Effect of addition of 2% chlorhexidine or 10% doxycycline on antimicrobial activity of biodentine.

AIM: The purpose of this in vitro study was to determine whether the addition of 2% chlorhexidine gluconate or 10% doxycycline would enhance the antimicrobial activity of Biodentine against Staphylococcus aureus (ATCC-25923), Enterococcus faecalis (ATCC-29212), Candida albicans (ATCC-90028), and Streptococcus mutans (MTCC-497).
MATERIALS AND METHODS: Three wells of 4 mm diameter and 4 mm depth on each plate were prepared on the agar medium with standardized suspensions of each microorganism. Biodentine powder mixed with 2% chlorhexidine (0.06 g) or 10% doxycycline (0.30 g) in its liquid or liquid alone was placed to fill each well. Plates were incubated at 37°C as required for microbial growth. A blinded, independent observer measured zones of inhibition. The data were analyzed using independent "t" test to compare the differences among the three cement preparations for different micro-organisms.
RESULTS: All Biodentine samples inhibited microbial growth. The highest mean diameters of zone of inhibition for all the micro-organisms were found around Biodentine/chlorhexidine (13.417) followed by Biodentine alone (12.236) and Biodentine/doxycycline (11.25).
CONCLUSION: In conclusion, adding 2% chlorhexidine gluconate in liquid of Biodentine enhanced the antimicrobial activity of Biodentine against all the tested micro-organisms except Candida albicans, while addition of 10% doxycycline decreased the antimicrobial activity of Biodentine. The differences were significant statistically (P < 0.05).

INTRODUCTION

Development and progression of pulpal and periapical diseases and endodontic failure has been associated with micro-organisms. Varieties of disease-causing micro-organisms have been isolated from the infected root canals e.g. Enterococcus, Actinomyces, Propionibacterium, Yeasts, Streptococcus etc.[1] Although shaping, cleaning, and disinfection protocols are aimed to eliminate bacteria from root canals, bacteria remain in the root canal after chemo-mechanical preparation of root canal, probably due to complexities of root canal.[23] Facultative anaerobic micro-organisms such as Enterococcus faecalis, Staplylococcus aureus, and Candida albicans are considered to have the highest resistance in the oral cavity, with the potential to cause failure of root canal treatment.[4]

Success of endodontic treatment not only depends on sealing of all portal of entries to prevent future contamination of root canal system but also elimination of infected tissues and micro-organisms as re-infection of root canal after treatment is not desirable.[5] Therefore, an ideal root-end filling material should not only be dimensionally stable, radiopaque, non-reasonable, non-toxic, and biocompatible but also bactericidal or bacteriostatic and provide impervious seal.

A new material Biodentine (Septodont, Saint Maur des Fossés, France) has been introduced into dentistry in recent years. According to Research and development department of said manufacturer, this material could conciliate high mechanical properties with excellent biocompatibility and bioactive behavior.[6] Biodentine is based on calcium silicate, and it is packaged in single use capsules that contain powder and single use ampoules of liquid. It is indicated for crown and root dentin repair treatment, indirect and direct pulp capping, pulpotomy, repair of perforations or resorptions, apexification, and root-end fillings,[7] but there are no studies to evaluate its antibacterial activity barring only one conducted by Valyi E et al.[8]

Chlorhexidine is a chemical antiseptic and has a broad antibacterial action, thus was initially used for general disinfection. Later, its inhibitory action on dental caries and formation of dental plaque was discovered. Various in vitro studies concluded chlorhexidine to be effective against S. aureus, E. faecalis, S. mutans, C. albicans, S. salivarius, A. viscous, and Spyogenes.[910] Other than mouthwash, it is also frequently used as root canal irrigant and inter-appointment filling material (e.g. Chlorhexidine-impregnated gutta-percha point, the Active point by Roeko, Langenau, Germany). The purpose for all these uses remained same that is disinfection. Holt et al.,[11] advocated substituting 0.12% chlorhexidine gluconate for water to enhance the antimicrobial activity of MTA.

Doxycycline, a hydroxyl derivative of tetracycline, is a very potent anticollagenase antibiotic among the tetracycline.[12] Its ability to eliminate biofilm more potently than calcium hydroxide inside human root canals has been well documented.[13]

Although substantivity and evidences are available for both chlorhexidine and doxycycline, no studies so far have been designed to determine whether the addition of these two substances will enhance the antimicrobial properties of Biodentine.

The aim of the present study was to explore the effect of adding one of these substances chlorhexidine and doxycycline to a new root end filling material, Bio dentine and determine its effect against relevant micro-organisms S. mutans, E. faecalis, C. albicans, and S. aureus using agar diffusion method.

MATERIALS AND METHODS

The antimicrobial activities of the freshly mixed cements were evaluated by the agar diffusion method against four reference strains: Staphylococcus aureus (ATCC-25923), Enterococcus faecalis (ATCC-29212), Candida albicans (ATCC-90028), and Streptococcus mutans (MTCC-497).

