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Year : 2019  |  Volume : 22  |  Issue : 2  |  Page : 227-231

Short-term antibacterial activity and compressive strength of biodentine containing chlorhexidine/cetirimide mixtures

1 Department of Pediatric Dentistry, Faculty of Dentistry, Cukurova University, Adana, Turkey
2 Department of Pediatric Dentistry, Faculty of Dentistry, Karadeniz Technical University, Trabzon, Turkey
3 Department of Pediatric Dentistry, Faculty of Dentistry, Gazi University, Ankara, Turkey
4 Department of Microbiology, Faculty of Medicine, Karadeniz Technical University, Trabzon, Turkey

Date of Acceptance13-Nov-2018
Date of Web Publication7-Feb-2019

Correspondence Address:
Dr. C Deveci
Cukurova University Faculty of Dentistry, Department of Pediatric Dentistry, Adana
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/njcp.njcp_436_18

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Background and Purpose: Aim is to evaluate the antibacterial activity and physical properties of Biodentine containing chlorhexidine (CHX)/cetrimide (CT) mixtures at 24 h. Materials and Methods: Powder formulations of CHX (2.5%) and CT (2.5%) were added to the powder of Biodentine and served as experimental groups (EXP). Antibacterial free groups were designed as control (CNT). The antibacterial acitivity (AB) (n = 5; for each) of disc-shaped specimens against Streptococcus mutans and Lactobacillius casei and compressive strength (CS) (n = 10; for each) tests used both for groups after 24 h after polymerization. Statistical Analysis Used: Normality of data were tested with the Shapiro–Wilk test. Significant differences were analyzed with the Mann–Whintey U test for both the methods (agar disk diffusion and compressive strength) at a significance level of P < 0.05. Results: Significantly higher antibacterial effects were obtained in EXP groups compared to the CNTs for both bacteria after 24 h, distinctly (P < 0.01). No differences were found between groups regarding the CS values (P > 0.05). Conclusion: Since the incorporation of 5% CHX/CT to the Biodentine exhibited acceptable antibacterial activity without hampering compressive strength values at 24 h, this combination could be a promising alternative for eliminating the residual bacteria for indirect pulp-capping therapies.

Keywords: Antimicrobial activity, Biodentine, cetrimide, chlorhexidine

How to cite this article:
Deveci C, Tuzuner T, Cinar C, Odabas M E, Buruk C K. Short-term antibacterial activity and compressive strength of biodentine containing chlorhexidine/cetirimide mixtures. Niger J Clin Pract 2019;22:227-31

How to cite this URL:
Deveci C, Tuzuner T, Cinar C, Odabas M E, Buruk C K. Short-term antibacterial activity and compressive strength of biodentine containing chlorhexidine/cetirimide mixtures. Niger J Clin Pract [serial online] 2019 [cited 2022 Aug 14];22:227-31. Available from:

