Influence of surfactants addition on the properties of calcium hypochlorite solutions

Abstract Objectives The aim of this study was to evaluate the influence of surfactants 0.2% or 0.1% cetrimide (Cet) or 0.008% benzalkonium chloride (BAK) on 2.5% calcium hypochlorite (Ca(OCl)2), and compare to sodium hypochlorite (NaOCl), regarding the properties of pH, free chlorine content, surface tension, contact angle, pulp dissolution and antimicrobial activity. Material and Methods The pH and free chlorine content were evaluated by digital pHmeter and by titration, respectively. Surface tension was measured by the platinum ring technique with a Du Noüy tensiometer. The solution's contact angle in human dentin surfaces was checked by Drop Shape Analyzer software. Bovine pulps were used for pulp dissolution analysis and the dissolving capacity was expressed by percent weight loss. Antimicrobial activity over Enterococcus faecalis was evaluated by the agar diffusion method. Results Surfactants addition to Ca(OCl)2 and NaOCl did not alter the pH, free chlorine content and pulp dissolution properties. Ca(OCl)2 had the highest surface tension among all tested solutions. When surfactants were added to Ca(OCl)2 and NaOCl, there was a significant reduction of surface tension and contact angle values. The addition of 0.2% or 0.1% Cet enhanced antimicrobial activity of both Ca(OCl)2 and NaOCl. Conclusion Surfactant addition to 2.5% Ca(OCl)2 has shown acceptable outcomes for pH, free chlorine content, surface tension, contact angle, pulp dissolution and antimicrobial activity. Furthermore, the addition of 0.2% Cet showed better results for all tested properties.


Introduction
Root canals present a complex internal anatomy with irregularities and isthmus, which may not be accessible during instrumentation. Hence, irrigation is an important step of the root canal treatment and should allow the irrigant to reach those areas. 1 The ideal root canal irrigant should have antimicrobial spectrum, including action against biofilms, dissolve pulp and necrotic tissue, inactivate endotoxin, prevent/remove smear layer and not injure the periapical tissue in case of accidental contact. 1 Moreover, irrigants with lower surface tension may penetrate more in dental tubules, with greater antimicrobial activity. 2 Sodium hypochlorite (NaOCl) is the most used irrigant solution in clinical practice, because it has the unique capacity to dissolve necrotic tissue and organic components of the smear layer. 1 However, its chemical instability can influence the availability of chlorine ions and interfere with the desired characteristics of the irrigant. 3 In this regard, other auxiliary chemicals should be studied.
Calcium hypochlorite (Ca(OCl) 2 ) is a white powder that can be dissolved in distilled water, used for industrial sterilization and purifying water treatment. 4 It is relatively stable and shows more available chlorine content than NaOCl. 3,5 Studies demonstrated the potential of Ca(OCl) 2 solutions to dissolve tissue 5,6 and antimicrobial action against E. faecalis 7 . Also, Ca(OCl) 2 showed favorable results of viability and induced a low-level inflammatory response when compared with NaOCl. 8 Nevertheless, aqueous solutions of NaOCl and Ca(OCl) 2 have high surface tension values. 3,7,9 Surface tension is related to the wetting ability, surface free energy or capillarity effects. [10][11][12] It allows solution penetration into both the main and lateral canals and into the dentinal tubules. 1,12 On the other hand, surface free energy is the result of intermolecular interactions between the surface and the liquid. It can be expressed as the contact angle between a drop of liquid on a solid surface. 10,11 The lower the surface tension and contact angle, the better the wettability potential of the solution. 11 Cetrimide (Cet) is a cationic surfactant (quaternary ammonium salt) available as a white odorless powder and, according to the manufacturer, it is highly hygroscopic. It has the capacity of lowering surface tension, 12 and antimicrobial activity. 13-15 Benzalkonium chloride (BAK) is also a cationic surfactant, already used in dental clinical practice and associated with some endodontic irrigants. 16 The antibacterial potential of BAK is associated with changes promoted on the ionic resistance of the cell membrane. 17 The addition of surfactants to NaOCl as an alternative to decrease their high surface tension has been reported in the past. 9 Rio Grande do Sul, Brazil), was mixed with 100 mL of sterile distilled water. All solutions were prepared under constant agitation and were stored in bottles identified with random numbers for blinding.

