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ORIGINAL ARTICLE
Year : 2021  |  Volume : 13  |  Issue : 6  |  Page : 1538-1543  

Comparative evaluation of antimicrobial efficacy of calcium hydroxide, triple antibiotic paste, and 2% chlorhexidine combined with 0.5% cetrimide against Enterococcus faecalis biofilm-infected dentin model: An In vitro study


Department of Conservative Dentistry and Endodontics, Saraswati Dental College, Lucknow, Uttar Pradesh, India

Date of Submission29-Mar-2021
Date of Decision27-Apr-2021
Date of Acceptance01-May-2021
Date of Web Publication10-Nov-2021

Correspondence Address:
Gaurav Jain
Associate Professor, Department of Conservative Dentistry and Endodontics, Saraswati Dental College and Hospital, 233, Tiwari Ganj, Faizabad Road, Lucknow - 227 105, Uttar Pradesh
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jpbs.jpbs_277_21

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   Abstract 


Background: Enterococcus faecalis is the most common and important microorganism found in infected root canals associated with persistent periapical periodontitis and failing endodontically treated tooth. Intracanal medicaments used after chemomechanical preparation of an infected root canal play a vital in eradication of this microorganism and pave the way for long-term success of endodontic therapy. Hence, the present in vitro study was conducted to assess the antimicrobial efficacy of calcium hydroxide (Ca(OH)2), triple antibiotic paste (metronidazole 400 mg + minocycline 100 mg + ciprofloxacin 500 mg), and 2% chlorhexidine (CHX) combined with 0.5% cetrimide on eradication of E. faecalis biofilm. Materials and Methods: Eighty dentin specimens were taken and infected extraorally with E. faecalis to induce microbial colonization. The specimens were then divided into four groups of twenty each based on medicaments used and further subdivided into two subgroups based on assessment of live cells done either immediately after the elimination of the medicament or after 24-h incubation in brain–heart infusion (BHI) medium: Group I specimens were treated with Ca(OH)2, Group II with triple antibiotic paste, Group III with 2% CHX combined with 0.5% cetrimide, and Group IV with saline (control) for 7 days at 37°C. Assessment of live cells was done using confocal microscope. Results: 2% CHX combined with 0.5% cetrimide (Group III) and triple antibiotic paste (Group II) showed a statistically significant result with high antimicrobial efficacy and lower percentage of live cells as compared to Ca(OH)2 (Group I). The mean percentage of live cells in Group I immediately after elimination of medicaments was 64.7%, in Group II was 1.52%, in Group III was 1.49%, and in Group IV was 83.4%. After 24 h of incubation in BHI medium, 2% CHX combined with 0.5% cetrimide (Group III) showed a statistically significant (p < 0.05) result of 1.27% mean live cells as compared to 84.2% in Ca(OH)2 (Group I), 1.82% in triple antibiotic paste (Group II), and 94.2% in saline (Group IV control). Conclusion: 2% CHX combined with 0.5% cetrimide exhibited maximum antimicrobial efficacy with least number of mean live cells followed by triple antibiotic paste as compared to Ca(OH)2. Based on these findings, 2% CHX combined with 0.5% cetrimide was most effective in eradicating E. faecalis from the extraorally infected dentine biofilm.

Keywords: Cetrimide, chlorhexidine, endodontic therapy, intracanal medicament, triple antibiotic paste


How to cite this article:
Shreya, Jain G, Srinkhala, Singh P, Agarwal K. Comparative evaluation of antimicrobial efficacy of calcium hydroxide, triple antibiotic paste, and 2% chlorhexidine combined with 0.5% cetrimide against Enterococcus faecalis biofilm-infected dentin model: An In vitro study. J Pharm Bioall Sci 2021;13, Suppl S2:1538-43