   Introduction Top

The calcium silicate based cements are being considered as the succesful materials that stimulate the dentin repair potential as well as providing pulpal healing under optimal conditions.[1],[2],[3] As similar with the mineral trioxide aggregate (MTA), one of the popular biocompatible calcium silicate based agent known as Biodentine have been used for the vital dentin-pulp regeneration therapies such as indirect pulp-capping treatments (IPT).[2],[3],[4],[5],[6],[7] The powder of material contains tri- and di-calcium silicates, calcium carbonate, zirconium dioxide, and also the liquid comprises calcium chloride aqueous solution that provides mechanically stronger and less soluble structure with the less setting time features.[4],[7],[8],[9],[10] The another advantage of this therapeutic agent is that the material can be implemented into the cavity as a bulk material without any surface preparation treatment on the cavity walls which clearly produces reperative dentin formation with the alkaline caustic effect that leads to elevated mineral precipitation.[1],[4],[6],[7],[8],[9],[10],[11],[12],[13] From that point of view, the provisional usage of material in IPT for deep caries lesions seems to have beneficial effects under clinical conditions.[11],[14] Specifically, in pediatric dentistry, IPT includes the removal of softened and demineralized infected dentine near the pulp tissue to reduce the risk of pulp exposure in symptom-free deep carious lesions of primary teeth. This method with the term of “minimally invasive management” strategies could be applied particularly for un-cooperative children.[15],[16],[17],[18] However, residual caries problem should not be overlooked in that type of treatments.[17],[18],[19] Additionally, Biodentine can be used as a cavity-lining material particulary under the composite materials as for the IPT purposes.[11],[20],[21] However, bacterial microleakage problem was also found as for the Biodentine at the dentine to material interface.[21] It has been indicated as the porosity of unhydrated cement particles of tricalcium silicate based cements might cause microspaces filling with the water when the cements are being hydrated. However, if the water/cement ratio is excessively increased during the mixing, the porosity could be increased which improperly caused a microleakage.[22] Such studies revealed the changeable but better antimicrobial effects of Biodentine against cariogenic microorganisms compared to the MTA and other cements, whereas others did not report any significant antimicrobial action.[1],[4],[21],[23],[24] Thus, to enhance the antibacterial activity in calcium silicate based cements, the idea of adding antibacterials such as chlorhexidine (CHX) to the original cement, has been introduced as a beneficial method in terms of reducing the bacterial count under the restorations.[25],[26],[27] Moreover, the usage of CHX and/or cetrimide (CT) with dental materials seemed to have strong antibacterial actions against the specific bacteria in the previous findings.[28],[29],[30],[31] According to our knowledge, there is no current findings related with the CHX + CT combination when added to the Biodentine powder.

Because of the above reasons, this study was designed to test two different below hypotheses:

  1. Antibacterial added Biodentine samples showed increased antimicrobial action against the carious associated bacteria compared to the antibacterial free ones
  2. Physicomechanical negative effects would be obtained after adding antibacterials.

   Materials and Methods Top

Details of the materials are listed in [Table 1]. The set disc-shaped specimens in control group did not have any antibacterials, whereas experimental group was prepared by adding 2.5% CHX + 2.5% CT (powder + powder) to the powder of Biodentine (w/w) manually.
Table 1: Details of the materials

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Agar disk diffusion test

About 10 mm in diameter; 2 mm depth were mixed according to the manufacturer's recommendations for both the groups (n = 5; for each group). The antibacterial effects against MS and LB of set specimens were assessed with agar-diffusion tests. The strains stored at −20°C were cultured on blood agar (Merck, Darmstadt, Germany) and Lactobacilli MRS Agar plates (for Lactobacillius casei, DifcoLab., MI, USA) at 37°C for 24 h in 5% CO2. Single colonies from plates were transferred into the Brain-Heart Infusion (BHI) broth (Merck) and Lactobacilli MRS broth (for L. casei, DifcoLab.) and incubated at 37°C for 24 h. Suspensions of the strains were prepared in phosphate-buffered saline (PBS) at c. 1.5 × 108 organisms/mL by using the McFarland 0.5 turbidity tubes and then were flood-inoculated onto the surface of BHI agar plates. Before placement of the set specimens, the surface of the plates was air dried by leaving them at 37°C for 15 min. Five specimens were used for all groups. The paste was then put into a mold, covered with a glass slide, and allowed to set at room temperature. All the specimens were then sterilized with UV before experiment. The set disc-shaped specimens were placed onto BHI agar plates. Inhibition zones around the specimens were calculated at three different points of the zones for each specimen and averaged at 24 h period by blinded independent observer.

