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ORIGINAL ARTICLE
Year : 2020  |  Volume : 38  |  Issue : 2  |  Page : 132-137
 

Comparative evaluation of calcium release of the apical plugs formed by mineral trioxide aggregate, Biodentine, and EndoSequence root repair material with and without 2% triple antibiotic powder: An in vitro study


Department of Paedodontics and Preventive Dentistry, School of Dental Sciences, Krishna Institute of Medical Sciences Deemed University, Karad, Satara, Maharashtra, India

Date of Submission13-Feb-2020
Date of Acceptance03-Jun-2020
Date of Web Publication28-Jun-2020

Correspondence Address:
Dr. Pooja Nitin Mapara
Department of Paedodontics and Preventive Dentistry, School of Dental Sciences, Krishna Institute of Medical Sciences Deemed University, Karad, Satara - 415 110, Maharashtra
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/JISPPD.JISPPD_74_20

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   Abstract 


Background: Apical plug formation by mineral trioxide aggregate (MTA), Biodentine, and EndoSequence root repair material (RRM) is an excellent alternative technique to the conventional apexification procedure. Several antimicrobial agents have been incorporated in MTA and Biodentine to boost their antimicrobial efficacy. Considering the polymicrobial nature of root canal infection, a combination of potent antimicrobials like triple antibiotic powder (TAP) would be needed to address the diverse flora encountered. Calcium release is the consequential factor in the clinical excellence of these cements. Aim: The aim of the study was to evaluate and compare the calcium released from the apical plugs formed by MTA, Biodentine, and EndoSequence RRM with and without incorporation of 2% TAP. Methods: Ninety single-rooted teeth were divided into (n = 15) Group A: MTA + 2% TAP, Group B: MTA, Group C: Biodentine + 2% TAP, Group D: Biodentine, Group E: EndoSequence RRM + 2% TAP, and Group F: EndoSequence RRM to form the 4 mm apical plugs. Each sample tooth was then immersed in 10 ml of deionized water. Evaluation of calcium release was done on days 7, 15, and 30 using an atomic absorption spectrophotometer. Data were analyzed using one-way analysis of variance, post hoc test, and unpaired t-test. Results: Calcium released was maximum for Group E compared to Group F (P < 0.05), maximum for Group C compared to Group D, and was maximum for Group A compared to Group B (P < 0.05) at days 7, 15 and 30. Conclusion: Incorporation of 2% TAP resulted in increased calcium ions released from MTA, Biodentine, and EndoSequence RRM.


Keywords: Apexification, apical plug, atomic absorption spectrophotometry, bioceramic cements, Biodentine, calcium release, EndoSequence root repair material, immature tooth, mineral trioxide aggregate, triple antibiotic powder


How to cite this article:
Mapara PN, Shashikiran N D, Gugawad S, Gaonkar N, Hadakar S, Taur S, Khade D. Comparative evaluation of calcium release of the apical plugs formed by mineral trioxide aggregate, Biodentine, and EndoSequence root repair material with and without 2% triple antibiotic powder: An in vitro study. J Indian Soc Pedod Prev Dent 2020;38:132-7

How to cite this URL:
Mapara PN, Shashikiran N D, Gugawad S, Gaonkar N, Hadakar S, Taur S, Khade D. Comparative evaluation of calcium release of the apical plugs formed by mineral trioxide aggregate, Biodentine, and EndoSequence root repair material with and without 2% triple antibiotic powder: An in vitro study. J Indian Soc Pedod Prev Dent [serial online] 2020 [cited 2020 Sep 21];38:132-7. Available from: http://www.jisppd.com/text.asp?2020/38/2/132/288231





   Introduction Top


An eruption of a tooth in the oral cavity simulates “iceberg” phenomenon. Although the crown appears to be developed, the root formation is still incomplete and the tooth is yet in the stages of development. Gradual root development and closure of the apex perpetuate until 2–3 years postclinical eruption of a tooth. This critical stage of tooth development is vulnerably susceptible to the minute amount of mechanical, physical, and microbiological insults. Any incidence of trauma, extensive caries, and any pulpal pathosis during this critical period leads to a halt in dentin formation which ultimately results to have a tooth with an open apex. Pulp therapies to rescue such immature teeth with necrotic pulp are challenging due to the lack of apical barrier. The conventional treatment of choice in such cases is apexification with calcium hydroxide due to its high pH and antimicrobial activity.[1] Calcium hydroxide apexification technique has certain drawbacks such as prolonged treatment duration, need for multiple appointments, and long-term treatment with calcium hydroxide which makes the tooth susceptible to root fractures.[2] Hence, one-step apical plug technique of a single appointment to create a hermetic seal at the apex of a tooth with an open apex is the need of the day.

