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Table of Contents   
ORIGINAL ARTICLE  
Year : 2022  |  Volume : 25  |  Issue : 5  |  Page : 536-540
Effectiveness of polyhexamethylene biguanide, chlorhexidine, and calcium hydroxide intracanal medicament against intraradicular mature polymicrobial biofilm: A microbiological study


1 Department of Conservative Dentistry and Endodontics, Subharti Dental College and Hospital, Swami Vivekanand Subharti University, Meerut, Uttar Pradesh, India
2 Department of Microbiology, Subharti Medical College and Hospital, Swami Vivekanand Subharti University, Meerut, Uttar Pradesh, India

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Date of Submission15-May-2022
Date of Decision05-Jun-2022
Date of Acceptance06-Jun-2022
Date of Web Publication12-Sep-2022
 

   Abstract 

Aim: To compare the effectiveness of different intracanal medicaments against polymicrobial biofilm formed by Enterococcus faecalis, Staphylococcus aureus, and Candida albicans.
Materials and Methods: Eighty mature human roots with intraradicular polymicrobial biofilm were randomly assigned into four groups (n = 20). Intracanal medicaments 0.2% polyhexamethylene biguanide (PHMB), 2% chlorhexidine (CHX), and calcium hydroxide (CH) were applied into the root canals. Collected dentine samples were tested at 7th, 15th, and 30th day for microbial growth, and the colony-forming units per ml (CFU/ml) were determined.
Results: The CFU/ml data were analyzed using unpaired t-test and one-way ANOVA-F comparison test. All medicaments resulted in a significant reduction (P < 0.05) in microbial growth at all time intervals compared to the control group. CHX and PHMB showed a similar reduction in CFU/ml at 7th and 15th day but significantly more than CH at all time intervals. At 30th day, PHMB caused a significantly more reduction in CFU/ml than CHX.
Conclusions: All the three tested intracanal medicaments such as CH, CHX, and PHMB Gel were effective in reducing the microbial count. CH has a limited antimicrobial effect against the polymicrobial biofilm found inside the root canal. 2% CHX gel has a time-dependent antimicrobial effect. PHMB has a superior antimicrobial effect in comparison with CHX and CH.

Keywords: Biofilm, calcium hydroxide, Candida albicans, chlorhexidine, Enterococcus faecalis, intracanal medicament, polyhexamethylene biguanide, Staphylococcus aureus.

How to cite this article:
Khan A, Nikhil V, Pandey A, Chaturvedi P. Effectiveness of polyhexamethylene biguanide, chlorhexidine, and calcium hydroxide intracanal medicament against intraradicular mature polymicrobial biofilm: A microbiological study. J Conserv Dent 2022;25:536-40

How to cite this URL:
Khan A, Nikhil V, Pandey A, Chaturvedi P. Effectiveness of polyhexamethylene biguanide, chlorhexidine, and calcium hydroxide intracanal medicament against intraradicular mature polymicrobial biofilm: A microbiological study. J Conserv Dent [serial online] 2022 [cited 2022 Sep 25];25:536-40. Available from: https://www.jcd.org.in/text.asp?2022/25/5/536/355910

   Introduction Top


Root canal infections are caused by biofilms which are polymicrobial in nature.[1] Bacteria inside biofilm are 100–1000 times more resistant to the antimicrobial agents than planktonic forms. Enterococcus faecalis, a Gram-positive anaerobic facultative coccus, has been reported as one of the most frequently isolated microorganisms from the root canals at the time of retreatment. It is known to penetrate deeply into dentinal tubules 250–400 μm, binding to collagen and hydroxyapatite, which makes its complete elimination difficult.[2] Candida albicans is a frequently isolated fungus, which can form biofilm even in nutrient-deprived conditions and can colonize dentinal tubules to depths approximately 150 μm.[3] Staphylococcus aureus is a Gram-positive facultative anaerobic coccus found commonly in postendodontic treatment lesions.[4] They have the capacity to withstand extreme conditions for long periods and resist changes in temperature and dehydration.

Reduction of endodontic microbiota has been achieved by a series of antimicrobial strategies that include root canal preparation, irrigating solutions, intracanal dressing, and root canal filling.