Compressive strength

Experimental and control group samples were prepared with laser cut plexiglass cylindrical molds of 6 mm diameter and 4 mm thickness. The material compacted in plexiglass molds with amalgam plugger to be sure that the surface is leveled. All discs examined according to any cracks on the surface and seven discs in each group selected for compressive strength testing. All discs immersed in the Eppendorf tubes containing PBS and incubated at 37°C with 100% humidity for 7 days. Afterwards the discs have been tested in universal compressive strength machine (Instron Model 1011, UK) with a crosshead speed of 1 mm/s. The force required to break the samples (N/mm 2) was evaluated.

Statistical analysis

Statistical analysis was performed with the SPSS Statistics version 17.0 (IBM, Armonk, NY, USA). Normality of data were tested with the Shapiro–Wilk test. Significant differences were analyzed with the Mann–Whintey U test for both the methods (agar disk diffusion and compressive strength) at a significance level of P < 0.05.

   Results Top

Significantly higher antibacterial effects were obtained in experimental compared to the control group for both Streptococcus mutans and Lactobacillius casei at 24 h period (P < 0.01) [Table 2]. Experimental groups showed increased antibacterial action for LB compared to the MS. Higher, but insignificant differences were obtained in the experimental group compared to the control according to the compressive strength values (P > 0.05) [Table 3].
Table 2: Antibacterial activity of groups against Streptoccocus mutans and Lactobacillius casei. Inhibition zones in disc diffusion agar given in mm

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Table 3: Compressive strength values of the groups at 24 h

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   Discussion Top

This study accepted the first hypothesis that Biodentine + CHX + CT mixtures showed significantly higher antibacterial effects, whereas rejected the second one which indicates the proper compressive strength test values.

Even providing the viability of tested microorganisms is limited, the agar disk diffusion test method that was used in this study allowed to obtain the direct effects of Biodentine on cariogenic bacteria with some certain advantages such as less expensive, rapid, and realistic properties.[20],[21],[23],[24],[25],[26] Since the tested microorganisms (S. mutans and L. casei) and their toxins have been considered as the predominant types in infected/demineralized dentin tissue in IPT, these microorganisms were used for an in-vitro model.[20],[21],[22],[23],[24],[25],[26] Previous reports, indicated increased antibacterial effects of CHX and/or CT when used with glass ionomers or tri- di-calcium silicate cements.[26],[28],[29],[30] Nikhil et al. reported such significantly higher antibacterial effects on S. mutans, Staphylococcus aureus, Enterococcus faecalis, and Candida albicans of Biodentine +2% CHX samples compared to the antibacterial free ones. They also exhibited the limited antimicrobial action of antibacterial free Biodentine samples which could be clearly related with the high pH alkaline properties.[26] Also, in a recent study, the antimicrobial effect of light curable pulp-capping materials, Therecal and Biodentine have been investigated against S. mutans, Staphylococcus gordonii, and Staphylococcus sorbinus and found to have similar antimicrobial activity.[32] Similar studies have been conducted with new bioceramic pulp-capping materials such as MTA and Dycal and showed that MTA has a better antimicrobial activity than Dycal.[33] However, contraversial findings of antibacterial free Biodentine studies also showed presence or absence of inhibiton zones against various microrganisms according to the agar disk diffusion test method.[23],[24] The variations of the antimicrobial actions could be explained by the diffusion capacity, contact properties, molecular weight, size, type, and concentration of Biodentine or such properties of agar medium as stated previously.[24] In a very recent study, Jardine et al. investigated the antimicrobial effect of bioceramic cements on biofilm and stated that all tested materials including Biodentin is not effective on multispecies microcosm biofilm.[34] Previous findings also indicated such acceptable antibacterial action of CHX + CT mixtures with dental materials with the same concentration as a total of 5% (2.5% +2.5%) or in various concentrations.[28],[29],[30] In the present results, even control group (antibacterial free) has also shown such antibacterial effects on the tested microorganisms, the Biodentine + CHX + CT combination showed significantly higher and better antibacterial effects on both bacteria (MS and LB). Also, the antibacterial added group showed much better inhibitor effects on LB compared to the MS. Thus, the idea of combining the suitable antibacterials versus single usage at different ratios would elevate the antibacterial action by killing different cell types of various bacteria.[35] From that point of view, it could be beneficial to use the combination of tested antibacterial agents versus individual usage of them with Biodentine. Because of the above reasons, these results might indicate an acceptable range of activity against the carious associated bacteria for IPT therapies with the specific inherent potency and sensitivity of tested mixture. In another study, the antimicrobial action of Biodentine +2% CHX gluconate or 10% doxycycline was evaluated for 24, 48, and 72 h and showed a decreasing pattern.[26] This result also consistent with the previous reports for dental material–antibacterial combinations. Commonly, the antibacterial effects could decrease from the beginning to the later time periods in that type of studies.[26],[28],[29] Thus, the need for prolonged antibacterial effects should not be overlooked in terms of inhibiting bacteria under the restorative materials specifically when the carious tissue is not eliminated completely.[28],[29] Since, we have only calculated the short-term inhibitor effect, it might be considered as a limitation factor of this study. However, the present results obtained in Biodentine + CHX + CT groups revealed much more higher inhibiton zones compared to the previous findings for the MS.[26] Thus, the present promising results would also be analyzed regarding the antibacterial activity on different microrganisms for longer time periods in future study designs. Additionally, cytotoxic effects of antibacterials should not be overlooked on human cells as stated previously.[36] Even antibacterial actions were found optimal in this study, the potential cytotoxic pattern of Biodentine + CHX + CT combination should also be tested with biologic tissues for the long-term periods. Thus, this handicap could be considered as another limitation factor of the present study.

