Effect of proanthocyanidin and bamboo salt on the push-out bond strength of an epoxy resin sealer to sodium hypochlorite-treated root dentin: An in vitro study.

BACKGROUND: In endodontic therapy, final irrigation is often done with sodium hypochlorite (NaOCl). This jeopardizes the bond strength between the epoxy resin sealer, used subsequently in obturation and radicular dentin. This study aimed to analyze the effect of natural antioxidants, 6.5% proanthocyanidin (PA) and 25% bamboo salt (BS) on the reversal of NaOCl-induced reduced bond strength of an epoxy resin sealer to dentin.
MATERIALS AND METHODS: Thirty-three single-rooted extracted human teeth were randomly divided into three groups based on the final irrigation protocol: group 1 (saline), Group 2 (6.5% PA), and Group 3 (25% BS). The canals were cleaned, shaped, and obturated with gutta-percha and AH Plus sealer. 1.5 mm-thick root slices made from coronal, middle, and apical thirds of the canal were subjected to push-out bond strength (PBS) testing. The data were statistically analyzed using Kruskal-Wallis and Dunn's post hoc test (P < 0.05).
RESULTS: 5.25% NaOCl significantly decreased the bond strength of AH Plus to dentin (P < 0.05). Both 6.5% PA and 25% BS were capable of reversing the compromised PBS of AH Plus to NaOCl-treated dentin.
CONCLUSION: Final irrigation with antioxidants such as PA and BS eliminates the risk of reduced bond strength of AH Plus to root canal walls, which ensues following the use of NaOCl as an irrigant.

INTRODUCTION

Adhesion between the root dentin, core material, and the sealer is a desirable outcome of root canal obturation.[1] This aids in reducing coronal and apical leakage, thereby improving the health of the periapical tissues.[2] Cleaning and shaping of the root canal involve the use of chemical substances to disinfect the canal as well as to remove the smear layer, to make it more receptive to the obturating material. Sodium hypochlorite (NaOCl) is a widely used root canal disinfectant. It is effective in dissolving the organic component of the smear layer.[3] However, remnants of this strong oxidizing agent or its oxidative by-products such as hypochlorous acid and hypochlorite ions are reported to inhibit the free radical polymerization of methacrylate resins and adhesion of epoxy resins used in the canal.[45] This eventually compromises the bond strength of the sealer to root dentin and hinders its sealing ability.[6]

Studies have shown that pretreatment of the root canal with antioxidants or neutralizing agents before adhesive bonding of resin sealers is capable of reversing the NaOCl-induced reduction in bond strength.[789] Sodium ascorbate and proanthocyanidins (PA) are some of the most researched antioxidants in dentistry.[1011] The free radical scavenging ability of PA is well-documented and is shown to be more effective than sodium ascorbate.[11] PAs are naturally occurring plant metabolites, which are a part of group of polyphenolic compounds known as flavonoids, widely present in grape seed, cranberries, leaves of bilberry and birch, and bark of several trees.[11] PAs from grape seed extract (GSE) are composed primarily of 36% oligomeric and polymeric procyanidins, namely, catechin or epicatechin and <5% of flavan-3-ol monomeric catechins.[12]

Bamboo salt (BS) is one such natural product with antimicrobial, antioxidant, and anti-inflammatory properties. It is obtained by packing and repeatedly (nine times) roasting sun-dried salt, in bamboo trunks sealed by yellow soil, using pinewood and pine resin as fuel. BS contains a wide array of macro- and micro-nutrients (B, Fe, Sr, Cu, V, and Mn) and trace elements. It is composed of 29.3% Na, 1.4% K, 0.4% S, 0.1% Ca, and 0.08% Mg as macronutrients.[13] It finds therapeutic application in the prevention and treatment of various diseases such as inflammation, allergies, gastritis, circulatory disorders, and cancers;[14151617] whether it is capable of reversing NaOCl-induced reduction of root dentin-resin sealer bond strength is not yet known. Hence, the aim of this in vitro study was to determine the effectiveness of 25% BS in comparison with 6.5% PA, on the push-out bond strength (PBS) of AH Plus, an epoxy resin sealer to root canal dentin irrigated with 5.25% NaOCl. The null hypothesis was that neither the antioxidant agent applied nor the different regions of the canal (coronal, middle, or apical) would influence the PBS of the sealer to dentin.

