Evaluation of the actual chlorine concentration and the required time for pulp dissolution using different sodium hypochlorite irrigating solutions.

INTRODUCTION: The goal of root canal treatment is to shape and clean the endodontic space, reducing the bacterial load and removing the pulp tissue. Obviously, the action of the endodontic instruments is limited to the main canals, regardless of the complexity of the endodontic space. Consequently, finding the best possible cleaning technique, which can be obtained chemically using irrigation solutions, is a fundamental aid in endodontic therapy. One of the most commonly used root canal irrigants is sodium hypochlorite (NaOCl), available in various commercial formulations. The effectiveness of NaOCl is undeniable. However, the action of dissolution of the pulp tissue is merely dependent on the concentration and the characteristics of the irrigant itself. AIM: The aim of this study is to evaluate the effective concentration of different commercial formulas of NaOCl, by evaluating the percentage of total chlorine in each product. The dissolution capacity of the pulp tissue of each of the tested products was then analyzed by measuring the required time.
MATERIALS AND METHODS: Three commercial types of NaOCl were selected for this study: 5% NaOCl (ACE, Procter and Gamble), 5% NaOCl (N5, Simit Dental), and 6% NaOCl (CanalPro, Coltene). For each product, 10 packages were used, from which samples of the product were taken and 30 ml × 5 ml tubes were filled. All samples were divided into three groups and were analyzed using the DIN EN ISO 7393-2 method and the percentage of total chlorine (expressed as a percentage) was calculated. Forty samples of vital pulp were obtained from teeth freshly extracted for periodontal reasons and stored in physiological solution. In order to unify the size and weight of the samples (0.0001 mg), a microtome and a precision balance (Pro Explorer Ohaus) were used. Each sample, carefully examined by stereomicroscope (×40), was placed in artificial plastic containers and submerged in 0.1 ml of irrigating solution at room temperature (26°C). A fourth control group used saline solution as irrigant. Simultaneously with the insertion of the irrigating solution, a digital stopwatch was activated and the time necessary for the complete dissolution of the pulp sample was measured. The data obtained was subjected to statistical analysis.
RESULTS: The average percentages of chlorine detected for each group were: 4.26% (ACE), 5.16% (N5), and 5.97% (CanalPro). The Kruskal-Wallis test showed statistically significant differences between the different commercial formulations of hypochlorite (P < 0.05). CanalPro showed the lowest values, whereas ACE showed the highest values of dissolution time of the pulp. DISCUSSION: The analysis of the total chlorine percentage found that the actual concentration of the NaOCl in the samples is close to the values declared by the manufacturers both in the case of N5 and CanalPro. On the contrary, the concentration detected in the samples of common bench bleach (ACE) is significantly lower, which has average values <5%. This explains the longer time taken for the complete dissolution of the pulp tissue. The average dissolution time of the pulp samples was in fact inversely proportional to the concentration detected in the tested irrigants and hence that a lower time corresponds to a higher concentration.

INTRODUCTION

Root canal treatment has to be focused on cleaning, shaping, and sealing the endodontic space[1] to achieve the whole dissolution of the pulp, the removal of microorganisms and the prevention of recontamination.[2345] Endodontic anatomy is complex, and the mechanical action of the files is limited only to the main canals: instruments are made to shape the canal, but the effective cleaning is guaranteed by irrigating solutions. These chemical agents require bacteriostatic or bactericidal potentials and organic/inorganic tissues dissolving effects, so the debridement, the cleaning, and the disinfection of the root canal space can be obtained: for this reason, they are essential for the success of each treatment.[678910] Manual or rotary files are not able to eliminate bacteria from the root canal space.[1112] During instrumentation, irrigating solutions favor flushing out of debris, lubricating the canal, and dissolving organic and inorganic tissue; at the same time, they guarantee an adequate antimicrobial effect.[13] Sodium hypochlorite (NaOCl) is the most commonly used solution, thanks to its high bacteriostatic action, but it is not able to remove the smear layer from the dentin walls.[1415161718192021] NaOCl shows antiseptic properties due to the formation of hypochlorous acid and the subsequent release of chlorine, which is a very active bactericide.[1] Free chlorine in NaOCl dissolves necrotic tissue by breaking down proteins into amino acids; to obtain this effect concentrations ranging from 0.5% to 5.25% have been recommended.[1] Manipulations that enhance the efficacy of NaOCl include warming the solution: increasing the temperature from 22°C to 45°C has been shown to improve both tissue dissolution ability and antibacterial action.[212223]

