Sealing Ability of Obturation with Different Sealers Applied Over Apically Separated Rotary File - Retrograde Bacterial Leakage Study.

Background and Aims: . Retrieval of separated rotary file may not be possible many times. Then sealing of coronal root canal system depends on sealing ability of root canal sealers. The type of seal to be obtained should be bacterial-tight seal. Hence it is important to evaluate the sealing ability of obturation with different newer sealers with separated file in apical 3rd of root canals with bacterial penetration method using e.fecalis.
Methods: Forty six human mandibular molars extracted for periodontal reasons were collected. After Decoronation of teeth Protaper rotary S1 files (Dentsply Maillefer) were intentionally separated in apical third of canal and obturated with gutta-percha (Prime Dental Products, India) along with zinc oxide eugenol sealer (Dental products of India-Mumbai) in Group I, Nano Zinc oxide eugenol (Nano Research Lab, Jharkand, India) in Group II and MTA sealer (Angelus, Londrina, Brazil) in Group III. Samples were assessed for bacterial leakage with E fecalis for 48 hours. The number of colony forming units was assessed.
Results: The values obtained were subjected to one way ANOVA test and significance level was set to 0.05. Group I scored the highest mean value of (6.58), followed by Group II (3.91). The lowest Mean value was with Group III (2.91). However there was no statistical significant difference in Mean values among groups with (p-value 0.05).
Conclusion: The mean value of colony forming units in Group III was 2.91, in Group II 3.91 and 6.58 in Group I. None of the groups demonstrated complete bacteria-tight seal. Copyright:

BACK GROUND AND AIMS

Separation of Endodontic instrument is a nightmare to a Root canal specialist as it complicates the entire root canal procedure. Depending upon the location of the separation and condition of the root canal at that time, different treatment modalities can be adopted.

When the instrument is separated in apical 3rd, it can be best left in the canal owing to the complexity of the region. About 3.3% of treated teeth have retained the endodontic file in the canal. Some studies suggested that a retained fracture instrument can be incorporated into the obturation.[1] The root canal space coronal to the separated file has to be prepared and sealed meticulously.

The contemporary obturation procedure involves compaction of guttapercha with an appropriate sealer. As previous studies have proved the lateral compacted technique presents a risk of void formation and difficult in curved canals, warm vertical compaction is considered as better option.[2]

Traditional Zinc oxide eugenol sealer has certain disadvantages such as prolonged setting time, shrinkage on setting, high solubility, and may not be able to seal the space.[3] Hence sealer of smaller particle size and bioactivity are needed.

Nano technology has revolutionized medicine and dentistry by producing materials of nano size with better properties. A study, tested zinc oxide nanoparticles as sealers for sealing ability. The result showed apical microleakage with zinc oxide nanopowder sealer was less, and it was stated that these sealers are suitable for root canal therapy. The microleakage observed was least with zinc oxide nanopowders as sealing ability will be better by using smaller nanopowders particles.[4]

MTA based sealer has an optimal sealing property and used as an apical seal, apical plug, and perforation repair. It is biocompatible and nontoxic and has bactericidal properties. Along with bioactivity property, it might seal the separated file in the dentin. MTA is hydrophilic endodontic cement capable of penetrating into small dentinal tubules. Also, they have the setting expansion, which results in their better adaptation to canal walls. Moreover, MTA cement forms hydroxyapatite and provides a better seal between dentinal walls and root canal filling material.[5]

Enterococcus fecalis is a persistent organism that, despite making up a small proportion of the flora in untreated canals, plays a major role in the etiology of persistent peri radicular lesions after root canal treatment. It is commonly found in a high percentage of root canal failures, and it is able to survive in the root canal as a single organism or as a significant component of the flora.[6]

Nano Zinc Oxide Eugenol sealer and MTA sealer have been proved superior in sealing ability of normal root canal therapy. However they have not been evaluated for sealing ability in canals with separated file.

This study was designed to evaluate the sealing ability of obturation with different newer sealers with a separated file in apical 3rd of root canals with the null hypothesis that, newer sealers will not have better effect in sealing ability.

Sealing ability was evaluated using a bacterial penetration method using E.fecalis

METHODS

Sample size calculation

Sample size was calculated to be 12 in each group considering = 95, power of 90% and for penetration of e Fecalis into canals filled with MTA based sealer and Zinc oxide nanoparticles into the canal to ensure the reliability of results, 12 teeth were included in each group. The teeth were selected using random sampling method (random.org).