Contact angle measurement
Thirty single-rooted extracted human teeth were obtained. The crown was removed at the cementoenamel junction, and the roots were longitudinally sectioned in two halves, mesial and distal. Each half was transversely divided in 2 pieces, cervical and apical.
Four dentin specimens were obtained from each tooth.
A total of 120 specimens (10 per group) were obtained, and stratified so that each group received 5 cervical and 5 apical specimens. The surfaces were polished under running water using 80-, 100-, 120-, 150-and 180-grit abrasive papers to obtain a flat wide dentin surface. 10,16,20 Contact angle measurements were achieved at 22°C by using a drop shape analysis system DSA100 (Kruss; Hamburg, Hamburg, Germany). A drop with 2 µl volume of tested solution was carefully placed with a micropipette on each dentin surface. After 30 seconds, the image of the droplet was obtained and the contact angle was calculated. 10,16,20 Three parallel measurements were performed with each tested solution on both cervical and apical thirds.
Dentin specimens were polished to create a smooth and flat dentin surface, 16 otherwise the program would not be able to measure contact angle inside the concave surface of root canal walls. The samples did not receive any superficial treatment, in order to simulate the presence of smear layer during root canal preparation and to observe solutions ability to penetrate in dentine tubules.

Pulp dissolution test
Bovine pulp tissue was employed for the experiment.
Teeth were extracted and stored at -20°C until required.
The crowns were removed at the cementoenamel junction and the pulp tissue was removed. One-hundred recording, the specimens were randomly divided.
The pulp fragment was placed in a cell culture well and each specimen was irrigated with a total volume of 10 mL of solution, for 10 minutes. One mL of the solution was replaced every minute. At the end of the irrigation time, the specimens were removed from the wells and left in contact with a sterile absorbent paper for 30 seconds to remove the excess solution. The specimens were weighed to assess their final weight.
The percentage of weight loss was determined for each sample. [23][24][25] All specimens were weighed by a single investigator, which was unaware of how each was to be treated.

Antimicrobial test
The agar diffusion method was used to measure the antimicrobial activity. The test was performed in

Contact angle measurement
Representative images of the analyzed groups (DW, NaOCl, Ca(OCl) 2 and BAK) can be found in Figure 1. There was no statistical difference among cervical and apical specimens when the same solution was evaluated (p>0.05). Statistical difference was observed between Ca(OCl) 2 and BAK, in both cervical (p=0.042) and apical (p=0.041) thirds. Ca(OCl) 2 presented higher contact angle than BAK (Table 4).

Pulp dissolution test
NaOCl solutions, with and without surfactant addition, dissolved more pulpal tissue than all other tested solutions. Ca(OCl) 2 solutions presented pulpal dissolution ability. Surfactants alone did not have dissolution ability, as well as DW (Table 5).

Antimicrobial test
Ampicillin (positive control) had the highest antimicrobial inhibition zone. All solutions, except DW,  had antimicrobial activity against E. faecalis. Addition of 0.2 or 0.1% Cet to NaOCl and Ca(OCl) 2 enhanced their inhibition zone (p<0.05) ( Table 6).

