A Confocal Microscopic Study on Percentage Penetration of Different Sealers into Dentin.

CONTEXT: Three-dimensional seal of the root canal space has always been challenging. The incorporation of gutta-percha and sealer prevents microleakage by bacteria, ensuring a shielded root canal space. However, the penetration of sealer to various depths within the root canal has always been looked with curiosity. AIMS: Thus, the present in vitro study was undertaken to evaluate the percentage and average depth of penetration of Endoflas F. S., AH Plus, and Epiphany sealers into dentinal tubules among the coronal, middle, and apical thirds of the roots following obturation with a lateral compaction technique using Confocal Laser Scanning Microscopy. SETTINGS AND
DESIGN: The study is an in vitro randomized control trial. SUBJECTS AND METHODS: Thirty sound central incisors were decoronated at the level of the cementoenamel junction. Working length determination was done followed by a meticulous cleaning, shaping, and under copious irrigation. The teeth were then randomly divided into three groups: Endoflas FS sealer, AH Plus sealer, and Epiphany sealer. On fluorescence treatment, the teeth were sectioned at the midpoint of coronal, middle, and apical third of each root and viewed under confocal laser scanning microscope. STATISTICAL ANALYSIS USED: The results were analyzed using one-way ANOVA, and the significant difference between groups was analyzed with post hoc Tukey test.
RESULTS: Epiphany sealer provided with better percentage and depth penetration in comparison to Endoflas FS and AH Plus sealers. Furthermore, the coronal third of the root had better percentage and sealer penetration than the middle and apical thirds.
CONCLUSIONS: Sealers tend to provide a firm bond between the tooth and the gutta-percha. They bind, lubricate, and seal the gutta-percha cones to fill the accessory canals. Within the limitations of this study, the superior flow and enhanced setting time Epihany sealers provide better percentage and depth of penetration than AH Plus and Endoflas FS. Copyright:

INTRODUCTION

Successful endodontic therapy includes instrumentation of the root canal to obtain a surface free from debris and organic matter and obturation to achieve a well-sealed root canal.[1] Prevention of periapical fluids from seeping into the canal, entombing bacterial remnants, and preventing coronal leakage are the three major functions a root canal filling must possess.[2] Microleakage may occur in both apical and coronal directions, creating voids at dentinal wall and obturating agent junction or inside the obturating material itself, adversely affecting the outcome of endodontic therapy.[3]

Bacteria being the primary cause of pulpal or periapical pathosis either caused due to ineffective root debridement or poor coronal or periapical seal.[34] Bacteria could also persist within the dentinal tubules and lateral canals, playing a pivotal role in persistent periapical disease.[5] The penetrability of the materials into dentinal tubules plays a pivotal role in the prevention of reinfection related to the root.[6]

Although gutta-percha is universally recognized obturating agent, it is nonadhesive to the dentin irrespective of the obturation method.[7] Therefore, sealers provide an impermeable seal amid the core material and the canal walls. It provides both lateral and apical and adapts well to the root canal dentine.[6] Hence, sealers help to bind, lubricate, and seal the gutta-percha and to plug the lateral canals.[7] The ingredients from the sealer exhibit antibacterial property when in close proximation with the bacteria.[45] If this is not achieved, microleakage may occur subsequently, leading to persistent apical periodontitis.[4] None of the existing obturation procedures has proven to deliver an sufficient seal in spite of the development of many obturation systems.[3] Thus, materials with adhesive properties have been advocated for a better seal between the gutta-percha and root dentin.[6]

Endoflas F. S. (Sanlor Laboratories, USA) is a zinc-oxide based, antibacterial, hydrophilic sealer and indicated for use in comparatively moist canals. AH Plus (Dentsply, Maillefer) is a two-paste, polymerizable epoxy-based sealer. It has high radiopacity, biocompatibility, low shrinkage, and no formaldehyde byproduct.[8] Recently, Resilon-Epiphany system (Pentron Clinical Technologies, Wallingford) is introduced with a promising tubular adhesion.[9] Epiphany forms a “Monoblock” by adhering equally to the primer and the resilon cones.[10]