Mineral trioxide aggregate (MTA) is widely used to form an apical barrier as an alternative to the calcium hydroxide apexification technique. It is a material with excellent biocompatibility and good sealing properties. MTA also sets in the presence of moisture and blood. There are some drawbacks of MTA such as slow setting kinetics, dentin discoloration, and complicated handling properties.[3] With the unceasing need for more new dental materials, bioceramic cements got introduced in dentistry. Biodentine is termed as “dentin in a capsule” due to its high physical and mechanical properties simulating tooth's dentin. It has ease of handling with excellent biocompatibility and bioactive nature. Biodentine has faster setting time, and also, metal impurities are eliminated, so it does not cause dentin discoloration which is an advantage over MTA.[4] EndoSequence root repair material (RRM) introduced by Brasseler company is a new generation bioceramic material indicated for apical plug in immature teeth. It has ease of handling with biocompatible and bioactive nature. It has ability to induce tissue regeneration by its osteoinductive and osteoconductive properties.

MTA, Biodentine, and EndoSequence RRM form calcium hydroxide as their reaction product, which further dissociates into calcium and hydroxyl ions. This results in high alkaline pH, which creates an unfavorable environment for bacterial growth leading to the disinfection of hard and soft tissues. Thus, calcium release is the consequential factor in the mechanism of action of MTA, Biodentine, and EndoSequence RRM.[5]

Root canal infection is polymicrobial in nature.[6],[7] Abundant efforts have been done to boost the antimicrobial efficacy of MTA and Biodentine by incorporating antimicrobial additives like 2% chlorhexidine, 10% doxycycline, and 2% silver zeolite powder. Considering the polymicrobial nature of the root canal infection, it is unlikely that incorporation of any single antimicrobial agent in apical plug forming dental material could result in effective sterilization of the root canal.[8] More likely, a combination of potent antimicrobials like triple antibiotic powder (TAP) would be needed to address the diverse flora encountered.[9],[10] Thus, the incorporation of 2% TAP in apical plug forming MTA, Biodentine, and EndoSequence RRM to manage critical cases of immature teeth is a novel concept.[11]

TAP is well known for its lesion sterilization and tissue repair property. The conventional TAP consists of ciprofloxacin, metronidazole, and minocycline/doxycycline. Doxycycline, a drug of the tetracycline group, has a tendency to form chelates with available free calcium ions which are released from the apical plugs. Thus, doxycycline can alter the effectiveness of the apical plugs formed by MTA, Biodentine, and EndoSequence RRM.[12] Modified TAP formed by replacing doxycycline with cefaclor was thus considered in the present study.[13]

Hence, the present study was conducted to evaluate and compare the calcium release potential of MTA, Biodentine, and EndoSequence RRM and also to evaluate whether the incorporation of 2% TAP in MTA, Biodentine, and EndoSequence RRM affects their cardinal calcium release property.


   Methods Top


This was anin vitro study conducted after due approval from the ethical committee with protocol number 0255/2017-2018. In this study, MTA (MTA-Angelus®, Angelus, Londrina, PR, Brazil), Biodentine (Septodont, Saint-Maur-des-Fosses, France) and EndoSequence RRM (EndoSequence RRM, BC RRM-Fast Set Putty™, Brasseler, USA) were used to form apical plugs in teeth prepared to simulate immature teeth with open apices. These novel endodontic cements were selected on the basis of their clinical excellence, biocompatibility, and biosmart behavior.

Ninety intact, caries-free human single-rooted teeth freshly extracted for the therapeutic purpose were included. Teeth with root fracture, preexisting external defects, or cracks on the root surface and teeth with anatomical irregularities were excluded from the study.

Once the teeth were collected, they were kept in a 0.5% chloramine T solution. Prior to use, the teeth were cleaned with a scaler tip for removing the debris and polished with prophylaxis paste using a polishing brush with a low-speed handpiece.

