接剂固化样本中溶出发挥抗菌作用的可能,明确了DMAE-CB改性粘接剂固化后具有接触性抗菌性能。
4.5 DMAE-CB改性粘接剂可以影响所接触细菌的胞膜完整性,由此部分解释固定于树脂基质中阳离子抗菌基团的抗菌机制。
4.6 DMAE-CB改性粘接剂和含有抗菌单体MDPB的阳性对照粘接剂的固化表面可以抑制变形链球菌生物膜和全菌生物膜的形成。
4.7老化处理后,DMAE-CB改性粘接剂和阳性对照的固化表面对变形链球菌生物膜和全菌生物膜形成的抑制作用无明显衰减。
4.8 DMAE-CB改性粘接剂表面可下调变链生物膜和全菌生物膜的活性。
4.9 DMAE-CB改性粘接剂和阳性对照的固化表面可以选择性下调生物膜变链的gtf基因表达,由此部分解释固定于树脂基质中阳离子抗菌基团抑制变链生物膜形成的机制。
4.10抗菌单体改性材料表面对gtf基因表达的影响局限于材料表面的变链生物膜,排除了抗菌单体从粘接样本中溶出发挥抗菌作用的可能,明确了DMAE-CB改性粘接剂具有接触性抗菌性能。
综上,可聚合季铵盐抗菌单体DMAE-CB可以用于牙本质粘接剂抗菌改性,改性材料固化后,阳离子抗菌基团固定于树脂基质网络,影响变形链球菌生长、粘附、胞膜完整性,以及影响生物膜的形成和活性,选择性下调变形链球菌gtf基因表达水平,发挥抗菌、抑制生物膜形成的作用,赋予粘接剂持久稳定的接触性抗菌活性。
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.
目前,开发具有抗菌功能的复合树脂和粘接系统受到了国内外学者的广泛关注,希望借此减少继发龋的发生。有研究将抗菌剂添加至树脂基质,依赖抗菌剂的释放获得抗菌功能。但抗菌成分的添加和释放会影响材料的力学性能和美学效果,且获得的抗菌性能难以稳定持久。为克服上述抗菌改性方式的不足,研究者尝试开发可聚合抗菌单体用于树脂材料抗菌改性,将抗菌功能基团接枝共聚于树脂基质,发挥稳定的接触性抗菌作用,且无损材料的机械性能,具有良好的应用前景。据此,本课题组合成了多种可聚合季铵盐抗菌功能单体,其中甲基丙烯酰氧乙基一正十六烷基一二甲基氯化铵(methacryloxylethyl cetyl dimethyl ammonium chloride,DMAE-CB),对口腔常见细菌具有较强的杀菌作用,因此作为备选单体用于树脂材料抗菌改性。
评价修复材料抗菌效果的研究多局限于应用琼脂平板弥散法对抑菌环的观察,材料浸提液对细菌悬液的作用,以及材料对浮游状态细菌生长的影响等。上述实验均可用于检测材料的溶出性抗菌性能,但无法准确评价含有季铵盐抗菌单体的高分子牙科材料的接触性抗菌性能。此外,实验中采用浮游状态细菌进行抗菌研究,同口腔环境中的菌斑生物膜相差甚远。细菌在形成菌斑生物膜后,基因表达和生物学行为发生改变,耐药性显著提高。因此,体外培养生物膜,研究抗菌材料表面对生物膜形成和生态特征的影响,对于准确评估材料的临床应用前景尤为重要。
1主要研究目的:
将季铵盐抗菌单体DMAE-CB添加到商品化牙本质粘接剂Single Bond 2中,获得改性粘接剂作为实验组,并以Single Bond 2作为阴性对照,含有抗菌单体MDPB的粘接系统Clearfil Protect Bond作为阳性对照。评价DMAE-CB改性牙本质粘接剂固化后的抗菌效果,及其对菌斑生物膜形成和活性的影响,初步探索固定于高分子网络的季铵盐基团抑制细菌生长和生物膜形成的机理。
2主要研究方法:
2.1采用接触抑菌实验评价改性粘接剂的固化表面对变形链球菌生长的影响。
2.2研究老化处理和唾液处理对改性粘接剂固化表面抗菌性能的影响。
2.3分析变形链球菌在粘接剂浸提液中的生长动力学和倍增时间,检测材料浸提液的抗菌活性。
2.4利用荧光指示剂和激光共聚焦扫描显微镜评价改性粘接剂固化表面对变形链球菌胞膜完整性的影响,初步探索固定于高分子网络的季铵盐基团的抗菌机理。
2.5改性粘接剂固化表面进行变链生物膜和全菌生物膜的体外培养,通过光密度值评价改性材料对变链生物膜和全菌生物膜形成的影响。
2.6采用光密度值分析,评价改性粘接剂的固化表面经老化处理后对体外变链生物膜和全菌生物膜形成的影响。
2.7扫描电镜观察改性粘接剂表面变链生物膜和全菌生物膜初步形成和成熟过程,验证改性材料对生物膜形成的影响。
2.8应用荧光指示剂和激光共聚焦扫描显微镜研究改性材料表面变链生物膜和全菌生物膜的三维活性结构。
2.9改性材料表面培养变链生物膜,收集生物膜变链和孵育液中浮游变链,进行RNA抽提和实时定量RT-PCR分析,检测改性材料表面生物膜变链和孵育液中浮游变链的gtf基因表达水平,初步探索固定于高分子网络的季铵盐基团抑制生物膜形成的机理。
3主要研究结果:
3.1固化后的DMAE-CB改性粘接剂可抑制表面变形链球菌的生长,生长抑制率约为99%(P < 0.05)。
3.2老化处理或唾液处理后,改性材料表面仍表现出抗菌活性:细菌生长抑制率分别约为99% (P < 0.05)和90% (P < 0.05)。
3.3改性粘接剂的浸提液无抗菌活性:实验组和阴性对照组浸提液中变形链球菌指数生长期拟合直线的斜率及细菌的倍增时间均无显著差异(P > 0.05)。
3.4荧光染色标记后,细胞膜完整的细菌染为绿色,胞膜受损的细菌染为红色。阴性对照粘接剂固化表面细菌密集;改性粘接剂和阳性对照粘接剂表面细菌附着较少。
3.5荧光强度分析显示各组的总荧光强度FIt和红绿荧光强度比值FIr/FIg的差异显著(P < 0.05);阴性对照组的FIt值最高而FIr/FIg最低(P < 0.05),提示改性粘接剂组和阳性对照可以损伤变形链球菌胞膜的完整性,减少细菌粘附。
3.6同阴性对照相比,改性粘接剂和阳性对照组的固化表面可抑制表面变链生物膜和全菌生物膜的形成(P < 0.05)。
3.7老化处理后改性材料和阳性对照的表面,变链生物膜和全菌生物膜形成量较老化前均无显著提高(P > 0.05),仍表现出抑制表面生物膜形成的作用(P < 0.05)。
3.8扫描电镜观察发现:改性粘接剂表面变链生物膜孵育4 h,可见变链和细胞外基质散在分布;孵育24 h,形成松散的变链生物膜。改性粘接剂表面的全菌生物膜孵育4 h,可见细菌残骸;孵育24 h后,形成全菌生物膜。
3.9改性粘接剂表面的变链生物膜和全菌生物膜的厚度和总体活性均较阴性对照组下降。
3.10粘接剂表面生物膜变链的gtfBCD基因表达,三组之间有所差异:阳性对照组和改性粘接剂组的生物膜变链gtfB表达分别下降为阴性对照的23%和8.6% (P < 0.05),gtfC的表达分别下降为阴性对照的35.9 %和14.1 % (P < 0.05);而阳性对照组和改性粘接剂组gtfD的表达同阴性对照之间没有显著差异(P > 0.05)。
3.11试件孵育液中的浮游态细菌的gtfB、gtfC和gtfD的RNA拷贝数,三组之间均无显著差异(P > 0.05)。
4主要结论:
4.1 DMAE-CB改性粘接剂固化后可以抑制表面细菌生长和粘附。
4.2老化处理后DMAE-CB改性材料仍表现出抗菌活性,提示季铵盐单体改性牙本质粘接剂可以长期发挥抗菌作用。
4.3唾液预处理后DMAE-CB改性材料仍表现出抗菌活性,提示季铵盐单体改性粘接剂可在口腔环境中发挥抗菌作用,具有临床应用前景。
4.4排除了抗菌单体从改性粘接剂固化样本中溶出发挥抗菌作用的可能,明确了DMAE-CB改性粘接剂固化后具有接触性抗菌性能。
4.5 DMAE-CB改性粘接剂可以影响所接触细菌的胞膜完整性,由此部分解释固定于树脂基质中阳离子抗菌基团的抗菌机制。
4.6 DMAE-CB改性粘接剂和含有抗菌单体MDPB的阳性对照粘接剂的固化表面可以抑制变形链球菌生物膜和全菌生物膜的形成。
4.7老化处理后,DMAE-CB改性粘接剂和阳性对照的固化表面对变形链球菌生物膜和全菌生物膜形成的抑制作用无明显衰减。
4.8 DMAE-CB改性粘接剂表面可下调变链生物膜和全菌生物膜的活性。
4.9 DMAE-CB改性粘接剂和阳性对照的固化表面可以选择性下调生物膜变链的gtf基因表达,由此部分解释固定于树脂基质中阳离子抗菌基团抑制变链生物膜形成的机制。
4.10抗菌单体改性材料表面对gtf基因表达的影响局限于材料表面的变链生物膜,排除了抗菌单体从粘接样本中溶出发挥抗菌作用的可能,明确了DMAE-CB改性粘接剂具有接触性抗菌性能。
综上,可聚合季铵盐抗菌单体DMAE-CB可以用于牙本质粘接剂抗菌改性,改性材料固化后,阳离子抗菌基团固定于树脂基质网络,影响变形链球菌生长、粘附、胞膜完整性,以及影响生物膜的形成和活性,选择性下调变形链球菌gtf基因表达水平,发挥抗菌、抑制生物膜形成的作用,赋予粘接剂持久稳定的接触性抗菌活性。
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.
