|Year : 2017 | Volume
| Issue : 1 | Page : 16-22
Effect of chelating agents on sealing ability of Biodentine and mineral trioxide aggregate
Shubha Chhaparwal1, Nidambur Vasudev Ballal1, Nympha Deena Menezes2, Shobha Ullas Kamath1
1 Department of Conservative Dentistry and Endodontics, Manipal College of Dental Sciences, Manipal University, Manipal, Karnataka, India
2 Department of Biochemistry, Kasturba Medical College, Manipal University, Manipal, Karnataka, India
|Date of Web Publication||10-Jan-2017|
Nidambur Vasudev Ballal
Department of Conservative Dentistry and Endodontics, Manipal College of Dental Sciences, Manipal University, Manipal - 576 104, Karnataka
Source of Support: None, Conflict of Interest: None
Aim: To evaluate the effect of 17% ethylenediaminetetraacetic acid (EDTA) and 7% maleic acid (MA) irrigation on microleakage of mineral trioxide aggregate (MTA) and Biodentine® (BD) when used as a root-end filling material.
Materials and Methods: Sixty human anterior teeth were decoronated and subjected to root canal instrumentation. 3 mm of apical root portion was resected, and root-end cavities were prepared using ultrasonic tips. Teeth were then randomly divided into two groups (n = 30). Group 1 - root end cavity to be filled with MTA; Group 2 - root end cavity to be filled with BD. Each group was further divided into three subgroups A, B, and C based on irrigation regimen. Group A - 17% EDTA; Group B - 7% MA; Group C - 0.9% saline. About 5 mL of all irrigating solutions were used for 1 min. After final irrigation, root-end cavities were filled with respective root end filling material. Specimens were then subjected to microleakage analysis at 24 h, 7 days and 14 days using glucose filtration technique. For smear layer evaluation, six central incisors were subjected to root canal instrumentation and irrigation with 7% MA and 17% EDTA. Then, these samples were analyzed under the standard error of the mean data was analyzed using one-way ANOVA, Bonferroni test, Mann–Whitney U-test and Kruskal–Wallis test.
Results: Saline group demonstrated significant higher leakage than that of 17% EDTA and 7% MA in both MTA and BD groups. However, there was no significant difference between 17% EDTA and 7% MA group when MTA was used as root-end filling material. In BD group, 17% EDTA showed more leakage than 7% MA. 7% MA was able to remove the smear layer better than 17% EDTA.
Conclusion: MTA had a better sealing ability as compared to that of BD when root-end cavities were irrigated with 7% MA.
Keywords: Biodentine, maleic acid, mineral trioxide aggregate, root end filling, smear layer
|How to cite this article:|
Chhaparwal S, Ballal NV, Menezes ND, Kamath SU. Effect of chelating agents on sealing ability of Biodentine and mineral trioxide aggregate. Saudi Endod J 2017;7:16-22
|How to cite this URL:|
Chhaparwal S, Ballal NV, Menezes ND, Kamath SU. Effect of chelating agents on sealing ability of Biodentine and mineral trioxide aggregate. Saudi Endod J [serial online] 2017 [cited 2020 Feb 19];7:16-22. Available from: http://www.saudiendodj.com/text.asp?2017/7/1/16/197983
| Introduction|| |
The goal of the endodontic therapy is to provide fluid tight seal of all avenues of communication between intraradicular space and periradicular tissues. The preferred treatment of failing endodontic cases is nonsurgical retreatment. This treatment usually results in successful outcomes. However, because of the complexity of root canal systems, inadequate instrumentation and presence of physical barriers (anatomical, post and core restoration, separated instruments, etc.), successful outcomes may be difficult to achieve with a nonsurgical approach. Surgical endodontic therapy then becomes the first alternative. The objective of periapical surgery is to eliminate diseased tissues and obtain an apical seal to prevent the ingress of residual irritants into the periradicular area. Instrumentation of the root canal dentin surface during periradicular surgery produces smear layer, which consists of organic and inorganic material, microorganisms, and their endotoxins. It has been reported that application of acids or chelating agents, can remove smear layer and improve the adhesion and penetration of root-end filling materials. This makes the root surface more biocompatible, optimizing periodontal healing without interfering with apical root end filling seal., The combination of ethylenediaminetetraacetic acid (EDTA) and sodium hypochlorite (NaOCl) is most commonly used for the smear layer removal. However, it has been reported that 17% EDTA is not efficient in removal of smear layer especially in the apical third of the root canal system. It has also been reported to be cytotoxic. 7% maleic acid (MA) is a mild organic acid which has shown to remove the smear layer significantly better than 17% EDTA in apical third of root canal system when used as a root canal irrigant.