The main rationale behind intracanal medicament is to kill the bacteria inside the root canal, prevent growth of residual microbes, and thus avert interappointment reinfection. Calcium hydroxide (CH) has low solubility in water (around 1.2 g L) and has the dissociation coefficient of CH (0.17), which controls the slow release of both calcium and hydroxyl ions responsible for its hard-tissue deposition and antimicrobial property.[5] This low solubility is a useful clinical characteristic as an extended period is necessary before it becomes solubilized when in direct contact with fluids from vital tissues.[6]

Chlorhexidine (CHX) has antimicrobial activity against Gram-negative and Gram-positive microorganisms due to its cationic molecule binding to negatively charge bacterial cell walls, thereby altering the bacterial cell's osmotic equilibrium.[7]

Polyhexamethylene biguanide (PHMB) is a cationic biguanide polymer, which is utilized in a wide variety of antibacterial application.[8]

Thus, the present study tested the effectiveness of 0.2% PHMB, 2% CHX, and CH intracanal medicament against polymicrobial biofilm composed of E. faecalis, C. albicans, and S. aureus. The null hypothesis tested was that the different intracanal medicaments have no effect on polymicrobial biofilm.


   Materials and Methods Top


The calculated sample size (n) was 18.2 using standard deviation (1.09 from a previous study[9]), value from the table of probabilities of standard normal distribution for the desired confidence interval (Z = 1.96 for 95% confidence), and margin of error (0.05). Thus, 20 samples were used for each group.

Eighty extracted and disinfected single-canal single-rooted teeth were decoronated to obtain a standardized root length of 8 mm. Root canals were enlarged first with K-fles (#15–40, Dentsply Maillefer, Ballaigues, Switzerland) and then Gates–Glidden drills (#2,3 and 4, Mani Inc., Tachigi-ken, Japan) to standardize the diameter of root canal. Each instrumentation was followed by 3 ml of 5% NaOCl irrigation, and on completion, 3 ml of liquid ethylenediaminetetraacetic acid (Avue Prep, Dental Avenue) was irrigated for 3 min to remove smear layer. Normal saline (5 ml) was used to neutralize the residual irrigant. Outer surfaces of the specimens were covered with two coats of nail varnish and the specimens were autoclaved at 121°C for 10 min at 15 psi. Each specimen was put in separate sterile McCartney bottles (HiMedia, India) containing 5 ml sterile BHI solution (HiMedia, India).

Formation of polymicrobial biofilm

The pure culture isolates of E. faecalis (ATCC 29212), S. aureus (ATCC 25923), and C. albicans (ATCC 90028) were grown on 5% sheep blood agar.

After 24 h of culture, the isolated colonies of all the three microorganisms were inoculated in 35 McCartney bottles each containing 5.0 ml of BHI when matching the turbidity of 0.5 McFarland. The bottles containing specimens were opened under laminar flow to remove 2 ml of sterile BHI and replaced with 2 ml of bacterial inoculum with sterile pipette. Bottles were closed before giving a short vortex and kept at 37°C for 2 weeks with replacement of 1 ml of contaminated BHI for 1 ml of freshly prepared BHI after every 2 days to avoid medium saturation. The turbidity of the medium during the incubation period indicated bacterial growth. The purity of the cultures was confirmed by Gram staining and colony morphology on blood agar. The organism's ability to form biofilm was detected by tissue culture plate method, a quantified test described by Christensen et al.[10] Optical density (OD) of stained adherent biofilm was obtained using a micro-enzyme-linked immunosorbent assay autoreader (Robonik) at a wavelength of 570 nm. The interpretation of biofilm production was done according to the criteria of Stepanovic et al.[11]

Antibacterial assessment

Following the contamination period, each specimen was removed under aseptic conditions and the canal was irrigated with 5.0 ml of sterile saline and dried with sterile paper points. Specimens were randomly divided into 3 experimental groups and 1 control group according to the type of intracanal medicament used.

  • Group 1 (n = 20): CH paste (RC Cal, Prime Dental)
  • Group 2 (n = 20): 2% CHX gel (2% Gluco Chex, Cerkamed)
  • Group 3 (n = 20): 0.2% PHMB gel
  • Group 4 (n = 20): No intracanal medicament only BHI suspension.


The specimens were fixed at the bottom of wells of 96-well cell culture plates (HiMedia, India) (well volume 3.2 ml) with sticky wax (pyrax) that also obliterated the apical surface of the specimens. The vacant portion of the wells at the circumference of specimens were filled with silicone type putty material (Flexceed, GC).

Preparation of 0.2% polyhexamethylene biguanide gel

0.2% PHMB gel was prepared with 2% PHMB solution in a gel base of 1% hydroxyethyl cellulose (Natrasol, Ashland). Preparation was carried out in the College of Pharmacy.

The intracanal medicaments were applied to the canal lumen with the help of sterile 3.0 ml plastic syringes and 27-gauge needles, until the canals were full and all specimens were incubated at 37°C, for the periods of 1 week.

