【作者】 李锋；

【导师】 陈吉华；

【作者基本信息】 第四军医大学 ， 口腔临床医学， 2010， 博士

【摘要】 窝沟封闭作为口腔防龋的有效手段已具有四十多年的应用历史。众多的研究表明,应用窝沟封闭剂是预防口腔窝沟龋最有效的方法之一。目前,常用的窝沟封闭剂主要包括复合树脂和玻璃离子两大类。玻璃离子类封闭剂由于本身机械强度较差,保留率较低,所以使用相对较少;相比之下,复合树脂类封闭剂凭借其合理的流动性、良好的润湿性和物理性能等众多优点,在临床上应用最为广泛。但是复合树脂类材料由于存在聚合收缩,可使牙体结构和材料间产生微裂隙,口腔细菌则会趁机侵入裂隙并且繁殖生长,从而形成继发龋并导致窝沟封闭的失败。另外,一些早期龋坏的牙体组织可能在临床操作中被覆盖于窝沟封闭剂下方,其中的致龋菌可能存活甚至继续生长繁殖,导致龋坏的进一步发展。由此可见,赋予窝沟封闭剂一定的抗菌性能十分必要。氟是公认的防龋活性物质,并且已经被广泛应用于商品化的窝沟封闭剂中。它通过抑制牙体硬组织脱矿、促进再矿化、干扰菌斑的形成以及抑制细菌的生长代谢等机制影响龋坏的形成。但是在牙科材料中添加氟存在一些缺点:首先氟的释放表现为初期的“突释效应”,随后释放浓度就会很快降低,氟释放的长期有效性值得怀疑。其次,尚缺乏充足的证据证明含氟封闭剂的临床防龋效果优于不含氟的封闭剂,而且仅有个别的含氟封闭剂被报道具备有效的抗菌活性。另外,由于氟的潜在毒性,在口腔环境中长期应用含氟材料的安全性尚有待于进一步的研究。除氟化物以外,抗生素、无机抗菌剂等抗菌活性物质也被加入牙科修复材料或粘接剂中。但是由于添加的抗菌剂都是简单分散在树脂基质中的,仅仅是依靠抗菌成分的溶出而发挥抗菌作用,所以抗菌的持久性无法得到保证。所以如果能将抗菌功能基团以化学键形式键合到牙科高分子材料中应该是更好的途径。基于以上目的,Imazato等合成了一种具有抗菌活性的季铵盐单体,甲基丙烯酰氧十二烷基溴吡啶(MDPB),并将其加入到自酸蚀粘接剂中合成了具有抗菌功能的粘接剂,发现该材料在固化后具有接触性抑菌作用。本课题组已经合成一种可聚合季铵盐抗菌功能单体—甲基丙烯酰氧乙基-正十六烷基-二甲基氯化铵(methacryloxylethyl cetyl dimethyl ammonium chloride,DMAE-CB),实验证明其对口腔常见致病菌具有较强的杀菌活性,添加DMAE-CB单体的改性粘接剂固化后具有明显的接触抑菌效果。1主要研究目的:将DMAE-CB添加到商品窝沟封闭剂Helioseal中获得DMAE-CB改性封闭剂作为实验组;未添加抗菌单体的Helioseal则作为阴性对照,具备氟释放功能的窝沟封闭剂Helioseal F作为阳性对照,评价抗菌单体DMAE-CB的添加对窝沟封闭剂抗菌性能的影响,推测改性封闭剂的抗菌机制,并且研究封闭剂的机械性能是否会因为抗菌单体的添加而受到削弱,初步评价抗菌改性窝沟封闭剂的临床应用前景。2主要研究方法:2.1采用接触抑菌实验评价改性封闭剂的固化表面对变形链球菌生长的影响,并在老化处理和唾液处理后重复上述实验以评价材料表面的抗菌效果。2.2制备三组试件的浸提液,研究变形链球菌在浸提液中的生长动力学和倍增时间,以考察材料浸提液的抗菌活性。2.3在封闭剂的固化表面进行变形链球菌生物膜的体外培养,通过光密度值分析改性材料对生物膜形成的影响;利用扫描电镜观察DMAE-CB改性封闭剂表面变链生物膜的形成过程,考察抗菌材料表面对变链生物膜形成的影响。2.4采用荧光染色技术结合激光共聚焦显微镜观察,评价不同组封闭剂的固化表面对变形链球菌生存状态(死菌/活菌)的影响,推测细菌胞膜完整性的情况,进一步明确季铵盐改性牙科树脂材料的抗菌机理。2.5测定比较三组封闭剂材料在牙釉质表面的接触角,评价DMAE-CB抗菌单体的添加对窝沟封闭剂渗透性的影响。2.6采用傅立叶变换红外光谱技术,通过检测三组封闭剂材料光照固化前后的C=C双键伸缩振动,评价材料的双键转化率,推测DMAE-CB的添加对窝沟封闭剂中树脂单体聚合转化率的影响。2.7研究三组封闭剂光固化后的表面显微硬度,进一步评价DMAE-CB抗菌单体的添加对窝沟封闭剂本身固化能力的影响。2.8通过窝沟封闭剂微渗漏实验和对封闭剂与牙釉质粘接界面的结构观察,探讨抗菌单体DMAE-CB的添加对窝沟封闭剂与牙面密合性的影响。2.9将DMAE-CB添加到封闭剂Helioseal F中,考察这种季铵盐阳离子单体的添加对含氟封闭剂在体外环境下氟离子释放的影响。3主要研究结果:3.1 DMAE-CB改性封闭剂固化后表面可以抑制变形链球菌的生长,老化处理或唾液处理后改性材料表面仍表现出一定的抗菌活性。作为阳性对照的含氟窝沟封闭剂Helioseal F和阴性对照组相比未表现出明显的抗菌活性(P > 0.05)。3.2三组封闭剂浸提液的细菌生长曲线均同空白对照组相似。四组浸提液细菌生长回归线的截距值没有显著差异(P > 0.05);DMAE-CB改性封闭剂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倍增时间均无显著差异(P > 0.05)。3.3 DMAE-CB改性封闭剂固化后表面可抑制变形链球菌生物膜的形成。扫描电镜观察发现:孵育4 h,改性封闭剂表面仅见有非常少量细菌单个或穗状散在分布,很少的细胞外基质结构附着于材料表面,且结构塌陷;孵育24 h,改性封闭剂表面已有变链生物膜形成,但结构较为松散。3.4细菌经荧光染色后,激光共聚焦显微镜下死菌显示为红色,活菌显示为绿色。阴性对照组和阳性对照组固化表面均显示有大量的红色和绿色荧光斑块;而实验封闭剂组表面无论红色或者绿色的荧光斑块均较稀疏。3.5荧光强度分析表明实验封闭剂组的总荧光强度FIt明显要比阴性对照组和阳性对照组低(P < 0.05),红绿荧光强度比值FIr/FIg实验封闭剂组则为最高(P < 0.05)。3.6实验封闭剂组剂和阴性对照组的接触角均显著小于阳性对照组(P < 0.05),而且实验封闭剂组和阴性对照组的接触角没有显著的统计学差异(P > 0.05);各组酸蚀处理亚组的接触角均小于未酸蚀亚组的(P < 0.05)。3.7实验封闭剂组和阴性对照组的聚合转化率并没有显著的统计学差异(P > 0.05);相比较阴性对照组和实验封闭剂组,阳性对照组的聚合转化率最高(P < 0.05)。3.8实验封闭剂组和阴性对照组的硬度值并没有显著的统计学差异(P > 0.05);相比较阴性对照组和实验封闭剂组,阳性对照组的硬度值最高(P < 0.05)。3.9实验封闭剂组和阴性对照组在微渗漏程度上无显著统计学差异(P > 0.05);而阳性对照组微渗漏的发生率明显高于阴性对照组和实验封闭剂组(P < 0.05).3.10两组封闭剂Helioseal F与Helioseal F(1% w/w DMAE-CB)表现出类似的氟释放方式,初期的大量释放和较长时间的低水平持续释放,而且在不同时间点测得的两组氟释放量之间均没有显著性的统计学差异(P > 0.05)。