|Year : 2016 | Volume
| Issue : 1 | Page : 23-26
Effect of addition of 2% chlorhexidine gluconate, calcium hydroxide, and tetracycline powder on antimicrobial activity of mineral trioxide aggregate
Natasha Gupta1, Neelam Singh1, Bindiya Thapar2
1 Department of Conservative Dentistry and Endodontics, Jamia Millia Islamia, New Delhi, India
2 Department of Endodontics and Conservative Dentistry, Saveetha Dental College, Saveetha University, Chennai, Tamil Nadu, India
|Date of Web Publication||21-Jun-2016|
Department of Conservative Dentistry and Endodontics, Jamia Millia Islamia, New Delhi
Source of Support: None, Conflict of Interest: None
Aim: To evaluate the effect on antimicrobial property of mineral trioxide aggregate (MTA) against Enterococcus faecalis when mixed with 2% chlorhexidine gluconate, calcium hydroxide, and tetracycline powder.
Materials and Methods: Pellets of MTA, MTA with 2% chlorhexidine gluconate, MTA with calcium hydroxide, and MTA with tetracycline powder were prepared. The antimicrobial activity was tested on Sheep Blood Agar. Wells were made and the mixed material was placed in these wells. The diameter of zones of inhibition was measured after 24 h and 48 h incubation at 37°C. The data were analyzed to compare the antimicrobial efficacy of all the groups by ANOVA test.
Results: The zones of inhibition were measured using precision ruler. This study showed that the best antimicrobial efficacy against E. faecalis was exhibited by the group in which MTA was mixed with 2% chlorhexidine gluconate followed by calcium hydroxide group and MTA group. The tetracycline group was least effective.
Conclusion: The study concludes that the addition of 2% chlorhexidine gluconate to MTA enhances the antimicrobial property and is more effective than MTA when used as a root end filling material alone.
Clinical Significance: In cases where nonsurgical treatment of root canal fails and need of surgical intervention is a must to salvage the tooth, role of root end filling material becomes important. It should not only provide a good seal but also be antimicrobial in nature so that chances of re infection re least.
Keywords: 2% chlorhexidine gluconate; antimicrobial efficacy; calcium hydroxide; Enterococcus faecalis; mineral trioxide aggregate; tetracycline.
|How to cite this article:|
Gupta N, Singh N, Thapar B. Effect of addition of 2% chlorhexidine gluconate, calcium hydroxide, and tetracycline powder on antimicrobial activity of mineral trioxide aggregate. Endodontology 2016;28:23-6
|How to cite this URL:|
Gupta N, Singh N, Thapar B. Effect of addition of 2% chlorhexidine gluconate, calcium hydroxide, and tetracycline powder on antimicrobial activity of mineral trioxide aggregate. Endodontology [serial online] 2016 [cited 2019 Nov 21];28:23-6. Available from: http://www.endodontologyonweb.org/text.asp?2016/28/1/23/184326
| Introduction|| |
Microorganisms have always been an influencing element in the outcome of success of root canal treatment.  Few that have been isolated from infected root canals are Enterococcus, Actinomyces, Propionibacterium, Yeasts and Streptococcus. Although biomechanical preparation is done with the aim to eliminate all the bacteria still, they may remain in the canal due to its complex anatomy. 
When the microorganism egress to periradicular tissues and infection persists in bony area around the tooth structure, surgical intervention becomes a necessity. To prevent reinfection of surgically treated tooth root end filling is done.  Some of the ideal properties are that it should be nontoxic, biocompatible, moisture resistant, dimensionally stable, antibacterial, and provide a hermetic seal. 
Enterococcus faecalis a normal commensal of oral cavity possess a concern for endodontist as it is associated with persistent, asymptomatic endodontic infections. The occurrence in such infections ranges from 22% to 74%. 
Mineral trioxide aggregate (MTA), contains tricalcium silicate, tricalcium aluminate, bismuth oxide and has hydrophilic fine particles which when come in contact of moisture or blood tends to harden. 
Two percent chlorhexidine gluconate has been repeatedly proven to be the most effective method to remove or completely eliminate E. faecalis from dentinal tubules up to 100 μm. ,
Tetracycline is a crystalline powder yellow in color, acidic in nature and primarily bacteriostatic and odorless. It has been used as broad spectrum antibiotic. It has been known to bind with dentine surface. 
Calcium hydroxide is an amorphous matrix with crystalline fillers having initial bactericidal then bacteriostatic action. Its alkaline pH has therapeutic effect. It dissolves necrotic tissues.  Its nonspecific bactericidal effect destroys cell membranes and protein structure. 
