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ORIGINAL RESEARCH  
Year : 2011  |  Volume : 22  |  Issue : 1  |  Page : 66-70
Antimicrobial effectiveness of different preparations of calcium hydroxide


Department of Pedodontics and Preventive Dentistry, Faculty of Dental Sciences, King George's Medical University, Lucknow, India

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Date of Submission22-Dec-2009
Date of Decision03-May-2010
Date of Acceptance03-Oct-2010
Date of Web Publication25-Apr-2011
 

   Abstract 

Background: Intracanal medicaments have an antibacterial effect on the root canal flora. The effectiveness of such vehicles has to be tested and substantiated against the normal bacterial flora. The various vehicles that were tested for their effectiveness were commercially prepared calcium hydroxide (Metapex), saline, glycerine, CMCP, and Rexidine-M gel. The bacterial cultures tested for sensitivity were aerobes (Staphylococcus aureus, Streptococcus viridans, Streptococcus pyogens, Enterococcus faecalis), anaerobes (Lactobacillus, Bacteroides melaninogenicus) and a commonly found fungus (Candida albicans). One hundred and twenty-five samples were divided into five experimental groups consisting of 21 samples each and one control group of the same size.
Aim: The aim of this study was to investigate in vitro the influence of four different vehicles on the effectiveness of calcium hydroxide against the commonly found aerobic and anaerobic bacteria in endodontic infections.
Materials and Methods: The samples were taken and transported using standardized techniques and pure growth of each bacterium was isolated. Petri dishes were prepared for the various calcium hydroxide combinations for each bacterium and incubated. The inhibition zone was recorded at three intervals of 24, 96and 168 hours. Results were tabulated and sent for statistical analysis.
Results and Conclusions: It was seen that calcium hydroxide and CMCP combination showed the maximum zone of inhibition, and maximum inhibitory effect was seen at 24 hours. The bacteria most susceptible was found to be S. aureus and the least susceptible was E. faecalis. Further clinical studies are required to substantiate these results.

Keywords: Calcium hydroxide, in vitro, Staphylococcus aureus, Enterococcus faecalis

How to cite this article:
Gangwar A. Antimicrobial effectiveness of different preparations of calcium hydroxide. Indian J Dent Res 2011;22:66-70

How to cite this URL:
Gangwar A. Antimicrobial effectiveness of different preparations of calcium hydroxide. Indian J Dent Res [serial online] 2011 [cited 2020 Jul 10];22:66-70. Available from: http://www.ijdr.in/text.asp?2011/22/1/66/79986
The most common cause of dental pulp injury is bacterial assault. Bacteria or bacterial products may enter the pulp via various routes.

It has been well established that the endodontic infections are polymicrobial in nature. The microorganisms most frequently isolated from the infected root canals are streptococci and micrococci. The development of various new techniques for the growth and isolation of obligate anaerobes has shown that anaerobic microorganisms are much more prevalent root canal species than were earlier thought to be. The primary goal of an endodontic treatment is to achieve a bacteria free environment in the root canal in order to achieve clinical success.

Normally, the infected tissue removal is achieved by irrigating the canal with proteolytic disinfecting solution and thorough biomechanical cleaning. After all the infected and necrotic pulp tissue is removed, the root canal is disinfected, enlarged and shaped to allow optimal root canal filling. However, with the use of conventional chemomechanical technique of root canal treatment preparation, neither the removal of soft tissue is complete nor does the elimination of bacteria seem to be sufficient. Inability to completely eradicate the microorganisms and/or provide bacteria proof seal can result in root canal therapy failure. Hence, the use of intracanal medicaments has been advocated to further reduce the number of microorganisms.

Hermann [1] introduced calcium hydroxide and its various preparations for clinical application. Initially, calcium hydroxide was used in vital pulp therapy; its emergence as an intracanal medicament has almost became a panacea in multibacterial infections of root canals. Its various effects include: (1) microbial control, (2) healing periapical inflammation, (3) arresting inflammatory root resorption, (4) stimulating hard tissue formation in apexification procedure and (5) serving as a temporary obturating material between appointments.

An antibacterial intracanal medicament must have a wide spectrum of activity and a reasonable duration of action to eliminate all the bacteria in the root canal. However, till date, no intracanal medicament has proved to be active against the whole spectrum of microorganisms. The combination therapy of antimicrobial agents chosen should cover the most common known or suspected pathogen in polymicrobial infection in order to obtain enhanced inhibition.

