|Year : 2013 | Volume
| Issue : 1 | Page : 18-21
Effectiveness of calcium hydroxide plus points and chlorhexidine activ points against Enterococcus faecalis by agar diffusion test: An in-vitro study
Venkatesh Alagarsamy1, AV Rajesh Ebenezar2, MR Srinivasan3, Ajit George Mohan4, Senthil Kumar5
1 Department of Conservative Dentistry and Endodontics, Sree Balaji Dental College & Hospital, Narayanapuram, India
2 Department of Conservative Dentistry and Endodontics, SRM Dental College, Chennai, India
3 Department of Conservative Dentistry and Endodontics, Sri Venkateshwara Dental College, Chennai, India
4 Dr. Ajit's Dental and Medical Centre, Angamaly, Cochin, Kerala, India
5 Department of Conservative Dentistry and Endodontics, Saveetha Dental College, Chennai, India
|Date of Web Publication||29-Apr-2013|
Department of Conservative Dentistry and Endodontics, Sree Balaji Dental College and Hospital, Narayanapuram, Chennai
Source of Support: None, Conflict of Interest: None
Aims/Objectives: The aim of this study was to evaluate the antimicrobial effectiveness of two intracanal medicaments and their combination against Enterococcus faecalis, which are commonly found in root canals. Materials and Methods: In this study, the antimicrobial effect of chlorhexidine activ points, calcium hydroxide plus points and combination of both points after 1 st day, 2 nd day, 3 rd day, 7 th day, and 14 th day were evaluated using agar diffusion test. Anti-bacterial activity as evidenced by inhibitory zones of clearance around the medicament was observed and diameters (in mm) measured. Results: Chlorhexidine activ points show the maximum inhibition against E. faecalis calcium hydroxide points alone and when combined with chlorhexidine shows the minimal antibacterial efficacy. Conclusion: Chlorhexidine activ points obtained the highest antimicrobial activity compared with other medicament and can be the medicament of choice.
Keywords: Antimicrobial effect, calcium hydroxide plus point, chlorhexidine activ point, Enterococcus faecalis, intracanal medicament
|How to cite this article:|
Alagarsamy V, Rajesh Ebenezar A V, Srinivasan M R, Mohan AG, Kumar S. Effectiveness of calcium hydroxide plus points and chlorhexidine activ points against Enterococcus faecalis by agar diffusion test: An in-vitro study. J Res Dent 2013;1:18-21
|How to cite this URL:|
Alagarsamy V, Rajesh Ebenezar A V, Srinivasan M R, Mohan AG, Kumar S. Effectiveness of calcium hydroxide plus points and chlorhexidine activ points against Enterococcus faecalis by agar diffusion test: An in-vitro study. J Res Dent [serial online] 2013 [cited 2020 Sep 29];1:18-21. Available from: http://www.jresdent.org/text.asp?2013/1/1/18/111228
| Introduction|| |
Bacteria have a major role in the success of an endodontic treatment as mechanical preparation alone cannot effectively eliminate bacteria from dentinal tubules and other irregularities in the root canal.  Few remnant micro-organisms can multiply between appointments often reaching the same level as at the start of the previous session. These observations call for an effective intra-canal medication that will help to disinfect the root canal system. This medication should be effective throughout its application and eliminate bacteria from the main canal and from irregular spaces such as dentinal tubules, lateral canals, apical deltas, and isthmus. 
Calcium hydroxide has been used in dentistry from the 1930s. It has been considered to be the material of choice as an intra-canal medicament because of its superior activity and reduced cytotoxicity to the periradicular tissue. Action of calcium hydroxide on common endodontic pathogen is due to its high alkalinity. It damages the bacterial cytoplasmic membrane.  Chlorhexidine has been used in dentistry from the 1950s chlorhexidine has been found to have a broad spectrum antimicrobial action and relative absence of toxicity. The antimicrobial mechanism is related to its cationic bisguanide molecular structure. It acts by adsorbing on to the micro-organism cell wall and causing intracellular component leakage. Calcium hydroxide when combined with chlorhexidine has been shown to possess better antimicrobial properties than Ca(OH) 2 when used alone. ,,
Enterococcus faecalis, gram-positive bacteria is often associated with persistent root canal infections. It has the ability to invade deeply into the dentinal tubules and can resist most of the bactericidal medicaments used in endodontics. ,
Calcium hydroxide points are ready to use, firm, yet flexible for easy introduction in to the canal. The constituents of calcium hydroxide plus points include 52% calcium hydroxide, 42% gutta-percha, sodium chloride, surfactant, and coloring agents. Pure calcium hydroxide is homogenously distributed throughout the gutta-percha matrix. Sodium chloride and the surfactant improve the solubility of calcium hydroxide and the mobility of the ions. The points are ISO standard and are light brown in color for easy identification. A drop of sterile water is used together with calcium hydroxide points for the initial release of ions. After inserting into the canal, sufficient fluid flows into the space between the point and canal wall from dentinal tubules and apical area to activate the calcium hydroxide. The moisture is sufficient to cause dissociation of ions from the point. 
