Comparative Evaluation of Nano Inorganic Metal Oxides as Intracanal Medicaments for Cytotoxicity and Antimicrobial Activity in the Root Canal System
Corresponding Author: Prerna Barge, Department of Pediatric and Preventive Dentistry, Yogita Dental College and Hospital, Khed, Ratnagiri, Maharashtra India, Phone: +91 9689997636, e-mail: firstname.lastname@example.org
Aim and objective: To evaluate and compare the cytotoxicity and antimicrobial activity of various inorganic metal oxide nanoparticles along with vehicles when used as intracanal medicaments in the root canal system.
Materials and methods: The study included triplicates (n = 36 times) that were subjected to n calcium oxide (CaO), n zinc oxide (ZnO), n magnesium oxide (MgO), and metapaste as intracanal medicaments. The efficacy of novel intracanal medicaments was evaluated for biocompatibility assay using 3-(4,5-dimethylthiazol-2-yl)—2,5-diphenyl tetrazolium bromide (MTT) reagent following antimicrobial efficacy against Enterococcus faecalis (E. faecalis) was evaluated using zone of inhibition (ZOI) and minimum inhibitory concentration (MIC). The statistical analysis Kruskal–Wallis test, student t-test, and analysis of variance (ANOVA) using Statistical Package for the Social Sciences (SPSS) software (v.20.0).
Results: The order of proliferative activity of experimental groups on L929 mouse fibroblast cells using MTT assay was: metapaste > nCaO > nMgO > nZnO). After evaluation of antimicrobial efficacy, group I: nCaO showed maximum ZOI and MIC against E. faecalis, which showed high statistically significant differences between all four groups after ANOVA (p < 0.0001*).
Conclusion: n calcium oxide (CaO) mixed with propylene glycol (PPG) 400 has a potential role as an intracanal medicament with minimum cytotoxic effect and maximum antimicrobial activity against endodontic pathogens.
Clinical significance: Nanoparticles-based intracanal medicament can provide a promising future in reducing endodontic flareups when used as intracanal medicament.
How to cite this article: Barge P, Gugawad S, Devendrappa SN, et al. Comparative Evaluation of Nano Inorganic Metal Oxides as Intracanal Medicaments for Cytotoxicity and Antimicrobial Activity in the Root Canal System. Int J Clin Pediatr Dent 2023;16(S-2):S168–S175.
Source of support: Nil
Conflict of interest: None
Keywords: Antimicrobial activity, Calcium hydroxide, Endodontic flareups, Enterococcus faecalis, Fibroblast, Intracanal medicaments, Laboratory research, Nanoparticles, 3-(4,5-dimethylthiazol-2-yl)—2,5-diphenyl tetrazolium bromide assay
Carious lesions cause demineralization of organic and inorganic components of enamel and dentin, causing cavitation on the tooth surface. Carious lesions containing microbial infection, if left untreated, can lead to pulpal injury that necessitates endodontic therapy. This, over some time, induces inflammatory changes in the pulp. The dominant microorganisms responsible for causing pulpal infections are anaerobic bacteria, of which the most resistant organisms are Enterococcus faecalis (E. faecalis), followed by Streptococcus mitis, Streptococcus sanguinis, along certain Actinomyces species, Fusobacterium, Spirochetes, and Prevotella species.1 Endodontic pathogen is the main reason for the progression or failure of endodontic treatments. This sheds light on the increasing demand for improved endodontic therapies to deal with the most resistant microbial flora. Traditionally, intracanal medicaments have gone hand-in-glove with endodontics. They are considered to be an integral part of treatment and an important part of the success of root canal therapies.
Grossman, in 1951 used polyantibiotic paste, whereas, in 1920, Hermann introduced calcium hydroxide [Ca(OH)2], which became the first choice for multi-visit root canal therapy. In the romp of modern endodontics, nanoparticles (NPs) were introduced in endodontics to treat infections because of their desirable properties and various mechanisms for destroying resistant bacteria. NPs, due to this effect, have the potential to be used as an intracanal medicament. In addition to its antimicrobial effect against resistant microbial flora, it helps to repair the periapical tissue, reducing endodontic flareups.
Although the majority of clinical trials are associated with conventional multi-visit endodontics, limited comparative literature on NPs as intracanal medicaments paves the need for comprehensive experimental trials. Hence, this in vitro study aims to create newer combinations of intracanal medicaments that have maximum antimicrobial efficacy with a minimum cytotoxic effect.