As per the available literature, various calcium hydroxide preparations have been tried so as to utilize its antibacterial potential. The complex synergistic interaction between microbiota and pathological process has called for scientific research to study the antimicrobial properties of therapeutic agent, which would further add to the benefits of therapy. Keeping the above fact in view, the present in vitro study was undertaken.

This study aims to evaluate the antimicrobial properties of different preparations of calcium hydroxide on specific, most commonly associated microorganisms found within infected root canal.


   Materials and Methods Top


This "in vitro" study was conducted on 125 samples in the outpatient Department of Pedodontics and Preventive Dentistry.

Criteria for collection of samples

  1. After thorough oral prophylaxis, the patient was advised to maintain oral hygiene properly.
  2. All the samples were collected under rubber dam isolation to avoid any contamination in test samples.
Collection of sample

The selection of tooth for sample was made after thorough history taking, clinical, radiological examination, routine investigations, and proper oral hygiene maintenance. None of the patients selected for the study received antibiotic therapy within previous 15 days or during the course of treatment so that the antibiotic does not alter the growth of bacteria in present study. Precaution was taken to see to that the selected patient did not present with any subjective and objective signs of systemic or chronic debilitating diseases. Permanent uniradicular teeth exhibiting no vitality were selected for sampling, irrespective of sex and socioeconomic status. The patients' age ranged from 10 to 14 years. The whole procedure was carried out under sterile conditions to avoid any contamination.

One hundred teeth were selected for the collection of test samples. Special care was exercised to use aseptic technique for the access opening and procuring the samples from the root canals without the use of any irrigant. Access to all selected cases of root canal treatment was gained using the standardized technique of root canal preparation. A sterile paper point was then placed to full length of the root canal for 60 seconds. The paper point sample from the root canal was preserved in tight screw capped bottle containing the transportation media. These samples were then immediately transferred to the Bacteriology Laboratory of the Department of Microbiology. The test samples were inoculated in Robertson's cooked meat broth and incubated at temperature of 37 ± 1°C for 24 hours in aerobic and anaerobic atmosphere before subjecting them to various experimental procedures.

Materials and instruments

Media

  1. Transportation media

    • Thioglycollate broth
    • Robertson's cooked meat broth
  2. Calcium hydroxide powder® assay 96% (Rolex Chemical Limited, Mumbai, India)
  3. Metapex (Meta Dental New York, Elmhurst, USA)
  4. Sterile N-saline -sodium chloride injection IP (0.9% w/v) (Core Health Care Limited, Ahemdabad, Gujrat, India.)
  5. "Excela R" (Qualigens Fine Chemicals Limited, Mumbai, India)
  6. Camphorated paramonochlorophenol (trade name Cresophene) (Septodont Saint Maur des - Sosses Cedex, France)
  7. Rexidine-M gel (Indoco Remedies Ltd., Maharashtra, India)
Instruments

  1. Sterile paper point (Dentsply India Pvt. Ltd., Delhi, India)
  2. Platinum loop
  3. Incubator
  4. Test tube stand
Procedure

Incubation of samples

Samples collected from the patient were inoculated in Robertson's cooked media and incubated at a temperature of 37 ± 1 o C.

Experimental aerobic and anaerobic bacterial isolates used in this study were:

Aerobes

Staphylococcus aureus

Streptococcus pyogens

Streptococcus viridans

Enterococcus faecalis

Candida albicans (fungi)


Anaerobes

Lactobacillus species

The aerobeswere isolated from clinical trials. The anaerobes were obtained from Department of Microbiology, Sanjay Gandhi Postgraduate Institute of Medical Sciences, Lucknow.

Each microbial strain was evaluated against calcium hydroxide formulation prepared with different vehicles as follows [Figure 1]:

  1. Metapex
  2. Calcium hydroxide + N-saline
  3. Calcium hydroxide + glycerine Excela "R"
  4. Calcium hydroxide + camphorated paramonochlorphenol
  5. Calcium hydroxide + Rexidine-M gel (chlorhexidine + metronidazole)
  6. Control group (no preparation used)
Figure 1: Distribution of preparations used

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The antimicrobial effect of various calcium hydroxide formulations was evaluated at various time intervals, i.e., 1 hour, 24, 96 and 168 hours. The antimicrobial effect was evaluated by measuring the zone of inhibition.

The various calcium hydroxide pastes were prepared using calcium hydroxide powder. The consistency of various calcium hydroxide pastes was similar to that of tooth paste, with the viscosity of 3501 cP at 0.1 rpm and pH of 12.5 as determined by a digital pH meter.