Chlorhexidine impregnated gutta-percha points (chlorhexidine activ points) are ready to use, firm, yet flexible for easy introduction in to the canals. The ingredients include chlorhexidine diacetate 5%, gutta-percha, zinc oxide, barium sulfate and coloring agents. The chlorhexidine diacetate is homogeneously distributed throughout the gutta-percha matrix. The ISO points are colored orange to prevent confusion with gutta-percha or calcium hydroxide points. Chlorhexidine diacetate is released into the fluid which flows in to the canal through the dentinal tubules and apex immediately after drying. The point allows sufficient chlorhexidine diacetate to be applied effectively in the canal. According to the manufacturer, when chlorhexidine comes in contact with moisture it releases cations which combines with the anionic molecules on the surface of cell wall of bacteria causing osmosis to malfunction.  The objective of this study was to evaluate and compare the antimicrobial activity of calcium hydroxide points and chlorhexidene points and their combinations against Enterococcus faecalis at the end of 1 st day, 2 nd day, 3 rd day, 7 th day, and 14 th day.
| Materials and Methods|| |
Calcium hydroxide plus points, chlorhexidine activ points and combination of both were studied for their antibacterial effect. The E. faecalis (ATCC) 27812 strains was inoculated for 4 hours and adjusted to 0.5 Macfarland's standard. The test strain adjusted to 0.5 Macfarland's standard was inoculated in the Mueller Hinton agar. In the Mueller Hinton agar the test materials calcium hydroxide plus points, chlorhexidine activ points, their combinations with a drop of saline for their release of material from those points and vancomycin 30 mcg (positive control) were placed on the surface of the agar and the plates were incubated at 37°C. The zone of inhibition around the points and the positive control were measured at the end of 1 st day, 2 nd day, 3 rd day, 7 th day, and 14 th day, respectively. The measured zones were recorded. And the interpretation was made according to the zones of inhibition which was measured in millimeters.
The data were statistically analyzed using one-way analysis of variance followed by Tukey-Honestly significant difference HSD-procedure. Statistical significance was defined as P < 0.05. The significance between the groups was determined by modified least significant difference LSD (Bonferroni) test with significance level 0.05 [Table 1].
- Group A: Chlorhexidine activ points.
- Group B: Calcium hydroxide plus points.
- Group C: Combination of chlorhexidine activ points and calcium hydroxide plus points.
- Group D: Vancomycin (control).
|Table 1: The multiple range tests: Modified least significant difference (Bonferroni) test with significance level 0.05|
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| Results|| |
[Table 2] shows the mean value of zone of inhibition measured in millimeter after 1 st day, 2 nd day, 3 rd day, 7 th day, and 14 th day. The mean value of zone of inhibition gradually increases as the day progresses and at the 14 th day it reaches the maximum value for all the three groups. Among the groups, Group II (chlorhexidine activ points) shows the maximum zone of inhibition followed by Group III (combination of chlorhexidine activ points and calcium hydroxide activ point). Group I (calcium hydroxide plus point) shows the least value in the zone of inhibition among all the groups.
|Table 2: The mean value of zone of inhibition measured in millimeter in 10 samples per group after 1st day, 2nd day, 3rd day, 7th day, and 14th day|
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Anti-bacterial activity as evidenced by inhibitory zones of clearance around the medicament were observed and diameter (in mm) measured and recorded after 1 st day, 2 nd day, 3 rd day, 7 th day, and 14 th day. Based on the zones of inhibition the antibacterial effect of the medicaments were tabulated and ranked from strongest rank (1) to weakest rank (4).
- Positive control (Group D) - vancomycin: Rank 1.
- Chlorhexidine "activ points" points (Group A): Rank 2.
- Combination (Group C): Rank 3
- Calcium hydroxide plus points (Group B): Rank 4.
| Discussion|| |
Bacteria and their products play a major role in initiation and perpetuation of pulpo-periapical pathosis (i) root canal infections are polymicrobial infections in which anaerobic bacteria can make up as much as 90% of the cultivable flora and (ii) polymicrobial infections often are more pathogenic than those involving single organisms due to bacterial synergy. 
