An in vitro comparison of the antimicrobial efficacy of positive pressure and negative pressure irrigation techniques in root canals infected with Enterococcus faecalis.

AIM: The aim of this in vitro study was to compare the efficacy of irrigation with the negative pressure and the traditional positive pressure in eradicating Enterococcus faecalis from the root canal.
MATERIALS AND METHODS: A total of 40 extracted mandibular premolars were sterilized and divided into four groups with 10 samples each. Then, they were prepared to apical size #35 (Group 1, 3) and 45 (Group 2, 4) and inoculated with E. faecalis for 7 days and then irrigated with 3% sodium hypochlorite and 17% ethylenediaminetetraacetic acid. The positive pressure irrigation was performed in Group 1 and Group 2 while the negative pressure irrigation (NPI) using the Endovac in Group 3 and Group 4. Samples taken after the root canal irrigation procedures were cultured and the colony-forming units were counted. Data were statistically analyzed using the Fisher's exact test.
RESULTS: Results were statistically significant when comparing apical negative-pressure irrigation size 45 (Group 4) to positive-pressure irrigation size 35 (Group 1).
CONCLUSION: The results of this in vitro study showed that the apical NPI method (Endovac) was more effective in removing E. faecalis from the root canal at the larger apical preparation size.

INTRODUCTION

The primary aim of endodontic therapy in necrotic teeth is the reduction in the number of bacteria and the other microbes in the root canal system that prevent healing of periradicular periodontitis.[1]

The complex root canal morphology makes complete mechanical debridement a clinical challenge.[2] It has been shown that 35% or more of the canal system remains untouched by endodontic instruments.[3] This finding highlights the importance of the root canal irrigation and the chemical debridement and disinfection of the root canal system.

Sodium hypochlorite (NaOCl) is the most commonly used root canal irrigant due to its antimicrobial properties. It is introduced passively into the canal through a notched end or side-vented needle which is placed 2–3 mm short of the working length. However, if the needle is placed too close to the apical foramen or the irrigant is forcibly expressed, the chances of extrusion of the solution increases.[4]

These concerns advocate search of newer irrigation delivery systems which place the irrigant up to working length without any risk of periapical extrusion. To address these objectives; Endovac (Discus Dental, Culver City, CA, USA) an apical negative-pressure irrigation was developed. Various studies using the Endovac have shown the increased efficiency of irrigation at working length, increased sealer penetration and to avoid extrusion, and complications.[567]

Among the different microorganisms, Enterococcus faecalis is the most difficult bacteria to eradicate from the root canals. It is found in 4%–40% of primary endodontic infections.[8] In root-filled teeth with periradicular lesions it has a prevalence ranging from 30% to 78%.[910]

Various studies[111213] have been conducted to check the antimicrobial efficacy of the negative pressure irrigation (NPI) system but with conflicting results. While some studies have shown better results, others have reported no or little effect. This study was conducted with the aim of comparing the antimicrobial efficacy of the positive pressure and the NPI techniques in root canals infected with E. faecalis.

MATERIALS AND METHODS

Forty intact human single-rooted teeth with one root canal and one apical foramen (mandibular premolars) were collected and stored in distilled water until further use. The presence of a single canal was verified radiographically by buccolingual and mesiodistal radiographs. All teeth crowns were flattened so that only 5 mm of coronal structure remained and the root canal length remains at 16–17 mm. All teeth were divided into four groups of 10 samples each.

Group 1: Positive pressure irrigation (PPI) with apical preparation up to no. 35

Group 2: PPI with apical preparation up to no. 45

Group 3: NPI with apical preparation up to no. 35

Group 4: NPI with apical preparation up to no. 45.

Procedure

The simplified outline of complete procedure is presented in flowchart of methodology (Flowchart 1). Cultivation of E. faecalis: pure isolated colonies of E. faecalis (ATCC 29212) were grown on sheep blood agar plates. These colonies were suspended in brain–heart infusion (BHI) broth and incubated for 48 h at 37°C to form the inoculum [Figure 1].

