Evaluation of effect of addition of 2% chlorhexidine on the sealing ability of Biodentine: An in vitro study.

AIM: To evaluate the effect of the addition of 2% chlorhexidine on the sealing ability of Biodentine.
MATERIALS AND METHODS: Forty-six extracted human premolar teeth with single canal and apical foramen were selected, cleaned, and decoronated to standardize the length of 17 mm. Canals were prepared using ProTaper rotary files till size F4. The samples were divided into 2 experimental groups of 20 samples each on the basis of absence/presence of 2% chlorhexidine in liquid: Group BM = Biodentine mixed with the provided liquid, Group BC = Biodentine mixed with 2% chlorhexidine in provided liquid. Three samples, each were assigned to control groups: Group BP (positive control) = No root end filling was placed, Group BN (negative control) = Root ends were filled as in Group BM, and entire external surface was coated with sticky wax. The samples were then evaluated for the apical sealing using fluid filtration method.
RESULTS: Results were analyzed using Student's t-test (P ≤ 0.05). Group BC showed the better sealing ability (3.06) as compared to Group BM (3.85). However, the difference was statistically insignificant (P > 0.05).
CONCLUSION: Addition of 2% chlorhexidine to the liquid enhanced the sealing ability of Biodentine.

INTRODUCTION

Imperfect apical seal has been quoted as the most frequent (75%) cause of failing endodontic cases.[1] Cases where conventional root canal therapy alone fails to achieve fluid tight seal of the root canal, a surgical approach is the only reasonable alternative to extraction. Sealing ability refers to the materials ability to resist micro-leakage through the entire thickness of the material. Root end fillings seal the contents of root canal system within the canal and prevent the ingress of microbes and their byproducts, or toxic materials into the surrounding periradicular tissues. Over the years, a large number of materials have been tested as retro-filling materials for their ability to three-dimensionally seal the root canal. Amalgam,[23] composite resin,[3] glass-ionomer cements (GIC),[24] zinc oxide eugenol cements, mineral trioxide aggregate (MTA),[234] and many other dental materials have been tried and tested as root end endodontic filling materials. However, to date no material has been found to satisfy all the requirements of an ideal root-end filling material.

Biodentine with active biosilicate technology was introduced by Septodont as a calcium silicate-based dentin replacement material that is bioactive, biocompatible, and has excellent sealing properties.[5] Septodont claims that Biodentine has features as an endodontic repair material that are superior to MTA. In addition, it has better consistency, handling characteristics, and faster setting time.[6]

Ravi Chandra et al.[7] compared the marginal adaptation of Bio dentine, MTA, and GIC and found the lowest marginal gaps in Bio dentine compared to MTA and GIC. Similarly, Kokate and Pawar,[8] Radeva et al.[9] and Sulaiman[10] also concluded that Biodentine has the better sealing ability.

A study conducted by Valyi et al.[11] concluded that the antibacterial activity of Biodentine is although comparable to other Ca-based cement, it is more dependent on strain type. In addition to a recent study, it was observed that adding 2% chlorhexidine gluconate in liquid of Biodentine enhanced the antimicrobial activity of Biodentine.[12] To the best of our knowledge, no study so far has been designed to determine the effect of combining chlorhexidine on sealing ability of Biodentine.

Techniques advocated for detection and evaluation of micro leakage around the root end filling material are dye penetration,[2] scanning electron microscope,[13] electrochemical,[14] bacterial penetration method,[3] fluid filtration,[1516] and glucose penetration model[15] are currently used for leakage tests. Among the different methods, fluid filtration method has certain advantage over other methods which includes that it is nondestructive, it does require the addition of a tracer molecule and repeated measurements can be performed over a period of time.

Therefore, the purpose of this study was to evaluate the effect of the addition of 2% chlorhexidine on the sealing ability of Biodentine when used as root-end filling material using fluid filtration method. The null hypothesis was that there is no difference in the sealing ability of Biodentine to the root canal walls when 2% chlorhexidine is added.

MATERIALS AND METHODS

Freshly extracted human premolars with single canal, and apical foramen were collected and stored in 0.1% thymol solution. The storage period was not more than 3 months.