The patency of the root canal was confirmed by inserting a 25 mm size 10 Kerr file (K file) and working length was determined. The biomechanical preparation was done such that the apical part of the root canal was prepared up to size 40 K file and coronal flaring was done by size 80 K file. Root canals were irrigated with 2 ml of 5.25% sodium hypochlorite (NaOCl) between each instrumentation used. While the samples were being prepared, the roots of the teeth were separated from their crowns by a transverse section through the cementoenamel junction with the help of a diamond disc. Simulation of an open apex was done by removing apical 2 mm of root with the help of a diamond disc with a rapidly rotating handpiece. All the specimens were standardized to have 12 mm root length. After preparation, root canals were irrigated with 17% ethylenediaminetetraacetic acid and 5.25% NaOCl to remove the smear layer, followed by a final flush of distilled water for the total removal of NaOCl from the canals. Then, the canals were dried using proper paper points. All the prepared sample teeth were autoclaved and stored in sterilized airtight jars in an incubator at 37°C.

After the sample teeth preparation, modified TAP was prepared by replacing the doxycycline of conventional TAP (ciprofloxacin + metronidazole + doxycycline) with cefaclor. Generic medications were used in their powdered form to standardize the grain size and consistency of the mixture. Prepared modified TAP (ciprofloxacin + metronidazole + cefaclor) was stored in an airtight container.

Subsequently, all prepared root specimens were randomly separated into six experimental groups, and apical plugs (4 mm) were formed in each sample as follows (n = 15):

  • Group A: MTA + 2% TAP (4 mm of apical plug formed using MTA with the incorporation of 2% TAP)
  • Group B: MTA (4 mm of apical plug formed using MTA)
  • Group C: Biodentine + 2% TAP (4 mm of apical plug formed using Biodentine with the incorporation of 2% TAP)
  • Group D: Biodentine (4 mm of apical plug formed using Biodentine)
  • Group E: EndoSequence RRM + 2% TAP (4 mm of apical plug formed using EndoSequence RRM with the incorporation of 2% TAP)
  • Group F: EndoSequence RRM (4 mm of apical plug formed using EndoSequence RRM).


For apical plug formation in samples of Group B, Group D, and Group E, MTA, Biodentine, and EndoSequence RRM were strictly manipulated according to the manufacturer's instructions and 4 mm of the apical plug was formed using endodontic pluggers.

For apical plug formation in samples of Group A, Group C, and Group F, 2% by weight of TAP was incorporated in MTA, Biodentine, and EndoSequence RRM that were strictly manipulated according to the manufacturer's instructions to form 4 mm of apical plug using endodontic pluggers.

After formation of the apical plugs in all root specimens, nail varnish was applied on the external root surface except for the 2 mm around the apical foramen to standardize the evaluation of calcium ions released only through the apical opening of the apical plugs.

After the final setting of the apical plugs, each specimen was immersed in a test tube containing 10 ml of deionized water. Tubes were sealed and were incubated at 370°C. Evaluation of calcium release was done on days 7, 15, and 30 with atomic absorption spectrophotometer (15 samples which were there in each group were further subdivided, i.e., n = 5 into three testing intervals, i.e., on days 7, 15, and 30). The values obtained were analyzed using one-way analysis of variance (ANOVA), Post hoc test, and unpaired t-test. All the statistical analyses were carried out using the Statistical Package for the Social Sciences (SPSS) software version (Version 21.0. Armonk, NY: IBM Corp).


   Results Top


Apical plugs formed by MTA, Biodentine, and EndoSequence RRM and apical plugs formed with 2% TAP incorporation in MTA, Biodentine, and EndoSequence RRM showed calcium release over 30 days of the study period. All the apical plugs released the highest calcium ions up to day 7 which gradually decreased on days 15–30.

One-way ANOVA and post hoc test, accepting P < 0.05, showed that the mean value of calcium ions released was maximum for Group F (EndoSequence RRM) on days 7, 15, and 30 as compared to Group D (Biodentine) and Group B (MTA) (P < 0.05) [Table 1].
Table 1: One-way analysis of variance and post hoc test for the comparison of the mean calcium released (mg/L) from the apical plugs formed by mineral trioxide aggregate (Group B), Biodentine (Group D), and EndoSequence root repair material (Group F) on days 7, 15, and 30

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One-way ANOVA and post hoc test, accepting P < 0.05, showed that the mean value of calcium ions released was maximum for Group E (EndoSequence RRM + 2% TAP) on days 7, 15, and 30 as compared to Group C (Biodentine + 2% TAP) and Group A (MTA + 2% TAP) (P < 0.05) [Table 2].
Table 2: One-way analysis of variance and post hoc test for the comparison of the mean calcium released (mg/L) from the apical plugs formed by mineral trioxide aggregate, Biodentine, and EndoSequence root repair material with incorporating 2% triple antibiotic powder on days 7, 15, and 30