Flow Chart 1

Flowchart of methodology

Figure 1

Inoculation and Enterococcus faecalis colonies. (a) Inoculation of infected brain heart infusion broth on sheep blood agar plate. (b) Colonies of Enterococcus faecalis on sheep blood agar plates

Group I

Conventional access cavities were prepared and working length was determined using no. 10 K file. All teeth were prepared to an apical size 35 using the step-back technique. The samples were than sterilized by autoclaving. To check the sterility of the sample each sample was dipped in a test tube containing BHI broth. These test tubes were incubated at 37°C and checked for turbidity after 24 h. If no turbidity was present it was further incubated for another 24 h. If still there was no turbidity, the sample was considered sterile. In case turbidity was present the sample was reprocessed. The sample was held with the help of sterile tweezer. BHI broth containing E. faecalis (see the cultivation of E. faecalis) was taken in a sterile insulin syringe and injected into the prepared canal of the sample until it started trickling from the periapical opening. This infected sample was dipped in a test tube containing 5 ml of sterile BHI broth and incubated at 37°C for 1-week. Then, it was rinsed with freshly prepared distilled water and its external surface was wiped with gauge dipped in alcohol. This sample was then dipped into 5 ml of sterile BHI broth and incubated at 37°C. The test tubes were checked for turbidity every 24 h for a maximum period of 72 h. The appearance of turbidity indicated that the sample is infected with E. faecalis. In this case, no turbidity appeared it was reinfected.

Irrigation of the infected samples

For Group 1 irrigation was performed using the positive pressure technique using 3% NaOCl and 17% ethylenediaminetetraacetic acid (EDTA). Irrigation was performed with a 30-G needle and a 5 ml syringe [Figure 2a]. Irrigation was performed with 3% NaOCl for 2 min, followed by 17% EDTA for 1 min, and 3% NaOCl for 2 min. The total final irrigation time was 5 min.

Figure 2

Positive and negative pressure irrigation. (a) Positive pressure irrigation with 30 G needle. (b) Endovac irrigation

Checking the antimicrobial efficacy of irrigation

With the help of sterile H-file dentinal shavings were collected from the irrigated sample. The portion of the H-file with the shavings was cut into a small pieces of approximately 2 mm and dipped into sterile BHI broth and incubated for 48 h at 37°C. In case no turbidity appeared within this period, it was an indicator that the sample has been completely sterilized by the irrigation. In case of turbidity 20 μL of this suspension was inoculated on to the sheep blood agar plates which were incubated at 37°C for 48 h. Colony-forming units were counted after the purity of cultures was confirmed by Gram staining and colony morphology. Observations were recorded and put to the statistical analysis.

Group 2 – All the steps were same as Group 1 except that all samples in this group were prepared to an apical size of no. 45.

Group 3 – All the steps were similar to those of Group 1 except irrigation of infected samples.

Irrigation was done using Endovac (or the negative pressure technique) using 3% NaOCl and 17% EDTA following the manufacturer's recommendations [Figure 2b]. Macroirrigation of each canal was accomplished with 3% NaOCl over 30 s. The open-ended macrocannula was moved up and down in the canal from a point where it started to bind to a point only below the orifice. The macrocannula was then removed quickly so that no air was drawn into the canal space and the canal was left full of irrigant. Microirrigation began immediately after the macro-irrigation with three cycles of irrigation. During a cycle of microirrigation, the pulp chamber was kept full with irrigant, whereas the microcannula was taken to the full working length for 6 s, then lifted 2 mm coronally for 6 s, and then moved back to the working length for 6 s. The second cycle of irrigation used 17% EDTA and the third used 6% NaOCl again. The total final irrigation time was 3 min 30 s for the Endovac groups. The total volume of irrigant used was 10 ml in both groups.

Group 4 – All the steps were same as Group 3, except that the biomechanical preparation for the samples was done up to no. 45.

