The effect of NaOCl gel activated by ultrasonic device on bovine dental pulp dissolution: an in vitro study

INTRODUCTION

The main objective of pulp therapy is to prevent and treat lesions [1]. Dissolving the pulp tissues and cleaning the dental canals is considered an essential requirement to achieve this objective.

Sodium hypochlorite (NaOCl) became the primary and most preferred irrigation solution in the daily practice of dental clinics due to its antiseptic, antibacterial, and antifungal properties, in addition to its ability to dissolve pulp tissues [2]. The efficacy of NaOCl is related to its concentration, application time, temperature, and movement [3, 4]. However, an increased concentration of NaOCl is associated with an increased risk of toxicity [5]. Therefore, increasing the intensity by activating the liquid through stirring and raising the temperature of the liquid, decreases this time [6].

The activation of NaOCl solution is more effective when washing root canals, so the use of ultrasound device with NaOCl will help to wash the root canal system [7]. The vibrations caused by an ultrasound device provide a continuous flow of fluid. Thus, allows to remove residues and raise the fluid temperature [8], which improves tissue dissolution properties through its cavitation and acoustic streaming effect [9].

Zand et al. reported that NaOCl gel could be used instead of the solution in an attempt to reduce correlated problems [10]. The gel was significantly effective in removing a smear layer in synergy with EDTA [11] and easily- controlled [12].

To the best of our knowledge, this is the first study to evaluate the potential pulp tissue dissolution with NaOCl gel with or without ultrasonic activation.

MATERIAL AND METHODS

Sample size was calculated based on a pilot study using the program (G* Power 3.1.7 software, Heinrich-Hein- Universität Düsseldorf, Germany; http://www.gpower. hhu.de/), and a total of 50 dental pulp samples were divided into five groups, with 10 samples per group.
This in vitro, randomized, single-blinded experiment was conducted after an approval from the affiliate institutional scientific research ethics committee.

STUDY GROUPS
The study consisted of 50 pieces of dental pulp removed from bovine teeth, divided into five equal groups (n = 10).
Groups by subject:
• group 1: NaOCl solution 2.2% (Clorox®, USA);
• group 2: NaOCl gel 2.2% (Clorox®, USA);
• group 3: NaOCl solution 2.2% (Clorox®, USA) activated by an ultrasonic device;
• group 4: NaOCl gel 2.2% (Clorox®, USA) activated by an ultrasonic device;
• group 5: saline, the negative control.

PREPARATION AND PRESERVATION OF THE SAMPLE
Upper molars were obtained from a butcher shop. Therefore, the study did not have any effect on the animals’ lives and this research is consistent with the ethical principles of the Helsinki Declaration published in 2016 [13].

After extraction, the teeth were washed immediately with running water. The blood and soft tissues attached to the teeth were removed. The teeth were then kept in sealed plastic containers with 0.5% chloramine-T solution for disinfection. After cutting the molars horizontally with separating discs, the pulps were removed from the upper molars. The dental pulp was cut to obtain appropriate samples using a punch of 2 mm in diameter and 2 mm in length. Pulp pieces were placed in tightly sealed containers submerged in distilled water, and preserved at 4°C until their use [14]. The ambient temperature was 25°C when the experiment was initiated.

RANDOMIZATION AND BLINDING
An assistant doctor was requested to give a number from 1 to 50 for each sample using random sampling of samples at https://www.randomizer.org. He was also asked to place the samples in plastic containers and submerge them into one of the five studied materials without the knowledge of the researcher. The groups identity was not revealed until the completion of statistical process in order to maintain the credibility of work without any bias.

MEASURING THE TIME REQUIRED FOR PULP DISSOLUTION
The experiment was filmed with a digital camera (Nikon®, D3200) to determine the time of dissolution of the sample accurately and thoroughly. The samples in all groups were transferred to plastic tested tubes with 3 ml of tested materials. The samples were vortexed with the use of a wooden stick, each for 2 minutes until complete dissolution of the pulp. For groups with an ultrasonic activation, the head of the ultrasound P25 activation device K25 (P5 Booster, Acteon, India) was placed in a tube, and then operated at speed 7, activated for a whole 60 seconds, and repeated for 2 minutes.

STATISTICAL ANALYSIS
IBM SPSS Statistics 23.0 (IBM Corp., Armonk, USA) was used to analyze data at 95% confidence interval. Kolmogorov-Smirnov test showed that the distribution of groups’ data was normal, allowing one-way ANOVA analysis and post-hoc test for LSD.

