Effect of a calcium hydroxide-based intracanal medicament containing N-2-methyl pyrrolidone as a vehicle against Enterococcus faecalis biofilm

Abstract This study investigated the effect of a calcium hydroxide (CH) paste (CleaniCal®) containing N-2-methyl pyrrolidone (NMP) as a vehicle on Enterococcus faecalis (E. faecalis) biofilms compared with other products containing saline (Calasept Plus™) or propylene glycol (PG) (Calcipex II®). Methodology Standardized bovine root canal specimens were used. The antibacterial effects were measured by colony-forming unit counting. The thickness of bacterial microcolonies and exopolysaccharides was assessed using confocal laser scanning microscopy. Morphological features of the biofilms were observed using field-emission scanning electron microscopy (FE-SEM). Bovine tooth blocks covered with nail polish were immersed into the vehicles and dispelling was observed. The data were analyzed using one-way analysis of variance and Tukey tests (p<0.05). Results CleaniCal® showed the highest antibacterial activity, followed by Calcipex II® (p<0.05). Moreover, NMP showed a higher antibacterial effect compared with PG (p<0.05). The thickness of bacteria and EPS in the CleaniCal® group was significantly lower than that of other materials tested (p<0.05). FE-SEM images showed the specimens treated with Calasept Plus™ were covered with biofilms, whereas the specimens treated with other medicaments were not. Notably, the specimen treated with CleaniCal® was cleaner than the one treated with Calcipex II®. Furthermore, the nail polish on the bovine tooth block immersed in NMP was completely dispelled. Conclusions CleaniCal® performed better than Calasept Plus™ and Calcipex II® in the removal efficacy of E. faecalis biofilms. The results suggest the effect might be due to the potent dissolving effect of NMP on organic substances.


Introduction
Enterococcus faecalis (E. faecalis) is a grampositive facultative anaerobe mainly related to failed root canal treatment. 1 It can resist very harsh environmental conditions by invading dentinal tubules, enduring prolonged periods of starvation, and forming intraradicular biofilms that are more resistant to antimicrobial agents. [2][3][4] Among these, the ability to form biofilms is one of the main mechanisms for the survival of bacterial species in the root canal system.
Microbes in biofilms are much harder to eradicate using antimicrobial agents compared with corresponding planktonic forms. 5 In this respect, it is necessary to use intracanal medicaments in interappointment such as calcium hydroxide (CH) for further disinfection in addition to mechanical instrumentation with irrigants during endodontic treatment to reduce microorganisms in the root canal system. However, the disinfection efficacy of CH is hindered in the presence of biofilms; 4,6 therefore, enhanced efficacy in biofilm removal of intracanal medicaments has been demanded nowadays.
CH has a long history of use as an endodontic intracanal medicament due to its antibacterial effect. 7 Thus, liquid vehicles are required for CH to facilitate the delivery of dry powder and the release of hydroxyl ions. 8 The vehicles are also used to improve antibacterial effect and biocompatibility. 9,10 Basically, three main types of vehicle exist: (i) water-soluble substances including water, saline, and methylcellulose; (ii) viscous vehicles such as glycerin, polyethylene glycol (PEG), and propylene glycol (PG); (iii) oil-based vehicles such as olive oil, silicone oil, and some fatty acids.

Removal effect of nail polish on bovine tooth blocks
The following experiment using nail polish and bovine tooth blocks was performed to verify whether NMP can dispel organic substances from inorganic tooth surfaces. The bovine tooth block (5 mm x 5 mm) was obtained from the crown portion of bovine teeth.
Then, red-colored nail polish was varnished on the enamel surface of the block. The blocks were immersed in water, saline, PG, or NMP, respectively. After 2 h, the surface was observed using a stereo microscope (MZ16FA; Leica Microsystems, Wetzlar, Germany) to verify whether the nail polish was dispelled.

Statistical analysis
The data were statistically analyzed using Kolmogorov-Smirnov test for determination of normal distribution, and one-way analysis of variance (ANOVA) and Tukey tests were used to detect any significance. These analyses were performed using the SPSS software (SPSS 12.0 K for Windows; SPSS Inc., Chicago, IL, USA). A p-value lower than 0.05 was considered statistically significant.

