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 Table of Contents  
Year : 2022  |  Volume : 34  |  Issue : 1  |  Page : 16-21

Sealing ability of mineral trioxide aggregate, Biodentine, and EndoSequence RRM putty used as retrograde restorative material: An in vitro bacterial leakage model study

1 Department Of Conservative Dentistry and Endodontics, Kerala University of Health Sciences, Ernakulam, Kerala, India
2 Department of Conservative Dentistry and Endodontics, Annoor Dental College and Hospital, Kerala University of Health Science, Ernakulam, Kerala, India

Date of Submission22-Sep-2021
Date of Decision15-Dec-2021
Date of Acceptance17-Jan-2022
Date of Web Publication25-Mar-2022

Correspondence Address:
Dr. Jesmy K Antony
Choozhikunnel House, Pulluvazhy P. O., Ernakulam - 683 541, Kerala
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/endo.endo_176_21

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Aim: the present study aimed to evaluate and compare the bacterial sealing ability of ProrootMTA, Biodentine and EndoSequenceRRM putty as retrograde filling material using E-faecalis leakage model over a period of 6weeks.
Materials and Method: Forty single rooted human premolar teeth were collected, disinfected and decoronated to a standardized of 16mm. Cleaning and shaping was done up to 40#K-file apical preparation and step back method. The roots were sectioned 3 mm from the apex. Root end cavities were prepared using ultrasonic tips (Woodpecker EMS EndoScaler TipE3D) and restored using the three different materials. Specimens were divided in to four groups with a sample size of n =10. Group 1–ProRootMTA, Group2– Biodentine, Group3– EndoSequenceRRM putty, Group 4-Control without any filling. Specimens were then mounted on the E-faecalis (ATCCstrain29212) bacterial leakage models. The bacterial leakage was assessed by measuring the optical density of the broth at every 7th day for a 6 weeks period.
Results: In the baseline reading there was significant difference between all the groups. In the 2nd week, Group1 has got significantly higher values than Group 3 and not with Group 2. In the 3rd, 4th, 5th and 6th weeks, Group1 show significantly higher turbidity values than Group 2 and 3. No significant difference between Group2 and 3.
Conclusion: Within limitations of this study it was concluded that, both ESRRMputty and Biodentine have got comparable sealing properties. ProrootMTA has got higher microleakage than others in the 3rd, 4th, 5th and 6th week. The microleakage for ProrootMTA, Biodentine and ESRRM putty was increasing up to the third week following which it is static.

Keywords: Proroot MTA, Biodentine, Endosequence RRM putty, Bacterial sealing, Retrogarde restorations

How to cite this article:
Antony JK, George L, Mathew J, Joy A. Sealing ability of mineral trioxide aggregate, Biodentine, and EndoSequence RRM putty used as retrograde restorative material: An in vitro bacterial leakage model study. Endodontology 2022;34:16-21

How to cite this URL:
Antony JK, George L, Mathew J, Joy A. Sealing ability of mineral trioxide aggregate, Biodentine, and EndoSequence RRM putty used as retrograde restorative material: An in vitro bacterial leakage model study. Endodontology [serial online] 2022 [cited 2022 Aug 8];34:16-21. Available from: https://www.endodontologyonweb.org/text.asp?2022/34/1/16/340836

  Introduction Top

The objective of endodontic therapy is to clean, shape, and obturate the root canal system three dimensionally.[1] The communication between the main body of the root canal and the periodontal ligament space is through the channels of lateral canals and the accessory canals which are found at the apical delta.[2] These channels can be a niche for microorganisms. The main goal of the surgical endodontic treatment is to prevent the invasion of bacteria and their byproducts from the root canal system into the periradicular tissues by sealing it with adequate and efficient root-end filling material.[3] These irritants from the root canal that egress into the periradicular tissues result in apical periodontitis. Periradicular surgery is indicated in case of repeated failures of root canal treatment. This involves exposure of the apex, root resection, root-end preparation, and root-end filling to obtain a tight apical seal to keep the microorganism and their byproducts out of the periapical region.[4]

Numerous materials have been proposed for root-end fillings of which mineral trioxide aggregate (MTA) is the first calcium silicate-based bioceramic material which is successfully used as a retrograde restorative material. Its excellent biocompatibility, sealing ability, cementogenesis, and several other advantages made it a landmark in the history of endodontics.[5] Biodentine is similar to MTA in terms of basic composition, and the manufacturers claim that the modification in its powder composition by addition of setting accelerators and softeners largely improved the physical properties of the material, making it more user-friendly.[6] EndoSequence Root Repair Material (RRM) (ESRRM) putty is a premixed bioceramic material available as injectable paste or putty consistency. Endo Sequence RRM, with more of a putty consistency, is reported as being easier to physically manage and to place where it is necessary.[7]

The aim of this study was to compare and evaluate the sealing ability of three retrograde filling materials ProRoot MTA, Biodentine, and EndoSequence RRM putty using bacterial leakage models.