Hence, this study has been undertaken to evaluate the antimicrobial efficacy of the MTA against E. faecalis if mixed with 2% chlorhexidine gluconate, tetracycline hydrochloride, and calcium hydroxide.
| Materials and Methods|| |
The materials that were used in this study were ProRoot MTA (DENTSPLY Tulsa Dental Specialties, DENTSPLY, USA), 2% chlorhexidine gluconate (Dentochlor, Amrit Chemicals and Mineral Agencies, Punjab, India), tetracycline HCl 250 mg (Zydus Cadila, Gujrat, India), calcium hydroxide powder (Pulpdent Corporation, India). The results of this study showed that the most efficacy against E. faecalis was exhibited by Group 2 followed by Group 4, Group 1 and least effective being Group 3 after 24 and 48 has shown in [Figure 1].
Bacterial strain and media
The test strain of E. faecalis that was used was ATCC 29212. With the help of wire loop, four morphologically similar colonies were transferred to test tube which contained 4 ml of sterile peptone water. The antimicrobial activity was tested by agar diffusion test and medium used was Sheep Blood Agar (Hi-Media Pvt. Ltd., Mumbai, India). The lawn culture was done on blood agar plate after inoculums were adjusted to 0.5 McFarland tube.
The materials were divided into four study groups.
- Group 1: MTA - control group (0.12 g of MTA was taken)
- Group 2: MTA mixed with 2% chlorhexidine gluconate (0.12 g of MTA mixed with 90 μL of 2% chlorhexidine gluconate)
- Group 3: MTA mixed with 1% tetracycline hydrochloride (0.12 g of MTA mixed with 90 μL of 1% tetracycline hydrochloride solution)
- Group 4: MTA mixed with calcium hydroxide (0.12 g of MTA mixed with 0.12 grm of calcium hydroxide powder).
Four wells of 6 mm × 4 mm were made in each dish after the medium solidified. The wells were placed at equidistant points using sterile straws. These wells were immediately filled with test materials. The mixing of the material was carried out according to the manufacturer's direction. The plates were kept at room temperature for 2 h so as to let the prediffusion of the materials occur. They were then incubated at 37°C under appropriate gaseous conditions for 24 h and 48 h. The diameters of zones of inhibition were measured after 24 h and 48 h using a precision ruler.
| Results|| |
Zones of inhibition analysis
The diameters of zones of inhibition were measured, and the group that showed larger zone was considered as having the most efficient antimicrobial activity. The results of this study showed that the most efficacy against E. faecalis was exhibited by Group 2 followed by Group 4, Group 1 and least effective being Group 3 after 24 and 48 h.
| Discussion|| |
The main objective of root end filling material lies in providing a good apical seal so as to prevent the egress of bacteria and its products into the periapical tissue. The materials despite having a perfect seal tend to leave microscopic space at the interface of material and the cavity of root end. This space acts perfectly for microorganisms to ingress and proliferate. As a result, it is important for root-end filling materials to just not be biocompatible and provide a perfect seal but also possess some antimicrobial property. 
MTA was developed to fulfill a need of an ideal root-end filling material for surgical endodontic treatment at the Loma Linda University, California, USA.  It has a can induce bone, periodontal ligament and cementum formation thus leading to regeneration rather than repair. 
It works on the same principal as calcium hydroxide, which is that it releases hydroxyl ions and the produces high pH environment. The component playing a major role is calcium oxide when in contact with moisture is converted into calcium hydroxide thereby raising the pH to 12.5. This high pH is an unfavorable environment for microorganisms to thrive and grow. Hence, it has antibacterial property. The advantage over calcium hydroxide is the decreased solubility and thus maintaining the hard and excellent marginal seal.  MTA showed zones of inhibition which depicts a self-inherent antimicrobial property. This property is associated with the high pH it possesses. Initial pH is 10.2 which rise to 12.5 in 3 h.  The high pH of 12 is known to halt the growth of E. faecalis. 
E. faecalis a normal inhabitant of oral cavity is a Gram-positive cocci, facultative anaerobe, know to survive in harsh environments. It penetrates deep into dentinal tubules and thrives as it secretes serine protease, gelatinase, and collagen-binding protein (Ace). The serum secreted by periodontal ligament and alveolar bone also helps it to bind to dentin. The root canal treated teeth are nine times more likely to be having E. faecalis than the untreated infected teeth. 
Tetracycline binds to 30S ribosomal subunit leading to inhibition of protein synthesis thus having antimicrobial effect. It is a broad spectrum antibiotic. When mixed with acidic solution of pH below 2 its potency is reduced and destroyed when mixed in alkali hydroxide solution. 
This study employed agar diffusion method to evaluate the antimicrobial activity as several studies previously have used this technique.  Although it is a reliable method  formation of zones of inhibition may be altered due to variation in agar medium, bacterial strains, cellular density, and diffusion capacity of the agent. 