Endodontic therapy may fail when pathogen microorganism survive in the root canal system.[24] Bacteria, in fact, can survive in adverse conditions: they are able to infiltrate dentinal tubules along canal walls and[2526] and to organize as biofilms, with higher resistance to chemotherapeutic agents and medications or cements than planktonic equivalents.[27] The persistence of bacterial load at the end of the therapy, before obturation, is firmly related to the failure of the treatment.[2829] For this reason, it is necessary to increase the efficacy of disinfecting protocols in endodontic therapy, also considering the complexity of endodontic anatomy. Additional techniques/strategies for obtaining a complete tri-dimensional cleaning have been proposed and tested. New dedicated devices for endodontic irrigation and activation of irrigating solutions have been introduced to minimize or eliminate residual bacteria in the root canal.

5% NaOCl is considered a gold standard as irrigating solution thanks to its well-known bactericidal effect, to its efficacy in dissolving organic matter and in aiding the mechanical flushing of debris upward from root canals.[30] NaOCl effectively eliminates bacteria.[7891011] No agreement has been reached regarding its correct concentration for the use in endodontics, because has to be a compromise between its antibacterial activity and its cytotoxicity. Byström and Sundqvist in 1983[15] suggested the use of 0.5% NaOCl to minimize cytotoxicity, but some doubt has been underlined on its effectiveness NaOCl.[19] The antimicrobial effect depends on the concentration, which ranges from 0.5% to 5.25%. Radcliffe et al.[30] tested the antimicrobial activity of varying concentrations versus different pathogens and observed that Enterococcus faecalis, one of the most resistant to NaOCl, reduced its CFU/ml to zero when exposed to 0.5% NaOCl for 30 min, to 1.0% NaOCl for 10 min, to 2.5% NaOCl for 5 min, or to 5.25% NaOCl for 2 min. It means that the higher is the concentration the higher is its efficacy (considered as a time to kill all the bacteria). Gomes et al.[31] obtained similar results, showing that 0.5% NaOCl required 30 min to destroy bacterial cells of E. faecalis, whereas 5.25% NaOCl <30 s to obtain the same results. That is why they concluded that 5.25% is the ideal and the most effective concentration.

This study aimed to evaluate the effective concentration of different commercial formulations of NaOCl, by evaluating the percentage of total chlorine in each product. For each of the tested products, the dissolution capacity of the pulp tissue was then analyzed, measuring the necessary time.

MATERIALS AND METHODS

Three commercial types of NaOCl were selected for this study: 5% NaOCl (ACE, Procter and Gamble, USA), 5% NaOCl (N5, Simit Dental, Italy) and 6% NaOCl (CanalPro, Coltene, Swiss). For each product, ten packages were used, from which samples of the product were taken and 30 ml × 5 ml tubes were filled. All samples were divided into three groups and were analyzed using the DIN EN ISO 7393-2 method and the percentage of total chlorine (expressed as a percentage) was calculated.

