An in-vitro evaluation of cytotoxicity of fungal derived nanosilver particle endodontic irrigant on human periodontal ligament fibroblast cells.

Rahul S Halkai; Kiran R Halkai; Shishir Ram Shetty; Raghavendra M Shetty; Sunaina Shetty; Prathibha Prasad

doi:10.4103/sej.sej_223_20

ORIGINAL ARTICLE

Year : 2021 | Volume : 11 | Issue : 3 | Page : 364-368

An in-vitro evaluation of cytotoxicity of fungal derived nanosilver particle endodontic irrigant on human periodontal ligament fibroblast cells.

Rahul S Halkai¹, Kiran R Halkai¹, Shishir Ram Shetty², Raghavendra M Shetty³, Sunaina Shetty⁴, Prathibha Prasad⁵
¹ Department of Conservative Dentistry and Endodontics, Al- Badar Rural Dental College and Hospital, Kalaburgi, Karnataka, India
² Department of Oral &Craniofacial Health Sciences, College of Dental Medicine, University of Sharjah, Sharjah, United Arab Emirates
³ Department of Growth and Development, College of Dentistry, Ajman University, Ajman, United Arab Emirates
⁴ Department of Preventive and Restorative Dentistry, College of Dental Medicine, University of Sharjah, Sharjah, United Arab Emirates
⁵ Department of Oral pathology, Ras Al Khaimah College of Dental Sciences, Ras Al Khaimah Medical and Health Sciences University, Ras Al Khaimah, United Arab Emirates

Date of Submission	26-Aug-2020
Date of Decision	28-Oct-2020
Date of Acceptance	28-Nov-2020
Date of Web Publication	3-Sep-2021

Correspondence Address:
Dr. Kiran R Halkai
Reader, Department of Conservative Dentistry and Endodontics, Al-Badar Rural Dental College & Hospital, Kalaburgi, Karnataka
India

Source of Support: None, Conflict of Interest: None

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DOI: 10.4103/sej.sej_223_20

Abstract

Introduction: Recently, silver nanoparticles were indicated for root canal irrigation. The cytotoxicity evaluation of these agents helps to ascertain confident clinical use. This article aims at evaluating the cytotoxic effect of fungal-derived nanosilver particle irrigant on human periodontal ligament fibroblast (hPDLF) cells using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay.
Materials and Methods: The test “nanoparticle” irrigant was produced using the fungi “Fusarium semitectum.” The cells were cultured in Dulbecco's Modified Eagle's Medium solution and adjusted to 5 × 10³ cells/ml. About 100 μl of cells were seeded into a 96 well microplate and 100 μl test irrigant ranging from 5 to 640 μg/ml concentrations was added to a microplate and incubated at 37°C, 5% CO₂ humidified conditions for 24 h. Untreated cells were used as a control group. About 5 mg/ml MTT was added to the plates and incubated at 37°C in 5% CO₂ conditions for 4 h. The viability of cells and the percentage inhibition of cell were determined.
Results: The 50% inhibition of cells was found to be around 320 μg/ml concentration. Cytotoxicity was found to be dose dependent and increased with higher concentrations of the nanosolution.
Conclusion: Fungal-derived nanosilver irrigant is safe to hPDLF cells “in vitro” at a concentration of <320 μg/ml.

Keywords: Biosynthesized silver nanoparticles, cell culture, cytotoxicity, endodontic disinfection, periodontal ligament fibroblast cells

How to cite this article:
Halkai RS, Halkai KR, Shetty SR, Shetty RM, Shetty S, Prasad P. An in-vitro evaluation of cytotoxicity of fungal derived nanosilver particle endodontic irrigant on human periodontal ligament fibroblast cells. Saudi Endod J 2021;11:364-8

How to cite this URL:
Halkai RS, Halkai KR, Shetty SR, Shetty RM, Shetty S, Prasad P. An in-vitro evaluation of cytotoxicity of fungal derived nanosilver particle endodontic irrigant on human periodontal ligament fibroblast cells. Saudi Endod J [serial online] 2021 [cited 2022 May 18];11:364-8. Available from: https://www.saudiendodj.com/text.asp?2021/11/3/364/325400