The microbiota associated with failed cases differs markedly from that reported in untreated teeth. In untreated teeth primary endodontic infection is typically a mixed infection. In primary infection, Gram-negative bacteria are dominant whereas secondary endodontic infection is usually due to few bacterial species, generally Gram-positive bacteria. 
The microbial flora of the re-treatment cases, however, has been characterized as mono infections of predominantly Gram-positive micro-organisms with approximately equal proportions of facultative and obligate anaerobes. E. faecalis, a facultative Gram-positive coccus, is the most frequently isolated species and may also sometimes be the only isolate. 
In this study, E. faecalis was chosen as the test species because of its implication as a possible microbial factor in therapy resistant apical periodontitis. It is a non-fastidious microbe that is easy to culture and has been shown in vitro to predictably penetrate deeply into human dentinal tubules. The ability of cells of E. faecalis to cause periapical disease and chronic failure of an endodontically treated tooth may be due to their ability to invade dentinal tubules and remain viable with in the tubule. E. faecalis remain viable and maintain the capability to invade dentinal tubules and adhere to collagen in the presence of human serum. This mechanism of E. faecalis within the radicular dentinal tubules acts as a pathogen in failed endodontically treated teeth. 
Vancomycin were ranked 1 and showed very good zone of inhibition in this study. Hence it was used as positive control. Vancomycin is a unique glycopeptide structurally unrelated to any currently available antibiotic. It also has a unique mode of action inhibiting the second stage of cell wall synthesis of susceptible bacteria. There is also evidence that vancomycin alters the permeability of the cell membrane and selectively inhibits ribonucleic acid synthesis. Induction of bacterial L phase variants from susceptible organisms with vancomycin is extremely difficult, and such variants are unstable. Stable L phase variants induced by other agents are susceptible to vancomycin. Vancomycin is active against a large number of species of Gram-positive bacteria, such as Staphylococcus aureus (including methicillin-resistant strains), Staphylococcus epidermidis luding multiple resistant strains), Streptococcus pneumoniae luding multiple resistant strains) Streptococcus pyogenes, Streptococcus agalactiae, Streptococcus bovis, Streptococcus mutans, Viridans streptococci, Enterococci, Clostridium species, Diptheriods, Listeria monocytogenes, Actinomyces species, and Lactobacillus species. There has been no increase in resistance to vancomycin during the past three decades.  The results of this study also showed good response with zone of inhibition, being maximum for vancomycin.
Calcium hydroxide plus points were ranked 4 and showed very less zone of inhibition in this study. In this study, the medium used was agar, a solid medium which could have made the diffusion of hydroxyl ions from calcium hydroxide more difficult. The absence of bacterial growth inhibition by calcium hydroxide in agar plates may have been due to its low diffusibility in agar or to the buffering ability of the artificial media reducing its pH.  Holland et al. stated that it could be hypothesized that calcium hydroxide on the surface of gutta-percha points could react with carbon dioxide, that will form calcium carbonate, a product that will not produce the same effect as calcium hydroxide.  Azabal-Arroyo et al. stated that the low alkanising power of calcium hydroxide, gutta-percha points could be due to the disposition of calcium in the gutta-percha matrix that somehow impedes the release of the calcium hydroxyl ions. 
Chlorhexidine activ points were ranked 2 and showed very good zone of inhibition. Chlorhexidine acts by absorbing onto the cell wall of the micro-organisms and causing leakage of intracellular components. At low concentrations, small molecular weight substances will leak out especially potassium and phosphorous resulting in a bacteriostatic effect. At higher concentration chlorhexidine has a bactericidal effect due to precipitation and coagulation of cytoplasm probably caused by protein cross-linking. Lin et al. stated that activ points seem to be an effective intra canal medicament with high penetration ability in to dentinal tubules and strong anti-bacterial properties. 
Combination of chlorhexidine activ points and calcium hydroxide plus points were ranked 3 and showed minimal zone of inhibition. The combination proved to be superior to calcium hydroxide alone. According to Lin et al. combination of calcium hydroxide and chlorhexidine should demonstrate additive or even synergistic antibacterial action reaching a broader spectrum of endodontal pathogens than the single substances. However, the pH dependent solubility of chlorhexidine could have reduced the potential benefit of such a combination. 
| Conclusion|| |
Based on the current experimental design, the following conclusions could be made:
- Chlorehexidine activ points obtained the highest antimicrobial activity compared with other medicament.
- Combination of both shows higher antimicrobial activity when compared with calcium hydroxide plus points alone.
- Calcium hydroxide plus points reveals significantly less antimicrobial activity.
- Chlorehexidine points can be the medicament of choice since they are more effective against the microbes that are routinely found in the infected root canals. Further they can exert substantial antimicrobial activity.
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[Table 1], [Table 2]