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 Table of Contents  
Year : 2022  |  Volume : 22  |  Issue : 3  |  Page : 233-239

Bacterial microleakage in endodontically treated teeth following two methods of postspace preparation at two-time intervals: An in vitro study

1 Department of Prosthodontics, School of Dentistry, Tehran University of Medical Sciences, Tehran, Iran
2 Dentist, Private Practice, Tehran University of Medical Sciences, Tehran, Iran
3 Board Certified Endodontist, Private Practice, Tehran University of Medical Sciences, Tehran, Iran
4 Dental Research Center, Dentistry Research Institute, Tehran University of Medical Sciences, Tehran, Iran

Date of Submission10-Sep-2021
Date of Decision06-Mar-2022
Date of Acceptance12-Mar-2022
Date of Web Publication18-Jul-2022

Correspondence Address:
Azam S Mostafavi
Department of Prosthodontics, School of Dentistry, Tehran University of Medical Sciences, North Kargar Avenue, Tehran
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/jips.jips_441_21

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Aims: The goal of this study was to analyze the bacterial microleakage following two methods (heat vs. rotary) of postspace preparation after two-time intervals (immediate vs. 1 week later).
Setting and Design: In vitro-comparative study.
Materials and Methods: Eighty-two single-rooted teeth were decoronated at the cementoenamel junction. Root canals were prepared using rotary files. After root canal obturation, specimens were randomly allocated to 4 experimental groups based on the method of postspace preparation (heat or peeso reamer) and time interval (immediate or 1 week later) (n = 18). Group 1: Peeso reamer-immediate, Group 2: Heat-immediate, Group 3: Peeso reamer-1 week later, Group 4: Heat-1 week later. 10 specimens were considered as positive and negative controls (n = 5 each). Custom-made dual-chamber devices were used to appraise the bacterial microleakage for 60 days.
Statistical Analysis Used: Data were analyzed with Chi-Square and Log-Rank tests and Cox regression.
Results: All through the experimental period, there was no significant difference (P = 0.41) between the studied groups. Groups 2 and 4 had the highest microleakage and the lowest survival rate (55.56% ± 11.71%). Group 1 showed the lowest microleakage and the highest survival rate (77.8% ± 9.80%).
Conclusions: The applied techniques for postspace preparation and the time intervals (neither independently nor simultaneously) showed no significant difference in the field of bacterial leakage.

Keywords: Dental instruments, dental leakage, heat, post and core technique, time

How to cite this article:
Mostafavi AS, Rasoulzadehsheikh M, Meraji N, Pourhajibagher M. Bacterial microleakage in endodontically treated teeth following two methods of postspace preparation at two-time intervals: An in vitro study. J Indian Prosthodont Soc 2022;22:233-9

How to cite this URL:
Mostafavi AS, Rasoulzadehsheikh M, Meraji N, Pourhajibagher M. Bacterial microleakage in endodontically treated teeth following two methods of postspace preparation at two-time intervals: An in vitro study. J Indian Prosthodont Soc [serial online] 2022 [cited 2022 Dec 7];22:233-9. Available from: https://www.j-ips.org/text.asp?2022/22/3/233/351283