Agar diffusion method

Incubation of trypticase soy blood agar and Muller Hinton agar: To test the effectiveness of various preparations of calcium hydroxide by agar diffusion method, respective agar plates inoculated with aerobic bacteria were incubated at 37°C. For anaerobes, respective anaerobic agar plates were placed in anaerobic jars (CO 2 incubator) in an atmosphere of 10% carbon dioxide gas + 10% hydrogen gas + 80% nitrogen gas and incubated at 37°C.

Observations were made to detect the zone of bacterial inhibition aroundeach medicament at various time intervals, i.e., 1, 24, 96 and 168 hours. Zones of inhibition were measured using Vernier Calipers and divider. Inhibitory growth of 21 microorganisms was evaluated against each preparation of calcium hydroxide.

Statistical analysis was done using the analysis of variance (ANOVA) test and the Student's t test


   Results Top


Analysis of zone of inhibition around each medicament against microorganisms was done [Figure 2] and [Figure 3].The zone showed no changes after 1 hour and no inhibition of bacteria was seen in the control group.
Figure 2: Slide showing E. faecalis growth after 24 hours

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Figure 3: Slide showing S. aureus growth after 24 hours

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The mean inhibition scores for different groups after 24 hours were as follows:

Group D > Group C > Group E > Group A > Group B

The orders of mean inhibition scores for different groups after 96 hours were as follows:

Group D > Group C > Group E > Group A > Group B

The mean inhibition scores for different groups after 168 hours were as follows:

Group D > Group C > Group E > Group A > Group B

[Figure 4] shows the overall scores for different bacterial strain inhibition as against all the experimental medicaments. S. aureus shows the maximum score of inhibition at all time zones while E. faecalis consistently shows the least inhibition at all time zones.
Figure 4: Bar graph showing bacterial growth against all calcium hydroxide combinations

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[Figure 5] shows the overall inhibition scores for different medicaments against all the bacterial strains under study. Group D showed the maximum effectiveness at all time periods while Group B showed the least effectiveness.
Figure 5: Bar graph showing calcium hydroxide combinations against bacterial growth

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   Discussion Top


In the past, numerous antimicrobial agents had been used as root canal medicaments. These included traditional phenolic and fixative agents such as camphorated monochlorophenol, formocresol, eugenol, metacresylacetate and halides (iodine, potassium iodide).

The current intracanal dressing of choice is calcium hydroxide, a white odorless powder with high pH and low solubility in water. Delivery of dry calcium hydroxide powder alone is difficult or impossible to use in smaller or curved canals. In most cases, calcium hydroxide must be mixed with a liquid to facilitate placement. Calcium hydroxide is normally used as slurry of calcium hydroxide in a water base.

A sterile paper point, as per the method of M. Georgopolou et al.[2] was inserted into canal and sample was taken. This was done to ensure standardization of technique for collection of samples.

For in vitro preparations, the most common method for antimicrobial activity assessment is agar diffusion method, as also supported by previous studies. [3]

The zones of bacterial inhibition around each medicament was used as the criteria for comparison of the effectiveness of the medicament. In control cases, no formulation of calcium hydroxide preparation was used for bacterial counteraction.

All the preparations of calcium hydroxide showed inhibitory growth but it was found that S. aureus showed maximum inhibition, whereas E. faecalis was the least inhibited bacterial strain. Among the different preparations of calcium hydroxide, calcium hydroxide with camphorated paramonochlorophenol showed the best result, while calcium hydroxide with saline showed minimum zone of inhibition.

In this study, inhibitory effect of calcium hydroxide preparation was seen optimum at 24 hours with slight change occurring at 96 hours. However, at 168 hours, some bacterial growth was observed. Acidic pH favors microbial growth; however, after application of calcium hydroxide, alkaline pH is obtained. Due to disassociation of calcium hydroxide into Ca ++ and OH- ions, these ions exert lethal effect on bacteria. With time, calcium hydroxide diffuses into agar media to a great extent, thereby resulting in dilution of Ca ++ and OH- ions, which in return decreases the pH of the media. This reduced pH is responsible for further growth of microorganisms.

Castagnola and Orlay [4] reported that iodoform pastes destroy the microorganisms in tissue remnants and render them unsuitable for supporting microbial life.