4主要结论:4.1 DMAE-CB改性封闭剂固化后可以抑制变形链球菌的生长,其抗菌活性在老化处理和唾液处理后没有明显的衰减,说明季铵盐单体改性窝沟封闭剂可以在唾液环境中发挥较持久的抗菌作用,具有临床应用的可行性。4.2改性封闭剂材料在发挥抗菌功能的同时并没有抗菌阳离子单体的溶出。4.3 DMAE-CB改性窝沟封闭剂固化后可以抑制表面变形链球菌生物膜的形成,材料老化处理后抑制作用并无明显衰减。4.4 DMAE-CB改性封闭剂固化后的材料表面通过破坏细菌细胞膜的完整性,可以杀死吸附于材料表面的细菌,同时影响细菌在其表面的黏附。4.5少量抗菌单体DMAE-CB的添加并没有改变窝沟封闭剂在牙釉质表面的接触角;牙釉质表面的酸蚀处理可以降低封闭剂在其表面的接触角。4.6少量抗菌单体DMAE-CB的添加并没有改变窝沟封闭剂材料本身的聚合转化率,聚合能力没有受到影响;无机填料比例的增加会提高光固化型复合树脂材料的聚合转化率。4.7少量抗菌单体DMAE-CB的添加并没有改变窝沟封闭剂材料本身固化后的表面硬度,也间接说明其聚合固化能力没有受到影响。4.8少量抗菌单体DMAE-CB的添加并没有对窝沟封闭剂与牙面间的密合性造成影响,没有改变微渗漏的情况;无填料的封闭剂产生的微渗漏更少。4.9窝沟封闭剂Helioseal F中少量DMAE-CB的添加并没有影响在体外环境下材料固化后氟离子的释放情况;通过添加季铵盐抗菌单体以改善含氟封闭剂的抗菌性能具备一定的可行性。综上所述,DMAE-CB改性封闭剂固化后具备一定的抗菌活性,可以抑制变形链球菌的生长和黏附,破坏细菌胞膜的完整性,老化处理和唾液预处理后改性封闭剂材料仍表现出一定的抗菌活性,发挥抗菌功能的同时并没有抗菌阳离子单体的溶出,表现为接触性抑菌;另外,少量抗菌单体DMAE-CB的添加并没有对窝沟封闭剂本身的机械性能造成影响,封闭剂在牙釉质表面的接触角、聚合转化率、硬度和微渗漏情况均没有明显的变化。更多还原

【Abstract】 Pit and fissure sealing is considered to be one of the most effective method of protecting teeth from dental caries. The composite resin and glass ionomer are main materials of sealant. Because the retention of the glass ionomer sealant as compared with the composite resin is poor, the composite resin is the most commonly used material in this procedure, due to its reasonable flowability, good wettability and improved physical properties. However, polymerization shrinkage of composite resin-based materials might facilitate the formation of gaps between the filling and cavity wall, providing space for bacterial invasion and proliferation, thereby leading to secondary caries and the failure of restorations. Additionally, incipient caries may inadvertently be sealed in with dental sealants and the fate of those bacteria within carious lesions is of significance. Therefore, if sealants could provide additional antibacterial protection, especially against cariogenic bacteria such as Streptococcus mutans (S. mutans), additional protection could be afforded to prevent subsequent deterioration at the sealant-tooth interface and caries initiation.Fluoride is well-known for its anticarious activity by inhibiting the biosynthetic metabolism of bacteria. Fluoride released from dental restorative materials affects caries formation by reducing demineralization, enhancing remineralization, interfering with plaque formation and inhibiting microbial metabolism. The addition of fluoride to pit and fissure sealants has been widely applied in dental materials. However, the dynamic release of fluoride is unstable, exhibiting a higher initial release and subsequent leaching-out at very low levels, thereby making the permanence and validity of fluoride release suspicious. Moreover, there is some debate about whether adding fluoride to a sealant actually prevents caries more effectively than the sealant alone. Aside from fluoride, some other antibacterial components, such as