Freshly extracted human mandibular molars (n = 13) were chosen for this study based on a protocol approved by the Institutional Review Board and Ethics Committee. Forty samples of vital pulp were obtained from these teeth freshly extracted for periodontal reasons and stored in physiological solution. Informed consent was obtained from the patients. Within 2 h from the extraction, the crown of the teeth was removed (using a laboratory diamond disk) and the pulp tissue was removed (using microtweezers). To unify the size and weight of the samples (0.0001 mg), a microtome and a precision balance (Pro Explorer Ohaus) were used [Figures 1 and 2]. Each sample, carefully examined by stereomicroscope (×40), was placed in artificial plastic containers and submerged in 0.1 ml of irrigating solution at room temperature (26°C) [Figures 3-8]. A fourth control group used saline solution as irrigant. Simultaneously with the insertion of the irrigating solution, a digital stopwatch was activated and the time necessary for the complete dissolution of the pulp sample was measured.

Figure 1

0.1 mg of pulp tissue

Figure 2

Professional balance

Figure 3

0.1 mg of pulp tissue before inserting the irrigant

Figure 8

Increased magnification showing the complete dissolution of the pulp tissue and sodium hypochlorite in action

0.1 mg of pulp tissue during dissolution

The dissolution of the pulp tissue

Partial dissolution of the pulp tissue

Complete dissolution of the pulp tissue

The data obtained was subjected to statistical analysis using the Stata 12 software (StataCorp. 2011. Stata Statistical Software: Release 12. College Station, TX: StataCorp LP.). The Pearson's correlation index was calculated between the percentage values of total chlorine present in each irrigant and the time values necessary for the complete dissolution of the pulp. The dissolution time values were analyzed with the Kruskal–Wallis nonparametric ANOVA test to evaluate any statistically significant differences between the different commercial formulations of NaOCl (P < 0.05).

RESULTS

The average percentages of chlorine detected for each group were: 4.26% (ACE), 5.16% (N5), and 5.97% (CanalPro). The average dissolution time of the samples of pulp measured was: 1331.76 s (22 min and 12 s) for ACE, 1133.88 s (18 min and 54 s) for N5, and 1011.71 s (16 min and 52 s) for CanalPro. Samples from the control group (saline) showed no dissolution reactions from the pulp. The Kruskal–Wallis test showed statistically significant differences between the different commercial formulations of hypochlorite (P < 0.05). CanalPro showed the lowest values, whereas ACE showed the highest values of dissolution time of the pulp (Tables 1-2).

Table 1

Chlorine values detected for each sample

Group 1ACE	Group 2N5	Group 3CanalPro
4.3	5.2	5.9
4.3	5.1	6.0
4.2	5.2	6.0
4.2	5.2	6.0
4.2	5.2	6.0
4.2	5.1	6.0
4.3	5.2	5.9
4.3	5.1	5.9
4.3	5.2	6.0
4.3	5.1	6.0
Average - 4.26	Average - 5.16	Average - 5.97

Table 2

Dissolution time of pulp samples (expressed in seconds)

Group 1ACE	Group 2N5	Group 3CanalPro	Group 4Control
1317.54	1137.54	1014.32	/
1344.45	1114.25	1018.52	/
1365.22	1143.38	1035.44	/
1312.44	1135.23	1022.34	/
1354.55	1124.43	1005.28	/
1287.24	1107.14	980.12	/
1334.15	1124.25	998.51	/
1304.12	1193.28	1025.14	/
1342.44	1175.13	1012.14	/
1355.45	1084.13	1005.28	/
Average - 1331.76	Average - 1133.88	Average - 1011.71	/

/: seconds

DISCUSSION AND CONCLUSIONS

The main purpose of endodontic treatment is the removal, as complete as possible, of damaged tissues and bacteria from the complex endodontic system. The endodontic system is not simple but must be esteemed as a three-dimensional (3D) entity. In fact, it is not only composed of the main canal but also of lateral canals, ramifications, loops, isthmuses, deltas, and dentinal tubules. Hence the cleaning phase must be active, it also must be in a 3D manner. This 3D cleaning is to ensure that the irrigants can penetrate and act on pulp tissue and bacteria in most of the complex root anatomies.