Gartner and Dom proposed that an ideal root-end filling material should be easy to manipulate, radiopaque, dimensionally stable, nonabsorbable, insensitive to moisture, adhesive to dentine, nontoxic, and biocompatible. To date, several different materials have been proposed to seal the root-end cavity, including amalgam, Gutta-percha, zinc oxide-eugenol cement, composite resin with and without dentin-bonding adhesives, polycarboxylate cement, glass ionomer cement, compomers, resin-modified glass ionomers, resin cement and mineral trioxide aggregate (MTA).,, MTA has been investigated as one of the best root end filling material in endodontics. Severalin vitro andin vivo studies have shown that MTA prevents microleakage, is biocompatible, promotes regeneration of the original tissues when placed in contact with periradicular tissues  and also provides a better seal than amalgam, IRM, and EBA cement when used as a root-end filling material. Biodentine ® (BD) which is a tricalcium silicate material is used for crown and root dentin repair, repair of perforations or resorptions, apexification, and root-end fillings. Laurent et al. evaluated the genotoxicity, cytotoxicity, and effects on target cells specific functions of BD. They concluded that BD is a biocompatible material. The effect of smear layer removal agents on BD, when used as root-end filling material, is currently lacking. Hence, the aim of this study was to evaluate the effect of 17% EDTA and 7% MA irrigation on the microleakage of MTA and BD when used as a root-end filling material.
| Materials and Methods|| |
Sixty human maxillary central incisors with relative similar dimensions as examined by intraoral periapical radiographs (buccolingual and mesiodistal directions) extracted for periodontal reasons were selected based on the inclusion (caries free, single root canal, and completely formed apex) and exclusion criteria's (open apex or incompletely formed apex, root resorptions, cracks, and endodontic restorations). Ethical clearance was obtained from institutional review board (IEC 573/2012). Teeth were thoroughly cleaned using ultrasonics and stored in 0.2% of sodium azide (Sigma Chemical Co, St. Louis, MO) at 4°C until the experiment. The crowns of all teeth were decoronated using a diamond disc (Horico, Germany) at the cementoenamel junction so that the coronal surface was perpendicular to the long axis of the root and the remaining root length was adjusted to 15 mm. The working length was established by inserting a no. 10 K file (Mani Inc., Tochigi-Ken, Japan) into each root canal until it was just visible at apical foramen (observed by magnifying loupes) and by subtracting 1 mm from this point. After working length determination, the canal was enlarged to size F3 using rotary protaper files (Dentsply Maillefer, Ballaigues, Switzerland). About 5 mL of 2.5% NaOCl (KMC Pharmacy, India) was used as irrigant for 1 min between each instrument change. Final irrigation was done with 5 mL of distilled water for 1 min, and the canals were then dried using paper points (Dentsply Maillefer, Ballaigues, Switzerland). Irrigation was performed using a disposable syringe and 28 gauge needles (Navi-Tip, Ultradent, South Jordan, UT, USA). 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 (Horico, Berlin, Germany) under water coolant at a high-speed. 3-mm deep root-end cavities were prepared using the ultrasonic tip (JT-4B, size - 0.3 mm, B and L Biotech, USA) at a low power setting using water coolant. Cutting with the ultrasonic tips were performed using back and forth motion with the tip enveloped in water spray. Sixty samples were then randomly assigned to two groups (n = 30): Group 1 - root-end cavities to be filled with MTA (ProRoot MTA, Dentsply, Tulsa Dental Specialties, USA). This group was further divided into 3 subgroups A, B and C (n = 10).
Group A: Root-end cavity irrigated with 17% EDTA (Merck, Dermstadt, Germany).
Group B: Root-end cavity irrigated with 7% MA (KMC Pharmacy, India).
Group C: Root-end cavity irrigated with 0.9% saline.
In Group 2, root-end cavities to be filled with BD (Septodont, Saint Maur des Fosses, France). This group was further divided into three subgroups (n = 10) similar to Group 1.
