|
 
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| ORIGINAL RESEARCH |
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| Year : 2023 | Volume
: 15
| Issue : 4 | Page : 384-390 |
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Cytotoxicity evaluation of three different types of intracanal medications
Soha Adel Abdou1, Amira Ibrahim Mohamed2, Yousra Aly2
1 Dental Department, Research Institute of Ophthalmology (RIO), Giza, Egypt
2 Restorative and Dental Materials Department, National Research Centre, Dokki, Egypt
| Date of Submission | 22-Feb-2023 |
| Date of Decision | 10-Jul-2023 |
| Date of Acceptance | 10-Jul-2023 |
| Date of Web Publication | 31-Aug-2023 |
Correspondence Address:
Dr. Soha Adel Abdou
Dental Department, Research Institute of Ophthalmology (RIO), El Mokattam, Cairo 11571
Egypt
 Source of Support: None, Conflict of Interest: None  | Check |
DOI: 10.4103/jioh.jioh_50_23
Aim: The purpose of this research was to compare the cytotoxicity effect of two naturally based intracanal medications, which are silver nanoparticles (AgNPs) and AgNPs with curcumin, to the commonly utilized calcium hydroxide (Ca(OH)2). Materials and Methods: Evaluation was done using cell line in vitro. The cell line used in our study was Gingival Fibroblast cells (ATCC PCS-201-012). After 48 h of exposure, the water soluble tetrazolium salt (WST-1) assay was used to assess the vitality of the cells by Abcam kit. Fifty-four samples were categorized randomly into three groups in accordance with the type of intracanal medicament applied: group A—18 samples of AgNPs; group B—18 samples of AgNPs with curcumin; and group C—18 samples of Ca(OH)2. Each main group was subdivided randomly into six subgroups; three samples for each subgroup according to concentrations (Conc) used, which were 0.01, 0.1, 1, 10, and 100, and normal cells as a control. Statistical analysis was done using Shapiro–Wilk and Kolmogorov–Smirnov tests, paired sample t test, and one-way analysis of variance. Results: In Conc 0.01, the highest viability percentage of living cells with the least cytotoxicity percentage was found in the control group, then in group A, and then in group C, whereas the lowest viability percentage was detected in group B. I the water soluble tetrazolium salt (WST-1) assay was used n Conc 0.1, Conc 1, Conc 10, and Conc 100, the highest viability percentage of living cells was detected in the control group, then in group C, and then in group A; however, the lowest was detected in group B. Conclusion: All three tested intracanal medications were noncytotoxic. Keywords: Calcium Hydroxide, Curcumin, Cytotoxicity, Intracanal Medication, Silver Nanoparticles
How to cite this article:
Abdou SA, Mohamed AI, Aly Y. Cytotoxicity evaluation of three different types of intracanal medications. J Int Oral Health 2023;15:384-90 |
How to cite this URL:
Abdou SA, Mohamed AI, Aly Y. Cytotoxicity evaluation of three different types of intracanal medications. J Int Oral Health [serial online] 2023 [cited 2023 Sep 25];15:384-90. Available from: https://www.jioh.org/text.asp?2023/15/4/384/384663 |
| Introduction | |  |
The primary target of the endodontic treatment is to remove microorganisms of root canals.[1],[2] Root canal ramifications resulted in incomplete disinfection by mechanical preparation only.[3],[4] Therefore, intracanal medicaments use is an important step for achieving a complete disinfection process.[5]
The ideal properties of intracanal medicaments are to have high antimicrobial activity and low cytotoxicity to get rid of microorganisms without damaging periapical tissues.[6],[7]
Cytotoxicity was measured using many tests. One of the most accurate tests was water soluble tetrazolium salt (WST-1) assay. The succinate tetrazolium reductase in the mitochondria powers this second-generation tetrazolium derivative test. WST-1 is metabolized to products that do not need to solubilize, because the products were nontoxic, water-soluble, and membrane-permeable.[8]
Nanotechnology has deployed massively in the last decade, becoming a major component of medical sciences.[9],[10] Therefore, using silver nanoparticle (AgNP) products and silver-based products has become widespread in medicine and dental fields related to their antibacterial properties.[11]
Nanosilver means silver particles that have a proportion of less than one hundred nm on a three-dimensional scramble.[12] It has unrivaled chemical and physical characteristics such as nanoscale, spate particular surface area, powerful surface reaction, and innervate reaction through particles,[13] that leads to the vast use of nanosilver in multiple scopes, such as diagnostics, dentistry, and medicine.[14]
In comparison to the plain silver, the nanosilver has special biological characteristics, like high antimicrobial leverage. Adding nanosilver to toothpaste helps in the decontamination of the oral cavity and could be designed in jelly consistency to cure inflammations.[15] However, people are not conscious of all the poisonous hazards of nanosilver, techniques implicated in their toxic influence, and possibilities to alter their cytotoxic action.
