粪肠球菌在根管内定植模式的体外研究

Patterns of Enterococcus faecalis in infected root canals:An in vitro study

目的:建立体外粪肠球菌感染根管模型,观察根管内粪肠球菌的感染状态及其分布特征。方法:选择因正畸减数拔除的健康前磨牙10例,随机分为两组,将其中5例高压灭菌后分别浸泡于含有1.5 mL无菌脑心浸液(brain heart infusion,BHI)培养基和0.1 mL麦氏比浊度为5的粪肠球菌菌液的Eppendorf管中,于CO2孵育箱中培养21 d建成体外粪肠球菌根管感染模型,其余5例则作为对照组。将所有样本沿牙长轴近远中向劈为两半,一半作组织学Brown&Brenn染色观察,另一半置扫描电镜下观察粪肠球菌在根管内的感染状态。结果:组织学结果显示,在所有研究组样本中,粪肠球菌均不同程度地侵入牙本质小管中,测微尺测量深度可达330～1 000μm。所有对照组样本的根管壁表面干净,牙本质小管排列有序,无细菌染色颗粒。扫描电镜结果显示,在所有研究组样本的根尖1/3段,粪肠球菌与无定形基质形成典型的生物膜结构,而在根管的冠1/3和中1/3段,粪肠球菌则成对或短链状排列在根管壁表面,呈现浮游状态。对照组的根管壁表面洁净,牙本质小管和管间牙本质结构清晰可见,未见细菌侵入。结论:粪肠球菌可单独定植于根管内,形成生物膜并侵入牙本质小管深部。此模型可用于体外评价各种机械预备和化学消毒药物对根管内感染的清除效果,从而为临床选择治疗操作提供参考。

Objective：To establish an in vitro root canal model infected by Enterococcus faecalis and to observe the morphology,distribution and relative position of Enterococcus faecalis in infected root canals.Methods： Ten human healthy premolars extracted for orthodontic reasons were collected.Following sterilization,a total of 5 specimens were aseptically transferred to separate Eppendorf tubes containing 1.5 mL brain-heart infusion broth（BHI） inoculated with 0.1 mL Enterococcus faecalis suspension that had been adjusted to Mcfarland 5,and were incubated at 37 ℃ for 21 days.The other 5 specimens were as controls.The roots of all specimens were then split into two halves along the mesiodistal axis.One half was processed with light microscopic（Brown Brenn stain） to check the bacteria in dentinal tubules,and the other was observed with SEM to investigate the bacterial status in infected root canals.Results： Enterococcus faecalis could penetrate into the dentinal tubules about 330-1 000 μm.A dense bacterial aggregation composed of Enterococcus faecalis and amorphous matrix was observed in the apical third of the root canals,whereas Enterococcus faecalis were seen free-floating or planktonic in the crown and middle third of the root canals.No microorganisms were found in the root canals of the controls.Conclusion： Enterococcus faecalis could form bacterial biofilm on the root canal walls and penetrate into the dentinal tubules.The in vitro model designed was simple,and had good practicability to make a further comparative evaluation of various antimicrobial methods in the reduction of intracanal bacteria.