MATERIALS AND METHODS
This in vitro study was carried out in the Department of Pedodontics and Preventive Dentistry, School of Dental Science, Krishna Vishwa Vidyapeeth (Deemed to be University), Karad, Maharashtra, India. Ethical clearance was obtained from the committee before the initiation of the study (protocol number-163/2019-2020).
The sample size for the study was derived by a statistician with a 95% confidence level and 95% power to obtain statistically significant results. All the triplicates (n = 36) were divided into two parameters that included cytotoxicity using 3-(4,5-dimethylthiazol-2-yl)—2,5-diphenyl tetrazolium bromide (MTT) assay (n = 12 triplicates) and antimicrobial activity using zone of inhibition (ZOI) (n = 12 triplicates) and minimum inhibitory concentration (MIC) (n = 12 triplicates). Study materials were as follows: group I: n calcium oxide (CaO) + propylene glycol (PPG) 400, group II: n zinc oxide (ZnO) + PPG400, group III: n magnesium oxide (MgO) + PPG400, group IV: metapaste (control).
Culture preparation to evaluate MTT assay: the flask containing the L929 mouse fibroblast cell line was procured from National Centre for Cell Science (NCCS) repository center, Pune. The T25 flask containing fibroblast and fetal bovine serum (FBS) media was placed in a CO2 incubator for 4 hours at 37°C. The FBS medium was removed, leaving 4 mL in the T25 flask. Multi-layered fibroblastic cells were seen when the T25 flask was viewed under 10× magnification. After 12 hours, 4 mL of FBS media was completely removed and replaced with Dulbecco’s Modified Eagle Medium supplemented with 10% FBS and 1% Pen-Strep solution. This was further incubated at 37°C in a CO2 incubator for 24 hours. The subculturing of L929 mouse fibroblast cells was done by splitting fibroblast cells up to 49 generations, 1:3 flask initially, and later from three T25 flasks to 13 flasks. The subculturing up to the 49th generation was performed using 1 mL of 0.25% trypsin which was neutralized by adding fresh medium into the flask each time, and cells were stripped from the flask bottom followed by a CO2 incubator at 37°C for 5–10 minutes. An eluate was prepared by mixing a 100 µL solution containing suspended fibroblast cells from a T25 flask and 100 µL of 0.5% trypan blue solution for validation of good viable cells. The quantification of L929 mouse fibroblast cells was done using a hemocytometer. For quantification, the cells from four T25 flasks were collected, and the cell number was adjusted to a cell density of 104 cells/0.1 mL (200 µL). The viable fibroblast cells, after staining with trypan blue, were viewed under 60× using a hemocytometer.
Quantitative evaluation of the effect of different intracanal medicaments using cell proliferation assay (MTT assay): a total of 200 µL L929 mouse fibroblast cells were transferred to each well of 96 multi-well plates followed by 37°C CO2 incubation for 24 hours. A 50 µL of group I: nCaO, group II: nZnO, group III: nMgO, and group IV: metapaste, respectively, were added to the 96 wells in different concentration of 1500 ng (6 ng/µL), 1000 ng (4 ng/µL), 500 ng (2 ng/µL), 250 ng (1 ng/µL), 100 ng (0.4 ng/µL), 50 ng (0.2 ng/µL), 25 ng (0.1 ng/µL), 10 ng (0.04 ng/µL), 1 ng (0.004 ng/µL) followed by incubation of 96 well plates at 37°C in a CO2 incubator for 48 hours. After incubation MTT [3-(4,5-dimethylthiazol-2-yl)—2,5-diphenyltetrazolium bromide] reagent (10% of the total volume of the culture, which was 25 µL) was added to the plates. These wells were incubated for 24 hours at 37°C CO2 incubators to allow mitochondrial succinate dehydrogenases in viable cells to reduce intracellular soluble yellow tetrazolium dye MTT to insoluble violet formazan dye. The plates were viewed under an inverted microscope for the proliferative growth of fibroblast cells (Figs 1234).
A 100 µL of dissolution buffer was added to 96 well plates and kept in a biosafety sterile cabinet for 3 hours before taking absorbance at 570 nm. This MTT assay was performed in triplicates to get statistically significant results.
Sample Preparation to Evaluate Antimicrobial Activity
The entire in vitro experimental study was done in triplicate, where a standard strain of E. Faecalis (ATCC 29212) strain procured from Sigma Aldrich with a 0.5 McFarland scale was used to test intracanal medicaments.
