Effect of final irrigation with sodium hypochlorite at different temperatures on postoperative pain level and antibacterial activity: a randomized controlled clinical study

Abstract Objective To evaluate the effect of final irrigation of root canals with NaOCl solution at different temperatures on postoperative pain level and antimicrobial activity. Methodology 45 patients were randomly divided into three groups using a web program according to the irrigation selected: NaOCl 2ºC, NaOCl 25ºC and NaOCl 45ºC. First root canal samples were collected before treatment (S1). After chemo-mechanical preparation, final irrigation was performed with the selected irrigant (NaOCl 2ºC, NaOCl 25ºC and NaOCl 45ºC) and second samples were collected (S2). Samples were subjected to quantitative real-time polymerase chain reaction to evaluate the levels of total bacteria. The root canal treatments were completed and the participants were given instructions to record postoperative pain levels at 24, 48 and 72 hours, 5 days and 1 week after treatment using a visual analog scale (VAS). Results The reduction in the number of total bacterial cell equivalents from S1 to S2 was statistically significant in all groups (p<0.001). The NaOCl 2˚C group reported significantly less postoperative pain than the NaOCl 45˚C group (p<0.05). Postoperative analgesic intake was significantly higher in the NaOCl 45˚C group than in the NaOCl 2˚C group (p<0.05). Conclusion We conclude that final irrigation with NaOCl at different temperatures results in similar antibacterial effectiveness. Final irrigation with cold NaOCl (2˚C) is better than NaOCl 45˚C when comparing postoperative pain levels.