  Introduction Top

The three-dimensional (3D) seal of the root canals is one of the most important achievements of endodontic treatment, and by disrupting it, the possibility of treatment failure increases. Teeth with this problem often require difficult and complex treatments. According to studies in endodontic treatment, canal contamination is possible in two stages; lack of proper cleansing of the canal and subsequent remaining infection or coronal leakage after treatment.[1],[2] It is noteworthy that coronal leakage after treatment is the most common cause of endodontic treatment failure.[3] Therefore, proper restorative reconstruction will increase the success rate of dental treatment remarkably. After endodontic treatment, the teeth need to return to their function. In most cases, a significant amount of coronal tooth structure is missing and needs to be reconstructed with a postcore and an extra-coronal restoration. A dowel is considered to provide the crown retention that commonly would have been gained from coronal tooth structure. Since endodontic retreatment after postcementation would be so complicated, maintenance of the seal of the filling material which can be jeopardized during postspace preparation is of great importance.[4],[5] Therefore, besides successful endodontic treatment, the 3D seal of the root canal system should not be compromised during postspace preparation. Studies have displayed that the accuracy of the remaining filling material depends on several elements such as; the amount of residual Gutta-percha,[6] the type of obturation material,[7] the method of root canal filling,[5] the technique and time of postspace preparation,[8] placement of temporary restoration[9] and the sealer used.[10] Throughout the preparation of the postspace, the remaining gutta-percha can be displaced and ultimately jeopardize the apical seal.[11] As a result, choosing a more appropriate and safer method for preparing the postspace can reduce the likelihood of leakage which consequently may fail. It is demonstrated that the leakage of endodontically treated teeth would be initiated within 7 to 28 days after postspace preparation depending on temporary restoration existence.[9] In addition to the method, the time of postspace preparation is also controversial. It can be done immediately after root canal treatment or sometime after endo treatment. Immediate postspace preparation is definitely less time-consuming, but it would threaten the apical seal due to the incomplete setting of sealer. Impaired apical seal before postcementation has consequences that are difficult to compensate. Therefore, it is necessary to try to prevent the probable leakage until receiving the permanent restoration by scrutinizing the factors in which we can intervene and selecting more appropriate alternatives. There are different techniques to provide postspace such as mechanical (bur/drills), physical (heat instrument), and chemical (solvents). Heated appliances and peeso reamers are among the most common and widely used preparation methods.[12] Failure of endo treatment is due to the penetration of bacteria or their products into the canal and the apical region. In Torabinejad et al.'s study, more than 50% of the root canals exposed to Staphylococcus epidermis and 50% of the canals exposed to proteus vulgaris were completely infected after 19 and 42 days orderly.[13] In previous studies about microleakage subsequent to postspace preparation, contradictory results have been stated that may be due to the use of different materials and measurement methods.[7],[14],[15],[16],[17] The goal of this study was to investigate the bacterial microleakage of endodontically treated teeth following two methods (peeso reamer vs. heat plugger) of postspace preparation at different timings (immediately vs. 1 week after endodontic treatment). The null hypothesis was that neither techniques nor timing of postspace preparation would affect the bacterial microleakage of endodontically treated teeth.

  Materials and Methods Top

Eighty-two recently extracted single-rooted human teeth were chosen according to the inclusion criteria (at least 15 mm root length, absence of root caries, cracks, curve, and structural imperfections) for the present in vitro study. A power analysis was performed based on the results of a previous study by Grecca et al.[17] The sample size was calculated as 18 specimens per group for a significance level of α = 0.05 and power of 0.80. The selected teeth were extracted due to orthodontic and periodontal reasons, also this project was accepted by the ethical committee of Tehran University of Medical Sciences (the ethical code: IR. TUMS. DENTISTRY. REC.1397.096). The teeth were immersed in 5.25% sodium hypochlorite solution for 24 h and then maintained in normal saline 0.9%. Single-rooted teeth were verified by mesiodistal and buccolingual radiographs. Different types of teeth (central, canines, and premolars) were marked to be divided equally into different groups. The crowns were removed at the cementoenamel junction level by a high-speed fissure bur; the length of all specimens was adjusted using a #10 k-file to 14 mm. The teeth with the same mark were randomly allocated into four groups (n = 18). Two groups (n = 5) were also considered positive and negative controls.

Root canal treatment

The root length of the specimens was determined by inserting a manual No. 10 k-file (Dentsply, Maillefer, Ballaigues, Switzerland) into the root canal until the tip could be seen from the root tip and reducing it by 1 mm. The desired length was confirmed by digital radiography. Root canal preparation was achieved by using rotary files (SP1 V-Taper, China) up to #40.06 according to the manufacturer's instructions. RC Perp (Master-Dent RC Lube, Dentonics Inc, USA) was used as a lubricant, and smear layer was removed by 17% EDTA (MD-Cleanser™, Meta Biomed Co. Ltd., Cheongju City, Chungbuk, Korea). Rinsing of root canals was done with Sodium hypochlorite solution (2.5%) followed by saline. Root canal obturation was done by lateral compaction method using gutta-percha (Meta Biomed Co., Ltd., Cheongju City, Chungbuk, Korea) and AH26 sealer (Dentsply DeTrey GmbH, Konstanz, Germany). Finally, the quality of the root canal filling was confirmed by radiography. Specimens in the positive control group (n = 5) were not filled after root canal preparation and the ones in the negative control group (n = 5), were not prepared for the postspace after filling the root canal. As a result, the specimens were divided randomly into four experimental groups (n = 18) according to the method and time of the postspace preparation, and two groups of 5 roots were prepared as control groups. The studied groups are depicted in [Table 1]. Specimens in groups 2 and 4 were stored at 37°c and 100% humidity for 7 days in the incubator. To avoid leakage through dentinal tubules, root surface of the specimens was covered with two coats of nail polish except in the apical section.
Table 1: The studied groups in this study