引文
[1]. Skjorland KK. Plaque accumulation on different dental filling materials. Scand J Dent Res 1973;81:538-42.
[2]. Skjorland KK. Bacterial accumulation on silicate and composite materials. J Biol Buccale 1976;4:315-22.
[3]. Weitman RT, Eames WB. Plaque accumulation on composite surfaces after various finising procedures. J Am Dent Assoc 1975;91:101-6.
[4]. Skjorland KK, Sonju T. Effect of sucrose rinses on bacterial colonization on amalgam and composite. Acta Odontol Scand 1982;40:193-6.
[5]. Svanberg M, Mjor IA, Orstavik D. Mutans streptococci in plaque from margins of amalgam, composite, and glass-ionomer restorations. J Dent Res 1990;69:861-4.
[6]. Beyth N, Domb AJ, Weiss EI. An in vitro quantitative antibacterial analysis of amalgam and composite resins. J Dent 2007;35:201-6.
[7]. Fontana M, Gonzalez-Cabezas C. Secondary caries and restoration replacement: an unresolved problem. Compend Contin Educ Dent 2000;21:15-8, 21-4, 6 passim; quiz 30.
[8]. Mjor IA, Moorhead JE, Dahl JE. Reasons for replacement of restorations in permanent teeth in general dental practice. Int Dent J 2000;50:361-6.
[9]. Syafiuddin T, Hisamitsu H, Toko T, Igarashi T, Goto N, Fujishima A, Miyazaki T. In vitro inhibition of caries around a resin composite restoration containing antibacterial filler. Biomaterials 1997;18:1051-7.
[10]. Yamamoto K, Ohashi S, Aono M, Kokubo T, Yamada I, Yamauchi J. Antibacterial activity of silver ions implanted in SiO2 filler on oral streptococci. Dent Mater 1996;12:227-9.
[11]. Yap AU, Khor E, Foo SH. Fluoride release and antibacterial properties of new-generation tooth-colored restoratives. Oper Dent 1999;24:297-305.
[12]. Syafiuddin T, Igarashi T, Shimomura H, Hisamitsu H, Goto N. Bacteriological and mechanical evaluation of antibacterial filler containing composite resins. J Showa Univ Dent Soc 1993;13:443-9.
[13]. Syafiuddin T, Igarashi T, Toko T, Hisamitsu H, Goto N. Bacteriological and mechanical evaluation of resin composites containing antibacterial filler (Apacider). J Showa Univ Dent Soc 1995;15:119-25.
[14]. Imazato S, Torii M, Tsuchitani Y, McCabe JF, Russell RR. Incorporation of bacterial inhibitor into resin composite. J Dent Res 1994;73:1437-43.
[15]. Imazato S, McCabe JF. Influence of incorporation of antibacterial monomeron curing behavior of a dental composite. J Dent Res 1994;73:1641-5.
[16]. Imazato S, Russell RR, McCabe JF. Antibacterial activity of MDPB polymer incorporated in dental resin. J Dent 1995;23:177-81.
[17]. Xiao YH, Chen JH, Fang M, Xing XD, Wang H, Wang YJ, Li F. Antibacterial effects of three experimental quaternary ammonium salt (QAS) monomers on bacteria associated with oral infections. Journal of Oral Science 2008;50:323-7.
[18].肖玉鸿,陈吉华,沈丽娟,方明,邢晓东,王辉,王迎捷.三种可聚合季铵盐单体对口腔病原菌的抗菌活性.实用口腔医学杂志2008;24:189-92.
[19]. Boston DW, Graver HT. Histobacteriological analysis of acid red dye-stainable dentin found beneath intact amalgam restorations. Oper Dent 1994;19:65-9.
[20]. Kidd EA, Joyston-Bechal S, Beighton D. The use of a caries detector dye during cavity preparation: a microbiological assessment. Br Dent J 1993;174:245-8.
[21]. Bergenholtz G, Cox CF, Loesche WJ, Syed SA. Bacterial leakage around dental restorations: its effect on the dental pulp. J Oral Pathol 1982;11:439-50.
[22]. Qvist V. Resin restorations: leakage, bacteria, pulp. Endod Dent Traumatol 1993;9:127-52.
[23]. Schmalz G. Der Einfluss verschiedener Frontzahnfullungsmaterialien auf das In-vitro-Wachstum von Streptococcus mutans [The effect of various front tooth filling materials on the in vitro growth of Streptococcus mutans]. Dtsch Zahnarztl Z 1977;32:575-9.
[24]. Jedrychowski JR, Caputo AA, Kerper S. Antibacterial and mechanical properties of restorative materials combined with chlorhexidines. J Oral Rehabil 1983;10:373-81.
[25]. Takemura K SY, Staninec M. Antibacterial activity of a bis-GMA based composite resin and antibacterial effect of chlorhexidine incorporation. Jpn J Conserv Dent 1983;26:540-7.
[26]. Nurit Beyth AJD, Ervin I. Weiss. An in vitro quantitative antibacterial analysis of amalgam and composite resins. J Dent 2007;35:201-6.
[27]. Kawai K. Effects of eluate from composite resins on Gtase and growth of Streptococcus mutans. Jpn J Conserv Dent 1988;31:332-51.
[28]. Addy M, Thaw M. In vitro studies into the release of chlorhexidine acetate, prednisolone sodium phosphate, and prednisolone alcohol from cold cure denture base acrylic. J Biomed Mater Res 1982;16:145-57.
[29]. Wilson SJ, Wilson HJ. The release of chlorhexidine from modified dental acrylic resin. J Oral Rehabil 1993;20:311-9.
[30]. Forss H, Jokinen J, Spets-Happonen S, Seppa L, Luoma H. Fluoride and mutans streptococci in plaque grown on glass ionomer and composite. Caries Res 1991;25:454-8.
[31]. Xu X, Burgess JO. Compressive strength, fluoride release and recharge of fluoride-releasing materials. Biomaterials 2003;24:2451-61.