Canal number and curvature (0-10°) were verified by exposing each tooth to X-ray in two directions, mesiodistally, and faciolingually utilizing RVG software (Gendex, Visualix, USA). Canal curvature was measured by the method described by Schneider.[17]

The samples were then decoronated to obtain a standardized length of 17 mm. Working length was determined by viewing the tip of #10 K-file at the apical foramen and then subtracting 1 mm from the length. Biomechanical preparation was carried out using ProTaper universal rotary instruments (Dentsply Malliefer, Ballaigues, Switzerland) till size F4 ProTaper using 2 mL of 3% sodium hypochlorite solution and 2 mL of 17% EDTA (AMMDENT, Mohali, India) alternatively after each file.

Apical 3 mm of the roots were resected perpendicular to the long axis of the tooth with a high-speed tapered fissure bur (TR-13, Mani, Japan) under water. A master Gutta-percha cone was inserted (without sealer) into the canal and pulled through the resected root surface until tug-back was achieved. The apically extruded Gutta-percha was sectioned. A 3 mm deep root-end preparation was made by using a straight fissure bur (SF-41, Mani, Japan) of 1 mm diameter. The final diameter of the preparation was determined by the radius of the tip of the straight fissure diamond bur. The prefitted Gutta-percha cone served as a barrier for the condensation of the root end filling materials. All the preparations were washed with distilled water for 5 s and then excess moisture was removed with paper points. The samples were randomly divided into two experimental groups each containing 20 samples and 2 positive and 2 negative control groups each consisting of three samples.

Group BM (n = 20): Biodentine mixed with the provided liquid as per manufacturer's instructions.

Group BC (n = 20): Biodentine was mixed as per manufacturer's instructions with 2% chlorhexidine in provided liquid.

Group BP (n = 3): Root end preparations were left empty. These samples were used as positive control.

Group BN (n = 3): Root end preparations were filled as in Group BM and their entire external root surface were covered with 3 coats of nail polish and sticky wax. These samples served as negative control.

The volume of liquid provided with Biodentine (Septodont, Saint Maur des Fosses, France) is 5 drops (0.3 ml). Thus to make 2% chlorhexidine solution, 6 mg of chlorhexidine gluconate powder was mixed with the 0.3 ml liquid. Samples were then wrapped in moist cotton and stored in airtight bottles for 24 h at 37°C to allow the materials to set. After that, the Gutta-percha was removed from each root canal. All the external surfaces of teeth except for 1 mm around the retro-filling materials and canal orifice area were covered with 3 coats of nail polish. The sample was fixed in fluid filtration model, and the apical leakage was evaluated.

Fluid filtration method

The apparatus consisted of a two-neck bottle with openings: One for gas (oxygen) and other for a micropipette with a two-way tube (internal diameter 1 mm). The oxygen cylinder and micropipette were connected to the two-neck bottle with glass tubes. The glass tube connecting micropipette was immersed in distilled water placed in the two-neck bottle while the other glass tube connecting oxygen cylinder was fixed above the fluid level. The three-way tube provided the attachment for the tooth sample and two syringes. Test samples were mounted on a fluid transport model one at a time. Coronal end of each root was then connected to the filtration apparatus by cyanoacrylate and parafilm. All pipettes, syringes, and the glass tubes of the device were filled with distilled water. Using a syringe, water was sucked back into the open end of the glass capillary, and an air bubble was created. A headspace pressure of 0.07 kg/cm2 was supplied from the inlet side, through the micropipette to the coronal end of the canal, through the voids along the filling, thus displacing the air bubble in the capillary tube. After 3 h, the movement of an air bubble was recorded at 2 min intervals for 8 min (2 min, 4 min, 6 min, and 8 min) for every sample. The linear movement of an air bubble in the micropipette was measured with a digital vernier caliper. The mean of the four readings was calculated and considered.

RESULTS

The data thus collected was statistically analyzed using Student's t-test.

According to fluid filtration test, Group BN (negative group) showed zero leakage as no movement of air bubble was observed, while in Group BP (positive group), the air bubble moved away from the ruler in <1 s. Experimental groups (Group BC and Group BM) showed significantly more leakage than negative group (Group BN) and less than positive control (Group BP). The mean value of the leakage of Group BC was less leakage as compared to Group BM [Table 1]. However, the results were not statistically significant (P > 0.05).

