【作者】 杨焰；

【导师】 曹颖光；

【作者基本信息】 华中科技大学 ， 口腔临床医学， 2016， 博士

【摘要】 临床上对感染根管的消毒方式主要包括,直接去除感染组织的根管机械预备,以及起化学消毒作用的根管内冲洗和封药。本论文的第一部分评价了操作者经验对WaveOne单根锉根管预备效果的影响。显微计算机断层扫描(micro-CT)分析结果显示,在预备离体下颌磨牙近中根管时,无论操作者有无熟练使用经验,根管体积与表面积扩大程度以及根管偏移大小均没有统计学差异。无操作经验者完成根管预备所需时间显著多于有熟练经验者,但经一个月培训后操作时间明显缩短,接近于熟练操作者所需时间,说明无经验操作者能快速掌握WaveOne单根锉根管预备技术。根管封药是根管化学预备过程的一个组成部分,但根管常用封药氢氧化钙糊剂不易从根管内清除,可能影响根管永久充填的密封效果而引起微渗漏,致使根管内残留细菌增殖导致根管治疗失败,目前已报导的所有根管冲洗方法都很难将氢氧化钙彻底清除。本论文第二部分采用micro-CT的方法评价了三种根管冲洗方式对离体下颌磨牙根管内氢氧化钙糊剂的清理效果：GentleWave系统、传统器械预备结合超声荡洗或针管冲洗的方式。结果显示GentleWave系统仅以蒸馏水为冲洗液,作用90秒即能有效去除下颌磨牙所有根管内的氢氧化钙,其根管清理效果明显优于其它两种根管冲洗方式。虽然目前大多根管冲洗方法都不能彻底清除根管内氢氧化钙糊剂,成为根管治疗失败的隐患,但如若能直接有效提高根管消毒的效果,也能从根本上为根管治疗的成功打下坚实的基础。定植于牙本质小管内的细菌可能躲避根管机械与化学预备作用而幸存,是导致根管治疗失败的主要危险因素之一。建立标准化的体外牙本质小管感染模型,将有助于更有效地评价根管消毒措施对牙本质小管内细菌的灭活效果。牙本质小管内感染的细菌种类及生物膜成熟程度均是建模时需重点考虑的因素。离心法能快速制得多个标准化的牙本质小管重度感染样本,可重复性高,但已报导的相关研究中都只使用了单一菌株感染牙本质小管。本项目首次采用离心的方法制备牙本质小管内同一菌种不同株系单菌种生物膜模型以及不同菌斑来源的多菌种生物膜模型,并评价牙本质小管内细菌来源及生物膜成熟程度对其敏感性的影响。本论文第三部分,从根尖周炎患牙根管中分离出在浮游状态具有不同敏感性的两种粪肠球菌株系VP3-181和Gel 31,在离心力作用下分别感染牙本质小管。感染样本在37℃厌氧环境中培养1周或3周后,分别由无菌水、2%洗必泰(CHX)和2%次氯酸钠(NaOCl)处理3分钟,采用细菌活力荧光染色结合共聚焦激光扫描显微镜(CLSM)的方法,评价牙本质小管内不同株系粪肠球菌生物膜在不同成熟状态时对冲洗液的敏感性。结果显示通过离心法成功构建两个粪肠球菌株系感染牙本质小管的模型,虽然年轻的Gel 31生物膜对2%NaOCl的抵抗力不如成熟生物膜,没有统计学差异(P>0.05),但其它组牙本质小管内3周龄生物膜对冲洗液的抵抗力均明显大于1周龄生物膜(P<0.05)。除了在3周龄时2%NaOCl对粪肠球菌VP3-181和Gel 31生物膜表现出相近的灭菌效果(P>0.05),VP3-181生物膜的敏感性比Gel 31生物膜高(P<0.05)。2%CHX和2%NaOCl对相同年龄相同株系粪肠球菌生物膜的灭菌效果相近(P>0.05)。牙本质小管内粪肠球菌单菌种生物膜对冲洗液的敏感性与株系种类和生物膜的成熟程度有关,成熟的生物膜比年轻的生物膜抵抗力更强。本论文第四部分,利用离心力使来自于三个不同志愿者的牙菌斑分别侵入牙本质小管,采用与第三部分相同的实验分组与分析方法,评价感染牙本质的菌种来源及生物膜成熟程度对牙本质小管内多菌种生物膜敏感性的影响。结果显示本实验能成功通过离心法将牙菌斑悬液中的细菌推入牙本质小管建立多菌种生物膜。三个菌斑来源的牙本质小管多菌种生物膜对冲洗液的敏感性没有统计学差异(P>0.05),且在3周龄时的抵抗力都明显强于年轻生物膜(P<0.05),在相同成熟程度时对2%CHX的抵抗力都大于2%NaOCl (P<0.05),但本实验中检测的两种冲洗液灭菌效果均欠佳。菌斑制备的牙本质小管内多菌种生物膜对冲洗液的敏感性与菌斑来源无关,但受生物膜的成熟程度影响,成熟的生物膜比年轻的生物膜抵抗力更强。更多还原