[32]. Arends J, Dijkman GE, Dijkman AG. Review of fluoride release and secondary caries reduction by fluoridating composites. Adv Dent Res 1995;9:367-76.
[33]. Hicks J, Garcia-Godoy F, Donly K, Flaitz C. Fluoride-releasing restorative materials and secondary caries. J Calif Dent Assoc 2003;31:229-45.
[34]. Karantakis P, Helvatjoglou-Antoniades M, Theodoridou Pahini S, Papadogiannis Y. Fluoride release from three glass ionomers, a compomer, and a composite resin in water, artificial saliva, and lactic acid. Oper Dent 2000;25:20-5.
[35]. Vermeersch G, Leloup G, Vreven J. Fluoride release from glass-ionomer cements, compomers and resin composites. J Oral Rehabil 2001;28:26-32.
[36]. Asmussen E, Peutzfeldt A. Long-term fluoride release from a glass ionomer cement, a compomer, and from experimental resin composites. Acta Odontol Scand 2002;60:93-7.
[37]. Preston AJ, Agalamanyi EA, Higham SM, Mair LH. The recharge of esthetic dental restorative materials with fluoride in vitro-two years' results. Dent Mater 2003;19:32-7.
[38]. Vermeersch G, Leloup G, Delmee M, Verven J. Antibacterial activity of glass-ionomer cements, compomers and resin composites: relationship between acidity and material setting phase. J Oral Rehabil 2005;32:368-74.
[39]. Uchida M. Antimicrobial zeolite and its application. Chem Ind 1995;46:48-54.
[40]. Grier N. Silver and its compounds, disinfection, sterilization and preservation. Philadelphia: Lea and Febiger 1983:375-89.
[41]. Schreurs WJ, Rosenberg H. Effect of silver ions on transport and retention of phosphate by Escherichia coli. J-Bacteriol 1982;152:7-13.
[42]. Modak SM, Fox CL, Jr. Binding of silver sulfadiazine to the cellular components of Pseudomonas aeruginosa. Biochem Pharmacol 1973;22:2391-404.
[43]. Ghandour W, Hubbard JA, Deistrung J, Hughes MN, Poole RK. The uptake of silver ions by Escherichia coli: toxic effects and interactions with copper ions. Appl Microbiol Biotechnol. 1988;28:559-65.
[44]. Bragg PD, Rainnie DJ. The effect of silver ions on the respiratory chain of Escherichia coli. Can J Microbiol 1974;20:883-9.
[45]. Alt V, Bechert T, Steinrucke P, Wagener M, Seidel P, Dingeldein E, Domann E, Schnettler R. An in vitro assessment of the antibacterial properties and cytotoxicity of nanoparticulate silver bone cement. Biomaterials 2004;25:4383–91.
[46]. Kawashita M, Tsuneyama S, Miyaji F, Kokubo T, Kozuka H, Yamamoto K. Antibacterial silver-containing silica glass prepared by sol-gel method.Biomaterials 2000;21:393-8.
[47]. Kawahara K, Tsuruda K, Morishita M, Uchida M. Antibacterial effect of silver-zeolite on oral bacteria under anaerobic conditions. Dent Mater 2000;16:452-5.
[48]. Imazato S, Ebi N, Tarumi H, Russell RR, Kaneko T, Ebisu S. Bactericidal activity and cytotoxicity of antibacterial monomer MDPB. Biomaterials 1999;20:899-903.
[49]. Imazato S, Tarumi H, Kato S, Ebisu S. Water sorption and colour stability of composites containing the antibacterial monomer MDPB. J Dent 1999;27:279-83.
[50]. Franklin TJ, Snow GA. Biochemistry of Antimicrobial Action. London: Chapman&Hall; 1981. p. 58.
[51]. Ebi N, Imazato S, Noiri Y, Ebisu S. Inhibitory effects of resin composite containing bactericide-immobilized filler on plaque accumulation. Dent Mater 2001;17:485-91.
[52]. Beyth N, Yudovin-Farber I, Bahir R, Domb AJ, Weiss EI. Antibacterial activity of dental composites containing quaternary ammonium polyethylenimine nanoparticles against Streptococcus mutans. Biomaterials 2006;27:3995-4002.
[53]. Yoshida K, Tanagawa M, Atsuta M. Characterization and inhibitory effect of antibacterial dental resin composites incorporating silver-supported materials. J Biomed Mater Res 1999;47:516-22.
[54]. Tanagawa M, Yoshida K, Matsumoto S, Yamada T, Atsuta M. Inhibitory effect of antibacterial resin composite against Streptococcus mutans. Caries Res 1999;33:366-71.
[55]. Brannstrom M. Infection beneath composite resin restorations: can it be avoided? Oper Dent 1987;12:158-63.
[56]. Settembrini L, Boylan R, Strassler H, Scherer W. A comparison of antimicrobial activity of etchants used for a total etch technique. Oper Dent 1997;22:84-8.
[57]. Luglie PF, Delitala PP, Zanetti S, Sanna S. Studio batteriologico in vivo sugli effetti della mordenzatura acida nei fondi cavitari.[An in-vivo bacteriological study on the effects of acid etching at the bottom of cavities]. Minerva Stomatol 1998;47:19-26.
[58]. Walshaw PR, McComb D. SEM evaluation of the resin-dentin interface with proprietary bonding agents in human subjects. J Dent Res 1994;73:1079-87.
[59]. Goracci G, Mori G, Bazzucchi M. Marginal seal and biocompatibility of a fourth-generation bonding agent. Dent Mater 1995;11:343-7.
[60]. Perdigao J, Lambrechts P, Van-Meerbeek B, Braem M, Yildiz E, Yucel T,Vanherle G. The interaction of adhesive systems with human dentin. Am J Dent 1996;9:167-73.
[61]. Emilson CG, Bergenholtz G. Antibacterial activity of dentinal bonding agents. Quintessence Int 1993;24:511-5.
[62]. Prati C, Fava F, Di Gioia D, Selighini M, Pashley DH. Antibacterial effectiveness of dentin bonding systems. Dent Mater 1993;9:338-43.
[63]. Meiers JC, Miller GA. Antibacterial activity of dentin bonding systems, resin-modified glass ionomers, and polyacid-modified composite resins. Oper Dent 1996;21:257-64.
[64]. Imazato S, Imai T, Ebisu S. Antibacterial activity of proprietary self-etching primers. Am J Dent 1998;11:106-8.
[65]. Herrera M, Carrion P, Bravo M, Castillo A. Antibacterial activity of four dentin bonding systems. Int J Antimicrob Agents 2000;15:305-9.
[66]. Schmidlin PR, Zehnder M, Gohring TN, Waltimo TM. Glutaraldehyde in bonding systems disinfects dentin in vitro. J Adhes Dent 2004;6:61-4.
[67]. Baseren M, Yazici AR, Ozalp M, Dayangac B. Antibacterial activity of different generation dentin-bonding systems. Quintessence Int 2005;36:339-44.
[68]. Ohmori K, Maeda N, Kohno A. Evaluation of antibacterial activity of three dentin primers using an in vitro tooth model. Oper Dent 1999;24:279-85.
[69]. Imazato S, Kuramoto A, Kaneko T, Ebisu S, Russell RR. Comparison of antibacterial activity of simplified adhesive systems. Am J Dent 2002;15:356-60.
[70]. Bapna MS, Murphy R, Mukherjee S. Inhibition of bacterial colonization by antimicrobial agents incorporated into dental resins. J Oral Rehabil 1988;15:405-11.
[71]. Ozer F, Unlu N, Karakaya S, Ergani O, Hadimli HH. Antibacterial activities of MDPB and fluoride in dentin bonding agents. Eur J Prosthodont Restor Dent 2005;13:139-42.
[72]. Kudou Y, Obara K, Kawashima T, Kubota M, Abe S, Endo T, Komatsu M, Okuda R. Addition of antibacterial agents to MMA-TBB dentin bonding systems--influence on tensile bond strength and antibacterial effect. Dent Mater J 2000;19:65-74.
