Evaluation of antimicrobial efficacy of three calcium silicate-based materials using tube dilution: An in vitro study

Background: Periapical pathosis is considered as a persistent microbial infection throughout the treatment period of root canal procedures. Recently the prevalence of endodontic failures is huge and this primarily accounts for the periapical pathogens; mainly Enterococcus faecalis and Candida albicans. Thus, with the advent of newer materials; identification of the most appropriate sealing material with highest antimicrobial efficacy is essential for endodontic success. Aim and objectives: To evaluate and compare the antimicrobial efficacy of MTA Angelus, Biodentine and Theracal LC against Enterococcus faecalis and Candida albicans. Materials and Methods: An in-vitro study was conducted using three silicate-based cements; MTA Angelus, Biodentine and Theracal LC. Their antimicrobial efficacy was evaluated using Tube dilution method and their Minimum inhibitory concentration (MIC) and Bacterial counts (CFU’s) were assessed and compared. Statistical analysis was done using Paired t-test and ANOVA at 95% confidence interval (p value <0.05). Results: The MIC was highest and CFU’s were lowest with Biodentine in comparison with other two materials. There were statistically significant differences noted between Biodentine and MTA angelus. Conclusion: Our study results conclude that Biodentine was found to be more anti-bacterial as compared to MTA angelus and theracal LC. © 2020 Published by Innovative Publication. This is an open access article under the CC BY-NC license (https://creativecommons.org/licenses/by-nc/4.0/)


Introduction
Microorganisms play an essential role in pulpal and periapical diseases. 1 Periapical pathosis is considered an endogenous infection caused by the oral microflora. Therefore, many investigators have attempted to isolate and identify various microorganisms from root canals or periapical regions. 2 Later, Fabricius et al, 3 in 1982 showed that strict anaerobes succeed over primarily facultative ones in the canals because of the predominant ecology of root canal. Gram positive microorganisms are predominant in re-treatment diseased teeth. They can survive in difficult and less-nutrient environment in obturated teeth. A high prevalence was seen of Enterococci about 29 to 77% in those teeth with persistent and recurrent disease.
Enterococcus faecalis can withstand high pH of intracanal dressings like calcium hydroxide, and is hence found in higher concentration in reinfection cases. 4 Enterococci survive very harsh environments including extreme alkaline pH & salt conc. Other common organism identified in post treatment apical pathology is Candida albicans. Both survive as mono-infection and invade dentinal tubules. 5 Candida albicans also showed the ability of colonization root canal walls and penetration into dentinal tubules. The colonization of fungi in the root canals and various other factors affecting this colonisation are not fully understood.
The past literature studies have hypothesized that the specific diminution of bacteria in the root canals during endodontic treatment allows overgrowth of fungi in basic environment of low nutrition. 6 Due of coronal leakage, many fungi especially Candida albicans can gain access to the apex of the root canals and lead to initial failures of endodontic treatment. Orthograde retreatment and/or endodontic surgery has helped for the recovery, in such cases. To avoid recontamination in the future and primarily enhance the success of treatment; complete elimination of the infected tissue and the microorganisms is essential to attain the overall root canal system seal. 7 About 5.5% of the procedures involve perforation repair and endodontic surgery from majority of the 24 million treatments carried out on an annual basis. 8 Those surgeries performed at the apex involve the placement of the material at apical end of the root to seal the root canal from the periapex. Various materials used from amalgam, zinc oxide eugenol, composite resins, & glass ionomer cements. With reference to all these materials, none was able to satisfy the requirements of an ideal root-end filling material. Later in 1990s, mineral trioxide aggregate (MTA) introduced as a root-end filling material. Till date (MTA) used as a material of choice. 9 The hydraulic self-setting cements involve calcium silicate-based cements (CSC), such as mineral trioxide aggregate (MTA). The powdered form of CSC contains tricalcium and dicalcium silicate. The reaction involves mixing of the powder with water. This leads to the formation of calcium silicate hydrate Ca(OH) 2 primarily. Further, this mix results into a colloidal sticky gel i.e. calcium silicate hydrate gel. This gel solidifies to form a hard substance. The regeneration procedures and repair procedures like rootend filling, apexogenesis, pulpotomy, apexification, pulp capping and perforation repair involves usage of calcium silicate-based cements. 10,11 Their biocompatibility and sealing ability synergistic to the physicochemical reaction and interaction with the surrounding local environment contribute to be the specific factors in achieving suitability in difficult clinical situations. Pulp-capping agents have been developed over the years. Most commonly used are calcium hydroxide-based materials.
Till recently, Mineral Trioxide Aggregate (MTA) is also used commonly. But the most popular is calcium hydroxide for the treatment involving direct and/or indirect pulp capping. 12 The high pH (12.5) gives calcium hydroxide an important antimicrobial activity. As stated by Siqueira, the main bactericidal action of Ca(OH) 2 primarily depends on the maximum release of hydroxyl ions in a clean aqueous environment According to. Siqueira and Lopes evaluated that hydroxyl ions are highly oxidant free radicals that show extreme reactivity with several biomolecules, causing denaturation of proteins and damages to the bacterial cytoplasmic membrane. 13 The only disadvantage of MTA is the long setting time and poor handling properties. Two new materials MTA like materials; MTA ANGELUS and a calcium silicatebased material; Biodentine can be used as a root-end filling material.A newer calcium silicate-based material for the treatment of pulp capping is Theracal LC. This has found to be advantageous over many conflicting reports and studies stating the antifungal and antibacterial properties of MTA. However, MTA seems to have limited antimicrobial effect against some microorganisms. Thus. the aim of our study was to evaluate and compare the antimicrobial efficacy of three new materials MTA Angelus, Biodentine and Theracal LC using tube dilution method.

