Evaluation of the effect of pre-treatment of intra-radicular dentin with various agents on bio-dentine’s sealing ability in immature roots: an ex vivo study

Introduction

Any insult caused to developing tooth can lead to cessation in root development, resulting in an incompletely formed root apex with thin dentinal walls and wide-open apex. Restoration of such tooth is a challenging task. Many treatment strategies for such cases have been proposed in the literature. Apexification is one of the options, involving formation of an apical barrier to provide apical seal [1].
From the historical point of view, calcium hydroxide was the material of choice for the formation of apical calcific barrier in apexification. However, a variety of issues, including the necessity of multiple visits, patient compliance, and more porous apical barrier, have led to the introduction of a single-visit apexification that can be carry out with the help of mineral trioxide aggregate (MTA) [2]. Even though MTA demonstrated to provide an apical stop with satisfactory sealing, it revealed some drawbacks, such as the possibility of tooth staining, difficulties in handling, etc. In the year 2010, bio-dentine was introduced, which not only decreased the chances of discoloration but also improved handling properties [3].
Instrumentation of the root canal space forms a collection of debris with organic and in-organic contents, known as the smear layer. Numerous studies concluded that the removal of smear layer results in better penetration of irrigating solution, and the sealer inhibits the growth of micro-organisms, thereby providing the pro­per seal [4, 5].
Previous research have compared the effect of seve­ral agents, showing that chelating agents possess the capability of increasing the apical seal of filling material [6-8]. Agents, such as 17% ethylenediaminetetraacetic acid (EDTA), maleic acid, phytic acid, etidronic acid, citric acid, etc., have been studied previously to eva­luate their effects on the sealing ability of MTA and bio-dentine used for apexification procedure [9]. Furthermore, the role of lasers in the field of endodontics increased many folds. Currently, diode lasers are popular in utilization due to their compactness and ability to penetrate into deeper layers [10]. The efficiency of dio­de lasers in removing the smear layer has been stu­died by Amin et al. [11], who observed melting and evaporation of the layer, leading to its complete removal. Parirokh et al. [12] also found positive effects of diode lasers treatments for eradication of the smear layer, specifically in the apical third part of the root.

Objectives

Most in vitro studies compared the sealing ability ofbio-dentine with other root end filling materials. How­ever, to the best of our knowledge, the effects of chelating agents and diode laser achieving the apical seal as well as on the sealing ability after formation of apical bar­rier of bio-dentine, have not been discussed in the past. Therefore, the current study evaluated the effects of pre-treatment of intra-radicular dentin on the sealing ability of bio-dentine in immature roots. The null hypo­thesis was that there is no effect of pre-treatment of intra-radicular dentin with various agents on the sealing ability of bio-dentine in immature roots.