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Microbial process

All the specimens were sterilized by gamma radiation (activity: 6450 ci and dose rate: 1.54 G/sec) so that external microbial contamination would not be a source for errors in the results during the microbiological process.[9] To test the bacterial penetration, a modified method similar to Torabinejad et al. set-up which was a customized dual-chamber device was used.[13] The upper chamber containing artificial saliva and contaminated with Enterococcus faecalis (1.5 × 108 colony forming units/mL of EF ATCC 29212, Iranian Biological Resource Center, Tehran, Iran) was placed on the coronal part of the specimens, and the lower chamber containing the Trypticase Soy Broth (TSB, Merck KGaA, Darmstadt, Germany) was in contact with the apical part of the roots; all the connections between two chambers were sealed so that the only connection between two chambers was through the root canal. It was planned to renew the suspension every 3 days. The assemblies were stored at 37°C and the culture medium (TBS broth) was checked daily over the next 60 days. The sterile yellow TBS medium is amber and transparent, which would become cloudy in case of microorganisms' growth. Therefore, specimens with the opaque chamber (culture medium) were considered positive in terms of microleakage and the day was recorded. The phenol-red reagent was added to the lower chamber for photographic resolution of the specimens and to facilitate their detection. The culture medium in which the bacteria penetrated was acidic due to bacterial metabolism and the reagent changed color to red [Figure 1]. 10 μL of infected TBS broth was then cultured in bile esculin agar plates as a selective and differential medium which is used to identify the E. faecalis strain and the plates were incubated at 37°C. After 24 h, the microbial colonies were counted using the Miles et al. method.[18] At the end of the test period, microleakage information of the specimens was recorded and statistical analysis was performed.
Figure 1: The color of culture medium (a) turned into red (b) in case of bacterial penetration (due to existence of phenol red reagent)

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Statistical analysis

Data pertinent to microbial microleakage were reported as numbers and percentages on different days. Analysis was performed with SPSS software (IBM Corp. Released 2017. IBM SPSS Statistics for Windows, Version 25.0. Armonk, NY, USA: IBM Corp). Microleakage percentage during 60 days was examined among the groups by Chi-square test. The survival rate without microleakage was tested using the Kaplan–Meier curve and the Log-Rank test. The interaction of time and method of postspace preparation was examined using Cox regression and hazard ratio (HR) was reported accordingly.

  Results Top

The number of specimens contaminated during daily examinations is presented in [Table 2]. All the specimens in positive control group became infected during the 1st week but the negative control group did not show any sign of microleakage till the end of the survey. Groups 2 and 4 had the highest microleakage and the lowest survival rate (55.56% ± 11.71%). Group 1 showed the least microleakage and the highest survival rate (77.8% ± 9.80%). There was no significant difference among studied groups in terms of bacterial microleakage (P = 0.37) and survival rate (P = 0.41) [Figure 2]. Furthermore, according to the Cox regression, two examined factors (time and method of postpreparation) had no significant interaction (P = 0.71); besides these two factors had no significant effect on the obtained results (HR = 0.76, 95% confidence interval [CI]: 0.35–1.67, P = 0.49 and HR = 0.57, 95% CI: 0.25–1.29, P = 0.17, respectively).
Table 2: Number of infected specimens in each group during the determined weeks

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Figure 2: Bacterial leakage and survival rate based on two factors of time and method of post space preparation