Materials and Methods
This is an in-vitro study conducted after obtaining relevant permissions from the Scientific Advisory Committee and Institutional ethics committee number-1672/2016-17 dated 23/11/2016. Three materials were used in the study: MTA Angelus, Biodentine and Theracal LC. The antimicrobial efficacy was tested against Candida albicans and Enterococcus faecalis using tube dilution method. The culture media used were Brain heart infusion broth, Mac Conkey's agar, sabouraud dextrose broth, and sabouraud dextrose.
Ten microliter of standardized Enterococcus faecalis was added to tube no. 1-10. These tubes were then incubated at 37 • C. Subcultures on Mac Conkey's agar were made on each of the tubes. At the end of 24 hours of incubation, the readings were noted. The antibacterial efficacy of these materials was evaluated in a similar manner, wherein ten different strains of Enterococcus faecalis were analysed. For antifungal efficacy, similar methodology was followed. For dilutions and culture Sabouraud's dextrose agar (SDA) and Sabouraud's dextrose broth (SDB) were used. For the evaluation of antifungal efficacy of these materials, ten different strains of Candida albicans were used.

Laboratory analysis
The outcomes measured were the Minimum Inhibitory Concentration (MIC) and minimal bacterial count {Colony forming units (CFU's)} for all three-calcium silicate material against Enterococcus fecalis and candida albicans.

Statistical analysis
The data entry and sorting was done in Microsoft Excel 2013. Frequency analysis was done of demographic status by using Statistical Package for Social Sciences (IBM SPSS Statistics V21.0) statistical software. Mean and standard deviation of the MIC and Colony forming units was derived. For inter-group comparison; One-way ANOVA was used to determine the difference between the groups followed by Tukey's post hoc test at 95% confidence intervals    considering p value <0.05 as significant.