The microleakage from the obturated canals has been studied extensively with techniques such as linear measurement of tracer (dye or isotope), fluid filtration models, bacterial leakage models, electro-chemical models, spectrophotometry, and scanning electron microscopy (SEM).[11] SEM is a common method used to evaluate sealer-dentin interface. However, this technique needs a special processing of the specimens, the microscopic images show the reduced thickness of the sealer, and artifacts may also be present.[3612]

Marvin Minsky presented the model of confocal microscopy in 1950s to overcome the drawbacks of SEM with the advent in digital and laser technology.[13.14] Confocal laser scanning microscope (CLSM) produced indestructible images with lower artifacts.[5] Even at a magnification as low as 50x – 100x, overall sealer penetration into dentin was evident, which is not possible in SEM.[4]

Thus, the present in vitro experiment was undertaken to estimate the proportion and mean penetration depth of Endoflas F. S., AH Plus, and Epiphany sealers into dentin. The root sections after obturation with a lateral compaction technique were studied using CLSM.

SUBJECTS AND METHODS

Thirty maxillary noncarious extracted central incisors were collected on appropriate consent from the institutional ethics committee. The teeth were disinfected using 3% sodium hypochlorite solution for half an hour and then kept in biological saline. Tooth crown was trimmed up to the cementoenamel junction using a rotary diamond disk. The authors used number 10 K-file for canal instrumentation till it was visible from the apical end. The authors determined the working length following instrumentation by deducting 1 mm from the original measurement. Protaper rotary files (Dentsply Sirona, USA) and reduction gear hand piece (Anthogyr, Dentsply, USA) up to F3 file size were used to enlarge the canals. 10 ml of 3% sodium hypochlorite (NaOCl) irrigation was performed during change of each file. The smear layer was removed by flushing with 10 ml neutralized 17% EDTA and 10 ml NaOCl. 10 ml sterile water was used to eliminate remnants of NaOCl. The authors used sterile paper points to dry the canals.

Group 1

To enable fluorescence for CLSM, Endoflas F. S. sealer was combined with 0.1% Acridine orange fluorescent dye (maximum absorption: 570 nm, maximum emission: 720 nm). A K-file was used to coat the mixture of fluorescent dye and sealer in anticlockwise direction along the canal. Gutta-percha F3 size cones were selected as master cones, and the fit was verified by placing it to the working length. The authors performed lateral condensation technique for obturation along with the use of gutta-percha accessory cones. A heated instrument trimmed the excess cone and hand pluggers aided in vertical compaction. Radiographs ensured the absence of voids. The access cavity was sealed with Cavit-G (3M ESPE, USA).

Group 2

A mix of AH Plus sealer and 0.1% Acridine orange fluorescent dye (maximum absorption: 570 nm, maximum emission: 720 nm) facilitated florescence for CLSM. The same procedure as in Group 1 was followed until the sealing of the access cavity was achieved.

Group 3

An epiphany primer-coated paper point was placed to length and the point was left to wick the primer to the apex. Fluorescence mixture formulation and its placement were similar to Group 1. Resilon size 30, 0.06 taper cones were selected as master cones, and the fit was verified by placing it to the working length. A lateral condensation technique was used for obturation along with the use of Resilon accessory cones. After removing excess cone and vertical compaction, the orifice cement was light polymerized for 40 s. Radiographs ensured the absence of voids. Access cavity sealing was achieved in a similar way as in Group 1.

Preparation of specimen for microscopy

Roots in all the three groups were sectioned at the mid-point of the coronal, middle, and apical third to obtain ten sections each. A Zeiss Pascal Laser Scanning Microscope (Carl Zeiss, Germany) was used to scrutinize the samples. CLSM pictures were documented in an oil immersion fluorescent mode under ×10 magnification. Individual specimen was scrutinized for a constant fluorescent ring round the canal wall representing sealer and root dentin. Sealer penetration depth was analyzed through Adobe Photoshop CS3. After importing the image to photoshop, a lasso tool was used to determine the circumference of the canal. The area along the canal walls was measured, in which sealer infiltrated. The ratio of sealer penetration was analyzed using the outlined distances by the canal circumference. The statistical analysis was performed by ANOVA test and the significance difference between groups was calculated using Tukey post hoc procedure. The depth penetration was recorded at four standard points on individual section using ruler tool. The canal wall functioned as the onset position, and the penetration depth was assessed to an utmost depth of 1000 μm. It marks the external boundary of sealer penetration evident inside the microscopic field. The mean and standard deviation were calculated. ANOVA and Tukey post hoc tests were used to average the depths of penetration for each group and to record the significant difference between groups.