Dentine shavings

At the end of the experimental periods, the specimens were removed from the culture wells and the canals were irrigated with 5.0 ml sterile saline to remove all medicaments and were dried with sterile paper points. Peeso Reamers #4 and #5 (Mani Inc., Tachigi-ken, Japan), held in a low-speed torque control endomotor (Canal pro CL2, Coltene, Switzerland), were used for the sequential removal of dentine weighing approximately 4 mg. Produced dentine shavings were immediately collected into individual McCartney bottles containing 3.0 ml of freshly prepared BHI and neutralizers to avoid continued action of the medicaments. The neutralizer for CH was 0.5% citric acid (Qualigens Fine Chemicals, Mumbai, India), while 0.5% Tween 80 (Rankem) mixed with 0.07% lecithin (Sigma Aldrich, India) was used for CHX and PHMB. The bottles were incubated at 37°C for 7 days and observed for turbidity representing microbial growth. The specimens were again incubated in sterile BHI with neutralizer at 37°C.

The same procedure was conducted after 15th and 30th day. The intracanal medicaments were compared for their antibiofilm potential by observing the microbial growth seen on culture plate on 7th, 15th, and 30th day, and the colony-forming units (CFU)/ml count was done for samples which had microbial growth. The data were collected and subjected to statistical analysis using Statistical Package for the Social Sciences Version 23 (IBM Corp, New York, USA).


   Results Top


The mean and standard deviation of CFU/ml in all four groups on 7th, 15th, and 30th days was calculated [Table 1]. Comparison at successive time points with the help of unpaired t-test was done [Table 2]. Intergroup comparison was done with ANOVA test and the significance was established at 5% level [Table 3]. Antibacterial activity of all experimental intracanal medicaments reduced progressively with time. PHMB gel and CHX gel inhibited the 100% growth of the microorganisms at 7th and 15th day and showed mild growth on 30th day in few samples, whereas CH was ineffective to inhibit microbial growth at all experimental times. The control group showed viable bacteria at all experimental times with maximum CFU/ml. Among experimental groups at all time intervals, CH showed maximum and PHMB showed minimum CFU/ml.
Table 1: Mean and standard deviation of colony-forming units per ml in four groups on 7th, 15th and 30th day

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Table 2: Comparison by un-paired t-test between successive time points in four groups for colony-forming units per ml

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Table 3: Comparison by one-way ANOVA-F test among four groups on each time points for colony-forming units per ml

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   Discussion Top


Intracanal medicament is generally considered an integral part of treatment of infected root canal as it helps in eradication of microbial biofilm. The biofilm model used in the current study is based on the model developed by Haapasalo and Orstavik[12] as it attempts to replicate the heterogeneous nature of an in vivo biofilm, and the versatility and high reproducibility of this model makes it a reliable method to study the effects of treatment on biofilm removal.[13]

The choice for microorganisms used in the present study was based on microorganism's level of virulence, resistance to antimicrobial agents, and their prevalence in root canals after failed endodontic treatments. The influence of biofilm age on the effectiveness of an antimicrobial treatment has been investigated in the range of studies and it is incontestable that the use of intracanal medicament is ineffective against mature biofilm. Stojicic et al. found that the biofilm bacteria need 2–3 weeks to reach the maturation stage.[14] Hence, in this study, 2 weeks were given for the bacteria to reach the maturation stage.

Dentine was used as a substrate material for the growth of in vitro biofilm, as it signifies the biofilm's natural habitat.[15]

The most commonly used concentration of microorganisms in biofilms is 108 CFU/ml (ranging from 104 to 1010 CFU/ml). Hence, the turbidity of the final inoculum was set at 0.5 MacFarland. Colorimetric method has been used in the present study to evaluate the formation of biofilms inside the canal, as it is a rapid semiquantitative method that assesses the entire biofilm-covered surface.[16]

On comparison, the OD values obtained with polymicrobial biofilm were higher as compared to monomicrobial biofilm. Colony counting was used to judge the antibacterial efficacy of intracanal medicaments as this would signify the quantity of live residual bacteria present in the root canals. Upadhyay et al. tested the antimicrobial activity of intracanal medicaments using the culture method.[17] The advantage of this method is that it permits direct comparison of the materials against the organisms, indicating which material has the potential to eliminate bacteria in the native microenvironment of the root canal system.

The null hypothesis was rejected in the present study. The use of intracanal medicament has a significant antimicrobial effect (P < 0.05) against intraradicular polymicrobial biofilm as they were able to inhibit the microbial growth at each time interval tested as compared to the control group in which 100% of samples exhibited microbial growth. This clearly establishes the role of intracanal medicaments as an essential step in killing/inhibiting the microbes in root canals.

