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 Table of Contents  
Year : 2022  |  Volume : 34  |  Issue : 3  |  Page : 162-167

Comparative evaluation of cerium oxide nanoparticles and calcium hydroxide as intracanal medicament against Enterococcus faecalis on tooth substrate: An in vitro study

1 Department of Restorative Dentistry and Prosthodontics, University of Rwanda, Kigali, Rwanda
2 Department of Conservative Dentistry and Endodontics, Madha Dental College, Kundrathur, Tamil Nadu, India
3 Department of Restorative Dentistry, Majmaah University, Al Zulfi, Saudi Arabia
4 Department of Preventive Dental Sciences, College of Dentistry, Majmaah University, Al Zulfi, Saudi Arabia
5 Dr. Rao Dental Clinic, Chennai, Tamil Nadu, India

Date of Submission29-Jan-2022
Date of Decision01-Mar-2022
Date of Acceptance22-Mar-2022
Date of Web Publication30-Sep-2022

Correspondence Address:
Prof. Veronica Aruna Kumari
Department of Conservative Dentistry and Endodontics, Madha Dental College and Hospitals, Kundrathur, Chennai - 600 069, Tamil Nadu
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/endo.endo_25_22

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Aim: The aim of this study is to compare the antibacterial efficacy of cerium oxide nanoparticles and calcium hydroxide as intracanal medicaments against Enterococcus faecalis on tooth substrate – an in vitro study.
Materials and Methods: The experiment was conducted in three stages. In Stage 1, antibacterial susceptibility test of three test materials, cerium oxide nanoparticles, calcium hydroxide, and vancomycin against E. faecalis was done using disc diffusion method. In Stage 2, determination of minimal inhibitory concentration and minimum bactericidal concentration of the cerium oxide nanoparticles was done by tube dilution method. In Stage 3, determination of CFU was carried out using dentinal blocks at 1-day and 5-day intervals at two different dentinal depths (200 μm and 400 μm) for calcium hydroxide (Group 1), cerium oxide nanoparticle dispersion (Group 2), a mixture of calcium hydroxide and cerium oxide nanoparticles (Group 3), and sterile water (Group 4).
Results: The reduction of E. faecalis achieved by cerium oxide nanoparticles dispersion on day 5 was 66.9% as against 46.7% as shown by mixture of the two medicaments and 34.8% by calcium hydroxide paste alone on day 5 at 400-μm depth.
Conclusion: Cerium oxide nanoparticles show a significantly higher antibacterial efficacy when compared to calcium hydroxide and its combination.

Keywords: Antibacterial, cerium oxide nanoparticles, intracanal medicament

How to cite this article:
Sanju D, Kumari VA, Thomas T, Thomas JT, Sujeer R. Comparative evaluation of cerium oxide nanoparticles and calcium hydroxide as intracanal medicament against Enterococcus faecalis on tooth substrate: An in vitro study. Endodontology 2022;34:162-7

How to cite this URL:
Sanju D, Kumari VA, Thomas T, Thomas JT, Sujeer R. Comparative evaluation of cerium oxide nanoparticles and calcium hydroxide as intracanal medicament against Enterococcus faecalis on tooth substrate: An in vitro study. Endodontology [serial online] 2022 [cited 2022 Nov 30];34:162-7. Available from: https://www.endodontologyonweb.org/text.asp?2022/34/3/162/357701

  Introduction Top

Microbes are the primary etiologic factor of persistent endodontic infections.[1],[2] Bacteria present in biofilm survive in a nutrient-depleted environment for a prolonged period.[3] Enterococcus faecalis is the most common microorganism which persists in endodontically treated teeth.[4],[5] Successful endodontic therapy depends on the complete elimination of bacteria from the root canal system and preventing its recolonization.[6],[7] This can be achieved by placing intracanal medicament after cleaning and shaping. The most popular medicament used in endodontics is calcium hydroxide. It releases hydroxyl ion, which has lethal effects on bacterial cells,[8],[9] including those of protein denaturation,[10] and damage to the bacterial cytoplasmic membranes,[11] and DNA.[12],[13] Studies have shown dentin exudate from the periapical area, and microbial biomass results in the inactivation of calcium hydroxide.[14]

Numerous studies focus on the use of nanoparticulate materials to disinfect root canals.[15] Nano particulates by nature have the greater surface area and charge density which helps them to interact with bacterial cells which are negatively charged.[16] Nanoparticles can be delivered into complex anatomical spaces because of their nanometric sizes, allowing them to penetrate deeper and show a good antibacterial property.[17] Cerium oxide nanomaterials can adjust its electronic configuration and change its oxidative state based on its requirement, this catalytic properties can be used for biomedical application. It can be used as an antioxidant agent; it has oxygen valencies on its surface, and Ce3+ and Ce4+ are its two oxidation states. Cerium oxide nanoparticles have been shown to have antibacterial effects against Bacillus subtilis,[18] Staphylococcus aureus,[19] and  Escherichia More Details coli.[20] The aim of the study is to compare the antibacterial efficacy of cerium oxide nanoparticles, calcium hydroxide, and combination of cerium oxide and calcium hydroxide as intracanal medicaments against E. faecalis on tooth substrate.