[73]. Kazuno T, Fukushima T, Hayakawa T, Inoue Y, Ogura R, Kaminishi H, Miyazaki K. Antibacterial activities and bonding of MMSA/TBB resin containing amphiphilic lipids. Dent Mater J 2005;24:244-50.
[74]. Imazato S, Kinomoto Y, Tarumi H, Torii M, Russell RR, McCabe JF. Incorporation of antibacterial monomer MDPB into dentin primer. J Dent Res 1997;76:768-72.
[75]. Imazato S, Ehara A, Torii M, Ebisu S. Antibacterial activity of dentine primer containing MDPB after curing. J Dent 1998;26:267-71.
[76]. Imazato S, Torii Y, Takatsuka T, Inoue K, Ebi N, Ebisu S. Bactericidal effect of dentin primer containing antibacterial monomer methacryloyloxydodecylpyridinium bromide (MDPB) against bacteria in human carious dentin. J Oral Rehabil 2001;28:314-9.
[77]. Imazato S, Kinomoto Y, Tarumi H, Ebisu S, Tay FR. Antibacterial activity and bonding characteristics of an adhesive resin containing antibacterial monomer MDPB. Dent Mater 2003;19:313-9.
[78]. Imazato S, Kuramoto A, Takahashi Y, Ebisu S, Peters MC. In vitro antibacterial effects of the dentin primer of Clearfil Protect Bond. Dent Mater 2006;22:527-32.
[79]. Imazato S, Tay FR, Kaneshiro AV, Takahashi Y, Ebisu S. An in vivo evaluation of bonding ability of comprehensive antibacterial adhesive system incorporating MDPB. Dent Mater 2007;23:170-6.
[80]. Tziafas D, Koliniotou-Koumpia E, Tziafa C, Papadimitriou S. Effects of a new antibacterial adhesive on the repair capacity of the pulp-dentine complex in infected teeth. Int Endod J 2007;40:58-66.
[81]. Atac AS, Cehreli ZC, Sener B. Antibacterial activity of fifth-generation dentin bonding systems. J Endod 2001;27:730-3.
[82]. Herrera M, Castillo A, Bravo M, Liebana J, Carrion P. Antibacterial activity of resin adhesives, glass ionomer and resin-modified glass ionomer cements and a compomer in contact with dentin caries samples. Oper Dent 2000;25:265-9.
[83]. Takahashi Y, Imazato S, Kaneshiro AV, Ebisu S, Frencken JE, Tay FR. Antibacterial effects and physical properties of glass-ionomer cements containing chlorhexidine for the ART approach. Dent Mater 2006;22:647-52.
[84]. Imazato S, Imai T, Russell RR, Torii M, Ebisu S. Antibacterial activity of cured dental resin incorporating the antibacterial monomer MDPB and an adhesion-promoting monomer. J Biomed Mater Res 1998;39:511-5.
[85]. Imazato S, Ebi N, Takahashi Y, Kaneko T, Ebisu S, Russell RR. Antibacterial activity of bactericide-immobilized filler for resin-based restoratives. Biomaterials 2003;24:3605-9.
[86]. Ozer F, Karakaya S, Unlu N, Erganis O, Kav K, Imazato S. Comparison of antibacterial activity of two dentin bonding systems using agar well technique and tooth cavity model. J Dent 2003;31:111-6.
[87]. Costerton JW, Lewandowski Z, DeBeer D, Caldwell D, Korber D, James G. Biofilms, the customized microniche. J Bacteriol 1994;176:2137-42.
[88]. Costerton JW, Lewandowski Z, Caldwell DE, Korber DR, Lappin-Scott HM.Microbial biofilms. Annu Rev Microbiol 1995;49:711-45.
[89]. Bradshaw DJ, Marsh PD, Hodgson RJ, Visser JM. Effects of glucose and fluoride on competition and metabolism within in vitro dental bacterial communities and biofilms. Caries Res 2002;36:81-6.
[90]. Guggenheim B, Guggenheim M, Gmur R, Giertsen E, Thurnheer T. Application of the Zurich biofilm model to problems of cariology. Caries Res 2004;38:212-22.
[91]. Filoche SK, Soma KJ, Sissons CH. Caries-related plaque microcosm biofilms developed in microplates. Oral Microbiol Immunol 2007;22:73-9.
[92]. Marsh PD, Hunter JR, Bowden GH, Hamilton IR, McKee AS, Hardie JM, Ellwood DC. The influence of growth rate and nutrient limitation on the microbial composition and biochemical properties of a mixed culture of oral bacteria grown in a chemostat. J Gen Microbiol 1983;129:755-70.
[93]. Marsh PD. Are dental diseases examples of ecological catastrophes? Microbiology 2003;149:279-94.
[94]. McBain AJ, Sissons C, Ledder RG, Sreenivasan PK, De Vizio W, Gilbert P. Development and characterization of a simple perfused oral microcosm. J Appl Microbiol 2005;98:624-34.
[95]. Pigman W, Elliott HC, Jr., Laffre RO. An artificial mouth for caries research. J Dent Res 1952;31:627-33.
[96]. Coulter WA, Russell C. A miniature continuous culture system for controlled production of simulated bacterial dental plaque. Arch Oral Biol 1976;21:333-4.
[97]. Coulter WA, Russell C. pH and Eh in single and mixed culture bacterial plaque in an artificial mouth. J Appl Bacteriol 1976;40:73-87.
[98]. Russell C, Coulter WA. Continuous monitoring of pH and Eh in bacterial plaque grown on a tooth in an artificial mouth. Appl Microbiol 1975;29:141-4.
[99]. Shu M, Wong L, Miller JH, Sissons CH. Development of multi-species consortia biofilms of oral bacteria as an enamel and root caries model system. Arch Oral Biol 2000;45:27-40.
[100]. Pearce EI, Wakefield JS, Sissons CH. Therapeutic mineral enrichment of dental plaque visualized by transmission electron microscopy. J Dent Res 1991;70:90-4.
[101]. Sissons CH, Cutress TW, Hoffman MP, Wakefield JS. A multi-station dental plaque microcosm (artificial mouth) for the study of plaque growth, metabolism, pH, and mineralization. J Dent Res 1991;70:1409-16.
[102]. Bibby BG, Huang CT, Zero D, Mundorff SA, Little MF. Protective effect of milk against in vitro caries. J Dent Res 1980;59:1565-70.
[103]. Noorda WD, Purdell-Lewis DJ, van Montfort AM, Weerkamp AH. Developmentaland metabolic aspects of a monobacterial plaque of Streptococcus mutans C 67-1 grown on human enamel slabs in an artificial mouth model. II. Enamel data. Caries Res 1986;20:308-14.
[104]. Noorda WD, Purdell-Lewis DJ, van Montfort AM, Weerkamp AH. Monobacterial and mixed bacterial plaques of Streptococcus mutans and Veillonella alcalescens in an artificial mouth: development, metabolism, and effect on human dental enamel. Caries Res 1988;22:342-7.
[105]. Noorda WD, van Montfort AM, Purdell-Lewis DJ, Weerkamp AH. Developmental and metabolic aspects of a monobacterial plaque of Streptococcus mutans C 67-1 grown on human enamel slabs in an artificial mouth model. I. Plaque data. Caries Res 1986;20:300-7.
[106].卢滇楠,周轩榕,邢晓东.表面接枝季铵盐型聚合物的纤维素纤维-灭菌机理研究.高分子学报2004;14:107-13.
[107]. Sumitomo T, Nagamune H, Maeda T, Kourai H. Correlation between the bacterioclastic action of a bis-quaternary ammonium compound and outer membrane proteins. Biocontrol Sci 2006;11:115-24.
[108]. Yoshimatsu T, Hiyama K. Mechanism of the action of didecyldimethylammonium chloride (DDAC) against Escherichia coil and morphological changes of the cells. Biocontrol Sci 2007;12:93-9.
[109]. Beyth N, Domb AJ, Weiss EI. An in vitro quantitative antibacterial analysis of amalgam and composite resins. J Dent 2007;35:201-6.
[110]. Khalichi P, Cvitkovitch DG, Santerre JP. Effect of composite resin biodegradation products on oral streptococcal growth. Biomaterials 2004;25:5467-72.