How to cite this URL:
Shreya, Jain G, Srinkhala, Singh P, Agarwal K. Comparative evaluation of antimicrobial efficacy of calcium hydroxide, triple antibiotic paste, and 2% chlorhexidine combined with 0.5% cetrimide against Enterococcus faecalis biofilm-infected dentin model: An In vitro study. J Pharm Bioall Sci [serial online] 2021 [cited 2022 Aug 13];13, Suppl S2:1538-43. Available from: https://www.jpbsonline.org/text.asp?2021/13/6/1538/330061




   Introduction Top


Root canal therapy provides an opportunity to maintain an endodontically compromised tooth due to infection or trauma in proper form and function in oral cavity. Unlike mature and fully developed permanent tooth, it is well established through previous available literatures that endodontic management of an infected, necrotic immature permanent tooth with incomplete root formation poses a great challenge during treatment due to the presence of thin fragile roots and blunderbuss root canals.[1],[2],[3],[4],[5],[6] The reason for the limited treatment via mechanical debridement is the presence of thin dentinal root canal walls,[7] as mechanical cleaning with endodontic instruments further weakens the already thin root canal walls, leaving the root weakened and more prone to fracture.[8],[9]

Due to difficulty in instrumentation, the use of irrigating solutions and intracanal medicaments can be the only reliable method for disinfection.[10] Further, the obturation of such root canals having immature root and open apex without extending the obturating material into the periapical tissue is itself clinically challenging, as large apical opening does not provide any mechanical stop.[8] To overcome this clinical situation, many clinical strategies have been proposed which include conventional apexification using calcium hydroxide (Ca(OH)2), apical plug formation using mineral trioxide aggregate, or more recently introduced regenerative endodontic procedure involving revascularization.[2],[8],[11],[12] Although conventional procedures allow root apex closure by forming hard tissue barrier at root apex, but they do not reinforce already thin and weakened roots as they fail in stimulating root development.[1],[2] However, revascularization allows replacement of damaged pulp with a noninfected living tissue to initiate physiologic root maturation by increasing the root thickness and length and allowing apex closure.[13]

Numerous intracanal medicaments are widely used in the process of apexification of immature root. Among these Ca(OH)2 is the most common and extensively used medicament, that aid in formation of hard tissue barrier at open root apex.[14] By the process of apexification, an environment is created that helps in placement of root canal obturating material well within the root canal space. The only limitation of this method is fracture of radicular portion, as Ca(OH)2 is found to significantly reduce dentin flexure strength, microhardness, and root resistance to fracture owing to its failure to deposit dentin at the radicular dentin wall.[15],[16],[17],[18]

Hence, for these reasons, endodontic regeneration involving revascularization has gained popularity over years. For a successful revascularization process and stimulation of residual stem cells to achieve continued root development along with hard tissue deposition in an immature tooth, adequate root canal disinfection creating aseptic condition is a prerequisite.[8] The American Association of Endodontists recommends the use of sodium hypochlorite (NaOCl) as root canal irrigant for debridement of necrotic pulp followed by application of intracanal medicament which is considered as an essential component in achieving complete root canal disinfection.[19] The intracanal medicaments used for this procedure should not negatively affect the physical and mechanical properties of radicular dentin.[20]

Literatures suggest that disinfection and subsequent placement of matrix in root canal helps in cell ingrowth.[21] use of irrigating solutions such as NaOCl followed by placement of intracanal medicaments like triple antibiotic paste promote root canal disinfection thereby creating a favorable environment for proliferation of pulp-like tissue after blood induction within the canal, thus aiding in revascularization.[20] Although the use of triple antibiotic paste as intracanal medicament provides an effective root canal disinfection, it also possesses disadvantages such as discoloration and particularly stem cell toxicity because of its acidic nature, which may hinder cell proliferation needed for successful revascularization.[22],[23],[24]