Recently, intensive researches on curcumin have proved it to have antibacterial, antiviral, antifungal, and anti-inflammatory effects.[16],[17]
They were safe when used in therapeutic purposes, but their application was intricate. They have disadvantages such as fast metabolism, bad bioavailability, and incomplete absorption.[18] Therefore, we need to combine the benefits of AgNPs and curcumin in one intracanal medication (AgNPs with curcumin) and compare its cytotoxicity effect with AgNPs when used alone, and with a more commonly utilized intracanal medication calcium hydroxide (Ca(OH)2). Our study’s null hypothesis was that three tested intracanal medicaments were noncytotoxic.
| Materials and Methods | | |
Calculating the sample size
Calculation of sample size was done instituted on a prior study[19] as a guide. Using P.S. Power version 3.1.2, the minimum acceptable sample size for our research was 3 for each group when the response within each group had a normal distribution with a standard deviation of 1.39, an estimated mean difference was 4 when power was 80%, and type I error probability was 0.05.
Cell culture
Evaluation was done using fibroblast cells of animal gingiva (ATCC PCS-201-012) in vitro. The holding company for biological products and vaccines (VACSERA, Cairo, Egypt) laboratory’s Tissue Culture Department provided the cells. Cells were kept alive in Dulbecco’s modified Eagle medium at 37°C in a humidified 5% (v/v) CO2 atmosphere with 100 units/mL of penicillin, 100 mg/mL of streptomycin, and 10% of heat-inactivated fetal bovine serum. Refreshment process was done to medium every 2 days, then it became twice weekly when the outgrowth of the cells was recorded. The duration of the study was 5 weeks. Preparation of the cells takes 4 weeks, and the testing procedure takes 1 week.
Grouping and study design of tested materials
Our study was a prospective study. There were no inclusion or exclusion criteria for study sample collection. Fifty-four samples were divided randomly into three main groups according to the applied type of intracanal medicament: group A, 18 samples of AgNPs (Nanogate company, Cairo, Egypt); group B, 18 samples of AgNPs with curcumin (Nanogate company); and group C, 18 samples of Ca(OH)2 (Metapaste, META BIOMED, Korea). Each main group was subdivided randomly into six subgroups; three samples for each subgroup according to concentrations (Conc) used, which were 0.01, 0.1, 1, 10, and 100, and normal cells as a control.
Each of group A, group B, and group C was subdivided into six subgroups: control group (three samples), Conc 0.01 (three samples), Conc 0.1 (three samples), Conc 1 (three samples), Conc 10 (three samples), and Conc 100 (three samples).
Metapaste is composed of Ca(OH)2, iodoform, and silicon oil.
The manufacturing of AgNPs and AgNPs with curcumin
According to Pal et al.,[20] AgNPs have been manufactured by a chemical reduction process. By microwave irradiating a silver nitrate (AgNO3) solution in ethanolic medium and utilizing polyvinyl pyrrolidone as a stabilizing factor, AgNPs were produced. It has been seen that ethanol acts as a reducing factor in the existence of microwave. The solvent was steamed at room temperature to obtain the silver (Ag) in powder form. After that, the suspension was diluted with carboxymethyl cellulose (CMC) paste, 5% by volume (1 mL Ag suspension + 4 mL CMC), to obtain 100 ppm semipaste.
AgNPs with curcumin preparation
A total of 5 mg of curcumin was sprinkled within 1 mL silver suspension before increment of CMC paste.
Characterization
Obtaining ultraviolet-visible spectroscopy absorption spectra was achieved by using Ocean Optics USB2000+VIS-NIR fiber optics spectrophotometer.
Transmission electron microscope was used for the measurement of shape and size. It was performed on JEOL JEM-2100 high resolution with an accelerating voltage of 200 kV.
Properties
AgNPs with curcumin and AgNPs alone were yellow in color and in the form of semipaste. Their size was 12 ± 3 nm and spherical in shape with optical properties: λmax ~405 nm.