About 5 mL of all the irrigating solutions were used for 1 min using a disposable syringe and 28 gauge needle. Final irrigation in all the groups was performed with 5 mL of distilled water for 1 min. After final irrigation, root-end cavities were dried with paper points. MTA and BD were mixed according to the manufacturer's instructions, and the root-end cavities were filled with the respective material. A customized endodontic hand plugger was placed in the root canal 3 mm short of root apex, which formed a coronal matrix before filling with root-end material. Excess material from root-end cavity was removed with wet cotton pellets, and the roots were kept in 100% humidity at 37°C for 48 h.
Evaluation of microleakage
Microleakage through the restored root-end cavities was evaluated using glucose penetration model. The coronal part of each root was glued to one end of a modified plastic dropper (in which both ends were cut to accommodate specimen and the glass tube) using cyanoacrylate. Care was taken so that, glue does not cover the coronal orifice of root. Leakage at this connection was eliminated by the use of sticky wax. Through other end, a glass tube of 15 cm in length was connected. A seal was obtained using cyanoacrylate glue and sticky wax. The assembly was then placed in a sterile 5 ml glass bottle covered with paraffin sheet and sealed with sticky wax [Figure 1]. The tracer used was a 1 mol/L glucose solution (pH = 7.0), whose density was 1.09 × 103 g/L and viscosity 1.18 × 10–3 Pas at 37°C. Glucose has a low molecular weight of 180 Da and is hydrophilic and chemically stable. About 5 ml of glucose solution, containing 0.2% sodium azide (NaN3), was injected into the modified dropper from glass tube until the top of the solution was 14 cm higher than the top of specimen, which created a hydrostatic pressure of 1.5 kPa (15 cm H2O). The glass bottle contained 1 ml of 0.2% aqueous solution of NaN3, in which glucose that leaked through the restored canal was collected.
Measurement of microleakage
A 100-µL aliquot of the solution was drawn from the glass beaker using a micropipette after 24 h, 7 days and 14 days. After drawing the sample, 100 µL of fresh 0.2% NaN3 was added to glass bottle reservoir to maintain a constant volume of 1 mL. If there was any decrease in the volume in the control bottle due to evaporation, a corresponding amount of sterile deionized water was added to the glass beaker. The sample was then analyzed with a glucose kit in a colorimeter at 500 nm wavelength. Two blinded independent evaluators conducted the colorimetric determination of glucose concentration. The results of leakage in all groups were calculated as mmol/L from the respective optical density observed in colorimeter.
Scanning electron microscopic analysis
Six human single-rooted maxillary central incisors were selected and prepared as mentioned previously. Once the samples were prepared, they were divided into two groups (n = 3). Samples in Group 1 were irrigated with 5 mL of 17% EDTA solution for 1 min. Similarly, samples in Group 2 were irrigated with 5 mL of 7% MA solution for 1 min. Finally, all the samples were irrigated with 5 mL of distilled water for 1 min. The apical third of all the samples were then horizontally cut. Then, longitudinal grooves were prepared on the buccal and lingual surfaces of each of the apical segment using a diamond disc at a slow speed without penetrating the root canal. The apical segment were then split into two halves using a straight chisel and stored in deionized water at 37°C until standard error of the mean analysis. The specimens were dehydrated using ascending grades of ethyl alcohol and were then mounted on metallic stubs, gold sputtered using an ion sputter, and examined under scanning electron microscope (LEO 440i, Carl Zeiss, Tokyo, Japan) for presence or absence of smear layer. Photomicrographs were taken to observe the surface morphology at ×1500 magnification and 10 KV of the canal walls. Two independent evaluators who were unaware of the experimental groups evaluated the images obtained. The images were scored according to the criteria given by Torabinejad et al.
1 = no smear layer (no smear layer on the surface of the root canal; all tubules were clean and open); 2 = moderate smear layer (no smear layer on the surface of the root canal, but tubules contained debris); and 3 = heavy smear layer (smear layer covered the root canal surface and the tubules).
Five representative samples from each group: (a) 17% EDTA and MTA; (b) 7% MA and MTA; (c) 17% EDTA and BD; and (d) 7% MA and BD were selected and sectioned horizontally using diamond disc under water spray. The samples were then observed under stereomicroscope (Leica microsystems, Wetzlar, Germany) at ×10 magnification for the marginal adaptation of root-end filling material to the root canal walls.