MATERIALS AND METHODS

Preparation of 6.5% proanthocyanidin solution

About 6.5 g of GSE in the form of powder (Puritans Pride Inc., Oakdale, NY, USA) was collected from the capsules and dissolved in 100 ml of distilled water to make 6.5% PA solution.

Preparation of 25% bamboo salt solution

About 25 g of BS (Koreasalt Co. Ltd., Gyeongsangnam, Korea) was dissolved in 100 ml of distilled water to make 25% BS solution.

Sample preparation

Human teeth samples were collected in conformation with the provisions of the Declaration of Helsinki. Thirty-three teeth extracted for periodontal and/or orthodontic reasons with single roots and single canals were collected and stored in distilled water until use. The teeth were decoronated at the cementoenamel junction and standardized to a length of 12 mm with the help of a low-speed diamond disk under water spray. Patency of each root canal was checked using a K-file (#15) (Mani Inc., Tochigi, Japan) and working length was established 1 mm short of the apex. Cleaning and shaping were performed by crown-down technique, using ProTaper rotary shaping and finishing files (Dentsply, Maillefer, Ballaigues, Switzerland), up to size F4. During the preparation of the canal, a total of 5 ml of 5.25% NaOCl (Nice Chemicals (p) Limited, Kerala, India) was used for irrigation between instruments. After instrumentation, a final rinse with 5 ml of 17% ethylenediaminetetraacetic acid (EDTA) (RC Help, Prime Dental Products, Thane, India) was done to remove the smear layer. Finally, the canals were irrigated with 5 ml of 5.25% NaOCl. The samples were divided into three groups (n = 11) based on the final treatment protocol: group 1 (saline), Group 2 (6.5% PA), and Group 3 (25% BS). The canals were flushed with their respective solutions, keeping a standardized quantity and contact time of 5 ml and 5 min, respectively. The root canals of the samples in Groups 2 and 3 were additionally rinsed with 5 ml of distilled water and the canals of all the samples were dried with paper points (Dentsply, Maillefer, Ballaigues, Switzerland). The canals were coated with AH Plus sealer (Dentsply, De Trey, Konstanz, Germany) with the aid of a lentulospiral (Mani Inc., Tochigi, Japan) and obturated using #F4 gutta-percha (Dentsply DeTrey, Konstanz, Germany). The root samples were coronally sealed with intermediate restorative material (IRM, Dentsply DeTrey, Konstanz, Germany) and were stored in distilled water for 7 days.

Push-out bond strength testing

A cylindrical mold was filled with autopolymerizing acrylic resin and the roots were vertically embedded in it, until the acrylic resin set. Acrylic blocks were removed from the mold, and 1.5 mm thick slice was obtained from coronal, middle, and apical third of each embedded root sample using a rotating diamond disk under water-cooling. Each slice was subjected to PBS test in a universal testing machine (Tecsol, Chennai, India) using a metallic indentor with a round cross-section and diameters of 0.5 mm, 0.7 mm, and 0.9 mm customized to test slices from apical, middle, and coronal thirds, respectively. A crosshead speed of 1 mm/min was used. The fractured samples were viewed under a stereomicroscope at 40× magnification to analyze the mode of failure. The classification of failure modes was adopted from Jainaen et al.[18]

Data and statistical analysis

The data were tabulated and statistically analyzed using SPSS Statistics V22.0 (IBM, USA). The PBS data were subjected to Kruskal–Wallis and Dunn's post hoc test. The significance was set at P < 0.05.

RESULTS

The mean and standard deviation of the PBS of all the groups at three different levels of the canal (in MPa) is given in Table 1. The PBS of samples in control group (Group 1) was the least at all the three levels. 6.5% PA (Group 2) and 25% BS (Group 3) treated samples showed significantly higher bond strength values compared to Group 1 (P < 0.05). There was no significant difference between Groups 2 and 3 (P > 0.05). Intragroup comparisons showed no significant difference in Groups 1 and 3 (P > 0.05), whereas in Group 2, the PBS values were significantly lesser at the apical third, compared to coronal and middle thirds of the root (P < 0.05). The failure modes of the samples are given in Table 2. Adhesive failure at the dentin-sealer interface was the predominant mode of failure in Group 1, whereas in Groups 2 and 3, mixed failures were mostly seen.

