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 Table of Contents  
Year : 2021  |  Volume : 33  |  Issue : 3  |  Page : 144-148

Evaluation and comparison of the apical seal obtained with Biodentine after conditioning of root end with three different solutions using ultraviolet-visible spectrophotometer: An in vitro study

1 Department of Conservative Dentistry and Endodontics, Annoor Dental College, Muvattupuzha, Kerala, India
2 Kerala University of Health Sciences, Kerala, India

Date of Submission30-Apr-2021
Date of Decision12-Jun-2021
Date of Acceptance05-Sep-2021
Date of Web Publication30-Sep-2021

Correspondence Address:
Dr, Sethuparvathi Anitha
Alpha Dental Clinic, Pala, Kottayam, Kerala
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/endo.endo_119_20

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Aim: The aim of the study is to evaluate and compare the apical seal obtained with Biodentine after conditioning of root end with three different solutions (HEBP, chitosan, and ethylenediaminetetraacetic acid [EDTA]).
Materials and Method: Forty-eight maxillary incisors were selected and decoronated. Instrumentation was done with protaper rotary files up to F4 file and obturated with AH plus sealer (Dentsply, Germany) and Protaper gutta-percha cone using lateral condensation technique. The apical part of each root was resected at 90° to the long axis of the root for 3 mm, and retrograde cavity preparation was done in standardized dimensions. The teeth were randomly divided into four groups with 12 samples in each group and later subjected to a standard regimen of 3 ml of respective solutions for 5 min: Group I – 18% HEBP, Group II – 0.2% chitosan, Group III – 17% EDTA, Group IV – saline (control). All teeth were then restored with Biodentine (Septodont, USA). Samples were coated with two coats of nail varnish except in the apical 3 mm which will be immersed in 5 ml of 2% methylene blue dye and stored in incubator for 72 h. The nail varnish will be removed later, and the teeth will be immersed in 35% nitric acid for 72 h. The solutions were then filtered using a fine-grit filter paper and centrifuged at 2000 rpm for 1 min. The solutions thus collected will be used to determine absorbency in ultraviolet-visible spectrophotometer.
Results: The mean values of absorbance were greatest for Group IV (saline) followed by Group III (17% EDTA), Group II (0.2% chitosan), and Group I (18% HEBP). Group IV (saline) and Group III (17% EDTA) have got statistically significant difference with all the other groups. Group II has got statistically significant difference with all the other groups except Group I.
Conclusion: Within the limitations of the study, it was concluded that irrigation with newer agents significantly influenced the sealing ability of biodentine. Root-end irrigation with 18% HEBP and 0.2% chitosan showed the least microleakage when compared to 17% EDTA.

Keywords: Biodentine, chitosan, HEBP

How to cite this article:
Anitha S, George L, Mathew J, Paul S, Varghese T, Vineet R V. Evaluation and comparison of the apical seal obtained with Biodentine after conditioning of root end with three different solutions using ultraviolet-visible spectrophotometer: An in vitro study. Endodontology 2021;33:144-8

How to cite this URL:
Anitha S, George L, Mathew J, Paul S, Varghese T, Vineet R V. Evaluation and comparison of the apical seal obtained with Biodentine after conditioning of root end with three different solutions using ultraviolet-visible spectrophotometer: An in vitro study. Endodontology [serial online] 2021 [cited 2022 Aug 8];33:144-8. Available from: https://www.endodontologyonweb.org/text.asp?2021/33/3/144/327267

  Introduction Top

The goal of an ideal endodontic therapy is to seal all pathways of communication between the pulp and periodontium. The apical seal obtained by obturation and coronal seal obtained by postendodontic restoration prevent the percolation of fluids thus preventing the recontamination of the root canals.[1] The presence of smear layer acts as an avenue for leakage and source for bacterial growth and ingress, particularly following retrograde preparation and also support growth of bacteria remaining in the dentinal tubules.[2] Hence, it is important to remove the debris created during retrograde preparation from the dentine surface of the canal wall and the dentine tubules.

