【作者】 张珂；

【导师】 白玉兴；

【作者基本信息】 首都医科大学 ， 口腔正畸学， 2013， 博士

【摘要】 复合树脂及其粘接系统因为具有优良的美学效果，以及简便的临床操作过程等优势，近年来广泛应用于修复牙体缺损和改善牙齿外观。但相较其他口腔修复材料，复合树脂更容易在表面堆积菌斑生物膜，同时复合树脂的聚合收缩和粘接不良易导致边缘微渗漏，成为唾液和细菌进出的通道，即菌斑生物膜的入侵途径。菌斑生物膜是引起继发龋和修复失败的主要原因，一方面会直接引起某些口腔软硬组织感染性疾病，如继发龋、义齿性口炎，另一方面则能造成材料的老化粗糙，导致材料性能下降，从而更易吸引细菌黏附。这样的恶性循环最终将会造成修复失败，甚至对全身产生不良影响。修复失败的再修复所花费的时间占到牙医操作时间的50-70%，而全美每年再修复的花费更是超过200亿美金。正因为复合树脂缺乏抗菌作用，赋予复合树脂本身，及将复合树脂与牙体粘接起来的粘接系统抗菌作用，就显得尤为必要。具有抗菌性能的复合树脂和粘接系统能减小对口腔微生态环境的影响，改善修复体的长期临床效果，维护口腔微生态环境的健康。各国研究人员在研制开发具有抗菌作用的复合树脂和粘接系统等材料方面进行了大量的研究，以期达到抑制细菌生长，防止菌斑生物膜的附着，预防继发龋的发生，防治修复失败和因修复体引发的感染和炎症的目的。本课题主要从以下四个部分进行研究：第一部分抗菌粘接系统的研发和检测:季铵盐和纳米银颗粒改性牙本质粘接剂对粘接强度和牙菌斑生物膜的影响;第二部分添加无定型磷酸钙纳米颗粒，赋予粘接剂再矿化性能：新型含纳米银和无定形磷酸钙的牙本质粘接剂；第三部分烷基链长度的影响：含有新型抗菌季铵盐单体甲基丙烯酸十二烷基二甲铵的牙本质粘接系统；第四部分长期效果：水老化对抗菌粘接系统的机械性能及抗菌性能影响。第一部分季铵盐和纳米银颗粒改性牙本质粘接剂对粘接强度和牙菌斑生物膜的影响目的：抗菌牙本质粘接剂在抑制残留细菌和阻止细菌入侵方面有很重要的作用。本研究的目的是开发一种含有新型季铵盐单体二甲基丙烯酸季铵（quaternary ammonium dimethacrylate， QADM）和纳米银颗粒（nanoparticles of silver, NAg）的抗菌牙本质粘接剂，并对含有QADM-NAg的粘接剂及底胶对牙本质粘接强度和牙菌斑生物膜的影响进行研究。方法：商品化的牙本质粘接剂及底胶Scotchbond Multi-Purpose作为本实验的对照组。实验组粘接剂和底胶是在对照组中加入QADM和NAg。测试了10组人牙本质剪切粘接强度（n=10）。使用人唾液培养所得到牙菌斑全菌生物膜模型来研究生物膜的代谢活性，菌落形成单位（colony forming unit, CFU）计数，产乳酸量，死/活细菌染色（n=6）。结果：添加了QADM和NAg的牙本质粘接剂和底胶，其牙本质剪切粘接强度没有下降，仍介于30至35兆帕间（P>0.1）。扫描电子显微镜（scanningelectron microscope, SEM）照片显示有大量树脂突形成，这在对照组和添加了QADM和NAg的实验组中是相同的。与对照组相比，添加QADM和NAg的对照组菌斑生物膜的代谢活性显著降低。在粘接剂里同时添加QADM和NAg比添加单一的抗菌成分具有更强的抗菌作用（P<0.05）。与对照组相比，同时在粘接剂和底胶中添加QADM和NAg具有最强的抗菌活性，最大程度降低了菌斑生物膜代谢活性，减少了CFU计数和一个数量级的产乳酸量。意义：在不影响牙本质粘接强度和树脂突形态的前提下，本研究第一次证实了含有QADM和NAg的牙本质粘接剂和底胶，对牙菌斑生物膜具有较强的抗菌作用。含有QADM和NAg的牙本质粘接剂和底胶在抑制窝洞中残留细菌和阻止细菌入侵方面有很重要的作用，因此可以预防继发龋的的发生。QADM和NAg添加到其他牙本质粘接系统可能同样具有广泛的应用前景。第二部分新型含纳米银和无定形磷酸钙的牙本质粘接剂目的：抗菌牙本质粘接剂在抑制窝洞中残留细菌和在修复体边缘阻止细菌入侵方面发挥重要的作用。钙（Ca）和磷酸根离子（PO4）的释放曾被证明能够促进病变牙的再矿化。本研究的目标是将纳米银颗粒NAg和无定形磷酸钙纳米颗粒（Ca3[PO4]2, nanoparticles of amorphous calcium phosphate, NACP）添加到牙本质粘接剂中，以研究其对牙本质粘接强度和牙菌斑生物膜的影响。方法：SBMP粘接系统作为本实验的对照组。NAg按质量分数0.1%被添加到对照粘接剂和底胶中。NACP则按质量分数10％，20％，30％和40％与对照粘接剂混合。使用人唾液培养所得到牙菌斑全菌生物膜模型来研究生物膜，让其附着在由复合树脂，粘接剂和底胶构成的试件上进行生长。测定了牙菌斑生物膜的代谢活性，CFU计数和产乳酸量。结果：人牙本质剪切粘接强度介于26至34兆帕间，说明添加了NAg和NACP后粘接强度没有显著下降（P>0.1）。与对照组相比，添加了NAg和NACP牙本质粘接剂和底胶大大减少了生物膜活力和代谢活性。添加了NAg和NACP的实验组全菌，总链球菌，变形链球菌CFU计数较对照组降低了近一个数量级。添加了NAg和NACP的实验组产乳酸量减少到对照组的1/4。 SEM观察发现牙本质小管内充填有大量树脂突，并且牙本质小管和粘接剂层内都渗透了大量的NACP。意义：本研究第一次证实了含有NAg和NACP的粘接剂可以显著降低菌斑生物膜活力和产乳酸量，并且不损害牙本质粘接强度。这种添加NAg和NACP的方法可能在其他牙本质粘接系统中同样具有广泛的应用前景。第三部分含有新型抗菌季铵盐单体甲基丙烯酸十二烷基二甲铵（dimethylaminododecyl methacrylate, DMADDM）的牙本质粘接系统目的：树脂修复失败的主要原因是菌斑生物膜产酸引起的继发龋。修复失败的再修复花费的时间占到牙医操作时间的50-70%。本研究的目标是把新的季铵盐单体DMADDM和纳米银颗粒NAg添加到牙本质粘接剂和底胶中，探讨其对粘接系统抗菌性能和粘接强度的影响。方法：SBMP粘接系统作为本实验的对照组。合成DMADDM并将其与NAg添加到粘接剂和底胶中。使用人唾液培养所得到牙菌斑全菌生物膜模型来研究生物膜，检测生物膜代谢活力，CFU计数，产乳酸量，并测量人牙本质剪切粘接强度。