Result of the present study is in accordance with that of Tchaou et al[5] They showed that there is no/minimal antibacterial effect with Metapex/Vitapex. Pabla et al[6] reported least antimicrobial activity of Metapex/Vitapex against aerobic and anaerobic bacteria in comparison to zinc oxide, eugenol, KRI paste and MAISTO paste in their study.

The results of our study showed that combination of calcium hydroxide and saline provides the minimum inhibition zones for all strains of microorganisms.

In this study, the inhibitory effect of calcium hydroxide with glycerine was found to be inferior to that of calcium hydroxide with camphorated paramonochlorophenol combination, as also reported by Gomes et al.[7] They concluded that antibacterial effect of calcium hydroxide in combination with glycerine was lesser than that of calcium hydroxide in combination with camphorated paramonochlorophenol. This may be due to the release of camphorated paramonochlorophenol from the paste that increases the calcium hydroxide diffusibility. [8] Furthermore, when calcium hydroxide is mixed with camphorated paramonochlorophenol, it yields calcium paramonochlorophenolate (PMC), which is a weak salt that progressively releases paramonochlorophenol and hydroxyl ions to the surrounding medium, which are both bactericidal. The present experiment showed that the combination of both (calcium hydroxide + camphorated paramonochlorophenol) was found to be more lethal for bacteria.

Combination of calcium hydroxide with Rexidine-M (combination of metronidazole and chlorhexidine) showed inhibition of bacterial zone in all microbial strains at different time intervals, but minimal range of inhibition in comparison to calcium hydroxide + camphorated paramonochlorphenol and calcium hydroxide + glycerine Excela"R".


   Conclusions Top


The following conclusions can be drawn from the study conducted:

  • Calcium hydroxide + camphorated paramonochlorophenol showed the best results in inhibiting microbial growth.
  • Among the microorganisms tested, S. aureus was found to be most susceptible to the bactericidal action of calcium hydroxide preparations, whereas E. faecalis was found to be least susceptible.
  • According to the time taken in achieving the antimicrobial efficacy, maximum inhibitory effect was noticed after 24 hours.
The results of this study are encouraging and we can safely conclude that although the present study is an in vitro one, yet taking a clue from this, further in vivo studies are recommended, the results of which must be carefully analyzed before their extrapolation to clinical conditions with caution.


   Acknowledgments Top


The author would like to thank Dr. Pandey, Dr. Loomba, Dr. Amita Jain and Dr. Vandana Tiwari for all their assistance in thsis endeavor. The author also would like to acknowledge the help of all her Post graduate colleagues, and teaching staffs and non-teaching staffs of Department of Pediatric Dentistry.

 
   References Top

1.Hermann BW. Calcium hydroxyl als mittel zum behandein und fullen von wurzelkanalen. Dissertation, Germany: University of Wurzberg, 1920.  Back to cited text no. 1
    
2.Georgopoulou M, Kontakiotis E, Nakau M. In vitro evaluation of effectiveness of Calcium hydroxide and paramono-chlorophenol on anaerobic bacteria from the root canal. Endod Dent Traumatol 1993;9:249-53.  Back to cited text no. 2
    
3.Siqueira JF Jr, de Uzeda M. Influence of different vehicles on the antibacterial effects of calcium hydroxide. J Endod 1998;24:663-5.  Back to cited text no. 3
[PUBMED]  [FULLTEXT]  
4.Castagnola L, Orlay HG. Treatment of gangrene of the pulp. Br Dent J 1952;93:93-102.  Back to cited text no. 4
    
5.Tchauo WS, Turing BF, Minan GE, Coll JA. In vitro inhibition of bacteria from root canals of primary teeth by various dental materials. Pediatr Dent 1995;17:351-5.  Back to cited text no. 5
    
6.Pabla T, Gulati MS, Mohan U. Evaluation of antimicrobial efficiency of various root canal filling materials for primary teeth. J Indian Soc Pedod Prev Dent 1997;15:134 40.  Back to cited text no. 6
    
7.Gomes BP, Ferraz CC, Rosalin PL. Susceptibility of selected micro-organisms to intracanal medicaments. Int Endo J 2000;33:729.  Back to cited text no. 7
    
8.Holland R, Souza V, Nery MJ, Filho JA. A histological study of the effect of Ca(OH) 2 in the treatment of Pulpless teeth of dogs. Int Endo J Br Soc 1979;12:15-23.  Back to cited text no. 8
    

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Correspondence Address:
Anshul Gangwar
Department of Pedodontics and Preventive Dentistry, Faculty of Dental Sciences, King George's Medical University, Lucknow
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0970-9290.79986

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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]

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