The effect of natural reducing agents on push-out bond strength of AH plus and BioRoot RCS to sodium hypochlorite treated root dentin.

AIMS: To evaluate the effect of natural anticoagulants 6.5% proanthocyanidin (PA) and 25% bamboo salt on push-out bond strength (PBS) of AH Plus and BioRoot RCS to dentin. SUBJECTS AND METHODS: 30 single-rooted extracted human teeth were collected. After establishing the working length samples were prepared up to size F3. 5 ml of 3% NaOCl was used as irrigant during instrumentation followed by rinse with 5 ml of 17% ethylenediaminetetraacetic acid. Samples were randomly divided into groups based on the final irrigation solution: Group I - AH plus sealer group, Ia - Saline group, Ib - PA group, Ic - Bamboo salt (BS) group. Group II - BioRoot RCS group, IIa - Saline group, IIb - PA group, IIc - BS group. After obturation, samples were embedded in self-cure acrylic resin and 2 mm thick root slices were made at coronal middle and apical 3rd. These slices were subjected to PBS testing followed by stereomicroscopic examination for checking the mode of failure. STATISTICAL ANALYSIS USED: Kruskal-Wallis and Dunn's post hoc test.
RESULTS: 3% NaOCl significantly decreased the bond strength of AH Plus as compared to BioRoot RCS to dentin (P < 0.05). Both PA and BS were capable of increasing the PBS of AH Plus and BioRoot RCS to NaOCl-treated dentin.
CONCLUSIONS: Final irrigation with antioxidants such as PA and BS eliminates the risk of reduced bond strength of sealer to root canal walls, which ensues following the use of NaOCl as an irrigant. Copyright:

INTRODUCTION

The purpose of endodontic therapy is to ensure a fluid tight seal thereby creating a single block configuration which seals the canal space hermetically. Sealers are used to create a seal between the core material and dentinal walls. It should fill imperfections and increase the adaptation of the root filling material to the canal walls.[12]

Adhesion between the root dentin, core material, and the sealer is a desirable outcome of root canal obturation.[2] This aids in reducing coronal and apical leakage, thereby improving the health of the periapical tissues.[3] 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.[4]

The disadvantages of NaOCl include toxicity,[5] promotion of structural changes in organic dentin components (mainly collagen),[6] and its effects on the mechanical properties of dentin, such as reduced flexural strength and the elastic modulus.[78]

AH Plus (Dentsply DeTrey, Konstanz, Germany) is a hydrophobic, epoxy resin-based sealer that is widely used because of its particular physical properties.[9] The adhesiveness of AH Plus to root dentin is based on covalent bonding between the open epoxide ring and the exposed side-chain amine groups of the collagen network.[10] Dentin surface treatments with different irrigation protocols predominantly cause alterations to the collagen fibrils of dentin, which may compromise the adhesiveness of endodontic sealers to the dentin surfaces. Many studies have demonstrated that NaOCl reduces the bond strength between adhesive materials and dentin.[111213] Furthermore, it potentially reduces the bonding strength of AH Plus sealer.[1415] This may be attributable to the presence of residual oxygen species on the dentin, which affects the setting time of the adhesive material.[15]

BioRoot RCS (Septodont, St. Maur-des-Fossés, France) is a calcium silicate-based sealer with a good biocompatibility.[16] The release of calcium hydroxide after hydration and the contact with phosphate from tissue fluids leads to the precipitation of calcium phosphate or calcium carbonate on the surface.[1718] The formation of hydroxyapatite on BioRoot RCS after contact with phosphate-buffered saline solution was reported.[18] Calcium silicates form an interfacial layer at the dentin called the “mineral infiltration zone” with increased mineralization.[1920]

The compromised bond strength of NaOCl-treated dentin could be restored by the application of antioxidants before the adhesive procedure;[21] these agents interact with the by-products of NaOCl,[21] resulting in the neutralization and reversal of the oxidizing effects of NaOCl on the dentin surface.[21] Sodium ascorbate and proanthocyanidins (PA) are some of the most researched antioxidants in dentistry.[21] Naturally derived reducing agents have the ability to chemically modify collagen without damaging biological tissues and moreover, improve dentin matrix properties.[22232425] The application of these provided redox potential as reducing agents for NaOCl-treated dentin, and can potentially improve the quality of collagen modified by NaOCl.