  Materials and Methods Top

This in vitro study was completed in the Department of Conservative Dentistry and Endodontics (Ethical committee approval no: IHEC Ref No: 018-A/04). Forty single-rooted human premolar teeth were collected. The collected teeth were disinfected with 6% hydrogen peroxide,[8] followed by sample preparation.

The samples were decoronated and the lengths of all the roots were standardized to 16 mm. Cleaning and shaping was done along with intermittent irrigation using 2 ml of 5% of sodium hypochlorite (Chemdent) alternating with 17% ethylenediaminetetraacetic acid (Meta MD Cleanser, Biomed).[9] Irrigation was done using side-vented needles. The apical preparation was done up to 40 #Kfile (Mani, INC.) and step back preparation was done up to size 55#K file (Mani, INC.).

The roots were sectioned 3 mm from the apex. The root end cavities were prepared using ultrasonic tips (Woodpecker EMS Endo Scaler Tip E3D) with a standard measurement of 3 mm depth and 1 mm diameter. The preparations were done using ultrasonic tips on which 3 mm markings were made.

Specimens were then randomly divided into four groups:

  • Group 1 – ProRoot MTA (n = 10)
    • (Dentsply Sirona Endodontics)
  • Group 2 – Biodentine (n = 10)
    • (Septodont, Saint Maur des Fosses, France)
  • Group 3 – EndoSequence root repair putty (n = 10)
    • (Brasseler USA, Savannah, GA)
  • Group 4 – Control group– without any filling (n = 10).

A gutta percha point of 60 size and 2% taper was then placed in the root canal just above the root end cavity against which the samples condensed with the tested materials.

Retrograde cavities were filled with the three different materials according to the guidelines. The samples were then coated with two coats of three different colored nail varnish except on the apical 3 mm. The restorations were confirmed for the absence of voids with radiographs. Specimens were then stored for a period of 3 weeks in the presence of moisture in order to achieve the complete setting of the materials. Specimens were then mounted on the Enterococcus faecalis (ATCC strain 29,212) bacterial leakage models in which 2–3 mm of the root tip was in contact with the brain heart infusion (BHI) broth. The whole models were placed in ultraviolet (UV) chamber for sterilization (Uniclave 5G UV chamber BioWarrior). Each root canal was then aseptically inoculated with 15 μL freshly prepared culture and incubated in a humid environment at 37°C for 6 weeks. The turbidity (optical density [OD]) generated by the bacteria present in the BHI broth was measured using a UV spectrophotometer. The measurements were taken at every 7th day for a 6-week period. The sample obtained from the broth was then Gram stained for confirmation of cell morphology.

The data were subjected to statistical analysis using one-way ANOVA to compare the difference in OD value between the materials in the 6-week period. Repeated-measure ANOVA was used to assess the changes in the OD value obtained for each material over 1–6 weeks. Post hoc was done using Bonferroni test. P < 0.05 was considered statistically significant.

IBM SPSS Version 25 – International Business Machines Corp. (IBM) 2017 Statistical package for Social Science (SPSS) statistics Version 25 (Armonk, NY: IBM Corp).

  Results Top

One-way ANOVA was used to compare the OD value between the materials in each week (baseline, 1st, 2nd, 3rd, 4th, 5th, and 6th). There was a statistically significant difference between the materials in all the 6 weeks [Table 1].
Table 1: One-way ANOVA

Click here to view

Results of Post hoc test-bonferroni

  1. Baseline reading – There is a statistically significant difference between all the groups (Group 4> Group 1> Group 2> Group 3)
  2. First week – There is a statistically significant difference of the three materials with the control group only. No significant difference is found between the materials
  3. Second week – Group 1 has got statistically significant difference with Group 3 and not with Group 2. Group 2 and 3 have got no significant difference
  4. Third, 4th, 5th, and 6th weeks – Group 1 showed significantly higher value than Group 2 and 3. There is no significant difference between Groups 2 and 3.