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The unpaired t-test, accepting P < 0.05, showed that the calcium ions released by Group A (MTA + 2% TAP), Group C (Biodentine + 2% TAP), and Group E (EndoSequence RRM + 2%TAP) were greater than calcium ions released by Group B (MTA), Group D (Biodentine), and Group F (EndoSequence RRM), respectively, on days 7, 15, and 30 (P < 0.05) [Table 3].
Table 3: Comparison of calcium release (mg/L) from the apical plugs formed by mineral trioxide aggregate, Biodentine, and EndoSequence root repair material to apical plugs formed by incorporating 2% triple antibiotic powder in mineral trioxide aggregate +2% triple antibiotic powder, biodentine +2% triple antibiotic powder, and EndoSequence root repair material +2% triple antibiotic powder, respectively, on days 7, 15, and 30

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Thus, the results suggested that the incorporation of 2% TAP in MTA, Biodentine, and EndoSequence RRM significantly improved their calcium ions release potential.


   Discussion Top


Incomplete root development as a result of any trauma, caries, or other pulpal pathosis that occurred during the critical developmental phase of a tooth requires special attention and treatment. The efficacy of biomechanical instrumentation is compromised due to thin dentinal walls. The divergent apex architecture makes complete canal debridement uncertain, and the lack of apical constriction generates a constant risk for endodontic irrigants, medicaments, root canal fillings, bacteria, and their by-products to extrude beyond the apex. Management of a nonvital tooth with an open apex necessitates stimulating the formation of an apical barrier which can function as a stop for the endodontic materials. MTA, Biodentine, and EndoSequence RRM with their excellent physical, mechanical properties, and their ability to overcome the disadvantages of conventional dental materials have become an excellent alternative to long-term apexification procedure. Hence, these novel bioactive materials were selected in the present study.

The root canal infection is polymicrobial in nature.[6],[7] Some resistant bacteria like  Enterococcus faecalis Scientific Name Search ound residing in root canals even after canal disinfection procedures. Enterococcus faecalis can withstand alkaline conditions and glucose starvation and thus survive in root canals for a prolonged duration. Thus, they are termed as “exceptional survivors” of the root canals.[14] Hence, the inclusion of Enterococcus faecalis (ATCC 29212) as experimental bacteria in the present study was justified.

To achieve utmost sterilization of the infected root canals and periradicular region in an immature tooth, abundant effort has been made to boost the antimicrobial efficacy of apical plugs by incorporation of antimicrobial additives such as 2% chlorhexidine, 10% doxycycline, and 2% silver zeolite powder.[8] Similarly, 2% TAP known for its lesion sterilization and tissue repair capacity was incorporated in MTA, Biodentine, and EndoSequence RRM to form the apical plugs in immature teeth.

It is well known that the cytocompatibility, biocompatibility, and regenerative/reparative potential are assumed to be inherent to MTA, Biodentine, and EndoSequence RRM by virtue of their cardinal calcium ion release mechanism.[15] Hence, the present study was conducted to evaluate calcium release potential of MTA, Biodentine, and EndoSequence RRM and also to evaluate whether the incorporation of 2% TAP in MTA, Biodentine, and EndoSequence RRM affects their cardinal calcium release property.

In accordance with Jacinto et al., 10 ml deionized water at neutral pH was chosen for specimen immersion to obtain accurate measurements of calcium ions released without ion contamination from the immersion liquid on days 7, 15, and 30 using atomic absorption spectrophotometry.[5]

In previous studies to evaluate calcium ions release, materials were incorporated in polyethylene tubes and were immersed in 10 ml deionized water. In our study, we had used root samples, in which apical plugs were formed. The methodology used in the present study closely mimics the clinical situation and makes calcium release evaluation more relevant. Atomic absorption spectrophotometry is a known successful modality used in earlier studies for analyzing calcium ions released through dental materials.[16],[17]

The presentin vitro study demonstrated that calcium ions released by apical plugs formed with EndoSequence RRM and EndoSequence + 2% TAP were greater than other study groups. The present study also documented that the incorporation of 2% TAP in MTA, Biodentine, and EndoSequence RRM significantly improved their calcium ion release potential.