The free radical scavenging ability of PA is well-documented and is shown to be more effective than sodium ascorbate.[26] PAs are naturally occurring plant metabolites, which are a part of the group of polyphenolic compounds known as flavonoids, widely present in grape seed, cranberries, leaves of bilberry and birch, and bark of several trees.[26] PAs from grape seed extract (GSE) are composed primarily of 36% oligomeric and polymeric procyanidins, namely, catechin or epicatechin and <5% flavan-3-ol monomeric catechins.[27]

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.[28] It finds therapeutic application in the prevention and treatment of various diseases such as inflammation, allergies, gastritis, circulatory disorders, and cancers.[29303132] It has been found to be as efficacious as PA in reinstating the reduced bond strength of AH Plus to NaOCl-treated dentin.[33]

Hence, the aim of this in vitro study was to determine the effectiveness of natural anticoagulants 25% Bamboo Salt and 6.5% PA, on the push-out bond strength (PBS) of AH Plus and BioRoot RCS to root canal dentin irrigated with 3% 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 sealers to dentin.

SUBJECTS AND METHODS

Preparation of 6.5% proanthocyanidin solution

About 6.5 g of GSE in the form of powder (Medlife Essentials, Medlife Wellness Retail Private Limited, Bengaluru, India) 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 Bamboo Salt (GOKOCO, Korea) was dissolved in 100 ml of distilled water to make 25% BS solution.

Sample preparation

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 F3. During the preparation of the canal, a total of 5 ml of 3% NaOCl (Septodont Healthcare India Pvt., Ltd., Panvel, India) was used for irrigation between instruments. After instrumentation, a final rinse with 5 ml of 17% ethylenediaminetetraacetic acid (Canalarge, Ammdent, Mohali, India) was done to remove the smear layer. Finally, the canals were irrigated with 5 ml of 3% NaOCl. The samples were divided into two groups (n = 15) based on the type of sealer used for obturation: Group 1 (AH Plus sealer), Group 2 (BioRoot RCS). They are further divided into three subgroups each based on the final treatment protocol: Group 1a (saline), Group 1b (6.5% PA), Group 1c (25% Bamboo Salt), Group 2a (saline), Group 2b (6.5% PA), and Group 2c (25% bamboo Salt). 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 1b, 1c, 2b, and 2c 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 their respective sealers with the aid of a lentulospiral (Dentsply DeTrey, Konstanz, Germany) and obturated using #F3 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.

Pushout 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 2 mm thick slice was obtained from the 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 the 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.[34]

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 Group 1a, 2a was the least at all three levels. 6.5% PA (Group 1b, 2b) and 25% BS (Group 1c, 2c) treated samples showed significantly higher bond strength values compared to Groups 1a and Group 2a (P < 0.05). There was a significant difference between Groups 1 and 2 (P > 0.05). 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 1a, whereas in Groups 1b, 1c, 2a, 2b, and 2c, 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	n	Mean Score±standard deviation			P
		Coronal	Middle	Apical
Group Ia	5	0.34±0.15*	0.68±1.06*	0.53±0.22*	<0.001**
Group IIa	5	2.22±0.73	1.99±0.33	1.16±0.17
Group IIIa	5	1.65±0.34	1.84±0.55	1.42±0.30
Group Ib	5	0.90±0.20	0.82±0.15	0.77±0.22
Group IIb	5	3.75±0.40	3.12±0.30	2.56±0.19
Group IIIb	5	2.64±0.39	2.73±0.45	2.12±0.25