Discussion
Root canal irrigation plays a fundamental role in clinical endodontic success. The main irrigant should be able to spread along all root canal structures, enabling the clinician to promote better cleaning and shaping.
The addition of surfactants to the Ca(OCl) 2 solution reduced its surface tension, possibly enhancing its wetting ability, which may enable a better diffusion of the irrigant on dentin walls, 16 leading to improved action of the solution during endodontic treatment.
A digital pHmeter was used to determine the pH of solutions, as already described by other studies. 3,12,15,[26][27][28] Ca(OCl) 2 and NaOCl are strongly alkaline solutions. 3,15,18,26 Even though the surfactants showed a lower pH than Ca(OCl) 2 and NaOCl, their addition to the solutions did not alter their pH, agreeing with other study. 18 This could be related to the lower concentration of surfactants compared to Ca(OCl) 2 and NaOCl (two and three orders of magnitude lower for cetrimide and BAK, respectively).
Since surfactants did not alter the pH of solutions, they may not affect the properties directly related to it, as antimicrobial activity and pulp dissolution.
Additionally, the non-polar long tail group of the surfactants and the low concentration used in the present work allow dissolution into a low surface free energy interface, such as the pulp. At lower pH, available chlorine is in the form of hypochlorous acid, 1 which has greater antimicrobial activity and is more cytotoxic. 26 Moreover, solution becomes more unstable, losing a significant amount of free chlorine content and, consequently, its action potential. 29 On the other hand, at higher pH values, available chlorine is in the form of hypochlorite ions, 1 which has less antimicrobial activity but is also less cytotoxic. 29 The hypochlorite concentration can be affected by many factors, such as temperature and storage conditions, 3,15 and for this reason it is important to know quantitatively the free chlorine content before use. In the present work the standard thiosulphate titration method was used to obtain accuracy in actual hypochlorite concentration. 3,29 The titration results showed that surfactant addition did not alter Ca(OCl) 2 and NaOCl free chlorine content, as already described for NaOCl solutions. 16,18,27 This could be explained by the low concentration of surfactants used in this study, which would not be able to affect chlorine concentration of Ca(OCl) 2

and
NaOCl. In addition, the titration results showed that all hypochlorite solutions prepared had a higher amount of chlorine than 2.5%, demonstrating the importance of the thiosulphate titration method to know the actual hypochlorite concentration. Also, analysis of the free chlorine content of solutions allows knowing their ability to act against microorganisms and organic tissue.
Facultative bacteria, such as Enterococus faecalis have been considered one of the most critical endodontic pathogens in endodontic infections, and its presence can lead to root canal treatment failure. 30 Although Ca(OCl) 2 has more hypochlorous acid release, 5 there was no difference in antimicrobial activity when compared to NaOCl, agreeing with other authors. 7 The theoretical concentrations of free OClused in the present study for Ca(OCl) 2     However, this is one of the most common tests used for evaluating antibacterial activity against a chemical agent, and, also, it can be considered a relevant protocol. 32 Dissolution has been studied in bovine pulp tissue, 2 human pulp tissue, 33 bovine muscle tissue. 5,20 Concerning the irrigant, few studies evaluated the tissue dissolution ability of Ca(OCl) 2 . 5,6 None of the tested surfactants had pulp dissolving ability, which can be explained by their absence of free chlorine content. Therefore, surfactant addition did not alter the dissolution ability, agreeing with other authors, who tested NaOCl at different concentrations. 24,27,33 However, other authors found that surfactant addition resulted in better NaOCl dissolution ability. 20,27 The results showed that Ca(OCl) 2 presented lower dissolution ability than NaOCl. One of the concerns related to NaOCl dissolution ability is the harm to periapical tissues in case of accidental outflow. 1 It has been suggested that Ca(OCl) 2 could be less aggressive to periapical tissues. 5 In this regard, Ca(OCl) 2 could have favorable results of viability and induced low-level inflammatory response, mainly in infected immature teeth treatment.
The present study observed that specimens immersed in Ca(OCl) 2 developed a white surface coating on the entire pulp fragment, as already described. [3][4][5] According to Dutta, et al. 5  The value is calculated by the equipment and was expressed in mN/m, but it can also be expressed in other measurement unit such as dyne/cm or mJ/m 2 .
Since the ring method does not consider the dentin surface, 16 it is important to analyze not only the surface tension but also the contact angle. Surface tension reduction and lower contact angle values can improve the contact between irrigant and the dentin walls. 9,12 If the contact angle value is lower than 90°, the substrate is wetted by the liquid; if the contact angle is greater than 90°, the liquid is considered nonwetting. A contact angle value equal to zero represents complete wetting. 11 Thus, the lower the contact angle, the faster the liquid will spread through the dental surface. 10 The addition of surfactants in both Ca(OCl) 2 and NaOCl resulted in complete wetting of the dentin specimens. Among surfactants, only BAK solution had a contact angle measured by the software, yet this did not occur when it was added to hypochlorite. irregularities and natural moisture of the dentin, will influence this behavior. This is the first study to evaluate surfactant addition to Ca(OCl) 2  Furthermore, the addition of 0.2% Cet showed better results for all tested properties.