Alternatively, 2% chlorhexidine (CHX), with recognizable antimicrobial and anti-inflammatory property, showing effective and promising disinfection of root canal with significant clinical relevance has been frequently advocated to be used in endodontic therapy of immature tooth.[11],[20],[25] It possesses high antibacterial property with low toxicity.[26] However, microorganism associated with persistent root canal infection comprises multiple bacterial species, and the most important one among them is Enterococcus faecalis. Its resistance to the most intracanal medicaments is due to its capacity to grow as biofilm on root canal walls. Moreover, CHX is a more potent planktonic and less effective against microbial biofilm cultures. Hence, certain potent antimicrobial agents that act on the bacterial biofilm and decrease its mechanical stability need to be studied. Cetrimide is one such effective cationic surfactant having ability to decrease bacterial biofilm stability with potent bactericidal activity.[25]

For these reasons, the aim of the present in vitro study was to assess and compare the antimicrobial efficacy of Ca(OH)2, triple antibiotic paste, and 2% CHX combined with 0.5% cetrimide on eradication of E. faecalis biofilm.


   Materials and Methods Top


In the present in vitro study, 80 sterile dentin sections of anatomically sound, extracted impacted third molar teeth were taken. The size of each specimen was 2 mm × 2 mm × 2 mm. All specimens were treated for 2 min with CanalPro™ EDTA (ethylenediaminetetraacetic acid) 17% (Coltene Whaledent, USA) so as to remove smear layer produced during tooth sectioning. These dentin specimens were infected extraorally with E. faecalis. Standard strains of E. faecalis ATCC 29212 were used for the study. The specimens were incubated under aerobic conditions in 2-ml brain–heart infusion (BHI) medium to induce microbial colonization at 37°C for 24 h, considered as sufficient time for obtaining required density of microbial biofilm.[27],[28] Later, specimens were washed off with 2 ml of normal saline (NS) (Nirlife, Nirma Ltd. [Healthcare Division], Gujarat, India) to remove the remnants of culture medium and nonadherent microbial cells.

The medicaments studied for their antimicrobial efficacy against E. faecalis were prepared as follows: Ca(OH)2 (Vishal Dentocare Ltd., Gujarat, India) paste was prepared by mixing 1-g powder in 1 ml of distilled water (Sadbhavna Chemicals, Gujarat, India). For triple antibiotic paste, metronidazole 400 mg (Metron 400 [Alkem Laboratories Ltd, Mumbai, India]), ciprofloxacin 500 mg (Ciplox 500 [Cipla Ltd., Sikkim, India]), and minocycline 100 mg (Minoz™ 100 [Sun Pharmaceutical Ind. Ltd., Mumbai, India]) tablets were taken, crushed to powder, and mixed in propylene glycol (AgriLabs, USA) (used as a carrier) to achieve a creamy consistency. For third medicament paste, 0.5% cetrimide 1-g powder (Trends Pharma Pvt. Ltd., Mumbai, India) was mixed with 1 ml of 2% CHX digluconate liquid (CanalPro™ CHX 2% [Coltene Whaledent, USA]).

The infected dentin specimens were divided into four groups of twenty samples each based on medicaments used [Table 1]. The specimens were treated by immersing them in the medicaments studied as per the groups, namely Group I specimens were treated with Ca(OH)2, Group II with triple antibiotic paste, Group III with 2% CHX combined with 0.5% cetrimide, and Group IV with saline (control), in a tissue culture plate for 7 days at 37°C. For live cell assessment, done immediately after elimination of medicament, ten samples, randomly selected from each group, were washed with NS for elimination of any remaining medicament and later subjected to confocal analysis for live microbial cells.
Table 1: Specimen distribution in different groups based on medicaments used in the study

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For further assessment of live cells in the remaining ten samples of each group, the samples were again incubated in BHI medium for 24 h, to test whether residual microorganism has ability to recover from antimicrobial stress after being subject to medicaments for 7 days and are able to recolonize or not. The samples were then washed with NS for elimination of any remaining medicament and later subjected to confocal analysis for live microbial cells. Assessment of live cells was done using confocal microscope. Data thus found were compiled for evaluation and comparison.