Cytotoxicity assay
Cell viability was performed in NAWAH Scientific lab (Cairo, Egypt) and assessed by WST-1 assay using Abcam kit (United Kingdom) (ab 155902 WST-1 cell proliferation reagent). This test depends on the splitting of a tetrazolium salt to soluble formazan dye because of the mitochondrial dehydrogenase of living cells. Equal amounts of 50-μL cell suspension (3 × 103 cells) were cultured in 96-well plates and placed in an incubator for 24 h. Each one of the tested intracanal medicaments was tested by 103 animal gingival fibroblast cells. Another aliquot of 50-μL media containing the three tested intracanal medications (AgNPs with Curcumin, AgNPs, and Ca(OH)2) at five serial concentrations (0.01, 0.1, 1, 10, 100) was put on the cells. Cells were treated with 10 μL of WST-1 reagent after 48 h of drug exposure, and a BMG LABTECH-FLUO star Omega microplate reader Allmendgrün, Ortenberg, scaled the absorbance after an hour at 450 nm.
Statistical analysis
In every group, mean values and standard deviations were computed. Study results were checked for their normality via Shapiro–Wilk and Kolmogorov–Smirnov tests that revealed normal ranking. The comparison among two groups in relative samples was done by paired sample t test. In unrelated samples, one-way analysis of variance was employed to compare data from more than two groups. The significance level was defined at P ≤ 0.05. With IBM SPSS (USA) Statistics Version 20 for Windows, statistical analyses were carried out.
| Results | | |
Effect of different concentrations
Group A: (AgNPs)
Significant statistical disparity existed among Conc 0.01, Conc 0.1, Conc 1, Conc 10, and Conc 100 where P = 0.011 [Table 1] and [Figure 1][Figure 2][Figure 3][Figure 4]. | Table 1: Standard deviation and the mean values of cytotoxicity of tested groups
Click here to view |
 | Figure 4: Cells by electron microscope (×100): (A) normal cells, (B) cells after treatment with 10 conc. AgNPs, (C) cells after treatment with 10 conc. AgNPs with curcumin, and (D) cells after treatment with 10 conc. Ca(OH)2
Click here to view |
Significant statistical disparity existed among Conc 0.01 and each of Conc 0.1, Conc 1, Conc 10, and Conc 100 where P = 0.041, P = 0.032, P = 0.018, and P = 0.014 respectively.
There was no significant statistical difference among Conc 0.1, Conc 1, Conc 10, and Conc 100.
The highest viability percentage of living cells with the least cytotoxicity percentage was found in Conc 0.01, whereas the least viability percentage of living cells with the highest cytotoxicity percentage was found in Conc 100.
Group B: (AgNPs with curcumin) and group C: (Ca(OH)2)
There was no significant statistical difference among different concentrations where P = 0.942 and P = 0.095.
In both groups, the highest viability percentage of living cells with the least cytotoxicity percentage was found in Conc 0.01, whereas the least viability percentage of living cells with the highest cytotoxicity percentage was found in Conc 100.
Effect of different groups
In Conc 0.01
No significant statistical difference was found among normal cells (control group) and every of AgNPs (group A) and Ca(OH)2 (group C) as P = 0.671 and P = 0.168, whereas a significant statistical difference was detected among normal cells (control group) and AgNPs with curcumin (group B) as P < 0.001 [Table 1].
Significant statistical disparity existed among AgNPs with curcumin (group B) and each of AgNPs (group A), and Ca(OH)2 (group C) as P < 0.001 and P = 0.001 sequentially.
No significant statistical difference was found between AgNPs group (group A) and Ca(OH)2 group (group C) as P = 0.651.
The highest viability percentage of living cells was detected in normal cells (control group), then in AgNPs group (group A), followed by Ca(OH)2 group (group C), whereas the least viability percentage of living cells with the highest cytotoxicity percentage was found in AgNPs with curcumin (group B).
In Conc 0.1 and Conc 1
Significant statistical disparity existed among normal cells (control group) and every of AgNPs (group A) and AgNPs with curcumin (group B) as P = 0.014 and P = 0.001 in Conc 0.1, whereas P = 0.005 and P < 0.001 in Conc 1. However, no significant statistical difference was detected among normal cells (control group) and Ca(OH)2 (group C) as P = 0.195 in Conc 0.1, whereas P = 0.079 in Conc 1.
No significant statistical difference was found among AgNPs (group A) and each of AgNPs with curcumin (group B), and Ca(OH)2 (group C) as P = 0.210 and P = 0.308 in Conc 0.1, whereas P = 0.116 and P = 0.250 in Conc 1.
There was a significant statistical difference among AgNPs with curcumin and Ca(OH)2 as P = 0.016 in Conc 0.1 and P = 0.007 in Conc 1.
In Conc 10 and Conc 100
Significant statistical disparity existed among the control group and every of group A, group B, and group C as P < 0.001.
No significant statistical difference was found among group A and each of group B and group C as P = 0.059 and P = 0.639 in Conc 10, whereas P = 0.055 and P = 0.836 in Conc 100.