Table 1

Amount of microleakage in experimental groups and analysis of variance for amount of microleakage among different groups

DISCUSSION

In the present study, addition of 2% chlorhexidine enhanced the sealing ability of the Biodentine, thus the null hypothesis was rejected. Biodentine (Septodont, Saint-Maur-des-Fosses, France) consists of a powder in a capsule, and liquid in a pipette. The powder mainly contains tricalcium and dicalcium silicate, the principal component of Portland cement, as well as calcium carbonate. Zirconium dioxide serves as a contrast medium. The liquid consists of calcium chloride (1.2%) in aqueous solution with an admixture of polycarboxylate.

2% chlorhexidine is a disinfecting agent that is effective against a variety of micro-organisms, including Enterococcus faecalis.[8] Nikhil et al.[12] observed that the addition of 2% chlorhexidine gluconate in liquid of Biodentine enhanced the antimicrobial activity of Biodentine against all the tested micro-organisms (Staphylococcus aureus, E. faecalis and Streptococcus mutans) except Candida albicans. Although 2% chlorhexidine increased the antimicrobial activity of Biodentine, but its effect on the sealing ability required investigation. Hence, the present study was planned to evaluate the sealing ability of Biodentine when mixed with 2% chlorhexidine solution of provided liquid using fluid filtration method.

Traditional method of micro leakage assessment requires the sectioning of the samples, thus associated with loss of some of the tooth structure due to the thickness of cutting blade, and the sectioning process, which ultimately affects the accuracy of the measurements of the dye penetration. Fluid filtration studies present the several advantages over such traditional micro leakage studies.[15] The samples are not destroyed, allowing repeated measurements over a period of time. In fluid filtration technique, no tracer is needed with the related problem of molecular size and affinity for dentin and no intricate materials are required as in the bacterial penetration studies or radioactive tracer studies. However, the fluid filtration technique is not able to discern the root canal level at which the microleakage is located.

In the present study, fluid filtration model similar to that given by Javidi et al.[18] was used. The duration of application of pressure was 3 h per sample as given by Shemesh et al.[15] The fluid conductive system was used at a hydraulic pressure of 6.9 kPa (0.07 kg/cm2) to measure the bubble movement, which is in accordance with other studies.[16]

The proposed mechanism of action of Biodentine is that the calcium silicate interacts with water leading to the setting and hardening of the cement. This hydration of the tricalcium silicate (3CaO.SiO2=C3S) produces a hydrated calcium silicate gel (CSH gel), and calcium hydroxide (Ca(OH)2). Such dissolution process occurs at the surface of each grain of calcium silicate. The hydrated calcium silicate gel and the excess of (Ca(OH)2) tend to precipitate at the surface of the particles and in the pores of the powder, due to the saturation of the medium. This precipitation process is reinforced in systems with low water content. The unreacted tricalcium silicate grains are surrounded by the layers of calcium silicate hydrated gel, which are relatively impermeable to water, thereby slowing down the effects of further reactions. The hydrated calcium silicate gel formation is due to the permanent hydration of the tricalcium silicate, which gradually fills in the spaces between the tricalcium silicate grains. The hardening process results from the formation of crystals that are deposited in a supersaturated solution.[19]

The addition of chlorhexidine increased the sealing ability of Biodentine, but the difference was statistically insignificant (P > 0.05). As there is no previous study conducted on Biodentine mixed with chlorhexidine, thus the comparison cannot be done. The reason for this increase is not understood. But it is assumed since Biodentine and MTA both are Portland cement based material, and addition of 2% chlorhexidine to MTA resulted in faster setting of MTA, similarly, mixing chlorhexidine with Biodentine might have resulted in faster setting and crystal growth of Biodentine, which might have influenced the sealing ability of Biodentine. However, further study is required to find out the actual reason.

CONCLUSION

Within the limitations of the study conducted, 2% chlorhexidine, when incorporated in Biodentine enhanced the sealing ability of Biodentine. But further in vitro studies with larger sample size and in vivo studies must be conducted before its clinical use.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.