A total of forty-six human mandibular molars extracted for periodontal reasons, were collected from the department of the Oral and Maxillofacial surgery, with the consent of ethical committee (VDC/IEC/2017/35). Selected teeth were cleaned of the remaining connective tissue and debris with an ultrasonic scaler. Teeth were then rinsed with distilled water and stored in 0.1% w/v thymol until use at room temperature.

Teeth were examined under Labomed dental operating microscope at 2.5X magnification for the presence of surface defects and morphological anomalies.

Inclusion criteria: Presence of noncarious teeth, No development anomalies, anatomically and morphologically well-defined teeth.

Exclusion criteria: Severe dilacerations Calcified canals Presence of Pulp stones The appearance of any fracture lines.

Methodology

The samples were divided into 3 groups.

Group I: Zinc oxide Eugenol.

Group II: Nano Zinc Oxide.

Group III: MTA.

To standardize the root length, all the specimens were decoronated to a length of 13 mm by using a Double-faced diamond disc (KG Sorensen, Barueri, SP, Brazil) mounted in a straight handpiece running at a speed of 300 rpm and attached to the micro motor unit.

The roots were mounted in the wax model and pre operative radiograph was taken. Working length was determined by using a 21 mm, 10k hand file (Dentsply Maillefer) into the root canal until it was absolutely visible at the apical foramen. The final working length was determined by subtracting 0.5mm with the help of endo gauge (Dentsply-Maillefer, Tulsa, OK, USA).

All the roots were prepared with Protaper Universal file (Dentsply Maillefer) and X smart Endomotor (Dentsply Maillefer). The canals were prepared for glide path with 10 and 15k files. Initially SX instrument was used up to one-third of the working length with X-smart Endomotor at 300 rpm and a torque of 5.10Ncm and proceeded with S1 up to the working length.

In all the groups the rotary file S1 was intentionally separated by keeping notch at apical 3 mm of the file and file was to attached to endodontic hand piece and inserted into canal for preparation and file had got separated in the canal and was confirmed with radiograph.

The remaining coronal 3rd of root canals were prepared up to F3 as per manufacturer instructions. Irrigation was done with 5 ml of 3% Sodium hypochlorite (Prime dental PVT LTD., India). Apical patency was maintained by using 15K-file (Dentsply Malliefer) for recapitulation between files. To remove the smear layer, all canals were irrigated with 3 ml of 17% ethylenediaminetetraacetic acid (Prime Dental Product) for 1 minute, followed by 5 ml of using 5 mL of distilled water to remove any remaining irrigating solution. All the irrigation procedure was followed using a side vented needle placed 1 mm short of the apical foramen. The canals were dried with # 30 sterile absorbent paper points (Prime dental PVT LTD., India.) after irrigation.

Obturation

Primary non standardized greater taper guttapercha corresponding to last instrument was taken. Canal was coated with thin layer of Root canal sealer with guttapercha. Primary guttapercha cone was inserted to the working length. Coronal end of the cone was cut off with heated plugger. Heated plugger was forced into the coronal third of the guttapercha. Coronal guttapercha seared off by the plugger was removed from the canal. Vertical pressure with heated plugger of size 3 was applied to plasticize and compact the guttapercha apically. Remaining canal was obturated with additional cones with warm vertical compaction. Quality of obturation was assessed using radiograph [Figure 1]. The teeth were sealed with intermediate restorative material (cavit) at the coronal end.

Figure 1

Radiograph showing separated file and obturation

Sample preparation for microleakage with E fecali

Bacterial leakage study was done in special certified microbiological research centre under the guidance of experienced microbiologist. In the negative control group (5) entire root with apical foramen was sealed with nail varnish. Samples were autoclaved before starting the obturation of the canal. The procedure warm vertical compaction was done under sterile conditions. After obturation, the samples were autoclaved before inoculating the bacteria that is E fecalis. All the process was done in the Laminar flow chamber. After completion of obturation, the samples were transferred to Effendroff tubes. By using a BP blade, a hole was made at the centre of rubber stop through which the tooth was inserted under pressure up to cementoenamel junction so that the crown was outside and root tip was inside the vial [Figure 2]. Different dilutions were plated in triplicate on m- Enterococcus agar culture medium and carried into the Effendroff tubes with the help of an insulin syringe and placed over apical 3rd portion of the root for 48 hrs.