[111]. Filoche SK, Coleman MJ, Angker L, Sissons CH. A fluorescence assay to determine the viable biomass of microcosm dental plaque biofilms. J Microbiol Methods 2007;69:489-96.
[112]. van der Mei HC, White DJ, Atema-Smit J, van de Belt-Gritter E, Busscher HJ. A method to study sustained antimicrobial activity of rinse and dentifrice components on biofilm viability in vivo. J Clin Periodontol 2006;33:14-20.
[113]. Montanaro L, Campoccia D, Rizzi S, Donati ME, Breschi L, Prati C, Arciola CR. Evaluation of bacterial adhesion of Streptococcus mutans on dental restorative materials. Biomaterials 2004;25:4457-63.
[114]. Ahlstrom B, Chelminska-Bertilsson M, Thompson RA, Edebo L. Long-chain alkanoylcholines, a new category of soft antimicrobial agents that are enzymatically degradable. Antimicrob Agents Chemother 1995;39:50-5.
[115]. Calas M, Ancelin ML, Cordina G, Portefaix P, Piquet G, Vidal-Sailhan V, Vial H. Antimalarial activity of compounds interfering with Plasmodium falciparum phospholipid metabolism: comparison between mono- andbisquaternary ammonium salts. J Med Chem 2000;43:505-16.
[116]. Lindstedt M, Allenmark S, Thompson RA, Edebo L. Antimicrobial activity of betaine esters, quaternary ammonium amphiphiles which spontaneously hydrolyze into nontoxic components. Antimicrob Agents Chemother 1990;34:1949-54.
[117]. Ajdic D, McShan WM, McLaughlin RE, Savic G, Chang J, Carson MB, Primeaux C, Tian R, Kenton S, Jia H, Lin S, Qian Y, Li S, Zhu H, Najar F, Lai H, White J, Roe BA, Ferretti JJ. Genome sequence of Streptococcus mutans UA159, a cariogenic dental pathogen. Proc Natl Acad Sci U S A 2002;99:14434-9.
[118]. Feuerstein O, Matalon S, Slutzky H, Weiss EI. Antibacterial properties of self-etching dental adhesive systems. J Am Dent Assoc 2007;138:349-54; quiz 96-8.
[119]. Hope CK, Wilson M. Analysis of the effects of chlorhexidine on oral biofilm vitality and structure based on viability profiling and an indicator of membrane integrity. Antimicrob Agents Chemother 2004;48:1461-8.
[120]. Rolland SL, McCabe JF, Robinson C, Walls AW. In vitro biofilm formation on the surface of resin-based dentine adhesives. Eur J Oral Sci 2006;114:243-9.
[121]. Hamada S, Slade HD. Biology, immunology, and cariogenicity of Streptococcus mutans. Microbiol Rev 1980;44:331-84.
[122]. Loesche WJ. Role of Streptococcus mutans in human dental decay. Microbiol Rev 1986;50:353-80.
[123]. Yamashita Y, Bowen WH, Burne RA, Kuramitsu HK. Role of the Streptococcus mutans gtf genes in caries induction in the specific-pathogen-free rat model. Infect Immun 1993;61:3811-7.
[124]. Kuramitsu HK. Virulence factors of mutans streptococci: role of molecular genetics. Crit Rev Oral Biol Med 1993;4:159-76.
[125]. Fujiwara T, Hoshino T, Ooshima T, Hamada S. Differential and quantitative analyses of mRNA expression of glucosyltransferases from Streptococcus mutans MT8148. J Dent Res 2002;81:109-13.
[126]. Shemesh M, Tam A, Steinberg D. Differential gene expression profiling of Streptococcus mutans cultured under biofilm and planktonic conditions. Microbiology 2007;153:1307-17.
[127]. Shemesh M, Tam A, Feldman M, Steinberg D. Differential expression profiles of Streptococcus mutans ftf, gtf and vicR genes in the presence of dietary carbohydrates at early and late exponential growth phases. Carbohydr Res 2006;341:2090-7.
[128]. Cury JA, Koo H. Extraction and purification of total RNA from Streptococcus mutans biofilms. Anal Biochem 2007;365:208-14.
[129]. Ooshima T, Matsumura M, Hoshino T, Kawabata S, Sobue S, Fujiwara T.Contributions of three glycosyltransferases to sucrose-dependent adherence of Streptococcus mutans. J Dent Res 2001;80:1672-7.
[130]. Li Y, Burne RA. Regulation of the gtfBC and ftf genes of Streptococcus mutans in biofilms in response to pH and carbohydrate. Microbiology 2001;147:2841-8.
[131]. Goodman SD, Gao Q. Characterization of the gtfB and gtfC promoters from Streptococcus mutans GS-5. Plasmid 2000;43:85-98.
[132]. Chen PM, Chen JY, Chia JS. Differential regulation of Streptococcus mutans gtfBCD genes in response to copper ions. Arch Microbiol 2006;185:127-35.
[133]. Koo H, Seils J, Abranches J, Burne RA, Bowen WH, Quivey RG, Jr. Influence of apigenin on gtf gene expression in Streptococcus mutans UA159. Antimicrob Agents Chemother 2006;50:542-6.
[2]. Skjorland KK. Bacterial accumulation on silicate and composite materials. J Biol Buccale 1976;4:315-22.
[3]. Weitman RT, Eames WB. Plaque accumulation on composite surfaces after various finising procedures. J Am Dent Assoc 1975;91:101-6.
[4]. Skjorland KK, Sonju T. Effect of sucrose rinses on bacterial colonization on amalgam and composite. Acta Odontol Scand 1982;40:193-6.
[5]. Svanberg M, Mjor IA, Orstavik D. Mutans streptococci in plaque from margins of amalgam, composite, and glass-ionomer restorations. J Dent Res 1990;69:861-4.
[6]. Beyth N, Domb AJ, Weiss EI. An in vitro quantitative antibacterial analysis of amalgam and composite resins. J Dent 2007;35:201-6.
[7]. Fontana M, Gonzalez-Cabezas C. Secondary caries and restoration replacement: an unresolved problem. Compend Contin Educ Dent 2000;21:15-8, 21-4, 6 passim; quiz 30.
[8]. Mjor IA, Moorhead JE, Dahl JE. Reasons for replacement of restorations in permanent teeth in general dental practice. Int Dent J 2000;50:361-6.
[9]. Syafiuddin T, Hisamitsu H, Toko T, Igarashi T, Goto N, Fujishima A, Miyazaki T. In vitro inhibition of caries around a resin composite restoration containing antibacterial filler. Biomaterials 1997;18:1051-7.
[10]. Yamamoto K, Ohashi S, Aono M, Kokubo T, Yamada I, Yamauchi J. Antibacterial activity of silver ions implanted in SiO2 filler on oral streptococci. Dent Mater 1996;12:227-9.
[11]. Yap AU, Khor E, Foo SH. Fluoride release and antibacterial properties of new-generation tooth-colored restoratives. Oper Dent 1999;24:297-305.
[12]. Syafiuddin T, Igarashi T, Shimomura H, Hisamitsu H, Goto N. Bacteriological and mechanical evaluation of antibacterial filler containing composite resins. J Showa Univ Dent Soc 1993;13:443-9.
[13]. Syafiuddin T, Igarashi T, Toko T, Hisamitsu H, Goto N. Bacteriological and mechanical evaluation of resin composites containing antibacterial filler (Apacider). J Showa Univ Dent Soc 1995;15:119-25.
[14]. Imazato S, Torii M, Tsuchitani Y, McCabe JF, Russell RR. Incorporation of bacterial inhibitor into resin composite. J Dent Res 1994;73:1437-43.
[15]. Imazato S, McCabe JF. Influence of incorporation of antibacterial monomeron curing behavior of a dental composite. J Dent Res 1994;73:1641-5.
[16]. Imazato S, Russell RR, McCabe JF. Antibacterial activity of MDPB polymer incorporated in dental resin. J Dent 1995;23:177-81.