Since measuring compressive strength values of the setting process of tricalcium cements is to have much more importance for providing valuable information about clinical durability in vital pulp therapies, we used this method to test the in-vitro mechanical strength of Biodentine.[8],[9] Although, the dental material + antibacterial combination have such benefits over certain microorganisms, the addition of antimicrobial particles might have negative roles on the basic physicomechanical properties of cement nature.[28],[29],[37] Holt et al. revealed that the addition of CHX to the MTA would show lower compressive strength values compared to the MTA mixed with sterile water even those were not significantly different. They also reported if the cement failed to set, the antibacterial added material might have brittle properties which indicates the antibacterial efficiacy of combination would be considered as negligible.[36] In this study, the antibacterial free group showed lower but insignificant values compared to the antibacteral added group and we did not obtain any deformation while removing the samples from the mold. With the advent of these unexpected outcomes for this kind of study, the setting reaction of Biodentine would probably have not been affected by the foreign antibacterial particles. The acceptable dimensionally stable setting and compressive strength properties of antibacterial added specimens obtained in the present study could be attributed to the lower water/cement ratio, various particle size distribution, or high retention of the strong chemical affinity of CHX + CT to the original Biodentine. Thus, these mentioned indicators can be considered as promising factors for setting reaction process.[8],[10] Nevertheless, mechanical strength measurements may not always detect the exact setting reaction occurring in the dental material and cannot always explain the real clinical longevity because of certain factors such as saliva, pH changes, food, liquids, and masticatory functions in the oral environment.[38] Thus, to gain the exact results, future studies should be focused on the compressive strength and other mechanical tests that could be further evaluated within different time periods including various storage mediums of in-vitro conditions.

   Conclusion Top

In view of the results and limitations of the present study, the addition of CHX + CT combination at a total of 5% concentration to the powder of Biodentine may constitute a short-term antibacterial effect against the MS and LB bacteria without seriously deteriorating compressive strength values of the cement. This combination could have a chance to provide beneficial antibacterial effects in IPT therapies. However, longer-lasting antibacterial and physicochemical alterations should be warranted by future in-vitro, in-situ, and/or in-vivo studies.

Financial support and sponsorship


Conflicts of interest

There are no conflicts of interest.

   References Top

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  [Table 1], [Table 2], [Table 3]


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