The effect of temperature on the tissue dissolution capacity of 10% calcium hypochlorite solution

INTRODUCTION

Disinfection of root canal is essential for the successful outcome of endodontic therapy. Root canal space is complex with many ramifications which are not amenable for instrumentation. This makes the use of chemicals in the form of root canal irrigants mandatory during cleaning and shaping of the root canal.¹ An ideal irrigant should be microbicidal, low in toxicity and possess the ability to flush debris and dissolve organic matter.^2,3 The most universally accepted irrigant for the enclosed root canal space containing infected organic matter is sodium hypochlorite (NaOCl).⁴ Sodium hypochlorite is antibacterial and has the ability to dissolve pulp tissue in healthy or necrotic state.^5-7 The tissue dissolution ability is dependent on its concentration and on the total available chlorine content.^8-10 Despite the benefits, high cytotoxicity of 5.25% Sodium hypochlorite has always been a serious concern.¹¹ Several chemical agents like chlorhexidine gluconate, MTAD have been investigated in the quest for a less toxic, but equally potential substitute for Sodium hypochlorite.

Calcium Hypochlorite was widely used in industries for disinfection, purification of water and as household bleach. Prior research has proven that calcium hypochlorite causes less tissue irritation and contains more available chlorine than NaOCl. Based on these findings, calcium hypochlorite could have been suggested as a viable alternative to sodium hypochlorite for intra canal irrigation. But, the rate of tissue dissolution of NaOCl is superior to Ca(OCl)2 when used at room temperature.¹²

Increasing the temperature of the solution prior to use may enhance the tissue dissolution rate of Ca(OCl)2 . The purpose of this study was to assess the effect of rise in temperature on the rate of tissue dissolution by Ca(OCl)2 solution.

MATERIALS AND METHODS

This study was conducted in the Department of Conservative Dentistry and Endodontics, Government Dental College and Research Institute, Bangalore. The duration of the study was about 4 weeks including the time taken for sample collection. Ethical clearance for this study was obtained from the institutional ethic committee and review board of Government Dental College and Research Institute, Bangalore

Calcium Hypochlorite Solution

Ca(ClO)2 solution was made freshly from Ca(ClO)2 granules (Sigma-Aldrich, Bangalore, India) at the time of the experiment. A solution of 10% strength was prepared by mixing 10 grams of Ca(ClO)2 in distilled water to make 100 ml of solution using a magnetic stirrer for 10 minutes. The free available chlorine was assessed using iodine, thiosulphate titration assay.¹

Temperature measurements

Forty four 10-ml syringes were filled with the Ca(ClO)2 solutions of 250 C and heated in a syringe warming device (Aerne Analytic) until they reached 450 C and 600 C, respectively. The irrigant temperatures in the syringes were measured using a calibrated electronic thermometer.

The amount of available chlorine in solution after heating was reassessed using iodine titration method. Ca(ClO)² solution of 25o C was kept in a water bath to maintain the temperature constant.

Sample preparation

Thirty five human single rooted mandibular premolars extracted for orthodontic reasons were collected from the Department of Oral and Maxillofacial surgery, Government Dental College, Bangalore. Patients volunteered to donate their teeth, which required extraction as per their individual treatment strategy, for research. Informed consent was taken from all patients. Teeth were immediately stored in a freezer at a temperature of -27o C, until further use. Teeth were not treated with any chemicals to eliminate any possible interference with the dissolution assay.

Tissue Dissolution Assay

The teeth were thawed in sterile 0.9% saline before sectioning. Pilot grooves were prepared in the crowns, buccal and lingual aspects without entering the pulp chamber using a diamond- coated fissure bur (SS White). Teeth were then carefully split into two fragments using a micro chisel and the pulp tissue was removed in toto using a spoon excavator. Before immersion in test solutions, the pulps were blotted dry with a cellulose membrane, was trimmed using no.15 BP blade and weighed using a precision balance (AT261 Delta range, Mettler Toledo). The specimen weight was standardized to 9.5mg. Thirty pulp tissues were divided into 3 groups (n=10) and each pulp was transferred into individual test tubes tempered to a temperature of 32oC containing 2ml of the test solutions, Ca(ClO)2 solution at 25o C, at 45o C and at 60o C. The remaining five specimens were immersed in physiologic saline solution which served as the control. The test tubes were agitated manually. The tissue was then carefully removed, blotted dry on absorbent paper, and weighed on the precision balance after five minutes. The solution was discarded and the sample re immersed in a fresh two ml of test solution. This process was repeated at regular intervals of five minutes until complete dissolution or up to 60 minutes whichever was appropriate. Absence of visible remnants was considered as complete dissolution. The percentage difference in weights for each sample after each time interval was calculated and the mean percentage loss determined.

STATISTICAL ANALYSIS

The sample size for the present study was computed by using Power analysis at confidence interval 95% with level of significance set at P<0.05. The results were subjected to statistical analysis using one –way analysis of variance to determine if there were significant differences in weight loss among groups at each time interval. Intergroup comparison at different time intervals was done using Bonferroni test. SPSS version 21 was used for the analysis.

RESULTS

Table 1 shows the concentrations of available chlorine. There was no difference in the total available chlorine between the solution at room temperature(250 C) and preheated solutions. The mean percentage loss in weight of the samples is shown in Table 2. The graphical representation of the mean tissue dissolution for test solutions is denoted in Fig. 1.