The inoculum was prepared by making a direct brain heart infusion broth suspension of the isolated colonies from an overnight growth on blood agar plate. The suspension was adjusted to achieve a turbidity equivalent to 0.5 Mc Farland opacity standards. A sterile cotton swab was dipped into the adjusted suspension. The swab was rotated several times and pressed finally on the side wall of the tube above the fluid level to remove excess fluid from the swab. The dried surface of an agar plate was inoculated by streaking the swab over the entire sterile agar surface. This procedure was repeated by streaking two more times, rotating the plate approx. 60° each time to ensure an even distribution of inoculums. S. aureus and C. albicans suspension were inoculated on to Muller Hinton agar plates and E. faecalis and S. mutans on blood agar plates.

Wells were prepared on the medium with the help of puncher of 4 mm diameter and 4 mm depth (three on each plate) and were then immediately filled with the freshly prepared test materials. The volume of liquid provided with Biodentine powder is 5 drops (0.3 ml), thus to make 2% chlorhexidine solution of this liquid, 0.06 g of chlorhexidine gluconate powder was mixed. Similarly, for 10% doxycycline solution, 0.30 g of doxycycline powder was mixed. The test materials were manipulated in accordance with the manufacturer's instructions in aseptic condition. A total number of 12 plates were employed; three each for four strains. After pre-diffusion of the test materials for 2 h. at room temperature, all the plates were incubated at 37°C in an incubator directly, whereas the S. mutans blood agar plate was kept in candle jar at 37°C in the incubator.

All the above procedures were performed in the class II Biosafety Cabinet in Microbiology Department. A blinded, independent observer then evaluated the plates at 24 h, 48 h, and 72 h. Microbial inhibition zones were measured with a precision ruler. The results were expressed as the mean and standard deviation of three independent experiments. The data for each group were subjected to independent “t” test to determine if significant differences in zones of inhibition occurred between different groups: Confidence level was set at 5%.

RESULTS

The antibacterial activities of listed test material determined by the means and standard deviation of zones of inhibition in millimeters on all test micro-organisms are shown in Tables 1 and 2. Biodentine was always inhibitory regardless of the addition of antimicrobial agent; however, the inhibition zones were larger when antimicrobials were added with the exception of the Biodentine/doxycycline for S. aureus, E. faecalis, and C. albicans and Biodentine/chlorhexidine for C. albicans. These micro-organisms showed slightly larger zones of inhibition with Biodentine alone. The antimicrobial action of Biodentine/chlorhexidine on all micro-organisms tested was superior to that of Biodentine/doxycycline or Biodentine alone showing inhibition zones ranging from 8-26 mm, except for C. albicans, which was better inhibited by Biodentine alone. Decreasing order of inhibition zones produced by Biodentine/chlorhexidine, Biodentine alone, and Biodentine/doxycycline on all micro-organisms ranged from 8.33-24.33, 7.5-26.17, and 6.33-24.17, respectively. All the tested groups were more effective against C. albicans than against S. aureus, E. faecalis, and S. mutans. Direct comparisons between the groups by independent “regeneration”’ test for different micro-organisms revealed statistically significant discrepancies, among the 3 groups (P < .05), except for C. albicans and group 2 and group 3 for S. mutans [Table 3].

Table 1

Zones of inhibition in mm of the tested materials against tested micro-organisms at various time intervals

Table 2

Mean and standard deviation of zones of inhibition in mm of different micro-organisms in three groups

Table 3

Comparison between groups for their antibacterial efficacy/action against different microorganisms

DISCUSSION

Infected root canals contain a variety of micro-organisms, which include aerobes, anaerobes, and fungus. Root canal-treated teeth are about nine times more likely to harbor E. faecalis than cases of primary infections.[4] Similarly, C. albicans has been demonstrated in root-filled teeth with chronic apical periodontitis. Therefore, different strains of micro-organisms were included in this study.

Biodentine, an endodontic repair material, possesses several advantageous properties that include good sealing capability, biocompatibility, and antibacterial activity. However, study conducted by Valyi et al.,[8] concluded that the antibacterial activity of Biodentine (a tricalcium silicate based cement) is although comparable to other Ca-based cements, it is more dependent on strain type.

Chlorhexidine, a broad-spectrum antibacterial agent, has shown its effectiveness on various microbes, particularly against both E. faecalis and C. albicans.[10] Doxycycline, a member of the tetracycline antibiotics group, is commonly used to treat a variety of infections. It is antibacterial, antiprotozoal, and antihelminthic. In dentistry, it is used in the treatment of periodontitis.

Therefore, Biodentine alone as well as mixtures of Biodentine/chlorhexidine and Biodentine/doxycycline were included to evaluate their effect on these stains of micro-organisms.

The zone of inhibition appeared on the surface of the agar as a zone immediately around the Biodentine or its mixture with antimicrobial agents plug containing no micro-organisms colonies. In the present study, zones of inhibition were seen in all the three groups against all the micro-organisms irrespective of whether antimicrobial added or not, with the former being larger.

