Effect of accelerants on the immediate and the delayed sealing ability of mineral trioxide aggregate when used as an apical plug: An in vitro study.

AIM: To evaluate and compare the influence of various accelerators, 15% disodium hydrogen phosphate (Na2HPO4), 10% calcium chloride (CaCl2) and 23.1 wt% calcium lactate gluconate (CLG), on the immediate (after 72 h) and delayed (after 2 months) sealing ability of white ProRoot mineral trioxide aggregate (WMTA) when it is used as an apical plug.
MATERIALS AND METHODS: Eighty, single-rooted mandibular premolars were instrumented and standardized artificial open apices were created. The samples were then randomly assigned into four experimental groups and two control groups. WMTA was mixed with the respective accelerators and an apical plug of 4-mm thickness was fabricated. The remaining canal spaces were then backfilled. The samples were stored for the stipulated time periods and then immersed in 0.2% Rhodamine B solution for 72 h. Dye leakage was analyzed using a stereomicroscope.
RESULTS: Mean microleakage values of all experimental groups revealed that MTA + 23.1 wt% CLG showed the least leakage, followed by MTA + 15% Na2HPO4 and MTA + 10% CaCl2 with MTA + deionized water showing the maximum leakage at both the time intervals (P < 0.001). All the samples stored for a period of 2 months showed less leakage as compared with the samples stored for 72 h (P < 0.05).
CONCLUSIONS: It was found that all three accelerators significantly accelerated the set of WMTA, of which 23.1 wt% CLG showed the best results, followed by 15% Na2HPO4 and 10% CaCl2. The sealing ability of all the experimental groups was significantly superior after 2 months as compared with that after 72 h.

RCT Entities:   Population Interventions Outcomes

INTRODUCTION

Treatment of nonvital immature permanent teeth with calcium hydroxide (Ca(OH)2) is associated with some difficulties such as weakened tooth fracture, root canal reinfection and long treatment time.[1] Mineral trioxide aggregate (MTA) has overcome all the disadvantages of Ca(OH)2 and has addressed the need for an apical seal by inducing hard tissue barriers.[2]

However, MTA is difficult to use because of its longer setting time and poor handling properties,[3] necessitating a second appointment for the completion of the procedures such as apexification. To overcome this problem, various accelerators such as (2%, 5%, 10%) calcium chloride, (1-10%) calcium nitrate, (25-50%) calcium nitrite, (20%) calcium formate, low-dose citric acid and lactic acid have been added to MTA. Various studies showed that the addition of CaCl2 to MTA decreased the setting time and improved its sealing ability.[45] Recently, disodium hydrogen orthophosphate[6] and calcium lactate gluconate[3] have been added to MTA to reduce its setting time to a significant extent. But, their effect on the sealing ability of MTA is not yet known.

There have been no studies reported till date to evaluate and compare the effect of 10% CaCl2, 15% Na2HPO4 and 23.1 wt% calcium lactate gluconate (CLG) on the sealing ability of MTA when used as an apical plug. Thus, the purpose of this study was to evaluate and compare the effect of these accelerants on the immediate and delayed sealing ability of MTA when used as an apical plug.

MATERIALS AND METHODS

Preparation of accelerants

15% Disodium hydrogen phosphate (Na2HPO4) was prepared by taking 1.5 g of powder and mixing with 10 mL of deionized water. For the 10% calcium chloride (CaCl2) preparation, 1 g of powder was mixed with 10 mL of deionized water. To prepare 23.1 wt% CLG, 27.0 g of lactic acid (molecular weight of 90.1 g/mol), 35.6 g of glucono delta lactone (molecular weight of 178.0 g/mol) and 14 g of calcium oxide (molecular weight of 56.0 g/mol) were mixed with 100 mL of deionized water. The respective volumetric flasks were then kept in a sonicator for 500 s for the complete mixing of the powders with the deionized water.

Eighty, single-rooted human mandibular premolars, extracted for orthodontic reasons, were included in the study. The teeth were stored in 10% formalin and were kept moist before the experiment.