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  Discussion Top

In this study, two methods of postspace preparation (peeso reamer vs. heat) at two different times (immediately after obturation vs. 1 week later) were investigated in terms of bacterial microleakage. According to insignificant obtained results among the studied groups, the null hypothesis of the research was confirmed. It has been demonstrated that the length of remaining filling material is inversely related to apical leakage after postspace preparation; according to the literature, the appropriate length of 5 mm was considered in the present study. Since most of the pulp and peri-radicular diseases are related to microorganisms, using them for microleakage evaluation is more precise and similar to the oral condition compared to other methods such as fluid filtration,[7],[10] dye penetration,[4],[5] electrochemical,[8],[11] SEM analysis,[19] radioactive tracer assay,[6] micro-computed tomography scans.[20] In addition, saliva seems to be the most suitable carrier for the microorganisms used due to its similarity to clinical conditions and viscosity. In this study, E. faecalis ATCC29212 was selected, which is one of the commonly known bacteria in dental infections and is associated with periapical infections. To standardize the root canal preparation, rotary files were used and due to the routine use of lateral compaction technique, the canals were filled with this method in the present study. Root canal sealers are used to fill the remaining gaps between the gutta-percha and root canal walls. In addition, sealer may trap the residual bacteria in dentinal tubules. AH 26 is an epoxy resin-based sealer applied in this research because of its good sealing properties and widespread use. There was no statistically significant difference between the study groups in terms of bacterial microleakage neither in different methods of postspace preparation nor in different timing. There is still no consensus about the superior method of gutta-percha removal regarding apical seal in the literature. Mattison et al. showed no significant difference in leakage between hot pluggers and Gates-Gliddens.[11] This finding has been confirmed with other studies[6],[17],[21],[22] and also the current study. On the other hand, according to Haddix et al. survey,[23] heat instruments were safer than rotary ones (Gates-Glidden drill and GPX instruments) in terms of the apical seal. Although, heat instruments could be applied without the risk of stripping or perforation; rotary instruments are faster, easier, and commonly used.[8] It has been declared that applying heat instruments would result in gutta-percha expansion and therefore improve the seal. In addition, the resulting frictional heat during the use of heat instruments would lead to plasticized deformation of gutta-percha which diminishes the gap between the filling material and dentinal wall, moreover, hinder excessive pulling of gutta-percha and results in a better seal.[11],[23] Haddix et al. considered the speed of rotary instruments as an influential factor in this context.[23] Nonetheless, Balto et al. revealed less leakage for peeso reamers compared to hot pluggers.[12] The authors attributed gutta-percha shrinkage to more leakage of heat instruments. It has been suggested that insufficient or inaccurately condensed obturation material, would be displaced during postspace preparation.[24] Therefore, the obturation phase and also condensing of the remaining filling material after preparation of the root canal are of great importance. The existed heterogeneity among studies could be due to different filling techniques, exitances of coronal dressing, follow-up times, and analyzing methods.

In contrary to the nonsignificant results for time of postspace preparation in this study, Padmanabhan et al. reported less apical microleakage in immediate postspace preparation groups than delayed ones.[25] The authors argued that in the immediate method, the setting time of sealer is not completed yet while the hot plugger or peeso reamers enter the root canal. Therefore, microfractures do not cause a gap between the sealer/gutta-percha or sealer/dental tissue. Whereas, after the completion of setting time, the entrance of tools into the root canal may cause the gutta-percha to move and break the interfaces.[25] Although the filling technique in the recent study was single cone obturation and the leakage was evaluated using dye penetration. Salim declared similar outcomes with the same method.[26] Methylene blue, like India ink, is not very accurate due to its small molecules, different viscosity, and structural differences with saliva, and its results are questionable. According to Torabinejad et al., if a filling material does not allow small molecules (such as the ink molecule) to pass through, it is very likely to prevent the penetration of larger particles like bacteria and their products.[27] Based on this theory, it can be accepted that the nonpassage of dye molecules means the nonpassage of bacteria, but the leakage of dye does not mean the passage of bacteria and its byproducts which shadows the obtained results of dye-based studies. The results of Padmanbhan et al. study were in accordance with the observations of Dhaded et al. research in which the seal and adaptation of Gutta-percha filling material to the dentinal walls and the interface were examined by SEM analysis.[19] Furthermore, other studies have suggested immediate root canal preparation due to the less following leakage.[7],[14],[28],[29],[30],[31] Abramovitz et al. stated no significant difference between immediate postspace preparation with a hot plugger and late removal with rotatory instruments which was in line with the results of the present study.[6] They increased the sensitivity of the assay by using a pressure system, but this question arises that this system does not simulate the intra-oral condition. The result of Madison and Zakariasen study was similar in terms of apical dye leakage with immediate or delayed (48 h later) removal of gutta-percha either with heated pluggers or peeso reamers.[21] In confirmation of recent results, Grecca et al. reported that immediate and delayed postspace preparation either with heated plugger or LA Axxess rotary instruments yielded similar outcomes regarding the canal seal.[17] Furthermore, no significant result was addressed in Aydemir et al. study in which the leakage analysis performed by electrochemical method and postspace was prepared after 30 days in the delayed group.[8] The same outcome was presented in other studies.[12],[16],[20],[32],[33],[34],[35],[36],[37],[38],[39] Although Chen and Chang also came to the same conclusion, the authors suggested postponding the time of postspace preparation until the complete setting of sealer and close to the time of restoration installation.[4] The reason for this precaution is mentioned to sustain the integrity of filling material and apical seal. In contrast to the previous studies, Nagas et al. addressed better apical seal by delayed postspace preparation compared to immediate postpreparation.[10] The authors claimed that polymerization of sealers may also be a reason for better sealing of delayed postpreparations. However, a modified fluid transport system was applied to measure the leakage in their study. Chen et al. expressed optimal results in case of waiting for the complete reaction and settlement of filling materials before any preparations.[5] In addition, Ibrahim et al.'s study was in consistent with the previous studies.[40] Lack of consensus in this context could be due to different methodology, filling material and sealer, filling and removal technique, presence of coronal dressing, leakage analysis, and following time in performed studies.