Table 1

Mean±standard deviation of the push-out bond strength of all the groups at different levels of the canal (MPa)

Groups	Level of root
	Coronal	Middle	Apical
1 (5.25% NaOCl)	0.34±0.15*	0.68±1.06*	0.53±0.22*
2 (6.5% PA)	2.22±0.73	1.99±0.33	1.16±0.17#
3 (25% BS)	1.65±0.34	1.84±0.55	1.42±0.30

*Intergroup comparison denotes significantly lesser PBS in Group 1 compared to Groups 2 and 3 (P<0.05), #Intragroup comparison denotes significantly lesser PBS at apical third compared to coronal and middle thirds of the root in Group 2 (P<0.05). NaOCl: Sodium hypochlorite, PA: Proanthocyanidin, BS: Bamboo salt, PBS: Push-out bond strength

Table 2

Failure modes of all the groups in percentage

Code	Failure mode	Groups
		1 (5.25% NaOCl)	2 (6.5% PA)	3 (25% BS)
A	Adhesive failure at dentin-sealer interface	48.48% (16)	-	-
B	Adhesive failure at sealer-GP interface	12.12% (4)	3.03% (1)	6.06% (2)
C	Combination adhesive failure showing features of both A and B	6.06% (2)	6.06% (2)	3.03% (1)
D	Cohesive failure involving the sealer	9.09% (3)	24.24% (8)	33.33% (11)
E	Mixed failure involving combinations of A, B, and D	24.24% (8)	66.66% (22)	57.57% (19)

NaOCl: Sodium hypochlorite, PA: Proanthocyanidin, BS: Bamboo salt, PBS: Push-out bond strength, GP: Gutta-percha

DISCUSSION

A controversy exists regarding the filling of canals with either the sealer or sealer/core material in PBS studies. Few authors support their decision to use only the sealer while testing as it eliminates a dual interface that could arise if a core material is used. Another reason suggested is that this technique produces a better seal in the canal, as epoxy resins, especially do not shrink; rather they expand and seal the canal. However, in a clinical scenario, the canals are always filled with a core material, as obturating with resin sealer alone makes retreatment of such canals difficult.[16] Hence, in the present study, to closely mimic the clinical scenario, gutta-percha was used in combination with AH Plus to fill the canals. The varying diameters of indenters used matched the differing lumen diameters of specimens from three levels of the canal.

The use of NaOCl decreased the bond strength of AH Plus to dentin. The results of the present study are in accordance with the previous studies.[4567] The use of NaOCl during instrumentation removes the organic debris from the canal. In due course, it also causes degradation of root dentin collagen. On contact with organic matter in the root canal, NaOCl breaks down into chloramines and protein-derived radical intermediates.[19] Daumer et al. observed that these breakdown products are capable of having an adverse effect on the pyridinoline cross-links occurring in type I collagen.[20] Thus, irrigation with NaOCl results in a structurally compromised collagen in the root dentin. Collagen is essential for adhesion of epoxy resin sealer, as the latter has been shown to chemically bond to the amino groups of dentin.[21] This adhesion mechanism is hindered when NaOCl is used as a final rinse before the use of AH Plus in the canal. Another logical reason is that the oxygen bubbles formed following the use of NaOCl prevent the penetration of the epoxy resin sealer into the fine apertures of dentinal tubules.[6722] These could be the reasons behind the significantly lower bond strength in Group 1 (control). This finding was further confirmed by the increased number of adhesive failures at the dentin-sealer interface in Group 1 compared to Group 2 and 3.

Irrigation with 6.5% PA significantly improved the PBS of AH Plus to dentin. Studies have proven that PAs have excellent radical scavenging and antioxidant potential.[823] The multiple electron donor sites of PAs donate hydrogen atoms, thereby binding free radical molecules.[11] This property is directly proportional to the content of flavanols and their degree of polymerization.[24] The pH of GSE solution used in this study is 6. This slightly acidic pH could have also improved the bond strength of AH Plus to dentin. These could be the reasons behind the enhanced bond strength seen in specimens irrigated with PA. The results are in accordance with several other studies.[825] A first of its kind study by Manimaran et al. showed that 5% GSE could significantly improve the bond strength of resin cement to NaOCl-treated root dentin. They also showed that PAs were more effective than sodium ascorbate.[8] Drawing conclusions from their study, in the present study, only PAs were used for comparative purposes and not sodium ascorbate.

