The effect of three different antioxidants on the dentinal tubular penetration of Resilon and Real Seal SE on sodium hypochlorite-treated root canal dentin: An in vitro study.

OBJECTIVE: The effect of 10% ascorbic acid, 10% tannic acid, and 10% gallic acid on the dentinal tubular penetration of Resilon and Real Seal SE on sodium hypochlorite-treated root canal dentin was evaluated.
MATERIALS AND METHODS: Fifty human premolars were decoronated to attain 14-mm root length and divided into five groups of 10 teeth each. Biomechanical preparation was done with rotary instruments. Group I specimens were irrigated with saline and 17% ethylenediaminetetraacetic acid (EDTA). Specimens from groups II, III, IV, and V were irrigated with 5.25% sodium hypochlorite and 17% EDTA. Specimens from groups III, IV, and V underwent additional irrigation with antioxidants-10% ascorbic acid, 10% tannic acid, and 10% gallic acid, respectively. Following obturation with Resilon and Real Seal SE, scanning electron microscope (SEM) analysis was done to note the maximum dentinal tubular penetration at the cervical, middle, and apical thirds of each specimen. The data were statistically analyzed using one-way analysis of variance (ANOVA), Post hoc and Dunnett's test.
RESULTS: Maximum dentinal tubular penetration of Resilon and Real Seal SE was obtained following irrigation with 10% gallic acid.
CONCLUSION: 10% gallic acid was superior among the antioxidant irrigants that enabled the increased dentinal tubular penetration of Resilon and Real Seal SE.

INTRODUCTION

Successful endodontic treatment outcome depends on the proper diagnosis, access cavity preparation, complete chemomechanical preparation, debridement, and three-dimensional hermetic seal of the root canal system.[12] Since its introduction in 1848, gutta-percha has been the most commonly used root canal obturating material. One of the main disadvantages of gutta-percha is the inability to adhere to the dentinal wall, which can lead to microleakage and failure of the endodontic treatment.[3]

In 2004, a thermoplastic, biodegradable, synthetic polyester root canal obturating material called polycapronolactone was introduced in the market as Resilon, which was to be used with its Epiphany sealer. Resilon, along with Epiphany, was said to create what is called as a “monoblock effect.”[4] Sodium hypochlorite (0.5-6%) is an irreplaceable irrigant in endodontic treatment, which not only eliminates microorganisms but also dissolves proteins and is relatively cheap with a reasonable shelf life.[5] One of the disadvantages of this irrigant is that it interferes with the polymerization of bonding resin including root canal sealers because of residual oxygen left in the dentinal tubules after irrigation. This decreases the bond strength of resin obturating materials with root canal dentin. This can be reversed with the use of antioxidants such as 10% ascorbic acid, which works on the principle of oxidation reduction or redox reactions.[678] Tannic acid is also a known antioxidant with related dental research to prove its ability to increase mechanical properties of the dentin and increase the resin-dentin bond strength.[9] Gallic acid and its esters are hydroxybenzoic derivatives, which are used as antioxidant additives in both the food and pharmaceutical industries.[10]

In this study, three different antioxidants, namely, 10% ascorbic acid, 10% tannic acid, and 10% gallic acid were used to analyze their antioxidant effect on the dentinal tubular penetration of Resilon and Real Seal SE on sodium hypochlorite-treated root canal dentin. The use of antioxidants may increase the bond strength of resin-obturating materials due to enhanced polymerization, which may lead to more dentinal tubular penetration of Resilon and Real Seal SE. This may provide a better seal of the root canal space. Therefore, this study was undertaken. The study hypothesis was that an antioxidant application on sodium hypochlorite-treated root canal dentin would enhance the dentinal tubular penetration of Resilon and Real Seal SE.

MATERIALS AND METHODS

Tooth preparation

Fifty single-rooted mandibular premolars freshly extracted for orthodontic purpose and which were free of cracks, caries, restorations, resorptive defects, and open apices were chosen using a dental operating microscope (Pico, Carl Zeiss, Jena, Thuringia, Germany) at 20x magnification. The teeth were disinfected in 0.5% chloramine-T solution and washed under running water and stored in saline and used within 3 months of extraction. They were decoronated using a diamond disc (SS White, NJ, USA) to attain a standardized 14-mm root length from the apex. The teeth were then randomly divided into five groups of 10 teeth each. The apex of the teeth was sealed with sticky wax (Dental Products of India, Mumbai, Maharashtra, India) to prevent the extrusion of irrigants. Root canal treatment was initiated in all teeth with patency instrument no. 10 K-file (Mani Inc., Utsunomiya, Tochigi, Japan). Working length was established 1 mm short of the apex and confirmed with radiographs. Crown-down technique of instrumentation was performed using HERO Shaper (Micro Mega, rue du Tunnel, Besancon Cedex, France) nickel-titanium (NiTi) rotary instruments. The canals were enlarged till the International Organization for Standardization (ISO) 30 size and 6% taper.