  Subjects and Methods Top

Forty-eight extracted human maxillary incisors were selected and were decoronated with a diamond disc to standardize the root length to 14 mm from the root apex. The working length was established using a 10#k file, and the root canals were instrumented with protaper rotary files till F4. Intermittent irrigation was done with saline followed by 5.25% sodium hypochlorite (NaOCl) and a final rinse of 17% ethylenediaminetetraacetic acid (EDTA) solution. The canals were coated with AH plus sealer and obturated with protaper F4 gutta-percha cone. The coronal opening was sealed with temporary restoration (ORAFIL-G). The apical 3 mm of the root was measured with a divider and was standardized by marking with a pencil on all the samples. The root ends were resected at 90° to the long axis of the root with tungsten carbide bur (#699) using a slow-speed micromotor handpiece. The apical root-end cavity of 3 mm depth and 1.2 mm diameter were prepared with a slow speed carbide bur (#57). The samples were randomly divided into 4 groups with 12 teeth in each group, and the prepared root-end cavity was subjected to a standard regimen of 3 ml of respective solution with a 30 gauge needle in each group for 5 min.

Group I – 18% HEBP irrigation (n = 12)

Group II – 0.2% chitosan irrigation (n = 12)

Group III – 17% EDTA irrigation (n = 12)

Group IV – Saline irrigation (n = 12)

After irrigation, the root-end cavity of all teeth was air dried and all the samples were restored with Biodentine. Samples from all the groups were coated with two coats of nail varnish except on the apical 3 mm, which were then immersed in 5 ml of 2% methylene blue. The nail varnish was later removed and all the samples were rinsed for 10 min under tap water. The samples were immersed in 35% nitric acid for 72 h and the solutions were filtered using a fine-grit filter paper and centrifuged at 2000 rpm for 1 min. The solution thus collected was used to determine absorbancy in ultraviolet (UV) visible spectrophotometer at 670 nm. The absorbancy calculated is directly proportional to the microleakage.

  Results Top

The statistical analysis was done using one-way ANOVA and post hoc test.

The results of the one-way ANOVA suggested that there is significant difference in absorbance values among the groups [Table 1].
Table 1: One-way ANOVA of mean values of the absorbance

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The results of the Tukey's post hoc analysis suggested that there is significant difference between all groups except Group 1 (18% HEBP) and Group 2 (0.2% chitosan) [Table 2].
Table 2: Tukey's post hoc analysis (multiple comparisons) of mean values of absorbance

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Group 1 (18% HEBP) has got statistically significant difference with all other groups except Group 2 (0.2% chitosan).

The bar diagram [Graph 1] shows that the mean values of absorbance are in the order of Group 4 (saline) > Group 3 (17% EDTA) > Group 2 (0.2% chitosan) > Group 1 (18% HEBP). Group 1 (18% HEBP) showed the least microleakage.

  Discussion Top

Microorganisms play a major role in pulpoperiapical diseases, and therefore, the primary aim of a root canal therapy is the removal of the pathological microflora in the canals and also to provide a fluid-tight seal to prevent the ingress of microorganisms or toxins to and from the canal system and periapical tissues. In some cases, conventional endodontic treatment may not be sufficient to resolve the lesion and surgical intervention is required.[3] This goal is achieved by root-end resection, root-end cavity preparation, conditioning of the retrograde cavity, and a bacteria-tight closure of the root-canal system at the cut root end with a retrograde restorative material.[4]

Cavity preparation creates smear layer and studies have pointed out that its presence is detrimental to the proper marginal adaptation of the restorative material.[5] In a study by Ballal, they suggested that total removal of the smear layer is essential to facilitate the penetration of filling materials into the dentinal tubules and to prevent microleakage.[6] George et al. in his study also pointed out that smear layer should be removed since it may act as a substrate for bacteria, allowing its deeper penetration into the dentinal tubules.[7]

EDTA has been a commonly used agent for smear layer removal. Etidronate and chitosan are two chelating agents introduced recently in endodontics. Chelating agents primarily act on the inorganic component of smear layer, aiding in its removal. They induce changes in calcium and phosphorous ion concentration in the root canal dentin.[8] The demineralizing effect of chelators acts simultaneously on the smear layer and the root canal dentin, resulting in collagen exposure and reduction of dentin microhardness.