Statistical analysis for inter-group comparison was done using one-way ANOVA and Bonferroni test, and that of intra-group comparison was done using Mann–Whitney U-test and Kruskal–Wallis test. In the smear layer evaluation, the inter-examiner's reliability was verified using the Kappa test. The level of statistical significance was set at P < 0.05.
| Results|| |
In MTA group, saline (control) showed maximum leakage at all the time intervals (P < 0.001) as compared to that of 17% EDTA and 7% MA. There was no significant difference between 17% EDTA and 7% MA [Figure 2]. Similarly, in BD group, saline (control group) showed the maximum leakage at all the time intervals (P < 0.001) as compared to that of 17% EDTA and 7% MA groups. However, there was a significant difference between 17% EDTA and 7% MA group at 48 h (P = 0.008), 7 days (P < 0.001) and 14 days (P = 0.002), with 17% EDTA demonstrating higher leakage [Figure 3].
|Figure 2: Bar graph demonstrating comparison of microleakage using different irrigants in mineral trioxide aggregate group|
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|Figure 3: Bar graph demonstrating comparison of microleakage using different irrigants in Biodentine group|
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On intercomparison at different time, at 48 h in 17% EDTA group, BD showed significant higher leakage than that of MTA (P < 0.001). However, in 7% MA (P = 0.172) and saline group (P = 0.65) there were no significant difference between BD and MTA [Figure 4]. At 7 and 14 days, 17% EDTA (P < 0.001), 7% MA (P < 0.001), and saline (P = 0.034) groups demonstrated significant more leakage in BD as compared to MTA group [Figure 5] and [Figure 6].
|Figure 4: Bar graph demonstrating comparison of microleakage between mineral trioxide aggregate and Biodentine at 48 h|
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|Figure 5: Bar diagram showing comparison of microleakage between mineral trioxide aggregate and Biodentine at 7 days|
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|Figure 6: Bar diagram showing comparison of microleakage between mineral trioxide aggregate and Biodentine at 14 days|
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Kappa test results for smear layer evaluation showed that there was no statistical significant difference between the two examiners for both 7% MA and 17% EDTA groups. About 7% MA was able to remove the smear layer better than 17% EDTA. There was no smear layer on root canal walls, and the tubules were open in 7% MA group. In 17% EDTA-treated specimens, tubules were partially obliterated with smear layer [Figure 7].
|Figure 7: Standard error of the mean photomicrographs of root-.end cavities irrigated with 17% ethylenediaminetetraacetic acid and 7% maleic acid|
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The stereomicroscopic evaluation of the adaptation of BD and MTA to root canal walls treated with 17% EDTA and 7% MA demonstrated that there was a poor marginal adaptation of BD to root canal walls treated with 17% EDTA as compared to 7% MA. The adaptation of MTA to the root canal walls treated with 17% EDTA and 7% MA was found to be good [Figure 8].
|Figure 8: Stereomicroscope photograph demonstrating adaptation of root end filling material to root-.end cavity. (a) 17% ethylenediaminetetraacetic acid and mineral trioxide aggregate; (b) 7% maleic acid and mineral trioxide aggregate; (c) 17% ethylenediaminetetraacetic acid and Biodentine; (d) 7% maleic acid and Biodentine|
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| Discussion|| |
The aim of placing a root-end filling material following root-end resection is to establish an effective barrier between the root canal and the periapical tissues. It has been demonstrated that insufficient apical seal is a one of the major causes of endodontic surgical failure.
In this study, all irrigation groups both in MTA and BD showed microleakage over time, but saline demonstrated significant higher leakage than that of 17% EDTA and 7% MA. However, there was no significant difference in the microleakage between 17% EDTA and 7% MA group when MTA was used as root-end filling material. This could be attributed to the good adaptation of MTA to root canal walls as reported by various studies., In BD group, there was a significant difference in microleakage between 17% EDTA and 7% MA, with 17% EDTA group showing more leakage. This could be attributed to the improper marginal adaptation of BD to root canal walls treated with 17% EDTA. It has been reported that the adhesion of BD to the root canal walls is most likely through the tag like structures formed within the dentinal tubules leading to micromechanical retention. Han and Okiji demonstrated that calcium and silicon ion uptake into dentin leading to the formation of tag like structures was higher in BD than MTA. It has been reported that dentin in apical third of root canal system is sclerosed. Hence, in the present study, in BD group, there may not have been complete tag formation inside the apical sclerosed root canal dentin which would have led to its improper marginal adaptation. This was evaluated in this study by using stereomicroscope which demonstrated, poor marginal adaptation of BD to root canal walls in 17% EDTA-treated group as compared to 7% MA. Similar results of poor marginal adaptation of BD have been reported by Soundappan et al. stating that, MTA and IRM were significantly superior to BD in terms of marginal adaptation when used as root-end filling material.