antibiotics and inorganic agents, have also been added into the dental materials. However, the release kinetics of those antibacterial agents is difficult to control strictly. Moreover, the constant release of antibacterial agents may damage the physical and chemical properties of the carrier material.In order to avoid the defects caused by solubility of the agent, the dental material with antibacterial activity could be produced by binding the polymerizable monomers into polymer matrix. Several reports have described the incorporation of a quaternary ammonium monomer, methacryloyloxydodecyl pyridinium bromide (MDPB), in composite resin or an adhesive system as a strategy for obtaining antibacterial properties. Accordingly, methacryloxylethyl cetyl dimethyl ammonium chloride (DMAE-CB), a polymerizable cationic monomer containing a quaternary ammonium for antibacterial effect, was synthesized by our research group, and the DMAE-CB-incorporated adhesive was demonstrated to exert antibacterial activity and an anti-biofilm effect against S. mutans.In the present study, DMAE-CB was incorporated into a commercial resin-based sealant material to obtain an experimental DMAE-CB-incorporated sealant for evaluation of antibacterial activity and various other properties. The hypotheses were that the mobilized DMAE-CB in the resin-based sealant would exhibit a stable and long-lasting antibacterial effect against S. mutans and that the addition of a small amount (1% w/w) of DMAE-CB would not affect the properties of the parent material.1 The main objectives:DMAE-CB was incorporated at 1% (w/w) into a clinically used sealant (Helioseal). Helioseal without DMAE-CB served as a negative control. Helioseal F, containing a fluoride-releasing resin, was used as a positive control. This aim of this study was to evaluate their antibacterial activity and properties including surface contact angle, degree of conversion, hardness and microleakage characteristics.2 The main methods:2.1 The inhibitory effect of the cured sealant on bacterial growth was evaluated by the film contact method, and the influence of aging treatment or saliva treatment of the modified sealant was also evaluated by the same method.2.2 The bacterial growth in the eluent of the modified sealant was investigated with spectrophotometry and growth kinetic analysis.2.3 The effects of the cured DMAE-CB-incorporated sealant on the S. mutans biofilm accumulation of in vitro investigated with spectrophotometry,and the microcosm of S. mutans biofilm were observed with scanning electron microscope spectrophotometry.2.4 The effects of the cured sealants on the membrane integrity of S. mutans were investigated using confocal laser scanning microscopy (CLSM) in conjunction with fluorescent indicators.2.5 The contact angles of the sealants on the enamel surface were measured with a camera based goniometer.2.6 The degree