Comparative evaluation of the antimicrobial efficacy of different concentrations of the novel root canal filling material for primary teeth: A microbiological study

Lavanya Govindaraju; Ganesh Jeevanandan

doi:10.4103/jioh.jioh_121_22

ORIGINAL RESEARCH

Year : 2023 | Volume : 15 | Issue : 1 | Page : 84-88

Comparative evaluation of the antimicrobial efficacy of different concentrations of the novel root canal filling material for primary teeth: A microbiological study

Lavanya Govindaraju, Ganesh Jeevanandan
Saveetha Dental College and Hospitals, Saveetha Institute of Medical and Technical Sciences, Saveetha University, Chennai, India

Date of Submission	31-May-2022
Date of Decision	07-Dec-2022
Date of Acceptance	09-Dec-2022
Date of Web Publication	28-Feb-2023

Correspondence Address:
Dr. Lavanya Govindaraju
Saveetha Dental College and Hospitals, Saveetha Institute of Medical and Technical Sciences, Saveetha University, Chennai
India

Source of Support: None, Conflict of Interest: None

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DOI: 10.4103/jioh.jioh_121_22

Abstract

Aim: To determine the antimicrobial efficacy of the different concentrations of a novel root canal filling material and to proclaim the best concentration with maximal antimicrobial effect against Streptococcus mutans and Enterococcus faecalis. Materials and Methods: The present study is an in vitro microbiological study to check the antimicrobial efficacy of different concentrations of the novel obturating material which was prepared using calcium hydroxide, zinc oxide cement, and metronidazole against S. mutans and E. faecalis using Muller Hilton Agar for 24 hours at 37°C. A total of 30 samples were tested (15 for S. mutans and 15 for E. faecalis). The zone of inhibition was measured in millimeters after 24 hours. The values were entered in a Microsoft Excel spreadsheet and subjected to statistical analysis using Friedman test and Kruskal–Wallis test. Results: The mean zone of inhibition was statistically greater against S. mutans at 100 µL with 2% and 3% concentration of the novel root canal filling material (P = 0.01). Comparison in between the groups at different concentrations shows no statistically significant difference among the three different concentrations; however, the zone of inhibition is greater with 3% metronidazole. Conclusion: The formulation obtained with 3% metronidazole at 80-20 (calcium hydroxide–zinc oxide) concentration of the novel obturating material at 100 µL showed better antimicrobial activity against both S. mutans and E. faecalis.

Keywords: Child Dentistry, Novel, Obturating Material, Pediatric Endodontics, Pulpectomy

How to cite this article:
Govindaraju L, Jeevanandan G. Comparative evaluation of the antimicrobial efficacy of different concentrations of the novel root canal filling material for primary teeth: A microbiological study. J Int Oral Health 2023;15:84-8

How to cite this URL:
Govindaraju L, Jeevanandan G. Comparative evaluation of the antimicrobial efficacy of different concentrations of the novel root canal filling material for primary teeth: A microbiological study. J Int Oral Health [serial online] 2023 [cited 2023 Apr 1];15:84-8. Available from: https://www.jioh.org/text.asp?2023/15/1/84/370741

Introduction

Primary teeth with pulpal and periapical issues should be retained in the dental arch until its exfoliation for the normal growth and functioning in children.^[1],[2] Endodontics has become the role change in preserving these primary teeth. Success of the endodontic treatment in primary teeth depends on the complete elimination of the causative micro-organisms. Pulpal infection in primary teeth certitudes to be poly microbial in nature as the complexity of the root canal morphology in primary teeth harbors various pathogens. Also, complexity and torturous morphology of primary root canals hinder thorough debridement of the canals via chemo-mechanical preparation.^[3] This necessitates an ideal root canal filling material in primary teeth with antimicrobial properties to inhibit the growth of remanent bacteria within the canals, contributing to the success of the endodontic treatment.

Zinc oxide eugenol and Calcium hydroxide + iodoform paste commercially available as Metapex, most commonly used materials for obturation in primary teeth. However, a study done by Kriplani et al., showed that Metapex was ineffective against all the test micro-organisms isolated from the infected primary root canals.^[4] Other studies in the literature also demonstrated that calcium hydroxide and iodoform pastes exhibited no antimicrobial properties.^[5],[6],[7] Though the literature has documented that Zinc oxide eugenol shows greater inhibitory effects against micro-organisms found in primary root canals, the eugenol component has been declared to be irritant to the periapical tissues, causing necrosis of bone and cementum.^[8],[9] With endodontic treatment being the only option to maintain the integrity of the primary dentition until its physiological exfoliation, the search for an ideal root canal filling material with antimicrobial property continues.

In the present study, a novel root canal filling material was developed using zinc oxide, calcium hydroxide, and metronidazole. Apart from having maximal antibacterial activity, an ideal root canal filling material in primary teeth should also resorb at the same rate of the primary teeth without exerting any deleterious effects on the permanent teeth and the periapical tissues.^[10],[11] Hence, calcium hydroxide and zinc oxide were mixed at 80-20 concentration, respectively, as the evidences demonstrate that zinc oxide has the tendency to resorb slowly while calcium hydroxide resorbs much faster than the resorption rate of the tooth.^[12],[13] The combination of these materials can overcome the disadvantages of one material over the other. 1%, 2%, 3% of metronidazole—an antimicrobial agent was added to this mixture and was subjected for antimicrobial activity tests.