RESULTS

Figures 1–3 provide details of CLSM at the coronal, middle, and apical thirds for Group 1, Group 2, and Group 3, respectively. It can be observed from the tables that the percent and the mean depth of penetration were highest for Epiphany followed by AH Plus. It was least for Endoflas F. S. at all levels of the roots. The results of ANOVA test and Tukey post hoc procedure are elaborated in Tables 1 and 2. The results were statistically significant. The highest percent and mean depth of penetration were found at the coronal sections, reducing at the middle and the apical sections, respectively, in all the three groups. Figure 4 explains the calculations of (a) percentage of penetration (%) and (b) depth of penetration (μm).

Figure 1

Group 1 sections under confocal laser scanning microscope: (a) Coronal third (b) middle third (c) apical third

Figure 3

Group 3 sections under Confocal laser scanning microscope: (a) Coronal third (b) middle third (c) apical third

Table 1

Percentage of penetration: Comparison by ANOVA

Sides	SV	Sum of squares	df	Mean square	F	P
Coronal	Between groups	6748.54	2	3374.2720	159.5590	0.0000*
	Within groups	570.98	27	21.1470
	Total	7319.53	29
Middle	Between groups	9624.36	2	4812.1810	208.5690	0.0000*
	Within groups	622.95	27	23.0720
	Total	10247.32	29
Apical	Between groups	2991.27	2	1495.6350	72.9710	0.0000*
	Within groups	553.40	27	20.4960
	Total	3544.67	29

*P<0.05. SV= Standard Variation

Table 2

Percentage of penetration: Pairwise comparison by post hoc Tukey test

Side	Groups	Groups	Mean difference	Standard error	P
Coronal	Group 1	Group 2	−13.0590	2.0566	0.0000*
		Group 3	−36.2680	2.0566	0.0000*
	Group 2	Group 3	−23.2090	2.0566	0.0000*
Middle	Group 1	Group 2	−18.0140	2.1481	0.0000*
		Group 3	−43.6520	2.1481	0.0000*
	Group 2	Group 3	−25.6380	2.1481	0.0000*
Apical	Group 1	Group 2	−7.2440	2.0247	0.0040*
		Group 3	−23.8540	2.0247	0.0000*
	Group 2	Group 3	−16.6100	2.0247	0.0000*

*P<0.05

Figure 4

Calculations: (a) Percentage of penetration (%) (b) depth of penetration (μm)

Group 2 sections under confocal laser scanning microscope: (a) Coronal third (b) middle third (c) apical third

DISCUSSION

Although Gutta-percha presents with favorable properties, it lacks bond to the internal tooth structure resulting in incomplete seal. It results in bacterial microleakage and results in failure of the root canal therapy.[5678] Hence, a sealer is important to plug the gap amid the dentin wall and the obturating core interface, sealing the cavities and asymmetries inside the canal, to plug lateral and accessory canals, and to permeate into the tubules.[678] Thorough penetration of sealer not only improves the seal but also retains the material by mechanical interlocking.[5] Sealers also possess antibacterial properties, thereby preventing bacterial repopulation or promotes inactivation in case of an accidental negligible leakage.[25] A thinner sealer mix facilitates better seal and deeper infiltration.[24] Sealers with good flow results in thorough canal wetting, better adaptation, and a hermetic seal.

A disadvantage of zinc-oxide-based sealers (Endoflas F. S.) is that they shrivel upon set and disintegrate over time. Resin-based sealers (AH Plus) bond well with the canals.[8] Resilon/Epiphany (R/E) system involves Resilon core material, Epiphany (a resin-based sealer), and a primer. Epiphany/Resilon was noticed to deliver enhanced coronal seal,[210] resist microleakage, and increase fracture resistance of an endodontically treated tooth.[615] Built on these assuring results, Resilon, with the Epiphany primer and sealer system was successively described to as the Resilon monoblock system. A study with SEM analysis found good bond of Epiphany sealer to the canal walls with visible tags in dentinal tubules.[9]

Thirty maxillary central incisors were selected so as to eliminate variations that may be present in root canal anatomy. As AH Plus, Endoflas F. S., and Epiphany sealers have no fluorescent properties, it was essential to incorporate a fluorescent acridine orange dye to the sealers for visualization under the CLSM. Therefore, analysis of microscopic results was proportional to the infiltration of the fluorescent marked sealers.