The teeth were decoronated so as to have a standardized length of 14 mm. The apical 3 mm of the root tips were then resected. Access to the root canal was gained and the apical patency was established with a size of 10 K–file. The specimens were then sequentially prepared by K-files (Dentsply Maillefer, Ballaigues, Switzerland) to an ISO size file #80, which could be visualized 1 mm beyond the apex. Irrigation was performed with 1 mL of 5.25% NaOCl solution subsequent to instrumentation. Final irrigation was performed with 1 mL of 17% EDTA for 5 min followed by 5 mL of 5.25% NaOCl. The canals were then instrumented with a Peeso Reamer (Dentsply-Maillefer) up to #4, with each passing 1 mm beyond the apex. This was followed by drying of the canals and final flushing with 1 mL of sterile water.

The samples were then randomly assigned into four experimental groups consisting of 16 samples each (n = 64) and two control groups with eight samples each (n = 16).

In the negative control group, ProRoot white MTA (WMTA) (Dentsply Tulsa Dental, Tulsa, OK, USA) was mixed in deionized water and in the positive control group, no apical plug was fabricated.

Group 1: ProRoot WMTA mixed with15% Na2HPO4 in deionized water

Group 2: ProRoot WMTA mixed with 10% CaCl2 in deionized water

Group 3: ProRoot WMTA mixed with 23.1 wt% CLG in deionized water

Group 4: ProRoot WMTA mixed in deionized water alone.

In Group 1, WMTA powder was mixed with 15% Na2HPO4 in deionized water in a 3:1 powder–liquid ratio and was placed using a Micro Apical Placement (MAP) System. WMTA was then condensed with a hand condenser and plugger to achieve a 4-mm-thick apical plug. Radiographs were taken to ensure the proper placement of the material and a moistened cotton pellet was placed into the canal just above the apical plug material for 3 h to allow the material to set. After 48 h, the root canals were obturated using Obtura II (Obtura Spartan, Fenton, MO, USA) and AH Plus sealer (Dentsply Tulsa Dental).

In groups 2, 3 and 4, the apical plug was fabricated in a manner similar to that described above.

To simulate a periapical environment, the samples were inserted into a flower foam arrangement, moistened with distilled water. A cotton pellet was fitted into the apical portion to prevent the foam from penetrating into the root canal.

Eight samples from each experimental group and four samples from each control group were randomly stored for 72 h and the remaining samples were stored for 2 months in an incubator at 37°C and 100% humidity.

After this, the root surfaces of all the samples of the experimental groups were coated with two coats of nail varnish, leaving 1 mm of the root surface around the tooth and an apical plug material interface at the apex. The apical and coronal openings of the positive control group samples were devoid of any impermeable coating. The entire root surface of the specimens of the negative control group was coated with two coats of nail varnish.

The samples were then immersed in 0.2% Rhodamine B solution at a pH of 7 for 72 h at 37°C and 100% humidity in an incubator. The teeth were then kept in running water, dried and sectioned longitudinally in a buccolingual direction using a safe-sided diamond cutting disc at low speed.

Leakage was determined from the most apical part to the most cervical portion of the apical plug, which was evaluated under a 30X stereomicroscope (Olympus, Spectro Analytical Laboratory, Delhi, India) and calibrated with the help of the Measurement and Image Analysis Software. The measurement was made by taking the mean of the two walls. If the leakage was greater than the full length of the MTA apical plug, the measurement was stopped at the coronal most point of the plug.

Statistical analysis

One-way analysis of variance (ANOVA) with Tukey's HSD was applied to determine the significance (P < 0.001) among all groups. Paired “t” test was applied to determine the significant comparison of change in microleakage between the two time intervals (P < 0.05).

RESULTS

All the positive control group samples showed complete dye penetration and the negative control group samples did not show any leakage at both the time intervals. The mean microleakage values of all experimental groups revealed that WMTA + 23.1 wt% CLG showed the least leakage, followed by WMTA + 15% Na2HPO4 and WMTA + 10% CaCl2, with WMTA + deionized water showing the maximum leakage at both the time intervals. The 2-month stored samples showed less leakage than the 72-h samples. A statistically significant difference existed between all the groups (P < 0.001) and at both the time intervals (P < 0.05). The mean dye penetration and its standard deviation along with intergroup comparison are given in Table 1.

Table 1

Intergroup comparison of microleakage at both the intervals, i.e., after 72 h and after 2 months

DISCUSSION

In the present study, the sealing ability of WMTA mixed with hydration accelerators was significantly better than that of WMTA mixed with deionized water at both the time intervals.