Statistical analysis

The data were tabulated and statistically analyzed using analysis of variance. A comparative assessment of live cells between different groups was performed using Mann–Whitney U-test. The selected level of significance was set at a p < 0.05. Data were tabulated in MS Excel sheet, and statistical analysis was done using SPSS 19 (IBM Corporation, Chicago, IL, USA).


   Results Top


[Table 2] and [Graph 1] show that the mean percentage of live bacterial cells after 7 days immediately after elimination of medicaments in Group I was 64.7%, in Group II was 1.52%, in Group III was 1.49%, and in Group IV was 83.4%, whereas the mean percentage of live bacterial cells after 7 days followed by 24-h incubation in BHI medium was 84.2% in Group I, 1.82% in Group II, 1.27% in Group III, and 94.2% in Group IV. The difference was found to be significant (p < 0.05).
Table 2: Mean±standard deviation percentage of live bacterial cells

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   Discussion Top


A successful endodontic therapy requires complete eradication of multiple species of microorganism present within the root canal space. This is achieved by adequate mechanical instrumentation along with simultaneous use of chemical irrigants and followed by rational use of a suitable intracanal medicament.[11],[29],[30],[31] The most common microorganism associated with persistent root canal infections and causing endodontic failure is E. faecalis. Its resistance is largely due to its ability to form biofilm along the dentinal wall of root canal. Biofilms are communities of microorganism embedded in self-produced extracellular polymeric substance matrix that is attached to the surface. This matrix not only provides mechanical stability to the microbial biofilm but also prevents its direct contact with antimicrobial agents, thus making it resistant to them and diminishing the antimicrobial efficacy of commonly used root canal medicaments.[25] Hence, in the present in vitro study, we used E. faecalis strains to compare the antimicrobial efficacy of different medicaments.

Although an effective root canal disinfection is attained by an ideal chemomechanical preparation of root canal space along with the use of intracanal medicaments, immature vital tooth with incomplete root formation poses the biggest challenge for dentists, as root canal decontamination is restricted to the use of irrigating solutions and intracanal medicaments only due to the presence of thin fragile root canal walls.[11],[29],[30],[31] Therefore, use of special intracanal medicament is required, which can fulfill the purpose of disinfection for these immature teeth.[32] Hence, the present in vitro study compared antimicrobial efficacy of Ca(OH)2, triple antibiotic paste, and 2% CHX combined with 0.5% cetrimide, used as intracanal medicaments for root canal disinfection during revascularization procedure in an immature tooth.

In the present study, we found that the mean percentage of live cells in Group I immediately after elimination of medicaments was 64.7%, in Group II was 1.52%, in Group III was 1.49%, and in Group IV was 83.4%, whereas, after 24 h of further specimen incubation in BHI medium, the mean percentage of live cells was 84.2% in Group I, 1.82% in Group II, 1.27% in Group III and 94.2% in Group IV.

Ca(OH)2 in Group I was significantly able to reduce the number of live cells when compared to NS (Group IV). This can be explained by the fact that freshly mixed Ca(OH)2 has an high alkaline pH of 12.5 approximately, which is highly toxic towards the bacterial cells, making it an effective bactericidal.[8] However, studies have shown that this high pH is also toxic to vital human cells, damages epithelial cell rests of Malassez, hindering cell proliferation, and ingrowth of pulp-like tissue with odontogenic potential during revascularization.[10],[33] Moreover, the percentage of live cells significantly increased from 64.7% to 84.2% in Ca(OH)2 treated specimens after 24 h of further specimen incubation in BHI medium during secondary assessment. This can be attributed to the fact that the initial high alkalinity of freshly mixed Ca(OH)2 paste is neutralized over time making it less potent to microorganism and also the residual microorganism has ability to recover from antimicrobial stress and is able to recolonize when exposed to new nutrients.[34]