CLSM and FE-SEM analysis
The CLSM images of the biofilms after the medicament treatments showed bacterial microcolonies and EPS thickness. As shown in Figure 2, the bacterial and EPS thickness of Calcipex II ® and CleaniCal ® groups were significantly lower than those of the control and Calasept Plus™ groups (p<0.05).
In addition, CleaniCal ® showed lower microcolonies and EPS thickness than Calcipex II ® (p<0.05). FE-SEM images showed that the root canal surfaces of both control and Calasept Plus™-treated group were fully covered by biofilms, whereas the canal surfaces treated with Calcipex II ® and CleaniCal ® were not (Figure 3a-d). Notably, the specimen treated with CleaniCal ® was cleaner than the one treated with Calcipex II ® . Furthermore, the nail polish on the enamel surface of the bovine tooth block immersed in NMP was completely dispelled after 24 h, whereas the nail polish was not dispelled at all in the other groups ( Figure 3a'-d').  Figure 1a, all the tested medicaments showed lower CFU values compared with the control. However, CleaniCal showed the lowest value. CH releases hydroxyl ion, which is responsible for the antibacterial activity and dissolution of organic tissues. Therefore, the content of CH in the medicament and hydroxyl ion releasing efficacy are the main key attributes to remove biofilms. According to the manufacturer's information, the CH content of the products tested are different (Calasept Plus™ ≈ 41%, Calcipex II ®"" ≈ 24%, CleaniCal ® ≈ 30%). Although Calasept Plus™ contains more CH, the antibacterial effect on E. faecalis was lower than that of other products that contain less CH. In this respect, the conclusion was that CH content could not explain the CFU results. The ability of releasing hydroxyl ions from CH in a given product is more critical than CH content itself. Furthermore, in general, water-based CH paste releases more hydroxyl ions and raises the mouth pH relative to the other pastes containing viscous vehicles, although controversies remain in the literature. 7,9,10,14,15 Therefore, it was assumed that the vehicle per se might be the factor and the antibacterial effect of each vehicle including saline, PG, and NMP was assessed.
In this study NMP showed lower CFU values than PG ( Figure 1B). Phaechamud,et al. 16 (2012)   other medicaments containing water or PG. These results also indicate that the structure of biofilms was dispersed and dispelled easily by irrigation.
Lastly, according to the CLSM results, the morphological features of the biofilms were investigated after treating the root canal wall with various medicaments. FE-SEM images showed the specimens treated with Calasept Plus™ were covered by biofilms, whereas the specimens treated with other medicaments were not (Figure 3a-d). Furthermore, the specimen treated with CleaniCal ® had a cleaner image than the one that had Calcipex II ® treatment, suggesting that CleaniCal ® was completely removed from the specimen. Biofilms are organic layers present on root canal walls in nature that consist of bacteria, EPS, proteins, and lipids. Therefore, it was speculated that the viscous vehicle, which is an organic solvent, might contribute to dispelling biofilms composed of organic substrates. A supplementary experiment was performed using nail polish to support this speculation.
The experiment consists of a film-forming polymer that can be dissolved by an organic solvent. In fact, NMP is used for pain stripping, graffiti removal, and industrial cleanup. It has been approved by Food and Drug Administration (FDA) as a biodegradable solvent and is listed as generally recognized as safe (GRAS). 18 In this respect, it was assumed that nail polish could model a biofilm and NMP could serve as the "stripper" intracanal medicament. As shown in Figure 3a'-d', the nail polish immersed in NMP solution was completely removed from the bovine tooth surface. However, the nail polish was not removed in other groups. Lim,et al. 19 (2017) demonstrated that CleaniCal ® was removed more effectively from the human root canal compared with ApexCal ® (Ivoclar Vivadent, Schaan, Liechtenstein), which contains PEG and Calcipex II ® .
They suggested the favorable effect might be due to the higher solubilizing efficiency of NMP compared with PEG or PG. Thus, the null hypothesis was rejected.

Conclusions
Overall, CleaniCal ® performed better than Calasept Plus™ and Calcipex II ® in the removal efficacy of E. faecalis biofilms. The results also suggest that the effect might be due to the potent dissolving effect of NMP on organic substances. Within the limitations of this study, CleaniCal ® has the potential to be used as a recommended intracanal medicament.