  Materials and Methods Top

This study was done under guidelines approved by the Ethical Committee and Scientific Review Board of Saveetha Dental College and Hospital (SRB Ref NO. SRB/SDMDS11ODS2 dated October 16, 2013). Eighty single rooted human premolar teeth which were extracted for orthodontic reason were obtained for the study. Single-rooted human permanent teeth with one canal having fully formed apices were included in this study. Chipped or fractured teeth, single-rooted teeth with more than one canal, and teeth with calcified canals were excluded.

Sample size determination

A pilot study was done on a total of 18 samples with 6 samples allocated in each group. Antibacterial susceptibility test was done by standard disc diffusion method; mean zone of inhibition was measured between calcium hydroxide, cerium oxide nanoparticles, and vancomycin after 24 h. The effect size was evaluated to be large (Cohen's d > 0.8) Depending on the pilot study, a total sample of 80 dentinal blocks 20 in each group was used for antimicrobial assessment study. Teeth were infected for 21 days, medicaments were placed in the canals at the end of 1 and 5 days; an assessment of microbial cells was carried out with 10 specimens at each time interval. Harvesting of dentin was carried out at two depths 200 and 400 μm. Sample size determination was done using G Power 3.1(Heinrich Heine University, Dusseldorf, North Rhine-Westphalia), assuming alpha- and beta-errors at 0.05 level power of 90%. Further, the 20 samples assigned to check the colony-forming unit in each group were as follows:

  • Group 1: Calcium hydroxide
  • Group 2: Cerium oxide nanoparticles
  • Group 3: Combination of cerium oxide nanoparticle and calcium hydroxide
  • Group 4: Unmedicated.

Disc diffusion method

In disc diffusion method, Mueller Hinton (MH) agar was used (HiMedia, Mumbai, India); an uncontaminated pure culture of E. faecalis (ATCC 29212) (IBMS, Chennai) was grown in it. Antibacterial activity of cerium oxide nanoparticle against E. faecalis was carried out using Kirby-Bauer disk diffusion susceptibility method.[21] Sterile blank discs 6-mm diameter (Himedia, Mumbai, India) were placed separately in each of the prepared test solutions calcium hydroxide, cerium oxide nanoparticle, and vancomycin for 5 min; the discs were carefully removed using sterile forceps. After the bacterial suspension was uniformly plated on MH agar plates, discs containing test materials were placed on the plates, and plates were incubated at 37°C for 24 h. Zone of inhibition was observed after 24 h of incubation.

Minimal inhibitory concentration

Two-fold serial dilution of cerium oxide nanoparticles was carried out in MH broth at 100, 50, 10, 5, 1, 0.5, 0.1, and 0.01 mg/ml concentrations of cerium oxide nanoparticles. To each test tube, 10 × 5 CFU/ml of actively growing bacterial culture was inoculated and incubated at 37°C for 24 h. The lowest concentration in the series of dilution which did not permit the growth of susceptible bacteria was considered to be minimal inhibitory concentration (MIC). After the incubation, the tubes were checked for the growth of bacteria, and the minimum bactericidal concentration (MBC) of test samples was determined.

Antimicrobial assessment study

Preparation of dentine specimens

The in vitro model suggested by Haapasalo M et al., 1987,[22] has been modified to assess the efficacy of endodontic medicaments in the disinfection of dentinal tubules. Eighty single-rooted teeth were selected; teeth were decoronated below the cementoenamel junction and apical part to obtain 6 mm of the middle third of the root. Root surface was prepared by removing the cementum. Gates Glidden drills no. 3 (Mani Inc., Tochigi-ken, Japan) in a slow-speed handpiece was used to standardize the internal diameter of the root canals. 17% EDTA was used to remove the inorganic debris for 5 min, followed by 3% NaOCl for 5 min to remove organic debris, later washed with distilled water for 5 min, and sterilized in an autoclave for two cycles. The first cycle was at 121°C, and the second was with the specimens immersed in 1 mL of MH broth in individual microcentrifuge tubes.