Introduction
Sodium hypochlorite (NaOCl) is the most common irrigant used in endodontics because of its antibacterial and physicochemical properties 1 and its unique ability to dissolve necrotic tissue remnants. 2 Mechanical instrumentation and NaOCl irrigation have shown to dramatically reduce the bacterial load in the root canal system. 3 Unfortunately, irrigation with NaOCl during chemo-mechanical debridement does not always result in bacteria-free root canals. 4 Achieving bacteria-free root canals is critically important, since several studies reported that the presence of bacteria at the time of root filling significantly influences the outcome of endodontic treatment. [5][6][7] Therefore, several attempts have been made to increase NaOCl antimicrobial effectiveness, such as passive ultrasonic irrigation as a supplementary disinfecting step 8 and using NaOCl with a higher concentration, 9 volume 10 or temperature. 11 Previously, Sirtes, et al. (2005) reported that NaOCl at 45˚C has better antibacterial efficacy than at 20˚C. 11 However, preheated NaOCl superior antibacterial action and effect on postoperative pain level have not yet been clinically demonstrated; one clinical study reported that the temperature of preheated NaOCl (60˚C) starts to decrease as soon as the solution touches the root canal wall, reaching body temperature (35.7˚C) within a few minutes. 12 Due to the ability of NaOCl to kill microorganisms in seconds, 13 and the lack of information about the clinical antibacterial efficacy of preheated NaOCl, it is important to investigate if preheated NaOCl exerts an additional antibacterial effect and affects the postoperative pain level clinically, since the temperature of the irrigation solution is known to affect postoperative pain. 14,15 Consequently, there are no clinical studies to support the use of preheated NaOCl for root canal irrigation. Our randomized controlled clinical study aimed to evaluate the effect of final irrigation of root canals with NaOCl solution at different temperatures on postoperative pain level and antimicrobial activity. Teeth with necrotic pulps, clinically confirmed by pulp sensibility tests and the absence of bleeding on accessing the pulp chamber, no previous history of endodontic treatment, a pocket depth of <3 mm and having a periapical lesion with a periapical index score of 3, 4 or 5 (Ørstavik, et al. (1986)) were included. 17 Exclusion criteria: patients that underwent treatment with antibiotics or NSAIDs within 1 month before the study; patients with any systemic disease; teeth that had received previous endodontic treatment; teeth with extensive destruction of the crown that prevented proper rubber dam isolation; and the presence of internal or external resorption. A web program (www.randomizer.org) was used to randomly assign the 45 participants into three groups (n=15).
After randomization, the number of each group and of each patient were recorded. All the treatments were performed by one clinician (N.A.). Blinding the clinician to the groups was not feasible because of the recognizable temperature of the syringe. However, the patients were blinded to the groups.
Root canal samples were collected under strict aseptic technique. Before treatment, an oral rinse was performed with 0.12% chlorhexidine and before isolation of the teeth with the rubber dam, supragingival scaling was performed to remove plaque, followed by cleansing with pumice. Before the access cavity preparation, 30% H 2 O 2 and 2.5% NaOCl for 30 s was used for disinfection of the crowns and surrounding structures (dam and clamp). After the Effect of final irrigation with sodium hypochlorite at different temperatures on postoperative pain level and antibacterial activity: a randomized controlled clinical study J Appl Oral Sci. 2021;29:e20200502 3/8 disinfection protocol, the NaOCl was inactivated using 5% sodium thiosulfate. Access cavities were prepared using sterile round burs under cooling with sterile saline solution. The disinfection protocol described previously was performed again after completing access cavity preparation. To prevent the penetration of disinfectants into the pulp chamber and root canals, a sterile cotton pellet was placed on the floor of the pulp chamber. Sodium thiosulfate was used to neutralize NaOCl effectiveness and first sterility control samples were taken with sterile paper points from the coronal surface of the tooth, rubber dam, clamp and access cavity walls. Paper points were transferred to Eppendorf tubes containing a Tris-EDTA buffer (10 mmol/L Tris hydrochloride and 1 mmol/L EDTA, pH 7.6) and then the samples were kept at -80ºC until bacterial presence was assessed with quantitative polymerase chain reaction (qPCR). The teeth with negative sterility control samples for bacterial presence in the qPCR assay were included in our study.
After working length determination with an electronic apex locator (Raypex 6; VDW, Munich, Germany), the root canals were filled with sterile saline solution, not allowing them to overflow, and then a gentle filling motion was performed with a sterile #15 K-file (Mani, Tochigi, Japan). To obtain the first bacteriological sample (S1), paper points (Dentsply Maillefer, Ballaigues, Switzerland) were used to soak up the intracanal fluid in the root canal. The sampling procedure was repeated using three paper points and each paper point was left in the root canal for at least 60 seconds. Then the paper points were transferred to a tube containing a Tris-EDTA buffer. Any contact between the paper points and the cavity walls was avoided to prevent contamination during transfer of the paper points into the tubes.
Root canals were prepared using Reciproc files (R25, R40 or R50) (VDW, Munich, Germany), according to the manufacturer recommendations. During preparation, root canal irrigation was performed using 1 mL of 1% NaOCl between three pecking motions of the file and final irrigation was performed with 5 mL of 17% EDTA followed by 5 mL of 1% NaOCl (at different temperatures). According to the final irrigation, the groups were divided as follows: NaOCl 2ºC: Syringes filled with 5 mL of 1% NaOCl were kept in a fridge at -2 ºC. Before the irrigation procedure, the syringe was removed from the fridge and when the temperature of the syringe, which was checked with a thermometer, increased to 2ºC, root canal irrigation was performed for 1 minute (the room temperature was 24.2ºC).
NaOCl 25ºC: A CanalPro syringe heater (Coltene, Altstätten, Switzerland) was used to heat a syringe filled with 5 mL of 1% NaOCl. The syringe was left in the device for 30 seconds to reach an average temperature of 26±0.2ºC, because the device cannot be set to a specific temperature. Then the syringe was removed from the device and when the temperature of the syringe, which was checked with a thermometer, decreased to 25ºC, root canal irrigation was performed for 1 minute (the room temperature was 24.2ºC).
NaOCl 45ºC: A CanalPro syringe heater (Coltene, Altstätten, Switzerland) was used to heat a syringe filled with 5 mL of 1% NaOCl. The syringe was left in the device for 20 minutes to reach an average temperature of 48±0.2ºC, because the device cannot be set to a specific temperature. Then the syringe was removed from the device and when the temperature of the syringe, which was checked with a thermometer, decreased to 45 º C, root canal irrigation was performed for 1 minute (the room temperature was 24.2ºC).
After that, 2 mL of 0.5% sodium thiosulfate was used to inactivate the NaOCl and then the root canals were finally irrigated with distilled water. Second samples (S2) were taken as described above.
Next, paper points were used to dry the root canals