Data were statistically analyzed using the Fisher's exact test with the two-sided P value and level of significance established at P < 0.05.

RESULTS

Results are tabulated in Table 1. No statistically significant difference was found between Groups 1 and 2, between Groups 3 and 4, and Group 1 and Group 3. Nonsignificant results were obtained when Group 2 and 3 were compared. The statistically significant difference was observed between Groups 1 and 4 with a value of P < 0.01.

Table 1

Results showing positive and negative samples obtained after irrigation

DISCUSSION

Microorganisms and their end products are the main cause of pulpal and periapical pathosis. Owing to the complex root canal system mechanical debridement only reduces the half of bacterial load from the root canals. Along with irrigation, this bacterial load decreases by 50%–80%.[14] This underscores the importance of irrigation in endodontics.

Syringe irrigation (PPI) is the most common and widely used method of irrigant delivery.[15] This method is economical, requires a minimal apparatus and has universal availability. Limitations of syringe irrigation include its inability to clean beyond the main root canal,[16] vapor lock,[17] apical extrusion of irrigant,[18] or hypochlorite accidents.[19] Endovac or the NPI was developed to minimize the risks associated with syringe irrigation. Although costly in comparison it is found to be effective in removing debris from the apical third, smear layer removal, and minimize the risk of apical extrusion of irrigant.[2021] However, conflicting results have been obtained by the studies reporting its clinical antimicrobial efficacy.[111213]

The present study was conducted to ascertain the antimicrobial efficacy of the positive and negative irrigation techniques. E. faecalis was selected for the purpose of the present study, because it is believed to be one of the intracanal bacteria which is most resistant to elimination by disinfecting agents.[22]

The current study found the nonsignificant difference between syringe irrigation groups (Group 1 and 2) although a number of negative cultures were more in Group 2. This is expected as preparing the apical portion of the root canal to the larger instrument size (#45 in Group 2) has been shown to enhance root canal disinfection by reaching inaccessible areas.[23] This provides the potential to remove the intratubular bacteria and open the dentinal tubules to allow antimicrobials to penetrate more effectively. Same holds true for the nonsignificant difference between Group 3 and 4.

Similarly nonsignificant difference was seen between Group 1 and Group 3 which could be due to the reason that although the Endovac microcannula effectively aspirates irrigants in the most apical area of the canal, the clogging of its small perforations by debris may reduce its disinfection effect by reducing the fluid flow in the apical canal.[1224]

Significant results are observed in Group 1 (PPI #30) and Group 4 (NPI #45). The study results are in concurrence with the study of Hockett et al.[11] who found the significant reduction in E. faecalis numbers when compared to the positive control needle group. This can be due to the improved efficiency of irrigation with an increase in apical size preparation,[23] and superior removal of debris leading to better disinfection.[20] These results are in conflict with Brito et al.[12] and Miller and Baumgartner[13] who found no significant differences among the conventional irrigation and Endovac irrigation. In Brito et al. the root canals were instrumented to apical size 25 only. Furthermore, paper points were used to assess the microbial load in vitro which has known limitations.[25] In Miller et al. different methodology was used. They bypassed sampling and pulverized the apical 5 mm of each specimen.

Comparison of Group 2 and 4 also yielded nonsignificant results which indicates that preparing a canal to the larger size may leads to better disinfection[22] irrespective of the method of the delivery of irrigant.

The study design may be favorable to the negative-pressure irrigation groups since the microcannula was introduced to working length while the positive-pressure irrigation needle was introduced 2 mm short of working length. However, this simulates the clinical situation that most practitioners feel comfortable and safe with when using syringe irrigation. Thus, the Endovac can act as a better option when cleaning teeth with the large apical preparations but its high cost may act as a deterrent.

CONCLUSION

Within the limitations of this in vitro study, an apical negative pressure method (Endovac) may be more effective in removing E. faecalis from the root canal at the larger apical preparation size (no. 45).

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.