The diameters of zone of inhibition in this study around MTA with 2% chlorhexidine gluconate were found to be larger than other groups and also statistically significant. Chlorhexidine enhanced the antimicrobial property of MTA which could be attributed to the fact that it can be adsorbed onto and subsequently be released from cement hence substantivity makes chlorhexidine gluconate a potent antimicrobial agent.  The group where tetracycline was added to MTA showed not much difference in diameters of zones of inhibition in comparison to MTA. 13.8% of isolates of E. faecalis has reported to develop resistance against tetracycline which could have been the reason for reduced zones of inhibition.  The resistance, when developed for any member of this class, leads to cross-resistance to tetracycline.  This study has differed results when compared to Estrela et al., who showed that MTA had no antimicrobial activity against E. faecalis.
| Conclusion|| |
Within the limitation of this study 2% chlorhexidine gluconate when mixed with MTA showed to enhance its antimicrobial property.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| References|| |
Fouad AF, Zerella J, Barry J, Spångberg LS. Molecular detection of Enterococcus
species in root canals of therapy resistant endodontic infections. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2005;99:112-8.
Byström A, Sundqvist G. Bacteriologic evaluation of the efficacy of mechanical root canal instrumentation in endodontic therapy. Scand J Dent Res 1981;89:321-8.
Hasan Zarrabi M, Javidi M, Naderinasab M, Gharechahi M. Comparative evaluation of antimicrobial activity of three cements: New endodontic cement (NEC), mineral trioxide aggregate (MTA) and Portland. J Oral Sci 2009;51:437-42.
Asgary S, Kamrani FA. Antibacterial effects of five different root canal sealing materials. J Oral Sci 2008;50:469-74.
Haapasalo M, Udnæs T, Endal U. Persistent, recurrent, and acquired infection of the root canal system post treatment. Endod Topics 2003;6:29-56.
Karale R, Thakore A, Shetty V. An evaluation of antibacterial efficacy of 3% sodium hypochlorite, high-frequency alternating current and 2% chlorhexidine on Enterococcus faecalis
: An in vitro
study. J Conserv Dent 2011;14:2-5.
Alves FR, Almeida BM, Neves MA, Moreno JO, Rôças IN, Siqueira JF Jr. Disinfecting oval-shaped root canals: Effectiveness of different supplementary approaches. J Endod 2011;37:496-501.
Mohammadi Z, Giardino L, Palazzi F, Shalavi S, Farahani MF. Substantivity of three concentrations of tetraclean in bovine root dentin. Chonnam Med J 2012;48:155-8.
Schäfer E, Bössmann K. Antimicrobial efficacy of chlorhexidine and two calcium hydroxide formulations against Enterococcus faecalis
. J Endod 2005;31:53-6.
Priyanka SR, Veronica. A literature review of root-end filling materials. IOSR J Dent Med Sci 2013;9:20-5.
Tawil PZ, Duggan DJ, Galicia JC. Mineral trioxide aggregate (MTA): Its history, composition, and clinical applications. Compend Contin Educ Dent 2015;36:247-52.
Baek SH, Plenk H Jr., Kim S. Periapical tissue responses and cementum regeneration with amalgam, SuperEBA, and MTA as root end filling materials. J Endod 2005;31:444-9.
Chambers HF. Antimicrobial agents: Protein synthesis inhibitors and miscellaneous antibacterial agents. In: Hardman JG, Limbird LE, Gilman AG, editors. Goodman and Gilman′s the Pharmacological Basis of Therapeutics. 10 th
ed. New York, USA: McGraw Hill; 2001. p. 1239-72.
Torabinejad M, Hong CU, Pitt Ford TR, Kettering JD. Antibacterial effects of some root end filling materials. J Endod 1995;21:403-6.
McHugh CP, Zhang P, Michalek S, Eleazer PD. pH required to kill Enterococcus faecalis in vitro
. J Endod 2004;30:218-9.
Peciuliene V, Reynaud AH, Balciuniene I, Haapasalo M. Isolation of yeasts and enteric bacteria in root filled teeth with chronic apical periodontitis. Int Endod J 2001;34:429-34.
Dahlén G, Samuelsson W, Molander A, Reit C. Identification and antimicrobial susceptibility of enterococci isolated from the root canal. Oral Microbiol Immunol 2000;15:309-12.
Fridland M, Rosado R. MTA solubility: A long term study. J Endod 2005;31:376-9.
Estrela C, Bammann LL, Estrela CR, Silva RS, Pécora JD. Antimicrobial and chemical study of MTA, Portland cement, calcium hydroxide paste, Sealapex and Dycal. Braz Dent J 2000;11:3-9.
Parsons GJ, Patterson SS, Miller CH, Katz S, Kafrawy AH, Newton CW. Uptake and release of chlorhexidine by bovine pulp and dentin specimens and their subsequent acquisition of antibacterial properties. Oral Surg Oral Med Oral Pathol 1980;49:455-9.
Mohammadi Z, Dummer PM. Properties and applications of calcium hydroxide in endodontics and dental traumatology. Int Endod J 2011;44:697-730.