of conversion of the sealants was measured using Fourier transform infrared reflectance spectroscopy.2.7 Vickers microhardness of the cured sealants was measured using a Micro Hardness Tester.2.8 30 extracted sound human molar teeth were randomly divided into three groups. After application of the three groups of materials, the specimens were subjected to thermocycling and then immersed in 1% methylene-blue dye. Following sectioning, specimens were examined under a sterecmicroscope and microleakage scores were assigned.2.9 Fluoride release from Helioseal F and Helioseal F(1% DMAE-CB) was evaluated at time intervals of 4, 8, 12, and 24 hours and 2, 3, 7, 14, 28, 56, and 112 days. Seven disks of each material were made and stored for equilibration in double distilled water at 37°C for the time of each measurement. The solution was analyzed for fluoride with a fluoride combination electrode.3 The main results:3.1 The bacterial growth was inhibited significantly by the DMAE-CB -incorporated sealant (P < 0.05); no significant difference was found between the negative control and the positive control (P > 0.05).3.2 The bacterial growth curves of S. mutans in the eluents of the three sealant groups were similar to that of the blank control in fresh BHI. There was no statistical difference in the intercept values among the four groups (P > 0.05). The slope values of the three sealant groups were significantly lower than that of the blank control, but the difference was not statistically significant (P > 0.05).3.3 The cured DMAE-CB-incorporated dental sealant could inhibit the formation of S. mutans biofilm on the material surface. After 4-h incubation, on the surface of DMAE-CB incorporated sealant, S. mutans were sporadically scattered with blurred contours; extracelluar matrix was scanty and deformed. After 24-h incubation the biofilm of DMAE-CB-incorporated group was incompact with long chains of S. mutans attached on extracellular scaffold.3.4 CLSM images showed that the live bacteria were dyed as green and those dead bacteria were red. The DMAE-CB-incorporated sealant retained fewer red and green patches.3.5 The cured DMAE-CB-incorporated sealant could inhibit the adherence of S. mutans by destroying bacterial membrane integrity (P < 0.05).3.6 The contact angles of the positive control were higher than those of the DMAE-CB-incorporated group and the negative control (P < 0.05), and the difference between the latter two groups was not significant (P > 0.05). Moreover, the contact angle with etching was lower than that without etching (P < 0.05).3.7 The degree of conversion of the positive control was higher than that of the DMAE-CB-incorporated group and the negative control (P < 0.05). There was no significant difference between the DMAE-CB-incorporated group and the negative control (P > 0.05).3.8 The microhardness of the positive control was