Furthermore, the results of the present in vitro study showed that triple antibiotic paste in Group II was significantly able to reduce the number of bacterial live cells in both immediate and secondary assessment when compared to Group I (Ca(OH)2). Studies show that the alkaline antimicrobial effect of Ca(OH)2 is ineffective in eliminating bacteria present in biofilm,[35-38] whereas triple antibiotic paste possesses high bactericidal efficacy,[39] with ability to eliminate colonizing bacteria in biofilm.[20] Triple antibiotic paste consists of mainly minocycline and metronidazole. It has shown promising results in cases of persisting periodontitis where significant reduction of supra and subgingival microbial flora has been found after its application.[34] Literatures show that minocycline, a tetracycline derivative, presents a known substantivity effect toward microbes with potent antimicrobial efficacy.[40],[41],[42] Our results are in agreement with previous studies by Hoshino et al.[39] and Sato et al.,[43] which showed that triple antibiotic paste is an effective bactericidal agent and capable of sterilizing infected dentin, with less percentage of live microbial cells seen within the bacterial biofilm as compared to Ca(OH)2.

Group III (2% CHX combined with 0.5% cetrimide) showed highly significant (p ≤ 0.01) antimicrobial efficacy with less percentage of live cells at both time intervals of assessment (1.49% and 1.27%) as compared to Group I and Group IV. It also showed good antibacterial activity when compared to Group II. The present results may be attributed to the fact that antimicrobial efficacy of 2% CHX against bacterial biofilms is enhanced when it is combined with 0.5% cetrimide, a known cationic surfactant belonging to quaternary ammonium group, which reduces the surface tension of CHX, weakens bacterial biofilms cohesive forces, and facilitates its entry into bacterial extracellular polymeric substance matrix, causing bacterial cell lysis.[25],[44]

The use of 2% CHX alone as antimicrobial agents is under scanner. Few studied revealed marked efficacy while others showed its limited effect.[22] In a previous study by Shen et al.,[45] they assessed antibacterial efficacy of 2% CHX and CHX-Plus on collagen-coated hydroxyapatite and uncoated hydroxyapatite disks inoculated with dispersed subgingival plaque. The results of the study revealed that CHX-Plus resulted in higher reduction in microbes at both exposure times compared to 2% CHX alone. Similarly, Gomes et al.[46] studied antimicrobial effect of 2% CHX, Ca(OH)2, and 2% CHX combined with Ca(OH)2 on 180 E. faecalis-infected dentine tubes and found that combination of CHX gluconate and Ca(OH)2 led to 100% antimicrobial effect, whereas 2% CHX and Ca(OH)2 when used alone failed to eliminate bacteria completely. Hence, in consensus to previous findings, an interesting finding of the present in vitro study was the fact that 2% CHX combined with 0.5% cetrimide showed the highest antimicrobial efficacy with observed lowest number and percentage of viable live bacterial cells when compared to triple antibiotic paste and Ca(OH)2.


   Conclusion Top


To enhance the efficacy of intracanal medicaments used for root canal disinfection during revascularization, using combination of distinct antimicrobials agents is an effective strategy to eliminate bacterial biofilms' extracellular polymeric substance matrix, as well as eradication of multiple bacterial cell type within single species microbial biofilm. Although triple antibiotic paste showed good antimicrobial efficacy against E. faecalis, its proven toxicity on stem cells makes it a less viable option. Hence, a more palliative antimicrobial agent like 2% CHX combined with 0.5% cetrimide might provide desired results.

Limitations

The present in vitro study involved only one type of bacteria while assessing antimicrobial efficacy of different medicaments. Other species of bacteria found in infected root canal of immature tooth also need to be examined in order to validate the antimicrobial efficacy of medicaments studied. Further, to maximize stem cell viability, toxicity of studied medicaments on human cells should be assessed.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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