Table 2

Failure modes of all the groups in percentage

Code	Failure mode	Group Ia (%)	Group Ib (%)	Group Ic (%)	Group IIa (%)	Group IIb (%)	Group IIc (%)
A	Adhesive failure at dentinsealer interface	47.76	-	-	-	-	-
B	Adhesive failure at sealer-GP interface	11.5	3.02	6.10	20.18	14.11	16.33
C	Combination adhesive failure showing features of both A and B	7.20	5.78	3.05	6.21	2.33	5.23
D	Cohesive failure involving the sealer	9.33	23.66	32.32	67.20	77.67	73.20
E	Mixed failure involving combinations of A, B, and D	25.38	67.66	58.58	7.02	6.11	6.10

DISCUSSION

The aim of endodontic therapy is not only to eliminate microorganisms by cleaning and shaping the root canal but also to ensure that the root canal system is fluid-free and that a single block configuration is created that seals hermetically the canal space. Because of the poor adhesiveness of gutta-percha, the use of sealers has been considered mandatory.

The major function of a root canal sealer is to fill imperfections and increase adaptation of the root filling material to the canal walls, failing which the chances of leakage and failure increase.[3536] Improved bond strength minimizes the risk of filling detachment from dentin during restorative procedures or the masticatory function, ensuring that seal is maintained and consequently, clinical success of endodontic treatment.[37]

The bond (sealer and canal walls) through frictional retention or micromechanical adhesion may be beneficial in maintaining the integrity of this crucial interface between dentine and cement. The force is applied in apico-coronal direction to avoid interference due to canal taper, during dislodgement of the filling material.[38]

Leakage studies have drawbacks, as does the micro-tensile method, which can result in premature bond failure when cutting the specimens so, in this study, a PBS test was used.[39]

The ability of the push-out test to evaluate the bonding strength surpasses that of other tests because it generates parallel fractures in the interfacial area of dentin bonding. However, a limitation of the push-out test is that it creates nonuniform stress distribution.[40] We prevented this limitation in this study by using 2 mm thick slices.

In the present study, Group 2 (BioRoot RCS) showed the higher bond strength with a statistically significant difference (P < 0.05) than Group 1 (AH Plus) irrespective of the irrigant used. This may be due to its true self-adhesive nature, which forms a chemical bond (through the production of HA during setting) with dentine.

The use of NaOCl decreased the bond strength of AH Plus to dentin more as compare to BioRoot RCS. The results of the present study are in accordance with the previous studies.[414243] 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.[44] 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.[45] Thus, irrigation with NaOCl results in a structurally compromised collagen in the root dentin. Collagen is essential for the adhesion of epoxy resin sealer, as the latter has been shown to chemically bond to the amino groups of dentin.[46] 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.[47] These could be the reasons behind the lower bond strength in Group 1a. This finding was further confirmed by the increased number of adhesive failures at the dentin-sealer interface in Group 1a compared to other groups.

However, the results of this study contrast with another study by Donnenmayer et al. where maximum PBS values were exhibited by AH Plus as compared to BioRoot RCS and GuttaFlow 2 using different final irrigation protocols[48].

Irrigation with 6.5% PA significantly improved the PBS of AH Plus and BioRoot RCS to dentin. Studies have proven that PAs have excellent radical scavenging and antioxidant potential.[49] The multiple electron donor sites of PAs donate hydrogen atoms, thereby binding free radical molecules.[49] This property is directly proportional to the content of flavanols and their degree of polymerization.[50] 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 and BioRoot RCS 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.[51]

Previous studies have shown that significantly higher bond strength is recorded at the coronal and middle thirds of the canal compared to the apical third.[52] 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 and BioRoot RCS to NaOCl-treated dentin. This could be attributed to the antioxidant potential of BS. Om and Jeong studied the 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.[30]

The results correlated well with fracture analysis, with the fractured specimens in Group 1b, 1c, 2a, 2b, and 2c showing no adhesive failures and predominantly mixed failures compared to Group 1a.

BS and PA prove to be effective in bonding an epoxy resin sealer AH Plus and calcium silicate sealer BioRoot RCS to dentin irrigated with NaOCl. Future studies should also aim at the ultramorphological study of root surface irrigated with BS.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.