【Abstract】 Disinfecting approaches of infected dentin in root canals include instrumentation, irrigation and interappointment medication. In the first part of this study, the influence of operator’s experience on the quality of instrumentation with WaveOne Primary Files by an experienced and an inexperienced operator was evaluated using micro-computed tomographic scanning (micro-CT). The results showed that the changes in canal volume and surface area after treatment, percentages of uninstrumented surface area, and the canal transportations were similar between experimental groups. The inexperienced operator without training used significantly more time for instrumentation than the experienced operator, after training for one month the instrumentation time by the same inexperienced operator was shorter, close to the experienced operator. WaveOne instrumentation technique required a short learning curve for inexperienced user in order to master this technology.Interappointment intracanal medication is a common chemical disinfection method. But failure to remove all Calcium hydroxide [Ca(OH)2], a most common used interappointment medicament, from the root canal before the canal is filled with a permanent root filling can interfere with the sealing ability of the root filling and result in leakage, even root canal therapy (RCT) failure. Therefore, in the second part of current study we evaluated the removal of Ca(OH)2 from root canals of mandibular molars by means of the GentleWave system, conventional instrumentation with passive ultrasonic activation, and conventional instrumentation with needle irrigation using micro-CT imaging as the assessment approach. The study confirmed the difficulty to remove Ca(OH)2 from root canals using conventional methods. The GentleWave system removed the paste within 90 seconds using water irrigation alone. Although most of irrigation methods couldn’t remove Ca(OH)2 from canals completely, which may lead to RCT failure, improvement of root canal disinfection effectiveness would facilitate to achieve eventual RCT success.Bacteria colonizing in dentinal tubules may survive from root canal mechanical and chemical preparation, which is thought to be a main cause of RCT failure. A standardized in vitro dentinal tubule infection model could be aiding in the effectiveness evaluation of various dentinal canals disinfecting ways. It is important to take bacterial diversity and biofilm maturation into consideration when establishing the infected dentine model. In previous studies, the centrifugation has been proved to achieve homogenous and dense presence of bacteria in different specimens deep in dentin, but only one strain of bacteria was used in each investigation. Therefore, the present project established monospecies dentin canal biofilm model with two Enterococcus faecalis (E. faecalis) strains separately and multispecies dentin canal biofilm model with plaque bacteria using centrifugation. Furthermore, the effect of the source of biofilm bacteria and the level of biofilm maturation on their susceptibility in dentinal tubules was also evaluated.In the third part, E. faecalis VP3-181 and Gel 31 were introduced into dentinal tubules individually to form monospecies biofilms by centrifugation. After 1-week and 3-week incubation, the samples from each group were subjected to sterile water,2% chlorhexidine (CHX), and 2% sodium hypochlorite (NaOCl) respectively. Following 3-minute exposure, the proportions of killed bacteria in dentinal canals were assessed by LIVE/DEAD viability staining and confocal laser scanning microscopy (CLSM). The proportion of killed bacteria in mature (3 weeks) biofilms was lower than in young biofilms (1 week) after treatment (P <0.05), except no significant differences were detected for young and mature Gel 31 biofilms exposure to 2% NaOC1. E. faecalis Gel 31 biofilms were more resistant than VP3-181 biofilms except exposure to 2% NaOC1 of 3-week-old biofilms (P<0.05). No significant difference was detected between 2% CHX and 2% NaOC1 against the E. faecalis strains. The susceptibility of the monospecies E. faecalis dentin biofilm showed strain related differences. Mature monospecies biofilms in dentinal tubules were more resistant to disinfectants than young biofilms.In the fourth part, three specimens of pooled plaque bacteria collected from different donors were centrifuged into dentin to grow multispecies biofilms in dentin canals. And the following experimental procedures were the same as those in the third part. The results indicated that 3-week-old biofilms were more resistant than 1-week-old biofilms (P< 0.05). No differences in the susceptibilities to the disinfecting agents of the three multispecies biofilms were detected. The 2% NaOC1 was more effective against multispecies biofilms in dentin than 2% CHX (P<0.05). The multispecies biofilms from different donors showed similar susceptibility. Mature multispecies biofilms in dentinal tubules were more resistant to disinfectants than young biofilms.更多还原