The fact that Biodentine alone showed zones of inhibition implies that Biodentine itself has some limited anti-microbial effects and might be attributed to Biodentine high pH. E. faecalis can survive in extreme alkaline environments up to pH of 11.1.[14] Perhaps the inherent, persistent alkalinity of Biodentine is just enough to overwhelm the E. faecalis. Biodentine powder is mainly composed of tricalcium silicate, calcium carbonate, and zirconium oxide as the radio-pacifier, whilst Biodentine liquid contains calcium chloride as the setting accelerator and water as reducing agent. The addition of up to 30% calcium carbonate, calcium sulfate, and calcium chloride resulted in improvement in physical properties of tricalcium silicate cement.[1516] as well as enhancing the bioactivity and degradability of the resultant composite material. Hydration of tricalcium silicate results in the formation of calcium silicate hydrate gel, calcium hydroxide, and un-reacted tricalcium silicate.[17] The calcium hydroxide produced from the tricalcium silicate hydration possesses antibacterial and anti-inflammatory properties[16] mainly due to the high (alkaline) pH of the surrounding environment after it dissolves. The pH level remained stable over time at a value of around 11-12.[17]

Both MTA and Biodentine demonstrate bioactivity, i.e. both form a bone-like hydroxyapatite layer on the surface when immersed in physiological solution; this is a common characteristic observed in calcium-silicate based materials. This layer is also instrumental in maintaining the bone — biomaterial interface when implanted in the body.[15] Furthermore, calcium silicate cements including tricalcium silicate were found to have advantageously shortened setting time compared with MTA, and postulated to be suitable for replacement of the cement component of MTA due to their similar composition and bioactivity.[18] Tricalcium silicate cement possesses good injectability, good bioactivity, and moderate in vitro degradability, meaning that ultimately the body may be able to replace the implanted cement by natural tissue.[15]

In this study, Biodentine/chlorhexidine mixture produced greater zones of inhibition than the Biodentine/doxycycline mixtures or Biodentine alone, and the difference was statistically significant (P < 0.05) except for C. albicans.

In contrast to the other three microorganisms, the Biodentine alone seemed to have a greater inhibitory effect than the Biodentine/chlorhexidine mixture on C. albicans, although the magnitude of the difference was very small [Table 1]. Additional studies may clarify this anomaly. The results of this experiment show that chlorhexidine enhanced the antimicrobial efficacy of Biodentine. The reason may be that in addition to its immediate action on bacteria, chlorhexidine can be adsorbed onto and subsequently released from the cement as reported for dental tissues, resulting in substantive antibacterial activity or “substantivity.”[19]

Various reports have concluded differing results on potential toxicity of chlorhexidine, when in contact with biologic tissues for the long-term. Chlorhexidine was shown to be highly cytotoxic to human fibroblasts in vitro,[20] but chlorhexidine has been used in the treatment of severe burns without reports of adverse effects on healing.[21] In addition, it has been successfully used in periodontal surgery where more rapid healing with less inflammation is reported; when chlorhexidine is used as a post-surgical rinse and MTA mixed with 0.12%, chlorhexidine appeared to be biocompatible when implanted subcutaneously in rats.[22] Gabler et al. claimed that serum present during the initial healing period seems to provide significant protection against these cytotoxic effects.[23] However, because a principle benefit of Biodentine is its alleged biocompatibility and bioactivity, adding chlorhexidine could potentially be detrimental to the host cells.

In the present study, mixture of Biodentine and doxycycline showed smaller zones of inhibition for all the strains than mixture of Biodentine and chlorhexidine and Biodentine alone, except for S. mutans, in which it was equal to Biodentine alone. This means that instead of enhancement of antibacterial activity of Biodentine, doxycycline reduced its antibacterial activity. The reason may be that when doxycycline combines with dairy, antacids, or calcium supplements, any of these foods and supplements may decrease doxycycline's effectiveness and on hydration, Biodentine forms calcium hydroxide, which later on dissociate into Ca++ and OH-, this calcium is responsible for antibacterial activity of Biodentine, but quite possible doxycycline forms chelates with it, resulting into decrease in antibacterial activity.

CONCLUSION

Within the limitations of the current study, the following conclusions were drawn:

The antimicrobial activity of Biodentine was clear against tested bacteria and fungi.

Susceptibility to the Biodentine or its mixture with 2% chlorhexidine or 10% doxycycline was different for all tested bacterial strains. C. albicans was most susceptible while E. faecalis was least.

Addition of 2% chlorhexidine to Biodentine enhanced the antibacterial activity of Biodentine alone, while addition of 10% doxycycline to Biodentine decreased the antibacterial activity of Biodentine alone.

In vitro antimicrobial susceptibility testing per se has its limitations, thus correlating in vitro results with the in vivo activity is difficult.[24] Further research is warranted on the physical properties, e.g. setting and working times after addition of antimicrobials to the Biodentine, as well as on testing the anti-microbial effect of already set Biodentine samples as the results could be influenced by the solubility and diffusibility of the test agent through the agar.[25] Research directed toward investigating the cellular response to the Biodentine/chlorhexidine mixture would also be needed before advocating its clinical use.