Introduction

Rapid advancements in nanotechnology have led to a significant increase in the application of nanoparticles, especially the silver nanoparticles (AgNPs) in various fields including dentistry.^[1] AgNPs possess unique physicochemical and biological properties. Owing to the smaller particle size (usually 1–100 nm) and large surface area, the antimicrobial properties of AgNPs are significantly superior to the solid form.^[2],[3] AgNPs penetrate tissues due to their small particle size and impart antimicrobial activity for a longer duration with a considerably less amount of the agent.^[4] This advantage is generally not achievable with conventional antimicrobial agents.^[5] Recently, a rapid inclination toward the biosynthesized AgNPs is seen in the field of nanoparticles due to their eco-friendly, nonhazards nature, cost-effectiveness, and inherent medicinal values.^[2],[6]

Biosynthesized AgNPs are produced using natural entities such as plants, bacteria, fungi, algae, yeast, and viruses.^[7] Among these, fungal-derived AgNPs are used as effective antimicrobial agents.^[8] Among the many advantages of fungal-derived products is the ease with which large scale culture and production can be achieved in a shorter time. Besides, there are added advantages such as an as simple process and lower technique sensitivity.^[9] Another distinct advantage is the fungal-derived enzymes such as nitrate reductase, that act as naturally occurring reducing and capping agents for AgNPs and thus help in the stabilization of nanoparticles.^[10]

Several nanoparticles have been recommended as root canal irrigants and intracanal medicaments.^[1],[6] Recently, studies have revealed that fungal-derived AgNPs are exhibit effective antimicrobial activity against resistant microorganisms such as Enterococcus faecalis, Porphyromonas gingivalis, and their biofilms. These studies have recommended the application of AgNPs for root canal disinfection as irrigant and intracanal medication.^[8],[11] However, literature regarding the biocompatibility/cytotoxicity studies of biosynthesized AgNPs in endodontics is relatively sparse.

In an attempt to fill in this lacuna of knowledge, this in vitro study was conducted to evaluate the cytotoxic effects of the fungal-derived nanosilver particles solution on primary human periodontal ligament fibroblast (hPDLF) cells using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay.

Materials and Methods

The present study was approved by the Institutional Ethical Committee (Reference no HKES/SNDCG/2015-2016/1610) and performed accordingly. The fungal-derived nanosilver solution was biosynthesized using freshly isolated colonies of endophytic fungi, Fusarium semitectum, and characterized by using ultraviolet spectrum (T90+ UV–Vis, USA), transmission electron microscope (JEOL/JEM 2100, USA), fourier transform infrared spectroscopy (Thermo Nicolet Avatar 370, USA) as described earlier in previous studies.^[8],[11] The nanoparticles were found to be 1–10 nm in size and used as a solution. The pH of the solution was measured as 7.4 and stored in a refrigerator at 4°C until use. Before being added to the cell suspension, the temperature of the prepared nanosilver solution was elevated to 37°C in a warm water bath.

The cytotoxic effect of nanosilver solution was evaluated by MTT assay on hPDLF cells. The cells were procured from the National Center for cell lines, Pune, Maharashtra, India. Dulbecco's Modified Eagle's Medium solution (DMEM), MTT, and trypsin were obtained from Sigma Aldrich Co, St Louis, USA. Dimethyl sulfoxide (DMSO) and propanol were obtained from E. Merck Ltd., Mumbai, Maharashtra, India. Glucose, ethylenediaminetetraacetic acid (EDTA), antibiotics, and the remaining all other chemicals were obtained from Hi-Media Laboratories Ltd., Mumbai, Maharashtra, India.

For the preparation of nanoparticle test solution, the freshly prepared AgNPs test irrigant was dissolved in distilled DMSO, and the volume was made up with DMEM solution supplemented with 2% inactivated fetal bovine serum (FBS) for obtaining a stock solution of 1 mg/ml concentration. Serial two-fold dilutions were prepared from this to carry out the cytotoxic studies of different concentration of test solution ranging from 5 to 640 μg/ml.