Contamination of the specimens

E. faecalis was used in this study, which is a Gram-positive facultative anaerobic bacterium that is common in root-filled teeth; it (ATCC 29212) was grown in MH agar for 24 h. The culture was suspended in 5 mL of MH broth and incubated for 4 h at 37°C. 0.5 McFarland standard was its turbidity. Microcentrifuge tubes were sterilized before starting the experiment, and dentine block was placed in the tubes containing 1 mL of the TS broth. The inoculum of E. faecalis which was about 50 μl was transferred into each of the microcentrifuge tubes. Toward the end of 24 h, specimens that had dentine were transferred into fresh broth containing E. faecalis. Entire procedures were carried out under laminar flow. The culture medium was replenished every 48 h with a fresh medium. The purity of the culture was checked by subculturing 5 μL of the broth from the incubated dentine specimens in MH broth on MH agar plates. The dentine specimens were contaminated for 21 days.

Antimicrobial assessment

The specimens were irrigated with 5 ml of sterile saline at the end of 21 days to remove the incubation broth. They were assigned into four groups: 20 dentine blocks in each group. Group 1 consists of 1.5 weight per volume calcium hydroxide in 1 weight per volume of sterile water, Group 2 consists of 0.5 mg of cerium oxide nanoparticle dispersion in 1 ml, Group 3 consists of ratio of 3 portions of calcium hydroxide and 1 portion of cerium oxide nanoparticles in dispersion, and Group 4 consists of unmedicated teeth irrigated with sterile water. Calcium hydroxide (Sigma Aldrich, Mumbai, India) was mixed with sterile water to get a smooth paste-like consistency.[23] Based on the MBC value of cerium oxide nanoparticles, the concentration of Group 2 and the ratio in which calcium hydroxide and nanoparticles should be combined in Group 3, were ascertained. Cerium oxide nanoparticles (<25 nm average particle size, Sigma Aldrich, Mumbai, India) were dispersed in double-distilled water at a concentration of 0.5 mgl/ml and were ultrasonicated for 1 min to get a uniform dispersion. Methylcellulose was added as a thickening agent in Group 2 and Group 3.

The medicaments were placed inside the canals and sealed at both ends with sticky wax. They were incubated in an anaerobic environment for 24 h at 37°C. At the end of 1 and 5 days, an assessment of microbial cells was carried out with 10 specimens at each time interval. Harvesting of dentin was carried out at two depths 200 and 400 μm with Gates Glidden drills size 4 and 5, respectively. The collected dentin shavings were transferred into 1 mL of sterile MH broth and incubated in an anaerobic environment at 37°C for 24 h. After 24 h, the contents of each tube were serially diluted, 100 μl of the broth in 100 μl of sterile saline five times. Fifty microliters of the dilution were then plated on MH agar plates and incubated for 24 h. Colonies were counted and readings were tabulated. They were checked for 1 day and 5 days at two dentinal depths 200 μm and 400 μm.

Statistical analysis

Data analysis for this study was done using IBM SPSS statistics 20.0( IBM Corporation, Armonk,NY, USA). MIC and MBC were first determined. Comparison of the zone of inhibition among all the groups was done using the Kruskal–Wallis test and Mann–Whitney test with the Bonferroni correction. Statistical analyses for CFU were performed on log10 converted data. The data were statistically analyzed with a one-way analysis of variance to compare mean log CFU between different groups for day 1 and day 5 at depth 200 μm and 400 μm.

  Results Top

In disc diffusion method, the zone of inhibition are shown in [Table 1], maximum mean zone of inhibition was seen for Group III (vancomycin), followed by Group II (cerium oxide nanoparticles), and no zone of inhibition in Group I (calcium hydroxide). A significant difference (P < 0.001) is seen among all the three groups in terms of mean diameter of zone of inhibition (mm). Cerium oxide nanoparticles were able to inhibit bacterial growth of E. faecalis at a minimum concentration of 0.1 mg/ml. Cerium oxide nanoparticles were bactericidal against E. faecalis at a minimum concentration of 0.5 mg/ml.
Table 1: Comparison of diameter of zone of inhibition (mm) between three groups

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In antibacterial assessment study as shown in [Graph 1] and [Graph 2] for day 1 as well as day 5 at depth 200 μm and 400 μm, there is a statistically significant difference among all groups in terms of mean log CFU, with Group 2 showing the least mean log CFU followed by Group 3, Group 1, and Group 4 in the increasing order. Mean log CFU between different groups for day 1 and day 5 at depth 200 μm and 400 μm was compared which shows statistical siginificant difference as shown in [Table 2] and [Table 3] with (P<0.05) in bacterial count (log CFU) among all the groups.
Table 2: One-way ANOVA (to compare mean log colony-forming unit between different groups, for day 1 and day 5 at depth 200 µM)