Material and methods

Freshly extracted, due to periodontal or orthodontic reasons, teeth with single root and single canal were collected for the study. Teeth with caries, restorations, fractures, resorption, multiple canals, developmental anomalies, pulp calcifications, and previous endodontic treatments, were excluded. The selected teeth were cleaned with ultrasonic scaler, autoclaved, and stored in physiologic saline until further use. Sample size was estimated using G*Power software, version 3.1.9.7, and its calculation assumed a large effect size, based on a previous study by Thakkar et al. [13], who detected substantial differences in the sealing ability of bio-dentine using a glucose filtration model. Assuming f = 0.75, a power = 0.80, and a significance level of a = 0.01, the required total sample size for four groups was calculated as 36. Therefore, a total of 40 teeth were included in the study.
Samples were de-coronated at cemento-enamel junction in order to standardize the root length of 16 mm. The teeth’ apical thirds were resected (Figure 1), and canals were enlarged using Peeso reamers from #1 to #5 by taking them beyond the apex, so that open apex can be simulated mimicking immature roots with thin dentinal walls (Figure 2). Working length was determined by visualizing the tip of K file at the apex, and subtracting 0.5 mm from the length noted. Minimal instrumentation was done with #K files, and irrigation was carried out using 5% sodium hypochlorite, followed by 17% EDTA treatment. Lentulo spiral fillers were applied to the root canal space with water-based calcium hydro­xide paste, and the samples were then incubated at 37℃ and 100% humidity for 7 days. After that, the teeth were withdrawn from the incubator, and calcium hydroxide was removed. The samples were divided randomly into four groups (n = 10) as follows: group 1: pre-treatment with 3 ml of 17% EDTA was applied for 5 minutes; group 2: diode laser, working at 980 nm wavelength, set at continuous mode at 2W power for 20 seconds was used to irradiate the apical end of the samples; group 3: pre-treatment with 3 ml of 10% citric acid was applied for 5 minutes; group 4 (control): samples were irrigated with 0.9% physiologic saline for 5 minutes.
Using sterile absorbent paper points, the root canals were dried and bio-dentine was administered as per the manufacturer’s recommendations, with an apical plug of 4 mm modelled and confirmed radiographically. All samples were left intact until bio-dentine setting, after which they were coated with different colors of nail varnishes, except at the apical 3 mm of the root, for group differentiation.
Mounting of the samples was done by cutting the end of Eppendorf vial. The apical third of root samples was glued to the vial with cyano-acrylate. Leakage at this interface was eliminated by using sticky wax. A plastic tube of at least 15 mm was connected in close approximation with the root orifice through a hole created in the cap of Eppendorf vial. The entire assembly was placed in a 5 ml vacutainer, with the samples glued in apico-coronal direction. Complete assembly was checked for any type of leakage by pouring few milliliters of water in the vacutainer. To maintain atmospheric pressure, a 10 mM sucrose solution containing 0.2% sodium aside as preservative was injected into the vacutainer through a plastic tube, until the top of solution was 13.6 cm higher than the root canal orifice. Using a pipette, 1 ml of 0.2% NaN3 was placed in the vacutainer, in which the root specimens were immersed. Sucrose that leaked through the samples and passed through the canals was collected in the vacutainer (Figure 3).
A 10 ml of the solution was drawn from the vacu­tainer using a micro-pipette at 24 hours, 3 days, 15 days, 30 days, and 60 days’ time intervals. After drawing the sample, 10 ml of fresh 0.2% NaN3 was added to the vacutainer reservoir to maintain a constant volume of 1 ml. If there was any decrease in volume of the solution in the vacutainer due to evaporation, a corresponding amount of sterile deionized water was added to the vacutainer. The withdrawn sample was then analyzed to estimate the concentration of sucrose leaked. Spectrophotometric analysis was done to check the absorbance of standard sample and tested sample. From the absorbance value, the concentration of the sucrose was calculated using the following formula: Sucrose concentration = (Absorbance − 0.0622)/92148.9. Then, the percentage of micro-leakage was estimated as follows: % leakage = (Sucrose concentration calculated × 100)/Initial sucrose concentration.

Statistical analysis

SPSS software (IBM SPSS Statistics for Windows, version 25.0, Armonk, NY: IBM Corp., USA) was the statistical analysis method used in the study. One-way analysis of variance (ANOVA) F-test and independent Student’s t-test were performed for data analyses. Level of significance was set at 1%.

Results

Table 1 and Figure 4 illustrate the mean values of the mean percentage of sucrose leakage that occurred from the samples during observation time. From the results, group 2 had the least percentage of sucrose leakage, followed by group 1 and group 3. Group 4 (control) showed the maximum percentage of sucrose leakage during the tested time intervals. The comparison between the four investigated groups at all time intermissions was performed using one-way ANOVA F-test (Table 2). Statistically significant difference was found between the groups (p < 0.01). The difference between the groups at each time intervals of the observation was compared using independent t-test. The p-values between the groups are shown in Table 3. There was a significant difference between the groups at all time intervals (p < 0.01).