(Group 4> Group 1> Group 2 and Group 3).

Repeated-measures ANOVA was used to assess the changes in the OD value obtained for each material over a period of 1–6 weeks. For all the groups, there was a statistically significant difference in the OD value over the 6-week period [Table 2].
Table 2: Repeated-measures ANOVA

Click here to view

Post hoc test results

For Groups 1, 2, and 3, the baseline reading was significantly lower compared to 3rd, 4th, 5th, and 6th weeks.

For Group 3, the mean difference of OD between 1st versus 3rd, 4th, 5th, and 6th weeks is statistically significant with P < 0.05.

  Discussion Top

Provision of an effective and three-dimensional apical seal is one of the most important factors that is to be considered while selecting a retrograde restorative material.[10] In this study, the sealing ability of white ProRoot MTA, which is a widely used retrograde filling material, was compared with Biodentine and the newer generation calcium phosphate silicate cement, EndoSequence RRM putty, for a time period of 6 weeks. Several in vitro methods have been proposed for assessment of the sealing ability of restorative materials. Bacterial leakage models were used in this study as it is found to better simulate the clinical conditions when comparaed to dye leakage method because of the smaller size of dye particles (Timpawat et al).[11] While doing root-end resection, at least 3 mm of the root-end must be eliminated to reduce 98% of the apical ramifications and 93% of the lateral canals and that perpendicular resection minimizes the number of exposed dentinal tubules.[12] As the angle of the bevel increases the apical leakage also increases. This is due to the exposure of wider apical surface and greater number of dentinal tubules which leads to the increased permeability.[13] So a 90° angle was used for root end resection. Sonic and ultrasonic devices have been demonstrated to produce cleaner, well-centered, and conservative root-end cavities (Khabbaz et al).[14]

According to the results obtained, the turbidity values of the control group were higher than the three tested materials in all the 6 weeks which depicts the presence of active growth in the inoculum and the BHI broth. The bacterial inoculum is in direct contact with the tested materials present in the root end cavities and all the three tested materials have been shown to possess some antibacterial effect.[15],[16] ProRoot MTA and Biodentine have demonstrated good antifungal and antibacterial properties when studied using tube dilution methods. The antimicrobial efficacy may be due to the release of calcium ions and hydroxyl ions which results in an increase in pH.[17] Biodentine showed a higher release of free calcium ions when compared to ProRoot MTA due to the presence of both calcium silicate and calcium chloride.[18] Hence Biodentine has got higher alkalinizing capacity and improved antibacterial efficacy. But it was demonstrated that the elevated pH of white MTA was sustained for a longer period of time, as the material continues to cure over a period of several weeks (Hansen et al).[19] EndoSequence RRM putty and ProRoot MTA have demonstrated similar antimicrobial efficacy against clinical Isolates of E. faecalis. (Lovato et al).[20]

In the 3rd, 4th, 5th, and 6th weeks, the leakage values were significantly higher for ProRoot MTA when compared to both Biodentine and ESRRM putty. Both the materials demonstrated comparable sealing ability in this study. Sealing ability of MTA-Angelus, Biodentine, and EndoSequence RRMputty in furcation perforations was studied using protein leakage assessment and concluded that Biodentine had the least leakage followed by EndoSequence, and MTA had the highest leakage (Kakani and Veeramachaneni).[21] The methodologies used in these studies could have contributed to the variation in the results.

In this in vitro analysis, ProRoot MTA showed the highest leakage. The composition of Biodentine and MTA was analyzed by Camilleri et al. and it was found that tricalcium silicate was the main constituent of Biodentine and no dicalcium silicate or calcium oxide was detected.[22]

