|Year : 2016 | Volume
| Issue : 1 | Page : 19-24
A comparative evaluation of the microleakage of blood-contaminated mineral trioxide aggregate and Biodentine as root-end filling materials: An in vitro study
Lisha Alphonsa Mathew1, Sandya Kini1, Shashi Rashmi Acharya1, Shobha Kamath2, Nympha Deena Menezes2, Sheetal Rao1
1 Department of Conservative Dentistry and Endodontics, Manipal College of Dental Sciences, Manipal, Karnataka, India
2 Department of Biochemistry, Kasturba Medical College Manipal, Manipal University, Manipal, Karnataka, India
|Date of Web Publication||10-Aug-2016|
Lisha Alphonsa Mathew
Department of Conservative Dentistry and Endodontics, Manipal College of Dental Sciences, Manipal, Karnataka
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Context: Success of a periradicular surgery depends on the attainment of a fluid tight apical seal with a well-adapted root-end restoration. Since achieving a dry field is not always possible, the study design was aimed at evaluating the sealing ability of test materials in blood-contaminated field which is usually the clinical scenario. Aims: To evaluate and compare the microleakage of blood-contaminated mineral trioxide aggregate (MTA) and Biodentine as root-end filling materials. Settings and Design: After decoronating, forty recently extracted single-rooted teeth were selected. The canal was enlarged to size #50 using hand files and the rest of the canal was prepared to #80 file at 1 mm increments. The canal was copiously irrigated with 2.5% sodium hypochlorite, ethylenediaminetetraacetic acid, and normal saline between instrument changes. After root-end resection, 3 mm of root-end preparation was done. Subjects and Methods: The root-end filling materials were placed in the following manner. Group 1: Root-end cavity was filled with MTA, Group 2: Root-end cavity was filled with Biodentine, Group 3: Root-end cavity was filled with MTA with blood contamination, and Group 4: Root-end cavity was filled with Biodentine with blood contamination. All samples were incubated for 24 h at 37°C and 100% humidity, and the microleakage was evaluated using a glucose filtration model. Statistical Analysis Used: Statistical analysis was done using an SPSS version 17 software. Data were analyzed with repeated-measures ANOVA and post hoc Bonferroni test. Results: Mean values of microleakage of the different groups in day 1, 4, and 7 were recorded. It was seen that the leakage was increasing with days irrespective of the material and no significant difference in the rate of increase was observed between the different materials. It was also seen that the blood-contaminated samples showed less leakage than the corresponding dry samples except the blood-contaminated Biodentine which showed more leakage on the day 1. However, overall, marginal means indicate no significant difference in the microleakage between materials. Conclusions: The sealing ability of Biodentine showed comparable results with that of MTA in dry and blood-contaminated environments and hence Biodentine can be used as an alternative to MTA for root-end filling procedures in a blood-contaminated environment.
Clinical Relevance to Interdisciplinary Dentistry
- With the movement toward evidence-based dental healthcare, interdisciplinary approach has been gaining ground quickly. Of which, endodontics forms an integral part of interdisciplinary dentistry
- Integrating biochemical analysis with endodontics forms basis to research and is the foundation for a successful dental outcome
- Biodentine shows to be bioactive, which helps to restore the tooth and periradicular structures back to normal form and function.