Irrigation protocol for groups

For group I (negative control), irrigation was done with saline (Nirlife, Nirma Ltd., Ahmedabad, Gujarat, India) and 17% ethylenediaminetetraacetic acid (EDTA) (Desmear, Anabond Stedman Pharma Research Ltd., Chennai, Tamil Nadu, India). For groups II (positive control), III, IV, and V, the irrigation protocol followed was 5 mL of 5.25% sodium hypochlorite (Novo Dental Products Pvt. Ltd., Mumbai, Maharashtra, India) between the change of each instrument and 5 mL of 17% EDTA for 1 min after biomechanical preparation. Final irrigation and flushing of the canals were done using distilled water.

Antioxidant preparation

10% ascorbic acid (HiMedia, Mumbai, Maharashtra, India), 10% tannic acid (Sigma-Aldrich, Bangalore, Karnataka, India) and 10% gallic acid (Sigma-Aldrich, Bangalore, Karnataka, India) were prepared by mixing 10 g of powder of the respective antioxidants in 100 mL of distilled water.

Antioxidant application

Root canals of groups III, IV, and V were further irrigated with 10% ascorbic acid, 10% tannic acid, and 10% gallic acid, respectively, for 10 min. This was followed by irrigation with distilled water.

Obturation with Resilon and sealer Real Seal SE

Following irrigation, the canals of the specimens were dried with paper points (Dentsply, Chemin du Verger, Ballaigues, Maillefer, Switzerland). Resilon cones, (SybronEndo Corporation, Orange County, CA, USA) ISO 30 size and 6% taper were used to obturate the canals. Real Seal SE (SybronEndo Corporation, Orange County, CA, USA) was automixed and applied to the root canal with a lentulo spiral (Mani Inc., Tochigi, Japan). The Resilon cones were coated with the sealer and placed into the canals. Lateral condensation was done with ISO 25 size and 2% taper Resilon cones using a 25-size spreader (Mani Inc., Tochigi, Japan). Vertical heat condensation was done with a hot instrument and curing was done for 40 s with a light-emitting diode (LED) light (Dentsply, Milford, Detroit, MI, USA) of intensity 500 mw/cm2. Radiographs were taken to assess the quality of obturation. The canal orifices were sealed with sticky wax. All the specimens were stored in 100% relative humidity at 37°C for 24 h to ensure a complete set of the obturating material.

Scanning electron microscope analysis

The analysis of the scanning electron microscope (SEM) samples was nonblinded. The samples were sectioned longitudinally with the help of a diamond disc in a micromotor handpiece (NSK, Tochigi, Japan) under copious irrigation with distilled water in such a way that one portion retained the obturating material. The root sections that retained the obturating material were soaked in 17% EDTA solution for 10 min followed by soaking in 5.25% sodium hypochlorite for 10 min and then washed thoroughly with distilled water. The sections were dehydrated by placing them sequentially in 50%, 75%, and 100% ethyl alcohol for a total of 8 h and left overnight in a drying chamber maintained at 60°C. They were gold sputtered (No. E-1010 Ion sputter, Hitachi, Japan) for SEM evaluation at the cervical, middle, and apical thirds. A SEM (NO. S-2400, Hitachi, Omeshi, Tokyo, Japan) was used at 500x magnification [Figure 1].

Figure 1

Scanning electron microscope (SEM) images of the cervical, middle, and apical thirds of the root (500×): 1a, 1b, 1c (10% ascorbic acid irrigation), 2a, 2b, 2c (10% tannic acid irrigation), 3a, 3b, 3c (10% gallic acid irrigation)

Evaluation of Resilon and Real Seal SE penetration

The samples were evaluated for dentinal tubular penetration of Resilon and Real Seal SE at three levels — cervical, middle, and apical portions of the root. The maximum tubular penetration at each level was recorded in micrometers (μm). At each level, five points were marked corresponding to the maximum penetration. The distance to these points was measured from the dentin-obturating material interface and the depth of penetration was calculated. The mean of the five readings was taken at each level and tabulated.

Statistical analysis

Data entry was done in Microsoft Excel. The values obtained were statistically analyzed using computer software Statistical Package for the Social Sciences (SPSS) (16.0) (SPSS Inc, Chicago, USA). The data were expressed with the mean and standard deviation. One-way analysis of variance (ANOVA) was applied for statistical analysis. Post hoc test followed by Dunnett's test was used to find the statistical significance between the groups. P value less than 0.05 (P < 0.05) was considered to be statistically significant at 95% confidence interval.

RESULTS

Maximum dentinal tubular penetration value was observed in group V at all the three levels, i.e., cervical, middle, and apical thirds of the root and was statistically significant compared to the other groups (P < 0.05). Minimum dentinal tubular penetration value was observed in group II at all the three levels and was statistically significant compared to the other groups (P < 0.05). The mean dentinal tubular penetration value of group I was statistically significant compared to the other groups (P < 0.05). The mean value of group III was statistically significant compared to the other groups (P < 0.05). The mean value of group III (cervical and apical thirds) was not statistically significant compared to group IV (cervical and apical thirds) (P > 0.05). The mean value of group III (middle third) was statistically significant compared to group IV (middle third) (P < 0.05). The mean value of group IV was statistically significant compared to all the groups except group III (cervical and apical thirds) (P < 0.05) [Table 1].