结果：DMADDM的最低抑菌浓度（minimal inhibitory concentration, MIC）和最低杀菌浓度（minimal bactericidal concentration, MBC）较原先的QADM降低了多个数量级。未固化的加入DMADDM的底胶，其琼脂平板抑菌环的直径远大于加入QADM的底胶所形成的抑菌环直径（P<0.05）。而固化的加入了DMADDM-NAg的粘接剂和底胶大大降低了菌斑生物膜代谢活性（P <0.05）。同时加入DMADDM和NAg的实验组，CFU计数的结果少于单独加入DMADDM的实验组（P<0.05）。与对照组相比，加入了DMADDM-NAg的实验组，菌斑生物膜产乳酸量减少了20倍。在粘接系统中加入DMADDM和NAg对牙本质粘接强度没有不利影响。意义：本研究合成了一种新的抗菌单体DMADDM，并第一次将其添加到牙本质粘接剂和底胶中。抗菌牙本质粘接剂在抑制残留细菌和阻止细菌入侵方面有很重要的作用。DMADDM和NAg在牙本质粘接系统和复合树脂修复方面，有良好的应用前景。第四部分水老化对含有新型单体dimethylaminododecyl methacrylate的牙本质粘接剂粘接强度和抗生物膜活性的影响目的：本研究的目的是将新的抗菌单体DMADDM以及纳米银颗粒NAg和无定形磷酸钙纳米颗粒NACP添加到牙本质粘接系统中，开发出新的具有抗菌性能的牙本质粘接系统，并第一次通过6个月的水老化方法，检测其牙本质粘接强度和抗菌性能的长期效果。方法：检测了以下四组粘接系统：SBMP对照组；SBMP+5％DMADDM；SBMP+5％DMADDM+0.1％NAg和SBMP+5％DMADDM+0.1％NAg，粘接剂另加入20％NACP。试件在37℃下水老化1天和6个月，然后测量牙本质的剪切粘接强度。使用人唾液培养所得到牙菌斑全菌生物膜模型来研究生物膜，并测量生物膜代谢活性，CFU计数，和产乳酸量。结果：SBMP对照组经过6个月的水老化后，牙本质粘接强度降低了35％（n=10），相比之下，加入抗菌粘接剂的实验组没有出现粘接强度下降。含有DMADDM-NAg-NACP的实验组表现出了强的DMADDM抗菌效果，菌斑生物膜活力大大降低，代谢活动和产酸均显著减少。与对照组相比，CFU计数减少超过两个数量级。此外，含有DMADDM-NAg-NACP的粘接系统具有长期的抗菌性能，1天和6个月的抗菌效果没有显着差异（P>0.1）。结论：添加DMADDM-NAg-NACP的牙本质粘接系统具有强效和持久的抗菌性能，经过6个月水老化后的粘接强度远大于商品化对照组。这种新型抗菌粘接系统能有效抑制菌斑生物膜的生长和修复体边缘的继发龋。本方法在其他牙本质粘接系统，正畸粘接剂和复合树脂的改性方面具有广泛的适用性。更多还原

【Abstract】 Resin composites and resin-based bonding system are increasingly popularin dental clinics because of their excellent esthetic effects and direct-fillingcapabilities. However, compared to other restorative materials, compositesaccumulated more biofilms/plaques. And the polymerization shrinkage of resincomposites causes marginal microleakage, leading to the formation of channelfor dental plaque invasion. Dental plaque biofilm is on the one hand, a directresult of certain oral infectious diseases, such as secondary caries, denturestomatitis, on the other hand can cause the material aging or degradation, thusmore likely to attract bacterial accumulation. This vicious circle will ultimatelylead to restoration failing, and even impact negatively on the whole body. Dentalplaques contribute to secondary caries, which is a main reason for restorationfailure. Replacing the failed restorations consumes50-70%of the dentist’s time.Replacement dentistry costs$5billion annually in the United States. Extensivestudies have been performed to improve the fillers, resins, and polymerizationproperties. Besides composites, it is also important to develop novelantibacterial and remineralizing adhesives because composite restorations arebonded to tooth structure via adhesives. Resin composites and resin-basedbonding system with antibacterial properties can stabilize oral micro-ecologicalenvironment, improve the long-term clinical effect of the restoration andmaintain the health of the oral microbial environment.Part1Effect of