[17]. Xiao YH, Chen JH, Fang M, Xing XD, Wang H, Wang YJ, Li F. Antibacterial effects of three experimental quaternary ammonium salt (QAS) monomers on bacteria associated with oral infections. Journal of Oral Science 2008;50:323-7.
[18].肖玉鸿,陈吉华,沈丽娟,方明,邢晓东,王辉,王迎捷.三种可聚合季铵盐单体对口腔病原菌的抗菌活性.实用口腔医学杂志2008;24:189-92.
[19]. Boston DW, Graver HT. Histobacteriological analysis of acid red dye-stainable dentin found beneath intact amalgam restorations. Oper Dent 1994;19:65-9.
[20]. Kidd EA, Joyston-Bechal S, Beighton D. The use of a caries detector dye during cavity preparation: a microbiological assessment. Br Dent J 1993;174:245-8.
[21]. Bergenholtz G, Cox CF, Loesche WJ, Syed SA. Bacterial leakage around dental restorations: its effect on the dental pulp. J Oral Pathol 1982;11:439-50.
[22]. Qvist V. Resin restorations: leakage, bacteria, pulp. Endod Dent Traumatol 1993;9:127-52.
[23]. Schmalz G. Der Einfluss verschiedener Frontzahnfullungsmaterialien auf das In-vitro-Wachstum von Streptococcus mutans [The effect of various front tooth filling materials on the in vitro growth of Streptococcus mutans]. Dtsch Zahnarztl Z 1977;32:575-9.
[24]. Jedrychowski JR, Caputo AA, Kerper S. Antibacterial and mechanical properties of restorative materials combined with chlorhexidines. J Oral Rehabil 1983;10:373-81.
[25]. Takemura K SY, Staninec M. Antibacterial activity of a bis-GMA based composite resin and antibacterial effect of chlorhexidine incorporation. Jpn J Conserv Dent 1983;26:540-7.
[26]. Nurit Beyth AJD, Ervin I. Weiss. An in vitro quantitative antibacterial analysis of amalgam and composite resins. J Dent 2007;35:201-6.
[27]. Kawai K. Effects of eluate from composite resins on Gtase and growth of Streptococcus mutans. Jpn J Conserv Dent 1988;31:332-51.
[28]. Addy M, Thaw M. In vitro studies into the release of chlorhexidine acetate, prednisolone sodium phosphate, and prednisolone alcohol from cold cure denture base acrylic. J Biomed Mater Res 1982;16:145-57.
[29]. Wilson SJ, Wilson HJ. The release of chlorhexidine from modified dental acrylic resin. J Oral Rehabil 1993;20:311-9.
[30]. Forss H, Jokinen J, Spets-Happonen S, Seppa L, Luoma H. Fluoride and mutans streptococci in plaque grown on glass ionomer and composite. Caries Res 1991;25:454-8.
[31]. Xu X, Burgess JO. Compressive strength, fluoride release and recharge of fluoride-releasing materials. Biomaterials 2003;24:2451-61.
[32]. Arends J, Dijkman GE, Dijkman AG. Review of fluoride release and secondary caries reduction by fluoridating composites. Adv Dent Res 1995;9:367-76.
[33]. Hicks J, Garcia-Godoy F, Donly K, Flaitz C. Fluoride-releasing restorative materials and secondary caries. J Calif Dent Assoc 2003;31:229-45.
[34]. Karantakis P, Helvatjoglou-Antoniades M, Theodoridou Pahini S, Papadogiannis Y. Fluoride release from three glass ionomers, a compomer, and a composite resin in water, artificial saliva, and lactic acid. Oper Dent 2000;25:20-5.
[35]. Vermeersch G, Leloup G, Vreven J. Fluoride release from glass-ionomer cements, compomers and resin composites. J Oral Rehabil 2001;28:26-32.
[36]. Asmussen E, Peutzfeldt A. Long-term fluoride release from a glass ionomer cement, a compomer, and from experimental resin composites. Acta Odontol Scand 2002;60:93-7.
[37]. Preston AJ, Agalamanyi EA, Higham SM, Mair LH. The recharge of esthetic dental restorative materials with fluoride in vitro-two years' results. Dent Mater 2003;19:32-7.
[38]. Vermeersch G, Leloup G, Delmee M, Verven J. Antibacterial activity of glass-ionomer cements, compomers and resin composites: relationship between acidity and material setting phase. J Oral Rehabil 2005;32:368-74.
[39]. Uchida M. Antimicrobial zeolite and its application. Chem Ind 1995;46:48-54.
[40]. Grier N. Silver and its compounds, disinfection, sterilization and preservation. Philadelphia: Lea and Febiger 1983:375-89.
[41]. Schreurs WJ, Rosenberg H. Effect of silver ions on transport and retention of phosphate by Escherichia coli. J-Bacteriol 1982;152:7-13.
[42]. Modak SM, Fox CL, Jr. Binding of silver sulfadiazine to the cellular components of Pseudomonas aeruginosa. Biochem Pharmacol 1973;22:2391-404.
[43]. Ghandour W, Hubbard JA, Deistrung J, Hughes MN, Poole RK. The uptake of silver ions by Escherichia coli: toxic effects and interactions with copper ions. Appl Microbiol Biotechnol. 1988;28:559-65.
[44]. Bragg PD, Rainnie DJ. The effect of silver ions on the respiratory chain of Escherichia coli. Can J Microbiol 1974;20:883-9.
[45]. Alt V, Bechert T, Steinrucke P, Wagener M, Seidel P, Dingeldein E, Domann E, Schnettler R. An in vitro assessment of the antibacterial properties and cytotoxicity of nanoparticulate silver bone cement. Biomaterials 2004;25:4383–91.
[46]. Kawashita M, Tsuneyama S, Miyaji F, Kokubo T, Kozuka H, Yamamoto K. Antibacterial silver-containing silica glass prepared by sol-gel method.Biomaterials 2000;21:393-8.
[47]. Kawahara K, Tsuruda K, Morishita M, Uchida M. Antibacterial effect of silver-zeolite on oral bacteria under anaerobic conditions. Dent Mater 2000;16:452-5.
[48]. Imazato S, Ebi N, Tarumi H, Russell RR, Kaneko T, Ebisu S. Bactericidal activity and cytotoxicity of antibacterial monomer MDPB. Biomaterials 1999;20:899-903.
[49]. Imazato S, Tarumi H, Kato S, Ebisu S. Water sorption and colour stability of composites containing the antibacterial monomer MDPB. J Dent 1999;27:279-83.
[50]. Franklin TJ, Snow GA. Biochemistry of Antimicrobial Action. London: Chapman&Hall; 1981. p. 58.
[51]. Ebi N, Imazato S, Noiri Y, Ebisu S. Inhibitory effects of resin composite containing bactericide-immobilized filler on plaque accumulation. Dent Mater 2001;17:485-91.
[52]. Beyth N, Yudovin-Farber I, Bahir R, Domb AJ, Weiss EI. Antibacterial activity of dental composites containing quaternary ammonium polyethylenimine nanoparticles against Streptococcus mutans. Biomaterials 2006;27:3995-4002.
[53]. Yoshida K, Tanagawa M, Atsuta M. Characterization and inhibitory effect of antibacterial dental resin composites incorporating silver-supported materials. J Biomed Mater Res 1999;47:516-22.
[54]. Tanagawa M, Yoshida K, Matsumoto S, Yamada T, Atsuta M. Inhibitory effect of antibacterial resin composite against Streptococcus mutans. Caries Res 1999;33:366-71.
[55]. Brannstrom M. Infection beneath composite resin restorations: can it be avoided? Oper Dent 1987;12:158-63.
[56]. Settembrini L, Boylan R, Strassler H, Scherer W. A comparison of antimicrobial activity of etchants used for a total etch technique. Oper Dent 1997;22:84-8.
[57]. Luglie PF, Delitala PP, Zanetti S, Sanna S. Studio batteriologico in vivo sugli effetti della mordenzatura acida nei fondi cavitari.[An in-vivo bacteriological study on the effects of acid etching at the bottom of cavities]. Minerva Stomatol 1998;47:19-26.