To determine the ZOI, 25 mL of Mueller-Hinton agar (MHA) was melted to 100°C and cooled to 55°C. The agar was poured into a 90 mm diameter Petri plate. A single colony of E. Faecalis was added to 5 mL Luria broth and incubated at 37°C for 16 hours to form bacterial inoculum, and the turbidity was adjusted to 0.5. The bacterial inoculum was spread on an MHA plate, and wells of 5 mm diameter were bored at 3 mm apart using a sterile borer. The experimental groups of intracanal medicaments were mixed into PPG400 (50 µL) in concentrations of 30, 20, 10, 5, 2, 1, 0.5, 0.2, and 0.02 ng/µL and were transferred into the wells. The disks impregnated with cephalothin, cefpodoxime, ampicillin, and amikacin were placed on an MHA plate as positive control and were incubated at 37°C for 24 hours. The zones of inhibition (clear, circular halo without bacteria surrounding the sample of material) were measured from the periphery of the disc after 24 hours. Zone diameters were measured in millimeters by holding a vernier caliper on the back of the Petri plate illuminated with reflected light. This procedure was repeated three times for each group, and the mean value of the inhibition zones was reported.
To determine the MIC, a single colony of E. faecalis was inoculated into a 5 mL brain heart infusion (BHI) broth medium and was incubated for 16 hours. A 5% subculture (10 mL culture was added to 190 mL BHI broth) was prepared and placed in a shaking incubator. The absorbance was checked for culture at optical density (OD) 600, which was 0.432. A total of 2 mL of prepared broth was transferred into test tube followed by adding test groups in concentration range of 1500 (6 ng/µL), 1000 (4 ng/µL), 500 (2 ng/µL), 250 (1 ng/µL), 100 (0.4 ng/µL), 50 (0.2 ng/µL), 25 (0.1 ng/µL), 10 (0.04 ng/µL), and 1 ng (0.004 ng/µl). The 5 µl of overnight inoculated E. faecalis was transferred into each test tube following the incubation was done at 37°C for 24 hours. A 200 µL of inoculated media was removed from these test tubes and plated in a Petri plate following incubation absorbance at 600 nm was taken using a spectrophotometer.
Data were recorded, tabulated, and entered in Microsoft Excel (v.2021). Statistical analysis was performed using Statistical Package for the Social Sciences (SPSS) software (v.20.0). All statistical tests were performed at 95% confidence intervals, keeping a p-value of <0.05 as statistically significant. Descriptive statistics were performed for experimental parameters assessing amongst experimental groups.
In intragroup comparison, the order of proliferative activity of experimental groups on L929 mouse fibroblast cells using MTT assay was: metapaste > nCaO > nMgO > nZnO which showed higher statistically significant differences after analysis of variance (ANOVA) (p < 0.0002*) (Table 1). The student t-test was used for intergroup comparison of experimental groups during the evaluation of cytotoxic potential using MTT assay. It was observed that there was a higher statistically significant difference observed when group IV: metapaste was compared to other groups that included nCaO (p < 0.0021*), nZnO (p < 0.0001*), and nMgO (p < 0.0001*). The comparison between group I: nCaO and group III: nZnO showed significance (p: 0.0026*). Group I: nCaO, when compared with group III: nMgO (p: 0.05) and group II: nZnO with group III: nMgO, no statistically significant difference was not observed (p: 0.16) (Table 2). The proliferative activity of fibroblast was measured by absorbance (nm) of different concentrations of the experimental groups where group I: nCaO did not show cytotoxicity up to a concentration of 100 ng (0.4 ng/µL) above this concentration, cell proliferative activity was reported with a mean of 1.17 and standard deviation (SD) of 0.17 at an absorbance of 570 nm. Group II: nZnO showed a cytotoxic effect at 10 ng (0.04 ng/µL); above this concentration, there was a slight increase in OD value with a mean of 1.49 and SD of 0.21 at 570 nm. Group III: nMgO showed a cytotoxic effect at 10 ng (0.04 ng/µL); above this concentration, up to 500 ng (2 ng/µL), nMgO showed an inhibitory effect, but above 1000 ng (4 ng/µL) proliferative effect was reported with a mean of 1.35 and SD of 0.19 at an absorbance of 570 nm. Group IV: metapaste did not show any effect on treated cells up to a concentration of 500 ng (2 ng/µL); above this concentration, proliferative activity was seen. In addition, the cytotoxic activity of metapaste was not seen below 500 ng (2 ng/µL) (Fig. 5).