quaternary ammonium and silver nanoparticle-containingadhesives on dentin bond strength and dental plaque microcosm biofilmsObjective: Antibacterial bonding agents are promising to hinder the residualand invading bacteria at the tooth–restoration interfaces. The objectives of thisstudy were to develop an antibacterial bonding agent by incorporation of quaternary ammonium dimethacrylate (QADM) and nanoparticles of silver(NAg), and to investigate the effect of QADM-NAg adhesive and primer on dentinbond strength and plaque microcosm biofilm response for the first time.Methods: Scotchbond Multi-Purpose adhesive and primer were used as control.Experimental adhesive and primer were made by adding QADM and NAg intocontrol adhesive and primer. Human dentin shear bond strengths weremeasured (n=10). A dental plaque microcosm biofilm model with human salivaas inoculum was used to investigate biofilm metabolic activity, colony-formingunit (CFU) counts, lactic acid production, and live/dead staining assay (n=6).Results: Adding QADM and NAg into adhesive and primer did not compromisethe dentin shear bond strength which ranged from30to35MPa (p>0.1).Scanning electron microscopy (SEM) examinations revealed numerous resintags, which were similar for the control and the QADM and NAg groups. AddingQADM or NAg markedly reduced the biofilm viability, compared to adhesivecontrol. QADM and NAg together in the adhesive had a much strongerantibacterial effect than using each agent alone (p<0.05). Adding QADM and NAgin both adhesive and primer had the strongest antibacterial activity, reducingmetabolic activity, CFU, and lactic acid by an order of magnitude, compared tocontrol.Significance: Without compromising dentin bond strength and resin tagformation, the QADM and NAg containing adhesive and primer achieved strongantibacterial effects against microcosm biofilms for the first time. QADM-NAgadhesive and primer are promising to combat residual bacteria in tooth cavityand invading bacteria at the margins, thereby to inhibit secondary caries. QADMand NAg incorporation may have a wide applicability to other dental bondingsystems. Part2Novel dental adhesives containing nanoparticles of silver and amorphouscalcium phosphateObjective: Antibacterial bonding agents are promising to inhibit residualbacteria in the prepared tooth cavity and invading bacteria along the margins.The release of calcium (Ca) and phosphate (PO4) ions was previously shown toremineralize tooth lesions. The objectives of this study were to incorporatenanoparticles of silver (NAg) and nanoparticles of amorphous calciumphosphate (NACP) into a bonding agent for the first time, and to investigate theeffects on dentin