Despite nonsignificant difference between studied groups in the present study, it is noteworthy that in groups with immediate removal, leakage was seen in no specimens till the 24th day, while in delayed preparation groups, leakage was recorded from the first of the second week. Therefore, it can be concluded that in immediate postspace preparation, apical leakage would occur later and exactly this time is needed to form the postpattern, laboratory process, and postcementation. Based on the obtained results, regardless of the time and technique for postspace preparation, apical leakage occurred in all groups. Therefore, it is of great importance to maintain the coronal seal of the access cavity with an accurate material after endodontic treatment. Furthermore, immediate removal of filling material would have advantages in terms of postponed leakage compared to the delayed method, which needs to be confirmed by further studies. Lack of turbidity in the negative group indicates the importance of accurate filling to prevent leakage through the root canal. The main determining factor in the success of the root canal treatment was proved to be the quality of the root canal filling in Tronstad et al. study rather than the quality of the coronal restoration.[41] However, contamination of all specimens in the positive group during the 1st week indicates the importance of appropriate remaining obturation material and coronal seal. Regarding the obtained findings, the remaining filling material cannot hinder bacterial leakage in root canals exposed to the oral environment, which asserts to expedite the prosthesis delivery. Providing postspace by an endodontist or dentist who has performed the root canal treatment has several benefits; this clinician is more acquainted with the anatomy of root canals and the working length; root canal preparation would be done under the same aseptic condition as endodontic treatment.[42] Besides, it prevents accidents such as ledge, stripping, or perforation. On the other hand, higher postoperative pain has been reported when postspace preparation and postinstallation were performed in the same appointment.[43] However, based on nonsignificant obtained results in the present study, dentists can make the decision about the time and method of postspace preparation based on each individual's situation. One of the limitations of this study was the lack of intra-oral conditions like thermocycling and cyclic loading, furthermore, coronal temporization was not considered which commonly is applied for patients to reduce coronal leakage during treatment sessions. Furthermore, it is recommended to implement this project as an in vivo research with follow-up sessions to essay the experimented factors in the clinical situations.

  Conclusions Top

Based on the statistically nonsignificant results, the practitioner can choose each of the removal techniques (peeso reamer or hot plugger) and time intervals (immediate or 1 week later) based on the patient's condition, ease of work, clinical factors, and time management related to the patient.


This research was supported by the grant number 97-02-69-38991 from Tehran University of Medical Sciences.

Number of pages: 13, Number of photographs: 2, Word counts for abstract: 245, Word count for text: 3119, word counts for introduction + discussion: 2110.

Financial support and sponsorship


Conflicts of interest

There are no conflicts of interest.

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Babaier RS, Basudan SO. Do dentists practice what they know? A cross-sectional study on the agreement between dentists' knowledge and practice in restoring endodontically treated teeth. BMC Oral Health 2021;21:110.  Back to cited text no. 42
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  [Figure 1], [Figure 2]

  [Table 1], [Table 2]


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