In vitro comparison of apical microleakage by spectrophotometry in simulated apexification using White Mineral Trioxide Aggregate, TotalFill Bioceramic Root Repair material, and BioDentine.

AIM: The purpose of this study was to compare the sealing ability of various calcium silicate-based root-filling materials with a glucose leakage model after orthograde obturation using an open apex model.
MATERIALS AND METHODS: Thirty-two recently extracted human maxillary anterior teeth with single, straight root canals were selected and divided into four groups: Group 1 (G1), White Mineral Trioxide Aggregate; the material was delivered into the canal using the MAP system and further compacted with a specific plugger. Group 2 (G2), TotalFill bioceramic Root Repair material: the material was injected directly into the middle half of the root canal. Group 3 (G3), BioDentine; the material was delivered into the canal using the Hawe composite gun and prefitted pluggers. STATISTICAL ANALYSIS: Statistical analysis was performed using the SPSS 23.0 statistical software package. The Kruskal-Wallis nonparametric test was applied to compare the differences in glucose microleakage.
RESULTS: There were no significant differences among the three experimental groups. The results showed a tendency for leakage to increase from the 1st day to the end of experimental period.
CONCLUSIONS: Within the parameters of this in vitro study, it may be concluded that the three materials evaluated present similar apical microleakage when treating teeth with open apices requiring orthograde delivery of an apical barrier.

INTRODUCTION

The main objective of a root-end filling material is to provide an apical seal that prevents the movement of bacteria and the diffusion of bacterial products from the root canal system into the periapical tissues.[12] In this context, endodontic management of the teeth with open apices often presents a challenge as a blunderbuss configuration of the apex makes the delivery of root-filling materials difficult, potentially leading to overextension and/or overfilling of the root canal.[3]

Conventionally, long-term apexification procedures with calcium hydroxide (Ca[OH]2) have been used to establish apical closure by the induction of a hard tissue barrier. As an alternative to conventional Ca (OH)2 apexification, artificial apical barriers made from a variety of materials have been proposed. In 1999, Torabinejad and Chivian published an article recommending the use of Mineral Trioxide Aggregate (MTA) as an artificial apical barrier. It has since become the material of choice in artificial apical barrier procedures.[4]

Although MTA is a material that has been extensively investigated and has proven biocompatible and offers an excellent sealing capacity, it suffers certain clinical disadvantages, namely its handling properties and lengthy setting time.[56789]

Recently, new calcium silicate-based materials have been introduced such as TotalFill Root Repair material (FKG, Brasseler, Savannah, USA) and BioDentine (Septodont, St. Maurdes Fossés, France), which offer improved color stability and handling characteristics, while exhibiting physical and chemical properties comparable with MTA. More importantly, these materials have been shown to release the calcium and phosphate ions essential for hydroxyapatite deposition.[3]

However, to date, no research has been published that evaluates apical microleakage in the orthograde obturation of teeth with open apices. Hence, the purpose of this study was to compare the sealing ability of various calcium silicate-based root-filling materials after orthograde placement in an open apex model using the glucose leakage method. The null hypothesis of this study was that none of the studied materials presented a tightly seal.

MATERIALS AND METHODS

Specimen selection and preparation

Thirty-two recently extracted human maxillary anterior teeth with single, straight root canals were selected for the study. Any root with cracks, open apices, resorptive defects, or large carious lesions approaching the pulp was excluded. The study was approved by the bioethics committee of the University of Barcelona (Spain).

The coronal portions of all teeth were removed with diamond disks so that each specimen was 15-mm long. A standard access was prepared with a diamond burr to gain straight-line entry to the root canal, and apical patency was confirmed with a size 15 K-file (Dentsply Sirona, Ballaigues, Switzerland). A working length of 1 mm from the apex was determined.

Instrumentation used was the ProTaper Next system (files X1, X2, and X3) (Dentsply Sirona). After each instrument, the canal was irrigated with 1 mL of freshly prepared 5.25% sodium hypochlorite solution using a 27G needle. A final rinse was performed with 1 mL 10% citric acid to remove the smear layer and 1 mL 2% chlorhexidine.