bond strength and dental plaque microcosm biofilm response.Methods: Scotchbond Multi-Purpose adhesive and primer were used as control.NAg were incorporated into the control primer and adhesive at0.1%by mass.NACP were mixed into adhesive at10%,20%,30%and40%. A microcosmbiofilm model with human saliva as inoculum was used on disks with primercovering the adhesive on the top of a composite. Biofilm metabolic activity,colony-forming unit (CFU) and lactic acid production were measured.Results: Human dentin shear bond strengths ranged from26to34MPa; addingNAg and NACP did not significantly decrease the bond strength (p>0.1). Bondingagents containing NAg and NACP greatly reduced the biofilm viability andmetabolic activity, compared to the control. CFU for total microorganisms, totalstreptococci, and mutans streptococci on bonding agents with NACP and NAgwere an order of magnitude less than those of control. Lactic acid production forgroups containing NACP and NAg were reduced to1/4of that of control. SEMexamination revealed the prevalence of resin tags from well-filled dentinaltubules, and numerous NACP infiltrated in the adhesive layer as well as indentinal tubules. Significance: In conclusion, microcosm biofilm viability and acid productionwere greatly reduced on bonding agents containing NAg and NACP nanoparticlesfor the first time, without compromising dentin bond strength. The method ofincorporating a remineralizing agent NACP and an antibacterial agent NAg may Part3Dental primer and adhesive containing a new antibacterial quaternaryammonium monomer dimethylaminododecyl methacrylateObjectives: The main reason for restoration failure is secondary caries causedby biofilm acids. Replacing the failed restorations accounts for50–70%of alloperative work. The objectives of this study were to incorporate a newquaternary ammonium monomer (dimethylaminododecyl methacrylate,DMADDM) and nanoparticles of silver (NAg) into a primer and an adhesive, andto investigate their effects on antibacterial and dentin bonding properties.Methods: Scotchbond Multi-Purpose (SBMP) served as control. DMADDM wassynthesized and incorporated with NAg into primer/adhesive. A dental plaquemicrocosm biofilm model with human saliva was used to investigate metabolicactivity, colony-forming units (CFU), and lactic acid. Dentin shear bond strengthswere measured.Results: Minimum inhibitory concentration (MIC) and minimum bactericidalconcentration (MBC) of the new DMADDM were orders of magnitude lower thanthose of a previous quaternary ammonium dimethacrylate (QADM). Uncuredprimer with DMADDM had much larger inhibition zones than QADM (p <0.05).Cured primer/adhesive with DMADDM-NAg greatly