Keywords: Biodentine, blood contamination, mineral trioxide aggregate
|How to cite this article:|
Mathew LA, Kini S, Acharya SR, Kamath S, Menezes ND, Rao S. A comparative evaluation of the microleakage of blood-contaminated mineral trioxide aggregate and Biodentine as root-end filling materials: An in vitro study. J Interdiscip Dentistry 2016;6:19-24
|How to cite this URL:|
Mathew LA, Kini S, Acharya SR, Kamath S, Menezes ND, Rao S. A comparative evaluation of the microleakage of blood-contaminated mineral trioxide aggregate and Biodentine as root-end filling materials: An in vitro study. J Interdiscip Dentistry [serial online] 2016 [cited 2019 Aug 17];6:19-24. Available from: http://www.jidonline.com/text.asp?2016/6/1/19/188159
| Introduction|| |
The goal of an ideal endodontic therapy is to hermetically seal all pathways of communication between the pulp and periodontium. Apical seal obtained by obturation and coronal seal obtained by postendodontic restoration prevent the percolation of any oral fluids thus preventing the recontamination of the root canals.  A nonhealing or a persistent periapical lesion shows a failure of orthograde root canal therapy or nonsurgical endodontics. These cases are best treated by apisectomy followed by root-end restoration.  The main objective of a root-end restoration is to provide a seal that prevents the leakage of microorganisms from the pulp to periapical tissues and vice versa. Apical seal is the crucial factor for the success of surgical endodontics.  An ideal root-end filling material should be impervious to moisture, antibacterial, nontoxic, nonresorbable, easy to manipulate, radiopaque, easily adaptable, biocompatible, provide good seal, and promote regeneration of the periodontal apparatus.  Among the various root-end filling materials, mineral trioxide aggregate (MTA) had shown the most promising results because of its biocompatibility, mineralized tissue formation, less apical leakage, better marginal adaptation, and the possibility of using in a humid field.  Hence, it is considered as the gold standard material for comparing other materials.  However, it also possesses some disadvantages like difficulty to handle and insert into a retrograde cavity and wash out due to long setting time. , Recently, a calcium silicate-based material was introduced into the market by Septodont (France) called Biodentine. The product file of Biodentine states that the powder component of the material consists of tricalcium silicate, dicalcium silicate, calcium carbonate, oxide filler, iron oxide shade, and zirconium oxide. Tricalcium silicate and dicalcium silicate are indicated as main and second core materials, respectively, whereas zirconium oxide serves as a radio pacifier. The liquid contains calcium chloride as an accelerator and a hydrosoluble polymer that serves as a water reducing agent. The fast setting time, one unique characteristic of the product, is achieved by increasing particle size, adding calcium chloride to the liquid component, and decreasing the liquid content. The setting period of the material is as short as 9-12 min. This shorter setting time is an improvement compared to other calcium silicate materials.  It also has the added advantage of better handling and mechanical properties and biocompatibility and ability to induce odontoblast differentiation mineralization.  The manufacturer claims that this material can be used for pulp capping, pulpotomy, apexification, root perforation, internal and external resorption, and also as a root-end filling material in periapical surgery. It has also been proved that Biodentine shows a better marginal adaptation when compared with the commonly used root-end filling materials.  It is inevitable that moisture including blood can contaminate the root end when filling material is placed and may affect its sealing ability.  Farhad et al. and Gondim et al. proved that the seal of MTA was not affected by blood contamination. , However, the sealing properties of Biodentine in blood-contaminated environments have not been evaluated. Hence, the purpose of this study was to evaluate and compare the microleakage of blood-contaminated MTA and Biodentine as root-end filling materials using glucose filtration method.
| Subjects and methods|| |
Institutional ethical clearance was obtained. Forty single-rooted freshly extracted human anterior teeth were used in the study. The selection criteria were the presence of a single root canal, no evidence of cracks, fractures, root caries or restoration, and mature apices and straight canals without calcification. After extraction, teeth were kept in 10% buffered formalin and stored in saline prior to instrumentation. Teeth were decoronated at the cementoenamel junction with the diamond disc at high speed with water spray coolant. Working length (WL) was determined by placing a #10K file into each root canal until it was just visible at the apical foramen and then subtracting 1 mm from this point. After WL determination, the canal was enlarged to size #50 using hand files and the rest of the canal was prepared to #80 file at 1 mm increments. The canal was copiously irrigated with 2.5% sodium hypochlorite, ethylenediaminetetraacetic acid, and normal saline between instrument changes. Final irrigation was done with 5 ml of distilled water and the canal was dried with paper points. Apical root resections were done by removing 3 mm of the apex at a 90° angle to the long axis of the root with a diamond bur under water coolant at a high speed. The root-end cavities were prepared ultrasonically with Piezon Suprasson P5 unit (SBL, Satelect, France). A 3 mm deep root-end cavity was made in the resected root-end at the highest frequency setting. Cutting was performed using back and forth motion with the tip enveloped in water spray [Figure 1].