Naturally, bacteria are able to live either as independent free-floating cells (planktonic state) or be members of colonized surface-attached microbial communities known as biofilms. Biofilms consist of microorganisms that are imbedded in a self-produced extracellular matrix which bind cells together.[323334]

Biofilms have major clinical importance as they provide bacteria with shielding environments against physical traumas, immune responses of the host, antibacterial agents, and antibiotics.[3536] After several years of thorough research, it is now well proven that biofilm formation is a developmental process that begins when a cell attaches to a surface and it is strictly regulated in response to environmental conditions.[36] Briefly, the formation of a bacterial biofilm is a progressive process that begins when a cell attaches to a surface. The formation of microbial biofilms includes several steps that can be divided into two main parts: the initial interactions of cells with the substrate and growth and development of the biofilm.

In root canals of teeth, biofilms have been confirmed by examinations of extracted teeth associated with periapical lesions,[37] sections were viewed by transmission electron microscopy, showed dense aggregates of cocci and rods embedded in an extracellular matrix were observed along the walls.[38] The manner in which biofilm grows contributes to resistance to host defenses, and within the biofilm, there are formed subpopulations of cells that are phenotypically highly resistant to antibiotics and biocides.[39404142] Although there is no overall agreement on mechanism to explain this broad resistance to antimicrobials, the extent of the problem in endodontics is significant.

To explain, biofilms initiate formation when a cell in the planktonic state is deposited on a substratum coated with an organic conditioning polymeric matrix or “conditioning film.”

When the first bacterial cells arrive, there is a weak and reversible contact between the cell and the conditioning film.[43] The next step is when the adhesion of the cell to the substrate is now irreversible. This is partly due to surface attachments overcoming the repulsive forces between the two surfaces and also helped by the sticky exopolymers secreted by the cells. The second part of the formation of a biofilm includes its growth and development. Development of a biofilm occurs as a result of adherent cells replicating and by additional cells adhering to the biofilm.[44]

Biofilm bacteria usually have an increased resistance to antimicrobial agents, in some cases up to 1000-fold greater than that of the same microorganisms living in liquid suspension.[4546]

The reasons for the increased resistance of bacteria when forming a biofilm are believed to be multiple: physical and acquired mechanisms. The physical protection is mainly related to the impaired penetration of antibiotics through the biofilm matrix. While, acquired resistance consists of differentiation of cells with low metabolic activity, differentiation of cells that actively respond to stress, and differentiation of cells with a very high persistent phenotype.

Until now, the most common and efficient antibiofilm strategy used in root canal therapy is the removal with mechanical instrumentation and irrigant activation.[47484950]

Activation of irrigants through sonic, ultrasonic, internal heating, or laser devices has shown great improvement in the cleaning and disinfection of the root canal system and should be considered an important fundamental step in nonsurgical endodontic therapy.[5152535455]

Torabinejad and Walton[1] outlined the ideal properties of an endodontic irrigating solution: organic and inorganic tissue solvent, antimicrobial action, nontoxicity to periapical tissues, low surface tension, and lubricant action. No one solution as yet possesses all the properties of an ideal irrigant. NaOCl is still the most preferred irrigating solution, thanks to its numerous advantages: it is an excellent antibacterial agent, capable of dissolving necrotic tissue, vital pulp tissue, and the organic components of dentin and biofilm; in addition, it is inexpensive, with a long shelf life, and it is easily available.[114]

In the current study, the analysis of the total chlorine percentage found that the actual concentration of the NaOCl in the samples is close to the values declared by the manufacturers both in the case of N5 and CanalPro. On the contrary, the concentration detected in the samples of common bench bleach (ACE) is significantly lower, which has average values <5%. This explains the longer time taken for the complete dissolution of the pulp tissue. The average dissolution time of the pulp samples was in fact inversely proportional to the concentration detected in the tested irrigants and hence that a lower time corresponds to a higher concentration.

In conclusion, it is important to use techniques that activate irrigants and above all, based on this study, use NaOCl products manufactured exclusively for Endodontics.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.