SEM has been a commonly employed testing method to evaluate sealer-dentin interface. However, this technique needs a special processing of the specimens, and this may produce artifacts.[51213] CLSM allows the users to form a histotomographic image of the dentin bulk in a nondehydrated sample, at the subsurface of a bulky sample.[2] Furthermore, it produces comprehensive data regarding the existence and dispersal of the sealers within the tubules at a low magnification (50x–100x) by incorporation of fluorescent dye marked sealers.[4] Our results were consistent with the results of Patel et al.[6] and Sevimay and Dalat,[7] wherein they found lower depth and low sealer penetrations in the apical third compared to middle and coronal thirds.

Factors affecting sealer penetrability are the presence of smear layer, dentin permeability, and differences in sealer properties. In all the groups, sealer diffusion was maximum in the coronal section followed by the middle and apical sections. It can be attributed to significantly high density of dentinal tubules in the coronal third with a larger diameter in contrast to the middle and the apical sections.[351216] This was consistent with findings by Mjör et al., who stated that tubule density was significantly lower in the middle and apical sections.[17] This phenomenon can be because of the existence of sclerotic dentin, an occluded tubule, and/or the manual variations by the clinician while obturating the canal.[416] For effective bonding and removal of smear layer, a combination of 3 ml of 17% EDTA for 3 min and a 3 ml rinse with 1% NaOCl is adequate.[3] The efficacy of smear layer elimination procedures is decreased nearer to the apical third as compared to the coronal third. Thus, sealer penetration into tubules decreases from the coronal to apical thirds.[57]

Furthermore, the greater depths the spreader extends (to within 0.5–1.0 mm), the use of hand spreaders and the use of semi-rotary motion during compaction may influence the degree of sealer penetration.[4] The lateral compaction forces during obturation are likely greater in the smaller apical region than the coronal third. The rebound or displacement of the compressed gutta-percha by the apical condensing force of the spreader leaves the area without sealer.[418] This result is in unison with the results of Facer and Walton and Allan et al. The authors of these studies reported the absence of sealer in the canal walls on completion of the lateral condensation procedure.[1819]

The sealers penetrate the tubules due to capillary pressure and not due to hydraulic forces formed while restoring. This explains why AH Plus with a longer setting time of 8 h showed considerably deeper penetration than Endoflas F. S. with a setting time of 4 h.[5] The setting time of Resilon in an anaerobic environment is 30 min. However, in case of an aerobic environment, it may extend beyond 3 weeks.[20]

The rheological properties of the AH Plus sealer are influenced by the presence of resin monomers and the particle size of the fillers. The incorporation of bisphenol A and bisphenol F epoxy resins to AH Plus provides it with longer setting time and highest mean flow in comparison to Endoflas F. S. Whereas Endoflas F. S. possesses highest film thickness in comparison to AH Plus. AH Plus contains of finely powdered calcium tungstate and zirconium oxide, with an average particle dimension of 8 μm and 1.5 μm, respectively. The low filler dimension had an important impact on the film thickness of the AH Plus.[8] However, the primer of the Epiphany-Rresilon system acts as a water chaser and draws in the adhesive component of the sealer deeper into dentinal tubules. Given its hydrophilic nature and low film thickness, the primer and sealer can effectively penetrate deeper into dentinal tubules.[3] This advantage is lacking with AH-Plus technique which only relies on its good flow and chemical bonding to dentin to promote penetration into dentinal tubules during lateral compaction.[21] Souza et al. documented that RealSeal (Epiphany) exhibited greater flow when compared to AH Plus.[22] The increased flow is a significant factor for the deeper infiltration of RealSeal (Epiphany) into the dentin. This validates the results of our study.

CONCLUSION

Thus, we conclude that the sealers significantly influence the outcome of an endodontic therapy. Sealers not only exert antibacterial properties but also stop any noncompliant bacteria present in the tubules and render them harmless. This prevents reinfection of the dentinal tubules, thereby influencing the outcome of endodontic therapy. R/E sealer with longer setting time and better flow proved to possess a greater depth of penetration and a greater percentage of depth in comparison to AH-Plus and Endoflas F. S.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.