Later, the forty samples were randomly assigned to four groups (n = 10 each):
A customized plugger was placed in the root canal 3 mm short of resected root apex before filling with root-end material [Figure 2].  Later, root-end cavity was dried with paper points and filled with root-end filling material.
- Group 1: Root-end cavity was filled with MTA
- Group 2: Root-end cavity was filled with Biodentine
- Group 3: Root-end cavity was filled with MTA with blood contamination
- Group 4: Root-end cavity was filled with Biodentine with blood contamination.
Groups 1 and 2 were filled dry. Prior to root-end filling of Group 3 and 4, 1 ml of human blood was obtained by phlebotomy procedure using a 23-gauge needle from the observer by trained authorized personnel and root ends were filled with whole, fresh human blood which was removed by aspirating with the syringe to leave a coating of blood on the inner wall of cavity [Figure 3].  Materials were mixed as per manufacturer's instructions.
Excess material was removed with wet cotton pellets, and the specimen was kept in a 100% humid environment at 37°C for 24 h.  Twenty-four hours after root-end filling, the roots' surface was coated with three layers of nail varnish. The varnish was applied onto the entire root surface, except for the area corresponding to the resected apical surface, and root canal orifices and was left for drying.
Preparation of specimen for glucose filtration
The resected coronal part of each root was glued to the end of a modified plastic dropper in which both ends were cut to accommodate the specimen and the glass tube using cyanoacrylate glue. Care was taken so that the glue does not cover the coronal orifice of the root. Leakage at this connection was eliminated by the generous use of sticky wax.
Through the other end, a glass tube of 15 cm in length was connected. Seal was obtained using cyanoacrylate glue and sticky wax. The assembly was placed in a sterile 5 ml glass beaker covered with paraffin sheet and sealed with sticky wax [Figure 4].
The tracer used in the study was a 1 mol/L glucose solution (pH = 7.0). About 5 ml of the glucose solution, containing 0.2% NaN 3 , was injected into the modified dropper through the glass tube until the top of the solution was 14 cm higher than the top of root-end filling in the canal which created a hydrostatic pressure of 1.5 kPa (15 cm H 2 O).
The glass beaker contained 1 ml 0.2% solution of NaN 3 , in which glucose that leaked through the canal was collected.
Measurement of microleakage
A 100 μL of solution was drawn from the glass beaker using a micropipette at 24 h, 4 days, and 7 days. After drawing the sample, 100 μL of fresh 0.2% NaN 3 was added to the glass beaker reservoir to maintain a constant volume of 1 ml. The sample was then analyzed with a glucose kit in a ultraviolet-visible recording spectrophotometer at 500 nm wavelength.  Two blinded independent evaluators conducted the colorimetric determination of glucose concentration. The results in all groups were calculated as mmol/L from the respective optical density observed in a colorimeter, and the results were statistically analyzed.
| Results|| |
Data were analyzed with SPSS software version 15 (IBM SPSS Statistics). Repeated-measures ANOVA was conducted. [Table 1] shows the mean values of microleakage of the different groups in days 1, 4, and 7. It was seen that the leakage was increasing with increase in days irrespective of the material and no significant difference in the rate of increase was observed between the different materials (P = 0.38). It was also seen that the blood-contaminated samples showed less leakage than the corresponding dry samples except the blood-contaminated Biodentine which showed more leakage on day 1 [Graph 1].
|Table 1: The mean values of microleakage of different groups in day 1, 4 and 7 |
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[Table 2] of marginal mean indicate no significant difference in the microleakage between materials (P = 0.42) [Graph 2].
|Table 2: Marginal Means of MTA and Biodetine in dry and blood contaminates environment |
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| Discussion|| |
The success of a periradicular surgery depends on the attainment of a fluid tight apical seal with a well-adapted root-end restoration. Since achieving a dry field is not always possible, the study design was aimed at evaluating the sealing ability of test material in blood-contaminated field which is usually the clinical scenario. 