Root-end resection and root-end filling

The apical 3 mm was resected perpendicular to the root's long axis by means of a diamond disc. Then, the apical portion of the canal was instrumented to a size 60 master file using the balanced force technique and a K-file.

The prepared roots were randomly divided into three experimental groups (n = 10) according to the obturation material applied and two control groups (n = 2). Canals were dried with paper points, and a 5-mm apical plug of root filling material was incrementally placed in orthograde direction. Each material was prepared according to the respective manufacturer's recommendations. Three groups were formed as follows:

Group 1 (G1), White MTA (ProRoot MTA, Dentsply Tulsa Dental): The material was delivered into the canal using the MAP system (Roydent, Johnson City, TN, USA) and further compacted with a prefitted plugger

Group 2 (G2), TotalFill BC Root Repair Material (FKG, Brasseler USA): The material was injected directly into the middle half of the root canal

Group 3 (G3), BioDentine (Septodont, St. Maurdes Fossés, France): The material was delivered into the canal using the Hawe composite gun (Hawe Neos Dental, Bioggio, Switzerland) and prefitted pluggers.

Positive control specimens were not filled with any material. Specimens in the negative control group, including the root canal orifice and apical foramen, were completely coated with nail polish. Final radiographs were taken to confirm uniform and dense obturation in the experimental groups. Immediately after filling, all samples were stored in saline-moistened gauze for 72 h to allow the sealing materials to fully set.

Specimen preparation

The roots of the experimental groups and positive control group were coated with nail polish, except for the coronal access cavities and apical apices.

Teeth were placed into a device designed by Xu et al.[10] to measure glucose leakage. Teeth were glued to the end of an eppendorf tube using cyanoacrylate. A plastic tube of at least 15 mm was connected to the root canal root. The assembly was then placed in a sterile 5 mL glass bottle.

The tracer used in the study was a 1 mol/L glucose solution (pH 7.0), with a density of 1.09 g/L × 103 g/L and viscosity 1.18 × 10−3(at 37°C). Approximately 5 mL of the glucose solution, containing 0.2% NaN3, was injected into the plastic tube until the top of the solution was 14 cm higher than the top of the root canal, which created a hydrostatic pressure of 1.5 kPa (15 cmH2O). The glass bottle contained 1 mL 0.2% solution of NaN3, in which glucose that passed through the obturated canal would be collected; NaN3 was used here to inhibit the proliferation of microorganisms that might decompose the glucose.

The model was transferred to an incubator that provided 100% humidity and 37°C temperature for the duration of the experimental period. To determine whether there was any evaporation of solution in the tube, another such assembly containing 1 mL 0.2% NaN3 was placed in the incubator under the same conditions and weighted every day during the experimental period.

Sample evaluation

A 10 μL aliquot of solution was drawn from the glass bottle using a micropipette at 1, 2, 7, 15, 21, and 30 days. Each sample was analyzed using a glucose kit in a UV/VIS spectrophotometer (Shimadzu; Kyoto, Japan) performing a kinetic assay at 480-nm wavelength. All the readings were taken in duplicate, and the mean value was considered for statistical analysis. The results were expressed as mmol/L.

Statistical analysis

Statistical analysis was performed using the SPSS 23.0 statistical software package (IBM Corp., Armonk, NY, USA). The Kruskal–Wallis nonparametric test was applied to compare the differences in glucose microleakage. Statistical significance was established as P < 0.05.

RESULTS

After the observation periods, no signs of glucose were detected in the glass bottle in the negative control group. However, the positive control group showed high glucose leakage values from the 1st day. These control results indicated that test system seal was reliable and effective.

Mean microleakage scores and standard deviations for each root repair material are summarized in Table 1. The Kruskall–Wallis test (P > 0.05) showed no significant differences among the three experimental groups. The results showed a tendency for leakage to increase from the 1st day to the end of experimental period.