RESULTS

After the evaluation of descriptive statistics, the values of mean, standard deviation, minimum, and maximum within the 95% confidence interval of the time of dental pulp dissolution were assessed (Table 1). All pulp tissue samples were completely dissolved within the 1-hour test time, except for the negative control. When performing one-way ANOVA analysis between different durations of the groups, the value of significance was p = 0.000 (Table 1).

The post-hoc LSD test showed significantly higher dissolution for all NaOCl groups compared to the negative control. NaOCl solution had significantly lower dissolution time in comparison to the gel type (p = 0.000), and the ultrasonic activation significantly shortened the time of dissolution in both NaOCl solution and gel (p = 0.000) (Table 2).

DISCUSSION

This study aimed to evaluate the effect of commercial sodium hypochlorite gel on the breakdown of dental pulp tissues. Sodium hypochlorite is used by many dentists as irrigation solution, with concentrations ranging from 0.5% to 6% due to its antibacterial properties and its ability to breakdown the pulp tissue [15, 16]. Several studies have shown that an increase of concentration of sodium hypochlorite leads to an increased ability to dissolve pulp tissues [6, 17]. However, at the same time, it increases its side effects. The NaOCl gel was selected not only because of its availability, but because of its transparent texture, which facilitates the performance of experiment. Clorox® gel used in our study is reported to consists of 2.2% sodium hypochlorite and 0.25% of sodium hydroxide solutions with viscosity enhancers. One of the positive characteristics of sodium hypochlorite gel is its ability for easy management and control [12], which is an important factor, particularly in pediatric dentistry.

When treating the canals of primary teeth, low viscosity solutions should be used in irrigation in order not to cross the apical foramen and damage the buds of permanent teeth [18]. NaOCl gel showed an equal antibacterial efficacy in concentration of 2.5% as compared to the same concentration of NaOCl solution [12]. Al Nesser and Bshara study indicated that NaOCl gel can significantly reduce the apical extrusion as compared to a solution when the diameter of apical foramen was ≤ 2.5 mm [19]. The present study showed that the complete dissolution of pulp tissue with 2.2% NaOCl solution took 6.25 min. This was significantly lower than NaOCl gel, which took 30.32 min for a complete lysis. Such a significantly longer dissolution time in NaOCl gel could be attributed to its higher viscosity. A study done by Almeida et al. [20] demonstrated that adding surfactants to NaOCl regardless of its concentration, enhances its efficacy of tissue dissolution, and this might be due to lower viscosity that allows for better contact of NaOCl particles on the surface of tissues. Activating both NaOCl solution and gel significantly lowered the time of dissolution to approximately its half-time, which was consistent with Stojicic et al. study [6]. Ultrasonic activation promotes tissue dissolution effects of NaOCl solution through rising its temperature, and by the improvement of irrigant contact to root canal walls, in addition to its cavitation and acoustic streaming effects; moreover, the rapid movement of this device enhances the shear stress of pulp tissue [21, 22]. A study by Niewierowski et al. also reported reduced dissolution time to its half with an ultrasonic device [23], which is in agreement with the present study. Additionally, it was reported that ultrasonic activation of NaOCl can accelerate chemical reactions and promote superior cleaning action [24]. Even though Leichtweis et al. showed a lower dissolution time when NaOCl solution was activated with ultrasonic device when compared to manual agitation, no significant difference was observed between these two investigated methods [25]. This difference in the results could be attributed to higher agitation time with ultrasonic used in the present study when compared to their research. Based on the aforementioned, we conclude that sodium hypochlorite solution yielded better results with a concentration of 2.2% than sodium hypochlorite gel with a concentration of 2.2%. The activation via ultrasonic device was an efficient way to decrease the time to almost half.

CONCLUSIONS

The 2.2% sodium hypochlorite gel was inferior to the sodium hypochlorite solution and its ability to dissolve the bovine’s pulp tissue. In addition, the activation of ultrasonic device was able to shorten the duration of time to approximately more than half the period for the samples for complete dissolution.

ACKNOWLEDGEMENT

The authors would like to acknowledge the Damascus University for academic support.