Figure 2

Effendroff tube with tooth

Evaluation of sample by using E fecalis

Samples were placed into a 5 ml syringe in an inverted direction, and syringes were placed in the Effendroff tubes. The bacteria were leaked in the apical third of the samples for 48 hrs. After 48 hrs bacteria leakage was observed from the apical to the coronal part of the tooth. Then the bacteria which were leaked into the coronal part of the tooth were calculated as a number of colony-forming units.

RESULTS

Samples were subjected to observe the apical microleakage with e Fecalis from retrograde and were evaluated for microleakage for 2 days. The number of bacteria leaked into coronal portion of the tooth was calculated by number of colony forming units. The Apical microleakage was noted in the following table [Table 1].

Table 1

The number of colony forming units in the groups

Groups	1	2	3	4	5	6	7	8	9	10	11	12
GP I [ZOE]	NG	10	10	8	NG	20	10	NG	10	10	1	NG
GP II [NANO ZOE]	10	20	15	NG	NG	1	NG	NG	1	NG	NG	NG
GPIII [MTA]	NG	NG	NG	NG	NG	NG	NG	20	NG	NG	NG	15

The values obtained after bacterial leakage through colony forming units were subjected to one way ANOVA test and significance level was set to 0.05. Group I scored the highest mean value of (6.58), followed by Group II (3.916667). The lowest Mean value was with Group III (2.91) [Figure 3]. However there was no statistical significant difference in Mean values among groups with (p-value 0.05) [Table 2].

Figure 3

Mean values of Apical Microleakage in three groups

Table 2

Comparison of mean colony forming units

Groups	N	Mean	Standard Deviation	F	Significance
GP I [ZOE]	12	6.58	6.35	1.440	0.238
GP II[NAOZOE]	12	3.91	7.02
GP III [MTA]	12	2.91	6.82

DISCUSSION

Efficient obturation must provide a bacteria-tight or fluid impervious seal to prevent re entry of microorganisms. An optimal apical seal plays an important role in success of endodontic treatment and health of periapical tissues and can increase the success of endodontic treatment by up to 97%. Absence of apical seal, apical leakage, has been reported as the most common cause of endodontic treatment failure.[2]

Root canals can be prepared with hand or rotary files. Rotary files enable faster canal preparation and those made of nickel titanium (NiTi) can even be used in narrow curved canals due to high flexibility and fracture strength. However, risk of fatigue fracture or breakage due to shear stresses still exists.[7]

Evidence shows that a broken instrument remained in the root canal does not have a significant adverse effect on the quality of root canal seal by filling materials and success of endodontic treatment mainly depends on coronal seal and cleaning of the middle and coronal thirds.[8]

The primary objective of obturation is to achieve a bacteria-tight seal not only in apical, lateral and coronal sections of root canal system and also in accessory and lateral canals.[9] These canal complexities are very fine in dimension hence can be sealed with root canal sealers. Similarly the space around the separated instrument will be very minimal and might hinder the flow of guttapercha. Any material with low viscosity and better flow should be able to occupy the space to obtain fluid tight seal.

Sealers have been extensively studied in the past and play an important role in root canal obturation; however, an ideal sealer that meets all properties has not been found. Grossmans states that a good sealer adheres strongly to the dentin and the core material; it must also have strength to hold the obturation together. Sealers should be antimicrobial, biocompatible and one that can be dissolved in solvent if retreatment is warranted. Sealers should also have minimal toxicity that may be not irritating to the tissues. The sealer must have some degree of radio-opacity to be visible on x-rays.[10]

Traditionally sealing ability was evaluated using dye penetration methodology using a variety of dyes, such as methylene blue, Indian ink and Pelikan. But the reliability, reproducilibility and clinical relevance are questionable. Some of the dyes have low molecular weight and can penetrate into sites where protein and bacteria cannot, so the exaggerated leakage can be seen.[11] Barthel et al.[12] had done a study comparing bacterial leakage versus dye leakage in obturated canals. The factors such as ionic charge, PH, temperature changes and the ability of viable microbes to change their shape and size and move actively, duplicate or grow, which might play a role in the root canal and which cannot be represented by an aqueous dye solution. Hence in present study bacterial leakage was taken for sealing ability.