Several authors have demonstrated that infection in primary root canals is polymicrobial in nature.^[14],[15] However, Streptococcus mutans and Enterococcus faecalis were declared to be the most predominantly isolated organisms from root canal infections in primary teeth.^[16],[17] The aim of the present study was to evaluate the anti-microbial efficacy of three different compositions of the novel obturating material (1%, 2%, 3% of metronidazole + 80:20 calcium hydroxide:zinc oxide) at three different concentrations (25, 50, 100 µL) against S. mutans and E. faecalis and determine the most efficacious composition of the novel obturating material.

Materials and Methods

Setting and design

The present microbiological analysis was designed as an in vitro study and was conducted in the micro-biological lab of Saveetha Dental College and Hospitals.

Ethical approval

The clearance was obtained from the institutional review board (IHEC/SDC/PEDO-2125/21/537) of Saveetha Dental College and Hospitals prior to the start of the study.

Methodology

Preparation of the obturating material

A digital weighing machine was used to exactly measure 800 mg of calcium hydroxide powder [Prevest DenPro] and 200 mg of Zinc oxide powder [Prevest DenPro], which was then mixed manually and was kept in magnetic stirrer for 1 hour to form 80 to 20 concentration of the mixture.

A motor and pestle was used to crush 400 mg of Metronidazole tablet and make it into a fine powder. 1% of calcium hydroxide—zinc oxide—metronidazole powder was prepared by mixing 2 mg of metronidazole powder to 198 mg of the calcium hydroxide—zinc oxide mixture. Similarly, 2% and 3% calcium hydroxide—zinc oxide—metronidazole powder was prepared by mixing 4 and 6 mg of metronidazole powder to 196 and 194 mg of zinc-oxide- calcium hydroxide mixture, respectively. The exact amount of the powders were dispensed by using a digital weighing machine. The samples were mixed with 2 ml of distilled water measured with micropipette, placed in vortex for 15 minutes and then in mixer for 24 hours.

Preparation of the culture medium

Agar well diffusion method was employed to evaluate the antimicrobial activity of the different concentrations of the novel obturating material against S. mutans and E. faecalis. Mueller Hilton Agar was prepared and sterilized at 121°C for 15 minutes. The media was poured into 30 sterilized plates (15 for S. mutans and 15 for E. faecalis) and was allowed for solidification. The wells were cut using the well cutter and the test organisms were swabbed. 1%, 2% and 3% of the Novel obturating material were loaded at 25, 50, 100 µL and were incubated at 37°C for 1 day. The diameters of the inhibition zone were measured in millimeters after 24 hours using vernier caliper [Figure 1] and [Figure 2].

Figure 1: Zone of inhibition observed for S. mutans after 24 hours at different concentration of the novel obturating material

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Figure 2: Zone of inhibition observed for E. faecalis after 24 hours at different concentration of the novel obturating material

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Study methods

Data were entered in Microsoft Excel spreadsheet and analyzed using SPSS software (IBM SPSS Statistics, Version 20.0, IBM Corp., Arming, NY). Descriptive statistics were used for data summarization and presentation. Friedman test and Kruskal–Wallis test were used to compare the different concentrations of the Novel obturating materials against S. mutans and E. faecalis.

Results

The mean zone of inhibition was greater against S. mutans and E. faecalis at 100 µL irrespective of the concentration of the metronidazole [Table 1] and [Table 2]. However statistical significance was noted against S. mutans with 2% and 3% concentration of the novel root canal filling material (P = 0.01). Comparison in between the groups at different concentrations shows no statistically significant difference between the three different concentrations; however, the zone of inhibition is greater with 3% metronidazole + 80:20 calcium hydroxide:zinc oxide [Table 3] and [Table 4].

Table 1: Comparison of different concentrations of the novel obturating material against Streptococcus mutans

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Table 2: Comparison of different concentrations of the novel obturating material against E. faecalis

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Table 3: Comparison between 1%, 2%, 3% 80-20 concentration of the novel obturating material against Streptococcus mutans at 25, 50, 100 µL

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Table 4: Comparison between 1%, 2%, 3% 80-20 concentration of the novel obturating material against E. faecalis at 25, 50, 100 µL

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Discussion

Pulpectomy being the one and only option to prevent pre-mature loss of primary teeth in children, understanding the anatomy and root canal morphology of primary teeth plays a cardinal role in determining the success of the treatment. The apical foramen of the primary teeth keeps changing continuously and simultaneously the deposition of secondary dentin can divide into separate canals which hinders the complete debridement of the root canals in primary teeth making it more prone to re-infection and failure of the endodontic treatment.^[18] Consequently, the success of the endodontic treatment depends on the quality of the obturating material used, as it remains in contact with the remanent pulpal tissues and entrapped micro-organisms. Thus, an ideal obturating material in primary teeth should have maximum antimicrobial property to neutralize the remaining pulp tissue and micro-organisms and increase the longevity of the endodontically treated primary teeth. Primary root canals being polymicrobial in nature, search for an ideal root canal filling material with maximal antimicrobial property continues.^[19]