For the determination of cytotoxicity by MTT assay, the stock cells were cultured in DMEM solution supplemented with 100 mg/ml of streptomycin, 100 IU/mL of penicillin, inactivated 10% FBS in a humidified atmosphere of 5% CO₂ at 37°C in a CO₂ incubator (Thermo Scientific BBD 6220, USA) until lavish growth. The cells were then suspended in a phosphate-buffered solution containing 0.05% glucose, 0.2% trypsin, and 0.02% EDTA to dissociate and disaggregate the adherent cells. The cells were washed by resuspending in 5 mL of sterile phosphate-buffered solution. The cells were centrifuged in a benchtop centrifuge (Thermo Scientific, Heraeus Multifuge X1, USA) at 200 relative centrifugal forces for 5 min and subcultured in a T-75 cm² tissue culture flask (Sigma Aldrich Co, Mumbai, Maharashtra, India). The cell viability was checked under an inverted microscope (Model IX73, Olympus Corporation, Tokyo, Japan) at × 40 magnification. The cell count was adjusted to 5 × 10⁵ cells/mL using DMEM solution containing 10% FBS, and about 100 ml of the diluted cells (approximately about 50,000 cells/well) were placed in 96 well microtiter plates (Tarsons India Pvt. Ltd., Kolkata, West Bengal, India) and incubated for 24 h in a humidified atmosphere of 5% CO₂ at 37°C. Since the experiment was carried out in triplets for different concentrations of test irrigant altogether 27 wells were used including the control group. After 24 h, a monolayer was formed on the culture microplates. The supernatant was discarded, and the monolayer of cells was washed with the DMEM solution and 100 μl of each of the different test concentrations of nano solution were then inoculated into the 96 well microplates containing the monolayer and incubation was carried out in 5% CO₂ atmosphere for 24 h at 37°C. An equal volume of cells in culture media without test irrigant (untreated cells) was used as a control group.

After 24 h, the tested solutions in the microplate wells were discarded, and the culture medium containing 5 mg/ml MTT was added to each microplate well and further incubated for about 4 h at 37°C. The superficial layer was discarded and 100 ml of DMSO was added to the microplates and slightly shaken to dissolve the formed formazan. The cells were observed under a microscope at ×40 magnification [Figure 1]a, [Figure 1]b, [Figure 1]c. A microplate reader (BMR-100, Boeco, Hamburg, Germany) was used to measure the absorbance and optical density at 540 nm wavelength. The experiment was repeated in triplets, and the average of three readings was calculated as the final reading. The concentration of the sample tolerated by 50% of the cultures exposed (CTC ₅₀) “lethal dose” of nanoirrigant that inhibits the cellular growth by 50% was determined using the following formula, and the percentage growth inhibition was calculated.

Figure 1: Microscopic observation of human periodontal ligament cells. (a) Normal cell line (b) At 320 μg/ml conc. of nanosilver irrigant (CTC50) cells were seen with normal morphology with a spindle shaped and few cells exhibited cell lysis (c) At 640 μg/ml concentration of nano silver irrigant, most of the cells were oval in shape with altered cell structure with reduced cytoplasmic processes, loss of cellular components exhibiting cell lysis

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Results

The control group showed no change in cellular activity as no test solution was added [Table 1]. The CTC₅₀ that prevents cell growth by 50% was recorded to be around 320 μg/ml with 46.8% inhibition. Highest percentage inhibition 64.2% was found at 640 μg/ml. At a dose of 160 μg/ml, 30.2% inhibition was seen, and the lowest of 1.8% at 5 μg/ml. The cytotoxic effects were higher for the nanoparticle solution with higher concentration range at 640 μg/ml. The microscopic observations of cells before treatment with nanoparticle solution revealed the cells were flat with dendroid or spindle shape, arranged in monolayer with normal cell spacing and full cellular structures, clear nuclei and two or three cytoplasmic processes [Figure 1]a. A change in morphology of the cells such as change in the shape from spindle to oval, detachment of the cells, cell exhibited contraction and became sparse with reduced cytoplasmic processes and loss of cellular components and cell lysis at higher dose of 640 μg/ml with the appearance of formazan crystals within the cells [Figure 1]c. At 320 μg/ml (CTC₅₀) cells were seen with normal morphology with a spindle shaped and few cells exhibited cell lysis [Figure 1]b.

Table 1: Percentage inhibition of human periodontal ligament cells by nanosilver particle at different concentration

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Discussion

Apical periodontitis is usually caused by bacteria and bacterial products in the root canal system extending into periapical tissues. This makes the periodontal tissues in the periapical area susceptible to pulpo-periapical lesions.^[12]

The primary objective of the endodontic treatment comprises merely of complete eradication of the microorganisms and to prevent reinfection.^[13] Factors such as root canal complexities, biofilm-mediated endodontic infection, the persistence of the bacteria like E. faecalis into dentinal tubules, pose a challenge for the success of endodontic therapy.^[13] Another major disadvantage is the inability of most of the irrigants to reach these difficult areas. All these factors cause the persistence of bacteria within the root canal system leading to persistent infections and reinfections.^[12],[13]