The superior sealing ability of WMTA with 23.1 wt% CLG than the deionized water group as seen in our study might be attributed to the calcium ions released from it during its hydration. Further, the shortening of the setting time of WMTA with the addition of 23.1 wt% CLG[78] might be responsible for its antiwashout resistance.

Many studies have proven that Na2HPO4 solution could be used as a cement liquid to accelerate the setting of the cement prepared from α-tricalcium phosphate as a cement powder.[910] The presence of HPO4 ions embedded in 15% Na2HPO4 accelerator might lead to the more apatite crystal formation. This could be one of the reasons for MTA with 15% Na2HPO4 to show a better sealing ability when compared with deionized water alone in our study. This is supported by the studies showing a less bacterial penetration in MTA when placed in phosphate-buffered solution.[11] Reports have also shown that MTA alone was also able to form hydroxyapatite deposits on its surface when it was in contact with HPO4 ions.[12] The rate of hydroxyapatite formation over a period of time has been suggested as an indicator of the setting reaction.[13] In one study,[6] it was found that the setting time of MTA was 151 min, which reduced to 26 min with the addition of 15% Na2HPO4, increasing the washout resistance of MTA over a period of time.

With the addition of 10% CaCl2, an improved sealing ability of MTA was seen in the study as compared with the deionized water group. A possible explanation for the results observed is the penetration of CaCl2 in the pores of cements, which strongly accelerates the hydration of silicates, leading to their faster crystallization and shorter setting time.[14] The continuous release of Ca ions by CaCl2[15] might have a role to play in shortening of the setting time. In a previous study,[16] 10% and 15% CaCl2 were added to Portland cement, resulting in a reduced hardening time. This in turn might accelerate the onset and final setting of the cement, reduce the incorporation of water and allow the cement to resist hydrostatic pressure at early stages, avoiding its leaching out.

At both the time intervals, the 23.1 wt% CLG group showed a significantly better sealing ability than 10% CaCl2. Both 23.1 wt% CLG and 10% CaCl2 solution supplied adequate calcium ions to accelerate the initial setting of the cement, but the crystalline nature of calcium chloride tends to precipitate out as sand-like particles and offers less enhancement of cement cohesiveness. This might have resulted in the inferior sealing ability of CaCl2 when compared with CLG.

The results demonstrated that less dye penetration was seen with 23.1 wt% CLG when compared with 15% Na2HPO4 solution. On the other hand, 15% Na2HPO4 solution showed less leakage when compared with 10% CaCl2. These findings could not be compared with any other study as no study comparing the above two groups has been performed till date.

Regarding the two variables analyzed (both time intervals), significantly lesser (P < 0.01) leakage was observed in all samples stored for 2 months, showing that the leakage decreased with time. Slight expansion of the material upon setting[17] could be one of the reasons for the improved sealing ability of MTA, without any accelerator, after 2 months of storage. Another explanation could be the formation of apatite-like materials,[1819] filling the MTA–dentin interfacial space, accompanied with tag-like structures that extend into the dentinal tubules.[18] It has also been established that the ProRoot White MTA and the MTA Angelus released calcium and maintained an elevated pH even after 126 h.[20]

In case of 15% Na2HPO4, the formation of apatite crystals after the contact of HPO4 ions, embedded in the MTA-Na2HPO4 cement system, with the moisture over a period of time, might be responsible for the statistically decreased microleakage after 2 months. An increase in sample weight after 30 days, which might be due to the formation of apatite, has also been shown.[21]

The improved sealing ability of 10% CaCl2 after 2 months might be due to the release of more Ca ions leading to biomineralization over a period of time. It has been shown that MTAs’ sealing ability could be improved over a period of time by the addition of CaCl2.[7] Also, a study[14] showed that the addition of CaCl2 provided a reduction in the setting time of MTA, which might lead to its antiwashout resistance. This study also showed that WMTA with CaCl2 exhibited a weight gain, which might be due to the apatite precipitation over a period of time.

The increased precipitation of amorphous 23.1 wt% CLG with time could be the reason for it to show a better sealing ability at 2 months. However, we cannot compare these results with those of others in the literature as no study has been done yet.

CONCLUSION

Within the parameters of this study, it could be concluded that the sealing ability of WMTA mixed with hydration accelerators was superior to WMTA with deionized water alone at both the time intervals. The sealing ability of all the experimental groups was significantly superior after 2 months as compared with that after 72 h.