Table 1

Microleakage of glucose in three experimental groups (mM/L)

Time (days)	MTA	Totalfill RRM	Biodentine
1	0.10±0.49	0.15±0.52	0.12±0.31
2	0.11±0.45	0.16±0.50	0.13±0.30
7	0.15±0.42	0.18±0.49	0.14±0.31
15	0.20±0.53	0.21±0.55	0.18±0.35
20	0.23±0.51	0.24±0.54	0.22±0.38
30	0.25±0.53	0.27±0.55	0.23±0.37

DISCUSSION

Microleakage tests of an apical barrier may be considered controversial. Nevertheless, in agreement with Tran et al.[3] and Hachmeister et al.,[11] we believed that it is important to evaluate the apical microleakage of silicate-based materials and MTA barriers because the success of these treatment techniques depends on accurate placement, which can present a challenge. The sealing ability of MTA and other root repair materials has been examined for both root-end filling[251213] and perforation repair material,[1415] obtaining successful results. Recent studies have reported successful long-term clinical outcomes associated with MTA apexification procedures.[1617] Moreover, MTA has been extensively studied as a root repair material in open apices, obtaining favorable outcomes.[11121819] However, studies comparing the performance of MTA with other commercially available calcium silicate-based products are scarce.[3] For this reason, MTA was compared with two calcium silicate-based materials because both these BC materials contain hydroxyapatite, with similar capacities to MTA for preventing leakage. The results showed no significant differences between the three calcium silicate-based materials when used as a root repair material in apexification. Hence, the three materials may be considered suitable for orthograde obturation.

BioDentine was evaluated because it is a new bioactive cement with the capacity to induce the formation of new cementum and periodontal ligament, making it biologically acceptable for closing root canals with open apices.[20] However, Lee et al.[21] have recommended the use of BioDentine as well as MTA as root-end filling materials because, in contact with mesenchymal stem cells, they induce osteoblast differentiation. TotalFill Root Repair (previously known as EndoSequence Root Repair material) is a BC material selected because experimental studies have reported favorable characteristics in terms of its cytotoxicity, antibacterial activity, marginal adaptation, and biomineralization potential.[22]

Several methods have been developed for testing the sealability and the apical microleakage[101213] of apical root repair materials. These methods include bacterial penetration,[5] fluid transport,[18] and glucose leakage models.[13] None of them is universally accepted as each method presents different limitations.[13] A glucose leakage model was chosen for this study because it was possible to quantify endodontic microleakage continuously over time. The total amount of microleakage was the cumulative value of leaked glucose.

According to Xu et al.[10] and Leal et al.,[13] glucose has a low molecular weight (180 Da) and the glucose method has more clinical relevance than the other tracers used in microleakage tests. It has been reported that the glucose molecule is a known nutrient source for bacteria that might remain in the root system. In the present study, the quantitative analysis of leakage was conducted by determining the concentration of glucose in the apical reservoir that leaked through the filled root canal; this concentration was defined with colorimetric determination, based upon two-coupled enzymatic reactions and using o-dianisidine (ODD) as a colorimetric substrate. This reaction is more sensitive than bubble movement (minimum 1 μ) in the fluid transport method,[23] and hence, the glucose leakage method is now considered to overcome many of the limitations suffered by other methods.[101323] The method involves two reactions. First, glucose oxidase catalyzed oxidation of glucose to gluconic acid and H2O2. Second, catalyzation by peroxidase, in which 2e- is transferred from H2O2 to ODD; in its oxidized state, this changes from a colorless state to a brown color and so can be quantified by spectrophotometry.

The results of the present study are in agreement with previous studies that have shown that leakage was not significantly different when comparing MTA with calcium silicate-based materials.[313]

Nevertheless, further studies are needed to corroborate the results of this study and to investigate the behavior of these materials when exposed to in vivo conditions. In addition, we would speculate that lower apical microleakage would be observed in nonexperimental settings in which a root canal sealer is used with gutta-percha backfill after performing apical plugging. The seal would probably be further improved with the adjunctive use of an appropriate material along the filled canal orifice, capable of providing a reliable coronal seal.[18]

CONCLUSIONS

Within the parameters of this in vitro study, it may be concluded that for treating teeth with open apices requiring orthograde delivery of an apical barrier, the three materials evaluated (White MTA, TotalFill BC Root Repair Material, and BioDentine) obtain similar apical microleakage.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.