reduced biofilm metabolicactivity (p <0.05). Combining DMADDM with NAg in primer/adhesive resultedin less CFU than DMADDM alone (p <0.05). Lactic acid production by biofilms was reduced by20-fold via DMADDM-NAg, compared to control. Incorporationof DMADDM and NAg into primer/adhesive did not adversely affect dentin bondstrength.Significance: A new antibacterial monomer DMADDM was synthesized andincorporated into primer/adhesive for the first time. The bonding agents arepromising to combat residual bacteria in tooth cavity and invading bacteria attooth-restoration margins to inhibit caries. DMADDM and NAg are promising foruse into a wide range of dental adhesive systems and restoratives. Part4Effect of water-aging on dentin bond strength and anti-biofilm activity ofbonding agent containing new monomer dimethylaminododecyl methacrylateObjectives: The objectives of this study were to develop bonding agentcontaining a new antibacterial monomer dimethylaminododecyl methacrylate(DMADDM) as well as nanoparticles of silver (NAg) and nanoparticles ofamorphous calcium phosphate (NACP), and to investigate the effects of water-aging for6months on dentin bond strength and anti-biofilm properties for thefirst time.Methods: Four bonding agents were tested: Scotchbond Multi-Purpose (SBMP)primer and adhesive control; SBMP+5%DMADDM; SBMP+5%DMADDM+0.1%NAg; and SBMP+5%DMADDM+0.1%NAg with20%NACP in adhesive.Specimens were water-aged for1day and6months at37oC. Then the dentinshear bond strengths were measured. A dental plaque microcosm biofilm modelwas used to inoculate bacteria on water-aged specimens and to measuremetabolic activity, colony-forming units (CFU), and lactic acid production.Results: Dentin bond strength showed a35%loss in6months of water-agingfor SBMP control (mean±sd; n=10); in contrast, the new antibacterial bonding agents showed no strength loss. The DMADDM-NAg-NACP containing bondingagent imparted a strong antibacterial effect by greatly reducing biofilm viability,metabolic activity and acid production. The biofilm CFU was reduced by morethan two orders of magnitude, compared to SBMP control. Furthermore, theDMADDM-NAg-NACP bonding agent exhibited a long-term antibacterialperformance, with no significant difference between1d and6months (p>0.1).Conclusions: Incorporating DMADDM-NAg-NACP in bonding agent yieldedpotent and long-lasting antibacterial properties, and much stronger bondstrength after6months of water-aging than a commercial control. The newantibacterial bonding agent is promising to inhibit biofilms and caries at themargins. The method of DMADDM-NAg-NACP incorporation may have a wideapplicability to other adhesives, cements and composites.更多还原