The rate of tissue dissolution increased with time in all the three test groups. In the control group the percentage weight loss was 0% as was expected. Ca(ClO)2 at elevated temperature showed faster tissue dissolution ability than that at room temperature. Among the preheated solutions, Ca(ClO)2 at 600 C completely dissolved fresh pulp tissue in approximately 35 minutes. At all intervals the rate of dissolution with this solution was significantly higher than that of solution at 450 C and 250 C (P<0.05). The average time taken for complete dissolution of tissue was 53 min for the solution preheated to 450 C. Calcium hypochlorite at 450 C dissolved the tissue quicker than the solution at 250 C and the difference was statistically significant (P<0.05). The rate of dissolution of Calcium hypochlorite at 250 C increased after first half an hour, but still was significantly lower than that of the other groups. Also in the 250 C group, complete dissolution was observed in only four out of the ten samples after 1 hour.

DISCUSSION

Ability to dissolve human pulp tissue is a requisite for an ideal root canal irrigant. In this study preheating of calcium hypochlorite solution was done anticipating an enhancement in its pulp dissolution ability. At room temperature, Ca(ClO)2 dissociates in aqueous solution to form Hypochlorous acid and Calcium Hydroxide.

Ca(ClO)2 + 2H2 O → 2HOCl + Ca(OH)2 HOCl ↔ H+ + OCl

At a PH above 8.5 there is predominance of hypochlorite ions where as at a lower pH below 7, hypochlorous acid predominates. Hypochlorous acid is more active than hypochlorite ion and is responsible for chloramination reaction leading to amino acid degradation.¹³ Previous tissue dissolution studies have shown that the rate of dissolution is slower initially with Ca(ClO)2 than NaOCl, when used at room temperature. Dutta A and Saunders WP compared the tissue dissolution capacity of sodium hypochlorite solutions and calcium hypochlorite solutions at room temperature and concluded that the initial dissolution rate for sodium hypochlorite was higher than that of the latter. The reason was attributed to the slower formation of hypochlorous acid owing to the excess hydroxyl ion formation during dissociation of Ca(ClO)2 , which is twice the amount of that formed during dissociation of NaOCl 12. In the present study also the rate of soft tissue dissolution of calcium hypochlorite at room temperature was slow initially.

Taneja etal studied the tissue dissolution ability of various irrigants and concluded that the rate of dissolution by Ca(ClO)2 increased with time and concentration which is consistent with the present study.¹⁴

There is evidence in literature that the rate of decomposition of commercial calcium hypochlorite (used as high grade bleach) is enhanced at elevated temperatures.15 The solutions were replaced every five minutes to simulate the clinical situation where the irrigation is done periodically during instrumentation. The time of evaluation was set as 60 minutes which approximates the time taken for instrumentation during endodontic procedure. In this study human pulp tissue was used to better correlate with the clinical situation, unlike many other studies in which bovine tissue was used.^1,7,10,16,17 The weight of the tissue was standardized to 9.5mg by trimming it, so that variations don’t affect the dissolution rate.^1,5,16,18,19 The test tubes were tempered to a temperature of 320 C as it was the average intra canal temperature recorded after access opening.²⁰ The concentration of available chlorine was found to be unchanged after subjecting the solution to heating, which was in accordance with the previous studies.²¹

The results of the present study showed that, the tissue dissolution capacity of Ca(ClO)2 preheated to 600 was significantly higher than the other study groups. The solution at 450 had a significantly better dissolution capacity than that at room temperature. The complete dissolution of pulp tissue by Ca(ClO)2 at 45o was observed after a mean time of 53minutes where as with solution at 60o the average time taken for the same was 35minutes; the latter is in par with the action of NaOCl.¹² The reason for the rapid action of preheated solutions might be because of the faster rate of dissociation of Ca(ClO)2 at elevated temperatures. The Ca(OH)2 formed during the reaction formed a white coating over the specimens. The hydroxyl ions participating in saponification and amino acid neutralization reactions might have got depleted faster due to the increased rate of reaction in the heated solutions as compared to the solution at room temperature. The resultant drop in PH would have allowed the predominance of hypochlorous acid leading to rapid tissue dissolution.

Souza etal used passive ultrasonic agitation to enhance the tissue dissolution by various irrigants and found a favourable effect with Ca(ClO)2 . One of the reasons attributed to increased dissolution with agitation was the increase in the temperature of the solution with agitation.²²

The results of this study favour the use of Ca(ClO)2 at 600 C as an equally potential substitute for NaOCl in terms of tissue dissolution capacity. A matter of concern is the potential damage to the periodontal tissues by the elevated temperature of the irrigant used. Prior investigations have indicated that the solution will achieve temperature equilibrium rapidly.²⁰

Macedo RG etal studied the change in temperature of preheated solutions used for endodontic therapy and concluded that there is a rapid drop in the temperature of the solution after it is delivered into the canal.²³

The results of the present study favour the use of preheated solutions of Ca(ClO)2 as an equally potential substitute for NaOCl, during root canal preparation.

Limitations of the study

As the present study was conducted in an in vitro setting simulating the clinical situation, the results cannot be exactly predictive of the clinical condition. Further in vivo studies need to be conducted to corroborate the above results. Additionally, the effect of temperature on the cytotoxicity of Ca(ClO)2 needs to be evaluated before conducting further in vivo trials.

CONCLUSION

Within the limitations of this study, preheating the solution enhanced the dissolution ability of 10% calcium hypochlorite. Preheating to 600 C resulted in complete dissolution of tissue in an average time of 35 minutes which was comparable to the dissolution ability of sodium hypochlorite as shown in previous studies. 10% Ca(ClO)2 solution at 60oC can be used as an efficient tissue dissolving irrigant if further invivo research corroborates with the present study results. Future studies should be directed towards assessing the cytotoxicity of preheated calcium hypochlorite solutions.

Conflicts of interest: None