[58]. Walshaw PR, McComb D. SEM evaluation of the resin-dentin interface with proprietary bonding agents in human subjects. J Dent Res 1994;73:1079-87.
[59]. Goracci G, Mori G, Bazzucchi M. Marginal seal and biocompatibility of a fourth-generation bonding agent. Dent Mater 1995;11:343-7.
[60]. Perdigao J, Lambrechts P, Van-Meerbeek B, Braem M, Yildiz E, Yucel T,Vanherle G. The interaction of adhesive systems with human dentin. Am J Dent 1996;9:167-73.
[61]. Emilson CG, Bergenholtz G. Antibacterial activity of dentinal bonding agents. Quintessence Int 1993;24:511-5.
[62]. Prati C, Fava F, Di Gioia D, Selighini M, Pashley DH. Antibacterial effectiveness of dentin bonding systems. Dent Mater 1993;9:338-43.
[63]. Meiers JC, Miller GA. Antibacterial activity of dentin bonding systems, resin-modified glass ionomers, and polyacid-modified composite resins. Oper Dent 1996;21:257-64.
[64]. Imazato S, Imai T, Ebisu S. Antibacterial activity of proprietary self-etching primers. Am J Dent 1998;11:106-8.
[65]. Herrera M, Carrion P, Bravo M, Castillo A. Antibacterial activity of four dentin bonding systems. Int J Antimicrob Agents 2000;15:305-9.
[66]. Schmidlin PR, Zehnder M, Gohring TN, Waltimo TM. Glutaraldehyde in bonding systems disinfects dentin in vitro. J Adhes Dent 2004;6:61-4.
[67]. Baseren M, Yazici AR, Ozalp M, Dayangac B. Antibacterial activity of different generation dentin-bonding systems. Quintessence Int 2005;36:339-44.
[68]. Ohmori K, Maeda N, Kohno A. Evaluation of antibacterial activity of three dentin primers using an in vitro tooth model. Oper Dent 1999;24:279-85.
[69]. Imazato S, Kuramoto A, Kaneko T, Ebisu S, Russell RR. Comparison of antibacterial activity of simplified adhesive systems. Am J Dent 2002;15:356-60.
[70]. Bapna MS, Murphy R, Mukherjee S. Inhibition of bacterial colonization by antimicrobial agents incorporated into dental resins. J Oral Rehabil 1988;15:405-11.
[71]. Ozer F, Unlu N, Karakaya S, Ergani O, Hadimli HH. Antibacterial activities of MDPB and fluoride in dentin bonding agents. Eur J Prosthodont Restor Dent 2005;13:139-42.
[72]. Kudou Y, Obara K, Kawashima T, Kubota M, Abe S, Endo T, Komatsu M, Okuda R. Addition of antibacterial agents to MMA-TBB dentin bonding systems--influence on tensile bond strength and antibacterial effect. Dent Mater J 2000;19:65-74.
[73]. Kazuno T, Fukushima T, Hayakawa T, Inoue Y, Ogura R, Kaminishi H, Miyazaki K. Antibacterial activities and bonding of MMSA/TBB resin containing amphiphilic lipids. Dent Mater J 2005;24:244-50.
[74]. Imazato S, Kinomoto Y, Tarumi H, Torii M, Russell RR, McCabe JF. Incorporation of antibacterial monomer MDPB into dentin primer. J Dent Res 1997;76:768-72.
[75]. Imazato S, Ehara A, Torii M, Ebisu S. Antibacterial activity of dentine primer containing MDPB after curing. J Dent 1998;26:267-71.
[76]. Imazato S, Torii Y, Takatsuka T, Inoue K, Ebi N, Ebisu S. Bactericidal effect of dentin primer containing antibacterial monomer methacryloyloxydodecylpyridinium bromide (MDPB) against bacteria in human carious dentin. J Oral Rehabil 2001;28:314-9.
[77]. Imazato S, Kinomoto Y, Tarumi H, Ebisu S, Tay FR. Antibacterial activity and bonding characteristics of an adhesive resin containing antibacterial monomer MDPB. Dent Mater 2003;19:313-9.
[78]. Imazato S, Kuramoto A, Takahashi Y, Ebisu S, Peters MC. In vitro antibacterial effects of the dentin primer of Clearfil Protect Bond. Dent Mater 2006;22:527-32.
[79]. Imazato S, Tay FR, Kaneshiro AV, Takahashi Y, Ebisu S. An in vivo evaluation of bonding ability of comprehensive antibacterial adhesive system incorporating MDPB. Dent Mater 2007;23:170-6.
[80]. Tziafas D, Koliniotou-Koumpia E, Tziafa C, Papadimitriou S. Effects of a new antibacterial adhesive on the repair capacity of the pulp-dentine complex in infected teeth. Int Endod J 2007;40:58-66.
[81]. Atac AS, Cehreli ZC, Sener B. Antibacterial activity of fifth-generation dentin bonding systems. J Endod 2001;27:730-3.
[82]. Herrera M, Castillo A, Bravo M, Liebana J, Carrion P. Antibacterial activity of resin adhesives, glass ionomer and resin-modified glass ionomer cements and a compomer in contact with dentin caries samples. Oper Dent 2000;25:265-9.
[83]. Takahashi Y, Imazato S, Kaneshiro AV, Ebisu S, Frencken JE, Tay FR. Antibacterial effects and physical properties of glass-ionomer cements containing chlorhexidine for the ART approach. Dent Mater 2006;22:647-52.
[84]. Imazato S, Imai T, Russell RR, Torii M, Ebisu S. Antibacterial activity of cured dental resin incorporating the antibacterial monomer MDPB and an adhesion-promoting monomer. J Biomed Mater Res 1998;39:511-5.
[85]. Imazato S, Ebi N, Takahashi Y, Kaneko T, Ebisu S, Russell RR. Antibacterial activity of bactericide-immobilized filler for resin-based restoratives. Biomaterials 2003;24:3605-9.
[86]. Ozer F, Karakaya S, Unlu N, Erganis O, Kav K, Imazato S. Comparison of antibacterial activity of two dentin bonding systems using agar well technique and tooth cavity model. J Dent 2003;31:111-6.
[87]. Costerton JW, Lewandowski Z, DeBeer D, Caldwell D, Korber D, James G. Biofilms, the customized microniche. J Bacteriol 1994;176:2137-42.
[88]. Costerton JW, Lewandowski Z, Caldwell DE, Korber DR, Lappin-Scott HM.Microbial biofilms. Annu Rev Microbiol 1995;49:711-45.
[89]. Bradshaw DJ, Marsh PD, Hodgson RJ, Visser JM. Effects of glucose and fluoride on competition and metabolism within in vitro dental bacterial communities and biofilms. Caries Res 2002;36:81-6.
[90]. Guggenheim B, Guggenheim M, Gmur R, Giertsen E, Thurnheer T. Application of the Zurich biofilm model to problems of cariology. Caries Res 2004;38:212-22.
[91]. Filoche SK, Soma KJ, Sissons CH. Caries-related plaque microcosm biofilms developed in microplates. Oral Microbiol Immunol 2007;22:73-9.
[92]. Marsh PD, Hunter JR, Bowden GH, Hamilton IR, McKee AS, Hardie JM, Ellwood DC. The influence of growth rate and nutrient limitation on the microbial composition and biochemical properties of a mixed culture of oral bacteria grown in a chemostat. J Gen Microbiol 1983;129:755-70.
[93]. Marsh PD. Are dental diseases examples of ecological catastrophes? Microbiology 2003;149:279-94.
[94]. McBain AJ, Sissons C, Ledder RG, Sreenivasan PK, De Vizio W, Gilbert P. Development and characterization of a simple perfused oral microcosm. J Appl Microbiol 2005;98:624-34.
[95]. Pigman W, Elliott HC, Jr., Laffre RO. An artificial mouth for caries research. J Dent Res 1952;31:627-33.
[96]. Coulter WA, Russell C. A miniature continuous culture system for controlled production of simulated bacterial dental plaque. Arch Oral Biol 1976;21:333-4.