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Table 3: One-way ANOVA (to compare mean log colony-forming unit between different groups, for day 1 and day 5 at depth 400 µm)

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  Discussion Top

The primary focus of root canal treatment is to eliminate the bacterial biofilm from the root canal system.[24] The complex anatomy of a root canal, along with the composition of dentine structure, contributes to limitations in endodontic disinfection.[24] Intracanal medicament placement reduces the count or eliminates bacteria in the root canal system and increases the success rate.[25] Bacterial exposure to the disinfectants for a longer time period might induce resistance to subsequent exposure at levels that might normally be lethal.[26] Diffusion of hydroxyl ions (OH) from calcium hydroxide results in a highly alkaline environment which kills microorganisms. However, dentin mud would inactivate the bacterial destroying effect of calcium hydroxide.[14] Calcium hydroxide medication is found to be ineffective against E. faecalis infection of root canals.[27],[28]

In recent times, nanoparticles have been shown to have higher antibacterial activity than antibacterial powder.[17] There have been numerous nanoparticles of chitosan, silver, zinc oxide, and bioactive glass used for testing antibacterial activity against E. faecalis. Shreshta et al. highlighted the antibacterial efficacy of chitosan nanoparticles to reduce the number of viable E. faecalis.

Present disc diffusion study maximum mean zone of inhibition was seen for Group III (vancomycin), followed by Group II (cerium oxide nanoparticles). Group I (calcium hydroxide) showed almost no zone of inhibition. In this study we estimated the MIC and MBC for cerium oxide nanoparticles. Antibacterial efficacy for all the groups using CFU was determined and the data were analysed. There is no previous study on cerium oxide nanoparticle; hence, we checked this nanoparticle. The combination of calcium hydroxide and cerium oxide nanoparticles was mixed to check whether there is any synergistic effect when used together. The dispersion medium used for cerium oxide nanoparticles was double-distilled water as used in the method suggested by Jones et al.

Cerium oxide nanoparticles (Group 2) showed maximum antibacterial efficacy followed by combination of cerium oxide nanoparticles and calcium hydroxide (Group 3), and then, calcium hydroxide (Group 1) least was seen with unmedicated (Group 4). The reason may be attributed to the mechanism of resistance of E. faecalis to calcium hydroxide. At pH 11.5 or greater, E. faecalis does not survive, yet it can survive at a pH below 11.5.[25] Because of the buffering effect of dentine,[14],[29],[30] it is unlikely that the high pH of calcium hydroxide (>11.5) is attained within dentinal tubules where E. faecalis has the capacity, at least in vitro, to penetrate deeply.[22],[27],[31],[32],[33] In radicular dentine, alkalinity may only reach pH 10.3 after dressing the canal with calcium hydroxide.[30],[34] A functioning proton pump with the capacity to acidify the cytoplasm is responsible for the survival of E. faecalis at high pH.[26]

In this study a statistical difference was observed in the antibacterial activity of cerium oxide nanoparticles (Group 2), when compared to a combination of cerium oxide nanoparticles with calcium hydroxide (Group 3) and calcium hydroxide alone (Group 1). This can be attributed to the over production of reactive oxygen species(ROS) and cell death due to DNA degeneration. Kuang et al., in 2011 showed 7nm(7-CeO2), 25 nm(25-CeO2) and their bulk counterpart (b-CeO2) had antibacterial efficacy against  E.coli Scientific Name Search . The rise in intracellular ROS level induced by direct contact of particles with the surface of E. coli will impair the integrity of outer membrane could be the mechanism for antibacterial activity of CeO2 NPs. The antibacterial property against E. faecalis could be due to the same. A combination of cerium oxide nanoparticles and calcium hydroxide (Group 3) showed significantly lower antibacterial efficacy when compared to cerium oxide nanoparticles (Group 2) when used alone, which may be due to the lower concentration of cerium oxide nanoparticles. Studies have shown that pH-controlled cerium oxide nanoparticles inhibit both Gram-positive and Gram-negative bacteria.[35]

  Conclusion Top

Within the limitations of this study, it was found that cerium oxide nanoparticle dispersion showed the best antibacterial efficacy, followed by the combination of cerium oxide nanoparticles and calcium hydroxide, and then followed by calcium hydroxide alone. Further studies are encouraged to determine the cytotoxicity of cerium oxide nanoparticles before it can be used in vivo as an intracanal medicament by being an alternative to calcium hydroxide or as an adjunct to calcium hydroxide to produce a synergistic effect.

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Conflicts of interest

There are no conflicts of interest.

  References Top

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  [Table 1], [Table 2], [Table 3]


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