|MTT assay||Absorbance at 570 nm||ANOVA||p-value|
|Group I nCaO||1.17||0.17||19.97||0.0002*|
|Group II nZnO||1.49||0.21|
|Group III nMgO||1.35||0.19|
|Group IV Metapaste||0.8||0.25|
The data obtained from measuring the clear ZOI showed that group I: nCaO had the highest ZOI against E. Faecalis within the experimental groups, followed by group II–nZnO and group III: nMgO at a concentration of 30 ng/µL. No significant ZOI was shown by group IV: metapaste. In comparison with standard amikacin 30 µg, antibiotic-impregnated disk better results were obtained with group I: nCaO and group II: nZnO. The mixing vehicle PPG400 exhibits antimicrobial activity and is similar to group II: nZnO. In an intragroup comparison of the ZOI (mm), group I: nCaO showed a maximum ZOI with an average mean of 2.29, while in group II: nZnO mean average of 2.07, which was more in comparison to group III: nMgO with 0.93 and lowest ZOI was observed with group IV: metapaste with 0.89 and showed higher statistically significant differences between all four groups after ANOVA (p < 0.0001) (Table 3). Intergroup comparison showed a higher statistically significant difference using Kruskal–Wallis test when group I: nCaO when compared with group III: nMgO and group II: nZnO with group III: nMgO were seen to be highly significant (p < 0.0001*) (Table 4).
|Antimicrobial activity||Group I: nCaO||Group II: nZnO||Group III: nMgO||Group IV: metapaste||Test of significance||p-value|
|MIC (600 nm)||1.06||0.61||1.24||0.8||0.62||0.13||0.7||0.14||KW-value||0.0484*|
The data obtained from measuring MIC at 600 nm absorbance showed that group I: nCaO showed the highest antimicrobial activity against E. faecalis at a concentration of 1500 ng, where the bactericidal effect was observed. The bacteriostatic effect can be observed even at 1 ng, whereas in group II: nZnO showed the bactericidal effect at 1500 ng and a mild bacteriostatic effect from 1 to 100 ng, which was at the lower side when compared with nMgO and metapaste. Group III: nMgO showed the bactericidal effect at 1500 ng, and the bacteriostatic effect was observed at 50 ng and group IV: metapaste showed the bactericidal effect at 1500 ng, and the bacteriostatic effect was observed with 50 ng; this was at the lower side when compared with nCaO (Table 3) The intergroup comparison of MIC showed higher statistically significant difference using student t-test when group I: nCaO was compared group II: nZnO (p: 0.0036*) (Table 5 and Fig. 6). There was no statistical difference observed when other groups were compared.
The most commonly used Ca(OH)2-based medicaments release hydroxyl ions that lead to an increase in pH value within the canals and exhibit a direct effect on the cytoplasmic membrane, deoxyribonucleic acid (DNA), and enzymes of pathogens bacteria showing antimicrobial activity against endodontic pathogens, but when these medicaments come in contact with periapical cells, it causes irritation and lethal effect on these cells. Some controversies have been associated with Ca(OH)2 used against E. faecalis and Candida albicans in a study done by Mattigatti et al., there is decreased antimicrobial effect of Ca(OH)2 when compared with chlorhexidine and other agents.2 While in the experimental study conducted by Chen et al. showed that Ca(OH)2 was ineffective against biofilms containing E. faecalis.3 Various groups of NPs, like, organic, inorganic, and carbon-based, are used in the medical field, in which inorganic metal oxide NPs are commonly used in dental sciences.4
The intracanal medications containing NPs have to be biocompatible that are evaluated with colorimetric assay by using an MTT reagent. This is possible because MTT (3-[4, 5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) reagent is water-soluble yellow tetrazolium salt, which is converted to insoluble purple formazan by cleavage of tetrazolium ring by enzyme succinate dehydrogenase present within the mitochondria of fibroblast cells. These formazan crystals are impermeable to the cell membrane of fibroblast, causing them to accumulate within healthy cells.5 The degree of light absorption is dependent on the degree of formazan concentration accumulated on the surface and within the fibroblast cell. The higher concentration of purple formazan will show deeper purple color with higher absorbance.6 Hence, in our study, we have evaluated cytotoxicity following the international standard ISO 10993-5 (ISO 10993. 2009) using MTT assay.