Statistical analysis
The Poisson regression model, which is the basic approach for modeling bacterial count data, was used as previously described. 4 For intragroup analysis, the Mann-Whitney U test was used to compare reduction of counts of total bacteria between the two samples (S1 and S2), since the data were not normally distributed.
The chi-square test was used to compare the number of root canals positive for bacteria in S1 and S2 among the NaOCl 2ºC, NaOCl 25ºC and NaOCl 45ºC groups.
The number of bacteria in S1 and S2 samples and the reduction (%) in the number of total bacteria from S1 to S2 among the groups were compared using the Total bacterial counts Table 2 shows total bacterial counts, which were analyzed using universal 16S rRNA gene-based primers, for S1 and S2 samples according to the groups. In the NaOCl 2˚C, 25˚C and 45˚C groups, a mean number of 6.47×10 6 , 5.18×10 6 and 5.76×10 6 bacterial counts in S1 was decreased to a mean of 3.66×10 5 , 2.7×10 5 and 3.31×10 5 bacterial counts in S2, respectively. The reduction in the number of total bacterial cell equivalents from S1 to S2 was statistically significant in all groups (p<0.001). The percentage of reduction was 94.7, 95 and 93.9 for the NaOCl 2˚C, 25˚C and 45˚C groups, respectively. There was no statistically significant difference among the groups in terms of percentage of reduction of total bacterial counts (p>0.05).
The number of root canals positive for bacteria was also evaluated. Whereas all the S1 samples were positive for bacteria in all groups, 3 root canals from the NaOCl 2˚C group, 3 root canals from the NaOCl 25˚C group and 2 root canals from the NaOCl 45˚C group became negative for bacteria in S2 samples (Table 3). The difference among the groups in the number of root canals positive for bacteria was not statistically significant (p>0.05).

Postoperative pain
Linear regression analyses revealed that postoperative pain level on day 1 was only influenced by the group (p<0.05). Sex, age and tooth number did not influence the postoperative pain level on day 1 (p>0.05). (Table 4).
T h e m e a n p o s t o p e ra t i ve p a i n l e ve l wa s 6.67±10.722, 16.87±27.604 and 40.0±50.709 for the NaOCl 2˚C, 25˚C and 45˚C groups, respectively.
(   pain than the NaOCl 45˚C group (p<0.05). However, there was no statistically significant difference between the NaOCl 2˚C and NaOCl 25˚C groups and between the NaOCl 25˚C and NaOCl 45˚C groups in postoperative pain level on day 1 (p>0.05). Moreover, there was a statistically significant difference among the groups in postoperative analgesic intake. The number of patients that needed analgesic was 0, 2 and 5 for the NaOCl 2˚C, NaOCl 25˚C and NaOCl 45˚C groups, respectively. Postoperative analgesic intake was significantly higher in the NaOCl 45˚C group than in the NaOCl 2˚C group (p<0.05). However, there was no statistically significant difference between the NaOCl 2˚C and NaOCl 25˚C groups and between the NaOCl 25˚C and NaOCl 45˚C groups in postoperative analgesic intake (p>0.05).

