【作者】 李芳；

【导师】 陈吉华；

【作者基本信息】 第四军医大学 ， 口腔临床医学， 2009， 博士

【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。更多还原

【Abstract】 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.更多还原