As it takes nearly 7 days for the CH to raise the pH of the environment to 9.0, a level at which many bacteria do not grow, thus first evaluation was done on 7th day.[18] On the 7th day, CH had shown microbial inhibition in 60% of samples as compared to CHX and PHMB where 100% growth inhibition was observed. Previously published reports have conflicting results related to effectiveness of CHX and CH against biofilm. The possible reasons are the differences in the methods and materials used, including microbiological assessments, concentrations and physical forms of CHX, periods of experiments, strains and concentrations, and methods of bacterial inoculation. 2% CHX gel was significantly more effective than CH in preventing microbial growth, which can be explained by its wide antimicrobial spectrum, as it is effective against both Gram-positive and Gram-negative bacteria as well as yeast. It acts by electrostatic interaction resulting in cell death.[19]

On testing the efficacy of intracanal medicament on the 15th day, CHX and PHMB group showed 100% negative culture, establishing their superior and sustained antimicrobial effectiveness compared to CH in which the mean CFU/ml values showed an increase. CH portraits its antimicrobial efficiency due to its alkaline pH. The decrease in antimicrobial effectiveness of CH against intracanal biofilm may be because of the buffering action of dentin, neutralizing the pH of CH affected dentin.

As very limited literature is available for the antibiofilm potential for PHMB, thus a comparison can be made based on only the available published literature. To the extent, the authors believe only a single study by Zaugg et al., exists in endodontic literature where the effect of PHMB as intracanal medicament has been evaluated on intraradicular biofilm.[20] However, Zaugg et al. used monomicrobial biofilm and the commercial preparation for PHMB, in contrast to 2 week polymicrobial biofilm of the present study and freshly prepared gel. Nonetheless, the results were supportive of significant antibiofilm potential of 2% CHX and PHMB and hence could be matched to this study result.

The result obtained in the present study regarding the antimicrobial effectiveness of CHX on the 7th and 15th day is in agreement with the study by Siqueira et al.,[21] although the increase in colony count was seen on the 30th day as mature biofilms develop the localized environment that dictate the metabolic activities of cells and protect them to some extent against changes in the environment. The PHMB displayed its potency with excellent results by eliminating biofilm at all times interval tested (P < 0.05) as compared to CHX and CH even at the 30th day. The bactericidal property of PHMB is the result of the interaction of positively charged polyhexanide molecule with a negative charge on DNA and EPS within biofilms. PHMB showed greater substantivity than CHX and similar results were seen in a study by Chandki et al.[22] that compared the substantivity of 0.2% PHMB and 2% CHX on root canal dentin at 1 h, 24 h, 7-days and 21 days and came to a similar conclusion.

The antimicrobial activity presented a time-dependent effect, especially in the case of PHMB and CHX where the CFU/ml count increased on the 30th day and a significant difference (P < 0.05) was obtained comparing their antibiofilm effectiveness on the 7th and 30th day and 15th and 30th day but there was no significant difference when the values at 7th and 15th day were compared.

The limitations of the present study are the use of a defined engineered biofilm model as known laboratory reference strains of microbial species were combined in the present study. Root canal infection is a dynamic process whereby the characteristics of the biofilm change over time. Changing environmental conditions, such as oxygen tension and nutrient availability, entail a shift in the composition of the microbiota. These variables oral conditions could not be simulated in the present in vitro study.


   Conclusions Top


All the three tested intracanal medicaments such as CH, 2% CHX gel, and 0.2% PHMB gel were effective in reducing the microbial count. CH has a limited antimicrobial effect against the polymicrobial biofilm found inside the root canal. 2% CHX gel has a time-dependent antimicrobial effect. PHMB gel has a superior antimicrobial effect in comparison with 2% CHX gel and CH when used as an intracanal medicament.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
   References Top

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[PUBMED]  [Full text]  
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20.
Zaugg LK, Zitzmann NU, Hauser-Gerspach I, Waltimo T, Weiger R, Krastl G. Antimicrobial activity of short- and medium-term applications of polyhexamethylene biguanide, chlorhexidine digluconate and calcium hydroxide in infected immature bovine teeth in vitro. Dent Traumatol 2014;30:326-31.  Back to cited text no. 20
    
21.
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22.
Chandki R, Nikhil V, Kalyan SS. Comparative evaluation of substantivity of two biguanides-0.2% polyhexanide and 2% chlorhexidine on human dentin. J Conserv Dent 2020;23:46-50.  Back to cited text no. 22
  [Full text]  

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Correspondence Address:
Dr. Vineeta Nikhil
Subharti Dental College and Hospital, Swami Vivekanand Subharti University, NH-58 Bypass Road, Meerut - 250 005, Uttar Pradesh
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jcd.jcd_288_22

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