Within the BD group, the 17% EDTA irrigation group showed more microleakage as compared to that of the 7% MA group. This could be attributed to the effective removal of smear layer in the apical third of the root canal dentin by 7% MA when compared to that of 17% EDTA, which was evaluated in the present study using scanning electron microscope. The previous study reported by Ballal et al. has demonstrated that smear layer removal ability of 7% MA is better than 17% EDTA, especially in the apical third of root canal system. It has also been reported that the postobturation apical seal following irrigation with 7% MA or 17% EDTA evaluated using dye leakage under vacuum method was better with 7% MA than 17% EDTA. The authors attributed the minimal leakage with 7% MA irrigation due to its efficient smear layer removal in the apical third. In the present study, MTA was used as one of root end filling material because it has shown to have higher biocompatibility and sealing ability. In addition, MTA demonstrates superior marginal adaptation over the other materials. The other material which was tested as a root-end filling material in the present study is BD. It is a relatively new material introduced as a dentin substitute. BD has demonstrated biocompatibility and the ability to induce odontoblast differentiation and mineralization in cultured pulp cells. The main benefits of BD over other calcium silicate based materials are the reduced setting time, better handling, and mechanical properties.
Traditionally, root end cavity is prepared by means of a round bur on a contra-angled slow-speed handpiece. However, in the clinical practice, this technique of apical preparation may exhibit numerous drawbacks such as nonparallel cavity walls, difficulty in reaching the root tip, and lingual perforation of the root. Hence, in the present study ultrasonic (US) retro tip was preferred to prepare the root end cavity as US retro tips have many advantages over traditional round bur. US retro tips produced cleaner, well-centered, and more conservative root-end cavities. In this study, 7% MA was used because it has been reported that, MA, when used at a higher concentration than 7%, caused damage to the intertubular dentin. The irrigation time in the present study was set to 1 min because irrigation with 17% EDTA for more than 1 min has shown to cause excessive peritubular and intertubular dentinal erosion.
Several methods have been employed to assess sealing ability of root end filling materials such as dye leakage, fluid filtration, bacterial penetration, radiolabelled isotopes, and electromechanical tests. The dye penetration test is one of the most popular technique used in endodontic leakage studies due to its simplicity and cost effectiveness. However, it has got disadvantages like, it can lead to observers bias, cannot be reproducible and comparable. The use of bacterial species for the leakage analysis may be more relevant than the use of dyes. However the results might vary with bacterial species used and maintaining aseptic conditions may pose a problem. Radioisotope labeling and electrochemical methods pose a radiation hazard and require sophisticated materials and hence these techniques are less frequently used in endodontic research. The fluid filtration is the most commonly used technique in endodontic leakage studies. It has advantages of being sensitive, nondestructive and can be used repeatedly for observing the same specimen overtimes. However, it possess disadvantages like, lack of standardization of the measurement time, applied pressure, diameter of the tube containing the bubble, and the length of the bubble. All these factors may influence the results of the leakage studies.
In the present study, glucose filtration technique which was introduced by Xu et al. for quantitative testing of endodontic leakage was used. This quantitative technique is sensitive, nondestructive and clinically relevant. The rationale for using glucose as a tracer in this study was, due to its small molecular size and it is a known nutrient for the bacteria. Hence, if it could enter the root canal system from oral cavity, the bacteria that might have survived after root canal preparation and obturation could multiply and potentially lead to periapical inflammation. Hence, the use of glucose as a tracer in endodontic microleakage studies would be more clinically relevant than using other tracers.
Shemesh et al. compared glucose penetration and fluid filtration method for measuring leakage and found that glucose penetration technique was more sensitive in detecting leakage along root fillings. Different time periods for evaluation of microleakage of various root-end-filling materials have been used. In the present study, the leakage was evaluated at 48 h followed by 7 days and 14 days, which was in accordance with Chogle et al. They reported that microbial leakage was reduced significantly as setting time increased from 4 h to 2 days or 1 week. There was no significant reduction in leakage when the setting time was increased from 2 days to 1 week. Hence, in this study, the maximum period for the assessment of leakage was taken as 14 days.
| Conclusion|| |
Within the limitations of this study, it can be concluded that MTA had a better sealing ability as compared to that of BD when root-end cavities were irrigated with 7% MA.
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], [Figure 6], [Figure 7], [Figure 8]