The results of the present study demonstrate that the zone of inhibition was greater at 100 µL against both S. mutans and E. faecalis with all the three different concentration of the Novel Obturating material with statistical significance noted only at 2% and 3% concentrations against S. mutans and no statistically significant difference noted against E. faecalis. Inter-group comparisons at 100 μm show no statistically significant difference among the three different concentrations; however, the zone of inhibition was maximum at 3% against both S. mutans and E. faecalis. Thus, all the three different concentrations of the Novel obturating material show maximal anti-microbial effect at 100 μm with 3% being the most effective against both S. mutans and E. facecalis followed by 1% and 2%.

In the present study, different concentrations of the newly developed root canal filling material were subjected to antimicrobial testing so as to obtain a novel root canal filling material with optimum antimicrobial property. The novel root canal filling material comprises of zinc oxide, calcium hydroxide, and metronidazole at 80:20 concentration of calcium hydroxide and zinc oxide powder. Two factors were considered while fixing the concentration of calcium hydroxide and zinc oxide—(a) the antimicrobial quality of the materials; (b) the rate of resorption of the materials. Studies have shown that zinc oxide powder as such does not have antimicrobial property and its addition of eugenol showed inhibitory effect on micro-organisms.^[1] A study done by Ibrahim et al. also concluded that the antimicrobial effect of obturating material depends on the concentration of Eugenol.^[20] On the other hand, Calcium hydroxide when mixed with aqueous vehicle has shown greater zone of inhibition due to the ionic dissociation in calcium and hydroxyl ions.^[21] Also a recent Micro computed tomography study that evaluated the quality of obturation with various obturating materials under different techniques have shown calcium hydroxide to be a better choice as it reduces the incidence of void formation within the canals of the primary teeth.^[22] But Calcium hydroxide has the tendency to resorb faster than the rate of resorption of the primary root resulting in hollow tube effect. At the same time the rate of resorption of Zinc oxide is much slower.^[12],[13] Hence, the novel root canal filling material was developed with greater concentration of calcium hydroxide and lesser concentration of zinc oxide powder assuming the combination of these materials can overcome the disadvantages of each other. Metronidazole was the antimicrobial agent added to these materials as it is known to be effective against facultative anaerobes by diffusing into the organism, eventually inhibiting the protein synthesis, consequently resulting in the cell death of the susceptible organisms.^[23] Hence 1%, 2%, 3% of metronidazole powder was added to the 80-20 concentration of the calcium hydroxide and zinc oxide powder and was subjected to micro-biological evaluation.

Agar well diffusion method was used in the present study as this method of evaluating the antimicrobial efficacy was more reliable and easier to perform. Previous studies that evaluated the antimicrobial efficacy of various obturating materials have also employed Agar well diffusion method.^{[4],[18],[24]} This method directly compares the efficacy of different obturating material against the test microorganisms, thus determining the most effective material. S. mutans and E. faecalis serve as a standard for testing the antimicrobial efficacy of any root canal medicament and hence the novel root canal filling material was tested against S. mutans and E. faecalis in the present study.

The potential limitation of the present study could be that the antimicrobial efficacy of the test material was evaluated in in vitro conditions which can alter in clinical scenarios in the presence of saliva, serum, dentin, and other micro-organisms. Antimicrobial property being the most important factor to be considered for preventing re-growth of the bacteria, however, requires further studies to evaluate the safety of this concentration of the novel root canal filling material for clinical applicability. Future in vivo studies can confirm and validate these findings. The biocompatibility and antimicrobial efficacy with other pathogenic organisms need to be examined. Studies need to examine any alteration in the physical or mechanical properties of the obturating material with the addition of metronidazole. Future trials should consider using a combination of antibiotics for antimicrobial effect. Pulpectomy is a technique sensitive procedure, whose success depends on several factors. The use of this novel obturating material with proper diagnosis and operative technique may raise the odds of a successful outcome.

Conclusion

The formulation obtained with 3% metronidazole at 80-20 (calcium hydroxide-zinc oxide) concentration of the Novel Obturating material at 100 µL showed better antimicrobial activity against both S. mutans and E. faecalis.

Acknowledgement

None.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

Authors’ contributions

Lavanya Govindaraju contributed to the conception, design, data acquisition and interpretation, performed all statistical analyses, drafted the manuscript.

Ganesh Jeevanandan contributed to the conception, design and critically revised the manuscript.

All authors gave their final approval and agree to be accountable for all aspects of the work.

Ethical policy and institutional review board statement

The clearance was obtained from the institutional review board (IHEC/SDC/PEDO-2125/21/537) of Saveetha Dental College and Hospitals prior to the start of the study.

Patient declaration of consent

Not applicable.

Data availability statement

Not applicable.

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