Department of Oral Health Sciences, Section of Endodontology, Ghent University, Ghent, Belgium.
Laboratory of Pharmaceutical Microbiology, Ghent University, Ghent, Belgium.
Int Endod J. 2021 Sep;54(9):1557-1570. doi: 10.1111/iej.13544. Epub 2021 Jul 12.
To evaluate in a laboratory setting the influence of several model system parameters on the sodium hypochlorite (NaOCl) susceptibility of endodontic biofilms. Based on these findings, a relevant in vitro endodontic biofilm model is proposed.
In vitro biofilms were cultured, varying the following experimental model parameters: biofilm composition (monospecies Enterococcus faecalis and a multispecies biofilm including E. faecalis, Fusobacterium nucleatum, Prevotella intermedia and Porphyromonas gingivalis), incubation time (24 h or 11 days), incubation atmosphere (aerobically or anaerobically) and biofilm substrate (polystyrene microtiter plate wells, hydroxyapatite or dentine). Biofilms were subjected to treatment with NaOCl (0.025%, 0.1%, 0.5%, 2.5%) for 1 min, control groups included treatment with purified water. Biofilms were harvested and the number of surviving cells was determined by plate counting using general (monospecies biofilms) or selective (multispecies biofilms) media. A two-way ANOVA was used to explore the effect of the model parameters on biofilm eradication. Finally, the most physiologically relevant biofilm model (11-day-old multispecies biofilm grown anaerobically on dentine discs) was characterized by selective media plate counting, NaOCl susceptibility testing, scanning and transmission electron microscopy.
There was no difference in NaOCl eradication between the anaerobically and aerobically grown E. faecalis biofilms. One-day-old biofilms of E. faecalis were more susceptible to most tested NaOCl concentrations than 11-day-old biofilms (p < .05). When grown in a multispecies biofilm, E. faecalis was significantly less susceptible to NaOCl treatment than in a monospecies biofilm (p < .05). E. faecalis in a multispecies biofilm grown in a MTP was more susceptible to NaOCl (0.025% and 0.1%) than when grown on hydroxyapatite or dentine. No difference in biofilm NaOCl susceptibility was seen between hydroxyapatite and dentine. The multispecies biofilm proved to be a reproducible model with high NaOCl resistance, complex structure and organization.
The parameters biofilm age, biofilm composition and substrate had a significant influence on the NaOCl susceptibility of E. faecalis biofilms. Older biofilms, multispecies biofilms and biofilms grown on dentine and hydroxyapatite had reduced NaOCl susceptibility. These findings emphasize the importance of selecting relevant parameters when designing a laboratory biofilm model system for the evaluation of antimicrobial treatments.
在实验室环境中评估几个模型系统参数对根管生物膜对次氯酸钠（NaOCl）敏感性的影响。基于这些发现，提出了一种相关的体外根管生物膜模型。
体外生物膜培养，改变以下实验模型参数：生物膜组成（单物种粪肠球菌和包括粪肠球菌、梭杆菌、中间普氏菌和牙龈卟啉单胞菌的多物种生物膜）、孵育时间（24 小时或 11 天）、孵育气氛（需氧或厌氧）和生物膜底物（聚苯乙烯微孔板孔、羟基磷灰石或牙本质）。将生物膜用 NaOCl（0.025%、0.1%、0.5%、2.5%）处理 1 分钟，对照组用纯净水处理。收获生物膜，用普通（单物种生物膜）或选择性（多物种生物膜）培养基平板计数法测定存活细胞数。采用双因素方差分析探讨模型参数对生物膜清除的影响。最后，通过选择性培养基平板计数、NaOCl 敏感性试验、扫描和透射电子显微镜对最具生理相关性的生物膜模型（11 天龄厌氧生长于牙本质片上的多物种生物膜）进行了表征。
厌氧和需氧培养的粪肠球菌生物膜对 NaOCl 的清除率无差异。与 11 天龄生物膜相比，1 天龄粪肠球菌生物膜对大多数测试的 NaOCl 浓度更敏感（p<0.05）。在多物种生物膜中生长时，粪肠球菌对 NaOCl 处理的敏感性明显低于单物种生物膜（p<0.05）。在 MTP 中生长的多物种生物膜中的粪肠球菌对 NaOCl（0.025%和 0.1%）的敏感性高于在羟基磷灰石或牙本质上生长时。在羟基磷灰石和牙本质上，生物膜对 NaOCl 的敏感性无差异。多物种生物膜被证明是一种具有高耐 NaOCl 性、复杂结构和组织的可重复模型。
生物膜年龄、生物膜组成和底物等参数对粪肠球菌生物膜的 NaOCl 敏感性有显著影响。较老的生物膜、多物种生物膜以及在牙本质和羟基磷灰石上生长的生物膜对 NaOCl 的敏感性降低。这些发现强调了在设计用于评估抗菌处理的实验室生物膜模型系统时选择相关参数的重要性。