CONFLICT OF INTEREST

The authors declare no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

REFERENCES

Trope M. The vital tooth – its importance in the study and practice of endodontics. Endod Top 2003; 5: 1-5.
Zahed M. Sodium hypochlorite in endodontics: an update review. Int Dent J 2008; 58: 329-341.
Rahman S, Whitworth J, Dummer P. Carisolv: an alternative to NaOCl in immature root canals? Int Endod J 2005; 38: 448-455.
Garcia A, Kuga M, Palma-Dibb R, et al. Effect of sodium hypochlorite under several formulations on root canal dentin microhardness. J Investig Clin Dent 2013; 4: 229-232.
Maciel Filho M, Zotarelli-Filho I, Linhares de Castro F. Main predictors of root canal endodontical treatment: systematic review. Int J Dent Oral Sci 2018; 5: 595-600.
Stojicic S, Zivkovic S, Qian W, Zhang H, Haapasalo M. Tissue dissolution by sodium hypochlorite: effect of concentration, temperature, agitation, and surfactant. J Endod 2010; 36: 1558-1562.
van der Sluis L, Versluis M, Wu M, Wesselink P. Passive ultrasonic irrigation of the root canal: a review of the literature. Int Endod J 2007; 40: 415-426.
Ferreira R, Marchesan M, Silva-Sousa Y, Sousa-Neto M. Effectiveness of root canal debris removal using passive ultrasound irrigation with chlorhexidine digluconate or sodium hypochlorite individually or in combination as irrigants. J Contemp Dent Pr 2008; 9: 68-75.
Jiang LM, Verhaagen B, Versluis M, Langedijk J, Wesselink P, Van Der Sluis LWM. The influence of the ultrasonic intensity on the cleaning efficacy of passive ultrasonic irrigation. J Endod 2011; 37: 688-692.
Zand V, Lotfi M, Soroush MH, Abdollahi AA, Sadeghi M, Mojadadi A. Antibacterial efficacy of different concentrations of sodium hypochlorite gel and solution on Enterococcus faecalis biofilm. Iran Endod J 2016; 11: 315-319.
Zand V, Lotfi M, Rahimi S, Mokhtari H, Kazemi A, Sakhamanesh V. A comparative scanning electron microscopic investigation of the smear layer after the use of sodium hypochlorite gel and solution forms as root canal irrigants. J Endod 2010; 36: 1234-1237.
Shamsi PN, Yeganeh LAB, Saberi BV, Parast KF, Kashani AT. Antibacterial Effect of Sodium Hypochlorite Gel and Solution on Enterococcus faecalis. J Dentomaxillofacial Radiol Pathol Surg 2017; 6: 27-30.
Chaudhary DK. Mandatory of Helsinki Declaration and consideration of ethical aspects in human involvement research. EC Microbiology 2016; 6: 801-802.
Kaur R, Singh R, Sethi K, Garg S, Miglani S. Review article irrigating solutions in pediatric dentistry : literature review and update. J Adv Med Dent Sci Res 2014; 2: 104-115.
Mohammadi Z, Shalavi S, Moeintaghavi A, Jafarzadeh H. A review over benefits and drawbacks of combining sodium hypochlorite with other endodontic materials. Open Dent J 2017; 11: 661-669.
Frais S, Ng Y, Gulabivala K. Some factors affecting the concentration of available chlorine in commercial sources of sodium hypochlorite. Int Endod J 2001; 34: 206-215.
Abou-Rass M, Oglesby S. The effects of temperature, concentration, and tissue type on the solvent ability of sodium hypochlorite. J Endod 1981; 7: 376-377.
Nizami SK, Chaudhary P, Lodhi R, Syed M, Nagpal M, Thukral S. Irrigating solutions in pediatric dentistry – a review. World J Pharm Pharm Sci 2018; 7: 357-368.
Al Nesser SF, Bshara NG. Evaluation of the apical extrusion of sodium hypochlorite gel in immature permanent teeth: an in vitro study. Dent Med Probl 2019; 56: 149-153.
de Almeida LHS, e Silva Leonardo NG, Gomes APN, Giardino L, Souza EM, Pappen FG. Pulp tissue dissolution capacity of sodium hypochlorite combined with cetrimide and polypropylene glycol. Braz Dent J 2013; 24: 477-481.
Wright PP, Walsh LJ. Optimizing antimicrobial agents in endodontics. In: Kumavath RN (ed.) Antimicrobial Agents. Croatia: InTech; 2017, pp. 87-107.
Raies Ar, Almarrawi K, Al Nesser S. Evaluation of penetration depth of sodium hypochlorite into dentinal tubules after passive ultrasonic irrigation compared to er;yag laser activation. An in-vitro study. Cumhur Dent J 2020; 23: 5-12.
Niewierowski RS, Scalzilli LR, Dornelles R. Bovine pulp tissue dissolution ability of irrigants associated or not to ultrasonic agitation. Braz Dent J 2015; 26: 537-540.
Plotino G, Cortese T, Grande NM, et al. New technologies to improve root canal disinfection. Braz Dent J 2016; 27: 3-8.
Leichtweis AL, Melo TAF de, Kunert GG. Analysis of the time required for dissolving the pulp tissue according to different methods of sodium hypochlorite activation. Rev Sul-Brasileira Odontol 2015; 12: 8-11.