Discussion
Our study compared the effect of NaOCl with different temperatures on elimination of bacteria from root canals and postoperative pain level. Since there was no significant difference among the groups in antibacterial effectiveness, but the temperature of the solution affected the postoperative pain level, the null hypothesis was partially rejected. According to the results of our study, chemo-mechanical preparation and final irrigation with EDTA + NaOCl was highly effective in significantly reducing the intracanal bacterial counts, irrespective of the NaOCl temperature. This is in agreement with previous studies, which reported statistically significant reduction of bacterial counts by chemo-mechanical preparation. 4,10,18 However, there was no statistically significant difference among the groups, when comparing NaOCl with different temperatures, in the removal of bacteria from root canals. Several studies investigated the antibacterial effect of NaOCl with different temperatures and conflicting results have been reported 11,[19][20][21]et al. 11 (2005)  Moreover, they incubated the bacteria in the NaOCl solution for 10 minutes. Giardino, et al. 20 (2016) also assessed the NaOCl antibacterial efficacy after a 10-minute contact, which is a relatively longer contact time when compared with clinical conditions. Moreover, both studies were culture-based; which are less sensitive than the qPCR, which can detect as-yetuncultivated bacteria. 24 The methodological differences mentioned above could explain the difference in the findings of our study and the previous ones. Our findings could be explained by the strong antibacterial effect and the NaOCl concentration (1%) used in our study was efficient for killing sufficient bacteria in the root canal system. NaOCl can kill bacteria even at concentrations lower than 0.1%. Additionally, and consistent with our results, et al. 19 (2015) compared NaOCl solutions with a concentration of 1% at different temperatures (22°C and 37°C) in terms of antibacterial efficacy and concluded that the temperature variation of the NaOCl is not relevant in killing or dissolving bacterial biofilms. Likewise, Gulsahi,et al. 21 (2014)   be speculated that NaOCl with a concentration of 1% exerts a strong antibacterial efficacy that provides a substantial reduction in bacterial counts in the root canal system, regardless of the solution temperature.
Our study also evaluated the effect of the NaOCl at different temperatures on the level of postoperative pain and showed that root canal irrigation with NaOCl at 45°C resulted in a significantly higher postoperative pain value than NaOCl at 2°C. This finding is in accordance with previous studies that reported less postoperative pain with the application of intracanal cryotherapy. 14, 25 Cryotherapy leads to reduced cellular metabolism by dropping local temperature, which causes reduced blood flow. 26 Consequently, the effect of cryotherapy on limiting inflammation may explain the reduced postoperative pain value in NaOCl 2°C group. 27 However, the most important finding of our study was that, although there was no statistically significant difference between the NaOCl 25°C and NaOCl 2°C groups, postoperative pain reached the highest level in the NaOCl 45°C group, with a significant difference when compared with the NaOCl 2°C group. This means that using preheated NaOCl for root canal irrigation results in a higher postoperative pain value. Additionally, in the NaOCl 45°C group, the need for analgesic intake was significantly higher than in the NaOCl 2°C group. There are no previous studies evaluating the effect of intracanal irrigation with preheated NaOCl on the level of postoperative pain, therefore, a direct comparison cannot be performed.
However, the postoperative pain-enhancing effect of the preheated NaOCl can be explained by the fact that heat increases tissue temperature, which results in vasodilatation. 28 Vasodilatation increases blood flow and allows leukocytes and plasma proteins to exit the circulation, which may cause an increase in inflammatory response, thus increasing postoperative pain.
One of the limitations of our study is that the temperature of the solution in the root canal was not constant during the irrigation procedure. De Hemptinne, et al. 12 (2015) showed that the solution temperature (45°C) decreased to body temperature (37°C) in 60 seconds when the solution was used for root canal irrigation in vivo. However, since the NaOCl exerts its antibacterial efficacy in seconds, 13 60 seconds is enough to assess the antibacterial efficacy of the preheated NaOCl solution. Moreover, it can be claimed that a decrease in the temperature of the solution had no significant effect, since the preheated NaOCl group showed higher postoperative pain values than the NaOCl 2°C group. This means that the effect of the heat was clearly shown in our study.

Conclusion
We conclude that preheating NaOCl does not provide any extra antibacterial effect and results in a higher postoperative pain value than the cold NaOCl when used for final irrigation of root canals of teeth with asymptomatic apical periodontitis.

Conflict of interest
The authors declare no conflict of interest.