Significant statistical disparity existed among group B and group C as P = 0.011 in Conc 10 and P = 0.017 in Conc 100.
In Conc 0.1, Conc 1, Conc 10 and Conc 100
The highest viability percent of living cells was detected in normal cells (control group), then in Ca(OH)2 group (group C), followed by AgNPs group (group A), whereas the least viability percentage of living cells with the highest cytotoxicity percentage was found in AgNPs with curcumin group (group B).
| Discussion | | |
Our research was targeting the comparison of the cytotoxicity effect of two naturally based intracanal medications, which are AgNPs and AgNPs with curcumin to the commonly used Ca(OH)2.
Compounds containing silver were used for antimicrobial purposes,[15] as they have high stability, strong antimicrobial activity, and broad antibacterial spectrum.
Newly, it was proved that silver ions reduce the growth of microorganisms by linking to thiol units (-SH) in enzymes leading to their crippling. Silver creates steady S-Ag linked with compounds consisting of thiol in the cell membrane, which are implicated in the reproduction of energy and ion transmission.[11]
Nanotechnology has started a new beginning in material amelioration for better clinical outcomes. Adding nanosilver to the obturation products inhibits the spread of bacteria in root canals because of its antimicrobial activity and biocompatibility.[21]
Previous study[22] reported that AgNPs and AgNPs with curcumin showed an antibacterial effect superior than Ca(OH)2 when utilized as intracanal medication. That is why our research was carried on to assess the cytotoxicity of these intracanal medications compared with Ca(OH)2.
ISO (10993-5, 2009) recommends using established cell lines for in vitro cytotoxicity tests for better reproducibility.[23] Animal fibroblast cells were selected for this research.[19],[24],[25],[26],[27]
In our research, WST-1 assay was chosen to examine the cells' viability. As its technique is easy, unharmed, owing a high reproducibility, it is vastly utilized giving accurate results. Moreover, phenol red index in a medium containing cell culture does not intervene with the dye response and the colored dye that was generated at the terminus of test is water dissolvable and nontoxic.[28]
The finding of our research clarified that the highest viability percentage of living cells with the least cytotoxicity percentage was found in AgNPs Conc 0.01 (group A) followed by Ca(OH)2 Conc 0.01 (group C) This could be a result of choosing the exact concentration of AgNPs, as silver has to be utilized with wariness as its toxicity usually depends on its concentration.[29] Panáček et al.[30] demonstrated that AgNPs used for medical purposes in low concentrations were not cytotoxic to mammalian tissues. The cytotoxicity of AgNPs was related to their amount of free Ag ions.[31] Takamiya et al.[25] evaluated AgNPs cytotoxicity on mouse fibroblasts and proved that AgNPs with a concentration less than 25 µg/mL were not cytotoxic. A recent review by Mallineni et al.[32] stated that AgNPs in dentistry are well characterized by preventing bacteria from making biofilms and that they showed a great capability to stop bacterial metabolism. They concluded that AgNPs can be used safely for humans as they were found to be biocompatible with mammalian cells.
This result was in accordance with the results of Gomes-Filho et al.[33] who examined tissue’s reaction to surgically implanted polyethylene tubes containing fibrin sponge and immersed in a dispersion of low concentration of AgNPs. The results showed that AgNPs dispersion was biocompatible and produced a mild tissue reaction. Also, this result confirmed Zand et al.[34] results, which proved that the biocompatibility comparison between the mineral trioxide aggregate (MTA) and MTA with AgNPs was of no significant difference. Furthermore, this result was in coincidence with Zhang et al.[26] conclusions that compared toxicity of nanosilver base antibacterial agents at different concentrations. It was found that nanosilver base antibacterial agents are more biocompatible and less cytotoxic as the concentration decreased. Results reported by Chan et al.[27] also revealed that nanosilver irrigant has noncytotoxic effect on mouse fibroblast cell line NIH 3T3 and human periodontal ligament stem cells. Dose-response correlation was detected between nanosilver irrigant dilutions and the cell viability of the tested cells.
The highest viability percentage of living cells with the least cytotoxicity percentage was found in Ca(OH)2 0.1, 1, 10, and 100 (group C) followed by AgNPs 0.1, 1, 10, and 100 (group A). This may be related to using high concentrations of AgNPs as the high concentration of AgNPs is a key parameter in evaluating the applicability. It may give rise to inverse health impact.[29] Many in vitro researches proved that AgNPs in a high concentration could cause an oxidative pressure that produces free radicals, which was gathered in the cell nuclei and cytoplasm and impair the function of mitochondria of human tissues.[29],[35] Owing to its nano size, which results in more contact area, AgNPs had the ability to change the regular activity of bioactive particles and eukaryotic tissues.[36] Our outcome was consistent with Afkhami et al.[37] results, which proved that the concentration of AgNPs plays a major role in its cytotoxicity as using low concentrations of AgNPs in the endodontic field showed less cytotoxicity.