The collagen cross-linking ability of PAs could be another possible reason behind the improved bond strength. The proline-rich proteins present in collagen have high affinity for PAs and form strong hydrogen bonds with them, resulting in increased inter- and intra-molecular crosslinks.[2627] This helps in stabilizing the collagen, which is essentially exposed during irrigation of the canal using EDTA. Kalra et al. studied the effect of PAs on the biodegradation resistance of demineralized root dentin and the bond strength of a resin sealer to dentin. They observed that PAs enhance biodegradation resistance and they also maintain the interfacial seal between resin sealer and root dentin.[25]

Ethanol and acetone are commonly used as solvents in the extraction of PAs from GSE.[28] In dentistry, they find the use as dentin-cleaning agents and water chasers in bonding agents. Sarac et al. observed that these solvents could effectively be used to remove water from the dentin surface and facilitate better penetration of resin into the dentin. This could have also enhanced the action of PAs on the root dentin.[29]

Previous studies have shown that significantly higher bond strength is recorded at the coronal and middle thirds of the canal compared to apical third.[6830] Such a pattern was observed only in specimens irrigated with PAs. In control and BS groups, no significant difference could be observed in intragroup comparisons. Hence, the null hypothesis stands rejected, as both the antioxidant applied and the varying regions of the canal influence the PBS of the sealer to dentin.

The results of the current study showed that no significant difference existed between PA- and BS-treated specimens. BS was as efficacious as PA in reinstating the reduced bond strength of AH Plus to NaOCl-treated dentin. This could be attributed to the antioxidant potential of BS. Om and Jeong studied superoxide dismutase activity of BS and inferred that BS was a potent antioxidant and it inhibited ROS formation. The efficacy of BS was found to be 4.5–7.2 times higher than table salt and 3.3–7.1 times higher than Vitamin E, a known antioxidant.[15] Zhao et al. (2012) studied the antioxidant activity of BS using DPPH assay. They observed that 25% BS exhibited superior radical scavenging ability (81.4%) than solar salt (5%) and purified salt (2%). BS has a pH of 11.4. The abundance of available – OH groups and the alkaline pH of BS contribute to its higher reduction potential.[31] The high concentrations of minerals such as iron, silicon, potassium, calcium, magnesium, and manganese present in BS also confer it with antioxidant properties.[16] These could be the reasons behind the improved bond strength seen in BS-treated specimens.

The results correlated well with fracture analysis, with the fractured specimens in Group 2 and 3 showing no adhesive failures and predominantly mixed failures compared to the control (Group 1). Jainaen et al. studied the PBS of three resin sealers to dentin in root canals filled with and without a main cone. They observed that the failure pattern of sealer was influenced by the presence or absence of a main cone. Cohesive failure of the sealer was observed in canals filled using a main cone, whereas adhesive failure at the dentin-sealer interface was predominant in specimens, which were filled only with sealer. When placed as thin film, like in the case of sealer in combination with gutta-percha, the resin part of the sealer penetrates the tubules extensively. This leaves behind a weak, resin depleted and filler particle-enriched layer, which are too big to penetrate the tubules, thus making this thin film susceptible to cohesive failure.[18] Similar findings were observed in PA- and BS-treated groups in this study, wherein an increased number of cohesive failures were seen in the sealer layer.

Surface tension (ST) of irrigants used influences their penetration into the root canal. A lower ST ensures superior contact and wetting of the root canal by irrigants. ST of GSE is 48.4 mJ/m2, which is closer to that of NaOCl (49 mJ/m2) and EDTA (46.8 mJ/m2).[3233] However, the ST of water is 72 mJ/m2, which further increases linearly with the addition of inorganic salts.[34] Thus, the ST of BS is expected to be higher than water. This might serve as a deterrent in achieving complete penetration of BS into the root canal and adequate wetting of the dentinal surface. However, this warrants further studies.

BS and PA prove to be effective in bonding an epoxy resin sealer to dentin irrigated with NaOCl; whether these antioxidants have a similar effect on methacrylate resin-based sealers needs to be evaluated. Future studies should also aim at the ultramorphological study of root surface irrigated with BS.

CONCLUSION

Within the limitations of this in vitro study, it can be concluded that:

Use of NaOCl as a final irrigant significantly decreases the dislocation resistance of AH Plus to root canal dentin

Use of PA and BS as final irrigating solutions reverses the compromised PBS of AH Plus to NaOCl-treated dentin

BS was as good as PA in improving the adhesion of epoxy resin sealer to root dentin.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.