The present study evaluated the effect of smear layer removal on the marginal adaptation of retrograde filling material to root dentin. It was observed that there was significant difference in the sealing ability of biodentine after root-end irrigation with 18% HEBP (Group 1) and 0.2% chitosan (Group 2) to that of EDTA (Group 3) and saline (control group) (P > 0.05).

In Group 1, HEBP (18%) was used for smear layer removal. HEBP (1-hydroxyethylidene-1, 1-bisphosphonate [BP]), also known as etidronic acid or etidronate, has been proposed as a potential alternative to EDTA or citric acid because this agent shows no short-term reactivity with NaOCl.[9] Studies have shown that the use of HEBP as a chelating agent has optimal effects on Ca/P ratio, dentin surface roughness, and microhardness with no erosive effects on dentin wall as compared to the other agents. Tartari et al. in his study reported that HEBP caused the removal of phosphate, exposure of the collagen matrix, and an increase in the amide III/phosphate ratio, which was concentration dependent.[10] Another study by Yadav et al. reported that the chelating capacity of 18% HEBP was found to be better than 9% HEBP because of high concentration.[11] BPs have a calcium chelating property and have similar structure as the natural pyrophosphate and contain two phosphonate (PO3) groups. These two phosphonate groups attached to a central carbon replace oxygen in pyrophosphate. This three-dimensional structure of pyrophosphate can chelate the divalent cations.[12] The results of the present study showed that root-end irrigation with 18% HEBP gave the lowest microleakage scores with a mean value of 0.6460. The lowest microleakage scores with HEBP group in the present study were in accordance with results of the study done by Ulusoy et al.[13] This could be attributed to the possible low surface tension of the solution, increasing its diffusion into the root canals.[13]

In Group 2, 0.2% chitosan was used as a chelating agent which is a natural polysaccharide that has multiple applications in the field of dentistry because of its properties such as biodegradability, biocompatibility, bioadhesion, and no toxicity.[14] The mean value of Group 2 after irrigation with 0.2% chitosan is 0.6805, greater than Group 1 (18% HEBP) but this difference is not statistically significant. The comparable chelating property of chitosan could be attributed to two theories based on the mechanism involved in chelating with chitosan. The bridge model theory is based on the mechanism where two or more amino groups on the chitosan chain bound the same metal ion.[15] The second theory claims that only one of the amino groups on the chitosan chain is anchored to the metal ion.[16] Another study revealed that chitosan and metal ion complexes are a result of ion exchange, chelation, and adsorption.[17] Chitosan acts with its functional phosphate groups reacting with dentin calcium ions leading to the formation of calcium phosphate layer. Pimenta et al. in their study suggested the application of 0.2% of chitosan solution for 5 min as the most viable combination for conditioning of root dentin.[18] Hence, in this study, 0.2% chitosan was chosen as chelating agent. The results of present study are in accordance with Darrag that the use of 0.2% chitosan solution has obtained better results compared to 17% EDTA at the three levels of the root.[19]

In Group 3, 17% EDTA was used for root-end conditioning and the mean value of microleakage was 0.775 which is greater when compared Group 1 and Group 2 where 18% HEBP and 0.2% chitosan were used, respectively, and was statistically significant. Ethylenediaminetetraacetic acid was commonly used for smear layer removal which is achieved by acting on the inorganic matter.[20] Von der in his study pointed out that its reaction with calcium ions in dentine results in calcium chelation, promoting decalcification of dentine at approximate depths of 20–30 μm within 5 min.[21] Spanó et al. in his study recommended EDTA in combination with different concentrations of NaOCl for the complete elimination of the smear layer.[22] The relationship between the concentration of the chelating agent and its application time seems to be important as it was found that highly concentrated solutions applied for a long period, cause roughness of dentin surface.[23] Few literatures reveal that EDTA can cause harm to the periapical tissues with its increasing frequency of usage.[22]

In Group 4, saline was used as the control and the results showed a higher microleakage value with a mean score of 1.034 which is higher than the other 3 groups. This high score suggests the effectiveness of chelating agents in the removal of smear layer.