Results
An in-vitro study was conducted with the aim to to evaluate and compare the antimicrobial efficacy of three new materials MTA Angelus, Biodentine and Theracal LC using tube dilution method. Inter-group comparison was done to determine the CFU counts using candida oneway ANOVA test. This comparison showed statistically significant differences (p value <0.05) between the three groups. (Table 1). Pair-wise comparison was done using multiple Tukey's post hoc test. A statistically significant difference was noted between Bio-dentine and MTA Angelus. (Table 2) Intergroup comparison was done to determine the CFU counts using Enterococcus faecalis one-way ANOVA test. This comparison showed statistically significant differences (p value <0.05) between the three groups. (Table 3).
Pair-wise comparison was done using multiple Tukey's post hoc test. A statistically significant difference was noted between Bio-dentine and MTA Angelus. (Table 4) Thus, after evaluating minimal inhibitory concentration and bacterial count (CFU's) for these three-calcium silicate material against Enterococcus fecalis and Candida albicans with tube dilution method; Biodentine found to be more significant in comparison with MTA angelus and theracal LC in our study.

Discussion
Our study has evaluated and compared the anti-microbial efficacy of three silicate-based cements. In endodontic disease the primary aetiological agents are represented by microorganisms. In our study Candida albicans and Enterococcus faecalis were primarily focussed upon because Candida albicans is regarded as a potent pathogen which infects the periapical lesions and also has been associated to those infections, which are resistant endodontic non-surgical therapy. It adapts to an extreme range of pH, low oxygen and nutritional environment. 14 Endodontic failure mainly occurs as major cause because of the persistence of microbial infection in the root canal system. Therefore, to prevent such failures it is important to eliminate the microorganisms, including fungi, from the complex root canal systems. Considering some bacteria, like Enterococcus faecalis and fungi like Candida albicans, it has been found that they are primarily resistant to the antibiotics that are present in irrigating solutions. Thus, causing failure of the endodontic treatment. 15 The antibacterial activity of dental materials has been widely evaluated with the tube dilution test. This test allows direct comparisons of materials against tested microorganisms. But there is a disadvantage that it is not able to distinguish the between micro-biostatic and microbicidal materials. Furthermore, the control and standardisation of the dilution, evaluation of results, and reading point of inhibition haloes are restricting factors affecting the dynamics and variability of dilution tests. 16 Various studies were conducted to evaluate the antimicrobial efficacy of root canal materials.
Similar to the results of our study, were the conclusions of Gomes et al 17 which showed that Enterococcus faecalis was more frequently recovered from the canals in later appointments after biomechanical treatment procedures; whereas, Sundqvist et al reported that from nine cases in which Enterococcus faecalis was isolated in initial samples during root-canal re-treatment. Molander et al 18 19 wherein the Candida species are predominantly resistant to the disinfectants and antiseptics, which are commonly used as endodontic medicaments and irrigants. S.Arikan et al stated that MIC value recorded is defined as the lowest concentration of the assayed antimicrobial agent that inhibits the visible growth of the microorganism tested and concluded the lowest MIC for biodentine. These results were similar to those of our study.
According to E. Canton et al; MBC is defined as the lowest concentration of antimicrobial agent needed to kill 99.9% of the final inoculum after incubation for 24h under a Standardized set of conditions. In the study of Zhang et al 20 the antibacterial effect of MTA and Biodentine at low concentrations was investigated with their suspensions. These showed similar bacterial killing and completely destroyed all bacteria in 1 hour. These results were similar to our study. In our study, biodentine was found to have the highest antibacterial effect as compared to MTA. Similar results were found by Torabinejad et al, 21 which concluded that MTA had no antibacterial activity against E.faecalis and has no effect on the strict anaerobic bacteria.

Conclusion
The results of our study can be extrapolated to other population within the country. According to the statistical evaluation within the limitation of this study biodentine has shown to have good antifungal and antibacterial property as compared to MTA Angelus and theracal Lc. Further studies should be conducted using different parameters to evaluate the antibacterial properties of the cements with larger sample size. Moreover, the research studies can be conducted on newer cements to reduce the chances of endodontic failure. Therefore, the evaluation of newer materials will show more biocompatibility of materials when used in vivo.

Source of Funding
None.

Conflict of Interest
None.