Density and porosity are critical factors which determine the amount of leakage and outcome of the treatment because a larger pore diameter results in increased leakage which corresponds to the ingress and transmission of microorganisms and hence compromised three-dimensional seal.[23] Porosity is an intrinsic characteristic of tricalcium silicate-based cements and occurs as a result of the spaces between the un-hydrated cement grains. These spaces are filled with water once the material hydrates. As the hydration reaction progresses, the hydration products fill these gaps and the porosity decreases. However, if the water-to-cement ratio is too high during mixing, excess water eventually dries off and leaves voids that are not filled by hydration products. Thus, porosity is found to increase with an increase in water-to-cement ratio and decreases as the cement ages.[24],[25] A significant increase in solubility and porosity of ProRoot MTA with the increase in water-to-powder ratio has been reported.[26] This may be a reason for the leakage values which was increasing up to the 3rd week in all groups, after which it became static, whereas it continued to increase in the control group up to the 6th week. When the porosity of Biodentine and ProRoot MTA was compared using micro-computed tomography characterization, no significant differences were found in porosity between the two materials. But due to low water content in the mixing stage, Biodentine exhibited lower porosity than MTA.[27] It was demonstrated that ambient conditions and material additives such as calcium carbonate in Biodentine affect the porosity and root dentine to material interface of root-end filling materials (Camilleri et al.). Furthermore, it was found that dry storage of Biodentine resulted in changes in the material microstructure and cracks at the root dentine to Biodentine interface, and to reduce this, the specimens were stored in moisture before inoculation. Evaluation of solubility of RRMs can also give some information about their sealing ability. High levels of Ca2+ ions release have been found in different bioceramic RRMs, and this could be a reason for the high solubility observed for these materials.[27]

Marginal adaptation has correlation with the sealing ability of dental material and, hence effect on clinical success rate.[28] In the interface between MTA, Biodentine, and dentine, tag-like microstructures were detected in the fractured samples in both confocal and scanning electron microscopy studies.[29] Confocal studies of Biodentine demonstrated a “mineral infiltration zone” (MIZ), which may be associated with an altered intertubular microstructure leading to a change in the optical properties of the interfacial dentin (Atmeh et al.). This may be due to the high alkalinity (pH 12) of hydrated Biodentine that induces a caustic denaturation and formation of a porous dentin structure which facilitates the permeation of high concentrations of Ca2+, OH, and (CO3)2− ions, leading to increased mineralization in this region. This alkaline caustic effect or “caustic etching” has virtually no effect on the highly mineralized peritubular dentin due to its lower collagen content. This “MIZ” along dentin-cement interface helps to impart a better seal. Biodentine has a more prominent biomineralization ability than MTA, with wider calcium and silicon-rich layer at the materialdentine interface (Han and Okiji).[29] The better marginal adaptation of Biodentine was attributed to the small size of Biodentine particles that enhance the adaptation at the cavity surface and dentine interface (Alazrag et al.).[30] In this study, the leakage value obtained for ProRoot MTA was higher than Biodentine in all the 6 weeks.

ESRRM putty displayed the least leakage value in all the 6 weeks and after the 3rd week, both Biodentine and ESRRM showed similar results and lesser leakage than ProRoot MTA. ESRRM putty is calcium phosphate silicate cement and consists of phosphate salts in addition to hydraulic calcium silicates.[31] It is mainly composed of calcium silicates, monobasic calcium phosphate, zirconium oxide, tantalum oxide, proprietary fillers, and thickening agents.[32] It has been developed as ready-to-use premixed bioceramic material which overcomes the difficulty in handling of the other powder/liquid bioceramic products. The moisture present in the dentinal tubules is adequate to allow the material to set.[33] This also eliminates the potential of heterogeneous consistency during mixing as the material is premixed with nonaqueous but water-miscible carriers. It will not set during storage and hardens only on exposure to an aqueous environment.[34] This least leakage value for ESRRM which was attained in the first reading itself may be due to the smaller particle size and the homogeneous consistency of the premixed material. This can also be attributed to the higher pH which will be attained earlier due to the lower setting time.

The particle size distribution also affects the sealing properties of these bioceramic powders. Smaller particles may penetrate tubules and hydrate faster than larger particles because of their higher surface-to-volume ratio.[35] ESRRM putty has nanosphere particles with a maximum diameter of 1 × 10-3μm that allow for the material to enter dentinal tubules, be moistened by dentine liquid, and create a mechanical bond upon setting which may be another reason for its increased sealing property.[36]

From the results obtained it can be concluded that use of retrograde materials reduced microleakage to a significant amount. Among the different biomaterials used for retrograde restoration, ESRRM putty showed the lowest leakage in the baseline reading. As it reaches the 6th week, both ESRRM putty and Biodentine have got comparable leakage values. In all the weeks, ProRoot MTA has got the highest microleakage. Hence, both Biodentine and ESRRM putty can be used interchangeably as retrograde restorative materials. However, ESRRM putty is available as premixed form and has improved handling characteristics when compared to the powder liquid form of Biodentine and ProRoot MTA.

Financial support and sponsorship


Conflicts of interest

There are no conflicts of interest.

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  [Table 1], [Table 2]


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