Discussion

Endodontic treatment of immature necrotic permanent teeth is a challenging task due to the presence of wide apex and thin dentinal walls, creating difficulty in achieving apical seal. A wide variety of options are available for management of such teeth. Apexification offers the advantage of formation of apical barrier, over which the root canal space can be filled while maintaining the integrity of the teeth [1].
Bio-dentine demonstrated a satisfactory apical seal when compared with MTA at single-visit apexification procedure. Bio-dentine is a calcium silicate-based material with the property of bio-compatibility. The feature of marginal seal is provided by the calcium and hydroxyl ions, which are released once the material sets, resulting in the formation of a crystalline precipitate [14]. In addition to chemical characteristics of bio-dentine, it has enhanced physio-chemical advantages, such as 12 minutes shorter setting time than MTA and better mechanical strength, thereby demonstrating better handling properties [15]. A study by Bani et al. [16] have shown the efficiency of bio-dentine when used as an apical plug-forming material.
Originally introduced by Xu et al. [17], the glucose filtration technique is used for the quantitative testing of endodontic leakage, which is sensitive, non-destructive, and clinically relevant method. However, in this study, sucrose was employed as a tracer agent instead of glucose. Since sucrose not only shows similar outcomes as glucose by being non-toxic, but is also relatively non-reactive due to the lack of unsubstituted hemiacetal group present in reducing disaccharides. Therefore, sucrose is more stable tracer agent than glucose [18].
There are immense studies demonstrating the increased sealing ability of root end filling materials treated with different agents, improving bonding and preparing a contamination-free dentin surface. These studies reported that when the dentin surface is treated with different agents prior to formation of apical barrier, it shows better adaptation of the material, which amplify the material’s sealing ability. However, the effects eva­luated in this study were not compared directly with the previous ones.
The present study was performed to evaluate the effect of pre-treatment of various agents on the sealing ability of bio-dentine. Since the results of the study revealed statistically significant difference of the effect of pre-treatment on the sealing ability of bio-dentine, the null hypothesis was rejected.
According to the results, group 2, i.e., diode laser-treated group, showed the least amount of leakage, thereby enhancing the sealing ability of bio-dentine to the maximum. This could be due to the compactness and thin flexible fibre tip of diode laser, which increased its penetration depth, so that it could reach to the apical third of the root more conveniently, as explained by Wang et al. [19]. Additionally, diode laser helps in the opening of the dentinal tubules and alter the tubules’ diameter, which aid in its ability to provide the apical seal. A study by Amin et al. [11] demonstrated that diode lasers can efficiently remove the smear layer, helping a greater adaptation of filling material to the dentinal walls, thus enhancing the seal of the material. Similarly, Reza et al. [20] found that diode lasers can cause melting and evaporation of the smear layer, hence more cleaned canal wall can be formed after their treatment, which could have a greater role in providing the maximum of sealing ability.
Agents used in group 1 and group 3, i.e., 17% EDTA and 10% citric acid as chelating agents, have the potential to remove in-organic debris. The application of these chelating agents in the apical third can result in the elimination of the smear layer, thereby improving the adhesion and facilitating the penetration of the root end filling materials. However, some studies reported possible drawbacks of EDTA, such as being cyto-toxic to periapical tissues. Zeid et al. [21] noticed that EDTA can cause reduction of the calcium ions, and can decrease the micro-hardness of radicular dentin, thus weakening the tooth. Their extrusion into the periapical tissues demonstrated causing inflammation and severe allergies [21]. Therefore, citric acid, a more bio-compatible option, was compared with other agents in this study. 17% EDTA was found to have a greater ability to remove the smear layer, as shown by Scelza et al. [22], who compared it with citric acid. This could be due to better sealing ability of EDTA than citric acid. However, different concentrations of citric acid might affect the smear layer removal potential, and hence can affect the results, as noted by Mancini et al. [23], where 42% citric acid was used, showing a better smear layer removal potential as compared with 17% EDTA agent.
In the current study, a significant difference was observed in the leakage when the samples were treated with agent from group 4 (control), where no pre-treatment was applied; therefore, it can be hypothesized that the utilization of these chelating agents or diode laser can enhance the sealing ability of bio-dentine.
There are few limitations of the study to acknowledge, including inability in simulating the immature roots to the most accurate details. The procedure lacked to replicate the tissue composition and physical characteristics [24]. Also, as an ex vivo study, it could not replicate oral condition, which can affect the leakage from the teeth. Lastly, the leakage was analyzed from the apical plug formed only without completely filling the root canal space, which is not practical in a real clinical scenario.

Conclusions

Within the limitations of the study, it can be concluded that: 1) the leakage was observed in all investigated groups irrespective of intra-radicular dentin pre-treatment evaluated in this study; 2) the maximum sealing ability of bio-dentine was found when the root surface was pre-treated with diode laser for 20 seconds; 3) 17% EDTA and 10% citric acid improved apical seal more than 0.9% physiologic saline used in no pre-treatment group.

Disclosures

1. Institutional review board statement: Not applicable.
2. Assistance with the article: None.
3. Financial support and sponsorship: None.
4. Conflicts of interest: None.

References