Up to 90% of Enterococcal infections are by Enterococcus fecalis. Initial flora of untreated non vital teeth comprises of small portion of Enterococcus fecalis but more than 70% of E fecalis is found in secondary infections and from periapical lesions refractory to Endodontic treatment. This is because E Fecalis can withstand harsh environmental conditions and also adapt to adverse conditions. The starving E fecalis cells can remain vital for extended period and become resistant to sterilization procedures.[13]

A study has demonstrated that S1 files exhibit 12 increasingly larger tapers that range from. 0 to 0.11. S1 when reaches the apical third fractures with lesser fragments, this could be because of the larger diameter of the file in the coronal third and the smaller diameter in apical third where tightening will occur.[14]

Warm vertical compaction is one the best recommended in such cases. Warm vertical compaction produces consistently dense, dimensionally stable, canal Fillings and has been developed to fill canal irregularities more effectively than is Possible with cold gutta-percha. Although the warm filling technique improves the compaction of gutta-percha into root canals, it is still necessary to use a sealer because any small voids between the gutta-percha and canal wall irregularities or canal Fins have to be filled. It has been reported that warm gutta-percha root fillings without sealer leak more than those with sealer.[15]

Traditionally Zinc oxide eugenol is used for root canal obturation since 100 years. The advantage of zinc oxide eugenol sealer is its antimicrobial activity and popularity among clinicians, especially when used with thermo plasticized obturation technique. However Zinc oxide eugenol sealer has certain disadvantages such as prolonged setting time, shrinkage on setting, high solubility, and can stain the tooth structure.[3]

In the bacterial leakage was maximum in this group with mean value of 6.58. The particle size of Zinc oxide powders is 10-50 micrometer and film thickness is 10-15 microns. A study of comparison of apical seal and tubular penetration of zinc oxide eugenol sealer showed, less apical seal of zinc oxide eugenol with less tubular penetration. The factors affecting the tubular penetration are surface activity, the contact angle between the sealer and dentinal tubules.[5]

Maryam Javidi prepared a new experimental endodontic sealer with nano particles of zinc oxide. Javidi et al.[4] had done a study on Zinc oxide nano particles comparing the sealing ability of Zinc oxide nano particles and AH26 and found that there is significant difference in micro leakage. Hence nano zinc oxide eugenol sealer was used as one of the experimental group.

In the present study nano zinc oxide eugenol sealer produced better bacteria tight apical seal compared with convention zinc oxide eugenol sealer with mean leakage value of 3.91.

The probable reason could be the smaller size of the nano zinc oxide particle which could penetrate the small space available between fractured instrument and root canal dentin. Very scanty literature is available on nano zinc oxide eugenol sealer. Conventional Zinc Oxide eugenol sealer has antimicrobial property, the nano size of ZOE may have higher antibacterial property owing to increase surface area to volume ratio. Zinc oxide nano particle damage the bacterial cell wall and then penetrate and interfere with metabolic functions of microbes causing their death[16]

Manufacturer claims that MTA Fillapex has good sealing ability and biocompatibility, slow setting time, sufficient working time, perfect flow to allow the fillings of accessory canals and low solubility. Some studies are available regarding sealing and dentinal tubule penetration ability of MTA Fillapex. However there are no studies were sealing ability of MTA Fillapex was researched along with separated endodontic instrument.

The better sealing ability of MTA Fillapex demonstrated in the present study is in accordance with the following literature. MTA Fillapex has shown lowest mean value of 2.91. According to Asawaworarit et al.[17] MTA Fillapex demonstrated better sealing ability after 4 weeks, as MTA Fillapex contains high ratio of Salicylate causing the long chemical reaction time.

A comparative study effect of apical sealibility of MTA Fillapex and Endosequence BC sealer showed no statistical significant difference between both the groups. The presence of nano silicate particles in MTA Fillapex enables a homogenous mixture, better flow and less film thickness of the sealer. The alkaline PH of the bioceramic sealer might have caused denaturing of the dentinal collagen fibers and facilitated the deeper penetration into the dentinal tubules.[18]

As the comparison of the results amongst the group by ANOVAs did not show significant difference the null hypothesis is accepted.

The oral cavity dynamics including pH, temperature and occlusal loading was not simulated in the present experiment. Hence ex vivo and long term in vivo studies are indicated.

CONCLUSION

The results of this study showed that MTA Fillapex has shown less microleakage compared to Zinc oxide Nano particles and Zinc oxide Eugenol sealer.

Further researches should focus on long term clinical follow up in accidental file separation and the limitations and the potential unknown risks involved in the use of ZnO nano-powders as a medical material should be considered to verify their safety.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.