Hirschman et al. used human gingival fibroblasts cell lines to assess pulp capping agents as these cells have proliferative potential and can be easily procured from periapical tissues and the periodontal ligament.7 Martínez-Cortés et al. used human gingival fibroblast (HGF) and L929 mouse fibroblast cells to evaluate the cytotoxicity and found that L929 mouse cell line fibroblasts show high sensitivity to toxic products and can be subcultured easily when compared to the HGF cell line.8 so in our study we have used the L929 mouse fibroblast cell line which was procured from NCCS, Pune. The cytotoxic of experiment groups is due to the potential of NPs to cause oxidative stress has been implicated as the main mechanism behind nanoparticle toxicity according to Nel et al.9 Another proposed mechanism by Singh et al. cytotoxicity could be due to DNA directly and inflammatory responses within the cell.10 In the study by Mahmoud et al., nMgO caused 50% inhibition in cell viability using an MTT assay in which maximum cell death (14.8%) was seen even at the highest concentration (500 µg/mL).11 Alsalleeh et al. reported the cytotoxic potential of metapaste on periodontal ligament cells viability in concentrations range of 1.0, 0.5, 0.25, and 0.125 mg/mL. The percentage of cell death seen with metapaste after 24 and 48 hours was a maximum of 0.125 mg/mL.12 whereas in our present study, it was found that metapaste did not show any effect on treated cells up to a concentration of 500 ng (2 ng/μL); above this concentration, proliferative activity was seen. In addition, the cytotoxic activity of metapaste was not seen below 500 ng (2 ng/μL).
The antibacterial mechanism of NPs is by damaging bacterial cell membranes where NPs bind to target bacterial cell membranes via electrostatic forces. This leads to changes in membrane potential, causing loss of polarity. The functions of microbial cell-like respiration, transportation of nutrients, and disturbance of energy transduction are disturbed that cause cell death. The second mechanism is the release of oxygen free radicals like reactive oxygen species that block the protein function and cause excess radical production, which leads to DNA damage.13 Another mechanism includes protein and enzyme dysfunction along with a disturbance in the regulation of metabolic functions of microbes when metal-based NPs are used; this causes retardation of the bacterial cell due to unrepairable destruction of bacterial DNA that leads to the death of bacteria. Other than all these mechanisms, NPs interconnect with the biopolymers through their electrical configurations, where it negatively influences the replication process of chromosome and plasmid DNA in the bacterial cell resulting in the inhibition of signal transduction.14
The study conducted by Sobrinho et al. concluded that E. faecalis was found to be persistent facultative anaerobes in periradicular infections and was seen in 4–40% of primary endodontic infections. The presence is nine-time higher in an endodontic flareup.15 The resistance of E. Faecalis to medicaments may be due to its ability to form a single-species biofilm and its penetration within dentinal tubules, as these species can undergo prolonged starvation and survival in alkaline environments at more alkaline pH.16 In our present study, we have used E. faecalis strain to evaluate the antimicrobial activity of novel intracanal medicaments. The study by Louwakul et al. reported the antibacterial efficiency of 50 µL of nCaO for 1 week and concluded that nCaO could be effectively used in the elimination of E. faecalis from dentinal tubules.17 Similar results were found in our study, where the inhibition zone of 2.29 mm for nCaO and 2.07 with nZnO; this was in agreement with a study conducted by Yousef et al. evaluated nZnO against different oral pathogens with MIC where nZnO showed to exhibit a good bacteriostatic effect but a poor bactericidal effect towards all tested microbial pathogens.18 In the study by Jhamb et al., the ZOI was highest for metapaste plus hexidine when compared to Ca(OH)2 against E. faecalis.19 Similarly, our study showed an inhibition zone of 0.89 mm for metapaste against E. faecalis. However, further studies are recommended if antibacterial effects have to be improved, and more in vivo evaluation is also required. To the best of our knowledge, the first time this combination of intracanal medicaments was tested and compared for their cytotoxicity, and antimicrobial efficacy.
Within the limitations of this study, the following conclusions were drawn:
The metapaste did not show any effect on treated L929 fibroblast cells up to a concentration of 500 ng, while for nCaO, there was no cytotoxic effect up to 100 ng. The cytotoxic effect was present at a concentration of 10 ng with nZnO and nMgO.
The highest ZOI against E. faecalis was seen with nCaO, and the lowest zone was observed with metapaste. The vehicle PPG400 also exhibits antimicrobial efficacy. The MIC of nCaO showed the highest antimicrobial activity at a concentration of 1500 ng and was lowest with nZnO and nMgO.
Nanoparticle mentioned in this study exhibit properties that are similar to the ideal properties of intracanal material hence using such modified medicament can help to combat E. faecalis and increases the success rate of endodontic therapy.
Prerna Barge https://orcid.org/0000-0002-4149-2174
Dr RS Santosh, Director, Phytocom Pharmaceuticals Pvt Ltd, Kalamassery, Kerala.
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