Table 1

Multiple comparison of mean dentinal tubular penetration values (in micrometers) among the groups at the cervical, middle, and apical thirds

DISCUSSION

The teeth specimens treated with the antioxidants showed deeper tubular penetration of Resilon and Real Seal SE at the cervical, middle, and apical thirds of the root canal dentin. This confirmed our study hypothesis that an antioxidant application following sodium hypochlorite irrigation on root canal dentin enhances the dentinal tubular penetration of Resilon and Real Seal SE.

A concentration of 10% of the three antioxidants was used to irrigate the root canals of the respective groups for a time period of 10 min. In the studies conducted by Manimaran et al. and Shreshta et al., 10% ascorbic acid and 10% sodium ascorbate solutions, respectively, were used as irrigants for 10 min to counter the effects of sodium hypochlorite on the root canal dentin.[68] Bedran-Russo et al. demonstrated that 10% tannic acid irrigation for 10 min can improve the elastic modulus and bond strength of demineralized dentin.[9]

The teeth sections were soaked in 17% EDTA and 5.25% sodium hypochlorite alternatively for 10 min to remove the smear layer formed during sectioning. An SEM was chosen for evaluation as it allows a highly descriptive and detailed observation of the dentinal tubules and the obturating material and the penetration depth could be calculated with great accuracy throughout the section.[11]

Ascorbic acid is a naturally produced water soluble organic compound. It is a potent antioxidant capable of quenching reactive free radicals in biological systems. Ascorbic acid and its salts such as sodium ascorbate can neutralize the adverse effects of oxidative solutions via redox reactions.[12] The increased dentinal tubular penetration of Resilon and Real Seal SE following the use of 10% ascorbic acid may be due to the fact that this antioxidant with its radical scavenging activity has the ability to remove residual oxygen and reverse the effect of sodium hypochlorite on root dentin, thus leading to better sealer penetration into the tubules. This was in accordance with the findings of the study by Chadha et al.[11] A study by Morris et al. has shown that 10% ascorbic acid and 10% sodium ascorbate can reverse the action of 5% sodium hypochlorite and RC prep on the resin-dentin bond strength.[13] Gonulol et al. has also proved that a 10% sodium salt of ascorbic acid increased the bond strength to root dentin when it was used following oxygen-releasing irrigants such as sodium hypochlorite and hydrogen peroxide.[14]

Tannic acid is a polyphenol and a weak acid. It is used as an antioxidant, astringent, desensitizing agent, and also as surface treatment for the removal of smear layer. It is a naturally occurring cross-linking agent comprising a complex mixture of polygalloyl glucose esters.[9]

Following the use of tannic acid as an irrigant, better penetration of Resilon and Real Seal SE into the dentinal tubules could be attributed to the antioxidant potential of tannic acid whereby residual oxygen generated by sodium hypochlorite is removed. According to Bedran-Russo et al., tannic acid improved the mechanical properties (elastic modulus) of demineralized dentin as well as the bond strength of resin-dentin interface.[9]

Gallic acid is a naturally occurring phenolic acid found in green tea, red wine, grape seed, gall nuts, sumac, witch hazel, tea leaves, oak bark, and other plants. It has a primary antioxidant activity and also has anticarcinogenic potential, antimicrobial activity, and cholesterol reduction capability.[15161718] The redox potential of gallic acid depends on the number of hydroxyl moieties that are attached to the aromatic ring of the benzoic acid molecule. Maximum dentinal tubular penetration of Resilon and Real Seal SE following 10% gallic acid irrigation may be due to the fact that gallic acid with three hydroxyl groups is a very active phenolic acid with highest radical scavenging activity in the group of phenolic acids.[19] The redox potential of gallic acid may help to remove the residual oxygen following the use of sodium hypochlorite and thus, enhance dentinal tubular penetration of Resilon and Real Seal SE. However, no studies on gallic acid as an irrigant for root canal treatment have been reported.

Since in this study it was found that irrigation with 10% gallic acid resulted in maximum dentinal tubular penetration of Resilon and Real Seal SE, this may be used as an antioxidant irrigant following the use of sodium hypochlorite, which may improve the bonding between resin obturating material and root dentin.

A number of factors affect the success of endodontic treatment, especially when obturation is based on technique-sensitive adhesive procedures. Moreover, since the antioxidant irrigants are acidic in nature, they may further weaken the sodium hypochlorite-treated root dentin. This being an in vitro study, it cannot absolutely mimic the in vivo conditions of the oral cavity Secondary tests and clinical trials are the ultimate in deciding the outcome of in vitro studies. Hence, further clinical trials are necessary to substantiate the results and findings.

CONCLUSION

Within the limitations of this in vitro study it was observed that among the antioxidant irrigants, 10% gallic acid was superior in enhancing the dentinal tubular penetration of Resilon and Real Seal SE on sodium hypochlorite-treated root canal dentin.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.