[97]. Coulter WA, Russell C. pH and Eh in single and mixed culture bacterial plaque in an artificial mouth. J Appl Bacteriol 1976;40:73-87.
[98]. Russell C, Coulter WA. Continuous monitoring of pH and Eh in bacterial plaque grown on a tooth in an artificial mouth. Appl Microbiol 1975;29:141-4.
[99]. Shu M, Wong L, Miller JH, Sissons CH. Development of multi-species consortia biofilms of oral bacteria as an enamel and root caries model system. Arch Oral Biol 2000;45:27-40.
[100]. Pearce EI, Wakefield JS, Sissons CH. Therapeutic mineral enrichment of dental plaque visualized by transmission electron microscopy. J Dent Res 1991;70:90-4.
[101]. Sissons CH, Cutress TW, Hoffman MP, Wakefield JS. A multi-station dental plaque microcosm (artificial mouth) for the study of plaque growth, metabolism, pH, and mineralization. J Dent Res 1991;70:1409-16.
[102]. Bibby BG, Huang CT, Zero D, Mundorff SA, Little MF. Protective effect of milk against in vitro caries. J Dent Res 1980;59:1565-70.
[103]. Noorda WD, Purdell-Lewis DJ, van Montfort AM, Weerkamp AH. Developmentaland metabolic aspects of a monobacterial plaque of Streptococcus mutans C 67-1 grown on human enamel slabs in an artificial mouth model. II. Enamel data. Caries Res 1986;20:308-14.
[104]. Noorda WD, Purdell-Lewis DJ, van Montfort AM, Weerkamp AH. Monobacterial and mixed bacterial plaques of Streptococcus mutans and Veillonella alcalescens in an artificial mouth: development, metabolism, and effect on human dental enamel. Caries Res 1988;22:342-7.
[105]. Noorda WD, van Montfort AM, Purdell-Lewis DJ, Weerkamp AH. Developmental and metabolic aspects of a monobacterial plaque of Streptococcus mutans C 67-1 grown on human enamel slabs in an artificial mouth model. I. Plaque data. Caries Res 1986;20:300-7.
[106].卢滇楠,周轩榕,邢晓东.表面接枝季铵盐型聚合物的纤维素纤维-灭菌机理研究.高分子学报2004;14:107-13.
[107]. Sumitomo T, Nagamune H, Maeda T, Kourai H. Correlation between the bacterioclastic action of a bis-quaternary ammonium compound and outer membrane proteins. Biocontrol Sci 2006;11:115-24.
[108]. Yoshimatsu T, Hiyama K. Mechanism of the action of didecyldimethylammonium chloride (DDAC) against Escherichia coil and morphological changes of the cells. Biocontrol Sci 2007;12:93-9.
[109]. Beyth N, Domb AJ, Weiss EI. An in vitro quantitative antibacterial analysis of amalgam and composite resins. J Dent 2007;35:201-6.
[110]. Khalichi P, Cvitkovitch DG, Santerre JP. Effect of composite resin biodegradation products on oral streptococcal growth. Biomaterials 2004;25:5467-72.
[111]. Filoche SK, Coleman MJ, Angker L, Sissons CH. A fluorescence assay to determine the viable biomass of microcosm dental plaque biofilms. J Microbiol Methods 2007;69:489-96.
[112]. van der Mei HC, White DJ, Atema-Smit J, van de Belt-Gritter E, Busscher HJ. A method to study sustained antimicrobial activity of rinse and dentifrice components on biofilm viability in vivo. J Clin Periodontol 2006;33:14-20.
[113]. Montanaro L, Campoccia D, Rizzi S, Donati ME, Breschi L, Prati C, Arciola CR. Evaluation of bacterial adhesion of Streptococcus mutans on dental restorative materials. Biomaterials 2004;25:4457-63.
[114]. Ahlstrom B, Chelminska-Bertilsson M, Thompson RA, Edebo L. Long-chain alkanoylcholines, a new category of soft antimicrobial agents that are enzymatically degradable. Antimicrob Agents Chemother 1995;39:50-5.
[115]. Calas M, Ancelin ML, Cordina G, Portefaix P, Piquet G, Vidal-Sailhan V, Vial H. Antimalarial activity of compounds interfering with Plasmodium falciparum phospholipid metabolism: comparison between mono- andbisquaternary ammonium salts. J Med Chem 2000;43:505-16.
[116]. Lindstedt M, Allenmark S, Thompson RA, Edebo L. Antimicrobial activity of betaine esters, quaternary ammonium amphiphiles which spontaneously hydrolyze into nontoxic components. Antimicrob Agents Chemother 1990;34:1949-54.
[117]. Ajdic D, McShan WM, McLaughlin RE, Savic G, Chang J, Carson MB, Primeaux C, Tian R, Kenton S, Jia H, Lin S, Qian Y, Li S, Zhu H, Najar F, Lai H, White J, Roe BA, Ferretti JJ. Genome sequence of Streptococcus mutans UA159, a cariogenic dental pathogen. Proc Natl Acad Sci U S A 2002;99:14434-9.
[118]. Feuerstein O, Matalon S, Slutzky H, Weiss EI. Antibacterial properties of self-etching dental adhesive systems. J Am Dent Assoc 2007;138:349-54; quiz 96-8.
[119]. Hope CK, Wilson M. Analysis of the effects of chlorhexidine on oral biofilm vitality and structure based on viability profiling and an indicator of membrane integrity. Antimicrob Agents Chemother 2004;48:1461-8.
[120]. Rolland SL, McCabe JF, Robinson C, Walls AW. In vitro biofilm formation on the surface of resin-based dentine adhesives. Eur J Oral Sci 2006;114:243-9.
[121]. Hamada S, Slade HD. Biology, immunology, and cariogenicity of Streptococcus mutans. Microbiol Rev 1980;44:331-84.
[122]. Loesche WJ. Role of Streptococcus mutans in human dental decay. Microbiol Rev 1986;50:353-80.
[123]. Yamashita Y, Bowen WH, Burne RA, Kuramitsu HK. Role of the Streptococcus mutans gtf genes in caries induction in the specific-pathogen-free rat model. Infect Immun 1993;61:3811-7.
[124]. Kuramitsu HK. Virulence factors of mutans streptococci: role of molecular genetics. Crit Rev Oral Biol Med 1993;4:159-76.
[125]. Fujiwara T, Hoshino T, Ooshima T, Hamada S. Differential and quantitative analyses of mRNA expression of glucosyltransferases from Streptococcus mutans MT8148. J Dent Res 2002;81:109-13.
[126]. Shemesh M, Tam A, Steinberg D. Differential gene expression profiling of Streptococcus mutans cultured under biofilm and planktonic conditions. Microbiology 2007;153:1307-17.
[127]. Shemesh M, Tam A, Feldman M, Steinberg D. Differential expression profiles of Streptococcus mutans ftf, gtf and vicR genes in the presence of dietary carbohydrates at early and late exponential growth phases. Carbohydr Res 2006;341:2090-7.
[128]. Cury JA, Koo H. Extraction and purification of total RNA from Streptococcus mutans biofilms. Anal Biochem 2007;365:208-14.
[129]. Ooshima T, Matsumura M, Hoshino T, Kawabata S, Sobue S, Fujiwara T.Contributions of three glycosyltransferases to sucrose-dependent adherence of Streptococcus mutans. J Dent Res 2001;80:1672-7.
[130]. Li Y, Burne RA. Regulation of the gtfBC and ftf genes of Streptococcus mutans in biofilms in response to pH and carbohydrate. Microbiology 2001;147:2841-8.
[131]. Goodman SD, Gao Q. Characterization of the gtfB and gtfC promoters from Streptococcus mutans GS-5. Plasmid 2000;43:85-98.
[132]. Chen PM, Chen JY, Chia JS. Differential regulation of Streptococcus mutans gtfBCD genes in response to copper ions. Arch Microbiol 2006;185:127-35.
[133]. Koo H, Seils J, Abranches J, Burne RA, Bowen WH, Quivey RG, Jr. Influence of apigenin on gtf gene expression in Streptococcus mutans UA159. Antimicrob Agents Chemother 2006;50:542-6.