The ideal apical preparation should comply with a series of requirements, walls parallel to the longitudinal axis of the tooth, 3 mm depth, and central location with respect to the root. 
In the current study, root-end resections were carried out 3 mm from the apex and perpendicular to the long axis of tooth. Mjör et al. showed that in 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. 
Ultrasonic tips allow us to follow the longitudinal axis of the tooth, while conserving the morphology of the root canal. The apical cavities conform much easier, safer, and with greater precision, the cavities are smaller and more centrally located, reducing the risk of root perforation, and finally better cleaning of the cavity walls, reducing the volume of dentinal residues. ,, Diamond-coated tips help in preventing crack propagation. 
Biodentine was chosen as the test group in the current study because of its better handling properties and shorter setting time than MTA.  Mechanism of action of Biodentine is by the formation of hydrated calcium silicate which precipitates on the cement particles and forms a matrix. 
Various methods have been developed to assess sealing ability of root canal filling materials, usually based on the same principle, that is, to evaluate the penetration of a tracer along the obturated canal of an extracted tooth. Several tracers, such as dye, radioisotope, and bacteria and their products, had been used for evaluation of microleakage. In the current study, glucose filtration was chosen because quantitative analysis of the microleakage was possible by determination of the concentration of glucose that leaked through the canal into the apical reservoir.  Glucose was chosen because of its small molecular size and it is a nutrient for bacteria. If glucose could enter the canal from the oral cavity, bacteria that might survive root canal preparation and obturation could multiply and potentially lead to periapical inflammation. Glucose, therefore, was thought to be more clinically relevant than other tracers used in microleakage tests. To determine the concentration of glucose, the enzymatic glucose oxidase method was chosen because it provided the ultimate degree of specificity and high sensitivity when compared with other methods, such as copper or ferricyanide methods. With this method, glucose is oxidized by the enzyme glucose oxidase in the presence of oxygen to gluconic acid with formation of hydrogen peroxide. Then, in the presence of a peroxidase enzyme, a chromogenic oxygen acceptor (4-aminoantipyrine and phenol) is oxidized by the hydrogen peroxide, resulting in the formation of a red product (oxidized chromogen) [Figure 5]. The quantity of this oxidized chromogen is proportional to the glucose present initially in the first reaction, which quantity is determined by spectrophotometry. With this model, it was possible to quantify the endodontic microleakage continuously over time. The amount of microleakage was the cumulative value of leaked glucose. In addition, the coronal low pressure used in the study could help rule out entrapped air or fluid and seemed to be sufficient for a device with high sensitivity. 
Various microleakage studies have demonstrated the superiority of MTA over the commonly used root-end filling materials and so MTA is used as the gold standard for comparing the sealing ability of Biodentine. ,,,
Torabinejad et al. assessed root-end dye microleakage in dry and blood-contaminated environments and reported that MTA sealing ability was significantly better than amalgam, intermediate restorative material, and super ethoxy benzoic acid (EBA). Their study also showed that MTA mean dye leakage in blood-contaminated environment was less than in a dry environment. 
MTA hydration in the presence of solutions containing phosphate ions, such as tissue fluid or blood, results in the precipitation of hydroxyapatite crystals  and formation of tag-like structures at the junction of MTA/dentine interface  and the expansion on exposure to blood proteins  and block the porosities, retarding hydration, and increasing expansion. 
The sealing ability of Biodentine and MTA shows comparable although the values are higher for Biodentine which is in accordance with other studies  which can be attributed to its higher particle size.
Sealing ability of Biodentine is not affected by blood contamination, and the values of microleakage were lower in blood-contaminated samples than the dry field. The reason for this can be attributed to the same as that for MTA because Biodentine is calcium silicate cement like MTA.
| Conclusion|| |
Within the ambit of this study it can be concluded that, the sealing ability of Biodentine is comparable with that of MTA in dry and blood-contaminated environments and hence Biodentine can be used as an alternative to MTA for root-end filling procedures in a blood-contaminated environment.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]
[Table 1], [Table 2]