Ca(OH)2 is the gold criterion intracanal medicament, which has antimicrobial activity, and promotes mineralized tissue formation.[5],[38] Low concentration of Ca(OH)2 controls the extracellular phosphorylated signal-correlated kinases that is an indicator for the multiplying of stem cells of periodontal ligaments and teeth pulp.[39] Alsalleeh et al.[40] proved that there was a correlation among the cytotoxicity, concentration, and potential of hydrogen (PH). Based on that, Ca(OH)2 in low concentration had low PH and, hence, low cytotoxicity. The findings of this research agreed with the outcomes of Bhandi et al.[41] who declared that the exposure of 25 µg/mL Ca(OH)2 to dental pulp stem cells for 48 h did not involve its viability. Also, our findings agreed with the results of Gonçalves et al.[24] who stated that Ca(OH)2 was not cytotoxic when tested on fibroblast cells after 1 and 3 days of exposure. Moreover, Da Silva et al.[42] stated that medications that contained Ca(OH)2 or not were biocompatible and noncytotoxic when implanted in the subcutaneous tissues of experimental rats. Our result was disagreeing with the results of Hussein et al.[7] who declared that Ca(OH)2 presented moderate cytotoxicity when used as intracanal medicaments at a concentration of 0.5 mg/mL. This may be due to the difference in concentration of Ca(OH)2 used and difference in technique used for testing the toxicity.
Also, least viability percent of living cells with highest cytotoxicity percentage was found in AgNPs with curcumin (group B) at all five concentrations (0.01, 0.1, 1, 10, and 100), but it was considered to be biocompatible and noncytotoxic as its viability percentage was more than 70% as recommended by the ISO (10993-5, 2009).[43] The anticytotoxic effect of curcumin was produced by altering oxidative stress elements as reactive oxygen species and releasing antioxidant genes. There was a direct correlation between curcumin concentration and the appearance of antioxidant enzymes such as superoxide dismutase and catalase.[44] The results of Mandroli et al.[45] and this finding were comparable, which confirmed that curcumin induced cell viability and promoted dental pulp fibroblast proliferation. And with findings of Samiei et al.,[44] which proved that curcumin nanocrystals did not exhibit a cytotoxic effect on dental pulp stem cells DPSCs in various concentrations and tested time periods.
At all concentrations of curcumin, no cytotoxic effect was revealed. Ownership of these wished biological actions combined with its safety use, and low cost makes it a charming agent for further research to be employed for regenerative endodontic treatment on permanent teeth as well as vital pulp therapy on both primary and permanent teeth. As curcumin enhances cells’ vitality and primary pulp fibroblast proliferating, it could be considered a dependable medication for vital pulp therapy.[46]
Within the limitations of our study, AgNPs alone or with curcumin can be considered as a better intracanal medicaments compared with Ca(OH)2 paste regarding cytotoxicity. Our future scope was to make further evaluations in animal models for AgNPs alone and with curcumin for usage tests. As we are aiming to have new intracanal medicaments with superior properties to increase the percentage of the success rate of endodontic treatment, we recommend evaluating the genotoxicity and cytotoxicity of our materials using other procedures to evaluate our results. The hypothesis of the study was confirmed because our study proved that three tested intracanal medicaments were noncytotoxic.
| Conclusions | | |
- 1All three tested intracanal medications were noncytotoxic.
- 2The percentage of cell viability of AgNPs with curcumin group was lower than AgNPs group and Ca(OH)2 group, but it is still noncytotoxic.
Acknowledgement
Not applicable.
Financial support and sponsorship
Nil.
Conflicts of interest
There were no conflicts of interest.
Authors contributions
SAA and YA: Conceiving the idea and designing the study. SAA: data acquisition, preparing the samples, performing the test, interpretation of the statistical results, and manuscript writing. SAA, YA and AIM: Manuscript revision. The manuscript has been read and approved by all the authors. Finally, all authors had given approval for the manuscript publication.
Ethical policy and institutional review board statement
Our experimental research was approved by the Medical Research Ethics Committee MREC, National Research Centre NRC, Giza, Egypt, on September 2, 2021. Approval number 3439102021.
Patient declaration of consent
Not applicable.
Data availability statement
Raw data are available from the corresponding author upon reasonable request.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4]
[Table 1]
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