According to Jacinto et al., bioceramic, endodontic materials show excellent ability to release calcium ions for longer duration owing to their structural characteristic that calcium silicate present in them gets hydrated to produce a calcium silicate hydrate gel and calcium hydroxide. The calcium hydroxide dissociates and releases calcium ions and hydroxyl ions into the medium. These ions react with the phosphate ions from tissue fluid to precipitate hydroxyapatite and water. The water continues to react with the calcium silicates to precipitate additional gel-like calcium silicate hydrate and the reaction progresses, resulting in continued and extended release of calcium ions and precipitation of more hydroxyapatite crystals.[5] Similarly, in the present study, all of the tested study materials released calcium ions throughout the 30 days of the study period.

EndoSequence RRM group showed statistically highest release of calcium ions, followed by Biodentine and least with MTA group on days 7, 15, and 30. Similarly, EndoSequence RRM + 2% TAP showed statistically highest release of calcium ions followed by Biodentine + 2% TAP, and least calcium ions release was by MTA + 2% TAP. Similar results were observed by Abu Zeid and Mokeem Saleh and Candeiro et al. that strong and prolonged alkalinity of EndoSequence RRM was synchronized with its greatest calcium release than other materials.[18],[19]

The results of the present study were in accordance with Aprillia et al. that Biodentine released more calcium ions compared to MTA-Angelus.[20] The justification put forward by Gandolfi et al. was that the ion release phenomenon depends on a variety of factors including structure and constituent mineral particles of the materials. These are ultimately responsible for water absorption and solubility of material. Biodentine forms calcium phosphate particles measuring < 1 μ, resulting in a more compact surface layer. MTA Angelus, on the other hand, has a larger particle size (diameter: 1–5 μ). This is one of the factors responsible for greater calcium ion release from Biodentine compared to that from MTA, as seen in the present study.[21]

The addition of antibacterial agents to dental materials can alter the physical properties of material, which may affect their clinical performance. Thus, the incorporation of such antimicrobial agents in dental materials is only justified when it improves the antimicrobial efficacy without compromising the excellent peculiar physical and morphological properties of that dental material. In the present study, the incorporation of 2% TAP to MTA, Biodentine, and EndoSequence RRM increased their calcium release ability.

Appreciating that calcium release property of bioceramic materials is worth a million dollar in their mechanism of action, the present study documented that calcium release property of MTA, Biodentine and EndoSequence RRM with 2% TAP showed a statistically significant increase than the calcium ions released by materials alone. Thus, in critical cases of apexification, apical plugs can be formed with incorporating 2% TAP in MTA, Biodentine, and EndoSequence RRM to improve their antimicrobial efficacy without affecting and rather to improve their cardinal calcium release mechanism.

Furtherin vivo studies have to be done to prove the effect and efficacy of combination formed by incorporating 2% modified TAP containing cefaclor in MTA, Biodentine and EndoSequence RRM to form one step apical plug in a necrotic immature tooth with an open apex.


   Conclusion Top


EndoSequence RRM showed the highest potential of calcium ion release than Biodentine and MTA at all study intervals. EndoSequence RRM + 2% TAP showed highest calcium ions released through apical plug than Biodentine + 2% TAP and MTA + 2% TAP. The incorporation of 2% by weight of modified TAP containing cefaclor in MTA, Biodentine, and EndoSequence RRM to form apical plug during endodontic treatment of an immature necrotic tooth increases their cardinal calcium release potential.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
   References Top

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Farhad AR, Barekatain B, Allameh M, Narimani T. Evaluation of the antibacterial effect of calcium hydroxide in combination with three different vehicles: Anin vitro study. Dent Res J (Isfahan) 2012;9:167-72.  Back to cited text no. 1
    
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Jacinto RC, Linhares-Farina G, Sposito Oda S, Zanchi CH, Cenci MS. Influence of 2% chlorhexidine on pH, calcium release and setting time of a resinous MTA-based root-end filling material. Braz Oral Res 2015;29:1-6.  Back to cited text no. 5
    
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Gandolfi MG, Siboni F, Polimeni A, Bossù M, Riccitiello F, Rengo S,et al. In vitro screening of the apatite-forming ability, biointeractivity and physical properties of a tricalcium silicate material for endodontics and restorative dentistry. Dent J 2013;1:41-60.  Back to cited text no. 21
    



 
 
    Tables

  [Table 1], [Table 2], [Table 3]



 

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