higher than that of the DMAE-CB-incorporated group and the negative control (P < 0.05). There was no significant difference between the DMAE-CB-incorporated group and the negative control (P > 0.05).3.9 The incorporation of the antibacterial monomer DMAE-CB showed no more microleakage compared to the commercial sealant in which no antibacterial composite was used (P > 0.05). The microleakage score for the positive control was higher than that of the other two groups (P < 0.05).3.10 Fluoride was released from the evaluated sealants with a similar pattern. Both the materials released measurable amounts of fluoride throughout the test period, with considerable amounts of fluoride released in the first 24 h and especially during the first 4 h.4 The main conclusions:4.1 The DMAE-CB-incorporated sealant could inhibit the bacterial growth and the antibacterial effect was also showed after 2-month aging process or saliva treatment.4.2 The eluent of DMAE-CB-incorporated sealant did not have any greater influence on bacterial growth than its parent material and bioactive components didn’t release into the medium.4.3 DMAE-CB-incorporated sealant was demonstrated to have inhibitory effects on in vitro biofilm accumulation of S. mutans. 4.4 The DMAE-CB-incorporated sealant could destroy bacterial membrane integrity and even killing bacteria.4.5 The incorporation of a small amount of DMAE-CB to the sealant Helioseal had no significant influence on the surface contact angle. The etching of the enamel surface is necessary to ensure the effectiveness of the sealant.4.6 The incorporation of DMAE-CB had no adverse influence on the curing behaviour of the Bis-GMA/TEGDMA-based sealant. The degree of conversion increases with increasing filler content of composite resin material.4.7 The incorporation of the antibacterial monomer DMAE-CB had no influence on the microhardness and the curing level of the cured sealants.4.8 The incorporation of the antibacterial monomer DMAE-CB had no influence on the ability to prevent microleakage. The unfilled fissures showed less microleakage than filled fissures.4.9 The incorporation of DMAE-CB had no adverse influence on the fluoride release from Helioseal F which containing a fluoride-releasing resin. The antibacterial activity of long-term and effectiveness by mean of the incorporation of antibacterial monomer is feasible way.Overall, the DMAE-CB-incorporated sealant after polymerization showed a contact antibacterial effect against S. mutans, which could inhibit the bacterial growth, biofilm accumulation and destroy bacterial membrane integrity. The antibacterial activity could be maintained after aging process or saliva treatment. Additionally, the incorporation of small amounts of the monomer DMAE-CB showed no influence on the contact angle, degree of conversion, hardness, microleakage characteristic of the parental material.更多还原