Different retrograde filling materials have been tried in surgical procedures after root-end resection and preparation, in an attempt to prevent microleakage and promote bone healing. von Arx reported that the success of periapical surgery is attained not only by applying the correct procedure and indications but also by the use of biocompatible retrograde filling material, which provides good apical seal thereby prevents the penetration of periapical fluids into the root canal system.[4] Hence, Biodentine was chosen as a retrograde filling material in this study. Hindlekar and Raghavendra in his study stated that the tricalcium oxide in the cement of biodentine reacts with the tissue fluid and stimulates dentine regeneration by inducing odontoblast differentiation from pulp progenitor cells.[24] Further, Malhotra and Hegde proposed that the smaller size of Biodentine particles aids in enhanced adaptation at the cavity surface and filling interface.[25] The decreased pore volume and porosity of biodentine as compared to MTA resulted in better sealing ability.[25] The modified composition of the Biodentine powder such as the absence of calcium aluminate, calcium sulfate, and presence of calcium chloride in liquid has improved its physical properties mainly handling and the sealing ability. The faster setting of Biodentine would have prevented the prolonged leakage thereby reducing the bacterial contamination. In Biodentine, the formation of biomineralization-tag (apatite forming ability in the presence of phosphate solution) has improved the sealing ability of Biodentine.[26]

Maxillary incisors were selected to simulate the clinical scenario and to obtain predictable results. The apical 3 mm of the roots, with an angle of 90°, was resected in the present study. This would allow for a better quality of cut, reduce apical ramifications by 98%, and lateral canals by 93%.[11] The preparation depth of 3 mm decreases the leakage which is attributed to the occlusion of apical dentinal tubules by retrograde filling materials.

The present study utilized a dye extraction method since it provides more reliable results than dye penetration study because of its ability to measure all of the dyes taken up in the root. In this method, the teeth are dissolved in acids, releasing all the dyes from the interfacial areas and a spectrophotometer determines the optical density (OD) of the solution.[27] Thus, it quantitatively measures dye penetration through the margins of restoration.[28] Methylene blue dye was used because it was an accepted dye in many in vitro studies and also it was inexpensive, has a high degree of staining, and has a molecular weight lower than that of bacterial toxins. The SEM analysis was not carried out since dehydration and drying procedures during sample preparation may create artifacts in hard tissues.[29] Janda in his study compared “direct” and “indirect” SEM analysis in which indirect approach is carried out by taking impressions with appropriate materials but cannot provide the detailed information of the tooth structure.[30]

In the present study, the results were recorded as a measure of absorbance of light. According to the Beer-Lamberts law, the absorbency of the solution is directly proportional to the concentration of absorbing species in the solution and path length. UV visible spectroscopy can be used to determine the concentration of the absorber in the solution for a fixed path length.[31] Hence, it can be interpreted that absorbance of the solution is directly related to the amount of microleakage. The absorption or reflectance in the visible range directly affects the perceived color of the chemicals involved.[32]

The results of the present study emphasize on the removal of smear layer for the better adhesion of retrograde filling material to root dentine. 18% HEBP and 0.2% chitosan have shown better property of smear layer removal and can be used as an alternative irrigant. Further research is necessary to establish it as a potential irrigant since many in vivo environment could not be simulated in this study.

  Conclusion Top

Within the limitations of this study, it was concluded that:

  1. The use of root conditioning agents improved the sealing ability of Biodentine as a retrograde restorative material.
  2. Root-end conditioning with 18% HEBP (1-hydroxyethylidene-1, 1-bisphosphonate) and 0.2% chitosan showed the least microleakage when compared to 17% EDTA.

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Conflicts of interest

There are no conflicts of interest.

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