In the present study, cytotoxicity was determined by using MTT assay as it is considered to be the gold standard for determining the cell proliferation and viability/cytotoxicity of the new test drugs in initial stages.^[14] It is appropriate for high-throughput screening with high accuracy and reproducibility of the obtained results.^[15] It is commonly employed to determine the cytotoxicity of dental materials.^[16] It has been widely used due to simple, rapid procedure, with low cost and high precision. The major advantage of this method is that it requires fewer cells and multiple sample concentrations can be used on a single 96 microwell plate; hence, it is very easy to perform. Since the reduction of MTT occurs in living cells and cellular reduction is catalyzed only by metabolically active cells; hence, it allows to distinguish nonviable from viable cells by microscopic analysis.^[17] It measures the formation of insoluble purple formazan by mitochondrial succinate dehydrogenase of the viable cells reducing the yellow-colored tetrazolium salt MTT which dissolves in water. The insoluble formazan can be dissolved by DMSO or solvents such as isopropanol and propanol. The resulting purple solution is measured under a spectrophotometer. The change in the cell numbers is dependent on the amount of formazan formed, which indicates the degree of cytotoxic effects of the experimental agent.^[16]

Primary/diploid human cells, mainly oral fibroblasts, are considered to be biologically relevant cell lines for cytotoxicity evaluation for dental materials.^[18] Anatomically, the root is surrounded by the periodontal ligament and during root canal irrigation the irrigating solution might accidentally penetrates into the periapical area causing tissue irritation. Hence, evidence about the effect of root canal irrigants on periapical tissues is essential for the effective use of novel irrigants.^[19] The present study focused on hPDLF cells for cytotoxic evaluation. According to the recommendations of ISO (10,993–52,009) if the number of viable cells is <70%, then the testing material/agent is regarded to be cytotoxic to that specific cell type and considered to be as nonbiocompatible.^[19] In the present study, fungal derived nanosilver particle endodontic irrigant was found to be least cytotoxic to the hPDLF cells. It was found that the amount of cytotoxic effects directly correlates with the dose of AgNP nanoirrigant. The solution was biocompatible at lower doses, which is in accordance with the previous study showing AgNPs to be safe to the cells “in vitro” at a concentration lower than 260 micrograms/milliliter.^[20]

Chan et al. showed that nanosilver irrigant was biocompatible to hPDLF cells. The 50% lethal doses of nanosilver solution for hPDLF cells were found to be 0.608 after direct exposure to the testing agent for 48 h.^[19] The cytotoxic effect of silver includes the chemical interactions of silver atoms with the functional groups of intracellular protein and with the nitrogen and phosphate groups of DNA and physical interactions with the cell wall and the intracellular components leading to the disintegration of the cell wall and nuclear components leading ultimately to cell death.^[21]

The microscopic observations in the present study revealed morphological changes of the cells after treatment with different doses of nano solution. At CTC₅₀ most of the cells were seen with normal morphology and few cells exhibited cell lysis as described above. These findings are in accordance to Chan et al.^[19]

The present study shows that fungal-derived nanosilver solution is biocompatible at lower doses to the periodontal cells and can be recommended as an alternative endodontic irrigant for root canal disinfection. However, there is a further need for studies for possible silver dissolution of fungal derived nanosilver solution and its effect on root dentin. Its ability to remove the smear layer, its effect on the strength of root canal dentin, any discoloration of root dentin, possible interactions with other root canal irrigants, need further in vitro and in vivo studies for these novel solution can be effectively and confidently used for root canal disinfection.

Limitations of the study

In spite of being commonly employed for cytotoxic evaluation of the new drugs or agents on the cells, the MTT assay has certain limitations that might affect the accuracy of results which must be considered such as it shows the highest variation of the CTC₅₀ concentrations when observed in the linear range, it causes decrease in nicotinamide adenine dinucloetide (NADH) or NADH phosphate or D-glucose in culture medium might result in decreased formazan production which is directly related to the number of viable cells. MTT might activate the factors related to apoptosis or hasten the cell content leakage after the formation of formazan. The culture techniques and medium used and sterilization measures to prevent the contamination could also affect the results. Therefore, this method has to be carefully performed, which otherwise leads to the underestimation of cell viability. Since, it is a basic quantitative technique often used for initial studies to select the range of concentrations (CTC₅₀) and the effective drugs hence, used for the present study. However, other cytotoxic evaluation techniques and further in vivo and in vitro studies should be considered further for effective clinical application of this newer nanoparticle solution.

Conclusion

Fungal-derived nanosilver solution is safe to hPDLF cells “in vitro” at a concentration of <320 μg/ml.

Acknowledgments

The authors would like to thank Dr. Vandana Rathod, Department of Microbiology, Gulbarga University, Karnataka for the laboratory support and Sophisticated Test and Instrumentation Centre, Cochin University of Science and Technology, Cochin, India for technical support.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

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